Effect of Co-exposure to Heat and Psychological Stressors on Sperm DNA and Semen Parameters.

The present study aims to investigate the effects of co-exposure to heat and psychological stress on sperm DNA and semen parameters among male rats. The study was conducted on 40 healthy adult male Wistar rats. The rats were randomly categorized into four groups of same size consisting of a control group, a heat stress, psychological and co-exposure groups. The heat stress group was exposed to a temperature of 36 °C at 20% relative humidity. The psychological stress exposure group was subjected to three stressors including exposure to strobe light, noise and tilting cage. According the results,the co-exposure group had lower mean sperm parameters including sperm count (17.22 ± 4.22 106/ml), motility (42.63 ± 12.95 %), viability (48.50 ± 23.25 %), normal morphology (56 ± 7.5%), progressive motility (11.61 ± 7.81%), non-progressive motility (31.18 ± 7.77%), curvilinear velocity (24.11 ± 3.81 µm/s) and straight-line velocity (3.2 ± 1.4 µm/s) when compared with those of the other groups (P = 0.001). Mean sperm immobility (57.36 ± 12.95%) and non-progressive motility (37.93 ± 11.15%) in the co-exposure group was higher compared to the other groups (P = 0.001 and P = 0.333, respectively). Assessment of damage to sperm DNA revealed that the heat exposure group had a higher percentage of sperm DNA damage (9.44 ± 6.80 %) compared to others (P = 0.185). In case of all of exposure scenario, the chance that the semen quality decreased compared to the control group has been increased. In general the combined stress had a greater significant effect on sperm parameters compared to other exposure groups, except for DNA damage.

Cholesterol-associated lysosomal disorder triggers cell death of hematological malignancy: Dynamic analysis on cytotoxic effects of LW-218.

The integrity of lysosomes is of vital importance to survival of tumor cells. We demonstrated that LW-218, a synthetic flavonoid, induced rapid lysosomal enlargement accompanied with lysosomal membrane permeabilization in hematological malignancy. LW-218-induced lysosomal damage and lysosome-dependent cell death were mediated by cathepsin D, as the lysosomal damage and cell apoptosis could be suppressed by depletion of cathepsin D or lysosome alkalization agents, which can alter the activity of cathepsins. Lysophagy, was initiated for cell self-rescue after LW-218 treatment and correlated with calcium release and nuclei translocation of transcription factor EB. LW-218 treatment enhanced the expression of autophagy-related genes which could be inhibited by intracellular calcium chelator. Sustained exposure to LW-218 exhausted the lysosomal capacity so as to repress the normal autophagy. LW-218-induced enlargement and damage of lysosomes were triggered by abnormal cholesterol deposition on lysosome membrane which caused by interaction between LW-218 and NPC intracellular cholesterol transporter 1. Moreover, LW-218 inhibited the leukemia cell growth in vivo. Thus, the necessary impact of integral lysosomal function in cell rescue and death were illustrated.

Neurodegeneration-related beta-amyloid as autocatabolism-attenuator in a micro-in vivo system.

Investigation of human neurodegeneration-related aggregates of beta-amyloid 1-42 (Aß42) on bdelloid rotifers is a novel interdisciplinary approach in life sciences. We reapplied an organ size-based in vivo monitoring system, exploring the autocatabolism-related alterations evoked by Aß42, in a glucose-supplemented starvation model. The experientially easy-to-follow size reduction of the bilateral reproductive organ (germovitellaria) in fasted rotifers was rescued by Aß42, serving as a nutrient source- and peptide sequence-specific attenuator of the organ shrinkage phase and enhancer of the regenerative one including egg reproduction. Recovery of the germovitellaria was significant in comparison with the greatly shrunken form. In contrast to the well-known neurotoxic Aß42 (except the bdelloids) with specific regulatory roles, the artificially designed scrambled version (random order of amino acids) was inefficient in autocatabolism attenuation, behaving as negative control. This native Aß42-related modulation of the 'functionally reversible organ shrinkage' can be a potential experiential and supramolecular marker of autocatabolism in vivo.

Binding site identification of anticancer drug gefitinib to HSA and DNA in the presence of five different probes.

This study was carried out to evaluate the binding interaction of gefitinib (GEF) with human serum albumin (HSA) and calf thymus DNA (ct-DNA) using fluorescence, UV-Visible, zeta potential measurements and molecular docking methods in order to understand its pharmacokinetic mechanism. By increasing the temperature, a steady decrease in Stern-Volmer quenching constants was observed for HSA binding properties; this indicates a static type of fluorescence quenching. Negative values were calculated for Gibbs free energy (ΔG), enthalpy (ΔH), and entropy (ΔS) changes, indicating that the reaction is spontaneous and enthalpy-driven. Probe competitive experimental results showed that GEF contains the same binding site as warfarin and are consistent with modeling results. The zeta potential of the HSA increased with increasing GEF, which represents the presence of electrostatic interactions in the system. DNA binding properties were investigated in the presence of three probes. The experimental results showed that by increasing GEF to DNA-AO (acridine-orange) and DNA-MB (methylene-blue) system, the fluorescence intensity and absorbance spectra had no considerable change. Furthermore, with the addition of GEF to DNA, the zeta potential decreased gradually, indicating that the hydrophobic interaction between the GEF and the bases of DNA is the major factor. Thus, GEF can bind to DNA via a groove binding mode. It was also found that GEF entered into the minor groove in the A-T rich region of DNA fragment and bind via van der-Waals forces and three H-bond with double strands of DNA. This is in good agreement with experimental results.

A systematic review on sperm DNA fragmentation in male factor infertility: Laboratory assessment.

OBJECTIVE: To review sperm DNA fragmentation (SDF) testing as an important sperm function test in addition to conventional semen analysis. High SDF is negatively associated with semen quality, the fertilisation process, embryo quality, and pregnancy outcome. Over recent decades, different SDF assays have been developed and reviewed extensively to assess their applicability and accuracy as advanced sperm function tests. Amongst them, the standardisation of the terminal deoxynucleotidyl transferased UTP nick-end labelling (TUNEL) assay with a bench top flow cytometer in clinical practice deserves special mention with a threshold value of 16.8% to differentiate infertile men with DNA damage from fertile men. MATERIALS AND METHODS: A systematic literature search was performed through the PubMed, Medline, and ScienceDirect databases using the keywords 'sperm DNA fragmentation' and 'laboratory assessment'. Non-English articles were excluded and studies related to humans were only included. RESULTS: Of the 618 identified, 87 studies (original research and reviews) and in addition eight book chapters meeting the selection criteria were included in this review. In all, 366 articles were rejected in the preliminary screening and a further 165 articles related to non-human subjects were excluded. CONCLUSION: There are pros and cons to all the available SDF assays. TUNEL is a reliable technique with greater accuracy and as an additional diagnostic test in Andrology laboratories along with basic semen analysis can predict fertility outcome, and thus direct the choice of an assisted reproductive technology procedure for infertile couples. Also, the TUNEL assay can be used as a prognostic test and results are beneficial in deciding personalised treatment for infertile men.

Mesoporous silica nanoparticles for drug and gene delivery.

Mesoporous silica nanoparticles (MSNs) are attracting increasing interest for potential biomedical applications. With tailored mesoporous structure, huge surface area and pore volume, selective surface functionality, as well as morphology control, MSNs exhibit high loading capacity for therapeutic agents and controlled release properties if modified with stimuli-responsive groups, polymers or proteins. In this review article, the applications of MSNs in pharmaceutics to improve drug bioavailability, reduce drug toxicity, and deliver with cellular targetability are summarized. Particularly, the exciting progress in the development of MSNs-based effective delivery systems for poorly soluble drugs, anticancer agents, and therapeutic genes are highlighted.

The impact of coexisting sperm DNA fragmentation and seminal oxidative stress on the outcome of varicocelectomy in infertile patients: A prospective controlled study.

OBJECTIVES: To study the relationship between sperm DNA fragmentation (SDF) and reactive oxygen species (ROS) levels in infertile patients with varicocele, and to examine the beneficial effect of varicocelectomy and elucidate predictors of improvement after repair. PATIENTS SUBJECTS AND METHODS: We prospectively studied 60 patients with varicocele and abnormal semen variables who attended the outpatient clinic complaining of infertility for ≥12 months. In all, 25 patients (41.7%) had bilateral varicoceles and 35 (58.3%) had left varicoceles. The DNA fragmentation index (DFI%, percentage of sperm with denatured nuclei), ROS and total non-enzymatic antioxidant capacity (TAC) were measured. Inguinal varicocelectomy was performed in all patients. At 3-6 months postoperatively, all measurements were repeated. A control group, comprised of 20 normozoospermic fertile men, was included. Regression analysis was used to examine predictors of improvement. RESULTS: The mean (SD) DFI% in the 60 infertile patients with varicocele was 29.9 (8.3) and 7.56 (2.84)% in the controls; ROS levels were 4.49 (0.9) in patients and 2.62 (0.8) photons/min in controls; and the TAC was 0.97 (0.4) in patients and 1.5 (0.5) mM in controls; with highly significant differences between the patients and controls. The DFI% showed a positive correlation with ROS levels, whilst the total motile sperm count (TMSC) had a significant negative correlation with DFI%, ROS levels and grade of varicocele, whilst there was significant positive correlation with TAC. The grade of varicocele and duration of infertility were related to the presence of higher levels of ROS and increased of DFI%. Postoperatively, improvement (measured as a >50% increase in TMSC) occurred in 40 of 55 (73%) patients available at follow-up, with a significant reduction in the mean (SD) DFI% from 29.49 (8.58) to 18.78 (7.23)%, ROS levels from 4.49 (0.88) to 3.27 (1.3) photons/min (both P < 0.001), and a significant increase in the mean (SD) TAC from 1.01 (0.44) to 2.05 (0.51) mM (P < 0.001). Responders had a shorter infertility duration and lower preoperative DFI% and ROS levels. Regression analysis showed that DFI% is a predictor of improvement after varicocelectomy. CONCLUSION: SDF was shown to have a negative impact on improvement after varicocelectomy. Hence, DFI% could be recommended as a prognostic test in infertile patients with varicocele to help decision-making as regards the necessity and the anticipated outcome of varicocelectomy in patients with infertility.

Cholesterol-associated lysosomal disorder triggers cell death of hematological malignancy: Dynamic analysis on cytotoxic effects of LW-218

The integrity of lysosomes is of vital importance to survival of tumor cells. We demonstrated that LW-218, a synthetic flavonoid, induced rapid lysosomal enlargement accompanied with lysosomal membrane permeabilization in hematological malignancy. LW-218-induced lysosomal damage and lysosome-dependent cell death were mediated by cathepsin D, as the lysosomal damage and cell apoptosis could be suppressed by depletion of cathepsin D or lysosome alkalization agents, which can alter the activity of cathepsins. Lysophagy, was initiated for cell self-rescue after LW-218 treatment and correlated with calcium release and nuclei translocation of transcription factor EB. LW-218 treatment enhanced the expression of autophagy-related genes which could be inhibited by intracellular calcium chelator. Sustained exposure to LW-218 exhausted the lysosomal capacity so as to repress the normal autophagy. LW-218-induced enlargement and damage of lysosomes were triggered by abnormal cholesterol deposition on lysosome membrane which caused by interaction between LW-218 and NPC intracellular cholesterol transporter 1. Moreover, LW-218 inhibited the leukemia cell growth

p8 attenuates the apoptosis induced by dihydroartemisinin in cancer cells through promoting autophagy.

Dihydroartemisinin (DHA) exhibits anticancer activities in a variety of cancer cells, but DHA alone are not effective enough for cancer therapy. In this study we found the stress-regulated protein p8 was obviously increased after DHA treatment in several cancer cells, which further to induce autophagy by the upregulation of endoplasmic reticulum stress-related protein ATF4 and CHOP. Furthermore, when we silenced p8 by siRNA in cancer cells, the apoptosis induced by DHA were notably increased, whereas the overexpression of p8 in cancer cells leaded to the resistance to DHA-induced apoptosis. Moreover, we found the inhibition of autophagy with chloroquine (CQ) can enhance the anticancer effect of DHA both in vitro and in vivo. In conclusion, we found that p8-mediated autophagy attenuates DHA-induced apoptosis in cancer cells, which provides evidence to support the use p8 as a cancer therapeutic target, and suggests that the combination treatment with DHA and autophagy inhibitor might be an effective cancer therapeutic strategy.

Shiga toxins induce autophagic cell death in intestinal epithelial cells via the endoplasmic reticulum stress pathway.

Shiga toxins (Stxs) are a family of cytotoxic proteins that lead to the development of bloody diarrhea, hemolytic-uremic syndrome, and central nervous system complications caused by bacteria such as S. dysenteriae, E. coli O157:H7 and E. coli O104:H4. Increasing evidence indicates that macroautophagy (autophagy) is a key factor in the cell death induced by Stxs. However, the associated mechanisms are not yet clear. This study showed that Stx2 induces autophagic cell death in Caco-2 cells, a cultured line model of human enterocytes. Inhibition of autophagy using pharmacological inhibitors, such as 3-methyladenine and bafilomycin A1, or silencing of the autophagy genes ATG12 or BECN1 decreased the Stx2-induced death in Caco-2 cells. Furthermore, there were numerous instances of dilated endoplasmic reticulum (ER) in the Stx2-treated Caco-2 cells, and repression of ER stress due to the depletion of viable candidates of DDIT3 and NUPR1. These processes led to Stx2-induced autophagy and cell death. Finally, the data showed that the pseudokinase TRIB3-mediated DDIT3 expression and AKT1 dephosphorylation upon ER stress were triggered by Stx2. Thus, the data indicate that Stx2 causes autophagic cell death via the ER stress pathway in intestinal epithelial cells.

Single-cell analysis challenges the connection between autophagy and senescence induced by DNA damage.

Autophagy and senescence have been described as central features of cell biology, but the interplay between these mechanisms remains obscure. Using a therapeutically relevant model of DNA damage-induced senescence in human glioma cells, we demonstrated that acute treatment with temozolomide induces DNA damage, a transitory activation of PRKAA/AMPK-ULK1 and MAPK14/p38 and the sustained inhibition of AKT-MTOR. This produced a transient induction of autophagy, which was followed by senescence. However, at the single cell level, this coordinated transition was not observed, and autophagy and senescence were triggered in a very heterogeneous manner. Indeed, at a population level, autophagy was highly negatively correlated with senescence markers, while in single cells this correlation did not exist. The inhibition of autophagy triggered apoptosis and decreased senescence, while its activation increased temozolomide-induced senescence, showing that DNA damage-induced autophagy acts by suppressing apoptosis.

PAWR-mediated suppression of BCL2 promotes switching of 3-azido withaferin A (3-AWA)-induced autophagy to apoptosis in prostate cancer cells.

An active medicinal component of plant origin with an ability to overcome autophagy by inducing apoptosis should be considered a therapeutically active lead pharmacophore to control malignancies. In this report, we studied the effect of concentration-dependent 3-AWA (3-azido withaferin A) sensitization to androgen-independent prostate cancer (CaP) cells which resulted in a distinct switching of 2 interrelated conserved biological processes, i.e. autophagy and apoptosis. We have observed 3 distinct parameters which are hallmarks of autophagy in our studies. First, a subtoxic concentration of 3-AWA resulted in an autophagic phenotype with an elevation of autophagy markers in prostate cancer cells. This led to a massive accumulation of MAP1LC3B and EGFP-LC3B puncta coupled with gradual degradation of SQSTM1. Second, higher toxic concentrations of 3-AWA stimulated ER stress in CaP cells to turn on apoptosis within 12 h by elevating the expression of the proapoptotic protein PAWR, which in turn suppressed the autophagy-related proteins BCL2 and BECN1. This inhibition of BECN1 in CaP cells, leading to the disruption of the BCL2-BECN1 interaction by overexpressed PAWR has not been reported so far. Third, we provide evidence that pawr-KO MEFs exhibited abundant autophagy signs even at toxic concentrations of 3-AWA underscoring the relevance of PAWR in switching of autophagy to apoptosis. Last but not least, overexpression of EGFP-LC3B and DS-Red-BECN1 revealed a delayed apoptosis turnover at a higher concentration of 3-AWA in CaP cells. In summary, this study provides evidence that 3-AWA is a strong anticancer candidate to abrogate protective autophagy. It also enhanced chemosensitivity by sensitizing prostate cancer cells to apoptosis through induction of PAWR endorsing its therapeutic potential.

A novel quinazolinone derivative induces cytochrome c interdependent apoptosis and autophagy in human leukemia MOLT-4 cells.

Crosstalk between apoptosis and autophagy is budding as one of the novel strategies in the cancer therapeutics. The present study tinted toward the interdependence of autophagy and apoptosis induce by a novel quinazolinone derivative 2,3-dihydro-2-(quinoline-5-yl) quinazolin-4(1H)-one structure [DQQ] in human leukemia MOLT-4 cells. DQQ induces cytochrome c arbitrated apoptosis and autophagy in MOLT-4 cells. Apoptosis induces by DQQ was confirmed through a battery of assay e.g. cellular and nuclear microscopy, annexin-V assay, cell cycle analysis, loss of mitochondrial membrane potential and immune-expression of cytochrome c, caspases and PARP. Furthermore, acridine orange staining, LC3 immunofluorescence and western blotting of key autophagy proteins revealed the autophagic potential of DQQ. A universal caspase inhibitor, Z-VAD-FMK and cytochrome c silencing, strongly inhibited the DQQ induce autophagy and apoptosis. Beclin1 silencing through siRNA partially reversed the cell death, which was not as significant as by cytochrome c silencing. Although, it partially reversed the PARP cleavage induced by DQQ, indicating the role of autophagy in the regulation of apoptosis. The present study first time portrays the negative feedback potential of cytochrome c regulated autophagy and the importance of quinazolinone derivative in discovery of novel anticancer therapeutics.

Acidocalcisome is required for autophagy in Trypanosoma brucei.

Lysosomes play important roles in autophagy, not only in autophagosome degradation, but also in autophagy initiation. In Trypanosoma brucei, an early divergent protozoan parasite, we discovered a previously unappreciated function of the acidocalcisome, a lysosome-related organelle characterized by acidic pH and large content of Ca(2+) and polyphosphates, in autophagy regulation. Starvation- and chemical-induced autophagy is accompanied with acidocalcisome acidification, and blocking the acidification completely inhibits autophagosome formation. Blocking acidocalcisome biogenesis by depleting the adaptor protein-3 complex, which does not affect lysosome biogenesis or function, also inhibits autophagy. Overall, our results support the role of the acidocalcisome, a conserved organelle from bacteria to human, as a relevant regulator in autophagy.

Mesoporous silica nanoparticles for drug and gene delivery

Mesoporous silica nanoparticles (MSNs) are attracting increasing interest for potential biomedical applications. With tailored mesoporous structure, huge surface area and pore volume, selective surface functionality, as well as morphology control, MSNs exhibit high loading capacity for therapeutic agents and controlled release properties if modified with stimuli-responsive groups, polymers or proteins. In this review article, the applications of MSNs in pharmaceutics to improve drug bioavailability, reduce drug toxicity, and deliver with cellular targetability are summarized. Particularly, the exciting progress in the development of MSNs-based effective delivery systems for poorly soluble drugs, anticancer agents, and therapeutic genes are highlighted.

Dihydroartemisinin induces autophagy and inhibits the growth of iron-loaded human myeloid leukemia K562 cells via ROS toxicity.

Dihydroartemisinin (DHA), an active metabolite of artemisinin derivatives, is the most remarkable anti-malarial drug and has little toxicity to humans. Recent studies have shown that DHA effectively inhibits the growth of cancer cells. In the present study, we intended to elucidate the mechanisms underlying the inhibition of growth of iron-loaded human myeloid leukemia K562 cells by DHA. Mitochondria are important regulators of both autophagy and apoptosis, and one of the triggers for mitochondrial dysfunction is the generation of reactive oxygen species (ROS). We found that the DHA-induced autophagy of leukemia K562 cells, whose intracellular organelles are primarily mitochondria, was ROS dependent. The autophagy of these cells was followed by LC3-II protein expression and caspase-3 activation. In addition, we demonstrated that inhibition of the proliferation of leukemia K562 cells by DHA is also dependent upon iron. This inhibition includes the down-regulation of TfR expression and the induction of K562 cell growth arrest in the G2/M phase.