Bio-orthogonal Fluorescent Labelling of Biopolymers through Inverse-Electron-Demand Diels–Alder Reactions

Bio-ort hogona llabellin gschemes based on inverse-elec tron- deman dDiels–Ald er (IEDDA) cycloa ddition have attracted much attention in chem ical biology recently .The appeal ing features of this reactio n, such as the fast reactio nkinetics, fully bio-ort hogonal nature and high selectiv ity,have helped chem i- cal biologists gain deeper understandi ng of biochemic al pro- cesses at the molecular level.Listing the compo nents and dis- cussing the possib ilities andlimitations of thesereagent s, we provid earecent snapshot of the field of IEDDA -based biomo- lecular manipulatio nwith special focus on fluores cent modula- tion approaches throug hthe use of bio-orthogon alized build- ing blocks. At the end, we discuss challenges that need to be addres sed for further develop ments in order to overcome recent limita tions and to enabl eresearchers to answer biomo - lecular quest ions in more detail

Reactivity of Biarylazacyclooctynones in Copper-Free Click Chemistry

The 1,3-dipolar cycloaddition of cyclooctynes with azides, also called "copper-free click chemistry", is a bioorthogonal reaction with widespread applications in biological discovery. The kinetics of this reaction are of paramount importance for studies of dynamic processes, particularly in living subjects. Here we performed a systematic analysis of the effects of strain and electronics on the reactivity of cyclooctynes with azides through both experimental measurements and computational studies using a density functional theory (DFT) distortion/interaction transition state model. In particular, we focused on biarylazacyclooctynone (BARAC) because it reacts with azides faster than any other reported cyclooctyne and its modular synthesis facilitated rapid access to analogues. We found that substituents on BARAC's aryl rings can alter the calculated transition state interaction energy of the cycloaddition through electronic effects or the calculated distortion energy through steric effects. Experimental data confirmed that electronic perturbation of BARAC's aryl rings has a modest effect on reaction rate, whereas steric hindrance in the transition state can significantly retard the reaction. Drawing on these results, we analyzed the relationship between alkyne bond angles, which we determined using X-ray crystallography, and reactivity, quantified by experimental second-order rate constants, for a range of cyclooctynes. Our results suggest a correlation between decreased alkyne bond angle and increased cyclooctyne reactivity. Finally, we obtained structural and computational data that revealed the relationship between the conformation of BARAC's central lactam and compound reactivity. Collectively, these results indicate that the distortion/interaction model combined with bond angle analysis will enable predictions of cyclooctyne reactivity and the rational design of new reagents for copper-free click chemistry

Impact of CD4 and CD8 dynamics and viral rebounds on loss of virological control in HIV controllers

Objective: HIV controllers (HICs) spontaneously maintain HIV viral replication at low level without antiretroviral therapy (ART), a small number of whom will eventually lose this ability to control HIV viremia. The objective was to identify factors associated with loss of virological control. Methods: HICs were identified in COHERE on the basis of \ue2\u89\ua55 consecutive viral loads (VL) \ue2\u89\ua4500 copies/mL over \ue2\u89\ua51 year whilst ART-naive, with the last VL \ue2\u89\ua4500 copies/mL measured \ue2\u89\ua55 years after HIV diagnosis. Loss of virological control was defined as 2 consecutive VL >2000 copies/mL. Duration of HIV control was described using cumulative incidence method, considering loss of virological control, ART initiation and death during virological control as competing outcomes. Factors associated with loss of virological control were identified using Cox models. CD4 and CD8 dynamics were described using mixed-effect linear models. Results: We identified 1067 HICs; 86 lost virological control, 293 initiated ART, and 13 died during virological control. Six years after confirmation of HIC status, the probability of losing virological control, initiating ART and dying were 13%, 37%, and 2%. Current lower CD4/CD8 ratio and a history of transient viral rebounds were associated with an increased risk of losing virological control. CD4 declined and CD8 increased before loss of virological control, and before viral rebounds. Discussion: Expansion of CD8 and decline of CD4 during HIV control may result from repeated low-level viremia. Our findings suggest that in addition to superinfection, other mechanisms, such as low grade viral replication, can lead to loss of virological control in HICs

No need for secondary Pneumocystis jirovecii pneumonia prophylaxis in adult people living with HIV from Europe on ART with suppressed viraemia and a CD4 cell count greater than 100 cells/µL.

INTRODUCTION: Since the beginning of the HIV epidemic in resource-rich countries, Pneumocystis jirovecii pneumonia (PjP) is one of the most frequent opportunistic AIDS-defining infections. The Collaboration of Observational HIV Epidemiological Research Europe (COHERE) has shown that primary Pneumocystis jirovecii Pneumonia (PjP) prophylaxis can be safely withdrawn in patients with CD4 counts of 100 to 200 cells/µL if plasma HIV-RNA is suppressed on combination antiretroviral therapy. Whether this holds true for secondary prophylaxis is not known, and this has proved difficult to determine due to the much lower population at risk. METHODS: We estimated the incidence of secondary PjP by including patient data collected from 1998 to 2015 from the COHERE cohort collaboration according to time-updated CD4 counts, HIV-RNA and use of PjP prophylaxis in persons >16 years of age. We fitted a Poisson generalized additive model in which the smoothed effect of CD4 was modelled by a restricted cubic spline, and HIV-RNA was stratified as low (10,000copies/mL). RESULTS: There were 373 recurrences of PjP during 74,295 person-years (py) in 10,476 patients. The PjP incidence in the different plasma HIV-RNA strata differed significantly and was lowest in the low stratum. For patients off prophylaxis with CD4 counts between 100 and 200 cells/µL and HIV-RNA below 400 copies/mL, the incidence of recurrent PjP was 3.9 (95% CI: 2.0 to 5.8) per 1000 py, not significantly different from patients on prophylaxis in the same stratum (1.9, 95% CI: 0.1 to 3.7). CONCLUSIONS: HIV viraemia importantly affects the risk of recurrent PjP. In virologically suppressed patients on ART with CD4 counts of 100 to 200/µL, the incidence of PjP off prophylaxis is below 10/1000 py. Secondary PjP prophylaxis may be safely withheld in such patients. While European guidelines recommend discontinuing secondary PjP prophylaxis only if CD4 counts rise above 200 cells/mL, the latest US Guidelines consider secondary prophylaxis discontinuation even in patients with a CD4 count above 100 cells/µL and suppressed viral load. Our results strengthen and support this US recommendation

Healthcare Access and Quality Index based on mortality from causes amenable to personal health care in 195 countries and territories, 1990-2015 : a novel analysis from the Global Burden of Disease Study 2015

Background National levels of personal health-care access and quality can be approximated by measuring mortality rates from causes that should not be fatal in the presence of effective medical care (ie, amenable mortality). Previous analyses of mortality amenable to health care only focused on high-income countries and faced several methodological challenges. In the present analysis, we use the highly standardised cause of death and risk factor estimates generated through the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) to improve and expand the quantification of personal health-care access and quality for 195 countries and territories from 1990 to 2015. Methods We mapped the most widely used list of causes amenable to personal health care developed by Nolte and McKee to 32 GBD causes. We accounted for variations in cause of death certification and misclassifications through the extensive data standardisation processes and redistribution algorithms developed for GBD. To isolate the effects of personal health-care access and quality, we risk-standardised cause-specific mortality rates for each geography-year by removing the joint effects of local environmental and behavioural risks, and adding back the global levels of risk exposure as estimated for GBD 2015. We employed principal component analysis to create a single, interpretable summary measure-the Healthcare Quality and Access (HAQ) Index-on a scale of 0 to 100. The HAQ Index showed strong convergence validity as compared with other health-system indicators, including health expenditure per capita (r= 0.88), an index of 11 universal health coverage interventions (r= 0.83), and human resources for health per 1000 (r= 0.77). We used free disposal hull analysis with bootstrapping to produce a frontier based on the relationship between the HAQ Index and the Socio-demographic Index (SDI), a measure of overall development consisting of income per capita, average years of education, and total fertility rates. This frontier allowed us to better quantify the maximum levels of personal health-care access and quality achieved across the development spectrum, and pinpoint geographies where gaps between observed and potential levels have narrowed or widened over time. Findings Between 1990 and 2015, nearly all countries and territories saw their HAQ Index values improve; nonetheless, the difference between the highest and lowest observed HAQ Index was larger in 2015 than in 1990, ranging from 28.6 to 94.6. Of 195 geographies, 167 had statistically significant increases in HAQ Index levels since 1990, with South Korea, Turkey, Peru, China, and the Maldives recording among the largest gains by 2015. Performance on the HAQ Index and individual causes showed distinct patterns by region and level of development, yet substantial heterogeneities emerged for several causes, including cancers in highest-SDI countries; chronic kidney disease, diabetes, diarrhoeal diseases, and lower respiratory infections among middle-SDI countries; and measles and tetanus among lowest-SDI countries. While the global HAQ Index average rose from 40.7 (95% uncertainty interval, 39.0-42.8) in 1990 to 53.7 (52.2-55.4) in 2015, far less progress occurred in narrowing the gap between observed HAQ Index values and maximum levels achieved; at the global level, the difference between the observed and frontier HAQ Index only decreased from 21.2 in 1990 to 20.1 in 2015. If every country and territory had achieved the highest observed HAQ Index by their corresponding level of SDI, the global average would have been 73.8 in 2015. Several countries, particularly in eastern and western sub-Saharan Africa, reached HAQ Index values similar to or beyond their development levels, whereas others, namely in southern sub-Saharan Africa, the Middle East, and south Asia, lagged behind what geographies of similar development attained between 1990 and 2015. Interpretation This novel extension of the GBD Study shows the untapped potential for personal health-care access and quality improvement across the development spectrum. Amid substantive advances in personal health care at the national level, heterogeneous patterns for individual causes in given countries or territories suggest that few places have consistently achieved optimal health-care access and quality across health-system functions and therapeutic areas. This is especially evident in middle-SDI countries, many of which have recently undergone or are currently experiencing epidemiological transitions. The HAQ Index, if paired with other measures of health-systemcharacteristics such as intervention coverage, could provide a robust avenue for tracking progress on universal health coverage and identifying local priorities for strengthening personal health-care quality and access throughout the world. Copyright (C) The Author(s). Published by Elsevier Ltd.Peer reviewe

Pseudo-dynamic combinatorial chemistry

Pseudo-dynamic combinatorial chemistry (pDCC) combines the synthesis, screening and destruction of combinatorial libraries to kinetically resolve inhibitors based on their affinity for a target. In our proof-of-principle studies, a library of dipeptides was formed in the presence of a target, carbonic anhydrase (CA), and a destruction mechanism, a protease. Since the target and the protease were separated by a dialysis membrane, only the proportion of dipeptides that was not bound to the target was available for destruction and therefore, the rate of hydrolysis of the pseudo-dynamic combinatorial library (pDCL) could be correlated to the librarie's relative affinity for the target. The first set of proof-of-principle pDCC experiments were found to be flawed. Rather than reflecting the binding affinity of the library for the target, the final product distribution reflected the protease's substrate specificity. The main problems with these pDCLs were insufficient and imbalanced rate of destruction of the peptides in the absence of the target, pH drift, and insufficient permeability across 1000 MWCO cellulose ester membranes. A dipeptide amide based pDCL that was efficiently cleaved by thermolysin was designed. The new pDCL could be used in pH 7.5 75 mM HEPES, 16.6 mM CaCl2 buffer, provided that the chambers were limited by 3500 MWCO membranes. A 4th generation pDCC experiment that evolved reflecting the library's affinity for the target was performed. In order to study pDCC behavior a simplified pDCC mimic (pDCCm) was designed. In pDCCm, synthesis was replaced by a static library of compounds and destruction by dilution. The design of a pDCCm based kinetic model led to aLa chimie combinatoire pseudo dynamique (pDCC) combine la synthèse, le criblage et la destruction de libraires combinatoires afin de sélectionner cinétiquement des inhibiteurs sur la base de leur affinité pour une cible. Nos études préliminaires ont été basées sur une librairie de dipeptides formés en présence de l'anhydrase carbonique (CA) comme cible et détruits grâce à une protéase. Étant donné que la cible et la protéase étaient séparées par une membrane de dialyse, seuls les dipeptides non complexés avec la cible étaient susceptibles d'être détruits. Ainsi, la vitesse d'hydrolyse des membres de la librairie combinatoire pseudo-dynamique (pDCL) peut correspondre aux affinités relatives des molécules pour la cible. Les premières expériences prototypes de pDCC étaient défectueuses. Plutôt que de refléter l'affinité des membres de la librairie pour la cible, la distribution finale des produits reflétait la préférence de la protéase pour les substrats. Les principaux problèmes de ces pDCL étaient: une vitesse de destruction des peptides insuffisante et inégale en l'absence de la cible, une déviation progressive du pH et une perméabilité insuffisance au travers les membranes d'ester de cellulose 1000 MWCO. Une pDCL basée sur des amides dipeptidiques, aptes à être efficacement clivés par thermolysine a été élaborée, et utilisée avec succès. Afin d'étudier les pDCC, une expérience modèle simplifiée (pDCCm) a été créée. Dans ce modèle, la synthèse a été remplacée par une librairie statique de molécules et la destruction par une dilution. Le design d'un modèle cinétique basé sur la pDCCm a mené à la c