Biology subjects

Shepherd, P. R.

Publications and source records attributed to Shepherd, P. R..

5 recordsLinked to original sources

bioRxiv2025

Targeting PI3K Signaling for Broad Inhibition of β-Coronavirus Infection

The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays a central role in regulating key cellular processes such as survival, metabolism, and immune responses. Aberrant activation of this pathway is associated with tumorigenesis, and several PI3K inhibitors have been developed as anticancer agents. Emerging evidence suggests that viruses, including {beta}-coronaviruses, have evolved mechanisms to exploit host PI3K signaling for their replication and immune evasion. In this study, we evaluated the antiviral efficacy of a panel of PI3K inhibitors against {beta}-coronaviruses, including mouse hepatitis virus (MHV), human OC43 (HuCoV-OC43) and four major SARS-CoV-2 variants using both cell line and organoid models. Our findings reveal that these compounds exhibit low micromolar potency in inhibiting viral replication. Notably, the inhibitor C20 (PWT33597) demonstrated broad-spectrum activity against multiple {beta}-coronaviruses, including SARS-CoV-2, MHV, and HuCoV-OC43, in conventional cell lines as well as in air-liquid interface (ALI)-cultured, differentiated primary human nasal and bronchial epithelial cells. Given that cytokine storm is a major contributor to SARS-CoV-2-related multiorgan failure and mortality, we further explored the impact of PI3K inhibition on host inflammatory responses. We found that MHV infection markedly increased cytokine expression in 17CL-1 fibroblasts and RAW264.7 macrophages. Interestingly, treatment with C20 further amplified cytokine production in this context, suggesting complex immunomodulatory effects that warrant further investigation. Together, our findings support the therapeutic potential of repurposing PI3K inhibitors as broad-spectrum antivirals. These compounds not only suppress viral replication but may also influence host immune responses, providing a promising avenue for intervention against current and emerging coronavirus threats.

microbiology↗

bioRxiv2021

Glucose regulates expression of pro-inflammatory genes IL-1 and IL-12 through a mechanism involving hexosamine biosynthesis pathway dependent regulation of α-E catenin in the RAW 264.7 macrophage cell line

High glucose levels are associated with changes in macrophage polarization and evidence indicates that the sustained or even short-term high glucose levels modulate inflammatory responses in macrophages. However, the mechanism by which macrophages can sense the changes in glucose levels are not clearly understood. We find that high glucose levels rapidly increase the -E catenin protein level in RAW264.7 macrophages. We also find an attenuation of glucose induced increase of -E catenin when hexosamine biosynthesis pathway is inhibited either with glutamine depletion or with the drugs azaserine and tunicamycin. This indicates the involvement of hexosamine biosynthesis pathway in this process. Then, we investigated the potential role of -E catenin in glucose induced macrophage polarization. We find that the reduction of -E catenin level using siRNA attenuates the glucose induced change of IL-1{beta} mRNA level under LPS stimulated condition. Further, we identified that the depletion of -E catenin also decreases the IL-12 gene expression in basal glucose conditions leading to a reduction of glucose induced changes in IL-12. Together this indicates that -E catenin can sense the changes in glucose levels in macrophages via hexosamine biosynthesis pathway and also can modulate the glucose induced gene expression of inflammatory markers such as IL-1-{beta} and IL-12. This identifies a new part of the mechanism by which macrophages are able to respond to changes in glucose levels.

cell biology↗

bioRxiv2021

Leptin regulates glucose homeostasis via the canonical WNT pathway.

Leptin is a body weight regulatory hormone, but it is arguably even more potent at regulating blood glucose levels. To further our understanding of the molecular mechanisms by which leptin controls glucose homeostasis, we have used transgenic zebrafish models and conditional deletion of beta catenin in the mediobasal hypothalamus of adult mice to show that Wnt signalling in the brain mediates glucoregulatory effects of leptin. In zebrafish, under normal feeding conditions, leptin regulates glucose homeostasis but not adipostasis. In times of nutrient excess, we found that leptin also regulates body weight and size in this species. Using a Wnt signalling reporter fish, we show that leptin directly activates the canonical Wnt pathway in vivo. Pharmacological inhibition of this pathway prevented the leptin-induced improvement in glucose tolerance. In adult mice, conditional deletion of the key Wnt effector molecule, {beta}-catenin, in the mediobasal hypothalamus of male mice confirmed the essential role of the Wnt pathway in mediating leptin action and the neuroendocrine regulation of glucose homeostasis. Adult-onset {beta}-catenin deletion in the mediobasal hypothalamus led to glucose intolerance, exacerbation of caloric intake and body weight gain under high fat diet, as well as resistance to exogenous leptin.

physiology↗

bioRxiv2020

Cetuximab produced from a goat mammary gland expression system is equally efficacious as innovator cetuximab in model systems of cancer.

Humanised monoclonal antibodies have proven a very effective mode of therapy for a wide range of conditions. With many of the monoclonal antibody drugs now coming off patent there is an increasing interest in developing biosimilar, or even biobetter, forms of these drugs. With the commercial competition associated with such generic products there is increasing demand for improved production and purification system for biosimilars. Cetuximab, also known as Erbitux, is widely used in cancer therapy, especially in advanced colorecal cancer, metastatic non-small cell lung cancer and head and neck cancer and it will come off patent in the near future. We have previously reported on a genetically engineered goat system to produce cetuximab (gCetuximab) in milk. Herein we now report on the further charactization of the gCetuximab produced utilizing additional and more sophisticated biological assays. There is similar bioactivity of the gCetuximab compared with the commercial product produced in mammalian cell culture. In particular both cetuximab antibodies selectively target EGFR and induce its internalization to down regulate EGFR signaling. Both forms have very similar half life in animals and in a HT29 colorectal cancer xenograft model have similar efficacy. We also show that the toxin MMAE can be conjugated to gCetuximab, that this targets it to cells and that this results in direct cell killing in HT29 cells. This demonstrates that the gCetuximab will also be a viable vehicle for antibody drug conjugate based therapies. Taken together, this shows that the goat milk monoclonal antibody production system is an effective way of producing a biosimilar form of cetuximab.

bioengineering↗

bioRxiv2017

Discordant association of the CREBRF rs373863828 minor allele with increased body mass index and protection from type 2 diabetes in Maori and Pacific (Polynesian) people living in New Zealand

Aim/HypothesesThe minor allele of CREBRF rs373863828 associates with increased body mass index (BMI) and reduced risk of type 2 diabetes (T2D) in the Samoan population of Samoa and American Samoa. Our aim was to test rs373863828 for association with BMI and odds of T2D, gout and chronic kidney disease (CKD) in M[a]ori and Pacific (Polynesian) people living in Aotearoa New Zealand in 2,286 adults.\n\nMethodsAssociation analyses were performed by linear and logistic regression with BMI, log-transformed BMI, waist circumference, T2D, gout and CKD. Analyses were adjusted for age, sex, the first four genome-wide principal components, and (when appropriate) BMI, waist circumference and T2D.\n\nResultsFor the minor allele of rs373863828 the effect size for log-transformed BMI was 0.038 (95% CI [0.022-0.055], P=4.8x10-6) and for T2D was OR=0.59 (95% CI [0.47-0.73], P=1.9x10-6). There was no evidence for association of genotype with variance in BMI (P=0.13). Nor was there evidence for association with serum urate ({beta}=0.012 mmol/L, Pc=0.10), gout (OR=1.00, P=0.98) or CKD (OR=0.91, P=0.59).\n\nConclusions/interpretationOur results replicated, with very similar effect sizes, association of the minor allele of rs373863828 with higher BMI but lower odds of T2D among New Zealand Polynesian adults, as in Samoan adults living in Samoa and American Samoa.

genetics↗