title: Influence of basal media composition on barrier fidelity within human pluripotent stem cell-derived blood-brain barrier models

abstract: It is increasingly recognized that brain microvascular endothelial cells (BMECs), the principle component of the blood-brain barrier (BBB), are highly sensitive to soluble cues from both the bloodstream and the brain. This concept extends in vitro, where the extracellular milieu can also influence BBB properties in cultured cells. However, the extent to which baseline culture conditions can affect BBB properties in vitro remains unclear, which has implications for model variability and reproducibility, as well as downstream assessments of molecular transport and disease phenotypes. Here, we explore this concept by examining BBB properties within human induced pluripotent stem cell (iPSC)-derived BMEC-like cells cultured under serum-free conditions in different basal media with fully defined compositions. We demonstrate notable differences in both passive and active BBB properties as a function of basal media composition. Further, RNA sequencing and phosphoproteome analyses revealed alterations to various signaling pathways in response to basal media differences. Overall, our results demonstrate that baseline culture conditions can have a profound influence on the performance of in vitro BBB models, and these effects should be considered when designing experiments that utilize such models for basic research and preclinical assays.


title: Reduced antibody cross-reactivity following infection with B.1.1.7 than with parental SARS-CoV-2 strains

abstract: We examined the immunogenicity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant B.1.1.7 that arose in the United Kingdom and spread globally. Antibodies elicited by B.1.1.7 infection exhibited significantly reduced recognition and neutralisation of parental strains or of the South Africa B.1.351 variant, than of the infecting variant. The drop in cross-reactivity was more pronounced following B.1.1.7 than parental strain infection, indicating asymmetric heterotypic immunity induced by SARS-CoV-2 variants.

category: immunology


title: Negligible impact of SARS-CoV-2 variants on CD4+ and CD8+ T cell reactivity in COVID-19 exposed donors and vaccinees.

abstract: The emergence of SARS-CoV-2 variants highlighted the need to better understand adaptive immune responses to this virus. It is important to address whether also CD4+ and CD8+ T cell responses are affected, because of the role they play in disease resolution and modulation of COVID-19 disease severity. Here we performed a comprehensive analysis of SARS-CoV-2-specific CD4+ and CD8+ T cell responses from COVID-19 convalescent subjects recognizing the ancestral strain, compared to variant lineages B.1.1.7, B.1.351, P.1, and CAL.20C as well as recipients of the Moderna (mRNA-1273) or Pfizer/BioNTech (BNT162b2) COVID-19 vaccines. Similarly, we demonstrate that the sequences of the vast majority of SARS-CoV-2 T cell epitopes are not affected by the mutations found in the variants analyzed. Overall, the results demonstrate that CD4+ and CD8+ T cell responses in convalescent COVID-19 subjects or COVID-19 mRNA vaccinees are not substantially affected by mutations found in the SARS-CoV-2 variants.


title: C4-dicarboxylate metabolons: Interaction of C4-dicarboxylate transporters of Escherichia coli with cytosolic enzymes and regulators

abstract: Metabolons represent the structural organization of proteins for metabolic or regulatory pathways. Here the interaction of enzymes fumarase FumB and aspartase AspA with the C4-DC transporters DcuA and DcuB of Escherichia coli was tested by a bacterial two-hybrid (BACTH) assay in situ, or by co-chromatography (mSPINE). DcuB interacted strongly with FumB and AspA, and DcuA with AspA. The fumB-dcuB and the dcuA-aspA genes encoding the respective proteins are known for their colocalization on the genome and the production of co-transcripts. The data consistently suggest the formation of DcuB/FumB, DcuB/AspA and DcuA/AspA metabolons in fumarate respiration for the uptake of L-malate, or L-aspartate, conversion to fumarate and excretion of succinate after reduction. The DcuA/AspA metabolon catalyzes L-Asp uptake and fumarate excretion in concerted action also to provide ammonia for nitrogen assimilation.

category: microbiology


title: C4-dicarboxylate metabolons: Interaction of C4-dicarboxylate transporters of Escherichia coli with cytosolic enzymes


title: Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell

abstract: Established in vitro models for SARS-CoV-2 infection are limited and include cell lines of non-human origin and those engineered to overexpress ACE2, the cognate host cell receptor. We identified human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of ACE2. Infection of H522 cells required the SARS-CoV-2 spike protein, though in contrast to ACE2-dependent models, spike alone was not sufficient for H522 infection. Temporally resolved transcriptomic and proteomic profiling revealed alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type-I interferon signaling. Focused chemical screens point to important roles for clathrin-mediated endocytosis and endosomal cathepsins in SARS-CoV-2 infection of H522 cells. These findings imply the utilization of an alternative SARS-CoV-2 host cell receptor which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.


title: The E3 Ubiquitin Ligase RNF5 Facilitates SARS-CoV-2 Membrane Protein-Mediated Virion Release

abstract: As enveloped virus, SARS-CoV-2 membrane protein (M) mediates viral release from cellular membranes, but the molecular mechanisms of SARS-CoV-2 virions release remain poorly understood. Here, we performed RNAi screening and identified the E3 ligase RNF5 which mediates ubiquitination of SARS-CoV-2 M at residue K15 to enhance the interaction of viral envelope (E) with M. M-E complex ensures the uniform size of viral particles for viral maturation and mediates viral release. Moreover, overexpression of M induces complete autophagy which is dependent on RNF5-mediated ubiquitin modification. M inhibits the activity of lysosome protease, and uses autolysosomes for virion release. Consequently, all these results demonstrate that RNF5 mediates ubiquitin modification of SARS-CoV-2 M to stabilize the M-E complex and induce autophagy for virion release.


title: Interaction of thyroid hormones and gonadotropin inhibitory hormone in the multifactorial control of zebrafish (Danio rerio) spermatogenesis

abstract: Reproduction is under multifactorial control of neurohormones, pituitary gonadotropins, as well as a number of gonadal hormones including sex steroids and growth factors. Gonadotropin-inhibitory hormone (Gnih), a novel RFamide neuropeptide, was shown to be involved in the control of pituitary gonadotropin production, as well as being involved as a paracrine factor in the regulation of gonadal function. In this context, recent studies have demonstrated that Gnih inhibited gonadotropin-induced spermatogenesis in the zebrafish testicular explants. Thyroid hormones are known to interact with the reproductive axis, and are, in particular, involved in the regulation of testicular function. Based on this background, we investigated the interaction between Gnih and thyroid hormones in the control of zebrafish spermatogenesis. To this end, zebrafish adult males were treated with the goitrogen methimazole (1mM for 21 days) in order to generate a hypothyroid model organism. Subsequently, a factorial design using an ex vivo testis culture system in combination with histomorphometrical and FACScan cell cycle analyses were adopted. Our results showed that methimazole treatment affected both basal and gonadotropin-induced spermatogenesis, in particular, meiosis and spermiogenesis. Moreover, the goitrogen treatment nullified the inhibitory actions of Gnih on the gonadotropin-induced spermatogenesis, specifically in the haploid cell population. We have demonstrated that thyroid hormones interaction with gonadotropin and Gnih are important components for the regulation of zebrafish spermatogenesis. The results provide a support for the hypothesis that thyroid hormones are important contributors in multifactorial control of spermatogenesis in zebrafish.

category: cell biology


title: An evolutionary genomic approach reveals both conserved and species-specific genetic elements related to human disease in closely related Aspergillus fungi

abstract: Aspergillosis is an important opportunistic human disease caused by filamentous fungi in the genus Aspergillus. Roughly 70% of infections are caused by Aspergillus fumigatus, with the rest stemming from approximately a dozen other Aspergillus species. Several of these pathogens are closely related to A. fumigatus and belong in the same taxonomic section, section Fumigati. Pathogenic species are frequently most closely related to non-pathogenic ones, suggesting Aspergillus pathogenicity evolved multiple times independently. To understand the repeated evolution of Aspergillus pathogenicity, we performed comparative genomic analyses on 18 strains from 13 species, including 8 species in section Fumigati, which aimed to identify genes, both ones previously connected to virulence as well as ones never before implicated, whose evolution differs between pathogens and non-pathogens. We found that most genes were present in all species, including approximately half of those previously connected to virulence, but a few genes were section- or species-specific. Evolutionary rate analyses identified hundreds of genes in pathogens that were faster-evolving than their orthologs in non-pathogens. For example, over 25% of all single-copy genes examined in A. fumigatus were faster-evolving. Functional testing of deletion mutants of 17 transcription factor-encoding genes whose evolution differed between pathogens and non-pathogens identified eight genes that affect either fungal survival in a model of phagocytic killing, host survival in an animal model of fungal disease, or both. These results suggest that the evolution of pathogenicity in Aspergillus involved both conserved and species-specific genetic elements, illustrating how an evolutionary genomic approach informs the study of fungal disease.

category: evolutionary biology


title: Circular RNA repertoires are associated with evolutionarily young transposable elements

abstract: AbstractCircular RNAs (circRNAs) are found across eukaryotes and can function in post-transcriptional gene regulation. Their biogenesis through a circle-forming backsplicing reaction is facilitated by reverse-complementary repetitive sequences promoting pre-mRNA folding. Orthologous genes from which circRNAs arise, overall contain more strongly conserved splice sites and exons than other genes, yet it remains unclear to what extent this conservation reflects purifying selection acting on the circRNAs themselves. Our analyses of circRNA repertoires from five species representing three mammalian lineages (marsupials, eutherians: rodents, primates) reveal that surprisingly few circRNAs arise from orthologous exonic loci across all species. Even the circRNAs from orthologous loci are associated with young, recently active and species-specific transposable elements, rather than with common, ancient transposon integration events. These observations suggest that many circRNAs emerged convergently during evolution - as a byproduct of splicing in orthologs prone to transposon insertion. Overall, our findings argue against widespread functional circRNA conservation.


title: Arabidopsis ROOT UV-B SENSITIVE 1 and 2 Interact with Aminotransferases to Regulate Vitamin B6 Homeostasis

abstract: Pyridoxal-5-phosphate (PLP), the enzymatic cofactor form of Vitamin B6 (vitB6), is a versatile compound that has essential roles in metabolism. Cellular PLP homeostasic regulation is currently not well understood. Here we report that in Arabidopsis, biosynthesized PLP is sequestered by specific aminotransferases (ATs), and that the proteins ROOT UV-B SENSITIVE 1 (RUS1) and RUS2 function with ATs to regulate PLP homeostasis. The stunted growth phenotypes of rus1 and rus2 mutants were previously shown to be rescuable by exogenously supplied vitB6. Specific residue changes near the PLP-binding pocket in ASPARTATE AMINOTRANSFERASE2 (ASP2) also rescued rus1 and rus2 phenotypes. In this study, saturated suppressor screens identified 14 additional suppressor of rus (sor) alleles in four aminotransferase genes (ASP1, ASP2, ASP3, or ALANIN AMINOTRANSFERASE1 (AAT1)), which suppressed the rus phenotypes to varying degrees. Each of the sor mutations altered an amino acid in the PLP-binding pocket of the protein, and sor proteins were found to have reduced levels of PLP conjugation. Genetic data revealed that the availability of PLP normally requires both RUS1 and RUS2, and that increasing the number of sor mutants additively enhanced the suppression of rus phenotypes. Biochemical results showed that RUS1 and RUS2 physically interacted with ATs. Our studies suggest a mechanism in which RUS1, RUS2 and specific ATs work together to regulate PLP homeostasis in Arabidopsis.

category: genetics


title: A Comparison of Performance for Different SARS-Cov-2 Sequencing Protocols

abstract: SARS-Cov-2 genome sequencing has been identified as a fundamental tool for fighting the COVID-19 pandemic. It is used, for example, for identifying new variants of the virus and for elaborating phylogenetic trees that help to trace the spread of the virus. In the present study we provide a comprehensive comparison between the quality of the assemblies obtained from different sequencing protocols. We demonstrate how some protocols actively promoted by different high-level administrations are inefficient and how less-used alternative protocols show a significant increased performance. This increase of performance could lead to cheaper sequencing protocols and therefore to a more convenient escalation of the sequencing efforts around the world.


title: PRDM9 directs recombination hotspots to prevent competition with meiotic transcription

abstract: PRDM9 drives recombination hotspots in some mammals, including mice and apes. Non-functional orthologs of PRDM9 are present in a wide variety of vertebrates, but why it is functionally maintained in some lineages is not clear. One possible explanation is that PRDM9 plays a role in ensuring that meiosis is successful. During meiosis, available DNA may be a limiting resource given the tight packaging of chromosomes and could lead to competition between two key processes: meiotic transcription and recombination. Here we explore this potential competition and the role that PRDM9 might play in their interaction. Leveraging existing mouse genomic data, we use resampling schemes that simulate shuffled features along the genome and models that account for the rarity of features in the genome, to test if PRDM9 influences interactions between recombination hotspots and meiotic transcription in a whole genome framework. We also explored possible DNA sequence motifs associated to clusters of hotspots not tied to transcription or PRDM9. We find evidence of competition between meiotic transcription and recombination, with PRDM9 appearing to relocate recombination to avoid said conflict. We also find that retrotransposons may be playing a role in directing hotspots in the absence of other factors.

category: genomics


title: The aging DNA methylome reveals environment-by-aging interactions in a model teleost

abstract: The rate at which individuals age underlies variation in life history and attendant health and disease trajectories. Age specific patterning of the DNA methylome ("epigenetic aging") is strongly correlated with chronological age in humans and can be modeled to produce epigenetic age predictors. However, epigenetic age estimates vary among individuals of the same age, and this mismatch is correlated to the onset of age-related disease and all-cause mortality. Yet, the origins of epigenetic-to-chronological age discordance are not resolved. In an effort to develop a tractable model in which environmental drivers of epigenetic aging can be assessed, we investigate the relationship between aging and DNA methylation in a small teleost, medaka (Oryzias latipes). We find that age-associated DNA methylation patterning occurs broadly across the genome, with the majority of age-related changes occurring during early life. By modeling the stereotypical nature of age-associated DNA methylation dynamics, we built an epigenetic clock, which predicts chronological age with a mean error of 29.1 days (~4% of average lifespan). Characterization of clock loci suggests that aspects of epigenetic aging are functionally similar across vertebrates. To understand how environmental factors interact with epigenetic aging, we exposed medaka to four doses of ionizing radiation for seven weeks, hypothesizing that exposure to such an environmental stressor would accelerate epigenetic aging. While the epigenetic clock was not significantly affected, radiation exposure accelerated and decelerated patterns of normal epigenetic aging, with radiation-induced epigenetic alterations enriched at loci that become hypermethylated with age. Together, our findings advance ongoing research attempting to elucidate the functional role of DNA methylation in integrating environmental factors into the rate of biological aging.


title: Day-night and seasonal variation of human gene expression across tissues

abstract: Circadian and circannual cycles trigger physiological changes whose reflection on human transcriptomes remains largely uncharted. We used the time and season of death of 932 individuals from GTEx to jointly investigate transcriptomic changes associated with those cycles across multiple tissues. Overall, most variation across tissues during day-night and among seasons was unique to each cycle. Although all tissues remodeled their transcriptomes, brain and gonadal tissues exhibited the highest seasonality, whereas those in the thoracic cavity showed stronger day-night regulation. Core clock genes displayed marked day-night differences across multiple tissues, which were largely conserved in baboon and mouse, but adapted to their nocturnal or diurnal habits. Seasonal variation of expression affected multiple pathways and it was enriched among genes associated with the immune response, consistent with the seasonality of viral infections. Furthermore, they unveiled cytoarchitectural changes in brain regions. Altogether, our results provide the first combined atlas of how transcriptomes from human tissues adapt to major cycling environmental conditions.


title: Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Native Top-Down Mass Spectrometry

abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes an extensively glycosylated surface spike (S) protein to mediate host cell entry and the S protein glycosylation is strongly implicated in altering viral binding/function and infectivity. However, the structures and relative abundance of the new O-glycans found on the S protein regional-binding domain (S-RBD) remain cryptic because of the challenges in intact glycoform analysis. Here, we report the complete structural characterization of intact O-glycan proteoforms using native top-down mass spectrometry (MS). By combining trapped ion mobility spectrometry (TIMS), which can separate the protein conformers of S-RBD and analyze their gas phase structural variants, with ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR) MS analysis, the O-glycoforms of the S-RBD are comprehensively characterized, so that seven O-glycoforms and their relative molecular abundance are structurally elucidated for the first time. These findings demonstrate that native top-down MS can provide a high-resolution proteoform-resolved mapping of diverse O-glycoforms of the S glycoprotein, which lays a strong molecular foundation to uncover the functional roles of their O-glycans. This proteoform-resolved approach can be applied to reveal the structural O-glycoform heterogeneity of emergent SARS-CoV-2 S-RBD variants, as well as other O-glycoproteins in general.

category: biochemistry


title: Deep learning tools and modeling to estimate the temporal expression of E2Fs over the cell cycle from 2D still images

abstract: Automatic characterization of fluorescent labeling in intact mammalian tissues remains a challenge due to the lack of quantifying techniques capable of segregating densely packed nuclei and intricate tissue patterns. Here, we describe a powerful deep learning-based approach that couples remarkably precise nuclear segmentation with quantitation of fluorescent labeling intensity within segmented nuclei, and then apply it to the analysis of cell cycle dependent protein concentration in mouse tissues using 2D fluorescent still images. First, several existing deep learning-based methods were evaluated to accurately segment nuclei using different imaging modalities with a small training dataset. Next, we developed a deep learning-based approach to identify and measure fluorescent labels within segmented nuclei, and created an ImageJ plugin to allow for efficient manual correction of nuclear segmentation and label identification. Lastly, using fluorescence intensity as a readout for protein concentration, a three-step global estimation method was applied to the characterization of the cell cycle dependent expression of E2F proteins in the developing mouse intestine.

category: bioinformatics


title: Deep learning tools and modeling to estimate the temporal expression of cell cycle proteins from 2D still images


title: Expression levels of long noncoding natural antisense transcripts overlapping the UGT73C6 gene affect rosette size of Arabidopsis thaliana

abstract: Natural antisense long noncoding RNAs (lncNATs) are involved in the regulation of gene expression in plants, modulating different relevant developmental processes and responses to various stimuli. We identified and characterized two lncNATs (NAT1UGT73C6 and NAT2UGT73C6, collectively NATsUGT73C6) in Arabidopsis thaliana that are transcribed from a gene overlapping UGT73C6, a member of the UGT73C subfamily of genes encoding UDP-glycosyltransferases (UGTs). Expression of both NATsUGT73C6 is developmentally controlled and occurs independently of the transcription of UGT73C6 in cis. Downregulation of NATsUGT73C6 levels through artificial microRNAs results in a reduction of the rosette area, while constitutive overexpression of NAT1UGT73C6 or NAT2UGT73C6 leads to the opposite phenotype, an increase in rosette size. This activity of NATsUGT73C6 relies on its RNA sequence, and, although modulation of UGT73C6 in cis cannot be excluded, the observed phenotypes are not a consequence of the regulation of UGT73C6 in trans. The NATsUGT73C6 levels were shown to affect cell proliferation and thus individual leaf size. Consistent with this concept, our data suggest that the NATsUGT73C6 modulate the expression levels of key transcription factors involved in regulating leaf growth by modulating cell proliferation. These findings thus reveal an additional regulatory layer on the process of leaf growth.

category: plant biology


title: Cinnamoyl-CoA Reductase 1 (CCR1) and CCR2 Function Divergently in Tissue Lignification, Flux Control and Cross-talk with Glucosinolate Pathway in Brassica napus

abstract:
Cinnamoyl-CoA reductase (CCR) is the entry point of lignin pathway and a crucial locus in dissection and manipulation of associated traits, but its functional dissection in Brassicaceae plants is largely lagged behind though Arabidopsis thaliana CCR1 has been characterized to certain extent. Here, 16 CCR genes are identified from Brassica napus and its parental species B. rapa and B. oleracea. Brassicaceae CCR genes are divided into CCR1 subfamily and CCR2 subfamily with divergent organ-specificity, yellow-seed trait participation and stresses responsiveness. CCR1 is preferential in G- and H-lignins biosynthesis and vascular development, while CCR2 has a deviation to S-lignin biosynthesis and interfascicular fiber development. CCR1 has stronger effects on lignification-related development, lodging resistance, phenylpropanoid flux control and seed coat pigmentation, whereas CCR2 controls sinapates levels. CCR1 upregulation could delay bolting and flowering time, while CCR2 upregulation weakens vascular system in leaf due to suppressed G lignin accumulation. Besides, CCR1 and CCR2 are deeply but almost oppositely linked with glucosinolates metabolism through inter-pathway crosstalk. Strangely, upregulation of both CCR1 and CCR2 could not enhance resistance to UV-B and S. sclerotiorum though CCR2 is sharply induced by them. These results provide systemic dissection on Brassica CCRs and CCR1-CCR2 divergence in Brassicaceae.

HighlightBrassicaceae contains two types of Cinnamoyl-CoA reductase. As revealed in Brassica napus, they are divergently involved in lignin monomer biosynthesis, tissue lignification, phenylpropanoid flux control, and inter-pathway crosstalk with glucosinolates.


title: Two Types of Cinnamoyl-CoA Reductase Function Divergently in Tissue Lignification, Phenylpropanoids Flux Control, and Inter-pathway Cross-talk with Glucosinolates as Revealed in Brassica napus


title: Meta-Research: Citation needed? Wikipedia and the COVID-19 pandemic

abstract:
BackgroundWith the COVID-19 pandemics outbreak, millions flocked to Wikipedia for updated information. Amid growing concerns regarding an "infodemic", ensuring the quality of information is a crucial vector of public health. Investigating if and how Wikipedia remained up to date and in line with science is key to formulating strategies to counter misinformation. Using citation analyses, we asked: which sources informed Wikipedias COVID-19-related articles before and during the pandemics first wave (January-May 2020).

ResultsWe found that coronavirus-related articles referenced trusted media sources and high-quality academic research. Moreover, despite a surge in COVID-19 preprints, Wikipedia had a clear preference for open-access studies published in respected journals and made little use of preprints. Building a timeline of English COVID-19 articles from 2001-2020 revealed a nuanced trade-off between quality and timeliness. It further showed how preexisting articles on key topics related to the virus created a framework for integrating new knowledge. Supported by a rigid sourcing policy, this "scientific infrastructure" facilitated contextualization and regulated the influx of new information. Lastly, we constructed a network of DOI-Wikipedia articles, which showed the shifting landscape of pandemic-related knowledge on Wikipedia and how academic citations create a web of shared knowledge supporting topics like COVID-19 vaccine development.

ConclusionsUnderstanding how scientific research interacts with the digital knowledge-sphere during the pandemic provides insight into how Wikipedia can facilitate access to science. It also reveals how, aided by what we term its "citizen encyclopedists", it successfully fended off COVID-19 disinformation and how this unique model may be deployed in other contexts.

category: scientific communication and education


title: Citation needed? Wikipedia and the COVID-19 pandemic


title: Citation needed?Wikipedia bibliometrics during the first waveof the COVID-19 pandemic.


title: Practical Identifiability in the Frame of Nonlinear Mixed Effects Models: the Example of the in vitro Erythropoiesis

abstract: Nonlinear mixed effects models provide a way to mathematically describe experimental data involving a lot of inter-individual heterogeneity. In order to assess their practical identifiability and estimate confidence intervals for their parameters, most mixed effects modelling programs use the Fisher Information Matrix. However, in complex nonlinear models, this approach can mask practical unidentifiabilities. Herein we rather propose a multistart approach, and use it to simplify our model by reducing the number of its parameters, in order to make it identifiable. Our model describes several cell populations involved in the in vitro differentiation of chicken erythroid progenitors grown in the same environment. Inter-individual variability observed in cell population counts is explained by variations of the differentiation and proliferation rates between replicates of the experiment. Alternatively, we test a model with varying initial condition. We conclude by relating experimental variability to precise and identifiable variations between the replicates of the experiment of some model parameters.

category: systems biology


title: Characterization of induction methods for Drosophila seizure mutations

abstract:
Epilepsy is one of the most common neurological disorders. Around one third of patients do not respond to current medications. This lack of treatment indicates a need for better understanding of the underlying mechanisms and, importantly, the identification of novel targets for drug manipulation. The fruitfly Drosophila melanogaster has a fast reproduction time, powerful genetics, and facilitates large sample sizes, making it a strong model of seizure mechanisms. However, there has not yet been a systematic analysis of the wide range of behavioral and physiological phenotypes observed across major fly seizure genotypes. To understand this, we systematically measured seizure severity and secondary behavioral phenotypes at both the larval and adult stage. Comparison of several seizure-induction methods; specifically electrical, mechanical and heat-induction, show that larval electroshock is the most effective at inducing seizures across a wide range of seizure-prone mutants. Locomotion in adults and larvae was found to be non-predictive of seizure susceptibility. Recording activity in identified larval motor neurons revealed variations in action potential patterns, across different genotypes, but these patterns did not correlate with seizure susceptibility. To conclude, while there is wide variation in mechanical induction, heat induction, and secondary phenotypes, electroshock is the most consistent method of seizure induction across known major seizure genotypes in Drosophila.

Significance StatementEpilepsy is a neurological disorder affecting 1 in 130 people globally, with a significant impact on patients, families, and society. Approximately one third of epileptics do not respond to currently available medication. Thus, better insights into underlying disease mechanisms and identification of new drugs are needed. Fruit flies (Drosophila melanogaster) are a powerful genetic model: a number of single gene mutant flies exhibit seizures, phenotypes that have been shown to respond to established antiepileptic drugs. We compare methods of seizure induction and their utility, to establish which induction method is the most consistent across a range of different seizure-inducing genetic backgrounds. Adopting a common method for seizure analysis in this model will, we predict, speed identification of novel anti-convulsive treatments.

category: neuroscience


title: Comparison of cerebrospinal fluid biomarkers relevant to neurodegenerative diseases in healthy cynomolgus and rhesus macaque monkeys

abstract:
Structured AbstractO_ST_ABSINTRODUCTIONC_ST_ABSNon-human primates are important translational models of neurodegenerative disease. We characterized how species, sex, age, and site of sampling affected concentrations of key biomarkers of neurodegeneration.

METHODSAmyloid-beta (A{beta}40, A{beta}42), tau (tTau, pTau), and neurofilament light (NFL) in CSF were measured in 82 laboratory-housed naive cynomolgus and rhesus macaques of both sexes.

RESULTSA{beta}40, A{beta}42, and NFL were significantly higher in rhesus compared with cynomolgus macaques. tTau and NFL were higher in males. pTau was not affected by species or sex. Site of acquisition only affected NFL, with NFL being higher in CSF acquired from lumbar compared with cisterna magna puncture.

DISCUSSIONNormative values for key neurodegeneration biomarkers were established for laboratory housed cynomolgus and rhesus macaque monkeys. Differences were observed as a function of species, sex and site of CSF acquisition that should be considered when employing primate models.

Research In ContextO_LISystematic review: We reviewed reports characterizing CSF biomarkers of neurodegenerative diseases in non-human primates - an increasingly important model of disease - revealing that studies with laboratory housed macaque monkeys were of small sample size, with a paucity of data about how biomarkers varied as a function of species, sex, age, and site of acquisition.
C_LIO_LIInterpretation: To address this gap, we collected CSF from 82 naive laboratory housed male and female macaques of two species and measured A{beta}40, A{beta}42, tTau, pTau, and NFL. In addition to providing normative statistics for concentrations of these biomarkers, we revealed various species and sex differences.
C_LIO_LIFuture directions: Establishing normative values of biomarkers is an important step to the efficient development of cynomolgus and rhesus macaques as models of neurodegenerative disorders such as Alzheimers disease. Reference values reduce the need for large control groups by which to compare with disease model animals.
C_LI


title: Non-canonical Hedgehog signaling through L-type voltage gated Ca2+ channels controls CD8+ T cell killing

abstract:
Cytotoxic CD8+ T lymphocytes (CTLs) are critical to the immune response against intracellular pathogens and cancer and act by eliminating infected and malignant cells through targeted secretion of cytotoxic granules. Hedgehog (Hh) signaling has been shown to be critical for CTL killing. Interestingly, Hh signaling in CD8+ T cells is not induced by extracellular Hh ligands but is initiated upon T cell receptor (TCR) engagement. How the TCR induces the Hh pathway independently of extracellular Hh ligands is unknown. Here we show that the Hh transcription factor Gli1 is essential for efficient CTL function and is induced downstream of the TCR by an extracellular Ca2+ influx selectively controlled by L-type voltage gated Ca2+ channels localized at the plasma membrane. We demonstrate that this novel mode of Hh signaling induction is independent of the canonical Hh pathway and represents the primary mechanism of Gli1 induction in naive CD8+ T cells, while CTLs can also activate Gli1 via MAP Kinase signaling. Importantly, we show that this L-type voltage gated Ca2+ channel-controlled Gli1 induction is functionally required for CTL killing in mice and humans. Gli inhibitors are currently in clinical trials against various cancers and our observations indicate that they likely inhibit the anti-tumor response.

Significance statementCytotoxic CD8+ T cells (CTLs) kill infected and malignant cells by targeted secretion of cytotoxic granules. Hedgehog signaling is critical for effective CTL killing and is activated by the T cell receptor (TCR) independently of exogenous Hedgehog ligands. This study shows that Hedgehog transcription factor Gli1 is required for CTL killing and identifies L-type voltage gated Ca2+ channels (Cav1) as essential regulators of CTL killing in mouse and human, by virtue of their ability to activate Gli1 downstream of the TCR. This Cav1-Gli1 axis operates independently of canonical Hedgehog signaling. Our work suggests that caution is required when using Gli inhibitors, currently in trials as anti-cancer therapeutics, since they may dampen the anti-tumor response.


title: CD47 as a potential biomarker for the early diagnosis of severe COVID-19

abstract: The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Most SARS-CoV-2 infections are mild or even asymptomatic. However, a small fraction of infected individuals develops severe, life-threatening disease, which is caused by an uncontrolled immune response resulting in hyperinflammation. Antiviral interventions are only effective prior to the onset of hyperinflammation. Hence, biomarkers are needed for the early identification and treatment of high-risk patients. Here, we show in a range of model systems and data from post mortem samples that SARS-CoV-2 infection results in increased levels of CD47, which is known to mediate immune escape in cancer and virus-infected cells. Systematic literature searches also indicated that known risk factors such as older age and diabetes are associated with increased CD47 levels. High CD47 levels contribute to vascular disease, vasoconstriction, and hypertension, conditions which may predispose SARS-CoV-2-infected individuals to COVID-19-related complications such as pulmonary hypertension, lung fibrosis, myocardial injury, stroke, and acute kidney injury. Hence, CD47 is a candidate biomarker for severe COVID-19. Further research will have to show whether CD47 is a reliable diagnostic marker for the early identification of COVID-19 patients requiring antiviral therapy.


title: Polymer physics and machine learning reveal a combinatorial code linking chromatin 3D architecture to 1D epigenetics

abstract: The mammalian genome has a complex 3D organization, serving vital functional purposes, yet it remains largely unknown how the multitude of specific DNA contacts, e.g., between transcribed and regulatory regions, is orchestrated by chromatin organizers, such as Transcription Factors. Here, we implement a method combining machine learning and polymer physics to infer from only Hi-C data the genomic 1D arrangement of the minimal set of binding sites sufficient to recapitulate, through only physics, 3D contact patterns genome-wide in human and mouse cells. The inferred binding sites are validated by their predictions on how chromatin refolds in a set of duplications at the Sox9 locus against available independent cHi-C data, showing that their different phenotypes originate from distinct enhancer hijackings in their 3D structure. Albeit derived from only Hi-C, our binding sites fall in epigenetic classes that well match chromatin states from epigenetic segmentation studies, such as active, poised and repressed states. However, the inferred binding domains have an overlapping, combinatorial organization along chromosomes, missing in epigenetic segmentations, which is required to explain Hi-C contact specificity with high accuracy. In a reverse approach, the epigenetic profile of binding domains provides a code to derive from only epigenetic marks the DNA binding sites and, hence, the 3D architecture, as validated by successful predictions of Hi-C matrices in an independent set of chromosomes. Overall, our results shed light on how complex 3D architectural information is encrypted in 1D epigenetics via the related, combinatorial arrangement of specific binding sites along the genome.

category: molecular biology


title: Leveraging three-dimensional chromatin architecture for effective reconstruction of enhancer-target gene regulatory network

abstract:
A growing amount of evidence in literature suggests that germline sequence variants and somatic mutations in non-coding distal regulatory elements may be crucial for defining disease risk and prognostic stratification of patients, in genetic disorders as well as in cancer. Their functional interpretation is challenging because genome-wide enhancer-target gene (ETG) pairing is an open problem in genomics. The solutions proposed so far do not account for the most updated knowledge on chromatin three-dimensional (3D) architecture, which is organized in a hierarchy of structural domains.

Here we introduce a paradigm shift based on the definition of multi-scale structural chromatin domains, integrated in a statistical framework to define ETG pairs. In this work i) we develop a computational and statistical framework to reconstruct a comprehensive ETG regulatory network leveraging functional genomics data; ii) we demonstrate that the incorporation of chromatin 3D architecture information improves ETG pairing accuracy; and iii) we use multiple experimental datasets to extensively benchmark our method against previous solutions for the genome-wide reconstruction of ETG pairs. This solution will facilitate the annotation and interpretation of sequence variants in distal non-coding regulatory elements. We expect this to be especially helpful in clinically oriented applications of whole genome sequencing in cancer and undiagnosed genetic diseases research.


title: Identify phage hosts from metaviromic short reads based on deep learning and Markov chain model

abstract: Phages - viruses that infect bacteria and archaea - are dominant in the virosphere and play an important role in the microbial community. It is very important to identify the host of a given phage fragment from metavriome data for understanding the ecological impact of phage in a microbial community. State-of-the-art tools for host identification only present reliable results on long sequences within a narrow candidate host range, while there are a large number of short fragments in real metagenomic data and the taxonomic composition of a microbial community is often complicated. Here, we present a method, named HoPhage, to identify the host of a given phage fragment from metavirome data at the genus level. HoPhage integrates two modules using the deep learning algorithms and the Markov chain model, respectively. By testing on both the artificial benchmark dataset of phage contigs and the real virome data, HoPhage demonstrates a satisfactory performance on short fragments within a wide candidate host range at every taxonomic level. HoPhage is freely available at http://cqb.pku.edu.cn/ZhuLab/HoPhage/.


title: Cryo-EM structure of lipid embedded human ABCA7 at 3.6 &Aring resolution

abstract: Phospholipid extrusion by ABC subfamily A (ABCA) exporters is central to cellular physiology, although the specifics of the underlying substrate interactions and transport mechanisms remain poorly resolved at the molecular level. Here we report cryo-EM structures of lipid-embedded human ABCA7 in an open state and a nucleotide-bound, closed state at resolutions between 3.6-4.0 [A]. The former reveals an ordered patch of bilayer lipids traversing the transmembrane domain (TMD), while the latter reveals a lipid-free, closed TMD with a small extracellular opening. These structures offer a structural framework for both substrate entry and exit from the ABCA7 TMD and highlight conserved rigid-body motions that underlie the associated conformational transitions. Combined with functional analysis and molecular dynamics (MD) simulations, our data also shed light on lipid partitioning into the ABCA7 TMD and localized membrane perturbations that underlie ABCA7 function and have broader implications for other ABCA family transporters.

category: biophysics


title: Capturing the interplay of membrane lipids and structural transitions in human ABCA7


title: Copy number variant heterogeneity among cancer types reflects inconsistent concordance with diagnostic classifications

abstract:
Due to frequent genome instability and accumulation of mutations during the neoplastic process, malignant tumors present with patterns of somatic genome variants on diverse levels of heterogeneity. The delineation of pathophysiological consequences of these patterns remains one of the main challenges in cancer prognosis and treatment.

Although continuous efforts aim for better characterization of cancer entities through inclusion of molecular characteristics, current ontology systems still heavily rely on clinico-pathological features. Traditionally, malignant diseases have been classified using domain-specific or generalized classification systems, based on histopathological features and clinical gestalt. Aside from the general purpose "International Classification for Diseases in Oncology" (ICD-O; WHO), hierarchical terminologies such as NCIt promote data interoperability and ontology-driven computational analysis.

To evaluate two prominent, general cancer classification systems (NCIt and ICD-O) towards their concordance with genomic mutation patterns we have performed a data-driven meta-analysis of 83505 curated cancer samples with genome-wide CNA (copy number aberration) profiles from our Progenetix database. The analysis provides a basis to assess the correspondence level of existing classification systems with respect to homogeneous molecular groups, and how individual codes represent an adequately detailed classification.

category: cancer biology


title: Phosphate transporter genes: genome-wide identification and characterization in Camelina sativa

abstract: Phosphorus is known as a key element associated with growth, energy, and cell signaling. In plants, phosphate transporters (PHTs) are responsible for moving and distributing phosphorus in cells and organs. PHT genes have been genome-wide identified and characterized in various plant species, however, these genes have not been widely identified based on available genomic data in Camellia sativa, which is an important oil seed plant. In the present study, we found 66 PHT genes involved in phosphate transporter/translocate in C. sativa. The recognized genes belonged to PHTs1, PHTs2, PHTs4, PHOs1, PHO1 homologs, glycerol-3-PHTs, sodium dependent PHTs, inorganic PHTs, xylulose 5-PHTs, glucose-6-phosphate translocators, and phosphoenolpyruvate translocators. Our finding revealed that PHT proteins are divers based on their physicochemical properties such as Isoelectric point (pI), molecular weight, GRAVY value, and exon-intron number(s). Besides, the expression profile of PHT genes in C. sativa based on RNA-seq data indicate that PHTs are involved in response to abiotic stresses such as cold, drought, salt, and cadmium. The tissue specific expression high expression of PHO1 genes in root tissues of C. sativa. In additions, four PHTs, including a PHT4;5 gene, a sodium dependent PHT gene, and two PHO1 homolog 3 genes were found with an upregulation in response to aforementioned studied stresses. In the current study, we found that PHO1 proteins and their homologs have high potential to post-translation modifications such as N-glycosylation and phosphorylation. Besides, different cis-acting elements associated with response to stress and phytohormone were found in the promoter region of PHT genes. Overall, our results show that PHT genes play various functions in C. Sativa and regulate Camellia responses to external and intracellular stimuli. The results can be used in future studies related to the functional genomics of C. sativa.


title: Analysis of Classic Tomato Mutants Reveals Influence of Leaf Vein Density on Fruit BRIX

abstract: Tomato bipinnate (bip) is a classic leaf mutant, with highly increased leaf complexity resulting from the loss of function of a BEL-LIKE HOMEODAMAIN (BELL) gene. Here, we analyzed several bip mutants and their isogenic wildtype backgrounds for a suite of leaf morphology traits, ranging from leaf complexity, leaflet shape and size, to leaf vascular density to investigate how changes in leaf morphology influence fruit traits. Our analyses showed an unexpected relationship between leaf vein density and fruit sugar levels, where leaf vein density was negatively correlated with fruit BRIX. RNA-Seq analysis suggested variation in Glucose-6-phosphate translocator2 (GPT2) gene expression caused correlated changes in leaf vein density and BRIX when bip mutant and wildtype were compared, suggesting that the correlation between leaf vein density and fruit sugar may result from the genes regulating leaf vein development that are also involved in regulating leaf sugar biosynthesis. Our results provide a resource for further exploration of the genetic basis for the complex relationship between fruit quality and leaf traits in natural populations.


title: The Fkh1 Forkhead Associated Domain Promotes ORC Binding to a Subset of DNA Replication Origins in Budding Yeast

abstract: The pioneer event in eukaryotic DNA replication is binding of chromosomal DNA by the origin recognition complex (ORC), which directs the formation of origins, the specific chromosomal regions where DNA will be unwound for the initiation of DNA synthesis. In all eukaryotes, incompletely understood features of chromatin promote ORC-DNA binding. Here, we uncover a role for the Fkh1 (forkhead homolog) protein, and, in particular, its forkhead associated (FHA) domain in promoting ORC-origin binding and origin activity at a subset of origins in Saccharomyces cerevisiae. The majority of the FHA-dependent origins within the experimental subset examined contain a distinct Fkh1 binding site located 5 of and proximal to their ORC sites (5-FKH-T site). Epistasis experiments using selected FHA-dependent origins provided evidence that the FHA domain promoted origin activity through Fkh1 binding directly to this 5 FKH-T site. Nucleotide substitutions within two of these origins that enhanced the affinity of their ORC sites for ORC bypassed these origins requirement for their 5 FKH-T sites and for the FHA domain. Significantly, direct assessment of ORC-origin binding by ChIPSeq provided evidence that this mechanism affected ~25% of yeast origins. Thus, this study reveals a new mechanism to enhance ORC-origin binding in budding yeast that requires the FHA domain of the conserved cell-cycle transcription factor Fkh1.


title: RNA-sequencing indicates immune cell signaling and inflammatory gene expression in cardiac fibroblasts increases with developmental age

abstract:
BackgroundCardiac fibroblasts are responsible for extracellular matrix turnover and repair in the cardiac environment and serve to help facilitate immune responses. However, it is well established that they have significant phenotypic heterogeneity with respect to location, physiological conditions, and developmental age. The goal of this study was to provide an in-depth transcriptomic profile of cardiac fibroblasts derived from rat hearts at fetal, neonatal, and adult developmental ages to ascertain variations in gene expression that may drive functional differences in these cells at these specific stages of development.

ResultsWe performed RNA-seq of cardiac fibroblasts isolated from fetal, neonatal, and adult rats was performed and compared to the rat genome. Principal component analysis of RNA-seq data suggested data variance was predominantly due to developmental age. Differential expression and Gene set enrichment analysis against Gene Ontology and Kyoto Encyclopedia of Genes and Genomes datasets indicated an array of differences across developmental ages, including significant decreases in cardiac development and cardiac function-associated genes with age, and a significant increase in immune and inflammatory-associated functions - particularly immune cell signaling, and cytokine and chemokine production - with respect to increasing developmental age.

ConclusionThese results reinforce established evidence of diverse phenotypic heterogeneity of fibroblasts with respect to developmental age. Further, based on our analysis of gene expression, age-specific alterations in cardiac fibroblasts may play a crucial role in observed differences in cardiac inflammation and immune response observed across developmental ages.


title: A drastic shift in the energetic landscape of toothed whale sperm cells

abstract: Mammalia spermatozoa are a notable example of energetic compartmentalization. While mitochondrial oxidative phosphorylation is restricted to the midpiece, sperm-specific glycolysis operates in the flagellum. Consequently, these highly specialized cells exhibit a clear adaptability to fuel substrates. This plasticity is essential to ensure sperm motility, and is known to vary among species. Here we describe an extreme example of spermatozoa-energetics adaptation. We show that toothed whales exhibit impaired sperm glycolysis, due to gene and exon erosion, and demonstrate that dolphin spermatozoa motility depends uniquely on endogenous fatty acid {beta}-oxidation, but not carbohydrates. Our findings substantiate the observation of large mitochondria in spermatozoa, possibly boosting ATP production from endogenous fatty acids. This unique energetic rewiring emphasizes the physiological body reorganisation imposed by the carbohydrate-depleted marine environment.


title: MoBiFC: development of a modular bimolecular fluorescence complementation toolkit for the analysis of chloroplast protein-protein interactions.

abstract: The bimolecular fluorescence complementation (BiFC) assay has emerged as one of the most popular methods for analysing protein-protein interactions (PPIs) in plant biology. This includes its increasing use as a tool for dissecting the molecular mechanisms of chloroplast function. However, the construction of chloroplast fusion proteins for BiFC can be difficult, and the availability and selection of appropriate controls is not trivial. Furthermore, the challenges of performing BiFC in restricted cellular compartments has not been specifically addressed. Here we describe the development of a flexible modular cloning-based toolkit (MoBiFC) for chloroplast BiFC and proximity labelling using synthetic biology principles. The approach facilitates the cloning process for chloroplast-targeted proteins, allows robust ratiometric quantification, and the toolkit comes with model positive and negative controls. Our study also highlights many potential pitfalls including the choice of fluorescent protein (FP) split, negative controls, cell type, and reference FP. Finally, we provide an example of how users can enrich the toolset by providing functional proximity labelling modules, and we discuss how MoBiFC could be further improved and extended to other compartments of the plant cell.


title: Periodic synchronization of dengue epidemics in Thailand: the roles played by temperature and immunity

abstract:
The spatial distribution of dengue and its vectors (spp. Aedes) may be the widest it has ever been, and projections suggest that climate change may allow the expansion to continue. However, the largest impacts of climate change on dengue might be in regions where the pathogen is already endemic. In these areas, the waxing and waning of immunity has a large impact on temporal dynamics of cases of dengue haemorrhagic fever. Here, we use 51 years of data across 72 provinces and characterise spatio-temporal patterns of dengue in Thailand, where dengue has caused almost 1.5 million cases over the last thirty years, and examine the roles played by temperature and dynamics of immunity in giving rise to those patterns. We find that timescales of multiannual oscillations in dengue vary in space and time and uncover an interesting spatial phenomenon: Thailand has experienced multiple, periodic synchronization events. We show that patterns in synchrony of dengue are consistent with those observed in temperature. Applying a temperature-driven dengue model, we explore how dynamics of immunity interact with temperature to produce the observed multiannual dynamics and patterns in synchrony. While multiannual oscillations are readily produced by immunity in absence of multiannual timescales in temperature, synchrony in temperature can synchronise dengue dynamics in different locations. However, at higher mean temperatures and lower seasonal variation, immune dynamics become more predominant, and dengue dynamics become more insensitive to multiannual fluctuations in temperature. These findings can help underpin predictions of disease patterns as global temperatures rise.

Author summary

category: ecology


title: Species-wide transposable element repertoires retrace the evolutionary history of the Saccharomyces cerevisiae host

abstract:
Transposable elements (TE) are an important source of genetic variation with a dynamic and content that greatly differ in a wide range of species. The origin of the intraspecific content variation is not always clear and little is known about the precise nature of it. Here, we surveyed the species-wide content of the Ty LTR-retrotransposons in a broad collection of 1,011 Saccharomyces cerevisiae natural isolates to understand what can stand behind the variation of the repertoire, i.e. the type and number of Ty elements. We have compiled an exhaustive catalog of all TE variants present in the S. cerevisiae species by identifying a large set of new variants. The characterization of the TE content in each isolate clearly highlighted that each subpopulation exhibits a unique and specific repertoire, retracing the evolutionary history of the species. Most interestingly, we have shown that ancient interspecific hybridization events had a major impact in the birth of new variants and therefore in the shaping of the TE repertoires. We also investigated the transpositional activity of these elements in a large set of natural isolates, and we found a broad variability related to the level of ploidy as well as the genetic background. Overall, our results pointed out that the evolution of the Ty content is deeply impacted by clade-specific events such as introgressions and therefore follows the population structure. In addition, our study lays the foundation for future investigations to better understand the transpositional regulation and more broadly the TE-host interactions.

Authors summaryMobile DNA elements are widely distributed in the genomes of many eukaryotes, but their contents greatly vary between species, populations and even individuals. In fact, little is known about the origin of this variation of transposable element (TE) content across individuals of the same species. Here, we surveyed the Ty LTR-retrotransposon content in a broad collection of 1,011 Saccharomyces cerevisiae yeast natural isolates. We have defined an exhaustive and precise catalog of the TE variants present in the S. cerevisiae species. We found that the TE content follows the evolutionary history of the species because each subpopulation has a unique and specific content. Interestingly, our results highlighted that ancient interspecific hybridization events led to the appearance of new TE variants and therefore had a strong impact on the variation of the TE repertoires in this species. We also investigated the transpositional activity of these elements and found a wide variability related to the genetic background diversity. Altogether, our results have led to a better understanding of the variability of TE content at a species level.


title: Self-Assembling Peptide Hydrogels - PeptiGels(R) as a Platform for Hepatic Organoid Culture

abstract: A major challenge in advancing preclinical studies is the lack of robust in vitro culture systems that fully recapitulate the in vivo scenario together with limited clinical translational to humans. Organoids, as 3-dimensional (3D) self-replicating structures are increasingly being shown as powerful models for ex vivo experimentation in the field of regenerative medicine and drug discovery. Organoid formation requires the use of extracellular matrix (ECM) components to provide a 3D platform. However, the most commonly used ECM, essential for maintaining organoid growth is Matrigel and is derived from a tumorigenic source which limits its translational ability. PeptiGels(R) which are self-assembling peptide hydrogels present as alternatives to traditional ECM for use in 3D culture systems. Synthetic PeptiGels(R) are non-toxic, biocompatible, biodegradable and can be tuneable to simulate different tissue microenvironments. In this study, we validated the use of different types of PeptiGels(R) for porcine hepatic organoid growth. Hepatic organoids were assessed morphologically and using molecular techniques to determine the optimum PeptiGel(R) formulation. The outcome clearly demonstrated the ability of PeptiGel(R) to support organoid growth and offer themselves as a technological platform for 3D cultured physiologically and clinically relevant data.

category: bioengineering


title: DNMT family induced down-regulation of NDRG1 via DNA methylation and clinicopathological significance in gastric cancer

abstract:
BackgroundAberrant DNA methylation of tumor suppressor genes is a common event in the development and progression of gastric cancer(GC). Our previous study showed NDRG1, which could suppress cell invasion and migration, was frequently down-regulated by DNA methylation of its promoter in GC.

Purpose and MethodsTo analyze the relationship between the expression and DNA methylation of NDRG1 and DNA methyltransferase (DNMT) family. We performed a comprehensive comparison analysis using 407 patients including sequencing analysis data of GC from TCGA.

ResultsNDRG1 was negatively correlative to DNMT1 (p =0.03), DNMT3A(p =0.01), DNMT3B(p =0.88), respectively. Whereas, the DNA methylation of NDRG1 was positively correlative to DNMT family(DNMT1 p<0.01, DNMT3A p<0.001, DNMT3B p=0.57, respectively). NDRG1 expression was significantly inverse correlated with invasion depth (p =0.023), and DNMT1 was significantly positive correlated with the degree of tumor cell differentiation (p =0.049). DNMT3B was significantly correlated with tumor cell differentiation (p =0.030). However, there was no association between the expression of DNMT3A and clinicopathological features. The univariate analysis showed that NDRG1and DNMTs had no association with prognosis of GC patients. But, multivariate analysis showed DNMT1 was significantly correlated with prognosis of GC patients.

ConclusionThese data suggest that down-regulation of NDRG1 in gastric cancer is due to DNA methylation of NDRG1 gene promoter via DNMT family. The demethylating agent maybe a potential target drug for GC patients.


title: The time to peak blood bicarbonate (HCO3-), pH, and strong ion difference (SID) following sodium bicarbonate (NaHCO3) ingestion in highly trained adolescent swimmers

abstract:
BackgroundContemporary research suggests that the optimal timing of sodium bicarbonate (NaHCO3) should be based upon an individual time in which bicarbonate (HCO3-) or pH peaks within the blood. However, the mechanisms surrounding acidosis on exercise performance are contested, therefore it is plausible that the ergogenic effects of NaHCO3 are instead a result of an increased strong ion difference (SID) following ingestion. Since the post-ingestion time course of the SID is currently unknown, the purpose of this study was to investigate the pharmacokinetics of the SID in direct comparison to HCO3- and pH.

MethodsTwelve highly trained, adolescent swimmers (age: 15.9 {+/-} 1.0 yrs, body mass: 65.3 {+/-} 9.6 kg) consumed their typical pre-competition nutrition before ingesting 0.3 g{middle dot}kg BM-1 NaHCO3 in gelatine capsules. Capillary blood samples were then taken during quiet, seated rest on nine occasions (0, 60, 75, 90, 105, 120, 135, 150, and 165 min post-ingestion) for the assessment of time course changes in HCO3-, pH, and the SID.

ResultsOn a group mean level, no differences were found in the time in which each variable peaked within the blood (HCO3- = 130 {+/-} 35 min, pH = 120 {+/-} 38 min, SID = 96 {+/-} 35 min; p = 0.06). A large effect size was calculated between the timing of peak HCO3- and the SID (g = 0.91), however, suggesting that a difference may occur between these two measures in practice.

ConclusionsA time difference between peak HCO3- and the SID presents an interesting avenue for further research since an approach based upon individual increases in extracellular SID has yet to be investigated. Future studies should therefore compare these dosing strategies directly to elucidate whether either one is more ergogenic for exercise performance.

category: physiology


title: Biotransformation of agro-industrial waste to wealth in Africa through mushroom farming

abstract: Discarding Waste in open drainages, water bodies and unauthorized dumpsites is a common practice in Africa that is deleterious to the global ecosystem. Global warming, flooding and land encroachment are direct impact of these actions. Therefore, a proposed eco-friendly solution to efficient waste management in Africa is "Mushroom Farming". The region between Latitude 6.43-7.95{degrees}N and Longitude 2.88-5.24{degrees}E (Nigeria) was the research focus. Mushroom samples were collected, identified, coded and geo-tagged in the field, while Laboratory analyses and mushroom cultivation was conducted in NIHORT and University of Ibadan. The result showed that postharvest waste (rice straw) was best for spawning (Ek1-8[->]23days, LA1-8[->]21days, OG1-8[->]20days, and OY1-8[->]22days), while agro-waste from Gliricidium sepium facilitated early pin head emergence (26days) and fruiting (28days). Also, agro-waste from Cedrela odorata facilitated early maturation (3days after fruiting), and those from Mangifera indica, G. sepium and C. odorata, improved yield (9.05g), pileus size (4.24g) and dry matter (3.01g), respectively. The Laboratory analyses showed that LA1-8 had the best N2, Na and Ca contents (13.59, 70.49 and 61.90mg/100g, respectively), while fat, fibre and carbohydrate contents were highest in samples from EK1-8 (6.60%), OS1-8 (25.13%) and OG1-8 (54.23%), respectively. Mushroom farming is the key to efficient transformation of waste to wealth in Africa.


title: A role for the immune system-released activating agent (ISRAA) in the ontogenetic development of brain astrocytes

abstract: The Immune System-Released Activating Agent (ISRAA) was discovered as a novel molecule that functions as a mediator between the nervous and immune systems in response to a nervous stimulus following an immune challenge. This research investigated the role of ISRAA) in promoting the ontogeny of the mouse brain astrocytes. Astrocyte cultures were prepared from two-month-old BALB/c mice. Recombinant ISRAA protein was used to stimulate astrocyte cultures. Immunohistochemistry and ELISA were utilized to measure ISRAA and IFN-{gamma} levels, IFN-{gamma}R expression and STAT1 nuclear translocation. MTT-assay was used to evaluate cellular survival and proliferation. To assess astrocyte cell lysates and tyrosine-phosphorylated proteins, SDS-PAGE and western blot were used. ISRAA was highly expressed in mouse embryonic astrocytes, depending on cell age. Astrocytes aged seven days (E7) showed increased proliferation and diminished differentiation, while 21-day-old (E21) astrocytes depicted reversed effects. ISRAA stimulated the tyrosine phosphorylation of numerous cellular proteins and the nuclear translocation of STAT1. IFN-{gamma} was involved in the ISRAA action as ISRAA induced IFN-{gamma} in both age groups, but only E21 astrocytes expressed IFN-{gamma}R. The results suggest that ISRAA is involved in mouse brain development through the cytokine network involving IFN-{gamma}.


title: Sandalwood, an Indian medicinal plant attenuates the microbial growth and influence up/down regulation of the metabolites

abstract: The methanolic extract of sandalwood (SwME) was prepared by soxhlet apparatus and the antibacterial assay was performed. Further, the metabolite profiling of SwME and lysates of E. coli and E. coli grown in the presence of SwME was generated. SwME showed maximum inhibition against E. coli (MTCC 443) i.e. 82.71%, and minimal against B. subtilis (MTCC 736) i.e. 26.82%. The metabolome profiles of E. coli and SwME were generated using gas chromatography-mass spectrometry (GC-MS) technique. Comparative studies were done to understand to what extent metabolite modifications differ between SwME, E. coli lysate and the E. coli strain grown in presence of extract. Result revealed 23 peaks with major compounds present in E. coli were 9-Octadecenoic Acid (Z)-, Methyl Ester (26.85%), Hexadecanoic Acid, methyl ester (20.5%) and Hexadecanoic acid, trimethylsilyl ester (15.79%). When E. coli was grown in the presence of SwME, GC-MS analysis showed 25 peaks with major compounds such as 9-Octadecenoic Acid, Methyl Ester (21.97%), Hexadecanoic Acid, Methyl Ester (17.03%), and Hexadecanoic Acid, Trimethylsilyl Ester (14.96%). Correlating the metabolic profiles with the changes occurring is essential to progression their comprehension and in the development of new approaches to identify the metabolomics regulation in E. coli in response to SwME.

category: microbiology


title: Protein phosphatase SppA regulates apical growth and dephosphorylates cell polarity determinant DivIVA in Streptomyces coelicolor

abstract:
Bacteria that exhibit polar growth, i.e. build their peptidoglycan cell walls in restricted zones at cell poles, often show large morphological diversity and plasticity. However, their mechanisms for regulation of cell shape and cell wall assembly are poorly understood. The Gram-positive Streptomyces bacteria, like other Actinobacteria, depend on the essential coiled coil protein DivIVA for establishment of cell polarity and direction of polar growth. Streptomycetes grow as filamentous hyphae that exhibit tip extension. New hyphal tips are generated by lateral branching. Cell shape is largely determined by the control of cell wall growth at these hyphal tips. The Ser/Thr protein kinase AfsK is involved in controlling polar growth and directly phosphorylates DivIVA. Here, we identify a protein phosphatase in Streptomyces coelicolor, SppA, that dephosphorylates DivIVA in vivo and in vitro and affects growth and cell shape. An sppA mutant shows reduced rate of hyphal tip extension, altered hyphal branching patterns, and exhibits frequent spontaneous hyphal growth arrests, all contributing to the unusually dense mycelial structure and slow growth rate that characterize sppA mutants. These phenotypes are largely suppressed in an afsK sppA double mutant, showing that AfsK and SppA partially affect the same regulatory pathway and share target proteins that are involved control of polar growth in S. coelicolor. Strains with a non-phosphorylatable mutant DivIVA were constructed and confirm that the effect of afsK on hyphal branching during normal growth is mediated by DivIVA phosphorylation. However, the phenotypic effects of sppA deletion are independent of DivIVA phosphorylation and must be mediated via other substrates. Altogether, this study identifies a PPP-family protein phosphatase directly involved in the control of polar growth and cell shape determination in S. coelicolor and underscore the importance of eukaryotic-type Ser/Thr phosphorylation in regulation of growth and cell envelope biogenesis in Actinobacteria.

Author summaryGram-positive bacteria often use eukaryotic-like Ser/Thr protein phosphorylation in the regulation of central processes related to growth and cell cycle progression. Members of the Actinobacteria, including pathogenic mycobacteria and antibiotic-producing streptomycetes, exhibit a distinctive mode of polar growth, with cell wall synthesis being restricted to zones at cell poles and directed by the essential cell polarity determinant DivIVA. Previously, we have shown that Streptomyces coelicolor modulates its machinery for polar growth and cell shape determination via the Ser/Thr kinase AfsK, which phosphorylates DivIVA. Here, we identify a phosphoprotein phosphatase that targets DivIVA and reverses the AfsK-mediated phosphorylation. We show that this phosphatase has strong effects on polar growth and cell shape, including hyphal branching and the rate of tip extension. The effects are to a large extent dependent on the kinase AfsK but do not require DivIVA phosphorylation as such. The results identify a new regulator of polar growth in streptomycetes, and highlight the importance of Ser/Thr phosphorylation in regulation of actinobacterial growth and cell shape determination.


title: Derivation and Characterization of an inositol phosphate-independent HIV-1

abstract:
A critical step in the HIV-1 replication cycle is the assembly of Gag proteins to form virions at the plasma membrane. Virion assembly and maturation is facilitated by the cellular polyanion inositol hexaphosphate (IP6), which is proposed to stabilize both the immature Gag lattice and the mature capsid lattice by binding to rings of primary amines at the center of Gag or capsid protein (CA) hexamers. The amino acids comprising these rings are critical for proper virion formation and their substitution results in assembly deficits or impaired infectiousness. To better understand the nature of the deficits that accompany IP6-deficiency, we passaged HIV-1 mutants that had substitutions in IP6-coordinating residues to select for compensatory mutations. We found a mutation, a threonine to isoleucine substitution at position 371 (T371I) in Gag, that restored replication competence to an IP6-binding-deficient HIV-1 mutant. Notably, unlike wild-type HIV-1, the assembly and infectiousness of resulting virus was not impaired when IP6 biosynthetic enzymes were genetically ablated. Surprisingly, we also found that the maturation inhibitor Bevirimat (BVM) could restore the assembly and replication of an IP6-binding deficient mutant. Moreover, using BVM-dependent mutants we were able to image the BVM-inducible assembly of individual HIV-1 particles assembly in living cells. Overall these results suggest that IP6-Gag and Gag-Gag contacts are finely tuned to generate a Gag lattice of optimal stability, and that under certain conditions BVM can functionally replace IP6.

Author SummaryA key step in HIV-1 replication is the assembly of virions that are released from the infected cell. Previous work has suggested that a small molecule called IP6 is critical role in this process, promoting both HIV-1 assembly and the stability of mature fully infectious virions. Since IP6 is required for multiple steps in HIV-1 assembly and maturation, it is a candidate for the development of anti-retroviral therapies. Here, we identify an HIV-1 mutant that replicates independently of IP6, and show that a different small molecule can functionally substitute for IP6 under certain conditions. These findings suggest that IP6 regulates the stability of protein interactions during virion assembly and that the precise degree stability of these interactions is finely tuned and important for generating infectious virions. Finally, our work identifies an inducible virion assembly system that can be utilized to visualize HIV-1 assembly events using live cell microscopy.


title: Disruption of the Pseudomonas aeruginosa Tat system perturbs PQS-dependent quorum sensing and biofilm maturation through loss of the Rieske cytochrome bc1 sub-unit

abstract:
Extracellular DNA (eDNA) is a major constituent of the extracellular matrix of Pseudomonas aeruginosa biofilms and its release is regulated via the pseudomonas quinolone signal (PQS) dependent quorum sensing (QS). By screening a P. aeruginosa transposon library to identify factors required for DNA release, mutants with insertions in the twin-arginine translocation (Tat) pathway were identified as exhibiting reduced eDNA release, and defective biofilm architecture with enhanced susceptibility to tobramycin. P. aeruginosa tat mutants showed substantial reductions in pyocyanin, rhamnolipid and membrane vesicle (MV) production consistent with perturbation of 2-heptyl-3-hydroxy-4-quinolone (PQS) dependent QS as demonstrated by changes in pqsA expression and 2-alkyl-4-quinolone (AQ) production. Provision of exogenous PQS to the tat mutants did not return pqsA, rhlA or phzA1 expression or pyocyanin production to wild type levels. However, transformation of the tat mutants with the AQ-independent pqs effector pqsE restored phzA1 expression and pyocyanin production. Since mutation or inhibition of Tat prevented PQS-driven auto-induction, we sought to identify the Tat secretion substrate responsible. A pqsA::lux fusion was introduced into each of 34 validated P. aeruginosa Tat substrate deletion mutants. Analysis of each mutant for reduced bioluminescence revealed that the signalling defect was associated with the Rieske iron-sulfur subunit of the cytochrome bc1 complex. In common with the parent strain, a Rieske mutant exhibited defective PQS signalling, AQ production, rhlA expression and eDNA release that could be restored by genetic complementation. Thus, lack of the Rieske sub-unit export is clearly responsible for the Tat-mediated perturbation of PQS-dependent QS, the loss of virulence factor production, biofilm eDNA and the tobramycin tolerance of P. aeruginosa biofilms.

Author SummaryPseudomonas aeruginosa is a highly adaptable human pathogen responsible for causing chronic biofilm-associated infections. Biofilms are highly refractory to host defences and antibiotics and thus difficult to eradicate. The biofilm extracellular matrix incorporates extracellular DNA (eDNA). This stabilizes biofilm architecture and helps confer tolerance to antibiotics. Since mechanisms that control eDNA release are not well understood, we screened a P. aeruginosa mutant bank for strains with defects in eDNA release and discovered a role for the twin-arginine translocation (Tat) pathway that exports folded proteins across the cytoplasmic membrane. Perturbation of the Tat pathway resulted in defective biofilms susceptible to antibiotic treatment as a consequence of perturbed pseudomonas quinolone (PQS) signalling. This resulted in the failure to produce or release biofilm components including eDNA, phenazines and rhamnolipids as well as microvesicles. Furthermore, we discovered that perturbation of PQS signalling was a consequence of the inability of tat mutants to translocate the Rieske subunit of the cytochrome bc1 complex involved in electron transfer and energy transduction. Given the importance of PQS signalling and the Tat system to virulence and biofilm maturation in P. aeruginosa, our findings underline the potential of the Tat system as a drug target for novel antimicrobial agents.


title: ASSOCIATION STUDY OF SIX CANDIDATE GENES WITH MAJOR DEPRESSIVE DISORDER IN THE NORTH-WESTERN POPULATION OF PAKISTAN

abstract: People around the world are currently affected by Major Depressive Disorder (MDD). Despite its many aspects, symptoms, manifestations and impacts, efforts have been made to identify the root causes of the disorder. In particular, genetic studies have concentrated on identifying candidate genes for MDD and exploring associations between these genes and some specific group of individuals. The aim of this research was to find out the association between single nucleotide polymorphisms in 6 candidate genes linked to the neurobiology of major depressive disorder in the North-Western population of Pakistan. We performed a case-control analysis, with 400 MDD and 232 controls. A trained psychiatrist or clinical psychologists evaluated the patients. Six polymorphisms were genotyped and tested for allele and genotype association with MDD. There were no statistical variations between MDD patients and healthy controls for genotypic and allelic distribution of all the polymorphisms observed. Thus, our analysis does not support the major role of these polymorphisms in contributing to MDD susceptibility, although it does not preclude minor impact. The statistically significant correlation between six polymorphisms and major depressive disorder in the studied population was not observed. There are inconsistencies in investigations around the world. Future research, including GWAS and association analysis on larger scale should be addressed for further validation and replication of the present findings.


title: The impact of mutations on the structural and functional properties of SARS-CoV-2 proteins: A comprehensive bioinformatics analysis

abstract: An in-depth analysis of first wave SARS-CoV-2 genome is required to identify various mutations that significantly affect viral fitness. In the present study, we have performed comprehensive in-silico mutational analysis of 3C-like protease (3CLpro), RNA dependent RNA polymerase (RdRp), and spike (S) proteins with the aim of gaining important insights into first wave virus mutations and their functional and structural impact on SARS-CoV-2 proteins. Our integrated analysis gathered 3465 SARS-CoV-2 sequences and identified 92 mutations in S, 37 in RdRp, and 11 in 3CLpro regions. The impact of those mutations was also investigated using various in silico approaches. Among these 32 mutations in S, 15 in RdRp, and 3 in 3CLpro proteins are found to be deleterious in nature and could alter the structural and functional behavior of the encoded proteins. D614G mutation in spike and P323L in RdRp are the globally dominant variants with a high frequency. Most of them have also been found in the binding moiety of the viral proteins which determine their critical involvement in the host-pathogen interactions and drug targets. The findings of the current study may facilitate better understanding of COVID-19 diagnostics, vaccines, and therapeutics.


title: Plasma concentrations of progesterone in the preceding estrous cycle are associated with the intensity of estrus and fertility of Holstein cows

abstract: The aim of this study was to determine the association between concentrations of progesterone (P4) during previous the estrous cycle with the intensity of spontaneous or estrogen-induced estrous expression and pregnancy per artificial insemination (P/AI). A total of 1,953 AI events from lactating Holstein cows were used, consisting of 1,289 timed AI events from experiment 1 (Exp. 1) and 664 AI events from experiment 2 (Exp. 2). In Exp. 1, cows were bred after a timed AI protocol based on estradiol and P4. In Exp. 2 animals were bred upon spontaneous estrus detection. In both experiments cows were continuously monitored by an automated activity monitor (AAM). The intensity of estrous expression was determined for each event and classified as either high or low intensity using the median of each experiment. Blood samples were collected for P4 analysis in Exp. 1 at -4 d, 0 d, and 7 d relative to timed AI, and in Exp. 2 immediately following estrus (0 d), 7 d, 14 d, and 21 d post-AI. Concentration of P4 was classified as greater or lower according to the median value in each experiment. Cows with lower concentrations of P4 at AI had greater estrous expression in Exp. 1 (363.6 {+/-} 5.2 vs. 275.9 {+/-} 8.0 % relative increase) and Exp. 2 (76.7 {+/-} 1.9 vs. 67.4 {+/-} 4.7 index; and 12.5 {+/-} 0.5 vs. 9.3 {+/-} 1.8 h). Cows with a greater intensity of estrous expression at timed AI had greater concentrations of P4 at -4 d than cows with lower intensity estrus or no estrous expression (4.6 {+/-} 0.2 vs. 3.6 {+/-} 0.2 vs. 3.7 {+/-} 0.2 ng/mL). Cows with greater concentrations of P4 at -4 d had greater P/AI (32.8 {+/-} 4.4 vs. 22.4 {+/-} 4.5 %), whereas cows with lesser concentrations of P4 at d0 for either timed AI (35.2 {+/-} 3.4 vs. 19.6 {+/-} 4.6 %) or spontaneous estrus (31.8 {+/-} 2.8 vs. 23.4 {+/-} 3.2 %) had greater P/AI. Cows with greater concentrations of P4 7 d post-timed AI had greater P/AI compared with cows that had lower concentration of P4 (39.1 {+/-} 2.9 vs. 24.7 {+/-} 2.6 %). Similarly, cows that had lower concentrations of P4 at 7 d, 14 d and 21 d post-spontaneous estrus tended to have lower P/AI when compared with cows with greater concentrations of P4. Overall, concentrations of P4 prior to and at AI were associated with greater estrous intensity and P/AI at both spontaneous and timed AI events.


title: Effects of accumulated temperature on growth curve of Gynostemma pentaphyllum stem and leaf under the conditions of Simulated light under the forest

abstract: The effects of accumulated temperature on the growth curve and leaf number growth curve of Gynostemma pentaphyllum were studied. The growth curve of twigs and strands were studied by curve regression analysis. The results showed that the growth of Gynostemma pentaphyllum Leaf growth curve was optimized, and the growth curve of stem and leaf of Gynostemma pentaphyllum was established, and the curve was fitted and analyzed. Three - dimensional model of the effect of accumulated temperature on the stem length and leaf number of Gynostemma pentaphyllum. The results show that the stem length L and the accumulated temperature t of the strands are in the logistic function, the mathematical model was L=-112.69/(1+(t/892.1)3.31)+130.54, the number of gibbere leaf number n is logistic function with the accumulated temperature t, The model was n=-27.86/(1+(t/1159.77)0.26)+30.37, and the growth model could reflect the dynamic growth of Gynostemma pentaphyllum growth. The experimental results provide a theoretical basis for choosing suitable habitat for Gynostemma pentaphyllum under the forest.


title: EHF is essential for epidermal and colonic epithelial homeostasis and suppresses Apc-initiated colonic tumorigenesis

abstract:
BackgroundEts homologous factor (EHF) is a member of the epithelial-specific Ets (ESE) transcription factors. EHF is specifically expressed in epithelial tissues, however its role in development and epithelial homeostasis is largely uncharacterized.

MethodsWe generated a novel mouse strain in which the Ets DNA binding domain (exon 8) of Ehf was flanked by loxP sites (EhfLox/Lox). To inactivate Ehf in the whole body, EhfLox/Lox mice were crossed to CMVCre mice, which were then bred out to generate germline Ehf null (Ehf-/-) mice. To inactivate Ehf specifically in the intestinal epithelium, EhfLox/Lox mice were bred to tamoxifen-inducible VillinCre-ERT2 mice. EhfLox/Lox mice were also crossed to tamoxifen-inducible Cdx2CreERT2; ApcLox/+ mice to determine the impact of Ehf deletion on Apc-initiated colon cancer development.

ResultsTranscripts encoding the Ets binding domain of EHF were effectively deleted in all tissues in Ehf-/- mice. Ehf-/- mice were born at the expected Mendelian ratio, but showed reduced body weight gain and developed a series of pathologies during their lifespan that led the majority of Ehf-/- mice to reach an ethical endpoint within one year of age. Most prominent of these were the development of papillomas in the chin, and abscesses in the preputial glands (males) or vulvae (females) which showed evidence of Staphylococcus and Proteus infection. Consistent with the development of papillomas, the epidermis of Ehf-/- mice showed evidence of mild hyperplasia. A subset of Ehf-/- mice also developed cataracts and corneal ulcers. EHF is highly expressed in the colonic epithelium and Ehf-/- mice displayed increased susceptibility to dextran sodium sulphate-induced colitis. This phenotype was confirmed in intestinal-specific Ehf knockout mice, and histopathological analyses revealed reduced numbers of goblet cells and extensive transcriptional reprogramming in the colonic epithelium. Finally, colon-specific deletion of Ehf enhanced Apc-initiated adenoma development, unveiling a novel, tumour suppressive role for EHF in colorectal cancer.

ConclusionThe Ets DNA-binding domain of EHF is essential for post-natal homeostasis of the epidermis and colonic epithelium, and functions as a tumour suppressor in the colon.

category: cell biology


title: Scaling of subcellular structures with cell length through decelerated growth

abstract:
How cells tune the size of their subcellular parts to scale with cell size is a fundamental question in cell biology. Until now, most studies on the size control of organelles and other subcellular structures have focused on scaling relationships with cell volume, which can be explained by limiting pool mechanisms. Here, we uncover a distinct scaling relationship with cell length rather than volume, revealed by mathematical modeling and quantitative imaging of yeast actin cables. The extension rate of cables decelerates as they approach the rear of the cell, until cable length matches cell length. Further, the deceleration rate scales with cell length. These observations reveal a new mode of scaling that senses the linear dimensions of a cell.

One Sentence SummaryAs actin cables in yeast cells grow longer, their extension rate decelerates, enabling the cable length to match cell length.


title: Imaging-based evaluation of pathogenicity by novel DNM2 variants associated with centronuclear myopathy

abstract:
Centronuclear myopathy (CNM) is characterized clinically by muscle weakness and pathologically by the presence of centralized nuclei and disarrangement of T-tubules in muscle fibers. DNM2 which encodes a large GTPase dynamin 2 have been identified as a causative gene for CNM. Nevertheless, the identification of DNM2 variants may not always lead to the definitive diagnosis as their pathogenicity is often unknown.

In this study, by imaging T-tubule-like structures reconstituted in cellulo, we demonstrated that aberrant membrane remodeling by mutant dynamin 2 is tightly associated with gain-of-function features of DNM2 variants. This simple in cellulo assay provided quantitative data required for accurately evaluating pathogenicity of reported and novel DNM2 variants identified from CNM patients in our cohort. Our approaches combining the in cellulo assay with clinical information of the patients enabled to explain the course of a disease progression by pathogenesis of each variant in DNM2-associated CNM.


title: The SWI/SNF chromatin remodeling assemblies BAF and PBAF differentially regulate cell cycle exit and cellular invasion in vivo

abstract:
Chromatin remodelers such as the SWI/SNF complex coordinate metazoan development through broad regulation of chromatin accessibility and transcription, ensuring normal cell cycle control and cellular differentiation in a lineage-specific and temporally restricted manner. Mutations in genes encoding the structural subunits of chromatin, such as histone subunits, and chromatin regulating factors (CRFs) are associated with a variety of disease mechanisms including cancer metastasis, in which cancer co-opts cellular invasion programs functioning in healthy cells during development. Here we utilize Caenorhabditis elegans anchor cell (AC) invasion as an in vivo model to identify the suite of chromatin agents and CRFs that promote cellular invasiveness. We demonstrate that the SWI/SNF ATP-dependent chromatin remodeling complex is a critical regulator of AC invasion, with pleiotropic effects on both G0 cell cycle arrest and activation of invasive machinery. Using targeted protein degradation and enhanced RNA interference (RNAi) vectors, we show that SWI/SNF contributes to AC invasion in a dose-dependent fashion, with lower levels of activity in the AC corresponding to aberrant cell cycle entry and increased loss of invasion. Our data specifically implicate the SWI/SNF BAF assembly in the regulation of the G0 cell cycle arrest in the AC, whereas the SWI/SNF PBAF assembly promotes AC invasion via cell cycle-independent mechanisms, including attachment to the basement membrane (BM) and activation of the pro-invasive fos-1/FOS gene. Together these findings demonstrate that the SWI/SNF complex is necessary for two essential components of AC invasion: arresting cell cycle progression and remodeling the BM. The work here provides valuable single-cell mechanistic insight into how the SWI/SNF assemblies differentially contribute to cellular invasion and how SWI/SNF subunit-specific disruptions may contribute to tumorigeneses and cancer metastasis.

SUMMARY STATEMENTCellular invasion through the basement membrane by the C. elegans anchor cell requires both BAF and PBAF SWI/SNF assemblies to arrest the cell cycle and promote the expression of pro-invasive genes.

category: developmental biology


title: ZFP207 controls pluripotency by multiple post-transcriptional mechanisms

abstract: The pluripotent state is not solely governed by the action of the core transcription factors Oct4, Sox2, and Nanog, but also by a series of co-transcriptional and post-transcriptional events, including alternative splicing (AS) and the interaction of RNA-binding proteins (RBPs) with defined subpopulations of RNAs. Zinc Finger Protein 207 (ZFP207) is an essential transcription factor for mammalian embryonic development. Here, we employ multiple functional analyses to characterize the role of ZFP207 in mouse embryonic stem cells (ESCs). We find that ZFP207 plays a pivotal role in ESC maintenance, and silencing of Zfp207 leads to severe neuroectodermal differentiation defects. In striking contrast to human ESCs, ZFP207 does not transcriptionally regulate stem cell and neuronal-related genes but exerts its effects by control AS networks and acting as an RBP. Our study expands the role of ZFP207 to maintain ESC identity, and underscores ZFP207 functional versatility with key roles in neural fate commitment.


title: Embryonic requirements for Tcf12 in the development of the mouse coronal suture

abstract: A major feature of Saethre-Chotzen syndrome is coronal craniosynostosis, the fusion of the frontal and parietal bones at the coronal suture. It is caused by heterozygous loss-of-function mutations in the basic HLH transcription factors TWIST1 and TCF12. While compound heterozygous Tcf12; Twist1 mice display severe coronal synostosis, the individual role of Tcf12 has remained unexplored. Here we show that Tcf12 controls several key processes in calvarial development, including the rate of frontal and parietal bone growth, and the boundary between sutural and osteogenic cells. Genetic analysis supports an embryonic requirement for Tcf12 in suture formation, as combined deletion of Tcf12 in the embryonic neural crest and mesoderm, but not in the postnatal suture mesenchyme, disrupts the coronal suture. We also detect asymmetric distribution of Grem1 + mesenchymal cells on opposing sides of the wild-type frontal and parietal bones, which prefigures later bone overlap at the sutures. In Tcf12 mutants, reduced asymmetry correlates with lack of bone overlap. Our results indicate a largely embryonic function of Tcf12 in controlling the rate and asymmetrical growth of calvarial bones and establishment of suture boundaries, which together ensure the proper formation of the overlapping coronal suture.


title: Development of a yeast-based assay for bioavailable phosphorous

abstract: Preventing eutrophication of inland freshwater ecosystems requires quantifying the phosphorus (P) content of the streams and rivers that feed them. Typical methods for measuring P assess soluble reactive P (SRP) or total P (TP) and require expensive analytical techniques that produce hazardous waste. Here we present a novel method for measuring the more relevant bioavailable P (BAP); this assay utilizes the growth of familiar bakers yeast, avoids production of hazardous waste, and reduces cost relative to measurements of SRP and TP. The yeast BAP (yBAP) assay takes advantage of the observation that yeast density at saturating growth increases linearly with provided P. We show that this relationship can be used to measure P in freshwater in concentration ranges relevant to eutrophication. In addition, we measured yBAP in water containing known amounts of fertilizer and in samples from agricultural waterways. We observed that the majority of yBAP values were between those obtained from standard SRP and TP measurements, demonstrating that the assay is compatible with real-world settings. The cost-effective and nonhazardous nature of the yeast-based assay suggests that it could have utility in a range of settings, offering added insight to identify water systems at risk of eutrophication from excess phosphorus.


title: Development of a yeast-based assay for bioavailable phosphorus


title: The dynamic interactive network of long non-coding RNAs and chromatin accessibility facilitates erythroid differentiation

abstract:
Erythroid differentiation is a dynamic process regulated by multiple factors, while the interaction between long non-coding RNAs and chromatin accessibility and its influence on erythroid differentiation remains unclear. To elucidate this interaction, we employed hematopoietic stem cells, multipotent progenitor cells, common myeloid progenitor cells, megakaryocyte-erythroid progenitor cells, and erythroblasts from human cord blood as an erythroid differentiation model to explore the coordinated regulatory functions of lncRNAs and chromatin accessibility in erythropoiesis by integrating RNA-Seq and ATAC-Seq data. We revealed that the integrated network of chromatin accessibility and LncRNAs exhibits stage-specific changes throughout the erythroid differentiation process, and that the changes at the EB stage of maturation are dramatic. We identified a subset of stage-specific lncRNAs and transcription factors (TFs) that coordinate with chromatin accessibility during erythroid differentiation, in which lncRNAs are key regulators of terminal erythroid differentiation via a lncRNA-TF-gene network. LncRNA PCED1B-AS1 was revealed to regulate terminal erythroid differentiation by coordinating GATA1 dynamically binding to the chromatin during erythroid differentiation. DANCR, another lncRNA that is highly expressed at the MEP stage, was verified to promote erythroid differentiation by compromising megakaryocyte differentiation and coordinating with chromatin accessibility and TFs, such as RUNX1. Overall, our results identified the interactive network of lncRNAs and chromatin accessibility in erythropoiesis and provide novel insights into erythroid differentiation and abundant resources for further study.

Key PointsO_LILncRNAs regulate erythroid differentiation through coordinating with chromatin accessibility.
C_LIO_LIThe integrative multi-omics analysis reveals stage-specific interaction network of LncRNAs and chromatin accessibility in erythropoiesis.
C_LI