Molecular characterization of the unusual peptide CORO1C-47aa encoded by the circular RNA and docking simulations with its binding partner.

Circular RNAs, which have covalently closed ends, are in the class of non-coding RNAs. Recent studies reveal that they are associated with various biochemical pathways. One such involvement of circular RNAs is in the onset of different types of cancers. Though the circular RNAs are known as non-coding RNAs, some of them are found to possess the capacities to code for proteins. One such circular RNA is hsa-circ-0000437 which is known to code for a short peptide referred to as CORO1C-47aa. The peptide has anti-angiogenic activity and is associated with the prevention of endometrial cancer. The peptide binds to the PAS-B domain of the Aryl hydrocarbon Receptor Nuclear Translocator (ARNT). However, till date only the amino acid sequence of the peptide is known and no structural details of the peptide are available. Therefore, in this work, our aim was to predict how the peptide would fold and what could be its possible ligand binding sites. We used computational tools to determine the structure of the peptide refined further by molecular dynamics simulations. We then performed molecular docking simulations of the peptide with its known binding partner ARNT to gain an insight into the modes of binding as the process is associated with endometrial cancer. The possible ligand binding sites along-with the natures of the possible other different ligands of the peptide were analyzed further. From this structure function analysis study, we tried to elucidate the plausible mechanism of the involvements of the peptide in the onset of endometrial cancer. This is the first report on the structural characterization of the peptide and its modes of interactions with the partner protein ARNT. This study may therefore be useful in determining the structures of new drug candidates for the treatment of endometrial cancer.

Effects of two potential probiotic Lactobacillus bacteria on adipogenesis in vitro.

AIMS: Overweight is a major global health problem. Various methodologies to get rid of the extra fat are available, but usually, those are associated with adverse side effects. Probiotics, on the contrary, seem to have the potential to help reduce fat accumulation without much apparent adversity. In this study, we have evaluated a pair of well-documented probiotics for their anti-obesogenic effects. MAIN METHODS: We used strains of Lactobacillus acidophilus (LA) and a cocktail (LDB-ST) of Lactobacillus delbruckei sp. bulgaricus (LDB) and Streptococcus thermophilus (ST) in this study. The murine pre-adipocyte cell line 3T3-L1 was terminally differentiated to matured adipocytes to use as a model to evaluate the bacteria's anti-obesogenic effects. The optimal dose for treatment of both the probiotics was determined using a cell viability assay. We assessed the probiotic internalization potential of differentiated 3T3-L1 cells by flow cytometry, fluorescence microscopy, and cell lysis method. We determined the lipolytic and anti-adipogenic potential of probiotics by intracellular lipid staining, spectrophotometry, and gene expression analysis. KEY FINDINGS: Both probiotics were effective lipolytic agents as revealed by reducing cellular lipids and down-regulation of mammalian adipogenesis marker genes in terminally differentiated 3T3-L1 cells. SIGNIFICANCE: Previous studies from our group had proven the immune-modulatory properties of these probiotics on an immune-biased mouse model. The present study demonstrates LA and LDB-ST to be effective against adipogenesis. Further in vivo studies will be conducted to strengthen this claim.

Macrophage plays important role in cortisol and serotonin induced adipogenesis in vitro.

Psychological stress is an important cause to induce various metabolic disorders such as obesity, type II diabetes, and cardiovascular disorders by affecting the visceral adipose tissue. Pathophysiology of these diseases is often accompanied by the hyperactive immune system. The hyperactive immune system causes immune cells to infiltrate in the adipose tissue to increase the severity of metabolic disorders and to affect the levels of stress associated hormones, such as cortisol and serotonin. Cortisol and serotonin, alone or together, could regulate several aspects of the metabolic and immunological deregulations by manipulating the lipid accumulation or adipogenesis in cells. During adipogenesis, macrophages are recruited. Previous reports from the Aich laboratory established the roles of cortisol and serotonin to influence adipogenesis in pre-adipocytes 3T3-L1 in the presence and absence of macrophages. In the current study, we reported the role of macrophage RAW264.7, especially its polarized states, on differentiated murine adipocytes 3T3-L1 in the presence or absence of cortisol and serotonin. The current study also compares the differential role of macrophage recruitment on pre- and differentiated adipocytes.

Probiotics L. acidophilus and B. clausii Modulate Gut Microbiota in Th1- and Th2-Biased Mice to Ameliorate Salmonella Typhimurium-Induced Diarrhea.

Gut microbiota play important role in maintaining health. Probiotics are believed to augment it further. We aimed at comparing effects of probiotics, Lactobacillus acidophilus (LA) and Bacillus clausii (BC) (a) on the gut microbiota abundance and diversity and (b) their contributions to control intestinal dysbiosis and inflammation in Th1- and Th2-biased mice following Salmonella infection. We report how could gut microbiota and the differential immune bias (Th1 or Th2) of the host regulate host responses when challenged with Salmonella typhimurium in the presence and absence of either of the probiotics. LA was found to be effective in ameliorating the microbial dysbiosis and inflammation caused by Salmonella infection, in Th1 (C57BL/6) and Th2 (BALB/c)-biased mouse. BC was able to ameliorate Salmonella-induced dysbiosis and inflammation in Th2 but not in Th1-biased mouse. These results may support probiotics LA as a treatment option in the case of Salmonella infection.

Comparative efficacy analysis of anti-microbial peptides, LL-37 and indolicidin upon conjugation with CNT, in human monocytes.

BACKGROUND: Antimicrobial peptides (AMPs) have the potential to serve as an alternative to antibiotic. AMPs usually exert bactericidal activity via direct killing of microbial pathogens. Reports have proposed that by harnessing innate immune activation, AMPs can regulate pathogen invasion and may control infection. It has been reported that AMPs could be utilized to activate the innate mucosal immune response in order to eliminate pathogenic infections. This way of controlling pathogen infection, by activating host immunity, confers the potential to the select AMPs to alleviate the problem of antibiotic resistance. Among various AMPs tested LL-37 and indolicidin, showed promise to be potential candidates for eliciting enhanced host innate immune responses. LL-37 and indolicidin had exhibited substantial innate immune activation in both human and murine macrophages. Dosage for each of the AMPs, however, was high with adverse side effects. RESULTS: In this study, we reported that upon conjugation with carbon nanotubes (CNT), each AMP remained biologically functional at a concentration that was 1000-fold less than the dosage required for free AMP to remain active in the cells. CONCLUSIONS: Current study also revealed that while indolicidin induced signalling events mediated through the TNFRSF1A pathway in THP1 cells, followed by activation of NFκB and c-JUN pathways, treatment of cells with LL-37 induced signalling events by activating IL1R, with subsequent activation of NFκB and NFAT2. Thp1 cells, primed with CNT conjugated LL-37 or indolicidin, are protected against Salmonella typhimurium infection at 16 h post challenge.

Comparative Analysis of the Effects of Two Probiotic Bacterial Strains on Metabolism and Innate Immunity in the RAW 264.7 Murine Macrophage Cell Line.

Probiotic and potential probiotic bacterial strains are routinely prescribed and used as supplementary therapy for a variety infectious diseases, including enteric disorders among a wide range of individuals. While there are an increasing number of studies defining the possible mechanisms of probiotic activity, a great deal remains unknown regarding the diverse modes of action attributed to these therapeutic agents. More precise information is required to support the appropriate application of probiotics. To address this objective, we selected two probiotics strains, Lactobacillus acidophilus MTCC-10307 (LA) and Bacillus clausii MTCC-8326 (BC) that are frequently prescribed for the treatment of intestinal disorders and investigated their effects on the RAW 264.7 murine macrophage cell line. Our results reveal that LA and BC are potent activators of both metabolic activity and innate immune responses in these cells. We also observed that LA and BC possessed similar activity in preventing infection simulated in vitro in murine macrophages by Salmonella typhimurium serovar enterica.

Influence of ultraviolet C bystander effect on inflammatory response in A375 cell on subsequent exposure to ultraviolet C or hydrogen peroxide.

Ultraviolet C (UVC) irradiation (λ: 200-280 nm) causes release of several secretory cytokines responsible for inflammation. Our objective was to investigate whether inflammatory response was also induced in bystander cells. For this purpose, the conditioned medium containing the released factors from UVC irradiated A375 cells was used in this study to evaluate the expression of inflammatory markers, such as tumour necrosis factor alpha (TNFα), nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) and p38 mitogen-activated protein kinase (p38 MAPK) in its bystander cells. Inflammatory responses in bystander cells subjected to further irradiation by UVC or other damaging agent like H2O2 were also examined. It was observed that TNFα, NFκB and p38 MAPK were not induced in UVC-bystander cells, but their expression was suppressed in the UVC-bystander cells treated with UVC or H2O2. This lowering in inflammatory response might be due to smaller depletion in the reduced glutathione (GSH) content present in these treated bystander cells. The study indicated that UVC-induced bystander effect was an intrinsic protective response in cells, capable of suppressing inflammation induced in cells on exposure to damaging agents.

Antioxidant enzymes and the mechanism of the bystander effect induced by ultraviolet C irradiation of A375 human melanoma cells.

Irradiated cells generate dynamic responses in non-irradiated cells; this signaling phenomenon is known as the bystander effect (BE). Factors secreted by the irradiated cells communicate some of these signals. Conditioned medium from UVC-irradiated A375 human melanoma cells was used to study the BE. Exposure of cells to conditioned medium induce cell-cycle arrest at the G2/M transition. Although conditioned medium treatment, by itself, did not alter cell viability, treated cells were more resistant to the lethal action of UVC or H2O2. This protective effect of conditioned medium was lost within 8h. Apoptotic or autophagic cell death was not involved in this resistance. Exposure to conditioned medium did not influence the rate of DNA repair, as measured by NAD(+) depletion. The activities of catalase and superoxide dismutase were elevated in cells exposed to conditioned medium, but returned to normal levels by 8h post-treatment. These results indicate a close correlation between BE-stimulated antioxidant activity and cellular sensitivity. Cell-cycle arrest and stimulation of antioxidant activity may account for the resistance to killing that was observed in bystander cells exposed to UVC or H2O2 treatment and are consistent with the role of the BE as a natural defense function triggered by UVC irradiation.

Some UV-bystander effects are mediated through induction of antioxidant defense in mammalian cells.

Bystander effect is the communication of signals from irradiated to unexposed neighboring cells which is often mediated through factors released from irradiated cells. We have attempted to investigate whether UV-bystander phenomenon can modulate the sensitivity of A375 cells and its mechanism. For this purpose, the conditioned medium from UVC-irradiated cells, which contained these released factors, was used to treat non-exposed cells. These cells were then subsequently treated with UVC or another genotoxicant H2O2. Cell viability was determined by Trypan blue-exclusion assay, DNA damage by flow cytometry analysis, ROS production by flow cytometry and microscopic analysis. Lipid peroxidation and antioxidant defense were assayed biochemically. Our findings revealed that exposure of non-irradiated cells to these factors induced increased in SOD and catalase activities which reverted to normal levels by 8 h. During this period, the released factors-treated cells were resistant to killing by UVC or H2O2 and induced DNA damage and lipid peroxidation were also lowered. This protection from cell killing was not present 8 h after exposure to these released factors. Our results suggested UV-bystander effect increased viability of cells through induction of antioxidant defense. This indicated UV-bystander phenomenon triggers protective response in cells.

UV released factors induce antioxidant defense in A375 cells.

UV light leads to release of different secretory factors from irradiated cells of which some of them have been characterized. We have reported earlier that cells exposed to the supernatant medium from irradiated cells were resistant to killing by some genotoxic agents. In this study, we present our finding that demonstrates DNA damage induced by UV or H(2)O(2) is lowered on prior exposure to the UV released factors (UVRF). Production of ROS in cells and lipid peroxidation was also lowered. It was found that treatment of unexposed cells with UVRF present in the supernatant medium altered the antioxidant defense activity in cells. Significant was the increase in catalase (CAT) and Cu-Zn superoxide dismutase (SOD) activity, whereas glutathione peroxidase (GPx) and reduced glutathione (GSH) levels remained unaffected. Cells exposed to UVRF prior to UV or H(2)O(2) treatment also experienced such upregulation; however, the remarkable increase in the GPx activity exhibited by these cells was not observed in cells exposed to H(2)O(2) or UV alone. It appears that exposure to UVRF tinkered with antioxidant defense in cells to facilitate its proliferation upon assault by an agent that can produce oxidative damage.

9-phenyl acridine exhibits antitumour activity by inducing apoptosis in A375 cells.

Several acridine derivatives have been screened for their therapeutic potential and some are already established as antiprotozoan, antibacterial or anticancer agents. However, phenyl derivative at C-9 position of acridine had remained unexplored for their biological activity so far. In this report, we present our findings with 9-phenyl acridine (ACPH) as an anticancer agent. Both A375 and HeLa, two human cancer cell lines, were more sensitive to ACPH than normal cells namely human lymphocytes and also Chinese hamster V79 cells. ACPH also led to regression of tumour volume in mice. In A375 cells, we have shown that it caused DNA damage and blocked cell cycle progression at G(2)-M phase. Treatment with ACPH led to lowering of mitochondrial potential, upregulation of bax, release of cytochrome C and activation of caspase 3. As a new agent showing lower toxicity to normal cells and greater sensitivity towards cancerous cell line, ACPH shows promise as an effective cancer chemotherapeutic agent. ACPH treatment resulted in apoptotic death of cells through mitochondria-mediated caspase-dependent pathway.