In vitro maturation using an agarose matrix with incorporated extracellular matrix proteins improves porcine oocyte developmental competence by enhancing cytoplasmic maturation.

Here, we present a novel in vitro maturation (IVM) system comprising an agarose matrix supplemented with extracellular matrix (ECM) proteins for enhanced maturation of immature oocytes within cumulus-oocyte complexes (COCs) derived from porcine medium antral follicles (MAFs). Immunocytochemical analyses of integrin subunit α2 , α5 , α6 , ß1 , and ß4 expression suggested that integrin α2 ß1 , α5 ß1 , α6 ß1 , and α6 ß4 play pivotal roles in IVM of porcine immature oocytes. Combinatorial supplementation of fibronectin interacting with integrin α5 ß1 , collagen interacting with integrin α2 ß1 , and laminin interacting with integrin α6 ß1 and α6 ß4 to the agarose matrix had no significant effect on nuclear maturation. However, the number of parthenogenetic embryos that developed into blastocysts increased when oocytes were matured using agarose IVM matrices supplemented with fibronectin, collagen, or laminin. Furthermore, significant increases in cytoplasmic maturation-related parameters (BMP15 level, cumulus cell expansion score, intra-oocyte ATP level, and index of cortical granule distribution) were observed in COCs matured in vitro using ECM protein-incorporated agarose matrices. Our data suggest that mature porcine oocytes with enhanced developmental competence and high-quality cytoplasm can be generated via IVM using agarose matrices supplemented with fibronectin, collagen, or laminin.

Persister Escherichia coli Cells Have a Lower Intracellular pH than Susceptible Cells but Maintain Their pH in Response to Antibiotic Treatment.

Persister and viable but non-culturable (VBNC) cells are two clonal subpopulations that can survive multidrug exposure via a plethora of putative molecular mechanisms. Here, we combine microfluidics, time-lapse microscopy, and a plasmid-encoded fluorescent pH reporter to measure the dynamics of the intracellular pH of individual persister, VBNC, and susceptible Escherichia coli cells in response to ampicillin treatment. We found that even before antibiotic exposure, persisters have a lower intracellular pH than those of VBNC and susceptible cells. We then investigated the molecular mechanisms underlying the observed differential pH regulation in persister E. coli cells and found that this is linked to the activity of the enzyme tryptophanase, which is encoded by tnaA. In fact, in a ΔtnaA strain, we found no difference in intracellular pH between persister, VBNC, and susceptible E. coli cells. Whole-genome transcriptomic analysis revealed that, besides downregulating tryptophan metabolism, the ΔtnaA strain downregulated key pH homeostasis pathways, including the response to pH, oxidation reduction, and several carboxylic acid catabolism processes, compared to levels of expression in the parental strain. Our study sheds light on pH homeostasis, proving that the regulation of intracellular pH is not homogeneous within a clonal population, with a subset of cells displaying a differential pH regulation to perform dedicated functions, including survival after antibiotic treatment. IMPORTANCE Persister and VBNC cells can phenotypically survive environmental stressors, such as antibiotic treatment, limitation of nutrients, and acid stress, and have been linked to chronic infections and antimicrobial resistance. It has recently been suggested that pH regulation might play a role in an organism's phenotypic survival to antibiotics; however, this hypothesis remains to be tested. Here, we demonstrate that even before antibiotic treatment, cells that will become persisters have a more acidic intracellular pH than clonal cells that will be either susceptible or VBNC upon antibiotic treatment. Moreover, after antibiotic treatment, persisters become more alkaline than VBNC and susceptible E. coli cells. This newly found phenotypic feature is remarkable because it distinguishes persister and VBNC cells that have often been thought to display the same dormant phenotype. We then show that this differential pH regulation is abolished in the absence of the enzyme tryptophanase via a major remodeling of bacterial metabolism and pH homeostasis. These new whole-genome transcriptome data should be taken into account when modeling bacterial metabolism at the crucial transition from exponential to stationary phase. Overall, our findings indicate that the manipulation of the intracellular pH represents a bacterial strategy for surviving antibiotic treatment. In turn, this suggests a strategy for developing persister-targeting antibiotics by interfering with cellular components, such as tryptophanase, that play a major role in pH homeostasis.

Starvation induces shrinkage of the bacterial cytoplasm.

Environmental fluctuations are a common challenge for single-celled organisms; enteric bacteria such as Escherichia coli experience dramatic changes in nutrient availability, pH, and temperature during their journey into and out of the host. While the effects of altered nutrient availability on gene expression and protein synthesis are well known, their impacts on cytoplasmic dynamics and cell morphology have been largely overlooked. Here, we discover that depletion of utilizable nutrients results in shrinkage of E. coli's inner membrane from the cell wall. Shrinkage was accompanied by an ∼17% reduction in cytoplasmic volume and a concurrent increase in periplasmic volume. Inner membrane retraction after sudden starvation occurred almost exclusively at the new cell pole. This phenomenon was distinct from turgor-mediated plasmolysis and independent of new transcription, translation, or canonical starvation-sensing pathways. Cytoplasmic dry-mass density increased during shrinkage, suggesting that it is driven primarily by loss of water. Shrinkage was reversible: upon a shift to nutrient-rich medium, expansion started almost immediately at a rate dependent on carbon source quality. A robust entry into and recovery from shrinkage required the Tol-Pal system, highlighting the importance of envelope coupling during shrinkage and recovery. Klebsiella pneumoniae also exhibited shrinkage when shifted to carbon-free conditions, suggesting a conserved phenomenon. These findings demonstrate that even when Gram-negative bacterial growth is arrested, cell morphology and physiology are still dynamic.

Myocardial protection properties of parishins from the roots of Gastrodia elata Bl.

Parishins, important constituents of Gastrodia elata (G. elata), are known to exhibit a number of biological and pharmacological properties. However, their role and mechanisms of action in myocardial ischemia are unknown. The present study investigated the potential protective effects and mechanisms of parishins extracted from G. elata on hypoxia/reoxygenation (H/R) injury in H9c2 cardiomyocytes. The results demonstrated that parishins had significant protective effects on myocardial cells with parishins J and B providing greater cardioprotection through down-regulation of the level of cleaved-caspase-3 and cytochrome c in the cytoplasm and Bax, and up-regulation of cytochrome c in the mitochondria and Bcl-2 than induced by the positive control gastrodin. Additional study of the mechanisms of action indicated that the myocardial protection provided by parishin J was due to inhibition of JNK1 phosphorylation levels, down-regulation of c-jun and ATF-2 phosphorylation levels, a decrease in the phosphorylation of 14-3-3 and an increase in its binding to Bax. Therefore, parishin J was revealed to be a promising candidate as a novel treatment for myocardial protection.

Amentoflavone, a novel cyanobacterial killing agent from Selaginella tamariscina.

Harmful cyanobacterial bloom (HCB) by Microcystis aeruginosa is increasingly becoming a serious concern to the environment and human health alike. Currently, many physical, chemical, and biological controls are underway to eliminate HCB, but natural chemicals are rarely used. To find a control agent with low environmental toxicity and high potential for practical use, 60 plant extracts were screened. Only Selaginella tamariscina extract killed all four Microcystis aeruginosa strains, but not the other tested bacteria. Chloroform fraction of S. tamariscina extract (CSE) showed the highest killing activity. The effects of CSE on M. aeruginosa were monitored using differential interference contrast microscopy and flow-cytometry analysis, scanning electron microscopy, and transmission electron microscopy. The images showed that CSE-treated cells were abnormally altered, with damaged cell membranes, peptidoglycan layers, and cytoplasm. Quadrupole time-of-flight liquid chromatography-mass spectrometry was used to identify amentoflavone as a major active compound. Pure amentoflavone, even at low concentrations showed a powerful killing effect on M. aeruginosa, but not on other non-cyanobacteria. Overall, in this study, we have highlighted the potentials of S. tamariscina extracts and amentoflavone as selective HCB control agents.

Vitamin E but Not GSH Decreases Reactive Oxygen Species Accumulation and Enhances Sperm Production during In Vitro Maturation of Frozen-Thawed Prepubertal Mouse Testicular Tissue.

Freezing-thawing procedures and in vitro culture conditions are considered as a source of stress associated with increased reactive oxygen species (ROS) generation, leading to a damaged cell aerobic metabolism and consequently to oxidative stress. In the present study, we sought to investigate whether vitamin E (Vit E) or reduced glutathione (GSH) enhances sperm production by decreasing ROS accumulation during in vitro maturation of prepubertal mice testes. Testes of prepubertal mice were cryopreserved using a freezing medium supplemented or not supplemented with Vit E and were cultured after thawing. In presence of Rol alone in culture medium, frozen-thawed (F-T) testicular tissues exhibited a higher ROS accumulation than fresh tissue during in vitro culture. However, Vit E supplementation in freezing, thawing, and culture media significantly decreased cytoplasmic ROS accumulation in F-T testicular tissue during in vitro maturation when compared with F-T testicular tissue cultured in the presence of Rol alone, whereas GSH supplementation in culture medium significantly increased ROS accumulation associated with cytolysis and tissue disintegration. Vit E but not GSH promoted a better in vitro sperm production and was a suitable ROS scavenger and effective molecule to improve the yield of in vitro spermatogenesis from F-T prepubertal mice testes. The prevention of oxidative stress in the cytoplasmic compartment should be regarded as a potential strategy for improving testicular tissue viability and functionality during the freeze-thaw procedure and in vitro maturation.

Ginsenoside Rg3 regulates DNA damage in non-small cell lung cancer cells by activating VRK1/P53BP1 pathway.

Lung cancer is the leading cause of cancer-related deaths. Ginsenoside Rg3 is the main ingredient of Ginseng which is used to treat non-small cell lung cancer (NSCLC). It has been found to enhance the efficiency of chemotherapy thereby reducing its side effects. Previous studies found that ginsenoside Rg3 can reduce the occurrence of NSCLC by inducing DNA damage. Yet, its anti-DNA damaging effects and mechanisms in tumor cells are still not fully understood. This study explored the effect of ginsenoside Rg3 on DNA repair and VRK1/P53BP1 signaling pathway. Ginsenoside Rg3 treatment significantly decreased the incidence and invasionin a mouse model of lung cancer induced by urethane. The results of cell survival assay and single cell gel electrophoresis showed that ginsenoside Rg3 protected lung adenocarcinoma cells from DNA damage as well as inhibited the proliferation of tumor cells. Ginsenoside Rg3 increased the mRNA and protein expression of VRK1 in NSCLC cells as measured by RT-qPCR and western blot, respectively. These findings suggests that ginsenoside Rg3 regulates VRK1 signaling. Immunofluorescence assays showed that P53BP1 and VRK1 protein level increased, and the VRK1 protein translocated between the nuclei and cytoplasm. Finally, this conclusion was confirmed by the reverse validation in VRK1-knockdown cells. Taken together, these results show that ginsenoside Rg3 upregulate VRK1 expression and P53BP1 foci formation in response to DNA damage thereby inhibiting the tumorigenesis and viability of cancer cells. These findings reveal the role of Rg3 in lung cancer and provides therapeutic targets for developing new drugs in the prevention and treatment of lung cancer.

An in situ high-throughput screen identifies inhibitors of intracellular Burkholderia pseudomallei with therapeutic efficacy.

Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm) are Tier-1 Select Agents that cause melioidosis and glanders, respectively. These are highly lethal human infections with limited therapeutic options. Intercellular spread is a hallmark of Burkholderia pathogenesis, and its prominent ties to virulence make it an attractive therapeutic target. We developed a high-throughput cell-based phenotypic assay and screened ∼220,000 small molecules for their ability to disrupt intercellular spread by Burkholderia thailandensis, a closely related BSL-2 surrogate. We identified 268 hits, and cross-species validation found 32 hits that also disrupt intercellular spread by Bp and/or Bm Among these were a fluoroquinolone analog, which we named burkfloxacin (BFX), which potently inhibits growth of intracellular Burkholderia, and flucytosine (5-FC), an FDA-approved antifungal drug. We found that 5-FC blocks the intracellular life cycle at the point of type VI secretion system 5 (T6SS-5)-mediated cell-cell spread. Bacterial conversion of 5-FC to 5-fluorouracil and subsequently to fluorouridine monophosphate is required for potent and selective activity against intracellular Burkholderia In a murine model of fulminant respiratory melioidosis, treatment with BFX or 5-FC was significantly more effective than ceftazidime, the current antibiotic of choice, for improving survival and decreasing bacterial counts in major organs. Our results demonstrate the utility of cell-based phenotypic screening for Select Agent drug discovery and warrant the advancement of BFX and 5-FC as candidate therapeutics for melioidosis in humans.

In vitro antibacterial activity of plumbagin isolated from Plumbago zeylanica L. against methicillin-resistant Staphylococcus aureus.

Plumbagin (5-hydroxy-2-methyl-1,4-napthoquinone) is a bicyclic naphthoquinone, found in three major plant families viz. Plumbaginaceae, Ebenceae and Droseraceae. The phytochemical is reported to exhibit various pharmacological properties. In this study, plumbagin isolated from Plumbago zeylanica L. was investigated for its in vitro activity against methicillin-resistant Staphylococcus aureus (MRSA). Against 100 MRSA isolates that included multi-drug-resistant phenotypes, plumbagin showed consistent activity with a narrow minimum inhibitory concentration (MIC) range of 4-8 µg ml-1 . The time-kill study revealed 99% kill of a reference MRSA strain, 8 h after exposure to plumbagin. In the combination MIC study using the reference MRSA strain, plumbagin showed synergistic effect with ciprofloxacin and piperacillin while additive or indifference effect with other commonly used antibiotics. The transmission electron micrograph of the reference MRSA strain treated with plumbagin confirmed cell wall and cytoplasmic changes. Our results demonstrated potent anti-MRSA activity of plumbagin which was not impacted by multi-drug resistance. This is a first ever study that evaluated in vitro anti-MRSA activity of plumbagin employing large number of MRSA isolates. The findings of this study support the need for the further investigation on this phytochemical agent for therapeutic application. SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed phytochemical plumbagin's potent and consistent in vitro antibacterial activity against clinically problematic methicillin-resistant Staphylococcus aureus (MRSA) including multi-drug-resistant (MDR) phenotypes. The study results support further research to assess the clinical scope of plumbagin.

Fluorescent nanoparticles in the popular pizza: properties, biodistribution and cytotoxicity.

Food-borne nanoparticles that are generated during the thermal processing of various consumed foods are of great concern due to their unique properties. In this study, the presence of fluorescent nanoparticles (FNPs) in pizza, their biodistribution and cytotoxicity were investigated. The spherical FNPs have a diameter of about 3.33 nm. X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy analysis revealed that they contained 68.21% C, 27.44% O, 2.75% N and 1.60% S, and the functional groups on their surface included -OH, -COOH, C[double bond, length as m-dash]C, -NH2 and C[double bond, length as m-dash]O. In vitro and in vivo biodistribution of pizza FNPs was evaluated using normal rat kidney (NRK) cells, onion epidermal cells, Caenorhabditis elegans and mice. The fluorescence microscopy images clearly indicate that the pizza FNPs appear to be localized within the cytoplasm. However, the FNPs remained restricted to the extracellular space of the onion epithelium and did not enter the onion cell cytoplasm because of the cell wall. The FNPs were swallowed by the Caenorhabditis elegans worms when exposed to food OP50 and distributed within the pharynx, intestine and anus. Obvious fluorescence of the FNPs in the stomach, intestine, liver, lung and kidney was observed for the FNPs in mouse organs, but not the brain, heart, and spleen. Furthermore, the produced FNPs were found to cause cell cycle arrest at the G0/G1 phase in NRK cells, and resulted in cell apoptosis at high doses. The outcome of this research offers an important insight into the nature of thermal processing-induced nanoparticles and their in vivo and in vitro biological effects.

Saponin-rich fraction from Agave sisalana: effect against malignant astrocytic cells and its chemical characterisation by ESI-MS/MS.

Astrocytic tumour cells derived from human (GL-15) and rat (C6) gliomas, as well as non-tumoural astrocytic cells, were exposed to the saponin-rich fraction (SF) from Agave sisalana waste and the cytotoxic effects were evaluated. Cytotoxicity assays revealed a reduction of cell viability that was more intensive in glioma than in non-tumoural cells. The SF induced morphological changes in C6 cells. They were characterised by cytoplasmic vacuole formation associated with increase in the formation of acidic lysosomes. The SF was subjected to purification on Sephadex LH-20, which characterised three probable steroidal saponins (sisalins) by electrospray ionisation mass spectrometry multistage (ESI-MSn). Sisalins from sisal may be responsible for the cytotoxicity, which involves cytoplasmatic vacuole formation and selective action for glioma cells.

Differential ratios of fish/corn oil ameliorated the colon carcinoma in rat by altering intestinal intraepithelial CD8+ T lymphocytes, dendritic cells population and modulating the intracellular cytokines.

Intraepithelial lymphocytes (IELs) impart a crucial role in maintaining intestinal homeostasis, yet their role in colon cancer pathogenesis remains unknown. Here, we posited that the modulation of intestinal immune response via dietary interventions might be an implacable strategy in restraining colon carcinoma. In the above context, we studied the effect of differential ratios of fish oil (FO) and corn oil (CO) on the gut immune response in experimentally induced colon cancer. Male Wistar rats were divided into six groups: Group I obtained purified diet while Groups II and III were fed on the diet supplemented with differential ratios of FO and CO i.e. 1:1 and 2.5:1, respectively. The groups were further subdivided into control and carcinogenic group, treated with ethylenediaminetetraacetic acid (EDTA) or N,N'-dimethylhydrazine dihydrochloride (DMH), respectively. Initiation phase comprised the animals sacrificed 48 h after the last injection whereas, the post -initiation phase was constituted by animals sacrificed 12 weeks after the treatment regimen. CD8+ T cells, CD8/αß TCR cells, dendritic cells increased significantly on treatment with DMH as compared to control. However, on treatment with differential ratios of FO and CO these cells decreased significantly. The intracellular cytokine i.e. interferon gamma (IFN-γ) and cytotoxic granules component i.e Perforin and Granzyme decreased significantly in the initiation phase but in the post-initiation phase IFN-γ and Perforin increased considerably on carcinogen treatment as compared to the control group. On treatment with FO and CO in the initiation phase the IFN-γ, Perforin and Granzyme expression increased significantly. However, in the post-initiation phase treatment with differential ratios of FO and CO led to a significant decrease in the IFN-γ, Perforin and increase in Granzyme was observed in these groups. Altogether, FO supplementation appeared to activate the immune response that may further attenuate the process of carcinogenesis, in a dose and time-dependent manner.

Melatonin improves the fertilization capacity and developmental ability of bovine oocytes by regulating cytoplasmic maturation events.

Melatonin is a well-characterized antioxidant that has been successfully used to protect oocytes from reactive oxygen species during in vitro maturation (IVM), resulting in improved fertilization capacity and development ability. However, the mechanism via which melatonin improves oocyte fertilization capacity and development ability remains to be determined. Here, we studied the effects of melatonin on cytoplasmic maturation of bovine oocytes. In the present study, bovine oocytes were cultured in IVM medium supplemented with 0, 10-7 , 10-9 , and 10-11  mol/L melatonin, and the cytoplasmic maturation parameters of MII oocytes after IVM were investigated, including redistribution of organelles (mitochondria, cortical granules [CGs], and endoplasmic reticulum [ER]), intracellular glutathione (GSH) and ATP levels, expression of endogenous antioxidant genes (Cat, Sod1, and GPx), and fertilization-related events (IP3R1 distribution and expression of CD9 and Juno). Our results showed that melatonin significantly improved the cytoplasmic maturation of bovine oocytes by improving the normal distribution of organelles, increasing intracellular GSH and ATP levels, enhancing antioxidant gene expression levels, and modulating fertilization-related events, all of which resulted in increased fertilization capacity and developmental ability. Meanwhile, melatonin also increased the mRNA and protein expression levels of the Tet1 gene and decreased the Dnmt1 gene mRNA and protein levels in bovine oocytes, indicating that melatonin regulates the expression of the detected genes via demethylation. These findings shed insights into the potential mechanisms by which melatonin improves oocyte quality during IVM.

Intracellular investigation on the differential effects of 4 polyphenols on MCF-7 breast cancer cells by Raman imaging.

The past decades have seen significant interest in the study of polyphenolic compounds as potential therapeutic agents in medicine because they display a vast array of cellular effects beneficial to treat or manage a plethora of chronic diseases including inflammatory diseases, cardiovascular abnormalities and several types of cancer. These compounds act at different stages of carcinogenesis but deciphering their mode of action is a complex task. Live MCF-7 breast cancer cells were investigated using Raman imaging to evaluate the perturbations induced after incubating cells with four different polyphenols: EGCG, gallic acid, resveratrol and tannic acid. First, clear spectral changes could be observed between the spectra of the cytoplasm and the nucleus of live MCF-7 cancer cells demonstrating a difference in their respective global chemical composition. The treatments induced significant modifications in the cells but no clear common pattern of modifications from the 4 drugs could be observed in the cell spectra in the 1800-600 cm-1 region. The high spatial resolution of Raman confocal microscopy enabled both the nucleus and cytoplasm to be independently targeted to study the impact of the polyphenols on the cell line. Positive spectral variations at 2851 cm-1 and 2920 cm-1 as well as in the 1460-1420 cm-1 and 1660-1650 cm-1 spectral regions inside cell cytoplasm reflected an increase of the lipid content after exposure to polyphenols. Lipid accumulation appears to be an early biomarker of drug-induced cell stress and subsequent apoptosis. Interestingly an increase of cytochrome c into the cytosol was also induced by EGCG. These multiple events are possibly associated with cell apoptosis. In conclusion, Raman micro-spectroscopy provides a complementary spectroscopic method to realize biological investigations on live cancer cells and to evaluate the effects of polyphenols at the subcellular level.

Supplementation action with ascorbic acid in the morphology of the muscular layer and reactive acetylcholinesterase neurons of ileum of mdx mice.

The Duchenne Muscular Dystrophy (DMD) is a genetic disorder characterized by the absence of dystrophin protein, causing severe myopathy from increases of oxidative stress. Injuries of intestinal muscle can compromise the myenteric plexus. This study aimed to evaluate the disorders occurred in the muscular layer and in the acetylcholinesterase myenteric neurons (ACHE-r) of ileum of mdx mice, and the effects of supplementation with ascorbic acid (AA) in both components. 30 male mice C57BL/10, and 30 male mice C57BL/10Mdx were separated according to the age and treatment (n=10/group): 30-days-old control group (C30); 30-days-old dystrophic group (D30); 60-days-old control group (C60); 60-days-old dystrophic group (D60); 60-days-old control group supplemented with AA (CS60); and 60-days-old dystrophic group supplemented with AA (DS60). The animals were euthanized and the ileum was collected and processed. Semi-serial sections were stained by Masson's trichrome, and acetylcholinesterase histochemical technique in whole-mounts preparations to identify the myenteric neurons. The muscular layer thickness and the area of smooth muscle of ileum were lower in dystrophic groups, especially in D30 group. The DS60 group showed the muscular layer thickness similar to C60. The density of ACHE-r neurons of myenteric plexus of ileum was lower in D30 animals; however, it was similar in animals of 60-days-old without treatment (C60 and D60) and, higher in DS60. The cell body profile area of ACHE-r neurons was similar in C30-D30 and C60-D60; however, it was higher in DS60. DMD caused damage to the ileum's musculature and myenteric plexus, and the AA prevented the ACHE-r neuronal loss.

Direct Involvement of Androgen Receptor in Oxytocin Gene Expression: Possible Relevance for Mood Disorders.

Oxytocin (OXT), synthesized in the hypothalamic paraventricular nucleus (PVN) and then released into different brain areas, may play a crucial role in various behaviors and neuropsychiatric disorders, including depression. Testosterone has been proposed by clinical studies to have the opposite effect of oxytocin in these disorders. We began by studying, in the postmortem hypothalamus of fifteen patients with mood disorders and fifteen matched controls, the expression of OXT in the PVN by means of immunocytochemistry (ICC) and the co-localization of OXT and androgen receptor (AR) by means of double labeling ICC. Subsequently, the regulatory effect of AR on OXT gene expression was studied in vitro. We found a higher expression of PVN OXT in the mood disorder patients than in the control subjects, and observed a clear co-localization of AR in OXT-expressing neurons, both in the cytoplasm and in the nucleus. In addition, a significant decrease in OXT-mRNA levels was observed after pre-incubation of the SK-N-SH cells with testosterone. A further potential androgen-responsive element in the human OXT gene promotor was revealed by electrophoretic mobility shift assays and co-transfections in neuroblastoma cells. Finally, in vitro studies demonstrated that AR mediated the down-regulation of OXT gene expression. These results suggest that the fact that OXT and testosterone appear to have opposite effects in neuropsychiatric disorders might be based upon a direct inhibition of AR on OXT transcription, which may provide a novel target for therapeutic strategies in depression.

VEGF alleviates ALS-CSF induced cytoplasmic accumulations of TDP-43 and FUS/TLS in NSC-34 cells.

Cytoplasmic mislocalisation and aggregation of TDP-43 and FUS/TLS proteins in spinal motor neurons contribute to the pathogenesis of the highly fatal disorder amyotrophic lateral sclerosis (ALS). We investigated the neuroprotective effect of VEGF on expression of these proteins in the motor neuronal cell line NSC-34 modelled to reminisce sporadic form of ALS. We studied the expression of TDP-43 and FUS/TLS proteins after exposure to ALS-CSF and following VEGF supplementation by quantitative confocal microscopy and electron microscopy. ALS-CSF caused cytoplasmic overexpression of both the proteins and stress-granule formation in the cells. These alterations were alleviated by VEGF supplementation. The related ultrastructural changes like nuclear membrane dysmorphism and p-bodies associated changes were also reversed. However the protein expression did not completely translocate to the nucleus, as some cells continued to show to cytoplasmic mislocalisation. Thus, the present findings indicate that VEGF alleviates TDP43 and FUS pathology by complimenting its role in controlling apoptosis and reversing choline acetyl transferase expression. Hence, VEGF appears to target multiple pathogenic processes in the neurodegenerative cascade of ALS.

Evaluation of Parameters Impacting Drug Susceptibility in Intracellular Trypanosoma cruzi Assay Protocols.

In order to understand the key parameters influencing drug susceptibility, different Trypanosoma cruzi assay protocols were evaluated using a comparative assay design. The assays compared in this study were an image-based intracellular T. cruzi assay quantified through an image-mining algorithm and an intracellular assay utilizing a ß-galactosidase-expressing T. cruzi strain. Thirty-one reference compounds known to exhibit activities against intracellular T. cruzi were used as benchmarks. Initial comparison using EC50 values from two assays showed a very poor correlation, with an R2 value of 0.005. Nitroheterocyclics and CYP51 inhibitors were inactive in an image-based assay, but were highly active in a colorimetric assay. In order to identify the differentiating factor, we synchronized the compound-parasite incubation times or the sequential cell and compound seeding schemes between assays, but the correlation remained low. A high correlation ( R2 = 0.86) was observed only after both compound incubation time and cell seeding were synchronized between assays. Further analysis of EC50 and maximum inhibition values showed that nitroheterocyclics and CYP51 inhibitors exhibit relatively large deviations in activity between experimental protocols routinely used for in vitro intracellular T. cruzi assays. These findings suggest that the factors mentioned are critical when designing an intracellular T. cruzi assay.

Calycosin suppresses expression of pro-inflammatory cytokines via the activation of p62/Nrf2-linked heme oxygenase 1 in rheumatoid arthritis synovial fibroblasts.

The activation of synovial fibroblasts (SFs) and the subsequent production and expression of pro-inflammatory cytokines play a crucial role in the pathogenesis and progression of rheumatoid arthritis (RA). In the current study, rheumatoid arthritis synovial fibroblasts (RASFs) isolated from the joint of the patients were used to evaluate the suppressive effects of calycosin (CAL), a compound derived from the Chinese medicinal herb Radix Astragali, on the expression of pro-inflammatory cytokines in RASFs. The results demonstrated that increased mRNA expression levels of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-25 (IL-25), interleukin-33(IL-33) were significantly inhibited by CAL. Furthermore, the compound obviously suppressed IL-6 and IL-33 secretion. The key inflammatory mediator, cyclooxygenase-2 (COX-2) was significantly attenuated by CAL. A mechanistic study showed that the antioxidant enzymes heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase quinone 1(NQO1) and Nrf2 of RASFs were markedly activated by CAL. Furthermore, CAL potentiated the accumulation of sequestosome 1 (SQSTM1, p62) and the degradation of Kelch-like ECH-associated protein 1 (Keap1), thereby inducing Nrf2 translocation from the cytoplasm to the nucleus. Thus, CAL suppresses the expression of pro-inflammatory cytokines via p62/Nrf2-linked HO-1 induction in RASFs, which suggests that the compound should be further investigated as a candidate anti-inflammatory and anti-arthritic agent.

Validation of chemical compound library screening for transcriptional co-activator with PDZ-binding motif inhibitors using GFP-fused transcriptional co-activator with PDZ-binding motif.

Transcriptional co-activator with PDZ-binding motif (TAZ) plays versatile roles in cell proliferation and differentiation. It is phosphorylated by large tumor suppressor kinases, the core kinases of the tumor-suppressive Hippo pathway. Phosphorylation induces the cytoplasmic accumulation of TAZ and its degradation. In human cancers, the deregulation of the Hippo pathway and gene amplification enhance TAZ activity. TAZ interacts with TEA domain family members (TEAD), and upregulates genes implicated in epithelial-mesenchymal transition. It also confers stemness to cancer cells. Thus, TAZ activation provides cancer cells with malignant properties and worsens the clinical prognosis. Therefore, TAZ attracts attention as a therapeutic target in cancer therapy. We applied 18 606 small chemical compounds to human osteosarcoma U2OS cells expressing GFP-fused TAZ (GFP-TAZ), monitored the subcellular localization of GFP-TAZ, and selected 33 compounds that shifted GFP-TAZ to the cytoplasm. Unexpectedly, only a limited number of compounds suppressed TAZ-mediated enhancement of TEAD-responsive reporter activity. Moreover, the compounds that weakened TEAD reporter activity did not necessarily decrease the unphosphorylated TAZ. In this study, we focused on three compounds that decreased both TEAD reporter activity and unphosphorylated TAZ, and treated several human cancer cells with these compounds. One compound did not show a remarkable effect, whereas the other two compounds compromised the cell viability in certain cancer cells. In conclusion, the GFP-TAZ-based assay can be used as the first screening for compounds that inhibit TAZ and show anticancer properties. To develop anticancer drugs, we need additional assays to select the compounds.