Context Free and Context-Dependent Conceptual Representation in the Brain How concepts are coded in the brain is a core issue in cognitive neuroscience. Studies have focused on how individual concepts are processed, but the way in which conceptual representation changes to suit the context is unclear. We parametrically manipulated the association strength between words, presented in pairs one word at a time using a slow event-related fMRI design. We combined representational similarity analysis and computational linguistics to probe the neurocomputational content of these trials. Individual word meaning was maintained in supramarginal gyrus (associated with verbal short-term memory) when items were judged to be unrelated, but not when a linking context was retrieved. Context-dependent meaning was instead represented in left lateral prefrontal gyrus (associated with controlled retrieval), angular gyrus and ventral temporal lobe (regions associated with integrative aspects of memory). Analyses of informational connectivity, examining the similarity of activation patterns across trials between sites, showed that control network regions had more similar multivariate responses across trials when association strength was weak, reflecting a common controlled retrieval state when the task required more unusual associations. These findings indicate that semantic control and representational sites amplify contextually-relevant meanings in trials judged to be related.
Mathematical Relationships between Spinal Motoneuron Properties Our understanding of the behaviour of spinal alpha-motoneurons (MNs) in mammals partly relies on our knowledge of the relationships between MN membrane properties, such as MN size, resistance, rheobase, capacitance, time constant, axonal conduction velocity and afterhyperpolarization period. Qualitative relations between some of these properties are often assumed based on scattered but converging evidence from experimental studies and review papers.

We reprocessed the data from 40 experimental studies in adult cat, rat and mouse MN preparations in vivo to empirically derive precise mathematical relationships between MN electrophysiological and anatomical properties. This validated mathematical framework, which demonstrates that the MN membrane properties are all related to each other, expands our knowledge in neural electrophysiology beyond available literature data. For example, it extends the Hennemans size principle by the association between MN size and all other MN properties.

The derived mathematical relationships provide a convenient tool for neuroscientists and experimenters to estimate non-measurable MN properties, to explore relationships between pairs of MN properties never concurrently observed in previous experiments, to support future experimental investigations, or to investigate inter-mammalian-species variations in MN membrane properties. Using this mathematical framework, which supports the classic description of a MN as a membrane equivalent electrical circuit, modelers can build profiles of inter-consistent MN-specific properties to scale advanced models of MNs, with consequences on our understanding of the neural control of movement.
CryoEM of endogenous mammalian V-ATPase interacting with the TLDc protein mEAK-7 V-ATPases are rotary proton pumps that serve as signaling hubs with numerous proposed binding partners in cells. We used cryoEM combined with exhaustive focused classification to detect endogenous proteins that associate with V-ATPase from porcine kidney. A super-stoichiometric copy of subunit C was found in ~3% of complexes, while an additional ~1.6% of complexes bound mEAK-7, a protein with proposed roles in dauer formation in nematodes and mTOR signaling in mammals. High-resolution cryoEM of porcine kidney V-ATPase with recombinant mEAK-7 shows that mEAK-7s TLDc domain interacts with V-ATPases stator while its C-terminal  helix binds V-ATPases rotor. This crosslink would be expected to inhibit rotary catalysis. However, unlike inhibition of yeast V-ATPase by the TLDc protein Oxr1p, exogenous mEAK-7 does not inhibit V-ATPase and mEAK-7 overexpression in cells does not alter lysosomal or phagosomal pH. Instead, cryoEM suggests that interaction of mEAK-7 with V-ATPase is disrupted by ATP-induced rotation of the rotor. Comparison of Oxr1p and mEAK-7 binding explains this difference. Together, these results show that differences in V-ATPase binding by TLDc domain-containing proteins can lead to effects ranging from strong inhibition to formation of labile interactions that are sensitive to the enzymes activity. A protease-activatable luminescent biosensor andreporter cell line for authentic SARS-CoV-2 infection Efforts to define serological correlates of protection against COVID-19 have been hampered by the lack of a simple, scalable, standardised assay for SARS-CoV-2 infection and antibody neutralisation. Plaque assays remain the gold standard, but are impractical for high-throughput screening. In this study, we show that expression of viral proteases may be used to quantitate infected cells. Our assays exploit the cleavage of specific oligopeptide linkers, leading to the activation of cell-based optical biosensors. First, we characterise these biosensors using recombinant SARS-CoV-2 proteases. Next, we confirm their ability to detect viral protease expression during replication of authentic virus. Finally, we generate reporter cells stably expressing an optimised luciferase-based biosensor, enabling viral infection to be measured within 24 h in a 96- or 384-well plate format, including variants of concern. We have therefore developed a luminescent SARS-CoV-2 reporter cell line, and demonstrated its utility for the relative quantitation of infectious virus and titration of neutralising antibodies.

Author summaryTechniques for measuring infection with SARS-CoV-2 in the laboratory are laborious and time-consuming, and different laboratories use different approaches. There is therefore no generally agreed way to quantitate neutralising antibodies against SARS-CoV-2, which block infection with the virus and protect people from COVID-19. In this study, we describe a new way to measure SARS-CoV-2 infection, which is much simpler and faster than existing methods. It relies on the production of a specific protease enzyme by the virus, which is able to cleave and activate an engineered protein biosensor in infected cells. This biosensor emits light in the presence of viral infection, and the amount of light released is used as a readout for the amount of infectious SARS-CoV-2 present. The signal is very sensitive, so the number of infected cells required is very small, and the method can be scaled-up to test many samples at once. In particular, we demonstrate how it can be used to detect different variants of SARS-CoV-2, and quantitate neutralising antibodies against these viruses. Conserved organ-specific microbial assemblages in different populations of a terrestrial crab Brachyuran crabs originated in the oceans and evolved specific morphological and physiological adaptations to live in freshwater, intertidal and even terrestrial habitats but the role of a selection mechanism involving symbiotic microorganisms long these colonization processes are not known. In this work we investigated the associated microbiota of three populations of a terrestrial brachyuran crab, Chiromantes haematocheir, to find evidence of a conserved crab-specific microbiome unrelated to the population of origin and dissimilar from environmental microbial assemblages. Bacterial 16S rRNA gene and fungal ITS sequences were obtained from selected crab organs and environmental matrices to profile microbial communities. In spite of the presence of truly marine larval stages and no gregarious behaviour, favouring microbiota exchanges, we found common, organ-specific microbiota, associated to the gut and the gills of the crabs (with more than 15% of the genera detected specifically enriched only in one organ). Our results suggest an early establishment of a new common, stable microbiota in the transition from water to land. "Adopt a Microorganism" methodology enhances the discourse richness of high school students in both hybrid learning and remote education The Internet has changed the way teachers and students access information and build knowledge. The recent COVID-19 pandemic has created challenges for both teachers and students and a demand for new methodologies of remote learning. In the life sciences, mixing online content with practical activities represents an even greater challenge. In microbiology, the implementation of an active teaching methodology, the #Adopt project, based on the social network Facebook(R), represents an excellent option for connecting remote education with classroom activities. In 2020, the version applied in high school, "Adopt a Microorganism", was adapted to meet the demands of emergency remote education owing to the suppression of face-to-face activities caused by the pandemic. In the present study, we assessed how the change in methodology impacted the discourse richness of students from high school integrated with technical education in the Business Administration program of the Federal Institute of Sao Paulo, Sorocaba Campus. Three questionnaires related to the groups of microorganisms (Archaea, Bacteria, Virus, Fungi, and Protozoan) were applied. The students responses in the 2019 and 2020 classes were compared concerning content richness and multiplicity of concepts through the application of the Shannon diversity index, an approach that is generally used to assess biodiversity in different environments. The observed results suggest that remote learning provided students with a conceptual basis and richness of content equivalent to that achieved by students subjected to the hybrid teaching model. In conclusion, this study suggests that the #Adopt project methodology increases students discourse richness in microbiology even without face-to-face traditional classes. The compound of baicalein, wogonin, and oroxylin-A inhibiting EMT in A549 cell line via PI3K/AKT-TWIST1-glycolysis pathway Non-small cell lung cancer (NSCLC) is a worldwide disease with high morbidity and mortality, which is most derived from its metastasis. Some researches show that the epithelial-mesenchymal transition (EMT) process promotes lung cancer cells migration and invasion, leading to NSCLC metastasis. Total Flavonoid Aglycones Extract (TFAE) isolated from Scutellaria baicalensis was reported to inhibit tumor growth and induce apoptosis. In this study, we found that baicalein, wogonin, and oroxylin-A were the active compounds of TFAE. After reconstructing with these three compounds (baicalein (65.8%), wogonin (21.2%), and oroxylin-A (13.0%)), the reconstructed TFAE (reTFAE) present inhibitory effect on the EMT process of A549 cells. Then, bioinformatic technology was employed to elucidate the potential pharmacodynamic mechanism network of reTFAE. We identified the relationship between reTFAE and PI3K/Akt signaling pathways, with TWIST1 as the key protein. LY294002, the inhibitor of PI3K/Akt signaling pathway, and knock-down TWIST1 could significantly enhance the efficacy of reTFAE, with increasing expression of epithelial markers and decreasing expression of mesenchymal markers in A549 cells at the same time. Furthermore, stable isotope dimethyl-labeled proteomics technology was conducted to complement the follow-up mechanism that the EMT-inhibition process may be realized through glycolysis pathway. In conclusion, we claim that TWIST1-targeted flavonoids could provide a new strategy to inhibit EMT progress for the treatment of NSCLC. Analyzing Assay Specificity in Metabolomics using Unique Ion Signature Simulations Targeted, untargeted and data-independent acquisition (DIA) metabolomics workflows are often hampered by ambiguous identification based on either MS1 information alone or relatively few MS2 fragment ions. While DIA methods have been popularized in proteomics, it is less clear whether they are suitable for metabolomics workflows due to their large precursor isolation windows and complex co-isolation patterns. Here, we quantitatively investigate the conditions necessary for unique metabolite detection in complex backgrounds using precursor and fragment ion mass-to-charge separation, comparing three benchmarked MS methods (MS1, MRM, DIA). Our simulations show that DIA outperformed MS1-only and MRM-based methods with regards to specificity by a factor of ~2.8-fold and ~1.8-fold, respectively. Additionally, we show that our results are not dependent on the number of transitions used or the complexity of the background matrix. Finally, we show that collision energy is an important factor in unambiguous detection and that a single collision energy setting per compound cannot achieve optimal pairwise differentiation of compounds. Our analysis demonstrates the power of using both high resolution precursor and high resolution fragment ion m/z for unambiguous compound detection. This work also establishes DIA as an emerging MS acquisition method with high selectivity for metabolomics, outperforming both DDA and MRM with regards to unique compound identification potential.



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org.highwire.dtl.DTLVardef@142b100org.highwire.dtl.DTLVardef@1728a1corg.highwire.dtl.DTLVardef@c953c8org.highwire.dtl.DTLVardef@19b856a_HPS_FORMAT_FIGEXP  M_FIG C_FIG Glucose inhibits haemostasis and accelerates diet-induced hyperlipidaemia in zebrafish larvae Hyperglycaemia damages the microvasculature in part through the reduced recruitment of immune cells and interference with platelet signalling, leading to poor wound healing and accelerated lipid deposition in mammals. We investigated the utility of zebrafish larvae to model the effect of glucose on neutrophil and macrophage recruitment to a tail wound, wound-induced haemostasis, and chicken egg yolk feed challenge-induced hyperlipidaemia by supplementing larvae with exogenous glucose by immersion or injection. Neither method of glucose supplementation affected the recruitment of neutrophils and macrophages following tail transection. Glucose injection reduced thrombocyte retention and fibrin plug formation while only thrombocyte retention was reduced by glucose immersion following tail transection. We observed accelerated lipid accumulation in glucose-injected larvae challenged with high fat chicken egg yolk feeding. Our study identifies conserved and divergent effects of high glucose on inflammation, haemostasis, and hyperlipidaemia in zebrafish larvae compared to mammals. Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly. Unevolved proteins from modern and prebiotic amino acids manifest distinct structural profiles Natural proteins represent numerous but tiny structure/function islands in a vast ocean of possible protein sequences, most of which has not been explored by either biological evolution or research. Recent studies have suggested this uncharted sequence space possesses surprisingly high structural propensity, but development of an understanding of this phenomenon has been awaiting a systematic high-throughput approach.

Here, we designed, prepared, and characterized two combinatorial protein libraries consisting of randomized proteins, each 105 residues in length. The first library constructed proteins from the entire canonical alphabet of 20 amino acids. The second library used a subset of only 10 residues (A,S,D,G,L,I,P,T,E,V) that represent a consensus view of plausibly available amino acids through prebiotic chemistry. Our study shows that compact conformations resistant to proteolysis are (i) abundant (up to 40%) in random sequence space, (ii) independent of general Hsp70 chaperone system activity, and (iii) not granted solely by "late" and complex amino acid additions. The Hsp70 chaperone system effectively increases solubility and refoldability of the canonical alphabet but has only a minor impact on the "early" library. The early alphabet proteins are inherently more soluble and refoldable, possibly assisted by the cell-like environment in which these assays were performed.

Our work indicates that both early and modern amino acids are predisposed to supporting protein structure (either in forms of oligomers or globular/molten globule structures) and that protein structure may not be a unique outcome of evolution. TSC22D4 interacts with Akt1 in response to metabolic and stress signals Transforming Growth Factor {beta} 1 Stimulated Clone 22 D4 (TSC22D4) is an intrinsically disordered protein that regulates cellular and physiological processes such as cell proliferation, cellular senescence as well as hepatic glucose and lipid metabolism. The molecular mechanism of TSC22D4 action in these cellular and metabolic functions, however, remains largely elusive. Here, we identified TSC22D4 as a novel protein kinase B/Akt1 interacting protein, a critical mediator of insulin/PI3K signaling pathway implicated in diverse set of diseases including type 2 diabetes, obesity and cancer. TSC22D4 interacts with Akt1 not constitutively but rather in a regulatory manner. While glucose and insulin stimulation of cells or refeeding of mice impair the hepatic TSC22D4-Akt1 interaction, inhibition of mitochondria and oxidative stress, promote it; indicating that extra- and intra-cellular cues play a key role in controlling TSC22D4-Akt1 interaction. Our results also demonstrate that together with its dimerization domain, i.e. the TSC box, TSC22D4 requires its intrinsically disordered region (D2 domain) to interact with Akt1. To understand regulation of TSC22D4 function further, we employed tandem mass spectrometry and identified 15 novel phosphorylation sites on TSC22D4. Similar to TSC22D4-Akt1 interaction, TSC22D4 phosphorylation also responds to environmental signals such as starvation, mitochondrial inhibition and oxidative stress. Interestingly, 6 out of the 15 novel phosphorylation sites lie within the TSC22D4 D2 domain, which is required for TSC22D4-Akt1 interaction. Characterization of the regulation and function of these novel phosphorylation sites, in the future, will shed light on our understanding of the role of TSC22D4-Akt1 interaction in both cell biological and physiological functions. Overall, our findings postulate a model whereby TSC22D4 acts as an environmental sensor and interacts with Akt1 to regulate cell proliferation, cellular senescence as well as maintain metabolic homeostasis. The physics of liquid-to-solid transitions in multi-domain protein condensates Many RNA-binding proteins (RBPs) that assemble into membraneless organelles, have a common architecture including disordered prion-like domain (PLD) and folded RNA-binding domain (RBD). An enrichment of PLD within the condensed phase gives rise to formation, on longer time scales, amyloid-like fibrils (aging). In this study, we employ coarse-grained Langevin dynamics simulations to explore the physical basis for the structural diversity in condensed phases of multi-domain RBPs. We discovered a highly cooperative first order transition between disordered structures and an ordered phase whereby chains of PLD organize in fibrils with high nematic orientational order. An interplay between homo-domain (PLD-PLD) and hetero-domain (PLD-RBD) interactions results in variety of structures with distinct spatial architectures. Interestingly, the different structural phases also exhibit vastly different intra-cluster dynamics of proteins, with diffusion coefficients 5 (disordered structures) to 50 times (ordered structures) lower than that of the dilute phase. Cooperativity of this liquid-solid transition makes fibril formation highly malleable to mutations or post-translational modifications. Our results provide a mechanistic understanding of how multi-domain RBPs could form assemblies with distinct structural and material properties.

Significance StatementAssembly of proteins and nucleic acids into dense, liquid-like pockets is associated with several key functions including stress response, gene-regulation, DNA-repair and RNA processing. Several RNA binding proteins such as FUS are known to form liquid-like condensates that progressively harden into more dynamically, solid-like structures, a phenomenon that gets accelerated by disease mutations. In this study, we discover the mechanistic origins of this transition and show that small mutational or posttranslational modifications could result in sharp disorder-order transitions that could characterize accelerated liquid-solid transition in disease mutants. 5S-IGS rDNA in wind-pollinated trees ( Fagus  L.) encapsulates 55 million years of reticulate evolution and hybrid origins of modern species Standard models of plant speciation assume strictly dichotomous genealogies in which a species, the ancestor, is replaced by two offspring species. The reality in wind-pollinated trees with long evolutionary histories is more complex: species evolve from other species through isolation when genetic drift exceeds gene flow; lineage mixing can give rise to new species (hybrid taxa such as nothospecies and allopolyploids). The multi-copy, potentially multi-locus 5S rDNA is one of few gene regions conserving signal from dichotomous and reticulate evolutionary processes down to the level of intra-genomic recombination. Therefore, it can provide unique insights into the dynamic speciation processes of lineages that diversified tens of millions of years ago. Here, we provide the first high-throughput sequencing (HTS) of the 5S intergenic spacers (5S-IGS) for a lineage of wind-pollinated subtropical to temperate trees, the Fagus crenata - F. sylvatica s.l. lineage, and its distant relative F. japonica. The observed 4,963 unique 5S-IGS variants reflect a complex history of hybrid origins, lineage sorting, mixing via secondary gene flow, and intra-genomic competition between two or more paralogous-homoeologous 5S rDNA lineages. We show that modern species are genetic mosaics and represent a striking case of ongoing reticulate evolution during the past 55 million years.

Significance statementThe evolution of extra-tropical wind-pollinated tree genera involves dynamic speciation processes. High-throughput sequencing of the multi-copy, potentially multi-locus 5S rDNA reveals a complex history of hybrid origins, lineage sorting and mixing, and intra-genomic competition between paralogous-homeologous loci in the core group of Eurasian beech trees (genus Fagus) and their distant relative, F. japonica. The modern species are genetic mosaics and represent a striking case of at least 55 million years of ongoing reticulate evolution. CellKb Immune: a manually curated database of hematopoietic marker gene sets from 7 species for rapid cell type identification Using single-cell RNA sequencing, it is now possible to get the detailed gene expression pattern in each cell for millions of cells in a single experiment under varying conditions. Annotating cells by assigning a type or state to them is important for correct interpretation of results. In spite of current advances in technology, assigning cell types in single-cell datasets remains a bottleneck due to the lack of a comprehensive reference database and an accurate search method in a single tool. CellKb Immune is a knowledgebase of manually collected, curated and annotated marker gene sets representing cell types in the immune response in mouse. It allows users to find matching cell types in literature given a list of genes. We evaluated the contents and search methodology of CellKb Immune using a leave-one-out approach. We further used CellKb Immune to annotate previously defined marker gene sets from a bulk RNA-seq dataset to confirm its accuracy and coverage. CellKb Immune provides an easy-to-use online tool with a reliable method to find matching cell types and annotate cells in single-cell experiments. It is available at https://www.cellkb.com/immune. Multivariate genome-wide association study on tissue-sensitive diffusion metrics identifies key molecular pathways for axonal growth, synaptogenesis, and astrocyte-mediated neuroinflammation The molecular determinants of tissue composition of the human brain remain largely unknown. Recent genome-wide association studies (GWAS) on this topic have had limited success due to methodological constraints. Here, we apply advanced whole-brain analyses on multi-shell diffusion imaging data and multivariate GWAS to two large scale imaging genetic datasets (UK Biobank and the Adolescent Brain Cognitive Development study) to identify and validate genetic association signals. We discovered 503 unique genetic loci that have impact on multiple regions of human brain. Among them, more than 79% were validated in either of two large-scale independent imaging datasets. Key molecular pathways involved in axonal growth, astrocyte-mediated neuroinflammation, and synaptogenesis during development were found to significantly impact the measured variations in tissue-specific imaging features. Our results shed new light on the biological determinants of brain tissue composition and their potential overlap with the genetic basis of neuropsychiatric disorders. Land use change through the lens of macroecology: insights from Azorean arthropods and the Maximum Entropy Theory of Ecology Human activity and land management practices, in particular land use change, have resulted in the global loss of biodiversity. These types of disturbance affect the shape of macroecological patterns, and therefore analyzing these patterns can provide insights into how ecosystems are affected by land use change. We here use arthropod census data from 96 sites at Terceira Island in the Azores archipelago across four different land uses of increasing management intensity: native forest, exotic forest, semi-natural pasture, and intensive pasture, to examine the effects of land use type on three macroecological patterns: the species abundance distribution, the metabolic rate distribution of individuals, and the species-area relationship. The Maximum Entropy Theory of Ecology (METE) has successfully predicted these patterns across habitats and taxa in undisturbed ecosystems, and thus provides a null expectation for their shapes. Across these patterns, we find that the forest habitats are the best fit by METE predictions, while the semi-natural pasture is consistently the worst fit, and the intensive pasture is intermediately well fit. We show that the direction of failure of the METE predictions at the pasture sites is likely due to the hyper-dominance of introduced spider species present there. We hypothesize that the particularly poor fit for the semi-natural pasture is due to the mix of arthropod communities out of equilibrium, leading to greater heterogeneity in composition and complex dynamics that violate METEs assumption of static state variables. The comparative better fit for the intensive pasture plausibly results from more homogeneous arthropod communities that are well adapted to intensive management, and thus whose state variables are less in flux. Analyzing deviations from theoretical predictions across land use type provides useful information about how land use and disturbance affect ecosystems, and such comparisons could be useful across other habitats and taxa. The C. elegans gonadal sheath Sh1 cells extend asymmetrically over a differentiating germ cell population in the proliferative zone The C. elegans adult hermaphrodite germ line is surrounded by a thin tube formed by somatic sheath cells that support germ cells as they mature from the stem-like mitotic state through meiosis, gametogenesis and ovulation. Recently, we discovered that the distal-most Sh1 sheath cells associate with mitotic germ cells as they exit the niche. Here we report that these distal sheath-associated germ cells differentiate first in animals with temperature-sensitive mutations affecting germ cell state, and stem-like germ cells are maintained distal to the Sh1 boundary. We analyze several markers of the distal sheath, which is best visualized with endogenously tagged membrane proteins, as overexpressed fluorescent proteins fail to localize to distal membrane processes and can cause gonad morphology defects. However, such reagents with highly variable expression can be used to determine the relative positions of the two Sh1 cells, one of which often extends further distal than the other. New Segmentation and Feature Extraction Algorithm for Classification of White Blood Cells in Peripheral Smear Images This article addresses a new method for the classification of white blood cells (WBCs) using image processing techniques and machine learning methods. The proposed method consists of three steps: detecting the nucleus and cytoplasm, extracting features, and classification. At first, a new algorithm is designed to segment the nucleus. For the cytoplasm to be detected, only a part of it located inside the convex hull of the nucleus is involved in the process. This attitude helps us overcome the difficulties of segmenting the cytoplasm. In the second phase, three shapes and four novel color features are devised and extracted. Finally, by using an SVM model, the WBCs are classified. The segmentation algorithm can detect the nucleus with a dice similarity coefficient of 0.9675. The proposed method can categorize WBCs in Raabin-WBC, LISC, and BCCD datasets with accuracies of 94.65 %, 92.21 %, and 94.20 %, respectively. It is worth mentioning that the hyperparameters of the classifier are fixed only with the Raabin-WBC dataset, and these parameters are not readjusted for LISC and BCCD datasets. The obtained results demonstrate that the proposed method is robust, fast, and accurate. ZIKV disrupts placental ultrastructure and drug transporter expression in mice Congenital Zika virus (ZIKV) infection can induce fetal brain abnormalities. Here, we investigated whether maternal ZIKV infection affects placental physiology and metabolic transport potential and impacts the fetal outcome, regardless of viral presence in the fetus at term. Low (103 PFU-ZIKVPE243; low ZIKV) and high (5x107 PFU-ZIKVPE243; high ZIKV) virus titers were injected into immunocompetent (ICompetent C57BL/6) and immunocompromised (ICompromised A129) mice at gestational day (GD) 12.5 for tissue collection at GD18.5 (term). High ZIKV elicited fetal death rates of 66% and 100%, whereas low ZIKV induced fetal death rates of 0% and 60% in C57BL/6 and A129 dams, respectively. All surviving fetuses exhibited intrauterine growth restriction (IUGR) and decreased placental efficiency. High-ZIKV infection in C57BL/6 and A129 mice resulted in virus detection in maternal spleens and placenta, but only A129 fetuses presented virus RNA in the brain. Nevertheless, pregnancies in both strains produced fetuses with decreased head sizes (p<0.05). Low-ZIKV-A129 dams had higher IL-6 and CXCL1 levels (p<0.05), and their placentas showed increased CCL-2 and CXCL-1 contents (p<0.05). In contrast, low-ZIKV-C57BL/6 dams had an elevated CCL2 serum level and increased type I and II IFN expression in the placenta. Notably, less abundant microvilli and mitochondrial degeneration were evidenced in the placental labyrinth zone (Lz) of ICompromised and high-ZIKV-ICompetent mice but not in low-ZIKV-C57BL/6 mice. In addition, decreased placental expression of the drug transporters P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) and the lipid transporter Abca1 was detected in all ZIKV-infected groups, but Bcrp and Abca1 were only reduced in ICompromised and high-ZIKV ICompetent mice. Our data indicate that gestational ZIKV infection triggers specific proinflammatory responses and affects placental turnover and transporter expression in a manner dependent on virus concentration and maternal immune status. Placental damage may impair proper fetal-maternal exchange function and fetal growth/survival, likely contributing to congenital Zika syndrome. Activation of the type 3 secretion system of enteropathogenic E. coli leads to remodeling of its membrane composition The cell envelope of gram-negative bacteria is a complex structure, essential for bacterial survival and for resistance to many antibiotics. Channels that cross the bacterial envelope and the host cell membrane form secretion systems that are activated upon attachment to host, enabling bacteria to inject effector molecules into the host cell, required for bacterial-host interaction. The type III secretion system (T3SS) is critical for the virulence of several pathogenic bacteria, including enteropathogenic E. coli (EPEC). The EPEC T3SS activation is associated with repression of carbon storage regulator (CsrA), resulting in gene expression remodeling, which is known to affect EPEC central carbon metabolism and contributes to the adaptation to a cell-adherent lifestyle in a poorly understood manner. We reasoned that the changes in bacterial envelope upon attachment to host and the activation of a secretion system may involve a modification of the lipid composition of bacterial envelope. Accordingly, we performed a lipidomics analysis on mutant strains that simulate T3SS activation. We saw a shift in glycerophospholipid metabolism towards the formation of lysophospholipids, attributed to corresponding upregulation of the phospholipase pldA and the acyltransferase ygiH upon T3SS activation in EPEC. We also detected a shift from menaquinones and ubiquinones to undecaprenyl lipids, concomitant to abnormal synthesis of O-antigen. The remodeling of lipid metabolism is mediated by CsrA and associated with increased bacteria cell size and Zeta potential, and a corresponding alteration in EPEC permeability to vancomycin, increasing the sensitivity of T3SS-activated strains and of adherent wild type EPEC to the antibiotic.

ImportanceThe characterization of EPEC membrane lipid metabolism upon attachment to host is an important step towards a better understanding the shift of EPEC, a notable human pathogen, from a planktonic to adherent life style. It may also apply to other pathogenic bacteria that use this secretion system. We predict that upon attachment to host cells the lipid remodeling upon T3SS activation contributes to bacterial fitness and promotes host colonization, and show that it is associated with increased cell permeability and higher sensitivity to vancomycin.

To the best of our knowledge, this is the first demonstration of a bacterial lipid remodeling due to activation of a secretion system. Developing High-Affinity Decoy Receptor Optimized for Multiple Myeloma and Diffuse Large B Cell Lymphoma Treatment Recent T Cell therapies have been effective in the treatment of hematological cancers. However, immunotoxicity and treatment relapse pose significant clinical challenges. Here, we revealed distinctive requirement for neutralizing TNF receptor ligands APRIL and BAFF in MM and DLBCL. Furthermore, we investigated the use of BCMA decoy receptor (sBCMA-Fc) as a therapeutic inhibitor of ARPIL and BAFF. While wild-type sBCMA-Fc successfully blocked APRIL signaling with picomolar binding affinity, inhibiting tumor growth in MM models, it lacked efficacy in inhibiting DLBCL progression due to its weak binding for BAFF. To expand the therapeutic utility of sBCMA-Fc, using a protein engineering approach, we generated an affinity enhanced mutant sBCMA-Fc fusion molecule (sBCMA-Fc V3) with 4-folds and 500-folds enhancement in binding to APRIL and BAFF respectively. The sBCMA-Fc V3 clone significantly enhanced antitumor activity against both MM and DLBCL. Importantly, sBCMA-Fc V3 was proven to be a viable clinical candidate by showing adequate toxicity profile and on-target mechanism of action in nonhuman primates.

SUMMARYThis study demonstrates the dichotomous function of APRIL and BAFF in MM and DLBCL, that can be safely targeted by an engineered fusion protein designed to trap APRIL and BAFF with ultra-high binding affinity. Adapting to extremes: reconstructing evolution in response to changing climate over time and space in the diverse Australian plant genus Acacia AimMacroevolutionary analysis is increasingly being used to study biodiversity responses to climate change, for example by using phylogenetic node ages to infer periods of diversification, or phylogenetic reconstruction of traits to infer adaptation to particular stresses. Here we apply a recently developed macroevolutionary method to investigate the responses of a diverse plant genus, Acacia, to increasing aridity and salinity in Australia from the Miocene to the present. We ask whether increase in tolerance of aridity and salinity coincided with periods of aridification, and if it allowed the radiation of Acacia into a wide range of niches.

TaxonAcacia

LocationAustralia

MethodsWe applied the Niche Evolution Model (NEMo), which combines Environmental (or Ecological) Niche Modelling (ENM) with phylogenetic comparative methods (PCM) in a single statistical framework, to a large database of Acacia presence-only records and presence-absence survey sites in order to infer current environmental tolerances of Australia Acacia species and reconstruct the evolution of environmental tolerance to increasing aridity and salinity.

ResultsWe find that patterns in evolution of Acacia, over time and across different habitat types, are consistent with the aridification history of Australia and suggests substantial ability to adapt to high aridity and salinity.

Main conclusionsOur results suggest that many Acacia lineages have been able to exploit new environments created during the aridification of Australia through evolution of environmental tolerance, resulting in their current dominance of many habitats across the continent. This study demonstrates that phylogenetic studies of the evolution of responses to changing environment can move beyond application of simple trait-based models, allowing the underlying processes of speciation, adaptation and dispersal to be explicitly modelled in a macroecological and macroevolutionary context.

Statement of significanceAcacia species are found throughout Australia, from rainforests to deserts, and are striking in their environmental adaptability, so they are a perfect case study for understanding evolution of tolerance to environmental extremes in a changing climate. We use the largest database of spatial distribution records yet assembled, using both surveys and atlas data, and a new analytical method that combines the strengths of environmental niche modelling with phylogenetic comparative methods, to demonstrate rapid evolution in aridity and salinity tolerance in response to aridification of the Australian continent during the Neogene and Quaternary. Microtubule poleward flux promotes chromosome centering on the spindle Chromosome alignment at the spindle equator promotes proper chromosome segregation and depends on pulling forces exerted at kinetochore fiber tips together with polar ejection forces. However, kinetochore fibers are also subjected to forces driving their poleward flux. Here we introduce a flux-driven centering model that relies on flux generated by forces within the overlaps of bridging and kinetochore fibers. This centering mechanism works so that the longer kinetochore fiber fluxes faster than the shorter one, moving the kinetochores towards the center. We developed speckle microscopy in human spindles and confirmed the key prediction that kinetochore fiber flux is length-dependent. Kinetochores are better centered when overlaps are shorter and the kinetochore fiber flux markedly slower than the bridging fiber flux. We identify Kif18A and Kif4A as overlap and flux regulators and NuMA as a fiber coupler. Thus, length-dependent sliding forces exerted by the bridging fiber onto kinetochore fibers promote chromosome alignment. Long term analysis of social structure: evidence of age-based consistent associations in male Alpine ibex Despite its recognized importance for understanding the evolution of animal sociality as well as for conservation, long term analysis of social networks of animal populations is still relatively uncommon. We investigated social network dynamics in males of a gregarious mountain ungulate (Alpine ibex, Capra ibex) over ten years focusing on groups, sub-groups and individuals, exploring the dynamics of sociality over different scales. Despite the social structure changing between seasons, the Alpine ibex population was highly cohesive: fission-fusion dynamics lead almost every male in the population to associate with each other male at least once. Nevertheless, we found that male Alpine ibex showed preferential associations that were maintained across seasons and years. Age seemed to be the most important factor driving preferential associations while other characteristics, such as social status, appeared less crucial. We also found that centrality measures were influenced by age and were also related to individual physical condition. The multi-scale and long-term frame of our study helped us show that ecological constrains, such as resource availability, may play a role in shaping associations in a gregarious species, but they cannot solely explain sociality and preferential association that are likely also to be driven by life-history linked physiological and social needs. Our results highlight the importance of long-term studies based on individually recognizable subjects to help us build on our understanding of the evolution of animal sociality. Statistical Analysis of Three-Dimensional Chromatin Packing Domains Determined by Chromatin Scanning Transmission Electron Microscopy (ChromSTEM) Chromatin organization over a wide range of length scales plays a critical role in the regulation of transcription and deciphering the interplay of these processes requires high-resolution, three-dimensional, quantitative imaging of chromatin structure in vitro. Herein, we introduce ChromSTEM, a method that utilizes high angle annular dark-field imaging and tomography in scanning transmission electron microscopy combined with DNA-specific staining for electron microscopy. We utilized ChromSTEM to quantify chromatin structure in cultured cells and the statistical packing behavior of the chromatin polymer. Using chromatin mass and density analysis, we observed that chromatin forms spatially well-defined higher-order domains which are around 100 nm in radius, with a radially decreasing mass-density from the center to the periphery. Although the morphological properties of the domains vary within the same cell line, they seem to exhibit greater heterogeneity across cell lines, underlying a potential role of statistical chromatin packing in regulating cell-type-specific gene expression.