title: Novel Roles of G-quadruplexes on Enhancers in human chromatin

abstract: G-quadruplexes (G4), stable four-stranded non-canonical DNA structures, are highly related to function of promoters and initiation of gene transcription. We found that G4 structures were also enriched in the enhancers across different cell lines. However, the relationship between G4 structures and enhancer activity remains unknown. Here, we proved that G4 structures on enhancers lead to the re-positioning of nucleosomes create nucleosome depleted regions (NDRs). Moreover, stable NDRs and special secondary structures of G4 help enhancers to recruit abundant TFs to co-bind, especially for architectural proteins including CTCF, RAD21, and SMC3. These architectural proteins, which play critical roles in the formation of higher-order chromatin organization, further influenced the chromatin interactions of G4 enhancers. Additionally, we revealed that G4 enhancers harbored significantly higher enrichment of eQTLs than typical enhancers, suggesting G4 enhancers displayed more enhancer regulatory activity. We found that most super enhancers (SEs) contain G4 structures. Even though the enrichment of chromatin accessibility and histone modifications around G4-containing SEs are not significantly higher than those around other SEs, G4-containing SEs still possess much more TFs across different cell lines. According to these results, we proposed a model in which the formation of G4 structures on enhancer exclude nucleosome occupancy and recruit abundant TFs which lead to the stable chromatin interaction between G4 enhancers and their target genes. Because of the relevance between G4 structures and enhancers, we hypothesized that G4 structures may be a potential markers indicating enhancer regulatory activity.


title: Z-DNA as a Touchstone for Additive Empirical Force Fields and a Refinement of the Alpha/Gamma DNA torsions for AMBER

abstract: Although current AMBER force fields are relatively accurate for canonical B-DNA, many non-canonical structures are still described incorrectly. As non-canonical motifs are attracting increasing attention due to the role they play in living organisms, further improvement is desirable. Here, we have chosen Z-DNA molecule, can be considered a touchstone of the universality of empirical force fields, since the non-canonical  and {gamma} backbone conformations native to Z-DNA are also found in protein-DNA complexes, i-motif DNA and other non-canonical DNAs. We show that spurious /{gamma} conformations occurring in simulations with current AMBER force fields, OL15 and bsc1, are largely due to inaccurate /{gamma} parameterization. Moreover, stabilization of native Z-DNA substates involving {gamma} = trans conformations appears to be in conflict with the correct description of the canonical B-DNA structure. Because the balance of the native and spurious conformations is influenced by non-additive effects, this is a difficult case for an additive dihedral energy scheme such as AMBER. We propose new /{gamma} parameters, denoted OL21, and show that they improve the stability of native /{gamma} Z-DNA substates while keeping the canonical DNA description virtually unchanged, and thus represent a reasonable compromise within the additive force field framework. Although further extensive testing is needed, the new modification appears to be a promising step towards a more reliable description of non-canonical DNA motifs and provides the best performance for Z-DNA molecules among current AMBER force fields.

category: biophysics


title: Adsorption of Lysozyme Into a Charged Confining Pore

abstract: Several applications arise from the confinement of proteins on surfaces since their stability and biological activity are enhanced. It is also known that the way a protein adsorbs on the surface is important for its biological function since its active sites should not be obstructed. In this study, the adsorption properties of hen egg-white Lysozyme, HEWL, into a negatively charged silica pore is examined employing a coarse-grained model and constant-pH Monte Carlo simulations. The role of electrostatic interactions is taken into account when including the Debye-HAijckel potentials into the C structure-based model. We evaluate the effects of pH, salt concentration, and pore radius on the protein preferential orientation and spatial distribution of its residues regarding the pore surface. By mapping the residues that stay closer to the pore surface, we find the increase of pH leads to orientational changes of the adsorbed protein when the solution pH gets closer to the HEWL isoelectric point. At these conditions, the pKa shift of these important residues caused by the adsorption into the charged confining surface results in a HEWL charge distribution that stabilizes the adsorption in the observed protein orientation. We compare our observations to the results of pKa shift for HEWL available in the literature and to some experimental data.


title: Identification of RNA base pairs and complete assignment of nucleobase resonances by proton-detected solid-state NMR spectroscopy at 100 kHz MAS

abstract: Knowledge of RNA structure, either in isolation or in complex, is fundamental to understand the mechanism of cellular processes. Solid-state NMR (ssNMR) is applicable to high molecular-weight complexes and does not require crystallization; thus, it is well-suited to study RNA as part of large multicomponent assemblies. Recently, we solved the first structures of both RNA and an RNA-protein complex by ssNMR using conventional 13C- and 15N-detection. This approach is limited by the severe overlap of the RNA peaks together with the low sensitivity of multidimensional experiments. Here, we overcome the limitations in sensitivity and resolution by using 1H-detection at fast MAS rates. We develop experiments that allow the identification of complete nucleobase spin-systems together with their site-specific base pair pattern using sub-milligram quantities of one uniformly labelled RNA sample. These experiments provide rapid access to RNA secondary structure by ssNMR in protein-RNA complexes of any size.


title: The co-evolution of the genome and epigenome in colorectal cancer

abstract: Colorectal malignancies are a leading cause of cancer death. Despite large-scale genomic efforts, DNA mutations do not fully explain malignant evolution. Here we study the co-evolution of the genome and epigenome of colorectal tumours at single-clone resolution using spatial multi-omic profiling of individual glands. We collected 1,373 samples from 30 primary cancers and 9 concomitant adenomas and generated 1,212 chromatin accessibility profiles, 527 whole-genomes and 297 whole-transcriptomes. We found positive selection for DNA mutations in chromatin modifier genes and recurrent chromatin changes in regulatory regions of cancer drivers with otherwise no mutation. Genome-wide alterations in transcription factor binding accessibility involved CTCF, downregulation of interferon, and increased accessibility for SOX and HOX, indicating developmental genes reactivation. Epigenetic aberrations were heritable, distinguishing adenomas from cancers. Mutational signature analysis showed the epigenome influencing DNA mutation accumulation. This study provides a map of (epi)genetic tumour heterogeneity, with fundamental implications for understanding colorectal cancer biology.

category: cancer biology


title: Amino acid availability is not essential for lifespan extension by dietary restriction in the fly

abstract: Dietary restriction (DR) is one of the most potent ways to extend health- and lifespan. Key progress in understanding the mechanisms of DR, and ageing more generally, was made when dietary protein, and more specifically essential amino acids (EAA), were identified as the key dietary component to restrict to obtain DRs health and lifespan benefits. This role of dietary amino acids has strongly influenced work on ageing mechanisms, especially in nutrient sensing, e.g. Tor and insulin(-like) signalling networks. Experimental biology in Drosophila melanogaster has been instrumental in generating and confirming the now dominant hypothesis that EAA availability is central to ageing. Here, we expand on previous work testing the involvement of EAA in DR through large scale (N=6,238) supplementation experiments across four diets and two genotypes in female flies. Surprisingly, we find that EAA are not essential to DRs lifespan benefits. Importantly, we do identify the fecundity benefits of EAA supplementation suggesting the supplemented EAA were bioavailable. Furthermore, we find that the effects of amino acids on lifespan vary by diet and genetic line studied and that at our most restricted diet fecundity is constrained by other nutrients than EAA. We suggest that DR for optimal health is a concert of nutritional effects, orchestrated by genetic, diet and environmental interactions. Our results question the universal importance of amino acid availability in the biology of ageing and DR.

category: physiology


title: Remodeling of organelles and microtubules during spermiogenesis in the liverwort Marchantia polymorpha

abstract:
Gametogenesis is an essential biological event for sexual reproduction in various organisms. Bryophytes and some other plants employ motile sperms (spermatozoids) as male gametes, which self-locomote to the egg cells to accomplish fertilization. Spermatozoids of bryophytes harbor distinctive morphological characteristics, including the cell body with a helical slender shape and two motile flagella at the anterior edge. During transformation from a spermatid to spermatozoid (spermiogenesis), the shape and cellular contents of spermatids are dynamically reorganized. However, how each organelle is reorganized during plant spermiogenesis remains obscure. In this study, we classified the developmental processes during spermiogenesis in the liverwort Marchantia polymorpha according to cellular and nuclear shapes and flagella development. We then examined the remodeling of microtubules and reorganization of endomembrane organelles during spermiogenesis. The results indicate that the state of post-translational modification of tubulin is dynamically changed during the formation of the flagella and spline, and the plasma membrane and endomembrane organelles are drastically reorganized in a precisely regulated manner during spermiogenesis. These findings are expected to provide useful indexes to classify developmental and subcellular processes of spermiogenesis in bryophytes.

Summary statementWe classified developmental processes of spermatozoids into 1 + 5 stages and characterized remodeling of organelles and microtubules during spermiogenesis in the liverwort Marchantia polymorpha.

category: plant biology


title: Non-invasive imaging of tau-targeted probe uptake by whole brain multi-spectral optoacoustic tomography

abstract:
AimAbnormal tau accumulation plays an important role in tauopathy diseases such as Alzheimers disease and Frontotemporal dementia. There is a need for high-resolution imaging of tau deposits at the whole brain scale in animal models. Here, we demonstrate non-invasive whole brain imaging of tau-targeted PBB5 probe in P301L model of 4-repeat tau at 130 m resolution using volumetric multi-spectral optoacoustic tomography (vMSOT).

MethodsThe binding properties of PBB5 to 4-repeat K18 tau and A{beta}42 fibrils were assessed by using Thioflavin T assay and surface plasmon resonance assay. We identified the probe PBB5 suitable for vMSOT tau imaging. The imaging performance was first evaluated using postmortem human brain tissues from patients with Alzheimers disease, corticobasal degeneration and progressive supranuclear palsy. Concurrent vMSOT and epi-fluorescence imaging of in vivo PBB5 targeting (i.v.) was performed in P301L and wild-type mice. Ex vivo measurements on excised brains along with multiphoton microscopy and immunofluorescence staining of tissue sections were performed for validation. The spectrally-unmixed vMSOT data was registered with MRI atlas for volume-of-interest analysis.

ResultsPBB5 showed specific binding to recombinant K18 tau fibrils, Alzheimers disease brain tissue homogenate by competitive binding against [11C]PBB3 and to tau deposits (AT-8 positive) in post-mortem corticobasal degeneration and progressive supranuclear palsy brain. i.v. administration of PBB5 in P301L mice led to retention of the probe in tau-laden cortex and hippocampus in contrast to wild-type animals, as also confirmed by ex vivo vMSOT, epi-fluorescence and multiphoton microscopy results.

ConclusionvMSOT with PBB5 facilitates novel 3D whole brain imaging of tau in P301L animal model with high-resolution for future mechanistic studies and monitoring of putative treatments targeting tau.

category: neuroscience


title: A dynamic pink noise paradigm to assess spatial-temporal attention without biasing perceptual processing

abstract: Psychophysical paradigms measure visual attention via localized test items to which observers must react or whose features have to be discriminated. These items, however, potentially interfere with the intended measurement as they bias observers spatial and temporal attention to their location and presentation time. Furthermore, visual sensitivity for conventional test items naturally decreases with retinal eccentricity, which prevents direct comparison of central and peripheral attention assessments. We developed a stimulus that overcomes these limitations. A brief oriented discrimination signal is seamlessly embedded into a continuously changing 1/f noise field, such that observers cannot anticipate potential test locations or times. Using our new paradigm, we demonstrate that local orientation discrimination accuracy for 1/f filtered signals is largely independent of retinal eccentricity. Moreover, we show that items present in the visual field indeed shape the distribution of visual attention, suggesting that classical studies investigating the spatiotemporal dynamics of visual attention via localized test items may have obtained a biased measure. We recommend our paradigm as an efficient method to evaluate the spatial and temporal spread of visual attention.


title: Interhemispheric functional connectivity in the primary motor cortex distinguishes between training on a physical and a virtual surgical simulator

abstract:
Functional brain connectivity using functional near-infrared spectroscopy (fNIRS) during a pattern cutting (PC) task was investigated in physical and virtual simulators.

14 right-handed novice medical students were recruited and divided into separate cohorts for physical (N=8) and virtual (N=6) PC training. Functional brain connectivity measured were based on wavelet coherence (WCOH) from task-related oxygenated hemoglobin (HBO2) changes from baseline at left and right prefrontal cortex (LPFC, RPFC), left and right primary motor cortex (LPMC, RPMC), and supplementary motor area (SMA). HBO2 changes within the neurovascular frequency band (0.01-0.07Hz) from long-separation channels were used to compute average inter-regional WCOH metrics during the PC task. The coefficient of variation (CoV) of WCOH metrics and PC performance metrics were compared. WCOH metrics from short-separation fNIRS time-series were separately compared.

Partial eta squared effect size (Bonferroni correction) between the physical versus virtual simulator cohorts was found to be highest for LPMC-RPMC connectivity. Also, the percent change in magnitude-squared WCOH metric was statistically (p<0.05) different for LPMC-RPMC connectivity between the physical and the virtual simulator cohorts. Percent change in WCOH metrics from extracerebral sources was not different at the 5% significance level. Also, higher CoV for both LPMC-RPMC magnitude-squared WCOH metric and PC performance metrics were found in physical than a virtual simulator.

We conclude that interhemispheric connectivity of the primary motor cortex is the distinguishing functional brain connectivity feature between the physical versus the virtual simulator cohorts. Brain-behavior relationship based on CoV between the LPMC-RPMC magnitude-squared WCOH metric and the FLS PC performance metric provided novel insights into the neuroergonomics of the physical and virtual simulators that is crucial for validating Virtual Reality technology.


title: A connectome-based neuromarker of the non-verbal number acuity and arithmetic skills

abstract: The approximate number system (ANS) is vital for survival and reproduction in animals and crucial in constructing abstract mathematical abilities in humans. Most previous neuroimaging studies focused on identifying discrete brain regions responsible for the ANS and characterizing their functions in numerosity perception. However, there lacks a neuromarker to characterize an individuals ANS acuity, especially one based on the whole-brain functional connectivity (FC). Here, we identified a distributed brain network (i.e., numerosity network) using a connectome-based predictive modeling (CPM) analysis on the resting-state functional magnetic resonance imaging (rs-fMRI) data based on a large sample size. The summed strength of all FCs within the numerosity network could reliably predict individual differences of the ANS acuity in behavior. Furthermore, in an independent dataset from the Human Connectome Project (HCP), we found that the summed FC strength within the numerosity network could also predict individual differences in arithmetic skills. Our findings illustrate that the numerosity network we identified could be an applicable neuromarker of the non-verbal number acuity and might serve as the neural basis underlying the known link between the non-verbal number acuity and mathematical abilities.


title: Consistent differences among individuals more influential than energetic trade-offs in life history variation in grey seals (Halichoerus grypus)

abstract: Life history variation is thought to be mainly a result of energetic trade-offs among fitness components; however, detecting these trade-offs in natural populations has yielded mixed results. Individual quality and environmental variation may mask expected relationships among fitness components because some higher quality individuals may be able to acquire more resources and invest more in all functions. Thus, life history variation may be more affected by variation in individual quality than varying strategies to resolve energetic trade-offs, e.g. costs of reproduction. Here, we investigated whether variation in female quality or costs of reproduction is a larger factor in shaping differences in life history trajectories by assessing the relationship between survival and individual reproductive performance using a 32-year longitudinal data set of repeated reproductive measurements from 273 individually marked, known-aged female grey seals (Halichoerus grypus) from the Sable Island breeding colony. We defined individual reproductive performance using two traits: reproductive frequency (a females probability of breeding) and provisioning performance (provisions given to young measured by offspring mass), computed using mixed effects models separately for (1) all reproductive events, and (2) an age-class specific reproductive investment. Individual differences contributed a large portion of the variance in reproductive traits, with individuals displaying a range in individual reproductive frequencies from 0.45 to 0.94, and a range of average pup weaning masses from 34.9 kg to 61.8 kg across their lifetime. We used a Cormack-Jolly-Seber open-population model to estimate the effect of these reproductive performance traits on adult survival probability. Our approach estimated a positive relationship between reproductive performance and survival, where individuals that consistently invest well in their offspring survive longer. The best supported model estimated survival as a function of age-class specific provisioning performance, where late-life performance was quite variable and had the greatest impact on survival, possibly indicating individual variation in senescence. There was no evidence to support a trade-off in reproductive performance and survival at the individual level. These results suggest that in grey seals, individual quality is a stronger driver in life history variation than varying strategies to mitigate trade-offs among fitness components.

category: ecology


title: Reproductive success is influenced by early development: the covariation of natal body size and reproductive performance in grey seals (Halichoerus grypus)

abstract: An individuals size when young may be an important source individual variation in lifetime reproductive performance, as size effects on ontogenetic development can have cascading physiological and behavioral consequences throughout life. Here, we explored how natal size influences subsequent reproductive performance in grey seals (Halichoerus grypus) using mark-recapture and repeated measures reproductive data on a marked sample of 363 females that were measured for standard length at roughly 4 weeks of age and eventually recruited to the Sable Island breeding colony. Two reproductive traits were considered: provisioning performance (mass of weaned offspring), modeled using linear mixed effects models; and reproductive frequency (the rate at which a female returns to breed), modeled using mixed-effects multistate mark-recapture models. After accounting for female age, experience, and offspring sex, we found a positive association between natal length and our measures of reproductive performance. Mothers with the longest natal lengths produced pups nearly 8 kg heavier and were 20% more likely to breed in a given year than mothers with the shortest natal lengths. Because correlation in individual body length between natal and adult life stages is weak (animals that were longer than average as pups do not always grow to be longer than average adults), the covariation between natal length and future reproductive performance reported here may act more as a carry-over effect from the size advantages afforded in the juvenile stage that allow for greater adult performance, rather than a physical trait that is maintained throughout life.


title: A multiple hypotheses framework to study movement ecology and diet in a migratory songbird

abstract: Whether and how migratory organisms exhibit inter-individual behavioral and/or physiological variation across movement strategies remains an open question. The Dark-eyed Junco (Junco hyemalis) is a migratory songbird known for its intra-species variation displayed in relation to morphology, song repertoires, and migration. Thus, studies focusing on juncos can reveal how migratory strategy may covary with other individual-scale factors and, therefore, identify the selective forces driving intra-species variations throughout its distribution. We used Dark-eyed Junco hydrogen stable isotope feather values ({delta}2H) and implemented a Bayesian framework to infer the breeding and molting origin of migratory juncos captured on their winter grounds in Oklahoma, United States (U.S.). We modeled the distribution of feather hydrogen stable isotope values as a function of five morphological variables including body mass and fat deposition measured in Oklahoma during the winter. We then investigated the trade-off between longer and more energetically costly migration strategies in relation to diet preferences through carbon ({delta}13C) and nitrogen ({delta}15N) stable isotope analysis from feather values. Dark-eyed Juncos wintering in south central U.S. likely originate from multiple breeding populations in northern U.S. and Canada. Body condition at the wintering ground (e.g., mass) had no effect on feather hydrogen stable isotope abundance. However, we found a positive correlation between nitrogen and hydrogen stable isotopes, suggesting that a trophic level shift towards insect consumption might occur in individuals migrating from southern latitudes. Increased insect-derived protein consumption might be explained by reduced fatty-acid reserves necessary to complete a shorter migratory journey.


title: The functional form of specialized predation dramatically affects whether Janzen-Connell effects can prevent competitive exclusion

abstract: Janzen Connell Effects (JCEs), specialized predation of seeds and seedlings near conspecific trees, are hypothesized to promote high species richness. While past modeling studies show JCEs can maintain higher diversity than a neutral community, recent theoretical work indicates JCEs may weakly inhibit competitive exclusion when species exhibit interspecific fitness variation. However, recent models make somewhat restrictive assumptions about the functional form of specialized predation - that JCEs occur at a fixed rate when seeds/seedlings are within a fixed distance of a conspecific tree. Using a theoretical model, I show that the functional form of JCEs largely impacts their ability to promote coexistence. If specialized predation pressure increases additively with adult tree density and decays exponentially with distance, JCEs maintain considerably higher diversity than predicted by recent models. Parameterizing the model with values from a Panamanian tree community indicates JCEs can maintain high diversity in communities exhibiting high interspecific fitness variation.


title: A recombinant protein containing influenza viral conserved epitopes and superantigen induces broad-spectrum protection

abstract: Influenza pandemic poses public health threats annually for lacking vaccine which provides cross-protection against novel and emerging influenza viruses. Combining conserved antigens inducing cross-protective antibody response with epitopes activating cross-protective cytotoxic T-cells would offer an attractive strategy for developing universal vaccine. In this study, we constructed a recombinant protein NMHC consisting of influenza viral conserved epitopes and superantigen fragment. NMHC promoted the mature of bone marrow-derived dendritic cells and induced CD4+ T cells to differentiate into Th1, Th2 and Th17 subtypes. Mice vaccinated with NMHC produced high level of immunoglobulins which cross-bound to HA fragments from six influenza virus subtypes with high antibody titers. Anti-NMHC serum showed potent hemagglutinin inhibition effects to highly divergent group 1 (H1 subtypes) and group 2 (H3 subtype) influenza virus strains. And purified anti-NMHC antibodies could bind to multiple HAs with high affinities. NMHC vaccination effectively protected the mice from infection and lung damage challenged by two subtypes of H1N1 influenza virus. Moreover, NMHC vaccination elicited CD4+ and CD8+ T-cell responses to clear the virus from infected tissue and prevent virus spreading. In conclusion, this study provided proof of concept for triggering both B cells and T cells immune responses against multiple influenza virus infection, and NMHC may be a potential candidate of universal broad-spectrum vaccine for various influenza virus prevention and therapy.

category: epidemiology


title: Suppressed eusocial reproduction supports evolutionary convergence over co-option

abstract: Eusocial insects are characterized by the presence of division of labour between reproductive (i.e. workers) and non-reproductive (i.e. queens) individuals. In eusocial insects, such as wasps, ants, honeybees and termites, such reproductive division of labour is mediated by the use of unique pheromones. In eusocial animals, pheromones control two distinct aspects of the division of labour: reproduction and division of tasks amongst workers. It has been suggested that eusociality arose independently on several occasions from pheromone-mediated co-option of pathways regulating the reproductive life cycle of solitary insects. Transcriptomic analysis has supported the co-option of similar pathways in each independently evolved eusocial insect. Using a solitary insect model, we sought to determine if these similar transcriptomic signals resulted from the co-option of similar pathways or varying pathways subject to convergent evolution. We measured the transcriptomic and physiological response of Drosophila melanogaster to pheromones from bumblebees, honey bees, and termites. In each case we observed a strong physiological response - the reduction of ovary size. However, employing conventional differential gene expression analysis and several forms of network analysis, we were unable to detect any conserved pathway or genes acting as a primer for eusociality. This strongly suggests that pheromone-mediated eusociality is the result of convergent evolution. Wherein a physiological response, such as reduced reproductive capacity, acts as the primer for eusociality and is subsequently refined.

category: evolutionary biology


title: Evolution with private resources reverses some changes from long-term evolution with public resources

abstract: A population under selection to improve one trait may evolve a sub-optimal state for another trait due to tradeoffs and other evolutionary constraints. How this evolution affects the capacity of a population to adapt when conditions change to favor the second trait is an open question. We investigated this question using isolates from a lineage spanning 60,000 generations of the Long-Term Evolution Experiment (LTEE) with Escherichia coli, where cells have access to a shared pool of resources, and have evolved increased competitive ability and a concomitant reduction in numerical yield. Using media-in oil emulsions we shifted the focus of selection to numerical yield, where cells grew in isolated patches with private resources. We found that the time spent evolving under shared resources did not affect the ability to re-evolve toward higher numerical yield. The evolution of numerical yield commonly occurred through mutations in the phosphoenolpyruvate phosphotransferase system. These mutants exhibit slower uptake of glucose, making them poorer competitors for public resources, and produce smaller cells that release less carbon as overflow metabolites. Our results demonstrate that mutations that were not part of adaptation under one selective regime may enable access to ancestral phenotypes when selection changes to favor evolutionary reversion.


title: Antigenic evolution of human influenza H3N2 neuraminidase is constrained by charge balancing

abstract: As one of the main influenza antigens, neuraminidase (NA) in H3N2 virus has evolved extensively for more than 50 years due to continuous immune pressure. While NA has emerged as an effective vaccine target recently, biophysical constraints on the antigenic evolution of NA remain largely elusive. Here, we apply deep mutational scanning to characterize the local fitness landscape in an antigenic region of NA in six different human H3N2 strains that were isolated around 10 years apart. The local fitness landscape correlates well among strains and the pairwise epistasis is highly conserved. Our analysis further demonstrates that local net charge governs the pairwise epistasis in this antigenic region. In addition, we show that residue coevolution in this antigenic region can be predicted by charge states and pairwise epistasis. Overall, this study demonstrates the importance of quantifying epistasis and the underlying biophysical constraint for building a predictive model of influenza evolution.


title: Independent adoptions of a set of proteins found in the matrix of the mineralized shell-like eggcase of Argonaut octopuses

abstract: The Argonaut octopus, commonly called the paper nautilus, has a spiral-coiled shell-like eggcase. As the main characteristics, the eggcase has no internal septum, is composed entirely of calcite with chitosan being the main polycarbonate and is reportedly formed by organic materials secreted from the membranes of the arms. Meanwhile, the biomineralized external "true" shells of the Mollusks, which includes the Cephalopods, are secreted from the mantle tissue. Therefore, the histological origin of the two shells is completely different. The question of how the Argonauts, which phylogenetically diverged from the completely shell-less octopuses, could form a converging shell-like external structure has thus intrigued biologists for a long time. To answer this question, we performed a multi-omics analysis of the transcriptome and proteome of the two congeneric Argonaut species, Argonauta argo and A. hians. Our result indicates that the shell-like eggcase is not a homolog of the shell, even at the protein level, because the Argonauts apparently recruited a different set of protein repertoires to as eggcase matrix proteins (EcMPs). However, we also found the homologs of three shell matrix proteins (SMPs) of the Conchiferan Mollusks, Pif-like, SOD, and TRX, in the eggcase matrix. The proteins were also found in the only surviving shelled Cephalopods, the nautiloid Nautilus pompilius. Phylogenetic analysis revealed that homologous genes of the Conchiferan SMPs and EcMPs were found in the draft genome of shell-less octopuses. Our result reported here thus suggests that the SMP-coding genes are conserved in both shelled and shell-less Cephalopods. Meanwhile, the Argonauts adopted some of the SMP-coding genes and other non-SMP-coding genes, to form a convergent, non-homologous biomineralized external structure, the eggcase, which is autapomorphic to the group.


title: High-resolution estimates of crossover and noncrossover recombination from a captive baboon colony

abstract:
Homologous recombination has been extensively studied in humans and a handful of model organisms. Much less is known about recombination in other species, including non-human primates. Here we present a study of crossovers and non-crossover (NCO) recombination in olive baboons (Papio anubis) from two pedigrees containing a total of 20 paternal and 17 maternal meioses, and compare these results to linkage-disequlibrium (LD) based recombination estimates from 36 unrelated olive baboons. We demonstrate how crossovers, combined with LD-based recombination estimates, can be used to identify genome assembly errors. We also quantify sex-specific differences in recombination rates, including elevated male crossover and reduced female crossover rates near telomeres. Finally, we add to the increasing body of evidence suggesting that while most NCO recombination tracts in mammals are short (e.g., < 500 bp), there are a non-negligible fraction of longer (e.g., > 1 Kb) NCO tracts. We fit a mixture-of-two-geometric distributions model to the NCO tract length distribution and estimate that >99% of all NCO tracts are very short (mean 24 bp), but the remaining tracts can be quite long (mean 11 Kb). A single geometric distribution model for NCO tract lengths is incompatible with the data, suggesting that LD-based methods for estimating NCO recombination rates that make this assumption may need to be modified.

SignificanceMost homologous recombination events are noncrossovers (NCO), but little is known about NCO conversion tract lengths. Here we utilize whole-genome sequence data from large baboon pedigrees to estimate the NCO tract length distribution and to study other aspects of recombination.

category: genomics


title: Multiple shifts in gene network interactions shape phenotypes of Drosophila melanogaster selected for long and short night sleep duration

abstract: All but the simplest phenotypes are believed to result from interactions between two or more genes forming complex networks of gene regulation. Sleep is a complex trait known to depend on the system of feedback loops of the circadian clock, and on many other genes; however, the main components regulating the phenotype and how they interact remain an unsolved puzzle. Genomic and transcriptomic data may well provide part of the answer, but a full account requires a suitable quantitative framework. Here we conducted an artificial selection experiment for sleep duration with RNA-seq data acquired each generation. The phenotypic results are robust across replicates and previous experiments, and the transcription data provides a high-resolution, time-course data set for the evolution of sleep-related gene expression. In addition to a Hierarchical Generalized Linear Model analysis of differential expression that accounts for experimental replicates we develop a flexible Gaussian Process model that estimates interactions between genes. 145 gene pairs are found to have interactions that are different from controls. Our method not only is considerably more specific than standard correlation metrics but also more sensitive, finding correlations not significant by other methods. Statistical predictions were compared to experimental data from public databases on gene interactions.


title: Gluconobacter oxydans Knockout Collection Finds Improved Rare Earth Element Extraction

abstract: Rare earth elements (REE) are critical components of our technological society and essential for renewable energy technologies. Traditional thermochemical processes to extract REE from mineral ores or recycled materials are costly and environmentally harmful1, and thus more sustainable extraction methods require exploration. Bioleaching offers a promising alternative to conventional REE extraction2-4, and is already used to extract 5% of the worlds gold, and {approx} 15% of the worlds copper supply5,6. However, the performance of REE bioleaching lags far behind thermochemical processes2,7-9. Despite this, to the best of our knowledge no genetic engineering strategies have yet been used to enhance REE bioleaching, and little is known of the genetics that confer this capability. Here we build a whole genome knockout collection for Gluconobacter oxydans B58, one of the most promising organisms for REE bioleaching10, and use it to comprehensively characterize the genomics of REE bioleaching. In total, we find 304 genes that notably alter production of G. oxydans acidic biolixiviant, including 165 that hold up under statistical comparison with wild-type. The two most impactful groups of genes involved in REE bioleaching have opposing influences on acid production and REE bioleaching. Disruption of genes underlying synthesis of the cofactor pyrroloquinoline quinone (PQQ) and the PQQ-dependent membrane-bound glucose dehydrogenase all but eliminates bioleaching. In contrast, disruption of the phosphate-specific transport system accelerates acid production and enhances bioleaching. We identified 6 disruption mutants, that increase bioleaching by at least 11%. Most significantly, disruption of pstC, encoding part of the phosphate-specific transporter, pstSCAB, enhances bioleaching by 18%. Taken together, these results give a comprehensive roadmap for engineering multiple sites in the genome of G. oxydans to further increase its bioleaching efficiency.


title: A statistical framework for differential pseudotime analysis with multiple single-cell RNA-seq samples

abstract: Pseudotime analysis with single-cell RNA-sequencing (scRNA-seq) data has been widely used to study dynamic gene regulatory programs along continuous biological processes. While many computational methods have been developed to infer the pseudo-temporal trajectories of cells within a biological sample, methods that compare pseudo-temporal patterns with multiple samples (or replicates) across different experimental conditions are lacking. Lamian is a comprehensive and statistically-rigorous computational framework for differential multi-sample pseudotime analysis. It can be used to identify changes in a biological process associated with sample covariates, such as different biological conditions, and also to detect changes in gene expression, cell density, and topology of a pseudotemporal trajectory. Unlike existing methods that ignore sample variability, Lamian draws statistical inference after accounting for cross-sample variability and hence substantially reduces sample-specific false discoveries that are not generalizable to new samples. Using both simulations and real scRNA-seq data, including an analysis of differential immune response programs between COVID-19 patients with different disease severity levels, we demonstrate the advantages of Lamian in decoding cellular gene expression programs in continuous biological processes.


title: Inferring the demographic history of tetraploid species from genomic data

abstract: Genomic patterns of diversity and divergence are impacted by certain life history traits, reproductive systems and demographic history. The latter is characterised by fluctuations in population sizes over time, as well as by temporal patterns of introgression. For a given organism, identifying a demographic history that deviates from the standard neutral model allows a better understanding of its evolution, but also helps to reduce the risk of false positives when screening for molecular targets of natural selection. Tetraploid organisms and beyond have demographic histories that are complicated by the mode of polyploidisation, the mode of inheritance and different scenarios of gene flow between subgenomes and diploid parental species. Here we provide guidelines for experimenters wishing to address these issues through a flexible statistical framework: approximate Bayesian computation (ABC). The emphasis is on the general philosophy of the approach to encourage future users to exploit the enormous flexibility of ABC beyond the limitations imposed by generalist data analysis pipelines.


title: CADD-SV - a framework to score the effects of structural variants in health and disease

abstract:
While technological advances improved the identification of structural variants (SVs) in the human genome, their interpretation remains challenging. Several methods utilize individual mechanistic principles like the deletion of coding sequence or 3D genome architecture disruptions. However, a comprehensive tool using the broad spectrum of available annotations is missing. Here, we describe CADD-SV, a method to retrieve and integrate a wide set of annotations to predict the effects of SVs.

Previously, supervised learning approaches were limited due to a small number and biased set of annotated pathogenic or benign SVs. We overcome this problem by using a surrogate training-objective, the Combined Annotation Dependent Depletion (CADD) of functional variants. We use human and chimpanzee derived SVs as proxy-neutral and contrast them with matched simulated variants as proxy-pathogenic, an approach that has proven powerful for SNVs.

Our tool computes summary statistics over diverse variant annotations and uses random forest models to prioritize deleterious structural variants. The resulting CADD-SV scores correlate with known pathogenic and rare population variants. We further show that we can prioritize somatic cancer variants as well as non-coding variants known to affect gene expression. We provide a website and offline-scoring tool for easy application of CADD-SV (https://cadd-sv.bihealth.org/).

category: bioinformatics


title: Enhancing Precision in Bioprinting Utilizing Fuzzy Systems

abstract: Bioprinting facilitates the generation of complex, three-dimensional (3D), cell-based constructs for a variety of applications. Although multiple bioprinting technologies have been developed, extrusion-based systems have become the dominant technology due to the diversity of substrate materials (bioinks) that can be accommodated, either individually or in combination. However, each bioink has unique material properties and extrusion characteristics that limit bioprinting precision, particularly when generating constructs from different bioinks. Here, we aimed to achieve high precision (i.e. repeatability) across samples by generating bioink-specific printing parameters using a systematic approach. We hypothesized that a fuzzy system could be used as a "black box" method to tackle the inherent vagueness and imprecision in 3D bioprinting data and uncover the optimal printing parameters for a specific bioink that would result in high accuracy and precision. Our fuzzy model was used to approximate and quantify the precision and ease of printability for two common bioinks - type I collagen and Pluronic F127, with or without dilution in MEM culture media. The model consisted of three inputs (pressure, speed, and dilution percent of bioink) and a single output (layer width). Using this system, we introduce the Bioink Precision Index (BPI), a metric that can be used to quantify and compare the precision of any bioink. Here, we show that printing with parameters optimized using BPI increases the precision for collagen (+15%) and Pluronic F127 (+29%) as compared to the manufacturers recommended printing parameters.


title: Power Calculator for Detecting Allelic Imbalance Using Hierarchical Bayesian Model

abstract: Allelic imbalance (AI) is the differential expression of the two alleles in a diploid. AI can vary between tissues, treatments, and environments. Statistical methods for testing in this area exist, with impacts of explosive type I error in the presence of bias well understood. However, for study design, the more important and understudied problem is the type II error and power. As the biological questions for this type of study explode, and the costs of the technology plummet, what is more important: reads or replicates? How small of an interaction can be detected while keeping the type I error at bay? Here we present a simulation study that demonstrates that the proper model can control type I error below 5% for most scenarios. We find that a minimum of 2400, 480, and 240 allele specific reads divided equally among 12, 5, and 3 replicates is needed to detect a 10%, 20%, and 30%, respectively, deviation from allelic balance in a condition with power >80%. A minimum of 960 and 240 allele specific reads is needed to detect a 20% or 30% difference in AI between conditions with comparable power but these reads need to be divided amongst 8 replicates. Higher numbers of replicates increase power more than adding coverage without affecting type I error. We provide a Python package that enables simulation of AI scenarios and enables individuals to estimate type I error and power in detecting AI and differences in AI between conditions tailored to their own specific study needs.


title: Evolution of a confluent gut epithelium under cyclic stretching

abstract: The progress of food in the gastrointestinal (GI) tract is driven by a peristaltic motion generated by the muscle belt surrounding the GI tract. In turn, the response of the intestinal epithelial cells to the peristaltic stresses affects the dynamics of the epithelial structure. In this work, we study the effect of cyclic stretching (0.125 Hz, 10% strain) on the spatial organization of the intestinal epithelium using intestinal cells deposited on a flat elastomeric substrate to mimic the peristaltic motion in vitro. A confluent monolayer of Caco-2 cells is grown on a PDMS chip to probe the morphological and orientational response of the tissue to cyclic stretching. The PDMS chips are either covalently or non-covalently coated with laminin to recapitulate the basement membrane. We observe a significant orientational response where the cells rearrange their long axes perpendicular to the stretching direction for both coating conditions. The experiment is modeled by a vertex model where the cells store elastic energy with varying strain and effectively have a rotational diffusive motion through rearrangements of their shapes. The model also predicts a transition between the perpendicular orientation and orientation at an oblique angle determined by the level of the cell elastic anisotropy. It provides a general framework to study cell response and relaxation dynamics under cyclic stretching across different cell types. We also discuss potential relevance of peristalsis in determining planar cell polarity in 3D architectures.


title: Odorization of Natural Gas: What are the Challenges?

abstract: Modern natural gas (NG) has little or no odor, so other compounds, usually mercaptans and thiols, are added as warning odorants. Federal regulations state that NG must be odorized so that it is readily detectable by people with normal senses of smell at one fifth the lower explosive limit, but regulations dont define "readily detectable" or "normal senses of smell." Methods to measure human odor detection have been available for decades. However, most previous work on NG odorants has underestimated human sensitivity, and measurements need to be repeated using the latest methods. More work is also needed to determine how odor sensitivity measured under optimal laboratory conditions is affected by real-world factors such as distraction and exposure to other odors in the environment. Regarding a "normal sense of smell," healthy people vary over orders of magnitude in the concentrations they can detect, so samples of subjects should be chosen to reflect the range of differences in the population.


title: A novel non-antibiotic selectable marker GASA6 for plant transformation

abstract: Selectable markers help the transformed cell/tissue to survive in an otherwise lethal exposure of an antibiotic or herbicide. Unfortunately, almost all the traditional selectable markers are antibiotic and herbicide resistance genes, which are controversial on human health concerns and environmental impact. Novel plant-derived, non-antibiotic, and non-herbicide selectable markers are urgently needed in plant transformation. Our previous work showed that the seedlings of overexpression Arabidopsis lines of AtGASA6 survived on medium with a high concentration of sugar, which leads to the hypothesis that AtGASA6 could be a selectable marker on media with high or low sugar content. In this study, leaf explants of AtGASA6 overexpression tobacco lines regenerated shoots on sugar-free shooting medium while those of wild type could not. Moreover, the seeds of AtGASA6 overexpression tobacco lines germinated and grew into normal seedlings on sugar-free MS medium while those of WT could not. Attractively, no developmental defects were observed in AtGASA6 transgenic progenies. Using AtGASA6 as a selectable marker, overexpression tobacco lines of GAI, which restrains plant size, were created on sugar-free media. The GAI overexpression lines had a smaller plant size than that of control. Considering its plant-derived and non-antibiotic nature, GASA6 is promising to be used as a selectable marker in plant transformation.


title: Membrane activity of a DNA-based ion channel depends on the stability of its double-stranded structure.

abstract: Structural DNA nanotechnology has emerged as a promising method for designing spontaneously-inserting and fully-controllable synthetic ion channels. However, both insertion efficiency and stability of existing DNA-based ion channels leave much room for improvement. Here, we demonstrate an approach to overcoming the unfavorable DNA-lipid interactions that hinder the formation of a stable transmembrane pore. Our all-atom MD simulations and experiments show that the insertion-driving cholesterol modifications, when introduced at an end of a DNA strand, are likely to cause fraying of the terminal base pairs as the DNA nanostructure adopts its energy-minimum configuration in the membrane. We also find that fraying of base pairs distorts nicked DNA constructs when embedded in a lipid bilayer. Here, we show that DNA nanostructures that do not have discontinuities (nicks) in their DNA backbones form considerably more stable DNA-induced conductive pores and insert into lipid membranes with a higher efficiency than the equivalent nicked constructs. Moreover, lack of nicks allows to design and maintain membrane-spanning helices in a tilted orientation within lipid bilayer. Thus, reducing the conformational degrees of freedom of the DNA nanostructures enables better control over their function as synthetic ion channels.

category: synthetic biology


title: The Automated Preprocessing Pipe-Line for the Estimation of Scale-wise Entropy from EEG Data (APPLESEED): Development and validation for use in pediatric populations

abstract: It is increasingly understood that moment-to-moment brain signal variability - traditionally modeled out of analyses as mere "noise" - serves a valuable function role and captures properties of brain function related to development, cognitive processing, and psychopathology. Multiscale entropy (MSE) - a measure of signal irregularity across temporal scales - is an increasingly popular analytic technique in human neuroscience. MSE provides insight into the time-structure and (non)linearity of fluctuations in neural activity and network dynamics, capturing the brains moment-to-moment complexity as it operates on multiple time scales. MSE is emerging as a powerful predictor of developmental processes and outcomes. However, differences in data preprocessing and MSE computation make it challenging to compare results across studies. Here, we (1) provide an introduction to MSE for developmental researchers, (2) demonstrate the effect of preprocessing procedures on scale-wise entropy estimates, and (3) establish a standardized EEG preprocessing and entropy estimation pipeline that generates scale-wise entropy estimates that are reliable and capable of differentiating developmental stages and cognitive states. This novel pipeline - the Automated Preprocessing Pipe-Line for the Estimation of Scale-wise Entropy from EEG Data (APPLESEED) is fully automated, customizable, and freely available for download from https://github.com/mhpuglia/APPLESEED. The dataset used herein to develop and validate the pipeline is available for download from https://openneuro.org/datasets/ds003710.


title: Dynamics of glioma-associated microglia and macrophages reveals their divergent roles in the immune response of brain

abstract: Glioma microenvironment contains numerous myeloid cells, including brain-resident microglia and recruited monocytes and macrophages (Mo/M{varphi}). When studied collectively, these cells presented pro-tumor effects. Yet, little is known about the differences among these myeloid populations. Using single-cell sequencing analysis, we studied the phenotypic characteristics, spatial variances, and dynamic changes of these relatively heterogeneous cell populations. Microglia populations with distinct spatial distribution presented different functional states, including tumor-associated subsets with phagocytic and lipid metabolism signature. Notably, this subset of glioma-associated microglia shared similar trait in a diverse spectrum of neuropathogenesis. In contrast, Mo/M{varphi} highly expressed genes related to angiogenesis, tumor invasion, and immune evasion. Moreover, identifying the Mo/M{varphi} subsets had prognostic and classificatory value in clinical application. These results thus eliminate the long-existing ambiguity about the role of microglia and Mo/M{varphi} in glioma pathogenesis, and reveal their prognostic and therapeutic value for glioma patients.

category: immunology


title: Regulation of positive and negative selection and TCR signaling during thymic T cell development by capicua

abstract: Central tolerance is achieved through positive and negative selection of thymocytes mediated by T cell receptor (TCR) signaling strength. Thus, the dysregulation of the thymic selection process often leads to autoimmunity. Here, we show that capicua (CIC), a transcriptional repressor that suppresses autoimmunity, controls the thymic selection process. Loss of CIC prior to T-cell lineage commitment impaired both positive and negative selection of thymocytes. CIC deficiency attenuated TCR signaling in CD4+CD8+ double-positive (DP) cells, as evidenced by a decrease in CD5 and phospho-ERK levels and calcium flux. We identified Spry4, Dusp4, Dusp6, and Spred1 as CIC target genes that could inhibit TCR signaling in DP cells. Furthermore, impaired positive selection and TCR signaling were partially rescued in Cic and Spry4 double mutant mice. Our findings indicate that CIC is a transcription factor required for thymic T cell development and suggest that CIC acts at multiple stages of T cell development and differentiation to prevent autoimmunity.


title: Ecology and transmission of a dengue virus serotype 4 identified in wild Aedes aegypti in Florida

abstract:
1Dengue virus is the most prevalent mosquito-borne virus, causing approximately 390 million infections and 25,000 deaths per year. Aedes aegypti, the primary mosquito vector of dengue virus, is well established throughout the state of Florida, USA. Autochthonous transmission of dengue virus to humans in Florida has been increasing since 2009, alongside consistent importation of dengue cases. However, most cases of first infection with dengue are asymptomatic and the virus can be maintained in mosquito populations, complicating surveillance and leading to an underestimation of disease risk. Metagenomic sequencing of Aedes aegypti mosquitoes in Manatee County, Florida revealed the presence of dengue virus serotype 4 (DENV-4) genomes in mosquitoes from multiple trapping sites over 2 years, in the absence of a human DENV-4 index case and even though a locally acquired case of DENV-4 has never been reported in Florida. This finding suggested that: i) DENV-4 may circulate amongst humans undetected, ii) the virus was being maintained in the mosquito population, or iii) the detected complete genome sequence may not represent a viable virus. This study demonstrates that an infectious clone generated from the Manatee County DENV-4 (DENV-4M) sequence is capable of infecting mammalian and insect tissue culture systems, as well as adult female Aedes aegypti mosquitoes when fed in a blood meal. However, the virus is subject to a dose dependent infection barrier in mosquitoes, and has a kinetic delay compared to a phylogenetically related wild-type (WT) control virus from a symptomatic child, DENV-4H (strain Homo sapiens/Haiti-0075/2015, GenBank accession MK514144.1). DENV-4M disseminates from the midgut to the ovary and saliva at 14 days post-infection. Viral RNA was also detectable in the adult female offspring of DENV-4M infected mosquitoes. These results demonstrate that the virus is capable of infecting vector mosquitoes, is transmissible by bite, and is vertically transmitted, indicating a mechanism for maintenance in the environment without human-mosquito transmission. These findings suggest undetected human-mosquito transmission and/or long-term maintenance of the virus in the mosquito population is occurring in Florida, and underscore the importance of proactive surveillance for viruses in mosquitoes.

Graphical AbstractIn order to better assess the public health risk posed by a detection of DENV-4 RNA in Manatee County, FL Aedes aegypti, we produced an infectious clone using the sequence from the wild-caught mosquitoes and characterized it via laboratory infections of mosquitoes and mosquito tissues.

category: microbiology


title: Circuit motifs and graph properties of connectome development in C. elegans

abstract: Network science is a powerful tool that can be used to better explore the complex structure of brain networks. Leveraging graph and motif analysis tools, we interrogate C. elegans connectomes across multiple developmental time points and compare the resulting graph characteristics and substructures over time. We show the evolution of the networks and highlight stable invariants and patterns as well as those that grow or decay unexpectedly, providing a substrate for additional analysis.


title: Active components of commonly prescribed medicines affect influenza A virus-host cell interaction: a pilot study

abstract:
BackgroundEvery year, millions of people are hospitalized, and thousands die from influenza A virus (FLUAV) infection. Most cases of hospitalizations and death occur among elderly. Many of these elderly patients are reliant on medical treatment of underlying chronic diseases, such as arthritis, diabetes, and hypertension. We hypothesized that the commonly prescribed medicines for treatment of underlying chronic diseases can affect host responses to FLUAV infection, and thus contribute to morbidity and mortality associated with influenza. Therefore, the aim of this study was to examine whether commonly prescribed medicines could affect host responses to virus infection in vitro.

MethodsWe first identified 45 active compounds of commonly prescribed medicines. Then we constructed a drug-target interaction network and identified potential implication of these interactions for FLUAV-host cell interplay. Finally, we tested the effect of 45 drugs on viability, transcription and metabolism of mock- and FLUAV-infected human retinal pigment epithelial (RPE) cells.

ResultsIn silico drug-target interaction analysis revealed that many drugs, such as atorvastatin, candesartan, and hydroxocobalam, could target and modulate FLUAV-host cell interaction. In vitro experiments showed that these and other compounds at non-cytotoxic concentrations differently affected transcription and metabolism of mock- and FLUAV-infected cells.

ConclusionMany commonly prescribed drugs modulate FLUAV-host cell interactions in silico and in vitro and, therefore, could affect their interplay in vivo, thus, contributing to morbidity and mortality of patients with influenza virus infections.

category: pharmacology and toxicology


title: Spatio-Temporal Recruitment Of Adult Neural Stem Cells For Transient Neurogenesis During Pregnancy

abstract: Neural stem cells (NSCs) in the adult mouse brain contribute to lifelong brain plasticity. NSCs in the adult ventricular-subventricular zone (V-SVZ) are heterogeneous and, depending on their location in the niche, give rise to different subtypes of olfactory bulb interneurons. Here, we show that during pregnancy multiple regionally-distinct NSCs are dynamically recruited at different times. Coordinated temporal activation of these NSC pools generates sequential waves of short-lived olfactory bulb interneuron subtypes that mature in the mother around birth and in the perinatal care period. Concomitant with neuronal addition, oligodendrocyte progenitors also transiently increase in the olfactory bulb. Thus, life experiences, such as pregnancy, can trigger transient neurogenesis and gliogenesis under tight spatial and temporal control, and may provide a novel substrate for brain plasticity in anticipation of temporary physiological demand.


title: Chemically Induced Senescence in Human Stem Cell-Derived Neurons Promotes Phenotypic Presentation of Neurodegeneration

abstract: Modeling age-related neurodegenerative disorders with human stem cells is difficult due to the embryonic nature of stem cell derived neurons. We developed a chemical cocktail to induce senescence of iPSC-derived neurons to address this challenge. We first screened small molecules that induce embryonic fibroblasts to exhibit features characteristic of aged fibroblasts. We then optimized a cocktail of small molecules that induced senescence in fibroblasts and cortical neurons without causing DNA damage. The utility of the "senescence cocktail" was validated in motor neurons derived from ALS patient iPSCs which exhibited protein aggregation and axonal degeneration substantially earlier than those without cocktail treatment. Our "senescence cocktail" will likely enhance the manifestation of disease-related phenotypes in neurons derived from iPSCs, enabling the generation of reliable drug discovery platforms.


title: Effects of Frontoparietal Theta tACS on Verbal Working Memory: Behavioral and Neurophysiological Analysis

abstract: Left-lateralized frontoparietal theta oscillations are thought to play an important role in verbal working memory. We causally tested this idea by stimulating the frontoparietal theta network at individual theta frequencies (4 to 8 Hz) during verbal working memory and observing the subsequent behavioral and neurophysiological effects. Weak electric currents were delivered via two 4x1 HD electrode arrays centered at F3 and P3. Three stimulation configurations, including in-phase, anti-phase, or sham, were tested on three different days in a cross-over design. On each test day, the subject underwent three experimental sessions: pre-, during- and post-stimulation sessions. In all sessions, the subject performed a Sternberg verbal working memory task with three levels of memory load (load 2, 4 and 6), imposing three levels of cognitive demand. Analyzing behavioral, EEG, and pupillometry data from the post-stimulation sessions, we report three results. First, in-phase stimulation improved task performance only in subjects with higher working memory capacity (WMC) and under higher memory load (load 6). Second, in-phase stimulation enhanced frontoparietal theta synchrony during working memory retention only in subjects with higher WMC under higher memory loads (load 4 and load 6), and the enhanced frontoparietal theta synchronization is mainly driven by enhanced frontal[->]parietal theta Granger causality. Third, the pupil diameter was not different irrespective of whether the preceding stimulation was in-phase, anti-phase, or sham. These findings suggest that theta tACS effects on verbal working memory were load- and subject-dependent, rooted in tACS-induced changes in frontoparietal network interactions, and not driven by changes in arousal levels.


title: Altered costimulatory signals and hypoxia support chromatin landscapes limiting the functional potential of exhausted T cells in cancer

abstract: Immunotherapy has changed cancer treatment with major clinical successes, but response rates remain low due in part to elevated prevalence of dysfunctional, terminally exhausted T cells. However, the mechanisms promoting progression to terminal exhaustion remain undefined. We profiled the histone modification landscape of tumor-infiltrating CD8 T cells throughout differentiation, finding terminally exhausted T cells possessed chromatin features limiting their transcriptional potential. Active enhancers enriched for bZIP/AP-1 transcription factor motifs lacked correlated gene expression, which were restored by immunotherapeutic costimulatory signaling. Epigenetic repression was also driven by an increase in histone bivalency, which we linked directly to hypoxia exposure. Our study is the first to profile the precise epigenetic changes during intratumoral differentiation to exhaustion, highlighting their altered function is driven by both improper costimulatory signals and environmental factors. These data suggest even terminally exhausted T cells remain poised for transcription in settings of increased costimulatory signaling and reduced hypoxia.


title: Niclosamide reverses SARS-CoV-2 control of lipophagy

abstract: The global effort to combat COVID-19 rapidly produced a shortlist of approved drugs with anti-viral activities for clinical repurposing. However, the jump to clinical testing was lethal in some cases as a full understanding of the mechanism of antiviral activity as opposed to pleiotropic activity/toxicity for these drugs was lacking. Through parallel lipidomic and transcriptomic analyses we observed massive reorganization of lipid profiles of infected Vero E6 cells, especially plasmalogens that correlated with increased levels of virus replication. Niclosamide (NIC), a poorly soluble anti-helminth drug identified for repurposed treatment of COVID-19, reduced the total lipid profile that would otherwise amplify during virus infection. NIC treatment reduced the abundance of plasmalogens, diacylglycerides, and ceramides, which are required for virus production. Future screens of approved drugs may identify more druggable compounds than NIC that can safely but effectively counter SARS-CoV-2 subversion of lipid metabolism thereby reducing virus replication. However, these data support the consideration of niclosamide as a potential COVID-19 therapeutic given its modulation of lipophagy leading to the reduction of virus egress and the subsequent regulation of key lipid mediators of pathological inflammation.


title: Genetic slippage after sex maintains diversity for parasite resistance in a natural host population

abstract: Although parasite-mediated selection is thought to be a major driver of host evolution, its influence on genetic variation for parasite resistance is not yet well understood. We monitored a large population of the planktonic crustacean Daphnia magna over eight years, as it underwent yearly epidemics of the bacterial pathogen Pasteuria ramosa. We observed a cyclical pattern of resistance evolution: resistant phenotypes increased in frequency throughout the epidemics, but susceptibility was restored each spring when hosts hatched from sexual resting stages, a phenomenon described as genetic slippage in response to sex. Collecting and hatching D. magna resting stages across multiple seasons showed that largely resistant host populations can produce susceptible offspring through recombination. Resting stages produced throughout the planktonic season accurately represent the hatching population cohort of the following spring. A genetic model of resistance developed for this host-parasite system, based on multiple loci and strong epistasis, is in partial agreement with these findings. Our results reveal that, despite strong selection for resistance in a natural host population, genetic slippage after sexual reproduction has the potential to maintain genetic diversity of host resistance.


title: Evolutionary footprints of a cold relic in a rapidly warming world

abstract: With accelerating global warming, understanding the evolutionary dynamics of plant adaptation to environmental change is increasingly urgent. Here we reveal the enigmatic history of the genus Cochlearia (Brassicaceae), a Pleistocene relic that originated from a drought-adapted Mediterranean sister genus during the Miocene. Cochlearia rapidly diversified and adapted to circum-Arctic regions and other cold-characterized habitat types during the Pleistocene. This rapid change in ecological preferences was accompanied by a highly complex, reticulate polyploid evolution, which was apparently triggered by the impact of repeated Pleistocene glaciation cycles. Our results illustrate that two early diversified arctic-alpine diploid gene pools contributed differently to the evolution of this young polyploid genus now captured in a cold-adapted niche. Metabolomics revealed ancestral central carbon metabolism responses to cold in diverse ecotypes, likely due to continuous connections to cold habitats that we hypothesize facilitated widespread parallel adaptation to alpine and subalpine habitats, and which we speculate were coopted from existing drought adaptations. Given the growing scientific interest in adaptive evolution of temperature-related traits, our results provide much-needed taxonomic and phylogenomic resolution of a model system as well as first insights into the origins of its adaptation to cold.


title: High rate of mutational events in SARS-CoV-2 genomes across Brazilian geographical regions, February 2020 to June 2021

abstract: Brazil has been considered as one of the emerging epicenters of the coronavirus pandemic in 2021, experiencing over 3,000 daily deaths caused by the virus at the peak of the second wave. In total, the country has more than 19.2 million confirmed cases of Covid-19, including over 533,509 fatalities. A set of emerging variants arose in the country, some of them posing new challenges for COVID-19 control. The goal of this study was to describe mutational events across samples from Brazilian SARS-CoV-2 sequences openly publicly obtainable on the Global Initiative on Sharing Avian Influenza Data-EpiCoV (GISAID-EpiCoV) platform and generate an index of new mutant by each genome. A total of 16,953 SARS-CoV-2 genomes were obtained and are not proportionally representative of the five Brazilian geographical regions. A comparative sequence analysis was conducted to identify common mutations located at 42 positions of the genome (38 were in coding regions whereas two in 5 and two in 3 UTR). Moreover, 11 were synonymous and 27 missense variants, and more than 44.4% were located in the spike gene. Across the total of single nucleotide variations (SNVs) identified, 32 were found in genomes obtained from all five Brazilian regions. While a high genomic diversity is reported in Europe given the large number of sequenced genomes, Africa is emerging as a hotspot for new variants. In South America, Brazil and Chile, rates are similar to those found in South Africa and India, giving enough space to generate new viral mutations. Genomic surveillance is the central key to identifying the emerging variants of SARS-CoV-2 in Brazil and has shown that the country is one of the "hotspots" in the generation of new variants.


title: Evaluating de novo assembly and binning strategies for time-series drinking water metagenomes.

abstract: Reconstructing microbial genomes from metagenomic short-read data can be challenging due to the unknown and uneven complexity of microbial communities. This complexity encompasses highly diverse populations which often includes strain variants. Reconstructing high-quality genomes is a crucial part of the metagenomic workflow as subsequent ecological and metabolic inferences depend on their accuracy, quality, and completeness. In contrast to microbial communities in other ecosystems, there has been no systematic assessment of genome-centric metagenomic workflows for drinking water microbiomes. In this study, we assessed the performance of a combination of assembly and binning strategies for time-series drinking water metagenomes that were collected over a period of 6 months. The goal of this study was to identify the combination of assembly and binning approaches that results in high quality and quantity metagenome-assembled genomes (MAGs), representing most of the sequenced metagenome. Our findings suggest that the metaSPAdes co-assembly strategies had the best performance as they resulted in larger and less fragmented assemblies with at least 85% of the sequence data mapping to contigs greater than 1kbp. Furthermore, a combination of metaSPAdes co-assembly strategies and MetaBAT2 produced the highest number of medium-quality MAGs while capturing at least 70% of the metagenomes based on read recruitment. Utilizing different assembly/binning approaches also assist in the reconstruction of unique MAGs from closely related species that would have otherwise collapsed into a single MAG using a single workflow. Overall, our study suggests that leveraging multiple binning approaches with different metaSPAdes co-assembly strategies may be required to maximize the recovery of good-quality MAGs, which more accurately capture the microbial diversity of drinking water samples.


title: SARS-CoV-2 Spike Protein Mutations and Escape from Antibodies: a Computational Model of Epitope Loss in Variants of Concern

abstract:
The SARS-CoV-2 spike (S) protein is exposed on the viral surface and is the first point of contact between the virus and the host. For these reasons it represents the prime target for Covid-19 vaccines. In recent months, variants of this protein have started to emerge. Their ability to reduce or evade recognition by S-targeting antibodies poses a threat to immunological treatments and raises concerns for their consequences on vaccine efficacy.

To develop a model able to predict the potential impact of S-protein mutations on antibody binding sites, we performed unbiased multi-microsecond molecular dynamics of several glycosylated S-protein variants and applied a straightforward structure-dynamics-energy based strategy to predict potential changes in immunogenic regions on each variant. We recover known epitopes on the reference D614G sequence. By comparing our results, obtained on isolated S-proteins in solution, to recently published data on antibody binding and reactivity in new S variants, we directly show that modifications in the S-protein consistently translate into the loss of potentially immunoreactive regions. Our findings can thus be qualitatively reconnected to the experimentally characterized decreased ability of some of the Abs elicited against the dominant S-sequence to recognize variants. While based on the study of SARS-CoV-2 Spike variants, our computational epitope-prediction strategy is portable and could be applied to study immunoreactivity in mutants of proteins of interest whose structures have been characterized, helping the development/selection of vaccines and antibodies able to control emerging variants.


title: Wall-associated Receptor Kinase and The Expression Profiles in Wheat Responding to Fungal Stress

abstract: Cell wall-associated kinases (WAKs), which are encoded by conserved gene families in plants, are crucial for development and responses to diverse stresses. However, the wheat (Triticum aestivum L.) WAKs have not been systematically classified, especially those involved in protecting plants from disease. Here, we classified 129 WAK proteins (encoded by 232 genes) and 75 WAK-Like proteins (WAKLs; encoded by 109 genes) into four groups, via a phylogenetic analysis. An examination of protein sequence alignment revealed diversity in the GUB-domain of WAKs structural organization, but it was usually characterized by a PYPFG motif followed by CxGxGCC motifs, while the EGF-domain was usually initiated with a YAC motif, and eight cysteine residues were spliced by GNPY motif. The expression profiles of WAK-encoding homologous genes varied in response to Blumeria graminis f. sp. tritici (Bgt), Puccinia striiformis f. sp. tritici (Pst) and Puccinia triticina (Pt) stress. A quantitative real-time polymerase chain reaction (qRT-PCR) analysis proved that TaWAK75 and TaWAK76b were involved in wheat resistance to Bgt. This study revealed the structure of the WAK-encoding genes in wheat, which may be useful for future functional elucidation of wheat WAKs responses to fungal infections.


title: Collaborations and deceptions in strategic interactions revealed by hyperscanning fMRI

abstract: Despite its ubiquity, deceiving as a social phenomenon is scarcely addressed with fMRI, partly due to the spontaneity and individual differences in cheating, and the contextual variability that fosters lying. In this hyperscanning fMRI study, the participant pairs (n=33) from Taipei and Tainan joined an opening-treasure-chest (OTC) game, where the dyads took alternative turns as senders (to inform) and receivers (to decide) for guessing the right chest. The cooperation condition was achieved by, upon successful guessing, splitting the $200NTD trial reward, thereby promoting mutual trust. The competition condition, in contrast, was done by, also upon winning, the latter receivers taking all the $150NTD reward, thereby encouraging strategic interactions. One key fMRI finding was the negative correlations between the connectivity of the right temporo-parietal junction (rTPJ), known as the theory-of-mind function, and amygdala, parahippocampal gyrus, and rostral anterior cingulate (rACC), to senders behavioral lying rates. Furthermore, the Multi-Voxel Pattern Analysis (MVPA) over multiple searchlight-identified Region-Of-Interests (ROIs), in classifying either the "truthful vs. lying in $150" or the "truthful in $200 vs. truthful in $150" conditions achieved 61% and 84.5% accuracy, respectively, reflecting the idiosyncratic brain networks involved in distinguishing the social trust vs. deceptions in the dyadic interactions.


title: Double nicking by RNA-directed Cascade-nCas3 for high-efficiency large-scale genome engineering

abstract: New CRISPR-based genome editing technologies are developed to continuedly drive advances in life sciences, which, however, are predominantly derived from systems of Type II CRISPR-Cas9 and Type V CRISPR-Cas12a for eukaryotes. Here we report a novel CRISPR-n(nickase)Cas3 genome editing tool established upon an endogenous Type I system of Zymomonas mobilis. We demonstrate that nCas3 variants can be created by alanine-substituting any catalytic residue of the Cas3 helicase domain. While nCas3 overproduction via plasmid shows severe cytotoxicity; an in situ nCas3 introduces targeted double-strand breaks, facilitating genome editing, without visible cell killing. By harnessing this CRISPR-nCas3, deletion of genes or genomic DNA stretches can be consistently accomplished with near-100% efficiencies, including simultaneous removal of two large genomic fragments. Our work describes the first establishment of a CRISPR-nCas3-based genome editing technology, thereby offering a simple, easy, yet useful approach to convert many endogenous Type I systems into advanced genome editing tools. We envision that many CRISPR-nCas3-based toolkits would be soon available for various industrially important non-model bacteria that carry active Type I systems to facilitate high-throughput prokaryotic engineering.


title: Pathogenic and transcriptomic differences of emerging SARS-CoV-2 variants in the Syrian golden hamster model

abstract: Following the discovery of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its rapid spread throughout the world, new viral variants of concern (VOC) have emerged. There is a critical need to understand the impact of the emerging variants on host response and disease dynamics to facilitate the development of vaccines and therapeutics. Syrian golden hamsters are the leading small animal model that recapitulates key aspects of severe coronavirus disease 2019 (COVID-19). In this study, we show that intranasal inoculation of SARS-CoV-2 into hamsters with the ancestral virus (nCoV-WA1-2020) or VOC first identified in the United Kingdom (B.1.1.7) and South Africa (B.1.351) led to similar gross and histopathologic pulmonary lesions. Although differences in viral genomic copy numbers were noted in the lungs and oral swabs of challenged animals, infectious titers in the lungs were comparable. Antibody neutralization capacities varied, dependent on the original challenge virus and cross-variant protective capacity. Transcriptional profiling indicated significant induction of antiviral pathways in response to all three challenges with a more robust inflammatory signature in response to B.1.1.7. Furthermore, no additional mutations in the spike protein were detected at peak disease. In conclusion, the emerging VOC showed distinct humoral responses and transcriptional profiles in the hamster model compared to the ancestral virus.


title: PROTEOMIC SIGNATURES OF MICROBIAL ADAPTATION TO THE HIGHEST UV -IRRADIATION ON EARTH: LESSONS FROM A SOIL ACTINOBACTERIUM

abstract: In the Puna region, the total solar irradiation and the UV incidence is the highest on Earth, thus, restraining the physiology of individual microorganisms and the composition of microbial communities. UV-resistance of microbial strains thriving in High-Altitude Andean Lakes was demonstrated and their mechanisms were partially characterized by genomic analysis, biochemical and physiological assays. In this work, we present the molecular events involved in the adaptive response of the model HAAL extremophilic actinobacterium Nesterenkonia sp. Act20 under artificial UV-B radiation, herein called as UV-resistome. Proteomic profiles of cultures exposed to different UV-experimental conditions showed that the leading systems for adaptation to the UV-challenge in-vitro are DNA repair and antioxidant mechanisms.

category: molecular biology


title: The retrotransposon R2 maintains Drosophila ribosomal DNA repeats

abstract:
Ribosomal DNA (rDNA) loci contain the hundreds of tandemly repeated copies of ribosomal RNA genes needed to support cellular viability. This repetitiveness makes it highly susceptible to copy loss, threatening multi-generational maintenance of rDNA. How this threat is counteracted to avoid extinction of the lineage has remained unclear. Here, we show that the rDNA-specific retrotransposon R2 is essential for rDNA copy number (CN) maintenance in the Drosophila male germline, despite the perceived disruptive nature of transposable elements. Depletion of R2 led to defective rDNA CN maintenance, causing a decline in fecundity over generations and eventual extinction. This study reveals that retrotransposons can provide a benefit to their hosts, contrary to their reputation as genomic parasitic, which may contribute to their widespread success throughout taxa.

One Sentence SummaryThe retrotransposon R2 initiates restoration of ribosomal DNA copies to trans-generationally mantain essential locus.

category: cell biology


title: Artemisinin acts by inhibiting Plasmodium falciparum Ddi1, a retropepsin, resulting into the accumulation of ubiquitinated proteins

abstract: Reduced sensitivity of the human malaria parasite, Plasmodium falciparum, to Artemisinin and its derivatives (ARTs) threatens the global efforts towards eliminating malaria. ARTs have been shown to cause ubiquitous cellular and genetic insults, which results in the activation of the unfolded protein response (UPR) pathways. The UPR restores protein homeostasis, which otherwise would be toxic to cellular survival. Here, we interrogated the role of DNA-damage inducible protein 1 (PfDdi1), a unique proteasome-interacting retropepsin in mediating the actions of the ARTs. We demonstrate that PfDdi1 is an active A2 family protease that hydrolyzes ubiquitinated substrates. We further show that treatment with ARTs lead to the accumulation of ubiquitinated proteins in the parasites and blocks the destruction of the ubiquitinated substrates by PfDdi1. Besides, whereas the PfDdi1 is predominantly localised in the cytoplasm, exposure of the parasites to ARTs leads to DNA fragmentation and increased recruitment of the PfDdi1 into the nucleus. Furthermore, Ddi1 knock-out Saccharomyces cerevisiae cells are more suceptible to ARTs and the PfDdI1 protein robustly restores the corresponding functions in the knock-out cells. Together, these results show that ARTs act by inducing DNA and protein damage, and impairing the damage recovery by inhibiting the activity of PfDdi1, an essential ubiquitin-proteasome retropepsin.


title: Lentiviral vector optimization enhances the expression and cytotoxicity of chimeric antigen receptors

abstract: Adoptive chimeric antigen receptor (CAR)-modified T or NK cells (CAR-T/NK) have emerged as a novel form of disease treatment. Lentiviral vectors (LVs) are commonly employed to engineer T/NK cells for the efficient expression of CARs. This study reported for the first time the influence of single-promoter and dual-promoter LVs on the CAR expression and cytotoxicity of engineered NK cells. Our results demonstrated that the selected CAR exhibits both a higher expression level and a higher coexpression concordance with the GFP reporter in HEK-293T or NK92 cells by utilizing the optimized single-promoter pCDHsp rather than the original dual-promoter pCDHdp. After puromycin selection, the pCDHsp produces robust CAR expression and enhanced in vitro cytotoxicity of engineered NK cells. Therefore, infection with a single-promoter pCDHsp lentivector is recommended to prepare CAR-engineered cells. This research will help to optimize the production of CAR-NK cells and improve their functional activity, to provide CAR-NK cell products with better and more uniform quality.


title: ERK-mediated Curvature Feedback Regulates Branching Morphogenesis in Lung Epithelial Tissue

abstract: Intricate branching patterns emerge in internal organs because of the repetitive presence of simple deformations in epithelial tissues. During murine lung development, epithelial cells in distal tips of a single tube require fibroblast growth factor (FGF) signals generated by their surrounding mesenchyme to form repetitive tip bifurcations. However, it remains unknown how the cells employ FGF signaling to convert their behaviors to achieve the recursive branching processes. Here we show a self-sustained epithelial regulatory system during the murine lung branching morphogenesis, mediated by extracellular signal-regulated kinase (ERK), which acts as a downstream driver of FGF signaling. We found that tissue-scale curvature regulates ERK activity in the lung epithelium using two-photon live cell imaging and mechanical perturbations. ERK is activated specifically in epithelial tissues with a positive curvature, regardless of whether the change in curvature was attributable to morphogenesis or artificial perturbations. Moreover, we found that ERK activation accelerates actin polymerization specifically at the apical side of cells, and mechanically contributes to the extension of the apical membrane, leading to a decrease in epithelial tissue curvature. These results indicate the existence of a negative feedback loop between tissue curvature and ERK activity beyond scale. We confirmed that this regulation was sufficient to generate the recursive branching processes by a mathematical model. Taken together, we propose that ERK mediates the curvature feedback loop underlying the process of branching morphogenesis in developing lungs.

category: developmental biology


title: I alternate therefore I generalize: how the intrinsic resistance risk of fungicides counterbalances their durability

abstract:
The evolution of resistance to pesticides is a major burden in agriculture. Resistance management involves maximizing selection pressure heterogeneity, particularly by combining active ingredients with different modes of action. We tested the hypothesis that the temporal alternation of active ingredients may delay the build-up of resistance not only by spreading selection pressure over longer periods, but also by decreasing the rate of evolution of resistance to alternated fungicides. Here, we applied an original experimental evolution approach to the economically important crop pathogen Zymoseptoria tritici. We observed the dynamics of Z. tritici resistance in 56 independent lines subjected to 14 continuous or alternation regimes of 3 fungicides contrasting for their mode of action, at their EC95 selection doses. For the first time in a plant pathogen, our results show that alternation is either neutral or slows the evolution of resistance, relative to continuous fungicide use, but results in higher levels of generalism in evolved lines. We demonstrate that the mode of action of resistance of fungicides drivingly underlies this trade-off, more so than the number of fungicides and the frequency of alternation. This trade-off is also dynamic over the course of resistance evolution, as shown by the change in population phenotype structure and the relative impact of selection drivers. These findings open up new possibilities for tailoring resistance management effectively while optimizing smart interplay between alternation components. They also confirm experimental evolution as an untapped but promising approach to dissect adaptation in phytopathogenic fungi.

Author summaryThe efficacy of pesticides has been compromised by the generalization of their use, leading to the rapid and widespread evolution of resistance. This constitutes a major economic and environmental burden in agriculture. The temporal alternation (or cycling) of active ingredients is a management strategy that induces temporal variation of selection of pathogens. Here, we dissected how it can be optimized according to the number and nature of alternated modes of action and to the rhythm of their application and sought to understand how these drivers determine the performance of alternation strategies. We used an approach original in plant pathology, experimental evolution, applied on an economically important fungus, Zymoseptoria tritici. We concluded that alternation can delay the rate of resistance selection with performance depending most likely on the mode of action of alternated antifungals, more so than on other drivers. But we also highlighted that alternation regimes select generalist resistance, that is the ability to resist a large diversity of antifungals. This trade-off was dynamic over time. Our study provides new insights for the informed management of pesticides and the reduction of their side effects.


title: Delayed but generalist resistance in alternation: a trade-off shaped by the mode of action of antifungals, as revealed by experimental evolution in a plant pathogen.


title: Native MOWChIP-seq: Genome-wide profiles of key protein bindings reveal functional differences among various brain regions

abstract: Genome-wide profiling of interactions between genome and various functional proteins is critical for understanding regulatory processes involved in development and diseases. Conventional assays require a large number of cells and high-quality data on tissue samples are scarce. Here we optimized a low-input chromatin immunoprecipitation followed by sequencing (ChIP-seq) technology for profiling RNA polymerase II (Pol II), transcription factor (TF), and enzyme binding at the genome scale. The new approach, termed native MOWChIP-seq, produces high-quality binding profiles using 1000-50,000 cells. We used the approach to examine the binding of Pol II and two TFs (EGR1 and MEF2C) in cerebellum and prefrontal cortex of mouse brain and found that their binding profiles are highly reflective of the functional differences between the two brain regions. Our analysis reveals the potential for linking genome-wide TF or Pol II profiles with neuroanatomical origins of brain cells.


title: Mapping the nuclear microenvironment of genes at agenome-wide scale

abstract: The nuclear folding of chromosomes relative to nuclear bodies is an integral part of gene function. Here, we demonstrate that population-based modeling--from ensemble Hi-C data--can provide a detailed description of the nuclear microenvironment of genes and its role on gene function. We define the microenvironment by the subnuclear positions of genomic regions with respect to nuclear bodies, local chromatin compaction, and preferences in chromatin compartmentalization. These structural descriptors are determined in single cell models on a genome-wide scale, thereby revealing the structural variability between cells. We demonstrate that the structural microenvironment of a genomic region is linked to its functional potential in gene transcription, replication and chromatin compartmentalization. Some chromatin regions are distinguished by their strong preferences to a single microenvironment, due to associations to specific nuclear bodies in most cells. Other chromatin shows high structural variability, which is a strong indicator of functional heterogeneity. Moreover, we identify specialized nuclear microenvironments, which distinguish chromatin in different functional states and reveal a key role of nuclear speckles in chromosome organization. We demonstrate that our method produces highly predictive 3-dimensional genome structures, which accurately reproduce data from TSA-seq, DamID, GPSeq and super-resolution imaging. Thus, our method considerably expands the range of Hi-C data analysis and is widely applicable.


title: Population-based structure modeling reveals key roles of nuclear microenvironment in gene functions


title: Chemoproteomic profiling reveals cellular targets of nitro-fatty acids

abstract: Nitro-fatty acids are a class of endogenous electrophilic lipid mediators with anti-inflammatory and cytoprotective effects in a wide range of inflammatory and fibrotic disease models. While these beneficial biological effects of nitro-fatty acids are mainly attributed to their ability to form covalent adducts with proteins, only a small number of proteins are known to be nitro-alkylated and the scope of protein nitro-alkylation remains undetermined. Here we describe the synthesis and application of a clickable nitro-fatty acid probe for the detection and first global identification of mammalian proteins that are susceptible to nitro-alkylation. 184 high confidence nitro-alkylated proteins were identified in human macrophages, majority of which are novel targets of nitro-fatty acids, including Extended synaptotagmin 2 (ESYT2), Signal transducer and activator of transcription 3 (STAT3), Toll-like receptor 2 (TLR2), Retinoid X receptor alpha (RXR) and Glucocorticoid receptor (NR3C1). In particular, we showed that 9-nitro-oleate covalently modified and inhibited dexamethasone binding to NR3C1. Bioinformatic analyses revealed that nitro-alkylated proteins are highly enriched in endoplasmic reticulum and transmembrane proteins, and are overrepresented in lipid metabolism and transport pathways. This study significantly expands the scope of protein substrates targeted by nitro-fatty acids in living cells and provides a useful resource towards understanding the pleiotropic biological roles of nitro-fatty acids as signaling molecules or as multi-target therapeutic agents.

category: biochemistry


title: Longitudinal in vivo changes in radial peripapillary capillaries and optic nerve head structure in non-human primates with early experimental glaucoma

abstract:
PurposeThere is conflicting evidence as to whether a loss of radial peripapillary capillaries (RPCs) precedes neuronal loss in glaucoma. We examined the time-course of in vivo changes in RPCs, optic nerve head (ONH) structure, and retinal nerve fiber layer thickness (RNFLT) in experimental glaucoma (EG).

MethodsSpectral domain optical coherence tomography images were acquired before and approximately every 2 weeks after inducing unilateral EG in 9 rhesus monkeys to quantify mean anterior lamina cribrosa surface depth (ALCSD), minimum rim width (MRW), and RNFLT. Perfused RPC density was measured from adaptive optics scanning laser ophthalmoscope images acquired on the temporal half of the ONH. The time of first significant change was quantified as when values fell and remained outside of the 95% confidence interval established from control eyes.

ResultsMean ALCSD and/or MRW were the first parameters to change in 8 EG eyes. RPC density changed first in the 9th. At their first points of change, mean ALCSD posteriorly deformed by 100.2 {+/-} 101.2 m, MRW thinned by 82.3 {+/-} 65.9 m, RNFLT decreased by 25 {+/-} 14 m, and RPC density decreased by 4.5 {+/-} 2.1%. RPC density decreased before RNFL thinning in 5 EG eyes. RNFLT decreased before RPC density decreased in 2 EG eyes, while 2 EG eyes had simultaneous changes.

ConclusionsIn most EG eyes, RPC density decreased before (or simultaneous with) a change in RNFLT, suggesting that vascular factors may play a role in axonal loss in some eyes in early glaucoma.

category: bioengineering