Automated nuclear image morphometry on fine needle aspiration smears of malignant round cell tumors

OBJECTIVE: To analyze nuclear image morphometry in fine needle aspiration cytology smears of different groups of malignant round cell tumors (MRCTs) to evaluate its diagnostic role. STUDY DESIGN: In this study there were 55 cases of MRCT, consisting of 18 Ewing's sarcoma (EW), 10 neuroblastoma (NB), 5 non-Hodgkin's lymphoma (NHL), 6 rhabdomyosarcoma (RMS), 4 peripheral neuroectodermal tumor (PNET), 8 Wilm's tumor (WT), 2 retinoblastoma (RB) and 2 undifferentiated round cell tumor (URCT). A Leica image cytometer with Quantimet 600 software (Leica, Cambridge, U.K) was used to measure nuclear area, nuclear diameter, nuclear perimeter, nuclear convex perimeter (CP), nuclear roundness and nuclear convex area on hematoxylin and eosin-stained cytologic smears. At least 100 cells were studied in each case. RESULTS: The RB group of tumors showed the highest mean nuclear area (NA), convex area (CA), CP, diameter (D), perimeter (P) and roundness (R). RMS had the highest mean CA, and URCT had the highest mean roundness. ANOVA was performed on the tumors and showed significant differences for all the variables in all the groups (P < .000). All the morphometric data (except roundness) were significantly different in RMS versus all other MRCTs except RB. Similarly, morphometric data on WT were also significantly different from that on NHL. Most of the morphometric data (except CA and R) showed significant differences between RB and all other MRCTs except RMS. PNET, EW and NB could not be differentiated with those variables. CONCLUSION: RMS and RB could successfully be differentiated from all other MRCTs with the help of morphometry. It was not possible to differentiate RMS and RB by image cytometry (ICM) since the ICM data overlapped in those two groups. It was possible to differentiate WT and NHL with ICM. Nuclear ICM was not significantly different in the NB, PNET and ES groups, and probably ICM would not be very helpful to differentiate these groups of MRCT.     

Intracoronary boluses of adenosine and sodium nitroprusside in combination reverses slow/no-reflow during angioplasty: a clinical scenario of ischemic preconditioning

No or slow reflow following percutaneous coronary intervention (PCI), despite the presence of a patent epicardial vessel, is a serious complication resulting in increased morbidity and mortality. In the present study, we have evaluated the combination therapy of adenosine and sodium nitroprusside administered as sequential intracoronary (IC) boluses on no-reflow during PCI. Seventy-five high risk acute coronary syndrome patients who underwent PCI with evidence of initial less than TIMI (thrombolysis in myocardial infarction) III flow or developed deterioration in TIMI flow during the procedure were randomized to prophylactic administration of multiple boluses of IC saline solution, adenosine (12 microg/bolus) or the combination of adenosine (12 microg/bolus) and sodium nitroprusside (50 microg/bolus), sequentially. Assessment of TIMI and the TMP (tissue myocardial perfusion) grade was done and major adverse cardiac events (MACE) were assessed at the end of 6 months. Slow or no-reflow was persistent in 70% patients receiving saline solution, 31% patients receiving adenosine, and 4% patient receiving the combination. IC injection with saline solution did not produce improvement in TIMI flow or TMP grade. IC injection with combination resulted in greater improvement of TIMI flow and TMP grade. The crossover of patients with no-reflow in saline solution group or adenosine with combination treatment was associated with reestablishment of TIMI II in 4 and TIMI III in 20 patients. Our data suggest that combination therapy of adenosine and nitroprusside is safe and provides better improvement in coronary flow and MACE as compared with IC adenosine alone in cases of impaired flow during coronary interventions.     

Effect of pioglitazone and its combination with statins in coronary artery disease patients with hyperinsulinemia

The objective of the study was to demonstrate the effect of pioglitazone and pioglitazone in combination with statin on East Indian patients with hyperinsulinemia and hyperlipidemia. It was a randomized, placebo-controlled, double-blind study with a parallel-group design comprising 83 patients. Patients of either sex with cardiac complications, including hyperlipidemia and (or) diabetes mellitus with or without hyperinsulinemia, were enrolled. Patients over 70 years of age, with renal or hepatic failure, or with severe diabetes mellitus (total glucose >400 mg/dL) were excluded from the study. Enrolled patients were randomly assigned to 4 groups that received placebo, pioglitazone, atorvastatin, or both. Blood samples were collected before and after treatment for analysis of serum glucose, insulin, lipid profile, apolipoprotein (apo) A1, apo B, and fibrinogen. Data were compared with that of patients with normal insulin or hyperinsulinemia. The patients with hyperinsulinemia receiving only pioglitazone showed a significant decrease in insulin levels compared with those with normal insulin levels. These patients also showed a significant increase in HDL levels. However, no significant change was observed in patients treated with both atorvastatin and pioglitazone. Pioglitazone was also found to increase significantly the apo A1 levels in patients with hyperinsulinemia, but there was no significant increase in patients given both atorvastatin and pioglitazone. Our data suggests that pioglitazone should be given preferably to the patients with hyperinsulinemia and statin should not be coadministered.     

Genomic prediction for rust resistance in diverse wheat landraces

Key message

We have demonstrated that genomic selection in diverse wheat landraces for resistance to leaf, stem and strip rust is possible, as genomic breeding values were moderately accurate. Markers with large effects in the Bayesian analysis confirmed many known genes, while also discovering many previously uncharacterised genome regions associated with rust scores.

Abstract

Genomic selection, where selection decisions are based on genomic estimated breeding values (GEBVs) derived from genome-wide DNA markers, could accelerate genetic progress in plant breeding. In this study, we assessed the accuracy of GEBVs for rust resistance in 206 hexaploid wheat (Triticum aestivum) landraces from the Watkins collection of phenotypically diverse wheat genotypes from 32 countries. The landraces were genotyped for 5,568 SNPs using an Illumina iSelect 9 K bead chip assay and phenotyped for field-based leaf rust (Lr), stem rust (Sr) and stripe rust (Yr) responses across multiple years. Genomic Best Linear Unbiased Prediction (GBLUP) and a Bayesian Regression method (BayesR) were used to predict GEBVs. Based on fivefold cross-validation, the accuracy of genomic prediction averaged across years was 0.35, 0.27 and 0.44 for Lr, Sr and Yr using GBLUP and 0.33, 0.38 and 0.30 for Lr, Sr and Yr using BayesR, respectively. Inclusion of PCR-predicted genotypes for known rust resistance genes increased accuracy more substantially when the marker was diagnostic (Lr34/Sr57/Yr18) for the presence-absence of the gene rather than just linked (Sr2). Investigation of the impact of genetic relatedness between validation and reference lines on accuracy of genomic prediction showed that accuracy will be higher when each validation line had at least one close relationship to the reference lines. Overall, the prediction accuracies achieved in this study are encouraging, and confirm the feasibility of genomic selection in wheat. In several instances, estimated marker effects were confirmed by published literature and results of mapping experiments using Watkins accessions.     

Mapping of durable stripe rust resistance in a durum wheat cultivar Wollaroi

Wollaroi, an Australian durum wheat cultivar, produced a low stripe rust response and the alternative parent Bansi was highly susceptible. The Wollaroi/Bansi recombinant inbred line (RIL) population was phenotyped across three consecutive crop seasons. A genetic map of the Wollaroi/Bansi RIL population comprising 799 markers (diversity arrays technology and simple sequence repeat markers) was used to determine the genomic location of stripe rust resistance genes carried by the cultivar Wollaroi. Composite interval mapping detected three consistent quantitative trait loci (QTL) in chromosomes 2A, 3B and 5B. These QTL were named QYr.sun-2A, QYr.sun-3B and QYr.sun-5B. Another QTL, QYr.sun-1B, was detected only in the 2009 crop season. QTL in chromosomes 1B, 2A, 3B and 5B explained on average 6, 9.3, 26.7 and 8.7 %, respectively, of the variation in stripe rust response. All QTL were contributed by Wollaroi. RILs carrying these QTL singly produced intermediate stripe rust severities ranging from 46.2 to 55.7 %, whereas RILs with all four QTL produced the lowest disease severity (34.3 %). The consistently low stripe rust response of Wollaroi for 20 years demonstrated the durability of the resistance loci involved. The QTL combination detected in this study is being transferred to common wheat.     

Genotyping of Indian <Emphasis Type="Italic">Yersinia pestis</Emphasis> strains by MLVA and repetitive DNA sequence based PCRs

India experienced two plague outbreaks in Gujarat and Maharastra during 1994 and then in the Shimla district of Himachal Pradesh during 2002. Yersinia pestis strains recovered from rodents and pneumonic patients during the 1994 outbreaks, pneumonic patients from the 2002 Shimla outbreak and rodents trapped on the Deccan Plateau during a surveillance activity carried out in 1998 were characterized by MLVA, ERIC-PCR and ERIC-BOX-PCR. MLVA genotyping of Indian Y. pestis strains revealed strains of 2 Orientalis, 1 Mediaevalis and 1 Antiqua genotypes distributed in three distinct branches corresponding to their biovar. The Orientalis genotype strains recovered from the 1994 outbreaks and 1998 surveillance activity clustered in one branch while the Antiqua biovar strains from the Shimla outbreak and the Mediaevalis strain recovered from a rodent trapped on the Deccan Plateau region during surveillance formed the other branches. The Orientalis Y. pestis strains recovered from rodents and patients from the 1994 plague outbreaks exhibited similar MLVA, ERIC-PCR and ERIC-BOX-PCR profiles and these were closely related to the Orientalis strains recovered from the rodents trapped on the Deccan Plateau. These data provide evidence for the possible linkage between the Y. pestis strains resident in the endemic region and those that were associated with the 1994 plague outbreaks. Mediaevalis and Antiqua biovars also were recovered from the environmental reservoir on the Deccan Plateau and from the pneumonic patients of 2002 plague outbreak. Therefore, as in Central Asian and African regions, Antiqua and Mediaevalis biovars seem to be well established in the Indian subcontinent as well. ERIC-PCR DNA fingerprinting delineated genotypes similar to those defined by MLVA. Thus ERIC-PCR appears to have the potential to be used as a molecular marker in the molecular epidemiological investigations of plague.     

Generation and Characterization of Murine Monoclonal Antibodies to Recombinant YopM, YopB and LcrV Proteins of Yersinia pestis

Summary YopM, an effector, YopB, a translator, and LcrV, a regulator, are proteins forming important componants of type III secretion system of Yersinia pestis. Recombinant truncated YopM of 32 kDa, YopB of 28 kDa and LcrV of 31 kDa sizes were utilized for priming BALB/c mice for the generation of monoclonal antibodies following standard poly-ethylene glycol (PEG) fusion protocol. Nine, 10 and 6 stabilized hybridoma cell lines could be generated against YopM, YopB and LcrV proteins, respectively. All these monoclonal antibodies were found reactive to Y. pestis strain A1122 and did not show any cross-reactivity to Y. enterocolitica, Y. pseudotuberculosis, Y. kristensenii, Y. frederiksenii, Y. intermedia, Klebsiella pneumoniae, Escherichia coli, Salmonella typhi, Salmonella abortus-equi and Staphylococcus aureus tested by ELISA and Western blotting. Monoclonal antibodies also exhibited reactivity to their corressponding native protein antigens in Y. pestis i.e. 42 kDa for YopM, 41 kDa for YopB and 37 kDa for LcrV in immunoblotting. Reactivity of monoclonal antibodies was further assessed on 26 Y. pestis isolates including 18 from 1994 plague outbreak regions (11 from pneumonic patients, 7 from rodents) and 8 from rodents of Deccan plateau of Southern India by Western blotting as well as by sandwich ELISA. The monoclonal antibodies could specifically locate the expression of yopM, yopB and lcrV genes among these Indian Y. pestis strains as well. Results obtained with sandwich ELISA and Western blot were identical to those observed by PCR. Monoclonal antibodies to Yops, therefore, can be employed for an early and reliable identification of virulent Y. pestis strains.