title: Enhanced metanephric specification to functional proximal tubule enables toxicity screening and infectious disease modelling in kidney organoids

abstract: While pluripotent stem cell-derived kidney organoids represent a promising approach for the study of renal disease, renal physiology and drug screening, the proximal nephron remains immature with limited evidence for key functional solute channels. This may reflect early mispatterning of the nephrogenic mesenchyme or insufficient maturation. In this study, prolonged differentiation and modification of media conditions to enhance metanephric nephron progenitor specification resulted in the induction of nephrons containing elongated and aligned proximal nephron segments together with SLC12A1+ loops of Henle. Nephron proximal segments showed superior HNF4A gene and protein expression, as well as upregulation of key functional transporters, including SLC3A1/2, SLC47A1, and SLC22A2. The striking proximo-distal orientation of nephrons was shown to result from localised WNT antagonism originating from the centre of the organoid. Functionality of such transporters was evidenced by albumin and organic cation uptake, as well as appropriate KIM-1 upregulation in response to the nephrotoxicant, cisplatin. PT-enhanced organoids also possessed improved expression of receptors associated with SARS-CoV2 entry, rendering these organoids susceptible to infection and able to support viral replication without co-location of ACE2 and TMPRSS2. These PT-enhanced organoids provide an accurate model with which to study human proximal tubule maturation, inherited and acquired proximal tubular disease, and drug and viral responses.

category: bioengineering


title: Pneumonia Detection with Semantic Similarity Scores

abstract: X-ray images have been widely used for medical diagnoses of cardiothoracic and pulmonary abnormalities due to its noninvasiveness. Advancement in computer-aided diagnostic technologies, such as deep supervised methods, can help radiologists with a reliable early treatment and reduce diagnosis time. Nevertheless, these methods are prone to the small number of labeled samples and are limited to a specific abnormality. In this paper we combined a self-supervised contrastive method with a Mahalanobis distance score to develope an abnormality detection method that uses only healthy images during the training procedure. We were able to outperform previous unsupervised methods for the task of Pneumonia detection. We show that representation learned by the self-supervised method improves the supervised tasks for Pneumonia detection.

category: bioinformatics


title: PSEA: A phenotypic similarity ensemble approach for prioritizes candidate genes to aid mendelian disease diagnosis

abstract:
MotivationNext-generation sequencing (NGS) is increasingly applied to the molecular diagnosis of genetic disorders. However, challenges for the interpretation of NGS data remain given the massive number of variants produced by NGS. Careful assessment is required to identify the most likely disease-causing variants that best match the patients clinical phenotypes, which is highly experience-dependent and of low cost-effectiveness.

ResultsThe human phenotype ontology (HPO) together with the information content (IC) are widely used for phenotypic similarity evaluation. Here, we introduce PSEA, a new phenotypic similarity evaluation tool capable of quantifying groups of HPO terms unbiasedly. By comparing with other methods, PSEA show optimal performance and show a higher tolerance to phenotypic noise or incompleteness. We also developed a web server for disease-causing gene prioritization and HPO-gene weighted linkage visualization.

AvailabilitySource code and Web service are free available at https://github.com/zhonghua-wang/psea and https://phoenix.bgi.com/psea, respectively.

Contactwangzhonghua@genomics.cn

Supplementary informationSupplementary data are available at Bioinformatics online.


title: Structural rearrangement of the intracellular gate of the serotonin transporter induced by Thr276 phosphorylation

abstract: The reuptake of the neurotransmitter serotonin from the synaptic cleft by the serotonin transporter, SERT, is essential for proper neurological signaling. Biochemical studies have shown Thr276 of transmembrane helix 5 is a site of PKG-mediated SERT phosphorylation, which has been proposed to shifts the SERT conformational equlibira to promote inward-facing states, thus enhancing 5HT transport. Recent structural and simulation studies have provided insights into the conformation transitions during substrate transport but have not shed light on SERT regulation via post-translational modifications. Using molecular dynamics simulations and Markov state models, we investigate how Thr276 phosphorylation impacts the SERT mechanism and its role in enhancing transporter stability and function. Our simulations show that Thr276 phosphorylation alters the hydrogen-bonding network involving residues on transmembrane helix 5. This in turn decreases the free energy barriers for SERT to transition to the inward-facing state, thus facilitating 5HT transport. The results provide atomistic insights into in vivo SERT regulation and can be extended to other pharmacologically important transporters in the solute carrier superfamily.

category: biophysics


title: ERα-LBD, a novel isoform of estrogen receptor alpha, promotes breast cancer proliferation and endocrine resistance

abstract:
Estrogen receptor alpha (ER) drives mammary gland development and breast cancer (BC) growth through an evolutionarily conserved linkage of DNA binding and hormone activation functions. Therapeutic targeting of the hormone binding pocket is a widely utilized and successful strategy for breast cancer prevention and treatment. However, resistance to this endocrine therapy is frequently encountered and may occur through bypass or reactivation of ER-regulated transcriptional programs. We now identify the induction of a novel ER isoform, ER-LBD, that is encoded by an alternative ESR1 transcript and lacks the activation function and DNA binding domains. Despite lacking the transcriptional activity, ER-LBD is found to promote breast cancer growth and resistance to the ER antagonist fulvestrant. ER-LBD is predominantly localized to the cytoplasm and mitochondria of BC cells and leads to enhanced glycolysis, respiration and stem-like features. Intriguingly, ER-LBD expression and function does not appear to be restricted to cancers that express full length ER but also promotes growth of triple negative breast cancers and ER-LBD transcript (ESR1-LBD) is also present in BC samples from both ER(+) and ER(-) human tumors. These findings point to ER-LBD as a potential mediator of breast cancer progression and therapy resistance.

SIGNIFICANCE STATEMENTEndocrine resistant and metastatic breast cancer (BC) is a clinically significant problem. Our study of fulvestrant resistant cancer cells led to the discovery of a novel ER isoform which we call ER-LBD. Encoded by a truncated transcript variant (ESR1-LBD) and lacking the N-terminal domains (activation of transcription and DNA binding), ER-LBD displays a unique role in BC tumorigenesis and progression by mechanisms that may involve metabolic and cell growth advantages, stemness and therapy resistance. Importantly, ESR1-LBD is preferentially expressed in human breast tumor tissues and may be used as prognostic marker in BC.

category: cancer biology


title: EEG-based clusters differentiate psychological distress, sleep quality and cognitive function in adolescents

abstract:
1IntroductionTo better understand the relationships between brain activity, cognitive function and mental health risk in adolescence there is value in identifying data-driven subgroups based on measurements of brain activity and function, and then comparing cognition and mental health symptoms between such subgroups.

MethodsHere we implement a multi-stage analysis pipeline to identify data-driven clusters of 12-year-olds (M = 12.64, SD = 0.32) based on frequency characteristics calculated from resting state, eyes-closed electroencephalography (EEG) recordings. EEG data was collected from 59 individuals as part of their baseline assessment in the Longitudinal Adolescent Brain Study (LABS) being undertaken in Queensland, Australia. Applying multiple unsupervised clustering algorithms to these EEG features, we identified well-separated subgroups of individuals. To study patterns of difference in cognitive function and mental health symptoms between core clusters, we applied Bayesian regression models to probabilistically identify differences in these measures between clusters.

ResultsWe identified 5 core clusters which were associated with distinct subtypes of resting state EEG frequency content. EEG features that were influential in differentiating clusters included Individual Alpha Frequency, relative power in 4 Hz bands up to 16 Hz, and 95% Spectral Edge Frequency. Bayesian models demonstrated substantial differences in psychological distress, sleep quality and cognitive function between these clusters. By examining associations between neurophysiology and health measures across clusters, we have identified preliminary risk and protective profiles linked to EEG characteristics.

ConclusionIn this work we have developed a flexible and scaleable pipeline to identify subgroups of individuals in early adolescence on the basis of resting state EEG activity. These findings provide new clues about neurophysiological subgroups of adolescents in the general population, and associated patterns of health and cognition that are not observed at the whole group level. This approach offers potential utility in clinical risk prediction for mental and cognitive health outcomes throughout adolescent development.

category: neuroscience


title: The PRO model accounts for the anterior cingulate cortex role in risky decision-making and monitoring

abstract: The anterior cingulate cortex (ACC) has been implicated in a number of functions including performance monitoring and decision making involving effort. The prediction of responses and outcomes (PRO) model has provided a unified account of much human and monkey ACC data involving anatomy, neurophysiology, EEG, fMRI, and behavior. Here we explore the computational nature of ACC with the PRO model, extending it to account specifically for both human and macaque monkey decision-making under risk, including both behavioral and neural data. We show that the PRO model can account for a number of additional effects related to outcome prediction, decision-making under risk, gambling behavior, and we show that the ACC represents the variance of uncertain outcomes, suggesting a link between ACC function and mean-variance theories of decision making. The PRO model provides a unified account of a large set of data regarding the ACC.


title: Lin28a rejuvenates muscle stem cells via mitochondrial optimization

abstract: The well-conserved correlation between juvenility and tissue regeneration was first discussed by Charles Darwin. Ectopic Lin28 is known to play an important role in somatic reprogramming and tissue regeneration, but endogenous Lin28s role in tissue homeostasis and juvenility had remained unclear. Through lineage tracing, we found that a rare subset of muscle stem cells (MuSCs) expressing Lin28a can respond to acute injury by proliferating as Pax3+ or Pax7+ MuSCs, and contribute to all types of myofibers during muscle regeneration. Compared with conventional Pax7+ MuSCs, Lin28a+ MuSCs express more Pax3 and show enhanced myogenicity in vitro. In terms of the epigenetic clock, adult Lin28a+ MuSCs lie between adult Pax7+ MuSCs and embryonic Pax7+ myoblasts according to their DNA methylation profiles. We found that Lin28a+ MuSCs upregulate several embryonic limb bud mesoderm transcription factors and could maintain a juvenile state with enhanced stem cell self-renewal and stress-responsiveness in vitro and in vivo. When combined with telomerase and TP53 inhibition to biomimic mouse Lin28a+ MuSCs, we found that Lin28a can rejuvenate and dedifferentiate aged human primary myoblasts into engraftable, self-renewing MuSCs. Mechanistic studies revealed that Lin28a activated the HIF1A pathway by optimizing mitochondrial ROS (mtROS), thereby rejuvenating MuSC self-renewal and muscle regeneration. Our findings connect the stem cell factor Lin28, mtROS metabolism and stress response pathways to the process of stem cell rejuvenation and tissue regeneration.

category: cell biology


title: Oxalic acid binds to gustatory receptor Gr23a and inhibits feeding in the brown planthopper

abstract:
Plants produce diverse secondary compounds as natural protection against microbial and insect attack. Most of these compounds, including bitters and acids, are sensed by insect gustatory receptors (Grs). Acids are potentially toxic to insects, but there are few reports on sour compounds as ligands of insect Grs. Here, using two different heterologous expression systems, the insect Sf9 cell line and the mammalian HEK293T cell line, we started from crude extracts of rice (Oryza sativa) and successfully identified oxalic acid (OA) as a ligand of NlGr23a, a Gr in the brown planthopper Nilaparvata lugens. The antifeedant effect of OA on the brown planthopper was dose dependent, and NlGr23a is essential for OAs antifeedant activity in both artificial diets and rice plants. NlGr23a is also indispensable for tarsal OA sensing. To our knowledge, OA is the first identified ligand starting from plant crude extracts and the first known strong acid for insect Grs. These findings on rice-planthopper interactions will be of broad interest for pest control in agriculture and also for better understanding of how insects select host plants.

Research organismNilaparvata lugens

category: ecology


title: Quantifying the scale of genetic diversity extinction in the Anthropocene

abstract: More species than ever before are at risk of extinction due to anthropogenic habitat loss and climate change. But even species that are not threatened have seen reductions in their populations and geographic ranges, likely impacting their genetic diversity. Although preserving genetic diversity is key to maintaining adaptability of species, we lack predictive tools and global estimates of genetic diversity loss across ecosystems. By bridging theories of biodiversity and population genetics, we introduce a mathematical framework to understand the loss of naturally occurring DNA mutations within decreasing habitat within a species. Analysing genome-wide variation data of 10,095 geo-referenced individuals from 20 plant and animal species, we show that genome-wide diversity follows a power law with geographic area (the mutations-area relationship), which can predict genetic diversity loss in spatial computer simulations of local population extinctions. Given pre-21st century values of ecosystem transformations, we estimate that over 10% of genetic diversity may already be lost, surpassing the United Nations targets for genetic preservation. These estimated losses could rapidly accelerate with advancing climate change and habitat destruction, highlighting the need for forecasting tools that facilitate implementation of policies to protect genetic resources globally.


title: Genetic diversity loss in the Anthropocene


title: Functional screen of Inflammatory Bowel Disease genes reveals key epithelial functions

abstract:
BackgroundGenetic studies have been tremendously successful in identifying genomic regions associated with a wide variety of phenotypes, although the success of these studies in identifying causal genes, their variants, and their functional impacts have been more limited.

MethodsWe identified 145 genes from IBD-associated genomic loci having endogenous expression within the intestinal epithelial cell compartment. We evaluated the impact of lentiviral transfer of the open reading frame (ORF) of these IBD genes into the HT-29 intestinal epithelial cell line via transcriptomic analyses. Comparing the genes whose expression was modulated by each ORF, as well as the functions enriched within these gene lists, identified ORFs with shared impacts and their putative disease-relevant biological functions.

ResultsAnalysis of the transcriptomic data for cell lines expressing the ORFs for known causal genes such as HNF4a, IFIH1 and SMAD3 identified functions consistent for what is known for these genes. These analyses also identified two major cluster of genes: Cluster 1 contained the known IBD causal genes IFIH1, SBNO2, NFKB1 and NOD2, as well as genes from other IBD loci (ZFP36L1, IRF1, GIGYF1, OTUD3, AIRE and PITX1), whereas Cluster 2 contained the known causal gene KSR1 and implicated DUSP16 from another IBD locus. Our analyses highlight how multiple IBD gene candidates impact on epithelial structure and function, including the protection of the mucosa from intestinal microbiota, and demonstrate that DUSP16, acts a regulator of MAPK activity and contributes to mucosal defense, in part via its regulation of the polymeric immunoglobulin receptor, involved in the protection of the intestinal mucosa from enteric microbiota.

ConclusionsThis functional screen, based on expressing IBD genes within an appropriate cellular context, in this instance intestinal epithelial cells, resulted in changes to the cells transcriptome that are relevant to their endogenous biological function(s). This not only helped in identifying likely causal genes within genetic loci but also provided insight into their biological functions. Furthermore, this work has highlighted the central role of intestinal epithelial cells in IBD pathophysiology, providing a scientific rationale for a drug development strategy that targets epithelial functions in addition to the current therapies targeting immune functions.

category: genetics


title: Genome sequencing and comparative analysis of Ficus benghalensis and Ficus religiosa trees reveal evolutionary mechanisms of longevity

abstract: Ficus benghalensis (Indian banyan tree) and Ficus religiosa (Peepal) trees are well-known for their long lifespan, traditional significance, and medicinal properties. Therefore, to understand the genomic and evolutionary aspect of these characteristics the whole genomes of these two Ficus species were sequenced using 10x Genomics and Oxford Nanopore sequencing platforms. The draft genome assemblies of F. benghalensis (392.89 Mbp genome containing 25,016 high-confidence coding genes), and F. religiosa (332.97 Mbp genome containing 23,929 high-confidence coding genes) were constructed. We also established the genome-wide phylogenetic position of the two Ficus trees with respect to 46 other Angiosperm plant species and studied the comparative population demographic history of these two species to show a population bottleneck event ~0.8 Mya for both the species. We also identified 7,468 orthogroups across 16 phylogenetically closer Eudicot plant species including F. benghalensis and F. religiosa. Comparative evolutionary analyses using these orthogroups, gene family expansion/contraction analysis, and gene duplication analysis showed adaptive evolution in genes involved in cellular pathways and mechanisms that are central to plant growth and development and provide genomic insights into longevity and ecological significance of these large woody trees.

category: genomics


title: Transcriptomic responses of Galapagos finches to avian pox virus infection

abstract: Emerging pathogens can have devastating effects on naive hosts, but disease outcomes often vary among hosts. Comparing the cellular response of different host species to infection can provide insight into mechanisms of host defense and the basis of host susceptibility to disease. Here, we used RNA-seq to characterize the transcriptomic response of Darwins finches to avian poxvirus, which is introduced to the Galapagos Islands. We tested whether gene expression differs between infected and uninfected birds, and whether transcriptomic differences were related either to known antiviral mechanisms and/or the co-option of the host cellular environment by the virus. We compared two species, the medium ground finch (Geospiza fortis) and the vegetarian finch (Platyspiza crassirostris), to determine whether related species have similar responses to the same novel pathogen. We found that medium ground finches had a strong transcriptomic response to infection, upregulating genes involved in the innate immune response including interferon production, inflammation, and other immune signaling pathways. In contrast, vegetarian finches had a more limited response to infection. Our results also revealed evidence of viral manipulation of the hosts cellular function and metabolism, providing insight into the ways in which poxviruses affect their hosts. Many of the transcriptomic responses to infection mirrored known processes seen in model and in-vitro studies of poxviruses indicating that many pathways of host defense against poxviruses are conserved among vertebrates and present even in hosts without a long evolutionary history with the virus. At the same time, the variation we observed between closely related species indicates that some endemic species of Galapagos finch may be more susceptible to avian pox than others.


title: The 4D Nucleome Data Portal: a resource for searching and visualizing curated nucleomics data

abstract: The 4D Nucleome (4DN) Network aims to elucidate the complex structure and organization of chromosomes in the nucleus and the impact of their disruption in disease biology. We present the 4DN Data Portal (https://data.4dnucleome.org/), a repository for datasets generated in the 4DN network and relevant external datasets. Datasets were generated with a wide range of experiments, including chromosome conformation capture assays such as Hi-C and other innovative sequencing and microscopy-based assays probing chromosome architecture. All together, the 4DN data portal hosts more than 1800 experiment sets and 34000 files. Results of sequencing-based assays from different laboratories are uniformly processed and quality-controlled. The portal interface allows easy browsing, filtering, and bulk downloads, and the integrated HiGlass genome browser allows interactive visualization and comparison of multiple datasets. The 4DN data portal represents a primary resource for chromosome contact and other nuclear architecture data for the scientific community.


title: Interpretable Pairwise Distillations for Generative Protein Sequence Models

abstract: AO_SCPLOWBSTRACTC_SCPLOWMany different types of generative models for protein sequences have been proposed in literature. Their uses include the prediction of mutational effects, protein design and the prediction of structural properties. Neural network (NN) architectures have shown great performances, commonly attributed to the capacity to extract non-trivial higher-order interactions from the data. In this work, we analyze three different NN models and assess how close they are to simple pairwise distributions, which have been used in the past for similar problems. We present an approach for extracting pairwise models from more complex ones using an energy-based modeling framework. We show that for the tested models the extracted pairwise models can replicate the energies of the original models and are also close in performance in tasks like mutational effect prediction.


title: Methods for analyzing continuous conformational variability of biomolecules in cryo electron subtomograms: HEMNMA-3D vs. traditional classification

abstract: Cryogenic electron tomography (cryo-ET) allows studying biological macromolecular complexes in cells by three-dimensional (3D) data analysis. The complexes continuously change their shapes (conformations) to achieve biological functions. The shape heterogeneity in the samples imaged in the cryo electron microscope is a bottleneck for comprehending biological mechanisms and developing drugs. Low signal-to-noise ratio and spatial anisotropy (missing wedge artefacts) make cryo-ET data particularly challenging for resolving the shape variability. Other shape variability analysis techniques simplify the problem by considering discrete rather than continuous conformational changes of complexes. Recently, HEMNMA-3D was introduced for cryo-ET continuous shape variability analysis, based on elastic and rigid-body 3D registration between simulated shapes and cryo-ET data. The simulated motions are obtained by normal mode analysis of a high- or low-resolution 3D reference model of the complex under study. The rigid-body alignment is achieved via fast rotational matching with missing wedge compensation. HEMNMA-3D provides a visual insight into molecular dynamics by grouping and averaging subtomograms of similar shapes and by animating movies of registered motions. This article reviews the method and compares it with existing literature on a simulated dataset for nucleosome shape variability.


title: Labkit: Labeling and Segmentation Toolkit for Big Image Data

abstract: We present LO_SCPLOWABKITC_SCPLOW, a user-friendly Fiji plugin for the segmentation of microscopy image data. It offers easy to use manual and automated image segmentation routines that can be rapidly applied to single- and multi-channel images as well as to timelapse movies in 2D or 3D. LO_SCPLOWABKITC_SCPLOW is specifically designed to work efficiently on big image data and enables users of consumer laptops to conveniently work with multiple-terabyte images. This efficiency is achieved by using ImgLib2 and BigDataViewer as the foundation of our software. Furthermore, memory efficient and fast random forest based pixel classification inspired by the Waikato Environment for Knowledge Analysis (Weka) is implemented. Optionally we harness the power of graphics processing units (GPU) to gain additional runtime performance. LO_SCPLOWABKITC_SCPLOW is easy to install on virtually all laptops and workstations. Additionally, LO_SCPLOWABKITC_SCPLOW is compatible with high performance computing (HPC) clusters for distributed processing of big image data. The ability to use pixel classifiers trained in LO_SCPLOWABKITC_SCPLOW via the ImageJ macro language enables our users to integrate this functionality as a processing step in automated image processing workflows. Last but not least, LO_SCPLOWABKITC_SCPLOW comes with rich online resources such as tutorials and examples that will help users to familiarize themselves with available features and how to best use LO_SCPLOWABKITC_SCPLOW in a number of practical real-world use-cases.


title: The chromatin regulator HMGA1a undergoes phase separation in the nucleus

abstract: The protein high mobility group A1 (HMGA1) is an important regulator of chromatin organization and function. However, the mechanisms by which it exerts its biological function are not fully understood. Here, we report that the HMGA isoform, HMGA1a, nucleates into foci that display liquid-like properties in the nucleus, and that the protein readily undergoes phase separation to form liquid condensates in vitro. By bringing together machine-leaning modelling, cellular and biophysical experiments and multiscale simulations, we demonstrate that phase separation of HMGA1a is critically promoted by protein-DNA interactions, and has the potential to be modulated by post-transcriptional effects such as phosphorylation. We further show that the intrinsically disordered C-terminal tail of HMGA1a significantly contributes to its phase separation through cation-{pi} and electrostatic interactions. Our work sheds light on HMGA1 phase separation as an emergent biophysical factor in regulating chromatin structure.


title: MspA Porin as a Local Nanopore Probe for Membrane-bound Proteins

abstract: Nanopore sensing is based on detection and analysis of nanopore transient conductance changes induced by analyte capture. We have recently shown that -Synuclein (Syn), an intrinsically disordered, membrane-active, neuronal protein implicated in Parkinson disease, can be reversibly captured by the VDAC nanopore. The capture process is a highly voltage dependent complexation of the two proteins where transmembrane potential drives the polyanionic C-terminal domain of Syn into VDAC--exactly the mechanism by which generic nanopore-based interrogation of proteins and polynucleotides proceeds. The complex formation, and the motion of Syn in the nanopore, thus may be expected to be only indirectly dependent on the pore identity. Here, we confirm this prediction by demonstrating that when VDAC is replaced with a different transmembrane pore, the engineered mycobacterial porin M2MspA, all the qualitative features of the Syn/nanopore interaction are preserved. The rate of Syn capture by M2MspA rises exponentially with the applied field, while the residence time displays a crossover behavior, indicating that at voltages >50 mV M2MspA-bound Syn largely undergoes translocation to the other side of the membrane. The translocation is directly confirmed using the selectivity tag method, in which the polyanionic C-terminal and neutral N-terminal regions of Syn alter the selectivity of the M2MspA channel differently, allowing direct discrimination of translocation vs retraction for single Syn molecules. We thus prove that the physical model of the motion of disordered protein chains in the nanopore confinement and the selectivity tag technique are not limited to VDAC but are broadly applicable to nanopore-based protein detection, analysis, and separation technologies.


title: γδ T cells are effectors of immune checkpoint blockade in mismatch repair-deficient colon cancers with antigen presentation defects

abstract: DNA mismatch repair deficient (MMR-d) cancers present an abundance of neoantigens that likely underlies their exceptional responsiveness to immune checkpoint blockade (ICB)1,2. However, MMR-d colon cancers that evade CD8+ T cells through loss of Human Leukocyte Antigen (HLA) class I-mediated antigen presentation3-6, frequently remain responsive to ICB7 suggesting the involvement of other immune effector cells. Here, we demonstrate that HLA class I-negative MMR-d cancers are highly infiltrated by {gamma}{delta} T cells. These {gamma}{delta} T cells are mainly composed of V{delta}1 and V{delta}3 subsets, and express high levels of PD-1, activation markers including cytotoxic molecules, and a broad repertoire of killer-cell immunoglobulin-like receptors (KIRs). In vitro, PD-1+ {gamma}{delta} T cells, isolated from MMR-d colon cancers, exhibited a cytolytic response towards HLA class I-negative MMR-d colon cancer cell lines and {beta}2-microglobulin (B2M)-knockout patient-derived tumor organoids (PDTOs), which was enhanced as compared to antigen presentation-proficient cells. This response was diminished after blocking the interaction between NKG2D and its ligands. By comparing paired tumor samples of MMR-d colorectal cancer patients obtained before and after dual PD-1 and CTLA-4 blockade, we found that ICB profoundly increased the intratumoral frequency of {gamma}{delta} T cells in HLA class I-negative cancers. Taken together, these data indicate that {gamma}{delta} T cells contribute to the response to ICB therapy in patients with HLA class I-negative, MMR-d colon cancers, and illustrate the potential of {gamma}{delta} T cells in cancer immunotherapy.


title: Integrating Subclonal Response Heterogeneity to Define Cancer Organoid Therapeutic Sensitivity

abstract: Tumor heterogeneity is predicted to confer inferior clinical outcomes, however modeling heterogeneity in a manner that still represents the tumor of origin remains a formidable challenge. Sequencing technologies are limited in their ability to identify rare subclonal populations and predict response to the multitude of available treatments for patients. Patient-derived organotypic cultures have significantly improved the modeling of cancer biology by faithfully representing the molecular features of primary malignant tissues. Patient-derived cancer organoid (PCO) cultures contain numerous individual organoids with the potential to recapitulate heterogeneity, though PCOs are most commonly studied in bulk ignoring any diversity in the molecular profile or treatment response. Here we demonstrate the advantage of evaluating individual PCOs in conjunction with cellular level optical metabolic imaging to characterize the largely ignored heterogeneity within these cultures to predict clinical therapeutic response, identify subclonal populations, and determine patient specific mechanisms of resistance.


title: A live yeast supplementation to gestating ewes improves bioactive molecules composition in colostrum with no impact on its bacterial composition and beneficially affects immune status of the offspring

abstract:
Colostrum quality is of paramount importance in the management of optimal ruminant growth and infectious disease prevention in early life. Live yeast supplementation effect during the last month of gestation was evaluated on ewes colostrum composition. Two groups of ewes (n=14) carrying twin lambs were constituted and twins were separated into groups (mothered or artificially-fed) 12h after birth. Nutrient, oligosaccharides (OS), IgG and lactoferrin concentrations were measured over 72h after lambing, and bacterial community was described in colostrum collected at parturition (T0). Immune passive transfer was evaluated through IgG measurement in lamb serum. In both groups, colostral nutrient, OS concentrations and IgG concentrations in colostrum and lamb serum decreased over time, (p < 0.01) except for lactose, which slightly increased (p < 0.001) and lactoferrin which remained stable. Bacterial population was stable over time with high relative abundances of Aerococcaceae, Corynebacteriaceae, Moraxellaceae and Staphylococcaceae in T0-colostrum. No effect of supplementation was observed in nutrient and lactoferrin concentrations. In supplemented ewes, colostral IgG level was higher at T0 and a higher level of serum IgG was observed in lambs born from supplemented mothers and artificially-fed, while no effect of supplementation was observed in the mothered lambs groups. Using a metabolomic approach, we showed that supplementation affected OS composition with significantly higher levels of colostral Neu-5Gc compounds up to 5h after birth. No effect of supplementation was observed on bacterial composition. Our data suggest that live yeast supplementation offsets the negative impact of early separation and incomplete colostrum feeding in neonate lambs.

Graphical abstract

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category: physiology


title: Glucose uptake in mammalian cells measured by ICP-MS

abstract: We developed a sensitive, ratiometric method to measure simultaneously 13C-labeled glucose and rubidium in biological samples using ICP-MS. The method uses probe-assisted ultra-sonication with water to extract 13C-[6C]-labeled-D-glucose and other polar analytes from mammalian tissues. It extracts >80% of the reference value for Rb and >95 % of 13C in a CRM spiked with 13C-[6C]-labeled-D-glucose in the micro-molar range. Using optimized instrument conditions, the method achieves a stable 13C/12C signal without spectral interferences. The 13C/12C signal is independent of sample composition and depends linearly on the concentration of 13C-[6C]-labeled-D-glucose in spiked samples. Overall, the method achieves a limit of detection of 10 {micro}M for 6-C-labeled 13C glucose in biological tissues. This detection capability for carbon in biological matrices by ICP-MS opens a wider range of applications for ICP-MS in biomedical research. As proof-of-principle, we combined 13C detection with the multi-channel capability of ICP-MS to measure glucose and rubidium uptake in the same contracting skeletal muscles. Multi-isotope detection is needed to study many biological processes, including coupled membrane transport. These results demonstrate a capability for carbon detection by ICP-MS that can significantly advance studies of complex biological processes that require multi-isotope detection.

category: systems biology


title: Sex Differences in Cortical Morphometry during Ageing: Examining the Interplay between Lifestyle and Reproductive Factors

abstract: Sex differences in neurodegenerative disorder prevalence have been attributed to life expectancy, modifiable risk factors related to lifestyle and the impact of changes in sex hormones and the reproductive system. Although these factors are known to interact with one another, they are often studied in isolation. Here, we used a multivariate approach to investigate how lifestyle, along with menopause and the number of children, interacts with cortical thickness (CT) in healthy adults. Using CT measures from T1-weighted scans (MPRAGE, 1 mm3 voxels; 124 participants; 67 females; 40-70 years old) from the Cam-CAN dataset. Using a partial least squares decomposition, we identified patterns of covariance between CT and lifestyle factors, menopause and the number of children. In women, we identified significant patterns that linked education, socioeconomic status, social contact and length of reproductive period to CT in the left prefrontal cortex, as well as alcohol consumption, physical activity and menopausal status to CT in the frontal poles. Contrastingly, the results in men were driven by education and anxiety, and involved increased CT in the temporal poles. Our findings suggests that sex differences in cortical anatomy during brain ageing might be driven by interactions between contrasting lifestyles and the female-specific endocrine environment.


title: Visual space curves before eye movements

abstract: In most experiments within the field of cognitive and systems neuroscience, fixation is often required prior to the onset of an experimental trial. However, the term "fixation" is rather misleading since our eyes are constantly moving. One type of transient miniature movement ubiquitously observed during fixation is commonly referred to as fixational eye movements or microsaccades. Perimicrosaccadic compression of visual space -- the ability of retinotopic cells to transiently exhibit predictive spatiotemporal retinotopic compressive shifts toward the target of an impending microsaccade -- is known to dramatically alter visual perception. However, whether perimicrosaccadic compressive shifts can become spatially asymmetric, that is, continuously directed toward a specific foveal region over another (e.g., an upper over a lower region in the fovea) and for what purpose, remains poorly understood. Assuming that these transient shifts are indeed asymmetric under certain conditions, the perceptual and oculomotor consequences such asymmetricity might accompany across visual space is poorly understood. Equally unaccounted for is a mechanistic account of the neural computation and architecture that could support these transient asymmetric shifts while the visual system actively maintains retinotopic organization. Here, we systematically measured visual sensitivity in human subjects to faint probes presented during fixation and around the time of saccadic eye movement at geometrically symmetric retinotopic locations in the foveal, parafoveal, and peripheral regions of visual space. Remarkably, we observed transient local asymmetric visual sensitivity differences between these symmetric retinotopic locations where none should be observed. Equally surprising, we observed the trajectories of saccadic eye movements, which are expected to travel along a linear path, routinely deviate along a curved path toward orthogonal eccentric locations. To provide a mechanistic account of the neural computation and architecture that may explain our results, we proposed a novel neurobiologically inspired phenomenological force field model in which underlying attentional and oculomotor signals are modulated by transient eccentric error signals that manifest as temporally overlapping predictive forces and impinge on the retinotopic visual cortex. These forces, which transiently bias putative population sensitivity toward an orthogonal retinotopic foveal region and, around the time of a saccadic eye movement, along an axis orthogonal to the saccade direction toward a mislocalized peripheral region, succinctly capture the essence of our empirical observations.


title: Stable modulation of neuronal oscillations produced through brain-machine equilibrium

abstract: Brain stimulation is predominantly delivered independent of ongoing activity, whereas closed-loop systems can modify activity through interaction. They have the unrealised potential to continuously bind external stimulation to specific dynamics of a neural circuit. Such manipulations are particularly suited to rhythmic activities, where neuronal activity is organised in oscillatory cycles. Here, we developed a fast algorithm that responds on a cycle-by-cycle basis to stimulate basal ganglia nuclei at predetermined phases of successive cortical beta cycles in parkinsonian rats. Using this approach, we demonstrate a stable brain-machine interaction. An equilibrium emerged between the modified brain signal and feedback-dependent stimulation pattern, which led to sustained amplification or suppression of the oscillation. Sustained beta amplification slowed movement speed by altering the mode of locomotion. Integrating an external stimulus with network activity in this way could be used to correct maladaptive activities and to define the role of oscillations in fundamental brain functions.


title: Functional and structural features of L2/3 pyramidal cells continuously covary with pial depth in mouse visual cortex

abstract:
Pyramidal cells of neocortical layer 2/3 (L2/3 PyrCs) integrate signals from numerous brain areas and project throughout the neocortex. Within L2/3, PyrCs show functional and structural specializations depending on their pial depth, indicating participation in different functional microcircuits. However, it is unknown whether these depth-dependent differences result from separable L2/3 PyrC subtypes or whether functional and structural features represent a continuum while correlating with pial depth. Here, we assessed the stimulus selectivity, electrophysiological properties, dendritic morphology, and excitatory and inhibitory synaptic connectivity across the depth of L2/3 in the binocular visual cortex (bV1) of female mice. We find that the structure of the apical but not the basal dendritic tree varies with pial depth, which is accompanied by differences in passive but not active electrophysiological properties. PyrCs in lower L2/3 receive increased excitatory and inhibitory input from L4, while upper L2/3 PyrCs receive a larger proportion of intralaminar input. Complementary in vivo calcium imaging revealed a systematic change in visual responsiveness, with deeper L2/3 PyrCs showing more robust responses than superficial PyrCs. Furthermore, deeper L2/3 PyrCs are more strongly driven by contralateral than ipsilateral eye stimulation. In contrast, orientation- and direction-selectivity of L2/3 PyrCs are not dependent on pial depth. Importantly, the transitions of the various properties are gradual, and cluster analysis does not support the classification of L2/3 PyrCs into discrete subtypes. These results show that L2/3 PyrCs multiple functional and structural properties systematically correlate with their depth within L2/3, forming a continuum rather than representing discrete subtypes.

SIGNIFICANCE STATEMENTNeocortical pyramidal cells in layer 2/3 (L2/3 PyrCs) are crucial for cortical computation and display heterogenous properties. We investigated whether and how these properties vary across the depth of L2/3 and whether L2/3 PyrCs can be subdivided into distinct subtypes. This is important for a better understanding of the coding strategy and information integration processes within L2/3. We find that multiple properties such as morphology, physiology, connectivity, and functional in vivo responses of L2/3 PyrCs correlate with cortical depth in mouse visual cortex. These variations are continuous and do not support classification of L2/3 PyrCs into discrete subtypes. In contrast to L5 and L6, PyrCs in L2/3 therefore process information based on a continuous property space.


title: Short-term plasticity in the visual thalamus

abstract: While there is evidence that the visual cortex retains a potential for plasticity in adulthood, less is known about the subcortical stages of visual processing. Here we asked whether short-term ocular dominance plasticity affects the visual thalamus. We addressed this question in normally sighted adult humans, using ultra-high field (7T) magnetic resonance imaging combined with the paradigm of short-term monocular deprivation. With this approach, we previously demonstrated transient shifts of perceptual eye dominance and ocular dominance in visual cortex (Binda et al., 2018). Here we report evidence for short-term plasticity in the ventral division of the pulvinar (vPulv), where the deprived eye representation was enhanced over the non-deprived eye. This pulvinar plasticity effect was similar as previously seen in visual cortex and it was correlated with the ocular dominance shift measured behaviorally. In contrast, there was no short-term plasticity effect in Lateral Geniculate Nucleus (LGN), where results were reliably different from vPulv, despite their spatial proximity. We conclude that the visual thalamus retains potential for short-term plasticity in adulthood; the plasticity effect differs across thalamic subregions, possibly reflecting differences in their cortical connectivity.


title: Endothelial Cell-Specific Molecule-1 Inhibits Albuminuria in Diabetic Mice

abstract: Diabetic kidney disease (DKD) is the most common cause of kidney failure in the world, and novel predictive biomarkers and molecular mechanisms of disease are needed. Endothelial cell-specific molecule-1 (Esm-1) is a secreted proteoglycan that attenuates inflammation. We previously identified that a glomerular deficiency of Esm-1 associates with more pronounced albuminuria and glomerular inflammation in DKD-susceptible relative to DKD-resistant mice, but its contribution to DKD remains unexplored. In this study, we show that lower circulating Esm-1 predicts progressive stages of albuminuria in patients with diabetes. In DKD-susceptible mice, Esm-1 inversely correlates with albuminuria and glomerular leukocyte infiltration. Using hydrodynamic tail-vein injection, we show that over-expression of either mouse or human Esm-1 reduces diabetes-induced albuminuria relative to saline-injected controls independent of leukocyte infiltration. Using a complementary approach, we find that constitutive deletion of Esm-1 in DKD-resistant mice increases the degree of diabetes-induced albuminuria versus wild-type controls. Mechanistically, over-expression of Esm-1 attenuates diabetes-induced podocyte injury. By glomerular RNAseq, we identify that Esm-1 attenuates diabetes-induced up-regulation of interferon-stimulated genes, and Esm-1 inhibits expression of kidney disease-promoting and interferon-related genes, including Ackr2 and Cxcl11. In conclusion, we demonstrate that Esm-1 protects against diabetes-induced albuminuria, and podocytopathy, possibly through select interferon signaling.

category: immunology


title: Shell shape as an indicator of phenotypic stocks of Tehuelche scallop (Aequipecten tehuelchus) in Northern Patagonia, Argentina

abstract: Tehuelche scallop, Aequipecten tehuelchus, is a commercially exploited species in Northern Patagonia, Argentina. Without genetic differentiation at the species level, A. tehuelchus presents three morphotypes: tehuelchus, madrynensis, and a non-common variant felipponei. The main goal of this study was to analyze the shell shape variation of Tehuelche scallop to differentiate and identify the phenotypic stocks. The shape differences between and within the two main morphotypes (tehuelchus and madrynensis) were assessed using geometric morphometrics in nine localities. The shell shape presented variability at geographic scale, with the morphologic traits that maximized the differentiation among localities between the tehuelchus and madrynensis morphotypes. Scallops from madrynensis morphotype presented higher and circular shell discs with smaller auricles than those from tehuelchus morphotype. Morphometric differentiation was also detected among localities of each morphotype, wherein most of the variability was related to the disc circularity and the symmetry of the auricles. The presence of morphologic variation in San Matias and San Jose gulfs, wherein a single genetic pool is shared, evidenced the plastic nature of the species. Given the distribution of this resource in distinct provincial jurisdictions, the differentiation of phenotypic stocks has relevance in the context of fishery management, especially if zoning and rotational strategies are implemented.


title: Coral niche construction: coral recruitment increases along a coral-built structural complexity gradient

abstract: Niche construction is the process through which organisms modify environmental states in ways favourable to their own fitness. Here, we test experimentally whether scleractinian corals can be considered niche constructors. In particular, we demonstrate a positive feedback involved in corals building structures which facilitate recruitment. Coral larval recruitment is a key process for coral reef persistence. Larvae require low flow conditions to settle from the plankton, and hence the presence of colony structures that can break the flow is expected to facilitate coral recruitment. Here, we show an increase in settler presence on artificial tiles deployed in the field along a gradient of coral-built structural complexity. Structural complexity had a positive effect on settlement, with an increase of 15,71% of settler presence probability along the range of structural complexity considered. This result provides evidence that coral built structural complexity creates conditions that facilitate coral settlement, while demonstrating that corals meet the criteria for ecological niche construction.


title: scSPLAT, a scalable plate-based protocol for single cell WGBS library preparation

abstract:
DNA methylation is a central epigenetic mark that has diverse roles in gene regulation, development, and maintenance of genome integrity. 5 methyl cytosine (5mC) can be interrogated at base resolution in single cells by using bisulfite sequencing (scWGBS). Several different scWGBS strategies have been described in recent years to study DNA methylation in single cells. However, there remain limitations with respect to cost-efficiency and yield. Herein, we present a new development in the field of scWGBS library preparation; single cell Splinted Ligation Adapter Tagging (scSPLAT). scSPLAT employs a pooling strategy to facilitate sample preparation at a higher scale and throughput than previously possible. We demonstrate the accuracy and robustness of the method by generating data from 225 single K562 cells and from 309 single liver nuclei and compare scSPLAT against other scWGBS methods.

MotivationscWGBS library preparation in a one-cell-per-library format presents practical and economical constraints to the number of cells that can be analyzed in a research project. In addition, most of the current scWGBS methods suffer from low read alignment rates. We present a scWGBS protocol which mitigates these issues, empowering single-cell DNA methylation analysis at an increased scale.


title: The β-amylase7 gene in Zea mays encodes a protein with structural and catalytic properties similar to Arabidopsis BAM2

abstract: Starch accumulates in the plastids of green plant tissue during the day to provide carbon for metabolism at night. Starch hydrolysis is catalyzed by members of the {beta}-amylase (BAM) family, which in Arabidopsis thaliana (At), includes nine structurally and functionally diverse members. One of these enzymes, AtBAM2, is a plastid-localized enzyme that is unique among characterized {beta}-amylases since it is tetrameric and exhibits sigmoidal kinetics. Sequence alignments show that the BAM domains of AtBAM7, a catalytically inactive, nuclear-localized transcription factor with an N-terminal DNA binding domain, and AtBAM2 are more closely related to each other than they are to any other AtBAM. Since BAM2 is found in more ancient lineages, it was hypothesized that BAM7 evolved from BAM2. However, analysis of the genomes of 48 flowering plants revealed 12 species that appear to have a BAM7 gene but lack a BAM2 gene. Upon closer inspection, these BAM7 proteins have a greater percent identity to AtBAM2 than to AtBAM7, and they share all of the AtBAM2 functional residues that BAM7 proteins normally lack. We hypothesize that these genes may encode a BAM2-like protein although they are currently annotated as BAM7-like genes. To test this hypothesis, we designed a cDNA of the short form of corn BAM7 (ZmBAM7-S) for expression in E. coli. Small Angle X-Ray Scattering data indicate that ZmBAM7-S has a tetrameric solution structure more similar to that of AtBAM2 than AtBAM1. In addition, partially purified ZmBAM7-S is catalytically active and exhibits sigmoidal kinetics. Together these data suggest that some BAM7 genes may encode a functional BAM2. Exploring and understanding {beta}-amylase gene structure could have impacts on the current annotation of genes.

category: biochemistry


title: In vivo mRNA delivery to virus-specific T cells by light-induced ligand exchange of MHC class I antigen-presenting nanoparticles

abstract:
Simultaneous delivery of mRNA to multiple populations of antigen (Ag)-specific CD8+ T cells is challenging given the diversity of peptide epitopes and polymorphism of class I major histocompatibility complexes (MHCI). We developed Ag-presenting nanoparticles (APNs) for mRNA delivery using pMHCI molecules that were refolded with photocleavable peptides to allow rapid ligand exchange by UV light and site-specifically conjugated with a lipid tail for post-insertion into preformed mRNA lipid nanoparticles. Across different TCR transgenic mouse models (P14, OT-1, Pmel), UV-exchanged APNs bound and transfected their cognate Ag-specific CD8+ T cells equivalent to APNs produced using conventionally refolded pMHCI molecules. In mice infected with PR8 influenza, multiplexed delivery of UV-exchanged APNs against three immunodominant epitopes led to ~50% transfection of a VHH mRNA reporter in cognate Ag-specific CD8+ T cells. Our data shows that UV-mediated peptide exchange can be used to rapidly produce APNs for mRNA delivery to multiple populations of Ag-specific T cells in vivo.

TeaserLight-induced rapid production of antigen-presenting nanoparticles for mRNA delivery to multiple virus-specific T cell populations.


title: Identification of antineoplastic agents for oral squamous cell carcinoma: an integrated bioinformatics approach using differential gene expression and network biology

abstract: Oral squamous cell carcinoma (OSCC) is the most common malignant epithelial neoplasm and anatomical subtype of head and neck squamous cell carcinoma (HNSCC) with an average 5-year survival rate of less than 50%. To improve the survival rate of OSCC, the discovery of novel anti-cancer drugs is urgently needed. In the present study, we performed metanalysis of 5 gene expression datasets (GSE23558, GSE25099, GSE30784, GSE37991 and TCGA-OSCC) that resulted in 1851 statistically significant DEGs in OSCC. The DEGs were involved in key biological pathways that drive the progression of OSCC. A comprehensive protein-protein interaction (PPI) network was constructed from the DEGs and the top protein clusters (modules) were extracted in Cytoscape. The DEGs from the top modules were searched for antineoplastic agents using L1000CDS2 server. The search resulted in a total of 37 perturbing agents from which 12 well-characterized antineoplastic agents were selected. The selected 12 antineoplastic agents namely Teniposide, Palbociclib, Etoposide, Fedratinib, Tivozanib, Afatinib, Vemurafenib, Mitoxantrone, Idamycin, Canertinib, Dovitinib and Selumetinib. These drugs showed interactions with the over expressed hub genes that regulate cellular proliferation and growth in OSCC progression. These identified antineoplastic agents are candidates for their potential role in treating OSCC.


title: Characterization of Cell-cell Communication in Autistic Brains with Single Cell Transcriptomes

abstract: Autism spectrum disorder is a neurodevelopmental disorder, affecting 1-2% of children. Studies have revealed genetic and cellular abnormalities in the brains of affected individuals, leading to both regional and distal cell communication deficits. Recent application of single cell technologies, especially single cell transcriptomics, has significantly expanded our understanding of brain cell heterogeneity and further demonstrated that multiple cell types and brain layers or regions are perturbed in autism. The underlying high-dimensional single cell data provides opportunities for multi-level computational analysis that collectively can better deconvolute the molecular and cellular events altered in autism. Here, we apply advanced computation and pattern recognition approaches on single cell RNA-seq data to infer and compare inter-cell-type signaling communications in autism brains and controls. Our results indicate that at a global level there are cell-cell communication differences in autism in comparison to controls, largely involving neurons as both signaling senders and receivers, but glia also contribute to the communication disruption. Although the magnitude of change is moderate, we find that excitatory and inhibitor neurons are involved in multiple intercellular signaling that exhibit increased strengths in autism, such as NRXN and CNTN signaling. Not all genes in the intercellular signaling pathways are differentially expressed, but genes in the pathways are enriched for axon guidance, synapse organization, neuron migration, and other critical cellular functions. Furthermore, those genes are highly connected to and enriched for genes previously associated with autism risks. Overall, our proof-of-principle computational study using single cell data uncovers key intercellular signaling pathways that are potentially disrupted in the autism brains, suggesting that more studies examining cross-cell type affects can be valuable for understanding autism pathogenesis.


title: Shared and distinct genetic features in human and canine B-cell lymphomas

abstract:
Animal models of human cancers are an important tool for the development and preclinical evaluation of therapeutics. Canine B-cell lymphoma (cBCL) is an appealing model for human mature B-cell neoplasms due to the high sequence similarity in cancer genes to humans and inactive telomerase in adult tissues. We performed targeted sequencing on 86 canine patients from the Canine Comparative Oncology Genomic Consortium, with 61 confirmed as B-cell lymphomas. We confirmed a high frequency of mutations in TRAF3 (45%) and FBXW7 (20%) as has been reported by our group and others. We also note a higher frequency of DDX3X (20%) and MYC (13%) mutations in our canine cohort.

We compared the pattern and incidence of mutations in cBCL to human diffuse large B-cell lymphoma (hDLBCL) and human Burkitt lymphoma (hBL). Canine MYC mutations displayed a focal pattern with 80% of mutations affecting the conserved phosphodegron sequence in MYC box 1, which are known to stabilize MYC protein. We also note that MYC and FBXW7 mutations do not co-occur in our cBCL cohort, leading to the hypothesis that these mutations represent alternative approaches to stabilize MYC in canine lymphoma.

We observed striking differences in the pattern of DDX3X mutations in canine lymphoma as compared to hBL and uncovered a sex-specific pattern of DDX3X mutations in hBL that is not consistent with those identified in canine lymphomas.

In sum, we describe key differences between cBCL and human mature B-cell lymphomas which may indicate differences in the biology of these cancers. This should be considered in future studies of cBCL as a model of human lymphomas.


title: Dysregulated Protein Translation Control in Hidradenitis Suppurativa: Implication for Lesion-associated Squamous Cell Carcinoma Development

abstract: Hidradenitis Suppurativa (HS) is a proinflammatory painful skin disorder. This chronic disease is often associated with aggressive squamous cell carcinoma (SCC). The molecular pathogenesis of this disease progression remains undefined. The translation initiation factor eIF4E/4G/4A1 complex is overexpressed in a variety of human malignancies. In this study, we found that the expression of eIF4E/4G/4A1 as well as phosphorylated eIF4E were upregulated in HS skin. In the global transcription profiles derived from two public database, we were able to enrich 734 eIF4F-related genes. GSEA pathway enrichment analysis further demonstrated that RAS/MEK/ERK oncogene signaling pathway associated with inflammation signaling were significantly activated in HS lesion. The increase expression of eIF4 protein components was associated with enhanced eIF4E translation targets Cyclin D1 and c-Myc. Confocal fluorescence microscopy analysis further revealed that Cyclin D1 and c-Myc specifically co-localized in nuclei of certain cells in HS epithelium. We also found that many of the PCNA positive hyperproliferative cells were also positive for c-Myc expression. These data demonstrate that 5-cap{square}dependent translation is a potential pathway underlying the SCC pathogenesis in chronic HS lesions. Furthermore, being a druggable target, inhibition of eIF4F may block lesion-associated lethal SCCs in HS patients.


title: Mutual Regulation between PFKP and VEGF Promotes Glioblastoma Tumor Growth

abstract: Glioblastoma (GBM) is highly vascular malignant brain tumor that overexpresses vascular endothelial growth factor (VEGF) and phosphofructokinase 1 platelet isoform (PFKP), which catalyzes a rate-limiting reaction in glycolysis. However, it remains unknown whether PFKP and VEGF are reciprocally regulated during GBM tumor growth. Here, we show that PFKP promotes EGFR activation-induced VEGF expression in HIF-1-dependent and -independent manners in GBM cells. Importantly, we demonstrate that EGFR-phosphorylated PFKP Y64 has critical roles in the AKT/SP1-mediated transcriptional expression of HIF-1 and in the AKT-mediated {beta}-catenin S552 phosphorylation, to fully enhance VEGF transcription and subsequent blood vessel formation and brain tumor growth. The levels of PFKP Y64 phosphorylation in human GBM specimens positively correlate with HIF-1 expression, {beta}-catenin S552 phosphorylation, and VEGF expression. Conversely, VEGF upregulates PFKP expression in a PFKP S386 phosphorylation-dependent manner, leading to increased PFK enzyme activity, aerobic glycolysis, and proliferation in GBM cells. These findings highlight a novel mechanism underlying the mutual regulation that occurs between PFKP and VEGF for promoting GBM tumor growth and provide the therapeutic potential of targeting the PFKP/VEGF regulatory loop for GBM patients.


title: Super-doses of dietary vitamin D3 intake in aged laying hens illustrates limitation of 24,25-dihydroxycholecalciferol conversion

abstract:
ABSRACTO_ST_ABSBackgroundC_ST_ABSHumans take vitamin D supplements to reduce risk of vitamin D deficiency and reduce the risk of osteoporosis. However, it is unclear how dietary super-doses (10,000x greater than requirement) can affect vitamin D status in aged animals. Aged laying hens could be a model to compare with women in peri- or postmenopausal stages of life. The hens bone health is physiologically taxed from egg production and they are highly susceptible to osteoporosis.

ObjectiveWe investigated dietary super-dose impacts of cholecalciferol (vitamin D3) on vitamin D status in aged laying hens in production.

MethodsForty-eight 68 wk old Hy-Line Brown laying hens were individually housed in cages with eight hens per dietary treatment for eleven weeks. Hens were randomly assigned to one of six groups of dietary vitamin D3 supplementation and fed ad libitum. Supplementation levels were 400, 800, 7,400, 14,000, 20,000, and 36,000 IU D3/kg of feed. At termination of the study, all hens were euthanized and we collected tissue samples and their feces. Tissue calcium, plasma and egg yolk vitamin D metabolites, and gene expression levels were measured.

ResultsWe observed that increasing dietary vitamin D3 increased plasma vitamin D3, 25-hydroxycholecalciferol, and 24,25-dihydroxycholecalciferol concentrations (p < 0.0001 for all 3 metabolites). Also, egg yolk vitamin D3 and 25-hydroxycholecalciferol showed a similar effect like plasma vitamin D metabolites (p < 0.0001 for both metabolites). We also observed super-dose fed hens had decreased kidney 24-hydroxylase expression (p = 0.0006).

ConclusionsDietary super-doses of vitamin D3 led to greater plasma and egg yolk vitamin D levels to show that aged laying hens can deposit excess vitamin D in egg yolk. We suggest future research should explore how 24-hydroxylation mechanisms are affected by vitamin D supplementation. Further understanding of 24-hydroxylation can help ascertain ways to reduce risk of vitamin D toxicity.


title: Predicting the Influence of Axon Myelination on Sound Localization Precision Using a Spiking Neural Network Model of Auditory Brainstem

abstract: Spatial hearing allows animals to rapidly detect and localize auditory events in the surrounding environment. The auditory brainstem plays a central role in processing and extracting binaural spatial cues through microsecond-precise binaural integration, especially for detecting interaural time differences (ITDs) of low-frequency sounds at the medial superior olive (MSO). A series of mechanisms exist in the underlying neural circuits for preserving accurate action potential timing across multiple fibers, synapses and nuclei along this pathway. One of these is the myelination of afferent fibers that ensures reliable and temporally precise action potential propagation in the axon. There are several reports of fine-tuned myelination patterns in the MSO circuit, but how specifically myelination influences the precision of sound localization remains incompletely understood. Here we present a spiking neural network model of the auditory brainstem with myelinated axons to investigate whether different axon myelination thicknesses alter the sound localization process. Our model demonstrates that axon myelin thickness along the contralateral pathways can substantially modulate ITD detection. Furthermore, optimal ITD sensitivity is reached when the MSO receives contralateral inhibition via thicker myelinated axons compared to contralateral excitation, a result that is consistent with previously reported experimental observations. Our results suggest specific roles of axon myelination for extracting temporal dynamics in ITD perception, especially in the pathway of the contralateral inhibition.


title: On the brain networks organization of individuals with high versus average fluid intelligence: a combined DTI and MEG study

abstract: The neural underpinning of human fluid intelligence (Gf) has gathered a large interest in the scientific community. Nonetheless, previous research did not provide a full understanding of such intriguing topic. Here, we studied the structural (from diffusion tensor imaging, DTI) and functional (from magnetoencephalography (MEG) resting state) connectivity in individuals with high versus average Gf scores. Our findings showed greater values in the brain areas degree distribution and higher proportion of long-range anatomical connections for high versus average Gfs. Further, the two groups presented different community structures, highlighting the structural and functional integration of the cingulate within frontal subnetworks of the brain in high Gfs. These results were consistently observed for structural connectivity and functional connectivity of delta, theta and alpha. Notably, gamma presented an opposite pattern, showing more segregation and lower degree distribution and connectivity in high versus average Gfs. Our study confirmed and expanded previous perspectives and knowledge on the "small-worldness" of the brain. Further, it complemented the widely investigated structural brain network of highly intelligent individuals with analyses on fast-scale functional networks in five frequency bands, highlighting key differences in the integration and segregation of information flow between slow and fast oscillations in groups with different Gf.


title: Impact of IgG isotype on the induction of antibody-dependent cellular phagocytosis of HIV by human milk leukocytes

abstract: Approximately 100,000 mother-to-child transmission (MTCT) events of HIV via human milk feeding occur each year (1). However, only about 15% of infants milk-fed by untreated HIV+ mothers become infected, suggesting a protective effect of the milk itself (1, 2). Infants ingest 105-108 maternal leukocytes daily via milk, which remain functional beyond ingestion (3-9). Such function may be elicited by maternal milk antibody (Ab). Though IgA is dominant in milk, most HIV-specific milk Abs are of the IgG subclass, highlighting the importance of investigating the function of each IgG isotype in the milk context (10-16). Though Ab effector function mediated by the constant (Fc) domain via interaction with Fc Receptors (FcRs), such as Ab-dependent cellular phagocytosis (ADCP), are critical in protecting against HIV infection, ADCP is largely unexplored as it relates to mitigation of MTCT (17-21). Presently we report the ADCP activity of milk leukocytes against HIV particles and immune complexes (ICs), using 57 unique samples from 34 women, elicited by IgG1/2/3/4 of monoclonal (m)Ab 246-D. Granulocyte ADCP of HIV was most potent compared to other phagocytes when elicited by IgG1/3/4. IgG1/3 activated granulocytes similarly, exhibiting 1.6x-4.4x greater activity compared to IgG2/4, and a preference for virus compared to ICs. Notably, CD16-monocyte ADCP of a given target were unaffected by isotype, and CD16+ monocytes were poorly stimulated by IgG1. IgG2/4 elicited potent IC ADCP, and in terms of total leukocyte IC ADCP, IgG4 and IgG3 exhibited similar function, with IgG4 eliciting 1.6x-2.1x greater activity compared to IgG1/IgG2, and CD16+ monocytes most stimulated by IgG2. These data contribute to a more comprehensive understanding of Fc-mediated functionality of milk leukocytes, which is critical in order to develop therapeutic approaches to eliminating this route of MTCT, including mucosal administration of mAbs and/or a maternal vaccination aimed to elicit a potent milk Ab response.


title: Annulment of antagonism shifts properties that are beneficial to plants in two-member consortia of Bacillus velezensis

abstract:
Bacillus spp. strains that are beneficial to plants are widely used in commercial biofertilizers and biocontrol agents for sustainable agriculture. Generally, functional Bacillus strains are applied as single strain communities since the principles of synthetic microbial consortia constructed with Bacillus strains remain largely unclear. Here, we demonstrated that the mutual compatibility directly affects the survival and function of two-member consortia composed of B. velezensis SQR9 and FZB42 in the rhizosphere. A mutation in the global regulator Spo0A of SQR9 markedly reduced the boundary phenotype with wild-type FZB42, and the combined use of the SQR9{triangleup}spo0A mutant and FZB42 improved biofilm formation, root colonization and the production of secondary metabolites that are beneficial to plants. We further confirmed the correlation between the swarm discrimination phenotype between the two consortia members and the effects that are beneficial to plants in a greenhouse experiment. Our results provide evidence that social interactions among bacteria could be an influencing factor in achieving a desired community-level function.

IMPORTANCEBacillus velezensis is one of the most widely applied bacteria in biofertilizers in China and Europe. Additionally, the molecular mechanisms of plant growth promotion and disease suppression by representative model strains are well established, such as B. velezensis SQR9 and FZB42. However, it remains extremely challenging to design efficient consortia based on these model strains. Here, we showed that swarm discrimination phenotype is one of the major determinants affects the performance of two-member Bacillus consortia in vitro and in the rhizosphere. Deletion in global regulatory gene spo0A of SQR9 reduced the strength of boundary formation with FZB42, result in the improved plant growth promotion performance of dual consortium. This knowledge provides new insights into efficient probiotics consortia design in Bacillus.

category: microbiology


title: Bacterial, phytoplankton, and viral dynamics of meromictic Lake Cadagno offer insights into the Proterozoic ocean microbial loop

abstract:
Lake Cadagno, a permanently stratified high-alpine lake with a persistent microbial bloom in its anoxic chemocline, has long been considered a model for the low-oxygen, high-sulfide Proterozoic ocean where early microbial life gave rise to Earths oxygenated atmosphere. Although the lake has been studied for over 25 years, the absence of concerted study of the bacteria, phytoplankton, and viruses, together with primary and secondary production, has hindered a comprehensive understanding of its microbial food web. Here, the identities, abundances, and productivity of microbes were evaluated in the context of Lake Cadagno biogeochemistry. Photo-synthetic pigments and chloroplast 16S rRNA gene phylogenies suggested high abundances of eukaryotic phytoplankton, primarily Chlorophyta, through the water column. Of these, a close relative of Ankyra judayi, a high-alpine adapted chlorophyte, peaked with oxygen in the mixolimnion, while Closteriopsis-related chlorophytes peaked in the chemocline and monimolimnion. Anoxygenic phototrophic sulfur bacteria, Chromatium, dominated the chemocline along with Lentimicrobium, a newly observed genus of known fermenters. Secondary production peaked in the chemocline, suggesting anoxygenic primary producers depended on heterotrophic nutrient remineralization. Virus-to-microbe ratios spanned an order of magnitude, peaking with high phytoplankton abundances and at a minimum at the peak of Chromatium, dynamic trends that suggest viruses may play a role in the modulation of oxygenic and anoxygenic photo- and chemosynthesis in Lake Cadagno. Through the combined analysis of bacterial, eukaryotic, viral, and biogeochemical dynamics of Lake Cadagno, this study provides a new perspective on the biological and geochemical connections that comprised the food webs of the Proterozoic ocean.

IMPORTANCEAs a window to the past, the study offers insights into the role of microbial guilds of Proterozoic ocean chemoclines in the production and recycling of organic matter of sulfur- and ammonia-containing ancient oceans. The new observations described here suggest that eukaryotic algae were persistent in the low oxygen upper-chemocline in association with purple and green sulfur bacteria in the lower half of the chemocline. Further, this study provides the first insights into Lake Cadagno viral ecology. High viral abundances suggested viruses may be essential components of the chemocline where their activity may result in the release and recycling of organic matter. The framework developed in this study through the integration of diverse geochemical and biological data types lays the foundation for future studies to quantitatively resolve the processes performed by discrete populations comprising the microbial loop in this early anoxic ocean analogue.


title: Hunting the plant surrender signal activating apoplexy in grapevines after Neofusicoccum parvum infection

abstract:
O_LIApoplectic breakdown from Grapevines Trunk Diseases (GTDs) has become a serious challenge to viticulture in consequence to drought stress. We hypothesise that fungal aggressiveness is controlled by a chemical communication between host and colonising fungus.
C_LIO_LIWe introduce the new concept of a "plant surrender signal" accumulating in host plants under stress and triggering aggressive behaviour of the strain Neofusicoccum parvum (Bt-67) causing Botryosphaeriaceae-related dieback in grapevines.
C_LIO_LIUsing a cell-based experimental system (Vitis cells) and bioactivity-guided fractionation, we identify trans-ferulic acid, a monolignol precursor, as "surrender signal". We show that this signal specifically activates secretion of the fungal phytotoxin Fusicoccin A. We show further that this phytotoxin, mediated by 14-3-3 proteins, activates programmed cell death in Vitis cells.
C_LIO_LIWe arrive at a model pinpointing the chemical communication driving apoplexy in Botryosphaeriaceae-Vitis interaction and define the channelling of phenylpropanoid pathway from the lignin precursor, trans-ferulic acid to the phytoalexin transresveratrol as target for future therapy.
C_LI


title: Characterization of pyridomycin B reveals the formation of functional groups in antimycobacterial pyridomycin

abstract:
Pyridomycin, a cyclodepsipeptide with potent antimycobacterial activity, specifically inhibits the InhA enoyl reductase of Mycobacteria tuberculosis. Structure-activity relationship studies indicated that the enolic acid moiety in pyridomycin core system is an important pharmacophoric group and the natural configuration of the C-10 hydroxyl contributes to the bioactivity of pyridomycin. The ring structure of pyridomycin was generated by the nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) hybrid system (PyrE-F-G). Bioinformatics analysis reveals that SDR family protein Pyr2 functions as a 3-oxoacyl ACP reductase in the pyridomycin pathway. Inactivation of pyr2 resulted in accumulation of pyridomycin B, a new pyridomycin analogue featured with enol moiety in pyridyl alanine moiety and a saturated 3-methylvaleric acid group. The elucidated structure of pyridomycin B suggests that rather than functioning as a post-tailoring enzyme, Pyr2 catalyzes ketoreduction to form the C-10 hydroxyl group in pyridyl alanine moiety and the double bond formation of the enolic acid moiety derived from isoleucine when the intermediate assembled by PKS-NRPS machinery is still tethered to the last NRPS module, in a special energy-saving manner. Ser-His-Lys residues constitute the active site of Pyr2, which is different from the typically conserved Tyr based catalytic triad in the majority of SDRs. Site-directed mutation identified that His154 in the active site is a critical residue for pyridomycin B production. These findings will improve our understanding of the pyridomycin biosynthetic logic, identify the missing link for the double bound formation of enol ester in pyridomycin and enable creating chemical diversity of pyridomycin derivatives.

ImportanceTuberculosis (TB) is one of the worlds leading causes of death by infection. Recently, pyridomycin, the antituberculous natural product from Streptomyces has garnered considerable attention for being determined as a target inhibitor of InhA enoyl reductase of Mycobacteria tuberculosis. In this study, we report a new pyridomycin analogue from mutant HTT12, demonstrate the essential role of a previously ignored gene pyr2 in pyridomycin biosynthetic pathway, and imply that Pyr2 functions as a trans ketoreductase (KR) contributing to the formation of functional groups of pyridomycin utilize a distinct catalytic mechanism. As enol moiety are important for pharmaceutical activities of pyridomycin, our work would expand the understanding the mechanism of SDR family proteins and set the stage for future bioengineering of new pyridomycin derivatives.


title: Clustered PHD domains in mixed lineage leukaemia proteins are attracted by acetylation-rich active promoters and enhancers

abstract: Histone lysine methyltransferase (KMT2) proteins form the core of COMPASS and COMPASS-like complexes that mediate transcriptional memory by methylating H3K4 at promoters and enhancers. KMT2A-D proteins, alternatively called mixed lineage leukaemia proteins (MLL1-4), contain highly conserved unique triplet and quartet of plant homeodomains (PHDs). Here, we show that clustered PHDs, expressed in isolation in HeLa cells, localize to well-defined loci of acetylation-rich active promoters and enhancers. Binding sites overlap with targets of full-length KMT2A (MLL1) and the COMPASS-like subunit WDR5, RbBP5 and with cell cycle and cancer-related genes. COSMIC data identify frequent variations in the PHDs of KMT2 proteins, particularly KMT2C, in a wide spectrum of malignancies. Changes are enriched at conserved positions within the PHDs, indicating that they cause loss-of-function mutations. Taken together, the biochemical and cancer data suggest that the PHDs contribute to KMT2A-D targeting to active promoters and enhancers.

category: molecular biology


title: Heat shock chaperone HSPB1 regulates cytoplasmic TDP-43 phase separation and liquid-to-gel transition

abstract: While the RNA binding protein TDP-43 reversibly phase separates within nuclei into complex droplets (anisosomes) with TDP-43-containing liquid outer shells and liquid centers of HSP70 family chaperones, cytoplasmic aggregates of TDP-43 are hallmarks of multiple neurodegenerative diseases, including ALS. Here we show that transient oxidative stress, proteasome inhibition, or inhibition of HSP70s ATP-dependent chaperone activity provokes reversible cytoplasmic TDP-43 de-mixing and transition from liquid to gel/solid, independent of RNA binding or stress granules. Isotope labeling mass spectrometry is used to identify that phase separated cytoplasmic TDP-43 is primarily bound by the small heat shock protein HSPB1. Binding is direct, mediated through TDP-43s RNA binding and low complexity domains. HSPB1 partitions into TDP-43 droplets, inhibits TDP-43 assembly into fibrils, and is essential for disassembly of stress-induced, TDP-43 droplets. Decrease of HSPB1 promotes cytoplasmic TDP-43 de-mixing and mislocalization. HSPB1 depletion is identified within ALS-patient spinal motor neurons containing aggregated TDP-43. These findings identify HSPB1 to be a regulator of cytoplasmic TDP-43 phase separation and aggregation.


title: Golgi structural defect impairs glycosaminoglycan synthesis, sulfation, and secretion

abstract: Synthesis of glycosaminoglycans such as heparan sulfate (HS) and chondroitin sulfate (CS) occurs in the lumen of the Golgi but the relationship between Golgi structural integrity and glycosaminoglycan synthesis is not clear. In this study, we disrupted the Golgi structure by knocking out GRASP55 and GRASP65 and determined its effect on the synthesis, sulfation, and secretion of HS and CS. We found that GRASP depletion increased HS synthesis while decreasing CS synthesis in cells, altered HS and CS sulfation, and reduced both HS and CS secretion. Using proteomics, RNA-seq and biochemical approaches, we identified EXTL3, a key enzyme in the HS synthesis pathway, whose level is upregulated in GRASP knockout cells; while GalNacT1, an essential CS synthesis enzyme, is robustly reduced. In addition, we found that GRASP depletion decreased HS sulfation via the reduction of PAPSS2, a bifunctional enzyme in HS sulfation. Our study provides the first evidence that Golgi structural defect may significantly alter the synthesis and secretion of glycosaminoglycans.


title: Extremely reduced supergroup F Wolbachia: transition to obligate insect symbionts

abstract: Wolbachia are widely distributed symbionts among invertebrates that manifest by a broad spectrum of lifestyles from parasitism to mutualism. Wolbachia Supergroup F is considered a particularly interesting group which gave rise to symbionts of both arthropods and nematodes, and some of its members are obligate mutualists. Further investigations on evolutionary transitions in symbiosis have been hampered by a lack of genomic data for Supergroup F members. In this study, we present genomic data for five new supergroup F Wolbachia strains associated with four chewing lice species. These new strains in different evolutionary stages show genomic characteristics well-illustrating the evolutionary trajectory which symbiotic bacteria experience during their transition to mutualism. Three of the strains have not yet progressed with the transition, the other two show typical signs of ongoing gene deactivation and removal (genome size, coding density, low number of pseudogenes). Particularly, wMeur1, a symbiont fixed in all Menacanthus eurysternus populations across four continents, possesses a highly reduced genome of 733,850 bp with a horizontally acquired capacity for pantothenate synthesis. Comparing with other strains showed wMeur1 genome as the smallest currently known among all Wolbachia and the first example of Wolbachia which has completed genomic streamlining known from the gammaproteobacterial obligate symbionts.

category: evolutionary biology


title: Genetic and environmental influences on the evolution of virulence in the HIV-associated opportunistic human fungal pathogen Cryptococcus neoformans

abstract: The fungus Cryptococcus neoformans is considered the leading cause of death in immunocompromised patients. Despite numerous investigations concerning its molecular epidemiology, there are only a few studies addressing the impacts of varying factors on genotype-phenotype correlations. It remains largely unknown whether genetic and environmental variabilities among isolates from different sources may have dramatic consequences on virulence. In this study, we analyzed 105 Chinese C. neoformans isolates, including 54 from HIV-infected patients, 44 from HIV-uninfected individuals and 7 from a natural environment, to investigate factors influencing the outcome of C. neoformans infection. MLST analysis clearly identified sequence type (ST) 5 as the prevalent sequence type in all clinical isolates and interestingly, genotypic diversities were observed in isolates from both HIV-uninfected individual and natural environment but not those from HIV-infected patients. Moreover, we found that compared to those from HIV-infected patients, the isolates from HIV-uninfected individuals exhibited enhanced virulence-associated traits including significantly elevated capsule production and melanin formation, increases in survival in human cerebrospinal fluid (CSF), less effective uptake by host phagocytes, and higher mortality in a mouse model of cryptococcosis. Consistently, pathogenic phenotypes were associated with CD4 counts of patients, implying environmental impact on within-host C. neoformans virulence. Importantly, a large-scale whole-genome sequencing analysis revealed that genomic variations within genes related to specific functions may act as a vital driving force of host intrinsic virulence evolution. Taken together, our results support a strong genotype-phenotype correlation suggesting that the pathogenic evolution of C. neoformans could be heavily affected by both genetic and environmental factors.


title: Diversity and evolution of nitric oxide reduction

abstract: Nitrogen is an essential element for life, with the availability of fixed nitrogen limiting productivity in many ecosystems. The return of oxidized nitrogen species to the atmospheric N2 pool is predominately catalyzed by microbial denitrification (NO3- [->] NO2- [->] NO [->] N2O [->] N2)1. Incomplete denitrification can produce N2O as a terminal product, leading to an increase in atmospheric N2O, a potent greenhouse and ozone-depleting gas2. The production of N2O is catalyzed by nitric oxide reductase (NOR) members of the heme-copper oxidoreductase (HCO) superfamily3. Here we use phylogenomics to identify a number of previously uncharacterized HCO families and propose that many of them (eNOR, sNOR, gNOR, and nNOR) perform nitric oxide reduction. These families have novel active-site structures and several have conserved proton channels, suggesting that they might be able to couple nitric oxide reduction to energy conservation. We isolated and biochemically characterized a member of the eNOR family from Rhodothermus marinus, verifying that it performs nitric oxide reduction both in vitro and in vivo. These newly identified NORs exhibit broad phylogenetic and environmental distributions, expanding the diversity of microbes that can perform denitrification. Phylogenetic analyses of the HCO superfamily demonstrate that nitric oxide reductases evolved multiple times independently from oxygen reductases, suggesting that complete denitrification evolved after aerobic respiration.


title: Speed fluctuations of bacterial replisomes

abstract: Replisomes are multi-protein complexes that replicate genomes with remarkable speed and accuracy. Despite their importance, their dynamics is poorly characterized, especially in vivo. In this paper, we present an approach to infer the replisome dynamics from the DNA abundance distribution measured in a growing bacterial population. Our method is sensitive enough to detect subtle variations of the replisome speed along the genome. As an application, we experimentally measured the DNA abundance distribution in Escherichia coli populations growing at different temperatures using deep sequencing. We find that the average replisome speed increases nearly five-fold between 17{degrees}C and 37{degrees}C. Further, we observe wave-like variations of the replisome speed along the genome. These variations correlate with previously observed variations of the mutation rate. We interpret this correlation as a speed-error trade-off in DNA replication. Our approach has the potential to elucidate replication dynamics in E. coli mutants and in other bacterial species.


title: Chemophoresis Engine: Universal Principle of ATPase-driven Cargo Transport

abstract:
Cell polarity regulates the orientation of the cytoskeleton members that directs intracellular transport for cargo-like organelles, using chemical gradients sustained by ATP or GTP hydrolysis. However, how cargo transports are directly mediated by chemical gradients remains unknown. We previously proposed a physical mechanism that enables directed movement of cargos, referred to as chemophoresis. According to the mechanism, a cargo with reaction sites is subjected to a chemophoresis force in the direction of the increased concentration. Based on this, we introduce an extended model, the chemophoresis engine, as a general mechanism of cargo motion, which transforms chemical free energy into directed motion through the catalytic ATP hydrolysis. We applied the engine to plasmid motion in a parABS system to demonstrate the the self-organization system for directed plasmid movement and pattern dynamics of ParA-ATP concentration, thereby explaining plasmid equi-positioning and pole-to-pole oscillation observed in bacterial cells and in vitro experiments. We mathematically show the existence and stability of the plasmid-surfing pattern, which allows the cargo-directed motion through the symmetry-breaking transition of the ParA-ATP spatiotemporal pattern. Finally, based on its generality, we discuss the chemophoresis engine as a universal principle of hydrolysis-driven intracellular transport.

SIGNIFICANCEThe formation of organelle/macromolecule patterns depending on chemical concentration under non-equilibrium conditions, first observed during macroscopic morphogenesis (1), has recently been observed at the intracellular level as well, and its relevance as intracellular morphogen has been demonstrated in the case of bacterial cell division. These studies have discussed how cargos maintain positional information provided by chemical concentration gradients/localization. However, how cargo transports are directly mediated by chemical gradients remains unknown. Based on the previously proposed mechanism of chemotaxis-like behavior of cargos (referred to as chemophoresis, (2, 3)), we introduce a chemophoresis engine as a physicochemical mechanism of cargo motion, which transforms chemical free energy to directed motion. The engine is based on the chemophoresis force to make cargoes move in the direction of the increasing ATPase(-ATP) concentration and an enhanced catalytic ATPase hydrolysis at the positions of the cargoes. Applying the engine to ATPase-driven movement of plasmid-DNAs in bacterial cells, we constructed a mathematical model to demonstrate the self-organization for directed plasmid motion and pattern dynamics of ATPase concentration, as is consistent with in vitro and in vivo experiments. We propose that this chemophoresis engine works as a universal principle of hydrolysis-driven intracellular transport.


title: Ultraviolet radiation modulates DNA methylation in melanocytes

abstract:
Ultraviolet radiation (UVR) is the principal causal factor for melanoma; albeit the underlying mechanisms remain unclear. While the mutagenic properties of UVR are irrefutable, the role of UVR-induced mutations in the initiation of melanoma is controversial which highlights the gap in our knowledge of the initial critical molecular mechanisms of UVR-induced melanomagenesis. To investigate the potential non-mutational mechanisms of UVR-induced melanomagenesis, we studied the role of UVR in modulating DNA methylation changes in melanocytes via next-generation sequencing-based methodologies. Here we show that UVR directly causes stable changes in the DNA methylome and transcriptome, one month after exposure. Genomic features associated with transcription were protected from 5mC alterations whereas CpG sites found in intergenic regions were more likely to be affected. Additionally, the long-term effects of UVR seem to perturb signaling pathways important for melanocyte biology. Interestingly, UVR-sensitive CpG sites were found to be prognostic of overall patient survival and highlighted a subset of CpG sites that may be relevant in melanomagenesis.

SignificanceWe report a novel finding that ultraviolet radiation (UVR) induces DNA methylation changes along with stable alterations in gene expression in cultured melanocytes. Our results provide experimental evidence of UVR-induced epigenetic rewiring, which may be implicated in the susceptibility to melanomagenesis, independently of its mutational effects. These findings offer novel insight into the role of UVR in the initiation and pathogenesis of melanoma via a currently underappreciated mechanism.


title: Integrated analysis of single-cell and transcriptome based RNA-seq multilayer network and WGCNA for construction and validation of an immune cell-related prognostic model in clear cell renal cell carcinoma

abstract: Clear cell renal cell carcinoma (ccRCC) is the most common type of renal cancer (RCC). The increasing incidence and poor prognosis of ccRCC after tumour metastasis makes the study of its pathogenesis extremely important. Traditional studies mostly focus on the regulation of ccRCC by single gene, while ignoring the impact of tumour heterogeneity on disease progression. The purpose of this study is to construct a prognostic risk model for ccRCC by analysing the differential marker genes related to immune cells in the single-cell database for providing help in clinical diagnosis and targeted therapy. Single-cell data and ligand-receptor relationship pair data were downloaded from related publications, and ccRCC phenotype and expression profile data were downloaded from TCGA and CPTAC. The DEGs and marker genes of the immune cell were combined and then intersected with the ligand-receptor gene data, and the 981 ligand-receptor relationship pairs obtained were intersected with the target gene of the transcription factor afterwards; 7,987 immune cell multilayer network relationship pairs were finally observed. Then, the genes in the network and the genes in TCGA were intersected to obtain 966 genes for constructing a co-expression network. Subsequently, 53 genes in black and magenta modules related to prognosis were screened by WGCNA for subsequent analysis. Whereafter, using the data of TCGA, 28 genes with significant prognostic differences were screened out through univariate Cox regression analysis. After that, LASSO regression analysis of these genes was performed to obtain a prognostic risk scoring model containing 16 genes, and CPTAC data showed that the effectiveness of this model was good. The results of correlation analysis between the risk score and other clinical factors showed that age, grade, M, T, stage and risk score were all significantly different (p < 0.05), and the results of prognostic accuracy also reached the threshold of qualification. Combined with clinical information, univariate and multivariate Cox regression analyses verified that risk score was an independent prognostic factor (p < 0.05). A nomogram constructed based on a predictive model for predicting the overall survival was established, and internal validation performed well. Our findings suggest that the predictive model built based on the immune cells scRNA-seq will enable us to judge the prognosis of patients with ccRCC and provide more accurate directions for basic relevant research and clinical practice.


title: KRAS is vulnerable to reversible switch-II pocket engagement in cells

abstract: Current small molecule inhibitors of KRAS(G12C) bind irreversibly in the switch-II pocket, exploiting the strong nucleophilicity of the acquired cysteine as well as the preponderance of the GDP-bound form of this mutant. Nevertheless, many oncogenic KRAS mutants lack these two features, and it remains unknown whether targeting the switch-II pocket is a practical therapeutic approach for KRAS mutants beyond G12C. Here we use NMR spectroscopy and a novel cellular KRAS engagement assay to address this question by examining a collection of SII-P ligands from the literature and from our own laboratory. We show that the switch-II pockets of many GTP hydrolysis-deficient KRAS hotspot (G12, G13, Q61) mutants are accessible using non-covalent ligands, and that this accessibility is not necessarily coupled to the GDP state of KRAS. The results we describe here emphasize the switch-II pocket as a privileged drug binding site on KRAS and unveil new therapeutic opportunities in RAS-driven cancer.