Optimized delivery of siRNA into 3D tumor spheroid cultures in situ.

3D tissue culture provides a physiologically relevant and genetically tractable system for studying normal and malignant human tissues. Despite this, gene-silencing studies using siRNA has proved difficult. In this study, we have identified a cause for why traditional siRNA transfection techniques are ineffective in eliciting gene silencing in situ within 3D cultures and proposed a simple method for significantly enhancing siRNA entry into spheroids/organoids. In 2D cell culture, the efficiency of gene silencing is significantly reduced when siRNA complexes are prepared in the presence of serum. Surprisingly, in both 3D tumour spheroids and primary murine organoids, the presence of serum during siRNA preparation rapidly promotes entry and internalization of Cy3-labelled siRNA in under 2 hours. Conversely, siRNA prepared in traditional low-serum transfection media fails to gain matrigel or spheroid/organoid entry. Direct measurement of CTNNB1 mRNA (encoding ß-catenin) from transfected tumour spheroids confirmed a transient but significant knockdown of ß-catenin when siRNA:liposome complexes were formed with serum, but not when prepared in the presence of reduced-serum media (Opti-MEM). Our studies suggest a simple modification to standard lipid-based transfection protocols facilitates rapid siRNA entry and transient gene repression, providing a platform for researchers to improve siRNA efficiency in established 3D cultures.

LGR5 expression is regulated by EGF in early colorectal adenomas and governs EGFR inhibitor sensitivity.

BACKGROUND: LGR5 serves as a co-receptor for Wnt/ß-catenin signalling and marks normal intestinal stem cells; however, its role in colorectal cancer (CRC) remains controversial. LGR5+ cells are known to exist outside the stem cell niche during CRC progression, and the requirement for epidermal growth factor (EGF) signalling within early adenomas remains to be fully elucidated. METHODS: Epidermal growth factor and gefitinib treatments were performed in EGF-responsive LGR5+ early adenoma RG/C2 cells. 2D growth assays were measured using an IncuCyte. LGR5 or MEK1/2 silencing studies were executed using siRNA and LGR5 expression was assessed by qRT-PCR and immunoblotting. Ki67 level and cell cycle status were analysed by flow cytometry. RESULTS: Epidermal growth factor suppresses expression of LGR5 at both the transcript and protein level in colorectal adenoma and carcinoma cells. Suppression of LGR5 reduces the survival of EGF-treated adenoma cells by increasing detached cell yield but also inducing a proliferative state, as evidenced by elevated Ki67 level and enhanced cell cycle progression. Repression of LGR5 further increases the sensitivity of adenoma cells to EGFR inhibition. CONCLUSIONS: LGR5 has an important role in the EGF-mediated survival and proliferation of early adenoma cells and could have clinical utility in predicting response of CRC patients to EGFR therapy.

Associations between single-nucleotide polymorphisms (+45T>G, +276G>T, -11377C>G, -11391G>A) of adiponectin gene and type 2 diabetes mellitus: a systematic review and meta-analysis.

AIMS/HYPOTHESIS: The associations between adiponectin polymorphisms and type 2 diabetes have been studied widely; however, results are inconsistent. METHODS: We searched electronic literature databases and reference lists of relevant articles. A fixed or random effects model was used on the basis of heterogeneity. Sub-group and meta-regression analyses were conducted to explore the sources of heterogeneity. RESULTS: There were no statistically significant associations between +45T>G (rs2241766), +276G>T (rs1501299), -11391G>A (rs17300539) and type 2 diabetes risk. However, for -11377C>G (rs266729), the pooled OR (95% CI) for G vs C allele was 1.07 (1.03-1.11, p = 0.001). Subgroup analysis by study design revealed that -11377C>G (rs266729) dominant model (CG+GG vs CC, p = 0.0008) and G vs C allele (p = 0.0004) might be associated with type 2 diabetes risk in population-based case-control studies. After stratification by ethnicity, we found that -11377C>G (rs266729) dominant model (CG+GG vs CC, p = 0.004) and G vs C allele (p = 0.001) might be associated with type 2 diabetes risk in white individuals. In individuals with a family history of diabetes, the presence of -11391G>A (rs17300539) dominant model (GA+AA vs GG) and A vs G allele might be associated with increased risk of type 2 diabetes. CONCLUSIONS/INTERPRETATION: The presence of +45T>G (rs2241766), +276G>T (rs1501299) and -11391G>A (rs17300539) do not appear to influence the development of type 2 diabetes. However, G vs C allele of -11377C>G (rs266729) might be a risk factor for type 2 diabetes.

Carotid artery angioplasty for restenosis following endarterectomy.

INTRODUCTION: The higher complication rate associated with the surgical treatment of restenosis following carotid endarterectomy (CEA) has led several authors to advocate angioplasty as the treatment of choice in the management of restenosis. We describe our experience with internal carotid artery angioplasty for post-endarterectomy restenosis over 7 years. PATIENTS AND METHODS: From January 1994 to April 2001, all patients with a >90% restenosis following CEA were considered for angioplasty. Thirty angioplasties were carried out in 25 patients, 80% (24/30), for asymptomatic recurrent stenosis. There was no difference between those who had intervention for recurrent stenosis (n=31) and those who did not (n=545) in age, sex, smoking status or incidence of diabetes or hypertension. A significantly greater number of patients who underwent angioplasty were hypercholesterolaemic (p<0.05, Chi-squared test). RESULTS: Mean time from surgery to angioplasty was 13 months (range 1-23). Angioplasty was technically successful in 29 cases (97%). Three patients (10%) experienced transient neurological symptoms during the procedure. There were no strokes. Ninety-six percent (28/29) of patients were followed up with duplex scanning. Mean follow-up was 20 months (range 2-48). Three patients developed a greater than 90% restenosis. CONCLUSION: Angioplasty is an acceptable alternative to surgery in the management of internal carotid artery restenosis following endarterectomy.

Diagnosis in chronic illness: disabling or enabling--the case of chronic fatigue syndrome.

This paper examines doctors' and patients' views on the consequences of an increasingly common symptomatic diagnosis, chronic fatigue syndrome (CFS). Two studies were conducted: the first comprised interviews with 20 general practitioners; the second was a longitudinal study, comprising three interviews over a period of 2 years with 50 people diagnosed with CFS. Contrasts were apparent between doctors' practical and ethical concerns about articulating a diagnosis of CFS and patients' experiences with and without such a diagnosis. Seventy per cent of the doctors were reluctant to articulate a diagnosis of CFS. They felt constrained by the scientific uncertainty regarding its aetiology and by a concern that diagnosis might become a disabling self-fulfilling prophecy. Patients, by contrast, highlighted the enabling aspects of a singular coherent diagnosis and emphasized the negative effects of having no explanation for their problems.

Multiple drug use by older people: exploring the health development and political economy perspectives.

The use of multiple medications by a large number of older people provides grounds for concern in terms of quality of life as well as cost. We argue that problems faced by older people are being over-medicalized in a manner that palliates and obscures social causes such as loss of income, the falling away of social support and a discounted role in society. Primary health care is a policy model for the development of health services which offers a credible strategy for addressing clinical problems associated with growing old in ways that also contribute to recognizing and addressing social and structural problems that may be expressed in people's private troubles.

The community development in health project.

This paper describes the purpose and process of a 15 month project established to develop resources to assist people who are seeking to use a community development approach in addressing health issues.

Distribution and diversity of hsd genes in Escherichia coli and other enteric bacteria.

We screened Salmonella typhimurium, Citrobacter freundii, Klebsiella pneumoniae, Shigella boydii, and many isolates of Escherichia coli for DNA sequences homologous to those encoding each of two unrelated type I restriction and modification systems (EcoK and EcoA). Both K- and A-related hsd genes were identified, but never both in the same strain. S. typhimurium encodes three restriction and modification systems, but its DNA hybridized only to the K-specific probe which we know to identify the StySB system. No homology to either probe was detected in the majority of E. coli strains, but in C. freundii, we identified homology to the A-specific probe. We cloned this region of the C. freundii genome and showed that it encoded a functional, A-related restriction system whose specificity differs from those of known type I enzymes. Sequences immediately flanking the hsd K genes of E. coli K-12 and the hsd A genes of E. coli 15T- were shown to be homologous, indicating similar or even identical positions in their respective chromosomes. E. coli C has no known restriction system, and the organization of its chromosome is consistent with deletion of the three hsd genes and their neighbor, mcrB.

CEPRIME (Continuing Education and Peer Review in Internal Medicine): linking quality assurance with continuing medical education.

This was a pilot project intended to evaluate the practicalities of a new method of linking Peer Review with Continuing Medical Education for consultant physicians (internists). Peer Review of the physicians' reports to the referring general practitioners on each consultation. Sixteen physicians were enrolled for varying periods of time. They submitted from 12 to 454 cases for review (mean 94 cases) overall. One half of the cases attracted comment from the reviewers. The main themes of critical comments have been identified. Appropriate study courses based on the results of the review were developed for some of the participants. The latter were in general agreement that the CEPRIME system has potential to achieve its purpose; but it is obvious that certain modifications would improve the flow of material and the value of the exercise to the physicians.

Quality assurance: what is the consumers' role?

We commenced with the question: what is the "proper" role for consumers in quality assurance? Quality assurance has been discussed as encompassing three phases: clinical review, linkage and drive. We have discussed a range of mechanisms which can contribute to expressing a consumer perspective or the consumers' interest in each of these phases of quality assurance. It is clear that the consumers' experience is a useful and legitimate input to clinical review. It is clear that mechanisms for listening more carefully to the consumers' perspective could be introduced more widely. It is clear that greater consumer understanding of an involvement in the whole quality assurance cycle is practicable and, from the point of view of many consumers, highly desirable. Whilst more detailed answers have not been brought forward, a useful framework for considering how to proceed from here has been presented.

Continuing education and peer review in internal medicine (CEPRIME).

Research in adult education shows that educational initiatives are most effective when they are related to perceived needs. The CEPRIME system was devised as a means of using peer review to reveal the educational needs of physicians. Sixteen physicians volunteered to test the system, by allowing a panel of specialists to review copies of their reports to referring general practitioners. The comments of the reviewers, both commendatory and critical, were referred back to the participants, and their evaluation of the system was sought. Although the method was found to have limitations relating both to the adequacy of the report as a document for review, and to the objectivity and relevance of the comments made about it, the results were such as to encourage further development of CEPRIME as a means of appraising physicians of their specific educational needs.

Computed tomographic arteriography in the pre-surgical evaluation of hepatic tumours.

Five patients with hepatic neoplasms considered suitable for surgical resection were examined by computed tomography (CT), hepatic angiography and then a combination of angiography and CT (CTA). In each patient the tumour was more clearly defined and its extent more accurately determined than by routine CT. Three patients were spared surgery when further hepatic tumour was shown by CTA. The left lobe was shown to be free of tumour in the other two patients and surgical resection was carried out. Examination by CTA is recommended in all patients with hepatic tumours considered suitable for hepatic resection.