title: B.1.1.529 escapes the majority of SARS-CoV-2 neutralizing antibodies of diverse epitopes

abstract: The SARS-CoV-2 B.1.1.529 variant (Omicron) contains 15 mutations on the receptor-binding domain (RBD). How Omicron would evade RBD neutralizing antibodies (NAbs) requires immediate investigation. Here, we used high-throughput yeast display screening1,2 to determine the RBD escaping mutation profiles for 247 human anti-RBD NAbs and showed that the NAbs could be unsupervised clustered into six epitope groups (A-F), which is highly concordant with knowledge-based structural classifications3-5. Strikingly, various single mutations of Omicron could impair NAbs of different epitope groups. Specifically, NAbs in Group A-D, whose epitope overlap with ACE2-binding motif, are largely escaped by K417N, G446S, E484A, and Q493R. Group E (S309 site)6 and F (CR3022 site)7 NAbs, which often exhibit broad sarbecovirus neutralizing activity, are less affected by Omicron, but still, a subset of NAbs are escaped by G339D, N440K, and S371L. Furthermore, Omicron pseudovirus neutralization showed that single mutation tolerating NAbs could also be escaped due to multiple synergetic mutations on their epitopes. In total, over 85% of the tested NAbs are escaped by Omicron. Regarding NAb drugs, the neutralization potency of LY-CoV016/LY-CoV555, REGN10933/REGN10987, AZD1061/AZD8895, and BRII-196 were greatly reduced by Omicron, while VIR-7831 and DXP-604 still function at reduced efficacy. Together, data suggest Omicron would cause significant humoral immune evasion, while NAbs targeting the sarbecovirus conserved region remain most effective. Our results offer instructions for developing NAb drugs and vaccines against Omicron and future variants.


title: Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies

category: immunology


title: Combinatorial mRNA vaccination enhances protection against SARS-CoV-2 delta variant

abstract: Emergence of SARS-CoV-2 variants of concern (VOC), including the highly transmissible delta strain, has posed challenges to current COVID-19 vaccines that principally target the viral spike protein (S). Here, we report a nucleoside-modified mRNA vaccine that expresses the more conserved viral nucleoprotein (mRNA-N). We show that mRNA-N alone was able to induce a modest but significant control of SARS-CoV-2 in mice and hamsters. Critically, by combining mRNA-N with the clinically approved S-expressing mRNA vaccine (mRNA-S-2P), we found that combinatorial mRNA vaccination (mRNA-S+N) led to markedly enhanced protection against the SARS-CoV-2 delta variant compared to mRNA-S. In a hamster model, we demonstrated that while mRNA-S alone elicited significant control of the delta strain in the lungs ([~]45-fold reduction in viral loads compared to un-vaccinated control), its effectiveness in the upper respiratory tract was weak, whereas combinatorial mRNA-S+N vaccination induced markedly more robust control of the delta variant infection in the lungs ([~]450-fold reduction) as well as in the upper respiratory tract ([~]20-fold reduction). Immune analyses indicated that induction of N-specific immunity as well as augmented S-specific T-cell response and neutralizing antibody activity were collectively associated the enhanced protection against SARS-CoV-2 delta strain by combinatorial mRNA vaccination. These findings suggest that the combined effects of protection in the lungs and upper respiratory tract could both reduce the risk of severe disease as well as of infection and transmission.

category: microbiology


title: Orally administered epeleuton inhibits SARS-CoV-2 viral load, replication and pathology in the Syrian Hamster model

abstract: Early treatment of patients with confirmed COVID-19 presenting mild symptoms can reduce the number that progress to more severe disease and require hospitalization. Considering the potential for the development of drug resistance to existing therapies and the emergence of new SARS-CoV-2 variants, there is a need for an expanded armamentarium of treatment options for COVID-19. Epeleuton is a novel orally administered second-generation n-3 fatty acid with potential direct antiviral and immunomodulatory actions, and a favourable clinical safety profile. In this study we show that epeleuton inhibits SARS-CoV-2 infectious viral load, replication and disease pathology in the lungs and upper airways in the Syrian hamster model of SARS-CoV-2 infection. These data support the potential utility of epeleuton in the early treatment and prevention of SARS-CoV-2 infection. Clinical trials are needed to evaluate the efficacy of epeleuton as an outpatient treatment and prevention of COVID-19.


title: Deep Mutational Engineering of broadly-neutralizing and picomolar affinity nanobodies to accommodate SARS-CoV-1 & 2 antigenic polymorphism

abstract: We report in this study the molecular engineering of nanobodies that bind with picomolar affinity to both SARS-CoV-1 and SARS-CoV-2 Receptor Binding Domains (RBD) and are highly neutralizing. We applied Deep Mutational Engineering to VHH72, a nanobody initially specific for SARS-CoV-1 RBD with little cross-reactivity to SARS-CoV-2 antigen. We first identified all the individual VHH substitutions that increase binding to SARS-CoV-2 RBD and then screened highly focused combinatorial libraries to isolate engineered nanobodies with improved properties. The corresponding VHH-Fc molecules show high affinities for SARS-CoV-2 antigens from various emerging variants and SARS-CoV-1, block the interaction between ACE2 and RBD and neutralize the virus with high efficiency. Its rare specificity across sarbecovirus relies on its peculiar epitope outside the immunodominant regions. The engineered nanobodies share a common motif of three amino acids, which contribute to the broad specificity of recognition. These nanobodies appears as promising therapeutic candidates to fight SARS-CoV-2 infection.

category: biochemistry


title: Mutations in the spike RBD of SARS-CoV-2 omicron variant may increase infectivity without dramatically altering the efficacy of current multi-dosage vaccinations

abstract: With the continuous evolution of SARS-CoV-2, variants of concern (VOCs) and their mutations are a focus of rapid assessment. Vital mutations in the VOC are found in spike protein, particularly in the receptor binding domain (RBD), which directly interacts with ACE2 on the host cell membrane, a key determinant of the binding affinity and cell entry. With the reporting of the most recent VOC, omicron, we performed amino acid sequence alignment of the omicron spike protein with that of the wild type and other VOCs. Although it shares several conserved mutations with other variants, we found that omicron has a large number of unique mutations. We applied the Hopp-Woods scale to calculate the hydrophilicity scores of the amino acid stretches of the RBD and the entire spike protein, and found 3 new hydrophilic regions in the RBD of omicron, implying exposure to water, with the potential to bind proteins such as ACE2 increasing transmissibility and infectivity. However, careful analysis reveals that most of the exposed domains of spike protein can serve as antigenic epitopes for generating B cell and T cell-mediated immune responses. This suggests that in the collection of polyclonal antibodies to various epitopes generated after multiple doses of vaccination, some can likely still bind to the omicron spike protein and the RBD to prevent severe clinical disease. In summary, while the omicron variant might result in more infectivity, it can still bind to a reasonable repertoire of antibodies generated by multiple doses of current vaccines likely preventing severe disease. Effective vaccines may not universally prevent opportunistic infections but can prevent the sequelae of severe disease, as observed for the delta variant. This might still be the case with the omicron variant, albeit, with increased frequency of infection.


title: A Graph Convolutional Network-based screening strategy for rapid identification of SARS-CoV-2 cell-entry inhibitors

abstract:
The cell entry of SARS-CoV-2 has emerged as an attractive drug development target. We previously reported that the entry of SARS-CoV-2 depends on the cell surface heparan sulfate proteoglycan (HSPG) and the cortex actin, which can be targeted by therapeutic agents identified by conventional drug repurposing screens. However, this drug identification strategy requires laborious library screening, which is time-consuming and often limited number of compounds can be screened. As an alternative approach, we developed and trained a graph convolutional network (GCN)-based classification model using information extracted from experimentally identified HSPG and actin inhibitors. This method allowed us to virtually screen 170,000 compounds, resulting in [~]2000 potential hits. A hit confirmation assay with the uptake of a fluorescently labeled HSPG cargo further shortlisted 256 active compounds. Among them, 16 compounds had modest to strong inhibitory activities against the entry of SARS-CoV-2 pseudotyped particles into Vero E6 cells. These results establish a GCN-based virtual screen workflow for rapid identification of new small molecule inhibitors against validated drug targets.

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category: bioinformatics


title: Structural Insights of SARS-CoV-2 Spike Protein from Delta and Omicron Variants

abstract: Given the continuing heavy toll of the COVID-19 pandemic and the emergence of the Delta (B.1.617.2) and Omicron (B.1.1.529) variants, the WHO declared both as variants of concern (VOC). There are valid concerns that the latest Omicron variant might have increased infectivity and pathogenicity. In addition, the sheer number of S protein mutations in the Omicron variant raise concerns of potential immune evasion and resistance to therapeutics such as monoclonal antibodies. However, structural insights that underpin the potential increased pathogenicity are unknown. Here we adopted an artificial intelligence (AI)-based approach to predict the structural changes induced by mutations of the Delta and Omicron variants in the spike (S) protein using Alphafold. This was followed by docking the human angiotensin-converting enzyme 2 (ACE2) with the predicted S proteins for Wuhan-Hu-1, Delta, and Omicron variants. Our in-silico structural analysis indicates that S protein for Omicron variant has a higher binding affinity to ACE-2 receptor, compared to Wuhan-Hu-1 and Delta variants. In addition, the recognition sites of the receptor binding domains for Delta and Omicron variants showed lower electronegativity compared to Wuhan-Hu-1. Importantly, further molecular insights revealed significant changes induced at fusion protein (FP) site, which may mediate enhanced viral entry. These results represent the first computational analysis of structural changes associated with Omicron variant using Alphafold, Collectively, our results highlight potential structural basis for enhanced pathogenicity of the Omicron variant, however further validation using X-ray crystallography and cryo-EM are warranted.

category: biophysics


title: Atypus karschi Dönitz, 1887 (Araneae: Atypidae): an Asian purse-web spider established in Pennsylvania, USA

abstract: The Mygalomorph spiders of the family Atypidae are among the most archaic spiders. The genus Atypus Latreille, 1804 occurs in Eurasia and northern Africa, with a single enigmatic species, Atypus snetsingeri Sarno, 1973, restricted to a small area in southeastern Pennsylvania in Eastern USA. This study was undertaken to learn more about genetics of that species, its habitat requirements and natural history. A close relationship to European species could be assumed based on A. snetsingeris occurrence on the eastern coast of the USA, however molecular markers (CO1 sequences) confirmed that A. snetsingeri is identical with Atypus karschi Donitz, 1887 native to East Asia; it is an introduced species. The specific epithet snetsingeri is therefore relegated to a junior synonym of A. karschi. The karyotype of A. karschi has 42 chromosomes in females and 41 in males (X0 sex chromosome system). Chromosomes were metacentric except for one pair, which exhibited submetacentric morphology. In Pennsylvania the above-ground webs are usually vertical and attached to the base of bushes, trees, or walls, although some webs are oriented horizontally near the ground. It was found in a variety of habitats from forests to suburban shrubbery, and over a wide range of soil humidity and physical parameters. Prey include millipedes, snails, woodlice, carabid beetles and earthworms. The number of juveniles in excavated female webs ranged from 70 to 201. Atypus karschi is the first known case of an introduced purse-web spider. It is rarely noticed but well-established within its range in southeastern Pennsylvania.

category: zoology


title: Delta breakthrough infections elicit potent, broad and durable neutralizing antibody responses

abstract: The SARS-CoV-2 Delta variant is currently responsible for most infections worldwide, including among fully vaccinated individuals. Although these latter infections are associated with milder COVID-19 disease relative to unvaccinated subjects, the specificity and durability of antibody responses elicited by Delta breakthrough cases remain unknown. Here, we demonstrate that breakthrough infections induce serum binding and neutralizing antibody responses that are markedly more potent, durable and resilient to spike mutations observed in variants of concern than those observed in subjects who were infected only or received only two doses of COVID-19 vaccine. However, wee show that Delta breakthrough cases, subjects who were vaccinated after SARS-CoV-2 infection and individuals vaccinated three times (without infection) have serum neutralizing activity of comparable magnitude and breadth indicate that multiple types of exposure or increased number of exposures to SARS-CoV-2 antigen(s) enhance spike-specific antibody responses. Neutralization of the genetically divergent SARS-CoV, however, was moderate with all four cohorts examined, except after four exposures to the SARS-CoV-2 spike, underscoring the importance of developing vaccines eliciting broad sarbecovirus immunity for pandemic preparedness.


title: A novel bacterial protease inhibitor adjuvant in RBD-based COVID-19 vaccine formulations increases neutralizing antibodies, specific germinal center B cells and confers protection against SARS-CoV-2 infection.

abstract: In this work we evaluated recombinant receptor binding domain (RBD) based vaccine formulation prototypes with potential for further clinical development. We assessed different formulations containing RBD plus Alum, AddaS03, AddaVax or the combination of Alum and U-Omp19: a novel Brucella spp. protease inhibitor vaccine adjuvant. Results show that the vaccine formulation composed of U-Omp19 and Alum as adjuvants have a better performance: it significantly increased mucosal and systemic neutralizing antibodies in comparison to antigen plus Alum, AddaVax or AddaS03. Antibodies induced with the formulation containing U-Omp19 not only increased their neutralization capacity against the wild-type virus but also cross neutralized alpha, lambda and gamma variants with similar potency. Also, addition of U-Omp19 to vaccine formulation increased the frequency of RBD-specific geminal center B cells and plasmablasts. Additionally, U-Omp19+Alum formulation induced RBD-specific Th1 and CD8+ T cell responses in spleens and lungs. Finally, this vaccine formulation conferred protection against an intranasal SARS-CoV-2 challenge of K18-hACE2 mice.


title: A co-opted endogenous retroviral envelope promotes cell survival by controlling SLC31A1/CTR1-mediated copper transport and homeostasis

abstract: Copper is a critical element for eukaryotic life, involved in numerous cellular functions and in redox balance but it can be toxic in excess. Therefore, tight regulation of copper acquisition and homeostasis is essential for cell physiology and survival. Here, we identified a unique mechanism for cell survival involving the regulation of copper homeostasis by an endogenous retroviral (ERV) envelope glycoprotein called Refrex1. We show that extracellular copper sensing by cells increases Refrex1 expression, which in turn regulates copper acquisition through interaction with the main copper transporter SLC31A1/CTR1. Downmodulation of Refrex1 resulted in intracellular copper accumulation leading to ROS production and subsequent apoptosis, which could be reverted by copper chelator treatment. Our results demonstrate that Refrex1 has been co-opted for its ability to regulate copper entry through CTR1 interaction in order to limit copper excess for a proper redox balance, and suggests that other ERV may have similar metabolic functions among vertebrates.


title: Preliminary Characterization of Phage-like Particles from the Male-Killing Mollicute Spiroplasma poulsonii (an Endosymbiont of Drosophila)

abstract: Viruses are vastly abundant and influential in all ecosystems, and are generally regarded as pathogens. Viruses of prokaryotes (themselves highly diverse and abundant) are known as bacteriophages or phages. Phages engage in diverse associations with their hosts, and contribute to regulation of biogeochemical processes, horizontal movement of genes, and control of bacterial populations. Recent studies have revealed the influential role of phage in the association of arthropods and their heritable endosymbiotic bacteria (e.g. the Proteobacteria genera Wolbachia and Hamiltonella). Despite prior studies ([~]30 years ago) documenting presence of phage in the mollicute Spiroplasma infecting Drosophila, genomic sequences of such phage are lacking, and their effects on the Spiroplasma-Drosophila interaction have not been comprehensively characterized. The present work isolated phage-like particles from the male-killing Spiroplasma poulsonii (strains NSRO and MSRO-Br) harbored by Drosophila melanogaster. Isolated particles were subjected to DNA sequencing, assembly, and annotation. Our results recovered three [~]19 kb phage-like contigs (two in NSRO and one in MSRO-Br), and two smaller non-phage-like contigs encoding a known Spiroplasma toxin and an insertion element. Whole or parts of the particle-derived contigs were found in the genome assemblies of members of the Spiroplasma poulsonii clade. Although our results do not allow us to distinguish whether the contigs obtained represent infective phage-like particles capable of transmitting their DNA to new hosts, their encoding of several typical phage genes suggests that they are at least remnants of functional phage. We discuss potential implications of our findings and suggest future directions.


title: Nitrogen metabolism in Pseudomonas putida: functional analysis using random barcode transposon sequencing

abstract:
Pseudomonas putida KT2440 has long been studied for its diverse and robust metabolisms, yet many genes and proteins imparting these growth capacities remain uncharacterized. Using pooled mutant fitness assays, we identified genes and proteins involved in the assimilation of 52 different nitrogen containing compounds. To assay amino acid biosynthesis, 19 amino acid drop- out conditions were also tested. From these 71 conditions, significant fitness phenotypes were elicited in 672 different genes including 100 transcriptional regulators and 112 transport-related proteins. We divide these conditions into 6 classes, and propose assimilatory pathways for the compounds based on this wealth of genetic data. To complement these data, we characterize the substrate range of three promiscuous aminotransferases relevant to metabolic engineering efforts in vitro. Furthermore, we examine the specificity of five transcriptional regulators, explaining some fitness data results and exploring their potential to be developed into useful synthetic biology tools. In addition, we use manifold learning to create an interactive visualization tool for interpreting our BarSeq data, which will improve the accessibility and utility of this work to other researchers.

IMPORTANCEUnderstanding the genetic basis of P. putidas diverse metabolism is imperative for us to reach its full potential as a host for metabolic engineering. Many target molecules of the bioeconomy and their precursors contain nitrogen. This study provides functional evidence linking hundreds of genes to their roles in the metabolism of nitrogenous compounds, and provides an interactive tool for visualizing these data. We further characterize several aminotransferases, lactamases, and regulators--which are of particular interest for metabolic engineering.


title: Biogenesis of a bacterial metabolosome for propanediol utilization

abstract: Bacterial metabolosomes are a family of protein organelles in bacteria. Elucidating how thousands of proteins self-assemble to form functional metabolosomes is essential for understanding their significance in cellular metabolism and pathogenesis. Here we investigate the de novo biogenesis of propanediol-utilization (Pdu) metabolosomes and characterize the roles of the key constituents in generation and intracellular positioning of functional metabolosomes. Our results demonstrate that the Pdu metabolosome undertakes both "Shell first" and "Cargo first" assembly pathways, unlike the {beta}-carboxysome structural analog which only involves the "Cargo first" strategy. Shell and cargo assemblies occur independently at the cell poles. The internal cargo core is formed through the ordered assembly of multiple enzyme complexes, and exhibits liquid-like properties within the metabolosome architecture. Our findings provide mechanistic insight into the molecular principles driving bacterial metabolosome assembly and expand our understanding of liquid-like organelle biogenesis.


title: Nanotrap Particles Improve Nanopore Sequencing of SARS-CoV-2 and Other Respiratory Viruses

abstract: Presented here is a magnetic hydrogel particle enabled workflow for capturing and concentrating SARS-CoV-2 from diagnostic remnant swab samples that significantly improves sequencing results using the Oxford Nanopore Technologies MinION sequencing platform. Our approach utilizes a novel affinity-based magnetic hydrogel particle, circumventing low input sample volumes and allowing for both rapid manual and automated high throughput workflows that are compatible with nanopore sequencing. This approach enhances standard RNA extraction protocols, providing up to 40x improvements in viral mapped reads, and improves sequencing coverage by 20-80% from lower titer diagnostic remnant samples. Furthermore, we demonstrate that this approach works for contrived influenza virus and respiratory syncytial virus samples, suggesting that it can be used to identify and improve sequencing results of multiple viruses in VTM samples. These methods can be performed manually or on a KingFisher Apex system.


title: Rec8 cohesin-mediated loop-axis chromatin architecture is required for meiotic recombination

abstract: During meiotic prophase, cohesin-dependent axial structures are formed in the synaptonemal complex (SC). However, the functional correlation between these structures and cohesion remains elusive. Here, we examined the formation of cohesin-dependent axial structures in the fission yeast Schizosaccharomyces pombe. This organism forms atypical SCs composed of linear elements (LinEs) resembling the lateral elements of SC but lacking the transverse filaments. Hi-C analysis using a highly synchronous population of meiotic S. pombe cells revealed that the axis-loop chromatin structure formed in meiotic prophase was dependent on the Rec8 cohesin complex. In contrast, the Rec8-mediated formation of the axis-loop structure occurred in cells lacking components of LinEs. To dissect the functions of Rec8, we identified a rec8-F204S mutant that lost the ability to assemble the axis-loop structure without losing cohesion of sister chromatids. This mutant showed defects in the formation of the axis-loop structure and LinE assembly and thus exhibited reduced meiotic recombination. Collectively, our results demonstrate that the Rec8-dependent axis-loop structure provides a structural platform essential for LinE assembly, facilitating meiotic recombination of homologous chromosomes, independently of its role in sister chromatid cohesion.

category: cell biology


title: Rec8 cohesin-mediated axis-loop chromatin architecture is required for meiotic recombination


title: Acute oral toxicity of zinc phosphide: an assessment for wild house mice in Australia

abstract:
BACKGROUNDThe efficacy of zinc phosphide (ZnP) for broadacre control of wild house mice in Australia is being reported by growers as increasingly variable. Have mice become less sensitive over time or are they taking a sub-lethal dose and developing aversion? In this laboratory study the sensitivity of groups of wild caught and an outbred laboratory strain of mice was assessed using oral gavage of a range of ZnP concentrations. The willingness of mice to consume ZnP-coated grains was then determined.

RESULTSEach mouse group had very similar LD50 values (72 to 79 mg ZnP per kg body weight) which are significantly higher than previously reported. Time to death post-gavage ranged between 2.5 to 48 h. ZnP coated grains (50 g ZnP per kg grain) presented in the absence of alternative food were consumed and 94 percent of wild mice died. Mice provided with alternative food and ZnP coated wheat grains (either 25 or 50 g ZnP per kg grain) consumed toxic and non-toxic grains, and mortality was lower (33 to 55 percent). If a sublethal amount of ZnP coated grain was consumed, aversion occurred mostly in the presence of alternative food.

CONCLUSIONSThe sensitivity of wild house mice to ZnP in Australia is significantly lower than previously assumed. Under laboratory conditions ZnP coated grains coated with a new higher dose (50 g ZnP per kg grain) were readily consumed. Consumption of toxic grain occurred when alternative food was available but was decreased. It is important to assess the efficacy of the higher ZnP dose per grain under natural field conditions, especially when background food is low.

category: ecology


title: Linked-read whole-genome sequencing resolves common and private structural variants in multiple myeloma

abstract:
Multiple myeloma (MM) is an incurable and aggressive plasma cell malignancy characterized by a complex karyotype with multiple structural variants (SVs) and copy number variations (CNVs). Linked-read whole-genome sequencing (lrWGS) allows for refined detection and reconstruction of SVs by providing long-range genetic information from standard short-read sequencing. This makes lrWGS an attractive solution for capturing the full genomic complexity of MM. Here we show that high-quality lrWGS data can be generated from low numbers of FACS sorted cells without DNA purification. Using this protocol, we analyzed FACS sorted MM cells from 37 MM patients with lrWGS. We found high concordance between lrWGS and FISH for the detection of recurrent translocations and CNVs. Outside of the regions investigated by FISH, we identified >150 additional SVs and CNVs across the cohort. Analysis of the lrWGS data allowed for resolving the structure of diverse SVs affecting the MYC and t(11;14) loci causing the duplication of genes and gene regulatory elements. In addition, we identified private SVs causing the dysregulation of genes recurrently involved in translocations with the IGH locus and show that these can alter the molecular classification of the MM. Overall, we conclude that lrWGS allows for the detection of aberrations critical for MM prognostics and provides a feasible route for providing comprehensive genetics. Implementing lrWGS could provide more accurate clinical prognostics, facilitate genomic medicine initiatives, and greatly improve the stratification of patients included in clinical trials.

KEY POINTS- Linked-read WGS can be performed without DNA purification and allows for resolving the diverse structural variants found in multiple myeloma.
- Linked-read WGS can, as a stand-alone assay, provide comprehensive genetics in myeloma and other diseases with complex genomes.

category: genetics


title: Fast in-vitro screening of FLT3-ITD inhibitors using silkworm-baculovirus protein expression system

abstract: We report expression and purification of a FLT3 protein with ITD mutation (FLT3-ITD) with a steady tyrosine kinase activity using a silkworm-baculovirus system, and its application as a fast screening system of tyrosine kinase inhibitors. The FLT3-ITD protein was expressed in Bombyx mori L. pupae infected by gene-modified nucleopolyhedrovirus, and was purified as an active state. We performed an inhibition assay using 17 potential kinase inhibitors, and succeeded in identifying two potent inhibitors for FLT3-ITD. The result has paved the way for screening FLT3-ITD inhibitors in a fast and easy manner, and also for structural studies.


title: Whole genome resequencing and comparative genome analysis of three Puccinia striiformis f. sp. tritici pathotypes prevalent in India

abstract: Stripe rust disease of wheat, caused by Puccinia striiformis f. sp. tritici, (Pst) is one of the most serious diseases of wheat worldwide. In India, virulent stripe rust races have been constantly evolving in the North-Western Plains Zone leading to the failure of some of the most widely grown resistant varieties in the region. With the goal of studying the recent evolution of virulent races in this region, we conducted whole-genome sequencing of three prevalent Indian Pst pathotypes Pst46S119, Pst78S84 and Pst110S119. We assembled 58.62, 58.33 and 55.78 Mb of Pst110S119, Pst46S119 and Pst78S84 genome, respectively. Pathotypes were found to be highly heterozygous. Comparative phylogenetic analysis indicated the recent evolution of pathotypes Pst110S119 and Pst78S84 from Pst46S119. Pathogenicity-related genes classes (CAZyme, proteases, effectors, and secretome proteins) were identified and found to be under positive selection. Higher rate of gene family expansion was also observed in the three pathotypes. A strong association between the effector genes and transposable elements may be the source of the rapid evolution of these strains. Phylogenetic analysis differentiated the Indian races in this study from other known US, European, African and Asian races. Diagnostic markers developed for the identification of different Pst pathotypes will help tracking of yellow rust at farmers field and strategizing resistance gene deployment.


title: SARS-CoV-2 Omicron Spike Glycoprotein Receptor Binding Domain Exhibits Super-Binder Ability with ACE2 but not Convalescent Monoclonal Antibody

abstract:
BackgroundSARS-CoV-2, the causative virus for COVID-19 has now super-mutated into the Omicron (Om) variant. On its spike glycoprotein alone, more than 30 substitutions have been characterized with 15 within the receptor binding domain (RBD); It therefore calls to question the transmissibility and antibody escapability of Omicron. This study was setup to investigate the Omicron RBDs interaction with ACE2 (host receptor) and a SARS-CoV-2 neutralizing monoclonal antibody (mAb).

MethodsIn-silico mutagenesis was used to generate the Om-RBD in complex with ACE2 or mAb from the wildtype. All-atom molecular dynamics (MD) simulation trajectories were analyzed for interaction.

ResultsMD trajectories showed that Omicron RBD has evolved into an efficient ACE2 binder, via pi-pi (Om-RBD-Y501/ACE2-Y41) and salt-bridge (Om-RBD-K493/ACE2-Y41) interactions. Conversely, in binding mAb, it has become less efficient (Center of mass distance of RBD from mAb complex, wildtype {approx} 30 [A], Omicron {approx} 41 [A]). Disruption of Om-RBD/mAb complex resulted from loose interaction between Om-RBD and the light chain complementarity-determining region residues.

ConclusionsOmicron is expected to be better transmissible and less efficiently interacting with neutralizing convalescent mAbs.

General significanceOur results elucidate the mechanisms for higher transmissibility in Omicron variant.


title: The Landscape-Based Protein Stability Analysis and Network Modeling of Multiple Conformational States of the SARS-CoV-2 Spike D614 Mutant: Conformational Plasticity and Frustration-Driven Allostery as Energetic Drivers of Highly Transmissible Spike Variant

abstract: The structural and functional studies of the SARS-CoV-2 spike protein variants revealed an important role of the D614G mutation that is shared across many variants of concern(VOCs), suggesting the effect of this mutation on the enhanced virus infectivity and transmissibility. The recent structural and biophysical studies provided important evidence about multiple conformational substates of the D614G spike protein. The development of a plausible mechanistic model which can explain the experimental observations from a more unified thermodynamic perspective is an important objective of the current work. In this study, we employed efficient and accurate coarse-grained simulations of multiple structural substates of the D614G spike trimers together with the ensemble-based mutational frustration analysis to characterize the dynamics signatures of the conformational landscapes. By combining the local frustration profiling of the conformational states with residue-based mutational scanning of protein stability and network analysis of allosteric interactions and communications, we determine the patterns of mutational sensitivity in the functional regions and sites of variants. We found that the D614G mutation may induce a considerable conformational adaptability of the open states in the SARS-CoV-2 spike protein without compromising folding stability and integrity of the spike protein. The results suggest that the D614G mutant may employ a hinge-shift mechanism in which the dynamic couplings between the site of mutation and the inter-protomer hinge modulate the inter-domain interactions, global mobility change and the increased stability of the open form. This study proposes that mutation-induced modulation of the conformational flexibility and energetic frustration at the inter-protomer interfaces may serve as an efficient mechanism for allosteric regulation of the SARS-CoV-2 spike proteins.


title: eIF5B and eIF1A remodel human translation initiation complexes to mediate ribosomal subunit joining

abstract: Joining of the ribosomal subunits at a translation start site on a messenger RNA during initiation commits the ribosome to synthesize a protein. Here, we combined single-molecule spectroscopy and structural methods using an in vitro reconstituted system to examine how the human ribosomal subunits join. Single-molecule fluorescence revealed when universally-conserved eukaryotic initiation factors (eIFs) eIF1A and eIF5B associate with and depart from initiation complexes. Guided by single-molecule dynamics, we examined initiation complexes that contained both eIF1A and eIF5B using single-particle electron cryo-microscopy. The resulting structure illuminated how eukaryote-specific contacts between eIF1A and eIF5B remodel the initiation complex to orient initiator tRNA in a conformation compatible with ribosomal subunit joining. Collectively, our findings provide a quantitative and architectural framework for the molecular choreography orchestrated by eIF1A and eIF5B during human translation initiation.


title: Nuclear NAD+ homeostasis is essential for naive and chemoresistant BRCA1/2-deficient tumor survival

abstract:
Resistance to PARP inhibitors (PARPi) is emerging as the major obstacle to their effectiveness for the treatment of BRCA1/2-mutated, also referred as homologous recombination (HR)-deficient, tumors (HRD). Over the years, mechanistic studies gained insights on effectors acting downstream of PARP1, lagging behind the understanding of earlier events upstream - and thus independent - of PARP1. Here, we investigated the role of nuclear NAD+, an essential cofactor for the activity of key DNA repair proteins, including PARP1 and sirtuins. We show that NMNAT1-the enzyme synthesizing nuclear NAD+ - is synthetically lethal with BRCA1/2 in a PARP1-independent but SIRT6-dependent manner. Consequently, inhibition of NMNAT1/SIRT6 axis not only kills naive but also PARPi-resistant HRD cancer cells. Our results unravel a unique vulnerability of HRD tumors, therapeutically exploitable even upon PARPi resistance development.

One-Sentence SummaryTargeting NMNAT1 kills chemoresistant and naive BRCA1/2-deficient tumors by disrupting SIRT6-dependent base excision repair.

category: cancer biology


title: Programmable in vivo CRISPR activation   elucidates the oncogenic and immunosuppressive role of MYC in lung adenocarcinoma

abstract: Conventional genetically engineered mouse models (GEMMs) are time consuming, laborious and offer limited spatio-temporal control. Here, we describe the development of a streamlined platform for in vivo gene activation using CRISPR activation (CRISPRa) technology. Unlike conventional GEMMs, our model system allows for flexible, sustained and timed activation of one or more target genes using single or pooled lentiviral guides. Using Myc and Yap1 as model oncogenes, we demonstrate gene activation in primary pancreatic organoid cultures in vitro and enhanced tumorigenic potential in Myc-activated organoids when transplanted orthotopically. By implementing our model as an autochthonous lung cancer model, we show that transduction-mediated Myc activation leads to accelerated tumor progression and significantly reduced overall survival relative to non-targeted tumor controls. Furthermore, we found that Myc-activation led to the acquisition of an immune suppressive "cold" tumor microenvironment. Through cross-species validation of our results using publicly available RNA/DNA-seq data sets, we were able to link MYC to a previously described, immunosuppressive transcriptomic subtype in patient tumors, thus identifying a patient cohort that may benefit from combined MYC/immune-targeted therapies. Overall, our work demonstrates how CRISPRa can be used for rapid functional validation of putative oncogenes and may allow for the identification and evaluation of potential metastatic and oncogenic drivers through competitive screening.


title: Effect of growth forms of bamboo on the mycorrhizal and fluorescent pseudomonas population

abstract: Bamboo, also called as the poor mans timber, is one of the fastest growing giant grass species. Having shallow root system and fast growth rate, the dependability of the plant on the rhizospheric microbial web cannot be denied. The study was conducted to explore the population and seasonal variations of indigenous mycorrhizal fungi as well as the functional diversity of plant growth promoting bacteria, especially fluorescent pseudomonas from the different growth forms of Dendrocalamus strictus, the most commonly present bamboo species in Indian sub-continent. In a past study, it was established that the growth forms of D. strictus which developed over time in their respective locations, were genetically varied. The present research further explores the variations in their respective rhizospheric microbes and looks for the role of plant selection phenomenon. Considerable variation in mycorrhizal structures and in the functional diversity of fluorescent pseudomonas was registered. It seems probable that selection pressure of nutrient deficient condition has created a condition that promoted occurrence of high numbers of P solubilisers which, in turn, boosted the mycorrhizal as well as bamboo growth.

category: plant biology


title: The Role of Mentoring in Promoting Diversity, Equity, and Inclusion in STEM Education and Research

abstract: Mentoring success is derived from active and respectful listening and the willingness to learn and accept opportunities for personal growth. Mentoring shapes every trainee and their career path in science, technology, engineering, and mathematics (STEM). Productive mentoring relationships cultivate rapport, stimulate moments of introspection, and provide constructive feedback. Effective mentoring in STEM allows trainees, especially underrepresented minorities (URMs), to flourish in welcoming and supportive environments. However, URM trainees often experience inadequate mentoring due to their mentors inexperience with URM groups, poor mentorship training, or a lack of understanding of their mentees journey. To promote diversity, equity, and inclusion in STEM education and research, it is essential for mentors and mentees to work together with creativity, authenticity, and networking. In this workshop, we will focus on mentees perspective on how mentors can enhance their training, professional and career development, and improve their focus. We analyzed data on feedback obtained from students interested in pursuing graduate education who attended a recent workshop. Our results show that despite low initial expectations for the workshop, many students were satisfied with the knowledge they learned. The future of increasing the URM representation in STEM lies in providing adequate community support and mentorship throughout the careers of URM professionals.

category: scientific communication and education


title: Strain variation in Clostridioides difficile toxin activity associated with genomic variation at both PaLoc and non-PaLoc loci

abstract:
Clinical disease from Clostridioides difficile infection can be mediated by two toxins and their neighboring regulatory genes encoded within the five-gene pathogenicity locus (PaLoc). We provide several lines of evidence that the toxin activity of C. difficile may be modulated by genomic variants outside of the PaLoc. We used a phylogenetic tree-based approach to demonstrate discordance between toxin activity and PaLoc evolutionary history, an elastic net method to show the insufficiency of PaLoc variants alone to model toxin activity, and a convergence-based bacterial genome-wide association study (GWAS) to identify correlations between non-PaLoc loci with changes in toxin activity. Combined, these data support a model of C. difficile disease wherein toxin activity may be strongly affected by many non-PaLoc loci. Additionally, we characterize multiple other in vitro phenotypes relevant to human infections including germination and sporulation. These phenotypes vary greatly in their clonality, variability, convergence, and concordance with genomic variation. Lastly, we highlight the intersection of loci identified by GWAS for different phenotypes and clinical severity. This strategy to identify the overlapping loci can facilitate the identification of genetic variation linking phenotypic variation to clinical outcomes.

IMPORTANCEClostridioides difficile has two major disease mediating toxins, A and B, encoded within the pathogenicity locus (PaLoc). In this study we demonstrate via multiple approaches that genomic variants outside of the PaLoc are associated with changes in toxin activity. These genomic variants may provide new avenues of exploration in the hunt for novel disease modifying interventions. Additionally, we provide insight into the evolution of several additional phenotypes also critical to clinical infection such as sporulation, germination, and growth rate. These in vitro phenotypes display a range of responses to evolutionary pressures and as such vary in their appropriateness for certain bacterial genome wide association study approaches. We used a convergence-based association method to identify the genomic variants most correlated with both changes in these phenotypes and disease severity. These overlapping loci may be important to both bacterial function and human clinical disease.


title: Variable oxygen environments and DNMT2 determine the DNA cytosine epigenetic landscape of Plasmodium falciparum

abstract: DNA cytosine methylation and its oxidized products are important epigenetic modifications in mammalian cells. Although 5-methylcytosine (5mC) was detected in the human malaria parasite Plasmodium falciparum, the presence of oxidized 5mC forms remain to be characterized. Here we establish a protocol to optimize nuclease-based DNA digestion for the extremely AT-rich genome of P. falciparum (>80% A+T) for quantitative LC-MS/MS analysis of 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). We demonstrate the presence of 5hmC, 5fC and 5caC cytosine modifications in a DNMT2-only organism and observe striking ratio changes between 5mC and 5hmC during the 48-hour blood stage parasite development. Parasite-infected red blood cells cultured in different physiological oxygen concentrations revealed a shift in the cytosine modifications distribution towards the oxidized 5hmC and 5caC forms. In the absence of the canonical C5-DNA methyltransferase (DNMT1 and DNMT3A/B) in P. falciparum, we show that all cytosine modifications depend on the presence of DNMT2. We conclude that DNMT2 and oxygen levels are critical determinants that shape the dynamic cytosine epigenetic landscape in this human pathogen.

category: molecular biology


title: Microbiome Analysis of Gut Bacterial Communities of Healthy and Diseased Malaysian Mahseer (Tor tambroides)

abstract:
AimsThe gut microbiota is referred to an  extra organ and is ciritical in assisting the host in terms of nutrition and immunity. Environmental stressors could alter gut microbial community and cause gut inflammation. This study aimed to investigate and compare the gut microbiota community between healthy and diseased Tor tambroides.

Methodology and resultsIn this study, such gut microbial alterations were explored using NGS-based 16S rDNA sequencing on the Malaysian mahseer (T. tambroides). Three adult healthy and three diseased adult Malaysian mahseers (showing signs of exophthalmia, coelomic distension and petechial haemorrhage) were obtained from LTT Aquaculture Sdn Bhd. Our results revealed significant differences in microbial diversity, composition and function between both populations of T. tambroides. Alpha diversity analysis depicts lower diversity of gut microbiota composition in diseased T. tambroides as compared to the healthy group. In particular, Enterobacteriaceae, Aeromonas, Bacteroides, Vibrio and Pseudomonas were found within gut microbiota of the diseased fishes. In addition, cellulose-degrading bacteria and protease-producing bacteria were identified from the gut of T. tambroides.

Conclusion significance and impact of studyThus, our findings emphasised on the association between the alteration in gut microbiota composition and infectious abdominal dropsy (IAD) in T. tambroides. This finding is important to provide basic information for further diagnosis, prevention and treatment of intestinal diseases in fish.


title: A small molecule reveals role of insulin receptor-insulin like growth factor-1 receptor heterodimers

abstract: The insulin receptor and insulin like growth factor-1 receptor are heterodimers consisting of two extracellular -subunits and two transmembrane {beta}-subunits. IR {beta} and IGF1R {beta} hemi-receptors can heterodimerize to form hybrids composed of one IR {beta} and one IGF1R {beta}. Widely distributed in mammalian tissues, in contrast to IR and IGF1R the physiological function of hybrids is unclear. To identify tool compounds that inhibit hybrid formation we performed a high-throughput small molecule screen based on a homology model of hybrid structure. Our studies unveil a first in class quinoline-containing heterocyclic small molecule that reduces hybrids by >50% in human umbilical vein endothelial cells with no effect on IR or IGF1R expression. Downstream of IR and IGF1R our small molecule led to reduced expression of the negative regulatory p85 subunit of phosphatidylinositol 3-kinase, an increase in phosphorylation of its downstream target Akt and enhanced insulin and shear-induced phosphorylation of Akt. We show that hybrids have a role in human endothelial cell physiology distinct from IR and IGF1R.


title: Toxoplasma ERK7 defends the apical complex from premature degradation

abstract: Accurate cellular replication balances the biogenesis and turnover of complex structures. Apicomplexan parasites such as Plasmodium and Toxoplasma replicate by forming daughter cells within an intact mother cell, creating additional challenges to ensuring fidelity of division. Critical to these parasites infectivity is an intricate cytoskeleton structure called the apical complex. Before the daughter apical complex can be inserted into the plasma membrane, the maternal material must be turned over. We previously identified the kinase ERK7 as required for the maturation of the apical complex in Toxoplasma gondii. Here we define the Toxoplasma ERK7 interactome, and identify a putative E3 ligase, CSAR1, as the downstream effector responsible for the phenotype. Genetic disruption of CSAR1 fully suppresses loss of the apical complex upon ERK7 knockdown. Furthermore, we show that CSAR1 is normally responsible for turnover of maternal cytoskeleton during cytokinesis, and that its aberrant function is driven by a mislocalization from the parasite residual body to the maternal and daughter apical complexes. These data identify a protein homeostasis pathway critical for Toxoplasma replication and fitness and suggest an unappreciated role for the parasite residual body in compartmentalizing processes that threaten the fidelity of parasite development.


title: Hierarchical length and sequence preferences establish a single major piRNA 3'-end

abstract:
PIWI-interacting RNAs (piRNAs) guard germline genomes against the deleterious action of retroviruses and other mobile genetic elements. How piRNAs faithfully discriminate between self and non-self to restrict all mobile elements while sparing essential genes remains a key outstanding question in genome biology. PiRNAs use extensive base-pairing to recognize their targets and variable 3ends could change the specificity and efficacy of piRNA silencing. Here, we identify conserved rules that ensure the generation of a single major piRNA 3end in flies and mice. Our data suggest that the PIWI proteins initially define a short interval on pre-piRNAs that grants access to the ZUC-processor complex. Within this Goldilocks zone, the preference of the ZUC-processor to cut in front of a Uridine determines the ultimate processing site. We observe a mouse-specific roadblock that relocates the Goldilocks zone and generates an opportunity for consecutive trimming by PNLDC1. Our data reveal a conserved hierarchy between length and sequence preferences that controls the piRNA sequence space. The unanticipated precision of 3end formation bolsters the emerging understanding that the functional piRNA sequence space is tightly controlled to ensure effective defense.

Key findingsO_LIEvery piRNA has a single major 3end
C_LIO_LILength preference trumps sequence preference during 3end formation.
C_LIO_LIRelocation of the Goldilocks zone generates opportunity for trimming of mouse piRNAs.
C_LI

category: developmental biology


title: Transcriptional response in a sepsis mouse model reflects transcriptional response in sepsis patients

abstract: Mortality due to sepsis remains unacceptably high, especially for septic shock patients. Murine models have been used to better understand pathophysiology mechanisms. However, the mouse model is still under debate. Here we investigated the transcriptional response of mice injected with lipopolysaccharide (LPS) and compared it to either human cells stimulated in vitro with LPS or to blood cells of septic patients. We identified a molecular signature composed of 2331 genes with an FDR median of 0%. This molecular signature is highly enriched in regulated genes in peritoneal macrophages stimulated with LPS. There is a significant enrichment in several inflammatory signaling pathways, and in disease terms, such as pneumonia, sepsis, systemic inflammatory response syndrome, severe sepsis, an inflammatory disorder, immune suppression, and septic shock. A significant overlap between the genes up-regulated in mouse and human cells stimulated with LPS has been demonstrated. Finally, genes up-regulated in mouse cells stimulated with LPS are enriched in genes up-regulated in human cells stimulated in vitro and in septic patients, who are at high risk of death. Our results support the hypothesis of common molecular and cellular mechanisms between mouse and human sepsis.

category: genomics


title: Ongoing transposition in cell culture reveals the phylogeny of diverse Drosophila S2 sub-lines.

abstract: Cultured cells are widely used in molecular biology despite poor understanding of how cell line genomes change in vitro over time. Previous work has shown that Drosophila cultured cells have a higher transposable element (TE) content than whole flies, but whether this increase in TE content resulted from an initial burst of transposition during cell line establishment or ongoing transposition in cell culture remains unclear. Here we sequence the genomes of 25 sub-lines of Drosophila S2 cells and show that TE insertions provide abundant markers for the phylogenetic reconstruction of diverse sub-lines in a model animal cell culture system. Analysis of DNA copy number evolution across S2 sub-lines revealed dramatically different patterns of genome organization that support the overall evolutionary history reconstructed using TE insertions. Analysis of TE insertion site occupancy and ancestral states support a model of ongoing transposition dominated by episodic activity of a small number of retrotransposon families. Our work demonstrates that substantial genome evolution occurs during long-term Drosophila cell culture, which may impact the reproducibility of experiments that do not control for sub-line identity.


title: Long read sequencing reveals sequential complex rearrangements driven by Hepatitis B virus integration

abstract: Integration of Hepatitis B virus (HBV) into human genome disrupts genetic structures and cellular functions. Here, we conducted multiplatform long read sequencing on two cell lines and five clinical samples of HBV-infected hepatocellular carcinomas (HCC). We resolved three types of viral integration-induced complex genome rearrangements (CGR) and proposed a model of  multi-hits and sequential-breaks to depict their formation process by differentiating inserted HBV copies with HiFi long reads. We deduced that all three complex types were initialized from focal replacement and fragile virus-human junctions triggered subsequent rearrangements. We further revealed that such rearrangements caused a prevalent loss-of-heterozygosity at chr4q, accounting for 19.5% of HCC samples in ICGC cohort and contributing to immune and metabolic dysfunction. Overall, our long read based analysis reveals novel sequential rearrangement processes initiated by HBV integration, hinting its structural and functional impact on HCC.


title: LitRev: A data driven method for quick literature review from PubMed

abstract:
SummaryLitRev is a novel robust data driven approach, developed for quick literature review on a particular topic of interest. This method identifies common biological phrases that follow a power law distribution and important phrases which have the normalized point wise mutual information score greater than zero.

Contactgourabdas0727@gmail.com


title: Protein Organization with Manifold Exploration and Spectral Clustering

abstract: We present a method to provide a biologically meaningful representation of the space of protein sequences. While billions of protein sequences are available, organizing this vast amount of information into functional categories is daunting, time-consuming and incomplete. We present our unsupervised approach that combines Transformer protein language models, UMAP graphs, and spectral clustering to create meaningful clusters in the protein spaces. To demonstrate the meaningfulness of the clusters, we show that they preserve most of the signal present in a dataset of manually curated enzyme protein families.


title: CXCL8/CXCR2 signaling mediates bone marrow fibrosis and represents a therapeutic target in myelofibrosis

abstract: Pro-inflammatory signaling is a hallmark feature of human cancer, including in myeloproliferative neoplasms (MPNs), most notably myelofibrosis (MF). Dysregulated inflammatory signaling contributes to fibrotic progression in MF; however, the individual cytokine mediators elicited by malignant MPN cells to promote collagen-producing fibrosis and disease evolution remain yet to be fully elucidated. Previously we identified a critical role for combined constitutive JAK/STAT and aberrant NF-{kappa}B pro-inflammatory signaling in myelofibrosis development. Using single-cell transcriptional and cytokine-secretion studies of primary MF patient cells and two separate murine models of myelofibrosis, we extend this previous work and delineate the role of CXCL8/CXCR2 signaling in MF pathogenesis and bone marrow fibrosis progression. MF patient hematopoietic stem/progenitor cells are enriched in a CXCL8/CXCR2 gene signature and display dose-dependent proliferation and fitness in response to exogenous CXCL8 ligand in vitro. Genetic deletion of Cxcr2 in the hMPLW515L adoptive transfer model abrogates fibrosis and extends overall survival, and pharmacologic inhibition of the CXCR1/2 pathway improves hematologic parameters, attenuates bone marrow fibrosis, and synergizes with JAK inhibitor therapy. Our mechanistic insights provide a rationale for therapeutic targeting of the CXCL8/CXCR2 pathway in MF patients at risk for continued fibrotic progression.


title: Quantitative Action Spectroscopy Reveals ARPE-19 Sensitivity to Long-Wave Ultraviolet Radiation at 350 nm and 380 nm

abstract:
The role of ultraviolet radiation (UVR) exposure in the pathology of age-related macular degeneration (AMD) has been debated for decades with epidemiological evidence failing to find a clear consensus for or against it playing a role. A key reason for this is a lack of foundational research into the response of living retinal tissue to UVR in regard to AMD-specific parameters of tissue function. We therefore explored the response of cultured retinal pigmented epithelium (RPE), the loss of which heralds advanced AMD, to specific wavelengths of UVR across the UV-B and UV-A bands found in natural sunlight.

Using a bespoke in vitro UVR exposure apparatus coupled with bandpass filters we exposed the immortalised RPE cell line, ARPE-19, to 10nm bands of UVR between 290 and 405nm. Physical cell dynamics were assessed during exposure in cells cultured upon specialist electrode culture plates which allow for continuous, non-invasive electrostatic interrogation of key cell parameters during exposure such as monolayer coverage and tight-junction integrity. UVR exposures were also utilised to quantify wavelength-specific effects using a rapid cell viability assay and a phenotypic profiling assay which was leveraged to simultaneously quantify intracellular reactive oxygen species (ROS), nuclear morphology, mitochondrial stress, epithelial integrity and cell viability as part of a phenotypic profiling approach to quantifying the effects of UVR.

Electrical impedance assessment revealed unforeseen detrimental effects of UV-A, beginning at 350nm, alongside previously demonstrated UV-B impacts. Cell viability analysis also highlighted increased effects at 350nm as well as 380nm. Effects at 350nm were further substantiated by high content image analysis which highlighted increased mitochondrial dysfunction and oxidative stress.

We conclude that ARPE-19 cells exhibit a previously uncharacterised sensitivity to UV-A radiation, specifically at 350nm and somewhat less at 380nm. If upheld in vivo, such sensitivity will have impacts upon geoepidemiological risk scoring of AMD.

category: pharmacology and toxicology


title: Geographic variation in larval cold tolerance and exposure across the invasion front of a widely established forest insect

abstract:
As the global climate changes, high and low temperature extremes can drive changes in species distributions. Across the range of a species, thermal tolerance can experience plasticity and may undergo selection, shaping resilience to temperature stress. In this study, we measured variation in the lower thermal tolerance of early instar larvae of an invasive forest insect, Lymantria dispar dispar L. (Lepidoptera: Erebidae), using populations sourced from the climatically diverse invasion of the Eastern United States. In two chill coma recovery experiments, we recorded recovery time following a period of exposure to a non-lethal cold temperature. A third experiment quantified growth responses after chill coma recovery to evaluate sublethal effects. Our results indicate that cold tolerance is linked to regional climate, with individuals from cold climate populations recovering faster from chill coma. While this geographic gradient is seen in many species, detecting this pattern is notable for an introduced species founded from a single point-source introduction. We demonstrate that the cold temperatures used in our experiments occur in nature from cold snaps after spring hatching, but negative impacts to growth and survival appear low. We expect that population differences in cold temperature performance manifest more from differences in temperature-dependent growth than acute exposure. Evaluating intraspecific variation in cold tolerance increases our understanding of the role of climatic gradients on the physiology of an invasive species, and contributes to tools for predicting further expansion.

Summary StatementChill coma recovery experiments demonstrate geographic variation in cold temperature tolerance for a widespread invasive species, and the risk of early season exposure differs across the invasion front.

category: physiology


title: Rapamycin induces autophagy and increases heat tolerance in Drosophila melanogaster

abstract: Autophagy is a physiological process that facilitates the recycling of intracellular cytosolic components as a response to diverse stressful conditions. By increasing the turnover of damaged structures and clearance of long-lived and larger protein aggregates, the induction of autophagy increases tolerance to abiotic stress in a range of organisms. However, the contribution of this process to heat-tolerance of insect models remains poorly studied to date. Here, we report that rapamycin exposure in Drosophila melanogaster induces autophagy in flies, which in turn correlates with an increase in heat tolerance and quicker recovery from heat-coma. This confirms the potentially important role of the autophagic process in heat tolerance mechanisms in this organism, opening the path to further characterization of its relationship to thermal acclimation and molecular level processes related to stress.


title: The need for unrealistic experiments in global change biology

abstract: Climate change is an existential threat, and our ability to conduct experiments on how organisms will respond to it is limited by logistics and resources, making it vital that experiments be maximally useful. The majority of experiments on phytoplankton responses to warming and CO2 use only two levels of each driver. However, to project the characters of future populations, we need a mechanistic and generalizable explanation for how phytoplankton respond to concurrent changes in temperature and CO2. This requires experiments with more driver levels, to produce response surfaces that can aid in the development of predictive models. We recommend prioritizing experiments or programmes that produce such response surfaces on multiple scales for phytoplankton.


title: Volatile-mediated signaling induces resistance of barley against infection with the biotrophic fungus Blumeria graminis f.sp. hordei

abstract:
Plants have evolved a vast variety of secondary metabolites to counteract biotic stress. Volatile organic compounds (VOCs) are carbon-based molecules induced by herbivore attack or pathogen infection. A mixture of plant VOCs is released for direct or indirect plant defense, plant-plant or plant-insect communication. Recent studies suggest that VOCs can also induce biotic stress resistance in distant organs and neighboring plants. Among other VOCs, green leaf volatiles (GLVs) are quickly released by plant tissue after the onset of herbivory or wounding.

We analysed VOCs emitted by 13-day old barley plants (Hordeum vulgare L.) after mechanical wounding using passive absorbers and TD-GC/MS detection. We investigated the influence of pure (Z)-3-hexenyl acetate (Z3HAC) as well as complex VOCs from wounded barley plants on the barley - powdery mildew interaction by pre-exposure in a static and a dynamic headspace connected to a powdery mildew susceptibility assay.

GLVs dominated the volatile profile of wounded barley plants with Z3HAC as the most prominent compound. Pre-exposure with Z3HAC resulted in induced resistance of barley against fungal infection. Barley complex volatiles emitted after mechanical wounding, similarly, enhanced resistance in receiver plants.

We found volatile-induced modification of the interaction towards an enhanced resistance against fungal infection. In addition, Z3HAC triggered a modulation of the alcohol dehydrogenase isoenzyme activity in receiver plants, a physiological response that possibly contributes to induced resistance. Plant-originated volatile metabolites could be a useful supplementation for future agronomic or horticultural practices.

HighlightVolatile-induced modification of the barley-powdery mildew interaction towards an enhanced resistance against fungal infection.


title: Rational domestication of a plant-based recombinant expression system expands its biosynthetic range.

abstract: Plant molecular farming aims to provide a green, flexible, and rapid alternative to conventional recombinant expression systems, capable of producing complex biologics such as enzymes, vaccines, and antibodies. Historically, the recombinant expression of therapeutic peptides in plants has proven difficult, largely due to their small size and instability. However, some plant species harbour the capacity for peptide backbone cyclization, a feature inherent in stable therapeutic peptides. One obstacle to realizing the potential of plant-based therapeutic peptide production is the proteolysis of the precursor before it is matured into its final stabilized form. Here we demonstrate the rational domestication of Nicotiana benthamiana within two generations to endow this plant molecular farming host with an expanded repertoire of peptide sequence space. The in planta production of molecules including an insecticidal peptide, a prostate cancer therapeutic lead and an orally active analgesic are demonstrated.

category: synthetic biology


title: Development and validation of inducible protein degradation and quantitative phosphoproteomics to identify kinase-substrate relationships.

abstract: Phosphorylation signaling is an essential post-translational regulatory mechanism that governs almost all eukaryotic biological processes and is controlled by an interplay between protein kinases and phosphatases. Knowledge of direct substrates of kinases provides evidence of mechanisms that relate activity to biological function. Linking kinases to their protein substrates can be achieved by inhibiting or reducing kinase activity and quantitative comparisons of phosphoproteomes in the presence and absence of kinase activity. Unfortunately, most of the human kinases lack chemical inhibitors with selectivity required to unambiguously assign protein substrates to their respective kinases. Here, we develop and validate a chemical proteomics strategy for linking kinase activities to protein substrates via targeted protein degradation and quantitative phosphoproteomics and apply it to the well-studied, essential mitotic regulator polo-like kinase 1 (Plk1). We leveraged the Tir1/auxin system to engineer HeLa cells with endogenously homozygous auxin-inducible degron (AID)-Plk1). We used HeLa cells and determined the impact of AID-tagging on Plk1 activity, localization, protein interactors, and substrate motifs. Using quantitative proteomics, we show that of over 8,000 proteins quantified, auxin addition is highly selective for degrading AID-Plk1 in mitotic cells. Comparison of phosphoproteome changes in response to chemical Plk1 inhibition to auxin-induced degradation revealed a striking degree of correlation. Finally, we explored basal protein turnover as a potential basis for clonal differences in auxin-induced degradation rates for AID-Plk1 cells. Taken together, our work provides a roadmap for the application of AID technology as a general strategy for the kinome-wide discovery of kinase-substrate relationships.

category: systems biology


title: Cylindromatosis Drives Synapse Pruning and Weakening by Promoting Macroautophagy through Akt-mTOR Signaling

abstract: The lysine-63 deubiquitinase cylindromatosis (CYLD) is long recognized as a tumor suppressor in immunity and inflammation and its loss-of-function mutations lead to familial cylindromatosis. However, recent studies reveal that CYLD is enriched in mammalian brain postsynaptic densities, and a gain-of-function mutation causes frontotemporal dementia (FTD), suggesting critical roles at excitatory synapses. Here we report that CYLD drives synapse elimination and weakening by acting on the Akt-mTOR-autophagy axis. Mice lacking CYLD display abnormal sociability, anxiety- and depression-like behaviors, and cognitive inflexibility. These behavioral impairments are accompanied by excessive synapse numbers, increased postsynaptic efficacy, augmented synaptic summation, and impaired NMDA receptor-dependent hippocampal long-term depression (LTD). Exogenous expression of CYLD results in removal of established dendritic spines from mature neurons in a deubiquitinase activity-dependent manner. In search of underlying molecular mechanisms, we find that CYLD knockout mice display marked overactivation of Akt and mTOR and reduced autophagic flux and, conversely, CYLD overexpression potently suppresses Akt and mTOR activity and promotes autophagy. Consequently, abrogating the Akt-mTOR-autophagy signaling pathway abolishes CYLD-induced spine loss, whereas enhancing autophagy in vivo by the mTOR inhibitor rapamycin rescues the synaptic pruning and LTD deficits in mutant mice. Our findings establish CYLD, via Akt-mTOR signaling, as a synaptic autophagy activator that exerts critical modulations on synapse maintenance, function, and plasticity.

category: neuroscience


title: Trapping of Nicotinic Acetylcholine Receptor Ligands Assayed by in vitro Cellular Studies and in vivo PET Imaging

abstract:
A question relevant to nicotine addiction is how nicotine and other nicotinic receptor membrane-permeant ligands, such as the anti-smoking drug varenicline (Chantix), distribute in the brain. Previously, we found that varenicline is trapped in intracellular acidic vesicles that contain 4{beta}2-type nicotinic receptors (4{beta}2Rs). Nicotine is not trapped but concentrates there. Here, combining subcellular methods with in vivo PET imaging, we present evidence that the 4{beta}2R PET ligand, 2-FA85380 (2-FA), is trapped within 4{beta}2R-containing acidic vesicles, while the PET ligand, Nifene, is not trapped. Additional evidence, using a fluorescent-tagged 4{beta}2R PET ligand, Nifrolidine, identified the trapping vesicles as Golgi satellites, an organelle regulated by nicotine in neurons where 4{beta}2Rs are expressed and traffics and processes 4{beta}2Rs in those neurons. Using PET imaging, 2-[18F]FA kinetics in high 4{beta}2R-expressing regions were much slower than ligand unbinding rates consistent with 2-FA trapping in Golgi satellites extending ligand residence time and 2-[18F]FA imaging of the Golgi satellites. Chloroquine, which dissipates acidic organelle pH gradients, reduced 2-[18F]FA distribution in vivo consistent with ligand trapping. In contrast, [18F]Nifene kinetics were rapid, consistent with ligand residence time reflecting ligand unbinding rates, and [18F]Nifene imaging all 4{beta}2R pools. Specific 2-[18F]FA and [18F]Nifene signals were eliminated in {beta}2 subunit knockout mice or by acute nicotine injections demonstrating binding to high-affinity sites on {beta}2-containing receptors. Altogether, we find that kinetic differences in 4{beta}2R PET ligands are consistent with their distribution among different 4{beta}2R pools in the brain, [18F]Nifene binding and imaging all ligand-binding 4{beta}2Rs and 2-[18F]FA imaging 4{beta}2Rs in Golgi satellites.

SignificanceMechanisms of nicotine addiction remain poorly understood. An earlier study using in vitro methods found that the anti-smoking nicotinic ligand, varenicline (Chantix) was trapped in 4{beta}2R-containing acidic vesicles. Using a fluorescent labeled high-affinity nicotinic ligand, this study provided evidence that these intracellular acidic vesicles were 4{beta}2R-containing Golgi satellites. In vivo PET imaging with F-18 labeled nicotinic ligands provided additional evidence that differences in PET ligand trapping in acidic vesicles were the cause of differences in PET ligand kinetics and subcellular distributions. These findings combining in vitro and in vivo imaging revealed new mechanistic insights into the kinetics of weak base PET imaging ligands and the subcellular mechanisms underlying nicotine addiction.


title: SV2A-Syt1 interaction controls surface nanoclustering and access to recycling synaptic vesicles

abstract: Following exocytosis, the recapture of vesicular proteins stranded at the plasma membrane in recycling synaptic vesicles (SVs) is essential to sustain neurotransmission. Nanoclustering is emerging as a mechanism through which proteins may be  pre-assembled prior to endocytosis, to ensure high fidelity of retrieval for subsequent rounds of vesicle fusion. Here, we used single molecule imaging to examine the nanoclustering of synaptotagmin-1 (Syt1) and synaptic vesicle protein 2A (SV2A). Syt1 forms surface nanoclusters through interaction of its C2B domain (K326/K328) with SV2A, as demonstrated by mutating Syt1 (K326A/K328A) and knocking down endogenous SV2A. Blocking cognate interaction with Syt1 (SV2AT84A) also decreased SV2A clustering. Impaired nanoclustering of Syt1 and SV2A leads to accelerated endocytosis of Syt1, altered intracellular sorting and decreased trafficking of Syt1 to a Rab5-positive endocytic pathway. We conclude that the interaction between SV2A and Syt1 locks both molecules into surface nanoclusters, controlling their entry into recycling SVs.


title: CD4+ and CD8+ regulatory T cells characterization in the rat using a unique transgenic Foxp3-EGFP model.

abstract:
BackgroundCD4+ and CD8+ regulatory T cells (Treg) in diverse species include different subsets from different origins. In all species, CD8+ Treg have been poorly characterized. CD4+ and CD8+ Treg in rats have only partially been characterized and there is no rat model in which FOXP3+ Treg are genetically tagged.

ResultsWe generated a rat transgenic line using the CRISPR/Cas9 system in which EGFP was inserted in frame on the 3 end of the Foxp3 gene using a 2A self-cleaving peptide. EGFP was exclusively expressed by CD4+ and CD8+ T cells in similar proportion as observed with anti-FOXP3 antibodies. CD4+EGFP+ Treg were 5-10 times more frequent than CD8+EGFP+ Treg. CD4+ and CD8+ EGFP+ Treg expressed both the CD25highCD127lowCD45RClow/- markers. The suppressive activity of CD4+ and CD8+ Treg was largely confined to EGFP+ cells. RNAseq analyses showed similarities but also differences among CD4+ and CD8+ EGFP+ cells and provided the first description of the natural FOXP3+ CD8+ Treg transcriptome. In vitro culture of CD4+ and CD8+ EGFP- cells with TGFbeta and IL-2 resulted in the induction of EGFP+ Treg. Preferential expansion of CD4+ and CD8+ EGFP+ Treg could be detected upon in vivo administration of a low dose of IL-2.

ConclusionsThis new and unique Foxp3-EGFP rat line constitutes a useful model to identify and isolate viable natural and induced CD4+ and CD8+ Treg. Additionally, it allows to identify new molecules expressed in CD8+ Treg that may allow to better define their phenotype and function not only in rats but also in other species.

category: immunology


title: The polyanionic drug suramin neutralizes histones and prevents endotheliopathy

abstract:
Drugs are needed to protect against the neutrophil-derived histones responsible for endothelial injury in acute inflammatory conditions such as trauma and sepsis. Heparin and other polyanions can neutralize histones but may cause secondary, deleterious effects such as excessive bleeding. Here, we demonstrate that suramin--a widely available polyanionic drug--completely neutralizes the toxic effects of histones. The sulfate groups on suramin form stable electrostatic interactions with hydrogen bonds in the histone octamer with a dissociation constant of 250 nM. In cultured endothelial cells (Ea.Hy926), histone-induced thrombin generation was significantly decreased by suramin. In isolated murine blood vessels, suramin abolished aberrant endothelial cell calcium signals and rescued impaired endothelial-dependent vasodilation caused by histones. Suramin significantly decreased pulmonary endothelial cell ICAM-1 expression and neutrophil recruitment caused by infusion of sub-lethal doses of histones in vivo. Suramin also prevented lung edema, intra-alveolar hemorrhage and mortality in mice receiving a lethal dose of histones. Protection of vascular endothelial function from histone-induced damage is a novel mechanism of action for suramin with therapeutic implications for conditions characterized by elevated histone levels.

Significance StatementPathologic levels of circulating histones cause acute endotheliopathy, characterized by widespread disruption of critical endothelial functions and thromboinflammation. We discovered that suramin binds histones and prevents histone-induced endothelial dysfunction, thrombin generation, lung injury, and death. Histone binding is a novel mechanism of action for suramin, considered among the safest and most effective drugs by the World Health Organization. These results support the use of suramin for protection of blood vessels in conditions exacerbated by circulating histones including trauma and sepsis.


title: Integrated computational and in vivo models reveal Key Insights into Macrophage Be-havior during bone healing

abstract:
Myeloid-derived monocyte and macrophages are key cells in the bone that contribute to remodeling and injury repair. However, their temporal polarization status and control of boneresorbing osteoclasts and bone-forming osteoblasts responses is largely unknown. In this study, we focused on two aspects of monocyte/macrophage dynamics and polarization states over time: 1) the injury-triggered pro- and anti-inflammatory monocytes/macrophages temporal profiles, 2) the contributions of pro-versus anti-inflammatory monocytes/macrophages in coordinating healing response. Bone healing is a complex multicellular dynamic process. While traditional in vitro and in vivo experimentation may capture the behavior of select populations with high resolution, they cannot simultaneously track the behavior of multiple populations. To address this, we have used an integrated a coupled ordinary differential equations (ODEs)-based framework describing multiple cellular species to in vivo bone injury data in order to identify and test various hypotheses regarding bone cell populations dynamics. Our approach allowed us to infer several biological insights including, but not limited to,: 1) anti-inflammatory macrophages are key for early osteoclast inhibition and pro-inflammatory macrophage suppression, 2) pro-inflammatory macrophages are involved in osteoclast bone resorptive activity, whereas osteoblasts promote osteoclast differentiation, 3) Pro-inflammatory monocytes/macrophages rise during two expansion waves, which can be explained by the anti-inflammatory macrophages-mediated inhibition phase between the two waves. In addition, we further tested the robustness of the mathematical model by comparing simulation results to an independent experimental dataset. Taken together, this novel comprehensive mathematical framework allowed us to identify biological mechanisms that best recapitulate bone injury data and that explain the coupled cellular population dynamics involved in the process. Furthermore, our hypothesis testing methodology could be used in other contexts to decipher mechanisms in complex multicellular processes.

Author SummaryMyeloid-derived monocytes/macrophages are key cells for bone remodeling and injury repair. However, their temporal polarization status and control of bone-resorbing osteoclasts and boneforming osteoblasts responses is largely unknown. In this study, we focused on two aspects of monocyte/macrophage population dynamics: 1) the injury-triggered pro- and anti-inflammatory monocytes/macrophages temporal profiles, 2) the contributions of pro-versus anti-inflammatory monocytes/macrophages in coordinating healing response. In order to test various hypotheses regarding bone cell populations dynamics, we have integrated a coupled ordinary differential equations-based framework describing multiple cellular species to in vivo bone injury data. Our approach allowed us to infer several biological insights including: 1) anti-inflammatory macrophages are key for early osteoclast inhibition and pro-inflammatory macrophage suppression, 2) pro-inflammatory macrophages are involved in osteoclast bone resorptive activity, whereas osteoblasts promote osteoclast differentiation, 3) Pro-inflammatory monocytes/macrophages rise during two expansion waves, which can be explained by the anti-inflammatory macrophages-mediated inhibition phase between the two waves. Taken together, this mathematical framework allowed us to identify biological mechanisms that recapitulate bone injury data and that explain the coupled cellular population dynamics involved in the process.


title: Discovery of a new species of trichomonasvirus in the human parasite Trichomonas vaginalis using transcriptome mining

abstract: Trichomonas vaginalis is the most common nonviral cause of sexually transmitted infections globally, with an estimated quarter of a billion people infected around the world. Infection by the protozoan parasite results in the clinical syndrome trichomoniasis, which manifests as an inflammatory syndrome with acute and chronic consequences. Half or more of these parasites are themselves infected with one or more dsRNA viruses which can exacerbate the inflammatory disease. Four distinct viruses have been found in T. vaginalis to date, Trichomonas vaginalis virus 1 through 4 (or TVVs). Despite the global prevalence of these viruses, few coding-complete genome sequences have been determined. We conducted viral sequence mining in publicly available transcriptomes across 60 RNA-seq datasets representing 13 distinct T. vaginalis isolates. We assembled sequences for 27 new trichomonasvirus strains across all known TVV species, with 17 of these assemblies representing coding-complete genomes. Using a strategy of de novo sequence assembly followed by taxonomic classification, we discovered a fifth species of TVV that we term Trichomonas vaginalis virus 5 (TVV5). Six strains of TVV5 were assembled, including two coding-complete genomes. These TVV5 sequences exhibit high sequence identity to each other, but low identity to any strains of TVV1-4. Phylogenetic analysis corroborates the species-level designation. These results substantially increase the number of coding-complete TVV genome sequences and demonstrate the utility of mining publicly available transcriptomes for the discovery of RNA viruses in a critical human pathogen.

category: microbiology


title: HBV enveloped particle secretion is positively regulated by GRP78 through direct interaction with preS1

abstract:
Hepatitis B virus (HBV) infection is a common cause of liver diseases worldwide. Existing drugs do not effectively eliminate HBV from infected hepatocytes; thus, novel curative therapies are needed. Enveloped-particle secretion is a key but poorly studied aspect of the viral life cycle. Here, we report that GRP78 positively regulates HBV enveloped-particle secretion. GRP78 is the specific target of preS1 binding; HBV can upregulate GRP78 in liver cell lines and sera from chronic hepatitis B patients. GRP78 promoted intact HBV-particle secretion in liver cell lines and an HBV transgenic-mouse model. Some peptides screened from preS1 via phage display could inhibit viral-particle secretion by interacting with GRP78 via hydrogen bonds and hydrophobic interactions, thereby disturbing the interaction with HBV particles. These results provide insight into enveloped-particle secretion in the HBV life cycle. GRP78 might be a potential target for HBV-infection treatment via restricting GRP78-preS1 interactions to block viral-particle secretion.

IMPORTANCEHBV is a major human pathogen. The virus More than 250 million n individuals globally are chronically infected with HBV and can result in the infected personsabout 800,000 die for HBV-related disease annually. The mature HBV enveloped particles containing HBs and the relaxed circular (RC) DNA genome could secreted extracellularly as virions. And these enveloped virions serves as the infectious HBV particles for initiating a complete life cycle of the virus. Here, we show the GRP78 is the specific target of preS1 binding, and it could positive regulate HBV enveloped particle secretion in cell and HBV transgenic mice model. Furthermore, several peptides screened from preS1 and Phage screening could inhibit viral particle secretion by interacting with GRP78 and disturbing its interaction with HBV, and then block the life cycle of the virus. This has important implications for our understanding of the mechanisms of antivirals that target intact HBV particles secretion.


title: Transcriptome-wide identification of coding and noncoding RNA-binding proteins defines the comprehensive RNA interactome of Leishmania mexicana

abstract:
Proteomic profiling of RNA-binding proteins in Leishmania is currently limited to polyadenylated mRNA-binding proteins, leaving proteins that interact with nonadenylated RNAs, including noncoding RNAs and pre-mRNAs, unidentified. Using a combination of unbiased orthogonal organic phase separation methodology and tandem mass tag-labelling-based high resolution quantitative proteomic mass spectrometry, we robustly identified 2,417 RNA-binding proteins, including 1289 putative novel non-poly(A)-RNA-binding proteins across the two main Leishmania life cycle stages. Eight out of twenty Leishmania deubiquitinases including the recently characterised L. mexicana DUB2 with an elaborate RNA-binding protein interactome were exclusively identified in the non-poly(A)-RNA-interactome. Additionally, an increased representation of WD40 repeat domains were observed in the Leishmania non-poly(A)-RNA-interactome, thus uncovering potential involvement of this protein domain in RNA-protein interactions in Leishmania. We also characterise the protein-bound RNAs using RNA-sequencing and show that in addition to protein coding transcripts ncRNAs are also enriched in the protein-RNA interactome. Differential gene expression analysis revealed enrichment of 145 out of 195 total L. mexicana protein kinase genes in the protein-RNA-interactome, suggesting important role of protein-RNA interactions in the regulation of the Leishmania protein kinome. Additionally, we characterise the quantitative changes in RNA-protein interactions in hundreds of Leishmania proteins following inhibition of heat shock protein 90 (Hsp90). Our results show that the Hsp90 inhibition in Leishmania causes widespread disruption of RNA-protein interactions in ribosomal proteins, proteasomal proteins and translation factors in both life cycle stages, suggesting downstream effect of the inhibition on protein synthesis and degradation pathways in Leishmania. This study defines the comprehensive RNA interactome of Leishmania and provides in-depth insight into the widespread involvement of RNA-protein interactions in Leishmania biology.

IMPORTANCEAdvances in proteomics and mass spectrometry have revealed the mRNA-binding proteins in many eukaryotic organisms, including the protozoan parasites Leishmania spp., the causative agents of leishmaniasis, a major infectious disease in over 90 tropical and sub-tropical countries. However, in addition to mRNAs, which constitute only 2 to 5% of the total transcripts, many types of non-coding RNAs participate in crucial biological processes. In Leishmania, RNA-binding proteins serve as primary gene regulators. Therefore, transcriptome-wide identification of RNA-binding proteins is necessary for deciphering the distinctive posttranscriptional mechanisms of gene regulation in Leishmania. Using a combination of highly efficient orthogonal organic phase separation method and tandem mass tag-labelling-based quantitative proteomic mass spectrometry, we provide unprecedented comprehensive molecular definition of the total RNA interactome across the two main Leishmania life cycle stages. In addition, we characterise for the first time the quantitative changes in RNA-protein interactions in Leishmania following inhibition of heat shock protein 90, shedding light into hitherto unknown large-scale downstream molecular effect of the protein inhibition in the parasite. This work provides insight into the importance of total RNA-protein interactions in Leishmania, thus significantly expanding our knowledge of the emergence of RNA-protein interactions in Leishmania biology.


title: A novel Alex3/Gαq protein complex regulating mitochondrial dynamics, dendritic complexity, and neuronal survival

abstract: In neurons, mitochondrial dynamics and trafficking are essential to provide the energy required for neurotransmission and neuronal activity. Recent studies point to GPCR and G proteins as important regulators of mitochondrial dynamics and energy metabolism. Here we show that activation of Gq negatively regulates mitochondrial dynamics and trafficking in neurons. Gq interacts with the mitochondrial trafficking protein Alex3. By generating a CNS-specific armcx3 knock-out mouse line, we demonstrate that Alex3 is required for Gq effects on mitochondrial dynamics and trafficking, and dendritic growth. Armcx3-deficient mice present decreased OXPHOS complex and ER stress response protein levels, which correlate with increased neuronal death, motor neuron and neuromuscular synaptic loss, and severe motor alterations. Finally, we show that Alex3 disassembles from the Miro1/Gq complex upon calcium rise. These data uncover a novel Alex3/Gq complex that regulates neuronal mitochondrial dynamics and neuronal death and allows the control of mitochondrial functions by GPCRs.


title: The luminal AAA+ ATPase torsinA mediates distinct mechanisms of nuclear-cytoplasmic communication by adopting different functional assembly states.

abstract: Chemical and mechanical nuclear-cytoplasmic communication across the nuclear envelope (NE) is largely mediated by the nuclear pore complex (NPC) and the linker of nucleoskeleton and cytoskeleton (LINC) complex, respectively. While NPC and LINC complex assembly are functionally related, the mechanisms responsible for this relationship remain poorly understood. Here, we investigated how the luminal ATPases associated with various cellular activities (AAA+) protein torsinA promotes NPC and LINC complex assembly using fluorescence fluctuation spectroscopy (FFS), quantitative photobleaching analyses, and functional cellular assays. We report that torsinA controls LINC complex-dependent nuclear-cytoskeletal coupling as a soluble hexameric AAA+ protein and interphase NPC biogenesis as a membrane-associated helical polymer. These findings help resolve the conflicting models of torsinA function that were recently proposed based on in vitro structural studies. Our results will enable future studies of the role of defective nuclear-cytoplasmic communication in DYT1 dystonia and other diseases caused by mutations in torsinA.


title: It ain't what you do, it's what drives why you do it: how the basis of growers' decision making affects the effectiveness of disease control

abstract: While the spread of plant disease depends strongly on biological factors driving transmission, it also has a human dimension. Disease control depends on decisions made by individual growers, who are in turn influenced by a broad range of factors. Despite this, human behaviour has rarely been included in plant epidemic models. Considering Cassava Brown Streak Disease, we model how the perceived increase in profit due to disease management influences participation in clean seed systems (CSS). Our models are rooted in game theory, with growers making strategic decisions based on the expected profitability of different control strategies. We find that both the information used by growers to assess profitability and the perception of economic and epidemiological parameters influence long-term participation in the CSS. Over-estimation of infection risk leads to lower participation in the CSS, as growers perceive that paying for the CSS will be futile. Additionally, even though good disease management can be achieved through the implementation of CSS, and a scenario where all controllers use the CSS is achievable when growers base their decision on the average of their entire strategy, CBSD is rarely eliminated from the system. These results are robust to stochastic and spatial effects. Our work highlights the importance of including human behaviour in plant disease models, but also the significance of how that behaviour is included.


title: How grower decision-making is modelled impacts the effect of plant disease control.


title: Estimating real snakebite incidence in Colombia by using mathematical modelling and statistical inference.

abstract: Snakebite envenoming is a Neglected Tropical Disease affecting mainly deprived populations. Its burden is normally underestimated because patients prefer to seek for traditional medicine. Thus, applying strategies to optimize disease management and treatment delivery is difficult. We propose a framework to estimate snakebite incidence at a fine political scale based on available data, testing it in Colombia. First, we produced snakebite fine-scale risk maps based on the most medically important venomous snake species (Bothrops asper and B. atrox). We validated them with reported data in the country. Then, we proposed a generalized mixed effect model that estimates total incidence based on produced risk maps, poverty indexes, and an accessibility score that reflects the struggle to reach a medical center. Finally, we calibrated our model with national snakebite reported data from 2010 to 2019 using a Markov chain Monte Carlo (MCMC) algorithm and estimated underreporting based on the total incidence estimation. Our results suggest that 10.3% of total snakebite cases are not reported in Colombia and do not seek medical attention. The Orinoco and Amazonian regions (east of Colombia) share a high snakebite risk with a high underreporting. Our work highlights the importance of multidisciplinary approaches to face snakebite.


title: Trade-offs among plant reproductive traits determine interactions with floral visitors

abstract: Plant life-history strategies are constrained by cost-benefit trade-offs that determine plant form and function. However, despite recent advances in the understanding of trade-offs for vegetative and physiological traits, little is known about plant reproductive economics and how they constrain plant life-history strategies and shape interactions with floral visitors. Here, we investigate plant reproductive trade-offs and how these drive interactions with floral visitors using a dataset of 17 reproductive traits for 1,506 plant species from 28 plant-pollinator studies across 18 countries. We tested whether a plants reproductive strategy predicts its interactions with floral visitors and if the different reproductive traits predict the plants role within the pollination network. We found that over half of all plant reproductive trait variation was explained by two independent axes that encompassed plant form and function. Specifically, the first axis indicated the presence of a trade-off between flower number and flower size, while the second axis indicated a pollinator dependency trade-off. Plant reproductive trade-offs helped explain partly the presence or absence of interactions with floral visitors, but not differences in visitation rate. However, we did find important differences in the interaction level among floral visitor guilds on the different axes of trait variation. Finally, we found that plant size and floral rewards were the most important traits in the understanding of the plant species network role. Our results highlight the importance of plant reproductive trade-offs in determining plant life-history strategies and plant-pollinator interactions in a global context.