Biology subjects

Shaw, B.

Publications and source records attributed to Shaw, B..

10 recordsLinked to original sources

bioRxiv2026

New Human IPSC Models of Late-onset Alzheimer's Disease Polygenic Risk Identify Multiple Impairments of Microglial Function

Genetic discoveries implicate microglia in late-onset Alzheimers disease (AD). We modelled AD in a powerful study of 51 human induced pluripotent stem cell (iPSC) microglia derived from high-polygenic risk AD or low-risk cognitively well individuals, sampled from a large cohort. We explored mitochondrial function, cytokine secretion, endocytosis, phagocytosis, lipid accumulation, calcium store release, and chemotaxis under basal conditions and immune challenge. High polygenic risk was independently associated with significant functional deficits in iPSC microglia under immune challenge, in mitochondrial ATP production (p=0.005, -13%), and inflammatory cytokine release (IL-6: p=0.018, -42%; TNF p=0.026, -38.5%). Furthermore, a selective deficit in amyloid-{beta} uptake was identified (p=0.00477, -5.9%). Deficits in inflammatory cytokine release were driven by APOE {varepsilon}4. These findings reflect primary changes in AD pathogenesis predating plaque formation and validate a human in vitro platform for late-onset AD to further understand disease mechanisms and screen drug or genetic therapies.

neuroscience↗

bioRxiv2024

Aspergillus nidulans Transcription Factor BrlA is Utilized in a Conidiation-Independent Response to Cell-Wall Stress

Under synchronized conidiation, over 2500 gene products show differential expression, including transcripts for both brlA and abaA, which increase steadily over time. In contrast, during wall-stress induced by the echinocandin micafungin, the brlA transcript is upregulated while the abaA transcript is not. In addition, when mpkA (last protein kinase in the cell wall integrity signaling pathway) is deleted, brlA expression is not upregulated in response to wall stress. Together, these data imply BrlA may play a role in a cellular stress-response which is independent of the canonical BrlA-mediated conidiation pathway. To test this hypothesis, we performed a genome-wide search and found 332 genes with a putative BrlA response element (BRE) in their promoter region. From this set, we identified 28 genes which were differentially expressed in response to wall-stress, but not during synchronized conidiation. This set included seven gene products whose homologues are involved in transmembrane transport and 14 likely to be involved in secondary metabolite biosynthesis. We selected six of these genes for further examination and find that they all show altered expression behavior in the brlA deletion strain. Together, these data support the idea that BrlA plays a role in various biological processes outside asexual development. ImportanceThe Aspergillus nidulans transcription factor BrlA is widely accepted as a master regulator of conidiation. Here, we show that in addition to this function BrlA appears to play a role in responding to cell-wall stress. We note that this has not been observed outside A. nidulans. Further, BrlA-mediated conidiation is highly conserved across Aspergillus species, so this new functionality is likely relevant in other Aspergilli. We identified several transmembrane transporters that have altered transcriptional responses to cell-wall stress in a brlA deletion mutant. Based on our observation, together with what is known about the brlA gene locus regulation, we identify brlA{beta} as the likely intermediary in function of brlA in the response to cell-wall stress.

cell biology↗

bioRxiv2024

The genetic origins and impacts of historical Papuan migrations into Wallacea

The tropical archipelago of Wallacea was first settled by anatomically modern humans (AMH) by 50 thousand years ago (kya), with descendent populations thought to have remained genetically isolated prior to the arrival of Austronesian seafarers around 3.5 kya. Modern Wallaceans exhibit a longitudinal countergradient of Papuan- and Asian-related ancestries widely considered as evidence for mixing between local populations and Austronesian seafarers, though converging multidisciplinary evidence suggests that the Papuan-related component instead comes primarily from back-migrations from New Guinea. Here, we reconstruct Wallacean population genetic history using more than 250 newly reported genomes from 12 Wallacean and three West Papuan populations and confirm that the vast majority of Papuan-related ancestry in Wallacea ([~]75-100%) comes from prehistoric migrations originating in New Guinea and only a minor fraction is attributable to the founding AMH settlers. Mixing between Papuan and local Wallacean lineages appear to have been confined to the western and central parts of the archipelago and likely occurred contemporaneously with the widespread introduction of genes from Austronesian seafarers--which now comprise between [~]40-85% of modern Wallacean ancestry--though dating historical admixture events remains challenging due to mixing continuing into the Historical Period. In conjunction with archaeological and linguistic records, our findings point to a dynamic Wallacean population history that was profoundly reshaped by the spread of Papuan genes, languages, and culture in the past 3,500 years.

evolutionary biology↗

bioRxiv2024

A novel PSMB8 isoform associated with multiple sclerosis lesions induces P-body formation

Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the central nervous system (CNS). Current therapies primarily target the inflammatory component of the disease and are highly effective in early stages of MS while limited therapies have an effect in the more chronic progressive stages of MS where resident glia have a larger role. MS lesions tend to be inflammatory even after the initial peripheral immune cell invasion has subsided and this inflammation is known to cause alternative splicing events. We used qPCR of normal-appearing white matter and white matter lesions from postmortem MS tissue, in vitro studies, and immunostaining in MS tissue to investigate the alternative splicing of one gene known to be important during recovery in an animal model of MS, PSMB8. We found a novel, intron-retained isoform which has not been annotated, upregulated specifically in MS patient white matter lesions. We found that this novel isoform activates the nonsense-mediated decay pathway in primary human astrocytes, the most populous glial cell in the CNS, and is then degraded. Overexpression of this isoform in astrocytes leads to an increased number of processing bodies in vitro, the primary site of mRNA decay. Finally, we demonstrated that MS white matter lesions have a higher burden of processing bodies compared to normal-appearing white matter, predominantly in GFAP-positive astrocytes. The increase in alternative splicing of the PSMB8 gene, the stress that this alternative splicing causes, and the observation that processing bodies are increased in white matter lesions suggests that the lesion microenvironment may lead to increased alternative splicing of many genes. This alternative splicing may blunt the protective or reparative responses of resident glia in and around white matter lesions in MS patients.

molecular biology↗

bioRxiv2023

Post-translational regulation of the Numb/Notch pathway in neurogenesis and cancer by Dlk2

Perturbations in fundamental developmental pathways have a profound influence on tumorigenesis. Numb plays a pivotal role in vertebrate development, including neurogenesis and is a key negative regulator of Notch signaling1, 2. Perturbation of Numb expression affects brain morphology and cell fate3. While extensive research has been conducted on canonical Notch ligands, regulation by vertebrate-specific non-canonical ligands is not understood. Here we identify Delta like non-canonical Notch ligand 2/EGFL9 (Dlk2) as a regulator of zebrafish neurogenesis with mutants exhibiting early increase and subsequent depletion of neural stem cells, decreased radial glial cells density, impaired neuronal cell distribution, and hypersensitivity to stimuli mimicking the embryonic murine Numb/Numblike null phenotype3. Numb function is inactivated by aberrant phosphorylation4, and we show that Dlk2 protein exhibits a high affinity direct interaction with Numb, with loss of Dlk2 in zebrafish telencephalon increasing Numb Ser276 phosphorylation with a concomitant increase in Notch signaling. Patients with tumors exhibiting reduced levels of Dlk2 have a poorer prognosis, while overexpression of Dlk2 in human cancer cell lines reduces cell proliferation. Our findings identify Dlk2 as a key partner of Numb, a gatekeeper of its activity, and an important player in a network of protein interactions regulating both neurogenesis and cancer with potential therapeutic implications.

cell biology↗

bioRxiv2021

Microenvironmental correlates of immune checkpoint inhibitor response in human melanoma brain metastases revealed by T cell receptor and single-cell RNA sequencing

Melanoma-derived brain metastases (MBM) represent an unmet clinical need due to central nervous system (CNS) progression as a frequent, end-stage site of disease. Immune checkpoint inhibition (ICI) represents a clinical opportunity against MBM; however, the MBM tumor microenvironment (TME) has not been fully elucidated in the context of ICI. To dissect unique MBM-TME elements and correlates of MBM-ICI response, we collected 32 fresh MBM and performed single cell RNA sequencing of the MBM-TME and T cell receptor clonotyping on T cells from MBM and matched blood and extracranial lesions. We observed myeloid phenotypic heterogeneity, most notably multiple distinct neutrophil states including an IL-8 expressing population that correlated with malignant cell epithelial-to-mesenchymal transition. Additionally, we observe significant relationships between intracranial T cell phenotypes and the distribution of T cell clonotypes intracranially and peripherally. We found that the phenotype, clonotype, and overall number of MBM-infiltrating T cells were associated with response to ICI, suggesting that ICI-responsive MBMs interact with peripheral blood in a manner similar to extracranial lesions. These data demonstrate unique features of the MBM-TME, which may represent potential targets to improve clinical outcomes for patients with MBM.

cancer biology↗

bioRxiv2021

Single cell based high-throughput Ig and TCR repertoire sequencing analysis in rhesus macaques

Recent advancements in microfluidics and high-throughput sequencing technologies have enabled recovery of paired heavy- and light-chains of immunoglobulins (Ig) and VDJ- and VJ-chains of T cell receptors (TCR) from thousands of single cells simultaneously in humans and mice. Despite rhesus macaques being one of the most well-studied model organisms for the human adaptive immune response, high-throughput single cell immune repertoire sequencing assays are not yet available due to the complexity of these polyclonal receptors. Here we employed custom primers that capture all known rhesus macaque Ig and TCR isotypes and chains that are fully compatible with a commercial solution for single cell immune repertoire profiling. Using these rhesus specific assays, we sequenced Ig and TCR repertoires in over 60,000 cells from cryopreserved rhesus PBMC, splenocytes, and FACS-sorted B and T cells. We were able to recover every Ig isotype and TCR chain, measure clonal expansion in proliferating T cells, and pair Ig and TCR repertoires with gene expression profiles of the same single cells. Our results establish the ability to perform high-throughput immune repertoire analysis in rhesus macaques at the single cell level.

immunology↗

bioRxiv2021

Pseudogene-mediated gene conversion after CRISPR-Cas9 editing demonstrated by partial CD33 conversion with SIGLEC22P

Although gene editing workflows typically consider the possibility of off-target editing, pseudogene-directed homology repair has not, to our knowledge, been reported previously. Here, we employed a CRISPR-Cas9 strategy for targeted excision of exon 2 in CD33 in U937 human monocyte cell line. Candidate clonal cell lines were screened by using a clinically relevant antibody known to label the IgV domain encoded by exon 2 (P67.6, gemtuzumab). In addition to the anticipated deletion of exon 2, we also found unexpected P67.6-negative cell lines which had apparently retained CD33 exon 2. Sequencing revealed that these lines underwent gene conversion from the nearby SIGLEC22P pseudogene during homology repair that resulted in three missense mutations relative to CD33. Ectopic expression studies confirmed that the P67.6 epitope is dependent upon these amino acids. In summation, we report that pseudogene-directed homology repair can lead to aberrant CRISPR gene editing.

cell biology↗

bioRxiv2021

DNA spike-ins enable confident interpretation of SARS-CoV-2 genomic data from amplicon-based sequencing

The rapid global spread and continued evolution of SARS-CoV-2 has highlighted an unprecedented need for viral genomic surveillance and clinical viral sequencing. Amplicon-based sequencing methods provide a sensitive, low-cost and rapid approach but suffer a high potential for contamination, which can undermine lab processes and results. This challenge will only increase with expanding global production of sequences by diverse research groups for epidemiological and clinical interpretation. We present an approach which uses synthetic DNA spike-ins (SDSIs) to track samples and detect inter-sample contamination through a sequencing workflow. Applying this approach to the ARTIC Consortiums amplicon design, we define a series of best practices for Illumina-based sequencing and provide a detailed characterization of approaches to increase sensitivity for low-viral load samples incorporating the SDSIs. We demonstrate the utility and efficiency of the SDSI method amidst a real-time investigation of a suspected hospital cluster of SARS-CoV-2 cases.

molecular biology↗

bioRxiv2018

Recording and reproducing the diurnal oviposition rhythms of wild populations of the soft- and stone- fruit pest Drosophila suzukii

Drosophila suzukii is a horticultural pest on a global scale which causes both yield and economic losses on a range of soft- and stone-fruit. Tackling this pest is problematic but exploiting behavioral rhythms could increase the impact of control. To do this, a better understanding of behavioral patterns is needed. Within this study we aimed to investigate rhythms in reproductive behavior of wild D. suzukii under natural conditions in the field. Environmental parameters were also recorded to decipher how they influence these rhythms. Assays were then performed on laboratory cultures, housed under artificial conditions mimicking the temperature and light cycles, to see if these patterns were reproducible and rhythmic. We were able to promote field like oviposition patterns within the laboratory using realistic temperature and light cycles regardless of variations in other factors including substrate, humidity, and lighting type. Locomotion activity was also recorded under these mimicked conditions to identify how this behavior interacts with oviposition rhythms. Both our field and laboratory assays show that oviposition behavior is likely under the control of the circadian clock and primarily occurs during the day, but these patterns will be disrupted by unfavorable temperature conditions. This was also found within locomotion rhythms. With an increased understanding of how these behaviors are influenced by environmental conditions, we highlight the importance of using realistic temperature and light cycles when investigating behavioral patterns. From an increased understanding of D. suzukii behavior we increase our ability to target the pest in the field.

animal behavior and cognition↗