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Objectives: Engineered Heart Tissue (EHT) can be used to investigate cardiomyocyte hypertrophy in vitro. In this model coherently beating rat cardiomyocytes in 3D tissue culture are acutely forced to work against an increased afterload. They subsequently display hypertrophy and functional and transcriptional alterations stereotypically associated with heart failure. In the present study we used the model to investigate DNA methylation patterns of cardiomyocyte hypertrophy. We assessed two different approaches: An unbiased microarray and bisulfite sequencing of promoter regions of genes associated with pathological hypertrophy.Methods: EHTs were cultured on hollow silicone posts in a 24-well format for 2 weeks until maturation. Afterload was then acutely increased (“afterload enhanced”, AE) by inserting metal braces into the hollow silicone posts for one week. Additional EHTs were treated with phenylephrine to induce hypertrophy (PE), control EHTs were left untreated. Methylated DNA fragments were enriched by MBD2 pull-down and quantified by microarray. Data was evaluated and mapped using DEVA, R and DAVID software. Additionally, DNA from EHTs was sodium bisulfite treated to convert all non-methylated cytosines to thymidines. Promoter region fragments of Nppa, Nppb, Acta-1 and Atp2a2 (ANP, BNP, alpha skeletal muscle actin and Serca2a) and methylation positive and negativ controls Oxt and Ube2b were PCR amplified and subjected to next generation sequencing (NGS). Single-base resolution methylation data was extracted by Bismark software.Results: In the microarray analysis PE and AE clustered together. Methylation changes among AE or PE and controls respectively mapped to pathways relevant for pathological hypertrophy. Further analyses identified previously unidentified differentially methylated regions and confirmed differential methylation in well-studied genes such as Serca2a, supporting the validity of the approach. Targeted NGS confirmed subtle but reproducible DNA methylation alterations in promoter regions of hypertrophy associated genes. A region at -3000 bp of the Serca2a transcriptional start site was identified as differentially methylated using the microarray approach and confirmed by NGS.Conclusion: Exposing EHTs to enhanced afterload rapidly and specifically alters DNA methylation in cardiac myocytes. In vitro hypertrophy is accompanied by a common DNA methylation signature irrespective of the cause of hypertrophy. Its probable regulatory function and high plasticity suggest DNA methylation as a possible drug target in cardiac hypertrophy. [ABSTRACT FROM AUTHOR]

Enzymatic degradation of algae cell wall carbohydrates by microorganisms is under increasing investigation as marine organic matter gains more value as a sustainable resource. The fate of carbon in the marine ecosystem is in part driven by these degradation processes. In this study, we observe the microbiome dynamics of the macroalga Fucus vesiculosus in 25-day-enrichment cultures resulting in partial degradation of the brown algae. Microbial community analyses revealed the phylum Pseudomonadota as the main bacterial fraction dominated by the genera Marinomonas and Vibrio. More importantly, a metagenome-based Hidden Markov model for specific glycosyl hydrolyses and sulphatases identified Bacteroidota as the phylum with the highest potential for cell wall degradation, contrary to their low abundance. For experimental verification, we cloned, expressed, and biochemically characterised two α-L-fucosidases, FUJM18 and FUJM20. While protein structure predictions suggest the highest similarity to a Bacillota origin, protein-protein blasts solely showed weak similarities to defined Bacteroidota proteins. Both enzymes were remarkably active at elevated temperatures and are the basis for a potential synthetic enzyme cocktail for large-scale algal destruction., (© 2024. The Author(s).)

Drug-based antiretroviral therapies (ART) efficiently suppress HIV replication in humans, but the virus persists as integrated proviral reservoirs in small numbers of cells. Importantly, ART cannot eliminate HIV from an infected individual, since it does not target the integrated provirus. Therefore, genome editing-based strategies that can inactivate or excise HIV genomes would provide the technology for novel curative therapies. In fact, the HIV-1 LTR-specific designer-recombinase Brec1 has been shown to remove integrated proviruses from infected cells and is highly efficacious on clinical HIV-1 isolates in vitro and in vivo, suggesting that Brec1 has the potential for clinical development of advanced HIV-1 eradication strategies in people living with HIV. In line with the preparation of a first-in-human advanced therapy medicinal product gene therapy trial, we here present an extensive preclinical evaluation of Brec1 and lentiviral vectors expressing the Brec1 transgene. This included detailed functional analysis of potential genomic off-target sites, assessing vector safety by investigating vector copy number (VCN) and the risk for potential vector-related insertional mutagenesis, as well as analyzing the potential of Brec1 to trigger an undesired strong T cell immune response. In conclusion, the antiviral designer-recombinase Brec1 is shown to lack any detectable cytopathic, genotoxic or T cell-related immunogenic effects, thereby meeting an important precondition for clinical application of the therapeutic lentiviral vector LV-Brec1 in novel HIV-1 curative strategies., Competing Interests: The author’s have read the journal’s policy and have the following competing interests: Jan Chemnitz, Joachim Hauber, Frank Buchholz, Ilona Hauber, Niklas Beschorner and Maike Voges are founding partners of PROVIREX Genome Editing Therapies GmbH, which is commercially developing Brec1 technology. Niklas Beschorner and Maike Voges are full-time employees of PROVIREX Genome Editing Therapies GmbH since 2022, and Jan Chemnitz, Frank Buchholz, Joachim Hauber and Ilona Hauber are part-time employees. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare., (Copyright: © 2024 Beschorner et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Biofilms are resistant to many traditional antibiotics, which has led to search for new antimicrobials from different and unique sources. To harness the potential of aquatic microbial resources, we analyzed the meta-omics datasets of microalgae-bacteria communities and mined them for potential antimicrobial and quorum quenching enzymes. One of the most interesting candidates (Dlh3), a dienelactone hydrolase, is a α/β-protein with predicted eight α-helices and eight β-sheets. When it was applied to one of the major fish pathogens, Edwardsiella anguillarum, the biofilm development was reproducibly inhibited by up to 54.5%. The transcriptome dataset in presence of Dlh3 showed an upregulation in functions related to self-defense like active genes for export mechanisms and transport systems. The most interesting point regarding the biotechnological potential for aquaculture applications of Dlh3 are clear evidence of biofilm inhibition and that health and division of a relevant fish cell model (CHSE-214) was not impaired by the enzyme., (© 2024. The Author(s).)

Many marine organisms produce bioactive molecules with unique characteristics to survive in their ecological niches. These enzymes can be applied in biotechnological processes and in the medical sector to replace aggressive chemicals that are harmful to the environment. Especially in the human health sector, there is a need for new approaches to fight against pathogens like Stenotrophomonas maltophilia which forms thick biofilms on artificial joints or catheters and causes serious diseases. Our approach was to use enrichment cultures of five marine resources that underwent sequence-based screenings in combination with deep omics analyses in order to identify enzymes with antibiofilm characteristics. Especially the supernatant of the enrichment culture of a stony coral caused a 40% reduction of S. maltophilia biofilm formation. In the presence of the supernatant, our transcriptome dataset showed a clear stress response (upregulation of transcripts for metal resistance, antitoxins, transporter, and iron acquisition) to the treatment. Further investigation of the enrichment culture metagenome and proteome indicated a series of potential antimicrobial enzymes. We found an impressive group of metalloproteases in the proteome of the supernatant that is responsible for the detected anti-biofilm effect against S. maltophilia. KEY POINTS: • Omics-based discovery of novel marine-derived antimicrobials for human health management by inhibition of S. maltophilia • Up to 40% reduction of S. maltophilia biofilm formation by the use of marine-derived samples • Metalloprotease candidates prevent biofilm formation of S. maltophilia K279a by up to 20., (© 2023. The Author(s).)

Infections in early life can elicit substantially different immune responses and pathogenesis than infections in adulthood. Here, we investigate the consequences of murine cytomegalovirus infection in newborn mice on NK cells. We show that infection severely compromised NK cell maturation and functionality in newborns. This effect was not due to compromised virus control. Inflammatory responses to infection dysregulated the expression of major transcription factors governing NK cell fate, such as Eomes, resulting in impaired NK cell function. Most prominently, NK cells from perinatally infected mice have a diminished ability to produce IFN-γ due to the downregulation of long non-coding RNA Ifng-as1 expression. Moreover, the bone marrow's capacity to efficiently generate new NK cells is reduced, explaining the prolonged negative effects of perinatal infection on NK cells. This study demonstrates that viral infections in early life can profoundly impact NK cell biology, including long-lasting impairment in NK cell functionality., (© 2023. Springer Nature Limited.)

In order to expand the knowledge of microbial ecosystems from deep-sea hydrothermal vent systems located on the Central and South-East Indian Ridge, we sampled hydrothermal fluids, massive sulfides, ambient water and sediments of six distinct vent fields. Most of these vent sites were only recently discovered in the course of the German exploration program for massive sulfide deposits and no previous studies of the respective microbial communities exist. Apart from typically vent-associated chemosynthetic members of the orders Campylobacterales , Mariprofundales , and Thiomicrospirales , high numbers of uncultured and unspecified Bacteria were identified via 16S rRNA gene analyses in hydrothermal fluid and massive sulfide samples. The sampled sediments however, were characterized by an overall lack of chemosynthetic Bacteria and the presence of high proportions of low abundant bacterial groups. The archaeal communities were generally less diverse and mostly dominated by members of Nitrosopumilales and Woesearchaeales , partly exhibiting high proportions of unassigned Archaea. Correlations with environmental parameters were primarily observed for sediment communities and for microbial species (associated with the nitrogen cycle) in samples from a recently identified vent field, which was geochemically distinct from all other sampled sites. Enrichment cultures of diffuse fluids demonstrated a great potential for hydrogen oxidation coupled to the reduction of various electron-acceptors with high abundances of Hydrogenovibrio and Sulfurimonas species. Overall, given the large number of currently uncultured and unspecified microorganisms identified in the vent communities, their respective metabolic traits, ecosystem functions and mediated biogeochemical processes have still to be resolved for estimating consequences of potential environmental disturbances by future mining activities., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Adam-Beyer, Laufer-Meiser, Fuchs, Schippers, Indenbirken, Garbe-Schönberg, Petersen and Perner.)

Previous studies have identified a unique Treg population, which expresses the Th17 characteristic transcription factor RORγt. These RORγt + Tregs possess enhanced immunosuppressive capacity, which endows them with great therapeutic potential. However, as a caveat, they are also capable of secreting pro-inflammatory IL-17A. Since the sum function of RORγt + Tregs in glomerulonephritis (GN) remains unknown, we studied the effects of their absence. Purified CD4 + T cell populations, containing or lacking RORγt + Tregs, were transferred into immunocompromised RAG1 knockout mice and the nephrotoxic nephritis model of GN was induced. Absence of RORγt + Tregs significantly aggravated kidney injury, demonstrating overall kidney-protective properties. Analyses of immune responses showed that RORγt + Tregs were broadly immunosuppressive with no preference for a particular type of T cell response. Further characterization revealed a distinct functional and transcriptional profile, including enhanced production of IL-10. Expression of the chemokine receptor CCR6 marked a particularly potent subset, whose absence significantly worsened GN. As an underlying mechanism, we found that chemokine CCL20 acting through receptor CCR6 signaling mediated expansion and activation of RORγt + Tregs. Finally, we also detected an increase of CCR6 + Tregs in kidney biopsies, as well as enhanced secretion of chemokine CCL20 in 21 patients with anti-neutrophil cytoplasmic antibody associated GN compared to that of 31 healthy living donors, indicating clinical relevance. Thus, our data characterize RORγt + Tregs as anti-inflammatory mediators of GN and identify them as promising target for Treg directed therapies., (Copyright © 2023 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

The tumor microenvironment is a complex niche enveloping a tumor formed by extracellular matrix, blood vessels, immune cells, and fibroblasts constantly interacting with cancer cells. Although tumor microenvironment is increasingly recognized as a major player in cancer initiation and progression in many tumor types, its involvement in Merkel cell carcinoma (MCC) pathogenesis is currently unknown. In this study, we provide a molecular and functional characterization of cancer-associated fibroblasts (CAFs), the major tumor microenvironment component, in patient-derived xenografts of patients with MCC. We show that subcutaneous coinjection of patient-derived CAFs and human MCC MKL-1 cells into severe combined immunodeficient mice significantly promotes tumor growth and metastasis. These fast-growing xenografts are characterized by areas densely populated with human CAFs, mainly localized around blood vessels. We provide evidence that the growth-promoting activity of MCC-derived CAFs is mediated by the aminopeptidase A/angiotensin II and III/angiotensin II type 1 receptor axis, with the expression of aminopeptidase A in CAFs being a triggering event. Together, our findings point to aminopeptidase A as a potential marker for MCC prognostic stratification and as a candidate for therapeutic intervention., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The ongoing coronavirus disease 2019 pandemic significantly burdens hospitals and other healthcare facilities. Therefore, understanding the entry and transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for effective prevention and preparedness measures. We performed surveillance and analysis of testing and transmission of SARS-CoV-2 infections in a tertiary-care hospital in Germany during the second and third pandemic waves in fall/winter 2020., Methods: Between calendar week 41 in 2020 and calendar week 1 in 2021, 40%, of all positive patient and staff samples (284 total) were subjected to full-length viral genome sequencing. Clusters were defined based on similar genotypes indicating common sources of infection. We integrated phylogenetic, spatial, and temporal metadata to detect nosocomial infections and outbreaks, uncover transmission chains, and evaluate containment measures' effectiveness., Results: Epidemiologic data and contact tracing readily recognize most healthcare-associated (HA) patient infections. However, sequencing data reveal that temporally preceding index cases and transmission routes can be missed using epidemiologic methods, resulting in delayed interventions and serially linked outbreaks being counted as independent events. While hospital-associated transmissions were significantly elevated at a moderate rate of community transmission during the second wave, systematic testing and high vaccination rates among staff have led to a substantial decrease in HA infections at the end of the second/beginning of the third wave despite high community transmissions., Conclusions: While epidemiologic analysis is critical for immediate containment of HA SARS-CoV-2 outbreaks, integration of genomic surveillance revealed weaknesses in identifying staff contacts. Our study underscores the importance of high testing frequency and genomic surveillance to detect, contain and prevent SARS-CoV-2-associated infections in healthcare settings., Competing Interests: Potential conflict of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Anthropogenic activities are modifying the oceanic environment rapidly and are causing ocean warming and deoxygenation, affecting biodiversity, productivity, and biogeochemical cycling. In coastal sediments, anaerobic organic matter degradation essentially fuels the production of hydrogen sulfide and methane. The release of these compounds from sediments is detrimental for the (local) environment and entails socio-economic consequences. Therefore, it is vital to understand which microbes catalyze the re-oxidation of these compounds under environmental dynamics, thereby mitigating their release to the water column. Here we use the seasonally dynamic Boknis Eck study site (SW Baltic Sea), where bottom waters annually fall hypoxic or anoxic after the summer months, to extrapolate how the microbial community and its activity reflects rising temperatures and deoxygenation. During October 2018, hallmarked by warmer bottom water and following a hypoxic event, modeled sulfide and methane production and consumption rates are higher than in March at lower temperatures and under fully oxic bottom water conditions. The microbial populations catalyzing sulfide and methane metabolisms are found in shallower sediment zones in October 2018 than in March 2019. DNA-and RNA profiling of sediments indicate a shift from primarily organotrophic to (autotrophic) sulfide oxidizing Bacteria, respectively. Previous studies using data collected over decades demonstrate rising temperatures, decreasing eutrophication, lower primary production and thus less fresh organic matter transported to the Boknis Eck sediments. Elevated temperatures are known to stimulate methanogenesis, anaerobic oxidation of methane, sulfate reduction and essentially microbial sulfide consumption, likely explaining the shift to a phylogenetically more diverse sulfide oxidizing community based on RNA., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Perner, Wallmann, Adam-Beyer, Hepach, Laufer-Meiser, Böhnke, Diercks, Bange, Indenbirken, Nikeleit, Bryce, Kappler, Engel and Scholz.)

Iron (Fe) is an essential trace element for life. In the ocean, Fe can be exceptionally scarce and thus biolimiting or extremely enriched causing microbial stress. The ability of hydrothermal plume microbes to counteract unfavorable Fe-concentrations up to 10 mM is investigated through experiments. While Campylobacterota (Sulfurimonas) are prominent in a diverse community at low to intermediate Fe-concentrations, the highest 10 mM Fe-level is phylogenetically less diverse and dominated by the SUP05 clade (Gammaproteobacteria), a species known to be genetically well equipped to strive in high-Fe environments. In all incubations, Fe-binding ligands were produced in excess of the corresponding Fe-concentration level, possibly facilitating biological Fe-uptake in low-Fe incubations and detoxification in high-Fe incubations. The diversity of Fe-containing formulae among dissolved organics (SPE-DOM) decreased with increasing Fe-concentration, which may reflect toxic conditions of the high-Fe treatments. A DOM-derived degradation index (I DEG ) points to a degradation magnitude (microbial activity) that decreases with Fe and/or selective Fe-DOM coagulation. Our results show that some hydrothermal microbes (especially Gammaproteobacteria) have the capacity to thrive even at unfavorably high Fe-concentrations. These ligand-producing microbes could hence play a key role in keeping Fe in solution, particularly in environments, where Fe precipitation dominates and toxic conditions prevail., (© 2022. The Author(s).)

Merkel cell polyomavirus is the causative agent for most Merkel cell carcinomas (MCCs). This highly aggressive skin cancer shows rapid progression, with metastasis being a significant challenge for patient therapy. Virus-positive MCCs show low mutation rates, and tumor cell proliferation is dependent on viral oncoproteins small T antigen (sT) and large T antigen. Although the role of sT and large T antigen in early events of tumorigenesis has been extensively studied, their role in tumor progression has been scarcely addressed. In this study, we investigate the possible mechanisms of how Merkel cell polyomavirus oncoproteins, particularly sTs, contribute to metastasis. We show that sT specifically affects selectin ligand binding and processing by altering the presentation of multiple MCC surface molecules, thereby influencing initial metastasis events and tumor cell immune recognition. Furthermore, we show that sT regulates the surface antigen CD47, which inhibits phagocytosis by macrophages. By applying either sT short hairpin RNAs, CD47-targeted small interfering RNAs, or a therapeutic anti-CD47 antibody, we show that immune recognition of MCC cells can be restored. Thus, CD47 is a promising therapeutic target on MCC cells. Blocking the CD47‒SIRPα interaction effectively promotes phagocytosis of MCC cells and might be a promising combinatorial immunotherapy approach together with PD-1/PD-L1 axis in MCC treatment., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Early life environmental influences such as exposure to cigarette smoke (CS) can disturb molecular processes of lung development and thereby increase the risk for later development of chronic respiratory diseases. Among the latter, asthma and chronic obstructive pulmonary disease (COPD) are the most common. The airway epithelium plays a key role in their disease pathophysiology but how CS exposure in early life influences airway developmental pathways and epithelial stress responses or survival is poorly understood. Using Drosophila melanogaster larvae as a model for early life, we demonstrate that CS enters the entire larval airway system, where it activates cyp18a1 which is homologues to human CYP1A1 to metabolize CS-derived polycyclic aromatic hydrocarbons and further induces heat shock protein 70. RNASeq studies of isolated airways showed that CS dysregulates pathways involved in oxidative stress response, innate immune response, xenobiotic and glutathione metabolic processes as well as developmental processes (BMP, FGF signaling) in both sexes, while other pathways were exclusive to females or males. Glutathione S-transferase genes were further validated by qPCR showing upregulation of gstD4, gstD5 and gstD8 in respiratory tracts of females, while gstD8 was downregulated and gstD5 unchanged in males. ROS levels were increased in airways after CS. Exposure to CS further resulted in higher larval mortality, lower larval-pupal transition, and hatching rates in males only as compared to air-exposed controls. Taken together, early life CS induces airway epithelial stress responses and dysregulates pathways involved in the fly's branching morphogenesis as well as in mammalian lung development. CS further affected fitness and development in a highly sex-specific manner., (Copyright © 2022. Published by Elsevier Ltd.)

Genetic variants in α-actinin-2 (ACTN2) are associated with several forms of (cardio)myopathy. We previously reported a heterozygous missense (c.740C>T) ACTN2 gene variant, associated with hypertrophic cardiomyopathy, and characterized by an electro-mechanical phenotype in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Here, we created with CRISPR/Cas9 genetic tools two heterozygous functional knock-out hiPSC lines with a second wild-type (ACTN2wt) and missense ACTN2 (ACTN2mut) allele, respectively. We evaluated their impact on cardiomyocyte structure and function, using a combination of different technologies, including immunofluorescence and live cell imaging, RNA-seq, and mass spectrometry. This study showed that ACTN2mut presents a higher percentage of multinucleation, protein aggregation, hypertrophy, myofibrillar disarray, and activation of both the ubiquitin-proteasome system and the autophagy-lysosomal pathway as compared to ACTN2wt in 2D-cultured hiPSC-CMs. Furthermore, the expression of ACTN2mut was associated with a marked reduction of sarcomere-associated protein levels in 2D-cultured hiPSC-CMs and force impairment in engineered heart tissues. In conclusion, our study highlights the activation of proteolytic systems in ACTN2mut hiPSC-CMs likely to cope with ACTN2 aggregation and therefore directs towards proteopathy as an additional cellular pathology caused by this ACTN2 variant, which may contribute to human ACTN2 -associated cardiomyopathies.

Microalgae are one of the most dominant forms of life on earth that is tightly associated with a distinct and specialized microbiota. We have previously shown that the microbiota of Scenedesmus quadricauda harbors less than 10 distinct microbial species. Here, we provide evidence that dominant species are affiliated with the genera of Variovorax, Porphyrobacter, and Dyadobacter. Experimental and transcriptome-based evidence implies that within this multispecies interaction, Dyadobacter is a key to alga growth and fitness and is highly adapted to live in the phycosphere. While presumably under light conditions the alga provides the energy source to the bacteria, Dyadobacter produces and releases mainly a large variety of polysaccharides modifying enzymes. This is coherent with high-level expression of the T9SS in alga cocultures. The transcriptome data further imply that quorum-quenching proteins (QQ) and biosynthesis of vitamins B 1 , B 2 , B 5 , B 6 , and B 9 are expressed by Dyadobacter at high levels in comparison to Variovorax and Porphyrobacter. Notably, Dyadobacter produces a significant number of leucine-rich repeat (LRR) proteins and enzymes involved in bacterial reactive oxygen species (ROS) tolerance. Complementary to this, Variovorax expresses the genes of the biosynthesis of vitamins B 2 , B 5 , B 6 , B 7 , B 9 , and B 12 , and Porphyrobacter is specialized in the production of vitamins B 2 and B 6 . Thus, the shared currency between partners are vitamins, microalgae growth-promoting substances, and dissolved carbon. This work significantly enlarges our knowledge on alga-bacteria interaction and demonstrates physiological investigations of microalgae and associated bacteria, using microscopy observations, photosynthetic activity measurements, and flow cytometry. IMPORTANCE The current study gives a detailed insight into mutualistic collaboration of microalgae and bacteria, including the involvement of competitive interplay between bacteria. We provide experimental evidence that Gram-negative bacteria belonging to the Dyadobacter, Porphyrobacter, and Variovorax are the key players in a Scenedesmus quadricauda alga-bacteria interaction. We impart strong evidence that Dyadobacter produces and releases polysaccharides degradation enzymes and leucine-rich repeat proteins; Variovorax supplies the consortium with auxins and vitamin B 12 , while Porphyrobacter produces a broad spectrum of B vitamins. We show not only that the microalgae collaborate with the bacteria and vice versa but also that the bacteria interact with each other via quorum-sensing and secretion system mechanisms. The shared currency between partners appears to be vitamins, microalgae growth-promoting substances, and dissolved carbon.

NK cells play a pivotal role in viral immunity, utilizing a large array of activating and inhibitory receptors to identify and eliminate virus-infected cells. Killer-cell immunoglobulin-like receptors (KIRs) represent a highly polymorphic receptor family, regulating NK cell activity and determining the ability to recognize target cells. Human leukocyte antigen (HLA) class I molecules serve as the primary ligand for KIRs. Herein, HLA-C stands out as being the dominant ligand for the majority of KIRs. Accumulating evidence indicated that interactions between HLA-C and its inhibitory KIR2DL receptors (KIR2DL1/L2/L3) can drive HIV-1-mediated immune evasion and thus may contribute to the intrinsic control of HIV-1 infection. Of particular interest in this context is the recent observation that HIV-1 is able to adapt to host HLA-C genotypes through Vpu-mediated downmodulation of HLA-C. However, our understanding of the complex interplay between KIR/HLA immunogenetics, NK cell-mediated immune pressure and HIV-1 immune escape is still limited. Therefore, we investigated the impact of specific KIR/HLA-C combinations on the NK cell receptor repertoire and HIV-1 Vpu protein sequence variations of 122 viremic, untreated HIV-1 + individuals. Compared to 60 HIV-1 - controls, HIV-1 infection was associated with significant changes within the NK cell receptor repertoire, including reduced percentages of NK cells expressing NKG2A, CD8, and KIR2DS4. In contrast, the NKG2C + and KIR3DL2 + NK cell sub-populations from HIV-1 + individuals was enlarged compared to HIV-1 - controls. Stratification along KIR/HLA-C genotypes revealed a genotype-dependent expansion of KIR2DL1 + NK cells that was ultimately associated with increased binding affinities between KIR2DL1 and HLA-C allotypes. Lastly, our data hinted to a preferential selection of Vpu sequence variants that were associated with HLA-C downmodulation in individuals with high KIR2DL/HLA-C binding affinities. Altogether, our study provides evidence that HIV-1-associated changes in the KIR repertoire of NK cells are to some extent predetermined by host KIR2DL/HLA-C genotypes. Furthermore, analysis of Vpu sequence polymorphisms indicates that differential KIR2DL/HLA-C binding affinities may serve as an additional mechanism how host genetics impact immune evasion by HIV-1., Competing Interests: CDS reports grants and personal fees from AbbVie, grants, fees and non-financial support from Gilead Sciences, grants and personal fees from Janssen-Cilag, grants and personal fees from MSD, grants from Cepheid, personal fees from GSK, grants and personal fees from ViiV Healthcare, during the conduct of the study; fees from AstraZeneca, other from Apeiron, grants, personal fees and non-financial support from BBraun Melsungen, grants, personal fees from BioNtech, personal fees from Eli Lilly, personal fees from Formycon, personal fees from Molecular partners, grants and personal fees from Eli Lilly, personal fees from Roche, personal fees from SOBI. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Vollmers, Lobermeyer, Niehrs, Fittje, Indenbirken, Nakel, Virdi, Brias, Trenkner, Sauer, Peine, Behrens, Lehmann, Meurer, Pauli, Postel, Roider, Scholten, Spinner, Stephan, Wolf, Wyen, Richert, Norman, Sauter, Schmidt, Hoelzemer, Altfeld and Körner.)

Background: Respiratory syncytial virus (RSV) causes community-acquired respiratory tract infections during winter. However, outbreaks in hospitals also occur repeatedly. In particular, patients with hematologic malignancies are at an increased risk for a severe and potentially fatal course of RSV infection. Here we present the investigation of an RSV outbreak in a hematology ward for adults following the ORION statement., Methods: An epidemiologic and molecular outbreak analysis was performed. We developed and employed a minimal oligonucleotide probe set in target capture probe sequencing that allows cost-effective RSV-A or -B capturing to reconstruct RSV genomes from clinical samples., Results: Four adult patients were involved in the outbreak caused by RSV-B in March 2019. The enforcement of the pre-existing infection control measures by effective training of hospital staff contributed to a successful containment. PCR-based RSV screening on the ward enabled early detection of new cases and rapid isolation measures. The molecular analysis demonstrated that the outbreak sequences were highly related and distinct to other RSV-B strains circulating at the same time., Conclusions: A multimodal infection control concept is essential for the timely detection and control of RSV outbreaks in patients with hematological disease. Among other measures, preventive screening for respiratory viruses is recommended. Furthermore, the integration of conventional and molecular epidemiology, such as whole-genome sequencing and variant calling, significantly contributes to the understanding of transmission pathways. Based on this, appropriate conclusions can be drawn for targeted prevention measures that have prepared us for the COVID-19 pandemic beyond the RSV approach described here., (© 2022. The Author(s).)

Hospitals are one of the main reservoirs of multi-resistant Enterobacterales (MRE). As MRE are resistant to the most frequently used antibiotics, therapy for patients with MRE infections is challenging. It has been previously described that MRE from hospital wastewater can pass into municipal wastewater and even surface water. In this study, we investigated the diversity and epidemiology of MRE in the wastewater of a large tertiary care hospital. Wastewater samples were collected for a four-day period and tested for the presence of Enterobacterales resistant to 3rd gen. cephalosporins. Representative isolates were further characterized by whole genome sequencing. In 120 β-glucuronidase-producing isolates, 68 Escherichia coli and, interestingly, also 52 Citrobacter freundii were identified. In 120 β-glucosidase-producing isolates 45 Serratia marcescens, 34 Klebsiella oxytoca, 32 Enterobacter cloacae and 9 Klebsiella pneumoniae were observed. For all species various MLST sequence types and different clusters of resistance genes were determined, showing a great diversity within the different Enterobacterales, further corroborated by clonal analysis performed by cgMLST. The most prominent clone was wastewater associated E. coli ST635, which accounted for 47.1% of all E. coli isolates. Interestingly, 45.6% of E. coli, 88.5% of C. freundii, 95.6% of S. marcescens, 91.2% of K. oxytoca, 96.9% of E. cloacae and 88.9% of K. pneumoniae isolates carried a carbapenemase gene, indicating a high burden with carbapenemase-producing Enterobacterales. Comparison with clinical isolates from the same hospital displayed few clonal matches. One wastewater isolate of K. pneumoniae was identified to be closely related compared to a clone that had been introduced into the hospital during an outbreak four years earlier. One E. coli isolate was identified as identical to an isolate from a patient, with inpatient stay during the sampling period. The data obtained in this study highlight the problem of antibiotic resistance of Enterobacterales in hospital wastewater. In particular, the clustered occurrence of carbapenemase genes is of great concern and underscores the problem of increasingly scarce antibiotic options against these bacteria., (Copyright © 2022 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The human skin and in particular its outermost layer, the epidermis, protects the body from potentially harmful substances, radiation as well as excessive water loss. However, the interference between the various stress responses of the epidermal keratinocytes, which often occur simultaneously, is largely unknown. The focus of this study was to investigate the interference between osmotic stress and DNA damage response. In addition to revealing the already well-described regulation of diverse gene sets, for example, cellular processes such as transcription, translation, and metabolic pathways (e.g., the KEGG citrate cycle and Reactome G2/M checkpoints), gene expression analysis of osmotically stressed keratinocytes revealed an influence on the transcription of genes also related to UV-induced DNA damage response. A gene network regulating the H2AX phosphorylation was identified to be regulated by osmotic stress. To analyze and test the interference between osmotic stress and DNA damage response, which can be triggered by UV stress on the one hand and oxidative stress on the other, in more detail, primary human keratinocytes were cultured under osmotic stress conditions and subsequently exposed to UV light and H 2 O 2 , respectively. γH2AX measurements revealed lower γH2AX levels in cells previously cultured under osmotic stress conditions.

We describe two outbreaks of SARS-CoV-2 in daycare centers in the metropolitan area of Hamburg, Germany. The outbreaks occurred in rapid chronological succession, in neighborhoods with a very similar sociodemographic structure, thus allowing for cross-comparison of these events. We combined classical and molecular epidemiologic investigation methods to study infection entry, spread within the facilities, and subsequent transmission of infections to households. Epidemiologic and molecular evidence suggests a superspreading event with a non-variant of concern (non-VOC) SARS CoV-2 strain at the root of the first outbreak. The second outbreak involved two childcare facilities experiencing infection activity with the variant of concern (VOC) B.1.1.7 (Alpha). We show that the index cases in all outbreaks had been childcare workers, and that children contributed substantially to secondary transmission of SARS-CoV-2 infection from childcare facilities to households. The frequency of secondary transmissions in households originating from B.1.1.7-infected children was increased compared to children with non-VOC infections. Self-reported symptoms, particularly cough and rhinitis, occurred more frequently in B.1.1.7-infected children. Especially in light of the rapidly spreading VOC B.1.617.2 (Delta), our data underline the notion that rigorous SARS-CoV-2 testing in combination with screening of contacts regardless of symptoms is an important measure to prevent SARS-CoV-2 infection of unvaccinated individuals in daycare centers and associated households., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

Microbial contamination of fuels, associated with a wide variety of bacteria and fungi, leads to decreased product quality and can compromise equipment performance by biofouling or microbiologically influenced corrosion. Detection and quantification of microorganisms are critical in monitoring fuel systems for an early detection of microbial contaminations. To address these challenges, we have analyzed six metagenomes, one transcriptome, and more than 1,200 fluid and swab samples taken from fuel tanks or kerosene. Our deep metagenome sequencing and binning approaches in combination with RNA-seq data and qPCR methods implied a metabolic symbiosis between fungi and bacteria. The most abundant bacteria were affiliated with α-, β-, and γ-Proteobacteria and the filamentous fungi Amorphotheca . We identified a high number of genes, which are related to kerosene degradation and biofilm formation. Surprisingly, a large number of genes coded enzymes involved in polymer degradation and potential bio-corrosion processes. Thereby, the transcriptionally most active microorganisms were affiliated with the genera Methylobacteria, Pseudomonas, Kocuria, Amorpotheka, Aspergillus, Fusarium, and Penicillium . Many not yet cultured bacteria and fungi appeared to contribute to the biofilm transcriptional activities. The largest numbers of transcripts were observed for dehydrogenase, oxygenase, and exopolysaccharide production, attachment and pili/flagella-associated proteins, efflux pumps, and secretion systems as well as lipase and esterase activity., Competing Interests: BH, TL, CS, and RR were employed by company Lufthansa Technik AG HAM. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Krohn, Bergmann, Qi, Indenbirken, Han, Perez-Garcia, Katzowitsch, Hägele, Lübcke, Siry, Riemann, Alawi and Streit.)

Viral vectors have emerged as a promising alternative to classical vaccines due to their great potential for induction of a potent cellular and humoral immunity. Cytomegalovirus (CMV) is an attractive vaccine vector due to its large genome with many non-essential immunoregulatory genes that can be easily manipulated to modify the immune response. CMV generates a strong antigen-specific CD8 T cell response with a gradual accumulation of these cells in the process called memory inflation. In our previous work, we have constructed a mouse CMV vector expressing NKG2D ligand RAE-1γ in place of its viral inhibitor m152 (RAE-1γMCMV), which proved to be highly attenuated in vivo . Despite attenuation, RAE-1γMCMV induced a substantially stronger CD8 T cell response to vectored antigen than the control vector and provided superior protection against bacterial and tumor challenge. In the present study, we confirmed the enhanced protective capacity of RAE-1γMCMV as a tumor vaccine vector and determined the phenotypical and functional characteristics of memory CD8 T cells induced by the RAE-1γ expressing MCMV. RNAseq data revealed higher transcription of numerous genes associated with effector-like CD8 T cell phenotype in RAE-1γMCMV immunized mice. CD8 T cells primed with RAE-1γMCMV were enriched in TCF1 negative population, with higher expression of KLRG1 and lower expression of CD127, CD27, and Eomes. These phenotypical differences were associated with distinct functional features as cells primed with RAE-1γMCMV showed inferior cytokine-producing abilities but comparable cytotoxic potential. After adoptive transfer into naive hosts, OT-1 cells induced with both RAE-1γMCMV and the control vector were equally efficient in rejecting established tumors, suggesting the context of latent infection and cell numbers as important determinants of enhanced anti-tumor response following RAE-1γMCMV vaccination. Overall, our results shed new light on the phenotypical and functional distinctness of memory CD8 T cells induced with CMV vector expressing cellular ligand for the NKG2D receptor., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Šustić, Cokarić Brdovčak, Lisnić, Materljan, Juranić Lisnić, Rožmanić, Indenbirken, Hiršl, Busch, Brizić, Krmpotić and Jonjić.)

During development, gene expression is tightly controlled to facilitate the generation of the diverse cell types that form the central nervous system. Brahma-related gene 1 (Brg1, also known as Smarca4) is the catalytic subunit of the SWItch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex that regulates transcription. We investigated the role of Brg1 between embryonic day 6.5 (E6.5) and E14.5 in Sox2-positive neural stem cells (NSCs). Being without major consequences at E6.5 and E14.5, loss of Brg1 between E7.5 and E12.5 resulted in the formation of rosette-like structures in the subventricular zone, as well as morphological alterations and enlargement of neural retina (NR). Additionally, Brg1-deficient cells showed decreased survival in vitro and in vivo. Furthermore, we uncovered distinct changes in gene expression upon Brg1 loss, pointing towards impaired neuron functions, especially those involving synaptic communication and altered composition of the extracellular matrix. Comparison with mice deficient for integrase interactor 1 (Ini1, also known as Smarcb1) revealed that the enlarged NR was Brg1 specific and was not caused by a general dysfunction of the SWI/SNF complex. These results suggest a crucial role for Brg1 in NSCs during brain and eye development., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Congenital human cytomegalovirus (cHCMV) infection of the brain is associated with a wide range of neurocognitive sequelae. Using infection of newborn mice with mouse cytomegalovirus (MCMV) as a reliable model that recapitulates many aspects of cHCMV infection, including disseminated infection, CNS infection, altered neurodevelopment, and sensorineural hearing loss, we have previously shown that mitigation of inflammation prevented alterations in cerebellar development, suggesting that host inflammatory factors are key drivers of neurodevelopmental defects. Here, we show that MCMV infection causes a dramatic increase in the expression of the microglia-derived chemokines CXCL9/CXCL10, which recruit NK and ILC1 cells into the brain in a CXCR3-dependent manner. Surprisingly, brain-infiltrating innate immune cells not only were unable to control virus infection in the brain but also orchestrated pathological inflammatory responses, which lead to delays in cerebellar morphogenesis. Our results identify NK and ILC1 cells as the major mediators of immunopathology in response to virus infection in the developing CNS, which can be prevented by anti-IFN-γ antibodies., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2021 Kveštak et al.)

Aberrant expression of the transcriptional modulator and early B-cell factor 1 (EBF1) antagonist ZNF423 has been implicated in B-cell leukemogenesis, but its impact on transcriptional circuitries in lymphopoiesis has not been elucidated in a comprehensive manner. Herein, in silico analyses of multiple expression data sets on 1354 acute leukemia samples revealed a widespread presence of ZNF423 in various subtypes of acute lymphoblastic leukemia (ALL). Average expression of ZNF423 was highest in ETV6-RUNX1, B-other, and TCF3-PBX1 ALL followed by BCR-ABL, hyperdiploid ALL, and KMT2A-rearranged ALL. In a KMT2A-AFF1 pro-B ALL model, a CRISPR-Cas9-mediated genetic ablation of ZNF423 decreased cell viability and significantly prolonged survival of mice upon xenotransplantation. For the first time, we characterized the genome-wide binding pattern of ZNF423, its impact on the chromatin landscape, and differential gene activities in a B-lineage context. In general, chromatin-bound ZNF423 was associated with a depletion of activating histone marks. At the transcriptional level, EBF1-dependent transactivation was disrupted by ZNF423, whereas repressive and pioneering activities of EBF1 were not discernibly impeded. Unexpectedly, we identified an enrichment of ZNF423 at canonical EBF1-binding sites also in the absence of EBF1, which was indicative of intrinsic EBF1-independent ZNF423 activities. A genome-wide motif search at EBF1 target gene loci revealed that EBF1 and ZNF423 co-regulated genes often contain SMAD1/SMAD4-binding motifs as exemplified by the TGFB1 promoter, which was repressed by ZNF423 outcompeting EBF1 by depending on its ability to bind EBF1 consensus sites and to interact with EBF1 or SMADs. Overall, these findings underscore the wide scope of ZNF423 activities that interfere with B-cell lymphopoiesis and contribute to leukemogenesis., (© 2021 by The American Society of Hematology.)

Atypical teratoid/rhabdoid tumors (ATRTs) are very aggressive childhood malignancies of the central nervous system. The underlying genetic cause are inactivating bi-allelic mutations in SMARCB1 or (rarely) in SMARCA4. ATRT-SMARCA4 have been associated with a higher frequency of germline mutations, younger age, and an inferior prognosis in comparison to SMARCB1 mutated cases. Based on their DNA methylation profiles and transcriptomics, SMARCB1 mutated ATRTs have been divided into three distinct molecular subgroups: ATRT-TYR, ATRT-SHH, and ATRT-MYC. These subgroups differ in terms of age at diagnosis, tumor location, type of SMARCB1 alterations, and overall survival. ATRT-SMARCA4 are, however, less well understood, and it remains unknown, whether they belong to one of the described ATRT subgroups. Here, we examined 14 ATRT-SMARCA4 by global DNA methylation analyses. We show that they form a separate group segregating from SMARCB1 mutated ATRTs and from other SMARCA4-deficient tumors like small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) or SMARCA4 mutated extra-cranial malignant rhabdoid tumors. In contrast, medulloblastoma (MB) samples with heterozygous SMARCA4 mutations do not group separately, but with established MB subgroups. RNA sequencing of ATRT-SMARCA4 confirmed the clustering results based on DNA methylation profiling and displayed an absence of typical signature genes upregulated in SMARCB1 deleted ATRT. In summary, our results suggest that, in line with previous clinical observations, ATRT-SMARCA4 should be regarded as a distinct molecular subgroup.

Objectives: Investigation whether in depth characterization of virus variant patterns can be used for epidemiological analysis of the first severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection clusters in Hamburg, Germany., Methods: Metagenomic RNA-sequencing and amplicon-sequencing and subsequent variant calling in 25 respiratory samples from SARS-CoV-2 infected patients involved in the earliest infection clusters in Hamburg., Results: Amplikon sequencing and cluster analyses of these SARS-CoV-2 sequences allowed the identification of the first infection cluster and five non-related infection clusters occurring at the beginning of the viral entry of SARS-CoV-2 in the Hamburg metropolitan region. Viral genomics together with epidemiological analyses revealed that the index patient acquired the infection in northern Italy and transmitted it to two out of 134 contacts. Single nucleotide polymorphisms clearly distinguished the virus variants of the index and other clusters and allowed us to track in which sequences worldwide these mutations were first described. Minor variant analyses identified the transmission of intra-host variants in the index cluster and household clusters., Conclusions: SARS-CoV-2 variant tracing allows the identification of infection clusters and the follow up of infection chains occurring in the population. Furthermore, the follow up of minor viral variants in infection clusters can provide further resolution on transmission events indistinguishable at a consensus sequence level., (Copyright © 2020 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

We describe a multifactorial investigation of a SARS-CoV-2 outbreak in a large meat processing complex in Germany. Infection event timing, spatial, climate and ventilation conditions in the processing plant, sharing of living quarters and transport, and viral genome sequences were analyzed. Our results suggest that a single index case transmitted SARS-CoV-2 to co-workers over distances of more than 8 m, within a confined work area in which air is constantly recirculated and cooled. Viral genome sequencing shows that all cases share a set of mutations representing a novel sub-branch in the SARS-CoV-2 C20 clade. We identified the same set of mutations in samples collected in the time period between this initial infection cluster and a subsequent outbreak within the same factory, with the largest number of confirmed SARS-CoV-2 cases in a German meat processing facility reported so far. Our results indicate climate conditions, fresh air exchange rates, and airflow as factors that can promote efficient spread of SARS-CoV-2 via long distances and provide insights into possible requirements for pandemic mitigation strategies in industrial workplace settings., (© 2020 The Authors Published under the terms of the CC BY 4.0 license.)

Colorectal cancer (CRC) patients suffer from the second highest mortality among all cancer entities. In half of all CRC patients, colorectal cancer liver metastases (CRLM) can be observed. Metastatic colorectal cancer is associated with poor overall survival and limited treatment options. Even after successful surgical resection of the primary tumor, metachronous liver metastases occur in one out of eight cases. The only available curative intended treatment is hepatic resection, but metachronous CRLM frequently recur after approximately 1 year. In this study, we performed a proteome analysis of three recurrent liver metastases of a single CRC patient by mass spectrometry. Despite surgical resection of the primary CRC and adjuvant chemotherapy plus cetuximab treatment, the patient developed three metachronous CRLM which occurred consecutively after 9, 21 and 31 months. We identified a set of 1132 proteins expressed in the three metachronous CRLM, of which 481 were differentially regulated, including 81 proteins that were associated with the extracellular matrix (ECM). 56 ECM associated proteins were identified as upregulated in the third metastasis, 26 (46%) of which were previously described as negative prognostic markers in CRC, including tenascin C, nidogen 1, fibulin 1 and vitronectin. These data may reflect an ascending trend of malignancy from the first to the third metachronous colorectal cancer liver metastasis. Additionally, the results indicate different ECM phenotypes for recurrent metachronous metastasis, associated with different grades of malignancy and highlights the importance of individual analysis of molecular features in different, consecutive metastatic events in a single patient.

Merkel Cell Polyomavirus (MCPyV) is the etiological agent of the majority of Merkel Cell Carcinomas (MCC). MCPyV positive MCCs harbor integrated, defective viral genomes that constitutively express viral oncogenes. Which molecular mechanisms promote viral integration, if distinct integration patterns exist, and if integration occurs preferentially at loci with specific chromatin states is unknown. We here combined short and long-read (nanopore) next-generation sequencing and present the first high-resolution analysis of integration site structure in MCC cell lines as well as primary tumor material. We find two main types of integration site structure: Linear patterns with chromosomal breakpoints that map closely together, and complex integration loci that exhibit local amplification of genomic sequences flanking the viral DNA. Sequence analysis suggests that linear patterns are produced during viral replication by integration of defective/linear genomes into host DNA double strand breaks via non-homologous end joining, NHEJ. In contrast, our data strongly suggest that complex integration patterns are mediated by microhomology-mediated break-induced replication, MMBIR. Furthermore, we show by ChIP-Seq and RNA-Seq analysis that MCPyV preferably integrates in open chromatin and provide evidence that viral oncogene expression is driven by the viral promoter region, rather than transcription from juxtaposed host promoters. Taken together, our data explain the characteristics of MCPyV integration and may also provide a model for integration of other oncogenic DNA viruses such as papillomaviruses., Competing Interests: The authors have declared that no competing interests exist.

Nitrification is a key process for N-removal in engineered and natural environments, but recent findings of novel nitrifying microorganisms with surprising features revealed that our knowledge of this functional guild is still incomplete. Especially nitrite oxidation - the second step of nitrification - is catalyzed by a phylogenetically diverse bacterial group, and only recently bacteria of the phylum Chloroflexi have been identified as thermophilic nitrite-oxidizing bacteria (NOB). Among these, Nitrolancea hollandica was isolated from a laboratory-scale nitrifying bioreactor operated at 35°C with a high load of ammonium bicarbonate. However, its distribution remains cryptic as very few closely related environmental 16S rRNA gene sequences have been retrieved so far. In this study, we demonstrate how such thermophilic NOB can be enriched using modified mineral media inoculated with samples from a wastewater side-stream reactor operated at 39.5°C. Distinct cultivation conditions resulted in quick and reproducible high enrichment of two different strains of Nitrolancea , closely related to Nl. hollandica . The same cultivation approach was applied to a complex nitrite-oxidizing pre-enrichment at 42°C inoculated with biomass from a geothermal spring in the Copahue volcano area in Neuquen, Argentina. Here, an additional distinct representative of the genus Nitrolancea was obtained. This novel species had 16S rRNA and nitrite oxidoreductase alpha subunit ( nxrA ) gene sequence identities to Nl. hollandica of 98.5% and 97.2%, respectively. A genomic average nucleotide identity between the Argentinian strain and Nl. hollandica of 91.9% indicates that it indeed represents a distinct species. All Nitrolancea cultures formed lancet-shaped cells identical to Nl. hollandica and revealed similar physiological features, including the capability to grow at high nitrite concentrations. Growth was optimal at temperatures of 35-37°C and was strongly enhanced by ammonium supplementation. Genomic comparisons revealed that the four Nitrolancea strains share 2399 out of 3387 orthologous gene clusters and encode similar key functions. Our results define general growth conditions that enable the selective enrichment of Nitrolancea from artificial and natural environments. In most natural habitats these NOB apparently are of low abundance and their proliferation depends on the balanced presence of nitrite and ammonium, with an optimal incubation temperature of 37°C., (Copyright © 2020 Spieck, Sass, Keuter, Hirschmann, Spohn, Indenbirken, Kop, Lücker and Giaveno.)

Scenedesmus acuminatus , also known as Tetradesmus acuminatus , is a promising green microalga for sustainable production of microalga products, including valuable compounds such as astaxanthin, β-carotene, and lutein, polysaccharides such as β-glucan, and polyunsaturated fatty acids. Here, we report the draft whole-genome sequence of Scenedesmus acuminatus SAG 38.81., (Copyright © 2020 Astafyeva et al.)

Here, we describe the complete genome sequence of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain isolated from an oropharyngeal swab sample from a female patient with COVID-19 who was infected in Hamburg, northern Germany., (Copyright © 2020 Pfefferle et al.)

Germline mutations in the basic helix-loop-helix transcription factor 4 (TCF4) cause the Pitt-Hopkins syndrome (PTHS), a developmental disorder with severe intellectual disability. Here, we report findings from a new mouse model with a central nervous system-specific truncation of Tcf4 leading to severe phenotypic abnormalities. Furthermore, it allows the study of a complete TCF4 knockout in adult mice, circumventing early postnatal lethality of previously published mouse models. Our data suggest that a TCF4 truncation results in an impaired hippocampal architecture affecting both the dentate gyrus as well as the cornu ammonis. In the cerebral cortex, loss of TCF4 generates a severe differentiation delay of neural precursors. Furthermore, neuronal morphology was critically affected with shortened apical dendrites and significantly increased branching of dendrites. Our data provide novel information about the role of Tcf4 in brain development and may help to understand the mechanisms leading to intellectual deficits observed in patients suffering from PTHS., (© 2020 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Merkel cell polyomavirus (MCPyV) is the only polyomavirus known to be associated with tumorigenesis in humans. Similarly to other polyomaviruses, MCPyV expresses a l arge t umor antigen (LT-Ag) that, together with a s mall t umor antigen (sT-Ag), contributes to cellular transformation and that is of critical importance for the initiation of the viral DNA replication. Understanding the cellular protein network regulated by MCPyV early proteins will significantly contribute to our understanding of the natural MCPyV life cycle as well as of the mechanisms by which the virus contributes to cellular transformation. We here describe KRAB-associated protein 1 (Kap1), a chromatin remodeling factor involved in cotranscriptional regulation, as a novel protein interaction partner of MCPyV T antigens sT and LT. Kap1 knockout results in a significant increase in the level of viral DNA replication that is highly suggestive of Kap1 being an important host restriction factor during MCPyV infection. Differently from other DNA viruses, MCPyV gene expression is unaffected in the absence of Kap1 and Kap1 does not associate with the viral genome. Instead, we show that in primary normal human dermal fibroblast (nHDF) cells, MCPyV DNA replication, but not T antigen expression alone, induces ataxia telangiectasia mutated (ATM) kinase-dependent Kap1 S824 phosphorylation, a mechanism that typically facilitates repair of double-strand breaks in heterochromatin by arresting the cells in G 2 We show that MCPyV-induced inhibition of cell proliferation is mainly conferred by residues within the origin binding domain and thereby by viral DNA replication. Our data suggest that phosphorylation of Kap1 and subsequent Kap1-dependent G 2 arrest/senescence represent host defense mechanisms against MCPyV replication in nHDF cells. IMPORTANCE We here describe Kap1 as a restriction factor in MCPyV infection. We report a novel, indirect mechanism by which Kap1 affects MCPyV replication. In contrast with from other DNA viruses, Kap1 does not associate with the viral genome in MCPyV infection and has no impact on viral gene expression. In MCPyV-infected nHDF cells, Kap1 phosphorylation (pKap1 S824) accumulates because of genomic stress mainly induced by viral DNA replication. In contrast, ectopic expression of LT or LT MCPyV mutants, previously shown to be important for induction of genotoxic stress, does not result in a similar extent of pKap1 accumulation. We show that cells actively replicating MCPyV accumulate pKap1 (in a manner dependent on the presence of ATM) and display a senescence phenotype reflected by G 2 arrest. These results are supported by transcriptome analyses showing that LT antigen, in a manner dependent on the presence of Kap1, induces expression of secreted factors, which is known as the senescence-associated secretory phenotype (SASP)., (Copyright © 2020 Siebels et al.)

Nitrifying microorganisms occur across a wide temperature range from 4 to 84 °C and previous studies in geothermal systems revealed their activity under extreme conditions. Archaea were detected to be responsible for the first step of nitrification, but it is still a challenging issue to clarify the identity of heat-tolerant nitrite oxidizers. In a long-term cultivation approach, we inoculated mineral media containing ammonium and nitrite as substrates with biofilms and sediments of two hot springs in Yellowstone National Park (USA). The nitrifying consortia obtained at 70 °C consisted mostly of novel Chloroflexi as revealed by metagenomic sequencing. Among these, two deep-branching novel Chloroflexi were identified as putative nitrite-oxidizing bacteria (NOB) by the presence of nitrite oxidoreductase encoding genes in their genomes. Stoichiometric oxidation of nitrite to nitrate occurred under lithoautotrophic conditions, but was stimulated by organic matter. Both NOB candidates survived long periods of starvation and the more abundant one formed miniaturized cells and was heat resistant. This detection of novel thermophilic NOB exemplifies our still incomplete knowledge of nitrification, and indicates that nitrite oxidation might be an ancient and wide-spread form of energy conservation.

CREB (cyclic AMP response element binding protein) binding protein (CBP, CREBBP) is a ubiquitously expressed transcription coactivator with intrinsic histone acetyltransferase (KAT) activity. Germline mutations within the CBP gene are known to cause Rubinstein-Taybi syndrome (RSTS), a developmental disorder characterized by intellectual disability, specific facial features and physical anomalies. Here, we investigate mechanisms of CBP function during brain development in order to elucidate morphological and functional mechanisms underlying the development of RSTS. Due to the embryonic lethality of conventional CBP knockout mice, we employed a tissue specific knockout mouse model (hGFAP-cre::CBP Fl/Fl , mutant mouse) to achieve a homozygous deletion of CBP in neural precursor cells of the central nervous system.Our findings suggest that CBP plays a central role in brain size regulation, correct neural cell differentiation and neural precursor cell migration. We provide evidence that CBP is both important for stem cell viability within the ventricular germinal zone during embryonic development and for unhindered establishment of adult neurogenesis. Prominent histological findings in adult animals include a significantly smaller hippocampus with fewer neural stem cells. In the subventricular zone, we observe large cell aggregations at the beginning of the rostral migratory stream due to a migration deficit caused by impaired attraction from the CBP-deficient olfactory bulb. The cerebral cortex of mutant mice is characterized by a shorter dendrite length, a diminished spine number, and a relatively decreased number of mature spines as well as a reduced number of synapses.In conclusion, we provide evidence that CBP is important for neurogenesis, shaping neuronal morphology, neural connectivity and that it is involved in neuronal cell migration. These findings may help to understand the molecular basis of intellectual disability in RSTS patients and may be employed to establish treatment options to improve patients' quality of life.

B-cell precursor (BCP) ALL carry a variety of classical V(D)J rearrangements as well as genomic fusions and translocations. Here, we assessed the value of genomic capture high-throughput sequencing (gc-HTS) in BCP ALL (n = 183) for the identification and implementation of targets for minimal residual disease (MRD) testing. For TRδ, a total of 300 clonal rearrangements were detected in 158 of 183 samples (86%). Beside clonal Vδ2-Dδ3, Dδ2-Dδ3, and Vδ2-Jα we identified a novel group of recurrent Dδ-Jα rearrangements, comprising Dδ2 or Dδ3 segments fused predominantly to Jα29. For IGH-JH, 329 clonal rearrangements were identified in 172 of 183 samples (94%) including novel types of V(D)J joining. Oligoclonality was found in ~1/3 (n = 57/183) of ALL samples. Genomic breakpoints were identified in 71 BCP-ALL. A distinct MRD high-risk subgroup of IGH-V(D)J-germline ALL revealed frequent deletions of IKZF1 (n = 7/11) and the presence of genomic fusions (n = 10/11). Quantitative measurement using genomic fusion breakpoints achieved equivalent results compared to conventional V(D)J-based MRD testing and could be advantageous upon persistence of a leukemic clone. Taken together, selective gc-HTS expands the spectrum of suitable MRD targets and allows for the identification of genomic fusions relevant to risk and treatment stratification in childhood ALL.

We describe DAMIAN, an open source bioinformatics tool designed for the identification of pathogenic microorganisms in diagnostic samples. By using authentic clinical samples and comparing our results to those from established analysis pipelines as well as conventional diagnostics, we demonstrate that DAMIAN rapidly identifies pathogens in different diagnostic entities, and accurately classifies viral agents down to the strain level. We furthermore show that DAMIAN is able to assemble full-length viral genomes even in samples co-infected with multiple virus strains, an ability which is of considerable advantage for the investigation of outbreak scenarios. While DAMIAN, similar to other pipelines, analyzes single samples to perform classification of sequences according to their likely taxonomic origin, it also includes a tool for cohort-based analysis. This tool uses cross-sample comparisons to identify sequence signatures that are frequently present in a sample group of interest (e.g., a disease-associated cohort), but occur less frequently in control cohorts. As this approach does not require homology searches in databases, it principally allows the identification of not only known, but also completely novel pathogens. Using samples from a meningitis outbreak, we demonstrate the feasibility of this approach in identifying enterovirus as the causative agent.

The microbial community composition and its functionality was assessed for hydrothermal fluids and volcanic ash sediments from Haungaroa and hydrothermal fluids from the Brothers volcano in the Kermadec island arc (New Zealand). The Haungaroa volcanic ash sediments were dominated by epsilonproteobacterial Sulfurovum sp. Ratios of electron donor consumption to CO 2 fixation from respective sediment incubations indicated that sulfide oxidation appeared to fuel autotrophic CO 2 fixation, coinciding with thermodynamic estimates predicting sulfide oxidation as the major energy source in the environment. Transcript analyses with the sulfide-supplemented sediment slurries demonstrated that Sulfurovum prevailed in the experiments as well. Hence, our sediment incubations appeared to simulate environmental conditions well suggesting that sulfide oxidation catalyzed by Sulfurovum members drive biomass synthesis in the volcanic ash sediments. For the Haungaroa fluids no inorganic electron donor and responsible microorganisms could be identified that clearly stimulated autotrophic CO 2 fixation. In the Brothers hydrothermal fluids Sulfurimonas (49%) and Hydrogenovibrio/Thiomicrospira (15%) species prevailed. Respective fluid incubations exhibited highest autotrophic CO 2 fixation if supplemented with iron(II) or hydrogen. Likewise catabolic energy calculations predicted primarily iron(II) but also hydrogen oxidation as major energy sources in the natural fluids. According to transcript analyses with material from the incubation experiments Thiomicrospira/Hydrogenovibrio species dominated, outcompeting Sulfurimonas . Given that experimental conditions likely only simulated environmental conditions that cause Thiomicrospira/Hydrogenovibrio but not Sulfurimonas to thrive, it remains unclear which environmental parameters determine Sulfurimonas' dominance in the Brothers natural hydrothermal fluids., (Copyright © 2019 Böhnke, Sass, Gonnella, Diehl, Kleint, Bach, Zitoun, Koschinsky, Indenbirken, Sander, Kurtz and Perner.)

The proliferative darkening syndrome (PDS) is a lethal disease of brown trout ( Salmo trutta fario ) which occurs in several alpine Bavarian limestone rivers. Because mortality can reach 100%, PDS is a serious threat for affected fish populations. Recently, Kuehn and colleagues reported that a high throughput RNA sequencing approach identified a piscine orthoreovirus (PRV) as a causative agent of PDS. We investigated samples from PDS-affected fish obtained from two exposure experiments performed at the river Iller in 2008 and 2009. Using a RT-qPCR and a well-established next-generation RNA sequencing pipeline for pathogen detection, PRV-specific RNA was not detectable in PDS fish from 2009. In contrast, PRV RNA was readily detectable in several organs from diseased fish in 2008. However, similar virus loads were detectable in the control fish which were not exposed to Iller water and did not show any signs of the disease. Therefore, we conclude that PRV is not the causative agent of PDS of brown trout in the rhithral region of alpine Bavarian limestone rivers. The abovementioned study by Kuehn used only samples from the exposure experiment from 2008 and detected a subclinical PRV bystander infection. Work is ongoing to identify the causative agent of PDS.

The RNA-binding protein TDP-43 is heavily implicated in neurodegenerative disease. Numerous patient mutations in TARDBP, the gene encoding TDP-43, combined with data from animal and cell-based models, imply that altered RNA regulation by TDP-43 causes Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. However, underlying mechanisms remain unresolved. Increased cytoplasmic TDP-43 levels in diseased neurons suggest a possible role in this cellular compartment. Here, we examined the impact on translation of overexpressing human TDP-43 and the TDP-43A315T patient mutant protein in motor neuron-like cells and primary cultures of cortical neurons. In motor-neuron like cells, TDP-43 associates with ribosomes without significantly affecting global translation. However, ribosome profiling and additional assays revealed enhanced translation and direct binding of Camta1, Mig12, and Dennd4a mRNAs. Overexpressing either wild-type TDP-43 or TDP-43A315T stimulated translation of Camta1 and Mig12 mRNAs via their 5'UTRs and increased CAMTA1 and MIG12 protein levels. In contrast, translational enhancement of Dennd4a mRNA required a specific 3'UTR region and was specifically observed with the TDP-43A315T patient mutant allele. Our data reveal that TDP-43 can function as an mRNA-specific translational enhancer. Moreover, since CAMTA1 and DENND4A are linked to neurodegeneration, they suggest that this function could contribute to disease.

Infections of fungi by mycoviruses are often symptomless but sometimes also fatal, as they perturb sporulation, growth, and, if applicable, virulence of the fungal host. Hypovirulence-inducing mycoviruses, therefore, represent a powerful means to defeat fungal epidemics on crop plants. Infection with Fusarium graminearum virus China 9 (FgV-ch9), a double-stranded RNA (dsRNA) chrysovirus-like mycovirus, debilitates Fusarium graminearum , the causal agent of fusarium head blight. In search for potential symptom alleviation or aggravation factors in F. graminearum , we consecutively infected a custom-made F. graminearum mutant collection with FgV-ch9 and found a mutant with constantly elevated expression of a gene coding for a putative mRNA-binding protein that did not show any disease symptoms despite harboring large amounts of virus. Deletion of this gene, named virus response 1 ( vr1 ), resulted in phenotypes identical to those observed in the virus-infected wild type with respect to growth, reproduction, and virulence. Similarly, the viral structural protein coded on segment 3 (P3) caused virus infection-like symptoms when expressed in the wild type but not in the vr1 overexpression mutant. Gene expression analysis revealed a drastic downregulation of vr1 in the presence of virus and in mutants expressing P3. We conclude that symptom development and severity correlate with gene expression levels of vr1 This was confirmed by comparative transcriptome analysis, showing a large transcriptional overlap between the virus-infected wild type, the vr1 deletion mutant, and the P3-expressing mutant. Hence, vr1 represents a fundamental host factor for the expression of virus-related symptoms and helps us understand the underlying mechanism of hypovirulence. IMPORTANCE Virus infections of phytopathogenic fungi occasionally impair growth, reproduction, and virulence, a phenomenon referred to as hypovirulence. Hypovirulence-inducing mycoviruses, therefore, represent a powerful means to defeat fungal epidemics on crop plants. However, the poor understanding of the molecular basis of hypovirulence induction limits their application. Using the devastating fungal pathogen on cereal crops, Fusarium graminearum , we identified an mRNA binding protein (named virus response 1, vr1 ) which is involved in symptom expression. Downregulation of vr1 in the virus-infected fungus and vr1 deletion evoke virus infection-like symptoms, while constitutive expression overrules the cytopathic effects of the virus infection. Intriguingly, the presence of a specific viral structural protein is sufficient to trigger the fungal response, i.e., vr1 downregulation, and symptom development similar to virus infection. The advancements in understanding fungal infection and response may aid biological pest control approaches using mycoviruses or viral proteins to prevent future Fusarium epidemics., (Copyright © 2018 American Society for Microbiology.)

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.

Bromodomain-containing protein 4 (BRD4) is a member of the bromo- and extraterminal (BET) domain-containing family of epigenetic readers which is under intensive investigation as a target for anti-tumor therapy. BRD4 plays a central role in promoting the expression of select subsets of genes including many driven by oncogenic transcription factors and signaling pathways. However, the role of BRD4 and the effects of BET inhibitors in non-transformed cells remain mostly unclear. We demonstrate that BRD4 is required for the maintenance of a basal epithelial phenotype by regulating the expression of epithelial-specific genes including TP63 and Grainy Head-like transcription factor-3 (GRHL3) in non-transformed basal-like mammary epithelial cells. Moreover, BRD4 occupancy correlates with enhancer activity and enhancer RNA (eRNA) transcription. Motif analyses of cell context-specific BRD4-enriched regions predicted the involvement of FOXO transcription factors. Consistently, activation of FOXO1 function via inhibition of EGFR-AKT signaling promoted the expression of TP63 and GRHL3. Moreover, activation of Src kinase signaling and FOXO1 inhibition decreased the expression of FOXO/BRD4 target genes. Together, our findings support a function for BRD4 in promoting basal mammary cell epithelial differentiation, at least in part, by regulating FOXO factor function on enhancers to activate TP63 and GRHL3 expression., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Pregnant women are at high risk for severe influenza disease outcomes, yet insights into the underlying mechanisms are limited. Here, we present models of H1N1 infection in syngenic and allogenic pregnant mice; infection in the latter mirrors the severe course of 2009 pandemic influenza in pregnant women. We found that the anti-viral immune response in the pregnant host was significantly restricted as compared to the non-pregnant host. This included a reduced type I interferon response as well as impaired migration of CD8 + T cells into the lung. The multi-faceted failure to mount an anti-viral response in allogenic pregnant mice resulted in a less stringent selective environment that promoted the emergence of 2009 H1N1 virus variants that specifically counteract type I interferon response and mediate increased viral pathogenicity. These insights underscore the importance of influenza vaccination compliance in pregnant women and may open novel therapeutic avenues., (Copyright © 2017 Elsevier Inc. All rights reserved.)