title: BH3 Profiling Identifies Selective BCL-2 Dependence of Adult Early T-cell Progenitor (ETP) Subtype of Acute Lymphoblastic Leukemia (ALL) Patients

abstract:
Early T-cell precursor (ETP) acute lymphoblastic leukemia (T-ALL) is a high-risk subtype of T-ALL and is associated with poor survival outcomes with chemotherapy. We previously showed that maturation stage of thymocytes distinguishes pro-survival dependencies of pediatric ETP-ALL (BCL-2 dependent) from non-ETP-ALL (BCL-XL dependent). Comparable data in adults are lacking. Our present study focuses on characterizing functional dependencies of adult ETP-ALL and T-ALL on BCL-2 family proteins. Using BH3 profiling on primary tumors, we report that similar to pediatric ALL, adult ETP-ALL is primarily dependent on BCL-2; however, unlike pediatric ALL, adult non-ETP-ALL is co-dependent on both BCL-2 and BCL-XL for survival. By measuring direct mitochondrial permeabilization and cell viability assays, we validated that BH3 profiling predicted on-target cytotoxicity of venetoclax in adult ETP-ALL and navitoclax plus venetoclax in adult T-ALL. These findings provide pre-clinical evidence for venetoclax and navitoclax as potentially efficacious combination therapy for adults with T-ALL.

Statement of SignificanceAdults with relapsed T-ALL have poor prognosis and represent an unmet need for novel treatments. We report that adult ETP-ALL is dependent on BCL-2 for survival, similar to pediatric ETP-ALL. However, adult non-ETP-ALL is heterogeneously dependent on both BCL-2 and BCL-XL. We characterize key dependencies on BCL-2 family proteins to direct BH3 mimetics therapy in T-ALL patients.


title: Cellulose synthase-like D (CSLD) proteins move in the plasma membrane and their targeting to cell tips, but not cell plates, depends on the actin cytoskeleton

abstract: Cellulose Synthase-Like D (CSLD) proteins are implicated in cell wall remodeling during tip growth and cell division in plants, and are known to generate {beta}-1,4-glucan. It is unknown whether they form complexes and move in the plasma membrane like members of the Cellulose Synthase (CESA) family. We used the genetically tractable moss Physcomitrium patens, which has a filamentous protonemal stage that undergoes both tip growth and cell division and is amenable to high resolution live cell imaging, to investigate CSLD function and intracellular trafficking. CSLD2 and CSLD6 are highly expressed in gametophores and are redundantly required for gametophore cellular patterning. Live cell imaging revealed that CSLD6 is also expressed in protonemata where it moves in the plasma membrane and localizes to cell plates and cell tips. Notably, delivery to the apical plasma membrane, but not the cell plate, depends on actin. By comparing the behavior of endogenously tagged CSLD6 and CESA10, we discovered that CSLD6 movements in the plasma membrane were significantly faster, shorter in duration and less linear than CESA10 movements and were insensitive to the cellulose synthesis inhibitor isoxaben. These data suggest that CSLD6 and CESA10 function within different structures and may thus produce structurally distinct cellulose microfibrils.

category: plant biology


title: High-throughput phenotyping of leaf discs infected withgrapevine downy mildew using shallow convolutionalneural networks

abstract: Objective and standardized recording of disease severity in mapping crosses and breeding lines is a crucial step in characterizing resistance traits utilized in breeding programs and to conduct QTL or GWAS studies. Here we report a system for automated high-throughput scoring of disease severity on inoculated leaf discs. As proof of concept, we used leaf discs inoculated with Plasmopara viticola causing grapevine downy mildew (DM). This oomycete is one of the major grapevine pathogens and has the potential to reduce grape yield dramatically if environmental conditions are favorable. Breeding of DM resistant grapevine cultivars is an approach for a novel and more sustainable viticulture. This involves the evaluation of several thousand inoculated leaf discs from mapping crosses and breeding lines every year. Therefore, we trained a shallow convolutional neural-network (SCNN) for efficient detection of leaf disc segments showing P. viticola sporangiophores. We could illustrate a high and significant correlation with manually scored disease severity used as ground truth data for evaluation of the SCNN performance. Combined with an automated imaging system, this leaf disc-scoring pipeline has the potential to reduce the amount of time during leaf disc phenotyping considerably. The pipeline with all necessary documentation for adaptation to other pathogens is freely available.


title: The active zone protein Clarinet regulates ATG-9 trafficking at synapses and presynaptic autophagy

abstract:
In neurons, autophagy is temporally and spatially regulated to occur near presynaptic sites. How trafficking of autophagy proteins is regulated to support synaptic autophagy is not well understood. From forward genetic screens, we identify a role for the long isoform of the active zone protein Clarinet (CLA-1L) in regulating trafficking of autophagy protein ATG-9 at synapses, and presynaptic autophagy. ATG-9 is a transmembrane protein that undergoes activity-dependent exo-endocytosis at synapses, and mutations in CLA-1L result in abnormal accumulation of ATG-9 into clathrin-rich endocytic intermediates. CLA-1L extends from the active zone to the periactive zone, and genetically interacts with periactive zone proteins required for clathrin-dependent endocytosis. We find that CLA-1L is specifically required for sorting of ATG-9 at synapses, likely via endosome-mediated endocytosis, and for activity-dependent presynaptic autophagy. Our findings provide mechanistic insights into how active zone proteins regulate key steps of ATG-9 exo-endocytosis, a process that could couple the activity state of the neuron and autophagy.

HighlightsO_LI* The long isoform of the active zone protein Clarinet (CLA-1L) regulates ATG-9 trafficking at synapses
C_LIO_LI* CLA-1L extends from the active zone to the periactive zone and cooperates with the periactive zone endocytic proteins EHS-1/EPS15 and ITSN-1/ intersectin 1 in ATG-9 trafficking during exo-endocytosis
C_LIO_LI* Mutations in CLA-1L, or in clathrin-associated adaptor molecules, result in abnormal accumulation of ATG-9 into clathrin-rich endocytic intermediates
C_LIO_LI* CLA-1L mutants which affect ATG-9 trafficking are also defective in activity-dependent presynaptic autophagy
C_LI

category: neuroscience


title: Immersion in Modified Reality: Sensitivity to the Slope (α) of the Amplitude Spectrum is Dependent on the α of Recently Viewed Environments

abstract: Scenes contain many statistical regularities that could benefit visual processing if accounted for by the visual system. One such statistic is the orientation-averaged slope () of the amplitude spectrum of natural scenes. Human observers show different discrimination sensitivity to : sensitivity is highest for  values between 1.0 and 1.2 and decreases as  is steepened or shallowed. The range of  for peak discrimination sensitivity is concordant with the average  of natural scenes, which may indicate that visual mechanisms are optimized to process information at  values commonly encountered in the environment. Here we explore the association between peak discrimination sensitivity and the most viewed s in natural environments. Specifically, we verified whether discrimination sensitivity depends on the recently viewed environments. Observers were immersed, using a Head-Mounted Display, in an environment that was either unaltered or had its average  steepened or shallowed by 0.4. Discrimination thresholds were affected by the average shift in , but this effect was most prominent following adaptation to a shallowed environment. We modeled these data with a Bayesian observer and explored whether a change in the prior or a change in the likelihood best explained the psychophysical effects. Change in discrimination thresholds following adaptation could be explained by a shift in the central tendency of the prior concordant with the shift of the environment, in addition to a change in the likelihood. Our findings suggest that expectations on the occurrence of  that result from a lifetime of exposure remain plastic and able to accommodate for the statistical structure of recently viewed environments.


title: Sensitivity to the slope (α) of the amplitude spectrum is dependent on the α of Recently Viewed Environments: A visual adaptation study in modified reality


title: Sensitivity to the slope of the amplitude spectrum is dependent on the spectral slopes of recently viewed environments: A visual adaptation study in modified reality


title: Modeling Neural Variability in Deep Networks with Dropout

abstract: Convolutional neural networks (CNNs) have been used to model the biological visual system. Compared to other models, CNNs can better capture neural responses to natural stimuli. However, previous successes are limited to modeling mean responses; while another fundamental aspect of cortical activity, namely response variability, is ignored. How the CNN models capture neural variability properties remains unknown. Previous computational neuroscience studies showed that the response variability can have a functional role, and found that the correlation structure (especially noise correlation) influences the amount of information in the population code. However, CNN models are typically deterministic, so noise (and correlations) in CNN models have not been studied. In this study, we developed a CNN model of visual cortex that includes neural variability. The model includes Monte Carlo dropout, namely a random subset of units is silenced at each presentation of the input image, inducing variability in the model. We found that our model captured a wide-range of neural variability findings in electrophysiology experiments, including that response mean and variance scale together, noise correlations are small but positive on average, both evoked and spontaneous noise correlation are larger for neurons with similar tuning, and the noise covariance is low-dimensional. Further, we found that removing the correlation can boost trial-by-trial decoding performance in the CNN model.


title: Corticostriatal suppression of appetitive Pavlovian conditioned responding

abstract:
The capacity to suppress learned responses is essential for animals to adapt in dynamic environments. Extinction is a process by which animals learn to suppress conditioned responding when an expected outcome is omitted. The infralimbic cortex (IL) to nucleus accumbens shell (NAcS) neural circuit is implicated in suppressing conditioned responding after extinction, especially in the context of operant cocaine-seeking behaviour. However, the role of the IL-to-NAcS neural circuit in the extinction of responding to appetitive Pavlovian cues is unknown and the psychological mechanisms involved in response suppression following extinction are unclear. We trained rats to associate a 10 s auditory conditioned stimulus (CS; 14 trials per session) with a sucrose unconditioned stimulus (US; 0.2 mL per CS) in a specific context and then, following extinction in a different context, precipitated a renewal of CS responding by presenting the CS alone in the original Pavlovian conditioning context. Unilateral, optogenetic stimulation of the IL-to-NAcS circuit selectively during CS trials suppressed renewal. In a separate experiment, IL-to-NAcS stimulation suppressed CS responding regardless of prior extinction and impaired extinction retrieval. Finally, IL-to-NAcS stimulation during the CS did not suppress the acquisition of Pavlovian conditioning but was required for the subsequent expression of CS responding. These results are consistent with multiple studies showing that the IL-to-NAcS neural circuit is involved in the suppression of operant cocaine-seeking, extending these findings to appetitive Pavlovian cues. The suppression of appetitive Pavlovian responding following IL-to-NAcS circuit stimulation does not, however, appear to require an extinction-dependent process.

SIGNIFICANCE STATEMENTExtinction is a form of inhibitory learning through which animals learn to suppress conditioned responding in the face of non-reinforcement. We investigated the role of infralimbic (IL) cortex inputs to the nucleus accumbens shell (NAcS) in the extinction of responding to appetitive Pavlovian cues and the psychological mechanisms involved in response suppression following extinction. Using in vivo optogenetics, we found that stimulating the IL-to-NAcS neural circuit suppressed context-induced renewal of conditioned responding after extinction. In a separate experiment, stimulating the IL-to-NAcS circuit suppressed conditioned responding in an extinction-independent manner. These findings can be leveraged by future research aimed at understanding how corticostriatal circuits contribute to behavioural flexibility and mental disorders that involve the suppression of learned behaviours.


title: Lacking social support is associated with structural divergences in hippocampus-default network co-variation patterns

abstract: Elaborate social interaction is a pivotal asset of the human species. The complexity of peoples social lives may constitute the dominating factor in the vibrancy of many individuals environment. The neural substrates linked to social cognition thus appear especially susceptible when people endure periods of social isolation: here, we zoom in on the systematic inter-relationships between two such neural substrates, the allocortical hippocampus (HC) and the neocortical default network (DN). Previous human social neuroscience studies have focused on the DN, while HC subfields have been studied in most detail in rodents and monkeys. To bring into contact these two separate research streams, we directly quantified how DN subregions are coherently co-expressed with specific HC subfields in the context of social isolation. A two-pronged decomposition of structural brain scans from [~]40,000 UK Biobank participants linked lack of social support to mostly lateral subregions in the DN patterns. This lateral DN association co-occurred with HC patterns that implicated especially subiculum, presubiculum, CA2, CA3, and dentate gyrus. Overall, the subregion divergences within spatially overlapping signatures of HC-DN co-variation followed a clear segregation divide into the left and right brain hemispheres. Separable regimes of structural HC-DN co-variation also showed distinct associations with the genetic predisposition for lacking social support at the population level.


title: A novel lineage-tracing mouse model for MmuPV1 infection enables in vivo studies in the absence of cytopathic effects

abstract:
Human papillomaviruses (HPVs) are DNA viruses that ubiquitously infect humans and have been associated with hyperproliferative lesions. The recently discovered mouse specific papillomavirus, MmuPV1, provides the opportunity to study papillomavirus infections in vivo in the context of a common laboratory mouse model (Mus musculus). To date, a major challenge in the field has been the lack of tools to identify, observe and characterize individually the papillomavirus hosting cells and also trace the progeny of these cells over time.

Here, we present the successful generation of an in vivo lineage-tracing model of MmuPV1-harboring cells and their progeny by means of genetic reporter activation. Following the validation of the system both in vitro and in vivo, we used it to provide a proof-of-concept of its utility. Using flow-cytometry analysis, we observed increased proliferation dynamics and decreased MHC-I cell surface expression in MmuPV1-treated tissues which could have implications in tissue regenerative capacity and ability to clear the virus. This model is a novel tool to study the biology of the MmuPV1 host-pathogen interactions.

category: microbiology


title: Single-cell transcriptomic assessment of cellular phenotype stability in human precision-cut lung slices

abstract: Precision-cut lung slices (PCLS) are increasingly utilized for ex vivo disease modeling, but a high-resolution characterization of cellular phenotype stability in PCLS has not been reported. Comparing the single-cell transcriptomic profile of human PCLS after five days of culture to freshly isolated human lung tissue, we found striking changes in endothelial cell and alveolar epithelial cell programs, reflecting both injury and pathways activated in static culture, while immune cell frequencies and programs remained largely intact and similar to the native lung. These cellular dynamics should be considered when utilizing PCLS as a model of the human lung.

category: cell biology


title: Isolation, omics characterization and organotypic culture of alveolar type II pulmonary epithelial cells

abstract: The alveolar type II (AT2) epithelial cell fraction includes the stem cells of the pulmonary alveoli, functioning in lung homeostasis and post-injury repair. AT2 cells have been characterized primarily in situ, in transgenic mouse models. We report a new methodology for their isolation, their "omics" characterization and stroma-cell-free organotypic culture. Our multi-omics analysis identified high expression of genes involved in oxidative phosphorylation and of AP-1 components, as well as new phosphorylation sites in AT2 biomarkers. Furthermore, we show that supplementation with KGF, FGF10 & HGF suffices for the in vitro proliferation of AT2 cells and formation of alveolar organoids, suggesting that AT2-based organotypic development depends on ligands of the c-Met and FGFR2 receptors. The reported methodology and in-depth molecular characterization provide new tools for the in vitro and in vivo functional analysis of pulmonary cells and of mouse models of lung disease.


title: A human stem cell resource to decipher the biochemical and cellular basis of neurodevelopmental defects in Lowe Syndrome

abstract: Human brain development is a complex process where multiple cellular and developmental events are co-ordinated to generate normal structure and function. Alteration in any of these events can impact brain development, manifesting clinically as neurodevelopmental disorders. Human genetic disorders of lipid metabolism often present with features of altered brain function. Lowe syndrome (LS), is a X-linked recessive disease with features of altered brain function. LS results from mutations in OCRL1 that encodes a phosphoinositide 5-phosphatase enzyme. However, the cellular mechanisms by which loss of OCRL1 leads to brain defects remain unknown. Human brain development involves several cellular and developmental features not conserved in other species and understanding such mechanisms remains a challenge. Rodent models of LS have been generated, but failed to recapitulate features of the human disease. Here we describe the generation of human stem cell lines from LS patients. Further, we present biochemical characterization of lipid metabolism in patient cell lines and demonstrate their use as a "disease-in-a-dish" model for understanding the mechanism by which loss of OCRL1 leads to altered cellular and physiological brain development.


title: Shaping coral traits: plasticity more than filtering

abstract: The structure of ecosystems is usually determined by the shape of the organisms that build it, commonly known as ecosystem engineers. Understanding to what extent plasticity and environmental filtering determine variation in ecosystem engineer physical structure is necessary to predict how ecosystem structure may change. Here, we explored coral survival and the plasticity of morphological traits that are critical for habitat provision in coral reefs. We conducted a reciprocal clonal transplant experiment in which branching corals from the genus Porites and Acropora were moved to and from a deep and a shallow site within a lagoon in the Maldives. Survival and trait analyses showed that transplant destination consistently induced the strongest changes, particularly among Acropora spp. The origin of the corals only marginally affected some of the traits. We also detected variation in the way individuals from the same species and site differentiate their shape, showing that traits linked to habitat provision are phenotypically plastic. The results suggest coral phenotypic plasticity plays a stronger role than environmental filtering, in determining zonation of coral morphologies, and consequently the habitats they provide for other taxa.

category: ecology


title: A generic sequential stimulation adapter for reducing muscle fatigue during functional electrical stimulation

abstract:
BackgroundClinical applications of conventional functional electrical stimulation (FES) administered via a single electrode is limited by rapid onset neuromuscular fatigue. "Sequential" (SEQ) stimulation, involving rotation of pulses between multiple active electrodes, has been shown to reduce fatigue compared to conventional FES. However, there has been limited adoption of SEQ in research and clinical settings.

MethodsThe SEQ adapter is a small, battery-powered device that transforms the output of any commercially available electrical stimulator into SEQ stimulation. We examined the output of the adaptor across a range of clinically relevant stimulation pulse parameters to verify the signal integrity preservation ability of the SEQ adapter. Pulse frequency, amplitude, and duration were varied across discrete states between 4-200 Hz, 10-100 mA, and 50-2000 s, respectively.

ResultsA total of 420 trials were conducted, with 80 stimulation pulses per trial. The SEQ adapter demonstrated excellent preservation of signal integrity, matching the pulse characteristics of the originating stimulator within 1% error. The SEQ adapter operates as expected at pulse frequencies up to 160 Hz, with a noted failure mode at 200 Hz.

ConclusionThe SEQ adapter represents an effective and low-cost solution to increase the utilization of SEQ in existing rehabilitation paradigms.

category: bioengineering


title: Multi view based imaging genetics analysis on Parkinson disease

abstract: Longitudinal studies integrating imaging and genetic data have recently become widespread among bioinformatics researchers. Combining such heterogeneous data allows a better understanding of complex diseases origins and causes. Through a multi-view based workflow proposal, we show the common steps and tools used in imaging genetics analysis, interpolating genotyping, neuroimaging and transcriptomic data. We describe the advantages of existing methods to analyze heterogeneous datasets, using Parkinsons Disease (PD) as a case study. Parkinsons disease is associated with both genetic and neuroimaging factors, however such imaging genetics associations are at an early investigation stage. Therefore it is desirable to have a free and open source workflow that integrates different analysis flows in order to recover potential genetic biomarkers in PD, as in other complex diseases.

category: bioinformatics


title: Tools and applications for integrative analysis of DNA methylation in social insects

abstract:
DNA methylation is a common epigenetic signaling tool and an important biological process which is widely studied in a large array of species. The presence, level, and function of DNA methylation varies greatly across species. In insects, DNA methylation systems are reduced, and methylation rates are often low. Low methylation levels probed by whole genome bisulfite sequencing require great care with respect to data quality control and interpretation. Here we introduce BWASP/R, a complete workflow that allows efficient, scalable, and entirely reproducible analyses of raw DNA methylation sequencing data. Consistent application of quality control filters and analysis parameters provides fair comparisons among different studies and an integrated view of all experiments on one species. We describe the capabilities of the BWASP/R workflow by re-analyzing several publicly available social insect WGBS data sets, comprising 70 samples and cumulatively 147 replicates from four different species. We show that the CpG methylome comprises only about 1.5% of CpG sites in the honeybee genome and that the cumulative data are consistent with genetic signatures of site accessibility and physiological control of methylation levels.

Significance StatementDNA methylation in the honeybee genome occurs almost entirely at CpG sites. Methylation rates are small compared to rates in mammalian or plant genomes. De novo analysis of all published honeybee methylation studies and statistical modeling suggests that the CpG methylome consists of about only 300,000 sites. The development of a fully reproducible, scalable, portable workflow allows for easy accessible updates of integrative views of all current experiments. The integrated results for the honeybee are consistent with genetic determination of methylation site accessibility by yet uncharacterized sequence features and physiological control of methylation levels at those sites.


title: MINK1 modulates 5FU resistance in OSCC through AKT/MDM2 mediated regulation of p53

abstract: Cisplatin, 5FU and docetaxel (TPF) are the most common chemotherapy regimen used for advanced OSCC. However, many cancer patients experience relapse, continued tumor growth, and spread due to drug resistance, which leads to treatment failure and metastatic disease. Here, using a CRISPR/Cas9 based kinome knockout screening, Misshapen-like kinase 1 (MINK1) is identified as an important mediator of 5FU resistance in OSCC. Analysis of clinical samples demonstrated significantly higher MINK1 expression in the tumor tissues of chemotherapy non-responder as compared to chemotherapy responders. The nude mice and zebrafish xenograft experiments indicate that knocking out MINK1 restores 5FU mediated cell death in chemoresistant OSCC. An antibody based phosphorylation array screen revealed MINK1 as a negative regulator of p53. Mechanistically, MINK1 modulates AKT phosphorylation at Ser473, which enables p-MDM2 (Ser 166) mediated degradation of p53. We also identified lestaurtinib as a potent inhibitor of MINK1 kinase activity. The patient derived TPF resistant cell based xenograft data suggest that lestaurtinib restores 5FU sensitivity and facilitates a significant reduction of tumor burden. Overall, our study suggests that MINK1 is a major driver of 5FU resistance in OSCC. The novel combination of MINK1 inhibitor lestaurtinib and 5FU needs further clinical investigation in advanced OSCC.

category: cancer biology


title: CRISPR-based kinome-screening revealed MINK1 as a druggable player to rewire 5FU-resistance in OSCC through AKT/MDM2/p53 axis


title: An siRNA-guided Argonaute protein directs RNA Polymerase V to initiate DNA methylation

abstract: In mammals and plants, cytosine DNA methylation is essential for the epigenetic repression of transposable elements and foreign DNA. In plants, DNA methylation is guided by small interfering RNAs (siRNAs) in a self-reinforcing cycle termed RNA-directed DNA methylation (RdDM). RdDM requires the specialized RNA Polymerase V (Pol V), and the key unanswered question is how Pol V is first recruited to new target sites without preexisting DNA methylation. We find that Pol V follows and is dependent upon the recruitment of an AGO4-clade ARGONAUTE protein, and any siRNA can guide the ARGONAUTE protein to the new target locus independent of preexisting DNA methylation. These findings reject long-standing models of RdDM initiation and instead demonstrate that siRNA-guided ARGONAUTE targeting is necessary, sufficient and first to target Pol V recruitment and trigger the cycle of RdDM at a transcribed target locus, thereby establishing epigenetic silencing.


title: Improving robustness of 3D multi-shot EPI by structured low-rank reconstruction of segmented CAIPI sampling for fMRI at 7T

abstract:
Three-dimensional (3D) encoding methods are increasingly being explored as alternatives to multi-slice two-dimensional (2D) acquisitions in fMRI, particularly in cases where high isotropic resolution is needed. 3D multi-shot EPI is the most popular 3D fMRI acquisition method, but is susceptible to physiological fluctuations which can induce inter-shot phase variations, and thus reducing the achievable tSNR, negating some of the benefit of 3D encoding. This issue can be particularly problematic at ultra-high fields like 7T, which have more severe off-resonance effects. In this work, we aim to improve the temporal stability of 3D multi-shot EPI at 7T by improving its robustness to inter-shot phase variations. We presented a 3D segmented CAIPI sampling trajectory ("seg-CAIPI") and an improved reconstruction method based on Hankel structured low-rank matrix recovery. Simulation and in-vivo results demonstrate that the combination of the seg-CAIPI sampling scheme and the proposed structured low-rank reconstruction is a promising way to effectively reduce the unwanted temporal variance induced by inter-shot physiological fluctuations, and thus improve the robustness of 3D multi-shot EPI for fMRI.

HighlightsO_LIA 3D multi-shot EPI sampling trajectory using interleaved ordering along kz and a CAIPI blipping pattern improves robustness to inter-shot phase variations
C_LIO_LIReconstruction based on Hankel structured low-rank matrix completion can significantly improve the temporal stability of 3D multi-shot acquisitions at 7T
C_LIO_LI1.5mm resolution brain fMRI data show that ~60% improvement in mean tSNR can be obtained using the proposed method compared to the conventional method
C_LIO_LI1.8mm resolution brain fMRI data demonstrate that the proposed method allows for 4-fold acceleration without loss of tSNR compared to conventional 3D EPI
C_LIO_LIPreliminary brainstem fMRI data show that ~40% improvement in mean tSNR can be obtained using the proposed sampling and reconstruction
C_LI


title: Parallel pathways for rapid odor processing in lateral entorhinal cortex: Rate and temporal coding by layer 2 subcircuits

abstract: Olfactory information is encoded in lateral entorhinal cortex (LEC) by two classes of layer 2 (L2) principal neurons: fan and pyramidal cells. However, the functional properties of L2 neurons are unclear. Here, we show in awake mice that L2 cells respond rapidly to odors during single sniffs and that LEC is essential for discrimination of odor identity and intensity. Population analyses of L2 ensembles reveals that while rate coding distinguishes odor identity, firing rates are weakly concentration-dependent and changes in spike timing represent odor intensity. L2 principal cells differ in afferent olfactory input and connectivity with local inhibitory circuits and the relative timing of pyramidal and fan cell spikes underlies odor intensity coding. Downstream, intensity is encoded purely by spike timing in hippocampal CA1. Together, these results reveal the unique processing of odor information by parallel LEC subcircuits and highlight the importance of temporal coding in higher olfactory areas.


title: Harbor porpoise losing its edges: genetic time series suggests a rapid population decline in Iberian waters over the last 30 years

abstract: Impact of climate changes on species is expected to be especially visible at the extremities of the species distribution, where they meet sub-optimal conditions. In Mauritania and Iberia, two genetically isolated populations of harbor porpoises form a distinct ecotype and are presumably locally adapted to the upwelling waters. By analyzing the evolution of mitochondrial genetic variation in the Iberian population between two temporal cohorts (1990-2002 vs. 2012-2015), we report a dramatic decrease in genetic diversity. Phylogenetic analyses including neighboring populations identified two porpoises in southern Iberia carrying a divergent haplotype close to the Mauritanian population, yet forming a distinctive lineage. This suggests that Iberian porpoises may not be as isolated as previously thought with immigration from Mauritania or an unknown population in between, but none from the northern ecotype. The rapid decline in the Iberian mitochondrial diversity may be driven by either genetic drift, or by a dramatic decline in census population size possibly resulting from environmental stochasticity, prey depletion, or acute fishery bycatches. These results illustrate the value of genetics time series to inform demographic trends and emphasize the urgent need for conservation measures to ensure the viability of this small harbor porpoise population in Iberia.

category: evolutionary biology


title: Gene expression in African Americans and Latinos reveals ancestry-specific patterns of genetic architecture

abstract: We explored the role of genetic ancestry in shaping the genetic architecture of whole blood gene expression using whole genome and RNA sequencing data from 2,733 African American and Hispanic/Latino children. We find that heritability of gene expression significantly increases with greater proportion of genome-wide African ancestry and decreases with higher levels of Indigenous American ancestry. Fine-mapping of expression quantitative trait loci (eQTLs) in individuals with predominantly African or Indigenous American ancestry revealed ancestry-specific eQTLs in over 30% of heritable genes. We leveraged our data to train genetically derived transcriptome prediction models, which identified significantly more associated genes when applied to 28 traits from a multi-ancestry population. Our findings underscore the importance of increasing representation from ancestrally diverse populations in genomic studies to enable new discoveries and ensure their equitable translation.

category: genetics


title: A scalable algorithm for clonal reconstruction from sparse time course genomic sequencing data

abstract: Long-term evolution experiments (LTEEs) reveal the dynamics of clonal compositions in an evolving bacterial population over time. Accurately inferring the haplotypes - the set of mutations that identify each clone, as well as the clonal frequencies and evolutionary history in a bacterial population is useful for the characterization of the evolutionary pressure on multiple correlated mutations instead of that on individual mutations. Here, we study the computational problem of reconstructing the haplotypes of bacterial clones from the variant allele frequencies (VAFs) observed during a time course in a LTEE. Previously, we formulated the problem using a maximum likelihood approach under the assumption that mutations occur spontaneously, and thus the likelihood of a mutation occurring in a specific clone is proportional to the frequency of the clone in the population when the mutation occurs. We also developed several heuristic greedy algorithms to solve the problem, which were shown to report accurate results of clonal reconstruction on simulated and real time course genomic sequencing data in LTEE. However, these algorithms are too slow to handle sparse time course data when the number of novel mutations occurring during the time course are much greater than the number of time points sampled. In this paper, we present a novel scalable algorithm for clonal reconstruction from sparse time course data. We employed a statistical method to estimate the sampling variance of VAFs derived from low coverage sequencing data and incorporated it into the maximum likelihood framework for clonal reconstruction on noisy sequencing data. We implemented the algorithm (named ClonalTREE2) and tested it using simulated and real sparse time course genomic sequencing data. The results showed that the algorithm was fast and achieved near-optimal accuracy under the maximum likelihood framework for the time course data involving hundreds of novel mutations at each time point. The source code of ClonalTREE2 is available at https://github.com/COL-IU/ClonalTREE2.


title: Learning the local landscape of protein structures with convolutional neural networks

abstract: The fundamental problem of protein biochemistry is to predict protein structure from amino acid sequence. The inverse problem, predicting either entire sequences or individual mutations that are consistent with a given protein structure, has received much less attention even though it has important applications in both protein engineering and evolutionary biology. Here, we ask whether 3D convolutional neural networks (3D CNNs) can learn the local fitness landscape of protein structure to reliably predict either the wild-type amino acid or the consensus in a multiple sequence alignment from the local structural context surrounding a site of interest. We find that the network can predict wild type with good accuracy, and that network confidence is a reliable measure of whether a given prediction is likely going to be correct or not. Predictions of consensus are less accurate, and are primarily driven by whether or not the consensus matches the wild type. Our work suggests that high-confidence mis-predictions of the wild type may identify sites that are primed for mutation and likely targets for protein engineering.


title: Vectorial Channeling as a Mechanism for Translational Control by Functional Prions and Condensates

abstract:
Translation of messenger RNA is regulated through a diverse set of RNA-binding proteins. A significant fraction of RNA-binding proteins contain prion-like domains which form functional prions. This raises the question of how prions can play a role in translational control. Local control of translation in dendritic spines by prions has been invoked in the mechanism of synaptic plasticity and memory. We show how channeling through diffusion and processive translation cooperate in highly ordered mRNA/prion aggregates as well as in less ordered mRNA/protein condensates depending on their sub-structure. We show the direction of translational control, whether it is repressive or activating, depends on the polarity of the mRNA distribution in mRNA/prion assemblies which determines whether vectorial channeling can enhance recycling of ribosomes. Our model also addresses the effect of changes of substrate concentration in assemblies that have been suggested previously to explain translation control by assemblies through the introduction of a potential of mean force biasing diffusion of ribosomes inside the assemblies. The results from the model are compared with the experimental data on translational control by two functional RNA-binding prions, CPEB involved in memory and Rim4 involved in gametogenesis.

Significance StatementmRNA/protein assemblies such as functional prions and condensates are involved in locally regulating translation in eukaryotic cells. The mode of regulation depends on the structure of these assemblies. We show that the vectorial processive nature of translation can couple to transport via diffusion so as to repress or activate translation depending on the structure of the RNA protein assembly. We find that multiple factors including diffusivity changes and free energy biases in the assemblies can regulate the translation rate of mRNA by changing the balance between substrate recycling and competition between mRNAs. We mainly focus on the example of CPEB, a functional prion that has been implicated in the mechanism of synaptic plasticity of neurons and in memory.

category: biophysics


title: Prospects of raising baobab (Adansonia digitata L.) to fruiting in two years

abstract: Baobab is a very important plant with several uses, serving as food security crop and has the potential to raise income of farmers. However, the plant is undomesticated. We have the objective of domesticating the plant and promote its cultivation and utilization. Seeds were treated with sulphuric acid for early germination and grafted with matured scions suspected to be fruiting for several years. Twenty-two of the seedlings were transplanted on the field at Ho Technical University campus. Unfortunately, only four survived bushfire and destruction by stray animals. Surprisingly, one of the four surviving trees anthesis within twenty-eight months after seed treatment (twenty months after transplanting). This is the earliest ever reported anthesis of baobab. With better agronomic practices such as supplementary irrigation, insect pest control, manuring and protection from stray animals, baobab could start fruiting within two years of planting. This would alleviate fear of long maturity period and motivate farmers to go into baobab plantation. This would help diversify agriculture in many African countries, increase income and food security and contribute towards the attainment of the sustainable development goals of the United Nations.


title: ITRAQ-based quantitative proteomic analysis of japonica rice seedling during cold stress

abstract: Low temperature is one of the important environmental factors that affect rice growth and yield. To better understand the japonica rice responses to cold stress, isobaric tags for relative and absolute quantification (iTRAQ) labeling based quantitative proteomics approach was used to detected changes in protein level. Two-week-old seedlings of the cold tolerance rice variety Kongyu131 were treated at 8{degrees}C for 24, 48 and 72 h, then the total proteins were extracted from tissues and used for quantitative proteomics analysis. A total of 5082 proteins were detected for quantitative analysis, of which 289 proteins were significantly regulated, consisting of 169 uniquely up-regulated proteins and 125 uniquely down-regulated proteins in cold stress groups relative to control group. Functional analysis revealed most of regulation proteins involved in photosynthesis, metabolic pathway, biosynthesis of secondary metabolites and carbon metabolism. Western blot analysis showed that protein regulation was consistent with the iTRAQ data. The corresponding genes of 25 regulation proteins were used for quantitative real time PCR analysis, and the results showed that the mRNA level was not always parallel to the corresponding protein level. The importance of our study is providing new insights into cold stress responses in rice on proteomic aspect.


title: Sugar Beet Extract Acts as a Natural Bio-Stimulant for Physio-Biochemical analysis of Hordeum Vulgare L. under Induced Salinity Stress

abstract:
Change in climate of the entire globe due to elevated temperature and minimum annual rainfall in barren zone frequently leads to salinity of soil. The current study was aimed to evaluate the importance of sugar beet extract (SBE) as a bio-stimulant to improve the adverse damages of induced salinity stress (40mM) on growth, oosmolytes and antioxidant defense system of barley (Hordeum vulgare L.). Pot experiment was carried in green house under different concentrations of SBE (10%, 20%, 30%, 40%, 50%) pre-soaked seeds of Hordeum vulgare for 5 hours SBE was analyzed for glycine betaine (100mmol/kg), betalains (1.3mg/l), phenolics (1.30g/100ml), flavonoids (0.59mg/ml), carotenoids (0.23ml/100ml), vitamin E (0.002%), vitamin C (8.04g/100ml), sugar (8g/100ml), protein (1.39mg/100ml), and oxalic acid (38mg/100ml) while Ca (13.72mg/l), Mg (7.121 mg/l) and K (11.45mg/l) contents were also determined. We found significant improvement in germination parameters of Hordeum vulgare L. via SB extract on coefficient of velocity of emergence (CVE), mean emergence time (MET), germination energy (GE), timson germination index (TGI), germination rate index (GRI) and time to 50% emergence (E50) under induced salinity stress. However, photosynthetic pigments, e.g., chlorophyll and carotenoids were enhanced using 40% SB extract, soluble sugar, protein, proline, POD, MDA with 50% SB extract while SOD and H2O2 in 20% SBE, respectively. Our findings suggested that SB extract promotes both agronomical and physiological attributes, is a positive way to enhance our economy by increasing crop yields in arid and semi-arid areas along with plant tolerance to under induced salinity stress.

HighlightsThe recent study is of utmost importance owing to the following reasons:[tpltrtarr] Changes in climatic condition results in salinity, droughts, floods, earthquakes, fluctuation in temperature and other environmental hazards.
[tpltrtarr]Salinity stress has myriad of damaging effects on crop growth and productivity and becoming a global issue of major concern especially in the Asian countries including Pakistan. Statistical analysis showed that 6.30 million of lands is affected with salinity.
[tpltrtarr]Barley (Hordeum Vulgare L.) being an important crop of economic and nutritional value has been facing salinity stress condition in Pakistan, resulting in productivity reduction and ultimately paving the way for food scarcity and crippling economy.
[tpltrtarr]The sole purpose of this research article is to assess and ameliorate the physio-biochemical responses via application of sugar beet extract as bio-stimulants in Barley (Hordeum Vulgare L.) grown under induced salinity stress; in order to cope with the dire consequences of salinity stress especially in the southern areas of Pakistan being badly affected by salt stress.
[tpltrtarr]Furthermore, there is a great need for adaptive measures suggested by the present research and other findings made by researchers in pursuit of tackling this alarming global menace.


title: Pre-vaccination Frequency of Circulatory Tfh is associated with Robust Immune Response to TV003 Dengue Vaccine

abstract:
It has been estimated that more than 390 million people are infected with Dengue virus every year; around 96 millions of these infections result in clinical pathologies. To date, there is only one licensed viral vector-based Dengue virus vaccine CYD-TDV approved for use in dengue endemic areas. While initially approved for administration independent of serostatus, the current guidance only recommends the use of this vaccine for seropositive individuals. Therefore, there is a critical need for investigating the influence of Dengue virus serostatus and immunological mechanisms that influence vaccine outcome. Here, we provide comprehensive evaluation of sero-status and host immune factors that correlate with robust immune responses to a Dengue virus vector based tetravalent vaccine (TV003) in a Phase II clinical cohort of human participants. We observed that sero-positive individuals demonstrate a much stronger immune response to the TV003 vaccine. Our multi-layered immune profiling revealed that sero-positive subjects have increased baseline/pre-vaccination frequencies of circulating T follicular helper (cTfh) cells and the Tfh related chemokine CXCL13/BLC. Importantly, this baseline/pre-vaccination cTfh profile correlated with the vaccinees ability to launch neutralizing antibody response against all four sero-types of Dengue virus, an important endpoint for Dengue vaccine clinical trials. Overall, we provide novel insights into the favorable cTfh related immune status that persists in Dengue virus sero-positive individuals that correlate with their ability to mount robust vaccine specific immune responses. Such detailed interrogation of cTfh cell biology in the context of clinical vaccinology will help uncover mechanisms and targets for favorable immuno-modulatory agents.

Author summaryDengue virus (DENV) is a worldwide threat that causes significant health and economic burden. Currently, there are several challenges in the development of a DENV vaccine including the existence of four different serotypes all; capable of causing disease and antibody dependent enhancement (ADE). For complete protection, a vaccine must be able to generate neutralizing antibodies against all 4 serotypes to avoid ADE. Currently, there is one licensed DENV vaccine, CYD-TDV (DENGVAXIATM). However, this vaccine is only efficacious in protecting against severe disease in DENV seropositive individuals therefore serostatus effect must be further studied for optimal vaccine design. A subset of CD4+ T cells called T-follicular helper (Tfh) cells have been well known to play a major role in aiding high affinity antibody production. Therefore, we chose to look at subsets of Tfh and the cytokines they produce in human blood that can serve as biomarkers for effective vaccine design. We found that DENV sero-positive participants had increased pre-vaccination frequencies of Tfh cells and higher levels of the Tfh related chemokine CXCL13/BLC that plays a role in directing antigen-specific responses. This pre-vaccination Tfh profile and CXCL13/BLC are then correlated positively with the vaccinees ability to produce neutralizing antibody against all four sero-types (breadth of the Response) of DENV, an important goal for all DENV vaccine trials.

category: immunology


title: The DNA sensors AIM2 and IFI16 are NET-binding SLE autoantigens

abstract: Nucleic acid binding proteins are frequently targeted as autoantigens in systemic lupus erythematosus (SLE) and other interferon (IFN)-linked rheumatic diseases. The AIM-like receptors (ALRs) are IFN-inducible innate sensors that form supramolecular assemblies along double-stranded DNA of various origins. Here, we identify the ALR Absent in melanoma 2 (AIM2) as a novel autoantigen in SLE, with similar properties to the established ALR autoantigen interferon-inducible protein 16 (IFI16). Our SLE cohort revealed a frequent co-occurrence of anti-AIM2, anti-IFI16 and anti-DNA antibodies, and higher clinical measures of disease activity in patients positive for antibodies against these ALRs. We examined neutrophil extracellular traps (NETs) as DNA scaffolds on which these antigens might interact in a pro-immune context, finding that both ALRs bind NETs in vitro and in SLE renal tissues. We demonstrate that ALR binding causes NETs to resist degradation by DNase I, suggesting a mechanism whereby extracellular ALR-NET interactions may promote sustained IFN signaling. Our work suggests that extracellular ALRs bind NETs, leading to DNase resistant nucleoprotein fibers that are targeted as autoantigens in SLE.


title: Assessment of Carbon Sequestration Potential of some Selected Urban Tree Species

abstract: Tree species carbon assessment and quantification remain the only opportunity to determine the position of forest in climate change amelioration potentials. Forest biomass constitutes the largest terrestrial carbon sink and accounts for approximately 90% of all living terrestrial biomass. The aim of this study is to assess tree species carbon sequestration potentials of selected urban tree species. The study was carried out in Adekunle Ajasin University Campus, Akungba Akoko, Ondo State, Nigeria. All trees species [≥]10 cm Diameter at Breast Height (Dbh) within the area were identified and their Dbh measured as well as other variables for volume computation such as height, diameters at the base, middle and top. Also, for density assessment; stem core samples were collected. Again, the coordinate of individual tree was recorded using a Global Positioning System (GPS) receiver. A total of 124 individual trees were encountered with varying growth variables as well as carbon values. The study area contains some indigenous and exotic tree species such as Acacia auriculiformis, Terminalia mantily, Gmelina arborea and Tectona grandis etc. but Acacia auriculiformis had the highest frequency. The tree species with highest carbon sequestration was Gmelina arborea as recorded for this study. The total carbon and carbon dioxide sequestered in the study area were reported as 47.94 kg and 176.03 kg respectively.


title: Analysis of migration of pathogenic drug-resistant bacteria to soils and groundwater after fertilization with sewage sludge

abstract: The paper discusses the analysis of the effect of using sewage sludge for fertilization on the level of soil and groundwater contamination with drug-resistant bacteria. Other sanitary contaminants in these environments were also analysed. Composted sewage sludge was introduced into the sandy soil over a period of 6 months. The examinations were conducted under conditions of a lysimetric experiment with the possibility of collecting soil leachates (in natural conditions). The following doses of sewage sludge were used: 0, 10, 20, 30 and 40 t/ha calculated per experimental object containing 10 kg of sandy soil. The research were carried out within the time frame of one year. Dactylis glomerata grass was grown on the fertilized soils. In soils and leachates from soils (which may have polluted groundwater) collected from fertilized experimental objects, the sanitary condition and quantity of drug-resistant bacteria (mainly from the families Enterobacteriaceae and Enterococcus) were analysed one year after fertilization. Their drug resistance to selected antibiotics was also analysed based on current recommendations.


title: Village in a dish: a model system for population-scale hiPSC studies

abstract: The mechanisms by which DNA alleles contribute to disease risk, drug response, and other human phenotypes are highly context-specific, varying across cell types and under different conditions. Human induced pluripotent stem cells (hiPSCs) are uniquely suited to study these context-dependent effects, but to do so requires cell lines from hundreds or potentially thousands of individuals. Village cultures, where multiple hiPSC lines are cultured and differentiated together in a single dish, provide an elegant solution for scaling hiPSC experiments to the necessary sample sizes required for population-scale studies. Here, we show the utility of village models, demonstrating how cells can be assigned back to a donor line using single cell sequencing, and addressing whether line-specific signaling alters the transcriptional profiles of companion lines in a village culture. We generated single cell RNA sequence data from hiPSC lines cultured independently (uni-culture) and in villages at three independent sites. We show that the transcriptional profiles of hiPSC lines are highly consistent between uni- and village cultures for both fresh (0.46 < R < 0.88) and cryopreserved samples (0.46 < R < 0.62). Using a mixed linear model framework, we estimate that the proportion of transcriptional variation across cells is predominantly due to donor effects, with minimal evidence of variation due to culturing in a village system. We demonstrate that the genetic, epigenetic or hiPSC line-specific effects on gene expression are consistent whether the lines are uni- or village-cultured (0.82 < R < 0.94). Finally, we identify the consistency in the landscape of cell states between uni- and village-culture systems. Collectively, we demonstrate that village methods can be effectively used to detect hiPSC line-specific effects including sensitive dynamics of cell states.

category: genomics


title: Spinal cord synaptic plasticity by GlyRβ release from receptor fields and syndapin-dependent uptake

abstract: Glycine receptor-mediated inhibitory neurotransmission is key for spinal cord function. Recent observations suggested that by largely elusive mechanisms also glycinergic synapses display synaptic plasticity. We here identify syndapin I as critical player. Interestingly, syndapin I cooperates but in part also competes with gephyrin. Syndapin I deficiency led to fragmentation of glycine receptor fields, more disperse receptors and increased receptor mobility. Kainate treatment highlighted syndapin Is importance even more. Our analyses unveiled that PKC-mediated S403 phosphorylation-mediated glycine receptor {beta} decoupling from gephyrin scaffolds simultaneously promoted syndapin I association. In line, kainate-treated syndapin I KO spinal cords showed even more severe receptor field fragmentation. Furthermore, syndapin I deficiency completely disrupted kainate-induced glycine receptor internalization. Together, this unveiled important mechanisms controlling the number and organization of glycine receptor fields at inhibitory postsynapses during both steady-state and kainate-induced synaptic rearrangement - principles organizing and fine-tuning synaptic efficacy of inhibitory synapses in the spinal cord.


title: Different adjuvanted pediatric HIV envelope vaccines induced distinct plasma antibody responses despite similar B cell receptor repertoires in infant rhesus macaques.

abstract: Different HIV vaccine regimens elicit distinct plasma antibody responses in both human and nonhuman primate models. Previous studies in human and non-human primate infants showed that adjuvants influenced the quality of plasma antibody responses induced by pediatric HIV envelope vaccine regimens. We recently reported that use of the 3M052-SE adjuvant and longer intervals between vaccinations are associated with higher magnitude of antibody responses in infant rhesus macaques. However, the impact of different adjuvants in HIV vaccine regimens on the developing infant B cell receptor (BCR) repertoire has not been studied. This study evaluated whether pediatric HIV envelope vaccine regimens with different adjuvants induced distinct antigen-specific memory B cell repertoires and whether specific immunoglobulin (Ig) immunogenetic characteristics are associated with higher magnitude of plasma antibody responses in vaccinated infant rhesus macaques. We utilized archived preclinical pediatric HIV vaccine studies PBMCs and tissue samples from 19 infant rhesus macaques immunized either with (i) HIV Env protein with a squalene adjuvant, (ii) MVA-HIV and Env protein coadministered using a 3-week interval, (iii) MVA-HIV prime/ protein boost with an extended 6-week interval between immunizations, or (iv) with HIV Env administered with 3M-052-SE adjuvant. Frequencies of vaccine-elicited HIV Env-specific memory B cells from PBMCs and tissues were similar across vaccination groups (frequency range of 0.06-1.72%). There was no association between vaccine-elicited antigen-specific memory B cell frequencies and plasma antibody titer or avidity. Moreover, the epitope specificity and Ig immunogenetic features of vaccine-elicited monoclonal antibodies did not differ between the different vaccine regimens. These data suggest that pediatric HIV envelope vaccine candidates with different adjuvants that previously induced higher magnitude and quality of plasma antibody responses in infant rhesus macaques were not driven by distinct antigen-specific memory BCR repertoires.


title: Determinants shaping the nanoscale architecture of the mouse rod outer segment

abstract:
The unique membrane organization of the rod outer segment (ROS), the specialized sensory cilium of rod photoreceptor cells, provides the foundation for phototransduction, the initial step in vision. ROS architecture is characterized by a stack of identically shaped and tightly packed membrane disks loaded with the visual receptor rhodopsin. A wide range of genetic aberrations compromise ROS ultrastructure, impairing photoreceptor viability and function. Yet, the structural basis giving rise to the remarkable long-range order of ROS membrane stacks and the molecular mechanisms underlying genetically inherited diseases remain elusive. Here, cryo-electron tomography (cryo-ET) performed on native ROS at molecular resolution provides insights into key structural determinants of ROS membrane architecture.

Our data reveal the existence of two molecular connectors/spacers which likely contribute to the nanometer-scale precise stacking of the ROS disks. We further show that the extreme radius of curvature at the disk rims is enforced by a continuous supramolecular assembly composed of peripherin-2 (PRPH2) and rod outer segment membrane protein 1 (ROM1) tetramers. We suggest that, together these molecular assemblies constitute the structural basis of the highly specialized ROS functional architecture. Cryo-ET therefore provides novel quantitative and structural information on the molecular architecture in ROS and insights into possible mechanisms underlying pathologies of certain PRPH2 mutations leading to blindness.


title: Tomato CRABS CLAW paralogues interact with chromatin remodelling factors to mediate carpel development and floral determinacy

abstract: CRABS CLAW (CRC) orthologues play a crucial role in floral meristem (FM) determinacy and gynoecium formation across angiosperms, key developmental processes for ensuring successful plant reproduction and crop production. Here, we revealed that the incomplete penetrance and variable expressivity of the carpel-inside-carpel phenotype observed in flowers of the tomato fruit iterative growth (fig) mutant is due to a lack of function of a homologue of the CRC gene, Solanum lycopersicum CRCa (SlCRCa). Likewise, a comprehensive functional analysis of SlCRCa and SlCRCb paralogues, including Arabidopsis complementation experiments, allowed us to propose that they operate as positive regulators of FM determinacy by acting in a compensatory and partially redundant manner to safeguard the proper formation of flowers and fruits. Furthermore, we provide the first evidence for the role of putative CRC orthologues as members of the chromatin remodelling complex that terminates floral stem cell activity by repressing WUSCHEL expression.

category: developmental biology


title: Signatures of adaptive evolution in platyrrhine primate genomes

abstract: The family Cebidae (capuchin and squirrel monkeys) form a remarkable platyrrhine clade exhibiting among the largest primate encephalisation quotients. Each cebid lineage is characterised by notable lineage-specific traits, with capuchins showing striking similarities to Hominidae including high sensorimotor intelligence with tool use, advanced cognitive abilities, and behavioural flexibility. Here, we take a comparative genomics approach, analysing five cebid branches including successive lineages, to infer a stepwise timeline for cebid adaptive evolution. We uncover candidate targets of selection across various periods of cebid evolution that may underlie the emergence of lineage-specific traits. Our analyses highlight shifting and sustained selective pressures on genes related to brain development, longevity, reproduction, and morphology, including evidence for cumulative and diversifying neurobiological adaptations over cebid evolutionary history. In addition to generating a new, high-quality reference genome assembly for robust capuchins, our results lend to a better understanding of the adaptive diversification of this distinctive primate clade.


title: Molecular basis for GIGYF-TNRC6 complex assembly in miRNA-mediated translational repression

abstract: The GIGYF proteins associate with 4EHP and RNA-associated proteins to elicit transcript-specific translational repression. However, the mechanism by which the GIGYF1/2-4EHP complex is recruited to its target transcripts remain unclear. Here we report the crystal structures of the GYF domains from GIGYF1 and GIGYF2 in complex with proline-rich sequences from miRISC-binding proteins TNRC6C and TNRC6A, respectively. The TNRC6 proline-rich motifs bind to a conserved array of aromatic residues on the surface of the GIGYF1/2 GYF domain, bridging 4EHP to Argonaute-miRNA mRNA targets. Our structures also reveal a phenylalanine residue conserved from yeast to human GYF domains that contributes to GIGYF2 thermostability. The molecular details we outline here are likely to be conserved between GIGYF1/2 and other RNA-binding proteins to elicit 4EHP-mediated repression in different biological contexts.

category: biochemistry


title: Liquid-liquid phase separation of florigen activation complex induces flowering

abstract: Floral transition, regulated by the systemic action of the mobile florigen protein FLOWERING LOCUS T (FT), is essential for successful plant reproduction1. How FT controls downstream gene expression remains incompletely understood, although it relies on the florigen activation complex (FAC), a core component of FT function2-4. The FAC is a nucleus-localized transcriptional activator of genes encoding MADS-box transcription factors critical to reproductive development and consists of florigen FT; a scaffold 14-3-3 protein that is a key component for complex assembly; and FD, a basic leucine-zipper protein that recruits the FAC to DNA. Here we report that the FAC exhibits phase separation. In rice shoot apical meristem cells, rice florigen Heading date 3a (Hd3a) fused to the green fluorescent protein formed speckles in the nucleus. The FAC speckle is formed in a FAC-dependent manner in tobacco cells. Recombinant Hd3a, but not OsFD1, phase-separated in vitro, and this effect was enhanced in the presence of 14-3-3 protein. Furthermore, mutations affecting functionally important residues in the pocket region or C-terminal disordered region of Hd3a affected FAC phase separation, providing a biochemical framework for the proteins effect on flowering. The ability to form condensates via phase transition represents a previously unknown mechanism for gene activation by the FAC.


title: Transcriptomic and proteomic retinal pigment epithelium signatures of age-related macular degeneration.

abstract: Induced pluripotent stem cells generated from patients with geographic atrophy as well as healthy individuals were differentiated to retinal pigment epithelium (RPE) cells. By integrating transcriptional profiles of 127,659 RPE cells generated from 43 individuals with geographic atrophy and 36 controls with genotype data, we identified 439 expression Quantitative Trait (eQTL) loci in cis that were associated with disease status and specific to subpopulations of RPE cells. We identified loci linked to two genes with known associations with geographic atrophy - PILRB and PRPH2, in addition to 43 genes with significant genotype x disease interactions that are candidates for novel genetic associations for geographic atrophy. On a transcriptome-only level, we identified molecular pathways significantly upregulated in geographic atrophy-RPE including in extracellular cellular matrix reorganisation, neurodegeneration, and mitochondrial functions. We subsequently implemented a large-scale proteomics analysis, confirming modification in proteins associated with these pathways. We also identified six significant protein (p) QTL that regulate protein expression in the RPE cells and in geographic atrophy - two of which share variants with cis-eQTL. Transcriptome-wide association analysis identified genes at loci previously associated with age-related macular degeneration. Further analysis conditional on disease status, implicated statistically significant RPE-specific eQTL. This study uncovers important differences in RPE homeostasis associated with geographic atrophy.


title: CD4+CCR6+ T cells dominate the BCG-induced transcriptional signature

abstract: The century-old Mycobacterium bovis Bacillus Calmette-Guerin (BCG) remains the only licensed vaccine against tuberculosis (TB). Despite this, there is still a lot to learn about the immune response induced by BCG, both in terms of phenotype and specificity. Here, we investigated immune responses in adult individuals pre and 8 months post BCG vaccination. We specifically determined changes in gene expression, cell subset composition, DNA methylome, and the TCR repertoire induced in PBMCs and CD4 memory T cells associated with antigen stimulation by either BCG or a Mycobacterium tuberculosis (Mtb)-derived peptide pool. Following BCG vaccination, we observed increased frequencies of CCR6+ CD4 T cells, which includes both Th1* and Th17 subsets, and mucosal associated invariant T cells (MAITs). A large number of immune response genes and pathways were upregulated post BCG vaccination with similar patterns observed in both PBMCs and memory CD4 T cells, thus suggesting a substantial role for CD4 T cells in the cellular response to BCG. These upregulated genes and associated pathways were also reflected in the DNA methylome. We described both qualitative and quantitative changes in the BCG-specific TCR repertoire post vaccination, and importantly found evidence for similar TCR repertoires across different subjects. The immune signatures defined herein can be used to track and further characterize immune responses induced by BCG, and can serve as reference for benchmarking novel vaccination strategies.


title: Selective estrogen receptor modulators limit alphavirus infection by targeting the viral capping enzyme nsP1

abstract: Alphaviruses cause animal or human diseases that are characterized by febrile illness, debilitating arthralgia, or encephalitis. Selective estrogen receptor modulators (SERMs), a class of FDA-approved drugs, have been shown to possess antiviral activities against multiple viruses, including Hepatitis C virus, Ebola virus, dengue virus, and vesicular stomatitis virus. Here, we evaluated three SERM compounds, namely 4-hydroxytamoxifen, tamoxifen, and clomifene, for plausible antiviral properties against two medically important alphaviruses, chikungunya virus (CHIKV) and Sindbis virus (SINV). In cell culture settings, these SERMs displayed potent activity against CHIKV and SINV at non-toxic concentrations with EC50 values ranging between 400 nM and 3.9 M. Further studies indicated that these compounds inhibit a post-entry step of the alphavirus life cycle, while enzymatic assays involving purified recombinant proteins confirmed that these SERMs target the enzymatic activity of non-structural protein 1 (nsP1), the capping enzyme of alphaviruses. Finally, tamoxifen treatment restrained CHIKV growth in the infected mice and diminished musculoskeletal pathologies. Combining biochemical, cell culture-based studies, and in vivo analyses, we strongly argue that SERM compounds, or their derivatives, may provide for attractive therapeutic options against alphaviruses.


title: Screening of surface exposed lipoproteins of Leptospira involved in modulation of host innate immune response

abstract: Leptospira, a zoonotic pathogen is capable of causing both chronic and acute infection in susceptible host. Surface exposed lipoproteins play major role in modulating the host immune response by activating the innate cells like macrophages and DCs or evading complement attack and killing by phagocytes like neutrophils to favour pathogenesis and establish infection. In this study we screened some of surface exposed lipoproteins which are known to be involved in pathogenesis for their possible role in immune modulation (innate immune activation or evasion). Surface proteins of Len family (LenB, LenD, LenE), Lsa30, Loa22 and Lipl21 were purified in recombinant form and then tested for their ability to activate macrophages of different host (mouse, human and bovine). These proteins were tested for binding with complement regulators (FH, C4BP), host protease (plasminogen, PLG) and as nucleases to access their possible role in innate immune evasion. Our results show that of various proteins tested Loa22 induced strong innate activation and Lsa30 was least stimulatory as evident from production of pro-inflammatory cytokines (IL-6, TNF-a) and expression of surface markers (CD80, CD86, MHCII). All the tested proteins were able to bind to FH, C4BP and PLG, however Loa22 showed strong binding to PLG correlating to plasmin activity. All the proteins except Loa22 showed nuclease activity albeit with requirement of different metal ions. The nuclease activity of these proteins correlated to in vitro degradation of Neutrophil extracellular trap (NET). These results indicate that these surface proteins are involved in innate immune modulation and may play critical role in assisting the bacteria to invade and colonize the host tissue for persistent infection.


title: In Situ Visualization of the pKM101-Encoded Type IV Secretion System Reveals a Highly Symmetric ATPase Energy Center

abstract:
Bacterial conjugation systems are members of the type IV secretion system (T4SS) superfamily. T4SSs can be classified as  minimized or  expanded based on whether they are composed of a core set of signature subunits or additional system-specific components. Prototypical  minimized systems mediating Agrobacterium tumefaciens T-DNA transfer and pKM101 and R388 plasmid transfer are built from subunits generically named VirB1-VirB11 and VirD4. We visualized the pKM101-encoded T4SS in the native cellular context by in situ cryoelectron tomography (CryoET). The T4SSpKM101 is composed of an outer membrane core complex (OMCC) connected by a thin stalk to an inner membrane complex (IMC). The OMCC exhibits 14-fold symmetry and resembles that of the T4SSR388 analyzed previously by single-particle electron microscopy. The IMC is highly symmetrical and exhibits 6-fold symmetry. It is dominated by a hexameric collar in the periplasm and a cytoplasmic complex composed of a hexamer of dimers of the VirB4-like TraB ATPase. The IMC closely resembles equivalent regions of three  expanded T4SSs previously visualized by in situ CryoET, but differs strikingly from the IMC of the purified T4SSR388 whose cytoplasmic complex instead presents as two side-by-side VirB4 hexamers. Analyses of mutant machines lacking each of the three ATPases required for T4SSpKM101 function supplied evidence that TraBB4 as well as VirB11-like TraG contribute to distinct stages of machine assembly. We propose that the VirB4-like ATPases, configured as hexamers-of-dimers at the T4SS entrance, orchestrate IMC assembly and recruitment of the spatially-dynamic VirB11 and VirD4 ATPases to activate the T4SS for substrate transfer.

SIGNIFICANCEBacterial type IV secretion systems (T4SSs) play central roles in antibiotic resistance spread and virulence. By cryoelectron tomography (CryoET), we solved the structure of the plasmid pKM101-encoded T4SS in the native context of the bacterial cell envelope. The inner membrane complex (IMC) of the in situ T4SS differs remarkably from that of a closely-related T4SS analyzed in vitro by single particle electron microscopy. Our findings underscore the importance of comparative in vitro and in vivo analyses of the T4SS nanomachines, and support a unified model in which the signature VirB4 ATPases of the T4SS superfamily function as a central hexamer of dimers to regulate early-stage machine biogenesis and substrate entry passage through the T4SS. The VirB4 ATPases are therefore excellent targets for development of intervention strategies aimed at suppressing the action of T4SS nanomachines.


title: Drosophila Filamin exhibits a mechano-protective role during nephrocyte injury via induction of hypertrophic growth

abstract: Podocytes are highly specialized epithelial cells of the kidney glomerulus and are an essential part of the filtration barrier. Due to their position and function in the kidney, they are exposed to constant biomechanical forces such as shear stress and hydrostatic pressure. These forces increase during disease, resulting in podocyte injury and loss. The mechanism by which biomechanical forces are sensed and transduced to elicit an adaptive and protective response remains largely unknown. Here we show, using the Drosophila nephrocyte model, that the mechanosensor Cheerio (dFilamin) is central to this  mechano-protective mechanism. We found expression of an activated mechanosensitive variant of Cheerio induced hypertrophy and rescued filtration function in injured nephrocytes. Additional analysis with human Filamin B confirmed this mechano-protective role. We delineated the mechano-protective pathway downstream of Cheerio and found activation of TOR and Yorkie induce nephrocyte hypertrophy, whereas their repression reversed the Cheerio-mediated hypertrophy. Although Cheerio/Filamin B pathway mediates a mechano-protective role in the face of injury, we found excessive activity resulted in a pathological phenotype, indicating activity levels must be tightly controlled. Taken together, our data suggest that Cheerio acts via the TOR and YAP pathway to induce hypertrophic growth, as a mechano-protective response to nephrocyte injury.


title: Evolutionary dynamics of piRNA clusters in Drosophila

abstract: Small RNAs produced from transposable element (TE) rich sections of the genome, termed piRNA clusters, are a crucial component in the genomic defense against selfish DNA. In animals it is thought the invasion of a TE is stopped when a copy of the TE inserts into a piRNA cluster, triggering the production of cognate small RNAs that silence the TE. Despite this importance for TE control, little is known about the evolutionary dynamics of piRNA clusters, mostly because these repeat rich regions are difficult to assemble and compare. Here we establish a framework for studying the evolution of piRNA clusters quantitatively. Previously introduced quality metrics and a newly developed software for multiple alignments of repeat annotations (Manna) allow us to estimate the level of polymorphism segregating in piRNA clusters and the divergence among homologous piRNA clusters. By studying 20 conserved piRNA clusters in multiple assemblies of four Drosophila species we show that piRNA clusters are evolving rapidly. While 70-80% of the clusters are conserved within species, the clusters share almost no similarity between species as closely related as D. melanogaster and D. simulans. Furthermore, abundant insertions and deletions are segregating within the Drosophila species. We show that the evolution of clusters is mainly driven by large insertions of recently active TEs, and smaller deletions mostly in older TEs. The effect of these forces is so rapid that homologous clusters often do not contain insertions from the same TE families.x


title: The effects of long-term translocation on population genetic structure of Ayu in Japan: Management implications for a recreationally fished iconic species

abstract: Ayu (Plecoglossus altivelis altivelis) is an important freshwater fisheries resource and popular recreational fishing species in Japan, lives for only one year, and has a single breeding season. To supplement increased recreational fishing demand for this species, huge numbers of wild-born landlocked juveniles have been translocated from Lake Biwa into most Japanese rivers for more than 100 generations. Hatchery-reared juveniles born from captive-reared parents for more than 30 generations have also been extensively released. Previous studies have reported that landlocked and amphidromous forms of Ayu easily hybridise, but survival of landlocked larvae could be very low in seawater, leading to a general consensus among scientists, hatchery managers, and commercial and recreational fishers that the reproductive success of landlocked Ayu is very low, or even 0 in translocated rivers. Despite this, limited information exists regarding the reproductive success of landlocked Ayu in translocated rivers, and no study has evaluated the effects of translocation on population structure. Demonstrating that hybridisation occurs between the two forms is central to future management and conservation of this specie. To address this issue, a comprehensive literature search was undertaken, and three published genetic data sets are analysed. Analyses provide strong evidence for very high gene flow between populations, but population structure has been retained in several regions, and several populations are nested. Allele frequencies are similar in amphidromous and landlocked forms. Genetic diversity is homogeneous in amphidromous populations. Bayesian admixture analysis infers widespread introgression in Japanese rivers, with a mean introgression proportion of 24 {+/-} 8%. Maximum likelihood admixture graphs detect two migration events from Lake Biwa to anadromous populations. Analyses consistently indicate that hybridisation between translocated landlocked juveniles and native amphidromous Ayu occurs throughout Japanese rivers.


title: Long-term translocation and hatchery release explain population genetic structure of a recreationally fished iconic species in Japan: A review and reanalysis


title: Long-term translocation explains population genetic structure of a recreationally fished iconic species in Japan: Combining current knowledge with reanalysis


title: Evolutionary history of mammalian UDP-glucuronosyltransferase (UGT)1 and UGT2 families: the emergence of UGT2B subfamily in eutherians after the diversification of flowering plants

abstract: The UDP-glucuronosyltransferase (UGT) gene family is responsible for the transfer of glucuronic acid to exogenous and endogenous chemicals. Based on the highly diversified number of genes, the mammalian UGT1A and UGT2B subfamily genes are believed to be involved in the conjugation reactions of xenobiotic metabolism. However, it is speculated that the UGT2 family genes are not involved in the xenobiotic metabolism of avian species due to the less diverse number of genes. In this study, we aimed to investigate the evolutionary history of mammalian UGT1 and UGT2 family genes and determine when the diversification of UGT2B genes occurred. We also attempted to identify the main factors responsible for the diversification of UGT genes. By examining the genomic information and feeding habits of 67 species representing each mammalian family, we discovered that the UGT2B genes emerged in the Eutheria on or after Cretaceous period and that their number were higher in plant-eating mammals (herbivore or omnivore) than in carnivorous mammals. We also found that the UGT2B genes in some herbivorous mammals underwent positive selection. In contrast, the diversity of the UGT1 family genes was inherited from the common ancestor of birds and mammals. Thus, our findings suggest that the emergence of angiosperms (flowering plants) and the occurrence of "animal-plant warfare" influenced the evolution of this gene family involved in the xenobiotic metabolism of eutherians. Furthermore, future research investigating the marsupials and birds that do not possess UGT2B genes is required to elucidate the mechanisms underlying the metabolism of chemical substances in these species.


title: NR4A3 is oxidative stress responsible transcription factor through HMOX1, and also controls cell cycle through CCNE1 and CDK2 in pancreatic islet derived 1.1B4 cells

abstract:
The mechanism of antioxidant defense system is still controversial. As islet {beta}-cell is weak in oxidative condition, that causes diabetes mellitus, therefore, antioxidant defense system of human pancreatic islet derived 1.1B4 cell was analyzed. Cells were exposed to H2O2 and comprehensive gene expression was analyzed by Agilent human microarray. HMOX1 and NR4A3, member of orphan receptor, were up-regulated. Therefore, NR4A3 was knocked down with siRNA, then analyzed gene expression by microarray, and found that the knocked down cells were weak in oxidative stress. HMOX1 expression was strongly inhibited by siRNA of NR4A3, and NR4A3 responsible sequence of aaggtca was found near the HMOX1 gene, suggesting NR4A3 is oxidative stress responsible transcription factor through HMOX1 expression. The expression of CCNE1 and CDK2 was also inhibited by knocked down of NR4A3, it is suggested NR4A3 is also important transcription factor for cell growth regulation.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/457070v1_ufig1.gif" ALT="Figure 1">
View larger version (32K):
org.highwire.dtl.DTLVardef@18d0371org.highwire.dtl.DTLVardef@dd1eacorg.highwire.dtl.DTLVardef@108b1b9org.highwire.dtl.DTLVardef@1cbf899_HPS_FORMAT_FIGEXP  M_FIG Hydrogen peroxide induces NR4A3 and binds to aaggtca sequence of HMOX1, and increased transcription of HMOX1. Resulting heme oxygenase produces biliverdin, antioxidants, from heme. NR4A3 also bind to aaggtca sequence of CDK2 and CCNE1, resulting CDK2 and Cyclin E. CDK2 bind to cyclin E and cell goes from G1 to S phase.

C_FIG


title: Assembly status transition offers an avenue for activity modulation of a supramolecular enzyme

abstract: Nature has evolved many supramolecular proteins assembled in certain, sometimes even seemingly oversophisticated, morphological manners. The rationale behind such evolutionary efforts is often poorly understood. Here we provide atomic-resolution insights into how the dynamic building of a structurally complex enzyme with higher-order symmetry offers amenability to intricate allosteric regulation. We have established the functional coupling between enzymatic activity and protein morphological states of glutamine synthetase (GS), an old multi-subunit enzyme essential for cellular nitrogen metabolism. Cryo-EM structure determination of GS in both the catalytically active and inactive assembly states allows us to reveal an unanticipated self-assembly-induced dynamics-driven allosteric paradigm, in which the remote interactions between two subcomplex entities significantly rigidify the otherwise structurally fluctuating active sites, thereby regulating activity. We further show in vivo evidences that how the enzyme morphology transitions could be modulated by cellular factors on demand. Collectively, our data present an example of how assembly status transition offers an avenue for allosteric modulation, and sharpens our mechanistic understanding of allostery, dynamics, cooperativity, and other complex functional and regulatory properties of supramolecular enzymes.


title: Acoustic light-sheet microscopy

abstract:
Light-sheet imaging of 3D objects with high spatial resolution remains an open challenge because of the trade-off between field-of-view (FOV) and axial resolution originating from the diffraction of light. We developed acoustic light-sheet microscopy (acoustic LSM), which actively manipulates the light propagation inside a large sample to obtain wide-field microscopic images deep inside a target. By accurately coupling a light-sheet illumination pulse into a planar acoustic pulse, the light-sheet can be continuously guided over large distances. We imaged a fluorescence-labeled transparent mouse brain for the FOVs of 19.3 x 12.4 mm2 and 9.7 x 5.9 mm2 with resolved microstructures and single cells deep inside the brain. Acoustic LSM creates new opportunities for the application of light-sheet in the field of industry to basic science.

One Sentence SummaryAn acoustic-optical method overcomes a trade-off between field-of-view and axial resolution in light-sheet microscopy.


title: Frequent Aneuploidy in Primary Human T Cells Following CRISPR-Cas9 cleavage

abstract: Multiple ongoing clinical trials use site-specific nucleases to disrupt T cell receptor (TCR) genes in order to allow for allogeneic T cell therapy1-5. In particular, the first U.S. clinical trial using CRISPR-Cas9 entailed the targeted disruption of the TCR chains and programmed cell death protein 1 (PDCD1) in T cells of refractory cancer patients6. Here, we used the same guide RNA sequences and applied single-cell RNA sequencing (scRNAseq) to more than 7000 primary human T cells, transfected with CRISPR-Cas9. Four days post-transfection, we found a loss of chromosome 14, harboring the TCR locus, in up to 9% of the cells, and a chromosome 14 gain in up to 1.4% of the cells. We further identified truncations of chromosome 7, harboring the TCR{beta} locus, in 9.9% of the cells. Loss of heterozygosity (LOH) was further validated using fluorescence in situ hybridization (FISH) and the temporal dynamics of cleavage and incomplete repair were monitored using digital droplet PCR (ddPCR). Aneuploidy was found among all T cell subsets and was associated with transcriptional signatures of reduced proliferation and metabolism as well as with induced p53 activation and cell death. We conclude that aneuploidy and chromosomal truncations are frequent outcomes of CRISPR-Cas9 cleavage in clinical protocols. Monitoring and minimizing these aberrant products is crucial for future applications of genome editing in T cell engineering and beyond.


title: MultiVI: deep generative model for the integration of multi-modal data

abstract: Jointly profiling the transcriptional and chromatin accessibility landscapes of single-cells is a powerful technique to characterize cellular populations. Here we present MultiVI, a probabilistic model to analyze such multiomic data and integrate it with single modality datasets. MultiVI creates a joint representation that accurately reflects both chromatin and transcriptional properties of the cells even when one modality is missing. It also imputes missing data, corrects for batch effects and is available in the scvi-tools framework: https://docs.scvi-tools.org/.


title: Nanomechanics combined with HDX reveal allosteric drug binding sites of CFTR NBD1

abstract: Cystic fibrosis is most frequently caused by the deletion of F508 ({Delta}F508) in CFTRs nucleotide binding domain 1 (NBD1), compromising CFTR folding, stability and domain assembly. The limitation of developing a successful therapy is due to the lack of molecules that synergistically facilitate folding by targeting distinct structural defects of {Delta}F508-CFTR. To improve drug efficacy by targeting the {Delta}F508-NBD1 folding and stability, and to study potential {Delta}F508-NBD1 allosteric corrector binding sites at the atomic level, we combined molecular dynamics (MD) simulations, atomic force spectroscopy (AFM) and hydrogen-deuterium exchange (HDX) experiments. These methods allowed us to describe unfolding intermediates and forces acting during NBD1 mechanical unfolding and to elucidate the stabilization mechanism of {Delta}F508-NBD1 by 5-bromoindole-3-acetic acid (BIA). An NBD1 region, including the -subdomain, was identified as a potentially important participant of the first folding steps, characterized by non-native interactions of F508, thus destabilized in the deletion mutant. The instability was counteracted by the low-potency corrector BIA, increasing the mechanical resistance of the {Delta}F508-NBD1 -subdomain, which was confirmed as a binding site by computational modeling and HDX experiments. Our results underline the complementarity of computational and experimental methods and provide a possible strategy to improve folding correctors.


title: Structures of topoisomerase V in complex with DNA reveal unusual DNA binding mode and novel relaxation mechanism

abstract: Topoisomerase V is a unique topoisomerase that combines DNA repair and topoisomerase activities. The enzyme has an unusual arrangement, with a small topoisomerase domain followed by 12 tandem (HhH)2 domains, which include three AP lyase repair domains. The unusual architecture of this enzyme bears no resemblance to any other known topoisomerase. Here we present structures of topoisomerase V in complex with DNA. The structures show that the (HhH)2 domains wrap around the DNA and in this manner appear to act as a processivity factor. There is a conformational change in the protein to expose the topoisomerase active site. The DNA bends sharply to enter the active site, which melts the DNA and probably facilitates relaxation. The structures show a DNA binding mode not observed before and provide information on the way this unusual topoisomerase relaxes DNA.


title: Sequence grammar underlying unfolding and phase separation of globular proteins

abstract:
Aberrant phase separation of globular proteins is associated with many diseases. Here, we use a model protein system to understand how unfolded states of globular proteins drive phase separation and the formation of unfolded protein deposits (UPODs). For UPODs to form, the concentrations of unfolded molecules must be above a threshold value. Additionally, unfolded molecules must possess appropriate sequence grammars to drive phase separation. While UPODs recruit molecular chaperones, their compositional profiles are also influenced by synergistic physicochemical interactions governed by the sequence grammars of unfolded proteins and sequence features of cellular proteins. Overall, we find that the driving forces for phase separation and the compositional profiles of UPODs are governed by the sequence grammar of unfolded proteins. Our studies highlight the need for uncovering the sequence grammars of unfolded proteins that drive UPOD formation and lead to gain-of-function interactions whereby proteins are aberrantly recruited into UPODs.

HighlightsO_LIUnfolded states of globular proteins phase separate to form UPODs in cells
C_LIO_LIThe fraction of unfolded molecules and the sticker grammar govern phase separation
C_LIO_LIHydrophobic residues act as stickers that engage in intermolecular interactions
C_LIO_LISticker grammar also influences gain-of-function recruitment into aberrant UPODs
C_LI


title: Exact distribution of threshold-crossing times for protein concentrations: Implication for biological timekeeping

abstract: Stochastic transcription and translation dynamics of protein accumulation up to some concentration threshold sets the timing of many cellular physiological processes. Here we obtain the exact distribution of first threshold-crossing times of protein concentration, in either Laplace or time domain, and its associated cumulants: mean, variance and skewness. The distribution is asymmetric and its skewness non-monotonically varies with the threshold. We study lysis times of E-coli cells for holin gene mutants of bacteriophage-{lambda} and find a good match with theory. Mutants requiring higher holin thresholds show more skewed lysis time distributions as predicted.


title: Type I PRMTs and PRMT5 Inversely Regulate Post-Transcriptional Intron Detention

abstract: Protein arginine methyltransferases (PRMTs) are required for the regulation of RNA processing factors. Type I enzymes catalyze mono- and asymmetric dimethylation; Type II enzymes catalyze mono- and symmetric dimethylation. To understand the specific mechanisms of PRMT activity in splicing regulation, we inhibited Type I and II PRMTs and probed their transcriptomic consequences. Using the newly developed SKaTER-seq method, analysis of co-transcriptional splicing revealed that PRMT inhibition resulted in slower splicing rates. Surprisingly, altered co-transcriptional splicing kinetics correlated poorly with ultimate changes in alternative splicing of polyadenylated RNA--particularly intron retention (RI). Investigation of RI following inhibition of nascent transcription demonstrated that PRMTs inversely regulate RI post-transcriptionally. Subsequent proteomic analysis of chromatin-associated polyadenylated RNA identified aberrant binding of the Type I substrate, CHTOP, and the Type II substrate, SmB. Targeted mutagenesis of all methylarginine sites in SmD3, SmB, and SmD1 recapitulated splicing changes seen with Type II PRMT inhibition. Conversely, mutagenesis of all methylarginine sites in CHTOP recapitulated the splicing changes seen with Type I PRMT inhibition. Closer examination of subcellular fractions indicated that RI were isolated to the nucleoplasm and chromatin. Together, these data demonstrate that PRMTs regulate the post-transcriptional processing of nuclear, detained introns through Sm and CHTOP arginine methylation.


title: Covalent inhibition of endoplasmic reticulum chaperone GRP78 disconnects the transduction of ER stress signals to inflammation and lipid accumulation in diet-induced obese mice

abstract: Targeting endoplasmic reticulum (ER) stress, inflammation and metabolic dysfunctions may halt the pathogenesis of obesity and thereby reduce the prevalence of diabetes, cardiovascular disesases and cancers. The present study was designed to elucidate the mechnaisms by which plant-derived celastrol ameliorated inflammation and lipid accumulation in obesity. The mouse model of diet-induced obesity was induced by feeding high-fat diet for 3 months and subsequently intervented with celastrol for 21 days. Hepatic and adipose tissues were analysed for lipid accumulation, macrophage activation and biomarker expression. As result, celastrol effectively reduced body weight, suppressed ER stress, inflammation and lipogenesis while promoted hepatic lipolysis. RNA-sequencing revealed that celastrol-loaded nanomicelles restored the expression of 49 genes that regulate ER stress, inflammation and lipid metabolism. On the other hand, celastrol-PEG4-alkyne was synthesized for identifying celastrol-bound proteins in RAW264.7 macrophages. ER chaperone GRP78 was identified by proteomics approach for celastrol binding to the residue Cys41. Upon binding and conjugation, celastrol diminished the chaperone activity of GRP78 by 130-fold and reduced ER stress in palmitate-challenged cells, while celastrol analogue lacking quinone methide failed to exhibit anti-obesity effects. Thus, covalent GRP78 inhibition may induce the reprograming of ER signaling, inflammation and metabolism against diet-induced obesity.

category: pharmacology and toxicology


title: Organ protection by caloric restriction depends on activation of the de novo NAD+ synthesis pathway

abstract: Therapeutic strategies to treat acute kidney injury (AKI) are lacking in clinical practice. Interestingly, preconditioning by hypoxia (HP) and caloric restriction (CR) is highly protective in rodent AKI models. However, the underlying molecular mechanisms of this process are unknown. A comparative transcriptome analysis of murine kidneys after HP and CR identified Kynureninase (KYNU) as a common downstream target. Using a newly generated KYNU-deficient mouse line, we show that KYNU strongly contributes to the protective effect of preconditioning. Metabolome, transcriptome and proteome analyses reveal the KYNU-dependent de novo nicotinamide adenine dinucleotide (NAD+) biosynthesis pathway as necessary for CR-associated maintenance of NAD+ levels. Importantly, the impact of CR on the de novo NAD+ biosynthesis pathway can be recapitulated in humans. These findings provide a valuable insight into the molecular mechanisms mediating protection upon preconditioning and point towards the de novo branch of NAD+ biosynthesis as a conserved target in nephroprotection.

category: physiology