Jellyfish Venom Peptides Targeting Human Potassium Channels Identified through Ligand Screening: Morphometric and Molecular Identification of the Species and Antibiotic Potential.

The relative lack of marine venom could be attributed to the difficulty in dealing with venomous marine animals. Moreover, the venom of marine animals consists of various bioactive molecules, many of which are proteins with unique properties. In this study, we investigated the potential toxic proteins of jellyfish collected for ligand screening to understand the mechanism of the toxic effects of jellyfish. Since taxonomic identification is problematic due to the lack of proper keys, we conducted morphological and molecular mitochondrial DNA sequencing from COI and ITS regions. The venom extract from nematocysts found along the bell margins was used for protein characterization using SDS-gel electrophoresis and nano-liquid chromatography-tandem mass spectrometry. Ligand screening for the most potent toxin and antibacterial and cytotoxicity assays were carried out. The phylogenetic tree showed distinct clustering from other Catostylus sp. The proteomic analysis revealed venom with many bioactive proteins. Only 13 venom proteins were identified with molecular weights ranging from 4318 to 184,923 Da, exhibiting the venom's complexity. The overall toxin protein composition of Catostylus sp. venom was dominated by potassium channel toxin alpha-KTx. Molecular docking of toxin alpha-KTx 1.13 revealed high specificity towards the human voltage-gated potassium channel Kv3 with a high fitness score and a minimum energy barrier of -17.9 kcal/mol. Disc diffusion and cytotoxicity assays revealed potent antibacterial activity against Klebsiella pneumoniae with no cytotoxicity. Further studies on detailed characterization and therapeutic potentials are warranted.

Integration of in vitro and in-silico analysis of Caulerpa racemosa against antioxidant, antidiabetic, and anticancer activities.

Marine algae are found to be excellent in their nutritional and potential therapeutic properties. This study explores the antidiabetic and anticancer potential of fractionated polyphenolic extract of Caulerpa racemosa, green macroalgae. Crude polyphenolic extract (CPE) of C. racemosa and its fractions (n-hexane, ethyl acetate, chloroform, and distilled water) were tested for its total phenol and flavonoid contents and antioxidant potential. The ethyl acetate fraction was subjected to gas chromatography/mass spectrometry (GC/MS). The in vitro antidiabetic activity was assessed by alpha-amylase, glucosidase inhibition and anti-glycation assays. Also, in-silico studies were conducted to test the binding affinities between caulerpin with alpha-glucosidase enzyme and estrogen receptor (ER) active sites. Each fraction was tested for its in vitroin vitroanticancer activity by CellTiter-Glo and MTT cell proliferation assays. The total phenolic and flavonoid contents and the antioxidant potential of the crude extract were observed to be dose dependent. The GC/MS analysis of the ethyl acetate fraction yielded 47 peaks, whereas n-hexadecanoic acid and hexadecanoic acid methyl ester showed the highest compatibility percentages of 99% and 96%, respectively. The CPE exhibited a higher potential in both alpha-amylase inhibitory and anti-glycation activities. The ethyl acetate fraction was more effective against alpha-glucosidase inhibition. Molecular docking revealed a high binding affinity between the alpha-glucosidase enzyme and caulerpin and showed high binding affinity toward caulerpin, with H-bond interactions. The in vitro anticancer analyses revealed that chloroform fraction and CPE exhibited moderate activity on the KAIMRC1 cell line. Also, the CPE exhibited high specificity compared to the standard drug in anticancer studies. Our findings evidence the pharmacological potential of the CPE of C. racemosa, and bioactive compounds of the species may be utilized as lead molecules to develop anti-diabetic and anti-cancer drugs.

A Novel In Silico Benchmarked Pipeline Capable of Complete Protein Analysis: A Possible Tool for Potential Drug Discovery.

Current in silico proteomics require the trifecta analysis, namely, prediction, validation, and functional assessment of a modeled protein. The main drawback of this endeavor is the lack of a single protocol that utilizes a proper set of benchmarked open-source tools to predict a protein's structure and function accurately. The present study rectifies this drawback through the design and development of such a protocol. The protocol begins with the characterization of a novel coding sequence to identify the expressed protein. It then recognizes and isolates evolutionarily conserved sequence motifs through phylogenetics. The next step is to predict the protein's secondary structure, followed by the prediction, refinement, and validation of its three-dimensional tertiary structure. These steps enable the functional analysis of the macromolecule through protein docking, which facilitates the identification of the protein's active site. Each of these steps is crucial for the complete characterization of the protein under study. We have dubbed this process the trifecta analysis. In this study, we have proven the effectiveness of our protocol using the cystatin C and AChE proteins. Beginning with just their sequences, we have characterized both proteins' structures and functions, including identifying the cystatin C protein's seven-residue active site and the AChE protein's active-site gorge via protein-protein and protein-ligand docking, respectively. This process will greatly benefit new and experienced scientists alike in obtaining a strong understanding of the trifecta analysis, resulting in a domino effect that could expand drug development.

Therapeutic Efficacy of Nyctanthes arbor-tristis Flowers to Inhibit Proliferation of Acute and Chronic Primary Human Leukemia Cells, with Adipocyte Differentiation and in Silico Analysis of Interactions between Survivin Protein and Selected Secondary Metabolites.

Although the antidiabetic efficacy of Nyctanthes arbor-tristis flowers has been reported, antiproliferative and anti-obesity activities are yet to be explored. We examined the anti-obesity and antiproliferative potentials of different fractions (hexane, chloroform, ethyl acetate, methanol) of N. abor-tristis flower extract for the first time using 3T3-L1 cells, primary peripheral blood mononuclear cells (PBMC) isolated from healthy and adult acute myeloid (AML) and chronic lymphocytic leukemia (CLL) patients, recombinant Jurkat T cells, and MCF7 cell lines. The in vitro hypoglycemic activity was evaluated using the inhibition of -amylase enzyme and glucose uptake by yeast cells. The percentage glucose uptake and -amylase inhibitory activity increased in a dose-dependent manner in the crude and the tested fractions (hexane and ethyl acetate). Inhibition of the 3T3-L1 cells' differentiation was observed in the ethyl acetate and chloroform fractions, followed by the hexane fraction. Antiproliferative analyses revealed that Nyctanthes exerted a high specific activity against anti-AML and anti-CLL PBMC cells, especially by the hexane and ethyl acetate fractions. The gas chromatography/mass spectrometry analysis indicated the presence of 1-heptacosanol (hexane fraction), 1-octadecene (hexane and chloroform fractions), and other organic compounds. Molecular docking demonstrated that phenol,2,5-bis(1,1-dimethylethyl) and 4-hydroxypyridine 1-oxide compounds showed specificity toward survivin protein, indicating the feasibility of N. abor-tristis in developing new drug leads against leukemia.

Potent antibacterial, antioxidant and toxic activities of extracts from Passiflora suberosa L. leaves.

Passiflora suberosa L. belonging to the family Passifloraceae is an important medicinal plant used in traditional medicinal system in Sri Lanka to treat diabetes, hypertension and skin diseases. We extracted P. suberosa leaves under reflux conditions using different solvents (hexane, chloroform, methanol and water), then subjected to phytochemical screening. Alkaloids, flavonoids and saponins and saponins and anthraquinones were present in hexane and chloroform extracts. Alkaloids, unsaturated sterols, triterpenes, saponins, flavonoids and tannins were observed in both methanol and aqueous extracts. Proanthocyanidins were observed only in the aqueous extract. Hence, aqueous and methanol extracts with most classes of phytochemicals present were subjected to antimicrobial, antioxidant, antihaemolytic activities and Brine shrimp lethality studies. Antibacterial activity and minimum inhibition concentrations were evaluated using three Gram-positive (Bacillus subtilis, Staphylococcus aureus and Enterococcus faecium) and three Gram-negative bacteria (Pseudumonas aeruginosa, Salmonella typhimuriam and Escherichia coli). The results indicated that only the methanol extract of P. suberosa exhibited antibacterial activities against all the strains of Gram-negative and Gram-positive bacterial with stronger activity against Gram-negative bacteria. DPHH (2,2-diphenyl-1-picrylhydrazy) scavenging assay was adopted to evaluate antioxidant properties while antihaemolytic and toxic activities were studied respectively using cow blood and Brine shrimp lethality assay. The IC50 values of the aqueous extract in both antioxidant and antihaemolytic assays were significantly lower than the standard ascorbic acid. Similar results were observed in the Brine shrimp lethality assay. In conclusion both aqueous and methanol extracts of P. suberosa leaves showed the presence of majority of phytochemicals including proanthocyanidins. Antibacterial activity was obtained only for methanol extract with better activity against Gram-negative bacteria. The aqueous extract showed better antioxidant, antihaemolytic and toxic activities than the methanol extract and their respective standards. Further investigations on the chemical composition and possible isolation of active ingredients is warranted.

Hypoglycemic and hypolipidemic activity of aqueous leaf extract of Passiflora suberosa L.

Leaves of Passiflora suberosa L. (Family: Passifloraceae; common name: wild passion fruit, devil's pumpkin) are used in Sri Lankan traditional medicine for treating diabetes. The present study investigated the in vivo ability of P. suberosa leaves to manage blood sugar status and associated cholesterol levels. Mechanisms of action and toxicity were also determined. Phytochemical screening of aqueous extracts of P. suberosa leaves and carbohydrate content of the leaves were determined according to previously published methods. In two group of male mice (n = 9), effects on fasting and random blood glucose levels (BGLs) of different acute doses (0, 25, 50, 100 and 200 mg/kg) of the aqueous leaf extract (ALE) were evaluated at 1, 3, and 5 h post-treatment. In another set of mice, the fasting BGL was evaluated following treatment of 0 or 50 mg/kg ALE (dose prescribed in traditional medicine) for 30 consecutive days. The lipid profile, some mechanism of ALE action (diaphragm glucose uptake, glycogen content in the liver and skeletal muscles) and its toxicity (behavioural observation, food and water intake, hepatoxicity) were also assessed following 30-day treatment. However, sucrose and glucose tolerance tests and intestinal glucose uptake were conducted to determine portion of mechanisms of action following single dose of 50 mg/kg ALE. Phytochemical screening revealed the presence of alkaloids, unsaturated sterols, triterpenes, saponins, flavonoids, tannins and proanthocyanidins. Carbohydrate content of the leaves was 12.97%. The maximum hypoglycemic effect was observed after 4 h of 50 and 100 mg/kg ALE administration. The extract decreased fasting BGL (18%) following an oral sucrose challenge and inhibited (79%) glucose absorption from the intestine. Correspondingly, the levels of glycogen in the liver (61%) and in the skeletal muscles (57%) were found be higher than that of the control group. The levels of total cholesterol (17%) and tri-glyceraldehyde levels (12%) found to be reduced in treated groups. Furthermore, no significant toxic effects were observed in treated groups. The present results suggest that the leaves of P. suberosa can be used to manage blood glucose and cholesterol levels. Isolation of active compounds are recommended for further analysis.

Low doses of chlorpyrifos interfere with spermatogenesis of rats through reduction of sex hormones.

Use of pesticides results in indirect effects on human health. We aimed to evaluate implications of toxicological effects of subchronic chlorpyrifos exposure on reproductive function in male rats. A total of 48 adult Wistar male rats were separated into four groups (n = 12). Animals were gavaged with 2.5 mg/kg (T1), 5 mg/kg (T2), or 10 mg/kg (T3) body weight of chlorpyrifos (CPF) or distilled water (control) daily for 30 days. Organ weights, epididymal sperm parameters, DNA integrity, sex hormonal (FHS and LH) levels, and alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), and creatinine concentrations were determined on day 31. Another two sets of (four groups/set; n = 10) animals were orally treated with the same doses of CPF, control animal groups were treated with distilled water only for 30 days, and fertility indices and blood plasma acetylcholine esterase (AchE) were determined on day 31. Exposure to CPF resulted in a significant (p < 0.05) decrease in weights of testis and epididymis. An increase in liver weight resulted in reduced sperm counts and sperm motility and an increase in sperm abnormalities. Significant reduction in serum testosterone (p < 0.01), luteinizing hormone (p < 0.05), and follicular stimulating hormone (p < 0.05) levels was evident in animals treated with the highest dose. A significant decrease in the number of viable implantation sites and pups was observed in female rats mated with the T3 (p < 0.01) and T2 (p < 0.05) males. The ALT, AST, GGT, and creatinine contents were significantly increased (p < 0.05 and p < 0.01, respectively) on CPF exposure. A significant (p < 0.01) reduction in blood plasma AchE enzyme was observed with the highest dose. Our results demonstrated that prolonged exposure of CPF induces spermatogenesis damage, possibly through interference with sex hormones and AchE enzyme resulting in reduction of fertility. Therefore, awareness programs on handling CPF (pesticides) to enhance safety warrant minimization of its hazards.

Hypoglycemic and hypolipidemic activities of aqueous extract of flowers from Nycantus arbor-tristis L. in male mice.

BACKGROUND: Boiled aqueous extract of flowers (AEF) from Nyctanthes arbor-tristis L. are used in Sri Lankan traditional Ayruvedic Medicine to manage diabetes mellitus. AEF has widely been used as a folk medicine for the treatment of various ailments due to its therapeutic activity. However, little is known concerning therapeutic activity of the extract as well as its underline mechanisms and safety. Diabetes is known to increase low-density cholesterol and decrease high-density cholesterol thus triggering coronary diseases. Hence, the primary objective of the present study is to investigate the hypoglycemic and hypolipidemic activities of the AEF. METHODS: AEF was prepared and male mice (n = 9 group) were gavaged either with 250, 500 and 750 mg/kg of AEF or distilled water (DW). Subsequently, fasting and random blood glucose concentrations were determined. To investigate mechanisms of actions of AEF, animals were orally administered with 500 mg/kg or the vehicle (DW) and glucose tolerance was performed before and after glucose challenge. For further studies, in vitro alpha-amylase assay and glucose absorption from the gastrointestinal tract were performed using 500 mg/kg of the extract. Additionally, glycogen content in the liver and skeletal muscles, a complete lipid profile assay, and toxicological and biochemical parameters were conducted after a chronic study. RESULTS: Five hundred mg/kg and 750 mg/kg of AEF significantly (p < 0.01) reduced fasting blood glucose levels respectively by 49 and 39% at 4 h post-treatment, while 500 mg/kg of AEF also decreased the random blood glucose level significantly (p < 0.01) by 32% at 4 h post-treatment. AEF significantly inhibited glucose absorption by 85% from the intestine and increased diaphragm uptake of glucose by 64%. The extract also exhibited inhibition (16.66%) of alpha-amylase enzyme activity. It also decreased the level of total cholesterol (by 44.8%), triglyceride (by 53%) and increased (by 57%) the high-density lipoprotein cholesterol. Treatment with AEF did not induce any overt signs of toxicity or hepatotoxicity. CONCLUSION: Results the present study indicated that AEF possess hypoglycemic and hypolipdemic properties. Therefore, AEF could be used as an alternative medicine in management of diabetes mellitus.

Does obesity increase the risk of injury or mortality in motor vehicle crashes? A systematic review and meta-analysis.

The objective of this review was to assess the risk of obesity in injuries and fatalities resulting from motor vehicle crash (MVC), as compared with individuals with a normal-range body mass index. A systematic review of the literature was conducted yielding 824 potential studies. Nine of these studies met our inclusion criteria. Meta-analyses examining obesity as a risk factor for various injury types and risk of fatality were conducted using data from these studies. Obesity was associated with higher fatality risk (odds ratio [OR] = 1.89, 95% confidence interval [CI] = 1.51-2.37, P = .0001; pooled estimate from 6 studies), and increased risk of lower extremity fractures (OR = 1.39, 95% CI = 1.18-1.65, P = .0001; pooled estimate from 2 studies). No significant differences were observed when considering abdominal injuries or pelvic fractures. Interestingly, for head injuries obesity was a protective factor (OR = 0.67, 95% CI = 0.46-0.97, P = .0001; pooled data from 3 studies). Evidence strongly supports the association of obesity with higher fatality and fractures of the lower extremities in MVCs. Contrary to our hypothesis, 3 studies showed that obesity was a protective factor in reducing head injuries. Furthermore, the review shows that obesity was not a risk factor of MVC-related pelvic fractures and abdominal injuries.

Bull bars and vulnerable road users.

OBJECTIVES: Pedestrian injuries are a leading cause of the global death and injury burden, accounting for 65 percent of the 1.2 million annual road deaths. The purpose of this brief literature review is to examine whether bull bars, a rigid aftermarket accessory fitted to the front end of passenger vehicles, increase the risk of severe and fatal injuries to vulnerable road users in the event of a collision. METHODS: Applicable peer-reviewed research, review papers, and grey literature were identified from a search of MEDLINE; the Transportation Research Board (TRB) database composed of Transportation Research Information Services (TRIS) and International Transport Research Documentation (TRID) databases; the Cochrane Database of Systematic Reviews; and Google Scholar. The following search terms were used: "bull bars" OR "nudge bars" OR "sahara bars" AND "pedestrians" OR "vulnerable road users" for 1948 to March 1, 2011. A secondary set of search terms was also included "bull bars" OR "nudge bars" OR "sahara bars" OR "vehicle frontal protective systems" AND "pedestrians" OR "vulnerable road users" for 1948 to March 1, 2011. RESULTS: Neither the MEDLINE search nor the Cochrane Review search returned any relevant literature. The TRID search returned 19 research articles, 9 of which were included. Searches using Google Scholar returned 110 items, of which 21 were included in the present review after excluding patents and citations. Seven of the articles from TRID were also found in the Google Scholar search, resulting in 23 unique articles being included in this review. The studies used included 12 real-world studies, 3 computer modeling studies, and 8 laboratory testing studies. Very few studies examined the road safety of pedal-cyclists and motorcyclists; therefore, we focused solely on studies examining pedestrian safety. CONCLUSIONS: The literature reviewed in this study indicates that vehicles fitted with bull bars, particularly those without deformable padding, concentrate crash forces over a smaller area of vulnerable road users during collisions compared to vehicles not fitted with a bull bar. Rigid bull bars, such as those made from steel or aluminum, stiffen the front end of vehicles and interfere with the vital shock absorption systems designed in vehicle fronts. These devices therefore significantly alter the collision dynamics of vehicles, resulting in an increased risk of pedestrian injury and mortality in crashes. This literature review shows that bull bars do indeed increase the severity of injuries to vulnerable road users in the event of a collision and highlights the need for current traffic safety policies to reflect the safety concerns surrounding the use of bull bars.

Sociodemographic factors associated with aggressive driving behaviors of 3-wheeler taxi drivers in Sri Lanka.

Little is known about the nature and scope of aggressive driving in developing countries. The objective of this study is to specifically examine the sociodemographic factors associated with aggressive driving behavior among 3-wheeler taxi drivers in Sri Lanka. Convenience samples of 3-wheeler taxi drivers from Rathnapura, Ahaliyagoda, Sri Lanka were surveyed from June to August 2006. Analyses included bivariate and multivariate logistic regression. Drivers with less than high school education were 3.5 times more likely to drive aggressively (odds ratio [OR] = 3.46; 95% confidence interval [CI] = 1.08, 11.1). Single drivers were 9 times more likely to run red lights (OR = 8.74; 95% CI = 2.18, 35.0), and being single was a major risk factor for drunk driving (OR = 4.80; 95% CI = 1.23, 18.7). Furthermore, high school completers were 4 times more likely to bribe a policeman (OR = 4.27; 95% CI = 1.23, 14.9) when caught violating the road rules. Aggressive driving and risk-taking behavior are amenable to policy initiatives, and preventive programs targeted at key groups could be used to improve road safety in Sri Lanka. This study demonstrates that aggressive driving behavior is associated with sociodemographic factors, including the level of education, marital status, and other socioeconomic factors. Hence, economic factors should be addressed to find solutions to traffic-related issues. It will be the government's and policy makers' responsibility to try and understand the economic factors behind risky road behavior and bribe-taking behavior prior to legislating or enforcing new laws.

Baseline knowledge on vehicle safety and head restraints among Fleet Managers in British Columbia Canada: a pilot study.

BACKGROUND: Whiplash is the most common injury type arising from motor vehicle collisions, often leading to long-term suffering and disability. Prevention of such injuries is possible through the use of appropriate, correctly positioned, vehicular head restraints. OBJECTIVE: To survey the awareness and knowledge level of vehicle fleet managers in the province of British Columbia, Canada, on the topics of vehicle safety, whiplash injury, and prevention; and to better understand whether these factors influence vehicle purchase/lease decisions. METHODS: A survey was administered to municipal vehicle fleet managers at a professional meeting (n = 27). RESULTS: Although many respondents understood the effectiveness of vehicle head restraints in the prevention of whiplash injury, the majority rarely adjusted their own headrests. Fleet managers lacked knowledge about the seriousness of whiplash injuries, their associated costs for Canada's healthcare system, and appropriate head restraint positions to mitigate such injuries. The majority of respondents indicated that fleet vehicle purchase/lease decisions within their organization did not factor whiplash prevention as an explicit safety priority. CONCLUSIONS: There is relatively little awareness and enforcement of whiplash prevention strategies among municipal vehicle fleet managers.

The extracellular calcium-sensing receptor reciprocally regulates the secretion of BMP-2 and the BMP antagonist Noggin in colonic myofibroblasts.

To understand whether postprandial extracellular Ca(2+) (Ca(o)(2+)) changes were related to intestinal epithelial homeostasis, we performed array analysis on extracellular calcium-sensing receptor (CaSR)-expressing colonic myofibroblasts (18Co cells) and observed increases in bone morphogenetic protein (BMP)-2 transcripts. The present experiments demonstrated that regulated secretion of BMP-2 occurs in response to CaSR activation of these cells and revealed a new property of BMP-2 on the intestinal barrier. Activation by Ca(o)(2+), spermine, GdCl(3), or neomycin sulfate of 18Co cells or primary isolates of myofibroblasts from the normal human colon stimulated both the synthesis (RT-PCR) and secretion (ELISA) of BMP-2. Transient transfection with short interfering RNA against CaSR completely inhibited BMP-2 secretion. Transient transfection with dominant negative CaSR (R185Q) increased the EC(50) of Ca(o)(2+) (5.7 vs. 2.3 mM). Upregulation of BMP-2 transcript and secretion occurring within 3 h of CaSR activation was prevented by actinomycin D. CaSR-mediated BMP-2 synthesis and secretion required phosphatidylinositol 3-kinase activation (as assessed by phospho-Akt generation). Exogenous BMP-2 and conditioned medium from CaSR-stimulated 18Co cells accelerated restitution in wounded postconfluent Caco-2 cells. Exogenous BMP-2 and conditioned medium from CaSR-stimulated 18Co cells increased the transepithelial resistance of low- and high-resistance T-84 epithelial monolayers. CaSR stimulation of T-84 epithelia and colonic myofibroblasts downregulated the BMP family antagonist Noggin, as assessed by RT-PCR and Western blot analysis. Together, our data suggest that the CaSR mediates the effective concentration of BMP-2 in the intestine, which leads to enhanced repair and barrier development.