Acidic pH derived from cancer cells as a double-edged knife modulates wound healing through DNA repair genes and autophagy.

Wound healing is a sequester program that involves diverse cell signalling cascades. Notwithstanding, complete signal transduction pathways underpinning acidic milieu derived from cancer cells is not clear, yet. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, fluorescein diacetate/propidium iodide staining, and cell cycle flow cytometry revealed that acidic media decreased cell viability and cell number along with enhanced dead cells and S-phase arrest in normal fibroblasts. Notably, the trends of intracellular reactive oxygen species production and lactate dehydrogenase release significantly increased with time. It seems the downregulation of Klf4 is in part due to acidosis-induced DNA damage. It promoted cells towards S-phase arrest and diminished cell proliferation. Klf4 downregulation had a direct correlation with the P53 level while acidic microenvironment promotes cells towards cell death mechanisms including apoptosis and autophagy. Noteworthily, the unchanged levels of Rb and Mlh1 indicated in those genes had no dominant role in the repairing of DNA damage in fibroblasts treated with the acidic microenvironment. Therefore, cells owing to not entering to mitosis and accumulation of DNA damage were undergone cell death to preserve cell homeostasis. Since acidic media decreased the level of tumour suppressor and DNA repair genes and altered the normal survival pathways in fibroblasts, caution should be exercised to not lead to cancer rather than wound healing.

Autophagic, apoptotic, and necrotic cancer cell fates triggered by acidic pH microenvironment.

Cancer as a multifactorial and smart disease is now considered a challenging problem. Despite many investigations on drug discovery, it remains incurable, in part, due to insufficient understanding of its special mechanisms. For the first time, we collaterally investigated the effect of acidosis on the contribution of apoptosis, necrosis, and autophagy in MDA-MB 231 cells. Our data showed that necrosis, apoptosis, and intracellular reactive oxygen species production drastically decreased from 48 to 72 hr while cell viability and autophagy increased along with a gap between the percentages. Eventually, the decrease of necrosis and apoptosis was related to upregulation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and fatty acid synthetase, respectively. It seems that at the early stage of cancer progression, apoptosis is the main mechanism of cell mortality and afterward autophagy would be the main mechanism of cell survival. Therefore, at the acute phase of cancer, apoptotic inducer medications would be effective while at the chronic phase of cancer progression, autophagy inhibitor medication would be added as well. This eventually means that autophagy acts as both cell death and survival mechanisms at the onset of cancer progression with the approach towards cell survival. Besides other unknown cell survival mechanisms are involved in cell viability, except for apoptosis and necrosis inhibition and autophagy improvement. This study reiterates the inefficaciousness of autophagy inhibitor's medication at the onset of disease. It also emphasizes discovering other cell death mechanisms for cancer cell adaptation at the onset of disease with the aim of their targeting in cancer invasion therapy.

Testicular piRNA Analysis Identified Dysregulated piRNAs in Non-obstructive Azoospermia.

Male infertility has remained idiopathic in a remarkable proportion of all cases. Gonadal expression of PIWI-interacting RNAs (piRNAs) has been shown to be vital to normal spermatogenesis, as they are expressed in almost all types of testicular germ cells. These molecules and their related Piwi proteins strictly regulate transposable elements' activity and gene expression. We aimed to identify dysregulated piRNAs in idiopathic non-obstructive azoospermic (NOA) testis by global expression analysis. Testis tissue samples from 18 azoospermic patients (ten NOA and eight OA) were studied by small RNA sequencing. To validate high-throughput sequencing data, quantitative real-time polymerase chain reactions for two differentially altered piRNAs were performed. Bioinformatics analyses were undertaken to identify pathways affected by piRNA dysregulation. In the NOA group, 1328 piRNAs were identified to be differentially expressed, of which 1322 were downregulated and 6 were upregulated. Bioinformatics analysis corroborated the involvement of dysregulated piRNA in spermatogenesis. We also identified 64 clusters of differentially expressed piRNAs, of which 42 clusters had a minimum of ten absolute piRNA hits. Our study suggests that piRNAs show significant dysregulation in infertility. Their target genes play a role in their self-biogenesis, probably by regulating their own production through a feedback mechanism. The downregulated piRNAs may find value as biomarkers for the presence of spermatozoa in the testis of azoospermic individuals, while the upregulated piRNAs are great candidates for further investigation of their precise functions in spermatogenesis.

Low cycle fatigue lifetime prediction of superplastic shape memory alloy structures: Application to endodontic instruments.

In this paper we propose a methodology for a fast numerical determination of low cycle fatigue lifetime of superelastic shape memory alloy structures. This method is based on the observation that generally, in low cycle fatigue, shape memory alloy (SMA) structures are subject to loadings that lead to a confined non-linear behaviour at stress concentration points, such as notches. Numerical fatigue lifetime prediction requires the computation of the mechanical state at critical points. However, classical computational methods, like the non-linear finite element method, lead to a prohibitive computation time in a non-linear cyclic framework. To overcome this issue, we propose to use fast prediction methods, based on localization laws. Following the determination of the stabilized behaviour, an energetic fatigue criterion is applied. The numerical fatigue life prediction model is validated experimentally on SMA endodontic instruments.

Protective effect of N-acetyl cysteine on the mitochondrial dynamic imbalance in temporal lobe epilepsy: Possible role of mTOR.

Understanding the underlying molecular mechanisms involved in epilepsy is critical for the development of more effective therapies. It is believed that mTOR (Mechanistic Target of Rapamycin kinases) activity and the mitochondrial dynamic balance change during epilepsy. mTOR affects mitochondrial fission by stimulating the translation of mitochondrial fission process 1 (MTFP1). In This study, the protective role of N-acetylcysteine was studied in temporal lobe epilepsy (TLE) through the regulation of mTOR and mitochondrial dynamic proteins. Rats received N-acetylcysteine (oral administration) seven days before induction of epilepsy, followed by one day after epilepsy. TLE was induced by microinjection of kainite into the left lateral ventricle. The total mTOR and Drp1 levels in the hippocampus were evaluated by western blotting. MFN1 was assessed using immunohistochemistry, and the expression of Fis.1 and MTFP1 (fission-related proteins) and OPA (fusion-related protein) were detected by real-time PCR. The mitochondrial membrane potential was measured by Rhodamin 123. The results showed that 72 h after induction of epilepsy, the mTOR protein level increased, and the balance of the mitochondrial dynamic was disturbed; however, oral administration of NAC decreased the mTOR protein level and improved the mitochondrial dynamic. These findings indicate that NAC plays a neuroprotective role in temporal lobe epilepsy, probably through decreasing the mTOR protein level, which can improve the imbalance in the mitochondrial dynamic.

Designing Two Synthetic Constructs for Real Time PCR Detection of Francisella tularensis and Ebola Virus.

BACKGROUND: Generally, timely diagnosis of micro-organisms is very important to prevent many diseases. Many methods can detect micro-organisms like culture-based methods and molecular methods. The molecular methods are usually preferred because they provide fast and reliable results. In some cases, microbial strains are not accessible, and there is no safety to work with them; therefore, synthetic constructs which are designed according to the available sequences in databases can be used as a positive control for detection of them. METHODS: In this study, a synthetic construct was designed for molecular detection of Francisella tularensis (F. tularensis) and the Ebola virus by multiplex real-time PCR reaction. For this, sequences were taken from databases and then multiple alignments were done by software. Also, conventional PCR and two models of real-time PCR (SYBR green and TaqMan) were applied. Finally, multiplex real-time PCR was performed. RESULTS: The synthetic construct was designed and used for conventional PCR and multiplex PCR. The results of common PCR showed a single band at 148 bp and 167 bp in 1.5% agarose gel stained by ethidium bromide for F. tularensis and Ebola virus, respectively. Also, a dual-band at 148 and 167 bp was observed in multiplex PCR. Results of real-time PCR showed a limit of detection about 0.1 pg of plasmid/µl. CONCLUSION: In conclusion, the designed construct can be used as a positive control for an accurate diagnosis of these micro-organisms without any biological danger for laboratory staff. So, this method is useful for diagnosis of these agents in food, water, and blood samples.

GSK-3ß as a target for apigenin-induced neuroprotection against Aß 25-35 in a rat model of Alzheimer's disease.

Glycogen synthase kinase-3 (GSK-3) is a critical molecule in Alzheimer's disease (AD) that modulates two histopathological hallmarks of AD: Amyloid beta (Aß) plaques and neurofibrillary tangles composed of aberrant hyper-phosphorylation of tau protein. This study was performed to investigate the protective effect of flavone apigenin through inhibition of GSK-3 and the involvement of this kinase in the inhibition of BACE1 expression and hyperphosphorylation of tau protein in an AD rat model. 15 nM of aggregated amyloid-beta 25-35 was microinjected into the left lateral ventricle of an AD rat. Apigenin (50 mg/kg) was administered orally 45 min before the Aß injection and continued daily for three weeks. Immunohistochemistry and western blot analysis showed that apigenin significantly reduced the hyperphosphorylation of tau levels in the hippocampus. Real-time PCR analysis revealed significant inhibition of the mRNA level of ß secretase (BACE1) and GSK-3ß, but Apigenin had no effect on the level of GSK-3α. The results demonstrate that apigenin has a protective effect against amyloid-beta 25-35 by decreasing the expression of GSK-3ß with the consequence of lowering the hyperphosphorylation of tau protein and suppressing BACE1 expression.

Valproic acid administration exerts protective effects against stress-related anhedonia in rats.

Anhedonia or inability to experience pleasure is the sign of various neuropsychiatric conditions. Current treatment options do not provide adequate control of anhedonia. The present study was conducted to evaluate the protective effects of valproic acid (VPA) as a nonspecific histone deacetylase (HDAC) inhibitor to reverse the effects of stress on induction of anhedonia and explore possible mechanisms. To induce anhedonia, a rat model of chronic unpredictable mild stress (CUMS) was established. Animals were assigned into no stress, stress (6 weeks of CUMS) and two treatment groups. VPA treatment was carried out for 4 continuous weeks (200 mg/kg/day). Behavioral assessments were performed using sucrose consumption (SCT) and new object recognition (NOR) tests. The expression of genes was evaluated using qRT-PCR. The cell density was determined using Nissl staining. Rats with CUMS showed depressive-like behaviors and impaired memory performance compared with the non-stressed group (p < 0.01). Moreover, they had significantly higher levels of HDAC3 and MC4R expression in the nucleus accumbens (NAc) compared to the non-stressed group (p < 0.01). The NAc cell density was significantly higher in the non-stressed rats (p < 0.05). Corticosterone plasma level was increased in the CUMS compared to the non-stressed group (p < 0.05). In the CUMS + VPA subgroup, the corticosterone (CORT) plasma level was lower compared with the CUMS + Saline and/or the CUMS groups (p < 0.05). These findings suggest that VPA can improve anhedonia and stress. Although the protective effect of VPA might link to decreasing HDAC3 and MC4R genes expression in NAc.