ORCID Profile
0000-0001-6636-2555
Current Organisations
University of Technology Sydney
,
Kids Research
,
Children's Medical Research Institute
,
WorldIONM
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Publisher: MDPI AG
Date: 11-10-2021
Abstract: Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are two structurally related immunosuppressive peptides. However, the underlying mechanisms through which these peptides regulate microglial activity are not fully understood. Using lipopolysaccharide (LPS) to induce an inflammatory challenge, we tested whether PACAP or VIP differentially affected microglial activation, morphology and cell migration. We found that both peptides attenuated LPS-induced expression of the microglial activation markers Iba1 and iNOS (### p 0.001), as well as the pro-inflammatory mediators IL-1β, IL-6, Itgam and CD68 (### p 0.001). In contrast, treatment with PACAP or VIP exerted distinct effects on microglial morphology and migration. PACAP reversed LPS-induced soma enlargement and increased the percentage of small-sized, rounded cells (54.09% vs. 12.05% in LPS-treated cells), whereas VIP promoted a phenotypic shift towards cell subpopulations with mid-sized, spindle-shaped somata (48.41% vs. 31.36% in LPS-treated cells). Additionally, PACAP was more efficient than VIP in restoring LPS-induced impairment of cell migration and the expression of urokinase plasminogen activator (uPA) in BV2 cells compared with VIP. These results suggest that whilst both PACAP and VIP exert similar immunosuppressive effects in activated BV2 microglia, each peptide triggers distinctive shifts towards phenotypes of differing morphologies and with differing migration capacities.
Publisher: American Society of Hematology
Date: 24-06-2022
DOI: 10.1182/BLOODADVANCES.2022007029
Abstract: Telomere biology disorders (TBDs) are a spectrum of multisystem inherited disorders characterized by bone marrow failure, resulting from mutations in the genes encoding telomerase or other proteins involved in maintaining telomere length and integrity. Pathogenicity of variants in these genes can be hard to evaluate, because TBD mutations show highly variable penetrance and genetic anticipation related to inheritance of shorter telomeres with each generation. Thus, detailed functional analysis of newly identified variants is often essential. Herein, we describe a patient with compound heterozygous variants in the TERT gene, which encodes the catalytic subunit of telomerase, hTERT. This patient had the extremely severe Hoyeraal-Hreidarsson form of TBD, although his heterozygous parents were clinically unaffected. Molecular dynamic modeling and detailed biochemical analyses demonstrate that one allele (L557P) affects association of hTERT with its cognate RNA component hTR, whereas the other (K1050E) affects the binding of telomerase to its DNA substrate and enzyme processivity. Unexpectedly, the data demonstrate a functional interaction between the proteins encoded by the two alleles, with wild-type hTERT rescuing the effect of K1050E on processivity, whereas L557P hTERT does not. These data contribute to the mechanistic understanding of telomerase, indicating that RNA binding in one hTERT molecule affects the processivity of telomere addition by the other molecule. This work emphasizes the importance of functional characterization of TERT variants to reach a definitive molecular diagnosis for patients with TBD, and, in particular, it illustrates the importance of analyzing the effects of compound heterozygous variants in combination, to reveal interallelic effects.
Publisher: Elsevier BV
Date: 2022
DOI: 10.1016/J.GIM.2021.09.001
Abstract: Genetic variants causing aberrant premessenger RNA splicing are increasingly being recognized as causal variants in genetic disorders. In this study, we devise standardized practices for polymerase chain reaction (PCR)-based RNA diagnostics using clinically accessible specimens (blood, fibroblasts, urothelia, biopsy). A total of 74 families with erse monogenic conditions (31% prenatal-congenital onset, 47% early childhood, and 22% teenage-adult onset) were triaged into PCR-based RNA testing, with comparative RNA sequencing for 19 cases. Informative RNA assay data were obtained for 96% of cases, enabling variant reclassification for 75% variants that can be used for genetic counseling (71%), to inform clinical care (32%) and prenatal counseling (41%). Variant-associated mis-splicing was highly reproducible for 28 cases with s les from ≥2 affected in iduals or heterozygotes and 10 cases with ≥2 biospecimens. PCR licons encompassing another segregated heterozygous variant was vital for clinical interpretation of 22 of 79 variants to phase RNA splicing events and discern complete from partial mis-splicing. RNA diagnostics enabled provision of a genetic diagnosis for 64% of recruited cases. PCR-based RNA diagnostics has capacity to analyze 81.3% of clinically significant genes, with long licons providing an advantage over RNA sequencing to phase RNA splicing events. The Australasian Consortium for RNA Diagnostics (SpliceACORD) provide clinically-endorsed, standardized protocols and recommendations for interpreting RNA assay data.
Publisher: MDPI AG
Date: 20-12-2021
Abstract: High-fat diet (HFD)-induced comorbid cognitive and behavioural impairments are thought to be the result of persistent low-grade neuroinflammation. Metformin, a first-line medication for the treatment of type-2 diabetes, seems to ameliorate these comorbidities, but the underlying mechanism(s) are not clear. Pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) are neuroprotective peptides endowed with anti-inflammatory properties. Alterations to the PACAP/VIP system could be pivotal during the development of HFD-induced neuroinflammation. To unveil the pathogenic mechanisms underlying HFD-induced neuroinflammation and assess metformin’s therapeutic activities, (1) we determined if HFD-induced proinflammatory activity was present in vulnerable brain regions associated with the development of comorbid behaviors, (2) investigated if the PACAP/VIP system is altered by HFD, and (3) assessed if metformin rescues such diet-induced neurochemical alterations. C57BL/6J male mice were ided into two groups to receive either standard chow (SC) or HFD for 16 weeks. A further HFD group received metformin (HFD + M) (300 mg/kg BW daily for 5 weeks) via oral gavage. Body weight, fasting glucose, and insulin levels were measured. After 16 weeks, the proinflammatory profile, glial activation markers, and changes within the PI3K/AKT intracellular pathway and the PACAP/VIP system were evaluated by real-time qPCR and/or Western blot in the hypothalamus, hippoc us, prefrontal cortex, and amygdala. Our data showed that HFD causes widespread low-grade neuroinflammation and gliosis, with regional-specific differences across brain regions. HFD also diminished phospho-AKT(Ser473) expression and caused significant disruptions to the PACAP/VIP system. Treatment with metformin attenuated these neuroinflammatory signatures and reversed PI3K/AKT and PACAP/VIP alterations caused by HFD. Altogether, our findings demonstrate that metformin treatment rescues HFD-induced neuroinflammation in vulnerable brain regions, most likely by a mechanism involving the reinstatement of PACAP/VIP system homeostasis. Data also suggests that the PI3K/AKT pathway, at least in part, mediates some of metformin’s beneficial effects.
Publisher: MDPI AG
Date: 31-08-2021
DOI: 10.20944/PREPRINTS202108.0559.V1
Abstract: Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are two structurally-related immunosuppressive peptides. However, the underlying mechanisms through which these peptides regulate microglial activity are not fully understood. Using lipopolysaccharide (LPS) to induce an inflammatory challenge, we tested whether PACAP or VIP differentially affected microglial activation, morphology and cell migration. We found that both peptides attenuated LPS-induced expression of the microglial activation markers Iba1 and iNOS (###p& .001), as well as the pro-inflammatory mediators IL-1& beta , IL-6, Itgam and CD68 (###p& .001). In contrast, treatment with PACAP or VIP exerted distinct effects on microglial morphology and migration. PACAP reversed LPS-induced soma enlargement and increased the percentage of small-sized, rounded cells (54.09% vs 12.05% in LPS-treated cells), whereas VIP promoted a phenotypic shift towards cell subpopulations with mid-sized, spindle-shaped soma (48.41% vs 31.36% in LPS-treated). Additionally, PACAP was more efficient than VIP in restoring LPS-induced impairment of cell migration and the expression of urokinase plasminogen activator (uPA) in BV2 cells compared with VIP. These results suggest that whilst both PACAP and VIP exert similar immunosuppressive effects in activated BV2 microglia, each peptide triggers distinctive shifts towards phenotypes of differing morphologies and with differing migration capacities.
Publisher: Springer Science and Business Media LLC
Date: 13-08-2020
No related grants have been discovered for Aram Niaz.