title: The Gulf Coast tick, Amblyomma maculatum (Ixodida: Ixodidae) and spotted fever group Rickettsia in the highly urbanized northeastern US

abstract: We report the multi-year collection of the Gulf Coast tick, Amblyomma maculatum Koch (Acaridae: Ixodida: Ixodidae) in Staten Island, New York City (NYC) as well as their detection in Brooklyn, NYC, and in Atlantic and Cumberland counties in southern NJ, USA. The first detections on all sites were of adults but in Freshkills Park on Staten Island larvae were collected in a following year. Based on known observations on birds of this tick species, it is likely A. maculatum are expanding north on migratory birds, which are now often seen in Freshkills Park. The presence of larvae indicates that adults are being successful at finding hosts in Staten Island. We describe the landscape features of the area in Staten Island where populations were highest and larvae were detected, which could have facilitated the establishment of A. maculatum. Notably, we also report the presence of human pathogens Rickettsia parkeri in 5/10 (50%) of adults tested and R. felis in 1/24 (4.17%) of larvae tested. In addition to established populations in Staten Island we found evidence of A. maculatum in NJ and other NYC boroughs, suggesting current or future establishment is possible. The failure thus far to detect established populations in these areas may be due to inherent difficulties in detecting low density, spatially heterogeneous incipient populations, which could require targeted surveillance efforts for this species. We discuss the consequences to public health of the establishment of A. maculatum and detection of two additional rickettsial pathogens in the densely populated Northeastern US.


title: Prior selfing occur in larger flowers in a mixed mating species with delayed selfing

abstract: Theory predicts that prior self-pollination (prior selfing) should not evolve in mixed mating species that enable delayed selfing. In this study, we test the hypotheais that prior selfing has evolved under severe pollinator limitation in the mixed mating species Commelina communis which can reproduce via delayed selfing. The hypothesis predicts that prior selfing occurs more frequently in populations with very low pollinator availability and/or in smaller flowers which receive infrequent visitations. We tested the predictions by comparing the degree of prior selfing among ten populations experiencing various levels of pollinator limitation and by examining a relationship between individual flower size and the occurrence of prior selfing. Populations with higher pollinator availability had higher prior selfing rate. Moreover, prior selfing occurs more frequently in larger flowers. These findings were totally opposite patterns of the predictions and the previous findings. We proposed new hypotheses that prior selfing has been maintained by the presence of reproductive interference from the congener and/or propotency in C. communis to explain our unexpected findings. We should verify potential effects of reproductive interference and propotency in future to elucidate the mystery of prior selfing in this mixed mating species with delayed selfing.

category: ecology


title: Lessons on fruiting body morphogenesis from genomes and transcriptomes of Agaricomycetes

abstract: Fruiting bodies of mushroom-forming fungi (Agaricomycetes) are among the most complex structures produced by fungi. Unlike vegetative hyphae, fruiting bodies grow determinately and follow a genetically encoded developmental program that orchestrates tissue differentiation, growth and sexual sporulation. In spite of more than a century of research, our understanding of the molecular details of fruiting body morphogenesis is limited and a general synthesis on the genetics of this complex process is lacking. In this paper, we aim to comprehensively identify conserved genes related to fruiting body morphogenesis and distill novel functional hypotheses for functionally poorly characterized genes. As a result of this analysis, we report 921 conserved developmentally expressed gene families, only a few dozens of which have previously been reported in fruiting body development. Based on literature data, conserved expression patterns and functional annotations, we provide informed hypotheses on the potential role of these gene families in fruiting body development, yielding the most complete description of molecular processes in fruiting body morphogenesis to date. We discuss genes related to the initiation of fruiting, differentiation, growth, cell surface and cell wall, defense, transcriptional regulation as well as signal transduction. Based on these data we derive a general model of fruiting body development, which includes an early, proliferative phase that is mostly concerned with laying out the mushroom body plan (via cell division and differentiation), and a second phase of growth via cell expansion as well as meiotic events and sporulation. Altogether, our discussions cover 1480 genes of Coprinopsis cinerea, and their orthologs in Agaricus bisporus, Cyclocybe aegerita, Armillaria ostoyae, Auriculariopsis ampla, Laccaria bicolor, Lentinula edodes, Lentinus tigrinus, Mycena kentingensis, Phanerochaete chrysosporium, Pleurotus ostreatus, and Schizophyllum commune, providing functional hypotheses for [~]10% of genes in the genomes of these species. Although experimental evidence for the role of these genes will need to be established in the future, our data provide a roadmap for guiding functional analyses of fruiting related genes in the Agaricomycetes. We anticipate that the gene compendium presented here, combined with developments in functional genomics approaches will contribute to uncovering the genetic bases of one of the most spectacular multicellular developmental processes in fungi.

category: evolutionary biology


title: The price of defence: toxins, visual signals and oxidative state in an aposematic butterfly

abstract: In a variety of aposematic species, the conspicuousness of an individuals warning signal and the quantity of its chemical defence are positively correlated. This apparent honest signalling in aposematism is predicted by resource competition models which assume that the production and maintenance of aposematic defences compete for access to antioxidant molecules that have dual functions as pigments directly responsible for colouration and in protecting against oxidative lipid damage. Here we study a model aposematic system--the monarch butterfly (Danaus plexippus) and make use of the variable phytochemistry of its larval host-plant, milkweeds (Asclepiadaceae), to manipulate the concentration of sequestered cardenolides. We test two fundamental assumptions of resource competition models: that (1) the possession of secondary defences is associated with costs in the form of oxidative lipid damage and reduced antioxidant defences; and (2) that oxidative damage or decreases in antioxidant defences can reduce the capacity of individuals to produce aposematic displays. Monarch caterpillars that sequestered the highest concentrations of cardenolides exhibited higher levels of oxidative lipid damage as adults. The relationship between warning signals, cardenolide concentrations and oxidative damage differed between the sexes. In male monarchs conspicuousness was explained by an interaction between oxidative damage and sequestration: as males sequester more cardenolides, those with high levels of oxidative damage become less conspicuous, while those that sequester lower levels of cardenolides equally invest in conspicuous with increasing oxidative damage. There was no significant effect of oxidative damage or concentration of sequestered cardenolides on female conspicuousness. Our results demonstrate physiological linkage between the production of coloration and protection from autotoxicity, that warning signals can be honest indicators of defensive capability, and that the relationships are different between the sexes.


title: Polynomial Mendelian Randomization reveals widespread non-linear causal effects in the UK Biobank

abstract: Causal inference is a critical step in improving our understanding of biological processes and Mendelian randomisation (MR) has emerged as one of the foremost methods to efficiently interrogate diverse hypotheses using large-scale, observational data from biobanks. Although many extensions have been developed to address the three core assumptions of MR-based causal inference (relevance, exclusion restriction, and exchangeability), most approaches implicitly assume that any putative causal effect is linear. Here we propose PolyMR, an MR-based method which provides a polynomial approximation of an (arbitrary) causal function between an exposure and an outcome. We show that this method provides accurate inference of the shape and magnitude of causal functions with greater accuracy than existing methods. We applied this method to data from the UK Biobank, testing for effects between anthropometric traits and continuous health-related phenotypes and found most of these (84%) to have causal effects which deviate significantly from linear. These deviations ranged from slight attenuation at the extremes of the exposure distribution, to large changes in the magnitude of the effect across the range of the exposure (e.g. a 1 kg/m2 change in BMI having stronger effects on glucose levels if the initial BMI was higher), to non-monotonic causal relationships (e.g. the effects of BMI on cholesterol forming an inverted U shape). Finally, we show that the linearity assumption of the causal effect may lead to the misinterpretation of health risks at the individual level or heterogeneous effect estimates when using cohorts with differing average exposure levels.

category: genetics


title: Deciphering the genetic links between NAFLD and co-occurring conditions using a liver gene regulatory network

abstract:
Background & AimsNon-alcoholic fatty liver disease (NAFLD) is a multi-system metabolic disease that co-occurs with various hepatic and extra-hepatic diseases. The phenotypic manifestation of NAFLD is primarily observed in the liver. Therefore, identifying liver-specific gene regulatory interactions between variants associated with NAFLD and multimorbid conditions may help to improve our understanding of underlying shared aetiology.

MethodsHere, we constructed a liver-specific gene regulatory network (LGRN) consisting of genome-wide spatially constrained expression quantitative trait loci (eQTLs) and their target genes. The LGRN was used to identify regulatory interactions involving NAFLD-associated genetic modifiers and their inter-relationships to other complex traits.

Results and ConclusionsWe demonstrate that MBOAT7 and IL32, which are associated with NAFLD progression, are regulated by spatially constrained eQTLs that are enriched for an association with liver enzyme levels. MBOAT7 transcript levels are also linked to eQTLs associated with cirrhosis, and other traits that commonly co-occur with NAFLD. In addition, genes that encode interacting partners of NAFLD-candidate genes within the liver-specific protein-protein interaction network were affected by eQTLs enriched for phenotypes relevant to NAFLD (e.g. IgG glycosylation patterns, OSA). Furthermore, we identified distinct gene regulatory networks formed by the NAFLD-associated eQTLs in normal versus diseased liver, consistent with the context-specificity of the eQTLs effects. Interestingly, genes targeted by NAFLD-associated eQTLs within the LGRN were also affected by eQTLs associated with NAFLD-related traits (e.g. obesity and body fat percentage). Overall, the genetic links identified between these traits expand our understanding of shared regulatory mechanisms underlying NAFLD multimorbidities.


title: Plasticity across levels: relating epigenomic, transcriptomic, and phenotypic responses to osmotic stress in a halotolerant microalga

abstract: Phenotypic plasticity, the ability of a given genotype to produce alternative phenotypes in response to its environment of development, is an important mechanism for coping with variable environments. While the mechanisms underlying phenotypic plasticity are diverse, their relative contributions need to be investigated quantitatively to better understand the evolvability of plasticity across biological levels. This requires relating plastic responses of the epigenome, transcriptome, and organismal phenotype, and how they vary with the genotype. Here we carried out this approach for responses to osmotic stress in Dunaliella salina, a green microalga that is a model organism for salinity tolerance. We compared two strains that show markedly different demographic responses to osmotic stress, and showed that these phenotypic responses involve strain- and environment-specific variation in gene expression levels, but a relative low - but significant - effect of strain x environment interaction. We also found an important genotype effect on the genome-wide methylation pattern, but little contribution from environmental conditions to the latter. However, we did detect a significant marginal effect of epigenetic variation on gene expression, beyond the influence of genetic differences on epigenetic state, and we showed that hypomethylated regions are correlated with higher gene expression. Our results indicate that epigenetic mechanisms are either not involved in the rapid plastic response to environmental change in this species, or involve only few changes in trans that are sufficient to trigger concerted changes in the expression of many genes, and phenotypic responses by multiple traits.

category: genomics


title: Temporal response characterization across individual multiomics profiles of prediabetic and diabetic subjects

abstract: Longitudinal deep multi-omics profiling, which combines biomolecular, physiological, environmental and clinical measures data, shows great promise for precision health. However, integrating and understanding the complexity of such data remains a big challenge. Here we propose a bottom-up framework starting from assessing single individuals multi-omics time series, and using individual responses to assess multi-individual grouping based directly on similarity of their longitudinal deep multi-omics profiles. We applied our method to individual profiles from a study profiling longitudinal responses in type 2 diabetes mellitus. After generating periodograms for individual subject omics signals, we constructed within-person omics networks and analyzed personal-level immune changes. The results showed that our method identified both individual-level responses to immune perturbation, and the clusters of individuals that have similar behaviors in immune response and which was associated to measures of their diabetic status.


title: Ancient DNA from a 2,700-year-old goitered gazelle (Gazella subgutturosa) confirms gazelle hunting in Iron Age Central Asia

abstract: Central Asia has been an important region connecting the different parts of Eurasia throughout history and prehistory, with large states developing in this region during the Iron Age. Archaeogenomics is a powerful addition to the zooarchaeological toolkit for understanding the relation of these societies to animals. Here, we present the genetic identification of a goitered gazelle specimen (Gazella subgutturosa) at the site Gazimulla-Tepa, in modern-day Uzbekistan, confirming hunting of the species in the region during the Iron Age. The sample was directly radiocarbon dated to 2724-2439 calBP. A phylogenetic analysis of the mitochondrial genome places the individual into the modern variation of G. subgutturosa. Our data does represent both the first ancient DNA and the first nuclear DNA sequences of this species. The lack of genomic resources available for this gazelle and related species prevented us from performing a more in-depth analysis of the nuclear sequences generated. Therefore, we are making our sequence data available to the research community to facilitate other research of this nowadays threatened species which has been subject to human hunting for several millennia across its entire range on the Asian continent.


title: Accurate measurement of microsatellite length by disrupting its tandem repeat structure

abstract: Replication of tandem repeats of simple sequence motifs, also known as microsatellites, is error prone and variable lengths frequently occur during population expansions. Therefore, microsatellite length variations could serve as markers for cancer. However, accurate error-free quantitation of microsatellite lengths is difficult with current methods because of a high error rate during amplification and sequencing. We have solved this problem by using partial mutagenesis to disrupt enough of the repeat structure so that it can replicate faithfully, yet not so much that the flanking regions cannot be reliably identified. In this work we use bisulfite mutagenesis to convert a C to a U, later read as T. Compared to untreated templates, we achieve three orders of magnitude reduction in the error rate per round of replication. By requiring two independent first copies of an initial template, we reach error rates below one in a million. We discuss potential clinical applications of this method.


title: A mathematical model for cell-induced gel contraction incorporating osmotic effects

abstract: Biological tissues are composed of cells surrounded by the extracellular matrix (ECM). The ECM can be thought of as a fibrous polymer network, acting as a natural scaffolding to provide mechanical support to the cells. Reciprocal mechanical and chemical interactions between the cells and the ECM are crucial in regulating the development of tissues and maintaining their functionality. Hence, to maintain in vivo-like behaviour when cells are cultured in vitro, they are often seeded in a gel, which aims to mimic the ECM. In this paper, we present a mathematical model that incorporate cell-gel interactions together with osmotic pressure to study the mechanical behaviour of biological gels. In particular, we consider an experiment where cells are seeded within a gel, which gradually compacts due to forces exerted on it by the cells. Adopting a one-dimensional Cartesian geometry for simplicity, we use a combination of analytical techniques and numerical simulations to investigate how cell traction forces interact with osmotic effects (which can lead to either gel swelling or contraction depending on the gels composition). Our results show that a number of qualitatively different behaviours are possible, depending on the composition of the gel (i.e. the chemical potentials) and the strength of the cell traction forces. We observe an unusual case where the gel oscillates between swelling and contraction. We also consider on how physical parameters like drag and viscosity affect the manner in which the gel evolves.

category: bioengineering


title: Label-free imaging to track reprogramming of human somatic cells

abstract: The process of reprogramming patient samples to human induced pluripotent stem cells (iPSCs) is stochastic, asynchronous, and inefficient leading to a heterogeneous population of cells. Here, we track the reprogramming status of single patient-derived cells during reprogramming with label-free live-cell imaging of cellular metabolism and nuclear morphometry to identify high-quality iPSCs. Erythroid progenitor cells (EPCs) isolated from human peripheral blood showed distinct patterns of autofluorescence lifetime for the reduced form of nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] and flavin adenine dinucleotide (FAD) during reprogramming. Random forest models classified starting EPCs, partially-reprogrammed intermediate cells, and iPSCs with [~]95% accuracy. Reprogramming trajectories resolved at the single cell level indicated significant reprogramming heterogeneity along different branches of cell state. This combination of micropatterning, autofluorescence imaging, and machine learning provides a unique non-destructive method to assess the quality of iPSCs in real-time for various applications in regenerative medicine, cell therapy biomanufacturing, and disease modeling.


title: An engineered nanocomplex with Photodynamic and Photothermal synergistic properties for cancer treatment

abstract:
Photodynamic therapy (PDT) and photothermal therapy (PTT) are promising therapeutic methods for cancer treatment; however, as single modality therapies, either PDT or PTT are still limited in their success rate. A dual application of both PDT and PTT, in a combined protocol, has gained immense interest. In this study, gold nanoparticles (AuNPs) are conjugated with a PDT agent, meso-tetrahydroxyphenylchlorin (mTHPC) photosensitizer, designed as nanotherapeutic agents that can activate a dual photodynamic/photothermal therapy.

The AuNP-mTHPC complex is biocompatible, soluable, and photostable. PDT efficiency is high because of immediate reactive oxygen species (ROS) production upon mTHPC activation by the 650 nm laser which decreased mitochondrial membrane potential ({Delta}{Psi}m). Likewise, the AuNP-mTHPC complex is used as a photoabsorbing (PTA) agent for PTT, due to efficient plasmon absorption and excellent photothermal conversion characteristics of AuNPs under laser irradiation at 532 nm. Under the laser irradiation of a PDT/PTT combination, a twofold phototoxicity outcome follows, compared to PDT-only or PTT-only treatment. This indicates that PDT and PTT have synergistic effects together as a combined therapeutic method. Hence, applying our AuNP-mTHPC may be a potential treatment of cancer in the biomedical field.

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title: Evolution of tunnels in alpha/beta-hydrolase fold proteins - what can we learn from studying epoxide hydrolases?

abstract:
The evolutionary variability of a proteins residues is highly dependent on protein region and function. Solvent-exposed residues, excluding those at interaction interfaces, are more variable than buried residues whereas active site residues are considered to be conserved. The abovementioned rules apply also to /{beta}-hydrolase fold proteins - one of the oldest and the biggest superfamily of enzymes with buried active sites equipped with tunnels linking the reaction site with the exterior. We selected soluble epoxide hydrolases as representative of this family to conduct the first systematic study on the evolution of tunnels. We hypothesised that tunnels are lined by mostly conserved residues, and are equipped with a number of specific variable residues that are able to respond to evolutionary pressure. The hypothesis was confirmed, and we suggested a general and detailed way of the tunnels evolution analysis based on entropy values calculated for tunnels residues. We also found three different cases of entropy distribution among tunnel-lining residues. These observations can be applied for protein reengineering mimicking the natural evolution process. We propose a  perforation mechanism for new tunnels design via the merging of internal cavities or protein surface perforation. Based on the literature data, such a strategy of new tunnel design could significantly improve the enzymes performance and can be applied widely for enzymes with buried active sites.

Author SummarySo far very little is known about proteins tunnels evolution. The goal of this study is to evaluate the evolution of tunnels in the family of soluble epoxide hydrolases - representatives of numerous /{beta}-hydrolase fold enzymes. As a result two types of tunnels evolution analysis were proposed (a general and a detailed approach), as well as a  perforation mechanism which can mimic native evolution in proteins and can be used as an additional strategy for enzymes redesign.

category: bioinformatics


title: InterPepScore: A Deep Learning Score for Improving the FlexPepDock Refinement Protocol

abstract:
MotivationInteractions between peptide fragments and protein receptors are vital to cell function yet difficult to experimentally determine the structural details of. As such, many computational methods have been developed to aid in peptide-protein docking or structure prediction. One such method is Rosetta FlexPepDock which consistently refines coarse peptide-protein models into sub-[A]ngstrom precision using Monte-Carlo simulations and statistical potentials. Deep learning has recently seen increased use in protein structure prediction, with graph neural network seeing use in protein model quality assessment.

ResultsHere, we introduce a graph neural network, InterPepScore, as an additional scoring term to complement and improve the Rosetta FlexPepDock refinement protocol. InterPepScore is trained on simulation trajectories from FlexPepDock refinement starting from thousands of peptide-protein complexes generated by a wide variety of docking schemes. The addition of InterPepScore into the refinement protocol consistently improves the quality of models created, and on an independent benchmark on 109 peptide-protein complexes its inclusion results in an increase in the number of complexes for which the top-scoring model had a DockQ-score of 0.49 (Medium quality) or better from 14.8% to 26.1%.

AvailabilityInterPepScore is available online at http://wallnerlab.org/InterPepScore.


title: Efficient pre-processing of Single-cell ATAC-seq data

abstract:
The primary tool currently used to pre-process 10X Chromium single-cell ATAC-seq data is Cell Ranger, which can take very long to run on standard datasets. To facilitate rapid pre-processing that enables reproducible workflows, we present a suite of tools called scATAK for pre-processing single-cell ATAC-seq data that is 18 times faster than Cell Ranger on human samples, and that uses 33% less RAM when 8 CPU threads are used. Our tool can also calculate chromatin interaction potential matrices and generate open chromatin signals and interaction traces for cell groups. We demonstrate the utility of scATAK in an exploration of the chromatin regulatory landscape of a healthy adult human brain and show that it can reveal cell-type-specific features.

scATAK is available at https://github.com/pachterlab/scATAK/.


title: Improved Transcriptome Assembly Using a Hybrid of Long and Short Reads with StringTie

abstract: Short-read RNA sequencing and long-read RNA sequencing each have their strengths and weaknesses for transcriptome assembly. While short reads are highly accurate, they are unable to span multiple exons. Long-read technology can capture full-length transcripts, but its high error rate often leads to mis-identified splice sites, and its low throughput makes quantification difficult. Here we present a new release of StringTie that performs hybrid-read assembly. By taking advantage of the strengths of both long and short reads, hybrid-read assembly with StringTie is more accurate than long-read only or short-read only assembly, and on some datasets it can more than double the number of correctly assembled transcripts, while obtaining substantially higher precision than the long-read data assembly alone. Here we demonstrate the improved accuracy on simulated data and real data from Arabidopsis thaliana, Mus musculus, and human. We also show that hybrid-read assembly is more accurate than correcting long reads prior to assembly while also being substantially faster. StringTie is freely available as open source software at https://github.com/gpertea/stringtie.


title: DISA tool: discriminative and informative subspace assessment with categorical and numerical outcomes

abstract:
MotivationPattern discovery and subspace clustering play a central role in the biological domain, supporting for instance putative regulatory module discovery from omic data for both descriptive and predictive ends. In the presence of target variables (e.g. phenotypes), regulatory patterns should further satisfy delineate discriminative power properties, well-established in the presence of categorical outcomes, yet largely disregarded for numerical outcomes, such as risk profiles and quantitative phenotypes.

ResultsDISA (Discriminative and Informative Subspace Assessment), a Python software package, is proposed to assess patterns in the presence of numerical outcomes using well-established measures together with a novel principle able to statistically assess the correlation gain of the subspace against the overall space. Results confirm the possibility to soundly extend discriminative criteria towards numerical outcomes without the drawbacks well-associated with discretization procedures. A case study is provided to show the properties of the proposed method.

AvailabilityDISA is freely available at https://github.com/JupitersMight/DISA under the MIT license.

Contact{leonardoalexandre@tecnico.ulisboa.pt,rmch@tecnico.ulisboa.pt} and {rs.costa@fct.unl.pt}


title: DeepRank-GNN: A Graph Neural Network Framework to Learn Patterns in Protein-Protein Interfaces

abstract:
Gaining structural insights into the protein-protein interactome is essential to understand biological phenomena and extract knowledge for rational drug design or protein engineering. We have previously developed DeepRank, a deep-learning framework to facilitate pattern learning from protein-protein interfaces using Convolutional Neural Network (CNN) approaches. However, CNN is not rotation invariant and data augmentation is required to desensitize the network to the input data orientation which dramatically impairs the computation performance. Representing protein-protein complexes as atomic- or residue-scale rotation invariant graphs instead enables using graph neural networks (GNN) approaches, bypassing those limitations.

We have developed DeepRank-GNN, a framework that converts protein-protein interfaces from PDB 3D coordinates files into graphs that are further provided to a pre-defined or user-defined GNN architecture to learn problem-specific interaction patterns. DeepRank-GNN is designed to be highly modularizable, easily customized, and is wrapped into a user-friendly python3 package. Here, we showcase DeepRank-GNNs performance for scoring docking models using a dedicated graph interaction neural network (GINet). We show that this graph-based model performs better than DeepRank, DOVE and HADDOCK scores and competes with iScore on the CAPRI score set. We show a significant gain in speed and storage requirement using DeepRank-GNN as compared to DeepRank.

DeepRank-GNN is freely available from https://github.com/DeepRank/DeepRank-GNN.

Contacta.m.j.j.bonvin@uu.nl


title: The role of surface adhesion on the macroscopic wrinkling of biofilms

abstract: Biofilms, bacterial communities of cells encased by a self-produced matrix, exhibit a variety of three-dimensional structures. Specifically, channel networks formed within the bulk of the biofilm have been identified to play an important role in the colonies viability by promoting the transport of nutrients and chemicals. Here, we study channel formation and focus on the role of the adhesion of the biofilm matrix to the substrate in Pseudomonas aeruginosa biofilms grown under constant flow in microfluidic channels. We perform phase contrast and confocal laser scanning microscopy to examine the development of the biofilm structure as a function of the substrates surface energy. The formation of the wrinkles and folds is triggered by a mechanical buckling instability, controlled by biofilm growth rate and the films adhesion to the substrate. The three-dimensional folding gives rise to hollow channels that rapidly increase the overall volume occupied by the biofilm and facilitate bacterial movement inside them. The experiments and analysis on mechanical instabilities for the relevant case of a bacterial biofilm grown during flow enable us to predict and control the biofilm morphology.

category: biophysics


title: Ion Transport, Selectivity, and Electronic Polarization in Fluoride Channels

abstract:
Fluoride channels (Fluc) export toxic F- from the cytoplasm. Crystallography and mutagenesis have identified several conserved residues crucial for fluoride transport, but the permeation mechanism at the molecular level has remained elusive. Herein we have applied constant-pH molecular dynamics and free energy sampling methods to investigate fluoride permeation through a Fluc protein from Escherichia coli. We find that fluoride is facile to permeate in its charged form, i.e., F-, by traversing through a non-bonded network. The extraordinary F- selectivity is gained by the hydrogen-bonding capability of the central binding site and the Coulombic filter at the channel entrance. The F- permeation rate calculated using an electronically polarizable force field is significantly more accurate compared to the experimental value than that calculated using a more standard additive force field, suggesting an essential role for electronic polarization in the F- - Fluc interactions.

O_TEXTBOXSIGNIFICANCE

A comprehensive atomistic-level computational study is presented of the mechanism of fluoride permeation through fluoride channels. The mechanism of fluoride permeation of the F- anion is established and the microscopic determinants of F- selectivity revealed. The essential nature of electronic polarization during F- permeation is also demonstrated through the computational modeling.

C_TEXTBOX


title: Ion Permeation, Selectivity, and Electronic Polarization in Fluoride Channels


title: Lipid droplets 3D full measurement by holographic in-flow tomography

abstract: The most recent discoveries in the biochemical field are highlighting the increasingly important role of lipid droplets (LDs) in several regulatory mechanisms in living cells. LDs are dynamic organelles and therefore their complete characterization in terms of number, size, spatial positioning and relative distribution in the cell volume can shed light on the roles played by LDs. Until now, fluorescence microscopy and transmission electron microscopy are assessed as the gold standard methods for identifying LDs due to their high sensitivity and specificity. However, such methods generally only provide 2D assays and partial measurements. Furthermore, both can be destructive and with low productivity, thus limiting analysis of large cell numbers in a sample. Here we demonstrate for the first time the capability of 3D visualization and the full LD characterization in high-throughput with a tomographic phase-contrast flow-cytometer, by using ovarian cancer cells and monocyte cell lines as models. A strategy for retrieving significant parameters on spatial correlations and LD 3D positioning inside each cell volume is reported. The information gathered by this new method could allow more in depth understanding and lead to new discoveries on how LDs are correlated to cellular functions.


title: The Effect of Ligands and Transducers on the Neurotensin Recep-tor 1 (NTS1) Conformational Ensemble

abstract: Using a discrete, intracellular 19F-NMR probe on transmembrane helix 6 (TM6) of the Neurotensin receptor 1 (NTS1), we aim to understand how ligands and transducers modulate the receptors structural ensemble in solution. For apo NTS1, 19F-NMR spectra reveal an ensemble of at least three conformational substates (one inactive and two active-like) in equilibrium that exchange on the ms-s timescale. Dynamic NMR experiments reveal that these substates follow a linear three-site exchange process that is both thermodynamically and kinetically remodeled by orthosteric ligands. As previously observed in other GPCRs, the full agonist is insufficient to completely stabilize the active-like state. The inactive substate is abolished upon coupling to {beta}-arrestin-1 or the C-terminal helix of Gq, which comprises 60% of the GPCR/G protein interface surface area. Whereas {beta}-arrestin-1 exclusively selects for pre-existing active-like substates, the Gq peptide induces a new substate. Both transducer molecules promote substantial line-broadening of active-like states suggesting contributions from additional s-ms exchange processes. Together, our study suggests i) the NTS1 allosteric activation mechanism may be alternatively dominated by induced fit or conformational selection depending on the coupled transducer, and ii) the available static structures do not represent the entire conformational ensemble observed in solution.


title: HDfleX: Software for flexible high structural resolution of hydrogen/deuterium-exchange mass spectrometry data

abstract: Hydrogen/deuterium-exchange mass spectrometry (HDX-MS) experiments on protein structures can be performed at three levels: (1) by enzymatically digesting labelled proteins and analyzing the peptides (bottom-up), (2) by further fragmenting peptides following digestion (middle-down), and (3) by fragmenting the intact labelled protein (top-down), using soft gas-phase fragmentation methods, such as electron transfer dissociation (ETD). However, to the best of our knowledge, the software packages currently available for the analysis of HDX-MS data do not enable the peptide- and ETD-levels to be combined - they can only be analyzed separately. Thus, we developed HDfleX - a standalone application for the analysis of flexible high structural resolution of HDX-MS data, which allows data at any level of structural resolution (intact protein, peptide, fragment) to be merged. HDfleX features rapid experimental data fitting, robust statistical significance analyses and optional methods for theoretical intrinsic calculations and a novel empirical correction for comparison between solution conditions.


title: WILD-seq: Clonal deconvolution of transcriptomic signatures of sensitivity and resistance to cancer therapeutics in vivo.

abstract: Tumour heterogeneity is thought to be a major barrier to successful cancer treatment due to the presence of drug resistant clonal lineages. However, identifying the characteristics of such lineages that underpin resistance to therapy has remained challenging. Here we present WILD-seq; Wholistic Interrogation of Lineage Dynamics by sequencing, a platform that leverages expressed barcodes to simultaneously map clonal identities and transcriptional states at single cell resolution. Our optimised pipeline ensures recurrent representation of clonal lineages across animals and samples, facilitating analysis of clonal dynamics under perturbation. Application of WILD-seq to two triple negative mammary carcinoma mouse models, identified changes in clonal abundance, gene expression and microenvironment in response to JQ1 or taxane chemotherapy. WILD-seq reveals oxidative stress protection as a major mechanism of taxane resistance that renders our tumour models collaterally sensitive to non-essential amino acid deprivation. In summary, WILD-seq enables facile coupling of lineage and gene expression in vivo to elucidate clone-specific pathways of resistance to cancer therapies.

category: cancer biology


title: Minor intron splicing efficiency increases with the development of lethal prostate cancer

abstract:
Here we explored the role of minor spliceosome (MiS) function and minor intron-containing gene (MIG) expression in prostate cancer (PCa). We show MIGs are enriched as direct interactors of cancer-causing genes and their expression discriminates PCa progression. Increased expression of MiS U6atac snRNA, including others, and 6x more efficient minor intron splicing was observed in castration-resistant PCa (CRPC) versus primary PCa. Notably, androgen receptor signalling influenced MiS activity. Inhibition of MiS through siU6atac in PCa caused minor intron mis-splicing and aberrant expression of MIG transcripts and encoded proteins, which enriched for MAPK activity, DNA repair and cell cycle. Single cell-RNAseq confirmed cell cycle defects and lineage dependency on the MiS from primary to CRPC and neuroendocrine PCa. siU6atac was [~]50% more efficient in lowering tumor burden of CRPC cells and organoids versus current state-of-the-art combination therapy. In all, MiS is a strong therapeutic target for lethal PCa and potentially other cancers.

Graphical AbstractU6atac expression, MiS activity, and minor intron splicing correlate with PCa therapy resistance and PCa progression to CRPC-adeno and transdifferentiation to CRPC-NE. One major MiS regulator during that process is the AR-axis, which is re-activated during CRPC-adeno and blocked in CRPC-NE. Molecularly, an increase in MiS dependent splicing promotes changes of transcriptome and proteome. This results in cell cycle activation, increased MAPK signalling and increased DNA repair. U6atac mediated MiS inhibition renders MiS splicing error-prone through increased intron retention and alternative splicing events, which results in cell cycle block and decreased MAPK signalling and DNA repair. MiS inhibition blocks all stages of PCa. Figure created with BioRender.com.

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org.highwire.dtl.DTLVardef@1c1c296org.highwire.dtl.DTLVardef@1ce1416org.highwire.dtl.DTLVardef@88d842org.highwire.dtl.DTLVardef@2ddf96_HPS_FORMAT_FIGEXP  M_FIG C_FIG


abstract:
The discovery and characterization of tumor associated antigens is increasingly important to advance the field of immuno-oncology. Data on the, 1) topology, 2) amino acid (a.a.) homology analyses and 3) cell surface localization are presented as evidence in support of labyrinthin as a pan-adenocarcinoma marker. Multiple bioinformatics tools were used to collectively predict calcium binding domain(s), N-myristoylation sites, kinase II phosphorylation sites, and transmembrane domain(s) with predicted N- and C-termini orientations. Sequence homology comparisons were found and alignments made for labyrinthin (255 a.a.) vs. the intracellular aspartyl/asparaginyl beta-hydroxylase (ASPH; 758 a.a.) and the ASPH-gene related protein junctate (299 a.a.), which are both type II proteins. The epitopes for commonly used MCA 44-3A6 anti-labyrinthin and FB-50 anti-ASPH/junctate antibodies were present in all three proteins. The selective expression of labyrinthin was investigated by third party fluorescent activated cell sorter (FACS) analyses of MCA 44-3A6 binding to non-permeabilized adenocarcinoma and normal cell line models. Labyrinthin was detected on non-permeabilized A549 human lung adenocarcinoma cells, but not on normal WI-38 human lung fibroblasts nor primary cultures of normal human glandular-related cells. Microscopic images of immunofluorescent labelled MCA 44-3A6 binding to A549 cells at random cell cycle stages complement the FACS results by further showing that labyrinthin persisted on the cell surfaces along with some cell internalization within 20 minutes. The dataset presented herein is related to research article: "Labyrinthin: A distinct pan-adenocarcinoma diagnostic and immunotherapeutic tumor specific antigen" by Babich et al., Heliyon, 2022 [1].

Specifications Table

O_TBL View this table:
org.highwire.dtl.DTLVardef@19222bborg.highwire.dtl.DTLVardef@1264bcdorg.highwire.dtl.DTLVardef@13a8158org.highwire.dtl.DTLVardef@1dcf0org.highwire.dtl.DTLVardef@503a9f_HPS_FORMAT_FIGEXP  M_TBL C_TBL Value of the data O_LIBecause identification and characterization of neoantigens [3, 4, 5] are rare yet increasingly important for diagnostics and therapeutics, the identification of labyrinthin as a pan-adenocarcinoma target is significant for further study by clinicians and cancer researchers.
C_LIO_LIThe data provide a basis to determine to what extent labyrinthin is a distinguishing marker for adenocarcinomas vs. conventional methods.
C_LIO_LIThe results indicate that therapeutic strategies can be employed that take advantage of labyrinthins convenient presence on adenocarcinoma cell surfaces.
C_LIO_LIThe discovery of a calcium binding sequence in labyrinthin provides a basis to research if it is key to explaining the long-known phenomena of elevated calcium and irregular signaling in cancer cells [8, 9].
C_LIO_LIThe data reveal a need to explore distinctions between ASPH and labyrinthin, in particular because ASPH [10, 11] and labyrinthin-based treatments have already been FDA approved for clinical trials.
C_LI


title: Topology and adenocarcinoma cell localization dataset on the labyrinthin biomarker.


title: Single-cell meta-analysis of cigarette smoking lung atlas

abstract: Single-cell RNA-seq (scRNA-seq) technologies have been broadly utilized to reveal the molecular mechanisms of respiratory diseases and physiology at single-cell resolution. Here, we constructed a cigarette smoking lung atlas by integrating data from 8 public datasets, including 104 lung scRNA-seq samples with patient state information. The cigarette smoking lung atlas generated by this single-cell meta-analysis (scMeta-analysis) revealed early carcinogenesis events and defined the alterations of single-cell gene expression, cell population, fundamental properties of biological pathways, and cell-cell interactions induced by cigarette smoking. In addition, we developed two novel scMeta-analysis methods incorporating clinical metadata: VARIED (Visualized Algorithms of Relationships In Expressional Diversity) and AGED (Aging-related Gene Expressional Differences). VARIED analysis revealed the expressional diversity associated with smoking carcinogenesis in each cell population. AGED analysis revealed differences in gene expression related to both aging and smoking states. Our scMeta-analysis provided new insights into the effects of smoking and into cellular diversity in the human lung at single-cell resolution.


title: A direct high-throughput protein quantification strategy facilitates discovery and characterization of a celastrol-derived BRD4 degrader

abstract: We describe a generalizable time-resolved Forster resonance energy transfer (TR-FRET)-based platform to profile the cellular action of heterobifunctional degraders (or proteolysis-targeting chimeras; PROTACs), capable of both accurately quantifying protein levels in whole cell lysates in less than 1 h and measuring small-molecule target engagement to en-dogenous proteins, here specifically for human bromodomain-containing protein 4 (BRD4). The detection mix consists of a single primary antibody targeting the protein of interest, a luminescent donor-labeled anti-species nanobody, and a fluorescent acceptor ligand. Importantly, our strategy can readily be applied to other targets of interest and will greatly facilitate the cell-based profiling of small molecule inhibitors and PROTACs in high-throughput format with unmodified cell lines. We further-more validate our platform in the characterization of celastrol, a p-quinone methide-containing pentacyclic triterpenoid, as a broad cysteine-targeting E3 ubiquitin ligase warhead for potent and efficient targeted protein degradation.

category: pharmacology and toxicology


title: Functional modulation of PTH1R activation and signalling by RAMP2

abstract:
Receptor-activity-modifying proteins (RAMPs) are ubiquitously expressed membrane proteins that associate with different G protein-coupled receptors (GPCRs) including the parathyroid hormone 1 receptor (PTH1R), a class B GPCR and an important modulator of mineral ion homeostasis and bone metabolism. However, it is unknown whether and how RAMP proteins may affect PTH1R function.

Using different optical biosensors to measure the activation of PTH1R and its downstream signalling, we describe here that RAMP2 acts as a specific allosteric modulator of PTH1R, shifting PTH1R to a unique pre-activated state that permits faster activation in a ligand-specific manner. Moreover, RAMP2 modulates PTH1R downstream signalling in an agonist-dependent manner, most notably increasing the PTH-mediated Gi3 signalling sensitivity. Additionally, RAMP2 increases both PTH- and PTHrP-triggered {beta}-arrestin2 recruitment to PTH1R. Employing homology modelling we describe the putative structural molecular basis underlying our functional findings.

These data uncover a critical role of RAMPs in the activation and signalling of a GPCR that may provide a new venue for highly specific modulation of GPCR function and advanced drug design.


title: Comparing Brain Asymmetries Independently of Brain Size

abstract:
Studies examining cerebral asymmetries typically divide the L-R Measure (e.g., Left- Right Volume) by the L+R Measure to obtain an Asymmetry Index (AI). However, contrary to widespread belief, such a division fails to render the AI independent from the L+R Measure and/or from total brain size. As a result, variations in brain size may bias correlation estimates with the AI or group differences in AI. We investigated how to analyze brain asymmetries in to distinguish global from regional effects, and report unbiased group differences in cerebral asymmetries.

We analyzed the extent to which the L+R Measure, Total Cerebral Measure (TCM, e.g., Total Brain Volume), and L-R TCM predict regional asymmetries. As a case study, we assessed the consequences of omitting each of these predictors on the magnitude and significance of sex differences in asymmetries.

We found that the L+R Measure, the TCM, and the L-R TCM predicted the AI of more than 89% of regions and that their relationships were generally linear. Removing any of these predictors changed the significance of sex differences in 33% of regions and the magnitude of sex differences across 13-42% of regions. Although we generally report similar sex and age effects on cerebral asymmetries to those of previous large-scale studies, properly adjusting for regional and global brain size revealed additional sex and age effects on brain asymmetry.

Highlights- The typical Asymmetry Index (AI) scales with the size of a region and brain size.
- Omitting the Left+Right Measure influences reported sex differences in asymmetries.
- Omitting brain size or asymmetry influences reported sex differences in asymmetries.
- We report sex and age effects on AIs independent of regional and global brain size.

category: neuroscience


title: Mapping of the autophagosomal degradome identifies IL-7Rα as key cargo in proliferating CD4+ T-cells

abstract: CD4+ T cells orchestrate both humoral and cytotoxic immune responses. While it is known that CD4+ T cell proliferation relies on autophagy, direct identification of the autophagosomal cargo involved is still missing. Here, we created a transgenic mouse model, which, for the first time, enables us to directly map the proteinaceous content of autophagosomes in any primary cell by LC3 proximity labelling. IL-7R, a cytokine receptor mostly found in naive and memory T cells, was reproducibly detected in autophagosomes of activated CD4+ T cells. Consistently, CD4+ T cells lacking autophagy showed increased IL-7R surface expression, while no defect in internalisation was observed. Mechanistically, excessive surface IL-7R sequestrates the common gamma chain, impairing the IL-2R assembly and downstream signalling crucial for T cell proliferation. This study provides proof-of-principle that key autophagy substrates can be reliably identified with this model to help mechanistically unravel autophagys contribution to healthy physiology and disease.

category: immunology


title: Single-cell profiling of healthy human kidney reveals features of sex-based transcriptional programs and tissue-specific immunity

abstract: Maintaining organ homeostasis requires complex functional synergy between distinct cell types, a snapshot of which is glimpsed through the simultaneously broad and granular analysis provided by single-cell atlases. Knowledge of the transcriptional programs underpinning the complex and specialized functions of human kidney cell populations at homeostasis is limited by difficulty accessing healthy, fresh tissue. Here, we present a single-cell perspective of healthy human kidney from 19 living donors, with equal contribution from males and females, profiling the transcriptome of 27677 high-quality cells to map healthy kidney at high resolution. Our sex-balanced dataset revealed sex-based differences in gene expression within proximal tubular cells, specifically, increased anti-oxidant metallothionein genes in females and the predominance of aerobic metabolism-related genes in males. Functional differences in metabolism were confirmed between male and female proximal tubular cells, with male cells exhibiting higher oxidative phosphorylation and higher levels of energy precursor metabolites. Within the immune niche, we identified kidney-specific lymphocyte populations with unique transcriptional profiles indicative of kidney-adapted functions and validated findings by flow cytometry. We observed significant heterogeneity in resident myeloid populations and identified an MRC1+ LYVE1+ FOLR2+ C1QC+ population as the predominant myeloid population in healthy kidney. This study provides a detailed cellular map of healthy human kidney, revealing novel insights into the complexity of renal parenchymal cells and kidney-resident immune populations.


title: Human cellular homeostasis buffers trans-acting translational effects of heterologous gene expression with very different codon usage bias

abstract: The frequency of synonymous codons in protein coding genes is non-random and varies both between species and between genes within species. Whether this codon usage bias (CUBias) reflects underlying neutral mutational processes or is instead shaped by selection remains an open debate, especially regarding the role of selection for enhanced protein production. Variation in CUBias of a gene (be it natural synonymous mutations or biotechnological synonymous recoding) can have an enormous impact on its expression by diverse cis- acting mechanisms. But expression of genes with extreme CUBias can also lead to strong phenotypic effects by altering the overall intracellular translation homeostasis via competition for ribosomal machinery or tRNA depletion. In this study, we expressed at high levels in human cells six different synonymous versions of a gene and used matched transcriptomic and proteomic data to evaluate the impact of CUBias of the heterologous gene on the translation of cellular transcripts. Our experimental design focused specifically on differences during translation elongation. Response to expression of the different synonymous sequences was assessed by various approaches, ranging from analyses performed on a per-gene basis to more integrated approaches of the cell as a whole. We observe that the transcriptome displayed substantial changes as a result of heterologous gene expression by triggering an intense antiviral and inflammatory response, but that changes in the proteomes were very modest. Most importantly we notice that changes in translation efficiency of cellular transcripts were not associated with the direction of the CUBias of the heterologous sequences, thereby providing only limited support for trans- acting effects of synonymous changes. We interpret that, in human cells in culture, changes in CUBias can lead to important cis- acting effects in gene expression, but that cellular homeostasis can buffer the phenotypic impact of overexpression of heterologous genes with extreme CUBias.

category: cell biology


title: Fitness of breeders in social Damaraland mole-rats is independent of group size

abstract:
Damaraland mole-rats (Fukomys damarensis) are usually viewed as a eusocial or obligate cooperative breeder in which successful reproduction is dependent on help from closely related group members. However, because longitudinal studies of mole-rats in their natural environment are uncommon, the extent to which successful reproduction by breeders relies on assistance from other group members is unclear, and for non-breeders, the immediate and delayed fitness consequences of staying and helping are poorly understood. Using data from a 7-year field study of marked individuals, we investigate whether increasing group size confers reproductive and survival benefits to breeders and non-breeders, and explore the different routes through which individuals acquire breeding positions. We show that solitary natal dispersal was the most common route to breeding for both sexes and that the inheritance of dominant breeding positions was uncommon in both sexes. After dispersing, females typically settled alone in new burrow systems where they enjoyed high survival rates and remained in good body condition - often for several years - before being joined by males. In contrast to most obligately cooperative species, pairs of potential breeders reproduced successfully without helpers and experimentally formed pairs had the same reproductive success as larger established groups. Though larger breeding groups recruited slightly more pups on average, our data suggest that neither survival nor reproduction depend on the presence of non-breeding helpers, indicating that Damaraland mole-rats are not obligate cooperative breeders. We suggest that extended philopatry and group living in Damaraland mole-rats have evolved because of the high costs and constraints of dispersal rather than because of strong indirect benefits accrued through cooperative behaviour and that similarities between their breeding systems and those of obligatorily eusocial insects have been over-emphasized.

Significance StatementThe social mole-rats are often considered eusocial mammals in which successful reproduction depends on assistance from non-breeding helpers. In this study we show that in wild Damaraland mole-rats, the presence of non-breeders is associated with both costs and benefits and that nascent breeding pairs show high reproductive success despite the lack of non-breeding helpers. These findings indicate that Damaraland mole-rats are not obligate cooperative breeders and suggest that similarities between their breeding systems and those of obligatorily eusocial insects have been over-emphasized.


title: Damaraland mole-rats are not obligate cooperative breeders


title: Exact breakpoints of In(1)wm4 rearrangement

abstract: In(1)wm4 was known for decades as a classic example of position effect variegation-causing rearrangement and was mentioned in hundreds of publications. Nevertheless, the euchromatin breakpoint position of the rearrangement was not precisely localized. We performed nanopore sequencing of DNA from In(1)wm4 homozygous flies and determined the exact position of euchromatic (chrX:2767875) and heterochromatic breakpoints of the rearrangement.


title: CRISPR knockouts of pmela and pmelb engineered a golden tilapia by regulating relative pigment cell abundance

abstract:
Premelanosome protein (pmel) is a key gene for melanogenesis in vertebrates. Mutations in this gene are responsible for white plumage in chicken, but its role in pigmentation of fish remains to be demonstrated. In this study we found that most fishes have two pmel genes arising from the teleost-specific whole genome duplication. Both pmela and pmelb were expressed at high levels in the eyes and skin of Nile tilapia. We mutated both genes in tilapia using CRISPR/Cas9 gene editing. Homozygous mutation of pmela resulted in yellowish body color with weak vertical bars and a hypo-pigmented retinal pigment epithelium (RPE) due to significantly reduced number and size of melanophores. In contrast, we observed an increased number and size of xanthophores in mutants compared to wild-type fish. Homozygous mutation of pmelb resulted in a similar, but milder phenotype than pmela-/- mutants, without effects on RPE pigmentation. Double mutation of pmela and pmelb resulted in loss of additional melanophores compared to the pmela-/- mutants, and also an increase in the number and size of xanthophores, producing a strong golden body color without bars in the trunk. The RPE pigmentation of pmela-/-;pmelb-/- was similar to pmela-/- mutants, with much less pigmentation than pmelb-/- mutants and wild-type fish. Taken together, our results indicate that, while both pmel genes are important for the formation of body color in tilapia, pmela plays a more important role than pmelb. To our knowledge, this is the first report on mutation of pmelb or both pmela;pmelb in fish. Studies on these mutants suggest new strategies for breeding golden tilapia, and also provide a new model for studies of pmel function in vertebrates.

Author SummaryMelanophores, the most common pigment cell type, have been studied for nearly 150 years. Many genes are involved in melanoblast migration, melanophore differentiation, and melanin biosynthesis. Pmel is fundamental for melanosome development by directing melanin biosynthesis and melanosome phase transition. Specifically, PMEL can form a fibrillar structure within the melanosome upon which melanin is deposited. We identified two pmel genes in Nile tilapia arising from the teleost-specific whole genome duplication. Disruption of either pmela or pmelb in tilapia leads to significant hypo-pigmentation. PMEL disrupted fish showed not only a reduction in melanin and tiny melanophores, but also a significant increase in the number of xanthophores, and even guanine-filled melanophores, which led to a golden tilapia with hypo-pigmented RPE. Our study confirmed the role of pmel in melanin biosynthesis and maturation, and also highlighted its effects on melanophore number and size. These results provide new insights into pigment cell biology and will help us better understand the mechanisms of color patterning in teleosts. Knockout of both pmela and pmelb provide a new strategy for engineering a golden tilapia, which might provide a foundation for developing new strains in the tilapia industry.


title: Host immune responses to enzootic and invasive pathogen lineages vary in magnitude, timing, and efficacy

abstract: Infectious diseases of wildlife continue to pose a threat to biodiversity worldwide, yet pathogens are far from monolithic in virulence. Within the same pathogen species, virulence can vary considerably depending on strain or lineage, in turn eliciting variable host responses. One pathogen that has caused extensive biodiversity loss is the amphibian-killing fungus, Batrachochytrium dendrobatidis (Bd), which is comprised of a globally widespread hypervirulent lineage (Bd-GPL), and multiple geographically restricted lineages. Whereas host immunogenomic responses to Bd-GPL have been characterized in a number of amphibian species, immunogenomic responses to geographically-restricted, enzootic Bd lineages are unknown. To examine lineage- specific host immune responses to Bd, we exposed a species of pumpkin toadlet, Brachycephalus pitanga, which is endemic to Brazils Southern Atlantic Forest, to either the Bd-GPL or the enzootic Bd-Asia-2/Brazil (hereafter Bd-Brazil) lineage. We quantified functional immunogenomic responses over the course of infection using differential gene expression tests and coexpression network analyses. Host immune responses varied significantly with Bd lineage. Toadlet responses to Bd-Brazil were weak at early infection (26 genes differentially expressed), peaked by mid-stage infection (435 genes) and were nearly fully resolved by late-stage disease (9 genes). In contrast, responses to Bd-GPL were magnified and delayed; toadlets differentially expressed 97 genes early, 86 genes at mid-stage infection, and 728 genes by late-stage infection. Given that infection intensity did not vary between mid- and late-stage disease, this suggests that pumpkin toadlets may be at least partially tolerant to the geographically-restricted Bd- Brazil lineage. In contrast, mortality was higher in Bd-GPL-infected toadlets, suggesting that late-stage immune activation against Bd-GPL was not protective and was consistent with immune dysregulation previously observed in other species. Our results demonstrate that both the timing of immune response and the particular immune pathways activated are specific to Bd lineage. Within regions where multiple Bd lineages co-occur, and given continued global Bd movement, these differential host responses may influence not only individual disease outcome, but transmission dynamics at the population and community levels.


title: A haplotype-resolved genome assembly of the Nile rat facilitates exploration of the genetic basis of diabetes

abstract: The Nile rat (Avicanthis niloticus) is an important animal model for biomedical research, including the study of diurnal rhythms and type 2 diabetes. Here, we report a 2.5 Gb, chromosome-level reference genome assembly with fully resolved parental haplotypes, generated with the Vertebrate Genomes Project (VGP). The assembly is highly contiguous, with contig N50 of 11.1 Mb, scaffold N50 of 83 Mb, and 95.2% of the sequence assigned to chromosomes. We used a novel workflow to identify 3,613 segmental duplications and quantify duplicated genes. Comparative analyses revealed unique genomic features of the Nile rat, including those that affect genes associated with type 2 diabetes and metabolic dysfunctions. These include 14 genes that are heterozygous in the Nile rat or highly diverged from the house mouse. Our findings reflect the exceptional level of genomic detail present in this assembly, which will greatly expand the potential of the Nile rat as a model organism for genetic studies.


title: Genome-wide identification and analyses of tomato (Solanum lycopersicum L.) high-affinity nitrate transporter 2 (NRT2) family genes and their responses to drought and salinity

abstract: High-affinity nitrate transporter 2 (NRT2) proteins have vital roles in nitrate (NO3-) uptake and translocation in plants. The gene families coding NRT2 proteins have been identified and functionally characterized in many plant species. However, no systematic identification of NRT2 family members have been reported in tomato (Solanum lycopersicum). There is also little known about their expression profiles under environmental stresses. Accordingly, the present study aimed to identify NRT2 gene family in the tomato genome; then, investigate them in detail through bioinformatics, physiological and expression analyses. As a result, four novel NRT2 genes were identified in the tomato genome, all of which contain the same domain belonging to the Major Facilitator Superfamily (PF07690). The co-expression network of SlNRT genes revealed that they were co-expressed with several other genes in many different molecular pathways including transport, photosynthesis, fatty acid metabolism and amino acid catabolism. Programming many crucial physiological and metabolic pathways, various numbers of phosphorylation sites were predicted in the NRT2 proteins.


title: Simultaneous quantification of ankle, muscle, and tendon impedance in humans

abstract:
ObjectiveRegulating the impedance of our joints is essential for the effective control of posture and movement. The impedance of a joint is governed mainly by the mechanical properties of the muscle-tendon units spanning it. Many studies have quantified the net impedance of joints but not the specific contributions from the muscles and tendons. The inability to quantify both muscle and tendon impedance limits the ability to determine the causes underlying altered movement control associated with aging, neuromuscular injury, and other conditions that have different effects on muscle and tendon properties. Therefore, we developed a technique to quantify joint, muscle, and tendon impedance simultaneously and evaluated this technique at the human ankle.

MethodsWe used a single degree of freedom actuator to deliver pseudorandom rotations to the ankle while measuring the corresponding torques. We simultaneously measured the displacement of the medial gastrocnemius muscle-tendon junction with B-mode ultrasound. From these experimental measurements, we were able to estimate ankle, muscle, and tendon impedance using non-parametric system identification.

ResultsWe validated our estimates by comparing them to previously reported muscle and tendon stiffness, the position-dependent component of impedance, to demonstrate that our technique generates reliable estimates of these properties.

ConclusionOur approach can be used to clarify the respective contributions from the muscle and tendon to the net mechanics of a joint.

SignificanceThis is a critical step forward in the ultimate goal of understanding how muscles and tendons govern ankle impedance during posture and movement.


title: Induced fit with replica exchange improves protein complex structure prediction

abstract:
Despite the progress in prediction of protein complexes over the last decade, recent blind protein complex structure prediction challenges revealed limited success rates (less than 20% models with DockQ score > 0.4) on targets that exhibit significant conformational change upon binding. To overcome limitations in capturing backbone motions, we developed a new, aggressive sampling method that incorporates temperature replica exchange Monte Carlo (T-REMC) and conformational sampling techniques within docking protocols in Rosetta. Our method, ReplicaDock 2.0, mimics induced-fit mechanism of protein binding to sample backbone motions across putative interface residues on-the-fly, thereby recapitulating binding-partner induced conformational changes. Furthermore, ReplicaDock 2.0 clocks in at 150-500 CPU hours per target (protein-size dependent); a runtime that is significantly faster than Molecular Dynamics based approaches. For a benchmark set of 88 proteins with moderate to high flexibility (unbound-to-bound iRMSD over 1.2 [A]), ReplicaDock 2.0 successfully docks 61% of moderately flexible complexes and 35% of highly flexible complexes. Additionally, we demonstrate that by biasing backbone sampling particularly towards residues comprising flexible loops or hinge domains, highly flexible targets can be predicted to under 2 [A] accuracy. This indicates that additional gains are possible when mobile protein segments are known.

Significance StatementProteins bind each other in a highly specific and regulated manner, and these associated dynamics of binding are intimately linked to their function. Conventional techniques of structure determination such as cryo-EM, X-ray crystallography and NMR are time-consuming and arduous. Using a temperature-replica exchange Monte Carlo approach that mimics the kinetic mechanism of "induced fit" binding, we improved prediction of protein complex structures, particularly for targets that exhibit considerable conformational changes upon binding (Interface root mean square deviation (unbound-bound) > 1.2 [A]. Capturing these binding-induced conformational changes in proteins can aid us in better understanding biological mechanisms and suggest intervention strategies for disease mechanisms.


title: scMARK an 'MNIST' like benchmark to evaluate and optimize models for unifying scRNA data

abstract: Todays single-cell RNA analysis tools provide enormous value in enabling researchers to make sense of large single-cell RNA (scRNA) studies, yet their ability to integrate different studies at scale remains untested. Here we present a novel benchmark dataset (scMARK), that consists of 100,000 cells over 10 studies and can test how well models unify data from different scRNA studies. We also introduce a two-step framework that uses supervised models, to evaluate how well unsupervised models integrate scRNA data from the 10 studies. Using this framework, we show that the Variational Autoencoder, scVI, represents the only tool tested that can integrate scRNA studies at scale. Overall, this work paves the way to creating large scRNA atlases and  off-the-shelf analysis tools.


title: Differential Compositional Variation FeatureSelection: A Machine Learning Framework with LogRatios for Compositional Metagenomic Data

abstract: The demand for tight integration of compositional data analysis and machine learning methodologies for predictive modeling in high-dimensional settings has increased dramatically with the increasing availability of metagenomics data. We develop the differential compositional variation machine learning framework (DiCoVarML) with robust multi-level log ratio bio-marker discovery for metagenomic datasets. Our framework makes use of the full set of pairwise log ratios, scoring ratios according to their variation between classes and then selecting out a small subset of log ratios to accurately predict classes. Importantly, DiCoVarML supports a targeted feature selection mode enabling researchers to define the number of predictors used to develop models. We demonstrate the performance of our framework for binary classification tasks using both synthetic and real datasets. Selecting from all pairwise log ratios within the DiCoVarML framework provides greater flexibility that can in demonstrated cases lead to higher accuracy and enhanced biological insight.


title: Skopi: a simulation package for diffractive imaging of noncrystalline biomolecules

abstract: X-ray free electron lasers (XFEL) have the ability to produce ultra-bright femtosecond X-ray pulses for coherent diffraction imaging of biomolecules. While the development of methods and algorithms for macromolecular crystallography is now mature, XFEL experiments involving aerosolized or solvated biomolecular samples offer new challenges both in terms of experimental design and data processing. Skopi is a simulation package that can generate single-hit diffraction images for reconstruction algorithms, multi-hit diffraction images of aggregated particles for training machine learning classification tasks using labeled data, diffraction images of randomly distributed particles for fluctuation X-ray scattering (FXS) algorithms, and diffraction images of reference and target particles for holographic reconstruction algorithms. We envision skopi as a resource to aid the development of on-the-fly feedback during non-crystalline experiments at XFEL facilities, which will provide critical insights into biomolecular structure and function.


title: NITROGEN LIMITATION ADAPTATION functions as a negative regulator of Arabidopsis immunity

abstract:
BackgroundPhosphorus is an important macronutrient required for plant growth and development. It is absorbed through the roots in the form of inorganic phosphate (Pi). To cope with Pi limitation, plants have evolved an array of adaptive mechanisms to facilitate Pi acquisition and protect them from stress caused by Pi starvation. The NITROGEN LIMITATION ADAPTION (NLA) gene plays a key role in the regulation of phosphate starvation responses (PSR), its expression being regulated by the microRNA miR827. Stress caused by Pi limiting conditions might also affect the plants response to pathogen infection. However, cross-talk between phosphate signaling pathways and immune responses remains unclear.

ResultsIn this study, we investigated whether NLA plays a role in Arabidopsis immunity. We show that loss-of-function of NLA and MIR827 overexpression causes an increase in phosphate (Pi) content which results in resistance to infection by the fungal pathogen Plectosphaerella cucumerina. The nla mutant plants accumulated callose in their leaves, a response that is also observed in wild-type plants that have been treated with high Pi. We also show that pathogen infection and treatment with fungal elicitors is accompanied by transcriptional activation of MIR827 and down-regulation of NLA. Upon pathogen challenge, nla plants exhibited higher levels of the phytoalexin camalexin compared to wild type plants. Camalexin level also increases in wild type plants treated with high Pi. Furthermore, the nla mutant plants accumulated salicylic acid (SA) and jasmonic acid (JA) in the absence of pathogen infection whose levels further increased upon pathogen.

ConclusionsThis study shows that NLA acts as a negative regulator of Arabidopsis immunity. Overaccumulation of Pi in nla plants positively affects resistance to infection by fungal pathogens. This piece of information reinforces the idea of signaling convergence between Pi and immune responses for the regulation of disease resistance in Arabidopsis.

category: plant biology


title: Improving Lab Culture Through Self-Assessment: A Case Study

abstract:
PurposeMotivated by perceived dissatisfaction within our labs changed working environment brought about by the COVID-19 pandemic, we performed a self-assessment of our lab culture through anonymous surveys and live sessions.

MethodsIn Survey 1, we asked each lab member to identify and rank up to 10 values that are important for a healthy lab environment. They were then asked to rate how well the lab embodied those values at two time points: before the COVID-19 pandemic while working onsite, and at the time of the survey while working remotely (10 months into the pandemic). In a series of live group sessions, we reviewed relevant literature and the survey results to finalize ten themes. We then reflected on each theme and proposed action items to address any deficiencies. Finally, we conducted Survey 2 after the self-assessment to judge the groups finalized themes, implemented changes, and overall satisfaction with the assessment process.

ResultsThemes identified were attitude, accountability, teamwork/collaboration, communication, diversity/inclusion, emotional intelligence, integrity, training, well-being, and adaptability in crisis-management. All lab members liked the self-assessment process and felt their voices were heard. On average, there was a 12% increase in satisfaction across all themes from the start to end of the lab assessment.

ConclusionWe successfully assessed the culture of our lab and subsequently improved lab member satisfaction. The success of this team project suggests that other scientific labs could benefit from similar interactive self-assessments.

category: scientific communication and education


title: The channel capacity of multilevel linguistic features constrains speech comprehension

abstract: Humans are expert at processing speech but how this feat is accomplished remains a major question in cognitive neuroscience. Capitalizing on the concept of channel capacity, we developed a unified measurement framework to investigate the respective influence of seven acoustic and linguistic features on speech comprehension, encompassing acoustic, sub-lexical, lexical and supra-lexical levels of description. We show that comprehension is independently impacted by all these features, but at varying degrees and with a clear dominance of the syllabic rate. Comparing comprehension of French words and sentences further reveals that when supra-lexical contextual information is present, the impact of all other features is dramatically reduced. Finally, we estimated the channel capacity associated with each linguistic feature and compared them with their generic distribution in natural speech. Our data point towards supra-lexical contextual information as the feature limiting the flow of natural speech. Overall, this study reveals how multilevel linguistic features constrain speech comprehension.


title: miR-124 regulates Notch and NeuroD1 to mediate transition states of neuronal development

abstract: MicroRNAs (miRNAs) regulate gene expression by destabilizing target mRNA and/or inhibiting translation in animal cells. The ability to mechanistically dissect the function of miR-124 during specification, differentiation, and maturation of neurons during development within a single system has not been accomplished. Using the sea urchin (Strongylocentrotus purpuratus) embryo, we take advantage of the manipulability of the embryo and its well-documented gene regulatory networks (GRNs). We incorporated NeuroD1 as part of the sea urchin neuronal GRN and determined that miR-124 inhibition resulted in decreased gut contractions, swimming velocity, and neuronal development. We further integrated post-transcriptional regulation of miR-124 into the neuronal GRN. Inhibition of miR-124 resulted in increased number of cells expressing transcription factors associated with progenitor neurons and a concurrent decrease of mature and functional neurons. Results revealed that miR-124 regulates undefined factors early in neurogenesis during neuronal specification and differentiation in the early blastula and gastrula stages. In the late gastrula and larval stages, miR-124 regulates Notch and NeuroD1. Specifically, miR-124 regulates the transition between neuronal differentiation and maturation, by directly suppressing NeuroD1. Removal of miR-124s suppression of NeuroD1 results in increased mature neurons with decreased Synaptagmin B-positive mature, functional neurons. By removing both miR-124 suppression of NeuroD1 and Notch, we were able to phenocopy miR-124 inhibitor induced defects. Overall, we have improved the neuronal GRN and identified miR-124 to play a prolific role in regulating various transitions of neuronal development.


title: The intrinsic spatiotemporal organization of the human brain - A multi-dimensional functional network atlas

abstract: The human brain is a complex, integrative and segregative network that exhibits dynamic fluctuations in activity across space and time. A canonical set of large-scale networks has been historically identified from resting-state fMRI (rs-fMRI), including the default mode, visual, somatomotor, salience, attention, and executive control. However, the methods used in identification of these networks have relied on assumptions that may inadvertently constrain their properties and consequently our understanding of the human connectome. Here we define a brain "network" as a functional component that jointly describes its spatial distribution and temporal dynamics, where neither domain suffers from unrealistic constraints. Using our recently developed BrainSync algorithm and the Nadam-Accelerated SCAlable and Robust (NASCAR) tensor decomposition, we identified twenty-three brain networks using rs-fMRI data from a large group of healthy subjects acquired by the Human Connectome Project. These networks are spatially overlapped, temporally correlated, and highly reproducible across two independent groups and sessions. We show that these networks can be clustered into six distinct functional categories and naturally form a representative functional network atlas for a healthy population. Using this atlas, we demonstrate that individuals with attention-deficit/hyperactivity disorder display disproportionate brain activity increases, relative to neurotypical subjects, in visual, auditory, and somatomotor networks concurrent with decreases in the default mode and higher-order cognitive networks. Thus, this work not only yields a highly reproducible set of spatiotemporally overlapped functional brain networks, but also provides convergent evidence that individual differences in these networks can be used to explain individual differences in neurocognitive functioning.


title: Shared mechanisms of auditory and non-auditory vocal learning in the songbird brain

abstract: Songbirds and humans share the ability to adaptively modify their vocalizations based on sensory feedback. Prior studies have focused primarily on the role that auditory feedback plays in shaping vocal output throughout life. In contrast, it is unclear whether and how non-auditory information drives vocal plasticity. Here, we first used a reinforcement learning paradigm to establish that non-auditory feedback can drive vocal learning in adult songbirds. We then assessed the role of a songbird basal ganglia-thalamocortical pathway critical to auditory vocal learning in this novel form of vocal plasticity. We found that both this circuit and its dopaminergic inputs are necessary for non-auditory vocal learning, demonstrating that this pathway is not specialized exclusively for auditory-driven vocal learning. The ability of this circuit to use both auditory and non-auditory information to guide vocal learning may reflect a general principle for the neural systems that support vocal plasticity across species.


title: Preserved representations and decodability of diverse cognitive functions across the cortex, cerebellum, and subcortex

abstract: Which part of the brain contributes to our complex cognitive processes? Studies have revealed contributions of the cerebellum and subcortex to higher-order cognitive functions; however it is unclear whether such functional representations are preserved across the cortex, cerebellum, and subcortex. In this study, we used functional magnetic resonance imaging data with 103 cognitive tasks and constructed three voxel-wise encoding and decoding models independently using cortical, cerebellar, and subcortical voxels. Representational similarity analysis revealed that the structure of task representations is preserved across the three brain parts. Principal component analysis visualized distinct organizations of abstract cognitive functions in each part of the cerebellum and subcortex. More than 90% of the cognitive tasks were decodable from the cerebellum and subcortical activities, even for the novel tasks not included in model training. Furthermore, we discovered that the cerebellum and subcortex have sufficient information to reconstruct activity in the cerebral cortex.


title: Boosting serotonin increases information gathering by reducing subjective cognitive costs

abstract: Serotonin is implicated in the valuation of aversive costs, such as delay or physical effort. However, its role in governing sensitivity to cognitive effort, for example deliberation costs during information gathering, is unclear. We show that week-long treatment with a serotonergic antidepressant enhances a willingness to gather information when trying to maximize reward. Using computational modelling, we show this arises from a diminished sensitivity to subjective deliberation costs during the sampling process. This result is consistent with the notion that serotonin alleviates sensitivity to aversive costs in a domain-general fashion, with implications for its potential contribution to a positive impact on motivational deficits in psychiatric disorders.


title: Age- and sex-related differences in baboon (Papio anubis) gray matter covariation

abstract: Age-related changes in cognition, brain morphology, and behavior are exhibited in several primate species. Baboons, like humans, naturally develop Alzheimers disease-like pathology and cognitive declines with age and are an underutilized model for studies of aging. To determine age-related differences in gray matter covariation of 89 olive baboons (Papio anubis), we used source-based morphometry (SBM) to analyze data from magnetic resonance images. We hypothesized that we would find significant age effects in one or more SBM components, particularly those which include regions influenced by age in humans and other nonhuman primates (NHPs). A multivariate analysis of variance revealed that individual weighted gray matter covariation scores differed across the age classes. Elderly baboons contributed significantly less to gray matter covariation components including the brainstem, superior parietal cortex, thalamus, and pallidum compared to juveniles, and middle and superior frontal cortex compared to juveniles and young adults (p<0.05). Future studies should examine the relationship between the changes in gray matter covariation reported here and age-related cognitive decline.


title: Primary infection by E. multilocularis induces distinct patterns of crosstalk between hepatic regulatory T and natural killer T cells in mice

abstract:
The larval stage of the helminthic cestode Echinococcus multilocularis can inflict tumor-like hepatic lesions that cause the parasitic disease alveolar echinococcosis in humans, with high mortality in untreated patients. Recently, opportunistic properties of the disease have been proposed based on the increased incidence in immunocompromised patients and mouse models, indicating that an appropriate adaptive immune response is required for the control of the disease. However, little is known about how the local hepatic immune responses modulate the infection with E. multilocularis. In a mouse model of oral infection that mimics the normal infection route in human patients, the adaptive immune response in the liver was assessed using single-cell RNA sequencing of isolated hepatic CD3+ T cells at different infection stages. We observed an early significant increase in regulatory T and natural killer T cells in parallel with an active downregulation of CD4+ and CD8+ T cells. Early interactions between regulatory T cells and natural killer T cells indicate a promotion of the formation of hepatic lesions and later contribute to suppression of the resolution of parasite-induced pathology. The obtained data provides a fresh insight on the adaptive immune responses and local regulatory pathways at different infection stages of E. multilocularis in mice.

Author summaryAlveolar echinococcosis is an endemic parasitic infection leading to slowly growing but potentially lethal liver lesions if untreated. Transmission by increasing populations of urban foxes and the raise of immunosuppressed patients in mainly industrializsed endemic zones are the main causes responsible for increased incidence of alveolar echinococcosis. Observations in humans and mice indicate that reactions of the adaptive immune system are required to control the disease and to protect from a chronic infection. Therefore, we analysed the responses of T cells in the liver at single-cell resolution in a murine model mimicking the typical route of infection in humans. This so called single-cell RNA sequencing revealed specific temporal changes of T cell subsets such as natural killer T cells and regulatory T cells, indicating that these two cell types are recruited in the early phase to try to protect from parasite proliferation and are subsequently inhibited in the late phase of infection thus indicating immune escape mechanisms of the parasite. This study shows temporal changes of the immune cell profile in the liver over the course of a natural infection with E. multilocularis at the single cell level and reveals putative targets for novel therapeutic approaches for human AE and possibly other (parasitic/helminthic) diseases.


title: Modulation of the Drosophila Transcriptome by Developmental Exposure to Alcohol

abstract:
Prenatal exposure to ethanol can cause fetal alcohol spectrum disorder (FASD), a prevalent, preventable pediatric disorder. Identifying genetic risk alleles for FASD is challenging since time, dose, and frequency of exposure are often unknown, and manifestations of FASD are diverse and evident long after exposure. Drosophila melanogaster is an excellent model to study the genetic basis of the effects of developmental alcohol exposure since many individuals of the same genotype can be reared under controlled environmental conditions. We used 96 sequenced, wild-derived inbred lines from the Drosophila melanogaster Genetic Reference Panel (DGRP) to profile genome-wide transcript abundances in young adult flies that developed on ethanol-supplemented medium or standard culture medium. We found substantial genetic variation in gene expression in response to ethanol with extensive sexual dimorphism. We constructed sex-specific genetic networks associated with alcohol-dependent modulation of gene expression that include protein-coding genes, Novel Transcribed Regions (NTRs, postulated to encode long non-coding RNAs) and female-specific coordinated regulation of snoRNAs that regulate pseudouridylation of ribosomal RNA. We reared DGRP lines which showed extreme upregulation or downregulation of snoRNA expression during developmental alcohol exposure on standard or ethanol supplemented medium and demonstrated that developmental exposure to ethanol has genotype-specific effects on adult locomotor activity and sleep. There is significant and sex-specific natural genetic variation in the transcriptional response to developmental exposure to ethanol in Drosophila that comprises networks of genes affecting nervous system development and ethanol metabolism as well as networks of regulatory non-coding RNAs.

Summary statementWe developed a Drosophila model for Fetal Alcohol Spectrum Disorder and found that developmental exposure to alcohol modulates expression of networks of genes affecting nervous system development and ethanol metabolism as well as networks of regulatory noncoding RNAs.


title: An epitope specific patient-derived LGI1-autoantibody enhances neuronal excitability by modulating the Kv1.1 channel.

abstract: Leucine-rich Glioma Inactivated protein 1 (LGI1) is expressed in the central nervous and genetic loss of function is associated with epileptic disorders. Also, patients with LGI1-directed autoantibodies have frequent focal seizures as a key feature of their disease. LGI1 is composed of a Leucine Rich Repeat (LRR) and an Epitempin (EPTP) domain. These domains are reported to interact with different aspects of the transsynaptic complex formed by LGI1 at excitatory synapses, including presynaptic Kv1 potassium channels. Patient-derived monoclonal antibodies (mAbs) are ideal reagents to study whether domain-specific LGI1-autoantibodies induce epileptiform activities in neurons, and their downstream mechanisms. To address this question, we measured the intrinsic excitability of CA3 pyramidal neurons in organotypic cultures from rat hippocampus treated with either a LRR- or an EPTP-reactive patient-derived mAb. The antibodies induced changes in neuronal intrinsic excitability which led us to measure their effects on Kv1-type potassium currents. We found an increase of intrinsic excitability correlated with a reduction of the sensitivity to a selective Kv1.1-channel blocker in neurons treated with the LRR mAb compared to the control, but not in neurons treated with the EPTP mAb. Our findings suggest LRR mAbs are able to modulate neuronal excitability that could account for epileptiform activities observed in patients.


title: 4-phenylbutyrate restored GABA uptake and reduced seizures in SLC6A1 variants-mediated disorders

abstract: We have previously studied the molecular mechanisms of solute carrier family 6 member 1 (SLC6A1) associated with a continuum of neurodevelopmental disorders, including various epilepsy syndromes, autism, and intellectual disability. Based on functional assays of variants in a large cohort with heterogenous clinical phenotypes, we conclude that partial or complete loss of GABA uptake function in the mutant GAT-1 is the primary etiology as identified in GABAA receptor mutation-mediated epilepsy and in cystic fibrosis. Importantly, we identified that there are common patterns of the mutant protein trafficking from biogenesis, oligomerization, glycosylation, and translocation to the cell membrane across variants with the conservation of this process across cell types. Conversely any approach to facilitate membrane trafficking would increase presence of the functional protein in the targeted destination in all involved cells. PBA is an FDA-approved drug for pediatric use and is orally bioavailable so it can be quickly translated to patient use. It has been demonstrated that PBA can correct protein misfolding, reduce ER stress, and attenuate unfolded protein response in neurodegenerative diseases, it has also showed promise in treatment of cystic fibrosis. The common cellular mechanisms shared by the mutant GAT-1 and the mutant cystic fibrosis transmembrane conductance regulator led us to test if PBA and other pharmaco-chaperones could be a potential treatment option for SLC6A1 mutations. We examined the impact of PBA and other small molecules in a library of variants and in cell and knockin mouse models. Because of the critical role of astrocytic GAT-1 deficit in seizures, we focused on astrocytes, and demonstrated that the existence of the mutant GAT-1 retained the wildtype GAT-1, suggesting aberrant protein oligomerization and trafficking caused by the mutant GAT-1. PBA increased GABA uptake in both mouse and human astrocytes bearing the mutations. Importantly, PBA increased GAT-1 expression and suppressed spike wave discharges (SWDS) in the heterozygous knockin mice. Although the detailed mechanisms of action for PBA are ambiguous, it is likely that PBA can facilitate the forward trafficking of the wildtype GAT-1 favoring over the mutant GAT-1, thus increasing GABA uptake. Since all patients with SLC6A1 mutations are heterozygous and carry one wildtype functional allele, this suggests a great opportunity for treatment development by leveraging the endogenous protein trafficking pathway to promote forward trafficking of the wildtype in combination with enhancing the disposal of the mutant allele as treatment mode. The study opens a novel avenue of treatment development for genetic epilepsy via drug repurposing.


title: Aneuploidy influences the gene expression profiles in Saccharomyces pastorianus group I and II strains during fermentation.

abstract: The lager yeasts, Saccharomyces pastorianus, are hybrids of Saccharomyces cerevisiae and Saccharomyces eubayanus and are divided into two broad groups, Group I and II. The two groups evolved from at least one common hybridisation event but have subsequently diverged with Group I strains losing many S. cerevisiae chromosomes while the Group II strains retain both sub-genomes. The complex genomes, containing orthologous alleles from the parental chromosomes, pose interesting questions regarding gene regulation and its impact on the fermentation properties of the strains. Superimposed on the presence of orthologous alleles are complexities of gene dosage due to the aneuploid nature of the genomes. We examined the contribution of the S. cerevisiae and S. eubayanus alleles to the gene expression patterns of Group I and II strains during fermentation. We show that the relative expression of S. cerevisiae and S. eubayanus orthologues is positively correlated with gene copy number. Despite the reduced S. cerevisiae content in the Group I strain, S. cerevisiae orthologues contribute to biochemical pathways upregulated during fermentation which may explain the retention of specific chromosomes in the strain. Conversely, S. eubayanus genes are significantly overrepresented in the upregulated gene pool in the Group II strain. Comparison of the transcription profiles of Group I and II strains during fermentation identified both common and unique gene expression patterns, with gene copy number being a dominant contributory factor. Thus, the aneuploid genomes create complex patterns of gene expression during fermentation with gene dosage playing a crucial role both within and between strains.