title: An MHV macrodomain mutant predicted to lack ADP-ribose binding activity is severely attenuated, indicating multiple roles for the macrodomain in coronavirus replication

abstract:
All coronaviruses (CoVs) contain a macrodomain, also termed Mac1, in non-structural protein 3 (nsp3) which binds and hydrolyzes ADP-ribose covalently attached to proteins. Despite several reports demonstrating that Mac1 is a prominent virulence factor, there is still a limited understanding of its cellular roles during infection. Currently, most of the information regarding the role of CoV Mac1 during infection is based on a single point mutant of a highly conserved asparagine-to-alanine mutation, which is known to largely eliminate Mac1 ADP-ribosylhydrolase activity. To determine if Mac1 ADP-ribose binding separately contributes to CoV replication, we compared the replication of a murine hepatitis virus (MHV) Mac1 mutant predicted to dramatically reduce ADP-ribose binding, D1329A, to the previously mentioned asparagine mutant, N1347A. D1329A and N1347A both replicated poorly in bone-marrow derived macrophages (BMDMs), were inhibited by PARP enzymes, and were highly attenuated in vivo. However, D1329A was significantly more attenuated than N1347A in all cell lines tested that were susceptible to MHV infection. In addition, D1329A retained some ability to block IFN-{beta} transcript accumulation compared to N1347A, indicating that these two mutants impacted distinct Mac1 functions. Mac1 mutants predicted to eliminate both binding and hydrolysis activities were unrecoverable, suggesting that the combined activities of Mac1 may be essential for MHV replication. We conclude that Mac1 has multiple roles in promoting the replication of MHV, and that these results provide further evidence that Mac1 could be a prominent target for anti-CoV therapeutics.

IMPORTANCEIn the wake of the COVID-19 epidemic, there has been a surge to better understand how CoVs replicate, and to identify potential therapeutic targets that could mitigate disease caused by SARS-CoV-2 and other prominent CoVs. The highly conserved macrodomain, also termed Mac1, is a small domain within non-structural protein 3. It has received significant attention as a potential drug target as previous studies demonstrated that it is essential for CoV pathogenesis in multiple animal models of infection. However, the various roles and functions of Mac1 during infection remain largely unknown. Here, utilizing recombinant Mac1 mutant viruses, we have determined that different biochemical functions of Mac1 have distinct roles in the replication of MHV, a model CoV. These results indicate that Mac1 is more important for CoV replication than previously appreciated, and could help guide the development of inhibitory compounds that target unique regions of this protein domain.


title: Unipolar peptidoglycan synthesis in the Rhizobiales requires an essential class A penicillin-binding protein

abstract: Members of the Rhizobiales are polarly-growing bacteria that lack homologs of the canonical rod complex. To investigate the mechanisms underlying polar cell wall synthesis, we systematically probed the function of cell wall synthesis enzymes in the plant-pathogen Agrobacterium tumefaciens. The development of fluorescent O_SCPLOWDC_SCPLOW-amino acid dipeptide (FDAAD) probes, which are incorporated into peptidoglycan by penicillin-binding proteins in A. tumefaciens, enabled us to monitor changes in growth patterns in the mutants. Use of these fluorescent cell wall probes and peptidoglycan compositional analysis convincingly demonstrate that a single class A penicillin-binding protein is essential for polar peptidoglycan synthesis. Furthermore, we find evidence of an alternative mode of cell wall synthesis that likely requires O_SCPLOWLDC_SCPLOW-transpeptidase activity. Genetic analysis and cell wall targeting antibiotics reveal that the mechanism of unipolar growth is conserved in Sinorhizobium and Brucella. This work provides insights into unipolar peptidoglycan biosynthesis employed by the Rhizobiales during cell elongation.

category: microbiology


title: Multi-omics approach to identify bacterial polyynes and unveil their antifungal mechanism against Candida albicans

abstract: Bacterial polyynes are highly active natural products with a broad-spectrum of antimicrobial activities. However, their detailed mechanism of action remains unclear. Through integrating comparative genomics, transcriptomics, functional genetics, and metabolomics analysis, we identified a unique polyyne resistance gene, masL (encoding acetyl-CoA acetyltransferase), from the biosynthesis gene cluster (BGC) dominant for the production of antifungal polyynes (massilin A, massilin B, collimonin C, and collimonin D) in Massilia sp. YMA4. Phylogenic and chemotaxonomic analyses characterized the core architecture of bacterial polyyne BGC. The crystallographic analysis of the MasL-collimonin C complex indicated that bacterial polyynes serve as a covalent inhibitor of acetyl-CoA acetyltransferase. Moreover, we confirmed that the bacterial polyynes disrupted cell membrane integrity and inhibited cell viability of Candida albicans by targeting ERG10 (homolog of MasL). Overall, understanding of the antifungal mechanism of bacterial polyynes presented herein will be useful for the development of polyynes for fungal infections.


title: Deep conservation of Hid-like RHG gene family homologs in winged insects

abstract: Together with sickle (skl), the Drosophila paralogs reaper (rpr), head involution defective (hid), and grim (RHG) control a critical switch in the induction of programmed cell death. RHG homologs have been identified in other dipteran and lepidopteran species but not beyond. Revisiting this issue with a "taxon hopping" BLAST search strategy in current genome and transcriptome resources, I detected high confidence RHG homologs in Coleoptera (beetles), Hymenoptera (bees+wasps), Hemiptera (true bugs), termites, and cockroaches. Analyses of gene structure and protein sequence conservation revealed a shared ancestral splicing pattern and highly conserved amino acid residues at both the N- and C-terminal ends that identify hid as the most ancestrally organized RHG gene family member in Drosophila. hid-like RHG homologs were also detected in mosquitoes, redefining their michelob_x (mx) genes as an expansion of derived RHG homologs. Only singleton homologs were detected in the large majority of other insect clades. Lepidopteran RHG homologs, however, stand out by producing an evolutionarily derived splice isoform, identified in previous work, in addition to the newly detected hid-like isoform. Exceptional sequence diversification of select RHG homologs at the family- and genus-level explain their elusiveness in important insect genome model species like the red flour beetle Tribolium castaneum and the pea aphid Acyrthosiphon pisum. Combined, these findings expand the minimal age of the RHG gene family by about 100 million years and open new avenues for molecular cell death studies in insects.

category: developmental biology


title: Hypsugo stubbei sp. nov., a novel cryptic bat species of the genus Hypsugo (Vespertilionidae, Chiroptera, Mammalia) from Mongolia. Results of the Mongolian-German Biological Expeditions Since 1962, No. 347.

abstract:
The occurrence of two members of the genus Hypsugo, namely H. alaschanicus and H. savii caucasicus, have been reported for Mongolia in the literature. Due to various taxonomic reassignments within and between genera, the number of records for the genus Hypsugo in Mongolia is quite scarce and sometimes not resolved at species or subspecies level. Despite recognition of the two above-mentioned species, recent reports based on genetic analyses describe only new and further records of H. alaschanicus. Thus, it exists a large uncertainty regarding the occurrence and distribution of H. savii caucasicus in Mongolia. Here, our efforts in gaining a deeper understanding towards the occurrence and distribution of Hypsugo species in Mongolia are described.

A combination of genetic and morphological analyses of collected material from Hypsugo specimens revealed the existence of a genetically largely distant Hypsugo clade. Therefore, a new and cryptic Hypsugo species is proposed which is named after Prof. Dr. Michael Stubbe for his continuous, long-standing and significant contributions into the biological exploration of Mongolia. Hypsugo stubbei sp. nov. differs by at least 8.4 % and 9 % to the closest Western Palearctic distributed H. cf. darwinii and H. savii as well as at least 11.3 % to the Easter Palearctic (including Mongolia) distributed H. alaschanicus based on the first 798 nucleotides of the gene encoding the mitochondrial ND1 (subunit one of NADH dehydrogenase). Neither a close proximity species based on the gene encoding the mitochondrial COI (cytochrome oxidase subunit one) could be found in publicly accessible nucleotide databases. While the cryptic H. stubbei sp. nov. reveals no obvious cranial and morphological differences, few external characteristics are dissimilar to both H. alaschanicus and H. savii (caucasicus). Currently, Hypsugo stubbei sp. nov. was found at four different locations in Mongolia. Among the 11 specimens captured, six facilitated a genetic assignment. Based on the current scarce data records, the species seems to occur mainly in the far west of Mongolia inhabiting semi-deserts and steppes up to high mountain areas. An overlapping distribution with H. alaschanicus cannot be excluded based on the limited data currently available.

category: ecology


title: Short-interval reburns in the boreal forest alter soil bacterial communities, reflecting increased pH and poor conifer seedling establishment

abstract: Increasing burn rates (percentage area burned annually) in some biomes are leading to fires burning in close succession, triggering rapid vegetation change as well as altering soil properties. Despite the importance of soil microbes for nutrient cycling and as plant symbionts, the effects of increased fire frequency on belowground microbial communities remain largely unknown. We present a study of the effects of short interval reburns (defined here as <20 years between fires) on soil bacterial communities in the boreal forest of northwestern Canada, using a paired site design that spans wetlands and uplands, with 50 sites total. We asked whether short interval reburns significantly alter soil bacterial community composition and richness, and which bacterial taxa are associated with greater or lower fire frequency. We found that, while short interval reburns had no significant effect on bacterial richness, there were significant changes in overall community composition. We did not find correlations between understory vegetation community dissimilarities and bacterial community dissimilarities, suggesting the primary drivers of changes induced by short interval reburns may differ between plants and microbes. We identified an abundant Blastococcus sp. that was consistently enriched in short interval reburns, in both wetlands and uplands, indicating its role as a strongly "pyrophilous" bacterium. We also identified an abundant Callaberonia sordidicola taxon as being consistently depleted in short interval reburns. This endophytic diazotrophic organism is a robust colonizer of pine and spruce seedlings and has the ability to increase seedling growth, due in part to large contributions of fixed nitrogen. Its depletion in short-interval reburn sites raises questions about whether this is contributing to - or merely reflects - poor conifer seedling recolonization post-fire at short-interval reburns.


title: Broad variation in rates of polyploidy and dysploidy across flowering plants is correlated with lineage diversification

abstract: Changes in chromosome number are considered an important driver of diversification in angiosperms. Single chromosome number changes caused by dysploidy may produce strong reproductive barriers leading to speciation. Polyploidy, or whole genome duplication, yields new species that are often reproductively isolated from progenitors and may exhibit novel morphology or ecology that may further facilitate diversification. Here, we examined the rates of polyploidy, dysploidy, and diversification across the angiosperms. Our analyses of nearly 30,000 taxa representing 46 orders and 147 families found that rates of polyploidy and dysploidy differed by two to three orders of magnitude. The rates of polyploidy and dysploidy were positively correlated with diversification rates, but relative importance analyses indicated that variation in polyploidy was better correlated with diversification rates than dysploidy. Our results provide an overview of angiosperm chromosomal evolution and a roadmap for future research on the complex relationships among polyploidy, dysploidy, and diversification.

category: evolutionary biology


title: Novel insights into joint estimations of demography, mutation rate, and selection using UV sex chromosomes

abstract: A central goal in evolutionary genomics is to understand the processes that shape genetic variation in natural populations. In anisogamous species, these processes may generate asymmetries between genes transmitted through sperm or eggs. The unique inheritance of sex chromosomes facilitates studying such asymmetries, but in many systems sex-biased mutation, demography, and selection are confounded with suppressed recombination in only one sex (the W in females, or the Y in males). However, in a UV sex-determination system, both sex chromosomes are sex-specific and experience suppressed recombination. Here we built a spatially-structured simulation to examine the effects of population density and sex-ratio on female and male effective population size in haploids and compare the results to polymorphism data from whole-genome resequencing of the moss Ceratodon purpureus. In the parameter space we simulated, males nearly always had a lower effective population size than females. Using the C. purpureus resequencing data, we found the U and V have lower nucleotide diversity than the autosomal mean, and the V is much lower than the U, however, we found no parameter set in the model that explained both the U/V and U/autosome ratios we observed. We next used standard molecular evolutionary analyses to test for sex-biased mutation and selection. We found that males had a higher mutation rate but that natural selection shapes variation on the UV sex chromosomes. All together the moss system highlights how anisogamy alone can exert a profound influence on genome-wide patterns of molecular evolution.


title: Extensive variation in the mouse intelectin gene family: recent duplications, deletions and inactivating variants result in diversity in laboratory strains

abstract: Intelectins are a family of multimeric secreted proteins that bind microbe-specific glycans. Both genetic and functional studies have suggested that intelectins have an important role in innate immunity and are involved in the etiology of various human diseases, including inflammatory bowel disease. Experiments investigating the role of intelectins in human disease using mouse models are limited by the fact that there is not a clear one-to-one relationship between intelectin genes in humans and mice, and that the number of intelectin genes varies between different mouse strains. In this study we show by gene sequence and gene expression analysis that human intelectin-1 (ITLN1) has multiple orthologues in mice, including a functional homologue Itln1; however, human intelectin-2 has no such orthologue or homologue. We confirm that all sub-strains of the C57-line have a large deletion resulting in retention of only one intelectin gene, Itln1. The majority of laboratory strains have a full complement of six intelectin genes, except wild-derived CAST, SPRET, SKIVE, MOLF and PANCEVO, which are derived from different mouse species/subspecies and encode different complements of intelectin genes. In wild mice, intelectin deletions are polymorphic in Mus musculus castaneus and Mus musculus domesticus. Further sequence analysis shows that Itln3 and Itln5 are polymorphic pseudogenes due to premature truncating mutations, and that mouse Itln1 has undergone recent adaptive evolution. Taken together, our study shows extensive diversity in intelectin genes in both laboratory and wild-mice, suggesting a pattern of birth-and-death evolution. In addition, our data provide a foundation for further experimental investigation of the role of intelectins in disease.

category: genetics


title: Chromatin accessibility changes induced by the microbial metabolite butyrate reveal possible mechanisms of anti-cancer effects

abstract:
Butyrate is a four-carbon fatty acid produced in large quantities by bacteria found in the human gut. It is the major source of colonic epithelial cell energy, can bind to and agonize short-chain fatty acid G-protein coupled receptors and functions as a histone deacetylase (HDAC) inhibitor. Anti-cancer effects of butyrate are attributed to a global increase in histone acetylation in colon cancer cells; however, the role that corresponding chromatin remodeling plays in this effect is not fully understood. We used longitudinal paired ATAC-seq and RNA-seq on HCT-116 colon cancer cells to determine how butyrate-related chromatin changes functionally associate with cancer. We detected distinct temporal changes in chromatin accessibility in response to butyrate with less accessible regions enriched in transcription factor binding motifs and distal enhancers. These regions significantly overlapped with regions maintained by the SWI/SNF chromatin remodeler, and were further enriched amongst chromatin regions that are associated with ARID1A/B synthetic lethality. Finally, we found that butyrate-induced chromatin regions were enriched for both colorectal cancer GWAS loci and somatic mutations in cancer. These results demonstrate the convergence of both somatic mutations and GWAS risk variants for colon cancer within butyrate-responsive chromatin regions, providing a molecular map of the mechanisms by which this microbial metabolite might confer anti-cancer properties.

HighlightsO_LIChromatin accessibility changes longitudinally upon butyrate exposure in colon cancer cells.
C_LIO_LIChromatin regions that close in response to butyrate are enriched among distal enhancers.
C_LIO_LIThere is strong overlap between butyrate-induced peaks and peaks associated with SWI/SNF synthetic lethality.
C_LIO_LIButyrate-induced peaks are enriched for colorectal cancer GWAS loci and somatic variation in colorectal cancer.
C_LI

category: genomics


title: CanDIG: Secure Federated Genomic Queries and Analyses Across Jurisdictions

abstract:
Rapid expansions of bioinformatics and computational biology have broadened the collection and use of -omics data including genomic, transcriptomic, methylomic and a myriad of other health data types, in the clinic and the laboratory. Both clinical and research uses of such data require co-analysis with large datasets, for which participant privacy and the need for data custodian controls must remain paramount. This is particularly challenging in multi-jurisdictional settings, such as Canada, where health privacy and security requirements are often heterogeneous. Data federation presents a solution to this, allowing for integration and analysis of large datasets from various sites while abiding by local policies.

The Canadian Distributed Infrastructure for Genomics platform (CanDIG) enables federated querying and analysis of -omics and health data while keeping that data local and under local control. It builds upon existing infrastructures to connect five health and research institutions across Canada, relies heavily on standards and tooling brought together by the Global Alliance for Genomics and Health (GA4GH), implements a clear division of responsibilities among its participants and adheres to international data sharing standards. Participating researchers and clinicians can therefore contribute to and quickly access a critical mass of -omics data across a national network in a manner that takes into account the multi-jurisdictional nature of our privacy and security policies. Through this, CanDIG gives medical and research communities the tools needed to use and analyze the ever-growing amount of -omics data available to them in order to improve our understanding and treatment of various conditions and diseases. CanDIG is being used to make genomic and phenotypic data available for querying across Canada as part of data sharing for five leading pan-Canadian projects including the Terry Fox Comprehensive Cancer Care Centre Consortium Network (TF4CN) and Terry Fox PRecision Oncology For Young peopLE (PROFYLE), and making data from provincial projects such as POG (Personalized Onco- Genomics) more widely available.


title: Tunable human myocardium derived decellularized extracellular matrix for 3D bioprinting and cardiac tissue engineering

abstract: The generation of 3D tissue constructs with multiple cell types and matching mechanical properties remains a challenge in cardiac tissue engineering. Recently, 3D bioprinting has become a powerful tool to achieve these goals. Decellularized extracellular matrix (dECM) is a common scaffold material due to providing a native biochemical environment. Unfortunately, dECMs low mechanical stability prevents usage for bioprinting applications alone. In this study, we developed bioinks composed of decellularized human heart ECM (dhECM) with either gelatin methacryloyl (GelMA) or GelMA- methacrylated hyaluronic acid (MeHA) hydrogels dual crosslinked with UV light and microbial Transglutaminase (mTGase). We characterized the bioinks mechanical, rheological, swelling, printability and biocompatibility properties. Composite GelMA-MeHA-dhECM (GME) hydrogels demonstrated improved mechanical properties by an order of magnitude, compared to GelMA-dhECM (GE) hydrogels. All hydrogels were extrudable and compatible with human induced pluripotent stem cells derived cardiomyocytes (iCMs) and human cardiac fibroblasts (hCFs). Tissue-like beating of the printed constructs with striated sarcomeric alpha-actinin and Connexin 43 expression was observed. The order of magnitude difference between the elastic modulus of these hydrogel composites offers applications in in vitro modelling of the myocardial infarct boundary. Here, as a proof of concept, we created an infarct boundary region with control over mechanical properties along with cellular and macromolecular content through printing iCMs with GE bioink and hCFs with GME bioink.

category: bioengineering


title: OME-NGFF: scalable format strategies for interoperable bioimaging data

abstract:
Biological imaging is one of the most innovative fields in the modern biological sciences. New imaging modalities, probes, and analysis tools appear every few months and often prove decisive for enabling new directions in scientific discovery. One feature of this dynamic field is the need to capture new types of data and data structures. While there is a strong drive to make scientific data Findable, Accessible, Interoperable and Reproducible (FAIR 1), the rapid rate of innovation in imaging impedes the unification and adoption of standardized data formats. Despite this, the opportunities for sharing and integrating bioimaging data and, in particular, linking these data to other "omics" datasets have never been greater. Therefore, to every extent possible, increasing "FAIRness" of bioimaging data is critical for maximizing scientific value, as well as for promoting openness and integrity.

In the absence of a common, FAIR format, two approaches have emerged to provide access to bioimaging data: translation and conversion. On-the-fly translation produces a transient representation of bioimage metadata and binary data but must be repeated on each use. In contrast, conversion produces a permanent copy of the data, ideally in an open format that makes the data more accessible and improves performance and parallelization in reads and writes. Both approaches have been implemented successfully in the bioimaging community but both have limitations. At cloud-scale, those shortcomings limit scientific analysis and the sharing of results. We introduce here next-generation file formats (NGFF) as a solution to these challenges.

category: bioinformatics


title: Differential heating of metal nanostructures by radio frequencies: a theoretical study

abstract: Nanoparticles with strong absorption of incident radio frequency (RF) or microwave irradiation are desirable for remote hyperthermia treatments. While controversy has surrounded the absorption properties of spherical metallic nanoparticles, other geometries such as prolate and oblate spheroids have not received sufficient attention for application in hyperthermia therapies. Here, we use the electrostatic approximation to calculate the relative absorption ratio of metallic nanoparticles in various biological tissues. We consider a broad parameter space, sweeping across frequencies from 1 MHz to 10 GHz, while also tuning the nanoparticle dimensions from spheres to high-aspect-ratio spheroids approximating nanowires and nanodiscs. We find that while spherical metallic nanoparticles do not offer differential heating in tissue, large absorption cross sections can be obtained from long prolate spheroids, while thin oblate spheroids offer minor potential for absorption. Our results suggest that metallic nanowires should be considered for RF- and microwave-based wireless hyperthermia treatments in many tissues going forward.

category: biophysics


title: Design principles of collateral sensitivity-based dosing strategies

abstract: Collateral sensitivity (CS)-based antibiotic treatments, where increased resistance to one antibiotic leads to increased sensitivity to a second antibiotic, may have the potential to limit the emergence of antimicrobial resistance. However, it remains unclear how to best design CS-based treatment schedules. To address this problem, we use mathematical modelling to study the effects of pathogen- and drug-specific characteristics for different treatment designs on bacterial population dynamics and resistance evolution. We confirm that simultaneous and one-day cycling treatments could supress resistance in the presence of CS. We show that the efficacy of CS-based cycling therapies depends critically on the order of drug administration. Finally, we find that reciprocal CS is not essential to suppress resistance, a result that significantly broadens treatment options given the ubiquity of one-way CS in pathogens. Overall, our analyses identify key design principles of CS-based treatment strategies and provide guidance to develop treatment schedules to suppress resistance.

category: pharmacology and toxicology


title: In silico analysis of cis-elements and identification of transcription factors putatively involved in the regulation of the OAS cluster genes SDI1 and SDI2

abstract: A. thaliana sulfur deficiency-induced 1 and sulfur deficiency-induced 2 (SDI1 and SDI2) are involved in partitioning sulfur among metabolite pools during sulfur deficiency and their transcription is strongly induced by this condition. However, little is currently known about the cis- and trans-factors that regulate SDI expression. To identify potential transcription factors and DNA sequence element regulators of SDI expression we performed a comparative in silico analysis of their promoter sequences cataloguing known and potentially new cis-elements. We further screened an arrayed library of Arabidopsis transcription factors (TF) for binding to the SDI1 and SDI2 promoters. In total 14 candidate TF regulators of SDIs were identified with yeast-one-hybrid analyses, of which five bound to both promoters, 4 were specific to SDI1, and 5 were specific SDI2. Direct association between particular cis-elements in these promoter regions and specific TFs was established via electrophoretic mobility shift assays. SLIM1 was shown to bind SURE cis-element(s) in the proximal promoter region of both SDI1 and SDI2. The bZIP core cis-element in the proximal promoter region of SDI2 was shown to be important for bZIP16, bZIP44, and HYH binding. GBF1 was shown to bind the E-box in the proximal promoter region of SDI2. Additionally, we performed a meta-analysis of expression changes of these 14 TF candidates in a variety of conditions that alter SDI expression. These data will allow for more detailed future analysis of the molecular factors required for transcriptional regulation of SDIs under a range of physiological and metabolic conditions, apart from sulfur deficiency.

category: plant biology


title: Genomic Analysis of Human Noroviruses Using Hybrid Illumina-Nanopore Data

abstract: Whole genome sequence (WGS) analysis of noroviruses is routinely performed by employing a metagenomic approach. While this methodology has several advantages, such as allowing for examination of co-infection, it has some limitations such as the requirement of high viral load to achieve full-length or near full-length genomic sequences. In this study, we used an amplification approach to obtain full-length genomic amplicons from 39 Canadian GII isolates followed by deep sequencing on Illumina and Oxford Nanopore platforms. This approach significantly reduced the required viral titre to obtain full-genome coverage. Herein, we compared the coverage and sequences obtained by both platforms and provided an in-depth genomic analysis of the obtained sequences, including the presence of single nucleotide variants (SNVs) and recombination events.


title: Temporal regulation of green and red cone specification in human retinas and retinal organoids

abstract:
Trichromacy is unique to primates among mammals, enabled by specification of blue (short/S), green (medium/M), and red (long/L) cones. During retinal development in humans and Old World monkeys, cones make a poorly understood choice between M and L cone subtype fates. Two nonexclusive models have been proposed: a stochastic model, in which a regulatory DNA element randomly loops to either the M- or L-opsin promoter to drive expression, and a temporal model, in which cone subtypes are generated in a developmental progression. Here we present data that support a temporal mechanism for M and L cone specification. M-opsin is expressed prior to L-opsin during fetal retinal development. Natural variation in the ratios of M and L cone subtypes is associated with a polymorphism in the NR2F2 gene, a mediator of retinoic acid (RA) signaling. RA-synthesizing enzymes are highly expressed in early development and then decrease, suggesting high RA signaling early. Human retinal organoids grown in standard culture conditions were enriched for L cones, whereas early addition of RA yielded M cone-rich organoids. Our data suggest that M cones are generated before L cones and that RA signaling induces M cones and suppresses L cones early during development. These studies advance human retinal organoids as a model to study developmental processes unique to humans and primates.

One sentence summaryTemporal specification of human cones


title: Microbial rhodopsins are increasingly favored over chlorophyll in High Nutrient Low Chlorophyll waters

abstract:
Microbial rhodopsins are simple light-harvesting complexes that, unlike chlorophyll photosystems, have no iron requirements for their synthesis and phototrophic functions. Here we report the first environmental concentrations of rhodopsin along the Subtropical Frontal Zone off New Zealand, where Subtropical waters encounter the iron-limited Subantarctic High Nutrient Low Chlorophyll (HNLC) region. Rhodopsin concentrations were highest in HNLC waters where chlorophyll-a concentrations were lowest. Furthermore, while the ratio of rhodopsin to chlorophyll-a photosystems was on average 20 along the transect, this ratio increased to over 60 in HNLC waters. We further show that microbial rhodopsins are abundant in both picoplankton (0.2-3m) and in the larger (>3m) size fractions of the microbial community containing eukaryotic plankton and/or particle-attached prokaryotes. These findings suggest that rhodopsin phototrophy could be critical for microbial plankton to adapt to resource-limiting environments where photosynthesis and possibly cellular respiration are impaired.

Originality-Significance statementHigh Nutrient Low Chlorophyll (HNLC) regimes cover approximately 30% of the global ocean surface and play a crucial role in the Earths carbon cycle. Here we show that microbial rhodopsins are particularly abundant in a HNLC region of the Subantarctic ocean, where chlorophyll abundance is relatively low and photosynthesis and respiration might be impaired due to iron limitation. These data suggest that rhodopsin phototrophy can contribute significantly to the energy budgets of HNLC regions, capturing meaningful amounts of light that cannot be channeled through photosynthesis.


title: Inhibition of MRN activity by a telomere protein motif

abstract: The MRN complex (MRX in Saccharomyces cerevisiae) initiates the repair of DNA double-stranded breaks (DSBs) and activates the Tel1/ATM kinase, which orchestrates the DNA damage response (DDR). Telomeres prevent DDR activation at chromosome ends, partly by keeping MRN-ATM in check. We show that the multiple activities of the MRX complex are disabled by telomeric protein Rif2 through the action of a short motif (MIN, MRN/X-inhibitory motif) at the N-terminal end of the protein. MIN executes telomeric suppression of Tel1, DDR and and non-homologous end joining (NHEJ) via direct biding to the N-terminal region of Rad50. A combination of biochemical and genetic data suggests that Rif2 promotes a transition within the MRX complex that is not conductive for endonuclease activity, DNA-end tethering or Tel1 kinase activation. We suggests that the MIN motif operates in the RIF2 paralog ORC4 (Origin Recognition Complex 4) in K. lactis and in telomeric protein Taz1 in Schizoccharomyces pombe, which is not evolutionarily related to Orc4/Rif2. These results highlight a potential Achilles heel in Rad50, the regulatory subunit of MRN, which we suggest has been targeted by different telomeric factors in multiple fungal lineages, raising the possibility that analogous approaches might be deployed in other Eukaryotes as well.


title: Gene name errors: lessons not learned

abstract: Erroneous conversion of gene names into other dates and other data types has been a frustration for computational biologists for years. We hypothesized that such errors in supplementary files might diminish after a report in 2016 highlighting the extent of the problem. To assess this, we performed a scan of supplementary files published in PubMed Central from 2014 to 2020. Overall, gene name errors continued to accumulate unabated in the period after 2016. An improved scanning software we developed identified gene name errors in 30.9% of articles with supplementary Excel gene lists; a figure significantly higher than previously estimated. This is due to gene names being converted not just to dates and floating-point numbers, but also to internal date format (five-digit numbers). These findings further reinforce that spreadsheets are ill-suited to use with large genomic data.


title: An automated feeding system for the African killifish reveals effects of dietary restriction on lifespan and allows scalable assessment of associative learning

abstract: The African turquoise killifish is an exciting new vertebrate model for aging studies. A significant challenge for any model organism is control over its diet in space and time. To address this challenge, we created an automated and networked fish feeding system. Our automated feeder is designed to be open-source, easily transferable, and built from widely available components. Compared to manual feeding, our automated system is highly precise and flexible. As a proof-of-concept for the feeding schedule flexibility of these automated feeders, we define a favorable regimen for growth and fertility for the African killifish and a dietary restriction regimen where both feeding time and quantity are reduced. We show that this dietary restriction regimen extends lifespan in males. Moreover, combining our automated feeding system with a video camera, we establish an associative learning assay for the killifish. This learning assay provides an integrative measure of cognitive decline during aging. The ability to precisely control food delivery in the killifish opens new areas to assess lifespan and cognitive behavior dynamics and to screen for dietary interventions and drugs in a high-throughput manner previously impossible with traditional vertebrate model organisms.

category: physiology


title: Robust and tunable signal processing in mammalian cells via engineered covalent modification cycles

abstract: Rewired and synthetic signaling networks can impart cells with new functionalities and enable efforts in engineering cell therapies and directing cell development. However, there is a need for tools to build synthetic signaling networks that are tunable, can precisely regulate target gene expression, and are robust to perturbations within the complex context of mammalian cells. Here, we use proteins derived from bacterial two-component signaling pathways to develop synthetic phosphorylation-based and feedback-controlled devices in mammalian cells with such properties. First, we isolate kinase and phosphatase proteins from the bifunctional histidine kinase EnvZ. We then use these proteins to engineer a synthetic covalent modification cycle, in which the kinase and phosphatase competitively regulate phosphorylation of the cognate response regulator OmpR, enabling analog tuning of OmpR-driven gene expression. Further, we show that the phosphorylation cycle can be extended by connecting phosphatase expression to small molecule and miRNA inputs in the cell, with the latter enabling cell-type specific signaling responses and accurate cell type classification. Finally, we implement a tunable negative feedback controller by co-expressing the kinase-driven output gene with the small molecule-tunable phosphatase. This negative feedback substantially reduces cell-to-cell noise in output expression and mitigates the effects of cell context perturbations due to off-target regulation and resource competition. Our work thus lays the foundation for establishing tunable, precise, and robust control over cell behavior with synthetic signaling networks.

category: synthetic biology


title: Functional architecture of the aging brain

abstract: The intrinsic functional organization of the brain changes into older adulthood. Age differences are observed at multiple spatial scales, from global reductions in modularity and segregation of distributed brain systems, to network-specific patterns of dedifferentiation. Whether dedifferentiation reflects an inevitable, global shift in brain function with age, circumscribed, experience dependent changes, or both, is uncertain. We employed a multi-method strategy to interrogate dedifferentiation at multiple spatial scales. Multi-echo (ME) resting-state fMRI was collected in younger (n=181) and older (n=120) healthy adults. Cortical parcellation sensitive to individual variation was implemented for precision functional mapping of each participant, while preserving group-level parcel and network labels. ME-fMRI processing and gradient mapping identified global and macroscale network differences. Multivariate functional connectivity methods tested for microscale, edge-level differences. Older adults had lower BOLD signal dimensionality, consistent with global network dedifferentiation. Gradients were largely age-invariant. Edge-level analyses revealed discrete, network-specific dedifferentiation patterns in older adults. Visual and somatosensory regions were more integrated within the functional connectome; default and frontoparietal control network regions showed greater connectivity; and the dorsal attention network was more integrated with heteromodal regions. These findings highlight the importance of multi-scale, multi-method approaches to characterize the architecture of functional brain aging.

category: neuroscience


title: Age differences in the functional architecture of the human brain


title: Impact of pubertal onset on region-specific Esr2 expression

abstract: In female rats, pubertal onset is associated with maturation of the medial prefrontal cortex (mPFC) and mPFC-mediated behaviors. These behavioral and anatomical changes are likely due to effects of estrogen at the nuclear estrogen receptor beta (ER{beta}), which is expressed at higher levels than the estrogen receptor alpha (ER) isoform in the adult mPFC. Researchers have previously quantified ER{beta} protein and Esr2 RNA in rodents during early postnatal development and adulthood, but an adolescent-specific trajectory of this receptor in the mPFC has not been documented. Given that levels of Esr2 can fluctuate in the presence or absence of estrogens, puberty and the subsequent rise in gonadal hormones could influence ER{beta} expression in the adolescent brain. To further explore this, we used RNAscope to quantify the amount of Esr2 mRNA in pre-pubertal adolescent, recently post-pubertal adolescent, and adult female rats. We show here that Esr2 expression decreases significantly in the mPFC, striatum and motor cortex between pre-pubertal adolescence and adulthood. In the mPFC, this decrease occurs rapidly at pubertal onset, with no significant decrease in Esr2 levels between the recently post-pubertal and adult cohort. In contrast in the striatum and motor cortex, there were no significant differences in the amount of Esr2 between pre- and post-pubertal females. Insofar as the amount of Esr2 is proportional to functional ER{beta}, these results suggest ER{beta} decreases in a region-specific pattern in response to pubertal onset and highlight a role for this receptor in the maturational events that occur in the female rat mPFC at puberty.


title: HuConTest: Testing human contamination in great ape samples

abstract:
Modern human contamination is a common problem in ancient DNA studies. We provide evidence that this issue is also present in studies in great apes, which are our closest living relatives, for example in non-invasive samples. Here, we present a simple method to detect human contamination in short read sequencing data from different species. We demonstrate its feasibility using blood and tissue samples from these species. This test is particularly useful for more complex samples (such as museum and non-invasive samples) which have smaller amounts of endogenous DNA, as we show here.

Significance statementHuman contamination can be a confounding factor in genomic studies, especially in the case of fecal, museum or ancient DNA from great apes. It is important for quality assessment, screening purposes and prioritization to identify and quantify such contamination. The tool presented here is a simple and versatile method for this purpose, and can be applied to a wide range of sample types.


title: Linking Diabetes mellitus to SARS-CoV-2 infection through differential targeting of the microRNAs in the Pancreas tissue

abstract: Coronavirus Disease 2019 (COVID-19) severity and Diabetes mellitus affect each other bidirectionally. The plus-sense single-stranded RNA (+ssRNA) genome of the SARS-CoV-2 virus can be targeted and suppressed by the host cells microRNAs (miRNAs). Using the differential gene expression analysis between the mock-infected and the SARS-CoV-2-infected pancreatic tissue, we report five Diabetes-associated genes that are upregulated due to SARS-CoV-2 infection in the hESC pancreas tissues. Ten miRNAs regulating these five genes can potentially target the SARS-CoV-2 genome. We hypothesize that the SARS-CoV-2 genome copies in the infected human pancreas cell compete with the host cells native genes in being regulated by the native miRNAs. It leads to the reduced miRNA-regulation and, thus, the upregulation of the Diabetes-associated native genes. Thus, the resultant new-onset or elevated Diabetic symptoms may worsen the condition of COVID-19 patients.


title: A spatial multi-scale fluorescence microscopy toolbox discloses entry checkpoints of SARS-CoV-2 variants in VeroE6 cells

abstract: We exploited a multi-scale microscopy imaging toolbox to address some major issues related to SARS-CoV-2 interactions with host cells. Our approach harnesses both conventional and super-resolution fluorescence microscopy and easily matches the spatial scale of single-virus/cell checkpoints. We deployed this toolbox to characterize subtle issues related to the entry phase of SARS-CoV-2 variants in Vero E6 cells. Our results suggest that in these cells the variant of concern B.1.1.7, (aka Alpha variant), became the predominant circulating variant in several countries by a clear transmission advantage. In fact, in these cells B.1.1.7 outcompetes its ancestor B.1.177 in terms of a much faster kinetics of entry. Given the cell-entry scenario dominated by the endosomal "late pathway", the faster internalization of B.1.1.7 could be directly related to the N501Y mutation in the S protein, which is known to strengthen the binding of Spike receptor binding domain with ACE2. Remarkably, we also directly observed the main role of clathrin as mediator of late-entry endocytosis, reconciling it with the membrane localization of the ACE2 receptor previously attributed to caveolin-enriched rafts. Overall, we believe that our fluorescence microscopy-based approach represents a fertile strategy to investigate the molecular features of SARS-CoV-2 interactions with cells.


title: A spatial multi-scale fluorescence microscopy toolbox discloses entry checkpoints of SARS-CoV-2 variants in Vero E6 cells


title: Quantification of durable CRISPR-based gene silencing activity

abstract: Development of CRISPR-based technologies for regulating gene expression stands to provide novel methods for the study and engineering of biological behavior. New tools capable of inducing long-lasting changes in gene expression will increase the utility of these techniques, providing durable effects from one-time doses of reagents. We describe here a reporter system for quantifying the ability of CRISPR-based effectors to induce stable gene repression. We observe a continuous gradation of the ability of these effectors to silence gene expression, depending on the domain composition and configuration. We also report the creation of a single CRISPR protein capable of producing durable gene silencing. This assay should allow for the continued development of enhanced gene repression tools which will be useful in a wide array of biological research and engineering applications.


title: Mobius Assembly for Plant Systems highlights promoter-terminator interaction in gene regulation

abstract: Plant synthetic biology is a fast-evolving field that employs engineering principles to empower research and bioproduction in plant systems. Nevertheless, in the whole synthetic biology landscape, plant systems lag compared to microbial and mammalian systems. When it comes to multigene delivery to plants, the predictability of the outcome is decreased since it depends on three different chassis: E. coli, Agrobacterium, and the plant species. Here we aimed to develop standardised and streamlined tools for genetic engineering in plant synthetic biology. We have devised Mobius Assembly for Plant Systems (MAPS), a user-friendly Golden Gate Assembly system for fast and easy generation of complex DNA constructs. MAPS is based on a new group of small plant binary vectors (pMAPs) that contains an origin of replication from a cryptic plasmid of Paracoccus pantotrophus. The functionality of the pMAP vectors was confirmed by transforming the MM1 cell culture, demonstrating for the first time that plant transformation is dependent on the Agrobacterium strains and plasmids; plasmid stability was highly dependent on the plasmid and bacterial strain. We made a library of new short promoters and terminators and characterised them using a high-throughput protoplast expression assay. Our results underscored the strong influence of terminators in gene expression, and they altered the strength of promoters in some combinations and indicated the presence of synergistic interactions between promoters and terminators. Overall this work will further facilitate plant synthetic biology and contribute to improving its predictability, which is challenged by combinatorial interactions among the genetic parts, vectors, and chassis.


title: Global Temporal Movement Control

abstract: Complex, goal-directed and time-critical movements require the processing of temporal features in sensory information as well as the fine-tuned temporal interplay of several effectors. Temporal estimates used to produce such behavior may thus be obtained through perceptual or motor processes. To disentangle the two options, we tested whether adaptation to a temporal perturbation in an interval reproduction task transfers to interval reproduction tasks with varying sensory information (visual appearance of targets, modality, virtual reality (VR) environment or real-world) or varying movement types (continuous arm movements or brief clicking movements). Halfway through the experiments we introduced a temporal perturbation, such that continuous pointing movements were artificially slowed down in VR, causing participants to adapt their behavior to sustain performance. In four experiments, we found that sensorimotor adaptation to temporal perturbations is independent of environment context and movement type, but modality specific. Our findings suggest that motor errors induced by temporal sensorimotor adaptation affect the modality specific perceptual processing of temporal estimates.


title: Temporal Perturbations Cause Global Sensorimotor Adaptation


title: Temporal perturbations cause movement-context independent but modality-specific sensorimotor adaptation


title: Understanding Others Requires Right Temporoparietal and Inferior Frontal Regions

abstract: The human ability to infer other peoples knowledge and beliefs, known as  theory of mind, is an essential component of social interactions. Theory of mind tasks activate frontal and temporoparietal regions of cortex in fMRI studies. However, it is unknown whether these regions are critical. We examined this question using multivariate voxel-based lesion symptom mapping in 22 patients with acute right hemisphere brain damage. Studies of acute patients eliminate questions of recovery and reorganization that plague long-term studies of lesioned patients. Damage to temporoparietal and inferior frontal regions impaired thinking about others perspectives. This impairment held even after adjustment for overall extent of brain damage and language comprehension, memory, comprehension, and attention abilities. These results provide evidence that right temporoparietal and inferior frontal regions are necessary for the human ability to reason about the knowledge and beliefs of others.


title: Differences in Regional Grey Matter Volume Predict the Extent to which Openness influences Judgments of Beauty and Pleasantness of Interior Architectural Spaces

abstract: Hedonic evaluation of sensory objects varies from person to person. While this variability has been linked to differences in experience and personality traits, little is known about why stimuli lead to different evaluations in different people. We used linear mixed effect models to determine the extent to which the openness, contour, and ceiling height of interior architectural spaces influenced the beauty and pleasantness ratings of 18 participants. Then, by analyzing structural brain images acquired for the same group of participants we asked if any regional grey matter volume (rGMV) co-varied with these differences in the extent to which openness, contour and ceiling height influence beauty and pleasantness ratings. Voxel-based morphometry analysis revealed that the influence of openness on pleasantness ratings correlated with rGMV in the anterior prefrontal cortex (BA 10), and the influence of openness on beauty ratings correlated with rGMV in the temporal pole (BA 38) and posterior cingulate cortex (BA 31). There were no significant correlations involving contour or ceiling height. Our results suggest that regional variance in grey matter volume may play a role in the computation of hedonic valuation, and account for differences in the way people weigh certain attributes of interior architectural spaces.


title: A new method for obtaining bankable and expandable adult-like microglial cells

abstract: The emerging role of microglia in neurological disorders requires a novel method for obtaining massive amounts of adult microglia because current in vitro methods for microglial study have many limitations, including a limited proliferative capacity, low cell yield, immature form, and too many experimental animals use. Here, we developed a new method for obtaining bankable and expandable adult-like microglial cells using the head neuroepithelial layer (NEL) of mouse E13.5. The NEL includes microglia progenitors that proliferate and ramify over time. Functional validation with a magnetic-activated cell sorting system using the NEL showed that the isolated CD11b-positive cells (NEL-MG) exhibited microglial functions, such as phagocytosis (microbeads, amyloid {beta}, synaptosome), migration, and inflammatory changes following lipopolysaccharide (LPS) stimulation. NEL was subcultured and the NEL-MG exhibited a higher expression of microglia signature genes than the neonatal microglia, a widely used in vitro surrogate. Banking or long-term subculture of NEL did not affect NEL-MG characteristics. Transcriptome analysis revealed that NEL-MG exhibited better conservation of microglia signature genes with a closer fidelity to freshly isolated adult microglia than neonatal microglia. This new method effectively contributes to obtaining adult-like microglial cells, even when only a small number of experimental animals are available, leading to a broad application in neuroscience-associated fields.


title: Antagonistic inhibitory subnetworks control cooperation and competition across cortical space

abstract: The cortical microcircuit can dynamically adjust to dramatic changes in the strength, scale, and complexity of its input. In the primary visual cortex (V1), pyramidal cells (PCs) integrate widely across space when signals are weak, but narrowly when signals are strong, a phenomenon known as contrast-dependent surround suppression. Theoretical work has proposed that local interneurons could mediate a shift from cooperation to competition of PCs across cortical space, underlying this computation. We combined calcium imaging and electrophysiology to constrain a stabilized supralinear network model that explains how the four principal cell types in layer 2/3 (L2/3) of mouse V1- somatostatin (SST), parvalbumin (PV), and vasoactive intestinal peptide (VIP) interneurons, and PCs- transform inputs from layer 4 (L4) PCs to encode drifting gratings of varying size and contrast. Using bidirectional optogenetic perturbations, we confirmed key predictions of the model. Our data and modeling showed that recurrent amplification drives a transition from a positive PC[->]VIP{dashv}SST{dashv}PC feedback loop at small size and low contrast to a negative PC[->]SST{dashv}PC feedback loop at large size and high contrast to contribute to this flexible computation. This may represent a widespread mechanism for gating competition across cortical space to optimally meet task demands.


title: A model of the reference frame of the ventriloquism aftereffect

abstract:
BackgroundVentriloquism aftereffect (VAE), observed as a shift in the perceived locations of sounds after audiovisual stimulation, requires reference frame (RF) alignment since hearing and vision encode space in different RFs (head-centered, HC, vs. eye-centered, EC). Experimental studies examining the RF of VAE found inconsistent results: a mixture of HC and EC RFs was observed for VAE induced in the central region, while a predominantly HC RF was observed in the periphery. Here, a computational model examines these inconsistencies, as well as a newly observed EC adaptation induced by AV-aligned audiovisual stimuli.

MethodsThe model has two versions, each containing two additively combined components: a saccade-related component characterizing the adaptation in auditory-saccade responses, and auditory space representation adapted by ventriloquism signals either in the HC RF (HC version) or in a combination of HC and EC RFs (HEC version).

ResultsThe HEC model performed better than the HC model in the main simulation considering all the data, while the HC model was more appropriate when only the AV-aligned adaptation data were simulated.

ConclusionVisual signals in a uniform mixed HC+EC RF are likely used to calibrate the auditory spatial representation, even after the EC-referenced auditory-saccade adaptation is accounted for.


title: Ankle muscles activate: mediolateral center of pressure control ensures stable gait

abstract: During steady-state walking, mediolateral gait stability can be maintained by controlling the center of pressure (CoP). The CoP modulates the moment of the ground reaction force, which brakes and reverses movement of the center of mass (CoM) towards the lateral border of the base of support. In addition to foot placement, ankle moments serve to control the CoP. We hypothesized that, during steady-state walking, single stance ankle moments establish a CoP shift to correct for errors in foot placement. We expected ankle muscle activity to be associated with this complementary CoP shift. During treadmill walking, full-body kinematics, ground reaction forces and electromyography were recorded in thirty healthy participants. We found a negative relationship between preceding foot placement error and CoP displacement during single stance; steps that were too medial were compensated for by a lateral CoP shift and vice versa, steps that were too lateral were compensated for by a medial CoP shift. Peroneus longus, soleus and tibialis anterior activity correlated with these CoP shifts. As such, we identified an (active) ankle strategy during steady-state walking. As expected, absolute explained CoP variance by foot placement error decreased when walking with shoes constraining ankle moments. Yet, contrary to our expectations that ankle moment control would compensate for constrained foot placement, the absolute explained CoP variance by foot placement error did not increase when foot placement was constrained. We argue that this lack of compensation reflects the interdependent nature of ankle moment and foot placement control. We suggest that single stance ankle moments do not only compensate for preceding foot placement errors, but also assist control of the subsequent foot placement. Foot placement and ankle moment control are  caught in a circular relationship, in which constraints imposed on one will also influence the other.


title: Ankle muscles drive mediolateral center of pressure control to ensure stable steady state gait


title: Neural basis of functional ultrasound signals

abstract: Functional ultrasound imaging (fUSI) is a popular method for measuring blood flow and thus infer brain activity, but it relies on the physiology of neurovascular coupling and requires extensive signal processing. To establish to what degree its trial-by-trial signals reflect neural activity, we performed simultaneous fUSI and neural recordings with Neuropixels probes in awake mice. fUSI signals strongly correlated with the slow (<0.3 Hz) fluctuations in local firing rate, and were closely predicted by the smoothed firing rate of local neurons, particularly putative inhibitory neurons. The optimal smoothing filter had width ~3 s, matched the hemodynamic response function of awake mouse, was invariant across mice and stimulus conditions, and similar in cortex and hippocampus. fUSI signals also matched neural firing spatially: firing rates were as highly correlated across hemispheres as fUSI signals. Thus, hemodynamic signals measured by ultrasound bear a simple and accurate relationship to neuronal firing.


title: Single-Cell Transcriptomic Analysis of Livers During NLRP3 Inflammasome Activation Reveals a Novel Immune Niche

abstract: The NOD-like receptor protein 3 (NLRP3) inflammasome is a central contributor to human acute and chronic liver disease, yet the molecular and cellular mechanisms by which its activation precipitates injury remain incompletely understood. Here, we present single cell transcriptomic profiling of livers from a global transgenic Tamoxifen-inducible constitutively-activated Nlrp3A350V mutant mouse, and we investigate the changes in parenchymal and non-parenchymal liver cell gene expression that accompany inflammation and fibrosis. Our results demonstrate that NLRP3 activation causes chronic extramedullary myelopoiesis marked by an increase in proliferating myeloid progenitors that differentiate into neutrophils, monocytes, and monocyte-derived macrophages, results that were corroborated by flow cytometry and histological staining. We observed prominent neutrophil infiltrates with increased Ly6gHI and Ly6gINT cells exhibiting transcriptomic signatures of granulopoiesis typically found in the bone marrow. This was accompanied by a marked increase in Ly6cHI monocytes differentiating into Cd11bHITim4HIClec4fHI macrophages that express proinflammatory transcriptional programs similar to macrophages of non-alcoholic steatohepatitis (NASH) models. NLRP3 activation also downregulated metabolic pathways in hepatocytes and shifted hepatic stellate cells towards an activated pro-fibrotic state based on expression of collagen and extracellular matrix (ECM) regulatory genes. These results, which highlight abundant neutrophils and extramedullary granulopoiesis define an inflamed and fibrotic hepatic single cell microenvironment, precipitated solely by NLRP3 activation. Clinically, our data support the notion that neutrophils and NLRP3 should be explored as therapeutic targets in NASH-like inflammation.

category: immunology


title: Candida albicans enhances the progression of oral squamous cell cancrinoma in vitro and in vivo

abstract: Oral squamous cell carcinoma (OSCC) is a serious health issue worldwide. OSCC is highly associated with oral candidiasis, although it is unclear whether the fungus promotes the genesis and progression of OSCC or cancer facilitates the growth of the fungus. Therefore, we investigated whether Candida could directly influence OSCC development and progression. Our in vitro results suggest that the presence of live C. albicans, but not C. parapsilosis, enhances the progression of OSCC by stimulating the production of matrix metalloproteinases, oncometabolites, pro-tumor signaling routes, and overexpression of prognostic marker genes associated with metastatic events. We also found that oral candidiasis triggered by C. albicans enhanced the progression of OSCC in vivo through the induction of inflammation and overexpression of metastatic genes and markers of epithelial-mesenchymal transition. Taken together, these results suggest that C. albicans actively participates in the complex process of OSCC progression.


title: A Broadly Conserved Deoxycytidine Deaminase Protects Bacteria from Phage Infection

abstract: The El Tor biotype of Vibrio cholerae is responsible for perpetuating the longest cholera pandemic in recorded history (1961-current). The genomic islands VSP-1 and -2 are two understudied genetic features that distinguish El Tor from previous pandemics. To understand their utility, we calculated the co-occurrence of VSP genes across bacterial genomes. This analysis predicted the previously uncharacterized vc0175, herein renamed deoxycytidylate deaminase Vibrio (dcdV), is in a gene network with dncV, a cyclic GMP-AMP synthase involved in phage defense. DcdV consists of two domains, a P-loop kinase and a deoxycytidylate deaminase, that are required for the deamination of dCTP and dCMP, inhibiting phage predation by corrupting cellular nucleotide concentrations. Additionally, DcdV is post-translationally inhibited by a unique noncoding RNA encoded 5 of the dcdV locus. DcdV homologs are conserved in bacteria and eukaryotes and our results identify V. cholerae DcdV as the founding member of a previously undescribed bacterial phage defense system.


title: A nascent polypeptide sequence modulates DnaA translation elongation in response to nutrient availability

abstract: The ability to regulate DNA replication initiation in response to changing nutrient conditions is an important feature of most cell types. In bacteria, DNA replication is triggered by the initiator protein DnaA, which has long been suggested to respond to nutritional changes, nevertheless the underlying mechanisms remain poorly understood. Here, we report a novel mechanism that adjusts DnaA synthesis in response to nutrient availability in Caulobacter crescentus. By performing a detailed biochemical and genetic analysis of the dnaA mRNA we identified a sequence downstream of the dnaA start codon that inhibits DnaA translation elongation upon carbon exhaustion. Our data show that the corresponding peptide sequence, but not the mRNA secondary structure or the codon choice, are critical for this response, suggesting that specific amino acids in the growing DnaA nascent chain tune translational efficiency. Our study provides new insights into DnaA regulation and highlights the importance of translation elongation as a regulatory target. We propose that nascent chain sequences, like the one described, might constitute a general strategy for modulating the synthesis rate of specific proteins under changing conditions.


title: Unprecedented Protein Divergence within a T3SS Family

abstract: Selection pressure drives rapid emergence of antibiotic resistance mechanisms promoting searches for therapeutic targets in bacterial processes needed for virulence, not viability, which include the Type Three Secretion System (T3SS). Distinct T3SS families evolved from the flagellar export apparatus where remaining homology hinders development of anti-T3SS specific therapies. Around 15 proteins that are highly-conserved within, but not between T3SS families yet such divergence is rarely leveraged, to promote understanding, due to unknown evolutionary histories. Here we document unprecedented divergence in two  LEE T3SS family members. Interchangeability studies uncover unusual LEE biology (eg 2-orf genes) and illustrate each T3SS protein can tolerate dramatic change. Functional defects (12 proteins) and novel phenotypes enabled studies that reveal i) pathotype-specific protein functionality, ii) T3SS crosstalk with other processes, and iii) potential therapeutic targets. The work provides resources and testable predictions for further discoveries and will promote comparable studies between distinct T3SS families.


title: Deletion of hypoxia-inducible factor prolyl 4-hydroxylase 2 in FoxD1-lineage mesenchymal cells leads to congenital truncal alopecia

abstract: Hypoxia-inducible factors (HIFs) induce hundreds of genes regulating oxygen homeostasis in tissues. Oxygen sensors of the cells, the HIF prolyl 4-hydroxylases (HIF-P4Hs), regulate the stability and activity of HIFs in an oxygen-dependent manner. In this study, we show that lack of Hif-p4h-2 in FoxD1-lineage mesodermal cells interferes the normal development of hair follicles (HF) in mice. The FoxD1-lineage cells were found to be mainly mesenchymal cells located in the dermis of truncal skin, including the cells composing the dermal papilla of the HF. Upon Hif-p4h-2 inactivation, HF development was disturbed during the first catagen leading to formation of large epithelial lined HF cysts filled by unorganized keratins, which eventually manifested as truncal alopecia. The depletion of Hif-p4h-2 led to HIF stabilization and dysregulation of multiple genes involved in keratin formation, HF differentiation, and HIF, TGF{beta} and Notch signaling. The failure of the controlled process of HF cycling is likely to be mechanistically caused by disruption of the precise and timely interplay of the HIF, TGF{beta} and Notch pathways. In summary, we show here for the first time that HIF-P4H-2 function in FoxD1-lineage cells is essential for the normal development and homeostasis of HFs.

category: molecular biology


title: Virulence shift in a sexual clade of Type X Toxoplasma infecting Southern Sea Otters

abstract:
How virulent protozoal pathogens capable of causing overt disease are maintained in nature is an important paradigm of eukaryotic pathogenesis. Here we used population genetics and molecular methods to study the evolution and emergence of a marine invasion of new genetic variants of Toxoplasma gondii, referred collectively as Type X (HG12). 53 Toxoplasma isolates were obtained from mustelids that stranded between 1998-2004 with toxoplasmosis (ranging from chronic infection to fatal encephalitis). Over 74% of the sea otters collected throughout their geographic range were infected with Type X as determined by multi-locus PCR-DNA sequencing. Depending on the locus investigated, Type X strains possessed one of three allelic types that had independently assorted across the strains examined; either genetically distinct alleles, referred to as "{gamma}" or "{delta}", or a Type II allele. Phylogenetic incongruence among locus-specific trees, genome-wide CGH array and WGS analyses confirmed that Type X is a sexual clade of natural recombinants that resemble F1 progeny from a genetic cross between Type II and a mosaic of two distinct "{gamma}" or "{delta}" ancestries. A single Type X genotype (19/53; 36%) had expanded in sea otters largely as subclinical chronic infections, but it was highly pathogenic to mice (LD100= 1 parasite). To determine whether murine virulence genes could be mapped within this naturally occurring population, we performed a genome scan and identified four QTLs with LOD scores greater than 4.0. Targeted disruption of ROP33, the strongest candidate from among 16 genes within the highest QTL on Chromosome VIIa established ROP33 as a murine virulence locus. The ability of this highly pathogenic clone to expand and cause the majority of sea otter infections supports a virulence shift model whereby generalist pathogens like Toxoplasma utilize their sexual cycles to produce new strains with an expanded biological potential. Such a trait enables pathogens to extend their host range or be naturally selected within their vast intermediate host range to maximize their transmission. Our work thus establishes a rationale for how virulent strains can be maintained cryptically in nature across a pathogens broad host range, and act as reservoirs for epidemic disease.

ImportanceWaterborne outbreaks of protozoal parasites are increasingly causing fatal disease in a wide range of animals, including humans. Population expansion of felids near marine estuarine environments has led to increased exposure of marine wildlife to highly infectious Toxoplasma gondii oocysts shed in the feces of cats that are dispersed by storm events. In North America Toxoplasma is thought to possess a highly clonal population structure dominated by 4 clonal lineages (I, II, III, and X). Population genetic analysis of 53 Toxoplasma isolates collected longitudinally from mustelids infected with Toxoplasma that stranded between 1998-2004 identified a majority of otters (74%) to be infected with Type X Toxoplasma, and that Type X is not a clonal lineage, but rather a recombinant clade of strains consistent with a recent genetic cross that produced at least 12 distinct haplotypes. Importantly, one Type X haplotype expanded in 36% of otters across their geographic range and caused relatively benign infections, however it was highly pathogenic to mice. A genome scan was performed to identify a new virulence locus, a secreted serine threonine kinase (ROP33), that is pathogenic in mice, but not sea otters. Our data support a virulence shift model whereby generalist pathogens like Toxoplasma utilize their sexual cycles to produce virulent strains that are maintained cryptically in nature, according to their differential capacity to cause disease within the pathogens broad intermediate host range. This type of "zoonotic selection" has important public health implications. Strains capable of causing fatal infections can persist in nature by circulating as chronic infections in intermediate host species that act as reservoirs for epidemic disease.


title: Structural basis for the tryptophan sensitivity of TnaC-mediated ribosome stalling

abstract: Free L-tryptophan (L-Trp) induces the expression of the Escherichia coli tryptophanase operon, leading to the production of indole from L-Trp. Tryptophanase operon expression is controlled via a mechanism involving the tryptophan-dependent stalling of ribosomes engaged in translation of tnaC, a leader sequence upstream of tnaA that encodes a 24-residue peptide functioning as a sensor for L-Trp. Although extensive biochemical characterization has revealed the elements of the TnaC peptide and the ribosome that are responsible for translational arrest, the molecular mechanism underlying the recognition and response to L-Trp by the TnaC-ribosome complex remains unknown. Here, we use a combined biochemical and structural approach to characterize a variant of TnaC (R23F) in which stalling by L-Trp is enhanced because of reduced cleavage of TnaC(R23F)-peptidyl-tRNA. In contrast to previous data originated from lower resolution structural studies, we show that the TnaC-ribosome complex captures a single L-Trp molecule to undergo tryptophan-dependent termination arrest and that nascent TnaC prevents the catalytic GGQ loop of release factor 2 from adopting an active conformation at the peptidyl transferase center. In addition, we show that the conformation of the L-Trp binding site is not altered by the R23F mutation. This leads us to propose a model in which rates of TnaC-peptidyl-tRNA cleavage by release factor and binding of the L-Trp ligand to the translating ribosome determine the tryptophan sensitivity of the wild-type and mutant TnaC variants. Thus, our study reveals a strategy whereby a nascent peptide assists the bacterial ribosome in sensing a small metabolite.


title: Molecular characterization of a complex of Apoptosis Inducing Factor 1 (AIFM1) with cytochrome c oxidase of the mitochondrial respiratory chain

abstract: Combining mass spectrometry based chemical cross-linking and complexome profiling, we analyzed the interactome of heart mitochondria. We focused on complexes of oxidative phosphorylation and found that dimeric apoptosis inducing factor 1 (AIFM1) forms a defined complex with ~10% of monomeric cytochrome c oxidase (COX), but hardly interacts with respiratory chain supercomplexes. Multiple AIFM1 inter-crosslinks engaging six different COX subunits provided structural restraints to build a detailed atomic model of the COX-AIFM12 complex. Application of two complementary proteomic approaches thus provided unexpected insight into the macromolecular organization of the mitochondrial complexome. Our structural model excludes direct electron transfer between AIFM1 and COX. Notably however, the binding site of cytochrome c remains accessible allowing formation of a ternary complex. The discovery of the previously overlooked COX-AIFM12 complex and clues provided by the structural model hint at a role of AIFM1 in OXPHOS biogenesis and in programmed cell death.


title: A tyrosine-based trafficking signal in the simian immunodeficiency virus envelope cytoplasmic domain is strongly selected for in pathogenic SIV infection

abstract: The HIV/SIV envelope glycoprotein (Env) cytoplasmic domain contains a highly conserved Tyr-dependent trafficking signal that mediates both clathrin-dependent endocytosis and polarized sorting of Env. Despite extensive characterization, the role of these functions in viral infection and pathogenesis is unclear. An SIV molecular clone (SIVmac239) in which the Tyr-based signal is inactivated by deletion of Gly-720 and Tyr-721 (SIVmac239{Delta}GY) replicates to high levels acutely in pigtail macaques (PTM) but is rapidly controlled. We previously reported that rhesus macaques and PTM can progress to AIDS following SIVmac239{Delta}GY infection in association with novel amino acid changes in the Env cytoplasmic domain. These included an R722G flanking the {Delta}GY deletion and a nine nucleotide deletion that encodes amino acids 734-736 ({Delta}QTH) and overlaps with the rev and tat open reading frames. We show that molecular clones containing these mutations reconstitute signals for both endocytosis and polarized sorting. In one PTM, a novel genotype was selected, which generated a new signal for polarized sorting but not endocytosis. This mutation by itself was sufficient to maintain high viral loads for several months when introduced into naive PTMs. These findings reveal, for the first time, strong selection pressure for Env endocytosis and, in particular, for polarized sorting during pathogenic SIV infection in vivo.

category: cell biology


title: Decreased adaptation at human disease genes as a possible consequence of interference between advantageous and deleterious variants

abstract: Advances in genome sequencing have dramatically improved our understanding of the genetic basis of human diseases, and thousands of human genes have been associated with different diseases. Despite our expanding knowledge of gene-disease associations, and despite the medical importance of disease genes, their evolution has not been thoroughly studied across diverse human populations. In particular, recent genomic adaptation at disease genes has not been well characterized, even though multiple evolutionary processes are expected to connect disease and adaptation at the gene level. Understanding the relationship between disease and adaptation at the gene level in the human genome is severely hampered by the fact that we dont even know whether disease genes have experienced more, less, or as much adaptation as non-disease genes during recent human evolution. Here, we compare the rate of strong recent adaptation in the form of selective sweeps between disease genes and non-disease genes across 26 distinct human populations from the 1,000 Genomes Project. We find that disease genes have experienced far less selective sweeps compared to non-disease genes during recent human evolution. This sweep deficit at disease genes is particularly visible in Africa, and less visible in East Asia or Europe, likely due to more intense genetic drift in the latter populations creating more spurious selective sweeps signals. Investigating further the possible causes of the sweep deficit at disease genes, we find that this deficit is very strong at disease genes with both low recombination rates and with high numbers of associated disease variants, but is inexistant at disease genes with higher recombination rates or lower numbers of associated disease variants. Because recessive deleterious variants have the ability to interfere with adaptive ones, these observations strongly suggest that adaptation has been slowed down by the presence of interfering recessive deleterious variants at disease genes. These results clarify the evolutionary relationship between disease genes and recent genomic adaptation, and suggest that disease genes suffer not only from a higher load of segregating deleterious mutations, but also an inability to adapt as much, and/or as fast as the rest of the genome.


title: CRISPR-mediated knock-in of transgenes into the malaria vector Anopheles funestus

abstract:
The ability to introduce mutations, or transgenes, of choice to precise genomic locations has revolutionised our ability to understand how genes and organisms work.

In many mosquito species that are vectors of various human disease, the advent of CRISPR genome editing tools has shed light on basic aspects of their biology that are relevant to their efficiency as disease vectors. This allows a better understanding of how current control tools work and opens up the possibility of novel genetic control approaches, such as gene drives, that deliberately introduce genetic traits into populations. Yet for the Anopheles funestus mosquito, a significant vector of malaria in sub-Saharan Africa and indeed the dominant vector species in many countries, transgenesis has yet to be achieved.

We describe herein an optimised transformation system based on the germline delivery of CRISPR components that allows efficient cleavage of a previously validated genomic site and preferential repair of these cut sites via homology-directed repair (HDR), which allows introduction of exogenous template sequence, rather than end-joining repair. The rates of transformation achieved are sufficiently high that it should be able to introduce alleles of choice to a target locus, and recover these, without the need to include additional dominant marker genes. Moreover, the high rates of HDR observed suggest that gene drives, which employ an HDR-type mechanism to ensure their proliferation in the genome, may be well suited to work in An. funestus.


title: Differential mutational profile of SARS-CoV-2 proteins across deceased and asymptomatic patients

abstract: The SARS-CoV-2 infection spread at an alarming rate with many places showed multiple peaks in incidence. Present study involves a total of 332 SARS-CoV-2 sequences from 114 Asymptomatic and 218 Deceased patients from twenty-one different countries. The mining of mutations was done using the GISAID CoVSurver (www.gisaid.org/epiflu-applications/covsurver-mutations-app) with the reference sequence  hCoV-19/Wuhan/WIV04/2019 present in NCBI with Accession number NC-045512.2. The impact of the mutations on SARS-CoV-2 proteins mutation was predicted using PredictSNP1(loschmidt.chemi.muni.cz/predictsnp1) which is a meta-server integrating six predictor tools: SIFT, PhD-SNP, PolyPhen-1, PolyPhen-2, MAPP and SNAP. The iStable integrated server (predictor.nchu.edu.tw/iStable) was used to predict shifts in the protein stability due to mutations. A total of 372 variants were observed in the 332 SARS-CoV-2 sequences with several variants incident in multiple patients accounting for a total of 1596 incidences. Asymptomatic and Deceased specific mutants constituted 32% and 62% of the repertoire respectively indicating their exclusivity. However, the most prevalent mutations were those present in both. Though some parts of the genome are more variable than others but there was clear difference between incidence and prevalence. NSP3 with 68 variants had total occurrence of only 105 whereas Spike protein had 346 occurrences with just 66 variants. For Deleterious variants, NSP3 had the highest incidence of 25 followed by NSP2 (16), ORF3a (14) and N (14). Spike protein had just 7 Deleterious variants out of 66. Deceased patients have more Deleterious than Neutral variants as compared to the symptomatic ones. Further, it appears that the Deleterious variants which decrease protein stability are more significant in pathogenicity of SARS-CoV-2.


title: The sleeping brain switches between working memory and long-term memory processing.

abstract:
We provide evidence that human sleep is a competitive arena where cognitive domains vie for limited resources. Using pharmacology and effective connectivity analysis, we demonstrate that long-term memory and working memory are served by distinct offline neural mechanisms that are mutually antagonistic. Specifically, we administered zolpidem to increase central sigma activity and demonstrated targeted suppression of autonomic vagal activity. With effective connectivity, we determined the central activity has greater causal influence over autonomic activity, and the magnitude of this influence during sleep produced a behavioral trade-off between offline long-term and working memory processing. These findings show the first evidence of a sleep switch mechanism that toggles between central sigma-dependent long-term memory and autonomic vagal-dependent working memory processing.

Significant StatementSleep facilitates both long-term episodic memory consolidation and short-term working memory functioning. However, the mechanism by which the sleeping brain performs both complex feats, and which sleep features are associated with these processes remain unclear. Using a pharmacological approach, we demonstrate that long-term and working memory are served by distinct offline neural mechanisms, and that these mechanisms are mutually antagonistic. We propose a Sleep Switch model in which the brain toggles between the two memory processes via a complex interaction at the synaptic, systems, and mechanistic level, with implications for research on cognitive disturbances observed in neurodegenerative disorders such as Alzheimers and Parkinsons disease, both of which involve the decline of sleep.


title: Competitive dynamics underlie cognitive improvements during sleep.


title: Balance training improves feedback control of perturbed balance in older adults

abstract: Recovering balance after perturbations becomes challenging with aging, but an effective balance training could reduce such challenges. In this study, we examined the effect of balance training on feedback control after unpredictable perturbations by investigating balance performance, recovery strategy, and muscle synergies. We assessed the effect of balance training on unipedal perturbed balance in twenty older adults (>65 years) after short-term (one session) and long-term (3-weeks) training. Participants were exposed to random medial and lateral perturbations consisting of 8-degree rotations of a robot-controlled balance platform. We measured full-body 3D kinematics and activation of 9 muscles (8 stance leg muscles, one trunk muscle) during 2.5 s after the onset of perturbation. The perturbation was divided into 3 phases: phase1 from the onset to maximum rotation of the platform, phase 2 from the maximum rotation angle to the 0-degree angle and phase 3 after platform movement. Balance performance improved after long-term training as evidenced by decreased amplitudes of center of mass acceleration and rate of change of body angular momentum. The rate of change of angular momentum did not directly contribute to return of the center of mass within the base of support, but it reoriented the body to an aligned and vertical position. The improved performance coincided with altered activation of synergies depending on the direction and phase of the perturbation. We concluded that balance training improves control of perturbed balance, and reorganizes feedback responses, by changing temporal patterns of muscle activation. These effects were more pronounced after long-term than short-term training.


title: Mechanisms involved in the active secretion of CTX-M-15 β-lactamase by pathogenic E. coli ST131

abstract:
Infections caused by antimicrobial resistant bacterial pathogens are fast becoming an important global health issue. Strains of Escherichia coli are common causal agents of urinary tract infection and can carry multiple resistance genes. This includes the gene blaCTX-M-15 that encodes for an extended spectrum beta-lactamase (ESBL). While studying antimicrobial resistance (AMR) in the environment we isolated several strains of E. coli ST131 downstream of a WWTP in a local river. These isolates were surviving in the river sediment and characterisation proved that a multi-resistant phenotype was evident. Here, we show that E. coli strain 48 (river isolate ST131), provided a protective effect against a third-generation cephalosporin (cefotaxime) for a susceptible E. coli strain 33 (river isolate ST3576) through secretion of a functional ESBL into the growth medium. Furthermore, extracellular ESBL activity was stable for at least 24 h after secretion. Proteomic and molecular genetic analyses identified CTX-M-15 as the major secreted ESBL responsible for the observed protective effect. In contrast to previous studies, OMVs were not the sole route for CTX-M-15 secretion. Indeed, mutation of the Type I secretion system led to a significant reduction in the growth of the ESBL-producing strain as well as a significantly reduced ability to confer protective effect. We speculate that CTX-M-15 secretion, mediated through active secretion using molecular machinery provides a public goods service by facilitating the survival of otherwise susceptible bacteria in the presence of cefotaxime.

Abstract importanceInfections caused by antimicrobial resistant bacterial pathogens have become an important global health issue. Wastewater treatment plants (WWTPs) have been identified as hotspots for the dissemination of antimicrobial resistant genes/bacteria into the environment. In this study, we investigated resistance enzyme secretion by a multi-drug resistant human pathogenic E. coli, isolated from a UK river, downstream of a WWTP. We present evidence that the resistant strain actively secreted an important resistance enzyme into the surrounding medium which degraded the antibiotic cefotaxime. This research provided evidence for the mechanism for secretion of this enzyme which could indicate a new target to tackle antibiotic resistance pathogens.

category: microbiology


title: JAG1 intracellular domain acts as a transcriptional cofactor that forms an oncogenic transcriptional complex with DDX17/SMAD3/TGIF2

abstract: Jagged1 (JAG1) is a Notch ligand that contact-dependently activates Notch receptors and regulates cancer progression. The JAG1 intracellular domain (JICD1) is generated from JAG1, such as the formation of NOTCH1 intracellular domain (NICD1), however, the role of JICD1 in tumorigenicity has not been comprehensively elucidated. Herein, we revealed that JICD1 induced astrocytes to acquire several cancer stem cell properties, including tumor formation, invasiveness, stemness, and resistance to chemotherapy and radiotherapy. The transcriptome, ChIP-sequencing, and proteomic analyses revealed that JICD1 increased SOX2 expression by forming a transcriptional complex with DDX17, SMAD3, and TGIF2. Furthermore, JICD1-driven tumorigenicity was directly regulated by SOX2. Therefore, our results demonstrated that, like NICD1, JICD1 acts as a transcriptional cofactor in the formation of the DDX17/SMAD3/TGIF2 transcriptional complex, leading to oncogenic transformation.


title: Large-scale identification of protein histidine methylation in human cells

abstract: Methylation can occur on histidine, lysine and arginine residues in proteins and often serves a regulatory function. Histidine methylation has recently attracted notable attention through the discovery of the human histidine methyltransferase enzymes SETD3 and METTL9. There are currently no methods to enrich histidine methylated peptides for mass spectrometry analysis and large-scale analyses of the modification are hitherto absent. In the present study we query ultra-comprehensive proteomic datasets to generate a resource of histidine methylation sites in human cells. We use this resource to explore the frequency, localization, targeted domains, protein types and sequence requirements of histidine methylation and benchmark all analyses to methylation events on lysine and arginine. Our results demonstrate that histidine methylation is widespread in human cells and tissues and that the modification is over-represented in regions of mono-spaced histidine repeats. We also report colocalization of the modification with functionally important phosphorylation sites and disease associated mutations to identify regions of likely regulatory and functional importance. Taken together, we here report a system level analysis of human histidine methylation and our results represent a comprehensive resource enabling targeted studies of individual histidine methylation events.


title: Predicting skeletal stature using ancient DNA

abstract:
ObjectivesAncient DNA provides an opportunity to separate the genetic and environmental bases of complex traits by allowing direct estimation of genetic values in ancient individuals. Here, we test whether genetic scores for height in ancient individuals are predictive of their actual height, as inferred from skeletal remains. We estimate the contributions of genetic and environmental variables to observed phenotypic variation as a first step towards quantifying individual sources of morphological variation.

Materials and MethodsWe collected stature estimates and femur lengths from West Eurasian skeletal remains with published genome-wide ancient DNA data (n=167, dating from 33,000-850 BP). We also recorded genetic sex, genetic ancestry, date and paleoclimate data for each individual, and{delta} 13C and{delta} 15N stable isotope values where available (n=67).

ResultsA polygenic score (PRS) for height predicts 6.8% of the variance in femur length in our data (n=117, SD=0.0068%, p<0.001), controlling for sex, ancestry, and date. This is consistent with the predictive power of height PRS in present-day populations and the low coverage of ancient samples. Comparatively, sex explains about 15% of the variance in femur length in our sample. Environmental effects also likely play a role in variation, independent of genetics, though with considerable uncertainty (longitude: R2=0.0317, SD=0.009, p=0.019).

DiscussionPolygenic scores explain a small but significant proportion of the variance in height in ancient individuals, though not enough to make useful predictions of individual phenotypes. However, environmental variables also contribute to phenotypic outcomes and understanding their interaction with direct genetic predictions will provide a framework with which to model how plasticity and genetic changes ultimately combine to drive adaptation and evolution.


title: An integrative skeletal and paleogenomic analysis of prehistoric stature variation suggests relatively reduced health for early European farmers

abstract: Human culture, biology, and health were shaped dramatically by the onset of agriculture ~12,000 years before present (BP). Subsistence shifts from hunting and gathering to agriculture are hypothesized to have resulted in increased individual fitness and population growth as evidenced by archaeological and population genomic data alongside a simultaneous decline in physiological health as inferred from paleopathological analyses and stature reconstructions of skeletal remains. A key component of the health decline inference is that relatively shorter statures observed for early farmers may (at least partly) reflect higher childhood disease burdens and poorer nutrition. However, while such stresses can indeed result in growth stunting, height is also highly heritable, and substantial inter-individual variation in the height genetic component within a population is typical. Moreover, extensive migration and gene flow were characteristics of multiple agricultural transitions worldwide. Here, we consider both osteological and ancient DNA data from the same prehistoric individuals to comprehensively study the trajectory of human stature variation as a proxy for health across a transition to agriculture. Specifically, we compared  predicted genetic contributions to height from paleogenomic data and  achieved adult osteological height estimated from long bone measurements on a per-individual basis for n=160 ancient Europeans from sites spanning the Upper Paleolithic to the Iron Age (~38,000-2,400 BP). We found that individuals from the Neolithic were shorter than expected (given their individual polygenic height scores) by an average of -4.47 cm relative to individuals from the Upper Paleolithic and Mesolithic (P=0.016). The average osteological vs. expected stature then increased relative to the Neolithic over the Copper (+2.67 cm, P=0.052), Bronze (+3.33 cm, P=0.032), and Iron Ages (+3.95 cm, P=0.094). These results were partly attenuated when we accounted for genome-wide genetic ancestry variation in our sample (which we note is partly duplicative with the individual polygenic score information). For example, in this secondary analysis Neolithic individuals were -3.48 cm shorter than expected on average relative to individuals from the Upper Paleolithic and Mesolithic (P=0.056). We also incorporated observations of paleopathological indicators of non-specific stress that can persist from childhood to adulthood in skeletal remains (linear enamel hypoplasia, cribra orbitalia, and porotic hyperostosis) into our model. Overall, our work highlights the potential of integrating disparate datasets to explore proxies of health in prehistory.