Biology subjects

Thapa, D.

Publications and source records attributed to Thapa, D..

10 recordsLinked to original sources

bioRxiv2026

Multimodal non-ionizing quantitative characterization of enamel hypomineralization

Enamel hypomineralization is clinically graded but difficult to quantify with visual and radiographic assessment. We evaluated whether optical coherence tomography (OCT) and lock-in thermography imaging (LITI) provide non-ionizing, quantitative lesion localization and phenotyping, using clinical mDDE classification as a practical benchmark and micro-CT ({micro}CT) mineral density as an in vitro reference in a subset. Twenty-five extracted first permanent molars were classified as control, hypomineralized (Type 1 white-cream; Type 2 yellow-brown), or other enamel defects. Co-localized OCT A-scan decay slopes and LITI lock-in phase-contrast were measured in predefined ROIs; six teeth underwent {micro}CT with hydroxyapatite calibration. Clinical scoring showed substantial agreement (inter-rater {kappa}=0.61; intra-rater {kappa}=0.70). OCT decay slopes differed across clinical groups (Kruskal-Wallis p=0.00028), separating controls from defect groups, while separation between hypomineralization and other defects was limited. LITI detected thermophotonic "hot spots" at many clinically identified sites (sensitivity 80%) but showed low specificity (20%) at a single modulation frequency, consistent with high anomaly sensitivity but limited etiologic discrimination. In the {micro}CT subset, hypomineralized ROIs showed reduced mineral density relative to adjacent enamel and spatial concordance with OCT and LITI contrast. Together, OCT+LITI support objective lesion mapping and motivate multi-frequency thermophotonics and expanded reference sampling to improve specificity for chairside translation.

biophysics↗

bioRxiv2023

Validation of GCN5L1/BLOC1S1/BLOS1 Antibodies Using Knockout Cells and Tissue

GCN5L1, also known as BLOC1S1 and BLOS1, is a small intracellular protein involved in a number of key biological processes. Over the last decade, GCN5L1 has been implicated in the regulation of protein lysine acetylation, energy metabolism, endo-lysosomal function, and cellular immune pathways. An increasing number of published papers have used commercially-available reagents to interrogate GCN5L1 function. However, in many cases these reagents have not been rigorously validated, leading to potentially misleading results. In this report we tested several commercially-available antibodies for GCN5L1, and found that two-thirds of those available did not unambiguously detect the protein by western blot in cultured mouse cells or ex vivo liver tissue. These data suggest that previously published studies which used these unverified antibodies to measure GCN5L1 protein abundance, in the absence of other independent methods of corroboration, should be interpreted with appropriate caution.

cell biology↗

bioRxiv2022

Reduced acetylation of TFAM promotes bioenergetic dysfunction in the failing heart

General Control of Amino-Acid Synthesis 5-like 1 (GCN5L1) was previously identified as a key regulator of protein lysine acetylation in mitochondria. Subsequent studies demonstrated that GCN5L1 regulates the acetylation status and activity of mitochondrial fuel substrate metabolism enzymes. However, the role of GCN5L1 in response to chronic hemodynamic stress is largely unknown. Here, we show that cardiomyocyte-specific GCN5L1 knockout mice (cGCN5L1 KO) display exacerbated pressure overload-induced heart failure progression following transaortic constriction (TAC). Mitochondrial DNA and mitochondrial electron transport chain protein levels were decreased in cGCN5L1 KO hearts after TAC, and isolated neonatal cardiomyocytes with reduced GCN5L1 expression had lower bioenergetic output in response to hypertrophic stress. Loss of GCN5L1 expression led to a decrease in the acetylation status of mitochondrial transcription factor A (TFAM) after TAC in vivo, which was linked to a reduction in mtDNA levels in vitro. Together, these data suggest that GCN5L1 may protect from hemodynamic stress by maintaining mitochondrial bioenergetic output. HighlightsO_LIReduced GCN5L1 expression in the failing heart promotes contractile dysfunction C_LIO_LIMitochondrial DNA (mtDNA) levels are reduced in cardiomyocyte-specific GCN5L1 knockout mice following hemodynamic stress C_LIO_LIGCN5L1 knockdown reduces, and GCN5L1 overexpression increases, bioenergetic output in hypertrophic cardiomyocytes C_LIO_LIGCN5L1-mediated acetylation of TFAM promotes increased mtDNA levels C_LI

physiology↗

bioRxiv2022

G-PROTEIN COUPLED RECEPTOR 19 (GPR19) KNOCKOUT MICE DISPLAY SEX-DEPENDENT METABOLIC DYSFUNCTION

G-protein coupled receptors (GPCRs) mediate signal transduction from the cellular surface to intracellular metabolic pathways. While the function of many GPCRs has been delineated previously, a significant number require further characterization to elucidate their cellular function. G-protein coupled receptor 19 (GPR19) is a poorly characterized class A GPCR which has been implicated in the regulation of circadian rhythm, tumor metastasis, and mitochondrial homeostasis. In this report, we use a novel knockout (KO) mouse model to examine the role of GPR19 in whole-body metabolic regulation. We show that loss of GPR19 promotes increased energy expenditure and decreased activity in both male and female mice. However, only male GPR19 KO mice display glucose intolerance in response to a high fat diet (HFD). Loss of GPR19 expression in male mice, but not female mice, resulted in diet-induced hepatomegaly, which was associated with decreased expression of key fatty acid oxidation genes in male GPR19 KO livers. Overall, our data suggest that loss of GPR19 impacts whole-body energy metabolism in diet-induced obese mice in a sex-dependent manner.

physiology↗

bioRxiv2021

Diet-induced obese mice are resistant to improvements in cardiac function resulting from short-term adropin treatment

Previous studies have shown that treatment with recombinant adropin, a circulating peptide secreted by the liver and brain, restores glucose utilization in the hearts of diet-induced obese mice. This restoration of fuel substrate flexibility, which is lost in obese and diabetic animals, has the potential to improve contractile function in the diabetic heart. Using an ex vivo approach, we examined whether short-term adropin treatment could enhance cardiac function in a mouse model of diet-induced obesity. Our study showed that acute adropin treatment reduces inhibitory phosphorylation of pyruvate dehydrogenase in primary neonatal cardiomyocytes, and leads to moderate improvements in ex vivo cardiac function in mice fed a low fat diet. Conversely, short-term exposure to adropin led to a small decrease in cardiac function in mice fed a long-term high fat diet. Insulin treatment did not significantly alter cardiac function in adropin treated hearts from either low or high fat diet mice, however acute adropin treatment did moderately restore downstream insulin signaling in high fat diet fed mice. Overall, these data suggest that in an ex vivo setting, acute adropin treatment alone is not sufficient to promote improved cardiac function in obese animals.

physiology↗

bioRxiv2021

GPER-dependent estrogen signaling increases cardiac GCN5L1 expression and MCAD activity

Reversible lysine acetylation regulates the activity of cardiac metabolic enzymes, including those controlling fuel substrate metabolism. Mitochondrial-targeted GCN5L1 and SIRT3 have been shown to regulate the acetylation status of mitochondrial enzymes, which results in alterations to the relative oxidation rates of fatty acids, glucose, and other fuels for contractile activity. However, the role that lysine acetylation plays in driving metabolic differences between male and female hearts is not currently known. In this study, we report that estrogens induce the expression of GCN5L1 via GPER agonism in cardiac cells, which increases the enzymatic activity and acetylation status of the fatty acid oxidation enzyme medium chain acyl-CoA dehydrogenase (MCAD).

physiology↗

bioRxiv2021

Inhibitory Acetylation of Pyruvate Dehydrogenase Promotes Diastolic Dysfunction in Mice

Left ventricular diastolic dysfunction is a structural and functional condition that precedes the development of heart failure with preserved ejection fraction (HFpEF). The etiology of diastolic dysfunction includes alterations in fuel substrate metabolism that negatively impact cardiac bioenergetics, and may precipitate the eventual transition to heart failure. To date, the molecular mechanisms that regulate early changes in fuel metabolism leading to diastolic dysfunction remain unclear. In this report, we use a diet-induced obesity model and quantitative acetylproteomics in aged mice to show that inhibitory lysine acetylation of the pyruvate dehydrogenase (PDH) complex promotes energetic deficits and diastolic dysfunction in mouse hearts. Cardiomyocyte-specific deletion of the mitochondrial lysine acetylation regulatory protein GCN5L1 prevented hyperacetylation of the PDH complex subunit PDHA1, allowing aged obese mice to continue using pyruvate as a bioenergetic substrate in the heart. Our findings suggest that changes in mitochondrial protein lysine acetylation represent a key metabolic component of diastolic dysfunction that precedes the development of heart failure.

physiology↗

bioRxiv2021

Dysfunctional TRPM8 signalling in the vascular response to environmental cold in ageing.

Ageing is associated with increased vulnerability to environmental cold exposure. Previously, we identified the role of the cold-sensitive transient receptor potential (TRP) A1, M8 receptors as vascular cold sensors in mouse skin. We hypothesised that this dynamic cold-sensor system may become dysfunctional in ageing. We show that behavioural and vascular responses to skin local environmental cooling are impaired with even moderate ageing, with reduced TRPM8 gene/protein expression especially. Pharmacological blockade of the residual TRPA1/TRPM8 component substantially diminished the response in aged, compared with young mice. This implies the reliance of the already reduced cold-induced vascular response in ageing mice on remaining TRP receptor activity. Moreover, sympathetic-induced vasoconstriction was reduced with downregulation of the 2c adrenoceptor receptor in ageing. The cold-induced vascular response is important for sensing cold and retaining body heat and health. These findings reveal that cold sensors, essential for this neurovascular pathway, decline as ageing onsets.

biochemistry↗

bioRxiv2021

An enhanced Bouma model fits a hundred people's visual crowding

AbstractCrowding is the failure to recognize an object due to surrounding clutter. Our visual crowding survey measured 13 crowding distances (or "critical spacings") twice in each of 50 observers. The survey included three eccentricities (0, 5, and 10 deg), four cardinal meridians, two orientations (radial and tangential), and two fonts (Sloan and Pelli). The survey also tested foveal acuity, twice. Remarkably, fitting a two-parameter model, the well- known Bouma law -- crowding distance grows linearly with eccentricity -- explains 82% of the variance for all 13 x 50 measured log crowding distances, cross-validated. An enhanced Bouma law, with factors for meridian, crowding orientation, target kind, and observer, explains 94% of the variance, again cross-validated. These additional factors reveal several asymmetries, consistent with previous reports, which can be expressed as crowding- distance ratios: 0.62 horizontal:vertical, 0.79 lower:upper, 0.78 right:left, 0.55 tangential:radial, and 0.78 Sloan font:Pelli font. Across our observers, peripheral crowding is independent of foveal crowding and acuity. Evaluation of the Bouma factor b (the slope of the Bouma law) as a biomarker of visual health would be easier if there were a way to compare results across crowding studies that use different methods. We define a standardized Bouma factor b that corrects for differences from Boumas 25 choice alternatives, 75% threshold criterion, and linearly symmetric flanker placement. For radial crowding on the right meridian, the standardized Bouma factor b is 0.24 for this study, 0.35 for Bouma (1970), and 0.30 for the geometric mean across five representative modern studies, including this one, showing good agreement across labs, including Boumas. We found that guaranteeing fixation by gaze-contingent display halved the standard deviation across observers of the estimated log b. The reduction in standard deviation is explained by a "peeking" model in which the observer looked near an anticipated target location in 50% of unmonitored-fixation trials. Individual differences are robust, as evidenced by the much larger 0.08 SD of log b across observers than the 0.03 SD of test-retest within observers. Crowdings ease of measurement enhances its promise as a biomarker for dyslexia and visual health.

neuroscience↗

bioRxiv2019

Cardiomyocyte-Specific Deletion of GCN5L1 Limits Cardiac Contractile Decline in Obese Pre-Diabetic Mice

Reduced substrate flexibility, and a shift towards increased cardiac fatty acid utilization, is a key feature of obesity and diabetes. We previously reported that increased acetylation of several mitochondrial FAO enzymes, regulated in part via increased abundance of the mitochondrial acetyltransferase GCN5L1, correlated with increased FAO enzyme activity in the heart. The focus of the current study was to investigate whether decreased acetylation, via cardiomyocyte-specific deletion of GCN5L1 (GCN5L1 cKO), regulates cardiac energy metabolism following exposure to a high fat diet (HFD). Excess dietary fat led to similar cardiac hypertrophy in wildtype and GCN5L1 cKO mice. We show that acetylation of pyruvate dehydrogenase (PDH) was significantly reduced in HFD GCN5L1 cKO hearts, which correlated with its increased enzymatic activity relative to HFD wildtype controls. The acetylation of both electron transport chain Complex I protein NDUFB8, and manganese superoxide dismutase 2 (SOD2), was significantly reduced by GCN5L1 deletion in HFD animals, resulting in decreased lipid peroxidation. Finally, we show that in contrast to wildtype mice, GCN5L1 cKO hearts maintain ex vivo contractility and workload in response to a HFD. In summary, we show that GCN5L1 deletion limits cardiac functional decline observed in HFD mice, by increasing fuel substrate flexibility and limiting reactive oxygen species damage.

physiology↗