Binding of Sulfamethazine to β-cyclodextrin and Methyl-β-cyclodextrin

β-cyclodextrin (βCD) and methyl-β-cyclodextrin (MβCD) complexes with sulfamethazine (SMT) were prepared and characterized by different experimental techniques, and the effects of βCD and MβCD on drug solubility were assessed via phase-solubility analysis. The phase-solubility diagram for the drug showed an increase in water solubility, with the following affinity constants calculated: 40.4 ± 0.4 (pH 2.0) and 29.4 ± 0.4 (pH 8.0) M⁻¹ with βCD and 56 ± 1 (water), 39 ± 3 (pH 2.0) and 39 ± 5 (pH 8.0) M⁻¹ with MβCD. According to ¹H NMR and 2D NMR spectroscopy, the complexation mode involved the aromatic ring of SMT included in the MβCD cavity. The complexes obtained in solid state by freeze drying were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, and thermal analysis. The amorphous complexes obtained in this study may be useful in the preparation of pharmaceutical dosage forms of SMT.     

Enhancement of bound-state residual dipolar couplings: conformational analysis of lactose bound to Galectin-3

Residual dipolar couplings (RDCs) have proven to be a valuable NMR tool that can provide long-range constraints for molecular structure determination. The constraints are orientational in nature and are, thus, highly complementary to conventional distance constraints from NOE data. This complementarity would seem to extend to the study of the geometry of ligands bound to proteins. However, unlike transferred NOEs, where collection, even with a large excess of free ligand, results in measurements dominated by bound contributions, RDCs of exchanging ligands can be dominated by free-state contributions. Here we present a strategy for enhancement of RDCs from bound states that is based on specifically enhancing the alignment of the protein to which a ligand will bind. The protein is modified by addition of a hydrophobic alkyl tail that anchors it to the bicelles that are a part of the ordering medium needed for RDC measurement. As an illustration, we have added a propyl chain to the C terminus of the carbohydrate recognition domain of the protein, Galectin-3, and report enhanced RDCs that prove consistent with known bound-ligand geometries for this protein.     

Rapid measurement of residual dipolar couplings for fast fold elucidation of proteins

It has been demonstrated that protein folds can be determined using appropriate computational protocols with NMR chemical shifts as the sole source of experimental restraints. While such approaches are very promising they still suffer from low convergence resulting in long computation times to achieve accurate results. Here we present a suite of time- and sensitivity optimized NMR experiments for rapid measurement of up to six RDCs per residue. Including such an RDC data set, measured in less than 24 h on a single aligned protein sample, greatly improves convergence of the Rosetta-NMR protocol, allowing for overnight fold calculation of small proteins. We demonstrate the performance of our fast fold calculation approach for ubiquitin as a test case, and for two RNA-binding domains of the plant protein HYL1. Structure calculations based on simulated RDC data highlight the importance of an accurate and precise set of several complementary RDCs as additional input restraints for high-quality de novo structure determination.     

Niosomal Gel of Lornoxicam for Topical Delivery: In vitro Assessment and Pharmacodynamic Activity

Lornoxicam is a potent oxicam class of non steroidal anti-inflammatory agent, prescribed for mild to moderate pain and inflammation. Niosomal gel of lornoxicam was developed for topical application. Lornoxicam niosomes (Lor-Nio) were fabricated by thin film hydration technique. Bilayer composition of niosomal vesicles was optimized. Lor-Nio dispersion was characterized by DSC, XRD, and FT-IR. Morphological evaluation was performed by scanning electron microscopy (SEM). Lor-Nio dispersion was incorporated into a gel using 2% w/w Carbopol 980 NF. Rheological and texture properties of Lor-Nio gel formulation showed suitability of the gel for topical application. The developed formulation was evaluated for in vitro skin permeation and skin deposition studies, occlusivity test and skin irritation studies. Pharmacodynamic activity of the Lor-Nio gel was performed by carragenan-induced rat paw model. Optimized Lor-Nio comprised of Span 60 and cholesterol in a molar ratio of 3:1 with 30 μM dicetyl palmitate as a stabilizer. It had particle size of 1.125 ± 0.212 μm (d₉₀), with entrapment efficiency of 52.38 ± 2.1%. DSC, XRD, and IR studies showed inclusion of Lor into niosomal vesicles. SEM studies showed spherical closed vesicular structure with particles in nanometer range. The in vitro skin permeation studies showed significant improvement in skin permeation and skin deposition for Lor-Nio gel (31.41 ± 2.24 μg/cm², 30.079 ± 1.2 μg/cm²) over plain lornoxicam gel (7.37 ± 1.27 μg/cm², 6.6 ± 2.52 μg/cm²). The Lor-Nio gel formulation showed enhanced anti-inflammatory activity by exhibiting mean edema inhibition (87.69 ± 1.43%) which was significantly more than the plain lornoxicam gel (53.84 ± 2.21%).KEY WORDS: anti-inflammatory activity, lornoxicam, niosomes, rheology, texture analysis     

Pressure‐induced structural transition of mature HIV‐1 protease from a combined NMR/MD simulation approach

We investigate the pressure‐induced structural changes in the mature human immunodeficiency virus type 1 protease dimer, using residual dipolar coupling (RDC) measurements in a weakly oriented solution. ¹D_NH RDCs were measured under high‐pressure conditions for an inhibitor‐free PR and an inhibitor‐bound complex, as well as for an inhibitor‐free multidrug resistant protease bearing 20 mutations (PR20). While PR20 and the inhibitor‐bound PR were little affected by pressure, inhibitor‐free PR showed significant differences in the RDCs measured at 600 bar compared with 1 bar. The structural basis of such changes was investigated by MD simulations using the experimental RDC restraints, revealing substantial conformational perturbations, specifically a partial opening of the flaps and the penetration of water molecules into the hydrophobic core of the subunits at high pressure. This study highlights the exquisite sensitivity of RDCs to pressure‐induced conformational changes and illustrates how RDCs combined with MD simulations can be used to determine the structural properties of metastable intermediate states on the folding energy landscape. Proteins 2015; 83:2117–2123. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.     

Effects of the binding of calcium ions on the structure and dynamics of the ΦX174 virus investigated using molecular dynamics

It is known that the presence of calcium ions (Ca^2 +) is necessary for the enterobacterial virus ΦX174 to inject its DNA into the host cell, and that some mutations in the major capsid proteins lead to better survivability at higher temperatures. Our goal in the current study is to determine the physical changes in both the wild-type and mutant virus due to the binding of Ca^2 +. Thus, we performed molecular dynamics simulations of the ΦX174 major capsid protein complex with and without Ca^2 + bound. Our results show that binding of Ca^2 + leads to energetic and dynamical changes in the virus proteins. In particular, the results suggest that binding of Ca^2 + is energetically favorable and that the mutation leads to increased fluctuations of the protein complex (especially with the calcium ions bound to the complex), which may increase the rate of genome packaging and ejection for ΦX174.     

Identification and analysis of hepatitis C virus NS3 helicase inhibitors using nucleic acid binding assays

Typical assays used to discover and analyze small molecules that inhibit the hepatitis C virus (HCV) NS3 helicase yield few hits and are often confounded by compound interference. Oligonucleotide binding assays are examined here as an alternative. After comparing fluorescence polarization (FP), homogeneous time-resolved fluorescence (HTRF®; Cisbio) and AlphaScreen® (Perkin Elmer) assays, an FP-based assay was chosen to screen Sigma’s Library of Pharmacologically Active Compounds (LOPAC) for compounds that inhibit NS3-DNA complex formation. Four LOPAC compounds inhibited the FP-based assay: aurintricarboxylic acid (ATA) (IC₅₀ = 1.4 μM), suramin sodium salt (IC₅₀ = 3.6 μM), NF 023 hydrate (IC₅₀ = 6.2 μM) and tyrphostin AG 538 (IC₅₀ = 3.6 μM). All but AG 538 inhibited helicase-catalyzed strand separation, and all but NF 023 inhibited replication of subgenomic HCV replicons. A counterscreen using Escherichia coli single-stranded DNA binding protein (SSB) revealed that none of the new HCV helicase inhibitors were specific for NS3h. However, when the SSB-based assay was used to analyze derivatives of another non-specific helicase inhibitor, the main component of the dye primuline, it revealed that some primuline derivatives (e.g. PubChem CID50930730) are up to 30-fold more specific for HCV NS3h than similarly potent HCV helicase inhibitors.     

Defining the Critical Material Attributes of Lactose Monohydrate in Carrier Based Dry Powder Inhaler Formulations Using Artificial Neural Networks

The study aimed to establish a function-based relationship between the physical and bulk properties of pre-blended mixtures of fine and coarse lactose grades with the in vitro performance of an adhesive active pharmaceutical ingredient (API). Different grades of micronised and milled lactose (Lactohale (LH) LH300, LH230, LH210 and Sorbolac 400) were pre-blended with coarse grades of lactose (LH100, LH206 and Respitose SV010) at concentrations of 2.5, 5, 10 and 20 wt.%. The bulk and rheological properties and particle size distributions were characterised. The pre-blends were formulated with micronised budesonide and in vitro performance in a Cyclohaler device tested using a next-generation impactor (NGI) at 90 l/min. Correlations between the lactose properties and in vitro performance were established using linear regression and artificial neural network (ANN) analyses. The addition of milled and micronised lactose fines with the coarse lactose had a significant influence on physical and rheological properties of the bulk lactose. Formulations of the different pre-blends with budesonide directly influenced in vitro performance attributes including fine particle fraction, mass median aerodynamic diameter and pre-separator deposition. While linear regression suggested a number of physical and bulk properties may influence in vitro performance, ANN analysis suggested the critical parameters in describing in vitro deposition patterns were the relative concentrations of lactose fines % < 4.5 μm and % < 15 μm. These data suggest that, for an adhesive API, the proportion of fine particles below % < 4.5 μm and % < 15 μm could be used in rational dry powder inhaler formulation design.KEY WORDS: critical material attributes, cohesive-adhesive balance, dry powder inhaler, lactose, quality-by-design     

NMR and small-angle scattering-based structural analysis of protein complexes in solution

Structural analysis of multi-domain protein complexes is a key challenge in current biology and a prerequisite for understanding the molecular basis of essential cellular processes. The use of solution techniques is important for characterizing the quaternary arrangements and dynamics of domains and subunits of these complexes. In this respect solution NMR is the only technique that allows atomic- or residue-resolution structure determination and investigation of dynamic properties of multi-domain proteins and their complexes. As experimental NMR data for large protein complexes are sparse, it is advantageous to combine these data with additional information from other solution techniques. Here, the utility and computational approaches of combining solution state NMR with small-angle X-ray and Neutron scattering (SAXS/SANS) experiments for structural analysis of large protein complexes is reviewed. Recent progress in experimental and computational approaches of combining NMR and SAS are discussed and illustrated with recent examples from the literature. The complementary aspects of combining NMR and SAS data for studying multi-domain proteins, i.e. where weakly interacting domains are connected by flexible linkers, are illustrated with the structural analysis of the tandem RNA recognition motif (RRM) domains (RRM1-RRM2) of the human splicing factor U2AF65 bound to a nine-uridine (U9) RNA oligonucleotide.     

Role of the JNK/c-Jun/AP-1 signaling pathway in galectin-1-induced T-cell death

Galectin-1 (gal-1), an endogenous β-galactoside-binding protein, triggers T-cell death through several mechanisms including the death receptor and the mitochondrial apoptotic pathway. In this study we first show that gal-1 initiates the activation of c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase kinase 4 (MKK4), and MKK7 as upstream JNK activators in Jurkat T cells. Inhibition of JNK activation with sphingomyelinase inhibitors (20 μM desipramine, 20 μM imipramine), with the protein kinase C-δ (PKCδ) inhibitor rottlerin (10 μM), and with the specific PKCθ pseudosubstrate inhibitor (30 μM) indicates that ceramide and phosphorylation by PKCδ and PKCθ mediate gal-1-induced JNK activation. Downstream of JNK, we observed increased phosphorylation of c-Jun, enhanced activating protein-1 (AP-1) luciferase reporter, and AP-1/DNA-binding in response to gal-1. The pivotal role of the JNK/c-Jun/AP-1 pathway for gal-1-induced apoptosis was documented by reduction of DNA fragmentation after inhibition JNK by SP600125 (20 μM) or inhibition of AP-1 activation by curcumin (2 μM). Gal-1 failed to induce AP-1 activation and DNA fragmentation in CD3-deficient Jurkat 31-13 cells. In Jurkat E6.1 cells gal-1 induced a proapoptotic signal pattern as indicated by decreased antiapoptotic Bcl-2 expression, induction of proapoptotic Bad, and increased Bcl-2 phosphorylation. The results provide evidence that the JNK/c-Jun/AP-1 pathway plays a key role for T-cell death regulation in response to gal-1 stimulation.     

The Control of Skin-Permeating Rate of Bisoprolol by Ion-Pair Strategy for Long-Acting Transdermal Patches

A moderate drug permeating rate (flux) is desirable for long-acting transdermal patches. In this work, a novel simple method of controlling bisoprolol (BSP) flux by ion-pair strategy was initiated. Different ion-pair complexes including bisoprolol maleate (BSP-M), bisoprolol tartarate, bisoprolol besilate, and bisoprolol fumarate were prepared and their fluxes through rabbit abdominal skin were determined separately in vitro. Furthermore, permeation behavior from isopropyl myristate, solubility index in pressure-sensitive adhesives, determined by DSC, and n-octanol/water partition coefficient (log P) were investigated to illustrate the mechanism of drug permeation rate controlling. The results showed that compared to free BSP (J = 25.98 ± 2.34 μg/cm²/h), all BSP ion-pair complexes displayed lower and controllable flux in the range of 0.11 to 4.19 μg/cm²/h. After forming ion-pair complexes, the capability of BSP to penetrate through skin was weakened due to the lowered log P and increased molecule weight. Accordingly, this study has demonstrated that the flux of BSP could be controlled by ion-pair strategy, and among all complexes investigated, BSP-M was the most promising candidate for long-acting transdermal patches.Key words: bisoprolol, flux, ion-pair, transdermal     

Micropropagtion of Terminalia bellerica from nodal explants of mature tree and assessment of genetic fidelity using ISSR and RAPD markers

The present study reports an efficient in vitro micropropagation protocol for a medicinally important tree, Terminalia bellerica Roxb. from nodal segments of a 30 years old tree. Nodal segments taken from the mature tree in March-April and cultured on half strength MS medium gave the best shoot bud proliferation response. Combinations of serial transfer technique (ST) and incorporation of antioxidants (AO) [polyvinylpyrrolidone, PVP (50 mg l⁻¹) + ascorbic acid (100 mg l⁻¹) + citric acid (10 mg l⁻¹)] in the culture medium aided to minimize browning and improve explant survival during shoot bud induction. Highest multiplication of shoots was achieved on medium supplemented with 6-benzyladenine (BA, 8.8 μM) and α-naphthalene acetic acid (NAA, 2.6 μM) in addition to antioxidants. Shoot elongation was obtained on MS medium containing BA (4.4 μM) + phloroglucinol (PG, 3.9 μM). Elongated shoots were transferred to half strength MS medium containing indole-3-butyric acid (IBA, 2.5 μM) for root development. The acclimatization of plantlets was carried out under greenhouse conditions. The genetic fidelity of the regenerated plants was checked using inter simple sequence repeats (ISSR) and randomly amplified polymorphic DNA (RAPD) analysis. Comparison of the bands among the regenerants and mother plant confirmed true-to-type clonal plants.     

An efficient in vitro shoot regeneration from immature inflorescence and ex vitro rooting of Arnebia hispidissima (Lehm). DC. - A red dye (Alkannin) yielding plant

Arnebia hispidissima, which belongs to the family Boraginaceae, is an important medicinal and dye yielding plant. The alkannin, a red dye, are root-specific secondary metabolites of A. hispidissima. Shoots were regenerated from callus derived from immature inflorescence explants obtained from field grown plants. MS medium containing 4.52 μM 2, 4-D and 3.33 μM BAP was found to be most effective for the proliferation of callus, induced on medium containing 4.52 μM 2, 4-D. Maximum number (43.1 ± 0.25) with average length (5.2 ± 0.23) of shoots regenerated when callus was transferred to MS medium supplemented with 1.11 μM BAP, 1.16 μM Kin and 0.57 μM IAA. About 75.5 % of in vitro regenerated shoots were rooted on half-strength MS medium supplemented with 9.84 μM of IBA and 200 mg l⁻¹ of activated charcoal. In comparison to in vitro, higher percent (90.2 %) of shoots were rooted under ex vitro conditions when treated with IBA (0.98 mM) for 5 min. Plantlets rooted in vitro as well as ex vitro were acclimatized successfully under the green house conditions. Ex vitro rooted plants exhibited higher survival percentage (75 %) as compared to in vitro rooted plantlets (60 %). Present study may be applicable in the large-scale root-specific red dye (alkannin) production via root induction under ex vitro condition.     

Increased In Situ Intestinal Absorption of Phytoestrogenic Diarylheptanoids from Curcuma comosa in Nanoemulsions

Curcuma comosa has long been used as a gynecological medicine. Several diarylheptanoids have been purified from this plant, and their pharmacological effects were proven. However, there is no information about the absorption of C. comosa components to support the formulation usage. In the present study, C. comosa hexane extract and the mixture of its two major compounds, (4E,6E)-1,7-diphenylhepta-4,6-dien-3-ol (DA1) and (6E)-1,7-diphenylhept-6-en-3-ol (DA2), were formulated into nanoemulsions. The physical properties of the nanoemulsions and the in situ intestinal absorptions of DA1 and DA2 were evaluated. The results demonstrated the mean particle sizes at 0.207 ± 0.001 and 0.408 ± 0.014 μm, and the zeta potential at −14.57 ± 0.85 and −10.47 ± 0.32 mV for C. comosa nanoemulsion (C.c-Nano) and mixture of diarlylheptanoid nanoemulsions (DA-Nano), respectively. The entrapments of DA1 and DA2 were 76.61% and 75.41%, and 71.91% and 71.63% for C.c-Nano and DA-Nano, respectively. The drug loading ratios of DA1 and DA2 were 351.47 and 614.53 μg/mg, and 59.48 and 126.72 μg/mg for C.c-Nano and DA-Nano. The intestinal absorption rates of DA1 and DA2 were 0.329 ± 0.015 and 0.519 ± 0.026 μg/min/cm² in C.c-Nano, and 0.380 ± 0.006 and 0.428 ± 0.036 μg/min/cm² in DA-Nano, which were five to ten times faster than those in oil. In conclusion, the formulation in nanoemulsion forms obviously increased the intestinal absorption rate of diarylheptanoids.KEY WORDS: Curcuma comosa, diarylheptanoids, intestinal absorption, nanoemulsion, phytoestrogen     

Structural basis for norovirus inhibition and fucose mimicry by citrate

Human noroviruses bind with their capsid-protruding domains to histo-blood-group antigens (HBGAs), an interaction thought to direct their entry into cells. Although human noroviruses are the major cause of gastroenteritis outbreaks, development of antivirals has been lacking, mainly because human noroviruses cannot be cultivated. Here we use X-ray crystallography and saturation transfer difference nuclear magnetic resonance (STD NMR) to analyze the interaction of citrate with genogroup II (GII) noroviruses. Crystals of citrate in complex with the protruding domain from norovirus GII.10 Vietnam026 diffracted to 1.4 Å and showed a single citrate bound at the site of HBGA interaction. The citrate interaction was coordinated with a set of capsid interactions almost identical to that involved in recognizing the terminal HBGA fucose, the saccharide which forms the primary conserved interaction between HBGAs and GII noroviruses. Citrate and a water molecule formed a ring-like structure that mimicked the pyranoside ring of fucose. STD NMR showed the protruding domain to have weak affinity for citrate (460 μM). This affinity, however, was similar to the affinities of the protruding domain for fucose (460 μM) and H type 2 trisaccharide (390 μM), an HBGA shown previously to be specifically recognized by human noroviruses. Importantly, competition STD NMR showed that citrate could compete with HBGA for norovirus binding. Together, the results suggest that citrate and other glycomimetics have the potential to block human noroviruses from binding to HBGAs.     

Mapping protein receptor-ligand interactions via in vivo chemical crosslinking, affinity purification, and differential mass spectrometry

Protein receptor-ligand interactions play important roles in mediating enzyme catalysis, signal transduction, and other protein functions. Immunoaffinity purification followed by mass spectrometry analysis is a common method for identifying protein receptor-ligand complexes. However, it is difficult to distinguish between specific protein binding partners and non-specifically bound proteins that co-purify with the complex. In addition, weakly interacting binding partners may dissociate from the protein receptor-ligand complexes during immunoaffinity purification. The combination of chemical crosslinking, affinity purification, and differential mass spectrometry analysis provides a direct method for capturing stable, weak, and transient protein interactions that occur in vivo and in vitro. This approach enables the identification of functional receptor-ligand binding partners with high confidence. Herein, we describe a differential mass spectrometry approach coupled with in situ chemical crosslinking and immunoaffinity purification for identifying receptor-ligand binding partners. In particular, we identified a functional, counter-ligand structure of the natural killer cell p30-related protein.     

In vitro propagation and withaferin A production in Withania ashwagandha,a rare medicinal plant of India

Withania ashwagandha, belonging to the family Solanaceae, is an important medicinal herb of India with restricted geographic distribution. It is a rich source of withaferin A (WA) and other bioactive withanolides. In the present study a rapid in vitro mass propagation protocol of W. ashwagandha was developed from nodal explants. Nodal explants were cultured on MS medium supplemented with various concentrations and combinations of plant growth regulators (PGRs). The highest number of regenerated shoots per ex-plant (33 ± 2.7) and highest WA (13.4 ± 1.15 mg/g of DW) production was obtained on MS medium supplemented with 5.0 μM 6-benzyladenine (BA) and 1.0 μM Kinetin (Kn). In vitro raised shoots were further rooted on half-strength MS medium containing 2.0 μM Indole-3-butyric acid (IBA) and analyzed for WA production. The rooted plantlets when transferred to poly bags in the greenhouse showed 90 % survival frequency. Levels of WA were higher in the in vitro and ex vitro derived shoot and root tissues as compared to field grown mother plants. In an attempt to further maximize WA production, shoot cultures were further grown in liquid MS medium supplemented with 5.0 μM 6-benzyladenine (BA) and 1.0 μM Kinetin (Kn). Root cultures were grown on half strength MS liquid medium fortified with 2.0 μM of IBA. WA production in the liquid cultures was significantly higher compared to the static composition of the same media. This protocol, first of its kind in this plant, can be successfully employed for conservation, proliferation and large-scale production of WA. The regenerated plants can also be used in traditional medicine as an alternative to naturally collected plants.     

Double blind, cluster randomised trial of low dose supplementation with vitamin A or beta carotene on mortality related to pregnancy in Nepal. The NNIPS-2 Study Group

ObjectiveTo assess the impact on mortality related to pregnancy of supplementing women of reproductive age each week with a recommended dietary allowance of vitamin A, either preformed or as β carotene.DesignDouble blind, cluster randomised, placebo controlled field trial.SettingRural southeast central plains of Nepal (Sarlahi district).Subjects44 646 married women, of whom 20 119 became pregnant 22 189 times.Intervention270 wards randomised to 3 groups of 90 each for women to receive weekly a single oral supplement of placebo, vitamin A (7000 μg retinol equivalents) or β carotene (42 mg, or 7000 μg retinol equivalents) for over 3½ years.ResultsMortality related to pregnancy in the placebo, vitamin A, and β carotene groups was 704, 426, and 361 deaths per 100 000 pregnancies, yielding relative risks (95% confidence intervals) of 0.60 (0.37 to 0.97) and 0.51 (0.30 to 0.86). This represented reductions of 40% (P<0.04) and 49% (P<0.01) among those who received vitamin A and β carotene. Combined, vitamin A or β carotene lowered mortality by 44% (0.56 (0.37 to 0.84), P<0.005) and reduced the maternal mortality ratio from 645 to 385 deaths per 100 000 live births, or by 40% (P<0.02). Differences in cause of death could not be reliably distinguished between supplemented and placebo groups.ConclusionSupplementation of women with either vitamin A or β carotene at recommended dietary amounts during childbearing years can lower mortality related to pregnancy in rural, undernourished populations of south Asia.

Key messages

• Maternal vitamin A deficiency, evident as night blindness or low serum retinol concentration during pregnancy, is widely prevalent in rural south Asia
• In Nepal, women of reproductive age who were given 7000 μg retinol equivalents of vitamin A on a weekly basis showed a reduction in mortality related to pregnancy of 40%
• Weekly dosing with 42 mg β carotene (also providing 7000 μg retinol equivalents) lowered their mortality by 49%
• Preventing maternal vitamin A deficiency in rural South Asia can lower the risk of mortality of women during and after pregnancy