title: cir-DNA sequencing revealed the landscape of extrachromosomal circular DNA in articular cartilage and the potential roles in osteoarthritis

abstract:
ObjectiveExtrachromosomal circular DNA (eccDNA) has been shown to be involved in several physiological and pathological processes including immunity, inflammation, aging and tumor. However, the expression of eccDNA in cartilage has been not reported until now. In this study, we aimed to investigate the landscape of eccDNA in articular cartilage and analyze the potential roles in Osteoarthritis (OA).

MethodsThe samples of articular cartilage were obtained from total knee arthroplasty (TKA) donors with OA. The mtDNAs (mitochondrial DNA) and the linear DNAs from chondrocytes of articular cartilage were removed. Then the eccDNAs were enriched for cir-DNA sequencing. After quality control evaluation, we systematically revealed the identified eccDNA data including size distribution, the size range and sequence pattern. Moreover, we explored and disscussed the potential roles of eccDNA in OA via motif analysis and GO/KEGG pathways analysis.

ResultsThe chondrocytes from OA cartilage contained an abundance of eccDNAs, which was termed as OC-eccDNAs (OA Cartilage-derived eccDNA). The characteristics of OC-eccDNAs were tissue specific, including the distribution, the size range and sequence pattern. Moreover, the functional analysis indicated that eccDNA may be involved in the homeostasis maintenance of chondrocytes and participated in the process of OA.

ConclutionsOur data firstly showed the landscape of eccDNA in articular cartilage, and preliminarily indicated the potential roles of eccDNA in OA.


title: Single-cell RNA sequencing identifies phenotypically, functionally, and anatomically distinct stromal niche populations in human bone marrow

abstract: Hematopoiesis is regulated by the bone marrow (BM) stroma. However, cellular identities and functions of the different BM stromal elements in humans remain poorly defined. Based on single-cell RNA sequencing, we systematically characterized the BM stromal compartment which led to the identification of six transcriptionally and functionally distinct stromal cell populations. Stromal cell differentiation hierarchy was recapitulated based on RNA velocity analysis, in vitro proliferation capacities and differentiation potentials. Potential key factors that govern the transition from stem and progenitor cells to fate-committed cells were identified. Cell-cell communication and in situ localization analysis demonstrated distinct hematopoietic stromal cell niches in specific BM locations, which used either the CXCL12 or SPP1 axis as the major hematopoiesis-regulating mechanism. These findings provide the basis for a comprehensive understanding of the cellular complexity of the human BM microenvironment and the intricate stroma-hematopoiesis crosstalk mechanisms, thus refining our current view on hematopoietic niche organization.

category: cell biology


title: An organoid CRISPRi screen revealed that SOX9 primes human fetal lung tip progenitors to receive WNT and RTK signals

abstract:
The balance between self-renewal and differentiation in human fetal lung epithelial progenitors controls the size and function of the adult organ. Moreover, progenitor cell gene regulation networks are employed by both regenerating and malignant lung cells, where modulators of their effects could potentially be of therapeutic value. Details of the molecular networks controlling human lung progenitor self-renewal remain unknown. We performed the first CRISPRi screen in primary human lung organoids to identify transcription factors controlling progenitor self-renewal. We show that SOX9 promotes proliferation of lung progenitors and inhibits precocious airway differentiation. Moreover, by identifying direct transcriptional targets using Targeted DamID we place SOX9 at the centre of a transcriptional network which amplifies WNT and RTK signalling to stabilise the progenitor cell state. In addition, the proof-of-principle CRISPRi screen and Targeted DamID tools establish a new approach for using primary human organoids to elucidate detailed functional mechanisms underlying normal development and disease.

HighlightsO_LIA pooled CRISPRi screen in human fetal lung organoids identified transcription factors controlling progenitor cell self-renewal.
C_LIO_LISOX9 promotes tip progenitor cell proliferation and supresses precocious airway differentiation.
C_LIO_LITargeted DamID (TaDa) identified SOX9 direct binding targets, revealing that SOX9 lies at the intersection of WNT and RTK signalling.
C_LIO_LISOX9 and ETVs co-regulate the human fetal lung progenitor self-renewal programme.
C_LI

category: developmental biology


title: Genetic Regulation of Circular RNA Expression in Human Aortic Smooth Muscle Cells and Vascular Traits

abstract:
BackgroundCircular RNAs (circRNAs) are a class of non-coding RNAs that have cell-type specific expression and are relevant in cardiovascular disease. Aortic smooth muscle cells (SMCs) play a crucial role in cardiovascular disease by differentiating from a quiescent to proliferative phenotype. The role of circRNAs in SMCs and their relevance to cardiovascular disease is largely unexplored.

ResultsIn this study, we employ a systems genetics approach to identify circRNA transcripts at a genome wide level and their relevance in cardiovascular traits. We quantified circRNA expression across 151 quiescent and proliferative human aortic SMCs from multiethnic donors. We identified 1,589 expressed circRNAs. Between quiescent and proliferative SMCs, we identified 173 circRNAs which were differentially expressed. To characterize the genetic regulation of circRNA expression, we associated the genotypes of 6.3 million single nucleotide polymorphisms (SNPs) with circRNA abundance and found 96 circRNAs which were associated with genetic loci. Three SNPs were associated with circRNA expression in proliferative SMCs but not in quiescent SMCs. We identified 6 SNPs which had distinct association directions with circRNA isoforms from the same gene. Lastly, to identify the relevance of circRNAs in cardiovascular disease, we overlapped genetic loci associated with circRNA expression with vascular disease related GWAS loci. We identified 7 blood pressure, 1 myocardial infarction, and 3 coronary artery disease loci which were associated with a circRNA transcript (circZKSCAN1, circFOXK2, circANKRD36, circLARP4, circCEP85L, circGTF3C2, circPDS5A, circSLC4A7, and chr17:42610108|42659552) but not mRNA transcript.

ConclusionsOverall, our results provide mechanistic insight into the regulation of circRNA expression and the genetic basis of cardiovascular disease.

category: genetics


title: Programmable eukaryotic protein expression with RNA sensors

abstract:
The diversity of cell types and states can be scalably measured and defined by expressed RNA transcripts. However, approaches to programmably sense and respond to the presence of specific RNAs within living biological systems with high sensitivity are lacking. RNA sensors that gate expression of reporter or cargo genes would have diverse applications for basic biology, diagnostics and therapeutics by enabling cell-state specific control of transgene expression. Here, we engineer a novel programmable RNA-sensing technology, Reprogrammable ADAR Sensors (RADARS), which leverages RNA editing by adenosine deaminases acting on RNA (ADAR) to gate translation of a protein payload on the presence of endogenous RNA transcripts. In mammalian cells, we engineer RADARS with diverse payloads, including luciferase and fluorescent proteins, with up to 164-fold activation and quantitative detection in the presence of target RNAs. We show RADARS are functional either expressed from DNA or as synthetic mRNA. Importantly, RADARS can function with endogenous cellular ADAR. We apply RADARS to multiple contexts, including RNA-sensing induced cell death via caspases, cell type identification, and in vivo control of synthetic mRNA translation, demonstrating RADARS as a tool with significant potential for gene and cell therapy, synthetic biology, and biomedical research.

One Sentence SummaryA new technology utilizing ADAR mediated RNA-editing enables robust reprogrammable protein expression based on target RNA transcripts in mammalian cells, leading to broad applications in basic science research, cell engineering, and gene therapy.

category: genomics


title: Pyrrole produced by Pseudomonas aeruginosa influences olfactory food choice of Caenorhabditis elegans

abstract: Sense of smell can influence dietary choices in animals. So far, most of the research has focused on how animals respond to distinct odors when they are introduced individually. However, it remains unclear how animals evaluate foods that contain a bouquet of olfactory cues with contrasting effects. Here, we utilize Caenorhabditis elegans as a bacterivore to ask if odors produced by dietary bacteria can regulate worms food preferences. We show that the bacterium Pseudomonas aeruginosa produces a relatively small quantity of a new attractant for C. elegans. We identify the odor as a heterocyclic compound called pyrrole. We find that pyrrole contributes to the sensory decision-making of worms in diet preference assays. Using specific neuronal ablation lines and calcium response assays, we show that AWA odor sensory neurons of worms are necessary for sensing pyrrole. In all, we show that specific odors produced by bacteria can influence food choice behavior of animals.

category: animal behavior and cognition


title: Permethylation of ribonucleosides provides enhanced mass spectrometry quantification of post-transcriptional modifications

abstract:
Chemical modifications of RNA are associated with fundamental biological processes such as RNA splicing, export, translation, degradation, as well as human disease states such as cancer. However, the analysis of ribonucleoside modifications is impeded due to the hydrophilicity of the ribonucleoside molecules. In this research, we used solid-phase permethylation to derivatize the ribonucleosides, and the permethylated ribonucleosides, which were then quantitively analyzed using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method. The solid-phase permethylation efficiently derivatized the ribonucleosides, and more than 60 RNA modifications were simultaneously monitored using ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS) performed in the dynamic multiple reaction monitoring (dMRM) mode. Because of the increased hydrophobicity of permethylated ribonucleosides, this method enhanced retention, separation, and ionization efficiency, resulting in improved detection and quantification when compared to existing analytical strategies of RNA modifications. We applied this new approach to measure the extent of cytosine methylation and hydroxymethylation in RNA obtained from mouse embryonic stem cells with genetic deficiencies in ten-eleven translocation (TET) enzymes. The results matched previously performed analyses and highlighted the sensitivity, efficacy, and robustness of the new method. The advantage of this method enables comprehensive analysis of RNA modifications in biological samples.

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category: biochemistry


title: Marine Bacteria Chemotaxis to Crude Oil Components with Opposing Effects

abstract: Marine microorganisms were critical to hydrocarbon removal from the Gulf of Mexico oil spill. Chemotaxis, a process attracting motile bacteria toward higher hydrocarbon concentrations, can increase biodegradation efficiency. However, crude oil also contains heavy metal ions that repel bacteria. Will bacteria migrate toward hydrocarbons or away from heavy metals? We exposed a marine isolate Halomonas sp. to decane and copper ions in a microfluidic device that maintained a constant concentration gradient across a channel. Bacterial distributions were used to quantify parameters in a mathematical model capturing bacteria motility and chemotaxis. This multi-scale model was adapted from the signal transduction mechanism of E. coli. For Halomonas sp., we used independent receptors for sensing attractant or repellent and chemotaxis parameter values were assessed. Predictions based on the multi-scale model correctly estimated the net attraction or repulsion responses of bacteria to the stimuli mixture. In some cases, the model yielded a stronger repulsion than what was observed experimentally, but still captured the general trends of bacteria distribution. Understanding how marine bacteria integrate information from multiple inputs to yield net migration toward or away from oil will improve predictions of hydrocarbon degradation rates.

category: bioengineering


title: Grandmaternal allergen exposure causes distinct epigenetic trajectories in offspring associated with airway hyperreactivity and inflammation.

abstract:
Maternal asthma increases childhood asthma risk through multiple mechanisms including epigenetic regulation of asthma-associated genes. DNA methylation is one form of epigenetic regulation that is both inherited and modified by environmental exposures throughout life. In this study, we tested whether grandmaternal house dust mite (HDM) allergen exposure altered airway physiology and inflammation, as well as DNA methylation in both airway epithelium and airway sensory neurons of second-generation offspring. Grandmaternal allergen exposure induced a limited number of epigenetic changes in offspring at baseline that were not associated with increased airway reactivity or inflammation. In contrast, grandmaternal allergen exposure significantly altered offsprings response to HDM sensitization and challenge, inducing airway hyperreactivity to inhaled serotonin, increased airway inflammation, and potentiated DNA methylation. Gene sequences susceptible to methylation after allergen sensitization, and their corresponding biological processes and enriched pathways, were unique in offspring from HDM-exposed founders, indicating that grandmaternal allergen exposure established an epigenetic trajectory in offspring at birth that directed epigenetic and physiologic responses to subsequent allergen sensitization and challenge, contributing to inheritance of asthma risk.

SUMMARYGrandmaternal allergen exposure establishes an intergenerational, tissue-specific epigenetic trajectory in offspring at birth, which uniquely directs responses to allergen sensitization and challenge later in life and contributes to inheritance of asthma risk.

category: physiology


title: Biocompatible fluorocarbon liquid underlays for in situ extraction of isoprenoids from microbial cultures

abstract: Microbial production of heterologous metabolites is now a mature technology in many host organisms, opening new avenues for green production processes for specialty chemicals. At lab scale, petroleum-based hydrophobic bio-compatible solvents like dodecane can be used as a second phase on top of microbial cultures to act as a physical sink for heterologous hydrocarbon products like isoprenoids. However, this approach has significant drawbacks at scale due to the difficulty of handling solvents and their potential contamination with unwanted byproducts of their manufacture. We discovered that synthetic perfluorocarbon liquids (FCs), commonly used for heat transfer, can also act as physical sinks for microbially produced isoprenoid compounds. FCs are stable, inert, and are amenable to direct liquid-liquid extraction with alcohols for rapid product isolation. These liquids are more dense than water and form a lower phase to microbial cultures rather than an upper phase as with other solvents. Their ability to form an under-layer or  underlay also enables the cultivation of microbes directly at the FC-culture medium interface via gravity settling, which could open their application for filamentous or mat-forming organisms. We present comparisons of the isoprenoid extraction potential of three commercial FCs: FC-3283, FC-40, and FC-770 with engineered green microalga cultures producing patchoulol, taxadiene, casbene, or 13R(+) manoyl oxide. We demonstrate that FCs are promising alternatives to traditional solvents and open new avenues in bio-process design for microbial heterologous metabolite milking.

category: synthetic biology


title: B-cell epitope mapping of TprC and TprD variants of Treponema pallidum subspecies

abstract: Several recent studies have focused on the identification, functional analysis, and structural characterization of outer membrane proteins (OMPs) of Treponema pallidum (Tp). The Tp species encompasses the highly related pallidum, pertenue, and endemicum subspecies of this pathogen, known to be the causative agents of syphilis, yaws, and bejel, respectively. These studies highlighted the importance of identifying surface-exposed OMP regions and the identification of B-cell epitopes that could be protective and used in vaccine development efforts. We previously reported that the TprC and TprD OMPs of Tp are predicted to contain external loops scattered throughout the entire length of the proteins, several of which show a low degree of sequence variability among strains and subspecies. In this study, these models were corroborated using AlphaFold2, a state-of-the-art protein structure modeling software. Here, we identified B-cell epitopes across the full-length TprC and TprD variants using the Geysan pepscan mapping approach with antisera from rabbits infected with syphilis, yaws, and bejel strains and from animals immunized with refolded recombinant TprC proteins from three syphilis strains. Our results show that the humoral response is primarily directed to sequences predicted to be on surface-exposed loops of TprC and TprD proteins, and that the magnitude of the humoral response to individual epitopes differs among animals infected with various syphilis strains and Tp subspecies. Rather than exhibiting strain-specificity, antisera showed various degrees of cross-reactivity with variant sequences from other strains. The data support the further exploration of TprC and TprD as vaccine candidates.

category: microbiology


title: Functional and evolutionary significance of unknown genes from uncultivated taxa

abstract: Most microbes on our planet remain uncultured and poorly studied. Recent efforts to catalog their genetic diversity have revealed that a significant fraction of the observed microbial genes are functional and evolutionary untraceable, lacking homologs in reference databases. Despite their potential biological value, these apparently unrelated orphan genes from uncultivated taxa have been routinely discarded in metagenomics surveys. Here, we analyzed a global multi-habitat dataset covering 151,697 medium and high-quality metagenome assembled genomes (MAGs), 5,969 single-amplified genomes (SAGs), and 19,642 reference genomes, and identified 413,335 highly curated novel protein families under strong purifying selection out of previously considered orphan genes. These new protein families, representing a three-fold increase over the total number of prokaryotic orthologous groups described to date, spread out across the prokaryote phylogeny, can span multiple habitats, and are notably overrepresented in recently discovered taxa. By genomic context analysis, we pinpointed thousands of unknown protein families to phylogenetically conserved operons linked to energy production, xenobiotic metabolism and microbial resistance. Most remarkably, we found 980 previously neglected protein families that can accurately distinguish entire uncultivated phyla, classes, and orders, likely representing synapomorphic traits that fostered their divergence. The systematic curation and evolutionary analysis of the unique genetic repertoire of uncultivated taxa opens new avenues for understanding the biology and ecological roles of poorly explored lineages at a global scale.


title: Simulated docking reveals putative channels for the transport of long-chain fatty acids in Vibrio cholerae

abstract:
Fatty acids (FA) play an important role in biological functions, such as membrane homeostasis, metabolism, and as signaling molecules. FadL is the only known protein that uptakes long-chain fatty acids in Gram-negative bacteria, and this uptake has traditionally been thought to be limited to fatty acids up to 18 carbon atoms in length. Recently however, it was found Vibrio cholerae has the ability to uptake fatty acids greater than 18 carbon atoms and this uptake corresponds to higher bacterial survivability. Using E. colis FadL as a template, V. cholerae FadL homologs vc1042, vc1043, and vca0862 have been folded, simulated on an atomistic level using molecular dynamics, and analyzed revealing the FadL transport channels. For vc1042 and vc1043 these transport channels have more structural accommodations for the many rigid unsaturated bonds of long-chain polyunsaturated fatty acids, while vca0862 was found to lack transport channels within the signature beta barrel of FadL proteins.

IMPORTANCEFatty acids are important precursors for membrane phospholipids as well as energy sources for bacteria. V. choleraes uptake of long-chain fatty acids has not been studied to the atomistic level at this point, and by doing so, the resulting putative pathways show important structural details of the transport protein. Not only do the transport channels have unique differences between one another, the predicted transport mechanics of V. choleraes protein homologs differ substantially from the E. coli FadLs pathway found from X-ray crystallography studies.

category: molecular biology


title: Tracking human skill learning with a hierarchical Bayesian sequence model

abstract:
Humans can implicitly learn complex perceptuo-motor skills over the course of large numbers of trials. This likely depends on our becoming better able to take advantage of ever richer and temporally deeper predictive relationships in the environment. Here, we offer a novel characterization of this process, fitting a non-parametric, hierarchical Bayesian sequence model to the reaction times of human participants responses over ten sessions, each comprising thousands of trials, in a serial reaction time task involving higher-order dependencies. The model, adapted from the domain of language, forgetfully updates trial-by-trial, and seamlessly combines predictive information from shorter and longer windows onto past events, weighing the windows proportionally to their predictive power. As the model defines a prior over window depths, we were able to determine the extent to which the internal predictions of individual participant depended on how many previous elements.

Already in the first session, the model showed that participants had begun to rely on two previous elements (i.e., trigrams), thereby successfully adapting to the most prominent higher-order structure in the task. The extent to which local statistical fluctuations influenced participants responses waned over subsequent sessions, as subjects forgot the trigrams less and evidenced skilled performance. By the eighth session, a subset of participants shifted their prior further to consider a context deeper than two previous elements. Finally, participants showed resistance to interference and slow forgetting of the old sequence when it was changed in the final sessions. Model parameters for individual subjects covaried appropriately with independent measures of working memory. In sum, the model offers the first principled account of the adaptive complexity and nuanced dynamics of humans internal sequence representations during long-term implicit skill learning.

Author summaryA central function of the brain is to predict. One challenge of prediction is that both external events and our own actions can depend on a variably deep temporal context of previous events or actions. For instance, in a short motor routine, like opening a door, our actions only depend on a few previous ones (e.g., push the handle if the key was turned). In longer routines such as coffee making, our actions require a deeper context (e.g., place the moka pot on the hob if coffee is ground, the pot is filled and closed, and the hob is on). We adopted a model from the natural language processing literature that matches humans ability to learn variable-length relationships in sequences. This model explained the gradual emergence of more complex sequence knowledge and individual differences in an experiment where humans practiced a perceptual-motor sequence over 10 weekly sessions.


title: Multi-modal and multi-subject modular organization of human brain networks

abstract: The human brain is a complex network of anatomically interconnected brain areas. Spontaneous neural activity is constrained by this architecture, giving rise to patterns of statistical dependencies between the activity of remote neural elements. The non-trivial relationship between structural and functional connectivity poses many unsolved challenges about cognition, disease, development, learning and aging. While numerous studies have focused on statistical relationships between edge weights in anatomical and functional networks, less is known about dependencies between their modules and communities. In this work, we investigate and characterize the relationship between anatomical and functional modular organization of the human brain, developing a novel multi-layer framework that expands the classical concept of multi-layer modularity optimization. By simultaneously mapping anatomical and functional networks estimated from different subjects into communities, this approach allows us to carry out a multi-subject and multi-modal analysis of the brains modular organization. Here, we investigate the relationship between anatomical and functional modules during resting state, finding unique and shared structures. The proposed framework constitutes a methodological advance in the context of multi-layer network analysis and paves the way to further investigate the relationship between structural and functional network organization in clinical cohorts, during cognitively demanding tasks, and in developmental or lifespan studies.

category: neuroscience


title: AICAR-mediated selection of karyotypically normal cells in Pallister Killian syndrome patient-derived skin fibroblasts

abstract:
Pallister-Killian Syndrome (PKS) is a mosaic aneuploidy syndrome typically caused by the presence of a supernumerary marker isochromosome containing two extra copies of the short arm of chromosome 12 (iso-chromosome 12p or tetrasomy 12p). This isochromosome is always present in a mosaicism state that has tissue limited distribution. PKS is characterized by severe neurodevelopmental delay, intellectual disability, multisystem involvement and congenital malformations including typical dysmorphic features and skin pigmentation anomalies.

Aneuploid cells, irrespective of the identity of the supernumerary chromosome, including cancer cells, yeast cells and mouse embryonic fibroblasts (MEFs), have been demonstrated to present a disruption of protein homeostasis and increased basal stress levels; resulting in a greater sensitivity to chemical compounds inducing cellular energy stress compared to euploid cell lines. The burden of trisomy 21 has also been recently shown to impair the proteostasis network in lymphoblastoid cell lines and fibroblasts obtained from individuals with Down syndrome.

In this study, we demonstrate that AICAR, 5-Aminoimidazole-4-carboxamide ribonucleotide, a known energy stress inducing drug with antiproliferative effects on aneuploidy cancer cells and MEFs, is also able to selectively eliminate cells carrying the isochromosme12p in PKS clones in a time and dosage dependent manner. Collectively, our results indirectly provide evidence of increased basal energy and proteotoxic stress in PKS cells carrying isochromosome 12p, and suggest a potential therapeutic drug-based strategy that, selectively acting as a stressor for aneuploid cells, may establish the euploid state in PKS and a broader spectrum of human mosaic disorders.


title: AICAR-mediated selection of karyotypically normal cells in Pallister-Killian syndrome patient-derived skin fibroblasts


title: Robust and reproducible neuronal differentiation of human embryonic stem cells for neurotoxicology

abstract:
Neuronal differentiation from pluripotent cells is commonly used to recapitulate events in early brain development. Neuronal precursors and developing neurons cultured in vivo, rely on attachment, density and cell-to-cell communication, and cell-passaging steps should be minimised to avert disruption of network cross-connectivity. This is crucial both for viability and maturation, and the former also applies to human embryonic stem cells (hESCs). Neuronal differentiation protocols from hESCs for neurotoxicology assessment studies should therefore provide standardized cell density requirements, and optimised coating matrix conditions. The effect of drug treatments may be masked by compound instability and degradation in cell culture medium but performing daily media changes can circumvent these issues. Here, we describe a robust neuronal differentiation protocol using dual SMAD/WNT signalling inhibitors LDN193189, SB431542 and XAV939 (LSX) for neural induction of hESCs, followed by self-patterning and cell maturation stages, for the generation of ventral telencephalic progenitors and neurons. We provide critical information on the optimized cell culture parameters and standardized methods of stage-specific validation. Using cell counts, immunofluorescence, qRT-PCR, and a proof of principle treatment with valproic acid to showcase issues with drug-induced cell toxicity, we facilitate reproducibility of the protocol.

Graphical abstract

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title: Transcription factor determines the sources of extracellular vesicle DNA

abstract: Extracellular vesicle DNA (evDNA) acts as a potential mechanism for intercellular communication and possesses an important diagnostic value for multiple diseases. In this study, we have provided the first example that transcription factor determines the sources of evDNA from chromatin DNA. Transcription factor FOXM1 mediates the loading of specific chromatin DNA fragments into extracellular vesicles (EVs). Specifically, certain chromatin DNA fragments are specified by FOXM1 and form LC3-FOXM1-DNA vesicles in cytoplasm during autophagy. Then the cytoplasmic LC3-FOXM1-DNAs are transported to EVs probably through the process of an autophagy-MVB transport. We have confirmed that LC3-FOXM1-DNAs can be absorbed by recipient cells along with the released EVs and certain evDNA fragments integrate into the genome of recipient cells. This discovery provides an understanding for studying the sources and functions of evDNA and helps to expand its application in the future.


title: A Complete Set of Equations and Parameters for the Computational Model of Mitochondrial Function for the Proximal Convoluted Tubule and Medullary Thick Ascending Limb Cells in the Rat Kidney

abstract: To investigate mitochondrial function of renal epithelial cells under different tissue oxygenation levels, we have developed and applied computational models of mitochondrial function of the proximal convoluted tubule and medullary thick ascending limb cells. The models predict several key cellular quantities, including ATP generation, P/O (phosphate/oxygen) ratio, proton motive force, electrical potential gradient, oxygen consumption, the redox state of important electron carriers, and ATP consumption. The complete set of model equations and parameters are presented here, together with local and global sensitivity analysis results.


title: Upregulation of Trem2 expression occurs exclusively on microglial contact with plaques

abstract: Using spatial cell-type-enriched transcriptomics, we compare plaque-induced gene (PIG) expression in microglia touching plaques, neighboring plaques, and far from plaques in 18-month-old APPNLF/NLF knock-in mice with and without the Alzheimers disease risk mutation Trem2R47H/R47H. We report that, in AppNLF/NLF mice, expression of 35/55 PIGs, is exclusively upregulated in microglia that are touching plaques. In 7 PIGs including Trem2 this upregulation is prevented by the Trem2R47H/R47H mutation. Unlike in young mice, knockin of the Trem2R47H/R47H mutation does not significantly decrease the Trem2 expression but decreases protein levels by 20% in the absence of plaques. On plaques, despite the mutation preventing increased gene expression, TREM2 protein levels increased by 1.6-fold (compared to 3-fold with Trem2WT/WT) and microglial density increased 20-fold compared to 30-fold. Hence microglia must touch plaques before Trem2 gene expression is increased but small changes in protein expression can increase microglia density without a change in gene expression.


title: Local PI(4,5)P2 generation controls fusion pore expansion during exocytosis

abstract: Phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) is an important signaling phospholipid that is required for regulated exocytosis and some forms of endocytosis. The two processes share a topologically similar pore structure that connects the vesicle lumen with the outside. Widening of the fusion pore during exocytosis leads to cargo release, while its closure initiates kiss&run or cavicapture endocytosis. We show here, using live cell TIRF microscopy of insulin granule exocytosis, that transient accumulation of PI(4,5)P2 at the release site recruits components of the endocytic fission machinery, and stalls the late fusion pore expansion that is required for peptide release. The absence of clathrin differentiates this mechanism from clathrin-mediated endocytosis. The PI(4,5)P2 transients result from local phosphatidylinositol-phosphate-5-kinase-1c (PIP5K1c) activity, and knockdown of PIP5K1c, or optogenetic ablation of PI(4,5)P2 promotes fusion pore expansion. Thus, local phospholipid signaling controls fusion pore expansion peptide release through an unconventional endocytic mechanism.


title: Robust, versatile DNA FISH probes for chromosome-specific repeats in Caenorhabditis elegans and Pristionchus pacificus

abstract: Repetitive DNA sequences are useful targets for chromosomal fluorescence in situ hybridization (FISH). We analyzed recent genome assemblies of Caenorhabditis elegans and Pristionchus pacificus to identify tandem repeats with a unique genomic localization. Based on these findings, we designed and validated sets of oligonucleotide probes for each species targeting at least one locus per chromosome. These probes yielded reliable fluorescent signals in different tissues and can easily be combined with immunolocalization of cellular proteins. Synthesis and labeling of these probes are highly cost-effective and require no hands-on labor. The methods presented here can be easily applied in other model and non-model organisms with a sequenced genome.


title: A pipeline to track unlabeled cells in wide migration chambers using pseudofluorescence

abstract:
Cell migration is a pivotal biological process, whose dysregulation is found in many diseases including inflammation and cancer. Advances in microscopy technologies allow now to study cell migration in vitro, within microenvironments that resemble in vivo conditions. However, when cells are observed within large 3D migration chambers at low magnification and for extended periods of time, data analysis becomes difficult. Indeed, cell detection and tracking are hampered due to the large pixel size, the possible low signal-to-noise ratio and distortions in the cell shape due to changes in the z-axis position. Although fluorescent staining can be used to facilitate cell detection, it may alter cell behavior and suffer from fluorescence loss over time (photobleaching).

Here we describe the application of an image analysis pipeline based on deep learning to convert the transmitted light signal from unlabeled lymphoma cells to pseudofluorescence. Such pipeline confers a significant improvement in tracking accuracy while not suffering from photobleaching. This is reflected in the possibility of tracking cells for three-fold longer periods of time.


title: Brain-keratinocyte communication suffices for epidermal daily homeostasis

abstract: In mammals, an integrated network of molecular oscillators drives daily rhythms of tissue-specific homeostatic processes. This network is required for maintaining health and can be compromised by physiological aging, disease, and lifestyle choices. However, critical elements and properties of this network, such as key signaling nodes, the mechanisms of communication, and how physiological coherence is maintained, remain undefined. To dissect this system, we constructed a minimal clock network comprising only two nodes: the peripheral epidermal clock and the central brain clock. We observe that communication between the brain and epidermal clock is sufficient for wild-type core clock activity and specific homeostatic processes in the epidermis, yet full daily physiology requires input from other clock nodes. Moreover, our results show that the epidermal clock selectively suppresses or interprets systemic signals to ensure coherence of specific homeostatic processes, such as the cell cycle, identifying a previously-unrecognized gatekeeper role for this peripheral clock. This novel approach for dissecting a tissues daily physiology reveals the specific rhythmic processes controlled by secondary tissues and niche components, opening the possibility of identifying the sources of age-related circadian disruption and potentially developing therapeutic interventions.


title: Puromycin is incorporated into regenerating flagella of Chlamydomonas reinhardtii as an indicator of nascent flagellar proteins

abstract: The unicellular alga Chlamydomonas reinhardtii assembles flagella after pH shock-induced deflagellation, in which process the cell vigorously synthesizes flagellar proteins. Previously, newly synthesized flagellar proteins were chased using radioisotopes. Here, we have developed an alternative, non-radioactive method using the surface sensing of translation (SUnSET) assay, an assay that takes advantage of the incorporation of the antibiotic puromycin into newly synthesized peptides. Just after deflagellation, puromycin-labeled proteins increased dramatically in the cytoplasm and newly assembled flagella. Axonemal incorporation of newly synthesized proteins occurred from the distal tip of the flagellum. Cycloheximide, a protein synthesis inhibitor, almost completely prevented the cellular and flagellar incorporation of puromycin, although, as reported previously, it allowed reassembly of half-length flagella from the cytoplasmic "flagellar precursor" proteins. In contrast to wild-type cells, a cycloheximide-resistant mutant act2 produced nearly full-length flagella containing puromycin-labeled proteins even in the presence of cycloheximide. These and other results demonstrate that the SUnSET method serves as a powerful tool for visualizing flagellar incorporation of nascent proteins in Chlamydomonas.


title: Spectral clustering of single-cell multi-omics data on multilayer graphs

abstract:
Single-cell sequencing technologies that simultaneously generate multimodal cellular profiles present opportunities for improved understanding of cell heterogeneity in tissues. How the multimodal information can be integrated to obtain a common cell type identification, however, poses a computational challenge. Multilayer graphs provide a natural representation of multi-omic single-cell sequencing datasets, and finding cell clusters may be understood as a multilayer graph partition problem.

We introduce two spectral algorithms on multilayer graphs, spectral clustering on multilayer graphs (SCML) and the weighted locally linear (WLL) method, to cluster cells in multi-omic single-cell sequencing datasets. We connect these algorithms through a unifying mathematical framework that represents each layer using a Hamiltonian operator and a mixture of its eigenstates to integrate the multiple graph layers, demonstrating in the process that the WLL method is a rigorous multilayer spectral graph theoretic reformulation of the popular Seurat weighted nearest neighbor (WNN) algorithm. Implementing our algorithms and applying them to a CITE-seq dataset of cord blood mononuclear cells yields results similar to the Seurat WNN analysis. Our work thus extends spectral methods to multimodal single-cell data analysis.

The code used in this study can be found at https://github.com/jssong-lab/sc-spectrum

category: bioinformatics


title: A high-throughput approach to identify reproductive toxicants among environmental chemicals using an in vivo evaluation of gametogenesis in budding yeast Saccharomyces cerevisiae

abstract:
BackgroundEnvironmental chemical exposures are likely making important contributions to current levels of infertility and its increasing incidence. Yet the US produces high volumes of industrial chemicals for which there is limited data on their potential human reproduction toxicity. Current assays typically used in policy and regulatory settings involve costly and timeconsuming whole-animal rodent tests which limit the rapidity with which one can assess the thousands of chemicals yet to be tested.

ObjectiveOur aim was to develop a fast and reliable strategy to evaluate a large number of chemicals for reproductive toxicity by developing a high-throughput toxicity assessment using the yeast S. cerevisiae.

MethodsYeast are chronically exposed to each environmental chemical at two doses, 30 M and 100 M, in a 96-well plate-based format throughout gametogenesis. Non-gametes are removed and chemicals are washed away before gamete viability is measured using absorbance at 600 nm to produce growth curves. The difference in time at half-maximal saturation with and without exposure is used to determine the extent of reproductive toxicity.

ResultsWe validated our assay using bisphenol A (BPA), a well-established mammalian reproductive toxicant. We find that BPA in yeast has similar detrimental effects in meiosis as shown in worms and mammals. Competition assays with BPA analogs reveal that two of out of 19 BPA analogs examined (bisphenol E and 17{beta}-estradiol) show synergistic effects with BPA at doses tested and none show antagonistic effects. Out of 179 additional environmental chemicals, we designated 57 chemicals as reproductively toxic. Finally, by comparing chemicals in our cohort that have been evaluated for reproductive toxicity in mammalian studies, we find a statistically significant association between toxic chemicals in yeast and mammals.

ConclusionWe show that a high-throughput assay using yeast may be a useful approach for rapidly and reliably identifying chemicals that pose a reproductive risk.

category: pharmacology and toxicology


title: Shade-induced transcription of PIF-Direct-Target Genes precedes H3K4-trimethylation chromatin modifications

abstract: The phytochrome (phy)-PIF (Phytochrome Interacting Factor) sensory module perceives and transduces light signals to Direct-Target Genes (DTGs), which then drive the adaptational responses in plant growth and development, appropriate to the prevailing environment. These signals include the first exposure of etiolated seedlings to sunlight upon emergence from subterranean darkness, and the change in color of the light that is filtered through, or reflected from, neighboring vegetation ( shade). Previously, we identified three broad categories of rapidly signal-responsive genes: those repressed by light and conversely induced by shade; those repressed by light, but subsequently unresponsive to shade; and those responsive to shade only. Here, we investigate the potential role of epigenetic chromatin modifications in regulating these contrasting patterns of phy-PIF module-induced expression of DTGs. Using RNA-seq and ChlP-seq, time-resolved profiling of transcript and histone 3 lysine 4 trimethylation (H3K4me3) levels, respectively, we show that, whereas the initial dark-to-light transition triggers a rapid, apparently temporally-coincident decline of both parameters, the light-to-shade transition induces similarly rapid increases in transcript levels that precede increases in H3K4me3 levels. Together with other recent findings, these data raise the possibility that, rather than being causal in the shade-induced expression changes, H3K4me3 may function to buffer the rapidly fluctuating shade/light switching that is intrinsic to vegetational canopies under natural sunlight conditions.

category: plant biology


title: Shade-induced transcription of PIF-Direct-Target Genes precedes H3K4-trimethylation chromatin modification rises


title: High performance liquid chromatography-based metabolomics of Psidium guajava Linn. leaf extracts

abstract:
BackgroundOver the years, a number of methods have been introduced in the field of herbal medicine. Amongst these, metabolomics has rapidly emerged as a method of choice due to its wide applicability and versatility. High performance liquid chromatography (HPLC) is a preferred method for fingerprinting with advantages such as easy and wide availability, and relatively low maintenance cost. The current study used HPLC profiling and attempted to correlate it to anti-diarrhoeal activity of Psidium guajava (guava) extracts.

MethodsNinety samples of guava leaves were collected from three locations in Maharashtra, India over three seasons. Hydroalcoholic extracts (50:50, ethanol:water) were prepared and the HPLC chromatogram of all 90 extracts obtained. A total of eight bioassays representing the important features of diarrhoeal pathogenesis were performed and the extracts were differentiated as  good or  poor according to cut-offs for each assay. The numerical data of the bioassays and the HPLC chromatogram was correlated using suitable mathematical models comprising Principal component analysis (PCA), orthogonal partial least squares (OPLS, regression) and orthogonal partial least squares discriminant analysis (OPLS-DA).

ResultsOPLS-DA showed good seasonal and regional segregation of extracts. For most of the bioassays, PCA was unsuccessful in showing significant discrimination. Hence, OPLS plots were further developed; differentiation of good and poor extracts among the 90 extracts was successfully demonstrated for antibacterial activity against Shigella flexneri and Vibrio cholerae, inhibition of invasion of enteropathogenic Escherichia coli into HEp-2 epithelial cells, and cholera toxin (CT) production by V. cholerae. However, for other assays, namely, inhibition of adherence of E. coli, invasion of S. flexneri into HEp-2 cells, and inhibition of E. coli labile toxin (LT), subsets of 90 extracts were selected to demonstrate a significant correlation.

ConclusionHPLC-based metabolomics has the potential to differentiate good and poor activities of guava leaf extracts. This approach can be extended for identifying phytoconstituents responsible for the anti-diarrhoeal activity of guava leaf and help standardization of crude extracts.

category: systems biology


title: Emodin Combined with Multiple Low-frequency Low-intensity Ultrasound to Relieve Osteomyelitis Through Sonoantimicrobial Chemotherapy

abstract: Treatment of osteomyelitis is still challenging as conventional antibiotic therapy is limited by the emergence of resistant strains and the formation of biofilms. Sonoantimicrobial chemotherapy (SACT) is a novel therapy of low-frequency and low-intensity ultrasound (LFLIU) combined with sonosensitizer. Therefore, in our study, a sonosensitizer named emodin (EM) was proposed to be combined with LFLIU to relieve acute osteomyelitis caused by Methicillin-resistant Staphylococcus aureus (MRSA) through synergistic antibacterial and anti-biofilm effects. The efficiency of different intensities of ultrasound, single (S-LFLIU, 15 min) and multiple ultrasound (M-LFLIU, 5 min every 4 h, three times) against bacteria and biofilm was compared, contributing to develop the best treatment regimen. Our results demonstrated that EM plus S-LFLIU or M-LFLIU (EM+S-LFLIU or EM+M-LFLIU) have significant synergetic bactericidal and anti-biofilm effects and EM+M-LFLIU exhibits superior performance in anti-biofilm. Furthermore, it was suggested that EM+M-LFLIU could produce a large amount of reactive oxygen species (ROS), destroy the integrity of bacterial membrane and wall, down-regulate the expression of oxidative stress, membrane wall synthesis, bacterial virulence and other related genes (agrB, PBP3, sgtB, GMK, zwf, msrA). In vivo study, micro-CT, H&E staining, ELISA assay and bacterial quantification of bone tissue indicated that EM+M-LFLIU could also relieve osteomyelitis of MRSA infection. Our work proffers an original treating bacterial osteomyelitis approach that weakens drug-resistant bacterial and suppresses biofilm formation through SACT, which may provide new prospects for clinical treatment.


title: LCK-14-3-3ζ-TRPM8 axis for regulating TRPM8 function/assembly promotes pancreatic cancer malignancy

abstract: The transient receptor potential melastatin 8 (TRPM8), function as a Ca2+-permeable channel in the plasma membrane (PM). Dysfunction of TRPM8 is associated with human pancreatic cancer and several other diseases in clinical patients, but with unclear underlying mechanisms. Here, we found lymphocyte-specific protein tyrosine kinase (LCK) directly interacts with TRPM8 and potentiates TRPM8 phosphorylation at Y1022. LCK positively regulated channel function characterized by increased TRPM8 currents densities through enhancing TRPM8 multimerization. Furthermore, 14-3-3{zeta} interacted with TRPM8 and positively modulated channel multimerization. LCK significantly enhanced the binding of 14-3-3{zeta} and TRPM8, whereas mutant TRPM8-Y1022F impaired TRPM8 multimerization and the binding of TRPM8 and 14-3-3{zeta}. Knockdown of 14-3-3{zeta} impaired the regulation of LCK on TRPM8 multimerization. Additionally, TRPM8 phosphotyrosine at Y1022 feedback regulated LCK activity by inhibition of Tyr505 phosphorylation and modulation of LCK ubiquitination. Finally, we revealed the importance of TRPM8 phosphorylation at Y1022 in the proliferation, migration and tumorigenesis of pancreatic cancer cells. Our findings demonstrate that LCK-14-3-3{zeta}-TRPM8 axis for regulating TRPM8 assembly, channel function, LCK activity and providing potential therapeutic targets for pancreatic cancer.


title: A universal insect trait tool (ITT, v1.0) for statistical analysis and evaluation of biodiversity research data

abstract: We present a unique data set of trait information for 586 insect families in Central Europe, covering the largest known part of described species (over 34,000 species). Life history information and major functional traits were evaluated with fuzzy coding and weighted according to the number of known species in Germany. An overall analysis of the German insect fauna is given and the data set is exemplarily applied to metabarcoding results of malaise trap samples. Due to the high functional and taxonomic diversity in insects, further developments and refinements of traits to be included will be an ongoing process with advancements of upcoming database versions to be subsequently published.

category: ecology


title: An exactly valid and distribution-free statistical significance test for correlations between time series

abstract: Computing a correlation between a pair of time series is a routine task in disciplines from biology to climate science. How do we test whether such a correlation is statistically significant (i.e. unlikely under the null hypothesis that the time series are independent)? This problem is made especially challenging by two factors. First, time series typically exhibit autocorrelation, which renders standard statistical tests invalid. Second, researchers are increasingly turning to nonlinear correlation statistics with no known analytical null distribution, thus rendering parametric tests inviable. Several statistical tests attempt to address these two challenges, but none is perfect: A few are valid only under restrictive data conditions, others have differing degrees of correctness depending on user-supplied parameters, and some are simply inexact. Here we describe the truncated time-shift procedure, which can be used with any correlation statistic to test for dependence between time series. We show that this test is exactly valid as long as one time series is stationary, a minimally restrictive requirement among nonparametric tests. Using synthetic data, we demonstrate that our test performs correctly even while other tests suffer high false positive rates. We apply the test to data sets from climatology and microbiome science, verifying previously discovered dependence relationships.


title: Inhibitor of the nuclear transport protein XPO1 enhances the anticancer efficacy of KRAS G12C inhibitors in preclinical models of KRAS G12C mutant cancers

abstract:
The identification of molecules that can bind covalently to KRAS G12C and lock it in an inactive GDP-bound conformation has opened the door to targeting KRAS G12C selectively. These agents have shown promise in preclinical tumor models and clinical trials. FDA has recently granted approval to sotorasib for KRAS G12C mutated non-small cell lung cancer (NSCLC). However, patients receiving these agents as monotherapy may not respond and generally develop drug resistance over time. This necessitates the development of multi-targeted approaches that can potentially sensitize tumors to KRAS inhibitors. We generated KRAS G12C inhibitor-resistant cell lines and observed that they exhibit sensitivity toward selinexor, a selective inhibitor of nuclear export protein exportin1 (XPO1), as a single agent. KRAS G12C inhibitor MRTX1257 in combination with selinexor suppressed the proliferation of KRAS G12C mutant cancer cell lines MiaPaCa-2 and NCI-H2122 in a synergistic manner. Moreover, combined treatment of selinexor with KRAS G12C inhibitors resulted in enhanced spheroid disintegration, reduction in the number and size of colonies formed by G12C mutant cancer cells. A combination of selinexor with KRAS G12C inhibitors potentiated the inhibition of KRAS expression in MiaPaCa-2 cells. NF-kB protein expression was also markedly reduced by selinexor and MRTX1257 combination. In an in vivo KRAS G12C cell-derived xenograft model, oral administration of a combination of selinexor and sotorasib was demonstrated to reduce tumor burden and enhance survival. In conclusion, we have shown that the nuclear transport protein XPO1 inhibitor can enhance the anticancer activity of KRAS G12C inhibitors in preclinical cancer models.

SignificanceIn this study, combining nuclear transport inhibitor selinexor with KRAS G12C inhibitors has resulted in potent antitumor effects in preclinical cancer models. This can be an effective combination therapy for cancer patients that do not respond or develop resistance to KRAS G12C inhibitor treatment.

category: cancer biology


title: The ALT pathway generates telomere fusions that can be detected in the blood of cancer patients

abstract: Telomere fusions (TFs) can trigger the accumulation of diverse genomic rearrangements and the acquisition of oncogenic alterations leading to malignant transformation and resistance to chemotherapy. Despite their relevance in tumour evolution, our understanding of the patterns and consequences of TFs in human cancer remains limited. Here, we have characterized the rates and spectrum of somatic TFs across >30 cancer types using whole-genome sequencing data. TFs are pervasive in human tumours with rates varying markedly across and within cancer types. In addition to end-to-end fusions, we find novel patterns of TFs that we mechanistically link to the activity of the alternative lengthening of telomeres (ALT) pathway. We show that TFs can be detected in the blood of cancer patients, which enables cancer detection with high specificity and sensitivity even for early-stage tumours and cancer types for which early detection remains a high unmet clinical need, such as pancreatic cancer and brain tumours. Overall, we report a novel genomic footprint that enables characterization of the telomere maintenance mechanism of tumours and liquid biopsy analysis, which has implications for early detection, prognosis, and treatment selection.


title: The CIC-ERF co-deletion underlies fusion independent activation of ETS family member, ETV1, to drive prostate cancer progression

abstract: The dysregulation of ETS family transcription factors drives human prostate cancer. The majority of prostate cancer is the result of chromosomal rearrangements that lead to aberrant ETS gene expression. The mechanisms that lead to fusion independent ETS factor upregulation and prostate oncogenesis remain unknown. Here, we show that two neighboring transcription factors, Capicua (CIC) and ETS2 repressor factor (ERF), which are co-deleted in human prostate tumors can drive prostate oncogenesis. Concurrent CIC and ERF loss commonly occurs through focal genomic deletions at chromosome 19q13.2. Mechanistically, CIC and ERF co-bind the proximal regulatory element and mutually repress the ETS transcription factor, ETV1. Targeting ETV1 in CIC and ERF deficient prostate cancer limits tumor growth. Thus, we have uncovered a fusion independent mode of ETS transcriptional activation defined by concurrent loss of CIC and ERF.


title: Deep Learning Characterization of Brain Tumours With Diffusion Weighted Imaging

abstract: Glioblastoma multiforme (GBM) is one of the most deadly forms of cancer. Methods of characterizing these tumours are valuable for improving predictions of their progression and response to treatment. A mathematical model called the proliferation-invasion (PI) model has been used extensively in the literature to model these tumours, though it relies on known values of two key parameters: the tumour cell diffusivity and proliferation rate. Unfortunately, these parameters are difficult to estimate in a patient-specific manner, making personalized tumour projections challenging. In this paper, we develop and apply a deep learning model capable of making accurate estimates of these key GBM-characterizing parameters while simultaneously producing a full projection of the tumour progression curve. Our method uses two sets of multi sequence MRI imaging in order to make predictions and relies on a preprocessing pipeline which includes brain tumour segmentation and conversion to tumour cellularity. We apply our deep learning model to both synthetic tumours and a dataset consisting of five patients diagnosed with GBM. For all patients, we derive evidence-based estimates for each of the PI model parameters and predictions for the future progression of the tumour. Discussion and implications for future work and clinical relevance are included.


title: Macrophage-derived VEGF-C decreases hematogenous metastatic dissemination by normalizing the tumor vasculature

abstract: AO_SCPLOWBSTRACTC_SCPLOWExpression levels of the pro-lymphangiogenic vascular endothelial growth factor (VEGF)-C in the primary tumor correlates to the occurrence of proximal lymph node metastasis for most solid cancers including those of the breast. However, the role of VEGF-C in regulating tumor cell dissemination to distant organs such as the lungs, is currently unclear. By using an experimental tumor model of breast cancer (BC), we show that perivascular tumor-associated macrophages (TAMs), key regulators of hematogenous spreading, expressing VEGF-C, decrease tumor cell dissemination to the lungs. The mechanism whereby TAM-derived VEGF-C hampered pulmonary mechanism was ascribed to a phenotypic change of TAMs that augmented VEGFR3 expression on tumor vessels facilitating tumor vessel normalization and functionality. In contrast, tumor cell-derived VEGF-C increases vessel abnormalization and pulmonary metastases corresponding to lack of VEGFR3 expression on tumor vessels. Further evidence that TAM-derived VEGF-C may play a role in hematogenous spreading was obtained from human breast cancer specimens. In line with our experimental data, perivascular TAM-derived VEGF-C is inversely correlated with malignant grade and the occurrence of three-cell complexes composed of a perivascular macrophage, a tumor cell and an endothelial cell (collectively defined as tumor microenvironment of metastasis, or TMEM), which are sites of breast cancer intravasation and dissemination. Instead, cancer cell-derived VEGF-C correlated with malignant grade and TMEM complexes. Thus, our study reveals novel and apparent paradoxical roles of TAM-derived VEGF-C in regulating hematogenous tumor spreading in both experimental models and clinical samples.


title: Divergent Golgi Trafficking Limits B cell-Mediated IgG Sialylation

abstract: The degree of 2,6-linked sialylation on IgG glycans is associated with a variety of inflammatory conditions and is thought to drive IgG anti-inflammatory activity. Previous findings revealed that ablation of {beta}-galactoside 2,6-sialyltransferase 1 (ST6Gal1) in B cells failed to alter IgG sialylation in vivo, yet resulted in the loss of B cell surface 2,6 sialylation, suggesting divergent pathways for IgG and cell surface glycoprotein glycosylation and trafficking. Employing both B cell hybridomas and ex vivo murine B cells, we discovered that IgG was poorly sialylated by ST6Gal1 and highly core fucosylated by 1,6-fucosyltransferase 8 (Fut8) in cell culture. In contrast, IgG-producing cells showed the opposite pattern by flow cytometry, with high cell surface 2,6 sialylation and low 1,6 fucosylation. Paired studies further revealed that ex vivo B cell-produced IgG carried significantly less sialylation compared to IgG isolated from the plasma of matched animals, providing evidence that sialylation increases after release in vivo. Finally, confocal analyses demonstrated that IgG poorly localized to subcellular compartments rich in sialylation and ST6Gal1, and strongly to regions rich in fucosylation and Fut8. These findings support a model in which IgG subcellular trafficking diverges from the canonical secretory pathway by promoting Fut8-mediated core fucosylation and limiting exposure to and modification by ST6Gal1, providing a mechanism for why B cell-expressed ST6Gal1 is dispensable for IgG sialylation in vivo.

category: immunology


title: Role of RIGI, MDA5 and interferon alpha of duck in Duck Plague infection, a novel report

abstract: Duck Plague (DP) or Duck viral enteritis is an acute contagious and highly fatal disease in water fowl commonly caused by Anatidalphavirus-1 belonging from Herpesviridae family and contains double stranded DNA as genetic material. Pathogen associated molecular pattern (PAMP)s when identified by Pathogen Recognition Receptor (PRR)s acts as effective immunity system action against the pathogen. Melanoma Differentiation-Associated protein 5 (MDA5) and Retionic Acid Inducible Gene I (RIG1) are protein sensor commonly sense for viral double stranded RNA and helps for pro-inflammatory cytokine expression. Gut Associated Lymphoid Tissue (GALT)s have important role in immune response. The current study depicts the important role of three important immune response genes as RIGI, MDA5 and INFalpha in duck plague infestation for the first time. In silico studies followed by differential mRNA expression of RIG1, MDA5 and INFalpha was employed to detect effectiveness of gut associated immune responsiveness in liver, where kupfer cells are the major immune response cells. This was further confirmed through histological section of liver, kupfer cell and immunohistochemistry. This will be helpful to identify molecular mechanism of host innate immunity through duck plague virus infection in indigenous duck. This information may be helful for production of duck with the inherent resistance against duck plague virus infection through suitable biotechnological approaches as gene editing.Due to this inherent nature of better immunity in terms of resistance to other common avian diseases, duck will evolve as one of the major sustainable poultry species.The current study explores the scope to study host immunity against herpes virus in animal model.


title: Microdynamics of lamin A Ig-fold domain regulates higher order assembly of the protein

abstract: Lamins maintain the shape and rigidity of the nucleus in the form of a proteinaceous scaffold underneath the inner nuclear membrane (INM) and provide anchorage to chromatin and other nuclear proteins. Mutations in the human LMNA gene encoding lamin A/C cause about 16 different diseases with distinct phenotypes collectively termed as laminopathies which affect primarily the muscle tissues as well as adipose tissues, neuromuscular junctions and multiple other organs in progeroid syndromes. Lamins contain several domains of which Ig-fold is one of the well characterized and structured domains that harbours many mutations leading to deleterious interactions with other nuclear proteins. In this work, we have elucidated the effects of 3 such mutations namely R453W, W498C and W498R on the dynamics and flexibility of the Ig-fold domain and the consequent effect on the assembly into lamina by live cell imaging, fluorescence correlation spectroscopy (FCS) and molecular dynamics (MD) simulations. From our experimental observations, we concluded that R453W has somewhat increased flexibility compared to the other mutants but lower than that of the wild type. This might be attributed due to an alteration of the salt bridges in the Ig fold domain of the mutant. This is the first report where such an alteration in the full length has been documented by gross changes in diffusional properties as a sequel to a mutation in the Ig fold domain


title: In silico analysis of the human milk oligosaccharide glycome reveals key enzymes of their biosynthesis

abstract: Human milk oligosaccharides (HMOs) form the third most abundant component of human milk and are known to convey several benefits to the neonate, including protection from viral and bacterial pathogens, training of the immune system, and influencing the gut microbiome. As HMO production during lactation is driven by enzymes that are common to other glycosylation processes, we adapted a model of mucin-type GalNAc-linked glycosylation enzymes to act on free lactose. We identified a subset of 11 enzyme activities that can account for 206 of 226 distinct HMOs isolated from human milk, and constructed a biosynthetic reaction network that identifies 5 new core HMO structures. A comparison of monosaccharide compositions demonstrated that the model was able to discriminate between two possible groups of intermediates between major subnetworks, and to assign possible structures to several previously uncharacterised HMOs. The effect of enzyme knockouts is presented, identifying {beta}-1,4-galactosyltransferase and {beta}-1,3-N-acetylglucosaminyltransferase as key enzyme activities involved in the generation of the observed HMO glycosylation patterns. The model also provides a synthesis chassis for the most common HMOs found in lactating mothers.


title: Innate immune cell response to host-parasite interaction in a human intestinal tissue microphysiological system

abstract:
Protozoan parasites that infect humans are widespread, and lead to varied clinical manifestations, including life-threatening illnesses in immunocompromised individuals. Animal models have provided insight into innate immunity against parasitic infections, however, species-specific differences and complexity of innate immune responses make translation to humans challenging. Thus, there is a need for novel in vitro systems that can elucidate mechanisms of immune control and parasite dissemination. We have developed a human microphysiological system of intestinal tissue to evaluate parasite-immune-specific interactions during infection, which integrates primary intestinal epithelial cells and immune cells to investigate the role of innate immune cells during epithelial infection by the protozoan parasite, Toxoplasma gondii, which affects billions of people worldwide. Our data indicate that epithelial-infection by parasites stimulates a broad range of effector functions in neutrophils and NK cell-mediated cytokine production that play immunomodulatory roles, demonstrating the potential of our system for advancing the study of human-parasite interactions.

TeaserNovel engineered model of human intestinal tissue for study of dissemination and immune control of parasitic infections.


title: ARZIMM: A Novel Analytic Platform for the Inference of Microbial Interactions and Community Stability from Longitudinal Microbiome Study

abstract: Dynamic changes of microbiome communities may play important roles in human health and diseases. The recent rise in longitudinal microbiome studies calls for statistical methods that can model the temporal dynamic patterns and simultaneously quantify the microbial interactions and community stability. Here, we propose a novel autoregressive zero-inflated mixed-effects model (ARZIMM) to capture the sparse microbial interactions and estimate the community stability. ARZIMM employs a zero-inflated Poisson autoregressive model to model the excessive zero abundances and the non-zero abundances separately, a random effect to investigate the underlining dynamic pattern shared within the group, and a Lasso-type penalty to capture and estimate the sparse microbial interactions. Based on the estimated microbial interaction matrix, we further derive the estimate of community stability, and identify the core dynamic patterns through network inference. Through extensive simulation studies and real data analyses we evaluated ARZIMM in comparison with the other methods.


title: NGSEP 4: Efficient and Accurate Identification of Orthogroups and Whole Genome Alignment

abstract: Whole-genome alignment allows researchers to understand the genomic structure and variations among the genomes. Approaches based on direct pairwise comparisons of DNA sequences require large computational capacities. As a consequence, pipelines combining tools for orthologous gene identification and synteny have been developed. In this manuscript, we present the latest functionalities implemented in NGSEP 4, to identify orthogroups and perform whole genome alignments. NGSEP implements functionalities for identification of clusters of homologus genes, synteny analysis and whole genome alignment, and visualization. Our results showed that the NGSEP algorithm for ortholog identification has competitive accuracy and better efficiency in comparison to commonly used tools. The implementation also includes a visualization of the whole genome alignment based on synteny of the orthogroups that were identified, and a reconstruction of the pangenome based on frequencies of the orthogroups among the genomes. Finally, our software includes a new graphical user interface. We expect that these new developments will be very useful for several studies in evolutionary biology and population genomics.


title: Dynamics of Human Serum Transferrin in Varying Physicochemical Conditions Explored by Using Molecular Dynamics Simulations

abstract: Conformational stability of human serum transferrin (Tf) at varying pH, salt, and excipient concentrations were investigated using molecular dynamics (MD) simulations and the results are compared with previously published small angle X-ray scattering (SAXS) experiments. SAXS study showed that at pH 5, Tf is predominantly present in partially open (PO) form, and the factions of PO differ based on the physicochemical condition and drifts towards closed form (HO) as the pH increases. Tf is a bilobal glycoprotein that is composed of homologous halves termed as N-lobe and C-lobe. The current study shows that the protonation of Y188 and K206 at pH 5 is the primary conformational drive into PO, which shifts towards the closed (HO) conformer as the pH increases. Furthermore, at pH 6.5, PO is unfavorable due to negative charge-charge repulsion at the N/C-lobe interface linker region causing increased hinge distance when compared to HO, which has favorable attractive electrostatics. Subsequently, the effect of salt concentration at 70 and 140 mM NaCl was studied. At 70 mM NaCl and pH 5, chloride ions bind strongly in the N-lobe iron-binding site, whereas these interactions are weak at pH 6.5. With increasing salt concentration at pH 5, regions surrounding the N-lobe iron-binding site are saturated and as a consequence sodium and chloride ions accumulate into the bulk. Additionally, protein-excipient interactions were investigated. At pH 5, excipients interact in similar loop regions, E89-T93, D416-D420, located in the C-lobe and N-lobe of the HO conformer, respectively. It is anticipated that interactions of additives in these two loop regions cause conformational changes that lead to iron coordinating residues in the N-lobe to drift away from iron and thus drive HO to PO conversion. Furthermore, at pH 6.5 and 140 mM histidine or phosphate, these interactions are negligible leading to the stabilization of HO.

category: biophysics


title: Parameterization and Application of the General Amber Force Field to Model Fluro Substituted Furanose Moieties and Nucleosides

abstract: Molecular mechanics force field calculations have historically shown significant limitations in modeling the energetic and conformational interconversions of highly substituted furanose rings. This is primarily due to the gauche effect that is not easily captured using pairwise energy potentials. In this study, we present a refinement to the set of torsional parameters in the General Amber Force Field (gaff) used to calculate the potential energy of mono, di-, and gem-fluorinated nucleosides. The parameters were optimized to reproduce the pseudorotation phase angle and relative energies of a diverse set of mono- and difluoro substituted furanose ring systems using quantum mechanics umbrella sampling techniques available in the IpolQ engine in the Amber suite of programs. The parameters were developed to be internally consistent with the gaff force field and the TIP3P water model. The new set of angle and dihedral parameters and partial charges were validated by comparing the calculated phase angle probability to those obtained from experimental nuclear magnetic resonance experiments.


title: Assessment of AlphaFold2 residue conformations for human proteins

abstract:
MotivationAs only 35% of human proteins feature (often partial) PDB structures, the protein structure prediction tool AlphaFold2 (AF2) could have massive impact on human biology and medicine fields, making independent benchmarks of interest. We studied AF2s ability to describe the backbone solvent exposure as an easily interpretable "natural coordinate" of protein conformation, using human proteins as test case.

ResultsAfter screening for appropriate comparative sets, we matched 1818 human proteins predicted by AF2 against 7585 unique experimental PDBs, and after curation for sequence overlap, we assessed 1264 comparative pairs comprising 115 unique AF2-structures and 652 unique experimental structures. AF2 performed markedly worse for multimers, whereas ligands, cofactors and experimental resolution were interestingly not very important for performance. AF2 performed excellently for monomer proteins. Challenges relating to specific groups of residues and multimers were analyzed. We identify larger errors for lower-confidence scores (pLDDT) and exposed residues, and polar residues (Asp, Glu, Asn e.g.) being less accurately described than hydrophobic residues. Proline conformations were the hardest to predict, probably due to common location in dynamic solvent-accessible parts. In summary, using solvent exposure as a natural metric of local conformation, we quantify the performance of AF2 for human proteins and provide estimates of the expected error as a function of ligand presence, multimer/monomer status, resolution, local residue solvent exposure, pLDDT, and amino acid type. Overall performance was found to be excellent.

Availability and ImplementationScripts used to perform benchmarking are available at https://github.com/ktbaek/AlphaFold.


title: Three-dimensional actuation of hummingbird wings requires diverse effects from the primary flight muscles

abstract: Hummingbirds have evolved to hover and maneuver with exceptional flight control. This is directly enabled by their musculoskeletal system that successfully exploits the agile motion of flapping wings. Here, we reveal novel principles of hummingbird wing actuation that provide insights into the evolution and robotic emulation of hummingbird flight. We develop a functional model of hummingbird musculoskeletal system, which predicts instantaneous, three-dimensional torque produced by primary (pectoralis and supracoracoideus) and combined secondary muscles. It also reveals primary muscle contractile behavior, including stress, strain, elasticity, and work. Results show that the primary muscles (i.e., the flight "engine") function as diverse effectors, as they do not simply power the stroke, but also actively deviate and pitch the wing. The secondary muscles produce controlled-tightening effects, by acting against primary muscles in deviation and pitching. The diverse effector capacity of pectoralis is associated with the evolution of a comparatively enormous bicipital crest on humerus.


title: STK25 inhibits PKA signaling by phosphorylating PRKAR1A

abstract: In the heart, Protein Kinase A (PKA) is critical for activating calcium handling and sarcomeric proteins in response to beta adrenergic stimulation leading to increased myocardial contractility and performance. The catalytic activity of PKA is tightly regulated by regulatory subunits which inhibit the catalytic subunit until released by cAMP binding. Phosphorylation of Type II regulatory subunits promotes PKA activation, however the role of phosphorylation in Type I regulatory subunits remain uncertain. Here we utilized human induced pluripotent stem cell cardiomyocytes (iPSC-CM) to identify STK25 as a kinase of the Type I regulatory subunit PRKAR1A. Phosphorylation PRKAR1A led to inhibition of PKA kinase activity and increased binding to the catalytic subunit in the presence of cAMP. Stk25 knockout in mice diminished Prkar1a phosphorylation, increased Pka activity and augmented contractile response to beta adrenergic stimulation. Together, these data support STK25 as a negative regulator of PKA signaling through phosphorylation of PRKAR1A.


title: FRIENDLY (FMT) is an RNA binding protein associated with cytosolic ribosomes at the mitochondrial surface

abstract:
The spatial organization of protein synthesis in the eukaryotic cell is essential for maintaining the integrity of the proteome and the functioning of the cell. Translation on free polysomes or on ribosomes associated with the endoplasmic reticulum has been studied for a long time. More recent data have revealed selective translation of mRNAs in other compartments, in particular at the surface of mitochondria. Although these processes have been described in many organisms, in particular in plants, the mRNA targeting and localized translation mechanisms remain poorly understood.

Here, the Arabidopsis thaliana Friendly (FMT) protein is shown to be a cytosolic RNA binding protein that associates with cytosolic ribosomes at the surface of mitochondria. Its knockdown delays seedling development and causes mitochondrial clustering. The mutation also disrupts the mitochondrial proteome and the localization of nuclear transcripts on the surface of mitochondria. These data indicate that FMT participates in the localization of mRNAs and their translation at the surface of mitochondria.


title: The T. ispahanicum elongated glume locus P2 maps to chromosome 6A and is associated with the ectopic expression of SVP-A1

abstract:
In rice and wheat, glume and floral organ length are positively correlated with grain size, making them an important target to increase grain size and potentially yield. The wheat subspecies Triticum ispahanicum is known to develop elongated glumes and floral organs as well as long grains. These multiple phenotypic effects are controlled by the P2 locus, which was previously mapped to wheat chromosome 7B. Using three mapping populations, we show that the long glume locus P2 does not map to chromosome 7B, but instead maps to a 1.68 Mbp interval on chromosome 6A. Within this interval, we identified SVP-A1, a MADS box transcription factor which is the direct ortholog of the maize gene underlying the  pod corn Tunicate locus and is a paralog to the T. polonicum elongated glume P1 gene. In T. ispahanicum, we identified a private allele which has a 482-bp deletion in the SVP-A1 promoter and is associated with ectopic and higher expression of SVP-A1 in the elongated glumes and floral organs. We used near-isogenic lines (NILs) to show that P2 has a consistent positive effect on the length of glume, lemma, palea, spike and grain. Based on the mapping data, natural variation, biological function of SVP genes in cereals and expression analyses, we propose the MADS-box transcription factor SVP-A1 as a promising candidate for P2.

Key messageWe propose the MADS-box transcription factor SVP-A1 as a promising candidate gene for the elongated glume locus P2, which maps to chromosome 6A instead of the previously proposed chromosome 7B.


title: A peptide-mediated, multilateral molecular dialogue for the coordination of pollen wall formation

abstract:
The surface of pollen grains is reinforced by pollen wall components produced non-cell autonomously by tapetum cells that surround developing pollen within the male floral organ, the anther. Here we show that tapetum activity is regulated by the GASSHO (GSO) receptor-like kinase pathway, controlled by two sulfated peptides, CASPARIAN STRIP INTEGRITY FACTOR 3 (CIF3) and CIF4, the precursors of which are expressed in the tapetum itself. Coordination of tapetum activity with pollen grain development depends on the action of subtilases, including AtSBT5.4, which are produced stage-specifically by developing pollen grains. Tapetum-derived CIF precursors are processed by subtilases, triggering GSO-dependent tapetum activation. We show that the GSO receptors act from the middle layer, a tissue surrounding the tapetum and developing pollen. Three concentrically organized cell types therefore cooperate to coordinate pollen wall deposition through a multilateral molecular dialog.

Significance StatementPollen viability depends on a tough external barrier called the pollen wall. Pollen wall components are produced by tapetum cells which surround developing pollen grains within the anther. Precise coordination of tapetum activity with pollen grain development is required to ensure effective pollen wall formation. Here we reveal that this is achieved through a multidirectional dialogue involving three distinct cell types. We show that peptide precursors from the tapetum are activated by proteases produced stage-specifically in developing pollen grains. Unexpectedly, we found that activated peptides are perceived not in the tapetum, but in the middle layer, which encloses the developing tapetum and pollen grains, revealing an unsuspected role for this enigmatic cell layer in the control of tapetum development.


title: A catalogue of resistance gene homologs and a chromosome-scale reference sequence support resistance gene mapping in winter wheat

abstract: A resistance gene atlas is an integral component of the breeders arsenal in the fight against evolving pathogens. Thanks to high-throughput sequencing, catalogues of resistance genes can be assembled even in crop species with large and polyploid genomes. Here, we report on capture sequencing and assembly of resistance gene homologs in a diversity panel of 907 winter wheat genotypes comprising ex situ genebank accessions and current elite cultivars. In addition, we use accurate long-read sequencing and chromosome conformation capture sequencing to construct a chromosome-scale genome sequence assembly of cv. Attraktion, an elite variety representative of European winter wheat. We illustrate the value of our resource for breeders and geneticists by (i) comparing the resistance gene complements in plant genetic resources and elite varieties and (ii) conducting genome-wide associations scans (GWAS) for the fungal diseases yellow rust and leaf rust using reference-based and reference-free GWAS approaches. The gene content under GWAS peaks was scrutinized in the assembly of cv. Attraktion.


title: Shared and distinct functional effects of patient-specific Tbr1 mutations on cortical development

abstract: Predicted loss-of-function and missense heterozygous de novo mutations of TBR1 are strongly associated with intellectual disability and autism. The functional effects of these heterogeneous mutations on cortical development and genotype-phenotype relationships have yet to be explored. We characterized mouse models carrying patient mutations A136PfsX80 and K228E, finding convergent and discordant phenotypes. The A136PfsX80 mutation is loss-of-function and allelic to the Tbr1 knockout. In contrast, K228E causes significant upregulation of TBR1. Heterozygosity of either mutation produces axon defects, including reduction of the anterior commissure, and CTIP2 downregulation in adult cortex. While mice lacking TBR1 show extensive cortical apoptosis and inverted layering, K228E homozygotes show normal apoptosis levels and a complex layering phenotype--suggesting partial, yet abnormal, function of the allele. The construct and face validity of these Tbr1 patient mutation mice suggests they will be valuable translational models for studying the function of this essential brain transcription factor.


title: Inflammasome mediated neuronal-microglial crosstalk: a therapeutic substrate in C9orf72-FTD/ALS.

abstract: Introductory ParagraphIntronic G4C2 hexanucleotide repeat expansions of C9orf72 are the most common cause of familial variants of frontotemporal dementia / amyotrophic lateral sclerosis (FTD/ALS)1,2. G4C2hexanucleotide repeat expansions (HREs) in C9orf72 undergo non-canonical repeat associated translation, producing dipeptide repeat (DPR) proteins, with various deleterious impacts on cellular homeostasis3. While five different DPRs are produced, poly(glycine-arginine) (GR) is amongst the most toxic, and is the only DPR to accumulate in the associated clinically relevant anatomical locations of the brain4,5. Previous work has demonstrated the profound effects of a poly(GR) model of c9FTD/ALS, including motor impairment, memory deficits, neurodegeneration, and neuroinflammation6. Neuroinflammation is hypothesized to be a driving factor in the disease course; microglia activation is present prior to symptom onset and persists throughout the disease7. Here, we establish the contributions of the NLRP3 inflammasome in the pathogenesis of FTD/ALS, resulting from a stress-induced neuronal-microglial crosstalk feedforward loop. In a mouse model of c9FTD/ALS, inflammasome-mediated inflammation was increased with microglial activation, cleavage of caspase-1, upregulation of Cxcl10, and production of IL-1{beta}. We find that genetic ablation of Nlrp3 protected behavioral deficits and prevented neurodegeneration as seen in C57BL6J Wild Type mouse model of c9FTD/ALS. Ultimately, survival was improved by the genetic ablation of Nlrp3. Moreover, we identified the process by which neuronal stress signals induced by hexanucleotide expansions initiate an inflammatory cascade in microglia. These findings provide evidence of the integral role of inflammasome-mediated innate immunity in c9FTD/ALS pathogenesis, and suggest the NLRP3 inflammasome as a therapeutic target.


title: A requirement for correctly scaled synapses in the regulation of motor circuit output

abstract: Neural networks tune synaptic and cellular properties to produce stable activity. One form of homeostatic regulation involves scaling the strength of synapses up or down in a global and multiplicative manner to oppose activity disturbances. In American bullfrogs, excitatory synapses scale up to regulate breathing motor function after inactivity in hibernation, connecting homeostatic compensation to motor behavior. In traditional models of homeostatic synaptic plasticity, inactivity is thought to increase synaptic strength via mechanisms that involve reduced Ca2+ influx through voltage-gated channels. Therefore, we tested whether pharmacological inactivity and inhibition of voltage-gated Ca2+ channels are sufficient to drive synaptic compensation in this system. For this, we chronically exposed in vitro brainstem preparations containing the intact respiratory network to tetrodotoxin (TTX) to stop activity and nimodipine to block L-type Ca2+ channels. We show that hibernation and TTX similarly increased motoneuron synaptic strength and that hibernation occluded the response to TTX. In contrast, block of L-type Ca2+ channels did not alter mean synaptic strength but disrupted the multiplicative scaling of synaptic compensation typically observed in response to hibernation. Thus, inactivity associated with hibernation drives up synaptic strength through mechanisms that do not rely on reduced L-type channel function, while Ca2+ signaling independently regulates the balance of synaptic weights. Altogether, these results point to multiple feedback signals for shaping synaptic compensation that gives rise to proper network function during environmental challenges in vivo.


title: Inactivity and Ca2+ signaling regulate synaptic compensation in motoneurons following hibernation in American bullfrogs


title: Sensing Echoes: Temporal misalignment as the Earliest Marker of Neurodevelopmental Derail

abstract: Sensory transduction and transmission delays operate and propagate along different time scales. From microseconds in the auditory domain, to hundreds of milliseconds in the visual, and kinesthetic domains, the brain must successfully align disparate delays arising from endogenously self-generated streams of motor and visceral sensorial information, with exogenous sensory inputs. To produce a cohesive response to environmental goals, constantly explore, adapt, and develop a sense of simultaneity, the brain must resolve this major feat and compensate for excessive delays in any sensory modality. Disruption in these processes may lead to altered perception of the self and others, and inadvertently affect social interactions. But how early such issues may emerge and be reliably detectable, remains a challenge. Here we assess in neonates, the transmission latencies of a sound wave that travels from the cochlear nerve to the brainstem on its way to the primary auditory cortex. Already at birth, we find systematic and cumulative delays in the propagation of this wave in neonates that later received a diagnosis of autism. Furthermore, we discover that the distributions of such temporal delays have far narrower bandwidth than those from neonates who did not receive the autism diagnosis. We identify associated codependent genes networks and define a reliable marker of neurodevelopment derail, detectable at birth. Under the precision autism model, we propose that the brainstem contains an endogenous clock anchoring and aligning disparate timescales critical for the emergence and maintenance of congruent percepts of the self and others.


title: Electrophysiological identification of daily rhythms in the prefrontal cortex

abstract: Circadian rhythms are ubiquitous in biology, from the molecular to behavioral levels. There is growing interest in understanding the functional implications of circadian oscillations in different cells and systems, including the brain. The prefrontal cortex (PFC) is heavily involved in myriad processes, including working memory, cognition, stress responses, and fear associated behaviors. Many PFC associated behaviors are time-of-day dependent, yet how time-of-day impacts the basic function of neurons in the PFC is not known. Here we use patch-clamp electrophysiology to record from layer 2/3 pyramidal neurons in the prelimbic (pl) PFC of male and female C57BL/6J mice at 4 separate bins of zeitgeber time (ZT): 0-4, 6-10, 12-16, and 18-22. We measured changes in membrane properties, inhibitory and excitatory inputs, ion channel function, and action potential kinetics. We demonstrate that the activity of plPFC neurons, their inhibitory inputs, and action potential dynamics are regulated by time-of-day. Further, we show that in males postsynaptic K+ channels play a central role in mediating these rhythms, suggesting the potential for an intrinsic gating mechanism mediating information throughput. These key discoveries in PFC physiology demonstrate the importance of understanding how daily rhythms contribute to the mechanisms underlying the basic functions of PFC circuitry.


title: Evolutionary convergence of a neural mechanism in the cavefish lateral line system

abstract: Animals can evolve dramatic sensory functions in response to environmental constraints, but little is known about the neural mechanisms underlying these changes. The Mexican tetra, Astyanax mexicanus, is a leading model to study genetic, behavioral, and physiological evolution by comparing eyed surface populations and blind cave populations. We compared neurophysiological responses of posterior lateral line afferent neurons and motor neurons across A. mexicanus populations to reveal how shifts in sensory function may shape behavioral diversity. These studies indicate differences in intrinsic afferent signaling and gain control across populations. Elevated endogenous afferent activity identified a lower response threshold in the lateral line of blind cavefish relative to surface fish. We next measured the effect of inhibitory corollary discharges from hindbrain efferent neurons onto afferents during locomotion. We discovered that three independently-derived cavefish populations have evolved persistent afferent activity during locomotion, suggesting for the first time that regression of the efferent system can be an evolutionary mechanism for neural adaptation of a vertebrate sensory system.


title: Retinal waves align the concentric orientation map in mouse superior colliculus to the center of vision

abstract: Neurons in the mouse superior colliculus (SC) are arranged in an orientation preference map that has a concentric organization, which is aligned to the center of vision and the optic flow experienced by the mouse. The developmental mechanisms that underlie this functional map remain unclear. Here, we propose that the spatiotemporal properties of spontaneous retinal waves during development provide a scaffold to establish the concentric orientation map in the mouse SC and its alignment to the optic flow. We test this hypothesis by modelling the orientation-tuned SC neurons that receive ON/OFF retinal inputs. Our results suggest that the stage III retinal wave properties, namely OFF delayed response and the wave propagation direction bias, are key factors that regulate the spatial organization of the SC orientation map. Specifically, the OFF delay mediates the establishment of orientation-tuned SC neurons by segregating their ON/OFF receptive subfields, the wave-like activities facilitate the formation of a concentric pattern, and the wave direction biases align the orientation map to the center of vision. Taken together, our model suggests that retinal waves may play an instructive role in establishing functional properties of SC neurons and provides a promising mechanism for explaining the correlations between the optic flow and the SC orientation map.


title: Decidual innate immune cell kinetics following lipopolysaccharide challenge

abstract:
In early pregnancy, macrophages (M{varphi}) and natural killer cells (NK) infiltrate and expand within the decidua to comprise 30% of all cellular content. These immune cell populations coordinate angiogenic and tissue remodeling processes that are needed for a healthy pregnancy. Importantly, decidual tissue-resident macrophages (trM{varphi}) and uterine NK retain immunosurveillance properties that facilitate the targeting of infections (e.g., viral, bacterial). The timing and severity of these infections, as well as the resulting immune response, can dictate pregnancy outcome. However, little is known about the kinetics and activities of uterine myeloid and NK populations following infections. To address this knowledge gap, we defined the stepwise changes of uterine myeloid and NK subpopulations following lipopolysaccharide (LPS) challenge in a mouse model of early pregnancy. Low (25 {micro}g/kg), moderate (50 {micro}g/kg), and high (200 {micro}g/kg) doses of LPS resulted in dose-dependent increases in peripheral and uterine inflammation, as well as a dose-dependent increase in the rate of fetal resorption. Compared with saline controls, mice exposed to LPS showed higher frequencies of immature monocytes, decreased TNF-producing monocytes and M{varphi}, and increased conventional (c)NK expression of granzyme B in the uterus. These changes were followed by alterations in overall uterine (u)NK frequencies with increased cNK and decreased tissue resident (tr)NK. Together, this work describes how discrete levels of LPS-induced inflammation shape the innate immune cell landscape of the decidua. These findings establish insight into the stepwise immunological changes following endotoxin challenge and provide a better understanding of how inflammation controls the activity of key decidual leukocytes.

Summary sentenceGraded LPS challenge in early pregnancy leads to a stepwise increase in fetal resorption and associates with distinct alterations in frequencies and activities of uterine immune cells.