Organometallic Bridge Diversification of Bicyclo[1.1.1]pentanes.

Bicyclo[1.1.1]pentane (BCP) derivatives have attracted significant recent interest in drug discovery as alkyne, tert-butyl and arene bioisosteres, where their incorporation is frequently associated with increased compound solubility and metabolic stability. While strategies for functionalisation of the bridgehead (1,3) positions are extensively developed, platforms allowing divergent substitution at the bridge (2,4,5) positions remain limited. Recent reports have introduced 1-electron strategies for arylation and incorporation of a small range of other substituents, but are limited in terms of scope, yields or practical complexity. Herein, we show the synthesis of diverse 1,2,3-trifunctionalised BCPs through lithium-halogen exchange of a readily accessible BCP bromide. When coupled with medicinally relevant product derivatisations, our developed 2-electron "late stage" approach provides rapid and straightforward access to unprecedented BCP structural diversity (>20 hitherto-unknown motifs reported). Additionally, we describe a method for the synthesis of enantioenriched "chiral-at-BCP" bicyclo[1.1.1]pentanes through a novel stereoselective bridgehead desymmetrisation.

Bridge Heteroarylation of Bicyclo[1.1.1]pentane Derivatives.

Herein, we report the decarboxylative Minisci heteroarylation of bicyclo[1.1.1]pentane (BCP) and 2-oxabicyclo[2.1.1]hexane (oBCH) derivatives at the bridge positions. In an operationally simple, photocatalyst-free process, free bridge carboxylic acids are directly coupled with nonprefunctionalized heteroarenes to provide rare examples of polysubstituted BCP and oBCH derivatives in synthetically useful yields. Additionally, the impact of the BCP core on the physicochemical properties of a representative example compared to those of its all-aromatic ortho- and meta-substituted analogues is evaluated.

Canary Seed (Phalaris canariensis L.) Peptides Prevent Obesity and Glucose Intolerance in Mice Fed a Western Diet.

Previous research showed that canary seed (Phalaris canariensis L.) peptides (CSP) possess robust in vitro antiobesity properties via inhibition of pancreatic lipase (PL). Nevertheless, no studies have yet explored their antiobesity properties in vivo. Consequently, we investigated the effects of CSP in C57BL/6J mice under a Western diet (WD). Mice were assigned into groups and fed a normal diet (ND) or a WD accompanied by an oral dose of CSP (250 or 500 mg/kg/day), orlistat (40 mg/kg/day), or distilled water. The results showed that consuming CSP can provide metabolic benefits, including preventing weight gain by up to 20%, increasing glucose tolerance, and reducing insulin, leptin, and LDL/VLDL levels in plasma. Conversely, total ghrelin was unaffected by CSP-500, but decreased by CSP-250, and amplified by orlistat. Surprisingly, CSP-250 was more effective in preventing weight gain and promoting satiety than CSP-500. Parallel to this, protein absorption in CSP-500 was decreased, supported by a rise in fecal crude protein (+3.5%). Similarly, fecal fat was increased by orlistat (38%) and was unaffected by CSP-250 (3.0%) and CSP (3.0%), comparatively to WD (2.5%). Despite this, both CSP treatments were equally effective in decreasing hepatic steatosis and avoiding hyperlipidemia. Furthermore, the enzymatic analysis showed that CSP-PL complexes dissociated faster (15 min) than orlistat-PL complexes (41 min). Lastly, CSP did not affect expression of hepatic lipid oxidation genes ACO and PPAR-α, but reduced the expression of the hydrolase gene LPL, and lipogenesis related genes FAS and ACC. Taken together, these results suggest that CSP antiobesity mechanism relies on lipid metabolism retardation to increase fat transit time and subsequently suppress hunger.

Oxidative Stress Protection by Canary Seed (Phalaris canariensis L.) Peptides in Caco-2 Cells and Caenorhabditis elegans.

During oxidative stress, degenerative diseases such as atherosclerosis, Alzheimer's, and certain cancers are likely to develop. Recent research on canary seed (Phalaris canariensis) peptides has demonstrated the high in vitro antioxidant potential. Thus, this study aimed to assess the cellular and in vivo antioxidant capacity of a low-molecular-weight (<3 kDa) canary seed peptide fraction (CSPF) using Caco-2 cells and the Caenorhabditis elegans model. The results show that the CSPF had no cytotoxicity effect on Caco-2 cells at any tested concentration (0.3−2.5 mg/mL). Additionally, the cellular antioxidant activity (CAA) of the CSPF was concentration-dependent, and the highest activity achieved was 80% by the CSPF at 2.5 mg/mL. Similarly, incubation with the CSPF significantly mitigated the acute and chronic oxidative damage, extending the lifespan of the nematodes by 88 and 61%, respectively. Furthermore, it was demonstrated that the CSPF reduced the accumulation of reactive oxygen species (ROS) to safe levels after sub-lethal doses of pro-oxidant paraquat. Quantitative real-time PCR revealed that the CSPF increased the expression of oxidative-stress-response-related gene GST-4. Overall, these results show that the CSPFs relied on GST-4 upregulation and scavenging of free radicals to confer oxidative stress protection and suggest that a CSPF can be used as a natural antioxidant in foods for health applications.

Bridge Functionalisation of Bicyclo[1.1.1]pentane Derivatives.

"Escaping from flatland", by increasing the saturation level and three-dimensionality of drug-like compounds, can enhance their potency, selectivity and pharmacokinetic profile. One approach that has attracted considerable recent attention is the bioisosteric replacement of aromatic rings, internal alkynes and tert-butyl groups with bicyclo[1.1.1]pentane (BCP) units. While functionalisation of the tertiary bridgehead positions of BCP derivatives is well-documented, functionalisation of the three concyclic secondary bridge positions remains an emerging field. The unique properties of the BCP core present considerable synthetic challenges to the development of such transformations. However, the bridge positions provide novel vectors for drug discovery and applications in materials science, providing entry to novel chemical and intellectual property space. This Minireview aims to consolidate the major advances in the field, serving as a useful reference to guide further work that is expected in the coming years.

Superior sagittal sinus-to-internal jugular vein bypass shunt with covered stent construct for intractable intracranial hypertension resulting from iatrogenic supratorcular sinus occlusion: technical note.

BACKGROUND: Acute occlusion of the posterior sagittal sinus may lead to dramatic increase in intracranial pressure (ICP), refractory to standard treatment. Hybrid vascular bypass of cranial venous outflow into the internal jugular vein (IJV) has seldom been described for this in recent neurosurgical literature. OBJECTIVE: To describe creation of a novel vascular bypass shunt from the superior sagittal sinus (SSS) to internal jugular vein (IJV) utilizing a covered stent-Dacron graft construct for control of refractory ICP. METHODS: We illustrate a patient with refractory ICP increases after acute sinus ligation that was performed to halt torrential bleeding from intraoperative injury. A temporary shunt was created that successfully controlled ICP. From the promising results of the temporary shunt, we utilized a prosthetic hybrid bypass graft to function as a shunt from the sagittal sinus to IJV. Yet the associated anticoagulation led to complications and a poor outcome. RESULTS: Rapid and sustained ICP reduction can be expected after sagittal sinus-to-jugular bypass shunt placement in acute sinus occlusion. Details of the surgical technique are described. Heparin anticoagulation, while imperative, is also associated with worrisome complications. CONCLUSION: Acute occlusion of posterior third of sagittal sinus carries a very malignant clinical course. Intractable intracranial hypertension from acute sinus occlusion may be effectively treated with a SSS-IJV bypass shunt. A covered stent construct provides an effective vascular bypass conduit. However, the anticoagulation risk can lead to fatal outcomes. The neurosurgeon must always strive for primary repair of an injured sinus.

Previous thoracic aortic repair is not associated with adverse outcomes after thoracic endovascular aortic repair.

BACKGROUND: As many as 20% of patients who have undergone previous thoracic aortic repair will require reintervention, which could entail thoracic endovascular aortic repair (TEVAR). A paucity of data is available on mortality and the incidence of spinal cord ischemia (SCI) and other postoperative complications associated with TEVAR after previous aortic repairs exclusive to the thoracic aorta. The aim of the present study was to assess the effect of previous thoracic aortic repair on the 30-day mortality and SCI outcomes for patients after TEVAR. METHODS: The Society for Vascular Surgery Vascular Quality Initiative database was queried for all cases of TEVAR from 2012 to 2018. Patients were excluded if they had undergone previous abdominal aortic repair, the TEVAR had extended beyond aortic zone 5, or SCI data were missing. The 3 cohorts compared were TEVAR with previous ascending aortic or aortic arch repair (group 1), TEVAR with previous descending thoracic aortic repair (group 2), and TEVAR without previous repair (group 3). The primary outcomes of interest were 30-day mortality and SCI. The secondary outcomes included stroke, myocardial infarction, cardiac complications, respiratory complications, postoperative length of stay, and reintervention. The patient variables were compared using χ2 tests, analysis of variance, or Kruskal-Wallis tests, as appropriate. Logistic regression analysis was performed to identify the predictors of 30-day mortality and SCI. RESULTS: A total of 4010 patients met the inclusion criteria, with 470 in group 1, 132 in group 2, and 3408 in group 3. The 30-day mortality was 4% (19 of 470) in group 1, 6% (8 of 132) in group 2, and 6% (213 of 3408) in group 3 (P = .17). The incidence of SCI was 3% (14 of 470) in group 1, 3% (4 of 132) in group 2, and 3.8% (128 of 3408) in group 3 (P = .65). Stroke, reintervention, myocardial infarction, and cardiac complications were not significantly different among the 3 groups. The incidence of respiratory complications was greatest for group 3 (11%; 360 of 3408) compared with groups 1 (9%; 44 of 470) and 2 (4%; 5 of 132; P = .034). Similarly, the postoperative length of stay was longest for group 3 (9.6 ± 19.4 days vs 8.2 ± 18.3 days for group 1 and 5.9 ± 8.6 days for group 2; P = .038). The independent predictors of 30-day mortality for all TEVAR patients included units of packed red blood cells transfused intraoperatively, urgent or emergent repairs, older age, increasing serum creatinine level, inability to perform self-care, total procedure time, occlusion of the left subclavian artery intraoperatively, distal endograft landing zone 5, and diabetes. The predictors of SCI included the total procedure time, urgent and emergent repairs, and increasing serum creatinine level. CONCLUSIONS: TEVAR after previous thoracic aortic repair was not associated with an increased risk of SCI or 30-day mortality compared with TEVAR without previous aortic repair.

The 21-gene Recurrence Score® assay predicts distant recurrence in lymph node-positive, hormone receptor-positive, breast cancer patients treated with adjuvant sequential epirubicin- and docetaxel-based or epirubicin-based chemotherapy (PACS-01 trial).

BACKGROUND: The 21-gene Recurrence Score (RS) result predicts outcome and chemotherapy benefit in node-negative and node-positive (N+), estrogen receptor-positive (ER+) patients treated with endocrine therapy. The purpose of this study was to evaluate the prognostic impact of RS results in N+, hormone receptor-positive (HR+) patients treated with adjuvant chemotherapy (6 cycles of FEC100 vs. 3 cycles of FEC100 followed by 3 cycles of docetaxel 100 mg/m2) plus endocrine therapy (ET) in the PACS-01 trial (J Clin Oncol 2006;24:5664-5671). METHODS: The current study included 530 HR+/N+ patients from the PACS-01 parent trial for whom specimens were available. The primary objective was to evaluate the relationship between the RS result and distant recurrence (DR). RESULTS: There were 209 (39.4%) patients with low RS (< 18), 159 (30%) with intermediate RS (18-30) and 162 (30.6%) with high RS (≥ 31). The continuous RS result was associated with DR (hazard ratio = 4.14; 95% confidence interval: 2.67-6.43; p <  0.001), adjusting for treatment. In multivariable analysis, the RS result remained a significant predictor of DR (p <  0.001) after adjustment for number of positive nodes, tumor size, tumor grade, Ki-67 (immunohistochemical status), and chemotherapy regimen. There was no statistically significant interaction between RS result and treatment in predicting DR (p = 0.79). CONCLUSIONS: After adjustment for clinical covariates, the 21-gene RS result is a significant prognostic factor in N+/HR+ patients receiving adjuvant chemoendocrine therapy. TRIAL REGISTRATION: Not applicable.

Multigene Expression Assay and Benefit of Radiotherapy After Breast Conservation in Ductal Carcinoma in Situ.

Background: Most women with ductal carcinoma in situ (DCIS) will receive breast-conserving surgery (BCS) and radiation (RT). RT can be omitted for women at low risk of local recurrence (LR). The Oncotype DX DCIS score (DS) predicts LR risk after BCS alone. This study assesses the impact of RT and DS on LR risk. Methods: Population-based cohort analysis of individuals with DCIS treated by BCS ± RT from 1994-2003. Treatment and outcomes were determined by linkage and chart review. We used a propensity score-adjusted multivariable model to examine associations between DS and LR and evaluate the impact of RT. All statistical tests were two-sided. Results: The cohort includes 571 individuals treated by BCS alone, 689 cases treated with BCS + RT. Median follow-up was 9.4 years. On multivariable analysis, factors associated with LR include RT, age at diagnosis, tumor size, and multifocality. Adjusting for these factors, the DS risk group was statistically significantly associated with LR risk (hazard ratio high/intermediate = 1.75, 95% confidence interval = 1.28 to 2.41, P < .001). Women with a low-risk DS treated by BCS alone had an LR risk of 10.6% at 10 years and a small benefit from RT, while those with a high DS had a higher risk of LR (25.4%) after BCS alone and greater benefit from RT. A subgroup of patients with favorable clinicopathological features had a high-risk DS; these patients had a higher than expected risk of LR after BCS alone and a greater benefit with RT. Conclusions: The DS molecular assay improves risk stratification and estimates of RT benefit in individuals with DCIS treated with breast-conserving therapy.

A population-based validation study of the DCIS Score predicting recurrence risk in individuals treated by breast-conserving surgery alone.

Validated biomarkers are needed to improve risk assessment and treatment decision-making for women with ductal carcinoma in situ (DCIS) of the breast. The Oncotype DX DCIS Score (DS) was shown to predict the risk of local recurrence (LR) in individuals with low-risk DCIS treated by breast-conserving surgery (BCS) alone. Our objective was to confirm these results in a larger population-based cohort of individuals. We used an established population-based cohort of individuals diagnosed with DCIS treated with BCS alone from 1994 to 2003 with validation of treatment and outcomes. Central pathology assessment excluded cases with invasive cancer, DCIS < 2 mm or positive margins. Cox model was used to determine the relationship between independent covariates, the DS (hazard ratio (HR)/50 Cp units (U)) and LR. Tumor blocks were collected for 828 patients. Final evaluable population includes 718 cases, of whom 571 had negative margins. Median follow-up was 9.6 years. 100 cases developed LR following BCS alone (DCIS, N = 44; invasive, N = 57). In the primary pre-specified analysis, the DS was associated with any LR (DCIS or invasive) in ER+ patients (HR 2.26; P < 0.001) and in all patients regardless of ER status (HR 2.15; P < 0.001). DCIS Score provided independent information on LR risk beyond clinical and pathologic variables including size, age, grade, necrosis, multifocality, and subtype (adjusted HR 1.68; P = 0.02). DCIS was associated with invasive LR (HR 1.78; P = 0.04) and DCIS LR (HR 2.43; P = 0.005). The DCIS Score independently predicts and quantifies individualized recurrence risk in a population of patients with pure DCIS treated by BCS alone.

SNP discovery and chromosome anchoring provide the first physically-anchored hexaploid oat map and reveal synteny with model species.

A physically anchored consensus map is foundational to modern genomics research; however, construction of such a map in oat (Avena sativa L., 2n = 6x = 42) has been hindered by the size and complexity of the genome, the scarcity of robust molecular markers, and the lack of aneuploid stocks. Resources developed in this study include a modified SNP discovery method for complex genomes, a diverse set of oat SNP markers, and a novel chromosome-deficient SNP anchoring strategy. These resources were applied to build the first complete, physically-anchored consensus map of hexaploid oat. Approximately 11,000 high-confidence in silico SNPs were discovered based on nine million inter-varietal sequence reads of genomic and cDNA origin. GoldenGate genotyping of 3,072 SNP assays yielded 1,311 robust markers, of which 985 were mapped in 390 recombinant-inbred lines from six bi-parental mapping populations ranging in size from 49 to 97 progeny. The consensus map included 985 SNPs and 68 previously-published markers, resolving 21 linkage groups with a total map distance of 1,838.8 cM. Consensus linkage groups were assigned to 21 chromosomes using SNP deletion analysis of chromosome-deficient monosomic hybrid stocks. Alignments with sequenced genomes of rice and Brachypodium provide evidence for extensive conservation of genomic regions, and renewed encouragement for orthology-based genomic discovery in this important hexaploid species. These results also provide a framework for high-resolution genetic analysis in oat, and a model for marker development and map construction in other species with complex genomes and limited resources.

Model SNP development for complex genomes based on hexaploid oat using high-throughput 454 sequencing technology.

BACKGROUND: Genetic markers are pivotal to modern genomics research; however, discovery and genotyping of molecular markers in oat has been hindered by the size and complexity of the genome, and by a scarcity of sequence data. The purpose of this study was to generate oat expressed sequence tag (EST) information, develop a bioinformatics pipeline for SNP discovery, and establish a method for rapid, cost-effective, and straightforward genotyping of SNP markers in complex polyploid genomes such as oat. RESULTS: Based on cDNA libraries of four cultivated oat genotypes, approximately 127,000 contigs were assembled from approximately one million Roche 454 sequence reads. Contigs were filtered through a novel bioinformatics pipeline to eliminate ambiguous polymorphism caused by subgenome homology, and 96 in silico SNPs were selected from 9,448 candidate loci for validation using high-resolution melting (HRM) analysis. Of these, 52 (54%) were polymorphic between parents of the Ogle1040 × TAM O-301 (OT) mapping population, with 48 segregating as single Mendelian loci, and 44 being placed on the existing OT linkage map. Ogle and TAM amplicons from 12 primers were sequenced for SNP validation, revealing complex polymorphism in seven amplicons but general sequence conservation within SNP loci. Whole-amplicon interrogation with HRM revealed insertions, deletions, and heterozygotes in secondary oat germplasm pools, generating multiple alleles at some primer targets. To validate marker utility, 36 SNP assays were used to evaluate the genetic diversity of 34 diverse oat genotypes. Dendrogram clusters corresponded generally to known genome composition and genetic ancestry. CONCLUSIONS: The high-throughput SNP discovery pipeline presented here is a rapid and effective method for identification of polymorphic SNP alleles in the oat genome. The current-generation HRM system is a simple and highly-informative platform for SNP genotyping. These techniques provide a model for SNP discovery and genotyping in other species with complex and poorly-characterized genomes.

New DArT markers for oat provide enhanced map coverage and global germplasm characterization.

BACKGROUND: Genomic discovery in oat and its application to oat improvement have been hindered by a lack of genetic markers common to different genetic maps, and by the difficulty of conducting whole-genome analysis using high-throughput markers. This study was intended to develop, characterize, and apply a large set of oat genetic markers based on Diversity Array Technology (DArT). RESULTS: Approximately 19,000 genomic clones were isolated from complexity-reduced genomic representations of pooled DNA samples from 60 oat varieties of global origin. These were screened on three discovery arrays, with more than 2000 polymorphic markers being identified for use in this study, and approximately 2700 potentially polymorphic markers being identified for use in future studies. DNA sequence was obtained for 2573 clones and assembled into a non-redundant set of 1770 contigs and singletons. Of these, 705 showed highly significant (Expectation < 10E-10) BLAST similarity to gene sequences in public databases. Based on marker scores in 80 recombinant inbred lines, 1010 new DArT markers were used to saturate and improve the 'Kanota' x 'Ogle' genetic map. DArT markers provided map coverage approximately equivalent to existing markers. After binning markers from similar clones, as well as those with 99% scoring similarity, a set of 1295 non-redundant markers was used to analyze genetic diversity in 182 accessions of cultivated oat of worldwide origin. Results of this analysis confirmed that major clusters of oat diversity are related to spring vs. winter type, and to the presence of major breeding programs within geographical regions. Secondary clusters revealed groups that were often related to known pedigree structure. CONCLUSION: These markers will provide a solid basis for future efforts in genomic discovery, comparative mapping, and the generation of an oat consensus map. They will also provide new opportunities for directed breeding of superior oat varieties, and guidance in the maintenance of oat genetic diversity.

Development of a multiplexed PCR detection method for Barley and Cereal yellow dwarf viruses, Wheat spindle streak virus, Wheat streak mosaic virus and Soil-borne wheat mosaic virus.

Barley and Cereal yellow dwarf viruses (B/CYDVs), Wheat spindle streak mosaic (WSSMV), Soil-borne wheat mosaic virus (SBWMV) and Wheat streak mosaic virus (WSMV) constitute the most economically important group of wheat viruses. In this paper, a multiplex reverse transcription polymerase chain reaction (M-RT-PCR) method was developed for the simultaneous detection and discrimination of eight viruses: five strains of B/CYDVs, WSSMV, SBWMV and WSMV. The protocol uses specific primer sets for each virus producing five distinct fragments 295, 175, 400, 237, and 365 bp, indicating the presence of two strains of BYDVs, -PAV, -MAV, CYDV-RPV and two unassigned Luteoviridae BYDV-SGV and -RMV, respectively. This system also readily detected WSSMV, SBWMV and WSMV specific amplicons at 154, 219 and 193 bp, respectively. The amplification specificity of these primers was tested against a range of field samples from different parts of United States. The protocol also utilizes fluorescently tagged primers that can streamline the detection of each virus through capillary electrophoresis. This study fulfills the need for a rapid and specific wheat virus diagnostic tool that also has the potential for investigating the epidemiology of these viral diseases.

Expression analysis of defense-related genes in wheat in response to infection by Fusarium graminearum.

Fusarium head blight (FHB), caused by the fungi Fusarium graminearum and Fusarium culmorum, is a worldwide disease of wheat (Triticum aestivum L.). The Chinese cultivar Ning 7840 is one of a few wheat cultivars with resistance to FHB. GeneCalling, an open-architecture mRNA-profiling technology, was used to identify differentially expressed genes induced or suppressed in spikes of Ning 7840 after infection by F. graminearum. One hundred and twenty-five cDNA fragments representing transcripts differentially expressed in wheat spikes were identified. Based on BLASTN and BLASTX analyses, putative functions were assigned to some of the genes: 28 were assigned functions in primary metabolism and photosynthesis, 7 were involved in defense response, 14 were involved in gene expression and regulation, 24 encoded proteins associated with structure and protein synthesis, 42 lacked homology to sequences in the database, and 3 were similar to cloned multidrug resistance or disease resistance proteins. Of particular interest in this study were genes associated with resistance and defense against pathogen infection. Real-time quantitative reverse-transcription PCR indicated that of 51 genes tested, 19 showed 2-fold or greater induction or suppression in infected Ning 7840 in comparison with the water-treated control. The remaining 32 genes were not significantly induced or suppressed in infected Ning 7840 compared with the control. Subsequently, these 19 induced or suppressed genes were examined in the wheat line KS24-1, containing FHB resistance derived from Lophopyrum elongatum, and Len, an FHB-susceptible wheat cultivar. The temporal expression of some of these sequences encoding resistance proteins or defense-related proteins showed FHB (resistance specific) induction, suggesting that these genes play a role in protection against toxic compounds in plant-fungus interactions. On the basis of comprehensive expression profiling of various biotic or abiotic stress response genes revealed by quantitative PCR in this study and other supporting data, we hypothesized that the plant-pathogen interactions may be highly integrated into a network of diverse biosynthetic pathways.

Timing of induction of osmotically controlled genes in Salmonella enterica Serovar Typhimurium, determined with quantitative real-time reverse transcription-PCR.

The signals that control the transcription of osmoregulated genes are not understood satisfactorily. The "turgor control model" suggested that the primary osmoregulatory signal in Enterobacteriaceae is turgor loss, which induces the kdp K+ transport operon and activates the Trk K+ permease. The ensuing increase in cytoplasmic K+ concentration was proposed to be the signal that turns on all secondary responses, including the induction of the proU (proline-glycine betaine transport) operon. The "ionic strength model" proposed that the regulatory signal for all osmotically controlled responses is the increase in the cytoplasmic ionic strength or macromolecular crowding after an osmotic upshift. The assumption in the turgor control model that the induction of kdp is a primary response to osmotic shock predicts that this response should precede all secondary responses. Both models predict that the induction of all osmotically activated responses should be independent of the chemical nature of the solute used to impose osmotic stress. We tested these predictions by quantitative real-time reverse transcription-PCR analysis of the expression of six osmotically regulated genes in Salmonella enterica serovar Typhimurium. After shock with 0.3 M NaCl, proU was induced at 4 min, proP and rpoS were induced at 4 to 6 min, kdp was induced at 8 to 9 min, and otsB and ompC were induced at 10 to 12 min. After an equivalent osmotic shock with 0.6 M sucrose, proU was induced with kinetics similar to those seen with NaCl, but induction of kdp was reduced 150-fold in comparison to induction by NaCl. Our results are inconsistent with both the turgor control and the ionic strength control models.

Induction of wheat defense and stress-related genes in response to Fusarium graminearum.

Fusarium head blight (FHB), caused by species of the fungus Fusarium, is a worldwide disease of wheat (Triticum aestivum L.). The Chinese T. aestivum 'Ning7840' is one of few wheat cultivars with resistance to FHB. To identify differentially expressed genes corresponding to FHB resistance, a cDNA library was constructed using pooled mRNA isolated from glumes of 'Ning7840' harvested at 2, 6, 12, 24, 36, 72, and 96 h after inoculation (hai) with a conidia spore suspension of Fusarium graminearum. Suppressive subtractive hybridization (SSH) cDNA subtraction was carried out using pooled glume mRNAs from the tester and the control. The cDNA library was differentially screened using the forward subtracted cDNAs and the reverse subtracted cDNAs as probes. Twenty-four clones with significant matches to either plant (16 sequences) or fungal (8 sequences) genes were isolated based on their specific hybridization with forward subtracted cDNA and not reverse subtracted cDNA. Six putative defense-related genes were confirmed by real-time quantitative reverse-transcriptase PCR. Many-fold higher induction of three clones (A3F8, B10H1, and B11H3) in the resistant genotypes compared with susceptible genotypes indicates a putative role in the resistance response to Fusarium graminearum. Transcript accumulations of P450, chitinase (Chi1), and one unknown gene (clone B8Q9) in both resistant and susceptible genotypes suggest an involvement in a generalized resistance response to F. graminearum. Nucleotide sequence analysis showed that cDNA clone A4C6 encodes a cytochrome P450 gene (CYP709C3v2), including 14 N-terminal amino acids that have a membrane-associated helical motif. Other domains characteristic of eukaryotic P450 are also present in CYP709C3v2. The deduced polypeptide of cDNA clone B2H2 encodes an acidic isoform of class I chitinase containing a 960-bp coding region. Southern hybridization using aneuploid lines of T. aestivum 'Chinese Spring' indicated that CYP709C3v2 was located on the short arm of chromosomes 2B and 2D.

EST-derived SSR markers from defined regions of the wheat genome to identify Lophopyrum elongatum specific loci.

Lophopyrum elongatum, a close relative of wheat, provides a source of novel genes for wheat improvement. Molecular markers were developed to monitor the introgression of L. elongatum chromosome segments into hexaploid wheat. Existing simple sequence repeats (SSRs) derived from genomic libraries were initially screened for detecting L. elongatum loci in wheat, but only 6 of the 163 markers tested were successful. To increase detection of L. elongatum specific loci, 165 SSRs were identified from wheat expressed sequence tags (ESTs), where their chromosomal positions in wheat were known from deletion bin mapping. Detailed sequence analysis identified 41 SSRs within this group as potentially superior in their ability to detect L. elongatum loci. BLASTN alignments were used to position primers within regions of the ESTs that have sequence conservation with at least 1 similar EST from another cereal species. The targeting of primers in this manner enabled 14 L. elongatum markers from 41 wheat ESTs to be identified, whereas only 2 from 124 primers designed in random positions flanking SSRs detected L. elongatum loci. Addition and ditelosomic lines were used to assign all 22 markers to specific chromosome locations in L. elongatum. Nine of these SSR markers were assigned to homoeologous chromosome locations based on their similar position in hexaploid wheat. The remaining markers mapped to other L. elongatum chromosomes indicating a degree of chromosome rearrangements, paralogous sequences and (or) sequence variation between the 2 species. The EST-SSR markers were also used to screen other wheatgrass species indicating further chromosome rearrangements and (or) sequence variation between wheatgrass genomes. This study details methodologies for the generation of SSRs for detecting L. elongatum loci.

The Basis for Thinopyrum-Derived Resistance to Cereal yellow dwarf virus.

ABSTRACT Incorporation of Thinopyrum intermedium-derived resistance genes into improved wheat germ plasm generated a wheat substitution line (P29) which is completely resistant to Cereal yellow dwarf virus (CYDV). The undetectable CYDV titer in P29 led many to conclude that resistance prevented viral replication. To determine whether CYDV replication or movement is inhibited, we examined inoculated leaves for replication and uninoculated leaves for systemic spread. CYDV subgenomic RNA, produced only during replication, was found within the inoculated area of P29 and T. intermedium leaves, demonstrating that viral replication occurred. Absence of CYDV from uninoculated, newly emerging leaves of inoculated P29 and T. intermedium plants indicated resistance via inhibition of viral systemic infection. Resistance was not effective if P29 was inoculated with 50 to 100 viruliferous aphids per plant at the first-leaf stage or younger, resulting in a systemic spread of CYDV. As these infected P29 seedlings continued to grow, the resistance phenotype was recovered. Our data suggested that T. intermedium-derived resistance to CYDV was primarily dosage dependent and could be developmentally regulated if the amount of inoculum was large enough.

Assessment of Her-2/Neu status by immunohistochemistry and fluorescence in situ hybridization in mammary Paget disease and underlying carcinoma.

HER-2/Neu overexpression is seen in 20% to 30% of invasive breast carcinomas and has been reported in as many as 80% of high-grade infiltrating carcinomas. Earlier studies have suggested that 100% of the tumor cells in mammary Paget disease show overexpression of HER-2 protein. We undertook this study to assess HER-2 status of mammary Paget disease and of the underlying breast carcinoma, when present, by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Formalin-fixed, paraffin-embedded tissue from 20 cases of mammary Paget disease were analyzed for HER-2 status by IHC and FISH. IHC for estrogen receptor (ER) was also performed. The patients ranged in age from 34 to 88 years, with a mean age of 62 years. Eighty percent of the cases showed strong overexpression (3+) of HER-2 protein by IHC, and all of these cases showed more than 5-fold amplification of the HER-2 gene by FISH. The remaining 4 cases, which were negative for HER-2/Neu by IHC, showed no amplification by FISH. All of the latter cases expressed ER, whereas no case that overexpressed HER-2 expressed ER. Sixteen cases had an underlying tumor, which was in situ in 6 cases. The underlying tumors were identical to the Paget disease with respect to their HER-2/Neu overexpression by both IHC and FISH. HER-2 overexpression was identified in 80% of our cases of Paget disease. There was 100% concordance between HER-2 protein overexpression by immunohistochemistry and gene amplification in both the Paget and the underlying tumor. Moreover, all of the cases negative for HER-2 overexpression expressed ER, whereas those positive for HER-2 did not.