Modulation of NLRP3 inflammasomes activation contributes to improved survival and function of mesenchymal stromal cell spheroids.

Mesenchymal stem cells (MSCs) are ubiquitous multipotent cells that exhibit significant therapeutic potentials in a variety of disorders. Nevertheless, their clinical efficacy is limited owing to poor survival, low rate of engraftment, and impaired potency upon transplantation. Spheroidal three-dimensional (3D) culture of MSCs (MSC3D) has been proven to better preserve their in vivo functional properties. However, the molecular mechanisms underlying the improvement in MSC function by spheroid formation are not clearly understood. NLRP3 inflammasomes, a key component of the innate immune system, have recently been shown to play a role in cell fate decision of MSCs. The present study examined the role of NLRP3 inflammasomes in the survival and potency of MSC spheroids. We found that MSC3D led to decreased activation of NLRP3 inflammasomes through alleviation of ER stress in an autophagy-dependent manner. Importantly, downregulation of NLRP3 inflammasomes signaling critically contributes to the enhanced survival rate in MSC3D through modulation of pyroptosis and apoptosis. The critical role of NLRP3 inflammasome suppression in the enhanced therapeutic efficacy of MSC spheroids was further confirmed in an in vivo mouse model of DSS-induced colitis. These findings suggest that 3D culture confers survival and functional advantages to MSCs by suppressing NLRP3 inflammasome activation.

Mapping Single-Molecule Protein Complexes in 3D with DNA Nanoswitch Calipers.

The ability to accurately map the 3D geometry of single-molecule complexes in trace samples is a challenging goal that would lead to new insights into molecular mechanics and provide an approach for single-molecule structural proteomics. To enable this, we have developed a high-resolution force spectroscopy method capable of measuring multiple distances between labeled sites in natively folded protein complexes. Our approach combines reconfigurable nanoscale devices, we call DNA nanoswitch calipers, with a force-based barcoding system to distinguish each measurement location. We demonstrate our approach by reconstructing the tetrahedral geometry of biotin-binding sites in natively folded streptavidin, with 1.5-2.5 Å agreement with previously reported structures.

Decreased water activity in nanoconfinement contributes to the folding of G-quadruplex and i-motif structures.

Due to the small size of a nanoconfinement, the property of water contained inside is rather challenging to probe. Herein, we measured the amount of water molecules released during the folding of individual G-quadruplex and i-motif structures, from which water activities are estimated in the DNA nanocages prepared by 5 × 5 to 7 × 7 helix bundles (cross-sections, 9 × 9 to 15 × 15 nm). We found water activities decrease with reducing cage size. In the 9 × 9-nm cage, water activity was reduced beyond the reach of regular cosolutes such as polyethylene glycol (PEG). With this set of nanocages, we were able to retrieve the change in water molecules throughout the folding trajectory of G-quadruplex or i-motif. We found that water molecules absorbed from the unfolded to the transition states are much fewer than those lost from the transition to the folded states. The overall loss of water therefore drives the folding of G-quadruplex or i-motif in nanocages with reduced water activities.

Effects of vitamin B12 supplementation on neurodevelopment and growth in Nepalese Infants: A randomized controlled trial.

BACKGROUND: Vitamin B12 deficiency is common and affects cell division and differentiation, erythropoiesis, and the central nervous system. Several observational studies have demonstrated associations between biomarkers of vitamin B12 status with growth, neurodevelopment, and anemia. The objective of this study was to measure the effects of daily supplementation of vitamin B12 for 1 year on neurodevelopment, growth, and hemoglobin concentration in infants at risk of deficiency. METHODS AND FINDINGS: This is a community-based, individually randomized, double-blind placebo-controlled trial conducted in low- to middle-income neighborhoods in Bhaktapur, Nepal. We enrolled 600 marginally stunted, 6- to 11-month-old infants between April 2015 and February 2017. Children were randomized in a 1:1 ratio to 2 µg of vitamin B12, corresponding to approximately 2 to 3 recommended daily allowances (RDAs) or a placebo daily for 12 months. Both groups were also given 15 other vitamins and minerals at around 1 RDA. The primary outcomes were neurodevelopment measured by the Bayley Scales of Infant and Toddler Development 3rd ed. (Bayley-III), attained growth, and hemoglobin concentration. Secondary outcomes included the metabolic response measured by plasma total homocysteine (tHcy) and methylmalonic acid (MMA). A total of 16 children (2.7%) in the vitamin B12 group and 10 children (1.7%) in the placebo group were lost to follow-up. Of note, 94% of the scheduled daily doses of vitamin B12 or placebo were reported to have been consumed (in part or completely). In this study, we observed that there were no effects of the intervention on the Bayley-III scores, growth, or hemoglobin concentration. Children in both groups grew on an average 12.5 cm (SD: 1.8), and the mean difference was 0.20 cm (95% confidence interval (CI): -0.23 to 0.63, P = 0.354). Furthermore, at the end of the study, the mean difference in hemoglobin concentration was 0.02 g/dL (95% CI: -1.33 to 1.37, P = 0.978), and the difference in the cognitive scaled scores was 0.16 (95% CI: -0.54 to 0.87, P = 0.648). The tHcy and MMA concentrations were 23% (95% CI: 17 to 30, P < 0.001) and 30% (95% CI: 15 to 46, P < 0.001) higher in the placebo group than in the vitamin B12 group, respectively. We observed 43 adverse events in 36 children, and these events were not associated with the intervention. In addition, 20 in the vitamin B12 group and 16 in the placebo group were hospitalized during the supplementation period. Important limitations of the study are that the strict inclusion criteria could limit the external validity and that the period of vitamin B12 supplementation might not have covered a critical window for infant growth or brain development. CONCLUSIONS: In this study, we observed that vitamin B12 supplementation in young children at risk of vitamin B12 deficiency resulted in an improved metabolic response but did not affect neurodevelopment, growth, or hemoglobin concentration. Our results do not support widespread vitamin B12 supplementation in marginalized infants from low-income countries. TRIAL REGISTRATION: ClinicalTrials.gov NCT02272842 Universal Trial Number: U1111-1161-5187 (September 8, 2014) Trial Protocol: Original trial protocol: PMID: 28431557 (reference [18]; study protocols and plan of analysis included as Supporting information).

Mesenchymal Stem Cell Capping on ECM-Anchored Caspase Inhibitor-Loaded PLGA Microspheres for Intraperitoneal Injection in DSS-Induced Murine Colitis.

Mesenchymal stem cells (MSCs) are considered as a promising alternative for the treatment of various inflammatory disorders. However, poor viability and engraftment of MSCs after transplantation are major hurdles in mesenchymal stem cell therapy. Extracellular matrix (ECM)-coated scaffolds provide better cell attachment and mechanical support for MSCs after transplantation. A single-step method for ECM functionalization on poly(lactic-co-glycolic acid) (PLGA) microspheres using a novel compound, dopamine-conjugated poly(ethylene-alt-maleic acid), as a stabilizer during the preparation of microspheres is reported. The dopamine molecules on the surface of microspheres provide active sites for the conjugation of ECM in an aqueous solution. The results reveal that the viability of MSCs improves when they are coated over the ECM-functionalized PLGA microspheres (eMs). In addition, the incorporation of a broad-spectrum caspase inhibitor (IDN6556) into the eMs synergistically increases the viability of MSCs under in vitro conditions. Intraperitoneal injection of the MSC-microsphere hybrid alleviates experimental colitis in a murine model via inhibiting Th1 and Th17 differentiation of CD4+ T cells in colon-draining mesenteric lymph nodes. Therefore, drug-loaded ECM-coated surfaces may be considered as attractive tools for improving viability, proliferation, and functionality of MSCs following transplantation.

A New Concentration Jump Strategy Reveals the Lifetime of i-Motif at Physiological pH without Force.

Concentration jumps for kinetics measurement remain a challenge for single-molecule techniques, which have demonstrated superior signal-to-noise levels compared to ensemble average approaches. Currently, all concentration jumps use mixing strategies. Here, we report a simple and drastically different jump strategy by rapid transportation of molecules between two side-by-side laminar streams in 80 ms. This allowed us to measure the lifetime of bioactive DNA i-motif structures at physiological pH without force. We placed a human telomeric i-motif inside a DNA hairpin-based mechanical reporter. Since the folded or unfolded state of the hairpin correlates with that of the i-motif, by recording hairpin transitions, a half-life of ∼3 s was found for the DNA i-motif at neutral pH without force. Such a lifetime is sufficient for i-motif to interact with proteins to modulate cellular processes. We anticipate this concentration jump offers a generic platform to investigate single-molecule kinetics.

Single-Molecule Mechanochemical pH Sensing Revealing the Proximity Effect of Hydroniums Generated by an Alkaline Phosphatase.

Due to the fast diffusion, small molecules such as hydronium ions (H3O+) are expected to be homogeneously distributed, even close to the site-of-origin. Given the importance of H3O+ in numerous processes, it is surprising that H3O+ concentration ([H3O+]) has yet to be profiled near its generation site with nanometer resolution. Here, we innovated a single-molecule method to probe [H3O+] in nanometer proximity of individual alkaline phosphatases. We designed a mechanophore with cytosine (C)-C mismatch pairs in a DNA hairpin. Binding of H3O+ to these C-C pairs changes mechanical properties, such as stability and transition distance, of the mechanophore. These changes are recorded in optical tweezers and analyzed in a multivariate fashion to reduce the stochastic noise of individual mechanophores. With this method, we found [H3O+] increases in the nanometer vicinity of an active alkaline phosphatase, which supports that the proximity effect is the cause for increased rates in cascade enzymatic reactions.

Maternal and infant vitamin B12 status during infancy predict linear growth at 5 years.

BACKGROUND: Many children worldwide have poor vitamin B12 status. The objective of this study was to estimate association between maternal and infant vitamin B12 status and long-term growth. METHODS: We randomly selected 500 Nepali mother-infant pairs and measured maternal intake and infant and maternal vitamin B12 status using plasma cobalamin, total plasma homocysteine, and methylmalonic acid concentrations. We revisited available children when they were 5 years old and measured growth. The associations between intake and maternal and infant markers of vitamin B12 and growth were estimated in multiple linear regression models adjusting for relevant confounders (n = 331). RESULTS: Maternal vitamin B12 intake and status and vitamin B12 status in infancy predicted linear growth at 5 years of age, but not during infancy. Each microgram increase in the vitamin B12 intake of the mother during infancy was associated with an increase in height of 0.4 (0.2, 0.6) height-for-age z-scores and 1.7 (0.7, 2.7) cm around the child's fifth birthday. CONCLUSION: Vitamin B12 status and intake in early life is an important determinant for linear growth at school age. Our findings should be verified in randomized, placebo controlled trials before translated into public health recommendations.

Erythrocyte fatty acid composition of Nepal breast-fed infants.

PURPOSE: Essential fatty acids play a critical role in the growth and development of infants, but little is known about the fatty acid status of populations in low-income countries. The objective was to describe the fatty acid composition of red blood cells (RBC) in breastfeed Nepali infants and a subsample of their mothers and to identify the main sources of fatty acids in the mother's diet, as well as the fatty acid composition of breast milk. METHODS: RBC fatty acid composition was analyzed in a random sample of 303 infants and 72 mother, along with 68 breastmilk samples. Fatty acid profiles of the most important dietary fat sources were analyzed. Information on mother's diet and intake of fat was collected by three 24-h dietary recalls. RESULTS: In infant RBC's, docosahexaenoic acid (DHA) was the main n-3 fatty acid, and arachidonic acid (AA) was the major n-6 fatty acid. Total n-6 PUFA was three times higher than total n-3 PUFA. Height-for-age (HAZ) was positively associated with DHA status and AA status in multivariable models. The concentration of all fatty acids was higher in children, compared to mothers, except Total n-6 PUFA and Linoleic acid (LA) where no differences were found. The mother's energy intake from fat was 13% and cooking oil (sesame, mustard, soybean or sunflower oil) contributed 52% of the fat intake. CONCLUSIONS: RBC-DHA levels in both infants and mother was unexpected high taking into account few dietary DHA sources and the low DHA concentrations in breastmilk.

Evaluating meteorological data from weather stations, and from satellites and global models for a multi-site epidemiological study.

BACKGROUND: Longitudinal and time series analyses are needed to characterize the associations between hydrometeorological parameters and health outcomes. Earth Observation (EO) climate data products derived from satellites and global model-based reanalysis have the potential to be used as surrogates in situations and locations where weather-station based observations are inadequate or incomplete. However, these products often lack direct evaluation at specific sites of epidemiological interest. METHODS: Standard evaluation metrics of correlation, agreement, bias and error were applied to a set of ten hydrometeorological variables extracted from two quasi-global, commonly used climate data products - the Global Land Data Assimilation System (GLDAS) and Climate Hazards Group InfraRed Precipitation with Stations (CHIRPS) - to evaluate their performance relative to weather-station derived estimates at the specific geographic locations of the eight sites in a multi-site cohort study. These metrics were calculated for both daily estimates and 7-day averages and for a rotavirus-peak-season subset. Then the variables from the two sources were each used as predictors in longitudinal regression models to test their association with rotavirus infection in the cohort after adjusting for covariates. RESULTS: The availability and completeness of station-based validation data varied depending on the variable and study site. The performance of the two gridded climate models varied considerably within the same location and for the same variable across locations, according to different evaluation criteria and for the peak-season compared to the full dataset in ways that showed no obvious pattern. They also differed in the statistical significance of their association with the rotavirus outcome. For some variables, the station-based records showed a strong association while the EO-derived estimates showed none, while for others, the opposite was true. CONCLUSION: Researchers wishing to utilize publicly available climate data - whether EO-derived or station based - are advised to recognize their specific limitations both in the analysis and the interpretation of the results. Epidemiologists engaged in prospective research into environmentally driven diseases should install their own weather monitoring stations at their study sites whenever possible, in order to circumvent the constraints of choosing between distant or incomplete station data or unverified EO estimates.

Erythrocyte DHA and AA in infancy is not associated with developmental status and cognitive functioning five years later in Nepalese children.

BACKGROUND: Long chain polyunsaturated fatty acids (LCPUFA) especially docosahexaenoic acid (DHA) and arachidonic acid (AA) are crucial for normal brain development in utero and in early infancy. Data on fatty acid status and cognitive development in infants and children from low-income countries are scarce. METHODS: We examined the association between the DHA and AA status in infancy (n = 320) and developmental status and cognitive functioning five years later. At five years of age, we measured development by the Ages and Stages Questionnaire 3rd. ed. (ASQ-3) and cognitive functioning by subtests from the neuropsychological test battery NEPSY II. In addition, infant fatty acid composition in red blood cells (RBC) was analyzed. In multiple linear and logistic regression models, we estimated the associations between DHA and AA status in infancy and scores on the ASQ-3 and the NEPSY II subtests. RESULTS: There were no notable associations between infant AA and DHA status, and the scores on the ASQ-3 and the NEPSY II subtests five years later. It should be noted that we found better than expected concentrations of erythrocyte DHA and AA among the infants, and the ASQ scores were left-skewed, which limited the ability to identify associations. CONCLUSION: DHA and AA status in infancy is seemingly not related to neurodevelopment measured 5 years later in this peri-urban population from Nepal.

Mechanical properties of DNA origami nanoassemblies are determined by Holliday junction mechanophores.

DNA nanoassemblies have demonstrated wide applications in various fields including nanomaterials, drug delivery and biosensing. In DNA origami, single-stranded DNA template is shaped into desired nanostructure by DNA staples that form Holliday junctions with the template. Limited by current methodologies, however, mechanical properties of DNA origami structures have not been adequately characterized, which hinders further applications of these materials. Using laser tweezers, here, we have described two mechanical properties of DNA nanoassemblies represented by DNA nanotubes, DNA nanopyramids and DNA nanotiles. First, mechanical stability of DNA origami structures is determined by the effective density of Holliday junctions along a particular stress direction. Second, mechanical isomerization observed between two conformations of DNA nanotubes at 10-35 pN has been ascribed to the collective actions of individual Holliday junctions, which are only possible in DNA origami with rotational symmetric arrangements of Holliday junctions, such as those in DNA nanotubes. Our results indicate that Holliday junctions control mechanical behaviors of DNA nanoassemblies. Therefore, they can be considered as 'mechanophores' that sustain mechanical properties of origami nanoassemblies. The mechanical properties observed here provide insights for designing better DNA nanostructures. In addition, the unprecedented mechanical isomerization process brings new strategies for the development of nano-sensors and actuators.

Predicting Undernutrition at Age 2 Years with Early Attained Weight and Length Compared with Weight and Length Velocity.

OBJECTIVE: To estimate the abilities of weight and length velocities vs attained growth measures to predict stunting, wasting, and underweight at age 2 years. STUDY DESIGN: We analyzed data from a community-based cohort study (The Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development study [MAL-ED] study) in Bhaktapur, Nepal. A total of 240 randomly selected children were enrolled at birth and followed up monthly up to age 24 months. Linear and logistic regression models were used to predict malnutrition at 2 years of age with growth velocity z scores at 0-3, 0-6, 3-6, 6-9, 6-12, and 9-12 months (using the World Health Organization Growth Standards) or attained growth at 0, 3, 6, and 12 months as predictors. RESULTS: At age 2 years, 4% of the children were wasted, 13% underweight, and 21% stunted. Children who were malnourished at age 2 years had lower mean growth z scores already at birth and throughout the study period. Anthropometric indicators in infancy were significant predictors for growth at the age of 2 years during most periods and at most ages in infancy. Weight-for-age z score, length-for-age z score, and weight-for-length z score at age 12 months had excellent areas under the curve (91-95) to predict the value of the same indicator at age 24 months. Maximum area under the curve values for weight and length velocity were somewhat lower (70-84). CONCLUSIONS: Growth measured at one time point in infancy was better correlated with undernutrition at age 2 years than growth velocity.

Vitamin D status is associated with treatment failure and duration of illness in Nepalese children with severe pneumonia.

BackgroundThere is no consensus on optimal Vitamin D status. The objective of this study was to estimate the extent to which vitamin D status predicts illness duration and treatment failure in children with severe pneumonia by using different cutoffs for vitamin D concentration.MethodsWe measured the plasma concentration of 25(OH)D in 568 children hospitalized with World Health Organization-defined severe pneumonia. The associations between vitamin D status, using the most frequently used cutoffs for vitamin D insufficiency (25(OH)D<50 and <75 nmol/l), and risk for treatment failure and time until recovery were analyzed in multiple logistic regression and Cox proportional hazards models, respectively.ResultsOf the 568 children, 322 (56.7%) had plasma 25(OH)D levels ≥75 nmol/l, 179 (31.5%) had levels of 50-74.9 nmol/l, and 67 (%) had levels <50 nmol/l. Plasma 25(OH)D <50 nmol/l was associated with increased risk for treatment failure and longer time until recovery.ConclusionOur findings indicate that low vitamin D status (25(OH)D<50 nmol/l) is an independent risk factor for treatment failure and delayed recovery from severe lower respiratory infections in children.

A Case of Fulminant Hepatic Failure Secondary to Congestive Heart Failure Without Evidence of Acute Cardiac Decompensation.

There are so far only a few reported cases of acute fulminant hepatic failure resulting from acute cardiomyopathy. This is a rare occurrence, especially in patients that do not exhibit any signs and symptoms of acute cardiac decompensation. We report a case of fulminant liver failure with nondiagnostic work up for the common causes of liver failure. This patient had concurrent history of congestive heart failure, but did not have acute decompensation. Right upper quadrant sonogram revealed hepatomegaly of 15 cm, trace amount of perihepatic ascites, pericholecystic fluid, and also thickened edematous gallbladder wall with no stones, no common bile duct stones, and no portal vein thrombosis. Echocardiogram revealed dilated left atrium and ventricle, severe mitral regurgitation, severe tricuspid regurgitation, grade 4 diastolic dysfunction, diffuse hypokinesis of left ventricle, and severely and newly reduced systolic function with an ejection fraction of 10 percent (decreased from 25 percent on last ECHO 18 months prior). Liver biopsy demonstrated marked centrilobular hepatocyte necrosis and dropout accompanied by congestion, some areas of bridging necrosis and focal confluent necrosis which was suggestive of severe congestive hepatopathy. With initiation of heart failure medications, liver function improved significantly.

Mechanochemical Sensing of Single and Few Hg(II) Ions Using Polyvalent Principles.

Sensitivity of biosensors is set by the dissociation constant (Kd) between analytes and probes. Although potent amplification steps can be accommodated between analyte recognition and signal transduction in a sensor to improve the sensitivity 4-6 orders of magnitude below Kd, they compromise temporal resolution. Here, we demonstrated mechanochemical sensing that broke the Kd limit by 9 orders of magnitude for Hg detection without amplifications. Analogous to trawl fishing, we introduced multiple Hg binding units (thymine (T)-T pairs) in a molecular trawl made of two poly-T strands. Inspired by dipsticks to gauge content levels, mechanical information (force/extension) of a DNA hairpin dipstick was used to measure the single or few Hg2+ ions bound to the molecular trawl, which was levitated by two optically trapped particles. The multivalent binding and single-molecule sensitivity allowed us to detect unprecedented 1 fM Hg ions in 20 min in field samples treated by simple filtrations.

Nascent RNA transcripts facilitate the formation of G-quadruplexes.

Recent discovery of the RNA/DNA hybrid G-quadruplexes (HQs) and their potential wide-spread occurrence in human genome during transcription have suggested a new and generic transcriptional control mechanism. The G-rich sequence in which HQ may form can coincide with that for DNA G-quadruplexes (GQs), which are well known to modulate transcriptions. Understanding the molecular interaction between HQ and GQ is, therefore, of pivotal importance to dissect the new mechanism for transcriptional regulation. Using a T7 transcription model, herein we found that GQ and HQ form in a natural sequence, (GGGGA)4, downstream of many transcription start sites. Using a newly-developed single-molecular stalled-transcription assay, we revealed that RNA transcripts helped to populate quadruplexes at the expense of duplexes. Among quadruplexes, HQ predominates GQ in population and mechanical stabilities, suggesting HQ may serve as a better mechanical block during transcription. The fact that HQ and GQ folded within tens of milliseconds in the presence of RNA transcripts provided justification for the co-transcriptional folding of these species. The catalytic role of RNA transcripts in the GQ formation was strongly suggested as the GQ folded >7 times slower without transcription. These results shed light on the possible synergistic effect of GQs and HQs on transcriptional controls.

Mechanochemical Sensing: A Biomimetic Sensing Strategy.

Existing biosensors employ two major components: analyte recognition and signal transduction. Although specificity is achieved through analyte recognition, sensitivity is usually enhanced through a chemical amplification stage that couples the two main units in a sensor. Although highly sensitive, the extra chemical amplification stage complicates the sensing protocol. In addition, it separates the two elements spatiotemporally, reducing the real-time response of the biosensor. In this review, we discuss the new mechanochemical biosensors that employ mechanochemical coupling strategies to overcome these issues. By monitoring changes in the mechanical properties of a single-molecule template upon analyte binding, single-molecule sensitivity is reached. As chemical amplification becomes unnecessary in this single-molecule mechanochemical sensing (SMMS) strategy, real-time sensing is achieved.

Low dietary diversity and micronutrient adequacy among lactating women in a peri-urban area of Nepal.

OBJECTIVE: The main objectives were to assess the adequacy of the micronutrient intakes of lactating women in a peri-urban area in Nepal and to describe the relationships between micronutrient intake adequacy, dietary diversity and sociodemographic variables. DESIGN: A cross-sectional survey was performed during 2008-2009. We used 24 h dietary recall to assess dietary intake on three non-consecutive days and calculated the probability of adequacy (PA) of the usual intake of eleven micronutrients and the overall mean probability of adequacy (MPA). A mean dietary diversity score (MDDS) was calculated of eight food groups averaged over 3 d. Multiple linear regression was used to identify the determinants of the MPA. SETTING: Bhaktapur municipality, Nepal. SUBJECTS: Lactating women (n 500), 17-44 years old, randomly selected. RESULTS: The mean usual energy intake was 8464 (sd 1305) kJ/d (2023 (sd 312) kcal/d), while the percentage of energy from protein, fat and carbohydrates was 11 %, 13 % and 76 %, respectively. The mean usual micronutrient intakes were below the estimated average requirements for all micronutrients, with the exception of vitamin C and Zn. The MPA across eleven micronutrients was 0·19 (sd 0·16). The diet was found to be monotonous (MDDS was 3·9 (sd 1·0)) and rice contributed to about 60 % of the energy intake. The multiple regression analyses showed that MPA was positively associated with energy intake, dietary diversity, women's educational level and socio-economic status, and was higher in the winter. CONCLUSIONS: The low micronutrient intakes are probably explained by low dietary diversity and a low intake of micronutrient-rich foods.

Bhaktapur, Nepal: the MAL-ED birth cohort study in Nepal.

The Etiology, Risk Factors and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) cohort study site in Nepal is located in the Bhaktapur municipality, 15 km east of Kathmandu, the capital city of Nepal. Bhaktapur, an ancient city famous for its traditional temples and buildings, is included on UNESCO's World Heritage List and is a major tourist attraction in Nepal. Nepal is a land-locked country located in South Asia between China and India with an area of 147 181 km(2), ranging from sea-level plains to Mount Everest, the world's highest peak. The total population as of the 2011 census was 26.6 million, with an average annual population growth rate of 1.4. Nepal is one of the world's least developed countries and is ranked 157 of 186 in the 2013 Human Development Report; one-third of the Nepali population lives below the poverty line. The current under-5 mortality rate is 54 per 1000 live births, the infant mortality rate is 46 per 1000 live births, and the neonatal mortality rate is 33 per 1000 live births. Vaccine coverage for all Expanded Program on Immunization vaccines is >80%. Among children, the most common diseases contributing to significant morbidity and mortality are acute respiratory infection and dehydration from severe diarrhea. In this article, we report on the geographic, demographic, and socioeconomic features of the Bhaktapur MAL-ED site and describe the data that informed our cohort recruitment strategy.