title: A mechanistic basis for understanding the dual activities of the bifunctional Azotobacter vinelandii mannuronan C-5 epimerase and alginate lyase AlgE7

abstract:
The functional properties of alginates are dictated by the monomer composition and molecular weight distribution. Mannuronan C-5 epimerases determine the former by epimerizing {beta}-D-mannuronic acid residues (M) into -L-guluronic acid residues (G). The molecular weight is affected by alginate lyases, which cleave alginate chains through {beta}-elimination. The reaction mechanisms for the epimerization and cleavage are similar and some enzymes can perform both. These dualistic enzymes share high sequence identity with mannuronan C-5 epimerases without lyase activity, and the mechanism behind their activity as well as the amino acids responsible for it are still unknown. In this study, we investigate mechanistic determinants of the bifunctional epimerase and lyase activity of AlgE7 from Azotobacter vinelandii. Based on sequence analyses, a range of AlgE7 variants were constructed and subjected to activity assays and product characterization by NMR. Our results show that the lyase activity of AlgE7 is regulated by the type of ion present: Calcium promotes it, whereas NaCl reduces it. By using defined poly-M and poly-MG substrates, the preferred cleavage sites of AlgE7 were found to be M{downarrow}XM and G{downarrow}XM, where X can be either M or G. By studying AlgE7 mutants, R148 was identified as an important residue for the lyase activity, and the point mutant R148G resulted in an enzyme with only epimerase activity. Based on the results obtained in the present study we suggest a unified catalytic reaction mechanism for both epimerase and lyase activities where H154 functions as the catalytic base and Y149 as the catalytic acid.

ImportancePost-harvest valorisation and upgrading of algal constituents is a promising strategy in the development of a sustainable bioeconomy based on algal biomass. In this respect, alginate epimerases and lyases are valuable enzymes for tailoring of the functional properties of alginate, a polysaccharide extracted from brown seaweed with numerous applications in food, medicine and material industries. By providing a better understanding of the reaction mechanism and of how the two enzyme reactions can be altered by changes in reaction conditions, this study opens for further applications of bacterial epimerases and lyases in enzymatic tailoring of alginate polymers.

category: biochemistry


title: Mechanistic basis for understanding the dual activities of the bifunctional Azotobacter vinelandii mannuronan C-5 epimerase and alginate lyase AlgE7


title: Emerging SARS-CoV-2 mutation hotspots associated with clinical outcomes

abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic. Understanding the influence of mutations in the SARS-CoV-2 gene on clinical outcomes and related factors is critical for treatment and prevention. Here, we analyzed 209,551 high-coverage complete virus sequences and 321 RNA-seq samples to mine the mutations associated with clinical outcome in the SARS-CoV-2 genome. Several important hotspot variants were found to be associated with severe clinical outcomes. Q57H variant in ORF3a protein were found to be associated with higher mortality rate, and was high proportion in severe cases (39.36%) and 501Y.V2 strains (100%) but poorly proportional to asymptomatic cases (10.04%). T265I could change nsp2 structure and mitochondrial permeability, and evidently higher in severe cases (20.12%) and 501Y.V2 strains (100%) but lower in asymptomatic cases (1.43%). Additionally, R203K and G204R could decrease the flexibility and immunogenic property of N protein with high frequency among severe cases, VUI 202012/01 and 484K.V2 strains. Interestingly, the SARS-CoV-2 genome was more susceptible to mutation because of the high frequency of nt14408 mutation (which located in RNA polymerase) and the high expression levels of ADAR and APOBEC in severe clinical outcomes. In conclusion, several important mutation hotspots in the SARS-CoV-2 genome associated with clinical outcomes was found in our study, and that might correlate with different SARS-CoV-2 mortality rates.

category: bioinformatics


title: Suppression of cdc13-2-associated senescence by pif1-m2 requires Ku-mediated telomerase recruitment

abstract: In Saccharomyces cerevisiae, recruitment of telomerase to telomeres requires an interaction between Cdc13, which binds single-stranded telomeric DNA, and the Est1 subunit of telomerase. A second pathway involving an interaction between the yKu complex and telomerase RNA (TLC1) contributes to telomerase recruitment, but cannot sufficiently recruit telomerase on its own to prevent replicative senescence when the primary Cdc13-Est1 pathway is abolished--for example, in the cdc13-2 mutant. In this study, we find that mutation of PIF1, which encodes a helicase that inhibits telomerase, suppresses the replicative senescence of cdc13-2 increasing reliance on the yKu-TLC1 pathway for telomerase recruitment. Our findings reveal new insight into telomerase-mediated telomere maintenance.

category: molecular biology


title: The effect of histone deacetylase inhibitor Trichostatin A on IL-17A-induced transformation of MRC5 into myofibroblasts and its mechanism

abstract:
ObjectiveTo elucidate potential IL-17A- and TSA-mediated regulation of fibroblasts transformation.

MethodsMTT assay, HDAC1 activity assay, cell immunofluorescence and Western blot were employed to detect the expression of related indicators.

ResultsMRC5 cells expressed only a small amount of Vimentin. IL-17A treatment upregulated MRC5 cell proliferation, in a concentration-dependent manner. TSA treatment, however, suppressed MRC5 cell proliferation. IL-17A treatment also upregulated HDAC1 activity in MRC5 cells, in a concentration-dependent manner. Using immunofluorescence, we demonstrated that IL-17A-treated MRC5 cells had markedly elevated Vimentin, Collagen-I and a-SMA levels, compared to controls. However, a combined treatment of IL-17A and TSA resulted in markedly reduced levels of the Vimentin, Collagen-I and a-SMA, compared to IL-17A alone, yet the amount was higher than controls. Using western blot analysis, we also revealed that the IL-17A-treated MRC5 cells had markedly elevated levels of Vimentin, a-SMA, HDAC1, p-Smad2, and p-Smad3, and markedly reduced level of Smad7, compared to controls. In TSA intervention group, the expression effect of the above protein was opposite. Moreover, no discernible difference was observed in the levels of Smad2 and Smad3 among the treated and un-treated groups.

ConclusionIL-17A stimulates proliferation of MRC5 cells and increases HDAC1 activity and protein expression. It also transforms MRC5 cells into myofibroblasts via activation of the TGF -{beta}1/ Smads signaling network. TSA, on the other hand, strongly suppresses TGF -{beta}1/ Smads pathway-mediated fibrosis by ceasing HDAC1 activity and protein expression.

category: cell biology


title: Towards deciphering the structure of long homozygous stretches in cattle genome

abstract: Up-to-day there is no as universally accepted software tool and threshold parameters to identify runs of homozygosity (ROH). The relative position of POH segments in the cattle genome has not been studied extensively. Specific objective of this study was to evaluate the effect of allowed missing and heterozygous SNPs in ROH on their number, on the estimate of inbreeding level, and on structure of ROH segments in the cattle genome. In this study 371 Holsteinized cows from six herds were genotyped with BovineSNP50 array. To identify ROH, the consecutive and sliding runs were carried out with detectRUNS and Plink tools. Neither effect was shown for missing SNPs genotype calls. Allowing even one heterozygous SNP resulted in significant bias of ROH data. Furthermore, the sliding runs identified less ROH than consecutive runs. The mean coefficient of inbreeding across herds was 0.111 {+/-} 0.003 and 0.104 {+/-} 0.003 based on consecutive and sliding runs respectively. It was shown how, using the heterozygous SNPs in ROH, may be possible to derive a distribution of ROH segments in the cow genome. We suggested it was similar to normal distribution. Furthermore, frequency of ROH in the chromosomes did not depend on their length. Of 29 chromosomes, the most abundant with ROH were BTA 14, BTA 7, and BTA 18. The result of this study confirmed more accurately identification of ROH with consecutive runs, uneven their distribution in the cattle genome, significant bias of the data due to allowing heterozygous SNPs in ROH.

category: genetics


title: Microfluidic delivery of cutting enzymes for fragmentation of surface-adsorbed DNA molecules

abstract: We describe a method for fragmenting, in-situ, surface-adsorbed and immobilized DNAs on polymethylmethacrylate(PMMA)-coated silicon substrates using microfluidic delivery of the cutting enzyme DNase I. Soft lithography is used to produce polydimethylsiloxane (PDMS) gratings which form microfluidic channels for delivery of the enzyme. Bovine serum albumin (BSA) is used to reduce DNase I adsorption to the walls of the microchannels and enable diffusion of the cutting enzyme to a distance of 10mm. Due to the DNAs being immobilized, the fragment order is maintained on the surface. Possible methods of preserving the order for application to sequencing are discussed.

category: biophysics


title: Are some commercial diets inadequate in essential nutrients?

abstract:
BackgroundCommercial manufacturers have formulated diets to promote not only weight reduction but also to reduce risks of chronic diseases. The objective of this study is to determine if these formulations satisfy requirements for essential nutrients.

MethodsWe have selected two established commercial diets, one low fat, high carbohydrate (diet 1) and the other, high fat, low carbohydrate (diet 2) and determined "representative meals" through use of recipes suggested in the manufacturers manuals. Nutrition Data System for Research (NDSR) software has been used to perform the most extensive nutrient analysis to date of these diets. Tables report macronutrients (energy), vitamins, minerals, essential amino acids, essential lipids and nutrient-related components for a total of 62entries.

ResultsDiet 1 satisfied requirements for 50 of these (81%) with only vitamin B12, vitamin D, and essential fatty acids not reaching recommended levels, while fiber and glycemic load exceeded suggested values. Diet 2 satisfied requirements for 46 of the components (71%) but had excess percentage of fat, especially saturated fat, sodium and cholesterol as well as decreased percentage of carbohydrate resulting in suboptimal intake of B-complex vitamins (B1, niacin and total folate) as well as fiber.

ConclusionsNeither diet satisfied adequacy for all reported nutrients. However, based on nutrient content alone diet 1, if supplemented or modified, could be sustained over the long term whereas diet 2 should not be encouraged for long term adaptation

category: scientific communication and education


title: Isolation and characterization of a novel Wolbachia bacteriophage from Allonemobius socius crickets in Missouri

abstract: Wolbachia are endosymbionts of numerous arthropod and some nematode species, are important for their development and if present can cause distinct phenotypes of their hosts. Prophage DNA has been frequently detected in Wolbachia, but particles of Wolbachia bacteriophages (phage WO) have been only occasionally isolated. Here, we report the characterization and isolation of a phage WO of the southern ground cricket, Allonemobius socius, and provided the first whole-genome sequence of phage WO from this arthropod family outside of Asia. We screened A. socius abdomen DNA extracts from a cricket population in eastern Missouri by quantitative PCR for Wolbachia surface protein and phage WO capsid protein and found a prevalence of 55% and 50%, respectively, with many crickets positive for both. Immunohistochemistry using antibodies against Wolbachia surface protein showed many Wolbachia clusters in the reproductive system of female crickets. Whole-genome sequencing using Oxford Nanopore MinION and Illumina technology allowed for the assembly of a high-quality, 55 kb phage genome containing 63 open reading frames (ORF) encoding for phage WO structural proteins and host lysis and transcriptional manipulation. Taxonomically important regions of the assembled phage genome were validated by Sanger sequencing of PCR amplicons. Analysis of the nucleotides sequences of the ORFs encoding the large terminase subunit (ORF2) and minor capsid (ORF7) frequently used for phage WO phylogenetics showed highest homology to phage WOKue of the Mediterranean flour moth Ephestia kuehniella (94.18% identity) and WOLig of the coronet moth, Craniophora ligustri (96.86% identity), respectively. Transmission electron microscopy examination of cricket ovaries showed a high density of phage particles within Wolbachia cells. Isolation of phage WO revealed particles characterized by 40-62 nm diameter heads and up to 190 nm long tails. This study provides the first detailed description and genomic characterization of phage WO from North America that is easily accessible in a widely distributed cricket species.

category: microbiology


title: A Tool for Reliable Detection of Aflatoxin Biosynthetic Gene Clusters in Aflatoxigenic and Atoxigenic Aspergillus flavus Isolates

abstract: Molecular techniques and phenotypic characterisation have been used to differentiate aflatoxigenic and atoxigenic Aspergillus flavus strains. However, there is a lack of a consistent and reliable tool for discrimination between these strains of A. flavus. Here we report, an optimised real-time qPCR-based tool for reliable differentiation between aflatoxigenic and atoxigenic strains of A. flavus. Accordingly, expression profiles and deletion patterns of genes responsible for aflatoxin production in five representative aflatoxigenic and atoxigenic A. flavus strains (KSM012, KSM014, HB021, HB026 and HB027) were examined using the optimised real-time qPCR tool. We observed that under induced conditions, aflP, aflS, aflR and aflO transcripts were the most upregulated genes across the tested isolates while aflS and aflO were always expressed in both induced and uninduced isolates. However, aflR and aflP did not give clear distinctions between non-toxin and toxin producing isolates. The deletion patterns were prominent for aflD and aflR whereas alfO, aflS and aflP had no deletions among the isolates. Significant variation in transcript abundance for aflD, aflR and aflS were observed for aflatoxigenic isolate KSM014 under induced and uninduced states. False detection of aflD gene transcript in atoxigenic strain KSM012 was evident in both induced and uninduced conditions. With the exception of KSM012, aflP gene did not exhibit significant variation in expression in the isolates between induced and uninduced conditions. One-way ANOVA and Post-test analysis for linear trends revealed that aflatoxin biosynthetic cluster genes show significant (P < 0.05) differences between atoxigenic and aflatoxigenic isolates. Our optimized qPCR-based tool reliably discriminated between aflatoxigenic and atoxigenic A. flavus isolates and could complement existing detection methods.


title: The unique neuronal structure and neuropeptide repertoire in the ctenophore Mnemiopsis leidyi shed light on the evolution of animal nervous systems

abstract: The ctenophore nerve net represents one of the earliest evolved nervous system of animals. Due to the uncertainties of their phylogenetic placement of ctenophores and the absence of several key bilaterian neuronal genes, it has been hypothesized that their neurons have evolved independently. Whether this is indeed the case remains unclear, and thus the evolutionary history of neurons is still contentious. Here, we have characterized the neuropeptide repertoire of the ctenophore Mnemiopsis leidyi. Using the machine learning NeuroPID tool1 129 new putative neuropeptide precursors were predicted. Sixteen of them are detected in the subepithelial nerve net (SNN), aboral organ (AO) and epithelial sensory cells (ESC) of early cydippid-stage M. leidyi by in situ hybridization (ISH) and immunohistochemistry (IHC). Four of these neuropeptides increase the animals swimming velocity in a behavioral essay. The new neuropeptides were used as markers to identify neuronal cell types in single cell transcriptomic data2. To unravel the neuronal architecture, we 3D reconstructed the SNN underlying the comb plates using serial block-face scanning electron microscopy (SBF-SEM). For the first time, we confirm a more than 100 years old hypothesis about anastomoses between neurites of the same cell in ctenophores and reveal that they occur through a continuous membrane. Our findings reveal the unique neuronal structure and neuropeptide repertoire of ctenophores and are important for reconstructing the evolutionary origin of animal neurons and nervous systems.

category: evolutionary biology


title: Connectivity and Synaptic Physiology in the Mouse and Human Cortex

abstract: To elucidate cortical microcircuit structure and synaptic properties we present a unique, extensive, and public synaptic physiology dataset and analysis platform. Through its application, we reveal principles that relate cell type to synapse properties and intralaminar circuit organization in the mouse and human cortex. The dynamics of excitatory synapses align with the postsynaptic cell subclass, whereas inhibitory synapse dynamics partly align with presynaptic cell subclass but with considerable overlap. Despite these associations, synaptic properties are heterogeneous in most subclass to subclass connections. The two main axes of heterogeneity are strength and variability. Cell subclasses divide along the variability axis, while the strength axis accounts for significant heterogeneity within the subclass. In human cortex, excitatory to excitatory synapse dynamics are distinct from those in mouse and short-term plasticity varies with depth across layers 2 and 3. With a novel connectivity analysis that enables fair comparisons between circuit elements, we find that intralaminar connection probability among cell subclasses exhibits a strong layer dependence.These and other findings combined with the analysis platform create new opportunities for the neuroscience community to advance our understanding of cortical microcircuits.

category: neuroscience


title: Local Connectivity and Synaptic Dynamics in Mouse and Human Neocortex


title: Precise and pervasive phasic bursting in locus coeruleus during maternal behavior

abstract: The noradrenergic locus coeruleus (LC) mediates key aspects of arousal, memory, and cognition in structured tasks, but its contribution to natural behavior remains unclear. Neuronal activity in LC is organized into sustained ( tonic) firing patterns reflecting global brain states and rapidly fluctuating ( phasic) bursts signaling discrete behaviorally significant events. LCs broad participation in social behavior including maternal behavior is well-established, yet the temporal relationship of its activity to sensory events and behavioral decisions in this context is unknown. Here, we made electrical and optical recordings from LC in female mice during maternal interaction with pups. We find that pup retrieval stably elicits precisely timed and pervasive phasic activation of LC that cant be attributed to sensory stimuli, motor activity, or reward. Correlation of LC activity with retrieval events shows that phasic events are most closely related to subsequent behavior. We conclude that LC likely drives goal-directed action selection during social behavior with globally-broadcast noradrenaline release.


title: Mild impairment of mitochondrial function increases longevity and pathogen resistance through ATFS-1-driven activation of p38-regulated innate immunity

abstract:
While mitochondrial function is essential for life in all multicellular organisms, a mild impairment of mitochondrial function can extend longevity. By understanding the molecular mechanisms involved, these pathways might be targeted to promote healthy aging. In studying two long-lived mitochondrial mutants in C. elegans, we found that disrupting subunits of the mitochondrial electron transport chain resulted in upregulation of genes involved in innate immunity, which we found to be dependent on not only the canonical p38-mediated innate immune signaling pathway but also on the mitochondrial unfolded protein response. Both of these pathways are absolutely required for the increased resistance to bacterial pathogens and extended longevity of the long-lived mitochondrial mutants, as is the FOXO transcription factor DAF-16. This work demonstrates that both the p38-mediated innate immune signaling pathway and the mitochondrial unfolded protein response can act on the same innate immunity genes to promote resistance to bacterial pathogens, and that input from the mitochondria can extend longevity by signaling through these two pathways. Combined, this indicates that multiple evolutionarily conserved genetic pathways controlling innate immunity also function to modulate lifespan.

Significance StatementIn this work, we explore the relationship between mitochondrial function, aging and innate immunity. We find that mild impairment of mitochondrial function results in upregulation of genes involved in innate immunity, increased resistance to bacterial pathogens and lifespan extension, all of which are dependent on two evolutionarily conserved signaling pathways. This work demonstrates how changes in functional status of the mitochondria can trigger activation of innate immunity, and that the underlying mechanisms are important for the longevity of the organism. This work advances our understanding of connections between metabolism and immunity. As the pathways studied here are conserved up to mammals, these insights may help us to understand the role of mitochondrial health, innate immunity and lifespan in humans.


title: The Maximum Entropy Formalism of statistical mechanics in a biological application: a quantitative analysis of tropical forest ecology

abstract: In a time of rapid global change, the question of what determines patterns in species abundance distribution remains a priority for understanding the complex dynamics of ecosystems. The constrained maximization of information entropy provides a framework for the understanding of such complex systems dynamics by a quantitative analysis of important constraints via predictions using least biased probability distributions. We apply it to over two thousand hectares of Amazonian tree inventories across seven forest types and thirteen functional traits, representing major global axes of plant strategies. Results show that constraints formed by regional relative abundances of genera explain almost ten times more of local relative abundances then constraints based on either directional or stabilizing selection for specific functional traits, although the latter does show clear signals of environmental dependency. These results provide a quantitative insight by inference from large-scale data using cross-disciplinary methods, furthering our understanding of ecological dynamics.

category: ecology


title: Prevalence and polymorphism of a mussel transmissible cancer in Europe

abstract: Transmissible cancers are parasitic malignant cell lineages that acquired the ability to infect new hosts from the same species, or sometimes related species. First described in dogs and Tasmanian devils, transmissible cancers were later discovered in some marine bivalves affected by a leukemia-like disease. In Mytilus mussels, two lineages of Bivalve Transmissible Neoplasia (BTN), both emerged in a M. trossulus founder individual, have been described to date (MtrBTN1 and MtrBTN2). Here, we performed an extensive screening of genetic chimerism, a hallmark of transmissible cancer, by genotyping hundred SNPs of thousands of European Mytilus mussels. The genetic analysis allowed us to simultaneously obtain the genotype of hosts -M. edulis, M. galloprovincialis or hybrids- and the genotype of tumors of heavily infected individuals. In addition, a subset of individuals were systematically genotyped and analysed by histology in order to screen for possible non-transmissible cancers. We detected MtrBTN2 at low prevalence in M. edulis, and also in M. galloprovincialis and hybrids although at a much lower prevalence. No MtrBTN1 or new BTN were found but a few individuals with non-transmissible neoplasia were observed at a single polluted site on the same sampling date. We observed a diversity of MtrBTN2 genotypes that appeared more introgressed or more ancestral than MtrBTN1 and reference healthy M. trossulus individuals. The observed polymorphism is most likely due to somatic null alleles caused by structural variations or point mutations in primer-binding sites leading to enhanced detection of the host alleles. Despite low prevalence, two divergent sublineages, confirmed by mtCOI sequences, are co-spreading in the same geographic area, suggesting a complex diversification of MtrBTN2 since its emergence and host species shift.


title: A Bioinspired Glycopolymer for Capturing Membrane Proteins in Native-Like Lipid-Bilayer Nanodiscs

abstract: Amphiphilic copolymers that directly extract membrane proteins and lipids from cellular membranes to form nanodiscs combine the advantages of harsher membrane mimics with those of a native-like membrane environment. Among the few commercial polymers that are capable of forming nanodiscs, alternating diisobutylene/maleic acid (DIBMA) copolymers have gained considerable popularity as gentle and UV-transparent alternatives to aromatic polymers. However, their moderate hydrophobicities and high electric charge densities render all existing aliphatic copolymers rather inefficient under near-physiological conditions. Here, we introduce Glyco-DIBMA, a bioinspired glycopolymer that possesses increased hydrophobicity and reduced charge density but nevertheless retains excellent solubility in aqueous solutions. Glyco-DIBMA outperforms established aliphatic copolymers in that it solubilizes lipid vesicles of various compositions much more efficiently, thereby furnishing smaller, more narrowly distributed nanodiscs that preserve a bilayer architecture and exhibit rapid lipid exchange. We demonstrate the superior performance of Glyco-DIBMA in preparative and analytical applications by extracting a broad range of integral membrane proteins from cellular membranes and further by purifying a membrane-embedded voltage-gated K+ channel, which was fluorescently labeled and analyzed with the aid of microfluidic diffusional sizing (MDS) directly within native-like lipid-bilayer nano-discs.


title: Effects of adhesive drying methods to reduce aerosol generation during resin bonding on enamel bond strength

abstract:
ObjectiveIn order to reduce the amount of aerosol during the use of dental adhesives, which are widely used in minimally invasive procedures, the effects of air stream-free adhesive drying methods on the enamel bond strength of resin adhesive systems were evaluated.

Materials and MethodsThe four adhesive drying techniques used were an air-stream, a micro-applicator, a cotton pellet and an absorbent paper. Adhesive systems were Single Bond Universal and Optibond All-in-one. The group in which the adhesive was not dried served as the negative control group. Enamel shear bond strength was performed with a universal tester with a crosshead speed of 1 mm/min (n=12). A two-way analysis of variance and the Tukey post-hoc test were used for analysis of the SBS data.

ResultsFor both adhesives, drying the adhesive with cotton pellet or micro-applicator provided a SBS mean values similar to air-stream drying, while statistically significantly lower SBS mean values were observed in the negative control group and in the absorbent paper-dry group compared to the air-drying group.

ConclusionsDrying the adhesive with micro-applicator and cotton pellets creates less aerosol and thus can be used in the COVID-19 pandemic as it provides enamel bonding strength similar to air drying.

CLINICAL SIGNIFICANCETo provide safer dental care during COVID-19 pandemic, it is highly recommended to use non-aerosol-generating alternatives, instead of aerosolgenerating dental procedures. In this study, we found that the aerosol generation-free methods for adhesive-drying such as cotton pellet and micro applicator drying methods provide similar enamel bonding with conventional drying method. We think that our findings will contribute to the realization of safer adhesive dentistry practice, especially during the COVID-19 pandemic.

category: bioengineering


title: Viola: a structural variant signature extractor with user-defined classifications

abstract:
Here, we present Viola, a Python package that provides structural variant (SV; large scale genome DNA variations that can result in disease, e.g., cancer) signature analytical functions and utilities for custom SV classification, merging multi-SV-caller output files, and SV annotation. We demonstrate that Viola can extract biologically meaningful SV signatures from publicly available SV data for cancer and we evaluate the computational time necessary for annotation of the data.

AvailabilityViola is available on pip (https://pypi.org/project/Viola-SV/) and on GitHub (https://github.com/dermasugita/Viola-SV).

Contactkdais-prm@m.u-tokyo.ac.jp


title: Methane, a gut bacteria-produced gas, does not affect arterial blood pressure in normotensive anaesthetized rats.

abstract: Methane is produced by carbohydrate fermentation in the gastrointestinal tract through the metabolism of methanogenic microbiota. Several lines of evidence suggest that methane exerts anti-inflammatory, anti-apoptotic and anti-oxidative effects. The effect of methane on cardiovascular system is obscure. The objective of the present study was to evaluate the hemodynamic response to methane. A vehicle or methane-rich saline were administered intravenously or intraperitoneally in normotensive anaesthetized rats. We have found no significant effect of the acute administration of methane-rich saline on arterial blood pressure and heart rate in anaesthetized rats. Our study suggests that methane does not influence the control of arterial blood pressure. However, further chronic studies may be needed to fully understand hemodynamic effects of the gas.

category: physiology


title: The Xanthomonas type-III effector protein XopS stabilizes CaWRKY40a to regulate defense hormone responses and preinvasion immunity in pepper (Capsicum annuum)

abstract: A critical component of plant immunity against invading pathogens is the rapid transcriptional reprogramming of the attacked cell to minimize virulence. Many adapted plant bacterial pathogens use type III effector (T3E) proteins to interfere with plant defense responses, including the induction of immunity genes. The elucidation of effector function is essential to understanding bacterial pathogenesis. Here, we show that XopS, a T3E of Xanthomonas campestris pv. vesicatoria (Xcv), interacts with and inhibits the proteasomal degradation of the transcriptional regulator of defense gene expression WRKY40. Virus-induced gene silencing of WRKY40 in pepper enhanced plant tolerance towards Xcv infection, indicating it represses immunity. Stabilization of WRKY40 by XopS reduces the expression of its targets including salicylic acid (SA)-responsive genes and the jasmonic acid (JA) signaling repressor JAZ8. Xcv bacteria lacking XopS display significantly reduced virulence when surface inoculated onto susceptible pepper leaves. XopS delivery by Xcv, as well as ectopic expression of XopS in Arabidopsis or Nicotiana benthamiana prevented stomatal closure in response to bacteria and biotic elicitors in a WRKY40 dependent manner. This suggests that XopS interferes with preinvasion as well as with apoplastic defense by manipulating WRKY40 stability and gene expression eventually altering phytohormone crosstalk to promote pathogen proliferation.

category: plant biology


title: Three Dimensions of Association Link Migraine Symptoms and Functional Connectivity

abstract: Migraine is a heterogeneous disorder with variable symptoms and responsiveness to therapy. Due to previous analytic shortcomings, variance in migraine symptoms has been weakly and inconsistently related to brain function. Taking advantage of neural network organization measured through resting-state functional connectivity (RSFC) and advanced statistical analysis, sophisticated symptom-brain mapping can now be performed. In the current analysis we used data from two sites (n=102 and 41), and performed Canonical Correlation Analysis (CCA), relating RSFC with a broad range of migraine symptoms ranging from headache characteristics to sleep abnormalities. This identified three dimensions of covariance between symptoms and RSFC. Importantly, none of these dimensions bore any relationship with subject motion. The first dimension was related to headache intensity, headache frequency, pain catastrophizing, affect, sleep disturbances, and somatic abnormalities, and was associated with frontoparietal and dorsal attention network connectivity, both of which are major cognitive networks. Additionally, RSFC scores from this dimension - both the baseline value and the change from baseline to post-intervention - were associated with clinical responsiveness to mind-body therapy. The second dimension was related to an inverse association between pain and anxiety, and to default mode network connectivity. The final dimension was related to pain catastrophizing, and salience, sensorimotor and default mode network connectivity. These unique symptom/brain-mappings over three dimensions provide novel network targets to modify specific ensembles of symptoms. In addition to performing CCA, we evaluated the current clustering of migraine patients into episodic and chronic subtypes, and found no evidence to support this clustering. However, when using RSFC scores from the three significant dimensions, we identified a novel clustering of migraine patients into four biotypes with unique functional connectivity patterns. These findings provide new insight into individual variability in migraine, and could serve as the foundation for novel therapies that take advantage of migraine heterogeneity.


title: Effect of rapamycin on mitochondria and lysosomes in fibroblasts from patients with mtDNA mutations

abstract: Maintaining mitochondrial function and dynamics is crucial for cellular health. In muscle, defects in mitochondria result in severe myopathies where accumulation of damaged mitochondria causes deterioration and dysfunction. Importantly, understanding the role of mitochondria in disease is a necessity to determine future therapeutics. One of the most common myopathies is mitochondrial encephalopathy lactic acidosis stroke-like episodes (MELAS), which has no current treatment. Recently, MELAS patients treated with rapamycin exhibited improved clinical outcomes. However, the cellular mechanisms of rapamycin effects in MELAS patients are currently unknown. In this study, we used cultured skin fibroblasts as a window into the mitochondrial dysfunction evident in MELAS cells, as well as to study the mechanisms of rapamycin action, compared to control, healthy individuals. We observed that mitochondria from patients were fragmented, had a 3-fold decline in the average speed of motility, a 2-fold reduced mitochondrial membrane potential and a 1.5-2-fold decline in basal respiration. Despite the reduction in mitochondrial function, mitochondrial import protein Tim23 was elevated in patient cell lines. MELAS fibroblasts had increased MnSOD, p62 and lysosomal function when compared to healthy controls. Treatment of MELAS fibroblasts with rapamycin for 24 hrs resulted in increased mitochondrial respiration compared to control cells, a higher lysosome content, and a greater localization of mitochondria to lysosomes. Our studies suggest that rapamycin has the potential to improve cellular health even in the presence of mtDNA defects, primarily via an increase in lysosomal content.


title: Photoswitchable epothilone-based microtubule stabilisers allow GFP imaging-compatible, optical control over the microtubule cytoskeleton

abstract: Optical methods to modulate microtubule stability and dynamics are promising approaches to reach the micron- and millisecond-scale resolution needed to decrypt the diverse roles of the microtubule cytoskeleton in biology. However, such optical methods have until now focussed nearly exclusively on microtubule destabilisation. Here, we introduce "STEpos" as light-responsive epothilone reagents, designed to photoswitchably bind to tubulin and stabilise lateral contacts in the microtubule lattice. Using a novel styrylthiazole photoswitch, designed to allow the hydrogen-bonding that is key to epothilone potency, we have created the first set of GFP-orthogonal photoswitchable microtubule stabilisers. The STEpos can photocontrol microtubule polymerisation, cell division, and cellular microtubule dynamics with micron- and second-scale spatiotemporal precision. STEpos offer substantial improvements of potency, solubility, and ease-of-use compared to the only previous photopharmaceuticals for microtubule stabilisation. The intriguing structure-photoswitching-activity relationship insights from this work will also assist future developments of improved STEpo reagents, and we anticipate that these will contribute greatly to high-precision cytoskeleton research across the fields of biophysics, cargo transport, cell motility, cell division, development, and neuroscience.


title: Liquid-to-solid phase transition of oskar RNP granules is essential for their function in the Drosophila germline

abstract: Asymmetric localization of oskar RNP granules to the oocyte posterior is crucial for abdominal patterning and germline formation of the Drosophila embryo. We show that oskar RNP granules in the oocyte are condensates with solid-like physical properties. Using purified oskar RNA and scaffold proteins Bruno and Hrp48, we confirm in vitro that oskar granules undergo a liquid-to-solid phase transition. Whereas the liquid phase allows RNA incorporation, the solid phase precludes incorporation of additional RNA while allowing RNA-dependent partitioning of specific proteins. Genetic modification of scaffold granule proteins, or tethering the intrinsically disordered region of human Fused in Sarcoma to oskar mRNA, allowed modulation of granule material properties in vivo. The resulting liquid-like properties impaired oskar localization and translation with severe consequences on embryonic development. Our study reflects how physiological phase transitions shape RNA-protein condensates to regulate localization and expression of a maternal RNA that instructs germline formation.


title: Pkd1 and Wnt5a genetically interact to control lymphatic vascular morphogenesis in mice

abstract: Lymphatic vascular development is regulated by well-characterised signalling and transcriptional pathways. These pathways regulate lymphatic endothelial cell (LEC) migration, motility, polarity and and morphogenesis. Canonical and non-canonical WNT signalling pathways are known to control LEC polarity and development of lymphatic vessels and valves. PKD1, encoding Polycystin-1, is the most commonly mutated gene in polycystic kidney disease but has also been shown to be essential in lymphatic vascular morphogenesis. The mechanism by which Pkd1 acts during lymphangiogenesis remains unclear. Here we find that loss of non-canonical WNT signalling components Wnt5a and Ryk phenocopy lymphatic defects seen in Pkd1 knockout mice. To investigate genetic interaction, we generated Pkd1/Wnt5a double knockout mice. Loss of Wnt5a suppressed phenotypes seen in the lymphatic vasculature of Pkd1-/- mice and Pkd1 deletion suppressed phenotypes observed in Wnt5a-/- mice. Thus, we report mutually suppressive roles for Pkd1 and Wnt5a, with developing lymphatic networks restored to a more wild-type state in double mutant mice. This genetic interaction between Pkd1 and the non-canonical WNT signalling pathway ultimately controls LEC polarity and the morphogenesis of developing vessel networks. Our work suggests that Pkd1 acts at least in part by regulating non-canonical WNT signalling during the formation of lymphatic vascular networks.

category: developmental biology


title: Microfluidic-based imaging of complete C. elegans larval development

abstract: Several microfluidic-based methods for long-term C. elegans imaging have been introduced in recent years, allowing real-time observation of previously inaccessible processes. The existing methods either permit imaging across multiple larval stages without maintaining a stable worm orientation, or allow for very good immobilization but are only suitable for shorter experiments. Here, we present a novel microfluidic imaging method, which allows parallel live-imaging across multiple larval stages, while delivering excellent immobilization and maintaining worm orientation and identity over time. This is achieved by employing an array of microfluidic trap channels carefully tuned to maintain worms in a stable orientation, while allowing growth and molting to occur. Immobilization is supported by an active hydraulic valve, which presses worms onto the cover glass during image acquisition, with the animals remaining free for most of an experiment. Excellent quality images can be acquired of multiple worms in parallel, with little impact of the imaging method on worm viability or developmental timing. The capabilities of this methodology are demonstrated by observing the hypodermal seam cell divisions and, for the first time, the entire process of vulval development from induction to the end of morphogenesis. Moreover, we demonstrate RNAi on-chip, which allows for perturbation of dynamic developmental processes, such as basement membrane breaching during anchor cell invasion.


title: Functional, metabolic and transcriptional maturation of stem cell derived beta cells

abstract: Transplantation of pancreatic islet cells derived from human pluripotent stem cells is a promising treatment for diabetes. Despite progress in stem cell-derived islet (SC-islet) generation, detailed characterization of their functional properties has not been conducted. Here, we generated functionally mature SC-islets using an optimized protocol and comprehensively benchmarked them against primary adult islets. Biphasic glucose stimulated insulin secretion developed during in vitro maturation, associated with cytoarchitectural reorganization and increased alpha cells. Electrophysiology and exocytosis of SC-islets were comparable to adult islets. Glucose-responsive insulin secretion was achieved despite differences in glycolytic and mitochondrial glucose metabolism. Single-cell transcriptomics of SC-islets in vitro and throughout 6 months of murine engraftment revealed a continuous maturation trajectory culminating in a transcriptional landscape closely resembling that of primary islets. Our thorough evaluation of SC-islet maturation highlights their advanced degree of functionality and supports their use in further efforts to understand and combat diabetes.


title: Uncoupling of ribosome biogenesis and Tor activation by TRIM-NHL proteins promotes terminal differentiation

abstract: Proper tissue development and homeostasis depends on the balance between growth and terminal differentiation, but the mechanisms coordinating these processes remain elusive. Accumulating evidence indicates that ribosome biogenesis (RiBi) and protein synthesis, two of the most energy-consuming cellular processes sustaining growth, are tightly regulated and yet can be uncoupled during stem cell fate transitions. Here, using the Drosophila adult female germline stem cell (GSC) and larval neuroblast (NB) systems, we show that Mei-P26 and Brat, two Drosophila TRIM-NHL paralogs of the mammalian TRIM32 protein family, are responsible for uncoupling RiBi and protein synthesis during GSC and NB differentiation, respectively. Mei-P26 and Brat modify the metabolism of differentiating cells by activating the Target of rapamycin (Tor) kinase to promote translation, while concomitantly repressing RiBi. Depletion of Mei-P26 or Brat results in excessive cellular growth and defective terminal differentiation, which can be rescued by ectopic activation of Tor together with suppression of RiBi. Our results indicate that the anabolic reprogramming established by TRIM-NHL activity by uncoupling RiBi and translation activities creates the conditions required for terminal differentiation.


title: Simultaneous suppression of ribosome biogenesis and Tor activation by TRIM-NHL proteins promotes terminal differentiation


title: Rapid mosaic brain evolution under artificial selection for relative telencephalon size in the guppy (Poecilia reticulata)

abstract: The vertebrate brain displays enormous morphological variation and the quest to understand the evolutionary causes and consequences of this variation has spurred much research. The mosaic brain evolution hypothesis, stating that brain regions can evolve relatively independently, is an important idea in this research field. Here we provide experimental support for this hypothesis through an artificial selection experiment in the guppy (Poecilia reticulata). After four generations of selection on relative telencephalon volume (relative to brain size) in replicated up-selected, down-selected and control-lines, we found substantial changes in telencephalon size, but no changes in other regions. Comparisons revealed that up-selected lines had larger telencephalon while down-selected lines had smaller telencephalon than wild Trinidadian populations. No cost of increasing telencephalon size was detected in offspring production. Our results support that independent evolutionary changes in specific brain regions through mosaic brain evolution can be important facilitators of cognitive evolution.


title: Standing covariation between genomic and epigenomic patterns as source for natural selection in wild strawberry plants

abstract: Adaptive evolution is generally thought to be the result of natural selection predominantly acting upon pre-existing DNA sequence polymorphisms through gene-environment interactions. Epigenetic inheritance is, however, recently considered an additional molecular force driving adaptive evolution independent of DNA sequence variation. Through comparative analyses of genome-wide genetic (SNPs) and epigenetic (DMCs) variation of wild strawberry plants raised under distinct drought settings, we demonstrate intermediate levels of genome-wide covariation between SNPs and DMCs. Cases of high SNP-DMC covariation were significantly associated with (i) applied stress, (ii) non-adaptive SNPs, and (iii) solitary DMCs (as opposed to DMC islands). We also found that DMCs, drought-responsive DMCs in particular, typically co-vary with hundreds of SNPs, indicating high genomic redundancy as a basis for polygenic adaptation. Our findings suggest that stress-responsive DMCs initially co-vary with many associated SNPs under increased environmental stress (cfr. co-gradient plasticity), and that natural selection acting upon these SNPs subsequently reduce standing covariation with stress-responsive DMCs. In addition, the degree of covariation between SNPs and DMCs appears independent of their respective genomic distance, indicating that trans-acting associations between SNPs and DMCs are as likely as cis-acting associations. Our study is in favor of DNA methylation profiles representing complex quantitative traits rather than independent evolutionary forces, but further research is required to fully rule out SNP-independence of genome-wide DMCs. We provide a conceptual framework for polygenic regulation and adaptation shaping genome-wide methylation patterns.


title: Penguin: A Tool for Predicting Pseudouridine Sites in Direct RNA Nanopore Sequencing Data

abstract:
Pseudouridine is one of the most abundant RNA modifications, occurring when uridines are catalyzed by Pseudouridine synthase proteins. It plays an important role in many biological processes and also has an importance in drug development. Recently, the single-molecule sequencing techniques such as the direct RNA sequencing platform offered by Oxford Nanopore technologies enable direct detection of RNA modifications on the molecule that is being sequenced, but to our knowledge this technology has not been used to identify RNA Pseudouridine sites. To this end, in this paper, we address this limitation by introducing a tool called Penguin that integrates several developed machine learning (ML) models (i.e., predictors) to identify RNA Pseudouridine sites in Nanopore direct RNA sequencing reads. Penguin extracts a set of features from the raw signal measured by the Oxford Nanopore and the corresponding basecalled k-mer. Those features are used to train the predictors included in Penguin, which in turn, is able to predict whether the signal is modified by the presence of Pseudouridine sites. We have included various predictors in Penguin including Support vector machine (SVM), Random Forest (RF), and Neural network (NN). The results on the two benchmark data sets show that Penguin is able to identify Pseudouridine sites with a high accuracy of 93.38% and 92.61% using SVM in random split testing and independent validation testing respectively. Thus, Penguin outperforms the existing Pseudouridine predictors in the literature that achieved an accuracy of 76.0 at most with an independent validation testing. A GitHub of the tool is accessible at https://github.com/Janga-Lab/Penguin.

HIGHLIGHTSO_LIPenguin integrates several developed ML learning models (i.e., predictors) to identify RNA {Psi} sites in Nanopore direct RNA sequencing reads.
C_LIO_LIThe pipeline of penguin automates the data preprocessing including Nanopore direct RNA reads alignment using Minimap2, and Nanopore signal extraction using Nanopolish, feature extraction from raw Nanopore signal for training ML predictors integrated in its platform, and the prediction of RNA {Psi} sites with those predictors.
C_LIO_LIPenguin can predict {Psi} sites with a performance that outperforms the performance of the state-of-the-art research methods existing in the literature.
C_LIO_LIPenguin platform can be adopted to be used for predicting other/various types of RNA modification.
C_LIO_LIThere are 6137606 U-mers samples predicted by penguin best ML model (SVM) as {Psi} ones from a total of 67491289 U-mers samples in the complete Hek293 cell line with 556813 unique genomic location of {Psi}.
C_LIO_LIThere are 1193192 U-mers samples predicted by penguin best ML model (SVM) as {Psi} ones from a total of 229637931 U-mers samples in the complete Hela cell line with 39384 unique genomic locations of {Psi}.
C_LIO_LIThere is a small fraction of 0.01% (6482 unique genomic locations) of {Psi} that are common (overlapped) between both Hek293 and Hela cell lines.
C_LIO_LIThe extend of {Psi} modification (the number of U-mers samples predicted as {Psi} samples to the total number of U-mer samples in the complete RNA sequence of the cell line) in RNA sequence of Hek293 cell line is much greater than its counterpart for Hela cell line (9% for Hek293 versus 0. 5 % for Hela cell line).
C_LI

category: genomics


title: Genome-wide association study on 13,167 individuals identifies regulators of hematopoietic stem and progenitor cell levels in human blood

abstract: Understanding how hematopoietic stem and progenitor cells (HSPCs) are regulated is of central importance for the development of new therapies for blood disorders and stem cell transplantation. To date, HSPC regulation has been extensively studied in vitro and in animal models, but less is known about the mechanisms in vivo in humans. Here, in a genome-wide association study on 13,167 individuals, we identify 9 significant and 2 suggestive DNA sequence variants that influence HSPC (CD34+) levels in human blood. The identified loci associate with blood disorders, harbor known and novel HSPC genes, and affect gene expression in HSPCs. Interestingly, our strongest association maps to the PPM1H gene, encoding an evolutionarily conserved serine/threonine phosphatase never previously implicated in stem cell biology. PPM1H is expressed in HSPCs, and the allele that confers higher blood CD34+ cell levels downregulates PPM1H. By functional fine-mapping, we find that this downregulation is caused by the variant rs772557-A, which abrogates a MYB transcription factor binding site in PPM1H intron 1 that is active in specific HSPC subpopulations, including hematopoietic stem cells, and interacts with the promoter by chromatin looping. Furthermore, rs772557-A selectively increases HSPC subpopulations in which the MYB site is active, and PPM1H shRNA- knockdown increased CD34+ and CD34+90+ cell proportions in umbilical cord blood cultures. Our findings represent the first large-scale association study on a stem cell trait, illuminating HSPC regulation in vivo in humans, and identifying PPM1H as a novel inhibition target that can potentially be utilized clinically to facilitate stem cell harvesting for transplantation.


title: Identification of cardiac afterload dynamics from data

abstract: The prospect of ex vivo functional evaluation of donor hearts is considered. Particularly, the dynamics of a synthetic cardiac afterload model are compared to those of normal physiology. A method for identification of continuous-time transfer functions from sampled data is developed and verified against results from the literature. The method relies on exact gradients and Hessians obtained through automatic differentiation. This also enables straightforward sensitivity analyses. Such analyses reveal that the 4-element Windkessel model is not practically identifiable from representative data while the 3-element model underfits the data. Pressure-volume (PV) loops are therefore suggested as an alternative for comparing afterload dynamics.


title: PCRedux: A Data Mining and Machine Learning Toolkit for qPCR Experiments

abstract:
MotivationQuantitative Real-time PCR (qPCR) is a widely used -omics method for the precise quantification of nucleic acids, in which the result is associated with the presence/absence or quantity of a specific nucleic acid sequence. As the amount of qPCR data increases worldwide, the manual assessment of results becomes challenging and difficult to reproduce. To overcome this, some automatable characteristics of amplification curves have been described in the literature, often with an appropriate "rule of thumb".

ResultsWe developed PCRedux to analyze and calculate 90 numerical qPCR amplification curve descriptors (  features") from large datasets of qPCR amplification curves that are aimed for interpretable machine learning and development of decision support systems. In a case study of a diverse dataset with 3181 positive, negative and ambiguous amplification curves, as assessed by three human raters, we demonstrate a sensitivity >99 % and specificity >97 % in detecting positive and negative amplification. PCRedux is unique as it goes beyond traditional qPCR analysis to capture curvature properties that improve the characterization and classification of amplification curves. The calculation of the features is reproducible and objective, since R is used as a controllable working environment. PCRedux is not a black box, but open source software following on the principle of mathematically interpretable features. These can be combined with user-defined labels for automatic multi-category classification and regression in machine learning.

Availabilityhttps://cran.r-project.org/package=PCRedux. Web server: http://shtest.evrogen.net/PCRedux-app/. Documentation: https://PCRuniversum.github.io/PCRedux/.


title: Glycosylated Antibiotics: New Promising Bacterial 1 Efflux Pumps Inhibitors

abstract:
BackgroundAntimicrobial resistance is considered a major concern problem; bacteria have evolved mechanisms to overcome antibiotics action through evolutionary process. One main resistance mechanism that bacteria developed is the pumping of the antibiotics out of bacterial cells by transmembrane transporter proteins known as efflux pumps.

Materials and methodsTo overcome bacterial resistance guided by efflux pumps, efflux pumps inhibitors (EPIs) are small molecules that obstruct efflux pumps binding sites and its structural assembly leading to disability in the efflux pumps normal function, new EPIs which under the current study are created by modifying the chemical structure of most common antibiotics including Ampicillin, Penicillin, Chloramphenicol, Ciprofloxacin and Tetracycline, such antibiotics are modified by adding N-acetyl glucose amine moiety to acceptor OH group of the respective antibiotic, the newly modified antibiotics are glycosylated EPIs. To test the effectiveness of the new EPIs in inhibiting AcrB-TolC and MexA-OprM efflux pumps functions, ADME properties for all of glycosylated antibiotics have been measured through applying Lipinskis role of 5, docking and simulation studies have been included as well.

ResultsDocked glycosylated tetracycline has given the highest binding energy in the active sites of both pumps, with -9.4 against AcrB and -8.8 against MexA. The simulation study has confirmed the binding of the glycosylated tetracycline in the active sites of both pumps, as well as its stability during the biological dynamicity of both pumps (opening and closing channels).

ConclusionThe results validation requires a long simulation time about 50 ns or more which was un applicable due to cost limitation, however, the newly glycosylated antibiotics have promising results that might make it eligible as drug candidates to overcome bacterial resistance.


title: Glycosylated Antibiotics: New Promising Bacterial Efflux Pumps Inhibitors


title: MetaProClust-MS1: A tool for clustering metaproteomes using rapid MS1 profiling

abstract: Metaproteomics is used to explore the composition, dynamics and function of microbial communities. How-ever, acquiring data by tandem mass spectrometry is time consuming and resource intensive. To mediate this challenge, we present MetaProClust-MS1, a computational framework for microbiome screening developed to reduce the time required for data acquisition by mass spectrometry. In this proof-of-concept study, we tested MetaProClust-MS1 on data acquired using short 15 minute MS1-only mass spectrometry gradients and compared the results to those produced using data acquired by a traditional tandem mass spectrometry approach. MetaProClust-MS1 identified robust microbiome shifts caused by xenobiotics in both datasets. Cluster topologies were also significantly correlated. We demonstrate that MetaProClust-MS1 is able to rapidly screen microbiomes using only short MS1 profiles. This approach can be used to prioritize samples for deep metaproteomic analysis and will be especially useful in large-scale metaproteomic screens or in clinical settings where rapid results are required.


title: Racial differences in androgen metabolism and receptor signaling in prostate cancer

abstract:
Dihydrotestosterone (DHT) and testosterone (T) mediated androgen receptor (AR) nuclear translocation initiates transcription of AR target genes that are pivotal for prostate cancer (PrCa) development and progression. Here we provide data indicating that in contrast to European American (EA) men, African American (AA) men with localized PrCa can exploit an alternative progesterone-androsterone-5-androstanedione pathway for DHT biosynthesis. Enzymes that are involved in alternate pathways of DHT biosynthesis are elevated in PrCa tissues from AA men, compared to EA men, and also correlated with increased serum DHT levels. In addition, higher serum DHT levels reflect increased RNA expression of AR target genes in PrCa tissues from AA men. Interestingly, serum T but not DHT levels are significantly lower in AA men compared to EA men with PrCa. Furthermore, serum progesterone and related intermediate metabolites levels that are produced during alternate pathways of DHT biosynthesis are significantly lower in AA men with PrCa and associated with a shorter time to disease progression. These data highlight that androgen biosynthesis is altered in therapy naive localized PrCa in AA men, and can potentially serve as prognostic indicators of disease progression.

SignificanceOur work provides a rationale to examine potential pharmacological interventions that target androgen biosynthesis and AR signaling earlier in the disease continuum in AA men with PrCa. Additionally, our study lays the groundwork for developing serum measurements of intermediate androgen metabolites as PrCa prognostic biomarkers.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/437727v1_ufig1.gif" ALT="Figure 1">
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category: cancer biology


title: Muscle-secreted neurturin couples myofiber oxidative metabolism and slow motor neuron identity.

abstract:
Endurance exercise promotes skeletal muscle vascularization, oxidative metabolism, fiber-type switching, and neuromuscular junction integrity. Importantly, the metabolic and contractile properties of the muscle fiber must be coupled to the identity of the innervating motor neuron (MN). Here, we show that muscle-derived neurturin (NRTN) acts on muscle fibers and MNs to couple their characteristics. Using a muscle-specific NRTN transgenic mouse (HSA-NRTN) and RNA-sequencing of MN somas, we observed that retrograde NRTN signaling promotes a shift towards a slow MN identity. In muscle, NRTN increased capillary density, oxidative capacity, and induced a transcriptional reprograming favoring fatty acid metabolism over glycolysis. This combination of effects on muscle and MNs, makes HSA-NRTN mice lean with remarkable exercise performance and motor coordination. Interestingly, HSA-NRTN mice largely recapitulate the phenotype of mice with muscle-specific expression of its upstream regulator PGC-11. This work identifies NRTN as a myokine that couples muscle oxidative capacity to slow MN identity.

HIGHLIGHTSO_LINRTN is a myokine induced by physical exercise.
C_LIO_LIMuscle-derived NRTN promotes a slow motor neuron identity.
C_LIO_LIMuscle-derived NRTN enhances muscle oxidative metabolism.
C_LIO_LINRTN improves systemic metabolism, exercise performance and motor coordination.
C_LI


title: A Hierarchical Process Model Links Behavioral Aging and Lifespan in C. elegans

abstract: Individuals who remain vigorous longer tend to live longer, supporting the design of predictive behavioral biomarkers of aging. In C. elegans, the timing of age-associated vigorous movement cessation (VMC) and lifespan correlate strongly between individuals. However, many genetic and pharmaceutical interventions that alter aging produce disproportional effects on VMC and lifespan, appearing to "uncouple" the rate of behavioral aging and lifespan. To study the causal structure underlying such uncoupling, we developed a high-throughput, automated imaging platform to quantify behavioral aging and lifespan at an unprecedented scale. Our method reveals an inverse correlation between each individuals vigorous movement span and their remaining lifespan. Robust across many lifespan-altering interventions including a new RNA-polymerase II auxin-inducible degron system, our data shows that individual C. elegans experience at least two distinct but coupled physical declines--one governing VMC and the other governing lifespan. Through simulations and modeling, we clarify the causal relationship between these two "biological ages" and highlight a crucial but often untested assumption in conventional aging biomarker research: predictive biomarkers may not always report on the same biological age as that which determines long-term health outcomes.

category: systems biology


title: CD150-dependent hematopoietic stem cells sensing of Brucella instructs myeloid commitment

abstract:
So far, hematopoietic stem cells (HSC) are considered the source of mature immune cells, the latter being the only ones capable of mounting an immune response. Recent evidence shows HSC can also directly sense cytokines released upon infection/inflammation and pathogen-associated molecular pattern interaction, while keeping a long-term memory of previous encountered signals. Direct sensing of danger signals by HSC induces early myeloid commitment, increases myeloid effector cell numbers and contributes to an efficient immune response. Here, using specific genetic tools on both host and pathogen sides, we show that HSC can directly sense B. abortus pathogenic bacteria within the bone marrow via the interaction of the cell surface protein CD150 with the bacterial outer membrane protein Omp25, inducing efficient functional commitment of HSC to the myeloid lineage. This is the first demonstration of a direct recognition of a live pathogen by HSC via CD150, which attests of a very early contribution of HSC to immune response.

SUMMARYThis work provides first evidence HSC directly sense Brucella abortus via the bacterial outer membrane protein Omp25 and the HSC surface receptor CD150, leading to functional commitment of HSC to myeloid lineage and very early initiation of immune response.

category: immunology


title: IL-21 and IFN-α have both opposite and redundant role on human innate precursors and memory B-cell differentiation.

abstract: Immunological memory is essential for effective immune protection upon antigen rechallenge. Memory B cells encompass multiple subsets, heterogeneous in terms of phenotypes, origins and precursors, anatomical localization, and functional responses. B-cell responses are conditioned by micro-environmental signals, including cytokines. Here, we analyzed in vitro the effects of two cytokines implicated in B-cell differentiation, interferon-alpha (IFN-) and interleukin (IL)-21, on the early functional response of four different mature B-cell subsets (IgD- CD27- naive, IgD+ CD27+ unswitched, IgD- CD27+ switched and double-negative B cells). The dual response of naive and memory B cells to IL-21 allowed us to uncover a unique IgD+ CD27- CD10- B-cell population (referred to as NARB+) characterized by the expression of marginal zone B-cell markers CD45RB and CD1c. Similar to memory B cells, NARB+ cells were in a pre-activated state, allowing them to rapidly differentiate into plasmablasts upon innate signals while maintaining their susceptibility to IL-21 activation-induced apoptosis as observed for the naive compartment. Both in-depth phenotypic analysis of circulating B cells, and identification of these cells in spleen, tonsil and gut-associated lymphoid tissues, supported that NARB+ are uncommitted precursors of human marginal zone B cells.


title: A heterochromatic knob reducing the flowering time in maize

abstract: Maize flowering time is an important agronomic trait, which has been associated with variations in the genome size and heterochromatic knobs content. We integrated three steps to show this association. Firstly, we selected inbred lines varying for heterochromatic knob composition at specific sites in the homozygous state. Then, we produced homozygous and heterozygous hybrids for knobs. Second, we measured the genome size and flowering time for all materials. Knob composition did not affect the genome size and flowering time. Finally, we developed an association study and identified a knob marker on chromosome 9 showing the strongest association with flowering time. Indeed, modelling allele substitution and dominance effects could offer only one heterochromatic knob locus that could affect flowering time, making it earlier rather than the knob composition.


title: The genetic basis of Drosophila melanogaster defense against Beauveria bassiana explored through evolve and resequence and quantitative trait locus mapping

abstract: Many of the molecular mechanisms for antifungal immunity in Drosophila melanogaster have been defined, but relatively little is known about the genetic basis for variation in antifungal immunity in natural populations. Using two population genetic approaches, Quantitative Trait Locus (QTL) Mapping and Evolve and Resequence (E&R), we explored the genetics underlying D. melanogaster immune defense against infection with the fungus Beauveria bassiana. Immune defense was highly variable both in the recombinant inbred lines from the Drosophila Synthetic Population Resource used for our QTL Mapping and in the synthetic outbred populations used in our E&R study. Survivorship of infection improved dramatically over just 10 generations in the E&R study, and continued to increase for an additional 9 generations, revealing a trade-off with uninfected longevity. Populations selected for increased defense against B. bassiana evolved cross resistance to a second, distinct B. bassiana strain but not to bacterial pathogens. The QTL mapping study revealed that sexual dimorphism in defense depends on host genotype, and the E&R study indicated that dimorphism also depends on the specific pathogen to which the host is exposed. Both the QTL Mapping and E&R experiments generated lists of potentially causal candidate genes, although these lists were non-overlapping.


title: Mechanical and molecular activation lead to structurally analogous MscL states

abstract: The MscL channel gates in response to membrane tension changes to allow the exchange of molecules through its pore. Lipid removal from transmembrane pockets leads to a MscL response. However, it is unknown whether there is correlation between the tension mediated state and the state derived by pocket delipidation in the absence of tension. Transitions between MscL states may follow a similar pathway to cover the available conformational space but may not necessarily sample the same discrete intermediates. Here, we combined pulsed-EPR and HDX-MS measurements on MscL, coupled with molecular dynamics under membrane tension, to investigate the changes associated with the distinctively derived states. Whether it is tension or pocket delipidation, we find that MscL samples a similar expanded state, which is the final step of the delipidation pathway but only an intermediate stop of the tension mediated path. Our findings hint at synergistic modes of regulation in mechanosensitive channels.


title: Tension mediated mechanical activation and pocket delipidation lead to an analogous MscL state


title: The Time Complexity of Self-Assembly

abstract: Time efficiency of self-assembly is crucial for many biological processes. Moreover, with the advances of nanotechnology, time efficiency in artificial self-assembly becomes ever more important. While structural determinants and the final assembly yield are increasingly well understood, kinetic aspects concerning the time efficiency, however, remain much more elusive. In computer science, the concept of time complexity is used to characterize the efficiency of an algorithm and describes how the algorithms runtime depends on the size of the input data. Here we characterize the time complexity of non-equilibrium self-assembly processes by exploring how the time required to realize a certain, substantial yield of a given target structure scales with its size. We identify distinct classes of assembly scenarios, i.e.  algorithms to accomplish this task, and show that they exhibit drastically different degrees of complexity. Our analysis enables us to identify optimal control strategies for non-equilibrium self-assembly processes. Furthermore, we suggest an efficient irreversible scheme for the artificial self-assembly of nanostructures, which complements the state-of-the-art approach using reversible binding reactions and requires no fine-tuning of binding energies.


title: How does a small molecule bind at a cryptic binding site?

abstract: Protein-protein interactions (PPIs) are ubiquitous biomolecular processes that are central to virtually all aspects of cellular function. Identifying small molecules that modulate specific disease-related PPIs is a strategy with enormous promise for drug discovery. The design of drugs to disrupt PPIs is challenging, however, because many potential drug-binding sites at PPI interfaces are "cryptic": When unoccupied by a ligand, cryptic sites are often flat and featureless, and thus not readily recognizable in crystal structures, with the geometric and chemical characteristics of typical small-molecule binding sites only emerging upon ligand binding. The rational design of small molecules to inhibit specific PPIs would benefit from a better understanding of how such molecules bind at PPI interfaces. To this end, we have conducted unbiased, all-atom MD simulations of the binding of four small-molecule inhibitors (SP4206 and three SP4206 analogs) to interleukin 2 (IL2)--which performs its function by forming a PPI with its receptor--without incorporating any prior structural information about the ligands binding. In multiple binding events, a small molecule settled into a stable binding pose at the PPI interface of IL2, resulting in a protein-small-molecule binding site and pose virtually identical to that observed in an existing crystal structure of the IL2-SP4206 complex. Binding of the small molecule stabilized the IL2 binding groove, which when the small molecule was not bound emerged only transiently and incompletely. Moreover, free energy perturbation (FEP) calculations successfully distinguished between the native and non-native IL2-small-molecule binding poses found in the simulations, suggesting that binding simulations in combination with FEP may provide an effective tool for identifying cryptic binding sites and determining the binding poses of small molecules designed to disrupt PPI interfaces by binding to such sites.


title: New metabolic alterations and predictive marker pipecolic acid in sera for esophageal squamous cell carcinoma

abstract: Esophageal squamous cell carcinoma (ESCC) is a major histological subtype of esophageal cancer with dismal prognosis. Although several serum metabolomic investigations have been reported, ESCC tumor-associated metabolic alterations along with predictive biomarkers in sera were not defined. Here we enrolled 34 treatment-naive ESCC patients and collected their pre-and post-esophagectomy sera together with sera from 34 healthy volunteers for metabolomic survey. Our comprehensive analysis discerned ESCC tumor-associated metabolic alterations as represented by a panel of 12 serum metabolites. Notably, postoperative abrosia and parenteral nutrition significantly perturbed the serum metabolome. Furthermore, we performed examination using sera from carcinogen-induced mice at dysplasia and ESCC stages, and identified three ESCC tumor-associated metabolites conserved between mice and humans. Notably, among these metabolites, pipecolic acid was progressively increased in mouse sera from dysplasia to cancerization, and it could accurately discriminate between mice at dysplasia stage and healthy control mice. Furthermore, this metabolite was essential for ECSS cells to oppose oxidative stress-induced DNA damage and cell proliferation arrest. Together, this study uncovered 12 ESCC tumor-associated serum metabolites with potential for monitoring therapeutic efficacy and disease relapse, presented evidence for refining parenteral nutrition composition, and highlighted serum pipecolic acid as an attractive biomarker for prediction of ESCC tumorigenesis.


title: Acylation of the incretin peptide exendin-4 directly impacts GLP-1 receptor signalling and trafficking

abstract:
The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor and mainstay therapeutic target for the treatment of type 2 diabetes and obesity. Recent reports have highlighted how biased agonism at the GLP-1R affects sustained glucose-stimulated insulin secretion through avoidance of desensitisation and downregulation. A number of GLP-1R agonists (GLP-1RAs) feature a fatty acid moiety to promote albumin binding in order to prolong their pharmacokinetics, but the potential for these ligand changes to influence GLP-1R signalling has rarely been investigated beyond potency assessments for cyclic adenosine monophosphate (cAMP). In this work we directly compare the prototypical GLP-1RA exendin-4 with its C-terminally acylated analogue, exendin-4-C16, for their relative propensities to recruit and activate G proteins and {beta}-arrestins, endocytic and post-endocytic trafficking profiles, and interactions with model and cellular membranes. Both ligands had similar cAMP potency but the exendin-4-C16 showed [~]2.5-fold bias towards G protein recruitment and a [~]60% reduction in {beta}-arrestin-2 recruitment efficacy compared to exendin-4, as well as reduced GLP-1R endocytosis and preferential targeting towards recycling pathways. These effects were associated with a reduced ability to promote the movement of the GLP-1R extracellular domain, as determined using a conformational biosensor approach, and a [~]70% increase in insulin secretion. Interactions with plasma membrane lipids were enhanced by the acyl chain. Exendin-4-C16 showed extensive albumin binding and was highly effective for lowering of blood glucose in mice over at least 72 hours. Overall, our study highlights the importance of a broad approach to the evaluation of GLP-1RA pharmacology.

Significance statementAcylation is a common strategy to enhance the pharmacokinetics of peptide-based drugs. Our work shows how acylation can also affect various other pharmacological parameters, including biased agonism, receptor trafficking and interactions with the plasma membrane, which may be therapeutically important.

category: pharmacology and toxicology


title: Tissue specific requirements for ECSIT in mitochondrial complex I assembly

abstract: Here we describe a mutation in the mitochondrial complex I assembly factor (Evolutionarily conserved signalling intermediate in Toll pathway) ECSIT which reveals tissue specific requirements for this factor in complex I assembly. Mitochondrial complex I assembly is a multi-step process dependant on assembly factors that organise and arrange the individual subunits, allowing for their incorporation into the complete enzyme complex. We have identified an ENU induced mutation in ECSIT (N209I) that exhibits a profound effect on complex I assembly only in heart tissue resulting in hypertrophic cardiomyopathy in the absence of other phenotypes. Mitochondrial function was reduced by 98% in mitochondria isolated from cardiac tissue but mitochondria from other tissues such as skeletal muscle, brain, liver, and kidney were unaffected. This data suggests the mechanisms underlying complex I assembly are tissue specific and has implications in understanding the pathogenesis of cardiomyopathy.

category: cell biology


title: Two timescales control the creation of large protein aggregates in cells

abstract:
Protein aggregation is of particular interest due to its connection with many diseases and disorders. Many factors can alter the dynamics and result of this process, one of them being the diffusivity of the monomers and aggregates in the system. Here, we study experimentally and theoretically an aggregation process in cells, and we identify two distinct physical timescales that set the number and size of aggregates. The first timescale involves fast aggregation of small clusters freely diffusing in the cytoplasm, while, in the second one, the aggregates are larger than the pore size of the cytoplasm and thus barely diffuse, and the aggregation process is slowed down. However, the process is not entirely halted, potentially reflecting a myriad of active but random forces forces that stir the aggregates. Such slow timescale is essential to account for the experimental results of the aggregation process. These results could also have implications in other processes of spatial organization in cell biology, such as phase-separated droplets.

O_TEXTBOXSIGNIFICANCEProtein aggregation is a physico-chemical process that underlies many diseases and disorders, such as Alzheimers or Huntingtons disease. Here, we study experimental and theoretically the effect of a sharp decrease of diffusivity in the aggregation dynamics, such as the one that could happen in the cell due to the presence of obstacles. We find that two different timescales are important in setting the size of large aggregates and we give an estimate of the size of the aggregate at which this dramatic change in behaviour occurs, which could not be exclusive of protein aggregation but affect many other intracellular processes.

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title: To knot and not: Multiple conformations of the SARS-CoV-2 frameshifting RNA element

abstract: The SARS-CoV-2 frameshifting RNA element (FSE) is an excellent target for therapeutic intervention against Covid-19. This small gene element employs a shifting mechanism to pause and backtrack the ribosome during translation between Open Reading Frames 1a and 1b, which code for viral polyproteins. Any interference with this process has profound effect on viral replication and propagation. Pinpointing the structures adapted by the FSE and associated structural transformations involved in frameshifting has been a challenge. Using our graph-theory-based modeling tools for representing RNA secondary structures, "RAG" (RNA-As-Graphs), and chemical structure probing experiments, we show that the 3-stem H-type pseudoknot (3_6 dual graph), long assumed to be the dominant structure has a viable alternative, an HL-type 3-stem pseudoknot (3_3) for longer constructs. In addition, an unknotted 3-way junction RNA (3_5) emerges as a minor conformation. These three conformations share Stems 1 and 3, while the different Stem 2 may be involved in a conformational switch and possibly associations with the ribo-some during translation. For full-length genomes, a stem-loop motif (2_2) may compete with these forms. These structural and mechanistic insights advance our understanding of the SARS-CoV-2 frameshifting process and concomitant virus life cycle, and point to three avenues of therapeutic intervention.

category: biochemistry


title: To knot or not to knot: Multiple conformations of the SARS-CoV-2 frameshifting RNA element


title: SARS-CoV-2 immune evasion by variant B.1.427/B.1.429

abstract: SARS-CoV-2 entry is mediated by the spike (S) glycoprotein which contains the receptor-binding domain (RBD) and the N-terminal domain (NTD) as the two main targets of neutralizing antibodies (Abs). A novel variant of concern (VOC) named CAL.20C (B.1.427/B.1.429) was originally detected in California and is currently spreading throughout the US and 29 additional countries. It is unclear whether antibody responses to SARS-CoV-2 infection or to the prototypic Wuhan-1 isolate-based vaccines will be impacted by the three B.1.427/B.1.429 S mutations: S13I, W152C and L452R. Here, we assessed neutralizing Ab responses following natural infection or mRNA vaccination using pseudoviruses expressing the wildtype or the B.1.427/B.1.429 S protein. Plasma from vaccinated or convalescent individuals exhibited neutralizing titers, which were reduced 3-6 fold against the B.1.427/B.1.429 variant relative to wildtype pseudoviruses. The RBD L452R mutation reduced or abolished neutralizing activity of 14 out of 35 RBD-specific monoclonal antibodies (mAbs), including three clinical-stage mAbs. Furthermore, we observed a complete loss of B.1.427/B.1.429 neutralization for a panel of mAbs targeting the N-terminal domain due to a large structural rearrangement of the NTD antigenic supersite involving an S13I-mediated shift of the signal peptide cleavage site. These data warrant closer monitoring of signal peptide variants and their involvement in immune evasion and show that Abs directed to the NTD impose a selection pressure driving SARS-CoV-2 viral evolution through conventional and unconventional escape mechanisms.


title: The neutralization potency of anti-SARS-CoV-2 therapeutic human monoclonal antibodies is retained against novel viral variants

abstract:
A wide range of SARS-CoV-2 neutralizing monoclonal antibodies (mAbs) were reported to date, most of which target the spike glycoprotein and in particular its receptor binding domain (RBD) and N-terminal domain (NTD) of the S1 subunit. The therapeutic implementation of these antibodies has been recently challenged by emerging SARS-CoV-2 variants that harbor extensively mutated spike versions. Consequently, the re-assessment of mAbs, previously reported to neutralize the original early-version of the virus, is of high priority.

Four previously selected mAbs targeting non-overlapping epitopes, were evaluated for their binding potency to RBD versions harboring individual mutations at spike positions 417, 439, 453, 477, 484 and 501. Mutations at these positions represent the prevailing worldwide distributed modifications of the RBD, previously reported to mediate escape from antibody neutralization. Additionally, the in vitro neutralization potencies of the four RBD-specific mAbs, as well as two NTD-specific mAbs, were evaluated against two frequent SARS-CoV-2 variants of concern (VOCs): (i) the B.1.1.7 variant, emerged in the UK and (ii) the B.1.351 variant, emerged in South Africa. Variant B.1.351 was previously suggested to escape many therapeutic mAbs, including those authorized for clinical use. The possible impact of RBD mutations on recognition by mAbs is addressed by comparative structural modelling. Finally, we demonstrate the therapeutic potential of three selected mAbs by treatment of K18-hACE2 transgenic mice two days post infection with each of the virus strains.

Our results clearly indicate that despite the accumulation of spike mutations, some neutralizing mAbs preserve their potency against SARS-CoV-2. In particular, the highly potent MD65 and BL6 mAbs are shown to retain their ability to bind the prevalent novel viral mutations and to effectively protect against B.1.1.7 and B.1.351 variants of high clinical concern.


title: Limiting the priming dose of a SARS CoV-2 vaccine improves virus-specific immunity

abstract: Since late 2019, SARS-CoV-2 has caused a global pandemic that has infected 128 million people worldwide. Although several vaccine candidates have received emergency use authorization (EUA), there are still a limited number of vaccine doses available. To increase the number of vaccinated individuals, there are ongoing discussions about administering partial vaccine doses, but there is still a paucity of data on how vaccine fractionation affects vaccine-elicited immunity. We performed studies in mice to understand how the priming dose of a SARS CoV-2 vaccine affects long-term immunity to SARS CoV-2. We first primed C57BL/6 mice with an adenovirus-based vaccine encoding SARS CoV-2 spike protein (Ad5-SARS-2 spike), similar to that used in the CanSino and Sputnik V vaccines. This prime was administered either at a low dose (LD) of 106 PFU or at a standard dose (SD) of 109 PFU, followed by a SD boost in all mice four weeks later. As expected, the LD prime induced lower immune responses relative to the SD prime. However, the LD prime elicited immune responses that were qualitatively superior, and upon boosting, mice that were initially primed with a LD exhibited significantly more potent immune responses. Overall, these data demonstrate that limiting the priming dose of a SARS CoV-2 vaccine may confer unexpected benefits. These findings may be useful for improving vaccine availability and for rational vaccine design.


title: Screening of HLA-A restricted T cell epitopes of SARS-CoV-2 and induction of CD8+ T cell responses in HLA-A transgenic mice

abstract: While SARS-CoV-2-specific T cells have been characterized to play essential roles in host immune protection in COVID-19 patients, few researches focus on the functional validation of T cell epitopes and development of vaccines inducing specific T cell responses. In this study, 120 CD8+ T cell epitopes from E, M, N, S and RdRp proteins were validated. Among them, 110 epitopes have not been reported previously; 110, 15, 6, 14 and 12 epitopes were highly homologous with SARS-CoV, OC43, NL63, HKU1, and 229E, respectively; 4 epitopes from S protein displayed one amino acid distinct from the current variants of SARS-CoV-2. Thirty-one epitopes restricted by HLA-A2 molecule were used to generate peptide cocktail vaccines in combination with Poly(I:C), R848 or polylactic-co-glycolic acid nanoparticles, which elicited robust specific CD8+ T cell responses in wild-type and HLA-A2/DR1 transgenic mice. Seven of the 31 epitopes were found to be cross-presented by HLA-A2 and H-2K/Db molecules. Unlike previous researches, this study established a modified cell co-culture system of DC-peptide-PBL using healthy donors PBMCs to validate the CD8+ T cell epitope on-silicon predicted; provided a library of CD8+ T cell epitopes restricted by a series of high-frequency HLA-A allotypes which covering broad Asian populations; identified the HLA-A cross-restrictions of these CD8+ T cell epitopes using competitive binding experiments with HMy2.CIR cell lines expressing indicated HLA-A molecules; and initially confirmed the in vivo feasibility of 9 or 10-mer peptide cocktail vaccines of SARS-CoV2. These data will facilitate the development of vaccines inducing antiviral CD8+ T cell responses.


title: Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry

abstract: Coagulopathy is a significant aspect of morbidity in COVID-19 patients. The clotting cascade is propagated by a series of proteases, including factor Xa and thrombin. While certain host proteases, including TMPRSS2 and furin, are known to be important for cleavage activation of SARS-CoV-2 spike to promote viral entry in the respiratory tract, other proteases may also contribute. Using biochemical and cell-based assays, we demonstrate that factor Xa and thrombin can also directly cleave SARS-CoV-2 spike, enhancing viral entry. A drug-repurposing screen identified a subset of protease inhibitors that promiscuously inhibited spike cleavage by both transmembrane serine proteases as well as coagulation factors. The mechanism of the protease inhibitors nafamostat and camostat may extend beyond inhibition of TMPRSS2 to coagulation-induced spike cleavage. Anticoagulation is critical in the management of COVID-19, and early intervention could provide collateral benefit by suppressing SARS-CoV-2 viral entry. We propose a model of positive feedback whereby infection-induced hypercoagulation exacerbates SARS-CoV-2 infectivity.


title: An alphavirus replicon-based vaccine expressing a stabilized Spike antigen induces sterile immunity and prevents transmission of SARS-CoV-2 between cats

abstract:
Early in the global SARS-CoV-2 pandemic concerns were raised regarding infection of other animal hosts and whether these could play a significant role in the viral epidemiology. Infection of animals could be detrimental by causing clinical disease but also of concern if they become a viral reservoir allowing further mutations, plus having the potential to infect other animals or humans. The first reported animals to be infected both under experimental conditions and from anecdotal field evidence were cats described in China early in 2020. Given the concerns this finding raised and the close contacts between humans and cats, we aimed to determine whether a vaccine candidate could be developed that was suitable for use in multiple susceptible animal species and whether this vaccine could reduce infection of cats in addition to preventing spread to other cats.

Here we report that a Replicon Particle (RP) vaccine based on Venezuelan equine encephalitis virus (VEEV), known to be safe and efficacious for use in a variety of animals, expressing a stabilised Spike antigen, could induce neutralising antibody titers in guinea pigs and cats. After two intramuscular vaccinations, virus neutralising antibodies were detected in the respiratory tract of the guinea pigs and a cell mediated immune response was induced. The design of the SARS-CoV-2 antigen was shown to be critical in developing a strong neutralising antibody response. Vaccination of cats was able to induce a serum neutralising antibody response which lasted for the course of the experiment. Interestingly, in contrast to control animals, infectious virus could not be detected in oropharyngeal or nasal swabs of vaccinated cats after challenge. Moreover, the challenged control cats spread the virus to in-contact cats whereas the vaccinated cats did not transmit virus. The results show that the RP vaccine induces sterile immunity preventing SARS-CoV-2 infection and transmission. This data suggests that this RP vaccine could be a multi-species vaccine useful for preventing spread to and between other animals should that approach be required.


title: Hydrogel-based slow release of a receptor-binding domain subunit vaccine elicits neutralizing antibody responses against SARS-CoV-2

abstract: The development of effective vaccines that can be rapidly manufactured and distributed worldwide is necessary to mitigate the devastating health and economic impacts of pandemics like COVID-19. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, which mediates host cell entry of the virus, is an appealing antigen for subunit vaccines because it is efficient to manufacture, highly stable, and a target for neutralizing antibodies. Unfortunately, RBD is poorly immunogenic. While most subunit vaccines are commonly formulated with adjuvants to enhance their immunogenicity, we found that clinically-relevant adjuvants Alum, AddaVax, and CpG/Alum were unable to elicit neutralizing responses following a prime-boost immunization. Here we show that sustained delivery of an RBD subunit vaccine comprising CpG/Alum adjuvant in an injectable polymer-nanoparticle (PNP) hydrogel elicited potent anti-RBD and anti-spike antibody titers, providing broader protection against SARS-CoV-2 variants of concern compared to bolus administration of the same vaccine and vaccines comprising other clinically-relevant adjuvant systems. Notably, a SARS-CoV-2 spike-pseudotyped lentivirus neutralization assay revealed that hydrogel-based vaccines elicited potent neutralizing responses when bolus vaccines did not. Together, these results suggest that slow delivery of RBD subunit vaccines with PNP hydrogels can significantly enhance the immunogenicity of RBD and induce neutralizing humoral immunity.


title: Structural dynamics of the β-coronavirus Mpro protease ligand binding sites

abstract: {beta}-coronaviruses alone have been responsible for three major global outbreaks in the 21st century. The current crisis has led to an urgent requirement to develop therapeutics. Even though a number of vaccines are available, alternative strategies targeting essential viral components are required as a back-up against the emergence of lethal viral variants. One such target is the main protease (Mpro) that plays an indispensible role in viral replication. The availability of over 270 Mpro X-ray structures in complex with inhibitors provides unique insights into ligand-protein interactions. Herein, we provide a comprehensive comparison of all non-redundant ligand-binding sites available for SARS-CoV2, SARS-CoV and MERS-CoV Mpro. Extensive adaptive sampling has been used to explore conformational dynamics employing convolutional variational auto encoder-based deep learning, and investigates structural conservation of the ligand binding sites using Markov state models across {beta}-coronavirus homologs. Our results indicate that not all ligand-binding sites are dynamically conserved despite high sequence and structural conservation across {beta}-coronavirus homologs. This highlights the complexity in targeting all three Mpro enzymes with a single pan inhibitor.

category: biophysics


title: Interactions during growth eclipse interactions during flowering for determining plant fecundity in pollinator-sharing annual plants

abstract: Density dependent interactions are fundamental to community ecology, but studies often reduce the complex nature of species interactions. In plant ecology, interactions during vegetative growth and flowering are often considered separately, though both can affect reproductive output. Here we use communities of annual flowering plants in the genus Clarkia to ask how interactions during growth and flowering contribute to density dependence in plant seed production, and if pollinator behaviors explain apparent patterns in plant interactions during flowering. We measure seed set (seed number per ovule) and total fecundity (whole-plant seed production) of Clarkia focal plants in experimental interaction plots with the effect of pollinators experimentally removed through supplemental pollination or retained. We also observe pollinator behaviors in the plots and experimental arrays to document pollinator preference, constancy and joint attraction. During flowering, pollinators significantly changed the density dependent effects of Clarkia interactions on seed set in 31% of species interactions, and these changes corresponded to pollinator behaviors. Total fecundity, however, did not depend on interactions between Clarkia; instead, earlier-flowering, non-Clarkia forbs limited total fecundity. Our study shows that interactions during vegetative growth can preclude the effect of pollinator- mediated interactions on fecundity by limiting potential reproductive output. Simultaneously studying different types of species interactions allows for understanding the contingency of ecological outcomes.


title: Density dependent interactions during growth mask the effect of interactions during flowering in pollinator-sharing annual plants