Modulation of autophagy as the target of mesenchymal stem cells-derived conditioned medium in rat model of myocardial ischemia/reperfusion injury

Molecular Biology Reports - Human amniotic membrane mesenchymal stem cells-derived conditioned medium (hAM-MSCs-CM) has positive effects against myocardial ischemia/reperfusion (MI/R) injury....     

Long dwell-time passage of DNA through nanometer-scale pores: kinetics and sequence dependence of motion

A detailed understanding of the kinetics of DNA motion though nanometer-scale pores is important for the successful development of many of the proposed next-generation rapid DNA sequencing and analysis methods. Many of these approaches require DNA motion through nanopores to be slowed by several orders of magnitude from its native translocation velocity so that the translocation times for individual nucleotides fall within practical timescales for detection. With the increased dwell time of DNA in the pore, DNA-pore interactions begin to play an increasingly important role in translocation kinetics. In previous work, we and others observed that when the DNA dwell time in the pore is substantial (>1 ms), DNA motion in α-hemolysin (α-HL) pores leads to nonexponential kinetics in the escape of DNA out of the pore. Here we show that a three-state model for DNA escape, involving stochastic binding interactions of DNA with the pore, accurately reproduces the experimental data. In addition, we investigate the sequence dependence of the DNA escape process and show that the interaction strength of adenine with α-HL is substantially lower relative to cytosine. Our results indicate a difference in the process by which DNA moves through an α-HL nanopore when the motion is fast (microsecond timescale) as compared with when it is slow (millisecond timescale) and strongly influenced by DNA-pore interactions of the kind reported here. We also show the ability of wild-type α-HL to detect and distinguish between 5-methylcytosine and cytosine based on differences in the absolute ionic current through the pore in the presence of these two nucleotides. The results we present here regarding sequence-dependent (and dwell-time-dependent) DNA-pore interaction kinetics will have important implications for the design of methods for DNA analysis through reduced-velocity motion in nanopores.     

Up-regulation of lymphocytic growth hormone secretion during the luteal phase of cycle and early pregnancy

Growth hormone (GH) has been shown to be produced and secreted by peripheral immune cells. Therefore, we studied the release of GH by lymphocytes, during various stages of pregnancy and estrous cycle in the cow. The effect of leptin on the lymphocytic GH release was also investigated. Estradiol-17β and progesterone concentrations in plasma were measured in all animals to confirm their reproductive status. Growth hormone levels measured in cell cultures during early pregnancy (days 60-80) and during the luteal phase were greater (p ≤ 0.01) than levels during follicular phase or mid (days 100-160) and late (days 240-245) pregnancy. Leptin treatment stimulated (p ≤ 0.05) lymphocytic GH release during mid-pregnancy when the basal GH levels were low. Changes in lymphocytic GH release and elevation of lymphocytic GH secretion by leptin during pregnancy and the absence of such effects in estrous cycle may indicate that leptin modulation of lymphocytic GH plays a role in the regulation of immune response during pregnancy.     

Mechanistic role of microRNA-146a in endotoxin-induced differential cross-regulation of TLR signaling

Human TLRs are critical sensors for microbial components leading to the production of proinflammatory cytokines that are controlled by various mechanisms. Monocytes pretreated with LPS exhibit a state of hyporesponsiveness, referred to as cross-tolerance, to both homologous and heterologous ligands, which play a broader role in innate immunity. To date, LPS-induced cross-tolerance has not been examined regarding microRNA expression kinetics. In this study, THP-1 monocytes treated with various inflammatory ligands showed a continuous amplification of microRNA (miR)-146a over 24 h that is inversely correlated to TNF-α production. In contrast, inhibition of miR-146a showed a reciprocal effect. Thus, the characteristic upregulation of miR-146a in LPS-exposed THP-1 monocytes was studied for cross-tolerance. Strikingly, in LPS-tolerized THP-1 monocytes, only miR-146a showed a continuous overexpression, suggesting its crucial role in cross-tolerance. Similarly, peptidoglycan-primed THP-1 cells showed homologous tolerance associated with miR-146a upregulation. Subsequently, interchangeable differential cross-regulation was observed among non-LPS ligands. TLR2 and TLR5 ligands showed both homologous and heterologous tolerance correlated to miR-146a overexpression. More importantly, inflammatory responses to TLR4, TLR2, and TLR5 ligands were reduced due to knockdown of miR-146a targets IL-1R-associated kinase 1 or TNFR-associated factor 6, suggesting the regulatory effect of miR-146a on these TLRs signaling. Transfection of miR-146a into THP-1 cells caused reduction of TNF-α production, mimicking LPS-induced cross-tolerance. Aside from individual ligands, a whole bacterial challenge in LPS-primed THP-1 monocytes was accompanied by less TNF-α production, which is conversely correlated to miR-146a expression. Our studies have thus demonstrated that miR-146a plays a crucial role for in vitro monocytic cell-based endotoxin-induced cross-tolerance.     

Efficiency of Plasmocin™ on various mammalian cell lines infected by mollicutes in comparison with commonly used antibiotics in cell culture: a local experience

Mycoplasma contamination is a deleterious event for cell culture laboratories. Plasmocin™ is used to prevent and eradicate mycoplasma infections from cell. In this study, 80 different mammalian cell lines from various sources; human, monkey, mice, hamster and rat were used to study and evaluate plasmocin™ efficiency and compare it to commonly used antibiotics such as BM-cyclin, ciprofloxacin and mycoplasma removal agent (MRA). It was shown that mycoplasma infections were eradicated by plasmocin™, BM-cyclin, ciprofloxacin and MRA in 65%, 66.25%, 20%, and 31.25%, respectively, of infected cell cultures. However, re-infection with mycoplasmas after the period of 4 months occurred in 10–80% of the studied cell lines. Cell cytotoxicity and culture death was observed in 25, 17.5 and 10% of the treated cells, for plasmocin™, BM-cyclin and MRA, respectively. In this study, Plasmocin™ showed strong ability to eradicate mollicutes from our cell lines with minimal percentage of regrowth. However, due to its high cell cytotoxicity it should be used with caution especially when dealing with expensive or hard-to-obtain cell lines. Amongst the antibiotics tested, BM-cyclin was shown to remove mycoplasma with the highest efficiency.     

SMAD2 rs4940086 heterozygosity increases the risk of cervical cancer development among the women in Bangladesh

Molecular Biology Reports - SMAD2 is a critical signal transducer molecule in the TGFβ- SMAD pathway which is also known for its tumor suppressor role. Genetic variations in SMAD2 render cells...     

The Blepharis persica seed hydroalcoholic extract synergistically enhances the apoptotic effect of doxorubicin in human colon cancer and gastric cancer cells

The goal of this survey is to evaluate the anti-proliferative effects of the hydroalcholic extract of Blepharis persica seeds and its synergic effect on doxorubicin (DOX) in human colon cancer (HT-29) and gastric cancer cell (AGS) lines. 70% Ethanol was used for extraction of B. persica seed. Aluminum–chloride colorimetric and Folin–Ciocalteu reagent methods were used to measure total flavonoid and total phenolic contents of the extract respectively. Gas chromatography-mass spectrometry (GC-MS) analysis of the B. persica extract was performed on GC-MS equipment after silylation. HT-29, AGS, and human fibroblast (SKM) cell lines were treated by different concentration of the B. persica extract, (DOX) and the combination of extraction and DOX. The cytotoxicity was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay while the apoptosis induction was monitored using flowcytometry by annexin-V FITC/PI double-staining. The changes in expression levels of BAX and BCL-2 were determined using Real-Time RT-qPCR. GC-MS analysis of the hydroalcoholic extract from B. persica seeds revealed 24 major components. The MTT assay revealed the cytotoxicity against three cell lines and also it was shown that 125 ng/mL of DOX and 0.625 mg/mL of B. persica extract had synergistic behavior against HT29 cell line. These results showed B. persica extract induced apoptosis in AGS and HT29 cells and its extract caused dose-dependent increase in up-regulation of BAX level (p?<?0.05) and down-regulation of BCL₂ (p?<?0.05). B. persica showed the synergistic effect in combination with DOX on HT29 cell line. These findings demonstrated a basis for further studies on the characterization and mechanistic evaluation of the bioactive compounds of B. persica extract which had antiproliferative effects on cancer cell lines.

     

TBC1D14 regulates autophagosome formation via Rab11- and ULK1-positive recycling endosomes

Autophagy is a bulk degradation process characterized by the formation of double membrane vesicles called autophagosomes. The exact molecular mechanism of autophagosome formation and the origin of the autophagosomal membrane remain unclear. We screened 38 human Tre-2/Bub2/Cdc16 domain-containing Rab guanosine triphosphatase-activating proteins (GAPs) and identified 11 negative regulators of starvation-induced autophagy. One of these putative RabGAPs, TBC1D14, colocalizes and interacts with the autophagy kinase ULK1. Overexpressed TBC1D14 tubulates ULK1-positive recycling endosomes (REs), impairing their function and inhibiting autophagosome formation. TBC1D14 binds activated Rab11 but is not a GAP for Rab11, and loss of Rab11 prevents TBC1D14-induced tubulation of REs. Furthermore, Rab11 is required for autophagosome formation. ULK1 and Atg9 are found on Rab11- and transferrin (Tfn) receptor (TfnR)-positive recycling endosomes. Amino acid starvation causes TBC1D14 to relocalize from REs to the Golgi complex, whereas TfnR and Tfn localize to forming autophagosomes, which are ULK1 and LC3 positive. Thus, TBC1D14- and Rab11-dependent vesicular transport from REs contributes to and regulates starvation-induced autophagy.     

Mitochondrial dysfunction and genetic heterogeneity in chronic periodontitis

We performed an extensive study on mitochondrial dysfunction in chronic periodontitis (CP). Electron microscopic analysis of gingival cells revealed abnormal mitochondria in 60% of the patients. Mitochondrial membrane potential and oxygen consumption of gingival cells were reduced by 4 fold and 5.8 fold, respectively; whereas ROS production was increased by 18%. The genetic analysis by complete mitochondrial DNA sequencing revealed the identification of 14 novel mutations only in periodontal tissues but not in the blood, suggesting a role of oxidative stress on periodontal tissues. Thus, our functional and genetic analysis provided an evidence for the mitochondrial dysfunction in CP.