Biology subjects

Ross, S.

Publications and source records attributed to Ross, S..

12 recordsLinked to original sources

bioRxiv2024

An analysis of RNA quality metrics in human brain tissue

Human brain tissue studies have historically used a range of metrics to assess RNA quality. However, few large-scale cross-comparisons of pre-sequencing quality metrics with RNA-seq quality have been published. Here, we analyze how well metrics gathered before RNA sequencing (post-mortem interval (PMI) and RNA integrity number RIN) relate to analyses of RNA quality after sequencing (Percent of counts in Top Ten genes (PTT), 5 bias, and 3 bias) as well as with individual gene counts across the transcriptome. We conduct this analysis across four different human cortical brain tissue collections sequenced with varying library preparation protocols. PMI and RIN have a low inverse correlation, and both PMI and RIN show consistent and opposing correlations with PTT. Unlike PMI, RIN shows strong consistent correlations with measurements of 3 and 5 bias, and RIN also correlates with 3,933 genes across datasets, in comparison to 138 genes for PMI. Neuronal and immune response genes correlate positively and negatively with RIN respectively, suggesting that different gene sets have divergent relationships with RIN in brain tissue. In summary, these analyses suggest that conventional metrics of RNA quality have varying degrees of value, and that PMI has an overall minimal but reproducible effect on RNA quality.

neuroscience↗

bioRxiv2024

Laboratory in vitro replication of Ostreid Herpes Virus (OsHV-1) using Pacific oyster tissue explants

Pacific oysters (Crassostrea or Magallana gigas) are one of the most economically important aquaculture species globally. Over the past two decades, ostreid herpesvirus (OsHV-1), has become a major pathogen of cultured Pacific oysters resulting in widespread mortality with a global distribution. Experimental use of OsHV-1 is challenging for many reasons, including both complexity and relative obscurity of host pathogen dynamics, and a lack of functioning model systems. The goal of this study was to improve the tools available for working with OsHV-1 in both whole animals and in tissue explants established ex vivo from oysters and maintained in controlled laboratory conditions. Tissue explants were taken from oysters originating from two different sources that have different levels of mortality in OsHV-1 challenges and were used in disease challenges alongside whole animals for comparison. Quantitative PCR, histology and electron microscopy were used to confirm that the explants were capable of replicating OsHV-1. Furthermore, the quantitative PCR results suggests that the source of the oysters was significant in determining the outcome of infection in the explants, supporting the validity of the explant model for OsHV-1 infection. This approach for studying OsHV-1 allows for the control of confounding factors in disease outcome that is not possible in whole animal challenges, providing a new tool for studying OsHV-1 in Pacific oysters.

microbiology↗

bioRxiv2023

Distinct TCR repertoire diversity, clonality and MHC-restriction in foetal and adult thymus

Here we sequenced rearranged TCR{beta} and TCR chain sequences in CD4+CD8+ double positive (DP), CD4+CD8-single positive (SP4) and CD4-CD8+ (SP8) thymocyte populations from the foetus and young adult mouse. We found that life-stage had a greater impact on TCR{beta} and TCR gene segment usage than cell-type. Foetal repertoires showed bias towards 3TRAV and 5TRAJ rearrangements in all populations, whereas adult repertoires used more 5TRAV gene segments, suggesting that progressive TCR rearrangements occur less frequently in foetal DP cells. When we synchronised young adult DP thymocyte differentiation by hydrocortisone treatment the new recovering DP thymocyte population showed more foetal-like 3TRAV and 5TRAJ gene segment usage. In foetus we identified less influence of MHC-restriction on -chain and {beta}-chain combinatorial VxJ usage and CDR1xCDR2 (V region) usage in SP compared to adult, indicating weaker impact of MHC-restriction on the foetal TCR repertoire. The foetal TCR{beta} repertoire was less diverse, less evenly distributed, with fewer non-template insertions, and all foetal populations contained more clonotypic expansions than adult. The differences between the foetal and adult thymus TCR repertoires are consistent with the foetal thymus producing {beta}T-cells with properties and functions that are distinct from adult T-cells: their repertoire is less governed by MHC-restriction, with preference for particular gene segment usage, less diverse with more clonotypic expansions, and more closely encoded by genomic sequence.

immunology↗

bioRxiv2022

Dynamic changes in the niche and transcription trigger early murine and human pluripotent stem cell-derived liver organogenesis

The shift from collective migration to differentiation is a crucial process in epithelial biology but recreating this intricate transition has thus far proved elusive. We provide experimental, mechanistic, in vivo, and bioinformatic data supporting an undoubtable link between human pluripotent stem cell (hPSC)- derived collectively migrating hepatoblasts (MHB), and transcriptionally mature, functional hPSC- hepatocytes (HEPs), which incorporates two unrecognized steps. The protocol induces FOXA-dependent induction of HBs, leading to TBX3-positive, YAP-TEAD active MHBs which provide a transcriptional match with murine liver E9.5 MHBs. Simple cultivation changes trigger MHBs to rapidly form functional day 18 HEPs, predicted by a deep-learning designed gene circuit, resulting in a [~]236% fold- increase in maturation (PACNet), on par with the highest score, but with enhanced global transcriptional shaping. Overall, incorporating the MHB to HEP transition establishes a new, unrecognized, and highly efficient mechanism for differentiation that can be cumulatively integrated with existing methods to overcome barriers to maturation.

bioengineering↗

bioRxiv2022

Bcl-xL is a key mediator of apoptosis following KRASG12C inhibition in KRASG12C mutant colorectal cancer.

PurposeNovel covalent inhibitors of KRASG12C have shown limited response rates in KRASG12C mutant (MT) colorectal cancer (CRC) patients. Thus, novel KRASG12C inhibitor combination strategies that can achieve deep and durable responses are needed. Experimental designSmall molecule KRASG12C inhibitors AZ1569 and AZ8037 were employed. To identify novel candidate combination strategies for AZ1569, we performed RNA sequencing, siRNA and high-throughput drug screening. Top hits were validated in a panel of KRASG12CMT CRC cells and in vivo. AZ1569-resistant CRC cells were generated and characterised. ResultsResponse to AZ1569 was heterogeneous across the KRASG12CMT models. AZ1569 was ineffective at inducing apoptosis when used as single-agent or combined with chemotherapy or agents targeting the EGFR/KRAS/AKT axis. Using a systems biology approach, we identified the anti-apoptotic BH3-family member BCL2L1/Bcl-xL as a top hit mediating resistance to AZ1569. Further analyses identified acute increases in the pro-apoptotic protein BIM following AZ1569 treatment. ABT-263 (Navitoclax), a pharmacological Bcl-2 family-inhibitor that blocks the ability of Bcl-xL to bind and inhibit BIM, led to dramatic and universal apoptosis when combined with AZ1569. Furthermore, this combination also resulted in dramatically attenuated tumour growth in KRASG12CMT xenografts. Finally, AZ1569-resistant cells showed amplification of KRASG12C, EphA2/c-MET activation, increased pro-inflammatory chemokine profile and cross-resistance to several targeted agents. Importantly, KRAS amplification and AZ1569-resistance were reversible upon drug withdrawal, arguing strongly for the use of drug holidays in the case of KRAS amplification. ConclusionsCombinatorial targeting of Bcl-xL and KRASG12C is highly effective, suggesting a novel therapeutic strategy for KRAS G12CMT CRC patients.

cancer biology↗

bioRxiv2021

Recombinant Lloviu virus as a model to study inaccessible zoonotic viruses

Next generation sequencing has revealed the presence of many RNA viruses in animal reservoir hosts, including many closely related to known human pathogens. Despite their zoonotic potential, many of these viruses remain understudied due to not yet being cultured. While reverse genetic systems can facilitate virus rescue, this is often hindered by missing viral genome ends. A prime example is Lloviu virus (LLOV), an uncultured filovirus that is closely related to the highly pathogenic Ebola virus. Using minigenome systems, we complemented the missing LLOV genomic ends and identified cis-acting elements required for LLOV replication that were lacking in the published sequence. We leveraged these data to generate recombinant full-length LLOV clones and rescue infectious virus. Recombinant LLOV (rLLOV) displays typical filovirus features, as shown by electron microscopy. Known target cells of Ebola virus, including macrophages and hepatocytes, are permissive to rLLOV infection, suggesting that humans could be potential hosts. However, inflammatory responses in human macrophages, a hallmark of Ebola virus disease, are not induced by rLLOV. We also used rLLOV to test antivirals targeting multiple facets of the replication cycle. Rescue of uncultured viruses of pathogenic concern represents a valuable tool in our arsenal against pandemic preparedness.

microbiology↗

bioRxiv2021

Resource selection by the Endangered Arabian tahr: identifying critical habitats for conservation and climate change adaptation.

The Arabian tahr is an Endangered mountain ungulate endemic to the Hajar Mountains of Arabia. The Arabian tahr population is in decline and threats to tahr habitat are intensifying, in addition new potential challenges from climate change are emerging. Fundamental to future conservation planning is understanding tahr habitat selection patterns, so we can prioritise habitat protection, and understand how habitat may be used to provide thermal refuge and allow adaptation to climate change impacts. We used GPS collars and resource selection functions to characterise Arabian tahr habitat preferences in Wadi Sareen Nature Reserve, Oman. We found tahr habitat selection was dependent on scale, sex and season. Vegetation resources were only selected at the smallest scales of selection and avoided at other scales. Habitat providing low heat load and thermal refuge were intensely selected at small and medium scales, by both sexes and in both seasons, suggesting the importance of thermal refuges in facilitating thermoregulation. Higher elevations, steep slopes and rugged habitats were selected across all scales tested here, and in previous landscape-scale studies, indicating the fundamental importance of these habitats in supporting Arabian tahr populations. Our results identified critical habitats required to sustain Arabian tahr, and demonstrated the importance of thermal refuges to species living in the hot climates such as the Arabian Peninsula. Given the accessibility of habitat layers, and ease in which the identified habitats can be mapped using a geographical information system, understanding the habitat selection of tahr and other species is a crucial step to increasing conservation management capacity of threatened species. Given our uncertainty of how to conserve wildlife under future climate change, understanding the availability and distribution of wildlife habitat is an important baseline from where we can plan, connect and preserve the resources necessary for wildlife conservation.

ecology↗

bioRxiv2021

Txikispora philomaios n. sp., n. g., a Micro-Eukaryotic Pathogen of Amphipods, Reveals Parasitism and Hidden Diversity in Class Filasterea

This study provides a morphological, ultrastructural, and phylogenetic characterization of a novel micro-eukaryotic parasite (2.3-2.6 {micro}m) infecting genera Echinogammarus and Orchestia. Longitudinal studies across two years revealed that infection prevalence peaked in late April and May, reaching 64% in Echinogammarus sp. and 15% in Orchestia sp., but was seldom detected during the rest of the year. The parasite infected predominantly haemolymph, connective tissue, tegument, and gonad, although hepatopancreas and nervous tissue were affected in heavier infections, eliciting melanization and granuloma formation. Cell division occurred inside walled parasitic cysts, often within host haemocytes, resulting in haemolymph congestion. Small subunit (18S) rRNA gene phylogenies including related environmental sequences placed the novel parasite as a highly divergent lineage within Class Filasterea, which together with Choanoflagellatea represent the closest protistan relatives of Metazoa. We describe the new parasite as Txikispora philomaios n. sp. n. g., the first confirmed parasitic filasterean lineage, which otherwise comprises four free-living flagellates and a rarely observed endosymbiont of snails. Lineage-specific PCR probing of other hosts and surrounding environments only detected T. philomaios in the platyhelminth Procerodes sp. We expand the known diversity of Filasterea by targeted searches of metagenomic datasets, resulting in 13 previously unknown lineages from environmental samples.

pathology↗

bioRxiv2020

APOBEC3B expression generates an immunogenic model of Kras mutant lung cancer

Mutations in oncogenes such as KRAS and EGFR cause a high proportion of lung cancers. Drugs targeting these proteins cause tumour regression but ultimately fail to cure these cancers, leading to intense interest in how best to combine them with other treatments, such as immunotherapies. However, preclinical systems for studying the interaction of lung tumours with the host immune system are inadequate, in part due to the low tumour mutational burden in genetically engineered mouse models. Here we set out to develop mouse models of mutant KRAS-driven lung cancer with an elevated tumour mutational burden by expressing the human DNA cytosine deaminase, APOBEC3B, to mimic the mutational signature seen in human lung cancer. This failed to substantially increase clonal tumour mutational burden and autochthonous tumours remained refractory to immunotherapy. However, by establishing clonal cell lines from these tumours we generated an immunogenic syngeneic transplantation model of KRAS mutant lung adenocarcinoma that was sensitive to immunotherapy. Unexpectedly, we found that anti-tumour immune responses were not directed against neoantigens but instead targeted derepressed endogenous retroviral antigens. The ability of KRASG12C inhibitors to cause regression of KRASG12C-expressing versions of these tumours was markedly potentiated by the adaptive immune system, providing a unique opportunity for the study of combinations of targeted and immunotherapies in immune-hot lung cancer.

cancer biology↗

bioRxiv2020

Mesenchyme modulates three-dimensional, collective cell migration of liver cells in vitro - a role for TGFβ pathway

Three dimensional (3D) collective cell migration (CCM) is critical for improving liver cell therapies, eliciting mechanisms of liver disease, and modeling human liver development/ organogenesis. Here, we modeled liver organogenesis to induce 3D CCM and improve existing models. The liver diverticulum, normally surrounded by septum transversum mesenchyme (STM) at E8.5, was modeled with a miniature liver spheroid surrounded by mesenchymal cells and matrix. In mixed spheroid models with both liver and uniquely MRC5 (fetal lung) fibroblasts, we observed co-migration of cells, and a significant increase in length and number of liver spheroid protrusions, and this was highly sensitive to TGFB1 stimulation. To understand paracrine effects between MRC-5 cells and liver, we performed conditioned medium (M-CM) experiments. Interestingly, the addition of M-CM increased liver 3D CCM, with thin, 3D, dose-dependent branching morphogenesis, an upregulation of Twist1, and a sensitivity to a broad TGFB inhibitor. To test the effects of cell-cell interactions of 3D CCM, the STM was modeled with a spheroid of MRC-5 cells, and we performed co-spheroid culture of liver with MRC-5. We observed a complex morphogenesis, whereby thin, linear, 3D liver cell strands attach to the MRC-5 spheroid, anchor, and thicken to form permanent and thick anchoring contacts between the two spheroids. We also observed spheroid fusion, a form of interstitial migration. In conclusion, we present several novel cultivation systems that induce distinct features of 3D CCM, as judged by the presence of branching, linearity, thickness, and interstitial migration. These methodologies will greatly improve our molecular, cellular, and tissue-scale understanding of liver organogenesis, liver diseases, and liver cell therapy, and will serve as a tool to bridge conventional 2D studies and preclinical in vivo studies.

cell biology↗

bioRxiv2020

A novel RNA virus, Macrobrachium rosenbergii Golda virus (MrGV), linked to mass mortalities of the larval giant freshwater prawn in Bangladesh

1Mass mortalities of the larval stage of the giant freshwater prawn, Macrobrachium rosenbergii, have been occurring in Bangladesh since 2011. Mortalities can reach 100% and have resulted in an 80% decline in the number of hatcheries actively producing M. rosenbergii. To investigate a causative agent for the mortalities, a disease challenge was carried out using infected material from a hatchery experiencing mortalities. Moribund larvae from the challenge were prepared for metatranscriptomic sequencing. De novo virus assembly revealed a 29 kb single-stranded positive-sense RNA virus with similarities in key protein motif sequences to yellow head virus (YHV), an RNA virus that causes mass mortalities in marine shrimp aquaculture, and other viruses in the Nidovirales order. Primers were designed against the novel virus and used to screen cDNA from larvae sampled from hatcheries in the South of Bangladesh from two consecutive years. Larvae from all hatcheries screened from both years were positive by PCR for the novel virus, including larvae from a hatchery that at the point of sampling appeared healthy, but later experienced mortalities. These screens suggest that the virus is widespread in M. rosenbergii hatchery culture in southern Bangladesh, and that early detection of the virus can be achieved by PCR. The hypothesised protein motifs of MrGV suggest that it is likely to be a new species within the Nidovirales order. Biosecurity measures should be taken in order to mitigate global spread through the movement of post-larvae within and between countries, which has previously been linked to other virus outbreaks in crustacean aquaculture.

molecular biology↗

bioRxiv2019

High resolution, dynamic imaging of early mouse and human liver bud morphogenesis in three dimensions

BackgroundLiver organogenesis has thus far served as a paradigm for solid organ formation. The developing liver bud is a well established model of organogenesis, and murine genetic studies demonstrate key molecules involved in key morphogenetic changes. However, the analysis of the liver bud is typically limited to 2D tissue sections, which precludes extensive visualization, quantitation, and analysis. Further, the lack of human liver bud data has further hindered our understanding of human liver organogenesis. Therefore, new analytical and visualization approaches are needed to elicit further morphogenetic details of liver organogenesis and to elucidate differences between mouse and human liver bud growth. ResultsTo address this need, we focused on high resolution imaging, visualization, and analysis of early liver growth by using available online databases for both mouse (EMAP, Toronto Phenogenomics center) and human (3D Atlas of Human Embryology), noninvasive multimodality imaging studies of the murine embryo, and mouse/human liver weight data. First, we performed three-dimensional (3D reconstructions) of stacked, digital tissue sections that had been initially segmented for the liver epithelium and the septum transversum mesenchyme (STM). 3D reconstruction of both mouse and human data sets enabled visualization and analysis of the dynamics of liver bud morphogenesis, including hepatic cord formation and remodeling, mechanisms of growth, and liver-epithelial STM interactions. These studies demonstrated potentially under-appreciated mechanisms of growth, including rapid exponential growth that is matched at the earliest stages by STM growth, and unique differences between mouse and human liver bud growth. To gain further insight into the exponential liver bud growth that was observed, we plotted volumetric data from 3D reconstruction together with fetal liver growth data from multimodality (optical projection tomography, magnetic resonance imaging, micro-CT) and liver weight data to compose complete growth curves during mouse (E8.5-E18) and human (day 25-300) liver development. For further analysis, we performed curve fitting and parameter estimation, using Gompertzian models, which enables the comparison between mouse and human liver bud growth, as well as comparisons to processes like liver regeneration. To demonstrate the importance of mesenchyme in rapid liver bud growth and morphogenesis in the human liver bud, we performed functional analysis in which human pluripotent stem cell (hPSC)-derived hepatic organoids were used to model collective migration that occurs in vivo, demonstrating that migration is strongly dependent upon mesenchyme. DiscussionOur data demonstrates improved visualization with 3D images, under-appreciated and potentially new mechanisms of growth, complete liver growth curves with quantitative analysis through embryonic and fetal stages, and a new functional human stem cell-derived liver organoid assay demonstrating mesenchyme-driven collective migration. These data enhance our understanding of liver organogenesis.

developmental biology↗