title: Bimodal Endocytic Probe for Three-Dimensional Correlative Light and Electron Microscopy

abstract: Correlative light and electron microscopy (CLEM) can infer molecular, functional and dynamic information to ultrastructure by linking information of different imaging modalities. One of the main challenges, especially in 3D-CLEM, is the accurate registration of fluorescent signals to electron microscopy (EM). Here, we present fluorescent BSA-gold (fBSA-Au), a bimodal endocytic tracer as fiducial marker for 2D and 3D CLEM applications. fBSA-Au consists of colloidal gold (Au) particles stabilized with fluorescent bovine serum albumin (BSA). The conjugate is efficiently endocytosed and distributed throughout the 3D endo-lysosomal network of the cells, and has an excellent visibility both in fluorescence microscopy (FM) and EM. We demonstrate the use of fBSA-Au in several 2D and 3D CLEM applications using Tokuyasu cryosections, resin-embedded material, and cryo-EM. As a fiducial marker, fBSA-Au facilitates rapid registration of regions of interest between FM and EM modalities and enables accurate (50-150 nm) correlation of fluorescence to EM data. Endocytosed fBSA-Au benefits from a homogenous 3D distribution throughout the endosomal system within the cell, and does not obscure any cellular ultrastructure. The broad applicability and visibility in both modalities makes fBSA-Au an excellent endocytic fiducial marker for 2D and 3D (cryo-)CLEM applications.


title: Sequence Determinants of Human Junctophilin-2 Protein Nuclear Localization and Phase Separation

abstract: Junctophilin-2 (JPH2) was conventionally considered as a structural membrane binding protein. Recently, it was shown that proteolytically truncated mouse JPH2 variants are imported into nucleus to exert alternative functions. However, the intranuclear behaviors of human JPH2 (hJPH2) and underlying molecular determinants have not been explored. Here, we demonstrate that full-length hJPH2 is imported into nucleus in human cells by two nuclear localization signals (NLSs), including a newly discovered one at the C-terminus. Importantly, unlike the JPH2 N-terminal truncation which diffuses throughout the nucleus, full-length hJPH2 forms nuclear bodies behaving like liquid-liquid phase separated droplets that are separated from chromatin. The C-terminal transmembrane domain is required for the formation of hJPH2 droplets. Oxidation mimicking substitution of residues C678 and M679 augments the formation of hJPH2 nuclear droplets, suggesting nuclear hJPH2 liquid-liquid phase separation could be modulated by oxidative stress. Mutation A405D, which introduces a negatively charged residue into an intrinsic disordered region (IDR) of hJPH2, turns liquid-like droplets into amyloid-like aggregates. Depletion of an Alanine Rich Region in the IDR recapitulates the liquid-amyloid phase transition. The MORN repeat regions of hJPH2 encodes intrinsic tendency to form amyloid-like structure. Together, these data for the first time revealed the novel intrinsic properties of hJPH2 to form nuclear liquid droplets, and identified critical functional domains and residues encoding these properties. We propose that hJPH2 droplets could function as membrane-less organelles participating in nuclear regulatory processes.

category: biochemistry


title: Compartmap enables inference of higher-order chromatin structure in individual cells from scRNA-seq and scATAC-seq

abstract: Single-cell profiling of chromatin structure remains a challenge due to cost, throughput, and resolution. We introduce compartmap to reconstruct higher-order chromatin domains in individual cells from transcriptomic (RNAseq) and epigenomic (ATACseq) assays. In cell lines and primary human samples, compartmap infers higher-order chromatin structure comparable to specialized chromatin capture methods, and identifies clinically relevant structural alterations in single cells. This provides a common lens to integrate transcriptional and epigenomic results, linking higher-order chromatin architecture to gene regulation and to clinically relevant phenotypes in individual cells.

category: genomics


title: Subcellular localization of PKA catalytic subunits provides a basis for their distinct functions in the retina

abstract: PKA signaling is essential for numerous processes but the subcellular localization of specific PKA isoforms has yet to be explored comprehensively in tissues. Expression of the C{beta} protein, in particular, has not been mapped previously at the tissue level. In this study we used retina as a window into PKA signaling in the brain and characterized localization of PKA C, C{beta}, RII, and RII{beta} subunits. Each subunit presented a distinct localization pattern. C and C{beta} were localized in all tissue layers, while RII and RII{beta} were enriched in the photoreceptor cells in contrast to the cell body and retinal portion of retinal ganglion cells. Only C was observed in photoreceptor outer segments and the cilia transition zone, while C{beta} was localized primarily to mitochondria and was especially prominent in the ellipsoid of the cone cells. In contrast to C, C{beta} also never colocalized with RII or RII{beta}. Using BaseScope technology to track expression of the C{beta} isoforms we find that C{beta}4 and C{beta}4ab are prominently expressed and, therefore, likely code for mitochondrial-C{beta} proteins. Our data indicates that PKA subunits are functionally nonredundant in the retina and suggesting that C{beta} might be important for mitochondrial-associated neurodegenerative diseases previously linked to PKA dysfunction.

category: neuroscience


title: Interplay between meropenem and human serum albumin on expression of carbapenem resistance genes and natural competence in Acinetobacter baumannii

abstract:
Acinetobacter baumannii A118, a mostly susceptible strain, and AB5075, carbapenem-resistant, were cultured in L broth or L broth with different supplements: 3.5% human serum albumin (HSA), human serum (HS), meropenem, or meropenem plus 3.5% HSA. Natural transformation levels were enhanced in A. baumannii A118 and AB5075 cultured in medium supplemented with 3.5 % HSA. Addition of meropenem plus 3.5% HSA caused synergistic enhancement of natural transformation in A. baumannii A118. Medium containing 3.5% HSA or meropenem enhanced the expression levels of the competence and type IV pilus associated genes. The combination meropenem plus 3.5% HSA produced a synergistic augmentation in the expression levels of many of these genes. The addition of HS, which has a high content of HSA, was also an inducer of these genes. Cultures in medium supplemented with HS or 3.5% HSA also affected resistance genes, which were expressed at higher or lower levels depending on the modification required to enhance resistance. The inducing or repressing activity of these modulators also occurred in three more carbapenem-resistant strains tested. An exception was the A. baumannii AMA16 blaNDM-1 gene, which was repressed in the presence of 3.5% HSA. In conclusion, HSA produces an enhancement of natural transformation and a modification in expression levels of competence genes and antibiotic resistance. Furthermore, when HSA is combined with carbapenems, which may produce stronger cellular stress, the A. baumannii responds increasing the levels of expression of genes involved in natural competence. This process may favor the acquisition of foreign DNA and accelerate evolution.

ImportanceAcinetobacter baumannii causes a variety of nosocomial- and community-infections that are usually resistant to multiple antimicrobial agents. As new strains acquire more resistance genes, these infections become harder to treat, and mortality can reach up to 39%. The high genomic plasticity exhibited by A. baumannii must be the consequence of numerous mechanisms that include acquiring foreign DNA and recombination. Here, we describe the ability of A. baumannii to induce competence genes when exposed to environments that resemble those found in the human body during untreated infection or after administration of carbapenems. In this latter scenario expression of genes related to resistance also modify their expression levels such that resistance is increased. The contributions of this article are two-pronged. First, when A. baumannii is exposed to substances present during infection, it responds, augmenting the ability to capture DNA and accelerate evolution. Second, in those conditions, the bacterium also modifies the expression of resistance genes to increase its resistance levels. In summary, recognition of substances that are naturally (HSA) or artificially (treatment with carbapenems) induces A. baumannii to defend, enhancing resistance and increasing the chances of acquiring new resistance mechanisms.

category: microbiology


title: Viability of MS2 and Phi6 Bacteriophages on Carpet and Dust

abstract:
Respiratory viral illnesses are commonly spread in the indoor environment through multiple transmission routes, including droplets, aerosols, and direct/indirect contact. Indoors, resuspension of dust from flooring is a major source of human exposure. However, it is critical to determine viral persistence on dust and flooring to better characterize human exposure. The goal of this work is to determine viral viability on two carpet types (cut and looped) and house dust over time and after four different cleaning methods. MS2 and Phi6 bacteriophages were used to represent non-enveloped and enveloped viruses, respectively. These viral surrogates were placed in an artificial saliva solution and nebulized onto carpet or dust. Viability was measured at various time points (0, 1, 2, 3, 4, 24, and 48 hours) and after cleaning (vacuuming, hot water extraction with stain remover, steam, and a disinfection spray). Viability decay was modeled as first-order. MS2 bacteriophages showed slower viability decay rates in dust (-0.11 hr-1), cut carpet (-0.20 hr-1), and looped carpet (-0.09 hr-1) compared to Phi6 (-3.36 hr-1, -1.57 hr-1, and - 0.20 hr-1 respectively). The difference between phages was statistically significant in dust and cut carpet (p<0.05). Viral RNA demonstrated minimal degradation that in most cases was not statistically different from zero over the 48 hours measured (p>0.05). Viable viral concentrations were reduced to below the detection limit for steam and disinfection for both MS2 and Phi6 (p<0.05), while vacuuming and hot water extraction with stain remover showed no significant changes in concentration from uncleaned carpet (p>0.05). This study used viral surrogates and did not model risk of viral transmission via dust. Overall, these results demonstrate that MS2 and Phi6 bacteriophages can remain viable in carpet and dust for several hours to days, and cleaning techniques with heat and disinfectants may be more effective than standard vacuuming for viral removal. Future work should model risk from exposure via dust and flooring for various viruses such as influenza, SARS-CoV-2, and RSV.

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title: Categorical edge-based analyses of phylogenomic data reveal conflicting signals for difficult relationships in the avian tree

abstract: Phylogenetic analyses of large-scale datasets sometimes fail to yield a satisfactory resolution of the relationships among taxa for a number of nodes in the tree of life. This has even been true for genome-scale datasets, where the failure to resolve relationships is unlikely to reflect limitations in the amount of data. Gene tree conflicts are particularly notable in studies focused on these contentious nodes in the tree of life, and taxon sampling, different analytical methods, and/or data-type effects are thought to further confound analyses. Observed conflicts among gene trees arise from both biological processes and artefactual sources of noise in analyses. Although many efforts have been made to incorporate biological conflicts, few studies have curated individual genes for their efficiency in phylogenomic studies. Here, we conduct an edge-based analysis of Neoavian evolution, examining the phylogenetic efficacy of two recent phylogenomic bird datasets and three datatypes (ultraconserved elements [UCEs], introns, and coding regions). We assess the potential causes for biases in signal-resolution for three difficult nodes: the earliest divergence of Neoaves, the position of the enigmatic Hoatzin (Opisthocomus hoazin), and the position of owls (Strigidae). We observed extensive conflict among genes for all data types and datasets even after we removed potentially problematic loci. Edge-based analyses increased congruence and examined the impact of data type, GC content variation (GCCV), and outlier genes on analyses. These factors had different impact on each of nodes we examined. First, outlier gene signals appeared to drive different patterns of support for the relationships among the earliest diverging Neoaves. Second, the position of Hoatzin was highly variable, but we found that data type was correlated with the signals that support different placements of the Hoatzin. However, the resolution with the most support in our analyses was Hoatzin + shorebirds. Finally, GCCV, rather than data type (i.e., coding vs non-coding) per se, was correlated with an owl + Accipitriformes signal. Eliminating high GCCV loci increased the signal for an owl + mousebird relationship. Difficult edges (i.e., characterized by deep coalescence and high gene-tree estimation error) are hard to recover with all methods (including concatenation, multispecies coalescent, and edge-based analyses), whereas "easy" edges (e.g., flamingos + grebes) can be recovered without ambiguity. Thus, the nature of the edges, rather than the methods, is the limiting factor. Categorical edge-based analyses can reveal the nature of each edge and provide a way to highlight especially problematic branches that warrant further examination in future phylogenomic studies. We suggest that edge-based analyses provide a tool that can increase our understanding about the parts of the avian tree that remain unclear, even with large-scale data. In fact, our results emphasize that the conflicts associated with edges that remain contentious in the bird tree may be even greater than appreciated based on previous studies.

category: evolutionary biology


title: Epigenetic predictors of maximum lifespan and other life history traits in mammals

abstract: Maximum lifespan of a species is the oldest that individuals can survive, reflecting the genetic limit of longevity in an ideal environment. Here we report methylation-based models that accurately predict maximum lifespan (r=0.89), gestational time (r=0.96), and age at sexual maturity (r=0.87), using cytosine methylation patterns collected from over 12,000 samples derived from 192 mammalian species. Our epigenetic maximum lifespan predictor corroborated the extended lifespan in growth hormone receptor knockout mice and rapamycin treated mice. Across dog breeds, epigenetic maximum lifespan correlates positively with breed lifespan but negatively with breed size. Lifespan-related cytosines are located in transcriptional regulatory regions, such as bivalent chromatin promoters and polycomb-repressed regions, which were hypomethylated in long-lived species. The epigenetic estimators of maximum lifespan and other life history traits will be useful for characterizing understudied species and for identifying interventions that extend lifespan.


title: Genome-wide association study revealed loci linked to post-drought recovery and traits related to persistence of smooth bromegrass (Bromus inermis)

abstract:
Association analysis has been proved as a powerful tool for genetic dissection of complex traits. This study was conducted to identify marker-trait associations for recovery, persistence, and as well as finding stable associations. In this study, a diverse panel of polycross derived progenies of smooth bromegrass was phenotyped under normal and water stress, during three consecutive years. Association analysis was performed between nine important agronomic traits along with three seasonal growth activity indices based on 535 SRAP markers. Population structure analysis identified five main subpopulations possessing significant genetic differences. Association analysis using mixed linear mode1 identified 339 and 233 marker-trait associations under normal and water stress environments, respectively. Some of these markers were associated with more than one trait; which can be attributed to pleiotropic effects or to a number of tightly linked genes affecting several traits. If the effectiveness of these markers in genetic control of these traits is validated, they could be potentially used for initiation of marker-assisted selection and targeted trait introgression of smooth bromegrass under normal and water stress environments.

HighlightIn this study, stable marker-trait associations (MTAs) between years and moisture regimes (normal and water stress environments) were identified in a diverse panel of polycross derived progenies of smooth bromegrass.

category: genetics


title: Application of augmented topic model to predicting biomarkers and therapeutic targets using multiple human disease-omics datasets

abstract: Human diseases are multifactorial - hence it is important to characterize diseases on the basis of multiple disease-omics. However, the capability of the existing methods is largely limited to classifying diseases based on a single type or a few closely related omics data. Herein, we report a topic model framework that allows for characterizing diseases according to their multiple omics data. We also show that this method can be utilized to predict potential biomarkers and/or therapeutic targets. In this study, we illustrate a computational concept of this augmented topic model and demonstrate its prediction performance by a leave one-disease features out cross-validation scheme. Furthermore, we exploit this method together with human disease tissue/organ-transcriptome data and identify putative biomarkers and/or therapeutic targets across 79 diseases. In conclusion, this method and the prediction framework shown reported herein provide important tools for understanding complex human diseases and also facilitate diagnostic and/or therapeutic development.

category: bioinformatics


title: KmerKeys: a web resource for searching indexed genome assemblies and variants

abstract: K-mers are short DNA sequences that are used for genome sequence analysis. Applications that use k-mers include genome assembly and alignment. Despite these current applications, the wider bioinformatic use of k-mers in has challenges related to the massive scale of genomic sequence data. A single human genome assembly has billions of these short sequences. The sheer amount of computation for effective use of k-mer information is enormous, particularly when involving multiple genome assemblies. To address these issues, we developed a new k-mer indexing data structure based on a hash table tuned for the lookup of k-mer keys. This web application, referred to as KmerKeys (https://kmerkeys.dgi-stanford.org/), provides performant, rapid query speeds for cloud computation on genome assemblies. We enable fuzzy as well as exact k-mer-based searches of assemblies. To enable robust and speedy performance, the website implements cache-friendly hash tables, memory mapping and massive parallel processing. Our method employs a scalable and efficient data structure that can be used to jointly index and search a large collection of human genome assembly information. One can include variant databases and their associated metadata such as the gnomAD population variant catalog. This feature enables the incorporation of future genomic information into sequencing analysis.


title: Trend, Population Structure And Trait Mapping From 15 Years Of National Varietal Trials Of UK Winter Wheat

abstract: There are now a rich variety of genomic and genotypic resources available to wheat researchers and breeders. However, the generation of high-quality and field-relevant phenotyping data which is required to capture the complexities of gene x environment interactions remains a major bottleneck. Historical datasets from national variety performance trials (NVPT) provide sufficient dimensions, in terms of numbers of years and locations, to examine phenotypic trends and study gene x environment interactions. Using NVPT for winter wheat varieties grown in the UK between 2002 - 2017, we examined temporal trends for eight traits related to yield, adaptation, and grain quality performance. We show a non-stationary linear trend for yield, grain protein content, HFN and days to ripening. Our data also show high environmental stability for yield, grain protein content and specific weight in UK winter wheat varieties and high environmental sensitivity for Hagberg Falling Number. Using the historical NVPT data in a genome-wide association analysis, we uncovered a significant marker-trait association peak on wheat chromosome 6A spanning the NAM-A1 gene that have been previously associated with early senescence. Together our results show the value of utilizing the data routinely collected during variety evaluation process for examining breeding progress and the genetic architecture of important traits.

category: plant biology


title: When rare meets common: Treatable genetic diseases are enriched in the general psychiatric population

abstract: 1.1Mental illnesses are one of the biggest contributors to the global disease burden. Despite the increased recognition, diagnosis and ongoing research of mental health disorders, the etiology and underlying molecular mechanisms of these disorders are yet to be fully elucidated. Moreover, despite many treatment options available, a large subset of the psychiatric patient population is non-responsive to standard medications and therapies. There has not been a comprehensive study to date examining the burden and impact of treatable genetic disorders (TGDs) that can present with neuropsychiatric features in psychiatric patient populations. In this study, we test the hypothesis that TGDs that present with psychiatric symptoms are more prevalent within psychiatric patient populations compared to the general population by performing targeted next-generation sequencing (NGS) of 129 genes associated with 108 TGDs in a cohort of 2301 psychiatric patients. In total, 72 putative affected and 293 putative carriers for TGDs were identified, with known or likely pathogenic variants in 78 genes. Despite screening for only 108 genetic disorders, this study showed an approximately four-fold (4.13%) enrichment for genetic disorders within the psychiatric population relative to the estimated 1% cumulative prevalence of all single gene disorders globally. This strongly suggests that the prevalence of these, and most likely all, genetic diseases are greatly underestimated in psychiatric populations. Increasing awareness and ensuring accurate diagnosis of TGDs will open new avenues to targeted treatment for a subset of psychiatric patients.


title: Comparative and systems analyses of Leishmania spp. non-coding RNAs through developmental stages

abstract:
Leishmania spp. is the causal agent of several diseases called leishmaniases, neglected diseases that seek to be eradicated in the coming years. We aimed to study the genomic structure and function of non-coding RNAs (ncRNAs) from Leishmania spp. and to get insights into its RNAome. We studied 26 strains corresponding to 16 different species of Leishmania genus. RNAome analysis revealed the presence of several ncRNAs that are shared through different species, allowing us to differentiate between subgenus and as well as species that are canonically related to visceral leishmaniasis. We found coexpression relationships within coding genes and ncRNAs, thus suggesting possible functional relationships between different coding genes-ncRNAs. Expression analysis in the metacyclic developmental stage comparison for Leishmania braziliensis and Leishmania major reveals the presence of shared coexpressed ncRNAs to several other coding genes in both species involved in chromatin structure and host interaction. This work constitutes the first effort to characterize the Leishmania RNAome, supporting further approaches to better understand the role of ncRNAs in the gene regulation, infective process and host-parasite interaction.

AUTHOR SUMMARYLeishmaniasis is a worldwide tropical neglected disease, which is endemic in several countries of America, Africa, Asia and Europe. Leishmania parasites are transmitted to mammal hosts by the bite of female sandflies. Currently, more than one million of the new infections are reported mainly in the Americas, the Mediterranean basin, the Middle East and Central Asia. The genomic knowledge of these parasites have increased since the first draft genome was published in 2005, however the role of non-coding RNAs in pathogenesis and development is still an open avenue. Here we use a system and comparative genomic approach to characterize and predict the function of the whole ncRNA repertoire in Leishmania parasites. Although Leishmania presents a large amount of ncRNA, there is a low conservation rate, however it is directly proportional to phylogenetic relationships. We also found that this conserved ncRNA was related to histone regulation and proteophosphoglycan ppg3 co-expression. Our study provides new insights in our knowledge about function and expression patterns of ncRNA and their relationship with Leishmania chromatin regulation and virulence.


title: NEOage clocks - Epigenetic clocks to estimate post-menstrual and postnatal age in preterm infants

abstract:
Epigenetic clocks based on DNA methylation (DNAm) can to accurately predict chronological age and are thought to capture biological aging. A variety of epigenetic clocks have been developed for different tissue types and age ranges, but none has focused on age prediction for preterm infants. Epigenetic estimators of biological age might be especially informative in epidemiologic studies of neonates, particularly those born preterm, since this is a key developmental window. Neonatal DNAm is dynamic and preterm infants are at heightened risk of developmental impairments. We aimed to fill this gap by developing epigenetic clocks for neonatal aging in preterm infants.

As part of the Neonatal Neurobehavior and Outcomes in Very Preterm Infants (NOVI) study, buccal cells were collected at NICU discharge to profile DNAm levels in 542 very preterm infants. We applied elastic net regression to identify four epigenetic clocks (NEOage) predictive of post-menstrual and postnatal age, compatible with the Illumina EPIC and 450K arrays. We observed high correlations between predicted and reported ages (0.93 - 0.94) with root mean squared errors (1.28 - 1.63 weeks).

Epigenetic estimators of neonatal aging in preterm infants can be useful tools to evaluate biological maturity and associations with neonatal and long-term morbidities.


title: Structure Activity Relationship of USP5 Allosteric Inhibitors

abstract:
USP5 is a deubiquitinase that has been implicated in a range of diseases, including cancer, but no USP5-targeting chemical probe has been reported to date. Here, we present the progression of a chemical series that occupies the C-terminal ubiquitin-binding site of a poorly characterized zinc-finger ubiquitin binding domain (ZnF-UBD) of USP5 and allosterically inhibits the catalytic activity of the enzyme. Systematic exploration of the structure-activity relationship, complemented with crystallographic characterization of the ZnF-UBD bound to multiple ligands, led to the identification of 64, which binds to the USP5 ZnF-UBD with a KD of 2.8 {micro}M. 64 is selective over the structurally similar ZnF-UBD domain of HDAC6 and inhibits USP5 catalytic activity in vitro with an IC50 of 26 {micro}M. This study provides a chemical and structural framework for the discovery of a chemical probe to delineate USP5 function in cells.

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title: Development of a Low-Cost System for Simultaneous Longitudinal Biological Imaging

abstract: Simultaneous longitudinal imaging across multiple conditions and replicates has been crucial for scientific studies aiming to understand biological processes and disease. Yet, imaging systems capable of accomplishing these tasks are economically unattainable for most academic and teaching laboratories around the world. Here we propose the Picroscope, which is the first low cost system for simultaneous longitudinal biological imaging made primarily using off-the-shelf and 3D-printed materials. The Picroscope is compatible with standard 24-well cell culture plates and captures 3D z-stack image data. The Picroscope can be controlled remotely, allowing for automatic imaging with minimal intervention from the investigator. Here we use this system in a range of applications. We gathered longitudinal whole organism image data for frogs, zebrafish and planaria worms. We also gathered image data inside an incubator to observe 2D monolayers and 3D mammalian tissue culture models. Using this tool, we can measure the behavior of entire organisms or individual cells over long time periods.

category: bioengineering


title: Modulating the selective utilization of carbon sources by engineering the 3rd and 4th helices of the DNA-binding domain of catabolite control protein A (CcpA) in Bacillus licheniformis

abstract:
The gram-positive bacterium Bacillus licheniformis exhibits obvious selective utilization on carbon sources. This process is mainly governed by the global regulator catabolite control protein A (CcpA), which can recognize and bind to multiple target genes widely distributed in metabolic pathways. Although the DNA-binding domain of CcpA has been predicted, the influence of key amino acids on target gene recognition and binding remains elusive. In this study, the impact of Lys31, Ile42 and Leu56 on in vitro protein-DNA interactions and in vivo carbon source selective utilization was investigated. The results showed that alanine substitution of Lys31 and Ile42, located within the 3rd helices of the DNA-binding domain, significantly weakened the binding strength between CcpA and target genes. These mutations also lead to alleviated repression of xylose utilization in the presence of glucose. On the other hand, the Leu56Arg mutant in the 4th helices exhibited enhanced binding affinity compared with that of the wild-type one. When this mutant was used to replace the native one in B. licheniformis cells, the selective utilization of glucose over xylose increased. The above research results are helpful for a deep understanding of how microorganisms can flexibly sense and adapt to changes in the external environment. Additionally, they can provide important theoretical basis for the rational design of biomass utilization and environmental adaptability of B. licheniformis cell factories.

ImportanceBacillus licheniformis is widely used in producing various valuable products, such as  enzymes, industrial chemicals and biocides. The carbon catabolite regulation process in the utilization of raw materials is crucial to maximizing the efficiency of this microbial cell factory. CcpA plays an important role in this process. This study represents a new paradigm to investigate the structure-function relationship in CcpA by fluorescence polarization experiments in vitro. The results also uncover key amino acids in the DNA-binding domain that affect the selective utilization of carbon sources. These results provide a theoretical basis for the rational design of industrial microorganisms.


title: Toughening mechanisms for the attachment of architectured materials: The mechanics of the tendon enthesis

abstract:
Architectured materials offer tailored mechanical properties but are limited in engineering applications due to challenges in maintaining toughness across their attachments. The enthesis connects tendon and bone, two vastly different architectured materials, and exhibits toughness across a wide range of loadings. Understanding the mechanisms by which this is achieved could inform the development of engineered attachments. Integrating experiments, simulations, and novel imaging that enabled simultaneous observation of mineralized and unmineralized tissues, we identified putative mechanisms of enthesis toughening in a mouse model and then manipulated these mechanisms via in vivo control of mineralization and architecture. Imaging uncovered a fibrous architecture within the enthesis that controls trade-offs between strength and toughness. In vivo models of pathology revealed architectural adaptations that optimize these trade-offs through cross-scale mechanisms including nanoscale protein denaturation, milliscale load-sharing, and macroscale energy absorption. Results suggest strategies for optimizing architecture for tough bimaterial attachments in medicine and engineering.

TeaserThe architecture of the tendon-to-bone attachment is designed for toughness.


title: Inference of tissue relative proportions of the breast epithelial cell types luminal progenitor, basal, and luminal mature

abstract: Single-Cell Analysis has revolutionised genomic science in recent years. However, due to cost and other practical considerations, single-cell analyses are impossible for studies based on medium or large patient cohorts. For example, a single-cell analysis usually costs thousands of euros for one tissue sample from one volunteer, meaning that typical studies using single-cell analyses are based on very few individuals. While single-cell genomic data can be used to examine the phenotype of individual cells, cell-type deconvolution methods are required to track the quantities of these cells in bulk-tissue genomic data. Hormone receptor negative breast cancers are highly aggressive, and are thought to originate from a subtype of epithelial cells called the luminal progenitor. In this paper, we show how to quantify the number of luminal progenitor cells as well as other epithelial subtypes in breast tissue samples using DNA and RNA based measurements. We find elevated levels of cells which resemble these hormone receptor negative luminal progenitor cells in breast tumour biopsies of hormone receptor negative cancers, as well as in healthy breast tissue samples from BRCA1 (FANCS) mutation carriers. We also find that breast tumours from carriers of heterozygous mutations in non-BRCA Fanconi Anaemia pathway genes are much more likely to be hormone receptor negative. These findings have implications for understanding hormone receptor negative breast cancers, and for breast cancer screening in carriers of heterozygous mutations of Fanconi Anaemia pathway genes.


title: Characterization of the subunit composition and structure of native adult glycine receptors

abstract: The strychnine-sensitive pentameric Glycine Receptor (GlyR) mediates fast inhibitory neurotransmission in the mammalian nervous system. Only heteromeric GlyRs mediate synaptic transmission, since they contain the {beta} subunit that permits clustering at the synapse through its interaction with scaffolding proteins. Here we show that 2 and {beta} subunits assemble with an unexpected 4:1 stoichiometry to produce GlyR with native electrophysiological properties. We determined structures in multiple functional states at 3.6 - 3.8 [A] resolutions and show 2{beta} GlyR assembly mechanism. Furthermore, we show that one single {beta} subunit in each GlyR gives rise to the characteristic electrophysiological properties of heteromeric GlyR, while more {beta} subunits renders GlyR non-conductive. A single {beta} subunit ensures a univalent GlyR-scaffold linkage, which means the scaffold alone regulates the cluster properties.

category: biophysics


title: Rescuable effect of R-salbutamol in LPS-induced immune dysfunction of sepsis

abstract: Sepsis is a severe life-threatening condition caused by a dysregulated host response to infection. So far, there are no pharmacotherapies to stop sepsis. Salbutamol, a commonly used {beta}2-adrenoreceptor agonist, has found to be potential in regulating immune response dysfunction and exert anti-inflammatory effect. However, salbutamol exists two isomers. R-isomer exhibits the therapeutic effect and clinical benefit, while S-isomer proves to be detrimental rather than benign. So, in this study, we investigated the preventive and therapeutic effect of R-salbutamol (R-sal), S-salbutamol (S-sal) or racemic mixture in a mouse model of lipopolysaccharide (LPS)-induced sepsis. Dexamethasone (Dex) was set as comparison. The results showed that R-sal markedly improved seven-day survival rate of septic mice both administered before or after LPS. Whereas Dex showed toxic and accelerated the death of septic mice when given before LPS injection. Lung histological examination and lung function assay revealed that LPS challenge resulted in acute lung damage, including inflammatory cell infiltration, thickened alveolar septa and congestion, and decreased minute volume in septic mice. R-sal pretreatment efficiently inhibited these changes, accompanying by markedly reduced lung MPO level, serum cytokines levels and lactate release and significantly restored the lymphocytes and suppressed the percentage of monocytes. Racemic mixture exhibited diminished effects while S-sal showed enhanced cytokines release. In addition, R-sal pretreatment showed a better improvement in prognostic pulmonary function at day4 in survived mice than that of Rac-sal. Collectively, our results indicate the potential benefit of R-sal for sepsis and sepsis-induced lung injury.

category: pharmacology and toxicology


title: HLA Gene Expression Mediates Tumor Immunogenicity and Escape

abstract:
Human Leukocyte Antigen (HLA) expression contributes to the activation of anti-tumor immunity through interactions with T cell receptors. However, pan-cancer HLA-mediated immunogenicity and immunoediting mechanisms has not been systematically studied. In a retrospective analysis of 33 tumor types from the Cancer Genome Atlas, we uncovered HLA class I and class II differential expression, which outperformed traditional clinical metrics in predicting patient survival. We also characterized the distribution of HLA supertypes across cancers and showed that patients with high HLA allelic diversity and gene expression had better prognosis. Immune microenvironments with varied survival outcomes could be predicted using a neural network model trained on HLA expression data. Furthermore, we identified a subset of tumors which upregulated HLA class I but not class II genes and exploited HLA-mediated escape strategies. Our results suggest the potential of using HLA expression data to predict patient prognosis. Taken together, we emphasize the crucial role of HLA upregulation in shaping prolonged anti-tumor immunity.

SynopsisIn a retrospective analysis of 11080 patients of 33 TCGA cancer types, we showed HLA class I and class II differential expression shape various immune microenvironments and tumor immunoediting mechanisms, predicting tumor immunogenicity and patient survival.

category: systems biology


title: Pan-cancer HLA Gene-mediated Tumor Immunogenicity and Immune Evasion


title: DiversityScanner: Robotic discovery of small invertebrates with machine learning methods

abstract: Invertebrate biodiversity remains poorly explored although it comprises much of the terrestrial animal biomass, more than 90% of the species-level diversity and supplies many ecosystem services. The main obstacle is specimen- and species-rich samples. Traditional sorting techniques require manual handling and are slow while molecular techniques based on metabarcoding struggle with obtaining reliable abundance information. Here we present a fully automated sorting robot, which detects each specimen, images and measures it before moving it from a mixed invertebrate sample to the well of a 96-well microplate in preparation for DNA barcoding. The images are then used by a newly trained convolutional neural network (CNN) to assign the specimens to 14 particularly common, usually family-level "classes" of insects in Malaise trap samples and an "other-class" (N=15). The average assignment precision for the classes is 91.4% (75-100%). In order to obtain biomass information, the specimen images are also used to measure specimen length and estimate body volume. We outline how the DiversityScanner robot can be a key component for tackling and monitoring invertebrate diversity. The robot generates large numbers of images that become training sets for CNNs once the images are labelled with identifications based on DNA barcodes. In addition, the robot allows for taxon-specific subsampling of large invertebrate samples by only removing the specimens that belong to one of the 14 classes. We conclude that a combination of automation, machine learning, and DNA barcoding has the potential to tackle invertebrate diversity at an unprecedented scale.

category: zoology


title: Basal Ganglia Responses to Electrical Stimulation of the Posterior Hypothalamic Nucleus

abstract: Electrical or chemical stimulation of the posterior hypothalamic nucleus (PH) elicits highly adaptive locomotion, demonstrating both evidence of flexibility and variety in exhibited motor behaviours. However, the neural substrates of PH stimulation elicited behavioural changes are poorly understood. The basal ganglia are postulated to be critically involved in the process of action selection in conjunction with thalamo-cortical systems. The present study examines changes in basal ganglia activities in response to the high-frequency stimulation of the PH. Under urethane anaesthesia, ensemble and single-unit recordings were obtained from the striatum (STR), globus pallidus externa (GPe), entopeduncular nucleus (EP), subthalamic nucleus (STN) and the substantia nigra pars reticulata (SNr). Upon PH stimulation, increases in firing rates were observed in the STR, GPe, and STN, a decrease was observed in the SNr and no changes were seen in the EP. The increase in spike rate in the STR and GPe was dependent on the stimulation intensity but not duration. Despite the differences in the direction of firing changes during PH stimulation, all examined areas including those not part of the basal ganglia demonstrated an elevated spiking rate upon stimulus train termination. Taking into account the known anatomical connections between the PH and the basal ganglia, it is hypothesized responses seen during PH stimulus trains are mediated through thalamic and cortical relays whereas the overall post-stimulus excitatory response is related to the impact of the PH on brainstem arousal systems.


title: Astrocyte Ca2+ Signaling is Facilitated in an Scn1a+/- Mouse Model of Dravet Syndrome

abstract: Dravet syndrome (DS) is an infantile-onset epileptic encephalopathy. More than 80% of DS patients have a heterozygous mutation in SCN1A, which encodes a subunit of the voltage-gated sodium channel, Nav1.1, in neurons. The roles played by astrocytes, the most abundant glial cell type in the brain, have been investigated in the pathogenesis of epilepsy; however, the specific involvement of astrocytes in DS has not been clarified. In this study, we evaluated Ca2+ signaling in astrocytes using genetically modified mice that have a loss-of-function mutation in Scn1a. We found that the slope of spontaneous Ca2+ spiking was increased without a change in amplitude in Scn1a+/- astrocytes. In addition, ATP-induced transient Ca2+ influx and the slope of Ca2+ spiking were also increased in Scn1a+/- astrocytes. These data indicate that perturbed Ca2+ dynamics in astrocytes may be involved in the pathogenesis of DS.


title: Deconstructing the Cortical Sources of Frequency Following Responses to Speech: A Cross-species Approach

abstract:
Time-varying pitch is a vital cue for human speech perception. Neural processing of time-varying pitch has been extensively assayed using scalp-recorded frequency-following responses (FFRs), an electrophysiological signal thought to reflect integrated phase-locked neural ensemble activity from subcortical auditory areas. Emerging evidence increasingly points to a putative contribution of auditory cortical ensembles to the scalp-recorded FFRs. However, the properties of cortical FFRs and precise characterization of laminar sources are still unclear. Here we used direct human intracortical recordings as well as extra- and intracranial recordings from macaques and guinea pigs to characterize the properties of cortical sources of FFRs to time-varying pitch patterns. We found robust FFRs in the auditory cortex across all species. We leveraged representational similarity analysis as a translational bridge to characterize similarities between the human and animal models. Laminar recordings in animal models showed FFRs emerging primarily from the thalamorecepient layers of the auditory cortex. FFRs arising from these cortical sources significantly contributed to the scalp-recorded FFRs via volume conduction. Our research paves the way for a wide array of studies to investigate the role of cortical FFRs in auditory perception and plasticity.

Significance StatementFrequency following responses (FFRs) to speech are scalp-recorded neural signals that inform the fidelity of sound encoding in the auditory system. FFRs, long believed to arise from brainstem and midbrain, have shaped our understanding of sub-cortical auditory processing and plasticity. Non-invasive studies have shown cortical contributions to the FFRs, however, this is still actively debated. Here we employed direct cortical recordings to trace the cortical contribution to the FFRs and characterize the properties of these cortical FFRs. With extra-cranial and intra-cranial recordings within the same subjects we show that cortical FFRs indeed contribute to the scalp-recorded FFRs, and their response properties differ from the sub-cortical FFRs. The findings provide strong evidence to revisit and reframe the FFR driven theories and models of sub-cortical auditory processing and plasticity with careful characterization of cortical and sub-cortical components in the scalp-recorded FFRs.


title: Attentional modulation of intrinsic timescales in visual cortex and spatial networks

abstract: Neural activity fluctuates endogenously on timescales varying across the neocortex. The variation in these intrinsic timescales relates to the functional specialization of cortical areas and their involvement in the temporal integration of information. Yet, it is unknown whether the timescales can adjust rapidly and selectively to the demands of a cognitive task. We measured intrinsic timescales of local spiking activity within columns of area V4 while monkeys performed spatial attention tasks. The ongoing spiking activity unfolded across at least two distinct timescales--fast and slow--and the slow timescale increased when monkeys attended to the receptive fields location. A recurrent network model shows that multiple timescales in local dynamics arise from spatial connectivity mimicking vertical and horizontal interactions in visual cortex and that slow timescales increase with the efficacy of recurrent interactions. Our results reveal that targeted neural populations integrate information over variable timescales following the demands of a cognitive task and propose an underlying network mechanism.


title: Spatiotemporal context modulates encoding and retrieval of overlapping events

abstract: Overlap between events can lead to interference due to a tradeoff between encoding the present event and retrieving the past event. Temporal context information -  when something occurred, a defining feature of episodic memory - can cue retrieval of a past event. However, the influence of temporal overlap, or proximity in time, on the mechanisms of interference are unclear. Here, by identifying brain states using scalp electroencephalography (EEG) from male and female human subjects, we show the extent to which temporal overlap promotes interference and induces retrieval. In this experiment, subjects were explicitly directed to either encode the present event or retrieve a past, overlapping event while perceptual input was held constant. We find that the degree of temporal overlap between events leads to selective interference. Specifically, greater temporal overlap between two events leads to impaired memory for the past event selectively when the top-down goal is to encode the present event. Using pattern classification analyses to measure neural evidence for a retrieval state, we find that greater temporal overlap leads to automatic retrieval of a past event, independent of top-down goals. Critically, the retrieval evidence we observe likely reflects a general retrieval mode, rather than retrieval success or effort. Collectively, our findings provide insight into the role of temporal overlap on interference and memory formation.


title: Δ133p53α Protects Human Astrocytes from Amyloid-Beta Induced Senescence and Neurotoxicity

abstract:
Cellular senescence is an important contributor to aging and age-related diseases such as Alzheimers disease (AD). Senescent cells are characterized by a durable cell proliferation arrest and the acquisition of a proinflammatory senescence-associated secretory phenotype (SASP), which participates in the progression of neurodegenerative disorders. Clearance of senescent glial cells in an AD mouse model prevented cognitive decline suggesting pharmacological agents targeting cellular senescence might provide novel therapeutic approaches for AD. {Delta}133p53, a natural protein isoform of p53, was previously shown to be a negative regulator of cellular senescence in primary human astrocyte, with clinical implications from its diminished expression in brain tissues from AD patients. Here we show that treatment of proliferating human astrocytes with amyloid-beta oligomers (A{beta}), an endogenous pathogenic agent of AD, results in reduced expression of {Delta}133p53, as well as induces the cells to become senescent and express proinflammatory SASP cytokines such as IL-6, IL-1{beta} and TNF. Our data suggest that A{beta}-induced astrocyte cellular senescence is associated with accelerated DNA damage, and upregulation of full-length p53 and its senescence-inducing target gene p21WAF1. We also show that exogenously enhanced expression of {Delta}133p53 rescues human astrocytes from A{beta}-induced cellular senescence and SASP through both protection from DNA damage and dominantnegative inhibition of full-length p53, leading to inhibition of A{beta}-induced, astrocyte-mediated neurotoxicity. The results presented here demonstrate that {Delta}133p53 manipulation could modulate cellular senescence in the context of AD, possibly opening new therapeutic avenues.

HIGHLIGHTSO_LIA{beta} diminishes the expression of the p53 isoform {Delta}133p53, and induces cellular senescence, SASP, and DNA damage in human astrocytes.
C_LIO_LI{Delta}133p53 protects human astrocytes from A{beta}-induced DNA damage and cellular senescence.
C_LIO_LI{Delta}133p53 prevents astrocyte-mediated neurotoxicity to neuro-progenitor cells.
C_LI


title: Cortical Oscillatory Dysrhythmias in Visual Snow Syndrome: A MEG Study

abstract:
Visual Snow (VS) refers to the persistent visual experience of static in the whole visual field of both eyes. It is often reported by patients with migraine and co-occurs with conditions like tinnitus and tremor. The underlying pathophysiology of the condition is poorly understood. Previously we hypothesised, that VSS may be characterised by disruptions to rhythmical activity within the visual system1.

To test this, data from 18 patients diagnosed with visual snow syndrome (VSS), and 16 matched controls, were acquired using Magnetoencephalography (MEG). Participants were presented with visual grating stimuli, known to elicit decreases in alpha-band (8-13Hz) power and increases in gamma-band power (40-70Hz).

Data were mapped to source-space using a beamformer. Across both groups, decreased alpha power and increased gamma power localised to early visual cortex. Data from primary visual cortex (V1) were compared between groups. No differences were found in either alpha or gamma peak frequency or the magnitude of alpha power, p>.05. However, compared with controls, our VSS cohort displayed significantly increased V1 gamma power, p=.035. This new electromagnetic finding concurs with previous fMRI and PET findings suggesting that in VSS, the visual cortex is hyper-excitable. The coupling of alpha-phase to gamma amplitude (i.e., phase-amplitude coupling, PAC) within V1 was also quantified. Compared with controls, the VSS group had significantly reduced alpha-gamma PAC, p<.05, indicating a potential excitation-inhibition imbalance in VSS, as well as a potential disruption to top-down "noise-cancellation" mechanisms.

Overall, these results suggest that rhythmical brain activity in primary visual cortex is both hyperexcitable and disorganised in VSS, consistent with visual snow being a condition of thalamocortical dysrhythmia.


title: Rat sensitivity to multipoint statistics is predicted by efficient coding of natural scenes

abstract: Efficient processing of sensory data requires adapting the neuronal encoding strategy to the statistics of natural stimuli. Previously, in Hermundstad et al. 2014, we showed that local multipoint correlation patterns that are most variable in natural images are also the most perceptually salient for human observers, in a way that is compatible with the efficient coding principle. Understanding the neuronal mechanisms underlying such adaptation to image statistics will require performing invasive experiments that are impossible in humans. Therefore, it is important to understand whether a similar phenomenon can be detected in animal species that allow for powerful experimental manipulations, such as rodents. Here we selected four image statistics (from single- to four-point correlations) and trained four groups of rats to discriminate between white noise patterns and binary textures containing variable intensity levels of one of such statistics. We interpreted the resulting psychometric data with an ideal observer model, finding a sharp decrease in sensitivity from 2- to 4-point correlations and a further decrease from 4- to 3-point. This ranking fully reproduces the trend we previously observed in humans, thus extending a direct demonstration of efficient coding to a species where neuronal and developmental processes can be interrogated and causally manipulated.


title: Synaptic Plasticity in the Agranular Insular Cortex Predicts Escalated Ethanol Consumption

abstract: The Agranular Insular Cortex (AIC) is implicated in alcohol use disorder and pharmacologically relevant concentrations of acute ethanol inhibit N-methyl-D-aspartate receptor (NMDAR)-mediated glutamatergic synaptic transmission and plasticity onto layer 2/3 AIC pyramidal neurons. However, it is not known whether the actions of ethanol on glutamatergic synapses are means by which chronic ethanol alters mechanisms of learning and memory in AIC as alcohol drinking transitions from controlled to problematic. We utilized the chronic intermittent ethanol (CIE) vapor model of ethanol exposure in adult male mice, alone or in combination with voluntary ethanol consumption, to determine whether glutamatergic synapses on layer 2/3 AIC pyramidal neurons are differentially regulated by different durations and intensities of chronic ethanol exposure. We observed evidence of both ethanol- and age-related metaplasticity of AIC layer 2/3 glutamatergic synapses, as only young adult, ethanol-naive mice exhibited NMDAR-dependent long term depression ex vivo. Our findings also indicated that voluntary ethanol consumption alone can elicit glutamatergic plasticity in vivo. We found that the ratio of NMDAR- to AMPAR-mediated postsynaptic currents was reduced not only in CIE-treated, but also in air-treated, chronically drinking mice relative to ethanol-naive controls. Furthermore, lower NMDA/AMPA ratios were predictive of greater escalation of ethanol consumption. These findings suggest that even moderate exposure to ethanol may elicit plasticity in the agranular insular cortex that contributes to the progression toward uncontrolled drinking.


title: Tafazzin regulates the function of lipopolysaccharide activated B lymphocytes in mice

abstract: B lymphocytes are responsible for humoral immunity and play a key role in the immune response. Optimal mitochondrial function is required to support B cell activity during activation. We examined how deficiency of tafazzin, a cardiolipin remodeling enzyme required for mitochondrial function, alters the metabolic activity of B cells and their response to activation by lipopolysaccharide in mice. B cells were isolated from 3 month old wild type or tafazzin knockdown mice and incubated for up to 72 h with lipopolysaccharide and cell proliferation, expression of cell surface markers, secretion of antibodies and chemokines, proteasome and immunoproteasome activities, and metabolic function determined. In addition, proteomic analysis was performed to identify altered levels of proteins involved in survival, immunogenic, proteasomal and mitochondrial processes. Compared to wild type lipopolysaccharide activated B cells, lipopolysaccharide activated tafazzin knockdown B cells exhibited significantly reduced proliferation, lowered expression of cluster of differentiation 86 and cluster of differentiation 69 surface markers, reduced secretion of immunoglobulin M antibody, reduced secretion of keratinocytes-derived chemokine and macrophage-inflammatory protein-2, reduced proteasome and immunoproteasome activities, and reduced mitochondrial respiration and glycolysis. Proteomic analysis revealed significant alterations in key protein targets that regulate cell survival, immunogenicity, proteasomal processing and mitochondrial function consistent with the findings of the above functional studies. The results indicate that the cardiolipin transacylase enzyme tafazzin plays a key role in regulating mouse B cell function and metabolic activity during activation.

category: immunology


title: Differential surface competition and biofilm invasion strategies of Pseudomonas aeruginosa PA14 and PA01

abstract:
Pseudomonas aeruginosa strains PA14 and PAO1 are among the two best characterized model organisms used to study the mechanisms of biofilm formation, while also representing two distinct lineages of P. aeruginosa. Our previous work showed that P. aeruginosa PA14 and PAO1 use distinct strategies to initiate biofilm growth. Using differentially-labeled strains and microfluidic devices, we show that PAO1 can outcompete PA14 in a head-to-head competition during early colonization of a surface, can do so in constant and perturbed environments, that this advantage is specific to biofilm growth and requires production of the Psl polysaccharide. In contrast, the P. aeruginosa PA14 exhibits a competitive fitness advantage when invading a pre-formed biofilm and is better able to tolerate starvation than PAO1 in the biofilm context. These data support the model that while P. aeruginosa PAO1 and PA14 are both able to effectively colonize surfaces, these strains use distinct strategies that are advantageous under different environmental settings.

ImportanceRecent studies indicate that P. aeruginosa PAO1 and PA14 use distinct strategies to initiate biofilm formation, with PAO1 committing to the surface through a processive mode of attachment, while PA14 uses a non-processive surface engagement strategy. We investigated whether their respective colonization strategies impact their ability to effectively compete under different biofilm-forming regimes. Our work shows that these different strategies do indeed impact how these strains colonize the surface: PAO1 dominates during colonization of a naive surface, while PA14 is more effective in colonizing a pre-formed biofilm or withstanding starvation conditions. These data suggest that even for very similar microbes there may be distinct strategies to successfully colonize and persist on surfaces during the biofilm life cycle.


title: Prophylactic inhibition of colonization by Streptococcus pneumoniae with the secondary bile acid metabolite deoxycholic acid

abstract: Streptococcus pneumoniae (Spn) colonizes the nasopharynx of children and the elderly but also kills millions worldwide yearly. The secondary bile acid metabolite, deoxycholic acid (DoC), affects the viability of human pathogens but also plays multiple roles in host physiology. We assessed in vitro the antimicrobial activity of DoC and investigated its potential to eradicate Spn colonization using an ex vivo model of human nasopharyngeal colonization and an in vivo mouse model of colonization. At a physiological concentration DoC (0.5 mg/ml; 1.27 mM) killed all tested Spn strains (N=48) two h post-inoculation. The ex-vivo model of nasopharyngeal colonization showed that DoC eradicated colonization by Spn strains as soon as 10 min post-exposure. The mechanism of action did not involve activation of autolysis since the autolysis-defective double mutants {Delta}lytA{Delta}lytC and{Delta} spxB{Delta}lctO were as susceptible to DoC as was the wild-type (WT). Oral streptococcal species (N=20), however, were not susceptible to DoC (0.5 mg/ml). Unlike trimethoprim, whose spontaneous resistance frequency (srF) for TIGR4 or EF3030 was [≥]1x10-9, no spontaneous resistance was observed with DoC (srF[≥]1x10-12). Finally, the efficacy of DoC to eradicate Spn colonization was assessed in vivo using a topical route via intranasal (i.n.) administration and as a prophylactic treatment. Mice challenged with Spn EF3030 carried a median of 4.05x105 cfu/ml four days post-inoculation compared to 6.67x104 cfu/ml for mice treated with DoC. Mice in the prophylactic group had a [~]99% reduction of the pneumococcal density (median, 2.61 x103 cfu/ml). Thus, DoC, an endogenous human bile salt, has therapeutic potential against Spn.


title: Biochemical Barriers on the Path to Ocean Anoxia?

abstract:
The kinetics of microbial respiration suggest that, if excess organic matter is present, oxygen should fall to nanomolar levels, in the range of the Michaelis-Menten constants (Km). Yet even in many biologically productive coastal regions, lowest observed O2 concentrations often remain several orders of magnitude higher than respiratory Km values. We propose the Hypoxic Barrier Hypothesis (HBH) to explain this apparent discrepancy. The HBH postulates that oxidative enzymes involved in organic matter catabolism are kinetically limited by O2 at concentrations far higher than the thresholds for respiration. We found support for the HBH in a meta-analysis of 1137 O2 Km values reported in the literature: the median value for terminal respiratory oxidases was 350 nM, but for other oxidase types the median value was 67 M. The HBH directs our attention to the kinetic properties of an important class of oxygen-dependent reactions that could help explain the trajectories of ocean ecosystems experiencing O2 stress.

IMPORTANCEDeclining ocean oxygen associated with global warming and climate change is impacting marine ecosystems across scales from microscopic planktonic communities to global fisheries. We report a fundamental dichotomy in the affinity of enzymes for oxygen. The importance of this observation has yet to be fully assessed, but it is predicted to impact the rate at which organic matter is oxidized in hypoxic ecosystems, and the types of organic matter that accumulate. Competition between intracellular enzymes for oxygen may also have impacted microbial strategies of adaptation to suboxia.


title: Maf/ham1-like pyrophosphatases, host-specific partners of viral RNA-dependent RNA polymerases

abstract: Cassava brown streak disease (CBSD), dubbed the "Ebola of plants", is a serious threat to food security in Africa caused by two viruses of the family Potyviridae: cassava brown streak virus (CBSV) and Ugandan (U)CBSV. Intriguingly, U/CBSV, along with another member of this family and one secoviridae, are the only known RNA viruses encoding a protein of the Maf/ham1-like family, a group of widespread pyrophosphatase of non-canonical nucleotides (ITPase) expressed by all living organisms. Despite the socio-economic impact of CDSD, the relevance and role of this atypical viral factor has not been yet established. Here, using an infectious cDNA clone and reverse genetics, we demonstrate that UCBSV requires the ITPase activity for infectivity in cassava, but not in the model plant Nicotiana benthamiana. HPLC MS/MS experiments showed that, quite likely, this host-specific constraint is due to an unexpected high concentration of non-canonical nucleotides in cassava. Finally, protein analyses and experimental evolution of mutant viruses indicated that keeping a fraction of the yielded UCBSV ITPase covalently bound to the viral RNA-dependent RNA polymerase (RdRP) optimizes viral fitness, and this seems to be a feature shared by the other members of the Potyviridae family expressing Maf/ham1-like proteins. All in all, our work (i) reveals that the over-accumulation of non-canonical nucleotides in the host might have a key role in antiviral defense, and (ii) provides the first example of an RdRP-ITPase partnership, reinforcing the idea that RNA viruses are incredibly versatile at adaptation to different host setups.

category: molecular biology


title: Maf/ham1-like pyrophosphatases of non-canonical nucleotides are host-specific partners of viral RNA-dependent RNA polymerases


title: A systematic comparison of fibroblasts derived from postmortem human dura mater versus dermal epithelium for neurodegenerative disease modeling

abstract: Patient-derived cells hold great promise for precision medicine approaches in human health. Fibroblast cells have been a major source of human cells for reprogramming and differentiating into specific cell types for disease modeling. Such cells can be isolated at various stages during life (presymptomatic, symptomatic, and postmortem) and thus can potentially be used to model different phases of disease progression. In certain circumstances, however, tissues are not collected during life and only postmortem tissues are the only available source of fibroblasts. Fibroblasts cultured from postmortem human dura mater of individuals with neurodegenerative diseases have been suggested as a primary source of cells for in vitro modeling of neurodegenerative diseases. Although fibroblast-like cells from human and mouse dura mater have been previously described, their utility for reprogramming and direct differentiation protocols requires further characterization. In this study, cells derived from dermal biopsies performed in living subjects were compared to cells derived from postmortem dura mater. In two instances, we have isolated and compared dermal and dural cell lines from the same subject. Notably, striking differences between the dermis and dura mater-derived cell lines were found. Compared to dermal fibroblasts, postmortem dura mater-derived cells demonstrated different morphology, exhibited slower growth rates, failed to express fibroblast protein markers, and exhibited significant differences in gene expression profiles. In addition, dura mater-derived cells were found to exhibit a high rate of chromosomal abnormalities, particularly in the loss of the Y chromosome. Our study highlights potential limitations of postmortem human dura mater-derived cells for disease modeling, argues for rigorous karyotyping prior to reprograming, and brings into question the identity of dura mater-derived cells as belonging to a fibroblast lineage.

category: cell biology


title: Polarized Dishevelled dissolution and condensation drives embryonic axis specification in oocytes

abstract: The organismal body axes that are formed during embryogenesis are intimately linked to intrinsic asymmetries established at the cellular scale in oocytes [1]. Here, we report an axis-defining event in meiotic oocytes of the sea star Patiria miniata. Dishevelled is a cytoplasmic Wnt pathway effector required for axis development in diverse species [2-4], but the mechanisms governing its function and distribution remain poorly defined. Using time-lapse imaging, we find that Dishevelled localizes uniformly to puncta throughout the cell cortex in Prophase I-arrested oocytes, but becomes enriched at the vegetal pole following meiotic resumption through a dissolution-condensation mechanism. This process is driven by an initial disassembly phase of Dvl puncta, followed by selective reformation of Dvl assemblies at the vegetal pole. Rather than being driven by Wnt signaling, this localization behavior is coupled to meiotic cell cycle progression and influenced by Lamp1+ endosome association and Frizzled receptors pre-localized within the oocyte cortex. Our results reveal a cell cycle-linked mechanism by which maternal cellular polarity is transduced to the embryo through spatially-regulated Dishevelled dynamics.


title: Unsupervised deep learning method for cell segmentation

abstract: Advances in the artificial neural network have made machine learning techniques increasingly more important in image analysis tasks. Recently, convolutional neural networks (CNN) have been applied to the problem of cell segmentation from microscopy images. However, previous methods used a supervised training paradigm in order to create an accurate segmentation model. This strategy requires a large amount of manually labeled cellular images, in which accurate segmentations at pixel level were produced by human operators. Generating training data is expensive and a major hindrance in the wider adoption of machine learning based methods for cell segmentation. Here we present an alternative strategy that uses unsupervised learning to train CNNs without any human-labeled data. We show that our method is able to produce accurate segmentation models. More importantly, the algorithm is applicable to both fluorescence and bright-field images, requiring no prior knowledge of signal characteristics and requires no tuning of parameters.


title: Feral and managed honey bees, Apis mellifera (Hymenoptera: Apidae), in southern California have similar levels of viral pathogens

abstract: Bees provide critical pollination services but are threatened by multiple stressors, including viral pathogens. Most studies of pollinator health focus on managed honey bees (Apis mellifera Linnaeus) (MHB) or native bee species, but a third player, the feral honey bee (FHB), requires further study. Spillover and spillback of viral pathogens between these managed, feral, and native bees is generating increasing interest. In this case study, we provide evidence suggesting that FHB colonies play an important role in viral pathogen dynamics of southern California pollinator communities because they act as reservoirs, of viral pathogens such as acute bee paralysis virus (ABPV), black queen cell virus (BQCV), and deformed wing virus (DWV). Surprisingly, even though FHB are not treated for diseases or parasites, they harbor similar pathogen loads to MHB, which are usually highly treated, suggesting the need for future studies to determine if FHB resist or are more resilient to viruses.

category: ecology


title: Urbanization, Bird Species Richness And Abundance Within Ibadan Metropolis, Nigeria

abstract:
Urbanisation is considered as one of the most profound threat to wildlife, with habitat loss and fragmentation being predominant. This study assessed the impacts of urbanisation on richness, abundance of bird species within Ibadan metropolis, Nigeria. A uniform grid of 500 square meters was installed on the map of Ibadan Metropolis using QGIS to produce 499 grid points distributed across the five urban local governments. 100 grids were selected randomly, identified with mapinR software application and surveyed with 5 point counts within each gird, established at 200m interval to avoid double counting. Each point count was observed for 5 minutes using a pair of 8x42mm binoculars within a 50m radius. Habitat variables like number of buildings, trees, paved roads, communication masts were also recorded.

A total number of 56 species of birds were observed at the end of the assessment, classified into 30 families. The test of statistics showed that there was no statistically significant difference in bird species richness between the Local Governments. The test of statistics showed that there was no statistically significant difference in bird species abundance between the local governments. The test of between-subjects effects revealed that there were no statistical significant effects when all the habitat variables were computed in the model on species richness. The number of paved roads and number of vehicles showed a significant effect on bird species abundance while others variables in the model did not exact statistically significant effects on bird species abundance.

The study therefore concluded that habitat actions due to urbanisation have not affected the richness and abundance of birds species found in all the local governments. The only habitat variables that have currently exacted significant effect on species abundance within the metropolis are number of vehicles and paved roads.


title: Genomic inference of a human super bottleneck in the Early Stone Age

abstract: The demographic history is a foundation of human evolutionary studies. However, the ancient demographic history during the Mid-Pleistocene is poorly investigated while it is essential for understanding the early origin of humankind. Here we present the fast infinitesimal time coalescent (FitCoal) process, which allows the analytical calculation of the composite likelihood of a site frequency spectrum and provides the precise inference of demographic history. We apply it to analyze 3,154 present-day human genomic sequences. We find that African populations have passed through a population super bottleneck, a small effective size of approximately 1,280 breeding individuals between 930 and 813 thousand years ago. Further analyses confirm the existence of the super bottleneck on non-African populations although it cannot be directly inferred. This observation, together with simulation results, indicates that confounding factors, such as population structure and selection, are unlikely to affect the inference of the super bottleneck. The time interval of the super bottleneck coincides with a gap in the human fossil record in Africa and possibly marks the origin of Homo heidelbergensis. Our results provide new insights into human evolution during the Mid-Pleistocene.


title: Genomic inference of a human super bottleneck in Mid-Pleistocene transition


title: A Cre-dependent massively parallel reporter assay allows for cell-type specific assessment of the functional effects of genetic variants in vivo

abstract: Human genetic studies have identified a large number of disease-associated de novo variants in presumptive regulatory regions of the genome that pose a challenge for interpretation of their effects: the impact of regulatory variants is highly dependent on the cellular context, and thus for psychiatric diseases these would ideally be studied in neurons in a living brain. Furthermore, for both common and rare variants, it is expected that only a subset fraction will affect gene expression. Massively Parallel Reporter Assays (MPRAs) are molecular genetic tools that enable functional screening of hundreds of predefined sequences in a single experiment. These assays have been used for functional screening of several different types of regulatory sequences in vitro. However, they have not yet been adapted to query specific cell types in vivo in a complex tissue like the mouse brain. Here, using a test-case 3'UTR MPRA library with variants from ASD patients, we sought to develop a method to achieve reproducible measurements of variant effects in vivo in a cell type-specific manner. We implemented a Cre-dependent design to control expression of our library and first validated our system in vitro. Next, we measured the effect of >500 3'UTR variants in excitatory neurons in the mouse brain. Finally, we report >40 variants with significant effects on transcript abundance in the context of the brain. This new technique should enable robust, functional annotation of genetic variants in the cellular contexts most relevant to psychiatric disease.


title: An Open-Access Simultaneous Electrocardiogram and Phonocardiogram Database

abstract: The electro-phono-cardiogram (EPHNOGRAM) project focused on the development of low-cost and low-power devices for recording simultaneous electrocardiogram (ECG) and phonocardiogram (PCG) data, with auxiliary channels for capturing environmental audio noise, which could be used for PCG quality enhancement through signal processing. The current database, recorded by version 2.1 of the developed hardware, has been acquired from 24 healthy adults aged between 23 and 29 (average: 25.4 {+/-} 1.9 years) in 30 min stress-test sessions during resting, walking, running and biking conditions, using indoor fitness center equipment. The dataset also contains several 30 s sample records acquired during rest conditions. This data is useful for simultaneous multi-modal analysis of ECG and PCG. It provides interesting insights into the inter-relationship between the mechanical and electrical mechanisms of the heart, under rest and physical activity. The database is provided online on PhysioNet.


title: Intratumoural heterogeneity and immune modulation in lung adenocarcinoma of female smokers and never smokers

abstract: Lung cancer is still the leading cause of cancer death worldwide despite declining smoking prevalence in industrialised countries. Although lung cancer is highly associated with smoking status, a significant proportion of lung cancer cases develop in patients who never smoked, with an observable bias towards female never smokers. A better understanding of lung cancer heterogeneity and immune system involvement during tumour evolution and progression in never smokers is therefore highly warranted. We employed single nucleus transcriptomics of surgical lung adenocarcinoma (LADC) and normal lung tissue samples from patients with or without smoking history. Immune cells as well as fibroblasts and endothelial cells respond to tobacco smoke exposure by inducing a highly inflammatory state in normal lung tissue. In the presence of LADC, we identified differentially expressed transcriptional programmes in macrophages and cancer-associated fibroblasts, providing insight into how the niche favours tumour progression. Within tumours, we distinguished eight subpopulations of neoplastic cells in female smokers and never smokers. Through pseudotemporal ordering, we inferred a trajectory towards two differentiated tumour cell states implicated in cancer progression and invasiveness. A proliferating cell population sustaining tumour growth exhibits differential immune modulating signatures in both patient groups. Our results resolve cellular heterogeneity and immune interactions in LADC, with a special emphasis on female never smokers and implications for the design of therapeutic approaches.

category: cancer biology


title: The human hepatocyte TXG-MAPr: WGCNA transcriptomic modules to support mechanism-based risk assessment

abstract: Mechanism-based risk assessment is urged to advance and fully permeate into current safety assessment practices, possibly at early phases of drug safety testing. Toxicogenomics is a promising source of comprehensive and mechanisms-revealing data, but analysis tools to interpret mechanisms of toxicity and specific for the testing systems (e.g. hepatocytes) are lacking. In this study we present the TXG-MAPr webtool (available at https://txg-mapr.eu/WGCNA_PHH/TGGATEs_PHH/), an R-Shiny-based implementation of weighted gene co-expression networks (WGCNA) obtained from the Primary Human Hepatocytes (PHH) TG-GATEs dataset. Gene co-expression networks (modules) were annotated with functional information (pathway enrichment, transcription factor) to reveal their mechanistic interpretation. Several well-known stress response pathways were captured in the modules, are perturbed by specific stressors and show preserved in rat systems (rat primary hepatocytes and rat in vivo liver), highlighting stress responses that translate across species/testing systems. The TXG-MAPr tool was successfully applied to investigate the mechanism of toxicity of TG-GATEs compounds and using external datasets obtained from different hepatocyte cells and microarray platforms. Additionally, we suggest that module responses can be calculated from targeted RNA-seq data therefore imputing biological responses from a limited gene. By analyzing 50 different PHH donors responses to a common stressor, tunicamycin, we were able to suggest modules associated with donors traits, e.g. pre-existing disease state, therefore connected to donors variability. In conclusion, we demonstrated that gene co-expression analysis coupled to an interactive visualization environment, the TXG-MAPr, is a promising approach to achieve mechanistic relevant, cross-species and cross-platform evaluation of toxicogenomic data.


title: Cortical network structure mediates response to stimulation: an optogenetic study in non-human primates

abstract:
As aberrant network-level functional connectivity underlies a variety of neural disorders, the ability to induce targeted functional reorganization would be a profound development towards therapies for neural disorders. Brain stimulation has been shown to alter large-scale network-wide functional connectivity, but the mapping from stimulation to the modification is unclear. Here, we leverage advances in neural interfaces, interpretable machine learning, and graph theory to arrive at a model which accurately predicts stimulation-induced network-wide functional reorganization. The model jointly considers the stimulation protocol and the cortical network structure, departing from the standard approach which only considers the stimulation protocol. We validate our approach in the primary sensorimotor cortex of non-human primates using paired optogenetic stimulation through a large-scale optogenetic interface. We observe that the stimulation protocol only predicts a small portion of the induced functional connectivity changes while the network structure predicts much more, indicating that the underlying network is the primary mediator of the response to stimulation. We extract the relationships linking the stimulation and network characteristics to the functional connectivity changes and observe that the mappings diverge over frequency bands and successive stimulations. Finally, we uncover shared processes governing real-time and longer-term effects of stimulation. Our framework represents a paradigm shift for targeted neural stimulation and can be used to interrogate, improve, and develop stimulation-based interventions for neural disorders.

TeaserBrain stimulation rewires the brain, but the pre-existing network structure of the brain controls the rewiring.


title: Network-level modeling of stimulation-induced functional connectivity change: An optogenetic study in non-human primate cortex


title: Cortical network structure mediates response to stimulation: An optogenetic study in non-human primate cortex


title: The mirror neuron system compensates for amygdala dysfunction-associated social deficits in individuals with higher autistic traits

abstract: The amygdala is a core node in the social brain which exhibits structural and functional abnormalities in Autism spectrum disorder and there is evidence that the mirror neuron system (MNS) can functionally compensate for impaired emotion processing following amygdala lesions. In the current study, we employed an fMRI paradigm in 241 subjects investigating MNS and amygdala responses to observation, imagination and imitation of dynamic facial expressions and whether these differed in individuals with higher as opposed to lower autistic traits. Results indicated that individuals with higher compared to lower autistic traits showed reduced left amygdala responses to imitation and enhanced responses in the left superior temporal sulcus (STS) of the MNS to observation, imagination and imitation. Additionally, functional connectivity between the left amygdala and the left STS as well as some other MNS regions was increased in individuals with higher autistic traits, especially during imitation of fearful expressions. The amygdala-MNS connectivity significantly moderated autistic group differences on recognition memory for fearful faces and real-life social network indices, indicating that increased amygdala-MNS connectivity could diminish the social behavioral differences between higher and lower autistic trait groups. Overall, findings demonstrate decreased imitation-related amygdala activity in individuals with higher autistic traits in the context of increased cortical MNS activity and amygdala-MNS connectivity which may functionally compensate for amygdala dysfunction and social deficits. Training targeting the MNS may capitalize on this compensatory mechanism for therapeutic benefits in Autism spectrum disorder.


title: Mobile Footprinting: Linking Individual Distinctiveness in Mobility Patterns to Mood, Sleep, and Brain Functional Connectivity

abstract: Mapping individual differences in behavior is fundamental to personalized neuroscience. Here, we establish that statistical patterns of smartphone-based mobility features represent unique "footprints" that allow individual identification. Critically, mobility footprints exhibit varying levels of person-specific distinctiveness and are associated with individual differences in affective instability, circadian irregularity, and brain functional connectivity. Together, this work suggests that real-world mobility patterns may provide an individual-specific signature linking brain, behavior, and mood.


title: Fiberoptic Probe For Hemodynamic Spectroscopy in Freely Moving Mice Shows Cerebrovascular Dysregulation In Alzheimers and Glioma Models

abstract:
SignificanceCerebral vascular reactivity is critical parameters of brain homeostasis in health and disease, but the investigational value of brain oxymetry is diminished by anesthesia and mechanical fixation of the mouse scull.

AimWe needed to reduce the physical restrictivity of hemodynamic spectroscopy to enable cancer and Alzheimers disease (AD) studies in freely-moving mice.

ApproachWe combined spectroscopy, spectral analysis software and a magnetic, implantable device to measure vascular reactivity in unanesthetized, freely-moving mice. We measured cerebral blood volume fraction (CBVF) and oxygen saturation (SO2).

ResultsCBVF and SO2 demonstrated delayed cerebrovascular recovery from hypoxia in an orthotopic xenograft glioma model and we found increased CBVF during hypercapnia in a mouse model of AD compared to wild-type littermates.

ConclusionsOur optomechanical approach to reproducibly getting light into and out of the brain enabled us to successfully measure CBVF and SO2 during hypercapnia in unanesthetized freely-moving mice. We present hardware and software enabling oximetric analysis of metabolic activity, which provides a safe and reliable method for rapid assessment of vascular reactivity in murine disease models as well as CBVF and SO2.


title: miR-2 contributes to WSSV infection by targeting Caspase 2 in mud crab (Scylla paramamosain)

abstract: As we known, Caspase 2 is widely studied for its apoptosis regulatory function in mammals. However, despite the fundamental role of apoptosis during the anti-viral immune response, the relationship between Caspase 2 and virus infection has not been extensively explored in invertebrates, whether miRNAs are involved in this process also remains unclear. To address this issue, the miRNA-mediated regulation of Caspase 2 in mud crab Scylla paramamosain was characterized in this study. The results suggested that Sp-Caspase 2 could suppress white spot syndrome virus (WSSV) infection via apoptosis induction. The further data showed that Caspase 2 was directly targeted by miR-2 in mud crab. Silencing or overexpression of miR-2 could affect apoptosis and WSSV replication through regulating the expression level of Caspase 2. Taken together, all these results demonstrated the crucial role of miR-2-Caspase 2 pathway in the innate immunity of mud crab and revealed a novel mechanism during anti-viral immune response in marine invertebrates.


title: Systematic characterization of short intronic splicing-regulatory elements

abstract: Intronic splicing enhancers and silencers (ISEs and ISSs) are two groups of splicing-regulatory elements (SREs) that play critical roles in determining splice-site selection, particularly for alternatively spliced introns or exons. SREs are often short motifs; their mutation or dysregulation of their cognate proteins frequently causes aberrant splicing and results in disease. To date, however, knowledge about SRE sequences and how they regulate splicing remains limited. Here, using an SMN2 minigene, we generated a complete pentamer-sequence library that comprises all possible combinations of 5 nucleotides in intron 7, at a fixed site downstream of the 5' splice site. We systematically analyzed the effects of all 1023 mutant pentamers on exon 7 splicing, in comparison to the wild-type minigene, in HEK293 cells. Our data show that the majority of pentamers significantly affect exon 7 splicing: 584 of them are stimulatory and 230 are inhibitory. To identify actual SREs, we utilized a motif set enrichment analysis (MSEA), from which we identified groups of stimulatory and inhibitory SRE motifs. We experimentally validated several strong SREs in SMN1/2 and MAPT minigene settings. Our results provide a valuable resource for understanding how short RNA sequences regulate splicing. Many novel SREs can be explored further to elucidate their mechanism of action.


title: Succession comprises a sequence of threshold-induced community assembly processes towards multidiversity

abstract: Research on successions and community assembly both address the same processes such as dispersal, species sorting, and biotic interactions but lack unifying concepts. Recent theoretical advances integrated both research lines proposing a sequence of stochastic and niche-based processes along successional gradients. Shifts in assembly processes are predicted to occur abruptly once abiotic and biotic factors dominate over dispersal as main driver. Considering the multidiversity composed of five organismal groups including plants, animals, and microbes, we empirically show that stochastic dispersal-dominated community assembly is replaced by environmental filters and biotic interactions after around 60 years of succession in a glacier forefield. The niche-based character of later successional processes is further supported by a decline in multi-beta-diversity. Our results may update concepts of community assembly by considering multiple taxa, help to bridge the gap between research on successions and community assembly, and provide new insights into the emergence of multidiverse and complex ecosystems.


title: A mutation-selection model of protein evolution under persistent positive selection

abstract:
We use first principles of population genetics to model the evolution of proteins under persistent positive selection (PPS). PPS may occur when organisms are subjected to persistent environmental change, during adaptive radiations, or in host-pathogen interactions. Our mutation-selection model indicates protein evolution under PPS is an irreversible Markov process, and thus proteins under PPS show a strongly asymmetrical distribution of selection coefficients among amino acid substitutions. Our model shows the criteria{omega} > 1 (where{omega} is the ratio of non-synonymous over synonymous codon substitution rates) to detect positive selection is conservative and indeed arbitrary, because in real proteins many mutations are highly deleterious and are removed by selection even at positively-selected sites. We use a penalized-likelihood implementation of our model to successfully detect PPS in plant RuBisCO and influenza HA proteins. By directly estimating selection coefficients at protein sites, our inference procedure bypasses the need for using{omega} as a surrogate measure of selection and improves our ability to detect molecular adaptation in proteins.

Significance StatementUnderstanding how natural selection acts on proteins is important as it can inform studies from adaptive radiations to host-pathogen co-evolution. Traditionally, selection on proteins is inferred indirectly by measuring the non-synonymous to synonymous rate ratio,{omega} , with{omega} > 1, = 1, and < 1 indicating positive (adaptive) selection, neutral evolution, and negative (purifying) selection respectively. However, the theoretical underpinnings of this ratio are not well understood. Here we use first-principles of population genetics to work out how persistent changes in selection affect protein evolution and we use our new model to detect adaptive sites in plant and influenza proteins. We show measuring selection directly improves detection of adaptation in proteins.


title: Y recombination arrest and degeneration in the absence of sexual dimorphism

abstract:
Current theory proposes degenerated sex chromosomes evolve via three successive steps: recombination arrest, which links male-beneficial alleles to the Y chromosome; degeneration of these regions due to the inefficacy of natural selection in the absence of recombination; and lastly, the evolution of dosage compensation to correct the resulting low expression of X-linked genes in males. Here we investigate new models of sex chromosome evolution incorporating the coevolution of cis- and trans-regulators of gene expression. We show that the early emergence of dosage compensation favors the maintenance of Y-linked inversions by creating sex-antagonistic regulatory effects. This is followed by inversion degeneration caused by regulatory divergence between the X and Y chromosomes. In stark contrast to the current theory, the whole process occurs without any selective pressure related to sexual dimorphism.

One Sentence SummaryTurning sex chromosome theory on its head: early evolution of dosage compensation can maintain successively forming Y chromosome strata that undergo genetic degeneration.


title: Using Machine Learning to design AAV2 with high likelihood of viral assembly

abstract: We study the application of machine learning in designing adeno-associated virus (AAV) capsid sequences with high likelihood of viral assembly, i.e. capsid viability. Specifically, we design and implement Origami, a model-based optimization algorithm, to identify highly viable capsid sequences within the vast space of 2033 possibilities. Our evaluation shows that Origami performs well in terms of optimality and diversity of model-designed sequences. Moreover, these sequences are ranked according to their viability score. This helps designing experiments given budget constraint.


title: Using machine learning to design adeno-associated virus capsids with high likelihood of viral assembly


title: Rules for designing protein fold switches and their implications for the folding code

abstract:
We have engineered switches between the three most common small folds, 3, 4{beta}+, and /{beta}-plait, referred to here as A, B, and S, respectively. Mutations were introduced into the natural S protein until sequences were created that have a stable S-fold in their longer ([~]90 amino acid) form and have an alternative fold (either A or B) in their shorter (56 amino acid) form. Five sequence pairs were designed and key structures were determined using NMR spectroscopy. Each protein pair is 100% identical in the 56 amino acid region of overlap. Several rules for engineering switches emerged. First, designing one sequence with good native state interactions in two folds requires care but is feasible. Once this condition is met, fold populations are determined by the stability of the embedded A- or B-fold relative to the S-fold and the conformational propensities of the ends that are generated in the switch to the embedded fold. If the stabilities of the embedded fold and the longer fold are similar, conformation is highly sensitive to mutation so that even a single amino acid substitution can radically shift the population to the alternative fold. The results provide insight into why dimorphic sequences can be engineered and sometimes exist in nature, while most natural protein sequences populate single folds. Proteins may evolve toward unique folds because dimorphic sequences generate interactions that destabilize and can produce aberrant functions. Thus, two-state behavior may result from natures negative design rather than being an inherent property of the folding code.

Significance StatementWe establish general rules for designing protein fold switches by engineering dimorphic sequences that link the three most common small folds. The fact that switches can be engineered in arbitrary and common protein folds, sheds light on several important questions: 1) What is the generality of fold switching? 2) What types of folds are amenable to switching? 3) What properties are shared by sequences that can fold into two completely different structures? This work has implications for understanding how amino acid sequence encodes structure, how proteins evolve, how mutation is related to disease, and how function is annotated to sequences of unknown structure.

ClassificationBiological Sciences: Biochemistry; Physical Sciences: Biophysics and Computational Biology