Identification of heat responsive genes in pea stipules and anthers through transcriptional profiling.

Field pea (Pisum sativum L.), a cool-season legume crop, is known for poor heat tolerance. Our previous work identified PR11-2 and PR11-90 as heat tolerant and susceptible lines in a recombinant inbred population. CDC Amarillo, a Canadian elite pea variety, was considered as another heat tolerant variety based on its similar field performance as PR11-2. This study aimed to characterize the differential transcription. Plants of these three varieties were stressed for 3 h at 38°C prior to self-pollination, and RNAs from heat stressed anthers and stipules on the same flowering node were extracted and sequenced via the Illumina NovaSeq platform for the characterization of heat responsive genes. In silico results were further validated by qPCR assay. Differentially expressed genes (DEGs) were identified at log2 |fold change (FC)| ≥ 2 between high temperature and control temperature, the three varieties shared 588 DEGs which were up-regulated and 220 genes which were down-regulated in anthers when subjected to heat treatment. In stipules, 879 DEGs (463/416 upregulation/downregulation) were consistent among varieties. The above heat-induced genes of the two plant organs were related to several biological processes i.e., response to heat, protein folding and DNA templated transcription. Ten gene ontology (GO) terms were over-represented in the consistently down-regulated DEGs of the two organs, and these terms were mainly related to cell wall macromolecule metabolism, lipid transport, lipid localization, and lipid metabolic processes. GO enrichment analysis on distinct DEGs of individual pea varieties suggested that heat affected biological processes were dynamic, and variety distinct responses provide insight into molecular mechanisms of heat-tolerance response. Several biological processes, e.g., cellular response to DNA damage stimulus in stipule, electron transport chain in anther that were only observed in heat induced PR11-2 and CDC Amarillo, and their relevance to field pea heat tolerance is worth further validation.

Development of genome wide transposable elements based repeat junction markers in Jatropha (Jatropha curcas L.).

Jatropha curcas is a potential biodiesel crop and a highly adaptable species to various agro-climatic conditions. In this study, we have utilized transposable elements' (TE) repeat junctions (RJs) which are an important constituent of the genome, used to form a genome-wide molecular marker platform owing to its use in genomic studies of plants. We screened our previously generated Jatropha hybrid genome assembly of size 265 Mbp using RJPrimers pipeline software and identified a total of 1274 TE junctions. For the predicted RJs, we designed 2868 polymerase chain reaction (PCR) based RJ markers (RJMs) flanking the junction regions. In addition to marker design, the identified RJs were utilized to detect 225,517 TEs across the genome. The different types of transposable repeat elements mainly were scattered into Retro, LTR, Copia and Gypsy categories. The efficacy of the designed markers was tested by utilizing a subset of RJMs selected randomly. We have validated 96 randomly selected RJ primers in a group of 32 J. curcas genotypes and more than 90% of the markers effectively intensified as amplicons. Of these, 10 primers were shown to be polymorphic in estimating genetic diversity among the 32 Jatropha lines. UPGMA cluster analysis revealed the formation of two clusters such as A and B exhibiting 85.5% and 87% similarity coefficient respectively. The various RJMs identified in this study could be utilized as a significant asset in Jatropha functional genomics including genome determination, mapping and marker-assisted selection.

Development of a Sequence-Based Reference Physical Map of Pea (Pisum sativum L.).

Whole genome profiling (WGP) is a sequence-based physical mapping technology and uses sequence tags generated by next generation sequencing for construction of bacterial artificial chromosome (BAC) contigs of complex genomes. The physical map provides a framework for assembly of genome sequence and information for localization of genes that are difficult to find through positional cloning. To address the challenges of accurate assembly of the pea genome (∼4.2 GB of which approximately 85% is repetitive sequences), we have adopted the WGP technology for assembly of a pea BAC library. Multi-dimensional pooling of 295,680 BAC clones and sequencing the ends of restriction fragments of pooled DNA generated 1,814 million high quality reads, of which 825 million were deconvolutable to 1.11 million unique WGP sequence tags. These WGP tags were used to assemble 220,013 BACs into contigs. Assembly of the BAC clones using the modified Fingerprinted Contigs (FPC) program has resulted in 13,040 contigs, consisting of 213,719 BACs, and 6,294 singleton BACs. The average contig size is 0.33 Mbp and the N50 contig size is 0.62 Mbp. WGPTM technology has proved to provide a robust physical map of the pea genome, which would have been difficult to assemble using traditional restriction digestion based methods. This sequence-based physical map will be useful to assemble the genome sequence of pea. Additionally, the 1.1 million WGP tags will support efficient assignment of sequence scaffolds to the BAC clones, and thus an efficient sequencing of BAC pools with targeted genome regions of interest.

De Novo Sequencing and Hybrid Assembly of the Biofuel Crop Jatropha curcas L.: Identification of Quantitative Trait Loci for Geminivirus Resistance.

Jatropha curcas is an important perennial, drought tolerant plant that has been identified as a potential biodiesel crop. We report here the hybrid de novo genome assembly of J. curcas generated using Illumina and PacBio sequencing technologies, and identification of quantitative loci for Jatropha Mosaic Virus (JMV) resistance. In this study, we generated scaffolds of 265.7 Mbp in length, which correspond to 84.8% of the gene space, using Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis. Additionally, 96.4% of predicted protein-coding genes were captured in RNA sequencing data, which reconfirms the accuracy of the assembled genome. The genome was utilized to identify 12,103 dinucleotide simple sequence repeat (SSR) markers, which were exploited in genetic diversity analysis to identify genetically distinct lines. A total of 207 polymorphic SSR markers were employed to construct a genetic linkage map for JMV resistance, using an interspecific F2 mapping population involving susceptible J. curcas and resistant Jatropha integerrima as parents. Quantitative trait locus (QTL) analysis led to the identification of three minor QTLs for JMV resistance, and the same has been validated in an alternate F2 mapping population. These validated QTLs were utilized in marker-assisted breeding for JMV resistance. Comparative genomics of oil-producing genes across selected oil producing species revealed 27 conserved genes and 2986 orthologous protein clusters in Jatropha. This reference genome assembly gives an insight into the understanding of the complex genetic structure of Jatropha, and serves as source for the development of agronomically improved virus-resistant and oil-producing lines.

Construction of high-density linkage maps for mapping quantitative trait loci for multiple traits in field pea (Pisum sativum L.).

BACKGROUND: The objective of this research was to map quantitative trait loci (QTLs) of multiple traits of breeding importance in pea (Pisum sativum L.). Three recombinant inbred line (RIL) populations, PR-02 (Orb x CDC Striker), PR-07 (Carerra x CDC Striker) and PR-15 (1-2347-144 x CDC Meadow) were phenotyped for agronomic and seed quality traits under field conditions over multiple environments in Saskatchewan, Canada. The mapping populations were genotyped using genotyping-by-sequencing (GBS) method for simultaneous single nucleotide polymorphism (SNP) discovery and construction of high-density linkage maps. RESULTS: After filtering for read depth, segregation distortion, and missing values, 2234, 3389 and 3541 single nucleotide polymorphism (SNP) markers identified by GBS in PR-02, PR-07 and PR-15, respectively, were used for construction of genetic linkage maps. Genetic linkage groups were assigned by anchoring to SNP markers previously positioned on these linkage maps. PR-02, PR-07 and PR-15 genetic maps represented 527, 675 and 609 non-redundant loci, and cover map distances of 951.9, 1008.8 and 914.2 cM, respectively. Based on phenotyping of the three mapping populations in multiple environments, 375 QTLs were identified for important traits including days to flowering, days to maturity, lodging resistance, Mycosphaerella blight resistance, seed weight, grain yield, acid and neutral detergent fiber concentration, seed starch concentration, seed shape, seed dimpling, and concentration of seed iron, selenium and zinc. Of all the QTLs identified, the most significant in terms of explained percentage of maximum phenotypic variance (PVmax) and occurrence in multiple environments were the QTLs for days to flowering (PVmax = 47.9%), plant height (PVmax = 65.1%), lodging resistance (PVmax = 35.3%), grain yield (PVmax = 54.2%), seed iron concentration (PVmax = 27.4%), and seed zinc concentration (PVmax = 43.2%). CONCLUSION: We have identified highly significant and reproducible QTLs for several agronomic and seed quality traits of breeding importance in pea. The QTLs identified will be the basis for fine mapping candidate genes, while some of the markers linked to the highly significant QTLs are useful for immediate breeding applications.

In vitro and in vivo metabolism of a novel cannabinoid-1 receptor inverse agonist, taranabant, in rats and monkeys.

The metabolism and excretion of taranabant (MK-0364, N-[(1S,2S)-3-(4-chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2{[5-(trifluoromethyl)pyridine-2-yl]oxy}propanamide), a potent cannabinoid-1 receptor inverse agonist, were evaluated in rats and rhesus monkeys. Following administration of [¹4C]taranabant, the majority of the radioactivity was excreted within 72 h. In both rats and rhesus monkeys, taranabant was eliminated primarily via oxidative metabolism, followed by excretion of metabolites into bile. Major pathways of metabolism that were common to rats and rhesus monkeys included hydroxylation at the benzylic carbon adjacent to the cyanophenyl ring to form a biologically active circulating metabolite M1, and oxidation of one of the two geminal methyl groups of taranabant or M1 to the corresponding diastereomeric carboxylic acids. Oxidation of the cyanophenyl ring, followed by conjugation with glutathione or glucuronic acid, was a major pathway of metabolism only in the rat and was not detected in the rhesus monkey. Metabolism profiles of taranabant in liver microsomes in vitro were qualitatively similar in rats, rhesus monkeys and humans and included formation of M1 and oxidation of taranabant or M1 to the corresponding carboxylic acids via oxidation of a geminal methyl group. In human liver microsomes, metabolism of taranabant was mediated primarily by CYP3A4.

Plant cysteine proteases that evoke itch activate protease-activated receptors.

BACKGROUND: Bromelain, ficin and papain are cysteine proteases from plants that produce itch upon injection into skin. Their mechanism of action has not been considered previously. OBJECTIVES: To determine the mechanism by which these proteases function. METHODS: The ability of these proteases to activate protease-activated receptors was determined by ratiometric calcium imaging. RESULTS: We show here that bromelain, ficin and papain activate protease-activated receptors 2 and 4. CONCLUSIONS: Bromelain, ficin and papain function as signalling molecules and activate protease-activated receptors. Activation of these receptors is the likely mechanism by which these proteases evoke itch.

Fine-needle aspiration of extranodal and extramedullary hematopoietic malignancies.

There is relatively little information concerning the use of fine-needle aspiration (FNA) to diagnose extranodal and extramedullary hematopoietic malignancies. Seventy-one such cases diagnosed by FNA form the basis of this study. Seventy-one cases of FNAs performed between 1988 and 1998 on extranodal and extramedullary hematopoietic malignancies were reviewed in order to evaluate the usefulness of this technique in diagnosing these entities as well as to assess patterns of relapse. There were 45 male and 26 female patients ranging in age from 29-86 years (mean, 68 years). Sixty-six patients had a previous history of a hematopoietic malignancy. Aspirates from 65 of these patients were consistent with the patient's known primary. One aspirate of a paravertebral mass from a multiple myeloma patient showed extramedullary hematopoiesis. The remaining five aspirates were cases of multiple myeloma that first presented as soft tissue masses. The most common malignancies were lymphoma: 52 cases (73%), 48 large cell lymphomas, four mixed small and large cell lymphoma; followed by multiple myeloma: 12 cases (17%); leukemia: four cases (5.4%); Hodgkin disease: two cases (2.8%); and one case of extramedullary hematopoiesis. The aspirate sites were soft tissue: 23 cases (32%); bone: 17 cases (24%); kidney: 14 cases (20%); liver: 11 cases (15%); lung: three cases (4%); adrenal: two cases (3%); and eye: one case. The interval between primary diagnosis and FNA was 1-36 months (mean, 13 months). In conclusion, 98% of the aspirates were neoplastic in patients with a known history of hematopoietic malignancies. The most common site of involvement was soft tissue in 23 (32%) cases. In five patients with multiple myeloma, the FNA diagnosis prompted a work-up to find the primary site of involvement. FNA is a useful technique in assessing extranodal and extramedullary hematopoietic malignancies.

Fine-needle aspiration biopsy of the liver: a multicenter study of 602 radiologically guided FNA.

With improved radiologic techniques fine-needle aspiration (FNA) is becoming a rapid, effective diagnostic method in evaluating a wide range of liver masses. Review of six hundred two radiologically guided liver aspirates performed over a ten-year period forms the basis of this report.

Chrysoptin is a potent glycoprotein IIb/IIIa fibrinogen receptor antagonist present in salivary gland extracts of the deerfly.

Salivary gland lysates of the deerfly (genus Chrysops) contain chrysoptin, an inhibitor of ADP-induced platelet aggregation, which presumably assists the fly in obtaining a blood meal. Chrysoptin has now been isolated, and its cDNA has been cloned and expressed. Chrysoptin was purified to homogeneity using anion exchange and hydrophobic interaction chromatography and found to be a protein with a molecular mass of 65 kDa as determined by gel electrophoresis. N-terminal amino acid sequencing allowed for the synthesis of degenerate oligonucleotides that led to cloning, from salivary gland specific mRNA, of the cDNA encoding this platelet inhibitor. No RGD sites are present in the predicted sequence. A search of GenBank(TM) did not reveal significant sequence homology between chrysoptin and other proteins. The molecular mass predicted from the cDNA was 59 kDa. Predicted glycosylation and phosphorylation sites may account for this difference in molecular mass, as recombinant chrysoptin expressed in Sf21 cells had a molecular mass of 65 kDa, matching that of the natural protein. Chrysoptin functions by inhibiting the binding of fibrinogen to the fibrinogen/glycoprotein IIb/IIIa receptor on platelets with an IC(50) of 95 pmol. These results reveal that insect salivary glands are a source of fibrinogen receptor antagonists.

Role of fine-needle aspiration in the clinical management of solid organ transplant recipients: a review.

BACKGROUND: We evaluated the clinical course of the solid-organ transplant population at our institutions to determine the role of fine-needle aspiration (FNA) in the clinical management of this subgroup of patients. METHODS: 1196 allograft recipients (522 liver, 288 cardiac, 250 renal, 131 lung, 5 heart and lung) were reviewed. A total of 62 (5.2%) (32 liver, 23 heart, 6 lung, and 1 renal) transplant patients underwent an FNA procedure. Thirty-seven males and 25 females were included, ranging in age from 18 to 71 years (mean 50 years). RESULTS: Of the 62 fine-needle aspirates, 29 (47%) were neoplastic. The most common malignancies aspirated were malignant solid tumors (15 cases)-including 8 epithelial malignancies, 5 hepatocellular carcinomas, and 2 mesenchymal neoplasms-followed by posttransplant lymphoproliferative disorders (14 cases). Thirteen (21%) aspirates were inflammatory. The remaining 20 (32%) cases were benign aspirates from various sites (9 liver, 3 breast, 2 thyroid, 2 soft tissue, 2 lung, and 2 vertebral body). Surgical and/or autopsy material was available in 34 cases (55%). There was agreement between the tissue diagnosis and FNA material in 33 cases (97%). One case (3%) was a false negative. No false-positive cases were recorded. CONCLUSIONS: This study showed that over 50% of the aspirates were benign, justifying a conservative approach in the clinical management of these patients. Histologic correlation was available in 54% of the cases with an overall specificity of 100% and a sensitivity of 97%. We conclude that FNA is a highly sensitive and specific technique in the evaluation of lesions occurring in posttransplant patients. Cancer (Cancer Cytopathol)

Utilization of fine-needle aspiration in the diagnosis of metastatic tumors to the kidney.

Renal masses secondary to metastases are not common. Few comprehensive reviews exist, which consist primarily of autopsy and radiologic reports. The purpose of this study was to review the types and incidences of various neoplasms which metastasize to the kidney and to determine the usefulness of fine-needle aspiration (FNA) in diagnosing them. Two hundred and sixty-one radiologically guided FNAs of renal lesions over a 9-yr period were reviewed. The diagnoses of the 261 renal FNAs were as follows: 136 (52%) were malignant, 111 (43%) were benign, and 14 (5%) were unsatisfactory. Of the 136 positive FNAs, 28 (21%) revealed metastatic tumors. The overall incidence of renal FNAs displaying metastatic tumors was 11%. Among the 28 patients with metastases to the kidney, 23 patients were men and 5 were women, with the mean age being 58 yr. Twenty-five patients (89%) had prior history of a primary malignancy, including lung carcinoma (11 cases, 39%), lymphoma (8 cases, 29%), hepatocellular carcinoma (3 cases, 11%), and one case each of breast, pancreatic, and cervical cancer. In the remaining 3 patients (11%), with metastatic adenocarcinoma (2 cases) and squamous-cell carcinoma (1 case), the primary tumor site remained unknown despite an extensive clinical workup. Overall survival after FNA was poor, with a mean of 9.8 mo. FNA is useful in the diagnosis of masses in the kidney secondary to metastatic disease. This information is of clinical importance, principally in the exclusion of a primary malignancy, but also to avoid unnecessary surgery and to plan for subsequent patient care.

Low-stage carcinoma of the male breast. A histologic, immunohistochemical, and flow cytometric comparison with localized female breast carcinoma.

Male breast carcinoma (MBC) accounts for only 1% of total mammary carcinomas. Controversy exists about whether MBC differs clinically and pathologically from female breast carcinoma (FBC). We compared 10 archival cases with 75 stage-matched FBCs. Clinical data, histologic details, immunostains for mammary lineage markers, and results of several putative "prognostic" analyses were addressed, including DNA ploidy and expression of c-erbB-2 (neu) oncoprotein and p53 protein. Cumulative literature data on 2,530 MBCs were contrasted with information from 135 institutional cases of FBC. A statistically significant difference in grade 3 lesions at low stage persisted when MBCs of all stages were compared with similar FBCs. For stages I and IIA, 5-year survival was 60% and 86% for MBCs and FBCs, respectively (also statistically significant). This difference disappeared when all stages were compared. A similar number of MBCs and FBCs, regardless of stage, demonstrated DNA aneuploidy with or without synthesis of S-100 protein, gross cystic disease fluid protein-15, c-erbB-2 protein, and p53 protein. Hormone receptor positivity was more common in MBC than in FBC at high tumor stages. Low-stage MBC and FBC differ biologically; MBCs tend to manifest at a higher grade with lessened 5-year survival. However, aside from distinctions in hormone receptor proteins, broader comparison of MBC and FBC at stages IIB and higher shows no significant differences in 5-year survival or expression of breast cancer-associated gene products.

Utilization of core wash material in the diagnosis of breast lesions by stereotactic needle biopsy.

BACKGROUND: Several reports have compared the results of fine-needle aspiration and stereotactic core needle biopsy in nonpalpable breast lesions. In this study the authors describe a simple method to retrieve cytologic material from a core breast biopsy sample that provides the diagnosis within 1 hour of the procedure. METHODS: Two hundred and eleven nonpalpable breast lesions were biopsied. Each core needle biopsy sample was placed in a mesh bag, and the bag and needle notch were washed in Cytolyt solution to obtain a monolayer using a commercial ThinPrep processor. The cytologic diagnoses were divided into four categories: benign, suspicious, malignant, and unsatisfactory, which then were compared with core needle biopsy results. RESULTS: Cytology reports of 211 lesions were as follows: 169 lesions (80%) were benign, 16 lesions (7.6%) were suspicious, 11 lesions (5.2%) were malignant, and 15 lesions (7.1%) were unsatisfactory. Core needle biopsy showed 165 of 169 samples (98%) to be benign and 4 to be malignant. Of the 16 suspicious smears, 10 were invasive carcinoma, 2 were in situ lesions, 3 were hyperplasias, and 1 was fibrosis. Of the 11 malignant smears, 10 were confirmed on core needle biopsy and 1 was read as atypia on the first core needle biopsy sample and malignant on a second, separate, follow-up core needle biopsy. Of the 15 unsatisfactory samples, 14 were found to be benign and 1 was found to be malignant on a separate, follow-up core needle biopsy. CONCLUSIONS: The core wash technique was 85% sensitive and 98% specific for malignancy. Only 7% of specimens were insufficient for diagnosis, and 93% of these were proven to be benign. This technique is useful for immediate (within 1 hour) diagnosis of breast lesions, alleviating patient anxiety and supplementing the diagnostic yield of the core biopsy.

The yield of bone marrow biopsy and culture compared with blood culture in the evaluation of HIV-infected patients for mycobacterial and fungal infections.

PURPOSE: To compare the clinical utility of bone marrow biopsy and culture specimens with blood cultures for mycobacterial and fungal infections among human immunodeficiency virus (HIV)-infected patients. PATIENTS AND METHODS: All bone marrow biopsies obtained from HIV-infected patients at the University of Alabama at Birmingham (UAB) Medical Center during 1993 to 1995 were blindly reviewed in a standardized format. Bone marrow culture results and blood culture results obtained within 6 weeks of each bone marrow study were compiled. Medical records were reviewed to determine indications for performing bone marrow biopsies, empiric or prophylactic antimicrobial therapies preceding the biopsy, and CD4 counts. RESULTS: Eighty-two bone marrow studies were obtained from 76 patients. Most were performed during the evaluation of fever, cytopenia, or weight loss. Of 55 bone marrow mycobacterial cultures, 13 yielded Mycobacterium avium complex (MAC) and 2 yielded M tuberculosis (MTB). Of 51 bone marrow fungal cultures performed, 2 yielded Cryptococcus neoformans and 1 Histoplasma capsulatum. All patients with a bone marrow culture positive for MAC had a CD4 count of 20 cells/mm3 or less. The mean CD4 count in this group (+/-95% confidence interval) (8+/-3 cells/mm3) was lower than that of culture-negative cases (41+/-25 cells/mm3); P <0.015). When bone marrow cultures and mycobacterial blood cultures were concurrently obtained, results were usually in agreement between the two sites. The mean time until the report of positive mycobacterial bone marrow cultures (22+/-5 days) was similar to that for blood cultures (24+/-3 days). Most (84%) patients with multiple mycobacterial cultures had completely concordant results (all positive or all negative). When blood or bone marrow culture yielded mycobacteria, only 29% of the corresponding bone marrow examinations revealed stainable acid-fast bacilli (AFB). In contrast, all 3 cases with positive fungal bone marrow cultures also had stainable organisms on histologic examination. CONCLUSIONS: The combined use of bone marrow biopsy and culture as well as blood cultures provide the maximum diagnostic yield when evaluating patients with AIDS for mycobacterial or fungal infections. However, when mycobacterial infections were diagnosed, bone marrow results seldom provided more immediate or specific information than lysis centrifugation blood cultures. A single lysis centrifugation blood culture should be the first step in the routine evaluation of HIV-infected patients when disseminated MAC infection is suspected.

Posttransplant lymphoproliferative disorders: a fine-needle aspiration biopsy study.

Fine-needle aspiration biopsy (FNAB) has been used with high sensitivity and specificity in the diagnosis of both Hodgkin's and non-Hodgkin's lymphoma. However, studies of FNAB of posttransplant lymphoproliferative disorders (PTLDs) are rare. The clinical course of 593 allograft recipients (cardiac, 288; renal, 250; lung, 50; and heart/lung, 5) was reviewed. Twenty-six patients developed PTLD with an overall incidence of 4.4%. Of these patients, 12 underwent FNAB. Their age ranged from 33-67 yr (mean, 55 yr). The interval between transplantation and FNAB ranged between 2-14 mo (average, 8.4 mo). The lungs were the most common site aspirated (7 cases), followed by lymph nodes (3 cases) and other extranodal sites (2 cases, liver and paraspinal mass). The cytologic features of these aspirates could be classified into two categories: a polymorphous smear composed of a spectrum of mature and immature lymphocytes with scattered plasma cells and histiocytes; and a monotonous population of large lymphoid cells consistent with malignant lymphoma, large-cell type. Surgical biopsies were available in 10 (83.3%) cases and confirmed the FNAB diagnosis. In summary, FNAB appears to be a highly sensitive and specific diagnostic tool in patients with PTLD.