Insights into the Age-Dependent Variation in Nutrition-Related Trace Elements in Diabetes Blood Using Total Reflection X-Ray Fluorescence.

The prevalence of diabetes has reached alarming levels in India, making it essential to understand the concentration of nutritional-trace elements (Fe, Cu, Zn, Cr. and Se) in blood samples from diabetic adults. In this study, 208 whole blood samples from diabetic (n = 104) and non-diabetic (n = 104) adults across various age groups were analyzed using total reflection X-ray fluorescence (TXRF) spectroscopy with a sample dilution method. Statistical analysis was performed to assess descriptive statistics and determine a significant correlation between elemental concentrations in the blood samples of diabetic and non-diabetic adults. The mean concentration of nutritional-related trace elements in diabetic blood was as follows: Fe (46 ± 5) > Zn (1.28 ± 0.14) > Cu (0.10 ± 0.01) > Cr (0.05 ± 0.004) > Se (0.013 ± 0.001) in mg/L, respectively. Additionally, this study investigated the influence of nutrition-related trace element concentrations across various age groups such as 25-40 years (young adults), 41-55 years (middle-aged adults), and 56-70 years (older adults). In this investigation, Zn (p < 0.001) and Cr (p < 0.05) concentrations differed significantly between diabetic and non-diabetic adults aged 56-70 years. These findings will help us to understand age-dependent changes in element concentrations, clarify their role in diabetes, and improve risk factor management associated with diabetes.

Ultrasound-driven facile fabrication of Pd doped SnO2 hierarchical superstructures: Structural, growth mechanism, dermatoglyphics, and anti-cancer activity.

This research introduces a novel method that leverages Spirulina extract (S.E) as a bio-surfactant in the ultrasound-assisted synthesis (UAS) of Pd3+ (0.25-10 mol%) doped tin oxide (SnO2) self-assembled superstructures. Nanotechnology has witnessed significant advancements in recent years, driven by the exploration of novel synthesis methods and the development of advanced nanomaterials tailored for specific applications. Metal oxide nanoparticles, particularly SnO2, have garnered considerable attention due to their versatile properties and potential applications in various fields, including gas sensing, catalysis, and biomedical engineering. The study explores how varying influential parameters like S.E concentration, sonication time, pH, and sonication power can influence the resulting superstructures' morphology, size, and shape. A theoretical model for forming different hierarchical superstructures (HS) is proposed. X-ray diffraction (XRD) analysis confirms the crystalline tetragonal rutile phase of the SnO2:Pd HS. Raman spectroscopy reveals a red shift in the A1g mode, indicating phonon confinement due to various defects in the SnO2 structure. Further characterization using transmission electron microscopy (TEM), scanning electron microscopy (SEM), and X-ray photoelectron spectroscopy (XPS) provides insights into particle size, surface morphology, elemental composition, and binding energy. The study also demonstrates the application of optimized SnO2:3Pd HS in developing latent fingerprints (LFPs) on different surfaces using a simple powder dusting (PD) method, with the fingerprints (FPs) visualized under normal light. A mathematical model developed in Python-based software is used to analyze various features of the developed FPs, including pore properties such as number, position, inter-spacing, area, and shape. Additionally, an in vitro MTT assay shows concentration-dependent anticancer activity of SnO2:3Pd nanoparticles (NPs) on MCF7 cell lines, highlighting their potential as a promising cancer treatment option. Overall, the study suggests that the optimized HS can serve as multifunctional platforms for biomedical and dermatoglyphics applications, demonstrating the versatility and potential of the synthesized materials.

Elevation of gene expression of calcineurin, calmodulin and calsyntenin in oxidative stress induced PC12 cells.

In normal physiological conditions, reactive oxygen and nitrogen species are used as important signaling molecules in the cell. However, in excess it causes the disruption of cell resulting in their death. Oxidative stress causes influx in intracellular calcium levels leading to higher concentrations of calcium in the cell. This accelerated calcium affects both the mitochondria and nuclei leading to excitotoxicity in neurons. Intracellular calcium levels are controlled by voltage dependent calcium channels located in the plasma membrane, calcium stores like endoplasmic/sarcoplasmic reticulum and majorly by calcium binding proteins. Our study was aimed at analyzing the gene expression of major calcium binding proteins namely calcineurin, calmodulin, calreticulin, synaptotagamin and calsyntenin in stress induced PC 12 cells. Rotenone (1 µM), Peroxynitrite (10 µM), H2O2 (100 µM) and High glucose (33 mM) were used to induce oxidative stress in PC12 cells. Results obtained from the study suggest that calcineurin, calmodulin and calsyntenin gene expression were enhanced compared to the control due to oxidative stress. However, synaptotagmin and calreticulin gene expression were down regulated. Further, Akt protein expression (stress marker) was enhanced in PC12 cells with all other stress inducers except in hyperglycemic condition.

Peroxynitrite-induced conformational changes in DNA that lead to cell death: UV, CD spectral, molecular dynamics simulation and FACS analysis.

Peroxynitrite is known to react with biomolecules leading to their structural and function alteration. Structural alteration in DNA induced by peroxynitrite is not clearly known. The current study was carried out to decipher the changes induced by peroxynitrite using UV-Vis spectra, circular dichrometry, molecular dynamics simulation followed by restriction digestion. Apoptotic markers Bax, Bcl-2 and caspase genes were also studied by FACS in peroxynitrite induced PC12 cells. The results obtained showed that PXN binds to DNA leading to hyperchromicity of DNA in the presence of PXN over a period of time and the same was established by In silico studies where PXN modifies the DNA to accommodate itself into the stacking and brings about the significant structural alterations. Further, FACS studies reveal that Bcl-2 gene expression was down regulated whereas BAXand caspase genes were up regulated compared to control concluding that PXN induces apoptotic cell death in PC12 cells.

Forskolin and Phorbol 12-myristate 13-acetate modulates the expression pattern of AP-1 factors and cell cycle regulators in estrogen-responsive MCF-7 cells.

Activator protein-1 (AP-1) transcription factor is a key component of many signal transduction pathways involved in the regulation of cellular processes and controls rapid responses of mammalian cells when exposed to the variety of stimulus. The phorbol 12-myristate 13-acetate and Forskolin (Fo) are well-known kinase activators/stimulators of Protein Kinase C (PKC) and Protein Kinase A (PKA) respectively. Importantly, these kinases are found to be present in transitional points of many cell signaling pathways, especially those involved in proliferation. The stimulating effect of PKC and PKA on the expression of AP-1 factors in MCF-7 breast cell proliferation is not well characterized. Hence, the role of PKC by PMA treatment and the role of PKA by using Fo in MCF-7 cells is investigated. Where, cells treated with PMA showed increased cell proliferation, while Fo had no effect, but inhibited the PMA induced proliferation. The RT-PCR results showed the PMA induced c-Jun, c-Fos and Fra-1 expressions compared to control and Fo. However, Fo in combination with PMA, inhibit the PMA induced above mRNA expressions where Fo alone has no effect. Western blot studies validated the c-Jun expressions in PMA treated MCF-7 cells. Further, PMA increases the mRNA expression of Cyclin-E1, Cyclin-D1, and CDK-4, whereas Fo decreases their expressions. Thus, mitogenic effect of PMA and inhibitory action of Fo on MCF-7 cells is probably enhanced via activation of AP-1 factors and concomitant action of cell cycle regulators in the downstream singling cascade.

Genetic Screening of Selected Disease-Causing Mutations in Glutaryl-CoA Dehydrogenase Gene among Indian Patients with Glutaric Aciduria Type I.

Glutaric aciduria type I (GA-I) is an organic aciduria caused by glutaryl-CoA dehydrogenase (GCDH) deficiency. There are limited studies on GA-I from India. A total of 48 Indian GA-I patients were screened for selected disease-causing mutations such as R402W, A421V, A293T, R227P, and V400M using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). Among these patients, 9 (18.8%) had R402W mutation, and none had A421V, A293T, R227P, or V400M mutation. One low excretor mutation (P286S) and several novel mutations (I152M, Q144P, and E414X) were also found in this study. We conclude that among selected mutations, R402W is the most common mutation found among Indian GA-I patients.

Discovery of a latent calcineurin inhibitory peptide from its autoinhibitory domain by docking, dynamic simulation, and in vitro methods.

Autoinhibitory domain (AID) of calcineurin (CN) was discovered two decades ago. Fewer investigations are reported to find out shortest possible peptide from the AID for CN inhibition. Hence, this study has focused on screening of nearly 150 peptide fragments derived from the AID using in silico method. Therefore, we have employed docking studies, aiming to analyze the best pose of AID-derived peptides on CN active site. We also analyzed binding free energy (ΔG) of docked complex using molecular mechanics/generalized Born surface area (MM/GBSA). MM/GBSA predicts two short peptides P1 and P2 found to be lowest binding free energy. Two peptides exhibit better binding affinity with CN, suggests that the possible candidates for potential CN inhibition. Further, the stability of the docked complex was analyzed using molecular dynamic (MD) simulation. MD study shows that CNA:P2 is the most stable complex than CN A:P1 and CN A:AID. Besides, we have synthesized and purified P1 and P2 peptides over high performance liquid chromatography (HPLC) found to be 90.31% and 98.93% of purity, respectively. In addition, AID peptides were characterized over mass spectral analysis. Peptides were subjected to CN inhibitory assay using malachite green method. Where, P1 and P2 exhibit CN inhibition better than AID. In particular, shortest peptide P2 shows highest inhibitory activity than AID. Enzyme assay reveals CN inhibitory activity of P2 peptide is consistent within silico results. In silico and in vitro, results corroborated each other to confirm short peptide P2 can be used as a potential CN inhibitor.

A novel assay method for calcium calmodulin dependent phosphatase from bovine brain extract.

Calcium calmodulin dependent protein ser/thr phosphatase, also referred to as protein phosphatase 2B (PP2B), is rich in neural tissue, and plays an important role in the overall function of the nervous system. Routinely phosphatase assay employs, para-Nitrophenlylphosphate (p-NPP), as a substrate, is also extended to assay PP2B. However, in the present study, the differential spectral characterstic property of tyrosine and phopshotyrosine has been exploited to employ the latter as a candidate substrate for the PP2B assay. The specific activity of PP2B using phosphortyrosine in bovine Bos Taurus indicus brain extract (Bos Taurus indicus), was measured in presence of different metal ions like Ca(2+), Mn(2+) and Mg(2+). Further modulators like dithiothreitol (DTT), calmodulin (CaM) and metal chelators such as EGTA and EDTA were applied to confirm the role of divalent cations and to determine calcium calmodulin dependent phoshphatase activity. PP2B activity was higher with phosphotyrosine in presence of Ca(2+) than with p-NPP. Further experiments, involving calmodulin as a modulator, confirmed phosphotyrosine as a better substrate over p-NPP. Calmodulin further enhanced the effect of phosphotyrosine as a potential substrate confirming calcium calmodulin dependent phosphatase activity. Phosphotyrosine is proposed as a better substrate in assaying calcium dependent phosphatase activity when compared to para-nitrophenylphosphate.

Effect of estrogen and tamoxifen on the expression pattern of AP-1 factors in MCF-7 cells: role of c-Jun, c-Fos, and Fra-1 in cell cycle regulation.

The activated transcription factor ERα plays an important role in the breast development and progression of cancer. In a non-classical pathway ER interacts with other transcription factors AP-1, NFkB, SP1, etc. AP-1 transcription factors control rapid responses of mammalian cells to stimuli that impact proliferation, differentiation, and transformation. AP-1 factors are leucine zipper proteins belonging to members of the Jun family (c-Jun, JunB, and JunD) and Fos family (c-Fos, FosB, Fra-1, and Fra-2) proteins. Although AP-1 factors are well characterized, not much is known about the expression pattern of the AP-1 factors in breast cancer cells. Hence to determine which AP-1 factors are expressed and regulated by estrogen, we used human breast cancer MCF-7 cells as in vitro model system. The MCF-7 cells were treated with or without estradiol-17ß (E2) or antiestrogen tamoxifen (TMX) and the cell proliferation and viability was assessed by MTT assay. The expression of different AP-1 factors was analyzed by semi-quantitative RT-PCR. The cells treated with E2 found to increase the cell proliferation by more than 35 % and TMX an antiestrogen decreased by 29 % compared to control. The E2 found to induce the expression of c-Jun, Fra-1, and c-Fos, while TMX decreased the expression. In addition TMX also decreased the mRNA levels of Jun-D and Fra-2. These results suggest that the AP-1 factors c-Jun, c-Fos, and Fra-1 may be involved in the proliferation and transformation of MCF-7 cells. E2 also found to induce cyclin D1 and cyclin E1 mRNA transcripts of cell cycle regulators while TMX significantly decreased compared to control. Further E2 induced the anti-apoptotic Bcl-2 and TMX decreased mRNA transcripts. The data presented here support the E2-ERα-mediated MCF-7 cell proliferation and confirms the role of AP-1 factors in cell cycle regulation.

A sensitive assay for ornithine amino transferase in rat brain mitochondria by ninhydrin method.

To establish/develop an assay method for measuring Ornithine Aminotransferase (EC.2.6.1.13) activity using rat brain mitochondria as a source of enzyme in presence and absence of Pyridoxal Phosphate (PLP). The modified method, with the improved sensitivity, is adopted for the assay of ornithine amino transferase activity in rat brain mitochondria. The enzyme activity was measured at 620 nm, the study showed that reaction was optimum at 37°C for 30 minutes. The assay is sensitive enough to detect activity at the order of nanomoles pyrroline-5-carboxylate/mg protein/minute and can be compared as an alternative to the radio isotopic method which is more cumbersome and aminobenzaldehyde method which is less sensitive. The K(m) & V(max) shows maximum activity in the presence of Pyridoxal Phosphate (Coenzyme) concentration at 0.05mM when compared with absence of Pyridoxal Phosphate as higher the concentration of Pyridoxal Phosphate affects the affinity of the enzyme to substrate. The OAT activity in different tissues of the rat was also studied and highest activity was found in liver and kidney.

Effect of pH on spectral characteristics of P5C-ninhydrin derivative: Application in the assay of ornithine amino transferase activity from tissue lysate.

Currently available method(s) for assaying pyrroline-5-carboxylate (P5C), an important intermediate metabolite of ornithine, proline and glutamate metabolic pathways, are cumbersome or not sensitive enough for microanalysis. The present study involving the synthesis of P5C followed by purity check, molecular mass (amu =113.1) determination by mass spectrometry and spectral characterization of P5C-ninhydrin derivative (λ max: 510 nm) confirmed the authenticity of the preparation. Studies on the effect of pH on spectral characteristics of P5C ninhydrin derivative demonstrated a significant change with respect to λ max (620 nm) and several ∼ 12 fold increase in molar extinction coefficient (ε: 1.96 × 10(5)) in alkaline conditions (pH:7.0-8.0) as compared to the reported Molar ε of 1.65 × 10(4) at max λ 510 nm in ethanolic solution. The modified method, with the improved sensitivity, is adopted for the assay of ornithine amino transferase activity in WBC's/platelets lysate(s) from human blood.