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1.  HOXC11–SRC-1 regulation of S100beta in cutaneous melanoma: new targets for the kinase inhibitor dasatinib 
British Journal of Cancer  2011;105(1):118-123.
Background:
Cutaneous melanoma is an aggressive disease. S100beta is an established biomarker of disease progression; however, the mechanism of its regulation in melanoma is undefined.
Methods:
Expression of HOXC11 and SRC-1 was examined by immunohistochemistry and immunofluorescence. Molecular and cellular techniques were used to investigate regulation of S100beta, including, western blot, qPCR, ChIP and migration assays.
Results:
Expression levels of the transcription factor HOXC11 and its coactivator SRC-1 were significantly elevated in malignant melanoma in comparison with benign nevi (P<0.001 and P=0.017, respectively, n=80), and expression of HOXC11 and SRC-1 in the malignant tissue associated with each other (P<0.001). HOXC11 recruitment to the promoter of S100beta was observed in the primary melanoma cell line SKMel28. S100beta expression was found to be dependant on both HOXC11 and SRC-1. Treatment with the Src/Abl inhibitor, dasatinib, reduced HOXC11–SRC-1 interaction and prevented recruitment of HOXC11 to the S100beta promoter. Dasatinib inhibited both mRNA and protein levels of S100beta and reduced migration of the metastatic cell line MeWo.
Conclusion:
We have defined a signalling mechanism regulating S100beta in melanoma, which can be modulated by dasatinib. Profiling patients for expression of key markers of this network has the potential to increase the efficacy of dasatinib treatment.
doi:10.1038/bjc.2011.193
PMCID: PMC3137411  PMID: 21654685
HOXC11; SRC-1; S100beta; melanoma; dasatinib
2.  Development of a questionnaire (EORTC module) to measure quality of life in patients with cholangiocarcinoma and gallbladder cancer, the EORTC QLQ-BIL21 
British Journal of Cancer  2011;104(4):587-592.
Background:
Quality of life measurement in cholangiocarcinoma and gallbladder cancer involves the assessment of patient-reported issues related to the symptoms, disease and treatment of these tumours. This study describes the development of the disease-specific quality of life (QoL) questionnaire for patients with cholangiocarcinoma and gallbladder cancer to supplement the European Organization for Research and Treatment of Cancer (EORTC)-QLQ C30 core cancer questionnaire.
Methods:
Phases 1–3 of the guidelines for module development published by the EORTC were followed, with adaptations for incorporation of questions from existing modules.
Results:
A total of 47 QoL issues (questions) were identified; 44 questions from the two related validated questionnaires, the EORTC QLQ-PAN26 (pancreatic module) and the EORTC QLQ-LMC21 (liver metastases module), two from the Functional Assessment of Cancer Therapy hepatobiliary module questionnaire in the literature search and one from healthcare professional interviews. Following phase 1 and 2 interviews with patients (n=101) and health care professionals (n=6), a 23-question provisional questionnaire was formulated. There were five questions from PAN26, 15 from LMC21 and three extra questions. In phase 3, the provisional item list was pre-tested in 52 patients in four languages and this resulted in a 21-item module.
Conclusion:
This is the only disease-specific QoL questionnaire for patients with cholangiocarcinoma and gallbladder cancer, and initial assessments show it to be accurate and acceptable to patients in reflecting QoL in these diseases.
doi:10.1038/sj.bjc.6606086
PMCID: PMC3049590  PMID: 21266979
cholangiocarcinoma; gall bladder cancer; quality of life
4.  Identification of Components of the Sigma B Regulon in Listeria monocytogenes That Contribute to Acid and Salt Tolerance ▿  
Applied and Environmental Microbiology  2008;74(22):6848-6858.
Sigma B (σB) is an alternative sigma factor that controls the transcriptional response to stress in Listeria monocytogenes and is also known to play a role in the virulence of this human pathogen. In the present study we investigated the impact of a sigB deletion on the proteome of L. monocytogenes grown in a chemically defined medium both in the presence and in the absence of osmotic stress (0.5 M NaCl). Two new phenotypes associated with the sigB deletion were identified using this medium. (i) Unexpectedly, the strain with the ΔsigB deletion was found to grow faster than the parent strain in the growth medium, but only when 0.5 M NaCl was present. This phenomenon was independent of the carbon source provided in the medium. (ii) The ΔsigB mutant was found to have unusual Gram staining properties compared to the parent, suggesting that σB contributes to the maintenance of an intact cell wall. A proteomic analysis was performed by two-dimensional gel electrophoresis, using cells growing in the exponential and stationary phases. Overall, 11 proteins were found to be differentially expressed in the wild type and the ΔsigB mutant; 10 of these proteins were expressed at lower levels in the mutant, and 1 was overexpressed in the mutant. All 11 proteins were identified by tandem mass spectrometry, and putative functions were assigned based on homology to proteins from other bacteria. Five proteins had putative functions related to carbon utilization (Lmo0539, Lmo0783, Lmo0913, Lmo1830, and Lmo2696), while three proteins were similar to proteins whose functions are unknown but that are known to be stress inducible (Lmo0796, Lmo2391, and Lmo2748). To gain further insight into the role of σB in L. monocytogenes, we deleted the genes encoding four of the proteins, lmo0796, lmo0913, lmo2391, and lmo2748. Phenotypic characterization of the mutants revealed that Lmo2748 plays a role in osmotolerance, while Lmo0796, Lmo0913, and Lmo2391 were all implicated in acid stress tolerance to various degrees. Invasion assays performed with Caco-2 cells indicated that none of the four genes was required for mammalian cell invasion. Microscopic analysis suggested that loss of Lmo2748 might contribute to the cell wall defect observed in the ΔsigB mutant. Overall, this study highlighted two new phenotypes associated with the loss of σB. It also demonstrated clear roles for σB in both osmotic and low-pH stress tolerance and identified specific components of the σB regulon that contribute to the responses observed.
doi:10.1128/AEM.00442-08
PMCID: PMC2583506  PMID: 18806006
5.  Proteomic Analyses of a Listeria monocytogenes Mutant Lacking σB Identify New Components of the σB Regulon and Highlight a Role for σB in the Utilization of Glycerol▿  
In Listeria monocytogenes the alternative sigma factor σB plays important roles in both virulence and stress tolerance. In this study a proteomic approach was used to define components of the σB regulon in L. monocytogenes 10403S (serotype 1/2a). Using two-dimensional gel electrophoresis and the recently developed isobaric tags for relative and absolute quantitation technique, the protein expression profiles of the wild type and an isogenic ΔsigB deletion strain were compared. Overall, this study identified 38 proteins whose expression was σB dependent; 17 of these proteins were found to require the presence of σB for full expression, while 21 were expressed at a higher level in the ΔsigB mutant background. The data obtained with the two proteomic approaches showed limited overlap (four proteins were identified by both methods), a finding that highlights the complementarity of the two technologies. Overall, the proteomic data reaffirmed a role for σB in the general stress response and highlighted a probable role for σB in metabolism, especially in the utilization of alternative carbon sources. Proteomic and physiological data revealed the involvement of σB in glycerol metabolism. Five newly identified members of the σB regulon were shown to be under direct regulation of σB using reverse transcription-PCR (RT-PCR), while random amplification of cDNA ends-PCR was used to map four σB-dependent promoters upstream from lmo0796, lmo1830, lmo2391, and lmo2695. Using RT-PCR analysis of known and newly identified σB-dependent genes, as well as proteomic analyses, σB was shown to play a major role in the stationary phase of growth in complex media.
doi:10.1128/AEM.01921-07
PMCID: PMC2227729  PMID: 18065622
6.  Cancer, cardiovascular disease and diabetes mortality among women with a history of endometrial cancer 
British Journal of Cancer  2007;96(11):1747-1749.
Among 7182 women with endometrial cancer in Scotland, standardised mortality ratios (and 95% confidence intervals (CI)) were 6.38 (5.74–7.15) for all cancers and 1.10 (1.00–1.22) for circulatory diseases as underlying cause of death and 2.81 (2.19–3.70) for diabetes as underlying/contributory cause of death.
doi:10.1038/sj.bjc.6603761
PMCID: PMC2359928  PMID: 17453005
endometrial cancer; mortality; breast cancer; ovarian cancer; cardiovascular disease; diabetes
8.  Fetal origins of adult disease: epidemiology and mechanisms 
Journal of Clinical Pathology  2000;53(11):822-828.
The past 10 years have provided unequivocal evidence that there are associations between birth size measures and future development of adult diseases, such as type 2 diabetes and coronary artery disease. Despite initial concern that bias or residual confounding in the analyses had produced these rather bizarre associations, the findings have now been reproduced in different cohorts by independent investigators from many parts of the world. The challenge for the next decade must be to discover the cellular and molecular mechanisms giving rise to these associations. If this aim is accomplished, it might be possible to devise strategies to reduce the impact of these disabling, chronic, and expensive diseases. The purpose of this review is to describe some of the relevant, important, and more recent epidemiological studies, and also to discuss potential mechanisms underpinning the associations.J Clin Pathol(J Clin Pathol 2000;53:822–828)
Key Words: atherosclerotic vascular disease • type 2 diabetes • birth weight
doi:10.1136/jcp.53.11.822
PMCID: PMC1731115  PMID: 11127263
9.  Characterization of Escherichia coli O157:H7 from Downer and Healthy Dairy Cattle in the Upper Midwest Region of the United States 
While cattle in general have been identified as a reservoir of Escherichia coli O157:H7, there are limited data regarding the prevalence and clonality of this pathogen in downer dairy cattle and the potential impact to human health that may occur following consumption of meat derived from downer dairy cattle. In the present study, conducted at two slaughter facilities in Wisconsin between May and October of 2001, we established a higher prevalence of E. coli O157:H7 in fecal and/or tissue samples obtained aseptically from intact colons of downer dairy cattle (10 of 203, 4.9%) than in those from healthy dairy cattle (3 of 201, 1.5%). Analyses of 57 isolates, representing these 13 positive samples (one to five isolates per sample), by pulsed-field gel electrophoresis, revealed 13 distinct XbaI restriction endonuclease digestion profiles (REDP). Typically, isolates from different animals displayed distinct REDP and isolates from the same fecal or colon sample displayed indistinguishable REDP. However, in one sample, two different, but highly related, REDP were displayed by the isolates recovered. Antimicrobial susceptibility testing indicated that 10 of the 57 isolates, recovered from 2 (1 downer and 1 healthy animal) of the 13 positive samples, were resistant to at least 1 of 18 antimicrobials tested. However, there was no appreciable difference in the frequency of resistance of isolates recovered from downer and healthy dairy cattle, and not all isolates with the same REDP displayed the same antimicrobial susceptibility profile. Lastly, it was not possible to distinguish between isolates recovered from downer and healthy cattle based on their XbaI REDP or antimicrobial susceptibility. These results indicate that downer cattle had a 3.3-fold-higher prevalence of E. coli O157:H7 than healthy cattle within the time frame and geographic scope of this study.
doi:10.1128/AEM.69.8.4683-4688.2003
PMCID: PMC169068  PMID: 12902258
10.  Randomized controlled trial of general practitioner versus usual medical care in a suburban accident and emergency department using an informal triage system. 
We determined if care provided by general practitioners (GPs) to non-emergency patients, in a suburban accident and emergency (A&E) department using an informal triage system, differs significantly from care provided by usual A&E staff. One thousand eight hundred and seventy-eight patients participated. By comparison with usual A&E staff, GPs prescribed significantly more often (percentage relative difference [% RD] = 12 [95% confidence interval = 1-23]) and referred more patients to hospital (% RD = 21 [95% CI = 9-33]). This is the first study to report that sessional GPs working in an A&E department utilize similar or more resources than usual A&E staff. It emphasizes the need for the continued audit of initiatives that have been introduced into new settings.
PMCID: PMC1313317  PMID: 10622016
11.  Surviving social assistance: 12-month prevalence of depression in sole-support parents receiving social assistance 
BACKGROUND: Although it is generally recognized that poverty and depression can coexist among single parents receiving social assistance, there is insufficient research on this topic. The goals of this study therefore were to investigate the prevalence, correlates and health care expenditures associated with depression among sole-support parents receiving social assistance. METHODS: Sole-support parents who had applied for social assistance in 2 regions of southwestern Ontario were included in the study. Depression was diagnosed with the 1994 University of Michigan Composite International Diagnostic Interview short forms. RESULTS: The 12-month prevalence rate of depressive disorder among the parents interviewed was 45.4% (345/760). A total of 247 (32.5%) had major depressive disorder alone, 19 (2.5%) had dysthymia, and 79 (10.4%) had both major depressive disorder and dysthymia ("double depression"). Those with major depressive disorder, particularly double depression, had significantly higher rates of coexisting psychiatric disorder than those without depressive disorders. Parents with depression reported higher rates of developmental delay and behaviour problems in their children than parents without depression. Expenditures for health care services were higher for parents with depression and for their children than for parents without depressive disorder and their children. INTERPRETATION: Single parents receiving social assistance have high rates of depression. Such parents with depression also have higher rates of other psychiatric disorders and higher expenditures for health care services, and their children have higher rates of developmental delay and behaviour problems.
PMCID: PMC1229183  PMID: 9559013
13.  Triglycerides and disease. 
Postgraduate Medical Journal  1993;69(815):679-695.
PMCID: PMC2399798  PMID: 8255832
14.  Duration of incubation of fungal cultures. 
Journal of Clinical Microbiology  1996;34(6):1583-1585.
To determine the optimum duration of incubation for recovery of fungi, the results of 2,173 consecutive clinical cultures were reviewed. Overall, 94% of fungal isolates were detected by day 7 and 98% were detected by day 14. Yeasts were usually (98%) detected within the first week of incubation. Recovery of molds required more time, but 81% were detected by day 7 and more than 96% were detected by day 14.
PMCID: PMC229071  PMID: 8735127
15.  The spv virulence operon of Salmonella typhimurium LT2 is regulated negatively by the cyclic AMP (cAMP)-cAMP receptor protein system. 
Journal of Bacteriology  1994;176(3):905-912.
The cyclic AMP (cAMP) receptor protein (CRP) was found to play a role in the growth phase regulation of the spv operon on the high-molecular-weight virulence plasmid of Salmonella typhimurium LT2. By using a lacZ reporter transcriptional fusion to the spvB structural gene on the single-copy virulence plasmid, it was found that while spvB transcription was induced in stationary-phase cultures, the induced level of expression was lower than that reported for the spv system in other serovars of Salmonella. Surprisingly, inactivation of the gene encoding the positive activator SpvR resulted in only a threefold reduction in spvB transcription. In contrast, spvB transcription in stationary-phase cultures was enhanced by 10-fold in mutants deficient in crp-encoded CRP or cya-encoded adenylate cyclase. Wild-type (i.e., 10-fold-lower) levels of spvB expression were restored by providing active copies of crp or cya on recombinant plasmids. Enhanced spvB transcription was not seen in crp or cya mutants in the absence of a functional spvR positive regulatory gene, showing that the cAMP-CRP system acted on spvB expression either in conjunction with or via SpvR. A lacZ transcriptional fusion to spvR could not be induced in stationary-phase cultures in the absence of functional SpvR, regardless of the cAMP-CRP status of the cells. When SpvR was provided in trans, transcription of the spvR-lacZ fusion was induced to similar levels in stationary-phase cultures with and without cAMP-CRP. These data are consistent with spvR being poorly transcribed from the single-copy virulence plasmid in S. typhimurium LT2 and with a suppression of this defect via inactivation of the cAMP-CRP system. The physiological significance of cAMP-CRP involvement in spv expression is discussed.
PMCID: PMC205128  PMID: 8300543
16.  Introduction and expression of the bacterial genes cysE and cysK in eukaryotic cells. 
The coding sequences of the cysE and cysK genes from Escherichia coli, which encode the enzymes of the cysteine biosynthetic pathway, namely, serine acetyltransferase (EC 2.3.1.30) and O-acetylserine sulfhydrylase (or cysteine synthase [EC 4.2.99.8]), were modified for expression in eukaryotic cells and introduced into murine L cells. A number of fusion genes comprising the cysE or cysK coding sequences joined to the promoter of the ovine metallothionein-Ia (MT-Ia) gene and various portions of the ovine growth hormone (GH) gene were prepared. Significant differences in the level of transcription were observed, depending on the amount and arrangement of the GH gene sequences used, the highest levels being obtained with the constructs MTCE10 and MTCK7, which contained only the GH 3' untranslated gene sequences. These two constructs were fused to produce the gene MTCEK1. In this single DNA sequence, each bacterial gene is under independent MT-Ia promoter control. Expression of the cysK sequence in this construct (MT-Ia promoter-cysE-3' GH sequence-MT-Ia promoter-cysK-3' GH sequence) was elevated compared with expression of the cysK gene in MTCK7. However, expression of the cysE sequence in MTCEK1 was only 40% of that of the cysE gene cloned into MTCE10. The double-promoter configuration, which enhances the expression of the second gene in MTCEK1, is proposed as a model for the modification of bacterial genes in general.
Images
PMCID: PMC202204  PMID: 7683185
17.  Probing keratinocyte and differentiation specificity of the human K5 promoter in vitro and in transgenic mice. 
Molecular and Cellular Biology  1993;13(6):3176-3190.
Keratins K5 and K14 form the extensive intermediate filament network of mitotically active basal cells in all stratified epithelia. We have explored the regulatory mechanisms governing cell-type-specific and differentiation stage-specific expression of the human K5 gene in transiently transfected keratinocytes in vitro and in transgenic mice in vivo. Six thousand base pairs of 5' upstream K5 sequence directed proper basal cell-specific expression in all stratified epithelia. Surprisingly, as few as 90 bp of the K5 promoter still directed expression to stratified epithelia, with expression predominantly in epidermis, hair follicles, and tongue. Despite keratinocyte-preferred expression, the truncated K5 promoter displayed departures from basal to suprabasal expression in epidermis and from outer root sheath to inner root sheath expression in the follicle, with some regional variations in expression as well. To begin to elucidate the molecular controls underlying the keratinocyte specificity of the truncated promoter, we examined protein-DNA interactions within this region. A number of keratinocyte nuclear proteins bind to a K5 gene segment extending from -90 to +32 bp and are functionally involved in transcriptional regulation in vitro. Interestingly, several of these factors are common to both the K5 and K14 promoters, although they appear to be distinct from those previously implicated in keratinocyte specificity. Mutagenesis studies indicate that factors binding in the vicinity of the TATA box and transcription initiation are responsible for the cell type specificity of the truncated K5 promoter.
Images
PMCID: PMC359757  PMID: 7684490
18.  Impaired glucose tolerance and height. 
BMJ : British Medical Journal  1991;303(6810):1134.
PMCID: PMC1671307  PMID: 1809269
20.  Role of community pharmacies in prevention of AIDS among injecting drug misusers: findings of a survey in England and Wales. 
BMJ : British Medical Journal  1989;299(6707):1076-1079.
OBJECTIVE--To determine the current and potential roles of community pharmacists in the prevention of AIDS among misusers of injected drugs. DESIGN--Cross sectional postal survey of a one in four random sample of registered pharmacies in England and Wales. SETTING--Project conducted in the addiction research unit of the Institute of Psychiatry, London. SUBJECTS--2469 Community pharmacies in the 15 regional health authorities in England and Wales. MAIN OUTCOME MEASURES--Willingness of pharmacists to sell injecting equipment to known or suspected misusers of drugs; pharmacists' attitudes to syringe exchange schemes, keeping a "sharps" box for use by misusers of drugs, and offering face to face advice and leaflets; and opinions of community pharmacists on their role in AIDS prevention and drug misuse. RESULTS--1946 Questionnaires were returned, representing a response rate of 79%. This fell short of the target of one in four pharmacies in each family practitioner committee area in England and Wales, and total numbers of respondents were therefore weighted in inverse proportion to the response rate in each area. The findings disclosed a substantial demand for injecting equipment by drug misusers. After weighting of numbers of respondents an estimated 676 of 2434 pharmacies were currently selling injecting equipment and 65 of 2415 (3%) were participating in local syringe exchange schemes; only 94 of 2410 pharmacies (4%) had a sharps box for used equipment. There was a high degree of concern among pharmacists about particular consequences of drug misusers visiting their premises, along with a widespread acceptance that the community pharmacist had an important part to play. CONCLUSIONS--Promoting the participation of community pharmacists in the prevention of AIDS among misusers of injected drugs is a viable policy, but several problems would need to be overcome before it was implemented.
PMCID: PMC1837978  PMID: 2511969
21.  DNA sequences of the cysK regions of Salmonella typhimurium and Escherichia coli and linkage of the cysK regions to ptsH. 
Journal of Bacteriology  1988;170(7):3150-3157.
Nucleotide sequences of the cysK regions of Salmonella typhimurium and Escherichia coli have been determined. A total of 3,812 and 2,595 nucleotides were sequenced from S. typhimurium and E. coli, respectively. Open reading frames of 323 codons were found in both species and were identified as those of cysK by comparison of deduced amino acid sequences with amino- and carboxyl-terminal amino acid analyses of the S. typhimurium cysK gene product O-acetylserine (thiol)-lyase A. The two cysK DNA sequences were 85% identical, and the deduced amino acid sequences were 96% identical. The major transcription initiation sites for cysK were found to be virtually identical in the two organisms, by using primer extension and S1 nuclease protection techniques. The -35 region corresponding to the major transcription start site was TTCCCC in S. typhimurium and TTCCGC in E. coli. The deviation of these sequences from the consensus sequence TTGACA may reflect the fact that cysK is subject to positive control and requires the cysB regulatory protein for expression. Sequences downstream of cysK were found to include ptsH and a portion of ptsI, thus establishing the exact relationship of cysK with these two genes. A 290-codon open reading frame, which may represent the cysZ gene, was identified upstream of cysK.
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PMCID: PMC211262  PMID: 3290198
22.  Assessment of prognostic factors in patients undergoing surgery for non-rheumatic mitral regurgitation. 
British Heart Journal  1980;44(4):369-380.
Twenty-four patients who had undegone mitral valve surgery for pure non-rheumatic mitral regurgitation were studied non-invasively six months to six years postoperatively. The long-term results of operation were assessed on the basis of clinical history, echocardiography, and treadmill stress testing using a points scoring system. The score so obtained was used to divide the patients into those with a good response to surgery (group 1) and those responding poorly (group 2). The effects on the long-term surgical outcome of several intraoperative and preoperative factors were then analysed both together and separately. A short symptomatic history (less than 1 year), a normal left ventricular end-diastolic volume index (less than or equal to 100 ml per m2), and a large post-ectopic potentiation of KV max (greater than 50 s-1) were found to be favourable prognostic factors when analysed independently. An angiographic ejection fraction less than 0.5 was uniformly associated with a poor outcome, and 71 per cent of patients in atrial fibrillation at the time of operation also responded badly. In those patients with good long-term function, cold potassium cardioplegia was more commonly used than intermittent aortic cross clamping as the means of intraoperative myocardial preservation, though this difference did not reach conventional significance. A standard analysis of variance allowed assessment of length of history, left ventricular end-diastolic volume index, and type of valve prosthesis simultaneously. This indicated that both length of history and left ventricular end-diastolic volume index were highly significant prognostic factors. The use of a Björk-Shiley as opposed to a Starr-Edwards prosthesis also emerged as significantly favouring a good long-term result. The state of the left ventricular myocardium before operation and the type of valve prosthesis used were thus shown to be the prime determinants of surgical outcome in these patients. The optimum time for operation was shown to be within one year of the onset of symptoms, and before the left left ventricular end-diastolic volume index exceeds 100 ml per m2, or the ejection fraction falls to less than 0.5. At such a time, irreversible changes in myocardial function sufficient to negate the beneficial effects of mitral valve surgery have not yet occurred.
PMCID: PMC482413  PMID: 7426197

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