Prevalence, Awareness and Control of Hypertension in Greece before and after the COVID-19 Pandemic: May Measurement Month Survey 2019-2022.

BACKGROUND: The COVID-19 pandemic had an adverse impact on several cardiovascular risk factors. This study investigated the prevalence, awareness and treatment of hypertension in Greece before and after the pandemic. Data were collected in the context of the May Measurement Month (MMM) global survey initiated by the International Society of Hypertension. METHODS: Adult volunteers (age ≥18 years) were recruited through opportunistic screening in public areas across cities in Greece in 2019 and 2022. Medical history and triplicate sitting blood pressure (BP) measurements were taken using validated automated upper-arm cuff devices. The data were uploaded to the international MMM cloud platform. Hypertension was defined as systolic BP ≥140 mmHg and/or diastolic ≥90 mmHg, and/or self-reported use of drugs for hypertension. The same threshold was used to define uncontrolled BP in treated individuals. RESULTS: Data from 12,080 adults were collected (5,727/6,353 in MMM 2019/2022; men 46/49%, p<0.01; mean age 52.7±16.6/54.8±16.2, p<0.001; smokers, 24.7/30.5, p<0.001; diabetics 12/11.5%, p=NS; cardiovascular disease 5/5.8%, p=NS). The prevalence of hypertension was 41.6/42.6% (MMM 2019/2022, p=NS), with 21.3/27.5% of hypertensives being unaware of their condition (p<0.001), 5.6/2.4% aware untreated (p<0.001), 24.8/22.1% treated uncontrolled (p<0.05), and 48.3/47.8% treated controlled (p=NS). CONCLUSIONS: In Greece, the COVID-19 pandemic did not appear to affect the prevalence and control of hypertension, However, the rate of undiagnosed hypertension was higher after the pandemic. National strategies need to be implemented for the early detection and optimal management of hypertension in the general population in Greece.

Enhancing ferryl accumulation in H2O2-dependent cytochrome P450s.

A facile strategy is presented to enhance the accumulation of ferryl (iron(IV)-oxo) species in H2O2 dependent cytochrome P450s (CYPs) of the CYP152 family. We report the characterization of a highly chemoselective CYP decarboxylase from Staphylococcus aureus (OleTSA) that is soluble at high concentrations. Examination of OleTSA Compound I (CpdI) accumulation with a variety of fatty acid substrates reveals a dependence on resting spin-state equilibrium. Alteration of this equilibrium through targeted mutagenesis of the proximal pocket favors the high-spin form, and as a result, enhances Cpd-I accumulation to nearly stoichiometric yields.

Excision of a Protein-Derived Amine for p-Aminobenzoate Assembly by the Self-Sacrificial Heterobimetallic Protein CADD.

Chlamydia protein associating with death domains (CADD), the founding member of a recently discovered class of nonheme dimetal enzymes termed hemeoxygenase-like dimetaloxidases (HDOs), plays an indispensable role in pathogen survival. CADD orchestrates the biosynthesis of p-aminobenzoic acid (pABA) for integration into folate via the self-sacrificial excision of a protein-derived tyrosine (Tyr27) and several additional processing steps, the nature and timing of which have yet to be fully clarified. Nuclear magnetic resonance (NMR) and proteomics approaches reveal the source and probable timing of amine installation by a neighboring lysine (Lys152). Turnover studies using limiting O2 have identified a para-aminobenzaldehyde (pABCHO) metabolic intermediate that is formed on the path to pABA formation. The use of pABCHO and other probe substrates shows that the heterobimetallic Fe/Mn form of the enzyme is capable of oxygen insertion to generate the pABA-carboxylate.

A Ferric-Superoxide Intermediate Initiates P450-Catalyzed Cyclic Dipeptide Dimerization.

The cytochrome P450 (CYP) AspB is involved in the biosynthesis of the diketopiperazine (DKP) aspergilazine A. Tryptophan-linked dimeric DKP alkaloids are a large family of natural products that are found in numerous species and exhibit broad and often potent bioactivity. The proposed mechanisms for C-N bond formation by AspB, and similar C-C bond formations by related CYPs, have invoked the use of a ferryl-intermediate as an oxidant to promote substrate dimerization. Here, the parallel application of steady-state and transient kinetic approaches reveals a very different mechanism that involves a ferric-superoxide species as a primary oxidant to initiate DKP-assembly. Single turnover kinetic isotope effects and a substrate analog suggest the probable nature and site for abstraction. The direct observation of CYP-superoxide reactivity rationalizes the atypical outcome of AspB and reveals a new reaction manifold in heme enzymes.

Dimer-assisted mechanism of (un)saturated fatty acid decarboxylation for alkene production.

The enzymatic decarboxylation of fatty acids (FAs) represents an advance toward the development of biological routes to produce drop-in hydrocarbons. The current mechanism for the P450-catalyzed decarboxylation has been largely established from the bacterial cytochrome P450 OleTJE. Herein, we describe OleTPRN, a poly-unsaturated alkene-producing decarboxylase that outrivals the functional properties of the model enzyme and exploits a distinct molecular mechanism for substrate binding and chemoselectivity. In addition to the high conversion rates into alkenes from a broad range of saturated FAs without dependence on high salt concentrations, OleTPRN can also efficiently produce alkenes from unsaturated (oleic and linoleic) acids, the most abundant FAs found in nature. OleTPRN performs carbon-carbon cleavage by a catalytic itinerary that involves hydrogen-atom transfer by the heme-ferryl intermediate Compound I and features a hydrophobic cradle at the distal region of the substrate-binding pocket, not found in OleTJE, which is proposed to play a role in the productive binding of long-chain FAs and favors the rapid release of products from the metabolism of short-chain FAs. Moreover, it is shown that the dimeric configuration of OleTPRN is involved in the stabilization of the A-A' helical motif, a second-coordination sphere of the substrate, which contributes to the proper accommodation of the aliphatic tail in the distal and medial active-site pocket. These findings provide an alternative molecular mechanism for alkene production by P450 peroxygenases, creating new opportunities for biological production of renewable hydrocarbons.

Beta-Blockers in Pregnancy: Clinical Update.

PURPOSE OF REVIEW: The aim of this review was to determine the anticipated benefits and adverse effects of beta-blockers in pregnant women with hypertension. The other issue was to assess the possible adverse effects of beta-blockers for their babies and provide current consensus recommendations for appropriate selection and individualized antihypertensive treatment with beta-blockers in pregnancy-associated hypertension. RECENT FINDINGS: Hypertensive disorders of pregnancy are a major cause of maternal and fetal morbidity, with consequences later in life. Certain beta-blockers are useful for ameliorating hypertension in pregnancy and may have a protective role in endothelial dysfunction. However, some aspects of beta-blocker use in pregnancy are contentious among providers. Evidence on their safety, although well documented, is variable, and recent research reveals areas of controversy. Besides intrauterine growth restriction, other neonatal and obstetric complications remain a concern and should be explored thoroughly. Attention is necessary when treating pregnancy-associated hypertensive disorders with beta-blockers. Specific beta-blockers are considered safe in pregnancy, although the associated effects in the fetus are not clearly known and evidence is lacking for many safety outcomes, other than intrauterine growth restriction. Nevertheless, beta-blockers with specific indications in pregnancy under individualized selection and monitoring may confer substantial improvements in pregnant women with hypertension.

Self-sacrificial tyrosine cleavage by an Fe:Mn oxygenase for the biosynthesis of para-aminobenzoate in Chlamydia trachomatis.

Chlamydia protein associating with death domains (CADD) is involved in the biosynthesis of para-aminobenzoate (pABA), an essential component of the folate cofactor that is required for the survival and proliferation of the human pathogen Chlamydia trachomatis. The pathway used by Chlamydiae for pABA synthesis differs from the canonical multi-enzyme pathway used by most bacteria that relies on chorismate as a metabolic precursor. Rather, recent work showed pABA formation by CADD derives from l-tyrosine. As a member of the emerging superfamily of heme oxygenase-like diiron oxidases (HDOs), CADD was proposed to use a diiron cofactor for catalysis. However, we report maximal pABA formation by CADD occurs upon the addition of both iron and manganese, which implicates a heterobimetallic Fe:Mn cluster is the catalytically active form. Isotopic labeling experiments and proteomics studies show that CADD generates pABA from a protein-derived tyrosine (Tyr27), a residue that is ∼14 Šfrom the dimetal site. We propose that this self-sacrificial reaction occurs through O2 activation by a probable Fe:Mn cluster through a radical relay mechanism that connects to the "substrate" Tyr, followed by amination and direct oxygen insertion. These results provide the molecular basis for pABA formation in C. trachomatis, which will inform the design of novel therapeutics.

Experimental and Theoretical Examination of the Kinetic Isotope Effect in Cytochrome P450 Decarboxylase OleT.

Using a combination of experimental studies, theory, simulation, and modeling, we investigate the hydrogen atom transfer (HAT) reaction by the high-valent ferryl cytochrome P450 (CYP) intermediate known as Compound I, a species that is central to innumerable and important detoxification and biosynthetic reactions. The P450 decarboxylase known as OleT converts fatty acids, a sustainable biological feedstock, into terminal alkenes and thus is of high interest as a potential means to produce fungible biofuels. Previous experimental work has established the intermediacy of Compound I in the C─C scission reaction catalyzed by OleT and an unprecedented ability to monitor the HAT process in the presence of bound fatty acid substrates. Here, we leverage the kinetic simplicity of the OleT system to measure the activation barriers for CYP HAT and the temperature dependence of the substrate 2H kinetic isotope effect. Notably, neither measurement has been previously accessible for a CYP to date. Theoretical analysis alludes to the significance of substrate fatty acid coordination for generating the hydrogen donor/acceptor configurations that are most conducive for HAT to occur. The analysis of the two-dimensional potential energy surface, based on multireference electronic wave functions, illustrates the uncoupled character of the hydrogen motion. Quantum dynamics calculations along the hydrogen reaction path demonstrate that hydrogen tunneling is essential to qualitatively capture the experimental isotope effect, its temperature dependence, and appropriate activation energies. Overall, a more fundamental understanding of the OleT reaction coordinate contributes to the development of biomimetic catalysts for controlled C─H bond activation, an outstanding current challenge for (bio)synthetic chemistry.

Stimuli-Modulated Metal Oxidation States in Photochromic MOFs.

Tuning metal oxidation states in metal-organic framework (MOF) nodes by switching between two discrete linker photoisomers via an external stimulus was probed for the first time. On the examples of three novel photochromic copper-based frameworks, we demonstrated the capability of switching between +2 and +1 oxidation states, on demand. In addition to crystallographic methods used for material characterization, the role of the photochromic moieties for tuning the oxidation state was probed via conductivity measurements, cyclic voltammetry, and electron paramagnetic resonance, X-ray photoelectron, and diffuse reflectance spectroscopies. We confirmed the reversible photoswitching activity including photoisomerization rate determination of spiropyran- and diarylethene-containing linkers in extended frameworks, resulting in changes in metal oxidation states as a function of alternating excitation wavelengths. To elucidate the switching process between two states, the photoisomerization quantum yield of photochromic MOFs was determined for the first time. Overall, the introduced noninvasive concept of metal oxidation state modulation on the examples of stimuli-responsive MOFs foreshadows a new pathway for alternation of material properties toward targeted applications.

Structural and functional characterization of ß-cyanoalanine synthase from Tetranychus urticae.

Tetranychus urticae is a polyphagous spider mite that can feed on more than 1100 plant species including cyanogenic plants. The herbivore genome contains a horizontally acquired gene tetur10g01570 (TuCAS) that was previously shown to participate in cyanide detoxification. To understand the structure and determine the function of TuCAS in T. urticae, crystal structures of the protein with lysine conjugated pyridoxal phosphate (PLP) were determined. These structures reveal extensive TuCAS homology with the ß-substituted alanine synthase family, and they show that this enzyme utilizes a similar chemical mechanism involving a stable α-aminoacrylate intermediate in ß-cyanoalanine and cysteine synthesis. We demonstrate that TuCAS is more efficient in the synthesis of ß-cyanoalanine, which is a product of the detoxification reaction between cysteine and cyanide, than in the biosynthesis of cysteine. Also, the enzyme carries additional enzymatic activities that were not previously described. We show that TuCAS can detoxify cyanide using O-acetyl-L-serine as a substrate, leading to the direct formation of ß-cyanoalanine. Moreover, it catalyzes the reaction between the TuCAS-bound α-aminoacrylate intermediate and aromatic compounds with a thiol group. In addition, we have tested several compounds as TuCAS inhibitors. Overall, this study identifies additional functions for TuCAS and provides new molecular insight into the xenobiotic metabolism of T. urticae.

BesC Initiates C-C Cleavage through a Substrate-Triggered and Reactive Diferric-Peroxo Intermediate.

BesC catalyzes the iron- and O2-dependent cleavage of 4-chloro-l-lysine to form 4-chloro-l-allylglycine, formaldehyde, and ammonia. This process is a critical step for a biosynthetic pathway that generates a terminal alkyne amino acid which can be leveraged as a useful bio-orthogonal handle for protein labeling. As a member of an emerging family of diiron enzymes that are typified by their heme oxygenase-like fold and a very similar set of coordinating ligands, recently termed HDOs, BesC performs an unusual type of carbon-carbon cleavage reaction that is a significant departure from reactions catalyzed by canonical dinuclear-iron enzymes. Here, we show that BesC activates O2 in a substrate-gated manner to generate a diferric-peroxo intermediate. Examination of the reactivity of the peroxo intermediate with a series of lysine derivatives demonstrates that BesC initiates this unique reaction trajectory via cleavage of the C4-H bond; this process represents the rate-limiting step in a single turnover reaction. The observed reactivity of BesC represents the first example of a dinuclear-iron enzyme that utilizes a diferric-peroxo intermediate to capably cleave a C-H bond as part of its native function, thus circumventing the formation of a high-valent intermediate more commonly associated with substrate monooxygenations.

Opportunistic screening for hypertension in the general population in Greece: International Society of Hypertension May Measurement Month 2019.

Hypertension remains a major public health issue with inadequate control worldwide. The May Measurement Month (MMM) initiative by the International Society of Hypertension was implemented in Greece in 2019 aiming to raise hypertension awareness and control. Adult volunteers (≥18 years) were recruited through opportunistic screening in five urban areas. Information on medical history and triplicate sitting blood pressure (BP) measurements were obtained using validated automated upper-arm devices. Hypertension was defined as systolic BP ≥140 mmHg and/or diastolic ≥90 mmHg, and/or self-reported use of drugs for hypertension. A total of 5727 were analysed [mean age 52.7 (SD 16.6) years, men 46.5%, 88.3% had BP measurement in the last 18 months]. The prevalence of hypertension was (41.6%) and was higher in men and in older individuals. Among individuals with hypertension, 78.7% were diagnosed, 73.1% treated, and 48.3% controlled. Awareness, treatment, and control of hypertension were higher in women and in older individuals. Hypertensives had a higher body mass index (BMI) and were more likely to have diabetes, myocardial infarction and stroke, and less likely to smoke than normotensives (all P < 0.001). Among treated hypertensives, 65.1% were on monotherapy, and with increasing number of antihypertensive drugs the BP levels were higher and hypertension control rates lower. The prevalence of hypertension in Greece is high, with considerable potential for improving awareness, treatment, and control. Screening programmes, such as MMM, need to be widely implemented at the population level, together with training programmes for healthcare professionals aiming to optimise management and control.

Joint ESH excellence centers' national meeting on renal sympathetic denervation: A Greek experts' survey.

OBJECTIVE: The efficacy of renal sympathetic denervation (RDN) has been affirmed by a number of recent clinical studies, despite controversies in this field over the last five years. Therefore, it is of paramount importance that hypertension experts debate the merits of RDN by revealing and expressing their personal beliefs and perspectives regarding this procedure. METHODS: A cross-sectional survey was conducted among Greek leaders of the Hypertension Excellence Centers with the use of a closed-type questionnaire specifically designed to elicit information and evaluate the respondent's views and perspectives about RDN efficacy, safety and ideal target patient population. RESULTS: A total of 36 participants completed the survey. Based on the results, RDN was considered efficient (91.7%) and safe (94.5%), while the overwhelming majority of the participants felt confident in the long-term efficacy (88.9%) of the intervention and that it lacks reliable predictors of blood pressure response (94.5%). Patients with resistant (91.7%), ultra-resistant (94.4%), and uncontrolled hypertension (80.6%) were suggested as ideal candidates for RDN. Establishing a close co-operation between interventionalists and hypertension experts was considered essential to ensure the efficacy (97.2%) as well as the safety (97.3%) of the procedure. CONCLUSION: The vast majority of Greek hypertension experts surveyed were convinced of the efficacy and safety of RDN based on the preponderance of available scientific and clinical data. Identification of the ideal patient group remains controversial. Respondents generally agreed on the necessity of building close collaborative relationships between interventionalists and hypertension experts in order to improve RDN clinical outcome.

A single-center, prospective, observational study on maternal smoking during pregnancy in Greece: The HELENA study.

INTRODUCTION: The unequivocal association between exposure to smoke and numerous complications of pregnancy, demonstrated in the last decades, has led to a significant decrease of smoking rates in pregnancy. The aim of the present study was to determine the prevalence of maternal smoking and to elucidate factors predisposing to it among pregnant women in Athens, Greece. METHODS: A population of 1700 pregnant women (mean age: 31.2±5.5 years) who visited consecutively the Cardiology Department of Helena Venizelou Maternity Hospital in Athens, Greece, between September 2016 and August 2017, was prospectively analyzed. Data regarding changes in the future mother's smoking habit as well as different sociodemographic factors potentially related to these changes were recorded. RESULTS: Of the 1700 participants, 704 (41.4%) were smokers, and of those 52.4% quit smoking after knowledge of their pregnancy status. The overall prevalence of smoking in pregnancy was 19.7%. Prevalence was higher in women who were aged <20 years (p=0.038), were multipara (p=0.032), had ≤12 years of education (p=0.044) and had a partner who was a smoker (p=0.047). Women aged ≤20 years were more likely to be persistent smokers at the beginning of pregnancy and demonstrated a higher prevalence of smoking during pregnancy (42.2% vs 19.7% in the overall study population). CONCLUSIONS: Our data demonstrate that maternal smoking during pregnancy still remains a major public health issue in Greece with a prevalence higher than most other industrialized countries.

Beta Blockers and Chronic Obstructive Pulmonary Disease (COPD): Sum of Evidence.

Approximately half a century has passed since the discovery of beta-blockers. Then, their prime therapeutic purpose was to treat angina and cardiac arrhythmias; nowadays, beta-blockers' usage and effectiveness are extended to treat other cardiovascular diseases, such as hypertension, congestive heart failure, and coronary artery disease. Safety concerns were raised about beta- blockers and their use for chronic obstructive pulmonary disease (COPD) patients with concurrent cardiovascular disease. After thorough research of the literature, this review summarizes the evidence proving that beta-blockers not only might be well tolerated in COPD patients, but they might also have a beneficial effect in this group of patients.

Direct Identification of Mixed-Metal Centers in Metal-Organic Frameworks: Cu3(BTC)2 Transmetalated with Rh2+ Ions.

Raman spectroscopy was used to establish direct evidence of heterometallic metal centers in a metal-organic framework (MOF). The Cu3(BTC)2 MOF HKUST-1 (BTC3- = benzenetricarboxylate) was transmetalated by heating it in a solution of RhCl3 to substitute Rh2+ ions for Cu2+ ions in the dinuclear paddlewheel nodes of the framework. In addition to the Cu-Cu and Rh-Rh stretching modes, Raman spectra of (CuxRh1-x)3(BTC)2 show the Cu-Rh stretching mode, indicating that mixed-metal Cu-Rh nodes are formed after transmetalation. Density functional theory studies confirmed the assignment of a Raman peak at 285 cm-1 to the Cu-Rh stretching vibration. Electron paramagnetic resonance spectroscopy experiments further supported the conclusion that Rh2+ ions are substituted into the paddlewheel nodes of Cu3(BTC)2 to form an isostructural heterometallic MOF, and electron microscopy studies showed that Rh and Cu are homogeneously distributed in (CuxRh1-x)3(BTC)2 on the nanoscale.

Antidiabetic drugs and blood pressure changes.

New era antidiabetic drugs are characterized by cardiovascular safety, including specific outcome benefits observed in randomized clinical trials (RCTs). It has been postulated that the favorable effects of new antidiabetic agents are related both to better control of blood pressure (BP) levels and to activation of multiple anti-atherosclerotic properties. In this review, we aimed to assess whether antidiabetic drugs have a pressor effect in glucose control and outcome-oriented RCTs, and to summarize the activated pathophysiological mechanisms relevant to BP control following the use of different antidiabetic drug classes. We also tried to determine which, if any, are the BP-lowering effects of more intense vs less intense glucose-lowering strategy irrespectively of trial antidiabetic regimen. To provide more robust results and evidence-based argumentation, a meta-analysis of placebo-controlled antidiabetic drug RCTs was undertaken to estimate the ongoing BP reduction for all considered and each separate drug class alone. This quantitative synthesis might be helpful for the clinician 1) to select or avoid the use of some classes of antidiabetic agents with a potential favorable or adverse pressor effect, respectively 2) to organize the overall drug regimen in patients with diabetes mellitus and minimize side effects because of concomitant use of drugs with established pressor effect (i.e. antihypertensive agents). This review was also organized to indicate whether BP change associated with different antidiabetic treatments may explain the specific macrovascular outcome benefits. Between all antidiabetic drugs including exogenous insulin, only sodium-glucose cotransporter 2 inhibitors produce a clinically important BP-lowering effect, but this BP reduction alone cannot explain the observed cardiovascular benefit.

Enhanced P450 fatty acid decarboxylase catalysis by glucose oxidase coupling and co-assembly for biofuel generation.

Cytochrome P450 OleT is a fatty acid decarboxylase that uses hydrogen peroxide (H2O2) to catalyze the production of terminal alkenes, which are industrially important chemicals with biofuel and synthetic applications. Despite its requirement for large turnover levels, high concentrations of H2O2 may cause heme group degradation, diminishing enzymatic activity and limiting broad application for synthesis. Here, we report an artificial enzyme cascade composed of glucose oxidase (GOx) and OleTSA from Staphylococcus aureus for efficient terminal alkene production. By adjusting the ratio of GOx to OleTSA, the GOx-based tandem catalysis shows significantly improved product yield compared to the H2O2 injection method. Moreover, the co-assembly of the GOx/OleTSA enzymes with a polymer, forming polymer-dual enzymes nanoparticles, displays improved activity compared to the free enzyme. This dual strategy provides a simple and efficient system to transform a naturally abundant feedstock to industrially important chemicals.

Microfluidic-assisted polymer-protein assembly to fabricate homogeneous functionalnanoparticles.

Functional polymer-protein nanoparticles (NPs) have broad applications in biotechnology and nanotechnology. In principle, controllable and vigorous mixing is required to fabricate homogeneous NPs, which remains a challenge via conventional bulk synthetic methods. In this study, an electrokinetics (EK) based microfluidic reactor with fast mixing is explored to assemble functional proteins with polymers in an ethanol/water co-solvent system. The resultant NPs show significantly improved size distribution by comparison with the ones prepared using conventional bulk method, while the NPs size can be tuned by adjusting the mass ratio of polymer to protein. The functionalities of the assembled proteins are sustained upon the EK based microfluidic mixing, indicating the application potential of our method in the controlled assembly of different functional proteins.

The Diiron Monooxygenase CmlA from Chloramphenicol Biosynthesis Allows Reconstitution of ß-Hydroxylation during Glycopeptide Antibiotic Biosynthesis.

ß-Hydroxylation plays an important role in the nonribosomal peptide biosynthesis of many important natural products, including bleomycin, chloramphenicol, and the glycopeptide antibiotics (GPAs). Various oxidative enzymes have been implicated in such a process, with the mechanism of incorporation varying from installation of hydroxyl groups in amino acid precursors prior to adenylation to direct amino acid oxidation during peptide assembly. In this work, we demonstrate the in vitro utility and scope of the unusual nonheme diiron monooxygenase CmlA from chloramphenicol biosynthesis for the ß-hydroxylation of a diverse range of carrier protein bound substrates by adapting this enzyme as a non-native trans-acting enzyme within NRPS-mediated GPA biosynthesis. The results from our study show that CmlA has a broad substrate specificity for modified phenylalanine/tyrosine residues as substrates and can be used in a practical strategy to functionally cross complement compatible NRPS biosynthesis pathways in vitro.