Evaluation of Xpert® MTB/RIF and ustar easyNAT™ TB IAD for diagnosis of tuberculous lymphadenitis of children in Tanzania : a prospective descriptive study

Fine needle aspiration biopsy has become a standard approach for diagnosis of peripheral tuberculous lymphadenitis. The aim of this study was to compare the performance of Xpert MTB/RIF and Ustar EasyNAT TB IAD nucleic acid amplification assays, against acid-fast bacilli microscopy, cytology and mycobacterial culture for the diagnosis of TB lymphadenitis in children from a TB-endemic setting in Tanzania.; Children of 8 weeks to 16 years of age, suspected of having TB lymphadenitis, were recruited at a district hospital in Tanzania. Fine needle aspirates of lymph nodes were analysed using acid-fast bacilli microscopy, liquid TB culture, cytology, Xpert MTB/RIF and EasyNAT. Latent class analysis and comparison against a composite reference standard comprising "culture and/or cytology" was done, to assess the performance of Xpert MTB/RIF and EasyNAT for the diagnosis of TB lymphadenitis.; Seventy-nine children were recruited; 4 were excluded from analysis. Against a composite reference standard of culture and/or cytology, Xpert MTB/RIF and EasyNAT had a sensitivity and specificity of 58 % and 93 %; and 19 % and 100 % respectively. Relative to latent class definitions, cytology had a sensitivity of 100 % and specificity of 94.7 %.; Combining clinical assessment, cytology and Xpert MTB/RIF may allow for a rapid and accurate diagnosis of childhood TB lymphadenitis. Larger diagnostic evaluation studies are recommended to validate these findings and on Xpert MTB/RIF to assess its use as a solitary initial test for TB lymphadenitis in children

GMOs in animal agriculture: time to consider both costs and benefits in regulatory evaluations

In 2012, genetically engineered (GE) crops were grown by 17.3 million farmers on over 170 million hectares. Over 70% of harvested GE biomass is fed to food producing animals, making them the major consumers of GE crops for the past 15 plus years. Prior to commercialization, GE crops go through an extensive regulatory evaluation. Over one hundred regulatory submissions have shown compositional equivalence, and comparable levels of safety, between GE crops and their conventional counterparts. One component of regulatory compliance is whole GE food/feed animal feeding studies. Both regulatory studies and independent peer-reviewed studies have shown that GE crops can be safely used in animal feed, and rDNA fragments have never been detected in products (e.g. milk, meat, eggs) derived from animals that consumed GE feed. Despite the fact that the scientific weight of evidence from these hundreds of studies have not revealed unique risks associated with GE feed, some groups are calling for more animal feeding studies, including long-term rodent studies and studies in target livestock species for the approval of GE crops. It is an opportune time to review the results of such studies as have been done to date to evaluate the value of the additional information obtained. Requiring long-term and target animal feeding studies would sharply increase regulatory compliance costs and prolong the regulatory process associated with the commercialization of GE crops. Such costs may impede the development of feed crops with enhanced nutritional characteristics and durability, particularly in the local varieties in small and poor developing countries. More generally it is time for regulatory evaluations to more explicitly consider both the reasonable and unique risks and benefits associated with the use of both GE plants and animals in agricultural systems, and weigh them against those associated with existing systems, and those of regulatory inaction. This would represent a shift away from a GE evaluation process that currently focuses only on risk assessment and identifying ever diminishing marginal hazards, to a regulatory approach that more objectively evaluates and communicates the likely impact of approving a new GE plant or animal on agricultural production systems

Electrofuels Program

Advanced Research Projects Agency-Energy project sheet summarizing general information about the Electrofuels program including critical needs, innovation and advantages, impacts, and contact information. This sheet discusses engineering a microorganism to create a liquid fuel from hydrogen and carbon dioxide as part of the "Novel Biological Conversion of Hydrogen and Carbon Dioxide Directly into Free Fatty Acids" project

Stochastic Modeling of Polymerase Chain Reaction and

Introduction The polymerase chain reaction (PCR) is a method that uses test tubes in biological laboratories for producing large amount of identical copies of a specific gene from small amount of complex molecules. Here we present our work on the stochastic modeling and theoretical studies of PCR, sexual PCR (DNA shu#ing) and their applications to in vitro evolution: an experimental method to evolve proteins with improved functions. 2. The Polymerase Chain Reaction The PCR uses the mechanism of DNA replication. There are three steps in a PCR cycle. In the first step, the double-stranded DNA molecules are heated to near boiling temperature so that the double-stranded DNA molecules are separated completely into two single-stranded sequences. This process is called denaturing. The single-stranded sequences generated by denaturing are used as templates for the primers and the DNA polymerase. In the second step, the temperature is lowered such that the primers anneal to the templates. T

Development of Endogenous and Synthetic CHO Promoter Expression Systems for Recombinant Protein Production

Chinese hamster ovary (CHO) cells are the most commonly used mammalian cell expression system for the industrial production of biotherapeutic recombinant proteins. Mammalian expression systems are a popular choice for the production of complex biopharmaceuticals due to their ability to correctly fold and assemble recombinant proteins and to carry out necessary human-like post-translational modifications. Traditionally, viral promoters are used to drive transgene expression in mammalian cells. This study reports on the investigation of endogenous CHO promoters as potential alternatives to the commonly used strong viral promoters such as SV40 and CMV. Although these promoters provide high recombinant gene expression, this is not always favourable as constitutive transgene over expression can ultimately lead to cellular stress. Furthermore, viral promoters have been reported to undergo silencing mechanisms over long culture periods, compromising product titers. Endogenous and synthetic CHO putative promoter sequences were investigated for their abilities to drive reporter and recombinant gene expression both in stable and transient systems. Initially a panel of ten putative promoters were identified from the literature, these being identified for this study as the associated gene or protein had been reported to have high transcript or protein amounts in CHO cells. The sequence 400 bp upstream of the transcript or translation start site of these genes was cloned into a promoterless eGFP reporter vector and their ability to drive transient gene expression examined. The ability of these putative promoters to drive eGFP expression was generally inferior to that of the viral SV40 promoter and always many-fold lower than that observed from the viral CMV with enhancer promoter. Regions 2 kb upstream from the transcript or translation site for each gene were then cloned into the promoterless eGFP reporter system to assess for promoter activity further upstream. For one of the 2 kb sequences, eGFP expression was enhanced above that observed from the equivalent 400 bp sequence and was greater than that from the SV40 promoter. A further nine target putative promoters were then identified from published RNAseq studies and sequences 500 bp upstream of the transcriptional start site of identified genes were cloned. Of the nine 500 bp sequences, three displayed promoter activity equivalent to or above that of SV40 and so regions 2 kb upstream of the transcriptional start site of these genes were investigated for further promoter activity. The use of the 2 kb segments resulted in an increase in stable but not transient eGFP expression from that observed from the 500 bp sequences alone. When placed directly downstream of the CMV enhancer two of the 2 kb sequences showed higher eGFP expression than when the CMV enhancer was not present. The CMV enhancer had no impact when upstream of the two other 2 kb sequences investigated showing this to be a sequence dependent effect. The 2 kb sequence which exhibited the strongest promoter activity of targets when driving transient eGFP expression, in addition to the two sequences with the CMV enhancer which showed enhanced transient eGFP expression, were investigated for their ability to drive IgG heavy and light chain, and hence IgG, stable expression in CHO cells compared to the CMV promoter with enhancer. Upon generation of pools and mini pools stably producing IgG under the control of the various promoters, the CHO endogenous 2 kb promoter outperformed the synthetic candidates, the CMV with enhancer and an industrially relevant control promoter. Indeed, the endogenous CHO promoter sequence was shown to achieve product titers of up to 3-fold greater than those from the commonly used CMV promoter with enhancer in CHO cells, demonstrating the potential for endogenous promoters to replace typically used viral promoters for recombinant gene expression. Further, colonies emerged faster after transfection and selection when using this promoter compared to the others investigated and a larger range of higher-expressing pools were available for investigation. In summary, this study has identified an endogenous CHO promoter sequence able to drive IgG expression beyond that of current widely used viral promoters and has generated additional promoters exhibiting a range of abilities to drive recombinant gene expression to varying amounts that could be applied to cell engineering approaches in the future

RPE SUB

Asia Network for Sustainable Agriculture and Bioresources (ANSAB) organized a national level policy workshop at Nagark, in Nepal, which included representatives from many institutions, aiming to identify policy-related opportunities and challenges in Non-Timber Forest Products (NTFP) sub-sector, and to make policy recommendations addressing social, economic and environmental concerns. This paper details activities of the workshop, including recommendations for an enabling policy environment, that would have simple rules and straightforward implementation. For instance: “Implementation of one- window system for tax collection, and a timely review of existing revenue rate of NTFP...