The phosphorylation of serine55 in enamelin is essential for murine amelogenesis.

Amelogenesis imperfecta (AI) is an inherited developmental enamel defect affecting tooth masticatory function, esthetic appearance, and the well-being of patients. As one of the major enamel matrix proteins (EMPs), enamelin (ENAM) has three serines located in Ser-x-Glu (S-x-E) motifs, which are potential phosphorylation sites for the Golgi casein kinase FAM20C. Defects in FAM20C have similarly been associated with AI. In our previous study of EnamRgsc514 mice, the Glu57 in the S55-X56-E57 motif was mutated into Gly, which was expected to cause a phosphorylation failure of Ser55 because Ser55 cannot be recognized by FAM20C. The severe enamel defects in ENAMRgsc514 mice reminiscent of Enam-knockout mouse enamel suggested a potentially important role of Ser55 phosphorylation in ENAM function. However, the enamel defects and ENAM dysfunction may also be attributed to distinct physicochemical differences between Glu57 and Gly57. To clarify the significance of Ser55 phosphorylation to ENAM function, we generated two lines of Enam knock-in mice using CRISPR-Cas9 method to eliminate or mimic the phosphorylation state of Ser55 by substituting it with Ala55 or Asp55 (designated as S55A or S55D), respectively. The teeth of 6-day or 4-week-old mice were subjected to histology, micro-CT, SEM, TEM, immunohistochemistry, and mass spectrometry analyses to characterize the morphological, microstructural and proteomic changes in ameloblasts, enamel matrix and enamel rods. Our results showed that the enamel formation and EMP expression in S55D heterozygotes (Het) were less disturbed than those in S55A heterozygotes, while both homozygotes (Homo) had no mature enamel formation. Proteomic analysis revealed alterations of enamel matrix biosynthetic and mineralization processes in S55A Hets. Our present findings indicate that Asp55 substitution partially mimics the phosphorylation state of Ser55 in ENAM. Ser55 phosphorylation is essential for ENAM function during amelogenesis.

Detection and Quantitation of Label-Retaining Cells in Mouse Incisors using a 3D Reconstruction Approach after Tissue Clearing.

The murine incisor is an organ that grows continuously throughout the lifespan of the mouse. The epithelial and mesenchymal stem cells residing in the proximal tissues of incisors give rise to progeny that will differentiate into ameloblasts, odontoblasts, and pulp fibroblasts. These cells are crucial in supporting the sustained turnover of incisor tissues, making the murine incisor an excellent model for studying the homeostasis of adult stem cells. Stem cells are believed to contain long-living quiescent cells that can be labeled by nucleotide analogs such as 5-ethynyl-2´-deoxyuridine (EdU). The cells retain this label over time and are accordingly named label-retaining cells (LRCs). Approaches for visualizing LRCs in vivo provide a robust tool for monitoring stem cell homeostasis. In this study, we described a method for visualizing and analyzing LRCs. Our innovative approach features LRCs in mouse incisors after tissue clearing and whole-mount EdU staining followed by confocal microscopy and a 3-dimensional (3D) reconstruction with the imaging software. This method enables 3D imaging acquisition and non-biased quantitation compared to traditional LRCs analysis on sectioned slides.

FAM20B-catalyzed glycosaminoglycans control murine tooth number by restricting FGFR2b signaling.

BACKGROUND: The formation of supernumerary teeth is an excellent model for studying the molecular mechanisms that control stem/progenitor cell homeostasis needed to generate a renewable source of replacement cells and tissues. Although multiple growth factors and transcriptional factors have been associated with supernumerary tooth formation, the regulatory inputs of extracellular matrix in this regenerative process remains poorly understood. RESULTS: In this study, we present evidence that disrupting glycosaminoglycans (GAGs) in the dental epithelium of mice by inactivating FAM20B, a xylose kinase essential for GAG assembly, leads to supernumerary tooth formation in a pattern reminiscent of replacement teeth. The dental epithelial GAGs confine murine tooth number by restricting the homeostasis of Sox2(+) dental epithelial stem/progenitor cells in a non-autonomous manner. FAM20B-catalyzed GAGs regulate the cell fate of dental lamina by restricting FGFR2b signaling at the initial stage of tooth development to maintain a subtle balance between the renewal and differentiation of Sox2(+) cells. At the later cap stage, WNT signaling functions as a relay cue to facilitate the supernumerary tooth formation. CONCLUSIONS: The novel mechanism we have characterized through which GAGs control the tooth number in mice may also be more broadly relevant for potentiating signaling interactions in other tissues during development and tissue homeostasis.

Usability and acceptability of an automated respiratory rate counter to assess childhood pneumonia in Nepal.

AIM: Pneumonia is the leading cause of child death after the neonatal period, resulting from late care seeking and inappropriate treatment. Diagnosis involves counting respiratory rate (RR); however, RR counting remains challenging for health workers and miscounting, and misclassification of RR is common. We evaluated the usability of a new automated RR counter, the Philips Children's Respiratory Monitor (ChARM), to Female Community Health Volunteers (FCHVs), and its acceptability to FCHVs and caregivers in Nepal. METHODS: A cross-sectional study was conducted in Jumla district, Nepal. About 133 FCHVs were observed between September and December 2018 when using ChARM during 517 sick child consultations, 264 after training and 253 after 2 months of routine use of ChARM. Acceptability of the ChARM was explored using semi-structured interviews. RESULTS: FCHV adherence to guidelines after 2 months of using ChARM routinely was 52.8% (95% CI 46.6-58.9). The qualitative findings suggest that ChARM is acceptable to FCHVs and caregivers; however, capacity constraints such as older age and low literacy and impacted device usability were mentioned. CONCLUSION: Further research on the performance, cost-effectiveness and implementation feasibility of this device is recommended, especially among low-literate CHWs.

The performance and yield of tuberculosis testing algorithms using microscopy, chest x-ray, and Xpert MTB/RIF.

SETTING: The introduction of Xpert MTB/RIF (Xpert) and renewed interest in chest x-ray (CXR) for tuberculosis testing has provided additional choices to the smear-based diagnostic algorithms used by TB programs previously. More programmatic data is needed to better understand the implications of possible approaches. OBJECTIVE: We sought to evaluate how different testing algorithms using microscopy, Xpert and CXR impacted the number of people detected with TB in a district hospital in Nepal. DESIGN: Consecutively recruited patients with TB-related symptoms were offered smear microscopy, CXR and Xpert. We tested six hypothetical algorithms and compared yield, bacteriologically positive (Bac+) cases missed, and tests conducted. RESULTS: Among 929 patients, Bac+ prevalence was 17.3% (n = 161). Smear microscopy detected 121 (75.2% of Bac+). Depending on the radiologists' interpretation of CXR, Xpert testing could be reduced by (31%-60%). Smear microscopy reduced Xpert cartridge need slightly, but increased the overall diagnostic tests performed. CONCLUSION: Xpert detected a large proportion of Bac+ TB cases missed by microscopy. CXR was useful in greatly reducing the number of diagnostic tests needed even among presumptive TB patients. Loose CXR readings should be used to identify more people for TB testing. More analysis of costs and standardized CXR reading should be considered.

Predictors of micronutrient powder (MNP) knowledge, coverage, and consumption during the scale-up of an integrated infant and young child feeding (IYCF-MNP) programme in Nepal.

Large-scale programmes using micronutrient powders (MNPs) may not achieve maximum impact due to limited/inappropriate MNP coverage, consumption, and use. We identify predictors of MNP coverage, maternal knowledge of appropriate use, and child MNP consumption in Nepal. A cross-sectional survey was conducted in 2,578 mother-child pairs representative of children 6-23 months in two districts that were part of the post-pilot, scale-up of an integrated infant and young child feeding-MNP (IYCF-MNP) programme. Children aged 6-23 months were expected to receive 60 MNP sachets every 6 months from a female community health volunteer (FCHV) or health centre. Outcomes of interest were MNP coverage (ever received), maternal knowledge of appropriate use (correct response to seven questions), repeat coverage (receipt ≥ twice; among children 12-23 months who had received MNP at least once, n = 1342), and high intake (child consumed ≥75% of last distribution, excluding those with recent receipt/insufficient time to use 75% at recommended one-sachet-per-day dose, n = 1422). Multivariable log-binomial regression models were used to identify predictors of the four outcomes. Coverage, knowledge of appropriate use, and repeat coverage were 61.3%, 33.5%, and 45.9%, respectively. Among MNP receivers, 97.9% consumed MNP at least once and 38.9% of eligible children consumed ≥75% of last distribution. FCHV IYCF-MNP counselling was positively associated with knowledge, coverage, repeat coverage, and high intake; health worker counselling with knowledge and coverage indicators; and radio messages with coverage indicators only. FCHV counselling had the strongest association with knowledge, coverage, and high intake. Community-based counselling may play a vital role in improving coverage and intake in MNP programmes.

Changes in growth, anaemia, and iron deficiency among children aged 6-23 months in two districts in Nepal that were part of the post-pilot scale-up of an integrated infant and young child feeding and micronutrient powder intervention.

There is limited research on integrated infant and young child feeding (IYCF) and micronutrient powders (MNPs) programmes operating at scale, despite widespread implementation. This study uses cross-sectional baseline (n = 2,542) and endline (n = 2,578) surveys representative of children 6-23 months in two districts in Nepal that were part of a post-pilot scale-up of a IYCF-MNP programme. Multivariable log-binomial regression models were used to estimate prevalence ratios (PRs) for stunting (length-for-age z-score <-2), wasting (weight-for-length z-score <-2), underweight (weight-for-age z-score <-2), anaemia (altitude-adjusted haemoglobin <110 µg/L), moderate or severe anaemia (altitude-adjusted haemoglobin <100 g/L), iron deficiency (inflammation-adjusted ferritin <12 µg/L), and iron deficiency anaemia (iron deficiency + anaemia [IDA]) at endline versus baseline and also to compare children in the endline survey based on frequency of mothers' interactions with female community health volunteers (FCHVs; >1× per month or monthly vs. <1× per month) and MNP coverage (1 or ≥2 distributions vs. none among children 12-23 months). Endline children were significantly less likely to be stunted than baseline children in both districts (multivariable-adjusted PR [95% CI]: 0.77 [0.69, 0.85], P < 0.001 and 0.82 [0.75, 0.91], P < 0.001 in Kapilvastu and Achham, respectively); however, only Achham had significantly lower prevalences of underweight, moderate/severe anaemia, iron deficiency, and IDA at endline. At endline, 53.5% and 71.4% of children had tried MNP in Kapilvastu and Achham districts, respectively, consuming an average of 24 sachets from the last distribution. Frequent maternal-FCHV interactions were associated with a reduced risk of stunting and underweight at endline, whereas repeat MNP coverage was associated with reduced risk of anaemia and IDA. Future research using experimental designs should verify the potential of integrated IYCF-MNP programmes to improve children's nutritional status.

Free Newborn Care Services: A New Initiative in Nepal.

BACKGROUND: Nepal has made a significant progress in reducing child mortality. However, the annual rate of reduction in neonatal mortality is not satisfactory. As safeguarded by constitution of Nepal and to address neonatal mortality due to poverty and inequity, government has introduced free newborn care (FNC) package. This study aims to assess the status of FNC services in all the public hospitals. METHODS: Child Health Division organized 5 workshops region-wise with the theme of newborn care services in March/April 2018 to cover all the public hospitals in the country. A template was designed comprising of duration of FNC implementation, number of newborns admitted since implementation, morbidities pattern, and number of babies served. It was circulated and all hospitals were advised to fill it and present in the review. Later, the data were compiled and analyzed. RESULTS: Only 58 presentations out of 93 participated hospitals were included in this study. The total admitted cases were 8564 newborns. The common causes of admission were neonatal sepsis (44.5%) followed by asphyxia (14.29%) and hyperbilirubinemia (11.4%). A total of 1573 neonates received services of FNC package C, 3722 package B, 3081 received package A. The main challenges faced in implementation reported were lack of infrastructure and human resources to provide services and the reimbursement is not enough. CONCLUSIONS: Free newborn care is a new initiative taken to reduce neonatal mortality. This package is very helpful to serve sick newborns. However, the package should be revised taking into consideration the appropriate reimbursement and extra staffs to provide this service.

Nepal Scale up Chlorhexidine as Part of Essential Newborn Care: Country Experience.

Chlorhexidine is a broad-spectrum antiseptic, effective on gram positive and gram negative bacteria as well as some viruses, having strong skin binding effect. Randomized controlled trials conducted in South Asian countries have proven that the use of chlorhexidine (4% weight/weight) for cord care can reduce neonatal mortality and prevent severe cord infections. Between 2011 and 2017, Nepal completed nationwide scale-up of the use of chlorhexidine by integrating with ongoing maternal and neonatal health programs, under the leadership of the Child Health Division. The chlorhexidine coverage and compliance study (2017) has revealed that the country has achieved 59% coverage of the intervention to date, with lowest use among home births. The strategy should be further strengthened to ensure that every newborn in need is reached with chlorhexidine. Keywords: Chlorhexidine; cord care; Nepal experience, Newborn.

Infant and Young Child Feeding (IYCF) Practices Improved in 2 Districts in Nepal during the Scale-Up of an Integrated IYCF and Micronutrient Powder Program.

BACKGROUND: Three-quarters of the ≥50 programs that use micronutrient powders (MNPs) integrate MNPs into infant and young child feeding (IYCF) programs, with limited research on impacts on IYCF practices. OBJECTIVE: This study assessed changes in IYCF practices in 2 districts in Nepal that were part of a post-pilot scale-up of an integrated IYCF-MNP program. METHODS: This analysis used cross-sectional surveys (n = 2543 and 2578 for baseline and endline) representative of children aged 6-23 mo and their mothers in 2 districts where an IYCF program added MNP distributions through female community health volunteers (FCHVs) and health workers (HWs). Multivariable log-binomial models estimated prevalence ratios comparing reported IYCF at endline with baseline and at endline on the basis of exposure to different sources of IYCF information. Mothers who received FCHV-IYCF counseling with infrequent (≤1 time/mo) and frequent (>1 time/mo) interactions were compared with mothers who never received FCHV-IYCF counseling. The receipt of HW-IYCF counseling and receipt of MNPs from an FCHV (both yes or no) were also compared. RESULTS: The prevalence of minimum dietary diversity (MDD) and minimum acceptable diet (MAD) was significantly higher at endline than at baseline. In analyses from endline, compared with mothers who never received FCHV counseling, only mothers in the frequent FCHV-IYCF counseling group were more likely to report feeding the minimum meal frequency (MMF) and MAD, with no difference for the infrequent FCHV-IYCF counseling group in these indicators. HW-IYCF counseling was not associated with these indicators. Mothers who received MNPs from their FCHV were more likely to report initiating solid foods at 6 mo and feeding the child the MDD, MMF, and MAD compared with mothers who did not, adjusting for HW- and FCHV-IYCF counseling and demographic covariates. CONCLUSIONS: Incorporating MNPs into the Nepal IYCF program did not harm IYCF and may have contributed to improvements in select practices. Research that uses experimental designs should verify whether integrated IYCF-MNP programs can improve IYCF practices.

Implementation of a psychosocial support package for people receiving treatment for multidrug-resistant tuberculosis in Nepal: A feasibility and acceptability study.

BACKGROUND AND OBJECTIVES: People receiving treatment for multidrug-resistant tuberculosis (MDR-TB) have high rates of depression. Psychosocial support in general, and treatments for depression in particular, form an important but neglected area of patient-centred care, and a key pillar in the global End TB strategy. We assessed the feasibility and acceptability of a psychosocial support package for people receiving treatment for MDR-TB in Nepal. METHODS: This feasibility study used a mixed quantitative and qualitative approach. We implemented the intervention package in two National Tuberculosis Programme (NTP) MDR-TB treatment centres and 8 sub-centres. We screened patients monthly for depression and anxiety (cut-off ≥24 and ≥17 respectively on the Hopkins Symptom Checklist) and also for low social support (cut-off ≤3 on the Multidimensional Scale of Perceived Social Support). Those who screened positive on either screening tool received the Healthy Activity Program (HAP), which uses brief counselling based on behavioural activation theory. Other aspects of the psychosocial package were information/education materials and group interactions with other patients. RESULTS: We screened 135 patients, of whom 12 (9%) received HAP counselling, 115 (85%) received information materials, 80 (59%) received an education session and 49 (36%) received at least one group session. Eight group sessions were conducted in total. All aspects of the intervention package were acceptable to patients, including the screening, information, group work and counselling. Patients particularly valued having someone to talk to about their concerns and worries. We were able to successfully train individuals with no experience of psychological counselling to deliver HAP. CONCLUSION: This psychosocial support package is acceptable to patients. The information materials we developed are feasible to deliver in the current NTP. However, the structured psychological counselling (HAP), is not feasible in the current NTP due to time constraints. This requires additional investment of counsellors in TB clinics.

Trends and predictors of appropriate complementary feeding practices in Nepal: An analysis of national household survey data collected between 2001 and 2014.

There is evidence that suboptimal complementary feeding contributes to poor child growth. However, little is known about time trends and determinants of complementary feeding in Nepal, where the prevalence of child undernutrition remains unacceptably high. The objective of the study was to examine the trends and predictors of suboptimal complementary feeding in Nepali children aged 6-23 months using nationally representative data collected from 2001 to 2014. Data from the 2001, 2006, and 2011 Nepal Demographic and Health Surveys and the 2014 Multiple Indicator Cluster Survey were used to estimate the prevalence, trends and predictors of four WHO-UNICEF complementary feeding indicators: timely introduction of complementary foods (INTRO), minimum meal frequency (MMF), minimum dietary diversity (MDD), and minimum acceptable diet (MAD). We used multilevel logistic regression models to identify independent factors associated with these indicators at the individual, household and community levels. In 2014, the weighted proportion of children meeting INTRO, MMF, MDD, and MAD criteria were 72%, 82%, 36% and 35%, respectively, with modest average annual rate of increase ranging from 1% to 2%. Increasing child age, maternal education, antenatal visits, and community-level access to health care services independently predicted increasing odds of achieving MMF, MDD, and MAD. Practices also varied by ecological zone and sociocultural group. Complementary feeding practices in Nepal have improved slowly in the past 15 years. Inequities in the risk of inappropriate complementary feeding are evident, calling for programme design and implementation to address poor feeding and malnutrition among the most vulnerable Nepali children.

Scaling up quality improvement intervention for perinatal care in Nepal (NePeriQIP); study protocol of a cluster randomised trial.

INTRODUCTION: Nepal Perinatal Quality Improvement Project (NePeriQIP) intends to scale up a quality improvement (QI) intervention for perinatal care according to WHO/National guidelines in hospitals of Nepal using the existing health system structures. The intervention builds on previous research on the implementation of Helping Babies Breathe-quality improvement cycle in a tertiary healthcare setting in Nepal. The objective of this study is to evaluate the effect of this scaled-up intervention on perinatal health outcomes. METHODS/DESIGN: Cluster-randomised controlled trial using a stepped wedged design with 3 months delay between wedges will be conducted in 12 public hospitals with a total annual delivery rate of 60 000. Each wedge will consist of 3 hospitals. Impact will be evaluated on intrapartum-related mortality (primary outcome), overall neonatal mortality and morbidity and health worker's performance on neonatal care (secondary outcomes). A process evaluation and a cost-effectiveness analysis will be performed to understand the functionality of the intervention and to further guide health system investments will also be performed. DISCUSSION: In contexts where resources are limited, there is a need to find scalable and sustainable implementation strategies for improved care delivery. The proposed study will add to the scarce evidence base on how to scale up interventions within existing health systems. If successful, the NePeriQIP model can provide a replicable solution in similar settings where support and investment from the health system is poor, and national governments have made a global pledge to reduce perinatal mortality. TRIAL REGISTRATION NUMBER: ISRCTN30829654.