Structure-Guided Design of an Anti-dengue Antibody Directed to a Non-immunodominant Epitope.

Dengue is the most common vector-borne viral disease, causing nearly 400 million infections yearly. Currently there are no approved therapies. Antibody epitopes that elicit weak humoral responses may not be accessible by conventional B cell panning methods. To demonstrate an alternative strategy to generating a therapeutic antibody, we employed a non-immunodominant, but functionally relevant, epitope in domain III of the E protein, and engineered by structure-guided methods an antibody directed to it. The resulting antibody, Ab513, exhibits high-affinity binding to, and broadly neutralizes, multiple genotypes within all four serotypes. To assess therapeutic relevance of Ab513, activity against important human clinical features of dengue was investigated. Ab513 mitigates thrombocytopenia in a humanized mouse model, resolves vascular leakage, reduces viremia to nearly undetectable levels, and protects mice in a maternal transfer model of lethal antibody-mediated enhancement. The results demonstrate that Ab513 may reduce the public health burden from dengue.

Ferric Carboxymaltose in Heart Failure with Iron Deficiency.

BACKGROUND: Ferric carboxymaltose therapy reduces symptoms and improves quality of life in patients who have heart failure with a reduced ejection fraction and iron deficiency. Additional evidence about the effects of ferric carboxymaltose on clinical events is needed. METHODS: In this double-blind, randomized trial, we assigned ambulatory patients with heart failure, a left ventricular ejection fraction of 40% or less, and iron deficiency, in a 1:1 ratio, to receive intravenous ferric carboxymaltose or placebo, in addition to standard therapy for heart failure. Ferric carboxymaltose or placebo was given every 6 months as needed on the basis of iron indexes and hemoglobin levels. The primary outcome was a hierarchical composite of death within 12 months after randomization, hospitalizations for heart failure within 12 months after randomization, or change from baseline to 6 months in the 6-minute walk distance. The significance level was set at 0.01. RESULTS: We enrolled 3065 patients, of whom 1532 were randomly assigned to the ferric carboxymaltose group and 1533 to the placebo group. Death by month 12 occurred in 131 patients (8.6%) in the ferric carboxymaltose group and 158 (10.3%) in the placebo group; a total of 297 and 332 hospitalizations for heart failure, respectively, occurred by month 12; and the mean (±SD) change from baseline to 6 months in the 6-minute walk distance was 8±60 and 4±59 m, respectively (Wilcoxon-Mann-Whitney P = 0.02; unmatched win ratio, 1.10; 99% confidence interval, 0.99 to 1.23). Repeated dosing of ferric carboxymaltose appeared to be safe with an acceptable adverse-event profile in the majority of patients. The number of patients with serious adverse events occurring during the treatment period was similar in the two groups (413 patients [27.0%] in the ferric carboxymaltose group and 401 [26.2%] in the placebo group). CONCLUSIONS: Among ambulatory patients who had heart failure with a reduced ejection fraction and iron deficiency, there was no apparent difference between ferric carboxymaltose and placebo with respect to the hierarchical composite of death, hospitalizations for heart failure, or 6-minute walk distance. (Funded by American Regent, a Daiichi Sankyo Group company; HEART-FID ClinicalTrials.gov number, NCT03037931.).

Structural basis for proenzyme maturation, substrate recognition, and ligation by a hyperactive peptide asparaginyl ligase.

Peptide ligases are versatile enzymes that can be utilized for precise protein conjugation for bioengineering applications. Hyperactive peptide asparaginyl ligases (PALs), such as butelase-1, belong to a small class of enzymes from cyclotide-producing plants that can perform site-specific, rapid ligation reactions after a target peptide asparagine/aspartic acid (Asx) residue binds to the active site of the ligase. How PALs specifically recognize their polypeptide substrates has remained elusive, especially at the prime binding side of the enzyme. Here we report crystal structures that capture VyPAL2, a catalytically efficient PAL from Viola yedoensis, in an activated state, with and without a bound substrate. The bound structure shows one ligase with the N-terminal polypeptide tail from another ligase molecule trapped at its active site, revealing how Asx inserts in the enzyme's S1 pocket and why a hydrophobic residue is required at the P2' position. Besides illustrating the anchoring role played by P1 and P2' residues, these results uncover a role for the Gatekeeper residue at the surface of the S2 pocket in shifting the nonprime portion of the substrate and, as a result, the activity toward ligation or hydrolysis. These results suggest a picture for proenzyme maturation in the vacuole and will inform the rational design of peptide ligases with tailored specificities.

In-Hospital Risk Factors for Reintervention and Amputation in Brachial Arterial Trauma.

INTRODUCTION: Brachial artery trauma is a rare but potentially devastating injury. There is little data regarding risk factors for reintervention and amputation prevention in this population, as well as anticoagulant (AC) and antiplatelet (AP) regimens and outcomes after discharge in trauma patients with vascular injuries requiring repair. This study aims to identify in-hospital risk factors for reintervention and amputation and stratify outcomes of follow-up by discharge AC or AP regimen. METHODS: The AAST Prospective Observational Vascular Injury Trial database was queried for all patients who underwent traumatic brachial arterial repair from 2013 to 2022. Patients were evaluated by need for reintervention, amputation, and outcomes at follow-up by AC or AP regimen. RESULTS: Three hundred and eleven patients required brachial repair, 28 (9%) required reoperation, and 8 (2.6%) required amputation. High injury severity score and an increased number of packed red blood cells and platelets showed a significant increase for reoperation and amputation. Damage control and shunt use were significant for the need to reoperate. Seventy-four percent (221/298) of patients were discharged with postoperative AC or AP regimens. There was no significant difference of short-term follow-up by type of AC or AP regimen. CONCLUSIONS: Damage control and temporary shunt may lead to additional operations but not an increase in amputations. However, anticoagulation intraoperatively and postoperatively does not appear to play a significant role in reducing reintervention. It also suggests that there is no increase in short-term follow-up complications with or without AC or AP therapy.

Unravelling the kinetics, isotherms, thermodynamics, and mass transfer behaviours of Zeolite Socony Mobil - 5 in removing hydrogen sulphide resulting from a dark fermentative biohydrogen production process.

Research into the speciation of sulfur and hydrogen molecules produced through the complex process of thermophilic dark fermentation has been conducted. Detailed surface studies of solid-gas systems using real biogas (biohydrogen) streams have unveiled the mechanisms and specific interactions between these gases and the physicochemical properties of a zeolite as an adsorbent. These findings highlight the potential of zeolites to effectively capture and interact with these molecules. In this study, the hydrogen sulphide removal analysis was conducted using 0.8 g of the adsorbent and at various reaction temperatures (25-125 °C), a flow rate of 100 mL min-1, and an initial concentration of approximately 5000 ppm hydrogen sulphide. The reaction temperature has been observed to be an essential parameter of Zeolite Socony Mobil - 5 adsorption capacity. The optimum adsorption capacity attains a maximum value of 0.00890 mg g-1 at an optimal temperature of 25 °C. The formation of sulphur species resulting from the hydrogen sulphide adsorption on the zeolite determines the kinetics, thermodynamics, and mass transfer behaviours of Zeolite Socony Mobil - 5 in hydrogen sulphide removal and Zeolite Socony Mobil - 5 is found to improve the quality of biohydrogen produced in thermophilic environments. Biohydrogen (raw gas) yield was enhanced from 2.48 mol H2 mol-1 hexose consumed before adsorption to 2.59 mol H2 mol-1 hexose consumed after adsorption at a temperature of 25 °C. The Avrami kinetic model was fitted for hydrogen sulphide removal on Zeolite Socony Mobil - 5. The process is explained well and fitted using the Temkin isotherm model and the investigation into thermodynamics reveals that the adsorption behaviour is exothermic and non-spontaneous. Furthermore, the gas molecule's freedom of movement becomes random. The adsorption phase is restricted by intra-particle diffusion followed by film diffusion during the transfer of hydrogen sulphide into the pores of Zeolite Socony Mobil - 5 prior to adsorption on its active sites. The utilisation of Zeolite Socony Mobil - 5 for hydrogen sulphide removal offers the benefit of reducing environmental contamination and exhibiting significant applications in industrial operations.

Roles of ß-adrenoceptor Subtypes and Therapeutics in Human Cardiovascular Disease: Heart Failure, Tachyarrhythmias and Other Cardiovascular Disorders.

ß-Adrenoceptors (ß-ARs) provide an important therapeutic target for the treatment of cardiovascular disease. Three ß-ARs, ß1-AR, ß2-AR, ß3-AR are localized to the human heart. Activation of ß1-AR and ß2-ARs increases heart rate, force of contraction (inotropy) and consequently cardiac output to meet physiological demand. However, in disease, chronic over-activation of ß1-AR is responsible for the progression of disease (e.g. heart failure) mediated by pathological hypertrophy, adverse remodelling and premature cell death. Furthermore, activation of ß1-AR is critical in the pathogenesis of cardiac arrhythmias while activation of ß2-AR directly influences blood pressure haemostasis. There is an increasing awareness of the contribution of ß2-AR in cardiovascular disease, particularly arrhythmia generation. All ß-blockers used therapeutically to treat cardiovascular disease block ß1-AR with variable blockade of ß2-AR depending on relative affinity for ß1-AR vs ß2-AR. Since the introduction of ß-blockers into clinical practice in 1965, ß-blockers with different properties have been trialled, used and evaluated, leading to better understanding of their therapeutic effects and tolerability in various cardiovascular conditions. ß-Blockers with the property of intrinsic sympathomimetic activity (ISA), i.e. ß-blockers that also activate the receptor, were used in the past for post-treatment of myocardial infarction and had limited use in heart failure. The ß-blocker carvedilol continues to intrigue due to numerous properties that differentiate it from other ß-blockers and is used successfully in the treatment of heart failure. The discovery of ß3-AR in human heart created interest in the role of ß3-AR in heart failure but has not resulted in therapeutics at this stage.

Eye growth pattern of myopic children wearing spectacle lenses with aspherical lenslets compared with non-myopic children.

INTRODUCTION: To evaluate eye growth of children wearing spectacle lenses with highly aspherical lenslets (HAL), slightly aspherical lenslets (SAL) and single-vision lenses (SVL) compared to eye growth patterns in non-myopes in Wenzhou, China. METHODS: The randomised trial had 170 myopic children (aged 8-13 years) randomly assigned to the HAL, SAL or SVL group. Normal eye growth was examined using 700 non-myopic schoolchildren (aged 7-9 years) in the Wenzhou Medical University-Essilor Progression and Onset of Myopia (WEPrOM) cohort study using logistic function models. Slow, normal and fast eye growth was defined as range of values <25th, 25th-75th and >75th percentiles, respectively. RESULTS: The predicted upper limits of slow eye growth (25th percentile) among non-myopes aged 7-10 years and 11-13 years were 0.20-0.13 and 0.08-0.01 mm (after 2-year period; 0.37-0.33 and 0.29-0.14 mm), respectively, while the upper limits of normal eye growth (75th percentile) were 0.32-0.31 and 0.28-0.10 mm (after 2-year period; 0.58-0.55 and 0.50-0.24 mm), respectively. The 2-year trial had 157 children, 96 of whom wore their lenses full time (everyday ≥12 h/day). The mean 2-year axial length change for HAL, SAL and SVL was 0.34, 0.51 and 0.69 mm (0.28, 0.46 and 0.69 mm in full-time wear), respectively. Slow eye growth was found in 35%, 17% and 2% (44%, 29% and 3% in full-time wear); normal eye growth in 35%, 26% and 12% (44%, 32% and 9% in full-time wear) and fast eye growth in 30%, 57% and 86% (12%, 39% and 88% in full-time wear), respectively (p < 0.001). CONCLUSIONS: The eye growth pattern in approximately 90% wearing HAL full time (compared with about 10% wearing SVL full time) was similar or slower than that of non-myopic children both after 1- and 2-year periods.

Towards environmentally-friendly solutions for real textile-dyeing wastewater with energy recovery: Effluent recirculation and Rubia plant-derived purpurin electron mediator in microbial fuel cell.

Escalating global water pollution exacerbated by textile-dyeing wastewater (TDW) poses significant environmental and health concerns due to the insufficient treatment methods being utilized. Thus, it is imperative to implement more effective treatment solutions to address such issues. In this research, different environmentally-friendly strategies involving effluent recirculation (ER) and Rubia cordifolia plant-derived purpurin electron mediator (EM) were introduced to enhance the treatment of real TDW and bioelectricity generation performance of an anti-gravity flow microbial fuel cell (AGF-MFC). The results revealed that optimum performance was achieved with a combination of hydraulic retention time (HRT) of 48 h with a recirculation ratio of 1, where the reduction efficiency of biochemical oxygen demand (BOD5), chemical oxygen demand (COD), ammonium (NH4+), nitrate (NO3-), sulphate (SO42-), ammonia nitrogen (NH3-N), colour and turbidity were 82.17 %, 82.15 %, 85.10 %, 80.52 %, 75.91 %, 59.52 %, 71.02 % and 93.10 %, respectively. In terms of bioelectricity generation performance, AGF-MFC showed a maximum output voltage and power density of 404.72 mV and 65.16 mW/m2, respectively. Moreover, the results also signified that higher treatment performance of TDW was obtained with natural purpurin from Rubia cordifolia plant than synthetic purpurin as EM. The reduced reactivity of highly stable synthetic purpurin EM for mediating the electron transfer was a contributing factor to the outperformance of plant-derived purpurin. Additionally, detailed electron-mediating mechanisms of purpurin were proposed to unravel the underlying electron transfer pathway involved in AGF-MFC. This research offers insight into the development of more sustainable solutions for managing TDW, and consequently reducing environmental pollution.

Baseline characteristics of patients in the randomized study to investigate the efficacy and safety of ferric carboxymaltose as treatment for heart failure with iron deficiency: HEART-FID trial.

BACKGROUND: Prior clinical trials have investigated intravenous iron in patients with heart failure (HF) and iron deficiency, but the safety and efficacy of this therapy remains unclear. METHODS: We report the baseline demographics and clinical characteristics of patients enrolled in the HEART-FID study and compare HEART-FID participants with patients within other contemporary clinical trials of patients with HF with reduced ejection fraction (HFrEF), including other intravenous iron trials. RESULTS: In the 3,065 participants randomized in HEART-FID, median (IQR) age was 69.7 (62.0-76.5) years, 1,037 (33.8%) were female, 322 (10.5%) were Black, median ejection fraction was 32% (25%-37%), 1,837 (60.0%) had ischemic etiology, and baseline median NT-proBNP was 1,462 (721-2,966) pg/mL. Median baseline hemoglobin was 12.6 (11.6-13.6) g/dL, and median 6-minute walk test distance was 272 (196-350) m, similar to prior intravenous iron HFrEF trials. Common comorbidities included atrial fibrillation/flutter (43.7%), and type 2 diabetes (45.2%). Compared with several recent HFrEF trials, patients enrolled in HEART-FID had similar baseline demographics and clinical characteristics, though a greater proportion of women and Black participants were recruited in HEART-FID. In HEART-FID, HFrEF therapy included a beta-blocker in 92.5%, angiotensin-converting enzyme inhibitor/angiotensin receptor blocker/angiotensin receptor-neprilysin inhibitors (ARNI) in 86.1% (with 29.7% ARNI), and a mineralocorticoid antagonist (MRA) in 55.6%. CONCLUSIONS: Patients enrolled in HEART-FID were similar to those enrolled in other contemporary HFrEF trials and registries, including trials of intravenous iron in HFrEF. However, the HEART-FID cohort is substantially larger and more racially diverse than prior trials of intravenous iron in HFrEF. TRIAL REGISTRATION: ClinicalTrials.gov (NCT03037931).

Myopia and Its Association with Near Work, Outdoor Time, and Housing Type among Schoolchildren in South India.

SIGNIFICANCE: In this comprehensive assessment of environmental associations with refractive status among schoolchildren in India, outdoor time was the key modifiable risk factor associated with myopia rather than time spent on near work. PURPOSE: This study aimed to investigate the environmental risk factors associated with myopia among adolescent schoolchildren in South India. METHODS: Children in grades 8 to 10 from 11 schools in Tamil Nadu, South India, underwent eye examination and risk factor assessments through a modified version of the Sydney myopia questionnaire. Time spent on near work and outdoors was analyzed after division into three groups based on tertiles. Mixed-effects logistic regression was performed to assess the factors associated with myopia. RESULTS: A total of 3429 children (response rate, 78.4%) provided both questionnaire and refraction data. The mean (standard deviation) age was 14 (0.93) years with an equal distribution of sexes. Myopia was present among 867 children (noncycloplegic spherical equivalent refraction, ≤-0.75 D). Refraction was not associated with near work tertiles ( P = .22), whereas less time outdoors was associated with higher myopic refractions ( P = .01). Refraction shifted toward increased myopia with an increase in the near-work/outdoor time ratio ( P = .005). Children living in apartment housing had a higher prevalence of myopia compared with other types of housing ( P < .001). In multivariate analysis, increased time outdoors was a protective factor against myopia (odds ratio, 0.79; 95% confidence interval, 0.63 to 0.99; P = .04), whereas living in apartment housing (odds ratio, 1.27; 95% confidence interval, 1.04 to 1.55; P = .02) was a significant risk factor. CONCLUSIONS: In this cohort of Indian children, outdoor time, increased near-work/outdoor time ratio, and type of housing were the factors associated with myopia. Policies should target implementing a balance between near-work and outdoor time among children.

Exploring the potential of thermophilic anaerobic co-digestion between agro-industrial waste and water hyacinth: operational performance, kinetic study and degradation pathway.

Anaerobic co-digestion (co-AD) of agro-industrial waste, namely, palm oil mill effluent (POME) and sugarcane vinasse (Vn), with water hyacinth (WH) as co-substrate was carried out in two separate Anaerobic Suspended Growth Closed Bioreactors (ASGCBs) under thermophilic (55 °C) conditions. The highest chemical oxygen demand (COD) and soluble COD reduction in co-AD of POME-WH (78.61%, 78.86%) is slightly higher than co-AD of Vn-WH (75.75%, 78.24%). However, VFA reduction in co-AD of POME-WH (96.41%) is higher compared to co-AD of Vn-WH (85.94%). Subsequently, biogas production peaked at 13438 mL/day values and 16122 mL/day for co-AD of POME-WH and Vn-WH, respectively. However, the methane content was higher in the co-AD of POME-WH (72.04%) than in the co-AD of Vn-WH (69.86%). Growth yield (YG), maximum specific substrate utilization rate (rx,max) and maximum specific biomass growth rate (µmax) are higher in co-AD of POME-WH, as supported by the higher mixed liquor volatile suspended solids (MLVSS) and COD reduction efficiency compared to co-AD of Vn-WH. However, methane yield ([Formula: see text]) reported in the co-AD of POME-WH and Vn-WH are 0.2748 and 0.3112 L CH4/g CODreduction, respectively, which suggests that WH is a more suitable co-substrate for Vn compared to POME.

Intermolecular degradation of aromatic compound and its derivatives via combined sequential and hybridized process.

The under-treated wastewater, especially remaining carcinogenic aromatic compounds in wastewater discharge has been expansively reported, wherein the efficiency of conventional wastewater treatment is identified as the primary contributor source. Herein, the advancement of wastewater treatments has drawn much attention in recent years. In the current study, combined sequential and hybridized treatment of thermolysis and coagulation-flocculation provides a novel advancement for environmental emerging pollutant (EP) prescription. This research is mainly demonstrating the mitigation efficiency and degradation pathway of pararosaniline (PRA) hybridized and combined sequential wastewater treatment. Notably, PRA degradation dominantly via a linkage of reaction: thermal cleavage, deamination, silication and diazene reduction. Thermolysis acts as an initiator for the PRA decomposition through thermally induced bond dissociation energy (BDE) for molecular fragmentation whilst coagulation-flocculation facilitates the formation of organo-bridged silsesquioxane as the final degradation product. Different from conventional treatment, the hybridized treatment showed excellent synergistic degradability by removing 99% PRA and its EPs, followed by combined sequential treatment method with 86% reduction. Comprehensive degradation pathway breakdown of carcinogenic and hardly degradable aromatic compounds provides a new insight for wastewater treatment whereby aniline and benzene are entirely undetectable in effluent. The degradation intermediates, reaction derivatives and end products were affirmed by gas chromatography-mass spectrometry, Fourier transform infrared spectroscopy and ultraviolet-visible spectrophotometry (GC-MS, FTIR and UV-Vis). This finding provides valuable guidance in establishing efficient integrated multiple-step wastewater treatments.

Prevalence of myopia among urban and suburban school children in Tamil Nadu, South India: findings from the Sankara Nethralaya Tamil Nadu Essilor Myopia (STEM) Study.

PURPOSE: To report the baseline prevalence of myopia among school children in Tamil Nadu, South India from a prospective cohort study. METHODS: Children between the ages of 5 and 16 years from 11 schools in two districts of Tamil Nadu underwent vision screening. All children underwent visual acuity assessment using a Pocket Vision Screener followed by non-cycloplegic open-field autorefraction (Grand Seiko WAM-5500). Myopia was defined as a spherical equivalent (SE) refraction of ≤-0.75 D and high myopia was defined as SE ≤ -6.00 D. Distribution of refraction, biometry and factors associated with prevalence of myopia were the outcome measures. RESULTS: A total of 14,699 children completed vision screening, with 2% (357) of them having ocular abnormalities other than refractive errors or poor vision despite spectacle correction. The remaining 14,342 children (7557 boys; 52.69%) had a mean age of 10.2 (Standard Deviation [SD] 2.8) years. A total of 2502 had myopia in at least one eye, a prevalence of 17.5% (95% CI: 14.7-20.5%), and 74 (0.5%; 95% CI: 0.3-0.9%) had high myopia. Myopia prevalence increased with age (p < 0.001), but sex was not associated with myopia prevalence (p = 0.24). Mean axial length (AL; 23.08 (SD = 0.91) mm) and mean anterior chamber depth (ACD; 3.45 (SD = 0.27) mm) positively correlated with age (p < 0.001). The mean flat (K1; 43.37 (SD = 1.49) D) and steep (K2; 44.50 (SD = 1.58) D) corneal curvatures showed negative correlation with age (p = 0.02 and p < 0.001, respectively). In the multivariable logistic regression, older age and urban school location had higher odds for prevalence of myopia. CONCLUSION: The baseline prevalence of myopia among 5- to 16-year-old children in South India is larger than that found in previous studies, indicating that myopia is becoming a major public health problem in this country.

Structural determinants for peptide-bond formation by asparaginyl ligases.

Asparaginyl endopeptidases (AEPs) are cysteine proteases which break Asx (Asn/Asp)-Xaa bonds in acidic conditions. Despite sharing a conserved overall structure with AEPs, certain plant enzymes such as butelase 1 act as a peptide asparaginyl ligase (PAL) and catalyze Asx-Xaa bond formation in near-neutral conditions. PALs also serve as macrocyclases in the biosynthesis of cyclic peptides. Here, we address the question of how a PAL can function as a ligase rather than a protease. Based on sequence homology of butelase 1, we identified AEPs and PALs from the cyclic peptide-producing plants Viola yedoensis (Vy) and Viola canadensis (Vc) of the Violaceae family. Using a crystal structure of a PAL obtained at 2.4-Å resolution coupled to mutagenesis studies, we discovered ligase-activity determinants flanking the S1 site, namely LAD1 and LAD2 located around the S2 and S1' sites, respectively, which modulate ligase activity by controlling the accessibility of water or amine nucleophile to the S-ester intermediate. Recombinantly expressed VyPAL1-3, predicted to be PALs, were confirmed to be ligases by functional studies. In addition, mutagenesis studies on VyPAL1-3, VyAEP1, and VcAEP supported our prediction that LAD1 and LAD2 are important for ligase activity. In particular, mutagenesis targeting LAD2 selectively enhanced the ligase activity of VyPAL3 and converted the protease VcAEP into a ligase. The definition of structural determinants required for ligation activity of the asparaginyl ligases presented here will facilitate genomic identification of PALs and engineering of AEPs into PALs.

Crystal structure and catalytic mechanism of the essential m1G37 tRNA methyltransferase TrmD from Pseudomonas aeruginosa.

The tRNA (m1G37) methyltransferase TrmD catalyzes m1G formation at position 37 in many tRNA isoacceptors and is essential in most bacteria, which positions it as a target for antibiotic development. In spite of its crucial role, little is known about TrmD in Pseudomonas aeruginosa (PaTrmD), an important human pathogen. Here we present detailed structural, substrate, and kinetic properties of PaTrmD. The mass spectrometric analysis confirmed the G36G37-containing tRNAs Leu(GAG), Leu(CAG), Leu(UAG), Pro(GGG), Pro(UGG), Pro(CGG), and His(GUG) as PaTrmD substrates. Analysis of steady-state kinetics with S-adenosyl-l-methionine (SAM) and tRNALeu(GAG) showed that PaTrmD catalyzes the two-substrate reaction by way of a ternary complex, while isothermal titration calorimetry revealed that SAM and tRNALeu(GAG) bind to PaTrmD independently, each with a dissociation constant of 14 ± 3 µM. Inhibition by the SAM analog sinefungin was competitive with respect to SAM (Ki = 0.41 ± 0.07 µM) and uncompetitive for tRNA (Ki = 6.4 ± 0.8 µM). A set of crystal structures of the homodimeric PaTrmD protein bound to SAM and sinefungin provide the molecular basis for enzyme competitive inhibition and identify the location of the bound divalent ion. These results provide insights into PaTrmD as a potential target for the development of antibiotics.

Non-nucleoside Inhibitors of Zika Virus RNA-Dependent RNA Polymerase.

Zika virus (ZIKV) remains a potentially significant public health concern because it can cause teratogenic effects, such as microcephaly in newborns and neurological disease, like Guillain-Barré syndrome. Together with efforts to develop a vaccine, the discovery of antiviral molecules is important to control ZIKV infections and to prevent its most severe symptoms. Here, we report the development of small nonnucleoside inhibitors (NNIs) of ZIKV RNA-dependent RNA polymerase (RdRp) activity. These NNIs target an allosteric pocket (N pocket) located next to a putative hinge region between the thumb and the palm subdomains that was originally described for dengue virus (DENV) RdRp. We first tested the activity of DENV RdRp N-pocket inhibitors against ZIKV RdRp, introduced chemical modifications into these molecules, and assessed their potency using both enzymatic and cell-based assays. The most potent compound had a 50% inhibitory concentration value of 7.3 µM and inhibited ZIKV replication in a cell-based assay with a 50% effective concentration value of 24.3 µM. Importantly, we report four high-resolution crystal structures detailing how these NNIs insert into the N pocket of ZIKV RdRp. Our observations point to subtle differences in the size, shape, chemical environment, and hydration of the N pocket from ZIKV RdRp from those of the N pocket from DENV RdRp that are crucial for the design of improved antiviral inhibitors with activity against ZIKV.IMPORTANCE Zika virus belongs to the Flavivirus genus, which comprises several important human pathogens. There is currently neither an approved vaccine nor antiviral drugs available to prevent infection by ZIKV. The nonstructural protein 5 (NS5) polymerase, which is responsible for replicating the viral RNA genome, represents one of the most promising targets for antiviral drug development. Starting from compounds recently developed against dengue virus NS5, we designed and synthesized inhibitors targeting Zika virus NS5. We show that these novel compounds inhibit viral replication by targeting the polymerase activity. High-resolution X-ray crystallographic structures of protein-inhibitor complexes demonstrated specific binding to an allosteric site within the polymerase, called the N pocket. This work paves the way for the future structure-based design of potent compounds specifically targeting ZIKV RNA polymerase activity.

Anti-inflammatory potential of simvastatin loaded nanoliposomes in 2D and 3D foam cell models.

Atherosclerosis is a multifactorial disease triggered and sustained by risk factors such as high cholesterol, high blood pressure and unhealthy lifestyle. Inflammation plays a pivotal role in atherosclerosis pathogenesis. In this study, we developed a simvastatin (STAT) loaded nanoliposomal formulation (LIPOSTAT) which can deliver the drug into atherosclerotic plaque, when administered intravenously. This formulation is easily prepared, stable, and biocompatible with minimal burst release for effective drug delivery. 2D and 3D in vitro models were examined towards anti-inflammatory effects of STAT, both free and in combination with liposomes. LIPOSTAT induced greater cholesterol efflux in the 2D foam cells and significantly reduced inflammation in both 2D and 3D models. LIPOSTAT alleviated inflammation by reducing the secretion of early and late phase pro-inflammatory cytokines, monocyte adherence marker, and lipid accumulation cytokines. Additionally, the 3D foam cell spheroid model is a convenient and practical approach in testing various anti-atherosclerotic drugs without the need for human tissue.

Pyruvate kinase from Plasmodium falciparum: Structural and kinetic insights into the allosteric mechanism.

During its intra-erythrocytic growth phase, the malaria parasite Plasmodium falciparum relies heavily on glycolysis for its energy requirements. Pyruvate kinase (PYK) is essential for regulating glycolytic flux and for ATP production, yet the allosteric mechanism of P. falciparum PYK (PfPYK) remains poorly understood. Here we report the first crystal structure of PfPYK in complex with substrate analogues oxalate and the ATP product. Comparisons of PfPYK structures in the active R-state and inactive T-state reveal a 'rock-and-lock' allosteric mechanism regulated by rigid-body rotations of each subunit in the tetramer. Kinetic data and structural analysis indicate glucose 6-phosphate is an activator by increasing the apparent maximal velocity of the enzyme. Intriguingly, the trypanosome drug suramin inhibits PfPYK, which points to glycolysis as a set of potential therapeutic targets against malaria.

Bilirubin analogues as model compounds for exciton coupling.

A series of phycobilin analogues have been investigated in terms of coupled excitonic systems. These compounds consist of a monomer, a tetrapyrrole structurally similar to bilirubin (bR), and two conjugated bR analogues. Spectroscopic and computational methods have been used to investigate the degree of interchromophore coupling. We find the synthesised bR analogue shows stronger excitonic coupling than bR, owing to a different molecular geometry. The excitonic coupling in the conjugated molecules can be controlled by modifying the bridge side-group. New computed energy levels for bR using the DFT/MRCI method are also presented, which improve on published values and re-assign the character of excited singlet states.

Impact of Patient and Model of Care Factors on Titration and Tolerability of Sacubitril/Valsartan: An Early Australian Real-World Experience.

BACKGROUND: Sacubitril/valsartan was shown to be superior to enalapril in the Prospective Comparison of angiotensin receptor neprilysin inhibitor with an angiotensin converting enzyme inhibitor to Determine Impact on Global Mortality and Morbidity in Heart Failure (PARADIGM-HF) study. However, the study design raised uncertainty about the potential real-world tolerability amongst less well selected cohorts. We aimed to examine the real-world tolerability and factors associated with successful titration of sacubitril/valsartan. METHODS: We performed a retrospective single centre analysis in a tertiary referral centre of 235 consecutive patients prescribed sacubitril/valsartan between August 2016 and January 2018. RESULTS: At baseline, our patients were younger, had lower baseline systolic blood pressure (SBP), reduced ischaemic aetiology and a higher rate of mineralocorticoids receptor antagonist compared to PARADIGM-HF. At last assessment, 120 patients (51%) reached target dose (97/103 mg bi-daily [BD]), 67 patients (29%) were stable on a mid-range dose (≥49/51 mg BD), 22 patients (9%) tolerated the low dose (24/26 mg BD) and 26 patients (11%) discontinued, comparable to PARADIGM-HF. Adverse effects were similar to PARADIGM-HF and hypotension remained the primary reason of sub-maximal titration. Several baseline characteristics were associated with successful titration to target dose including; higher baseline body mass index, systolic blood pressure (SBP) and sodium, male gender and treatment coordinated by multidisciplinary heart failure (HF) clinic. CONCLUSION: Comparable results to PARADIGM-HF in attaining target dose of sacubitril/valsartan and tolerability profile can be achieved in a real-world setting. Several baseline characteristics involving patient factors, markers of disease severity and systems of care predict successful titration to the target dose 97/103 mg BD.