Profiling the Hsp70 Chaperone Network in Heat-Induced Proteotoxic Stress Models of Human Neurons.

Heat stroke is among the most hazardous hyperthermia-related illnesses and an emerging threat to humans from climate change. Acute brain injury and long-lasting brain damage are the hallmarks of this condition. Hyperthermic neurological manifestations are remarkable for their damage correlation with stress amplitude and long-term persistence. Hyperthermia-induced protein unfolding, and nonspecific aggregation accumulation have neurotoxic effects and contribute to the pathogenesis of brain damage in heat stroke. Therefore, we generated heat-induced, dose-responsive extreme and mild proteotoxic stress models in medulloblastoma [Daoy] and neuroblastoma [SH-SY5Y] and differentiated SH-SY5Y neuronal cells. We show that heat-induced protein aggregation is associated with reduced cell proliferation and viability. Higher protein aggregation in differentiated neurons than in neuroblastoma precursors suggests a differential neuronal vulnerability to heat. We characterized the neuronal heat shock response through RT-PCR array analysis of eighty-four genes involved in protein folding and protein quality control (PQC). We identify seventeen significantly expressed genes, five of which are Hsp70 chaperones, and four of their known complementing function proteins. Protein expression analysis determined the individual differential contribution of the five Hsp70 chaperones to the proteotoxic stress response and the significance of only two members under mild conditions. The co-expression analysis reveals significantly high co-expression between the Hsp70 chaperones and their interacting partners. The findings of this study lend support to the hypothesis that hyperthermia-induced proteotoxicity may underlie the brain injury of heat stroke. Additionally, this study presents a comprehensive map of the Hsp70 network in these models with potential clinical and translational implications.

Fetal Nucleated Red Blood Cells Preferable Than Cell-Free Fetal DNA for Early Determination of Gender Among Invasive and Non-Invasive Source Using Novel Four Genes Multiplex PCR.

BACKGROUND: Deoxyribonucleic acid from invasive, non-invasive and 9th week embryo can be a resource for the determination of fetal sex using highly sensitive and specific multiplex PCR. METHODS: A total of 402 DNA samples were used to test the newly developed novel multiplex PCR including male specific (3 genes: SRY, DAZ2 and TSPY1) Y-biomarkers and internal control, ACTB. The study isolated cffDNA (Cell-free fetal DNA; n = 73) from mother's plasma, serum and urine, fetal DNA from 9th week embryo and cord blood, and fetal DNA from CD71+ve nucleated red blood cells (fNRBC; n = 73). Paternal and maternal DNA from buccal cells (n = 20) and blood (n = 232) used for male and female confirmation. RESULTS: The study observed that SRY alone cannot be a suitable Y-biomarker. Confirmation from any two Y-biomarkers is mandatory for male fetus identification. Direct sequencing of the gel eluted multiplex and single amplicons confirmed the specific sequences. Presence of two out of 3 Y-biomarkers OR single Y-biomarker with >1,000,000 intensity is considered positive for male. The multiplex PCR is suitable for determining sex from all source of fetal DNA including highly degraded cffDNA and can detect the sex using 0.5ng DNA. Individual marker-based real-time qPCR followed by combined melt curve analysis showed distinguished melt curve peaks for the markers. CONCLUSION: The multiplex PCR achieved 100% accuracy on fetal DNA from fNRBC for early determinations (<13 weeks) of gender. The developed novel and simple multiplex PCR and individual qPCR can be adopted in all types of laboratories for determining human fetal gender using fetal DNA from fNRBC. Early identification of gender can support to prepare for possible X-linked analysis, reduce anxiety in mother, strengthen a bond between mother and fetus, and effective decision making. Non-invasive source of fetal DNA from fNRBC preferred for identifying gender to reduce the risk of invasive procedures in early (8-13 weeks) pregnancy.

Quantification of the Lamin A/C Transcript Variants in Cancer Cell Lines by Targeted Absolute Quantitative Proteomics and Correlation with mRNA Expression.

Lamin A/C proteins have key roles in nuclear structural integrity and chromosomal stability. Lamin A/C cumulative protein expression of all variants is reported by semi-quantitative Western blotting. To date, there have not been specific antibodies for the individual Lamin A/C transcript variants. We developed a mass spectrometric approach for the quantification of Lamin A/C transcript variants. A signature peptide for each specific splice variant of Lamin A/C was selected. A LC-MS/MS assay based on the selected signature peptides and their labeled internal standards was established to measure the expression of Lamin A/C transcript variant concentrations. The method validation was carried out according to Food and Drug Administration (FDA) guidelines. The expression levels of the Lamin A/C transcript variants were measured in samples derived from MCF7 and U937 cell lines. RT-qPCR assay was also used to quantitate and compare the mRNA expression of splice variants of Lamin A/C. The established and validated method showed a great linearity, sensitivity, and precision. The different expressed Lamin A/C variants in different cell lines were measured and their levels were in concordance with qRT-PCR results. The developed method is reproducible, reliable, and sensitive for measuring different Lamin A/C transcript variants in different cell lines.

Chromosomal aberration analysis among underground water well workers in Saudi Arabia.

In the absence of permanent rivers or bodies of water, half of the Saudi Arabia domestic water consumption is provided through desalination. The other half is derived from groundwater. Groundwater from the Disi aquifer is already used for drinking water in parts of Jordan and, more extensively, in Saudi Arabia, where it is known as the Saq aquifer. Some of the geological analyses of the host sandstone aquifer rocks show (228)Ra and (226)Ra. The usefulness of chromosomal aberrations analysis as a bioindicator for ionising radiation effect was tested in underground water well workers of Saudi Arabia in this industry producing technologically enhanced naturally occurring radioactive material. The incidence of chromosomal aberrations was evaluated using the metaphase analysis method in the lymphocytes of peripheral blood of 10 persons working in underground water well. The age range of the workers was 25-40 y and their duration of service ranged from 3-7 y. For comparison, blood samples were also collected from 10 subjects (controls) who belonged to same age and socioeconomic status. Subjects in the both groups were non-smokers and non-alcoholics. Results showed that the mean frequencies of dicentrics and acentrics in underground water well workers are significantly higher than those in controls. The higher frequency of chromosomal aberration in lymphocytes of underground water well workers compared with controls could be due to the accumulative effect of radiation. The results of this study demonstrated that occupational exposure to radiation leads to a significant induction of cytogenetic damage in peripheral lymphocytes of workers engaged in underground water well.

In vivo cytogenetic studies on aspartame.

Aspartame (a-Laspartyl-L-phenylalanine 1-methylester) is a dipeptide low-calorie artificial sweetener that is widely used as a nonnutritive sweetener in foods and drinks. The safety of aspartame and its metabolic breakdown products (phenylalanine, aspartic acid and methanol) was investigated in vivo using chromosomal aberration (CA) test and sister chromatid exchange (SCE) test in the bone marrow cells of mice. Swiss Albino male mice were exposed to aspartame (3.5, 35, 350 mg/kg body weight). Bone marrow cells isolated from femora were analyzed for chromosome aberrations and sister chromatid exchanges. Treatment with aspartame induced dose dependently chromosome aberrations at all concentrations while it did not induce sister chromatid exchanges. On the other hand, aspartame did not decrease the mitotic index (MI). However, statistical analysis of the results show that aspartame is not significantly genotoxic at low concentration.