Analysis of mosquito bloodmeals using RFLP markers

An important variable in the amplification of arthropod vector-borne diseases is the degree of contact between human hosts and mosquito vectors. To analyze this interaction, a DNA based method was developed to differentiate human bloodmeals from other sources in the mosquito Anopheles stephensi (Diptera: Culicidae) Liston. A portion of the host mitochondrial DNA cytochrome B genes were PCR amplified and classified to the species level based on their restriction fragment length polymorphism (RFLP). The cytochrome B sequences showed sufficient interspecific polymorphism to distinguish between human, cow, sheep, chicken, and guinea pig hosts. XhoI could distinguish human from other vertebrates whereas TaqI alone could separate the others. The importance of these results in epidemiological studies of malaria and other vector borne diseases is discussed.     

The human spindle assembly checkpoint protein Bub3 is required for the establishment of efficient kinetochore-microtubule attachments

The spindle assembly checkpoint monitors the status of kinetochore-microtubule (K-MT) attachments and delays anaphase onset until full metaphase alignment is achieved. Recently, the role of spindle assembly checkpoint proteins was expanded with the discovery that BubR1 and Bub1 are implicated in the regulation of K-MT attachments. One unsolved question is whether Bub3, known to form cell cycle constitutive complexes with both BubR1 and Bub1, is also required for proper chromosome-to-spindle attachments. Using RNA interference and high-resolution microscopy, we analyzed K-MT interactions in Bub3-depleted cells and compared them to those in Bub1- or BubR1-depleted cells. We found that Bub3 is essential for the establishment of correct K-MT attachments. In contrast to BubR1 depletion, which severely compromises chromosome attachment and alignment, we found Bub3 and Bub1 depletions to produce defective K-MT attachments that, however, still account for significant chromosome congression. After Aurora B inhibition, alignment defects become severer in Bub3- and Bub1-depleted cells, while partially rescued in BubR1-depleted cells, suggesting that Bub3 and Bub1 depletions perturb K-MT attachments distinctly from BubR1. Interestingly, misaligned chromosomes in Bub3- and Bub1-depleted cells were found to be predominantly bound in a side-on configuration. We propose that Bub3 promotes the formation of stable end-on bipolar attachments.     

Characterization of human HtrA2, a novel serine protease involved in the mammalian cellular stress response.

Human HtrA2 is a novel member of the HtrA serine protease family and shows extensive homology to the Escherichia coli HtrA genes that are essential for bacterial survival at high temperatures. HumHtrA2 is also homologous to human HtrA1, also known as L56/HtrA, which is differentially expressed in human osteoarthritic cartilage and after SV40 transformation of human fibroblasts. HumHtrA2 is upregulated in mammalian cells in response to stress induced by both heat shock and tunicamycin treatment. Biochemical characterization of humHtrA2 shows it to be predominantly a nuclear protease which undergoes autoproteolysis. This proteolysis is abolished when the predicted active site serine residue is altered to alanine by site-directed mutagenesis. In human cell lines, it is present as two polypeptides of 38 and 40 kDa. HumHtrA2 cleaves beta-casein with an inhibitor profile similar to that previously described for E. coli HtrA, in addition to an increase in beta-casein turnover when the assay temperature is raised from 37 to 45 degrees C. The biochemical and sequence similarities between humHtrA2 and its bacterial homologues, in conjunction with its nuclear location and upregulation in response to tunicamycin and heat shock suggest that it is involved in mammalian stress response pathways.     

Impact of DNA repair genes polymorphism (XPD and XRCC1) on the risk of breast cancer in Egyptian female patients

The genes involved in DNA repair system play a crucial role in the protection against mutations. It has been hypothesized that functional deficiencies in highly conserved DNA repair processes resulting from polymorphic variation may increase genetic susceptibility to breast cancer (BC). The aim of the present study was to evaluate the association of genetic polymorphisms in 2 DNA repair genes, XPD (Asp312Asn) and XRCC1 (A399G), with BC susceptibility. We further investigated the potential combined effect of these DNA repair variants on BC risk. Both XPD (xeroderma pigmentosum group D) and XRCC1 (X-ray repair cross-complementing group 1) polymorphisms were characterized in 100 BC Egyptian females and 100 healthy women who had no history of any malignancy by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) method and PCR with confronting two-pair primers (PCR-CTPP), using DNA from peripheral blood in a case control study. Our results revealed that the frequencies of AA genotype of XPD codon 312 polymorphism were significantly higher in the BC patients than in the normal individuals (P ≤ 0.003), and did not observe any association between the XRCC1 Arg399Gln polymorphism and risk of developing BC. Also, no association between both XPD Asp312Asn and XRCC1 A399G polymorphisms and the clinical characteristics of disease. Finally, the combination of AA(XPD) + AG(XRCC1) were significantly associated with BC risk. Our results suggested that, XPD gene is an important candidate gene for susceptibility to BC. Also, gene–gene interaction between XPD(AA) + XRCC1(AG) polymorphism may be associated with increased risk of BC in Egyptian women.     

Molecular phylogeny and divergence times of Onosma (Boraginaceae s.s.) based on nrDNA ITS and plastid rpl32‐trnL(UAG) and trnH–psbA sequences

We analysed 87 species of Onosma (Boraginaceae) from throughout its distribution range to investigate its evolutionary history. Using nrDNA ITS and two plastid (rpl32‐trnL_(UAG) and trnH–psbA) markers, we reconstructed phylogenetic relationships within Onosma by conducting maximum parsimony, maximum likelihood, Bayesian, and BEAST analyses. The analyses revealed that Onosma as currently circumscribed is not monophyletic. However, the vast majority of Onosma species appear to belong to a single clade, the so‐called Onosma s.s. Outside of this core clade is a clade containing O. rostellata, a subclade of Sino‐Indian species and Maharanga emodii. Podonosma orientalis (as O. orientalis) appear only distantly related to Onosma but is more closely related to Alkanna, as also suggested in previous molecular studies. The Onosma s.s. clade includes all representatives of O. sect. Onosma, and encompasses three subsections, i.e. Onosma, Haplotricha and Heterotricha, corresponding to asterotrichous, haplotrichous and heterotrichous groups, respectively, but none of these subsections was retrieved as monophyletic. We observed significant incongruence between nuclear and chloroplast phylogenies regarding the phylogenetic status of the heterotrichous group. A dozen of the Iranian haplotrichous species formed a lineage which may not hybridize with asterotrichous species. Divergence time estimates suggested that the early radiation of Onosma s.l. took place at the Oligocene‐Miocene boundary and the diversification within Onosma s.s. occurred during middle to late Miocene and Pliocene.     

Inheritance of restriction fragment length polymorphisms and random amplified polymorphic DNAs in coastal Douglas-fir

A total of 225 new genetic loci [151 restriction fragment length polymorphisms (RFLP) and 74 random amplified polymorphic DNAs (RAPD)] in coastal Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco var. menziesii] have been identified using a three-generation outbred pedigree. The Mendelian inheritance of 16 RFLP loci and 29 RAPD loci was demonstrated based on single-locus segregation in a sample of F₂ progeny. One RFLP locus, PtIFG2025, showed segregation distortion. Probe pPtIFG2025 is a loblolly pine cDNA probe encoding for rbcS. The 16 RFLP loci and 23 allozyme loci were also assayed in a sample of 16 Douglas-fir seed-orchard clones. Allelism was determined at 11 of the 16 RFLP loci. RFLPs were able to detect slightly more variation (4.0 alleles per locus) than allozymes (3.1 alleles per locus). The inheritance of an additional 80 RAPD loci was determined based on haploid segregation analysis of megagametophytes from parent tree 013-1. Once 200–300 markers are identified and placed on a genetic map, quantitative trait loci affecting bud phenology will be mapped.     

Selection of fatty acid auxotrophs from the oleaginous yeast Cryptococcus curvatus and production of cocoa butter equivalents in batch culture

Summary A search was carried out for mutants, defective in the conversion of stearic acid to oleic acid in effort to improve the quality of lipids produced by Cryptococcus curvatus ATCC, 20509. Mutants were selected as unsaturated fatty acid (Ufa) auxotrophs. After treatment of parent organism with Ethyl methanesulfonate (EMS), 11 oleate-requiring auxotrophs were isolated. Only 3 of them were real unsaturated fatty acid (Ufa) mutants, while the other 8 were designated as fatty acid synthetase (Fas) mutants. The amount of saturated fatty acid (SFA) was about 65.2 % in the lipids extracted from an Ufa mutant named UfaM3 and it was significantly higher than that of the wild-type (WT) (46.6 %) and similar to that of cocoa butter (60.4 %).     

Self‐Powered Wireless Sensor Node Enabled by a Duck‐Shaped Triboelectric Nanogenerator for Harvesting Water Wave Energy

This paper presents a fully enclosed duck‐shaped triboelectric nanogenerator (TENG) for effectively scavenging energy from random and low‐frequency water waves. The design of the TENG incorporates the freestanding rolling mode and the pitch motion of a duck‐shaped structure generated by incident waves. By investigating the material and structural features, a unit of the TENG device is successfully designed. Furthermore, a hybrid system is constructed using three units of the TENG device. The hybrid system achieves an instantaneous peak current of 65.5 µA with an instantaneous output power density of up to 1.366 W m^?2. Following the design, a fluid–solid interaction analysis is carried out on one duck‐shaped TENG to understand the dynamic behavior, mechanical efficiency, and stability of the device under various water wave conditions. In addition, the hybrid system is experimentally tested to enable a commercial wireless temperature sensor node. In summary, the unique duck‐shaped TENG shows a simple, cost‐effective, environmentally friendly, light‐weight, and highly stable system. The newly designed TENG is promising for building a network of generators to harvest existing blue energy in oceans, lakes, and rivers.