Error-prone and error-restrictive mutations affecting ribosomal protein S12

Ribosomal protein S12 plays a pivotal role in decoding functions on the ribosome. X-ray crystallographic analyses of ribosomal complexes have revealed that S12 is involved in the inspection of codon-anticodon pairings in the ribosomal A site, as well as in the succeeding domain rearrangements of the 30S subunit that are essential for accommodation of aminoacyl-tRNA. A role for S12 in tRNA selection is also well supported by classical genetic analyses; mutations affecting S12 are readily isolated in bacteria and organelles, since specific alterations in S12 confer resistance to the error-inducing antibiotic streptomycin, and the ribosomes from many such streptomycin-resistant S12 mutants display decreased levels of miscoding. However, substitutions that confer resistance to streptomycin likely represent a very distinct class of all possible S12 mutants. Until recently, the technical difficulties in generating random, unselectable mutations in essential genes in complex operons have generally precluded the analysis of other classes of S12 alterations. Using a recombineering approach, we have targeted the Escherichia coli rpsL gene, encoding S12, for random mutagenesis and screened the resulting mutants for effects on decoding fidelity. We have recovered over 40 different substitutions located throughout the S12 protein that alter the accuracy of translation without substantially affecting the sensitivity to streptomycin. Moreover, this collection includes mutants that promote miscoding, as well as those that restrict decoding errors. These results affirm the importance of S12 in decoding processes and indicate that alterations in this essential protein can have diverse effects on the accuracy of decoding.     

Simple System for Isothermal DNA Amplification Coupled to Lateral Flow Detection

Infectious disease diagnosis in point-of-care settings can be greatly improved through integrated, automated nucleic acid testing devices. We have developed an early prototype for a low-cost system which executes isothermal DNA amplification coupled to nucleic acid lateral flow (NALF) detection in a mesofluidic cartridge attached to a portable instrument. Fluid handling inside the cartridge is facilitated through one-way passive valves, flexible pouches, and electrolysis-driven pumps, which promotes a compact and inexpensive instrument design. The closed-system disposable prevents workspace amplicon contamination. The cartridge design is based on standard scalable manufacturing techniques such as injection molding. Nucleic acid amplification occurs in a two-layer pouch that enables efficient heat transfer. We have demonstrated as proof of principle the amplification and detection of Mycobacterium tuberculosis (M.tb) genomic DNA in the cartridge, using either Loop Mediated Amplification (LAMP) or the Exponential Amplification Reaction (EXPAR), both coupled to NALF detection. We envision that a refined version of this cartridge, including upstream sample preparation coupled to amplification and detection, will enable fully-automated sample-in to answer-out infectious disease diagnosis in primary care settings of low-resource countries with high disease burden.     

The adequacy of informed consent forms in genetic research in Oman: a pilot study

Genetic research presents ethical challenges to the achievement of valid informed consent, especially in developing countries with areas of low literacy. During the last several years, a number of genetic research proposals involving Omani nationals were submitted to the Department of Research and Studies, Ministry of Health, Oman. The objective of this paper is to report on the results of an internal quality assurance initiative to determine the extent of the information being provided in genetic research informed consent forms. In order to achieve this, we developed checklists to assess the inclusion of basic elements of informed consent as well as elements related to the collection and future storage of biological samples. Three of the authors independently evaluated and reached consensus on seven informed consent forms that were available for review. Of the seven consent forms, four had less than half of the basic elements of informed consent. None contained any information regarding whether genetic information relevant to health would be disclosed, whether participants may share in commercial products, the extent of confidentiality protections, and the inclusion of additional consent forms for future storage and use of tissue samples. Information regarding genetic risks and withdrawal of samples were rarely mentioned (1/7), whereas limits on future use of samples were mentioned in 3 of 7 consent forms. Ultimately, consent forms are not likely to address key issues regarding genetic research that have been recommended by research ethics guidelines. We recommend enhanced educational efforts to increase awareness, on the part of researchers, of information that should be included in consent forms.     

Discovery of novel biaryl sulfonamide based Mcl-1 inhibitors

Mcl-1 is an anti-apoptotic protein overexpressed in hematological malignancies and several human solid tumors. Small molecule inhibition of Mcl-1 would offer an effective therapy to Mcl-1 mediated resistance. Subsequently, it has been the target of extensive research in the pharmaceutical industry. The discovery of a novel class of Mcl-1 small molecule inhibitors is described beginning with a simple biaryl sulfonamide hit derived from a high through put screen. A medicinal chemistry effort aided by SBDD generated compounds capable of disrupting the Mcl-1/Bid protein-protein interaction in vitro. The crystal structure of the Mcl-1 bound ligand represents a unique binding mode to the BH3 binding pocket where binding affinity is achieved, in part, through a sulfonamide oxygen/Arg263 interaction. The work highlights the some of the key challenges in designing effective protein-protein inhibitors for the Bcl-2 class of proteins.     

Non-dipping blood pressure in the metabolic syndrome among Arabs of the Oman family study

Objective: The objective was to examine the circadian changes in blood pressure and their relation to the metabolic syndrome and its components in Omani Arabs. Research Methods and Procedures: Ambulatory blood pressure (ABPM) was recorded in 1124 subjects from 5 large, extended, consanguineous, and young Arab pedigrees. According to the International Diabetes Federation's definition, 264 subjects had the metabolic syndrome, a prevalence of 23%. Subjects were defined as non‐dippers when their nocturnal systolic blood pressure (SBP) fell by <10% from daytime SBP. Results: Non‐dippers with the metabolic syndrome were 131 of 264 (50%), compared with 265 of 860 (31%) without the metabolic syndrome. Of the non‐dippers, 99 of 131 (76%) were females and 32 of 131 (24%) were males. Daytime and nighttime SBP and DBP and nighttime pulse pressure were significantly higher in non‐dipper subjects with the metabolic syndrome. The important determinants of a non‐dipping BP in this cohort were high BMI and high serum triglycerides. Discussion: We hypothesize that obesity and nocturnal volume‐dependent hypertension may be involved in the pathophysiology of non‐dipping in the metabolic syndrome. This study showed that non‐dipping BP was common in subjects with the metabolic syndrome. Higher 24‐hour blood pressure load may add to the indices of the overall cardiovascular burden already associated with the metabolic syndrome.     

Biological Control of <Emphasis Type="Italic">Fusarium</Emphasis> sp. NBRI-PMSF12 Pathogenic to Cultivated Betelvine by <Emphasis Type="Italic">Bacillus</Emphasis> sp. NBRI-W9, a Potential Biological Control Agent

Betelvine is prone to several fungal diseases including leaf spots, foot and root rot caused by Fusarium spp. due to humid conditions prevailing in fields. In the present study, a potent antagonistic bacterial endophyte and a virulent fungal pathogen were selected after rigorous screening of isolates from different betelvine varieties to provide an efficient biocontrol strategy in cultivation of betelvine. Wild varieties of crops are a rich source of untapped endophytes. Of the four betelvine varieties used for isolations and screening, the wild variety was richest in endophytic populations. Using 16S rRNA sequencing, the selected antagonist was identified as Bacillus sp. (NBRI-W9). The pathogen, virulent against cultivated varieties, was identified as Fusarium sp. (NBRI-PMSF12) using ITS 1 and 2 region sequencing. Under in vitro and field conditions, NBRI-W9 was able to induce early rooting, provide plant growth promotion, increase leaf size and yield (leaf number) and provide biocontrol against the Fusarium sp. infection. NBRI-W9 treatments showed bacterial colonization on the leaf surface preferably in the vicinity of pearl glands and the collenchyma region in scanning electron microscope (SEM) studies. NBRI-W9 was observed to directly enter the leaf by degrading cell walls and colonize the subcellular layers. SEM analysis showed direct confrontation of NBRI-W9 with Fusarium on the leaf surface and in the collenchyma region as one of the probable modes of biocontrol.     

Transgenic Tobacco and Apple Plants Expressing Biotin-binding Proteins are Resistant to two Cosmopolitan Insect Pests,Potato Tuber Moth and Lightbrown Apple Moth,Respectively

Tobacco (Nicotiana tabacum cv. Samsun) and apple (Malus x domestica cv. Royal Gala) plants expressing avidin or strepavidin were produced using Agrobacterium tumefaciens-mediated transformation. ELISA assays showed that avidin expression ranged from 3.1 to 4.6 microM in tobacco and from 1.9 to 11.2 microM in apple and streptavidin expression ranged from 11.4 to 24.5 microM in tobacco and from 0.4 to 14.6 microM in apple. Expressed at these levels, both biotin-binding proteins conferred a high level of insect resistance on transformed tobacco plants to larval potato tuber moth (PTM), Phthorimaea operculella (Zeller) (fam. Gelechiidae) and on apple plants to larvae of the lightbrown apple moth (LBAM) Epiphyas postvittana (Walker) (fam. Tortricidae). More than 90% of PTM larvae died on tobacco plants expressing either avidin or streptavidin genes within 9 days of inoculation. Mortality of LBAM larvae was significantly higher (P < 0.05) on three avidin-expressing (89.6, 84.9 and 80.1%) and two streptavidin-expressing (90 and 82.5%) apple plant lines than on non-transformed control plants (14.1%) after 21 days. Weight of LBAM larvae was also significantly reduced by feeding on all apple shoots expressing avidin and on apple shoots expressing streptavidin at levels of 3.8 microM and above.