title: Mutation Rate Evolution Drives Immune Escape In Mismatch Repair-Deficient Cancer

abstract: Mutation rate optimisation drives evolution and immune evasion of bacteria and lentiviral strains, including HIV. Whether evolving cancer lineages similarly adapt mutation rates to increase tumour cell fitness is unknown. Here, by mapping the clonal topography of mismatch repair-deficient (MMRd) colorectal cancer, we show that genomic MMRd mutability co-evolves with neoantigen selection to drive intratumour diversification and immune escape. Mechanistically, we find that microsatellite instability modulates subclonal DNA repair by toggling two hypermutable mononucleotide homopolymer runs in the mismatch repair genes MSH6 and MSH3 (C8 and A8, respectively) through stochastic frameshift switching. Spontaneous mutation and reversion at these evolvability switches modulates subclonal mutation rate, mutation bias, and clonal HLA diversity during MMRd cancer evolution. Combined experimental and simulation studies demonstrate that subclonal immune selection favours incremental MMR mutations. MMRd cancers thus fuel intratumour heterogeneity by adapting subclonal mutation rate and mutation bias to immune selection, revealing a conserved co-evolutionary arms race between neoantigen selection and adaptive genomic mutability. Our work reveals layers of mutational complexity and microsatellite biology in MMRd cancer evolution previously hidden in bulk analyses.


title: A mass spectrometry-based method for the determination of in vivo biodistribution of tumor targeting small molecule-metal conjugates

abstract: Nuclear medicine plays a key role in modern diagnosis and cancer therapy. The development of tumor targeting radionuclide conjugates (also named Small Molecule-Radio Conjugates - SMRCs) represents a significant improvement over the clinical use of metabolic radiotracers (e.g., [18F]-Fluorodeoxyglucose) for imaging and over the application of biocidal external beam radiations for therapy. During the discovery of SMRCs, molecular candidates must be carefully evaluated typically by performing biodistribution assays in preclinical tumor models. Quantification methodologies based on radioactive counts are typically demanding due to safety concerns, availability of radioactive material, and infrastructures. In this article, we report the development of a mass spectrometry (MS)-based method for the detection and quantification of small molecule-metal conjugates (SMMCs) as cold surrogates of SMRCs. We applied this methodology for the evaluation of the biodistribution of a particular class of tumor-targeting drug candidates based on natLu, natGa, natF and directed against Fibroblast Activation Protein (FAP). The reliability of the LC-MS analysis was validated by direct comparison of MSbased and radioactivity-based biodistribution data. Results show that MS biodistribution of stable isotope metal conjugates is an orthogonal tool for the preclinical characterization of different classes of radiopharmaceuticals.

category: pharmacology and toxicology


title: Blinding efficacy and adverse events following repeated transcranial alternating current, direct current, and random noise stimulation.

abstract: As transcranial electrical stimulation (tES) protocols advance, assumptions underlying the technique need to be retested to ensure they still hold. Whilst the safety of stimulation has been demonstrated mainly for a small number of sessions, and small sample size, adverse events (AEs) following multiple sessions remain largely untested. Similarly, whilst blinding procedures are typically assumed to be effective, the effect of multiple stimulation sessions on the efficacy of blinding procedures also remains under question. This is especially relevant in multisite projects where small unintentional variations in protocol could lead to inter-site difference. We report AE and blinding data from 1,019 participants who received up to 11 semi-consecutive sessions of active or sham transcranial alternating current stimulation (tACS), direct current stimulation (tDCS), and random noise stimulation (tRNS), at 4 sites in the UK and US. We found that AEs were often best predicted by factors other than tES, such as testing site or session number. Results from the blinding analysis suggested that blinding was less effective for tDCS and tACS than tRNS. The occurrence of AEs did not appear to be linked to tES despite the use of smaller electrodes or repeated delivery. However, blinding efficacy was impacted in tES conditions with higher cutaneous sensation, highlighting a need for alternative stimulation blinding protocols. This may be increasingly necessary in studies wishing to deliver stimulation with higher intensities.

category: neuroscience


title: Soluble P2X7 receptor is elevated in the plasma of COVID-19 patients and correlates with disease severity

abstract: Inflammation is a tightly coordinated response against bacterial and viral infections, triggered by the production of pro-inflammatory cytokines. SARS-CoV-2 infection induces COVID-19 disease, characterized by an inflammatory response mediated through the activation of the NLRP3 inflammasome, which results in the production of IL-1{beta} and IL-18 along with pyroptotic cell death. The NLRP3 inflammasome could be also triggered by sterile danger signals such as extracellular ATP triggering the purinergic P2X7 receptor. Severe inflammation in the lungs of SARS-CoV-2 infected individuals is associated with pneumonia, hypoxia and acute respiratory distress syndrome, these being the causes of death associated with COVID-19. Both the P2X7 receptor and NLRP3 have been considered as potential pharmacological targets for treating inflammation in COVID-19. However, there is no experimental evidence of the involvement of the P2X7 receptor during COVID-19 disease. In the present study we determined the concentration of different cytokines and the P2X7 receptor in the plasma of COVID-19 patients and found that along with the increase in IL-6, IL-18 and the IL-1 receptor antagonist in the plasma of COVID-19 patients, there was also an increase in the purinergic P2X7 receptor. Increase in COVID-19 severity and C-reactive protein concentration positively correlated with increased concentration of the P2X7 receptor in the plasma, but not with IL-18 cytokine. The P2X7 receptor was found in the supernatant of human peripheral blood mononuclear cells after inflammasome activation. Therefore, our data suggest that determining levels of the P2X7 receptor in the plasma could be a novel biomarker of COVID-19 severity.

category: immunology


title: Chimeric mRNA based COVID-19 vaccine elicits potent neutralizing antibodies and protection against Omicron and Delta

abstract: The emerging SARS-CoV-2 variants of concern (VOCs) exhibit enhanced transmission and immune escape, reducing the efficacy and effectiveness of the two FDA-approved mRNA vaccines. Here, we explored various strategies to develop novel mRNAs vaccines to achieve safer and wider coverage of VOCs. Firstly, we constructed a cohort of mRNAs that feature a furin cleavage mutation in the spike (S) protein of predominant VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1) and Delta (B.1.617.2). Not present in the mRNA vaccines currently in use, the mutation abolished the cleavage between the S1 and S2 subunits, potentially enhancing the safety profile of the immunogen. Secondly, we systematically evaluated the induction of neutralizing antibodies (nAb) in vaccinated mice, and discovered that individual VOC mRNAs elicited strong neutralizing activity in a VOC-specific manner. Thirdly, the IgG produced in mice immunized with Beta-Furin and Washington (WA)-Furin mRNAs showed potent cross-reactivity with other VOCs, which was further corroborated by challenging vaccinated mice with the live virus of VOCs. However, neither WA-Furin nor Beta-Furin mRNA elicited strong neutralizing activity against the Omicron variant. Hence, we further developed an Omicron-specific mRNA vaccine that restored protection against the original and the sublineages of Omicron variant. Finally, to broaden the protection spectrum of the new Omicron mRNA vaccine, we tested the concept of bivalent immunogen. Instead of just fusing two RBDs head-to-tail, we for the first time constructed an mRNA-based chimeric immunogen by introducing the RBD of Delta variant into the entire S antigen of Omicron. The resultant chimeric mRNA was capable of inducing potent and broadly acting nAb against Omicron (both BA.1 and BA.2) and Delta, which paves the way to develop new vaccine candidate to target emerging variants in the future.


title: Chimeric mRNA based COVID-19 vaccine induces protective immunity against Omicron and Delta


title: Elevated HLA-E and NKG2A as a consequence of chronic immune activation defines resistance to M. bovis BCG immunotherapy in Non-Muscle-Invasive Bladder Cancer

abstract: Mycobacterium bovis Bacillus Calmette-Guerin (BCG), the first-line treatment for non-muscle invasive bladder cancer (NMIBC), promotes the production of inflammatory cytokines, particularly interferon (IFN)-{gamma}. Prolonged inflammation and IFN-{gamma} exposure are known to cause an adaptive immune response, enabling immune escape and proliferation by tumor cells. We investigated HLA-E and NKG2A, a novel T and NK cell checkpoint pathway, as a driver of adaptive resistance in BCG unresponsive NMIBC. We observed ubiquitous inflammation in all patients after BCG immunotherapy, regardless of recurrence status. IFN-{gamma} was shown to drive tumor expression of HLA-E and PD-L1. Further, NKG2A-expressing NK and CD8 T cells were enriched in BCG unresponsive tumors and with enhanced capacity for cytolytic functions. Strikingly, in situ spatial analyses revealed that HLA-EHIGH tumors are activated to recruit NK and T cells via chemokine production, potentially sparing HLA-ELOW tumors that would otherwise be susceptible to lysis. Finally, blood-derived NK cells retained anti-tumor functions at the time of tumor recurrence. These data support combined NKG2A and PD-L1 blockade for BCG unresponsive disease.


title: Crippled Coronavirus: 5'-PolyU targeted Oligo prevents development of infectious Virions

abstract: Current RNA viral pandemic of COVID-19 has been worsened by rapidly spreading viral variants. To inhibit mutation-based development of new escape variants, elements that are indispensable for the virus may be targeted. The 5-polyU tract of the antigenome offers one such target. Host cells do not harbor 5-polyU tracts on any of their transcripts, making the tract an attractive virus-specific target. We hypothesize that inhibiting the 5-polyU by complementary oligonucleotide can limit the use of the tract as template for virus to generate 3 polyA tails of RNA viruses. Here, we used a frameshift-inducing DNA oligonucleotide with 3 polyAs to target the 5-polyU tract of mouse coronavirus (MHV-A59). Results from assays for double stranded RNA (dsRNA) synthesis, infectivity of released virions, and syncytium formation indicate that the oligonucleotide treatment prevented generation of infectious virions. Our results show a unique mode of action of the designed 3-polyA oligonucleotide against mouse coronavirus which leaves host cells unaffected. This strategy can be adopted for the development of novel classes of oligonucleotide-based drugs that inhibit the production of infectious RNA viruses, including the coronaviruses. Since the 5-polyU tract is conserved and is essential for variants of coronaviruses, this strategy can potentially address coronavirus variant emergence as well.

category: microbiology


title: Mycobacterial virulence regulator integrates cAMP homeostasis with stress response and virulence

abstract: Survival of M. tuberculosis within the host macrophages requires presence of the virulence regulator PhoP, but the underlying mechanism is yet to be understood. We discovered a signalling pathway which controls mycobacterial cAMP-inducible gene expression and cAMP homeostasis. We show that the level of intra-mycobacterial cAMP, one of the most widely used second messengers, is regulated by the virulence regulator PhoP, which recruits the cAMP responsive protein, CRP. We provide evidence to show that PhoP -dependent repression of cAMP specific phosphodiesterase Rv0805, which degrades cAMP hydrolytically, accounts for mycobacterial cAMP homeostasis. In keeping with these findings, genetic manipulation to inactivate PhoP-Rv0805-cAMP pathway leads to disruption of cAMP homeostasis, increased stress sensitivity and most critically, reduced mycobacterial survival in macrophages and animal models. Together, PhoP-dependent cAMP inducible gene expression and cAMP homeostasis represent a molecular checkpoint during intra-phagosomal survival and growth program of mycobacteria.


title: Low expression of EXOSC2 protects against clinical COVID-19 and impedes SARS-CoV-2 replication

abstract: New therapeutic targets are a valuable resource in the struggle to reduce the morbidity and mortality associated with the COVID-19 pandemic, caused by the SARS-CoV-2 virus. Genome-wide association studies (GWAS) have identified risk loci, but some loci are associated with co-morbidities and are not specific to host-virus interactions. Here, we identify and experimentally validate a link between reduced expression of EXOSC2 and reduced SARS-CoV-2 replication. EXOSC2 was one of 332 host proteins examined, all of which interact directly with SARS-CoV-2 proteins; EXOSC2 interacts with Nsp8 which forms part of the viral RNA polymerase. Lung-specific eQTLs were identified from GTEx (v7) for each of the 332 host proteins. Aggregating COVID-19 GWAS statistics for gene-specific eQTLs revealed an association between increased expression of EXOSC2 and higher risk of clinical COVID-19 which survived stringent multiple testing correction. EXOSC2 is a component of the RNA exosome and indeed, LC-MS/MS analysis of protein pulldowns demonstrated an interaction between the SARS-CoV-2 RNA polymerase and the majority of human RNA exosome components. CRISPR/Cas9 introduction of nonsense mutations within EXOSC2 in Calu-3 cells reduced EXOSC2 protein expression, impeded SARS-CoV-2 replication and upregulated oligoadenylate synthase (OAS) genes, which have been linked to a successful immune response against SARS-CoV-2. Reduced EXOSC2 expression did not reduce cellular viability. OAS gene expression changes occurred independent of infection and in the absence of significant upregulation of other interferon-stimulated genes (ISGs). Targeted depletion or functional inhibition of EXOSC2 may be a safe and effective strategy to protect at-risk individuals against clinical COVID-19.

category: genetics


title: Discovery of Potent Pyrazoline-Based Covalent SARS-CoV-2 Main Protease Inhibitors

abstract: While vaccines and antivirals are now being deployed for the current SARS-CoV-2 pandemic, we require additional antiviral therapeutics to not only effectively combat SARS-CoV-2 and its variants, but also future coronaviruses. All coronaviruses have relatively similar genomes that provide a potential exploitable opening to develop antiviral therapies that will be effective against all coronaviruses. Among the various genes and proteins encoded by all coronaviruses, one particularly "druggable" or relatively easy-to-drug target is the coronavirus Main Protease (3CLpro or Mpro), an enzyme that is involved in cleaving a long peptide translated by the viral genome into its individual protein components that are then assembled into the virus to enable viral replication in the cell. Inhibiting Mpro with a small-molecule antiviral would effectively stop the ability of the virus to replicate, providing therapeutic benefit. In this study, we have utilized activity-based protein profiling (ABPP)-based chemoproteomic approaches to discover and further optimize cysteine-reactive pyrazoline-based covalent inhibitors for the SARS-CoV-2 Mpro. Structure-guided medicinal chemistry and modular synthesis of di- and tri-substituted pyrazolines bearing either chloroacetamide or vinyl sulfonamide cysteine-reactive warheads enabled the expedient exploration of structure-activity relationships (SAR), yielding nanomolar potency inhibitors against Mpro from not only SARS-CoV-2, but across many other coronaviruses. Our studies highlight promising chemical scaffolds that may contribute to future pan-coronavirus inhibitors.

category: biochemistry


title: Potent universal-coronavirus therapeutic activity mediated by direct respiratory administration of a Spike S2 domain-specific human neutralizing monoclonal antibody

abstract: Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) marks the third novel {beta}-coronavirus to cause significant human mortality in the last two decades. Although vaccines are available, too few have been administered worldwide to keep the virus in check and to prevent mutations leading to immune escape. To determine if antibodies could be identified with universal coronavirus activity, plasma from convalescent subjects was screened for IgG against a stabilized pre-fusion SARS-CoV-2 spike S2 domain, which is highly conserved between human {beta}-coronavirus. From these subjects, several S2-specific human monoclonal antibodies (hmAbs) were developed that neutralized SARS-CoV-2 with recognition of all variants of concern (VoC) tested (Beta, Gamma, Delta, Epsilon, and Omicron). The hmAb 1249A8 emerged as the most potent and broad hmAb, able to recognize all human {beta}-coronavirus and neutralize SARS-CoV and MERS-CoV. 1249A8 demonstrated significant prophylactic activity in K18 hACE2 mice infected with SARS-CoV-2 lineage A and lineage B Beta, and Omicron VoC. 1249A8 delivered as a single 4 mg/kg intranasal (i.n.) dose to hamsters 12 hours following infection with SARS-CoV-2 Delta protected them from weight loss, with therapeutic activity further enhanced when combined with 1213H7, an S1-specific neutralizing hmAb. As little as 2 mg/kg of 1249A8 i.n. dose 12 hours following infection with SARS-CoV Urbani strain, protected hamsters from weight loss and significantly reduced upper and lower respiratory viral burden. These results indicate in vivo cooperativity between S1 and S2 specific neutralizing hmAbs and that potent universal coronavirus neutralizing mAbs with therapeutic potential can be induced in humans and can guide universal coronavirus vaccine development.


title: Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause severe disease

abstract: Pan-betacoronavirus neutralizing antibodies may hold the key to developing broadly protective vaccines against coronaviruses that cause severe disease, for anticipating novel pandemic-causing viruses, and to respond more effectively to SARS-CoV-2 variants. The emergence of the Omicron variant of SARS-CoV-2 has illustrated the limitations of solely targeting the receptor binding domain (RBD) of the envelope Spike (S)-protein. Here, we isolated a large panel of broadly neutralizing antibodies (bnAbs) from SARS-CoV-2 recovered-vaccinated donors that target a conserved S2 region in the fusion machinery on betacoronavirus spikes. Select bnAbs show broad in vivo protection against all three pathogenic betacoronaviruses, SARS-CoV-1, SARS-CoV-2 and MERS-CoV, that have spilled over into humans in the past 20 years to cause severe disease. The bnAbs provide new opportunities for antibody-based interventions and key insights for developing pan-betacoronavirus vaccines.


title: mRNA-based vaccine candidate COReNAPCIN(R) induces robust humoral and cellular immunity in mice and non-human primates

abstract: At the forefront of biopharmaceutical industry, the messenger RNA (mRNA) technology offers a flexible and scalable platform to address the urgent need for world-wide immunization in pandemic situations. This strategic powerful platform has recently been used to immunize millions of people proving both of safety and highest level of clinical efficacy against infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we provide preclinical report of COReNAPCIN(R); a vaccine candidate against SARS-CoV-2 infection. COReNAPCIN(R) is a nucleoside modified mRNA-based vaccine formulated in lipid nanoparticles (LNPs) for encoding the full-length prefusion stabilized SARS-CoV-2 spike glycoprotein on the cell surface. Vaccination of C57BL/6 and BALB/c mice and rhesus macaque with COReNAPCIN(R) induced strong humoral responses with high titers of virus-binding and neutralizing antibodies. Upon vaccination, a robust SARS-CoV-2 specific cellular immunity was also observed in both mice and non-human primate models. Additionally, vaccination protected rhesus macaques from symptomatic SARS-CoV-2 infection and pathological damage to the lung upon challenging the animals with high viral loads of up to 2x108 live viral particles. Overall, our data provide supporting evidence for COReNAPCIN(R) as a potent vaccine candidate against SARS-CoV-2 infection for clinical studies.


title: Intragenomic rearrangements of SARS-CoV-2 and other β-coronaviruses

abstract:
Variation of the betacoronavirus SARS-CoV-2 has been the bane of COVID-19 control. Documented variation includes point mutations, deletions, insertions, and recombination among closely or distantly related coronaviruses. Here, we describe yet another aspect of genome variation by beta- and alphacoronaviruses. Specifically, we report numerous genomic insertions of 5-untranslated region sequences into coding regions of SARS-CoV-2, other betacoronaviruses, and alphacoronaviruses. To our knowledge this is the first systematic description of such insertions. In many cases, these insertions change viral protein sequences and further foster genomic flexibility and viral adaptability through insertion of transcription regulatory sequences in novel positions within the genome. Among human Embecorivus betacoronaviruses, for instance, from 65% to all of the surveyed sequences in publicly available databases contain 5-UTR-derived inserted sequences. In limited instances, there is mounting evidence that these insertions alter the fundamental biological properties of mutant viruses. Intragenomic rearrangements add to our appreciation of how variants of SARS-CoV-2 and other beta- and alphacoronaviruses may arise.

SignificanceUnderstanding mechanisms of variation in coronaviruses is vital to control of their associated diseases. Beyond point mutations, insertions, deletions and recombination, we here describe for the first time intragenomic rearrangements and their relevance to changes in transmissibility, immune escape and/or virulence documented during the SARS-CoV-2 pandemic.

category: molecular biology


title: Intragenomic rearrangements of SARS-CoV-2, other betacoronaviruses, and alphacoronaviruses


title: Predicted binding interface between coronavirus nsp3 and nsp4

abstract: Double membrane vesicles (DMVs) in coronavirus-infected cells feature pores that span both membranes. DMV pores were observed to have six-fold symmetry and include the nsp3 protein. Co-expression of SARS-CoV nsp3 and nsp4 induces DMV formation, and elements of nsp3 and nsp4 have been identified that are essential for membrane disruption. I describe a predicted luminal binding interface between nsp3 and nsp4 that is membrane-associated, conserved in SARS-CoV-2 during the COVID-19 pandemic and in diverse coronaviruses, and stable in molecular dynamics simulation. Combined with structure predictions for the full-length nsp4 monomer and cryo-EM data, this suggests a DMV pore model in which nsp4 spans both membranes with nsp3 and nsp4 inserted into the same bilayer. This approach may be able to identify additional protein-protein interactions between coronavirus proteins.


title: SARS-CoV-2 triggers Golgi fragmentation via downregulation of GRASP55 to facilitate viral trafficking

abstract: The ongoing COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an enveloped RNA virus. Despite the high economic and life losses caused by SARS-CoV-2, the detailed viral cycle, especially how it assembles and traffics in the secretory pathway, remains largely unknown. Here, we show that SARS-CoV-2 infection induces global alterations of the host endomembrane system, including dramatic Golgi fragmentation. Disrupting Golgi function with small molecules strongly inhibits viral infection. Furthermore, expression of several SARS-CoV-2 proteins individually is sufficient to trigger Golgi fragmentation. Significantly, SARS-CoV-2 infection down-regulates GRASP55 but up-regulates TGN46 expression, while expression of GRASP55 or knockdown of TGN46 reduces the infection rate of both USA-WA1 and Delta variants of SARS-CoV-2. Our study reveals that SARS-CoV-2 modulates Golgi structure and function via altering GRASP55 and TGN46 expression to facilitate viral trafficking, indicating the Golgi as a novel therapeutic target to block SARS-CoV-2 infection.

category: cell biology


title: SARS-CoV-2 triggers Golgi fragmentation via down-regulation of GRASP55 to facilitate viral trafficking


title: Cetacean "gas-bubble thromboembolic polycystic liver disease": "Budd-Chiari-like syndrome" in dolphins?

abstract: Nearly two decades ago, pathologic examination results suggested acoustic factors, such as mid-frequency active naval military sonar (MFAS) could be the cause of acute decompression-like sickness in stranded beaked whales. Acute systemic gas embolism in these beaked whales was published together with enigmatic cystic liver lesions (CLL), characterized by intrahepatic encapsulated gas-filled cysts, tentatively interpreted as "gas-bubble" lesions in various cetacean species. Here we provide a pathologic reinterpretation of CLL in cetaceans. From 1,200 cetaceans necropsied, CLL were only observed in striped dolphins (Stenella coeruleoalba), with a low prevalence (2%), and recapitulated pathologic features of Budd-Chiari syndrome in humans. Our results strongly suggest that CLL are the result of the combination of pre-existing or concomitant hepatic vascular disorder (e.g., severe hepatobiliary trematodiasis) superimposed and exacerbated by gas bubbles, and clearly differ from acute systemic gas embolism in stranded beaked whales linked to MFAS.

category: pathology


title: Morphofunctional changes at the active zone during synaptic vesicle exocytosis

abstract: The fusion of synaptic vesicles (SVs) with the plasma membrane (PM) proceeds through intermediate steps that remain poorly resolved. Additionally, the effect of persistent high or low exocytosis activity on intermediate steps remains unknown. Through time-resolved cryo-electron tomography, we ordered events into a sequence. Following stimulation, proximal tethered SVs rapidly form additional tethers with the PM. Simultaneously, fusion initiation occurs by membrane curvature ( buckling) of the SV and PM. It is followed by the formation of a fusion pore, and the collapse of SV membrane. At this time, membrane-proximal, but not membrane-distal, vesicles lose their interconnections, allowing them to move towards the PM. Two mutations of SNAP-25 that arrests or disinhibit spontaneous release, respectively, both caused a loss of interconnectors, while the disinhibiting mutant also caused a loss of membrane proximal multiple-tethered SVs. Overall, tether formation and connector dissolution is triggered by stimulation and respond to the spontaneous fusion rate. These morphological observations likely correspond to the transition of SVs from one functional pool to another.


title: Diurnal rhythm of temporal reproduction in the visual field.

abstract: The aim of this study was to elucidate how temporal reproduction changed in a diurnal time course. The overestimated reproduction showed a smaller difference to the standard in the morning and evening and a larger difference in the afternoon, and the underestimated reproduction showed an inverse change. No eccentricity effect in temporal production was observed, which indicates homogenous temporal processing shared in the perifoveal and peripheral visual fields.


title: Effect of repeated explicit instructions on visuomotor adaptation and intermanual transfer

abstract: The aim of the present study was to investigate the effect of repeated explicit instructions on visuomotor adaptation, awareness, and intermanual transfer. In a comprehensive study design, 48 participants performed center-out reaching movements before and during exposure to a 60{degrees} rotation of visual feedback. Awareness and intermanual transfer were then determined. Twelve participants each were assigned to one of the following adaptation conditions: gradual adaptation, sudden adaptation without instructions, sudden adaptation with a single instruction before adaptation, and sudden adaptation with multiple instructions before and during adaptation. The explicit instructions explained the nature of the visual feedback perturbation and were given using an illustration of a clock face. Analysis of adaptation indices revealed neither increased nor decreased adaptation after repeated instructions compared with a single instruction. Our data also show less adaptation with gradual than with sudden adaptation and less adaptation without than with instruction. These differences were pronounced at the beginning of adaptation; however, by the end of adaptation, all four groups reached similar levels of adaptation. In addition, we found significant group differences for the awareness index, with lower awareness after gradual adaptation than after sudden, instructed adaptation. Intermanual transfer did not differ between groups. However, we found a significant correlation between the awareness and intermanual transfer indices. We conclude that the magnitude of the explicit process cannot be further increased by repeated instruction and that intermanual transfer appears to be largely related to the explicit adaptation process.


title: A guide to examining intramuscular fat formation and its cellular origin in skeletal muscle

abstract: Fibro-adipogenic progenitors (FAPs) are mesenchymal stromal cells that play a crucial role during skeletal muscle homeostasis and regeneration. FAPs build and maintain the extracellular matrix that acts as a molecular myofiber scaffold. In addition, FAPs are indispensable for myofiber regeneration as they secrete a multitude of beneficial factors sensed by the muscle stem cells (MuSCs). In diseased states, however, FAPs are the cellular origin of intramuscular fat and fibrotic scar tissue. This fatty fibrosis is a hallmark of sarcopenia and neuromuscular diseases, such as Duchenne Muscular Dystrophy. One significant barrier in determining why and how FAPs differentiate into intramuscular fat is effective preservation of adipocytes, especially in frozen tissue sections. Conventional methods of skeletal muscle tissue processing, such as snap-freezing, do not properly preserve the morphology of individual adipocytes, thereby preventing accurate visualization and quantification. Here, we describe a protocol that provides robust preservation of adipocyte morphology in skeletal muscle sections allowing visualization, imaging, and quantification of intramuscular fat. We also outline how to process a portion of muscle tissue for RT-qPCR, enabling users to confirm observed changes in fat formation by viewing differences in expression of adipogenic genes. Additionally, we will describe how our protocol can be adopted to visualize adipocytes by whole mount immunofluorescence of muscle samples. Finally, we will outline how to combine this protocol with genetic lineage tracing of Pdgfr-expressing FAPs to study the adipogenic conversion of FAPs. Our protocol consistently yields high-resolution and morphologically accurate immunofluorescent images of adipocytes that, along with confirmation by RT-qPCR, allows for robust, rigorous, and reproducible visualization and quantification of intramuscular fat. Together, our analysis pipeline is the first step to improve our understanding of how FAPs differentiate into intramuscular fat and provides a framework to validate novel interventions to prevent fat formation.

category: developmental biology


title: Anthroponumbers.org: A Quantitative Database Of Human Impacts on Planet Earth

abstract:
The Human Impacts Database (www.anthroponumbers.org) is a curated searchable resource housing quantitative data relating to the diverse anthropogenic impacts on our planet, with topics ranging from sea level rise, to livestock populations, greenhouse gas emissions, fertilizer use, and beyond. Each entry in the database relates a quantitative value (or a time-series of values) along with clear referencing of the primary source, the method of measurement or estimation, an assessment of uncertainty, links to the underlying data, as well as a permanent identifier called an Human Impacts ID ("HuID"). While there are other databases that house some of these values, they are typically focused on a single topic area like energy usage or greenhouse gas emissions. The Human Impacts Database provides centralized access to quantitative information about the myriad ways in which humans impact the Earth, giving links to more specialized databases for interested readers. Here, we outline the structure of the database and describe our curation procedures. Finally, we use this database to generate a graphical summary of the current state of human impacts on the Earth, illustrating both their numerical values and their dense interconnections.

The Bigger PictureOver the last 10,000 years, human activities have transformed the Earth through farming, forestry, mining and industry. The complex results of these activities are now observed and quantified as "human impacts" on Earths atmosphere, oceans, biosphere and geochemistry. While myriad studies have explored facets of human impacts on the planet, they are necessarily technical and often tightly-focused. Thus, finding reliable quantitative information requires a significant investment of time to assess each quantity, its methods of determination, and associated uncertainty. We present the Human Impacts Database (www.anthroponumbers.org), which houses a diverse array of such quantities. We review a subset of these values and how they help build intuition for understanding the Earth-human system. While collation alone does not tell us how to best ameliorate human impacts, we contend that any future plans should be made in light of a quantitative understanding of the interconnected ways in which humans impact the planet.

category: ecology


title: Whole-genome sequencing of Bacillus thuringiensis strain SY49.1 reveals the detection of novel candidate pesticidal and bioactive compounds isolated from Turkey

abstract:
BackgroundBacillus thuringiensis SY49.1 (Bt SY49.1) strain is a new strain isolated from a soil sample in Adana city which is nestled in the most fertile agricultural area in Turkey. This strain has insecticidal activity against insect pests from different orders. Also, it is characterized by its significant activity against plant fungal infections and as plant growth promotion.

AimsTo report the whole genome sequencing (WGS) and annotation of the Bt SY49.1 strain.

Materials and MethodsThe Bt SY49.1 strain was isolated from the soil sample in Adana city by using a sodium acetate enriched medium. Bacterial DNA was extracted then sequenced using Illumina HiSeq technology. After data filtering and quality control, de novo assembly and genomic annotation were applied. Evolutionary and comparative genomic analysis and variant calling were performed using various in silico tools.

ResultsThe WGS of strain Bt SY49.1 is 6. 32 Mbp long with a GC content of 34.68%. It reveals large numbers of single nucleotide polymorphisms (SNPs) and InDels. The assembled genome contains 6,562 protein-encoding genes. In addition, it encodes various gene inventories for the biosynthesis of bioactive compounds such as insecticidal delta endotoxins, growth stimulatory deaminase and phosphatases, fungicidal thuricin, bacillibactin, petrobactin, fengycin / plipastin, and zwittermycin A.

ConclusionThe strain Bt SY49.1 could have several potential utilities as a source of antibiotics compounds, plant growth promoting metabolites, and biocontrol agents for fungal phytopathogens, and insects. We expect that the draft genome of the Bt SY49.1 strain may provide a model for proper understanding and studying of antimicrobial compound mining, genetic diversity among the B. cereus group, and pathogenicity against insect pests and plant diseases, and growth-promoting activity.

category: genomics


title: Genetic heritage of the BaPhuthi highlights an over ethnicised notion of 'Bushman' in the Maloti-Drakensberg, Southern Africa

abstract: Using contemporary people as proxies for ancient communities is a contentious but necessary practice in anthropology. In Southern Africa, the distinction between the Cape KhoeSan and eastern KhoeSan remains unclear as ethnicity labels are continually changed through time and most communities were extirpated. The eastern KhoeSan may reflect an  essentialistic biological distinction from neighbouring Bantu-speaking communities or it may not be tied to  race and instead denote communities with a nomadic  life-way distinct from agro-pastoralism. The BaPhuthi of the 1800s in the Maloti-Drakensberg, Southern Africa had a substantial San constituency and a life-way of nomadism, cattle raiding, and horticulture. The BaPhuthi heritage could provide insights into the history of the eastern KhoeSan. We examine for the first time genetic affinities of 23 BaPhuthi to distinguish if KhoeSan ancestry reflects biologically distinct heritage or a shared life-way. Data were merged with 52 global populations. The Principle Component Analysis, ADMIXTURE clustering and F3 tests show no support for a unique eastern KhoeSan ancestry distinct from other KhoeSan or southern Bantu-speaking communities. The BaPhuthi have strong affinities with Nguni communities, as the non-Nguni show strong evidence of recent African admixture possibly related to late-iron age migrations. The BaPhuthi may have an interesting connection to the early iron-age Bantu-speaking communities as MALDER detected no signals for late-iron age admixture. We demonstrate how the  essentialistic understanding of references in historic literature creates misconstrued notions of ethnic/biological distinctions when  San and  Bushman may have reflected ambiguous references to the non-sedentary polities and practices.


title: Colitis-associated intestinal microbiota regulates brain glycine and host behavior in mice.

abstract: Inflammatory bowel diseases (IBD) are chronic and relapsing inflammatory disorders of the gastrointestinal tract with complex etiology and no strategies for complete cure. IBD are often complicated by mental disorders like anxiety and depression, indicating substantial shifts in the gut-brain axis. However, the mechanisms connecting IBD to mental diseases are still under debate. Here we use Muc2 mutant mouse model of chronic colitis to uncouple the effects of the intestinal microbiota on host behavior from chronic inflammation in the gut. Muc2 mutant male mice exhibit high exploratory activity, reduced anxiety-related behaviors, impaired sensorimotor gating, and altered social preference towards males and females. Microbial transfer to wild-type mice via littermate co-housing shows that colitis-associated microbiota rather than inflammation per se defines behavioral features in Muc2 colitis model. Metagenomic profiling and combination of antibiotic treatments revealed that bacterial species Akkermansia muciniphila is associated with the behavioral phenotype in mutants, and that its intestinal abundance correlates with social preference towards males. Metabolomic analysis together with pharmacological inhibition of Gly and NMDA receptors helped us to determine that brain glycine is responsible for the behavioral phenotype in Muc2 mice. Blood and brain metabolic profiles suggest that microbiota-dependent changes in choline metabolism might be involved in regulation of central glycine neurotransmission. Taken together, our data demonstrates that colitis-associated microbiota controls anxiety, sensorimotor gating and social behavior via metabolic regulation of the brain glycinergic system, providing new venues to combat neurological complications of IBD.

category: animal behavior and cognition


title: Types of anomalies in two-dimensional video-based gait analysis in uncontrolled environments

abstract:
Two-dimensional video-based pose estimation is a technique that can estimate human skeletal coordinates from video data alone. It is also being applied to gait analysis and, particularly, due to its simplicity of measurement, it has the potential to be applied to the gait analysis of large populations. In contrast, it is considered difficult to completely homogenize the environment and settings during the measurement of large populations. Therefore, it is necessary to appropriately deal with technical errors that are not related to the biological factors of interest. In this study, by analyzing a large cohort database, we have identified four major types of anomalies that occur during gait analysis using OpenPose in uncontrolled environments: anatomical, biomechanical, and physical anomalies and errors due to estimation. We have also developed a workflow for identifying and correcting those anomalies and confirmed that the workflow is reproducible through simulation experiments. Our results will help obtain a comprehensive understanding of the anomalies to be addressed in a pre-processing for 2D video-based gait analysis of large populations.

Author summaryGait is one of the important biomarkers of health conditions. With developing mobile health technologies, it is becoming easier to measure our health. However, to realize preventive medicine, establishing evidence is a critical issue, and we need to collect data from a large population. Two-dimensional video-based pose estimation can be a solution for the gait analysis of such a population. However, the technical accuracy and limitations of this analysis have not yet been sufficiently discussed. In this study, by analyzing the largest database currently available, we systematically identified four types of technical anomalies that occur during gait measurement: anatomical, biomechanical, and physical anomalies and errors dues to estimation. We have also shown how to deal with these issues and made solutions available as software so that researchers can reproduce them. In the future, increasing number of studies will use 2D video-based pose estimation to research health-related gait among large populations. We believe that our work will provide a guideline for researchers and clinicians involved in these studies to discuss design and algorithms.

category: physiology


title: The Metabolomics Workbench File Status Website: A Metadata Repository Promoting FAIR Principles of Metabolomics Data

abstract:
MotivationAn updated version of the mwtab Python package for programmatic access to the Metabolomics Workbench (MetabolomicsWB) data repository was released at the beginning of 2021. Along with updating the package to match the changes to MetabolomicsWBs  mwTab file format and to enhance the packages functionality, the package included format validation facilities which were used to detect and catalog file inconsistencies and errors across all publically available datasets in MetabolomicsWB.

ResultsThe Metabolomics Workbench File Status website was developed to provide continuous validation of MetabolomicsWB data files and a useful interface to find all inconsistencies and errors that are found. This list of detectable issues/errors include format parsing errors, format compliance issues, access problems via MetabolomicsWBs REST interface, and other small inconsistencies that can hinder reusability. The website uses the mwtab Python package to pull down and validate each available analysis file and then generates an html report. The website is updated on a weekly basis. Moreover, the Python website design utilizes GitHub and GitHub.io, providing an easy to replicate template for implementing other metadata, virtual, and meta-repositories.

AvailabilityMetabolomics Workbench file status website can be accessed at: https://moseleybioinformaticslab.github.io/mwFileStatusWebsite/. The mwtab Python library is available on GitHub: https://github.com/MoseleyBioinformaticsLab/mwtab, PyPI: https://pypi.org/project/mwtab/, and documentation is available on ReadTheDocs: https://mwtab.readthedocs.io/. Metabolomics Workbench analysis data files used for analysis presented here along with the generated HTML files of the website are available on FigShare: https://doi.org/10.6084/m9.figshare.19221159.

Contacthunter.moseley@uky.edu

Supplementary informationSupplementary data are available at FigShare online.

category: systems biology


title: Anti-phasic oscillatory development for speech and noise processing in cochlear implanted toddlers

abstract:
Human brain demonstrates amazing readiness for speech and language learning at birth, but the auditory development preceding such readiness remains unknown. Cochlear implanted (CI) children with prelingual deafness provide a unique opportunity to study this developmental stage. Using functional near-infrared spectroscopy, we revealed that the brain of CI children was nearly irresponsive to sounds at CI hearing onset. With increasing CI experiences up to 32 months, the brain demonstrated function, region and hemisphere specific development. Most strikingly, the left anterior temporal lobe showed an oscillatory trajectory, changing in opposite phases for speech and noise. In addition, speech responses increased linearly in left sylvian parieto-temporal area and right inferior frontal gyrus, and noise responses changed in U shape in right supramarginal gyrus. Such cortical development predicted behavioral improvement. The study provides the first longitudinal brain imaging evidence for early auditory development preceding speech acquisition in the human brain.

Graphic abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/483211v1_ufig1.gif" ALT="Figure 1">
View larger version (39K):
org.highwire.dtl.DTLVardef@d84974org.highwire.dtl.DTLVardef@1be9ba0org.highwire.dtl.DTLVardef@db5fb7org.highwire.dtl.DTLVardef@68f420_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LIThe brain of cochlear implanted toddlers is functionally inferior to neonates
C_LIO_LIHearing experiences drive functional development in auditory related cortical areas
C_LIO_LISpeech and noise processing develop in anti-phasic oscillatory trajectories
C_LIO_LIAuditory cortical development predicts post-implantation behavioral improvement
C_LI


title: Targeting neuronal homeostasis to prevent seizures

abstract: Manipulating neuronal homeostasis, which enables neurons to regulate their intrinsic excitability, offers an attractive opportunity to prevent seizures. However, no anticonvulsant compounds have yet been reported that directly manipulate neuronal homeostasis. Here, we describe a novel class of anticonvulsant compounds, based on 4-tert-butyl-benzaldehyde (4-TBB), with a mode-of-action that includes increased expression of the homeostatic regulator Pumilio (Pum). In Drosophila and mouse we use a pentylenetetrazole (PTZ) induced seizure model, and an electrically induced seizure model for refractory seizures to evaluate anticonvulsant efficacy. The pyrazole analogue (RAB216) demonstrates best efficacy, protecting 50% of mice from PTZ-induced seizure. Knock-down of Pum, in Drosophila, blocks anticonvulsive effects, whilst analysis of validated Pum targets show significant reductions following exposure of mouse brain to 4-TBB. This study provides proof-of-principle that anticonvulsant effects can be achieved through regulation of neuronal homeostasis and identifies a chemical lead compound for future development.


title: Evaluation of the antimicrobial, anti-adhesion, anti-biofilm and cell proliferation assay of a de-novo goji berry extract against periodontal pathogens: a comparative in-vitro study

abstract: Lycium barbarum, commonly known as goji berry, Chinese berry, or Tibetian berry, is emerging as a popular "superfood" with anti-inflammatory and antioxidant properties. Goji berry is being used for the treatment of various cancers, gastrointestional disorders, stroke, diabetes, Alzheimers disease, and glaucoma. However, its use for management of oral inflammatory diseases has not been explored. Therefore, the present study aims to evaluate the antimicrobial, anti-adhesion, and anti-biofilm, and cytotoxic properties of an ethanolic extract of Lycium barbarum (LBE) against oral and periodontal pathogens. The antimicrobial properties of LBE against five microorganisms were tested and compared against Chlorhexidine and doxycycline along with cytotoxicity and cell viability on the gingival fibroblast and modified keratinocyte cell lines. The anti-adhesion and anti-biofilm properties of LBE against Porphyromonas gingivalis, at its minimal bactericidal value, were evaluated. The antimicrobial, anti-adhesion and antibiofilm properties of LBE were found to be comparable to chlorhexidine but less than that of doxycycline. The LBE extract was also compactible to gingival fibroblast tissues and oral keratinocytes at 1 mg/ml. The results proved that goji berry is as effective as chlorhexidine and can be used as a promising natural herb for the management of inflammatory diseases of oral cavity.


title: Zoonotic Tick Borne Pathogens in Ticks at Human-Wildlife-Livestock interfaces in Kwale County, Kenya

abstract:
BackgroundTicks are vectors that can harbor pathogens like viruses, protozoa and bacteria that can cause zoonotic diseases in human. Human gets infected through tick bites where the pathogens are passed to the human blood. One tick bite can lead to transmission of more than one disease. Ticks are obligate hematophagous, infest most vertebrates globally. Lack of surveillance information about the tick-borne pathogens and diseases has made it impossible to assess its impact to the community.

Materials and MethodsWe morphologically identified ticks collected from two game reserves in Kenya, locations near the Shimba Hills National Reserve (SHNR) and Tsavo National Reserve (TNR). We identified tick-borne pathogens by HRM analysis and sequencing of specific RT-PCR products of Anaplasma, Ehrlichia, and Rickettsia.

ResultsA total of 317 (281 adult ticks and 36 nymphs) was collected at near Tsavo National Reserve in Taita Taveta County, which includes seven species. Amblyomma was the most sampled genus with Amblyomma gemma being the most sampled species (n=135, 42.6%). Other Amblyomma species sampled was Amblyomma Variegatum (n= 40, 12.62%). Greatest species diversity was identified in Rhipicephalus genus with four species identified that includes; Rhipicephalus appendiculatus (n=44, 13.9%), Rhipicephalus Averts (n=1, 0.31%), Rhipicephalus Decoloratus (n=5, 1.6%), Rhipicephalus Pulchellus (n= 91, 28.7%). A single species of Hyalomma sp. was sampled. From near Shimba Hill game reserve (SHNR), a total of 240 adults ticks were sampled that comprises of eight species. Amblyomma was the most sampled genus and again Amblyomma gemma being the most sampled species (n=156, 65 %). Other Amblyomma species sampled includes; Amblyomma Lepidum (n= 5, 2.1 %), Amblyomma Variegatum (n= 15, 6.3 %). Greatest species diversity was also identified in Rhipicephalus genus with four species identified that includes; Rhipicephalus appendiculatus (n=18, 7.5 %), Rhipicephalus Averts (n=6, 2.5 %), Rhipicephalus Decoloratus (n=4, 1.7 %), Rhipicephalus Pulchellus (n= 34, 14.2 %). The least sampled species was a single species of Hyalomma Scupense (n=2, 0.83 %). At near Tsavo National Reserve (TNR) in Taita Taveta County, a total of three pools of Rhipicephaline appendiculatus were positive for Theileria parva, two pools of Rhipicephaline evertsi for Anaplasma platys and one pool of Amblyomma variegatum nymphs for Rickettsia africae. From near Shimba Hill game reserve (SHNR), Kwale County, Rickettsia africae pathogen was detected in two pools of Am. variegatum and one pool of Am. Gemma. Rickettsia sp. and Anaplasma sp. were detected in Am. Gemma and Rh. evertsi respective. R. aeschlimannii was detected in a pool of Am. Gemma. These findings highlight the risk of transmission of zoonotic R. africae and unclassified Rickettsia spp. to humans causing African tick-bite fever and other spotted-fever group rickettsioses.


title: Comparing lipid remodeling in mouse adipose and liver tissue with quantitative Raman microscopy

abstract: Brown adipose tissue (BAT) consists of highly metabolically active adipocytes that catabolize nutrients to produce heat. Playing an active role in triacylglycerol (TAG) clearance, research has shown that dietary fatty acids can modulate the TAG chemistry deposition in BAT after weeks-long dietary intervention, similar to what has been shown in white adipose tissue (WAT). Our objective was to compare the influence of sustained, non-chronic dietary intervention (a one-week interval) on WAT and BAT lipid metabolism and deposition in situ. We use quantitative, label-free chemical microscopy to show that one week of HFD intervention results in dramatically larger lipid droplet (LD) growth in BAT (and liver) compared to LD growth in inguinal WAT (IWAT). Moreover, BAT showed lipid remodeling as increased unsaturated TAGs in LDs, resembling the dietary lipid composition, while WAT (and liver) did not show lipid remodeling on this time scale. Concurrently, expression of genes involved in lipid metabolism, particularly desaturases, was reduced in BAT and liver from HFD-fed mice after one week. Our data show that BAT lipid chemistry remodels exceptionally fast to dietary lipid intervention compared WAT, which further points towards a role in TAG clearance.


title: Rapid phenotypic differentiation and local adaptation in Japanese knotweed s.l. (Reynoutria japonica and R. x bohemica, Polygonaceae) invading novel habitats

abstract:
PREMISEMany plant invaders like the Japanese knotweeds are thought to colonize new habitats with low genetic diversity. Such species provide an opportunity to study rapid adaptation to complex environmental conditions.

METHODSUsing replicate reciprocal transplants of clones across three habitats, we described patterns of phenotypic response and assessed degree of local adaptation.

KEY RESULTSWe found plants from beach habitats had decreased height, number of leaves, leaf area, and biomass allocation to roots and shoots compared to plants from marsh and roadside habitats when grown in their home habitat. In the marsh habitat, marsh plants were generally larger than beach plants, but not different from roadside plants. There were no differences among plants from different habitats grown in the roadside habitat. Despite this evidence of differentiation in beach and marsh habitats, we found mixed evidence for local adaptation. In their "home site" plants from the marsh habitat had greater biomass than plants from the beaches but not compared to plants from roadsides. Biomass comparisons in other habitats were either maladaptive or not significant. However, plants from the roadside had greater survival in their "home site" compared to foreign plants. There were no differences in survival in the other habitats.

CONCLUSIONSWe found phenotypic differentiation associated with habitats despite the low reported genetic diversity for these populations. Our results partially support the hypothesis of local adaptation in marsh and roadside habitats. Identifying whether these patterns of differentiation result from genetic or heritable non-genetic mechanisms will require further work.


title: An updated map of GRCh38 linkage disequilibrium blocks based on European ancestry data

abstract: A map of approximately independent linkage disequilibrium (LD) blocks has many uses in statistical genetics. Current LD block maps are based on sparse recombination maps and only available for GRCh37 (hg19) and prior genome assemblies. We generated LD blocks for European (EUR) ancestry populations using a new large recombination map in GRCh38.


title: Candidates for Drug Repurposing to Address the Cognitive Symptoms in Schizophrenia

abstract: In the protein-protein interactome, we have previously identified a significant overlap between schizophrenia risk genes and genes associated with cognitive performance. Here, we further studied this overlap to identify potential candidate drugs for repurposing to treat the cognitive symptoms in schizophrenia. We first defined a cognition-related schizophrenia interactome from network propagation analyses, and identified drugs known to target more than one protein within this network. Thereafter, we used gene expression data to further select drugs that could counteract schizophrenia-associated gene expression perturbations. Additionally, we stratified these analyses by sex to identify sex-specific pharmacological treatment options for the cognitive symptoms in schizophrenia. After excluding drugs contraindicated in schizophrenia, we identified eight drug candidates, most of which have anti-inflammatory and neuroprotective effects. Due to gene expression differences in male and female patients, four of those drugs were also selected in our male-specific analyses, and the other four in the female-specific analyses. Based on our bioinformatics analyses of disease genetics, we suggest eight candidate drugs that warrant further examination for repurposing to treat the cognitive symptoms in schizophrenia, and suggest that these symptoms could be addressed by sex-specific pharmacological treatment options.


title: Medial septum activation improves strategy switching once strategies are well learned via bidirectional regulation of dopamine neuron population activity

abstract:
Strategy switching is a form of cognitive flexibility that requires inhibiting a previously successful strategy and then switching to a new strategy of a different categorical modality. It is dependent on dopamine (DA) receptor activation and release in ventral striatum and prefrontal cortex, two primary targets of ventral tegmental area (VTA) DA projections. Although the circuitry that underlies strategy switching early in learning has been studied, few studies have examined it after extended discrimination training. This may be important as DA activity and release patterns change across learning, with several studies demonstrating a critical role for substantia nigra pars compacta (SNc) DA activity and release once behaviors are well learned.

Our previous studies demonstrated that medial septum (MS) activation simultaneously increased VTA and decreased SNc DA population activity, as well as improved reversal learning via these actions on DA population activity. We hypothesized that MS activation would improve strategy switching both early in learning and after extended training through its ability to increase VTA DA population activity and decrease SNc DA population activity, respectively. To test this, we activated the MS of male and female rats with designer receptors exclusively activated by designer drugs and measured their performance on an operant-based strategy switching task, following 1, 10, or 15 days of discrimination training. Contrary to our hypothesis, MS activation did not affect strategy switching after 1 day of discrimination training. MS activation improved strategy switching after 10 days of discrimination training, but only in females. MS activation improved strategy switching in both sexes after 15 days of discrimination training. This improvement in strategy switching was attenuated by intra-ventral subiculum bicuculline infusion, which selectively inhibited the MS-mediated decrease in SNc DA population activity, and prevented by infusion of both bicuculline and scopolamine, which inhibited both the MS-mediated decrease in SNc and increase in VTA DA population activity. These data indicate that MS activation improves strategy switching, but only once the original strategy has been sufficiently well learned. They also suggest that the mechanism by which this occurs is likely via the MSs regulation of DA neuron responsivity, primarily via its ability to down-regulate DA population activity in the SNc.


title: Physiologically Based Multiphysics Pharmacokinetic Model for Determining the Temporal Biodistribution of Targeted Nanoparticles

abstract:
Nanoparticles (NP) are being increasingly explored as vehicles for targeted drug delivery because they can overcome free therapeutic limitations by drug encapsulation, thereby increasing solubility and transport across cell membranes. However, a translational gap exists from animal to human studies resulting in only several NP having FDA approval. Because of this, researchers have begun to turn toward physiologically based pharmacokinetic (PBPK) models to guide in vivo NP experimentation. However, typical PBPK models use an empirically derived framework that cannot be universally applied to varying NP constructs and experimental settings. The purpose of this study was to develop a physics-based multiscale PBPK compartmental model for determining continuous NP biodistribution. We successfully developed two versions of a physics-based compartmental model, models A and B, and validated the models with experimental data. The more physiologically relevant model (model B) had an output that more closely resembled experimental data as determined by normalized root mean squared deviation (NRMSD) analysis. A branched model was developed to enable the model to account for varying NP sizes. With the help of the branched model, we were able to show that branching in vasculature causes enhanced uptake of NP in the organ tissue. The models were solved using two of the most popular computational platforms, MATLAB and Julia. Our experimentation with the two suggests the highly optimized ODE solver package DifferentialEquations.jl in Julia outperforms MATLAB when solving a stiff system of ordinary differential equations (ODEs). We experimented with solving our PBPK model with a neural network using Julias Flux.jl package. We were able to demonstrate that a neural network can learn to solve a system of ODEs when the system can be made non-stiff via quasi-steady-state approximation (QSSA). In the future, this model will incorporate modules that account for varying NP surface chemistries, multiscale vascular hydrodynamic effects, and effects of the immune system to create a more comprehensive and modular model for predicting NP biodistribution in a variety of NP constructs.

Author summaryNanoparticles (NP) have been used in various drug delivery contexts because they can target specific locations in the body. However, there is a translational gap between animals and humans, so researchers have begun toward computational models to guide in vivo NP experimentation. Here, we present several versions of physics-based multiscale physiologically based pharmacokinetic models (PBPK) for determining NP biodistribution. We successfully developed two versions of ODE-based compartmental models (models A and B) and an ODE-based branched vascular model implemented in MATLAB and Julia and validated models with experimental data. Additionally, we demonstrated using a neural network to solve our ODE system. In the future, this model can integrate different NP surface chemistries, immune system effects, multiscale vascular hydrodynamic effects, which will enhance the ability of this model to guide a variety of in vivo experiments.

category: bioengineering


title: CF-Seq, An Accessible Web Application for Rapid Re-Analysis of Cystic Fibrosis Pathogen RNA Sequencing Studies

abstract: Researchers studying cystic fibrosis (CF) pathogens have produced numerous RNA-seq datasets which are available in the gene expression omnibus (GEO). Although these studies are publicly available, substantial computational expertise and manual effort are required to compare similar studies, visualize gene expression patterns within studies, and use published data to generate new experimental hypotheses. Furthermore, it is difficult to filter available studies by domain-relevant attributes such as strain, treatment, or media, or for a researcher to assess how a specific gene responds to various experimental conditions across studies. To reduce these barriers to data re-analysis, we have developed an R Shiny application called CF-Seq, which works with a compendium of 147 studies and 1,446 individual samples from 13 clinically relevant CF pathogens. The application allows users to filter studies by experimental factors and to view complex differential gene expression analyses at the click of a button. Here we present a series of use cases that demonstrate the application is a useful and efficient tool for new hypothesis generation. (CFSeq: http://scangeo.dartmouth.edu/CFSeq/)

category: bioinformatics


title: Alterations of the axon initial segment in multiple sclerosis

abstract: Grey matter damage has been established as a key contributor to disability progression in multiple sclerosis. Aside from neuronal loss and axonal transections, which predominate in cortical demyelinated lesions, synaptic alterations have been detected in both demyelinated plaques and normal-appearing grey matter, resulting in functional neuronal damage. The axon initial segment is a key element of neuronal function, responsible for action potential initiation and maintenance of neuronal polarity. Despite several reports of profound axon initial segment alterations in different pathological models, among which experimental auto-immune encephalomyelitis, whether the axon initial segment is affected in multiple sclerosis is still unknown. Using immunohistochemistry, we analyzed axon initial segments from control and multiple sclerosis tissue, focusing on layer 5/6 pyramidal neurons in the neocortex and Purkinje cells in the cerebellum and performed analysis on the parameters known to control neuronal excitability, i.e., axon initial segment length and position. We found that the axon initial segment length was unchanged among the different multiple sclerosis samples, and not different from controls. In contrast, in both cell types, the axon initial segment position was altered, with an increased soma-axon initial segment gap, in both active and inactive demyelinated lesions. In addition, using a computational model, we show that this increased gap between soma and axon initial segment might increase neuronal excitability. Taken together, these results show for the first time changes of axon initial segments in multiple sclerosis, in active as well as inactive grey matter lesions in both neocortex and cerebellum, which might alter neuronal function.


title: Heterotrimeric G proteins regulate planarian regeneration and behavior

abstract: G protein-coupled receptors (GPCRs) play broad roles in development and stem cell biology, but few roles for GPCR signaling in complex tissue regeneration have been uncovered. Planarian flatworms robustly regenerate all tissues and provide a model with which to explore potential functions for GPCR signaling in somatic regeneration and pluripotent stem cell biology. As a first step toward exploring GPCR function in planarians, we investigated downstream signal transducers that work with GPCRs, called heterotrimeric G proteins. Here, we characterized the complete heterotrimeric G protein complement in Schmidtea mediterranea for the first time and found that seven heterotrimeric G protein subunits promote regeneration. We further characterized two subunits critical for regeneration, Gq1 and G{beta}1-4a, finding that they promote the late phase of anterior polarity re-establishment, likely through anterior pole-produced Follistatin. Incidentally, we also found that five heterotrimeric G proteins modulate planarian behavior. We further identified a putative serotonin receptor, gcr052, that we propose works with G{beta}x2 in planarian locomotion, demonstrating the utility of our strategy for identifying relevant GPCRs. Our work provides foundational insight into roles of heterotrimeric G proteins in planarian biology and serves as a useful springboard towards broadening our understanding of GPCR signaling in adult tissue regeneration.


title: Proteomic Profiling of Hypoplastic Lungs Suggests an Underlying Inflammatory Response in the Pathogenesis of Abnormal Lung Development in Congenital Diaphragmatic Hernia

abstract: The pathogenesis of lung hypoplasia in congenital diaphragmatic hernia (CDH), a common birth defect, is poorly understood. The diaphragmatic defect can be repaired surgically, but the abnormal lung development contributes to a high mortality in these patients. To better understand the underlying pathobiology, we used the nitrofen rat model of CDH and characterized the proteome of hypoplastic CDH lungs at the alveolar stage (E21). Amongst the 218 significantly altered proteins between CDH and control lungs were Tenascin C, CREBBP, LYN and STAT3. We showed that Tenascin C was decreased around the distal airway branches in nitrofen and human fetal CDH lungs. In contrast, STAT3 was significantly increased in the airway epithelium of nitrofen lungs at E21. STAT3 inhibition after direct nitrofen exposure to fetal rat lung explants (E14.5) partially reversed the hypoplastic lung phenotype ex vivo by increasing peripheral lung budding. Moreover, we demonstrated that several STAT3 associated cytokines (IL-15, IL-9, IL-2) are increased in fetal tracheal aspirates of CDH survivors compared to non-survivors after fetoscopic tracheal occlusion. Using pathway analysis for significantly altered proteins in our proteomic analysis, we observed an enrichment in inflammatory response associated with Epstein Barr Virus and cytokine signaling in nitrofen CDH lungs. However, we were unable to detect EBV mRNA via in-situ Hybridization in human CDH lungs. With our unbiased proteomics approach, we show for the first time that inflammatory processes are likely underlying the pathogenesis of abnormal lung development in CDH.


title: Canine saliva is a source of interspecies antimicrobial resistance gene transfer

abstract: While the One Health issues of intensive animal farming are commonly discussed, keeping companion animals is less associated with the interspecies headway of antimicrobial resistance. With the constant advance of veterinary standards, antibiotics are regularly applied in companion animal medicine. Due to the close coexsistance of dogs and humans, dog bites and other casual encounters with dog saliva (e.g. licking the owner) are common. According to our metagenome studies based on 26 new generation sequencing canine saliva datasets from 2020 and 2021 reposited in NCBI SRA by The 10,000 Dog Genome Consortium and the Broad Institute within Darwins Ark project, canine saliva is rich in bacteria with predictably transferable antimicrobial resistance genes (ARGs). In the genom of potentially pathogenic Bacteroides, Capnocytophaga, Corynebacterium, Fusobacterium, Pasteurella, Porphyromonas, Staphylococcus and Streptococcus species, that are some of the most relevant bacteria in dog bite infections, ARGs against aminoglycosides, carbapenems, cephalosporins, glycylcyclines, lincosamides, macrolides, oxazolidinone, penams, phenicols, pleuromutilins, streptogramins, sulfonamides and tetracyclines could be identfied. Several ARGs, including ones against amoxicillin-clavulanate, the most commonly applied antibiotic by dog bites, was predicted to be potentially transferable based on their association with mobile genetic elements (e.g. plasmids, phages, integrated mobile genetic elements). According to our findings canine saliva may be a source of transfer of ARG-rich bacteria, that can either colonize the human body or transport ARGs to the host bacteriota and thus can be considered as a risk in the spread of antimicrobial resistance.


title: SARS-CoV-2 Spike evolution influences GBP and IFITM sensitivity

abstract: SARS-CoV-2 spike requires proteolytic processing for viral entry. The presence of a polybasic furin-cleavage site (FCS) in spike, and evolution towards an optimised FCS by dominant variants of concern (VOCs), are linked to enhanced infectivity and transmission. Guanylate binding proteins (GBP) are interferon-inducible restriction factors that target furin-mediated processing of viral envelope proteins and limit infectivity. Here we investigated whether GBPs restrict SARS-CoV-2 infection, and whether VOCs have evolved spikes that escape restriction. We show that GBP2 and 5 interfere with cleavage of the spike proteins of Wuhan-Hu-1, Alpha, Delta and Omicron, consistent with furin inhibition by GBPs. However, while GBP2/5 restrict Wuhan-Hu-1 infectivity, Alpha and Delta escape restriction. GBP exposure in producer cells influences viral entry route into target cells, with a shift towards endosomal entry. We therefore investigated whether GBP-targeting of spike alters sensitivity to endosomal restriction factors, IFITMs. We find IFITM1, but not IFITM 2 or 3, inhibit infection of naturally-permissive epithelial cells by early-lineage SARS-CoV-2, as well as Alpha and Delta, however GBPs did not sensitise to IFITM restriction. Strikingly, we find Omicron is unique amongst VOCs, being sensitive to restriction by GBP2/5, and also IFITM1, 2 and 3. We conclude evolution of Alpha and Delta spikes have conferred resistance to GBP restriction, but this is not solely due to acquisition of an enhanced FCS. Notably, Omicron, which has evolved under different selective pressures, has selected for changes in spike that not only mediate antibody escape, and shift in cell tropism and entry, but also impact the sensitivity of Omicron to innate immunity, potentially contributing to altered pathogenesis.


title: Erythritol synthesis in human cells is elevated in response to oxidative stress and regulated by the non-oxidative pentose phosphate pathway

abstract: Erythritol is a predictive biomarker of cardiometabolic diseases and is produced from glucose metabolism through the pentose phosphate pathway (PPP). Little is known regarding the regulation of endogenous erythritol synthesis in humans. In the present study, we investigated the stimuli that promote erythritol synthesis in human cells and characterized potential points of regulation along the PPP. Human A549 lung carcinoma cells were chosen for their known ability to synthesize erythritol. A549 cells were treated with potential substrates for erythritol production, including glucose, fructose, and glycerol. Using siRNA knockdown, we assessed the necessity of enzymes G6PD, TKT, TALDO, and SORD for erythritol synthesis. We also used position-specific 13C-glucose tracers to determine whether the carbons for erythritol synthesis are derived directly from glycolysis or through the oxidative PPP. Finally, we assessed if erythritol synthesis responds to oxidative stress using chemical and genetic models. Intracellular erythritol was directly associated with media glucose concentration. In addition, siRNA knockdown of TKT or SORD inhibited erythritol synthesis, whereas siG6PD did not. Both chemically induced oxidative stress and constitutive activation of the antioxidant response transcription factor NRF2 elevated intracellular erythritol. Our findings indicate that erythritol synthesis is proportional to flux through the PPP and is regulated by non-oxidative PPP enzymes.


title: Angiotensin converting enzyme 2 (ACE2): Virus accomplice or host defender

abstract:
The current coronavirus disease-19 (COVID-19) caused by the acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has seriously disrupted the daily life of human, mainly attributed to the fact that we know too little about SARS-CoV-2. Increasing studies show that viral infection alters host cells glucose metabolism, which is crucial for viral nucleic acid replication. Here, we integrated RNA-sequencing results and found that SARS-CoV-2 infection alters the aerobic glycolysis, pentose phosphate pathway (oxiPPP), and DNA replication in lung tissues and cells. However, the direction of metabolic flux and DNA replication were dominated by angiotensin-converting enzyme 2 (ACE2), a host cell-expressed viral receptor protein. More interesting, although hosts with high expression of ACE2 are more likely to be infected with SARS-CoV-2, the invading virus cannot perform nucleic acid replication well due to the restriction of glucose metabolism, and eventually resulting prolonged infection-cycle or infection failure. Our findings, after a typical epidemiological investigation and modeling analysis, preliminarily explain the reasons for the emergence of asymptomatic infections or lower copy virus at early stage in host with higher ACE2 levels, which will provide important help for the development of more accurate and effective detection methods for diagnosing COVID-19.

Graphical abstract

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title: In silico screening and testing of FDA approved small molecules to block SARS-CoV-2 entry to the host cell by inhibiting Spike protein cleavage

abstract: The COVID-19 pandemic began in 2019, but it is still active. The development of an effective vaccine reduced the number of deaths; however, a treatment is still needed. Here, we aimed to inhibit viral entry to the host cell by inhibiting Spike (S) protein cleavage by several proteases. We develop a computational pipeline to repurpose FDA-approved drugs to inhibit protease activity and thus prevent S protein cleavage. We tested some of our drug candidates and demonstrated a decrease in protease activity. We believe our pipeline will be beneficial in identifying a drug regimen for COVID-19 patients.

category: biophysics


title: Low memory T cells blood counts and high naive regulatory T cells percentage at relapsing remitting multiple sclerosis diagnosis

abstract:
ObjectiveTo assess the peripheral immune system of newly diagnosed relapsing remitting multiple sclerosis (RRMS) patients and compare it to healthy controls (HC).

MethodsCross-sectional study with 30 treatment-naive newly diagnosed RRMS patients, and 33 sex and age-matched HC. Their peripheral blood mononuclear cells were analysed regarding: i) thymic function surrogates [T cell receptor excision circles (TRECs) and recent thymic emigrants (RTEs)]; ii) naive and memory CD4+ and CD8+ T cells subsets; iii) T helper (Th) phenotype and chemokine receptors expression on T cells subsets; iv) regulatory T cell (Tregs) phenotype; and vi) expression of activating/inhibitory receptors by natural killer (NK) and NKT cells. Analyses were controlled for age, sex, and human cytomegalovirus (HCMV) IgG seroprevalence.

ResultsNewly diagnosed RRMS patients and HC have equivalent thymic function as determined by similar numbers of RTEs, and levels of sjTRECs, DJ{beta}TRECs and sj/DJ{beta}TREC ratio. In the CD8+ T cells compartment RRMS patients have a higher naive/memory ratio and lower memory cell counts in blood, specifically of effector memory and TemRA CD8+ T cells. Among CD4+ T cells lower blood counts of effector memory cells are found in patients upon controlling for sex, age and HCMV IgG seroprevalence. RRMS patients have higher percentage of naive Tregs comparing to HC. Percentages of immature CD56bright NK cells expressing the inhibitory receptor KLRG1, and of mature CD56dimCD57+ NK cells expressing NKp30 are higher in patients. No major alterations are observed on NKT cells. MS severity and time from relapse correlate with immune cells alterations.

ConclusionCharacterization of the peripheral immune system of treatment-naive newly diagnosed RRMS patients unveiled immune features present at clinical onset including lower memory T cells blood counts, particularly among CD8+ T cells, higher percentage of naive Tregs and altered percentages of NK cells subsets expressing inhibitory or activating receptors. These findings might set the basis to better understand disease pathogenesis.


title: Cell Chromatography: Biocompatible chromatographic separation and interrogation of microbial cells

abstract: The isolation of pure, single colonies lies at the heart of experimental microbiology. However, a microbial colony typically contains around one million cells at all stages of the life cycle. Here we describe a novel, cell chromatography method that facilitates the capture, purification and interrogation of microbial cells from both single and mixed cultures. The method described relies on, but is not limited to, differences in surface charge to separate bacterial strains. The method is fully biocompatible, leading to no significant loss of cell viability. The chromatographic capture of cells, combined with selective elution methods facilitates a greater level of experimental control over the sample inputs required for downstream high throughput and high sensitivity, analytical methods. The application of the method for interrogating the antibiotic resistance of bacterial strains and for the separation of bacteria from environmental samples is illustrated.


title: Droplet size and surface hydrophobicity enhance bacterial plasmid transfer rates in microscopic surface wetness

abstract: Conjugal plasmids constitute a major engine for horizontal gene transfer in bacteria, and are key drivers of the spread of antibiotic resistance, virulence, and metabolic functions. Bacteria in terrestrial habitats often inhabit surfaces that are not constantly water-saturated, where microscopic surface wetness (MSW), comprised of thin liquid films and microdroplets, permanently or intermittently occurs. How physical properties of microdroplets, and of the surfaces they reside on, affect plasmid transfer rates is not well understood. Here, building on microscopy-based microdroplet experiments, we examined the relation between droplet properties (size and spread) and plasmid transfer rates at single-cell and individual droplet resolution, using Pseudomonas putida as a model species. We show that transfer rates increase with droplet size, due to higher densities of cells on the surface in larger droplets, resulting from lower ratio between the area of the liquid-solid interface and droplet volumes. We further show that surface hydrophobicity promotes transfer rates via the same mechanism. Our results provide new insights into how physical properties of surfaces and MSW affect plasmid transfer rates, and more generally, interactions mediated by cell-to-cell contact, with important implications for our understanding of the ecology and evolution of bacteria in unsaturated environments.


title: Attenuation of a DNA Cruciform by a Conserved Regulator Directs T3SS-1 mediated virulence in Vibrio parahaemolyticus

abstract: Pathogenic Vibrio species account for 3-5 million annual life-threatening human infections. Virulence is driven by bacterial hemolysin and toxin gene expression often positively regulated by the winged helix-turn-helix (wHTH) HlyU transcriptional regulator family and silenced by Histone-like nucleoid structural protein (H-NS). In the case of Vibrio parahaemolyticus, HlyU is required for virulence gene expression associated with Type 3 Secretion System-1 (T3SS-1) although its mechanism of action is not understood. Here, we provide evidence for DNA cruciform attenuation mediated by HlyU binding to support concomitant virulence gene expression. Genetic and biochemical experiments revealed that upon HlyU mediated DNA cruciform attenuation, an intergenic cryptic promoter became accessible allowing for exsA mRNA expression and initiation of an ExsA autoactivation feedback loop at a separate ExsA-dependent promoter. Using a heterologous E. coli expression system, we reconstituted the dual promoter elements which revealed that HlyU binding and DNA cruciform attenuation were strictly required to initiate the ExsA autoactivation loop. The data indicate that HlyU acts to attenuate a transcriptional repressive DNA cruciform to support T3SS-1 virulence gene expression and reveals a non-canonical extricating gene regulation mechanism in pathogenic Vibrio species.


title: Enhanced cultured diversity of the mouse gut microbiota enables custom-made synthetic communities

abstract: Microbiome research is hampered by the fact that many bacteria are still unknown and by the lack of publicly available isolates. Fundamental and clinical research is in need of comprehensive and well-curated repositories of cultured bacteria from the intestine of mammalian hosts. In this work, we expanded the mouse intestinal bacterial collection (www.dsmz.de/miBC) to 212 strains, all publicly available and taxonomically described. This includes the study of strain-level diversity, small-sized bacteria, and the isolation and characterization of the first cultured members of one novel family, 10 novel genera, and 39 novel species. We demonstrate the value of this collection by performing two studies. First, metagenome-educated design allowed establishing custom synthetic communities (SYNs) that reflect different susceptibilities to DSS-induced colitis. Second, nine phylogenetically and functionally diverse species were used to amend the Oligo-Mouse Microbiota (OMM)12 model [Brugiroux et al. 2016 Nat Microbiol]. These strains compensated for differences observed between gnotobiotic OMM12 and specific pathogen-free (SPF) mice at multiple levels, including body composition and immune cell populations (e.g., T-cell subtypes) in the intestine and associated lymphoid tissues. Ready-to-use OMM stocks are available to the community for use in future studies. In conclusion, this work improves our knowledge of gut microbiota diversity in mice and enables functional studies via the modular use of isolates.


title: Moonlighting proteins activate transformation in multidrug-resistant Streptococcus pneumoniae epigenetic phase variants

abstract: Transformation of Streptococcus pneumoniae GPSC1 has enabled vaccine evasion and the acquisition of antibiotic resistance. Epigenetic phase variants of GPSC1 isolate RMV7, differentiated by rearrangements at the tvr restriction-modification locus, differed [~]100-fold in their transformation efficiency. This variation was recapitulated in knock-in mutants of the relevant tvr alleles. RNA-seq showed the difference was due to blocking of the early competence regulatory cascade. The more transformable variant upregulated expression of manLMN, encoding a carbon source importer. This was shown to be necessary for efficient competence induction, despite being dispensable for growth in rich media. Transformation was promoted by import of N-acetylglucosamine, which activated competence through an orthologue of the gram-negative competence regulator TfoX, and an enzyme likely involved in nucleotide-mediated signalling. A mobile genetic element was more active in the less transformable variant, which limited competence induction through inhibiting CIRCE motif binding by the chaperone regulator HrcA. Correspondingly, both heat shock and decreased Ca2+ concentrations reduced competence by limiting HrcA binding regulatory sequence motifs. Hence both ManLMN and HrcA moonlighted as activators of competence. Such proteins may be important in potentiating horizontal DNA exchange, as selection on their primary function likely constrains them from mutating into alleles that selfishly downregulate transformation.


title: Moonlighting proteins activate transformation in epigenetically-differentiated phase variants of multidrug-resistant Streptococcus pneumoniae


title: SUMO conjugation regulates the activity of the Integrator complex

abstract: RNA pol II transcribes snRNA genes in close proximity to Cajal bodies, subnuclear compartments that depend on the SUMO isopeptidase USPL1 for their assembly. We show here that overexpression of USPL1 alters snRNA 3-end cleavage, a process carried out by the Integrator complex. Beyond its role in snRNA biogenesis, this complex is responsible for regulating the expression of different non-coding and coding transcripts. We validated several subunits of the complex as SUMO conjugation substrates, and found that the SUMOylation of INTS11 subunit is regulated by USPL1. We defined Lys 381, Lys 462 and Lys 475 as bona fide SUMO attachment sites within INTS11 and observed that SUMOylation of this protein is required for efficient Integrator activity. Moreover, while an INTS11 SUMOylation deficient mutant is still capable of interacting with INTS4 and INTS9, its interaction with other subunits of the complex is affected. This mutant also shows a more cytoplasmatic localization than the wild type protein. These findings point to a regulatory role of SUMO conjugation on Integrator activity and suggest the involvement of INTS11 SUMOylation in the assembly of the complex. Furthermore, this work adds the Integrator-dependent RNA processing to the growing list of cellular processes regulated by SUMO conjugation.


title: Structural basis of Ty1 integrase tethering on RNA polymerase III for targeted DNA integration

abstract: The yeast Ty1 retrotransposon integrates upstream of genes transcribed by RNA polymerase III (Pol III). Specificity of integration is mediated by an interaction between the Ty1 integrase (IN1) and Pol III, currently uncharacterized at the atomic level. Here, we report cryo-EM structures of Pol III in complex with IN1, revealing a 16-residue segment at the IN1 C-terminus that contacts Pol III subunits AC40 and AC19, an interaction that we validate by in vivo mutational analysis. Unexpectedly, IN1 binding associates with insertion of subunit C11 C-terminal Zn-ribbon into the Pol III funnel, which provides atomic evidence for a two-metal mechanism during RNA cleavage. Moreover, unstructured regions of subunits C53 and C37 reorganize close to C11, likely explaining the connection between the C37/C53 heterodimer and C11 during transcription reinitiation. Our results suggest that IN1 binding induces a Pol III configuration that favors chromatin residence, thus improving the likelihood of Ty1 integration.


title: Cryo-EM structures reveal the activation and substrate recognition mechanism of human enteropeptidase

abstract: The enteropeptidase (EP) initiates the intestinal digestion by proteolytic processing of trypsinogen, generating catalytic active trypsin. The dysfunction of EP will cause a series of pancreatic diseases, the most severe of which is acute necrotizing pancreatitis. However, the molecular mechanism of EP activation and substrate recognition remain elusive due to the lack of structural information, hampering the structure-based research of EP and even further EP-targeted drug design. Here we report cryo-EM structures of human EP in multiple states, covering the functional cycle spanning from inactive to active state and eventually to the substrate binding state, with the inactive core region reached an atomic 2.7-[A]-resolution. The heavy chain of EP exhibits a clamping configuration with the CUB2 domain serving for substrate recognition. The N-terminus of light chain induces the surface loop remodeling from inactive to active conformation, resulting in a highly dynamic and active EP. Then the heavy chain performs like a hinge to ensure the flexibility of light chain for substrate recruitment and subsequent cleavage. Our study provides structural insights of EP remodeling and heavy chain dynamics while performing enzymatic function, facilitating our understanding of the pathogenies of EP-related pancreatitis and the EP-targeted treatment of pancreatitis.


title: Generation and Maturation of Human iPSC-derived Cardiac Organoids in Long Term Culture

abstract:
Cardiovascular diseases remain the leading cause of death worldwide; hence there is an increasing focus on developing physiologically relevant in vitro cardiovascular tissue models suitable for studying personalized medicine and pre-clinical tests. Despite recent advances, models that reproduce both tissue complexity and maturation are still limited.

We have established a scaffold-free protocol to generate multicellular, beating and self-organized human cardiac organoids (hCO) in vitro from hiPSCs that can be cultured for long term. This is achieved by differentiation of hiPSC in 2D monolayer culture towards cardiovascular lineage, followed by further aggregation on low-attachment culture dishes in 3D. The generated human cardiac organoids (hCOs) containing multiple cell types that physiologically compose the heart, gradually self-organize and beat without external stimuli for more than 50 days. We have shown that 3D hCOs display improved cardiac specification, survival and maturation as compared to standard monolayer cardiac differentiation. We also confirmed the functionality of hCOs by their response to cardioactive drugs in long term culture. Furthermore, we demonstrated that hCOs can be used to study chemotherapy-induced cardiotoxicity.

This study could help to develop more physiologically-relevant cardiac tissue models, and represent a powerful platform for future translational research in cardiovascular biology.


title: A hypomorphic mutation in Pold1 disrupts the coordination of embryo size expansion and morphogenesis during gastrulation

abstract:
Formation of a properly sized and patterned embryo during gastrulation requires a well-coordinated interplay between cell proliferation, lineage specification and tissue morphogenesis. Following transient physical or pharmacological manipulations, pre-gastrulation stage mouse embryos show remarkable plasticity to recover and resume normal development. However, it remains unclear how mechanisms driving lineage specification and morphogenesis respond to defects in cell proliferation during and after gastrulation. Null mutations in DNA replication or cell-cycle related genes frequently lead to cell cycle arrest and reduced cell proliferation, resulting in developmental arrest before the onset of gastrulation; such early lethality precludes studies aiming to determine the impact of cell proliferation on lineage specification and morphogenesis during gastrulation. From an unbiased ENU mutagenesis screen, we discovered a mouse mutant, tiny siren (tyrn), that carries a hypomorphic mutation producing an aspartate to tyrosine (D939Y) substitution in Pold1, the catalytic subunit of DNA polymerase {delta}. Impaired cell proliferation in the tyrn mutant leaves anterior-posterior patterning unperturbed during gastrulation but results in an overall reduction in embryo size and in severe morphogenetic defects. Our analyses show that the successful execution of morphogenetic events during gastrulation requires that lineage specification and the ordered production of differentiated cell types occur in concordance with embryonic growth.

Summary statementPold1 hypomorphic mutation caused reduced size and abnormal morphology of gastrulating mouse embryos, supporting the importance of coordinated embryo size, lineage specification and tissue morphogenesis for normal embryogenesis.


title: Lhx2 in germ cells suppresses endothelial cell migration in the developing ovary

abstract:
LIM-homeobox genes play multiple roles in developmental processes, but their roles in gonad development are not completely understood. Herein, we report that Lhx2, Ils2, Lmx1a, and Lmx1b are expressed in a sexually dimorphic manner in mouse, rat, and human gonads during sex determination. Amongst these, Lhx2 has female biased expression in the developing gonads of species with environmental and genetic modes of sex determination. Single-cell RNAseq analysis revealed that Lhx2 is exclusively expressed in the germ cells of the developing mouse ovaries. To elucidate the roles of Lhx2 in the germ cells, we analyzed the phenotypes of Lhx2 knockout XX gonads. While the gonads developed appropriately in Lhx2 knockout mice and the somatic cells were correctly specified in the developing ovaries, transcriptome analysis revealed enrichment of genes in the angiogenesis pathway. There was an elevated expression of several pro-angiogenic factors in the Lhx2 knockout ovaries. The elevated expression of pro-angiogenic factors was associated with an increase in numbers of endothelial cells in the Lhx2-/-ovaries at E13.5. Gonad recombination assays revealed that the increased numbers of endothelial cells in the XX gonads in absence of Lhx2 was due to ectopic migration of endothelial cells in a cell non-autonomous manner. We also found that, there was increased expression of several endothelial cell-enriched male-biased genes in Lhx2 knockout ovaries. Also, in absence of Lhx2, the migrated endothelial cells formed an angiogenic network similar to that of the wild type testis, although the coelomic blood vessel did not form. Together, our results suggest that Lhx2 in the germ cells is required to suppress vascularization in the developing ovary. These results suggest a need to explore the roles of germ cells in the control of vascularization in developing gonads.

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org.highwire.dtl.DTLVardef@59ca47org.highwire.dtl.DTLVardef@ef193eorg.highwire.dtl.DTLVardef@162651corg.highwire.dtl.DTLVardef@dc3d0f_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightO_LIMultiple LIM-HD genes are expressed in developing gonads during the window of sex determination with Lhx2 having female dominating expression in an evolutionarily conserved manner
C_LIO_LILhx2 is expressed in the germ cells of developing mouse ovaries
C_LIO_LILoss of Lhx2 in the developing ovaries alters the expression of genes involved in various pathways including angiogenesis
C_LIO_LILhx2 in germ cells suppress endothelial cell migration in the developing ovaries
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