Biology subjects

Howard, L.

Publications and source records attributed to Howard, L..

10 recordsLinked to original sources

bioRxiv2026

Identification of drug candidates for rescue of SOX17 gene targets in pulmonary arterial hypertension

BackgroundBoth rare and common variants in the SRY-Box Transcription Factor 17 (SOX17) locus are associated with pulmonary arterial hypertension (PAH). SOX17 dysregulation leads to pulmonary artery endothelial cell (PAEC) dysfunction and the obstructive remodelling that characterises PAH. HypothesisImpaired SOX17 expression contributes to the pathogenesis of PAH. Restoring the function of SOX17 or its downstream targets using compounds that mimic its transcriptomic signature will rescue PAEC dysfunction and prevent PAH development. Methods and ResultsWe defined thousands of genes with direct SOX17 genomic binding sites and identified important potential binding partners, including ETS-transcription factors such as ERG by ChIP-seq in PAECs. Through the integration of three PAEC RNA-seq datasets involving overexpression and silencing of SOX17, we defined a robust SOX17 transcriptomic signature. In PAH patients, circulating plasma protein levels of 10 SOX17 signature genes were associated with the SOX17 common risk variants. This included EFNB2 and UNC5B; knockdown of these genes altered the viability and apoptosis of PAECs in response to TNF treatment. The drug-transcriptome database Connectivity Map (CMap) was used to predict novel potential therapeutic compounds to correct the SOX17 transcriptomic signature. Five compounds were selected for in vitro testing and were able to partially reinstate SOX17 target gene expression in PAECs. One compound, BX-912, was selected for in vivo testing as it corrected the levels of multiple target genes, including suppressing Runt-related transcription factor-1 (RUNX1). BX-912 blocked the development of pulmonary hypertension in mice lacking the SOX17 enhancer associated with human disease. ConclusionWe have demonstrated the therapeutic potential of targeting SOX17 in PAH through correction of its gene targets, identifying BX-912 as a lead compound with in vivo efficacy.

pharmacology and toxicology↗

bioRxiv2026

Contrasting Species-Level and Genus Level Disparity Patterns within the ammonoid family Acanthoceratidae

Paleobiologists commonly use genera as a proxy for species in biodiversity studies. However, a lingering concern is that patterns among genera might not always faithfully reflect patterns among species. To date, the concern has focused chiefly on measured patterns of richness over time and on implied origination and extinction rates. However, similar issues might arise for studies of morphological disparity. Moreover, there potentially are additional implications of disparity patterns among species versus those among genera concerning the range of observable anatomical characters and whether disparity within genera is comparable to disparity among genera. If clades have some relatively slowly changing characters that workers have used to denote different genera, then we would expect to see congeneric species to cluster in morphospace; however, if such characters are rare, then within-genus disparity might approach among-genus disparity. Here, we use genus-level and species-level disparity patterns among acanthoceratid ammonoids from the Late Cretaceous. In particular, we examine whether these different level imply different evolutionary dynamics over a major ecological event (Ocean Anoxic Event 2) and how disparity within genera (i.e., among congeneric species) compares to disparity among genera. We find genus-level disparity somewhat inflates early acanthoceratid disparity but implies similar patterns over the OAE2. We also find that within-genus disparity is slightly lower than among-genus, but not hugely so. The combined results suggest that acanthoceratoid shell anatomy does not really show "genus" level characters, even if congeneric species do tend to be more similar to each other than to species in other genera. Thus, this might provide more of a warning for other types of studies using anatomical data (e.g., phylogenetic studies) than for disparity studies. Non-technical SummaryMany paleobiologists use genera to examine scientific questions. This leads to questions over whether this broader approach misses important species-level patterns. This study uses acanthoceratid ammonoids from the Late Cretaceous to examine disparity patterns at both the genus-level and the species-level. We specifically examine the disparity at both levels of this group over a time of high stress for this group, Ocean Anoxic Event 2 (OAE2). Our results show that genus-level disparity slightly exaggerates early acanthoceratid disparity but lowers to a similar pattern to the species-level disparity during OAE2. Within-genus disparity is shown to be slightly lower than among-genus, but not enough to be startling. Together, these results indicate that while some species within the same genus tend to be more alike to each other than those in other genera, there isnt a set of true "genus" level characters. This outcome leads to a warning against using anatomical data in phylogenetic studies, but less so for disparity studies.

paleontology↗

bioRxiv2025

Stromal cells modulate innate immune cell phenotype and function in colorectal cancer via the Sialic acid/Siglec axis

BackgroundThe immunosuppressive tumour microenvironment (TME) reduces immune response effectiveness in stromal-rich tumours, including CMS4 colorectal cancer (CRC). Mesenchymal stromal cells (MSCs), precursors to cancer-associated fibroblasts (CAFs), promote cancer progression by suppressing anti-tumour immune responses. Hypersialylation of glycans on tumours engages Siglec receptors on immune cells, driving immune dysfunction, but its role in stromal-mediated immunosuppression remains unclear. MethodsSialic acids and Siglec ligands were measured on CRC tissue, primary human CAFs, and tumour-conditioned-mesenchymal stromal cells (MSCTCS) and CAF using immunohistochemistry and flow cytometry. The effect of stromal cell sialylation on macrophages and NK cells was assessed in ex vivo primary stromal and immune cell co-cultures and expression of Siglec-10 and immune cell phenotype markers and function were measured by flow cytometry. Using an immunocompetent Balb/c CT26 mouse model, we induced tumours with/without conditioned stromal cells, with/without pre-treatment of stromal cells with sialyltransferase inhibitor (3FAX) or sialidase (E610). We assessed the effect of stromal cell sialylation on macrophages and NK cells in the tumour and secondary lymphoid tissues by flow cytometry. ResultsStromal cells, including CAFs, in CRC tumours are highly sialylated compared to epithelial cancer cells and are associated with high expression of ST6GalNAC6. Genetic knockdown of ST6GalNAC6 reduced the expression of stromal cell Siglec-10 ligands in MSCs. CAFs and MSCTCS induced Siglec-10 on macrophages and NK cells and impaired NK cell cytotoxicity. Sialidase treatment reduced Siglec-10 expression, restoring NK cell function. In vivo, desialylation of stromal cells increased macrophage activation (CD11b+CD80+) and reduced immunosuppressive marker expression (CD206, PD-L1, Siglec-G) in lymphoid tissues, indicating sustained systemic anti-tumour immunity. Intratumoural NK cells exhibited high Siglec-G expression and impaired cytotoxicity, and granzyme B expression significantly increased with sialidase treatment of stromal cells. In an inflammatory tumour model, inflammatory tumour-conditioned (iTCS) MSCs promoted metastasis and Siglec-G induction on NK cells and macrophages, both reversed by sialyltransferase inhibition, underscoring the effects of stromal modulation of innate immune cell function in inflammatory tumours. ConclusionStromal cell sialylation modulates innate immune suppression in CRC via the sialic acid/Siglec axis. Targeting stromal sialylation restores NK cytotoxicity and macrophage activation, offering a novel therapeutic strategy for immunosuppressive stromal-rich tumours. What is already known on this topicO_LIThe tumour microenvironment of consensus molecular subtype 4 (CMS4) colorectal cancer (CRC) is associated with high stromal burden, poor immune infiltration, poor response to anti-cancer therapies and thus poor patient prognosis. Immune checkpoint inhibitors (ICIs) have limited impact on stromal-rich CRC tumours, therefore highlighting the need to discover and target novel mechanisms of tumour immune evasion. C_LIO_LIEmerging studies have highlighted that stromal cells in CRC and pancreatic ductal adenocarcinoma (PDAC) are highly sialylated, expressing even higher levels of sialic acid on their cell surface than epithelial cancer cells. Targeting stromal cell sialylation has unveiled promising data in restoring the anti-tumour activity of T cells and macrophages. There is a need to explore the effects of targeting stromal cell sialylation on other immune cells of the TME and to evaluate the Siglec/sialic acid axis of stromal and immune cells in resistant CRC tumours. C_LI What this study addsO_LIWe reveal ST6GalNAC6 as a sialyltransferase enzyme that regulates the production of Siglec-10 ligands in CRC stromal cells. Overexpression of ST6GalNAC6 and Siglec-10 correlated with poor survival in CRC and mesenchymal CRC tumours. C_LIO_LIWe show for the first time an induction of Siglec-10 expression on macrophages and NK cells in stromal-immune co-culture experimental models with hypersialylated MSCs and CAFs in vitro and ex vivo. Targeting stromal cell sialylation increased NK cell cytotoxicity of CRC cells, indicating a direct functional role for stromal cell sialylation in immunosuppression. C_LIO_LIAn immunogenic mouse model of CRC was used to evaluate the potential therapeutic efficacy of targeting stromal cell sialylation in overcoming stromal cell-mediated immunosuppression in CRC. Sialic acid-targeting of stroma slowed tumour growth and reduced inflammation-driven metastasis. This was associated with greater infiltration and activation of macrophages and NK cells with stromal cell sialic acid depletion, highlighting stromal cell sialylation as a mechanism of innate immune cell suppression in stromal-rich CRC. C_LI How this study might affect Research, Practice or PolicyO_LIOur research provides insight into a novel mechanism of stromal cell-mediated immunosuppression of innate immune cells in CRC and may open up new avenues of research for targeting stromal cells in stromal-rich TMEs such as pancreatic, breast and ovarian cancers. C_LIO_LIOur research identifies a stromal cell effect of enhancing Siglec expression on tumour infiltrating innate immune cells as a novel immune checkpoint, which may be useful in identifying potential novel immunotherapeutic combinations in future. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/642985v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@cbafc3org.highwire.dtl.DTLVardef@80474forg.highwire.dtl.DTLVardef@1c9d050org.highwire.dtl.DTLVardef@8b6551_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

ATP13A3 Variants Promote Pulmonary Arterial Hypertension by Disrupting Polyamine Transport

AimsPotential loss-of-function variants of ATP13A3, the gene encoding a P5B-type transport ATPase of undefined function, were recently identified in pulmonary arterial hypertension (PAH) patients. ATP13A3 is implicated in polyamine transport but its function has not been fully elucidated. Here, we sought to determine the biological function of ATP13A3 in vascular endothelial cells and how PAH-associated mutations may contribute to disease pathogenesis. We also generated mice harbouring an Atp13a3 variant analogous to a human disease-associated variant to establish whether these mice develop PAH. Methods and ResultsWe studied the impact of ATP13A3 deficiency and overexpression in endothelial cell (EC) models (human pulmonary ECs, blood outgrowth ECs (BOECs) and HMEC-1 cells), including a PAH patient-derived BOEC line harbouring an ATP13A3 variant (LK726X). ATP13A3 localised to the recycling endosomes of human ECs. Knockdown of ATP13A3 in ECs generally reduced the basal polyamine content, consistently reduced putrescine uptake, and altered the expression of enzymes involved in polyamine metabolism. Conversely, overexpression of wild-type ATP13A3 increased polyamine uptake, with an overall preference of putrescine > spermidine > spermine. Functionally, loss of ATP13A3 was associated with reduced EC proliferation, increased apoptosis in serum starvation and increased monolayer permeability to thrombin. Assessment of five PAH-associated missense ATP13A3 variants (L675V, M850I, V855M, R858H, L956P) confirmed loss-of-function phenotypes represented by impaired polyamine transport and dysregulated EC function. Furthermore, mice carrying a heterozygous germ-line Atp13a3 frameshift variant representing a human mutation spontaneously developed a PAH phenotype, with increased pulmonary pressures, right ventricular remodelling and muscularisation of pulmonary vessels. ConclusionWe identify ATP13A3 as a polyamine transporter, deficiency of which leads to EC dysfunction and predisposes to PAH. This suggests a need for targeted therapies to alleviate the imbalances in polyamine homeostasis and EC dysfunction in PAH. Translational perspectiveRare missense ATP13A3 disease-associated variants have been identified in patients with pulmonary arterial hypertension (PAH), though their pathogenicity has not been confirmed as the function of ATP13A3 is not known. We have identified ATP13A3 as a polyamine transporter, showing that ATP13A3 deficiency impaired polyamine homeostasis and uptake, and drove endothelial dysfunction. Conversely, overexpression increased polyamine uptake and rescued the proapoptotic phenotype of cells harbouring a disease-associate variant. Mice heterozygous for a disease-associated Atp13a3 mutation spontaneously develop PAH. These findings support the rationale for exploring dysregulated polyamine homeostasis in PAH and suggest a potential for therapeutic targeting of polyamine pathways in PAH.

cell biology↗

bioRxiv2022

Application of Quasimetagenomics Methods to Define Microbial Diversity and Subtype Listeria monocytogenes in Dairy and Seafood Production Facilities

Microorganisms frequently colonize surfaces and equipment within food production facilities. Listeria monocytogenes is a ubiquitous foodborne pathogen widely distributed in food production environments and is the target of numerous control and prevention procedures. Detection of L. monocytogenes in a food production setting requires culture dependent methods, but the complex dynamics of bacterial interactions within these environments and their impact on pathogen detection remains largely unexplored. To address this challenge, we applied both 16S rRNA and shotgun quasimetagenomic (enriched microbiome) sequencing of swab culture enrichments from seafood and dairy production environments. Utilizing 16S rRNA amplicon sequencing, we observed variability between samples taken from different production facilities and a distinctive microbiome for each environment. With shotgun quasimetagenomic sequencing, we were able to assemble L. monocytogenes metagenome assembled genomes (MAGs) and compare these MAGSs to their previously sequenced whole genome sequencing (WGS) assemblies, which resulted in two polyphyletic clades (lineages I and II). Using these same datasets together with in silico downsampling to produce a titration series of proportional abundances of L. monocytogenes, we were able to begin to establish limits for Listeria detection and subtyping using shotgun quasimetagenomics. This study contributes to the understanding of microbial diversity within food production environments and presents insights into how many reads or relative abundance is needed in a metagenome sequencing dataset to detect, subtype, and source track at a SNP level, as well as providing an important foundation for utilizing metagenomics to mitigate unfavorable occurrences along the farm to fork continuum. IMPORTANCEIn developed countries, the human diet is predominantly food commodities, which have been manufactured, processed, and stored in a food production facility. It is well known that the pathogen Listeria monocytogenes is frequently isolated from food production facilities and can cause serious illness to susceptible populations. Multistate outbreaks of L. monocytogenes over the last 10 years have been attributed to food commodities manufactured and processed in production facilities, especially those dealing with dairy products such as cheese and ice cream. A myriad of recalls due to possible L. monocytogenes contamination have also been issued for seafood commodities originating from production facilities. It is critical to public health that the means of growth, survival and spread of Listeria in food production ecosystems is investigated with developing technologies, such as 16S rRNA and quasimetagenomic sequencing, to aid in the development of effective control methods.

microbiology↗

bioRxiv2022

Indigenous Cultural Safety Training for Applied Health, Social Work and Education Professionals: A PRISMA Scoping Review

BackgroundAnti-Indigenous racism is a widespread social problem in health, social work, and education systems in English-speaking Colonized countries such as Canada, with profound negative impacts to the health and education of Indigenous peoples. In 2015, Canadas Truth and Reconciliation Commission recognized the legacy and impact of Colonization and recommended training programs for these professions on cultural competency and curricula, and on the colonial history of Canada. Yet there is little evidence on best practices for such training, highlighting the need to synthesize existing findings on how these training programs are developed, implemented, and evaluated. MethodsThis scoping review explored the academic literature on Indigenous cultural safety and competence training in the health, social work, and education fields. Medline, EMBASE, CINAHL, ERIC and ASSIA were searched for articles published between 1996-2020 in Canada, United States, Australia, and New Zealand. The Joanna Briggs Institutes three-step search strategy was used as was the PRISMA extension for Scoping Reviews. Data was charted and synthesized in three stages. Results134 were included in this review. Data was extracted on four themes: 1) Article Characteristic; 2) Cultural Safety Concepts, Critiques and Rationale; 3) Characteristics of Cultural Safety Training; and 4) Evaluation Details of Cultural Safety Training. Findings suggest that research on cultural safety training programs in health, social work and education has grown significantly. Nursing and medicine professions have received a significant proportion of cultural training programs, compared with general/allied health, social work, and education. Across fields, professionals and students were targeted equally by training programs. Only half of evaluations of cultural safety and related intervention identified methodological limitations. ImplicationsConsidering, comparing, and contrasting literature on cultural safety and related concepts and how they are applied in practice would advance this scholarly work, as would more robust evaluations of cultural safety and similar training interventions to understand their impact at the individual level. Finally, commitment to meaningfully engage Indigenous communities to develop, implement and evaluate such programs is urgently needed.

scientific communication and education↗

bioRxiv2022

Serum metabolomic analysis of men on a low-carbohydrate diet for biochemically recurrent prostate cancer reveal the potential role of ketogenesis to slow tumor growth: A secondary analysis of the CAPS2 diet trial

BackgroundSystemic treatments for prostate cancer (PC) have significant side effects. Thus, newer alternatives with fewer side effects are urgently needed. Animal and human studies suggest the therapeutic potential of low carbohydrate diet (LCD) for PC. To test this possibility, Carbohydrate and Prostate Study 2 (CAPS2) trial was conducted in PC patients with biochemical recurrence (BCR) after local treatment to determine the effect of a 6-month LCD intervention vs. usual care control on PC growth as measured by PSA doubling time (PSADT). We previously reported the LCD intervention led to significant weight loss, higher HDL, and lower triglycerides and HbA1c with a suggested longer PSADT. However, the metabolic basis of these effects are unknown. MethodsTo identify the potential metabolic basis of effects of LCD on PSADT, serum metabolomic analysis was performed using baseline, month 3, and month 6 banked sera to identify the metabolites significantly altered by LCD and that correlated with varying PSADT. ResultsLCD increased the serum levels of ketone bodies, glycine and hydroxyisocaproic acid. Reciprocally, LCD reduced the serum levels of alanine, cytidine, asymmetric dimethylarginine (ADMA) and 2-oxobutanoate. As high ADMA level is shown to inhibit nitric oxide (NO) signaling and contribute to various cardiovascular diseases, the ADMA repression under LCD may contribute to the LCD-associated health benefit. Regression analysis of the PSADT revealed a correlation between longer PSADT with higher level of 2-hydroxybutyric acids, ketone bodies, citrate and malate. Longer PSADT was also associated with LCD reduced nicotinamide, fructose-1, 6-biphosphate (FBP) and 2-oxobutanoate. ConclusionThese results suggest a potential association of ketogenesis and TCA metabolites with slower PC growth and conversely glycolysis with faster PC growth. The link of high ketone bodies with longer PSADT supports future studies of ketogenic diets to slow PC growth.

cancer biology↗

bioRxiv2021

Non-canonical role of Hippo tumor suppressor serine/threonine kinase 3 STK3 in prostate cancer

Serine/threonine kinase 3 (STK3) is an essential member of the highly conserved Hippo Tumor suppressor pathway which regulates Yes 1 Associated protein (YAP1) and TAZ. STK3 and its paralog STK4 initiate a phosphorylation cascade that regulate YAP1/TAZ activation and degradation, which is important for regulated cell growth and organ size. Deregulation of this pathway leads to hyper-activation of YAP1 in various cancers. Counter to the canonical tumor suppression role of STK3, we report that in the context of prostate cancer (PC), STK3 has a pro-tumorigenic role. Our investigation started with the observation that STK3, but not STK4, is frequently amplified in PC. A high STK3 expression is associated with decreased overall survival and positively correlates with androgen receptor (AR) activity in metastatic castrate resistant PC. XMU-MP-1, an STK3/4 inhibitor, slowed cell proliferation, spheroid growth and matrigel invasion in multiple models. Genetic depletion of STK3 decreased proliferation in several PC cell lines. In a syngeneic allograft model, STK3 loss slowed tumor growth kinetics in vivo and biochemical analysis suggest a mitotic growth arrest phenotype. To further probe the role of STK3 in PC, we identified and validated a new set of selective STK3 inhibitors, with enhanced kinase selectivity relative XMU-MP-1, that inhibited tumor spheroid growth and invasion. Consistent with the canonical role, inhibition of STK3 induced cardiomyocyte growth and had chemo-protective effects. Our results contend that STK3 has a non-canonical role in PC progression and inhibition of STK3 may have therapeutic potential for PC that merits further investigation. SignificanceOur findings illuminate a new actionable target for PC therapy that would traditionally be overlooked due to its canonical role as a tumor suppressor in other cancer types.

cancer biology↗

bioRxiv2018

Endothelial Cells Filopodia Participation In The Anastomosis Of CNS Capillaries

By combining the classic Golgi method and the electron microscope, we have gained a better understanding of the anastomosis of CNS blood capillaries. The participation of growing capillary leading endothelial cells filopodia in the anastomotic process is described. The two approaching capillaries leading endothelial cells filopodia intermingle and interact forming complex conglomerates with narrow spaces filled with proteinaceous material (possibly basal lamina) secreted by them. The presence of tight junctions among the filopodia corroborates their vascular nature. Their presence also suggests a different endothelial cells origin as will those from the two approaching capillaries. The original narrow spaces coalesce into larger ones leading to the eventual formation of a single one that will interconnect (anastomose) the two capillaries. The newly formed post-anastomotic CNS capillaries are rather small with irregular and narrow lumina that might permit the passage of fluid but not yet of blood cells. Eventually, the new capillaries lumina will enlarge permitting the passage of blood cells.\n\nFunding informationThe M. M-P. Golgi studies were supported by a \"Jacob Javits Neurosciences Investigator Award\". NIH Grant NS-22897. And the L. H. EM studies were supported by the Gilman Fund/Class of 1978 Life Sciences Center. M. M-P. is Emeritus Professor of Pathology and Pediatrics and L. H. is a Consulting Electron Microscopists. Both from the Geisel School of Medicine at Dartmouth. Hanover, NH 03755, USA

neuroscience↗

bioRxiv2017

Novel causative genes for heritable pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a rare disorder with a poor prognosis. Deleterious variation within components of the transforming growth factor-{beta} pathway, particularly the bone morphogenetic protein type 2 receptor (BMPR2), underlie most heritable forms of PAH. Since the missing heritability likely involves genetic variation confined to small numbers of cases, we performed whole genome sequencing in 1038 PAH index cases and 6385 PAH-negative control subjects. Case-control analyses revealed significant overrepresentation of rare variants in novel genes, namely ATP13A3, AQP1 and SOX17, and provided independent validation of a critical role for GDF2 in PAH. We provide evidence for familial segregation of mutations in SOX17 and AQP1 with PAH. Mutations in GDF2, encoding a BMPR2 ligand, led to reduced secretion from transfected cells. In addition, we identified pathogenic mutations in the majority of previously reported PAH genes, and provide evidence for further putative genes. Taken together these findings provide new insights into the molecular basis of PAH and indicate unexplored pathways for therapeutic intervention.

genetics↗