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.     

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.     

Microsatellite mapping identifies TTKST-effective stem rust resistance gene in wheat cultivars VL404 and Janz

Wheat cultivar VL404 carries seedling resistance to Puccinia graminis f. sp. tritici pathotype TTKST. Monogenic segregation for seedling resistance was observed in a VL404/WL711 recombinant inbred line population and the resistance locus was temporarily designated SrVL. Bulked segregant analysis using Diversity Arrays Technology markers located SrVL on chromosome 2BL. Detailed simple sequence repeat mapping placed SrVL between gwm120 and wmc175, both at genetic distances of 3.3 cM. Based on adult plant responses of Janz and VL404 in India and Kenya, we expected these cultivars to carry the same gene against TTKST. A subset of Diamondbird/Janz doubled haploid (DH) population showed monogenic segregation, when tested against TTKST and the locus was temporarily named SrJNZ. SrVL-linked markers gwm120 and wmc175 flanked SrJNZ at a similar genetic distance, thereby confirming our hypothesis. Chromosome 2BL carries Sr9, Sr16 and Sr28. Sr9 is a multi-allelic locus and all known alleles of Sr9 and Sr16 are ineffective against TTKSK and its derivatives. A recombination value of 16.7 cM between Sr9g-linked stripe rust resistance gene Yr7 and SrJNZ in Diamondbird/Janz DH population suggested that SrJNZ is not an allele at the Sr9 locus. Based on comparison of published genetic distances between Lr13, Sr9, Sr28 and Sr16 with that observed in this study, we concluded SrVL and SrJNZ to be Sr28. This gene was contributed by a common parent Gabo, which also exhibited resistance against TTKST. Sr28-linked markers gwm120 and wmc175 confirmed the presence of this gene in a high proportion of Australian cultivars that showed stem rust resistance in Kenya. These markers can be used for marker-assisted pyramiding of Sr28 with other stem rust resistance genes.     

Molecular characterization of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> isolates from cholera outbreaks in north India

Vibrio cholerae isolates recovered from cholera outbreaks in Bhind district of Madhya Pradesh and Delhi, Northern India were characterized. The O1 serogroup isolates from Bhind outbreak were of Inaba serotype whereas both Ogawa and Inaba serotypes were recovered from Delhi. PCR analysis revealed that only O1 serogroup V. cholerae isolates carried the virulence-associated genes like ctxA, tcpA, ace, and zot. Molecular typing by repetitive sequence based ERIC, VCR1, and VC1 PCR’s revealed similar DNA profile for both Inaba and Ogawa serotypes. A discrete VC1-PCR band identified among the El Tor strains had greater similarity (>97%) to the V. cholerae genome sequence and therefore has the potential to be used as a marker for the identification of the V. cholerae strains. Non-O1 strains recovered from Bhind region differed among themselves as well as from that of the O1 isolates. All the O1 serogroup isolates possessed SXT element and were uniformly resistant to the antibiotics nalidixic acid, polymyxin-B, furazolidone, cloxacilin, trimethoprim-sulfamethaxazole, and vibriostatic agent 0129. Inaba strains from both Delhi and Bhind differed from Ogawa strains by their resistance to streptomycin despite sharing similar DNA patterns in all the three rep-PCRs. Though Delhi and Bhind are separate geographical regions in Northern India, Inaba strains from both these places appear to be closely related owing to their similarity in antibiogram and genetic profile.     

Inter-simple-sequence repeat (ISSR)-PCR for the identification of saprophytic strains of Leptospira

The inter-simple-sequence repeat (ISSR) primers that anneal to a simple repeat of various length and at non-repetitive motifs at 3 and 5 end were attempted for PCR amplification of Leptospira genome. Of the six ISSR primers tested, namely, (AG)₈T, (AG)₈C, (AG)₈G, (CA)₈A, (TG)₈C and (TG)₈G, only primer (AG)₈T produced amplification of 1000 bp in the two non-pathogenic Leptospira species tested, viz; Leptospira biflexa serovar patoc and L. meyeri serovar ranarum, with no amplification in any of the 16 standard pathogenic serovars tested. The remaining five ISSR primers did not exhibit any amplification of the Leptospira genome in either pathogenic or non-pathogenic species. From among 35 Leptospira isolates recovered from hospitalized patients with pyrexia of unknown origin and/or febrile jaundice (12 in number) and from different environmental water sources (23 in number), (AG)₈T ISSR-PCR correctly identified all the 22 isolates from water sources that were confirmed to be non-pathogenic by conventional tests. The results therefore, confirmed the ability of a primer, based on simple-sequence repeat motif, to produce a fragment that is useful as a group genetic marker in Leptospira species. The single nucleotide anchor, T, at the 3 end of the primer appeared to play an important role in differentiation of pathogenic and non-pathogenic species of Leptospira. Multiplex PCR, using ISSR primer, (AG)₈T and the reported 16S rRNA gene primers, specific for pathogenic Leptospira species, or the 23S rRNA Leptospira genus specific primers, provided clear identification of serovars and isolates into pathogenic or non-pathogenic groups.     

Effect of thidiazuron in germinating tamarind seedlings

Summary Tamarind, a multipurpose tropical tree species, is economically important for sustainable development of wasteland due to its hardy nature and adaptability to various agroclimatic ocnditions. Reports on in vitro morphogenesis in this species are limited, due to its recalcitrant and callogenic nature. To overcome these limitations, an attempt was made to induce meristematic activity in seedling explants. Seedlings were germinated in medium with or without thidiazuron (4.54, 9.08, 13.12, 18.16 μM). This growth regulator restricted the differentiation of the apical meristem to form shoots. It triggered proliferation of the meristematic tissue at the cotyledonary node and a large number of meristematic buds appeared in a ridial pattern around the node. The meristematic activity extended to the junction of the epicotyl and hypocotyl, giving rise to buds in the form of protuberances in all sides of the junction. These buds differentiated to form shoot primordia and subsequently to shoots in medium devoid of growth regulators. Plants developed by micrografting of these shoots on seedling-derived rootstocks survived in soil.     

Regulation of cell proliferation and morphogenesis by amino acids inBrassica tissue cultures and its correlation with threonine deaminase

The effect of amino acids has been investigated with respect to the capacity ofBrassica cultures to undergo proliferation and differentiation. Hormone medium without any amino acid resulted in 6% shoot formation. Addition of optimal concentrations of L-leucine and L-isoleucine enhanced shoot formation upto 30% and 60%, respectively. L-methionine, L-threonine and pyruvic acid supported only proliferation but no differentiation. Amino acids had a marked effect on the activity of enzyme threonine deaminase (TD), bothin vivo andin vitro. TD in proliferating callus cultures was 3-fold higher than in differentiating cultures. Amino acids which induced cell proliferation increased TD while those which supported differentiation repressed it. Amino acids which did not alter TD activity had no effect on morphogenesis. The results suggest that amino acids play a regulatory role inBrassica morphogenesis which can be correlated with the activity of threonine deaminase.     

Adult plant stripe rust resistance gene <Emphasis Type="Italic">Yr71</Emphasis> maps close to <Emphasis Type="Italic">Lr24</Emphasis> in chromosome 3D of common wheat

Australian cultivar Sunco carries three adult plant stripe rust resistance genes. One of these genes corresponded to Yr18 in chromosome 7DS; the second, YrCK, was mapped on chromosome 2D. Here, we describe the characterization of the third adult plant resistance (APR) gene from Sunco. Sunco/2*Avocet S-derived lines SA65 (resistant) and SA67 (susceptible) were crossed and a recombinant inbred line F₆ population was generated. Monogenic segregation among SA65/SA67-derived RIL population was demonstrated and the resistance locus was designated YrSA3. Selective genotyping using an iSelect 90 K Infinium SNP array and SSR markers located YrSA3 on chromosome 3D. Development of KASP markers for SNP loci showing association with YrSA3 allowed construction of a genetic map harboring the resistance gene. Ten KASP markers (KASP_8306, KASP_9142, KASP_10438, KASP_16434, KASP_17207, KASP_20836, KASP_23518, KASP_23615, KASP_57983 and KASP_63653), one SSR marker (gwm114b) and Lr24/Sr24 were mapped 1.8 cM distal to YrSA3. Comparison of marker data indicated that the previously named seedling stripe rust resistance gene Yr45 was located proximal to YrSA3, and therefore the latter was formally designated Yr71. Two recombinants carrying Lr24/Sr24 and Yr71 in combination were identified for use as donor sources in wheat breeding programs. The robustness of gwm114b, KASP_16434, KASP_17207 and KASP_20836 for marker-assisted selection of these genes was demonstrated through tests on 74 Australian wheat cultivars.