Deoxynucleotidyl Transferase-mediated Deoxyuridine Triphosphate Research Articles

Protective effects of hesperidin in gastric damage caused by experimental ischemia-reperfusion injury model in rats.

This study evaluated the protective effect of hesperidin on injury induced by gastric ischemia-reperfusion. Fifty male Sprague Dawley rats (250-300g) were divided into five groups: control (C), sham (S), ischemia (I), ischemia-reperfusion (I/R) and hesperidin + ischemia-reperfusion (Hes + I/R). Hesperidin was injected intraperitoneally at the dose of 100mg/kg one hour before the experimental stomach ischemia-reperfusion. Celiac artery was ligated. After 45minutes ischemia and 60minutes reperfusion period, blood samples were obtained under anesthesia. Then, animals were sacrificed, stomach tissues were excised for biochemical, and histopathological analyses were performed. Malondialdehyde levels and superoxide dismutase, glutathione peroxidase activities and total antioxidant status (TAS), total oxidant status (TOS), protein, total thiol parameters were measured in plasma, and tissue homogenate samples. H + E, periodic acid-Schiff, hypoxia inducible factor, terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end-labeling (TUNEL), and proliferating cell nuclear antigen (PCNA) for cell proliferation as immunohistochemical parameters were determined. Upon biochemical and histopathological assessment, hesperidin decreased stomach tissue changes in comparison with IR group. Ischemia-reperfusion injury led to a considerably increase in malondialdehyde, protein, and TOS levels (p < 0.001) in stomach tissue. Hesperidin treatment significantly decreased malondialdehyde, protein, and TOS levels (p < 0.001). Hesperidin increased superoxide dismutase, TAS, total thiol and glutathione peroxidase activities in comparison with IR group. Hesperidin reduced damage and also increased TUNEL and PCNA immunoreactivity in stomach tissue. Hesperidin was able to decrease I/R injury of the stomach tissue due to inhibition of lipid peroxidation and protein oxidation, duration of antioxidant, and free radical scavenger properties. Consequently, hesperidin can provide a beneficial therapeutic choice for preventing stomach tissue ischemia-reperfusion injury in clinical application.

Incomplete Caspase 3 Activation and Mitigation of Apoptosis in Hibernating Ground Squirrels, Spermophilus lateralis.

AbstractHibernating golden-mantled ground squirrels, Spermophilus [Callospermophilus] lateralis, tolerate proapoptotic conditions, such as low body temperature, anorexia, acidosis, and ischemia/reperfusion. Avoiding widespread apoptosis is critical for hibernator survival. Caspase 3, the key executioner of apoptosis, cleaves a majority of apoptotic targets. Under proapoptotic conditions, inactive procaspase 3 (32 kDa) is activated when cleaved into 17- and 12-kDa fragments (p32, p17, and p12, respectively). Caspase 3 activation results in extreme enzymatic activation. Activity increases >10,000-fold followed by apoptotic execution. Is widespread apoptosis occurring during the proapoptotic hibernation season? Western blots showed p17 increased ∼2-fold during hibernation, indicating caspase 3 activation. However, in vitro caspase 3 activity assays found no extreme increases in activity. Downstream caspase 3 targets ICAD (inhibitor of caspase-activated deoxyribonuclease) and PARP (poly (ADP-ribose) polymerase) did not experience elevated cleavage during hibernation, which is inconsistent with caspase 3 activation. TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling) assays from multiple tissues found only 0.001%-0.009% of cells were TUNEL positive during winter, indicating negligible apoptosis during hibernation. Typically, caspase 3 activation generates a strong commitment toward apoptosis. We found that despite a ∼2-fold increase in active caspase 3, hibernators experience no downstream caspase 3 activity or widespread apoptosis. A systems-level approach suggests an incomplete signaling cascade wherein some caspase 3 activation during hibernation does not necessarily lead to bona fide apoptosis.

Proliferative and apoptotic evaluations of renal preventive effects of coenzyme Q10 in radioiodine-131 induced renal damage

The aim of this study was to investigated anti-proliferative and anti-apoptotic effects of coenzyme Q10 (CoQ10) in the prevention of radioiodine-131 (RAI) (I131) induced kidney damage. A total of 24 Wistar albino rats were separated into equal three groups (n = 8/group): Group 1 (control): untreated group; Group 2 (RAI): 3 mCi/kg RAI oral route; Group 3 (RAI+CoQ10): 3 mCi/kg RAI oral route and intraperitoneally 30 mg/kg/day CoQ10. CoQ10 treatment was started two days before RAI administration and was continued five days once daily after RAI. Pathomorphological parameters of kidneys were measured using hematoxylin–eosin and Masson’s trichrome staining. Immunohistochemically; proliferating cell nuclear antigen (PCNA), caspase 8, caspase 9 and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) were used to determine proliferation and apoptosis. With the exception of the control group, varying degrees of inflammation, degeneration, necrosis, and interstitial/perivascular fibrosis were detected in the kidneys of all rats. This histopathological damage was found to be significantly less in CoQ10 group versus RAI group (P&amp;lt;0.05). The all immunohistochemical examinations demonstrated that administration of CoQ10 had reduced proliferation and apoptosis (P&amp;lt;0.05). The results of kidney histopathology and immunohistochemistry demonstrated that administration of CoQ10 had reduced inflammation, proliferation, and apoptosis. These findings show CoQ10 can play an important role in the radioprotection of kidneys against RAI-induced damage

High-Dose Vitamin C Administration Inhibits the Invasion and Proliferation of Melanoma Cells in Mice Ovary.

Several studies have been conducted to explore the anticancer effects of vitamin C (VC). However, the effect of high-dose VC administration on melanoma is still unknown. Therefore, in this study, we investigated the effects of high-dose VC (4 g/kg) on the invasion and proliferation of melanoma cells in various organs of mice. B16 melanoma cells (1 × 106 cells/100 µL) were intravenously injected into the tails of female mice, and VC solution (4 g/kg) was orally administered once a day for 14 d. On the 15th day, samples from the liver, lungs, jejunum, and ovaries were collected and analyzed for invasion and proliferation of melanoma cells. Oral VC administration decreased the number of dihydroxyphenylalanine (DOPA)-positive cells and gp100-positive melanoma cells in the ovaries and suppressed the invasion and proliferation of melanoma. Compared to melanoma-administered mice, macrophage inflammatory protein-2 levels and number of neutrophils were increased in the VC + melanoma-administered mice. Furthermore, the concentrations of VC, iron, and hydrogen peroxide, and the number of terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling (TUNEL)-positive cells were significantly increased in the ovaries of VC + melanoma-administered mice compared to those of melanoma-administered mice. These results suggest that VC can reduce the invasion and proliferation of melanoma cells in the ovaries, and neutrophils in the ovaries play an important role in achieving this melanoma-suppressive effect.

A decrease in the tear secretion volume in a mouse model with ulcerative colitis

Purpose Dry eye syndrome is known to develop from several systemic inflammatory diseases. Although dry eye may develop due to extraintestinal complications of ulcerative colitis (UC), the pathogenesis is not well-known. This study aimed to investigate whether there was decrease in the tear secretion volume in a mice model with UC; the difference between the control and dextran sodium sulphate (DSS)-treated group was also determined. Materials and methods This study included a mice model with UC induced by the oral administration of 5.0% DSS for 7 days. Following the DSS treatment, the tear volume was measured using the Schirmer’s test. The colon and ocular tissues, including the lacrimal gland, were evaluated using histological and protein analyses. Additionally, tumour necrosis factor (TNF)-α and interleukin (IL)-6 in the plasma were determined. Differences between groups (DSS-treated versus control mice) were determined using Student’s t-test. Results The tear volume in DSS-treated mice was decreased compared to that in the control mice. Plasma levels of TNF-α and IL-6 in DSS-treated mice was higher than that of control. Morphological change was observed with the invasion of the inflammatory cell in the lacrimal gland of DSS-treated mice. Terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labelling (TUNEL)-positive cells were increased in the lacrimal glands of DSS-treated mice compared with control group. The distribution of aquaporin-5 expressed in the lacrimal gland of DSS-treated mice was decreased compared to that in the control group. Conclusions These findings suggest that a decrease in the tear volume in UC was associated with a functional decline in the inflamed lacrimal gland. This result therefore provides useful information that could contribute to the development of treatment approaches for dry eye associated with UC.

Early prediction of tumor response after radiotherapy in combination with cetuximab in nasopharyngeal carcinoma using 99m Tc-duramycin imaging

Purpose99mTc-duramycin imaging enables specific visualization of cell death qualitatively and quantitatively. This study aimed to investigate the potential of 99mTc-duramycin imaging in the early prediction of the curative effect of radiotherapy in combination with or without cetuximab in a nasopharyngeal carcinoma (NPC) model. MethodsMale BALB/c mice bearing NPC xenografts were randomized into four groups (six mice each group). Group 1 received radiotherapy (RT, 15 Gy/mouse) in combination with cetuximab (CTX, 2 mg/mouse), group 2 received RT (15 Gy/mouse), group 3 was treated using CTX (2 mg/mouse), and group 4, the control group, was treated using a vehicle. 99mTc-duramycin imaging was performed before treatment and 24 h after treatment to evaluate tumor response. Tumor uptake of 99mTc-duramycin was validated ex vivo using γ-counting. Treatment response was further validated by cleaved caspase-3 (CC3) and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL). Another four groups were treated parallelly under the same conditions to observe treatment response by tumor volume changes. ResultsAfter 24 h treatment, 99mTc-duramycin uptake in the NPC tumor models were significantly higher in group 1 than in group 2 (P < 0.05), group 3 (P < 0.05), or group 4 (P < 0.05); the uptake also increased notably in comparison with baseline values (P < 0.05). Compared with group 4, group 2 and group 3 both showed significant 99mTc-duramycin uptake in the tumors (P < 0.05). Although the 99mTc-duramycin uptake of group 2 was moderately higher than group 3, there were no significant differences between these two groups (P >0.05). There was a strong positive correlation between tumor 99mTc-duramycin uptake and CC3 (r = 0.893, p < 0.0001) and TUNEL (r = 0.918, P < 0.0001). Tumor volume decreased remarkably in the RT in combination with CTX group on day 5, in the RT alone group on day 7, and was inhibited on day 8 in the CTX alone group, whereas the tumors grew continuously in the control group. ConclusionsWe demonstrated that RT in combination with CTX treatment significantly improved disease control in a NPC xenograft model compared with monotherapy with either. 99mTc-duramycin imaging might be able to reliably identify response to RT in combination with CTX as early as 24 h after therapy initiation in NPC xenograft models. This might help to isolate non-responding patients in a timely manner and avoid unnecessary side effects in the clinic in the future.

Prototheca zopfii genotype II induces mitochondrial apoptosis in models of bovine mastitis

Prototheca zopfii is an alga increasingly isolated from bovine mastitis. Of the two genotypes of P. zopfii (genotype I and II (GT-I and -II)), P. zopfii GT-II is the genotype associated with acute mastitis and decreased milk production, although its pathogenesis is not well known. The objective was to determine inflammatory and apoptotic roles of P. zopfii GT-II in cultured mammary epithelial cells (from cattle and mice) and murine macrophages and using a murine model of mastitis. Prototheca zopfii GT-II (but not GT-I) invaded bovine and murine mammary epithelial cells (MECs) and induced apoptosis, as determined by the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling assay. This P. zopfii GT-II driven apoptosis corresponded to mitochondrial pathways; mitochondrial transmembrane resistance (ΔΨm) was altered and modulation of mitochondrion-mediated apoptosis regulating genes changed (increased transcriptional Bax, cytochrome-c and Apaf-1 and downregulated Bcl-2), whereas caspase-9 and -3 expression increased. Apoptotic effects by P. zopfii GT-II were more pronounced in macrophages compared to MECs. In a murine mammary infection model, P. zopfii GT-II replicated in the mammary gland and caused severe inflammation with infiltration of macrophages and neutrophils and upregulation of pro-inflammatory genes (TNF-α, IL-1β and Cxcl-1) and also apoptosis of epithelial cells. Thus, we concluded P. zopfii GT-II is a mastitis-causing pathogen that triggers severe inflammation and also mitochondrial apoptosis.

Antitumor efficacy of arsenic/interferon in preclinical models of chronic myeloid leukemia resistant to tyrosine kinase inhibitors.

Tyrosine kinase inhibitors (TKIs) are the standard treatment for chronic myeloid leukemia (CML). Despite their clinical success, TKIs are faced with challenges such as treatment resistance, which may be driven by kinase domain mutations, and frequent disease relapse upon the cessation of treatment. The combination of arsenic trioxide (ATO) and interferon-α (IFN) was previously demonstrated to inhibit proliferation and induce apoptosis in CML cell lines, prolong the survival of primary wild-type CML mice, and dramatically decrease the activity of leukemia-initiating cells (LICs). The ATO/IFN combination was tested in vitro on imatinib (IMN)-resistant K562-R and Ar230-R cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays were used to evaluate proliferation and apoptosis, respectively. The acridine orange assay was used to assess autophagy, and quantitative reverse transcription-polymerase chain reaction was used to assess the involvement of the hedgehog (Hh) pathway. In vivo, a retroviral transduction/transplantation T315I BCR-ABL CML mouse model was used to assay the effect of the treatment on survival, tumor burden (histopathology and blood counts), and LIC activity (secondary transplantation). In vitro, ATO/IFN synergized to inhibit proliferation and induce apoptosis of IMN-resistant cells with variant modes of resistance. Furthermore, the preclinical effects of ATO/IFN were associated with induction of autophagy along with inhibition of the Hh pathway. Most remarkably, ATO/IFN significantly prolonged the survival of primary T315I-CML mice and displayed a dramatic impairment of disease engraftment in secondary mice, which reflected decreased LIC activity. Collectively, the ATO/IFN strategy has been demonstrated to have the potential to lead to durable remissions in TKI-resistant CML preclinical models and to overcome various TKI-specific mechanisms of resistance.

Depicting Changes in Tumor Biology in Response to Cetuximab Monotherapy or Combination Therapy by Apoptosis and Proliferation Imaging Using 18F-ICMT-11 and 18F-FLT PET.

Imaging biomarkers must demonstrate their value in monitoring treatment. Two PET tracers, the caspase-3/7–specific isatin-5-sulfonamide 18F-ICMT-11 (18F-(S)-1-((1-(2-fluoroethyl)-1H-[1,2,3]-triazol-4-yl)methyl)-5-(2(2,4-difluoro-phenoxymethyl)-pyrrolidine-1-sulfonyl)isatin) and 18F-FLT (3′-deoxy-3′-18F-fluorothymidine), were used to detect early treatment-induced changes in tumor biology and determine whether any of these changes indicate a response to cetuximab, administered as monotherapy or combination therapy with gemcitabine. Methods: In mice bearing cetuximab-sensitive H1975 tumors (non–small lung cancer), the effects of single or repeated doses of the antiepidermal growth factor receptor antibody cetuximab (10 mg/kg on day 1 only or on days 1 and 2) or a single dose of gemcitabine (125 mg/kg on day 2) were investigated by 18F-ICMT-11 or 18F-FLT on day 3. Imaging was also performed after 2 doses of cetuximab (days 1 and 2) in mice bearing cetuximab-insensitive HCT116 tumors (colorectal cancer). For imaging–histology comparison, tumors were evaluated for proliferation (Ki-67 and thymidine kinase 1 [TK1]), cell death (cleaved caspase-3 and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling [TUNEL]), and target engagement (epidermal growth factor receptor expression) by immunohistochemistry, immunofluorescence, and immunoblotting, respectively. Tumor and plasma were analyzed for thymidine and gemcitabine metabolites by liquid chromatography–mass spectrometry. Results: Retention of both tracers was sensitive to cetuximab in H1975 tumors. 18F-ICMT-11 uptake and ex vivo cleaved caspase-3 staining notably increased in tumors treated with repeated doses of cetuximab (75%) and combination treatment (46%). Although a single dose of cetuximab was insufficient to induce apoptosis, it did affect proliferation. Significant reductions in tumor 18F-FLT uptake (44%–50%; P < 0.001) induced by cetuximab monotherapy and combination therapy were paralleled by a clear decrease in proliferation (Ki-67 decrease, 72%–95%; P < 0.0001), followed by a marked tumor growth delay. TK1 expression and tumor thymidine concentrations were profoundly reduced. Neither imaging tracer depicted the gemcitabine-induced tumor changes. However, cleaved caspase-3 and Ki-67 staining did not significantly differ after gemcitabine treatment whereas TK1 expression and thymidine concentrations increased. No cetuximab-induced modulation of the imaging tracers or other response markers was detected in the insensitive model of HCT116. Conclusion: 18F-ICMT-11 and 18F-FLT are valuable tools to assess cetuximab sensitivity depicting distinct and time-variant aspects of treatment response.

CAAT/enhancer binding protein-homologous protein deficiency attenuates liver ischemia/reperfusion injury in mice.

Ischemia/reperfusion injury (IRI) is one of the main causes of liver dysfunction after liver surgery. Involvement of endoplasmic reticulum (ER) stress in various diseases has been demonstrated, and CAAT/enhancer binding protein-homologous protein (CHOP) is a transcriptional regulator that is induced by ER stress. It is also a key regulator of ER stress-mediated apoptosis. The aim of this study was to investigate the role of CHOP in liver IRI. Wild type (WT) and CAAT/enhancer binding protein-homologous protein knockout (CHOP-/-) mice were subjected to 70% liver warm ischemia/reperfusion for 60 minutes. At different times after reperfusion, liver tissues and blood samples were collected for evaluation. Induction of ER stress including CHOP expression was ascertained. Liver damage was evaluated based on serum liver enzymes, liver histology, and neutrophil infiltration. Hepatocyte death including apoptosis was assessed. Liver warm IRI induced ER stress in both WT and CHOP-/- mice. In addition, CHOP expression was up-regulated in WT mice. At 6 hours after reperfusion, liver damage was attenuated in CHOP-/- mice. On the basis of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining, apoptotic and necrotic cells were significantly reduced in CHOP-/- mice. CHOP deficiency also reduced the cleavage of caspase 3 and expression of the proapoptotic protein B cell lymphoma 2-associated X protein. Liver IRI induces CHOP expression, and CHOP deficiency attenuates liver IRI by inhibiting apoptosis. Elucidation of the function of CHOP in liver IRI may contribute to further investigation for a therapy against liver IRI associated with the ER stress pathway. Liver Transplantation 24 645-654 2018 AASLD.

Apoptosis and cell proliferation in short-term and long-term effects of radioiodine-131-induced kidney damage: an experimental and immunohistochemical study.

Radioiodine-131 is a radionuclide that is used for therapeutic purposes in hyperthyroidism and thyroid cancer. The aim of this study was to evaluate apoptotosis and proliferative changes in radioiodine-related kidney damage. Three groups (n=10/group) of rats were used as follows: the rats were in group 1 untreated, and the rats in groups 2 and 3 were treated once with oral radioiodine (111 MBq). The animals in group 2 were killed at the end of the seventh day and the rats in group 3 were killed at the end of the 10th week. The kidneys were removed and evaluated immunohistochemically. The presence of radioiodine in the kidneys was shown by the Na+/I-symporter antibody and proliferating cell nuclear antigen, Ki-67, caspase-3, caspase-8, caspase-9, and terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling assay were used to detect cell proliferation and apoptosis. Na+/I-symporter protein accumulation in the kidneys was observed to be significantly greater in group 2 than in group 3 (P<0.05). All the immunohistochemical analyses showed that cell proliferation and apoptosis began on the seventh day and peaked in the 10th week. The proliferating cell nuclear antigen, Ki-67, and caspase expressions and terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling values were all found to be statistically significantly increased in group 3 compared with the other groups (P<0.05). Radioiodine caused cell proliferation and apoptosis as shown by immunohistochemistry.

Apoptosis-mediated neurotoxicity and altered gene expression induced by silver nanoparticles.

Although not fully recognized, the neurotoxic effects of silver nanoparticles (Ag-NPs) are thought to occur through induction of oxidative stress and apoptosis. To investigate the exact underlying molecular mechanism, we aimed to explore the apoptotic effects of intraperitoneal injection of Ag-NPs and investigated the possible attributed changes in the mRNA expression of Bcl-2 and Bax genes in the rat hippocampus. Two in vivo sets of experiments, one to demonstrate apoptosis and the other to assess gene expression, were conducted on male Wistar rats. In each set, the first group, acting as control, received saline and the other three groups received Ag-NP at doses of 100, 200, and 400 ppm for five successive days. Ten days after the last injection, hippocampal tissue of the first set of rats was assessed for apoptosis using terminal deoxynucleotidyl transferase-mediated deoxy uridine triphosphate nick-end labeling staining. In the second set of experiments, mRNA expression of Bcl-2 and Bax genes was evaluated using real-time polymerase chain reaction. Ag-NP treatment was shown to induce apoptosis in a dose-dependent manner. Furthermore, Ag-NP reduced mRNA level of Bcl-2 in the rat hippocampal cells at all investigated doses compared to the control group ( p < 0.001). The mRNA level of Bax, on the other hand, was increased in these cells. The increase was significant compared to the control group at the doses of 200 ppm ( p < 0.05) and 400 ppm ( p < 0.001). Our results show that Ag-NPs reduce Bcl-2 and increase Bax genes expression, resulting in increased Bax/Bcl-2 ratios in rat hippocampal cells. This altered gene expression induces cell apoptosis and contributes to the neurotoxicity of Ag-NPs.

Anti-tumor effect of cisplatin in human oral squamous cell carcinoma was enhanced by andrographolide via upregulation of phospho-p53 in vitro and in vivo.

Oral squamous cell carcinoma is one of the most common neoplasm in the world. Despite the improvements in diagnosis and treatment, the outcome is still poor now. Thus, the development of novel therapeuticapproaches is needed. The aim of this study is to assess the synergistic anti-tumor effect of andrographolide with cisplatin (DDP) in oral squamous cell carcinoma CAL-27 cells in vitro and in vivo. We performed Cell Counting Kit-8 proliferation assay, apoptosis assay, and western blotting on CAL-27 cells treated with andrographolide, DDP or the combination in vitro. In vivo, we also treated CAL-27 xenografts with andrographolide or the combination, and performed terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay and immunohistochemical analysis of Ki-67. The results showed the combination of andrographolide and DDP synergistically inhibited CAL-27 cell proliferation in vitro and caused tumor regression in vivo in the CAL-27 xenografts. In addition, the synergistic anti-tumor effect of andrographolide with synergistic was due to an enhanced apoptosis. Moreover, the combination therapy upregulated the expression level of p-p53 in vitro and decreased Ki-67 expression in vivo. Our data indicate that the combination treatment of andrographolide and DDP results in synergistic anti-tumor growth activity against oral squamous cell carcinoma CAL-27 in vitro and in vivo. These results demonstrated that combination of andrographolide with DDP was likely to represent a potential therapeutic strategy for oral squamous cell carcinoma.

Measuring Sperm DNA Fragmentation and Clinical Outcomes of Medically Assisted Reproduction: A Systematic Review and Meta-Analysis.

Sperm DNA fragmentation has been associated with reduced fertilization rates, embryo quality, pregnancy rates and increased miscarriage rates. Various methods exist to test sperm DNA fragmentation such as the sperm chromatin structure assay (SCSA), the sperm chromatin dispersion (SCD) test, the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay and the single cell gel electrophoresis (Comet) assay. We performed a systematic review and meta-analysis to assess the value of measuring sperm DNA fragmentation in predicting chance of ongoing pregnancy with IVF or ICSI. Out of 658 unique studies, 30 had extractable data and were thus included in the meta-analysis. Overall, the sperm DNA fragmentation tests had a reasonable to good sensitivity. A wide variety of other factors may also affect the IVF/ICSI outcome, reflected by limited to very low specificity. The constructed hierarchical summary receiver operating characteristic (HSROC) curve indicated a fair discriminatory capacity of the TUNEL assay (area under the curve (AUC) of 0.71; 95% CI 0.66 to 0.74) and Comet assay (AUC of 0.73; 95% CI 0.19 to 0.97). The SCSA and the SCD test had poor predictive capacity. Importantly, for the TUNEL assay, SCD test and Comet assay, meta-regression showed no differences in predictive value between IVF and ICSI. For the SCSA meta-regression indicated the predictive values for IVF and ICSI were different. The present review suggests that current sperm DNA fragmentation tests have limited capacity to predict the chance of pregnancy in the context of MAR. Furthermore, sperm DNA fragmentation tests have little or no difference in predictive value between IVF and ICSI. At this moment, there is insufficient evidence to recommend the routine use of sperm DNA fragmentation tests in couples undergoing MAR both for the prediction of pregnancy and for the choice of treatment. Given the significant limitations of the evidence and the methodological weakness and design of the included studies, we do urge for further research on the predictive value of sperm DNA fragmentation for the chance of pregnancy after MAR, also in comparison with other predictors of pregnancy after MAR.

Lithium posttreatment confers neuroprotection through glycogen synthase kinase-3β inhibition in intracerebral hemorrhage rats

OBJECTIVE Inflammation and apoptosis are two key factors contributing to secondary brain injury after intracerebral hemorrhage (ICH). The objective of this study was to evaluate the effects of lithium posttreatment on behavior, brain atrophy, inflammation, and perihematomal cell death. Furthermore, the authors aimed to determine the role of the pro-apoptotic glycogen synthase kinase-3β (GSK-3β) after experimental ICH. METHODS Male Sprague-Dawley rats (n = 108) were subjected to intracerebral infusion of semicoagulated autologous blood. Window of opportunity and dose optimization studies of lithium on ICH-induced injury were performed by measuring neurological deficits. Animals with ICH received vehicle administration or lithium posttreatment (60 mg/kg) for up to 21 days. Hemispheric atrophy was evaluated. Perihematomal cell death was quantified through terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL). The number of myeloperoxidase (MPO)-positive neutrophils and OX42-positive microglia in the perihematomal areas were calculated. Western blotting was used for the quantification of GSK-3β, heat shock protein 70 (HSP70), nuclear factor-κB p65 (NF-κB p65), and cy-clooxygenase-2 (COX-2). RESULTS Lithium, at a dose of 60 mg/kg initiated from 2 hours after injury, exhibited the best effects of improving neurological outcomes 3, 5, 7, 14, 21, and 28 days after ICH, reduced the hemispheric atrophy at 42 days after surgery, and reduced the number of TUNEL-positive cells, MPO-positive neutrophils, and OX42-positive microglia in the perihematomal areas. Furthermore, lithium posttreatment modulated GSK-3β, increased HSP70, and decreased NF-κB p65 and COX-2 expression in the ipsilateral hemisphere. CONCLUSIONS Lithium posttreatment at a dose of 60 mg/kg, initiated beginning 2 hours after injury, improves functional and morphological outcomes, and inhibits inflammation and perihematomal cell death in a rat model of semicoagulated autologous blood ICH through inactivation of GSK-3β.

Proliferation, Apoptosis and Invasion effects of mistletoe alkali on human osteosarcoma U2OS in vitro.

To evaluate the effects of mistletoe alkali on human Osteosarcoma cells (U2OS) in vitro. Osteosarcoma is the most common primary malignant tumor of bone tumor, although there are a lot of therapies such as surgery, radiotherapy and chemotherapy, its prognosis is still very poor. There is increasing interest in the protective biological function of natural antioxidants contained in Chinese medicinal herbs, which are candidates for the prevention of tumors. Mistletoe alkali is one of the compounds extracted from Viscum coloratum (Komar.) Nakai, one kind of mistletoe, whose extracts contribute to the improvement of the prognosis of patients with malignancies. The effect of mistletoe alkali on the growth of U2OS cells was compared with 5-FU, using a Cell Counting Kit-8 (CCK-8). The influence of mistletoe alkali on U2OS's proliferation and apoptosis were tested by TUNEL staining and immunocytochemical (ICC) staining of caspase 3 and proliferating cell nuclear antigen (PCNA). Additionally, the invasion ability of U2OS cells was detected using a Boyden chamber trans-well migration assay. CCK-8 assays gave an IC50 of 7μg/ml for mistletoe alkali. Compared to 5-FU, mistletoe alkali inhibited U2OS proliferation and induced apoptosis more effectively. The invasion ability of U2OS was also weaker in mistletoe alkali than in 5-FU. Mistletoe alkali significantly inhibited growth and invasion abilities of U2OS cells and induced their apoptosis in vitro. Mistletoe alkali may be a more effective drug for Human Osteosarcoma than the standard chemotherapeutic drug 5-FU.

MP70-20 VALIDATION OF MEASURING OXIDATION-REDUCTION POTENTIAL IN FRESH AND FROZEN SEMEN SAMPLES WITH MIOXSYS DIAGNOSTIC SYSTEM

INTRODUCTION AND OBJECTIVES: Urocortin (UCN), which is a member of corticotropin-releasing factor (CRF) family peptide, is known to provide cardioprotection by reducing the ischemia-reperfusion (I/R) caused apoptotic cardiomyocyte cells loss. Recently, UCN has been reported to be expressed in male germ cells of human and rat, but only limited data on the role of UCN in germ cells are reported. In this study, we sought to investigate the role of UCN in the apoptosis of germ cells using the experimental testicular I/R model in the rat. METHODS: The left testes of Sprague-Dawley rats were rotated clockwise by 720 for 1 hour, and then the testes were harvested 0, 1, 3, 6 and 24 hours after detorsion (each group; n1⁄45). Realtime quantitative reverse transcription polymerase chain reaction (qRTPCR) and Western Blotting were used to investigate the change of expression levels of UCN. Terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate biotin nick end labeling (TUNEL) staining was performed to evaluate the association between the expression levels of UCN and germ cell apoptosis. In another series, a total of 5 rats underwent intratesticular injection of Astressin, which is a CRF receptor antagonist, 15 minutes before detorsion following the testicular torsion. Then the testes were harvested 3 hours after detorsion, and TUNEL staining was performed to evaluate the direct effect of UCN on the apoptosis of germ cells after I/R injury. RESULTS: The expression levels of UCN increased for 1, 3 and 6 hours after reperfusion, but significantly decreased after 24 hours. In contrast, the number of TUNEL-positive germ cells per seminiferous tubule did not increase after 1, 3 and 6 hours, but significantly increased after 24 hours compared to controls. The injection of Astressin significantly increased the TUNEL-positive germ cells 3 hours after detorsion, which suggests that UCN plays a role in the apoptosis of germ cells. CONCLUSIONS: Our data suggest that UCN is involved in cytoprotection of germ cells after experimental testicular I/R in the rat. Further examination is expected to elucidate the possible mechanisms of UCN-associated cytoprotection in spermatogenesis.

Successful transplantation in canines after long-term coronary sinus machine perfusion preservation of donor hearts

Machine perfusion is a promising strategy for donor heart preservation, but delivery of perfusate through the aorta may be limited by aortic valve incompetence. We hypothesized that retrograde machine perfusion preservation through the coronary sinus avoided this issue and allowed for recovery of donor hearts after long-term storage. Canine hearts were procured after arrest with 1 liter University of Wisconsin Machine Perfusion Solution (UWMPS) and preserved for 14 hours by static hypothermic storage (Static group, n = 5) or retrograde machine perfusion through the coronary sinus (RP group, n = 5). Myocardial oxygen consumption (MVo2) and lactate were monitored in perfused hearts. Hearts were implanted and reperfused for 6 hours. The pre-load recruitable stroke work was determined as a measure of myocardial function. Cardiac enzyme release was quantified. Cell death was evaluated by TUNEL (terminal deoxynucleotidyltransferase-mediated deoxy uridine triphosphate nick-end label). MVo2 decreased initially then stabilized. Lactate accumulation was low in RP hearts. All RP hearts separated from cardiopulmonary bypass. All Static hearts required a return to bypass (p < .05). Pre-load recruitable stroke work in RP hearts was increased (55 ± 7 mm Hg) compared with Static (20 ± 11 mm Hg, p < .05) and did not differ from baseline values. Creatine kinase release was greater in Static group hearts (102 ± 16 IU/liter/g) than in RP hearts (51 ± 8 IU/liter/g, p < .05). The fraction of TUNEL-positive cells was higher in the Static group, but this difference was not significant. Retrograde machine perfusion can preserve donor hearts for long intervals. Cardiac function after implantation suggested excellent myocardial protection. Retrograde machine perfusion appears promising for extending the donor ischemic interval and improving results of heart transplantation.

Relationship between Eimeria tenella development and host cell apoptosis in chickens

Coccidiosis causes considerable economic losses in the poultry industry. At present, the pathology of coccidiosis is preventable with anticoccidials and vaccination, although at considerable cost to the international poultry industry. The purpose of the present study was to elucidate the relationship between Eimeria tenella development and host cell apoptosis in chickens, which provides a theoretical basis for further study of the injury mechanism of E. tenella and the prevention and treatment of coccidiosis. Cecal epithelial cells from chick embryo were used as host cells in vitro. In addition, flow cytometry, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate (dUTP) nick-end labeling, and histopathological assays were used to detect the dynamic changes in E. tenella infection rates, DNA injury rates, and apoptosis rates in groups treated with and without the caspase-9 inhibitor Z-LEHD-FMK. Following E. tenella infection, we demonstrated that untreated cells had less apoptosis at 4 h and, inversely, more apoptosis at 24 to 120 h compared with control cells. Furthermore, after the application of Z-LEHD-FMK, terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling assays, and translation of phosphatidyl serines to the host cell plasma membrane surface, the treated group chick embryo cecal epithelial cells exhibited decreased apoptosis and DNA injuries (P < 0.01) at 24 to 120 h. However, light microscopy showed that E. tenella infection rates of treated cells were higher (P < 0.01) than untreated cells during the whole experimental period. Together, these observations suggest that E. tenella can protect host cells from apoptosis at early stages of development but can promote apoptosis during the middle to late stages. In addition, the inhibition of host cell apoptosis can be beneficial to the intracellular growth and development of E. tenella.

Altered Mitochondrial Dynamics Contributes to Propofol-induced Cell Death in Human Stem Cell-derived Neurons.

Studies in developing animals have shown that anesthetic agents can lead to neuronal cell death and learning disabilities when administered early in life. Development of human embryonic stem cell-derived neurons has provided a valuable tool for understanding the effects of anesthetics on developing human neurons. Unbalanced mitochondrial fusion and fission lead to various pathological conditions including neurodegeneration. The aim of this study was to dissect the role of mitochondrial dynamics in propofol-induced neurotoxicity. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate in situ nick-end labeling staining was used to assess cell death in human embryonic stem cell-derived neurons. Mitochondrial fission was assessed using TOM20 staining and electron microscopy. Expression of mitochondrial fission-related proteins was assessed by Western blot, and confocal microscopy was used to assess opening time of the mitochondrial permeability transition pore (mPTP). Exposure to 6 h of 20 μg/ml propofol increased cell death from 3.18 ± 0.17% in the control-treated group to 9.6 ± 0.95% and led to detrimental increases in mitochondrial fission (n = 5 coverslips per group) accompanied by increased expression of activated dynamin-related protein 1 and cyclin-dependent kinase 1, key proteins responsible for mitochondrial fission. Propofol exposure also induced earlier opening of the mPTP from 118.9 ± 3.1 s in the control-treated group to 73.3 ± 1.6 s. Pretreatment of the cells with mdivi-1, a mitochondrial fission blocker rescued the propofol-induced toxicity, mitochondrial fission, and mPTP opening time (n = 75 cells per group). Inhibiting cyclin-dependent kinase 1 attenuated the increase in cell death and fission and the increase in expression of activated dynamin-related protein 1. These data demonstrate for the first time that propofol-induced neurotoxicity occurs through a mitochondrial fission/mPTP-mediated pathway.