title: Large-scale mRNA translation and the intricate effects of competition for the finite pool of ribosomes

abstract: We present a new theoretical framework for large-scale mRNA translation using a network of models called the ribosome flow model with Langmuir kinetics (RFMLK), interconnected via a pool of free ribosomes. The input to each RFMLK depends on the pool density, and it affects the initiation rate and the internal ribosome entry rates at each site along each RFMLK. Ribosomes that detach from an RFMLK due to termination or premature drop-off are fed back into the pool. We prove that the network always converges to a steady-state, and study its sensitivity to variations in the parameters. For example, we show that if the drop-off rate at some site in some RFMLK is increased then the pool density increases and consequently the steady-state production rate in all the other RFMLKs increases. Surprisingly, we also show that modifying a parameter of a certain RFMLK can lead to arbitrary effects on the densities along the modified RFMLK, depending on the parameters in the entire network. We conclude that the competition for shared resources generates an indirect and intricate web of mutual effects between the mRNA molecules, that must be accounted for in any analysis of translation.

category: systems biology


title: A generalisable approach to drug susceptibility prediction for M. Tuberculosis using machine learning and whole-genome sequencing

abstract: Rapid and up-to-date drug susceptibility testing is urgently needed to address the threat of multidrug resistant tuberculosis. We developed a composite machine learning system to predict susceptibility from whole-genome sequences for 13 anti-tuberculosis drugs. We trained, validated and externally tested the system, and assessed its performance against a previously validated mutation catalogue, existing molecular assays, and World Health Organization Target Product Profiles. 174,492 phenotypes and 26,328 isolates from 34 countries were studied. The sensitivity of the model was greater than 90% for all drugs except ethionamide, clofazimine and linezolid. Specificity was greater than 95% for all drugs except ethambutol, ethionamide, bedaquiline, delamanid and clofazimine. The machine learning system was more sensitive than the mutation catalogue and molecular assays. For rifampicin-resistant samples, it correctly predicted a pan-susceptible second-line regimen with 98% accuracy. The proposed system can help guide therapy and be updated automatically as new resistance determinants emerge.

category: microbiology


title: Acidity and sulfur oxidation intermediate concentrations controlled by O2-driven partitioning of sulfur oxidizing bacteria in a mine tailings impoundment

abstract: Acidification of freshwater in mining impacted areas is a major global environmental problem catalyzed by sulfur-oxidizing bacteria (SOB). To date, little is known about the active bacteria in mine tailings impoundments and their environmental niches. Here, biological sulfur oxidation was investigated over four years in a mine tailings impoundment, integrating sulfur geochemistry, genome-resolved metagenomics and metatranscriptomics. We demonstrated oxygen driven niche partitioning of SOB and their metabolic pathways that explain acidity generation and thiosulfate persistence. Four chemolithoautotrophic SOB, Halothiobacillus, Thiobacillus, Sulfuricurvum and Sediminibacterium comprised 37% to 73% of the analyzed communities. The impoundment waters alternated between the dominance of Halothiobacillus versus a Thiobacillus, Halothiobacillus, Sulfuricurvum and Sediminibacterium consortia. Halothiobacillus dominance was associated with lower pH values ([~]4.3), higher [H+]/[SO42-] and lower [S2O32-], collectively indicative of extensive sulfur oxidation. Halothiobacillus, which couple sulfur oxidation via the Sox pathway to aerobic respiration or NO2- reduction, were present throughout the depth profile, yet their expression of sox genes occurred only in upper highly oxygenated waters. Conversely, when consortia of Thiobacillus, Halothiobacillus, Sulfuricurvum and Sediminibacterium dominated, recycling/disproportionating reactions were more prevalent. Thiobacillus, which dominated deeper micro-oxic/anoxic waters, oxidized sulfur primarily through the rDSR pathway, coupled to NO3-/NO2- reduction, resulting in lower [H+]/[SO42-] and higher [S2O32-] relative to upper waters. These field results mirror the Sox/rDSR-geochemical patterns of experimental SOB enrichments and reveal opportunities for biological treatments of recalcitrant reduced sulfur compounds, as well as gene-based monitoring and in situ RNA detection to predict the onset of problematic geochemistry.


title: Paving the way towards understanding the inflammatory pathways triggered by giant viruses in mammalian cells: effect of mimivirus-cell interactions on IκBα degradation

abstract: Even after two decades since the identification of the first giant virus, the Acanthamoeba polyphaga mimivirus (APMV), it still elude scientists. Their gigantic size and genome are unique in the whole virosphere, and many aspects of their biology are still unknown, including their possible hosts. They are cultivated in laboratories using Acanthamoeba cells as hosts, but little is known about the infectivity of these giant viruses in vertebrate cells. However, there is evidence of the possible involvement of APMV in pneumonia and activation of inflammatory pathways. Among the hundreds of prospected giant viruses members is Tupanvirus, isolated in Brazil. Its particles have a characteristically large size varying between 1.2 to 2 m and are covered by fibrils. In the present work, we aim to study the consequences of the incubation of APMV and Tupanvirus with mammalian cells. These cells express Toll-like receptors (TLR) that are capable of recognizing lipopolysaccharides, favoring the internalization of the antigen and activation of the inflammatory system. We used a lineage of human lung adenocarcinoma cells (A549) to evaluate possible effects of TLR activation by the giant viruses and if we could detect the probable cause of the said giant-virus dependent pneumonia. Our results show that APMV and Tupanvirus (TPV) activate cellular receptors related to the Toll-like 4 type-induced inflammatory response and that the A549 cells are capable of internalizing the latter virus. Therefore, this study brings new insights into the possible interactions established between mimiviruses (here represented by APMV and Tupanvirus) and members of the innate cellular immune response.


title: Regulating expression of mistranslating tRNAs by readthrough RNA polymerase II transcription

abstract: Transfer RNA (tRNA) variants that alter the genetic code increase protein diversity and have many applications in synthetic biology. Since the tRNA variants can cause a loss of proteostasis, regulating their expression is necessary to achieve high levels of novel protein. Mechanisms to positively regulate transcription with exogenous activator proteins like those often used to regulate RNA polymerase II (RNAP II) transcribed genes are not applicable to tRNAs as their expression by RNA polymerase III requires elements internal to the tRNA. Here, we show that tRNA expression is repressed by overlapping transcription from an adjacent RNAP II promoter. Regulating the expression of the RNAP II promoter allows inverse regulation of the tRNA. Placing either Gal4 or TetR-VP16 activated promoters downstream of a mistranslating tRNASer variant that mis-incorporates serine at proline codons in Saccharomyces cerevisiae allows mistranslation at a level not otherwise possible because of the toxicity of the unregulated tRNA. Using this inducible tRNA system, we explore the proteotoxic effects of mistranslation on yeast cells. High levels of mistranslation cause cells to arrest in G1 phase. These cells are impermeable to propidium iodide, yet growth is not restored upon repressing tRNA expression. High levels of mistranslation increase cell size and alter cell morphology. This regulatable tRNA expression system can be applied to study how native tRNAs and tRNA variants affect the proteome and other biological processes. Variations of this inducible tRNA system should be applicable to other eukaryotic cell types.

category: molecular biology


title: FGF21-FGFR4 signaling in cardiac myocytes promotes concentric cardiac hypertrophy in mouse models of diabetes

abstract: Fibroblast growth factor (FGF) 21, a hormone that increases insulin sensitivity, has shown promise as a therapeutic agent to improve metabolic dysregulation. Here we report that FGF21 directly targets cardiac myocytes by binding {beta}-klotho and FGF receptor (FGFR) 4. In combination with high glucose, FGF21 induces cardiac myocyte growth in width mediated by extracellular signal-regulated kinase 1/2 (ERK1/2) signaling. While short-term FGF21 elevation can be cardio-protective, we find that in type 2 diabetes (T2D) in mice, where serum FGF21 levels are elevated, FGFR4 activation induces concentric cardiac hypertrophy. As T2D patients are at risk for heart failure with preserved ejection fraction (HFpEF), we propose that induction of concentric hypertrophy by elevated FGF21-FGFR4 signaling constitutes a novel mechanism promoting T2D-associated HFpEF and that FGFR4 blockade might serve as a cardio-protective therapy in T2D. In addition, potential adverse cardiac effects of FGF21 mimetics currently in clinical trials should be investigated.


title: Determination of m6A frequency utilizing 4SedTTP-RT Ligation Assisted PCR (SLAP) in viral and cellular long non-coding RNAs

abstract:
N6-methyladenosine is one of the most abundant epitranscriptomic signatures that can affect every aspect of RNA biology, from structure and stability to intra- and intermolecular interactions. The accurate quantitative assessment of RNA stoichiometry at single-nucleotide resolution is a prerequisite to evaluate the biological significance of m6A in the context of specific RNA. We have developed a new method, termed 4-Selenothymidine 5-triphosphate reverse transcription and Ligation Assisted PCR analysis (SLAP), for quantitative and unbiased assessment of the m6A fraction on target RNA. The inclusion of thymidine triphosphate derivative during reverse transcription discourages base pair formation with m6A resulting in the reactions cessation, while maintaining normal A-T base pairing. The site-specific ligation of the resulting cDNAs with adapters, followed by amplification, generates two distinct products that reflect the modified and unmodified fraction of the analyzed RNA. These PCR products are subsequently separated by gel electrophoresis and quantified using densitometric analysis. We applied the SLAP to verify the position and assess the frequency of m6A sites present on two exemplary long non-coding RNAs. We assessed the SLAP specificity, accuracy, and sensitivity, proving the applicability of this method for the m6A analysis on less abundant transcripts. Overall, this method constitutes an extension of the birds-eye view of RNA m6A landscape provided by epitranscriptome-wide analyses by delivering quantitative assessment of modification frequency and can therefore aid the understanding of the consequences of m6A on biological processes.

Graphical Abstract

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title: Large-scale allosteric switch in the 7SK RNA regulates transcription in response to growth and stress

abstract: 7SK is a highly conserved non-coding RNA that regulates eukaryotic transcription by sequestering positive transcription elongation factor b (P-TEFb). 7SK regulatory function likely entails changes in RNA structure, but characterizing dynamic RNA-protein complexes in cells has remained an unsolved challenge. We describe a new chemical probing strategy (DANCE-MaP) that uses maximum likelihood deconvolution and probabilistic read assignment to define simultaneously (i) per-nucleotide reactivity profiles, (ii) direct base pairing interactions, and (iii) tertiary and higher-order interactions for each conformation of multi-state RNA structural ensembles, all from a single experiment. We show that human 7SK RNA, despite significant heterogeneity, intrinsically codes for a large-scale structural switch that couples dissolution of the P-TEFb binding site to structural remodeling at distal release factor binding sites. The 7SK structural equilibrium is regulated by cell type, shifts dynamically in response to cell growth and stress, and can be exogenously targeted to modulate transcription in cells. Our data support that the 7SK structural ensemble functions as an integrator of diverse cellular signals to control transcription elongation in environment and cell specific ways, and establishes DANCE-MaP as a powerful strategy for comprehensively defining RNA structure and dynamics in cells.

category: biochemistry


title: Single-molecule Dynamic In-Solution Inhibition Assay: A Method for Full Kinetic Profiling of Drug Candidate Binding to GPCRs in Native Membranes

abstract: Kinetic profiling of drug-target interactions using surface-based label-free technologies is well established for water-soluble pharmaceutical targets but is difficult to execute for membrane proteins in general and G-protein-coupled receptors (GPCRs) in particular. That is because surface immobilization of GPCRs tends to alter their configuration and function, leading to low target coverage and non-specific binding. We here describe a novel assay for kinetic profiling of drug binding to the GPCR human beta 2 adrenergic receptor ({beta}2AR). The assay involves temporally-resolved imaging of the binding of individual {beta}2AR-containing cell membrane-derived liposomes to a surface-immobilized ligand in the presence of screened drugs. This approach allowed to determine association and dissociation constants of {beta}2AR and suspended alprenolol (antagonist) and fenoterol (agonist). The set-up combines a 384 well-plate sensor chip with automated liquid handling and the assay takes minutes to complete, making it well adapted for drug screening campaigns.

category: biophysics


title: Feeding ecology of broadbill swordfish (Xiphias gladius) in the California Current

abstract: The feeding ecology of broadbill swordfish (Xiphias gladius) in the California Current was described based on analysis of stomach contents collected by federal fishery observers aboard commercial drift gillnet boats from 2007 to 2014. Prey were identified to the lowest taxonomic level and diet composition was analyzed using univariate and multivariate methods. Of 299 swordfish sampled (74 to 245 cm eye-to-fork length), 292 non-empty stomachs contained remains from 60 prey taxa. Diet consisted mainly of cephalopods but also included epipelagic and mesopelagic teleosts. Jumbo squid (Dosidicus gigas) and Gonatopsis borealis were the most important prey based on the geometric index of importance. Swordfish diet varied with body size, location and year. Jumbo squid, Gonatus spp. and Pacific hake (Merluccius productus) were more important for larger swordfish, reflecting the ability of larger specimens to catch large prey. Jumbo squid, Gonatus spp. and market squid (Doryteuthis opalescens) were more important in swordfish diet in inshore waters, while G. borealis and Pacific hake predominated offshore. Jumbo squid was more important from 2007-2010 than in 2011-2014, with Pacific hake the most important prey item in the latter period. Diet variation by area and year probably reflects differences in swordfish preference, prey availability, prey distribution, and prey abundance. The range expansion of jumbo squid that occurred during the first decade of this century may particularly explain their prominence in swordfish diet from 2007-2010. Some factors that may influence dietary variation in swordfish were identified. Standardization could make future studies more comparable for conservation monitoring purposes.

category: ecology


title: Cytokine gene polymorphism and parasite susceptibility in free-living rodents: importance of non-coding variants

abstract: Associations between genetic variants and susceptibility to infections have long been studied in free-living hosts to infer contemporary evolutionary forces shaping genetic polymorphisms of the immunity genes. Despite extensive studies of receptors, such as MHC or TLR, little is known about efferent arm of the immune system. Cytokines are signalling molecules that trigger and modulate the immune response, acting as a crucial link between innate and adaptive immunity. In the present study we investigated how genetic variation in cytokines affects susceptibility to parasitic diseases in bank voles. We focused on three cytokines: tumour necrosis factor (TNF), lymphotoxin alpha (LT), and interferon beta (IFN{beta}1). Two SNPs in LT and two in IFN{beta}1 significantly affected susceptibility to nematodes, and was of them was also associated with susceptibility to microbial pathogen Bartonella. All these variants displayed signatures of selection. One of the variants was synonymous, and one was located in an intron. Our study shows that cytokines are prone to parasite-driven selection, and non-coding variants may play an important role in susceptibility to infections in wild systems.

category: evolutionary biology


title: Development of a machine learning model to estimate biotic ligand model-based predicted no-effect concentrations for copper in freshwater

abstract: The copper biotic ligand model (BLM) has been used for environmental risk assessment by taking into account the bioavailability of copper in freshwater. However, the BLM-based environmental risk of copper has been assessed only in Europe and North America, with monitoring datasets containing all of the BLM input variables. For other areas, it is necessary to apply surrogate tools with reduced data requirements to estimate the BLM-based predicted no-effect concentration (PNEC) from commonly available monitoring datasets. To develop an optimized PNEC estimation model based on an available monitoring dataset, an initial model that considers all BLM variables, a second model that requires variables excluding alkalinity, and a third model using electrical conductivity as a surrogate of the major cations and alkalinity have been proposed. Furthermore, deep neural network (DNN) models have been used to predict the nonlinear relationships between the PNEC (outcome variable) and the required input variables (explanatory variables). The predictive capacity of DNN models in this study was compared with the results of other existing PNEC estimation tools using a look-up table and multiple linear and multivariate polynomial regression methods. Three DNN models, using different input variables, provided better predictions of the copper PNECs compared with the existing tools for four test datasets, i.e., Korean, United States, Swedish, and Belgian freshwaters. The adjusted r2 values in all DNN models were higher than 0.95 in the test datasets, except for the Swedish dataset (adjusted r2 > 0.87). Consequently, the most applicable model among the three DNN models could be selected according to the data availability in the collected monitoring database. Because the most simplified DNN model required only three water quality variables (pH, dissolved organic carbon, and electrical conductivity) as input variables, it is expected that the copper BLM-based risk assessment can be applied to monitoring datasets worldwide.

category: pharmacology and toxicology


title: Comparative transcriptomics of seven Impatiens species

abstract: Impatiens is a genus containing more than 1000 species. Thanks to its size, it is a unique system for studying species diversification in natural populations. This study focused on the characterization of novel transcriptomes from seven Impatiens species originating from Nepal. Leave transcriptome of Impatines balsamina L., I. racemosa DC., I. bicornuta Wall, I. falcifer Hook, I. devendrae Pusalkar, I. scullyi Hook and I. scabrida DC were sequenced and compared. Reads were de novo assembled and aligned to 92 035-226 081 contigs. We identified 14 728 orthology groups shared among all the species and 3 020 which were unique to a single species. In single species, 2536-3009 orthology groups were under selection from which 767 were common for all species. Six of the seven investigated species shared 77% of gene families with I. bicornuta being the most distinct species. Specific gene families involved in response to different environmental stimuli were closely described. Impatiens bicornuta selection profile shared selection on zing finger protein structures and flowering regulation and stress response proteins with the other investigated species. Overall, the study showed substantial similarity in patterns of selections on transcribed genes across the species suggesting similar evolutionary pressures. This suggests that the species group may have evolved via adaptive radiation.

category: plant biology


title: Kinetics of the inflammatory response during experimental Babesia rossi infection of beagle dogs

abstract: Babesia rossi causes severe morbidity and mortality in dogs in sub-Saharan Africa, and the complications associated with this disease are likely caused by an unfocused, excessive inflammatory response. During this experimental B. rossi study we investigated inflammatory marker and cytokine kinetics during infection and after treatment. We aimed to determine whether infectious dose and treatment would influence the progression of the inflammatory response and clinical disease. Five healthy male beagle dogs were infected with B. rossi, three with a high infectious dose (HD group) and two with a low infectious dose (LD group). Clinical examination, complete blood count (CBC) and C-reactive protein (CRP) were determined daily. Cytokines were quantified on stored plasma collected during the study, using a canine specific cytokine magnetic bead panel (Milliplex(C)). The experiment was terminated when predetermined endpoints were reached. Parasitemia occurred on day 1 and 3 in the HD group and LD group respectively. The rate of increase in parasitemia in the HD group was significantly faster than that seen in the LD group. Significant differences were found in heart rate, blood pressure, interferon gamma (INF{gamma}), keratinocyte chemoattractant (KC), INF{gamma}-induced protein 10 (IP10), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemoattractant protein 1 (MCP1), tumor necrosis factor alpha (TNF), interleukin 2 (IL-2), IL-6, IL-7, IL-8, IL-10 IL-15, IL-18, CRP, neutrophils and monocytes between groups at multiple time points during the course of the infection. Our findings suggest that the initiation of inflammation occurs before the onset of clinical disease in B. rossi infection and infectious dose influences the onset of the inflammatory response. Treatment not only fails to curb the inflammatory response but may enhance it. Finally, we found that there is an imbalance in pro/anti-inflammatory cytokine concentrations during infection which may promote parasite replication.

category: immunology


title: Genome-wide identification of genes important for growth of Dickeya dadantii and D. dianthicola in potato (Solanum tuberosum) tubers

abstract: Dickeya species are causal agents of soft rot diseases in many economically important crops, including soft rot disease of potato (Solanum tuberosum). Using random barcode transposon-site sequencing (RB-TnSeq), we generated genome-wide mutant fitness profiles of Dickeya dadantii 3937, Dickeya dianthicola ME23, and Dickeya dianthicola 67-19 isolates collected after passage through several in vitro and in vivo conditions. Tubers from the potato cultivars "Atlantic", "Dark Red Norland", and "Upstate Abundance" provided highly similar conditions for bacterial growth. Using the homolog detection software PyParanoid, we matched fitness values for orthologous genes in the three bacterial strains. Direct comparison of fitness among the strains highlighted shared and variable traits important for growth. Bacterial growth in minimal medium required many metabolic traits that were also essential for competitive growth in planta, such as amino acid, carbohydrate, and nucleotide biosynthesis. Growth in tubers specifically required the pectin degradation gene kduD. Disruption in three putative DNA-binding proteins had strain-specific effects on competitive fitness in tubers. Though the Soft Rot Pectobacteriaceae can cause disease with little host specificity, it remains to be seen the extent to which strain-level variation impacts virulence.


title: Intact reading ability in spite of a spatially distributed visual word form 'area' in an individual born without the left superior temporal lobe

abstract: The visual word form area (VWFA) is an experience-dependent region in the left ventral temporal cortex (VTC) of literate adults that responds selectively to visual words. Why does it emerge in this stereotyped location? Past research shows the VWFA is preferentially connected to the left-lateralized frontotemporal language network. However, it remains unclear whether the presence of a typical language network and its connections with VTC are critical for the VWFAs emergence, and whether alternative functional architectures may support reading ability. We explored these questions in an individual (EG) born without the left superior temporal lobe but exhibiting normal reading ability. We recorded fMRI activation to visual words, objects, faces, and scrambled words in EG and neurotypical controls. We did not observe word selectivity either in EGs right homotope of the VWFA (rVWFA)--the most expected location given that EGs language network is right-lateralized--or in her spared left VWFA (lVWFA), despite typical face selectivity in both the right and left fusiform face area (rFFA, lFFA). We replicated these results across scanning sessions (5 years apart). Moreover, in contrast with the idea that the VWFA is simply part of the language network that responds to general linguistic information, no part of EGs VTC showed selectivity to higher-level linguistic processing. Interestingly, multivariate pattern analyses revealed sets of voxels in EGs rVWFA and lVWFA that showed 1) higher within- than between-category correlations for words (e.g., Words-Words>Words-Faces), and 2) higher within-category correlations for words than other categories (e.g., Words-Words>Faces-Faces). These results suggest that a typical left-hemisphere language network may be necessary for the emergence of focal word selectivity within the VTC, and that orthographic processing can be supported by a distributed neural code.

category: neuroscience


title: Supervised Hierarchical Autoencoders for Multi-Omics Integration in Cancer Survival Models

abstract: With decreasing costs of high-throughput sequencing, more and more datasets providing omics profiles of cancer patients become available. Thus, novel survival analysis approaches integrating these differently sized and heterogeneous molecular and clinical groups of variables start being developed. Due to the difficulty of the task of multi-omics data integration, the Cox Proportional-Hazards (PH) model using clinical data has remained one of the best-performing techniques, barely outperformed by models using molecular data modalities. There is therefore a need for methods that can successfully perform multi-omics integration in survival analysis and outperform the clinical Cox PH model. Moreover, while certain deep learning methods have been shown to provide state-of-the-art accuracy of cancer survival prediction, most of them show no benefit or even decay in performance when integrating a larger number of modalities, further motivating a need to investigate how modality-specific representations should be integrated when using neural networks for multi-omics integration. We benchmarked multiple integration techniques for a neural network architecture, revealing that hierarchical autoencoder-based integration of modality-specific representations outperformed other methods such as max-pooling and was comparable with state-of-the-art statistical approaches for multi-omics integration. Further, we showed that the hierarchical autoencoder-based integration of modality-specific representations achieved increased performance through a soft modality selection mechanism, focusing on the most informative modalities for each cancer. We thus framed multiomics integration as a partial group-wise feature selection problem, highlighting that only those models performed well that could adequately weight important modalities in the presence of the high noise imposed by less important modalities.

category: bioinformatics


title: Hierarchical autoencoder-based integration improves performance in multi-omics cancer survival models through soft modality selection


title: Condensin compacts DNA in 15 nm steps and requires FACT for extrusion on chromatin

abstract: The authors have withdrawn this manuscript after detecting issues with two of the figures in the manuscript. Therefore, the authors do not wish this work to be cited as a reference. If you have any questions, please contact the corresponding authors.


title: Epigenetic Remodeling by Vitamin C Potentiates the Differentiation of Mouse and Human Plasma Cells

abstract: Ascorbate (vitamin C) is an essential micronutrient in humans. The chronic severe deficiency of ascorbate, termed scurvy, has long been associated with increased susceptibility to infections. How ascorbate affects the immune system at the cellular and molecular levels remained unclear. Here, from a micronutrient screen, we identified ascorbate as a potent enhancer for antibody response by facilitating the IL-21/STAT3-dependent plasma cell differentiation in mouse and human B cells. The effect of ascorbate is unique, as other antioxidants failed to promote plasma cell differentiation. Ascorbate is critical during early B cell activation by poising the cells to plasma cell lineage without affecting the proximal IL-21/STAT3 signaling and the overall transcriptome. Consistent with its role as a cofactor for epigenetic enzymes, ascorbate potentiates plasma cell differentiation by remodeling the epigenome via TET (Ten Eleven Translocation), the enzymes responsible for DNA demethylation by oxidizing 5-methylcytosines into 5-hydroxymethylcytosine (5hmC). Genome-wide 5hmC profiling identified ascorbate responsive elements (EAR) at the Prdm1 locus, including a distal element with a STAT3 motif overlapped with a CpG that was methylated and modified by TET in the presence of ascorbate. The results suggest that an adequate level of VC is required for antibody response and highlight how micronutrients regulate the activity of epigenetic enzymes to regulate gene expression. Our findings imply that epigenetic enzymes can function as sensors to gauge the availability of metabolites and influence cell fate decisions.


title: Immunoglobulin enhancers increase RNA polymerase stalling at somatic hypermutation target sequences

abstract: Somatic hypermutation (SHM) drives the genetic diversity of immunoglobulin (Ig) genes in activated B cells and supports the generation of antibodies with increased affinity for antigen. SHM is targeted to Ig genes by their enhancers (DIVACs; diversification activators), but how the enhancers mediate this activity is unknown. We show using chicken DT40 B cells that highly active DIVACs increase the phosphorylation of RNA polymerase 2 (Pol2) and Pol2 occupancy in the mutating gene with little or no accompanying increase in elongation-competent Pol2 or production of full-length transcripts, indicating accumulation of stalled Pol2. DIVAC has similar effect also in human Ramos Burkitt lymphoma cells. The DIVAC-induced stalling is weakly associated with an increase in the detection of single-stranded DNA bubbles in the mutating target gene. We did not find evidence for antisense transcription, or that DIVAC functions by altering levels of H3K27ac or the histone variant H3.3 in the mutating gene. These findings argue for a connection between Pol2 stalling and cis-acting targeting elements in the context of SHM and thus define a mechanistic basis for locus-specific targeting of SHM in the genome. Our results suggest that DIVAC elements render the target gene a suitable platform for AID-mediated mutation without a requirement for increasing transcriptional output.


title: A structured evaluation of cryopreservation in generating single cell transcriptomes from cerebrospinal fluid

abstract:
ImportanceA robust cerebrospinal fluid (CSF) cell cryopreservation protocol using high resolution single-cell (sc) transcriptomic data would enable the deployment of this important modality in multi-center translational research studies and clinical trials in which many sites do not have the expertise or resources to produce data from fresh samples. It would also serve to reduce technical variability in larger projects.

ObjectiveTo test a reliable cryopreservation protocol adapted for CSF cells, facilitating the characterization of these rare, fragile cells in moderate to large scale studies.

DesignDiagnostic lumbar punctures were performed on twenty-one patients at two independent sites. Excess CSF was collected and cells were isolated. Each cell sample was split into two fractions for single cell analysis using one of two possible chemistries: 3 sc-RNA-Sequencing or 5sc-RNA-Sequencing. One cell fraction was processed fresh while the second sample was cryopreserved and profiled at a later time after thawing.

SettingThe research protocol was deployed at two academic medical centers taking care of multiple sclerosis and other neurological conditions.

Participants21 subjects (age 24 - 72) were recruited from individuals undergoing a diagnostic lumbar puncture for suspected neuroinflammatory disease or another neurologic illness; they donated excess CSF.

FindingsOur comparison of fresh and cryopreserved data from the same individuals demonstrates highly efficient recovery of all known CSF cell types. The proportion of all cell types was similar between the fresh and the cryopreserved cells processed, and RNA expression was not significantly different. Results were comparable at both performance sites, and with different single cell sequencing chemistries. Cryopreservation also did not affect recovery of T and B cell clonotype diversity.

Conclusion and relevanceOur cryopreservation protocol for CSF-cells provides an important alternative to fresh processing of fragile CSF cells: cryopreservation enables the involvement of sites with limited capacity for experimental manipulation and reduces technical variation by enabling batch processing and pooling of samples.

Key pointsO_ST_ABSQuestionC_ST_ABSHow efficient is CSF cryopreservation for single-cell transcriptome analysis and can it be implemented in large multi-center translational and clinical trial settings?

FindingsWe compared single-cell transcriptomes of paired fresh and cryopreserved CSF from 21 patients at two independent sites. We validate the efficacy of a simple and cost effective CSF cryopreservation method that preserves the composition and the transcriptomes of CSF cells stored for weeks-months. The protocol is deployed in a large multicenter Phase 4 MS clinical trial.

MeaningA validated CSF cryopreservation method that would significantly advance basic science and biomarker research in neurological disorders by implementing single-cell transcriptome analyses in multi-center research and clinical trials.

category: genetics


title: Interpretable deep generative models for genomics

abstract: Deep neural networks implementing generative models for dimensionality reduction have been extensively used for the visualization and analysis of genomic data. One of their key limitations is lack of interpretability: it is challenging to quantitatively identify which input features are used to construct the embedding dimensions, thus preventing insight into why cells are organized in a particular data visualization, for example. Here we present a scalable, interpretable variational autoencoder (siVAE) that is interpretable by design: it learns feature embeddings that guide the interpretation of the cell embeddings in a manner analogous to factor loadings of factor analysis. siVAE is as powerful and nearly as fast to train as the standard VAE but achieves full interpretability of the embedding dimensions. Using siVAE, we exploit a number of connections between dimensionality reduction and gene network inference to identify gene neighborhoods and gene hubs, without the explicit need for gene network inference. We observe a systematic difference in the gene neighborhoods identified by dimensionality reduction methods and gene network inference algorithms in general, suggesting they provide complementary information about the underlying structure of the gene co-expression network. Finally, we apply siVAE to implicitly learn gene networks for individual iPSC lines and uncover a correlation between neuronal differentiation efficiency and loss of co-expression of several mitochondrial complexes, including NADH dehydrogenase, cytochrome C oxidase, and cytochrome b.

category: genomics


title: Metabotropic group II glutamate receptors mediate cue-triggered increases in incentive motivation for reward

abstract:
RationaleReward-associated cues can acquire incentive motivational properties and invigorate reward-seeking actions via Pavlovian-to-instrumental transfer (PIT). Glutamatergic neurotransmission mediates the appetitive effects of reward-associated cues. We characterized the expression of PIT and its mediation by metabotropic group II glutamate (mGlu2/3) receptor activity in female and male rats.

ObjectivesAcross the sexes, we used PIT procedures to determine i) cue-triggered increases in incentive motivation for water reward (Experiment 1), ii) the respective influences of the mGlu2/3 receptor agonist LY379268 and reward devaluation by satiation on this effect (Experiment 2).

MethodsWater-restricted male and female Sprague-Dawley rats learned to lever press for water. Separately, they learned that one of two auditory stimuli predicts free water (CS+ vs CS-). On PIT test days, the CS+ and CS- were presented non-contingently, and we measured effects on lever pressing under extinction (no water). In Experiment 1, we characterized PIT across the sexes. In Experiment 2, we measured PIT after systemic LY379268 administration (0, 0.3 and 1 mg/kg), and water satiation, respectively.

ResultsFemale and male rats showed similar PIT, with CS+ but not CS- presentations potentiating water-seeking behaviour. LY379268 (1 mg/kg) attenuated CS+ evoked increases in both water-associated lever pressing and conditioned approach to the water port. Reward devaluation attenuated both water-seeking and CS+ evoked conditioned approach behaviour.

ConclusionsThe sexes show similar cue-triggered increases in reward  wanting, and water devaluation suppresses both water seeking and cue-triggered anticipation of water reward. Finally, across the sexes, mGlu2/3 receptor activity mediates cue-triggered increases in reward  wanting.


title: Installation and imaging of thousands of minirhizotrons to phenotype root systems of field-grown plants

abstract:
BackgroundRoots are vital to plant performance because they acquire resources from the soil and provide anchorage. However, it remains difficult to assess root system size and distribution because roots are inaccessible in the soil. Existing methods to phenotype entire root systems range from slow, often destructive, methods applied to relatively small numbers of plants in the field to rapid methods that can be applied to large numbers of plants in controlled environment conditions. Much has been learned recently by extensive sampling of the root crown portion of field-grown plants. But, information on large-scale genetic and environmental variation in the size and distribution of root systems in the field remains a key knowledge gap. Minirhizotrons are the only established, non-destructive technology that can address this need in a standard field trial. Prior experiments have used only modest numbers of minirhizotrons, which has limited testing to small numbers of genotypes or environmental conditions. This study addressed the need for methods to install and collect images from thousands of minirhizotrons and thereby help break the phenotyping bottleneck in the field.

ResultsOver three growing seasons, methods were developed and refined to install and collect images from up to3038 minirhizotrons per experiment. Modifications were made to four tractors and hydraulic soil corers mounted to them. High quality installation was achieved at an average rate of up to 84.4 minirhizotron tubes per tractor per day. A set of four commercially available minirhizotron camera systems were each transported by wheelbarrow to allow collection of images of mature maize root systems at an average rate of up to 65.3 tubes per day per camera. This resulted in over 300,000 images being collected in as little as 11 days for a single experiment.

ConclusionThe scale of minirhizotron installation was increased by two orders of magnitude by simultaneously using four tractor-mounted, hydraulic soil corers with modifications to ensure high quality, rapid operation. Image collection can be achieved at the corresponding scale using commercially available minirhizotron camera systems. Along with recent advances in image analysis, these advances will allow use of minirhizotrons at unprecedented scale to address key knowledge gaps regarding genetic and environmental effects on root system size and distribution in the field.


title: Stabilization of spine Synaptopodin by mGluR1 is required for mGluR-LTD

abstract: Dendritic spines, actin-rich protrusions forming the postsynaptic sites of excitatory synapses, undergo activity-dependent molecular and structural remodeling. Activation of group 1 metabotropic glutamate receptors - mGluR1 and mGluR5 - by synaptic or pharmacological stimulation, induces LTD but whether this is accompanied with spine elimination remains unresolved. A subset of telencephalic mushroom spines contains the spine apparatus (SA), an enigmatic organelle composed of stacks of smooth endoplasmic reticulum, whose formation depends on the expression of the actin-bundling protein Synaptopodin. Allocation of Synaptopodin to spines appears governed by cell-intrinsic mechanisms as the relative frequency of spines harboring Synaptopodin is conserved in vivo and in vitro. Here we show that expression of Synaptopodin/SA in spines is required for induction of mGluR-LTD at Schaffer collateral-CA1 synapses. Post-mGluR-LTD, mushroom spines lacking Synaptopodin/SA are selectively lost whereas spines harboring it are preserved, a process dependent on activation of mGluR1 but not mGluR5. Mechanistically, we find that mGluR1 supports physical retention of Synaptopodin within excitatory spine synapses during LTD while triggering lysosome-dependent degradation of the protein residing in dendritic shafts. Together, these results reveal a cellular mechanism, dependent on mGluR1, which enables selective preservation of stronger spines containing Synaptopodin/SA while eliminating weaker ones and potentially countering spurious strengthening by de novo recruitment of Synaptopodin. Overall our results identify spines with Synaptopodin/SA as the locus of mGluR-LTD and underscore the importance of the molecular microanatomy of spines in synaptic plasticity.


title: Development of a new militarily-relevant whole-body low-intensity blast model for mild and subconcussive traumatic brain injury: Examination of acute neurological and multi-organ pathological outcomes

abstract:
This work describes a newly developed experimental mouse model reproducing features of blast-induced neurotrauma (BINT), induced in operationally relevant manner using a compressed air-driven shock tube. Mild BINT (smBINT) was induced by one exposure to a low-intensity blast (LIB), whereas subconcussive BINT (rscBINT) was caused by repeated exposures to LIB.

To mimic an operational scenario when a soldier is standing when exposed to blast using a quadruped experimental animal (mouse), a whole-body holder was developed to position mice in a bipedal stance, face-on toward the pressure wave generated in a shock tube. This restraint avoids  bobble head movement, thus prevents tertiary blast effects, and allows administration of fast-acting inhaled anesthetics via nose cone.

Using this model, we established and validated paradigms for primary blast-induced mild and repetitive traumatic brain injuries Our results showed that a single exposure to 69 kPa (10 psi) was capable of inducing smBINT, whereas three-rounds of exposure to 41 kPa (6 psi) caused rscBINT.

Mice recovered rapidly from both types of BINT without prolonged neurological dysfunction. Mild superficial pathology was found predominantly in the lungs 24h after injury, with equivalent pathology after smBINT or repetitive rscBINT. The Purkinje layer of the cerebellum exhibited neuronal damage persisting up to 7d. Similar to some other models as well as clinical findings, this model reproduces blast-induced cerebellar pathology. In conclusion, this model positioning mice in a bipedal stance and facing front-on toward the shockwave provides realistic representation of operational scenarios and reproduces militarily-relevant smBINT and rscBINT in the laboratory.


title: Seasonal temporal dynamics of marine protists communities in tidally mixed coastal waters

abstract: Major seasonal community reorganizations and associated biomass variations are landmarks of plankton ecology. However, the processes determining marine species and community turnover rates have not been fully elucidated so far. Here, we analyse patterns of planktonic protist community succession in temperate latitudes, based on quantitative taxonomic data from both microscopy counts and ribosomal DNA metabarcoding from plankton samples collected biweekly over 8 years (2009-2016) at the SOMLIT-Astan station (Roscoff, Western English Channel). Considering the temporal structure of community dynamics (creating temporal correlation), we elucidated the recurrent seasonal pattern of the dominant species and OTUs (rDNA-derived taxa) that drive annual plankton successions. The use of morphological and molecular analyses in combination allowed us to assess absolute species abundance while improving taxonomic resolution, and revealed a greater diversity. Overall, our results underpinned a protist community characterised by a seasonal structure, which is supported by the dominant OTUs. We detected that some were partly benthic as a result of the intense tidal mixing typical of the French coasts in the English Channel. While the occurrence of these microorganisms is driven by the physical and biogeochemical conditions of the environment, internal community processes, such as the complex network of biotic interactions, also play a key role in shaping protist communities.


title: Land use, macroecology, and the accuracy of the Maximum Entropy Theory of Ecology: A case study of Azorean arthropods

abstract: 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.


title: Land use change through the lens of macroecology: insights from Azorean arthropods and the Maximum Entropy Theory of Ecology


title: Interactive, visual simulation of a spatio-temporal model of gas exchange in the human alveolus

abstract: In interdisciplinary fields such as systems biology, close collaboration between experimentalists and theorists is crucial for the success of a project. Theoretical modeling in physiology usually describes complex systems with many interdependencies. On one hand, these models have to be grounded on experimental data. On the other hand, experimenters must be able to penetrate the model in its dependencies in order to correctly interpret the results in the physiological context. When theorists and experimenters collaborate, communicating results and ideas is sometimes challenging. We promote interactive, visual simulations as an engaging way to communicate theoretical models in physiology and to thereby advance our understanding of the process of interest. We defined a new spatio-temporal model for gas exchange in the human alveolus and implemented it in an interactive simulation software named Alvin. In Alvin, the course of the simulation can be traced in a three-dimensional rendering of an alveolus and dynamic plots. The user can interact by configuring essential model parameters. Alvin allows to run and compare multiple simulation instances simultaneously. The mathematical model was developed with the aim of visualization and the simulation software was engineered based on a requirements analysis. Our work resulted in an integrative gas exchange model and an interactive application that exceed the current standards. We exemplified the use of Alvin for research by identifying unknown dependencies in published experimental data. Employing a detailed questionnaire, we showed the benefits of Alvin for education. We postulate that interactive, visual simulation of theoretical models, as we have implemented with Alvin on respiratory processes in the alveolus, can be of great help for communication between specialists and thereby advancing research.


title: Long-term meditation training is associated with enhanced attention and stronger posterior cingulate - rostrolateral prefrontal cortex resting connectivity

abstract: Mindfulness meditation has been shown to increase resting state functional connectivity (rsFC) between the posterior cingulate cortex (PCC) and dorsolateral prefrontal cortex (DLPFC), which is thought to reflect improvements in attention to the present moment. However, prior research in long-term meditation practitioners lacked quantitative measures of attention that would provide a more direct behavioral correlate and interpretational anchor for PCC-DLPFC connectivity and was inherently limited by small sample sizes. Moreover, whether mindfulness meditation primarily impacts brain function locally, or impacts the dynamics of large-scale brain networks, remained unclear. Here, we sought to replicate and extend prior findings of increased PCC - DLPFC rsFC in a sample of 40 long-term meditators (average practice= 3759 hours) who also completed a behavioral assay of attention. In addition, we tested a network-based framework of changes in inter-regional connectivity by examining network-level connectivity. We found that meditators had stronger PCC-rostrolateral prefrontal cortex (PFC) rsFC, lower connector hub strength across the default mode network (DMN) relative to other functional networks, and better attention to task, compared to 124 meditation-naive controls. Orienting attention positively correlated with PCC- rostrolateral PFC connectivity, and negatively correlated with DMN connector hub strength. These findings provide novel evidence that posterior cingulate - rostrolateral PFC rsFC may support attention orienting, consistent with a role for rostrolateral PFC in meta-cognitive background awareness that is a core component of mindfulness meditation training. Our results further demonstrate that long-term mindfulness meditation may improve attention and strengthen the underlying brain networks.


title: The solute carrier MFSD1 decreases β1 integrins activation status and thus tumor metastasis

abstract: Solute carriers are increasingly recognized as participating in a plethora of pathologies, including cancer. We describe here the involvement of the orphan solute carrier MFSD1 in the regulation of tumor cell migration. Loss of MFSD1 enabled higher levels of metastasis in a mouse model. We identified an increased migratory potential in MFSD1-/- tumor cells which was mediated by increased focal adhesion turn-over, reduced stability of mature inactive {beta}1 integrin, and the resulting increased integrin activation index. We show that MFSD1 promoted recycling to the cell surface of endocytosed inactive {beta}1 integrin and thereby protected {beta}1 integrin from proteolytic degradation; this led to dampening of the integrin activation index. Furthermore, down-regulation of MFSD1 expression was observed during early steps of tumorigenesis and higher MFSD1 expression levels correlate with a better cancer patient prognosis. In sum, we describe a requirement for endolysosomal MFSD1 in efficient {beta}1 integrin recycling to suppress tumor spread.

category: cancer biology


title: The beta-tubulin isotype TUBB6 controls microtubule and actin dynamics in osteoclasts.

abstract: Osteoclasts are bone resorbing cells that participate in the maintenance of bone health. Pathological increase in osteoclast activity causes bone loss eventually resulting in osteoporosis. Actin cytoskeleton of osteoclasts organizes into a belt of podosomes, which sustains the bone resorption apparatus and is maintained by microtubules. Better understanding of the molecular mechanisms regulating osteoclast cytoskeleton is key to understand the mechanisms of bone resorption, in particular to propose new strategies against osteoporosis. We reported recently that {beta}-tubulin isotype TUBB6 is key for cytoskeleton organization in osteoclasts and for bone resorption. Here, using an osteoclast model CRISPR/Cas9 KO for Tubb6, we show that TUBB6 controls both microtubule and actin dynamics in osteoclasts. Osteoclasts KO for Tubb6 have reduced microtubule growth speed with longer growth life time, higher levels of acetylation and smaller EB1-caps. On the other hand, lack of TUBB6 increases podosome life time while the belt of podosomes is destabilized. Finally, we performed proteomic analyses of osteoclast microtubule-associated protein enriched fractions. This highlighted ARHGAP10 as a new microtubule-associated protein, which binding to microtubules appears to be negatively regulated by TUBB6. ARHGAP10 is a negative regulator of CDC42 activity, which participates in actin organization in osteoclasts. Our results suggest that TUBB6 plays a key role in the control of microtubule and actin cytoskeleton dynamics in osteoclasts. Moreover, by controlling ARHGAP10 association with microtubules, TUBB6 may participate in the local control CDC42 activity to ensure efficient bone resorption.

category: cell biology


title: IRE1α drives lung epithelial progenitor dysfunction to establish a niche for pulmonary fibrosis

abstract: Idiopathic pulmonary fibrosis (IPF) is a disease of progressive interstitial fibrosis, which leads to severe debilitation, respiratory failure, and death. In IPF, environmental exposures interact with genetic risk factors to engender critical patho-etiological events in lung epithelial cells, including endoplasmic reticulum (ER) stress and TGF{beta} signaling, but the interactions between these disparate pathways are not well understood. We previously showed that kinase inhibitors of the IRE1 bifunctional kinase/RNase--a central mediator of the unfolded protein response (UPR) to ER stress--protected mice from bleomycin-induced pulmonary fibrosis. Here we show that a nanomolar-potent, mono-selective kinase inhibitor of IRE1 (KIRA8) decreases ER-stress induced TGF{beta} signaling and the senescence-associated secretory phenotype (SASP) in the lung epithelium after bleomycin exposure. A recently-described subset of "damage-associated transient progenitors" (DATPs) display IRE1-regulated pathological gene signatures that are quelled by KIRA8, in vivo. After injury, these cells uniquely express integrin v{beta}6, a key activator of TGF{beta} in pulmonary fibrosis. KIRA8 inhibition of IRE1 decreases both DATP number and Itgb6 expression in remaining cells, with a decrease in local collagen accumulation. Single-cell RNA sequencing from IPF lungs revealed an analogous Itgb6+ cell population that may also be regulated by IRE1. These findings suggest that lung epithelial progenitor cells sit at the center of the fibrotic niche, and IRE1 signaling locks them into a dysfunctional state that establishes and perpetuates pathological fibrosis.


title: C-type natriuretic peptide facilitates autonomic Ca2+ entry in growth plate chondrocytes for stimulating bone growth

abstract: The growth plates are cartilage tissues found at both ends of developing bones, and vital proliferation and differentiation of growth plate chondrocytes are primarily responsible for bone growth. C-type natriuretic peptide (CNP) stimulates bone growth by activating natriuretic peptide receptor 2 (NPR2) which is equipped with guanylate cyclase on the cytoplasmic side, but its signaling pathway is unclear in growth plate chondrocytes. We previously reported that transient receptor potential melastatin-like 7 (TRPM7) channels mediate intermissive Ca2+ influx in growth plate chondrocytes, leading to activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) for promoting bone growth. In this report, we provide experimental evidence indicating a functional link between CNP and TRPM7 channels. Our pharmacological data suggest that CNP-evoked NPR2 activation elevates cellular cGMP content and stimulates big-conductance Ca2+-dependent K+ (BK) channels as a substrate for cGMP-dependent protein kinase (PKG). BK channel-induced hyperpolarization likely enhances the driving force of TRPM7-mediated Ca2+ entry and seems to accordingly activate CaMKII. Indeed, ex vivo organ culture analysis indicates that CNP-facilitated bone growth is abolished by chondrocyte-specific Trpm7 gene ablation. The defined CNP signaling pathway, the NPR2-PKG-BK channel-TRPM7 channel-CaMKII axis, likely pinpoints promising target proteins for developing new therapeutic treatments for divergent growth disorders.


title: Proteomic Characterization of Isolated Arabidopsis Clathrin-Coated Vesicles Reveals Evolutionarily Conserved and Plant Specific Components

abstract: In eukaryotes, clathrin-coated vesicles (CCVs) facilitate the internalization of material from the cell surface as well as the movement of cargo in post-Golgi trafficking pathways. This diversity of functions is partially provided by multiple monomeric and multimeric clathrin adaptor complexes that provide compartment and cargo selectivity. The adaptor- protein AP-1 complex operates as part of the secretory pathway at the trans-Golgi network, while the AP-2 complex and the TPLATE complex (TPC) jointly operate at the plasma membrane to execute clathrin-mediated endocytosis. Key to our further understanding of clathrin-mediated trafficking in plants will be the comprehensive identification and characterization of the network of evolutionarily conserved and plant- specific core and accessory machinery involved in the formation and targeting of CCVs. To facilitate these studies, we have analyzed the proteome of enriched trans-Golgi network/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis cells. Tandem mass spectrometry analysis results were validated by differential chemical labeling experiments to identify proteins co-enriching with CCVs. Proteins enriched in CCVs included previously characterized CCV components and cargos such as the vacuolar sorting receptors in addition to conserved and plant-specific components whose function in clathrin-mediated trafficking has not been previously defined. Notably, in addition to AP-1 and AP-2, all subunits of the AP-4 complex, but not AP-3 or AP-5, were found to be in high abundance in the CCV proteome. The association of AP-4 with suspension-cultured Arabidopsis CCVs is further supported via additional biochemical data.


title: Small molecule screen employing patient-derived iPS hepatocytes identifies LRRK2 as a novel therapeutic target for Alpha1 Antitrypsin Deficiency

abstract:
Alpha-1 antitrypsin deficiency is a life-threatening condition caused by inheritance of the SERPINA1 gene  Z variant. This single base pair mutation leads to protein misfolding, ER entrapment and gain of toxic function. Despite the significant unmet medical need presented by this disorder, there remain no approved medicines and the only curative option is liver transplantation. We hypothesized that an unbiased screen of human hepatocytes harbouring the Z mutation (ATZ) using small molecules targeted against protein degradation pathways would uncover novel biological insights of therapeutic relevance. Here we report the results of that screen performed in a patient-derived iPSC model of ATZ. Starting from 1,041 compounds we identified 14 targets capable of reducing polymer burden, including Leucine-rich repeat kinase-2 (LRRK2), a well-studied target in Parkinsons. Genetic deletion of LRRK2 in ATZ mice reduced polymers and associated fibrotic liver disease leading us to test a library of commercially available LRRK2 kinase inhibitors in both patient iPSC and CHO cell models. One of the molecules tested, CZC-25146, reduced polymer load, increased normal AAT secretion and reduced inflammatory cytokines with pharmacokinetic properties supporting its potential use for treating liver diseases. We therefore tested CZC-25146 in the ATZ mouse model and confirmed its efficacy for polymer reduction without signs of toxicity. Mechanistically, in both human and mouse models, our data show CZC-25146 inhibits LRRK2 kinase activity and induces autophagy. Cumulatively, these findings support the use of CZC-25146 and LRRK2 inhibitors in general in hepatic proteopathy disease research and as potential new treatment approaches for patients.

One Sentence SummaryA small molecule screen in patient iPSCs with in vivo validation in mice identifies LRRK2 as a new therapeutic target for Alpha-1 Antitrypsin Deficiency.


title: ZRC3308 monoclonal antibody cocktail shows protective efficacy in Syrian hamsters against SARS-CoV-2 infection

abstract: We have developed a monoclonal antibody (mAb) cocktail (ZRC-3308) comprising of ZRC3308-A7 and ZRC3308-B10 in the ratio 1:1 for COVID-19 treatment. The mAbs were designed to have reduced immune effector functions and increased circulation half-life. mAbs showed good binding affinities to non-competing epitopes on RBD of SARS-CoV-2 spike protein and were found neutralizing SARS-CoV-2 variants B.1, B.1.1.7, B.1.351, B.1.617.2 and B.1.617.2 AY.1 in vitro. The mAb cocktail demonstrated effective prophylactic and therapeutic activity against SARS-CoV-2 infection in Syrian hamsters. The antibody cocktail appears to be a promising candidate for the prophylactic use and for therapy in early COVID-19 cases which have not progressed to severe disease.


title: A novel hamster model of SARS-CoV-2 respiratory infection using a pseudotyped virus

abstract:
BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a biosafety level (BSL)-3 pathogen; therefore, its research environment is strictly limited. Pseudotyped viruses that mimic SARS-CoV-2 have been widely used for in vitro evaluation because they are available in BSL-2 containment laboratories; however, in vivo application is inadequate. Therefore, animal models that can be instigated with animal BSL-2 will increase opportunities for in vivo evaluations.

MethodsHamsters (6-to 10-week-old males) were intratracheally inoculated with luciferase-expressing vesicular stomatitis virus (VSV)-based SARS-CoV-2 pseudotyped virus. The lungs were harvested 24 h after inoculation, and luminescence was measured using an in vivo imaging system.

ResultsLung luminescence after inoculation with the SARS-CoV-2 pseudotyped virus increased in a dose-dependent manner. VSV-G (envelope [G]) pseudotyped virus also induced luminescence; however, a 100-fold concentration was required to reach a level similar to that of the SARS-CoV-2 pseudotyped virus.

ConclusionsThe SARS-CoV-2 pseudotyped virus is applicable to SARS-CoV-2 respiratory infections in a hamster model. Because of the single-round infectious virus, the model can be used to study the steps from viral binding to entry, which will be useful for future research regarding SARS-CoV-2 entry without using live SARS-CoV-2 or transgenic animals.


title: The nuts and bolts of SARS-CoV-2 Spike Receptor Binding Domain heterologous expression

abstract: COVID-19 is a highly infectious disease caused by a newly emerged coronavirus (SARS-CoV-2) that has rapidly progressed into a pandemic. This unprecedent emergency has stressed the significance of developing effective therapeutics to fight current and future outbreaks. The receptor-binding domain (RBD) of the SARS-CoV-2 surface Spike protein is the main target for vaccines and represents a helpful "tool" to produce neutralizing antibodies or diagnostic kits. In this work, we provide a detailed characterization of the native RBD produced in three major model systems: Escherichia coli, insect and HEK-293 cells. Circular dichroism, gel filtration chromatography and thermal denaturation experiments indicated that recombinant SARS-CoV-2 RBD proteins are stable and correctly folded. In addition, their functionality and receptor-binding ability were further evaluated through ELISA, flow cytometry assays and bio-layer interferometry.


title: RNANetMotif: identifying sequence-structure RNA network motifs in RNA-protein binding sites

abstract:
RNA molecules can adopt stable secondary and tertiary structures, which is essential in mediating physical interactions with other partners such as RNA binding proteins (RBPs) and in carrying out their cellular functions. In vivo and in vitro experiments such as RNAcompete and eCLIP have revealed in vitro binding preferences of RBPs to RNA oligomers and in vivo binding sites in cells. Analysis of these binding data showed that the structure properties of the RNAs in these binding sites are important determinants of the binding events; however, it has been a challenge to incorporate the structure information into an interpretable model. Here we describe a new approach, RNANetMotif, which takes predicted secondary structure of thousands of RNA sequences bound by an RBP as input and uses a graph theory approach to recognize enriched subgraphs. These enriched subgraphs are in essence shared sequence-structure elements that are important in RBP-RNA binding. To validate our approach, we performed RNA structure modeling via discrete molecular dynamics folding simulations for selected 4 RBPs, and RNA-protein docking for LIN28. The simulation results, e.g., solvent accessibility and energetics, further support the biological relevance of the discovered network subgraphs.

Author SummaryRNA binding proteins (RBPs) regulate every aspect of RNA biology, including splicing, translation, transportation, and degradation. High-throughput technologies such as eCLIP have identified thousands of binding sites for a given RBP throughout the genome. It has been shown by earlier studies that, in addition to nucleotide sequences, the structure and conformation of RNAs also play important role in RBP-RNA interactions. Analogous to protein-protein interactions or protein-DNA interactions, it is likely that there exist intrinsic sequence-structure motifs common to these RNAs that underlie their binding specificity to specific RBPs. It is known that RNAs form energetically favorable secondary structures, which can be represented as a graph, with nucleotides being nodes and backbone covalent bonds and base-pairing hydrogen bonds representing edges. We hypothesize that these graphs can be mined by graph theory approaches to identify sequence-structure motifs as enriched sub-graphs. In this article, we described the details of this approach, termed RNANetMotif and associated new concepts, namely EKS (Extended K-mer Subgraphs) and GraphK graph search algorithm. To test the utility of our approach, we conducted 3D structure modeling of selected RNA sequences through molecular dynamics (MD) folding simulation and evaluated the significance of the discovered RNA motifs by comparing their spatial exposure with other regions on the RNA. We believe that this approach has the novelty of treating the RNA sequence as a graph and RBP binding sites as enriched subgraph, which has broader applications beyond RBP-RNA interactions.


title: β-Catenin-NFκB-CFTR interactions in cholangiocytes regulate inflammation and fibrosis during ductular reaction

abstract: Expansion of biliary epithelial cells (BECs) during ductular reaction (DR) is observed in liver diseases including cystic fibrosis (CF), and associated with inflammation and fibrosis, albeit without complete understanding of underlying mechanism. Using two different genetic knockouts of {beta}-catenin, one with {beta}-catenin loss is hepatocytes and BECs (KO1), and another with loss in only hepatocytes (KO2), we demonstrate disparate long-term repair after an initial injury by 2-week choline-deficient ethionine- supplemented diet. KO2 show gradual liver repopulation with BEC-derived {beta}-catenin- positive hepatocytes, and resolution of injury. KO1 showed persistent loss of {beta}-catenin, NF-{kappa}B activation in BECs, progressive DR and fibrosis, reminiscent of CF histology. We identify interactions of {beta}-catenin, NF{kappa}B and CF transmembranous conductance regulator (CFTR) in BECs. Loss of CFTR or {beta}-catenin led to NF-{kappa}B activation, DR and inflammation. Thus, we report a novel {beta}-catenin-NF{kappa}B-CFTR interactome in BECs, and its disruption may contribute to hepatic pathology of CF.

category: pathology


title: Viscous effects and complex local flow behaviors dominate hemolymph circulation in the living wings of locusts

abstract: An insects living systems - circulation, respiration, and a branching nervous system - extend from the body into the wing.1,2. Hemolypmh circulation in the wing is critical for hydrating tissues, such as the highly elastic resilin3 that enhances wing flexibility, and for supplying nutrients to living systems, including sensory organs such as scent-producing patches, sound-receiving tympana, and wind-sensing sensilla distributed across the wing.4-7During flight, the presence of hemolymph in the wings reduces aerodynamic instabilities like flutter8,9, and faster hemolymph flows are induced by flapping.10 Despite the critical role of hemolymph circulation in maintaining healthy wing function, wings are often considered "lifeless" cuticle, and most measurements remain qualitative or employ coarse, bulk-flow techniques. While pioneering work in the 1960s mapped hemolymph flow direction in 100 insect species,11 half a century later we still only have quantitative measurements of flow within the wings of a few insects. Here, we focused on the North American locust Schistocerca americana, a well-studied agricultural pest species, and performed a detailed, quantitative study of global and local hemolymph flows in the densely venated fore and hind wings, along with key regions in the body and pumping organs. Through high-speed fluorescent microscopy, we measured 800 individual trajectories of neutrally buoyant fluorescent particles that move in sync with hemolymph, in the wings and body of 8 live, resting locusts. Our data show that overall flow within the wings is circuitous, but local flow behavior is highly complex, with three distinct types of flow (pulsatile, continuous, and "leaky") occurring in various combinations in different areas of the wing. We provide the first quantitative measurements of "leaky" flow into wing regions that act as sinuses, where hemolymph flows out of tubular veins and pools within thin membranous regions. We also calculate Peclet, Reynolds, and Womersley numbers, and find that viscous effects dominate flow regimes throughout the wing. Pumping organs and wing regions closest to the body display significantly faster flows and higher Reynolds numbers, but remain within the viscous flow regime. Given the central role of wings in sustaining ecologically important insect behaviors such as pollination, migration, and mating, along with the vast diversity of insect wings seen in nature, this first detailed, quantitative map of hemolymph flows across a wing provides a template for future studies investigating the dynamics of hemolymph flows critical to sustaining wing health among insects.

category: physiology


title: EXPLICIT-Kinase: a gene expression predictor for dissecting the functions of the Arabidopsis kinome

abstract: Protein kinases regulate virtually all cellular processes, but it remains challenging to determine the functions of all protein kinases, collectively called the kinome, in any species. We developed an approach called EXPLICIT-Kinase to predict the functions of the Arabidopsis kinome. Because the activities of many kinases can be regulated transcriptionally, their gene expression patterns provide clues to their functions. A universal gene expression predictor for Arabidopsis was constructed to predict the expression of 30,172 non-kinase genes based on the expression of 994 protein kinase genes. The model reconstituted highly accurate transcriptomes for diverse Arabidopsis samples. It identified the significant kinases as predictor kinases for predicting the expression of Arabidopsis genes and pathways. Strikingly, these predictor kinases were often known regulators of the related pathways, as exemplified by those involved in cytokinesis, tissue development, and stress responses. Comparative analyses have revealed that portions of these predictor kinases, including the novel ones, are shared and conserved between Arabidopsis and maize. The conservation between species provide additional evidence to support the novel predictor kinases as bona fide regulators of the pathways involved. Thus our approach enables the systematic dissection of the functions of the Arabidopsis kinome.

category: systems biology


title: High-order functional interactions in ageing explained via alterations in the connectome in a whole-brain model

abstract:
The human brain generates a rich repertoire of spatio-temporal activity patterns, which support a wide variety of motor and cognitive functions. These patterns of activity change with age in a multi-factorial manner. One of these factors is the variations in the brains connectomics that occurs along the lifespan. However, the precise relationship between high-order functional interactions and connnectomics, as well as their variations with age are largely unknown, in part due to the absence of mechanistic models that can efficiently map brain connnectomics to functional connectivity in aging. To investigate this issue, we have built a neurobiologically-realistic whole-brain computational model using both anatomical and functional MRI data from 161 participants ranging from 10 to 80 years old. We show that the age differences in high-order functional interactions can be largely explained by variations in the connectome. Based on this finding, we propose a simple neurodegeneration model that is representative of normal physiological aging. As such, when applied to connectomes of young participant it reproduces the age-variations that occur in the high-order structure of the functional data. Overall, these results begin to disentangle the mechanisms by which structural changes in the connectome lead to functional differences in the ageing brain. Our model can also serve as a starting point for modelling more complex forms of pathological ageing or cognitive deficits.

Author summaryModern neuroimaging techniques allow us to study how the human brains anatomical architecture (a.k.a. structural connectome) changes under different conditions or interventions. Recently, using functional neuroimaging data, we have shown that complex patterns of interactions between brain areas change along the lifespan, exhibiting increased redundant interactions in the older population. However, the mechanisms that underlie these functional differences are still unclear. Here, we extended this work and hypothesized that the variations of functional patterns can be explained by the dynamics of the brains anatomical networks, which are known to degenerate as we age. To test this hypothesis, we implemented a whole-brain model of neuronal activity, where different brain regions are anatomically wired using real connectomes from 161 participants with ages ranging from 10 to 80 years old. Analyzing different functional aspects of brain activity when varying the empirical connectomes, we show that the increased redundancy found in the older group can indeed be explained by precise rules affecting anatomical connectivity, thus emphasizing the critical role that the brain connectome plays for shaping complex functional interactions and the efficiency in the global communication of the human brain.


title: The Physiological Landscape and Specificity of Antibody Repertoires

abstract: Diverse antibody repertoires spanning multiple lymphoid organs (e.g., bone marrow, spleen, lymph nodes) form the foundation of protective humoral immunity. Changes in their composition across lymphoid organs are a consequence of B-cell selection and migration events leading to a highly dynamic and unique physiological landscape of antibody repertoires upon antigenic challenge (e.g., vaccination). However, to what extent B cells encoding identical or similar antibody sequences (clones) are distributed across multiple lymphoid organs and how this is shaped by the strength of a humoral response, remains largely unexplored. Here, we performed an in-depth systems analysis of antibody repertoires across multiple distinct lymphoid organs of immunized mice, and discovered that organ-specific antibody repertoire features (e.g., germline V-gene usage and clonal expansion profiles) equilibrated upon a strong humoral response (multiple immunizations and high serum titers). This resulted in a surprisingly high degree of repertoire consolidation, characterized by highly connected and overlapping B-cell clones across multiple lymphoid organs. Finally, we revealed distinct physiological axes indicating clonal migrations and showed that antibody repertoire consolidation directly correlated with antigen-specificity. Our study uncovered how a strong humoral response resulted in a more uniform but redundant physiological landscape of antibody repertoires, indicating that increases in antibody serum titers were a result of synergistic contributions from antigen-specific B-cell clones distributed across multiple lymphoid organs. Our findings provide valuable insights for the assessment and design of vaccine strategies.


title: Delayed implantation induced by letrozole in mice

abstract: Implantation timing is key for a successful pregnancy. Short delay in embryo implantation caused by targeted gene ablation produced a cascading problem in the later stages of the pregnancy. Although several delayed implantation models have been established in wild mice, almost none of them is suitable for investigating the delay on the late events of pregnancy. Here, we report a new delayed implantation model established by the intraperitoneally administration of letrozole at 5 mg/kg body weight on the day 3 of pregnancy. In these mice, initiation of implantation was induced at will by the injection of estradiol (E2). When the estradiol (3 ng) was injected on day 4 of pregnancy (i.e., without delay), the embryo implantation restarted, and the pregnancy continued normally. However, high dose of estrogen (25 ng) caused compromised implantation. We also found that only 67% of the female mice could be pregnant normally and finally gave birth when the injection of estradiol (3 ng) was on day 5 of pregnancy (i.e., one day delay). Most of the failed pregnancies had impaired decidualization, decreased plasma progesterone levels and compromised angiogenesis. Progesterone supplementation could rescue decidualization failure in the mice. Collectively, we established a new model of delayed implantation by letrozole, which can be easily used to study the effect and mechanisms of delay of embryo implantation on the progression of late pregnancy events.

category: developmental biology


title: Origin, specification and differentiation of a rare supporting-like lineage in the developing mouse gonad

abstract:
Gonadal sex determination represents a unique model for studying cell fate decisions. However, a complete understanding of the different cell lineages forming the developing testis and ovary remains elusive. Here, we investigated the origin, specification and subsequent sex-specific differentiation of a previously uncharacterized population of supporting-like cells (SLC) in the developing mouse gonads. The SLC lineage is closely related to the coelomic epithelium and specified as early as E10.5, making it the first somatic lineage to be specified in the bipotential gonad. SLC progenitors are localized within the genital ridge at the interface with the mesonephros and initially co-express Wnt4 and Sox9. SLCs become sexually dimorphic around E12.5, progressively acquire a Sertoli- or granulosa-like identity and contribute to the formation of the rete testis and rete ovarii. Finally, we found that WNT4 is a crucial regulator of the SLC lineage and is required for the formation of the rete testis.

TeaserDescription of an uncharacterized multipotent gonadal cell lineage involved in testis and ovary development


title: The biogeography of the Mediterranean Taurus Mountains: Illustrating how complex topography and climatic conditions have shaped their biodiversity

abstract: Climate is well known as the main driver of species distributions. In this study, I focused on Geyik Mountains and surrounding areas to illustrate how complex topography and climatic conditions have shaped the distribution patterns of species/communities and therefore the biodiversity of the Mediterranean Taurus Mountains, one of the most biologically diverse areas in the Mediterranean Basin biodiversity hotspot. Accordingly, I used an ecological niche modelling approach, which has been widely used in recent biogeographic studies. I chose Taurus ground squirrels (Spermophilus taurensis) and coniferous forests as the representatives of high- and low-altitude species/communities, respectively. The results simply illustrate how complex topography and temperature and precipitation gradients have had a substantial role in shaping the distribution patterns of species/communities and therefore the biodiversity of the Mediterranean Taurus Mountains.


title: Variation in colour signals among Sarracenia pitcher plants and the potential role of areoles in the attraction of flying Hymenoptera

abstract: Sarracenia insectivorous plants show a diversity of visual features in their pitchers but their perception by insects and their role in attraction, have received little attention. They also vary in prey composition, with some species trapping more flying Hymenoptera, such as bees. To test the hypothesis of a link between visual signal variability and prey segregation ability, and to identify which signal could attract flying Hymenoptera, we characterised, the colour patterns of 32 pitchers belonging to four taxa, modelled their perception by flying Hymenoptera, and examined the prey they trapped. The pitchers of the four taxa differed in colour patterns, with notably two long-leaved taxa displaying clear areoles, which contrasted strongly in colour and brightness with the vegetative background and with other pitcher areas in the eyes of flying Hymenoptera. These taxa trapped high proportion of flying hymenoptera. This suggests that contrasting areoles may act as a visual lure for flying Hymenoptera, making plants particularly visible to these insects. Prey capture also differed according to pitcher stage, morphology, season and visual characteristics. Further studies on prey visitation are needed to better understand the link between prey capture and attraction feature.


title: Evolution of degrees of carnivory and dietary specialization across Mammalia and their effects on speciation

abstract: Conflation between omnivory and dietary generalism limits ecological and evolutionary analyses of diet, including estimating contributions to speciation and diversification. Additionally, categorizing species into qualitative dietary classes leads to information loss in these analyses. Here, we constructed two continuous variables - degree of carnivory (i.e., the position along the continuum from complete herbivory to complete carnivory) and degree of dietary specialization (i.e., the number and variety of food resources utilized) - to elucidate their histories across Mammalia, and to tease out their independent contributions to mammalian speciation. We observed that degree of carnivory significantly affected speciation rate across Mammalia, whereas dietary specialization did not. We further considered phylogenetic scale in diet-dependent speciation and saw that degree of carnivory significantly affected speciation in ungulates, carnivorans, bats, eulipotyphlans, and marsupials, while the effect of dietary specialization was only significant in carnivorans. Across Mammalia, omnivores had the lowest speciation rates. Our analyses using two different categorical diet variables led to contrasting signals of diet-dependent diversification, and subsequently different conclusions regarding diets macroevolutionary role. We argue that treating variables such as diet as continuous instead of categorical reduces information loss and avoids the problem of contrasting macroevolutionary signals caused by differential discretization of biologically continuous traits.


title: Sound localization in world and head-centeredspace in ferrets

abstract:
The location of sounds can be described in multiple coordinate systems that are defined relative to ourselves, or the world around us. Evidence from neural recordings in animals point towards the existence of both head-centered and world-centered representations of sound location in the brain; however, it is unclear whether such neural representations have perceptual correlates in the sound localization abilities of non-human listeners. Here, we establish novel behavioral tests to determine the coordinate systems in which ferrets can localize sounds. We found that ferrets could learn to discriminate between sound locations that were fixed in either world-centered or head-centered space, across wide variations in sound location in the alternative coordinate system. Using probe sounds to assess broader generalization of spatial hearing, we demonstrated that in both head and world-centered tasks, animals used continuous maps of auditory space to guide behavior. Single trial responses of individual animals were sufficiently informative that we could then model sound localization using speaker position in specific coordinate systems and accurately predict ferrets actions in held-out data. Our results indicate that an animal model in which neurons are known to be tuned to sound location in egocentric and allocentric reference frames can also localize sounds in multiple head and world-centered spaces.

Significance StatementHumans can describe the location of sounds either relative to themselves, or in the world, independent of their momentary position. These different spaces are also represented in the activity of neurons in animals, but its not clear whether non-human listeners also perceive both head and world-centered sound location. Here, we designed two behavioral tasks in which ferrets had to discriminate between two sounds using their position in the world, or relative to the head. Subjects learnt to solve both problems and showed the ability to generalize sound location in each space when presented with infrequent probe sounds. These findings reveal a perceptual correlate of neural sensitivity previously observed in the ferret brain and establish that, like humans, ferrets can access an auditory map of their local environment.

category: animal behavior and cognition


title: Sound localization in world and head-centered space in ferrets


title: Tuna-AI: tuna biomass estimation with Machine Learning models trained on oceanography and echosounder FAD data

abstract: Echo-sounder data registered by buoys attached to drifting FADs provide a very valuable source of information on populations of tuna and their behaviour. This value increases when these data are supplemented with oceanographic data coming from CMEMS. We use these sources to develop TO_SCPLOWUNAC_SCPLOW-AI, a Machine Learning model aimed at predicting tuna biomass under a given buoy, which uses a 3-day window of echo-sounder data to capture the daily spatio-temporal patterns characteristic of tuna schools. As the supervised signal for training, we employ more than 5000 set events with their corresponding tuna catch reported by the AGAC tuna purse seine fleet.


title: MPTHub: an open-source software for characterizing the transport of particles in biorelevant media

abstract: The study of the transport of particles in different environments plays an essential role in understanding interactions with humans and other living organisms. Importantly, obtained data can be directly used for multiple applications in fields such as fundamental biology, toxicology or medicine. Particle movement in biorelevant media can be readily monitored using microscopy and converted into time-resolved trajectories using freely available tracking software. However, translation into tangible and meaningful parameters is time-consuming and not always intuitive. Thus, we developed a new software - MPTHub - as an open-access, stand-alone, user-friendly tool for the rapid and reliable analysis of particle trajectories extracted from video microscopy. The software was programmed using Python and allowed to import and analyze trajectory data, and export relevant data such as individual and ensemble time-averaged mean square displacements and effective diffusivity, and anomalous transport exponent. Data processing was reliable, fast (total processing time of less than 10 sec) and required minimal memory resources (up to a maximum of around 150 MB in RAM). Demonstration of software applicability was conducted by studying the transport of different polystyrene nanoparticles (100-200 nm) in mucus surrogates. Overall, MPTHub represents a freely available software tool that can be used even by unexperienced users for studying the transport of particles in biorelevant media.


title: Intestinal epithelial IPMK protects mice from experimental colitis via governing colonic tuft cell development

abstract: As a pleiotropic signaling factor, inositol polyphosphate multikinase (IPMK) is involved in key biological events such as growth and innate immunity, acting either enzymatically to mediate the biosynthesis of inositol polyphosphates and phosphatidylinositol 3,4,5-trisphosphates, or noncatalytically to control key signaling target molecules. However, the functional significance of IPMK in regulating gut epithelial homeostasis remains largely unknown. Here we show that intestinal epithelial-specific deletion of IPMK aggravates dextran sulfate sodium (DSS)-induced colitis with higher clinical colitis scores and elevated epithelial barrier permeability. No apparent defects in PI3K-AKT signaling pathway and pro-inflammatory cytokine production were found in IPMK-deficient colons challenged by DSS treatment. RNA-sequencing and FACS analyses further revealed significantly decreased tuft cells in IPMK-deficient colons. Importantly, IPMK deletion in the gut epithelium was found to decrease choline acetyltransferase (ChAT) but not IL-25, suggesting selective loss of cholinergic signaling. Thus, these findings identify IPMK as a physiological determinant of tuft cell differentiation and highlight the critical function of IPMK in the control of gut homeostasis.


title: Multimodal Detection of Dopamine by Sniffer Cells Expressing Genetically Encoded Fluorescence Sensors

abstract: Dopamine serves an important role in supporting both locomotor control and higher brain functions such as motivation and learning. Dopaminergic dysfunction is implicated in an equally multidimensional spectrum of neurological and neuropsychiatric diseases. Extracellular dopamine levels are known to be tightly controlled by presynaptic dopamine transporters (DAT), which is also a main target of psychostimulants. Still, detailed data on dopamine dynamics in space and time is needed to fully understand how dopamine signals are encoded and translated into cellular and behavioral responses, and to uncover the pathological effects of dopamine-related diseases. The recently developed genetically encoded fluorescent dopamine sensors enable unprecedented monitoring of dopamine dynamics and have changed the field of in vivo dopamine recording. However, the potential of these sensors to be used for in vitro and ex vivo assays remains unexplored. Here, we demonstrate a generalizable blueprint for making "sniffer" dopamine cells for multimodal detection of dopamine in vitro and ex vivo. We generated sniffer cell lines with inducible expression of six different dopamine sensors and performed a head-to-head comparison of sensor properties to guide users in sensor selection. In proof-of-principle experiments, we show how the sniffer cells can be applied to measure release of endogenous dopamine from cultured neurons and striatal slices, and for determining total dopamine content in striatal tissue. Furthermore, we use the sniffer cells to quantify DAT-mediated dopamine uptake, and AMPH-induced and constitutive dopamine efflux as a radiotracer free, high-throughput alternative to electrochemical- and radiotracer-based assays. Importantly, the sniffer cells framework can readily be applied to other transmitter systems for which the list of genetically encoded fluorescent sensors is rapidly growing.


title: Principles governing the topological organization of object selectivities in ventral temporal cortex

abstract: To achieve the computational goal of rapidly recognizing miscellaneous objects in the environment despite large variations in their appearance, our mind represents objects in a high-dimensional object space to provide separable category information and enable the extraction of different kinds of information necessary for various levels of the visual processing. To implement this abstract and complex object space, the ventral temporal cortex (VTC) develops different object-selective regions with certain topological organization as the physical substrate. However, the principle that governs the topological organization of object selectivities in the VTC remains unclear. Here, equipped with the wiring cost minimization principle constrained by the wiring length of neurons in human temporal lobe, we constructed a hybrid self-organizing map (SOM) model as an artificial VTC (VTC-SOM) to explain how the abstract and complex object space is faithfully implemented in the brain. In two in silico experiments with the empirical brain imaging and single-unit data, our VTC-SOM predicted the topological structure of fine-scale functional regions (face-, object-, body-, and place-selective regions) and the boundary (i.e., middle Fusiform Sulcus) in large-scale abstract functional maps (animate vs. inanimate, real-word large-size vs. small-size, central vs. peripheral), with no significant loss in functionality (e.g., categorical selectivity, a hierarchy of view-invariant representations). These findings illustrated that the simple principle utilized in our model, rather than multiple hypotheses such as temporal associations, conceptual knowledge, and computational demands together, was apparently sufficient to determine the topological organization of object-selectivities in the VTC. In this way, the high-dimensional object space is implemented in a two-dimensional cortical surface of the brain faithfully.