Chronic neurohormonal and mechanical stresses are central features of heart disease. Increasing evidence supports a role for the transient receptor potential canonical channels TRPC3 and TRPC6 in ...this pathophysiology. Channel expression for both is normally very low but is increased by cardiac disease, and genetic gain- or loss-of-function studies support contributions to hypertrophy and dysfunction. Selective small-molecule inhibitors remain scarce, and none target both channels, which may be useful given the high homology among them and evidence of redundant signaling. Here we tested selective TRPC3/6 antagonists (GSK2332255B and GSK2833503A; IC ₅₀, 3–21 nM against TRPC3 and TRPC6) and found dose-dependent blockade of cell hypertrophy signaling triggered by angiotensin II or endothelin-1 in HEK293T cells as well as in neonatal and adult cardiac myocytes. In vivo efficacy in mice and rats was greatly limited by rapid metabolism and high protein binding, although antifibrotic effects with pressure overload were observed. Intriguingly, although gene deletion of TRPC3 or TRPC6 alone did not protect against hypertrophy or dysfunction from pressure overload, combined deletion was protective, supporting the value of dual inhibition. Further development of this pharmaceutical class may yield a useful therapeutic agent for heart disease management.
In France, 2300 adolescents and young adults (AYAs, 15-25 years old) are diagnosed with cancer each year. As soon as the disease is diagnosed, a number of physical, psychological and social needs may ...arise. The aim of this study is to develop a tool to measure unmet needs that will allow the specificities of AYAs to be understood while allowing health care staff to mobilise the necessary actors to resolve them.
We developed the Questionnaire nEEd Cancer AYAs (QUEEC-AYAs questionnaire), from two existing questionnaires: the Cancer Needs Questionnaire Young People and the Needs Assessment & Service Bridge. A main sample of 103 AYAs then received and completed the questionnaire in order to conduct an exploratory factor analysis.
The final structure of the QUEEC-AYAs is composed of 7 dimensions and 48 items: information (8 items), cancer care team (6 items), Physical health (4 items), Emotional health (14 items), Sexual & reproductive health (6 items), Health behaviors & wellness (4 items), Daily life (6 items). The questionnaire has a good acceptability and all domains have a Cronbach's alphas value above 0.80.
The QUEEC-AYAs is the first measure of the psychosocial needs of AYAs available in French. Its systematic use in health care services should improve the coordination of care required by AYAs during and after treatment.
This study was approved by the ethics committee of the Paoli-Calmettes Institute (IRB # IPC 2021-041, 2021 May 20).
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•2D NMR provides fingerprints of the higher-order-structure of protein therapeutics.•Fingerprint of 1Hn-1Hα correlations acquired with 2D J-correlated NMR experiments.•Selective TOCSY ...(TACSY) enables selective J-correlation and optimal sensitivity.
The higher order structure (HOS) of protein therapeutics is essential for drug safety and efficacy and can be evaluated by two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy at atomic resolution. 1Hn-15N amide correlated and 1H-13C methyl correlated NMR spectroscopies at natural isotopic abundance have been demonstrated as feasible on protein therapeutics as large as monoclonal antibodies and show great promise for use in establishing drug substance structural consistency across manufacturing changes and in comparing a biosimilar to an originator reference product. Spectral fingerprints from 1Hn-1Hα correlations acquired using 2D homonuclear proton-proton J-correlated NMR experiments provide a complementary approach for high-resolution assessment of the HOS of lower molecular weight (<25 kDa) protein therapeutics. Here, we evaluate different pulse sequences (COSY, TOCSY and TACSY) used to generate proton-proton J-correlated NMR spectral fingerprints and appraise the performance of each method for application to protein therapeutic HOS assessment and comparability.
Lentiviral (LV) vectors have emerged as powerful tools for transgene delivery ex vivo but in vivo gene therapy applications involving LV vectors have faced a number of challenges, including the low ...efficiency of transgene delivery, a lack of tissue specificity, immunogenicity to both the product encoded by the transgene and the vector, and the inactivation of the vector by the human complement cascade. To mitigate these issues, several engineering approaches, involving the covalent modification of vector particles or the incorporation of specific protein domains into the vector’s envelope, have been tested. Short synthetic oligonucleotides, including aptamers bound to the surface of LV vectors, may provide a novel means with which to retarget LV vectors to specific cells and to shield these vectors from neutralization by sera. The purpose of this study was to develop strategies to tether nucleic acid sequences, including short RNA sequences, to LV vector particles in a specific and tight fashion. To bind short RNA sequences to LV vector particles, a bacteriophage lambda N protein-derived RNA binding domain (λN), fused to the measles virus hemagglutinin protein, was used. The λN protein bound RNA sequences bearing a boxB RNA hairpin. To test this approach, we used an RNA aptamer specific to the human epidermal growth factor receptor (EGFR), which was bound to LV vector particles via an RNA scaffold containing a boxB RNA motif. The results obtained confirmed that the EGFR-specific RNA aptamer bound to cells expressing EGFR and that the boxB containing the RNA scaffold was bound specifically to the λN RNA binding domain attached to the vector. These results show that LV vectors can be equipped with nucleic acid sequences to develop improved LV vectors for in vivo applications.
Abstract
The ongoing COVID-19 pandemic highlights the necessity for a more fundamental understanding of the coronavirus life cycle. The causative agent of the disease, SARS-CoV-2, is being studied ...extensively from a structural standpoint in order to gain insight into key molecular mechanisms required for its survival. Contained within the untranslated regions of the SARS-CoV-2 genome are various conserved stem-loop elements that are believed to function in RNA replication, viral protein translation, and discontinuous transcription. While the majority of these regions are variable in sequence, a 41-nucleotide s2m element within the genome 3′ untranslated region is highly conserved among coronaviruses and three other viral families. In this study, we demonstrate that the SARS-CoV-2 s2m element dimerizes by forming an intermediate homodimeric kissing complex structure that is subsequently converted to a thermodynamically stable duplex conformation. This process is aided by the viral nucleocapsid protein, potentially indicating a role in mediating genome dimerization. Furthermore, we demonstrate that the s2m element interacts with multiple copies of host cellular microRNA (miRNA) 1307-3p. Taken together, our results highlight the potential significance of the dimer structures formed by the s2m element in key biological processes and implicate the motif as a possible therapeutic drug target for COVID-19 and other coronavirus-related diseases.
The recent introduction of the “precision medicine” concept in oncology pushed cancer research to focus on dynamic measurable biomarkers able to predict responses to novel anticancer therapies in ...order to improve clinical outcomes. Recently, the involvement of extracellular vesicles (EVs) in cancer pathophysiology has been described, and given their release from all cell types under specific stimuli, EVs have also been proposed as potential biomarkers in cancer. Among the techniques used to study EVs, flow cytometry has a high clinical potential. Here, we have applied a recently developed and simplified flow cytometry method for circulating EV enumeration, subtyping, and isolation from a large cohort of metastatic and locally advanced nonhaematological cancer patients (N = 106); samples from gender- and age-matched healthy volunteers were also analysed. A large spectrum of cancer-related markers was used to analyse differences in terms of peripheral blood circulating EV phenotypes between patients and healthy volunteers, as well as their correlation to clinical outcomes. Finally, EVs from patients and controls were isolated by fluorescence-activated cell sorting, and their protein cargoes were analysed by proteomics. Results demonstrated that EV counts were significantly higher in cancer patients than in healthy volunteers, as previously reported. More interestingly, results also demonstrated that cancer patients presented higher concentrations of circulating CD31+ endothelial-derived and tumour cancer stem cell-derived CD133 + CD326- EVs, when compared to healthy volunteers. Furthermore, higher levels of CD133 + CD326− EVs showed a significant correlation with a poor overall survival. Additionally, proteomics analysis of EV cargoes demonstrated disparities in terms of protein content and function between circulating EVs in cancer patients and healthy controls. Overall, our data strongly suggest that blood circulating cancer stem cell-derived EVs may have a role as a diagnostic and prognostic biomarker in cancer.
Aims The benefits of adjunctive mechanical devices to prevent distal embolization in patients with acute myocardial infarction (AMI) are still a matter of debate. Growing interests are on manual ...thrombectomy devices as compared with other mechanical devices. In fact, they are inexpensive and user-friendly devices, and thus represent an attractive strategy. The aim of the current study was to perform an updated meta-analysis of randomized trials conducted with adjunctive manual thrombectomy devices to prevent distal embolization in AMI. Methods and results The literature was scanned by formal searches of electronic databases MEDLINE, CENTRAL, EMBASE, and The Cochrane Central Register of Controlled trials (http://www.mrw.interscience.wiley.com/cochrane/Cochrane_clcentral_articles_ fs.html) from January 1990 to May 2008, the scientific session abstracts (from January 1990 to May 2008) and oral presentation and/or expert slide presentations (from January 2002 to May 2008) on transcatheter coronary therapeutics (TCT), AHA (American Heart Association), ESC (European Society of Cardiology), ACC (American College of Cardiology) and EuroPCR websites. We examined all randomized trials on adjunctive mechanical devices to prevent distal embolization in AMI. The following keywords were used: randomized trial, myocardial infarction, reperfusion, primary angioplasty, rescue angioplasty, thrombectomy, thrombus aspiration, manual thrombectomy, Diver catheter, Pronto catheter, Export catheter, thrombus vacuum aspiration catheter. Information on study design, type of device, inclusion and exclusion criteria, number of patients, and clinical outcome was extracted by two investigators. Disagreements were resolved by consensus. A total of nine trials with 2417 patients were included 1209 patients (50.0%) in the manual thrombectomy device group and 1208 (50%) in the control group. Adjunctive manual thrombectomy was associated with significantly improved postprocedural TIMI (thrombolysis in myocardial infarction) 3 flow (87.1 vs. 81.2%, P < 0.0001), and postprocedural MBG 3 (myocardial blush grade 3) (52.1 vs. 31.7%, P < 0.0001), less distal embolization (7.9 vs. 19.5%, P < 0.0001), and significant benefits in terms of 30-day mortality (1.7 vs. 3.1%, P = 0.04). Conclusion This meta-analysis demonstrates that, among patients with AMI treated with percutaneous coronary intervention, the use of adjunctive manual thrombectomy devices is associated with better epicardial and myocardial perfusion, less distal embolization and significant reduction in 30-day mortality. Thus, adjunctive manual thrombectomy devices, if not anatomically contraindicated, should be routinely used among STEMI (ST-segment elevation myocardial infarction) patients undergoing primary angioplasty.
Abstract Background Coronary artery disease (CAD) is the leading cause of mortality among diabetic patients, and the neutrophil-to-lymphocyte ratio (NLR) has recently emerged from among inflammatory ...parameters as a potential indicator of vascular complications and poorer outcome in patients with diabetes. This study aimed to evaluate: 1) the impact of diabetes on NLR; and 2) the role of NLR on the extent of CAD among diabetic patients undergoing coronary angiography. Methods Consecutive patients undergoing coronary angiography were included. Diabetic status and main chemistry parameters were assessed at the time of admission. Significant CAD was defined as at least one vessel with stenosis > 50%, while severe CAD was left main and/or three-vessel disease, as evaluated by quantitative coronary angiography (QCA). Results Diabetes was observed in 1377 of 3756 patients (36.7%); they were older, and displayed higher-risk cardiovascular profile and more complex CAD. Diabetic status was also associated with a significant increase in NLR ( P = 0.004). Among diabetics, higher NLR tertile values were related to ageing ( P < 0.001), dyslipidaemia ( P < 0.001), renal failure ( P < 0.001), body mass index ( P < 0.001), previous percutaneous coronary revascularization ( P = 0.004) and cerebrovascular events ( P = 0.003), acute presentation ( P < 0.001), treatment at admission with beta-blockers/statins/ASA (all P < 0.001), diuretics ( P = 0.01) or clopidogrel ( P = 0.04), platelet count ( P = 0.03), white blood cell count, creatinine, glycaemia and C-reactive protein ( P < 0.001), and inversely related to haemoglobin, triglyceride levels ( P < 0.001) and smoking ( P = 0.03). NLR was associated with multivessel disease ( P < 0.001), degree of stenosis ( P = 0.01), type C lesions ( P = 0.02), coronary calcifications and intracoronary thrombus ( P < 0.001), but inversely with in-stent restenosis ( P = 0.003) and TIMI flow grade ( P = 0.02). Also, NLR was directly related to CAD prevalence ( P < 0.001; adjusted OR 95% CI: 1.62 1.27–2.07, P < 0.001) and CAD severity ( P < 0.001; adjusted OR 95% CI: 1.19 1.00–1.43, P = 0.05). Conclusion NLR is increased among diabetic patients and, in such patients, is independently associated with the prevalence and severity of CAD. Further studies are now needed to confirm present results and to evaluate the underlying pathophysiological mechanisms behind our findings.
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