Supplementation with rumen-inert fat in the growing phase altered adipogenic gene expression and the size and number of adipocytes in Hanwoo steers.

We hypothesized that the provision of rumen-inert fat (RIF) to growing cattle (9 to 13 mo of age) would affect the expression of genes involved in lipid metabolism and thereby affect the size and number of adipocytes of steers slaughtered at 30 mo of age. Thirty steers with an average initial body weight (BW) of 239 ±â€…25 kg were allocated to six pens, balanced for BW and genetic merit for marbling, and assigned to one of two treatment groups: control (only basal diet) or test diet (basal diet with 200 g of RIF per day, on an as-fed basis) for 5 mo. Biopsy samples of longissimus lumborum (LM) muscle were then collected for analysis of fatty acid composition and gene expression. Both groups were then fed the same basal diets during the early and late fattening phases, without RIF, until slaughter (average shrunk BW = 759 kg). Supplementation with RIF increased the longissimus thoracis (LT) intramuscular fatty acid concentration at slaughter (P = 0.087) and numerically increased the quality grade score (P = 0.106). The LM intramuscular relative mRNA expression of genes such as PPARα, ZFP423 and SREBP1, FASN, SCD, FABP4, GPAT1, and DGAT2 were downregulated (P < 0.1) following RIF supplementation. Supplementation of RIF decreased (P < 0.1) diameter and concomitantly increased intramuscular adipocytes per viewing section at slaughter. This likely was caused by promotion of triacylglycerol hydrolysis during the growing phase. Another possible explanation is that the relative mRNA expression of gene ATGL was upregulated by RIF supplementation during the growing (P < 0.1) and the fattening phases (P < 0.05), while the genes associated with fatty acid uptake (FABP4) and esterification (DGAT2) were downregulated during the growing phase and upregulated (P < 0.1) during the fattening phase. This implies that the lipid turnover rate was higher for steers during the growing than fattening phase. This study demonstrated that RIF supplementation during the growing phase induced a carryover effect on the lipogenic transcriptional regulation involved in adipocyte lipid content of intramuscular adipose tissue; increased triacylglycerol hydrolysis during the growing phase subsequently was followed by increased lipid accumulation during the fattening phases.

Rumen inert fat (RIF) is a type of fat supplement that is used in the diets of beef cattle as early as 6 mo of age in calves and continues through the finishing period to improve the dietary energy density which can be used by the animal to deposit more lipid in the muscle tissue. However, for Hanwoo beef cattle, the precise time of RIF supplementation has not yet been determined. This study hypothesized that supplementing RIF at the growing phase (9 to 13 mo of age) would have a positive influence on the marbling characteristics of meat at slaughter. The growth rate and performance of steers were not improved by RIF supplementation, however, an increase in intramuscular fatty acid content was noted that was accompanied by the increased number of intramuscular adipocytes and decreased intramuscular adipocyte diameter. Supportively, upregulation of the genes associated with fatty acid uptake and esterification during the fattening phase of RIF-fed animals was noted. Overall, supplementing RIF at the growing stage could improve the lipid content of the meat which is supported by the increased lipid hydrolysis during the growing phase and followed by increased lipid accumulation during the fattening phases.

Cinnamaldehyde inhibits Enterococcus faecalis biofilm formation and promotes clearance of its colonization by modulation of phagocytes in vitro.

The nosocomial pathogen, Enterococcus faecalis plays a crucial role in the pathogenesis of variety of infections including endocarditis, urinary tract, and recurrent root canal infections. Primary virulence factors of E. faecalis such as biofilm formation, gelatinase production and suppression of host innate immune response can severely harm host tissue. Thus, novel treatments are needed to prevent E. faecalis biofilm development and pathogenicity due to the worrisome rise in enterococcal resistance to antibiotics. The primary phytochemical in cinnamon essential oils, cinnamaldehyde, has shown promising efficacy against a variety of infections. Here, we looked into how cinnamaldehyde affected the growth of biofilms, the activity of the enzyme gelatinase, and gene expression in E. faecalis. In addition, we looked at the influence of cinnamaldehyde on RAW264.7 macrophages' interaction with biofilm and planktonic E. faecalis in terms of intracellular bacterial clearance, NO generation, and macrophage migration in vitro. According to our research, cinnamaldehyde attenuated the biofilm formation potential of planktonic E. faecalis and gelatinase activity of the biofilm at non-lethal concentrations. The expression of the quorum sensing fsr locus and its downstream gene gelE in biofilms were also found to be significantly downregulated by cinnamaldehyde. Results also demonstrated that cinnamaldehyde treatment increased NO production, intracellular bacterial clearance, and migration of RAW264.7 macrophages in presence of both biofilm and planktonic E. faecalis. Overall these results suggest that cinnamaldehyde has the ability to inhibit E. faecalis biofilm formation and modulate host innate immune response for better clearance of bacterial colonization.

The synergic impact of lignin and Lactobacillus plantarum on DSS-induced colitis model via regulating CD44 and miR 199a alliance.

Chronic or recurrent immune system activation and inflammation inside the gastrointestinal tract is characterized by inflammatory bowel disease (IBD). Due to the lack of safety and efficacy of traditional medications, the use of food supplements for IBD management is on the rise. Numerous studies reported that, certain food supplements have a variety of therapeutic benefits for IBD. In the present study, a mouse model of IBD was used to the anti-colitis effects of lignin supplementation with Lactobacillus plantarum (L. plantarum) on intestinal inflammation. The animal model was treated with dextran sodium sulphate (DSS), the illness index increased, and colon length and body weight declined, but these effects were reversed when lignin and L. plantarum treated groups. In addition, lignin and L. plantarum supplementation inhibited the DSS induced increase in levels of cytokines TNF-α (250 pg/mL), INF-γ (180 pg/mL), IL-1ß (70 pg/mL) and TGF- ß (72 pg/mL). Gene and protein expression study revealed that Lignin and L. plantarum supplementation restored the expression of E-cad and suppressed the expression of STAT3 in DSS induced colitis model. Lignin and L. plantarum supplementation also suppressed CD44 expression (1.2 fold) by up regulating the expression of miR199a (1 fold) over DSS induced colitis. Our study suggests that Lactobacillus, lignin, and their synergistic treatments have protective roles against inflammatory bowel disease through changes in inflammatory cytokines, and miR 199a expression in DSS-induced colitis.

Thidiazuron, a phenyl-urea cytokinin, inhibits ergosterol synthesis and attenuates biofilm formation of Candida albicans.

Candida albicans is a common human fungal pathogen that colonizes mucosa and develops biofilm in the oral cavity that causes oral candidiasis. It has been reported that cytochrome P450 enzyme (CYP51), a vital part of the ergosterol synthesis cascade, is associated with Candida infections and its biofilm formation. Thidiazuron, a phenyl-urea cytokinin, exhibits anti-senescence and elicitor activity against fungal infection in plants. However, how Thidiazuron impacts C. albicans biofilm formation is still uncertain. Here, we aimed to investigate the effects of a Thidiazuron against the growth and biofilm formation properties of C. albicans using in silico and in vitro experimental approaches. A preliminary molecular docking study revealed potential interaction between Thidiazuron and amino acid residues of CYP51. Further in vitro antifungal susceptibility test, scanning electron microscopy (SEM) and time kill analysis revealed the anti-fungal activity of Thidiazuron in both dose and time-dependent manner. Crystal violet staining, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay revealed 50% inhibition in C. albicans biofilm by Thidiazuron at concentrations 11 and 19 µM respectively. Acridine orange staining assay visually confirmed the biofilm inhibitory potential of Thidiazuron. The gene expression study showed that Thidiazuron treatment down regulated the expression of genes involved in ergosterol synthesis (ERG3, ERG11, ERG25), cell adhesion (ASL3, EAP1), and hyphae development (EFG1, HWP1, SAP5) in C. albicans. Wherease, the expression of negative transcription regulator of hyphae (NRG1) was upregulated (5.7-fold) by Thidiazuron treatment. Collectively, our data suggest that Thidiazuron is a robust antifungal compound and an outstanding biofilm inhibitor, which may promise further therapeutic development due to CYP51 binding and inhibition of ergosterol formation against C. albicans.

Brassica oleracea L. var. botrytis Leaf Extract Alleviates Gentamicin-Induced Hepatorenal Injury in Rats-Possible Modulation of IL-1ß and NF-κB Activity Assisted with Computational Approach.

BACKGROUND: Recently, crop byproducts are considered a hot topic and can be converted into beneficial products. Cauliflower is well-known for its protective effects against oxidative stress-induced damage. The current study aimed to investigate the chemical profile and the ameliorative effects of cauliflower leaf extract (CL) on gentamicin-induced renal and hepatic injuries in rats. METHODS: Cauliflower leaf was extracted with methanol to give the total methanol extract (TME) followed by the determination of total phenolic contents (TPC). Rats were divided into five groups; Group I was assigned as the control group, while the other groups were injected with gentamicin for ten days. Group II was given distilled water. Rats in groups III and IV were treated with oral CL (200 mg/kg and 400 mg/kg, respectively). Group V received L-cysteine (as a positive control). The functions of the kidneys and liver; oxidative stress and morphological and apoptotic changes of renal and hepatic tissues were assessed. RESULTS: The TME was subjected to chromatographic techniques to yield ferulic acid, vanillic acid, p-coumaric acid and quercetin. TPC was 72.31 mg GAE/g of dried extract. CL treatment dose-dependently ameliorated gentamicin-induced impaired kidney and liver functions and improved the histopathological appearance of both organs. It also reduced gentamicin-induced oxidative stress. CL demonstrated downregulation of mRNA and protein expressions of IL-1ß and NF-κB compared to nontreated rats. In silico interaction of the isolated compounds with amino acid residues of IL-1ß and NF-κB might explain the current findings. CONCLUSION: Taken together, this study raises the waste-to-wealth potential of cauliflower to mitigate gentamicin-induced hepatorenal injury and convert the waste agromaterials into valuable products.

Ameliorative Effect of Ocimum forskolei Benth on Diabetic, Apoptotic, and Adipogenic Biomarkers of Diabetic Rats and 3T3-L1 Fibroblasts Assisted by In Silico Approach.

Diabetes mellitus (DM) is a complicated condition that is accompanied by a plethora of metabolic symptoms, including disturbed serum glucose and lipid profiles. Several herbs are reputed as traditional medicine to improve DM. The current study was designed to explore the chemical composition and possible ameliorative effects of Ocimum forskolei on blood glucose and lipid profile in high-fat diet/streptozotocin-induced diabetic rats and in 3T3-L1 cell lines as a first report of its bioactivity. Histopathological study of pancreatic and adipose tissues was performed in control and treatment groups, along with quantification of glucose and lipid profiles and the assessment of NF-κB, cleaved caspase-3, BAX, and BCL2 markers in rat pancreatic tissue. Glucose uptake, adipogenic markers, DGAT1, CEBP/α, and PPARγ levels were evaluated in the 3T3-L1 cell line. Hesperidin was isolated from total methanol extract (TME). TME and hesperidin significantly controlled the glucose and lipid profile in DM rats. Glibenclamide was used as a positive control. Histopathological assessment showed that TME and hesperidin averted necrosis and infiltration in pancreatic tissues, and led to a substantial improvement in the cellular structure of adipose tissue. TME and hesperidin distinctly diminished the mRNA and protein expression of NF-κB, cleaved caspase-3, and BAX, and increased BCL2 expression (reflecting its protective and antiapoptotic actions). Interestingly, TME and hesperidin reduced glucose uptake and oxidative lipid accumulation in the 3T3-L1 cell line. TME and hesperidin reduced DGAT1, CEBP/α, and PPARγ mRNA and protein expression in 3T3-L1 cells. Moreover, docking studies supported the results via deep interaction of hesperidin with the tested biomarkers. Taken together, the current study demonstrates Ocimum forskolei and hesperidin as possible candidates for treating diabetes mellitus.

Metabolite Profile, Ruminal Methane Reduction, and Microbiome Modulating Potential of Seeds of Pharbitis nil.

We identified metabolites in the seeds of Pharbitis nil (PA) and evaluated their effects on rumen methanogenesis, fiber digestibility, and the rumen microbiome in vitro and in sacco. Four rumen-cannulated Holstein steers (mean body weight 507 ± 32 kg) were used as inoculum donor for in vitro trial and live continuous culture system for in sacco trial. PA was tested in vitro at doses ranging from 4.5 to 45.2% dry matter (DM) substrate. The in sacco trial was divided into three phases: a control phase of 10 days without nylon bags containing PA in the rumen, a treatment phase of 11 days in which nylon bags containing PA (180 g) were placed in the rumen, and a recovery phase of 10 days after removing the PA-containing bags from the rumen. Rumen headspace gas and rumen fluid samples were collected directly from the rumen. PA is enriched in polyunsaturated fatty acids dominated by linoleic acid (C18:2) and flavonoids such as chlorogenate, quercetin, quercetin-3-O-glucoside, and quinic acid derivatives. PA decreased (p < 0.001) methane (CH4) production linearly in vitro with a reduction of 24% at doses as low as 4.5% DM substrate. A quadratic increase (p = 0.078) in neutral detergent fiber digestibility was also noted, demonstrating that doses < 9% DM were optimal for simultaneously enhancing digestibility and CH4 reduction. In sacco, a 50% decrease (p = 0.087) in CH4 coupled with an increase in propionate suggested increased biohydrogenation in the treatment phase. A decrease (p < 0.005) in ruminal ammonia nitrogen (NH3-N) was also noted with PA in the rumen. Analysis of the rumen microbiome revealed a decrease (p < 0.001) in the Bacteroidetes-to-Firmicutes ratio, suggesting PA to have antiprotozoal potential. At the genus level, a 78% decrease in Prevotella spp. and a moderate increase in fibrolytic Ruminococcus spp. were noted in the treatment phase. In silico binding of PA metabolites to cyclic GMP-dependent protein kinase of Entodinium caudatum supported the antiprotozoal effect of PA. Overall, based on its high nutrient value and antiprotozoal activity, PA could probably replace the ionophores used for CH4 abatement in the livestock industry.

Effects of Dietary Protein Concentration on Lipid Metabolism Gene Expression and Fatty Acid Composition in 18-23-Month-Old Hanwoo Steers.

The present study evaluated the influence of dietary protein level on growth performance, fatty acid composition, and the expression of lipid metabolic genes in intramuscular adipose tissues from 18- to 23-month-old Hanwoo steers, representing the switching point of the lean-to-fat ratio. Forty steers with an initial live weight of 486 ± 37 kg were assigned to one of two treatment groups fed either a concentrate diet with 14.5% CP and or with 17% CP for 6 months. Biopsy samples of intramuscular tissue were collected to analyze the fatty acid composition and gene expression at 23 months of age. Throughout the entire experimental period, all steers were restrained twice daily to allow individual feeding. Growth performance, blood metabolites, and carcass traits, according to ultrasonic measurements, were not affected by the experimental diets. The high-protein diet significantly increased the expression of intramuscular PPARα (p < 0.1) and LPL (p < 0.05) but did not affect genes involved in fatty acid uptake (CD36 and FABP4) nor lipogenesis (ACACA, FASN, and SCD). In addition, it downregulated intramuscular VLCAD (p < 0.01) related to lipogenesis but also GPAT1 (p = 0.001), DGAT2 (p = 0.016), and SNAP23 (p = 0.057), which are involved in fatty acid esterification and adipocyte size. Hanwoo steers fed a high-protein diet at 18-23 months of age resulted in a relatively lower lipid turnover rate than steers fed a low-protein diet, which could be responsible for shortening the feeding period.

MicroRNA-7188-5p and miR-7235 regulates Multiple sclerosis in an experimental mouse model.

The short non-coding microRNAs (miRNAs) have emerged as reliable modulators of various pathological conditions including autoimmune diseases in mammals. The current study, aims to identify new potential differential expressed miRNAs and their downstream mRNA targets of the autoimmune disease, Multiple sclerosis (MS). The study identifies a new set of miRNA(s) that are probably implicated in MS using computational tools. The study further carried-out different in vivo and in vitro experiments to check these identified miRNAs could be role in as therapeutic and prognostic applications. Preliminary insilico screening revealed that miR-659-3p, miR-659-5p, miR-684, miR-3607-3p, miR-3607-5p, miR-3682-3p, miR-3682-5p miR-4647, miR-7188-3p, miR-7188-5p and miR-7235 are specifically elevated in the secondary lymphoid cells of EAE mice. In addition, expression of the downstream target mRNA of these miRNAs such as FXBO33, SGMS-1, ZDHHC-9, GABRA-3, NRXN-2 were reciprocal to miRNA expression in lymphoid cells. These confirmed by applying the mimic and silencing miRNA models, suggesting new inflammatory target genes of these promising miRNA markers. The in vivo adoptive transfer model revealed that the suppression of miRNA-7188-5p and miR-7235 changed the pattern of astrocytes and CNS pathophysiology. The current study opens a new miRNA and their mRNA targets in MS disease. The absence of miRNA-7188-5p and miR-7235 enhanced the disease alleviation, confirms the regulatory effect of these targets. These optimized results highlights new set of miRNA's with therapeutic potential in experimental MS. Further studies are required to confirm these miRNA as therapeutic biomarker.

Feeding Systems and Host Breeds Influence Ruminal Fermentation, Methane Production, Microbial Diversity and Metagenomic Gene Abundance.

Our previous research revealed the advantages of separate feeding (SF) systems compared to total mixed ration (TMR) in terms of ruminal methane (CH4) production. The purpose of this experiment was to confirm the advantage of SF as a nutritional strategy for CH4 mitigation, and to determine the effects of different feeding systems (TMR and SF) on the rumen microbiome and associated metagenome of two different breeds and on CH4 emissions. We randomly allocated four Holstein (305 ± 29 kg) and four Hanwoo steers (292 ± 24 kg) to two groups; the steers were fed a commercial concentrate with tall fescue (75:25) as TMR or SF, in a crossover design (two successive 22-day periods). Neither feeding systems nor cattle breeds had an effect on the total tract digestibility of nutrients. The TMR feeding system and Hanwoo steers generated significantly more CH4 (P < 0.05) and had a higher yield [g/d and g/kg dry matter intake (DMI)] compared to the SF system and Holstein steers. A larger rumen acetate:propionate ratio was observed for the TMR than the SF diet (P < 0.05), and for Hanwoo than Holstein steers (P < 0.001), clearly reflecting a shift in the ruminal H2 sink toward CH4 production. The linear discriminant analysis (LDA) effect size (LEfSe) revealed a greater abundance (α < 0.05 and LDA > 2.0) of operational taxonomic units (OTUs) related to methanogenesis for Hanwoo steers compared to Holstein steers. Kendall's correlation analysis revealed wide variation of microbial co-occurrence patterns between feeding systems, indicating differential H2 thermodynamics in the rumen. A metagenome analysis of rumen microbes revealed the presence of 430 differentially expressed genes, among which 17 and 27 genes exhibited positive and negative associations with CH4 production, respectively (P < 0.001). A strong interaction between feeding system and breed was observed for microbial and metagenomic abundance. Overall, these results suggest that the TMR feeding system produces more CH4, and that Hanwoo cattle are higher CH4 emitters than SF diet and Holstein cattle, respectively. Interestingly, host-associated microbial interactions differed within each breed depending on the feeding system, which indicated that breed-specific feeding systems should be taken into account for farm management.

Anti-Allergic Potential of Cinnamaldehyde via the Inhibitory Effect of Histidine Decarboxylase (HDC) Producing Klebsiella pneumonia.

Allergy is an immunological disorder that develops in response to exposure to an allergen, and histamines mediate these effects via histidine decarboxylase (HDC) activity at the intracellular level. In the present study, we developed a 3D model of Klebsiella pneumoniae histidine decarboxylase (HDC) and analyzed the HDC inhibitory potential of cinnamaldehyde (CA) and subsequent anti-allergic potential using a bacterial and mammalian mast cell model. A computational and in vitro study using K. pneumonia revealed that CA binds to HDC nearby the pyridoxal-5'-phosphate (PLP) binding site and inhibited histamine synthesis in a bacterial model. Further study using a mammalian mast cell model also showed that CA decreased the levels of histamine in the stimulated RBL-2H3 cell line and attenuated the release of ß-hexoseaminidase and cell degranulation. In addition, CA treatment also significantly suppressed the levels of pro-inflammatory cytokines TNF-α and IL-6 and the nitric oxide (NO) level in the stimulated mast cells. A gene expression and Western blotting study revealed that CA significantly downregulated the expressions of MAPKp38/ERK and its downstream pro-allergic mediators that are involved in the signaling pathway in mast cell cytokine synthesis. This study further confirms that CA has the potential to attenuate mast cell activation by inhibiting HDC and modifying the process of allergic disorders.

Synergistic effect of cytokinin and gibberellins stimulates release of dormancy in tea (Camellia sinensis (L.) O. Kuntze) bud.

Reactivation of dormant meristem in banjhi (dormant) shoots is important to enhance the quality and quantity of tea production. The field grown tea bushes were subjected to treatment with dormancy breaking agents such as potassium nitrate (KNO3), thiourea, sodium nitro prusside (SNP), the phytohormones kinetin (Kn) and gibberellins (GA). The efficacy of Kn and GA were comparatively lesser than KNO3 while the combination of Kn and GA (50 and100 ppm respectively) resulted in better dormancy reduction in tea buds. This observation was supported by our results from gene expression study where accumulation patterns of mRNAs corresponding to histones (H2A, H2B, H3 and H4), cyclins (B2, D1 and D3), cyclin-dependent kinase (CDKA), ubiquitination enzymes (FUS, EXT CE2), cyclophilin, E2F, and tubulin were analyzed during growth-dormancy cycles in tea apical buds under the influence of Kn, GA and their combinations. The level of these mRNAs was low in dormant buds, which was significantly increased by foliar application of GA and Kn combination. The present study indicated that the foliar application of GA in combination with Kn will help to improve quality and quantity of tea production by breaking dormancy and stimulating the bud growth.

Inhibitory potential of EGCG on Streptococcus mutans biofilm: A new approach to prevent Cariogenesis.

Dental caries is a common cause for tooth loss and Streptococcus mutans is identified as the etiologic pathogen. This study evaluates the inhibitory potential of Epigallocatechin gallate (EGCG) on S.mutans glucansucrase enzyme and its biofilm. Glucansucrase binding and the inhibitory potential of EGCG was validated using AutoDock tool and enzyme inhibitory assay. Biofilm inhibitory potential was also confirmed using Scanning Electron Microscopic (SEM) analysis in human tooth samples. Molecular docking revealed that EGCG interacted with GLU 515 and TRP 517 amino acids and binds to glucansucrase. SEM analysis revealed inhibition of S.mutans biofilm by various concentrations of EGCG on surfaces of tooth samples. Bioinformatics and biological assays confirmed that EGCG potentially binds to the S. mutans glucansucrase and inhibits its enzymatic activity. Enzymatic inhibition of glucansucrase attenuated biofilm formation potential of S. mutans on tooth surface. Thus, we conclude that EGCG inhibitory potential of S. mutans biofilm on the tooth surface is a novel approach in prevention of dental caries.

Anticancer potential of NF-κB targeting apoptotic molecule "flavipin" isolated from endophytic Chaetomium globosum.

BACKGROUND: Anticancer compounds from natural sources have drawn attention due to their structural diversity and relatively lesser side effects. Endophytic fungi are one such natural resource from, which plethoras of anticancerous compounds have been isolated. PURPOSE: The objective of the study was to isolate and characterize the bioactive metabolite from Chaetomium globosum that exhibits astonishing antiproliferative activity against cancerous cell lines. METHODS: Flavipin was isolated by bioassay-guided fractionation and identified using FT-IR, EI-MS and NMR studies. MTT assay was used to determine the cytotoxicity. Fluorescent staining (AO/EB) and DNA fragmentation studies confirmed the occurrence of apoptosis. Real time PCR and Western blotting were used to analyze the expression of apoptosis related genes and its proteins, respectively. RESULTS: Flavipin inhibited proliferation of A549, HT-29 and MCF-7 cancer cells in dose dependent manner with an IC50 concentration of 9.89 µg/ml, 18 µg/ml and 54 µg/ml, respectively, whereas it was comparatively less sensitive (IC50 = 78.89 µg/ml) against normal cell line (CCD-18Co). At IC50 concentration cancerous cells exhibited cell shrinkage and fragmentation of DNA, which indicated that flavipin induced apoptotic cell death. In treated cells there is an up-regulation of p53 gene and its associated protein, whereas reciprocal expression was observed in BCL-2 gene and its protein. Furthermore, western blotting results also showed down-regulation of NFκB. CONCLUSION: This is the first report on the antiproliferative activity of flavipin isolated from endophytic C. globosum and also proposed that interaction of flavipin with NFкB could be a possible mechanism for this activity. Flavipin induced apoptosis at low concentrations in cancer cell lines (A549, HT-29) and exhibited itself as a potential anticancer agent.

Identification of Expressed miRNAs in Human Rheumatoid Arthritis Using Computational Approach - Discovery of a New miR-7167 from Human.

BACKGROUND: Rheumatoid Arthritis (RA) is a chronic inflammatory and autoimmune disease leading to bones and joints destruction. It is one of the major causes of lifetime disability and mortality among humans in the developing and developed countries. It was evident that epigenetic dysregulation is related to the pathogenesis of RA. MicroRNAs (miRNAs) are small non-coding RNAs that are epigenetic regulators for diverse biological processes and also provided novel molecular insights in the formation of arthritis. OBJECTIVE: The influences of miRNAs in the alteration of gene regulation during the pathogenesis of arthritis were exposed in recent years. METHOD: The computational approach to identify miRNA through EST-based homology is more powerful, economical and time-efficient. In this study, we applied EST-based homology search to identify miRNAs responsible for the development of arthritis in human beings. RESULTS: Our study on 36519 ESTs in human RA condition revealed the expression of four miRNAs, HSA-miR-198, HSA-miR-4647, has-miR-7167-5p and has-miR-7167-3p. The present study is the first report about has-miR-7167 that was homologous to Macaca mulatta. CONCLUSION: The predicted targets of these identified miRNAs revealed many biological functions in the pathogenesis of RA. Further elaborated studies on these miRNAs will help to understand their function in the development of RA and the use of miRNAs as therapeutic targets in the future.

Molecular Characterization of Mosquitocidal Toxin (Surface Layer Protein, SLP) from Bacillus cereus VCRC B540.

A marine Bacillus cereus (VCRC B540) with mosquitocidal effect was recently reported from red snapper fish (Lutjanus sanguineous) gut and surface layer protein (S-layer protein, SLP) was reported to be mosquito larvicidal factor. In this present study, the gene encoding the surface layer protein was amplified from the genomic DNA and functionally characterized. Amplification of SLP-encoding gene revealed 1,518 bp PCR product, and analysis of the sequence revealed the presence of 1482 bp open reading frame with coding capacity for a polypeptide of 493 amino acids. Phylogenetic analysis revealed with homology among closely related Bacillus cereus groups of organisms as well as Bacillus strains. Removal of nucleotides encoding signaling peptide revealed the functional cloning fragment of length 1398 bp. Theoretical molecular weight (51.7 kDa) and isoelectric point (5.99) of the deduced functional SLP protein were predicted using ProtParam. The amplified PCR product was cloned into a plasmid vector (pGEM-T), and the open reading frame free off signaling peptide was subsequently cloned inpET-28a(+) and expressed in Escherichia coli BL21 (DE3). The isopropyl-ß-D-thiogalactopyranoside (IPTG)-induced recombinant SLP was confirmed using western blotting, and functional SLP revealed mosquito larvicidal property. Therefore, the major findings revealed that SLP is a factor responsible for mosquitocidal activity, and the molecular characterization of this toxin was extensively studied.

Pinocembrin, a novel histidine decarboxylase inhibitor with anti-allergic potential in in vitro.

Pinocembrin (5, 7- dihydroxy flavanone) is the most abundant chiral flavonoid found in propolis, exhibiting antioxidant, antimicrobial and anti-inflammatory properties. However, the effect of Pinocembrin on allergic response is unexplored. Thus, current study aimed at investigating the effects of Pinocembrin on IgE-mediated allergic response in vitro. A special emphasis was directed toward histidine decarboxylase (HDC) and other pro-allergic and pro-inflammatory mediators. Preliminary studies, using a microbiological model of Klebsiella pneumoniae, provided first evidences that suggest Pinocembrin as a potential thermal stable inhibitor for HDC. Applying docking analysis revealed possible interaction between Pinocembrin and mammalian HDC. In vitro studies validated the predicted interaction and showed that Pinocembrin inhibits HDC activity and histamine in IgE-sensitized RBL-2H3 in response to dinitrophenol (DNP)-bovine serum albumin (BSA) stimulation. In addition, Pinocembrin mitigated the damage in the mitochondrial membrane, formation of cytoplasmic granules and degranulation as indicated by lower ß-hexoseaminidase level. Interestingly, it reduced range of pro-inflammatory mediators in the IgE-mediated allergic response including tumor necrosis factor (TNF)-α, interleukin (IL)-6, nitric oxide (NO), inducible NO synthase (iNOS), phosphorylation of inhibitory kappa B (IкB)-α, prostaglandin (PGE)-2 and cyclooxygenase (COX)-2. In conclusion, current study suggests Pinocembrin as a potential HDC inhibitor, and provides the first evidences it is in vitro anti-allergic properties, suggesting Pinocembrin as a new candidate for natural anti-allergic drugs.

Novel Aryl Hydrocarbon Receptor Agonist Suppresses Migration and Invasion of Breast Cancer Cells.

BACKGROUND: Despite the remarkable progress to fight against breast cancer, metastasis remains the dominant cause of treatment failure and recurrence. Therefore, control of invasiveness potential of breast cancer cells is crucial. Accumulating evidences suggest Aryl hydrocarbon receptor (Ahr), a helix-loop-helix transcription factor, as a promising target to control migration and invasion in breast cancer cells. Thus, an Ahr-based exploration was performed to identify a new Ahr agonist with inhibitory potentials on cancer cell motility. METHODS: For prediction of potential interactions between Ahr and candidate molecules, bioinformatics analysis was carried out. The interaction of the selected ligand with Ahr and its effects on migration and invasion were examined in vitro using the MDA-MB-231 and T47D cell lines. The silencing RNAs were transfected into cells by electroporation. Expressions of microRNAs (miRNAs) and coding genes were quantified by real-time PCR, and the protein levels were detected by western blot. RESULTS: The in silico and in vitro results identified Flavipin as a novel Ahr agonist. It induces formation of Ahr/Ahr nuclear translocator (Arnt) heterodimer to promote the expression of cytochrome P450 family 1 subfamily A member 1 (Cyp1a1). Migration and invasion of MDA-MB-231 and T47D cells were inhibited with Flavipin treatment in an Ahr-dependent fashion. Interestingly, Flavipin suppressed the pro-metastatic factor SRY-related HMG-box4 (Sox4) by inducing miR-212/132 cluster. Moreover, Flavipin inhibited growth and adhesion of both cell lines by suppressing gene expressions of B-cell lymphoma 2 (Bcl2) and integrinα4 (ITGA4). CONCLUSION: Taken together, the results introduce Flavipin as a novel Ahr agonist, and provide first evidences on its inhibitory effects on cancer cell motility, suggesting Flavipin as a candidate to control cell invasiveness in breast cancer patients.

In Silico Identification of Human miR 3654 and its Targets Revealed its Involvement in Prostate Cancer Progression.

BACKGROUND: MicroRNAs (miRNAs) are non-coding RNAs known to control a broad range of biological functions such as cellular proliferation, differentiation and programmed cell death. Recent reports showed that miRNAs can act as oncogenes or tumor suppressors, thereby, playing an important role in cancer initiation and progression. Moreover, we know that Expressed sequence tags (ESTs) are random single pass sequence reads, which displays the condition/tissue specific transcripts (coding and non-coding) of an organism. METHODS: In the present study, we have applied the bioinformatics approach to identify miRNA from prostate cancer using EST resource and its expressions were analyzed by quantitative reverse transcription PCR (qRT-PCR). RESULTS: Analysis of transcriptomics resource from the LNCaP cells revealed the presence of an EST encoding hsa-miR-3654. Presence of the premature candidate of miR-3654, demonstrates its expression in LNCaP cells. We further indentified that the expression level (Fold Induction) of miR-3654 in LNCaP was higher than the normal and androgen insensitive prostate cancer cell lines (PNT1A, PC-3). CONCLUSION: we have identified the miR-3654 involved in prostate cancer progression using computational approach and hypothesized that the down regulation of miR-3654 could be responsible for a solid tumor to get cancer stem-like cell phenotype. Further studies are required to investigate the molecular mechanisms behind the STAT3 mediated miR-3654 repression and the associated metastasis.

Significance of microRNA 21 in gastric cancer.

Despite promising developments of treatment, the mortality due to gastric cancer remains high and the mechanisms of gastric cancer initiation and the development also remains elusive. It has been reported that patients with positive serologic tests for H. pylori have a higher risk of the development of gastric cancer. microRNAs (miRNAs) are short non-coding RNA molecules consisting of 21-25 nucleotides (nt) in length. The miRNAs silence their cognate target genes by inhibiting mRNA translation or degrading the mRNA molecules by binding to their 3'-untranslated (UTR) regions and plays a very important role in cancer biology. Recent evidences indicate that miR-21 is overexpressed in tumour tissue, including gastric cancer and plays a vital role in tumour cell proliferation, apoptosis, invasion and angiogenesis. Elevated levels of miR-21 is associated with downregulation of tumour suppressor genes, such as programmed cell death 4 (PDCD4), tissue inhibitor of metalloproteinase 3, phosphatase and tensin homolog (PTEN), tropomyosin 1, ras homolog gene family member B, and maspin. Silencing of miR-21 through the use of a miR-21 inhibitor affected cancer cell viability, induced cell cycle arrest and increased chemosensitivity to anticancer agents indicating that miR-21 functions as an oncogene. Although an increased expression level of miR-21 has been observed in gastric cancer, studies related to the role of miR-21 in gastric cancer progression is very limited. The main thrust of this mini review is to explain the potency of miR-21 as a prognostic and/or diagnostic biomarker and as a new target for clinical therapeutic for interventions of gastric cancer progression.