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Displacement loops (D-loops) are signature intermediates formed during homologous recombination. Numerous factors regulate D-loop formation and disruption, thereby influencing crucial aspects of DNA repair, including donor choice and the possibility of crossover outcome. While D-loop detection methods exist, it is currently unfeasible to assess the relationship between D-loop editors and D-loop characteristics such as length and position. Here, we developed a novel in vitro assay to characterize the length and position of individual D-loops with near base-pair resolution and deep coverage, while also revealing their distribution in a population. Non-denaturing bisulfite treatment modifies the cytosines on the displaced strand of the D-loop to uracil, leaving a permanent signature for the displaced strand. Subsequent single-molecule real-time sequencing uncovers the cytosine conversion patch as a D-loop footprint. The D-loop Mapping Assay is widely applicable with different substrates and donor types and can be used to study factors that influence D-loop properties., Competing Interests: SS, SH, FC No competing interests declared, WH Reviewing editor, eLife, (© 2020, Shah et al.)

We have made available a database of over 1 billion compounds predicted to be easily synthesizable, called Synthetically Accessible Virtual Inventory (SAVI). They have been created by a set of transforms based on an adaptation and extension of the CHMTRN/PATRAN programming languages describing chemical synthesis expert knowledge, which originally stem from the LHASA project. The chemoinformatics toolkit CACTVS was used to apply a total of 53 transforms to about 150,000 readily available building blocks (enamine.net). Only single-step, two-reactant syntheses were calculated for this database even though the technology can execute multi-step reactions. The possibility to incorporate scoring systems in CHMTRN allowed us to subdivide the database of 1.75 billion compounds in sets according to their predicted synthesizability, with the most-synthesizable class comprising 1.09 billion synthetic products. Properties calculated for all SAVI products show that the database should be well-suited for drug discovery. It is being made publicly available for free download from https://doi.org/10.35115/37n9-5738.

Coronaviruses (CoVs) are members of the genus Betacoronavirus and the Coronaviridiae family responsible for infections such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and more recently, coronavirus disease-2019 (COVID-19). CoV infections present mainly as respiratory infections that lead to acute respiratory distress syndrome (ARDS). However, CoVs, such as COVID-19, also present as a hyperactivation of the inflammatory response that results in increased production of inflammatory cytokines such as interleukin (IL)-1β and its downstream molecule IL-6. The inflammasome is a multiprotein complex involved in the activation of caspase-1 that leads to the activation of IL-1β in a variety of diseases and infections such as CoV infection and in different tissues such as lungs, brain, intestines and kidneys, all of which have been shown to be affected in COVID-19 patients. Here we review the literature regarding the mechanism of inflammasome activation by CoV infection, the role of the inflammasome in ARDS, ventilator-induced lung injury (VILI), and Disseminated Intravascular Coagulation (DIC) as well as the potential mechanism by which the inflammasome may contribute to the damaging effects of inflammation in the cardiac, renal, digestive, and nervous systems in COVID-19 patients., (Copyright © 2020 de Rivero Vaccari, Dietrich, Keane and de Rivero Vaccari.)

Despite the shift in the demographics of traumatic spinal cord injury (SCI) with increased proportion of injuries in the elderly, little is known on the potential effects of old age on the pathobiology of SCI. Since there is an assumption that age adversely affects neural response to SCI, this study examines the clinically relevant question on whether age is a key determinant of inflammatory response, oligodendroglial apoptosis and axonal survival after traumatic SCI. This unique study includes post-mortem spinal cord tissue from 64 cases of SCI (at cervical or high-thoracic levels) and 38 control cases without CNS injury. Each group was subdivided into subgroups of younger and elderly individuals (65 years of age or older at the SCI onset). The results of this study indicate that age at the SCI onset does not adversely affect the cellular inflammatory response to, oligodendroglial apoptosis and axonal survival after SCI. These results support the conclusion that elderly individuals have similar neurobiological responses to SCI as younger people and, hence, treatment decisions should be based on an assessment of the individual patient and not an arbitrary assumption that "advanced age" should exclude patients with an acute SCI from access to advanced care and translational therapies., Competing Interests: Declaration of Competing Interest The authors declare no competing interests to disclose., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Food has a major impact on all aspects of health. Recent data suggest that food composition can also affect susceptibility to infections by enteropathogenic bacteria. Here, we discuss how food may alter the microbiota as well as mucosal defenses and how this can affect infection. Salmonella Typhimurium diarrhea serves as a paradigm, and complementary evidence comes from other pathogens. We discuss the effects of food composition on colonization resistance, host defenses, and the infection process as well as the merits and limitations of mouse models and experimental foods, which are available to decipher the underlying mechanisms.

The interplay between host and pathogen relies heavily on rapid protein synthesis and accurate protein targeting to ensure pathogen destruction. To gain insight into this dynamic interface, we combined Click chemistry with pulsed stable isotope labelling of amino acids in cell culture to quantify the host proteome response during macrophage infection with the intracellular bacterial pathogen Salmonella enterica Typhimurium. We monitored newly synthesized proteins across different host cell compartments and infection stages. Within this rich resource, we detected aberrant trafficking of lysosomal proteases to the extracellular space and the nucleus. We verified that active cathepsins re-traffic to the nucleus and that these are linked to cell death. Pharmacological cathepsin inhibition and nuclear targeting of a cellular cathepsin inhibitor (stefin B) suppressed S. enterica Typhimurium-induced cell death. We demonstrate that cathepsin activity is required for pyroptotic cell death via the non-canonical inflammasome, and that lipopolysaccharide transfection into the host cytoplasm is sufficient to trigger active cathepsin accumulation in the host nucleus and cathepsin-dependent cell death. Finally, cathepsin inhibition reduced gasdermin D expression, thus revealing an unexpected role for cathepsin activity in non-canonical inflammasome regulation. Overall, our study illustrates how resolution of host proteome dynamics during infection can drive the discovery of biological mechanisms at the host-microbe interface.

Antibiotic treatment failure is of growing concern. Genetically encoded resistance is key in driving this process. However, there is increasing evidence that bacterial antibiotic persistence, a non-genetically encoded and reversible loss of antibiotic susceptibility, contributes to treatment failure and emergence of resistant strains as well. In this Review, we discuss the evolutionary forces that may drive the selection for antibiotic persistence. We review how some aspects of antibiotic persistence have been directly selected for whereas others result from indirect selection in disparate ecological contexts. We then discuss the consequences of antibiotic persistence on pathogen evolution. Persisters can facilitate the evolution of antibiotic resistance and virulence. Finally, we propose practical means to prevent persister formation and how this may help to slow down the evolution of virulence and resistance in pathogens.

The tumor suppressor BRCA2 plays a key role in initiating homologous recombination by facilitating RAD51 filament formation on single-stranded DNA. The small acidic protein DSS1 is a crucial partner to BRCA2 in this process. In vitro and in cells (1,2), BRCA2 associates into oligomeric complexes besides also existing as monomers. A dimeric structure was further characterized by electron microscopic analysis (3), but the functional significance of the different BRCA2 assemblies remains to be determined. Here, we used biochemistry and electron microscopic imaging to demonstrate that the multimerization of BRCA2 is counteracted by DSS1 and ssDNA. When validating the findings, we identified three self-interacting regions and two types of self-association, the N-to-C terminal and the N-to-N terminal interactions. The N-to-C terminal self-interaction of BRCA2 is sensitive to DSS1 and ssDNA. The N-to-N terminal self-interaction is modulated by ssDNA. Our results define a novel role of DSS1 to regulate BRCA2 in an RPA-independent fashion. Since DSS1 is required for BRCA2 function in recombination, we speculate that the monomeric and oligomeric forms of BRCA2 might be active for different cellular events in recombinational DNA repair and replication fork stabilization., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

We have adopted and extended the CHMTRN language and used it for the knowledge base of a computer program to generate a large database of synthetically accessible, drug-like chemical structures, the Synthetically Accessible Virtual Inventory (SAVI) Database. CHMTRN is a powerful language originally developed in the LHASA (Logic and Heuristics Applied to Synthetic Analysis) project at Harvard University and used together with the chemical pattern description language, PATRAN, to describe chemical retro-reactions. The languages have proven to be useful beyond the design of retrosynthetic routes and have the potential for much wider use in chemistry; this paper describes CHMTRN and PATRAN as now reimplemented for the forward-synthetic SAVI project but able to describe both forward and retro-reactions.

The immunoglobulin-like domain containing receptor 2 (ILDR2), a type I transmembrane protein belonging to the B7 family of immunomodulatory receptors, has been described to induce an immunosuppressive effect on T-cell responses. Besides its expression in several nonlymphoid tissue types, we found that ILDR2 was also expressed in fibroblastic reticular cells (FRC) in the stromal part of the lymph node. These immunoregulatory cells were located in the T-cell zone and were essential for the recruitment of naïve T cells and activated dendritic cells to the lymph nodes. Previously, it has been shown that an ILDR2-Fc fusion protein exhibits immunomodulatory effects in several models of autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, and type I diabetes. Herein, we report the generation and characterization of a human/mouse/monkey cross-reactive anti-ILDR2 hIgG2 antibody, BAY 1905254, developed to block the immunosuppressive activity of ILDR2 for cancer immunotherapy. BAY 1905254 was shown to promote T-cell activation in vitro and enhance antigen-specific T-cell proliferation and cytotoxicity in vivo in mice. BAY 1905254 also showed potent efficacy in various syngeneic mouse cancer models, and the efficacy was found to correlate with increasing mutational load in the cancer models used. Additive or even synergistic antitumor effects were observed when BAY 1905254 was administered in combination with anti-PD-L1, an immunogenic cell death-inducing chemotherapeutic, or with tumor antigen immunization. Taken together, our data showed that BAY 1905254 is a potential drug candidate for cancer immunotherapy, supporting its further evaluation., (©2020 American Association for Cancer Research.)

Traumatic brain injury (TBI) is a leading cause of death and disability yet treatment strategies remain elusive. Advances in machine learning present exciting opportunities for developing personalized medicine and informing laboratory research. However, their feasibility has yet to be widely assessed in animal research where data are typically limited or in the TBI field where each patient presents with a unique injury. The Operation Brain Trauma Therapy (OBTT) has amassed an animal dataset that spans multiple types of injury, treatment strategies, behavioral assessments, histological measures, and biomarker screenings. This paper aims to analyze these data using supervised learning techniques for the first time by partitioning the dataset into acute input metrics (i.e. 7 days post-injury) and a defined recovery outcome (i.e. memory retention). Preprocessing is then applied to transform the raw OBTT dataset, e.g. developing a class attribute by histogram binning, eliminating borderline cases, and applying principal component analysis (PCA). We find that these steps are also useful in establishing a treatment ranking; Minocycline, a therapy with no significant findings in the OBTT analyses, yields the highest percentage recovery in our ranking. Furthermore, of the seven classifiers we have evaluated, Naïve Bayes achieves the best performance (67%) and yields significant improvement over our baseline model on the preprocessed dataset with borderline elimination. We also investigate the effect of testing on individual treatment groups to evaluate which groups are difficult to classify, and note the interpretive qualities of our model that can be clinically relevant.Clinical Relevance- These studies establish methods for better analyzing multivariate functional recovery and understanding which measures affect prognosis following traumatic brain injury.

Mild cognitive impairment (MCI) is characterized by memory loss in the absence of dementia and is considered the translational stage between normal aging and early Alzheimer's disease (AD). Patients with MCI have a greater risk of advancing to AD. Thus, identifying early markers of MCI has the potential to increase the therapeutic window to treat and manage the disease. Protein levels of the inflammasome signaling proteins apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and interleukin (IL)-18 were analyzed in the serum of patients with MCI, AD and healthy age-matched donors as possible biomarkers, as well as levels of soluble amyloid precursor proteins α/β (sAPP α/β) and neurofilament light (NfL). Cut-off points and positive and negative predictive values, as well as receiver operator characteristic (ROC) curves, likelihood ratios and accuracy were determined for these proteins. Although the levels of ASC were higher in MCI and AD than in age-matched controls, protein levels of ASC were higher in MCI than in AD cases. For control vs. MCI, the area under the curve (AUC) for ASC was 0.974, with a cut-off point of 264.9 pg/mL. These data were comparable to the AUC for sAPP α and β of 0.9687 and 0.9068, respectively, as well as 0.7734 for NfL. Moreover, similar results were obtained for control vs. AD and MCI vs. AD. These results indicate that ASC is a promising biomarker of MCI and AD., Competing Interests: J.P.d.R.V., R.W.K. and W.D.D. are cofounders and managing members of InflamaCORE, LLC and have licensed patents on inflammasome proteins as biomarkers of injury and disease, as well as on targeting inflammasome proteins for therapeutic purposes. J.P.d.R.V., R.W.K. and W.D.D. are Scientific Advisory Board Members of ZyVersa Therapeutics. All other authors declare that there are no conflicts of interest. Data presented in this manuscript is protected under US Patent Application: 62/696,549 (Method for Detecting Inflammasome Proteins as Biomarkers of Neurological Disorders).

Initial enteropathogen growth in the microbiota-colonized gut is poorly understood. Salmonella Typhimurium is metabolically adaptable and can harvest energy by anaerobic respiration using microbiota-derived hydrogen (H 2 ) as an electron donor and fumarate as an electron acceptor. As fumarate is scarce in the gut, the source of this electron acceptor is unclear. Here, transposon sequencing analysis along the colonization trajectory of S. Typhimurium implicates the C4-dicarboxylate antiporter DcuABC in early murine gut colonization. In competitive colonization assays, DcuABC and enzymes that convert the C4-dicarboxylates aspartate and malate into fumarate (AspA, FumABC), are required for fumarate/H 2 -dependent initial growth. Thus, S. Typhimurium obtains fumarate by DcuABC-mediated import and conversion of L-malate and L-aspartate. Fumarate reduction yields succinate, which is exported by DcuABC in exchange for L-aspartate and L-malate. This cycle allows S. Typhimurium to harvest energy by H 2 /fumarate respiration in the microbiota-colonized gut. This strategy may also be relevant for commensal E. coli diminishing the S. Typhimurium infection., Competing Interests: Declaration of Interests The authors have no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Intestinal epithelial organoids established from gut tissue have become a widely used research tool. However, it remains unclear how environmental cues, divergent microbiota composition and other sources of variation before, during and after establishment confound organoid properties, and how these properties relate to the original tissue. While environmental influences cannot be easily addressed in human organoids, mice offer a controlled assay-system. Here, we probed the effect of donor microbiota differences, previously identified as a confounding factor in murine in vivo studies, on organoids. We analysed the proteomes and transcriptomes of primary organoid cultures established from two colonised and one germ-free mouse colony of C57BL/6J genetic background, and compared them to their tissue of origin and commonly used cell lines. While an imprint of microbiota-exposure was observed on the proteome of epithelial samples, the long-term global impact of donor microbiota on organoid expression patterns was negligible. Instead, stochastic culture-to-culture differences accounted for a moderate variability between independently established organoids. Integration of transcriptome and proteome datasets revealed an organoid-typic expression signature comprising 14 transcripts and 10 proteins that distinguished organoids across all donors from murine epithelial cell lines and fibroblasts and closely mimicked expression patterns in the gut epithelium. This included the inflammasome components ASC, Naip1-6, Nlrc4 and Caspase-1, which were highly expressed in all organoids compared to the reference cell line m-IC c12 or mouse embryonic fibroblasts. Taken together, these results reveal that the donor microbiota has little effect on the organoid phenotype and suggest that organoids represent a more suitable culture model than immortalised cell lines, in particular for studies of intestinal epithelial inflammasomes., (© 2020 The Authors. Cellular Microbiology published by John Wiley & Sons Ltd.)

Alternative lengthening of telomeres (ALT) in human cells is a conserved process that is often activated in telomerase-deficient human cancers. This process exploits components of the recombination machinery to extend telomere ends, thus allowing for increased proliferative potential. Human MUS81 (Mus81 in Saccharomyces cerevisiae) is the catalytic subunit of structure-selective endonucleases involved in recombination and has been implicated in the ALT mechanism. However, it is unclear whether MUS81 activity at the telomere is specific to ALT cells or if it is required for more general aspects of telomere stability. In this study, we use S. cerevisiae to evaluate the contribution of the conserved Mus81-Mms4 endonuclease in telomerase-deficient yeast cells that maintain their telomeres by mechanisms akin to human ALT. Similar to human cells, we find that yeast Mus81 readily localizes to telomeres and its activity is important for viability after initial loss of telomerase. Interestingly, our analysis reveals that yeast Mus81 is not required for the survival of cells undergoing recombination-mediated telomere lengthening, i.e. for ALT itself. Rather we infer from genetic analysis that Mus81-Mms4 facilitates telomere replication during times of telomere instability. Furthermore, combining mus81 mutants with mutants of a yeast telomere replication factor, Rrm3, reveals that the two proteins function in parallel to promote normal growth during times of telomere stress. Combined with previous reports, our data can be interpreted in a consistent model in which both yeast and human MUS81-dependent nucleases participate in the recovery of stalled replication forks within telomeric DNA. Furthermore, this process becomes crucial under conditions of additional replication stress, such as telomere replication in telomerase-deficient cells., Competing Interests: The authors have declared that no competing interests exist.

Salmonella enterica serovar Typhimurium (S.Tm) infections of cultured cell lines have given rise to the ruffle model for epithelial cell invasion. According to this model, the Type-Three-Secretion-System-1 (TTSS-1) effectors SopB, SopE and SopE2 drive an explosive actin nucleation cascade, resulting in large lamellipodia- and filopodia-containing ruffles and cooperative S.Tm uptake. However, cell line experiments poorly recapitulate many of the cell and tissue features encountered in the host's gut mucosa. Here, we employed bacterial genetics and multiple imaging modalities to compare S.Tm invasion of cultured epithelial cell lines and the gut absorptive epithelium in vivo in mice. In contrast to the prevailing ruffle-model, we find that absorptive epithelial cell entry in the mouse gut occurs through "discreet-invasion". This distinct entry mode requires the conserved TTSS-1 effector SipA, involves modest elongation of local microvilli in the absence of expansive ruffles, and does not favor cooperative invasion. Discreet-invasion preferentially targets apicolateral hot spots at cell-cell junctions and shows strong dependence on local cell neighborhood. This proof-of-principle evidence challenges the current model for how S.Tm can enter gut absorptive epithelial cells in their intact in vivo context., Competing Interests: The authors have declared that no competing interests exist.

Background: The inflammasome adaptor apoptosis-associated speck-like protein containing a CARD (ASC) is involved in immune signaling by bridging the interactions between inflammasome sensors and caspase-1. Strong experimental evidence has shown that ASC -/- mice are protected from disease progression in animal models of multiple sclerosis (MS), suggesting that targeting inflammasome activation via ASC inhibition may be a promising therapeutic strategy in MS. Thus, the goal of our study is to test the efficacy of IC100, a novel humanized antibody targeting ASC, in preventing and/or suppressing disease in the experimental autoimmune encephalomyelitis (EAE) model of MS., Methods: We employed the EAE model of MS where disease was induced by immunization of C57BL/6 mice with myelin oligodendrocyte glycoprotein peptide 35-55 (MOG 35-55 ). Mice were treated with vehicle or increasing doses of IC100 (10, 30, and 45 mg/kg) and clinical disease course was evaluated up to 35 days post EAE induction. Immune cell infiltration into the spinal cord and microglia responses were assessed., Results: We show that IC100 treatment reduced the severity of EAE when compared to vehicle-treated controls. At a dose of 30 mg/kg, IC100 significantly reduced the number of CD4 + and CD8 + T cells and CD11b + MHCII + activated myeloid cells entering the spinal cord from the periphery, and reduced the number of total and activated microglia., Conclusions: These data indicate that IC100 suppresses the immune-inflammatory response that drives EAE development and progression, thereby identifying ASC as a promising target for the treatment of MS as well as other neurological diseases with a neuroinflammatory component.

Inflammasomes can prevent systemic dissemination of enteropathogenic bacteria. As adapted pathogens including Salmonella Typhimurium (S. Tm) have evolved evasion strategies, it has remained unclear when and where inflammasomes restrict their dissemination. Bacterial population dynamics establish that the NAIP/NLRC4 inflammasome specifically restricts S. Tm migration from the gut to draining lymph nodes. This is solely attributable to NAIP/NLRC4 within intestinal epithelial cells (IECs), while S. Tm evades restriction by phagocyte NAIP/NLRC4. NLRP3 and Caspase-11 also fail to restrict S. Tm mucosa traversal, migration to lymph nodes, and systemic pathogen growth. The ability of IECs (not phagocytes) to mount a NAIP/NLRC4 defense in vivo is explained by particularly high NAIP/NLRC4 expression in IECs and the necessity for epithelium-invading S. Tm to express the NAIP1-6 ligands-flagella and type-III-secretion-system-1. Imaging reveals both ligands to be promptly downregulated following IEC-traversal. These results highlight the importance of intestinal epithelial NAIP/NLRC4 in blocking bacterial dissemination in vivo, and explain why this constitutes a uniquely evasion-proof defense against the adapted enteropathogen S. Tm.

Objective: Neuroprosthetics hold tremendous promise to restore function through brain-computer interfaced devices. However, clinical applications of implantable microelectrodes remain limited given the challenges of maintaining neuronal signals for extended periods of time and with multiple biological mechanisms negatively affecting electrode performance. Acute and chronic inflammation, oxidative stress, and blood brain barrier disruption contribute to inconsistent electrode performance. We hypothesized that therapeutic hypothermia (TH) applied at the microelectrode insertion site will positively modulate both inflammatory and apoptotic pathways, promoting neuroprotection and improved performance in the long-term., Approach: A custom device and thermoelectric system were designed to deliver controlled TH locally to the cortical implant site at the time of microelectrode array insertion and immediately following surgery. The TH paradigm was derived from in vivo cortical temperature measurements and finite element modeling of temperature distribution profiles in the cortex. Male Sprague-Dawley rats were implanted with non-functional Utah microelectrodes arrays (UMEA) consisting of 4 × 4 grid of 1.5 mm long parylene-coated silicon shanks. In one group, TH was applied to the implant site for two hours following the UMEA implantation, while the other group was implanted under normothermic conditions without treatment. At 48 h, 72 h, 7 d and 14 d post-implantation, mRNA expression levels for genes associated with inflammation and apoptosis were compared between normothermic and hypothermia-treated groups., Main Results: The custom system delivered controlled TH to the cortical implant site and the numerical models confirmed that the temperature decrease was confined locally. Furthermore, a one-time application of TH post UMEA insertion significantly reduced the acute inflammatory response with a reduction in the expression of inflammatory regulating cytokines and chemokines., Significance: This work provides evidence that acutely applied hypothermia is effective in significantly reducing acute inflammation post intracortical electrode implantation.

We have collected 86 different transforms of tautomeric interconversions. Out of those, 54 are for prototropic (non-ring-chain) tautomerism, 21 for ring-chain tautomerism, and 11 for valence tautomerism. The majority of these rules have been extracted from experimental literature. Twenty rules, covering the most well-known types of tautomerism such as keto-enol tautomerism, were taken from the default handling of tautomerism by the chemoinformatics toolkit CACTVS. The rules were analyzed against nine differerent databases totaling over 400 million (non-unique) structures as to their occurrence rates, mutual overlap in coverage, and recapitulation of the rules' enumerated tautomer sets by InChI V.1.05, both in InChI's Standard and a Nonstandard version with the increased tautomer-handling options 15T and KET turned on. These results and the background of this study are discussed in the context of the IUPAC InChI Project tasked with the redesign of handling of tautomerism for an InChI version 2. Applying the rules presented in this paper would approximately triple the number of compounds in typical small-molecule databases that would be affected by tautomeric interconversion by InChI V2. A web tool has been created to test these rules at https://cactus.nci.nih.gov/tautomerizer.

Traumatic brain injury (TBI) is one of the leading causes of mortality and disability around the world. Mild TBI (mTBI) makes up approximately 80% of reported cases and often results in transient psychological abnormalities and cognitive disruption. At-risk populations for mTBI include athletes and other active individuals who may sustain repetitive concussive injury during periods of exercise and exertion when core temperatures are elevated. Previous studies have emphasized the impact that increased brain temperature has on adverse neurological outcomes. A lack of diagnostic tools to assess concussive mTBI limits the ability to effectively identify the post-concussive period during which the brain is uniquely susceptible to damage upon sustaining additional injury. Studies have suggested that a temporal window of increased vulnerability that exists corresponds to a period of injury-induced depression of cerebral glucose metabolism. In the current study, we sought to evaluate the relationship between repetitive concussion, local cerebral glucose metabolism, and brain temperature using the Marmarou weight drop model to generate mTBI. Animals were injured three consecutive times over a period of 7 days while exposed to either normothermic or hyperthermic temperatures for 15 min prior to and 1 h post each injury. A 14 C-2-deoxy-d-glucose (2DG) autoradiography was used to measure local cerebral metabolic rate of glucose (lCMR Glc ) in 10 diverse brain regions across nine bregma levels 8 days after the initial insult. We found that repetitive mTBI significantly decreased glucose utilization bilaterally in several cortical areas, such as the cingulate, visual, motor, and retrosplenial cortices, as well as in subcortical areas, including the caudate putamen and striatum, compared to sham control animals. lCMR Glc was significant in both normothermic and hyperthermic repetitive mTBI animals relative to the sham group, but to a greater degree when exposed to hyperthermic conditions. Taken together, we report significant injury-induced glucose hypometabolism after repetitive concussion in the brain, and additionally highlight the importance of temperature management in the acute period after brain injury.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality. Approximately 20-25% of TBI subjects develop Acute Lung Injury (ALI), but the pathomechanisms of TBI-induced ALI remain poorly defined. Currently, mechanical ventilation is the only therapeutic intervention for TBI-induced lung injury. Our recent studies have shown that the inflammasome plays an important role in the systemic inflammatory response leading to lung injury-post TBI. Here, we outline the role of the extracellular vesicle (EV)-mediated inflammasome signaling in the etiology of TBI-induced ALI. Furthermore, we evaluate the efficacy of a low molecular weight heparin (Enoxaparin, a blocker of EV uptake) and a monoclonal antibody against apoptosis speck-like staining protein containing a caspase recruitment domain (anti-ASC) as therapeutics for TBI-induced lung injury. We demonstate that activation of an EV-mediated Neural-Respiratory Inflammasome Axis plays an essential role in TBI-induced lung injury and disruption of this axis has therapeutic potential as a treatment strategy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Bacterial bone infections after revision surgeries and diseases, like osteomyelitis, are still a challenge with regard to surgical treatments. Local bone infections were treated with antibiotics directly or by controlled drug-releasing scaffolds, like polymethylmethacrylate (PMMA) spheres, which have to be removed at a later stage, but there is a risk of a bacterial contamination during the removement. Therefore, biomaterials loaded with antibiotics for controlled release could be the method of choice: The biomaterials degrade during the drug release, therefore, there is no need for a second surgery to remove the drug eluting agent. Even non-resorbable bone materials are available (e.g., hydroxyapatite (HA)) or resorbable bone graft materials (e.g., beta-tricalcium phosphate (β-TCP)) that will be replaced by newly formed bone. Composite materials with organic additives (e.g., collagen) supports the handling during surgery and enhances the drug loading capacity, as well as the drug releasing time. The purpose of this study was to investigate the loading capacity and the release rate of Vancomycin and Gentamicin on TCP and HA granules in the shape of a degradable scaffold compared to composite materials from TCP mixed with porcine collagen. Its antibacterial efficacy to a more elementary drug with eluting in aqueous solution was examined. The loading capacity of the biomaterials was measured and compared according to the Minimum Inhibition Concentration (MIC) and the Minimum Biofilm Eradication Concentration (MBEC) of a bacterial biofilm after 24 h aging. Antibiotic elution and concentration of gentamycin and vancomycin, as well as inhibition zones, were measured by using the Quantitative Microparticle Systems (QMS) immunoassays. The antibiotic concentration was determined by the automated Beckman Coulter (BC) chemistry device. For examination of the antibacterial activity, inhibition zone diameters were measured. Generally, the antibiotic release is more pronounced during the first couple of days than later. Both TCP granules and HA granules experienced a significantly decline of antibiotics release during the first three days. After the fourth day and beyond, the antibiotic release was below the detection threshold. The antibiotic release of the composite material TCP and porcine collagen declined less drastically and was still in the frame of the specification during the first nine days. There was no significant evidence of interaction effect between antibiotic and material, i.e., the fitted lines for Gentamycin and Vancomycin are almost parallel. During this first in vitro study, β-TCP-Collagen composites shows a significantly higher loading capacity and a steadily release of the antibiotics Gentamycin and Vancomycin, compared to the also used TCP and HA Granules.

DNA double-strand breaks are genotoxic lesions whose repair can be templated off an intact DNA duplex through the conserved homologous recombination (HR) pathway. Because it mainly consists of a succession of non-covalent associations of molecules, HR is intrinsically reversible. Reversibility serves as an integral property of HR, exploited and tuned at various stages throughout the pathway with anti- and pro-recombinogenic consequences. Here, we focus on the reversibility of displacement loops (D-loops), a central DNA joint molecule intermediate whose dynamics and regulation have recently been physically probed in somatic S. cerevisiae cells. From homology search to repair completion, we discuss putative roles of D-loop reversibility in repair fidelity and outcome.

The microbiota confers colonization resistance, which blocks Salmonella gut colonization 1 . As diet affects microbiota composition, we studied whether food composition shifts enhance susceptibility to infection. Shifting mice to diets with reduced fibre or elevated fat content for 24 h boosted Salmonella Typhimurium or Escherichia coli gut colonization and plasmid transfer. Here, we studied the effect of dietary fat. Colonization resistance was restored within 48 h of return to maintenance diet. Salmonella gut colonization was also boosted by two oral doses of oleic acid or bile salts. These pathogen blooms required Salmonella's AcrAB/TolC-dependent bile resistance. Our data indicate that fat-elicited bile promoted Salmonella gut colonization. Both E. coli and Salmonella show much higher bile resistance than the microbiota. Correspondingly, competitive E. coli can be protective in the fat-challenged gut. Diet shifts and fat-elicited bile promote S. Typhimurium gut infections in mice lacking E. coli in their microbiota. This mouse model may be useful for studying pathogen-microbiota-host interactions, the protective effect of E. coli, to analyse the spread of resistance plasmids and assess the impact of food components on the infection process.

Hypothesis: Application of localized, mild therapeutic hypothermia during cochlear implantation (CI) surgery is feasible for residual hearing preservation., Background: CI surgery often results in a loss of residual hearing. In preclinical studies, local application of controlled, mild therapeutic hypothermia has shown promising results as a hearing preservation strategy. This study investigated a suitable surgical approach to deliver local hypothermia in patients utilizing anatomical and radiologic measurements and experimental measurements from cadaveric human temporal bones., Methods: Ten human cadaveric temporal bones were scanned with micro-computed tomography and anatomical features and measurements predicting round window (RW) visibility were characterized. For each bone, the standard facial recess and myringotomy approaches for delivery of hypothermia were developed. The St. Thomas Hospital (STH) classification was used to record degree of RW visibility with and without placement of custom hypothermia probe. Therapeutic hypothermia was delivered through both approaches and temperatures recorded at the RW, RW niche, over the lateral semicircular canal and the supero-lateral mastoid edge., Results: The average facial recess area was 13.87 ± 5.52 mm. The introduction of the cooling probe through either approach did not impede visualization of the RW or cochleostomy as determined by STH grading. The average temperatures at RW using the FR approach reduced by 4.57 ± 1.68 °C for RW, while using the myringotomy approach reduced by 4.11 ± 0.98 °C for RW., Conclusion: Local application of therapeutic hypothermia is clinically feasible both through the facial recess and myringotomy approaches without limiting optimal surgical visualization.

Homologous recombination (HR) uses a homologous template to accurately repair DNA double-strand breaks and stalled replication forks to maintain genome stability. During homology search, Rad51 nucleoprotein filaments probe and interact with dsDNA, forming the synaptic complex that is stabilized on a homologous sequence. Strand intertwining leads to the formation of a displacement-loop (D-loop). In yeast, Rad54 is essential for HR in vivo and required for D-loop formation in vitro, but its exact role remains to be fully elucidated. Using electron microscopy to visualize the DNA-protein complexes, here we find that Rad54 is crucial for Rad51-mediated synaptic complex formation and homology search. The Rad54-K341R ATPase-deficient mutant protein promotes formation of synaptic complexes but not D-loops and leads to the accumulation of stable heterologous associations, suggesting that the Rad54 ATPase is involved in preventing non-productive intermediates. We propose that Rad51/Rad54 form a functional unit operating in homology search, synaptic complex and D-loop formation.

Hyperoxia plays a key role in the development of bronchopulmonary dysplasia (BPD), a chronic lung disease of preterm infants. Infants with BPD often have brain injury that leads to long-term neurodevelopmental impairment, but the underlying mechanisms that control BPD-induced neurodevelopmental impairment remain unclear. Our previous studies have shown that hyperoxia-induced BPD in rodents is associated with lung inflammasome activation. Here, we tested the hypothesis that hyperoxia-induced lung and brain injury is mediated by inflammasome activation, and that inhibition of caspase-1, a key component of the inflammasome, attenuates hyperoxia-induced lung and brain injury in neonatal mice. C57/BL6 mouse pups were randomized to receive daily intraperitoneal injections of Ac-YVAD-CMK, an irreversible caspase-1 inhibitor, or placebo during exposure to room air or hyperoxia (85% O 2 ) for 10 days. We found that hyperoxia activated the NLRP1 inflammasome, increased production of mature IL-1β, and upregulated expression of p30 gasdermin-D (GSDMD), the active form of GSDMD that is responsible for the programmed cell death mechanism of pyroptosis in both lung and brain tissue. Importantly, we show that inhibition of caspase-1 decreased IL-1β activation and p30 GSDMD expression, and improved alveolar and vascular development in hyperoxia-exposed lungs. Moreover, caspase-1 inhibition also promoted cell proliferation in the subgranular zone and subventricular zone of hyperoxia-exposed brains, resulting in lessened atrophy of these zones. Thus, the inflammasome plays a critical role in hyperoxia-induced neonatal lung and brain injury, and targeting this pathway may be beneficial for the prevention of lung and brain injury in preterm infants.

The emergence of antibiotic-resistant bacteria through mutations or the acquisition of genetic material such as resistance plasmids represents a major public health issue 1,2 . Persisters are subpopulations of bacteria that survive antibiotics by reversibly adapting their physiology 3-10 , and can promote the emergence of antibiotic-resistant mutants 11 . We investigated whether persisters can also promote the spread of resistance plasmids. In contrast to mutations, the transfer of resistance plasmids requires the co-occurrence of both a donor and a recipient bacterial strain. For our experiments, we chose the facultative intracellular entero-pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) and Escherichia coli, a common member of the microbiota 12 . S. Typhimurium forms persisters that survive antibiotic therapy in several host tissues. Here we show that tissue-associated S. Typhimurium persisters represent long-lived reservoirs of plasmid donors or recipients. The formation of reservoirs of S. Typhimurium persisters requires Salmonella pathogenicity island (SPI)-1 and/or SPI-2 in gut-associated tissues, or SPI-2 at systemic sites. The re-seeding of these persister bacteria into the gut lumen enables the co-occurrence of donors with gut-resident recipients, and thereby favours plasmid transfer between various strains of Enterobacteriaceae. We observe up to 99% transconjugants within two to three days of re-seeding. Mathematical modelling shows that rare re-seeding events may suffice for a high frequency of conjugation. Vaccination reduces the formation of reservoirs of persisters after oral infection with S. Typhimurium, as well as subsequent plasmid transfer. We conclude that-even without selection for plasmid-encoded resistance genes-small reservoirs of pathogen persisters can foster the spread of promiscuous resistance plasmids in the gut.

This work introduces a cost and time efficient procedure to specifically increase mesopore volume and specific surface area of biogenic silica (specific surface area: 147 m 2 g -1 and mesopore volume: 0.23 cm 3 g -1 ) to make it suitable for applications in adsorption or as catalyst support. The target values were a specific surface area of ~500 m 2 g -1 and a mesopore volume of ~0.40-0.50 cm 3 g -1 as these values are industrially relevant and are reached by potential concurring products such as precipitated silica, silica gel, and fumed silica. The applied process of partial pseudomorphic transformation was carried out as a single reaction step in a microwave reactor instead of commonly used convective heating. In addition, the conventionally used surfactant cetyltrimethylammonium bromide (CTABr) was substituted by the low-cost surfactant (Arquad® 16-29, cetyltrimethylammonium chloride (CTACl) aqueous solution). The influence of microwave heating, type of surfactant as well as the concentration of NaOH and CTACl on the textural and structural properties of the modified biogenic silica was investigated using nitrogen adsorption as well as scanning and transmission electron microscopy. The results show that the textural parameters of the modified biogenic silica can be exactly controlled by the amount of NaOH in the reaction solution. By variation of the NaOH concentration, specific surface areas in the range of 215-1,001 m 2 g -1 and mesopore volumes of 0.25-0.56 cm 3 g -1 were achieved after reaction at 393 K for 10 min. The presented microwave route using the low-cost surfactant solution decreases the reaction time by 99% and as shown in an example for German prices, lowers the costs for the surfactant by 76-99%.

Therapy of children with post-transplantation lymphoproliferative disorder (PTLD) after hematopoietic stem cell (HSCT) and solid organ transplantation (SOT) can be challenging. In this retrospective study, we investigated PD-L1 and PD1 expression in all PTLD categories of childhood and adolescence to see whether checkpoint inhibition with PD-L1/PD1 inhibitors may serve as a therapy option. We included 21 patients aged 19 years or younger (at date of transplant) with PTLD following SOT or HSCT having adequate tumor samples available (n = 29). Using immunohistochemistry, we evaluated PD-L1/PD1 expression on both tumor cells and cells of the microenvironment in all samples. Availability of consecutively matched tumor samples during 6 of 21 patients' disease courses also allowed an intra-individual assessment of PD-L1/PD1 expression. We observed lower PD-L1 and higher PD1 expression in non-destructive lesions, and higher PD-L1 and lower PD1 expression in polymorphic and, in particular, in monomorphic PTLD, mostly diffuse large B-cell lymphomas (DLBCL, n = 10/21). The amount of PD-L1- and PD1-positive cells changed in the opposite way in sequential biopsies of the same individual correlating well with the PTLD category. This is the first comprehensive pediatric study assessing PD-L1 and PD1 expression on tumor cells and in the microenvironment of PTLD including not only monomorphic, but also non-destructive early lesions. PD-L1 expression of the tumor cells inversely correlated with PD1 expression in surrounding tissues, with the highest expression in DLBCL. Since PTLD can be therapeutically challenging, our results indicate a potential efficacy of checkpoint inhibitors if standard immune- and/or chemotherapy fail or are impossible. We therefore recommend routine staining of PD-L1 and PD1 in all PTLD categories., (© 2019 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Saccharomyces cerevisiae Srs2, in addition to its well-documented antirecombination activity, has been proposed to play a role in promoting synthesis-dependent strand annealing (SDSA). Here we report the identification and characterization of an SRS2 mutant with a single amino acid substitution ( srs2-F891A ) that specifically affects the Srs2 pro-SDSA function. This residue is located within the Srs2-Rad51 interaction domain and embedded within a protein sequence resembling a BRC repeat motif. The srs2-F891A mutation leads to a complete loss of interaction with Rad51 as measured through yeast two-hybrid analysis and a partial loss of interaction as determined through protein pull-down assays with purified Srs2, Srs2-F891A, and Rad51 proteins. Even though previous work has shown that internal deletions of the Srs2-Rad51 interaction domain block Srs2 antirecombination activity in vitro , the Srs2-F891A mutant protein, despite its weakened interaction with Rad51, exhibits no measurable defect in antirecombination activity in vitro or in vivo Surprisingly, srs2-F891A shows a robust shift from noncrossover to crossover repair products in a plasmid-based gap repair assay, but not in an ectopic physical recombination assay. Our findings suggest that the Srs2 C-terminal Rad51 interaction domain is more complex than previously thought, containing multiple interaction sites with unique effects on Srs2 activity., (Copyright © 2019 by the Genetics Society of America.)

Immune responses need to be controlled tightly to prevent autoimmune diseases, yet underlying molecular mechanisms remain partially understood. Here, we identify biallelic mutations in three patients from two unrelated families in differentially expressed in FDCP6 homolog (DEF6) as the molecular cause of an inborn error of immunity with systemic autoimmunity. Patient T cells exhibit impaired regulation of CTLA-4 surface trafficking associated with reduced functional CTLA-4 availability, which is replicated in DEF6-knockout Jurkat cells. Mechanistically, we identify the small GTPase RAB11 as an interactor of the guanine nucleotide exchange factor DEF6, and find disrupted binding of mutant DEF6 to RAB11 as well as reduced RAB11 + CTLA-4 + vesicles in DEF6-mutated cells. One of the patients has been treated with CTLA-4-Ig and achieved sustained remission. Collectively, we uncover DEF6 as player in immune homeostasis ensuring availability of the checkpoint protein CTLA-4 at T-cell surface, identifying a potential target for autoimmune and/or cancer therapy.

Increasing concerns about the rising rates of antibiotic therapy failure and advances in single-cell analyses have inspired a surge of research into antibiotic persistence. Bacterial persister cells represent a subpopulation of cells that can survive intensive antibiotic treatment without being resistant. Several approaches have emerged to define and measure persistence, and it is now time to agree on the basic definition of persistence and its relation to the other mechanisms by which bacteria survive exposure to bactericidal antibiotic treatments, such as antibiotic resistance, heteroresistance or tolerance. In this Consensus Statement, we provide definitions of persistence phenomena, distinguish between triggered and spontaneous persistence and provide a guide to measuring persistence. Antibiotic persistence is not only an interesting example of non-genetic single-cell heterogeneity, it may also have a role in the failure of antibiotic treatments. Therefore, it is our hope that the guidelines outlined in this article will pave the way for better characterization of antibiotic persistence and for understanding its relevance to clinical outcomes.

Hidradenitis suppurativa (HS) (also designated acne inversa) is a chronic inflammatory disease characterized by painful purulent skin lesions and progressive destruction of skin architecture. Despite the high burden for the patients, pathogenetic pathways underlying HS alterations remain obscure. When we examined the HS cytokine pattern, IL-1β turned out to be a highly prominent cytokine, overexpressed even compared with psoriatic lesions. Analyses of IL-1β-induced transcriptome in various cell types showed overlapping profiles, with upregulations of molecules causing immune cell infiltration and extracellular matrix degradation, and of specific cytokines including IL-6, IL-32, and IL-36. Matching cellular IL-1 receptor levels, dermal fibroblasts showed both the strongest and broadest IL-1β response, which was not clearly shared or strengthened by other cytokines. The IL-1β signature was specifically present in HS lesions and could be reversed by application of IL-1 receptor antagonist. Search for blood parameters associated with IL-1β pathway activity in HS identified serum amyloid A, which was synergistically induced by IL-1β and IL-6 in hepatocytes. Consequently, strongly elevated blood serum amyloid A levels in HS correlated positively with the extent of inflammatory skin alterations. In summary, the IL-1β pathway represents a pathogenetic cascade, whose activity may be therapeutically targeted and monitored by blood SAA levels., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Mucus separates gut-luminal microbes from the tissue. It is unclear how pathogens like Salmonella Typhimurium (S.Tm) can overcome this obstacle. Using live microscopy, we monitored S.Tm interactions with native murine gut explants and studied how mucus affects the infection. A dense inner mucus layer covers the distal colon tissue, limiting direct tissue access. S.Tm performs near-surface swimming on this mucus layer, which allows probing for colon mucus heterogeneities, but can also entrap the bacterium in the dense inner colon mucus layer. In the cecum, dense mucus fills only the bottom of the intestinal crypts, leaving the epithelium between crypts unshielded and prone to access by motile and non-motile bacteria alike. This explains why the cecum is highly infection permissive and represents the primary site of S.Tm enterocolitis in the streptomycin mouse model. Our findings highlight the importance of mucus in intestinal defense and homeostasis., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Bacterial host cell invasion mechanisms depend on the bacterium's virulence factors and the properties of the target cell. The enteropathogen Salmonella enterica serovar Typhimurium ( S Tm) invades epithelial cell types in the gut mucosa and a variety of immune cell types at later infection stages. The molecular mechanism(s) of host cell entry has, however, been studied predominantly in epithelial cell lines. S Tm uses a type three secretion system (TTSS-1) to translocate effectors into the host cell cytosol, thereby sparking actin ruffle-dependent entry. The ruffles also fuel cooperative invasion by bystander bacteria. In addition, several TTSS-1-independent entry mechanisms exist, involving alternative S Tm virulence factors, or the passive uptake of bacteria by phagocytosis. However, it remains ill-defined how S Tm invasion mechanisms vary between host cells. Here, we developed an internally controlled and scalable method to map S Tm invasion mechanisms across host cell types and conditions. The method relies on host cell infections with consortia of chromosomally tagged wild-type and mutant S Tm strains, where the abundance of each strain can be quantified by qPCR or amplicon sequencing. Using this methodology, we quantified cooccurring TTSS-1-dependent, cooperative, and TTSS-1-independent invasion events in epithelial, monocyte, and macrophage cells. We found S Tm invasion of epithelial cells and monocytes to proceed by a similar MOI-dependent mix of TTSS-1-dependent and cooperative mechanisms. TTSS-1-independent entry was more frequent in macrophages. Still, TTSS-1-dependent invasion dominated during the first minutes of interaction also with this cell type. Finally, the combined action of the SopB/SopE/SopE2 effectors was sufficient to explain TTSS-1-dependent invasion across both epithelial and phagocytic cells. IMPORTANCE Salmonella enterica serovar Typhimurium ( S Tm) is a widespread and broad-host-spectrum enteropathogen with the capacity to invade diverse cell types. Still, the molecular basis for the host cell invasion process has largely been inferred from studies of a few selected cell lines. Our work resolves the mechanisms that Salmonellae employ to invade prototypical host cell types, i.e., human epithelial, monocyte, and macrophage cells, at a previously unattainable level of temporal and quantitative precision. This highlights efficient bacterium-driven entry into innate immune cells and uncovers a type III secretion system effector module that dominates active bacterial invasion of not only epithelial cells but also monocytes and macrophages. The results are derived from a generalizable method, where we combine barcoding of the bacterial chromosome with mixed consortium infections of cultured host cells. The application of this methodology across bacterial species and infection models will provide a scalable means to address host-pathogen interactions in diverse contexts., (Copyright © 2019 Di Martino et al.)

Immunoglobulin A is a class of antibodies produced by the adaptive immune system and secreted into the gut lumen to fight pathogenic bacteria. We recently demonstrated that the main physical effect of these antibodies is to enchain daughter bacteria, i.e. to cross-link bacteria into clusters as they divide, preventing them from interacting with epithelial cells, thus protecting the host. These links between bacteria may break over time. We study several models using analytical and numerical calculations. We obtain the resulting distribution of chain sizes, that we compare with experimental data. We study the rate of increase in the number of free bacteria as a function of the replication rate of bacteria. Our models show robustly that at higher replication rates, bacteria replicate before the link between daughter bacteria breaks, leading to growing cluster sizes. On the contrary at low growth rates two daughter bacteria have a high probability to break apart. Thus the gut could produce IgA against all the bacteria it has encountered, but the most affected bacteria would be the fast replicating ones, that are more likely to destabilize the microbiota. Linking the effect of the immune effectors (here the clustering) with a property directly relevant to the potential bacterial pathogeneicity (here the replication rate) could avoid to make complex decisions about which bacteria to produce effectors against., Competing Interests: The authors have declared that no competing interests exist.

Forming the outer body barrier, our skin is permanently exposed to pathogens and environmental hazards. Therefore, skin diseases are among the most common disorders. In many of them, the immune system plays a crucial pathogenetic role. For didactic and therapeutic reasons, classification of such immune-mediated skin diseases according to the underlying dominant immune mechanism rather than to their clinical manifestation appears to be reasonable. Immune-mediated skin diseases may be mediated mainly by T cells, by the humoral immune system, or by uncontrolled unspecific inflammation. According to the involved T cell subpopulation, T cell-mediated diseases may be further subdivided into T1 cell-dominated (e.g., vitiligo), T2 cell-dominated (e.g., acute atopic dermatitis), T17/T22 cell-dominated (e.g., psoriasis), and Treg cell-dominated (e.g., melanoma) responses. Moreover, T cell-dependent and -independent responses may occur simultaneously in selected diseases (e.g., hidradenitis suppurativa). The effector mechanisms of the respective T cell subpopulations determine the molecular changes in the local tissue cells, leading to specific microscopic and macroscopic skin alterations. In this article, we show how the increasing knowledge of the T cell biology has been comprehensively translated into the pathogenetic understanding of respective model skin diseases and, based thereon, has revolutionized their daily clinical management.

DNA polymerases influence genome stability through their involvement in DNA replication, response to DNA damage, and DNA repair processes. Saccharomyces cerevisiae possess four non-essential DNA polymerases, Pol λ, Pol η, Pol ζ, and Rev1, which have varying roles in genome stability. In order to assess the contribution of the non-essential DNA polymerases in genome stability, we analyzed the pol4Δ rev1Δ rev3Δ rad30Δ quadruple mutant in microhomology mediated repair, due to recent studies linking some of these DNA polymerases to this repair pathway. Our results suggest that the length and quality of microhomology influence both the overall efficiency of repair and the involvement of DNA polymerases. Furthermore, the non-essential DNA polymerases demonstrate overlapping and redundant functions when repairing double-strand breaks using short microhomologies containing mismatches. Then, we examined genome-wide mutation accumulation in the pol4Δ rev1Δ rev3Δ rad30Δ quadruple mutant compared to wild type cells. We found a significant decrease in the overall rate of mutation accumulation in the quadruple mutant cells compared to wildtype, but an increase in frameshift mutations and a shift towards transversion base-substitution with a preference for G:C to T:A or C:G. Thus, the non-essential DNA polymerases have an impact on the nature of the mutational spectrum. The sequence and functional homology shared between human and S. cerevisiae non-essential DNA polymerases suggest these DNA polymerases may have a similar role in human cells., (Copyright © 2019 Elsevier B.V. All rights reserved.)