title: Caenorhabditis nematodes colonize ephemeral resource patches in neotropical forests.

abstract: Factors shaping the distribution and abundance of species include life-history traits, population structure, and stochastic colonization-extinction dynamics. Field studies of model species groups help reveal the roles of these factors. Species of Caenorhabditis nematodes are highly divergent at the sequence level but exhibit highly conserved morphological uniformity, and many of these species live in sympatry on microbe-rich patches of rotten material. Here, we use field experiments and large-scale opportunistic collections to investigate species composition, abundance, and colonization efficiency of Caenorhabditis in two of the worlds best studied lowland tropical field sites: Barro Colorado Island in Panama and La Selva in Sarapiqui, Costa Rica. We observed seven species of Caenorhabditis, four of them known only from these collections. While these localities contain species from many parts of the phylogeny, both localities were dominated by globally distributed androdiecious species. We found that Caenorhabditis were able to colonize baits accessible only by phoresy, preferring to colonize baits making direct contact with the ground. We estimate founder numbers per colonization event to be low.


title: The salmon louse genome may be much larger than sequencing suggests

abstract: The genome size of organisms impacts their evolution and biology and is often assumed to be characteristic of a species. Here we present the first published estimates of genome size of the ecologically and economically important ectoparasite, Lepeophtheirus salmonis (Copepoda, Caligidae). Four independent L. salmonis genome assemblies of the North Atlantic subspecies Lepeophtheirus salmonis salmonis, including two chromosome level assemblies, yield assemblies ranging from 665 - 790 Mbps. These genome assemblies are congruent in their findings, and appear very complete with Benchmarking Universal Single-Copy Orthologs analyses finding >92% of expected genes and transcriptome datasets routinely mapping >90% of reads. However, two cytometric techniques, flow cytometry and Feulgen image analysis densitometry, yield measurements of 1.3-1.6 Gb in the haploid genome. Interestingly, earlier cytometric measurements reported genome sizes of 939 and 567 Mbps in L. salmonis salmonis samples from Bay of Fundy and Norway, respectively. Available data thus suggest that the genome sizes of salmon lice are variable. Current understanding of eukaryotic genome dynamics suggests that the most likely explanation for such variability involves repetitive DNA, which for L. salmonis makes up {approx}60% of the genome assemblies.

category: genetics


title: Meiosis in the human pathogen Aspergillus fumigatus has the highest known number of crossovers

abstract:
Evidence from both population genetics and a laboratory sexual cycle indicate that sex is common in the fungus Aspergillus fumigatus. However, the impact of sexual reproduction has remained unclear. Here, we show that meiosis in A. fumigatus involves the highest known recombination rate, producing ~29 crossovers per chromosome. This represents the highest known crossover rate for any Eukaryotic species. We validate this recombination rate by mapping resistance to acriflavine, a common genetic marker. We further show that this recombination rate can produce the commonly encountered TR34/L98H azole-resistant cyp51A haplotype in each sexual event, facilitating its rapid and global spread. Understanding the consequences of this unparalleled crossover rate will not only enrich our genetic understanding of this emergent human pathogen, but of meiosis in general.

One-Sentence SummaryGenetic exchange between chromosomes during sex in Aspergillus fumigatus is higher than in any other known organism.


title: Identification of a transcriptional signature found in multiple models of ASD and related disorders

abstract: Epigenetic regulation plays a critical role in many neurodevelopmental disorders, including Autism Spectrum Disorder (ASD). In particular, many such disorders are the result of mutations in genes that encode chromatin modifying proteins. However, while these disorders share many features, it is unclear whether they also share gene expression disruptions resulting from the aberrant regulation of chromatin. We examined 5 chromatin modifiers that are all linked to ASD despite their different roles in regulating chromatin. Specifically, we depleted Ash1L, Chd8, Crebbp, Ehmtl, and Nsd1 in parallel in a highly controlled neuronal culture system. We then identified sets of shared genes, or transcriptional signatures, that are differentially expressed following loss of multiple ASD-linked chromatin modifiers. We examined the functions of genes within the transcriptional signatures and found an enrichment in many neurotransmitter transport genes and activity-dependent genes. In addition, these genes are enriched for specific chromatin features such as bivalent domains that allow for highly dynamic regulation of gene expression. The downregulated transcriptional signature is also observed within multiple mouse models of neurodevelopmental disorders that result in ASD, but not those only associated with intellectual disability. Finally, the downregulated transcriptional signature can distinguish between neurons generated from iPSCs derived from healthy donors and idiopathic ASD patients through RNA-deconvolution, demonstrating that this gene set is relevant to the human disorder. This work identifies a transcriptional signature that is found within many neurodevelopmental syndromes, helping to elucidate the link between epigenetic regulation and the underlying cellular mechanisms that result in ASD.

category: genomics


title: Structural Basis for the Calmodulin-Mediated Activation of eEF-2K

abstract: Translation is a highly energy consumptive process1 tightly regulated for optimal protein quality2 and adaptation to energy and nutrient availability. A key facilitator of this process is the -kinase eEF-2K that specifically phosphorylates the GTP-dependent translocase eEF-2, thereby reducing its affinity for the ribosome and suppressing the elongation phase of protein synthesis3,4. eEF-2K activation requires calmodulin binding and auto-phosphorylation at the primary stimulatory site, T348. Biochemical studies have predicted that calmodulin activates eEF-2K through a unique allosteric process5 mechanistically distinct from other calmodulin-dependent kinases6. Here we resolve the atomic details of this mechanism through a 2.3 [A] crystal structure of the heterodimeric complex of calmodulin with the functional core of eEF-2K (eEF-2KTR). This structure, which represents the activated T348-phosphorylated state of eEF-2KTR, highlights how through an intimate association with the calmodulin C-lobe, the kinase creates a "spine" that extends from its N-terminal calmodulin-targeting motif through a conserved regulatory element to its active site. Modification of key spine residues has deleterious functional consequences.

category: biochemistry


title: Drug Contraindications in Comorbid Diseases: a Protein Interactome Perspective

abstract: Adverse drug reactions (ADRs) are leading causes of death and drug withdrawals and frequently cooccur with comorbidities. However, systematic studies on the effects of drugs in comorbidities are lacking. Drug interactions with the cellular protein-protein interaction (PPI) network give rise to ADRs. We selected 6 comorbid disease pairs, identified the drugs used in the treatment of the individual diseases  A and  B- 44 drugs in anxiety and depression, 128 in asthma and hypertension, 48 in chronic obstructive pulmonary disease and heart failure, 58 in type 2 diabetes and obesity, 58 in Parkinsons disease and schizophrenia, and 84 in rheumatoid arthritis and osteoporosis - and categorized them based on whether they aggravate the comorbid condition. We constructed drug target networks (DTNs) and examined their enrichment among genes in disease A/B PPI networks, expressed across 53 tissues and involved in ~1000 pathways. To pinpoint the biological features characterizing the DTNs, we performed principal component analysis and computed the Euclidean distance between DTN component scores and feature loading values. DTNs of disease A drugs not contraindicated in B were affiliated with proteins common to A/B networks or uniquely found in the B network, similarly regulated common pathways, and disease-B specific pathways and tissues. DTNs of disease A drugs contraindicated in B were affiliated with common proteins or those uniquely found in the A network, differentially regulated common pathways, and disease A-specific pathways and tissues. Hence, DTN enrichment in pathways, tissues, and PPI networks of comorbid diseases will help identify drugs contraindications in comorbidities.

category: bioinformatics


title: Structure-Based Survey of the Human Proteome for Opportunities in Proximity Pharmacology

abstract: Proximity pharmacology (ProxPharm) is a novel paradigm in drug discovery where a small molecule brings two proteins in close proximity to elicit a signal, generally from one protein onto another. The potential of ProxPharm compounds as a new therapeutic modality is firmly established by proteolysis targeting chimeras (PROTACs) that bring an E3 ubiquitin ligase in proximity to a target protein to induce ubiquitination and subsequent degradation of the target protein. The concept can be expanded to induce other post-translational modifications via the recruitment of different types of protein-modifying enzymes. To survey the human proteome for opportunities in proximity pharmacology, we systematically mapped non-catalytic drug binding pockets on the structure of protein-modifying enzymes available from the Protein Databank. In addition to binding sites exploited by previously reported ProxPharm compounds, we identified putative ligandable non-catalytic pockets in 188 kinases, 42 phosphatases, 26 deubiquitinases, 9 methyltransferases, 7 acetyltransferases, 7 glycosyltransferases, 4 deacetylases, 3 demethylases and 2 glycosidases, including cavities occupied by chemical matter that may serve as starting points for future ProxPharm compounds. This systematic survey confirms that proximity pharmacology is a versatile modality with largely unexplored and promising potential, and reveals novel opportunities to pharmacologically rewire molecular circuitries.


title: Benchmarking software to predict antibiotic resistance phenotypes in shotgun metagenomes using simulated data

abstract:
The use of shotgun metagenomics for AMR detection is appealing because data can be generated from clinical samples with minimal processing. Detecting antimicrobial resistance (AMR) in clinical genomic data is an important epidemiological task, yet a complex bioinformatic process. Many software tools exist to detect AMR genes, but they have mostly been tested in their detection of genotypic resistance in individual bacterial strains. It is important to understand how well these bioinformatic tools detect AMR genes in shotgun metagenomic data.

We developed a software pipeline, hAMRoaster (https://github.com/ewissel/hAMRoaster), for assessing accuracy of prediction of antibiotic resistance phenotypes. For evaluation purposes, we simulated a short read (Illumina) shotgun metagenomics community of eight bacterial pathogens with extensive antibiotic susceptibility testing profiles. We benchmarked nine open source bioinformatics tools for detecting AMR genes that 1) were conda or Docker installable, 2) had been actively maintained, 3) had an open source license, and 4) took FASTA or FASTQ files as input. Several metrics were calculated for each tool including sensitivity, specificity, and F1 at three coverage levels.

This study revealed that tools were highly variable in sensitivity (0.25 - 0.99) and specificity (0.2 - 1) in detection of resistance in our synthetic FASTQ files despite similar databases and methods implemented. Tools performed similarly at all coverage levels (5x, 50x, 100x). Cohens kappa revealed low agreement across tools.

ImportanceSoftware selection for metagenomic AMR prediction should be driven by the context of the clinical/research questions and tolerance for true and false negative results. As the prediction software and databases are in a state of constant refinement, the approach used here--creating synthetic communities containing taxa and phenotypes of interest along with using hAMRoaster to assess performance of candidate software--offers a template to aid researchers in selecting the most appropriate strategy.


title: Phase Separation of Shell Protein and Enzyme: An Insight into the Biogenesis of a Prokaryotic Metabolosome

abstract:
Bacterial microcompartments are substrate specific metabolic modules that are conditionally expressed in certain bacterial species. These all protein structures have size in the range of 100-150 nm and are formed by the self-assembly of thousands of protein subunits, all encoded by genes belonging to a single operon. The operon contains genes that encode for both enzymes and shell proteins. The shell proteins self-assemble to form the outer coat of the compartment and enzymes are encapsulated within. A perplexing question in MCP biology is to understand the mechanism which governs the formation of these small yet complex assemblages of proteins. In this work we use 1,2-propanediol utilization microcompartments (PduMCP) as a paradigm to identify the factors that drive the self-assembly of MCP proteins. We find that a major shell protein PduBB tend to self-assemble under macromolecular crowded environment and suitable ionic strength. Microscopic visualization and biophysical studies reveal phase separation to be the principle mechanism behind the self-association of shell protein in the presence of salts and macromolecular crowding. The shell protein PduBB interacts with the enzyme diol-dehydratase PduCDE and co-assemble into phase separated liquid droplets. The co-assembly of PduCDE and PduBB results in the enhancement of catalytic activity of the enzyme. A combination of spectroscopic and biochemical techniques shows the relevance of divalent cation Mg2+ in providing stability to intact PduMCP in vivo. Together our results suggest a combination of protein-protein interactions and phase separation guiding the self-assembly of Pdu shell protein and enzyme in solution phase.

Significance StatementPresent work shows how surrounding environment modulates the self-assembly behavior of a major shell protein of 1,2-propanediol utilization microcompartment (PduMCP). Appropriate ionic strength and macromolecular crowding bring about liquid-liquid phase separation of the shell protein. Under crowded environment Mg2+ displayed unique property to drive the formation of shell protein liquid condensates. The co-phase separation of a native enzyme along with the shell protein enables it to outperform the enzyme in isolation. This adds on to the existing concept of phase separation being the underlying principle behind the genesis of lipid free organelles in prokaryotes. Further, our results indicate that the divalent metal ion Mg2+ plays an intricate role in the outfitting of the structure-function integrity of PduMCPs.

Graphical Abstract

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category: biophysics


title: Role of the circadian clock "Death-Loop" in the DNA damage response underpinning cancer treatment resistance

abstract: The Circadian Clock (CC) drives the normal cell cycle and reciprocally regulates telomere elongation. However, it can be deregulated in cancer, embryonic stem cells (ESC), and the early embryo. Here, its role in the resistance of cancer cells to genotoxic treatments was assessed in relation to whole-genome duplication (WGD) and telomere regulation. We first evaluated the DNA damage response of polyploid cancer cells and observed a similar impact on the cell cycle to that seen in ESC - overcoming G1/S, adapting DNA damage checkpoints, tolerating DNA damage, and coupling telomere erosion to accelerated cell senescence, favouring transition by mitotic slippage into the ploidy cycle (reversible polyploidy). Next, we revealed a positive correlation between cancer WGD and deregulation of CC assessed by bioinformatics on 11 primary cancer datasets (rho=0.83; p<0.01). As previously shown, the cancer cells undergoing mitotic slippage cast off telomere fragments with TERT, restore the telomeres by recombination and return their depolyploidised mitotic offspring to TERT-dependent telomere regulation. Through depolyploidisation and the CC "death loop" the telomeres and Hayflick limit count are thus again renewed. This mechanism along with similar inactivity of the CC in early embryos support a life-cycle (embryonic) concept of cancer.

category: cancer biology


title: Mitochondrial and redox modifications in early stages of Huntington disease

abstract: Defects in mitochondrial function and mitochondrial-related redox deregulation have been attributed to Huntingtons disease (HD), a genetic neurodegenerative disorder largely affecting the striatum. However, whether these changes occur in early stages of the disease and can be detected in vivo is still unclear. Thus, in the present study, we analyzed changes in mitochondrial function and overreduced states associated with production of reactive oxygen species (ROS) at early stages and along disease progression. Studies were performed in vivo in human brain using positron emission tomography (PET) using [64Cu]-ATSM and ex vivo in human skin fibroblasts of premanifest and prodromal (Pre-M) and manifest HD patients; in vivo brain [64Cu]-ATSM PET and isolated mitochondria derived from striatum and cortex were also analyzed in YAC128 transgenic mouse at pre-symptomatic (3 month-old, mo) and symptomatic (6 to 12 mo) stages. Oxygen consumption rates were assessed by Seahorse analysis, hydrogen peroxide levels were determined using fluorescent probes and mitochondrial morphology by transmission electron microscopy in human skin fibroblasts and mouse striatal and cortical isolated mitochondria. Pre-M HD carriers exhibited enhanced whole-brain (with exception of caudate) [64Cu]-ATSM labelling, correlating with CAG repeat number. Fibroblasts from Pre-M showed enhanced basal and maximal respiration, proton (H+) leak and increased hydrogen peroxide levels, the later progressing to manifest HD; mitochondria from fibroblasts of Pre-M HD carriers also showed reduced roundness, while higher number of mitochondrial DNA copies correlated with maximal respiratory capacity. In vivo animal PET analysis showed increased accumulation of [64Cu]-ATSM in YAC128 mouse striatum. Pre-symptomatic YAC128 mouse striatal isolated mitochondria exhibited a rise in basal and maximal mitochondrial respiration and in ATP production, along with increased complex II and III activities; mouse HD mitochondria also showed enhanced mitochondrial hydrogen peroxide levels and roundness, as revealed by brain ultrastructure analysis, and defects in Ca2+ handling, supporting increased striatal susceptibility in YAC128 mouse brain. Data demonstrate both human and mouse mitochondrial overactivity and altered morphology at early HD stages, facilitating redox unbalance, the latter extending over manifest disease stages.

category: neuroscience


title: Plasma cell maintenance and antibody secretion are under the control of Sec22b-mediated regulation of organelle dynamics

abstract: Despite the essential role of plasma cells in health and disease, the cellular mechanisms controlling their survival and secretory capacity are still poorly understood. Here, we identified the SNARE Sec22b as a unique and critical regulator of plasma cell maintenance and function. In absence of Sec22b, plasma cells were barely detectable and serum antibody titres were dramatically reduced. Accordingly, Sec22b deficient mice fail to mount a protective immune response. At the mechanistic level, we demonstrated that Sec22b is indispensable for efficient antibody secretion but also for plasma cell fitness through the regulation of the morphology of the endoplasmic reticulum and mitochondria. Altogether, our results unveil a critical role for Sec22b-mediated regulation of plasma cell biology through the control of organelle dynamics.

category: immunology


title: Population genomic evidence of selection on structural variants in a natural hybrid zone

abstract: Structural variants (SVs) can promote speciation by directly causing reproductive isolation or by suppressing recombination across large genomic regions. Whereas examples of each mechanism have been documented, systematic tests of the role of SVs in speciation are lacking. Here, we take advantage of long-read (Oxford nanopore) whole-genome sequencing and a hybrid zone between two Lycaeides butterfly taxa (L. melissa and Jackson Hole Lycaeides) to comprehensively evaluate genome-wide patterns of introgression for SVs and relate these patterns to hypotheses about speciation. We found >100,000 SVs segregating within or between the two hybridizing species. SVs and SNPs exhibited similar levels of genetic differentiation between species, with the exception of inversions, which were more differentiated. We detected credible variation in patterns of introgression among SV loci in the hybrid zone, with 562 of 1419 ancestry-informative SVs exhibiting genomic clines that deviating from null expectations based on genome-average ancestry. Overall, hybrids exhibited a directional shift towards Jackson Hole Lycaeides ancestry at SV loci, consistent with the hypothesis that these loci experienced more selection on average then SNP loci. Surprisingly, we found that deletions, rather than inversions, showed the highest skew towards excess introgression from Jackson Hole Lycaeides. Excess Jackson Hole Lycaeides ancestry in hybrids was also especially pronounced for Z-linked SVs and inversions containing many genes. In conclusion, our results show that SVs are ubiquitous and suggest that SVs in general, but especially deletions, might contribute disproportionately to hybrid fitness and thus (partial) reproductive isolation.


title: Transverse and axial resolution of femtosecond laser ablation

abstract: Femtosecond lasers are capable of precise ablation that produce surgical dissections in vivo. The transverse and axial resolution of the laser damage inside the bulk are important parameters of ablation. The transverse resolution is routinely quantified, but the axial resolution is more difficult to measure and is less commonly performed. In some in vivo samples, fine dissections can also be difficult to visualize, but in vitro samples may allow clear imaging. Using a 1040-nm, 400-fs pulsed laser, we performed ablation inside agarose and glass, producing clear and persistent damage spots. Near the ablation threshold of both media, we found that the axial resolution is similar to the transverse resolution. We also ablated neuron cell bodies and fibers in C. elegans and demonstrate submicrometer resolution in both the transverse and axial directions, consistent with our results in agarose and glass. Using simple yet rigorous methods, we define the resolution of laser ablation in transparent media along all directions.

category: bioengineering


title: Bicarbonate Activation of Monomeric Photosystem II-PsbS/Psb27 Complex

abstract:
In thylakoid membranes, Photosystem II monomers from the stromal lamellae contain the subunits PsbS and Psb27 (PSIIm-S/27), while Photosystem II monomers from granal regions (PSIIm) lack these subunits. Here, we have isolated and characterised these two types of Photosystem II complexes. The PSIIm-S/27 showed enhanced fluorescence, the near-absence of oxygen evolution, as well as limited and slow electron transfer from QA to QB compared to the near-normal activities in the granal PSIIm. However, when bicarbonate was added to the PSIIm-S/27, water splitting and QA to QB electron transfer rates were comparable to those in granal PSIIm. The findings suggest that the binding of PsbS and/or Psb27 inhibits forward electron transfer and lowers the binding affinity for the bicarbonate. This can be rationalized in terms of the recently discovered photoprotection role played by bicarbonate binding via the redox tuning of the QA/QA*- couple, which controls the charge recombination route, and this limits chlorophyll triplet mediated 1O2 formation (Brinkert K et al. (2016) Proc Natl Acad Sci U S A. 113(43):12144-12149). These findings suggest that PSIIm-S/27 is an intermediate in the assembly of PSII in which PsbS and/or Psb27 restrict PSII activity while in transit, by using a bicarbonate-mediated switch and protective mechanism.

One sentence summaryA photosystem II monomer with PsbS and Psb27 as additional subunits, is inactive as isolated but activated by bicarbonate, and is attributed to be a late-stage intermediate in photoassembly.

category: plant biology


title: TGF-β-R2 is required for HBP induced acute lung injury and vascular leakage for TGF-/Smad/Rho signaling pathway activation

abstract: Heparin-binding protein (HBP), as a granule protein secreted by polymorphonuclear neutrophils (PMNs) participates in the pathophysiological process of sepsis. It has been reported that HBP is a biomarker of sepsis, which is related to the severity of septic shock and organ dysfunction. HBP binds to vascular endothelial cells as one of the primary target sites. However, it is still unclear whether HBP-binding protein receptors exist on the surface of ECs. The effect of HBP on vascular permeability in sepsis and its mechanism needs to be explored. We conducted in vivo and in vitro study. We demonstrated that HBP bound to transforming growth factor-{beta} receptor type 2 (TGF-{beta}-R2) as a ligand. GST pull-down analysis reveals that HBP mainly interacts with the extracellular domain of TGF-{beta}-R2. HBP induced acute lung injury (ALI) and vascular leakage via activation of TGF-{beta}/SMAD2/3 signaling pathway. Permeability assay suggests TGF-{beta}-R2 is necessary for HBP-induced increased permeability. We also defined the role of HBP and its potential membrane receptor TGF-{beta}-R2 in the blood-gas barrier in the pathogenesis of HBP-related ALI.

category: molecular biology


title: Partial sex linkage and linkage disequilibrium on the guppy sex chromosome

abstract: The guppy Y chromosome has been considered a model system for the evolution of suppressed recombination between sex chromosomes, and it has been proposed that complete sex-linkage has evolved across about 3 Mb surrounding this fishs sex-determining locus, followed by recombination suppression across a further 7 Mb of the 23 Mb XY pair, forming younger "evolutionary strata". Sequences of the guppy genome show that Y is very similar to the X chromosome, making it important to understand which parts of the Y are completely non-recombining, and whether there is indeed a large completely non-recombining region. Here, we describe new evidence that supports a different interpretation of the data that suggested the presence of such a region. We analysed PoolSeq data in samples from multiple natural populations from Trinidad. This yields evidence for linkage disequilibrium (LD) between sequence variants and the sex-determining locus. Downstream populations have higher diversity than upstream ones (which display the expected signs of bottlenecks). The associations we observe conform to predictions for a genome region with infrequent recombination that carries one or more sexually antagonistic polymorphisms. They also suggest the region in which the sex-determining locus must be located. However, no consistently male-specific variants were found, supporting the suggestion that any completely sex-linked region may be very small.

category: evolutionary biology


title: Metal-binding propensity in the mitochondrial dynamin-related protein 1

abstract: Dynamin-related protein1 (Drp1) functions to divide mitochondria and peroxisomes by binding specific adaptor proteins and lipids, both of which are integral to the limiting organellar membrane. In efforts to understand how such multivalent interactions regulate Drp1 functions, in vitro reconstitution schemes rely on recruiting soluble portions of the adaptors appended with genetically encoded polyhistidine tags onto membranes containing Ni2+-bound chelator lipids. These strategies are facile and circumvent the challenge in working with membrane proteins but assume that binding is specific to proteins carrying the polyhistidine tag. Here, we find using chelator lipids and chelator beads that both native and recombinant Drp1 directly bind Ni2+ ions. Metal-binding therefore represents a potential strategy to deplete or purify Drp1 from native tissue lysates. Importantly, high concentrations of the metal in solution inhibit GTP hydrolysis and renders Drp1 inactive in membrane fission. Together, our results emphasize a metal-binding propensity, which could significantly impact Drp1 functions.


title: Cryo-EM reconstruction of the human 40S ribosomal subunit at 2.15 A resolution

abstract: The chemical modification of ribosomal RNA and proteins is critical for ribosome assembly, for protein synthesis and may drive ribosome specialization in development and disease. However, the inability to accurately visualize these modifications has limited mechanistic understanding of the role of these modifications in ribosome function. Here we report the 2.15 [A] resolution cryo-EM reconstruction of the human 40S ribosomal subunit. We directly visualize post-transcriptional modifications within the 18S rRNA and post-translational modifications at the N-termini of two ribosomal proteins. Additionally, we interpret the solvation shells in the core regions of the 40S ribosomal subunit and reveal how potassium and magnesium ions establish both universally conserved and eukaryote-specific coordination to promote the stabilization and folding of key ribosomal elements. This work provides unprecedented structural details for the human 40S ribosomal subunit that will serve as an important reference for unraveling the functional role of ribosomal RNA modifications.


title: Structural Insights into Sphingosine-1-phosphate Receptor Activation

abstract: As a critical sphingolipid metabolite, sphingosine-1-phosphate (S1P) plays an essential role in immune and vascular systems. There are five S1P receptors, designated as S1PR1-5, encoded in the human genome, and their activities are governed by endogenous S1P, lipid-like S1P mimics, or non-lipid-like therapeutic molecules. Among S1PRs, S1PR1 stands out due to its non-redundant functions, such as the egress of T and B cells from the thymus and secondary lymphoid tissues, making it a potential therapeutic target. However, the structural basis of S1PR1 activation and regulation by various agonists remains unclear. Here we reported four atomic resolution cryo-EM structures of Gi-coupled human S1PR1 complexes: bound to endogenous agonist d18:1 S1P, benchmark lipid-like S1P mimic phosphorylated Fingolimod ((S)-FTY720-P), or non-lipid-like therapeutic molecule CBP-307 in two binding modes. Our results revealed the similarities and differences of activation of S1PR1 through distinct ligands binding to the amphiphilic orthosteric pocket. We also proposed a two-step "shallow to deep" transition process of CBP-307 for S1PR1 activation. Both binding modes of CBP-307 could activate S1PR1, but from shallow to deep transition may trigger the rotation of the N-terminal helix of Gi and further stabilize the complex by increasing the Gi interaction with the cell membrane. We combine with extensive biochemical analysis and molecular dynamic simulations to suggest key steps of S1P binding and receptor activation. The above results decipher the common feature of the S1PR1 agonist recognition and activation mechanism and will firmly promote the development of therapeutics targeting S1P receptors.


title: Escherichia coli response to subinhibitory concentration of Colistin: Insights from study of membrane dynamics and morphology.

abstract: Prevalence of wide spread bacterial infections bring forth a critical need in understanding the molecular mechanisms of the antibiotics as well as the bacterial response to those antibiotics. Improper usage of antibiotics, which can be in sub-lethal concentrations is one among the multiple reasons for acquiring antibiotic resistance which makes it vital to understand the bacterial response towards sub-lethal concentrations of antibiotics. In this work, we have used colistin, a well-known membrane active antibiotic used to treat severe bacterial infections and explored the impact of its sub-minimum inhibitory concentration (MIC) on the lipid membrane dynamics and morphological changes of E. coli. Upon investigation of live cell membrane properties such as lipid dynamics using fluorescence correlation spectroscopy, we observed that colistin disrupts the lipid membrane at sub-MIC by altering the lipid diffusivity. Interestingly, filamentation-like cell elongation was observed upon colistin treatment which led to further exploration of surface morphology with the help of atomic force spectroscopy. The changes in the surface roughness upon colistin treatment provides additional insight on the colistin-membrane interaction corroborating with the altered lipid diffusion. Although altered lipid dynamics could be attributed to an outcome of lipid rearrangement due to direct disruption by antibiotic molecules on the membrane or an indirect consequence of disruptions in lipid biosynthetic pathways, we were able to ascertain that altered bacterial membrane dynamics is due to direct disruptions. Our results provide a broad overview on the consequence of the cyclic polypeptide, colistin on membrane specific lipid dynamics and morphology of a live Gram-negative bacterial cell.


title: Asymmetric Pendrin Homodimer Reveals its Molecular Mechanism as Anion Exchanger

abstract:
Pendrin SLC26A4 is an anion exchanger expressed in apical membranes of selected epithelia. Pendrin ablation causes Pendred syndrome, a genetic disorder disease associated with sensorineural hearing loss, hypothyroid goiter, and reduced blood pressure. However, its molecular structure has remained unknown limiting our understanding. Here, we determined the structures of mouse pendrin with symmetric and characteristically asymmetric homodimer conformations by cryo-electron microscopy. The asymmetric homodimer consists of an inward-facing protomer and an intermediate-state protomer, representing the coincident uptake and secretion process, and exhibits the unique state of pendrin as an electroneutral exchanger. This previously unrevealed conformation, together with other conformations we captured, provides an inverted alternate-access mechanism for anion exchange. Furthermore, our structural and functional data disclosed the properties of anion exchange cleft and interpreted the important pathogenetic mutations. These investigations shed light on the pendrin exchange mechanism and extend our structure-guided understanding of pathogenetic mutations.

One-Sentence SummaryCryo-EM asymmetric Pendrin homodimer cracks its exchange mechanism and guides our understanding for pathogenetic mutations.


title: Kynurenine metabolism is altered in mdx mice: a potential muscle to brain connection

abstract: Regular exercise can direct muscle kynurenine (KYN) metabolism toward the neuroprotective branch of the kynurenine pathway thereby limiting the accumulation of neurotoxic metabolites in the brain and contributing to mental resilience. While the effect of regular exercise has been studied, the effect of muscle disease on KYN metabolism has not yet been investigated. Previous work has highlighted anxiety-like behaviors in approximately 25% of patients with DMD, possibly due to altered KYN metabolism. Here, we characterized KYN metabolism in mdx mouse models of Duchenne muscular dystrophy (DMD). Young (8-10 week old) DBA/2J (D2) mdx mice, but not age-matched C57BL/10 (C57) mdx mice, had lower levels of circulating KYNA and KYNA:KYN ratio compared with their respective wild-type (WT) controls. Moreover, only D2 mdx mice displayed signs of anxiety-like behaviour, spending more time in the corners of their cages during a novel object recognition test when compared with WT. Along with this, we found that muscles from D2 mdx mice had less peroxisome proliferator-activated receptor-gamma coactivator 1-alpha and kynurenine amino transferase-1 enzyme content as well as elevated expression of inflammatory cytokines compared with WT muscles. Thus, our pilot work shows that KYN metabolism is altered in D2 mdx mice, with a potential contribution from altered muscle health.

category: physiology


title: Endothelial Nitric Oxide Synthase (eNOS) S1176 phosphorylation status governs atherosclerotic lesion formation

abstract:
ObjectiveWe have previously demonstrated the in vivo importance of the Akt-eNOS substrate-kinase relationship, as defective postnatal angiogenesis characteristic of global Akt1-null mice is rescued when bred to  gain-of-function eNOS S1176D mutant mice. While multiple studies support the cardioprotective role of endothelial NO generation, the causal role of Akt1-dependent eNOS S1176 phosphorylation during atherosclerotic plaque formation is not yet clear.

Approach & ResultsWe herein bred congenic  loss-of-function eNOS S1176A and  gain-of-function eNOS S1176D mutant mice to the proatherogenic Akt1-/-; ApoE-/- double knockout mice to definitively test the importance of Akt-mediated eNOS S1176 phosphorylation during atherogenesis. We find that a single amino acid substitution at the eNOS S1176 phosphorylation site yields divergent effects on atherosclerotic plaque formation, as an eNOS phospho-mimic aspartate (D) substitution at S1176 leads to decreased indices of atherosclerosis, even when on a proatherogenic Akt1 global deletion background. Conversely, mice harboring an unphosphorylatable mutation to alanine (S1176A) result in increased lipid deposition and cellular apoptosis, phenocopying the physiological consequence of eNOS deletion and/or impaired enzyme function. Furthermore, gene expression analyses of whole aortas indicate a combinatorial detriment from NO deficiency and Western Diet challenge, as  loss-of-function eNOS SA mice on a high-fat and high-cholesterol diet present a unique expression pattern indicative of augmented T-cell activity when compared to eNOS S1176D mice.

ConclusionsBy using genetic epistasis approaches, we conclusively demonstrate that Akt-mediated eNOS S1176 phosphorylation and subsequent activation remains to be the most physiologically relevant method of NO production to promote cardioprotective effects.


title: NF-κB memory coordinates transcriptional responses to dynamic inflammatory stimuli

abstract: Many scenarios in cellular communication requires cells to interpret multiple dynamic signals. It is unclear how exposure to immune stimuli alters transcriptional responses to subsequent stimulus under inflammatory conditions. Using high-throughput microfluidic live cell analysis, we systematically profiled the NF-{kappa}B response to different signal sequences in single cells. We found that NF-{kappa}B dynamics stores the history of signals received by cells: depending on the dose and type of prior pathogenic and cytokine signal, the NF-{kappa}B response to subsequent stimuli varied widely, from no response to full activation. Using information theory, we revealed that these stimulus-dependent changes in the NF-{kappa}B response encode and reflect information about the identity and dose of the prior stimulus. Small-molecule inhibition, computational modeling, and gene expression profiling show that this encoding is driven by stimulus-dependent engagement of negative feedback modules. These results provide a model for how signal transduction networks process sequences of inflammatory stimuli to coordinate cellular responses in complex dynamic environments.

category: systems biology


title: B cells discriminate HIV-1 Envelope protein affinities by sensing antigen binding association rates

abstract: HIV-1 Envelope (Env) proteins designed to induce neutralizing antibody responses allow study of the role of affinities (equilibrium dissociation constant, KD) and kinetic rates (association/dissociation rates) on B cell antigen recognition. It is unclear whether affinity discrimination during B cell activation is based solely on Env protein binding KD, and whether B cells discriminate between proteins of similar affinities but that bind with different kinetic rates. Here we used a panel of Env proteins and Ramos B cell lines expressing IgM BCRs with specificity for CD4 binding-site broadly neutralizing (bnAb) or a precursor antibody to study the role of antigen binding kinetic rates on both early (proximal/distal signaling) and late events (BCR/antigen internalization) in B cell activation. Our results support a kinetic model for B cell activation in which Env protein affinity discrimination is based not on overall KD, but on sensing of association rate and a threshold antigen-BCR half-life.


title: ART duration and immunometabolic state determine efficacy of DC-based treatment restoring functional HIV- specific CD8+ T cells in Chronic PLWH.

abstract: Dysfunction of CD8+ T cells in people living with HIV-1 (PLWH) receiving anti-retroviral therapy (ART) has restricted the efficacy of dendritic cell (DC)-based immunotherapies against HIV-1. Heterogeneous immune exhaustion and metabolic states of CD8+ T cells might differentially associate with dysfunction. However, specific parameters associated to functional restoration of CD8+ T cells after DC treatment have not been investigated in detail. Here, we studied the association of ART duration with memory subsets, exhaustion and metabolic profiles of CD8+ T cells from PLWH and improvement of polyfunctional and effector HIV-1 specific responses after stimulation with Gag-adjuvant-primed DC. HIV-1-specific CD8+ T cell responses from a larger proportion PLWH on ART for more than 10 years (LT-ARTp) improved polyfunctionality and capacity to eliminate autologous p24+ infected CD4+ T cells in vitro. In contrast, CD8+ T cells from PLWH on ART for less than a decade (ST-ARTp) were less responsive to DC treatment and functional improvement was limited in this group. This was associated with lower frequencies of central memory CD8+ T cells, increased co-expression of PD1 and TIGIT and reduced mitochondrial respiration and glycolytic induction upon TCR activation. In contrast, CD8+ T cells from LT-ARTp showed increased frequencies of TIM3+PD1-cells and preserved induction of glycolysis. Treatment of dysfunctional CD8+ T cells from ST-ARTp with combined anti-PD1 and anti-TIGIT antibodies plus a glycolysis promoting drug restored their ability to eliminate infected CD4+ T cells. Together, our study identifies specific immunometabolic parameters for different PLWH subgroups potentially useful for future personalized DC-based HIV-1 vaccines.


title: ART duration and immunometabolic state determine efficacy of DC-based treatment restoring functional HIV- specific CD8+ T cells in PLWH.


title: Naive arthritogenic SKG T cells have a defect in anergy and a repertoire pruned by superantigen

abstract:
How autoreactive CD4 T cells develop to cause rheumatoid arthritis remains unknown. We used a reporter for antigen-receptor signaling in the SKG autoimmune arthritis model to profile a T cell subpopulation enriched for arthritogenic naive CD4 T cells before arthritis onset by bulk and single cell RNA and T cell antigen-receptor (TCR) sequencing. Our analyses reveal that despite their impaired proximal TCR signaling, a subset of SKG naive CD4 T cells that have recently encountered endogenous antigen upregulate gene programs associated with positive regulation of T cell activation and cytokine signaling at higher levels than wild type cells in the pre-disease state. These arthritogenic cells also induce genes associated with negative regulation of T cell activation but do so less efficiently than wild type cells. Furthermore, their TCR sequences exhibit a previously unrecognized biased peripheral TCR V{beta} repertoire likely driven by endogenous viral superantigens. These particular V{beta}s, known to recognize endogenous mouse mammary tumor virus (MMTV) superantigen, are further expanded in arthritic joints. Our results demonstrate that autoreactive naive CD4 T cells which recognize endogenous viral superantigens are poised to cause disease by their altered transcriptome.

Summary blurbSelf-reactive SKG T cells that escaped negative selection harbor an independent defect in anergy that, together with chronic antigen stimulation, sets the stage for disease. Moreover, we propose a novel role for endogenous mouse mammary tumor virus (MMTV) superantigen in promoting arthritogenic T cell responses.


title: Comprehensive assessment of functional effects of commonly used sweeteners on ex vivo human gut microbiome

abstract: The gut microbiome composition and function are associated with health and diseases. Sweeteners are widely used food additives, although many studies using animal models have linked sweetener consumption to gut microbial changes and health issues. Whether sweeteners directly change the human gut microbiome functionality remains largely unknown. In this study, we systematically investigated the responses of five human gut microbiomes to 21 common sweeteners, using an approach combining high-throughput ex vivo microbiome culturing and metaproteomics to quantify functional changes in different taxa. Hierarchical clustering based on metaproteomic responses of individual microbiomes resulted in two clusters. The first cluster was composed of non-caloric artificial sweeteners (NAS) and two sugar alcohols with shorter carbon backbones (4-5 carbon atoms), and the second cluster was composed of sugar alcohols with longer carbon backbones. The metaproteomic functional responses of the second cluster were similar to the prebiotic fructooligosaccharides and kestose, indicating that these sugar alcohol-type sweeteners have potential prebiotic functions. This study provides a comprehensive evaluation of the direct effects of commonly used sweeteners on the functions of the human gut microbiome using a functional metaproteomics approach, improving our understanding of the roles of sweeteners on microbiome-associated human health and disease issues.

category: microbiology


title: Microbiomes of hadal fishes contain similar taxa, obligate symbionts, and known piezophiles across trench habitats

abstract:
Hadal snailfishes are the deepest-living fishes in the ocean, inhabiting trenches from depths of [~]6,000 to 8,000 m. While the microbial communities in trench environments have begun to be characterized, the microbes associated with hadal megafauna remain relatively unknown. Here, we describe the gut microbiomes of two hadal snailfishes, Pseudoliparis swirei (Mariana Trench) and Notoliparis kermadecensis (Kermadec Trench) using 16S rRNA gene amplicon sequencing. We contextualize these microbiomes with comparisons to the abyssal macrourid Coryphaenoides yaquinae and the continental shelf-dwelling snailfish Careproctus melanurus. The microbial communities of the hadal snailfishes were distinct from their shallower counterparts and were dominated by the same sequences related to the Mycoplasmataceae and Desulfovibrionaceae. These shared taxa indicate that symbiont lineages may have remained similar to the ancestral symbiont since their geographic separation or that they are dispersed between geographically distant trenches and subsequently colonize specific hosts. The abyssal and hadal fishes contained sequences related to known, cultured piezophiles, microbes that grow optimally under high hydrostatic pressure, including Psychromonas, Moritella, and Shewanella. These taxa are adept at colonizing nutrient-rich environments present in the deep ocean, such as on particles and in the guts of hosts, and we hypothesize they could make a dietary contribution to deep-sea fishes by degrading chitin and producing fatty acids. We characterize the gut microbiota within some of the deepest fishes to provide new insight into the diversity and distribution of host-associated microbial taxa and the potential of these animals, and the microbes they harbor, for understanding adaptation to deep-sea habitats.

ImportanceHadal trenches, characterized by high hydrostatic pressures and low temperatures, are one of the most extreme environments on our planet. By examining the microbiome of abyssal and hadal fishes, we provide insight into both the physiology of the deepest-living vertebrates and the microbes which colonize them. Our findings show that there are similar microbial populations in fishes geographically separated by thousands of miles, reflecting strong selection for specific microbial lineages. Only a handful of psychropiezophilic taxa, which do not reflect the diversity of microbial life at great depth, have been successfully isolated in the laboratory. Our examination of deep-sea fish microbiomes shows that typical high-pressure culturing methodologies, which have largely remained unchanged since the pioneering work of Claude ZoBell in the 1950s, may simulate the chemical environment found in animal guts and helps explain why the same deep-sea genera are consistently isolated.


title: Culture and genomic analysis of Symbiopectobacterium purcellii, gen.nov. sp. nov., isolated from the leafhopper Empoasca decipiens

abstract: Bacterial endosymbionts are found in multiple arthropod species, where they play crucial roles as nutritional symbionts, defensive symbionts or reproductive parasites. Recent work has highlighted a new clade of heritable microbes within the gammaproteobacteria that enter into both obligate and facultative symbioses, with an obligately required unculturable symbiont recently given the name Cand. Symbiopectobacterium. In this study, we describe a culturable rod shaped non-flagellated bacterial symbiont from this clade isolated from the leafhopper Empoasca decipiens. The symbiont is related to the transovarially-transmitted  BEV bacterium that was first isolated from the leafhopper Euscelidius variegatus by Alexander Purcell, and we therefore name the symbiont Symbiopectobacterium purcellii sp. nov. gen. nov. We further report the closed genome sequence for S. purcellii. The genome is atypical for a heritable microbe, being large in size, without profound AT bias and with little evidence of pseudogenization. The genome is predicted to encode Type II, III and VI secretion systems and associated effectors and a non-ribosomal peptide synthase array likely to produce bioactive small molecules. Predicted metabolism is more complete than for other symbionts in the Symbiopectobacterium clade, and the microbe is predicted to synthesize a range of B vitamins. However, Biolog plate analysis indicate metabolism is depauperate compared to the sister clade, represented by Pectobacterium carotovorum. A quorum-sensing pathway related to that of Pectobacterium spp. (containing an overlapping expI-expR1 pair in opposite directions and a "solo" expR2) is evidenced, and LC-MS/MS analysis reveals the presence of 3-hydroxy-C10-HSL as the sole N-acylhomoserine lactone (AHL) in our strain. This AHL profile is profoundly divergent from that of other Erwinia and Pectobacterium spp., which produce mostly 3-oxo-C6- and 3-oxo-C8-HSL and could aid group identification. Thus, this microbe denotes one that has lost certain pathways associated with a saprophytic lifestyle but represents an important baseline against which to compare other members of the genus Symbiopectobacterium that show more profound integration into host biology.


title: Sugar shock: Probing Streptococcus pyogenes metabolism through bioluminescence imaging

abstract: Streptococcus pyogenes (S. pyogenes) can thrive in its host during an infection, and, as a result, it must be able to respond to external stimuli and available carbon sources. The preclinical use of engineered pathogens capable of constitutive light production may provide realtime information on microbial-specific metabolic processes. Here we mapped the central metabolism of a luxABCDE-modified S. pyogenes Xen20 (Strep. Xen20) to its de novo synthesis of luciferase substrates as assessed by the rate of light production in response to different environmental triggers. Previous characterization predicted that the lux operon was under the myo-inositol iolE promotor. Here we show that supplementation with myoinositol generated increased Xen20 luminescence. Surprisingly, when supplemented with infection-relevant carbon sources, such as glucose or glycine, light production was diminished. This was presumably due to the scavenging of pyruvate by L-lactate dehydrogenase (LDH). Inhibition of LDH by its inhibitor, oxamate, partially restored luminescent signal in the presence of glucose, presumably by allowing the resulting pyruvate to proceed to acetyl-coenzyme A (CoA). This phenomenon appeared specific to the lactic acid bacterial metabolism as glucose or glycine did not reduce signal in an analogous luxABCDE-modified Gram-positive pathogen, Staph. Xen29. The Strep. Xen20 cells produced light in a concentration-dependent manner, inversely related to the amount of glucose present. Taken together, our measures of microbial response could provide new information regarding the responsiveness of S. pyogenes metabolism to acute changes in its local environments and cellular health.


title: Outer membrane protein A (OmpA) deficient Salmonella Typhimurium displays enhanced susceptibility towards β-lactam antibiotics: third-generation cephalosporins (ceftazidime) and carbapenems (meropenem)

abstract: The invasive non-typhoidal serovar of Salmonella enterica, namely Salmonella Typhimurium ST313, causes bloodstream infection in sub-Saharan Africa. Like other bacterial pathogens, the development of antimicrobial resistance is a severe problem in curing non-typhoidal Salmonella infection. In this work, we have investigated the role of four prominent outer membrane porins of S. Typhimurium, namely OmpA, OmpC, OmpD, and OmpF, in resistance against broad-spectrum {beta}-lactam antibiotics-ceftazidime and meropenem. We found that deleting OmpA from Salmonella makes the bacteria susceptible to {beta}-lactam drugs. The MIC for both the antibiotics reduced significantly for STM{Delta} ompA compared to the wild-type and the ompA complemented strains. Despite the presence of antibiotics, the uninterrupted growth of STM{Delta} ompC,{Delta} ompD, and{Delta} ompF endorsed the dispensability of these three porins in antimicrobial resistance. The {beta}-lactam antibiotics caused massive depolarization in the outer membrane of the bacteria in the absence of OmpA. We have proved that none of the extracellular loops but the complete structure of perfectly folded OmpA is required by the bacteria for developing antimicrobial resistance. Our data revealed that STM{Delta} ompA consumed more antibiotics than the wild-type and the complemented strain, resulting in severe damage of the bacterial outer membrane and subsequent killing of the pathogen by antibiotic-mediated oxidative stress. Upon deleting ompA, the steady decrease in the relative proportion of antibiotic-resistant persisters and the clearance of the STM{Delta} ompA from the liver and spleen of C57BL/6 mice upon treatment with ceftazidime proved the role of OmpA in rendering protection against {beta}-lactam antibiotics.


title: The cytoplasmic incompatibility Cif proteins from prophage WO modify sperm genome integrity

abstract: Inherited microorganisms can selfishly manipulate host reproduction to drive through populations. In Drosophila melanogaster, germline expression of the native Wolbachia prophage WO proteins CifA and CifB cause cytoplasmic incompatibility (CI) in which embryos from infected males and uninfected females suffer catastrophic mitotic defects and lethality; however, in infected females, CifA expression rescues the embryonic lethality and thus imparts a fitness advantage to the maternally-transmitted Wolbachia. Despite widespread relevance to sex determination, evolution, and vector control, the mechanisms underlying when and how CI impairs male reproduction remain unknown and a topic of debate. Here we use cytochemical, microscopic, and transgenic assays in D. melanogaster to demonstrate that CifA and CifB proteins of wMel localize to nuclear DNA throughout the process of spermatogenesis. Cif proteins cause abnormal histone retention in elongating spermatids and protamine deficiency in mature sperms that travel to the female reproductive tract with Cif proteins. Notably, protamine gene knockouts enhance wild type CI. In ovaries, CifA localizes to germ cell nuclei and cytoplasm of early-stage egg chambers, however Cifs are absent in late-stage oocytes and subsequently in fertilized embryos. Finally, CI and rescue are contingent upon a newly annotated CifA bipartite nuclear localization sequence. Together, our results strongly support the Host Modification model of CI in which Cifs initially modify the paternal and maternal gametes to bestow CI-defining embryonic lethality and rescue.

category: cell biology


title: The Cif proteins from Wolbachia prophage WO modify sperm genome integrity to establish cytoplasmic incompatibility


title: A giant virus genome is densely packaged by stable nucleosomes

abstract: The doublet histones of Marseillevirus are distantly related to the four eukaryotic core histones and wrap DNA to form remarkably similar nucleosomes. By releasing Marseillevirus chromatin from virions into solution and performing genome-wide nuclease digestion and chemical cleavage assays, we find that the higher-order organization of Marseillevirus chromatin differs greatly from that of eukaryotes. Marseillevirus nucleosomes fully protect DNA within virions, without linker DNA or phasing along genes. Likewise, we observe that most nucleosomes reconstituted onto 3-copy tandem repeats of a nucleosome positioning sequence are tightly packed and fully wrapped. We also document repeat generation and instability during viral passage in amoeboid culture. Dense promiscuous packing of fully wrapped nucleosomes rather than  beads-on-a-string with genic punctuation suggests a viral genome protection function for doublet histones.


title: Genome assembly, structural variants, and genetic differentiation between Lake Whitefish young species pairs (Coregonus sp.) with long and short reads

abstract: Nascent pairs of ecologically differentiated species offer an opportunity to get a better glimpse at the genetic architecture of speciation. Of particular interest is our recent ability to consider a wider range of genomic variants, not only single-nucleotide polymorphisms (SNPs), thanks to long-read sequencing technology. We can now identify structural variants (SVs) like insertions, deletions, and other rearrangements, allowing further insights into the genetic architecture of speciation and how different types of variants are involved in species differentiation. Here, we investigated genomic patterns of differentiation between sympatric species pairs (Dwarf and Normal) belonging to the Lake Whitefish (Coregonus clupeaformis) species complex. We assembled the first reference genomes for both C. clupeaformis sp. Normal and C. clupeaformis sp. Dwarf, annotated the transposable elements, and analysed the genomes in the light of related coregonid species. Next, we used a combination of long-read and short-read sequencing to characterize SVs and genotype them at population-scale using genome-graph approaches, showing that SVs cover five times more of the genome than SNPs. We then integrated both SNPs and SVs to investigate the genetic architecture of species differentiation in two different lakes and highlighted an excess of shared outliers of differentiation. In particular, a large fraction of SVs differentiating the two species correspond to insertions or deletions of transposable elements (TEs), suggesting that TE accumulation may represent a key component of genetic divergence between the Dwarf and Normal species. Altogether, our results suggest that SVs may play an important role in speciation and that, by combining second and third generation sequencing, we now have the ability to integrate SVs into speciation genomics.


title: Ancestral sequence reconstructions evaluated by extant sequence cross-validation

abstract:
Ancestral sequence reconstruction (ASR) has become widely used to analyze the properties of ancient biomolecules and to elucidate the mechanisms of molecular evolution. By recapitulating the structural, mechanistic, and functional changes of proteins during their evolution, ASR has been able to address many fundamental and challenging evolutionary questions where more traditional methods have failed. Despite the tangible successes of ASR, the accuracy of its reconstructions is currently unknown, because it is generally impossible to compare resurrected proteins to the true ancient ancestors that are now extinct. Which evolutionary models are the best for ASR? How accurate are the resulting inferences? Here we answer these questions by applying cross-validation (CV) to sets of aligned extant sequences.

To assess the adequacy of a chosen evolutionary model for predicting extant sequence data, our column-wise CV method iteratively cross-validates each column in an alignment. Unlike other phylogenetic model selection criteria, this method does not require bias correction and does not make restrictive assumptions commonly violated by phylogenetic data. We find that column-wise CV generally provides a more conservative criterion than the AIC by preferring less complex models.

To validate ASR methods, we also apply cross-validation to each sequence in an alignment by reconstructing the extant sequences using ASR methodology, a method we term "extant sequence reconstruction" (ESR). We can thus quantify the accuracy of ASR methodology by comparing ESR reconstructions to the corresponding true sequences. We find that a common measure of the quality of a reconstructed sequence, the average probability of the sequence, is indeed a good estimate of the fraction of the sequence that is correct when the evolutionary model is accurate or overparameterized. However, the average probability is a poor measure for comparing reconstructions, because more accurate phylogenetic models typically result in reconstructions with lower average probabilities. In contrast, the entropy of the reconstructed distribution is a reliable indicator of the quality of a reconstruction, as the entropy provides an accurate estimate of the log-probability of the true sequence. Both column-wise CV and ESR are useful methods to validate evolutionary models used for ASR and can be applied in practice to any phylogenetic analysis of real biological sequences.


title: A candidate gene cluster for the bioactive natural product gyrophoric acid in lichen-forming fungi

abstract:
Natural products of lichen-forming fungi are structurally diverse and have a variety of medicinal properties. Despite this, they a have limited implementation in industry, because the corresponding genes remain unknown for most of the natural products. Here we implement a long-read sequencing and bioinformatic approach to identify the biosynthetic gene cluster of the bioactive natural product gyrophoric acid (GA). Using 15 high-quality genomes representing nine GA-producing species of the lichen-forming fungal genus Umbilicaria, we identify the most likely GA cluster and investigate cluster gene organization and composition across the nine species. Our results show that GA clusters are promiscuous within Umbilicaria, with only three genes that are conserved across species, including the PKS gene. In addition, our results suggest that the same cluster codes for different but structurally similar NPs, i.e., GA, umbilicaric acid and hiascic acid, bringing new evidence that lichen metabolite diversity is also generated through regulatory mechanisms at the molecular level. Ours is the first study to identify the most likely GA cluster, and thus provides essential information to open new avenues for biotechnological approaches to producing and modifying GA and similar lichen-derived compounds. We show that bioinformatics approaches are useful in linking genes and potentially associated natural products. Genome analyses help unlocking the pharmaceutical potential of organisms such as lichens, which are biosynthetically diverse but slow growing, and difficult to cultivate due to their symbiotic nature.

ImportanceThe implementation of natural products in the pharmaceutical industry relies on the possibility of modifying the natural product (NP) pathway to optimize yields and pharmacological effects. Characterization of genes and pathways underlying natural product biosynthesis is a major bottleneck for the use of natural products in the pharmaceutical industry. Genome mining is a promising and relatively cost- and time-effective approach to exploit unexplored NP resources for drug discovery. In this study, we identify the most likely gene cluster for the lichen-forming fungal depside gyrophoric acid in nine Umbilicaria species. This compound shows cytotoxic and antiproliferative properties against several cancer cell lines, and is also a broad-spectrum antimicrobial agent. We identify the putative GA cluster from nine Umbilicaria species. This information paves the way for generating GA analogs with modified properties by selective activation/deactivation of genes.


title: Integrative in situ mapping of single-cell transcriptional states and tissue histopathology in an Alzheimer's disease model

abstract: Amyloid-{beta} plaques and neurofibrillary tau tangles are the neuropathologic hallmarks of Alzheimers disease (AD), but the spatiotemporal cellular responses and molecular mechanisms underlying AD pathophysiology remain poorly understood. Here we introduce STARmap PLUS to simultaneously map single-cell transcriptional states and disease marker proteins in brain tissues of AD mouse models at subcellular resolution (200 nm). This high-resolution spatial transcriptomics map revealed a core-shell structure where disease-associated microglia (DAM) closely contact amyloid-{beta} plaques, whereas disease-associated astrocytes (DAA) and oligodendrocyte precursor cells (OPC) are enriched in the outer shells surrounding the plaque- DAM complex. Hyperphosphorylated tau emerged mainly in excitatory neurons in the CA1 region accompanied by the infiltration of oligodendrocyte subtypes into the axon bundles of hippocampal alveus. The integrative STARmap PLUS method bridges single-cell gene expression profiles with tissue histopathology at subcellular resolution, providing an unprecedented roadmap to pinpoint the molecular and cellular mechanisms of AD pathology and neurodegeneration.


title: Contrast-enhanced microCT evaluation of degeneration following partial and full width injuries to mouse lumbar intervertebral disc

abstract:
Study DesignPreclinical animal study

ObjectiveEvaluation of the degenerative progression resulting from either a partial- or full- width injury to the mouse lumbar intervertebral disc (IVD) using contrast-enhanced micro-computed tomography and histological analyses. We utilized a lateral-retroperitoneal surgical approach to access the lumbar IVD, and the injuries to the IVD were induced by either incising one side of the annulus fibrosus or puncturing both sides of the annulus fibrosus. The full-width injury caused dramatic reduction in nucleus pulposus hydration and significant degeneration. A partial-width injury produces localized deterioration around the annulus fibrosus that resulted in local tissue remodeling without gross degeneration to the IVD.

MethodsFemale C57BL/6J mice of 3-4 months age were used in this study. They were divided into three groups to undergo partial-width, full-width, or sham injuries. The L5/L6 and L6/S1 lumbar IVDs were surgically exposed using a lateral-retroperitoneal approach. The L6/S1 IVDs were injured using either a surgical scalpel (partial-width) or a 33G needle (full-width), with the L5/L6 serving as an internal control. These animals were allowed to recover and then sacrificed at 2-, 4-, or 8-weeks post-surgery. The IVDs were assessed for degeneration using contrast-enhanced microCT (CE{micro}CT) and histological analysis.

ResultsThe high-resolution 3D evaluation of the IVD confirmed that the respective injuries localized within one side of the annulus fibrosus or spanned the full width of the IVD. The full-width injury caused deteriorations in the nucleus pulposus after 2 weeks and progressed to significant degeneration at 8 weeks, while the partial width injury caused localized disruptions that remained limited to the annulus fibrosus.

ConclusionThe use of CE{micro}CT revealed distinct IVD degeneration profiles resulting from partial- and full- width injuries. The partial width injury may serve as an alternative for IVD degeneration resulting from localized annulus fibrosus injuries in humans.

category: animal behavior and cognition


title: Vexitoxins: a novel class of conotoxin-like venom peptides from predatory gastropods of the genus Vexillum

abstract: Venoms of predatory marine cone snails (the family Conidae, order Neogastropoda) are intensely studied because of the broad range of biomedical applications of the neuropeptides that they contain, conotoxins. Meanwhile anatomy in some other neogastropod lineages strongly suggests that they have evolved similar venoms independently of cone snails, nevertheless their venom composition remains unstudied. Here we focus on the most diversified of these lineages, the genus Vexillum (the family Costellariidae). We have generated comprehensive multi-specimen, multi-tissue RNA-Seq data sets for three Vexillum species, and supported our findings in two species by proteomic profiling. We show that venoms of Vexillum are dominated by highly diversified short cysteine-rich peptides that in many aspects are very similar to conotoxins. Vexitoxins possess the same precursor organization, display overlapping cysteine frameworks and share several common post-translational modifications with conotoxins. Some vexitoxins show detectable sequence similarity to conotoxins, and are predicted to adopt similar domain conformations, including a pharmacologically relevant inhibitory cysteine-know motif (ICK). The tubular gL of Vexillum is a notably more recent evolutionary novelty than the conoidean venom gland. Thus, we hypothesize lower divergence between the toxin genes, and their  somatic counterparts compared to that in conotoxins, and we find support for this hypothesis in the molecular evolution of the vexitoxin cluster V027. We use this example to discuss how future studies on vexitoxins can inform origin and evolution of conotoxins, and how they may help addressing standing questions in venom evolution.


title: OrganoID: a versatile deep learning platform for organoid image analysis

abstract: Organoids have immense potential as ex vivo disease models for drug discovery and personalized drug screening. Dynamic changes in individual organoid morphology, number, and size can indicate important drug responses, however these metrics are difficult and labor-intensive to obtain for high-throughput image datasets. Here, we present OrganoID, a robust image analysis platform that automatically recognizes, labels, and tracks single organoids, pixel-by-pixel, in brightfield and phase-contrast microscopy experiments. The platform was trained on images of pancreatic cancer organoids and validated on separate images of pancreatic, lung, colon, and adenoid cystic carcinoma organoids, which showed excellent agreement with manual measurements of organoid count (96%) and size (95%) without any parameter adjustments. Single-organoid tracking accuracy remained above 89% over a four-day time-lapse microscopy study. Automated single-organoid morphology analysis of a chemotherapy dose-response experiment identified decreased organoid circularity as an important morphological feature reflecting drug response. OrganoID enables straightforward, detailed, and accurate image analysis to accelerate the use of organoids in high-throughput, data-intensive biomedical applications.


title: OrganoID: a versatile deep learning platform for tracking and analysis of single-organoid dynamics


title: Memory effects and static disorder reduce information in single-molecule signals

abstract: A key theoretical challenge posed by single-molecule studies is the inverse problem of deducing the underlying molecular dynamics from the time evolution of low-dimensional experimental observables. Toward this goal, a variety of low-dimensional models have been proposed as descriptions of single-molecule signals, including random walks with or without conformational memory and/or with static or dynamics disorder. Differentiating among different models presents a challenge, as many distinct physical scenarios lead to similar experimentally observable behaviors such as anomalous diffusion and nonexponential relaxation. Here we show that information-theory-based analysis of single-molecule time series, inspired by Shannons work studying the information content of printed English, can differentiate between Markov (memoryless) and non-Markov single-molecule signals and between static and dynamic disorder. In particular, non-Markov time series are more predictable and thus can be compressed and transmitted within shorter messages (i.e. have a lower entropy rate) than appropriately constructed Markov approximations, and we demonstrate that in practice the LZMA compression algorithm reliably differentiates between these entropy rates across several simulated dynamical models.


title: Multiple parameters shape the 3D chromatin structure of single nuclei

abstract: The spatial organization of chromatin at the scale of topologically associating domains (TADs) and below displays large cell-to-cell variations. Up until now, how this heterogeneity in chromatin conformation is shaped by chromatin condensation, TAD insulation, and transcription has remained mostly elusive. Here, we used Hi-M, a multiplexed DNA-FISH imaging technique providing developmental timing and transcriptional status, to show that the emergence of TADs at the ensemble level partially segregates the conformational space explored by single nuclei during the early development of Drosophila embryos. Surprisingly, a substantial fraction of nuclei displayed strong insulation even before TADs emerged. Moreover, active transcription within a TAD led to minor changes to the local inter- and intra-TAD chromatin conformation in single nuclei and only weakly affected insulation to the neighboring TAD. Overall, our results indicate that multiple parameters contribute to shaping the chromatin architecture of single nuclei at the TAD scale.


title: Combining LSD1 and JAK-STAT inhibition targets Down syndrome-associated myeloid leukemia at its core

abstract: Children with Down syndrome (DS) are predisposed to developing megakaryoblastic leukemia (ML-DS) and often experience severe toxicities from chemotherapy, highlighting the need for targeted therapies with beneficial risk profiles. The genomic landscape of ML-DS is characterized by a combination of mutations in signaling pathway genes and epigenetic modifiers, while aberrant lysine specific demethylase 1 (LSD1) and JAK-STAT activation have both been implicated in leukemogenesis. Here, we demonstrate that combined LSD1 and JAK1/2 inhibition exerts synergistic anti-leukemic effects specifically in ML-DS, both in vitro and in patient derived xenografts in vivo. The JAK1/2 inhibitor ruxolitinib enhanced the LSD1 inhibitor-induced differentiation, proliferation arrest and apoptosis in patient-derived leukemic blasts. At the transcriptional level, the combination synergistically repressed gene expression signatures essential for cell division. We further observed an immunogenic gene expression pattern in the form of increased cytokine signaling, which - by sensitizing ML-DS blasts to the JAK-STAT signaling blockade induced by ruxolitinib - could explain the increased susceptibility of ML-DS blasts to combination therapy. Taken together, we establish combined LSD1 and JAK-STAT inhibition as an efficacious therapeutic regimen specifically designed to target important steps in ML-DS leukemogenesis, paving the way for targeted therapies in this entity.


title: Single-cell multi-omics of human clonal hematopoiesis reveals that DNMT3A R882 mutations perturb early progenitor states through selective hypomethylation

abstract: Somatic mutations in cancer genes have been ubiquitously detected in clonal expansions across healthy human tissue, including in clonal hematopoiesis. However, mutated and wildtype cells are morphologically and phenotypically similar, limiting the ability to link genotypes with cellular phenotypes. To overcome this limitation, we leveraged multi-modality single-cell sequencing, capturing the mutation with transcriptomes and methylomes in stem and progenitors from individuals with DNMT3A R882 mutated clonal hematopoiesis. DNMT3A mutations resulted in myeloid over lymphoid bias, and in expansion of immature myeloid progenitors primed toward megakaryocytic-erythroid fate. We observed dysregulated expression of lineage and leukemia stem cell markers. DNMT3A R882 led to preferential hypomethylation of polycomb repressive complex 2 targets and a specific sequence motif. Notably, the hypomethylation motif is enriched in binding motifs of key hematopoietic transcription factors, serving as a potential mechanistic link between DNMT3A R882 mutations and aberrant transcriptional phenotypes. Thus, single-cell multi-omics pave the road to defining the downstream consequences of mutations that drive human clonal mosaicism.


title: IL-33 regulates age-dependency of long-term immune dysfunction induced by sepsis

abstract: Sepsis survival in adults is commonly followed by immunosuppression and increased susceptibility to secondary infections. However, the long-term immune consequences of pediatric sepsis are unknown. Here, we compared the frequency of Tregs, the activation of the IL-33/ILC2s axis in M2 macrophages, and the DNA methylation of epithelial lung cells from post-septic infant and adult mice. In contrast to adults, infant mice were resistant to secondary infection and did not show impairment in tumour controls upon melanoma challenge. Mechanistically, increased IL-33 levels, Tregs expansion, and activation of ILC2s and M2-macrophages were observed in post-septic adults but not infant mice. Impaired IL-33 production in post-septic infant mice was associated with increased DNA-methylation on lung epithelial cells. Notably, IL-33 treatment boosted the expansion of Tregs and induced immunosuppression in infant mice. Clinically, adults but not pediatric post-septic patients exhibited higher counts of Tregs and sera IL-33 levels. Hence, we describe a crucial and age-dependent role for IL-33 in post-sepsis immunosuppression.


title: Evolution of assortative mating following selective introgression of pigmentation genes between two Drosophila species

abstract: Adaptive introgression is ubiquitous in animals but experimental support for its role in driving speciation remains scarce. In the absence of conscious selection, admixed laboratory strains of Drosophila asymmetrically and progressively lose alleles from one parental species and reproductive isolation against the predominant parent ceases after 10 generations. Here, we selectively introgressed during one year light pigmentation genes of D. santomea into the genome of its dark sibling D. yakuba, and vice versa. We found that the pace of phenotypic change differed between the species and the sexes, and identified through genome sequencing common as well as distinct introgressed loci in each species. Mating assays showed that assortative mating between introgressed flies and both parental species persisted even after four years ([~] 60 generations) from the end of the selection. Those results indicate that selective introgression of as low as 0.5% of the genome can beget morphologically-distinct and reproductively-isolated strains, two prerequisites for the delimitation of new species. Our findings hence represent a significant step towards understanding the genome-wide dynamics of speciation-through-introgression.


title: A comprehensive library of canonical and non-canonical MHC class I antigens for cancer vaccine development.

abstract: A longstanding disconnect between the growing number of MHC Class I immunopeptidomic studies and genomic medicine hinders cancer vaccine design. We develop COD-dipp to genomically map the full spectrum of detected canonical and non-canonical (non-exonic) MHC Class I antigens from 26 cancer studies. We demonstrate that patient mutations in regions overlapping physically identified antigens better predict immunotherapy response when compared to neoantigen predictions. We suggest a vaccine design approach using 140,966 highly immune-visible regions of the genome annotated by their expression and haplotype frequency in the human population. These regions tend to be highly conserved, mutated in cancer and harbor 7.8 times more immunogenicity. Intersecting pan-cancer mutations with these immune surveilled regions revealed a potential to create off-the-shelf multi-epitope vaccines against public neoantigens. Here we release COD-dipp, a cancer vaccine toolkit as a web-application (https://www.proteogenomics.ca/COD-dipp) and open-source high-throughput resource.


title: The immunopeptidome from a genomic perspective: Establishing immune-relevant regions for cancer vaccine design


title: Imaging cerebral arteries tortuosity and velocities by transcranial Doppler ultrasound is a reliable assessment of brain aneurysm in mouse models

abstract:
Background and PurposeIntracranial aneurysms (IAs) are common vascular abnormalities of the brain with a prevalence of 3.2% in the general population. In the past few decades, several pathophysiological processes leading to IA rupture were identified, including irregular IA shape, an altered hemodynamic stress within the IA and vessel wall inflammation. The use of preclinical models of IA and imaging tools are paramount to better understand the underlying disease mechanisms. Therefore, there is a need for imaging methods to monitor intracranial aneurysm formation.

MethodsWe used two established mouse models of IA and we analyzed the progression of the IA by magnetic resonance imaging (MRI), transcranial Doppler (TCD), and histological studies.

ResultsIn both models of IA, we observed by TCD a significant decrease of the blood velocities and wall shear stress of the internal carotid arteries (ICA). We also observed the formation of tortuous arteries in both models which were correlated with the presence of an aneurysm as confirmed by MRI and histological analysis. A high grade of tortuosity has been associated with a significant decrease of the mean blood flow velocities and a greater artery dilation.

ConclusionsTCD is robust and easy imaging method to evaluate the progression of IA. The decrease of the blood flow velocities and the tortuosity can be used as reliable readout for IA detection.

category: pathology


title: Systematic Review and Meta-Analysis: Task-based fMRI Studies in Youths with Irritability

abstract:
ObjectiveChildhood irritability, operationalized as disproportionate and frequent temper tantrums and low frustration tolerance relative to peers, is a transdiagnostic symptom across many pediatric disorders. Studies using task-dependent functional magnetic resonance imaging (fMRI) to probe neural dysfunction in irritability have increased. However, an integrated review summarizing the published methods and synthesized fMRI results remains lacking.

MethodWe conducted a systematic search using irritability terms and task functional neuroimaging in key databases in March 2021, and identified 30 studies for our systematic review. Sample characteristics and fMRI methods were summarized. A subset of 28 studies met the criteria for extracting coordinate-based data for quantitative meta-analysis. Ten activation-likelihood estimations were performed to examine neural convergence across irritability measures and fMRI task domains.

ResultsSystematic review revealed small sample sizes (median = 58, mean age range = 8-16 years) with heterogeneous sample characteristics, irritability measures, tasks, and analytical procedures. Meta-analyses found no evidence for neural activation convergence of irritability across neurocognitive functions related to emotional reactivity, cognitive control, and reward processing, nor within each domain. Sensitivity analyses partialing out variances driven by heterogeneous tasks, irritability measures, stimulus types, and developmental ages all yielded null findings.

ConclusionThe lack of neural convergence suggests a need for common, standardized irritability assessments and more homogeneous fMRI tasks. Thoughtfully designed fMRI studies probing commonly defined neurocognitive functions may be more fruitful to elucidate the neural mechanisms of irritability. Open science practices, data mining in large neuroscience databases, and standardized analytical methods promote meaningful collaboration in irritability research.


title: Age-induced changes in Mu Opioid Receptor signaling in the midbrain Periaqueductal Gray of male and female rats

abstract:
The analgesic effects of opioids are attenuated in aged rats. Opioids such as morphine have decreased analgesic potency (but not efficacy) in aged rodents compared to adults; however, the neural mechanisms underlying this attenuated response are not yet known. The present study investigated the impact of advanced age and biological sex on opioid signaling in the ventrolateral periaqueductal gray (vlPAG) in the presence of chronic inflammatory pain. Assays measuring mu-opioid receptor (MOR) radioligand binding, GTP{gamma}S binding, receptor phosphorylation, cAMP inhibition, and regulator of G-protein signaling (RGS) protein expression were performed on vlPAG tissue from adult (2-3mos) and aged (16-18mos) male and female rats. Persistent inflammatory pain was induced by intraplantar injection of Complete Freunds Adjuvant (CFA). Adult males exhibited the highest MOR binding potential and the highest G-protein activation (activation efficiency ratio) in comparison to aged males and females (adult and aged). No impact of advanced age or sex on MOR phosphorylation state was observed. DAMGO-induced cAMP inhibition was highest in the vlPAG of adult males compared to aged males and females (adult and aged). vlPAG levels of RGS4 and RGS9-2, critical for terminating G-protein signaling, were assessed using RNAscope. Adult rats (both males and females) exhibited lower levels of vlPAG RGS4 and RGS9-2 mRNA expression compared to aged males and females. The observed age-related reductions in vlPAG MOR binding potential, G-protein activation efficiency, and cAMP inhibition, along with the observed age-related increases in RGS4 and RGS9-2 vlPAG expression, provide potential mechanisms whereby the potency of opioids is decreased in the aged population. These results have significant implications for pain management in this population.

HighlightsO_LIAged males and females (adult and aged) exhibit reduced vlPAG -opioid receptor binding potential compared to adult males.
C_LIO_LIAged males and females (adult and aged) exhibit reduced opioid-induced vlPAG G-protein activation compared to adult males.
C_LIO_LIAged males and females (adult and aged) exhibit reduced vlPAG MOR mediated cAMP inhibition compared to adult males.
C_LIO_LIAged rats (males and females) exhibit increased vlPAG mRNA expression of Regulator of G-Protein Signaling (RGS) proteins RGS4 and RGS9-2 compared to adult rats (males and females), which may explain the reduced receptor signaling observed in aged animals.
C_LIO_LIThese coordinate decreases in opioid receptor signaling may explain the previously reported reduced potency of opioids to produce pain relief in females and aged rats.
C_LI


title: Distinct Inhibitory Pathways Control Velocity and Directional Tuning in the Retina

abstract: The sensory periphery is responsible for detecting ethologically relevant features of the external world, using compact, predominantly feedforward circuits. Visual motion is a particularly prevalent sensory feature, the presence of which can be a signal to enact diverse behaviors ranging from gaze stabilization reflexes, to predator avoidance or prey capture. To understand how the retina constructs the distinct neural representations required for these diverse behaviors, we investigated two circuits responsible for encoding different aspects of image motion: ON and ON-OFF direction selective ganglion cells (DSGCs). Using a combination of 2-photon targeted whole cell electrophysiology, pharmacology, and conditional knockout mice, we show that distinct inhibitory pathways independently control tuning for motion velocity and motion direction in these two cell types. We further employ dynamic clamp and numerical modeling techniques to show that asymmetric inhibition provides a velocity-invariant mechanism of directional tuning, despite the strong velocity dependence of classical models of direction selectivity. We therefore demonstrate that invariant representations of motion features by inhibitory interneurons act as computational building blocks to construct distinct, behaviorally relevant signals at the earliest stages of the visual system.


title: Maternal immune activation accelerates puberty initiation and alters mechanical allodynia in male and female C57BL6/J mice.

abstract: The mechanisms that link maternal immune activation (MIA) with the onset of neurodevelopmental disorders remain largely unclear. Accelerated puberty is also associated with a heightened risk for psychopathology in later life but there is a dearth of evidence on the impacts of maternal infection on pubertal timing. We examined the effects of MIA on reproductive development, mechanical allodynia, and sensorimotor gating in juvenile, adolescent, and adult male and female mice. Moreover, we investigated hypothalamic neural markers associated with the reproductive and stress axes. Finally, we tested the mitigating effects of environmental enrichment (EE), which has clinical relevancy in human rehabilitation settings. Our results show that administration of polyinosinic-polycytidylic acid (poly(I:C)) on gestational day 12.5 led to early preputial separation, vaginal openings, and age of first estrus in offspring. MIA exposure altered pain sensitivity across development and modestly altered prepulse inhibition. The downregulation of Nr3c1 and Oprk mRNA in the hypothalamus of juvenile mice suggests that MIAs effects may be mediated through disruption of hypothalamic-pituitary-adrenal axis activity. In contrast, life-long housing with EE rescued many of these MIA-induced consequences. Overall, our findings suggest that accelerated puberty may be associated with the deleterious effects of infection during pregnancy and the onset of psychopathology.


title: 3D-imaging reveals conserved cerebrospinal fluid drainage via meningeal lymphatic vasculature in mice and humans

abstract:
Meningeal lymphatic vessels (MLVs) contribute to waste product elimination and immune surveillance in brain tissues. MLVs were identified in the dorsal and caudo-basal regions of the dura mater, where they ensure the clearance of cerebrospinal fluid (CSF). Whether MLVs exist in the complex anterior part of the murine and human skull, and how they connect with the glymphatic system and extracranial lymphatic vasculature remained unclear. Here, we generated three-dimensional (3D) maps of MLV drainage by light-sheet fluorescence microscopy (LSFM) imaging of mouse whole-head preparations following fluorescent OVA-A555 tracer injections into the CSF. In humans, we performed real-time magnetic resonance vessel wall imaging (MR-VWI) after systemic gadobutrol injections. We observed a conserved 3D-anatomy of MLVs in mice and humans, and we discovered an extended anterior network around the dural cavernous sinus including multiple capillary beds and exit routes through the foramina of emissary veins. MR-VWI may provide a diagnostic tool for patients with CSF drainage defects and neurological diseases.

Short abstractWe established the 3D-anatomy of meningeal lymphatic vasculature and associated CSF drainage by postmortem light-sheet imaging in mice and by real-time magnetic resonance imaging in humans, demonstrating conserved lymphatic circuitries in contact with dural venous sinuses.


title: Comparison of neural population dynamics in the regression subspace between continuous and categorical task parameters

abstract: Neural population dynamics, presumably fundamental computational units in the brain, provide a key framework for understanding information processing in the sensory, cognitive, and motor functions. However, neural population dynamics is not explicitly related to the conventional analytic framework for single-neuron activity, i.e., representational models that analyze neuronal modulations associated with cognitive and motor parameters. In this study, we applied a recently developed state-space analysis to incorporate the representational models into the dynamic model in combination with these parameters. We compared neural population dynamics between continuous and categorical task parameters during two visual recognition tasks, using the datasets originally designed for a single-neuron approach. We successfully extracted neural population dynamics in the regression subspace, which represent modulation dynamics for both continuous and categorical task parameters with reasonable temporal characteristics. Furthermore, we combined the classical optimal-stimulus analysis paradigm for the single-neuron approach (i.e., stimulus identified as maximum neural responses) into the dynamic model, and found that the most prominent modulation dynamics at the lower dimension were derived from these optimal responses. Thus, our approach provides a unified framework for incorporating knowledge acquired with the single-neuron approach into the dynamic model as a standard procedure for describing neural modulation dynamics in the brain.


title: Red harvester ant (Order: Hymenoptera) preference for cover crop seeds in South Texas

abstract: Harvester ants are known to selectively forage seeds, potentially impacting nearby plant community composition. In agricultural areas, harvester ants may be viewed as pests by foraging on crop seeds or as beneficials by preferentially removing weed seeds. However, little work has been done on harvester ant preferences for cover crop seeds. Local observations suggest that ants may take cover crop seeds, but no studies have evaluated ant agricultural impacts or seed preferences in the Lower Rio Grande Valley (LRGV). We examined red harvester ant (Pogonomyrmex barbatus Smith) preferences for commonly used cover crop seeds in the LRGV (vetch, oat, fescue, sunn hemp, and radish with wheatgrass as a control) and a commonly used bacterial seed inoculation treatment meant to increase root nodulation. We tested seed sets using choice tests housed in seed depots located within the foraging range of ant colonies with no prior exposure to the selected seeds. Of the evaluated cover crop seeds, wheatgrass and oat were the first to be removed entirely from the depot, with vetch remaining after 24 h. When we inoculated the two most preferred seeds to determine if there was a preference for non-inoculated seeds, we found no difference between inoculated and non-inoculated seeds. There were also significant changes in activity over time for both trials. These data indicate that harvester ant foraging preferences and activity can inform grower management recommendations regarding the risks of using certain cover crops and months sowing should be conducted in fields with known harvester ant presence.

category: ecology


title: Chemical defense acquired via pharmacophagy can lead to herd protection in a sawfly

abstract: Predation is an important selection pressure acting on organisms, with organisms evolving diverse anti-predator strategies to combat it. One such widespread strategy is chemical defense in which organisms either synthesize or extrinsically acquire defensive chemicals. Little is known about the intraspecific transfer of such chemicals and if such chemicals acquired from conspecifics can also serve as defense against predation. Here, we used adults of the turnip sawfly, Athalia rosae, which can acquire neo-clerodane diterpenoids ( clerodanoids) via pharmacophagy after exposure to the plant, Ajuga reptans. We show that clerodanoid access mediates protection against predation by mantids for the sawflies, both in a no-choice feeding assay and a microcosm setup. Moreover, even indirect access to clerodanoids, via nibbling on conspecifics that had access to the plant, resulted in protection against predation albeit to a much lower degree than direct access. Furthermore, sawflies that had no direct access to clerodanoids were less consumed by mantids when they were grouped with conspecifics that had direct access. Most, but not all, of such initially undefended sawflies could acquire clerodanoids from conspecifics that had direct access to the plant, although in low quantities. Together our results demonstrate that clerodanoids serve as chemical defense that can be intraspecifically transferred. Moreover, the presence of chemically defended individuals in a group can confer protection onto conspecifics that had no direct access to clerodanoids, suggesting a  herd-protection effect.