Spike mimicry of thrombopoietin may induce thrombocytopenia in COVID-19 SARS-CoV-2 causes COVID-19, a disease curiously resulting in varied symptoms and outcomes, ranging from asymptomatic to fatal. Autoimmunity due to cross-reacting antibodies resulting from molecular mimicry between viral antigens and host proteins may provide an explanation. We computationally investigated molecular mimicry between SARS-CoV-2 Spike and known epitopes. We discovered molecular mimicry hotspots in Spike and highlight two examples with tentative autoimmune potential and implications for understanding COVID-19 complications. We show that a TQLPP motif in Spike and thrombopoietin shares similar antibody binding properties. Antibodies cross-reacting with thrombopoietin may induce thrombocytopenia, a condition observed in COVID-19 patients. Another motif, ELDKY, is shared in multiple human proteins such as PRKG1 and tropomyosin. Antibodies cross-reacting with PRKG1 and tropomyosin may cause known COVID-19 complications such as blood-clotting disorders and cardiac disease, respectively. Our findings illuminate COVID-19 pathogenesis and highlight the importance of considering autoimmune potential when developing therapeutic interventions to reduce adverse reactions.
Protein sequence profile prediction using ProtAlbert transformer Protein profiles have many applications in bioinformatics. To construct the profile from a protein sequence, the sequence is aligned with database. However, sometimes there are no similar sequences with the query. This paper proposes a method based on pre-trained ProtAlbert transformer to predict the profile for a single protein sequence without alignment. The performance of transformers on natural languages is impressive. Protein sequences can be viewed as a language; therefore, we can benefit from using these models. We analyze the attention heads in different layers of ProtAlbert to show that the transformer can capture five essential protein characteristics of the family from a single protein sequence. These assessments are performed on the CASP13 dataset to find representative heads for each of five protein characteristics. Then, these heads are investigated on one thermophilic and two mesophilic proteins as case studies. The results show the significant attention heads for protein family properties extracted from a single protein sequence. This analysis led us to propose an algorithm called PA_SPP for profile prediction using only a single protein sequence as input. In our algorithm, we apply the masked language modeling method of ProtAlbert. The results display high similarity between the predicted profiles and HSSP profiles.
Ultrahigh-Throughput Ambient MS: Direct Analysis at 22 Samples per Second by Infrared Matrix-Assisted Desorption Electrospray Ionization Mass Spectrometry Infrared Matrix-Assisted Laser Desorption Electrospray Ionization (IR-MALDESI) mass spectrometry is an ambient-direct sampling method being developed for high-throughput, label-free, biochemical screening of large-scale compound libraries. Here, we report the development of an ultrahigh-throughput continuous motion IR-MALDESI sampling approach capable of acquiring data at rates up to 22.7 samples per second in a 384-well microtiter plate. At top speed, less than 1% analyte carryover is observed from well-to-well and signal intensity relative standard deviations (RSD) of 11.5% and 20.9% for 3 M 1-hydroxymidazolam and 12 M dextrorphan, respectively, are achieved. The ability to perform parallel kinetics studies on 384 samples with ~30s time resolution using an isocitrate dehydrogenase 1 (IDH1) enzyme assay is shown. Finally, we demonstrate the repeatability and throughput of our approach by measuring 115,200 samples from 300 microtiter plate reads consecutively over 5.54 hours with RSDs under 8.14% for each freshly introduced plate. Taken together, these results demonstrate the use of IR-MALDESI at sample acquisition rates that surpass other currently reported direct sampling mass spectrometry approaches used for high throughput compound screening.

For Table of Contents Only

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/465730v3_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@5f916corg.highwire.dtl.DTLVardef@4473b4org.highwire.dtl.DTLVardef@82e8forg.highwire.dtl.DTLVardef@128b57b_HPS_FORMAT_FIGEXP  M_FIG C_FIG Plasmodium DDI1 is a potential dual-therapeutic target and essential chromatin-associated protein DDI1 proteins are involved in a variety of cellular processes, including proteasomal degradation of specific proteins. All DDI1 proteins contain a ubiquitin-like (UBL) domain and a retroviral aspartyl protease (RVP) domain. Some DDI1 proteins also contain a ubiquitin-associated (UBA) domain. The three domains confer distinct activities to DDI1 proteins. The presence of RVP domain makes DDI1 a potential target of HIV protease inhibitors, which also block the development of malaria parasites. Hence, we investigated the DDI1 of malaria parasites to identify its roles during parasite development and potential as a therapeutic target. DDI1 proteins of Plasmodium and other Apicomplexan parasites share the UBL-RVP domain architecture, and some also contain the UBA domain. Plasmodium DDI1 is expressed across all the major life cycle stages and is essential, as conditional depletion of DDI1 protein in the mouse malaria parasite Plasmodium berghei and the human malaria parasite Plasmodium falciparum compromised parasite development. Infection of mice with DDI1 knock-down P. berghei was self-limiting and protected the recovered mice from subsequent infection with homologous as well as heterologous parasites, indicating potential of DDI1 knock-down parasites as a whole organism vaccine. P. falciparum DDI1 (PfDDI1) is associated with chromatin and DNA-protein crosslinks. PfDDI1-depleted parasites accumulated DNA-protein crosslinks and showed enhanced susceptibility to DNA damaging chemicals, indicating a role of PfDDI1 in removal of DNA-protein crosslinks. Knock-down of PfDDI1 increased susceptibility to the retroviral protease inhibitor lopinavir and antimalarial artemisinin, which suggests that simultaneous inhibition of DDI1 could potentiate antimalarial activity of these drugs. Hence, Plasmodium DDI1 could be a dual-therapeutic target for malaria control. Sarcoplasmic reticulum calcium handling in young DBA/2J vs C57BL/10 mdx models of Duchenne muscular dystrophy The DBA/2J (D2) mdx mouse has emerged as a more severe model of Duchenne muscular dystrophy when compared to the traditional C57BL/10 (C57) mdx mouse. Here, we questioned whether sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) function would differ in muscles from young D2 and C57 mdx mice. In gastrocnemius muscles, both D2- and C57 mdx mice exhibited signs of impaired Ca2+ uptake, however, this was more severe in D2 mdx mice. Maximal SERCA activity was lowered only in D2 mdx gastrocnemius muscles and not C57 mdx muscles. Furthermore, in the left ventricle and diaphragm, Ca2+ uptake was impaired in C57 mdx muscles with lowered rates of Ca2+ uptake compared with C57 WT mice, whereas in muscles from D2 mdx mice, rates of Ca2+ uptake were unattainable due to the severe impairments in their ability to transport Ca2+. Overall, our study demonstrates that SERCA function is drastically impaired in young D2 mdx mice. Cluster-specific gene markers enhance Shigella and Enteroinvasive Escherichia coli in silico serotyping Shigella and enteroinvasive Escherichia coli (EIEC) cause human bacillary dysentery with similar invasion mechanisms and share similar physiological, biochemical and genetic characteristics. The ability to differentiate Shigella and EIEC from each other is important for clinical diagnostic and epidemiologic investigations. The existing genetic signatures may not discriminate between Shigella and EIEC. However, phylogenetically, Shigella and EIEC strains are composed of multiple clusters and are different forms of E. coli. In this study, we identified 10 Shigella clusters, 7 EIEC clusters and 53 sporadic types of EIEC by examining over 17,000 publicly available Shigella/EIEC genomes. We compared Shigella and EIEC accessory genomes to identify the cluster-specific gene markers or marker sets for the 17 clusters and 53 sporadic types. The gene markers showed 99.63% accuracy and more than 97.02% specificity. In addition, we developed a freely available in silico serotyping pipeline named Shigella EIEC Cluster Enhanced Serotype Finder (ShigEiFinder) by incorporating the cluster-specific gene markers and established Shigella/EIEC serotype specific O antigen genes and modification genes into typing. ShigEiFinder can process either paired end Illumina sequencing reads or assembled genomes and almost perfectly differentiated Shigella from EIEC with 99.70% and 99.81% cluster assignment accuracy for the assembled genomes and mapped reads respectively. ShigEiFinder was able to serotype over 59 Shigella serotypes and 22 EIEC serotypes and provided a high specificity with 99.40% for assembled genomes and 99.38% for mapped reads for serotyping. The cluster markers and our new serotyping tool, ShigEiFinder (https://github.com/LanLab/ShigEiFinder), will be useful for epidemiologic and diagnostic investigations.

Impact statementThe differentiation of Shigella strains from enteroinvasive E. coli (EIEC) is important for clinical diagnosis and public health epidemiologic investigations. The similarities between Shigella and EIEC strains make this differentiation very difficult as both share common ancestries within E. coli. However, Shigella and EIEC are phylogenetically separated into multiple clusters, making high resolution separation using cluster specific genomic markers possible. In this study, we identified 17 Shigella or EIEC clusters including five that were newly identified through examination of over 17,000 publicly available Shigella and EIEC genomes. We further identified an individual or a set of cluster-specific gene markers for each cluster using comparative genomic analysis. These markers can then be used to classify isolates into clusters and were used to develop an in silico pipeline, ShigEiFinder (https://github.com/LanLab/ShigEiFinder) for accurate differentiation, cluster typing and serotyping of Shigella and EIEC from Illumina sequencing reads or assembled genomes. This study will have broad application from understanding the evolution of Shigella/EIEC to diagnosis and epidemiology.

Data summarySequencing data have been deposited at the National Center for Biotechnology Information under BioProject number PRJNA692536.

RepositoriesRaw sequence data are available from NCBI under the BioProject number PRJNA692536. Statistical testing in gene transcriptomic-neuroimaging associations: an evaluation of methods that assess spatial and gene specificity Multiscale integration of gene transcriptomic and neuroimaging data is becoming a widely used approach for exploring the molecular underpinnings of large-scale brain structure and function. Proper statistical evaluation of computed associations between imaging-based phenotypic and transcriptomic data is key in these explorations, in particular to establish whether observed associations exceed  chance level of random, non-specific effects. Recent approaches have shown the importance of spatial null models to test for spatial specificity of effects to avoid serious inflation of reported statistics. Here, we discuss the need for examination of the second category of specificity in transcriptomic-neuroimaging analyses, namely that of gene specificity, examined using null models built upon effects that occur from sets of random genes. Through simple examples of commonly performed transcriptomic-neuroimaging analyses, we show that providing additional gene specificity on observed transcriptomic-neuroimaging effects is of high importance to avoid non-specific (potentially false-positive) effects. Through simulations we further show that the rate of reported non-specific effects (i.e., effects that are generally observed and cannot be specifically linked to a gene-set of interest) can run as high as 60%, with only less than 5% of transcriptomic-neuroimaging associations observed through ordinary linear regression analyses showing spatial and gene specificity. We explain that using proper null models that test for both spatial specificity and gene specificity is warranted. Internet of Things Architecture for Cellular Biology The Internet of Things (IoT) provides a simple framework to easily control online devices. IoT is now a commonplace tool used by technology companies, but it is rarely used in biology experiments. IoT can benefit research through alarm notifications, automation, and the real-time monitoring of experiments. We developed and implemented an IoT architecture to control biological devices used in experiments. We developed our own electrophysiology, microscopy, and microfluidic devices so that may be controlled through a unified IoT architecture. The system allows each device to be monitored and controlled through an online web tool. We present our IoT architecture so other labs may replicate it for their own experiments. Glypiated FGF directs contact-mediated bidirectional signaling to self-regulate tissue-specific dispersion How signaling proteins generate a multitude of information to organize tissue patterns is critical to understanding morphogenesis. In Drosophila, FGF produced in wing-disc cells regulates the development of the disc-associated air-sac-primordium (ASP). Here, we show that FGF is Glycosylphosphatidylinositol-anchored to the producing cell surface and that this modification both inhibits free FGF secretion and activates target-specific bidirectional FGF-FGFR signaling through cytonemes. FGF-source and recipient ASP cells extend cytonemes that present FGF and FGFR on their surfaces and reciprocally recognize each other over distance by contacting through CAM-like FGF-FGFR binding. Contact-mediated FGF-FGFR binding induces bidirectional signaling, which, in turn, promotes ASP and source cells to polarize cytonemes toward each other and reinforce signaling contacts. Subsequent un-anchoring of FGFR-bound-FGF from the source cell membrane dissociates cytoneme contacts and delivers FGF target-specifically to ASP cytonemes for paracrine functions. Thus, GPI-anchored FGF organizes both source and recipient cells and self-regulates its cytoneme-mediated tissue-specific dispersion and signaling. Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly. Alteration of gut microbiota with a broad-spectrum antibiotic does not impair maternal care in the European earwig. AO_SCPLOWBSTRACTC_SCPLOWThe microbes residing within the gut of an animal host often increase their own fitness by modifying their hosts physiological, reproductive, and behavioural functions. Whereas recent studies suggest that they may also shape host sociality and therefore have critical effects on animal social evolution, the impact of the gut microbiota on maternal care remains unexplored. This is surprising, as this behaviour is widespread among animals, often determines the fitness of both juveniles and parents, and is essential in the evolution of complex animal societies. Here, we tested whether life-long alterations of the gut microbiota with rifampicin - a broad-spectrum antibiotic - impair pre- and post-hatching maternal care in the European earwig. Our results first confirm that rifampicin altered the mothers gut microbial communities and indicate that the composition of the gut microbiota differs before and after egg care. Contrary to our predictions, however, the rifampicin-induced alterations of the gut microbiota did not modify pre- or post-hatching care. Independent of maternal care, rifampicin increased the females feces production and resulted in lighter eggs and juveniles. By contrast, rifampicin altered none of the other 21 physiological, reproductive and longevity traits measured over the 300 days of a females lifetime. Overall, these findings reveal that altering the gut microbiota with a large spectrum antibiotic such as rifampicin does not necessarily affect host sociality. They also emphasize that not all animals have evolved a co-dependence with their microbiota and call for caution when generalizing the central role of gut microbes in host biology. Low dose rate γ-irradiation protects Drosophila melanogaster chromosomes from double strand breaks and telomere fusions by modulating the expression of the esi-RNA biogenesis factor Loquacious. It is still continuously debated whether the low-dose/dose-rate (LDR) of ionizing radiation represents a hazard for humans. Model organisms, such as fruit flies, are considered valuable systems to reveal insights into this issue. We found that, in wild-type Drosophila melanogaster larval neuroblasts, the frequency of Chromosome Breaks (CBs), induced by acute {gamma}-irradiation, is significantly reduced when flies are previously exposed to a protracted dose of 0.4Gy delivered at a dose rate of 2.5mGy/h. This indicates that this exposure, which is associated with an increased expression of DNA damage response proteins, induces a radioadaptive response (RAR) that protects Drosophila from extensive DNA damage. Interestingly, the same exposure reduces the frequency of telomere fusions (TFs) from Drosophila telomere capping mutants suggesting that the LDR can generally promote a protective response on chromatin sites that are recognized as DNA breaks. Deep RNA sequencing revealed that RAR is associated with a reduced expression of Loquacious D (Loqs-RD) gene that encodes a well-conserved dsRNA binding protein required for esiRNAs biogenesis. Remarkably, loss of Loqs mimics the LDR-mediated chromosome protection as it decreases the IR-induced CBs and TF frequency. Thus, our molecular characterization of RAR identifies Loqs as a key factor in the cellular response to LDR and unveils unprecedented epigenetic routes involved in radioresistance. An AI-guided signature reveals the nature of the shared proximal pathways of host immune response in MIS-C and Kawasaki disease A significant surge in cases of multisystem inflammatory syndrome in children (MIS-C, also called Pediatric Inflammatory Multisystem Syndrome - PIMS) has been observed amidst the COVID-19 pandemic. MIS-C shares many clinical features with Kawasaki disease (KD), although clinical course and outcomes are divergent. We analyzed whole blood RNA sequences, serum cytokines, and formalin fixed heart tissues from these patients using a computational toolbox of two gene signatures, i.e., the 166-gene viral pandemic (ViP) signature, and its 20-gene severe (s)ViP subset that were developed in the context of SARS-CoV-2 infection and a 13-transcript signature previously demonstrated to be diagnostic for KD. Our analyses revealed that KD and MIS-C are on the same continuum of the host immune response as COVID-19. While both the pediatric syndromes converge upon an IL15/IL15RA-centric cytokine storm, suggestive of shared proximal pathways of immunopathogenesis, they diverge in other laboratory parameters and cardiac phenotypes. The ViP signatures also revealed unique targetable cytokine pathways in MIS-C, place MIS-C farther along in the spectrum in severity compared to KD and pinpoint key clinical (reduced cardiac function) and laboratory (thrombocytopenia and eosinopenia) parameters that can be useful to monitor severity. "Adopt a Microorganism" methodology enhances the discourse richness of high school students in both hybrid learning and remote education The Internet has changed the way teachers and students access information and build knowledge. The recent COVID-19 pandemic has created challenges for both teachers and students and a demand for new methodologies of remote learning. In the life sciences, mixing online content with practical activities represents an even greater challenge. In microbiology, the implementation of an active teaching methodology, the #Adopt project, based on the social network Facebook(R), represents an excellent option for connecting remote education with classroom activities. In 2020, the version applied in high school, "Adopt a Microorganism", was adapted to meet the demands of emergency remote education owing to the suppression of face-to-face activities caused by the pandemic. In the present study, we assessed how the change in methodology impacted the discourse richness of students from high school integrated with technical education in the Business Administration program of the Federal Institute of Sao Paulo, Sorocaba Campus. Three questionnaires related to the groups of microorganisms (Archaea, Bacteria, Virus, Fungi, and Protozoan) were applied. The students responses in the 2019 and 2020 classes were compared concerning content richness and multiplicity of concepts through the application of the Shannon diversity index, an approach that is generally used to assess biodiversity in different environments. The observed results suggest that remote learning provided students with a conceptual basis and richness of content equivalent to that achieved by students subjected to the hybrid teaching model. In conclusion, this study suggests that the #Adopt project methodology increases students discourse richness in microbiology even without face-to-face traditional classes. Sahul's megafauna were vulnerable to plant-community changes due to their position in the trophic network Extinctions stemming from environmental change often trigger trophic cascades and coextinctions. However, it remains unclear whether trophic cascades were a large contributor to the megafauna extinctions that swept across several continents in the Late Pleistocene. The pathways to megafauna extinctions are particularly unclear for Sahul (landmass comprising Australia and New Guinea), where extinctions happened earlier than on other continents. We investigated the role of bottom-up trophic cascades in Late Pleistocene Sahul by constructing pre-extinction (~ 80 ka) trophic network models of the vertebrate community of Naracoorte, south-eastern Australia. These models allowed us to predict vertebrate species vulnerability to cascading extinctions based on their position in the network. We tested whether the observed extinctions could be explained by bottom-up cascades, or if they should be attributed to other external causes. Species that disappeared from the community were more vulnerable, overall, to bottom-up cascades than were species that survived. The position of extinct species in the network - having few or no predators - also suggests they might have been particularly vulnerable to a new predator. These results provide quantitative evidence that trophic cascades and naivety to predators could have contributed to the megafauna extinction event in Sahul. Frequency modulated timer regulates mammalian hibernation Mammalian hibernators decrease basal metabolism and body temperature (Tb) to minimize energy expenditure in harsh seasons. During hibernation, Tb drops to low temperature (<10 {degrees}C) and remains constant for days, known as deep torpor in small mammalian hibernators. Spontaneous interbout arousals interrupt torpor bouts, when Tb recovers to euthermic state ~37 {degrees}C. Torpor-interbout arousal event repeats during several months of hibernation. However, little is known about mechanisms governing Tb fluctuation across torpor-interbout arousal cycles during hibernation. Recent improvement in data-logging techniques enables us to monitor Tb for more than hundred days with high precision, opening up new avenues for quantitative analysis to address the principle governing Tb fluctuation. Here, we analyzed Tb fluctuation across torpor-interbout arousal cycle of Syrian hamster, which can hibernate in response to chronic cold and short photoperiod under a laboratory condition, using generalized harmonic analysis and discovered a model with frequency modulation quantitatively reproducing Tb fluctuation. This analysis also identified an unexpectedly longer period of 120-430 days as the period that modulates another period of several days, generating Tb fluctuation for Syrian hamster. We propose that concerted action of two endogenous periods governs torpor-interbout arousal cycles during hibernation. Deep learning-based quantification of arbuscular mycorrhizal fungi in plant roots Soil fungi establish mutualistic interactions with the roots of most vascular land plants. Arbuscular mycorrhizal (AM) fungi are among the most extensively characterised mycobionts to date. Current approaches to quantifying the extent of root colonisation and the abundance of hyphal structures in mutant roots rely on staining and human scoring involving simple, yet repetitive tasks prone to variations between experimenters.

We developed AMFinder which allows for automatic computer vision-based identification and quantification of AM fungal colonisation and intraradical hyphal structures on ink-stained root images using convolutional neural networks.

AMFinder delivered high-confidence predictions on image datasets of roots of multiple plant hosts (Nicotiana benthamiana, Medicago truncatula, Lotus japonicus, Oryza sativa) and captured the altered colonisation in ram1-1, str and smax1 mutants. A streamlined protocol for sample preparation and imaging allowed us to quantify mycobionts from the genera Rhizophagus, Claroideoglomus, Rhizoglomus and Funneliformis via flatbed scanning or digital microscopy including dynamic increases in colonisation in whole root systems over time.

AMFinder adapts to a wide array of experimental conditions. It enables accurate, reproducible analyses of plant root systems and will support better documentation of AM fungal colonisation analyses. AMFinder can be accessed here: https://github.com/SchornacklabSLCU/amfinder.git Reliance of neuronal gene expression on cohesin scales with chromatin loop length Cohesin and CTCF are major drivers of 3D genome organization, but their role in neurons is still emerging. Here we show a prominent role for cohesin in the expression of genes that facilitate neuronal maturation and homeostasis. Unexpectedly, we observed two major classes of activity-regulated genes with distinct reliance on cohesin in primary cortical neurons. Immediate early genes remained fully inducible by KCl and BDNF, and short-range enhancer-promoter contacts at the Immediate early gene Fos formed robustly in the absence of cohesin. In contrast, cohesin was required for full expression of a subset of secondary response genes characterised by long-range chromatin contacts. Cohesin-dependence of constitutive neuronal genes with key functions in synaptic transmission and neurotransmitter signaling also scaled with chromatin loop length. Our data demonstrate that key genes required for the maturation and activation of primary cortical neurons depend cohesin for their full expression, and that the degree to which these genes rely on cohesin scales with the genomic distance traversed by their chromatin contacts. Fungicide resistance characterised across seven modes of action in a Botrytis cinerea isolated from Australian vineyards BACKGROUNDBotrytis bunch rot, caused by Botrytis cinerea, is an economically important disease of grapes in Australia and across grape growing regions worldwide. Control of this disease relies on canopy management and the application of fungicides. Fungicide application can lead to the selection of resistant B. cinerea populations, which has an adverse effect on management of the disease. Characterising the distribution and severity of resistant B. cinerea populations is needed to inform resistance management strategies.

RESULTSIn this study, 725 isolates were sampled from 75 Australian vineyards during 2013 - 2016 and were screened against seven fungicides with different modes of action (MOAs). The resistance frequencies for azoxystrobin, boscalid, fenhexamid, fludioxonil, iprodione, pyrimethanil and tebuconazole were 5, 2.8, 2.1, 6.2, 11.6, 7.7 and 2.9% respectively. Nearly half of the resistant isolates (43.8%) were resistant to more than one of the fungicides tested. The frequency of vineyards with at least one isolate simultaneously resistant to 1, 2, 3, 4 or 5 fungicides was 19.5, 7.8, 6.5, 10.4 and 2.6%. Resistance was associated with previously published genotypes in CytB (G143A), SdhB (H272R/Y), Erg27 (F412S), Mrr1 (D354Y), Bos1 (I365S, N373S + Q369P, I365S + D757N) and Pos5 (V273I, P319A, L412F/V). Novel genotypes were also described in Mrr1 (S611N, D616G) Pos5 (V273L) and Cyp51 (P347S). Expression analysis was used to characterise fludioxonil resistant isolates exhibiting overexpression (6.3-9.6-fold) of the ABC transporter gene AtrB (MDR1 phenotype).

CONCLUSIONResistance frequencies were lower when compared to most previously published surveys of B. cinerea resistance in grape and other crops. Nevertheless, continued monitoring of critical MOAs used in Australian vineyards is recommended. The M1 and pre-M1 segments contribute differently to ion selectivity in ASICs and ENaCs The ability to discriminate between different ionic species, termed ion selectivity, is a key feature of ion channels and forms the basis for their physiological function. Members of the degenerin/epithelial sodium channel (DEG/ENaC) superfamily of trimeric ion channels are typically sodium selective, but to a surprisingly variable degree. While acid-sensing ion channels (ASICs) are weakly sodium selective (sodium:potassium around 10:1), ENaCs show a remarkably high preference for sodium over potassium (>500:1). The most obvious explanation for this discrepancy may be expected to originate from differences in the pore-lining second transmembrane segment (M2). However, these show a relatively high degree of sequence conservation between ASICs and ENaCs and previous functional and structural studies could not unequivocally establish that differences in M2 alone can account for the disparate degrees of ion selectivity. By contrast, surprisingly little is known about the contributions of the first transmembrane segment (M1) and the preceding pre-M1 region. In this study, we use conventional and non-canonical amino acid-based mutagenesis in combination with a variety of electrophysiological approaches to show that the pre-M1 and M1 regions of mASIC1a channels are major determinants of ion selectivity. Mutational investigations of the corresponding regions in hENaC show that they contribute less to ion selectivity, despite affecting ion conductance. In conclusion, our work supports the notion that the remarkably different degrees of sodium selectivity in ASICs and ENaCs are achieved through different mechanisms. The results further highlight how M1 and pre-M1 are likely to differentially affect pore structure in these related channels. Genetic Impairment of Succinate Metabolism Disrupts Bioenergetic Sensing in Adrenal Neuroendocrine Cancer Metabolic dysfunction mutations can impair energy sensing and cause cancer. Loss of function of mitochondrial TCA cycle enzyme, succinate dehydrogenase B (SDHB) results in various forms of cancer typified by pheochromocytoma (PC). Here we delineate a signaling cascade where the loss of SDHB induces the Warburg effect in PC tumors, triggers dysregulation of Ca2+ homeostasis, and aberrantly activates calpain and the protein kinase Cdk5, through conversion of its cofactor from p35 to p25. Consequently, aberrant Cdk5 initiates a cascade of phospho- signaling where GSK3 inhibition inactivates energy sensing by AMP-kinase through dephosphorylation of the AMP-kinase {gamma} subunit, PRKAG2. Overexpression of p25-GFP in mouse adrenal chromaffin cells also elicits this phosphorylation signaling and causes PC tumor formation. A novel Cdk5 inhibitor, MRT3-007, reversed this phospho-cascade, invoking an anti- Warburg effect, cell cycle arrest, and senescence-like phenotype. This therapeutic approach halted tumor progression in vivo. Thus, we reveal an important novel mechanistic feature of metabolic sensing and demonstrate that its dysregulation underlies tumor progression in PC and likely other cancers.

HighlightsO_LILoss of SDHB function in pheochromocytoma causes Ca2+ dysregulation, calpain activation, and aberrant activation of the protein kinase Cdk5.
C_LIO_LIHyperactive Cdk5 deregulates a GSK3/PRKAG2/AMPK signaling cascade.
C_LIO_LIp25 overexpression and consequent aberrant Cdk5 activity in chromaffin cells causes pheochromocytoma.
C_LIO_LIInhibition of Cdk5 activates the PRKAG2/AMPK/p53 signaling to rescue cell senescence and block PC tumor progression.
C_LI

Graphical Abstract

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org.highwire.dtl.DTLVardef@16f0a48org.highwire.dtl.DTLVardef@10c1c7aorg.highwire.dtl.DTLVardef@21b100org.highwire.dtl.DTLVardef@1eb8674_HPS_FORMAT_FIGEXP  M_FIG C_FIG A network approach to genetic circuit designs As genetic circuits become more sophisticated, the size and complexity of data about their designs increases. This data captured goes beyond monolithic genetic sequences and towards circuit modularity and functional details, which are beneficial for analyzing circuit performance and establishing design automation techniques. However, the accessibility, visualisation and usability of design data (and metadata) have received relatively little attention to date. Here, we present a method to turn circuit designs into networks and showcase its potential to enhance the utility of design data. Since networks are dynamic structures, initial graphs can be interactively shaped into sub-networks of relevant information based on requirements such as abstraction, hierarchy and protein interactions. Additionally, several visual changes can be applied, such as colouring or clustering nodes based on types (e.g., genes or promoters), resulting in easier comprehension from a user perspective. This approach allows circuit designs to be coupled to other networks, such as metabolic pathways or implementation protocols captured in graph-like formats. Therefore, we advocate using networks to structure, access and improve synthetic biology information. Exploring functional protein covariation across single cells using nPOP Many biological processes, such as the cell division cycle, are reflected in protein covariation across single cells. This covariation can be quantified and interpreted by single-cell proteomics with sufficiently high throughput and accuracy. Toward this goal, we developed the nano-ProteOmic sample Preparation (nPOP) method for single-cell proteomics. nPOP uses piezo acoustic dispensing to isolate individual cells in 300 picoliter volumes and performs all subsequent preparation steps in small droplets on a fluorocarbon-coated slide. This design enables simultaneous sample preparation of thousands of single cells, including lysing, digesting, and labeling individual cells in volumes below 20 nl. We used nPOP to prepare 1,888 single cells and 128 negative controls in a single batch. Their analysis enabled quantifying the covariation between thousands of proteins and cell-cycle protein markers. Many protein sets covaried with the cell cycle similarly across all cell types and states, reflecting cell-type independent cell cycle functions. However, the cell cycle covariation of other protein sets differed markedly between cell types, even within subpopulation of melanoma cells expressing markers for drug-resistance priming. The cells expressing these markers accumulated in the G1 phase of the cell cycle and exhibited different covariation of enzymes catabolizing glucose. These results demonstrate that protein covariation across single cells may reveal functionally concerted biological differences between closely related cell states.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=59 SRC="FIGDIR/small/441211v3_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@1e23b1org.highwire.dtl.DTLVardef@b93faeorg.highwire.dtl.DTLVardef@3c00forg.highwire.dtl.DTLVardef@362b41_HPS_FORMAT_FIGEXP  M_FIG C_FIG Membrane insertion mechanism of the caveolae coat protein Cavin1 Caveolae are small plasma membrane invaginations, important for control of membrane tension, signaling cascades and lipid sorting. The caveolae coat protein Cavin1 is essential for shaping such high curvature membrane structures. Yet, a mechanistic understanding of how Cavin1 assembles at the membrane interface is lacking. Here, we used model membranes combined with biophysical dissection and computational modelling to show that Cavin1 inserts into membranes. We establish that initial PI(4,5)P2-dependent membrane adsorption of the trimeric helical region 1 (HR1) of Cavin1 mediates the subsequent partial separation and membrane insertion of the individual helices. Insertion kinetics of the HR1 is further enhanced by the presence of flanking negatively charged disordered regions, which was found important for the co-assembly of Cavin1 with Caveolin1 in living cells. We propose that this intricate mechanism potentiates membrane curvature generation and facilitates dynamic rounds of assembly and disassembly of Cavin1 at the membrane.

Significance statementCaveolae are cholesterol enriched membrane invaginations coupled to severe muscle and lipid disorders. Their formation is dependent on assembly of the protein Cavin1 at the lipid membrane interface driving membrane curvature. In this work, we dissect the mechanism for how Cavin1 binds and inserts into membranes using a combination of biochemical and biophysical characterization as well as computational modelling. The proposed model for membrane assembly potentiates dynamic switching between shielded and exposed hydrophobic helices used for membrane insertion and clarifies how Cavin1 can drive membrane curvature and the formation of caveolae. Outer hair cell electro-mechanical power conversion Outer hair cells are the cellular motors in the mammalian inner ear responsible for sensitive high-frequency hearing. Motor function requires expression of the protein prestin in the OHC lateral membrane and mechano-electrical transduction in the apical hair bundle. In the present report, electrical power consumption and mechanical power output are determined using previously published voltage clamp data from isolated OHCs and membrane patches. Results reveal that power output by the prestin-motor complex peaks at a best frequency much higher than implied by the low-pass character of nonlinear capacitance, and much higher than the whole-cell resistive-capacitive corner frequency. High frequency power output is enabled by a -90{degrees} shift in the phase of prestin-dependent electrical charge displacement, manifested electrically as emergence of imaginary-valued nonlinear capacitance. The initial response of females towards congeneric males matches the propensity to hybridise in Ophthalmotilapia. AO_SCPLOWBSTRACTC_SCPLOWCichlid radiations often harbour closely related species with overlapping niches and distribution ranges. Such species sometimes hybridise in nature, which raises the question how can they coexist. This also holds for the Tanganyika mouthbrooders Ophthalmotilapia ventralis and O. nasuta. Earlier studies found indications of asymmetrical hybridisation with females of O. ventralis accepting males of O. nasuta, but not the other way around. We hypothesised that this was due to differences in the capacity for species recognition. Given the higher propensity of O. ventralis females towards hybridisation, we expect a reduced ability for species recognition in O. ventralis females, compared to O. nasuta females. We staged two experiments, one focusing on 22 female O. nasuta and one on 21 female O. ventralis. These fish were placed in one half of a tank and briefly exposed to a conspecific or a heterospecific male, a conspecific female, or nothing (control). Female response was evaluated by scoring six tracking parameters and by noting the occurrence of ten discrete behaviours before and during the encounter. Females always responded to the presence of another fish by approaching it. Remarkably, for both O. nasuta and O. ventralis, we did not find a different response between encounters with conspecific males and females. However, in agreement with our hypothesis, O. nasuta females behaved differently towards conspecific or heterospecific males, whereas O. ventralis females did not. When presented with a heterospecific male, O. nasuta females performed a lower number of  ram behaviours. Additionally, they never displayed the  flee behaviour, a component of the species mating repertoire that was seen in all but one of the presentations with a conspecific male. Our findings show that differences in species recognition at first encounter predict to a large degree the outcome of the mating process, even in the absence of mating behaviour.