Copper-Dependent Trafficking of the Ctr4-Ctr5 Copper Transporting Complex

In Schizosaccharomyces pombe, copper uptake is carried out by a heteromeric complex formed by the Ctr4 and Ctr5 proteins. Copper-induced differential subcellular localization may play a critical role with respect to fine tuning the number of Ctr4 and Ctr5 molecules at the cell surface.We have developed a bimolecular fluorescence complementation (BiFC) assay to analyze protein-protein interactions in vivo in S. pombe. The assay is based on the observation that N- and C-terminal subfragments of the Venus fluorescent protein can reconstitute a functional fluorophore only when they are brought into tight contact. Wild-type copies of the ctr4(+) and ctr5(+) genes were inserted downstream of and in-frame with the nonfluorescent C-terminal (VC) and N-terminal (VN) coding fragments of Venus, respectively. Co-expression of Ctr4-VC and Ctr5-VN fusion proteins allowed their detection at the plasma membrane of copper-limited cells. Similarly, cells co-expressing Ctr4-VN and Ctr4-VC in the presence of Ctr5-Myc(12) displayed a fluorescence signal at the plasma membrane. In contrast, Ctr5-VN and Ctr5-VC co-expressed in the presence of Ctr4-Flag(2) failed to be visualized at the plasma membrane, suggesting a requirement for a combination of two Ctr4 molecules with one Ctr5 molecule. We found that plasma membrane-located Ctr4-VC-Ctr5-VN fluorescent complexes were internalized when the cells were exposed to high levels of copper. The copper-induced internalization of Ctr4-VC-Ctr5-VN complexes was not dependent on de novo protein synthesis. When cells were transferred back from high to low copper levels, there was reappearance of the BiFC fluorescent signal at the plasma membrane.These findings reveal a copper-dependent internalization and recycling of the heteromeric Ctr4-Ctr5 complex as a function of copper availability

Diagnostic accuracy of non-invasive tests for advanced fibrosis in patients with NAFLD: an individual patient data meta-analysis

Objective: Liver biopsy is still needed for fibrosis staging in many patients with non-alcoholic fatty liver disease. The aims of this study were to evaluate the individual diagnostic performance of liver stiffness measurement by vibration controlled transient elastography (LSM- VCTE), Fibrosis-4 index (FIB-4) and NAFLD Fibrosis Score (NFS) and to derive diagnostic strategies that could reduce the need for liver biopsies.Design: Individual patient data meta-analysis of studies evaluating LSM-VCTE against liver histology was conducted. FIB-4 and NFS were computed where possible. Sensitivity, specificity and area under the receiver operating curve (AUROC) were calculated. Biomarkers were assessed individu-ally and in sequential combinations.Results: Data were included from 37 primary studies (n=5735; 45% female; median age: 54 years; median BMI: 30 kg/m2; 33% had type 2 diabetes; 30% had advanced fibrosis). AUROCs of individual LSM-VCTE, FIB-4 and NFS for advanced fibrosis were 0.85, 0.76 and 0.73. Sequential combination of FIB-4 cut-offs

Breast cancer management pathways during the COVID-19 pandemic: outcomes from the UK ‘Alert Level 4’ phase of the B-MaP-C study

Abstract: Background: The B-MaP-C study aimed to determine alterations to breast cancer (BC) management during the peak transmission period of the UK COVID-19 pandemic and the potential impact of these treatment decisions. Methods: This was a national cohort study of patients with early BC undergoing multidisciplinary team (MDT)-guided treatment recommendations during the pandemic, designated ‘standard’ or ‘COVID-altered’, in the preoperative, operative and post-operative setting. Findings: Of 3776 patients (from 64 UK units) in the study, 2246 (59%) had ‘COVID-altered’ management. ‘Bridging’ endocrine therapy was used (n = 951) where theatre capacity was reduced. There was increasing access to COVID-19 low-risk theatres during the study period (59%). In line with national guidance, immediate breast reconstruction was avoided (n = 299). Where adjuvant chemotherapy was omitted (n = 81), the median benefit was only 3% (IQR 2–9%) using ‘NHS Predict’. There was the rapid adoption of new evidence-based hypofractionated radiotherapy (n = 781, from 46 units). Only 14 patients (1%) tested positive for SARS-CoV-2 during their treatment journey. Conclusions: The majority of ‘COVID-altered’ management decisions were largely in line with pre-COVID evidence-based guidelines, implying that breast cancer survival outcomes are unlikely to be negatively impacted by the pandemic. However, in this study, the potential impact of delays to BC presentation or diagnosis remains unknown

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

A review of the literature on family decision-making at end of life precipitating hospital admission

Around 70% of people would prefer to die at home, yet around 50% die in hospital, according to Dying Matters. In collaboration with a local hospice, a literature review was undertaken to address the question: ‘what factors precipitate admission to hospital in the last few days of a person's life for those who had expressed a preference to die at home?’ Four electronic databases were searched, with a date range of 2008 to 2018. After 80 articles were screened, 13 were included in the review. The findings identified a number of barriers experienced by people with non-cancer conditions nearing the end of life and their family carers, which inhibit the transition to end-of-life care. The findings suggest that hospice support for non-cancer patients with a deteriorating health trajectory needs to precede patient and family recognition that end-of-life care is needed

Laboratory Risk Indicator for Necrotizing Fasciitis score for early diagnosis of necrotizing fasciitis in Darwin.

Soft tissue infections are a major health burden in the Top End of the Northern Territory of Australia. Necrotizing fasciitis (NF) is associated with mortality rates from 8 to 40%. Early recognition and aggressive surgical debridement are the cornerstones of successful treatment. The Laboratory Risk Indicator for Necrotizing Fasciitis (LRINEC) score, developed by Wong et al., uses six routine biochemical variables to aid early diagnosis. We aim to assess the diagnostic efficacy of the LRINEC score in our population. A retrospective review of patients with NF between 2005 and 2013 was conducted. A time matched cohort of abscesses/cellulitis was selected. Admission bloods were used to calculate the LRINEC score. An intraoperative finding of NF was used as the gold standard definition for comparison. The diagnostic accuracy of the LRINEC score was assessed. Ninety-eight patients with NF and 205 control patients were identified. The area under the receiver operator curve for the LRINEC score in detecting NF was 0.925 (0.890-0.959, P < 0.001). The sensitivity of the LRINEC ?5 for NF was 76.3%, with a specificity of 93.1%. The positive and negative predictive values were 95.5 and 88.1%, respectively. The positive and negative likelihood ratios were 11 and 0.25. The LRINEC score is a useful, robust, non-invasive and easily calculated scoring system that can be used as an adjunct to early diagnosis of NF. However, a high degree of clinical suspicion remains the most important factor in early diagnosis of NF

Validity of multi-frequency bioelectric impedance methods to measure body composition in obese patients : A systematic review

Objective Excessive lean tissue loss following bariatric surgery may pose serious metabolic consequences. Accurate methods to assess body composition following bariatric surgery are required. This review aimed to investigate if multi-frequency bioelectric impedance (MF-BI) is a valid tool to determine body composition in obese patients. Methods MEDLINE, EMBASE, CINAHL and CENTRAL databases were searched until March 2017. Included studies were published in English with obese (body mass index (BMI) ≥ 30 kg/m2) adults measuring body composition with MF-BI methods in comparison with reference methods. Exclusions were pregnancy, animal studies, non-English language studies, single frequency BI. A total of 6395 studies were retrieved. Results Sixteen studies were eligible for inclusion. Sample sizes ranged from 15 to 157, with BMI 26–48 kg/m2. MF-BI underestimated fat mass (FM) in 11 studies and overestimated fat-free mass (FFM) in nine studies in comparison with reference methods. Correlations of absolute values from MF-BI and reference methods for FM and FFM were high, however, agreement was lower at an individual level. When adjustments for BMI were made to machine algorithms, measurement accuracy improved. Significant heterogeneity was evident among included studies. Conclusions This review found that MF-BI is reliable for use at a group level. Obese-specific adjustment of algorithms for MF-BI machines increases the accuracy of absolute measures of body composition in obese individuals, improving their utility in the clinical setting. Multiple variables contributed a lack of consistency among studies included, highlighting the need for more robust studies that control confounding variables to establish clear validity assessment