Three DNA polymerases, recruited by different mechanisms, carry out NER repair synthesis in human cells

Nucleotide excision repair (NER) is the most versatile DNA repair system that deals with the major UV photoproducts in DNA, as well as many other DNA adducts. The early steps of NER are well understood, whereas the later steps of repair synthesis and ligation are not. In particular, which polymerases are definitely involved in repair synthesis and how they are recruited to the damaged sites has not yet been established. We report that, in human fibroblasts, approximately half of the repair synthesis requires both polκ and polδ, and both polymerases can be recovered in the same repair complexes. Polκ is recruited to repair sites by ubiquitinated PCNA and XRCC1 and polδ by the classical replication factor complex RFC1-RFC, together with a polymerase accessory factor, p66, and unmodified PCNA. The remaining repair synthesis is dependent on polɛ, recruitment of which is dependent on the alternative clamp loader CTF18-RFC

Sequential gene promoter methylation during HPV-induced cervical carcinogenesis

We aimed to link DNA methylation events occurring in cervical carcinomas to distinct stages of HPV-induced transformation. Methylation specific-multiplex ligation-dependent probe amplification (MS-MLPA) analysis of cervical carcinomas revealed promoter methylation of 12 out of 29 tumour suppressor genes analysed, with MGMT being most frequently methylated (92%). Subsequently, consecutive stages of HPV16/18-transfected keratinocytes (n=11), ranging from pre-immortal to anchorage-independent phenotypes, were analysed by MS-MLPA. Whereas no methylation was evident in pre-immortal cells, progression to anchorage independence was associated with an accumulation of frequent methylation events involving five genes, all of which were also methylated in cervical carcinomas. TP73 and ESR1 methylation became manifest in early immortal cells followed by RARβ and DAPK1 methylation in late immortal passages. Complementary methylation of MGMT was related to anchorage independence. Analysis of nine cervical cancer cell lines, representing the tumorigenic phenotype, revealed in addition to these five genes frequent methylation of CADM1, CDH13 and CHFR. In conclusion, eight recurrent methylation events in cervical carcinomas could be assigned to different stages of HPV-induced transformation. Hence, our in vitro model system provides a valuable tool to further functionally address the epigenetic alterations that are common in cervical carcinomas

Adenoviral vector-mediated expression of a gene encoding secreted, EpCAM-targeted carboxylesterase-2 sensitises colon cancer spheroids to CPT-11

CPT-11 (irinotecan or 7-ethyl-10[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin) is an anticancer agent in use for the treatment of colon cancer. In order to be fully active, CPT-11 needs to be converted into SN-38 (7-ethyl-10-hydroxycamptothecin) by the enzyme carboxylesterase (CE). In humans, only a minority of CPT-11 is converted to SN-38. To increase the antitumour effect of CPT-11 by gene-directed enzyme prodrug therapy, we constructed a replication-deficient adenoviral vector Ad.C28-sCE2 containing a fusion gene encoding a secreted form of human liver CE2 targeted to the surface antigen epithelial cell adhesion molecule (EpCAM) that is highly expressed on most colon carcinoma cells. By targeting CE2 to EpCAM, the enzyme should accumulate specifically in tumours and leakage into the circulation should be minimised. Ad.C28-sCE2-transduced colon carcinoma cells expressed and secreted active CE that bound specifically to EpCAM-expressing cells. In sections of three-dimensional colon carcinoma spheroids transduced with Ad.C28-sCE2, it was shown that C28-sCE2 was capable of binding untransduced cells. Most importantly, treatment of these spheroids with nontoxic concentrations of CPT-11 resulted in growth inhibition comparable to treatment with SN-38. Therefore, Ad.C28-sCE2 holds promise in gene therapy approaches for the treatment of colon carcinoma

The effectiveness of the McKenzie method in addition to first-line care for acute low back pain: a randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>Low back pain is a highly prevalent and disabling condition worldwide. Clinical guidelines for the management of patients with acute low back pain recommend first-line treatment consisting of advice, reassurance and simple analgesics. Exercise is also commonly prescribed to these patients. The primary aim of this study was to evaluate the short-term effect of adding the McKenzie method to the first-line care of patients with acute low back pain.</p> <p>Methods</p> <p>A multi-centre randomized controlled trial with a 3-month follow-up was conducted between September 2005 and June 2008. Patients seeking care for acute non-specific low back pain from primary care medical practices were screened. Eligible participants were assigned to receive a treatment programme based on the McKenzie method and first-line care (advice, reassurance and time-contingent acetaminophen) or first-line care alone, for 3 weeks. Primary outcome measures included pain (0-10 Numeric Rating Scale) over the first seven days, pain at 1 week, pain at 3 weeks and global perceived effect (-5 to 5 scale) at 3 weeks. Treatment effects were estimated using linear mixed models.</p> <p>Results</p> <p>One hundred and forty-eight participants were randomized into study groups, of whom 138 (93%) completed the last follow-up. The addition of the McKenzie method to first-line care produced statistically significant but small reductions in pain when compared to first-line care alone: mean of -0.4 points (95% confidence interval, -0.8 to -0.1) at 1 week, -0.7 points (95% confidence interval, -1.2 to -0.1) at 3 weeks, and -0.3 points (95% confidence interval, -0.5 to -0.0) over the first 7 days. Patients receiving the McKenzie method did not show additional effects on global perceived effect, disability, function or on the risk of persistent symptoms. These patients sought less additional health care than those receiving only first-line care (<it>P </it>= 0.002).</p> <p>Conclusions</p> <p>When added to the currently recommended first-line care of acute low back pain, a treatment programme based on the McKenzie method does not produce appreciable additional short-term improvements in pain, disability, function or global perceived effect. However, the McKenzie method seems to reduce health utilization although it does not reduce patient's risk of developing persistent symptoms.</p> <p>Trial Registration</p> <p>Australian New Zealand Clinical Trials Registry: ACTRN12605000032651</p

Psychosocial factors and their predictive value in chiropractic patients with low back pain: a prospective inception cohort study

BACKGROUND: Being able to estimate the likelihood of poor recovery from episodes of back pain is important for care. Studies of psychosocial factors in inception cohorts in general practice and occupational populations have begun to make inroads to these problems. However, no studies have yet investigated this in chiropractic patients. METHODS: A prospective inception cohort study of patients presenting to a UK chiropractic practice for new episodes of non-specific low back pain (LBP) was conducted. Baseline questionnaires asked about age, gender, occupation, work status, duration of current episode, chronicity, aggravating features and bothersomeness using Deyo's 'Core Set'. Psychological factors (fear-avoidance beliefs, inevitability, anxiety/distress and coping, and co-morbidity were also assessed at baseline. Satisfaction with care, number of attendances and pain impact were determined at 6 weeks. Predictors of poor outcome were sought by the calculation of relative risk ratios. RESULTS: Most patients presented within 4 weeks of onset. Of 158 eligible and willing patients, 130 completed both baseline and 6-week follow-up questionnaires. Greatest improvements at 6 weeks were in interference with normal work (ES 1.12) and LBP bothersomeness (ES 1.37). Although most patients began with moderate-high back pain bothersomeness scores, few had high psychometric ones. Co-morbidity was a risk for high-moderate interference with normal work at 6 weeks (RR 2.37; 95% C.I. 1.15–4.74). An episode duration of >4 weeks was associated with moderate to high bothersomeness at 6 weeks (RR 2.07; 95% C.I. 1.19 – 3.38) and negative outlook (inevitability) with moderate to high interference with normal work (RR 2.56; 95% C.I. 1.08 – 5.08). CONCLUSION: Patients attending a private UK chiropractic clinic for new episodes of non-specific LBP exhibited few psychosocial predictors of poor outcome, unlike other patient populations that have been studied. Despite considerable bothersomeness at baseline, scores were low at follow-up. In this independent health sector back pain population, general health and duration of episode before consulting appeared more important to outcome than psychosocial factors

Using theory to improve low back pain care in Australian Aboriginal primary care: a mixed method single cohort pilot study

Background: Low back pain (LBP) care is frequently discordant with research evidence. This pilot study evaluated changes in LBP care following a systematic, theory informed intervention in a rural Australian Aboriginal Health Service. We aimed to improve three aspects of care; reduce inappropriate LBP radiological imaging referrals, increase psychosocial oriented patient assessment and, increase the provision of LBP self-management information to patients. Methods: Three interventions to improve care were developed using a four-step systematic implementation approach. A mixed methods pre/post cohort design evaluated changes in the three behaviours using a clinical audit of LBP care in a six month period prior to the intervention and then following implementation. In-depth interviews elicited the perspectives of involved General Practitioners (GPs). Qualitative analysis was guided by the theoretical domains framework. Results: The proportion of patients who received guideline inconsistent imaging referrals (GICI) improved from 4.1 GICI per 10 patients to 0.4 (95 % CI for decrease in rate: 1.6 to 5.6) amongst GPs involved in the intervention. Amongst non-participating GPs (locum/part-time GPs who commenced post-interventions) the rate of GICI increased from 1.5 to 4.4 GICI per 10 patients (95 % CI for increase in rate: .5 to 5.3). There was a modest increase in the number of patients who received LBP self-management information from participating GPs and no substantial changes to psychosocial oriented patient assessments by any participants; however GPs qualitatively reported that their behaviours had changed. Knowledge and beliefs about consequences were important behavioural domains related to changes. Environmental and resource factors including protocols for locum staff and clinical tools embedded in patient management software were future strategies identified. Conclusions: A systematic intervention model resulted in partial improvements in LBP care. Determinants of practice change amongst GPs were increased knowledge of clinical guidelines, education delivered by someone considered a trusted source of information, and awareness of the negative consequences of inappropriate practices, especially radiological imaging on patient outcomes. Inconsistent and non-evidence based practices amongst locum GPs was an issue that emerged and will be a significant future challenge. The systematic approach utilised is applicable to other services interested in improving LBP care

Herbivore benefits from vectoring plant virus through reduction of period of vulnerability to predation

Herbivores can profit from vectoring plant pathogens because the induced defence of plants against pathogens sometimes interferes with the induced defence of plants against herbivores. Plants can also defend themselves indirectly by the action of the natural enemies of the herbivores. It is unknown whether the defence against pathogens induced in the plant also interferes with the indirect defence against herbivores mediated via the third trophic level. We previously showed that infection of plants with Tomato spotted wilt virus (TSWV) increased the developmental rate of and juvenile survival of its vector, the thrips Frankliniella occidentalis. Here, we present the results of a study on the effects of TSWV infections of plants on the effectiveness of three species of natural enemies of F. occidentalis: the predatory mites Neoseiulus cucumeris and Iphiseius degenerans, and the predatory bug Orius laevigatus. The growth rate of thrips larvae was positively affected by the presence of virus in the host plant. Because large larvae are invulnerable to predation by the two species of predatory mites, this resulted in a shorter period of vulnerability to predation for thrips that developed on plants with virus than thrips developing on uninfected plants (4.4 vs. 7.9 days, respectively). Because large thrips larvae are not invulnerable to predation by the predatory bug Orius laevigatus, infection of the plant did not affect the predation risk of thrips larvae from this predator. This is the first demonstration of a negative effect of a plant pathogen on the predation risk of its vector