TdT-mediated dUTP-biotin Nick End Labeling Research Articles

Role of autophagy in the bimodal stage after spinal cord ischemia reperfusion injury in rats

Autophagy plays an important role in spinal cord ischemia reperfusion (I/R) injury, but its neuroprotective or neurodegenerative role remains controversial. The extent and persistence of autophagy activation may be the critical factor to explain the opposing effects. In this study, the different roles and action mechanisms of autophagy in the early and later stages after I/R injury were investigated in rats. Thespinal cord I/R injury was induced by 14-min occlusion of the aortic arch, after which rats were treated with autophagic inhibitor (3-methyladenine, 3-MA) or agonist (rapamycin) immediately or 48h following the injury. Autophagy markers, microtubule-associated protein light chain 3-II (LC3-II) and Beclin 1 increased and peaked at the early stage (8h) and the later stage (72h) after spinal cord I/R injury. Beclin 1 was mostly expressed in neurons, but was also expressed to an extent in astrocytes, microglia and vascular endothelial cells. 8h after injury, rats treated with 3-MA showed a decrease in the hind-limb Basso–Beattie–Bresnahan (BBB) motor function scores, surviving motor neurons, and B-cell lymphoma-2 (Bcl-2) expression, and increase in the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells, Bcl-2-associated X protein (Bax), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) expression, and activation of microglia, while those treated with rapamycin showed opposing effects. However, 72h after injury, rats treated with 3-MA improved the BBB scores, and the surviving motor neurons, and reduced the autophagic cell death, while those treated with rapamycin had adverse effects. These findings provide the first evidence that early activated autophagy alleviates spinal cord I/R injury via inhibiting apoptosis and inflammation; however later excessively elevated autophagy aggravates I/R injury through inducing autophagic cell death.

Search for New Pro‐apoptotic agents in Indigenous Antitumor Plants in Nigeria

Due to severe cytotoxicities exacerbated by cytotoxic drugs, newer therapies from natural products are desirable. This work aimed to partially purify pro‐apoptotic agents from indigenous natural products and to investigate the mechanism of action of the selected plant fractions.Sixteen indigenous medicinal plants were collected and fractionated into hexane (HF), chloroform (CF), ethylacetate (EAF), methanol (MF) and water (WF) fractions using solvent‐solvent partitioning and polyamide adsorption chromatography. The resultant ninety‐six test fractions were screened for lethality potential using brine shrimp lethality (BSL) assay. Fractions with LC50 less than 10 ug/ml were subjected to Water Soluble Tetrazolium (WST‐1) cytotoxicity assay and apoptosis evaluations using: morphological assessment, flow cytometric analysis of phosphatidylserine externalization (Annexin V) and propidium iodide (PI) staining, caspase 3 enzyme assay, immunoblot assessment of Poly ADP‐Ribose Polymerase‐1 (PARP‐1) Cleavage, carboxymethyl‐2′,7′‐dichlorodihydrofluorescein‐diacetate (CM‐H2DCFDA) fluorescent microscopic/microplate analysis of reactive oxygen species generation, and Terminal deoxynucleotidyl transferase (Tdt) mediated dUTP‐biotin Nick End Labelling (TUNEL) assessment of DNA fragmentation.Following BSL assay, C. afer (EAF), P. guineense (HF), P. guineense (CF), A. virides (EAF), C. roseus (CF), Z. zanthoxyloides (HF) and Z. zanthoxyloides (EAF) had LC50 values < 10 μg/ml. WST‐1 Cytotoxicity testing of these hit fractions on HeLa (Tumourigenic) and KMST‐6 (Non tumourigenic) cell Lines confirms their cytotoxic effects with the exception of Z. zanthoxyloides (HF). Significant (P<0.05) selective cytotoxic preference for HeLa to KMST‐6 Cell lines was exhibited by P. guineense (CF) and C. roseus (CF). Membrane blebs were observed in HeLa cells treated with the hit fractions in comparison to 17.42 μg/ml(50μM) Camptothecin. Annexin V‐PI analysis reveals that Z. zanthoxyloides (EAF) and P. guineense (HF) showed similar apoptotic effect in comparison to 21.8% apoptotic population in Camptothecin. A significant (P<0.05) increase in caspase‐3 enzyme activity was observed in C. roseus (CF) and Z. zanthoxyloides (EAF) in comparison with the negative control. PARP‐1 cleavage was confirmed in all the test groups except for A. virides (EAF) treated cells.Findings in this study suggest that all the hit test fractions elicited apoptotic effects but at varying degrees with Z. zanthoxyloides (EAF) showing the most prominent effect. While A. virides (EAF) elicits its cytotoxic effect via necrosis.

Sodium Selenite Acts as an Otoprotectant against Neomycin-Induced Hair Cell Damage in a Zebrafish Model.

Sodium selenite is a trace element essential for many physiological functions in the body. It is involved in various biological processes; it acts as a cofactor for antioxidant enzymes that protect against free radicals and is reported to limit metal-mediated oxidative DNA damage. In the present study, we investigated the effect of sodium selenite on neomycin ototoxicity in wild-type and transgenic zebrafish (Brn3C: EGFP). Five or six days post-fertilization, zebrafish larvae were co-exposed to 125 μM neomycin and various concentrations (10 μM, 100 μM, 250 μM, and 500 μM) of sodium selenite for 1 h. Hair cells within neuromasts of the supraorbital (SO1 and SO2), otic (O1), and occipital (OC1) lateral lines were analyzed by fluorescence microscopy (n = 10 fish per treatment). Hair cell survival was estimated as the ratio of the hair cell numbers in each group compared to those of the control group that were not exposed to neomycin. Apoptosis and hair cell damage of neuromasts were evaluated using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assay and 2-[4-(dimethylamino) styryl]-N-ethylpyridinium iodide (DASPEI) assay, respectively. Ultrastructural changes were evaluated using scanning electron microscopy and transmission electron microscopy. Neuromast hair cells were preserved in zebrafish exposed to 125 μM neomycin and 500 μM sodium selenite for 1 h. Sodium selenite protected against neomycin-induced hair cell loss of neuromasts, reduced apoptosis, and prevented zebrafish ultrastructural changes. We propose that sodium selenite protects against neomycin-induced hair cell damage by inhibiting apoptosis, decreasing the disarray of stereocilia, and preventing ultrastructural changes in the neuromast hair cells of the zebrafish.

A Novel and Sensitive Approach for the Evaluation of Liver Ischemia-Reperfusion Injury After Liver Transplantation.

The purpose of our study was to evaluate the potential of x-ray propagation-based phase-contrast imaging (PCI) computed tomography (CT) for the detection and characterization of early changes after ischemia-reperfusion (IR) in a standardized rat liver transplantation (LTx) model. Syngeneic orthotopic liver transplantation was performed in male Lewis rats. Ischemia-reperfusion injury (IRI)-induced changes of liver parenchyma were investigated in a time-dependent manner (2, 16, 24, and 32 hours). X-ray phase-contrast images of formalin-fixated liver specimens were acquired in CT mode by using a voxel size of 8 × 8 × 8 μm. Necrapoptotic cell death was visualized with the TdT-mediated dUTP-biotin nick end labeling technique, and alterations of liver graft microhemodynamics, that is, acinar and sinusoidal perfusion failure, were evaluated by in vivo fluorescence microscopy. Acquired and reconstructed PCI-CT images showed an increase in necrotic liver parenchyma dependent on cold storage time, measuring 5.7% ± 1.6% after 2 hours (comparable to 2.6% ± 0.4% for sham livers), 11.5% ± 2.1% (16 hours; P < 0.05 vs control), 23.0% ± 0.5% (24 hours; P < 0.001 vs control), and 31.3% ± 2.2% (32 hours; P < 0.001 vs control). There were a significant lower number of perfused acini in dependence on increasing cold storage time. The acinar perfusion index reached 0.970 ± 0.006 after 2 hours of cold ischemia (comparable to 0.960 ± 0.009 for sham livers) and declined continuously after 16, 24, and 32 hours cold ischemia (0.58 ± 0.03, 0.49 ± 0.02, 0.41 ± 0.03, each P < 0.0001 vs controls). Comparable results were found for sinusoidal perfusion, reaching 1.8% ± 0.4% of nonperfused sinusoids for 2 hours of cold ischemia and 8.2% ± 0.8% after 16 hours, 18.8% ± 1.4% after 24 hours, and 39.0% ± 2.4% after 32 hours (each P < 0.0001 vs controls). Prolonged cold ischemia was associated with an increasing number of TdT-mediated dUTP-biotin nick end labeling-positive cells (hepatocytes and sinusoidal lining cells), reaching 0.4 ± 0.1 (sham), 0.7 ± 0.4 (2 hours), 6.4 ± 1.1 (16 hours), 2.1 ± 0.3 (24 hours), and 14.7 ± 3.5 (32 hours; P = 0.002) for hepatocytes. X-ray PCI of histological liver specimens can detect IR-induced tissue necrosis and can provide detailed complementary 3-dimensional information to standard histopathologic findings.

Abstract TP258: Enoph1-ADI1-MT-MMP-1 Pathway Contributes to Ischemic Brain Microvascular Endothelial Cell Apoptosis in vivo and in vitro

Enolase-Phosphatase 1 (Enoph1), a newly discovered enzyme of the methionine salvage pathway, has been identified as a candidate gene involved in the modulation of stress responses. In the present study, we investigated the effect of Enoph1 on ischemic blood brain barrier (BBB) injury on an in vitro model of oxygen-glucose deprivation (OGD) and an in vivo rat model of middle cerebral artery occlusion (MCAO). Cell death and apoptosis were assessed by measuring 3-(4,5-Dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide formation, lactate dehydrogenase release and TdT-mediated dUTP-biotin nick end labeling staining. BBB disruption was evaluated based on fluorescein-labeled albumin (FITC-albumin) extravasation. ROS generation was assessed by DCFH staining. Real time-PCR and western blot were used to measure mRNA and protein expression, respectively. CRISPR-activated plasmid and siRNA were applied to manipulate Enoph1 expression in brain microvascular endothelial cells (BMVECs). Exposure of BMVECs to OGD for 6 hours markedly increased Enoph1 expression and decreased the protein levels of its substrate aci-reductone dioxygenase 1 (ADI1) protein, which was accompanied by membrane type 1 matrix metalloproteinase (MT1-MMP) upregulation, increased apoptotic cell death and ROS generation. Increased Enoph1 expression was also observed in ischemic cerebromicrovassels where FITC-albumin extravasation was observed, indicating the co-localization of BBB disruption and increased Enoph1 expression. Knockdown of Enoph1 expression with siRNA significantly inhibited OGD-induce apoptosis, ROS generation, ADI1 protein reduction and MT1-MMP upregulation in BMVECs, while overexpression of Enoph1 CRISPR-activated plasmids exacerbated OGD-induced cell death and ROS generation. Of note, overexpression of ADI1 or knockdown of MT1-MMP with siRNA significantly blocked OGD-induced BMVECs apoptosis. In conclusion, our data demonstrate for the first time that cerebral ischemia activates Enoph1-ADI1-MT1-MMP axis to mediate BMVEC apoptosis and BBB disruption, and Enoph1 may represent a new therapeutic target for ischemic stroke.

Erythropoietin reduces apoptosis of brain tissue cells in rats after cerebral ischemia/reperfusion injury: a characteristic analysis using magnetic resonance imaging.

Some in vitro experiments have shown that erythropoietin (EPO) increases resistance to apoptosis and facilitates neuronal survival following cerebral ischemia. However, results from in vivo studies are rarely reported. Perfusion-weighted imaging (PWI) and diffusion-weighted imaging (DWI) have been applied successfully to distinguish acute cerebral ischemic necrosis and penumbra in living animals; therefore, we hypothesized that PWI and DWI could be used to provide imaging evidence in vivo for the conclusion that EPO could reduce apoptosis in brain areas injured by cerebral ischemia/reperfusion. To validate this hypothesis, we established a rat model of focal cerebral ischemia/reperfusion injury, and treated with intra-cerebroventricular injection of EPO (5,000 U/kg) 20 minutes before injury. Brain tissue in the ischemic injury zone was sampled using MRI-guided localization. The relative area of abnormal tissue, changes in PWI and DWI in the ischemic injury zone, and the number of apoptotic cells based on TdT-mediated dUTP-biotin nick end-labeling (TUNEL) were assessed. Our findings demonstrate that EPO reduces the relative area of abnormally high signal in PWI and DWI, increases cerebral blood volume, and decreases the number of apoptotic cells positive for TUNEL in the area injured by cerebral ischemia/reperfusion. The experiment provides imaging evidence in vivo for EPO treating cerebral ischemia/reperfusion injury.

Interferon-Regulatory Factor 5-Dependent Signaling Restricts Orthobunyavirus Dissemination to the Central Nervous System.

Interferon (IFN)-regulatory factor 5 (IRF-5) is a transcription factor that induces inflammatory responses after engagement and signaling by pattern recognition receptors. To define the role of IRF-5 during bunyavirus infection, we evaluated Oropouche virus (OROV) and La Crosse virus (LACV) pathogenesis and immune responses in primary cells and in mice with gene deletions in Irf3, Irf5, and Irf7 or in Irf5 alone. Deletion of Irf3, Irf5, and Irf7 together resulted in uncontrolled viral replication in the liver and spleen, hypercytokinemia, extensive liver injury, and an early-death phenotype. Remarkably, deletion of Irf5 alone resulted in meningoencephalitis and death on a more protracted timeline, 1 to 2 weeks after initial OROV or LACV infection. The clinical signs in OROV-infected Irf5(-/-) mice were associated with abundant viral antigen and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells in several regions of the brain. Circulating dendritic cell (DC) subsets in Irf5(-/-) mice had higher levels of OROV RNA in vivo yet produced lower levels of type I IFN than wild-type (WT) cells. This result was supported by data obtained in vitro, since a deficiency of IRF-5 resulted in enhanced OROV infection and diminished type I IFN production in bone marrow-derived DCs. Collectively, these results indicate a key role for IRF-5 in modulating the host antiviral response in peripheral organs that controls bunyavirus neuroinvasion in mice. Oropouche virus (OROV) and La Crosse virus (LACV) are orthobunyaviruses that are transmitted by insects and cause meningitis and encephalitis in subsets of individuals in the Americas. Recently, we demonstrated that components of the type I interferon (IFN) induction pathway, particularly the regulatory transcription factors IRF-3 and IRF-7, have key protective roles during OROV infection. However, the lethality in Irf3(-/-) Irf7(-/-) (DKO) mice infected with OROV was not as rapid or complete as observed in Ifnar(-/-) mice, indicating that other transcriptional factors associated with an IFN response contribute to antiviral immunity against OROV. Here, we evaluated bunyavirus replication, tissue tropism, and cytokine production in primary cells and mice lacking IRF-5. We demonstrate an important role for IRF-5 in preventing neuroinvasion and the ensuing encephalitis caused by OROV and LACV.

Decreased expression of mitochondrial aldehyde dehydrogenase-2 induces liver injury via activation of the mitogen-activated protein kinase pathway.

The aim of this study was to determine the role of ALDH2 in the injury of liver from brain-dead donors. Using brain-dead rabbit model and hypoxia model, levels of ALDH2 and apoptosis in tissues and cell lines were determined by Western blot, flow cytometry (FCM), and transferase (TdT)-mediated biotin-16-dUTP nick-end labeling (TUNEL) assays. After the expression of ALDH2 during hypoxia had been inhibited or activated, the accumulations of 4-hydroxynonenal (4-HNE) and molecules involved in mitogen-activated protein kinase (MAPK) signaling pathway were analyzed using ELISA kit and Western blot. The low expression of phosphorylated ALDH2 in liver was time-dependent in the brain-dead rabbit model. Immunohistochemistry showed ALDH2 was primarily located in endothelial, and the rates of cell apoptosis in the donation after brain-death (DBD) rabbit groups significantly increased with time. Following the treatment of inhibitor of ALDH2, daidzein, in combination with hypoxia for 8 h, the apoptosis rate and the levels of 4-HNE, P-JNK, and cleaved caspase-3 significantly increased in contrast to that in hypoxic HUVECs; however, they all decreased after treatment with Alda-1 and hypoxia compared with that in hypoxic HUVECs (P < 0.05). Instead, the levels of P-P38, P-ERK, P-JNK, and cleaved caspase-3 decreased and the ratio of bcl-2/bax increased with ad-ALDH2 (10(6) pfu/ml) in combination with hypoxia for 8 h, which significantly alleviated in contrast to that in hypoxic HUVECs. We found low expression of ALDH2 and high rates of apoptosis in the livers of brain-dead donor rabbits. Furthermore, decreased ALDH2 led to apoptosis in HUVECs through MAPK pathway.

Intraarticular Injection of Allogenic Mesenchymal Stem Cells has a Protective Role for the Osteoarthritis.

Background:Researchers initially proposed the substitution of apoptotic chondrocytes in the superficial cartilage by injecting mesenchymal stem cells (MSCs) intraarticularly. This effect was termed as bio-resurfacing. Little evidence supporting the treatment of osteoarthritis (OA) by the delivery of a MSC suspension exists. The aim of this study was to investigate the effects of injecting allogenic MSCs intraarticularly in a rat OA model and to evaluate the influence of immobility on the effects of this treatment.Methods:We established a rat knee OA model after 4 and 6 weeks and cultured primary bone marrow MSCs. A MSC suspension was injected into the articular space once per week for 3 weeks. A subgroup of knee joints was immobilized for 3 days after each injection, while the remaining joints were nonimmobilized. We used toluidine blue staining, Mankin scores, and TdT-mediated dUTP-biotin nick end labeling staining to evaluate the therapeutic effect of the injections. Comparisons between the therapy side and the control side of the knee joint were made using paired t-test, and comparisons between the immobilized and nonimmobilized subgroups were made using the unpaired t-test. A P value < 0.05 was considered significant.Results:The three investigative approaches revealed less degeneration on the therapy sides of the knee joints than the control sides in both the 4- and 6-week groups (P < 0.05), regardless of immobilization. No significant differences were observed between the immobilized and nonimmobilized subgroups (P > 0.05).Conclusions:Therapy involving the intraarticular injection of allogenic MSCs promoted cartilage repair in a rat arthritis model, and 3-day immobility after injection had little effect on this therapy.

DNMT1 mediates chemosensitivity by reducing methylation of miRNA-20a promoter in glioma cells.

Although methyltransferase has been recognized as a major element that governs the epigenetic regulation of the genome during temozolomide (TMZ) chemotherapy in glioblastoma multiforme (GBM) patients, its regulatory effect on glioblastoma chemoresistance has not been well defined. This study investigated whether DNA methyltransferase (DNMT) expression was associated with TMZ sensitivity in glioma cells and elucidated the underlying mechanism. DNMT expression was analyzed by western blotting. miR-20a promoter methylation was evaluated by methylation-specific PCR. Cell viability and apoptosis were assessed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and TdT-mediated dUTP-biotin nick end labeling assays, respectively. The results showed that compared with parental U251 cells, DNMT1 expression was downregulated, miR-20a promoter methylation was attenuated and miR-20a levels were elevated in TMZ-resistant U251 cells. Methyltransferase inhibition by 5-aza-2′-deoxycytidine treatment reduced TMZ sensitivity in U251 cells. In U251/TM cells, DNMT1 expression was negatively correlated with miR-20a expression and positively correlated with TMZ sensitivity and leucine-rich repeats and immunoglobulin-like domains 1 expression; these effects were reversed by changes in miR-20a expression. DNMT1 overexpression induced an increase in U251/TM cell apoptosis that was inhibited by the miR-20a mimic, whereas DNMT1 silencing attenuated U251/TM cell apoptosis in a manner that was abrogated by miR-20a inhibitor treatment. Tumor growth of the U251/TM xenograft was inhibited by pcDNA-DNMT1 pretreatment and boosted by DNMT1-small hairpin RNA pretreatment. In summary, DNMT1 mediated chemosensitivity by reducing methylation of the microRNA-20a promoter in glioma cells.

Protective effects of probiotic Lactobacillus plantarum BJ0021 on liver and kidney oxidative stress and apoptosis induced by endosulfan in pregnant rats

Endosulfan (EDS) is one of the most widely organochlorine insecticide used in many parts of the world, although it is currently banned or severely restricted in use in some countries. EDS causes a variety of negative effects in non-target species including humans. Therefore, the aim of the present study was to investigate the possible protective effects of Lactobacillus plantarum BJ0021 on toxicity, oxidative stress, and apoptosis induced by EDS intoxication on liver and kidneys of pregnant rats. This pesticide induced a significant increase in total cholesterol, alanine-amino transferase (ALAT), aspartate-amino transferase (ASAT), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (GGT), urea and creatinine in serum, while urinary urea and creatinine were lower than those of the control group. In the liver and kidney, lipid peroxidation increased significantly, the antioxidant levels, such as superoxide dismutase (SOD) and catalase (CAT) were markedly depressed and TdT-mediated dUTP-biotin Nick End Labeling (TUNEL) revealed more apoptotic cells. In contrast, co-administration of L. plantarum BJ0021 to EDS-treated animals ameliorated most of these biochemical parameters, but the activity of antioxidant enzymes (SOD, CAT) did not modify and the number of apoptotic nuclei remained significantly raised in kidney compared to control. In conclusion, the administration of L. plantarum BJ0021 decreased apoptosis and might play a protective role in reducing toxicity of EDS in pregnant rats.

RETRACTED ARTICLE: Comparative analysis of the replication of bovine herpesvirus 1 (BHV1) and BHV5 in bovine-derived neuron-like cells

Members of the subfamily Alphaherpesvirinae use the epithelium of the upper respiratory and/or genital tract as preferential sites for primary replication. However, bovine herpesvirus 5 (BoHV5) is neurotropic and neuroinvasive and responsible for meningoencephalitis in cattle and in animal models. A related virus, BoHV1 has also been occasionally implicated in natural cases of neurological infection and disease in cattle. The aim of the present study was to assess the in vitro effects of BoHV1 and BoHV5 replication in neuron-like cells. Overall, cytopathic effects, consisting of floating rounded cells, giant cells and monolayer lysis, induced by both viruses at 48 h postinfection (p.i.) resulted in a loss of cell viability and high virus titres (r = 0.978). The BoHV1 Cooper strain produced the lowest titres in neuron-like cells, although viral DNA was detected in infected cells during all experiments. Virus replication in infected cells was demonstrated by immunocytochemistry, flow cytometry and qPCR assays. BoHV antigens were better visualized at 48 h p.i. and flow cytometry analysis showed that SV56/90 and Los Angeles antigens were present at higher levels. In spite of the fact that BoHV titres dropped at 48 h p.i, viral DNA remained detectable until 120 h p.i. Sensitive TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) and annexin V assays were used to identify apoptosis. BoHV5 induced death in approximately 50% of cells within 24 h p.i., similar to what has been observed for BoHV1 Los Angeles. Infection with the BoHV1 Cooper strain resulted in 26.37% of cells being in the early stages of apoptosis; 63.69% of infected cells were considered viable. Modulation of mitochondrial function, as measured by mitochondrial membrane depolarization, was synchronous with the virus replication cycle, cell viability and virus titres at 48 h p.i. Our results indicate that apoptosis plays an important role in preventing neuronal death and provides a bovine-derived in vitro system to study herpesvirus-neuron interactions.

Garcinia xanthochymus Benzophenones Promote Hyphal Apoptosis and Potentiate Activity of Fluconazole against Candida albicans Biofilms.

Xanthochymol and garcinol, isoprenylated benzophenones purified from Garcinia xanthochymus fruits, showed multiple activities against Candida albicans biofilms. Both compounds effectively prevented emergence of fungal germ tubes and were also cytostatic, with MICs of 1 to 3 μM. The compounds therefore inhibited development of hyphae and subsequent biofilm maturation. Xanthochymol treatment of developing and mature biofilms induced cell death. In early biofilm development, killing had the characteristics of apoptosis, including externalization of phosphatidyl serine and DNA fragmentation, as evidenced by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) fluorescence. These activities resulted in failure of biofilm maturation and hyphal death in mature biofilms. In mature biofilms, xanthochymol and garcinol caused the death of biofilm hyphae, with 50% effective concentrations (EC50s) of 30 to 50 μM. Additionally, xanthochymol-mediated killing was complementary with fluconazole against mature biofilms, reducing the fluconazole EC50 from >1,024 μg/ml to 13 μg/ml. Therefore, xanthochymol has potential as an adjuvant for antifungal treatments as well as in studies of fungal apoptosis.

Retinal Cell Death Caused by Sodium Iodate Involves Multiple Caspase-Dependent and Caspase-Independent Cell-Death Pathways.

Herein, we have investigated retinal cell-death pathways in response to the retina toxin sodium iodate (NaIO3) both in vivo and in vitro. C57/BL6 mice were treated with a single intravenous injection of NaIO3 (35 mg/kg). Morphological changes in the retina post NaIO3 injection in comparison to untreated controls were assessed using electron microscopy. Cell death was determined by TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining. The activation of caspases and calpain was measured using immunohistochemistry. Additionally, cytotoxicity and apoptosis in retinal pigment epithelial (RPE) cells, primary retinal cells, and the cone photoreceptor (PRC) cell line 661W were assessed in vitro after NaIO3 treatment using the ApoToxGlo™ assay. The 7-AAD/Annexin-V staining was performed and necrostatin (Nec-1) was administered to the NaIO3-treated cells to confirm the results. In vivo, degenerating RPE cells displayed a rounded shape and retracted microvilli, whereas PRCs featured apoptotic nuclei. Caspase and calpain activity was significantly upregulated in retinal sections and protein samples from NaIO3-treated animals. In vitro, NaIO3 induced necrosis in RPE cells and apoptosis in PRCs. Furthermore, Nec-1 significantly decreased NaIO3-induced RPE cell death, but had no rescue effect on treated PRCs. In summary, several different cell-death pathways are activated in retinal cells as a result of NaIO3.

Neuroprotective effect of geniposide on Parkinson's disease model mice

Objective To investigate the neuroprotective effect of geniposide on 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) induced Parkinson's disease (PD) model mice and possible mechanism. Methods A total of 48 male C57BL/6 mice were randomly divided into control, model, geniposide and MPTP + geniposide groups. The behaviors of C57BL/6 mice were assessed by using open field test, the tyrosine hydroxylase (TH) and Bcl-2 positive neurons in the midbrain substantia nigra of mice were detected by immunohistochemistry and the number of apoptosis neurons were observed with TdT-mediated dUTP-biotin nick end labeling (TUNEL). Results The number of mobile grid [(76.33 ± 8.59) times/5 min], standing [(19.58 ± 3.97) times/5 min], TH-positive neurons [(12.83 ± 2.32)/HPF] and Bcl-2-positive neurons [(10.83 ± 2.23)/HPF] in model group were significantly lower than those in the control group [(142.50 ± 11.65) times/5 min, (39.17 ± 4.75) times/5 min, (35.67 ± 1.75)/HPF, (20.67 ± 1.75)/HPF; P = 0.000, for all]. The apoptosis neurons in model group [(20.33 ± 2.58)/HPF] were significantly higher than that in control group [(3.83 ± 1.67) /HPF, P = 0.000). The number of mobile grid [(97.67 ± 13.15) times/5 min, P = 0.000], standing [(29.33 ± 2.90) times/5 min, P = 0.000], TH-positive neurons [(17.50 ± 2.07)/HPF, P = 0.002] and Bcl-2-positive neurons [(15.17 ± 2.79) /HPF, P = 0.003] in MPTP + geniposide group were significantly higher than those in model group. The number of apoptosis neurons [(14.67 ± 3.08) /HPF] in MPTP + geniposide group was significantly lower than that in model group ( P = 0.001). Conclusions Geniposide can protect dopaminergic neurons in MPTP-induced neurodegeneration and the mechanism may be associated with the inhibition of neuronal apoptosis. DOI: 10.3969/j.issn.1672-6731.2015.06.012

Decreased DNA Disruption in the Porcine Neocortex with Erythromycin Preconditioning during Prolonged Hypothermic Circulatory Arrest: Evidence for Neuroprotection.

We have previously reported that the neocortex is selectively vulnerable to injury in an acute porcine model of hypothermic circulatory arrest (HCA) at 18°C. In view of recent evidence showing that pharmacologic preconditioning with a single dose of erythromycin induces tolerance against transient global cerebral ischemia in rats, we hypothesized that erythromycin would reduce the number of apoptotic neurons in the neocortex in an acute porcine model of HCA at 18°C. Fourteen piglets underwent 75 min of HCA at 18°C following pretreatment with erythromycin (25 mg/kg, IV) (n = 8) or vehicle (Normal Saline 0.9%) (n = 6), applied 12 hr before arrest. Three served as normal controls. After gradual rewarming to a temperature of 36°C, treatment animals were sacrificed and brains were perfusion-fixed and cryopreserved. Neuronal apoptosis after HCA was observed morphologically with hematoxylin and eosin staining, and characterized by in situ DNA fragmentation using terminal deoxynucleotidyl-transferase-mediated biotin-dUTP nick end-labeling (TUNEL) histochemistry. Pre-ischemic conditioning with a single dose of the antibiotic erythromycin reduced neuronal apoptosis in the neocortex of the porcine brain. TUNEL-positive cells indicating DNA fragmentation and neuronal injury were significantly greater in the neocortex of animals treated with 18°C HCA (2.55 ± 1.17) compared to animals undergoing HCA after erythromycin preconditioning (1.76 ± 0.91) (p ≤ 0.001). These results suggest that cerebral protection during HCA may be achieved with erythromycin pharmacological preconditioning in the porcine model.

On the use of an appropriate TdT-mediated dUTP–biotin nick end labeling assay to identify apoptotic cells

Apoptosis is an essential cellular mechanism involved in many processes such as embryogenesis, metamorphosis, and tissue homeostasis. DNA fragmentation is one of the key markers of this form of cell death. DNA fragmentation is executed by endogenous endonucleases such as caspase-activated DNase (CAD) in caspase-dependent apoptosis. The TUNEL (TdT-mediated dUTP–biotin nick end labeling) technique is the most widely used method to identify apoptotic cells in a tissue or culture and to assess drug toxicity. It is based on the detection of 3′-OH termini that are labeled with dUTP by the terminal deoxynucleotidyl transferase. Although the test is very reliable and sensitive in caspase-dependent apoptosis, it is completely useless when cell death is mediated by pathways involving DNA degradation that generates 3′-P ends as in the LEI/L-DNase II pathway. Here, we propose a modification in the TUNEL protocol consisting of a dephosphorylation step prior to the TUNEL labeling. This allows the detection of both types of DNA breaks induced during apoptosis caspase-dependent and independent pathways, avoiding underestimating the cell death induced by the treatment of interest.

Latent herpes simplex virus 1 infection does not induce apoptosis in human trigeminal Ganglia.

Herpes simplex virus 1 (HSV-1) can establish lifelong latency in human trigeminal ganglia. Latently infected ganglia contain CD8(+) T cells, which secrete granzyme B and are thus capable of inducing neuronal apoptosis. Using immunohistochemistry and single-cell reverse transcription-quantitative PCR (RT-qPCR), higher frequency and transcript levels of caspase-3 were found in HSV-1-negative compared to HSV-1-positive ganglia and neurons, respectively. No terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay-positive neurons were detected. The infiltrating T cells do not induce apoptosis in latently infected neurons.

Oestrous cycle-dependent expression of Fas and Bcl2 family gene products in normal canine endometrium.

During the oestrous cycle canine endometrium undergoes cyclical cellular proliferation, apoptosis and differentiation. To study the regulation of endometrial apoptosis and proliferation events the expression of apoptosis-related genes was analysed by real-time polymerase chain reaction and cellular expression of their proteins was identified through immunohistochemistry. Cellular apoptosis and proliferation events were detected by TdT-mediated dUTP-biotin nick end labeling (TUNEL) and proliferation marker Ki67 immunostaining, respectively. The highest proliferative index was observed in the follicular phase (all endometrial cellular components) and at early dioestrus (basal glands). This was associated with a low apoptotic index and a strong expression of anti- (Bcl2) and pro-apoptotic proteins (Fas, FasL, Bax). Subsequently (Days 11-45 of dioestrus), basal glandular epithelium experienced the highest apoptotic index, coincidental with a decrease of Bcl2 expression and a low ratio of Bcl2/Bax transcription. An increase in the apoptotic index of crypts, stromal and endothelial cells was observed at late dioestrus and the beginning of anoestrus. These results indicate that pro- and anti-apoptotic proteins regulate the balance between cell proliferation and death in the canine endometrium during the oestrous cycle. High Bcl2 expression in both the follicular and early dioestrous phases stimulate glandular proliferation and prevent apoptosis but, in the non-pregnant uterus, a decrease in Bcl2 expression together with an increase in pro-apoptotic proteins induces apoptosis of basal glandular epithelium cells.

The secreted effector protein EspZ is essential for virulence of rabbit enteropathogenic Escherichia coli.

Attaching and effacing (A/E) pathogens adhere intimately to intestinal enterocytes and efface brush border microvilli. A key virulence strategy of A/E pathogens is the type III secretion system (T3SS)-mediated delivery of effector proteins into host cells. The secreted protein EspZ is postulated to promote enterocyte survival by regulating the T3SS and/or by modulating epithelial signaling pathways. To explore the role of EspZ in A/E pathogen virulence, we generated an isogenic espZ deletion strain (ΔespZ) and corresponding cis-complemented derivatives of rabbit enteropathogenic Escherichia coli and compared their abilities to regulate the T3SS and influence host cell survival in vitro. For virulence studies, rabbits infected with these strains were monitored for bacterial colonization, clinical signs, and intestinal tissue alterations. Consistent with data from previous reports, espZ-transfected epithelial cells were refractory to infection-dependent effector translocation. Also, the ΔespZ strain induced greater host cell death than did the parent and complemented strains. In rabbit infections, fecal ΔespZ strain levels were 10-fold lower than those of the parent strain at 1 day postinfection, while the complemented strain was recovered at intermediate levels. In contrast to the parent and complemented mutants, ΔespZ mutant fecal carriage progressively decreased on subsequent days. ΔespZ mutant-infected animals gained weight steadily over the infection period, failed to show characteristic disease symptoms, and displayed minimal infection-induced histological alterations. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining of intestinal sections revealed increased epithelial cell apoptosis on day 1 after infection with the ΔespZ strain compared to animals infected with the parent or complemented strains. Thus, EspZ-dependent host cell cytoprotection likely prevents epithelial cell death and sloughing and thereby promotes bacterial colonization.