Nucleosomes Correlate with In Vivo Progression Pattern of De Novo Methylation of p16 CpG Islands in Human Gastric Carcinogenesis

BACKGROUND: The exact relationship between nucleosome positioning and methylation of CpG islands in human pathogenesis is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we characterized the nucleosome position within the p16 CpG island and established a seeding methylation-specific PCR (sMSP) assay based on bisulfite modification to enrich the p16 alleles containing methylated-CpG at the methylation "seeding" sites within its intron-1 in gastric carcinogenesis. The sMSP-positive rate in primary gastric carcinoma (GC) samples (36/40) was significantly higher than that observed in gastritis (19/45) or normal samples (7/13) (P<0.01). Extensive clone sequencing of these sMSP products showed that the density of methylated-CpGs in p16 CpG islands increased gradually along with the severity of pathological changes in gastric tissues. In gastritis lesions the methylation was frequently observed in the region corresponding to the exon-1 coding-nucleosome and the 5'UTR-nucleosome; the methylation was further extended to the region corresponding to the promoter-nucleosome in GC samples. Only few methylated-CpG sites were randomly detected within p16 CpG islands in normal tissues. The significantly inversed relationship between the p16 exon-1 methylation and its transcription was observed in GC samples. An exact p16 promoter-specific 83 bp-MSP assay confirms the result of sMSP (33/55 vs. 1/6, P<0.01). In addition, p16 methylation in chronic gastritis lesions significantly correlated with H. pylori infection; however, such correlation was not observed in GC specimens. CONCLUSIONS/SIGNIFICANCE: It was determined that de novo methylation was initiated in the coding region of p16 exon-1 in gastritis, then progressed to its 5'UTR, and ultimately to the proximal promoter in GCs. Nucleosomes may function as the basic extension/progression unit of de novo methylation of p16 CpG islands in vivo

REDUCE (Reviewing long-term antidepressant use by careful monitoring in everyday practice) internet and telephone support to people coming off long-term antidepressants: protocol for a randomised controlled trial.

BACKGROUND: Around one in ten adults take antidepressants for depression in England, and their long-term use is increasing. Some need them to prevent relapse, but 30-50% could possibly stop them without relapsing and avoid adverse effects and complications of long-term use. However, stopping is not always easy due to withdrawal symptoms and a fear of relapse of depression. When general practitioners review patients on long-term antidepressants and recommend to those who are suitable to stop the medication, only 6-8% are able to stop. The Reviewing long-term antidepressant use by careful monitoring in everyday practice (REDUCE) research programme aims to identify safe and cost-effective ways of helping patients taking long-term antidepressants taper off treatment when appropriate. METHODS: Design: REDUCE is a two-arm, 1:1 parallel group randomised controlled trial, with randomisation clustered by participating family practices. SETTING: England and north Wales. POPULATION: patients taking antidepressants for longer than 1 year for a first episode of depression or longer than 2 years for repeated episodes of depression who are no longer depressed and want to try to taper off their antidepressant use. INTERVENTION: provision of 'ADvisor' internet programmes to general practitioners or nurse practitioners and to patients designed to support antidepressant withdrawal, plus three patient telephone calls from a psychological wellbeing practitioner. The control arm receives usual care. Blinding of patients, practitioners and researchers is not possible in an open pragmatic trial, but statistical and health economic data analysts will remain blind to allocation. OUTCOME MEASURES: the primary outcome is self-reported nine-item Patient Health Questionnaire at 6 months for depressive symptoms. SECONDARY OUTCOMES: depressive symptoms at other follow-up time points, anxiety, discontinuation of antidepressants, social functioning, wellbeing, enablement, quality of life, satisfaction, and use of health services for costs. SAMPLE SIZE: 402 patients (201 intervention and 201 controls) from 134 general practices recruited over 15-18 months, and followed-up at 3, 6, 9 and 12 months. A qualitative process evaluation will be conducted through interviews with 15-20 patients and 15-20 practitioners in each arm to explore why the interventions were effective or not, depending on the results. DISCUSSION: Helping patients reduce and stop antidepressants is often challenging for practitioners and time-consuming for very busy primary care practices. If REDUCE provides evidence showing that access to internet and telephone support enables more patients to stop treatment without increasing depression we will try to implement the intervention throughout the National Health Service, publishing practical guidance for professionals and advice for patients to follow, publicised through patient support groups. TRIAL REGISTRATION: ISRCTN:12417565. Registered on 7 October 2019

Applying an Empirical Hydropathic Forcefield in Refinement May Improve Low-Resolution Protein X-Ray Crystal Structures

BACKGROUND: The quality of X-ray crystallographic models for biomacromolecules refined from data obtained at high-resolution is assured by the data itself. However, at low-resolution, >3.0 Å, additional information is supplied by a forcefield coupled with an associated refinement protocol. These resulting structures are often of lower quality and thus unsuitable for downstream activities like structure-based drug discovery. METHODOLOGY: An X-ray crystallography refinement protocol that enhances standard methodology by incorporating energy terms from the HINT (Hydropathic INTeractions) empirical forcefield is described. This protocol was tested by refining synthetic low-resolution structural data derived from 25 diverse high-resolution structures, and referencing the resulting models to these structures. The models were also evaluated with global structural quality metrics, e.g., Ramachandran score and MolProbity clashscore. Three additional structures, for which only low-resolution data are available, were also re-refined with this methodology. RESULTS: The enhanced refinement protocol is most beneficial for reflection data at resolutions of 3.0 Å or worse. At the low-resolution limit, ≥4.0 Å, the new protocol generated models with Cα positions that have RMSDs that are 0.18 Å more similar to the reference high-resolution structure, Ramachandran scores improved by 13%, and clashscores improved by 51%, all in comparison to models generated with the standard refinement protocol. The hydropathic forcefield terms are at least as effective as Coulombic electrostatic terms in maintaining polar interaction networks, and significantly more effective in maintaining hydrophobic networks, as synthetic resolution is decremented. Even at resolutions ≥4.0 Å, these latter networks are generally native-like, as measured with a hydropathic interactions scoring tool

Combination Therapy Is Superior to Sequential Monotherapy for the Initial Treatment of Hypertension:A Double-Blind Randomized Controlled Trial

Background: Guidelines for hypertension vary in their preference for initial combination therapy or initial monotherapy, stratified by patient profile; therefore, we compared the efficacy and tolerability of these approaches. Methods and Results: We performed a 1‐year, double‐blind, randomized controlled trial in 605 untreated patients aged 18 to 79 years with systolic blood pressure (BP) ≥150 mm Hg or diastolic BP ≥95 mm Hg. In phase 1 (weeks 0–16), patients were randomly assigned to initial monotherapy (losartan 50–100 mg or hydrochlorothiazide 12.5–25 mg crossing over at 8 weeks), or initial combination (losartan 50–100 mg plus hydrochlorothiazide 12.5–25 mg). In phase 2 (weeks 17–32), all patients received losartan 100 mg and hydrochlorothiazide 12.5 to 25 mg. In phase 3 (weeks 33–52), amlodipine with or without doxazosin could be added to achieve target BP. Hierarchical primary outcomes were the difference from baseline in home systolic BP, averaged over phases 1 and 2 and, if significant, at 32 weeks. Secondary outcomes included adverse events, and difference in home systolic BP responses between tertiles of plasma renin. Home systolic BP after initial monotherapy fell 4.9 mm Hg (range: 3.7–6.0 mm Hg) less over 32 weeks (P&lt;0.001) than after initial combination but caught up at 32 weeks (difference 1.2 mm Hg [range: −0.4 to 2.8 mm Hg], P=0.13). In phase 1, home systolic BP response to each monotherapy differed substantially between renin tertiles, whereas response to combination therapy was uniform and at least 5 mm Hg more than to monotherapy. There were no differences in withdrawals due to adverse events. Conclusions: Initial combination therapy can be recommended for patients with BP &gt;150/95 mm Hg. Clinical Trial Registration URL: http://www.ClinicalTrials.gov. Unique identifier: NCT00994617

Neutrophilic airways inflammation in lung cancer: the role of exhaled LTB-4 and IL-8

Background: Recent advances in lung cancer biology presuppose its inflammatory origin. In this regard, LTB-4 and IL-8 are recognized to play a crucial role in neutrophil recruitment into airways during lung cancer.Notwithstanding the intriguing hypothesis, the exact role of neutrophilic inflammation in tumour biology remains complex and not completely known.The aim of this study was to give our contribution in this field by investigating LTB-4 and IL-8 in the breath condensate of NSCLC patients and verifying their role in cancer development and progression.Method: We enrolled 50 NSCLC patients and 35 controls. LTB-4 and IL-8 concentrations were measured in the breath condensate and the blood of all the subjects under study using EIA kits. Thirty NSCLC patients and ten controls underwent induced sputum collection and analysis.Results: LTB-4 and IL-8 resulted higher in breath condensate and the blood of NSCLC patients compared to controls. Significantly higher concentrations were found as the cancer stages progressed. A positive correlation was observed between exhaled IL-8 and LTB-4 and the percentage of neutrophils in the induced sputum.Conclusion: The high concentrations of exhaled LTB-4 and IL-8 showed the presence of a neutrophilic inflammation in the airways of NSCLC patients and gave a further support to the inflammatory signalling in lung cancer. These exhaled proteins could represent a suitable non-invasive marker in the diagnosis and monitoring of lung cancer. © 2011 Carpagnano et al; licensee BioMed Central Ltd

Radiotherapy Versus Inguinofemoral Lymphadenectomy as Treatment for Vulvar Cancer Patients With Micrometastases in the Sentinel Node: Results of GROINSS-V II

PURPOSE: The Groningen International Study on Sentinel nodes in Vulvar cancer (GROINSS-V)-II investigated whether inguinofemoral radiotherapy is a safe alternative to inguinofemoral lymphadenectomy (IFL) in vulvar cancer patients with a metastatic sentinel node (SN). METHODS: GROINSS-V-II was a prospective multicenter phase-II single-arm treatment trial, including patients with early-stage vulvar cancer (diameter < 4 cm) without signs of lymph node involvement at imaging, who had primary surgical treatment (local excision with SN biopsy). Where the SN was involved (metastasis of any size), inguinofemoral radiotherapy was given (50 Gy). The primary end point was isolated groin recurrence rate at 24 months. Stopping rules were defined for the occurrence of groin recurrences. RESULTS: From December 2005 until October 2016, 1,535 eligible patients were registered. The SN showed metastasis in 322 (21.0%) patients. In June 2010, with 91 SN-positive patients included, the stopping rule was activated because the isolated groin recurrence rate in this group went above our predefined threshold. Among 10 patients with an isolated groin recurrence, nine had SN metastases > 2 mm and/or extracapsular spread. The protocol was amended so that those with SN macrometastases (> 2 mm) underwent standard of care (IFL), whereas patients with SN micrometastases (≤ 2 mm) continued to receive inguinofemoral radiotherapy. Among 160 patients with SN micrometastases, 126 received inguinofemoral radiotherapy, with an ipsilateral isolated groin recurrence rate at 2 years of 1.6%. Among 162 patients with SN macrometastases, the isolated groin recurrence rate at 2 years was 22% in those who underwent radiotherapy, and 6.9% in those who underwent IFL (P = .011). Treatment-related morbidity after radiotherapy was less frequent compared with IFL. CONCLUSION: Inguinofemoral radiotherapy is a safe alternative for IFL in patients with SN micrometastases, with minimal morbidity. For patients with SN macrometastasis, radiotherapy with a total dose of 50 Gy resulted in more isolated groin recurrences compared with IFL

Altered Arterial Stiffness and Subendocardial Viability Ratio in Young Healthy Light Smokers after Acute Exercise

Studies showed that long-standing smokers have stiffer arteries at rest. However, the effect of smoking on the ability of the vascular system to respond to increased demands (physical stress) has not been studied. The purpose of this study was to estimate the effect of smoking on arterial stiffness and subendocardial viability ratio, at rest and after acute exercise in young healthy individuals.Healthy light smokers (n = 24, pack-years = 2.9) and non-smokers (n = 53) underwent pulse wave analysis and carotid-femoral pulse wave velocity measurements at rest, and 2, 5, 10, and 15 minutes following an exercise test to exhaustion. Smokers were tested, 1) after 12h abstinence from smoking (chronic condition) and 2) immediately after smoking one cigarette (acute condition). At rest, chronic smokers had higher augmentation index and lower aortic pulse pressure than non-smokers, while subendocardial viability ratio was not significantly different. Acute smoking increased resting augmentation index and decreased subendocardial viability ratio compared with non-smokers, and decreased subendocardial viability ratio compared with the chronic condition. After exercise, subendocardial viability ratio was lower, and augmentation index and aortic pulse pressure were higher in non-smokers than smokers in the chronic and acute conditions. cfPWV rate of recovery of was greater in non-smokers than chronic smokers after exercise. Non-smokers were also able to achieve higher workloads than smokers in both conditions.Chronic and acute smoking appears to diminish the vascular response to physical stress. This can be seen as an impaired 'vascular reserve' or a blunted ability of the blood vessels to accommodate the changes required to achieve higher workloads. These changes were noted before changes in arterial stiffness or subendocardial viability ratio occurred at rest. Even light smoking in young healthy individuals appears to have harmful effects on vascular function, affecting the ability of the vascular bed to respond to increased demands