Terminal Deoxynucleotidyl Transferase Nick-end Labeling Research Articles

Ataxia Telangiectasia-Mutated Kinase Deficiency Induces Cardiac Dysfunction with Increased Myocyte Apoptosis and Hypertrophy with Age

Background: Heart failure is the leading cause of death globally. The risk for heart disease increases exponentially with age. The incidence of heart disease is approximately 40% in humans that are 40-59 years old. Ataxia telangiectasia-mutated kinase (ATM), a cell cycle protein, controls growth and repair in response to double-stranded DNA damage. Mutations in the ATM gene lead to a multisystemic disease known as Ataxia Telangiectasia (A-T). These individuals exhibit signs of premature aging. A-T heterozygotes make up about 2% of the population and are more susceptible to ischemic heart disease and die 11 years earlier compared to non-carriers. Previous work has shown that ATM deficiency increases myocyte hypertrophy in 4-month-old (young) ATM heterozygous knockout (hKO) mice vs. age-matched wild-type (WT) mice. However, there was no difference in myocyte apoptosis or heart function between the two groups. Hypothesis: It is hypothesized that with age, ATM deficiency induces myocytes apoptosis and hypertrophy, and cardiac dysfunction. Methods: Heart function was measured in young and 14-month-old WT and hKO mice using echocardiography. Cross sections (5 μm) of heart were used to measure myocyte apoptosis and hypertrophy using terminal deoxynucleotidyl transferase nick end labeling (TUNEL) and rhodamine wheat germ agglutinin (WGA) staining, respectively. Results: Heart function, measured by fractional shortening (%FS) and ejection fraction (%EF), remained unchanged in young and 14-month-old WT mice. However, %FS and %EF were significantly lower in 14-month-old hKO mice vs. young hKO and age-matched WT mice. Myocyte apoptosis and cross-sectional area (hypertrophy) were significantly higher in 14-month-old hKO vs. age-matched WT mice. Conclusion: The data indicates that ATM deficiency induces cardiac dysfunction with increased myocyte apoptosis and hypertrophy with age. This research was supported by Department of Biological Sciences, Undergraduate Research Honors Program, Offce of Research and Sponsored Programs, Department of Biomedical Sciences, and Department of Health Sciences. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Antifungal activity of human antimicrobial peptides targeting apoptosis in Candida auris.

Introduction. Innovative antifungal therapies are of crucial importance to combat the potentially life-threatening infections linked to the multidrug-resistant fungal pathogen Candida auris. Induction of regulated cell death, apoptosis, could provide an outline for future therapeutics. Human antimicrobial peptides (AMPs), well-known antifungal compounds, have shown the ability to induce apoptosis in pathogenic fungi.Hypothesis/Gap Statement . Although it is known that AMPs possess antifungal activity against C. auris, their ability to induce apoptosis requires further investigations.Aim. This study evaluated the effects of AMPs on the induction of apoptosis in C. auris.Methods. Human neutrophil peptide-1 (HNP-1), human β-Defensins-3 (hBD-3) and human salivary histatin 5 (His 5) were assessed against two clinical C. auris isolates. Apoptosis hallmarks were examined using FITC-Annexin V/PI double labelling assay and terminal deoxynucleotidyl transferase deoxynucleotidyl transferase nick-end labelling (TUNEL) to detect phosphatidylserine externalization and DNA fragmentation, respectively. Then, several intracellular triggers were studied using JC-10 staining, spectrophotometric assay and 2',7'-dichlorofluorescin diacetate staining to measure the mitochondrial membrane potential, cytochrome-c release and reactive oxygen species (ROS) production, respectively.Results and conclusion. FITC-Annexin V/PI staining and TUNEL analysis revealed that exposure of C. auris cells to HNP-1 and hBD-3 triggered both early and late apoptosis, while His 5 caused significant necrosis. Furthermore, HNP-1 and hBD-3 induced significant mitochondrial membrane depolarization, which resulted in substantial cytochrome c release. In contrast to His 5, which showed minimal mitochondrial depolarization and no cytochrome c release. At last, all peptides significantly increased ROS production, which is related to both types of cell death. Therefore, these peptides represent promising and effective antifungal agents for treating invasive infections caused by multidrug-resistant C. auris.

Hyperglycemia disrupted the integrity of the blood-brain barrier following diffuse axonal injury through the sEH/NF-κB pathway.

We aimed to investigate the role of soluble epoxide hydrolase for hyperglycemia induced-disruption of blood-brain barrier (BBB) integrity after diffuse axonal injury (DAI). Rat DAI hyperglycemia model was established by a lateral head rotation device and intraperitoneal injection of 50% glucose. Glial fibrillary acidic protein, ionized calcium-binding adapter molecule-1, β-amyloid precursor protein, neurofilament light chain, and neurofilament heavy chain was detected by immunohistochemistry. Cell apoptosis was examined by terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) assay. The permeability of blood-brain barrier (BBB) was assessed by expression of tight junction proteins, leakage of Evans blue and brain water content. The soluble epoxide hydrolase (sEH) pathway was inhibited by 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) and the nuclear transcription factor kappa B (NF-κB) pathway was inhibited by pyrrolidine dithiocarbamate and activated by phorbol-12-myristate-13-acetate in vivo and/or vitro, respectively. The inflammatory factors were detected by enzyme-linked immunosorbent assay. Hyperglycemia could exacerbate axonal injury, aggravate cell apoptosis and glial activation, worsen the loss of BBB integrity, increase the release of inflammatory factors, and upregulate the expression of sEH and NF-κB. Inhibition of sEH could reverse all these damages and protect BBB integrity by upregulating the expression of tight junction proteins and downregulating the levels of inflammatory factors in vivo and vitro, while the agonist of NF-κB pathway abrogated the protective effects of TPPU on BBB integrity in vitro. sEH was involved in mediating axonal injury induced by hyperglycemia after DAI by disrupting BBB integrity through inducing inflammation via the NF-κB pathway.

Deletion of the gene for the African swine fever virus BCL-2 family member A179L increases virus uptake and apoptosis but decreases virus spread in macrophages and reduces virulence in pigs.

African swine fever virus (ASFV) causes a lethal disease of pigs with high economic impact in affected countries in Africa, Europe, and Asia. The virus encodes proteins that inhibit host antiviral defenses, including the type I interferon response. Host cells also activate cell death through a process called apoptosis to limit virus replication. We showed that the ASFV A179L protein, a BCL-2 family apoptosis inhibitor, is important in reducing apoptosis in infected cells since deletion of this gene increased cell death and reduced virus replication in cells infected with the A179L gene-deleted virus. Pigs immunized with the BeninΔA179L virus showed no clinical signs and a weak immune response but were not protected from infection with the deadly parental virus. The results show an important role for the A179L protein in virus replication in macrophages and virulence in pigs and suggest manipulation of apoptosis as a possible route to control infection.

Effect of Different Vitrification Techniques on Viability and Apoptotic Index of Domestic Cat Testicular Tissue Cells.

Vitrification is essential for successful tissue cryopreservation and biobanking in wild cats. This study aimed to compare different methods of vitrification (Ovarian Tissue Cryosystem-OTC, Straws-STW, and Solid Surface vitrification-SSV) for testicular fragment vitrification in tom cats. Testicular fragments were recovered from five adult tom cats and subjected to equilibrium vitrification using different cryovials and methods under the same conditions of vitrification solutions and cryoprotectants. The efficiencies of the methods were evaluated using histological analysis of spermatogonia and Sertoli cell nuclei, seminiferous tubular basement membrane detachment, and the gonadal epithelium shrinkage score scale. Cell viability was assessed using Hoechst PI and Terminal deoxynucleotidyl transferase nick end labeling (TUNEL) assay. The results showed that OTC is an effective vitrification method for maintaining the distinction between spermatogonia and Sertoli cells. OTC was similar to the control for basal membrane detachment parameters (p = 0.05). Epithelial shrinkage was low in the SSV group, which showed the highest percentage of viable cells among the vitrified groups (p = 0.0023). The OTC and SSV vitrification methods were statistically similar in terms of the percentage of TUNEL-positive cells (p = 0.05). Therefore, OTC and SSV provide favorable conditions for maintaining viable cat testicular tissue cells after vitrification.

Curcumin potentiates the anti-inflammatory effects of Tehranolide by modulating the STAT3/NF-κB signaling pathway in breast and ovarian cancer cell lines.

Studies have demonstrated thatnatural products, such as curcumin and artemisinin,possess anti-inflammatory effects, which can be beneficial for cancer treatment.Tehranolide, as a novel natural product, has a wide range of biological activities, including anti-cancer effects. However,many properties of Tehranolide, like its anti-inflammatory activity and its combination with curcumin, have not been investigated yet. This investigation examined the anti-inflammatory activity of Tehranolide, either alone or in combination with curcumin, via modulating the NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and STAT3 (signal transducer and activator of transcription 3) signaling pathways in MDA-MB-231 and SKOV3, breast and ovarian cancer cell lines. ELISA-based methods were employed to measure the pro-inflammatory cytokine levels and the NF-κB activity in lipopolysaccharide (LPS)-induced cells. The real-time PCR experiment and Griess test were performed to evaluate inducible nitric oxide synthase (iNOS) gene expression and nitrite levels, respectively. The STAT3 and NF-κB signaling pathways were investigated by Western blotting analysis. Tehranolide's anti-cancer activity was also assessed in a mouse model of breast cancer using the TUNEL (terminal deoxynucleotidyl transferase nick-end labeling) assay. Tehranolide diminished levels of pro-inflammatory cytokines in cancer cells. Additionally, it suppressed NF-κB DNA binding and STAT3 phosphorylation, reducing iNOS gene expression and nitrite production. Moreover, Western blotting showed that Tehranolide enhanced the inhibitory κB (IκBα) and Bcl-2 (B-cell lymphoma 2)-associated X (BAX) expression, and downregulated the expression of Bcl-2 proteins. Furthermore, the TUNEL assay demonstrated that Tehranolide induced apoptosis in a breast cancer mouse model. Curcumin potentiated all the anti-inflammatory effects of Tehranolide. This investigation indicated for the first time that Tehranolide, either alone or in combination with curcumin, exerted its anti-inflammatory effects by suppressing NF-κB and STAT3 signaling pathways in SKOV3 and MDA-MB-231 cells.

The Effects of Duration of the In Vitro Maturation Process on the Maturation Level and Apoptosis of Kacang Goat

The success of in vitro fertilization and embryo culture depends on the success of in vitro maturation. However, standard culture conditions usually increase reactive oxygen species (ROS), which have been implicated as a major cause for reduced embryonicdevelopment. It is well-known that higher than physiological levels of ROS trigger granulosa cell apoptosis and thereby reduce the transfer of nutrients and survival factors to oocytes, leading to apoptosis. This study aimed to determine the optimal timing ofoocyte maturation and its relationship to DNA fragmentation. Ovaries were collected from a slaughterhouse and the follicles aspirated. The cumulus oocyte complexes were divided into groups and transferred to a maturation medium, where they were maintained for 18 hours (P1), 22 hours (P2) and 24 hours (P3) to evaluated maturation rate. Matured oocytes were characterized as oocytes that reached the MII stage. Matured oocytes were counterstained with terminal deoxynucleotidyl transferase nick-end labelling (TUNEL). The results showed that the maturity rate of Kacang goat oocytes reached 46% after 18 hours, 77% after 22 hours, and 63% after 24 hours. However, the results showed that the expression of DNA fragmentation in P2 (2.4 ± 0.89) were significantly different from P1 (5.4 ± 2.61) and P3 (9.0 ± 2.12). In conclusion, the optimal timing of in vitro maturation of Kacang goat oocytes is 22 hours.

Anoectochilus roxburghii flavonoids extract ameliorated the memory decline and reduced neuron apoptosis via modulating SIRT1 signaling pathway in senescent mice

Ethnopharmacological relevanceAnoectochilus roxburghii (A. roxburghii) is a precious herb and folk medicine in many Asian countries. It has been used traditionally to treat diabetes, etc., and also used as a dietary therapy to delay senescence. Aim of the studyThis study was to evaluate the neuroprotective effects of A. roxburghii flavonoids extract (ARF) and whether its effects were due to the regulation of SIRT1 signaling pathway in senescent mice and in D-galactose (D-gal) induced aging in SH-SY5Y cells. Materials and methods18-month-old mice were randomly divided into senescent model, low-dose ARF, high-dose ARF and vitamin E group. 2-Month-old mice were as a control group. After 8 weeks treatment, Morris water maze (MWM) was performed. The levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), monoamine oxidase (MAO) and acetylcholinesterase (ACh-E) in the cortex were determined. Hippocampus morphologic changes were observed with haematoxylin and eosin (H&E), Nissl, senescence-associated-galactosidase (SA-β-gal) and terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) staining. Apoptosis-related molecular expressions in the hippocampus were performed by western blotting. Furthermore, after stimulated by EX527 (a SIRT1 inhibitor), the SIRT1-dependent neuroprotective effects of ARF were determined by measuring SRIT1 and p53 expression in SH-SY5Y aging cells induced by D-gal. ResultsARF could significantly ameliorate memory decline in senescent mice and reduce the generations of ROS, MDA and the activities of MAO and ACh-E, while increasing SOD activities in the cortex of aging mice. ARF obviously improved hippocampus pathological alterations, increased the number of Nissl bodies, while reducing senescent and apoptotic cells in senescent mice hippocampus. Further, ARF positively regulated SIRT1 expression, and reduced apoptosis-related molecules p53, p21 and Caspase-3 expression, while increasing the ratio of Bcl-2/Bax. In D-gal-induced SH-SY5Y cells, the effects of ARF on SIRT1 and p53, and the ability of scavenging ROS were mostly abolished after incubation with the EX527. ConclusionsARF, in a SIRT1-dependent manner, exerted neuroprotection via modulating SIRT1/p53 signaling pathway against memory decline and apoptosis due to age-induced oxidative stress damage in senescent mice.

Improved Maturation Rate of Bovine Oocytes Following Sericin Supplementation in Collection and Maturation Media

Sericin is a water-soluble protein produced by silk cocoons and known to have antioxidant activity. This study is aimed to analyze the nuclear maturation and the quality of bovine oocytes in the collection and in vitro maturation (IVM) medium supplemented with sericin. Bovine oocytes were collected using a collection medium supplemented with sericin in 0 (control) concentrations, 0.1%, 0.5%, and 1%. Selected oocytes were then matured for 24 h at 38.5 o C in 5% CO₂ and evaluated for nuclear maturation. In the subsequent experiment, oocytes were collected and matured with or without 0.1% sericin at 38.5 o C in 5% CO 2 . Matured oocytes were counterstained with terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) and Hoechst 33342. Matured oocytes were characterized by oocytes that reached the MII stage. The results showed that supplementation of 0.1% sericin in the collection medium increased the number of oocytes reaching the metaphase II (MII) stage compared to the control group (p<0.05). In the next experiment, sericin 0.1% in the collection and in vitro maturation media increased (p<0.05) the percentage of oocytes reaching the MII stage compared to control without sericin supplementation. Furthermore, the number of fragmented DNA in the oocytes showed no differences in all groups. It can be concluded that supplementation of 0.1% sericin in the collection and in vitro maturation media improved the nuclear status without affecting DNA fragmentation.

Selenium deficiency induces spleen pathological changes in pigs by decreasing selenoprotein expression, evoking oxidative stress, and activating inflammation and apoptosis

BackgroundThe immune system is one aspect of health that is affected by dietary selenium (Se) levels and selenoprotein expression. Spleen is an important immune organ of the body, which is directly involved in cellular immunity. However, there are limited reports on Se levels and spleen health. Therefore, this study established a Se-deficient pig model to investigate the mechanism of Se deficiency-induced splenic pathogenesis.MethodsTwenty-four pure line castrated male Yorkshire pigs (45 days old, 12.50 ± 1.32 kg, 12 full-sibling pairs) were divided into two equal groups and fed Se-deficient diet (0.007 mg Se/kg) or Se-adequate diet (0.3 mg Se/kg) for 16 weeks. At the end of the trial, blood and spleen were collected to assay for erythroid parameters, the osmotic fragility of erythrocytes, the spleen index, histology, terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) staining, Se concentrations, the selenogenome, redox status, and signaling related inflammation and apoptosis.ResultsDietary Se deficiency decreased the erythroid parameters and increased the number of osmotically fragile erythrocytes (P < 0.05). The spleen index did not change, but hematoxylin and eosin and TUNEL staining indicated that the white pulp decreased, the red pulp increased, and splenocyte apoptosis occurred in the Se deficient group. Se deficiency decreased the Se concentration and selenoprotein expression in the spleen (P < 0.05), blocked the glutathione and thioredoxin antioxidant systems, and led to redox imbalance. Se deficiency activated the NF-κB and HIF-1α transcription factors, thus increasing pro-inflammatory cytokines (IL-1β, IL-6, IL-8, IL-17, and TNF-α), decreasing anti-inflammatory cytokines (IL-10, IL-13, and TGF-β) and increasing expression of the downstream genes COX-2 and iNOS (P < 0.05), which in turn induced inflammation. In addition, Se-deficiency induced apoptosis through the mitochondrial pathway, upregulated apoptotic genes (Caspase3, Caspase8, and Bak), and downregulated antiapoptotic genes (Bcl-2) (P < 0.05) at the mRNA level, thus verifying the results of TUNEL staining.ConclusionsThese results indicated that Se deficiency induces spleen injury through the regulation of selenoproteins, oxidative stress, inflammation and apoptosis.

Liquiritin Attenuates Lipopolysaccharides-Induced Cardiomyocyte Injury via an AMP-Activated Protein Kinase-Dependent Signaling Pathway.

Background: Liquiritin (LIQ) is a traditional Chinese medicine that has been reported to regulate inflammation, oxidative stress and cell apoptosis. However, the beneficial effects of LIQ in lipopolysaccharides (LPS)-induced septic cardiomyopathy (SCM) has not been reported. The primary goal of this study was to investigate the effects of LIQ in LPS-induced SCM model. Methods: Mice were pre-treated with LIQ for 7 days before they were injected with LPS (10 mg/kg) for inducing SCM model. Echocardiographic analysis was used to evaluate cardiac function after 12 h of LPS injection. Thereafter, mice were sacrificed to collect hearts for molecular and histopathologic assays by RT-PCR, western-blots, immunohistochemical and terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) staining analysis respectively. AMPKα2 knockout (AMPKα2−/−) mice were used to elucidate the mechanism of LIQ Neonatal rat cardiomyocytes (NRCMs) treated with or without LPS were used to further investigate the roles and mechanisms of LIQ in vitro experiments. Results: LIQ administration attenuated LPS-induced mouse cardiac dysfunction and reduced mortality, based upon the restoration of EF, FS, LVEDs, heart rate, dp/dt max and dp/dt min deteriorated by LPS treatment. LIQ treatment also reduced mRNA expression of TNFα, IL-6 and IL-1β, inhibited inflammatory cell migration, suppressed cardiac oxidative stress and apoptosis, and improved metabolism. Mechanistically, LIQ enhanced the phosphorylation of AMP-activated protein kinase α2 (AMPKα2) and decreased the phosphorylation of mTORC1, IκBα and NFκB/p65. Importantly, the beneficial roles of LIQ were not observed in AMPKα2 knockout model, nor were they observed in vitro model after inhibiting AMPK activity with an AMPK inhibitor. Conclusion: We have demonstrated that LIQ exerts its protective effects in an SCM model induced by LPS administration. LIQ reduced inflammation, oxidative stress, apoptosis and metabolic alterations via regulating AMPKα2 dependent signaling pathway. Thus, LIQ might be a potential treatment or adjuvant for SCM treatment.

Improved preservation of ovarian tissue morphology that is compatible with antigen detection using a fixative mixture of formalin and acetic acid.

STUDY QUESTIONCan ovarian tissue morphology be better preserved whilst enabling histological molecular analyses following fixation with a novel fixative, neutral buffered formalin (NBF) with 5% acetic acid (referred to hereafter as Form-Acetic)?SUMMARY ANSWERFixation with Form-Acetic improved ovarian tissue histology compared to NBF in multiple species while still enabling histological molecular analyses.WHAT IS KNOWN ALREADYNBF fixation results in tissue shrinkage in various tissue types including the ovary. Components of ovarian tissue, notably follicles, are particularly susceptible to NBF-induced morphological alterations and can lead to data misrepresentation. Bouin’s solution (which contains 5% acetic acid) better preserves tissue architecture compared to NBF but is limited for immunohistochemical analyses.STUDY DESIGN, SIZE, DURATIONA comparison of routinely used fixatives, NBF and Bouin’s, and a new fixative, Form-Acetic was carried out. Ovarian tissue was used from three different species: human (n = 5 patients), sheep (n = 3; 6 ovaries; 3 animals per condition) and mouse (n = 14 mice; 3 ovaries from 3 different animals per condition).PARTICIPANTS/MATERIALS, SETTING, METHODSOvarian tissue from humans (aged 13 weeks to 32 years), sheep (reproductively young i.e. 3–6 months) and mice (10 weeks old) were obtained and fixed in 2 ml NBF, Bouin’s or Form-Acetic for 4, 8, and 24 h at room temperature. Tissues were embedded and sectioned. Five-micron sections were stained with haemotoxylin and eosin (H&E) and the percentage of artefact (clear space as a result of shrinkage) between ovarian structures was calculated. Additional histological staining using Periodic acid-Schiff and Masson’s trichrome were performed on 8 and 24 h NBF, Bouin’s and Form-Acetic fixed samples to assess the compatibility of the new fixative with stains. On ovarian tissue fixed for both 8 and 24 h in NBF and Form-Acetic, immunohistochemistry (IHC) studies to detect FOXO3a, FoxL2, collagen IV, laminin and anti-Müllerian hormone (AMH) proteins were performed in addition to the terminal deoxynucleotidyl transferase nick end labelling (TUNEL) assay to determine the compatibility of Form-Acetic fixation with types of histological molecular analyses.MAIN RESULTS AND THE ROLE OF CHANCEFixation in Form-Acetic improved ovarian tissue morphology compared to NBF from all three species and either slightly improved or was comparable to Bouin’s for human, mouse and sheep tissues. Form-Acetic was compatible with H&E, Periodic acid-Schiff and Masson’s trichrome staining and all proteins (FOXO3a, FoxL2, collagen IV and laminin and AMH) could be detected via IHC. Furthermore, Form-Acetic, unlike NBF, enabled antigen recognition for most of the proteins tested without the need for antigen retrieval. Form-Acetic also enabled the detection of damaged DNA via the TUNEL assay using fluorescence.LARGE SCALE DATAN/ALIMITATIONS, REASONS FOR CAUTIONIn this study, IHC analysis was performed on a select number of protein types in ovarian tissue thus encouraging further studies to confirm the use of Form-Acetic in enabling the detection of a wider range of protein forms in addition to other tissue types.WIDER IMPLICATIONS OF THE FINDINGSThe simplicity in preparation of Form-Acetic and its superior preservative properties whilst enabling forms of histological molecular analyses make it a highly valuable tool for studying ovarian tissue. We, therefore, recommend that Form-Acetic replaces currently used fixatives and encourage others to introduce it into their research workflow.STUDY FUNDING/COMPETING INTEREST(S)This work was supported by the Oxford Medical Research Council Doctoral Training Programme (Oxford MRC-DTP) grant awarded to B.D.B. (Grant no. MR/N013468/1), the Fondation Hoffmann supporting R.A. and the Petroleum Technology Development Fund (PTDF) awarded to B.V.A.

Discovery, Bioactivity Evaluation, Biosynthetic Gene Cluster Identification, and Heterologous Expression of Novel Albofungin Derivatives

The crude extract of Streptomyces chrestomyceticus exhibited strong and broad activities against most “ESKAPE pathogens.” We conducted a comprehensive chemical investigation for secondary metabolites from the S. chrestomyceticus strain and identified two novel albofungin (alb) derivatives, i.e., albofungins A (1) and B (2), along with two known compounds, i.e., albofungin (3) and chloroalbofungin (4). The chemical structures of the novel compounds were elucidated using HRMS, 1D and 2D NMR, and electronic circular dichroism spectroscopy. The draft genome of S. chrestomyceticus was sequenced, and a 72 kb albofungin (alb) gene cluster with 72 open reading frames encoding type II polyketide synthases (PKSs), regulators, and transporters, and tailoring enzymes were identified using bioinformatics analysis. The alb gene cluster was confirmed using the heterologous expression in Streptomyces coelicolor, which successfully produced the compounds 3 and 4. Furthermore, compounds 1–4 displayed remarkable activities against Gram-positive bacteria and antitumor activities toward various cancer cells. Notably, compounds 1 and 3 showed potent activities against Gram-negative pathogenic bacteria. The terminal deoxynucleotidyl transferase (dUTP) nick-end labeling and flow cytometry analysis verified that compound 1 inhibited cancer cell proliferation by inducing cellular apoptosis. These results indicated that albofungins might be potential candidates for the development of antibiotics and antitumor drugs.

Using TUNEL Assay to Quantitate p53-Induced Apoptosis in Mouse Tissues.

Critical to tumor surveillance in eukaryotic cells is the ability to perceive and respond to DNA damage. p53, fulfills its role as "guardian of the genome" by either arresting cells in the cell cycle in order to allow time for repair of DNA damage or regulating a process of programmed cell death known as apoptosis. This process will eliminate cells that have suffered severe damage from intrinsic or extrinsic factors such as X-ray irradiation or chemotherapeutic drug treatments that include doxorubicin, etoposide, cisplatin, and methotrexate. Assays designed to specifically detect cells undergoing programmed cell death are essential in defining the tissue specific responses to tumor therapy treatment, tissue damage, or degenerative processes. This chapter will delineate the TUNEL (terminal deoxynucleotidyl transferase nick-end labeling) assay that is used for the rapid detection of 3' OH ends of DNA that are generated during apoptosis.

Curcumin attenuates angiotensin II-induced podocyte injury and apoptosis by inhibiting endoplasmic reticulum stress.

Podocytes are an important component of the glomerular filtration barrier in the kidneys. The dysfunction and apoptosis of podocytes are important factors that can lead to the progression of chronic kidney disease (CKD). In CKD, angiotensin II is continuously elevated in circulation and is considered to have key roles in inducing podocyte injury and apoptosis. Curcumin is a hydrophobic polyphenolic compound extracted from turmeric. Increasing evidence demonstrates that curcumin has a protective effect on the kidneys in CKD. However, the mechanisms mediating this protective effect remain unclear. The aim of this study was to explore whether curcumin could protect against angiotensin II-induced injury and apoptosis of podocytes. We performed western blotting, immunofluorescence, phalloidin staining, and terminal deoxynucleotidyl transferase nick-end labeling staining to observe the expression level of podocyte-specific proteins, apoptosis-related proteins, and the arrangement of F-actin. We found that curcumin could reverse angiotensin II-induced podocyte injury and apoptosis in a dose-dependent manner. In addition, curcumin dose-dependently attenuated a pro-apoptotic pathway, activated by angiotensin II-induced endoplasmic reticulum stress. Conversely, the protective effects of curcumin were impaired upon addition of tunicamycin, an activator of endoplasmic reticulum stress. Thus, we speculate that curcumin protects against angiotensin II-induced podocyte injury and apoptosis, at least partly by inhibiting endoplasmic reticulum stress.

Slc7a14 Is Indispensable in Zebrafish Retinas.

Previous study has identified SLC7A14 as a new causative gene of retinitis pigmentosa (RP). However, the role of SLC7A14 has not been fully characterized. The goal of this study was to investigate the biological features of slc7a14 in zebrafish. To determine the expression of slc7a14 in developing zebrafish, we performed in situ hybridization (ISH) and quantitative real-time PCR. Morpholino knockdown and overexpression experiments were performed to study the role of slc7a14 in zebrafish retinas. Immunostaining was carried out to observe structural changes. Visual motor responses (VMR) and optokinetic responses (OKR) were analyzed to assess visual behaviors. Terminal deoxynucleotidyl transferase (dUTP) nick-end labeling (TUNEL) staining was performed to survey apoptotic retinal cells. We found that slc7a14 was highly expressed in neuronal tissues, including the brain, spinal cord and retina, and that the expression levels increased during early embryogenesis. Consistently, ISH showed a similar expression pattern. Knockdown of slc7a14 led to dose-dependent microphthalmia that was reversed by overexpression. The immunostaining results revealed that the rod-specific protein zpr-3 and the retinal pigment epithelium-specific protein zpr-2 (decreased to 44.48%) were significantly suppressed in the slc7a14-silenced morphants. Notably, visual behaviors (the VMR and the OKR) were severely impaired in the slc7a14-deficient morphant, especially the VMR OFF response. In addition, apoptotic cells were observed in the retina at 3 days post fertilization (dpf) and 5 dpf by TUNEL assay. Our results demonstrated that slc7a14 is essential for visually mediated behaviors in zebrafish. Temporary silencing of slc7a14 in larvae led to severe visual impairments, consistent with the manifestations observed in RP patients. Our findings provide further insights into the genetic mechanisms of RP predisposition caused by SLC7A14 mutations.

P38-Regulated/activated protein kinase plays a pivotal role in protecting heart against ischemia-reperfusion injury and preserving cardiac performance.

p38-Regulated/activated protein kinase (PRAK) plays a critical role in modulating cellular survival and biological function. However, the function of PRAK in the regulation of myocardial ischemic injury remains unknown. This study is aimed at determining the function of PRAK in modulating myocardial ischemia-reperfusion injury and myocardial remodeling following myocardial infarction. Hearts were isolated from adult male homozygous PRAK-/- and wild-type mice and subjected to global ischemia-reperfusion injury in Langendorff isolated heart perfusion. PRAK-/- mice mitigated postischemic ventricular functional recovery and decreased coronary effluent. Moreover, the infarct size in the perfused heart was significantly increased by deletion of PRAK. Western blot showed that deletion of PRAK decreased the phosphorylation of ERK1/2. Furthermore, the effect of deletion of PRAK on myocardial function and remodeling was also examined on infarcted mice in which the left anterior descending artery was ligated. Echocardiography indicated that PRAK-/- mice had accelerated left ventricular systolic dysfunction, which was associated with increased hypertrophy in the infarcted area. Deletion of PRAK augmented interstitial fibrosis and terminal deoxynucleotidyl transferase nick-end labeling (TUNEL)-positive myocytes. Furthermore, immunostaining analysis shows that CD31-postive vascular density and α-smooth muscle actin capillary staining decreased significantly in PRAK-/- mice. These results indicate that deletion of PRAK enhances susceptibility to myocardial ischemia-reperfusion injury, attenuates cardiac performance and angiogenesis, and increases interstitial fibrosis and apoptosis in the infarcted hearts.

In vitro evaluation of the neuroprotective effect of oligo-porphyran from Porphyra yezoensis in PC12 cells

Porphyran, derived from the marine red algae Porphyra and Pyropia, is a type of polysaccharide. Oligo-porphyran (OP) was prepared from porphyran (isolated from Pyropia yezoensis) through the acidolysis reaction containing a linear backbone of alternating 3-linked β-d-galactose and 4-linked α-l-galactose-6-sulfate. Our previous studies have shown that oligo-porphyran could attenuate MPTP-induced behavioral deficits in mice by inhibiting the tyrosine hydroxylase (TH) loss. In the present study, we further investigated how OP protects the dopamine neurons from loss in PC12 cells. OP significantly attenuated LDH release and the production of reactive oxygen species (ROS) lesioned by 6-OHDA. Moreover, OP alleviated the mitochondrial membrane potential (MMP) loss, the ratio of Bax/Bcl-2, and upregulated the PI3K/ Akt/PKC pathway, the level of tyrosine hydroxylase (TH), and dopamine transporter (DAT). Furthermore, OP decreased the production of proinflammatory cytokines. Terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) staining further indicated that OP treatment inhibited apoptosis. These results might contribute to the overall understanding of the potential health benefits of OP for food and drug applications.

Prostate-Confined Radiation Decreased Pelvic Ganglia Neuronal Survival and Outgrowth

BackgroundErectile dysfunction (ED) is common following radiation therapy (RT) for prostate cancer. Although the cause of RT-induced ED is unknown, damage to both the neuronal and vascular components supporting erections are often implicated. AimTo determine the effects of prostatic RT on erections, penile vascular physiology, and major pelvic ganglia (MPG) neuron growth and survival in a rat model. MethodsMale rats underwent 0 Gy or 22 Gy single fraction of prostate-confined, conformal RT. At 2 weeks or 10 weeks post-RT (n = 10/group), cavernous nerve stimulation was performed and erections were assessed. Tissue bath experiments were performed to assess both penile artery and internal pudendal artery (IPA) function. MPGs were dissociated and neurons grown in culture for 72 hours. Immunofluorescence staining was done to quantify neuron survival (terminal deoxynucleotidyl transferase nick-end labeling), outgrowth (beta-tubulin III), type (nitric oxide synthase [nNOS] and tyrosine hydroxylase [TH]), and nerve injury markers (small GTPase Rac1 and ninjurin-1 [Ninj-1]). Whole MPG real-time quantitative polymerase chain reaction (qPCR) was performed to measure expression of genes related to nerve type, neuron injury, repair, and myelination, such as Ninj-1, Rac1, ATF3, GAP43, GFAP, SOX10, and KROX20. OutcomesIntracavernosal pressure (ICP) to mean arterial pressure (MAP) ratio, smooth muscle contractility and relaxation, gene expression, neuritogenesis, and apoptosis. ResultsFollowing RT, ICP/MAP was unchanged at 2 weeks or 10 weeks. Nerve-mediated penile contraction was increased at 2 weeks, whereas adrenergic contraction was reduced at 10 weeks. Penile relaxation and IPA vasoreactivity were unchanged. Neuronal apoptosis was more than doubled both early and late post-RT. RT caused a progressive decrease in neurite branching but an early increase and then late decrease in neurite lengthening. RT reduced the numbers of nNOS-positive neurons both early and late and also decreased MPG nitrergic gene expression. TH neurons and gene expression were unchanged at 2 weeks; however, both were decreased after 10 weeks. Although most markers of gene injury and repair were unaffected early post-RT, MPG expression of Ninj1 and GFAP increased. After 10 weeks, Ninj1 and GFAP remained elevated while markers of neuron injury (ATF3), outgrowth (GAP43 and Rac1), and myelin regulation (SOX10) were decreased. Clinical TranslationRT-induced ED may result from damage to the ganglia controlling erections. Strengths & LimitationsThis study used a clinically relevant, prostate-confined model to examine neurovascular structures not accessible in human studies. Unfortunately, rats did not exhibit ED at this time point. ConclusionThis is the first study to demonstrate impaired health and regeneration potential of dissociated MPG neurons following RT. Neuronal injury was apparent early post-RT and persisted or increased over time but was insufficient to cause ED at the time points examined.Powers SA, Odom MR, Pak ES, et al. Prostate-Confined Radiation Decreased Pelvic Ganglia Neuronal Survival and Outgrowth. J Sex Med 2019;16:27–41.

Delineating the Cellular Mechanisms Associated with Skin Electroporation.

The immune responses elicited following delivery of DNA vaccines to the skin has previously been shown to be significantly enhanced by the addition of electroporation (EP) to the treatment protocol. Principally, EP increases the transfection of plasmid DNA (pDNA) into the resident skin cells. In addition to increasing the levels of in vivo transfection, the physical insult induced by EP is associated with activation of innate pathways which are believed to mediate an adjuvant effect, further enhancing DNA vaccine responses. We investigated the possible mechanisms associated with this adjuvant effect, primarily focusing on the cell death pathways associated with the skin EP procedure independent of pDNA delivery. Using the minimally invasive CELLECTRA®-3P intradermal electroporation device that penetrates the epidermal and dermal layers of the skin, we have investigated apoptotic and necrotic cell death in relation to the vicinity of the electrode needles and electric field generated. Employing the well-established terminal deoxynucleotidyl transferase nick-end labeling assay, we detected apoptosis beginning as early as one hour after EP and peaking at the 4 h time point. The majority of the apoptotic events were detected in the epidermal region directly adjacent to the electrode needle. Using a novel propidium iodide in vivo necrotic cell death assay, we detected necrotic events concentrated in the epidermal region adjacent to the electrode. Furthermore, we detected upregulation of calreticulin expression on skin cells after EP, thus labeling these cells for uptake by dendritic cells and macrophages. These results allow us to delineate the cell death mechanisms occurring in the skin following intradermal EP independently of pDNA delivery. We believe these events contribute to the adjuvant effect observed following electroporation at the skin treatment site.