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1.  Structure and evolution of barley powdery mildew effector candidates 
BMC Genomics  2012;13:694.
Protein effectors of pathogenicity are instrumental in modulating host immunity and disease resistance. The powdery mildew pathogen of grasses Blumeria graminis causes one of the most important diseases of cereal crops. B. graminis is an obligate biotrophic pathogen and as such has an absolute requirement to suppress or avoid host immunity if it is to survive and cause disease.
Here we characterise a superfamily predicted to be the full complement of Candidates for Secreted Effector Proteins (CSEPs) in the fungal barley powdery mildew parasite B. graminis f.sp. hordei. The 491 genes encoding these proteins constitute over 7% of this pathogen’s annotated genes and most were grouped into 72 families of up to 59 members. They were predominantly expressed in the intracellular feeding structures called haustoria, and proteins specifically associated with the haustoria were identified by large-scale mass spectrometry-based proteomics. There are two major types of effector families: one comprises shorter proteins (100–150 amino acids), with a high relative expression level in the haustoria and evidence of extensive diversifying selection between paralogs; the second type consists of longer proteins (300–400 amino acids), with lower levels of differential expression and evidence of purifying selection between paralogs. An analysis of the predicted protein structures underscores their overall similarity to known fungal effectors, but also highlights unexpected structural affinities to ribonucleases throughout the entire effector super-family. Candidate effector genes belonging to the same family are loosely clustered in the genome and are associated with repetitive DNA derived from retro-transposons.
We employed the full complement of genomic, transcriptomic and proteomic analyses as well as structural prediction methods to identify and characterize the members of the CSEPs superfamily in B. graminis f.sp. hordei. Based on relative intron position and the distribution of CSEPs with a ribonuclease-like domain in the phylogenetic tree we hypothesize that the associated genes originated from an ancestral gene, encoding a secreted ribonuclease, duplicated successively by repetitive DNA-driven processes and diversified during the evolution of the grass and cereal powdery mildew lineage.
PMCID: PMC3582587  PMID: 23231440
Host-pathogen interactions; Effector protein structure; Fungal proteomics; Proteogenomics
2.  Transcriptional changes related to secondary wall formation in xylem of transgenic lines of tobacco altered for lignin or xylan content which show improved saccharification 
Phytochemistry  2012;74(100):79-89.
Graphical abstract
An EST library derived from xylogenic cells has been used to direct transcriptional profiling of genetically engineered tobacco lines which show improved biomass saccharification.
► Description of a xylogenic EST. ► Cell wall consequences of down-regulation of lignin and xylan. ► Improved saccharification of secondary walls but not primary walls. ► Transcriptional analysis of cell wall biosynthesis genes in modified transgenic lines. ► Identification of transcription factors.
In this study, an EST library (EH663598–EH666265) obtained from xylogenic tissue cultures of tobacco that had been previously generated was annotated. The library proved to be enriched in transcripts related to the synthesis and modification of secondary cell walls. The xylem-specific transcripts for most of the genes of the lignification and xylan pathways were identified and several full-length sequences obtained. Gene expression was determined in available tobacco lines down-regulated for enzymes of the phenylpropanoid pathway: CINNAMATE 4-HYDROXYLASE (sc4h), CINNAMOYL-COA REDUCTASE (asccr) and lignification-specific peroxidase (asprx). In addition, lines down-regulated in the nucleotide-sugar pathway to xylan formation through antisense expression of UDP-GLUCURONIC ACID DECARBOXYLASE (asuxs) were also analysed. It is shown herein that most transcripts were down-regulated for both lignin and xylan synthesis pathways in these lines, while CELLULOSE SYNTHASE A3 was up-regulated in lignin-modified lines. The analysis indicates the existence of interdependence between lignin and xylan pathways at the transcriptional level and also shows that levels of cellulose, xylan and lignin are not necessarily directly correlated to differences in transcription of the genes involved upstream, as shown by cell wall fractionation and sugar analysis. It is therefore suggested that cell wall biosynthesis regulation occurs at different levels, and not merely at the transcriptional level. In addition, all lines analyzed showed improved enzymic saccharification of secondary but not primary walls. Nevertheless, this demonstrates potential industrial applicability for the approach undertaken to improve biomass utility.
PMCID: PMC3657182  PMID: 22119077
ADH, bifunctional alcohol/UDP glucose dehydrogenase; AIM, acetone-insoluble material; CESA3, cellulose synthase; CSLD, cellulose synthase-like D; PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; C3H, coumaroyl-ester-3-hydroxylase; COMT, caffeic acid O-methyl transferase; CCOMT, caffeoyl-CoA methyl-transferase; CCR, cinnamoyl-CoA reductase; CAD, cinnamyl alcohol dehydrogenase; HQT, hydroxycinnamoyl-CoA:quinate hydroxycinnamoyltransferase; SUSY, sucrose synthase; UGD, UDP-glucose dehydrogenase; UXS, UDP-glucuronate decarboxylase; Tobacco; Nicotiana tabacum; Solanaceae; Cell wall; Lignin; Xylan; Antisense; Saccharification
3.  Peroxidase-dependent apoplastic oxidative burst in Arabidopsis required for pathogen resistance 
The oxidative burst is an early response to pathogen attack leading to the production of reactive oxygen species (ROS) including hydrogen peroxide. Two major mechanisms involving either NADPH oxidases or peroxidases that may exist singly or in combination in different plant species have been proposed for the generation of ROS. We identified an Arabidopsis thaliana azide-sensitive but diphenylene iodonium-insensitive apoplastic oxidative burst that generates H2O2 in response to a Fusarium oxysporum cell-wall preparation. Transgenic Arabidopsis plants expressing an anti-sense cDNA encoding a type III peroxidase, French bean peroxidase type 1 (FBP1) exhibited an impaired oxidative burst and were more susceptible than wild-type plants to both fungal and bacterial pathogens. Transcriptional profiling and RT-PCR analysis showed that the anti-sense (FBP1) transgenic plants had reduced levels of specific peroxidase-encoding mRNAs, including mRNAs corresponding to Arabidopsis genes At3g49120 (AtPCb) and At3g49110 (AtPCa) that encode two class III peroxidases with a high degree of homology to FBP1. These data indicate that peroxidases play a significant role in generating H2O2 during the Arabidopsis defense response and in conferring resistance to a wide range of pathogens.
PMCID: PMC3233234  PMID: 16889645
apoplastic peroxidase; Arabidopsis; oxidative burst; reactive oxygen species
4.  Experience of first- and subsequent-line systemic therapy in the treatment of non-small cell lung cancer 
Introduction: The treatment of advanced non-small cell cancer (NSCLC) has changed with multiple new treatment algorithms proposed based on histological and molecular subtyping but low mutation rates will ensure the dominance of cytotoxic chemotherapy. Accordingly, we undertook a detailed review of our practice delivering multiple lines of systemic therapy.
Method: We undertook a retrospective review of consecutive patients presenting with advanced (stage IIIb/IV) NSCLC treated with systemic therapy at two UK hospitals during a 2-year period, January 2007 to December 2008.
Results: A total of 130 patients were identified, treated with predominantly carboplatin/gemcitabine (20 initially radically). Fifty of 110 patients (45%) treated with first-line systemic therapy subsequently received second-line therapy, of which 10 patients received third-line and two patients fourth-line therapy. Sixty three of 110 first-line patients (58%) achieved clinical benefit, 19 out of 50 (38%) in the second-line, 6 out of 10 (60%) in third-line but both patients progressed at fourth-line. Median overall survival for 110 patients was 10 months (95% confidence interval [CI] 8.6–11.4); but 16 months (95% CI 14-17.9) in those receiving multiple lines. Median survival from the first cycle of last-line treatment to death in the multiple therapy lines was 5 months (95% CI 2.6-7.3) and the majority of patients spent more time off treatment.
Conclusion: Overall our outcomes are consistent with published data and show good survival times can be achieved. The future of advanced NSCLC is in selecting the best treatment approach on a histological and genotypic basis.
PMCID: PMC3150065  PMID: 21904578
non-small cell lung cancer; retrospective analysis; systemic therapy
5.  Incidence and prognostic value of tumour cells detected by RT–PCR in peripheral blood stem cell collections from patients with Ewing tumour 
British Journal of Cancer  2006;95(10):1326-1333.
To retrospectively evaluate the incidence of tumour cell contamination of peripheral blood stem cell (PBSC) collections and to correlate these data with the clinical outcome after high-dose chemotherapy (HDCT) with stem cell rescue in patients with a high-risk Ewing tumour. Peripheral blood stem cell collections obtained from 171 patients were analysed. Tumour contamination was assessed by reverse transcriptase–polymerase chain reaction (RT–PCR). The files of 88 patients who underwent HDCT followed by PBSC reinfusion were reviewed in detail, and their outcome compared to the PBSC RT–PCR results. Seven of 88 PBSC collections (8%) contained tumour cells as detected by RT–PCR. Peripheral blood stem cells were collected after a median of five cycles of chemotherapy. No clinical factor predictive of tumour cell contamination of PBSC harvest could be identified. Event-free survival (EFS) and overall survival (OS) of the whole study population were 45.3 % and 51.8 % at 3 years from the date of the graft, respectively. Forty-five patients relapsed with a median time of 15 months after graft, only four of whom had tumour cell contamination of the PBSC harvest. Tumour cell contamination of PBSC collection is rare and does not seem to be associated with a significantly poorer EFS or OS in this high-risk population.
PMCID: PMC2360590  PMID: 17088915
ewing tumour; PBSC; tumour cell contamination; RT–PCR; outcome
6.  Qualitative network models and genome-wide expression data define carbon/nitrogen-responsive molecular machines in Arabidopsis 
Genome Biology  2007;8(1):R7.
Qualitative network models and genome-wide expression data define carbon/nitrogen-responsive molecular machines in Arabidopsis and indicate that regulation by carbon/nitrogen metabolites occurs at multiple levels.
Carbon (C) and nitrogen (N) metabolites can regulate gene expression in Arabidopsis thaliana. Here, we use multinetwork analysis of microarray data to identify molecular networks regulated by C and N in the Arabidopsis root system.
We used the Arabidopsis whole genome Affymetrix gene chip to explore global gene expression responses in plants exposed transiently to a matrix of C and N treatments. We used ANOVA analysis to define quantitative models of regulation for all detected genes. Our results suggest that about half of the Arabidopsis transcriptome is regulated by C, N or CN interactions. We found ample evidence for interactions between C and N that include genes involved in metabolic pathways, protein degradation and auxin signaling. To provide a global, yet detailed, view of how the cell molecular network is adjusted in response to the CN treatments, we constructed a qualitative multinetwork model of the Arabidopsis metabolic and regulatory molecular network, including 6,176 genes, 1,459 metabolites and 230,900 interactions among them. We integrated the quantitative models of CN gene regulation with the wiring diagram in the multinetwork, and identified specific interacting genes in biological modules that respond to C, N or CN treatments.
Our results indicate that CN regulation occurs at multiple levels, including potential post-transcriptional control by microRNAs. The network analysis of our systematic dataset of CN treatments indicates that CN sensing is a mechanism that coordinates the global and coordinated regulation of specific sets of molecular machines in the plant cell.
PMCID: PMC1839130  PMID: 17217541
7.  Genome-wide patterns of carbon and nitrogen regulation of gene expression validate the combined carbon and nitrogen (CN)-signaling hypothesis in plants 
Genome Biology  2004;5(11):R91.
Microarray analysis and the 'InterAct class' method were used to study interactions between carbon and nitrogen signaling in Arabidopsis.
Carbon and nitrogen are two signals that influence plant growth and development. It is known that carbon- and nitrogen-signaling pathways influence one another to affect gene expression, but little is known about which genes are regulated by interactions between carbon and nitrogen signaling or the mechanisms by which the different pathways interact.
Microarray analysis was used to study global changes in mRNA levels due to carbon and nitrogen in Arabidopsis thaliana. An informatic analysis using InterAct Class enabled us to classify genes on the basis of their responses to carbon or nitrogen treatments. This analysis provides in vivo evidence supporting the hypothesis that plants have a carbon/nitrogen (CN)-sensing/regulatory mechanism, as we have identified over 300 genes whose response to combined CN treatment is different from that expected from expression values due to carbon and nitrogen treatments separately. Metabolism, energy and protein synthesis were found to be significantly affected by interactions between carbon and nitrogen signaling. Identified putative cis-acting regulatory elements involved in mediating CN-responsive gene expression suggest multiple mechanisms for CN responsiveness. One mechanism invokes the existence of a single CN-responsive cis element, while another invokes the existence of cis elements that promote nitrogen-responsive gene expression only when present in combination with a carbon-responsive cis element.
This study has allowed us to identify genes and processes regulated by interactions between carbon and nitrogen signaling and take a first step in uncovering how carbon- and nitrogen-signaling pathways interact to regulate transcription.
PMCID: PMC545782  PMID: 15535867
8.  High-dose sequential epirubicin and cyclophosphamide with peripheral blood stem cell support for advanced breast cancer: results of a phase II study 
British Journal of Cancer  2001;85(9):1240-1246.
The aim of this study was to evaluate the feasibility of a high-dose intensity and high-dose density multicycle epirubicin and cyclophosphamide regimen with peripheral blood stem cells (PBSC) and haematopoietic growth factor (G-CSF) support in advanced breast cancer patients. From August 1994 to September 1999, 56 breast cancer patients (8 stage IIIB and 48 stage IV) received 205 courses of cyclophosphamide 3 g m−2and epirubicin 100 mg m−2every 14 days. G-CSF 5 μg kg−1day−1was administered from day 3 to neutrophil recovery. 4 courses were planned. PBSC were collected after course 1, and reinfused after courses 3 and 4, with ≥ 2 × 106CD34+ PBSC kg−1required for each reinfusion. 48 patients (86%) received all 4 planned courses. Early withdrawal was consecutive to infectious complications (n= 4), severe asthenia (n= 3), haemorrhagic cystitis (n= 1). A median number of 10.8 × 106CD34+ PBSC kg−1(range, 3–80) was harvested with 1 or 2 apheresis in 48 patients (94%). Median relative dose intensity was 91.3% (range, 72–102%). Grade 4 neutrophil toxicity was observed in 100% of patients. Febrile neutropenia was observed in 40% of courses (median duration 2 days). Red blood cells and platelets had to be transfused in 54% and 27% of courses, respectively. There were no toxic deaths. Objective response rate was 69% in stage IV patients (31/45 evaluable pts), with a 16% complete response rate. Their median progression-free and overall survivals were 22.5 and 37 months, respectively. This epirubicine-containing high-dose regimen appeared feasible, albeit with high toxicity. Time-related progression parameters exceed commonly reported ones. Controlled studies of upfront sequential high-dose chemotherapy are still needed to evaluate its real benefit. © 2001 Cancer Research Campaign
PMCID: PMC2375253  PMID: 11720455
breast cancer; advanced; chemotherapy; high-dose
9.  Prognostic value of persistent node involvement after neoadjuvant chemotherapy in patients with operable breast cancer 
British Journal of Cancer  2000;83(11):1480-1487.
Neoadjuvant chemotherapy is able to reduce the size of the majority of breast tumours and down-stage axillary-node status. The aim of this study was to assess the prognostic value of persistent node involvement after neoadjuvant chemotherapy. A total of 488 patients with T2–T3, N0–N1 breast cancer treated by neoadjuvant chemotherapy followed by tumour excision and axillary lymph-node dissection between 1981 and 1992 were selected from the Institut Curie database. Median follow-up was 7 years. Overall objective response rate before local treatment was 52% and breast tumour size was reduced in 83% of patients. No pathologic nodal involvement was observed in 46.5% of patients. Patients with ≥ eight positive nodes had a very poor median disease-free survival of only 20 months. Their 10-year disease-free survival rate was 7%, while the 10-year disease-free survival rate for patients with no node involvement was 64%. Median survival for patients with ≥ eight nodes positive was 48 months and the 10-year survival rate was 26% (P < 0.0001). On multivariate analysis, outcome was strongly correlated with pathological nodal status, tumour grade, hormonal receptor status and clinical response of the tumour. In conclusion, patients with extensive nodal involvement after neoadjuvant chemotherapy have a very poor outcome. Second-line treatment should be considered in this population. © 2000 Cancer Research Campaign
PMCID: PMC2363409  PMID: 11076657
neoadjuvant chemotherapy; breast cancer; pathological nodal metastasis
10.  Carbogen breathing with nicotinamide improves the oxygen status of tumours in patients. 
British Journal of Cancer  1995;72(1):198-205.
Nicotinamide and carbogen breathing are both effective radiosensitisers in experimental tumour models and are even more effective in combination. This study was to investigate the feasibility of using the agents in combination in patients and to measure their effect on tumour oxygenation. Twelve patients with advanced malignant disease were treated with 4-6 g of oral nicotinamide (NCT) in tablet formulation. Ten of these 12 patients breathed carbogen (95% oxygen, 5% carbon dioxide) for up to 20 min at presumed peak plasma NCT concentration (Cpeak) and had tumour oxygen partial pressure (pO2) measured using the Eppendorf pO2) histograph. The mean Cpeak values were 82, 115 and 150 micrograms ml-1 for NCT doses of 4, 5 and 6 g respectively and were dose dependent. The time of Cpeak was independent of dose with an overall mean of 2.4 h (range 0.7-4 h). NCT toxicity occurred in 9 out of 12 patients and was mild in all but one; carbogen was well tolerated in all patients. Following NCT only two patients had significant rises (P < 0.05) in tumour median pO2. During carbogen breathing, eight out of ten patients had early highly significant rises in pO2 (P < 0.0001), of which six continued to rise or remained in plateau until completion of gas breathing. Six patients had hypoxic pretreatment values less than 5 mmHg, which were completely abolished in three and reduced in two during carbogen breathing. In conclusion, the combination of NCT and carbogen breathing was generally well tolerated and gave rise to substantial rises in tumour pO2 which were maintained throughout gas breathing. These results should encourage further study of this potentially useful combination of agents as radiosensitisers in the clinic.
PMCID: PMC2034117  PMID: 7599052
11.  Use of mucin like cancer associated antigen (MCA) in the management of breast cancer. 
British Journal of Cancer  1991;63(6):1000-1004.
A study of the epithelial mucin marker MCA was made in 233 patients with breast cancer. Only 6% of 72 patients with Stage I-III disease had a raised MCA (greater than 15 U ml-1) when assessed following surgical treatment of the primary tumour. Raised levels of MCA occurred in one out of 20 (10%) patients with stable local recurrence, and six out of ten (60%) patients with progressive local recurrence. In 115 patients with metastases 89 (77%) had a raised MCA, tumour extent and disease activity both influenced the MCA level. The change of MCA level during the treatment of 11 cases of local recurrence and 55 cases of metastatic disease showed a 64 and 84% concordance respectively with the change in clinical status. Coincidental measurement of MCA and bone scans showed a raised MCA in one out of 63 (1.5%) patients with negative or equivocal scans, and 26 out of 35 (74%) with positive scans. MCA provides a useful marker for the monitoring of the treatment of local recurrence and metastatic disease, and an independent indicator of the effects of changes in treatment.
PMCID: PMC1972559  PMID: 2069833
12.  Hepatic metabolism of free fatty acids in normal and diabetic dogs 
Journal of Clinical Investigation  1970;49(3):537-547.
Fasted dogs prepared with catheters in the femoral artery, portal vein, and hepatic vein and infused intravenously with palmitate-1-14C were used to estimate uptake of free fatty acids in liver and their conversion to major metabolic products secreted into hepatic venous blood. Animals were studied under ordinary conditions and when fat mobilization was increased abruptly by infusing norepinephrine or for a prolonged period by withdrawing insulin from depancreatized dogs. 80% of hepatic blood flow was assumed to be derived from the portal vein.
Hepatic uptake was proportional to net outflow transport of plasma free fatty acids in the three groups and, in each, hepatic extraction fraction was about 25%. Since specific activity of free fatty acids entering and leaving the liver was equal and their composition was closely similar in the three sites sampled, it was concluded that palmitate is a representative tracer for free fatty acids entering the liver and that the liver does not release free fatty acids into the blood.
In norepinephrine-infused dogs, the fraction of free fatty acids secreted in triglycerides (13%) was similar to that of control animals, so that transport of triglycerides was increased. In diabetic dogs no increased transport could be demonstrated since an average of only 2% of free fatty acids was converted to plasma triglyceride fatty acids; the hyperlipemia uniformly observed therefore appeared to result from defective removal of triglycerides from the blood.
A similar fraction of free fatty acids was converted to ketones in normal and norepinephrine-infused dogs. This fraction was somewhat higher in diabetic animals and, in addition, a substantial quantity of ketones was derived from unlabeled precursors. Fractional conversion of free fatty acids to CO2 was similar in normal and norepinephrine-infused dogs, but reduced in the diabetics.
PMCID: PMC322502  PMID: 5415680

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