title: Novel AT1R-Allosteric Ligands Mask the Preeclampsia Auto-antibody Epitope and Decrease Angiotensin-induced Vasoconstriction

abstract: Maternal blood pressure regulation by the hormone angiotensin II (AngII) sustains fetal growth through feto-placental circulation. AngII binding to orthosteric pocket in the angiotensin type 1 receptor (AT1R) induces G protein and {beta}-arrestin signaling. AT1R blocking drugs and {beta}-arrestin biased ligands also bind to the orthosteric pocket but evoke different inactive and active states1-6. AT1R-directed auto-antibodies observed in preeclampsia bound outside the orthosteric pocket to extracellular loop-2 (ECL2) of AT1R7-9. How auto-antibodies modulate AT1R activity causing preeclampsia pathogenesis is unknown. Here we report a druggable cryptic allosteric pocket encompassing the preeclampsia epitope on ECL2. Using structure based high-throughput small molecule screening we discovered 18 ligands specific for AT1Rs allosteric pocket. After procuring these ligands we validated inhibition of preeclampsia epitope-specific antibody binding. We characterize their inhibitory effect on antibody and AngII-signaling in cells and vasoconstriction ex vivo. These novel AT1R allosteric ligands, thus act as dual action negative modulators of auto-antibody action and vasoconstriction. Our study demonstrates that positive allosteric modulator action of auto-antibody causes a disease linked to AT1R. We anticipate our findings to kindle structure-based discovery of AT1R allosteric ligands for intervention in maladies such as preeclampsia7-10, rejection of organ transplants11, vasodilatory shock12, 13 and metabolic syndrome14.

category: pharmacology and toxicology


title: Identification of residues in Lassa virus glycoprotein 1 involved in receptor switch

abstract:
Lassa virus (LASV) is an enveloped, negative-sense RNA virus that causes Lassa hemorrhagic fever, for which there are limited treatment options. Successful LASV entry requires the viral glycoprotein 1 (GP1) to undergo a receptor switch from its primary receptor alpha-dystroglycan (-DG) to its endosomal receptor lysosome-associated membrane protein 1 (LAMP1). A conserved histidine triad in LASV GP1 has been reported to be responsible for receptor switch. To test the hypothesis that other non-conserved residues also contribute to receptor switch, we constructed a series of GP1 mutant proteins and tested them for binding to LAMP1. Four residues, L84, K88, L107, and H170, were identified as critical for receptor switch. Substituting any of the four residues with the corresponding lymphocytic choriomeningitis virus residue (L84N, K88E, L10F, and H170S) reduced the binding affinity of GP1LASV for LAMP1. Moreover, all the mutations caused decreases in GPC-mediated membrane fusion at both pH 4.5 and 5.2. The infectivity of pseudotyped viruses bearing either GPCL84N or GPCK88E decreased sharply in multiple cell types, whereas L107F and H170S had only mild effects on infectivity. Notably, in LAMP1 knockout cells, all four mutants showed reduced pseudovirus infectivity. Using biolayer light interferometry assay, we found that all four mutants had decreased binding affinity to LAMP1, in the order L84N > L107F > K88E > H170S.

IMPORTANCELassa virus requires pH-dependent receptor switch to infect host cells; however, the underlying molecular mechanisms of this process are not well known. Here, we identify four residues, L84, K88, L107, and H170 that contribute to the interaction with the second receptor lysosome-associated membrane protein 1 (LAMP1). Mutant any of the four residues would impair the binding affinity to LAMP1, decrease the glycoprotein mediated membrane fusion, and reduce the pseudovirus infectivity.

category: microbiology


title: INfrastructure for a PHAge REference Database: Identification of large-scale biases in the current collection of phage genomes

abstract:
BackgroundWith advances in sequencing technology and decreasing costs, the number of bacteriophage genomes that have been sequenced has increased markedly in the last decade.

Materials and MethodsWe developed an automated retrieval and analysis system for bacteriophage genomes, INPHARED (https://github.com/RyanCook94/inphared), that provides data in a consistent format.

ResultsAs of January 2021, 14,244 complete phage genomes have been sequenced. The data set is dominated by phages that infect a small number of bacterial genera, with 75% of phages isolated only on 30 bacterial genera. There is further bias with significantly more lytic phage genomes than temperate within the database, resulting in ~54% of temperate phage genomes originating from just three host genera. Within phage genomes, putative antibiotic resistance genes were found in higher frequencies in temperate phages than lytic phages.

ConclusionWe provide a mechanism to reproducibly extract complete phage genomes and highlight some of the biases within this data, that underpins our current understanding of phage genomes.


title: Evaluation of Antibiotic Tolerance in Pseudomonas aeruginosa for Aminoglycosides and its Prediction of Resistance Development Through In-silico Transcriptomic Analysis

abstract: Pseudomonas aeruginosa causes chronic infections like cystic fibrosis, endocarditis, bacteremia and sepsis, which are life-threatening and difficult to treat. The lack of antibiotic response in P. aeruginosa is due to adaptive resistance mechanism, which prevents the entry of antibiotics into cytosol of the cell to achieve tolerance. Among different groups of antibiotics, aminoglycosides are used as a parental antibiotic for treatment of P. aeruginosa. This study aims to determine the kinetics of antibiotic tolerance and gene expression changes in P. aeruginosa exposed to amikacin, gentamicin, and tobramycin. These antibiotics were exposed to P. aeruginosa at their MICs and the experimental setup was monitored till 72 hours, followed by the measurement of optical density in the interval of every 12 hours. The growth of P. aeruginosa in MICs of antibiotics represents the kinetics of antibiotic tolerance in amikacin, gentamicin, and tobramycin. Transcriptomic profile of antibiotic exposed P. aeruginosa PA14 was taken from Gene Expression Omnibus (GEO), NCBI as microarray datasets. The gene expressions of two datasets were compared by test versus control. Tobramycin exposed P. aeruginosa failed to develop tolerance in MICs 0.5{micro}g/mL, 1{micro}g/mL and 1.5{micro}g/mL. Whereas amikacin and gentamicin treated P. aeruginosa developed tolerance in MICs. This depicts the superior in vitro response of tobramycin over the gentamicin and amikacin. Further, in silico transcriptomic analysis of tobramycin treated P. aeruginosa resulted in low expression of 16s rRNA Methyltransferase E, B & L, alginate biosynthesis genes and several proteins of Type 2 Secretory System (T2SS) and Type 3 Secretory System (T3SS). The Differentially Expressed Genes (DEGs) of alginate biosynthesis, and RNA Methyltransferases suggests increased antibiotic response and low probability of developing resistance. The use of tobramycin as a parental antibiotic with its synergistic combination might combat P. aeruginosa with increased response.


title: Evaluation of Antibiotic Tolerance in Pseudomonas aeruginosa for Aminoglycosides and its Predicted Gene Regulations Through In-silico Transcriptomic Analysis


title: Phylogenomic study of Staphylococcus aureus and Staphylococcus haemolyticus clinical isolates from Egypt

abstract:
Antibiotic resistant Staphylococcus infections are a global concern, with increasing cases of resistant Staphylococcus aureus and Staphylococcus haemolyticus found circulating in the Middle East. While extensive surveys have described the prevalence of resistant infections in Europe, Asia, and North America, the population structure of resistant staphylococcal Middle Eastern clinical isolates is poorly characterized. We performed whole genome sequencing of 56 S. aureus and 10 S. haemolyticus isolates from Alexandria Main University Hospital. Supplemented with additional publicly available genomes from the region (34 S. aureus and 6 S. haemolyticus), we present the largest genomic study of staphylococcal Middle Eastern isolates. These genomes include 20 S. aureus multilocus sequence typing (MLST) types and 9 S. haemolyticus MLSTs, including 3 and 1 new MLSTs, respectively. Phylogenomic analyses of each species core genome largely mirrored MLSTs, irrespective of geographical origin. The hospital-acquired spa t037/SCCmec III/MLST CC8 clone represented the largest clade, comprising 22% of S. aureus isolates. Similar to other regional genome surveys of S. aureus, the Middle Eastern isolates have an open pangenome, a strong indicator of gene exchange of virulence factors and antibiotic resistance genes with other reservoirs. We recommend stricter implementation of antibiotic stewardship and infection control plans in the region.

Impact StatementStaphylococci are under-studied despite their prevalence within the Middle East. Methicillin-resistant Staphylococcus aureus (MRSA) is endemic to hospitals in this region, as are other antibiotic-resistant strains of S. aureus and S. haemolyticus. To provide insight into the strains currently in circulation within Egypt, we performed whole genome sequencing of 56 S. aureus and 10 S. haemolyticus isolates from Alexandria Main University Hospital (AMUH). Through analysis of these genomes, as well as other genomes of isolates from the Middle East, we were able to produce a more complete picture of the current diversity than traditional molecular typing strategies. Furthermore, the S. haemolyticus genome analyses provide the first insight into strains found in Egypt. Our analysis of resistance and virulence mechanisms carried by these strains provides invaluable insight into future plans of antibiotic stewardship and infection control within the region.

Data SummaryRaw sequencing reads and assembled genomes can be found at BioProject Accession number PRJNA648411 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA648411).


title: Oligomerization-driven MLKL ubiquitylation antagonises necroptosis

abstract: Mixed lineage kinase domain-like (MLKL) is the executioner in the caspase-independent form of programmed cell death called necroptosis. Receptor Interacting serine/threonine Protein Kinase 3 (RIPK3) phosphorylates MLKL, triggering MLKL oligomerization, membrane translocation and membrane disruption. MLKL also undergoes ubiquitylation during necroptosis, yet neither the mechanism nor significance of this event have been demonstrated. Here we show that necroptosis-specific, multi-mono-ubiquitylation of MLKL occurs following its activation and oligomerization. Ubiquitylated MLKL accumulates in a digitonin insoluble cell fraction comprising plasma/organellar membranes and protein aggregates. This ubiquitylated form is diminished by a plasma membrane located deubiquitylating enzyme. MLKL is ubiquitylated on at least 4 separate lysine residues once oligomerized, and this correlates with proteasome- and lysosome-dependent turnover. Using a MLKL-DUB fusion strategy, we show that constitutive removal of ubiquitin from MLKL licences MLKL auto-activity independent of necroptosis signalling in mouse and human cells. Therefore, besides its role in the kinetic regulation of MLKL-induced death following an exogenous necroptotic stimulus, ubiquitylation also contributes to the restraint of basal levels of activated MLKL to avoid errant cell death.

category: molecular biology


title: Transcriptomics reveals the mycoparasitic strategy of the mushroom Entoloma abortivum on species of the mushroom Armillaria

abstract:
During mycoparasitism, a fungus--the host--is parasitized by another fungus--the mycoparasite. The genetic underpinnings of these relationships have been best characterized in ascomycete fungi. However, within basidiomycete fungi, there are rare instances of mushroom-forming species parasitizing the reproductive structures, or sporocarps, of other mushroom-forming species, which have been rarely investigated on a genetic level. One of the most enigmatic of these occurs between Entoloma abortivum and species of Armillaria, where hyphae of E. abortivum are hypothesized to disrupt the development of Armillaria sporocarps, resulting in the formation of carpophoroids. However, it remains unknown whether carpophoroids are the direct result of a mycoparasitic relationship. To address the nature of this unique interaction, we analyzed gene expression of field-collected Armillaria and E. abortivum sporocarps and carpophoroids. Transcripts in the carpophoroids are primarily from E. abortivum, supporting the hypothesis that this species is parasitizing Armillaria. Most notably, we identified differentially upregulated E. abortivum {beta}-trefoil-type lectins in the carpophoroid, which we hypothesize bind to Armillaria cell wall galactomannoproteins, thereby mediating recognition between the mycoparasite and the host. The most differentially upregulated E. abortivum transcripts in the carpophoroid code for oxalate decarboxylases--enzymes that degrade oxalic acid. Oxalic acid is a virulence factor in many plant pathogens, including Armillaria species, however, E. abortivum has evolved a sophisticated strategy to overcome this defense mechanism. The number of gene models and genes that code for carbohydrate-active enzymes in the E. abortivum transcriptome were reduced compared to other closely related species, perhaps as a result of the specialized nature of this interaction.

IMPORTANCEBy studying fungi that parasitize other fungi, we can understand the basic biology of these unique interactions. Studies focused on the genetic mechanisms regulating mycoparasitism between host and parasite have thus far concentrated on a single fungal lineage within the Ascomycota. The work presented here expands our understanding of mycoparasitic relationships to the Basidiomycota and represents the first transcriptomic study to our knowledge that examines fungal-fungal relationships in their natural setting. The results presented here suggest that even distantly related mycoparasites utilize similar mechanisms to parasitize their host. Given that species of the mushroom-forming pathogen Armillaria cause plant root-rot diseases in many agroecosystems, an enhanced understanding of this interaction may contribute to better control of these diseases through biocontrol applications.

category: ecology


title: Symbiont-conferred immunity interacts with the effects of parasitoid genotype and intraguild predation to shape pea aphid immunity in a clone-specific fashion

abstract: Host-parasite interactions represent complex co-evolving systems in which genetic variation within a species can significantly affect selective pressure on traits in the other (for example via inter-species indirect genetic effects). While often viewed as a two-species interaction between host and parasite species, some systems are more complex due to the involvement of symbionts in the host that influence its immunity, enemies of the host, and the parasite through intraguild predation. However, it remains unclear what the joint effects of intraguild predation, defensive endosymbiosis, within-species genetic variation and indirect genetic effects on host immunity are. We have addressed this question in an important agricultural pest system, the pea aphid Acyrthosiphon pisum, which shows significant intraspecific variability in immunity to the parasitoid wasp Aphidius ervi due to immunity conferring endosymbiotic bacteria. In a complex experiment involving a quantitative genetic design of the parasitoid, two ecologically different aphid lineages and the aphid lion Chrysoperla carnea as an intraguild predator, we demonstrate that aphid immunity is affected by intraspecific genetic variation in the parasitoid and the aphid, as well as by associated differences in the defensive endosymbiont communities. Using 16s rRNA sequencing, we identified secondary symbionts that differed between the lineages. We further show that aphid lineages differ in their altruistic behaviour once parasitised whereby infested aphids move away from the clonal colony to facilitate predation. The outcome of these complex between-species interactions not only shape important host-parasite systems but have also implications for understanding the evolution of multitrophic interactions, and aphid biocontrol.


title: Modularity and connectivity of nest structure scale with colony size

abstract: Large body sizes have evolved structures to facilitate resource transport. Like unitary organisms, social insect colonies must transport information and resources. Colonies with more individuals may experience transport challenges similar to large-bodied organisms. In ant colonies, transport occurs in the nest, which may consist of structures that facilitate movement. We examine three attributes of nests that might have evolved to mitigate transport challenges related to colony size: (1) subdivision - nests of species with large colonies are more subdivided to reduce crowd viscosity; (2) branching - nest tunnels increase branching in species with large colonies to reduce travel distances; and (3) short-cuts - nests of species with large colonies have cross-linking tunnels to connect distant parts of the nest and create alternative routes. We test these hypotheses by comparing nest structures of species with different colony sizes in phylogenetically controlled meta-analyses. Our findings support the hypothesis that nest subdivision and branching evolved to mitigate transport challenges related to colony size. Nests of species with large colonies contain more chambers and branching tunnels. The similarity in how ant nests and bodies of unitary organisms have evolved in response to increasing size suggests common solutions across taxa and levels of biological organization.

category: evolutionary biology


abstract:
The analysis of scaling relationships, allometric scaling, has a long history of importance for modelling and predicting biological phenomena. Individual organisms are not truly independent, and as a result phylogenetic corrections are necessary to increase the accuracy of scaling relationships. The relationships between body mass and gestation length have not been previously reported at the Family level, as it was previously thought species had insufficient time to diverge evolutionarily leaving phylogenetic corrections unnecessary. Using a Carnivora supertree, we perform a phylogenetically generalised least squares (PGLS) analysis using life history information largely from the Pantheria dataset. Our results suggest that allometric relationships are maintained in four families: Canidae, Felidae, Herpestidae and Otariidae. Conversely, several evolutionary mechanisms may contribute to the lack of a significant scaling parameter in other families, such as diverse reproductive strategies or positive selection for genes affecting adiposity. In addition, low sample sizes or the inclusion of paternal body masses could alter the presence of significant scaling. Our results suggests that PGLS analyses are informative at the family level, and the absence of scaling can provide insight to understanding of the evolutionary mechanisms that work on the family taxonomic level or below.

CCS CONCEPTS* Applied computing [->] Molecular evolution; * Computing methodologies [->] Modeling methodologies.

ACM Reference FormatStefanie Navaratnam and Julie Baker Phillips. 2021. Breaking the Scale: Allometric scaling analysis in Carnivoran families. In BCB: ACM Conference on Bioinformatics, Computational Biology, and Health Informatics, August 01-04, 2021, Virtual due to COVID-19. ACM, New York, NY, USA, 7 pages. https://doi.org/10.1145/1122445.1122456


title: In heart failure reactivation of RNA-binding proteins drives the transcriptome into a fetal state

abstract: Transcriptome-wide expression changes occur during heart failure, including reactivation of fetal-specific isoforms. However, the underlying molecular mechanisms and the extent to which a fetal gene program switch occurs remains unclear. Limitations hindering transcriptome-wide analyses of alternative splicing differences (i.e. isoform switching) in cardiovascular system (CVS) tissues between fetal and adult (healthy and diseased) stages have included both cellular heterogeneity across bulk RNA-seq samples and limited availability of fetal tissue for research. To overcome these limitations, we have deconvoluted the cellular compositions of 996 RNA-seq samples representing heart failure, healthy adult (heart and arteria), and fetal-like (iPSC-derived cardiovascular progenitor cells) CVS tissues. Comparison of the expression profiles revealed that RNA-binding proteins (RBPs) are highly overexpressed in fetal-like compared with healthy adult and are reactivated in heart failure, which results in expression of thousands fetal-specific isoforms. Of note, isoforms for 20 different RBPs were among those that reverted in heart failure to the fetal-like expression pattern. We determined that, compared with adult-specific isoforms, fetal-specific isoforms are more likely to bind RBPs, have canonical sequences at their splice sites and encode proteins with more functions. Our findings suggest targeting RBP fetal-specific isoforms could result in novel therapeutics for heart failure.

category: genomics


title: PAM-interacting domain swapping is extensively utilized in nature to evolve CRISPR-Cas9 nucleases with altered PAM specificities

abstract: It has been anticipated that protospacer adjacent motif (PAM) specificity of the CRISPR-Cas9 nucleases protospacer appears to be modular. Here we present the finding that naturally occurring domain swapping has been extensively involved in altering its PAM specificities. Sequence analysis of streptococcal Cas9 sequences revealed conservation of three distinct PAM-interacting motifs, with phylogenetic analysis of full-length Cas9 and the PID demonstrating that PAM-interacting domain (PID) domain swapping was extensively utilized to diversify its PAM specificity. An extended analysis of 582 representative Cas9 sequences revealed that this PID swapping was broadly present in most of the investigated genera. Mimicking the natural PID domain swapping, a functional chimeric enzyme, based on the scaffold of compact Staphylococcus aureus Cas9, with novel NNAAAA PAM specificity was developed. In summary, our findings shed new light on a rich source of exchangeable PID domains in Cas9 genes, which can be mined for domain swapping aiming to an effective PAM refinement.

category: biochemistry


title: High-density spinal cord stimulation selectively activates lower urinary tract afferents

abstract: Epidural spinal cord stimulation (SCS) has recently been reported as a potential intervention to improve limb and autonomic functions, with lumbar stimulation improving locomotion and thoracic stimulation regulating blood pressure. We asked whether sacral SCS could be used to target the lower urinary tract. Here we show that high-density epidural SCS over the sacral spinal cord and cauda equina of anesthetized cats evokes responses in nerves innervating the bladder and urethra and that these nerves can be activated selectively. Sacral epidural SCS always recruited the pelvic and pudendal nerves and selectively recruited these nerves in all but one animal. Individual branches of the pudendal nerve were always recruited as well. Electrodes that selectively recruited specific peripheral nerves were spatially clustered on the arrays, suggesting anatomically organized sensory pathways. This selective recruitment demonstrates a mechanism to directly modulate bladder and urethral function through known reflex pathways, which could be used to restore bladder and urethral function after injury or disease.

category: bioengineering


title: Development of an open source program to identify the size of objects using bright-field microscopy - an automated calibration tool

abstract: In light microscopy, eyepiece graticules are commonly used to gauge the size of objects at the micron scale. While this is a relatively simple tool to use, not all microscopes possess this feature. Furthermore, calibrating an eyepiece graticule with a stage micrometer can be time-consuming, particularly for inexperienced microscopists. Similarly, calculating the size of individual objects may also take some time. We present an open-source program to determine the size of objects under a microscope using Python and OpenCV. Taking photos of a stage micrometer under a microscope, we identify gradations on the micrometer and calculate the distance between lines on the micrometer in pixels. From this, we can infer the size of objects from bright-field microscopy images. We believe this will improve access to quantitative microscopy techniques and increase the speed at which samples may be analyzed by light microscopy. Future studies may aim to integrate this with machine learning for object identification.

category: bioinformatics


title: Linked machine learning classifiers improve species classification of fungi when using error-prone long-reads on extendedmetabarcodes

abstract:
BackgroundThe increased usage of error-prone long-read sequencing for metabarcoding of fungi has not been matched with adequate public databases and concomitant analysis approaches. We address this gap and present a proof-of-concept study for classifying fungal taxa using linked machine learning classifiers. We demonstrate the capability of linked machine learning classifiers to accurately classify species and strains using real-world and simulated fungal ribosomal DNA datasets, including plant and human pathogens. We benchmark our new approach in comparison to current alignment and k-mer based methods based on synthetic mock communities. We also assess real world applications of species identification in complex unlabelled datasets.

ResultsOur machine learning approach assigned individual nanopore long-read amplicon sequences to fungal species with high recall rates and low false positive rates. Importantly, our approach successfully distinguished between closely-related species and strains when individual read errors were higher than the genetic distance between individual taxa, which the alignment and k-mer methods could not do. The machine learning approach showed an ability to identify key species with high recall rates, even in complex samples of unknown species composition.

ConclusionsA proof of concept machine learning approach using a tree-descent approach on a decision tree of classifiers can identify known taxa with high accuracy, and precisely detect known target species from complex samples with high recall rates. We propose this approach is suitable for detecting the known knowns of pathogens or invasive species in any environment of mostly unknown composition, including agriculture and wild ecosystems.


title: Linked machine learning classifiers improve species classification of fungi when using error-prone long-reads on extended metabarcodes


title: Phylodynamic modelling of bacterial outbreaks using nanopore sequencing

abstract: Nanopore sequencing and phylodynamic modelling have been used to reconstruct the transmission dynamics of viral epidemics, but their application to bacterial pathogens has remained challenging. Here, we implement Random Forest models for single nucleotide polymorphism (SNP) polishing to estimate divergence and effective reproduction numbers (Re) of two community-associated, methicillin-resistant Staphylococcus aureus (MRSA) outbreaks in remote Far North Queensland and Papua New Guinea (n = 159). Successive bar-coded panels of S. aureus isolates (2 x 12 per MinION) sequenced at low-coverage (> 5x - 10x) provided sufficient data to accurately infer assembly genotypes with high recall when compared with Illumina references. De novo SNP calling with Clair was followed by SNP polishing using intra- and inter-species models trained on Snippy reference calls. Models achieved sufficient resolution on ST93 outbreak sequence types (> 70 - 90% accuracy and precision) for phylodynamic modelling from lineage-wide hybrid alignments and birth-death skyline models in BEAST2. Our method reproduced phylogenetic topology, geographical source of the outbreaks, and indications of sustained transmission (Re > 1). We provide Nextflow pipelines that implement SNP polisher training, evaluation, and outbreak alignments, enabling reconstruction of within-lineage transmission dynamics for infection control of bacterial disease outbreaks using nanopore sequencing.


title: A novel framework for single-cell Hi-C clustering based on graph-convolution-based imputation and two-phase-based feature extraction

abstract: The three-dimensional genome structure plays a key role in cellular function and gene regulation. Singlecell Hi-C technology can capture genome structure information at the cell level, which provides the opportunity to study how genome structure varies among different cell types. However, few methods are well designed for single-cell Hi-C clustering, because of high sparsity, noise and heterogeneity of single-cell Hi-C data. In this manuscript, we propose a novel framework, named ScHiC-Rep, for singlecell Hi-C data representation and clustering. ScHiC-Rep mainly contains two parts: data imputation and feature extraction. In the imputation part, a novel imputation workflow is proposed, including graph convolution-based, random walk with restart-based and genomic neighbor-based imputation. In the feature extraction part, a two-phase feature extraction method is proposed, including linear phase for chromosome level and non-linear phase for cell level feature extraction. The evaluation results show that the proposed framework outperforms existing state-of-the-art approaches on both human and mouse datasets.


title: Adolescents' dietary habits and meal patterns influence school performance in the Northern Finland Birth Cohort 1986: mendelian randomisation study

abstract: Several observational studies indicate that dietary habits in children and adolescents are associated with school performance. These associations are heavily confounded by socio-economic characteristics, such as household income and parents educational attainment, amongst other factors. In this study, we report observational and causal effects of habitual diet on school performance, using individual level data for 9,220 adolescents in the Northern Finland Birth Cohort 1986. For this purpose, we derived principal components for the dietary variables, meal patterns and school performance variables. The observational study showed a significant association of consumption of foods that are high in fat, salt and sugar (HFSS) with poor performance in all school subjects, and an association of consumption of healthy foods and traditional foods with good school performance in general subjects, science and physical education (PE). Moreover, a positive association was observed between not skipping breakfast and good performance in all school subjects. Mendelian randomisation analysis confirmed a negative effect of HFSS on school performance in general/science subjects (-0.080, -0.128 to -0.033) and a positive effect of healthy food on school performance in general/science subjects (0.071, 0.024 to 0.119) and PE (0.065, 0.021 to 0.110). To conclude, we identified compelling evidence that HFSS foods and healthy foods were causally affecting school performance.


title: Targetron-assisted delivery of exogenous DNA sequences into Pseudomonas putida through CRISPR-aided counterselection

abstract: Genome editing methods based on Group II introns (known as Targetron technology) have been long used as a gene knock-out strategy in a wide range of organisms in a fashion independent of homologous recombination. Yet, their utility as delivery systems has been typically suboptimal because of their reduced efficiency of insertion when they carry exogenous sequences. We show that this limitation can be tackled and Targetron adapted as a general tool in Gram-negative bacteria. To this end, a set of broad host range standardized vectors were designed for conditional expression of the Ll.LtrB intron. After testing the correct functionality of these plasmids in Escherichia coli and Pseudomonas putida, we created a library of Ll.LtrB variants carrying cargo DNA sequences of different lengths to benchmark the capacity of intron-mediated delivery in these bacteria. Next, we combined CRISPR/Cas9-facilitated counterselection to increase the chances of finding genomic sites inserted with the thereby engineered introns. By following this pipeline, we were able to insert exogenous sequences of up to 600 bp at specific genomic locations in wild-type P. putida KT2440 and its {Delta}recA derivative. Finally, we were able to apply this technology to successfully tag this strain with an orthogonal short sequence (barcode) that acts as a unique identifier for tracking this microorganism in biotechnological settings. The results with P. putida exemplified the value of the Targetron approach for unrestricted delivery of small DNA fragments to the genomes of Gram-negative bacteria for a suite of genome editing endeavours.

category: synthetic biology


title: Minimal phrase composition revealed by intracranial recordings

abstract:
The ability to comprehend phrases is an essential integrative property of the brain. Here we evaluate the neural processes that enable the transition from single word processing to a minimal compositional scheme. Previous research has reported conflicting timing effects of composition, and disagreement persists with respect to inferior frontal and posterior temporal contributions. To address these issues, 19 patients (10 male, 19 female) implanted with penetrating depth or surface subdural intracranial electrodes heard auditory recordings of adjective-noun, pseudoword-noun and adjective-pseudoword phrases and judged whether the phrase matched a picture. Stimulus-dependent alterations in broadband gamma activity, low frequency power and phase-locking values across the language-dominant left hemisphere were derived. This revealed a mosaic located in the posterior superior temporal sulcus (pSTS), in which closely neighboring cortical sites displayed exclusive sensitivity to either lexicality or phrase structure, but not both. Distinct timings were found for effects of phrase composition (210-300 ms) and pseudoword processing (approximately 300-700 ms), and these were localized to neighboring electrodes in pSTS. The pars triangularis and temporal pole encode anticipation of composition in broadband low frequencies, and both regions exhibit greater functional connectivity with pSTS during phrase composition. Our results suggest that the pSTS is a highly specialized region comprised of sparsely interwoven heterogeneous constituents that encodes both lower and higher level linguistic features. This hub in pSTS for minimal phrase processing may form the neural basis for the human-specific computational capacity for forming hierarchically organized linguistic structures.

SignificanceLinguists have claimed that the integration of multiple words into a phrase demands a computational procedure distinct from single word processing. Here, we provide intracranial recordings from a large patient cohort, with high spatiotemporal resolution, to track the cortical dynamics of phrase composition. Epileptic patients volunteered to participate in a task in which they listened to phrases ("red boat"), word-pseudoword or pseudoword-word pairs (e.g., "red fulg"). At the onset of the second word in phrases, greater broadband high gamma activity was found in posterior superior temporal sulcus in electrodes that exclusively indexed phrasal meaning, and not lexical meaning. These results provide direct, high-resolution signatures of minimal phrase composition in humans, a potentially species-specific computational capacity.

category: neuroscience


title: Preserved motor memory in Parkinson's disease

abstract: Patients with Parkinsons disease, who lose the dopaminergic projections to the striatum, are impaired in certain aspects of motor learning. Recent evidence suggests that, in addition to its role in motor performance, the striatum plays a key role in the memory of motor learning. Whether Parkinsons patients have impaired motor memory and whether motor memory is modulated by dopamine at the time of initial learning is unknown. To address these questions, we measured memory of a learned motor sequence in Parkinsons patients who were either On or Off their dopaminergic medications. We compared them to a group of older and younger controls. Contrary to our predictions, motor memory was not impaired in patients compared to older controls, and was not influenced by dopamine state at the time of initial learning. To probe post-learning consolidation processes, we also tested whether learning a new sequence shortly after learning the initial sequence would interfere with later memory. We found that, in contrast to younger adults, neither older adults nor patients were susceptible to this interference. These findings suggest that motor memory is preserved in Parkinsons patients and raise the possibility that motor memory in patients is supported by compensatory non-dopamine sensitive mechanisms. Furthermore, given the similar performance characteristics observed in the patients and older adults and the absence of an effect of dopamine, these results raise the possibility that aging and Parkinsons disease affect motor memory in similar ways.


title: Crossbill: an open access single objective light-sheet microscopy platform

abstract: We present an open access scanned oblique plane microscopy platform Crossbill. It combines a new optical configuration, open hardware assembly, a systematic alignment protocol, and dedicated control software to provide a compact, versatile, high resolution single objective light-sheet microscopy platform. The demonstrated configuration yields the most affordable sub-micron resolution oblique plane microscopy system to date. We add galvanometer enabled tilt-invariant lateral scan for multi-plane, multi-Hz volumetric imaging capability. A precision translation stage extends stitched field of view to centimeter scale. The accompanying open software is optimized for Crossbill and can be easily extended to include alternative configurations. Using Crossbill, we demonstrate large volume structural fluorescence imaging with sub-micron lateral resolution in zebrafish and mouse brain sections. Crossbill is also capable of multiplane functional imaging, and time-lapse imaging. We suggest multiple alternative configurations to extend Crossbill to diverse microscopy applications.


title: Intracortical probe arrays with silicon backbone and microelectrodes on thin polyimide wings enable long-term stable recordings in vivo

abstract:
ObjectiveRecording and stimulating neuronal activity across different brain regions requires interfacing at multiple sites using dedicated tools while tissue reactions at the recording sites often prevent their successful long-term application. This implies the technological challenge of developing complex probe geometries while keeping the overall footprint minimal, and of selecting materials compatible with neural tissue. While the potential of soft materials in reducing tissue response is uncontested, the implantation of these materials is often limited to reliably target neuronal structures across large brain volumes.

ApproachWe report on the development of a new multi-electrode array exploiting the advantages of soft and stiff materials by combining 7-m-thin polyimide wings carrying platinum electrodes with a silicon backbone enabling a safe probe implantation. The probe fabrication applies microsystems technologies in combination with a temporal wafer fixation method for rear side processing, i.e., grinding and deep reactive ion etching, of slender probe shanks and electrode wings. The wing-type neural probes are chronically implanted into the entorhinal-hippocampal formation in the mouse for in vivo recordings of freely behaving animals.

Main resultsProbes comprising the novel wing-type electrodes have been realized and characterized in view of their electrical performance and insertion capability. Chronic electrophysiological in vivo recordings of the entorhinal-hippocampal network in the mouse of up to 104 days demonstrated a stable yield of channels containing identifiable multi-unit and single-unit activity outperforming probes with electrodes residing on a Si backbone.

SignificanceThe innovative fabrication process using a process compatible, temporary wafer bonding allowed to realize new Michigan style probe arrays. The wing-type probe design enables a precise probe insertion into brain tissue and long-term stable recordings of unit activity due to the application of a stable backbone and 7-m-thin probe wings provoking locally a minimal tissue response and protruding from the glial scare of the backbone.


title: Dyrk1a gene dosage in glutamatergic neurons has key effects in cognitive deficits observed in mouse models of MRD7 and Down syndrome

abstract:
Perturbation of the excitation/inhibition (E/I) balance leads to neurodevelopmental diseases including to autism spectrum disorders, intellectual disability, and epilepsy. Mutation in the DYRK1A gene located on human chromosome 21 (Hsa21) leads to an intellectual disability syndrome associated with microcephaly, epilepsy, and autistic troubles (MRD7). Overexpression of DYRK1A, on the other hand, has been linked with learning and memory defects observed in people with Down syndrome (DS). Dyrk1a is expressed in both glutamatergic and GABAergic neurons, but its impact on each neuronal population has not yet been elucidated. Here we investigated the impact of Dyrk1a gene copy number variation in glutamatergic neurons using a conditional knockout allele of Dyrk1a crossed with the Tg(Camk2-Cre)4Gsc transgenic mouse. We explored this genetic modification in homozygotes, heterozygotes and combined with the Dp(16Lipi-Zbtb21)1Yey trisomic mouse model to unravel the consequence of Dyrk1a dosage from 0 to 3, to understand its role in normal physiology, and in MRD7 and DS. Overall, Dyrk1a dosage in glutamatergic neurons did not impact locomotor activity, working memory or epileptic susceptibility, but revealed that Dyrk1a is involved in long-term explicit memory. Molecular analyses pointed at a deregulation of transcriptional activity through immediate early genes and a role of DYRK1A at the glutamatergic post-synapse by deregulating and interacting with key post-synaptic proteins implicated in mechanism leading to long-term enhanced synaptic plasticity. Altogether, our work gives important information to understand the action of DYRK1A inhibitors and have a better therapeutic approach.

Author summaryThe Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A, DYRK1A, drives cognitive alterations with increased dose in Down syndrome (DS) or with reduced dose in mental retardation disease 7 (MRD7). Here we report that specific and complete loss of Dyrk1a in glutamatergic neurons induced a range of specific cognitive phenotypes and alter the expression of genes involved in neurotransmission in the hippocampus. We further explored the consequences of Dyrk1a dosage in glutamatergic neurons on the cognitive phenotypes observed respectively in MRD7 and DS mouse models and we found specific roles in long-term explicit memory with no impact on motor activity, short-term working memory, and susceptibility to epilepsy. Then we demonstrated that DYRK1A is a component of the glutamatergic post-synapse and interacts with several component such as NR2B and PSD95. Altogether our work describes a new role of DYRK1A at the glutamatergic synapse that must be considered to understand the consequence of treatment targeting DYRK1A in disease.


title: EEG biomarkers of free recall

abstract: Brain activity in the moments leading up to spontaneous verbal recall provide a window into the cognitive processes underlying memory retrieval. But these same recordings also subsume neural signals unrelated to mnemonic retrieval, such as response-related motor activity. Here we examined spectral EEG biomarkers of memory retrieval under an extreme manipulation of mnemonic demands: subjects either recalled items after a few seconds or after several days. This manipulation helped to isolate EEG components specifically related to long-term memory retrieval. In the moments immediately preceding recall we observed increased theta (4-8 Hz) power (+T), decreased alpha (8-20 Hz) power (-A), and increased gamma (40-128 Hz) power (+G), with this spectral pattern (+T-A+G) distinguishing the long-delay and immediate recall conditions. As subjects vocalized the same set of studied words in both conditions, we interpret the spectral +T-A+G as a biomarker of episodic memory retrieval.


title: Flow cytometry method for absolute counting and single-cell phenotyping of mycobacteria

abstract: Detection and accurate quantitation of viable Mycobacterium tuberculosis is fundamental to understanding mycobacterial pathogenicity, tuberculosis (TB) disease progression and outcomes; TB transmission; drug action, efficacy and drug resistance. Despite this importance, methods for determining numbers of viable bacilli are limited in accuracy and precision owing to inherent characteristics of mycobacterial cell biology - including the tendency to clump, and "differential" culturability - and technical challenges consequent on handling an infectious pathogen under biosafe conditions. We developed an absolute counting method for mycobacteria in liquid cultures using a bench-top flow cytometer, and the low-cost fluorescent dyes Calcein-AM (CA) and SYBR-gold (SG). During exponential growth CA+ cell counts are highly correlated with CFU counts and can be used as a real-time alternative to simplify the accurate standardisation of inocula for experiments. In contrast to CFU counting, this method can detect and enumerate cell aggregates in samples, which we show are a potential source of variance and bias when using established methods. We show that CFUs comprise a sub-population of intact, metabolically active mycobacterial cells in liquid cultures, with CFU-proportion varying by growth conditions. A pharmacodynamic application of the flow cytometry method, exploring kinetics of fluorescent probe defined subpopulations compared to CFU is demonstrated. Flow cytometry derived Mycobacterium bovis BCG time-kill curves differ for rifampicin and kanamycin versus isoniazid and ethambutol, as do the relative dynamics of discrete morphologically-distinct subpopulations of bacilli revealed by this high-throughput single-cell technique.


title: The host factor ANP32A is required for influenza A virus vRNA and cRNA synthesis

abstract:
Influenza A viruses are negative-sense RNA viruses that rely on their own viral replication machinery to replicate and transcribe their segmented single-stranded RNA genome. The viral ribonucleoprotein complexes in which viral RNA is replicated consist of a nucleoprotein scaffold around which the RNA genome is bound, and a heterotrimeric RNA-dependent RNA polymerase that catalyzes viral replication. The RNA polymerase copies the viral RNA (vRNA) via a replicative intermediate, called the complementary RNA (cRNA), and subsequently uses this cRNA to make more vRNA copies. To ensure that new cRNA and vRNA molecules are associated with ribonucleoproteins in which they can be amplified, the active RNA polymerase recruits a second polymerase to encapsidate the cRNA or vRNA. Host factor ANP32A has been shown to be essential for viral replication and to facilitate the formation of a dimer between viral RNA polymerases and differences between mammalian and avian ANP32A proteins are sufficient to restrict viral replication. It has been proposed that ANP32A is only required for the synthesis of vRNA molecules from a cRNA, but not vice versa. However, this view does not match recent molecular evidence. Here we use minigenome assays, virus infections, and viral promoter mutations to demonstrate that ANP32A is essential for both vRNA and cRNA synthesis. Moreover, we show that ANP32 is not only needed for the actively replicating polymerase, but also for the polymerase that is encapsidating nascent viral RNA products. Overall, these results provide new insights into influenza A virus replication and host adaptation.

IMPORTANCEZoonotic avian influenza A viruses pose a constant threat to global health and they have the potential to cause highly pathogenic pandemic outbreaks. Species variations in host factor ANP32A play a key role in supporting the activity of avian influenza A virus RNA polymerases in mammalian hosts. Here we show that ANP32A acts at two stages in the influenza A virus replication cycle, supporting recent structural experiments and in line with its essential role. Understanding how ANP32A supports viral RNA polymerase activity and how it supports avian polymerase function in mammalian hosts is important for understanding influenza A virus replication and the development of antiviral strategies against influenza A viruses.


title: Alternative somatic and germline gene-regulatory strategies during starvation-induced developmental arrest

abstract: Nutrient availability governs growth and quiescence, and many animals arrest development when starved. Somatic and germline cells have distinct functions and constraints, suggesting different regulatory mechanisms contribute to an integrated starvation response. Using C. elegans L1 arrest as a model, we show that gene expression changes deep into starvation. Surprisingly, relative expression of germline-enriched genes increases for days. We conditionally degraded the large subunit of RNA Polymerase II and found that early but not late somatic transcription is required for survival and that germline transcription does not affect survival. Expression analysis revealed that thousands of genes are continuously transcribed in the soma, though their absolute abundance declines, while the germ line is transcriptionally quiescent with extreme transcript stability. This work reveals alternative somatic and germline gene-regulatory strategies during starvation, with the soma relying on a robust transcriptional response to support immediate survival while the germ line relies on transcriptional quiescence and mRNA stability to maintain future reproductive success.

category: genetics


title: A tissue-level phenome-wide network map of colocalized genes and phenotypes in the UK Biobank

abstract:
BackgroundPhenome-wide association studies conducted in electronic health record (EHR)-linked biobanks have uncovered a large number of genomic loci associated with traits and diseases. However, interpretation of the complex relationships of associated genes and phenotypes is challenging.

ResultsWe constructed a tissue-level phenome-wide network map of colocalized genes and phenotypes. First, we generated colocalized expression quantitative trait loci from 48 tissues of the Genotype-Tissue Expression project and from publicly available genome-wide association study summary statistics from the UK Biobank. We identified 9,151 colocalized genes for 1,411 phenotypes across 48 tissues. Then, we constructed a bipartite network using the colocalized signals to establish links between genes and phenotypes in each tissue. The majority of links are observed in a single tissue whereas only a few are present in all tissues. Finally, we applied the biLouvain clustering algorithm in each tissue-specific bipartite network to identify co-clusters of non-overlapping genes and phenotypes. The majority of co-clusters contains a small number of genes and phenotypes, and 88.6% of co-clusters are found in only one tissue. To demonstrate functionality of the phenome-wide map, we tested if these co-clusters were enriched with known biological and functional gene classes and observed several significant enrichments. Furthermore, we observed that tissue-specific co-clusters are enriched with reported drug side effects for the corresponding drug target genes in clinical trial data.

ConclusionsThe phenome-wide map provides links between genes, phenotypes and tissues across a wide spectrum of biological classes and can yield biological and clinical discoveries. The phenome-wide map is publicly available at https://rstudio-connect.hpc.mssm.edu/biPheMap/.


title: Ultraspecific somatic SNV and indel detection in single neurons using primary template-directed amplification

abstract: Primary template-directed amplification (PTA) is an improved amplification technique for single-cell DNA sequencing. We generated whole-genome analysis of 76 single neurons and developed SCAN2, a computational method to accurately identify both clonal and non-clonal somatic (i.e., limited to a single neuron) single nucleotide variants (SNVs) and small insertions and deletions (indels) using PTA data. Our analysis confirms an increase in non-clonal somatic mutation in single neurons with age, but revises estimates for the rate of this accumulation to be 15 SNVs per year. We also identify artifacts in other amplification methods. Most importantly, we show that somatic indels also increase by at least 2 indels per year per neuron and that indels may have a larger impact on gene function than somatic SNVs in human neurons.


title: ACTIVATION LOOP PHOSPHORYLATION OF A NON-RD RECEPTOR KINASE INITIATES PLANT INNATE IMMUNE SIGNALING

abstract: Receptor kinases (RKs) play fundamental roles in extracellular sensing to regulate development and stress responses across kingdoms. In plants, leucine-rich repeat receptor kinases (LRR-RKs) function primarily as peptide receptors that regulate myriad aspects of plant development and response to external stimuli. Extensive phosphorylation of LRR-RK cytoplasmic domains is among the earliest detectable responses following ligand perception, and reciprocal transphosphorylation between a receptor and its co-receptor is thought to activate the receptor complex. Originally proposed based on characterization of the brassinosteroid receptor, the prevalence of complex activation via reciprocal transphosphorylation across the plant RK family has not been tested. Using the LRR-RK ELONGATION FACTOR TU RECEPTOR (EFR) as a model RK, we set out to understand the steps critical for activating RK complexes. While the EFR cytoplasmic domain is an active protein kinase in vitro and is phosphorylated in a ligand-dependent manner in vivo, catalytically deficient EFR variants are functional in anti-bacterial immunity. These results reveal a non-catalytic role for the EFR cytoplasmic domain in triggering immune signaling and indicate that reciprocal transphoshorylation is not a ubiquitous requirement for LRR-RK complex activation. Rather, our analysis of EFR along with a detailed survey of the literature suggests a distinction between LRR-RK complexes with RD- versus non-RD protein kinase domains. Based on newly identified phosphorylation sites that regulate the activation state of the EFR complex in vivo, we propose that LRR-RK complexes containing a non-RD protein kinase may be regulated by phosphorylation-dependent conformational changes of the ligand-binding receptor which could initiate signaling in a feed-forward fashion either allosterically or through driving the dissociation of negative regulators of the complex.


title: Simplifying the development of portable, scalable, and reproducible workflows

abstract: Command-line software plays a critical role in biology research. However, processes for installing and executing software differ widely. The Common Workflow Language (CWL) is a community standard that addresses this problem. Using CWL, tool developers can formally describe a tools inputs, outputs, and other execution details in a manner that fosters use of shared computational methods and reproducibility of complex analyses. CWL documents can include instructions for executing tools inside software containers--isolated, operating-system environments. Accordingly, CWL tools are portable--they can be executed on diverse computers--including personal workstations, high-performance clusters, or the cloud. This portability enables easier adoption of bioinformatics pipelines. CWL supports workflows, which describe dependencies among tools and using outputs from one tool as inputs to others. To date, CWL has been used primarily for batch processing of large datasets, especially in genomics. But it can also be used for analytical steps of a study. This article explains key concepts about CWL and software containers and provides examples for using CWL in biology research. CWL documents are text-based, so they can be created manually, without computer programming. However, ensuring that these documents confirm to the CWL specification may prevent some users from adopting it. To address this gap, we created ToolJig, a Web application that enables researchers to create CWL documents interactively. ToolJig validates information provided by the user to ensure it is complete and valid. After creating a CWL tool or workflow, the user can create "input-object" files, which store values for a particular invocation of a tool or workflow. In addition, ToolJig provides examples of how to execute the tool or workflow via a workflow engine.


title: Precision neoantigen discovery using large-scale immunopeptidomes and composite modeling of MHC peptide presentation

abstract: Major histocompatibility complex (MHC)-bound peptides that originate from tumor-specific genetic alterations, known as neoantigens, are an important class of anti-cancer therapeutic targets. Accurately predicting peptide presentation by MHC complexes is a key aspect of discovering therapeutically relevant neoantigens. Technological improvements in mass-spectrometry-based immunopeptidomics and advanced modeling techniques have vastly improved MHC presentation prediction over the past two decades. However, improvement in the sensitivity and specificity of prediction algorithms is needed for clinical applications such as the development of personalized cancer vaccines, the discovery of biomarkers for response to checkpoint blockade and the quantification of autoimmune risk in gene therapies. Toward this end, we generated allele-specific immunopeptidomics data using 25 mono-allelic cell lines and created Systematic HLA Epitope Ranking Pan Algorithm (SHERPA), a pan-allelic MHC-peptide algorithm for predicting MHC-peptide binding and presentation. In contrast to previously published large-scale mono-allelic data, we used an HLA-null K562 parental cell line and a stable transfection of HLA alleles to better emulate native presentation. Our dataset includes five previously unprofiled alleles that expand MHC binding pocket diversity in the training data and extend allelic coverage in underprofiled populations. To improve generalizability, SHERPA systematically integrates 128 mono-allelic and 384 multi-allelic samples with publicly available immunoproteomics data and binding assay data. Using this dataset, we developed two features that empirically estimate the propensities of genes and specific regions within gene bodies to engender immunopeptides to represent antigen processing. Using a composite model constructed with gradient boosting decision trees, multiallelic deconvolution and 2.15 million peptides encompassing 167 alleles, we achieved a 1.44 fold improvement of positive predictive value compared to existing tools when evaluated on independent mono-allelic datasets and a 1.15 fold improvement when evaluating on tumor samples. With a high degree of accuracy, SHERPA has the potential to enable precision neoantigen discovery for future clinical applications.


title: Crowdsourcing biocuration: the Community Assessment of Community Annotation with Ontologies (CACAO)

abstract:
Experimental data about known gene functions curated from the primary literature have enormous value for research scientists in understanding biology. Using the Gene Ontology (GO), manual curation by experts has provided an important resource for studying gene function, especially within model organisms. Unprecedented expansion of the scientific literature and validation of the predicted proteins have increased both data value and the challenges of keeping pace. Capturing literature-based functional annotations is limited by the ability of biocurators to handle the massive and rapidly growing scientific literature. Within the community-oriented wiki framework for GO annotation called the Gene Ontology Normal Usage Tracking System (GONUTS), we describe an approach to expand biocuration through crowdsourcing with undergraduates. This multiplies the number of high-quality annotations in international databases, enriches our coverage of the literature on normal gene function, and pushes the field in new directions. From an intercollegiate competition judged by experienced biocurators, Community Assessment of Community Annotation with Ontologies (CACAO), we have contributed nearly 5000 literature-based annotations. Many of those annotations are to organisms not currently well-represented within GO. Over a ten-year history, our community contributors have spurred changes to the ontology not traditionally covered by professional biocurators. The CACAO principle of relying on community members to participate in and shape the future of biocuration in GO is a powerful and scalable model used to promote the scientific enterprise. It also provides undergraduate students with a unique and enriching introduction to critical reading of primary literature and acquisition of marketable skills.

Significance StatementThe primary scientific literature catalogs the results from publicly funded scientific research about gene function in human-readable format. Information captured from those studies in a widely adopted, machine-readable standard format comes in the form of Gene Ontology annotations about gene functions from all domains of life. Manual annotations based on inferences directly from the scientific literature, including the evidence used to make such inferences, represents the best return on investment by improving data accessibility across the biological sciences. To supplement professional curation, our CACAO project enabled annotation of the scientific literature by community annotators, in this case undergraduates, which resulted in contribution of thousands of validated entries to public resources. These annotations are now being used by scientists worldwide.


title: CRISPR-Cas-mediated tethering recruits the yeast HMR mating-type locus to the nuclear periphery but fails to silence gene expression

abstract: To investigate the relationship between genome structure and function, we have developed a programmable CRISPR-Cas system for nuclear peripheral recruitment in yeast. We benchmarked this system at the HMR and GAL2 loci, both well-characterized model systems for localization to the nuclear periphery. Using microscopy and gene silencing assays, we demonstrate that CRISPR-Cas-mediated tethering can recruit the HMR locus but does not silence reporter gene expression. A previously reported Gal4-mediated tethering system does silence gene expression, and we demonstrate that the silencing phenotype has an unexpected dependence on the structure of the protein tether. The CRISPR-Cas system was unable to recruit GAL2 to the nuclear periphery. Our results reveal potential challenges for synthetic genome structure perturbations and suggest that distinct functional effects can arise from subtle structural differences in how genes are recruited to the periphery.


title: GhostiPy: an efficient signal processing and spectral analysis toolbox for large data

abstract: Recent technological advances have enabled neural recordings consisting of hundreds to thousands of channels. As the pace of these developments continues to grow rapidly, it is imperative to have fast, flexible tools supporting the analysis of neural data gathered by such large scale modalities. Here we introduce ghostipy (general hub of spectral techniques in Python), a Python open source software toolbox implementing various signal processing and spectral analyses including optimal digital filters and time-frequency transforms. ghostipy prioritizes performance and efficiency by using parallelized, blocked algorithms. As a result, it is able to outperform commercial software in both time and space complexity for high channel count data and can handle out-of-core computation in a user-friendly manner. Overall, our software suite reduces frequently encountered bottlenecks in the experimental pipeline, and we believe this toolset will enhance both the portability and scalability of neural data analysis.


title: Multimodal brain features at preschool age and the relationship with pre-reading measures one year later: an exploratory study

abstract: Pre-reading language skills develop rapidly in early childhood and are related to brain structure and function in young children prior to formal education. However, the early neurobiological development that support these skills is not well understood and has not been assessed longitudinally using multiple imaging approaches. Here we acquired anatomical, diffusion tensor imaging (DTI) and resting state functional MRI (rs-fMRI) from 35 children at 3.5 years of age. Children were assessed for pre-reading abilities using the NEPSY-II subtests one year later (4.5 years). We applied a data-driven linked independent component analysis to explore the shared co-variation of grey and white matter measures. Two sources of structural variation at 3.5 years of age demonstrated a weak relationship with Speeded Naming scores at 4.5 years of age. The first imaging component involved volumetric variability in reading-related cortical regions alongside microstructural features of the superior longitudinal fasciculus. The second component was dominated by cortical volumetric variations within the cerebellum and visual association area. In a subset of children with rs-fMRI data, we evaluated the inter-network functional connectivity of the left-lateralized fronto-parietal language (FPL) network and its relationship with pre-reading measures. Higher functional connectivity between the FPL functional network and the default mode and visual networks at 3.5 years predicted better Phonological Processing scores at 4.5 years. Together, these results suggest that the integration of functional networks, as well as the co-development of white and grey matter brain structures in early childhood, may support the emergence of pre-reading measures in preschool children.


title: Molecular and cell biological analysis of SwrB in Bacillus subtilis

abstract:
Swarming motility is flagellar-mediated movement over a solid surface and Bacillus subtilis cells require an increase in flagellar density to swarm. SwrB is a protein of unknown function required for swarming that is necessary to increase the number of flagellar hooks but not basal bodies. Previous work suggested that SwrB activates flagellar type III secretion but the mechanism by which it might perform this function is unknown. Here we show that SwrB likely acts sub-stoichiometrically as it localizes as puncta at the membrane in numbers fewer than that of flagellar basal bodies. Moreover the action of SwrB is likely transient as puncta of SwrB were not dependent on the presence of the basal bodies and rarely co-localized with flagellar hooks. Random mutagenesis of the SwrB sequence found that a histidine within the transmembrane segment was conditionally required for activity and punctate localization. Finally, three hydrophobic residues that precede a cytoplasmic domain of poor conservation abolished SwrB activity when mutated and caused aberrant migration during electrophoresis. Our data are consistent with a model in which SwrB interacts with the flagellum, changes conformation to activate type III secretion, and departs.

IMPORTANCEType III secretion systems (T3SS) are elaborate nanomachines that form the core of the bacterial flagellum and injectisome of pathogens. The machines not only secrete proteins like virulence factors but also secrete the structural components for their own assembly. Moroever, proper construction requires complex regulation to ensure that the parts are roughly secreted in the order in which they are assembled. Here we explore a poorly understood activator the flagellar T3SS activation in Bacillus subtilis called SwrB. To aid mechanistic understanding, we determine the rules for subcellular punctate localization, the topology with respect to the membrane, and critical residues required for SwrB function.


title: EOMES is responsible for WNT memory and can substitute for WNT in mesendoderm specification

abstract: Embryogenesis is guided by a limited set of signaling pathways dynamically expressed in different places. How a context dependent signaling response is generated has been a central question of developmental biology, which can now be addressed with in vitro models of human embryos that are derived from embryonic stem cells (hESCs) called gastruloids. Our previous work demonstrated that during early self-organization of gastruloids, cells chronicle signaling hierarchy. Only cells that have been exposed (primed) by WNT signaling can respond to subsequent Activin exposure and differentiate to mesendodermal (ME) fates. Here, we show that WNT priming does not alter SMAD2 binding nor its chromatin opening, but rather, acts by inducing the expression of the SMAD2 co-factor, EOMES. Expression of EOMES is sufficient to replace WNT upstream of Activin-mediated ME differentiation, thus unveiling the mechanistic basis for priming and cellular memory in early development.

category: developmental biology


title: Mechanisms Underlying WNT-mediated Priming of Human Embryonic Stem Cells


title: Hierarchical, Memory-based Movement Models for Translocated Elk (Cervus canadensis)

abstract: The use of spatial memory is well documented in many animal species and has been shown to be critical for the emergence of spatial learning. Adaptive behaviors based on learning can emerge thanks to an interdependence between the acquisition of information over time and movement decisions. The study of how spatio-ecological knowledge is constructed throughout the life of an individual has not been carried out in a quantitative and comprehensive way, hindered by the lack of knowledge of the information an animal already has of its environment at the time monitoring begins. Identifying how animals use memory to make beneficial decisions is fundamental to developing a general theory of animal movement and space use. Here we propose several mobility models based on memory and perform hierarchical Bayesian inference on 11-month trajectories of 21 elk after they were released in a completely new environment. Almost all the observed animals exhibited preferential returns to previously visited patches, such that memory and random exploration phases occurred. Memory decay was mild or negligible over the study period. The fact that individual elk rapidly become used to a relatively small number of patches was consistent with the hypothesis that they seek places with predictable resources and reduced mortality risks such as predation.

category: animal behavior and cognition


title: Diverse Hotspot Thermal Profiling Methods Detect Phosphorylation-Dependent Changes in Protein Stability

abstract: Hotspot thermal profiling (HTP) methods utilize modified-peptide level information in order to interrogate proteoform-specific stability inside of live cells. The first demonstration of HTP involved the integration of phosphopeptide enrichment into a TMT-based, single-LC separation thermal profiling workflow1. Here we present a new  label-fractionate-enrich (LFE)-HTP method that involves high-pH reverse phase fractionation of TMT-labeled peptides prior to phosphopeptide enrichment, followed by peptide detection and quantitation using multi-notch LC-MS3. We find that LFE-HTP, while more resource intensive, improves the depth and precision of (phospho)proteoform coverage relative to the initial published HTP workflow. The fraction of detected phosphorylation sites that are significantly perturbed in this new dataset are consistent with those seen in our previous study, as well as those published by others, when compared head-to-head with the same analysis pipelines. Likewise, many  hotspot phosphorylation sites identified in our paper are consistently reproduced by LFE-HTP as well as other modified HTP methods. The LFE-HTP dataset contains many novel  hotspot phosphorylation sites that regulate the stability of diverse proteins, including phosphosites in the central glycolytic enzyme Aldolase A that are associated with monomer-to-oligomer formation, enzymatic activity and metabolic regulation in cancer cells. Our comparative analyses confirm that several variants of the HTP method can track modified proteoforms in live cells to detect and prioritize PTM-dependent changes in protein stability that may be associated with function.


title: Generation of fate patterns via intercellular forces

abstract: Studies of fate patterning during development typically emphasize cell-cell communication via diffusible signals. Recent experiments on monolayer stem cell colonies, however, suggest that mechanical forces between cells may also play a role. These findings inspire a model of mechanical patterning: fate affects cell contractility, and pressure in the cell layer biases fate. Cells at the colony boundary, more contractile than cells at the center, seed a pattern that propagates via force transmission. In agreement with previous observations, our model implies that the width of the outer fate domain depends only weakly on colony diameter. We further predict and confirm experimentally that this same width varies non-monotonically with substrate stiffness. This finding supports the idea that mechanical stress can mediate patterning in a manner similar to a morphogen; we argue that a similar dependence on substrate stiffness can be achieved by a chemical signal only if strong constraints on the signaling pathways mechanobiology are met.

category: biophysics


title: Time-reversal symmetric component of the flagellar beat enhances the translational and rotational velocities of isolated Chlamydomonas flagella

abstract: The beating of cilia and flagella is essential to perform many important biological functions, including generating fluid flows on the cell surface or propulsion of micro-organisms. In this work, we analyze the motion of isolated and demembranated flagella from green algae Chlamydomonas reinhardtii, which act as ATP-driven micro-swimmers. The waveform of the Chlamydomonas beating flagella has an asymmetric waveform that is known to involve the superposition of a static component, corresponding to a fixed, intrinsic curvature, and a dynamic wave component traveling in the base-to-tip direction at the fundamental beat frequency, plus higher harmonics. Here, we demonstrate that these modes are not sufficient to reproduce the observed flagella waveforms. We find that two extra modes play an essential role to describe the motion: first, a time-symmetric mode, which corresponds to a global oscillation of the axonemal curvature, and second, a secondary tip-to-base wave component at the fundamental frequency that propagates opposite to the dominant base-to-tip wave, albeit with a smaller amplitude. Although the time-symmetric mode cannot, by itself, contribute to propulsion (scallop theorem), it does enhance the translational and rotational velocities of the flagellum by approximately a factor of 2. This mode highlights a long-range coupled on/off activity of force-generating dynein motors and can provide further insight into the underling biology of the ciliary beat.


title: The effect of habitat type on population distribution and abundance of Rothschilds Giraffe (Giraffa camelopardalis rothschildi) in Ruma National Park and Mwea National Reserve in Kenya

abstract: The Rothschilds giraffe is currently listed as vulnerable by the International Union for Conservation of Nature (IUCN). This is attributed to the loss of habitat due to human activities. This study examined the effect of habitat type on population structure and distribution of Rothschilds giraffe in Ruma National Park (RNP) and Mwea National Reserve (MNR) in Kenya. The study employed road transects to collect data on the number, age class and sex distribution in three habitat types, open, medium and closed. Data was collected along three road transects of equal lengths measuring 14.2 km in each site (RNP and MNR) for comparison. A driving speed of 20 km per hour was maintained along each transect for standardization of survey effort and a maximum giraffe detection rate. Photographic capture of the coat patterns of the right side of all the giraffes sighted within 500 m from the transect was done for identification of age classes. The field visits were replicated 12 times for each transect giving 36 replications for each site spread equally through wet and dry seasons from March 2017 to November 2018. The effect of habitat type on population structure and distribution was analysed using ANOVA and Tukey HSD to test for significant differences. T-test was used to compare the mean population size of giraffe across the wet and dry seasons. Coat pattern analysis for age class identification was done using WildID software. The findings indicated that MNR had more males to females compared to RNP that registered more females and calves. Habitat type had a significant effect on the distribution of giraffes. The giraffe population showed a preference for medium habitat types. The findings are key for the management of habitat quality for giraffe populations at the interface where conservation areas overlap with human land use.


title: Evolutionary dynamics of circular RNAs in primates

abstract: Many primate genes produce non-coding circular RNAs (circRNAs). However, the extent of circRNA conservation between closely related species remains unclear. By comparing tissue-specific transcriptomes across over 70 million years of primate evolution, we identify that within 3 million years circRNA expression profiles diverged such that they are more related to species identity than organ type. However, our analysis also revealed a subset of circRNAs with conserved neural expression across tens of millions of years of evolution. These circRNAs are defined by an extended downstream intron that has shown dramatic lengthening during evolution due to the insertion of novel retrotransposons. Our work provides comparative analyses of the mechanisms promoting circRNAs to generate increased transcriptomic complexity in primates.


title: Aplp1 and the Aplp1-Lag3 Complex facilitates transmission of pathologic alpha-synuclein

abstract:
Pathologic -synuclein (-syn) spreads from cell-to-cell, in part, through binding to the lymphocyte-activation gene 3 (Lag3). Here we report that amyloid {beta} precursor-like protein 1 (Aplp1) forms a complex with Lag3 that facilitates the binding, internalization, transmission, and toxicity of pathologic -syn. Deletion of both Aplp1 and Lag3 eliminates the loss of dopaminergic neurons and the accompanying behavioral deficits induced by -syn preformed fibrils (PFF). Anti-Lag3 prevents the internalization of -syn PFF by disrupting the interaction of Aplp1 and Lag3, and blocks the neurodegeneration induced by -syn PFF in vivo. The identification of Aplp1 and the interplay with Lag3 for -syn PFF induced pathology advances our understanding of the molecular mechanism of cell-to-cell transmission of pathologic -syn and provides additional targets for therapeutic strategies aimed at preventing neurodegeneration in Parkinsons disease and related -synucleinopathies.

One Sentence SummaryAplp1 forms a complex with Lag3 that facilitates the binding, internalization, transmission, and toxicity of pathologic -synuclein.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/442157v1_ufig1.gif" ALT="Figure 1">
View larger version (35K):
org.highwire.dtl.DTLVardef@1d746bforg.highwire.dtl.DTLVardef@65acb2org.highwire.dtl.DTLVardef@12cf2f0org.highwire.dtl.DTLVardef@19d6e_HPS_FORMAT_FIGEXP  M_FIG Aplp1 and the Aplp1-Lag3 complex facilitates transmission of pathologic -synuclein.

Aplp1 is a receptor that drives pathologic -syn transmission, and genetic depletion of Aplp1 can significantly reduce the -synuclein pathogenesis. Aplp1 and Lag3 forms an Aplp1-Lag3 complex that accounts for substantial binding of pathologic -syn to cortical neurons. Together Aplp1 and Lag3 play a major role in pathologic -syn internalization, transmission and toxicity. Double knockout of Aplp1 and Lag3 and or a Lag3 antibody that disrupts the Aplp1 and Lag3 complex almost completely blocks -syn PFF-induced neurodegeneration.

C_FIG


title: Activation of extrasynaptic kainate receptors drives hilar mossy cell activity

abstract:
Mossy cells (MCs) of the dentate gyrus (DG) are key components of an excitatory associative circuit established by reciprocal connections with dentate granule cells (GCs). MCs are implicated in place field encoding, pattern separation and novelty detection, as well as in brain disorders such as temporal lobe epilepsy and depression. Despite their functional relevance, little is known about the determinants that control MC activity. Here, we examined whether MCs express functional kainate receptors (KARs), a subtype of glutamate receptors involved in neuronal development, synaptic transmission and epilepsy. Using mouse hippocampal slices, we found that bath application of submicromolar and micromolar concentrations of the KAR agonist kainic acid induced inward currents and robust MC firing. These effects were abolished in GluK2 KO mice, indicating the presence of functional GluK2-containing KARs in MCs. In contrast to CA3 pyramidal cells, which are structurally and functionally similar to MCs, and express synaptic KARs at mossy fiber (MF) inputs (i.e., GC axons), we found no evidence for KAR-mediated transmission at MF-MC synapses, indicating that most KARs at MCs are extrasynaptic. Immunofluorescence and immunoelectron microscopy analyses confirmed the extrasynaptic localization of GluK2-containing KARs in MCs. Finally, blocking glutamate transporters, a manipulation that increases extracellular levels of endogenous glutamate, was sufficient to induce KAR-mediated inward currents in MCs, suggesting that MC-KARs can be activated by increases in ambient glutamate. Our findings provide the first direct evidence of functional extrasynaptic KARs at a critical excitatory neuron of the hippocampus.

Significance StatementHilar mossy cells (MCs) are an understudied population of hippocampal neurons that form an excitatory loop with dentate granule cells. MCs have been implicated in pattern separation, spatial navigation, and epilepsy. Despite their importance in hippocampal function and disease, little is known about how MC activity is recruited. Here, we show for the first time that MCs express extrasynaptic kainate receptors (KARs), a subtype of glutamate receptors critically involved in neuronal function and epilepsy. While we found no evidence for synaptic KARs in MCs, KAR activation induced strong action potential firing of MCs, raising the possibility that extracellular KARs regulate MC excitability in vivo and may also promote dentate gyrus hyperexcitability and epileptogenesis.


title: Convergence of proprioceptive and visual feedback on neurons in primary motor cortex

abstract: An important aspect of motor function is our ability to rapidly generate goal-directed corrections for disturbances to the limb or behavioural goal. Primary motor cortex (M1) is a key region involved in feedback processing, yet we know little about how different sources of feedback are processed by M1. We examined feedback-related activity in M1 to compare how different sources (visual versus proprioceptive) and types of information (limb versus goal) are represented. We found sensory feedback had a broad influence on M1 activity with [~]73% of neurons responding to at least one of the feedback sources. Information was also organized such that limb and goal feedback targeted the same neurons and evoked similar responses at the single-neuron and population levels indicating a strong convergence of feedback sources in M1.


title: Corticohippocampal circuit dysfunction in a mouse model of Dravet syndrome

abstract: Dravet syndrome (DS) is a neurodevelopmental disorder defined by treatment-resistant epilepsy, autism spectrum disorder, and sudden death, due to pathogenic variants in SCN1A encoding the Nav1.1 sodium channel subunit. Convergent data suggest hippocampal dentate gyrus (DG) pathology. We found that optogenetic stimulation of entorhinal cortex was ictogenic in DS (Scn1a+/-) but not wild-type mice in vivo. Two-photon calcium imaging in brain slice demonstrated profound impairment in filtering of perforant path input by DG in young adult Scn1a+/- mice due to enhanced excitatory input to granule cells. Excitability of parvalbumin interneurons (PV-INs) was near-normal and selective activation of PV-INs rescued circuit impairments. This demonstrates developmental reorganization of hippocampal circuitry that can be modulated by recruitment of functional PV-INs, suggesting potential therapeutic approaches towards seizure modulation. The identified circuit abnormality mirrors that seen in models of chronic temporal lobe epilepsy, suggesting convergent mechanisms linking genetic and acquired causes of temporal lobe-onset seizures.


title: Frizzled BRET sensors based on bioorthogonal labeling of unnatural amino acids reveal WNT-induced dynamics of the cysteine-rich domain.

abstract: Frizzleds (FZD1-10) comprise a class of G protein-coupled receptors containing an extracellular cysteine-rich domain (CRD) that binds lipoglycoproteins of the Wingless/Int-1 family (WNTs). Despite the prominent role of the WNT/FZD system in health and disease, our understanding of how WNT binding to the FZD CRD is translated into receptor activation and transmembrane signaling remains limited. Current hypotheses dispute the roles for conformational dynamics and the involvement of the linker domain connecting the CRD with the seven-helical transmembrane core of FZD. To clarify the mechanism of WNT binding to FZD and to elucidate how WNT/FZD complexes achieve signaling pathway specificity, we devised conformational FZD-CRD biosensors based on bioluminescence-resonance-energy-transfer (BRET). Using FZD engineered with N-terminal nanoluciferase and fluorescently-labeled unnatural amino acids in the linker domain and extracellular loop 3, we show that WNT-3A and WNT-5A induce similar CRD conformational rearrangements despite promoting distinct downstream signaling pathways, and that CRD dynamics are not required for WNT/{beta}-catenin signaling. Thus, the novel FZD-CRD biosensors we report provide insights into the stepwise binding, activation and signaling processes in FZDs. The sensor design is broadly applicable to explore fundamental events in signal transduction mediated by other membrane receptors.

category: cell biology


title: Foxd3 controls heterochromatin-mediated silencing of repeat elements in mouse embryonic stem cells and represses the 2-cell transcription program

abstract: Repeat element transcription plays a vital role in early embryonic development. Expression of repeats such as MERVL characterises mouse embryos at the 2-cell stage, and defines a 2-cell-like cell (2CLC) population in a mouse embryonic stem cell culture. Repeat element sequences contain binding sites for numerous transcription factors. We identify the forkhead domain transcription factor FOXD3 as a regulator of repeat element transcription in mouse embryonic stem cells. FOXD3 binds to and recruits the histone methyltransferase SUV39H1 to MERVL and major satellite repeats, consequentially repressing the transcription of these repeats by the establishment of the H3K9me3 heterochromatin modification. Notably, depletion of FOXD3 leads to the de-repression of MERVL and major satellite repeats as well as a subset of genes expressed in the 2-cell state, shifting the balance between the stem cell and 2-cell like population in culture. Thus, FOXD3 acts as a negative regulator of repeat transcription, ascribing a novel function to this transcription factor.


title: Group size and modularity interact to shape the spread of infection and information through animal societies

abstract:
Social interactions between animals can provide many benefits, including the ability to gain useful environmental information through social learning. However, these social contacts can also facilitate the transmission of infectious diseases through a population. Animals engaging in social interactions must therefore face a trade-off between the potential informational benefits and the risk of acquiring disease. In order to understand how this trade-off can influence animal sociality, it is necessary to quantify the effects of different social structures on individuals likelihood of acquiring information versus infection Theoretical models have suggested that modular social networks, associated with the formation of groups or sub-groups, can slow spread of infection by trapping it within particular groups. However these social structures will not necessarily impact the spread of information in the same way if its transmission is considered as a "complex contagion", e.g. through individuals copying the majority (conformist learning). Here we use simulation models to demonstrate that modular networks can promote the spread of information relative to the spread of infection, but only when the network is fragmented and group sizes are small. We show that the difference in transmission between information and disease is maximised for more well-connected social networks when the likelihood of transmission is intermediate. Our results have important implications for understanding the selective pressures operating on the social structure of animal societies, revealing that highly fragmented networks such as those formed in fission-fusion social groups and multilevel societies can be effective in modulating the infection-information trade-off for individuals within them.

Significance statementRisk of infection is commonly regarded as one of the costs of animal social behaviours, while the potential for acquiring useful information is seen as a benefit. Balancing this risk of infection with the potential to gain useful information is one of the key trade-offs facing animals that engage in social interactions. In order to better understand this trade-off, it is necessary to quantify how different social structures can promote access to useful information while minimising risk of infection. We used simulations of disease and information spread to examine how group sizes and social network fragmentation influences both these transmission processes. Our models find that more subdivided networks slow the spread of disease far more than infection, but only group sizes are small. Our results demonstrate that showing that fragmented social structures can be more effective in balancing the infection-information trade-off for individuals within them.


title: Protein secondary structure affects glycan clustering in native mass spectrometry

abstract: Infection with human noroviruses (hNoV) for the vast majority of strains requires attachment of the viral capsid to histo blood group antigens (HBGA). The HBGA binding pocket is formed by dimers of the protruding domain (P dimers) of the capsid protein VP1. Several studies have focused on HBGA binding to P dimers, reporting binding affinities and stoichiometries. However, nuclear magnetic resonance spectroscopy (NMR) and native mass spectrometry (MS) analyses yielded incongruent dissociation constants (KD) for binding of HBGAs to P dimers and, in some cases, disagreed whether glycans bind at all. We hypothesized that glycan clustering during electrospray ionization in native MS critically depends on the physicochemical properties of the protein studied. It follows that the choice of the reference protein is crucial. We analyzed carbohydrate clustering using various P dimers and eight non-glycan binding proteins serving as possible references. Data from native and ion mobility MS indicate that the mass fraction of {beta}-sheet has a strong influence on the degree of glycan clustering. Therefore, the determination of specific glycan binding affinities from native MS must be interpreted cautiously.


title: Fine-scale spatial genetic structure across the species range reflects recent colonization of high elevation habitats in silver fir (Abies alba Mill.)

abstract: Variation in genetic diversity across species ranges has long been recognized as highly informative for assessing populations resilience and adaptive potential. The spatial distribution of genetic diversity, referred to as fine-scale spatial genetic structure (FSGS), also carries information about recent demographic changes, yet it has rarely been connected to range scale processes. We studied eight silver fir (Abies alba Mill.) population pairs (sites), growing at high and low elevations, representative of the main genetic lineages of the species. A total of 1368 adult trees and 540 seedlings were genotyped using 137 and 116 single nucleotide polymorphisms (SNPs), respectively. Sites revealed a clear east-west isolation-by-distance pattern consistent with the post-glacial colonization history of the species. Genetic differentiation among sites (FCT=0.148) was an order of magnitude greater than between elevations within sites (FSC=0.031), nevertheless high elevation populations consistently exhibited a stronger FSGS. Structural equation modeling revealed that elevation and, to a lesser extent, post-glacial colonization history, but not climatic and habitat variables, were the best predictors of FGSG across populations. These results may suggest that high elevation habitats have been colonized more recently across the species range. Additionally, paternity analysis revealed a high reproductive skew among adults and a stronger FSGS in seedlings than in adults, suggesting that FSGS may conserve the signature of demographic changes for several generations. Our results emphasize that spatial patterns of genetic diversity within populations provide complementary information about demographic history and could be used for defining conservation priorities.


title: Ventilation-induced epithelial injury drives biological onset of lung trauma in vitro and is mitigated with anti-inflammatory therapeutics

abstract: Mortality rates among patients suffering from acute respiratory failure remain perplexingly high despite maintenance of blood homeostasis. The biotrauma hypothesis advances that mechanical forces from invasive ventilation trigger immunological factors that spread systemically. Yet, how these forces elicit an immune response remains unclear. Here we show that flow-induced stresses under mechanical ventilation can injure the bronchial epithelium of ventilated in vitro upper airway models and directly modulate inflammatory cytokine secretion associated with pulmonary injury. We identify site-specific susceptibility to epithelial erosion in airways from jet-flow impaction and measure an increase in cell apoptosis and modulated secretions of cytokines IL-6, 8 and 10. We find that prophylactic pharmacological treatment with anti-inflammatory therapeutics reduces apoptosis and pro-inflammatory signaling during ventilation. Our 3D in vitro airway platform points to a previously overlooked origin of lung injury and showcases translational opportunities in preclinical pulmonary research towards protective therapies and improved protocols for patient care.


title: RNA splicing programs define tissue compartments and cell types at single cell resolution

abstract: More than 95% of human genes are alternatively spliced. Yet, the extent splicing is regulated at single-cell resolution has remained controversial due to both available data and methods to interpret it. We apply the SpliZ, a new statistical approach that is agnostic to transcript annotation, to detect cell-type-specific regulated splicing in > 110K carefully annotated single cells from 12 human tissues. Using 10x data for discovery, 9.1% of genes with computable SpliZ scores are cell-type specifically spliced. These results are validated with RNA FISH, single cell PCR, and in high throughput with Smart-seq2. Regulated splicing is found in ubiquitously expressed genes such as actin light chain subunit MYL6 and ribosomal protein RPS24, which has an epithelial-specific microexon. 13% of the statistically most variable splice sites in cell-type specifically regulated genes are also most variable in mouse lemur or mouse. SpliZ analysis further reveals 170 genes with regulated splicing during sperm development using, 10 of which are conserved in mouse and mouse lemur. The statistical properties of the SpliZ allow model-based identification of subpopulations within otherwise indistinguishable cells based on gene expression, illustrated by subpopulations of classical monocytes with stereotyped splicing, including an un-annotated exon, in SAT1, a Diamine acetyltransferase. Together, this unsupervised and annotation-free analysis of differential splicing in ultra high throughput droplet-based sequencing of human cells across multiple organs establishes splicing is regulated cell-type-specifically independent of gene expression.