title: Cellular translational enhancer elements that recruit eukaryotic initiation factor 3

abstract: Translation initiation is a highly regulated process which broadly affects eukaryotic gene expression. Eukaryotic initiation factor 3 (eIF3) is a central player in canonical and alternative pathways for ribosome recruitment. Here we have investigated how direct binding of eIF3 contributes to the large and regulated differences in protein output conferred by different 5'-untranslated regions (5'-UTRs) of cellular mRNAs. Using an unbiased high-throughput approach to determine the affinity of budding yeast eIF3 for native 5'-UTRs from 4,252 genes, we demonstrate that eIF3 binds specifically to a subset of 5'-UTRs that contain a short unstructured binding motif, AMAYAA. eIF3 binding mRNAs have higher ribosome density in growing cells and are preferentially translated under certain stress conditions, supporting the functional relevance of this interaction. Our results reveal a new class of translational enhancer and suggest a mechanism by which changes in core initiation factor activity enact mRNA-specific translation programs.


title: Tillage intensity and plant rhizosphere selection shape bacterial-archaeal assemblage diversity and nitrogen cycling genes

abstract: In agriculture, adoption of reduced tillage practices is a widespread adaptation to global change. The cessation of plowing reduces erosion, slows soil organic matter oxidation, and promotes soil carbon accrual, but it can also result in the development of potential N2O spots from denitrification activity. In this study, we hypothesized that 16S rRNA-based composition of bacterial-archaeal assemblages would differ in agricultural soils subjected for forty years to a range of disturbance intensities, with annual moldboard plowing (MP) being the most intensive. No-till planting (NT) represented tillage management with the least amount of disturbance, while chisel-disking (CD), a type of conservation tillage, was intermediate. All long-term tillage plots had been planted with the same crops grown in a three-year crop rotation (corn-soybean-small grain+cover crop), and both bulk and rhizosphere soils were analyzed from the corn and soybean years. We also evaluated denitrification gene markers by quantitative PCR at multiple points (three growth stages of corn and soybean). Tillage intensity, soil compartment (bulk or rhizosphere), crop year, growth stage, and interactions all exerted effects on community diversity and composition. Compared to MP and CD, NT soils had lower abundances of denitrification genes, higher abundances of nitrate ammonification genes, and higher abundances of taxa at the family level associated with the inorganic N cycle processes of archaeal nitrification and anammox. Soybean rhizospheres exerted stronger selection on community composition and diversity relative to corn rhizospheres. Interactions between crop year, management, and soil compartment had differential impacts on N gene abundances related to denitrification and nitrate ammonification. Opportunities for managing hot spots or hot moments for N losses from agricultural soils may be discernible through improved understanding of tillage intensity effects, although weather and crop type are also important factors influencing how tillage influences microbial assemblages and N use.

category: microbiology


title: Structural and functional characterization of ubiquitin variant inhibitors for the JAMM-family deubiquitinases STAMBP and STAMBPL1

abstract: Ubiquitination is one of the most crucial post-translational protein modifications involved in a myriad of biological pathways. This process is reversed by deubiquitinases (DUBs) that deconjugate the ubiquitin (Ub) moiety or poly-Ub chains from substrates. In the past decade, tremendous efforts have been focused on targeting DUBs for drug discovery. However, most chemical compounds with inhibitory activity for DUBs suffer from mild potency and low selectivity. To overcome these obstacles, we developed a phage display-based protein engineering strategy for generating Ub variant (UbV) inhibitors and have previously successfully applied it to the Ub-specific protease (USP) family of cysteine proteases. In this work, we leveraged the UbV platform to target STAMBP, a member of the JAB1/MPN/MOV34 (JAMM) metalloprotease family of DUB enzymes. We identified two UbVs (UbVSP.1 and UbVSP.3) that bind to STAMBP with high affinity but differ in their selectivity for the closely related paralog STAMBPL1. We determined the STAMBPL1-UbVSP.1 complex structure by X-ray crystallography, revealing hotspots of the tight JAMM-UbV interaction. Finally, we show that UbVSP.1 and UbVSP.3 are potent inhibitors of the STAMBP isopeptidase activity, far exceeding the reported small-molecule inhibitor BC-1471. This work demonstrates that UbV technology is suitable to develop tool molecules for metalloproteases. These tools can be used to understand the cellular function of JAMM family DUBs.

category: biochemistry


title: Quantification of Myxococcus xanthus Aggregation and Rippling Behaviors: Deep-learning Transformation of Phase-contrast into Fluorescence Microscopy Images

abstract: Myxococcus xanthus bacteria are a model system for understanding pattern formation and collective cell behaviors. When starving, cells aggregate into fruiting bodies to form metabolically inert spores. During predation, cells self-organize into traveling cell-density waves termed ripples. Both phase-contrast and fluorescence microscopy are used to observe these patterns but each has its limitations. Phase-contrast images have higher contrast, but the resulting image intensities lose their correlation with cell density. The intensities of fluorescence microscopy images, on the other hand, are well correlated with cell density, enabling better segmentation of aggregates and better visualization of streaming patterns in between aggregates. However, fluorescence microscopy requires the engineering of cells to express fluorescent proteins and can be phototoxic to the cells. To combine the advantages of both imaging methodologies, we develop a generative adversarial network that converts phase-contrast into fluorescent images. By including an additional histogram-equalized output to the state-of-art pix2pixHD algorithm, our model generates accurate images of aggregates and streams, enabling the estimation of aggregate positions and sizes, but with small shifts of their boundaries. Further training on ripple patterns enables accurate estimation of the rippling wavelength. Our methods are thus applicable for many other phenotypic behaviors and pattern formation studies.

category: systems biology


title: The M2 Gene Is a Determinant of Reovirus-Induced Myocarditis

abstract:
Although a broad range of viruses cause myocarditis, the mechanisms that underlie viral myocarditis are poorly understood. Here, we report that the M2 gene is a determinant of reovirus myocarditis. The M2 gene encodes outer capsid protein 1, which mediates host membrane penetration during reovirus entry. We infected newborn C57BL/6 mice with reovirus strain type 1 Lang (T1L) or a reassortant reovirus in which the M2 gene from strain type 3 Dearing (T3D) was substituted into the T1L genetic background (T1L/T3DM2). T1L was non-lethal in wild-type mice, whereas greater than 90% of mice succumbed to T1L/T3DM2 infection. T1L/T3DM2 produced higher viral loads than T1L at the site of inoculation. In secondary organs, T1L/T3DM2 was detected with more rapid kinetics and reached higher peak titers than T1L. We found that hearts from T1L/T3DM2-infected mice were grossly abnormal, with large lesions indicative of substantial inflammatory infiltrate. Lesions in T1L/T3DM2-infected mice contained necrotic cardiomyocytes with pyknotic debris, and extensive lymphocyte and histiocyte infiltration. In contrast, T1L induced the formation of small purulent lesions in a small subset of animals, consistent with T1L being mildly myocarditic. Finally, more activated caspase-3-positive cells were observed in hearts from animals infected with T1L/T3DM2 compared to T1L. Together, our findings indicate that substitution of the T3D M2 allele into an otherwise T1L genetic background is sufficient to change a non-lethal infection into a lethal infection. Our results further indicate that T3D M2 enhances T1L replication and dissemination in vivo, which potentiates the capacity of reovirus to cause myocarditis.

IMPORTANCEReovirus is a non-enveloped virus with a segmented double-stranded RNA genome that serves as a model for studying viral myocarditis. The mechanisms by which reovirus drives myocarditis development are not fully elucidated. We found that substituting the M2 gene from strain type 3 Dearing (T3D) into an otherwise type 1 Lang (T1L) genetic background (T1L/T3DM2) was sufficient to convert the non-lethal T1L strain into a lethal infection in neonatal C57BL/6 mice. T1L/T3DM2 disseminated more efficiently and reached higher maximum titers than T1L in all organs tested, including the heart. T1L is mildly myocarditic and induced small areas of cardiac inflammation in a subset of mice. In contrast, hearts from mice infected with T1L/T3DM2 contained extensive cardiac inflammatory infiltration and more activated caspase-3-positive cells, which is indicative of apoptosis. Together, our findings identify the reovirus M2 gene as a new determinant of reovirus-induced myocarditis.


title: A subcellular biochemical model for T6SS dynamics reveals winning competitive strategies

abstract: The Type VI secretion system (T6SS) is a broadly distributed interbacterial weapon that can be used to eliminate competing bacterial populations. Although unarmed target populations are typically used to study T6SS function, bacteria most likely encounter other T6SS-armed competitors in nature. The outcome of such battles is not well understood, neither is the connection between the outcomes with the subcellular details of the T6SS. Here, we incorporated new biological data derived from natural competitors of Vibrio fischeri light organ symbionts to build a biochemical model for T6SS function at the single cell level. The model accounts for activation of structure formation, structure assembly, and deployment. By developing an integrated agent-based model (IABM) that incorporates strain-specific T6SS parameters, we replicated outcomes of biological competitions, validating our approach. We used the IABM to isolate and manipulate strain-specific physiological differences between competitors, in a way that is not possible using biological samples, to identify winning strategies for T6SS-armed populations. We found that a tipping point exists where the cost of building more T6SS weapons outweighs their protective ability. Furthermore, we found that competitions between a T6SS-armed population and a unarmed target had different outcomes dependent on the geometry of the battlefield: target cells survived at the edges of a range expansion scenario where unlimited territory could be claimed, while competitions within a confined space, much like the light organ crypts where natural V. fischeri compete, resulted in the rapid elimination of the unarmed competitor.


title: Pathogenic potential in catheter associated Escherichia coli is associated with separable biofilm and virulence gene determinants

abstract: Urinary catheterization facilitates Escherichia coli colonization of the urinary tract and increases infection risk. While specific pathotypes are well-recognized for some E. coli infections, it is unclear whether strain-specific characteristics among E. coli are associated with infection risk in catheterized patients. Here we used comparative genomics and a simulated catheter biofilm model to compare strains associated with catheter-associated urinary tract infection (CAUTI) and catheter-associated asymptomatic bacteriuria (CAASB). CAUTI was associated with a phylotype B2 sub-clade dominated by the multidrug resistant ST131 lineage, while CAASB isolates were genetically more diverse. Catheter-associated biofilm formation was widespread but quantitatively variable among isolates. Network community analysis resolved distinct groups of genes associated with infection or biofilm formation, with iron acquisition-associated genes prominent throughout. Using a reporter construct and targeted mutagenesis, we detected a biofilm phenotype for the ferric citrate transport (Fec) system, the most prominent correlate of high catheter biofilm formation in these patients. In mixed cultures, catheter biofilms formed by some CAASB strains suppressed catheter colonization by ST131 CAUTI isolates. These results are consistent with a paradigm in which catheter biofilm-associated genes increase infection risk in strains with a high pathogenic potential and decrease infection risk through niche exclusion in strains with low pathogenic potential.


title: Structure-guided bifunctional molecules hit a DEUBAD-lacking hRpn13 species upregulated in multiple myeloma

abstract: Proteasome substrate receptor hRpn13 is a promising anti-cancer target. By integrated in silico and biophysical screening, we identified a chemical scaffold that binds hRpn13 with non-covalent interactions that mimic the proteasome and a weak electrophile for Michael addition. hRpn13 Pru domain binds proteasomes and ubiquitin whereas its DEUBAD domain binds deubiquitinating enzyme UCHL5. NMR revealed lead compound XL5 to interdigitate into a hydrophobic pocket created by lateral movement of a Pru {beta}-hairpin with an exposed end for Proteolysis Targeting Chimeras (PROTACs). Implementing XL5-PROTACs as chemical probes identified a DEUBAD-lacking hRpn13 species (hRpn13Pru) present naturally with cell type-dependent abundance. XL5-PROTACs preferentially target hRpn13Pru, causing its ubiquitination. Gene-editing and rescue experiments established hRpn13 requirement for XL5-PROTAC-triggered apoptosis and increased p62 levels. These data establish hRpn13 as an anti-cancer target for multiple myeloma and introduce an hRpn13-targeting scaffold that can be optimized for preclinical trials against hRpn13Pru-producing cancer types.

category: molecular biology


title: microRNAs Over-expressed in Diabetic Foot Ulcers Healing - Computational Modeling of Molecular Structure

abstract:
BackgroundVasculopathy associated with diabetic neuropathy are important risk factors for the diabetic foot ulcers development. Diabetic foot ulcers is severe complication that occur in about 15% of people with diabetes, being able require hospitalization and amputation in its treatment.

ObjectiveDesign in silico the molecular structure of micro-ribonucleic acid (miRNA) overexpressed in diabetic foot ulcers healing.

MethodWe performed a careful search of the nucleotide sequence of 8 miRNAs over-expressed in diabetic foot ulcers, designing in silico the molecular structure of following miRNAs: miRNA-146a, miRNA-155, miRNA-132, miRNA-191, miRNA-21, miRNA-203a, miRNA-203b, and miRNA-210. The nucleotides were taken from GenBank of National Center for Biotechnology Information genetic sequence database. The sequences acquired were aligned with the Clustal W multiple alignment algorithms. The molecular modeling of structures was built using the RNAstructure, an automated miRNAs structure modelling server.

ResultsWe showed a search for nucleotide sequence and the design of the molecular structure of following miRNA over-expressed in diabetic foot ulcers healing: miRNA-146a, miRNA-155, miRNA-132, miRNA-191, miRNA-21, miRNA-203a, miRNA-203b, and miRNA-210. We produced a tutorial on a molecular model of the 8 miRNAs overexpressed in the diabetic foot by processing in silico projection of their molecular structures.

ConclusionWe show in silico secondary structures design of selected of 8 miRNAs over-expressed in diabetic foot ulcers healing by means of computational biology.


title: The adaptation front equation explains diversification hotspots and living-fossilization

abstract:
Taxonomic turnovers are common in the evolutionary histories of biological communities. Such turnovers are often associated with the emergence and diversification of groups that have achieved fundamental innovations beneficial in various ecological niches. In the present study, we show that such innovation-driven turnovers could be analyzed using an equation that describes the dynamics of zero-fitness isoclines in a two-dimensional trait space comprising a "fundamental trait" (describing fundamental innovation) and a "niche trait" (describing niche position) or with its higher-dimensional extensions. Our equation allows analytical prediction of evolutionary source-sink dynamics along the niche axis for an arbitrary unimodal (or multimodal with weak separation) carrying capacity distribution. The prediction was confirmed by numerical simulation under different assumptions for resource competition, reproduction, and mutation. In the simulated evolution, biodiversity sources are the central niches having higher carrying capacities than the outer niches, allowing species there the faster evolutionary advancement in fundamental traits and their repeated diversification into outer niches, which outcompete the indigenous less advanced species. The outcompeted species go extinct or evolve directionally toward the far outer niches of the far slower advancement because of the far lower carrying capacities. In consequence of this globally acting process over niches, species occupying peripheral (i.e., the outermost) niches can have significantly primitive fundamental traits and deep divergence times from their closest relatives, and thus, they correspond to living fossils. The extension of this analysis for multiple geographic regions showed that living fossils are also expected in geographically peripheral regions for the focal species group.

Significance StatementWe developed a new equation for analyzing the long-term coevolution of many species through their directional evolution, evolutionary branching, and extinction in an arbitrary trait space comprising traits describing niche positions and traits describing fundamental innovations. This equation describes the many coevolving species as a fluid, as in the study of galaxy dynamics in astronomical physics. This equation may be used to explain how innovation-driven taxonomic turnovers generate diversification hotspots and coldspots among niches or geographical regions, accompanied by continuous production of "living fossil" species in peripheries, in a logic equivalent to that deduced by Darwin and Darlington from empirical observations.

category: evolutionary biology


title: A model-based approach to characterize enzyme-mediated response to antibiotic treatments: going beyond the SIR classification

abstract: To design appropriate treatments, one must be able to characterize accurately the response of bacteria to antibiotics. When exposed to {beta}-lactam treatments, bacteria can be resistant and/or tolerant, and populations can exhibit resilience. Disentangling these phenomena is challenging and no consolidated understanding has been proposed so far. Because these responses involve processes happening at several levels, including the molecular level (e.g. antibiotic degradation), the cell physiology level (filamentation) and the population level (release of {beta}-lactamases into the environment), quantitative modelling approaches are needed. Here, we propose a model of bacterial response to {beta}-lactam treatments that accounts for bacterial resistance, tolerance, and population resilience. Our model can be calibrated solely based on optical density readouts, can predict the inoculum effect, and leads to a mechanistically relevant classification of bacterial response to treatments that goes beyond the classical susceptible / intermediate / resistant classification. Filamentation-mediated tolerance and collective enzyme-mediated antibiotic degradation are essential model features to explain the complex observed response of cell populations to antibiotic treatments.

category: biophysics


title: DNA sliding and loop formation by E. coli SMC complex: MukBEF

abstract: SMC (structural maintenance of chromosomes) complexes share conserved architectures and function in chromosome maintenance via an unknown mechanism. Here we have used single-molecule techniques to study MukBEF, the SMC complex in Escherichia coli. Real-time movies show MukB alone can compact DNA and ATP inhibits DNA compaction by MukB. We observed that DNA unidirectionally slides through MukB, potentially by a ratchet mechanism, and the sliding speed depends on the elastic energy stored in the DNA. MukE, MukF and ATP binding stabilize MukB and DNA interaction, and ATP hydrolysis regulates the loading/unloading of MukBEF from DNA. Our data suggests a new model for how MukBEF organizes the bacterial chromosome in vivo; and this model will be relevant for other SMC proteins.


title: TREM2 impacts brain microglia, oligodendrocytes and endothelial co-expression modules revealing genes and pathways important in Alzheimer s disease

abstract: A microglia response to pathogenic signals in diseases such as Alzheimers disease (AD) has long been recognised, but recent genetic findings have cemented their direct causal contribution to AD and thus the potential to target them or their effector pathways as a possible treatment strategy. TREM2 is a highly penetrant microglia risk gene for AD, which appears central to the coordination of the damage response by microglia in AD. Its absence has a negative impact on Tau and amyloid symptoms and pathologies. Full knowledge of its pathway and relationships with other brain cells in AD has not been fully characterised, but will be essential to fully evaluate the impact of manipulating this pathway for treatment development and to establish the best targets for achieving this. We used whole genome RNA sequencing of hippocampus and cortical brain samples from control, AD, and AD TREM2 risk carriers to identify TREM2-dependent genes driving changes in pathways, processes and cell types in AD. Through highly influential intra and intermodular hub genes and overall changes in the levels of gene expression, TREM2-DAP12 was found to strongly influence a number of other microglia, oligodendrocyte and endothelial genes, notably those involved in complement and Fc{gamma} receptor function, microglia-associated ribosomal genes and oligodendrocyte genes, particularly proteosomal subunits. These strong TREM2 centred co-expression relationships were significantly disrupted in AD cases with a TREM2 risk variant, revealing for the first time genes and pathways directly impacted by TREM2 in the brains of AD patients. Consistent with its function as a lipid sensor, our data supports a role for TREM2 in mediating oligodendrocyte and/or myelin clearance in AD which may be essential not only for preserving healthy tissue homeostasis but may also serve to minimise the persistence of antigenic peptides and lipids which may lead to detrimental pro-inflammatory sequelae. Further work should expand our knowledge of TREM2 on complement and Fc{gamma} receptor function and its impact on oligodendcrotye and myelin integrity and further evaluate the genes and pathways we have identified as possible treatment targets for AD.

category: neuroscience


title: Deep deconvolution of the hematopoietic stem cell regulatory microenvironment reveals a high degree of specialization and conservation between mouse and human.

abstract: Understanding the regulation of normal and malignant human hematopoiesis requires comprehensive cell atlas of the hematopoietic stem cell (HSC) regulatory microenvironment. Here, we develop a tailored bioinformatic pipeline to integrate public and proprietary single-cell RNA sequencing (scRNA-seq) datasets. As a result, we robustly identify for the first time 14 intermediate cell states and 11 stages of differentiation in the endothelial and mesenchymal BM compartments, respectively. Our data provide the most comprehensive description to date of the murine HSC-regulatory microenvironment and suggests a higher level of specialization of the cellular circuits than previously anticipated. Furthermore, this deep characterization allows to infer conserved features in human, suggesting that the layers of microenvironmental regulation of hematopoiesis may also be shared between species. Our resource and methodology are a steppingstone towards a comprehensive cell atlas of the BM microenvironment.

category: immunology


title: Identification of leptospiral protein antigens recognized by WC1+ γδ T cell subsets as target for development of recombinant vaccines

abstract: Pathogenic Leptospira species cause leptospirosis, a neglected zoonotic disease recognized as a global public health problem. It is also the cause of the most common cattle infection that results in major economic losses due to reproductive problems. {gamma}{delta} T cells play a role in the protective immune response in livestock species against Leptospira while human {gamma}{delta} T cells also respond to Leptospira. Thus, activation of {gamma}{delta} T cells has emerged as a potential component for optimization of vaccine strategies. Bovine {gamma}{delta} T cells proliferate and produce IFN-{gamma} in response to vaccination with inactivated leptospires and this response is mediated by a specific subpopulation of the WC1-bearing {gamma}{delta} T cells. WC1 molecules are members of the group B scavenger receptor cysteine rich (SRCR) superfamily and are composed of multiple SRCR domains, of which particular extracellular domains act as ligands for Leptospira. Since WC1 molecules function as both pattern recognition receptors and {gamma}{delta} TCR coreceptors, the WC1 system has been proposed as a novel target to engage {gamma}{delta} T cells. Here, we demonstrate the involvement of leptospiral protein antigens in the activation of WC1+ {gamma}{delta} T cells and identified two leptospiral outer membrane proteins able to interact directly with them. Interestingly, we show that the protein-specific {gamma}{delta} T cell response is composed of WC1.1+ and WC1.2+ subsets, although a greater number of WC1.1+{gamma}{delta} T cells respond. Identification of protein antigens will enhance our understanding of the role {gamma}{delta} T cells play in the leptospiral immune response and in recombinant vaccine development.


title: Proximity interactome analysis of Lassa polymerase reveals eRF3a/GSPT1 as a druggable target for host directed antivirals.

abstract:
Completion of the Lassa virus (LASV) life cycle critically depends on the activities of the virally encoded RNA-dependent RNA polymerase in replication and transcription of the viral RNA genome in the cytoplasm of infected cells. The contribution of cellular proteins to these processes remains unclear. Here, we applied proximity proteomics to define the interactome of LASV polymerase in cells, under conditions that recreate LASV RNA synthesis. We engineered a LASV polymerase-biotin ligase (TurboID) fusion protein that retained polymerase activity and successfully biotinylated the proximal proteome, which allowed the identification of 42 high-confidence LASV polymerase interactors. We subsequently performed an siRNA screen to identify those interactors that have functional roles in authentic LASV infection. As proof-of-principle, we characterized eukaryotic peptide chain release factor subunit 3a (eRF3a/GSPT1), which we found to be a proviral factor that physically associates with LASV polymerase. Targeted degradation of GSPT1 by a small molecule drug candidate, CC-90009, resulted in strong inhibition of LASV infection in cultured cells. Our work demonstrates the feasibility of using proximity proteomics to illuminate and characterize yet to be defined, host-pathogen interactome, which can reveal new biology and uncover novel targets for the development of antivirals against highly pathogenic RNA viruses.

Significance StatementLassa virus (LASV), the causative agent of Lassa fever (LF), represents an important public health problem in Western Africa. There is no FDA-approved therapeutic intervention to treat LF. Due to its limited genome coding capacity, LASV proteins are often multifunctional and orchestrate complex interactions with cellular factors to execute steps required to complete the viral life cycle. LASV polymerase is essential for replication and expression of the viral genome, and thus is an attractive target for antiviral intervention. Here we present the first host interactome of the LASV polymerase, which can guide identification of novel druggable host cellular targets for the development of cost-effective antiviral therapies for LF.


title: Increased microbial diversity and decreased prevalence of common pathogens in the gut microbiomes of wild turkeys compared to domestic turkeys

abstract:
Turkeys (Meleagris gallopavo) provide a globally important source of protein and constitute the second most important source of poultry meat in the world. Bacterial diseases are common in commercial poultry production causing significant production losses for farmers. Due to the increasingly recognized problems associated with large-scale/indiscriminant antibiotic use in agricultural settings, poultry producers need alternative methods to control common bacterial pathogens. In this study we compared the cecal microbiota of wild and domestic turkeys, hypothesizing that environmental pressures faced by wild birds may select for a disease-resistant microbial community. Sequence analysis of 16S rRNA genes amplified from cecal samples indicate that free-roaming wild turkeys carry a rich and variable microbiota compared to domestic turkeys raised on large-scale poultry farms. Wild turkeys also had very low levels of Staphylococcus, Salmonella and E. coli when compared to domestic turkeys. E. coli strains isolated from wild or domestic turkey cecal samples also belong to distinct phylogenetic backgrounds and differ in their propensity to carry virulence genes. E. coli strains isolated from factory-raised turkeys were far more likely to carry genes for capsule (kpsII, kpsIII) or siderophore (iroN, fyuA) synthesis than those isolated from wild turkeys. These results suggest that the microbiota of wild turkeys may provide colonization resistance against common poultry pathogens.

ImportanceDue to the increasingly recognized problems associated with antibiotic use in agricultural settings, poultry producers need alternative methods to control common bacterial pathogens. In this study we compare the microbiota of wild and domestic turkeys. Results suggest that free ranging wild turkeys carry a distinct microbiome when compared to farm raised turkeys. The microbiome of wild birds contains very low levels of poultry pathogens compared to farm raised birds. The microbiomes of wild turkeys may be used to guide development of new ways to control disease in large scale poultry production.


title: Novel Hendra virus variant detected by sentinel surveillance of Australian horses.

abstract: A novel Hendra virus (HeV) variant, not detected by routine testing, was identified and isolated from a Queensland horse that suffered acute, fatal disease consistent with HeV infection. Whole genome sequencing and phylogenetic analysis demonstrated the variant to have ~83% nucleotide identity to the prototype HeV strain. An updated RT-qPCR assay was designed for routine HeV surveillance. In silico and in vitro comparison of the receptor-binding protein with prototypic HeV showed that the human monoclonal antibody m102.4 used for post-exposure prophylaxis, as well as the current equine vaccine, should be effective against this variant. Genetic similarity of this virus to sequences detected from grey-headed flying-foxes suggests the variant circulates at-least in this species. Studies determining infection kinetics, pathogenicity, reservoir-species associations, viral-host co-evolution and spillover dynamics for this virus are urgently needed. Surveillance and biosecurity practices should be updated to appreciate HeV spillover risk across all regions frequented by flying foxes.


title: Interpreting Polygenic Score Effects in Sibling Analysis

abstract: Researchers often claim that sibling analysis can be used to separate causal genetic effects from the assortment of biases that contaminate most downstream genetic studies. Indeed, typical results from sibling models show large (>50%) attenuations in the associations between polygenic scores and phenotypes compared to non-sibling models, consistent with researchers expectations about bias reduction. This paper explores these expectations by using family (quad) data and simulations that include indirect genetic effect processes and evaluates the ability of sibling models to uncover direct genetic effects. We find that sibling models, in general, fail to uncover direct genetic effects; indeed, these models have both upward and downward biases that are difficult to sign in typical data. When genetic nurture effects exist, sibling models create "measurement error" that attenuate associations between polygenic scores and phenotypes. As the correlation between direct and indirect effect changes, this bias can increase or decrease. Our findings suggest that interpreting results from sibling analysis aimed at uncovering direct genetic effects should be treated with caution.

category: genetics


title: A reference translatome map reveals two modes of protein evolution

abstract: Ribosome profiling experiments demonstrate widespread translation of eukaryotic genomes outside of annotated protein-coding genes. However, it is unclear how much of this "noncanonical" translation contributes biologically relevant microproteins rather than insignificant translational noise. Here, we developed an integrative computational framework (iRibo) that leverages hundreds of ribosome profiling experiments to detect signatures of translation with high sensitivity and specificity. We deployed iRibo to construct a reference translatome in the model organism S. cerevisiae. We identified [~]19,000 noncanonical translated elements outside of the [~]5,400 canonical yeast protein-coding genes. Most (65%) of these non-canonical translated elements were located on transcripts annotated as non-coding, or entirely unannotated, while the remainder were located on the 5 and 3 ends of mRNA transcripts. Only 14 non-canonical translated elements were evolutionarily conserved. In stark contrast with canonical protein-coding genes, the great majority of the yeast noncanonical translatome appeared evolutionarily transient and showed no signatures of selection. Yet, we uncovered phenotypes for 53% of a representative subset of evolutionarily transient translated elements. The iRibo framework and reference translatome described here provide a foundation for further investigation of a largely unexplored, but biologically significant, evolutionarily transient translatome.

category: genomics


title: A strategy to suppress STAT1 signalling conserved in pathogenic poxviruses and paramyxoviruses

abstract: The induction of interferon-stimulated genes by signal transducer and activator of transcription (STAT) proteins, is a critical host defence to fight virus infections. Here, a highly expressed poxvirus protein 018 is shown to inhibit IFN-induced signalling by binding the SH2 domain of STAT1 to prevent STAT1 association with an activated IFN receptor. Despite the presence of additional inhibitors of IFN-induced signalling, a poxvirus lacking 018 was attenuated in mice. The 2.0 [A] crystal structure of the 018:STAT1 complex reveals a mechanism for a high-affinity, pTyr-independent mode of binding to an SH2 domain. Furthermore, the STAT1 binding motif of 018 shows sequence similarity to the STAT1-binding proteins from Nipah virus, which like 018, block the association of STAT1 with an IFN receptor. Taken together, these results provide detailed mechanistic insight into a potent mode of STAT1 antagonism, found to exist in genetically diverse virus families.


title: Defining the Sensitivity Landscape of 74,389 EGFR Variants to Tyrosine Kinase Inhibitors

abstract:
BackgroundTyrosine kinase inhibitors (TKIs) therapy is a standard treatment for patients with advanced non-small-cell lung carcinoma (NSCLC) when activating epidermal growth factor receptor (EGFR) mutations are detected. However, except for the well-studied EGFR mutations, most EGFR mutations lack treatment regimens.

MethodsWe constructed two EGFR variant libraries containing substitutions, deletions, or insertions using the saturation mutagenesis method. All the variants were located in the EGFR mutation hotspot (exons 18-21). The sensitivity of these variants to afatinib, erlotinib, gefitinib, icotinib, and osimertinib was systematically studied by determining their enrichment in massively parallel cytotoxicity assays using an endogenous EGFR-depleted cell line, PC9.

ResultsA total of 3,914 and 70,475 variants were detected in the constructed EGFR Substitution-Deletion (Sub-Del) and exon 20 Insertion (Ins) libraries, accounting for 99.3% and 55.8% of the designed variants, respectively. Of the 3,914 Sub-Del variants, 813 were highly enriched in the reversible TKI (erlotinib, gefitinib, icotinib) cytotoxicity assays and 51 were enriched in the irreversible TKI (afatinib, osimertinib) cytotoxicity assays. For the 70,475 Ins variants, insertions at amino acid positions 770-774 were highly enriched in all the five TKI cytotoxicity assays. Moreover, the top 5% of the enriched insertion variants included a glycine or serine insertion at high frequency.

ConclusionsWe present a comprehensive reference for the sensitivity of EGFR variants to five commonly used TKIs. The approach used here should be applicable to other genes and targeted drugs.


title: Knockout of E-cadherin in adult mouse epithelium results in emphysema and airway disease

abstract: Chronic obstructive pulmonary disease (COPD) is a devastating lung disease, characterized by a progressive decline in lung function, alveolar loss (emphysema), and airflow limitation due to excessive mucus secretion (chronic bronchitis), that can occur even after the injurious agent is removed. It is slated to rise to the 3rd leading cause of death due to chronic disease by 2030 globally, and the 4th leading cause of death due to chronic disease in the USA. While there is substantial evidence indicating loss of E-cadherin in the lung epithelium of patients with COPD, it is not known if this is causal to the disease. We investigated if loss of E-cadherin can result in lung disease using in both in vitro models of primary, differentiated human cells and in mouse models. Using a cell type-specific promoter using Cre/LoxP mice system to knock-out E- cadherin in ciliated and alveolar epithelial cell (Type 1 and Type 2) populations in adult mouse models, we determined that loss of E-cadherin caused airspace enlargement, as well as increased airway hyperresponsiveness indicating that it does have a causative role in causing COPD. Strategies to upregulate CDH1 (encodes for E-cadherin) in CHBEs and cigarette-smoke injured NHBEs can rescue the dysfunctional epithelium.

category: cell biology


title: NDC1 is necessary for stable assembly of the nuclear pore scaffold to establish nuclear transport in early C. elegans embryos

abstract: Nuclear pore complexes (NPCs) are large protein assemblies that facilitate transport of macromolecules across the nuclear envelope (NE) [1, 2]. How thousands of NPCs rapidly assemble after open mitosis to form a functional NE is not known. Recruitment of the Nup107-160 outer ring scaffold to chromatin initiates NPC assembly. The Nup53/93 complex bridges the outer ring to the central channel to form a functional pore [3-6]. Nup53 interacts with the conserved transmembrane nucleoporin Ndc1; however, how Ndc1 contributes to post-mitotic NPC assembly is unclear [7-9]. Here, we use C. elegans embryos to show that the timely formation of a functional NE after mitosis depends on Ndc1. Endogenously tagged Ndc1 is recruited early to the reforming NE and is highly mobile in the nuclear rim. 3D analysis of post-mitotic NE formation revealed a decreased NPC density in NEs of ndc1 deleted embryos - continuous nuclear membranes contained few holes where assembling NPCs are normally located. Nup160 is highly mobile in NEs depleted of Ndc1 and outer ring scaffold components are less enriched at the rim. When both ndc1 and nup53 are absent, nuclear assembly fails. Together, these data show that Ndc1 dynamically associates with the NE and promotes stable association of the outer ring scaffold in the NE to facilitate NPC assembly after open mitosis. Furthermore, Ndc1 and Nup53 function in parallel to drive nuclear assembly. We propose that Ndc1 is a dynamic membrane adaptor that helps recruit and promote the self-assembly of the nuclear pore scaffold to drive post-mitotic NPC assembly.


title: Genome-Wide Detection of Imprinted Differentially Methylated Regions Using Nanopore Sequencing

abstract: Imprinting is a critical part of normal embryonic development in mammals, controlled by defined parent-of-origin (PofO) differentially methylated regions (DMRs) known as imprinting control regions. As we and others have shown, direct nanopore sequencing of DNA provides a mean to detect allelic methylation and to overcome the drawbacks of methylation array and short-read technologies. Here we leverage publicly-available nanopore sequence data for 12 standard B-lymphocyte cell lines to present the first genome-wide mapping of imprinted intervals in humans using this technology. We were able to phase 95% of the human methylome and detect 94% of the well-characterized imprinted DMRs. In addition, we found 28 novel imprinted DMRs (12 germline and 16 somatic), which we confirmed using whole-genome bisulfite sequencing (WGBS) data. Analysis of WGBS data in mus musculus, rhesus macaque, and chimpanzee suggested that 12 of these are conserved. We also detected subtle parental methylation bias spanning several kilobases at seven known imprinted clusters. These results expand the current state of knowledge of imprinting, with potential applications in the clinic. We have also demonstrated that nanopore long reads, can reveal imprinting using only parent-offspring trios, as opposed to the large multi - generational pedigrees that have previously been required.


title: Amplification-free detection of viral RNA by super resolution imaging-based CRISPR/Cas13a System

abstract: RNA detection is crucial for biological research and clinical diagnosis. The current methods include both direct and amplification-based RNA detection. These methods require complicated procedures, suffering from low sensitivity, slow turnaround, and amplification bias. The CRISPR/Cas13a system is a direct RNA detection method via target RNA induced collateral cleavage activity. However, to detect low concentration RNA with CRISPR/Cas13a, target amplification is always required. Herein, we optimize the components of the CRISPR/Cas13a assay to enhance the sensitivity of viral RNA detection which improve the detection limit from 1 pM up to 100 fM. In addition, the integration of CRISPR/Cas13a biosensing and single molecule super resolution imaging is a novel strategy for direct and amplification-free RNA detection. After surface modification, fluorescent RNA reporters are immobilized on the glass coverslip surface and fluorescent signals are captured by total internal reflection fluorescence microscopy (TIRFM), shifting the measurement from spectroscopy to imaging. We quantify the fluorescence signal intensity before and after collateral cleavage of the CRISPR system when viral RNA is present and achieve a detection limit of 10 fM. Therefore, we provide a novel TIRFM-based system to visualize the CRISPR trans-cleavage for direct and robust RNA detection.

category: bioengineering


title: Specificity of Loxosceles α clade phospholipase D enzymes for choline-containing lipids: role of a conserved aromatic cage

abstract: Spider venom GDPD-like phospholipases D (SicTox) have been identified to be one of the major toxins in recluse spider venom. They are divided into two major clades: the  clade and the {beta} clade. Most  clade toxins present high activity against lipids with choline head groups such as sphingomyelin, while activities in {beta} clade toxins vary and include preference for substrates containing ethanolamine headgroups (Sicarius terrosus, St_{beta}IB1). A structural comparison of available PLDs structures reveals a conserved aromatic cage in the  clade. To test the potential influence of the aromatic cage on membrane-lipid specificity we performed molecular-dynamics (MD) simulations of the binding of several PLDs onto lipid bilayers containing choline headgroups; two SicTox from the  clade, Loxosceles intermedia IA1 (Li_IA) and Loxosceles laeta III1 (Ll_III1), and one from the {beta} clade, St_{beta}IB1. The simulation results reveal that the aromatic cage captures a choline-headgroup and suggest that the cage plays a major role in lipid specificity. We also simulated an engineered St_{beta}IB1, where we introduced the aromatic cage, and this led to binding with choline-containing lipids. Moreover, a multiple sequence alignment revealed the conservation of the aromatic cage among the  clade PLDs. Here, we confirmed the membrane binding site of  and {beta} clade PLDs on choline and ethanolamine-containing bilayers, respectively. Furthermore, our results suggest a major role in choline lipid recognition of the aromatic cage of the  clade PLDs. The MD simulation results are supported by in vitro liposome binding assay experiments.

category: bioinformatics


title: Autofluorescence microscopy as a label-free tool for renal histology and glomerular segmentation

abstract: Functional tissue units (FTUs) composed of multiple cells like the glomerulus in the kidney nephron play important roles in health and disease. Histological staining is often used for annotation or segmentation of FTUs, but chemical stains can introduce artefacts through experimental factors that influence analysis. Secondly, many molecular -omics techniques are incompatible with common histological stains. To enable FTU segmentation and annotation in human kidney without the need for histological staining, we detail here the use of widefield autofluorescence (AF) microscopy as a simple, label-free modality that provides detailed renal morphology comparable to periodic acid-Schiff (PAS) stained tissue in both formalin-fixed paraffin-embedded (FFPE) and fresh frozen samples and with no tissue processing beyond sectioning. We demonstrate automated deep learning-based glomerular unit recognition and segmentation on PAS and AF images of the same tissue section from 9 fresh frozen samples and 9 FFPE samples. All training comparisons were carried out using registered AF microscopy and PAS stained whole slide images originating from the same section, and the recognition models were built with the exact same training and test examples. Measures of recognition performance, such as the Dice-Sorensen coefficient, the true positive rate, and the positive predictive value differed less than 2% between standard PAS and AF microscopy for both preservation methods. These results demonstrate that AF is a potentially powerful tool to study human kidney tissue, that it can serve as a label-free source for automated and manual annotation of tissue structures.


title: The low and high postpubertal ethanol use: long-term effects on drinkers reproductive parameters and ethanol-naive offspring development

abstract: The relationship between adolescent ethanol uses and its impacts throughout life is not conclusive. Thus, we evaluated if the low and high consumption of ethanol during the postpuberty period interferes with reproduction in adulthood, the ethanol-naive offspring development and if there are dose-related effects. Females and males rats were divided into three groups: low drinker (L), with UChA rats fed with ethanol ad libitum drinking < 1.9 g / kg / day, high drinker (H), with UChB rats fed with ethanol ad libitum drinking from 2 to 5 g / kg / day, and control (C), with rats without access to ethanol. The L and H groups were exposed to ethanol 10% (v/ v) from 65 to 80 days, with withdrawal after this period. The study was conducted in two phases. The retrospective analysis (1st phase) verified the consumption of ethanol between sexes, the litter size, and the sex ratio of offspring. The gestational and reproductive parameters of parents and the development of pups were analyzed in the 2nd phase. We observed a higher consumption of ethanol in females and a reduced litter size in both drinkers groups. Body weight gain and gestational feed consumption were lower in L and H. The offsprings body weight was also lower associated with alteration in landmarks of physical development. The high postpubertal ethanol use accents the impacts on consumers and offspring. The paternal and maternal reproductive organs weight was altered in group H, with an increase in morphologically abnormal sperm. We conclude that low and high post-pubertal alcohol consumption impairs reproductive parameters, even after withdrawal with long-term effects. Ethanol-naive offspring are also harmed, with effects associated with the dose of ethanol.

category: pharmacology and toxicology


title: Deep neural language modeling enables functional protein generation across families

abstract: Bypassing natures evolutionary trajectory, de novo protein generation--defined as creating artificial protein sequences from scratch--could enable breakthrough solutions for biomedical and environmental challenges. Viewing amino acid sequences as a language, we demonstrate that a deep learning-based language model can generate functional artificial protein sequences across families, akin to generating grammatically and semantically correct natural language sentences on diverse topics. Our protein language model is trained by simply learning to predict the next amino acid for over 280 million protein sequences from thousands of protein families, without biophysical or coevolutionary modeling. We experimentally evaluate model-generated artificial proteins on five distinct antibacterial lysozyme families. Artificial proteins show similar activities and catalytic efficiencies as representative natural lysozymes, including hen egg white lysozyme, while reaching as low as 44% identity to any known naturally-evolved protein. The X-ray crystal structure of an enzymatically active artificial protein recapitulates the conserved fold and positioning of active site residues found in natural proteins. We demonstrate our language models ability to be adapted to different protein families by accurately predicting the functionality of artificial chorismate mutase and malate dehydrogenase proteins. These results indicate that neural language models successfully perform de novo protein generation across protein families and may prove to be a tool to shortcut evolution.

category: synthetic biology


title: Using a Quadruplet Codon to Expand the Genetic Code of an Animal

abstract: Genetic code expansion in multicellular organisms is currently limited to the use of repurposed amber stop codons. Here we introduce a system for the use of quadruplet codons to direct incorporation of non-canonical amino acids in vivo in an animal, the nematode worm Caenorhabditis elegans. We develop hybrid pyrrolysyl tRNA variants to incorporate non-canonical amino acids in response to the quadruplet codon UAGA. We demonstrate the efficiency of the quadruplet decoding system by incorporating photocaged amino acids into two proteins widely used as genetic tools. We use photocaged lysine to express photocaged Cre recombinase for the optical control of gene expression and photocaged cysteine to express photo-activatable caspase for light inducible cell ablation. Our approach will facilitate the routine adoption of quadruplet decoding for genetic code expansion in eukaryotic cells and multicellular organisms.


title: Impact of acute visual experience on development of LGN receptive fields in the ferret

abstract:
Selectivity for direction of motion is a key feature of primary visual cortical neurons. Visual experience is required for direction selectivity in carnivore and primate visual cortex, but the circuit mechanisms of its formation remain incompletely understood. Here we examined how developing lateral geniculate nucleus (LGN) neurons may contribute to cortical direction selectivity. Using in vivo electrophysiology techniques, we examined LGN receptive field properties of visually naive female ferrets before and after exposure to 6 hours of motion stimuli in order to assess the effect of acute visual experience on LGN cell development. We found that acute experience with motion stimuli did not significantly affect the weak orientation or direction selectivity of LGN neurons. In addition, we found that neither latency nor sustainedness or transience of LGN neurons significantly changed with acute experience. These results suggest that the direction selectivity that emerges in cortex after acute experience is computed in cortex and cannot be explained by changes in LGN cells.

Significance StatementThe development of typical neural circuitry requires experience-independent and experience-dependent factors. In the visual cortex of carnivores and primates, selectivity for motion arises as a result of experience, but we do not understand whether the major brain area that sits between the retina and the visual cortex - the lateral geniculate nucleus of the thalamus - also participates. Here we found that lateral geniculate neurons do not exhibit changes as a result of several hours of visual experience with moving stimuli, at a time when visual cortical neurons undergo a rapid change. We conclude that lateral geniculate neurons do not participate in this plasticity, and that changes in cortex are likely responsible for the development of direction selectivity in carnivores and primates.


title: Radical pairs can explain magnetic field and lithium effects on the circadian clock

abstract: AO_SCPLOWBSTRACTC_SCPLOWDrosophilas circadian clock can be perturbed by magnetic fields, as well as by lithium administration. Cryptochromes are critical for the circadian clock. Further, the radical pairs in cryptochrome also can explain magnetoreception in animals. Based on a simple radical pair mechanism model of the animal magnetic compass, we show that both magnetic fields and lithium can influence the spin dynamics of the naturally occurring radical pairs and hence modulate the circadian clocks rhythms. Using a simple chemical oscillator model for the circadian clock, we show that the spin dynamics influence a rate in the chemical oscillator model, which translates into a change in the circadian period. Our model can reproduce the results of two independent experiments, magnetic fields and lithium effects on the circadian clock. Our model predicts that stronger magnetic fields would shorten the clocks period. We also predict that lithium influences the clock in an isotope-dependent manner. Furthermore, our model also predicts that magnetic fields and hyperfine interactions modulate oxidative stress. The findings of this work suggest that quantum nature and entanglement of radical pairs might play roles in the brain, as another piece of evidence in addition to recent results on xenon anesthesia and lithium effects on hyperactivity.


title: Chloride oscillation in pacemaker neurons regulates circadian rhythms through a chloride-sensing WNK kinase signaling cascade

abstract: Central pacemaker neurons regulate circadian rhythms and undergo diurnal variation in electrical activity in mammals and flies. In mammals, circadian variation in the intracellular chloride concentration of pacemaker neurons has been proposed to influence the response to GABAergic neurotransmission through GABAA receptor chloride channels. However, results have been contradictory, and a recent study demonstrated circadian variation in pacemaker neuron chloride without an effect on GABA response. Therefore, whether and how intracellular chloride regulates circadian rhythms remains controversial. Here, we demonstrate a signaling role for intracellular chloride in the Drosophila ventral lateral (LNv) pacemaker neurons. In control flies, intracellular chloride increases in LNv neurons over the course of the morning. Chloride transport through the sodium-potassium-2-chloride (NKCC) and potassium-chloride (KCC) cotransporters is a major determinant of intracellular chloride concentrations. Drosophila melanogaster with loss-of-function mutations in the NKCC encoded by Ncc69 have abnormally low intracellular chloride six hours after lights on, and a lengthened circadian period. Loss of kcc, which is expected to increase intracellular chloride, suppresses the long-period phenotype of Ncc69 mutant flies. Activation of a chloride-inhibited kinase cascade, consisting of the WNK (With No Lysine (K)) kinase and its downstream substrate, Fray, is necessary and sufficient to prolong period length. Fray activation of an inwardly rectifying potassium channel, Irk1, is also required for the long-period phenotype. These results indicate that the NKCC-dependent rise in intracellular chloride in Drosophila LNv pacemaker neurons restrains WNK-Fray signaling and overactivation of an inwardly rectifying potassium channel to maintain normal circadian period length.


title: Ipsilateral stimulus encoding in primary and secondary somatosensory cortex of awake mice

abstract:
Lateralization is a hallmark of somatosensory processing in the mammalian brain. However, in addition to their contralateral representation, unilateral tactile stimuli also modulate neuronal activity in somatosensory cortices of the ipsilateral hemisphere. The cellular organization and functional role of these ipsilateral stimulus responses in awake somatosensory cortices, especially regarding stimulus coding, are unknown. Here, we targeted silicon probe recordings to the vibrissa region of primary (S1) and secondary (S2) somatosensory cortex of awake head-fixed mice of either sex while delivering ipsilateral and contralateral whisker stimuli. Ipsilateral stimuli drove larger and more reliable responses in S2 than in S1, and activated a larger fraction of stimulus-responsive neurons. Ipsilateral stimulus-responsive neurons were rare in layer 4 of S1, but were located in equal proportion across all layers in S2. Linear classifier analyses further revealed that decoding of the ipsilateral stimulus was more accurate in S2 than S1, while S1 decoded contralateral stimuli most accurately. These results reveal substantial encoding of ipsilateral stimuli in S1 and especially S2, consistent with the hypothesis that higher cortical areas may integrate tactile inputs across larger portions of space, spanning both sides of the body.

S1ignificance StatementTactile information obtained by one side of the body is represented in the activity of neurons of the opposite brain hemisphere. However unilateral tactile stimulation also modulates neuronal activity in the other, or ipsilateral, brain hemisphere. This ipsilateral activity may play an important role in the representation and processing of tactile information, in particular when the sense of touch involves both sides of the body. Our work in the whisker system of awake mice reveals that neocortical ipsilateral activity, in particular that of deep layer excitatory neurons of secondary somatosensory cortex (S2), contains information about the presence and the velocity of unilateral tactile stimuli, which supports a key role for S2 in integrating tactile information across both body sides.


title: APOE2, E3 and E4 differentially modulate cellular homeostasis, cholesterol metabolism and inflammatory response in isogenic iPSC-derived astrocytes

abstract:
Apolipoprotein E (APOE) is the principal lipid carrier in the CNS and mainly expressed by astrocytes. The three different APOE alleles (E2, E3, and E4) impose differential risk to Alzheimers disease (AD); E2 is protective, E3 is defined as average risk, while E4 is the major genetic risk factor for sporadic AD. Despite recent advances, the fundamental role of different APOE alleles in brain homeostasis is still poorly understood. To uncover the functional role of APOE in human astrocytes, we differentiated human APOE-isogenic iPSCs (E4, E3, E2 and APOE-knockout (KO)) to functional astrocytes (hereafter "iAstrocytes"), with a resting, non-proliferating phenotype. Functional assays indicated that polymorphisms in APOE (APOE4>E3>E2=KO) reduced iAstrocyte metabolic and clearance functions including glutamate uptake and receptor-mediated uptake of {beta}-amyloid aggregates. We performed unlabelled mass spectrometry-based proteomic analysis of iAstrocytes at baseline and after activation with interleukin-1{beta} (IL-1{beta}) showing a reduction of cholesterol and lipid metabolic and biosynthetic pathways, and an increase of immunoregulatory pathways at baseline (E4>E3>E2). Cholesterol efflux and biosynthesis were reduced in E4 iAstrocytes, and subcellular localization of cholesterol in lysosomes was increased. In APOE-KO iAstrocytes, APOE-independent mechanisms showed to be proficient in mediating cholesterol biosynthesis and efflux. Proteomic analysis of IL-1{beta}-treated iAstrocytes showed an increase of cholesterol/lipid metabolism and biosynthesis as well as inflammatory pathways. Furthermore, cholesterol efflux, which was reduced in APOE4 iAstrocytes at baseline, was alleviated in activated E4 iAstrocytes. Inflammatory cytokine release was exacerbated upon IL-1{beta} treatment in E4 iAstrocytes (E4>E3>E2>KO), in line with the proteomic data. Taken together, we show that APOE plays a major role in several physiological and metabolic processes in human astrocytes with APOE4 pushing iAstrocytes to a disease-relevant phenotype, causing dysregulated cholesterol/lipid homeostasis, increased inflammatory signalling and reduced {beta}-amyloid uptake while APOE2 iAstrocytes show opposing effects.

Our study provides a new reference for AD-relevant proteomic and metabolic changes, mediated by the three main APOE isoforms in human astrocytes.


title: Dynamic surveillance of mosquitoes and their viromes in Wuhan during 2020.

abstract: Mosquitoes are medically important arthropod vectors and harbor a great variety of viruses. The population density, species and virome of mosquitoes varies according to geography and climate. To investigate the dynamic changes in the species composition and diversity of mosquitoes and their viromes in Wuhan, China, a total of 2,345 adult mosquitoes collected from different habitats including an urban residential area, two hospitals, a scenic area, and a pig farm in a rural region from April to October 2020 were subjected to morphological identification, RT-qPCR and metagenomic sequencing. The results indicated that the dominant presence of Culex mosquitoes was observed in both urban regions (90.32%, 1538/1703) and the pig farm (54.98%, 353/642). Viromes of Culex showed dynamic changes during the collection time. Several viruses, such as Culex flavivirus, Alphamesonivirus 1, Hubei mosquito virus 2 and Hubei mosquito virus 4, had seasonal changes and unimodal increases or declines. Other viruses, such as Wuhan mosquito virus 6, Hubei virga-like virus 2 and Zhejiang mosquito virus 3, were stable in all collected Culex and should be potential members of "core viromes". This study improves the understanding of the dynamic composition of mosquitoes and the viromes they carry and provides useful information for informing mosquito control and mosquito-borne disease prevention strategies.


title: LENG8 regulation of mRNA processing, is responsible for the control of mitochondrial activity

abstract: The processing of mRNA is essential for the maintenance of cellular and tissue homeostasis. However, the precise regulation of this process in mammalian cells, remains largely unknown. Here we have found that LENG8 represents the mammalian orthologue of the yeast mRNA processing factor Thp3 and Sac3. We go on to demonstrate that LENG8 binds to mRNAs, associates with components of mRNA processing machinery (the TREX complex) and contributes to mRNA nuclear export to the cytoplasm. Loss of LENG8, leads to aberrant accumulation of poly (A)+ RNA in the nucleus, in both Hela cells and murine fibroblasts. Furthermore, the precipitation of LENG8, is associated with an enrichment of both mRNAs and lncRNAs, and approximately half of these are also bound by the TREX component, THOC1. However, LENG8 preferentially binds mRNAs encoding for mitochondrial proteins and depletion of this processing factor, causes a dramatic breakdown in mitochondrial ultrastructure and a reduction in mitochondrial respiratory activity. Conditional deletion of Leng8 in mouse adipose tissues lead to a decreased body weight, and increased adipose thermogenesis. Our work has found an evolutionarily conserved mRNA processing factor that can control mitochondrial activity.


title: A rapid F0 CRISPR screen in zebrafish to identify regulators of neuronal development in the enteric nervous system

abstract: The enteric nervous system (ENS) provides the intrinsic innervation of the gastrointestinal (GI) tract with millions of neurons and diverse neuronal subtypes and glial cells. The ENS regulates essential gut functions such as motility, nutrient uptake, and immune response, but basic information about the genes that control ENS neuronal specification and differentiation remains largely unknown. Deficits in ENS neuron numbers and composition cause gut dysfunction with debilitating GI symptoms, and are associated with e.g. Hirschsprung disease, inflammatory gut diseases, autism spectrum disorder, and neurodegenerative diseases such as Parkinsons disease. The genetic basis of most of these ENS disorders remains unknown. Recent transcriptomic analyses have identified many candidate genes for regulating ENS neurogenesis. However, functional evaluation of these candidate genes significantly lags because experimental testing of their role in ENS neurogenesis is time-consuming and expensive. Here, we have developed a rapid, scalable F0 CRISPR genome editing screen in zebrafish to functionally determine which candidate genes control neuronal development in the ENS. Proof-of-concept experiments targeting the known ENS regulators sox10 and ret phenocopy stable mutants with high efficiency and precision showing that our approach is reliable to identify regulators ENS neurogenesis using F0 guide RNA-injected larvae (F0 crispants). We then evaluate the role of 10 transcription factor genes for regulating ENS neurogenesis and function. Pools of guide RNAs targeting 2-3 candidate genes are co-injected with Cas9 protein into one-cell stage phox2bb:GFP transgenic zebrafish embryos to directly assess qualitative change in ENS neuron numbers compared to controls in 6-day old F0 crispants. Target genes from crispant pools exhibiting reduced ENS neuronal numbers were then tested individually to identify the responsible gene(s). We identify five transcription factors that show a reduction in ENS neurons indicating an influence on enteric progenitor cell differentiation into ENS neurons. Adding a simple and efficient test to further assess crispant gut motility, we find that loss-of-function of two of the transcription factor genes reduced intestinal transit of fluorescently labeled food through the gut. In summary, our novel, multistep, yet straight-forward CRISPR screening approach in zebrafish enables testing the genetic basis of ENS developmental and disease gene functions that will facilitate high-throughput evaluation of the manifold candidate genes emerging from transcriptomic, genome-wide association or other ENS-omics studies. Such in vivo ENS crispant screens will contribute to a better understanding of ENS neuronal development regulation in vertebrates and what goes awry in ENS disorders.

category: developmental biology


title: TAF4b transcription networks regulating early oocyte differentiation

abstract: Establishment of a healthy ovarian reserve is contingent upon numerous regulatory pathways during embryogenesis. Previously, mice lacking TBP-associated factor 4b (Taf4b) were shown to exhibit a diminished ovarian reserve. However, potential oocyte-intrinsic functions of TAF4b have not been examined. Here we use a combination of gene expression profiling and chromatin mapping to characterize the TAF4b gene regulatory network in mouse oocytes. We find that Taf4b-deficient oocytes display inappropriate expression of meiotic, chromatin, and X-linked genes, and unexpectedly we found a connection with Turner Syndrome pathways. Using Cleavage Under Targets and Release Using Nuclease (CUT&RUN), we observed TAF4b enrichment at genes involved in meiosis and DNA repair, some of which are differentially expressed in Taf4b-deficient oocytes. Interestingly, TAF4b target genes were enriched for Sp/KLF family motifs rather than TATA-box, suggesting an alternate mode of promoter interaction. Together, our data connects several gene regulatory nodes that contribute to the ovarian reserve.


title: Multiple stage hydra effect in a stage-structured prey-predator model

abstract: In this work, we show by means of numerical bifurcation that two alternative stable states exhibit a hydra effect in a continuous-time stage-structured predator-prey model. We denote this behavior as a stage multiple hydra effect. This concomitant effect can have significant implications in population dynamics as well as in population management.

category: ecology


title: Body size mediates latitudinal population differences in response to Bd infection in two amphibian species.

abstract: Populations of the same species may differ in their sensitivity to pathogens but the factors behind this variation are poorly understood. Moreover, infections may cause sub-lethal fitness effects even in species resistant or tolerant to disease. The chytrid fungus Batrachochytrium dendrobatidis (Bd), is a generalist pathogen which has caused amphibian population declines worldwide. In many species, Bd infection causes the disease chytridiomycosis, often leading to high mortality. We investigated how geographical origin affects tolerance to Bd by exposing newly metamorphosed individuals of two North European amphibians (moor frog Rana arvalis, common toad Bufo bufo) from two latitudinal regions to two different BdGPL strains. Bd exposure strongly lowered survival in B. bufo, and in both species survival was lower in the northern region, this difference being much stronger in B. bufo. Northern individuals were smaller in both species, and the survival difference between the regions was size-mediated with smaller individuals being more sensitive to Bd. In both species, Bd exposure led to sub-lethal effects in terms of reduced growth suggesting that even individuals surviving the infection may have reduced fitness mediated by smaller body size. Bd strain affected size-dependent mortality differently in the two regions. We discuss the possible mechanisms how body size and geographical origin can contribute to the present results.


title: Phosphorus availability and arbuscular mycorrhizal fungi limit soil C cycling and influence plant responses to elevated CO2 conditions.

abstract: Enhanced soil organic matter (SOM) decomposition and organic phosphorus (P) cycling may help sustain plant productivity under elevated CO2 (eCO2) and P-limiting conditions. P-acquisition by arbuscular mycorrhizal (AM) fungi and their impacts on SOM decomposition may become even more relevant in these conditions. Yet, experimental evidence of the interactive effect of AM fungi and P availability influencing altered SOM cycling under eCO2 is scarce and the mechanisms of this control are poorly understood. Here, we performed a pot experiment manipulating P availability, AM fungal presence and atmospheric CO2 levels and assessed their impacts on soil C cycling and plant growth. Plants were grown in chambers with a continuous 13C-input that allowed differentiation between plant- and SOM-derived fractions of respired CO2 (R), dissolved organic C (DOC) and microbial biomass (MBC) as relevant C pools in the soil C cycle. We hypothesised that under low P availability, increases in SOM cycling may support sustained plant growth under eCO2 and that AM fungi would intensify this effect. We found the impacts of CO2 enrichment and P availability on soil C cycling were generally independent of each other with higher root biomass and slight increases in soil C cycling under eCO2 occurring regardless of the P treatment. Contrary to our hypotheses, soil C cycling was enhanced with P addition suggesting that low P conditions were limiting soil C cycling. eCO2 conditions increased the fraction of SOM-derived DOC pointing to increased SOM decomposition with eCO2. Finally, AM fungi increased microbial biomass under eCO2 conditions and low-P without enhanced soil C cycling, probably due to competitive interactions with free-living microorganisms over nutrients. Our findings in this plant-soil system suggest that, contrary to what has been reported for N-limited systems, the impacts of eCO2 and P availability on soil C cycling are independent of each other.


title: Ankyrin2 is required for neuronal morphogenesis and long-term memory and interacts genetically with HDAC4

abstract: Dysregulation of HDAC4 expression and/or subcellular distribution results in impaired neuronal morphogenesis and long-term memory in Drosophila melanogaster. A recent genetic screen for genes that interact in the same molecular pathway as HDAC4 identified the cytoskeletal adapter Ankyrin2 (Ank2). Here we sought to investigate the role of Ank2 in neuronal morphogenesis, learning and memory, and to examine the nature of interaction with HDAC4. We found that Ank2 is expressed widely throughout the Drosophila brain where it localizes predominantly to axon tracts. Pan-neuronal knockdown of Ank2 in the mushroom body, a region critical for memory formation, resulted in defects in axon morphogenesis, and similarly reduction of Ank2 in lobular plate tangential neurons of the optic lobe disrupted dendritic branching and arborization. Conditional knockdown of Ank2 in the mushroom body of adult Drosophila significantly impaired long-term courtship memory, and this requirement for Ank2 was isolated to gamma ({gamma}) neurons of the mushroom body. As overexpression of HDAC4 in {gamma} neurons also impairs the formation of long-term courtship memory, this suggests that any functional relationship between these proteins during LTM likely occurs in {gamma} neurons. We determined that the genetic interaction requires the presence of nuclear HDAC4 and is not dependent on a conserved putative ankyrin-binding motif present in HDAC4. In summary, we provide the first characterization of the expression pattern of Ank2 in the adult Drosophila brain and demonstrate that Ank2 is critical for morphogenesis of the mushroom body and for the molecular processes required in the adult brain for formation of long-term memories.


title: Merfin: improved variant filtering and polishing via k-mer validation

abstract: Read mapping and variant calling approaches have been widely used for accurate genotyping and improving consensus quality assembled from noisy long reads. Variant calling accuracy relies heavily on the read quality, the precision of the read mapping algorithm and variant caller, and the criteria adopted to filter the calls. However, it is impossible to define a single set of optimal parameters, as they vary depending on the quality of the read set, the variant caller of choice, and the quality of the unpolished assembly. To overcome this issue, we have devised a new tool called Merfin (k-mer based finishing tool), a k-mer based variant filtering algorithm for improved genotyping and polishing. Merfin evaluates the accuracy of a call based on expected k-mer multiplicity in the reads, independently of the quality of the read alignment and variant callers internal score. Moreover, we introduce novel assembly quality and completeness metrics that account for the expected genomic copy numbers. Merfin significantly increased the precision of a variant call and reduced frameshift errors when applied to PacBio HiFi, PacBio CLR, or Nanopore long read based assemblies. We demonstrate the utility while polishing the first complete human genome, a fully phased human genome, and non-human high-quality genomes.


title: Finding Overlapping Rmaps via Gaussian Mixture Model Clustering

abstract: Optical mapping is a method for creating high resolution restriction maps of an entire genome. Optical mapping has been largely automated, and first produces single molecule restriction maps, called Rmaps, which are assembled to generate genome wide optical maps. Since the location and orientation of each Rmap is unknown, the first problem in the analysis of this data is finding related Rmaps, i.e., pairs of Rmaps that share the same orientation and have significant overlap in their genomic location. Although heuristics for identifying related Rmaps exist, they all require quantization of the data which leads to a loss in the precision. In this paper, we propose a Gaussian mixture modelling clustering based method, which we refer to as OMO_SCPLOWCLUSTC_SCPLOW, that finds overlapping Rmaps without quantization. Using both simulated and real datasets, we show that OMO_SCPLOWCLUSTC_SCPLOW substantially improves the precision (from 48.3% to 73.3%) over the state-of-the art methods while also reducing CPU time and memory consumption. Further, we integrated OMO_SCPLOWCLUSTC_SCPLOW into the error correction methods (Elmeri and O_SCPLOWCC_SCPLOWOMO_SCPLOWETC_SCPLOW) to demonstrate the increase in the performance of these methods. When OMO_SCPLOWCLUSTC_SCPLOW was combined with O_SCPLOWCC_SCPLOWOMO_SCPLOWETC_SCPLOW to error correct Rmap data generated from human DNA, it was able to error correct close to 3x more Rmaps, and reduced the CPU time by more than 35x. Our software is written in C++ and is publicly available under GNU General Public License at https://github.com/kingufl/OMclust


title: Probing the toxic interactions between the reactive dye Drimaren Red and Human Serum Albumin

abstract: Azo dyes like Drimaren Red CL-5B (DR, CI Reactive Red 241) represent a class of compounds extensively used in the textile industry and are extremely dangerous to the environment and human health. Therefore, understanding the binding characteristics between such substances and biological macromolecules is essential from a toxic-kinetic perspective. The molecular interaction between DR and Human Serum Albumin (HSA) was investigated through spectroscopic techniques and molecular docking approaches. The results indicate that DR quenches HSA fluorescence following a static mechanism (corroborated by UV-Vis studies) with a moderate interaction (Ka~105 M-1), guided by electrostatic interactions ({Delta}S{degrees}> 0 and {Delta}H{degrees}< 0). DR is 5.52 nm distant from fluorophore residue Trp-214 (according to FRET investigations), and the interaction is mainly related to Tyr residues (as revealed by synchronous fluorescence). The Ellman assay identified a decrease in the content of HSA free thiol. The results of the RLS demonstrate that there are HSA alterations, suggesting damage to the confirmation of the protein. Molecular docking suggests the binding site of DR was located in subdomain IIB HSA, corroborating the experimental properties. Finally, the results suggest a high potential for DR toxicity triggered by contact with key proteins, which affects the biomolecule functionalities.


title: Spatio-temporal dynamics of stress-induced network reconfigurations reflect negative affectivity

abstract:
BackgroundChronic stress is an important risk factor in the etiology of mood and anxiety disorders, but exact pathomechanisms remain to be understood. Mapping individual differences of acute stress-induced neurophysiological changes, especially on the level of neural activation and functional connectivity (FC), could provide important insights in how variation in the individual stress response is linked to disease risk.

MethodsUsing an established psycho-social stress task flanked by two resting-state scans, we measured subjective, physiological, and brain responses to acute stress and recovery in 217 unmedicated participants with and without mood and anxiety disorders. To estimate block-wise changes in stress-induced brain activation and FC, we used hierarchical mixed-effects models based on denoised timeseries within a predefined stress network. We predicted inter- and intra-individual differences in stress phases (anticipation vs. acute stress vs. recovery) and transdiagnostic dimensions of stress reactivity using elastic net and support vector machines.

ResultsWe identified four subnetworks showing distinct changes in FC over time. Subnetwork trajectories predicted the stress phase (accuracy: 71%, pperm<.001) and increases in pulse rate (R2=.10, pperm<.001). Critically, individual spatio-temporal trajectories of changes across networks also predicted negative affectivity ({Delta}R2=.08, pperm=.009), but not the presence or absence of a mood and anxiety disorder.

ConclusionsSpatio-temporal dynamics of brain network reconfiguration induced by stress reflect individual differences in the psychopathology dimension negative affectivity. These results support the idea that vulnerability for mood and anxiety disorders can be conceptualized best at the level of network dynamics, which may pave the way for improved prediction of individual risk.


title: 3D molecular phenotyping of cleared human brain tissues with light-sheet fluorescence microscopy

abstract: The combination of optical tissue transparency with immunofluorescence allows the molecular characterization of biological tissues in 3D. However, adult human organs are particularly challenging to become transparent because of the autofluorescence contributions of aged tissues. To meet this challenge, we optimized SHORT (SWITCH - H2O2 - antigen Retrieval - TDE), a procedure based on standard histological treatments in combination with a refined clearing procedure to clear and label portions of the human brain. 3D histological characterization with multiple molecules is performed on cleared samples with a combination of multi-colors and multi-rounds labeling. By performing fast 3D imaging of the samples with a custom-made inverted light-sheet fluorescence microscope (LSFM), we reveal fine details of intact human brain slabs at subcellular resolution. Overall, we proposed a scalable and versatile technology that in combination with LSFM allows mapping the cellular and molecular architecture of the human brain, paving the way to reconstruct the entire organ.


title: Regional cytoarchitecture of the adult and developing mouse enteric nervous system

abstract: The enteric nervous system (ENS) populates the gastrointestinal (GI) tract and controls GI function. In contrast to the central nervous system, macrostructure of the ENS has been largely overlooked. Here, we visually and computationally demonstrate that the ENS is organized in circumferential stripes that regionally differ in development and neuronal composition. This characterization provides a blueprint for future understanding of region-specific GI function and identifying ENS structural correlates of GI disorders.


title: Cortical spheroids display oscillatory network dynamics

abstract: Three-dimensional brain cultures can facilitate the study of central nervous system function and disease, and one of the most important components that they present is neuronal activity on a network level. Here we demonstrate network activity in rodent cortical spheroids while maintaining the networks intact in their 3D state. Networks developed by nine days in culture and became more complex over time. To measure network activity, we imaged neurons in rat and mouse spheroids labelled with a calcium indicator dye, and in mouse spheroids expressing GCaMP. Network activity was evident when we electrically stimulated spheroids, was abolished with glutamatergic blockade, and was altered by GABAergic blockade or partial glutamatergic blockade. We quantified correlations and distances between somas with micron-scale spatial resolution. Spheroids seeded at as few as 4,000 cells gave rise to emergent network events, including oscillations. These results are the first demonstration that self-assembled rat and mouse spheroids exhibit network activity consistent with in vivo network events. These results open the door to experiments on neuronal networks that require fewer animals and enable high throughput experiments on network-perturbing alterations in neurons and glia.


title: Interferon-Induced Transmembrane Proteins Inhibit Infection by the Kaposis Sarcoma-Associated Herpesvirus and the Related Rhesus Monkey Rhadinovirus in a Cell Type-Specific Manner.

abstract:
The interferon-induced transmembrane proteins (IFITMs) are broad-spectrum antiviral proteins that inhibit the entry of enveloped viruses. We analyzed the effect of IFITMs on the gamma2-herpesviruses Kaposis sarcoma-associated herpesvirus (KSHV) and the closely related rhesus monkey rhadinovirus (RRV). We used CRISPR/Cas9-mediated gene knockout to generate A549, human foreskin fibroblast (HFF) and human umbilical vein endothelial cells (HUVEC) with combined IFITM1/2/3 knockout and identified IFITMs as cell type-dependent inhibitors of KSHV and RRV infection in A549 and HFF but not HUVEC. IFITM overexpression revealed IFITM1 as the relevant IFITM that inhibits KSHV and RRV infection. Fluorescent KSHV particles did not pronouncedly colocalize with IFITM-positive compartments. However, we found that KSHV and RRV glycoprotein-mediated cell-cell fusion is enhanced upon IFITM1/2/3 knockout. Taken together, we identified IFITM1 as a cell type-dependent restriction factor of KSHV and RRV that acts at the level of membrane fusion. Strikingly, we observed that the endotheliotropic KSHV circumvents IFITM-mediated restriction in HUVEC despite high IFITM expression, while influenza A virus (IAV) glycoprotein-driven entry into HUVEC is potently restricted by IFITMs even in the absence of interferon.

IMPORTANCEIFITM proteins are the first line of defense against infection by many pathogens, which may also have therapeutic importance, as they, among other effectors, mediate the antiviral effect of interferons. Neither their function against herpesviruses nor their mechanism of action are well understood. We report here that in some cells, but not in, for example, primary umbilical vein endothelial cells, IFITM1 restricts KSHV and RRV, and that, mechanistically, this is likely effected by reducing the fusogenicity of the cell membrane. Further, we demonstrate potent inhibition of IAV glycoprotein-driven infection of cells of extrapulmonary origin by high constitutive IFITM expression.

category: microbiology


title: Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3

abstract: Type I CRISPR-Cas3 uses an RNA-guided multi Cas-protein complex, Cascade, which detects and degrades foreign nucleic acids via the helicase-nuclease Cas3 protein. Despite many studies using cryoEM and smFRET, the precise mechanism of Cas3-mediated cleavage and degradation of target DNA remains elusive. Here we reconstitute the CRISPR-Cas3 system in vitro to show how the Escherichia coli Cas3 (EcoCas3) with EcoCascade exhibits collateral non-specific ssDNA cleavage and target specific DNA degradation. Partial binding of EcoCascade to target DNA with tolerated mismatches within the spacer sequence, but not the PAM, elicits collateral ssDNA cleavage activity of recruited EcoCas3. Conversely, stable binding with complete R-loop formation drives EcoCas3 to nick the non-target strand (NTS) in the bound DNA. Helicase-dependent unwinding then combines with trans ssDNA cleavage of the target strand and repetitive cis cleavage of the NTS to degrade the target dsDNA substrate. High-speed atomic force microscopy demonstrates that EcoCas3 bound to EcoCascade repeatedly reels and releases the target DNA, followed by target fragmentation. Together, these results provide a revised model for collateral ssDNA cleavage and target dsDNA degradation by CRISPR-Cas3, furthering understanding of type I CRISPR priming and interference and informing future genome editing tools.


title: A comparative in vitro and in vivo study of osteogenicity by using two biomaterials and two human mesenchymal stem cell subtypes

abstract: Bone repair induced by stem cells and biomaterials may represent an alternative to autologous bone grafting. Here, we compared the efficiency of two biomaterials - biphasic calcium phosphate (BCP) and bioactive glass (BG) - when loaded with either adult bone marrow mesenchymal stem cells (BM-MSCs) or newborn nasal ecto-mesenchymal stem cells (NE-MSCs), the latter being collected for further repair of lip cleft-associated bone loss. Both cell types display the typical stem cell surface markers CD73+/CD90+/CD105+/nestin, and exhibit the MSC-associated osteogenic, chondrogenic and adipogenic multipotency. NE-MSCs produce less collagen and alkaline phosphatase than BM-MSCs. At the transcript level, NE-MSCs express more abundantly three genes coding for bone sialoprotein, osteocalcin and osteopontin, while BM-MSCs produce extra copies of RUNX2. BM-MSCs and NE-MSCs adhere and survive on BCP and BG. In vivo experiments reveal that bone formation is only observed with BM-MSCs transplanted on BCP biomaterial.


title: SprayNPray: user-friendly taxonomic profiling of genome and metagenome contigs

abstract: Shotgun sequencing of cultured microbial isolates/individual eukaryotes (whole-genome sequencing) and microbial communities (metagenomics) has become commonplace in biology. Very often, sequenced samples encompass organisms spanning multiple domains of life, necessitating increasingly elaborate software for accurate taxonomic classification of assembled sequences. While many software tools for taxonomic classification exist, SprayNPray offers a quick and user-friendly, semiautomated approach, allowing users to separate contigs by taxonomy (and other metrics) of interest. Easy installation, usage, and intuitive output, which is amenable to visual inspection and/or further computational parsing, will reduce barriers for biologists beginning to analyze genomes and metagenomes. This approach can be used for broad-level overviews, preliminary analyses, or as a supplement to other taxonomic classification or binning software. SprayNPray profiles contigs using multiple metrics, including closest homologs from a user-specified reference database, gene density, read coverage, GC content, tetranucleotide frequency, and codon-usage bias. The output from this software is designed to allow users to spot-check metagenome-assembled genomes, identify, and remove contigs from putative contaminants in isolate assemblies, identify bacteria in eukaryotic assemblies (and vice-versa), and identify possible horizontal gene transfer events.


title: A Sequence-based Antibody Paratope Prediction Model Through Combing Local-Global Information and Partner Features

abstract: Antibodies are proteins which play a vital role in the immune system by recognizing and neutralizing antigens. The region on the antibody binding to an antigen, known as paratope, mediates antibody-antigen interaction with high affinity and specificity. And the accurate prediction of those regions from antibody sequence contributes to the design of therapeutic antibodies and remains challenging. However, the experimental methods are time-consuming and expensive. In this article, we propose a sequence-based method for antibody paratope prediction by combing local and global information of antibody sequence and partner features from partner antigen sequence. Convolution Neural Networks(CNNs) and a sliding window approach on antibody sequence are used to extract local information. Attention-based Bidirectional Long Short-Term Memory (Att-BLSTM) on antibody sequence are used to extract global information. Also, the partner antigen is vital for paratope prediction, and we employ Att-BLSTM on the partner antigen sequence as well. The outputs of CNNs and Att-BLSTM networks are combined to predict antibody paratope by fully-connected networks. The experiments show that our proposed method achieves superior performance over the state-of-the-art sequenced-based antibody paratope prediction methods on benchmark datasets.


title: Machine-learning-based predictions of caloricrestriction associations across ageing-related genes

abstract: Caloric restriction (CR) is the most studied pro-longevity intervention; however, a complete understanding of its underlying mechanisms remains elusive, and new research directions may emerge from the identification of novel CR-related genes and CR-related genetic features. This work used a Machine Learning (ML) approach to classify ageing-related genes as CR-related or NotCR-related using 9 different types of predictive features: PathDIP pathways, two types of features based on KEGG pathways, two types of Protein-Protein Interactions (PPI) features, Gene Ontology (GO) terms, Genotype-Tissue Expression (GTEx) expression features, Gene-Friends co-expression features and protein sequence descriptors. Our findings suggested that features biased towards curated knowledge (i.e. GO terms and biological pathways), had the greatest predictive power, while unbiased features (mainly gene expression and co-expression data) have the least predictive power. Moreover, a combination of all the feature types diminished the predictive power compared to predictions based on curated knowledge. Feature importance analysis on the two most predictive classifiers mostly corroborated existing knowledge and supported recent findings linking CR to the Nuclear Factor Erythroid 2-Related Factor 2 (NRF2) signalling pathway and G protein-coupled receptors (GPCR). We then used the two strongest combinations of feature type and ML algorithm to predict CR-relatedness among ageing-related genes currently lacking CR-related annotations in the data, resulting in a set of promising candidate CR-related genes (GOT2, GOT1, TSC1, CTH, GCLM, IRS2 and SESN2) whose predicted CR-relatedness remain to be validated in future wet-lab experiments.


title: Modelling, characterization of data-dependent and process-dependent errors in DNA data storage

abstract:
MotivationUsing DNA as the medium to store information has recently been recognized as a promising solution for long-term data storage. While several system prototypes have been demonstrated, the error characteristics in DNA data storage are discussed with limited content. Due to the data and process variations from experiment to experiment, the error variation and its effect on data recovery remain to be uncovered. To close the gap, we systematically investigate the storage channel, i.e., error characteristics in the storage process.

ResultsWe first propose a new concept named sequence corruption to unify the error characteristics into the sequence level, easing the channel analysis. Then we derived the formulations of the data imperfection at the decoder including both sequence loss and sequence corruption, revealing the decoding demand and monitoring the data recovery. Furthermore, we extensively explored several data-dependent unevenness observed in the base error patterns and studied a few potential factors and their impacts on the data imperfection at the decoder both theoretically and experimentally. The results presented here introduce a more comprehensive channel model and offer a new angle towards the data recovery issue in DNA data storage by further elucidating the error characteristics of the storage process.

Contactpoh.chuehloo@nus.edu.sg


title: Assessing the polymer coil-globule state from the very first spectral modes

abstract: The determination of the coil-globule transition of a polymer is generally based on the reconstruction of scaling laws, implying the need for samples from a rather wide range of different polymer lengths N. The spectral point of view developed in this work allows for a very parsimonious description of all the aspects of the finite-size coil-globule transition on the basis of the first two Rouse (cosine) modes only, shedding new light on polymer theory. Capturing the relevant configuration path features, the proposed approach enables to determine the state of a polymer without the need of any information about the polymer length or interaction strength. Importantly, we propose an experimental implementation of our analysis that can be easily performed with modern fluorescent imaging techniques, and would allow differentiation of coil or globule conformations by simply recording the positions of three discernible loci on the polymer.


title: Selenium in wheat from farming to food

abstract:
Selenium (Se) plays an important role in human health. Approximately 80% of the worlds population does not consume enough Se which recommended by WHO (World Health Organization). Wheat is an important staple food and Se source for most people in the world. This article summarizes literatures about Se from 1936 to 2020 to investigate Se in wheat farming soil, wheat, and its derived foods. Se fortification and the recommended Se level in wheat were also discussed. Results showed that Se contents in wheat farming soil, grain, and its derived foods around the world were 3.8-552 (mean, 220.99), 0-8,270 (mean, 347.30), and 15-2,372 (mean, 211.86) g{middle dot}kg-1, respectively. Adopting suitable agronomic measures could effectively realize Se fortification in wheat. The contents in grain, flour, and its derived foods could be improved from 93.94 to 1,181.92, 73.06 to 1,007.75, and 86.90 to 587.61 g{middle dot}kg-1 in average after leaf Se fertilizer application in the field. There was a significant positive correlation between Se content in farming soil and grain, and it was extremely the same between foliar Se fertilizer concentration rate and grain Se increased rate. The recommended Se fortification level in cultivation of wheat in China, India, and Spain was 18.53-23.96, 2.65-3.37, and 3.93-9.88 g{middle dot}hm-2 respectively. Milling processing and food type could greatly affect Se content of wheat derived food and should be considered seriously to meet peoples Se requirement by wheat.

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category: plant biology