title: High fat diet initiates rapid adaptation of the intestine

abstract: While the systemic impacts of overnutrition are well-known to cause obesity and metabolic syndrome over the course of months, the immediate adaptive response of the intestinal epithelium to dietary changes remains poorly understood. Here we used physiological metrics and single cell analyses to interrogate the adaptive response of intestinal epithelial cells when moved to a high fat diet (HFD). Within 1 day of HFD exposure, mice exhibited altered feeding behavior, an increase in energy expenditure and an increase in intestinal epithelial proliferation. Single cell transcriptional analysis demonstrated several acute cellular changes on day 1, including a cell-stress response in intestinal crypts, de-granularization of Paneth cells, and a shift towards fatty acid cellular metabolism. By 3 days of HFD, there was an emergence of uncommitted progenitors with a transcriptional profile indicative of a shift from secretory populations and towards an absorptive fate. In enterocytes, genes regulating lipid transport and absorption increased over the first 3 days which paralleled a functional increase in lipid absorption in vivo over the course of 7 days on HFD. These findings demonstrate that intestinal epithelial cell populations respond rapidly to changes in diet through initial changes in cellular function followed by a shift in cellular composition.


title: Microtubules self-repair in living cells

abstract: Microtubule self-repair has been studied both in vitro and in vivo as an underlying mechanism of microtubule stability. The turnover of tubulin dimers along the microtubule network has challenged the pre-existing dogma that only growing ends are dynamic. However, although there is clear evidence of tubulin incorporation into the shaft of polymerized microtubules in vitro, the possibility of such events taking place in living cells remains uncertain. In this study, we investigated this possibility by microinjecting purified tubulin dimers labeled with a red fluorophore into the cytoplasm of cells expressing GFP-tubulin. We observed the appearance of red dots along pre-existing green microtubule network within minutes. We found that the fluorescence intensities of these red dots were inversely correlated with the green signal, suggesting that the red dimers were incorporated into the microtubules and replaced the pre-existing green dimers. We then characterized the size and spatial frequency of these incorporations as a function of injected tubulin concentration and post-injection delay. The saturation of these measurements contradicted the hypothesis of nonspecific adsorption along microtubules and suggested that the injected dimers incorporated into a finite number of damaged sites. By our low estimate, within a few minutes of the injections, free dimers incorporated into major repair sites every 70 micrometers of microtubules. Finally, we mapped the location of these sites in micropatterned cells and found that they were more concentrated in regions where the actin filament network was less dense and where microtubules exhibited greater lateral fluctuations. These results provide evidences that microtubules do self-repair in living cells, and they provide a quantitative characterization of the temporal and spatial dynamics of this process in PtK2 cells.

category: cell biology


title: TYMV has dual action on the plant RNA silencing defense through its VSR P69 and the host RNASE THREE LIKE1

abstract:
Post-Transcriptional Gene Silencing (PTGS) is a defense mechanism that targets invading nucleic acids of endogenous (transposons) or exogenous (pathogens, transgenes) origins. During plant infection by viruses, virus-derived primary siRNAs target viral RNAs, resulting in both destruction of single-stranded viral RNAs (execution step) and production of secondary siRNAs (amplification step), which maximize the plant defense. As a counter-defense, viruses express proteins referred to as Viral Suppressor of RNA silencing (VSR). Some viruses express VSRs that totally inhibit PTGS, whereas other viruses express VSRs that have limited effect. Here we show that infection with the Turnip yellow mosaic virus (TYMV) is enhanced in Arabidopsis ago1, ago2 and dcl4 mutants, which are impaired in the execution of PTGS, but not in dcl2, rdr1 and rdr6 mutants, which are impaired in the amplification of PTGS. Consistently, we show that the TYMV VSR P69 localizes in siRNA-bodies, which are the site of production of secondary siRNAs, and limits PTGS amplification. Moreover, TYMV induces the production of the host enzyme RNASE THREE-LIKE 1 (RTL1) to further reduce siRNA accumulation. Together, these results indicate that the inhibition of PTGS amplification through the dual action of P69 and RTL1 let the sole siRNAs produced by the primary plant PTGS defense to limit the infection by TYMV, explaining the mild symptoms Arabidopsis-infected TYMV. This could be considered as an elegant strategy used by viruses to limit the plant PTGS defense and propagate without killing their host.

Author summaryRNA silencing is a conserved defense mechanism directed against viruses in various eukaryotic kingdoms. As a counter-defense, viruses generally express proteins referred to as viral suppressor of RNA silencing (VSR), which promote infection by inhibiting one or the other component of the RNA silencing machinery. So far, most of the work on VSRs has concentrated on those that strongly inhibit RNA silencing, causing severe infections and plant death. However, situations where VSRs only partially inhibit RNA silencing could be considered as advantageous for both partners of the infection because infected plants survive, flower and produce seeds despite virus multiplication. In this study, we show that Turnip yellow mosaic virus (TYMV) encodes a weak VSR, P69, which partially inhibits the amplification but not the execution of RNA silencing. In addition, TYMV induces the expression of the endogenous enzyme RNASE THREE-LIKE 1 (RTL1) to further reduce siRNA accumulation, and this induction is proportional to the amount of virus. This appears a smart strategy for a virus to propagate without killing its host. We propose that the limited effect of P69 on RNA silencing together with the proportional induction of host RTL1 results in a tight balance between virus propagation and plant development. In the light of these results, one could consider the Arabidopsis-TYMV interaction as an elegant model for plant-virus coevolution.

category: plant biology


title: Structure-function conservation between the methyltransferases SETD3 and SETD6

abstract: Among the protein lysine methyltransferases family members, it appears that SETD6 is highly similar and closely related to SETD3. The two methyltransferases show high similarity in their structure, which raised the hypothesis that they share cellular functions. Using a proteomic screen, we identified 52 shared interacting-proteins. Gene Ontology (GO) analysis of the shared proteins revealed significant enrichment of proteins involved in transcription. Our RNA-seq data of SETD6 KO and SETD3 KO HeLa cells identified [~]100 up-regulated and down-regulated shared genes. We have also identified a substantial number of genes that changed dramatically in the double KO cells but did not significantly change in the single KO cells. GO analysis of these genes revealed enrichment of apoptotic genes. Accordingly, we show that the double KO cells displayed high apoptotic levels, suggesting that SETD6 and SETD3 inhibit apoptosis. Collectively, our data strongly suggest a functional link between SETD6 and SETD3 in the regulation of apoptosis.


title: Computational rewiring of allosteric pathways reprograms GPCR selective responses to ligands

abstract: G-protein-coupled receptors (GPCRs) are the largest class of cell surface receptors and drug targets, and respond to a wide variety of chemical stimuli to activate diverse cellular functions. Understanding and predicting how ligand binding triggers a specific signaling response is critical for drug discovery and design but remains a major challenge. Here, computational design of GPCR allosteric functions is used to uncover the mechanistic relationships between agonist ligand chemistry, receptor sequence, structure, dynamics and allosteric signaling in the dopamine D2 receptor. Designed gain of function D2 variants for dopamine displayed very divergent G-protein signaling responses to other ligand agonists that strongly correlated with ligand structural similarity. Consistent with these observations, computational analysis revealed distinct topologies of allosteric signal transduction pathways for each ligand-bound D2 pair that were perturbed differently by the designs. We leveraged these findings by rewiring ligand-specific pathways and designed receptors with highly selective ligand responses. Overall, our study suggests that distinct ligand agonists can activate a given signaling effector through specific "allosteric activator" moieties that engage partially independent signal transmission networks in GPCRs. The results provide a mechanistic framework for understanding and predicting the impact of sequence polymorphism on receptor pharmacology, informing selective drug design and rationally designing receptors with highly selective ligand responses for basic and therapeutic applications.

category: bioengineering


title: Ageing critically transforms the network connectivity and viscoelasticity of RNA-binding protein condensates but RNA can prevent it

abstract: Biomolecular condensates, some of which are liquid-like during health, can age over time becoming gel-like pathological systems. Ageing of RNA-binding protein condensates can emerge from the progressive accumulation of inter-protein {beta}-sheets. To bridge microscopic understanding of such time-dependent transformation with the modulation of FUS and hnRNPA1 condensate viscoelasticity, we develop a multiscale simulation approach. Our method integrates atomistic simulations with sequence-dependent coarse-grained modelling of condensates that age over time due to accumulation of inter-protein {beta}-sheets. We reveal that ageing notably increases condensate viscosity but does not transform the phase diagrams. Strikingly, the network of molecular connections within condensates is drastically altered during ageing and culminates in gelation when the network of strong inter-protein {beta}-sheets fully percolates. High concentrations of RNA decelerate the accumulation of inter-protein {beta}-sheets, abrogating the effects of ageing. Our study uncovers molecular and kinetic factors explaining condensate ageing, and suggests a potential mechanism to slow ageing down.

category: biophysics


title: Effects of organic fertilizer digestate on the growth of tomato (Lycopersicon esculentum Mill)

abstract: Tomatoes are known for their human health benefits. Tomato producers use chemical fertilizers to increase tomato production levels. However, the use of organic fertilizers such as digestate can achieve satisfactory yields while sustaining the soil environment. In this tomato study, the effect of three dilutions of digestate (25%, 50% and 75%) on plant height, leaf area and fruit number were compared to NPK 15 15 15. The 50% dilution of digestate had the most beneficial effect on the plants at the end of the experiment.


title: Neighbouring modifications interfere with the detection of phosphorylated alpha-synuclein at Serine 129: Revisiting the specificity of pS129 antibodies

abstract: Alpha-synuclein (aSyn) within Lewy bodies, Lewy neurites, and other pathological hallmarks of Parkinsons disease and synucleinopathies have consistently been shown to accumulate in aggregated and phosphorylated forms of the protein, predominantly at Serine 129 (S129). Antibodies against phosphorylated S129 (pS129) have emerged as the primary tools to investigate, monitor, and quantify aSyn pathology in the brain and peripheral tissues. However, most of the antibodies and immunoassays aimed at detecting pS129-aSyn were developed based on the assumption that neighbouring post-translational modifications (PTMs) either do not co-occur with pS129 or do not influence its detection. Herein, we demonstrate that the co-occurrence of multiple pathology-associated C-terminal PTMs (e.g., phosphorylation at Tyrosine 125 or truncation at residue 133 or 135) differentially influences the detection of pS129-aSyn species by pS129-aSyn antibodies. These observations prompted us to systematically reassess the specificity of the most commonly used pS129 antibodies against monomeric and aggregated forms of pS129-aSyn in mouse brain slices, primary neurons, mammalian cells and seeding models of aSyn pathology formation. We identified two antibodies that are insensitive to pS129 neighbouring PTMs. However, consistent with previous reports, most pS129 antibodies showed cross-reactivity towards other proteins and often detected low and high molecular weight bands in aSyn knock-out samples that could be easily mistaken for monomeric or High Molecular Weight aggregates of aSyn. Our observations suggest that the pS129 antibodies do not capture the biochemical and morphological diversity of aSyn pathology. They also underscore the need for more specific pS129 antibodies, more thorough characterization and validation of existing antibodies, and the use of the appropriate protein standards and controls in future studies.

category: neuroscience


title: Identification and characterization of the T cell receptor (TCR) repertoire of the Cynomolgus macaque (Macaca Fascicularis)

abstract:
BackgroundNon-human primates (NHP) are desirable as animal models of human disease because they share behavioral, physiological, and genomic traits with people. Hence, NHP recapitulate manifestations of disease not observed in other animal species. The Macaca fascicularis (i.e., Cynomolgus macaque) is an NHP species extensively used for biomedical research, but the TCR repertoire hasnt been characterized yet.

ResultWe used the genomic sequences to design primers to identify the expressed TCR repertoire by single cell RNAseq. The data analysis from 22 unique samples were used to assign a functional status to each TCR genes. We identified and analyzed the TRA/D, TRB and TRG loci of the Cynomolgus macaque.

ConclusionThe genomic organization of the Cynomolgus macaque has great similarity with Macaca mulatta (i.e., Rhesus macaque) and they shared >90% sequence similarity with the human TCR repertoire. These data will facilitate the analysis of T cell immunity in Cynomolgus macaques.

category: immunology


title: Resolution of glycogen and glycogen-degrading activities reveals correlates of Lactobacillus crispatus dominance in a cohort of young African women

abstract:
BackgroundA healthy vaginal microbiome is dominated by Lactobacillus species that produce lactic acid, lowering vaginal pH and limiting colonization by pathogens. Lactobacillus dominance (LD) is established during puberty, but many women, especially those of Black race, lose LD during their reproductive years. Glycogen is thought to be a key host nutrient that supports vaginal lactobacilli and their fermentative lactic acid production, but mechanisms of glycogen utilization by Lactobacillus species are incompletely understood. By partitioning glycogen and glycogen-derived maltodextrin, as well as the activity of glycogen-degrading pullulanase enzymes, this work refines understanding of vaginal glycogen catabolism and identifies correlates of LD.

ResultsVaginal swab samples were collected from a cohort of young women with limited sexual experience in Thika, Kenya (N=17, ages 17-20). Metagenomic profiling of the vaginal microbiome revealed that most samples exhibited LD, particularly dominant Lactobacillus crispatus. Amylopullulanase activity, cleavage of glycogen -1,4 and -1,6 linkages by individual/multifunctional enzymes, showed a significant positive correlation with glycogen-derived maltodextrin, but no relationship with L. crispatus dominance. Pullulanase activity, which specifically targets glycogen -1,6 linkages, was 3-fold higher in L. crispatus-dominated samples and significantly correlated with D-lactic acid levels. Metagenomics and targeted PCR revealed that 36% of L. crispatus-dominated metagenomes from our African cohort lacked a functional L. crispatus pullulanase (pulA) gene, a 3-fold higher frequency of gene loss than that seen in metagenomes from European and North American women. Our findings suggest pulA gene loss or inactivation may correspond with reductions in L. crispatus abundance, pullulanase activity and lactic acid levels compared to samples dominated by pulA-competent L. crispatus.

ConclusionsOur results indicate that although amylase activity drives the accumulation of glycogen catabolites in vaginal fluid, pullulanase appears to specifically contribute to maximal D-lactic acid production by L. crispatus. However, this is only possible when a functional pulA gene is present, which was not the case in a substantial proportion of young African women with dominant L. crispatus. Scaling this analysis to a larger cohort will address whether genomic and enzymatic indicators of L. crispatus pullulanase activity are predictive of sustained LD and vaginal health.

category: microbiology


title: Hypoxia-induced tracheal elasticity in vector beetle facilitates the loading of pinewood nematode

abstract: Many pathogens rely on their insect vectors for transmission. Such pathogens are under selection to improve vector competence for their transmission by employing various tissue or cellular responses of vectors. However, whether pathogens can actively cause hypoxia in vectors and exploit hypoxia responses to promote their vector competence is still unknown. Fast dispersal of pinewood nematode (PWN), the causal agent for the destructive pine wilt disease and subsequent infection of pine trees is characterized by the high vector competence of pine sawyer beetles (Monochamus spp.), and a single beetle can harbor over 200,000 PWNs in its tracheal system. Here, we demonstrate that PWN loading activates hypoxia in tracheal system of the vector beetles. Both PWN loading and hypoxia enhanced tracheal elasticity and thickened the apical extracellular matrix (aECM) of the tracheal tubes. We observed a notable upregulation of an aECM gene Muc91C, encoding a resilin-like mucin protein in PWN-loaded and hypoxic tracheal tubes. RNAi knockdown of Muc91C reduced tracheal elasticity and aECM thickness under hypoxia conditions and thus decreasing PWN loading. Our study suggests a crucial role of hypoxia-induced developmental responses in shaping vector tolerance to the pathogen and provides clues for potential molecular targets to control pathogen dissemination.

category: ecology


title: The Role of Replication-induced Chromosomal Copy Numbers in Spatio-temporal Gene Regulation and Evolutionary Chromosome Plasticity

abstract: For a coherent response to environmental changes, bacterial evolution has formed a complex transcriptional regulatory system comprising classical DNA binding proteins sigma factors and modulation of DNA topology. In this study, we investigate replication-induced gene copy numbers - a regulatory concept that is unlike the others not based on modulation of promoter activity but replication dynamics. We show that a large fraction of genes are predominantly affected by transient copy numbers and identify cellular functions and central pathways governed by this mechanism in Escherichia coli. Furthermore, we show quantitatively that the previously observed spatio-temporal expression pattern between different growth phases mainly emerges from transient chromosomal copy numbers. We extend the analysis to the plant pathogen Dickeya dadantii and the biotechnologically relevant organism Vibrio natriegens. The analysis reveals a connection between growth phase dependent gene expression and evolutionary gene migration in these species. Further extension to the bacterial kingdom shows that chromosome evolution is governed by growth rate related transient copy numbers.

category: genetics


title: A divide-and-conquer approach for genomic prediction in rubber tree using machine learning

abstract: Rubber tree (Hevea brasiliensis) is the main feedstock for commercial rubber; however, its long vegetative cycle has hindered the development of more productive varieties via breeding programs. With the availability of H. brasiliensis genomic data, several linkage maps with associated quantitative trait loci (QTLs) have been constructed and suggested as a tool for marker-assisted selection (MAS). Nonetheless, novel genomic strategies are still needed, and genomic selection (GS) may facilitate rubber tree breeding programs aimed at reducing the required cycles for performance assessment. Even though such a methodology has already been shown to be a promising tool for rubber tree breeding, increased model predictive capabilities and practical application are still needed. Here, we developed a novel machine learning-based approach for predicting rubber tree stem circumference based on molecular markers. Through a divide-and-conquer strategy, we propose a neural network prediction system with two stages: (1) subpopulation prediction and (2) phenotype estimation. This approach yielded higher accuracies than traditional statistical models in a single-environment scenario. By delivering large accuracy improvements, our methodology represents a powerful tool for use in Hevea GS strategies. Therefore, the incorporation of machine learning techniques into rubber tree GS represents an opportunity to build more robust models and optimize Hevea breeding programs.


title: Correlation and Path Coefficient Analyses in Hot Pepper (Capsicum annum L.)

abstract: To assess the correlation and find out the direct and indirect effect of yield attributing traits on yield, twenty six accessions and four varieties during the off-season period from November 2016 to May 2017 under irrigation. at Dire dawa, Ethiopia. The experiment was conducted using RCBD design with three replications. Plant height, primary branch per plot, days to fifty percent flowering, stem width, number of fruit per plant, fruit length, days to maturity and number of fruit per plant were positive and highly significantly correlated with dry fruit yield per plot at both genotypic and phenotypic correlation. Plant height (0.1081), number of fruit per plant (0.2610), fruit length (0.4293), stem width (0.4059), pedicel length (0.0122), days to maturity (0.0401) and internode length (0.0227) exerted positive direct effect on dry fruit yield per plot at phenotypic level. Genotypic path analysis showed that days to fifty percent flowering (0.0956), stem width (0.5867), fruit length (0.3671), plant height (0.0754), number of fruit per plant (0.2673) and internode length (0.0079)had positive direct effect. The direct effect of these characters on dry fruit yield per plot indicates that improvement on these traits may increase yield.


title: CUT&Tag recovers up to half of ENCODE ChIP-seq peaks

abstract: Techniques for genome-wide epigenetic profiling have been undergoing rapid development toward recovery of high quality data from bulk and single cell samples. DNA-protein interactions have traditionally been profiled via chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq), which has become the gold standard for studying histone modifications or transcription factor binding. Cleavage Under Targets & Tagmentation (CUT&Tag) is a promising new technique, which enables profiling of such interactions in situ at high sensitivity and is adaptable to single cell applications. However thorough evaluation and benchmarking against established ChIP-seq datasets are still lacking. Here we comprehensively benchmarked CUT&Tag for H3K27ac and H3K27me3 against published ChIP-seq profiles from ENCODE in K562 cells. Across a total of 30 new and 6 published CUT&Tag datasets we found that no experiment recovers more than 50% of known ENCODE peaks, regardless of the histone mark. We tested peak callers MACS2 and SEACR, identifying optimal peak calling parameters. Balancing both precision and recall of known ENCODE peaks, SEACR without retention of duplicates showed the best performance. We found that reducing PCR cycles during library preparation lowered duplication rates at the expense of ENCODE peak recovery. Despite the moderate ENCODE peak recovery, peaks identified by CUT&Tag represent the strongest ENCODE peaks and show the same functional and biological enrichments as ChIP-seq peaks identified by ENCODE. Our workflow systematically evaluates the merits of methodological adjustments and will facilitate future efforts to apply CUT&Tag in human tissues and single cells.

category: genomics


title: Gradient-k: Improving the performance of K-Means using the density gradient

abstract: We introduce Gradient-k, an upgrade of the k-means algorithm that improves clustering accuracy and reduces the number of iterations required for convergence. This is achieved by correcting the distance used in the k-means algorithm by a factor based on the angle between the density gradient and the direction to the cluster center. We show that this correction allows the algorithm to go beyond the traditional tessellation obtained by k-means, enabling the creation of nonlinear separation boundaries. The correction also provides the ability to distinguish clusters having non-normal shapes and of varying densities. Furthermore, Gradient-k has a lower computational complexity for datasets with many samples. We compare the performance of Gradient-k with that of k-means and of DBSCAN on several benchmark datasets, as well as on a more realistic dataset in the context of neural spike sorting.

category: bioinformatics


title: Deep learning to decompose macromolecules into independent Markovian domains

abstract: The increasing interest in modeling the dynamics of ever larger proteins has revealed a fundamental problem with models that describe the molecular system as being in a global configuration state. This notion limits our ability to gather sufficient statistics of state probabilities or state-to-state transitions because for large molecular systems the number of metastable states grows exponentially with size. In this manuscript, we approach this challenge by introducing a method that combines our recent progress on independent Markov decomposition (IMD) with VAMPnets, a deep learning approach to Markov modeling. We establish a training objective that quantifies how well a given decomposition of the molecular system into independent subdomains with Markovian dynamics approximates the overall dynamics. By constructing an end-to-end learning framework, the decomposition into such subdomains and their individual Markov state models are simultaneously learned, providing a dataefficient and easily interpretable summary of the complex system dynamics. While learning the dynamical coupling between Markovian subdomains is still an open issue, the present results are a significant step towards learning "Ising models" of large molecular complexes from simulation data.


title: Painters in chromatin: a unified quantitative framework to systematically characterize epigenome regulation and memory

abstract: In eukaryotes, many stable and heritable phenotypes arise from the same DNA sequence, owing to epigenetic regulatory mechanisms relying on the molecular cooperativity of "reader-writer" enzymes. In this work, we focus on the fundamental, generic mechanisms behind the epigenome memory encoded by post-translational modifications of histone tails. Based on experimental knowledge, we introduce a unified modeling framework, the painter model, describing the mechanistic interplay between sequence-specific recruitment of chromatin regulators, chromatin-state-specific reader-writer processes and long-range spreading mechanisms. A systematic analysis of the model building blocks highlights the crucial impact of tridimensional chromatin organization and state-specific recruitment of enzymes on the stability of epigenomic domains and on gene expression. In particular, we show that enhanced 3D compaction of the genome and enzyme limitation facilitate the formation of ultra-stable, confined chromatin domains. The model also captures how chromatin state dynamics impact the intrinsic transcriptional properties of the region, slower kinetics leading to noisier expression. We finally apply our framework to analyze experimental data, from the propagation of{gamma} H2AX around DNA breaks in human cells to the maintenance of heterochromatin in fission yeast, illustrating how the painter model can be used to extract quantitative information on epigenomic molecular processes.


title: In silico analysis of Bacopa monnieri (L.) Wettst. compounds for drug development against Neurodegenerative Disorders

abstract:
Neurotrophins play a crucial role in the development and regulation of neurons. Alterations in the functioning of these Neurotrophins leads to several Neurodegenerative Disorders. Albeit engineered medications which are accessible for the treatment of Neurodegenerative Disorders, due to their numerous side-effects, it becomes imperative to formulate and synthesize novel drug candidates. Plants could be utilized as an alternative for these manufactured medications because of their low incidental effects in contrast with the engineered drugs. Bacopa monnieri has been traditionally known to be utilized to treat Neurodegenerative Disorders. Therefore, in current study an in-silico based study was carried out to evaluate the pharmacological effect of Bacopa monnieri. Molecular Docking was carried out to screen the active phytochemicals of Bacopa monnieri which can act as potential drug candidates against the causative proteins of Neurodegenerative Disorders. A total of 105 biologically active phytochemicals from Bacopa monnieri were docked against the receptors of brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4, and nerve growth factor. Based on molecular docking study it was observed that the phytocompounds Vitamin E, Benzene propanoic acid, 3,5-bis(1,1dimethylethyl)4-hydroxy-, methyl ester (BPA), Stigmasterol, and Nonacosane of Bacopa monnieri significantly fits to the active residues of the four selected drug targets. Further Molecular Dynamics simulation study was performed to examine the stability of the binding of these phytochemicals with the selected targets. Drug likeness properties as well as related physico-chemical properties were analyzed through ADMETox study. Our findings suggested that the phytocompounds Vitamin E, BPA, Stigmasterol and Nonacosane significantly bind against brain-derived neurotrophic factor, neurotrophin-3, neurotrophin4, and nerve growth factor, respectively which may be the potential drug candidates for the treatment of neurodegenerative disorders.

Graphic Abstract

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category: pharmacology and toxicology


title: Hypercaloric diet triggers transient molecular rearrangements of astrocytes selectively in the arcuate nucleus

abstract: Hypothalamic astrocytes are particularly affected by energy-dense food consumption. How the anatomical location of these glial cells and their spatial molecular distribution in the arcuate nucleus of the hypothalamus (ARC) determine the cellular response to a high caloric diet remains unclear. In this study, we investigated their distinctive molecular responses following the exposure to a high-fat high-sugar (HFHS) diet, specifically in the ARC. Using RNA sequencing and proteomics, we showed that astrocytes have a distinct transcriptomic and proteomic profile dependent on their anatomical location, with a major proteomic reprogramming in hypothalamic astrocytes. By ARC single-cell sequencing, we observed that a HFHS diet dictates time- and cell-specific transcriptomic responses, revealing that astrocytes have the most distinct regulatory pattern compared to other cell types. Lastly, we topographically and molecularly characterized astrocytes expressing glial fibrillary acidic protein and/or aldehyde dehydrogenase 1 family member L1 in the ARC, of which the abundance was significantly increased, as well as the alteration in their spatial and molecular profiles, with a HFHS diet. Together, our results provide a detailed multi-omics view on the spatial and temporal changes of astrocytes particularly in the ARC during different time points of adaptation to a high caloric diet.


title: Fear memory in humans is consolidated over time independent of sleep

abstract:
BackgroundMany pathological fear memories rely on processes of fear conditioning upon an aversive event. These fear memories may continue to change after acquisition, but the factors contributing to these changes are not well understood. Sleep is known to consolidate, strengthen and change newly acquired declarative and procedural memories. However, evidence on the role of time and sleep in the consolidation of fear memories is inconclusive. Here, we used highly sensitive electrophysiological measures to examine the development of fear-conditioned responses over time and sleep in humans.

MethodsWe assessed event-related brain potentials (ERP) in 18 healthy young individuals during fear conditioning before and after a 2-hour afternoon nap or a corresponding wake interval in a counterbalanced within-subject design. The procedure involved pairing a neutral tone (CS+) with a highly unpleasant sound (US). As a control, another neutral tone (CS-) was paired with a neutral sound. Fear responses were examined before the interval during a habituation phase and an acquisition phase as well as after the interval during an extinction phase and a re-acquisition phase.

ResultsDifferential fear conditioning during acquisition was evidenced by a more negative slow ERP component preceding the CS+ compared to the CS-(stimulus-preceding negativity). This differential fear response was even stronger after the interval during re-acquisition compared to initial acquisition, but this effect was similarly pronounced after sleep and wakefulness.

ConclusionsThese findings suggest that fear memories are consolidated over time, with this effect being independent of intervening sleep.


title: Incertohypothalamic A13 dopaminergic neurons are involved in fine forelimb movements but not reward

abstract: Tyrosine hydroxylase (TH)-containing neurons of the dopamine (DA) cell group A13 are well-positioned to impact known dopamine-related functions since their descending projections innervate target regions that regulate vigilance, sensorimotor integration and execution. Despite this known connectivity little is known regarding the functionality of A13-DA circuits. Using TH-specific loss-of-function methodology and techniques to monitor population activity in transgenic rats in vivo we investigated the contribution of A13-DA neurons in reward and movement-related actions. Our work demonstrates a role for A13-DA neurons in grasping and handling of objects that is independent from reward. A13-DA neurons respond strongly when animals grab and manipulate food items while their inactivation or degeneration prevents animals from successfully doing so - a deficit partially attributed to a reduction in grip strength. In contrast, there was no relation between A13-DA activity and food-seeking behavior when animals were tested on a reward-based task that did not include a reaching/grasping response. Moreover, motivation for food was unaffected as goal-directed behavior for food items was in general intact following A13 neuronal inactivation/degeneration. These results demonstrate a functional role for A13-DA neurons in prehensile actions that are uncoupled from reward and as such position A13-DA neurons into the functional framework regarding centrally-located DA populations and their ability to coordinate movement.


title: Plant-invertebrate interactions across a forested retrogressive chronosequence

abstract: In the long-term absence of disturbance, ecosystems often enter a decline or retrogressive phase which leads to reductions in primary productivity, plant biomass, nutrient cycling and foliar quality. However, the consequences of ecosystem retrogression for higher trophic levels such as herbivores and predators, are less clear. Using a post-fire forested island-chronosequence across which retrogression occurs, we show that nutrient availability strongly controls invertebrate herbivore biomass when predators are few, but that there is a switch from bottom-up to top-down control when predators are common. This trophic flip in herbivore control probably arises because invertebrate predators respond to alternative energy channels from the adjacent aquatic matrix, which were independent of plant biomass. Our results suggest that effects of nutrient limitation, following ecosystem retrogression, on trophic cascades are modified by independent variation in predator abundance, which requires a more holistic approach to trophic ecology to better understand herbivore effects on plant communities.


title: Performance and limitations of linkage-disequilibrium-basedmethods for inferring the genomic landscape of recombination anddetecting hotspots: a simulation study

abstract: Knowledge of recombination rate variation along the genome provides important insights into genome and phenotypic evolution. Population genomic approaches offer an attractive way to infer the population-scaled recombination rate{rho} =4Ner using the linkage disequilibrium information contained in DNA sequence polymorphism data. Such methods have been used on a broad range of plant and animal species to build genome-wide recombination maps. However, the reliability of these inferences has only been assessed under a restrictive set of conditions. Here, we evaluated the ability of one of the most widely used coalescent-based approaches, LDhelmet, to infer a biologically-realistic genomic landscape of recombination. Using simulations, we specifically assessed the impact of empirical (sample size, block penalty) and evolutionary parameters (effective population size (Ne), mutation and recombination rates) on inferred map quality. We report reasonably good correlations between simulated and inferred landscapes, but point to limitations when it comes to detecting recombination hotspots. False positives and false negatives considerably confound fine-scale patterns of inferred recombination under a wide array of conditions, particularly when Ne is small and the mutation/recombination rate ratio is low, to the extent that maps inferred from populations sharing the same recombination landscape appear uncorrelated. We thus address a message of caution to users of such approaches, while also recognizing their importance and potential, particularly in species with less complex landscapes for which LD-based approaches should provide high quality recombination maps.


title: Variable Selection via Grace-AKO with Applications in Genomics

abstract:
MotivationVariable selection is a common statistical approach to detecting genes associated with clinical outcomes of scientific interest. There are hundreds of thousands of genes in genomic studies, while only a limited number of individual samples are available. Therefore, it is important to develop a method to identify genes associated with outcomes of interest that can control finite-sample false discovery rate (FDR) in high-dimensional data settings.

ResultsThis article proposes a novel method named Grace-AKO for graph-constrained estimation (Grace), which incorporates aggregation of multiple knockoffs (AKO) with the network-constrained penalty. Grace-AKO can control FDR in finite sample settings and improve stability. Simulation studies show that Grace-AKO has better performance in FDR control in finite sample settings than the original Grace model. We apply Grace-AKO to the prostate cancer data in the The Cancer Genome Atlas (TCGA) program by incorporating prostate-specific antigen (PSA) pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) as the prior information. Grace-AKO finally identifies 47 candidate genes associated with PSA level, and more than 75% of the detected genes can be validated.

Availability and implementationWe developed an R package for Grace-AKO available at: https://github.com/mxxptian/GraceAKO

Contactdoraz@hku.hk

Supplementary informationSupplementary data are available at Bioinformatics online.


title: Contributions of the N-terminal intrinsically disordered region of the SARS-CoV-2 nucleocapsid protein to RNA-induced phase separation

abstract:
SARS-CoV-2 nucleocapsid protein is an essential structural component of mature virions, encapsulating the genomic RNA and modulating RNA transcription and replication. Several of its activities might be associated with the proteins ability to undergo liquid-liquid phase separation. NSARS-CoV-2 contains an intrinsically disordered region at its N-terminus (NTE) that can be phosphorylated and is affected by disease-relevant mutations. Here we show that NTE deletion decreases the range of RNA concentrations that can induce phase separation of NSARS-CoV-2. In addition, deletion of the prion-like NTE allows NSARS-CoV-2 droplets to retain their liquid-like nature during incubation. We further demonstrate that RNA-binding engages multiple parts of the NTE and changes NTEs structural properties. The results form the foundation to characterize the impact of N-terminal mutations and post-translational modifications on the molecular properties of the SARS-CoV-2 nucleocapsid protein.

StatementThe nucleocapsid protein of SARS-CoV-2 plays an important role in both genome packaging and viral replication upon host infection. Replication has been associated with RNA-induced liquid-liquid phase separation of the nucleocapsid protein. We present insights into the role of the N-terminal part of the nucleocapsid protein in the proteins RNA-mediated liquid-liquid phase separation.


title: The dynamical Matryoshka model: 2. Modeling of local lipid dynamics at the sub-nanosecond timescale in phospholipid membranes

abstract:
Biological membranes are generally formed by lipids and proteins. Often, the membrane properties are studied through model membranes formed by phospholipids only. They are molecules composed by a hydrophilic head group and hydrophobic tails, which can present a panoply of various motions, including small localized movements of a few atoms up to the diffusion of the whole lipid or collective motions of many of them. In the past, efforts were made to measure these motions experimentally by incoherent neutron scattering and to quantify them, but with upcoming modern neutron sources and instruments, such models can now be improved. In the present work, we expose a quantitative and exhaustive study of lipid dynamics on DMPC and DMPG membranes, using the Matryoshka model recently developed by our group. The model is confronted here to experimental data collected on two different membrane samples, at three temperatures and two instruments. Despite such complexity, the model describes reliably the data and permits to extract a series of parameters. The results compare also very well to other values found in the literature.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/486370v1_fig1.gif" ALT="Figure 1">
View larger version (25K):
org.highwire.dtl.DTLVardef@374f9corg.highwire.dtl.DTLVardef@14ba37eorg.highwire.dtl.DTLVardef@dae67corg.highwire.dtl.DTLVardef@151f721_HPS_FORMAT_FIGEXP  M_FIG O_FLOATNOFigure 1:C_FLOATNO Graphical abstract.

C_FIG Highlights- The Matryoshka model brings a new general description of local lipid dynamics.
- Phospholipid membranes on various conditions are compared in this novel framework.
- Effects of main phase transition, membrane geometry or motion direction are probed.
- Despite high number of parameters, overfitting is avoided by a global fit strategy.


title: Mapping whole-brain projections of anatomically defined prefrontal neurons using combined 3D convolution networks

abstract: Long-range axonal projections provide the foundation for functional connectivity between brain regions and are critical in the modulation of behavior. Descending projections from medial prefrontal cortex (mPFC) to various target regions regulate critical behavioral functions including decision making, social behavior and mood. While specific mPFC projections have distinct behavioral roles, individual mPFC projection neurons can also innervate multiple target regions. Yet how mPFC projection neurons divide their axons across the brain is poorly understood. In this study, we mapped the axon collaterals of mPFC neurons that project to nucleus accumbens (NAc), ventral tegmental area (VTA), or contralateral mPFC (cmPFC) in mice. We used tissue clearing and light sheet fluorescence microscopy to visualize the 3-D structure of axonal arbors across the intact brain. While machine learning can automate analysis of axons in images of cleared tissue, it is challenging to train a model that generalizes to all axonal structures because the appearance of axons varies by target region. In this study, we present DeepTraCE (Deep learning-based image Tracing with Combined-model Enhancement), a new strategy for axon segmentation and quantification in images of cleared tissue. DeepTraCE is based on the deep-learning framework TRAILMAP; it achieves highly accurate axon detection by combining multiple machine learning models that are each applied to different brain regions. Using DeepTraCE, we find that cmPFC, NAc, and VTA-projecting mPFC neurons represent largely separable classes with unique axon collaterals in cortical, olfactory, and thalamic regions, respectively.


title: Hierarchical architecture of dopaminergic circuits enables second-order conditioning in Drosophila

abstract: Dopaminergic neurons with distinct projection patterns and physiological properties compose memory subsystems in a brain. However, it is poorly understood whether or how they interact during complex learning. Here, we identify a feedforward circuit formed between dopamine subsystems and show that it is essential for second-order conditioning, an ethologically important form of higher-order associative learning. The Drosophila mushroom body comprises a series of dopaminergic compartments, each of which exhibits distinct memory dynamics. We find that a slow and stable memory compartment can serve as an effective "teacher" by instructing other faster and transient memory compartments via a single key interneuron, which we identify by connectome analysis and neurotransmitter prediction. This excitatory interneuron acquires enhanced response to reward-predicting odor after first-order conditioning and, upon activation, evokes dopamine release in the "student" compartments. These hierarchical connections between dopamine subsystems explain distinct properties of first- and second-order memory long known by behavioral psychologists.


title: Fear extinction is regulated by long noncoding RNA activity at the synapse

abstract: Long noncoding RNAs (lncRNAs) represent a multidimensional class of regulatory molecules involved in many aspects of brain function. Emerging evidence indicates that lncRNAs are expressed at the synapse; however, a direct role for their activity in this subcellular compartment in memory formation has yet to be demonstrated. Using lncRNA capture-seq on synaptosomes, we identified a significant number of lncRNAs that accumulate at synapses within the infralimbic prefrontal cortex of adult male C57/Bl6 mice. Among these is a splice variant related to the stress-associated lncRNA, Gas5. RNA immunoprecipitation followed by mass spectrometry and single molecule imaging revealed that this Gas5 isoform, in association with the RNA binding proteins G3bp2 and Caprin1, regulates the activity-dependent trafficking and clustering of RNA granules in dendrites. In addition, we found that cell-type-specific, state-dependent, and synapse-specific knockdown of the Gas5 variant led to impaired fear extinction memory. These findings identify a new mechanism of fear extinction that involves the dynamic interaction between local lncRNA activity and the coordination of RNA condensates in the synaptic compartment.


title: Screening the Pathogen Box Compounds for Activity Against Plasmodium falciparum Sporozoite Motility

abstract: As the malaria parasite becomes resistant to every drug that we develop, identification and development of novel drug candidates is essential. Many studies have screened compounds designed to target the clinically important blood stages. However, if we are to shrink the malaria map, new drugs that block transmission of the parasite are needed. Sporozoites are the infective stage of the malaria parasite, transmitted to the mammalian host as mosquitoes probe for blood. Sporozoite motility is critical to their ability to exit the inoculation site and establish infection and drug-like compounds targeting motility are effective in blocking infection in the rodent malaria model. In this study, we established a moderate throughput motility assay for sporozoites of the human malaria parasite Plasmodium falciparum, enabling us to screen the 400 drug-like compounds from the Pathogen box provided by Medicines for Malaria Venture for their activity. Compounds exhibiting inhibitory effects on P. falciparum sporozoite motility were further assessed against transmission-blocking activity and asexual stage growth. Five compounds had a significant inhibitory effect on P. falciparum sporozoite motility at 1 M concentration and four of these compounds also showed significant inhibition on transmission of P. falciparum gametocytes to the mosquito and of these four, three had previously been shown to have inhibitory activity on asexual blood stage parasites. Our findings provide new antimalarial drug candidates that have multi-stage activity.


title: Conformational flexibility, lipid binding, and regulatory domains in CelTOS

abstract: CelTOS is an essential Plasmodium traversal protein and conserved in apicomplexan parasites. We showed that CelTOS forms pores in cell membranes to enable traversal of parasites through cells (Jimah et al., 2016). Here, we establish roles for the distinct regions of CelTOS, examine the mechanism of pore formation and evaluate the immunogenicity of engineered CelTOS variants. CelTOS dimer dissociation is required for pore formation as disulfide bridging between monomers inhibits pore formation and this inhibition is rescued by disulfide-bridge reduction. A helix destabilizing Pro127 allows CelTOS to undergo significant conformational changes to assemble into pores. The flexible C-terminus of CelTOS is a negative regulator that limits pore formation. Lipid binding is a pre-requisite for pore assembly as mutation of a phospholipid binding site in CelTOS resulted in loss of lipid binding and abrogated pore formation. The disulfide-locked and N-terminal deletion mutants showed improved immunogenicity relative to wild-type CelTOS in mice. These findings have implications for pore-forming proteins that are essential for diverse functions, identify critical regions in CelTOS, and will guide the design of effective CelTOS-targeting vaccines to combat infection and transmission of malaria and apicomplexan parasites.


title: Cross neutralization of Omicron BA.1 against BA.2 and BA.3 SARS-CoV-2

abstract: The Omicron SARS-CoV-2 has three distinct sublineages, among which sublineage BA.1 is responsible for the initial Omicron surge and is now being replaced by BA.2 world-wide, whereas BA.3 is currently at a low frequency. The ongoing BA.1-to-BA.2 replacement underscores the importance to understand the cross-neutralization among the three Omicron sublineages. Here we tested the neutralization of BA.1-infected human sera against BA.2, BA.3, and USA/WA1-2020 (a strain isolated in late January 2020). The BA.1-infected sera neutralized BA.1, BA.2, BA.3, and USA/WA1-2020 SARS-CoV-2s with geometric mean titers (GMTs) of 445, 107, 102, and 16, respectively. Thus, the neutralizing GMTs against heterologous BA.2, BA.3, and USA/WA1-2020 were 4.2-, 4.4-, and 28.4-fold lower than the GMT against homologous BA.1, respectively. These findings have implications in COVID-19 vaccine strategy.


title: Brequinar and Dipyridamole in Combination Exhibits Synergistic Antiviral Activity Against SARS-CoV-2 in vitro: Rationale for a host-acting antiviral treatment strategy for COVID-19

abstract: The continued evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has compromised the efficacy of currently available vaccines and monoclonal antibody (mAb)-based treatment options for COVID-19. The limited number of authorized small-molecule direct-acting antivirals present challenges with pill burden, the necessity for intravenous administration or potential drug interactions. There remains an unmet medical need for effective and convenient treatment options for SARS-CoV-2 infection. SARS-CoV-2 is an RNA virus that depends on host intracellular ribonucleotide pools for its replication. Dihydroorotate dehydrogenase (DHODH) is a ubiquitous host enzyme that is required for de novo pyrimidine synthesis. The inhibition of DHODH leads to a depletion of intracellular pyrimidines, thereby impacting viral replication in vitro. Brequinar (BRQ) is an orally available, selective, and potent low nanomolar inhibitor of human DHODH that has been shown to exhibit broad spectrum inhibition of RNA virus replication. However, host cell nucleotide salvage pathways can maintain intracellular pyrimidine levels and compensate for BRQ-mediated DHODH inhibition. In this report, we show that the combination of BRQ and the salvage pathway inhibitor dipyridamole (DPY) exhibits strong synergistic antiviral activity in vitro against SARS-CoV-2 by enhanced depletion of the cellular pyrimidine nucleotide pool. The combination of BRQ and DPY showed antiviral activity against the prototype SARS-CoV-2 as well as the Beta (B.1.351) and Delta (B.1.617.2) variants. These data support the continued evaluation of the combination of BRQ and DPY as a broad-spectrum, host-acting antiviral strategy to treat SARS-CoV-2 and potentially other RNA virus infections.


title: Methylene blue, Mycophenolic acid, Posaconazole, and Niclosamide inhibit SARS-CoV-2Omicron variant BA.1 infection of human airway epithelial explant cultures

abstract: Sublineages of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) Omicron variants continue to amass mutations in the spike (S) glycoprotein, which leads to immune evasion and rapid spread of the virus across the human population. Here we demonstrate the susceptibility of the Omicron variant BA.1 (B.1.1.529.1) to four repurposable drugs, Methylene blue (MB), Mycophenolic acid (MPA), Posaconazole (POS), and Niclosamide (Niclo) in post-exposure treatments of primary human airway cell cultures. MB, MPA, POS, and Niclo are known to block infection of human nasal and bronchial airway epithelial explant cultures (HAEEC) with the Wuhan strain, and four variants of concern (VoC), Alpha (B.1.1.7), Beta (B.1.351), Gamma (B.1.1.28), Delta (B.1.617.2) (1, 2). Our results not only show broad anti-coronavirus effects of MB, MPA, POS and Niclo, but also demonstrate that the Omicron variant BA.1 (B.1.1.529.1) sheds infectious virus from HAEEC over at least 15 days, and maintains both intracellular and extracellular viral genomic RNA without overt toxicity, suggesting viral persistence. The data underscore the broad effects of MB, MPA, POS, and Niclo against SARS-CoV-2 and the currently circulating VoC, and reinforce the concept of repurposing drugs in clinical trials against COVID-19.


title: Identification of C270 as a novel site for allosteric modulators of SARS-CoV-2 papain-like protease

abstract: The papain-like protease (PLpro) in coronavirus is one of key cysteine proteases responsible for the proteolytic processing of viral polyproteins, and plays an important role in dysregulation of host immune response. PLpro is a promising therapeutic target with a major challenge in inhibitor design due to the restricted S1/S2 sites for two consecutive glycine of substrates. Here we reported the discovery of two activators of the SARS-CoV-2 PLpro from a biochemical screening, and the identification of the unique residue, C270, as an allosteric and covalent regulation site for the activators. This site was also specifically modified by glutathione oxidized, resulting in the S-glutathionylation and activation of the protease. Furthermore, one compound was found to allosterically inhibit the protease by covalent binding to this crucial site. Together, these results elucidated an unrevealed molecular mechanism for allosteric modulation of the proteases activity, and provided a new strategy for discovery of allosteric inhibitors of the SARS-CoV-2 PLpro.

category: molecular biology


title: An in situ cut-and-paste genome editing platform mediated by CRISPR/Cas9 or Cas12a

abstract: Recombinant DNA technology mediated by restriction enzymes and ligases allows in vitro manipulation of a DNA segment isolated from the genome. Short overhangs generated by restriction enzymes facilitate efficient pasting together a DNA sequence and a vector. We adopted this recombinant DNA strategy to develop an in vivo recombinant-genome genome editing approach. Using the programmable endonuclease Cas9 or Cas12a as a restriction enzyme, we devised an in situ cut-and-paste (iCAP) genome editing method that was tested in both mouse germline and human cell line platforms. Mouse gene loci Slc35f2 and Slc35f6 were each edited with in-frame insertion of a large APEX2-Cre cassette and concurrent FRT3 insertion at a second location providing proof of principle for the iCAP method. Further, a de nova single nucleotide mutation associated with MED13L syndrome was efficiently corrected in patient cells. Altogether, the iCAP method provides a single genome editing platform with flexibility and multiutility enabling versatile and precise sequence alterations, such as insertion, substitution, and deletion, at single or multiple locations within a genomic segment in mammalian genomes.


title: Clinical mutations in WDR34 lead to diverse impacts on the assembly and function of dynein-2

abstract:
The primary cilium is a sensory organelle, receiving signals from the external environment and relaying them into the cell. Mutations in proteins required for transport in the primary cilium result in ciliopathies, a group of genetic disorders that commonly lead to the malformation of organs such as the kidney, liver and eyes and skeletal deformation. Motor proteins dynein-2 and kinesin-2 mediate retrograde and anterograde transport in the cilium. WDR34, a dynein-2 intermediate chain, is required for the maintenance of cilia function. Here, we investigated WDR34 mutations identified in Jeune syndrome, Short-rib polydactyly syndrome or Asphyxiating thoracic dysplasia patients by stably expressing the mutant proteins in WDR34 knockout cells. WDR34 mutations led to different spectrums of phenotypes. Each mutation results in a distinct profile of phenotypes. Quantitative proteomics demonstrated changes in dynein-2 assembly, whereas initiation and extension of the axoneme, IFT-B protein localization, transition zone integrity, and Hedgehog signalling were also affected.

Summary statementClinical mutations in WDR34 are found to have diverse impacts on ciliogenesis and cilia function following stable expression in a WDR34 knockout cell model.


title: Protein Quaternary Structures in Solution are a Mixture of Multiple forms

abstract: Over half the proteins form homo or hetero-oligomeric structures. Experimentally determined structures are often considered in determining a proteins oligomeric state, but static structures miss the dynamic equilibrium between different quaternary forms. The problem is exacerbated in homo-oligomers, where the oligomeric states are challenging to characterize. Here, we re-evaluated the oligomeric state of 17 different bacterial proteins across a broad range of protein concentrations and solutions by native mass-spectrometry (MS), mass photometry (MP), size exclusion chromatography (SEC), and small-angle x-ray scattering (SAXS), finding that most exhibit several oligomeric states. Surprisingly, many proteins did not show mass-action driven equilibrium between the oligomeric states. For approximately half the proteins, the predicted oligomeric forms described in publicly available databases underestimated the complexity of protein quaternary structures in solution. Conversely, AlphaFold Multimer provided an accurate description of the potential multimeric states for most proteins, suggesting that it could help resolve uncertainties on the solution state of many proteins.

category: biochemistry


title: Helical remodeling augments 5-lipoxygenase activity

abstract: The synthesis of pro-inflammatory leukotrienes implicated in asthma, allergic rhinitis, and atherosclerosis is initiated by the enzyme 5-lipoxygenase (5-LOX). The crystal structure of human Stable-5-LOX revealed a conformation where the catalytic iron was inaccessible to bulk solvent as two aromatic residues on a conserved helix-2 (H2) plugged the substrate access portal. Here, we present a new conformation of 5-LOX where H2 adopts an elongated conformation equivalent to that described in other animal lipoxygenase structures. The sigmoidal kinetic behavior of 5-LOX, which is indicative of positive cooperativity, is consistent with a substrate-induced conformational change that shifts the ensemble of enzyme populations to favor the catalytically competent state. Strategic point mutations along H2 designed to unlock the closed conformation and elongate H2 resulted in improved kinetic parameters, altered limited-proteolysis data, and a drastic reduction in the length of the lag phase yielding the most active 5-LOX enzyme to date. Structural predictions by AlphaFold2 of these variants statistically favor an elongated H2 and reinforce a model in which improved kinetic parameters correlate with a more readily adopted, open conformation.


title: Sensitive detection and structural characterisation of UV-induced cross-links in protein-RNA complexes using CLIR-MS

abstract: Cross-linking coupled with mass spectrometry is an increasingly popular methodology for elucidating structural information from biological complexes. Whilst protein-protein cross-linking workflows are widely used and well characterised, adoption of protein-RNA cross-linking workflows for structural studies is less widespread, and data produced from such experiments remains less well understood. The cross-linking of stable isotope labelled RNA coupled to mass spectrometry (CLIR-MS) workflow uses isotope labelled RNA to simultaneously confirm that peptides are cross-linked to RNA and aid cross-link localisation in an RNA sequence. For broader application of CLIR-MS as part of the structural analysis of ribonucleoproteins, the method must be sensitive, robust, and its reaction products need to be well characterised. We enhanced our previously published workflow, improving coverage and sensitivity. We used it to infer common properties of protein-RNA cross-links such as cross-linking distance, and to assess the impact of substitution of uracil with 4-thio-uracil in structural proteomics experiments. We profiled the compositional diversity of RNA-derived peptide modifications, and subsequently defined a more inclusive data analysis approach which more than doubles the number of cross-link spectrum matches compared with our past work. We defined distance restraints from these cross-links, and with the aid of visualisation software, demonstrated that on their own they provide sufficient information to localise an RNA chain to the correct position on the surface of a protein. We applied our enhanced workflow and understanding to characterise the binding interface of several protein-RNA complexes containing classical and uncommon RNA binding domains. The enhanced sensitivity and understanding demonstrated here underpin a wider adoption of protein-RNA cross-linking in structural biology.


title: Prediction of infectivity of SARS-CoV2: Mathematical Model with Docking Simulation analysis between Spike Protein and ACE2

abstract: Variants of a coronavirus (SARS-CoV-2) have been spreading in a global pandemic. Improved understanding of the infectivity of future new variants is important so that effective countermeasures against them can be quickly undertaken. In our research reported here, we aimed to predict the infectivity of SARS-CoV-2 by using a mathematical model with molecular simulation analysis, and we used phylogenetic analysis to determine the evolutionary distance of the spike protein gene (S gene) of SARS-CoV-2. We subjected the six variants and the wild type of spike protein and human angiotensin-converting enzyme 2 (ACE2) to molecular docking simulation analyses to understand the binding affinity of spike protein and ACE2. We then utilized regression analysis of the correlation coefficient of the mathematical model and the infectivity of SARS-CoV-2 to predict infectivity. The evolutionary distance of the S gene correlated with the infectivity of SARS-CoV-2 variants. The coefficient of the mathematical model obtained with results of molecular docking simulation also correlated with the infectivity of SARS-CoV-2 variants. These results suggest that the data from the docking simulation for the receptor binding domain of variant spike proteins and human ACE2 were valuable for prediction of SARS-CoV-2 infectivity. In addition, we developed a mathematical model for prediction of SARS-CoV-2 variant infectivity by using binding affinity obtained via molecular docking and the evolutionary distance of the S gene.


title: Prediction of infectivity of SARS-CoV2: Mathematical model with analysis of docking simulation for spike proteins and angiotensin-converting enzyme 2


title: Model guided design of enhanced bi-stable controllers to effectively switch cellular states

abstract: 1Bi-stable gene regulatory motifs are found in a wide variety of natural gene regulatory networks and typically effect transcriptional switching between stable phenotypic states in cells. In synthetic gene regulatory circuits, these architectures can be used to dynamically switch between distinct metabolic states for metabolic engineering and therapeutic applications. Though it has been over two decades since the first synthetic bi-stable switch was developed, the lack of modularity and predictability of these motifs in varying environments has limited widespread application, especially since the factors that affect switching characteristics are still unclear. In this work, we develop a mathematical model describing a bi-stable gene regulatory element using mass action kinetics. Using this model and a newly developed dynamical modeling and continuity analysis framework, we identify the changes in switching characteristics shown by the bi-stable motif over a range of biologically relevant model parameter values. Interestingly, there appears to be a trade-off between the robustness of the motif - the parameter ranges over which it retains bi-stable function, and the speed at which it effects a phenotypic change. Further, using E. coli as a model host, we constructed a large library of transcriptional switches that show a wide range of switching speeds, to experimentally demonstrate the presence of this trade-off. The presence of this trade-off has significant implications on the design of transcriptional switches for diverse applications and could potentially explain the circuit architecture of natural transcriptional switches as well. Additionally, we anticipate that our diverse library of bi-stable switches will be valuable to effect phenotypic changes with differing switching speed requirements for metabolic engineering applications.

category: synthetic biology


title: CIS calibrates GM-CSF signalling strength to regulate macrophage polarization via a STAT5-IRF8 axis

abstract: The cytokine granulocyte-macrophage-colony stimulating factor (GM-CSF) possesses the ability to differentiate macrophages (MOs) with opposing functions, namely proinflammatory M1-like and immunosuppressive M2-like. Despite the importance and opposing functional outcomes of these processes, the intrinsic mechanism that regulates the functional polarization of MOs under GM-CSF signaling remains elusive. Here we show that GM-CSF induced MOs polarisation resulted in the expression of the Cytokine-inducible SH2-containing protein (CIS), and that CIS deficiency diverted differentiation of monocytes into immunosuppressive M2-like MOs expression. CIS deficiency resulted in the hyperactivation of the JAK-STAT5 signaling pathway, consequently promoting the downregulation of the transcription factor Interferon Regulatory Factor 8 (IRF8). Loss and gain of function approaches highlighted IRF8 as a critical instructor of the M1-like polarisation program. In vivo, CIS deficiency led to skewing to M2-like macrophages, which induced strong Th2 immune responses characterised by the development of severe experimental asthma. Collectively, we reveal a CIS-censored mechanism interpreting the opposing actions of GM-CSF in MO differentiation and uncovering its role in controlling allergic inflammation.


title: cGASylation by a bacterial E1-E2 fusion protein primes antiviral immune signaling

abstract: In all organisms, innate immune pathways sense viral infection and rapidly activate potent immune responses while maintaining a high degree of specificity to prevent inappropriate activation (autoimmunity). In humans, the innate-immune receptor cGAS detects viral infection to produce the nucleotide second messenger cGAMP, which initiates STING-dependent antiviral signaling. Bacteria encode predecessors of the cGAS-STING pathway, termed cyclic oliogonucleotide-based antiphage signaling systems (CBASS), and bacterial cGAS detects bacteriophage infection to produce cGAMP. How bacterial cGAS activation is controlled, however, remains unknown. Here, we show that the CBASS-associated protein Cap2 primes bacterial cGAS for activation through a ubiquitin transferase-like mechanism. A cryoelectron microscopy structure of the Cap2-cGAS complex reveals Cap2 as an all-in-one ubiquitin transferase-like protein, with distinct domains resembling the eukaryotic E1 protein ATG7 and the E2 proteins ATG10 and ATG3. The structure captures a reactive-intermediate state with the cGAS C-terminus extending into the Cap2 E1 active site and conjugated to AMP. We find that Cap2 ligates the cGAS C-terminus to a target molecule in cells, a process we call cGASylation. cGASylation primes cGAS for a [~]50-fold increase in cGAMP production. We further demonstrate that Cap2 activity is balanced by a specific endopeptidase, Cap3, which deconjugates cGAS and antagonizes antiviral signaling. Our data demonstrate that bacteria control immune signaling using an ancient, minimized ubiquitin transferase-like system and provide insight into the evolution of E1 and E2 machinery across the kingdoms of life.


title: SARS-CoV-2 Omicron is specifically restricted in its replication in human lung tissue, compared to other variants of concern

abstract: SARS-CoV-2 Omicron variant has been characterized by decreased clinical severity, raising the question of whether early variant-specific interactions within the mucosal surfaces of the respiratory tract could mediate its attenuated pathogenicity. Here, we employed ex vivo infection of native human nasal and lung tissues to investigate the local-mucosal susceptibility and innate immune response to Omicron, compared to Delta and earlier SARS-CoV-2 variants of concern (VOC). We show that the replication of Omicron in lung tissues is highly restricted compared to other VOC, whereas it remains relatively unchanged in nasal tissues. Mechanistically, Omicron induced a much stronger antiviral interferon response in infected tissues compared to Delta and earlier VOC - a difference which was most striking in the lung tissues, where the innate immune response to all other SARS-CoV-2 VOC was blunted. Our data provide new insights to the reduced lung involvement and clinical severity of Omicron.


title: On the relationship between Pathogenic Potential and Infective Inoculum

abstract:
Pathogenic Potential (PP) is a mathematical description of an individual microbe, virus, or parasites ability to cause disease in a host, given the variables of inoculum, symptomology, mortality, and in some instances, median survival time of the host. We investigated the relationship between pathogenic potential (PP) and infective inoculum (I) using two pathogenic fungi in the wax moth Galleria mellonella with mortality as the relevant outcome. Our analysis for C. neoformans infection revealed negative exponential relationship between PP and I. Plotting the log(I) versus the Fraction symptomatic (Fs) over median host survival time (T) revealed a linear relationship, with a slope that varied between the different fungi studied and a y-intercept corresponding to the inoculum that produced no symptoms. The I vs Fs/T slope provided a measure of the pathogenicity of each microbial species, which we call the pathogenicity constant or kPath. The kPath provides a new parameter to quantitatively compare the relative virulence and pathogenicity of microbial species for a given host. In addition, we investigated the PP and Fs/T from values found in preexisting literature. Overall, the relationship between Fs/T and PP versus inoculum varied among microbial species and extrapolation to zero symptoms allowed the calculation of the lowest pathogenic inoculum (LPI) of a microbe. Microbes tended to fall into two groups: those with positive linear relationships between PP and Fs/T vs I, and those that had a negative exponential PP vs I relationship with a positive logarithmic Fs/T vs I relationship. The microbes with linear relationships tended to be bacteria, whereas the exponential-based relationships tended to be fungi or higher order eukaryotes. Differences in the type and sign of the PP vs I and Fs/T vs I relationships for pathogenic microbes suggest fundamental differences in host-microbe interactions leading to disease.

Author SummaryThe ability of a microbe, virus, or parasite to cause disease is dependent on multiple factors, virulence factors. host immune defenses, the infective inoculum, and the type of immune response. For many microbes their capacity for causing disease is highly dependent on the inoculum. The mathematical formula for Pathogenic Potential (PP) is a way to compare the ability of an organism to have a pathogenic effect, as measured by Fraction Symptomatic (Fs), mortality (M), and inoculum (I), and can include the median survival time of the host (T). Increasing inoculum of the fungus Cryptococcus neoformans for a moth host resulted in exponentially smaller pathogenic potential, and the Fs/T versus inoculum plot showed a logarithmic relationship. Together, these relationships show diminishing returns with increasing cryptococcal inoculum, in which each individual fungus plays a smaller role in pathogenicity. Literature data shows that other microbes, mostly bacteria, had linear Fs/T versus inoculum relationships, which indicate that each bacterium contributed an equal amount to pathogenicity. These differences in relationships can point to differences in host-microbe interactions and suggest new ways in which the organism causes disease.


title: Vaccination decreases the risk of influenza A virus reassortment with a concomitant increase in subgenomic genetic variation in pigs

abstract: Although vaccination is broadly used in North American swine breeding herds, managing swine influenza is challenging primarily due to the continuous evolution of influenza A virus (IAV) and the ability of the virus to transmit among vaccinated pigs. Studies that have simultaneously assessed the impact of vaccination on the emergence of IAV reassortment and genetic variation in pigs are limited. Here we directly sequenced 28 bronchoalveolar lavage fluid (BALF) samples collected from vaccinated and unvaccinated pigs co-infected with H1N1 and H3N2 IAV strains, and characterized 202 individual viral plaques recovered from 13 BALF samples. We identified 54 reassortant viruses that were grouped in 16 distinct and 18 mixed genotypes. Notably, we found that prime-boost vaccinated pigs had less reassortant viruses than non-vaccinated pigs, likely due to a reduction in the number of days pigs were co-infected with both challenge viruses. However, direct sequencing from BALF samples revealed limited impact of vaccination on viral variant frequency, evolutionary rates, and nucleotide diversity in any IAV coding regions. Overall, our results highlight the value of IAV vaccination not only at limiting virus replication in pigs but also at protecting public health by restricting the generation of novel reassortants with zoonotic and/or pandemic potential.


title: Small Molecules Restore Azole Activity Against Drug-Tolerant and Drug-Resistant Candida Isolates

abstract:
Each year, fungi cause more than 1.5 billion infections worldwide and have a devastating impact on human health, particularly in immunocompromised individuals or patients in intensive care units. The limited antifungal arsenal and emerging multidrug resistant species necessitate the development of new therapies. One strategy for combating drug resistant pathogens is the administration of molecules that restore fungal susceptibility to approved drugs. Accordingly, we carried out a screen to identify small molecules that could restore the susceptibility of pathogenic Candida species to azole antifungals. This screening effort led to the discovery of novel 1,4-benzodiazepines that restore fluconazole susceptibility in resistant isolates of Candida albicans, as evidenced by 100-1000-fold potentiation of fluconazole activity. This potentiation effect was also observed in azole-tolerant strains of C. albicans and in other pathogenic Candida species. The 1,4-benzodiazepines selectively potentiated different azoles, but not other antifungal drugs. A remarkable feature of the potentiation was that the combination of the 1,4-benzodiazepines with fluconazole was fungicidal, whereas fluconazole alone is fungistatic. Interestingly, the potentiators were not toxic to C. albicans in the absence of fluconazole, but inhibited virulence-associated filamentation of the fungus. We found that the combination of 1,4-benzodiazepines and fluconazole significantly enhanced host survival in a Galleria mellonella model of systemic fungal infection. Taken together, these observations validate a strategy wherein small molecules can restore the activity of highly used anti-infectives that have lost potency.

IMPORTANCEIn the last decade, we have been witnessing a higher incidence of fungal infections, an expansion of the fungal species capable of causing disease (e.g., Candida auris), as well as increased antifungal drug resistance. Among human fungal pathogens, Candida species are a leading cause of invasive infections and are associated with high mortality rates. Azoles are the most widely used antifungals against Candida infections, yet the emergence and expansion of drug-resistant species have narrowed their clinical utility. In this work, we describe a set of novel 1,4-benzodiazepines that potentiate fluconazole and restore the susceptibility of azole-resistant and azole-tolerant Candida isolates. Interestingly, the potentiating 1,4-benzodiazepines were not toxic to fungal cells but inhibited their virulence associated filamentous growth. Furthermore, combinations of 1,4-benzodiazepines and fluconazole enhanced host survival in the Galleria mellonella model of systemic fungal infections. Accordingly, we propose the use of novel antifungal potentiators as a powerful strategy to restore fungal susceptibility to clinically approved drugs.


title: Ultra-deep Sequencing of Hadza Hunter-Gatherers Recovers Vanishing Microbes

abstract:
The gut microbiome has been identified as a key to immune and metabolic health, especially in industrialized populations1. Non-industrialized individuals harbor more diverse microbiomes and distinct bacterial lineages2, but systemic under-sampling has hindered insight into the extent and functional consequences of these differences3. Here, we performed ultra-deep metagenomic sequencing and laboratory strain isolation on fecal samples from the Hadza, hunter-gatherers in Tanzania, and comparative populations in Nepal and California. We recover 94,971 total genomes of bacteria, archaea, bacteriophage, and eukaryotes, and find that 43% are novel upon aggregating with existing unified datasets4,5. Analysis of in situ growth rates, genetic pN/pS signatures, and high-resolution strain tracking reveal dynamics in the hunter-gatherer gut microbiome that are distinct from industrialized populations. Industrialized versus Hadza gut microbes are enriched in genes associated with oxidative stress, possibly a result of microbiome adaptation to inflammatory processes. We use phylogenomics to reveal that global spread of the spirochaete Treponema succinifaciens parallels historic human migration prior to its extinction in industrialized populations. When combined with a detailed definition of gut-resident strains that are vanishing in industrialized populations, our data demonstrate extensive perturbation in many facets of the gut microbiome brought on by the industrialized lifestyle.

Recognition of work with indigenous communitiesResearch involving indigenous communities is needed for a variety of reasons including to ensure that scientific discoveries and understanding appropriately represent all populations and do not only benefit those living in industrialized nations3,6. Special considerations must be made to ensure that this research is conducted ethically and in a non-exploitative manner. In this study we performed deep metagenomic sequencing on fecal samples that were collected from Hadza hunter-gatherers in 2013/2014 and were analyzed in previous publications using different methods2,7. A material transfer agreement with the National Institute for Medical Research in Tanzania specifies that stool samples collected are used solely for academic purposes, permission for the study was obtained from the National Institute of Medical Research (MR/53i 100/83, NIMR/HQ/R.8a/Vol.IX/1542) and the Tanzania Commission for Science and Technology, and verbal consent was obtained from the Hadza after the studys intent and scope was described with the help of a translator. The publications that first described these samples included several scientists and Tanzanian and Nepali field-guides as co-authors for the critical roles they played in sample collection, but as no new samples were collected in this study, only scientists who contributed to the analyses described here were included as co-authors in this publication. It is currently not possible for us to travel to Tanzania and present our results to the Hadza people, however we intend to do so once the conditions of the COVID-19 pandemic allow it.


title: Chaperone Requirements for De Novo Folding of Saccharomyces cerevisiae Septins

abstract: Polymers of septin protein complexes play cytoskeletal roles in eukaryotic cells. The specific subunit composition within complexes controls functions and higher-order structural properties. All septins have globular GTPase domains. The other eukaryotic cytoskeletal NTPases strictly require assistance from molecular chaperones of the cytosol, particularly the cage-like chaperonins, to fold into oligomerization-competent conformations. We previously identified cytosolic chaperones that bind septins and influence the oligomerization ability of septins carrying mutations linked to human disease, but it was unknown to what extent wild-type septins require chaperone assistance for their native folding. Here we use a combination of in vivo and in vitro approaches to demonstrate chaperone requirements for de novo folding and complex assembly by budding yeast septins. Individually purified septins adopted non-native conformations and formed non-native homodimers. In chaperonin- or Hsp70-deficient cells, septins folded slower and were unable to assemble post-translationally into native complexes. One septin, Cdc12, was so dependent on co-translational chaperonin assistance that translation failed without it. Our findings point to distinct translation elongation rates for different septins as a possible mechanism to direct a stepwise, co-translational assembly pathway in which general cytosolic chaperones act as key intermediaries.


title: Chemokine Receptor 1 and its associated immune pathway are downregulated in SF3B1MT blood and non-blood cancers

abstract: Mutation of the essential splicing factor SF3B1 is primarily associated with hematological cancers but also occurs in solid tumors. We edited the most common mutation, K700E, into human embryonic stem (ES) cells to determine the effects of this mutation alone in an undifferentiated/non-cancer background. Unexpectedly, >20% of the significantly upregulated genes in the SF3B1K700E ES lines have immune functions. Thus, SF3B1 may have an additional role in proper expression of immune genes in appropriate cell types. In striking contrast, we found that published RNA-seq data from SF3B1 blood (MDS, CLL, AML) and non-blood (BRCA, UVM) cancers exhibited the opposite, downregulation of a multitude of immune pathways with 7 of the pathways shared among all 5 of the SF3B1 cancers. One of these pathways, "leukocyte migration", is the 1st reported pathway shared among all splicing factor cancers, including the 5 SF3B1 cancers and MDS associated with U2AF1, SRSF2 and ZRSR2. Importantly, we identified CCR1, which is in the leukocyte migration pathway as the only shared downregulated gene in the 5 SF3B1 cancers and in U2AF1MT MDS. We conclude that downregulation of CCR1 and its associated immune pathway may play a key role in pathogenesis of these splicing factor cancers and are thus potential therapeutic targets.


title: Monitoring promoter activity by RNA editing based reporter

abstract: Traditional methods monitoring the promoter activity require the insertion of reporter protein (e.g. fluorescent protein) downstream of a targeted promoter. These approaches suffer from low sensitivity and potential interference to endogenous transcripts especially when the targeted transcript is a noncoding RNA. Here, we develop a mechanistically different reporter system to monitor promoter activity based on ribozyme processed ADAR engaging RNA directed editing (REDDIT). We show that REDDIT can be used to monitor the promoter activity of protein coding, long noncoding RNA, and microRNA (miRNA) genes. Furthermore, REDDIT reporter can also be adapted to use bioluminescence imaging to monitor the promoter activity, which is more suitable for in vivo live imaging. Finally, the reporter sensitivity may be further increased by the circularization of ADAR recruiting RNA. REDDIT provides a powerful platform that is promising in a variety of applications such as monitoring the promoter activity, cell lineage tracing, and processing and manipulating genetic information for synthetic biology applications.


title: Impact of hydrostatic pressure on organic carbon cycling of the deep-sea microbiome

abstract: Deep-sea microbial communities are exposed to high hydrostatic pressure. While some of these deep-sea prokaryotes are adapted to high-pressure conditions, the contribution of piezophilic (i.e., pressure-loving) and piezotolerant prokaryotes to the total deep-sea prokaryotic community remains unknown. Here we show that the metabolic activity of prokaryotic communities is increasingly inhibited with increasing hydrostatic pressure. At 4,000 m depth, the bulk heterotrophic prokaryotic activity under in situ hydrostatic pressure was only about one-third of that measured on the same community at atmospheric pressure conditions. Only [~]5% of the bathypelagic prokaryotic community are piezophilic while [~]85% of the deep-sea prokaryotes are piezotolerant. A small fraction ([~]10%) of the deep-sea prokaryotes is piezosensitive (mainly members of Bacteroidetes, Alteromonas) exhibiting specific survival strategies at meso- and bathypelagic depths. These piezosensitive bacteria elevated their activity by more than 100-fold upon depressurization. Hence, the consistently higher bulk metabolic activity of the deep-sea prokaryotic community measured upon depressurization is due to a rather small fraction of the prokaryotic community. Overall, the heterotrophic prokaryotic activity in the deep-sea is substantially lower than hitherto assumed with major impacts on the oceanic carbon cycling.


title: Recurrent but short-lived duplications of centromeric proteins in holocentric Caenorhabditis species

abstract: Centromeric histones (CenH3s) are essential for chromosome inheritance during cell division in most eukaryotes. CenH3 genes have rapidly evolved and undergone repeated gene duplications and diversification in many plant and animal species. In Caenorhabditis, two independent duplications of CenH3 (named hcp-3 for HoloCentric chromosome-binding Protein 3) have been previously identified: in C. elegans and C. remanei. Here, using phylogenomic analyses in Caenorhabditis, we find strict retention of the ancestral hcp-3 gene and eight additional independent hcp-3 duplications, most of which are only found in one or two species. hcp-3L (hcp-3-like) paralogs are expressed in both sexes (males and females/ hermaphrodites) and have a conserved histone fold domain. We identified novel N-terminal protein motifs, including putative kinetochore protein-interacting motifs and a potential separase cleavage site, which are well-conserved across Caenorhabditis HCP-3 proteins. Other N-terminal motifs vary in their retention across paralogs or species, revealing potential sub-functionalization or functional loss following duplication. C. afra encodes an unprecedented protein fusion, where the hcp-3 paralog fused to duplicated segments from hcp-4 (nematode CENP-C). Extending our analyses beyond CenH3, we found gene duplications of six inner and outer kinetochore genes in Caenorhabditis, including co-retention of different kinetochore protein paralogs in a few species. Our findings suggest that centromeric protein duplications occur frequently in Caenorhabditis nematodes, are selectively retained under purifying selection but only for short evolutionary periods, then degenerate or are lost entirely. We hypothesize that unique challenges associated with holocentricity in Caenorhabditis may lead to this rapid  revolving door of kinetochore protein paralogs.

category: evolutionary biology


title: Competition for H2A.Z between genes and repetitive elements establishes response to anti-viral immune activation

abstract:
Activation of endogenous retroviruses leads to widespread transcriptional reprograming, affecting innate immune activation, metabolic control, and development. We find that the histone variant H2A.Z plays a central role in orchestrating these responses. Stimulating retroviral expression in zebrafish embryos causes H2A.Z to exit developmental gene promoters, which become silent, and to accumulate specifically at  primed repetitive elements, which are pre-marked by H3K27ac and H3K9me3. Remarkably, this rewiring is greatly influenced by total H2A.Z abundance, and developmental consequences of retrovirus activation are mitigated by H2A.Z over-expression. Our results uncover mechanisms whereby H2A.Z levels determine sensitivity to retroviral activation, and repetitive elements function as a nuclear sink to dramatically influence total transcriptional output.

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title: A chromosome-length assembly of the Hawaiian Monk seal (Neomonarchus schauinslandi) confirms genomic stability in the Pinnipeds and a prolonged history of "genetic purging"

abstract: The Hawaiian monk seal (HMS) is the single extant species of tropical earless seals of the genus Neomonachus. The species survived a severe bottleneck in the late 19th century and experienced subsequent population declines until becoming the subject of a NOAA-led species recovery effort beginning in 1976 when the population was fewer than 1000 animals. Like other recovering species, the Hawaiian monk seal has been reported to have reduced genetic heterogeneity due to the bottleneck and subsequent inbreeding. Here we report a chromosomal reference assembly for a male animal produced using a variety of methods including linked-read sequencing, Hi-C contiguity mapping, optical genome mapping, and nanopore long read sequencing. The final assembly consisted of 16 autosomes, an X and portions of the Y chromosomes. We compared variants in the reference animals to nine other HMS and to the human reference NA12878 confirming a low level of variation within the species and one-eighth that of the human reference. A lack of variation in several MHC genes was documented suggesting that this species may be at risk for infectious disease. Lastly, the HMS chromosomal assembly confirmed significant synteny with other pinnipeds. This reference should be a useful tool for long-term management of HMS and evolutionary studies of other carnivorans.


title: Evaluating Client Protein Recovery by the Hsp40s DNAJB8 and DNAJB1 with AP-MS

abstract: Hsp40s play a central role in cellular protein homeostasis by promiscuously surveying the proteome for misfolded proteins. These misfolded client proteins are then delivered to Hsp70. Mutation of the Hsp40 J-domain blocks Hsp70 binding, inhibiting client protein release from Hsp40. We previously integrated misfolded protein recognition by Hsp40 into a platform to identify proteins that are destabilized by cellular stress. However, the dependences of Hsp40 interactions and client recovery on J-domain activity, Hsp40 identity, and crosslinking have not been addressed. Herein, we apply quantitative proteomics to systematically characterize the interactions networks of human Hsp40s DNAJB8 and DNAJB1 with intact or inactivated J-domains. We find that DNAJB8 irreversibly binds over a thousand protein interactors even in the absence of stress. Inactivation of the J-domain decreases interaction with Hsp70 family and associated proteins, but does not generally affect client binding. By contrast, J-domain inactivation and cellular crosslinking substantially increase the relative recovery of proteins from DNAJB1 co-immunoprecipitation. This advantage is completely offset by loss of DNAJB1 recovery under these conditions, making DNAJB1 a poor bait for client protein recovery as compared to DNAJB8. The J-domain inactivated DNAJB8H31Q has increased affinity to its client proteins under heat stress, while no such change in affinity is observed for the wild-type protein, despite their similar client binding profiles under basal conditions. Hence, we find that DNAJB8H31Q is an effective recognition element for the recovery of destabilized client proteins following cellular stress. Raw mass spectrometry data is accessible through the PRIDE archive (pxd030633).


title: A metabolic labeling-based chemoproteomic platform unravels the physiological roles of choline metabolites

abstract: Choline is an essential nutrient for mammalian cells. Our understanding of the cellular functions of choline and its metabolites, independent of their roles as choline lipid metabolism intermediates, remains limited. In addition to fundamental cellular physiology, this knowledge has implications for cancer biology because elevated choline metabolite levels are a hallmark of cancer. Here, we establish the mammalian choline metabolite-interacting proteome by utilizing a photocrosslinkable choline probe. To design this probe, we performed metabolic labeling experiments with structurally diverse choline analogs that resulted in the serendipitous discovery of a choline lipid headgroup remodeling mechanism involving sequential dealkylation and methylation steps. We demonstrate that phosphocholine inhibits the binding of one of the proteins identified, the attractive anticancer target, p32, to its endogenous ligands and to the promising p32-targeting anticancer agent, Lyp-1. Our results reveal that choline metabolites play vital roles in cellular physiology by serving as modulators of protein function.


title: Structural interplay between DNA-shape proteinrecognition and supercoiling: the case of IHF

abstract: The integration host factor (IHF) is a prominent example of indirect readout as it imposes one of the strongest bends on relaxed linear DNA. However, the relation between IHF and torsionally constrained DNA, as occurs physiologically, remains unclear. By using atomistic molecular dynamics simulations on DNA minicircles, we reveal, for the first time, the reciprocal influence between a DNA-bending protein and supercoiling. While the increased curvature of supercoiled DNA enhances wrapping around IHF, the protein pins the position of plectonemes, organizing the topology of the loop in a unique and specific manner. In addition, IHF restrains underor overtwisted DNA depending on whether the complex is formed in negatively or positively supercoiled DNA. This effectively enables IHF to become a  supercoiling buffer that dampens changes in the surrounding superhelical stress through DNA breathing around the protein or complex dissociation. We finally provide evidence of DNA bridging by IHF and reveal that these bridges divide DNA into independent topological domains. We anticipate that the crosstalk detected here between the  active DNA and the multifaceted IHF could be common to other DNA-protein complexes relying on the deformation of DNA.