Multiplex RT-nested PCR differentiation of gill-associated virus (Australia) from yellow head virus (Thailand) of Penaeus monodon

A multiplex RT-nested PCR has been developed to detect and differentiate the closely related prawn viruses, gill-associated virus (GAV) from Australia and yellow head virus (YHV) from Thailand. RT-PCR using primers to conserved sequences in the ORF1b gene amplified a 794 bp region of either GAV or YHV. Nested PCR using a conserved sense primer and either a GAV- or YHV-specific antisense primer to a divergent sequence differentially amplified a 277 bp region of the primary PCR amplicon. Multiplexing the YHV antisense primer with a GAV antisense primer to another divergent sequence allowed the viruses to be distinguished in a single nested PCR. Nested PCR enhanced detection sensitivity between 100- and 1000-fold and GAV or YHV RNA was detectable in approximately 10 fg lymphoid organ total RNA. The multiplex RT-nested PCR was also able to co-detect GAV and YHV RNA mixed over a wide range of concentrations to simulate potential dual-infection states. The robustness of the test was examined using RNA samples from Penaeus monodon prawns infected either chronically or acutely with GAV or YHV and collected at different locations in Eastern Australia and Thailand between 1994 and 1998. GAV- (406 bp) or YHV-specific (277 bp) amplicons were differentially generated in all cases, including five YHV RNA samples in which no primary RT-PCR amplicon was detected. Sequence analysis of GAV and YHV PCR amplicons identified minor variations in the regions targeted by the virus-specific antisense primers. However, none occurred at positions that critically affected the PCR.     

Effects of plane of nutrition and arginine on ovarian follicles in non-pregnant sheep: Cell proliferation,and expression of endothelial nitric oxide and its receptor

The aim of this study was to investigate the role of the nitric oxide (NO) system in ovarian function, by determining if arginine (Arg) supplementation impacts follicle number, cell proliferation, and expression of the NO system members in nutritionally compromised ewes. Ewes were randomly assigned into maintenance (C, 100% requirements), excess (O; 2xC), or restricted (U; 0.6xC) diets 8 weeks prior to Arg treatment. Ewes were individually fed twice daily with pelleted diets. Ewes from each nutritional group were randomly assigned to one of two treatments: saline or Arg, which was initiated on day 0 of the estrous cycle and administered 3 times per day. Ovaries were collected at the early-luteal, mid-luteal and late-luteal/follicular phases of the estrous cycle to determine 1) the number of surface follicles, 2) follicle cell proliferation marked by Ki67 protein expression, and 3) expression of endothelial nitric oxide (eNOS; NOS3) and soluble guanylyl cyclase beta (sGC; GUCY1B3) protein and mRNA in granulosa (G) and theca (T) layers using immunohistochemistry followed by image analysis and qPCR, respectively. During nutritional treatment, C maintained body weight, O gained 6±1.2 kg, and U lost 14±1.3 kg. Our data show that: 1) Ki67 was expressed in all ovarian compartments, eNOS protein was detected in blood vessels of T and stroma, and sGC protein was detected in T cells, and blood vessels of T layer and other ovarian compartments; 2) plane of nutrition affected the number of surface follicles, and thus folliculogenesis, cell proliferation in the T layer, eNOS and sGC protein expression in T, and NOS3 and GUCY1B3 mRNA expression in G; 3) Arg treatment affected cell proliferation in G and T, eNOS and sGC protein expression in T, mRNA expression of NOS3 in T in all groups, and GUCY1B3 in G depending on the stage of the estrous cycle; and 4) G and T cell proliferation, and expression of eNOS and sGC protein in T was affected by the stage of the estrous cycle. Our data demonstrated that plane of nutrition and Arg are involved in the regulation of follicular functions in non-pregnant sheep.     

In vivo gastric mucosal histopathology using endocytoscopy

AIM:To study the ability of endocytoscopy to identify normal gastric mucosa and to exclude Helicobacter pylori(H.pylori) infection.METHODS:Endocytoscopic examination of the gastric corpus and antrum was performed in 70 consecutive patients.Target biopsy specimens were also obtained from the assessed region and multiple H.pylori tests were performed.The normal endocytoscopy patterns of the corpus and antrum were divided into the normal pit-dominant type(n-Pit) or the normal papilladominant type(n-Pap), respectively characterized as either regular pits with capillary networks or round, smooth papillary structures with spiral capillaries.On the other hand, normal mucosa was defined as mucosa not demonstrating histological abnormalities, including inflammation and atrophy.RESULTS:The sensitivity and specificity of n-Pit for normal mucosa in the gastric corpus were 94.4%and 97.1%,respectively,whereas those of n-Pap for normal mucosa in the antrum were 92.0%and 86.7%,respectively.The positive predictive values of n-Pit and n-Pap for H.pylori-negative tissue were 88.6%and 93.1%,respectively,and their negative predictive values for H.pylori-negative tissues were 42.9%and41.5%,respectively.The inter-observer agreement for determining n-Pit and n-Pap for normal mucosa were0.857 and 0.769,respectively,which is considered reliable.CONCLUSION:N-Pit and n-Pap,seen using EC,are considered useful predictors of normal mucosa and theabsence of H.pylori infection.     

Estrogen receptor alpha (ERα) and progesterone receptor (PR) in the mammary gland of bitches during different stages of estrous cycle

The objective of this study was to evaluate the estrogen receptor alpha (ERα) and progesterone receptor (PR) expressions in normal mammary tissues of bitches during different stages of the estrous cycle. Mammary tissues were collected from five beagle bitches at six different estrous stages for each bitch, which were: anestrus, proestrus, estrus, early diestrus, mid diestrus, and late diestrus. The expressions of ERα and PR were evaluated by avidin–biotin–peroxidase complex (ABC) method, and ERα and PR scores were calculated. The lowest scores of ERα and PR were found in mammary tissue collected during mid diestrus, whereas the ERα and PR scores were significantly higher during estrus and proestrus. A significantly higher score of the PR during anestrus compared to during mid diestrus was also observed, but this was not found for ERα score. In addition, positive correlation between ERα and PR scores were found which indicated that the presence of ERα and PR may be under the same regulatory mechanism. In conclusion, these findings suggested that ovarian steroid hormones, estrogen and progesterone, which differed during the stages of the estrous cycle may have a regulatory role in ERα and PR expression in bitch mammary gland.     

Fractionation and characterization of lignin from sugarcane bagasse using a sulfuric acid catalyzed solvothermal process

Conversion of lignocellulosic residue to bioenergy and biofuel is a promising platform for global sustainability. Fractionation is an initial step for isolating lignocellulosic components for subsequent valorization. The aim of this research is to develop the solvothermal fractionation of sugarcane bagasse to produce high purity lignin. The physio-chemical structure of isolated lignin from this process was determined. In this study, a central composite design-based response surface methodology (RSM) was used to optimize an acid promoter for isolating lignin from sugarcane bagasse using a solvothermal fractionation process. The reaction was carried out with sulfuric acid, at a concentration of 0.01–0.02 M and a reaction temperature of 180–200 °C for 30–90 min. The optimal conditions for the experiment were obtained at the acid concentration of 0.02 M with a temperature of 200 °C for 90 min in methyl isobutyl ketone (MIBK)/methanol/water (35% : 25% : 40% v/v%). The results showed that 88% of lignin removal was done in the solid phase, while 87% of lignin recovery was conducted in the organic phase. Furthermore, the changes in the physico-chemical characteristics of solid residue and lignin recovery were analyzed using various techniques. GPC analysis of recovered lignin from the organic fraction showed a lower M_w (1374 g mol⁻¹) and polydispersity index (1.75) compared to commercial organosolv lignin. The major lignin degradation temperature of commercial organosolv lignin was estimated to be 410 °C, whereas BGL showed two main degradations at 291 °C and 437 °C, which could point to potential relationships with the degradation of β-O-4 cross-links. The results indicated that recovered lignin was mostly cross-linked by β-O-4 cross-links. In addition, Py-GC/MS and 2D HSQC NMR gave more information regarding the compositional and structural features of recovered lignin. The development of the sulfuric acid catalyzed solvothermal process in this study provides efficient extraction of high-value organosolv lignin from sugarcane bagasse and the production of recovered lignin in the organic phase with low contamination from other contents. The lignin characteristic data can contribute to the development of lignin valorization in value-added applications.

Conversion of lignocellulosic residue to bioenergy and biofuel is a promising platform for global sustainability.     

Butea superba Roxb. enhances penile erection in rats

This study aimed to investigate the effects of ethanol extracts of Butea superba in increasing intracavernous pressure (ICP) in vivo. The extracts were prepared from fresh and dried root cores and fresh and dried root barks. Penile erection was induced in aged rats by electrical stimulation of the cavernous nerve. Cavernous smooth muscle relaxation was also observed in vitro in the presence of the extract, cGMP or isobutyl-methylxanthine (IBMX) alone or the extract together with cGMP or IBMX. The dried root core extract from Phrae was the most effective in increasing the ICP. The dose-response relationship study revealed a bell-shape curve with the maximum effective dose at 1 mg/kg. The ICP of the control and 1 mg/kg extract-treated animals were 45.3 +/- 2.5 and 100.9 +/- 14.0 mmHg, respectively. The extract, cGMP and IBMX alone induced dose dependent muscle relaxation. B. superba significantly enhanced the effects of cGMP and IBMX. The results suggest that ethanol extracts of B. superba are effective in enhancing penile erection. The dried root core extract from Phrae is the most effective part with a maximal dose of 1 mg/kg. The results also suggest that B. superba may act through cAMP/cGMP pathways.     

Target for standard Thai PCR assay identical in 12 white spot syndrome virus (WSSV) types that differ in DNA multiple repeat length

A Thai PCR detection method (WSSV-232) yielding a 232 bp amplicon has been used for detection of white spot syndrome virus (WSSV) since 1996. It targets ORF 91 in the full sequence of the only Thai WSSV isolate at GenBank (AF369029). At the beginning of 2002, some Thai shrimp farmers complained that ponds stocked with WSSV-232 PCR negative post-larvae (PL) later suffered WSSV disease outbreaks. Although these outbreaks may have resulted from horizontal transmission of WSSV after stocking, it was also possible that they resulted from false negative PCR test results due to genetic changes at the PCR-assay target after the first appearance of WSSV in Thailand in 1995. Indeed, recent results have revealed at least 12 WSSV variants in Thailand that can be distinguished based on differences in DNA multiple repeat lengths in ORF 94 (GenBank AF369029). To test for variation in the WSSV-232 target sequence in ORF 91, 20 DNA extracts derived from field samples and representing 9 of the WSSV DNA multiple repeat groups were subjected to PCR amplification and sequencing using primers that generated a 403 bp amplicon covering the target for the WSSV-232 assay. An additional three repeat types were included from archived material. Analysis revealed that the 232 bp target sequence in ORF 91 was unchanged in all of the 12 types tested and that the original WSSV-232 detection system was still valid. Thus, any false negative PCR test results leading to farmer complaints would probably have arisen from small sample sizes and low sensitivity of the single-step PCR assay. If so, false negative results could be reduced by the use of nested PCR assays with larger PL sample sizes.