title: Clostridium difficile Toxin B activates the NLRP3 inflammasome in human macrophages, demonstrating a novel regulatory mechanism for the Pyrin inflammasome

abstract: Pyrin is a cytosolic immune sensor that forms an inflammasome when bacterial virulence factors inhibit RhoA, triggering the release of inflammatory cytokines, including IL-1{beta}. Gain of function mutations in the MEFV gene encoding Pyrin cause auto-inflammatory disorders, such as familial Mediterranean fever (FMF) and Pyrin associated auto-inflammation with Neutrophilic Dermatosis (PAAND). To precisely define the role of Pyrin in detecting pathogen virulence factors in relevant human immune cells, we investigated how the Pyrin inflammasome response was initiated and regulated in monocyte-derived macrophages (hMDM) compared to human monocytes. Unlike monocytes and murine macrophages, we determined that hMDM failed to activate Pyrin in response to known Pyrin activators Clostridioides difficile (C. difficile) toxins A or B (TcdA or TcdB). In contrast, TcdB activated the NLRP3 inflammasome in hMDM. Notably, we ascertained that the Pyrin inflammasome response could be re-enabled in hMDM by prolonged priming with either LPS or type I or II interferons, and required an increase in Pyrin expression. These data demonstrate an unexpected redundancy in detecting these toxins by inflammasome sensors.


title: SAFA facilitates chromatin opening of immune genes through interacting with nascent antiviral RNAs

abstract: Regulation of chromatin accessibility determines the transcription activities of genes, which endow the host with function-specific gene expression patterns. It remains unclear how chromatin accessibility is specifically directed, particularly, during host defense against viral infection. We previously reported that the nuclear matrix protein SAFA surveils viral RNA and regulates antiviral immune genes expression. However, how SAFA regulates the expression and what determines the specificity of antiviral immune genes remains unknown. Here, we identified that the depletion of SAFA specifically decreased the chromatin accessibility, activation and expression of virus induced genes in a genome-wide scale after VSV infection. SAFA exclusively bound with antiviral related RNAs, which mediated the specific opening of the according chromatin and robust transcription of these genes. Knockdown of these associated RNAs dampened the accessibility of corresponding genes in an extranuclear signaling pathway dependent manner. Moreover, VSV infection cleaved SAFA protein at the C-terminus which deprived its RNA binding ability for immune evasion. Thus, our results demonstrated that SAFA and the interacting RNA products during viral infection collaborate and remodel chromatin accessibility to facilitate antiviral innate immune response.

category: immunology


title: UPF3A and UPF3B are redundant and modular activators of nonsense-mediated mRNA decay in human cells

abstract: The paralogous human proteins UPF3A and UPF3B are involved in recognizing mRNAs targeted by nonsense-mediated mRNA decay (NMD). While UPF3B has been demonstrated to support NMD, contradicting reports describe UPF3A either as an NMD activator or inhibitor. Here, we present a comprehensive functional analysis of UPF3A and UPF3B in human cells using combinatory experimental approaches. Overexpression or knockout of UPF3A as well as knockout of UPF3B did not detectably change global NMD activity. In contrast, the co-depletion of UPF3A and UPF3B resulted in a marked NMD inhibition and a transcriptome-wide upregulation of NMD substrates, demonstrating a functional redundancy between both NMD factors. Although current models assume that UPF3 bridges NMD-activating exon-junction complexes (EJC) to the NMD factor UPF2, UPF3B exhibited only slightly impaired NMD activity in rescue experiments when UPF2 or EJC binding was impaired. Further rescue experiments revealed partially redundant functions of UPF3B domains in supporting NMD, involving both UPF2 and EJC interaction sites and the central region of UPF3. Collectively, UPF3A and UPF3B serve as fault-tolerant NMD activators in human cells.

category: molecular biology


title: High-throughput functional analysis of CFTR and other apically localized channels in iPSC derived intestinal organoids

abstract: Induced Pluripotent Stem Cells (iPSCs) can be differentiated into epithelial organoids that recapitulate the relevant context for CFTR and enable testing of therapies targeting Cystic Fibrosis (CF)-causing mutant proteins. However, to date, CF-iPSC-derived organoids have only been used to study pharmacological modulation of mutant CFTR channel activity and not the activity of other disease relevant membrane protein constituents. In the current work, we describe a high-throughput, fluorescence-based assay of CFTR channel activity in iPSC-derived intestinal organoids and describe how this method can be adapted to study other apical membrane proteins. In these proof-of-concept studies, we show how this fluorescence-based assay of apical membrane potential can be employed to study CFTR and ENaC channels and an electrogenic acid transporter in the same iPSC-derived intestinal tissue. This multiparameter phenotypic platform promises to expand CF therapy discovery to include strategies to target multiple determinants of epithelial fluid transport.


title: Concerted dynamics between conserved histidines in the N-extein region regulates Mycobacterium tuberculosis SufB intein cleavage

abstract: Inteins are auto-processing domains that implement a multi-step biochemical reaction termed protein splicing, marked by cleavage and formation of peptide bonds. They excise from a precursor protein, generating a functional protein via covalent bonding of flanking exteins. We report the kinetic study of splicing and cleavage reaction in [Fe-S] cluster assembly protein SufB from Mycobacterium tuberculosis. Although it follows a canonical intein splicing pathway, distinct features are added by extein residues present in the active site. Sequence analysis identified two conserved histidines in the N-extein region; His-5 and His-38. Kinetic analyses of His-5Ala and His-38Ala SufB mutants exhibited significant reductions in splicing and cleavage rates relative to the SufB wild-type precursor protein. Structural analysis and molecular dynamics simulations suggested that Mtu SufB displays a unique mechanism where two remote histidines work concurrently to facilitate N-terminal cleavage reaction. His-38 is stabilized by the solvent-exposed His-5, and can impact N-S acyl shift by direct interaction with the catalytic Cys1. Development of inteins as biotechnological tools or as pathogen specific novel antimicrobial targets requires a more complete understanding of such unexpected roles of conserved extein residues in protein splicing.


title: SufB intein splicing in Mycobacterium tuberculosis is influenced by two remote conserved N-extein Histidines


title: Rapid speciation and karyotype evolution in Orthoptera

abstract: To test the hypothesis that high speciation rate in groups is coupled with high rate of karyotype evolution but also that younger groups having a higher rate of karyotypic diversity, I estimated rates of speciation and rates of karyotype evolution in 1,177 species belonging to 26 families in the insect order Orthoptera. Rates of karyotype evolution were estimated using the diploid number and the number of chromosome arms (fundamental number) from published karyotypes of Orthoptera. Rates of speciation were quantified considering the number of species examined karyotypically in each family, the most recent common ancestor of each family and the information about extinction rate. The rate of speciation was strongly correlated with rate of karyotype evolution and the average rates of speciation was nearly ~177 times higher than the background rate estimated for Orthoptera based on acoustic communication using phylogenomic data, as well as 8.4 and 35.6 times higher than the estimated speciation rate in vertebrates and bivalve mollusks respectively, indicating that Orthoptera has evolved very fast at chromosomal level. The findings supported the hypothesis of a high speciation rate in lineages with high rate of chromosomal evolution but there were not evidences that younger groups tended to have higher rate of karyotypic diversity. Furthermore, rates of karyotype evolution most closely fitted the punctuational evolutionary model indicating the existence of long periods of stasis of karyotype change with most karyotype change occurring quickly over short evolutionary times. I discussed genetic drift, divergent selection and meiotic drive as potential biological mechanisms to explain karyotype evolution allowing or impeding for the fixation of chromosomal rearrangements and in turn speciation in orthopterans lineages.

category: evolutionary biology


title: Genome-wide Disease Screening in Early Human Embryos with Primary Template-Directed Amplification

abstract: Current preimplantation genetic testing (PGT) enables the selection of embryos based on fetal aneuploidy or the presence a small number of preselected disease-associated variants. Here we present a new approach that takes advantage of the improved genome coverage and uniformity of primary template-directed amplification (PTA) to call most early embryo genetic variants accurately and reproducibly from a preimplantation biopsy. With this approach, we identified clonal and mosaic chromosomal aneuploidy, de novo mitochondrial variants, and variants predicted to cause mendelian and non-mendelian diseases. In addition, we utilized the genome-wide information to compute polygenic risk scores for common diseases. Although numerous computational, interpretive, and ethical challenges remain, this approach establishes the technical feasibility of screening for and preventing numerous debilitating inherited diseases.

category: bioengineering


title: Oncogenic RABL6A promotes NF1-associated MPNST progression in vivo

abstract:
BackgroundMalignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas that display complex molecular and genetic alterations. Powerful tumor suppressors CDKN2A and TP53 are commonly disrupted in these lesions along with NF1, a gene that encodes a negative regulator of Ras. Many additional factors have been implicated in MPNST pathogenesis. A greater understanding of critical drivers of the disease is needed to guide more informed targeted therapies for patients. RABL6A is a newly identified driver of MPNST cell survival and proliferation whose in vivo role in the disease is unknown.

MethodsUsing CRISPR-Cas9 targeting of Nf1+Cdkn2a or Nf1+Tp53 in the sciatic nerve to form de novo MPNSTs, we investigated the biological significance of RABL6A in MPNST development. Molecular evaluation of terminal tumors (western blot, qRT-PCR, immunohistochemistry) yielded several insights.

ResultsMice lacking Rabl6 displayed slower tumor growth and extended survival relative to wildtype animals in both genetic contexts. YAP oncogenic activity was selectively downregulated in RABL6A-null, Nf1+Cdkn2a lesions but not in RABL6A-null, Nf1+Tp53 tumors. Regardless of genetic context, loss of RABL6A caused upregulation of the CDK inhibitor, p27 in tumors. Paradoxically, both models displayed elevated Myc protein expression and Ki67 staining in terminal tumors lacking RABL6A.

ConclusionsThese findings demonstrate RABL6A is required for optimal tumor progression of NF1 mutant MPNSTs in vivo in both Cdkn2a and p53 inactivated settings. However, sustained RABL6A loss may provide selective pressure for molecular alterations, such as Myc upregulation, that ultimately promote an unwanted, hyper-proliferative tumor phenotype akin to drug resistant lesions.

Importance of the StudyMPNSTs are aggressive, deadly, and challenging to treat tumors due to location around nerves and high mutational burden. Many factors implicated in MPNST genesis have yet to be fully tested for biological significance in disease formation. We establish a critical physiological role for a new oncoprotein, RABL6A, in promoting NF1-associated MPNST progression. We identify novel RABL6A-regulated pathways that likely contribute to tumor growth, specifically YAP and Myc signaling, and found that sustained RABL6A loss eventually yields more proliferative tumors. We liken RABL6A deficient tumors to those being treated with therapies targeting RABL6A effectors, such as CDKs. Therefore, those lesions should provide a powerful platform to uncover key mediators of drug resistance. Our data suggest oncogenic YAP and Myc could be such mediators of resistance. This study provides a novel system to examine one of the most pressing clinical challenges, drug resistant tumor growth and relapse, in cancer therapy.

category: cancer biology


title: Development of GPC2-directed chimeric antigen receptors using mRNA for pediatric brain tumors

abstract:
Pediatric brain tumors are the leading cause of cancer death in children with an urgent need for innovative therapies. Here we show that the cell surface oncoprotein glypican 2 (GPC2) is highly expressed on multiple lethal pediatric brain tumors, including medulloblastomas, embryonal tumors with multi-layered rosettes, other CNS embryonal tumors, as well as definable subsets of highly malignant gliomas. To target GPC2 on these pediatric brain tumors with adoptive cellular therapies and mitigate potential inflammatory neurotoxicity, we developed four mRNA chimeric antigen receptor (CAR) T cell constructs using the highly GPC2-specific fully human D3 single chain variable fragment. First, we validated and prioritized these CARs using in vitro cytotoxicity and T cell degranulation assays with GPC2-expressing neuroblastoma cells. Next, we expanded the testing of the two most potent GPC2-directed CAR constructs prioritized from these studies to GPC2-expressing medulloblastoma and high-grade glioma cell lines, showing significant GPC2-specific cell death in multiple models. Finally, locoregional delivery bi-weekly of two to four million GPC2-directed mRNA CAR T cells induced significant and sustained tumor regression in two orthotopic medulloblastoma models, and significantly prolonged survival in an aggressive orthotopic thalamic diffuse midline glioma model. No GPC2-directed CAR T cell related neurologic or systemic toxicity was observed. Taken together, these data show that GPC2 is a highly differentially expressed cell surface protein on multiple malignant pediatric brain tumors that can be targeted safely with local delivery of mRNA CAR T cells.

One Sentence SummaryGlypican 2 is expressed on the surface of multiple pediatric brain tumors and can be successfully targeted with mRNA chimeric antigen receptor T cells.


title: Single-cell profiling of the human primary motor cortex in ALS and FTLD

abstract: Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two devastating and fatal neurodegenerative conditions. While distinct, they share many clinical, genetic, and pathological characteristics1, and both show selective vulnerability of layer 5b extratelencephalic-projecting cortical populations, including Betz cells in ALS2,3 and von Economo neurons (VENs) in FTLD4,5. Here, we report the first high resolution single-cell atlas of the human primary motor cortex (MCX) and its transcriptional alterations in ALS and FTLD across ~380,000 nuclei from 64 individuals, including 17 control samples and 47 sporadic and C9orf72-associated ALS and FTLD patient samples. We identify 46 transcriptionally distinct cellular subtypes including two Betz-cell subtypes, and we observe a previously unappreciated molecular similarity between Betz cells and VENs of the prefrontal cortex (PFC) and frontal insula. Many of the dysregulated genes and pathways are shared across excitatory neurons, including stress response, ribosome function, oxidative phosphorylation, synaptic vesicle cycle, endoplasmic reticulum protein processing, and autophagy. Betz cells and SCN4B+ long-range projecting L3/L5 cells are the most transcriptionally affected in both ALS and FTLD. Lastly, we find that the VEN/Betz cell-enriched transcription factor, POU3F1, has altered subcellular localization, co-localizes with TDP-43 aggregates, and may represent a cell type-specific vulnerability factor in the Betz cells of ALS and FTLD patient tissues.

category: neuroscience


title: Bcl-3 suppresses Th9 differentiation by regulating glutamine utilization

abstract:
Bcl-3 is an atypical member of the I{kappa}B protein family that plays important and diverse roles in both innate and adaptive immunity, including Th17-dependent autoimmunity models in mice. When naive mouse splenic CD4+ T cells were cultured under Th17 conditions in vitro, we unexpectedly found that the most highly differentially expressed gene between wild type and Bcl-3-deficient (KO) Th17 cells encoded the cytokine IL-9. We therefore investigated the role of Bcl-3 in Th9 cell differentiation. When naive CD4+ T cells were cultured under Th9-polarizing conditions in vitro, the extent of Th9 differentiation observed in wild type cells was increased in Bcl-3 KO cells and conversely was decreased in cells overexpressing Bcl-3. The suppressive effect of Bcl-3 on Th9 differentiation was cell-autonomous, and NF-{kappa}B inhibitors abolished increased Th9 differentiation in Bcl-3 KO cells. Consistent with this, in the Th9 transfer model of OVA-induced allergic airway inflammation, mice receiving Bcl-3 KO cells had greater immune cell infiltration in the lung than mice receiving wild type cells.

Mechanistically, unsupervised transcriptomic analysis revealed differentially expressed genes in KO cells, including the glutamine transporter Slc1a5, which was downregulated. The functional significance of this was suggested by the ability of increasing concentrations of glutamine in the media to reduce the difference in Th9 differentiation between WT and KO cells. Our results suggest a novel role for Bcl-3 as a negative regulator of Th9 differentiation, in part by limiting glutamine accessibility through downregulation of Slc1a5.


title: Endosomal proteins NPC1 and NPC2 at African swine fever virus entry/fusion

abstract:
African swine fever virus (ASFV) infectious cycle starts with the viral adsorption and entry into the host cell. The virus is internalized via clathrin/dynamin mediated endocytosis and macropinocytosis. As several other viruses, ASF virion is then internalized and incorporated into the endocytic pathway. Endosomal maturation entails luminal acidification and the lowering of pH acting on the multi-layered virion structure dissolves the outer capsid. Upon decapsidation, the inner viral membrane is exposed to interact with the limiting membrane of the late endosome for fusion. Egress from endosome is related to cholesterol efflux, but it remains an intriguing process albeit essential for infection, specifically for the viral nucleic acid exit to the cytoplasm for replication. ASFV proteins E248R and E199L, with structural homology to the VACV proteins of the fusion complex, seem to have similar functions in ASFV. A direct interaction between these ASFV proteins with the cholesterol transporter protein NPC1 (Niemann-Pick C type 1) was observed, which was also shared by the E248R homologous protein L1R of VACV. Binding occurs between the transmembrane domain of E248R with the loop C of NPC1 at the same domain than EBOV binding site. These interactions suggest that these ASFV proteins are crucial for membrane fusion. CRISPR NPC1 KO Vero cells lacking NPC1 protein that were resistant to EBOV, reduced ASFV infection levels significantly. Reductions on ASFV infectivity and replication in NPC1 KO cells were accompanied by lesser viral factories of smaller size and lacking the typical cohesive morphology between endosomes and viral proteins. We observed a compensatory effect in NPC1 KO cells, elevating NPC2 levels while silencing NPC2 in Vero cells with shRNA, also reduced ASFV infection. Our findings pave the way to understand the role of these proteins at the membrane viral fusion step for several viruses.

Author SummaryAfrican swine fever virus (ASFV) causes a deadly disease of pigs and wild boars that was endemic in Africa but have extended over the last years to Europe, Asia and Oceania with high socioeconomic impact. ASFV enters the cell by endocytosis and has adapted to the endosomal conditions to acquire infectivity. Viral infectivity is dependent on cholesterol traffic at the endosomes, especially at the fusion step. Fusion of the internal viral membrane with the endosomal membrane is required for the exit of the viral DNA to the cytoplasm to start replication. ASF virion internal membrane proteins E248R and E199L were found to bind the Niemann Pick C1 (NPC1) receptor at the endosome. These proteins are highly conserved among ASFV isolates and resemble proteins of the VACV entry/fusion complex. The function of NPC1 is to regulate the efflux of dietary cholesterol efflux from the endosome to the endoplasmic reticulum, which appears to be necessary for viral fusion. NPC1 knockout cells by CRISPR reduced infection affecting infectivity and early replication. Also, removing the associated endosomal protein NPC2, further declined infectivity. These results show the relevance of NPC1 receptor in the viral infection actually shared by unrelated important viral families.

category: microbiology


title: KSTAR: An algorithm to predict patient-specific kinase activities from phosphoproteomic data

abstract: Kinase inhibitors are one of the largest classes of FDA-approved drugs and are major targets in oncology. Although kinase inhibitors have played an important role in improving cancer outcomes, major challenges still exist, including the development of resistance and failure to respond to treatments. Improvements for tumor profiling of kinase activity would be an important step in improving treatment outcomes and identifying effective kinase targets. Here, we present a graph- and statistics-based algorithm, called KSTAR, which harnesses the phosphoproteomic profiling of human cells and tissues by predicting kinase activity profiles from the observed phosphorylation of kinase substrates. The algorithm is based on the hypothesis that the more active a kinase is, the more of its substrates will be observed in a phosphoproteomic experiment. This method is error- and bias-aware in its approach, overcoming challenges presented by the variability of phosphoproteomic pipelines, limited information about kinase-substrate relationships, and limitations of global kinase-substrate predictions, such as training set bias and high overlap between predicted kinase networks. We demonstrate that the predicted kinase activities: 1) reproduce physiologically-relevant expectations and generates novel hypotheses within cell-specific experiments, 2) improve the ability to compare phosphoproteomic samples on the same tissues from different labs, and 3) identify tissue-specific kinase profiles. Global benchmarking and comparison to other algorithms demonstrates that KSTAR is particularly superior for predicting tyrosine kinase activities and, given its focus on utilizing more of the available phosphoproteomic data, significantly less sensitive to study bias. Finally, we apply the approach to complex human tissue biopsies in breast cancer, where we find that KSTAR activity predictions complement current clinical standards for identifying HER2-status - KSTAR can identify clinical false positives, patients who will fail to respond to inhibitor therapy, and clinically defined HER2-negative patients that might benefit from HER2-targeted therapy. KSTAR will be useful for both basic biological understanding of signaling networks and for improving clinical outcomes through improved clinical trial design, identification of new and/or combination therapies, and for identifying the failure to respond to targeted kinase therapies.

category: systems biology


title: Spatially resolved transcriptomics reveals unique gene signatures associated with human temporal cortical architecture and Alzheimer's pathology

abstract: Alzheimers disease (AD) is pathologically characterized by amyloid beta (A{beta}) plaques, neurofibrillary tangles (tau aggregates), and alterations in microglia, astrocytes and oligodendrocytes. The mesial temporal lobe is a vulnerable brain region in early AD; however, little is known about the transcriptome-scale gene expression in this region and its relation to AD pathology. Here we use the 10x Genomics Visium platform in combination with co-immunofluorescence staining of AD-associated pathological markers to define the spatial topography of gene expression in the middle temporal gyrus (MTG) from both early AD and age- and gender-matched control cases. We identify unique marker genes for six cortical layers and the adjacent white matter as well as gene expression patterns and alterations that showcase unique gene signatures and pathways associated with a range of AD pathology. Also, gene co-expression analyses of differentially expressed genes (DEGs) between AD and controls reveal four unique gene modules, which significantly change their co-expression patterns in the presence of variations of AD pathology. Furthermore, we validate the changes of key representative DEGs that are associated with AD pathology in neurons, microglia, astrocytes and oligodendrocytes using single-molecule fluorescent in situ hybridization. In summary, we provide a rich resource for the spatial transcriptomic profile of the human MTG, which will contribute to our understanding of the complex architecture and AD pathology of this vulnerable brain region.


title: Mapping the integration of sensory information across fingers in human sensorimotor cortex

abstract: The integration of somatosensory signals across fingers is essential for dexterous object manipulation. Previous experiments suggest that this integration occurs in neural populations in the primary somatosensory cortex (S1). However, the integration process has not been fully characterized, as previous studies have mainly used two-finger stimulation paradigms. Here, we addressed this gap by stimulating all 31 single- and multi-finger combinations. We measured population-wide activity patterns evoked during finger stimulation in human S1 and primary motor cortex (M1) using 7T functional magnetic resonance imaging (fMRI) in female and male participants. Using multivariate fMRI analyses, we found clear evidence of unique non-linear interactions between fingers. In Brodmann area (BA) 3b, interactions predominantly occurred between pairs of neighbouring fingers. In BA 2, however, we found equally strong interactions between spatially distant fingers, as well as interactions between finger triplets and quadruplets. We additionally observed strong interactions in the hand area of M1. In both M1 and S1, these non-linear interactions did not reflect a general suppression of overall activity, suggesting instead that the interactions we observed reflect rich, non-linear integration of sensory inputs from the fingers. We suggest that this non-linear finger integration allows for a highly flexible mapping from finger sensory inputs to motor responses that facilitates dexterous object manipulation.


title: Characterization of the radiation desiccation response regulon of the radioresistant bacterium Deinococcus radiodurans by integrative genomic analyses.

abstract:
Numerous genes are overexpressed in the radioresistant bacterium Deinococcus radiodurans after exposure to radiation or prolonged desiccation. The DdrO and IrrE proteins play a major role in regulating the expression of approximately predicted twenty of these genes. The transcriptional repressor DdrO blocks the expression of these genes under normal growth conditions. After exposure to genotoxic agents, the IrrE metalloprotease cleaves DdrO and relieves gene repression. Bioinformatic analyzes showed that this mechanism seems to be conserved in several species of Deinococcus, but many questions remain as such the number of genes regulated by DdrO. Here, by RNA-seq and CHiP-seq assays performed at a genome-wide scale coupled with bioinformatic analyses, we show that, the DdrO regulon in D. radiodurans includes many other genes than those previously described. These results thus pave the way to better understand the radioresistance mechanisms encoded by this bacterium.

Author SummaryThe main response pathway to genotoxic conditions in the radioresistant bacterium Deinococcus radiodurans is regulated by the constitutively expressed metalloprotease IrrE that cleaves the transcriptional repressor DdrO, leading to the expression of the genes repressed by DdrO. One of the major goals to better understand how pathways involved in radioresistance are coordinated into this fascinating bacterium is to highlight genes regulated by DdrO. In this study, we mapped in vivo the DdrO regulon in D. radiodurans by using two genome-scale approaches, ChIP-seq and RNA-seq analyses, coupled with bioinformatic analyses. As homologs of these two proteins are also found in many other bacteria, these results also pave the way to compare the stress-induced responses mediated by this couple of proteins in diverse bacteria.

category: genomics


title: Involvement of sensory neuron-TRPV4 in acute and chronic itch behaviors

abstract: Itch, particularly chronic itch, negatively impacts patients physical, social, and psychological well-being, leading to deterioration in their quality of life. Limited understanding of itch mechanisms hinders the development of effective antipruritic treatments.TRPV4, a multimodally activated nonselective cation channel, has been detected in sensory neurons of dorsal root and trigeminal ganglion (DRG, TG) and skin cells (e.g. keratinocytes, mast cells, and macrophages). Recent evidence from experimental and clinical relevant studies has implicated that TRPV4 in skin cells plays an important role in both acute and chronic itch. In contrast, little is known whether TRPV4 in sensory neurons directly contributes to scratching behaviors. Here we used sensory neuron-Trpv4 conditional knockout (cKO) mice to address this question. Our results showed that TRPV4 in sensory neurons contributes to scratching behavior evoked by histaminergic (histamine and 48/80) and partial histaminergic (5-HT), but not non-histaminergic (SLIGRL and CQ) pruritogens. Moreover, we observed that TRPV4 in sensory neurons is required for dry skin, but not allergic contact dermatitis, -associated chronic itch. These findings suggest that neuronal-TRPV4 might be specific for some forms of acute and chronic itch.


title: Spatial heterogeneity in forest carbon storage affects priorities for reforestation

abstract: Reforestation is an important strategy for nature-based climate solutions and identifying carbon storage potential of different locations is critical to its success. Applying average carbon values from forest inventories ignores the spatial heterogeneity in forest carbon and the effects of forest edges on carbon storage degradation. Here we show how spatially-explicit, predictive carbon modeling, that leverages satellite, social and biogeophysical datasets, can be used to identify more efficient restoration opportunities for climate mitigation than area-based carbon stock averages. Accounting for regeneration of forest edges, in addition to reforestation, boosts estimates of potential carbon gains by more than 20%. The total potential carbon gain that could be achieved through reforestation at the level indicated by the Bonn Challenge (350Mha) is 51 Gt CO2-eq, but the "missing carbon" in our current forests accounts for 64.6 Gt CO2-eq globally; the greatest potential carbon gains are found in areas of high fragmentation.

category: ecology


title: Impairment Of Aversive Episodic Memories During COVID-19 Pandemic: The Impact Of Emotional Context On Memory Processes

abstract: The threatening context of the COVID-19 pandemic provided a unique setting to study the effects of negative psychological symptoms on memory processes. Episodic memory is an essential function of the human being related to the ability to store and remember experiences and anticipate possible events in the future. Studying this function in this context is crucial to help understand what effects the pandemic will have on the formation of episodic memories. To study this, the formation of episodic memories was evaluated by free recall, recognition, and episode order tasks for an aversive and neutral content. The results indicated that aversive episodic memory is impaired both in the free recall task and in the recognition task. Even the beneficial effect that emotional memory usually has for the episodic order was undermined as there were no differences between the neutral and aversive condition. The present work adds to the evidence that indicates that the level of activation does not modify memory processes in a linear way, which also depends on the type of evocation that people are asked and the characteristics of the content to be encoded.

category: animal behavior and cognition


title: In silico analysis and molecular investigation of the nuclear shuttle protein-interacting kinase 1 (NIK1) in six Solanaceous species

abstract: The first layer of innate immunity in plants is initiated through the perception of microbe-associated molecular patterns (MAMPs) or damage-associated molecular patterns (DAMPs) by pattern recognition receptors (PRRs). MAMP/DAMP perception initiates downstream defense responses, a process which ultimately leads to pattern triggered immunity (PTI). In Arabidopsis, the nuclear shuttle protein-interacting kinase 1 (NIK1), among other PRRs, is one of the most important central components of PTI signaling and kinase signaling cascade, since it is involved in the plant antiviral response against geminiviruses. Despite the characterization of the structure and function of the NIK1 receptors made by some groups, studies related to NIK1 importance in the current gene-editing era are missing. By simple in silico analysis, in this study we investigated the NIK1 homologues from six Solanaceous plant species including: tomato (Solanum lycopersicum), potato (Solanum tuberosum), Solanum pennellii, eggplant (Solanum melongena), pepper (Capsicum annum), and Nicotiana benthamiana. The phylogenetic analyses of different NIK1 proteins from Arabidopsis and six Solanaceous plants revealed nine different clades. As expected, we found that these NIK1 orthologs have similar genomic structures suggesting a similar function. We could identify that SotubNIK1, SolyNIK1, SopenNIK1, CANIK1, NibenNIK1, SmeNIK1 have the highest sequence homology with AtNIK1. Additionally, the conserved protein kinase domain (PKD) that is present in NIK1 from Arabidopsis thaliana was bioinformatically analyzed and found in other species. As this highly conserved NIK1 region is present in several crops of economic importance, its potential is highlighted as a possible target site for gene editing, to develop crops tolerant to geminiviral infections.

category: bioinformatics


title: Yuel: Compound-Protein Interaction Prediction with High Generalizability

abstract: Virtual drug screening has the potential to revolutionize the stagnant drug discovery field due to its low cost and fast speed. Predicting binding affinities between small molecules and the protein target is at the core of computational drug screening. Deep learning-based approaches have recently been adapted to predict binding affinities and claim to achieve high prediction accuracy in their tests, however, we show that current approaches are not reliable for virtual drug screening due to the lack of generalizability, i.e. the ability to predict interactions between unknown proteins and unknown small molecules. To address this shortcoming, we develop a compound-protein interaction predictor, Yuel. Upon comprehensive tests on various datasets, we find that out of all the deep-learning approaches surveyed, only Yuel can predict interactions between unknown compounds and unknown proteins. Additionally, Yuel can also be utilized to identify compound atoms and proteins residues that are involved in the binding.


title: Straintables: An application that extracts sequences fromgenome assemblies and generates dissimilarity matrices

abstract:
Background and ObjectivesThe dissimilarity matrix (DM) is an important component of phylogenetic analysis, and many software packages exist to build and show DMs. However, as the common input for this type of software are sequences in FASTA file format, the process of extracting and aligning each set of sequences to produce a big number of matrices can be laborious. Additionally, existing software do not facilitate the comparison of clusters of similarity across several DMs built for the same group of individuals, using different genomic regions. To address our requirements of such a tool, we designed Straintables to extract specific genomic region sequences from a group of intraspecies genomic assemblies, using extracted sequences to build dissimilarity matrices.

MethodsA Python module with executable scripts was developed for a study on genetic diversity across strains of Toxoplasma gondii, being a general purpose system for DM calculation and visualization for preliminary phylogenetic studies. For automatic region sequence extraction from genomic assemblies we assembled a system that designs virtual primers using reference sequences located at genomic annotations, then matches those primers on genome files by using regex patterns. Extracted sequences are then aligned using Clustal Omega and compared to generate matrices.

ResultsUsing this software saves the user from manual preparation and alignment of the sequences, a process that can be laborious when a large number of assemblies or regions are involved. The automatic sequence extraction process can be checked against BLAST results using extracted sequence as queries, where correct results were observed for same-species pools for various organisms. The package also contains a matrix visualization tool focused on cluster visualization, capable of drawing matrices into image files with custom settings, and features methods of reordering matrices to facilitate the comparison of clustering patterns across two or more matrices.

ConclusionStraintables may replace and extend the functionality of existing matrix-oriented phylogenetic software, featuring automatic region extraction from genomic assemblies and enhanced matrix visualization capabilities emphasizing cluster identification. This module is open source, available at GitHub (https://github.com/Gab0/straintables) under a MIT license and also as a PIPY package.

HighlightsSimple in-silico protocol for generation, visualization and comparison of dissimilarity matrices.

Accurate automatic sequence extraction from multiple genomic assemblies by using virtual primers built from reference sequences in an annotation file.

Draws matrices as images, with enhanced cluster visualization and customized options.

Supports reordering of matrix indices to better visualize clustering pattern conservation across multiple regions.


title: Efficient gradient boosting for prognostic biomarker discovery

abstract:
MotivationGradient boosting decision tree (GBDT) is a powerful ensemble machine learning method that has the potential to accelerate biomarker discovery from high-dimensional molecular data. Recent algorithmic advances, such as Extreme Gradient Boosting (XGB) and Light Gradient Boosting (LGB), have rendered the GBDT training more efficient, scalable and accurate. These modern techniques, however, have not yet been widely adopted in biomarkers discovery based on patient survival data, which are key clinical outcomes or endpoints in cancer studies.

ResultsIn this paper, we present a new R package Xsurv as an integrated solution which applies two modern GBDT training framework namely, XGB and LGB, for the modeling of censored survival outcomes. Based on a comprehensive set of simulations, we benchmark the new approaches against traditional methods including the stepwise Cox regression model and the original gradient boosting function implemented in the package gbm. We also demonstrate the application of Xsurv in analyzing a melanoma methylation dataset. Together, these results suggest that Xsurv is a useful and computationally viable tool for screening a large number of prognostic candidate biomarkers, which may facilitate cancer translational and clinical research.

AvailabilityXsurv is freely available as an R package at: https://github.com/topycyao/Xsurv

Contactxuefeng.wang@moffitt.org

Supplementary informationSupplementary data are available at Bioinformatics online.


title: Approximation of the age distribution of cancer incidence using a mutational model

abstract:
The approximation of the age distributions of cancers was carried out using a complex mutational model presented in our work [1]. Datasets from the American National Cancer Institute (SEER program) were used.

We approximated the datasets for the age distributions of lung, stomach, colon and breast cancer in women; cancer of the lung, stomach, colon and prostate in men.

The average number of mutations (required for cancer formation) averaged over the four types of cancer is 5 mutations per cell in women and in men. The average (over the four types of cancer) mutation rate is estimated as 1.0 {middle dot} 10 -1 mutations per year per cell for women and 3.8 {middle dot} 10 -1 mutations per year per cell for men.

This article is a continuation of work [1].


title: Complete depletion of Arabidopsis linker histones impairs the correlations among chromatin compartmentalization, DNA methylation and gene expression

abstract: In eukaryotic cells, linker histone H1 anchors in and out ends of nucleosome DNA to promote chromatin to fold into the 30 nm fiber. However, if H1 plays a role in coordinating the three-dimensional (3D) chromatin architecture, DNA methylation, and transcriptional regulation is not clear. We engineered H1 knockout mutants in Arabidopsis thaliana which shows pleiotropic phenotypes. Using High-throughput Chromosome Conformation Capture (Hi-C), we found that H1 complete depletion dampens inter- and intra-chromosomal interactions, as well as intra- and inter-chromosomal arm interactions. MNase accessibility assays followed by sequencing (MNase-seq) showed that the nucleosome density decreases in centromeric regions and increases in chromosome arms. In contrast, DNA methylation level in CHG and CHH contexts increases in centromeric regions and decreases in chromosome arms as revealed by whole genome bisulfite sequencing (WGBS) in h1 mutant. Importantly, the functional link between DNA methylation and gene transcription is defected, and the extensive switches between chromatin compartment A and B are uncoupled from genome-wide DNA methylation and most of gene transcriptions upon H1 depletion. These results suggested that linker histone H1 works as linkers among chromatin compartmentalization, DNA methylation and transcription.

category: plant biology


title: Phloem transport limitation in Huanglongbing affected sweet orange is dependent on phloem-limited bacteria and callose

abstract: Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas), is a phloem-limited disease which disrupts citrus production in affected areas. In HLB-affected plants, phloem sieve plate pores accumulate callose, and leaf carbohydrate export is reduced. However, whether HLB causes a reduction in carbohydrate phloem translocation speed, and the quantitative relationships among callose, CLas population, and phloem translocation are still unknown. In this work, a procedure was developed to concurrently measure sugar transport, callose deposition, and relative pathogen population at different locations throughout the stem. Increasing quantities of CLas genetic material were positively correlated with quantity and density of callose deposits, and negatively correlated with phloem translocation speed. Callose deposit quantity was site- and rootstock dependent, and were negatively correlated with phloem translocation speed, suggesting a localized relationship. Remarkably, callose accumulation and phloem translocation disruption in the scion was dependent on rootstock genotype. Regression results suggested that the interaction of Ct values and number of phloem callose depositions, but not their size or density, explained the effects on translocation speed. Sucrose, starch, and sink 14C label allocation data support the interpretation of a transport pathway limitation by CLas infection. This work shows that the interaction of local accumulation of callose and CLas affect phloem transport. Further, the extent of this accumulation is attenuated by the rootstock and provides important information about the disease mechanism of phloem-inhabiting bacteria. Together, these results constitute the first example of a demonstrated transport limitation of phloem function by a microbial infection.


title: Arabidopsis Topless-related 1 mitigates physiological damage and growth penalties of induced immunity

abstract: Transcriptional corepressors of the Topless family are important regulators of plant hormone and immunity signaling. The lack of a genome-wide profile of their chromatin associations limits understanding of transcriptional regulation in plant immune responses. Chromatin immunoprecipitation with sequencing (ChIP-seq) was performed on GFP-tagged Topless-related 1 (TPR1) expressed in Arabidopsis thaliana lines with and without constitutive immunity dependent on Enhanced Disease Susceptibility 1 (EDS1). RNA-seq profiling of pathogen-infected tpl/tpr mutants and assessments of growth and physiological parameters were employed to determine TPL/TPR roles in transcriptional immunity and defense homeostasis. TPR1 bound to promoter regions of ~1,400 genes and ~10% of the detected binding required EDS1 immunity signaling. A tpr1 tpl tpr4 (t3) mutant displayed mildly enhanced defense-related transcriptional reprogramming upon bacterial infection but not increased bacterial resistance. Bacteria or pep1 phytocytokine-challenged t3 plants exhibited, respectively, photosystem II dysfunction and exacerbated root growth inhibition. Transgenic expression of TPR1 restored the t3 physiological defects. We propose that TPR1 and TPL-family proteins function in Arabidopsis to reduce detrimental effects associated with activated transcriptional immunity.


title: A cell-sorting-based nano-scale pipeline for cell-type-specific proteomics in plants

abstract: Multicellular organisms such as plants contain different cell types with specialized functions. Analyzing the characteristics of each cell type reveals specific cell functions and enhances understanding of organization and function at the organismal level. Here we report a highly-sensitive and efficient cell type-specific multiomics pipeline, combining simplified flow cytometry-based cell sorting for fluorescent protoplasts and optimized nanoscale proteomics and metabolomics methods, which allow in-depth analysis of the proteomes and metabolomes of a particular cell type. Using this method, we quantitatively compared the proteomes and metabolomes of guard cells and mesophyll cells and revealed that the enrichment of signal transduction-related proteins enables guard cells to respond rapidly to various environmental stimuli. We uncovered a guard cell-specific kinase cascade whereby RAF15 and OST1 mediate ABA-induced stomatal closure. This pipeline can be applied to various cell types in plant or non-plant systems to learn how cells function in highly organized multicellular organisms.


title: A cell type-specific multiomics uncovers a guard cell-specific RAF15-SnRK2.6/OST1 kinase cascade


title: Stimulus-induced narrowband gamma oscillations are test-retest reliable in healthy elderly in human EEG

abstract:
Visual stimulus-induced narrowband gamma oscillations in electroencephalogram (EEG) recordings have been recently shown to be compromised in subjects with Mild Cognitive Impairment or Alzheimers Disease (AD), suggesting that gamma could be an inexpensive and easily accessible biomarker for early diagnosis of AD. However, to use gamma as a biomarker, its characteristics should remain consistent across multiple recordings, even when separated over long intervals. Previous magnetoencephalography studies in young subjects have reported that gamma power remains consistent over recordings separated by a few weeks to months. Here, we assessed the consistency of slow (20-35 Hz) and fast gamma (36-66 Hz) oscillations induced by static full-field gratings in male (N=20) and female (N=20) elderly subjects (>49 years) in EEG recordings separated by more than a year, and tested the consistency in the magnitude of gamma power, its temporal evolution and spectral profile. Gamma oscillations had distinct spectral and temporal characteristics across subjects, which remained consistent across recordings (average intraclass correlation, ICC of [~]0.7). Alpha oscillations (8-12 Hz) and steady-state-visually-evoked-potentials (SSVEPs) were also found to be reliable. We further tested how EEG features can be used to identify two recordings as belonging to the same versus different subjects and found high classifier performance (area under ROC curve of [~]0.89), with the temporal evolution of slow gamma and spectral profile emerging as the most informative features. These results suggest that EEG gamma oscillations are reliable across recordings and can be used as a clinical biomarker as well as a potential tool for subject identification.

Significance statementWe demonstrate the reliability of stimulus-induced gamma oscillations in elderly humans for the first time in EEG. Since gamma has recently been shown to be compromised in patients with Mild Cognitive Impairment or early Alzheimers Disease (AD), together these results mark the first steps towards an EEG based clinical biomarker for early diagnosis of AD. We observed high reliability in the power spectrum, gamma power and its temporal characteristics, within the test-retest period of one year. Alpha and steady-state-visually-evoked potential power were also found to be reliable. These spectral and temporal features could also be used to identify EEG recordings as belonging to the same versus different subjects with high performance, suggesting a potentially key role in subject identification also.


title: Neural activity tracking identity and confidence in social information

abstract: Humans learn about the environment either directly by interacting with it or indirectly by seeking information about it from social sources such as conspecifics. The degree of confidence in the information obtained through either route should determine the impact that it has on adapting and changing behaviour. We examined whether and how behavioural and neural computations differ during non-social learning as opposed to learning from social sources. Trial-wise confidence judgments about non-social and social information sources offered a window into this learning process. Despite matching exactly the statistical features of social and non-social conditions, confidence judgments were more accurate and less changeable when they were made about social as opposed to non-social information sources. In addition to subjective reports of confidence, differences were also apparent in the Bayesian estimates of participants subjective beliefs. Univariate activity in dorsomedial prefrontal cortex (dmPFC) and posterior temporo-parietal junction (pTPJ) more closely tracked confidence about social as opposed to non-social information sources. In addition, the multivariate patterns of activity in the same areas encoded identities of social information sources compared to non-social information sources.


title: GLOBAL EFFECTS OF A PD RISK-SNP AT THE ALPHA-SYNUCLEIN LOCUS

abstract: SUMMARYOne of the most significant Parkinsons disease (PD) risk variants, rs356182, is located at the PD-associated locus near the alpha-synuclein encoding gene, SNCA. SNCA-proximal variants, including rs356182, are thought to function in PD via allele-specific regulatory effects on SNCA expression. However, this interpretation discounts the complex activity of genetic enhancers and possible nonconical effects of alpha-synuclein. Here we investigate a novel risk mechanism for rs356182. We use CRISPR-Cas9 in LUHMES cells, a model for dopaminergic neurons, to generate precise hemizygous lesions at rs356182. The PD-protective (A/-), PD-risk (G/-), and WT (A/G) strains are differentiated into dopaminergic neurons then compared transcriptionally and morphologically. We observe effects not typically ascribed to SNCA; hundreds of differentially expressed genes associated with neuronal differentiation and axonogenesis. Together, the data implicate a risk mechanism for rs356182 in which the risk-allele (G) is associated with abnormal neuronal differentiation. We speculate the disease-relevant effect originates as a diminished population of DA neurons leading to the predisposition for PD later in life.


title: A billion synthetic 3D-antibody-antigen complexes enable unconstrained machine-learning formalized investigation of antibody specificity prediction

abstract:
Machine learning (ML) is a key technology to enable accurate prediction of antibody-antigen binding, a prerequisite for in silico vaccine and antibody design. Two orthogonal problems hinder the current application of ML to antibody-specificity prediction and the benchmarking thereof: (i) The lack of a unified formalized mapping of immunological antibody specificity prediction problems into ML notation and (ii) the unavailability of large-scale training datasets. Here, we developed the Absolut! software suite that allows the parameter-based unconstrained generation of synthetic lattice-based 3D-antibody-antigen binding structures with ground-truth access to conformational paratope, epitope, and affinity. We show that Absolut!-generated datasets recapitulate critical biological sequence and structural features that render antibody-antigen binding prediction challenging. To demonstrate the immediate, high-throughput, and large-scale applicability of Absolut!, we have created an online database of 1 billion antibody-antigen structures, the extension of which is only constrained by moderate computational resources. We translated immunological antibody specificity prediction problems into ML tasks and used our database to investigate paratope-epitope binding prediction accuracy as a function of structural information encoding, dataset size, and ML method, which is unfeasible with existing experimental data. Furthermore, we found that in silico investigated conditions, predicted to increase antibody specificity prediction accuracy, align with and extend conclusions drawn from experimental antibody-antigen structural data. In summary, the Absolut! framework enables the development and benchmarking of ML strategies for biotherapeutics discovery and design.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/451258v2_ufig1.gif" ALT="Figure 1">
View larger version (81K):
org.highwire.dtl.DTLVardef@14295d2org.highwire.dtl.DTLVardef@7a12bdorg.highwire.dtl.DTLVardef@647102org.highwire.dtl.DTLVardef@375112_HPS_FORMAT_FIGEXP  M_FIG O_FLOATNOGraphical abstractC_FLOATNO The software framework Absolut! enables (A,B) the generation of virtually arbitrarily large numbers of in silico 3D-antibody-antigen structures, (C,D) the formalization of antibody specificity as machine learning (ML) tasks as well as the exploration of ML strategies for paratope-epitope prediction.

C_FIG Highlights- Software framework Absolut! to generate an arbitrarily large number of in silico 3D-antibody-antigen structures
- Generation of one billion in silico antigen-antibody structures reflecting biological layers of complexity that make ML predictions challenging
- Immunological antibody specificity prediction problems formalized as machine learning tasks for which the in silico complexes are immediately usable as benchmark.
- Exploration of machine learning architectures for paratope-epitope interaction prediction accuracy as a function of neural network depth, dataset size, and sequence-structure encoding


title: One billion synthetic 3D-antibody-antigen complexes enable unconstrained machine-learning formalized investigation of antibody specificity prediction


title: Harmonic Passive Motion Paradigm

abstract: How humans robustly interact with external dynamics is not yet fully understood. This work presents a hierarchical architecture of semi-autonomous controllers that can control the redundant kinematics of the limbs during dynamic interaction, even with delays comparable to the nervous system. The postural optimisation is performed via a non-linear mapping of the system kineto-static properties, and it allows independent control of the end-effector trajectories and the arms stiffness. The proposed architecture is tested in a physical simulator in the absence of gravity, presence of gravity, and with gravity plus a viscous force field. The data indicate that the architecture can generalise motor strategies to different environmental conditions. The experiments also verify the existence of a deterministic solution to the task-separation principle. The architecture is also compatible with Optimal Feedback Control and the Passive Motion Paradigm. The existence of a deterministic mapping implies that this task could be encoded in neural networks capable of generalisation of motion strategies to affine tasks.


title: Impact of Fkbp5 x Early Life Adversity x Sex in Humanized Mice on Multidimensional Stress Responses and Circadian Rhythmicity

abstract: The cumulative load of genetic predisposition, early life adversity (ELA) and lifestyle shapes the prevalence of psychiatric disorders. Single nucleotide polymorphisms (SNPs) in the human FKBP5 gene were shown to modulate disease risk. To enable investigation of disease-related SNPs in behaviorally relevant context, we generated humanized mouse lines carrying either the risk (AT) or the resiliency (CG) allele of the rs1360780 locus and exposed litters of these mice to maternal separation. Behavioral and physiological aspects of their adult stress responsiveness displayed interactions of genotype, early life condition and sex. In humanized females carrying the CG-but not the AT-allele, ELA led to altered HPA-axis functioning, exploratory behavior and sociability. These changes correlated with differential expression of genes in the hypothalamus, where synaptic transmission, metabolism, and circadian entrainment pathways were deregulated. Our data suggest an integrative role of FKBP5 in shaping the sex-specific outcome of ELA in adulthood.


title: SARS-CoV-2 Neurotropism and Single Cell Responses in Brain Organoids Containing Innately Developing Microglia

abstract: Neuropsychiatric manifestations are common in both the acute and post-acute phase of SARS-CoV-2 infection, but the mechanisms of these effects are unknown. In a newly established brain organoid model with innately developing microglia, we demonstrate that SARS-CoV-2 infection causes an extensive cell death and loss of post-synaptic termini. Despite limited neurotropism and a decelerating viral replication, we observe a threefold increase in microglial engulfment of postsynaptic termini after SARS-CoV-2 exposure. We define the microglial responses to SARS-CoV-2 infection by single cell transcriptomic profiling and observe an upregulation of interferon-responsive genes as well as genes promoting migration and synaptic stripping. To a large extent, SARS-CoV-2 exposed microglia display a transcriptomic profile previously observed in neurodegenerative disorders characterized by early a synapse loss and an increased incident risk after a Covid-19 infection. Our results reveal that brain organoids infected with SARS-CoV-2 display disruption in circuit integrity via microglia-mediated synapse elimination and identifies a potential novel mechanism contributing to cognitive impairments in patients recovering from Covid-19.


title: SARS-CoV-2 promotes microglial synapse elimination in human brain organoids


title: Area-based conservation planning in Japan: protected area network effectiveness to the post-2020 Global Biodiversity Framework

abstract: To reframe the imperfect review processes of nation-scale actions on area-based conservation through protected area (PA) networks, we first created novel infrastructure to visualize nation-level biodiversity information in Japan. We then assessed the performance of the existing PA network relative to land exploitation pressure and evaluated conservation effectiveness of PA expansion for the post-2020 Global Biodiversity Framework. The Zonation algorithm was used to spatially prioritize conservation areas to minimize biodiversity loss and the extinction risk for 8,500 Japanese vascular plant and vertebrate species under constraints of the existing PA network and land use. The spatial pattern of the identified priority areas, which were considered candidate areas for expansion of the current PA network, was influenced by land-use types according to the mask layers of non-PAs, and low-, middle-, and high-ranked PAs. The current PA network reduced the aggregate extinction risk of multiple species by 36.6%. Indeed, the percentage of built-up areas in the existing PAs was in general smaller than that in the areas surrounding PAs. Notably, high-ranked PAs fully restrained built-up pressure (0.037% per 10 years), whereas low-ranked PAs in the national park and wild-life protection areas did not (1.845% per 10 years). Conservation effects were predicted to substantially improve by expansion of high-ranked (legally strict) PAs into remote non-PAs without population/socio-economic activities, or expansion of medium-ranked PAs into agriculture forestry satoyama and urban areas. A 30% land conservation target was predicted to decrease extinction risk by 74.1% when PA expansion was implemented across remote areas, satoyama, and urban areas; moreover, PA connectivity almost doubled compared with the existing PA network. In contrast, a conventional scenario showed that placing national parks in state-owned and non-populated areas would reduce extinction risk by only 4.0%. The conservation prioritization analyses demonstrated an effectiveness of using a comprehensive conservation approach that reconciles land-sparing protection and land-sharing conservation in other effective area-based conservation measures (OECM) in satoyama and urban green spaces. Our results revealed that complementary inclusion of various PAs interventions related to their governance and land-use planning plays a critical role in effectively preventing biodiversity loss and makes it more feasible to achieve ambitious conservation targets.


title: Topology and habitat assortativity drive neutral and adaptive diversification in spatial graphs

abstract:
Biodiversity results from differentiation mechanisms developing within biological populations. Such mechanisms are influenced by the properties of the landscape over which individuals interact, disperse and evolve. Notably, landscape connectivity and habitat heterogeneity constrain the movement and survival of individuals, thereby promoting differentiation through drift and local adaptation. Nevertheless, the complexity of landscape features can blur our understanding of how they drive differentiation. Here, we formulate a stochastic, eco-evolutionary model where individuals are structured over a graph that captures complex connectivity patterns and accounts for habitat heterogeneity. Individuals possess neutral and adaptive traits, whose divergence results in differentiation at the population level. The modelling framework enables an analytical underpinning of emerging macroscopic properties, which we complement with numerical simulations to investigate how the graph topology and the spatial habitat distribution affect differentiation. We show that in the absence of selection, graphs with high characteristic length and high heterogeneity in degree promote neutral differentiation. Habitat assortativity, a metric that captures habitat spatial autocorrelation in graphs, additionally drives differentiation patterns under habitat-dependent selection. While assortativity systematically amplifies adaptive differentiation, it can foster or depress neutral differentiation depending on the migration regime. By formalising the eco-evolutionary and spatial dynamics of biological populations in complex landscapes, our study establishes the link between landscape features and the emergence of diversification, contributing to a fundamental understanding of the origin of biodiversity gradients.

Significance statementIt is not clear how landscape connectivity and habitat heterogeneity influence differentiation in biological populations. To obtain a mechanistic understanding of underlying processes, we construct an individualbased model that accounts for eco-evolutionary and spatial dynamics over graphs. Individuals possess both neutral and adaptive traits, whose co-evolution results in differentiation at the population level. In agreement with empirical studies, we show that characteristic length, heterogeneity in degree and habitat assortativity drive differentiation. By using analytical tools that permit a macroscopic description of the dynamics, we further link differentiation patterns to the mechanisms that generate them. Our study provides support for a mechanistic understanding of how landscape features affect diversification.


title: Feeding habits and the influence of pellet diameter on the feeding responses of the flathead grey mullet (Mugil cephalus) in captivity

abstract: The feeding habits and effect of the diameter of pelleted feeds on the feeding responses of wild juvenile and adult flathead grey mullet (Mugil cephalus) in captivity were examined. Optimal pellet size for feeding was defined according to the behavioural responses and ingestion of pellets with different diameters (2, 4, 6, 8 mm) that were dropped into the tank in a random sequence. Larger pellets (6 and 8 mm) were more attractive (lower reaction time, high percentage of capture), but the small to medium-sized pellets (2 and 4 mm) were consumed the most. The optimal size was the 2- and 4- mm pellet diameter for juvenile individuals (365.50 {+/-} 36.90 g; 28.8 {+/-} 0.84 cm) and the 4-mm diameter pellet for adults (937.49 {+/-} 146.54 g; 40 {+/-} 1.12 cm). The preferred feeding area of adult mullet was also studied to estimate preference in relation to pellet characteristics such as floating or sink. Two pellet types, floating or sinking, were offered simultaneously in the water column: at the surface, mid-water column and bottom of the tank. The flathead grey mullet had a preference to feed in the mid-water column and the bottom of the tanks indicating that sinking or slow-sinking pellets would be the optimal feed type in relation to mullet feeding behaviour.


title: The smooth transition from many-legged to bipedal locomotion: Gradual ground force reduction and its impact on total ground reaction forces, body dynamics and gait transitions

abstract:
Most terrestrial animals move with a specific number of propulsive legs, which differs between clades. The reasons for these differences are often unknown and rarely queried, despite the underlying mechanisms being indispensable for understanding the evolution of multilegged locomotor systems in the animal kingdom and the development of swiftly moving robots. Moreover, when speeding up, a range of species change their number of propulsive legs. The reasons for this behaviour have proven equally elusive. In animals and robots, the number of propulsive legs also has a decisive impact on the movement dynamics of the centre of mass. Here, I use the leg force interference model to elucidate these issues by introducing gradually declining ground reaction forces in locomotor apparatuses with varying numbers of leg pairs in a first numeric approach dealing with these measures impact on locomotion dynamics. The effects caused by the examined changes in ground reaction forces and timing thereof follow a continuum. However, the transition from quadrupedal to a bipedal locomotor system deviates from those between multilegged systems with different numbers of leg pairs. Only in quadrupeds do reduced ground reaction forces beneath one leg pair result in increased reliability of vertical body oscillations and therefore increased energy efficiency and dynamic stability of locomotion.

Significance statementThe model grants access to the effects of gradual ground force reduction on total ground reaction forces, body dynamics and gait transitions.

category: biophysics


title: Dual inhibition of anti-apoptotic proteins BCL-XL and MCL-1 enhances cytotoxicity of Nasopharyngeal carcinoma cells.

abstract: One of the many strategies that cancer cells use to evade cell death is through upregulation of the BCL-2 anti-apoptotic proteins. Hence, these proteins have become attractive therapeutic targets. Given that different cell population rely on different anti-apoptotic proteins for survival, it is crucial to determine which proteins are important for NPC survival. Here we determined the survival requirements for the NPC cells using combination of CRISPR/Cas9 technique and pharmacological approaches. A human apoptosis RT2 Profiler PCR Array was first employed to profile the anti-apoptotic gene expressions in NPC cell lines HK-1 and C666-1. The HK-1 cells expressed all the anti-apoptotic genes (MCL-1, BFL-1, BCL-2, BCL-XL, and BCL-w). Similarly, the C666-1 cells expressed all the anti-apoptotic proteins except BFL-1 (undetectable level). Notably, both cell lines highly expressed MCL-1. Deletion of MCL-1 sensitized cells to A-1331852 suggesting that MCL-1 and BCL-XL may be important for NPC cell survival. Co-inhibition of MCL-1 and BCL-2 with MCL-1 selective inhibitor S63845 and BCL-2 selective inhibitor ABT-199 inhibited NPC cell proliferation but the effect on cell viability was more profound with co-inhibition of MCL-1 and BCL-XL with S63845 and A-1331852, implying that MCL-1 and BCL-XL are crucial for NPC cell survival. Furthermore, co-inhibition of MCL-1 and BCL-XL inhibited the growth and invasion of NPC spheroids. Deletion of BFL-1 sensitized cells to A-1331852 suggesting that BFL-1 may play a role in NPC cell survival. Taken together co-inhibition of BCL-XL and MCL-1/BFL-1 could be potential treatment strategies for NPC.


title: Virtual meetings promise to eliminate the geographical and administrative barriers and increase accessibility, diversity, and inclusivity

abstract: The COVID-19 pandemic brought a new set of unprecedented challenges not only for healthcare, education, and everyday jobs but also in terms of academic conferences. In this study, we investigate the effect of the broad adoption of virtual platforms for academic conferences as a response to COVID-19 restrictions. We show that virtual platforms enable higher participation from underrepresented minority groups, increased inclusion, and broader geographic distribution. We also discuss emerging challenges associated with the virtual conference format resulting in a decreased engagement of social activities, limited possibilities of cross-fertilization between participants, and reduced peer-to-peer interactions. Lastly, we conclude that a novel comprehensive approach needs to be adopted by the conference organizers to ensure increased accessibility, diversity, and inclusivity of post-pandemic conferences. Our findings provide evidence favoring a hybrid format for future conferences, marrying the strength of both in-person and virtual platforms.

category: scientific communication and education


title: Hippocampal Astrocytes Encode Reward Location

abstract: Astrocytic calcium dynamics have been implicated in the encoding of sensory information, and modulating them has been shown to impact behavior. However, real-time calcium activity of astrocytes in the hippocampus of awake mice has never been investigated. We used 2-photon microscopy to chronically image CA1 astrocytes as mice ran in familiar or novel virtual environments and obtained water rewards. We found that astrocytes exhibit persistent ramping activity towards the reward location in a familiar environment, but not in a novel one. Using linear decoders, we could precisely predict the location of the mouse in a familiar environment from astrocyte activity alone. We could not do the same in the novel environment, suggesting astrocyte spatial activity is experience dependent. This is the first indication that astrocytes can encode location in spatial contexts, thereby extending their known computational capabilities, and their role in cognitive functions.


title: Structural-functional connectivity mapping of the insular cortex system: A combined data-driven and meta-analytic topic mapping approach

abstract: In this study, we examined structural and functional profiles of the insular cortex and mapped associations with well-described functional networks (FNs) throughout the brain using diffusion tensor imaging (DTI) and resting-state functional connectivity (RSFC) data. We used a data-driven method to independently estimate the structural-functional connectivity of the insular cortex. Data were obtained from the Human Connectome Project comprising 108 adult participants. Overall, we observed moderate to high associations between the structural and functional mapping scores of three different insular subregions: the posterior insula (associated with the sensorimotor network: RSFC, DTI = 50% and 72%, respectively), dorsal anterior insula (associated with ventral attention: RSFC, DTI = 83% and 83%, respectively), and ventral anterior insula (associated with the frontoparietal: RSFC, DTI = 42% and 89%, respectively). Further analyses utilized meta-analytic decoding maps to demonstrate specific cognitive and affective as well as gene expression profiles of the three subregions reflecting the core properties of the insular cortex. In summary, given the central role of the insular in the human brain, our results revealing correspondence between DTI and RSFC mappings provide a complementary approach and insight for clinical researchers to identify dysfunctional brain organization in various neurological disorders associated with insular pathology.


title: Structural-functional connectivity mapping of the insular cortex: A combined data-driven and meta-analytic topic mapping


title: Linking individual differences in cognitive control and perception: Meta-perception in older and younger adults

abstract: Visual perception is not only shaped by sensitivity, but also by confidence, i.e. the ability to estimate the accuracy of a visual decision. There is robust evidence that younger observers have access to a reliable measure of their own uncertainty when making visual decisions. This metacognitive ability might be challenged during aging due to increasing sensory noise and decreasing cognitive control resources. We investigated age effects on visual confidence using a confidence forced-choice paradigm. We determined discrimination thresholds for trials in which perceptual judgements were indicated as confident and for those in which they were declined as confident. Younger adults (19-38 years) showed significantly lower discrimination thresholds than older adults (60-78 years). In both age groups, perceptual performance was linked to confidence judgements, but overall results suggest reduced confidence efficiency in older adults. However, we observed substantial variability of confidence effects across all particpants. This variability was closely linked to individual differences in cognitive control capacities, i.e. executive function. Our findings provide evidence for age-related differences in meta-perceptual efficiency that present a specific challenge to perceptual performance in old age. We propose that these age effects are primarily mediated by cognitive control resources, supporting their crucial role for metacognitive efficiency.


title: Crossmodal metaperception: Visual and tactile confidence share a common scale

abstract: Perceptual decisions are typically accompanied by a subjective sense of (un)certainty. There is robust evidence that observers have access to a reliable estimate of their own uncertainty and can judge the validity of their perceptual decisions. However, there is still a debate to what extent these meta-perceptual judgements underly a common mechanism that can monitor perceptual decisions across different sensory modalities. It has been suggested that perceptual confidence can be evaluated on an abstract scale that is not only task-independent but also modality-independent. We aimed to scrutinize these findings by measuring visual contrast and tactile vibration discrimination thresholds in a confidence forced-choice task. A total of 56 participants took part in our study. We determined thresholds for trials in which perceptual decisions were chosen as confident and for those that were declined as confident. Confidence comparisons were made between perceptual decisions either within the visual and tactile modality, respectively, or across both modalities. Furthermore, we assessed executive functions to explore a possible link between cognitive control and meta-perceptual capacities. We found that perceptual performance was a good predictor of confidence judgments and that the threshold modulation was similarly pronounced in both modalities. Most importantly, participants compared their perceptual confidence across visual and tactile decisions with the same precision as within the same modality. Cognitive control capacities were not related to meta-perceptual performance. In conclusion, our findings corroborate that perceptual uncertainty can be accessed on an abstract scale, allowing for confidence comparisons across sensory modalities.


title: Autoimmunity increases susceptibility to and mortality from sepsis

abstract: Our prior publication detailing how sepsis influences subsequent development of EAE presented a conceptual advance in understanding the post-sepsis chronic immunoparalysis state (Jensen et al., 2020). However, the reverse scenario (autoimmunity prior to sepsis) defines a high-risk patient population whose susceptibility to sepsis remains poorly defined. Herein, we present a retrospective analysis of University of Iowa Hospital and Clinics patients demonstrating increased sepsis incidence among MS, relative to non-MS, patients. To interrogate how autoimmune disease influences host susceptibility to sepsis well-established murine models of MS and sepsis, EAE and CLP, respectively, were utilized. EAE, relative to non-EAE, mice were highly susceptible to sepsis-induced mortality with elevated cytokine storms. These results were further recapitulated in LPS and S. pneumoniae sepsis models. This work highlights both the relevance of identifying highly susceptible patient populations and expands the growing body of literature that host immune status at the time of septic insult is a potent mortality determinant.


title: Calpains are required for efficient microtubule detyrosination

abstract:
Detyrosination is a major post-translational modification of microtubules (MT), which has significant impact on MT function in cell division, differentiation, growth, migration, polarity, and intracellular trafficking. Detyrosination of -tubulin occurs via the recently identified complex of vasohibin 1/2 (vash1/2) and small vasohibin binding protein (SVBP). However, there is still remaining detyrosinating activity in the absence of vash1/2/SVBP, and little is known about the regulation of detyrosination. Using cellular and cell-free assays we showed that the calcium-dependent proteases calpains 1 and 2 regulate MT detyrosination. We identified new calpain cleavage sites in the N-terminal disordered region of vash1 using in vitro proteolysis followed by mass spectrometry. However, this cleavage did not affect the detyrosination activity of vasohibin. In conclusion, the regulation of MT detyrosination by calpains occurs via another yet unknown tubulin carboxypeptidase. Importantly, calpains calcium dependency could allow a fine regulation of MT detyrosination. Thus, identifying the calpain-regulated pathway of MT detyrosination can be of major importance for several basic and clinical research and should be focused on in future studies.

Summary StatementThe conventional calpains 1 and 2 play an important role in the regulation of microtubule detyrosination in a vasohibin independent way. Thus, they possibly control another still unknown tubulin carboxypeptidase.

category: cell biology


title: H3K9me2 genome-wide distribution in the holocentric insect Spodoptera frugiperda (Lepidoptera: Noctuidae)

abstract:
BackgroundEukaryotic genomes are packaged by Histone proteins in a structure called chromatin. There are different chromatin types. Euchromatin is typically associated with decondensed, transcriptionally active regions and heterochromatin to more condensed regions of the chromosomes. Methylation of Lysine 9 of Histone H3 (H3K9me) is a conserved biochemical marker of heterochromatin. In many organisms, heterochromatin is usually localized at telomeric as well as pericentromeric regions but can also be found at interstitial chromosomal loci. This distribution may vary in different species depending on their general chromosomal organization. Holocentric species such as Spodoptera frugiperda (Lepidoptera: Noctuidae) possess dispersed centromeres instead of a monocentric one and thus no observable pericentromeric compartment. To identify the localization of heterochromatin in such species we performed ChIP-Seq experiments and analyzed the distribution of the heterochromatin marker H3K9me2 in the Sf9 cell line and whole 4th instar larvae (L4) in relation to RNA-Seq data.

ResultsIn both samples we measured an enrichment of H3K9me2 at the (sub) telomeres, rDNA loci, and satellite DNA sequences, which could represent dispersed centromeric regions. We also observed that density of H3K9me2 is positively correlated with transposable elements and protein-coding genes. But contrary to most model organisms, H3K9me2 density is not correlated with transcriptional repression.

ConclusionThis is the first genome-wide ChIP-Seq analysis conducted in S. frugiperda for H3K9me2. Compared to model organisms, this mark is found in expected chromosomal compartments such as rDNA and telomeres. However, it is also localized at numerous dispersed regions, instead of the well described large pericentromeric domains, indicating that H3K9me2 might not represent a classical heterochromatin marker in Lepidoptera.


title: Genome binning of viral entities from bulk metagenomics data

abstract: Despite the accelerating number of uncultivated virus sequences discovered in metagenomics and their apparent importance for health and disease, the human gut virome and its interactions with bacteria in the gastrointestinal are not well understood. In addition, a paucity of whole-virome datasets from subjects with gastrointestinal diseases is preventing a deeper understanding of the viromes role in disease and in gastrointestinal ecology as a whole. By combining a deep-learning based metagenomics binning algorithm with paired metagenome and metavirome datasets we developed the Phages from Metagenomics Binning (PHAMB) approach for binning thousands of viral genomes directly from bulk metagenomics data. Simultaneously our methodology enables clustering of viral genomes into accurate taxonomic viral populations. We applied this methodology on the Human Microbiome Project 2 (HMP2) cohort and recovered 6,077 HQ genomes from 1,024 viral populations and explored viral-host interactions. We show that binning can be advantageously applied to existing and future metagenomes to illuminate viral ecological dynamics with other microbiome constituents.


title: All driven by energy demand? Integrative comparison of metabolism of Enterococcus faecalis wildtype and a glutamine synthase mutant

abstract: Lactic acid bacteria (LAB) play a significant role in biotechnology, e.g. food industry, but also in human health. Many LAB genera have developed a multidrug resistance in the past few years, becoming a serious problem in controlling hospital germs all around the world. Enterococcus faecalis accounts for a large part of the human infections caused by LABs. Therefore, studying its adaptive metabolism under various environmental conditions is particularly important. In this study, we investigated the effect of glutamine auxotrophy ({Delta}glnA mutant) on metabolic and proteomic adaptations of E. faecalis in response to a changing pH in its environment. Changing pH values are part of its natural environment in the human body, but also play a role in food industry. We compared the results to those of the wildtype. Our integrative method, using a genome-scale metabolic model, constrained by metabolic and proteomic data allows us to understand the bigger picture of adaptation strategies in this bacterium. The study showed that energy demand is the decisive factor in adapting to a new environmental pH. The energy demand of the mutant was higher at all conditions. It has been reported that {Delta}glnA mutants of bacteria are energetically less effective. With the aid of our data and model we are able to explain this phenomenon as a consequence of a failure to regulate glutamine uptake and the costs for the import of glutamine and the export of ammonium. Methodologically, it became apparent that taking into account the non-specificity of amino acid transporters is important for reproducing metabolic changes with genome-scale models since it affects energy balance.


title: Nonstoichiometric balanced complexes: Implications on the effective deficiency of the underlying metabolic network

abstract: The deficiency of a (bio)chemical reaction network can be conceptually interpreted as a measure of its ability to support exotic dynamical behavior and/or multistationarity. The classical definition of deficiency relates to the capacity of a network to permit variations of the complex formation rate vector at steady state, irrespective of the network kinetics. However, the deficiency is by definition completely insensitive to the fine details of the directionality of reactions as well as bounds on reaction fluxes. While the classical definition of deficiency can be readily applied in the analysis of unconstrained, weakly reversible networks, it only provides an upper bound in the cases where relevant constraints on reaction fluxes are imposed. Here we propose the concept of effective deficiency, which provides a more accurate assessment of the networks capacity to permit steady state variations at the complex level for constrained networks of any reversibility patterns. The effective deficiency relies on the concept of nonstoichiometric balanced complexes, which we have already shown to be present in real-world biochemical networks operating under flux constraints. Our results demonstrate that the effective deficiency of real-world biochemical networks is smaller than the classical deficiency, indicating the effects of reaction directionality and flux bounds on the variation of the complex formation rate vector at steady state.


title: Patch-seq of mouse DRG neurons reveals candidate genes for specific mechanosensory functions

abstract: A variety of mechanosensory neurons are involved in touch, proprioception and pain. Many molecular components of the mechanotransduction machinery subserving these sensory modalities remain to be discovered. Here, we combined recordings of mechanosensitive (MS) currents in mechanosensory neurons with single cell RNA sequencing. In silico combined analysis with a large-scale dataset enables assigning each transcriptome to DRG genetic clusters. Correlation of current signatures with single-cell transcriptomes provides a one-to-one correspondence between mechanoelectric properties and transcriptomically-defined neuronal populations. Moreover, gene expression differential comparison provides a set of candidate genes for mechanotransduction complexes. Piezo2 was expectedly found to be enriched in rapidly adapting MS current-expressing neurons, whereas Tmem120a and Tmem150c, thought to mediate slow-type MS currents, were uniformly expressed in all neuron subtypes, irrespective of their mechano-phenotype. Further knock-down experiments disqualified them as mediating DRG MS currents. This dataset constitutes an open-resource to explore further the cell-type-specific determinants of mechanosensory properties.


title: Shallow neural networks trained to detect collisions recover features of visual loom-selective neurons

abstract: Animals have evolved sophisticated visual circuits to solve a vital inference problem: detecting whether or not a visual signal corresponds to an object on a collision course. Such events are detected by specific circuits sensitive to visual looming, or objects increasing in size. Various computational models have been developed for these circuits, but how the collision-detection inference problem itself shapes the computational structures of these circuits remains unknown. Here, inspired by the distinctive structures of LPLC2 neurons in the visual system of Drosophila, we build an anatomically-constrained shallow neural network model and train it to identify visual signals that correspond to impending collisions. Surprisingly, the optimization arrives at two distinct, opposing solutions, only one of which matches the actual dendritic weighting of LPLC2 neurons. The LPLC2-like solutions are favored when a population of units is trained on the task, but not when units are trained in isolation. The trained model reproduces experimentally observed LPLC2 neuron responses for many stimuli, and reproduces canonical tuning of loom sensitive neurons, even though the models are never trained on neural data. These results show that LPLC2 neuron properties and tuning are predicted by optimizing an anatomically-constrained neural network to detect impending collisions.


title: A DEEP LEARNING APPROACH TO ESTIMATING INITIAL CONDITIONS OF BRAIN NETWORK MODELS IN REFERENCE TO MEASURED FMRI DATA

abstract: 1.Brain Network Models (BNMs) are a family of dynamical systems that simulate whole brain activity using neural mass models to represent local activity in different brain regions that influence each other via a global structural network. Research has been interested in using these network models to explain measured whole brain activity measured via resting state functional magnetic resonance imaging (rs-fMRI). Properties computed over longer periods of simulated and measured data such as average functional connectivity (FC), have shown to be comparable with similar properties estimated from measured rs-fMRI data. While this shows that these network models have similar properties over the dynamical landscape, it is unclear how well simulated trajectories compare with empirical trajectories on a timepoint-by-timepoint basis. Previous studies have shown that BNMs are able to produce relevant features at shorter timescales, but analysis of short-term trajectories or transient dynamics as defined by synchronized predictions from BNM made at the same timescale as the collected data has not yet been conducted. Relevant neural processes exist in the time frame of measurements and are often used in task fMRI studies to understand neural responses to behavioral cues. Therefore, it is important to investigate how much of these dynamics are captured by our current brain simulations. To test the nature of BNMs short term trajectories against observed data, we utilize a deep learning technique known as Neural ODE that based on an observed sequence of fMRI measurements, estimates the initial conditions such that the BNMs simulation is synchronized to produce the closest trajectory relative to the observed data. We test to see if the parameterization of a specific well studied BNM, the Firing Rate Model, calculated by maximizing its accuracy in reproducing observed short term trajectories matches with the parameterized model that produces the best average long-term metrics. Our results show that such an agreement between parameterization using long and short simulation analysis exists if also considering other factors such as the sensitivity in accuracy with relative to changes in structural connectivity. Therefore, we conclude that there is evidence that by solving for initial conditions, BNMs can be simulated in a meaningful way when comparing against measured data trajectories, although future studies are necessary to establish how BNM activity relate to behavioral variables or to faster neural processes during this time period.


title: Gating mechanism of human N-type voltage-gated calcium channel

abstract: N-type voltage-gated calcium (CaV) channels mediate Ca2+ influx at the presynaptic terminals in response to action potential and play vital roles in synaptogenesis, neurotransmitter releasing, and nociceptive transmission. Here we elucidate a cryo-electron microscopy (cryo-EM) structure of the human CaV2.2 complex at resolution of 2.8 [A]. This complex structure reveals how the CaV2.2, {beta}1, and 2{delta}1 subunits are assembled. In our structure, the second voltage-sensing domain (VSD) is stabilized at a resting-state conformation, which is distinct from the other three VSDs of CaV2.2 as well as activated VSDs observed in previous structures of CaV channels. The structure also shows that the intracellular gate formed by S6 helices is closed, and a W-helix from the DII-III linker is determined to act as a blocking-ball that causes closed-state inactivation in CaV2.2. Collectively, our structure provides previously unseen structural insights into fundamental gating mechanisms of CaV channels.


title: Structure of the integrin receptor M2 headpiece in complex with a function-modulating nanobody

abstract: The integrin receptor M{beta}2 mediates phagocytosis of complement-opsonized objects, adhesion to the extracellular matrix and trans-endothelial migration of leukocytes. Here we present the first atomic structure of the human M{beta}2 headpiece fragment in complex with the nanobody hCD11bNb1 determined at a resolution of 3.2 [A]. The receptor headpiece adopts the closed conformation expected to have low ligand affinity. The crystal structure advocates that in the R77H M variant associated with systemic lupus erythematosus, the modified allosteric coupling between ligand coupling and integrin outside-inside signalling is due to subtle conformational effects transmitted over 40 [A]. The nanobody binds to the I domain of the M subunit in an Mg2+ independent manner with low nanomolar affinity. Biochemical and biophysical experiments with purified proteins argue that the nanobody acts as a competitive inhibitor through steric hindrance exerted on the thioester domain of iC3b attempting to bind the M subunit. Surprisingly, the nanobody stimulates the interaction of cell-bound M{beta}2 with iC3b suggesting that it represents a novel high-affinity proteinaceous M{beta}2 specific agonist. We propose a model based on the conformational spectrum of the receptor to reconcile these conflicting observations regarding the functional consequences of hCD11bNb1 binding to M{beta}2. Furthermore, our data suggest that the iC3b-M{beta}2 complex may be more dynamic than predicted from the crystal structure of the core complex.


title: PlotXpress, a webtool for normalization and visualization of reporter expression data

abstract:
In molecular cell biology, reporter assays are frequently used to investigate gene expression levels. Reporter assays employ a gene that encodes a light-emitting protein, of which the luminescence is quantified as a proxy of gene expression. Commercial parties provide reporter assay kits that include protocols and specialized detection machinery. However, downstream analysis of the output data and their presentation are not standardized. We have developed plotXpress to fill this gap, providing a free, open-source platform for the semi-automated analysis and standardized visualisation of experimental gene reporter data. Users can upload raw luminescence data acquired from a reporter gene assay with an internal control. In plotXpress, the data is corrected for sample variation with the internal control and the average for each condition is calculated. When a reference condition is selected the fold change is calculated for all other conditions, based on the selected reference. The results are shown as dot plots with a statistical summary, which can be adjusted to create publication-grade plots without requiring coding skills. Altogether, plotXpress is an open-source, low-threshold, web-based tool, that promotes a standardized and reproducible analysis while providing an appealing visualization of reporter data. The webtool can be accessed at: https://huygens.science.uva.nl/PlotXpress/

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/451595v1_ufig1.gif" ALT="Figure 1">
View larger version (35K):
org.highwire.dtl.DTLVardef@1b499aborg.highwire.dtl.DTLVardef@1d4d375org.highwire.dtl.DTLVardef@186bac1org.highwire.dtl.DTLVardef@4ed7bc_HPS_FORMAT_FIGEXP  M_FIG C_FIG


title: LptB2FG is an ABC transporter with Adenylate Kinase activity regulated by LptC/A recruitment

abstract: Lipopolysaccharide (LPS) is an essential glycolipid covering the surface of gram-negative bacteria. Its transport involves a dedicated 7 protein transporter system, the Lpt machinery, that physically spans the entire cell envelope. LptB2FG complex is an ABC transporter that hydrolyses Adenosine Triphosphate (ATP) to extract LPS from the inner membrane (IM). LptB2FG was extracted directly from IM with its original lipid environment by Styrene-Maleic acids polymers(SMA). SMA-LptB2FG in nanodiscs displays ATPase activity and a previously uncharacterized Adenylate Kinase (AK) activity. It catalyzes phosphotransfer between two ADP molecules to generate ATP and AMP. ATPase and AK activities of LptB2FG are both stimulated by the interaction on the periplasmic side with LptC and LptA partners and inhibited by the presence of LptC transmembrane helix. Isolated ATPase module (LptB) has weak AK activity in absence of LptF and LptG, and one mutation, that weakens affinity for ADP, has AK activity similar to that of fully assembled complex. LptB2FG is thus capable of producing ATP from ADP depending on the assembly of the Lpt bridge and the AK activity might be important to ensure efficient LPS transport in fully assembled Lpt system.

category: biochemistry