Altered spin state equilibrium in the T309V mutant of cytochrome P450 2D6: a spectroscopic and computational study

Cytochrome P450 2D6 (CYP2D6) is one of the most important cytochromes P450 in humans. Resonance Raman data from the T309V mutant of CYP2D6 show that the substitution of the conserved I-helix threonine situated in the enzyme’s active site perturbs the heme spin equilibrium in favor of the six-coordinated low-spin species. A mechanistic hypothesis is introduced to explain the experimental observations, and its compatibility with the available structural and spectroscopic data is tested using quantum-mechanical density functional theory calculations on active-site models for both the CYP2D6 wild type and the T309V mutant

Activation of nuclear factor-κB in human prostate carcinogenesis and association to biochemical relapse

Nuclear factor (NF)-κB/p65 regulates the transcription of a wide variety of genes involved in cell survival, invasion and metastasis. We characterised by immunohistochemistry the expression of NF-κB/p65 protein in six histologically normal prostate, 13 high-grade prostatic intraepithelial neoplasia (PIN) and 86 prostate adenocarcinoma specimens. Nuclear localisation of p65 was used as a measure of NF-κB active state. Nuclear localisation of NF-κB was only seen in scattered basal cells in normal prostate glands. Prostatic intraepithelial neoplasias exhibited diffuse and strong cytoplasmic staining but no nuclear staining. In prostate adenocarcinomas, cytoplasmic NF-κB was detected in 57 (66.3%) specimens, and nuclear NF-κB (activated) in 47 (54.7%). Nuclear and cytoplasmic NF-κB staining was not correlated (P=0.19). By univariate analysis, nuclear localisation of NF-κB was associated with biochemical relapse (P=0.0009; log-rank test) while cytoplasmic expression did not. On multivariate analysis, serum preoperative prostate specific antigen (P=0.02), Gleason score (P=0.03) and nuclear NF-κB (P=0.002) were independent predictors of biochemical relapse. These results provide novel evidence for NF-κB/p65 nuclear translocation in the transition from PIN to prostate cancer. Our findings also indicate that nuclear localisation of NF-κB is an independent prognostic factor of biochemical relapse in prostate cancer

Effects of hospital facilities on patient outcomes after cancer surgery: an international, prospective, observational study

Background Early death after cancer surgery is higher in low-income and middle-income countries (LMICs) compared with in high-income countries, yet the impact of facility characteristics on early postoperative outcomes is unknown. The aim of this study was to examine the association between hospital infrastructure, resource availability, and processes on early outcomes after cancer surgery worldwide.Methods A multimethods analysis was performed as part of the GlobalSurg 3 study-a multicentre, international, prospective cohort study of patients who had surgery for breast, colorectal, or gastric cancer. The primary outcomes were 30-day mortality and 30-day major complication rates. Potentially beneficial hospital facilities were identified by variable selection to select those associated with 30-day mortality. Adjusted outcomes were determined using generalised estimating equations to account for patient characteristics and country-income group, with population stratification by hospital.Findings Between April 1, 2018, and April 23, 2019, facility-level data were collected for 9685 patients across 238 hospitals in 66 countries (91 hospitals in 20 high-income countries; 57 hospitals in 19 upper-middle-income countries; and 90 hospitals in 27 low-income to lower-middle-income countries). The availability of five hospital facilities was inversely associated with mortality: ultrasound, CT scanner, critical care unit, opioid analgesia, and oncologist. After adjustment for case-mix and country income group, hospitals with three or fewer of these facilities (62 hospitals, 1294 patients) had higher mortality compared with those with four or five (adjusted odds ratio [OR] 3.85 [95% CI 2.58-5.75]; p<0.0001), with excess mortality predominantly explained by a limited capacity to rescue following the development of major complications (63.0% vs 82.7%; OR 0.35 [0.23-0.53]; p<0.0001). Across LMICs, improvements in hospital facilities would prevent one to three deaths for every 100 patients undergoing surgery for cancer.Interpretation Hospitals with higher levels of infrastructure and resources have better outcomes after cancer surgery, independent of country income. Without urgent strengthening of hospital infrastructure and resources, the reductions in cancer-associated mortality associated with improved access will not be realised

Association of mechanical bowel preparation with oral antibiotics and anastomotic leak following left sided colorectal resection:an international, multi-centre, prospective audit

Introduction: The optimal bowel preparation strategy to minimise the risk of anastomotic leak is yet to be determined. This study aimed to determine whether oral antibiotics combined with mechanical bowel preparation (MBP+Abx) was associated with a reduced risk of anastomotic leak when compared to mechanical bowel preparation alone (MBP) or no bowel preparation (NBP). Methods: A pre-planned analysis of the European Society of Coloproctology (ESCP) 2017 Left Sided Colorectal Resection audit was performed. Patients undergoing elective left sided colonic or rectal resection with primary anastomosis between 1 January 2017 and 15 March 2017 by any operative approach were included. The primary outcome measure was anastomotic leak. Results: Of 3676 patients across 343 centres in 47 countries, 618 (16.8%) received MBP+ABx, 1945 MBP (52.9%) and 1099 patients NBP (29.9%). Patients undergoing MBP+ABx had the lowest overall rate of anastomotic leak (6.1%, 9.2%, 8.7% respectively) in unadjusted analysis. After case-mix adjustment using a mixed-effects multivariable regression model, MBP+Abx was associated with a lower risk of anastomotic leak (OR 0.52, 0.30–0.92, P = 0.02) but MBP was not (OR 0.92, 0.63–1.36, P = 0.69) compared to NBP. Conclusion: This non-randomised study adds ‘real-world’, contemporaneous, and prospective evidence of the beneficial effects of combined mechanical bowel preparation and oral antibiotics in the prevention of anastomotic leak following left sided colorectal resection across diverse settings. We have also demonstrated limited uptake of this strategy in current international colorectal practice

Evaluating the incidence of pathological complete response in current international rectal cancer practice

The mainstay of management for locally advanced rectal cancer is chemoradiotherapy followed by surgical resection. Following chemoradiotherapy, a complete response may be detected clinically and radiologically (cCR) prior to surgery or pathologically after surgery (pCR). We aim to report the overall complete pathological response (pCR) rate and the reliability of detecting a cCR by conventional pre-operative imaging.A pre-planned analysis of the European Society of Coloproctology (ESCP) 2017 audit was performed. Patients treated by elective rectal resection were included. A pCR was defined as a ypT0 N0 EMVI negative primary tumour; a partial response represented any regression from baseline staging following chemoradiotherapy. The primary endpoint was the pCR rate. The secondary endpoint was agreement between post-treatment MRI restaging (yMRI) and final pathological staging.Of 2572 patients undergoing rectal cancer surgery in 277 participating centres across 44 countries, 673 (26.2%) underwent chemoradiotherapy and surgery. The pCR rate was 10.3% (67/649), with a partial response in 35.9% (233/649) patients. Comparison of AJCC stage determined by post-treatment yMRI with final pathology showed understaging in 13% (55/429) and overstaging in 34% (148/429). Agreement between yMRI and final pathology for T-stage, N-stage, or AJCC status were each graded as 'fair' only (n = 429, Kappa 0.25, 0.26 and 0.35 respectively).The reported pCR rate of 10% highlights the potential for non-operative management in selected cases. The limited strength of agreement between basic conventional post-chemoradiotherapy imaging assessment techniques and pathology suggest alternative markers of response should be considered, in the context of controlled clinical trials

Marker discovery in volatolomics based on systematic alignment of GC-MS signals: Application to food authentication

International audienceStarting with an experiment to authenticate walnut oils based on GC-MS analysis of the volatolome, this paper aims to demonstrate the relevance of a two-step alignment-based strategy for the systematic research of VOC markers. The first step of the treatment consists of roughly reducing the time shifts with efficient, known warping techniques like COW (Correlation Optimized Warping). The second step relies on an accurate peak apex alignment in order to refine residual local misalignments and to enable further systematic marker research through univariate or multivariate data treatments. This two-step strategy was implemented on 117 GC-MS analyses of the volatolome of three vegetable oils with very similar composition. During the analysis campaign, the GC-MS system was intentionally subjected to instrumental drifts in order to generate realistic signal shifts. The first part of this study aims to assess the efficiency of the warping-based strategy in terms of signal alignment and sample discrimination. Whereas no distinction between the three oils was possible with unaligned raw GC-MS data, the application of COW enabled a significant but insufficient improvement of both reduction of temporal drifts and between-group separation with 79% of samples being well-classified according to Linear Discriminant Analysis (LDA). Applying the peak apex alignment procedure to COW-treated signals resulted in a suitable correction of the remaining local distortions and improved the proportion of well-classified samples in LDA to 100%. The second part of this study assesses the robustness of the discriminant markers highlighted in this approach by: (i) discussing the relevance of the best markers involved in the LDA model, where a close review of literature confirmed the consistency for two of them, and (ii) validating highlighted makers by retrieving the set of the 23 markers previously determined by manual processing among those automatically found. The third part shows the potential of the systematic approach for untargeted detection of 184 highly significant relevant markers from the oil volatolome. Finally, the fourth part presents a comparison of our hybrid alignment strategy with two reference alignment methods (iCoshift and STW) in order to assess quality alignment of the GC-MS data and to show the three methods' abilities to detect discriminant markers

The closed and compact domain organization of the 70-kDa human cytochrome P450 reductase in its oxidized state as revealed by NMR.

The NADPH cytochrome P450 reductase (CPR), a diflavin enzyme, catalyzes the electron transfer (ET) from NADPH to the substrate P450. The crystal structures of mammalian and yeast CPRs show a compact organization for the two domains containing FMN (flavin mononucleotide) and FAD (flavin adenine dinucleotide), with a short interflavin distance consistent with fast ET from the NADPH-reduced FAD to the second flavin FMN. This conformation, referred as "closed", contrasts with the alternative opened or extended domain arrangements recently described for partially reduced or mutant CPR. Internal domain flexibility in this enzyme is indeed necessary to account for the apparently conflicting requirements of having FMN flavin accessible to both the FAD and the substrate P450 at the same interface. However, how interdomain dynamics influence internal and external ETs in CPR is still largely unknown. Here, we used NMR techniques to explore the global, domain-specific and residue-specific structural and dynamic properties of the nucleotide-free human CPR in solution in its oxidized state. Based on the backbone resonance assignment of this 70-kDa protein, we collected residue-specific (15)N relaxation and (1)H-(15)N residual dipolar couplings. Surprisingly and in contrast with previous studies, the analysis of these NMR data revealed that the CPR exists in a unique and predominant conformation that highly resembles the closed conformation observed in the crystalline state. Based on our findings and the previous observations of conformational equilibria of the CPR in partially reduced states, we propose that the large-scale conformational transitions of the CPR during the catalytic cycle are tightly controlled to ensure optimal electron delivery