Chilean IPNV isolates: Robustness analysis of PCR detection

Background: The genomes of several infectious pancreatic necrosis viruses (IPNVs) isolated in Chile were sequenced with a single amplification approach for both segments A and B. The resulting sequences were then used to determine the conservation of the primer-binding regions used in polymerase chain reaction (PCR)-based diagnostic methods proposed in the literature. Thus, the robustness of each technique was studied, particularly the eventual effect of further mutations within the primer-binding sites. Results: On analysis, most methods currently used to detect Chilean IPNV varieties were deemed adequate. However, the primers were designed to be genogroup specific, implying that most detection methods pose some risk of detecting all strains prevalent in the country, due to the coexistence of genogroups 1 and 5. Conclusions: Negative resultsmust be interpreted carefully given the high genomic variability of IPNVs. Detection techniques (quantitative reverse transcription (qRT)-PCR) based on degenerate primers can be used to minimize the possibilities of false-negative detections.

Cloning of the ω-secalin gene family in a wheat 1BL/1RS translocation line using BAC clone sequencing

Background: Wheat 1BL/1RS translocation lines are planted around the world for their disease resistance and high yield. Most of them are poor in bread making, which is partially caused by ω-secalins that are encoded by the ω-secalin gene family, which is located on the short arm of rye chromosome 1R (1RS). However, information on the structure and evolution of the ω-secalin gene family is still limited. Results: We first generated a physicalmap of the ω-secalin gene family covering 195 kb of the Sec-1 locus based on sequencing three bacterial artificial chromosome (BAC) clones of the 1BL/1RS translocation wheat cultivar Shimai 15. A BAC contig was constructed spanning 168 kb of the Sec-1 locus on 1RS. Twelve ω-secalin genes were arranged in a head-to-tail fashion, separated by 8.2–21.6 kb spacers on the contig, whereas six other ω-secalin genes were arranged head-to-tail, separated by 8.2–8.4 kb of spacers on clone BAC125. The 18 ω-secalin genes can be classified into six types among which eight ω-secalin genes were expressed during seed development. The ω-secalin genes with the 1074-bp open reading frame (ORF) represented the main population. Except for two pseudogenes, the N-terminal of the ω-secalin gene was conserved, whereas variations in the C-terminal led to a change in ORF length. The spacers can be sorted into two classes. Class-1 spacers contained conserved and non-conservative sequences. Conclusion: The ω-secalin gene family consisted of at least 18 members in the 1BL/1RS translocation line cv. Shimai 15. Eight ω-secalin genes were expressed during seed development. Eighteen members may originate from a progenitor with a 1,074-bp ORF. The spacers differed in length and sequence conservation.

Comparing the expression of human DNA topoisomerase I in KM71H and X33 strains of Pichia pastoris

Background: Human is an essential cellular enzyme that is found in all human cells. As this enzyme is upregulated in cancer cells exceedingly, it is used as a target for cancer chemotherapeutic drug development. As such, producing the in-house enzyme for the purpose to speed up the search for more cost-effective and target specific hTopoI inhibitors is warranted. This study aims to compare the optimised conditions for the expression of hTopoI in KM71H (MutS) and X33 (Mut+) strains of Pichia pastoris P. pastoris transfected with an hTopoI recombinant vector was used for the optimization of a higher level of hTopoI expression. Results: In the process, fed-batch cultivation parameters that influence the expression of hTopoI, such as culture temperature, methanol induction and feeding strategy, were optimised in the transfected KM71H and X33 P. pastoris strains in a shake flask system. The cell density and total protein concentration (protein level) of transfected P. pastoris were compared to determine the optimum culture conditions for each transfected P. pastoris strain. A higher hTopoI level was observed in the transfected KM71H culture supernatant (2.26 ng/mL) when the culture was incubated in the optimum conditions. Conclusions: This study demonstrated that MutS strain (KM71H) expressed and secreted a higher level of hTopoI heterologous protein in the presence of methanol compared to the Mut+ strain; X33 (0.75 ng/mL). However, other aspects of optimization, such as pH, should also be considered in the future, to obtain the optimum expression level of hTopoI in P. pastoris.

Development, characterization and use of genomic SSR markers for assessment of genetic diversity in some Saudi date palm ( Phoenix dactylifera L.) cultivars

Background: The present study was undertaken towards the development of SSR markers and assessing genetic relationships among 32 date palm ( Phoenix dactylifera L.) representing common cultivars grown in different geographical regions in Saudi Arabia. Results: Ninety-three novel simple sequence repeat markers were developed and screened for their ability to detect polymorphism in date palm. Around 71% of genomic SSRs were dinucleotide, 25% tri, 3% tetra and 1% penta nucleotide motives. Twenty-two primers generated a total of 91 alleles with a mean of 4.14 alleles per locus and 100% polymorphism percentage. A 0.595 average polymorphic information content and 0.662 primer discrimination power values were recorded. The expected and observed heterozygosities were 0.676 and 0.763 respectively. Pair-wise similarity values ranged from 0.06 to 0.89 and the overall cultivars averaged 0.41. The UPGMA cluster analysis recovered by principal coordinate analysis illustrated that cultivars tend to group according to their class of maturity, region of cultivation, and fruit color. Analysis of molecular variations (AMOVA) revealed that genetic variation among and within cultivars were 27% and 73%, respectively according to geographical distribution of cultivars. Conclusions: The developed microsatellite markers are additional values to date palm characterization tools that can be used by researchers in population genetics, cultivar identification as well as genetic resource exploration and management. The tested cultivars exhibited a significant amount of genetic diversity and could be suitable for successful breeding program. Genomic sequences generated from this study are available at the National Center for Biotechnology Information (NCBI), Sequence Read Archive (Accession numbers. LIBGSS_039019).

The whole-cell immobilization of D-hydantoinase-engineered Escherichia coli for D-CpHPG biosynthesis

Background: D-Hydroxyphenylglycine is considered to be an important chiral molecular building-block of antibiotic reagents such as pesticides, and β-lactam antibiotics. The process of its production is catalyzed by D-hydantoinase and D-carbamoylase in a two-step enzyme reaction. How to enhance the catalytic potential of the two enzymes is valuable for industrial application. In this investigation, an Escherichia coli strain genetically engineered with D-hydantoinase was immobilized by calcium alginate with certain adjuncts to evaluate the optimal condition for the biosynthesis of D-carbamoyl-p-hydroxyphenylglycine (D-CpHPG), the compound further be converted to D-hydroxyphenylglycine (D-HPG) by carbamoylase. Result: The optimal medium to produce D-CpHPG by whole-cell immobilization was a modified Luria-Bertani (LB) added with 3.0% (W/V) alginate, 1.5% (W/V) diatomite, 0.05% (W/V) CaCl2 and 1.00 mM MnCl2. The optimized diameter of immobilized beads for the whole-cell biosynthesis here was 2.60 mm. The maximized production rates of D-CpHPG were up to 76%, and the immobilized beads could be reused for 12 batches. Conclusions: This investigation not only provides an effective procedure for biological production of D-CpHPG, but gives an insight into the whole-cell immobilization technology. © 2016 Pontificia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved.

Viability of probiotic bacteria and some chemical and sensory characteristics in cornelian cherry juice during cold storage

Background: Increased popularity of vegetarianism, lactose intolerance, and the high cholesterol content in dairy products, are all factors that have recently increased the demand for nondairy probiotic products. The objective of this study is to evaluate the effect of refrigeration on the viability of probiotics and asses someof the chemical and sensory characteristics in cornelian cherry juice. Results: The Iranian native probiotic strain (L. casei T4) showed greater viability compared to industrial types (viable count of 8.67 log cfu/mL versus <6.0 log cfu/mL at d 28). However, this most tolerant Iranian strain, could not withstand the conditions of ‘Natural juice’ at pH 2.6 for more than 7 d. Following a pH adjusted treatment (to pH ~3.5), the viability of the strain was improved to 28 d with some evidence of increased growth of the probiotic. However, the level of antioxidant activity, anthocyanin and phenolic compounds, revealed a slight decrease during cold storage. The changes in the chemical profile of the sample containing L. casei T4 indicated fermentation activity during cold storage. Sensory evaluation results showed significant differences between samples containing L. casei TD4 and other samples in taste, odor and overall acceptance in a complimentary way. Conclusions: The results showed that low pH and presence of inhibitor phenolic compounds of cornelian cherry juice have negative effect on viability of probiotics, especially industrial strains during refrigerated storage.

Oligomerization of Cry9Aa in solution without receptor binding, is not related with insecticidal activity

Background: Bacillus thuringiensis Cry toxins bind with different insect midgut proteins leading to toxin oligomerization, membrane insertion and pore formation. However, different Cry toxins had been shown to readily form high molecular weight oligomers or aggregates in solution in the absence of receptor interaction. The role of Cry oligomers formed in solution remains uncertain. The Cry9A proteins show high toxicity against different Lepidoptera, and no-cross resistance with Cry1A. Results: Cry9Aa655 protein formed oligomers easily in solution mediated by disulfide bonds, according to SDS-PAGE analysis under non-reducing and reducing conditions. However, oligomerization is not observed if Cry9Aa655 is activated with trypsin, suggesting that cysteine residues, C14 and C16, located in the N-terminal end that is processed during activation participate in this oligomerization. To determine the role of these residues on oligomerization and in toxicity single and double alanine substitution were constructed. In contrast to single C14A and C16A mutants, the double C14A–C16A mutant did not form oligomers in solution. Toxicity assays against Plutella xylostella showed that the C14A–C16A mutant had a similar insecticidal activity as the Cry9Aa655 protein indicating the oligomers of Cry9Aa formed in solution in the absence of receptor binding are not related with toxicity. Conclusions: The aggregation of Cry9Aa655 polypeptides was mediated by disulfide bonds. Cry9Aa655 C14 and C16C are involved in oligomerization in solution. These aggregate forms are not related to the mode of action of Cry9Aa leading to toxicity.

Indole acetic acid and ACC deaminase fromendophytic bacteria improves the growth of Solanum lycopersicum

Background: Endophytic bacteria are ubiquitous in all plant species contributing in host plant\'s nutrient uptake and helping the host to improve its growth. Moringa peregrina which is a medicinal plant, growing in arid region of Arabia, was assessed for the presence of endophytic bacterial strains. Results: PCR amplification and sequencing of 16S rRNA of bacterial endophytes revealed the 5 endophytic bacteria, in which 2 strains were from Sphingomonas sp.; 2 strains from Bacillus sp. and 1 from Methylobacterium genus. Among the endophytic bacterial strains, a strain of Bacillus subtilis LK14 has shown significant prospects in phosphate solubilization (clearing zone of 56.71 mm after 5 d), ACC deaminase (448.3 ± 2.91 nM α-ketobutyrate mg-1 h-1) and acid phosphatase activity (8.4 ± 1.2 nM mg-1 min-1). The endophytic bacteria were also assessed for their potential to produce indole-3-acetic acid (IAA). Among isolated strains, the initial spectrophotometry analysis showed significantly higher IAA production by Bacillus subtilis LK14. The diurnal production of IAA was quantified using multiple reactions monitoring method in UPLC/MS–MS. The analysis showed that LK14 produced the highest (8.7 μM) IAA on 14th d of growth. Looking at LK14 potentials, it was applied to Solanum lycopersicum , where it significantly increased the shoot and root biomass and chlorophyll (a and b) contents as compared to control plants. Conclusion: The study concludes that using endophytic bacterial strains can be bio-prospective for plant growth promotion, which might be an ideal strategy for improving growth of crops in marginal lands.

SSR genetic diversity assessment of popular pigeonpea varieties in Malawi reveals unique fingerprints

Background: Pigeonpea ( Cajanus cajan L. Millsp.) is a drought tolerant legume of the Fabaceae family and the only cultivated species in the genus Cajanus. It is mainly cultivated in the semi-arid tropics of Asia and Oceania, Africa and America. In Malawi, it is grown as a source of food and income and for soil improvement in intercropping systems. However, varietal contamination due to natural outcrossing causes significant quality reduction and yield losses. In this study, 48 polymorphic SSR markers were used to assess the diversity among all pigeonpea varieties cultivated in Malawi to determine if a genetic fingerprint could be identified to distinguish the popular varieties. Results: A total of 212 alleles were observed with an average of 5.58 alleles per marker and a maximum of 14 alleles produced by CCttc019 (Marker 40). Polymorphic information content (PIC), ranged from 0.03 to 0.89 with an average of 0.30. A neighbor-joining tree produced 4 clusters. The most commonly cultivated varieties, which include released varieties and cultivated land races, were well-spread across all the clusters observed, indicating that they generally represented the genetic diversity available in Malawi, although substantial variation was evident that can still be exploited through further breeding. Conclusion: Screening of the allelic data associated with the five most popular cultivated varieties, revealed 6 markers – CCB1, CCB7, Ccac035, CCttc003, Ccac026 and CCttc019 – which displayed unique allelic profiles for each of the five varieties. This genetic fingerprint can potentially be applied for seed certification to confirm the genetic purity of seeds that are delivered to Malawi farmers.

Selection of polyvalent bacteriophages infecting Salmonella enterica serovar Choleraesuis

Background: Ideally, bacteriophages of pathogenic bacterial hosts should be polyvalent to be able to replicate in an alternative nonpathogenic bacterium. Thus, accidental infection by the original host can be avoided when bacteriophage lysates are used in biocontrol protocols. Results: From 15 wastewater samples, collected at different sites in the V Region in Chile, we selected three bacteriophages (FC, FP, and FQ) capable of productively infecting Salmonella enterica serovar Choleraesuis. By transmission electron microscopy (TEM) observation, the bacteriophages were found to belong to the order Caudoviridae. Molecular analyses indicated that FC, FP, and FQ contained double-stranded DNA genomes, of sizes similar to bacteriophage P22, and distinct recognition sites for the restriction endonucleases HaeIII and HindIII. Assays of host range revealed that the bacteriophages were polyvalent and thus capable of infecting different strains of Escherichia coli and other serovars of Salmonella . Conclusion: We have isolated newbacteriophages of the serovar Choleraesuiswith various potential applications in relation to this pathogenic bacterium.

Thermotolerant fermenting yeasts for simultaneous saccharification fermentation of lignocellulosic biomass

Lignocellulosic biomass is the most abundant renewable source of energy that has been widely explored as second-generation biofuel feedstock. Despite more than four decades of research, the process of ethanol production from lignocellulosic (LC) biomass remains economically unfeasible. This is due to the high cost of enzymes, end-product inhibition of enzymes, and the need for cost-intensive inputs associated with a separate hydrolysis and fermentation (SHF) process. Thermotolerant yeast strains that can undergo fermentation at temperatures above 40°C are suitable alternatives for developing the simultaneous saccharification and fermentation (SSF) process to overcome the limitations of SHF. This review describes the various approaches to screen and develop thermotolerant yeasts via genetic and metabolic engineering. The advantages and limitations of SSF at high temperatures are also discussed. A critical insight into the effect of high temperatures on yeast morphology and physiology is also included. This can improve our understanding of the development of thermotolerant yeast amenable to the SSF process to make LC ethanol production commercially viable.

The impact of household size on poverty: An analysis of various low-income townships in the Northern Free State region, South Africa

Poverty is a multi-dimensional socio-economic problem in most sub-Saharan African countries. The purpose of this study is to analyse the relationship between household size and poverty in low-income communities. The Northern Free State region in South Africa was selected as the study region. A sample of approximately 2 900 households was randomly selected within 12 poor communities in the region. A poverty line was calculated and 74% of all households were found to live below the poverty line. The Pearson’s chi-square test indicated a positive relationship between household size and poverty in eleven of the twelve low-income communities. Households below the poverty line presented larger households than those households above the poverty line. This finding is in contradiction with some findings in other African countries due to the fact that South Africa has higher levels of modernisation with less access to land for subsistence farming. Effective provision of basic needs, community facilities and access to assets such as land could assist poor households with better quality of life. Poor households also need to be granted access to economic opportunities, while also receiving adult education regarding financial management and reproductive health.

Examining underlying determinants of fertility rates in Zambia: Evidence from the 2007 Zambia Demographic and Health Survey

Limited data exits on factors influencing fertility in Zambia. This study examined underlying determinants of fertility patterns and levels in Zambia. Data extracted from the 2007 Zambia Demographic and Health Survey was analysed using bivariate and multivariate logistic regression. Of 7146 women aged 15-49 years, age group 25-29 years experienced the highest prevalence of births (28.5%). Married women accounted for 27% of all births. Women with low education recorded more births (27%) than those with higher education (9.5%) (P<0.001). Fertility was higher among the poorest (28%) compared to the richest (12%) (P<0.001). Though not statistically significant, urban areas recorded more births (25%) than rural areas (15%). Education and wealth significantly influence fertility Zambia. Fertility management strategies should consider these factors and their fertility reducing effects. Improving education and wealth status of women can contribute to fertility reduction, particularly rural women. Lower fertility, with reduced mortality and migration, would provide less pressure on distribution of the limited economic resources of the country.

Association of type of birth attendant and place of delivery on infant mortality in sub-Saharan Africa.

Objective: To examine the association between type of birth attendant and place of delivery, and infant mortality (IM). Methods: This cross-sectional study used self-reported data from the Demographic Health Surveys for women in Ghana, Kenya, and Sierra Leone. Logistic regression estimated odds ratios (ORs) and95% confidence intervals. Results: In Ghana and Sierra Leone, odds of IM were higher for women who delivered at a health facility versus women who delivered at a household residence (OR=3.18, 95% confidence interval, CI: 1.29-7.83, p=0.01 and OR=1.62, 95% CI: 1.15-2.28, p=0.01, respectively). Compared to the use of health professionals, the use of birth attendants for assistance with delivery was not significantly associated with IM for women in Ghana or Sierra Leone (OR=2.17, 95% CI: 0.83-5.69, p=0.12 and OR=1.25, 95% CI: 0.92-1.70, p=0.15, respectively). In Kenya, odds of IM, though nonsignificant, were lower for women who used birth attendants than those who used health professionals to assist with delivery (OR=0.85, 95% CI: 0.51-1.41, p=0.46), and higher with delivery at a health facility versus a household residence (OR=1.29, 95% CI: 0.81-2.03, p=0.28). Conclusions: Women in Ghana and Sierra Leone who delivered at a health facility had statistically significant increased odds of IM. Birth attendant type-IM associations were not statistically significant.Future research should consider culturally-sensitive interventions to improve maternal health and help reduce IM.

Indirect child mortality estimation technique to identify trends of under-five mortality in Ethiopia.

Background: In sub-Saharan African countries, the chance of a child dying before the age of five years is high. The problem is similar in Ethiopia, but it shows a decrease over years. Methods: The 2000; 2005 and 2011 Ethiopian Demographic and Health Survey results were used for this work. The purpose of the study is to detect the pattern of under-five child mortality overtime. Indirect child mortality estimation technique is adapted to examine the under-five child mortality trend in Ethiopia. Results: From the result, it was possible to see the trend of under-five child mortality in Ethiopia. The under-five child mortality shows a decline in Ethiopia. Conclusion: From the study, it can be seen that there is a positive correlation between mother and child survival which is almost certain in any population. Therefore, this study shows the trend of under-five mortality in Ethiopia and decline over time.