Publications

• [ 2024 ]
• [ 2023 ]
• [ 2022 ]
• [ 2021 ]
• [ 2020 ]
• [ 2019 ]
• [ 2018 ]
• [ 2017 ]

• hnRNP R promotes O-GlcNAcylation of eIF4G and facilitates axonal protein synthesis. Zare, Abdolhossein; Salehi, Saeede; Bader, Jakob; Schneider, Cornelius; Fischer, Utz; Veh, Alexander; Arampatzi, Panagiota; Mann, Matthias; Briese, Michael; Sendtner, Michael. In Nature Communications, 15(1), p. 7430. 2024.
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  Motoneurons critically depend on precise spatial and temporal control of translation for axon growth and the establishment and maintenance of neuromuscular connections. While defects in local translation have been implicated in the pathogenesis of motoneuron disorders, little is known about the mechanisms regulating axonal protein synthesis. Here, we report that motoneurons derived from Hnrnpr knockout mice show reduced axon growth accompanied by lowered synthesis of cytoskeletal and synaptic components in axons. Mutant mice display denervated neuromuscular junctions and impaired motor behavior. In axons, hnRNP R is a component of translation initiation complexes and, through interaction with O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (Ogt), modulates O-GlcNAcylation of eIF4G. Restoring axonal O-GlcNAc levels rescued local protein synthesis and axon growth defects of hnRNP R knockout motoneurons. Together, these findings demonstrate a function of hnRNP R in controlling the local production of key factors required for axon growth and formation of neuromuscular innervations.
• Transcriptome-scale analysis uncovers conserved residues in the hydrophobic core of the bacterial RNA chaperone Hfq required for small regulatory RNA stability. McQuail, Josh; Krepl, Miroslav; Katsuya-Gaviria, Kai; Tabib-Salazar, Aline; Burchell, Lynn; Bischler, Thorsten; Gräfenhan, Tom; Brear, Paul; Šponer, Jiří; Luisi, Ben F.; Wigneshweraraj, Sivaramesh. In bioRxiv. Cold Spring Harbor Laboratory, 2024.
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  The RNA-chaperone Hfq plays crucial roles in bacterial gene expression and is a major facilitator of small regulatory RNA (sRNA) action. The toroidal molecular architecture of the Hfq hexamer contains three well characterised surfaces which allow it to bind sRNAs to stabilise them and engage target transcripts. Hfq-interacting sRNAs are categorised into two classes based on the surfaces they use to bind Hfq. By characterising a systematic alanine mutant library of Hfq to identify amino acid residues that impact survival of Escherichia coli experiencing nitrogen starvation, we corroborated the important role of the three RNA binding surfaces for Hfq function. Surprisingly, we uncovered two conserved residues, V22 and G34, in the hydrophobic core of Hfq, to have a profound impact on Hfq{\textquoteright}s RNA binding activity in vivo. Transcriptome-scale analysis revealed that V22A and G34A Hfq mutants cause widespread destabilisation of both sRNA classes. However, the alanine substitutions at these residues had no measurable impact on protein stability, structure or equilibrium binding to target sRNAs in vitro. We propose that V22 and G34 are key to the cooperative function among the RNA-binding surfaces of Hfq, a mechanism especially critical under cellular conditions when there is an increased demand for Hfq.Competing Interest StatementThe authors have declared no competing interest.
• Human dorsal root ganglia are either preserved or completely lost after deafferentation by brachial plexus injury. Sodmann, Annemarie; Degenbeck, Johannes; Aue, Annemarie; Schindehütte, Magnus; Schlott, Felicitas; Arampatzi, Panagiota; Bischler, Thorsten; Schneider, Max; Brack, Alexander; Monoranu, Camelia M.; Gräfenhan, Tom; Bohnert, Michael; Pham, Mirko; Antoniadis, Gregor; Blum, Robert; Rittner, Heike L. In British Journal of Anaesthesia. Elsevier BV, 2024.
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  Background Plexus injury results in lifelong suffering from flaccid paralysis, sensory loss, and intractable pain. For this clinical problem, regenerative medicine concepts set high expectations. However, it is largely unknown how dorsal root ganglia (DRG) are affected by accidental deafferentation. Methods Here, we phenotyped DRG of a clinically and MRI-characterised cohort of 13 patients with plexus injury. Avulsed DRG were collected during reconstructive nerve surgery. For control, we used DRG from forensic autopsy. The cellular composition of the DRG was analysed in histopathological slices with multicolour high-resolution immunohistochemistry, tile microscopy, and deep-learning-based bioimage analysis. We then sequenced the bulk RNA of corresponding DRG slices. Results In about half of the patients we found loss of the typical DRG units consisting of neurones and satellite glial cells. The DRG cells were replaced by mesodermal/connective tissue. In the remaining patients, the cellular units were well preserved. Preoperative plexus MRI neurography was not able to distinguish the two types. Patients with ‘neuronal preservation’ had less maximum pain than patients with ‘neuronal loss’. Arm function improved after nerve reconstruction, but severe pain persisted. Transcriptome analysis of preserved DRGs revealed expression of subtype-specific sensory neurone marker genes, but downregulation of neuronal attributes. Furthermore, they showed signs of ongoing inflammation and connective tissue remodelling. Conclusions Patients with plexus injury separate into two groups with either neuronal preservation or neuronal loss. The former could benefit from anti-inflammatory therapy. For the latter, studies should explore mechanisms of neuronal loss especially for regenerative approaches.
• Internal in-frame translation generates Cas11b, which is important for effective interference in an archaeal CRISPR-Cas system. Sailer, A.-L.; Brendel, J.; Chernev, A.; König, S.; Bischler, T.; Gräfenhan, T.; Urlaub, H.; Gophna, U.; Marchfelder, A. In bioRxiv. Cold Spring Harbor Laboratory, 2024.
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  CRISPR-Cas is a sophisticated defence system used by bacteria and archaea to fend off invaders. CRISPR-Cas systems vary in their Cas protein composition and have therefore been divided into different classes and types. Type I systems of bacteria have been shown to contain the small Cas11 protein as part of the interference complex. Here we show for the first time that an archaeal CRISPR-Cas type I system also contains a Cas11 protein. In addition, we show for the first time an internal in-frame translation of an archaeal protein. The Cas11b protein from the Haloferax volcanii type I-B system is encoded in the cas8b gene. Translation initiation at an internal methionine of the cas8b open reading frame results in synthesis of Cas11b. Cas11b is required for an effective interference reaction and without Cas11b fewer Cascade complexes form. Comparison of transcriptomes from wild type and a Cas11b less strain show that the depletion of Cas11b results in differential regulation of many genes. Taken together Cas11b is important for the defence reaction of the type I-B CRISPR-Cas system and seems to play an additional cellular role.Competing Interest StatementThe authors have declared no competing interest.
• Neuronal toxicity and recovery from early bortezomib-induced neuropathy: targeting the blood nerve barrier but not the dorsal root ganglion. Atalla, Mariam Sobhy; Bettenhausen, Anna-Lena; Verse, Julius M.; Cebulla, Nadine; Krug, Susanne M.; Sauer, Reine-Solange; Srivastava, Mugdha; Bischler, Thorsten; Chen, Jeremy T.C.; Kortüm, K. Martin; Kittel, Robert J.; Sommer, Claudia; Rittner, Heike L. In bioRxiv. Cold Spring Harbor Laboratory, 2024.
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  The use of the first in class proteasome inhibitor Bortezomib (BTZ) is highly effective in the treatment of multiple myeloma. However, it{\textquoteright}s long-term use is limited by the fact, that most treated patients develop dose limiting painful polyneuropathy. In some of the treated patients, pain resolves after variable timeframes, in others it persists, despite the discontinuation of treatment, with the underlying mechanisms poorly understood. One condition of neural toxicity is the ability to penetrate the blood nerve barrier. Here we present pathways involved in early bortezomib-induced polyneuropathy (BIPN) development and its resolution, in rats and in myeloma patients. One cycle of BTZ elicited transient mechanical hyperalgesia and cold allodynia in rats. Transcriptomic signature and network analysis revealed regulation of circadian, extracellular matrix, and immune genes within the nerve and modest changes in the dorsal root ganglia. Recovery processes resealed the small molecule leakiness of the perineurial barrier, reversed axonal swelling, and normalized small fiber density in the skin. Expression of the microtubule-associated cytoskeletal protein cortactin matched this process in the perineurium. Netrin-1 (Ntn1) as a known barrier sealer was also upregulated in pain resolution in nerve and skin. In patients with painful BIPN skin NTN1 was independent of axonal damage. In summary, our data demonstrate that early BTZ toxicity targets mainly the nerve and indicates that pain resolution could be supported by protective growth factors like Ntn1 for remodeling of the extracellular matrix and neuronal barriers.Summary Bortezomib leads to dose-limiting painful polyneuropathy. Already in the first cycle, BTZ toxicity weakens the blood nerve barrier which reseals upon upregulation of netrin-1.Competing Interest StatementThe authors have declared no competing interest.
• Cellular landscape of adrenocortical carcinoma at single-nuclei resolution. Tourigny, David S.; Altieri, Barbara; Secener, Kerim A.; Sbiera, Silviu; Schauer, Marc P.; Arampatzi, Panagiota; Herterich, Sabine; Sauer, Sascha; Fassnacht, Martin; Ronchi, Cristina L. In Molecular and Cellular Endocrinology, 590, p. 112272. Elsevier BV, 2024.
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  Adrenocortical carcinoma (ACC) is a rare yet devastating tumour of the adrenal gland with a molecular pathology that remains incompletely understood. To gain novel insights into the cellular landscape of ACC, we generated single-nuclei RNA sequencing (snRNA-seq) data sets from twelve ACC tumour samples and analysed these alongside snRNA-seq data sets from normal adrenal glands (NAGs). We find the ACC tumour microenvironment to be relatively devoid of immune cells compared to NAG tissues, consistent with known high tumour purity values for ACC as an immunologically "cold" tumour. Our analysis identifies three separate groups of ACC samples that are characterised by different relative compositions of adrenocortical cell types. These include cell populations that are specifically enriched in the most clinically aggressive and hormonally active tumours, displaying hallmarks of reorganised cell mechanobiology and dysregulated steroidogenesis, respectively. We also identified and validated a population of mitotically active adrenocortical cells that strongly overexpress genes POLQ, DIAPH3 and EZH2 to support tumour expansion alongside an LGR4+ progenitor-like or cell-of-origin candidate for adrenocortical carcinogenesis. Trajectory inference suggests the fate adopted by malignant adrenocortical cells upon differentiation is associated with the copy number or allelic balance state of the imprinted DLK1/MEG3 genomic locus, which we verified by assessing bulk tumour DNA methylation status. In conclusion, our results therefore provide new insights into the clinical and cellular heterogeneity of ACC, revealing how genetic perturbations to healthy adrenocortical renewal and zonation provide a molecular basis for disease pathogenesis.
• Plekhg5 controls the unconventional secretion of Sod1 by presynaptic secretory autophagy. Hutchings, Amy-Jayne; Hambrecht, Bita; Veh, Alexander; Giridhar, Neha Jadhav; Zare, Abdolhossein; Angerer, Christina; Ohnesorge, Thorben; Schenke, Maren; Selvaraj, Bhuvaneish T.; Chandran, Siddharthan; Sterneckert, Jared; Petri, Susanne; Seeger, Bettina; Briese, Michael; Stigloher, Christian; Bischler, Thorsten; Hermann, Andreas; Damme, Markus; Sendtner, Michael; Lüningschrör, Patrick. In Nature Communications, 15(1), p. 8622. 2024.
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  Increasing evidence suggests an essential function for autophagy in unconventional protein secretion (UPS). However, despite its relevance for the secretion of aggregate-prone proteins, the mechanisms of secretory autophagy in neurons have remained elusive. Here we show that the lower motoneuron disease-associated guanine exchange factor Plekhg5 drives the UPS of Sod1. Mechanistically, Sod1 is sequestered into autophagosomal carriers, which subsequently fuse with secretory lysosomal-related organelles (LROs). Exocytosis of LROs to release Sod1 into the extracellular milieu requires the activation of the small GTPase Rab26 by Plekhg5. Deletion of Plekhg5 in mice leads to the accumulation of Sod1 in LROs at swollen presynaptic sites. A reduced secretion of toxic ALS-linked SOD1G93A following deletion of Plekhg5 in SOD1G93A mice accelerated disease onset while prolonging survival due to an attenuated microglia activation. Using human iPSC-derived motoneurons we show that reduced levels of PLEKHG5 cause an impaired secretion of ALS-linked SOD1. Our findings highlight an unexpected pathophysiological mechanism that converges two motoneuron disease-associated proteins into a common pathway.
• Gene-environment interaction elicits dystonia-like features and impaired translational regulation in a DYT-TOR1A mouse model. Reinhold, Colette; Knorr, Susanne; McFleder, Rhonda L.; Rauschenberger, Lisa; Muthuraman, Muthuraman; Arampatzi, Panagiota; Gräfenhan, Tom; Schlosser, Andreas; Sendtner, Michael; Volkmann, Jens; Ip, Chi Wang. In Neurobiology of Disease, p. 106453. Elsevier BV, 2024.
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  DYT-TOR1A dystonia is the most common monogenic dystonia characterized by involuntary muscle contractions and lack of therapeutic options. Despite some insights into its etiology, the disease’s pathophysiology remains unclear. The reduced penetrance of about 30% suggests that extragenetic factors are needed to develop a dystonic phenotype. In order to systematically investigate this hypothesis, we induced a sciatic nerve crush injury in a genetically predisposed DYT-TOR1A mouse model (DYT1KI) to evoke a dystonic phenotype. Subsequently, we employed a multi-omic approach to uncover novel pathophysiological pathways that might be responsible for this condition. Using an unbiased deep-learning-based characterization of the dystonic phenotype showed that nerve-injured DYT1KI animals exhibited significantly more dystonia-like movements (DLM) compared to naive DYT1KI animals. This finding was noticeable as early as two weeks following the surgical procedure. Furthermore, nerve injured DYT1KI mice displayed significantly more DLM than nerve-injured wildtype (wt) animals starting at 6 weeks post injury. In the cerebellum of nerve-injured wt mice, multi-omic analysis pointed towards regulation in translation related processes. These observations were not made in the cerebellum of nerve-injured DYT1KI mice; instead, they were localized to the cortex and striatum. Our findings indicate a lack of effective translational rescue mechanisms in the cerebellum of phenotypic DYT1KI mice that exhibit DLM, while translation dysregulations in the cortex and striatum likely contribute to the dystonic phenotype.
• Single‐nucleus and spatial transcriptome reveal adrenal homeostasis in normal and tumoural adrenal glands. Altieri, Barbara; Secener, A. Kerim; Sai, Somesh; Fischer, Cornelius; Sbiera, Silviu; Arampatzi, Panagiota; Kircher, Stefan; Herterich, Sabine; Landwehr, Laura‐Sophie; Vitcetz, Sarah N.; Braeuning, Caroline; Fassnacht, Martin; Ronchi, Cristina L.; Sauer, Sascha. In Clinical and Translational Medicine, 14(8). Wiley, 2024.
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  The human adrenal gland is a complex endocrine tissue. Studies on adrenal renewal have been limited to animal models or human foetuses. Enhancing our understanding of adult human adrenal homeostasis is crucial for gaining insights into the pathogenesis of adrenal diseases, such as adrenocortical tumours. Here, we present a comprehensive cellular genomics analysis of the adult human normal adrenal gland, combining single-nuclei RNA sequencing and spatial transcriptome data to reconstruct adrenal gland homeostasis. As expected, we identified primary cells of the various zones of the adrenal cortex and medulla, but we also uncovered additional cell types. They constitute the adrenal microenvironment, including immune cells, mostly composed of a large population of M2 macrophages, and new cell populations, including different subpopulations of vascular-endothelial cells and cortical-neuroendocrine cells. Utilizing spatial transcriptome and pseudotime trajectory analysis, we support evidence of the centripetal dynamics of adrenocortical cell maintenance and the essential role played by Wnt/β-catenin, sonic hedgehog, and fibroblast growth factor pathways in the adult adrenocortical homeostasis. Furthermore, we compared single-nuclei transcriptional profiles obtained from six healthy adrenal glands and twelve adrenocortical adenomas. This analysis unveiled a notable heterogeneity in cell populations within the adenoma samples. In addition, we identified six distinct adenoma-specific clusters, each with varying distributions based on steroid profiles and tumour mutational status. Overall, our results provide novel insights into adrenal homeostasis and molecular mechanisms potentially underlying early adrenocortical tumorigenesis and/or autonomous steroid secretion. Our cell atlas represents a powerful resource to investigate other adrenal-related pathologies.
• Inactivity of Stat3 in sensory and non-sensory cells of the mature cochlea. Bieniussa, Linda; Stolte, Clara; Engert, Jonas; Arampatzi, Panagiota; Völker, Johannes; Hagen, Rudolf; Hackenberg, Stephan; Rak, Kristen. In Frontiers in Molecular Neuroscience, 17. 2024.
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  Signal transducer and activator of transcription 3 (Stat3) plays a role in various cellular processes such as differentiation, inflammation, cell survival and microtubule dynamics, depending on the cell type and the activated signaling pathway. Stat3 is highly expressed in the hair cells and supporting cells of the cochlea and is essential for the differentiation of mouse hair cells in the early embryonic stage. However, it is unclear how Stat3 contributes to the correct function of cells in the organ of Corti postnatally. To investigate this, an inducible Cre/loxp system was used to knock out Stat3 in either the outer hair cells or the supporting cells. The results showed that the absence of Stat3 in either the outer hair cells or the supporting cells resulted in hearing loss without altering the morphology of the organ of Corti. Molecular analysis of the outer hair cells revealed an inflammatory process with increased cytokine production and upregulation of the NF-kB pathway. However, the absence of Stat3 in the supporting cells resulted in reduced microtubule stability. In conclusion, Stat3 is a critical protein for the sensory epithelium of the cochlea and hearing and functions in a cell and function-specific manner.
• Sequential antigen loss and branching evolution in lymphoma after CD19- and CD20-targeted T-cell–redirecting therapy. Duell, Johannes; Leipold, Alexander M.; Appenzeller, Silke; Fuhr, Viktoria; Rauert-Wunderlich, Hilka; Da Via, Matteo; Dietrich, Oliver; Toussaint, Christophe; Imdahl, Fabian; Eisele, Florian; Afrin, Nazia; Grundheber, Lars; Einsele, Hermann; Weinhold, Niels; Rosenwald, Andreas; Topp, Max S.; Saliba, Antoine-Emmanuel; Rasche, Leo. In Blood, 143(8), pp. 685–696. 2024.
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  CD19 chimeric antigen receptor (CAR) T cells and CD20 targeting T-cell–engaging bispecific antibodies (bispecs) have been approved in B-cell non-Hodgkin lymphoma lately, heralding a new clinical setting in which patients are treated with both approaches, sequentially. The aim of our study was to investigate the selective pressure of CD19- and CD20-directed therapy on the clonal architecture in lymphoma. Using a broad analytical pipeline on 28 longitudinally collected specimen from 7 patients, we identified truncating mutations in the gene encoding CD20 conferring antigen loss in 80% of patients relapsing from CD20 bispecs. Pronounced T-cell exhaustion was identified in cases with progressive disease and retained CD20 expression. We also confirmed CD19 loss after CAR T-cell therapy and reported the case of sequential CD19 and CD20 loss. We observed branching evolution with re-emergence of CD20+ subclones at later time points and spatial heterogeneity for CD20 expression in response to targeted therapy. Our results highlight immunotherapy as not only an evolutionary bottleneck selecting for antigen loss variants but also complex evolutionary pathways underlying disease progression from these novel therapies.
• Atypical cellular responses mediated by intracellular constitutive active TrkB (NTRK2) kinase domains and a solely intracellular NTRK2-fusion oncogene. Gupta, Rohini; Dittmeier, Melanie; Wohlleben, Gisela; Nickl, Vera; Bischler, Thorsten; Luzak, Vanessa; Wegat, Vanessa; Doll, Dennis; Sodmann, Annemarie; Bady, Elena; Langlhofer, Georg; Wachter, Britta; Havlicek, Steven; Gupta, Jahnve; Horn, Evi; Lüningschrör, Patrick; Villmann, Carmen; Polat, Bülent; Wischhusen, Jörg; Monoranu, Camelia M.; Kuper, Jochen; Blum, Robert. In Cancer Gene Therapy. 2024.
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  Trk (NTRK) receptor and NTRK gene fusions are oncogenic drivers of a wide variety of tumors. Although Trk receptors are typically activated at the cell surface, signaling of constitutive active Trk and diverse intracellular NTRK fusion oncogenes is barely investigated. Here, we show that a high intracellular abundance is sufficient for neurotrophin-independent, constitutive activation of TrkB kinase domains. In HEK293 cells, constitutive active TrkB kinase and an intracellular NTRK2-fusion oncogene (SQSTM1-NTRK2) reduced actin filopodia dynamics, phosphorylated FAK, and altered the cell morphology. Atypical cellular responses could be mimicked with the intracellular kinase domain, which did not activate the Trk-associated MAPK/ERK pathway. In glioblastoma-like U87MG cells, expression of TrkB or SQSTM1-NTRK2 reduced cell motility and caused drastic changes in the transcriptome. Clinically approved Trk inhibitors or mutating Y705 in the kinase domain, blocked the cellular effects and transcriptome changes. Atypical signaling was also seen for TrkA and TrkC. Moreover, hallmarks of atypical pTrk kinase were found in biopsies of Nestin-positive glioblastoma. Therefore, we suggest Western blot-like immunoassay screening of NTRK-related (brain) tumor biopsies to identify patients with atypical panTrk or phosphoTrk signals. Such patients could be candidates for treatment with NTRK inhibitors such as Larotrectinhib or Entrectinhib.
• Circulating NK cells establish tissue residency upon acute infection of skin and mediate accelerated effector responses to secondary infection. Torcellan, Tommaso; Friedrich, Christin; Doucet-Ladevèze, Rémi; Ossner, Thomas; Solé, Virgínia Visaconill; Riedmann, Sofie; Ugur, Milas; Imdahl, Fabian; Rosshart, Stephan P.; Arnold, Sebastian J.; Gomez de Agüero, Mercedes; Gagliani, Nicola; Flavell, Richard A.; Backes, Simone; Kastenmüller, Wolfgang; Gasteiger, Georg. In Immunity, 57(1), pp. 124–140.e7. 2024.
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  Summary Natural killer (NK) cells are present in the circulation and can also be found residing in tissues, and these populations exhibit distinct developmental requirements and are thought to differ in terms of ontogeny. Here, we investigate whether circulating conventional NK (cNK) cells can develop into long-lived tissue-resident NK (trNK) cells following acute infections. We found that viral and bacterial infections of the skin triggered the recruitment of cNK cells and their differentiation into Tcf1hiCD69hi trNK cells that share transcriptional similarity with CD56brightTCF1hi NK cells in human tissues. Skin trNK cells arose from interferon (IFN)-γ-producing effector cells and required restricted expression of the transcriptional regulator Blimp1 to optimize Tcf1-dependent trNK cell formation. Upon secondary infection, trNK cells rapidly gained effector function and mediated an accelerated NK cell response. Thus, cNK cells redistribute and permanently position at sites of previous infection via a mechanism promoting tissue residency that is distinct from Hobit-dependent developmental paths of NK cells and ILC1 seeding tissues during ontogeny.
• Clonal competition assays identify fitness signatures in cancer progression and resistance in multiple myeloma. Haertle, Larissa; Munawar, Umair; Hernández, Hipólito N. C.; Arroyo‐Barea, Andres; Heckel, Tobias; Cuenca, Isabel; Martin, Lucia; Höschle, Carlotta; Müller, Nicole; Vogt, Cornelia; Bischler, Thorsten; del Campo, Paula L.; Han, Seungbin; Buenache, Natalia; Zhou, Xiang; Bassermann, Florian; Waldschmidt, Johannes; Steinbrunn, Torsten; Rasche, Leo; Stühmer, Thorsten; Martinez‐Lopez, Joaquin; Martin Kortüm, K.; Barrio, Santiago. In HemaSphere, 8(7). Wiley, 2024.
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  Multiple myeloma (MM) is a genetically heterogeneous disease and the management of relapses is one of the biggest clinical challenges. TP53 alterations are established high-risk markers and are included in the current disease staging criteria. KRAS is the most frequently mutated gene affecting around 20% of MM patients. Applying Clonal Competition Assays (CCA) by co-culturing color-labeled genetically modified cell models, we recently showed that mono- and biallelic alterations in TP53 transmit a fitness advantage to the cells. Here, we report a similar dynamic for two mutations in KRAS (G12A and A146T), providing a biological rationale for the high frequency of KRAS and TP53 alterations at MM relapse. Resistance mutations, on the other hand, did not endow MM cells with a general fitness advantage but rather presented a disadvantage compared to the wild-type. CUL4B KO and IKZF1 A152T transmit resistance against immunomodulatory agents, PSMB5 A20T to proteasome inhibition. However, MM cells harboring such lesions only outcompete the culture in the presence of the respective drug. To better prevent the selection of clones with the potential of inducing relapse, these results argue in favor of treatment-free breaks or a switch of the drug class given as maintenance therapy. In summary, the fitness benefit of TP53 and KRAS mutations was not treatment-related, unlike patient-derived drug resistance alterations that may only induce an advantage under treatment. CCAs are suitable models for the study of clonal evolution and competitive (dis)advantages conveyed by a specific genetic lesion of interest, and their dependence on external factors such as the treatment.
• Provirus deletion from Haloferax volcanii affects motility, stress resistance and CRISPR RNA expression. Di Cianni, Nadia; Bolsinger, Simon; Brendel, Jutta; Raabe, Monika; Koenig, Sabine; Mitchell, Laura; Bischler, Thorsten; Gräfenhan, Tom; Read, Clarissa; Erdmann, Susanne; Allers, Thorsten; Walther, Paul; Urlaub, Henning; Dyall-Smith, Mike; Pfeiffer, Friedhelm; Marchfelder, Anita. In bioRxiv. Cold Spring Harbor Laboratory, 2024.
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  Haloferax volcanii harbours four putative proviruses: Halfvol1, Halfvol2, Halfvol3 and Halfvol4. In this study we successfully deleted all four provirus genomes, demonstrating, that they are not essential. Transcriptome comparison between this strain (ΔHalfvol1-4) and a wild type strain reveals an increase in archaella and chemotaxis gene expression, resulting in higher swarming motility in ΔHalfvol1-4. Furthermore, ΔHalfvol1-4 cells show an elongated cell shape and a higher resistance to H2O2 stress compared to the wild type. RNA-seq also revealed down-regulation of CRISPR arrays in the provirus-free strain. Circularised genomes of Halfvol1, Halfvol2 and Halfvol3 were found in the culture supernatant. This confirms excision of the proviruses from the chromosome, which seems to happen more efficiently at low temperature (30°C). Electron microscopy revealed potential viral particles in the supernatant, and mass spectrometry analysis confirmed the presence of structural viral proteins of Halfvol1 and Halfvol3 in the isolated virus sample. These observations suggest that these proviruses are active and cause a chronic infection in Hfx. volcanii.
• Satellite glial cells from adult DRG dedifferentiate in vitro and can be reprogrammed into nociceptor-like neurons. Sodmann, Annemarie; Köhler, Niels; Esfahani, Nastaran M.; Schukraft, Nina; Aue, Annemarie; Jager, Sara E.; Bischler, Thorsten; Imdahl, Fabian; Gräfenhan, Tom; Leipold, Enrico; Rittner, Heike L.; Blum, Robert. In bioRxiv. Cold Spring Harbor Laboratory, 2024.
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  Loss of sensory neurons in the dorsal root ganglia (DRG) may be a cause of neuropathic pain following traumatic nerve lesion or surgery. To regenerate peripheral sensory neurons, satellite glial cells (SGCs) may be an attractive endogenous cell source. SGCs are known to acquire certain neural progenitor-like properties after injury and are derived from the same neural crest lineage as sensory neurons. Here, we found that adult mouse DRG harbor SGC-like cells that dedifferentiate into glial sensory progenitor cells in vitro. Surprisingly, forced coexpression of the early developmental transcription factors Neurog1 and Neurog2 was sufficient to induce neuronal and glial cell phenotypes. In the presence of nerve growth factor, the induced neurons developed a nociceptor phenotype characterized by functional expression of marker ion channels such as TrpA1, TrpV1 and TTX-resistant NaV channels. Our study demonstrates that glial cells harvested from the adult DRG have neural stem cell-like properties, are multipotent, and may be useful for future neural repair strategies in the peripheral nervous system.Summary statement The adult dorsal root ganglion carries a satellite glial cell source for generation of induced nociceptor-like neurons. The cells dedifferentiate in vitro and acquire properties of a multipotent peripheral neural progenitor.Competing Interest StatementThe authors have declared no competing interest.

• Microglia-mediated demyelination protects against CD8+ T cell-driven axon degeneration in mice carrying PLP defects. Groh, Janos; Abdelwahab, Tassnim; Kattimani, Yogita; Hörner, Michaela; Loserth, Silke; Gudi, Viktoria; Adalbert, Robert; Imdahl, Fabian; Saliba, Antoine-Emmanuel; Coleman, Michael; Stangel, Martin; Simons, Mikael; Martini, Rudolf. In Nature Communications, 14(1), p. 6911. 2023.
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  Axon degeneration and functional decline in myelin diseases are often attributed to loss of myelin but their relation is not fully understood. Perturbed myelinating glia can instigate chronic neuroinflammation and contribute to demyelination and axonal damage. Here we study mice with distinct defects in the proteolipid protein 1 gene that develop axonal damage which is driven by cytotoxic T cells targeting myelinating oligodendrocytes. We show that persistent ensheathment with perturbed myelin poses a risk for axon degeneration, neuron loss, and behavioral decline. We demonstrate that CD8+ T cell-driven axonal damage is less likely to progress towards degeneration when axons are efficiently demyelinated by activated microglia. Mechanistically, we show that cytotoxic T cell effector molecules induce cytoskeletal alterations within myelinating glia and aberrant actomyosin constriction of axons at paranodal domains. Our study identifies detrimental axon-glia-immune interactions which promote neurodegeneration and possible therapeutic targets for disorders associated with myelin defects and neuroinflammation.
• Cytotoxic CNS-associated Tcells drive axon degeneration by targeting perturbed oligodendrocytes in PLP1 mutant mice. Abdelwahab, Tassnim; Stadler, David; Knöpper, Konrad; Arampatzi, Panagiota; Saliba, Antoine-Emmanuel; Kastenmüller, Wolfgang; Martini, Rudolf; Groh, Janos. In iScience, 26(5), p. 106698. Elsevier BV, 2023.
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  Myelin defects lead to neurological dysfunction in various diseases and in normal aging. Chronic neuroinflammation often contributes to axon-myelin damage in these conditions and can be initiated and/or sustained by perturbed myelinating glia. We have previously shown that distinct PLP1 mutations result in neurodegeneration that is largely driven by adaptive immune cells. Here we characterize CD8+ CNS-associated T cells in myelin mutants using single-cell transcriptomics and identify population heterogeneity and disease-associated changes. We demonstrate that early sphingosine-1-phosphate receptor modulation attenuates T cell recruitment and neural damage, while later targeting of CNS-associated T cell populations is inefficient. Applying bone marrow chimerism and utilizing random X chromosome inactivation, we provide evidence that axonal damage is driven by cytotoxic, antigen specific CD8+ T cells that target mutant myelinating oligodendrocytes. These findings offer insights into neural-immune interactions and are of translational relevance for neurological conditions associated with myelin defects and neuroinflammation.
• An interferon gamma response signature links myocardial aging and immunosenescence. Ashour, DiyaaElDin; Rebs, Sabine; Arampatzi, Panagiota; Saliba, Antoine-Emmanuel; Dudek, Jan; Schulz, Richard; Hofmann, Ulrich; Frantz, Stefan; Cochain, Clément; Streckfuß-Bömeke, Katrin; Ramos, Gustavo Campos. In Cardiovascular Research. Oxford University Press (OUP), 2023.
  • [ Abstract ]
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  Aims: Aging entails profound immunological transformations that can impact myocardial homeostasis and predispose to heart failure. However, preclinical research in the immune-cardiology field is mostly conducted in young healthy animals, which potentially weakens its translational relevance. Herein, we sought to investigate how the aging T-cell compartment associates with changes in myocardial cell biology in aged mice. Methods and results: We phenotyped the antigen-experienced effector/memory T cells purified from heart-draining lymph nodes of 2-, 6-, 12-, and 18-month-old C57BL/6J mice using single-cell RNA/T cell receptor (TCR) sequencing (sc-seq). Simultaneously, we profiled all non-cardiomyocyte cell subsets purified from 2- and 18-month-old hearts and integrated our data with publicly available cardiomyocyte sc-seq datasets. Some of these findings were confirmed at the protein level by flow cytometry. With aging, the heart-draining lymph node and myocardial T cells underwent clonal expansion and exhibited an up-regulated pro-inflammatory transcription signature, marked by an increased interferon-γ (IFN-γ) production. In parallel, all major myocardial cell populations showed increased IFN-γ responsive signature with aging. In the aged cardiomyocytes, a stronger IFN-γ response signature was paralleled by the dampening of expression levels of transcripts related to most metabolic pathways, especially oxidative phosphorylation. Likewise, induced pluripotent stem cells-derived cardiomyocytes (iPSC-CM) exposed to chronic, low grade IFN-γ treatment showed a similar inhibition of metabolic activity. Conclusions: By investigating the paired age-related alterations in the T cells found in the heart and its draining lymph nodes, we provide evidence for increased myocardial IFN-γ signaling with age, which is associated with inflammatory and metabolic shifts typically seen in heart failure.
• Lymph node medulla regulates the spatiotemporal unfolding of resident dendritic cell networks. Ugur, Milas; Labios, R. Jacob; Fenton, Chloe; Knöpper, Konrad; Jobin, Katarzyna; Imdahl, Fabian; Golda, Gosia; Hoh, Kathrin; Grafen, Anika; Kaisho, Tsuneyasu; Saliba, Antoine-Emmanuel; Grün, Dominic; Gasteiger, Georg; Bajénoff, Marc; Kastenmüller, Wolfgang. In Immunity, 56(8), pp. 1778–1793.e10. Elsevier BV, 2023.
  • [ Abstract ]
  • [ DOI ]
  Unlike macrophage networks composed of long-lived tissue-resident cells within specific niches, conventional dendritic cells (cDCs) that generate a 3D network in lymph nodes (LNs) are short lived and continuously replaced by DC precursors (preDCs) from the bone marrow (BM). Here, we examined whether specific anatomical niches exist within which preDCs differentiate toward immature cDCs. In situ photoconversion and Prtn3-based fate-tracking revealed that the LN medullary cords are preferential entry sites for preDCs, serving as specific differentiation niches. Repopulation and fate-tracking approaches demonstrated that the cDC1 network unfolded from the medulla along the vascular tree toward the paracortex. During inflammation, collective maturation and migration of resident cDC1s to the paracortex created discontinuity in the medullary cDC1 network and temporarily impaired responsiveness. The decrease in local cDC1 density resulted in higher Flt3L availability in the medullary niche, which accelerated cDC1 development to restore the network. Thus, the spatiotemporal development of the cDC1 network is locally regulated in dedicated LN niches via sensing of cDC1 densities.
• Endothelial barrier dysfunction in systemic inflammation is mediated by soluble VE-cadherin interfering VE-PTP signaling. Knop, Juna-Lisa; Burkard, Natalie; Danesh, Mahshid; Kintrup, Sebastian; Dandekar, Thomas; Srivastava, Mugdha; Springer, Rebecca; Hiermaier, Matthias; Wagner, Nana-Maria; Waschke, Jens; Flemming, Sven; Schlegel, Nicolas. In iScience, 26(10), p. 108049. Elsevier BV, 2023.
  • [ Abstract ]
  • [ DOI ]
  Breakdown of endothelial barrier integrity determines organ dysfunction and outcome of patients with sepsis. Increased levels of soluble vascular endothelial (VE)-cadherin fragments (sVE-cadherin) have previously been linked with inflammation-induced loss of endothelial barrier function. We provide evidence for a causative role of sVE-cadherin to induce loss of endothelial barrier function. In patients with sepsis, sVE-cadherin levels were associated with organ dysfunction and the need for volume resuscitation. Similarly, LPS-induced systemic inflammation in rats with microvascular dysfunction was paralleled by augmented sVE-cadherin levels. Newly generated recombinant human sVE-cadherin (extracellular domains EC1-5) induced loss of endothelial barrier function in both human microvascular endothelial cells in vitro and in rat mesenteric microvessels in vivo and reduced microcirculatory flow. sVE-cadherinEC1-5 disturbed VE-cadherin-mediated adhesion and perturbed VE-protein tyrosine phosphatase (VE-PTP)/VE-cadherin interaction resulting in RhoGEF1-mediated RhoA activation. VE-PTP inhibitor AKB9778 and Rho-kinase inhibitor Y27632 blunted all sVE-cadherinEC1-5-induced effects, which uncovers a pathophysiological role of sVE-cadherin via dysbalanced VE-PTP/RhoA signaling.
• P-47 The role of immune cells in the brain-gut network in Parkinson’s Disease. McFleder, R.; Makhotkina, A.; Groh, J.; Alina, P.; Imdahl, F.; Vogel, J.; Volkmann, J.; Saliba, A.; Ip, C.W. In Clinical Neurophysiology, 148, pp. e29–e30. Elsevier BV, 2023.
  • [ DOI ]
• Vector-borne Trypanosoma brucei parasites develop in artificial human skin and persist as skin tissue forms. Reuter, Christian; Hauf, Laura; Imdahl, Fabian; Sen, Rituparno; Vafadarnejad, Ehsan; Fey, Philipp; Finger, Tamara; Jones, Nicola G.; Walles, Heike; Barquist, Lars; Saliba, Antoine-Emmanuel; Groeber-Becker, Florian; Engstler, Markus. In Nature Communications, 14(1), p. 7660. 2023.
  • [ Abstract ]
  • [ DOI ]
  Transmission of Trypanosoma brucei by tsetse flies involves the deposition of the cell cycle-arrested metacyclic life cycle stage into mammalian skin at the site of the fly's bite. We introduce an advanced human skin equivalent and use tsetse flies to naturally infect the skin with trypanosomes. We detail the chronological order of the parasites' development in the skin by single-cell RNA sequencing and find a rapid activation of metacyclic trypanosomes and differentiation to proliferative parasites. Here we show that after the establishment of a proliferative population, the parasites enter a reversible quiescent state characterized by slow replication and a strongly reduced metabolism. We term these quiescent trypanosomes skin tissue forms, a parasite population that may play an important role in maintaining the infection over long time periods and in asymptomatic infected individuals.
• A primary cell-based in vitro model of the human small intestine reveals host olfactomedin 4 induction in response to Salmonella Typhimurium infection. Däullary, Thomas; Imdahl, Fabian; Dietrich, Oliver; Hepp, Laura; Krammer, Tobias; Fey, Christina; Neuhaus, Winfried; Metzger, Marco; Vogel, Jörg; Westermann, Alexander J.; Saliba, Antoine-Emmanuel; Zdzieblo, Daniela. In Gut Microbes, 15(1). Informa UK Limited, 2023.
  • [ Abstract ]
  • [ DOI ]
  Infection research largely relies on classical cell culture or mouse models. Despite having delivered invaluable insights into host-pathogen interactions, both have limitations in translating mechanistic principles to human pathologies. Alternatives can be derived from modern Tissue Engineering approaches, allowing the reconstruction of functional tissue models in vitro. Here, we combined a biological extracellular matrix with primary tissue-derived enteroids to establish an in vitro model of the human small intestinal epithelium exhibiting in vivo-like characteristics. Using the foodborne pathogen Salmonella enterica serovar Typhimurium, we demonstrated the applicability of our model to enteric infection research in the human context. Infection assays coupled to spatiotemporal readouts recapitulated the established key steps of epithelial infection by this pathogen in our model. Besides, we detected the upregulation of olfactomedin 4 in infected cells, a hitherto unrecognized aspect of the host response to Salmonella infection. Together, this primary human small intestinal tissue model fills the gap between simplistic cell culture and animal models of infection, and shall prove valuable in uncovering human-specific features of host-pathogen interplay.
• Myocardial Milieu Favors Local Differentiation of Regulatory T Cells. Delgobo, Murilo; Weiß, Emil; Ashour, Diyaa ElDin; Richter, Leon; Popiolkowski, Lisa; Arampatzi, Panagiota; Stangl, Verena; Arias-Loza, Paula; Mariotti-Ferrandiz, Encarnita; Rainer, Peter P.; Saliba, Antoine-Emmanuel; Ludewig, Burkhard; Hofmann, Ulrich; Frantz, Stefan; Ramos, Gustavo Campos. In Circulation Research. Ovid Technologies (Wolters Kluwer Health), 2023.
  • [ Abstract ]
  • [ DOI ]
  Background: In the past years, several studies investigated how distinct immune cell subsets affects post-myocardial infarction repair. However, whether and how the tissue environment controls these local immune responses has remained poorly understood. We sought to investigate how antigen-specific T-helper cells differentiate under myocardial milieu's influence. Methods: We used a transgenic T cell receptor (TCR-M) model and major histocompatibility complex-II tetramers, both myosin-specific, combined with single-cell transcriptomics (single-cell RNA sequencing) and functional phenotyping to elucidate how the antigen-specific CD4+ T cells differentiate in the murine infarcted myocardium and influence tissue repair. Additionally, we transferred proinflammatory versus regulatory predifferentiated TCR-M-cells to dissect how they specially contribute to post-myocardial infarction inflammation. Results: Flow cytometry and scRNA-/ TCR-seq analyses revealed that transferred TCR-M cells acquired an induced regulatory phenotype (induced regulatory T cell) in the infarcted myocardium and blunted local inflammation. Myocardial TCR-M cells differentiated into 2 main lineages enriched with either cell activation and profibrotic transcripts (eg, Tgfb1) or suppressor immune checkpoints (eg, Pdcd1), which we also found in human myocardial tissue. These cells produced high levels of LAP (latency-associated peptide) and inhibited IL-17 (interleukin-17) responses. Endogenous myosin-specific T-helper cells, identified using genetically barcoded tetramers, also accumulated in infarcted hearts and exhibited a regulatory phenotype. Notably, TCR-M cells that were predifferentiated toward a regulatory phenotype in vitro maintained stable in vivo FOXP3 (Forkhead box P3) expression and anti-inflammatory activity whereas TH17 partially converted toward a regulatory phenotype in the injured myocardium. Overall, the myosin-specific Tregs dampened post-myocardial infarction inflammation, suppressed neighboring T cells, and were associated with improved cardiac function. Conclusions: These findings provide novel evidence that the heart and its draining lymph nodes actively shape local immune responses by promoting the differentiation of antigen-specific Tregs poised with suppressive function.
• Identification of NOTCH-driven matrisome-associated genes as prognostic indicators of multiple myeloma patient survival. Maichl, Daniela Simone; Kirner, Julius Arthur; Beck, Susanne; Cheng, Wen-Hui; Krug, Melanie; Kuric, Martin; Ade, Carsten Patrick; Bischler, Thorsten; Jakob, Franz; Hose, Dirk; Seckinger, Anja; Ebert, Regina; Jundt, Franziska. In Blood Cancer Journal, 13(1), pp. 1–6. 2023.
  • [ DOI ]
• Global Hfq-mediated RNA interactome of nitrogen starved Escherichia coli uncovers a conserved post-transcriptional regulatory axis required for optimal growth recovery. McQuail, Josh; Matera, Gianluca; Gräfenhan, Tom; Bischler, Thorsten; Haberkant, Per; Stein, Frank; Vogel, Jörg; Wigneshweraraj, Sivaramesh. In Nucleic Acids Research, 52(5), pp. 2323–2339. 2023.
  • [ Abstract ]
  • [ DOI ]
  The RNA binding protein Hfq has a central role in the post-transcription control of gene expression in many bacteria. Numerous studies have mapped the transcriptome-wide Hfq-mediated RNA–RNA interactions in growing bacteria or bacteria that have entered short-term growth-arrest. To what extent post-transcriptional regulation underpins gene expression in growth-arrested bacteria remains unknown. Here, we used nitrogen (N) starvation as a model to study the Hfq-mediated RNA interactome as Escherichia coli enter, experience, and exit long-term growth arrest. We observe that the Hfq-mediated RNA interactome undergoes extensive changes during N starvation, with the conserved SdsR sRNA making the most interactions with different mRNA targets exclusively in long-term N-starved E. coli. Taking a proteomics approach, we reveal that in growth-arrested cells SdsR influences gene expression far beyond its direct mRNA targets. We demonstrate that the absence of SdsR significantly compromises the ability of the mutant bacteria to recover growth competitively from the long-term N-starved state and uncover a conserved post-transcriptional regulatory axis which underpins this process.
• Th17.1 cell driven sarcoidosis-like inflammation after anti-BCMA CAR T cells in multiple myeloma. Leipold, Alexander M.; Werner, Rudolf A.; Düll, Johannes; Jung, Pius; John, Mara; Stanojkovska, Emilia; Zhou, Xiang; Hornburger, Hannah; Ruckdeschel, Anna; Dietrich, Oliver; Imdahl, Fabian; Krammer, Tobias; Knop, Stefan; Rosenwald, Andreas; Buck, Andreas; Sander, Leif Erik; Einsele, Hermann; Kortüm, K. Martin; Saliba, Antoine-Emmanuel; Rasche, Leo. In Leukemia. Springer Science and Business Media LLC, 2023.
  • [ Abstract ]
  • [ DOI ]
  Pseudo-progression and flare-up phenomena constitute a novel diagnostic challenge in the follow-up of patients treated with immune-oncology drugs. We present a case study on pulmonary flare-up after Idecabtagen Vicleucel (Ide-cel), a BCMA targeting CAR T-cell therapy, and used single-cell RNA-seq (scRNA-seq) to identify a Th17.1 driven autoimmune mechanism as the biological underpinning of this phenomenon. By integrating datasets of various lung pathological conditions, we revealed transcriptomic similarities between post CAR T pulmonary lesions and sarcoidosis. Furthermore, we explored a noninvasive PET based diagnostic approach and showed that tracers binding to CXCR4 complement FDG PET imaging in this setting, allowing discrimination between immune-mediated changes and true relapse after CAR T-cell treatment. In conclusion, our study highlights a Th17.1 driven autoimmune phenomenon after CAR T, which may be misinterpreted as disease relapse, and that imaging with multiple PET tracers and scRNA-seq could help in this diagnostic dilemma.
• Brain-to-gut trafficking of alpha-synuclein by CD11c+ cells in a mouse model of Parkinson’s disease. McFleder, Rhonda L.; Makhotkina, Anastasiia; Groh, Janos; Keber, Ursula; Imdahl, Fabian; Peña Mosca, Josefina; Peteranderl, Alina; Wu, Jingjing; Tabuchi, Sawako; Hoffmann, Jan; Karl, Ann-Kathrin; Pagenstecher, Axel; Vogel, Jörg; Beilhack, Andreas; Koprich, James B.; Brotchie, Jonathan M.; Saliba, Antoine-Emmanuel; Volkmann, Jens; Ip, Chi Wang. In Nature Communications, 14(1), p. 7529. 2023.
  • [ Abstract ]
  • [ DOI ]
  Inflammation in the brain and gut is a critical component of several neurological diseases, such as Parkinson’s disease (PD). One trigger of the immune system in PD is aggregation of the pre-synaptic protein, α-synuclein (αSyn). Understanding the mechanism of propagation of αSyn aggregates is essential to developing disease-modifying therapeutics. Using a brain-first mouse model of PD, we demonstrate αSyn trafficking from the brain to the ileum of male mice. Immunohistochemistry revealed that the ileal αSyn aggregations are contained within CD11c+ cells. Using single-cell RNA sequencing, we demonstrate that ileal CD11c+ cells are microglia-like and the same subtype of cells is activated in the brain and ileum of PD mice. Moreover, by utilizing mice expressing the photo-convertible protein, Dendra2, we show that CD11c+ cells traffic from the brain to the ileum. Together these data provide a mechanism of αSyn trafficking between the brain and gut.

• CD52 and OXPHOS—potential targets in ibrutinib-treated mantle cell lymphoma. Fuhr, Viktoria; Heidenreich, Shanice; Srivastava, Mugdha; Riedel, Angela; Düll, Johannes; Gerhard-Hartmann, Elena; Rosenwald, Andreas; Rauert-Wunderlich, Hilka. In Cell Death Discovery, 8(1), p. 505. 2022.
  • [ Abstract ]
  • [ DOI ]
  Altered features of tumor cells acquired across therapy can result in the survival of treatment-resistant clones that may cause minimal residual disease (MRD). Despite the efficacy of ibrutinib in treating relapsed/refractory mantle cell lymphoma, the obstacle of residual cells contributes to relapses of this mature B-cell neoplasm, and the disease remains incurable. RNA-seq analysis of an ibrutinib-sensitive mantle cell lymphoma cell line following ibrutinib incubation of up to 4 d, corroborated our previously postulated resistance mechanism of a metabolic switch to reliance on oxidative phosphorylation (OXPHOS) in surviving cells. Besides, we had shown that treatment-persisting cells were characterized by increased CD52 expression. Therefore, we hypothesized that combining ibrutinib with another agent targeting these potential escape mechanisms could minimize the risk of survival of ibrutinib-resistant cells. Concomitant use of ibrutinib with OXPHOS-inhibitor IACS-010759 increased toxicity compared to ibrutinib alone. Targeting CD52 was even more efficient, as addition of CD52 mAb in combination with human serum following ibrutinib pretreatment led to rapid complement-dependent-cytotoxicity in an ibrutinib-sensitive cell line. In primary mantle cell lymphoma cells, a higher toxic effect with CD52 mAb was obtained, when cells were pretreated with ibrutinib, but only in an ibrutinib-sensitive cohort. Given the challenge of treating multi-resistant mantle cell lymphoma patients, this work highlights the potential use of anti-CD52 therapy as consolidation after ibrutinib treatment in patients who responded to the BTK inhibitor to achieve MRD negativity and prolong progression-free survival.
• Stress signalling and STAT1 activation characterize the keratinocytic gene expression pattern in Hidradenitis suppurativa. Frings, V G; Jopp, L; Srivastava, M; Presser, D; Goebeler, M; Schmidt, M. In The Journal of the European Academy of Dermatology and Venereology, 36(12), pp. 2488–2498. 2022.
  • [ Abstract ]
  • [ DOI ]
  Background: The pathogenetic factors generating the innate immune signal necessary for T-cell activation, initiation and chronification of Hidradenitis suppurativa (HS, also known as Acne inversa) are still poorly understood. Emerging evidence suggests that a defective keratinocyte function critically contributes to HS disease development and progression. Objectives: To elucidate the role of keratinocytes in HS lesion formation, we compared the transcriptomes of lesional and perilesional epidermis isolated from HS patients by RNA sequencing (RNA Seq). Methods: Pairwise-matched lesional and perilesional HS skin samples of five different donors were obtained and epidermal keratinocytes freshly isolated and processed for RNA extraction and RNA seq. Lesionally regulated genes were analysed by large-scale promoter analysis and functional annotation clustering to identify epidermally overrepresented transcription factor binding sites and functionally related gene groups. Results were experimentally validated with independent epidermal isolates of patient-matched lesional and perilesional HS skin employing qRT-PCR, cell culture, immunoblot and immunostaining. Results: We show that HS is characterized by a strong epidermal stress state evident by a significant overrepresentation of an AP-1-driven gene signature and a substantial activation of the stress-activated cJun N-terminal kinase (JNK) pathway in lesional epidermis. Additionally, our data reveal a strong induction of STAT1 activation in lesional HS epidermis that likely results from IFNγ production and triggered expression of key inflammatory genes coordinating innate immune activation and the adaptive T-cell response in HS. Conclusions: Our data implicate a key role of stress signalling and JAK/STAT1 activation in disease progression of HS and suggest interference with JAK/STAT1 signalling as a potentially promising therapeutic approach for HS.
• hnRNP R negatively regulates transcription by modulating the association of P-TEFb with 7SK and BRD4. Ji, Changhe; Deng, Chunchu; Antor, Katharina; Bischler, Thorsten; Schneider, Cornelius; Fischer, Utz; Sendtner, Michael; Briese, Michael. In EMBO reports, p. e55432. 2022.
  • [ Abstract ]
  • [ DOI ]
  The P-TEFb complex promotes transcription elongation by releasing paused RNA polymerase II. P-TEFb itself is known to be inactivated through binding to the non-coding RNA 7SK but there is only limited information about mechanisms regulating their association. Here, we show that cells deficient in the RNA-binding protein hnRNP R, a known 7SK interactor, exhibit increased transcription due to phosphorylation of RNA polymerase II. Intriguingly, loss of hnRNP R promotes the release of P-TEFb from 7SK, accompanied by enhanced hnRNP A1 binding to 7SK. Additionally, we found that hnRNP R interacts with BRD4, and that hnRNP R depletion increases BRD4 binding to the P-TEFb component CDK9. Finally, CDK9 is stabilized upon loss of hnRNP R and its association with Cyclin K is enhanced. Together, our results indicate that hnRNP R negatively regulates transcription by modulating the activity and stability of the P-TEFb complex, exemplifying the multimodal regulation of P-TEFb by an RNA-binding protein.
• G6b-B regulates an essential step in megakaryocyte maturation. Becker, Isabelle C.; Nagy, Zoltan; Manukjan, Georgi; Haffner-Luntzer, Melanie; Englert, Maximilian; Heib, Tobias; Vögtle, Timo; Gross, Carina; Bharti, Richa; Dietrich, Sascha; Mott, Kristina; Heck, Johannes; Stegmaier, Sebastian; Baranowsky, Anke; Schinke, Thorsten; Schlegel, Nicolas; Heckel, Tobias; Stegner, David; Pleines, Irina; Ignatius, Anita; Schulze, Harald; Nieswandt, Bernhard. In Blood Advances, 6(10), pp. 3155–3161. American Society of Hematology, 2022.
  • [ Abstract ]
  • [ DOI ]
  G6b-B is a megakaryocyte lineage-specific immunoreceptor tyrosine-based inhibition motif-containing receptor, essential for platelet homeostasis. Mice with a genomic deletion of the entire Mpig6b locus develop severe macrothrombocytopenia and myelofibrosis, which is reflected in humans with null mutations in MPIG6B. The current model proposes that megakaryocytes lacking G6b-B develop normally, whereas proplatelet release is hampered, but the underlying molecular mechanism remains unclear. We report on a spontaneous recessive single nucleotide mutation in C57BL/6 mice, localized within the intronic region of the Mpig6b locus that abolishes G6b-B expression and reproduces macrothrombocytopenia, myelofibrosis, and osteosclerosis. As the mutation is based on a single-nucleotide exchange, Mpig6bmut mice represent an ideal model to study the role of G6b-B. Megakaryocytes from these mice were smaller, displayed a less-developed demarcation membrane system, and had a reduced expression of receptors. RNA sequencing revealed a striking global reduction in the level of megakaryocyte-specific transcripts, in conjunction with decreased protein levels of the transcription factor GATA-1 and impaired thrombopoietin signaling. The reduced number of mature MKs in the bone marrow was corroborated on a newly developed Mpig6b-null mouse strain. Our findings highlight an unexpected essential role of G6b-B in the early differentiation within the megakaryocytic lineage.
• Type 1 conventional dendritic cells maintain and guide the differentiation of precursors of exhausted T~cells in distinct cellular niches. Dähling, Sabrina; Mansilla, Ana Maria; Knöpper, Konrad; Grafen, Anika; Utzschneider, Daniel T.; Ugur, Milas; Whitney, Paul G.; Bachem, Annabell; Arampatzi, Panagiota; Imdahl, Fabian; Kaisho, Tsuneyasu; Zehn, Dietmar; Klauschen, Frederick; Garbi, Natalio; Kallies, Axel; Saliba, Antoine-Emmanuel; Gasteiger, Georg; Bedoui, Sammy; Kastenmüller, Wolfgang. In Immunity, 55(4), pp. 656–670.e8. Elsevier BV, 2022.
  • [ Abstract ]
  • [ DOI ]
  Reinvigoration of exhausted CD8+ T (Tex) cells by checkpoint immunotherapy depends on the activation of precursors of exhausted T (Tpex) cells, but the local anatomical context of their maintenance, differentiation, and interplay with other cells is not well understood. Here, we identified transcriptionally distinct Tpex subpopulations, mapped their differentiation trajectories via transitory cellular states toward Tex cells, and localized these cell states to specific splenic niches. Conventional dendritic cells (cDCs) were critical for successful αPD-L1 therapy and were required to mediate viral control. cDC1s were dispensable for Tpex cell expansion but provided an essential niche to promote Tpex cell maintenance, preventing their overactivation and T-cell-mediated immunopathology. Mechanistically, cDC1s insulated Tpex cells via MHC-I-dependent interactions to prevent their activation within other inflammatory environments that further aggravated their exhaustion. Our findings reveal that cDC1s maintain and safeguard Tpex cells within distinct anatomical niches to balance viral control, exhaustion, and immunopathology.
• Spacer prioritization in CRISPR–Cas9 immunity is enabled by the leader RNA. Liao, Chunyu; Sharma, Sahil; Svensson, Sarah L.; Kibe, Anuja; Weinberg, Zasha; Alkhnbashi, Omer S.; Bischler, Thorsten; Backofen, Rolf; Caliskan, Neva; Sharma, Cynthia M.; Beisel, Chase L. In Nature Microbiology. 2022.
  • [ Abstract ]
  • [ DOI ]
  CRISPR–Cas systems store fragments of foreign DNA, called spacers, as immunological recordings used to combat future infections. Of the many spacers stored in a CRISPR array, the most recent are known to be prioritized for immune defence. However, the underlying mechanism remains unclear. Here we show that the leader region upstream of CRISPR arrays in CRISPR–Cas9 systems enhances CRISPR RNA (crRNA) processing from the newest spacer, prioritizing defence against the matching invader. Using the CRISPR–Cas9 system from Streptococcus pyogenes as a model, we found that the transcribed leader interacts with the conserved repeats bordering the newest spacer. The resulting interaction promotes transactivating crRNA (tracrRNA) hybridization with the second of the two repeats, accelerating crRNA processing. Accordingly, disruption of this structure reduces the abundance of the associated crRNA and immune defence against targeted plasmids and bacteriophages. Beyond the S. pyogenes system, bioinformatics analyses revealed that leader-repeat structures appear across CRISPR–Cas9 systems. CRISPR–Cas systems thus possess an RNA-based mechanism to prioritize defence against the most recently encountered invaders.
• Liraglutide + PYY3-36 Combination Therapy Mimics Effects of Roux-en-Y Bypass on Early NAFLD Whilst Lacking-Behind in Metabolic Improvements. Metzner, Valentin; Herzog, Gloria; Heckel, Tobias; Bischler, Thorsten; Hasinger, Julia; Otto, Christoph; Fassnacht, Martin; Geier, Andreas; Seyfried, Florian; Dischinger, Ulrich. In Journal of Clinical Medicine, 11(3), p. 753. MDPI AG, 2022.
  • [ Abstract ]
  • [ DOI ]
  Background: Treatment options for NAFLD are still limited. Bariatric surgery, such as Roux-en-Y gastric bypass (RYGB), has been shown to improve metabolic and histologic markers of NAFLD. Glucagon-like-peptide-1 (GLP-1) analogues lead to improvements in phase 2 clinical trials. We directly compared the effects of RYGB with a treatment using liraglutide and/or peptide tyrosine tyrosine 3-36 (PYY3-36) in a rat model for early NAFLD. Methods: Obese male Wistar rats (high-fat diet (HFD)-induced) were randomized into the following treatment groups: RYGB, sham-operation (sham), liraglutide (0.4 mg/kg/day), PYY3-36 (0.1 mg/kg/day), liraglutide+PYY3-36, and saline. After an observation period of 4 weeks, liver samples were histologically evaluated, ELISAs and RNA sequencing + RT-qPCRs were performed. Results: RYGB and liraglutide+PYY3-36 induced a similar body weight loss and, compared to sham/saline, marked histological improvements with significantly less steatosis. However, only RYGB induced significant metabolic improvements (e.g., adiponectin/leptin ratio 18.8 ± 11.8 vs. 2.4 ± 1.2 in liraglutide+PYY3-36- or 1.4 ± 0.9 in sham-treated rats). Furthermore, RNA sequencing revealed a high number of differentially regulated genes in RYGB treated animals only. Conclusions: The combination therapy of liraglutide+PYY3-36 partly mimics the positive effects of RYGB on weight reduction and on hepatic steatosis, while its effects on metabolic function lack behind RYGB. Keywords: liraglutide; GLP-1; peptide tyrosine tyrosine (PYY); peptide tyrosine tyrosine 3-36 (PYY3-36); RYGB; gastric bypass; obesity; NASH; NAFLD
• Dynamics of monocyte-derived macrophage diversity in experimental myocardial infarction. Rizzo, Giuseppe; Gropper, Julius; Piollet, Marie; Vafadarnejad, Ehsan; Rizakou, Anna; Bandi, Sourish Reddy; Arampatzi, Panagiota; Krammer, Tobias; DiFabion, Nina; Dietrich, Oliver; Arias-Loza, Anahi Paula; Prinz, Marco; Mack, Matthias; Schlepckow, Kai; Haass, Christian; Silvestre, Jean Sébastien; Zernecke, Alma; Saliba, Antoine Emmanuel; Cochain, Clément. In Cardiovascular Research. Oxford University Press (OUP), 2022.
  • [ Abstract ]
  • [ DOI ]
  Aims: Macrophages have a critical and dual role in post-ischaemic cardiac repair, as they can foster both tissue healing and damage. Multiple subsets of tissue resident and monocyte-derived macrophages coexist in the infarcted heart, but their precise identity, temporal dynamics, and the mechanisms regulating their acquisition of discrete states are not fully understood. To address this, we used multi-modal single-cell immune profiling, combined with targeted cell depletion and macrophage fate mapping, to precisely map monocyte/macrophage transitions after experimental myocardial infarction. Methods and results: We performed single-cell transcriptomic and cell-surface marker profiling of circulating and cardiac immune cells in mice challenged with acute myocardial infarction, and integrated single-cell transcriptomes obtained before and at 1, 3, 5, 7, and 11 days after infarction. Using complementary strategies of CCR2+ monocyte depletion and fate mapping of tissue resident macrophages, we determined the origin of cardiac macrophage populations. The macrophage landscape of the infarcted heart was dominated by monocyte-derived cells comprising two pro-inflammatory populations defined as Isg15hi and MHCII+Il1b+, alongside non-inflammatory Trem2hi cells. Trem2hi macrophages were observed in the ischaemic area, but not in the remote viable myocardium, and comprised two subpopulations sequentially populating the heart defined as Trem2hiSpp1hi monocyte-to-macrophage intermediates, and fully differentiated Trem2hiGdf15hi macrophages. Cardiac Trem2hi macrophages showed similarities to 'lipid-associated macrophages' found in mouse models of metabolic diseases and were observed in the human heart, indicating conserved features of this macrophage state across diseases and species. Ischaemic injury induced a shift of circulating Ly6Chi monocytes towards a Chil3hi state with granulocyte-like features, but the acquisition of the Trem2hi macrophage signature occurred in the ischaemic tissue. In vitro, macrophages acquired features of the Trem2hi signature following apoptotic-cell efferocytosis. Conclusion: Our work provides a comprehensive map of monocyte/macrophage transitions in the ischaemic heart, constituting a valuable resource for further investigating how these cells may be harnessed and modulated to promote post-ischaemic heart repair.
• Neurodegeneration by α-synuclein-specific T cells in AAV-A53T-α-synuclein Parkinson’s disease mice. Karikari, Akua A.; McFleder, Rhonda L.; Ribechini, Eliana; Blum, Robert; Bruttel, Valentin; Knorr, Susanne; Gehmeyr, Mona; Volkmann, Jens; Brotchie, Jonathan M.; Ahsan, Fadhil; Haack, Beatrice; Monoranu, Camelia-Maria; Keber, Ursula; Yeghiazaryan, Rima; Pagenstecher, Axel; Heckel, Tobias; Bischler, Thorsten; Wischhusen, Jörg; Koprich, James B.; Lutz, Manfred B.; Ip, Chi Wang. In Brain, Behavior, and Immunity, 101, pp. 194–210. Elsevier BV, 2022.
  • [ Abstract ]
  • [ DOI ]
  Background: Antigen-specific neuroinflammation and neurodegeneration are characteristic for neuroimmunological diseases. In Parkinson's disease (PD) pathogenesis, α-synuclein is a known culprit. Evidence for α-synuclein-specific T cell responses was recently obtained in PD. Still, a causative link between these α-synuclein responses and dopaminergic neurodegeneration had been lacking. We thus addressed the functional relevance of α-synuclein-specific immune responses in PD in a mouse model. Methods: We utilized a mouse model of PD in which an Adeno-associated Vector 1/2 serotype (AAV1/2) expressing human mutated A53T-α-Synuclein was stereotactically injected into the substantia nigra (SN) of either wildtype C57BL/6 or Recombination-activating gene 1 (RAG1)-/- mice. Brain, spleen, and lymph node tissues from different time points following injection were then analyzed via FACS, cytokine bead assay, immunohistochemistry and RNA-sequencing to determine the role of T cells and inflammation in this model. Bone marrow transfer from either CD4+/CD8-, CD4-/CD8+, or CD4+/CD8+ (JHD-/-) mice into the RAG-1-/- mice was also employed. In addition to the in vivo studies, a newly developed A53T-α-synuclein-expressing neuronal cell culture/immune cell assay was utilized. Results: AAV-based overexpression of pathogenic human A53T-α-synuclein in dopaminergic neurons of the SN stimulated T cell infiltration. RNA-sequencing of immune cells from PD mouse brains confirmed a pro-inflammatory gene profile. T cell responses were directed against A53T-α-synuclein-peptides in the vicinity of position 53 (68-78) and surrounding the pathogenically relevant S129 (120-134). T cells were required for α-synuclein-induced neurodegeneration in vivo and in vitro, while B cell deficiency did not protect from dopaminergic neurodegeneration. Conclusions: Using T cell and/or B cell deficient mice and a newly developed A53T-α-synuclein-expressing neuronal cell culture/immune cell assay, we confirmed in vivo and in vitro that pathogenic α-synuclein peptide-specific T cell responses can cause dopaminergic neurodegeneration and thereby contribute to PD-like pathology. Keywords: Parkinson’s disease; T cells; antigen-specificity; neuroinflammation; α-synuclein.
• Genome-wide scan for potential CD4+ T-cell vaccine candidates in Candida auris by exploiting reverse vaccinology and evolutionary information. Gupta, Shishir K; Osmanoglu, Özge; Minocha, Rashmi; Bandi, Sourish Reddy; Bencurova, Elena; Srivastava, Mugdha; Dandekar, Thomas. In Frontiers in Medicine, 9, p. 1008527. 2022.
  • [ Abstract ]
  • [ DOI ]
  Candida auris is a globally emerging fungal pathogen responsible for causing nosocomial outbreaks in healthcare associated settings. It is known to cause infection in all age groups and exhibits multi-drug resistance with high potential for horizontal transmission. Because of this reason combined with limited therapeutic choices available, C. auris infection has been acknowledged as a potential risk for causing a future pandemic, and thus seeking a promising strategy for its treatment is imperative. Here, we combined evolutionary information with reverse vaccinology approach to identify novel epitopes for vaccine design that could elicit CD4+ T-cell responses against C. auris. To this end, we extensively scanned the family of proteins encoded by C. auris genome. In addition, a pathogen may acquire substitutions in epitopes over a period of time which could cause its escape from the immune response thus rendering the vaccine ineffective. To lower this possibility in our design, we eliminated all rapidly evolving genes of C. auris with positive selection. We further employed highly conserved regions of multiple C. auris strains and identified two immunogenic and antigenic T-cell epitopes that could generate the most effective immune response against C. auris. The antigenicity scores of our predicted vaccine candidates were calculated as 0.85 and 1.88 where 0.5 is the threshold for prediction of fungal antigenic sequences. Based on our results, we conclude that our vaccine candidates have the potential to be successfully employed for the treatment of C. auris infection. However, in vivo experiments are imperative to further demonstrate the efficacy of our design.
• Helicobacter pylori shows tropism to gastric differentiated pit cells dependent on urea chemotaxis. Aguilar, Carmen; Pauzuolis, Mindaugas; Pompaiah, Malvika; Vafadarnejad, Ehsan; Arampatzi, Panagiota; Fischer, Mara; Narres, Dominik; Neyazi, Mastura; Kayisoglu, Özge; Sell, Thomas; Blüthgen, Nils; Morkel, Markus; Wiegering, Armin; Germer, Christoph-Thomas; Kircher, Stefan; Rosenwald, Andreas; Saliba, Antoine-Emmanuel; Bartfeld, Sina. In Nature Communications, 13(1), p. 5878. 2022.
  • [ Abstract ]
  • [ DOI ]
  The human gastric epithelium forms highly organized gland structures with different subtypes of cells. The carcinogenic bacterium Helicobacter pylori can attach to gastric cells and subsequently translocate its virulence factor CagA, but the possible host cell tropism of H. pylori is currently unknown. Here, we report that H. pylori preferentially attaches to differentiated cells in the pit region of gastric units. Single-cell RNA-seq shows that organoid-derived monolayers recapitulate the pit region, while organoids capture the gland region of the gastric units. Using these models, we show that H. pylori preferentially attaches to highly differentiated pit cells, marked by high levels of GKN1, GKN2 and PSCA. Directed differentiation of host cells enable enrichment of the target cell population and confirm H. pylori preferential attachment and CagA translocation into these cells. Attachment is independent of MUC5AC or PSCA expression, and instead relies on bacterial TlpB-dependent chemotaxis towards host cell-released urea, which scales with host cell size.
• Temperature and livestock grazing trigger transcriptome responses in bumblebees along an elevational gradient. Brenzinger, Kristof; Maihoff, Fabienne; Peters, Marcell K.; Schimmer, Leonie; Bischler, Thorsten; Classen, Alice. In iScience, 25(10). 2022.
  • [ Abstract ]
  • [ DOI ]
  Climate and land-use changes cause increasing stress to pollinators but the molecular pathways underlying stress responses are poorly understood. Here, we analyzed the transcriptomic response of Bombus lucorum workers to temperature and livestock grazing. Bumblebees sampled along an elevational gradient, and from differently managed grassland sites (livestock grazing vs unmanaged) in the German Alps did not differ in the expression of genes known for thermal stress responses. Instead, metabolic energy production pathways were upregulated in bumblebees sampled in mid- or high elevations or during cool temperatures. Extensive grazing pressure led to an upregulation of genetic pathways involved in immunoregulation and DNA-repair. We conclude that widespread bumblebees are tolerant toward temperature fluctuations in temperate mountain environments. Moderate temperature increases may even release bumblebees from metabolic stress. However, transcriptome responses to even moderate management regimes highlight the completely underestimated complexity of human influence on natural pollinators.
• Eine Einzelzell-RNA-Sequenzierung identifiziert Metabolismus und CD52 als neue Angriffspunkte in Ibrutinib-persistenten Mantelzelllymphomzellen. Fuhr, Viktoria; Vafadarnejad, Ehsan; Dietrich, Oliver; Arampatzi, Panagiota; Riedel, Angela; Saliba, Antoine-Emmanuel; Rosenwald, Andreas; Rauert-Wunderlich, Hilka. In Die Pathologie. Springer Science and Business Media LLC, 2022.
  • [ Abstract ]
  • [ DOI ]
  Background: Ibrutinib improves the treatment of relapsed or refractory mantle cell lymphoma, a mature B cell neoplasm. However, relapses following treatment with this Bruton tyrosine kinase inhibitor occur frequently, and the outcome of affected patients is poor. Objectives: Single-cell RNA sequencing (scRNA-seq) can track trends in gene expression of mantle cell lymphoma cells across ibrutinib treatment and new therapeutic targets can be defined based on the detected resistance mechanisms. Materials and methods: The ibrutinib-sensitive mantle cell lymphoma cell line REC‑1 was treated with ibrutinib for 6 h and 48 h. Droplet-based scRNA-seq was performed to examine the transcriptomic alterations of surviving cells using the 10× Genomics platform. Extracellular flux analysis and flow cytometry were applied to further study the observed adaptations to ibrutinib treatment. Results: REC‑1 harbored a subpopulation with potential for crosstalk with microenvironment and therefore greater risk for aggressiveness and drug resistance. Following ibrutinib treatment, NF-κB signaling was turned off. In contrast, the cells upregulated B-cell receptor genes and surface antigens such as CD52, and switched their metabolism to increased dependence on oxidative phosphorylation. Conclusions: Targeting oxidative phosphorylation or CD52 in combination with or as follow-up to ibrutinib might overcome resistance and provide improved prognosis for mantle cell lymphoma patients.
• Postnatal expansion of mesenteric lymph node stromal cells towards reticular and CD34+ stromal cell subsets. Pezoldt, Joern; Wiechers, Carolin; Zou, Mangge; Litovchenko, Maria; Biocanin, Marjan; Beckstette, Michael; Sitnik, Katarzyna; Palatella, Martina; van Mierlo, Guido; Chen, Wanze; Gardeux, Vincent; Floess, Stefan; Ebel, Maria; Russeil, Julie; Arampatzi, Panagiota; Vafardanejad, Ehsan; Saliba, Antoine-Emmanuel; Deplancke, Bart; Huehn, Jochen. In Nature Communications, 13(1). Springer Science and Business Media LLC, 2022.
  • [ Abstract ]
  • [ DOI ]
  Gut-draining mesenteric lymph nodes (LN) provide the framework to shape intestinal adaptive immune responses. Based on the transcriptional signatures established by our previous work, the composition and immunomodulatory function of LN stromal cells (SC) vary according to location. Here, we describe the single-cell composition and development of the SC compartment within mesenteric LNs derived from postnatal to aged mice. We identify CD34+ SC and fibroblastic reticular stromal cell (FRC) progenitors as putative progenitors, both supplying the typical rapid postnatal mesenteric LN expansion. We further establish the location-specific chromatin accessibility and DNA methylation landscape of non-endothelial SCs and identify a microbiota-independent core epigenomic signature, showing characteristic differences between SCs from mesenteric and skin-draining peripheral LNs. The epigenomic landscape of SCs points to dynamic expression of Irf3 along the differentiation trajectories of FRCs. Accordingly, a mesenchymal stem cell line acquires a Cxcl9+ FRC molecular phenotype upon lentiviral overexpression of Irf3, and the relevance of Irf3 for SC biology is further underscored by the diminished proportion of Ccl19+ and Cxcl9+ FRCs in LNs of Irf3-/- mice. Together, our data constitute a comprehensive transcriptional and epigenomic map of mesenteric LNSC development in early life and dissect location-specific, microbiota-independent properties of non-endothelial SCs.

• Accumulation of cytotoxic T cells in the aged CNS leads to axon degeneration and contributes to cognitive and motor decline. Groh, Janos; Knöpper, Konrad; Arampatzi, Panagiota; Yuan, Xidi; Lößlein, Lena; Saliba, Antoine-Emmanuel; Kastenmüller, Wolfgang; Martini, Rudolf. In Nature Aging, 1(4), pp. 357–367. Springer Science and Business Media LLC, 2021.
  • [ Abstract ]
  • [ DOI ]
  Aging is a major risk factor for the development of nervous system functional decline, even in the absence of diseases or trauma. The axon–myelin units and synaptic terminals are some of the neural structures most vulnerable to aging-related deterioration1,2,3,4,5,6, but the underlying mechanisms are poorly understood. In the peripheral nervous system, macrophages—important representatives of the innate immune system—are prominent drivers of structural and functional decline of myelinated fibers and motor endplates during aging7. Similarly, in the aging central nervous system (CNS), microglial cells promote damage of myelinated axons and synapses8,9,10,11,12,13,14,15,16,17,18,19,20. Here we examine the role of cytotoxic CD8+ T lymphocytes, a type of adaptive immune cells previously identified as amplifiers of axonal perturbation in various models of genetically mediated CNS diseases21 but understudied in the aging CNS22,23,24,25. We show that accumulation of CD8+ T cells drives axon degeneration in the normal aging mouse CNS and contributes to age-related cognitive and motor decline. We characterize CD8+ T-cell population heterogeneity in the adult and aged mouse brain by single-cell transcriptomics and identify aging-related changes. Mechanistically, we provide evidence that CD8+ T cells drive axon degeneration in a T-cell receptor- and granzyme B-dependent manner. Cytotoxic neural damage is further aggravated by systemic inflammation in aged but not adult mice. We also find increased densities of T cells in white matter autopsy material from older humans. Our results suggest that targeting CD8+ CNS-associated T cells in older adults might mitigate aging-related decline of brain structure and function.
• Time-Resolved scRNA-Seq Tracks the Adaptation of a Sensitive MCL Cell Line to Ibrutinib Treatment. Fuhr, Viktoria; Vafadarnejad, Ehsan; Dietrich, Oliver; Arampatzi, Panagiota; Riedel, Angela; Saliba, Antoine-Emmanuel; Rosenwald, Andreas; Rauert-Wunderlich, Hilka. In International Journal of Molecular Sciences, 22(5), p. 2276. MDPI AG, 2021.
  • [ Abstract ]
  • [ DOI ]
  Since the approval of ibrutinib for relapsed/refractory mantle cell lymphoma (MCL), the treatment of this rare mature B-cell neoplasm has taken a great leap forward. Despite promising efficacy of the Bruton tyrosine kinase inhibitor, resistance arises inevitably and the underlying mechanisms remain to be elucidated. Here, we aimed to decipher the response of a sensitive MCL cell line treated with ibrutinib using time-resolved single-cell RNA sequencing. The analysis uncovered five subpopulations and their individual responses to the treatment. The effects on the B cell receptor pathway, cell cycle, surface antigen expression, and metabolism were revealed by the computational analysis and were validated by molecular biological methods. The observed upregulation of B cell receptor signaling, crosstalk with the microenvironment, upregulation of CD52, and metabolic reprogramming towards dependence on oxidative phosphorylation favor resistance to ibrutinib treatment. Targeting these cellular responses provide new therapy options in MCL.
• The healing myocardium mobilizes a distinct B-cell subset through a CXCL13-CXCR5-dependent mechanism. Heinrichs, Margarete; Ashour, DiyaaElDin; Siegel, Johanna; Büchner, Lotte; Wedekind, Georg; Heinze, Katrin G; Arampatzi, Panagiota; Saliba, Antoine-Emmanuel; Cochain, Clement; Hofmann, Ulrich; Frantz, Stefan; Ramos, Gustavo Campos. In Cardiovascular Research, 117(13), pp. 2664–2676. Oxford University Press (OUP), 2021.
  • [ Abstract ]
  • [ DOI ]
  Aims: Recent studies have revealed that B cells and antibodies can influence inflammation and remodelling following a myocardial infarction (MI) and culminating in heart failure-but the mechanisms underlying these observations remain elusive. We therefore conducted in mice a deep phenotyping of the post-MI B-cell responses in infarcted hearts and mediastinal lymph nodes, which drain the myocardium. Thereby, we sought to dissect the mechanisms controlling B-cell mobilization and activity in situ. Methods and results: Histological, flow cytometry, and single-cell RNA-sequencing (scRNA-seq) analyses revealed a rapid accumulation of diverse B-cell subsets in infarcted murine hearts, paralleled by mild clonal expansion of germinal centre B cells in the mediastinal lymph nodes. The repertoire of cardiac B cells was largely polyclonal and showed no sign of antigen-driven clonal expansion. Instead, it included a distinct subset exclusively found in the heart, herein termed 'heart-associated B cells' (hB) that expressed high levels of Cd69 as an activation marker, C-C-chemokine receptor type 7 (Ccr7), CXC-chemokine receptor type 5 (Cxcr5), and transforming growth factor beta 1 (Tgfb1). This distinct signature was not shared with any other cell population in the healing myocardium. Moreover, we detected a myocardial gradient of CXC-motif chemokine ligand 13 (CXCL13, the ligand of CXCR5) on Days 1 and 5 post-MI. When compared with wild-type controls, mice treated with a neutralizing CXCL13-specific antibody as well as CXCR5-deficient mice showed reduced post-MI infiltration of B cells and reduced local Tgfb1 expression but no differences in contractile function nor myocardial morphology were observed between groups. Conclusion: Our study reveals that polyclonal B cells showing no sign of antigen-specificity readily infiltrate the heart after MI via the CXCL13-CXCR5 axis and contribute to local TGF-ß1 production. The local B-cell responses are paralleled by mild antigen-driven germinal centre reactions in the mediastinal lymph nodes that might ultimately lead to the production of specific antibodies.
• Noncanonical crRNAs derived from host transcripts enable multiplexable RNA detection by Cas9. Jiao, Chunlei; Sharma, Sahil; Dugar, Gaurav; Peeck, Natalia L.; Bischler, Thorsten; Wimmer, Franziska; Yu, Yanying; Barquist, Lars; Schoen, Christoph; Kurzai, Oliver; Sharma, Cynthia M.; Beisel, Chase L. In Science, 372(6545), pp. 941–948. American Association for the Advancement of Science, 2021.
  • [ Abstract ]
  • [ DOI ]
  CRISPR-Cas systems recognize foreign genetic material using CRISPR RNAs (crRNAs). In Type II systems, a trans-activating crRNA (tracrRNA) hybridizes to crRNAs to drive their processing and utilization by Cas9. While analyzing Cas9-RNA complexes from Campylobacter jejuni, we discovered tracrRNA hybridizing to cellular RNAs, leading to formation of "noncanonical" crRNAs capable of guiding DNA targeting by Cas9. Our discovery inspired the engineering of reprogrammed tracrRNAs that link the presence of any RNA-of-interest to DNA targeting with different Cas9 orthologs. This capability became the basis for a multiplexable diagnostic platform termed LEOPARD (Leveraging Engineered tracrRNAs and On-target DNAs for PArallel RNA Detection). LEOPARD allowed simultaneous detection of RNAs from different viruses in one test and distinguished SARS-CoV-2 and its D614G variant with single-base resolution in patient samples.
• Fibromyalgia vs small fiber neuropathy: diverse keratinocyte transcriptome signature. Karl, Franziska; Bischler, Thorsten; Egenolf, Nadine; Evdokimov, Dimitar; Heckel, Tobias; Üçeyler, Nurcan. In PAIN, 162, pp. 2569–2577. 2021.
  • [ Abstract ]
  • [ DOI ]
  Damage to thinly myelinated and unmyelinated nerve fibers causes small fiber pathology, which is increasingly found in pain syndromes such as small fiber neuropathy (SFN) and fibromyalgia syndrome (FMS). The peripheral nerve endings of the small nerve fibers terminate within the epidermis, where they are surrounded by keratinocytes that may act as primary nociceptive transducers. We performed RNA sequencing of keratinocytes obtained from patients with SFN, FMS, and healthy controls. We found 141 deregulated protein coding genes between SFN patients and healthy controls and no differentially expressed genes between patients with FMS and healthy controls. When comparing patients with SFN with patients with FMS, we detected 167 differentially expressed protein coding genes (129 upregulated and 38 downregulated). Further analysis revealed enriched inflammatory pathways. Validation of selected candidates in an independent cohort confirmed higher expression of the proinflammatory mediators interleukin-8, C-X-C motif chemokine 3, endothelin receptor type A, and the voltage-gated sodium channel 1.7 in SFN compared with patients with FMS. We provide a diverse keratinocyte transcriptome signature between patients with SFN and patients with FMS, which may hint toward distinct pathomechanisms of small fiber sensitization in both entities and lay the basis for advanced diagnostics.
• Alveolar Regeneration in COVID-19 Patients: A Network Perspective. Gupta, SK; Srivastava, M; Minocha, R; Akash, A; Dangwal, S; Dandekar, T. In International Journal of Molecular Sciences, 22(20), p. 11279. 2021.
  • [ Abstract ]
  • [ DOI ]
  A viral infection involves entry and replication of viral nucleic acid in a host organism, subsequently leading to biochemical and structural alterations in the host cell. In the case of SARS-CoV-2 viral infection, over-activation of the host immune system may lead to lung damage. Albeit the regeneration and fibrotic repair processes being the two protective host responses, prolonged injury may lead to excessive fibrosis, a pathological state that can result in lung collapse. In this review, we discuss regeneration and fibrosis processes in response to SARS-CoV-2 and provide our viewpoint on the triggering of alveolar regeneration in coronavirus disease 2019 (COVID-19) patients.
• Homozygous BCMA gene deletion in response to anti-BCMA CAR T cells in a patient with multiple myeloma. Vià, Matteo C. Da; Dietrich, Oliver; Truger, Marietta; Arampatzi, Panagiota; Duell, Johannes; Heidemeier, Anke; Zhou, Xiang; Danhof, Sophia; Kraus, Sabrina; Chatterjee, Manik; Meggendorfer, Manja; Twardziok, Sven; Goebeler, Maria-Elisabeth; Topp, Max S.; Hudecek, Michael; Prommersberger, Sabrina; Hege, Kristen; Kaiser, Shari; Fuhr, Viktoria; Weinhold, Niels; Rosenwald, Andreas; Erhard, Florian; Haferlach, Claudia; Einsele, Hermann; Kortüm, K. Martin; Saliba, Antoine-Emmanuel; Rasche, Leo. In Nature Medicine, 27(4), pp. 616–619. Springer Science and Business Media LLC, 2021.
  • [ Abstract ]
  • [ DOI ]
  B cell maturation antigen (BCMA) is a target for various immunotherapies and a biomarker for tumor load in multiple myeloma (MM). We report a case of irreversible BCMA loss in a patient with MM who was enrolled in the KarMMa trial ( NCT03361748 ) and progressed after anti-BCMA CAR T cell therapy. We identified selection of a clone with homozygous deletion of TNFRSF17 (BCMA) as the underlying mechanism of immune escape. Furthermore, we found heterozygous TNFRSF17 loss or monosomy 16 in 37 out of 168 patients with MM, including 28 out of 33 patients with hyperhaploid MM who had not been previously treated with BCMA-targeting therapies, suggesting that heterozygous TNFRSF17 deletion at baseline could theoretically be a risk factor for BCMA loss after immunotherapy.
• Homeostatic calcium fluxes, ER calcium release, SOCE, and calcium oscillations in cultured astrocytes are interlinked by a small calcium toolkit. Schulte, Annemarie; Bieniussa, Linda; Gupta, Rohini; Samtleben, Samira; Bischler, Thorsten; Doering, Kristina; Sodmann, Philipp; Rittner, Heike; Blum, Robert. In Cell Calcium, 101, p. 102515. Elsevier BV, 2021.
  • [ Abstract ]
  • [ DOI ]
  How homeostatic ER calcium fluxes shape cellular calcium signals is still poorly understood. Here we used dual-color calcium imaging (ER-cytosol) and transcriptome analysis to link candidates of the calcium toolkit of astrocytes with homeostatic calcium signals. We found molecular and pharmacological evidence that P/Q-type channel Cacna1a contributes to depolarization-dependent calcium entry in astrocytes. For stimulated ER calcium release, the cells express the phospholipase Cb3, IP3 receptors Itpr1 and Itpr2, but no ryanodine receptors (Ryr1–3). After IP3-induced calcium release, Stim1/2 – Orai1/2/3 most likely mediate SOCE. The Serca2 (Atp2a2) is the candidate for refilling of the ER calcium store. The cells highly express adenosine receptor Adora1a for IP3-induced calcium release. Accordingly, adenosine induces fast ER calcium release and subsequent ER calcium oscillations. After stimulation, calcium refilling of the ER depends on extracellular calcium. In response to SOCE, astrocytes show calcium-induced calcium release, notably even after ER calcium was depleted by extracellular calcium removal in unstimulated cells. In contrast, spontaneous ER-cytosol calcium oscillations were not fully dependent on extracellular calcium, as ER calcium oscillations could persist over minutes in calcium-free solution. Additionally, cell-autonomous calcium oscillations show a second-long spatial and temporal delay in the signal dynamics of ER and cytosolic calcium. Our data reveal a rather strong contribution of homeostatic calcium fluxes in shaping IP3-induced and calcium-induced calcium release as well as spatiotemporal components of intracellular calcium oscillations.
• Terminally Differentiated CD4+ T Cells Promote Myocardial Inflammaging. Delgobo, Murilo; Heinrichs, Margarete; Hapke, Nils; Ashour, DiyaaElDin; Appel, Marc; Srivastava, Mugdha; Heckel, Tobias; Spyridopoulos, Ioakim; Hofmann, Ulrich; Frantz, Stefan; Ramos, Gustavo Campos. In Frontiers in Immunology, 12. Frontiers Media SA, 2021.
  • [ Abstract ]
  • [ DOI ]
  The cardiovascular and immune systems undergo profound and intertwined alterations with aging. Recent studies have reported that an accumulation of memory and terminally differentiated T cells in elderly subjects can fuel myocardial aging and boost the progression of heart diseases. Nevertheless, it remains unclear whether the immunological senescence profile is sufficient to cause age-related cardiac deterioration or merely acts as an amplifier of previous tissue-intrinsic damage. Herein, we sought to decompose the causality in this cardio-immune crosstalk by studying young mice harboring a senescent-like expanded CD4+ T cell compartment. Thus, immunodeficient NSG-DR1 mice expressing HLA-DRB1*01:01 were transplanted with human CD4+ T cells purified from matching donors that rapidly engrafted and expanded in the recipients without causing xenograft reactions. In the donor subjects, the CD4+ T cell compartment was primarily composed of naïve cells defined as CCR7+CD45RO-. However, when transplanted into young lymphocyte-deficient mice, CD4+ T cells underwent homeostatic expansion, upregulated expression of PD-1 receptor and strongly shifted towards effector/memory (CCR7- CD45RO+) and terminally-differentiated phenotypes (CCR7-CD45RO-), as typically seen in elderly. Differentiated CD4+ T cells also infiltrated the myocardium of recipient mice at comparable levels to what is observed during physiological aging. In addition, young mice harboring an expanded CD4+ T cell compartment showed increased numbers of infiltrating monocytes, macrophages and dendritic cells in the heart. Bulk mRNA sequencing analyses further confirmed that expanding T-cells promote myocardial inflammaging, marked by a distinct age-related transcriptomic signature. Altogether, these data indicate that exaggerated CD4+ T-cell expansion and differentiation, a hallmark of the aging immune system, is sufficient to promote myocardial alterations compatible with inflammaging in juvenile healthy mice.
• Effector differentiation downstream of lineage commitment in ILC1s is driven by Hobit across tissues. Friedrich, Christin; Taggenbrock, Renske L. R. E.; Doucet-Ladevèze, Rémi; Golda, Gosia; Moenius, Rebekka; Arampatzi, Panagiota; Kragten, Natasja A. M.; Kreymborg, Katharina; de Agüero, Mercedes Gomez; Kastenmüller, Wolfgang; Saliba, Antoine-Emmanuel; Grün, Dominic; van Gisbergen, Klaas P. J. M.; Gasteiger, Georg. In Nature Immunology, 22(10), pp. 1256–1267. Springer Science and Business Media LLC, 2021.
  • [ Abstract ]
  • [ DOI ]
  Innate lymphoid cells (ILCs) participate in tissue homeostasis, inflammation, and early immunity against infection. It is unclear how ILCs acquire effector function and whether these mechanisms differ between organs. Through multiplexed single-cell mRNA sequencing, we identified cKit+CD127hiTCF-1hi early differentiation stages of T-bet+ ILC1s. These cells were present across different organs and had the potential to mature toward CD127intTCF-1int and CD127-TCF-1- ILC1s. Paralleling a gradual loss of TCF-1, differentiating ILC1s forfeited their expansion potential while increasing expression of effector molecules, reminiscent of T cell differentiation in secondary lymphoid organs. The transcription factor Hobit was induced in TCF-1hi ILC1s and was required for their effector differentiation. These findings reveal sequential mechanisms of ILC1 lineage commitment and effector differentiation that are conserved across tissues. Our analyses suggest that ILC1s emerge as TCF-1hi cells in the periphery and acquire a spectrum of organ-specific effector phenotypes through a uniform Hobit-dependent differentiation pathway driven by local cues.
• The Rsm (Csr) post-transcriptional regulatory pathway coordinately controls multiple CRISPR–Cas immune systems. Campa, Aroa Rey; Smith, Leah M; Hampton, Hannah G; Sharma, Sahil; Jackson, Simon A; Bischler, Thorsten; Sharma, Cynthia M; Fineran, Peter C. In Nucleic Acids Research, 49(16), pp. 9508–9525. 2021.
  • [ Abstract ]
  • [ DOI ]
  CRISPR–Cas systems provide bacteria with adaptive immunity against phages and plasmids; however, pathways regulating their activity are not well defined. We recently developed a high-throughput genome-wide method (SorTn-seq) and used this to uncover CRISPR–Cas regulators. Here, we demonstrate that the widespread Rsm/Csr pathway regulates the expression of multiple CRISPR–Cas systems in Serratia (type I-E, I-F and III-A). The main pathway component, RsmA (CsrA), is an RNA-binding post-transcriptional regulator of carbon utilisation, virulence and motility. RsmA binds cas mRNAs and suppresses type I and III CRISPR–Cas interference in addition to adaptation by type I systems. Coregulation of CRISPR–Cas and flagella by the Rsm pathway allows modulation of adaptive immunity when changes in receptor availability would alter susceptibility to flagella-tropic phages. Furthermore, we show that Rsm controls CRISPR–Cas in other genera, suggesting conservation of this regulatory strategy. Finally, we identify genes encoding RsmA homologues in phages, which have the potential to manipulate the physiology of host bacteria and might provide an anti-CRISPR activity.
• Opposing Wnt signals regulate cervical squamocolumnar homeostasis and emergence of metaplasia. Chumduri, Cindrilla; Gurumurthy, Rajendra Kumar; Berger, Hilmar; Dietrich, Oliver; Kumar, Naveen; Koster, Stefanie; Brinkmann, Volker; Hoffmann, Kirstin; Drabkina, Marina; Arampatzi, Panagiota; Son, Dajung; Klemm, Uwe; Mollenkopf, Hans-Joachim; Herbst, Hermann; Mangler, Mandy; Vogel, Jörg; Saliba, Antoine-Emmanuel; Meyer, Thomas F. In Nature Cell Biology, 23(2), pp. 184–197. Springer Science and Business Media LLC, 2021.
  • [ Abstract ]
  • [ DOI ]
  The transition zones of the squamous and columnar epithelia constitute hotspots for the emergence of cancer, often preceded by metaplasia, in which one epithelial type is replaced by another. It remains unclear how the epithelial spatial organization is maintained and how the transition zone niche is remodelled during metaplasia. Here we used single-cell RNA sequencing to characterize epithelial subpopulations and the underlying stromal compartment of endo- and ectocervix, encompassing the transition zone. Mouse lineage tracing, organoid culture and single-molecule RNA in situ hybridizations revealed that the two epithelia derive from separate cervix-resident lineage-specific stem cell populations regulated by opposing Wnt signals from the stroma. Using a mouse model of cervical metaplasia, we further show that the endocervical stroma undergoes remodelling and increases expression of the Wnt inhibitor Dickkopf-2 (DKK2), promoting the outgrowth of ectocervical stem cells. Our data indicate that homeostasis at the transition zone results from divergent stromal signals, driving the differential proliferation of resident epithelial lineages.
• MAPS integrates regulation of actin-targeting effector SteC into the virulence control network of Salmonella small RNA PinT. Santos, Sara Correia; Bischler, Thorsten; Westermann, Alexander J.; Vogel, Jörg. In Cell Reports, 34(5), p. 108722. Elsevier BV, 2021.
  • [ Abstract ]
  • [ DOI ]
  A full understanding of the contribution of small RNAs (sRNAs) to bacterial virulence demands knowledge of their target suites under infection-relevant conditions. Here, we take an integrative approach to capturing targets of the Hfq-associated sRNA PinT, a known post-transcriptional timer of the two major virulence programs of Salmonella enterica. Using MS2 affinity purification and RNA sequencing (MAPS), we identify PinT ligands in bacteria under in vitro conditions mimicking specific stages of the infection cycle and in bacteria growing inside macrophages. This reveals PinT-mediated translational inhibition of the secreted effector kinase SteC, which had gone unnoticed in previous target searches. Using genetic, biochemical, and microscopic assays, we provide evidence for PinT-mediated repression of steC mRNA, eventually delaying actin rearrangements in infected host cells. Our findings support the role of PinT as a central post-transcriptional regulator in Salmonella virulence and illustrate the need for complementary methods to reveal the full target suites of sRNAs.
• Roux-en-Y Gastric Bypass and Caloric Restriction but Not Gut Hormone-Based Treatments Profoundly Impact the Hypothalamic Transcriptome in Obese Rats. Dischinger, Ulrich; Heckel, Tobias; Bischler, Thorsten; Hasinger, Julia; Königsrainer, Malina; Schmitt-Böhrer, Angelika; Otto, Christoph; Fassnacht, Martin; Seyfried, Florian; Hankir, Mohammed Khair. In Nutrients, 14(1), p. 116. MDPI AG, 2021.
  • [ Abstract ]
  • [ DOI ]
  Background: The hypothalamus is an important brain region for the regulation of energy balance. Roux-en-Y gastric bypass (RYGB) surgery and gut hormone-based treatments are known to reduce body weight, but their effects on hypothalamic gene expression and signaling pathways are poorly studied. Methods: Diet-induced obese male Wistar rats were randomized into the following groups: RYGB, sham operation, sham + body weight-matched (BWM) to the RYGB group, osmotic minipump delivering PYY3-36 (0.1 mg/kg/day), liraglutide s.c. (0.4 mg/kg/day), PYY3-36 + liraglutide, and saline. All groups (except BWM) were kept on a free choice of high- and low-fat diets. Four weeks after interventions, hypothalami were collected for RNA sequencing. Results: While rats in the RYGB, BWM, and PYY3-36 + liraglutide groups had comparable reductions in body weight, only RYGB and BWM treatment had a major impact on hypothalamic gene expression. In these groups, hypothalamic leptin receptor expression as well as the JAK-STAT, PI3K-Akt, and AMPK signaling pathways were upregulated. No significant changes could be detected in PYY3-36 + liraglutide-, liraglutide-, and PYY-treated groups. Conclusions: Despite causing similar body weight changes compared to RYGB and BWM, PYY3-36 + liraglutide treatment does not impact hypothalamic gene expression. Whether this striking difference is favorable or unfavorable to metabolic health in the long term requires further investigation. Keywords: PYY3-36; Roux-en-Y gastric bypass surgery; hypothalamic gene expression; liraglutide; obesity.

• The Influence of Met Receptor Level on {HGF}-Induced Glycolytic Reprogramming in Head and Neck Squamous Cell Carcinoma. Boschert, Verena; Klenk, Nicola; Abt, Alexander; Raman, Sudha Janaki; Fischer, Markus; Brands, Roman C.; Seher, Axel; Linz, Christian; Müller-Richter, Urs D. A.; Bischler, Thorsten; Hartmann, Stefan. In International Journal of Molecular Sciences, 21(2), p. 471. {MDPI} {AG}, 2020.
  • [ Abstract ]
  • [ DOI ]
  Head and neck squamous cell carcinoma (HNSCC) is known to overexpress a variety of receptor tyrosine kinases, such as the HGF receptor Met. Like other malignancies, HNSCC involves a mutual interaction between the tumor cells and surrounding tissues and cells. We hypothesized that activation of HGF/Met signaling in HNSCC influences glucose metabolism and therefore substantially changes the tumor microenvironment. To determine the effect of HGF, we submitted three established HNSCC cell lines to mRNA sequencing. Dynamic changes in glucose metabolism were measured in real time by an extracellular flux analyzer. As expected, the cell lines exhibited different levels of Met and responded differently to HGF stimulation. As confirmed by mRNA sequencing, the level of Met expression was associated with the number of upregulated HGF-dependent genes. Overall, Met stimulation by HGF leads to increased glycolysis, presumably mediated by higher expression of three key enzymes of glycolysis. These effects appear to be stronger in Methigh-expressing HNSCC cells. Collectively, our data support the hypothesized role of HGF/Met signaling in metabolic reprogramming of HNSCC.
• Single-cell transcriptomic profiling maps monocyte/macrophage transitions after myocardial infarction in mice. Rizzo, Giuseppe; Vafadarnejad, Ehsan; Arampatzi, Panagiota; Silvestre, Jean-Sebastien; Zernecke, Alma; Saliba, Antoine-Emmanuel; Cochain, Clement. In bioRxiv. Cold Spring Harbor Laboratory, 2020.
  • [ Abstract ]
  • [ DOI ]
  Rationale: Monocytes and macrophages have a critical and dual role in post-ischemic cardiac repair, as they can foster both tissue healing and damage. To decipher how monocytes/macrophages acquire heterogeneous functional phenotypes in the ischemic myocardium, we profiled the gene expression dynamics at the single-cell level in circulating and cardiac monocytes/macrophages following experimental myocardial infarction (MI) in mice. Methods and results: Using time-series single-cell transcriptome and cell surface epitope analysis of blood and cardiac monocytes/macrophages, as well as the integration of publicly available and independently generated single-cell RNA-seq data, we tracked the transitions in circulating and cardiac monocyte/macrophage states from homeostatic conditions up to 11 days after MI in mice. We show that MI induces marked and rapid transitions in the cardiac mononuclear phagocyte population, with almost complete disappearance of tissue resident macrophages 1 day after ischemia, and rapid infiltration of monocytes that locally acquire discrete and time-dependent transcriptional states within 3 to 7 days. Ischemic injury induced a shift of circulating monocytes towards granulocyte-like transcriptional features (Chil3, Lcn2, Prtn3). Trajectory inference analysis indicated that while conversion to Ly6Clow monocytes appears as the default fate of Ly6Chi monocytes in the blood, infiltrated monocytes acquired diverse gene expression signatures in the injured heart, notably transitioning to two main MI-associated macrophage populations characterized by MHCIIhi and Trem2hiIgf1hi gene expression signatures. Minor ischemia-associated macrophage populations with discrete gene expression signatures suggesting specialized functions in e.g. iron handling or lipid metabolism were also observed. We further identified putative transcriptional regulators and new cell surface markers of cardiac monocyte/macrophage states. Conclusions: Altogether, our work provides a comprehensive landscape of circulating and cardiac monocyte/macrophage states and their regulators after MI, and will help to further understand their contribution to post-myocardial infarction heart repair.Competing Interest StatementThe authors have declared no competing interest.
• {IL}-12 from endogenous {cDC}1, and not vaccine {DC}, is required for Th1 induction. Ashour, DiyaaElDin; Arampatzi, Panagiota; Pavlovic, Vladimir; Förstner, Konrad U.; Kaisho, Tsuneyasu; Beilhack, Andreas; Erhard, Florian; Lutz, Manfred B. In {JCI} Insight, 5(10). American Society for Clinical Investigation, 2020.
  • [ Abstract ]
  • [ DOI ]
  Success of DC vaccines relies on the quality of antigen presentation, costimulation, lymph node migration, and the release of IL-12, in case of Th1 priming. Here, we provide evidence for interaction between the injected vaccine DCs with endogenous lymph node–resident DCs for Th1 induction. While migration of the injected DCs was essential for antigen delivery to the lymph node, the injected DCs contributed only partially to Th0 priming and were unable to instruct Th1 generation. Instead, we provide evidence that the lymph node–resident XCR1+ DCs are activated by the injected DCs to present the cognate antigen and release IL-12 for Th1 polarization. The timing of interactions in the draining lymph nodes appeared step-wise as (a) injected DCs with cognate T cells, (b) injected DCs with bystander DCs, and (c) bystander DCs with T cells. The transcriptome of the bystander DCs showed a downregulation of Treg- and Th2/Th9-inducing genes and self-antigen presentation, as well as upregulation of MHC class II and genes required for Th1 instruction. Together, these data show that injected mature lymph node migratory DCs direct T cell priming and bystander DC activation, but not Th1 polarization, which is mediated by endogenous IL-12p70+XCR1+ resident bystander DCs. Our results are of importance for clinical DC-based vaccinations against tumors where endogenous DCs may be functionally impaired by chemotherapy.
• Dynamics of Cardiac Neutrophil Diversity in Murine Myocardial Infarction. Vafadarnejad, Ehsan; Rizzo, Giuseppe; Krampert, Laura; Arampatzi, Panagiota; Arias-Loza, Paula Anahi; Nazzal, Yara; Rizakou, Anna; Knochenhauer, Tim; Bandi, Sourish Reddy; Nugroho, Vallery Audy; Schulz, Dirk JJ; Roesch, Melanie; Alayrac, Paul; Vilar, Jos{{é}}; Silvestre, Jean-S{{é}}bastien; Zernecke, Alma; Saliba, Antoine-Emmanuel; Cochain, Clement. In Circulation Research. Ovid Technologies (Wolters Kluwer Health), 2020.
  • [ Abstract ]
  • [ DOI ]
  Rationale: After myocardial infarction, neutrophils rapidly and massively infiltrate the heart, where they promote both tissue healing and damage. Objective: To characterize the dynamics of circulating and cardiac neutrophil diversity after infarction. Methods and Results: We employed single-cell transcriptomics combined with cell surface epitope detection by sequencing (CITE-seq) to investigate temporal neutrophil diversity in the blood and heart after murine myocardial infarction. At day 1, 3, and 5 after infarction, cardiac Ly6G+ neutrophils could be delineated into six distinct clusters with specific time-dependent patterning and proportions. At day 1, neutrophils were characterized by a gene expression profile proximal to bone marrow neutrophils (Cd177, Lcn2, Fpr1), and putative activity of transcriptional regulators involved in hypoxic response (Hif1a) and emergency granulopoiesis (Cebpb). At 3 and 5 days two major subsets of Siglecfhi (enriched for e.g. Icam1 and Tnf) and Siglecflow (nSlpi, Ifitm1) neutrophils were found. CITE-seq analysis in blood and heart revealed that while circulating neutrophils undergo a process of ageing characterized by loss of surface CD62L and upregulation of Cxcr4, heart infiltrating neutrophils acquired a unique SiglecFhi signature. SiglecFhi neutrophils were absent from the bone marrow and spleen, indicating local acquisition of the SiglecFhi signature. Reducing the influx of blood neutrophils by anti-Ly6G treatment increased proportions of cardiac SiglecFhi neutrophils, suggesting accumulation of locally aged neutrophils. Computational analysis of ligand/receptor interactions revealed putative pathways mediating neutrophil to macrophage communication in the myocardium. Finally, SiglecFhi neutrophils were also found in atherosclerotic vessels, revealing that they arise across distinct contexts of cardiovascular inflammation. Conclusions: Altogether, our data provide a time-resolved census of neutrophil diversity and gene expression dynamics in the mouse blood and ischemic heart at the single-cell level, and reveal a process of local tissue specification of neutrophils in the ischemic heart characterized by the acquisition of a SiglecFhi signature.
• Tetrachloroethene respiration in Sulfurospirillum species is regulated by a two-component system as unraveled by comparative genomics, transcriptomics, and regulator binding studies. Esken, Jens; Goris, Tobias; Gadkari, Jennifer; Bischler, Thorsten; Förstner, Konrad U.; Sharma, Cynthia M.; Diekert, Gabriele; Schubert, Torsten. In {MicrobiologyOpen}. Wiley, 2020.
  • [ Abstract ]
  • [ DOI ]
  Energy conservation via organohalide respiration (OHR) in dehalogenating Sulfurospirillum species is an inducible process. However, the gene products involved in tetrachloroethene (PCE) sensing and signal transduction have not been unambiguously identified. Here, genome sequencing of Sulfurospirillum strains defective in PCE respiration and comparative genomics, which included the PCE-respiring representatives of the genus, uncovered the genetic inactivation of a two-component system (TCS) in the OHR gene region of the natural mutants. The assumption that the TCS gene products serve as a PCE sensor that initiates gene transcription was supported by the constitutive low-level expression of the TCS operon in fumarate-adapted cells of Sulfurospirillum multivorans. Via RNA sequencing, eight transcriptional units were identified in the OHR gene region, which includes the TCS operon, the PCE reductive dehalogenase operon, the gene cluster for norcobamide biosynthesis, and putative accessory genes with unknown functions. The OmpR-family response regulator (RR) encoded in the TCS operon was functionally characterized by promoter-binding assays. The RR bound a cis-regulatory element that contained a consensus sequence of a direct repeat (CTATW) separated by 17 bp. Its location either overlapping the -35 box or 50 bp further upstream indicated different regulatory mechanisms. Sequence variations in the regulator binding sites identified in the OHR gene region were in accordance with differences in the transcript levels of the respective gene clusters forming the PCE regulon. The results indicate the presence of a fine-tuned regulatory network controlling PCE metabolism in dehalogenating Sulfurospirillum species, a group of metabolically versatile organohalide-respiring bacteria.
• Integrative functional genomics decodes herpes simplex virus 1. Whisnant, Adam W.; Jürges, Christopher S.; Hennig, Thomas; Wyler, Emanuel; Prusty, Bhupesh; Rutkowski, Andrzej J.; L’hernault, Anne; Djakovic, Lara; Göbel, Margarete; Döring, Kristina; Menegatti, Jennifer; Antrobus, Robin; Matheson, Nicholas J.; Künzig, Florian W. H.; Mastrobuoni, Guido; Bielow, Chris; Kempa, Stefan; Liang, Chunguang; Dandekar, Thomas; Zimmer, Ralf; Landthaler, Markus; Grässer, Friedrich; Lehner, Paul J.; Friedel, Caroline C.; Erhard, Florian; Dölken, Lars. In Nature Communications, 11(1), p. 2038-. 2020.
  • [ Abstract ]
  • [ DOI ]
  The predicted 80 open reading frames (ORFs) of herpes simplex virus 1 (HSV-1) have been intensively studied for decades. Here, we unravel the complete viral transcriptome and translatome during lytic infection with base-pair resolution by computational integration of multi-omics data. We identify a total of 201 transcripts and 284 ORFs including all known and 46 novel large ORFs. This includes a so far unknown ORF in the locus deleted in the FDA-approved oncolytic virus Imlygic. Multiple transcript isoforms expressed from individual gene loci explain translation of the vast majority of ORFs as well as N-terminal extensions (NTEs) and truncations. We show that NTEs with non-canonical start codons govern the subcellular protein localization and packaging of key viral regulators and structural proteins. We extend the current nomenclature to include all viral gene products and provide a genome browser that visualizes all the obtained data from whole genome to single-nucleotide resolution.
• A Repeat-Associated Small {RNA} Controls the Major Virulence Factors of Helicobacter pylori. Eisenbart, Sara K.; Alzheimer, Mona; Pernitzsch, Sandy R.; Dietrich, Sascha; Stahl, Stephanie; Sharma, Cynthia M. In Molecular Cell. Elsevier {BV}, 2020.
  • [ Abstract ]
  • [ DOI ]
  Many bacterial pathogens regulate their virulence genes via phase variation, whereby length-variable simple sequence repeats control the transcription or coding potential of those genes. Here, we have exploited this relationship between DNA structure and physiological function to discover a globally acting small RNA (sRNA) regulator of virulence in the gastric pathogen Helicobacter pylori. Our study reports the first sRNA whose expression is affected by a variable thymine (T) stretch in its promoter. We show the sRNA post-transcriptionally represses multiple major pathogenicity factors of H. pylori, including CagA and VacA, by base pairing to their mRNAs. We further demonstrate transcription of the sRNA is regulated by the nickel-responsive transcriptional regulator NikR (thus named NikS for nickel-regulated sRNA), thereby linking virulence factor regulation to nickel concentrations. Using in-vitro infection experiments, we demonstrate NikS affects host cell internalization and epithelial barrier disruption. Together, our results show NikS is a phase-variable, post-transcriptional global regulator of virulence properties in H. pylori.
• Improved bacterial {RNA}-seq by Cas9-based depletion of ribosomal {RNA} reads. Prezza, Gianluca; Heckel, Tobias; Dietrich, Sascha; Homberger, Christina; Westermann, Alexander J; Vogel, Jörg. In {RNA}, p. rna.075945.120. Cold Spring Harbor Laboratory, 2020.
  • [ Abstract ]
  • [ DOI ]
  A major challenge for RNA-seq analysis of gene expression is to achieve sufficient coverage of informative non-ribosomal transcripts. In eukaryotic samples, this is typically achieved by selective oligo(dT)-priming of messenger RNAs to exclude ribosomal RNA (rRNA) during cDNA synthesis. However, this strategy is not compatible with prokaryotes in which functional transcripts are generally not polyadenylated. To overcome this, we adopted DASH (Depletion of Abundant Sequences by Hybridization), initially developed for eukaryotic cells, to improve both the sensitivity and depth of bacterial RNA-seq. DASH employs the Cas9 nuclease to remove unwanted cDNA sequences prior to library amplification. We report the design, evaluation, and optimization of DASH experiments for standard bacterial short-read sequencing approaches, including software for automated guide RNA (gRNA) design for Cas9-mediated cleavage in bacterial rDNA sequences. Using these gRNA pools, we effectively removed rRNA reads (56-86%) in RNA-seq libraries from two different model bacteria, the Gram-negative pathogen Salmonella enterica and the anaerobic gut commensal Bacteroides thetaiotaomicron. DASH works robustly, even with sub-nanogram amounts of input cDNA. Its efficiency, high sensitivity, ease of implementation, and low cost (~$5 per sample) render DASH an attractive alternative to rRNA removal protocols, in particular for material-constrained studies where conventional ribodepletion techniques fail.
• The minimal meningococcal ProQ protein has an intrinsic capacity for structure-based global RNA recognition. Bauriedl, Saskia; Gerovac, Milan; Heidrich, Nadja; Bischler, Thorsten; Barquist, Lars; Vogel, Jörg; Schoen, Christoph. In Nature Communications, 11(1), p. 2823-. 2020.
  • [ Abstract ]
  • [ DOI ]
  FinO-domain proteins are a widespread family of bacterial RNA-binding proteins with regulatory functions. Their target spectrum ranges from a single RNA pair, in the case of plasmid-encoded FinO, to global RNA regulons, as with enterobacterial ProQ. To assess whether the FinO domain itself is intrinsically selective or promiscuous, we determine in vivo targets of Neisseria meningitidis, which consists of solely a FinO domain. UV-CLIP-seq identifies associations with 16 small non-coding sRNAs and 166 mRNAs. Meningococcal ProQ predominantly binds to highly structured regions and generally acts to stabilize its RNA targets. Loss of ProQ alters transcript levels of >250 genes, demonstrating that this minimal ProQ protein impacts gene expression globally. Phenotypic analyses indicate that ProQ promotes oxidative stress resistance and DNA damage repair. We conclude that FinO domain proteins recognize some abundant type of RNA shape and evolve RNA binding selectivity through acquisition of additional regions that constrain target recognition.

• Cognate {Nonlytic} {Interactions} between {CD}8+ {T} {Cells} and {Breast} {Cancer} {Cells} {Induce} {Cancer} {Stem} {Cell}–like {Properties}. Stein, Roland G.; Ebert, Stefan; Schlahsa, Laura; Scholz, Claus J.; Braun, Matthias; Hauck, Petra; Horn, Evi; Monoranu, Camelia-Maria; Thiemann, Vincent J.; Wustrow, Michael P.; Häusler, Sebastian F.; Barrio, Itsaso Montalbán del; Stüber, Tanja N.; Wölfl, Matthias; Dietl, Johannes; Rosenwald, Andreas; Diessner, Joachim E.; Wöckel, Achim; Wischhusen, Jörg. In Cancer Research, 79(7), pp. 1507–1519. 2019.
  • [ Abstract ]
  • [ DOI ]
  Targeting of tumor immune escape mechanisms holds enormous therapeutic potential. Still, most patients progress under immune checkpoint blockade and some even become hyperprogressors. To investigate how cancer cells respond to activated but ineffective T cells, we challenged peptide-loaded MCF-7 breast cancer cells with antigen-specific CD8+ T cells in which lytic granules had been destroyed by pretreatment with Concanamycin A. Gene expression analysis after coculture revealed simultaneous induction of PD-L1, IDO1, CEACAM1, and further immunoregulatory checkpoints in breast cancer cells. Strikingly, we further observed gene signatures characteristic for dedifferentiation and acquisition of pluripotency markers including Yamanaka factors. Cognate interaction with nonlytic CD8+ T cells also increased the proportion of stem cell–like cancer cells in a cell-to-cell contact- or (at least) proximity-dependent manner in various cell lines and in primary breast cancer cell cultures; this induction of stem cell–like properties was confirmed by enhanced tumor-forming capacity in immunodeficient mice. Resulting tumors were characterized by enhanced cell density, higher proliferation rates, and increased propensity for lymphoid metastasis. These findings describe a widely underappreciated pathway for immune escape, namely immune-mediated dedifferentiation of breast cancer cells, which is associated with profound changes in gene expression and cellular behavior. As the enhanced malignant potential of cancer cells after nonlytic cognate interactions with CD8+ T cells enables increased tumor growth and metastasis in BALB/cnu/nu mice, the described mechanism may provide a possible explanation for the clinical phenomenon of hyperprogression in response to unsuccessful immunotherapy. Significance: This study shows that ineffective immune responses not only fail to clear a malignancy, but can also activate pathways in cancer cells that promote stemness and tumor-seeding capacity.
• Conditional Hfq Association with Small Noncoding RNAs in Pseudomonas aeruginosa Revealed through Comparative UV Cross-Linking Immunoprecipitation Followed by High-Throughput Sequencing. Chihara, K; Bischler, T; Barquist, L; Monzon, V A; Noda, N; Vogel, J; Tsuneda, S. In mSystems, 4(6). 2019.
  • [ Abstract ]
  • [ DOI ]
  Bacterial small noncoding RNAs (sRNAs) play posttranscriptional regulatory roles in cellular responses to changing environmental cues and in adaptation to harsh conditions. Generally, the RNA-binding protein Hfq helps sRNAs associate with target mRNAs to modulate their translation and to modify global RNA pools depending on physiological state. Here, a combination of in vivo UV cross-linking immunoprecipitation followed by high-throughput sequencing (CLIP-seq) and total RNA-seq showed that Hfq interacts with different regions of the Pseudomonas aeruginosa transcriptome under planktonic versus biofilm conditions. In the present approach, P. aeruginosa Hfq preferentially interacted with repeats of the AAN triplet motif at mRNA 5' untranslated regions (UTRs) and sRNAs and U-rich sequences at rho-independent terminators. Further transcriptome analysis suggested that the association of sRNAs with Hfq is primarily a function of their expression levels, strongly supporting the notion that the pool of Hfq-associated RNAs is equilibrated by RNA concentration-driven cycling on and off Hfq. Overall, our combinatorial CLIP-seq and total RNA-seq approach highlights conditional sRNA associations with Hfq as a novel aspect of posttranscriptional regulation in P. aeruginosaIMPORTANCE The Gram-negative bacterium P. aeruginosa is ubiquitously distributed in diverse environments and can cause severe biofilm-related infections in at-risk individuals. Although the presence of a large number of putative sRNAs and widely conserved RNA chaperones in this bacterium implies the importance of posttranscriptional regulatory networks for environmental fluctuations, limited information is available regarding the global role of RNA chaperones such as Hfq in the P. aeruginosa transcriptome, especially under different environmental conditions. Here, we characterize Hfq-dependent differences in gene expression and biological processes in two physiological states: the planktonic and biofilm forms. A combinatorial comparative CLIP-seq and total RNA-seq approach uncovered condition-dependent association of RNAs with Hfq in vivo and expands the potential direct regulatory targets of Hfq in the P. aeruginosa transcriptome.
• Efficient depletion of ribosomal RNA for RNA sequencing in planarians. Kim, Iana V.; Ross, Eric J.; Dietrich, Sascha; Döring, Kristina; Sánchez Alvarado, Alejandro; Kuhn, Claus-D. In BMC Genomics, 20(1), p. 909. 2019.
  • [ Abstract ]
  • [ DOI ]
  The astounding regenerative abilities of planarian flatworms prompt steadily growing interest in examining their molecular foundation. Planarian regeneration was found to require hundreds of genes and is hence a complex process. Thus, RNA interference followed by transcriptome-wide gene expression analysis by RNA-seq is a popular technique to study the impact of any particular planarian gene on regeneration. Typically, the removal of ribosomal RNA (rRNA) is the first step of all RNA-seq library preparation protocols. To date, rRNA removal in planarians was primarily achieved by the enrichment of polyadenylated (poly(A)) transcripts. However, to better reflect transcriptome dynamics and to cover also non-poly(A) transcripts, a procedure for the targeted removal of rRNA in planarians is needed.
• Integrative functional genomics decodes herpes simplex virus 1. Whisnant, Adam W.; J{ü}rges, Christopher S.; Hennig, Thomas; Wyler, Emanuel; Prusty, Bhupesh; Rutkowski, Andrzej J; L{\textquoteright}hernault, Anne; G{ö}bel, Margarete; D{ö}ring, Kristina; Menegatti, Jennifer; Antrobus, Robin; Matheson, Nicholas J.; K{ü}nzig, Florian W.H.; Mastrobuoni, Guido; Bielow, Chris; Kempa, Stefan; Chunguang, Liang; Dandekar, Thomas; Zimmer, Ralf; Landthaler, Markus; Gr{ä}sser, Friedrich; Lehner, Paul J.; Friedel, Caroline C.; Erhard, Florian; D{ö}lken, Lars. In bioRxiv. Cold Spring Harbor Laboratory, 2019.
  • [ Abstract ]
  • [ DOI ]
  Since the genome of herpes simplex virus 1 (HSV-1) was first sequenced more than 30 years ago, its predicted 80 genes have been intensively studied. Here, we unravel the complete viral transcriptome and translatome during lytic infection with base-pair resolution by computational integration of multi-omics data. We identified a total of 201 viral transcripts and 284 open reading frames (ORFs) including all known and 46 novel large ORFs. Multiple transcript isoforms expressed from individual gene loci explain translation of the vast majority of novel viral ORFs as well as N-terminal extensions (NTEs) and truncations thereof. We show that key viral regulators and structural proteins possess NTEs, which initiate from non-canonical start codons and govern subcellular protein localization and packaging. We validated a novel non-canonical large spliced ORF in the ICP0 locus and identified a 93 aa ORF overlapping ICP34.5 that is thus also deleted in the FDA-approved oncolytic virus Imlygic. Finally, we extend the current nomenclature to include all novel viral gene products. Taken together, this work provides a valuable resource for future functional studies, vaccine design and oncolytic therapies.
• The identification of patient-specific mutations reveals dual pathway activation in most patients with melanoma and activated receptor tyrosine kinases in {BRAF}/{NRAS} wild-type melanomas. Appenzeller, Silke; Gesierich, Anja; Thiem, Alexander; Hufnagel, Anita; Jessen, Christina; Kneitz, Hermann; Regensburger, Martina; Schmidt, Cornelia; Zirkenbach, Vanessa; Bischler, Thorsten; Schilling, Bastian; Siedel, Claudia; Goebeler, Maria-Elisabeth; Houben, Roland; Schrama, David; Gehrig, Andrea; Rost, Simone; Maurus, Katja; Bargou, Ralf; Rosenwald, Andreas; Schartl, Manfred; Goebeler, Matthias; Meierjohann, Svenja. In Cancer, 125(4), pp. 586–600. 2019.
  • [ Abstract ]
  • [ DOI ]
  Background Increasing knowledge of cancer genomes has triggered the development of specific targeted inhibitors, thus providing a valuable therapeutic pool. Methods In this report, the authors analyze the presence of targetable alterations in 136 tumor samples from 92 patients with melanoma using a comprehensive approach based on targeted DNA sequencing and supported by RNA and protein analysis. Three topics of high clinical relevance are addressed: the identification of rare, activating alterations; the detection of patient-specific, co-occurring single nucleotide variants (SNVs) and copy number variations (CNVs) in parallel pathways; and the presence of cancer-relevant germline mutations. Results The analysis of patient-matched blood and tumor samples was done with a custom-designed gene panel that was enriched for genes from clinically targetable pathways. To detect alterations with high therapeutic relevance for patients with unknown driver mutations, genes that are untypical for melanoma also were included. Among all patients, CNVs were identified in one-third of samples and contained amplifications of druggable kinases, such as CDK4, ERBB2, and KIT. Considering SNVs and CNVs, 60\% of patients with metastases exhibited co-occurring activations of at least 2 pathways, thus providing a rationale for individualized combination therapies. Unexpectedly, 9\% of patients carry potentially protumorigenic germline mutations frequently affecting receptor tyrosine kinases. Remarkably two-thirds of BRAF/NRAS wild-type melanomas harbor activating mutations or CNVs in receptor tyrosine kinases. Conclusions The results indicate that the integrated analysis of SNVs, CNVs, and germline mutations reveals new druggable targets for combination tumor therapy.
• Characterization of the transcriptome of Haloferax volcanii, grown under four different conditions, with mixed RNA-Seq. Laass, Sebastian; Monzon, Vivian A.; Kliemt, Jana; Hammelmann, Matthias; Pfeiffer, Friedhelm; Förstner, Konrad U.; Soppa, Jörg. In PLOS ONE, 14(4), pp. 1–24. Public Library of Science, 2019.
  • [ Abstract ]
  • [ DOI ]
  Haloferax volcanii is a well-established model species for haloarchaea. Small scale RNomics and bioinformatics predictions were used to identify small non-coding RNAs (sRNAs), and deletion mutants revealed that sRNAs have important regulatory functions. A recent dRNA-Seq study was used to characterize the primary transcriptome. Unexpectedly, it was revealed that, under optimal conditions, H. volcanii contains more non-coding sRNAs than protein-encoding mRNAs. However, the dRNA-Seq approach did not contain any length information. Therefore, a mixed RNA-Seq approach was used to determine transcript length and to identify additional transcripts, which are not present under optimal conditions. In total, 50 million paired end reads of 150 nt length were obtained. 1861 protein-coding RNAs (cdRNAs) were detected, which encoded 3092 proteins. This nearly doubled the coverage of cdRNAs, compared to the previous dRNA-Seq study. About 2/3 of the cdRNAs were monocistronic, and 1/3 covered more than one gene. In addition, 1635 non-coding sRNAs were identified. The highest fraction of non-coding RNAs were cis antisense RNAs (asRNAs). Analysis of the length distribution revealed that sRNAs have a median length of about 150 nt. Based on the RNA-Seq and dRNA-Seq results, genes were chosen to exemplify characteristics of the H. volcanii transcriptome by Northern blot analyses, e.g. 1) the transcript patterns of gene clusters can be straightforward, but also very complex, 2) many transcripts differ in expression level under the four analyzed conditions, 3) some genes are transcribed into RNA isoforms of different length, which can be differentially regulated, 4) transcripts with very long 5’-UTRs and with very long 3’-UTRs exist, and 5) about 30% of all cdRNAs have overlapping 3’-ends, which indicates, together with the asRNAs, that H. volcanii makes ample use of sense-antisense interactions. Taken together, this RNA-Seq study, together with a previous dRNA-Seq study, enabled an unprecedented view on the H. volcanii transcriptome.
• {MicroRNA} expression correlates with disease recurrence and overall survival in oral squamous cell carcinoma. Moratin, Julius; Hartmann, Stefan; Brands, Roman C.; Horn, Dominik; Fuchs, Andreas; Mutzbauer, Grit; Seher, Axel; Scholz, Claus; Müller-Richter, Urs D.A.; Linz, Christian. In Journal of Cranio-Maxillofacial Surgery, 47(3), pp. 523–529. Elsevier {BV}, 2019.
  • [ Abstract ]
  • [ DOI ]
  OBJECTIVES: Locoregional disease recurrence and metastatic events are the leading causes of death and the most important prognostic factors in patients with head and neck squamous cell carcinoma (HNSCC). A major goal of oncology is the identification of clinical and molecular parameters to evaluate the individual risk of recurrence. MicroRNAs (miRNAs) have been shown to correlate well with tumor size and differentiation. Therefore, they are candidate biomarkers for estimating clinical outcomes. MATERIALS AND METHODS: In this study, the expression levels of distinct miRNAs extracted from formalin-fixed, paraffin-embedded (FFPE) samples of oral squamous cell carcinoma were compared. RESULTS: Statistical analysis revealed significant correlations between distinct miRNAs and disease recurrence (miR-99*, miR-194*; p < 0.05) and overall survival (miR-99*; p < 0.05). The results were then validated via data from The Cancer Genome Atlas (TCGA). CONCLUSIONS: Our data show that miR-99* and miR-194* can possibly serve as biomarkers for clinical outcome in HNSCC. These findings may help to identify high-risk patients, who could profit from a more individualized treatment and follow-up.
• The small non-coding {RNA} {RsaE} influences extracellular matrix composition in {Staphylococcus} epidermidis biofilm communities. Schoenfelder, Sonja M. K.; Lange, Claudia; Prakash, Srinivasa Abishek; Marincola, Gabriella; Lerch, Maike F.; Wencker, Freya D. R.; Förstner, Konrad U.; Sharma, Cynthia M.; Ziebuhr, Wilma. In PLOS Pathogens, 15(3), p. e1007618. 2019.
  • [ Abstract ]
  • [ DOI ]
  RsaE is a conserved small regulatory RNA (sRNA) which was previously reported to represent a riboregulator of central carbon flow and other metabolic pathways in Staphylococcus aureus and Bacillus subtilis. Here we show that RsaE contributes to extracellular (e)DNA release and biofilm-matrix switching towards polysaccharide intercellular adhesin (PIA) production in a hypervariable Staphylococcus epidermidis isolate. Transcriptome analysis through differential RNA sequencing (dRNA-seq) in combination with confocal laser scanning microscopy (CLSM) and reporter gene fusions demonstrate that S. epidermidis protein- and PIA-biofilm matrix producers differ with respect to RsaE and metabolic gene expression. RsaE is spatiotemporally expressed within S. epidermidis PIA-mediated biofilms, and its overexpression triggers a PIA biofilm phenotype as well as eDNA release in an S. epidermidis protein biofilm matrix-producing strain background. dRNA-seq and Northern blot analyses revealed RsaE to exist as a major full-length 100-nt transcript and a minor processed species lacking approximately 20 nucleotides at the 5'-end. RsaE processing results in expansion of the mRNA target spectrum. Thus, full-length RsaE interacts with S. epidermidis antiholin-encoding lrgA mRNA, facilitating bacterial lysis and eDNA release. Processed RsaE, however, interacts with the 5'-UTR of icaR and sucCD mRNAs, encoding the icaADBC biofilm operon repressor IcaR and succinyl-CoA synthetase of the tricarboxylic acid (TCA) cycle, respectively. RsaE augments PIA-mediated biofilm matrix production, most likely through activation of icaADBC operon expression via repression of icaR as well as by TCA cycle inhibition and re-programming of staphylococcal central carbon metabolism towards PIA precursor synthesis. Additionally, RsaE supports biofilm formation by mediating the release of eDNA as stabilizing biofilm matrix component. As RsaE itself is heterogeneously expressed within biofilms, we consider this sRNA to function as a factor favoring phenotypic heterogeneity and supporting division of labor in S. epidermidis biofilm communities.
• The {Major} {RNA}-{Binding} {Protein} {ProQ} {Impacts} {Virulence} {Gene} {Expression} in {Salmonella} enterica {Serovar} {Typhimurium}. Westermann, Alexander J.; Venturini, Elisa; Sellin, Mikael E.; Förstner, Konrad U.; Hardt, Wolf-Dietrich; Vogel, Jörg. In mBio, 10(1), pp. e02504–18. 2019.
  • [ Abstract ]
  • [ DOI ]
  FinO domain proteins such as ProQ of the model pathogen Salmonella enterica have emerged as a new class of major RNA-binding proteins in bacteria. ProQ has been shown to target hundreds of transcripts, including mRNAs from many virulence regions, but its role, if any, in bacterial pathogenesis has not been studied. Here, using a Dual RNA-seq approach to profile ProQ-dependent gene expression changes as Salmonella infects human cells, we reveal dysregulation of bacterial motility, chemotaxis, and virulence genes which is accompanied by altered MAPK (mitogen-activated protein kinase) signaling in the host. Comparison with the other major RNA chaperone in Salmonella, Hfq, reinforces the notion that these two global RNA-binding proteins work in parallel to ensure full virulence. Of newly discovered infection-associated ProQ-bound small noncoding RNAs (sRNAs), we show that the 3′UTR-derived sRNA STnc540 is capable of repressing an infection-induced magnesium transporter mRNA in a ProQ-dependent manner. Together, this comprehensive study uncovers the relevance of ProQ for Salmonella pathogenesis and highlights the importance of RNA-binding proteins in regulating bacterial virulence programs. IMPORTANCE The protein ProQ has recently been discovered as the centerpiece of a previously overlooked “third domain” of small RNA-mediated control of gene expression in bacteria. As in vitro work continues to reveal molecular mechanisms, it is also important to understand how ProQ affects the life cycle of bacterial pathogens as these pathogens infect eukaryotic cells. Here, we have determined how ProQ shapes Salmonella virulence and how the activities of this RNA-binding protein compare with those of Hfq, another central protein in RNA-based gene regulation in this and other bacteria. To this end, we apply global transcriptomics of pathogen and host cells during infection. In doing so, we reveal ProQ-dependent transcript changes in key virulence and host immune pathways. Moreover, we differentiate the roles of ProQ from those of Hfq during infection, for both coding and noncoding transcripts, and provide an important resource for those interested in ProQ-dependent small RNAs in enteric bacteria.
• Hierarchy of mono- and biallelic {TP}53 alterations in multiple myeloma cell fitness. Munawar, Umair; Rasche, Leo; Müller, Nicole; Vogt, Cornelia; Da-Via, Matteo; Haertle, Larissa; Arampatzi, Panagiota; Dietrich, Sascha; Roth, Markus; Garitano-Trojaola, Andoni; Steinhardt, Maximilian Johannes; Strifler, Susanne; Gallardo, Miguel; Martinez-Lopez, Joaquin; Bargou, Ralf C.; Heckel, Tobias; Einsele, Hermann; Stühmer, Thorsten; Kortüm, K. Martin; Barrio, Santiago. In Blood, 134(10), pp. 836–840. 2019.
  • [ DOI ]
• {RAL} {GTPases} mediate multiple myeloma cell survival and are activated independently of oncogenic {RAS}. Seibold, Marcel; Stühmer, Thorsten; Kremer, Nadine; Mottok, Anja; Scholz, Claus-Jürgen; Schlosser, Andreas; Leich, Ellen; Holzgrabe, Ulrike; Brünnert, Daniela; Barrio, Santiago; Kortüm, K. Martin; Solimando, Antonio G.; Chatterjee, Manik; Einsele, Hermann; Rosenwald, Andreas; Bargou, Ralf C.; Steinbrunn, Torsten. In Haematologica, p. haematol.2019.223024. Ferrata Storti Foundation (Haematologica), 2019.
  • [ Abstract ]
  • [ DOI ]
  Oncogenic RAS provides crucial survival signaling for up to half of multiple myeloma cases, but has so far remained a clinically undruggable target. RAL is a member of the RAS superfamily of small GTPases and is considered to be a potential mediator of oncogenic RAS signaling. In primary multiple myeloma, we found RAL to be overexpressed in the vast majority of samples when compared with pre-malignant monoclonal gammopathy of undetermined significance or normal plasma cells. We analyzed the functional effects of RAL abrogation in myeloma cell lines and found that RAL is a critical mediator of survival. RNAi-mediated knockdown of RAL resulted in rapid induction of tumor cell death, an effect which was independent from signaling via mitogen-activated protein kinase, but appears to be partially dependent on Akt activity. Notably, RAL activation was not correlated with the presence of activating RAS mutations and remained unaffected by knockdown of oncogenic RAS. Furthermore, transcriptome analysis yielded distinct RNA expression signatures after knockdown of either RAS or RAL. Combining RAL depletion with clinically relevant anti-myeloma agents led to enhanced rates of cell death. Our data demonstrate that RAL promotes multiple myeloma cell survival independently of oncogenic RAS and, thus, this pathway represents a potential therapeutic target in its own right.
• Identification of Cholecystokinin by Genome-Wide Profiling as Potential Mediator of Serotonin-Dependent Behavioral Effects of Maternal Separation in the Amygdala. Weidner, Magdalena T.; Lardenoije, Roy; Eijssen, Lars; Mogavero, Floriana; Groodt, Lilian P. M. T. De; Popp, Sandy; Palme, Rupert; Förstner, Konrad U.; Strekalova, Tatyana; Steinbusch, Harry W. M.; Schmitt-Böhrer, Angelika G.; Glennon, Jeffrey C.; Waider, Jonas; van den Hove, Daniel L. A.; Lesch, Klaus-Peter. In Frontiers in Neuroscience, 13. Frontiers Media {SA}, 2019.
  • [ Abstract ]
  • [ DOI ]
  Converging evidence suggests a role of serotonin (5-hydroxytryptamine, 5-HT) and tryptophan hydroxylase 2 (TPH2), the rate-limiting enzyme of 5-HT synthesis in the brain, in modulating long-term, neurobiological effects of early-life adversity. Here, we aimed at further elucidating the molecular mechanisms underlying this interaction, and its consequences for socio-emotional behaviors, with a focus on anxiety and social interaction. In this study, adult, male Tph2 null mutant (Tph2 -/-) and heterozygous (Tph2 +/-) mice, and their wildtype littermates (Tph2 +/+) were exposed to neonatal, maternal separation (MS) and screened for behavioral changes, followed by genome-wide RNA expression and DNA methylation profiling. In Tph2 -/- mice, brain 5-HT deficiency profoundly affected socio-emotional behaviors, i.e., decreased avoidance of the aversive open arms in the elevated plus-maze (EPM) as well as decreased prosocial and increased rule breaking behavior in the resident-intruder test when compared to their wildtype littermates. Tph2 +/- mice showed an ambiguous profile with context-dependent, behavioral responses. In the EPM they showed similar avoidance of the open arm but decreased prosocial and increased rule breaking behavior in the resident-intruder test when compared to their wildtype littermates. Notably, MS effects on behavior were subtle and depended on the Tph2 genotype, in particular increasing the observed avoidance of EPM open arms in wildtype and Tph2 +/- mice when compared to their Tph2 -/- littermates. On the genomic level, the interaction of Tph2 genotype with MS differentially affected the expression of numerous genes, of which a subset showed an overlap with DNA methylation profiles at corresponding loci. Remarkably, changes in methylation nearby and expression of the gene encoding cholecystokinin, which were inversely correlated to each other, were associated with variations in anxiety-related phenotypes. In conclusion, next to various behavioral alterations, we identified gene expression and DNA methylation profiles to be associated with TPH2 inactivation and its interaction with MS, suggesting a gene-by-environment interaction-dependent, modulatory function of brain 5-HT availability.
• Contribution of {LTi} and {TH}17 cells to {B} cell aggregate formation in the central nervous system in a mouse model of multiple sclerosis. Schropp, Verena; Rohde, Jörn; Rovituso, Damiano M.; Jabari, Samir; Bharti, Richa; Kuerten, Stefanie. In Journal of Neuroinflammation, 16(1), p. 111. 2019.
  • [ Abstract ]
  • [ DOI ]
  In a subgroup of patients suffering from progressive multiple sclerosis (MS), which is an inflammation-mediated neurodegenerative disease of the central nervous system (CNS), B cell aggregates were discovered within the meninges. Occurrence of these structures was associated with a more severe disease course and cortical histopathology. We have developed the B cell-dependent MP4-induced experimental autoimmune encephalomyelitis (EAE) as a mouse model to mimic this trait of the human disease. The aim of this study was to determine a potential role of lymphoid tissue inducer (LTi) and TH17 cells in the process of B cell aggregate formation in the MP4 model.
• Time-resolved single-cell transcriptomics uncovers dynamics of cardiac neutrophil diversity in murine myocardial infarction. Vafadarnejad, Ehsan; Rizzo, Giuseppe; Krampert, Laura; Arampatzi, Panagiota; Nugroho, Vallery Audy; Schulz, Dirk; Roesch, Melanie; Alayrac, Paul; Vilar, Jose; Silvestre, Jean-S{é}bastien; Zernecke, Alma; Saliba, Antoine-Emmanuel; Cochain, Cl{é}ment. In bioRxiv. Cold Spring Harbor Laboratory, 2019.
  • [ Abstract ]
  • [ DOI ]
  Objective After myocardial infarction, neutrophils rapidly and massively infiltrate the heart, where they can promote both tissue healing and damage. Here, we investigated the dynamics of cardiac neutrophil heterogeneity after infarction.Methods and results We employed single-cell transcriptomics (scRNA-seq) to investigate temporal neutrophil heterogeneity in the heart after murine myocardial infarction. At day 1, 3, and 5 after infarction, neutrophils could be delineated into six distinct clusters with specific time-dependent patterning and proportions. While the majority of neutrophils at day 1 were characterized by high expression of chemokines (e.g. Cxcl3, Ccl6), and putative activity of transcriptional regulators involved in hypoxic response (Hif1a) and emergency granulopoiesis (Cebpb), two major subsets of Siglecfhi (enriched for e.g. Icam1 and Tnf) and Siglecflow (Slpi, Ifitm1) neutrophils were found at 3 and 5 days. Flow cytometry analysis confirmed the presence of LY6G+SIGLECFhi and LY6G+SIGLECFlow neutrophils in the heart from 3 days after infarction onwards. LY6G+SIGLECFhi neutrophils were absent from the bone marrow, blood and spleen, suggesting local acquisition of surface SIGLECF. Acquisition of the SIGLECFhi state was paralleled by features of neutrophil ageing and activation (ICAM1hiCXCR4hiCD49dhiCD62Llow). scRNA-seq of atherosclerotic aortas revealed two neutrophil subsets with gene expression patterns reminiscent of the major Siglecfhi and Siglecflow cardiac neutrophil subpopulations, revealing that these populations may be present across distinct contexts of cardiovascular inflammation.Conclusion Altogether, our data provide a time-resolved census of neutrophil diversity and gene expression dynamics in the mouse ischemic heart at the single-cell level, and suggests that temporal neutrophil heterogeneity is in part driven by local transition to a SIGLECFhi state.
• {scSLAM}-seq reveals core features of transcription dynamics in single cells. Erhard, Florian; Baptista, Marisa A. P.; Krammer, Tobias; Hennig, Thomas; Lange, Marius; Arampatzi, Panagiota; Jürges, Christopher S.; Theis, Fabian J.; Saliba, Antoine-Emmanuel; Dölken, Lars. In Nature, 571(7765), pp. 419–423. Springer Science and Business Media {LLC}, 2019.
  • [ Abstract ]
  • [ DOI ]
  Single-cell RNA sequencing (scRNA-seq) has highlighted the important role of intercellular heterogeneity in phenotype variability in both health and disease1. However, current scRNA-seq approaches provide only a snapshot of gene expression and convey little information on the true temporal dynamics and stochastic nature of transcription. A further key limitation of scRNA-seq analysis is that the RNA profile of each individual cell can be analysed only once. Here we introduce single-cell, thiol-(SH)-linked alkylation of RNA for metabolic labelling sequencing (scSLAM-seq), which integrates metabolic RNA labelling2, biochemical nucleoside conversion3 and scRNA-seq to record transcriptional activity directly by differentiating between new and old RNA for thousands of genes per single cell. We use scSLAM-seq to study the onset of infection with lytic cytomegalovirus in single mouse fibroblasts. The cell-cycle state and dose of infection deduced from old RNA enable dose-response analysis based on new RNA. scSLAM-seq thereby both visualizes and explains differences in transcriptional activity at the single-cell level. Furthermore, it depicts 'on-off' switches and transcriptional burst kinetics in host gene expression with extensive gene-specific differences that correlate with promoter-intrinsic features (TBP-TATA-box interactions and DNA methylation). Thus, gene-specific, and not cell-specific, features explain the heterogeneity in transcriptomes between individual cells and the transcriptional response to perturbations.
• Sensory profiles and immune-related expression patterns of patients with and without neuropathic pain after peripheral nerve lesion. Held, Melissa; Karl, Franziska; Vlckova, Eva; Rajdova, Aneta; Escolano-Lozano, Fabiola; Stetter, Christian; Bharti, Richa; Förstner, Konrad U.; Leinders, Mathias; Dušek, Ladislav; Birklein, Frank; Bednarik, Josef; Sommer, Claudia; Üçeyler, Nurcan. In {PAIN}, 160(10), pp. 2316–2327. Ovid Technologies (Wolters Kluwer Health), 2019.
  • [ Abstract ]
  • [ DOI ]
  In this multicenter cross-sectional study, we determined sensory profiles of patients with (NL-1) and without neuropathic pain (NL-0) after nerve lesion and assessed immune-related systemic gene expression. Patients and matched healthy controls filled in questionnaires and underwent neurological examination, neurophysiological studies, quantitative sensory testing, and blood withdrawal. Neuropathic pain was present in 67/95 (71%) patients (NL-1). Tactile hyperalgesia was the most prominent clinical sign in NL-1 patients (P < 0.05). Questionnaires showed an association between neuropathic pain and the presence of depression, anxiety, and catastrophizing (P < 0.05 to P < 0.01). Neuropathic pain was frequently accompanied by other chronic pain (P < 0.05). Quantitative sensory testing showed ipsilateral signs of small and large fiber impairment compared to the respective contralateral side, with elevated thermal and mechanical detection thresholds (P < 0.001 to P < 0.05) and lowered pressure pain threshold (P < 0.05). Also, more loss of function was found in patients with NL-1 compared to NL-0. Pain intensity was associated with mechanical hyperalgesia (P < 0.05 to P < 0.01). However, quantitative sensory testing did not detect or predict neuropathic pain. Gene expression of peptidylglycine α-amidating monooxygenase was higher in NL patients compared with healthy controls (NL-1, P < 0.01; NL-0, P < 0.001). Also, gene expression of tumor necrosis factor-α was higher in NL-1 patients compared with NL-0 (P < 0.05), and interleukin-1ß was higher, but IL-10 was lower in NL-1 patients compared with healthy controls (P < 0.05 each). Our study reveals that nerve lesion presents with small and large nerve fiber dysfunction, which may contribute to the presence and intensity of neuropathic pain and which is associated with a systemic proinflammatory pattern.

• {CRISPR} {RNA}-{Dependent} {Binding} and {Cleavage} of {Endogenous} {RNAs} by the {Campylobacter} jejuni {Cas}9. Dugar, Gaurav; Leenay, Ryan T.; Eisenbart, Sara K.; Bischler, Thorsten; Aul, Belinda U.; Beisel, Chase L.; Sharma, Cynthia M. In Molecular Cell, 69(5), pp. 893–905.e7. 2018.
  • [ Abstract ]
  • [ DOI ]
  Summary Cas9 nucleases naturally utilize CRISPR RNAs (crRNAs) to silence foreign double-stranded DNA. While recent work has shown that some Cas9 nucleases can also target RNA, RNA recognition has required nuclease modifications or accessory factors. Here, we show that the Campylobacter jejuni Cas9 (CjCas9) can bind and cleave complementary endogenous mRNAs in a crRNA-dependent manner. Approximately 100 transcripts co-immunoprecipitated with CjCas9 and generally can be subdivided through their base-pairing potential to the four crRNAs. A subset of these RNAs was cleaved around or within the predicted binding site. Mutational analyses revealed that RNA binding was crRNA and tracrRNA dependent and that target RNA cleavage required the CjCas9 HNH domain. We further observed that RNA cleavage was PAM independent, improved with greater complementarity between the crRNA and the RNA target, and was programmable in vitro. These findings suggest that C. jejuni Cas9 is a promiscuous nuclease that can coordinately target both DNA and RNA.
• Neonatally imprinted stromal cell subsets induce tolerogenic dendritic cells in mesenteric lymph nodes. Pezoldt, Joern; Pasztoi, Maria; Zou, Mangge; Wiechers, Carolin; Beckstette, Michael; Thierry, Guilhem R.; Vafadarnejad, Ehsan; Floess, Stefan; Arampatzi, Panagiota; Buettner, Manuela; Schweer, Janina; Fleissner, Diana; Vital, Marius; Pieper, Dietmar H.; Basic, Marijana; Dersch, Petra; Strowig, Till; Hornef, Mathias; Bleich, Andr{{é}}; Bode, Ulrike; Pabst, Oliver; Baj{{é}}noff, Marc; Saliba, Antoine-Emmanuel; Huehn, Jochen. In Nature Communications, 9(1). Springer Science and Business Media {LLC}, 2018.
  • [ Abstract ]
  • [ DOI ]
  Gut-draining mesenteric lymph nodes (mLNs) are important for inducing peripheral tolerance towards food and commensal antigens by providing an optimal microenvironment for de novo generation of Foxp3+ regulatory T cells (Tregs). We previously identified microbiota-imprinted mLN stromal cells as a critical component in tolerance induction. Here we show that this imprinting process already takes place in the neonatal phase, and renders the mLN stromal cell compartment resistant to inflammatory perturbations later in life. LN transplantation and single-cell RNA-seq uncover stably imprinted expression signatures in mLN fibroblastic stromal cells. Subsetting common stromal cells across gut-draining mLNs and skin-draining LNs further refine their location-specific immunomodulatory functions, such as subset-specific expression of Aldh1a2/3. Finally, we demonstrate that mLN stromal cells shape resident dendritic cells to attain high Treg-inducing capacity in a Bmp2-dependent manner. Thus, crosstalk between mLN stromal and resident dendritic cells provides a robust regulatory mechanism for the maintenance of intestinal tolerance.
• Explorative results from multistep screening for potential genetic risk loci of Alzheimer’s disease in the longitudinal VITA study cohort. Scholz, Claus-J{ü}rgen; Weber, Heike; Jungwirth, Susanne; Danielczyk, Walter; Reif, Andreas; Tragl, Karl-Heinz; Fischer, Peter; Riederer, Peter; Deckert, J{ü}rgen; Gr{ü}nblatt, Edna. In Journal of Neural Transmission, 125(1), pp. 77–87. 2018.
  • [ Abstract ]
  • [ DOI ]
  Alzheimer's disease (AD) is a neurodegenerative disorder that preferentially affects individuals of advanced age. Heritability estimates for AD range between 60 and 80{\%}, but only few genetic risk factors have been identified so far. In the present explorative study, we aimed at characterizing the genetic contribution to late-onset AD in participants of the Vienna Transdanube Aging (VITA) longitudinal birth cohort study in a two-step approach. First, we performed a genome-wide screen of pooled DNA samples (n = 588) to identify allele frequency differences between AD patients and non-AD individuals using life-time diagnoses made at the age of 80 (t = 60 months). This analysis suggested a high proportion of brain-expressed genes required for cell adhesion, cell signaling and cell morphogenesis, and also scored in known AD risk genes. In a second step, we confirmed associations using individual genotypes of top-ranked markers examining AD diagnoses as well as the dimensional scores: FULD and MMSE determined up to the age of 82.5 (t = 90 months). Taken together, our study proposes genes ANKS1B, ENST00000414107, LOC100505811, SLC22A14, QRFPR, ZDHHC8P1, ADAMTS3 and PPFIA1 as possible new candidates involved in the etiology of late-onset AD, with further research being needed to clarify their exact roles.
• {RNase} {E} cleavage shapes the transcriptome of {Rhodobacter} sphaeroides and strongly impacts phototrophic growth. Förstner, Konrad U.; Reuscher, Carina M.; Haberzettl, Kerstin; Weber, Lennart; Klug, Gabriele. In Life Science Alliance, 1(4), p. e201800080. 2018.
  • [ Abstract ]
  • [ DOI ]
  Bacteria adapt to changing environmental conditions by rapid changes in their transcriptome. This is achieved not only by adjusting rates of transcription but also by processing and degradation of RNAs. We applied TIER-Seq (transiently inactivating an endoribonuclease followed by RNA-Seq) for the transcriptome-wide identification of RNase E cleavage sites and of 5′ RNA ends, which are enriched when RNase E activity is reduced in Rhodobacter sphaeroides. These results reveal the importance of RNase E for the maturation and turnover of mRNAs, rRNAs, and sRNAs in this guanine-cytosine-rich α-proteobacterium, some of the latter have well-described functions in the oxidative stress response. In agreement with this, a role of RNase E in the oxidative stress response is demonstrated. A remarkably strong phenotype of a mutant with reduced RNase E activity was observed regarding the formation of photosynthetic complexes and phototrophic growth, whereas there was no effect on chemotrophic growth.
• Post-transcriptional gene regulation by an {Hfq}-independent small {RNA} in {Caulobacter} crescentus. Fröhlich, Kathrin S.; Förstner, Konrad U.; Gitai, Zemer. In Nucleic Acids Research, 46(20), pp. 10969–10982. 2018.
  • [ Abstract ]
  • [ DOI ]
  Bacterial small RNAs (sRNAs) are a heterogeneous group of post-transcriptional regulators that often act at the heart of large networks. Hundreds of sRNAs have been discovered by genome-wide screens and most of these sRNAs exert their functions by base-pairing with target mRNAs. However, studies addressing the molecular roles of sRNAs have been largely confined to gamma-proteobacteria, such as Escherichia coli. Here we identify and characterize a novel sRNA, ChvR, from the alpha-proteobacterium Caulobacter crescentus. Transcription of chvR is controlled by the conserved two-component system ChvI-ChvG and it is expressed in response to DNA damage, low pH, and growth in minimal medium. Transient over-expression of ChvR in combination with genome-wide transcriptome profiling identified the mRNA of the TonB-dependent receptor ChvT as the sole target of ChvR. Genetic and biochemical analyses showed that ChvR represses ChvT at the post-transcriptional level through direct base-pairing. Fine-mapping of the ChvR-chvT interaction revealed the requirement of two distinct base-pairing sites for full target regulation. Finally, we show that ChvR-controlled repression of chvT is independent of the ubiquitous RNA-chaperone Hfq, and therefore distinct from previously reported mechanisms employed by prototypical bacterial sRNAs. These findings have implications for the mechanism and evolution of sRNA function across bacterial species.
• Genome organization and {DNA} accessibility control antigenic variation in trypanosomes. Müller, Laura S. M.; Cosentino, Ra{{ú}}l O.; Förstner, Konrad U.; Guizetti, Julien; Wedel, Carolin; Kaplan, Noam; Janzen, Christian J.; Arampatzi, Panagiota; Vogel, Jörg; Steinbiss, Sascha; Otto, Thomas D.; Saliba, Antoine-Emmanuel; Sebra, Robert P.; Siegel, T. Nicolai. In Nature, 563(7729), pp. 121–125. Springer Science and Business Media {LLC}, 2018.
  • [ Abstract ]
  • [ DOI ]
  Many evolutionarily distant pathogenic organisms have evolved similar survival strategies to evade the immune responses of their hosts. These include antigenic variation, through which an infecting organism prevents clearance by periodically altering the identity of proteins that are visible to the immune system of the host1. Antigenic variation requires large reservoirs of immunologically diverse antigen genes, which are often generated through homologous recombination, as well as mechanisms to ensure the expression of one or very few antigens at any given time. Both homologous recombination and gene expression are affected by three-dimensional genome architecture and local DNA accessibility2,3. Factors that link three-dimensional genome architecture, local chromatin conformation and antigenic variation have, to our knowledge, not yet been identified in any organism. One of the major obstacles to studying the role of genome architecture in antigenic variation has been the highly repetitive nature and heterozygosity of antigen-gene arrays, which has precluded complete genome assembly in many pathogens. Here we report the de novo haplotype-specific assembly and scaffolding of the long antigen-gene arrays of the model protozoan parasite Trypanosoma brucei, using long-read sequencing technology and conserved features of chromosome folding4. Genome-wide chromosome conformation capture (Hi-C) reveals a distinct partitioning of the genome, with antigen-encoding subtelomeric regions that are folded into distinct, highly compact compartments. In addition, we performed a range of analyses-Hi-C, fluorescence in situ hybridization, assays for transposase-accessible chromatin using sequencing and single-cell RNA sequencing-that showed that deletion of the histone variants H3.V and H4.V increases antigen-gene clustering, DNA accessibility across sites of antigen expression and switching of the expressed antigen isoform, via homologous recombination. Our analyses identify histone variants as a molecular link between global genome architecture, local chromatin conformation and antigenic variation.
• A New Bioactive Compound From the Marine Sponge-Derived Streptomyces sp. {SBT}348 Inhibits Staphylococcal Growth and Biofilm Formation. Balasubramanian, Srikkanth; Skaf, Joseph; Holzgrabe, Ulrike; Bharti, Richa; Förstner, Konrad U.; Ziebuhr, Wilma; Humeida, Ute H.; Abdelmohsen, Usama R.; Oelschlaeger, Tobias A. In Frontiers in Microbiology, 9. Frontiers Media {SA}, 2018.
  • [ Abstract ]
  • [ DOI ]
  Staphylococcus epidermidis, the common inhabitant of human skin and mucosal surfaces has emerged as an important pathogen in patients carrying surgical implants and medical devices. Entering the body via surgical sites and colonizing the medical devices through formation of multi-layered biofilms leads to refractory and persistent device-related infections (DRIs). Staphylococci organized in biofilms are more tolerant to antibiotics and immune responses, and thus are difficult-to-treat. The consequent morbidity and mortality, and economic losses in health care systems has strongly necessitated the need for development of new anti-bacterial and anti-biofilm-based therapeutics. In this study, we describe the biological activity of a marine sponge-derived Streptomyces sp. SBT348 extract in restraining staphylococcal growth and biofilm formation on polystyrene, glass, medically relevant titan metal, and silicone surfaces. A bioassay-guided fractionation was performed to isolate the active compound (SKC3) from the crude SBT348 extract. Our results demonstrated that SKC3 effectively inhibits the growth (MIC: 31.25 μg/ml) and biofilm formation (sub-MIC range: 1.95-<31.25 μg/ml) of S. epidermidis RP62A in vitro. Chemical characterization of SKC3 by heat and enzyme treatments, and mass spectrometry (HRMS) revealed its heat-stable and non-proteinaceous nature, and high molecular weight (1258.3 Da). Cytotoxicity profiling of SKC3 in vitro on mouse fibroblast (NIH/3T3) and macrophage (J774.1) cell lines, and in vivo on the greater wax moth larvae Galleria mellonella revealed its non-toxic nature at the effective dose. Transcriptome analysis of SKC3 treated S. epidermidis RP62A has further unmasked its negative effect on central metabolism such as carbon flux as well as, amino acid, lipid, and energy metabolism. Taken together, these findings suggest a potential of SKC3 as a putative drug to prevent staphylococcal DRIs.
• A {Novel} {Mechanism} of {Inactivating} {Antibacterial} {Nitro} {Compounds} in the {Human} {Pathogen} {Staphylococcus} aureus by {Overexpression} of a {NADH}-{Dependent} {Flavin} {Nitroreductase}. El-Hossary, Ebaa M.; Förstner, Konrad U.; François, Patrice; Baud, Damien; Streker, Karin; Schrenzel, Jacques; Ohlsen, Knut; Holzgrabe, Ulrike. In Antimicrobial Agents and Chemotherapy, 62(2), pp. e01510–17. 2018.
  • [ Abstract ]
  • [ DOI ]
  Recently, the nitro-substituted bisquaternary bisnaphthalimides were reported to have substantial anti-infective activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Here, we selected resistant S. aureus clones by cultivation in increasing concentrations of the most active compound, MT02. Interestingly, MT02-resistant variants induced a diffusible red color of the broth. Chromatographic and spectroscopic investigations revealed a stepwise reduction of the bisquaternary bisnaphthalimides' nitro groups to amino groups. The corresponding derivatives were completely inactive against staphylococci. RNA sequencing experiments revealed a strong overexpression of a novel oxidoreductase in MT02-resistant strains. Deletion mutants of this enzyme did not produce the red color and were not able to develop resistance against bisquaternary bisnaphthalimides. Biochemical reactions confirmed an NADH-dependent deactivation of the nitro-substituted compounds. Thus, this is the first report of a nitroreductase-based antibiotic resistance mechanism in the human pathogen S. aureus.
• {hnRNP} {R} and its main interactor, the noncoding {RNA} 7SK, coregulate the axonal transcriptome of motoneurons. Briese, Michael; Saal-Bauernschubert, Lena; Ji, Changhe; Moradi, Mehri; Ghanawi, Hanaa; Uhl, Michael; Appenzeller, Silke; Backofen, Rolf; Sendtner, Michael. In Proceedings of the National Academy of Sciences, 115(12), pp. E2859-E2868. 2018.
  • [ Abstract ]
  • [ DOI ]
  Disturbed RNA processing and subcellular transport contribute to the pathomechanisms of motoneuron diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy. RNA-binding proteins are involved in these processes, but the mechanisms by which they regulate the subcellular diversity of transcriptomes, particularly in axons, are not understood. Heterogeneous nuclear ribonucleoprotein R (hnRNP R) interacts with several proteins involved in motoneuron diseases. It is located in axons of developing motoneurons, and its depletion causes defects in axon growth. Here, we used individual nucleotide-resolution cross-linking and immunoprecipitation (iCLIP) to determine the RNA interactome of hnRNP R in motoneurons. We identified ∼3,500 RNA targets, predominantly with functions in synaptic transmission and axon guidance. Among the RNA targets identified by iCLIP, the noncoding RNA 7SK was the top interactor of hnRNP R. We detected 7SK in the nucleus and also in the cytosol of motoneurons. In axons, 7SK localized in close proximity to hnRNP R, and depletion of hnRNP R reduced axonal 7SK. Furthermore, suppression of 7SK led to defective axon growth that was accompanied by axonal transcriptome alterations similar to those caused by hnRNP R depletion. Using a series of 7SK-deletion mutants, we show that the function of 7SK in axon elongation depends on its interaction with hnRNP R but not with the PTEF-B complex involved in transcriptional regulation. These results propose a role for 7SK as an essential interactor of hnRNP R to regulate its function in axon maintenance.
• Clinical implications of fracture-associated vascular damage in extremity and pelvic trauma. Gilbert, F.; Schneemann, C.; Scholz, C. J.; Kickuth, R.; Meffert, R. H.; Wildenauer, R.; Lorenz, U.; Kellersmann, R.; Busch, A. In BMC Musculoskeletal Disorders, 19(1), p. 404. 2018.
  • [ Abstract ]
  • [ DOI ]
  BACKGROUND: Vascular damage in polytrauma patients is associated with high mortality and morbidity. Therefore, specific clinical implications of vascular damage with fractures in major trauma patients are reassessed. METHODS: This comprehensive nine-year retrospective single center cohort study analyzed demography, laboratory, treatment and outcome data from 3689 patients, 64 patients with fracture-associated vascular injuries were identified and were compared to a control group. RESULTS: Vascular damage occurred in 7% of patients with upper and lower limb and pelvic fractures admitted to the trauma room. Overall survival was 80% in pelvic fracture and 97% in extremity fracture patients and comparable to non-vascular trauma patients. Additional arterial damage required substantial fluid administration and was visible as significantly anemia and disturbed coagulation tests upon admission. Open procedures were done in over 80% of peripheral extremity vascular damage. Endovascular procedures were predominant (87%) in pelvic injury. CONCLUSION: Vascular damage is associated with high mortality rates especially in combination with pelvic fractures. Initial anemia, disturbed coagulation tests and the need for extensive pre-clinical fluid substitution were observed in the cohort with vascular damage. Therefore, fast diagnosis and early interventional and surgical procedures are necessary to optimize patient-specific outcome.
• Panel {Sequencing} {Shows} {Recurrent} {Genetic} {FAS} {Alterations} in {Primary} {Cutaneous} {Marginal} {Zone} {Lymphoma}. Maurus, Katja; Appenzeller, Silke; Roth, Sabine; Kuper, Jochen; Rost, Simone; Meierjohann, Svenja; Arampatzi, Panagiota; Goebeler, Matthias; Rosenwald, Andreas; Geissinger, Eva; Wobser, Marion. In Journal of Investigative Dermatology, 138(7), pp. 1573–1581. 2018.
  • [ Abstract ]
  • [ DOI ]
  Primary cutaneous marginal zone lymphoma (PCMZL) represents an indolent subtype of non-Hodgkin lymphoma that is clinically characterized by slowly growing skin tumors with a very low propensity for systemic dissemination. The underlying genetic basis of PCMZL has not been comprehensively elucidated. To gain deeper insight into the molecular pathogenesis of PCMZL, we performed hybridization-based panel sequencing of 38 patients with well-characterized PCMZL. In 32 of the 38 patients, we identified genetic alterations within 39 selected target genes. The most frequently detected alterations (24/38 patients, 63.2\%) affected the FAS gene, of which 22 patients harbored alterations, which affect the functionally relevant death domain of the apoptosis-regulating FAS/CD95 protein in a dominant-negative manner. In addition, we identified highly recurrent mutations in three other genes, namely SLAMF1, SPEN, and NCOR2. Our molecular data suggest that apoptosis defects provide the molecular basis of the observed clinical features of PCMZL, which commonly presents with only slowly growing skin tumors, reflecting its invariably indolent behavior. From a diagnostic point of view, highly recurrent FAS mutations in PCMZL presumably separate this indolent lymphoma entity from pseudolymphoma, and this adds adjunctive discriminatory features at a molecular level.
• Global {Maps} of {ProQ} {Binding} {In} {Vivo} {Reveal} {Target} {Recognition} via {RNA} {Structure} and {Stability} {Control} at {mRNA} 3′ {Ends}. Holmqvist, Erik; Li, Lei; Bischler, Thorsten; Barquist, Lars; Vogel, Jörg. In Molecular Cell, 70(5), pp. 971–982.e6. 2018.
  • [ Abstract ]
  • [ DOI ]
  Summary The conserved RNA-binding protein ProQ has emerged as the centerpiece of a previously unknown third large network of post-transcriptional control in enterobacteria. Here, we have used in vivo UV crosslinking and RNA sequencing (CLIP-seq) to map hundreds of ProQ binding sites in Salmonella enterica and Escherichia coli. Our analysis of these binding sites, many of which are conserved, suggests that ProQ recognizes its cellular targets through RNA structural motifs found in small RNAs (sRNAs) and at the 3′ end of mRNAs. Using the cspE mRNA as a model for 3′ end targeting, we reveal a function for ProQ in protecting mRNA against exoribonucleolytic activity. Taken together, our results underpin the notion that ProQ governs a post-transcriptional network distinct from those of the well-characterized sRNA-binding proteins, CsrA and Hfq, and suggest a previously unrecognized, sRNA-independent role of ProQ in stabilizing mRNAs.
• Single-{Cell} {RNA}-{Seq} {Reveals} the {Transcriptional} {Landscape} and {Heterogeneity} of {Aortic} {Macrophages} in {Murine} {Atherosclerosis}. Cochain, Clément; Vafadarnejad, Ehsan; Arampatzi, Panagiota; Jaroslav, Pelisek; Winkels, Holger; Ley, Klaus; Wolf, Dennis; Saliba, Antoine-Emmanuel; Zernecke, Alma. In Circulation Research, p. CIRCRESAHA.117.312509. 2018.
  • [ Abstract ]
  • [ DOI ]
  Rationale: It is assumed that atherosclerotic arteries contain several macrophage subsets endowed with specific functions. The precise identity of these subsets is poorly characterized as they ha ve been defined by the expression of a restricted number of markers. Objective: We have applied single-cell RNA-seq as an unbiased profiling strategy to interrogate and classify aortic macrophage heterogeneity at the single-cell level in atherosclerosis. Methods and Results: We performed single-cell RNA sequencing of total aortic CD45+ cells extracted from the non-diseased (chow fed) and atherosclerotic (11 weeks of high fat diet) aorta of Ldlr-/- mice. Unsupervised clustering singled out 13 distinct aortic cell clusters. Among the myeloid cell populations, Resident-like macrophages with a gene expression profile similar to aortic resident macrophages were found in healthy and diseased aortae, whereas monocytes, monocyte-derived dendritic cells (MoDC), and two populations of macrophages were almost exclusively detectable in atherosclerotic aortae, comprising Inflammatory macrophages showing enrichment in Il1b, and previously undescribed TREM2hi macrophages. Differential gene expression and gene ontology enrichment analyses revealed specific gene expression patterns distinguishing these three macrophage subsets and MoDC, and uncovered putative functions of each cell type. Notably, TREM2hi macrophages appeared to be endowed with specialized functions in lipid metabolism and catabolism, and presented a gene expression signature reminiscent of osteoclasts, suggesting a role in lesion calcification. TREM2 expression was moreover detected in human lesional macrophages. Importantly, these macrophage populations were present also in advanced atherosclerosis and in Apoe-/- aortae, indicating relevance of our findings in different stages of atherosclerosis and mouse models. Conclusions: These data unprecedentedly uncovered the transcriptional landscape and phenotypic heterogeneity of aortic macrophages and MoDCs in atherosclerotic and identified previously unrecognized macrophage populations and their gene expression signature, suggesting specialized functions. Our findings will open up novel opportunities to explore distinct myeloid cell populations and their functions in atherosclerosis.
• The {Probiotic} {Escherichia} coli {Strain} {Nissle} 1917 {Combats} {Lambdoid} {Bacteriophages} stx and λ. Bury, Susanne; Soundararajan, Manonmani; Bharti, Richa; von Bünau, Rudolf; Förstner, Konrad U.; Oelschlaeger, Tobias A. In Frontiers in Microbiology, 9. 2018.
  • [ Abstract ]
  • [ DOI ]
  Shiga toxin (Stx) producing E. coli (STEC) such as Enterohemorrhagic E. coli (EHEC) are the major cause of foodborne illness in humans. In vitro studies showed the probiotic Escherichia coli strain Nissle 1917 (EcN) to efficiently inhibit the production of Stx. Life threatening EHEC strains as for example the serotype O104:H4, responsible for the great outbreak in 2011 in Germany, evolutionary developed from certain E. coli strains which got infected by stx2-encoding lambdoid phages turning the E. coli into lysogenic and subsequently Stx producing strains. Since antibiotics induce stx genes and Stx production, EHEC infected persons are not recommended to be treated with antibiotics. Therefore, EcN might be an alternative medication. However, because even commensal E. coli strains might be converted into Stx-producers after becoming host to a stx encoding prophage, we tested EcN for stx-phage genome integration. Our experiments revealed the resistance of EcN towards not only stx-phages but also against lambda-phages. This resistance was not based on the lack of or by mutated phage receptors. Rather it involved the expression of a phage repressor (pr) gene of a defective prophage in EcN which was able to partially protect E. coli K-12 strain MG1655 against stx and lambda phage infection. Furthermore, we observed EcN to inactivate phages and thereby to protect E. coli K-12 strains against infection by stx- as well as lambda-phages. Inactivation of lambda-phages was due to binding of lambda-phages to LamB of EcN whereas inactivation of stx-phages was caused by a thermostable protein of EcN. These properties together with its ability to inhibit Stx production make EcN a good candidate for the prevention of illness caused by EHEC and probably for the treatment of already infected people.
• Computational {Analysis} of {RNA}–{Protein} {Interactions} via {Deep} {Sequencing}. Li, Lei; Förstner, Konrad U.; Chao, Yanjie. In Transcriptome {Data} {Analysis}, pp. 171–182. Humana Press, New York, NY, 2018.
  • [ Abstract ]
  • [ DOI ]
  RNA-binding proteins (RBPs) function in all aspects of RNA processes including stability, structure, export, localization and translation, and control gene expression at the posttranscriptional level. To investigate the roles of RBPs and their direct RNA ligands in vivo, recent global approaches combining RNA immunoprecipitation and deep sequencing (RIP-seq) as well as UV-cross-linking (CLIP-seq) have become instrumental in dissecting RNA–protein interactions. However, the computational analysis of these high-throughput sequencing data is still challenging. Here, we provide a computational pipeline to analyze CLIP-seq and RIP-seq datasets. This generic analytic procedure may help accelerate the identification of direct RNA–protein interactions from high-throughput RBP profiling experiments in a variety of bacterial species.

• {sRNA}154 a newly identified regulator of nitrogen fixation in {Methanosarcina} mazei strain {Gö}1. Prasse, Daniela; Förstner, Konrad U.; Jäger, Dominik; Backofen, Rolf; Schmitz, Ruth A. In RNA Biology, 14(11), pp. 1544–1558. 2017.
  • [ Abstract ]
  • [ DOI ]
  Trans-encoded sRNA154 is exclusively expressed under nitrogen (N)-deficiency in Methanosarcina mazei strain Gö1. The sRNA154 deletion strain showed a significant decrease in growth under N-limitation, pointing toward a regulatory role of sRNA154 in N-metabolism. Aiming to elucidate its regulatory function we characterized sRNA154 by means of biochemical and genetic approaches. 24 homologs of sRNA154 were identified in recently reported draft genomes of Methanosarcina strains, demonstrating high conservation in sequence and predicted secondary structure with two highly conserved single stranded loops. Transcriptome studies of sRNA154 deletion mutants by an RNA-seq approach uncovered nifH- and nrpA-mRNA, encoding the α–subunit of nitrogenase and the transcriptional activator of the nitrogen fixation (nif)-operon, as potential targets besides other components of the N-metabolism. Furthermore, results obtained from stability, complementation and western blot analysis, as well as in silico target predictions combined with electrophoretic mobility shift-assays, argue for a stabilizing effect of sRNA154 on the polycistronic nif-mRNA and nrpA-mRNA by binding with both loops. Further identified N-related targets were studied, which demonstrates that translation initiation of glnA2-mRNA, encoding glutamine synthetase2, appears to be affected by sRNA154 masking the ribosome binding site, whereas glnA1-mRNA appears to be stabilized by sRNA154. Overall, we propose that sRNA154 has a crucial regulatory role in N-metabolism in M. mazei by stabilizing the polycistronic mRNA encoding nitrogenase and glnA1-mRNA, as well as allowing a feed forward regulation of nif-gene expression by stabilizing nrpA-mRNA. Consequently, sRNA154 represents the first archaeal sRNA, for which a positive posttranscriptional regulation is demonstrated as well as inhibition of translation initiation.
• Nimodipine fosters remyelination in a mouse model of multiple sclerosis and induces microglia-specific apoptosis. Schampel, Andrea; Volovitch, Oleg; Koeniger, Tobias; Scholz, Claus-Jürgen; Jörg, Stefanie; Linker, Ralf A.; Wischmeyer, Erhard; Wunsch, Marie; Hell, Johannes W.; Ergün, Süleyman; Kuerten, Stefanie. In Proceedings of the National Academy of Sciences, 114(16), pp. E3295-E3304. 2017.
  • [ Abstract ]
  • [ DOI ]
  Despite continuous interest in multiple sclerosis (MS) research, there is still a lack of neuroprotective strategies, because the main focus has remained on modulating the immune response. Here we performed in-depth analysis of neurodegeneration in experimental autoimmune encephalomyelitis (EAE) and in in vitro studies regarding the effect of the well-established L-type calcium channel antagonist nimodipine. Nimodipine treatment attenuated clinical EAE and spinal cord degeneration and promoted remyelination. Surprisingly, we observed calcium channel-independent effects on microglia, resulting in apoptosis. These effects were cell-type specific and irrespective of microglia polarization. Apoptosis was accompanied by decreased levels of nitric oxide (NO) and inducible NO synthase (iNOS) in cell culture as well as decreased iNOS and reactive oxygen species levels in EAE. In addition, increased numbers of Olig2+APC+ oligodendrocytes were detected. Overall, nimodipine application seems to generate a favorable environment for regenerative processes and therefore could be a treatment option for MS, because it combines features of immunomodulation with beneficial effects on neuroregeneration.
• The glucocorticoid receptor in monocyte-derived macrophages is critical for cardiac infarct repair and remodeling. Galuppo, Paolo; Vettorazzi, Sabine; Hövelmann, Julian; Scholz, Claus-Jürgen; Tuckermann, Jan Peter; Bauersachs, Johann; Fraccarollo, Daniela. In The FASEB Journal, 31(11), pp. 5122–5132. 2017.
  • [ Abstract ]
  • [ DOI ]
  Cell- and tissue-specific actions of glucocorticoids are mediated by the glucocorticoid receptor. Here, we demonstrate that the glucocorticoid receptor (GR) in macrophages is essential for cardiac healing after myocardial infarction. Compared with GRflox (wild-type controls), GRLysMCre mice that lacked GR in myeloid cells showed increased acute mortality as a result of cardiac rupture. Seven days after left coronary artery ligation, GRLysMCre mice exhibited worse cardiac function and adverse remodeling associated with impaired scar formation and angiogenic response to ischemic injury. Inactivation of GR altered the functional differentiation/maturation of monocyte-derived macrophages in the infarcted myocardium. Mechanistically, CD45+/CD11b+/Ly6G−/F4/80+ macrophages isolated from GRLysMCre infarcts showed deregulation of factors that control inflammation, neovascularization, collagen degradation, and scar tissue formation. Moreover, we demonstrate that cardiac fibroblasts sorted from the ischemic myocardium of GRLysMCre mice compared with cells isolated from injured GRflox hearts displayed higher matrix metalloproteinase 2 expression, and we provide evidence that the macrophage GR regulates myofibroblast differentiation in the infarct microenvironment during the early phase of wound healing. In summary, GR signaling in macrophages, playing a crucial role in tissue-repairing mechanisms, could be a potential therapeutic target during wound healing after ischemic myocardial injury.—Galuppo, P., Vettorazzi, S., Hövelmann, J., Scholz, C.-J., Tuckermann, J. P., Bauersachs, J., Fraccarollo, D. The glucocorticoid receptor in monocyte-derived macrophages is critical for cardiac infarct repair and remodeling.
• {GT}‐rich promoters can drive {RNA} pol {II} transcription and deposition of {H}2A.{Z} in {African} trypanosomes. Wedel, Carolin; Förstner, Konrad U.; Derr, Ramona; Siegel, T. Nicolai. In The EMBO Journal, 36(17), pp. 2581–2594. 2017.
  • [ Abstract ]
  • [ DOI ]
  Genome‐wide transcription studies are revealing an increasing number of “dispersed promoters” that, unlike “focused promoters”, lack well‐conserved sequence motifs and tight regulation. Dispersed promoters are nevertheless marked by well‐defined chromatin structures, suggesting that specific sequence elements must exist in these unregulated promoters. Here, we have analyzed regions of transcription initiation in the eukaryotic parasite Trypanosoma brucei, in which RNA polymerase II transcription initiation occurs over broad regions without distinct promoter motifs and lacks regulation. Using a combination of site‐specific and genome‐wide assays, we identified GT‐rich promoters that can drive transcription and promote the targeted deposition of the histone variant H2A.Z in a genomic context‐dependent manner. In addition, upon mapping nucleosome occupancy at high resolution, we find nucleosome positioning to correlate with RNA pol II enrichment and gene expression, pointing to a role in RNA maturation. Nucleosome positioning may thus represent a previously unrecognized layer of gene regulation in trypanosomes. Our findings show that even highly dispersed, unregulated promoters contain specific DNA elements that are able to induce transcription and changes in chromatin structure.
• Insights into Microalga and Bacteria Interactions of Selected Phycosphere Biofilms Using Metagenomic, Transcriptomic, and Proteomic Approaches. Krohn-Molt, Ines; Alawi, Malik; Förstner, Konrad U.; Wiegandt, Alena; Burkhardt, Lia; Indenbirken, Daniela; Thieß, Melanie; Grundhoff, Adam; Kehr, Julia; Tholey, Andreas; Streit, Wolfgang R. In Frontiers in Microbiology, 8. Frontiers Media {SA}, 2017.
  • [ Abstract ]
  • [ DOI ]
  Microalga are of high relevance for the global carbon cycling and it is well-known that they are associated with a microbiota. However, it remains unclear, if the associated microbiota, often found in phycosphere biofilms, is specific for the microalga strains and which role individual bacterial taxa play. Here we provide experimental evidence that Chlorella saccharophila, Scenedesmus quadricauda, and Micrasterias crux-melitensis, maintained in strain collections, are associated with unique and specific microbial populations. Deep metagenome sequencing, binning approaches, secretome analyses in combination with RNA-Seq data implied fundamental differences in the gene expression profiles of the microbiota associated with the different microalga. Our metatranscriptome analyses indicates that the transcriptionally most active bacteria with respect to key genes commonly involved in plant-microbe interactions in the Chlorella (Trebouxiophyceae) and Scenedesmus (Chlorophyceae) strains belong to the phylum of the α-Proteobacteria. In contrast, in the Micrasterias (Zygnematophyceae) phycosphere biofilm bacteria affiliated with the phylum of the Bacteroidetes showed the highest gene expression rates. We furthermore show that effector molecules known from plant-microbe interactions as inducers for the innate immunity are already of relevance at this evolutionary early plant-microbiome level.
• The regulation of tetraspanin 8 gene expression—{A} potential new mechanism in the pathogenesis of bipolar disorder. Schartner, Christoph; Scholz, Claus-Jürgen; Weber, Heike; Post, Antonia; Freudenberg, Florian; Grünewald, Lena; Reif, Andreas. In American Journal of Medical Genetics Part B: Neuropsychiatric Genetics, 174(7), pp. 740–750. 2017.
  • [ Abstract ]
  • [ DOI ]
  In a previous study, we identified the single nucleotide polymorphism (SNP) rs4500567, located in the upstream region of tetraspanin 8 (TSPAN8), to be associated with bipolar disorder (BD). Due to its proximal position, the SNP might have an impact on promoter activity, thus on TSPAN8 gene expression. We investigated the impact of rs4500567 on TSPAN8 expression in vitro with luciferase-based promoter assays in human embryonic kidney (HEK293) and neuroblastoma cells (SH-SY5Y), and its effect on expression of downstream associated genes by microarray-based transcriptome analyses. Immunohistochemical localization studies on murine brain slices served to identify possible target regions of altered TSPAN8 expression in the brain. Promoter assays revealed decreased TSPAN8 expression in presence of the minor allele. Transcriptome analyses of TSPAN8-knockdown cells, mirroring the effects of putatively reduced TSPAN8 expression in minor allele carriers, resulted in 231 differentially expressed genes with enrichments of relevant signaling pathways for psychiatric disorders and neuronal development. Finally, we demonstrate Tspan8 abundance in mouse cerebellum and hippocampus. These findings point to a role of TSPAN8 in neuronal function or development. Considering a rather protective effect of the minor allele of rs4500567, our findings reveal a possible novel mechanism that contributes to the development of BD.
• In {Vivo} {Cleavage} {Map} {Illuminates} the {Central} {Role} of {RNase} {E} in {Coding} and {Non}-coding {RNA} {Pathways}. Chao, Yanjie; Li, Lei; Girodat, Dylan; Förstner, Konrad U.; Said, Nelly; Corcoran, Colin; Śmiga, Michał; Papenfort, Kai; Reinhardt, Richard; Wieden, Hans-Joachim; Luisi, Ben F.; Vogel, Jörg. In Molecular Cell, 65(1), pp. 39–51. 2017.
  • [ Abstract ]
  • [ DOI ]
  Summary Understanding RNA processing and turnover requires knowledge of cleavages by major endoribonucleases within a living cell. We have employed TIER-seq (transiently inactivating an endoribonuclease followed by RNA-seq) to profile cleavage products of the essential endoribonuclease RNase E in Salmonella enterica. A dominating cleavage signature is the location of a uridine two nucleotides downstream in a single-stranded segment, which we rationalize structurally as a key recognition determinant that may favor RNase E catalysis. Our results suggest a prominent biogenesis pathway for bacterial regulatory small RNAs whereby RNase E acts together with the RNA chaperone Hfq to liberate stable 3′ fragments from various precursor RNAs. Recapitulating this process in vitro, Hfq guides RNase E cleavage of a representative small-RNA precursor for interaction with a mRNA target. In vivo, the processing is required for target regulation. Our findings reveal a general maturation mechanism for a major class of post-transcriptional regulators.
• Differential effects of {FTY}720 on the {B} cell compartment in a mouse model of multiple sclerosis. Bail, Kathrin; Notz, Quirin; Rovituso, Damiano M.; Schampel, Andrea; Wunsch, Marie; Koeniger, Tobias; Schropp, Verena; Bharti, Richa; Scholz, Claus-Juergen; Foerstner, Konrad U.; Kleinschnitz, Christoph; Kuerten, Stefanie. In Journal of Neuroinflammation, 14, p. 148. 2017.
  • [ Abstract ]
  • [ DOI ]
  BACKGROUND: MP4-induced experimental autoimmune encephalomyelitis (EAE) is a mouse model of multiple sclerosis (MS), which enables targeted research on B cells, currently much discussed protagonists in MS pathogenesis. Here, we used this model to study the impact of the S1P1 receptor modulator FTY720 (fingolimod) on the autoreactive B cell and antibody response both in the periphery and the central nervous system (CNS). METHODS: MP4-immunized mice were treated orally with FTY720 for 30 days at the peak of disease or 50 days after EAE onset. The subsequent disease course was monitored and the MP4-specific B cell/antibody response was measured by ELISPOT and ELISA. RNA sequencing was performed to determine any effects on B cell-relevant gene expression. S1P1 receptor expression by peripheral T and B cells, B cell subset distribution in the spleen and B cell infiltration into the CNS were studied by flow cytometry. The formation of B cell aggregates and of tertiary lymphoid organs (TLOs) was evaluated by histology and immunohistochemistry. Potential direct effects of FTY720 on B cell aggregation were studied in vitro. RESULTS: FTY720 significantly attenuated clinical EAE when treatment was initiated at the peak of EAE. While there was a significant reduction in the number of T cells in the blood after FTY720 treatment, B cells were only slightly diminished. Yet, there was evidence for the modulation of B cell receptor-mediated signaling upon FTY720 treatment. In addition, we detected a significant increase in the percentage of B220+ B cells in the spleen both in acute and chronic EAE. Whereas acute treatment completely abrogated B cell aggregate formation in the CNS, the numbers of infiltrating B cells and plasma cells were comparable between vehicle- and FTY720-treated mice. In addition, there was no effect on already developed aggregates in chronic EAE. In vitro B cell aggregation assays suggested the absence of a direct effect of FTY720 on B cell aggregation. However, FTY720 impacted the evolution of B cell aggregates into TLOs. CONCLUSIONS: The data suggest differential effects of FTY720 on the B cell compartment in MP4-induced EAE.
• {APRICOT}: an integrated computational pipeline for the sequence-based identification and characterization of {RNA}-binding proteins. Sharan, Malvika; Förstner, Konrad U.; Eulalio, Ana; Vogel, Jörg. In Nucleic Acids Research, 45(11), pp. e96-e96. 2017.
  • [ Abstract ]
  • [ DOI ]
  RNA-binding proteins (RBPs) have been established as core components of several post-transcriptional gene regulation mechanisms. Experimental techniques such as cross-linking and co-immunoprecipitation have enabled the identification of RBPs, RNA-binding domains (RBDs) and their regulatory roles in the eukaryotic species such as human and yeast in large-scale. In contrast, our knowledge of the number and potential diversity of RBPs in bacteria is poorer due to the technical challenges associated with the existing global screening approaches. We introduce APRICOT, a computational pipeline for the sequence-based identification and characterization of proteins using RBDs known from experimental studies. The pipeline identifies functional motifs in protein sequences using position-specific scoring matrices and Hidden Markov Models of the functional domains and statistically scores them based on a series of sequence-based features. Subsequently, APRICOT identifies putative RBPs and characterizes them by several biological properties. Here we demonstrate the application and adaptability of the pipeline on large-scale protein sets, including the bacterial proteome of Escherichia coli. APRICOT showed better performance on various datasets compared to other existing tools for the sequence-based prediction of RBPs by achieving an average sensitivity and specificity of 0.90 and 0.91 respectively. The command-line tool and its documentation are available at https://pypi.python.org/pypi/bio-apricot.
• Hypermethylation of the non-imprinted maternal MEG3 and paternal MEST alleles is highly variable among normal individuals. Haertle, Larissa; Maierhofer, Anna; Böck, Julia; Lehnen, Harald; Böttcher, Yvonne; Blüher, Matthias; Schorsch, Martin; Potabattula, Ramya; El Hajj, Nady; Appenzeller, Silke; Haaf, Thomas. In PLOS ONE, 12(8), pp. 1–16. Public Library of Science, 2017.
  • [ Abstract ]
  • [ DOI ]
  Imprinted genes show parent-specific activity (functional haploidy), which makes them particularly vulnerable to epigenetic dysregulation. Here we studied the methylation profiles of oppositely imprinted genes at single DNA molecule resolution by two independent parental allele-specific deep bisulfite sequencing (DBS) techniques. Using Roche (GSJunior) next generation sequencing technology, we analyzed the maternally imprinted MEST promoter and the paternally imprinted MEG3 intergenic (IG) differentially methylated region (DMR) in fetal cord blood, adult blood, and visceral adipose tissue. Epimutations were defined as paternal or maternal alleles with >50% aberrantly (de)methylated CpG sites, showing the wrong methylation imprint. The epimutation rates (range 2–66%) of the paternal MEST and the maternal MEG3 IG DMR allele, which should be completely unmethylated, were significantly higher than those (0–15%) of the maternal MEST and paternal MEG3 alleles, which are expected to be fully methylated. This hypermethylation of the non-imprinted allele (HNA) was independent of parental origin. Very low epimutation rates in sperm suggest that HNA occurred after fertilization. DBS with Illumina (MiSeq) technology confirmed HNA for the MEST promoter and the MEG3 IG DMR, and to a lesser extent, for the paternally imprinted secondary MEG3 promoter and the maternally imprinted PEG3 promoter. HNA leads to biallelic methylation of imprinted genes in a considerable proportion of normal body cells (somatic mosaicism) and is highly variable between individuals. We propose that during development and differentiation maintenance of differential methylation at most imprinting control regions may become to some extent redundant. The accumulation of stochastic and environmentally-induced methylation errors on the non-imprinted allele may increase epigenetic diversity between cells and individuals.
• An {RpoHI}-{Dependent} {Response} {Promotes} {Outgrowth} after {Extended} {Stationary} {Phase} in the {Alphaproteobacterium} {Rhodobacter} sphaeroides. Remes, B.; Rische-Grahl, T.; Müller, K. M. H.; Förstner, K. U.; Yu, Sung-Huan; Weber, L.; Jäger, A.; Peuser, V.; Klug, G. In Journal of Bacteriology, 199(14), pp. e00249–17. 2017.
  • [ Abstract ]
  • [ DOI ]
  Under unfavorable growth conditions, bacteria enter stationary phase and can maintain cell viability over prolonged periods with no increase in cell number. To obtain insights into the regulatory mechanisms that allow bacteria to resume growth when conditions become favorable again (outgrowth), we performed global transcriptome analyses at different stages of growth for the alphaproteobacterium Rhodobacter sphaeroides. The majority of genes were not differentially expressed across growth phases. After a short stationary phase (about 20 h after growth starts to slow down), only 7\% of the genes showed altered expression (fold change of {\textgreater}1.6 or less than −1.6, corresponding to a log2 fold change of {\textgreater}0.65 or less than −0.65, respectively) compared to expression at exponential phase. Outgrowth induced a distinct response in gene expression which was strongly influenced by the length of the preceding stationary phase. After a long stationary phase (about 64 h after growth starts to slow down), a much larger number of genes (15.1\%) was induced in outgrowth than after a short stationary phase (1.7\%). Many of those genes are known members of the RpoHI/RpoHII regulons and have established functions in stress responses. A main effect of RpoHI on the transcriptome in outgrowth after a long stationary phase was confirmed. Growth experiments with mutant strains further support an important function in outgrowth after prolonged stationary phase for the RpoHI and RpoHII sigma factors. IMPORTANCE In natural environments, the growth of bacteria is limited mostly by lack of nutrients or other unfavorable conditions. It is important for bacterial populations to efficiently resume growth after being in stationary phase, which may last for long periods. Most previous studies on growth-phase-dependent gene expression did not address outgrowth after stationary phase. This study on growth-phase-dependent gene regulation in a model alphaproteobacterium reveals, for the first time, that the length of the stationary phase strongly impacts the transcriptome during outgrowth. The alternative sigma factors RpoHI and RpoHII, which are important regulators of stress responses in alphaproteobacteria, play a major role during outgrowth following prolonged stationary phase. These findings provide the first insight into the regulatory mechanisms enabling efficient outgrowth.
• {dRNA}-seq transcriptional profiling of the {FK}506 biosynthetic gene cluster in Streptomyces tsukubaensis {NRRL}18488 and general analysis of the transcriptome. Bauer, Judith S.; Fillinger, Sven; Förstner, Konrad; Herbig, Alexander; Jones, Adam C.; Flinspach, Katrin; Sharma, Cynthia; Gross, Harald; Nieselt, Kay; Apel, Alexander K. In {RNA} Biology, 14(11), pp. 1617–1626. Informa {UK} Limited, 2017.
  • [ Abstract ]
  • [ DOI ]
  FK506 (tacrolimus) is a valuable immunosuppressant produced by several Streptomyces strains. In the genome of the wild type producer Streptomyces tsukubaensis NRRL18488, FK506 biosynthesis is encoded by a gene cluster that spans 83.5 (kb). A whole transcriptome differential shotgun sequencing (dRNA-seq) of S. tsukubaensis was performed to analyze transcription at 2 different time points; before and during active FK506 production. In total, 8,914 transcription start sites were identified in either condition, which enabled precise determination of the 5'-UTR length of the corresponding transcripts as well as the identification of 2 consensus sequence motifs in the promoter regions. The transcription start sites of all gene operons within the FK506 cluster were identified, including 3 examples of leaderless RNA transcripts. These data provide detailed insight into the transcription of the FK506 biosynthetic gene cluster to support future regulatory studies, genetic manipulation, and industrial production.
• Transcription {Profiling} of {Bacillus} subtilis {Cells} {Infected} with {AR}9, a {Giant} {Phage} {Encoding} {Two} {Multisubunit} {RNA} {Polymerases}. Lavysh, Daria; Sokolova, Maria; Slashcheva, Marina; Förstner, Konrad U.; Severinov, Konstantin. In mBio, 8(1), pp. e02041–16. 2017.
  • [ Abstract ]
  • [ DOI ]
  Bacteriophage AR9 is a recently sequenced jumbo phage that encodes two multisubunit RNA polymerases. Here we investigated the AR9 transcription strategy and the effect of AR9 infection on the transcription of its host, Bacillus subtilis. Analysis of whole-genome transcription revealed early, late, and continuously expressed AR9 genes. Alignment of sequences upstream of the 5′ ends of AR9 transcripts revealed consensus sequences that define early and late phage promoters. Continuously expressed AR9 genes have both early and late promoters in front of them. Early AR9 transcription is independent of protein synthesis and must be determined by virion RNA polymerase injected together with viral DNA. During infection, the overall amount of host mRNAs is significantly decreased. Analysis of relative amounts of host transcripts revealed notable differences in the levels of some mRNAs. The physiological significance of up- or downregulation of host genes for AR9 phage infection remains to be established. AR9 infection is significantly affected by rifampin, an inhibitor of host RNA polymerase transcription. The effect is likely caused by the antibiotic-induced killing of host cells, while phage genome transcription is solely performed by viral RNA polymerases. IMPORTANCE Phages regulate the timing of the expression of their own genes to coordinate processes in the infected cell and maximize the release of viral progeny. Phages also alter the levels of host transcripts. Here we present the results of a temporal analysis of the host and viral transcriptomes of Bacillus subtilis infected with a giant phage, AR9. We identify viral promoters recognized by two virus-encoded RNA polymerases that are a unique feature of the phiKZ-related group of phages to which AR9 belongs. Our results set the stage for future analyses of highly unusual RNA polymerases encoded by AR9 and other phiKZ-related phages.
• Insights into {Microalga} and {Bacteria} {Interactions} of {Selected} {Phycosphere} {Biofilms} {Using} {Metagenomic}, {Transcriptomic}, and {Proteomic} {Approaches}. Krohn-Molt, Ines; Alawi, Malik; Förstner, Konrad U.; Wiegandt, Alena; Burkhardt, Lia; Indenbirken, Daniela; Thieß, Melanie; Grundhoff, Adam; Kehr, Julia; Tholey, Andreas; Streit, Wolfgang R. In Frontiers in Microbiology, 8. 2017.
  • [ Abstract ]
  • [ DOI ]
  Microalga are of high relevance for the global carbon cycling and it is well-known that they are associated with a microbiota. However, it remains unclear, if the associated microbiota, often found in the phycosphere biofilms, is specific for the microalga strains and which role individual bacterial taxa play. Here we provide experimental evidence that Chlorella saccharophila, Scenedesmus quadricauda and Micrasterias crux-melitensis, maintained in strain collections, are associated with unique and specific microbial populations. Deep metagenome sequencing, binning approaches, secretome analyses in combination with RNA-seq data implied fundamental differences in the gene expression profiles of the microbiota associated with the different microalga. Our metatranscriptome analyses indicates that the transcriptionally most active bacteria with respect to key genes commonly involved in plant-microbe interactions in the Chlorella (Trebouxiophyceae) and Scenedesmus (Chlorophyceae) strains belong to the phylum of the α-Proteobacteria. In contrast, in the Micrasterias (Zygnematophyceae) phycosphere biofilm bacteria affiliated with the phylum of the Bacteroidetes showed the highest gene expression rates. We furthermore show that effector molecules known from plant-microbe interactions as inducers for the innate immunity are already of relevance at this evolutionary early plant-microbiome level.
• Pan-{Raf} co-operates with {PI}3K-dependent signalling and critically contributes to myeloma cell survival independently of mutated {RAS}. Müller, E.; Bauer, S.; Stühmer, T.; Mottok, A.; Scholz, C.-J.; Steinbrunn, T.; Brünnert, D.; Brandl, A.; Schraud, H.; Kreßmann, S.; Beilhack, A.; Rosenwald, A.; Bargou, R. C.; Chatterjee, M. In Leukemia, 31(4), pp. 922–933. 2017.
  • [ Abstract ]
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  Direct therapeutic targeting of oncogenic RAS is currently still impossible due to lack of suitable pharmacological inhibitors. Because specific blockade of druggable RAS effectors might represent an alternative treatment approach, we evaluated the role of the Raf complex for multiple myeloma (MM) pathobiology. We found frequent overexpression of the Raf isoforms (A-, B- and C-Raf) and downstream activation of MEK1,2/ERK1,2 in MM cells. Concomitant inhibition of all Raf isoforms (pan-Raf inhibition) by RNAi or pharmacological inhibitors was required to strongly induce apoptosis in human MM cell lines (HMCLs), in primary MM cells in vitro, and in a syngeneic MM mouse model in vivo. The anti-MM effect of pan-Raf inhibition did not correlate with the RAS mutation status, and functionally appeared to involve both MEK-dependent and -independent mechanisms. Furthermore, transcriptome analyses revealed that pan-Raf activity affects PI3K-dependent signalling, thus highlighting a functional link between the RAS/Raf and PI3K/mTOR/Akt pro-survival pathways. Accordingly, pharmacological inhibition of PI3K strongly enhanced the anti-MM effect of pan-Raf inhibition in MM cell lines and in primary MM cells in vitro and in vivo. Concomitant pan-Raf/PI3K inhibition was also effective in carfilzomib- and lenalidomide-resistant MM models underscoring that this attractive therapeutic anti-MM strategy is suitable for immediate clinical translation.
• Genetic Overlap Between Attention-Deficit/Hyperactivity Disorder and Bipolar Disorder: Evidence From Genome-wide Association Study Meta-analysis. van Hulzen, Kimm J.E.; Scholz, Claus J.; Franke, Barbara; Ripke, Stephan; Klein, Marieke; McQuillin, Andrew; Sonuga-Barke, Edmund J.; Kelsoe, John R.; Land{{é}}n, Mikael; Andreassen, Ole A.; Lesch, Klaus-Peter; Weber, Heike; Faraone, Stephen V.; Arias-Vasquez, Alejandro; Reif, Andreas. In Biological Psychiatry, 82(9), pp. 634–641. Elsevier {BV}, 2017.
  • [ Abstract ]
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  BACKGROUND: Attention-deficit/hyperactivity disorder (ADHD) and bipolar disorder (BPD) are frequently co-occurring and highly heritable mental health conditions. We hypothesized that BPD cases with an early age of onset (≤21 years old) would be particularly likely to show genetic covariation with ADHD. METHODS: Genome-wide association study data were available for 4609 individuals with ADHD, 9650 individuals with BPD (5167 thereof with early-onset BPD), and 21,363 typically developing controls. We conducted a cross-disorder genome-wide association study meta-analysis to identify whether the observed comorbidity between ADHD and BPD could be due to shared genetic risks. RESULTS: We found a significant single nucleotide polymorphism-based genetic correlation between ADHD and BPD in the full and age-restricted samples (rGfull = .64, p = 3.13 × 10-14; rGrestricted = .71, p = 4.09 × 10-16). The meta-analysis between the full BPD sample identified two genome-wide significant (prs7089973 = 2.47 × 10-8; prs11756438 = 4.36 × 10-8) regions located on chromosomes 6 (CEP85L) and 10 (TAF9BP2). Restricting the analyses to BPD cases with an early onset yielded one genome-wide significant association (prs58502974 = 2.11 × 10-8) on chromosome 5 in the ADCY2 gene. Additional nominally significant regions identified contained known expression quantitative trait loci with putative functional consequences for NT5DC1, NT5DC2, and CACNB3 expression, whereas functional predictions implicated ABLIM1 as an allele-specific expressed gene in neuronal tissue. CONCLUSIONS: The single nucleotide polymorphism-based genetic correlation between ADHD and BPD is substantial, significant, and consistent with the existence of genetic overlap between ADHD and BPD, with potential differential genetic mechanisms involved in early and later BPD onset.
• The enteric nervous system is a potential autoimmune target in multiple sclerosis. Wunsch, Marie; Jabari, Samir; Voussen, Barbara; Enders, Michael; Srinivasan, Shanthi; Cossais, François; Wedel, Thilo; Boettner, Martina; Schwarz, Anna; Weyer, Linda; Göcer, Oktay; Schroeter, Michael; Maeurer, Mathias; Woenckhaus, Matthias; Pollok, Karolin; Radbruch, Helena; Klotz, Luisa; Scholz, Claus-Jürgen; Nickel, Joachim; Friebe, Andreas; Addicks, Klaus; Ergün, Süleyman; Lehmann, Paul V.; Kuerten, Stefanie. In Acta Neuropathologica, 134(2), pp. 281–295. 2017.
  • [ Abstract ]
  • [ DOI ]
  Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) in young adults that has serious negative socioeconomic effects. In addition to symptoms caused by CNS pathology, the majority of MS patients frequently exhibit gastrointestinal dysfunction, which was previously either explained by the presence of spinal cord lesions or not directly linked to the autoimmune etiology of the disease. Here, we studied the enteric nervous system (ENS) in a B cell- and antibody-dependent mouse model of MS by immunohistochemistry and electron microscopy at different stages of the disease. ENS degeneration was evident prior to the development of CNS lesions and the onset of neurological deficits in mice. The pathology was antibody mediated and caused a significant decrease in gastrointestinal motility, which was associated with ENS gliosis and neuronal loss. We identified autoantibodies against four potential target antigens derived from enteric glia and/or neurons by immunoprecipitation and mass spectrometry. Antibodies against three of the target antigens were also present in the plasma of MS patients as confirmed by ELISA. The analysis of human colon resectates provided evidence of gliosis and ENS degeneration in MS patients compared to non-MS controls. For the first time, this study establishes a pathomechanistic link between the well-established autoimmune attack on the CNS and ENS pathology in MS, which might provide a paradigm shift in our current understanding of the immunopathogenesis of the disease with broad diagnostic and therapeutic implications.