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.     

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.

     

Comparative analysis of electron microscopy and immunocytochemistry in the cytologic diagnosis of malignant small round cell tumors

OBJECTIVE: To compare the contributions of electron microscopy (EM) and immunocytochemistry (ICC) as adjuncts in the cytodiagnosis of malignant small round cell tumors (MSRCT). STUDY DESIGN: This prospective study included 57 cases with a preliminary aspiration diagnosis of MSRCT. The contributions of EM and ICC in arriving at a specific diagnosis were evaluated. RESULTS: The 57 cases included 22 cases of Ewing's sarcoma/peripheral neuroectodermal tumor (PNET), 12 neuroblastomas, 8 Wilms' tumors, 6 rhabdomyosarcomas, 5 lymphomas, 2 retinoblastomas and 1 synovial sarcoma. One case remained unclassified. Electron microscopy was crucial to the diagnosis in 38.4% cases as against 39.2% of cases by ICC. The light microscopic diagnosis was confirmed in 42.3% and 53.5% cases by EM and ICC, respectively. EM and ICC were inconclusive for a specific diagnosis in 19.2% and 7.1% of cases, respectively. Technically unsatisfactory preparations in EM and ICC accounted for 5 and 1 cases, respectively. The overall efficiency in making a diagnosis was 80.7% for EM versus 92.8% for ICC. Aberrant expression of antigens led to difficulties in interpretation of ICC, and EM was particularly helpful. The ultrastructural demonstration of neural differentiation in Ewing's sarcoma/PNET tumors helped place tumors in the PNET category. CONCLUSION: While ICC is the ancillary method of choice in the cytologic diagnosis of MSRCT, EM contributes to the diagnosis and improves diagnostic accuracy.     

A haplotype map of allohexaploid wheat reveals distinct patterns of selection on homoeologous genomes

BackgroundBread wheat is an allopolyploid species with a large, highly repetitive genome. To investigate the impact of selection on variants distributed among homoeologous wheat genomes and to build a foundation for understanding genotype-phenotype relationships, we performed population-scale re-sequencing of a diverse panel of wheat lines.ResultsA sample of 62 diverse lines was re-sequenced using the whole exome capture and genotyping-by-sequencing approaches. We describe the allele frequency, functional significance, and chromosomal distribution of 1.57 million single nucleotide polymorphisms and 161,719 small indels. Our results suggest that duplicated homoeologous genes are under purifying selection. We find contrasting patterns of variation and inter-variant associations among wheat genomes; this, in addition to demographic factors, could be explained by differences in the effect of directional selection on duplicated homoeologs. Only a small fraction of the homoeologous regions harboring selected variants overlapped among the wheat genomes in any given wheat line. These selected regions are enriched for loci associated with agronomic traits detected in genome-wide association studies.ConclusionsEvidence suggests that directional selection in allopolyploids rarely acted on multiple parallel advantageous mutations across homoeologous regions, likely indicating that a fitness benefit could be obtained by a mutation at any one of the homoeologs. Additional advantageous variants in other homoelogs probably either contributed little benefit, or were unavailable in populations subjected to directional selection. We hypothesize that allopolyploidy may have increased the likelihood of beneficial allele recovery by broadening the set of possible selection targets.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0606-4) contains supplementary material, which is available to authorized users.     

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.     

The functional loss of human natural killer cell activity induced by K562 is reversible via an interleukin-2-dependent mechanism

In a recent study, we evaluated the functional status of human natural killer (NK) cells after their interaction with the NK-sensitive tumor target cell (TC), K562. We demonstrated that effector cells (EC), after treatment with K562 for 4 hr, lost greater than 90% of their original lytic activity. In this investigation, we examined whether this functional loss of NK cell activity represented an irreversible event in the NK lytic mechanism. Initial studies focused on the ability of K562-inactivated EC (ECi), which had been separated from their TC, to recover cytolytic activity following an 18-hr incubation. Our results indicated that ECi recovered 28% of their lytic activity in complete medium (CM) alone, 64% in CM containing interferon-beta (IFN-beta), and 91% in CM supplemented with interleukin 2 (IL-2). Analysis of the data revealed, however, that neither IFN-beta nor IL-2 simply boosted the lytic capacity of NK cells which initially escaped inactivation, but also, each cytokine affected the lytic capabilities of EC that were either truly inactivated by K562 or precursor NK (pre-NK) cells. Thus, to evaluate further the basis of IFN-beta and IL-2-induced ECi augmentation, we first treated the EC with IFN-beta or IL-2 prior to their interaction with K562 so that pre-NK cell subsets would be promoted to fully competent NK cells. Both pretreated EC preparations, after interacting with K562 for 4 hr, lost greater than 90% of their original lytic activities. NK inactivation did not result from cell death nor reflect alterations in conjugate formation or the percentages of Leu-7- and Leu-11-positive EC. IL-2-pretreated ECi, as did ECi, regained some lytic activity after incubation in CM alone, but recovered significantly more activity in CM containing IFN-beta or IL-2. In contrast to the restimulation profiles obtained for ECi and IL-2-pretreated ECi, IFN-pretreated ECi regained lytic function after incubation with IL-2, but not appreciably with IFN-beta or in CM alone. Overall, these findings suggest that EC, either untreated or pretreated with IFN-beta or IL-2, significantly lose their lytic capabilities following interaction with K562 while retaining their ability to bind to the TC; IFN-beta acts predominantly on pre-NK cells, but not on ECi; and IL-2 appears to play an important role in restoring lytic potential to functionally inactive NK cells.     

Effective cytochrome P450 (CYP) inhibitors isolated from tarragon (Artemisia dracunculus)

Two effective cytochrome P450 (CYP) inhibitors were isolated from tarragon, Artemisia dracunculus. Their structures were spectroscopically identified as 2E,4E-undeca-2,4-diene-8,10-diynoic acid isobutylamide (1) and 2E,4E-undeca-2,4-diene-8,10-diynoic acid piperidide (2). Both compounds had dose-dependent inhibitory effects on CYP3A4 activity with IC50 values of 10.0 ± 1.3 μM for compound 1 and 3.3 ± 0.2 μM for compound 2, and exhibited mechanism-based inhibition. This is the first reported isolation of effective CYP inhibitors from tarragon (Artemisia dracunculus) purchased from a Japanese market.     

Studies on the mechanism of human natural killer cell-mediated cytolysis: I. Modulation by dexamethasone and arachidonic acid

Natural killer (NK) cell-mediated cytotoxicity, as measured by the lysis of the human erythroleukemic cell line K562, is inhibited by the glucocorticosteroid dexamethasone (DEX). Kinetic analysis revealed that DEX inhibits an early event(s) in the lytic mechanism and that the inhibition is both transient and readily reversible if DEX is removed. The inhibition is not due to the production of a DEX-induced inhibitory protein or decreased target-cell binding. Attempts to counter the effects of DEX through the addition of inducers of NK activity were unsuccessful. Neither the calcium ionophore A23187 nor exogenous cyclic GMP was able to reverse the inhibition by DEX. The addition of arachidonic acid (AA), a pharmacologically active metabolite of phospholipase A-2 activation, was also unsuccessful in reversing the effects of DEX. In fact, AA itself inhibited NK activity in a dose-dependent fashion. This inhibition was not due to reduced target binding and was observed even in the presence of indomethacin. It is concluded that DEX blocks an early membrane-signaling event necessary to activate the lytic mechanism and that inhibition was not through some alternative mechanism. Inhibition of NK activity by arachidonic acid is not yet understood but most likely is not a result of enhanced prostaglandin synthesis. Hence, the study of DEX and AA inhibition provides a new approach to unravel some of the intricacies surrounding NK-mediated tumor target destruction.     

Molecular mapping of an adult plant stem rust resistance gene Sr56 in winter wheat cultivar Arina

Key message

This article covers detailed characterization and naming of QSr.sun - 5BL as Sr56 . Molecular markers linked with adult plant stem rust resistance gene Sr56 were identified and validated for marker-assisted selection.

Abstract

The identification of new sources of adult plant resistance (APR) and effective combinations of major and minor genes is well appreciated in breeding for durable rust resistance in wheat. A QTL, QSr.sun-5BL, contributed by winter wheat cultivar Arina providing 12–15 % reduction in stem rust severity, was reported in an Arina/Forno recombinant inbred line (RIL) population. Following the demonstration of monogenic segregation for APR in the Arina/Yitpi RIL population, the resistance locus was formally named Sr56. Saturation mapping of the Sr56 region using STS (from EST and DArT clones), SNP (9 K) and SSR markers from wheat chromosome survey sequences that were ordered based on synteny with Brachypodium distachyon genes in chromosome 1 resulted in the flanking of Sr56 by sun209 (SSR) and sun320 (STS) at 2.6 and 1.2 cM on the proximal and distal ends, respectively. Investigation of conservation of gene order between the Sr56 region in wheat and B. distachyon showed that the syntenic region defined by SSR marker interval sun209-sun215 corresponded to approximately 192 kb in B. distachyon, which contains five predicted genes. Conservation of gene order for the Sr56 region between wheat and Brachypodium, except for two inversions, provides a starting point for future map-based cloning of Sr56. The Arina/Forno RILs carrying both Sr56 and Sr57 exhibited low disease severity compared to those RILs carrying these genes singly. Markers linked with Sr56 would be useful for marker-assisted pyramiding of this gene with other major and APR genes for which closely linked markers are available.