Innate differences in the molecular signature of normal inferior & superior human parathyroid glands: potential implications for parathyroid adenoma.

Primary hyperparathyroidism is a common endocrine disorder. Interestingly, the majority (75%) of parathyroid tumors are localized to the inferior parathyroid glands. To date, the reason for this natural bias has not been investigated. We assessed the global gene expression profile of superior and inferior glands obtained from forensic autopsies. The genes with significant differential expression between superior and inferior parathyroids were further assessed by RT-PCR in 19 pairs. As an iterative approach, additional genes with an established role in parathyroid disorders, i.e., CASR, MAFB, PAX9, TBCE, TBX1, VDR, MEN1, CCND1, and CDC73 were also evaluated by RT-PCR in all 19 pairs of superior and inferior parathyroid glands. Seven homeobox genes, namely HOXA4, HOXA5, HOXBAS3, HOXB4, HOXB6, HOXB9, IRX1, and one encoding for ALDH1A2 showed a lower expression in the inferior parathyroid glands than in the superior. Conversely, SLC6A1 showed a higher expression in the inferior glands. Of the nine genes with significant differential mRNA expression among superior and inferior glands HOXB9, HOXB4 and IRX1 could be detected by western blotting/mass spectrometry. The study is the first to show the differential expression of nine genes HOXA4, HOXA5, HOXBAS3, HOXB4, HOXB6, HOXB9, IRX1, ALDH1A2, and SLC6A1 in inferior versus the superior parathyroid glands. This could have potential implications for the preferential localization of parathyroid tumors to the inferior parathyroid glands as observed in patients with primary hyperparathyroidism.

Effect of calcitriol and calcium on basal ganglia calcification in hypoparathyroidism: experimental models.

Basal ganglia calcification (BGC) is a common complication in hypoparathyroid patients, linked to hyperphosphatemia and altered vitamin-D and calcium homeostasis following conventional therapy. The pathogenesis of BGC in hypoparathyroidism is not clear. Recently, we developed an ex vivo model of BGC using rat-striatal cell culture in 10.0 mmol/L of ß-glycerophosphate (31.8 mg/dL phosphate). However, the effect of 1,25(OH)2 D, calcium, and milder phosphate excess on BGC in hypoparathyroidism is not known. This study describes two modified ex vivo models investigating pathogenesis of BGC in 'drug-naïve' and 'conventionally treated' hypoparathyroid state. The first modification involved striatal cells cultured in low concentration 1,25(OH)2D (16.0 pg/mL), ionized calcium(0.99 mmol/L), hPTH(1-34) (6.0 pg/mL), and 2.68 mmol/L (8.3 mg/dL) of phosphate akin to 'drug-naïve' state for 24 days. In second modification, striatal cells were exposed to 46.0 pg/mL of 1,25(OH)2D, normal ionized calcium of 1.17 mmol/L, and 2.20 mmol/L (6.8 mg/dL) of phosphate akin to 'conventionally treated' state. Striatal cell culture under 'drug-naïve' state showed that even 16.0 pg/mL of 1,25(OH)2D enhanced the calcification. In 'conventionally treated' model, striatal cell calcification was enhanced in 54% cases over 'drug-naïve' state. Calcification in 'conventionally treated' state further increased on increasing phosphate to 8.3 mg/dL, suggesting importance of phosphatemic control in hypoparathyroid patients. Striatal cells in 'drug-naïve' state showed increased mRNA expression of pro-osteogenic Wnt3a, Cd133,Vglut-1-neuronal phosphate-transporters, calcium-ion channel-Trvp2,Alp, and Collagen-1α and decreased expression of Ca-II. These models suggest that in 'drug-naïve' state, 1,25(OH)2D along with moderately elevated phosphate increases the expression of pro-osteogenic molecules to induce BGC. Although normalization of calcium in 'conventionally treated' state increased the expression of Opg, Osterix, Alp, and Cav2, calcification increased only in a subset, akin to variation in progression of BGC in hypoparathyroid patients on conventional therapy.

Modulation of protein phosphatase 1 gamma 2 during cell division of cervical cancer HeLa cells.

INTRODUCTION: Protein phosphatases (PP) and kinases are known to regulate the cell cycle dynamics. Although kinases have been studied extensively, most of the phosphatases are still unexplored. Therefore, the present study aimed to investigate the association of an isoform of PP1 family protein phosphatases 1 gamma 2 (PP1γ2) in the regulation of cervical cancer HeLa cell proliferation. MATERIAL AND METHODS: Expression of PP1γ2 transcript and protein was assessed in the cervical cancer cell line of HeLa cells through RT-PCR and western blotting. Flow cytometry was employed to confirm its expression quantitatively, and Immuno-fluorescence was done to evaluate the distribution of PP1γ2 in the dividing mononuclear and Taxol-induced multipolar HeLa cells. PP1γ2-specific siRNA-mediated silencing was done to understand downstream pathways. The effect of the hypoxic tumour microenvironment on PP1γ2 expression was also evaluated. RESULTS: RT-PCR and western blotting confirmed the expression of PP1γ2 in HeLa cells, and flow cytometry analysis established intracellular expression of PP1γ2. Immunofluorescence is localized PP1γ2 in the nucleus of mononuclear cells during interphase, whereas it is transiently redistributed to spindle poles throughout the cell division and localized back to the nucleus after complete karyokinesis. Taxol-induced multipolar HeLa cells also showed a temporal redistribution of PP1γ2 on the spindle poles. Hypoxic conditions upregulated PP1γ2 expression, but downregulated PP1γ2 levels through siRNA increased GSK3ß phosphorylation. CONCLUSIONS: Collectively, data suggests that PP1γ2 is modulated during HeLa cell division and regulates GSK3ß phosphorylation, which may regulate downstream signalling of cell division.

Osteogenic Mechanisms of Basal Ganglia Calcification and its ex vivo Model in the Hypoparathyroid Milieu.

CONTEXT: Basal-ganglia calcification (BGC) is common (70%) in patients with chronic hypoparathyroidism. Interestingly, cortical gray matter is spared from calcification. The mechanism of BGC, role of hyperphosphatemia, and modulation of osteogenic molecules by parathyroid hormone (PTH) in its pathogenesis is not clear. OBJECTIVE: We assessed the expression of a large repertoire of molecules with proosteogenic or antiosteogenic effects, including neuroprogenitor cells in caudate, dentate, and cortical gray matter from normal autopsy tissues. The effect of high phosphate and PTH was assessed in an ex vivo model of BGC using striatum tissue culture of the Sprague-Dawley rat. METHODS: The messenger RNA and protein expression of 39 molecules involved in multiple osteogenic pathways were assessed in 25 autopsy tissues using reverse-transcriptase polymerase chain reaction, Western blot, and immunofluorescence. The striatal culture was maintained in a hypoparathyroid milieu for 24 days with and without (a) high phosphate (10-mm ß-glycerophosphate) and (b) PTH(1-34) (50 ng/mL Dulbecco's modified Eagle's medium-F12 media) for their effect on striatal calcification and osteogenic molecules. RESULTS: Procalcification molecules (osteonectin, ß-catenin, klotho, FZD4, NT5E, LRP5, WNT3A, collagen-1α, and SOX2-positive neuroprogenitor stem cells) had significantly higher expression in the caudate than gray matter. Caudate nuclei also had higher expression of antiosteogenic molecules (osteopontin, carbonic anhydrase-II [CA-II], MGP, sclerostin, ISG15, ENPP1, and USP18). In an ex vivo model, striatum culture showed an increased propensity for calcified nodules with mineral deposition similar to that of bone tissue on Fourier-transformed infrared spectroscopy, alizarin, and von Kossa stain. Mineralization in striatal culture was enhanced by high phosphate and decreased by exogenous PTH through increased expression of CA-II. CONCLUSION: This study provides a conceptual advance on the molecular mechanisms of BGC and the possibility of PTH therapy to prevent this complication in a hypoparathyroid milieu.

Central Immune Tolerance of T and B Cells in Patients With Idiopathic Hypoparathyroidism, T1D, and Autoimmune Thyroiditis.

CONTEXT: Pathogenesis of idiopathic hypoparathyroidism (IH) is under investigation. Abnormalities in central immune tolerance have yet not been investigated in this condition. T-cell receptor excision circles (TRECs) and kappa-deleting recombination excision circles (KRECs), formed during receptor gene rearrangements, are tools to assess central T- and B-cell output. OBJECTIVE: We assessed the number of circulating TRECs and KRECs in patients with IH, autoimmune type 1 diabetes (T1D), and autoimmune thyroiditis (ATs) and healthy controls (HCs). DESIGN: Comparative case-control at tertiary care center. SUBJECTS AND METHODS: Absolute and relative TRECs and KRECs were measured in DNA extracted from whole blood of patients with IH (n = 181, 22 of whom were reassessed after a decade of follow-up) and T1D (n = 133), AT (n = 53), and HC (n = 135) using a quantitative real-time PCR/TaqMan® probe technique. RESULTS: Absolute and relative means of TRECs and KRECs in IH were comparable to HCs, and no differences were found between IH with and without calcium-sensing receptor antibodies or class I HLA-A*26:01 association. TRECs and KRECs did not change after a decade of follow-up. T1D had significantly higher absolute TRECs than IH, AT, and HCs, whereas AT patients showed lower TRECs and the highest KRECs; these levels showed no noteworthy correlation with thyroid dysfunctions. CONCLUSION: Patients with IH showed TRECs and KRECs comparable to HCs, indicating an intact mechanism of T- and B-cell central immune tolerance. Interestingly, absolute TRECs were significantly higher in T1D than HCs, suggesting impaired central immune tolerance in T1D.

Absence of vitamin D deficiency among common outdoor workers in Delhi.

BACKGROUND: There is reservation about accepting the notion of widespread vitamin D deficiency (VDD) in sunny countries because information base is largely urban indoors, and the cut-off serum 25(OH)D > 75.0 nmol/L to define sufficiency is perceived as high. OBJECTIVE: We assessed the vitamin D status of subjects engaged in six types of outdoor jobs with freedom to seek shade, when needed. DESIGN: Descriptive observational study. SUBJECTS AND METHODS: A total of 573 outdoors, (hawkers, n = 144; auto-rickshaw drivers, n = 113; manual rickshaw pullers, n = 49; fuel-station attendants, n = 84; gardeners, n = 96; traffic police personnel, n = 87) were assessed for serum 25(OH)D, iPTH and total calcium during summer and winter. Bank employees were indoor controls (n = 72). Serum 25(OH)D was defined as sufficient if ≥50.0 nmol/L and deficient when <30.0 nmol/L, as per 'Institute of Medicine'. RESULTS: Mean serum 25(OH)D of 573 outdoors was 44.8 ± 19.6 nmol/L and showed a physiological inverse relation with iPTH (P < 0.001). 77.5% of the outdoors did not have VDD. Hawkers, gardeners, fuel-station attendants and rickshaw pullers had sufficient or near sufficient serum 25(OH)D. The mean serum 25(OH)D (30.6 ± 23.2 nmol/L) of indoors though lower by 12.7 nmol/L than outdoors was above the cut-off of VDD. Proportions with supranormal iPTH were comparable between outdoors and indoors (14.0% vs 20.8%). Despite winter dip, the mean serum 25(OH)D (31.2 ± 14.3 nmol/l) of outdoors was not deficient. CONCLUSIONS: Vitamin D deficiency is not universal. Most urban outdoor workers do not have VDD.

Fluoride induced tissue hypercalcemia, IL-17 mediated inflammation and apoptosis lead to cardiomyopathy: Ultrastructural and biochemical findings.

An increased prevalence of cardiac complications has been observed in residents of fluorosis endemic areas chronically exposed to fluoride. Fluoride induces soft tissue injury due to oxidative stress, lipid peroxidation (LPO) and mitochondriopathy. It was hypothesized that chronic fluoride exposure induces apoptosis in cardiomyocytes due to inflammation, lysis of extra cellular matrix and altered calcium metabolism. This study was planned to evaluate the effects of chronic fluoride exposure and the mechanism of action in the cardiac muscle. Fifteen week old male Wistar rats were administered a human equivalent dose of fluoride (50 and 100 ppm ad-libitum, HED = 5 & 10 ppm in human) for 75-days. After 75-days of fluoride exposure, the animals were euthanized and fluoride, oxidative stress (SOD, GPX, Catalase activities) and LPO were measured. Histopathological and ultrastructural pathological examinations were conducted on the cardiac tissues using light, atomic force and electron microscopies. The cardiac tissues were also assessed for apoptosis (TUNEL/Caspase assays), and tissue calcium levels (Alizarin-assay and SEM-EDX). Tissue inflammation and expression of IL-17, MMP-9, Caspase-3 and Bcl-2 were evaluated. In the fluoride exposed groups, a significant (≤0.05) increase in levels of oxidative stress, LPO and apoptosis were observed. The IL-17, MMP-9 and Caspase-3 were significantly (≤0.05) higher in the cardiac muscle after chronic fluoride exposure. The fluoride seems to have induced inflammation in the cardiac tissues, as well as an increase in tissue calcium (≤0.05). There was significant damage to cardiac muscle fibres including, thinning, distortion and neo-vasculogenesis following chronic fluoride exposure. Mitochondriopathy, lysis of ground substance, oedema, and hyper-vacuolation was seen in fluoride treated groups. Remarkable levels of distortion and bending in Z band were observed under the AFM. Many of these observed changes mimic those occurring in cardiomegaly, cardiac hypertrophy and cardiomyopathies.

Vitamin D and calcium supplementation, skeletal muscle strength and serum testosterone in young healthy adult males: Randomized control trial.

BACKGROUND: Cholecalciferol and/or calcium supplementation might increase skeletal muscle strength and serum testosterone in young adult males. OBJECTIVE: We performed a randomized control trial assessing the effect of cholecalciferol/calcium on skeletal muscle strength and serum testosterone in vitamin D deficient young males. DESIGN: Two-by-two factorial RCT. SUBJECT AND INTERVENTION: Two-hundred and twenty-eight young males were block-randomized to (i) double-placebo, (ii) calcium/placebo, (iii) cholecalciferol/placebo and (iv) cholecalciferol/calcium. Doses for cholecalciferol were 60 000 IU/wk for 8 weeks followed by 60 000 IU/fortnightly, and doses for elemental calcium were 500 mg/twice daily for 6 months. A total of 180 subjects completed the study protocol. Their  ean age, body mass index and baseline 25(OH)D were 20.2 ± 2.2 years, 23.0 ± 3.6 kg/m2 and 21.5 ± 9.5 nmol/L, respectively. MEASUREMENTS: Handgrip (primary outcome), pinch-grip strength, distance walked in 6 minutes, dyspnoea-score, quality of life by Short Form 36, serum 25(OH)D, 1,25(OH)2 D, iPTH, total testosterone and free androgen index (FAI). RESULTS: After intervention, mean serum 25(OH)D was >75.0 nmol/L in cholecalciferol groups. However, the handgrip strength (29.7 ± 4.4, 29.3 ± 4.6, 30.6 ± 5.0 and 28.8 ± 4.3 kg, P = .28) was comparable in the 4 groups. Subgroups analysis among subjects with baseline serum 25OH)D < 25.0 and <12.0 nmol/L showed similar results. The mean serum testosterone decreased significantly at 6 months; however, delta change was similar in 4 groups. Change in handgrip strength and other outcomes was similar in 4 groups with and without adjustment for delta testosterone and FAI. CONCLUSIONS: Six months of cholecalciferol/calcium supplementation had no significant effect on skeletal muscle strength and serum testosterone in young adult males.

Identification of reference housekeeping-genes for mRNA expression studies in patients with type 1 diabetes.

Selection of appropriate housekeeping-genes as reference is important in mRNA expression-related experiments. It is more important in diabetes since hyperglycemia per se can influence expression of housekeeping-genes. RNA expression of Glyceraldehyde-3-phosphate-dehydrogenase, ß-actin and 18S-ribosomal-RNA, Hypoxanthine-phosphoribosyl-transferase (HPRT), Tyrosine-3-monooxygenase/tryptophan (YHWAZ), ß2-microglobin (ß2M), TATA-binding-protein (TBP), and Ubiquitin C and cytochrome1 (CYC1) assessed in circulating-lymphocytes-(PBMC) of patients with type-1-diabetes and healthy controls. The stability ('M' value <1.02) and number of housekeeping-genes required for normalization in qRT-PCR were determined by 'ge-norm software.' Vitamin-D-receptor (VDR) was used as a target gene. All the nine genes tested had sufficient 'M' value in diabetes and healthy controls. However, housekeeping-genes indicated a relatively higher stability of expression in healthy controls in comparison to diabetes. Use of single housekeeping-genes brought gross variation in the calculation of VDR-mRNA copies. The ge-norm software suggested geometric mean of five housekeeping-genes for ideal normalization in diabetes (CYC1, ß-actin, YHWAZ, HPRT, and ß2M) and only three in controls (CYC1, ß-actin, and TBP). HbA1c did not correlate with expression of any of the nine housekeeping-genes. Thus, geometric mean of CYC1, ß-actin, YHWAZ, HPRT, and ß2M needs to be used for ideal normalization of mRNA in type-1-diabetes. Similar studies are required in other population.

A new type of biocompatible fluorescent probe AFN for fixed and live cell imaging of intracellular lipid droplets.

We discovered a new class of nontoxic, highly fluorogenic and biocompatible probe AFN for selective staining of intracellular Lipid Droplets (LDs) in both fixed and live human cervical cancer cells (HeLa) and 3T3-L1 pre-adipocytes without any background artifacts. The salient features of the probe are its visible excitation maximum, aqueous compatibility, selectivity and remarkable stability (for more than a week) in live cells, even better than commercially available Nile red.

A Novel Methodology for Stripping and Reprobing of Western Blots Originally Developed with Colorimetric Substrate TMB.

Protein blotting is a widely used analytical technique for detection of specific protein(s) in a given sample of tissue or cell homogenate or extract. Both chemiluminescence (CL) and colorimetric detections can be used for imaging protein blots in western blotting. Here we describe a methodology for stripping such western blots, already developed with the colorimetric substrate TMB on nitrocellulose membrane and reprobing the blot (after stripping) for the detection of a second protein through CL. The stripping procedure utilizes a stripping buffer consisting of ß-mercaptoethanol, SDS, and Tris-HCl, while washing buffer consists of PBS added with 0.1 % Tween 20 and involves a series of steps facilitating accurate detection of the second protein in the stripped blot through CL.

A protocol for stripping and reprobing of Western blots originally developed with colorimetric substrate TMB.

Western blotting is a widely used analytical technique for detection of specific protein(s) in a given sample of tissue/cell homogenate or extract. Both chemiluminescence (CL) and colorimetric detections can be used for imaging Western blots. Colorimetric substrates offer background free, sensitive, and clean imaging results directly on the blotted membrane and provides more accurate profile with respect to prestained marker. However, blots stained with colorimetric substrates cannot be reused since no stripping protocols have been reported for such blots, thus limiting their reuse for detection of another protein. In the present study, for the first time, we report a novel method of stripping Western blots developed with the colorimetric substrate TMB for detection of a low-abundant protein and reprobing of these blots after stripping for detection of a more abundant protein through CL procedure. The stripping procedure utilizes a stripping buffer consisting of ß-mercaptoethanol, SDS, and Tris-HCl and a washing buffer consisting of PBS added with 0.1% Tween-20 involves a series of steps and facilitates accurate detection of the second protein (i.e., more abundant protein) in the stripped blot through CL. The protocol is reproducible and facilitates saving of precious clinical samples, in addition to saving cost and time as compared to the existing procedures.