Phytochelatin plays a role in UV-B tolerance in N2-fixing cyanobacterium Anabaena doliolum

To study the role of Cd-induced phytochelatins in UV-B tolerance, lipid peroxidation, antioxidative enzymes (superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase), glutathione arid phytochelatin contents were measured in buthionine sulphoximine treated and untreated cells of Anabaena doliolum. Cd-pretreatment of the cyanobacterium reduced the lipid peroxidation as well as the antioxidative enzymes in comparison to UV-B treatment alone, whereas the phytochelatin content demonstrated an increase. In contrast to this, buthionine sulphoximine-induced inhibition of phytochelatin synthase, dramatically decreased the Cd-induced co-tolerance against UV-B, hence demonstrating that phytochelatin not only protects the cyanobacterium from heavy metal but participates in UV-B tolerance as well.     

Characterization of outer membrane proteins of Yersinia pestis and Yersinia pseudotuberculosis strains isolated from India

The majority of virulence factors including the 12 Yersinia outer membrane proteins (Yops), 29 Yop secretion proteins (Ysc) and few specific Yop chaperone (Syc) are contributed by the 70 kb LCR middle plasmid of Yersinia pestis. Yersinia pestis isolates recovered during 1994 plague outbreak and rodent surveillance samples of Southern states of India were studied for the presence of important Yops by the conventional procedure of partially purifying outer membrane proteins (Omps) after cultivation in calcium deficient media. Prominent bands numbering 4-5 between 34-42 kDa region corresponding to important Yops were seen in all the isolates as well as in other Yersinia and non-Yersinia species by SDS-PAGE. Western blotting with the polyclonal antisera raised against these Omp preparations revealed few immuno-reactive bands that appeared to be shared among Y. pestis, Y. pseudotruberculosis, Y. enterocolitica, Y. fredrocksenii, Y. intermedia, Y. kristensenii and E. coli. Three recombinant Yop proteins namely, YopM, YopB and LcrV were produced and antisera to these proteins could reveal presence of these Yops in the Y. pestis Omp preparations. In order to further characterize the important Yops among Omps, attempts were made to generate monoclonal antibodies against Omp preparation. Three of the 4 stable reactive clones that were obtained, when tested, had extensive cross-reactions among pathogenic Yersinia species, Y. pestis and Y. pseudotuberculosis isolates, other Yersinia species and the members of Enterobacteriaceae in dot-ELISA and Western blotting. One of the monoclonal antibodies, YP1, exhibited reaction to all the pathogenic Yersinia species and the isolates, with restricted cross-reactivity to Y. intermedia, Y. kristensenii, K. pneumoniae. None of the 4 monoclonal antibodies had reactions with the 3 recombinant Yop proteins. It appears that under low calcium response, the Y. pestis not only activates secretion of Yops but also a large number of other proteins, which as per the present observations are cross-reactive within the family Enterobacteriaceae.     

Identification and mapping of resistance to stem rust in the European winter wheat cultivars Spark and Rialto

A mapping population of 126 doubled haploid (DH) lines derived from a cross between the English winter wheat cultivars Spark and Rialto was evaluated for response to Puccinia graminis f. sp. tritici in the greenhouse and in artificially inoculated field plots at two locations over 3 years (2011, 2012 and 2013). Genetic analysis indicated the involvement of two seedling genes (Sr5 and Sr31, contributed by Rialto) and three adult plant resistance genes. QTL analyses of field data showed the involvement of three consistent effects QTL on chromosome arms 1BS (contributed by Rialto), and 3BS and chromosome 5A (contributed by Spark) in the observed resistance to stem rust. These QTLs explained average phenotypic variation of 78.5, 9.0 and 5.9 %, respectively. With the presence of virulence for Sr5 and absence of Sr31 virulence in the field, the QTL detected on 1BS (QSr.sun-1BS) was attributed to the major seedling resistance gene Sr31. The QTL located on chromosome arm 3BS (QSr.sun-3BS) was closely associated with SSR marker gwm1034, and the QTL detected on 5A (QSr.sun-5A) was closely linked with SSR marker gwm443. DH lines carrying the combination of QSr.sun-3BS and QSr.sun-5A exhibited lower stem rust responses indicating the additive effects of the two APR genes in reducing disease severity. The markers identified in this study can be useful in pyramiding these QTLs with other major or minor genes and marker assisted selection for stem rust resistance in wheat.     

Draft Genome Sequences of Yersinia pestis Strains from the 1994 Plague Epidemic of Surat and 2002 Shimla Outbreak in India

We report the first draft genome sequences of the strains of plague-causing bacteria, Yersinia pestis, from India. These include two strains from the Surat epidemic (1994), one strain from the Shimla outbreak (2002) and one strain from the plague surveillance activity in the Deccan plateau region (1998). Genome size for all four strains is ~4.49 million bp with 139–147 contigs. Average sequencing depth for all four genomes was 21x.     

Reverse Line Blot Macroarray for Simultaneous Detection and Characterization of Four Biological Warfare Agents

The need for a rapid detection and characterization of biowarfare (BW) agents cannot be over emphasized. With diverse array of potential BW pathogen available presently, rapid identification of the pathogen is crucial, so that specific therapy and control measures can be initiated. We have developed a multiplex polymerase chain reaction based reverse line blot macroarray to simultaneously detect four pathogens of BW importance viz. Bacillus anthracis, Yersinia pestis, Brucella melitensis and Burkholderia pseudomallei. The multiplex PCR utilizes 14 pairs of primers targeting 18 specific markers. These markers include genes which are genus specific, species-specific chromosomal sequences and virulence markers of plasmid origin. The assay was evaluated on various human, environment and animal isolates. The assay w successful in simultaneous detection and characterization of isolates of the four pathogens on as a single platform with sensitivity ranging from 0.3 pg to 0.3 ng of genomic DNA. The assay was able to detect 5 × 10² cfu/ml for B. anthracis, 8 × 10² cfu/ml for Yersinia sp., 1.4 × 10² cfu/ml for B. melitensis and 4 × 10² cfu/ml for B. pseudomallei.     

A robust molecular marker for the detection of shortened introgressed segment carrying the stem rust resistance gene <Emphasis Type="Italic">Sr22</Emphasis> in common wheat

Stem rust resistance gene Sr22 transferred to common wheat from Triticum boeoticum and T. monococcum remains effective against commercially prevalent pathotypes of Puccinia graminis f. sp. tritici, including Ug99 and its derivatives. Sr22 was previously located on the long arm of chromosome 7A. Several backcross derivatives (hexaploid) possessing variable sized Sr22-carrying segments were used in this study to identify a closely linked DNA marker. Expressed sequenced tags belonging to the deletion bin 7AL-0.74–0.86, corresponding to the genomic location of Sr22 were screened for polymorphism. In addition, RFLP markers that mapped to this region were targeted. Initial screening was performed on the resistant and susceptible DNA bulks obtained from backcross derivatives carrying Sr22 in three genetic backgrounds with short T. boeoticum segments. A cloned wheat genomic fragment, csIH81, that detected RFLPs between the resistant and susceptible bulks, was converted into a sequence tagged site (STS) marker, named cssu22. Validation was performed on Sr22 carrying backcross-derivatives in fourteen genetic backgrounds and other genotypes used for marker development. Marker cssu22 distinguished all backcross-derivatives from their respective recurrent parents and co-segregated with Sr22 in a Schomburgk (+Sr22)/Yarralinka (−Sr22)-derived recombinant inbred line (RIL) population. Sr22 was also validated in a second population, Sr22TB/Lakin-derived F₄ selected families, containing shortened introgressed segments that showed recombination with previously reported flanking microsatellite markers.     

Synthesis of gold nanoparticles by various leaf fractions of Semecarpus anacardium L. tree

Gold nanoparticles (NPs) were synthesized using Semecarpus anacardium leaf extracts in water and the green biomass. Extract prepared at ambient condition by crushing the leaves in deionized water is identified as ‘green extract’, and that by boiling the leaf pieces as ‘boiled extract’. The mass remaining after separating the ‘green extract’ is identified as ‘green biomass’. These components triggered rapid reduction of Au(III) to Au (0) in HAuCl₄ solution indicating the natural ability of the leaves of S. anacardium to synthesize NPs in ambient conditions. Green extract produced more NPs compared to the boiled extract suggesting denaturization of some of the useful factors due to boiling. NPs were quantified using UV and ICP-AES analysis. These were characterized using Transmission electron microscopy, Fourier transform infrared spectroscopy and X-ray diffraction. TEM images of the particles formed with green extract, boiled extract and green biomass showed that the particles were of different shapes and sizes.     

Rapid multiplex PCR assay for the simultaneous detection of the Brucella Genus, B. abortus, B. melitensis, and B. suis

The routine identification and differentiation of Brucella species is a time-consuming and labor-intensive process, which frequently places personnel at risk of laboratoryacquired infection. Here, we describe the development of a rapid multiplex PCR assay for the confirmation of presumptive Brucella isolates. The assay was able to identify and differentiate major human pathogens, namely B. abortus, B. melitensis, and B. suis, in a single test of less than an hour and a half.     

Recellularization via the bile duct supports functional allogenic and xenogenic cell growth on a decellularized rat liver scaffold

Recent years have seen a proliferation of methods leading to successful organ decellularization. In this experiment we examine the feasibility of a decellularized liver construct to support growth of functional multilineage cells. Bio-chamber systems were used to perfuse adult rat livers with 0.1% SDS for 24 hours yielding decellularized liver scaffolds. Initially, we recellularized liver scaffolds using a human tumor cell line (HepG2, introduced via the bile duct). Subsequent studies were performed using either human tumor cells co-cultured with human umbilical vein endothelial cells (HUVECs, introduced via the portal vein) or rat neonatal cell slurry (introduced via the bile duct). Bio-chambers were used to circulate oxygenated growth medium via the portal vein at 37C for 5-7 days. Human HepG2 cells grew readily on the scaffold (n = 20). HepG2 cells co-cultured with HUVECs demonstrated viable human endothelial lining with concurrent hepatocyte growth (n = 10). In the series of neonatal cell slurry infusion (n = 10), distinct foci of neonatal hepatocytes were observed to repopulate the parenchyma of the scaffold. The presence of cholangiocytes was verified by CK-7 positivity. Quantitative albumin measurement from the grafts showed increasing albumin levels after seven days of perfusion. Graft albumin production was higher than that observed in traditional cell culture. This data shows that rat liver scaffolds support human cell ingrowth. The scaffold likewise supported the engraftment and survival of neonatal rat liver cell slurry. Recellularization of liver scaffolds thus presents a promising model for functional liver engineering.     

Tight repulsion linkage between <Emphasis Type="Italic">Sr36</Emphasis> and <Emphasis Type="Italic">Sr39</Emphasis> was revealed by genetic,cytogenetic and molecular analyses

Key message

The shortening of Aegilops speltoides segment did not facilitate recombination between stem rust resistance genes Sr36 and Sr39 . Robustness of marker rwgs28 for marker-assisted selection of Sr39 was demonstrated.

Abstract

Stem rust resistance genes Sr39 and Sr36 were transferred from Aegilops speltoides and Triticum timopheevii, respectively, to chromosome 2B of wheat. Genetic stocks RL6082 and RWG1 carrying Sr39 on a large and a shortened Ae. speltoides segments, respectively, and the Sr36-carrying Australian wheat cultivar Cook were used in this study. This investigation was planned to determine the genetic relationship between these genes. Stem rust tests on F₃ populations derived from RL6082/Cook and RWG1/Cook crosses showed tight repulsion linkage between Sr39 and Sr36. The genomic in situ hybridization analysis of heterozygous F₃ family from the RWG1/Cook population showed that the translocated segments do not overlap. Meiotic analysis on the F₁ plant from RWG1/Cook showed two univalents at the metaphase and anaphase stages in a majority of the cells indicating absence of pairing. Since meiotic pairing has been reported to initiate at the telomere, pairing and recombination may be inhibited due to very little wheat chromatin in the distal end of the chromosome arm 2BS in RWG1. The Sr39-carrying large Ae. speltoides segment transmitted preferentially in the RL6082/Cook F₃ population, whereas the Sr36-carrying T. timopheevii segment over-transmitted in the RWG1/Cook cross. Genotyping with the co-dominant Sr39- and Sr36-linked markers rwgs28 and stm773-2, respectively, matched the phenotypic classification of F₃ families. The RWG1 allele amplified by rwgs28 was diagnostic for the shortened Ae. speltoides segment and alternate alleles were amplified in 29 Australian cultivars. Marker rwgs28 will be useful in marker-assisted pyramiding of Sr39 with other genes.