Intron-containing hairpin RNA interference vector for OBP8 show promising mortality in peach potato aphid

Myzus persicae is a devastating pest affecting potato production. Intron-containing hairpin RNA (ihpRNA) silenced the odorant-binding protein 8 (OBP8) for enhanced protection against Myzus persicae in potatoes. OBP8 was isolated from M. persicae, sequenced, with the allotment of GenBank ID. ERNAi was used to design siRNA targets from OBP8 with no off-targets. Multiple Sequence Alignment show M. persicae OBP8 resemblance with Acyrthosiphon pisum, Rhopalosiphum maidis, Aphis fabae, and Sitobion avenae. dsRNA-OBP8 (7 µg/µL) oral feeding resulted in a 69% mortality, and 58% OBP8 reduced expression 8D post-feeding compared to control. Golden Gate cloning is used for RNA interference taking advantage of type IIs restriction enzyme Eco31I. ihpRNA-OBP8 introduced by agroinfiltration in Solanum tuberosum. Transgenic S. tuberosum fed Myzus show 57.6% mortality and 49% reduction in OBP8 expression 8D post-feeding, compared to control. This work proves OBP8 as promising ihpRNA targets in potato and related crops for whom Myzus is a destructive pest.

     

The Reverse Gyrase from Pyrobaculum calidifontis,a Novel Extremely Thermophilic DNA Topoisomerase Endowed with DNA Unwinding and Annealing Activities

Reverse gyrase is a DNA topoisomerase specific for hyperthermophilic bacteria and archaea. It catalyzes the peculiar ATP-dependent DNA-positive supercoiling reaction and might be involved in the physiological adaptation to high growth temperature. Reverse gyrase comprises an N-terminal ATPase and a C-terminal topoisomerase domain, which cooperate in enzyme activity, but details of its mechanism of action are still not clear. We present here a functional characterization of PcalRG, a novel reverse gyrase from the archaeon Pyrobaculum calidifontis. PcalRG is the most robust and processive reverse gyrase known to date; it is active over a wide range of conditions, including temperature, ionic strength, and ATP concentration. Moreover, it holds a strong ATP-inhibited DNA cleavage activity. Most important, PcalRG is able to induce ATP-dependent unwinding of synthetic Holliday junctions and ATP-stimulated annealing of unconstrained single-stranded oligonucleotides. Combined DNA unwinding and annealing activities are typical of certain helicases, but until now were shown for no other reverse gyrase. Our results suggest for the first time that a reverse gyrase shares not only structural but also functional features with evolutionary conserved helicase-topoisomerase complexes involved in genome stability.     

Does the MRCGP examination discriminate against Asian doctors?

OBJECTIVE--To ascertain whether the membership examination for the Royal College of General Practitioners (MRCGP) discriminates against doctors of Indian subcontinent ethnic origin ("Asian doctors"). DESIGN--Retrospective analysis of data from five administrations of the MRCGP examination (December 1988-December 1990). SETTING--United Kingdom national examination body. SUBJECTS--3686 doctors taking the examination for the first time, 244 of whom were classified as Asian, the remainder as non-Asian. MAIN OUTCOME MEASURES--Comparison of performance in each of the written and oral components of the examination between Asian doctors, identified by their names and classified into subgroups by countries of birth and primary medical training from data provided at registration, and non-Asian doctors. RESULTS--On written components of the examination (multiple choice paper mean score Asians versus non-Asians 42.3 v 48.6, modified essay paper 40.9 v 48.9, practice topic/critical reading paper 41.5 v 48.7, all p less than 0.001 by t testing). But analysis by countries of birth and primary training showed that these differences were due largely to poor performance by certain groups of Asian doctors, especially those born and trained in the Indian subcontinent or elsewhere outside the United Kingdom. Asian doctors born and trained in the United Kingdom and those born in Africa or the West Indies and trained in the United Kingdom performed similarly to the non-Asian doctors. CONCLUSIONS--The examination does not systematically discriminate against Asian doctors, but the poor performance of the two subgroups of Asians is cause for serious concern and requires investigation.     

Native Enzyme Mobility Shift Assay (NEMSA): a new method for monitoring carboxyl group modification of carboxymethylcellulase from Aspergillus niger

A simple, sensitive, accurate and more informative assay for determining the number of modified groups during the course of carboxyl group modification is described. Monomeric carboxymethylcellulase (CMCase) from Aspergillus niger was modified by 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of glycinamide. The different time-course aliquots were subjected to non-denaturing PAGE and the gel stained for CMCase activity. The number of carboxyl groups modified are directly read from the ladder of the enzyme bands developed at given time. This method showed that after 75 min of modification reaction there were five major species of modified CMCases in which 6 to 10 carboxyls were modified.     

Enhanced resistance against bacterial wilt in transgenic tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis>) lines expressing the <Emphasis Type="Italic">Xa21</Emphasis> gene

To enhance bacterial wilt resistance in tomato plants and simplify the protocol of Agrobacterium tumefaciens mediated gene transfer, parameters affecting transformation efficiency in tomato have been optimized. A. tumefaciens strain EHA101, harboring a recombinant binary expression vector pTCL5 containing the Xa21 gene under the control of the CaMV 35S promoter was used for transformation. Five cultivars of tomato (Rio Grande, Roma, Pusa Ruby Pant Bahr and Avinash) were tested for transformation. Transformation efficiency was highly dependent on preculture of the explants with acetosyringone, acetosyringone in co-cultivation media, shoot regeneration medium and pre-selection after co-cultivation without selective agent. One week of pre-selection following selection along with 400 μM acetosyringone resulted in 92.3% transient GUS expression efficiency in Rio Grande followed by 90.3% in Avinash. The presence and integration of the Xa21 gene in putative transgenic plants was confirmed by polymerase chain reaction (PCR) and Southern blot analyses with 4.5–42.12% PCR-positive shoots were obtained for Xa21 and hygromycin genes, respectively. Transgenic plants of the all lines showed resistance to bacterial wilt. T₁ plants (resulting from self-pollination of transgenic plants) tested against Pseudomonas solanacearum inoculation in glasshouse, showed Mendelian segregation.     

T-rosettes in hemodialysis patients and renal allograft recipients

Subcellular fractions, isolated from the lymphoid cell line IM-1, are capable of stimulating a weak proliferative response in allogeneic lymphocytes. They also stimulate the generation of cytotoxic effector lymphocytes. The proliferative response to subcellular fractions, as measured by ³H-thymidine incorporation, is only one-fourth to one-sixth as great as that to intact IM-1 cells, suggesting that a component(s) synthesized during the mixed lymphocyte reaction (MLR), or a short-lived cellular constituent, may be responsible for the ability of intact cells to stimulate a lymphocyte proliferative response. This component appears to be lacking or in limiting quantity in subcellular fractions, including the soluble fractions. In contrast to the decreased proliferative response to subcellular fractions, the cytotoxic capacity of the stimulated lymphocytes is comparable to that after stimulation by intact IM-1 cells. The data demonstrate that, in this system, cytotoxic effector lymphocytes can be generated in the absence of the extensive proliferative response normally observed in the MLR. The antigenic stimulus responsible for the generation of cytotoxic effector cells appears to reside on intracellular components as well as on plasma membrane. In these reactions, specificity is shown by the failure of the cytotoxic cells to release ⁵¹Cr from autologous target cells. In fact, reactivity of lymphocytes stimulated by subcellular fractions is more specific than the reactivity of cells stimulated by intact IM-1 as judged by their lytic capacity for another target cell, RPMI 4265.     

Improvement of Aspergillus oryzae for hyperproduction of endoglucanase: expression cloning of cmc-1 gene of Aspergillus aculeatus

FI-Carboxymethylcellulase (cmc1; family 12) is one of the endoglucanases of Aspergillus aculeatus and consists of single polypeptide chain of 221 amino acids. The cmc1 gene was expressed in Aspergillus oryzae niaD300 (niaD⁻) under promoter 8142. The plasmid pCMG14 carrying the cmc1 gene at PstI site was used as a source of the gene (920 bp) and Aspergillus oryzae was successfully transformed by the plasmid pNAN-cmc1 (harboring cmc1 gene). The plasmid was integrated in Aspergillus oryzae niaD300 genome at niaD locus and the transformed fungus constitutively produced very high amounts of endoglucanases when grown on glucose, maltose, soluble starch and wheat bran.     

Anatomy of the stimulative sequences flanking the ARS consensus sequence of chromosome VI in Saccharomyces cerevisiae

We have analyzed the relationship between autonomously replicating sequence (ARS) structure and function for three ARS (ARS605, ARS607 and ARS609) from chromosome VI of Saccharomyces cerevisiae by systematic XhoI-linker mutation in the ARS consensus sequence (ACS) and flanking sequences. All mutations that encroached upon the ACS destroyed ARS activity. DNA sequences stimulative for ARS function were identified on either side of the ACS of ARS605 and only on the 3'-side of the ACS of ARS607. In ARS609, however, no such stimulative sequences were observed. Base substitutions complementary to the wild-type sequence of those stimulative regions, in ARS605 and ARS607, that did not change the AG of unwinding nor affected ARS activity suggests that these regions have, at least, a function as DNA-unwinding elements (DUE). ARS605, ARS607 and ARS609 DNA are of low AG value and showed hypersensitivity to single-strand-specific nuclease when inserted in negatively supercoiled plasmid. Linker mutations inhibitory for ARS activity (5L11 and 7L14) also caused significant changes in local nucleotide (nt) sensitivity within the ACS and its adjoining regions. Complementary base substitutions, however, did not affect these changes in local nt sensitivity. These results imply that the stimulative regions flanking the ACS are necessary to produce an optimum conformation around the ACS which may be important for full ARS activity.     

Chemosensory responses of Aporcelaimellus nivalis (Nematoda: Dorylaimida) towards kairomones emitted by prey nematodes belonging to different trophic groups

Abstract

Observations were made on the attraction of Aporcelaimellus nivalis towards kairomones/attractants emitted by prey nematodes belonging to different trophic categories, viz., saprophagous, epidermal, migratory semi-endodermal, predatory nematodes, virus vectors and cortical feeders. Aporcelaimellus nivalis responded positively and significantly to prey kairomones, but showed variation in their individual behaviour. Predators are most attracted towards epidermal feeders and least attracted to virus vectors. The differential responses of A. nivalis towards different prey were attributed to the inert behaviour of predators, their preference for a particular species of prey, chemical composition, concentration, quality, quantity of prey attractant, formation of minimum perceptible attraction gradient of prey and minimum response threshold of predators. Various factors such as prey density, period of prey incubation, starvation of predators, temperature, agar concentration, agar thickness and distance of predators from the source of attraction (prey) govern chemosensory responses of predator. Aporcelaimellus nivalis maximum response was towards Hirschmanniella oryzae, when tested as 10 day starved predators in agar plates containing 2 mm thick layer of 1% water-agar with 200 prey individuals previously incubated for 16 h at 30°C. Prey kairomones were most attractive when A. nivalis were tested from a distance of 2 and 3 cm.     

Influenza Virus Surveillance in Pakistan during 2008-2011

Background

There is little information about influenza among the Pakistani population. In order to assess the trends of Influenza-like-Illness (ILI) and to monitor the predominant circulating strains of influenza viruses, a country-wide lab-based surveillance system for ILI and Severe Acute Respiratory Illness (SARI) with weekly sampling and reporting was established in 2008. This system was necessary for early detection of emerging novel influenza subtypes and timely response for influenza prevention and control.

Methods

Five sentinel sites at tertiary care hospitals across Pakistan collected epidemiological data and respiratory samples from Influenza-like illness (ILI) and severe acute respiratory illness (SARI) cases from January 2008 to December 2011. Samples were typed and sub-typed by Real-Time RT-PCR assay.

Results

A total of 6258 specimens were analyzed; influenza virus was detected in 1489 (24%) samples, including 1066 (72%) Influenza type A and 423 (28%) influenza type B viruses. Amongst influenza A viruses, 25 (2%) were seasonal A/H1N1, 169 (16%) were A/H3N2 and 872 (82 %) were A(H1N1)pdm09. Influenza B virus circulation was detected throughout the year along with few cases of seasonal A/H1N1 virus during late winter and spring. Influenza A/H3N2 virus circulation was mainly observed during summer months (August-October).

Conclusions

The findings of this study emphasize the need for continuous and comprehensive influenza surveillance. Prospective data from multiple years is needed to predict seasonal trends for vaccine development and to further fortify pandemic preparedness.