Antimicrobial effects of XF drugs against Candida albicans and its biofilms.

Compared with antibiotics for treating bacterial infections, there are a limited number of antifungal agents. This is due to several factors, including the difficulties of identifying suitable antifungals that target the fungal cell without damaging host cells, and the reduced rates of diagnosis of fungal infections compared with those caused by bacteria. The problem of treating fungal infections is exacerbated by an increasing incidence of antifungal resistance among human fungal pathogens. Three XF drugs (XF-73, XF-70, and DPD-207) have previously displayed innate bactericidal effects and a low propensity for microbial resistance, with XF-73 and XF-70 having a second, light-activated mechanism of action [known as photodynamic therapy (PDT)]. In an effort to expand the repertoire of antifungal agents, this research assessed the in vitro activity of XF drugs via both mechanisms of action against six strains of the fungal pathogen Candida albicans in both planktonic and biofilm cultures. In addition, this research examined the effects of XF drug treatment on biofilms of C. albicans in a reconstituted human oral epithelium model. All C. albicans strains tested were susceptible to XF-73 and XF-70, with minimum inhibitory concentrations (MICs) between 0.25 µg/mL and 2 µg/mL; DPD-207 was less potent, with MICs between 4 µg/mL and 16 µg/mL, and light activation did not enhance these MICs. Complete biofilm eradication was not reported at the tested XF drug concentrations. However, live and dead staining of C. albicans cells in biofilms after XF drug treatment demonstrated that XF-73 and XF-70 were active against most Candida biofilms tested from 64 µg/mL; again, light activation did not enhance anti-biofilm activity. Candida biofilms were more resistant to DPD-207, with fungicidal effects occurring from 256 µg/mL. XF-73 and XF-70 reduced penetration of C. albicans biofilm into reconstituted human oral epithelium (RHOE) and resulted in less damage (as determined by reduced lactate dehydrogenase release) than untreated biofilms. Overall, the results highlight the potential of XF drugs as new drugs for the management of topical infections caused by C. albicans. Further studies are warranted on the development of XF drugs as antifungals, particularly for XF-73 and XF-70.

An investigation of the features facilitating effective collaboration between public health experts and data scientists at a hackathon.

OBJECTIVES: The objective of this study is to explore facilitating factors for collaboration at hackathons, intensive events bringing together data scientists ('hackers') with experts in particular subject areas. STUDY DESIGN: This is a qualitative study. METHODS: Semistructured interviews were conducted with organisers before and after the event. The initial exploratory interviews influenced the content of questionnaires which were distributed to all participants asking about their motivations and experiences. Thematic analysis was used to explore key features of collaboration. RESULTS: Facilitating factors were clustered under the themes of preparation (the right amount of pre-event information, methods to maximise attendance and identification of suitable challenges), participants (enough people to progress and a mixture of skills and experience), working together (mutual understanding of the aim, getting the best out of each other, overcoming challenges together, effective facilitation and an enjoyable and valuable experience) and follow-up (recognised process for feedback and support for the development of prototypes). CONCLUSIONS: The findings of the study provide insight into fostering collaboration in this context and provide evidence that may be used to tailor future events for the effective delivery of technological and marketing-based solutions to public health challenges. Hackathons provide a methodological advance with potential for broad public health application.

Applying an extended theory of planned behaviour to predict breakfast consumption in adolescents.

BACKGROUND/OBJECTIVES: Breakfast skipping increases during adolescence and is associated with lower levels of physical activity and weight gain. Theory-based interventions promoting breakfast consumption in adolescents report mixed findings, potentially because of limited research identifying which determinants to target. This study aimed to: (i) utilise the Theory of Planned Behaviour (TPB) to identify the relative contribution of attitudes (affective, cognitive and behavioural) to predict intention to eat breakfast and breakfast consumption in adolescents and (ii) determine whether demographic factors moderate the relationship between TPB variables, intention and behaviour. SUBJECTS/METHODS: Questionnaires were completed by 434 students (mean 14±0.9 years) measuring breakfast consumption (0-2, 3-6 or 7 days), physical activity levels and TPB measures. Data were analysed by breakfast frequency and demographics using hierarchical and multinomial regression analyses. RESULTS: Breakfast was consumed everyday by 57% of students, with boys more likely to eat a regular breakfast, report higher activity levels and report more positive attitudes towards breakfast than girls (P<0.001). The TPB predicted 58% of the variation in intentions. Overall, the model was predictive of breakfast behaviours (P<0.001), but the relative contribution of TPB constructs varied depending on breakfast frequency. Interactions between gender and intentions were significant when comparing 0-2- and 3-6-day breakfast eaters only highlighting a stronger intention-behaviour relationship for girls. CONCLUSIONS: Findings confirm that the TPB is a successful model for predicting breakfast intentions and behaviours in adolescents. The potential for a direct effect of attitudes on behaviours should be considered in the implementation and design of breakfast interventions.

Regulation of self-renewal and differentiation in adult stem cell lineages: lessons from the Drosophila male germ line.

The ability to identify stem cells and trace their descendants in vivo has yielded insights into how self-renewal, proliferation, and differentiation are regulated in adult stem cell lineages. Analysis of male germ-line stem cells in Drosophila has revealed the importance of local signals from the microenvironment, the stem cell niche, in controlling stem cell behavior. Germ-line stem cells physically attach to the niche via localized adherens junctions that provide a polarity cue for orientation of centrosomes in interphase and the spindle in mitosis. As a result, stem cells divide asymmetrically: One daughter inherits attachment to the niche and remains within its embrace, whereas the other is displaced away and initiates differentiation. Strikingly, much as leukemia inhibitory factor (LIF) and transforming growth factor-beta (TGF-beta) signaling maintain mouse embryonic stem (ES) cells, maintenance of stem cell state in the Drosophila male germ line is regulated by cytokine-like signals from hub cells that activate the transcription factor STAT (signal transducer and activator of transcription) and TGF-beta class signals from surrounding support cells that repress expression of a key differentiation factor. Surprisingly, transit-amplifying cells can revert to the stem cell state if they reoccupy the niche. Upon cessation of mitosis and the switch to terminal differentiation, germ cells express cell-type- and stage-specific transcription machinery components that drive expression of terminal differentiation genes, in part by removing Polycomb transcriptional silencing machinery.

Tag SNP screening of the PDCD1 gene for association with Graves' disease.

OBJECTIVE: The Programmed Cell Death 1 gene (PDCD1) on chromosome 2q37.3 encodes PD-1 which is involved in providing a negative signal to activated T cells. Large case-control studies have shown association of PDCD1 with several autoimmune diseases although, to date, no such studies have been performed for Graves' disease (GD). The objective of our study was to investigate eight tag SNPs representing the majority of common variation in PDCD1 within a well-characterized large UK Caucasian GD dataset. DESIGN: A case control association study of eight polymorphisms. PATIENTS: 2671 Graves' disease patients and 864 controls. MEASUREMENTS: Tests for association with disease. RESULTS: No association with disease was seen for any of the +4163, +5049, +5318, +5640, +5678 and +7078 SNPs genotyped in this study. Association was detected between the +2375 SNP (P = 0.021, OR = 1.14 [95% CI = 1.01-1.29]) and GD and a small protective effect was seen with the +6799 SNP genotypes (P = 0.028, OR = 0.77 [95% CI = 0.58-1.03]). CONCLUSIONS: This study has, for the first time, shown that small effects within PDCD1 may contribute towards the development of GD, supporting the hypothesis that much of the currently unknown genetic contribution to GD could be due to several small genetic effects with ORs 1.2. Replication of this result is now needed to confirm our findings and justify more detailed fine mapping of a primary aetiological variant in this gene region.

Heat shock proteins form part of a danger signal cascade in response to lipopolysaccharide and GroEL.

An increasing number of cell types, including peripheral blood mononuclear cells (PBMCs), have been demonstrated to release heat shock proteins (Hsps). In this paper we investigate further the hypothesis that Hsps are danger signals. PBMCs and Jurkat cells released Hsp70 (0.22 and 0.7 ng/10(6) cells, respectively) into medium over 24 h at 37 degrees C. Release of Hsp70 was stimulated 10-fold by GroEL (P < 0.001) and more than threefold by lipopolysaccharide (LPS) (P < 0.001). Although Hsp60 could be detected in the medium of cells cultured at 37 degrees C for 24 h, the low rates of release were due probably to cell damage. Significant release of Hsp60 was observed when Jurkat cells were exposed to GroEL (2.88 ng/10(6) cells) or LPS (1.40 ng/10(6) cells). The data are consistent with the hypothesis that Hsp70 and Hsp60 are part of a danger signalling cascade in response to bacterial infection.

Retrospective comparison of mesh and sutured repair for adult umbilical hernias.

Adult umbilical and paraumbilical hernia repair is associated with a high recurrence rate of 10-30%. Mesh repair has been reported to be associated with low recurrence rates. This study aims to compare sutured repair with prosthetic mesh repair to evaluate recurrence and infection rates. A retrospective study was conducted over an 8-year period including all the umbilical and paraumbilical hernia repairs performed by one consultant surgeon. The hernias were repaired using interrupted suture, Mayo overlap, flat mesh and mesh plug techniques. The study was based on case-note review, telephone and postal questionnaire survey. A total of 100 patients were studied, of which 70 had paraumbilical hernias, 28 had umbilical hernias and 2 had both types of hernia. Median age was 56 years (range 19-90 years). A total of 61 patients had suture repair (50 interrupted suture repair, 11 Mayo) and 39 had prosthetic mesh repair (33 mesh plug, 6 flat mesh). The median body mass index (BMI) was 31.2 (range 23.4-44.5) in the suture repair group and 33.3 (range 24.1-59.1) in the mesh group, with no significant statistical difference in BMI between the two groups (P>0.05). Median follow-up was 4.5 years (range 1-8 years). Recurrence rates for the suture and mesh repair groups were 11.5 and 0%, respectively (P=0.007). Infection rates for the suture and mesh repair groups were 11.5 and 0%, respectively (P=0.007). Our data suggest that prosthetic mesh repair is ideal for managing primary and recurrent umbilical hernias in both obese and non-obese patients.

Eyes absent mediates cross-talk between retinal determination genes and the receptor tyrosine kinase signaling pathway.

Eyes absent (eya) encodes a member of a network of nuclear transcription factors that promotes eye development in both vertebrates and invertebrates. Despite extensive studies, the molecular mechanisms whereby cell-cell signaling pathways coordinate the function of this retinal determination gene network remain unknown. Here, we report that Drosophila Eya function is positively regulated by mitogen-activated protein kinase (MAPK)-mediated phosphorylation and that this regulation extends to developmental contexts independent of eye determination. In vivo genetic analyses, together with in vitro kinase assay results, demonstrate that Eya is a substrate for extracellular signal-regulated kinase, the MAPK acting downstream in the receptor tyrosine kinase (RTK) signaling pathway. Thus, phosphorylation of Eya appears to provide a direct regulatory link between the RTK/Ras/MAPK signaling cascade and the retinal determination gene network.

Functional enhancement of electrofusion-derived BRIN-BD11 insulin-secreting cells after implantation into diabetic mice.

Electrofusion-derived BRIN-BD11 cells are glucose-sensitive insulin-secreting cells which provide an archetypal bioengineered surrogate beta-cell for insulin replacement therapy in diabetes mellitus. 5x10(6) BRIN-BD11 cells were implanted intraperitoneally into severely hyperglycaemic (>24 mmol/l) streptozotocin-induced insulin-treated diabetic athymic nude (nu/nu) mice. The implants reduced hyperglycaemia such that insulin injections were discontinued by 5-16 days (<17 mmol/l) and normoglycaemia (<9 mmol/l) was achieved by 7-20 days. Implanted cells were removed after 28 days and re-established in culture. After re-culture for 20 days, glucose-stimulated (16.7 mmol/l) insulin release was enhanced by 121% (p<0.001) compared to non-implanted cells. Insulin responses to glucagon-like peptide-1 (10(-9) mol/l), cholecystokinin-8 (10(-8) mol/l) and L-alanine (10 mmol/l) were increased by 32%, 31% and 68% respectively (p<0.05-0.01). Insulin content of the cells was 148% greater at 20 days after re-culture than before implantation (p<0.001), but basal insulin release (at 5.6 mmol/l glucose) was not changed. After re-culture for 40 days, insulin content declined to 68% of the content before implantation (p<0.01), although basal insulin release was unchanged. However, the insulin secretory responses to glucose, glucagon-like peptide-1, cholecystokinin-8 and L-alanine were decreased after 40 days of re-culture to 65%, 72%, 73% and 42% respectively of the values before implantation (p<0.05-0.01). The functional enhancement of electrofusion-derived surrogate beta-cells that were re-cultured for 20 days after implantation and restoration of normoglycaemia indicates that the in vivo environment could greatly assist beta-cell engineering approaches to therapy for diabetes.

The immunohistochemical expression of desmoplakin and its role in vivo in the progression and metastasis of breast cancer.

Desmoplakin (DP) is a protein located at the inner plaque of desmosomes where it associates with the desmosomal cadherins to form a cell adhesion complex. Reduced expression of DP has been correlated with the progression of several cancers, but its role in in vivo breast cancer is yet to be established. The aim of this present paper was to determine the immunohistochemical (IHC) expression of DP in breast cancer specimens (n = 75) in comparison with ductal carcinoma in situ (DCIS) (n = 26), tumour associated normal (n = 29) and normal breast tissue (n = 7). DP expression was correlated with that of desmosomal cadherin, Desmoglein 2 (Dsg2) and other clinical and IHC prognostic markers. DP staining occurred at the sub-plasma membrane level. There was no significant correlation between the level of DP (as assessed by the H-score) and that of Dsg. Significantly stronger staining was demonstrated in normal breast tissue and well differentiated tumours compared with more moderately or poorly differentiated tumours (P = 0.04). A significant inverse correlation was seen between DP staining and tumour size (P = 0.01). In a limited series of 8 cases, primary tumours demonstrated significantly stronger staining than the matched metastatic lymph nodes (P = 0.046). Of all the IHC markers examined, only Ki-67 showed a significant inverse relationship with DP staining (P = 0.01). In summary, the data suggest that loss of DP may be of potential importance in progression of breast cancer in vivo from normal, DCIS, well differentiated through to poorly differentiated, large tumours. In addition, this loss may be associated with metastasis.

The National Breast Referral Guidelines have cut down inappropriate referrals in the under 50s.

AIMS: When the National Breast Referral Guidelines were applied to our local GPs letters immediately prior to their release in January 1996, it was shown that on the basis of the GPs own conclusions that 29% of symptomatic women could have been managed initially by their own GP without missing any carcinomas. We conducted this study at the Rapid Access Breast Clinic at the University Hospital of Wales to determine if the breast referral practices of local GPs have altered due to the breast referral guidelines. METHODS: We studied 2332 referrals from the inception of the Rapid Access Clinic in May 1995 to the issue of the guidelines, and 2421 referrals from May 1996 to the end of the year. Random samples of 600 patients were drawn from each year and the referral letters were scored as within or outside the guidelines. Family history patients were excluded. RESULTS: There was an 11% fall in referrals outside the guidelines in the under 50s (chi-squared=<0.001) but the 7% fall in the over 50s was not significant. CONCLUSIONS: The Breast Referral Guidelines seem to have been effective in reducing the higher level of inappropriate referrals in younger patients at less risk of carcinoma.

Prospects for insulin delivery by ex-vivo somatic cell gene therapy.

The principle of insulin delivery by ex-vivo somatic cell gene therapy involves the removal of non-B-cell somatic cells (e.g. fibroblasts) from a diabetic patient, and genetically altering them in vitro to produce and secrete insulin. The cells can be grown in culture and returned to the donor as a source of insulin replacement. Cells modified in this way could be evaluated before implantation, and reserve stocks could be cryopreserved. By using the patient's own cells, the procedure should obviate the need for immunosuppression and overcome the problem of tissue supply, while avoiding a recurrence of cell destruction. Ex-vivo somatic cell gene therapy requires an accessible and robust cell type that is amenable to multiple transfections and subject to controlled proliferation. Special problems associated with the use of non-B-cell somatic cells include the processing of proinsulin to insulin, and the conferment of sensitivity to glucose-stimulated proinsulin biosynthesis and regulated insulin release. Preliminary studies using fibroblasts, pituitary cells, kidney (COS) cells and ovarian (CHO) cells suggest that these challenges could be met, and that ex-vivo somatic cell gene therapy offers a feasible approach to insulin replacement therapy.

Is there a role for surgery in the treatment of mastalgia?

Breast pain is a common condition affecting most women at some stage in their reproductive life. Mastalgia is resistant to treatment in 6% of cyclical and 26% non-cyclical patients. Surgery is not widely used to treat this condition and only considered in patients with severe mastalgia resistant to medication. The aims of this study were to audit the efficacy of surgery in severe treatment resistant mastalgia and to assess patient satisfaction following surgery. This is a retrospective review of the medical records of all patients seen in mastalgia clinic in the University Hospital of Wales, Cardiff since 1973. A postal questionnaire was distributed to all patients who had undergone surgery. Results showed that of the 1054 patients seen in mastalgia clinic, 12 (1.2%) had undergone surgery. Surgery included 8 subcutaneous mastectomies with implants (3 bilateral, 5 unilateral), 1 bilateral simple mastectomy and 3 quadrantectomies (1 having a further simple mastectomy). The median duration of symptoms was 6.5 years (range 2-16 years). Five patients (50%) were pain free following surgery, 3 developed capsular contractures and 2 wound infections with dehiscence. Pain persisted in both patients undergoing quadrantectomy. We conclude that surgery for mastalgia should only be considered in a minority of patients. Patients should be informed of possible complications inherent of reconstructive surgery and warned that in 50% cases their pain will not be improved.

The long-term course of mastalgia.

Mastalgia is a common condition in women of reproductive years. We have assessed the long-term course in patients with severe mastalgia by distributing a postal questionnaire to 212 patients previously studied in 1983 who had attended the mastalgia clinic at the University Hospital of Wales, Cardiff. 175 patients (83%) responded, with an original diagnosis of cyclical mastalgia (CM) in 120 and non-cyclical mastalgia (NCM) in 55. The median age of onset of breast pain was 36 years (range 12-63 years). The average duration of pain was long (median 12 years), especially if it started in the second or third decade of life. Pain persisted in 68 (57%) of CM and 35 (64%) of NCM patients. In CM patients resolution was commonly associated with a 'hormonal' event, notably the menopause; in NCM patients it more often seemed to be spontaneous. Severe mastalgia ran a chronic relapsing course often requiring repeated drug treatments.

Expression of GLUT2 in insulin-secreting AtT20 pituitary cells.

The importance of the glucose transporter isoform, GLUT2, in the construction of glucose-sensitive surrogate insulin-secreting cells was evaluated using murine pituitary AtT20 cells. The cells were double transfected with cDNAs for human preproinsulin (hppI-1) driven by the cytomegalovirus promoter, and human GLUT2 driven by the beta-actin promoter. The stably transfected clone, AtTinsGLUT2.36, which strongly expressed both the hppI-1 and GLUT2 genes, constitutively released 7.5 ng/10(6) cells/24 h of immunoreactive insulin-like material, 75% of which was fully processed mature human insulin. Increasing glucose concentrations in the subphysiological range up to 50 microM increased insulin release, but greater glucose concentrations did not further increase insulin release. Suppression of the low-K(m) glucose-phosphorylating enzyme, hexokinase, with 2-deoxy-D-glucose increased glucose-stimulated insulin release by two- to threefold in the presence of subphysiological and physiological glucose concentrations up to 10 mM. Physiological glucose concentrations increased the amount of GLUT2 mRNA, indicating that the beta-actin promoter responds in a glucose-dependent manner. Implantation of 2 x 10(7) AtTinsGLUT2.36 cells intraperitoneally into streptozotocin-diabetic nude mice slowed the progression of hyperglycaemia. The implanted cells formed vascularised tumour-like cell aggregates attached to the peritoneum. The results demonstrate that the beta-actin promoter is partially regulated by glucose. Expression of GLUT2 enables glucose to enter the cell at high K(m), but high-K(m) glucose phosphorylation is also required to signal glucose-stimulated genes affecting insulin release.

Mitogen-activated protein kinase phosphatase 1 inhibits the stimulation of gene expression by hypertrophic agonists in cardiac myocytes.

The effect of constitutive expression of mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1) on gene expression in response to hypertrophic agonists was examined in cultured neonatal rat ventricular myocytes. Luciferase (LUX) reporter genes linked to promoters for atrial natriuretic factor, ventricular myosin light chain 2, beta-myosin heavy chain, skeletal muscle alpha-actin (SkM alpha-actin) and serum response element-regulated c-fos (c-fos-SRE) were transfected into cardiomyocytes. Phenylephrine (PE; 10 microM), phorbol 12-myristate 13-acetate (1 microM) and endothelin 1 (10 nM) stimulated the expression of these various reporter genes by 2. 5-20-fold. MKP-1 inhibited these effects by 60-85%. In contrast, MKP-1 had no effect on the expression of a constitutively active Rous sarcoma virus-LUX reporter gene. A catalytically inactive mutant MKP-1CS (cysteine-->serine mutation) and the broad-specificity protein tyrosine phosphatase 1B (PTP-1B) had no significant effect on any reporter gene tested. MKP-1 had much less effect on the morphological features accompanying agonist-induced cardiac hypertrophy. PE (10 microM) increased myocyte area by 59% but this effect was only decreased by one-third by MKP-1 and was also partly decreased (by 25%) by expression of PTP-1B. PE also altered cell shape but this was unaffected by MKP-1. There was also no clear effect of MKP-1 on the organization of the contractile apparatus into sarcomeric structures in the presence of 10 microM PE. We conclude that the transcriptional responses accompanying cardiac myocyte hypertrophy are dependent on an MKP-1-sensitive step, presumably the activation of one or members of the MAPK family, but that cell size, shape and myofibrillar organization are much less sensitive to inhibition by MKP-1.

Selection of specific phage-display antibodies using libraries derived from chicken immunoglobulin genes.

In chickens, single functional immunoglobulin variable and joining gene segments at each of the heavy and light chain loci undergo V(D)J rearrangement. Diversity is subsequently introduced by conversions templated by upstream pseudo V region genes in such a way that practically all V regions in mature B cells have identical ends. This greatly simplifies the representative amplification of V region genes. Furthermore, the entire naive repertoire of the adult chicken is produced in the bursa of Fabricius of the young bird. These special properties of the generation of immunoglobulin diversity in chickens have been exploited in the development of procedures to produce large libraries of diverse antibody combining sites derived from chicken Ig genes and expressed on filamentous bacteriophage. The utility of this library was assessed by selection of specifically binding phage using three solid phase-bound protein antigens, hen egg white lysozyme, bovine thyroglobulin and bovine serum albumin. The sequences of the V region genes thus isolated demonstrated that selection was specific and that the library contained useful diversity of binding sites. This library provides access to a repertoire whose diversity is based on a mechanism different from that underlying previously available libraries. The demonstrated feasibility of generating chicken phage antibodies may lead to the production of monoclonal reagents from immunised chickens, and the derivation of reagents for studying immunoglobulin mediated selection in avian B cell development.