Identification and Biochemical Characterization of Halisulfate 3 and Suvanine as Novel Inhibitors of Hepatitis C Virus NS3 Helicase from a Marine Sponge

Hepatitis C virus (HCV) is an important etiological agent that is responsible for the development of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV nonstructural protein 3 (NS3) helicase is a possible target for novel drug development due to its essential role in viral replication. In this study, we identified halisulfate 3 (hal3) and suvanine as novel NS3 helicase inhibitors, with IC₅₀ values of 4 and 3 µM, respectively, from a marine sponge by screening extracts of marine organisms. Both hal3 and suvanine inhibited the ATPase, RNA binding, and serine protease activities of NS3 helicase with IC₅₀ values of 8, 8, and 14 µM, and 7, 3, and 34 µM, respectively. However, the dengue virus (DENV) NS3 helicase, which shares a catalytic core (consisting mainly of ATPase and RNA binding sites) with HCV NS3 helicase, was not inhibited by hal3 and suvanine, even at concentrations of 100 µM. Therefore, we conclude that hal3 and suvanine specifically inhibit HCV NS3 helicase via an interaction with an allosteric site in NS3 rather than binding to the catalytic core. This led to the inhibition of all NS3 activities, presumably by inducing conformational changes.     

Landscape factors and how they influence whitefly pests in cassava fields across East Africa

Context

African production landscapes are diverse, with multiple cassava cultivars grown in small patches amongst a diversity of other crops. Studies on how diverse smallholder landscapes impact herbivore pest outbreak risk have not been carried out in sub-Saharan Africa.

Objectives

Bemisia tabaci is a cryptic pest species complex that cause damage to cassava through feeding and vectoring plant-virus diseases and are known to reach very high densities in certain contexts. However, the factors driving this phenomenon are unclear.

Methods

Bemisia density data in cassava across a large number of sites representing a geographic gradient across Uganda, Tanzania and Malawi were collected. We tested whether in-field or landscape factors associated with land-use patterns underpinned Bemisia density variability and parasitism.

Results

We found the B. tabaci SSA1 species dominated our study sites, although other species were also common in some cassava fields. Factors associated with the surrounding landscape were unimportant for explaining variability in adult density, but the in-field variables of cassava age and cultivar were very important. The density of nymphs and the parasitism of nymphs was heavily influenced by a diversity of landscape factors surrounding the field, including the size of focal cassava field, and area of cassava in the landscape. However, unlike the trend from many other studies on drivers of natural enemy populations, this pattern was not solely related to the amount of non-crop vegetation, or the diversity of crops grown in the landscape.

Conclusions

Our findings provide management options to reduce whitefly abundance, including describing the characteristics of landscapes with high parasitism. The choice of cassava cultivar by the farmer is critical to reduce whitefly outbreak risk at the landscape-scale.

     

Bacteria associated with golden pompano (<Emphasis Type="Italic">Trachinotus blochii</Emphasis>) broodstock from commercial hatchery in Malaysia with emphasis on their antibiotic and heavy metal resistances

This paper was the first report on pathogenic bacteria isolated from golden pompano (Trachinotus blochii) with the antibiogram, as well as the heavy metal resistance pattern. Golden pompano becomes popular among Malaysian fish farmers due to its high value and demand from local and oversea markets. However, the baseline information on antibiogram of pathogenic bacteria associated with golden pompano is not well established. Therefore, the information from this study may be useful to fish farmers in selecting appropriate antibiotic for treatment and prophylactive purpose in golden pompano culture. Isolation of bacterial isolates was carried out using 5% of Horse Blood agar, Tryptic Soy Agar (TSA), Mac Conkey, Thiosulphate Citrate Bile Salt (TCBS), Eosin Methylene Blue (EMB), Glutamate Starch Pseudomonas (GSP), Xylose Lysine Deoxycholate (XLD), and Baird Parker media. The bacterial isolates were then identified using conventional biochemical tests and confirmed by commercial bacterial identification kit. Antibiotic susceptibility test of bacterial isolates against 21 antibiotics (oxolinic acid 2 μg, ampicillin 10 μg, erythromycin 15 μg, furazolidone 15 μg, lincomycin 15 μg, oleandomycin 15 μg, amoxicillin 25 μg, colistin sulphate 25 μg, sulpha-methoxazole 25 μg, chloramphenicol 30 μg, doxycycline 30 μg, florfenicol 30 μg, flumequine 30 μg, kanamycin 30 μg, nalidixic acid 30 μg, novobiocin 30 μg, oxytetracycline 30 μg, tetracycline 30 μg, nitrofurantoin 50 μg, fosfomycin 50 μg, and spiramycin 100 μg) was determined using disk diffusion method, whereas the heavy metal resistance pattern (mercury Hg²⁺, cadmium Cd²⁺, chromium Cr^{6 +}, and copper Cu²⁺) of the bacterial isolates was characterized using two-fold agar dilution method. Five bacterial species were successfully found from 50 diseased golden pompano. They were Streptococcus spp. (n = 12), Escherichia coli (n = 30), Salmonella spp. (n = 20), Pseudomonas spp. (n = 36), and Vibrio spp. (n = 50). More than 80% of the bacterial isolates were found sensitive to 11 out of 21 antibiotics (tetracycline, nitrofurantoin, florfenicol, chloramphenicol, oxytetracycline, nalidixic acid, doxycycline, furazolidone, flumequine, fosfomycin, and oxolinic acid. However, all bacterial isolates were found resistant to all tested heavy metals except for copper and cadmium. The multiple antibiotic resistance (MAR) index of the present study indicated that the fish samples were under high risk exposure to the tested antibiotics. Florfenicol is suggested to be used as antimicrobial agent for golden pompano culture since all bacterial isolates were sensitive to it.     

Improvement of the Culture Conditions for In Vitro Production of Cattle Embryos in a Portable CO2 Incubator

The effects of different concentrations of growth hormone (GH) on in vitro maturation (IVM), fertilization (IVF) and culture (IVC) of bovine oocyte/embryos in CR1aa or CR2aa media using a simple CO₂ incubator were investigated. The IVM/IVF/IVC of oocytes were carried out in the presence of 0, 50, 100 and 200 ng/ml GH in the medium. The proportion of metaphase II oocytes was significantly higher (p < 0.05) in 200 ng/ml compared with 0 ng/ml GH in CR1aa medium (59 versus 85%, respectively), but this effect was not observed under CR2aa. Higher concentrations of GH yielded lower rates of unfertilized ova and thus superior cleavage rates (36.5 ± 0.2 and 63.5 ± 2.0% versus 17.5 ± 0.2 and 82.5 ± 1.5% or 40.4 ± 0.6 and 59.6 ± 1.4% versus 16.6 ± 1.2 and 83.4 ± 6.2% for 0 and 200 ng/ml GH in portable or ordinary incubator, respectively) in CR1aa. This dose‐dependent effect was also observed in the percentages of transferable embryos, although not statistically different (17.2 ± 1.7 versus 27.3 ± 1.8% and 16.6 ± 3.1 versus 26.0 ± 1.4%, for 0 versus 200 ng/ml GH in portable and ordinary incubator, respectively). In contrast to the CR1aa, different concentrations of GH in CR2aa medium did not increase either fertilization or cleavage rates. In fact, higher concentrations of GH in this medium negatively affected the rate of transferable embryos. Hence, percentages of transferable embryos obtained in the portable incubator under 0 or 50 ng/ml GH were higher (p < 0.05) compared with those obtained in 100 or 200 ng/ml GH (35.4 ± 5.7 or 40.5 ± 5.4% versus 22.4 ± 2.4 or 15.5 ± 2.1%, respectively). There was however, no significant difference in the rate of transferable embryos in an ordinary incubator employing CR2aa medium, but the trend was more or less similar to that observed in the portable incubator. Despite the fact that relatively fewer oocytes were employed for the culture in the ordinary incubator, overall results observed employing the simple portable CO₂ incubator were within the range of those obtained in an ordinary incubator; implying that the simple portable incubator can effectively be employed for the in vitro production of bovine embryos under field conditions.     

Fasciola hepatica: comparison of flukes from Korea and the United States by isoelectric focusing banding patterns of whole-body protein.

Comparisons were made between the flukes from Chonnam, Korea and Oregon, USA by isoelectric focusing (IEF) of whole-body protein. Adult Fasciola hepatica were recovered from bile ducts of Korean native cattle. Whole-body protein of the flukes was subjected to IEF, and the banding patterns of the fluke protein were compared with those of North American F. hepatica recovered from experimentally infected calves. The overall banding pattern of F. hepatica from Korea was essentially identical to that of F. hepatica from the United States. These results provide further support for the usefulness of this technique in differentiating Fasciola species in other geographical areas.     

Did transmission of Helicobacter pylori from humans cause a disease outbreak in a colony of Stripe-faced Dunnarts (Sminthopsis macroura)?

ABSTRACT: Since the discovery that Helicobacter pylori causes a range of pathologies in the stomachs of infected humans, it has become apparent that Helicobacters are found in a diverse range of animal species where they are frequently associated with disease. In 2003 and 2004, there were two outbreaks of increased mortality associated with gastric bleeding and weight-loss in a captive colony of the Australian marsupial, the Stripe-faced Dunnart (Sminthopsis macroura). The presence of gastric pathology led to an investigation of potential Helicobacter pathogenesis in these animals. Histological examination revealed the presence of gastritis, and PCR analysis confirmed the presence of Helicobacter infection in the stomachs of these marsupials. Surprisingly, sequencing of 16S rRNA from these bacteria identified the species as H. pylori and PCR confirmed the strain to be positive for the important pathogenesis factor, cagA. We therefore describe, for the first time, an apparent reverse zoonotic infection of Stripe-faced Dunnarts with H. pylori. Already prone to pathological effects of stress (as experienced during breeding season), concomitant H. pylori infection appears to be a possible essential but not sufficient co-factor in prototypic gastric bleeding and weight loss in these marsupials. The Stripe-faced Dunnart could represent a new model for investigating Helicobacter-driven gastric pathology. Infections from their human handlers, specifically of H. pylori, may be a potential risk to captive colonies of marsupials.     

Comparative observation of freeze–thaw‐induced damage in pig,rabbit, and human corneal stroma

Objective Although variations exist between species with respect to outcomes after cryopreservation, little is known about the differences in the susceptibility of the corneal stroma to cryoinjury. We performed this study to investigate freeze–thaw‐induced damage in keratocytes and collagen in rabbit, pig, and human corneas. Animals studied Rabbit, pig, and human. Procedures We prepared 250‐μm‐thick anterior stroma from rabbit, pig, and human corneas after scraping off the epithelium and endothelium. Each 250‐μm‐thick corneal stroma without epithelium was placed in a 50‐mL tube, frozen with liquid N₂ for 15 min and taken out to thaw rapidly at 37 °C. This procedure of rapid freezing and thawing was repeated three times. Differences between the species with respect to cells and collagen structures were examined using hematoxylin–eosin (H&E) staining, terminal deoxynucleotidyl transferase‐mediated nick end labeling (TUNEL) assay, and transmission electron microscopy (TEM). We orthotopically transplanted the pig and rabbit corneal transplants after the triple freeze–thaw cycle into rabbit eyes and evaluated graft survival. Results On gross examination, rabbit corneas became opaque after the triple freeze–thaw procedure, while pig and human corneas remained transparent. Histologically, keratocytes were apoptotic on TUNEL assay and TEM in rabbit, pig, and human corneas. Collagen fibrils were fragmented and the arrangement of collagen fibrils was severely disturbed in rabbit corneas on H&E staining and TEM; collagen was well preserved in pig and human corneas. Rabbit corneal stroma underwent autolysis after transplantation, whereas the pig corneal stroma remained clear for 1 month. Conclusions Our study showed that rabbit corneal stroma was more susceptible to freeze–thaw injury than pig and human corneas.     

Frond-bud propagation of Asplenium nidus L.

Asplenium nidus, a common epiphytic fern, has a short, erect rhizome and a rosette of simple fronds. Propagation is by spores as the plant does not reproduce vegetatively. Plants can be divided into two, four or even eight segments and potted individually to give these numbers of plantlets. A more productive method is described whereby individual fronds are dissected, each with a small piece of stem attached; the pieces are then planted in vermiculite. Up to 33 plantlets per plant and 13 plantlets per frond piece were produced by this method, which is easy and rapid. Sufficient plants are available to meet the limited demands for A. nidus, but the method is useful for multiplying some of the available and attractive cultivars.