Characterization of long and stable de novo single alpha-helix domains provides novel insight into their stability

Naturally-occurring single α-helices (SAHs), are rich in Arg (R), Glu (E) and Lys (K) residues, and stabilized by multiple salt bridges. Understanding how salt bridges promote their stability is challenging as SAHs are long and their sequences highly variable. Thus, we designed and tested simple de novo 98-residue polypeptides containing 7-residue repeats (AEEEXXX, where X is K or R) expected to promote salt-bridge formation between Glu and Lys/Arg. Lys-rich sequences (EK3 (AEEEKKK) and EK2R1 (AEEEKRK)) both form SAHs, of which EK2R1 is more helical and thermo-stable suggesting Arg increases stability. Substituting Lys with Arg (or vice versa) in the naturally-occurring myosin-6 SAH similarly increased (or decreased) its stability. However, Arg-rich de novo sequences (ER3 (AEEERRR) and EK1R2 (AEEEKRR)) aggregated. Combining a PDB analysis with molecular modelling provides a rational explanation, demonstrating that Glu and Arg form salt bridges more commonly, utilize a wider range of rotamer conformations, and are more dynamic than Glu-Lys. This promiscuous nature of Arg helps explain the increased propensity of de novo Arg-rich SAHs to aggregate. Importantly, the specific K:R ratio is likely to be important in determining helical stability in de novo and naturally-occurring polypeptides, giving new insight into how single α-helices are stabilized

Comet-assay parameters as rapid biomarkers of exposure to dietary/environmental compounds - An in vitro feasibility study on spermatozoa and lymphocytes

Twelve chemical compounds have been selected for the European NewGeneris study on the basis of their potential to damage DNA, in order to establish adequate and reliable biomarkers of exposure. These genotoxic chemicals include heterocyclic amines, organochlorines, polycyclic aromatic hydrocarbons, mycotoxins, lipid peroxidation products and alcohol. Damage in somatic cells such as lymphocytes could give rise to cancer, while damage in germ cells could not only give rise to cancer but also to heritable defects. The alkaline Comet assay, with and without metabolic activation, as well as the neutral Comet assay were used to assess DNA integrity in spermatozoa and lymphocytes after in vitro treatment with low, middle and high doses of each chemical. DNA-reactive aldehydes generated by lipid peroxidation, food mutagens such as heterocyclic amines, nitrosamine and benzo[a]pyrene produced the highest amounts of DNA damage, even without metabolic activation. Damage seen with the neutral Comet assay – detecting primarily double-strand breaks – was lower than with the alkaline assay. In general, there was increased damage in the spermatozoa by comparison with the lymphocytes, with altered slopes in the dose–response curves. The Comet assay with sperm was generally very sensitive in assessing genotoxic damage, with the Comet parameters being good biomarkers of induced DNA damage. Establishing reliable biomarkers of exposure for the evaluation of dietary/environmental carcinogens is of utmost importance to protect our health and the health of our offspring

Intravenous Oncolytic Vaccinia Virus Therapy Results in a Differential Immune Response between Cancer Patients

Pexa-Vec is an engineered Wyeth-strain vaccinia oncolytic virus (OV), which has been tested extensively in clinical trials, demonstrating enhanced cytotoxic T cell infiltration into tumours following treatment. Favourable immune consequences to Pexa-Vec include the induction of an interferon (IFN) response, followed by inflammatory cytokine/chemokine secretion. This promotes tumour immune infiltration, innate and adaptive immune cell activation and T cell priming, culminating in targeted tumour cell killing, i.e., an immunologically ‘cold’ tumour microenvironment is transformed into a ‘hot’ tumour. However, as with all immunotherapies, not all patients respond in a uniformly favourable manner. Our study herein, shows a differential immune response by patients to intravenous Pexa-Vec therapy, whereby some patients responded to the virus in a typical and expected manner, demonstrating a significant IFN induction and subsequent peripheral immune activation. However, other patients experienced a markedly subdued immune response and appeared to exhibit an exhausted phenotype at baseline, characterised by higher baseline immune checkpoint expression and regulatory T cell (Treg) levels. This differential baseline immunological profile accurately predicted the subsequent response to Pexa-Vec and may, therefore, enable the development of predictive biomarkers for Pexa-Vec and OV therapies more widely. If confirmed in larger clinical trials, these immunological biomarkers may enable a personalised approach, whereby patients with an exhausted baseline immune profile are treated with immune checkpoint blockade, with the aim of reversing immune exhaustion, prior to or alongside OV therapy

Insights into the Importance of Hydrogen Bonding in the γ-Phosphate Binding Pocket of Myosin: Structural and Functional Studies of Serine 236†,‡

The active site of myosin contains a group of highly conserved amino acid residues whose roles in nucleotide hydrolysis and energy transduction might appear to be obvious from the initial structural and kinetic analyses but become less clear on deeper investigation. One such residue is Ser236 (Dictyostelium discoideum myosin II numbering) which was proposed to be involved in a hydrogen transfer network during γ-phosphate hydrolysis of ATP, which would imply a critical function in ATP hydrolysis and motility. The S236A mutant protein shows a comparatively small decrease in hydrolytic activity and motility, and thus this residue does not appear to be essential. To understand better the contribution of Ser236 to the function of myosin, structural and kinetic studies have been performed on the S236A mutant protein. The structures of the D. discoideum motor domain (S1dC) S236A mutant protein in complex with magnesium pyrophosphate, MgAMPPNP, and MgADP·vanadate have been determined. In contrast to the previous structure of wild-type S1dC, the S236A·MgAMPPNP complex crystallized in the closed state. Furthermore, transient-state kinetics showed a 4-fold reduction of the nucleotide release step, suggesting that the mutation stabilizes a closed active site. The structures show that a water molecule approximately adopts the location of the missing hydroxyl of Ser236 in the magnesium pyrophosphate and MgAMPPNP structures. This study suggests that the S236A mutant myosin proceeds via a different structural mechanism than wild-type myosin, where the alternate mechanism is able to maintain near normal transient-state kinetic values

Quality of Chronic Kidney Disease Management in Canadian Primary Care.

Importance: Although patients with chronic kidney disease (CKD) are routinely managed in primary care settings, no nationally representative study has assessed the quality of care received by these patients in Canada. Objective: To evaluate the current state of CKD management in Canadian primary care practices to identify care gaps to guide development and implementation of national quality improvement initiatives. Design, Setting, and Participants: This cross-sectional study leveraged Canadian Primary Care Sentinel Surveillance Network data from January 1, 2010, to December 31, 2015, to develop a cohort of 46 162 patients with CKD managed in primary care practices. Data analysis was performed from August 8, 2018, to July 31, 2019. Main Outcomes and Measures: The study examined the proportion of patients with CKD who met a set of 12 quality indicators in 6 domains: (1) detection and recognition of CKD, (2) testing and monitoring of kidney function, (3) use of recommended medications, (4) monitoring after initiation of angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin II receptor blockers (ARBs), (5) management of blood pressure, and (6) monitoring for glycemic control in those with diabetes and CKD. The study also analyzed associations of divergence from these quality indicators. Results: The cohort comprised 46 162 patients (mean [SD] age, 69.2 [14.0] years; 25 855 [56.0%] female) with stage 3 to 5 CKD. Only 4 of 12 quality indicators were met by 75% or more of the study cohort. These indicators were receipt of an outpatient serum creatinine test within 18 months after confirmation of CKD, receipt of blood pressure measurement at any time during follow-up, achieving a target blood pressure of 140/90 mm Hg or lower, and receiving a hemoglobin A1c test for monitoring diabetes during follow-up. Indicators in the domains of detection and recognition of CKD, testing and monitoring of kidney function (specifically, urine albumin to creatinine ratio testing), use of recommended medications, and appropriate monitoring after initiation of treatment with ACEIs or ARBs were not met. Only 6529 patients (18.4%) with CKD received a urine albumin test within 6 months of CKD diagnosis, and 3954 (39.4%) had a second measurement within 6 months of an abnormal baseline urine albumin level. Older age (≥85 years) and CKD stage 5 were significantly associated with not satisfying the criteria for the quality indicators across all domains. Across age categories, younger patients (aged 18-49 years) and older patients (≥75 years) were less likely to be tested for albuminuria (314 of 1689 patients aged 18-49 years [18.5%], 1983 of 11 919 patients aged 75-84 years [61.6%], and 614 of 5237 patients aged ≥85 years [11.7%] received the urine albumin to creatinine ratio test within 6 months of initial estimated glomerular filtration rate <60 mL/min per 1.73 m2; P < .001). Patients aged 18 to 49 years were less commonly prescribed recommended medications (222 of 2881 [7.7%]), whereas patients aged 75 to 84 years were prescribed ACEIs or ARBs most frequently (2328 of 5262 [44.2%]; P < .001). Conclusions and Relevance: The findings suggest that management of CKD across primary care practices in Canada varies according to quality indicator. This study revealed potential priority areas for quality improvement initiatives in Canadian primary care practices

Prevalence and Demographics of CKD in Canadian Primary Care Practices: A Cross-sectional Study.

INTRODUCTION: Surveillance systems enable optimal care delivery and appropriate resource allocation, yet Canada lacks a dedicated surveillance system for chronic kidney disease (CKD). Using data from the Canadian Primary Care Sentinel Surveillance Network (CPCSSN), a national chronic disease surveillance system, this study describes the geographic, sociodemographic, and clinical variations in CKD prevalence in the Canadian primary care context. METHODS: This cross-sectional study included 559,745 adults in primary care in 5 provinces across Canada from 2010 through 2015. Data were analyzed by geographic (urban or rural residence), sociodemographic (age, sex, deprivation index), and clinical (medications prescribed, comorbid conditions) factors, using data from CPCSSN and the Canadian Deprivation Index. CKD stage 3 or higher was defined as 2 estimated glomerular filtration rate (eGFR) values of <60 ml/min per 1.73 m2 more than 90 days apart as of January 1, 2015. RESULTS: Prevalence of CKD was 71.9 per 1000 individuals and varied by geography, with the highest prevalence in rural settings compared with urban settings (86.2 vs. 68.4 per 1000). CKD was highly prevalent among individuals with 3 or more other chronic diseases (281.7 per 1000). Period prevalence of CKD indicated a slight decline over the study duration, from 53.4 per 1000 in 2010 to 46.5 per 1000 in 2014. CONCLUSION: This is the first study to estimate the prevalence of CKD in primary care in Canada at a national level. Results may facilitate further research, prioritization of care, and quality improvement activities to identify gaps and improvement in CKD care