Associations between antiretroviral regimen and changes in blood pressure: results from the D2EFT study.

In this randomised, controlled study in 14 low- and middle-income countries, individuals taking dolutegravir with darunavir/ritonavir for 48 weeks had a greater increase in systolic and diastolic blood pressure than individuals taking two nucleoside reverse transcriptase with darunavir/ritonavir. The difference remained significant after controlling for confounding factors including weight gain.

Evaluation of Simplified HCV Diagnostics in HIV/HCV Co-Infected Patients in Myanmar.

To evaluate a decentralised testing model and simplified treatment protocol of hepatitis C virus (HCV) infection to facilitate treatment scale-up in Myanmar, this prospective, observational study recruited HIV-HCV co-infected outpatients receiving sofosbuvir/daclatasvir in Yangon, Myanmar. The study examined the outcomes and factors associated with a sustained virological response (SVR). A decentralised "hub-and-spoke" testing model was evaluated where fingerstick capillary specimens were transported by taxi and processed centrally. The performance of the Xpert HCV VL Fingerstick Assay in detecting HCV RNA was compared to the local standard of care ( plasma HCV RNA collected by venepuncture). Between January 2019 and February 2020, 162 HCV RNA-positive individuals were identified; 154/162 (95%) initiated treatment, and 128/154 (84%) returned for their SVR12 visit. A SVR was achieved in 119/154 (77%) participants in the intent-to-treat population and 119/128 (93%) participants in the modified-intent-to-treat population. Individuals receiving an antiretroviral therapy were more likely to achieve a SVR (with an odds ratio (OR) of 7.16, 95% CI 1.03-49.50), while those with cirrhosis were less likely (OR: 0.26, 95% CI 0.07-0.88). The sensitivity of the Xpert HCV VL Fingerstick Assay was 99.4% (95% CI 96.7-100.0), and the specificity was 99.2% (95% CI 95.9-99.9). A simplified treatment protocol using a hub-and-spoke testing model of fingerstick capillary specimens can achieve an SVR rate in LMIC comparable to well-resourced high-income settings.

Quantifying Micromolar Crystallinity in Pharmaceutical Materials Utilizing 19F Solid-State NMR.

Quantifying low-level components in solid-state analysis presents a significant challenge for most thermal, diffractometric, vibrational, and spectroscopic techniques. In pharmaceutical analysis, identifying and quantifying the physical form of the drug substance in solid dosages is a critical task to ensure the quality of drug products. For example, recrystallization of active pharmaceutical ingredients in amorphous solid dispersions can compromise the stability and bioavailability of drug products. Herein, we have developed and demonstrated fluorine-19 solid-state nuclear magnetic resonance (19F ssNMR) methods and pushed the boundary to quantify minor crystalline contents in amorphous pharmaceuticals. Calibration curves suggest that 19F direct polarization and 1H-19F cross-polarization ssNMR can readily quantify 0.1% w/w crystalline compound I, a commercial fluorinated drug molecule developed by Merck & Co., Inc., Rahway, NJ, U.S.A., in its amorphous formulation. 1H-19F multiple cross-polarization (MultiCP) has been implemented, for the first time, and compared with conventional cross-polarization methods. Most importantly, a relaxation-filtered 19F ssNMR method was utilized to unambiguously identify and quantify as low as 0.04% w/w crystalline components, that is, 6 µmol in a 100 mg tablet at 25% drug loading, by suppressing the signal from the amorphous counterpart. Such a low level of detection offers high confidence and sensitivity to quantify trace amounts of phase change in pharmaceutical amorphous materials in the solid state, which can facilitate formulation development as well as quality control.

Golgi-localized putative S-adenosyl methionine transporters required for plant cell wall polysaccharide methylation.

Polysaccharide methylation, especially that of pectin, is a common and important feature of land plant cell walls. Polysaccharide methylation takes place in the Golgi apparatus and therefore relies on the import of S-adenosyl methionine (SAM) from the cytosol into the Golgi. However, so far, no Golgi SAM transporter has been identified in plants. Here we studied major facilitator superfamily members in Arabidopsis that we identified as putative Golgi SAM transporters (GoSAMTs). Knockout of the two most highly expressed GoSAMTs led to a strong reduction in Golgi-synthesized polysaccharide methylation. Furthermore, solid-state NMR experiments revealed that reduced methylation changed cell wall polysaccharide conformations, interactions and mobilities. Notably, NMR revealed the existence of pectin 'egg-box' structures in intact cell walls and showed that their formation is enhanced by reduced methyl esterification. These changes in wall architecture were linked to substantial growth and developmental phenotypes. In particular, anisotropic growth was strongly impaired in the double mutant. The identification of putative transporters involved in import of SAM into the Golgi lumen in plants provides new insights into the paramount importance of polysaccharide methylation for plant cell wall structure and function.

The impact of gender and the social determinants of health on the clinical course of people living with HIV in Myanmar: an observational study.

BACKGROUND: There is a growing recognition of the impact of gender and the social determinants of health on the clinical course of people living with HIV (PLHIV). However, the relative contribution of these factors to clinical outcomes of PLHIV is incompletely defined in many countries. This study was performed to gain a greater understanding of the non-clinical determinants of prognosis of PLHIV in Myanmar. METHODS: Selected demographic, behavioural and socioeconomic characteristics of outpatients at two specialist HIV hospitals and one general hospital in Yangon, Myanmar were correlated with their subsequent clinical course; a poor outcome was defined as death, hospitalisation, loss to follow-up or a detectable viral load at 6 months of follow-up. RESULTS: 221 consecutive individuals with advanced HIV commencing anti-retroviral therapy (ART) were enrolled in the study; their median CD4 T-cell count was 92 (44-158) cells/mm3, 138 (62.4%) were male. Socioeconomic disadvantage was common: the median (interquartile range (IQR) monthly per-capita income in the cohort was US$48 (31-77); 153 (69.9%) had not completed high school. However, in a multivariate analysis that considered demographic, behavioural, clinical factors and social determinants of health, male gender was the only predictor of a poor outcome: odds ratio (95% confidence interval): 2.33 (1.26-4.32, p = 0.007). All eight of the deaths and hospitalisations in the cohort occurred in males (p = 0.03). CONCLUSIONS: Men starting ART in Myanmar have a poorer prognosis than women. Expanded implementation of gender-specific management strategies is likely to be necessary to improve outcomes.

Xylan Structure and Dynamics in Native Brachypodium Grass Cell Walls Investigated by Solid-State NMR Spectroscopy.

The polysaccharide composition and dynamics of the intact stem and leaf cell walls of the model grass Brachypodium distachyon are investigated to understand how developmental stage affects the polysaccharide structure of grass cell walls. 13C enrichment of the entire plant allowed detailed analysis of the xylan structure, side-chain functionalization, dynamics, and interaction with cellulose using magic-angle-spinning solid-state NMR spectroscopy. Quantitative one-dimensional 13C NMR spectra and two-dimensional 13C-13C correlation spectra indicate that stem and leaf cell walls contain less pectic polysaccharides compared to previously studied seedling primary cell walls. Between the stem and the leaf, the secondary cell wall-rich stem contains more xylan and more cellulose compared to the leaf. Moreover, the xylan chains are about twofold more acetylated and about 60% more ferulated in the stem. These highly acetylated and ferulated xylan chains adopt a twofold conformation more prevalently and interact more extensively with cellulose. These results support the notion that acetylated xylan is found more in the twofold screw conformation, which preferentially binds cellulose. This in turn promotes cellulose-lignin interactions that are essential for the formation of the secondary cell wall.

Understanding molecular mechanisms of biologics drug delivery and stability from NMR spectroscopy.

Protein therapeutics carry inherent limitations of membrane impermeability and structural instability, despite their predominant role in the modern pharmaceutical market. Effective formulations are needed to overcome physiological and physicochemical barriers, respectively, for improving bioavailability and stability. Knowledge of membrane affinity, cellular internalization, encapsulation, and release of drug-loaded carrier vehicles uncover the structural basis for designing and optimizing biopharmaceuticals with enhanced delivery efficiency and therapeutic efficacy. Understanding stabilizing and destabilizing interactions between protein drugs and formulation excipients provide fundamental mechanisms for ensuring the stability and quality of biological products. This article reviews the molecular studies of biologics using solution and solid-state NMR spectroscopy on structural attributes pivotal to drug delivery and stability. In-depth investigation of the structure-function relationship of drug delivery systems based on cell-penetrating peptides, lipid nanoparticles and polymeric colloidal, and biophysical and biochemical stability of peptide, protein, monoclonal antibody, and vaccine, as the integrative efforts on drug product design, will be elaborated.

Time for pragmatic, prospective clinical trials to determine the role of empirical antibacterial therapy in critically ill adults hospitalized with malaria.

BACKGROUND: Children with severe falciparum malaria in malaria-endemic regions are predisposed to developing life-threatening bacterial co-infection. International guidelines therefore recommend empirical broad-spectrum antibacterial therapy in these children. Few studies have examined co-infection in adults, although it has been believed to be relatively rare; antibacterial therapy is therefore not routinely recommended in adults with falciparum malaria. DISCUSSION: However, the fundamental pathophysiology of falciparum malaria in adults and children is the same; it is therefore unclear why adults would not also be predisposed to bacterial infection. Indeed, recent studies have identified bacteraemia in >10% of adults hospitalized with malaria. Some have suggested that these adults probably had bacterial sepsis, with the parasitaemia an incidental finding. However, it is usually impossible in resource-limited settings to determine-at presentation-whether critically ill, parasitaemic adults have severe malaria, bacterial sepsis, or both. Given the significant case-fatality rates of severe malaria and bacterial sepsis, the pragmatic initial approach would be to cover both possibilities. CONCLUSIONS: Life-threatening bacterial co-infection may be more common in critically ill adults with malaria than previously believed. While further prospective data are awaited to confirm these findings, it might be more appropriate to provide empirical aantibacterial cover in these patients than current guidelines suggest.

Fast MAS 1H-13C correlation NMR for structural investigations of plant cell walls.

Plant cell walls consist of a mixture of polysaccharides that render the cell wall a strong and dynamic material. Understanding the molecular structure and dynamics of wall polysaccharides is important for understanding and improving the properties of this energy-rich biomaterial. So far, solid-state NMR studies of cell wall structure and dynamics have solely relied on 13C chemical shifts measured from 2D and 3D correlation experiments. To increase the spectral resolution, sensitivity and upper limit of measurable distances, it is of interest to explore 1H chemical shifts and 1H-detected NMR experiments for analyzing cell walls. Here we demonstrate 2D and 3D 1H-13C correlation experiments at both moderate and fast MAS frequencies of 10-50 kHz to resolve and assign 1H chemical shifts of matrix polysaccharides in Arabidopsis primary cell walls. Both 13C-detected and 1H-detected experiments are implemented and are shown to provide useful and complementary information. Using the assigned 1H chemical shifts, we measured long-range correlations between matrix polysaccharides and cellulose using 1H-1H instead of 13C-13C spin diffusion, and the 2D experiments can be conducted with either 13C or 1H detection.

The molecular structure of plant sporopollenin.

Sporopollenin is a ubiquitous and extremely chemically inert biopolymer that constitutes the outer wall of all land-plant spores and pollen grains1. Sporopollenin protects the vulnerable plant gametes against a wide range of environmental assaults, and is considered a prerequisite for the migration of early plants onto land2. Despite its importance, the chemical structure of plant sporopollenin has remained elusive1. Using a newly developed thioacidolysis degradative method together with state-of-the-art solid-state NMR techniques, we determined the detailed molecular structure of pine sporopollenin. We show that pine sporopollenin is primarily composed of aliphatic-polyketide-derived polyvinyl alcohol units and 7-O-p-coumaroylated C16 aliphatic units, crosslinked through a distinctive dioxane moiety featuring an acetal. Naringenin was also identified as a minor component of pine sporopollenin. This discovery answers the long-standing question about the chemical make-up of plant sporopollenin, laying the foundation for future investigations of sporopollenin biosynthesis and for the design of new biomimetic polymers with desirable inert properties.

Antibiotic Therapy in Adults with Malaria (ANTHEM): High Rate of Clinically Significant Bacteremia in Hospitalized Adults Diagnosed with Falciparum Malaria.

It has been believed that concomitant bacteremia is uncommon in adults hospitalized with falciparum malaria. Accordingly, the World Health Organization treatment guidelines presently only recommended additional antibacterial therapy in these patients if they have a clinical syndrome compatible with serious bacterial infection. Admission blood cultures were collected from 20 consecutive adults in Myanmar, hospitalized with a positive immunochromatographic test and blood film, suggesting a diagnosis of falciparum malaria; four (20%) had bacteremia with a clinically significant pathogen. These case series' data were pooled with a previously published multicenter study from Myanmar which had also collected blood cultures in adults hospitalized with a diagnosis of falciparum malaria. Among 87 patients in the two studies, 13 (15%) had clinically significant bacteremia on admission, with Gram-negative organisms in 10 (77%) and Staphylococcus aureus in the remaining three (23%). Bacteremic patients had more severe disease than non-bacteremic patients (median [interquartile range] respiratory coma acidosis malaria score 2 [1-4] versus 1 [1-2], P = 0.02) and were more likely to die (2/13 [15%] versus 1/74 [1%], P = 0.01). However, bacterial coinfection was suspected clinically in a minority of bacteremic patients (5/13 [38%] compared with 13/70 [19%] of non-bacteremic patients, P = 0.11). Concomitant bacteremia in adults diagnosed with falciparum malaria may be more common than previously believed and is difficult to identify clinically in resource-poor settings. Death is more common in these patients, suggesting that clinicians should have a lower threshold for commencing empirical antibacterial therapy in adults diagnosed with falciparum malaria in these locations than is presently recommended.

Direct Determination of Hydroxymethyl Conformations of Plant Cell Wall Cellulose Using 1H Polarization Transfer Solid-State NMR.

In contrast to the well-studied crystalline cellulose of microbial and animal origins, cellulose in plant cell walls is disordered due to its interactions with matrix polysaccharides. Plant cell wall (PCW) is an undisputed source of sustainable global energy; therefore, it is important to determine the molecular structure of PCW cellulose. The most reactive component of cellulose is the exocyclic hydroxymethyl group: when it adopts the tg conformation, it stabilizes intrachain and interchain hydrogen bonding, while gt and gg conformations destabilize the hydrogen-bonding network. So far, information about the hydroxymethyl conformation in cellulose has been exclusively obtained from 13C chemical shifts of monosaccharides and oligosaccharides, which do not reflect the environment of cellulose in plant cell walls. Here, we use solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy to measure the hydroxymethyl torsion angle of cellulose in two model plants, by detecting distance-dependent polarization transfer between H4 and H6 protons in 2D 13C-13C correlation spectra. We show that the interior crystalline portion of cellulose microfibrils in Brachypodium and Arabidopsis cell walls exhibits H4-H6 polarization transfer curves that are indicative of a tg conformation, whereas surface cellulose chains exhibit slower H4-H6 polarization transfer that is best fit to the gt conformation. Joint constraints by the H4-H6 polarization transfer curves and 13C chemical shifts indicate that it is unlikely for interior cellulose to have a significant population of the gt and gg conformation mixed with the tg conformation, while surface cellulose may adopt a small percentage of the gg conformation. These results provide new constraints to the structure and matrix interactions of cellulose in plant cell walls, and represent the first direct determination of a torsion angle in an important noncrystalline carbohydrate polymer.

Gradients in Wall Mechanics and Polysaccharides along Growing Inflorescence Stems.

At early stages of Arabidopsis (Arabidopsis thaliana) flowering, the inflorescence stem undergoes rapid growth, with elongation occurring predominantly in the apical ∼4 cm of the stem. We measured the spatial gradients for elongation rate, osmotic pressure, cell wall thickness, and wall mechanical compliances and coupled these macroscopic measurements with molecular-level characterization of the polysaccharide composition, mobility, hydration, and intermolecular interactions of the inflorescence cell wall using solid-state nuclear magnetic resonance spectroscopy and small-angle neutron scattering. Force-extension curves revealed a gradient, from high to low, in the plastic and elastic compliances of cell walls along the elongation zone, but plots of growth rate versus wall compliances were strikingly nonlinear. Neutron-scattering curves showed only subtle changes in wall structure, including a slight increase in cellulose microfibril alignment along the growing stem. In contrast, solid-state nuclear magnetic resonance spectra showed substantial decreases in pectin amount, esterification, branching, hydration, and mobility in an apical-to-basal pattern, while the cellulose content increased modestly. These results suggest that pectin structural changes are connected with increases in pectin-cellulose interaction and reductions in wall compliances along the apical-to-basal gradient in growth rate. These pectin structural changes may lessen the ability of the cell wall to undergo stress relaxation and irreversible expansion (e.g. induced by expansins), thus contributing to the growth kinematics of the growing stem.

Effects of Pectin Molecular Weight Changes on the Structure, Dynamics, and Polysaccharide Interactions of Primary Cell Walls of Arabidopsis thaliana: Insights from Solid-State NMR.

Significant cellulose-pectin interactions in plant cell walls have been reported recently based on 2D 13C solid-state NMR spectra of intact cell walls, but how these interactions affect cell growth has not been probed. Here, we characterize two Arabidopsis thaliana lines with altered expression of the POLYGALACTURONASE INVOLVED IN EXPANSION1 (PGX1) gene, which encodes a polygalacturonase that cleaves homogalacturonan (HG). PGX1AT plants overexpress PGX1, have HG with lower molecular weight, and grow larger, whereas pgx1-2 knockout plants have HG with higher molecular weight and grow smaller. Quantitative 13C solid-state NMR spectra show that PGX1AT cell walls have lower galacturonic acid and xylose contents and higher HG methyl esterification than controls, whereas high molecular weight pgx1-2 walls have similar galacturonic acid content and methyl esterification as controls. 1H-transferred 13C INEPT spectra indicate that the interfibrillar HG backbones are more aggregated whereas the RG-I side chains are more dispersed in PGX1AT cell walls than in pgx1-2 walls. In contrast, the pectins that are close to cellulose become more mobile and have weaker cross peaks with cellulose in PGX1AT walls than in pgx1-2 walls. Together, these results show that polygalacturonase-mediated plant growth is accompanied by increased esterification and decreased cross-linking of HG, increased aggregation of interfibrillar HG, and weaker HG-cellulose interactions. These structural and dynamical differences give molecular insights into how pectins influence wall dynamics during cell growth.

Multidimensional solid-state NMR spectroscopy of plant cell walls.

Plant biomass has become an important source of bio-renewable energy in modern society. The molecular structure of plant cell walls is difficult to characterize by most atomic-resolution techniques due to the insoluble and disordered nature of the cell wall. Solid-state NMR (SSNMR) spectroscopy is uniquely suited for studying native hydrated plant cell walls at the molecular level with chemical resolution. Significant progress has been made in the last five years to elucidate the molecular structures and interactions of cellulose and matrix polysaccharides in plant cell walls. These studies have focused on primary cell walls of growing plants in both the dicotyledonous and grass families, as represented by the model plants Arabidopsis thaliana, Brachypodium distachyon, and Zea mays. To date, these SSNMR results have shown that 1) cellulose, hemicellulose, and pectins form a single network in the primary cell wall; 2) in dicot cell walls, the protein expansin targets the hemicellulose-enriched region of the cellulose microfibril for its wall-loosening function; and 3) primary wall cellulose has polymorphic structures that are distinct from the microbial cellulose structures. This article summarizes these key findings, and points out future directions of investigation to advance our fundamental understanding of plant cell wall structure and function.

Modifier cation effects on (29)Si nuclear shielding anisotropies in silicate glasses.

We have examined variations in the (29)Si nuclear shielding tensor parameters of SiO4 tetrahedra in a series of seven alkali and alkaline earth silicate glass compositions, Cs2O·4.81 SiO2, Rb2O·3.96 SiO2, Rb2O·2.25 SiO2, K2O·4.48 SiO2, Na2O·4.74 SiO2, BaO·2.64 SiO2, and SrO·2.36 SiO2, using natural abundance (29)Si two-dimensional magic-angle flipping (MAF) experiments. Our analyses of these 2D spectra reveal a linear dependence of the (29)Si nuclear shielding anisotropy of Q((3)) sites on the Si-non-bridging oxygen bond length, which in turn depends on the cation potential and coordination of modifier cations to the non-bridging oxygen. We also demonstrate how a combination of Cu(2+) as a paramagnetic dopant combined with echo train acquisition can reduce the total experiment time of (29)Si 2D NMR measurements by two orders of magnitude, enabling higher throughput 2D NMR studies of glass structure.

High Frequency of Clinically Significant Bacteremia in Adults Hospitalized With Falciparum Malaria.

Background. African children with severe falciparum malaria commonly have concomitant Gram-negative bacteremia, but co-infection has been thought to be relatively rare in adult malaria. Methods. Adults with a diagnosis of falciparum malaria hospitalized at 4 tertiary referral hospitals in Myanmar had blood cultures collected at admission. The frequency of concomitant bacteremia and the clinical characteristics of the patients, with and without bacteremia, were explored. Results. Of 67 adults hospitalized with falciparum malaria, 9 (13% [95% confidence interval, 5.3%-21.6%]) were also bacteremic on admission, 7 (78%) with Gram-negative enteric organisms (Escherichia coli [n = 3], typhoidal Salmonella species [n = 3], nontyphoidal Salmonella [n = 1]). Bacteremic adults had more severe disease (median Respiratory Coma Acidosis Malaria [RCAM] score 3; interquartile range [IQR], 1-4) than those without bacteremia (median RCAM score 1; IQR, 1-2) and had a higher frequency of acute kidney injury (50% vs 16%, P = .03). Although 35 (52%) were at high risk of death (RCAM score ≥2), all 67 patients in the study survived, 51 (76%) of whom received empirical antibiotics on admission. Conclusions. Bacteremia was relatively frequent in adults hospitalized with falciparum malaria in Myanmar. Like children in high transmission settings, bacteremic adults in this low transmission setting were sicker than nonbacteremic adults, and were often difficult to identify at presentation. Empirical antibiotics may also be appropriate in adults hospitalized with falciparum malaria in low transmission settings, until bacterial infection is excluded.