Optimization protocol for the extraction of antioxidant components from Origanum vulgare leaves using response surface methodology

In the present work, the response surface methodology (RSM) based on a central composite rotatable design (CCRD), was used to determine optimum conditions for the extraction of antioxidant compounds from Origanum vulgare leaves. Four process variables were evaluated at three levels (31 experimental designs): methanol (70%, 80%, and 90%), the solute:solvent ratio (1:5, 1:12.5, 1:20), the extraction time (4, 10, 16 h), and the solute particle size (20, 65, 110 micron). Using RSM, a quadratic polynomial equation was obtained by multiple regression analysis for predicting optimization of the extraction protocol. Analysis of variance (ANOVA) was applied and the significant effect of the factors and their interactions were tested at 95% confidence interval. The antioxidant extract (AE) yield was significantly influenced by solvent composition, solute to solvent ratio, and time. The maximum AE was obtained at methanol (70%), liquid solid ratio (20), time (16 h), and particle size (20 micron). Predicted values thus obtained were closer to the experimental value indicating suitability of the model. Run 25 (methanol:water 70:30; solute:solvent 1:20; extraction time 16 h and solute particle size 20) showed highest TP contents (18.75 mg/g of dry material, measured as gallic acid equivalents) and DPPH radical scavenging activity (IC₅₀ 5.04 μg/mL). Results of the present study indicated good correlation between TP contents and DPPH radical scavenging activity. Results of the study indicated that phenolic compounds are powerful scavengers of free radical as demonstrated by a good correlation between TP contents and DPPH radical scavenging activity.     

In vitro investigations on extracellular proteins secreted by <Emphasis Type="Italic">Aphanomyces invadans</Emphasis>, the causative agent of epizootic ulcerative syndrome

Background

Proteases produced by many microorganisms, including oomycetes, are crucial for their growth and development. They may also play a critical role in disease manifestation. Epizootic ulcerative syndrome is one of the most destructive fish diseases known. It is caused by the oomycete Aphanomyces invadans and leads to mass mortalities of cultured and wild fish in many countries. The areas of concern are Australia, China, Japan, South and Southeast Asian countries and the USA. Extracellular proteases produced by this oomycete are believed to trigger EUS pathogenesis in fish. To address this activity, we collected the extracellular products (ECP) of A. invadans and identified the secreted proteins using SDS-PAGE and mass spectrometery. A. invadans was cultivated in liquid Glucose-Peptone-Yeats media. The culture media was ultra-filtered through 10 kDa filters and analysed using SDS-PAGE. Three prominent protein bands from the SDS gel were excised and identified by mass spectrometery. Furthermore, we assessed their proteolytic effect on casein and immunoglobulin M (IgM) of rainbow trout (Oncorhynchus mykiss) and giant gourami (Osphronemus goramy). Antiprotease activity of the fish serum was also investigated.

Results

BLASTp analysis revealed that the prominent secreted proteins were proteases, mainly of the serine and cysteine types. Proteins containing fascin-like domain and bromodomain were also identified. We could demonstrate that the secreted proteases showed proteolytic activity against the casein and the IgM of both fish species. The anti-protease activity experiment showed that the percent inhibition of the common carp serum was 94.2% while that of rainbow trout and giant gourami serum was 7.7 and 12.9%, respectively.

Conclusions

The identified proteases, especially serine proteases, could be the potential virulence factors in A. invadans and, hence, are candidates for further functional and host–pathogen interaction studies. The role of identified structural proteins in A. invadans also needs to be investigated further.

     

Quantitative phase imaging for medical diagnosis

Optical microscopy is an indispensable diagnostic tool in modern healthcare. As a prime example, pathologists rely exclusively on light microscopy to investigate tissue morphology in order to make a diagnosis. While advances in light microscopy and contrast markers allow pathologists to visualize cells and tissues in unprecedented detail, the interpretation of these images remains largely subjective, leading to inter‐ and intra‐observer discrepancy. Furthermore, conventional microscopy images capture qualitative information which makes it difficult to automate the process, reducing the throughput achievable in the diagnostic workflow. Quantitative Phase Imaging (QPI) techniques have been advanced in recent years to address these two challenges. By quantifying physical parameters of cells and tissues, these systems remove subjectivity from the disease diagnosis process and allow for easier automation to increase throughput. In addition to providing quantitative information, QPI systems are also label‐free and can be easily assimilated into the current diagnostic workflow in the clinic. In this paper we review the advances made in disease diagnosis by QPI techniques. We focus on the areas of hematological diagnosis and cancer pathology, which are the areas where most significant advances have been made to date.

[Image adapted from Y. Park, M. Diez‐Silva, G. Popescu, G. Lykotrafitis, W. Choi, M. S. Feld, and S. Suresh, Proc. Natl. Acad. Sci. 105, 13730–13735 (2008).]     

A longitudinal two-year survey of the prevalence of trypanosomes in domestic cattle in Ghana by massively parallel sequencing of barcoded amplicons

BackgroundAnimal African Trypanosomiasis (AAT) is one of the most economically important diseases affecting livestock productivity in sub-Saharan Africa. The disease is caused by a broad range of Trypanosoma spp., infecting both wild and domesticated animals through cyclical and mechanical transmission. This study aimed to characterize trypanosomes present in cattle at regular intervals over two years in an AAT endemic and a non-endemic region of Ghana.Methodology/Principal findingsGroups of cattle at Accra and Adidome were selected based on their geographical location, tsetse fly density, prevalence of trypanosomiasis and the breed of cattle available. Blood for DNA extraction was collected at approximately four to five-week intervals over a two-year period. Trypanosome DNA were detected by a sensitive nested PCR targeting the tubulin gene array and massively parallel sequencing of barcoded amplicons. Analysis of the data was a semi-quantitative estimation of infection levels using read counts obtained from the sequencing as a proxy for infection levels. Majority of the cattle were infected with multiple species most of the time [190/259 (73%) at Adidome and 191/324 (59%) at Accra], with T. vivax being the most abundant. The level of infection and in particular T. vivax, was higher in Adidome, the location with a high density of tsetse flies. The infection level varied over the time course, the timings of this variation were not consistent and in Adidome it appeared to be independent of prophylactic treatment for trypanosome infection. Effect of gender or breed on infection levels was insignificant.Conclusions/SignificanceMost cattle were infected with low levels of several trypanosome species at both study sites, with T. vivax being the most abundant. The measurements of infection over time provided insight to the importance of the approach in identifying cattle that could suppress trypanosome infection over an extended time and may serve as reservoir.     

Deciphering allelic variability and population structure in buckwheat: An analogy between the efficiency of ISSR and SSR markers

Food and nutritional security continue to be the issues of concern in developing countries like ours. Exploring the reservoir of high potential unexplored genetic resources could address the world’s food and nutritional insecurity. The availability of diverse data and the population structure of any crop germplasm is a valuable genetic resource for discovering genes that can help achieve food and nutritional stability. We used seven ISSR and seven SSR markers to investigate diversity among 63 buckwheat genotypes, including landraces from India''s northwestern Himalayas. Various parameters such as percent polymorphism, PIC, resolving power, and marker index was used to evaluate the inequitable efficacy of these markers. We foundthat both marker systems are effective in detecting polymorphism in buckwheat germplasm. Seven ISSRs produced 55 polymorphic bands, while seven SSRs produced 32bands. When compared to ISSRs, SSRs had a greater average PIC value (0.43) than that of (0.36). ISSRs, on the other hand, had a resolving power of (4.38) compared to (1.42) for SSRs. The hierarchical cluster analysis dendrogram divided genotypes into three major clusters. We found that both marker systems were equally accurate in grouping buckwheat genotypes according to their geographical origins. Using 7 ISSR and 7 SSR markers, the model-based STRUCTURE analysis established a population with two sub-populations that correspond to species-based groupings. Within the population, there was a high level of genetic diversity. These results have consequences for both buckwheat breeding and conservation efforts.Keyword: Buckwheat, SSR, ISSR, Genetic diversity, Population structure     

Daily number of bee louse (Braula coeca) in honey bee (Apis mellifera carnica and A. m. syriaca) colonies maintained under semi‐arid conditions

Experimental work was conducted at two apiaries located in Irbid district and in Shuna North, Jordan, during the years 2004–2006. The aims of these investigations were to estimate the seasonal changes in the infestation rates of the bee louse (Braula sp.) and to develop an easy and rapid method of estimating the infestation rate on workers with bee Braula. Two major honey bee subspecies are reared in Jordan; Apis mellifera carnica and Apis mellifera syriaca were used in this study. The results showed that the infestation rate began to increase rapidly in May, reaching the season's maximum rate of 16.2%, 15.8% and 17.4% for A. m. carnica and 22.6%, 23.9% and 22.9% for A. m. syriaca in December of 2004, 2005 and 2006, respectively. The maximum adult numbers of bees were found in April and June, whereas the minimum for the year was in January in both honey bee subspecies colonies during the study period. The actual population of the bee louse could be estimated by counting the daily dropped lice and multiplying by a factor of 158. This factor is valid for the experimental colonies of both subspecies kept for 3 years under semi‐arid Mediterranean conditions.     

Stimulation of food intake following opioid microinjection into the nucleus accumbens septi in rats

The involvement of opioid peptides in the regulation of food intake has been postulated. However, it is not known how they are involved in this regulation and which brain region is responsible for the mediation of their effects. We studied the effect of a microinjection of opioid agonists and antagonists into the nucleus accumbens septi (NAS) on the food intake in rats, as this area is known to be important for motivation. Male Wistar rats were implanted stereotaxically with guide cannulae. Rats were not allowed food prior to drug treatment and solutions (1 microliter) were microinjected bilaterally. Food intake was measured throughout a 2 hr period after the drug injection. Infusions into the NAS of 2, 5 and 10 nmol of morphine, D-ala2, D-Leu5-enkephalin (DADLE), and beta-endorphin (beta E), or of 5 and 10 nmol of alpha-neoendorphin (ANEO) induced a dose-dependent increase in the food intake. Dynorphin (DYN) also increased the food intake, but only at a 10 nmol dose. The new, highly selective delta agonist D-Pen2,5-enkephalin (DPDPE) induced a dose-dependent increase in the food intake. Naloxone in doses of 2 and 10 nmol antagonized the increased food intake induced by morphine, beta E, ANEO and DYN in a dose-dependent manner, but only partly antagonized the effect of DADLE on the food intake. The selective mu-receptor antagonist beta-funaltrexamine (beta-FNA), in a dose of 5 nmol completely blocked the increase in the food intake induced by morphine but not by DADLE.(ABSTRACT TRUNCATED AT 250 WORDS)