Search bioRxiv⌕ Search

Biology subjects

John, A.

Publications and source records attributed to John, A..

33 records · Page 2Linked to original sources

V-pipe 3.0: a sustainable pipeline for within-sample viral genetic diversity estimation

The large amount and diversity of viral genomic datasets generated by next-generation sequencing technologies poses a set of challenges for computational data analysis workflows, including rigorous quality control, adaptation to higher sample coverage, and tailored steps for specific applications. Here, we present V-pipe 3.0, a computational pipeline designed for analyzing next-generation sequencing data of short viral genomes. It is developed to enable reproducible, scalable, adaptable, and transparent inference of genetic diversity of viral samples. By presenting two large-scale data analysis projects, we demonstrate the effectiveness of V-pipe 3.0 in supporting sustainable viral genomic data science.

bioinformatics↗

Microclimate buffering varies across forest types during an extreme heat event

Increasing temperatures and extreme heat episodes have become more common with climate change. While forests are known to be buffered from increasing temperatures compared to non-forested areas, whether this buffering is maintained under extreme temperature events, how such events influence forests, and how forest organisms respond to extreme heat is relatively unknown. Here we assess the effects of an extreme heat event (the Pacific Northwest (PNW) heatdome in June 2021) on forest microclimates, forests, and the organisms living within them. We first asked how the PNW heatdome affected microclimates in forests with differing canopy cover (including non-forests) and found that the buffering capacity of forests is greater under denser canopies, even under extreme heat events. We then combined this information with organismal temperature tolerance curves for 12 relevant species and found that canopy buffering can minimize the negative impacts of even extreme heat events on understory organisms, with greater canopy density providing greater microclimate moderation. Finally, we analyzed seasonal NDVI trends in recent years, and found signs of canopy stress following the extreme 2021 heat event. In all, this suggests that although forest canopies may buffer the negative effects of extreme heat events on understory organisms, a greater frequency of extreme heat events may threaten this capacity by damaging forest canopies.

ecology↗

Topography influences diurnal and seasonal microclimate fluctuations in hilly terrain environments of coastal California

Understanding the topographic basis for microclimatic variation remains fundamental to predicting the site level effects of warming air temperatures. Quantifying diurnal fluctuation and seasonal extremes in relation to topography offer insight into the potential relationship between site level conditions and changes in regional climate. The present study investigated an annual understory temperature regime for 50 sites distributed across a topographically diverse area (>12 km2) comprised of mixed evergreen-deciduous woodland vegetation typical of California coastal ranges. We investigated the effect of topography and tree cover on site-to-site variation in near-surface temperatures using a combination of multiple linear regression and multivariate techniques. Sites in topographically depressed areas (e.g., valley bottoms) exhibited larger seasonal and diurnal variation. Elevation (at 10 m resolution) was found to be the primary driver of daily and seasonal variations, in addition to local topographic features that measure how depressed a site is compared to the surrounding area, canopy cover and northness. The elevation effect on seasonal mean temperatures was inverted, reflecting large-scale cold-air pooling in the study region, with elevated minimum and mean temperature at higher elevations. Additionally, several of our sites showed considerable buffering (dampened diurnal and seasonal temperature fluctuations) compared to average regional conditions measured by weather station. Results from this study help inform efforts to extrapolate temperature records across large landscapes and have the potential to improve our ecological understanding of fine-scale seasonal climate variation in coastal range environments.

ecology↗

A complete morphological characterization of all life stages of the phorid fly M. scalaris

Megaselia scalaris, commonly known as the scuttle fly, is a cosmopolitan species in the family Phoridae. It is an easily cultured fly species that is an emerging model organism in the fields of genetics and developmental biology. Its affinity for carrion and its predictable life cycle makes it useful in the field of forensic science for estimating the post-mortem interval (PMI) of human remains. Cases of human myasis caused by M. scalaris have also been reported in the medical literature. Despite its ubiquitous prevalence and its relevance across multiple fields, its morphology has not been adequately characterized. Here, we report the complete morphological characterization of all lifestages of M. scalaris, ranging from egg to adult. Scanning electron microscopy has enabled us to uncover morphological features and developmental processes that have previously not been reported in the literature. Our data lays the groundwork for future genetic studies: a morphological characterization of the wild type must be performed before mutants displaying different phenotypes can be identified. In this vein, we also report the observation of a acephalic, or headless, adult phenotype whose study could yield insights into the process of cephalogenesis.

zoology↗

Using photographs and deep neural networks to understand flowering phenology and diversity in mountain meadows

Mountain meadows are an essential part of the alpine-subalpine ecosystem; they provide ecosystem services like pollination and are home to diverse plant communities. Changes in climate affect meadow ecology on multiple levels, for example by altering growing season dynamics. Tracking the effects of climate change on meadow diversity through the impacts on individual species and overall growing season dynamics is critical to conservation efforts. Here, we explore how to combine crowd sourced camera images with machine learning to quantify flowering species richness across a range of elevations in alpine meadows located in Mt Rainier National Park, Washington, USA. We employed three machine learning techniques (Mask R-CNN, RetinaNet and YOLOv5) to detect wildflower species in images taken during two flowering seasons. We demonstrate that deep learning techniques can detect multiple species, providing information on flowering richness in photographed meadows. The results indicate higher richness just above the tree line for most of the species, which is comparable with patterns found using field studies. We found that the two-stage detector Mask R-CNN was more accurate than single-stage detectors like RetinaNet and YOLO, with the Mask R-CNN network performing best overall with mean average precision (mAP) of 0.67 followed by RetinaNet (0.5) and YOLO (0.4). We found that across the methods using anchor box variations in multiples of 16 led to enhanced accuracy. We also show that detection is possible even when pictures are interspersed with complex backgrounds and are not in focus. We found differential detection rates depending on species abundance, with additional challenges related to similarity in flower characteristics, labeling errors, and occlusion issues. Despite these potential biases and limitations in capturing flowering abundance and location-specific quantification, accuracy was notable considering the complexity of flower types and picture angles in this data set. We therefore expect that this approach can be used to address many ecological questions that benefit from automated flower detection, including studies of flowering phenology and floral resources, and that this approach can therefore complement a wide range of ecological approaches (e.g., field observations, experiments, community science, etc.). In all, our study suggests that ecological metrics like floral richness can be efficiently monitored by combining machine learning with easily accessible publicly curated datasets (e.g., Flickr, iNaturalist).

ecology↗

A network of CLAVATA receptors buffers auxin-dependent meristem maintenance

Plant body plans are elaborated in response to both environmental and endogenous cues. How these inputs intersect to promote growth and development remains poorly understood. During reproductive development, central zone stem cell proliferation in inflorescence meristems is negatively regulated by the CLAVATA3 (CLV3) peptide signaling pathway. In contrast, floral primordia formation on meristem flanks requires the hormone auxin. Here we show that CLV3 signaling is also necessary for auxin-dependent floral primordia generation and that this function is partially masked by both inflorescence fasciation and heat-induced auxin biosynthesis. Stem cell regulation by CLAVATA signaling is separable from primordia formation but is also sensitized to temperature and auxin levels. Additionally, we uncover a novel role for the CLV3 receptor CLAVATA1 in auxin- dependent meristem maintenance in cooler environments. As such, CLV3 signaling buffers multiple auxin-dependent shoot processes across divergent thermal environments with opposing effects on cell proliferation in different meristem regions.

plant biology↗

Differential remodelling in small and large murine airways revealed by novel whole lung airway analysis.

Airway remodelling occurs in chronic asthma leading to increased airway smooth muscle (ASM) mass and extra-cellular matrix (ECM) deposition. Whilst extensively studied in murine airways; studies report only selected larger airways at one time point meaning the spatial distribution and resolution of remodelling are poorly understood. Here we use a new method allowing comprehensive assessment of the spatial and temporal changes in ASM, ECM and epithelium in large numbers of murine airways after allergen challenge. Using image processing to analyse 20-50 airways from a whole lung section revealed increases in ASM and ECM after allergen challenge were greater in small and large rather than intermediate airways. ASM predominantly accumulated adjacent to the basement membrane whereas ECM was distributed across the airway wall. Epithelial hyperplasia was most marked in small and intermediate airways. Post challenge, ASM changes resolved over seven days whereas ECM and epithelial changes persisted. The new method suggests large and small airways remodel differently and the long-term consequences of airway inflammation may depend more on ECM and epithelial changes than ASM. The method reduces the number of animals needed, reveals important spatial differences in remodelling and could set new analysis standards for murine asthma models.

physiology↗

Embryo-scale epithelial buckling forms a propagating furrow that initiates gastrulation

Cell apical constriction driven by actomyosin contraction forces is a conserved mechanism during tissue folding in embryo development. While much effort has been made to better understand the molecular mechanisms responsible for apical constriction, it is still not clear if apical actomyosin contraction forces are necessary or sufficient per se to drive tissue folding. To tackle this question, we use the Drosophila embryo model system that forms a furrow on the ventral side, initiating mesoderm internalization. Past computational models support the idea that cell apical contraction forces may not be sufficient and that active or passive cell apico-basal forces may be necessary to drive cell wedging and tissue furrowing. By using 3D computational modelling and in toto embryo image analysis and manipulation, we now challenge this idea and show that embryo-scale force balance of the tissue surface, rather than cell-autonomous shape changes, is necessary and sufficient to drive a buckling of the epithelial surface forming a furrow which propagates and initiates embryo gastrulation.

developmental biology↗

Genetic architecture of a body color cline in Drosophila americana

Phenotypic variation within a species is often structured geographically in clines. In Drosophila americana, a longitudinal cline for body color exists within North America that appears to be due to local adaptation. The tan and ebony genes have been hypothesized to contribute to this cline, with alleles of both genes that lighten body color found in D. americana. These alleles are similar in sequence and function to the allele fixed in D. americanas more lightly pigmented sister species, Drosophila novamexicana. To test this hypothesis, we examined the frequency and geographic distribution of D. novamexicana-like alleles of tan and ebony in D. americana. Among alleles from over 100 strains of D. americana isolated from 21 geographic locations, we failed to identify additional alleles of tan or ebony with as much sequence similarity to D. novamexicana as the alleles previously described. However, using genetic analysis of 51 D. americana strains derived from 20 geographic locations, we identified one new allele of ebony and one new allele of tan segregating in D. americana that are functionally equivalent to the D. novamexicana allele. An additional 5 alleles of tan also showed marginal evidence of functional similarity. Given the rarity of these alleles, however, we conclude that they are unlikely to be driving the pigmentation cline. Indeed, phenotypic distributions of the 51 backcross populations analyzed indicate a more complex genetic architecture, with diversity in the number and effects of loci altering pigmentation observed both within and among populations of D. americana. This genetic heterogeneity poses a challenge to association studies and genomic scans for clinal variation, but might be common in natural populations.

evolutionary biology↗

Effect of prolonged captivity on the hemolymph profile of Tachypleus gigas

Horseshoe crabs amebocyte cells degranulate to form a gel clot when in contact with endotoxins. This phenomenon is the basis of both Horseshoe crab immune system and detection of endotoxin in biologicals. The present study investigates the amebocyte cells quality in Tachypleus gigas pre and post bleeding under captivity. Wild and captive horseshoe crabs (5 months captivity) were bled in 6 anticoagulant formulations (A, B, C, D, E and F). No profound difference in cell density between captive and wild groups with the mean value of 0.883x107 a cells/mL and 0.917x107 cells/mL, respectively. while, the cell viability of the captive group was significantly lower than the wild crabs (F=808.075, p<0.001). Anticoagulant formulation significantly affected cell viability and cell morphology in both captive and wild groups (p<0.001). Amebocyte cells collected from the wild T. gigas using optimum anticoagulant (formula C) showed 0.6 x107 cells/mL cell density and 86.9% cell viability, while morphology analysis revealed the percentage of contracted, granular flattened and degranulated flattened cells were 14.62%, 71.39% and 14%, respectively. The anticoagulant formulations showed varying capabilities in maintaining cell viability due to its buffering and chelating capacity. We conclude that captivity has a negative effect on the amebocyte cell quality. HIGHLIGHTSO_LICaptivity has a negative impact on amebocyte quality in horseshoe crab (Tachypleus gigas). C_LIO_LINo significant difference in cell density between captive and wild crabs. C_LIO_LIAnticoagulant formulations have significant impact on the cell density, viability and morphology C_LIO_LICaptive crabs were immunocompromised due to single specific feed intake. C_LI

systems biology↗

Horseshoe crab genomes reveal the evolutionary fates of genes and microRNAs after three rounds (3R) of whole genome duplication

Whole genome duplication (WGD) has occurred in relatively few sexually reproducing invertebrates. Consequently, the WGD that occurred in the common ancestor of horseshoe crabs ~135 million years ago provides a rare opportunity to decipher the evolutionary consequences of a duplicated invertebrate genome. Here, we present a high-quality genome assembly for the mangrove horseshoe crab Carcinoscorpius rotundicauda (1.7Gb, N50 = 90.2Mb, with 89.8% sequences anchored to 16 pseudomolecules, 2n = 32), and a resequenced genome of the tri-spine horseshoe crab Tachypleus tridentatus (1.7Gb, N50 = 109.7Mb). Analyses of gene families, microRNAs, and synteny show that horseshoe crabs have undergone three rounds (3R) of WGD, and that these WGD events are shared with spiders. Comparison of the genomes of C. rotundicauda and T. tridentatus populations from several geographic locations further elucidates the diverse fates of both coding and noncoding genes. Together, the present study represents a cornerstone for a better understanding of the consequences of invertebrate WGD events on evolutionary fates of genes and microRNAs at individual and population levels, and highlights the genetic diversity with practical values for breeding programs and conservation of horseshoe crabs.

evolutionary biology↗

Gene expression and in situ protein profiling of candidate SARS-CoV-2 receptors in human airway epithelial cells and lung tissue

In December 2019, SARS-CoV-2 emerged causing the COVID-19 pandemic. SARS-CoV, the agent responsible for the 2003 SARS outbreak, utilizes ACE2 and TMPRSS2 host molecules for viral entry. ACE2 and TMPRSS2 have recently been implicated in SARS-CoV-2 viral infection. Additional host molecules including ADAM17, cathepsin L, CD147, and GRP78 may also function as receptors for SARS-CoV-2. To determine the expression and in situ localization of candidate SARS-CoV-2 receptors in the respiratory mucosa, we analyzed gene expression datasets from airway epithelial cells of 515 healthy subjects, gene promoter activity analysis using the FANTOM5 dataset containing 120 distinct sample types, single cell RNA sequencing (scRNAseq) of 10 healthy subjects, immunoblots on multiple airway epithelial cell types, and immunohistochemistry on 98 human lung samples. We demonstrate absent to low ACE2 promoter activity in a variety of lung epithelial cell samples and low ACE2 gene expression in both microarray and scRNAseq datasets of epithelial cell populations. Consistent with gene expression, rare ACE2 protein expression was observed in the airway epithelium and alveoli of human lung. We present confirmatory evidence for the presence of TMPRSS2, CD147, and GRP78 protein in vitro in airway epithelial cells and confirm broad in situ protein expression of CD147 in the respiratory mucosa. Collectively, our data suggest the presence of a mechanism dynamically regulating ACE2 expression in human lung, perhaps in periods of SARS-CoV-2 infection, and also suggest that alternate receptors for SARS-CoV-2 exist to facilitate initial host cell infection.

immunology↗

Structural basis of substrate recognition and catalysis by fucosyltransferase 8

Fucosylation of the inner-most N-acetyl-glucosamine (GlcNAc) of N-glycans by fucosyltransferase 8 (FUT8) is an important step in the maturation of complex and hybrid N-glycans. This simple modification can have a dramatic impact on the activity and half-life of glycoproteins. These effects are relevant to understanding the invasiveness of some cancers, the development of monoclonal antibody therapeutics, and to a congenital disorder of glycosylation. The acceptor substrate preferences of FUT8 are well characterised and provide a framework for understanding N-glycan maturation in the Golgi, however the structural basis for these substrate preferences and the mechanism through which catalysis is achieved remains unknown. Here, we describe several structures of mouse and human FUT8 in the apo state and in complex with guanosine diphosphate (GDP), a mimic of the donor substrate, and a glycopeptide acceptor substrate. These structures provide insights into: a unique conformational change associated with donor substrate binding; common strategies employed by fucosyltransferases to coordinate GDP; features that define acceptor substrate preferences; and a likely mechanism for enzyme catalysis. Together with molecular dynamics simulations, the structures also reveal how FUT8 dimerisation plays an important role in defining the acceptor substrate binding site. Collectively, this information significantly builds on our understanding of the core-fucosylation process.

biochemistry↗

Trefoil factors share a lectin activity that defines their role in mucus

The trefoil factors (TFFs) are disulfide-rich mucosal peptides that protect the epithelium by promoting cell migration and increasing the viscoelasticity of the mucosa. Here we show that all TFFs are divalent lectins that recognise the GlcNAc--1,4-Gal disaccharide, which terminates type-III mucin-like O-glycans. Structural, mutagenic and biophysical data support a model of mucus viscoelasticity that features non-covalent cross-linking of glycoproteins by TFFs.

biochemistry↗

POFUT2-mediated O-glycosylation of MIC2 is dispensable for Toxoplasma gondii tachyzoites

Toxoplasma gondii is a ubiquitous obligate intracellular eukaryotic parasite that causes congenital birth defects, disease of the immunocompromised and blindness. Protein glycosylation plays an important role in the infectivity and evasion of immune response of many eukaryotic parasites and is also of great relevance to vaccine design. Here, we demonstrate that MIC2, the motility-associated adhesin of T. gondii, has highly glycosylated thrombospondin repeat domains (TSR). At least seven C-linked and three O-linked glycosylation sites exist within MIC2, with >95% occupancy at O-glycosylation sites. We demonstrate that the addition of O-glycans to MIC2 is mediated by a protein O-fucosyltransferase 2 homologue (TgPOFUT2) encoded by TGGT1_273550. While POFUT2 homologues are important for stabilizing motility associated adhesins and host infection in other apicomplexan parasites, in T. gondii loss of TgPOFUT2 has only a modest impact on MIC2 levels and the wider proteome. Consistent with this, both plaque formation and tachyzoite infectivity are broadly similar in the presence or absence of TgPOFUT2. These findings demonstrate that TgPOFUT2 O-glycosylates MIC2 and that this glycan is dispensable in T. gondii tachyzoites.

microbiology↗