title: A Computational Theory of Learning Flexible Reward-Seeking Behavior with Place Cells

abstract: An important open question in computational neuroscience is how various spatially tuned neurons, such as place cells, are used to support the learning of reward-seeking behavior of an animal. Existing computational models either lack biological plausibility or fall short of behavioral flexibility when environments change. In this paper, we propose a computational theory that achieves behavioral flexibility with better biological plausibility. We first train a mixture of Gaussian distributions to model the ensemble of firing fields of place cells. Then we propose a Hebbian-like rule to learn the synaptic strength matrix among place cells. This matrix is interpreted as the transition rate matrix of a continuous time Markov chain to generate the sequential replay of place cells. During replay, the synaptic strengths from place cells to medium spiny neurons (MSN) are learned by a temporal-difference like rule to store place-reward associations. After replay, the activation of MSN will ramp up when an animal approaches the rewarding place, so the animal can move along the direction where the MSN activation is increasing to find the rewarding place. We implement our theory into a high-fidelity virtual rat in the MuJoCo physics simulator. In a complex maze, the rat shows significantly better learning efficiency and behavioral flexibility than a rat that implements a neuroscience-inspired reinforcement learning algorithm, deep Q-network.


title: The structural basis of long-term potentiation in hippocampal synapses, revealed by EM-imaging of lanthanum-induced synaptic vesicle recycling

abstract: Hippocampal neurons in tissue-culture were exposed to the trivalent cation lanthanum for short periods (15 to 30 minutes) and prepared for electron microscopy (EM), to evaluate the stimulatory effects of this cation on synaptic ultrastructure. Not only were characteristic ultrastructural changes of exaggerated synaptic vesicle turnover seen within the presynapses of these cultures - - including synaptic vesicle depletion and proliferation of vesicle-recycling structures - - but the overall architecture of a large proportion of the synapses in the cultures was dramatically altered, due to large postsynaptic  bulges or herniations into the presynapses. Moreover, in most cases these postsynaptic herniations or protrusions produced by lanthanum were seen by EM to distort or break or  perforate the so-called postsynaptic densities (PSDs) that harbor receptors and recognition-molecules essential for synaptic function. These dramatic EM-observations lead us to postulate that such PSD-breakages or  perforations could very possibly create essential substrates or  tags for synaptic growth, simply by creating fragmented free-edges around the PSDs, into which new receptors and recognition-molecules could be recruited more easily, and thus they could represent the physical substrate for the important synaptic-growth process known as "long-term potentiation" (LTP). All of this was created simply in hippocampal tissue-cultures, and simply by pushing synaptic vesicle recycling way beyond its normal limits with the trivalent cation lanthanum; but we argue in this report that such fundamental changes in synaptic architecture - - given that they can occur at all - - could also occur at the extremes of normal neuronal activity, which are presumed to lead to learning and memory.

category: neuroscience


title: Precise temporal control of GEVI conformations enables the visualization of charge migration in a fluorescent protein resulting in an improved optical response

abstract: Electrodynamic pathways through the fluorescent protein domain enable the genetically encoded voltage indicator (GEVI) ArcLight and its derivatives to optically report conformational changes induced by membrane potential transients. These electrical pathways through the fluorescent protein domain of Super Ecliptic pHluorin exhibit remarkable precision as demonstrated by the divergent optical behavior of Gln versus Asn mutations. At position D147 on the external surface of the fluorescent protein domain, Gln exhibits a voltage-dependent behavior like other negatively charged acidic amino acids at that location while the Asn mutant exhibits a response similar to the charged positive amino acids at that position. Replacement of Thr with Gln at the 203 position with the amino acid side chain in close proximity to the internal chromophore does not affect the speed of the voltage-dependent optical signal, but replacement with Asn slows the voltage-dependent optical signal implicating a twisting of the chromophore in response to transient charge interactions on the exterior of the {beta}-can being altered during the voltage-induced conformational change. In addition to polar amino acids affecting these electrodynamic pathways, hydrophobic residues play a noticeable role in the migration of charge from the exterior of the fluorescent protein to the chromophore potentially by shielding/creating inducible dipoles on the surface of the fluorescent protein. Using these novel insights, a new GEVI that gets significantly brighter upon depolarization of the plasma membrane was developed enabling voltage imaging of population signals with low light levels at a frame rate of 5 kHz in acute brain slice as well as the reporting of neuronal activity in vivo.


title: The language network reliably 'tracks' naturalistic meaningful non-verbal stimuli

abstract: The language network, comprised of brain regions in the left frontal and temporal cortex, responds robustly and reliably during language comprehension but shows little or no response during many non-linguistic cognitive tasks (e.g., Fedorenko & Blank, 2020). However, one domain whose relationship with language remains debated is semantics--our conceptual knowledge of the world. Given that the language network responds strongly to meaningful linguistic stimuli, could some of this response be driven by the presence of rich conceptual representations encoded in linguistic inputs? In this study, we used a naturalistic cognition paradigm to test whether the cognitive and neural resources that are responsible for language processing are also recruited for processing semantically rich non-verbal stimuli. To do so, we measured BOLD responses to a set of [~]5-minute-long video and audio clips that consisted of meaningful event sequences but did not contain any linguistic content. We then used the inter-subject correlation (ISC) approach (Hasson et al., 2004) to examine the extent to which the language network  tracks these stimuli. Across all the regions of the language network, non-verbal meaningful stimuli elicited reliable ISCs. These ISCs were higher than the ISCs elicited by non-verbal stimuli without semantic content (e.g., a music clip), but substantially lower than the ISCs elicited by linguistic stimuli. Our results complement earlier findings from controlled experiments (e.g., Ivanova et al., 2021) in providing further evidence that the language network responds to meaningful content in non-verbal stimuli.


title: Toward Interpretable Digital Biomarkers of Walking and Reaching in Parkinson's Disease

abstract: Multimodal digital data registered with wearable biosensors has emerged as highly complementary of clinical pencil-and-paper criteria, offering new insights in ways to detect and diagnose various aspects of Parkinsons disease. A pressing question is how to combine both the clinical knowledge of PD and the new technology to create interpretable digital biomarkers easily obtainable with off-the-shelf technology. Several challenges concerning disparity in biophysical units, anatomical differences across participants, sensor positioning, and sampling resolution are addressed in this work, along with identification of optimal parameters to automatically differentiate patients with PD from controls. We combine data from a multitude of biosensors registering signals from the central (EEG) and peripheral (magnetometry, kinematics) nervous systems, inclusive of the autonomic nervous system (EKG), as the participants perform natural tasks requiring different levels of intentional planning and automatic control. We find that magnetometer data during walking, across a variety of amplitude and timing signals provide optimal separation of PD from neurotypical controls. We conclude that considering multimodal signals, while differentiating across levels of intent in natural actions can be revealing of important features of PD that otherwise escape the naked eye. Further we add that clinical criteria combined with such optimal digital parameter spaces offer a far more complete picture of PD than using either one of these pieces of data alone.


title: Genome-wide translocation events drive the evolution of Candida africana

abstract: Candida africana is emerging as an organism of interest. It is evolutionarily divergent from Candida albicans but has reduced virulence with a restricted ecological niche. This study aimed at comparing in silico the genome level to detect variations in the two species. Raw Illumina Hiseq data were downloaded from the European Nucleotide Archive (https://www.ebi.ac.uk/ena) with the accession number SRR6669859 and assembled using shovill (v. 1.0.9) the resulting genome was mapped against the haploid reference Candida albicans SC5314_A22 strain using the D-GENIES webtool and contigs were reordered based on the reference, then gap-filled using GapFiller (v1-10), and annotated using MAKER-P. Synima and progressive Mauve were used to compare the annotated genomes of Candida africana and Candida albicans for synteny. OrthoVenn2 webserver was used for the identification and comparison of orthologous clusters. Microsynteny variations within the genomes were determined using the GEvo. The study revealed the presence of insertions, deletions, and hypervariable regions within the genome of Candidia africana, showing a high level of synteny with Candida albicans. The genome of Candida africana is 14.04Mbp with a BUSCO score of 99.66%. The two species form a total of 5146 orthologous protein clusters and shared a total of 5124 protein, C. africana has a unique cluster protein cluster while C. albicans have 18 unique Protein clusters. The genome of C. africana has lots of structural variations and the presence of gene losses and gains. These genetic variations possibly play a role in the reduced virulence potential observed in C. africana.

category: microbiology


title: Jointly analyzing the association of human milk nutrients with cognition and temperament traits during the first 6 months of life

abstract: Early dietary exposure via human milk (HM) components offers a window of opportunity to support cognitive and temperamental development. While several studies have focused on associations of few pre-selected HM components with cognition and temperament, it is highly plausible that HM components synergistically and jointly support cognitive and behavioral development in early life. We aimed to discern the combined associations of a wide array of HM nutrients with cognition and temperament during the first six months of life and explore if there were persistent effects up to 18 months old, when HM is the primary source of an infants nutrition. The Mullen Scales of Early Learning and Infant Behavior Questionnaires-Revised were used to assess cognition and temperament, respectively, of fifty-four exclusively/predominantly breastfed infants in the first 6 months of life, whose follow-ups were conducted at 6-9, 9-12 and 12-18 months old. HM samples were obtained from the mothers of the participants at less than 6 months of life and analyzed for fatty acids (total monounsaturated fatty acids, polyunsaturated fatty acid, total saturated fatty acid (TSFA), arachidonic acid (ARA), docosahexaenoic acid (DHA), ARA/DHA, omega-6/omega-3 polyunsaturated fatty acids ratio (n-6/n-3)), phospholipids (phosphatidylcholine, phosphatidylethanolamine (PE), phosphatidylinositol (PI), sphingomyelin) and choline (free choline, phosphocholine (PCho), glycerophosphocholine). Feature selection was performed to select nutrients associated with cognition and temperament, respectively. The combined effects of selected nutrients were analyzed using multiple regression. A positive association between the arachidonic acid (ARA) and surgency was observed (p = 0.024). Significant effect of DHA, n-6/n-3, PE and TSFA concentrations on receptive language (R2 = 0.39, p = 0.025), and the elevated ARA, PCho, and PI with increased surgency (R2 = 0.43, p = 0.003) was identified, suggesting that DHA and ARA may have distinct roles for temperament and language functions. Furthermore, the exploratory association analyses suggest that the effects of HM nutrients on R.L. and surgency may persist beyond the first 6 months of life, particularly surgency at 12-18 months (p = 0.002). Our studies highlighted that various HM nutrients work together to support the development of cognition and temperament traits during early infancy.

category: developmental biology


title: Robust analysis of prokaryotic pangenome gene gain and loss rates with Panstripe

abstract: Horizontal gene transfer (HGT) plays a critical role in the evolution and diversification of many microbial species. The resulting dynamics of gene gain and loss can have important implications for the development of antibiotic resistance and the design of vaccine and drug interventions. Methods for the analysis of gene presence/absence patterns typically do not account for errors introduced in the automated annotation and clustering of gene sequences. In particular, methods adapted from ecological studies, including the pangenome gene accumulation curve, can be misleading as they may reflect the underlying diversity in the temporal sampling of genomes rather than a difference in the dynamics of HGT. Here, we introduce Panstripe, a method based on Generalised Linear Regression that is robust to population structure, sampling bias and errors in the predicted presence/absence of genes. We demonstrate using simulations that Panstripe can effectively identify differences in the rate and number of genes involved in HGT events, and illustrate its capability by analysing a number of diverse bacterial genome datasets representing major human pathogens. Panstripe is freely available as an R package at https://github.com/gtonkinhill/panstripe.

category: evolutionary biology


title: Structural variants in the barley gene pool: precision and sensitivity to detect them using short-read sequencing and their association with gene expression and phenotypic variation

abstract: In human genetics, several studies have shown that phenotypic variation is more likely to be caused by structural variants (SV) than by single nucleotide variants (SNV). However, accurate while cost-efficient discovery of SV in complex genomes remains challenging. The objectives of our study were to (i) facilitate SV discovery studies by benchmarking SV callers and their combinations with respect to their sensitivity and precision to detect SV in the barley genome, (ii) characterize the occurrence and distribution of SV clusters in the genomes of 23 barley inbreds that are the parents of a unique resource for mapping quantitative traits, the double round robin population, (iii) quantify the association of SV clusters with transcript abundance, and (iv) evaluate the use of SV clusters for the prediction of phenotypic traits. In our computer simulations based on a sequencing coverage of 25x, a sensitivity >70% and precision >95% was observed for all combinations of SV types and SV length categories if the best combination of SV callers was used. We observed a significant (P < 0.05) association of gene-associated SV clusters with global gene-specific gene expression. Furthermore, about 9% of all SV clusters that were within 5kb of a gene were significantly (P < 0.05) associated with the gene expression of the corresponding gene. The prediction ability of SV clusters was higher compared to that of single nucleotide polymorphisms from an array across the seven studied phenotypic traits. These findings suggest the usefulness of exploiting SV information when fine mapping and cloning the causal genes underlying quantitative traits as well as the high potential of using SV clusters for the prediction of phenotypes in diverse germplasm sets.

category: genomics


title: Direct N-Me Aziridination Reaction Enables Pinpointing C=C Bonds in Lipids with Mass Spectrometry

abstract: The biological functions of lipids largely depend on their chemical structures. The position of C=C bonds is an essential attribute that determines the structures of unsaturated lipids. Here, we developed a new type of chemical derivatization method for C=C bond using aziridination reaction. This new cyclization method for the C=C bonds in lipids based on the direct N-Me aziridination reaction of olefins using N-methyl-O-tosylhydroxylamine (TsONHCH3) as the aminating reagent. When combined with the tandem MS analysis, this novel activation approach for C=C bonds enables the accurate identification their positions in different kinds of unsaturated lipids. Furthermore, an integrated workflow has been established for comprehensively identifying the C=C bond positional isomers of lipids in complicated biological sample. This work provided a new chemical approach for the structural lipidomics.

category: biochemistry


title: DeepTMB: An uncertainty-aware deep calibration of tumor mutational burden with a synthetic tumor-only dataset

abstract:
BackgroundTumor mutational burden (TMB) is commonly investigated as a biomarker for immune checkpoint blockade (ICB) therapy, but lack of uniform standards in its calculation may be preventing consensus recommendations. Gene panels have mostly supplanted exome sequencing for calculating TMB, but these panels have a strong tendency to overestimate TMB. With TMB cutoffs being developed with panel-based estimates and suggestions to use The Cancer Genome Atlas (TCGA) exome data to model panel data, there is a need to understand how to properly compare values from different assays.

DesignOur approach to TMB calibration allows for a nonlinear fit and a heteroscedastic error distribution, utilizes a per-batch read depth augmentation to upsample the exomic data to what would be seen at panel depth, and is trained on a novel tumor-only synthetic dataset generated by utilizing germline as well as somatic calls.

ResultsWe were able to accurately model the distribution of tumor-normal data. Removing hotspots and including synonymous mutations resulted in better regression metrics, as did including NCIt label. Correcting for read depth resulted in a slight increase in the spread of the data but did not affect the model fit. When applying a model trained on tumor-normal data to synthetic tumor-only input there was a dramatic drop in the positive predictive value (PPV) at a TMB of 10. This was largely mitigated by training with synthetic tumor-only input (up to over a 200% improvement in PPV).

ConclusionsA deep learning model is capable of accurately modeling the tumor-normal TMB distribution. Using a synthetic tumor-only input for training appears to be a viable strategy for calibrating tumor-only panels.

category: bioinformatics


title: Loss of the chromatin remodeler, ATRX, promotes aggressive features of osteosarcoma with increased NF-κB signaling and integrin receptor binding

abstract: Osteosarcoma (OS) is a lethal disease with few known targeted therapies. Here we show that decreased ATRX expression is associated with more aggressive tumor cell phenotypes, including increased growth, migration, invasion, and metastasis. These phenotypic changes correspond with activation of NF-{kappa}B signaling, extracellular matrix remodeling, increased integrin v{beta}3 expression, and ETS family transcription factor binding. Here we characterize these changes in vitro, in vivo, and in a dataset of human OS patients. This increased aggression substantially sensitizes ATRX-deficient OS cells to integrin signaling inhibition. Thus, ATRX plays an important tumor suppression role in OS, and loss of function of this gene may underlie new therapeutic vulnerabilities. The relationship between ATRX expression and integrin binding, NF-{kappa}B activation, and ETS family transcription factor binding has not been described in previous studies and may impact the pathophysiology of other diseases with ATRX loss, including other cancers and the ATR-X alpha thalassemia mental retardation syndrome.

category: cancer biology


title: Unlocking the microblogging potential for science and medicine

abstract: Twitter is one of the most popular microblogging and social networking services, where users can post, retweet, comment, and engage in collaborative discussions. However, improper usage of Twitter can be detrimental to science and even have a negative impact on mental health. Thus, analyzing tweets and Twitter data of various researchers will help us to deduce appropriate ways of using Twitter to advance in our research careers. Existing literature has analyzed the activity of scientists on Twitter, such as studying the relationship between Twitter mentions and article citations, determining the benefits of Twitter in the development and distribution of scientific knowledge, relevant metrics for prediction of highly cited articles, and type of content that researchers tweet etc. For example, Eysenbach et al performed an analysis of tweets and citations and how one can predict citations using tweets. Most of the existing literature analyzed a limited number of researchers, compromising the generalizability of derived results. In our study, we have taken a comprehensive and systematic approach to analyze 167,000 scientists who published research papers on PubMed using data-driven methods. We observed various parameters like number of followers, number of friends, citation count and K-index, in the light of gender, ancestry and profession of the researchers.

category: scientific communication and education


title: From the ear flap to the nerve: A complete computer model of the peripheral auditory system

abstract: Computer models of the individual components of the peripheral auditory system - the outer, middle, and inner ears and the auditory nerve - have been developed in the past, with varying level of detail, breadth, and faithfulness of the underlying parameters. Here, we move modeling of the ear to the next level by presenting a complete, physiologically justified, bottom-up computer model based on up-to-date experimental data that integrates all these parts together in a seamless way. Such a model is instrumental for advancing quantitative understanding of the mechanism of hearing, as well as for devising next generation hearing aids and cochlear implants.


title: Decoding reward-curiosity conflict in decision-making from irrational behaviors

abstract: Humans and animals are not always rational. They not only rationally exploit rewards but also explore an environment, even if reward is less expected, owing to their curiosity. However, the mechanism of such curiosity-driven irrational behavior is largely unknown. Here, we developed a novel decision-making model for a two-choice task based on the free energy principle, which is a theory integrating recognition and action selection. The model successfully described irrational behaviors depending on the curiosity level. We then proposed a machine learning method to decode temporal curiosity from behavioral data, which enables us to quantitatively compare estimated curiosity and neural activities. By applying it to rat behavioral data, we found that the irrational choices sticking to one option was reflected to the negative curiosity level. Our decoding approach can be a fundamental tool for identifying the neural basis for reward-curiosity conflicts. Specifically, it could be effective in diagnosing mental disorders.


title: The association between working memory precision and the nonlinear dynamics of frontal and parieto-occipital EEG activity.

abstract: Electrophysiological working memory (WM) research has shown that distinct brain areas communicate through macroscopic oscillatory activities across multiple frequency bands. Such cross-frequency interactions generate nonlinear amplitude modulations (AM) in the observed signal. Traditionally, the AM of a signal is expressed as coupling strength between the signal and a pre-specified modulator at a lower frequency. Therefore, the idea of AM and coupling cannot be separately studied. This EEG study shows that the AM of parieto-occipital alpha/beta power and the coupling between frontal theta phase and parieto-occipital alpha/beta AM provide different information on WM processing. Thirty-three participants completed a color recall task with simultaneous EEG recording. The results showed that individual differences in WM precision are associated with frontal theta power enhancement and parieto-occipital alpha/beta power suppression. Furthermore, the AM of parieto-occipital alpha/beta power predicted WM precision after presenting a target-defining probe array. The phase-amplitude coupling (PAC) between frontal theta phase and parieto-occipital alpha/beta AM increased with WM load during the processing of incoming stimuli, but they did not predict the subsequent recall performance. These results indicate that the frontoparietal PAC reflects the executive control for selecting relevant WM representations, but whether the memorized information can be retrieved depends on the subsequent amplitude variation of parieto-occipital alpha/beta power. In conclusion, individuals with higher working memory precision are associated with enhanced frontal theta power and parieto-occipital alpha/beta power suppression.


title: Functional antibodies exhibit light chain coherence

abstract: The vertebrate adaptive immune system modifies the genome of individual B cells to encode antibodies binding particular antigens1. In most mammals, antibodies are composed of a heavy and a light chain which are sequentially generated by recombination of V, D (for heavy chains), J, and C gene segments. Each chain contains three complementarity-determining regions (CDR1-3), contributing to antigen specificity. Certain heavy and light chains are preferred for particular antigens2-21. We considered pairs of B cells sharing the same heavy chain V gene and CDRH3 amino acid sequence and isolated from different donors, also known as public clonotypes22,23. We show that for naive antibodies (not yet adapted to antigens), the probability that they use the same light chain V gene is [~]10%, whereas for memory (functional) antibodies it is [~]80%. This property of functional antibodies is a phenomenon we call light chain coherence. We also observe it when similar heavy chains recur within a donor. Thus, though naive antibodies appear to recur by chance, the recurrence of functional antibodies reveals surprising constraint and determinism in the processes of V(D)J recombination and immune selection. For most functional antibodies, the heavy chain determines the light chain.

category: immunology


title: First-in-human evaluation of cutaneous innate and adaptive immunomodulation by mosquito bites

abstract:
Mosquito-borne viruses are a growing global threat. Initial viral inoculation occurs in the skin via the mosquito  bite, eliciting immune responses that shape the establishment of infection and pathogenesis. To understand these phenomena, we assessed the cutaneous innate and adaptive immune responses via controlled Aedes aegypti feedings in humans living in an Aedes-endemic country. Gene expression profiling and immunophenotyping revealed induction of neutrophil degranulation and recruitment of skin-resident dendritic cells and M2-macrophages. As the immune reaction progressed over time, T cell priming and regulatory pathways were upregulated along with a shift to a Th2-driven response and CD8+ T cell activation. In accordance, participants bitten skin cells produced less pro-inflammatory cytokines when stimulated by Ae. aegypti salivary gland extract. These results identify key immune genes, cell types, and pathways in the human response to mosquito bites that can be leveraged to develop novel therapeutics and vector-targeted vaccine candidates to arboviral diseases.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/489145v1_ufig1.gif" ALT="Figure 1">
View larger version (57K):
org.highwire.dtl.DTLVardef@8c80ccorg.highwire.dtl.DTLVardef@5bdb9eorg.highwire.dtl.DTLVardef@af6c83org.highwire.dtl.DTLVardef@fd1a24_HPS_FORMAT_FIGEXP  M_FIG C_FIG


title: Similarities and differences in molecular epidemiology of third-generation cephalosporin-resistant Escherichia coli carried by dogs living in urban and nearby rural settings and associated behavioural risk factors

abstract:
SynopsisO_ST_ABSObjectivesC_ST_ABSOur aims were to compare faecal third-generation cephalosporin-resistant (3GC-R) Escherichia coli isolates from dogs living in a city and in a rural area ~30 km away; to compare isolates from dogs, cattle, and humans in these regions; to determine risk factors associated with 3GC-R E. coli carriage in these two cohorts of dogs.

Methods600 dogs were included, with faecal samples processed to recover 3GC-R E. coli using 2 mg/L cefotaxime. WGS was by Illumina; risk factor analyses were multivariable linear regression using the results of an owner-completed survey.

Results3GC-R E. coli were excreted by 20/303 rural and 31/297 urban dogs. Dog/human sharing was evident for the dominant canine 3GC-R sequence type, ST963(blaCMY-2). Cattle/dog sharing was evident for CTX-M-14 and CTX-M-32-producing E. coli from rural dogs, including sharing of plasmid pMOO-32, which is common on cattle farms in the area. Feeding raw meat was associated with carrying 3GC-R E. coli in rural dogs, but not in urban dogs, where swimming in rivers was a weak risk factor.

ConclusionsGiven clear zoonotic potential for resistant canine E. coli, our work suggests interventions that may reduce this threat. In rural dogs, carriage of 3GC-R E. coli, particularly CTX-M producers, was phylogenetically associated with interaction with local cattle and epidemiologically associated with feeding raw meat. In urban dogs, sources of 3GC-R E. coli appear to be more varied and include environments such as rivers.


title: LRRK2 Phosphorylates Neuronal Elav RNA-Binding Proteins to RegulatePhenotypes Relevant to Parkinson's Disease

abstract:
Parkinsons disease (PD) is characterized by accumulation of -synuclein and the loss of dopaminergic neurons. Mutations which cause an increase in the kinase activity of Leucine-Rich-Repeat Kinase-2 (LRRK2) are a major inherited cause of PD. Research continues to determine which targets LRRK2 phosphorylates to cause disease. Polymorphisms in the locus of ELAVL4, an RNA-binding protein are a risk-factor for Parkinsons disease and an ELAV family member was identified in Drosophila as required for pathology instigated by human mutant LRRK2. We discovered that three neuronal ELAVs including ELAVL4 (also known as HuD) are phosphorylated by LRRK2. This controls binding of neuronal ELAVs to mRNA and their post- transcriptional regulation of mRNA abundance and splicing in neuronal cell lines and the mouse midbrain. LRRK2 G2019S functionally inhibits neuronal ELAVs effects on mRNA abundance, while enhancing their effects on mRNA splicing. The combination of LRRK2 G2019S and ELAVL4-/- causes accumulation of LRRK2 and -synuclein, loss of dopaminergic neurons and motor deficits. Targets of neuronal ELAVs are also selectively misregulated in cells and tissues of PD patients. Together, this suggests that misregulation of neuronal ELAVs, triggered by LRRK2 mutations may contribute to the characteristic pathology of Parkinsons disease.

Brief SummaryLRRK2, a kinase linked to Parkinsons disease, phosphorylates the neuronal ELAV RNA-binding proteins to aggravate key hallmarks of Parkinsons disease including accumulation of -synuclein and motor deficits in mice.

category: cell biology


title: Identification of genetic markers for the discrimination of Bacillus thuringiensis within the Bacillus cereus group, in the context of foodborne outbreaks

abstract: Bacillus thuringiensis (Bt), belonging to the Bacillus cereus (Bc) group, is commonly used as a biopesticide worldwide, due to its ability to produce insecticidal protein crystals during sporulation. The use of Bt, especially subspecies aizawai and kurstaki, to control pests such as Lepidoptera generally involves spraying mixtures containing spores and crystals on crops intended for human consumption. Recent studies have suggested that the consumption of commercial Bt strains may be responsible for foodborne outbreaks (FBOs). However, its genetic proximity to Bc strains has hindered the development of routine tests to discriminate Bt from other Bc, especially Bacillus cereus sensu stricto (Bc ss), also responsible for FBOs. Here, to develop tools for the detection and the discrimination of Bt in food, we carried out a genome-wide association study (GWAS) on 286 complete genomes of Bc group strains to identify and validate in silico new molecular markers specific to different Bt subtypes. The analyses led to the determination and the validation in silico of 128 molecular markers specific to Bt, its subspecies aizawai, kurstaki and four previously described proximity clusters associated with these subspecies. We developed a command line tool (https://github.com/afelten-Anses/Bt_typing) based on a 14-marker workflow for in silico Bt identification of a putative Bc genome with the aim of facilitating the discrimination of Bt from other Bc and between Bt subspecies, especially in the context of FBOs. Collectively, these data provide key elements for investigating Bc/Bt-associated FBOs and for monitoring Bt in food.


title: Opposite polarity programs regulate asymmetric subsidiary cell divisions in grasses

abstract: Grass stomata recruit lateral subsidiary cells (SCs), which are key to the unique stomatal morphology and the efficient plant-atmosphere gas exchange in grasses. Subsidiary mother cells (SMCs) strongly polarise before an asymmetric division forms a SC. Yet apart from a proximal polarity module that includes PANGLOSS1 (PAN1) and guides nuclear migration, little is known regarding the developmental processes that form SCs. Using the genetic model grass Brachypodium distachyon, we identified BdPOLAR, which forms a novel, distal polarity domain in SMCs that is reciprocal to the proximal PAN1 domain. Both polarity domains are required for the formative SC division yet exhibit distinct roles in regulating pre-mitotic nuclear migration and SMC division plane orientation, respectively. Nonetheless, the domains are linked as the proximal domain controls polarisation of the distal domain. In summary, we identified two opposing polarity domains that coordinate the SC division, a process crucial for grass stomatal physiology.

category: plant biology


title: Ecology and Conservation status of Phoebe hainesiana Brandis: a data deficit timber species of Indo-Myanmar biodiversity hotspot

abstract: Phoebe hainesiana Brandis (Lauraceae) is an economically significant timber yielding forest species endemic to North-East India in Indo-Myanmar Biodiversity Hotspot (IMBH). The tree exhibits significant commercial importance in timber industries due to its peculiar wood qualities. The timbers strength, toughness, and durability make it in high demand for furniture making. Despite its significance, the botanical description of the species is not well documented due to the confined distribution of the species in IMBH region. The natural population is rare, but it is the  least concerned species in the IUCN Red List. Moreover, no taxonomical description of the species is available for its distinct identity among the other species from the genus. In the present study, we conducted a taxonomic, ecological, and forestry assessment of the species in its natural environment, researching the literature and conducting a field survey. A significant variation in intra-specific wood density (450 kg/m3 to 800 kg/m3) determines the scope of robust selection for the genetic improvement of the species. Its population has been severely fragmented due to over-harvesting in lack of sustainable management. According to our findings, the species is endangered due to a rapid decline in its population, a limited  extent of occurrence, and a small  area of occupancy. Among the other four species from the genus found in India, P. hainesiana can be identified based on its distinct lobes of the fruiting perianth. We recommend a sincere effort for the conservation of this key species of tropical evergreen forest in IMBH region.


title: FBXW7β isoform drives transcriptional activation of a proinflammatory TNF cluster in normal and malignant pro-B cells

abstract:
Non-canonical exon usage plays many important roles in cellular phenotypes, but its contribution to human B-cell development remains sketchily understood. To fill this gap, we collected various B-cell fractions from bone marrow and tonsil donors, performed RNA-seq, and examined transcript variants. We identified 150 genes that harbor local splicing variations in all pairwise comparisons. One of them encodes FBXW7, an E3 ubiquitin ligase implicated as a cancer driver in several blood cancers. Surprisingly, we discovered that in normal human pro-B cells, the predominant transcript utilized an alternative first exon to produce the poorly characterized FBXW7{beta} isoform, previously thought to be restricted to neural tissues. The FBXW7{beta} transcript was also abundant in cell lines and primary samples of pediatric B-cell acute lymphoblastic leukemia (B-ALL), which originates in the bone marrow. When overexpressed in a heterologous cell system, this transcript yielded the expected protein product, as judged by anti-FLAG immunoblotting and mass spectrometry. Furthermore, in REH B-ALL cells, FBXW7{beta} mRNA was the only FBXW7 isoform enriched in the polyribosome fraction. To shed light on possible functions of FBXW7{beta}, we utilized gain- and loss-of-function approaches and identified an FBXW7{beta}-dependent inflammatory gene signature, apparent in a subset of B-ALL with high FBXW7{beta} expression. This signature contained several members of the TNF superfamily, including those comprising the HLA Class III cluster (LTB, LST1, NCR3, LTA, and NFKBIL1). Our findings suggest that FBXW7{beta} expression drives proinflammatory responses, which could contribute to normal B-cell development, leukemogenesis and responses to anti-cancer therapies.

Key pointsO_LIPreviously thought to be restricted to neural tissues, FBXW7{beta} is the predominant FBXW7 isoform in normal and malignant human pro-B cells.
C_LIO_LIFBXW7{beta} promotes transcriptional activation of a proinflammatory gene cluster that contains TNF superfamily members.
C_LI


title: Effector-dependent activation and oligomerization of NRC helper NLRs by Rpi-amr3 and Rpi-amr1

abstract: Plant pathogens compromise crop yields. Plants have evolved robust innate immunity that depends in part on intracellular Nucleotide-binding, Leucine Rich-Repeat (NLR) immune receptors that activate defense responses upon detection of pathogen-derived effectors. Most "sensor" NLRs that detect effectors require the activity of "helper" NLRs, but how helper NLRs support sensor NLR function is poorly understood. Many Solanaceae NLRs require the NRC (NLR-Required for Cell death) class of helper NLRs. We show here that Rpi-amr3, a sensor NLR from Solanum americanum, detects AVRamr3 from the potato late blight pathogen, Phytophthora infestans, and activates oligomerization of the helper NLR NRC2 into a high-molecular weight resistosome. The NRC2 resistosome also forms upon recognition of P. infestans effector AVRamr1 by another sensor NLR, Rpi-amr1. The ATP-binding motif of Rpi-amr3 is required for NRC2 resistosome formation, but not for interaction with the cognate effector. The NRC2 resistosome can be activated by AVRamr3 homologs from other Phytophthora species. Mechanistic understanding of NRC resistosome formation will underpin engineering crops with durable disease resistance.


title: The small GTPase ARF3 controls metastasis and invasion modality by regulating N-cadherin levels.

abstract: ARF GTPases are central regulators of membrane trafficking that act by controlling local membrane identity and remodelling to facilitate vesicle formation. Unravelling ARF GTPase function is complicated by the overlapping association of ARFs with guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and numerous interactors. The extent to which redundancy is a major factor in ARF function or whether individual ARF GTPases make unique contributions to cellular behaviour remains unclear. Through a functional genomic screen of 3-Dimensional (3D) prostate cancer cell behaviour we explore the contribution of all known ARF GTPases, GEFs, GAPs, and a large selection of interactors to collective morphogenesis. This revealed that the ARF3 GTPase regulates the modality of invasion, acting as a switch between leader cell-led chains of invasion or collective sheet movement. Functionally, the ability of ARF3 to control invasion modality is dependent on association and subsequent control of the junctional adhesion molecule N-cadherin. In vivo, ARF3 levels acted as a rheostat for metastasis from intraprostatic tumour transplants and ARF3:N-cadherin expression can be used to identify prostate cancer patients with metastatic, poor-outcome disease. Our analysis defines a unique function for the ARF3 GTPase in controlling how cells collectively organise during invasion and metastasis.


title: A combined experimental-computational approach uncovers a role for the Golgi matrix protein Giantin in breast cancer progression

abstract: Few studies so far have investigated the impact of different cell migration traits on tumor progression. To address this, we developed a mathematical model wherein cells migrate in two-dimensional space, divide, die or intravasate into the vasculature. Exploring a wide range of speed and persistence combinations, we find that tumor growth positively correlates with increasing speed and higher persistence. As a biologically relevant example, we focused on Golgi fragmentation induced by depletion of Giantin, a Golgi matrix protein, the downregulation of which correlates with poor patient survival. Applying the migration and invasion traits of Giantin depleted cells to our mathematical model, we predict that loss of Giantin increases the number of intravasating cells. This prediction was validated, by showing that circulating tumor cells express significantly less Giantin than primary tumor cells. Altogether, our computational model identifies cell migration traits that regulate tumor progression and uncovers a role of Giantin in breast cancer progression.


title: SOCS3 limits endotoxin-induced endothelial dysfunction by blocking a required autocrine interleukin 6 signal in human umbilical vein endothelial cells

abstract: Increased circulating levels of soluble interleukin (IL)-6 receptor  (sIL-6R) are commonly observed during inflammatory responses, allowing for IL-6 signaling to occur in cells that express the ubiquitous receptor subunit gp130 but not IL-6R, such as endothelial cells. Activation of Toll-like receptor (TLR)-4 or the tumor necrosis factor (TNF) receptor leads to NF-{kappa}B-dependent increases in endothelial IL-6 expression. Thus, we hypothesize that danger signals may induce autocrine IL-6 signaling within the endothelium via sIL-6R-mediated trans-signaling. In support of this hypothesis, we recently demonstrated that conditional deletion in the endothelium of the IL-6 signaling inhibitor SOCS3 leads to rapid mortality in mice challenged with the TLR-4 agonist endotoxin through increases in vascular leakage, thrombosis, leukocyte adhesion, and a type I-like interferon response. Here, we sought to directly test a role for sIL-6R in LPS-treated human umbilical vein endothelial cells. We show that cotreatment with sIL-6R dramatically increases the loss of barrier function and the expression of COX2 and tissue factor mRNA levels induced by LPS. This co-treatment led to a strong activation of STAT1 and STAT3 while not affecting LPS-induced activation of p38 and NF-{kappa}B signaling. Similar results were obtained when sIL-6R was added to a TNF challenge. JAK inhibition by pretreatment with ruxolitinib or by SOCS3 overexpression blunted LPS and sIL-6R synergistic effects, while SOCS3 knockdown further increased the response. Together, these findings demonstrate that IL-6 signaling downstream of NF-kB activation leads to a strong endothelial activation and may explain the acute endotheliopathy observed during critical illness.


title: Unravelling the mechanisms of adaptation to high pressure in proteins

abstract: Life is thought to have appeared in the depth of the sea, under high hydrostatic pressure. Nowadays, it is known that the deep biosphere hosts a myriad of life forms thriving under high pressure conditions. However, the evolutionary mechanisms leading to their adaptation are still not known. Here we show the molecular bases of these mechanisms through a neutron scattering study of two orthologous proteins. We observed that pressure adaptation involves the decoupling of protein-water dynamics and the elimination of cavities in the protein core. This is achieved by an enrichment of acidic residues on the protein surface and by the use of bulkier hydrophobic residues in the core. These findings will be the starting point in the search of a complete genomic model explaining high pressure adaptation.

category: biophysics


title: The plant nucleoplasmin AtFKBP43 needs its extended arms for histone interaction

abstract: The nucleoplasmin family of histone chaperones is a key player in governing the dynamic architecture of chromatin, thereby regulating various DNA-templated processes. The crystal structure of the N-terminal domain of Arabidopsis thaliana FKBP43 (AtFKBP43), an FK506-binding immunophilin protein, revealed a characteristic nucleoplasmin fold, thus confirming it to be a member of the FKBP nucleoplasmin class. Small-Angle X-ray Scattering (SAXS) analyses confirmed its pentameric nature in solution, and additional studies confirmed the nucleoplasmin fold to be highly stable. The AtFKBP43 nucleoplasmin core domain could not interact with histones and required the acidic arms, C-terminal to the core, for histone association. Furthermore, SAXS generated low-resolution envelope structure, ITC, and AUC results revealed that an AtFKBP43 pentamer with C-terminal extensions interacts with H2A/H2B dimer and H3/H4 tetramer in an equimolar ratio. Put together, AtFKBP43 belongs to a hitherto unreported subclass of FKBP nucleoplasmins that requires the C-terminal acidic stretches emanating from the core domain for histone interaction.


title: Sensor NLR immune proteins activate oligomerization of their NRC helper

abstract: Nucleotide-binding domain and leucine-rich repeat (NLR) immune receptors are important components of plant and metazoan innate immunity that can function as individual units or as pairs or networks. Upon activation, NLRs form multiprotein complexes termed resistosomes or inflammasomes. Whereas metazoan paired NLRs, such as NAIP/NLRC4, activate into hetero-complexes, the molecular mechanisms underpinning activation of plant paired NLRs, especially whether they associate in resistosome hetero-complexes is unknown. In asterid plant species, the NLR required for cell death (NRC) immune receptor network is composed of multiple resistance protein sensors and downstream helpers that confer immunity against diverse plant pathogens. Here, we show that pathogen effector-activation of the NLR proteins Rx (confers virus resistance) and Bs2 (confers bacterial resistance) leads to oligomerization of the helper NLR NRC2. Activated Rx does not oligomerize or enter into a stable complex with the NRC2 oligomer and remains cytoplasmic. In contrast, activated NRC2 oligomers accumulate in membrane-associated puncta. We propose an activation-and-release model for NLRs in the NRC immune receptor network. This points to a distinct activation model compared to mammalian paired NLRs.


title: Flavivirus prM interacts with MDA5 and MAVS to inhibit RLR antiviral signaling

abstract:
Vector-borne flaviviruses, including tick-borne encephalitis virus (TBEV), Zika virus (ZIKA), West Nile virus (WNV), yellow fever virus (YFV), dengue virus (DENV), and Japanese encephalitis virus (JEV), pose a growing threat to public health worldwide, and have evolved complex mechanisms to overcome host antiviral innate immunity. However, the underlying mechanisms of flavivirus structural proteins to evade host immune response remain elusive. Here we show that TBEV structural proteins, including pre-membrane (prM), envelope, and capsid proteins, could inhibit type I interferon (IFN-I) production. Mechanically, TBEV prM interacted with both MDA5 and MAVS and interfered with the formation of MDA5-MAVS complex, thereby impeded the nuclear translocation and dimerization of IRF3 to inhibit RLR antiviral signaling. ZIKA and WNV prM was also demonstrated to interact with both MDA5 and MAVS, while dengue virus serotype 2 (DENV2) and YFV prM associated only with MDA5 or MAVS to suppress IFN-I production. In contrast, JEV prM could not suppress IFN-I production. Overexpression of TBEV and ZIKA prM significantly promoted the replication of Sendai virus. Our findings reveal the immune evasion mechanisms of flavivirus prM, which may contribute to understanding flavivirus pathogenicity, therapeutic intervention and vaccine development.

Author SummaryAll flaviviruses must overcome the antiviral innate immunity to infect vertebrate host. The non-structural proteins of flaviviruses are mainly responsible for viral replication and host innate immune escape, and the structural proteins are involved in the virus assembly, which are potential targets for prevention and treatment of flaviviral infections. Whether flavivirus structural proteins participate in host innate immune escape remains to be determined. Here, we found that tick-borne encephalitis virus structural proteins precursor membrane (prM), capsid, and envelope proteins can antagonize type I interferon production, in which prM interacts with both MDA5 and MAVS to inhibit RLR antiviral signaling. Additionally, Zika virus and West Nile virus prMs also interact with both MDA5 and MAVS, while dengue virus serotype 2 and yellow fever virus prMs associate only with MDA5 or MAVS to suppress type I interferon production. In contrast, Japanese encephalitis virus prM cannot antagonize type I interferon production. Our findings reveal that flavivirus prM inhibits type I interferon production via interacting with MDA5 and/or MAVS in a species-dependent manner, which may contribute to understanding flavivirus pathogenicity, therapeutic intervention, and vaccine development.


title: Memory loss at sleep onset

abstract: Every night, we pass through a transitory zone at the borderland between wakefulness and sleep, named the first stage of non-rapid eye movement sleep (N1). N1 sleep is associated with an increased hippocampal activity and dreamlike experiences that incorporate recent wake materials, suggesting that it may be associated with memory processing. Here, we investigated the specific contribution of N1 sleep in the processing of memory traces. Participants were asked to learn the precise locations of 48 objects on a grid and were then tested on their memory for these items before and after a 30-minute rest during which participants either stayed fully awake, transitioned toward N1 or deeper (N2) sleep. We showed that memory recall was lower (10% forgetting) after a resting period including only N1 sleep compared to N2 sleep. Furthermore, the ratio of alpha/theta power (an EEG marker of the transition towards sleep) correlated negatively with the forgetting rate when taking into account all sleepers (N1 and N2 groups combined), suggesting a physiological index for memory loss that transcends sleep stages. Our findings suggest that interrupting sleep onset at N1 may alter sleep-dependent memory consolidation and promote forgetting.


title: Isoform-specific O-glycosylation dictates Ebola virus infectivity

abstract: Ebola virus glycoprotein is one of the most heavily O-glycosylated viral envelope glycoproteins. The glycoprotein possesses a large mucin-like domain responsible for the cytopathic effect on infected cells, yet its structure or potential role in early entry events is poorly defined. To understand the importance of elongated O-glycans and the individual O-glycosylation sites for viral infectivity, we performed a comprehensive characterization of site-specific glycosylation governed by the three key GalNAc-transferases, GalNAc-T1, -T2, and -T3, initiating O-glycan biosynthesis. Using TMT isobaric labelling we performed quantitative differential O-glycoproteomics on proteins produced in wild type HEK293 cells and cell lines ablated for each of the three GalNAc-Ts, as well as compared it to patterns on wild type virus-like particles. In total we found 38 and 41 O-glycosites on virus like particle-derived and recombinant GP, respectively, with well correlated sites and site-specific structures. Examination of the isoform-specific glycosylation demonstrate selective initiation of a subset of O-glycosites by each enzyme, with GalNAc-T1 having the largest contribution. We next demonstrate that O-linked glycan truncation and perturbed initiation retarded the production of viral particles and decreased infectivity of progeny virus. This work represents a comprehensive site-specific analysis of EBOV GP and sheds light on differential regulation of EBOV GP glycosylation initiated by host GalNAc-Ts. Together with the effect on viral propagation it opens prospective avenues for tailored intervention approaches and means for modulating immunogen O-glycan density.


title: The salivary protein Saglin facilitates efficient midgut colonization of Anopheles mosquitoes by malaria parasites

abstract: Malaria is caused by the unicelullar parasite Plasmodium which is transmitted to humans through the bite of infected female Anopheles mosquitoes. To initiate sexual reproduction and to infect the midgut of the mosquito, Plasmodium gametocytes are able to recognize the intestinal environment after being ingested during blood feeding. A shift in temperature, pH change and the presence of the insect-specific compound xanthurenic acid have been shown to be important stimuli perceived by gametocytes to become activated and proceed to sexual reproduction. Here we report that the salivary protein Saglin, previously proposed to be a receptor for the recognition of salivary glands by sporozoites, facilitates Plasmodium colonization of the mosquito midgut, but does not contribute to salivary gland invasion. In mosquito mutants lacking Saglin, Plasmodium infection of Anopheles females is greatly reduced, resulting in impaired transmission of sporozoites at low infection densities. Interestingly, our results suggest that Saglin, present in mosquito saliva, must be ingested with the blood meal to affect Plasmodium development, indicating a previously unknown host-pathogen interaction. Furthermore, we were able to show that saglin deletion has no fitness cost in laboratory conditions, suggesting this gene would be an interesting target for gene drive approaches.


title: Ancient dental calculus preserves signatures of biofilm succession and inter-individual variation independent of dental pathology

abstract:
Dental calculus preserves oral microbes, enabling comparative studies of the oral microbiome and health through time. However, small sample sizes and limited dental health metadata have hindered health-focused investigations to date. Here we investigate the relationship between tobacco pipe smoking and dental calculus microbiomes. Dental calculus from 75 individuals from the 19th century Middenbeemster skeletal collection (Netherlands) were analyzed by metagenomics. Demographic and dental health parameters were systematically recorded, including the presence/number of pipe notches. Comparative data sets from European populations before and after the introduction of tobacco were also analyzed. Calculus species profiles were compared with oral pathology to examine associations between microbiome community, smoking behavior, and oral health status. The Middenbeemster individuals exhibited relatively poor oral health, with a high prevalence of periodontal disease, caries, heavy calculus deposits, and antemortem tooth loss. No associations between pipe notches and dental pathologies, or microbial species composition, were found. Calculus samples before and after the introduction of tobacco showed highly similar species profiles. Observed inter-individual microbiome differences were consistent with previously described variation in human populations from the Upper Paleolithic to the present. Dental calculus may not preserve microbial indicators of health and disease status as distinctly as dental plaque.

Research HighlightsO_LINo associations between calculus species profiles and oral health metrics were detected in a single large population
C_LIO_LIA minority of individuals have a dental calculus species profile characterized by low levels of Streptococcus and high levels of anaerobic taxa
C_LI


title: SOX15 regulates stem cell pluripotency and promotes neural fate during differentiation by activating Hes5

abstract: SOX2 and SOX15 are Sox family transcription factors enriched in embryonic stem cells (ESCs). The role of SOX2 in activating gene expression programs essential for stem cell self-renewal and acquisition of pluripotency during somatic cell reprogramming is well-documented. However, the contribution of SOX15 to these processes is unclear and often presumed redundant with SOX2 largely because overexpression of SOX15 can partially restore self-renewal in SOX2-deficient ESCs. Here, we show that SOX15 contributes to stem cell maintenance by cooperating with ESC-enriched transcriptional coactivators to ensure optimal expression of pluripotency-associated genes. We demonstrate that SOX15 depletion compromises reprogramming of fibroblasts to pluripotency which cannot be compensated by SOX2. Ectopic expression of SOX15 promotes the reversion of a post-implantation, epiblast stem cell state back to a pre-implantation, ESC-like identity even though SOX2 is expressed in both cell states. We also uncover a role of SOX15 in lineage specification, by showing that loss of SOX15 leads to defects in commitment of ESCs to neural fates. SOX15 promotes neural differentiation by binding to and activating a previously uncharacterized distal enhancer of a key neurogenic regulator, Hes5. Together, these findings identify a multifaceted role of SOX15 in induction and maintenance of pluripotency and neural differentiation.


title: Anticodon Table of the Chloroplast Genome and Identification of Putative Quadruplet Anticodons in Chloroplast tRNAs

abstract: The chloroplast genome of 5959 species was analyzed to construct the anticodon table of the chloroplast genome. Analysis of the chloroplast transfer ribonucleic acid (tRNA) revealed the presence of a putative quadruplet anticodon containing tRNAs in the chloroplast genome. The tRNAs with putative quadruplet anticodons were UAUG, UGGG, AUAA, GCUA, and GUUA, where the GUUA anticodon putatively encoded tRNAAsn. The study also revealed the complete absence of tRNA genes containing ACU, CUG, GCG, CUC, CCC, and CGG anticodons in the chloroplast genome from the species studied so far. The chloroplast genome was also found to encode tRNAs encoding N-formylmethionine (fMet), Ile2, selenocysteine, and pyrrolysine. The chloroplast genomes of mycoparasitic and heterotrophic plants have had heavy losses of tRNA genes. Furthermore, the chloroplast genome was also found to encode putative spacer tRNA, tRNA fragments (tRFs), tRNA-derived, stress-induced RNA (tiRNAs), and group I introns. An evolutionary analysis revealed that chloroplast tRNAs had evolved via multiple common ancestors and the GC% had more influence toward encoding the tRNA number in the chloroplast genome compared to the genome size.


title: Culture Associated DNA Methylation Changes Impact on Cellular Function of Human Intestinal Organoids

abstract:
Background & AimsHuman intestinal epithelial organoids (IEO) are a powerful tool to model major aspects of intestinal development, health and diseases, as patient derived cultures retain many features found in-vivo. A necessary aspect of the organoid model is the requirement to expand cultures in-vitro through several rounds of passaging. This is of concern, as the passaging of cells has been shown to affect cell morphology, ploidy, and function. In this study, we address concerns around long term passaging of IEO to better characterise and define effects on cell morphology and function.

MethodsHere we have analysed 173 human IEO from two sampling sites, terminal ileum and sigmoid colon and examined the effect of culture duration on DNA methylation (DNAm), gene expression and cellular function including their response to proinflammatory cytokines and in-vitro cell differentiation.

ResultsOur analyses revealed a major effect of culture duration on DNAm, leading to significant changes at 61,337 loci representing approximately 8% of all CpGs tested. Although global cellular functions such as gut segment-specific gene expression remained stable, a subset of methylation changes correlated with altered gene expression at baseline as well as in response to inflammatory cytokine exposure and in-vitro differentiation. Importantly, epigenetic changes were found to be enriched in genomic regions associated with colonic cancer and distant to the site of replication indicating similarities to malignant transformation.

ConclusionsOur study reveals culture-associated epigenetic, transcriptomic and functional changes in human mucosa derived IEO and highlights the importance of considering passage number as a potentially confounding factor.

SynopsisThis work describes cell culture induced changes to DNA methylation, gene expression and cellular function in human IEO. Globally organoids lost DNA methylation with time in culture while DNA methylation also became generally more variable. This work suggests a shifted epigenetic profile in organoids cultured long-term.


title: The whole blood microbiome of Indonesians reveals that environmental differences shape immune gene expression signatures

abstract: Pathogens found within local environments are a major cause of morbidity and mortality. This is particularly true in Indonesia, where infectious diseases such as malaria or dengue are a significant part of the disease burden within the country. One way to strengthen the control of infectious diseases is through better surveillance, however unequal investment in medical funding throughout Indonesia, particularly in rural areas, has resulted in under-reporting of cases. Here, we use transcriptome data from 117 healthy individuals living on the islands of Mentawai, Sumba, and the Indonesian side of New Guinea Island to explore which pathogens are present within whole blood. We are able to detect a broad range of taxa within RNA-sequencing data generated from whole blood, including bacteria, viruses, archaea, and eukaryotes. Using independent component analysis, we find that two of these pathogens--Flaviviridae and Plasmodium--have the most noticeable effects on expression profiles. We also identify specific genes linked with Plasmodium and Flavivirus abundance and find that both of these infections are most pronounced in the easternmost island within our Indonesian dataset. This study provides a framework for novel applications of RNA-seq as surveillance and a better understanding of environmental contributors affecting gene expression within Indonesia.


title: A Feed-forward Pathway Drives LRRK2 kinaseMembrane Recruitment and Apparent Activation

abstract: Activating mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) cause Parkinsons disease and previously we showed that activated LRRK2 phosphorylates a subset of Rab GTPases (Steger et al., 2017). Moreover, Golgi-associated Rab29 can recruit LRRK2 to the surface of the Golgi and activate it there for both auto- and Rab substrate phosphorylation. Here we define the precise Rab29 binding region of the LRRK2 Armadillo domain between residues 360-450 and show that this site, termed "Site #1", can also bind additional LRRK2 substrates, Rab8A and Rab10. Moreover, we identify a distinct, N-terminal, higher affinity interaction interface between LRRK2 phosphorylated Rab8 and Rab10 termed "Site #2", that can retain LRRK2 on membranes in cells to catalyze multiple, subsequent phosphorylation events. Kinase inhibitor washout experiments and mutation analysis demonstrate that rapid recovery of kinase activity in cells depends on the ability of LRRK2 to associate with phosphorylated Rab reaction products. Reconstitution of purified LRRK2 recruitment onto planar lipid bilayers decorated with Rab10 protein demonstrates cooperative association of only active LRRK2 with phospho-Rab10-containing membrane surfaces. These experiments reveal a feed-forward pathway that provides spatial control and apparent membrane activation of LRRK2 kinase activity.


title: Molecular Aggregation Induced Photoacoustics for NIR-II in vivo Imaging

abstract: Molecular aggregation induced photo-properties alteration has been found to play a crucial role in the light induced processes, such as aggregation induced emission (AIE) and J aggregation induced dramatic absorption red shift. The light induced acoustic process (photoacoustic) is also considered to be one of the most essential characters of the light absorbing molecules. However, to the best of our knowledge, the molecular aggregation induced photoacoustic effect (MAIPA) has never been reported. Herein, we report the first MAIPA effect for which the PA intensity is dominated by the molecular aggregation, rather than by absorbance as usual concerned. Molecular aggregation induces a strong electronic coupling effect, resulting in significant absorption suppression from the individual state to highly aggregated state (around 5 molecules aggregated). However, the corresponding PA efficiency was found to be about 2-orders of magnitude greater for the latter. A well-behaved linear correlation between the molecular aggregation level and MAIPA effect was observed. The surprisingly significant MAIPA effect was realized via novel NIR-II squaraine-benzothiopyrylium dyes. Excellent photophysical properties of the novel NIR-II dyes were achieved, such as large absorption extinction coefficient and high photostability. Thanks to the relatively narrow FWHM and the high PA efficiency of SQN2@PMAOPEG and ZC825@BSA, in vivo multiplex PA imaging was demonstrated for tumor tissue and macrophage cells, blood and lymphoid vessels.


title: Functional metagenomic screening in microfluidic droplets identifies a β-glucuronidase in an unprecedented sequence neighbourhood

abstract: The abundance of recorded protein sequence data stands in contrast to the small number of experimentally verified functional annotation. Here we screened a million-membered metagenomic library at ultrahigh throughput in microfluidic droplets for {beta}-glucuronidase activity. We identified SN243, a genuine {beta}-glucuronidase with little homology to previously studied enzymes of this type, as a glycoside hydrolase (GH) 3 family member. This GH family had no recorded evidence of {beta}-glucuronidases at the outset of this study, showing that a functional metagenomic approach can shed light on assignments that are currently  unpredictable by bioinformatics. Kinetic analyses of SN243 characterised it as a promiscuous catalyst and structural analysis suggests regions of divergence from homologous GH3 members creating a wide-open active site. With a screening throughput of >107 library members per day, picolitre volume microfluidic droplets enable functional assignments that complement current enzyme database dictionaries and provide bridgeheads for the annotation of unexplored sequence space.


title: Prediction Of The Impact Of Genetic Variability On Drug Sensitivity For Clinically Relevant EGFR Mutations

abstract: Mutations in the kinase domain of the Epidermal Growth Factor Receptor (EGFR) can be drivers of cancer and also trigger drug resistance in patients under chemotherapy treatment based on kinase inhibitors use. A priori knowledge of the impact of EGFR variants on drug sensitivity would help to optimize chemotherapy and to design new drugs effective against resistant variants. To this end, we have explored a variety of in silico methods, from sequence-based to  state-of-the-art atomistic simulations. We did not find any sequence signal that can provide clues on when a drug-related mutation appears and what will be the impact in drug activity. Low-level simulation methods provide limited qualitative information on regions where mutations are likely to produce alterations in drug activity and can predict around 70% of the impact of mutations on drug efficiency. High-level simulations based on non-equilibrium alchemical free energy calculations show predictive power. The integration of these  state-of-the-art methods in a workflow implementing an interface for parallel distribution of the calculations allows its automatic and high-throughput use, even for researchers with moderate experience in molecular simulations.


title: Spatial organization of Dectin-1 and TLR2 during synergistic crosstalk revealed by super-resolution imaging

abstract: Innate immune cells recognize and elicit responses against pathogens by integrating signals from different types of cell-surface receptors. How the receptors interact in the membrane to enable their signaling crosstalk is poorly understood. Here, we reveal the nanoscale organization of TLR2 and Dectin-1, a receptor pair known to cooperate in regulating antifungal immunity, through their synergistic signaling crosstalk at macrophage cell membranes. Using super-resolution single-molecule localization microscopy, we show that discrete non-colocalized nanoclusters of Dectin-1 and TLR2 are partially overlapped during their synergistic crosstalk. Compared to when one type of receptor is activated alone, the simultaneous activation of Dectin-1 and TLR2 leads to a higher percentage of both receptors being activated by their specific ligands, and consequently an increased level of tyrosine phosphorylation. Our results depict, in nanoscale detail, how Dectin-1 and TLR2 achieve synergistic signaling through the spatial organization of their receptor nanoclusters.


title: The bHLH transcription factor MpHYPNOS is a critical regulator of gemma dormancy in the liverwort Marchantia polymorpha.

abstract:
O_LIDormancy is a key process for land plants to adapt drastically changing terrestrial environment. The liverwort Marchantia polymorpha produce dormant propagules called gemmae for asexual reproduction. The plant hormone abscisic acid (ABA) plays significant roles in the regulation of dormancy in both seed of flowering plants and gemma of M. polymorpha.
C_LIO_LIBased on the previous transcriptome analysis, here we identified the basic helix-loop-helix transcription factor MpHYPNOS (MpHYP), as a key regulator of gemma dormancy.
C_LIO_LIThe knock-out mutants showed much higher germination rate of gemmae in gemma cup than ABA-related mutants did, while the growth and development were the same as wild type. Transient induction of MpHYP caused irreversible growth arrest in gemma and thallus. Transcriptome and qRT-PCR analyses revealed that MpHYP repressed cell-cycle related genes and induced the ABA biosynthesis and responsive genes. Indeed, ABA amounts were increased or decreased by overexpression or knock-out of MpHYP, respectively. However, the growth arrest caused by MpHYP overexpression was not suppressed by the mutation in the ABA receptor gene.
C_LIO_LIThese data suggest that MpHYP regulates gemma dormancy and thallus growth partially through the ABA pathway. Our findings would provide the clues to understand ABA-dependent and independent regulation of dormancy in land plants.
C_LI


title: DNA-based detection of Aphanomyces cochlioides in soil and sugar beet plants

abstract: Aphanomyces cochlioides, the causal agent of seedling damping-off and Aphanomyces root rot (ARR) of sugar beet, causes yield losses in major sugar beet growing regions. Currently, a 4-week soil bioassay and a 2-day culture-based assay are used to diagnose presence of A. cochlioides. However, these assays can be time-consuming and lack sensitivity. In this study we developed a sensitive, specific, and rapid assay to detect and quantify DNA of A. cochlioides. We developed a TaqMan qPCR assay targeting a region of the mitochondrial genome of A. cochlioides representing a unique gene order for Aphanomyces with genus-specific primers and a species-specific probe. The qPCR assay detected A. cochlioides in 12 naturally infested field soil samples with disease severity index (DSI) values of 48-100, in sugar beet seedlings 5-7 days after planting, and with as little as 1 fg of pure A. cochlioides DNA. Adult sugar beet roots with ARR symptoms were sampled to further validate this qPCR assay. Aphanomyces cochlioides was detected in 95% of these samples using this qPCR assay, while only 23% of the same samples were positive using a culture-based assay. This shows the improved sensitivity of this qPCR assay for disease diagnosis and could provide growers with ARR risk of a field, which would help them make informed disease management decisions. However, further research is required to translate the results of this study to growers fields to quantify A. cochlioides with a high degree of accuracy.


title: MECP2 expression coincides with age-dependent tactile sensory deficits in a female mouse model for Rett syndrome

abstract: Rett syndrome is characterized by an early period of typical development and then, regression of learned motor and speech skills in girls. The underlying mechanisms from normalcy to regression are unclear. Due to random X-chromosome inactivation, female patients of Rett syndrome, and female mouse models of Rett syndrome (Mecp2Heterozygous, Het) express a functional copy of wild-type MECP2 protein in half of all mature cells in brain and body. By analyzing wild-type MECP2 expression in the context of whisker tactile sensory perception, here we report compensatory increase in MECP2 protein expression in 6-week-old adolescent Het mice, which display normal levels of perineuronal net expression, mild tactile sensory perception deficit and efficient pup retrieval behavior. Comparatively, 12-week-old adult Het mice display decreased MECP2 expression and significant tactile sensory perception deficits. Thus, we have identified a period of normalcy and regression in this female mouse model, which coincide with variable wild-type expression of MECP2. We speculate that compensatory increases in MECP2 expression in Het brains allows for normal functioning, while the inability to maintain these expression level changes leads to behavioral deficits.


title: Experimental infection of Artibeus lituratus bats and no detection of Zika virus in neotropical bats from French Guyana, Peru, and Costa Rica, suggest a limited role of bats in Zika transmission.

abstract:
Bats are important natural reservoir hosts of a diverse range of viruses that can be transmitted to humans and have been suggested that could play an important role in the Zika virus (ZIKV) transmission cycle. However, the exact role of these animals as reservoirs for Flaviviruses is still controversial. To further expand our understanding of the role of bats in the ZIKV transmission cycle in Latin America, we carried an experimental infection in wild-caught Artibeus bats and sampled several free-living neotropical bats over three countries of the region. Experimental ZIKV infection was made in free-ranging adult bats (4 females and 5 males). The most relevant gross findings were hemorrhages in the bladder, stomach and patagium. Significant histological findings included inflammatory infiltrate consisting of a predominance of neutrophils and lymphocytes, in addition to degeneration in the reproductive tract of males and females. This suggests that bat reproduction might be at some level affected by ZIKV. Leukopenia was also observed in some inoculated animals. Hemorrhages, genital alterations, and leukopenia are suggestive to be caused by ZIKV, however, since these are wild-caught bats, we can not exclude other agents. Excretion of ZIKV by qPCR was detected (low titles) in only two urine samples in two inoculated animals. All other animals and tissues tested negative. Finally, no virus-neutralizing Abs were found in any animal. To determine ZIKV infection in nature, a total of 2056 bats were blood sampled for ZIKV detection by qPCR. Most of the sampled individuals belonged to the genus Pteronotus sp. (23%), followed by the species Carollia sp. (17%); Anoura sp. (14%), and Molossus sp. (13.7 %). No sample of any tested species resulted positive to ZIKV by qPCR.

These results together suggest that bats are not efficient amplifiers or reservoirs of ZIKV and may not have an important role in ZIKV transmission dynamics.

Author summaryIn previous works made in 2008-2009, we have found the presence of antibodies against Flaviviruses and viral RNA has been detected in Neotropical chiropterans of Mexico, which led us to support the hypothesis that these animals could be reservoirs of Flaviviruses. As controversial opinions have been exposed, and based on a previous (2019) experimental ZIKV infection made in Colorado State University using adult Artibeus males from a captive colony, in this work we also experimentally infected adult Artibeus males complementarily adding females and using free-living animals instead of laboratory bats. We also monitored a diverse range of natural bat populations in Latin America for the presence of viral RNA against ZIKV in blood. A plaque reduction seroneutralization test was used for the detection of antibodies against ZIKV. Similar to the previous work, we found histopathological alteration in male testicles but also in ovaries and oviducts of females, as well as gliosis and multifocal necrosis in pyramidal neurons and Purkinge cells of inoculated animals. Only two urine samples from inoculated animals showed viral RNA. Additionally, leukopenia and lymphoid follicular splenic hyperplasia were evidenced. Differing to what was reported, no neutralizing antibodies against ZIKV were detected in any sample. Viral RNA within the blood was not present in any of the 2056 bat samples collected in French Guyana, Peru and Costa Rica and proceeding from 33 bat genera. These results together suggest that bats are not efficient amplifiers or reservoirs of ZIKV and might not have an important role on ZIKV transmission dynamics.


title: Sequences and proteins that influence mRNA processing in Trypanosoma brucei: evolutionary conservation of SR-domain and PTB protein functions.

abstract: Spliced leader trans splicing is the addition of a short, capped sequence to the 5 end of mRNAs. It is widespread in eukaryotic evolution, but factors that influence trans splicing acceptor site choice have been little investigated. In Kinetoplastids, all protein-coding mRNAs are 5 trans spliced. A polypyrimidine tract is usually found upstream of the AG splice acceptor, but there is no branch point consensus; moreover, splicing dictates polyadenylation of the preceding mRNA. We here describe a trans splicing reporter system that can be used for studies and screens concerning the roles of sequences and proteins in processing site choice and efficiency. Splicing was poor with poly(U) tracts less than 9 nt long, and was influenced by nearby secondary structures. A screen for signals resulted in selection of sequences that were on average 45% U and 35% C. Tethering of either the splicing factor SF1, or the cleavage and polyadenylation factor CPSF3 within the intron stimulated processing in the correct positions, while tethering of two possible homologues of Opisthokont PTB inhibited processing. In contrast, tethering of SR-domain proteins RBSR1, RBSR2, or TSR1 or its interaction partner TSR1IP, promoted use of alternative signals upstream of the tethering sites. RBSR1 interacts predominantly with proteins implicated in splicing, whereas the interactome of RBSR2 is more diverse. These results suggest that the functions of PTB and SR-domain proteins in splice site definition were already present in the last eukaryotic common ancestor.

category: molecular biology


title: Initiator tRNA lacking 1-methyladenosine is unexpectedly targeted by the rapid tRNA decay pathway in evolutionarily distant yeast species

abstract:
All tRNAs have numerous modifications, lack of which often results in growth defects in the budding yeast Saccharomyces cerevisiae and neurological or other disorders in humans. In S. cerevisiae, lack of tRNA body modifications can lead to impaired tRNA stability and decay of a subset of the hypomodified tRNAs. Mutants lacking 7-methylguanosine at G46 (m7G46), N2,N2-dimethylguanosine (m2,2G26), or 4-acetylcytidine (ac4C12), in combination with other body modification mutants, target certain mature hypomodified tRNAs to the rapid tRNA decay (RTD) pathway, catalyzed by 5-3 exonucleases Xrn1 and Rat1, and regulated by Met22. The RTD pathway is conserved in the phylogenetically distant fission yeast Schizosaccharomyces pombe for mutants lacking m7G46. In contrast, S. cerevisiae trm6 mutants with reduced 1-methyladenosine (m1A58) specifically target pre-tRNAiMet(CAU) to the nuclear surveillance pathway for 3-5 exonucleolytic decay by the TRAMP complex and nuclear exosome.

We show here that the RTD pathway has an unexpected major role in the biology of m1A58 and tRNAiMet(CAU) in both S. pombe and S. cerevisiae. We find that S. pombe trm6{Delta} mutants lacking m1A58 are temperature sensitive due to decay of tRNAiMet(CAU) by the RTD pathway. Thus, trm6{Delta} mutants had reduced levels of tRNAiMet(CAU) and not of eight other tested tRNAs, overexpression of tRNAiMet(CAU) restored growth, and spontaneous suppressors that restored tRNAiMet(CAU) levels had mutations in dhp1/RAT1 or tol1/MET22. In addition, deletion of cid14/TRF4 in the nuclear surveillance pathway did not restore growth. Furthermore, re-examination of S. cerevisiae trm6 mutants revealed a major role of the RTD pathway in maintaining tRNAiMet(CAU) levels, in addition to the known role of the nuclear surveillance pathway. These findings provide evidence for the importance of m1A58 in the biology of tRNAiMet(CAU) throughout eukaryotes, and fuel speculation that the RTD pathway has a major role in quality control of body modification mutants throughout fungi and other eukaryotes.

Author SummarytRNA modifications are highly conserved, and their lack frequently results in growth defects in the yeast Saccharomyces cerevisiae and neurological disorders in humans. In S. cerevisiae lack of 1-methyladenosine at N58 (m1A58) in the tRNA body is lethal due to 3-5 decay of pre-tRNAiMet by the nuclear surveillance pathway. By contrast, lack of any of three other body modifications causes growth defects due to 5-3 decay of specific hypomodified tRNAs by the rapid tRNA decay (RTD) pathway. Despite their importance, little is known about either tRNAiMet quality control or tRNA decay pathways in eukaryotes other than S. cerevisiae.

Here we show an unexpected role of the RTD pathway in quality control of tRNAiMet lacking m1A58 in the phylogenetically distant yeast species Schizosaccharomyces pombe and S. cerevisiae. We find that S. pombe trm6{Delta} mutants, lacking m1A58, are temperature sensitive due to decay of tRNAiMet(CAU) primarily by the RTD pathway. Furthermore, re-investigation of S. cerevisiae trm6 mutants revealed a significant role of the RTD pathway, in addition to the nuclear surveillance pathway, in decay of tRNAiMet(CAU). Our results suggest that throughout eukaryotes the RTD pathway has a major role in decay of hypomodified tRNAs and that m1A58 is crucial to tRNAiMet(CAU) biology.

category: genetics


title: Epigenetic Control of Hundreds of Chromosome-Associated lncRNA Genes Essential for Replication and Stability

abstract: ASARs are long noncoding RNA genes that control replication timing of entire human chromosomes in cis. The three known ASAR genes are located on human chromosomes 6 and 15, and are essential for chromosome integrity. To identify ASARs on all human chromosomes we utilized a set of distinctive ASAR characteristics that allowed for the identification of hundreds of autosomal loci with epigenetically controlled, allele-restricted behavior in expression and replication timing of coding and noncoding genes, and is distinct from genomic imprinting. Disruption of noncoding RNA genes at five of five tested loci resulted in chromosome-wide delayed replication and chromosomal instability, validating their ASAR activity. In addition to the three known essential cis-acting chromosomal loci, origins, centromeres, and telomeres, we propose that all mammalian chromosomes also contain "Inactivation/Stability Centers" that display allele-restricted epigenetic regulation of protein coding and noncoding ASAR genes that are essential for replication and stability of each chromosome.


title: Linkage mapping reveals loci that underlie differences in C. elegans growth

abstract: Growth rate and body size are complex traits that contribute to the fitness of organisms. The identification of loci that underlie differences in these traits provides insights into the genetic contributions to development. Leveraging Caenorhabditis elegans as a tractable metazoan model for quantitative genetics, we can identify genomic regions that underlie differences in growth. We measured post-embryonic growth of the laboratory-adapted wild-type strain (N2) and a wild strain from Hawaii (CB4856), and found differences in body size. Using linkage mapping, we identified three distinct quantitative trait loci (QTL) on chromosomes IV, V, and X that are associated with variation in body size. We further examined these size-associated QTL using chromosome substitution strains and near-isogenic lines, and validated the chromosome X QTL. Additionally, we generated a list of candidate genes for the chromosome X QTL. These genes could potentially contribute to differences in animal growth and should be evaluated in subsequent studies. Our work reveals the genetic architecture underlying animal growth variation and highlights the genetic complexity of body size in C. elegans natural populations.


title: Inferring cell-type-specific causal gene regulatory networks during human neurogenesis

abstract:
BackgroundGenetic variation influences both chromatin accessibility, assessed in chromatin accessibility quantitative trait loci (caQTL) studies, and gene expression, assessed in expression QTL (eQTL) studies. Genetic variants can impact either nearby genes (local eQTLs) or distal genes (trans eQTLs). Colocalization between caQTL and eQTL, or local- and distant-eQTLs suggests that they share causal variants. However, pairwise colocalization between these molecular QTLs does not guarantee a causal relationship. Mediation analysis can be applied to assess the evidence supporting causality versus independence between molecular QTLs. Given that the function of QTLs can be cell-type-specific, we performed mediation analyses to find epigenetic and distal regulatory causal pathways for genes within two major cell types of the developing human cortex, progenitors and neurons.

ResultsWe found that expression of 168 and 38 genes were mediated by chromatin accessibility in progenitors and neurons, respectively. We also found that the expression of 781 and 200 downstream genes were mediated by upstream genes in progenitors and neurons. Moreover, we discovered that a genetic locus associated with inter-individual differences in brain structure showed evidence for mediation of SLC26A7 through chromatin accessibility, identifying molecular mechanisms of a common variant association to a brain trait.

ConclusionsIn this study, we identified cell-type-specific causal gene regulatory networks whereby the impacts of variants on gene expression were mediated by chromatin accessibility or distal gene expression. Identification of these causal paths will enable identifying and prioritizing actionable regulatory targets perturbing these key processes during neurodevelopment.


title: Method for quantifying the metabolic boundary fluxes of cell cultures in large cohorts by high resolution hydrophilic liquid chromatography mass spectrometry

abstract: Metabolomics is a mainstream approach for investigating the metabolic underpinnings of complex biological phenomena and is increasingly being applied to large scale studies involving hundreds or thousands of samples. Although metabolomics methods are robust in smaller scale studies, they can be challenging to apply in larger cohorts due to the inherent variability of liquid chromatography mass spectrometry (LC-MS). Much of this difficulty results from the time-dependent changes in the LC-MS system, which affects both the qualitative and quantitative performance of the instrument. Herein, we introduce an analytical strategy for addressing this problem in large-scale microbial studies. Our approach quantifies microbial boundary fluxes using two zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) columns that are plumbed to enable offline column equilibration. Using this strategy, we show that over 360 common metabolites can be resolved in 4.5 minutes per sample and that metabolites can be quantified with a median coefficient of variation of 0.127 across 1,100 technical replicates. We illustrate the utility of this strategy via an analysis of 960 strains of Staphylococcus aureus isolated from blood stream infections. These data capture the diversity of metabolic phenotypes observed in clinical isolates and provide an example of how large-scale investigations can leverage our novel analytical strategy.


title: CLASP2 stabilizes GDP-associated terminal tubulins to prevent microtubule catastrophe

abstract: CLASPs are ubiquitous stabilizers of microtubule dynamics but their molecular targets at the microtubule plus-end are not understood. Using DNA origami-based reconstructions we show that clusters of human CLASP2 form a load-bearing bond with terminal GDP-tubulins at the stabilized microtubule tip. This activity relies on the unconventional TOG2 domain of CLASP2, which releases its high-affinity bond with the GDP-dimers upon their conversion into polymerization-competent GTP-tubulin. By tethering dynamic microtubule ends near immobilized CLASP2, we show that the targets for CLASP2 binding at the polymerizing tip arise stochastically, leading to nanoscale disruptions in microtubule tip integrity. The ability of CLASP2 to recognize nucleotide-specific tubulin conformation and stabilize the catastrophe-promoting GDP-tubulins intertwines with the previously underappreciated exchange between GDP and GTP at terminal tubulins, providing a distinct molecular mechanism to suppress microtubule catastrophe without affecting tubulin incorporation.


title: Distinct complexes containing the cytosolic type III secretion system ATPase resolved by 3D single-molecule tracking in live Yersinia enterocolitica

abstract: The membrane-embedded injectisome, the structural component of the virulence-associated type III secretion system (T3SS), is used by gram-negative bacterial pathogens to inject species-specific effector proteins into eukaryotic host cells. The cytosolic injectisome proteins are required for export of effectors and display both stationary, injectisome-bound populations as well as freely-diffusing cytosolic populations. How the cytosolic injectisome proteins interact with each other in the cytosol and associate with membrane-embedded injectisomes remains unclear. Here, we utilize 3D single-molecule tracking to resolve distinct cytosolic complexes of injectisome proteins in living Yersinia enterocolitica cells. Tracking of the eYFP-labeled ATPase, YeSctN, and its regulator, YeSctL, reveals that these proteins form a cytosolic complex with each other and then further with YeSctQ. YeSctNL and YeSctNLQ complexes can be observed in both in wild type cells and in{Delta} sctD mutants, which cannot assemble injectisomes. In{Delta} sctQ mutants, the relative abundance of the YeSctNL complex is considerably increased. These data indicate that distinct cytosolic complexes of injectisome proteins can form prior to injectisome binding - a feature that could contribute to injectisome functional regulation.


title: Using genomes and evolutionary analyses to screen for host-specificity and arms-race dynamics in the plant pathogen Xylella fastidiosa

abstract: Xylella fastidiosa infects several economically important crops in the Americas, and it also recently emerged in Europe. It is not clear, however, whether X. fastidiosa isolates display specialization to plant hosts and, if so, what genetic determinants underlie the associations. Here, using a set of Xylella genomes reflective of the genus-wide diversity, we performed a pan-genome analysis based on both core and accessory genes. We first investigated evolutionary relationships among isolates, recapitulating the three recognized X. fastidiosa subspecies based on information from both core and accessory genes. We used these phylogenies as the basis to test associations between genetic divergence and host species, yielding significant evidence for specialization of X. fastidiosa to plant host species for core, but not accessory, genes. This observation places constraint on the hypothesis that host specificity is conferred by shifts in accessory gene content. We also analyzed the ratio of nonsynonymous to synonymous substitutions in both core and accessory genes to identify positively selected genes that are potentially involved in arms-race dynamics. Arms-race genes may contribute to interactions with the plant hosts or insect vectors, and they are thus candidates to act as virulence or pathogenicity factors. We uncovered evidence of positive selection across codons of 19.5% (200 of 1,205) core genes and 7.7% (181 of 2,349) accessory genes. Most of these genes had unknown functions, but we identified some tractable candidates including nagZ_2, which encodes a beta-glucosidase that is important for biofilm formation Neisseria gonorrhoeae, and cya, which modulates gene expression in pathogenic bacteria.


title: Spatial patterns of evolutionary diversity in Cactaceae show low ecological representation within protected areas

abstract: Mapping biodiversity patterns across taxa and environments is crucial to address the evolutionary and ecological dimensions of species distribution, suggesting areas of particular importance for conservation purposes. Within Cactaceae, spatial diversity patterns are poorly explored, as well as the abiotic factors that may predict these patterns. We gathered geographic and genetic data from 922 cactus species, which are tightly associated with drylands, to evaluate diversity patterns, such as phylogenetic diversity and endemism, paleo-, neo-, and superendemism, and the environmental predictor variables of such patterns in a global analysis. Hotspot areas of cacti diversity are scattered along the Neotropical and Nearctic regions, mainly in the desertic portion of Mesoamerica, Caribbean Island, and the dry diagonal of South America. The geomorphological features of these regions may create a complexity of areas that work as locally buffered zones over time, which triggers local events of diversification and speciation. Desert and dryland/dry forest areas comprise paleo- and superendemism and may act as both museums and cradles of species, displaying great importance for conservation. Past climates, topography, soil features, and solar irradiance seem to be the main predictors of distinct endemism types. The hotspot areas that encompass a major part of the endemism cells are outside or poorly covered by formal protection units. The current legally protected areas are not able to conserve cactus evolutionary history. Given the rapid anthropogenic disturbance, efforts must be reinforced to monitor biodiversity and the environment and to define/plan current and new protected areas.


title: Effects of axon branching and asymmetry between the branches on transport, mean age, and age density distributions of mitochondria in neurons

abstract: Mitochondria transport in a branched axon with two branches of different sizes is investigated; the results are compared with mitochondria transport in an axon with symmetric branches and in a straight (unbranched) axon. The interest in understanding mitochondria transport in branched axons is motivated by the large size of arbors of dopaminergic neurons, which die in Parkinsons disease. Since the failure of energy supply of multiple demand sites located in various axonal branches may be a possible reason for the death of these neurons, we were interested in investigating how branching affects mitochondria transport. Besides investigating mitochondria fluxes between the demand sites and mitochondria concentrations, we also studied how the mean age of mitochondria and mitochondria age densities depend on the distance from the soma. We established that if the axon splits into two branches of unequal length, the mean ages of mitochondria and age density distributions in the demand sites are affected by how the mitochondria flux splits at the branching junction (what portion of mitochondria enters the shorter branch and what portion enters the longer branch). However, if the axon splits into two branches of equal length, the mean ages and age densities of mitochondria are independent of how the mitochondria flux splits at the branching junction. This even holds for the case when all mitochondria enter one branch, which is equivalent to a straight axon. Because the mitochondrial membrane potential (which many researchers view as a proxy for mitochondrial health) decreases with mitochondria age, the independence of mitochondria age on whether the axon is symmetrically branched or straight (providing the two axons are of the same length), and on how the mitochondria flux splits at the branching junction, may explain how dopaminergic neurons can sustain very large arbors and still maintain mitochondrial health across branch extremities.

category: bioengineering