title: Depletion of ALMS1 inhibits TGF-β/SMAD3/SNAI1 axis altering normal cell migration capacity and epithelial-mesenchyme transition.

abstract:
BackgroundALMS1 is a ubiquitous gene associated with Alstrom syndrome (ALMS). The main symptoms of ALMS affect multiple organs and tissues, generating at last, multi-organic fibrosis in the lungs, kidneys and liver. TGF-{beta} is one of the main pathways implicated in fibrosis, controlling the cell cycle, apoptosis, cell migration, and epithelial-mesenchymal transition (EMT). Nevertheless, the role of ALMS1 gene in fibrosis generation and other implicated processes such as cell migration or epithelial-mesenchymal transition via the TGF-{beta} pathway has not been elucidated yet.

MethodsInitially, we evaluated how depletion of ALMS1 affects different processes like apoptosis, cell cycle and mitochondrial activity. Then, we performed proteomic profiling with TGF-{beta} stimuli in HeLa ALMS1 -/- cells and validated the results by examining different EMT biomarkers using qPCR. The expression of these EMT biomarkers was validated in hTERT-BJ-5ta. Finally, we also evaluated the SMAD3 phosphorylation in BJ-5ta model and its cell migration capacity.

ResultsDepletion of ALMS1 generated apoptosis resistance to thapsigargin (THAP) and C2-Ceramide (C2-C), and G2/M cell cycle arrest in HeLa cells. For mitochondrial activity, results did not show significant differences between ALMS1 +/+ and ALMS1 -/-. Proteomic results showed inhibition of downstream pathways regulated by TGF-{beta}. The protein-coding genes (PCG) were associated with processes like focal adhesion or cell-substrate adherens junction. EMT biomarkers VIM, DSP, and SNAI1 showed an opposite pattern to what would be expected when activating the EMT. Finally, inhibition of SNAI1 was consistent in BJ-5ta model, where a reduced activation of SMAD3 and a decrease in migratory capacity were also observed.

ConclusionALMS1 has a role in controlling the cell cycle and the apoptosis processes. Moreover, the depletion of ALMS1 inhibits the signal transduction through the TGF-{beta}/SMAD3/SNAI1, which could be affecting the cell migration capacity and EMT.


title: Percolation transition determines protein size limit for passive transport through the nuclear pore complex

abstract: Nuclear pore complexes (NPCs) control biomolecular transport in and out of the nucleus. Disordered nucleoporins in the complex"s central pore form a permeation barrier, preventing unassisted transport of large biomolecules. Here, we combine coarse-grained simulations of an experimentally-derived NPC structure with a theoretical model to determine the microscopic mechanism of passive transport. Brute-force simulations of protein diffusion through the NPC reveal telegraph-like behavior, where prolonged diffusion on one side of the NPC is interrupted by rapid crossings to the other. We rationalize this behavior using a theoretical model that reproduces the energetics and kinetics of permeation solely from statistical analysis of transient voids within the disordered mesh. As the protein size increases, the mesh transforms from a soft to a hard barrier, enabling orders-of-magnitude reduction in permeation rate for proteins beyond the percolation size threshold. Our model enables exploration of alternative NPC architectures and sets the stage for uncovering molecular mechanisms of facilitated nuclear transport.

category: biophysics


title: Lonafarnib improves cardiovascular function and survival in a mouse model of Hutchinson-Gilford Progeria Syndrome

abstract: Clinical trials have demonstrated that lonafarnib, a farnesyltransferase inhibitor, extends lifespan in patients afflicted by Hutchinson-Gilford progeria syndrome, a devastating condition that accelerates many characteristics of aging and results in premature death due to cardiovascular sequelae. The US Food and Drug Administration approved Zokinvy (lonafarnib) in November 2020 for treating these patients, yet a detailed examination of drug-associated effects on cardiovascular structure, properties, and function has remained wanting. In this paper, we report encouraging outcomes of daily post-weaning treatment with lonafarnib on the composition and biomechanical phenotype of elastic and muscular arteries as well as associated cardiac function in a well-accepted mouse model of progeria that exhibits severe end-stage cardiovascular disease. Lonafarnib resulted in 100% survival of the treated progeria mice to the study end-point (time of 50% survival of untreated mice), with associated improvements in arterial structure and function working together to significantly reduce pulse wave velocity and improve left ventricular diastolic function. By contrast, dual treatment with lonafarnib and rapamycin did not improve outcomes over that achieved with lonafarnib monotherapy.

category: pathology


title: Emergent Diversity and Persistent Turnover in Evolving Microbial Cross-Feeding Networks

abstract: A distinguishing feature of many ecological networks in the microbial realm is the diversity of substrates that could potentially serve as energy sources for microbial consumers. The microorganisms are themselves the agents of compound diversification via metabolite excretion or overflow metabolism. It has been suggested that the emerging richness of different substrates is an important condition for the immense biological diversity in microbial ecosystems. In this work, we study how complex cross-feeding networks (CFN) of microbial species may develop from a simple initial community given some elemental evolutionary mechanisms of resource-dependent speciation and extinctions using a network flow model. We report results of several numerical experiments and report an in-depth analysis of the evolutionary dynamics. We find that even in stable environments, the system is subject to persisting turnover, indicating an ongoing co-evolution. Further, we compare the impact of different parameters, such as the ratio of mineralization, as well as the metabolic versatility and variability on the evolving community structure. The results imply that high microbial and molecular diversity is an emergent property of evolution in cross-feeding networks, which affects transformation and accumulation of substrates in natural systems, such as soils and oceans, with potential relevance to biotechnological applications.

category: systems biology


title: An exploratory study on the microbiome of northern and southern populations of Ixodes scapularis ticks predicts changes and unique bacterial interactions

abstract: The black-legged tick (Ixodes scapularis) is the primary vector of Borrelia burgdorferi, the causative agent of Lyme disease in North America. However, the prevalence of Lyme borreliosis is clustered around the northern states of the United States of America. This study utilized a metagenomic sequencing approach to compare the microbial communities residing within Ix. scapularis populations from north and southern geographic locations in the USA. Using a SparCC network construction model, potential interactions between members of the microbial communities from Borrelia burgdorferi-infected tissues of unfed and blood-fed ticks were performed. A significant difference in bacterial composition and diversity among northern and southern tick populations was found between northern and southern tick populations. The network analysis predicted a potential antagonistic interaction between endosymbiont Rickettsia buchneri and Borrelia burgdorferi sensu lato. Network analysis, as expected, predicted significant positive and negative microbial interactions in ticks from these geographic regions, with the genus Rickettsia, Francisella, and Borreliella playing an essential role in the identified clusters. Interactions between Rickettsia buchneri and Borrelia burgdorferi sensu lato needs more validation and understanding. Understanding the interplay between the micro-biome and tick-borne pathogens within tick vectors may pave the way for new strategies to prevent tick-borne infections.

category: zoology


title: Identification of CDRH3 Loops in the Naive B Cell Receptor Repertoire that Can Be Engaged by Candidate Immunogens

abstract: B cell lineages that are the current focus of vaccine development efforts against HIV-1, influenza or coronaviruses, often contain rare features, such as long heavy chain complementarity determining regions (CDRH3) loops. These unusual characteristics may limit the number of available B cells in the natural immunoglobulin repertoire that can respond to pathogen vaccinations. To measure the ability of a given immunogen to engage naturally occurring B cell receptors of interest, here we describe a mixed experimental and bioinformatic approach for determining the frequency and sequence of CDRH3 loops in the immune repertoire that can be recognized by a vaccine candidate. By combining deep mutational scanning and B cell receptor database analysis, CDRH3 loops were found that can be engaged by two HIV-1 germline-targeting immunogens, thus illustrating how the methods described here can be used to evaluate candidate immunogens based on their ability to engage diverse B cell lineage precursors.

category: immunology


title: The Interferon Resistance of Transmitted HIV-1 is a Consequence of Enhanced Replicative Fitness

abstract: HIV-1 transmission via sexual exposure is a relatively inefficient process. When successful transmission does occur, newly infected individuals are colonized by either a single or a very small number of establishing virion(s). These transmitted founder (TF) viruses are more interferon (IFN) resistant than chronic control (CC) viruses present 6 months after transmission. To identify the specific molecular defences that make CC viruses more susceptible to the IFN-induced  antiviral state than TF viruses, we established a pair of fluorescent GFP-IRES-Nef TF and CC viruses and used arrayed interferon-stimulated gene (ISG) expression screening. The relatively uniform ISG resistance of transmitted HIV-1 directed us to investigate the underlying mechanism. Our subsequent in silico simulations, modelling, and in vitro characterisation of a model TF/CC pair (closely matched in replicative fitness), revealed that small differences in replicative growth rates can explain the broad IFN resistance displayed by transmitted HIV-1. We propose that the apparent IFN resistance of transmitted HIV-1 is a consequence of enhanced replicative fitness, as opposed to specific resistance to individual IFN-induced defences.

category: microbiology


title: A widespread family of WYL-domain transcriptional regulators co-localises with diverse phage defence systems and islands

abstract: AO_SCPLOWBSTRACTC_SCPLOWBacteria are under constant assault by bacteriophages and other mobile genetic elements. As a result, bacteria have evolved a multitude of systems that protect from attack. Genes encoding bacterial defence mechanisms can be clustered into "defence islands", providing a potentially synergistic level of protection against a wider range of assailants. However, there is a comparative paucity of information on how expression of these defence systems is controlled. Here, we functionally characterise a transcriptional regulator, BrxR, encoded within a recently described phage defence island from a multidrug resistant plasmid of the emerging pathogen Escherichia fergusonii. Using a combination of reporters and electrophoretic mobility shift assays, we discovered that BrxR acts as a repressor. We present the structure of BrxR to 2.15 [A], the first structure of this family of transcription factors, and pinpoint a likely binding site for ligands within the WYL-domain. Bioinformatic analyses demonstrated that BrxR homologues are widespread amongst bacteria. About half (48%) of identified BrxR homologues were co-localised with a diverse array of known phage defence systems, either alone or clustered into defence islands. BrxR is a novel regulator that reveals a common mechanism for controlling the expression of the bacterial phage defence arsenal.


title: In situ control of root-bacteria interactions using optical trapping in transparent soil

abstract: Bacterial attachment on root surfaces is an important step preceding the colonisation or internalisation and subsequent infection of plants by pathogens. Unfortunately, bacterial attachment is not well understood because the phenomenon is difficult to observe. Here we assessed whether this limitation could be overcome using optical trapping approaches. We have developed a system based on counter-propagating beams and studied its ability to guide Pectobacterium atrosepticum (Pba) cells to different root cell types within the interstices of transparent soils. Bacterial cells were successfully trapped and guided to root hair cells, epidermis cells, border cells and tissues damaged by laser ablation. Finally, we used the system to quantify the bacterial cell detachment rate of Pba cells on root surfaces following reversible attachment. Optical trapping techniques could greatly enhance our ability to deterministically characterise mechanisms linked to attachment and formation of biofilms in the rhizosphere.


title: Identification and characterization of BrxR as a regulatory gene in the BREX phage restriction system

abstract: Bacteriophage exclusion ( BREX) phage restriction systems are found in a wide range of bacteria. Various BREX systems encode unique combinations of proteins that usually include a site-specific methyltransferase; none appear to contain a nuclease. Here we describe the identification and characterization of a Type I BREX system from Acinetobacter and the effect of deleting each BREX ORF on growth, methylation, and restriction. We identified a previously uncharacterized gene in the BREX operon that is dispensable for methylation but involved in restriction. Biochemical and crystallographic analyses of this factor, which we term BrxR ( BREX Regulator), demonstrate that it forms a homodimer and specifically binds a DNA target site upstream of its transcription start site. Deletion of the BrxR gene causes cell toxicity, reduces restriction, and significantly increases the expression of BrxC. In contrast, the introduction of a premature stop codon into the BrxR gene, or a point mutation blocking its DNA binding ability, has little effect on restriction, implying that the BrxR coding sequence and BrxR protein play independent functional roles. We speculate that the BrxR coding sequence is involved in cis regulation of anti-phage activity, while the BrxR protein plays an additional regulatory role, perhaps during horizontal transfer.

category: molecular biology


title: Identification and characterization of the WYL BrxR protein and its gene asseparable regulatory elements of a BREX phage restriction system


title: Parent of origin DNA methylation as a potential mechanism for genomic imprinting in bees.

abstract: Genomic imprinting is defined as parent-of-origin allele-specific expression. In order for genes to be expressed in this manner an  imprinting mark must be present to distinguish the parental alleles within the genome. In mammals imprinted genes are primarily associated with DNA methylation. Genes exhibiting parent-of-origin expression have recently been identified in two species of Hymenoptera with functional DNA methylation systems; Apis mellifera and Bombus terrestris. We carried out whole genome bisulfite sequencing of parents and ofspring from reciprocal crosses of two B. terrestris subspecies in order to identify parent-of-origin DNA methylation. We were unable to survey a large enough proportion of the genome to draw a conclusion on the presence of parent-of-origin DNA methylation however we were able to characterise the sex- and caste-specific methylomes of B. terrestris for the first time. We find males difer significantly to the two female castes, with diferentially methylated genes involved in many histone modification related processes. We also analysed previously generated honeybee whole genome bisulfite data to see if genes previously identified as showing parent-of-origin DNA methylation in the honeybee show consistent allele-specific methylation in independent data sets. We have identified a core set of 12 genes in female castes which may be used for future experimental manipulation to explore the functional role of parent-of-origin DNA methylation in the honeybee. Finally, we have also identified allele-specific DNA methylation in honeybee male thorax tissue which suggests a role for DNA methylation in ploidy compensation in this species.

category: evolutionary biology


title: The genome of the Balearic shearwater (Puffinus mauretanicus), a Critically Endangered seabird: a valuable resource for evolutionary and conservation genomics

abstract: The Balearic shearwater (Puffinus mauretanicus) is the most threatened seabird in Europe. The fossil record suggests that human colonisation of the Balearic Islands resulted in a sharp decrease of the population size. Currently, populations continue to be decimated mainly due to predation by introduced mammals and bycatch in longline fisheries, and some studies predict their extinction by 2070. We present the first high-quality reference genome for the species which was obtained by a combination of short and long-read sequencing. Our hybrid assembly includes 4,169 scaffolds, with a scaffold N50 of 2.1 Mbp, a genome length of 1.2 Gbp, and BUSCO completeness of 96%, which is amongst the highest across sequenced avian species. This reference genome allowed us to study critical aspects relevant to the conservation status of the species, such as an evaluation of overall heterozygosity levels and the reconstruction of its historical demography. Our phylogenetic analysis using whole-genome information resolves current uncertainties in the order Procellariiformes systematics. Comparative genomics analyses uncover a set of candidate genes that may have played an important role into the adaptation to a pelagic lifestyle of Procellariiformes, including those for the enhancement of fishing capabilities, night vision and the development of natriuresis. This reference genome will be the keystone for future developments of genetic tools in conservation efforts for this Critically Endangered species.

category: genomics


title: Reviving ghost alleles: Genetically admixed coyotes along the American Gulf Coast are critical for saving the endangered red wolf

abstract:
The last red wolves were captured along the Gulf Coast in 1980, where they hybridized with coyote, to establish the captive breeding population. However, red wolf ancestry persists in local coyotes and could be leveraged by genomic innovations to support species persistence. We assessed genomic ancestry and morphology of coyotes in southwestern Louisiana, and find they carried 38-62% red wolf ancestry acquired in the last 30 years, which is enriched on land with minimal coyote hunting. These coyotes were also similar in ancestry to canids captured in the 1970s that initiated the red wolf captive breeding program. Further, we reported that coyotes with higher red wolf ancestry are larger in size. Our findings evidence the importance of hybrids as a reservoir of endangered species ancestry for contemporary conservation efforts. Admixed genomes are at the forefront of innovative solutions, with red wolf survival a prime candidate for this new paradigm.

TeaserCoyotes along the American Gulf Coast carry genetic variation critical for the survival of the endangered red wolf species.


title: Multiparameters dependance of tissue shape maintenance in myoblasts multicellular aggregates: the role of intermediate filaments.

abstract: Liquid and elastic behavior of tissues drives their morphology and their response to the environment. They appear as the first insight on tissue mechanics. We explore the role of individual cell properties on spheroids of mouse muscle precursor cells by developing a fully automated surface tension and Youngs modulus measurement system. Flattening multicellular aggregates under magnetic constraint, we show that rigidity and surface tension act as highly sensitive macroscopic reporters closely related to microscopic local tension and effective adhesion. Shedding light on the major contributions of acto-myosin contractility, actin organization and intercellular adhesions, we reveal the role of desmin organization on the macroscopic mechanics of this tissue model.


title: Phosphorylation of interfacial phosphosite leads to increased binding of Rap-Raf complex

abstract: The effect of phosphorylation of a serine residue in the Rap protein, residing at the complex interface of Rap-Raf complex is studied using atomistic molecular dynamics simulations. As the phosphosite of interest (SER39) is buried at the interface of the Rap-Raf complex, phosphorylation of only Rap protein was simulated and then complexed with the RBD of Raf for further analysis of complex stability. Our simulations reveal that the phosophorylation increases the binding of complex through strong electrostatic interactions and changes the charge distribution of the interface significantly. This is manifested as an increase in stable salt-bridge interactions between the Rap and Raf of the complex. Network analysis clearly shows that the phosphorylation of SER39 reorganizes the community network to include the entire region of Raf chain, including, Raf L4 loop potentially affecting downstream signalling.


title: DeepMed: A unified, modular pipeline for end-to-end deep learning in computational pathology

abstract: The interpretation of digitized histopathology images has been transformed thanks to artificial intelligence (AI). End-to-end AI algorithms can infer high-level features directly from raw image data, extending the capabilities of human experts. In particular, AI can predict tumor subtypes, genetic mutations and gene expression directly from hematoxylin and eosin (H&E) stained pathology slides. However, existing end-to-end AI workflows are poorly standardized and not easily adaptable to new tasks. Here, we introduce DeepMed, a Python library for predicting any high-level attribute directly from histopathological whole slide images alone, or from images coupled with additional meta-data (https://github.com/KatherLab/deepmed). Unlike earlier computational pipelines, DeepMed is highly developer-friendly: its structure is modular and separates preprocessing, training, deployment, statistics, and visualization in such a way that any one of these processes can be altered without affecting the others. Also, DeepMed scales easily from local use on laptop computers to multi-GPU clusters in cloud computing services and therefore can be used for teaching, prototyping and for large-scale applications. Finally, DeepMed is user-friendly and allows researchers to easily test multiple hypotheses in a single dataset (via cross-validation) or in multiple datasets (via external validation). Here, we demonstrate and document DeepMeds abilities to predict molecular alterations, histopathological subtypes and molecular features from routine histopathology images, using a large benchmark dataset which we release publicly. In summary, DeepMed is a fully integrated and broadly applicable end-to-end AI pipeline for the biomedical research community.

category: cancer biology


title: Skin resident memory cells generated by the L. major centrin gene deleted parasites mediate protective immune response analogous to leishmanization

abstract: Leishmaniasis is a vector-borne parasitic disease transmitted through the bite of a sand fly with no available vaccine for humans. Recently, we have developed a live attenuated Leishmania major centrin gene deleted parasite strain (LmCen-/-) that induced protection against a homologous and heterologous challenges. The protection is mediated by IFN{gamma} secreting CD4+ T effector cells and multifunctional T cells, which is analogous to leishmanization. Previously, skin tissue resident memory T cells (TRM cells) were shown to be crucial for host protection in a leishmanization model. In this study, we evaluated generation and function of skin TRM cells following immunization with LmCen-/- parasites and compared those with leishmanization. In the absence of recoverable LmCen-/- parasites, the skin of immunized mice showed functional TRM cells comparable to leishmanized mice. The generation of the skin TRM cells was supported by the induction of cytokines and chemokines essential for their production and survival. Following challenge infection with wild type L. major, TRM cells specific to L. major were rapidly recruited and proliferated at the site of infection in the immunized mice which was similar to leishmanization. Further, upon challenge, CD4+ TRM cells induced higher levels of IFN{gamma} and Granzyme B in the immunized and leishmanized mice than non-immunized mice. Taken together, our studies demonstrate that a genetically modified live attenuated Leishmania vaccine generates functional CD4+ TRM cells that mediate protection and can be a safer alternative to leishmanization.


title: Deep metabolic profiling assessment of tissue extraction protocols for three model organisms

abstract: Metabolic profiling harbors the potential to better understand various disease entities such as cancer, diabetes, Alzheimers, Parkinsons disease or COVID-19. Deciphering these intricate pathways in human studies requires large sample sizes as a means of reducing variability. While such broad human studies have discovered new associations between a given disease and certain affected metabolites, i.e. biomarkers, they often provide limited functional insights. To design more standardized experiments, reduce variability in the measurements and better resolve the functional component of such dynamic metabolic profiles, model organisms are frequently used. Standardized rearing conditions and uniform sampling strategies are prerequisites towards a successful metabolomic study. However, further aspects such as the choice of extraction protocol and analytical technique can influence the outcome drastically. Here, we employed a highly standardized metabolic profiling assay analyzing 630 metabolites across three commonly used model organisms (Drosophila, mouse and Zebrafish) to find the optimal extraction protocols for various matrices. Focusing on parameters such as metabolite coverage, metabolite yield and variance between replicates we compared seven extraction protocols. We found that the application of a combination of 75% ethanol and methyl tertiary-butyl ether (MTBE), while not producing the broadest coverage and highest yields, was the most reproducible extraction protocol. We were able to determine up to 530 metabolites in mouse kidney samples, 509 in mouse liver, 422 in Zebrafish and 388 in Drosophila and discovered a core overlap of 261 metabolites in these four matrices. To enable other scientists to search for the most suitable extraction protocol in their experimental context and interact with this comprehensive data, we have integrated our data set in the open-source shiny app "MetaboExtract". This will enable scientists to search for their metabolite or metabolite class of interest, compare it across the different tested extraction protocols and sample types as well as find reference concentrations.

category: biochemistry


title: anndata: Annotated data

abstract:
anndata is a Python package for handling annotated data matrices in memory and on disk (github.com/theislab/anndata), positioned between pandas and xarray. anndata offers a broad range of computationally efficient features including, among others, sparse data support, lazy operations, and a PyTorch interface.

Statement of needGenerating insight from high-dimensional data matrices typically works through training models that annotate observations and variables via low-dimensional representations. In exploratory data analysis, this involves iterative training and analysis using original and learned annotations and task-associated representations. anndata offers a canonical data structure for book-keeping these, which is neither addressed by pandas (McKinney, 2010), nor xarray (Hoyer & Hamman, 2017), nor commonly-used modeling packages like scikit-learn (Pedregosa et al., 2011).

category: bioinformatics


title: A Mathematical Model of Mitochondria in Proximal Tubule and Thick Ascending Limb Cells

abstract: Mitochondria are a key player in several kinds of tissue injury, and are even the ultimate cause of certain diseases. In this work we introduce new models of mitochondrial ATP generation in multiple tissues, including liver hepatocytes and the medullary thick ascending limb in the kidney. Using this model, we predict these tissues responses to hypoxia, uncoupling, ischemia-reperfusion, and oxidative phosphorylation dysfunction. Our results suggest mechanisms explaining differences in robustness of mitochondrial function across tissues.The medullary thick ascending limb and proximal tubule in the kidney both experience a high metabolic demand, while having lower baseline activity of oxidative phosphorylation relative to the liver. These factors make these tissues susceptible to dysfunction of ComplexIII. A lower baseline oxygen tension observed in the thick ascending limb makes it susceptible to Complex IV. On the other hand, since the liver lacks these risk factors, and has higher baseline rates of glycolysis, it is less susceptible to all kinds of oxidative phosphorylation dysfunction.

category: physiology


title: Effects of anesthesia on ozone-induced lung and systemic inflammation

abstract: Anesthetics are required for procedures that deliver drugs/biologics, infectious/inflammatory agents, and toxicants directly to the lungs. However, the possible confounding effects of anesthesia on lung inflammation and injury are underreported. Here, we tested the effects of brief isoflurane (Iso) or ketamine/xylazine/atipamezole (K/X/A) anesthesia prior to ozone exposure (4 hours, 3ppm) on lung inflammatory responses in mice. Anesthesia regimens modeled those used for non-surgical intratracheal instillations, and were administered 1-2 hours or 24 hours prior to initiating ozone exposure. We found that Iso given 1-2 hours prior to ozone inhibited inflammatory responses in the lung, and this effect was absent when Iso was given 23-24 hours prior to ozone. In contrast, K/X/A given 1-2 hours prior to ozone increased lung and systemic inflammation. Our results highlight the need to comprehensively evaluate anesthesia as an experimental variable in the assessment of lung inflammation in response to ozone and other inflammatory stimuli.


title: Optimizing dosage-specific treatments in a multi-scale model of a tumor growth

abstract: The emergence of cell resistance in cancer treatment is a complex phenomenon that emerges from the interplay of processes that occur at different scales. For instance, molecular mechanisms and population-level dynamics such as competition and cell-cell variability have been described as playing a key role in the emergence and evolution of cell resistances. Multi-scale models are a useful tool to study biology at a very different time and spatial scales, as they can integrate different processes that take place at the molecular, cellular and intercellular levels. In the present work, we use an extended hybrid multi-scale model of 3T3 fibroblast spheroid to perform a deep exploration of the parameter space of effective treatment strategies based on TNF pulses. To explore the parameter space of effective treatments in different scenarios and conditions, we have developed an HPC-optimized model exploration workflow based on EMEWS. We first studied the effect of the cells spatial distribution in the values of the treatment parameters by optimizing the supply strategies in 2D monolayers and 3D spheroids of different sizes. We later study the robustness of the effective treatments when heterogeneous populations of cells are considered. We found that our model exploration workflow can find effective treatments in all the studied conditions. Our results show that cells spatial geometry, as well as, population variability should be considered when optimizing treatment strategies in order to find robust parameter sets.


title: Insights from the IronTract challenge: optimal methods for mapping brain pathways from multi-shell diffusion MRI

abstract: Limitations in the accuracy of brain pathways reconstructed by diffusion MRI (dMRI) tractography have received considerable attention. While the technical advances spearheaded by the Human Connectome Project (HCP) led to significant improvements in dMRI data quality, it remains unclear how these data should be analyzed to maximize tractography accuracy. Over a period of two years, we have engaged the dMRI community in the IronTract Challenge, which aims to answer this question by leveraging a unique dataset. Macaque brains that have received both tracer injections and ex vivo dMRI at high spatial and angular resolution allow a comprehensive, quantitative assessment of tractography accuracy on state-of-the-art dMRI acquisition schemes. We find that, when analysis methods are carefully optimized, the HCP scheme can achieve similar accuracy as a more time-consuming, Cartesian-grid scheme. Importantly, we show that simple pre- and post-processing strategies can improve the accuracy and robustness of many tractography methods. Finally, we find that fiber configurations that go beyond crossing (e.g., fanning, branching) are the most challenging for tractography. The IronTract Challenge remains open and we hope that it can serve as a valuable validation tool for both users and developers of dMRI analysis methods.

category: neuroscience


title: Orbitofrontal cortex contributes to the comparison of values underlying economic choices

abstract:
Economic choices between goods entail the computation and comparison of subjective values. Previous studies examined neuronal activity in the orbitofrontal cortex (OFC) of monkeys choosing between different types of juices. Three groups of neurons were identified: offer value cells encoding the value of individual offers, chosen juice cells encoding the identity of the chosen juice, and chosen value cells encoding the value of the chosen offer. The encoded variables capture both the input (offer value) and the output (chosen juice, chosen value) of the decision process, suggesting that values are compared within OFC. Recent work demonstrates that choices are causally linked to the activity of offer value cells. Conversely, the hypothesis that OFC contributes to value comparison has not been confirmed. Here we show that weak electrical stimulation of OFC specifically disrupts value comparison without altering offer values. This result implies that neuronal populations in OFC participate in the decision process.

SignificanceEconomic choices encompass two mental stages: values are assigned to the available offers, and a decision is made by comparing values. Previous work showed that choices are causally related to offer values encoded in the orbitofrontal cortex (OFC). Conversely, the neural underpinnings of value comparison remain poorly understood. Here we show that weak electrical stimulation of OFC selectively disrupts the decision process without affecting offer values. Hence, neurons in OFC contribute to value comparison.


title: Large scale similarity search across digital reconstructions of neural morphology

abstract: Most functions of the nervous system depend on neuronal and glial morphology. Continuous advances in microscopic imaging and tracing software have provided an increasingly abundant availability of 3D reconstructions of arborizing dendrites, axons, and processes, allowing their detailed study. However, efficient, large-scale methods to rank neural morphologies by similarity to an archetype are still lacking. Using the NeuroMorpho.Org database, we present a similarity search software enabling fast morphological comparison of hundreds of thousands of neural reconstructions from any species, brain regions, cell types, and preparation protocols. We compared the performance of different morphological measurements: 1) summary morphometrics calculated by L-Measure, 2) persistence vectors, a vectorized descriptor of branching structure, 3) the combination of the two. In all cases, we also investigated the impact of applying dimensionality reduction using principal component analysis (PCA). We assessed qualitative performance by gauging the ability to rank neurons in order of visual similarity. Moreover, we quantified information content by examining explained variance and benchmarked the ability to identify occasional duplicate reconstructions of the same specimen. The results indicate that combining summary morphometrics and persistence vectors with applied PCA provides an information rich characterization that enables efficient and precise comparison of neural morphology. The execution time scaled linearly with data set size, allowing seamless live searching through the entire NeuroMorpho.Org content in fractions of a second. We have deployed the similarity search function as an open-source online software tool both through a user-friendly graphical interface and as an API for programmatic access.


title: Slice-direction geometric distortion evaluation and correction with reversed slice-select gradient acquisitions

abstract: Accurate spatial alignment of MRI data acquired across multiple contrasts in the same subject is often crucial for data analysis and interpretation, but can be challenging in the presence of geometric distortions that differ between acquisitions. It is well known that single-shot echo-planar imaging (EPI) acquisitions suffer from distortion in the phase-encoding direction due to B0 field inhomogeneities arising from tissue magnetic susceptibility differences and other sources, however there can be distortion in other encoding directions as well in the presence of strong field homogeneities. High-resolution ultrahigh-field MRI typically uses low bandwidth in the slice-encoding direction to acquire thin slices and, when combined with the pronounced B0 inhomogeneities, is prone to an additional geometric distortion in the slice direction as well. Here we demonstrate a presence of this slice distortion in high-resolution 7T EPI acquired with a novel pulse sequence allowing for the reversal of the slice-encoding gradient polarity that enables the acquisition of pairs of images with equal magnitudes of distortion in the slice direction but with opposing polarities. We also show that the slice-direction distortion can be corrected using gradient reversal-based method applying the same software used for conventional corrections of phase-encoding direction distortion.


title: Spontaneous Neuronal Oscillations in the Human Insula are Hierarchically Organized Traveling Waves

abstract: The insula plays a fundamental role in a wide range of adaptive human behaviors, but its electrophysiological dynamics are poorly understood. Here we used human intracranial electroencephalographic recordings to investigate the electrophysiological properties and hierarchical organization of spontaneous neuronal oscillations within the insula. We analyzed the neuronal oscillations of the insula directly and found that rhythms in the theta and beta frequency oscillations are widespread and spontaneously present. These oscillations are largely organized along the anterior-posterior axis of the insula. Both the left and right insula showed anterior-to-posterior decreasing gradients for the power of oscillations in the beta frequency band. The left insula also showed a posterior-to-anterior decreasing frequency gradient and an anterior-to-posterior decreasing power gradient in the theta frequency band. In addition to measuring the power of these oscillations, we also examined the phase of these signals across simultaneous recording channels and found that the insula oscillations in the theta and beta bands are traveling waves. The strength of the traveling waves in each frequency was positively correlated with the amplitude of each oscillation. However, the theta and beta traveling waves were uncoupled to each other in terms of phase and amplitude, which suggested that insula traveling waves in the theta and beta bands operate independently. Our findings provide new insights into the spatiotemporal dynamics and hierarchical organization of neuronal oscillations within the insula, which, given its rich connectivity with widespread cortical regions, indicates that oscillations and traveling waves have an important role in intra- and inter-insula communication.


title: Brain intrinsic connection patterns underlying tool processing in human adults are present in neonates and not in macaques

abstract: Tool understanding and use are supported by a dedicated left-lateralized, intrinsically connected network in the human adult brain. To examine this networks phylogenic and ontogenetic origins, we compared resting-state functional connectivity (rsFC) among regions subserving tool processing in human adults to rsFC among homologous regions in human neonates and macaque monkeys (adolescent and mature). These homologous regions formed an intrinsic network in human neonates, but not in macaques. Network topological patterns were highly similar between human adults and neonates, and significantly less so between humans and macaques. The premotor-parietal rsFC had most significant contribution to the formation of the neonate tool network. These results suggest that an intrinsic brain network potentially supporting tool processing exists in the human brain prior to individual tool use experiences, and that the premotor-parietal functional connection in particular offers a brain basis for complex tool behaviors specific to humans.


title: Sex-dependent effects of traumatic stress on social behavior and neuronal activation in the prefrontal cortex and amygdala

abstract: Social behavior is complex and fundamental, and deficits in social behavior are common pathological features for a variety of psychiatric disorders including anxiety, depression, and posttraumatic stress disorder. Acute stress has a negative impact on social behavior, and these effects may vary based on sex. The aim of this study was to explore the effect of footshock stress on the sociability of male and female C57Bl/6J mice. Animals were divided into two main groups of footshock exposure or context exposure control. Each group had mice that were treated with either the benzodiazepine alprazolam, or vehicle. Neuronal activation during social interaction was assessed using immunohistochemistry against the immediate early gene product cFos. Footshock stress induced a significantly increased latency to approach a social interaction counterpart in both sexes. Stress-induced increases in defensive tail-rattling behavior elicited during the sociability test were sex-dependent and alleviated by alprazolam. Alprazolam also lowered social exploration and neuronal activation in the infralimbic medial prefrontal cortex. Social interaction induced sex-dependent differences in cFos activation in the lateral subdivision of the central nucleus of the amygdala and ventromedial intercalated cell clusters. Overall, our results suggest that acute footshock stress induces alterations in sociability and patterns of cFos activation in a sex-dependent manner.


title: Dynamics of the habitat use of three predatory freshwater fish in a lentic ecosystem

abstract: To understand the conditions of coexistence in multiple-species predator community, we studied longitudinal and vertical movement of pike (Esox lucius), pikeperch (Sander lucioperca) and catfish (Silurus glanis) in the [R]imov Reservoir, using an autonomous telemetry system for 11 months. We found significant differences among these three species in movement and depth that varied considerably in time, with the greatest differences between warm (late spring and early autumn) and cold season (late autumn to early spring). Preference for different sections of the reservoir was stable for pike, while pikeperch and catfish frequently visited tributary during the warm season, and moved closer to the dam during the cold season. Pike longitudinal activity was highest in the cold season, pikeperch in the warm season, and catfish activity peaked in both the warm and cold seasons. Overlap in the depth used among species was higher in the warm season, when all species used the upper layer of the water column, and lower in cold season, when pikeperch and catfish used deeper areas. These results demonstrated ability of predators to actively inspect a large portion of the reservoir in both longitudinal and vertical dimensions, although differing in the timing of their habitat use and activity.

category: ecology


title: DIY caging apparatus to facilitate chronic and continuous stimulation or recording in an awake rodent

abstract: Chronic stimulation of and recording from the brain and brain diseases can require expensive apparatus and tedious cycles of inducing rodents with anesthesia. To resolve this, we have designed and fabricated a low-cost ([~]$75 vs. $450) DIY rodent caging apparatus consisting of commercially available and 3D-printed components. This apparatus is customizable and can be used to rapidly prototype devices with large rodent sample sizes. Importantly, it enables continuous and chronic stimulation of and recording from the brains of awake and freely moving rodents. It also opens the possibilities of trying complex paradigms of treatment (continuous, intermittent, variable, and chronic). We have successfully used this caging apparatus for chronic intratumoral hypothermia treatment and are currently using it while advancing electrotactic therapies.

category: bioengineering


title: The future of artificial intelligence in digital pathology - results of a survey across stakeholder groups

abstract: Artificial intelligence (AI) provides a powerful tool to extract information from digitized histopathology whole slide images. In the last five years, academic and commercial actors have developed new technical solutions for a diverse set of tasks, including tissue segmentation, cell detection, mutation prediction, prognostication and prediction of treatment response. In the light of limited overall resources, it is presently unclear for researchers, practitioners and policymakers which of these topics are stable enough for clinical use in the near future and which topics are still experimental, but worth investing time and effort into. To identify potentially promising applications of AI in pathology, we performed an anonymous online survey of 75 computational pathology domain experts from academia and industry. Participants enrolled in 2021 were queried about their subjective opinion on promising and appealing sub-fields of computational pathology with a focus on solid tumors. The results of this survey indicate that the prediction of treatment response directly from routine pathology slides is regarded as the most promising future application. This item was ranked highest in the overall analysis and in sub-groups by age and professional background. Furthermore, prediction of genetic alterations, gene expression and survival directly from routine pathology images scored consistently high across subgroups. Together, these data demonstrate a possible direction for the development of computational pathology systems in clinical, academic and industrial research in the near future.


title: A Model of Mitochondria in the Rat Hepatocyte

abstract: Mitochondria are a key player in several kinds of tissue injury, and are even the ultimate cause of certain diseases. In this work we introduce a new model of mitochondrial ATP generation in liver hepatocytes of the rat. Ischemia-reperfusion is an intriguing example of a non-equilibrium behaviour driven by a change in tissue oxygen tension. Ischemia involves prolonged hypoxia, followed by the sudden return of oxygen during reperfusion. During reperfusion, we predict that the build up of succinate causes the electron transport chain in the liver to temporarily be in a highly reduced state. This can lead to the production of reactive oxygen species. We accurately predict the timescale on which the electron transport chain is left in a reduced state, and we observe levels of reduction likely to lead to reactive oxygen species production. Aside from the above, we predict thresholds for ATP depletion from hypoxia, and we predict the consequences for oxygen consumption of uncoupling.


title: Response of Surface Water Quality Characteristics to Socio-economic Factors in Eastern-Central China

abstract: Following the implementation of the strictest water resource management system in China, it has become increasingly important to understand and improve the surface water quality and the rate at which water function zones reach the water quality standard. Based on the monthly monitoring data from 450 monitoring sites at the provincial borders of 27 provinces in China in 2019, the overall surface water quality at provincial boundaries in China was as follows: 61.7% of the water was classified under Class I-III; and 5%, 8.6%, and 12.2% of the water was classified under Class IV, V, and inferior V, respectively. The main standard items are DO, CODMn, COD, BOD5, NH3-N, and TP. The Canadian Council of Ministers of the Environment-water quality index (CCME-WQI) showed that the provincial boundary water quality exceeded the fair level, and F1 was the most influential factor. Then, 27 factors that directly or indirectly affect the water quality of surface water at the provincial boundaries of 27 provinces were identified, and the indirect influencing factors were integrated into the ecological environmental quality index and human activities quantitative index. Finally, the 27 factors were integrated into six factors, and the relationship between these indicators and CCME-WQI as well as the concentration of influencing elements with respect to regulatory standard limits were analyzed. The proportion of building land was the most significant factor affecting the quality of the aquatic environment in provincial boundaries. In addition, the economic development level, proportion of farmland, and degree of social development were identified as significant influencing factors. The six factors have different degrees of impact on the concentrations of major elements with respect to standard limits. This study basically explores water resource management and offers significant reference and guidelines for the improvement of the quality of surface water at provincial boundaries in China.


title: A Model Visualization-based Approach for Insight into Waveforms and Spectra Learned by CNNs

abstract:
Recent years have shown a growth in the application of deep learning architectures such as convolutional neural networks (CNNs), to electrophysiology analysis. However, using neural networks with raw time-series data makes explainability a significant challenge. Multiple explainability approaches have been developed for insight into the spectral features learned by CNNs from EEG. However, across electrophysiology modalities, and even within EEG, there are many unique waveforms of clinical relevance. Existing methods that provide insight into waveforms learned by CNNs are of questionable utility. In this study, we present a novel model visualization-based approach that analyzes the filters in the first convolutional layer of the network. To our knowledge, this is the first method focused on extracting explainable information from EEG waveforms learned by CNNs while also providing insight into the learned spectral features. We demonstrate the viability of our approach within the context of automated sleep stage classification, a well-characterized domain that can help validate our approach. We identify 3 subgroups of filters with distinct spectral properties, determine the relative importance of each group of filters, and identify several unique waveforms learned by the classifier that were vital to the classifier performance. Our approach represents a significant step forward in explainability for electrophysiology classifiers, which we also hope will be useful for providing insights in future studies.

Clinical RelevanceOur approach can assist with the development and validation of clinical time-series classifiers.


title: Prediction of high-risk liver cancer patients from their mutation profile: Benchmarking of mutation calling techniques

abstract: Identification of somatic mutations with high precision is one of the major challenges in prediction of high-risk liver-cancer patients. In the past, number of mutation calling techniques have been developed that include MuTect2, MuSE, Varscan2, and SomaticSniper. In this study, an attempt has been made to benchmark potential of these techniques in predicting prognostic biomarkers for liver cancer. Initially, we extracted somatic mutations in liver-cancer patients using VCF and MAF files from the cancer genome atlas. In terms of size, the MAF files are 42 times smaller than VCF files and containing only high-quality somatic mutations. Further, machine learning based models have been developed for predicting high-risk cancer patients using mutations obtain from different techniques. The performance of different techniques and data files have been compared based on their potential to discriminate high and low risk liver-cancer patients. Finally, univariate survival analysis revealed the prognostic role of highly mutated genes. Based on correlation analysis, we selected 80 genes negatively associated with the overall survival of the liver cancer patients. Single-gene based analysis showed that MuTect2 technique based MAF file has achieved maximum HRLAMC3 9.25 with p-value 1.78E-06. Further, we developed various prediction models using selected genes for each technique, and the results indicate that MuTect2 technique based VCF files outperform all other methods with maximum AUROC of 0.72 and HR 4.50 (p-value 3.83E-15). Eventually, VCF file generated using MuTect2 technique performs better among other mutation calling techniques to explore the prognostic potential of mutations in liver cancer. We hope that our findings will provide a useful and comprehensive comparison of various mutation calling techniques for the prognostic analysis of cancer patients.


title: Molecular identification of bats from  Wavulgalge cave, Wellawaya, Sri Lanka

abstract: This is the first report on the molecular identification and phylogeny of Rousettus leschenaultii, Rhinolophus rouxii, Hipposideros speoris, Hipposideros lankadiva, Miniopterus fuliginosus bat species in Sri Lanka, inferred from mitochondrially encoded cytochrome b gene sequences. Wellawaya Wavulgalge cave in Sri Lanka is one of the largest sympatric colonies found on the island, occupied by five species of bats. Recent research has indicated that bats show enormous cryptic genetic diversity. Moreover, even in the same species, acoustic properties of echolocation calls and morphological features such as fur colour could vary in different populations. Therefore, we have used molecular techniques for the accurate identification of five bat species recorded in one of the largest cave populations in Sri Lanka. Bats were caught using a hand net and saliva samples were collected non-invasively from each bat using a sterile oral swab. Nucleic acids were extracted from oral swab samples and mitochondrial DNA was amplified using primers targeting the mitochondrially encoded cytochrome b gene. This study identified the bat species recorded in the Wellawaya cave as Rousettus leschenaultii, Rhinolophus rouxii, Hipposideros speoris, Hipposideros lankadiva and Miniopterus fuliginosus. Our findings will contribute to future conservation and systematic studies of bats in Sri Lanka. This study will also provide the basis for a genetic database of Sri Lankan bats.


title: Reduced sera neutralization to Omicron SARS-CoV-2 by bothinactivated and protein subunit vaccines and the convalescents

abstract: Omicron variant continues to spread all over the world. There are lots of scientific questions remaining to be answered for such a devastating variant. There are a dozen of vaccines already in clinical use. The very urgent scientific question would be whether or not these vaccines can protect Omicron variant. Here, we tested the sera from both convalescents and vaccine recipients receiving either inactivated or protein subunits vaccines (CoronaVac from Sinovac, or BBIBP-CoV from Sinopharm, or ZF2001 from Zhifei longcom) for the binding antibody titers (ELISA) and neutralization antibodies titers (pseudovirus neutralization assay). We showed that Omicron do have severe immune escape in convalescents, with 15 of 16 were negative in neutralization. By contrast, in vaccinees who received three jabs of inactivated or protein subunit vaccine, the neutralizing activity was much better preserved. Especially in the ZF2001 group with an extended period of the second and third jab (4-6 months) remains 100% positive in Omicron neutralization, with only 3.1-folds reduction in neutralizing antibody (NAb) titer. In this case, we proposed that, the multi-boost strategy with an extended interval between the second and third jab for immune maturation would be beneficial for NAb against devastating variants such as Omicron.


title: In vitro and computational analysis of the putative furin cleavage site (RRARS) in the divergent spike protein of the rodent coronavirus AcCoV-JC34 (sub-genus luchacovirus)

abstract: The Coronaviridae is a highly diverse virus family, with reservoir hosts in a variety of wildlife species that encompass bats, birds and small mammals, including rodents. Within the taxonomic group alphacoronavirus, certain sub-genera (including the luchacoviruses) have phylogenetically distinct spike proteins, which remain essentially uncharacterized. Using in vitro and computational techniques, we analyzed the spike protein of the rodent coronavirus AcCoV-JC34 from the sub-genus luchacovirus, previously identified in Apodemus chevrieri (Chevriers field mouse). We show that AcCoV-JC34--unlike the other luchacoviruses--has a putative furin cleavage site (FCS) within its spike S1 domain, close to the S1/S2 interface. The pattern of basic amino acids within the AcCoV-JC34 FCS (-RR-R-) is identical to that found in "pre-variant" SARS-CoV-2--which is in itself atypical for an FCS, and suboptimal for furin cleavage. Our analysis shows that, while containing an -RR-R-motif, the AcCoV-JC34 spike "FCS" is not cleaved by furin (unlike for SARS-CoV-2), suggesting the possible presence of a progenitor sequence for viral emergence from a distinct wildlife host.


title: Changes of small non-coding RNAs by severe acute respiratory syndrome coronavirus 2 infection

abstract: The ongoing pandemic of coronavirus disease 2019 (COVID-19), which results from the rapid spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a significant global public health threat, with molecular mechanisms underlying its pathogenesis largely unknown. Small non-coding RNAs (sncRNAs) are known to play important roles in almost all biological processes. In the context of viral infections, sncRNAs have been shown to regulate the host responses, viral replication, and host-virus interaction. Compared with other subfamilies of sncRNAs, including microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), tRNA-derived RNA fragments (tRFs) are relatively new and emerge as a significant regulator of host-virus interactions. Using T4 PNK-RNA-seq, a modified next-generation sequencing (NGS), we recently found that nasopharyngeal swabs (NPS) samples from SARS-CoV-2 positive and negative subjects show a significant difference in sncRNA profiles. There are about 166 SARS-CoV-2-impacted sncRNAs. Among them, tRFs are the most significantly affected and almost all impacted tRFs are derived from the 5-end of tRNAs (tRF5). Using a modified qRT-PCR, which was recently developed to specifically quantify tRF5s by isolating the tRF signals from its corresponding parent tRNA signals, we validated that tRF5s derived from tRNA GluCTC (tRF5-GluCTC), LysCTT (tRF5-LysCTT), ValCAC (tRF5-ValCAC), CysGCA (tRF5-CysGCA) and GlnCTG (tRF5-GlnCTG) are enhanced in NPS samples of SARS-CoV2 patients and SARS-CoV2-infected airway epithelial cells. In addition to host-derived ncRNAs, we also identified several sncRNAs derived from the virus (svRNAs), among which a svRNA derived from CoV2 genomic site 346 to 382 (sv-CoV2-346) has the highest expression. The induction of both tRFs and sv-CoV2-346 has not been reported previously, as the lack of the 3-OH ends of these sncRNAs prevents them to be detected by routine NGS. In summary, our studies demonstrated the involvement of tRFs in COVID-19 and revealed new CoV2 svRNAs.


title: Neutralization and Stability of SARS-CoV-2 Omicron Variant

abstract: The SARS-CoV-2 B.1.1.529/Omicron variant was first characterized in South Africa and was swiftly designated a variant of concern1. Of great concern is its high number of mutations, including 30-40 mutations in the virus spike (S) protein compared to 7-10 for other variants. Some of these mutations have been shown to enhance escape from vaccine-induced immunity, while others remain uncharacterized. Additionally, reports of increasing frequencies of the Omicron variant may indicate a higher rate of transmission compared to other variants. However, the transmissibility of Omicron and its degree of resistance to vaccine-induced immunity remain unclear. Here we show that Omicron exhibits significant immune evasion compared to other variants, but antibody neutralization is largely restored by mRNA vaccine booster doses. Additionally, the Omicron spike exhibits reduced receptor binding, cell-cell fusion, S1 subunit shedding, but increased cell-to-cell transmission, and homology modeling indicates a more stable closed S structure. These findings suggest dual immune evasion strategies for Omicron, due to altered epitopes and reduced exposure of the S receptor binding domain, coupled with enhanced transmissibility due to enhanced S protein stability. These results highlight the importance of booster vaccine doses for maintaining protection against the Omicron variant, and provide mechanistic insight into the altered functionality of the Omicron spike protein.


title: Single Amino Acid Change Mutation in the Hydrophobic Core of the N-terminal Domain of P22 TSP affects the Proteins Stability.

abstract: The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has significantly shifted the attention of researchers to critically investigate most viruses to understand specific characteristics that impart their virulence. For instance, the SARS-CoV-2 has undergone several mutations, with some variants classified as "variants of concern", e.g., the Omicron and Delta variant of SARS-CoV-2 are known for their rapid transmission and antigenicity due to mutation in the Spike protein. P22 bacteriophage is a bacterial virus that has a tailspike protein (TSP) that performs similar functions as the Spike protein of SARS-COV-2. We previously carried out a site-directed mutagenesis of the P22 TSP to bear disruptive mutations in the hydrophobic core of the N-terminal Domain (NTD), then partially characterized the properties of the mutant TSPs. In this process, the valine patch (triple valine residues that formed a hydrophobic core) was replaced with charged amino acids (Asp or lysine) or hydrophobic amino acids (Leucine or isoleucine). Some of the mutant TSPs characterized showed significant differences in migration in both native and SDS-PAGE. Mutants with such disruptive mutation are known to show non-native properties, and as expected, most of these mutants obtained showed significantly different properties from the WT P22 TSP. In this work, we further characterized these mutant species by computational and in vitro assays to demonstrate the validity of our previous inference that the valine patch is a critical player in the stability of the N-terminal domain of the P22 TSP.


title: Modeling the dynamics of within-host viral infection and evolution predicts quasispecies distributions and phase boundaries separating distinct classes of infections

abstract:
We use computational modeling to study within-host viral infection and evolution. In our model, viruses exhibit variable binding to cells, with better infection and replication countered by a stronger immune response and a high rate of mutation. By varying host conditions (permissivity to viral entry T and immune clearance intensity A) for large numbers of cells and viruses, we study the dynamics of how viral populations evolve from initial infection to steady state and obtain a phase diagram of the range of cell and viral responses. We find three distinct replicative strategies corresponding to three physiological classes of viral infections: acute, chronic, and opportunistic. We show similarities between our findings and the behavior of real viral infections such as common flu, hepatitis, and SARS-CoV-2019. The phases associated with the three strategies are separated by a phase transition of primarily first order, in addition to a crossover region. Our simulations also reveal a wide range of physical phenomena, including metastable states, periodicity, and glassy dynamics. Lastly, our results suggest that the resolution of acute viral disease in patients whose immunity cannot be boosted can only be achieved by significant inhibition of viral infection and replication.

Author summaryVirus, in particular RNA viruses, often produce offspring with slightly altered genetic composition. This process occurs both across host populations and within a single host over time. Here, we study the interactions of viruses with cells inside a host over time. In our model, the viruses encounter host cell defenses characterized by two parameters: permissivity to viral entry T and immune response A). The viruses then mutate upon reproduction, eventually resulting in a distribution of related viral types termed a quasi-species distribution. Across varying host conditions (T, A), three distinct viral quasi-species types emerge over time, corresponding to three classes of viral infections: acute, chronic and opportunistic. We interpret these results in terms of real viral types such as common flu, hepatitis, and also SARS-CoV-2019. Analysis of viral of viral mutant populations over a wide range of permissivity and immunity, for large numbers of cells and viruses, reveals phase transitions that separate the three classes of viruses, both in the infection-cycle dynamics and at steady state. We believe that such a multiscale approach for the study of within-host viral infections, spanning individual proteins to collections of cells, can provide insight into developing more effective therapies for viral disease.


title: Structure of Vibrio phage XM1, a simple contractile DNA injection machine

abstract: Antibiotic resistance poses a growing risk to public health requiring new tools to combat pathogenic bacteria. Contractile injection systems, including bacteriophage tails, pyocins, and bacterial type VI secretion systems, can efficiently penetrate cell envelopes and become potential antibacterial agents. Bacteriophage XM1 is a dsDNA virus belonging to the Myoviridae family and infecting Vibrio bacteria. The XM1 virion, made of 18 different proteins, consists of an icosahedral head and a contractile tail, terminated with a baseplate. Here we report cryo-EM reconstructions of all components of the XM1 virion and describe atomic structures of 14 XM1 proteins. The XM1 baseplate is composed of a central hub surrounded by six wedge modules to which twelve spikes are attached. The XM1 tail contains a fewer number of smaller proteins compared with other reported phage baseplates, depicting the minimum requirements for building an effective cell-envelope-penetrating machine. We describe the tail sheath structure in the pre-infection post-infection states and its conformational changes during infection. In addition, we report, for the first time, the in situ structure of the phage neck region to near-atomic resolution. Based on these structures, we propose mechanisms of virus assembly and infection.


title: Proteome allocation and the evolution of metabolic cross-feeding

abstract: Metabolic cross-feeding (MCF) is a widespread type of ecological interaction where organisms share nutrients. In a common instance of MCF, an organism incompletely metabolizes sugars and releases metabolites that are used by another as a carbon source to produce energy. Why would the former waste edible food, and why does this preferentially occur at specific locations in the sugar metabolic pathway (acetate and glycerol are preferentially exchanged) have challenged evolutionary theory for decades. After showing that cells should in principle prioritise upstream reactions, we investigate how a special feature of these metabolites - their high diffusivity across the cell membrane - may trigger the emergence of cross feeding in a population. We explicitly model metabolic reactions, their enzyme-driven catalysis, and the cellular constraints on the proteome that may incur a cost to expressing all enzymes along the metabolic pathway. We find that up to high permeability coefficients of an intermediate metabolite, the expected evolutionary outcome is not a diversification that resembles cross-feeding but a single genotype that instead overexpresses the enzymes downstream the metabolite to limit its diffusion. Only at very high permeabilities and under restricted sets of parameters should the population diversify and MCF evolve.


title: Well-Lit: A programmable and customizable assistant for manual multi-well plate pipetting

abstract:
A very large number of biology and biochemistry laboratory protocols require transferring liquid aliquots from individual containers into individual wells of a multi-well plate, from plates to individual containers, or from one plate to another. Doing this by hand without errors, such as skipping wells, placing two samples in the same well, or swapping sample locations, especially when using plates with 96 wells or more, is difficult and requires enormous operator focus and/or a tedious manual error checking system. We present here a device built to facilitate error-free pipetting of samples from individual barcoded tubes to a multi-well plate or between multi-well plates (both 96 and 384 wells are supported). The device is programmable, modular and easily customizable to accommodate plates with different form-factors, and different protocols. The main components are only a 12.3" touch screen, a small form-factor PC, and a barcode scanner, combined with custom-made parts can be easily fabricated with a laser cutter and a hobby-grade 3D printer. The total cost is between approximately US$550 and US$600, depending on the configuration.

Specifications table

O_TBL View this table:
org.highwire.dtl.DTLVardef@8554f4org.highwire.dtl.DTLVardef@18c8878org.highwire.dtl.DTLVardef@153b050org.highwire.dtl.DTLVardef@15c7de6org.highwire.dtl.DTLVardef@14daeb7_HPS_FORMAT_FIGEXP  M_TBL C_TBL


title: Unexpected arabinosylation after humanization of plant protein N-glycosylation

abstract: As biopharmaceuticals, recombinant proteins have become indispensable tools in medicine. An increasing demand, not only in quantity but also in diversity, drives the constant development and improvement of production platforms. The N-glycosylation pattern on biopharmaceuticals plays an important role in activity, serum half-life and immunogenicity. Therefore, production platforms with tailored protein N-glycosylation are of great interest. Plant-based systems have already demonstrated their potential to produce pharmaceutically relevant recombinant proteins, although their N-glycan patterns differ from those in humans. Plants have shown great plasticity towards the manipulation of their glycosylation machinery, and some have already been glyco-engineered in order to avoid the attachment of plant-typical, putatively immunogenic sugar residues. This resulted in complex-type N-glycans with a core structure identical to the human one. Compared to humans, plants lack the ability to elongate these N-glycans with {beta}1,4-linked galactoses and terminal sialic acids. However, these modifications, which require the activity of several mammalian enzymes, have already been achieved for Nicotiana benthamiana and the moss Physcomitrella. Here, we present the first step towards sialylation of recombinant glycoproteins in Physcomitrella, human {beta}1,4-linked terminal N-glycan galactosylation, which was achieved by the introduction of a chimeric {beta}1,4-galactosyltransferase (FTGT). This chimeric enzyme consists of the moss 1,4-fucosyltransferase transmembrane domain, fused to the catalytic domain of the human {beta}1,4-galactosyltransferase. Stable FTGT expression led to the desired {beta}1,4-galactosylation. However, additional pentoses of unknown identity were also observed. The nature of these pentoses was subsequently determined by Western blot and enzymatic digestion followed by mass spectrometric analysis and resulted in their identification as -linked arabinoses. Since a pentosylation of {beta}1,4-galactosylated N-glycans was reported earlier, e.g. on recombinant human erythropoietin produced in glyco-engineered Nicotiana tabacum, this phenomenon is of a more general importance for plant-based production platforms. Arabinoses, which are absent in humans, may prevent the full humanization of plant-derived products. Therefore, the identification of these pentoses as arabinoses is important as it creates the basis for their abolishment to ensure the production of safe biopharmaceuticals in plant-based systems.


title: Environmental carcinogens disproportionally mutate genes implicated in neurodevelopmental disorders

abstract: De novo mutations contribute to a large proportion of sporadic psychiatric and developmental disorders, yet the potential role of environmental carcinogens as drivers of causal de novo mutations in neurodevelopmental disorders is poorly studied. We demonstrate that several mutagens, including polycyclic aromatic hydrocarbons (PAHs), disproportionately mutate genes related to neurodevelopmental disorders including autism spectrum disorders (ASD), schizophrenia, and attention deficit hyperactivity disorder (ADHD). Other disease genes including amyotrophic lateral sclerosis (ALS), Alzheimers disease, congenital heart disease, orofacial clefts, and coronary artery disease were generally not mutated more than expected. Our findings support a new paradigm of neurodevelopmental disease etiology driven by a contribution of environmentally induced rather than random mutations.


title: A deep learning approach to real-time HIV outbreak detection using genetic data

abstract:
Pathogen genomic sequence data are increasingly made available for epidemiological monitoring. A main interest is to identify and assess the potential of infectious disease outbreaks. While popular methods to analyze sequence data often involve phylogenetic tree inference, they are vulnerable to errors from recombination and impose a high computational cost, making it difficult to obtain real-time results when the number of sequences is in or above the thousands.

Here, we propose an alternative strategy to outbreak detection using genomic data based on deep learning methods developed for image classification. The key idea is to use a pairwise genetic distance matrix calculated from viral sequences as an image, and develop convolutional neutral network (CNN) models to classify areas of the images that show signatures of active outbreak, leading to identification of subsets of sequences taken from an active outbreak. We showed that our method is efficient in finding HIV-1 outbreaks with R0[≥] 3, and overall a specificity exceeding 85% and sensitivity better than 70%. We validated our approach using data from HIV-1 CRF01 in Europe, containing both endemic sequences and a well-known dual outbreak in intravenous drug users. Our model accurately identified known outbreak sequences in the background of slower spreading HIV. Importantly, we detected both outbreaks early on, before they were over, implying that had this method been applied in real-time as data became available, one would have been able to intervene and possibly prevent the extent of these outbreaks. This approach is scalable to processing hundreds of thousands of sequences, making it useful for current and future real-time epidemiological investigations, including public health monitoring using large databases and especially for rapid outbreak identification.

Author summaryThe analysis of pathogen genomic data to analyze epidemics at scale is constrained by the computational cost associated with phylogenetic tree reconstruction. As a fast and efficient alternative, we employed convolutional neural networks to analyze evolutionary pairwise distance matrices as images to perform classifications of the current epidemiological situation of a growing public health sequence database. We used simulated data to train and test our model, and as validation we accurately mapped the start and end of two linked well-documented HIV-1 outbreaks in the backdrop of ongoing slower HIV spread. Thus, our new approach is efficient, accurate, scalable, and can analyze data in real time.


title: isoformant: A visual toolkit for reference-free long-read isoform analysis at single-read resolution

abstract: isoformant is an analytical toolkit for isoform characterization of Oxford Nanopore Technologies (ONT) long-transcript sequencing data (i.e. direct RNA and cDNA). Deployment of these tools using Jupyter Notebook enables interactive analysis of user-defined region-of-interest (ROI), typically a gene. The core module of isoformant clusters sequencing reads by k-mer density to generate isoform consensus sequences without the requirement for a reference genome or prior annotations. The inclusion of differential isoform usage hypothesis testing based on read distribution among clusters enables comparison across multiple samples. Here, as proof-of-principle, we demonstrate the utility of isoformant for analyzing isoform diversity of commercially-available isoform standard mixtures. isoformant is available here: https://github.com/danledinh/isoformant.


title: A Single Molecule Investigation of I-Motif: Stability, Folding Kinetics, and Potential as an In-situ pH Sensor

abstract: We present a collection of single molecule work on the i-motif structure formed by the human telomeric sequence. Even though it was largely ignored in earlier years of its discovery due to its modest stability and requirement for physiologically low pH levels (pH<6.5), the i-motif has been attracting more attention recently as both a physiologically relevant structure and as a potent pH sensor. In this manuscript, we establish single molecule Forster resonance energy transfer (smFRET) as a tool to study the i-motif over a broad pH and ionic conditions. We demonstrate pH and salt dependence of i-motif formation under steady state conditions and illustrate the kinetics of i-motif folding in real time at the single molecule level. We also show the prominence of intermediate folding states and reversible folding/unfolding transitions. We present an example of using the i-motif as an in-situ pH sensor and use this sensor establish the time scale for the pH drop in a commonly used oxygen scavenging system.


title: MicroRNA-like snoRNA-derived RNAs (sdRNAs) promote castration resistant prostate cancer

abstract: We have identified 38 specifically excised, differentially expressed snoRNA fragments (sdRNAs) in TCGA prostate cancer (PCa) patient samples as compared to normal prostate controls. SnoRNA-derived fragments sdRNA-D19b and -A24 emerged among the most differentially expressed and were selected for further experimentation. We found that overexpression of either sdRNA significantly increased PC3 (a well-established model of castration-resistant prostate cancer (CRPC)) cell proliferation, and that sdRNA-D19b overexpression also markedly increased the rate of PC3 cell migration. In addition, both sdRNAs provided drug-specific resistances with sdRNA-D19b levels correlating with paclitaxel resistance and sdRNA-24A conferring dasatinib resistance. In silico and in vitro analyses revealed that two established PCa tumor suppressor genes, CD44 and CDK12, represent targets for sdRNA-D19b and sdRNA-A24 respectively. This outlines a biologically coherent mechanism by which sdRNAs downregulate tumor suppressors in AR-PCa to enhance proliferative and metastatic capabilities and to encourage chemotherapeutic resistance. Aggressive proliferation, rampant metastasis, and recalcitrance to chemotherapy are core characteristics of CRPC that synergize to produce a pathology that ranks 2nd in cancer-related deaths for men. This study defines sdRNA-D19b and -A24 as contributors to AR-PCa potentially providing novel biomarkers and therapeutic targets of use in PCa clinical intervention.


title: Efficient Design Of Peptide-Binding Polymers Using Active Learning Approaches

abstract: Active learning (AL) has become a subject of active recent research both in industry and academia as an efficient approach for rapid design and discovery of novel chemicals, materials, and polymers. The key advantages of this approach relate to its ability to (i) employ relatively small datasets for model development, (ii) iterate between model development and model assessment using small external datasets that can be either generated in focused experimental studies or formed from subsets of the initial training data, and (iii) progressively evolve models toward increasingly more reliable predictions and the identification of novel chemicals with the desired properties. Herein, we first compared various AL protocols for their effectiveness in finding biologically active molecules using synthetic datasets. We have investigated the dependency of AL performance on the size of the initial training set, the relative complexity of the task, and the choice of the initial training dataset. We found that AL techniques as applied to regression modeling offer no benefits over random search, while AL used for classification tasks performs better than models built for randomly selected training sets but still quite far from perfect. Using the best performing AL protocol, we have assessed the applicability of AL for the discovery of polymeric micelle formulations for poorly soluble drugs. Finally, the best performing AL approach was employed to discover and experimentally validate novel binding polymers for a case study of asialoglycoprotein receptor (ASGPR).

category: pharmacology and toxicology


title: Hepatocyte-specific activity of TSC22D4 triggers progressive NAFLD by impairing mitochondrial function

abstract:
ObjectiveFibrotic organ responses have recently been identified as long-term complication in diabetes. Indeed, insulin resistance and aberrant hepatic lipid accumulation represent driving features of progressive non-alcoholic fatty liver disease (NAFLD), ranging from simple steatosis and non-alcoholic steatohepatitis (NASH) to fibrosis. Effective pharmacological regimens to stop progressive liver disease are still lacking to-date.

MethodsBased on our previous discovery of transforming growth factor beta-like stimulated clone (TSC)22D4 as a key driver of insulin resistance and glucose intolerance in obesity and type 2 diabetes, we generated a TSC22D4-hepatocyte specific knockout line (TSC22D4-HepaKO) and exposed mice to control or NASH diet models. Mechanistic insights were generated by metabolic phenotyping and single cell liver sequencing.

ResultsHepatic TSC22D4 expression was significantly correlated with markers of liver disease progression and fibrosis in both murine and human livers. Indeed, hepatic TSC22D4 levels were elevated in human NASH patients as well as in several murine NASH models. Specific genetic deletion of TSC22D4 in hepatocytes led to reduced liver lipid accumulation, improvements in steatosis and inflammation scores and decreased apoptosis in mice. Single cell RNA sequencing revealed a distinct gene signature identifying an upregulation of mitochondrial-related processes. An enrichment of genes involved in the TCA cycle, mitochondrial organization, and triglyceride metabolism underscored the hepatocyte-protective phenotype and overall decreased liver damage as seen in mouse models.

ConclusionsTogether, our data uncover a new connection between targeted depletion of TSC22D4 and intrinsic metabolic processes in progressive liver disease. Cell-specific reduction of TSC22D4 improves hepatic steatosis, inflammation and promotes hepatocyte survival thus paving the way for further preclinical therapy developments.


title: Modeling spectral changes following stimulation: Is there an event-related shift in 1/f activity?

abstract: Neural activity contains oscillatory components (i.e., narrowband oscillations) and non-oscillatory components (e.g., event-related potentials [ERPs] and 1/f-like background activity). Here, surface-level EEG data was analyzed to investigate how the spectral content of neural activity below 25 Hz changes from before to after an event. We focused on changes in non-oscillatory background activity, a spectral component often assumed to be static across time in time-frequency analyses. During a simple auditory perception task (n = 46) and an auditory oddball task (n = 23), we found an apparent increase in the offset and a decrease in the slope of 1/f activity from before to after a tone. Importantly, however, these changes in non-oscillatory background activity were almost completely accounted for by the emergence of ERPs in response to the stimulus in frequencies below the alpha range (8-12 Hz). Our findings suggest that post-event spectral changes below 25 Hz can be modelled as the sum of pre-event non-oscillatory activity, the spectrum of the ERP, and an independent alpha component that is modulated in amplitude, but not elicited, by the event. Theta activity (4-8 Hz), however, was not present before the event and appeared to be phase-locked to it. The theoretical and methodological implications of our findings regarding the nature and origin of 1/f activity, and the interpretation of low-frequency activity in the time-frequency domain are discussed.


title: Electrobehavioral phenotype and seizure pharmacosensitivity in a novel mouse model of patient-derived SLC6A1 S295L mutation-associated neurodevelopmental epilepsy

abstract:
Solute carrier family 6 member 1 (SLC6A1) gene encodes GAT-1, a GABA transporter expressed on glia and presynaptic terminals of inhibitory neurons. Mutations in SLC6A1 are associated with myoclonic atonic epilepsy, absence epilepsy, autism, and intellectual disability. However, the mechanisms leading to these defects are unknown. Here, we used a novel mouse model harboring a point mutation (S295L) recently identified in the human SLC6A1 gene that results in impaired membrane trafficking of the GAT-1 protein. We performed chronic wireless telemetry recordings of heterozygous (GAT-1S295L/+) mice, and of mice lacking one or both copies of the gene (GAT-1+/- and GAT-1-/-). We assessed their behaviors and pharmacosensitivity, and investigated the relationship between seizure burden and behavioral performance. GAT-1S295L/+ mice exhibited frequent spikewave discharges (SWDs) associated with behavioral arrest, and there was a dose-effect relationship between GAT-1 gene copy number and the severity of electrocorticogram (ECoG) abnormalities. Seizure burden was inversely correlated with behavioral performance. Forelimb grip strength was reduced in female mice. Acute administration of GAT-1 antagonist NO-711 induced SWDs in wildtype mice, exacerbated the phenotype in GAT-1S295L/+ and GAT-1+/- mice, and had no effect on GAT-1-/- mice lacking the drug target. By contrast, ethosuximide normalized the ECoG in GAT-1S295L/+ and GAT-1+/- mice. In conclusion, GAT-1S295L/+ mice show haploinsufficiency with evidence of GAT-1 hypofunction. This mouse model reconstitutes major aspects of human disease and thus provides a useful preclinical model for drug screening and gene therapy.

Significance StatementThe SLC6A1 gene encodes for GAT-1, a major GABA transporter. Mutations in SLC6A1 lead to a spectrum of neurodevelopmental disorders and epilepsies collectively referred to as SLC6A1-related disorders (SRD). A critical contributor to disability in SRD patients is the burden of seizures and their sequelae. There is an urgent need to understand the mechanisms of SRD as they will inform therapeutic interventions. Mouse models can provide critical information allowing both the assessment of candidate therapies and the design of next generations therapies. Here we used behavioral assessments and wireless electrophysiology in a new mouse model of SRD to understand the disease pathogenesis and the association between seizure burden and behavioral deficits.


title: Mesoscale cortex-wide neural dynamics predict self-initiated actions in mice several seconds prior to movement

abstract: Volition - the sense of control or agency over ones voluntary actions - is widely recognized as the basis of both human subjective experience and natural behavior in non-human animals. To date, several human studies have found peaks in neural activity preceding voluntary actions, e.g. the readiness potential (RP), and some have shown upcoming actions could be decoded even before awareness. While these findings may pose a challenge to traditional accounts of human volition, some have proposed that random processes underlie and explain pre-movement neural activity. Here we seek to address part of this controversy by evaluating whether pre-movement neural activity in mice contains structure beyond that present in random neural activity. Implementing a self-initiated water-rewarded lever pull paradigm in mice while recording widefield [Ca++] neural activity we find that cortical activity changes in variance seconds prior to movement and that upcoming lever pulls or spontaneous body movements could be predicted between 1 second to more than 10 seconds prior to movement, similar to but even earlier than in human studies. We show that mice, like humans, are biased towards initiation of voluntary actions during specific phases of neural activity oscillations but that the pre-movement neural code in mice changes over time and is widely distributed as behavior prediction improved when using all vs single cortical areas. These findings support the presence of structured multi-second neural dynamics preceding voluntary action beyond that expected from random processes. Our results also suggest that neural mechanisms underlying self-initiated voluntary action could be preserved between mice and humans.


title: Sensory cortical dynamics during optical microstimulation training

abstract: Primary sensory cortex is a key locus of plasticity during learning. Exposure to novel stimuli often alters cortical activity, but isolating cortex-specific dynamics is challenging due to extensive pre-cortical processing. Here, we employ optical microstimulation of pyramidal neurons in layer (L) 2/3 of mouse primary vibrissal somatosensory cortex (vS1) to study cortical dynamics as mice learn to discriminate microstimulation intensity. Tracking activity over weeks using two-photon calcium imaging, we observe a rapid sparsification of the photoresponsive population, with the most responsive neurons exhibiting the largest declines in responsiveness. Following sparsification, the photoresponsive population attains a stable rate of neuronal turnover. At the same time, the photoresponsive population increasingly overlaps with populations encoding whisker movement and touch. Finally, we find that mice with larger declines in responsiveness learn the task more slowly than mice with smaller declines. Our results reveal that microstimulation-evoked cortical activity undergoes extensive reorganization during task learning and that the dynamics of this reorganization impact perception.


title: Targeting macrophages with CAR-T cells delays solid tumor progression and enhances anti-tumor immunity

abstract: Tumor-associated macrophages (TAMs) are one of the most abundant cell types in many solid tumors and typically exert protumor effects. This has led to an interest in macrophage-depleting agents for cancer therapy, but approaches developed to date have had limited success in clinical trials. Here, we report the development of a strategy for TAM depletion in mouse solid tumor models using chimeric antigen receptor (CAR) T cells targeting the macrophage marker F4/80 (F4.CAR-T). F4.CAR-T cells effectively killed macrophages in vitro and in vivo without toxicity. When injected into mice bearing orthotopic lung tumors, F4.CAR-T cells infiltrated tumor lesions and delayed tumor growth comparably to PD1 blockade, and significantly extended mouse survival. Anti-tumor effects were mediated by F4.CAR-T-produced IFN-{gamma}, which promoted upregulation of MHC molecules on cancer cells and tumor-infiltrating myeloid cells. Notably, F4.CAR-T promoted expansion of endogenous CD8 T cells specific for tumor-associated antigens and led to immune editing of highly antigenic tumor cell clones. Antitumor impact was also observed in mouse models of ovarian and pancreatic cancer. These studies provide proof-of-principle evidence to support CAR-T targeting of TAMs as a means to enhance antitumor immunity.


title: ILC homeostasis phenotyping in various tissues, aging, and sex differences

abstract: Innate lymphoid cells are the innate counterpart to CD4+ T cells that are mediated by the same transcription factors and produce similar cytokines. ILCs are being investigated in many different disease states, but the field current lacks foundational information on ILC representation whether it be in tissues, between males and females, or in aging as these are all vital components in disease etiology and severity. Our descriptive study used flow cytometry to characterize ILCs compared to the entire CD45+ (e.g., lymphocyte) and lineage negative (e.g., ILC) compartments to understand their homeostatic balance and plasticity. Moreover, we defined ILC2 expression and subsets based on their cytokine production and created several mathematical models to elucidate the correlation of extra- and intra-cellular ILC2 markers from least to most complex. ILC studies would benefit from more unbiased, holistic experiments including RNA-seq and mass spectroscopy to further define ILCs in steady state before adding more complex pathways like different disease states to enhance translational value and therapeutic targeting of these cells.


title: Recall of B cell memory depends on relative locations of prime and boost

abstract:
Re-entry of memory B cells to recall germinal centers (GCs) is essential for updating their B-cell antigen receptors (BCRs). Using single B-cell culture and fate-mapping, we have characterized BCR repertoires in recall GCs following boost immunizations at sites local or distal to the priming. Local boosts with homologous antigen recruit to recall GCs progeny of primary GC B cells more efficiently than do distal boosts. Recall GCs following local boosts contain significantly more B cells with elevated levels of Ig mutations and higher avidity BCRs. This local preference is unaffected by blockade of CD40:CD154 interaction that terminate active, primary GC responses. Local boosts with heterologous antigens elicit secondary GCs with B-cell populations enriched for cross-reactivity to the priming and boosting antigens; in contrast, cross-reactive GC B cells are rare following distal boosts. Our findings indicate the importance of locality in humoral immunity and inform serial vaccination strategies for evolving viruses.

One Sentence SummaryThe participation of memory B cells in recall germinal centers depends on whether the boost is local or distal to the priming site.


title: Hilltopping influences spatial dynamics in a patchy population of tiger moths

abstract: Dispersal is a key driver of spatial population dynamics. Dispersal behavior may be shaped by many factors, such as mate-finding, the spatial distribution of resources, or wind and currents, yet most models of spatial dynamics assume random dispersal. We examined the spatial dynamics of a day-flying moth species (Arctia virginalis) that forms mating aggregations on hilltops ( hilltopping) based on long-term adult and larval population censuses. Using time-series models, we compared spatial population dynamics resulting from empirically-founded hilltop-based connectivity indices, and modeled the interactive effects of temperature, precipitation, and density dependence. Model comparisons supported hilltop-based connectivity metrics over random connectivity, suggesting an effect of hilltopping behavior on dynamics. We also found strong interactive effects of temperature and precipitation on dynamics. Simulations based on fitted time series models showed lower patch occupancy and regional synchrony, and higher colonization and extinction rates when hilltopping was included, with potential implications for the probability of persistence of the patch network. Overall, our results show the potential for dispersal behavior to have important effects on spatial population dynamics and persistence, and we advocate inclusion of such non-random dispersal in metapopulation models.