In vitro and in silico screening of novel typhoid drugs from endangered herb (Enicostema axillare).

In the present study, antibacterial activity of swertiamarin from Enicostema axillare (Lam) was checked against three different human gram-negative pathogens namely Salmonella typhi, Klebsiella pneumoniae and Shigella flexneri. Minimum inhibitory concentration assay revealed low dose and efficient activity of swertiamarin on the above said pathogens. Though swertiamarin is a well-studied and characterized compound, there is no experimental proof available for its antibacterial activity. To gain more insight about the antibacterial efficiency of swertiamarin against typhoid causing S. typhi, a comparative molecular docking of S. typhi OmpF (3NSG) was performed with swertiamarin and other typhoid drugs available in the market which exposed better activity strength of swertiamarin compared with that of the other drugs. Further, molecular dynamics of S. typhi OmpF-swertiamarin shows good flexibility and stability at 100 ns. The outcome of this work will definitely provide an idea of using very low dose of swertiamarin as a potent and promising drug against typhoid fever.Communicated by Ramaswamy H. Sarma.

Effect of novel therapeutic medicine swertiamarin from Enicostema axillare in zebrafish infected with Salmonella typhi.

Herbal treatments have been practiced by humans over centuries and therefore possess time-proven safety. However, it is crucial to evaluate the toxic effects of herbal medicine to confirm their safety, particularly when developing therapeutic drugs. Use of laboratory animals such as mice, rat, and rabbits was considered as gold standard in herbal toxicity assessments. However, in the last few decades, the ethical consideration of using higher vertebrates for toxicity testing has become more controversial. As a possible alternative model involving lower vertebrates such as zebra fish were introduced. Hence in the present study, swertiamain compound isolated from E. axillare was assessed for it antimicrobial activity in zebra fish larvae againt S. typhi. The cumulative mortality rate and bacterial localization in zebra fish larvae were studied. Biochemical markers assays were performed to find the preventive role of the compound during the typhoid infection. The results showed that zebra fish can be successfully used as a model to study typhoid infection and the anti-bacterial compound swertiamarin used in this study clears the bacterial load and pathogenic symptoms to a great extent.

Developmental toxicity, antioxidant, and marker enzyme assessment of swertiamarin in zebrafish (Danio rerio).

A secoiridoid glycoside called swertiamarin has been widely used as a herbal medicine for many decades. In particular, swertiamarin from the Enicostema axillare herb has been used as a multipurpose drug to treat innumerable health problems. As this medicine is consumed orally, its toxicity level should be determined. To examine the safety of this compound, toxicology work was done in zebrafish, and this is the first report to describe swertiamarin toxicity in zebrafish. Zebrafish embryos were used in this swertiamarin toxicity study, and morphological changes were observed. Further, the compound was also studied in adult zebrafish to determine the impact of the compound on the fish liver. Enzyme profiling with superoxide dismutase, glutathione peroxidase, catalase, reduced glutathione levels, glutathione S-transferase, lactate dehydrogenase, glutamic oxaloacetic transaminases, lipid peroxidation, Na+ /K+ -ATPase, and glutamic pyruvic transaminases) was evaluated (p ≤ 0.05). Results suggest that swertiamarin is a safe drug only at a low concentration (40 µM). This study also shows that even herbal medicinal compounds may be toxic to humans at higher dosages. Hence, irrespective of whether a drug is synthetic or natural, it needs to be tested for its toxicity before use in humans.

Selenium nanoparticle synthesis from endangered medicinal herb (Enicostema axillare).

Green synthesis of selenium nanoparticles (SeNPs) was achieved by a simple biological procedure using the reducing power of E. axillare leaf extract. This method is capable of producing SeNPs in a size range of around 56.23-98.18 nm, under ambient lab condition. The synthesized nanoparticles were characterized by UV-Vis spectroscopy, Fourier-transform infrared spectroscopy (FTIR), Scanning electron microscopy (SEM) and Energy-dispersive X-Ray (EDX). To check the potential hazards of nanotoxicity median lethal concentration (LC50) and Na + /K + -ATPase activity was assessed on zebra fish which showed the LC50 of 258.72 ppm for 96 h and Na + /K + -ATPase seems to significantly decrease with increase in SeNPs concentration. Antibactericidal activity explored the use of SeNPs against wide range of pathogens. Cytotoxicity of SeNPs was assayed against human lung cancer cells (A549) confirmed that SeNPs are able to inhibit the cell growth by dose-dependent manner. In conclusion, green synthesized selenium nanoparticles are less toxic and harmless. This is the first report on green synthesized selenium nanoparticles using E. axillare possessing anti-bacterial and anti-cancer activity.

Evidence of post-transcriptional readthrough regulation in FGF5 gene of alpaca.

Two different phenotypes are described in alpaca, identified as suri and huacaya, which differ in the type of fleece. The huacaya fleece is characterized by compact, soft and highly crimped fibers, while the suri fleece is longer, straight, less-crimped and lustrous. In our study, the Fibroblast growth factor 5 (FGF5) was investigated as a possible candidate gene for hair length in alpaca (Vicugna pacos). As previously identified in other mammals, our results show that the alpaca FGF5 gene gives rise to a short (FGF5S) and a long (FGF5) isoform. Interestingly, in the long isoform, we observed a point mutation (i.e., a transition C>T at position 499 downstream of the ATG codon) that is able to generate a premature termination codon (PTC). The highly conserved nucleotide and amino acid sequence after PTC suggested a readthrough event (RT) that was confirmed by western blot analysis. The analysis of cDNA sequence revealed motifs and structures of mRNA undergoing RT. In fact, the event is positively influenced by particular signals harbored by the transcript. To the best of our knowledge, this is the first case of a readthrough event on PTC reported for the FGF5 gene and the first case of this translational mechanism in alpaca.

Immune response of olive flounder (Paralichthys olivaceus) infected with the myxosporean parasite Kudoa septempunctata.

This study evaluated the pathophysiological, biochemical, and immunological status of olive flounder (Paralichthys olivaceus) infected with the myxosporean parasite Kudoa septempunctata. Flounder fish collected from Kudoa-infected and uninfected farms were confirmed by microscopic and TaqMan probe-based quantitative PCR screening. Morphological, biochemical, histological, and immune gene expression analyses were performed on uninfected and infected hosts to assess the effect of K. septempunctata. Histological studies confirmed the presence of Kudoa myxospores in the trunk muscles of infected flounder fish. Serum biochemical parameters, including the levels of myeloperoxidase activity, superoxide dismutase activity, alanine aminotransferase, alkaline phosphatase, amylase, bilirubin, total protein, cholesterol, calcium, potassium, sodium, phosphorus, glucose, and galactose, were found to exhibit no significant variations (p > 0.05) between uninfected and infected flounder fish. However, immune-related genes such as Mx, lysozyme, signal transducer and activator of transcription 1, interferon-γ, interferon regulatory factor, and tumour necrosis factor showed significantly elevated expression (p < 0.05) in the trunk muscles of infected flounder fish while no significant differences were noted in uninfected fish trunk muscle and head-kidney of infected and uninfected flounder fish.

Isolation, Characterization, Antioxidant, Antimicrobial and Cytotoxic Effect of Marine Actinomycete, Streptomyces Carpaticus MK-01, against Fish Pathogens

ABSTRACT Present study aim to evaluate the antimicrobial, antioxidant and cytotoxic potential of crude extract of Marine Streptomyces carpaticus MK-01 isolated from seawater collected from Daejeong-cost of Jeju Island. About 24 actinomycetes strains were isolated and subjected to morphological and molecular analysis that confirmed the isolate as S. carpaticus MK-01. Crude ethyl acetate extract of MK-01 strain showed extensive antibacterial activity against Gram-positive fish pathogenic bacteria namely Streptococcus iniae and S. parauberis with a maximum zone of inhibition (0.92±0.03mm) was recorded against S. parauberis at the minimum extract concentration (3.12µg/ml). The MK-01 ethyl acetate extract shows dose dependant significant increase in antioxidant activity. The 50% cytotoxic concentration (CC50) of MK-01 ethyl acetate extract was attained at 53.71 μg/ml and the effective concentration 50 (EC50) against virus-infected Epithelioma papulosum cyprini cell lines was 8.72 μg/ml of S. carpaticus MK-01 crude ethyl acetate extract.

Meta-analysis Reveals That the Genus Pseudomonas Can Be a Better Choice of Biological Control Agent against Bacterial Wilt Disease Caused by Ralstonia solanacearum.

Biological control agents (BCAs) from different microbial taxa are increasingly used to control bacterial wilt caused by Ralstonia solanacearum. However, a quantitative research synthesis has not been conducted on the role of BCAs in disease suppression. Therefore, the present study aimed to meta-analyze the impacts of BCAs on both Ralstonia wilt disease suppression and plant (host) growth promotion. The analysis showed that the extent of disease suppression by BCAs varied widely among studies, with effect size (log response ratio) ranging from -2.84 to 2.13. The disease incidence and severity were significantly decreased on average by 53.7% and 49.3%, respectively. BCAs inoculation also significantly increased fresh and dry weight by 34.4% and 36.1%, respectively on average. Also, BCAs inoculation significantly increased plant yield by 66%. Mean effect sizes for genus Pseudomonas sp. as BCAs were higher than for genus Bacillus spp. Among antagonists tested, P. fluorescens, P. putida, B. cereus, B. subtilis and B. amyloliquefaciens were found to be more effective in general for disease reduction. Across studies, highest disease control was found for P. fluorescens, annual plants, co-inoculation with more than one BCA, soil drench and greenhouse condition were found to be essential in understanding plant responses to R. solanacearum. Our results suggest that more efforts should be devoted to harnessing the potential beneficial effects of these antagonists, not just for plant growth promoting traits but also in mode of applications, BCAs formulations and their field studies should be considered in the future for R. solanacearum wilt disease suppression.

Efficacy of formalin-killed Pseudomonas anguilliseptica vaccine on immune gene expression and protection in farmed olive flounder, Paralichthys olivaceus.

Formalin killed Pseudomonas anguilliseptica bacterial vaccine was prepared and administered to farm reared olive flounder, Paralichthys olivaceus reared at 17 °C and 20 °C for 4 weeks. Non-vaccinated fishes (n=150) served as positive control. Vaccinated fishes were divided into two groups (n=150 each in replicate). Both the vaccinated and non-vaccinated fishes were challenged intraperitoneally with P. anguilliseptica (1×10(7) CFU ml(-1)) isolates and PBS (negative control). Fishes were sampled from zero hour post injection (hpi) for 28 days (each hour and each day); the mean percent mortality and relative percent survival (RPS) were calculated for the challenged and control groups. The vaccinated fishes had a significant increase in RPS (69 and 89, respectively); the percentage mortality declined from 83±0.6 and 74±0.7 in challenged and control fishes to 25%±0.8% and 8%±0.8% in vaccinated and challenged fish groups, respectively. The immune gene expression assay was analyzed using real-time PCR. Vaccinated fishes registered a significant increase in the expression of TNFR-1, FasL, IRF7, TLR2, IL-1b and CD40 gene transcripts when compared to the control group. The upregulation of these genes along with the increased RPS values suggest that the formalin-killed cells of P. anguilliseptica could play an important role in immunizing olive flounder against P. anguilliseptica.

Dietary effect of Rubus coreanus ethanolic extract on immune gene expression in white leg shrimp, Penaeus vannamei.

The objective of this study was to evaluate the effect of dietary supplementation of a Rubus coreanus ethanolic extract on immunostimulatory response in white leg shrimp Penaeus vannamei. Shrimps with an average initial weight of 0.5 ± 0.04 g were collected and acclimatized for 10 days. Four experimental diets including a control diet, a probiotic diet and 0.25 and 0.5% of R. coreanus ethanolic extract (RcEE) diets were used to feed the shrimps. After 8 weeks of culture, shrimp fed with probiotic and 0.25% RcEE diet had showed significant enhancement in the growth while shrimp fed with 0.5% RcEE diet showed significantly increased expression of immune genes and antioxidant enzymes activities. One week of challenge experiments for all the four diets fed shrimps showed decreased cumulative mortality in the 0.5% RcEE diets fed shrimps, when compared with the probiotic and 0.25% RcEE diet fed shrimp groups. The results indicates that R. coreanus ethanolic extract could be used as a herbal immunostimulant for shrimps to increase its immunity and disease resistance against the bacterial pathogen, Vibrio alginolyticus.

Upregulation of the ß-form of 14-3-3 protein in telencephalon of goldfish (Carassius auratus): its possible role in spatial learning.

In the present study, we observed variations in the expression pattern of proteins isolated from the telencephalon of goldfish (Carassius auratus). The expression of a 28 kDa protein was elevated in the individuals trained in a spatial task when compared with the untrained individuals. The ∼28 kDa protein was analyzed using liquid chromatography and mass spectrometry; further, the data were analyzed using the MASCOT search engine. The analysis showed that the ∼28 kDa protein is a ß form of 14-3-3 protein with 35.1% identity. In addition, the semiquantitative PCR confirmed the variation in the expression of 14-3-3 between the trained and the untrained groups. Subsequently, we examined the effect of upregulation of 14-3-3 (ß) in the neurotransmitters; that is, serotonin (5-hydroxytryptamine, 5-HT) and dopamine (DA). Notably, the level of 5-HT and DA was found to be significantly elevated in the telencephalon of individuals trained in the spatial task than in the untrained individuals. Our results suggest that the spatial learning increases the expression of 14-3-3 (ß), which in turn leads to an increase in the level of 5-HT and DA. The upregulated 5-HT and DA may facilitate synapse formation during spatial learning in a novel environment.