Low birth weight (LBW) continues to be the primary cause of infant morbidity and mortality.This study was undertaken to identify the predictors of LBW in Nigeria.The data for this study was extracted from the 2013 Nigeria Demographic and Health Survey conducted by the National Population Commission. Several questionnaires were used in the survey, some covering questions on pregnancy characteristics. The inclusion criteria include mothers who gave birth to a child 5 years before the interview and aged 15–49 years who were either permanent residents or visitors present in the household on the night before the survey conducted. The birth weight of the infants was recorded from written records from the hospital cards or the mothers’ recall.The prevalence of LBW in this study was 7.3%. Multiple logistic regression analysis showed an adjusted significant odds ratio for mothers from North West region , twin pregnancy , primiparous mother , maternal weight of less than 70 kg , and manual paternal employment .The risk factors for LBW identified in this study are modifiable. In order to reduce this menace in Nigeria, holistic approaches such as health education, maternal nutrition, improvement in socio-economic indices, and increasing the quality and quantity of the antenatal care services are of paramount importance. Birth weight is one of the significant predictors of child mental development, future physical growth, and survival. It is one of the important risk factors for child morbidity and mortality –4. AccorThere is significant difference in the incidence of LBW between developed and developing countries and between various regions in a country. In developed countries, the occurrence is 7%, while in developing countries it is 15% . GloballThere are numerous maternal and fetal factors contributing to the LBW incident . LBW is Antenatal care (ANC) visits are important for maternal and fetus health. ANC refers to pregnancy-related healthcare services provided by skilled health personnel during pregnancy that monitor the well-being of both the mother and the unborn child. It is essential to the purposes of obtaining the best possible outcome and preventing any complications . The freLBW is one of the most important public health concerns worldwide and is still the leading cause of prenatal and neonatal deaths. Despite intense research conducted on LBW globally, the factors affecting LBW in Nigeria have not been adequately investigated. Identifying the predictors of LBW and addressing the best prevention strategies will help to avert early the childhood morbidity and mortality resulting from LBW. Therefore, this study was aimed at 1) determining the prevalence of LBW infants in Nigeria, 2) describing the socio-demographic and pregnancy characteristics of mothers who gave birth to LBW babies, and 3) exploring the predictors of LBW in Nigeria using the 2013 Nigeria Demographic and Health Survey (2013 NDHS).This manuscript had been developed by utilizing the 2013 NDHS data obtained from the Ministry of Health, Nigeria. NDHS was a cross-sectional survey with a stratified three-stage cluster design. It is the fifth in the series of national surveys implemented by the National Population Commission (NPC). This nationally representative survey covered all 36 states and the Federal Capital Territory in Nigeria. This survey was intended to provide the latest estimates on population and health in Nigeria. The list of enumerated areas prepared for the 2006 Population Census of the Federal Republic of Nigeria was used as to frame the samples. The selection of samples was based on stratified three-stage cluster design which consists of 372 clusters in urban areas and 532 clusters in rural areas, giving a total of 904 clusters. A total of 40,320 households were selected. The inclusion criteria for this study were mothers, aged 15–49 years, who gave birth to a child 5 years before the interview and were either permanent residents or current visitors in the household on the night before the survey conducted. Data collection was carried out between February and May 2013.A structured questionnaire was used for interviewing the mothers. The questionnaire captured information about the pregnancies such as maternal age, birth interval, parity, time of registration, and frequency of ANC visits from mothers. Socio-demographic details of the mothers such as highest educational attainment, wealth index, localities, and literacy levels were also obtained. Infant characteristics such as sex and mode of delivery were recorded. The questionnaire was translated into different Nigerian languages: Hausa, Igbo, and Yoruba. All questionnaires were pretested among 120 households in November 2012. The questionnaires were modified according to the country's requirement with the advice from health experts.Prior to the commencement of the study, a complete listing of households was obtained and a mapping exercise was conducted for each cluster from December 2012 until January 2013. Training on how to use the Global Positioning System (GPS) receivers to locate the coordinates of sample households was conducted for the enumerators. All participants were briefed on the objectives, procedures, expected outcomes, benefits, and risk associated with this study. An informed consent was obtained from the mothers prior to the interview. Ethical clearance to conduct NDHS was approved by the National Health Research Ethics Committee of Nigeria, Federal Ministry of Health, Abuja, Nigeria. NDHS data are public access data and were made available to us upon request.Birth weights of the infants were recorded based on the written records of the hospital cards or mothers’ recall. Birth weights less than 2,500 g were classified as LBW , 39–41. Birth order was recorded as continuous variable and then recoded as 1) first and 2) second or more. Information on the number of children ever born or parity was obtained. The period of time between two successive live births or birth interval was recorded. Place of birth was dichotomized into 1) delivery at health facility and 2) delivery at home. The frequency of ANC visits was grouped into 1) less than 4 visit and 2) ≥4 visits. The proportion of pregnant mothers who received four or more ANC visits has been used as a benchmark for adequate ANC . In addiP-value < 0.05 indicates significant association. The multivariate logistic regression procedure was used to determine the predictors of LBW babies in Nigeria. Significant predictors were identified based on 95% CI. All variables were tested in a simple model using logistic regression to obtain the unadjusted logistic regression. All factors for LBW were included in the model. The variables were entered using manual stepwise method. All significant variables reaching p<0.05 were retained in the model.The Statistical Package for the Social Sciences (SPSS) version 20 with a complex samples procedure was used for the analysis. Frequency and percentages with 95% confidence interval were used to describe the characteristics of the LBW babies. The chi-square analysis procedure in the complex samples add-on module in SPSS was used to test the associations between socio-demographic and pregnancy characteristics with birth weight of the babies. There were 5,189 babies who were weighed after delivery during the 5 years preceding the survey. The proportion of LBW in this study was 7.3%. Among LBW babies, 39.5% were from written records from the hospital cards.The results for the socio-demographic characteristics of mothers who delivered LBW babies are presented in Results for the pregnancy characteristics of mothers who gave birth to LBW babies are shown in The determinants of each variable were presented in Information on infants’ birth weight and size at birth is essential to avert the complications resulting from LBW. In the present study, 5,189 birth weights of children were measured in the 5 years preceding the survey. This is not surprising because majority of the births did not take place in the health facility, and children are less likely to be weighed at birth in a non-institutional setting. The prevalence of LBW in this study was 7.3%, which is lower than the global prevalence in developed countries (17%) . The preThis study has shown that maternal age and education, paternal education, wealth index, ethnicity, type of residence, and geopolitical zone were significantly associated with the birth weights of the infants. The association of birth weight with locality, wealth index, and maternal education observed in this study has also been reported by Jayant et al. . Lower wThe proportion of LBW children in the rural areas was higher than that of the urban areas in this study. It could be due to the fact that rural women are more susceptible to poor diet, infections during pregnancy, and inadequate ANC facilities . The proWith regards to pregnancy characteristics, our study found that number of earlier pregnancies, maternal height, weight and BMI, the experience any type of violence during pregnancy, and the frequency of ANC visits were significantly associated with the birth weights of the infants. The majority of the LBW infants were born to mothers who were late for their ANC registration. Inadequate ANC increases the risk of delivering LBW infants . Access Logistic regression analysis was computed to determine the predictors of LBW in Nigeria. Among the factors tested, five risk factors were identified as significant for LBW. The risk factors were geopolitical zone, primiparous women, twin pregnancy, maternal weight less than 70 kg, and paternal employment. The most important risk factor associated with LBW in Nigeria was pregnant mothers living in the North West region. This might result from the facts that the presence of skilled attendants at births is particularly low and the number of teenage pregnancy is high in this region . MothersPregnant women exposed to tobacco products were at high risk of delivering LBW babies . HoweverLBW is an indicator of infant's survival, growth, and psychosocial development . In the The result of the current study had provided valuable information on the significant risk factors associated with LBW infants, based on the recent national survey in Nigeria. The findings from this study will provide insight for public health professionals and policy makers to implement strategies or intervention programs to reduce the prevalence of LBW in the future. Our study has some limitations. Recall bias is possible during data collection. Birth weights of the infants were obtained from written records from the hospital cards or mothers’ recall. Moreover, a low number of LBW babies were recorded in health facilities, because most births do not take place in these facilities, which may have resulted in an underestimation of the prevalence of LBW in this study.Paternal employment, geopolitical zone, parity, number of pregnancies, and maternal weight were the significant factors for LBW in Nigeria. Even though LBW is influenced by a multiplicity of factors, the incidence of LBW could be reversed if maternal risk factors are detected earlier and appropriate prevention strategies are delivered to the high-risk group. From a public health perspective, it is an advantage that most of these factors can be modified.Improvement in the ANC services in Nigeria is needed. Multi-faceted approaches could deliver better services to the pregnant mothers in Nigeria. Such approaches would include health education, maternal nutrition, improvement in socio-economic indices, and more and better-quality ANC services.


The relationship between these common and rare variants has yet to be explored. In this study, we tested whether children with ADHD with (N=60) a large (>500 kb), rare (<1% frequency) CNV differ by polygenic risk scores for ADHD to children with ADHD without such CNVs (N=421). We also compared ADHD polygenic scores in ADHD children with and without CNVs with a group of population controls . The results show that children with ADHD with large, rare CNVs have lower polygenic scores than children without such CNVs (odds ratio (OR)=0.73, P=0.023). Although ADHD children without CNVs had higher scores than controls , this difference was not observed for ADHD children with CNVs . These results are consistent with a polygenic liability threshold model of ADHD with both common and rare variants involved.Attention deficit hyperactivity disorder (ADHD) is highly heritable. Genome-wide molecular studies show an increased burden of large, rare copy-number variants (CNVs) in children with ADHD compared with controls. Recent polygenic risk score analyses have also shown that  However, an aggregate score  of common genetic variants was found to be, on average, higher in ADHD cases than in controls.8 For now, the power of this analysis is weak, though it is likely to improve with score alleles derived from future larger discovery GWAS samples, as has been seen in genetic studies of other psychiatric conditions, for example schizophrenia.7Attention deficit hyperactivity disorder (ADHD) is a childhood-onset neurodevelopmental disorder characterized by developmentally inappropriate levels of hyperactivity, impulsivity and inattention. Twin studies consistently indicate that it is highly heritable.9 However, only a small proportion of affected children have such large deletions or duplications and only a few specific loci  have been found to be significantly enriched in ADHD cases relative to controls.9 Nevertheless, there is evidence that CNVs in children with ADHD are enriched in gene sets that are also enriched for common variant associations, suggesting that common and rare variants disrupt similar biological pathways in ADHD, such as those involved in cholesterol homeostasis or central nervous system development.2 Also, a pathway analysis of the top SNPs identified in ADHD GWAS has implicated a protein network related to neurite outgrowth, which also appears to be disrupted by several CNVs previously reported in ADHD cases.10In terms of rare variants, several studies have implicated an excess of large (>100 kb or >500 kb), rare (<1% population frequency) copy-number variants (CNVs) in ADHD cases compared with controls.8 polygenic risk scores in a sample of children with ADHD for whom we also had CNV data. Risk scores were based on association data from an independent ADHD case–control GWAS.6 We hypothesized that children without a CNV would have a higher polygenic risk score for ADHD than those carrying a large and rare deletion or duplication.A question that has not yet been addressed is how common and rare genetic variants jointly contribute to individual liability. A polygenic liability threshold model of disorder implies that affected individuals with large, rare CNVs  should, on average, have lower polygenic risk loading than affected individuals without such CNVs. To address this question, we analysed previously derived2 ADHD was assessed using the Child and Adolescent Psychiatric Assessment,11 a semi-structured interview with parents, administered by trained research psychologists, under the weekly supervision of a child psychiatrist. Children with comorbid epilepsy, autism spectrum disorder (ASD), neurological or genetic disorders, Tourette's syndrome or bipolar disorder were excluded. Children with comorbid intellectual disability (ID), as defined by an IQ<70, were included in the sample (N=45). Ethical approval was obtained from the North West England and Wales Multicentre Research Ethics Committees and written informed consent/assent was obtained from parents and children. Controls were provided by the Wellcome Trust Case Control Consortium-Phase 2, consisting of 3000 individuals born in the UK during 1 week in 1958  and 3000 individuals from the UK Blood Services collection.12 Controls were unscreened for ADHD or other psychiatric problems and were not matched with the cases on the basis of any phenotypes as such data were not available.Our cases were UK-based children with a research diagnosis of ADHD.2 Analyses were based on the 502 542 SNPs, which passed quality control. The polygenic score and CNV data have been used for previous analyses in this sample; as such, the method is presented in brief, with further information on quality control, CNV calling procedures and polygenic score generation available from the previously published work.8 Polygenic scores were calculated in this sample using the Psychiatric Genomics Consortium ADHD GWAS meta-analysis  as the discovery sample.6 The polygenic scores for our primary analysis were calculated in PLINK13 for LD-pruned SNPs reaching a P-value threshold of <0.5 in the main discovery sample, as previously published.8 Polygenic scores were additionally calculated at several other thresholds  using the same method.Quality control of SNP data has been described previously.3 CNVs >500 kb were called in the sample using PennCNV and limited to those CNVs which had a population (cases and controls) frequency of <1%. Primary analyses were limited to these CNVs because they have better concordance across different genotyping platforms, and are determined with greater accuracy and are more robustly associated with neurodevelopmental disorders.3 Data on CNVs and polygenic scores were available for N=481 children with ADHD, aged 6–17 years , of whom N=67 (13.9%) were female. Control samples consisted of N=5081 individuals with SNP data, of whom N=4670  had both polygenic score and CNV data available. Of note, 12.5% (N=60) of the children with ADHD and 8.5% (N=397) of the controls had a rare CNV>500 kb.As reported previously,N=46 156 risk alleles (at threshold P<0.5) and were standardized using z-score transformations. A logistic regression was used to determine whether polygenic risk scores were higher in children with ADHD with CNVs than in those without. Following on from previous work,8 logistic regressions were also used to test whether higher polygenic scores predicted case or control status, separately for children with ADHD with and without CNVs. In line with previous analyses, two principal components  were entered into all the regression analyses as covariates to control for population stratification.8 All analyses were also co-varied for gender. For each association test, the amount of variance explained was calculated as the difference of Nagelkerke pseudo-R2 in the full regression model , as compared with the reduced model (containing only the population covariates and gender). All analyses were performed in Stata version 13.1.The polygenic scores were based on a set of N children with=139; N children without=342). Additive and multiplicative models of CNV presence and polygenic risk scores were also tested.The main analyses (comparing children with ADHD with and without CNVs) were re-run using polygenic scores based on the different thresholds . The number of SNPs in each of these sets was: 37 078, 28 005, 18 723, 9519 and 4845, respectively. The analyses were also re-run expanding the list of CNVs in cases to any that were >200 kb . A two-tailed permutation test of CNV status in the case group (N=481) using 10 000 replicates supported these results =0.0015). 8 limiting the analysis to children with CNVs (N=60) shows no difference between cases and controls , although this may be due to the small sample size of children with ADHD with large, rare CNVs. On the other hand, restricting the analysis to children with ADHD without large CNVs (N=421) confirms that these children have higher polygenic scores as compared with controls .Children with a CNV>500 kb had a significantly lower polygenic risk score than those without such a CNV (odds ratio (OR)=0.73, 95% confidence intervals (CI)=0.55–0.96, P-value thresholds for calculating polygenic scores demonstrated that significant effects are seen only at the least stringent P-value threshold (P<0.5), a finding that is consistent with other work based on polygenic score analysis when risk alleles are derived from modest sample sizes.14 See P<0.5) and including smaller CNVs>200 kb also showed higher polygenic scores in those without CNVs . Significant results were additionally attained at more of the risk allele thresholds  did not alter the main results .Secondary analyses using different olds see . ExcludiP=0.009, R2=0.002) and CNV presence  showed independent associations with case status. A multiplicative model adding an interaction term of ‘CNV presence × polygenic risk score' showed a significant interaction term .To test an additive model including both common and rare variants, a multivariate logistic regression was run with both ADHD polygenic risk scores and CNV presence as predictors, all the covariates, and with case status as the outcome. Both polygenic scores  require less of a polygenic burden of common alleles to manifest the disorder. The polygenic risk score loading was relatively low in ADHD children with large, rare CNVs. However, the difference in polygenic loading between them and controls was not significant, although this may have resulted from low power. The results of this study are consistent with a polygenic liability threshold model of ADHD, with both common SNPs and rare CNVs involved in ADHD disease risk. Additive and multiplicative models support these results. Both CNV presence and higher polygenic risk scores were found to be independently associated with case status. Furthermore, the significant interaction term in the multiplicative model supports the main result of this study in that those with a CNV and lower polygenic score are more likely to be cases.15 This suggests that there may be no clear and distinct forms of ADHD but further work examining this issue is needed. Interestingly, CNVs found in children with ADHD appear to disrupt similar biological processes to those hit by top GWAS SNPs,10 which suggests that even if there are different aetiological routes to ADHD, they may act on similar biological mechanisms.Although these results could be interpreted to suggest that there may be common and rare variant forms of ADHD, it is important to bear in mind that the effect sizes of this and previous studies are quite small and it is not yet clear whether these are truly distinct variants of the disorder. We have previously shown that children with ADHD with a large, rare CNV do not differ clinically (beyond a greater likelihood of ID) or in terms of family history of psychiatric disorders or parental ADHD symptoms from children with ADHD without such CNVs.17 it is possible that some individuals with both a pathogenic CNV and a greater than average polygenic loading were not recruited to the ADHD sample by virtue of having another primary neurodevelopmental disorder, which may share aetiology with ADHD  and would have meant that they did not meet study inclusion criteria. These inclusion criteria are typical of ADHD studies and are intended to increase sample homogeneity. The recent publication of the DSM-5 saw the removal of previous diagnostic exclusions relevant to ADHD . As use of the DSM-5 becomes more widespread, research samples for genetic analyses of childhood disorders, such as ADHD may become more clinically representative. Although studies do find an increased burden of potentially pathogenic CNVs in children with ID and ASD18 and another study has reported an increased polygenic loading of ASD common variants in an independent sample of children with ASD,19 it is not yet known whether children with more complex neurodevelopmental problems  are more likely to have both types of risk variants (common and rare) than children with ADHD without these comorbidities.Given the known pleiotropy of genetic risks for various neurodevelopmental, psychiatric and neurological outcomes,Children with CNVs and high polygenic scores may have been less likely to be ascertained for other reasons. For example, those who are most severely affected may have not agreed to participate, whereas those families in which the genetic loading is highest may also have more chaotic family structures, which is likely to be a barrier for study participation.9 Similarly, as ADHD GWAS sample sizes increase, so will the power to robustly detect associated common variants. The current analyses are limited by existing small sample sizes and would therefore benefit from replication in larger future samples.Future ADHD studies should aim to determine whether the pattern of results observed in the current study is driven by particular CNV risk loci confirmed to be relevant to ADHD. Current ADHD sample sizes are too small to implicate many specific CNV loci in the disorder.Like other psychiatric conditions , ADHD is an aetiologically complex and highly heritable condition. The results of the present study provide the first empirical molecular genetic support for the hypothesis that risk for ADHD follows a polygenic liability threshold model, in which individuals with large, rare CNVs require less loading of multiple common genetic risk variants for developing ADHD. A similar pattern, where the liability threshold for disorder is shifted downwards, might also be seen for other types of rare genetic mutations and will require future investigation.


Bartonella. Here, we evaluated the presence of Bartonella in ixodid ticks and domestic animals from Palestine. We tested 633 partly engorged ticks and 139 blood samples from domestic animals  for Bartonella using ITS-PCR. Bartonella DNA was detected in 3.9% of the tested ticks. None of the ticks collected from sheep and goats were positive for Bartonella. Seventeen R. sanguineus ticks  collected from dogs were infected with B. rochalimae (n = 10), B. chomelii (n = 6), and B. koehlerae (n = 1). Four H. dromedarri ticks  obtained from camels were infected with B. bovis (n = 2) and B. rochalimae (n = 2). Among canine blood samples (n = 110), we found one asymptomatic female dog to be infected with B. rochalimae (0.9%). The detection of zoonotic Bartonella species in this study should raise awareness of these vector-borne diseases among physicians, veterinarians and public health workers and highlight the importance of surveillance and preventive measures in the region.Ticks play an important role in disease transmission as vectors for human and animal pathogens, including the Gram-negative pathogen  Bartonella, which infect erythrocytes and endothelial cells in humans  of fleas collected from various animal hosts   . Severalchalimae .Rickettsia from the spotted fever group, Babesia and Hepatozoon . Ticks were identified based on morphological characteristics , located in three zones in the central, northern and southern regions of the country (eristics .n = 19), sheep (n = 7), and goats (n = 3) from Jenin, Nablus, and Jericho, in January 2016. None of the animals showed clinical signs; all were apparently healthy at the time of sampling. The animal owners were verbally informed about the goals of this research and the sampling protocol. All owners gave their written informed consent to collect blood from their animals. The ethics committee at Al-Quds University approved the study.A total of 110 blood samples were collected from outdoor domestic dogs, with simultaneous tick collection from the locations mentioned above . All samples were collected in EDTA-anticoagulant tubes and stored at -20°C until further use. Study animals were selected irrespective of sex and age. In addition, 29 blood samples were obtained from camels  following the manufacturer’s instructions. The eluted DNA (100 μl) was stored at -20°C until used as templates for PCR amplifications. DNA was extracted from whole blood (200 μl) following the QIAamp animal blood and Tissue Kit procedure , adjusted in 200 μl of Tris- EDTA (TE) buffer and stored at -20°C until further use.n = 772) targeting the Intergenic Transcribed Spacer (ITS) locus, using the following forward and reverse primers: (321s: 5′AGATGATGATCCCAAGCCTTCTGG and H493as: 5′-TGAACCTCCGACCTCACGCTTATC) as previously described , one female dog sample was positive for Bartonella, yielding a PCR-amplified ITS sequence that showed 99% sequence identity to the reference sequence of B. rochalimae (FN645466.1). None of the blood samples from camels (n = 19), sheep (n = 7), and goats (n = 3) were positive for Bartonella.Among the canine blood samples , Haemaphysalis adleri (n = 13), Rhipicephalus turanicus (n = 91), Rhipicephalus sanguineus (n = 391), Rhipicephalus bursa (n = 7), Rhipicephalus sp.(n = 20), Hyalomma dromedarii (n = 63), and Hyalomma impeltatum (n = 5)] were screened for Bartonella DNA.A total of 633 hard ticks  were obtained from 188 animals , residing in nine districts throughout Palestine. The geographic distribution of collected ticks was shown in Bartonella DNA by ITS-PCR. Of these 21 ticks were collected from dogs and four from camels. None of the ticks from sheep or goats were positive for Bartonella (Table 1). Identification of Bartonella was successful for 21 positive samples (84%). The initial attempt to sequence four amplicons failed and no additional DNA samples were available. Seventeen R. sanguineus ticks were infected with B. rochalimae (n = 10), B. chomelii (n = 6), and B. koehlerae (n = 1). All these ticks were obtained from dogs.Overall, 25 ticks  were positive for H. dromedarii ticks obtained from a single camel in Hebron and from two camels in Jericho were found to be infected with B. bovis (n = 2) and B. rochalimae (n = 2), respectively (Table 2). Bartonella species identification was based on the highest scoring BLAST hit on GenBank. The comparison of the PCR-amplified ITS sequences from the positive tick samples showed 97–99% sequence identity and 100% coverage when aligned against the reference sequences of B. bovis (KR733201.1), B. chomelii (KM215714.1), B. rochalimae (FN645466.1), and B. koehlerae (AF312490.1). Representative partial sequences of the 16S–23S ribosomal RNA intergenic spacer identified in the present study were deposited in GenBank under the following accession numbers: two B. rochalimae from a tick and its host (dog) , B. chomelii (KX420617), B. bovis (KX420618), and B. koehlerae (KX420616).Four Bartonella DNA in ixodid ticks and blood samples from domestic animals from Palestine. The overall prevalence of Bartonella DNA in ticks (3.9%) was in agreement with previous screening undertaken worldwide including Czech Republic, United States, Italy and Thailand . We examined seven hard tick species for Bartonella and found evidence of the pathogen in two of them: the brown dog tick, Rhipicephalus sanguineus and the camel tick, Hyalomma dromedarii. These findings represent the first detection of Bartonella in Ixodid ticks from the Middle East. Cases of human parasitism by the brown dog tick—the most widespread tick in the world—are well documented . The dog that provided the sample was apparently healthy, showing no signs of bartonellosis. Detection of Bartonella in asymptomatic dogs has also been reported from Peru  than in the dog blood (0.9%) suggesting that Bartonella be carried in partially engorged adult ticks. These findings provide highly suggestive molecular evidence that R. sanguineus ticks can act as vectors of animal-associated Bartonella infection in Palestine. Furthermore, although the role of dogs as source of human Bartonella infection remains unclear, we speculate that they may present a risk for zoonotic transmission similar to that seen with B. henselae in cat scratch disease  appear to be at particular risk of exposure to rom Peru . We iden disease .Bartonella in camelids, which has been named Bartonella dromedarii sp. nov  in ticks obtained from camels, although we did not detect Bartonella DNA in blood samples from these camels. However, we cannot rule out bloodstream infections in the camels as the source of Bartonella in the ticks, as the blood samples were not taken at the same time as the ticks. Furthermore, even if camels are not reservoir hosts for these Bartonella species, they may have an important role as mechanical dispersers of infected ticks.Recently, a study conducted in Israel confirmed the presence of a novel species of  sp. nov . In our Bartonella in camel ticks from the species Hyalomma dromedarii is worrying because other species of hard ticks from the genus Hyalomma clearly bite humans (H. dromedarii can do so too2. Other lines of evidence support the role of ticks in the natural cycles of some Bartonella species including those pathogenic for humans (Bartonella. The pathogen can then survive in the midgut of ticks during molting and can be transmitted through feeding to an uninfected host.Our discovery of e humans  and therr humans . In partB. chomelii—a pathogen first isolated from French domestic cattle (Bartonella species infecting cattle grazing in Spain pastures (B. koehlerae in a R. sanguineus tick obtained from dog. This species was first isolated from the blood of two pet cats in California (In the present study, brown dog ticks were found to be infected with c cattle  and founpastures . We alsolifornia . Since tlifornia , has beelifornia . Howeverlifornia .Bartonella -positive ticks we obtained from a dog harbored Rickettsia, a pathogen that is known to be tick-transmitted (Bartonella is often found in ticks alongside well-known tick-transmitted organisms such as Anaplasma, Borrelia, and Rickettsia (Bartonella can be transmitted by ticks include a US report that dogs infected with Bartonella were also seropositive for Anaplasma phagocytophilum (Borrelia burgdorferi after tick bites also carried Bartonella DNA in their blood (One of nsmitted . This fickettsia . Other stophilum  and a huir blood .Bartonella species in this study should increase the awareness of these vector-borne diseases among physicians, veterinarians and public health workers and highlight the importance of surveillance and preventive measures in the region. Additional epidemiologic surveys are required to enhance our understanding of the transmission dynamics of Bartonella in Palestine and in other parts of the Middle East.The detection of zoonotic Conceived and designed the experiments: SE, AN, and ZA. Performed the experiments: SE, AA, and TZ. Analyzed the data: SE and AA-J. Wrote the first draft of the manuscript: SE. Directed, revised, and contributed to the writing of the manuscript: AN and V-TM. Final revision and approval of the manuscript to be published: SE, AN, V-TM, AA, AA-J, TZ, KA, and ZA.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Objective: In children with attention-deficit/hyperactivity disorder (ADHD), an increased theta/beta ratio in the resting EEG typically serves as a rationale to conduct theta/beta neurofeedback (NF) training. However, this finding is increasingly challenged. As NF may rather target an active than a passive state, we studied the EEG in a condition that requires attention.Methods: In children with ADHD of the DSM-IV combined type  and of the predominantly inattentive type  and in typically developing children (N = 19), EEG spectral analysis was conducted for segments during the attention network test (ANT) without processing of stimuli and overt behavior. Frontal , central  and parietal  electrodes were included in the statistical analysis. To investigate if EEG spectral parameters are related to performance measures, correlation coefficients were calculated.Results: Particularly in the ADHD-C group, higher theta and alpha activity was found with the most prominent effect in the upper-theta/lower-alpha (5.5–10.5 Hz) range. In the ADHD-I group, a significantly higher theta/beta ratio was observed at single electrodes  and a tendency for a higher theta/beta ratio when considering all electrodes (large effect size). Higher 5.5–10.5 Hz activity was associated with higher reaction time variability with the effect most prominent in the ADHD-C group. A higher theta/beta ratio was associated with higher reaction times, particularly in the ADHD-I group.Conclusions: (1) In an attention demanding period, children with ADHD are characterized by an underactivated state in the EEG with subtype-specific differences. (2) The functional relevance of related EEG parameters is indicated by associations with performance (reaction time) measures. (3) Findings provide a rationale for applying NF protocols targeting theta  activity and the theta/beta ratio in subgroups of children with ADHD. Theta/beta training belongs to the neurofeedback (NF) protocols which are frequently applied in children with ADHD; for review see Arns et al.  and GeveSpecificity of training effects is further supported by findings at the neurophysiological level  have been conducted since the 1980s and reviewed e.g., in Barry et al. . Consistca. 90%; Monastra et al., The theta/beta ratio measured at Cz was reported to discriminate reliably between children with ADHD and controls  participants.Applying theta/beta training was thought to “normalize” the cortical slowing. However, recent studies question if the major part of children with ADHD are actually characterized by an increased theta/beta ratio in the resting EEG. Arns et al.  conducteThis point of view is further supported by two studies, which were published after this meta-analysis and did not find differences between children with ADHD and typically developing children in any frequency band considered . For example, training may also comprise trials combined with tasks . In this respect, it appears to be more relevant to consider the EEG during task processing though it has to be kept in mind that the resting EEG does not only reflect a trait but also a state marker  alertness and free of stimulus processing. We expected that children with ADHD show increased theta activity and/or an increase theta/beta ratio across the scalp surface during an attentive state serving as a rationale to apply related protocols in NF training.We were also interested in comparing DSM-IV subtypes of ADHD. In order to learn more about the functional significance of the spectral EEG parameters, we studied associations (correlations) between these spectral EEG parameters and performance measures (particularly reaction time measures).Fifteen children with ADHD of the combined type , nine children with ADHD of the predominantly inattentive subtype (ADHD-I) and 19 typically developing children were included in the study. Children had to be aged 8–11 years and to have a full-scale IQ of at least 80. All children had normal or corrected-to-normal vision. Adequate task performance in the ANT and sufficient EEG data quality was also necessary to be included in this study (for details see below). The three groups  were comparable regarding age and sex  and took part in a medication trial  > 46.9; p < 0.001). For the two ADHD groups (ADHD-C vs. ADHD-I), the FBB-HKS total score (t(22) = 1.38, n.s.) and the score for the inattention subscale (t(22)= −0.20, n.s.) were comparable. However, the score for the hyperactivity/impulsivity subscale was higher in the ADHD-C group (t(21) = 2.45; p < 0.05).For all children, the German ADHD rating scale . During EEG preparation, the children could watch age-appropriate films. The ANT, which consisted of four blocks of 48 trials each, lasted about 15 min (including short breaks between the task blocks). During the test brain electrical activity was recorded. The children received standardized instructions before performing a practice block of 24 trials. After each task block, a summary of the task performance was shown on the screen.Children were instructed to “feed” a hungry fish that would appear above or below a fixation cross. If the fish pointed to the right (resp. left) side, the children had to press the right (resp. left) mouse button in order to feed the fish. This target fish was the center fish in a row of five fish with the flanking fish either looking in the same direction (congruent condition) or in the opposite direction (incongruent condition).One of three cue conditions  preceded the presentation of the fish: in the NeutralCue condition, an asterisk at the center of the screen indicated that the target fish was about to appear soon. In the SpatialCue condition, an asterisk was presented at the location of the target fish, indicating not only that the target was about to appear soon but also its location on the screen. In the NoCue condition, the fish were presented without a cue stimulus.A schematic illustration of the ANT as applied in the present study, including technical details is presented in Figure A Brainamp recording system  was used. Brain electrical activity was recorded from 23 sintered Ag/AgCl electrodes . Positions for reference and ground electrode were FCz and CPz, respectively. Vertical and horizontal electrooculogram was recorded from electrodes placed above and below the right eye and at the outer canthi. A sampling rate of 500 Hz was used. Filter bandwidth at recording was 0.016–120 Hz. Impedances were kept below 20 kΩ.For preprocessing and data analysis, the VisionAnalyzer software  was used. After applying a 50 Hz notch filter and bandpass filtering , eye movement artifacts were corrected using independent component analysis  = 2.13, p = 0.13).For each child, at least 20 artefact-free segments (followed by a correct response to the target stimulus) had to be available. The number of segments without artefacts were slightly but not significantly smaller in the ADHD groups , alpha 7.5–12.5 Hz), beta (12.5–20 Hz) band as well as the theta/beta ratio were calculated at different electrodes . In Figure .5 Hz, bet-tests were applied for post hoc analysis (pairwise comparisons of two groups) using Bonferroni-Holm correction to control for multiple comparisons.Performance data  were analyzed using a one-way ANOVA with between-subject factor GROUP . Post hoc analysis also comprised correction for multiple comparisons (Bonferroni-Holm). Corrected p-values are reported.For the different EEG frequency bands, repeated-measure ANOVAs were computed with between-subject factor GROUP and repeated-measurement (electrode) factors Y  and X  to test potential topography/laterality effects. Associations between EEG spectral parameters and performance measures were studied focusing on those EEG measures for which largest group-specific effects were obtained in the before-mentioned analysis. We controlled for age-related effects. However, as the portion of 8 year-old children was higher in the ADHD groups, controlling/correcting for age-related changes by considering the complete sample would lead to an overestimation of age-related changes at the cost of group-related effects. Instead, we decided to correct for age-related changes in the complete sample based on the regression coefficient of the control group. Pearson’s correlation coefficients were calculated for potentially age-corrected measures. If significant correlations were found for the complete sample, we also tested ADHD groups separately to exclude spurious correlations.IBM SPSS Statistics (Version 20.0) was used for statistical analysis.t(26) = −3.32; p (corr.) = 0.009; control vs. ADHD-C: t(32) = −2.61; p (corr.) = 0.03). For the median of reaction times, a GROUP effect was obtained due to higher reaction times in the ADHD-I group in comparison to the control group (control vs. ADHS-I: t(26) = −3.71; p (corr.) = 0.003) as well as the ADHD-C group (ADHD-C vs. ADHD-I: t(22) = −2.36; p (corr.) = 0.05).Results of performance measures are summarized in Table *Y related to the topography of the EEG activity in the different frequency bands. Theta, alpha and 5.5–10.5 Hz activity were highest at electrode Pz ; see also Figure Results of the ANOVAs for the different frequency bands are summarized in Table Post hoc analysis revealed that 5.5–10.5 Hz activity was higher particularly in the ADHD-C group (control vs. ADHD-C: t(32) = −3.35; p (corr.) = 0.006) and to a smaller extent in the ADHD-I extent in the ADHD-I group (control vs. ADHD-I: t(26) = −2.17; p (corr.) = 0.08, p (uncorr.) = 0.04).For theta activity, alpha activity and particularly upper-theta/lower-alpha (5.5–10.5 Hz) activity, significant group main effects indicated higher activity in the ADHD groups. η2 = 0.09) may indicate some effect which did not turn out to be significant due to the limited sample size. So, we decided to look at the theta/beta ratio in more detail in an exploratory way. At least a tendency for a higher theta/beta ratio (averaged over the nine electrodes) in the ADHD-I group (control vs. ADHD-I: t(26) = −2.0; p = 0.057; Cohen’s d = 0.8) was obtained whereas no effects were observed for the ADHD-C group (control vs. ADHD-C: t(32) = −1.02; n.s.). When group at single electrodes, significant effects were found for electrodes F3 and Fz (control vs. ADHD-I: t(26) ≤ −2.28; p ≤ 0.03).No significant main effect for the theta/beta ratio was found. However, a medium effect size : Higher activity in the 5.5–10.5 Hz band was associated with higher reaction time variability  and reaction time variability ; see Figure r = 0.54).A significant correlation was also obtained between the theta/beta ratio  and the median of reaction times  measures with deviations in the ADHD groups. It has to be noted that these correlations did not reach statistical significance in the respective ADHD group due to the small group sizes.In the present study, we conducted EEG spectral analysis during an attention demanding period in children with ADHD . Deviant EEG patterns were obtained with subtype-specific differences between the DSM-IV combined type and the predominantly inattentive subtype.In contrast to recent resting-EEG studies , we had already reported different distributions of cue-P3 single trial amplitudes for the two ADHD groups further strengthening this point of view.Interestingly, significant (positive) correlations between those spectral EEG parameters and reaction time measures  were found for which differences between the ADHD groups and the control group had been obtained. These associations suggest a functional relevance of the EEG parameters, particularly in the context of ADHD: a suboptimal neural state at stimulus presentation results in impaired performance. As we controlled for age effects and also considered the ADHD groups separately, it seems rather unlikely that the correlations obtained for our data reflect spurious correlations.g = 0.76). Increased activity in the upper-theta/lower-alpha band, which may be interpreted as an underactivated neural state, could reflect a neural mechanism underlying increased reaction time variability in ADHD besides top-down control and motor preparation processes . In this respect, findings of the present study may have the following implications for NF training in ADHD.In children with ADHD of the combined type, an upper-theta/lower-alpha (5.5–10.5 Hz) protocol associated with an attentive state may be more effective than theta/beta training. It will have to be studied if indication criteria for the use of a specific protocol based on  EEG characteristics at pre-training can be developed. As theta activity in the resting EEG at pre-training was found to be a predictor for the effects of theta/beta training .It has to be taken into consideration that frontal midline theta , it will have to be tested whether refined frequency bands and feedback parameters, respectively, may also be more characteristic for children with ADHD.Findings are limited by the generally small sample size. However, we’d see sample size more critical if findings had not turned out to be significant. Large effect sizes were obtained and effects were not just due to outliers suggesting clear differences in the distribution of control and ADHD groups. In any case, larger samples will have to be studied to see if results are confirmed and to what extend EEG-based subtypes can be found.We could not compare resting and active EEG conditions directly. Thus, it cannot be excluded that corresponding effect sizes could have also been found in the resting EEG of our sample. However, in our opinion, our findings complement/are compatible with results of recent studies that either report no significant global differences in recent resting EEG studies  range in children of the combined type, hints for an increased theta/beta ratio were found in children of the predominantly inattentive subtype. The functional relevance of these EEG parameters was indicated by associations with reaction time measures, which were pronounced most in the ADHD groups. Findings may provide a rationale for applying NF training protocols targeting theta activity and theta/beta ratio in subgroups of children with ADHD to achieve an attentive state. In this respect, it will be interesting if indication criteria for a specific protocol in an individual child can be developed which can be applied in clinical practice.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Objectives. (1) To assess the current presence of ADHD symptoms among patients seeking treatment for gambling disorder; (2) to explore clinical and sociodemographic differences between patients who score high and low on the measure of ADHD symptoms; (3) to analyze whether the presence of ADHD symptoms is associated with more severe psychopathology and with specific personality traits; (4) to analyze the mediating role of ADHD symptoms in the relationship between novelty seeking and gambling severity. Method. A total of 354 consecutive patients were administered an extensive battery assessing gambling behavior, psychopathology, and personality traits. Results. Male and female gamblers did not differ significantly in their mean scores on the ADHD measure. However, younger participants aged 18–35 scored higher. Higher ADHD scores were also associated with greater severity of gambling disorder and more general psychopathology. Regarding personality traits, high persistence and self-directedness were negatively related to ADHD scores, while in women alone a positive correlation was found between ADHD scores and scores on harm avoidance and self-transcendence. Conclusion. The presence of ADHD symptoms in both male and female gambling disorder patients may act as an indicator of the severity of gambling, general psychopathology, and dysfunctional personality traits. Diagnostic and Statistical Manual of Mental Disorders (DSM-5) ; p = .679).Finally, participants whose main problem was slot machines did not differ from those with other types of gambling problem . Regarding the level of psychopathology, higher ADHD scores were positively associated with higher SCL obsessive-compulsive symptom scores  and also with higher SCL GSI scores, although the latter relationship was only significant for middle-aged  and older patients . Finally, the TCI-R dimensions harm avoidance, in females , and self-transcendence  were positively related to ADHD scores , whereas persistence  and self-directedness  were negatively associated with ADHD scores .χ2 = 2.28 (p = .131), RMSEA = .063, CFI = .986, TLI = .900, and SRMR = .018. The global predictive capacity was also good (CD = .166).B = 0.306, SE = .050, z = 6.10, and p < .001), as well as an indirect effect mediated through the ADHD-total score: high novelty seeking scores were predictive of high ADHD scores , and high ADHD scores were related to high gambling severity . Patients' age was negatively related to ADHD symptoms , but age did not show a direct effect in relation to the DSM-total criteria . The path diagram shows that the ADHD-total score mediated the relationship between novelty seeking and gambling severity, as well as the relationship between age and gambling score.Novelty seeking showed a direct effect on gambling severity  were all more common among patients with high ADHD scores. Previous studies have similarly found an association between adult ADHD and substance abuse , 65, impRegarding personality traits, our results showed that high scores on harm avoidance were related to ADHD, although only in women, whereas the positive association between ADHD and the character trait self-transcendence was observed for both sexes. Several studies have also described the association between ADHD and specific personality traits such as neuroticism  anxiety The analysis also revealed that high levels of persistence and self-directedness were negatively associated with the presence of ADHD. Davtian et al.  also comThe study by Davtian et al.  also fouFinally, the path diagram analysis indicates that ADHD symptoms mediate the relationship between novelty seeking and severity of gambling behavior, as well as the relationship between the latter and age. These results support the findings of previous studies showing a direct relationship between ADHD and excitement-seeking . The conThis study has several limitations that need to be considered when interpreting the findings. First, the sample comprised treatment-seeking GD patients and therefore the results might not be generalizable to individuals with a GD who do not seek treatment. Second, the assessment procedures used did not allow for in-depth evaluation of comorbid disorders. Third, the cross-sectional design prevents us from establishing the direction of causality among the variables assessed. Fourth, the frequency of adult ADHD symptoms was estimated using a self-report screening instrument, which might explain the elevated prevalence of ADHD symptomatology in the study sample. Related to this limitation is the fact that the presence of childhood ADHD was not assessed, which would have been a prerequisite for an ADHD diagnosis in adulthood. Nevertheless, the ASRS has good psychometric properties to screen ADHD in adults, also in patients with comorbidities . Personality functioning was also based on self-report measures. While acknowledging these limitations, it is noteworthy that the study gathered information from a relatively broad clinical sample formed by consecutive treatment-seeking individuals with a GD and a path model was developed to describe the associations between sociodemographic characteristics, personality, ADHD, and GD severity.The presence of ADHD symptoms in gambling disorder patients could be an indicator of the severity of gambling, general psychopathology, and dysfunctional personality traits. Furthermore, the findings of the path model suggest that both ADHD and GD severity are driven by high levels of novelty seeking and young age. Intervention programs should focus on training in self-control strategies and skills for dealing with negative emotions, on ways of increasing tolerance to boredom and maintaining persistence, on improving planning skills and learning to delay both responses and rewards, and on encouraging alternative thought and decision-making processes. Moreover, these results confirm the importance of developing preventive and early detection strategies for GD in adolescence since, in this stage of life, self-control, and self-regulation skills have not yet been consolidated and teens have a higher risk and greater vulnerability to being exposed to activities such as gambling.


TANGO1 binds and exports Procollagen VII from the endoplasmic reticulum (ER). In this study, we report a connection between the cytoplasmic domain of TANGO1 and SLY1, a protein that is required for membrane fusion. Knockdown of SLY1 by siRNA arrested Procollagen VII in the ER without affecting the recruitment of COPII components, general protein secretion, and retrograde transport of the KDEL-containing protein BIP, and ERGIC53. SLY1 is known to interact with the ER-specific SNARE proteins Syntaxin 17 and 18, however only Syntaxin 18 was required for Procollagen VII export. Neither SLY1 nor Syntaxin 18 was required for the export of the equally bulky Procollagen I from the ER. Altogether, these findings reveal the sorting of bulky collagen family members by TANGO1 at the ER and highlight the existence of different export pathways for secretory cargoes one of which is mediated by the specific SNARE complex containing SLY1 and Syntaxin 18.DOI:http://dx.doi.org/10.7554/eLife.02784.001 Collagens are long proteins that join individual cells together to build tissues and organs. They also provide strength and elasticity to bones, tendons, and blood vessels. Like many other proteins, collagens are produced inside cells: they are folded in a compartment called the endoplasmic reticulum, and then packaged and transported to another compartment called the Golgi. Collagens are then directed from the Golgi to their final destination, which is typically the outside of the cell.Small proteins travel from the endoplasmic reticulum to the Golgi inside packages called vesicles. However it is not clear how large proteins like collagens are transported between these two compartments. It is known that a protein called TANGO1 is needed to direct a collagen called Procollagen VII to the outside of the cells. TANGO1 binds to Procollagen VII, and it is thought that TANGO1 delays the release of Procollagen VII from the endoplasmic reticulum, so that the vesicle can grow to a size that is able to accommodate such a bulky cargo.Nogueira, Erlmann et al. have now discovered that TANGO1 binds to another protein called SLY1, and that this protein must also be present if Procollagen VII is to be exported from the endoplasmic reticulum. In contrast, the transport of a different type of collagen—Collagen I—does not require TANGO1 or SLY1.SLY1 helps to fuse the membranes that enclose the structures involved in protein trafficking—such as the endoplasmic reticulum, the Golgi, and the vesicles—and this allows the cargoes of vesicles to pass from one compartment to another. Nogueira, Erlmann et al. also found that a second protein  is also required for the export of Procollagen VII.Nogueira, Erlmann et al. propose that collagen VII export involves TANGO1 delaying the release of collagen from the endoplasmic reticulum so that SLY1 and Syntaxin 18 can fuse other cellular membranes to the growing transport vesicle. Following this work, the next challenge is to uncover how different types of collagens are separated from each other, and identify which specific vesicles are involved in their export.DOI:http://dx.doi.org/10.7554/eLife.02784.002 Collagens are the most abundant secretory proteins, comprising 25–30% of the human body dry weight . They arDrosophila melanogaster  from the ER in tissue culture cells . The knonogaster . TANGO1 nogaster . On the nogaster , 2011. Wnogaster . Upon grnogaster , 2011. Unogaster . Sedlin nogaster . TANGO1 SLY1, an essential gene, has been described as a single copy suppressor of the YPT1 deletion (We now show the involvement of SLY1 (or SCFD1) in specific ER export events. SLY1 is a member of the STXBP/unc-18/SEC1 family of proteins that regulate the assembly or the activity of SNAREs in membrane fusion events . The yeadeletion  and impldeletion . In contdeletion . In mammdeletion  togetherdeletion  and Syntdeletion . SLY1 hadeletion . It is iWe describe in this study, our data that reveal the existence of different export routes for secretory cargoes from the ER: of specific interest is the finding that SLY1 and the ER specific t-SNARE STX18 are necessary for the export of PC VII but not of the equally bulky PC I from the ER.To search for proteins that interact with cytoplasmically oriented portions of TANGO1, we expressed a Myc-His tagged version of a truncated form of TANGO1 (TANGO1ct) that lacks the luminal domain in HeLa cells. After crosslinking with membrane permeable DSP and lysis, proteins were recovered on a Nickel agarose column and analyzed by mass spectrometry. Of interest was the finding of SLY1 in the pool of proteins cross-linked to TANGO1.To further ascertain the mass spectrometry data, we immunoprecipitated Myc-His-tagged TANGO1ct from transfected and crosslinked HeLa cells as described above and western blotted the bound material with anti-Myc and SLY1 antibodies, respectively. Our data show the presence of SLY1 in the TANGO1 immunoprecipitate . SLY1 isThe identification of SLY1 in a pool of proteins that are cross linked to TANGO1 prompted us to test its involvement in PC VII export from the ER. In addition to the RDEB/FB/C7 cell line in which Collagen VII was exogenously introduced, we tested the immortalized human esophageal cell line Het1a to monitor the involvement of SLY1 in the export of endogenous PC VII. RDEB/FB/C7 and Het1a cells were transfected with siRNA oligos specific for SLY1, TANGO1, or a scrambled oligo as a control. RDEB/FB/C7 cells were transfected twice to increase knockdown efficiency. The levels of SLY1 and TANGO1 after knockdown were monitored by western blotting and revealed an approximately 90% reduction compared with control cells. Knockdown of TANGO1 did not significantly alter SLY1 protein levels and vice versa . 48 hr aWe then transfected RDEB/FB/C7 or Het1a cells with SLY1 siRNA, TANGO1 siRNA, or a control siRNA, as described above, treated the cells with ascorbic acid and after 72 hr the cells were fixed and incubated with antibodies against Collagen VII, the ER-resident chaperone Hsp47 and GM130. In control cells, we did not detect intracellular PC VII because it is rapidly exported from the ER and secreted by the cells. In SLY1 knockdown cells, on the other hand, PC VII accumulated in large structures that also contained the collagen-specific protein chaperone Hsp47 . SimilarTo further ascertain the site of PC VII localization in SLY1 knockdown cells, we visualized PC VII by immunoelectron microscopy. In control cells, gold-conjugated secondary antibodies used to visualize anti-Collagen VII antibody were found distributed throughout the ER. In SLY1 knockdown cells, however, PC VII was observed in large patches within dilated ER  but smalWe then tested whether SLY1 knockdown affected the stability of proteins involved in the transport of cargoes between the ER and the Golgi. For this, we monitored the levels of COPI and COPII components in the lysates of SLY1, TANGO1 knockdown or control siRNA-treated RDEB/FB/C7 cells. The level of SAR1, the initiator of the COPII vesicle budding process, did not change upon SLY1 or TANGO1 knockdown. Also unchanged were the levels of the other COPII components that are required for the export of secretory cargo, including components of the extracellular matrix, from the ER, specifically SEC23A  and SEC3We tested the involvement of SLY1 in retrograde trafficking by monitoring the localization of a protein called GRP78/BIP that contains a KDEL sequence and cycles between the ER and the early Golgi cisternae. A block in retrograde transport leads to an accumulation of GRP78/BIP in the Golgi apparatus and its subsequent secretion from the cells . GRP78/BRetention of PC VII in the ER, upon knockdown of SLY1 or TANGO1, is therefore not because of a defect in cycling of a component that is specifically required for the constitutive trafficking between the ER and the early Golgi cisternae.35S-methionine for 20 min in methionine free medium, washed and then incubated in complete medium. Another control was included in which scrambled siRNA oligo transfected cells were incubated with Brefeldin A (BFA) during the chase period. The medium was collected after 2 hr and then analyzed by SDS-PAGE/autoradiography to identify the secreted polypeptides. Treatment with BFA, as expected, inhibited the secretion of newly synthesized proteins. Surprisingly, SLY1 knockdown did not have any appreciable effect on the secretion of newly synthesized proteins . A TANGO1 like protein called MIA2 that is expressed only in the liver and small intestine has also been identified . The carDrosophila S2 cells . The neck of this large PEB, concentrated in COPII coats, undergoes fission by the same mechanism as that of a standard COPII vesicle.Our surprising finding is that SLY1, which is known to interact with STX18, a t-SNARE for membrane fusion , is requThe PC VII enriched large membrane compartment separated from the ER also contains recycled ERGIC membranes. This membrane bound compartment in principle is the first post ER compartment that matures to move PC VII in the anterograde direction for secretion.Interestingly, Luini et al. have reported that export of PC I from the ER requires its concentration at a domain close to the ER exit sites, which they suggest protrudes and then separates from the ER to generate a PC I enriched carrier . Their mTANGO1 constructs were as described previously .SLY1 was amplified by RT-PCR from the total mRNA of HeLa cells. SLY1 was cloned into the retroviral vector pLJM1-GFP  using Gibson cloning strategy (www.snapgene.com) was used for molecular cloning procedures. The Ii-FRAP-HA plasmid has been previously described  or X-tremeGENE9  according to the manufacture's protocols. siRNAs were transfected in HeLa, RDEB/FB/C7, Het1a and Saos2 with Hiperfect  according to the manufacture's protocols. In the case of RDEB/FB/C7, siRNAs were transfected twice, on day 0 and on day 1. For lentiviral infection of SLY1-GFP into RDEB/FB/C7 cells, lentiviral particles were produced by co-tranfecting HEK293 cells with a third generation packaging vector pool and pJLM1-SLY1-GFP using TransIT-293 (Mirus Bio LLC). At 72 hr post transfection the viral supernatant was harvested, filtered, and directly added to RDEB/FB/C7 cells.RDEB/FB/C7 cells, HeLa, Het1a, and Saos2 cells were grown at 37°C with 5% COsiRNAs for SLY1, TANGO1, STX5, STX17, STX18, and SAR1 A and B were purchased from Eurofins MWG Operon : SLY1 siRNA- AGACUUAUUGAUCUCCAUA; TANGO1 siRNA- GAUAAGGUCUUCCGUGCUU; STX5 siRNA- GGACAUCAAUAGCCUCAAC; STX17 siRNA- GACUGUUGGUGGAGCAUUU; STX18 siRNA- CAGGACCGCUGUUUUGGAUUU; for knockdown of SAR1A+B a pool of 4 siRNAs were used as previously described . ControlAntibodies used in conventional western blotting, immunofluorescence, and immunoelectron microscopy are the following: SLY1, Collagen VII, Collagen I, GRP78/BIP, SEC23A ; TANGO1 ; SAR1 ; HSP47 ; SEC31, GM130 ; β-COP, myc, Tubulin, Actin ; HA ; ε-COP (a gift from Dr Rothman).g for 10 min at 4°C. The lysates were then incubated with Ni+-Agarose (Qiagen) beads for 2 hr at 4°C on a rotating platform. The beads were washed three times with lysis buffer and the specifically bound proteins eluted using PBS containing 500 mM Imidazol.HeLa cells were transfected with a plasmid encoding MycHis-tagged cytoplasmatic tail of TANGO1. 24 hr post transfection proteins were cross linked by incubation with the cell permeable cross linker DSP  for 30 min at room temperature. Residual cross linker was quenched by adding Tris-HCl to a final concentration of 150 mM. The cells were scraped into lysis buffer  and lysates were clarified by centrifugation at 16,000×RDEB/FB/C7 or Saos2 cells were transfected with siRNA as described above. 24 hr after the last siRNA transfection, the medium was replaced with a fresh medium containing 2 μg/ml ascorbic acid. Fresh medium containing ascorbic acid was added again to the cells 20 hr before being collected. Upon collection, the media were centrifuged at low speed to remove any cell in suspension and the supernatant boiled for 5 min with Laemmli SDS-sample buffer. For cell lysis, the cells were washed with PBS, lysed and centrifuged at 14,000 rpm for 15 min at 4°C. The supernatants were boiled for 5 min with Laemmli SDS-sample buffer. Both media and cell lysate were subjected to SDS-PAGE (6% acrylamide) and western blotting with Collagen VII, Collagen I, and Tubulin antibodies. ImageJ  was used for quantitation.Cells grown on coverslips were fixed either with cold methanol for 10 min at −20°C or with 4% paraformaldehyde in PBS for 10 min followed by permeabilization with 0.2% Triton X100 at room temperature, and then incubated with blocking reagent (Roche) for 30 min at room temperature. Primary antibodies were diluted in blocking reagent and incubated overnight at 4°C. Secondary antibodies conjugated with Alexa 488, Alexa 594 or Alexa 647 were diluted in blocking reagent and incubated for 1 hr at room temperature. For SLY1 localization studies, RDEB/FB/C7 expressing SLY1-GFP were transfected with the indicated siRNAs; after 72 hr after transfection cells were processed for immunofluorescence. Briefly, cells were washed twice with room temperature KHM buffer . Cells were then permeabilized by incubation in KHM containing 0.1% Saponin for 5 min on ice followed by wash for 7 min at room temperature with KHM buffer. Cells were subsequently fixed in 4% paraformaldehyde and processed for immunofluorescence microscopy. Images were taken with a Leica TCS SPE confocal with a 63x objective. SEC31 and TANGO1 localization images on 3 blocks. The blocks were infused with 2.3 M sucrose at 4°C overnight, frozen in liquid nitrogen and stored until cryo-ultramicrotomy. Sections (∼50 nm-thick) were cut at −120°C with a diamond knife in a Leica Ultracut T/FCS. Ultrathin sections were picked up in a mix of 1.8% methylcellulose and 2.3 M sucrose (1:1). tc\l 1 ‘Preparation of immunogold labeled cryosections’. Cryosections were collected on carbon and formvar-coated copper grids and incubated with rabbit polyclonal antibodies against Collagen VII, followed by protein A-gold. After labeling, the sections were treated with 1% glutaraldehyde, counterstained with uranyl acetate pH 7 and embedded in methyl cellulose–uranyl acetate pH 4 (9:1).Cryoimmunoelectron microscopy was performed as described previously . ControlHeLa cells were transfected with the respective siRNAs as mentioned above. Past 48 hr, the cells were transfected with FKBP-ERGIC53-GFP and Ii-FRAP-HA plasmids as described previously . 24 hr a35Smethionine  for 20 min. The cells were washed three times with PBS and chased with DMEM containing 10 mM unlabeled methionine for 2 hr. For BFA treatment, 10 µg/ml BFA was added to the medium for the last 10 min of incubation and kept for the whole experiment. Cells were lysed with PBS containing 1% Triton X-100. 20 µl of cell extracts was mixed with scintillation cocktail, and the radioactivity was determined for normalization. The collected medium was precipitated with TCA and analyzed by SDS-PAGE/autoradiography.Control and siRNA-treated cells were cultured in DMEM without L-methionine and L-cysteine for 60 min and pulsed with 100 µCi g for 15 min. For the chemiluminescence assay, 50 μl of medium and cell lysate were mixed with an ECL reagent (Thermo Scientific) and luminescence measured with a Victor 3 plate reader .HeLa cells stabily expressing ss-HRP were cultured in a 12-well plate and transfected with control siRNA, SLY1 or TANGO1-specific siRNA oligos. 72 hr later, cells were washed with medium and incubated with 1 ml of complete medium at 15°C for 2 hr. Then, cells were incubated with 500 μl of complete medium at 37°C in presence of 100 μM CHX. At the time points indicated, the medium and cells were harvested for HRP secretion assay. 50 μl of the medium was collected and cells were lysed in 100 μl of lysis buffer , 1% Nonidet P-40 (NP- 40) and 0.5% sodium deoxycholate) supplemented with protease inhibitors, 1 mM Na3VO4, and 25 mM sodium fluoride and centrifuged at 16,000× review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  that it acts after TANGO1. Syntaxin 5, but not 18, is required for collagen 1 secretion.eLife? Less is known than one would expect about the molecular basis of ER export in mammalian cells, and this work again highlights the interesting fact that multiple export pathways do indeed exist. If the paper were to teach us what Sly1 does in this process, there would be no hesitation. Nevertheless, the fundamental importance of the process and the molecular clarification of ER export sorting lean in favor of publication by eLife.Is this suitable for Issues that need to be addressed:1) The data are convincing regarding the phenotypes, but the possibility remains that this could be an indirect effect, despite the authors attempt to demonstrate otherwise. Certain cargos, like endogenous PC VII, and overexpressed cargos like ssHRP, do clearly have requirements for additional factors to aid in their incorporation into COPII vesicles. Indeed, TANGO1 is one of these factors. But given the established roles of SLY1, STX18, and other related proteins in mediating vesicle fusion, it has not yet been shown that SLY1 is directly involved in this process. An alternative explanation is that PCVII is an especially sensitive cargo, and its incorporation into COPII vesicles is hindered by general trafficking perturbations that do not affect most other cargos. Furthermore, how do the authors know that membranes are the limiting component for PCVII vesicle formation upon SLY1 knockdown? Might the recycling ERGIC membranes contain specific protein factors that might be important for this process? These are very tricky issues to address, but we offer a suggestion for how the authors might make their case stronger:Although the authors identify SLY1 as an interactor of TANGO1, this interaction is not explored functionally. Can the authors show that the interaction is important for PCVII to exit the ER? .2) The authors conclude with a speculative model suggesting that Sly1 helps fuse ERGIC derived membranes to build collagen VII carriers. Equally possible is a model in which the SNAREs drive fusion of the carriers with the target membranes and cannot form without packaging of the SNAREs. This should be discussed as well. Moreover, the authors should summarize what is known about all ER localized SNARE proteins, perhaps in a table, to highlight gaps in our understanding. The authors find that the SM protein SLY1 interacts with TANGO1, and find that SLY1 and STX18 are important for ER export of the large cargo PC VII. The key experimental results in this paper are that SLY1 knockdown dramatically affects the ER exit of PCVII and overexpressed ssHRP, but not ER exit of other cargos like PCI.3) If SLY1 is directly involved in PCVII ER export, one might expect that levels of SLY1 would be highest in those tissues or cells responsible for secreting PCVII. Is there any evidence in the literature  that SLY1 is particularly abundant in such tissues?4) One reviewer took issue with the authors' claim that SLY1 and STX18 act at a “post-COPII assembly step”. The evidence for this claim is based on the fact that SEC31 still forms punctae on the same ER compartments where PCVII is accumulating in SLY1-knockdown cells . However We are happy to receive the supporting comments on our findings describing the export of Procollagen VII from the Endoplasmic Reticulum (ER). The reviewers have requested that we show more on the interaction between TANGO1 and SLY1. Unfortunately, at present, this is beyond the scope of the paper. TANGO1 coprecipitates with SLY1 only in cells that are chemically crosslinked and we believe they are in a complex of a large number of other proteins. It is, therefore, unclear whether these two proteins bind directly. We were fortunate that we could identify this interaction but defining the mechanism of their binding is not possible at present. We have rewritten the paper extensively to address the reviewers’ concerns and better describe the data rather than a potential model based on our findings. Our data highlights the existing of separate pathways for the export of Procollagen VII and Procollagen I from the ER. In other words, size alone is not the reason for the separate pathways of procollagen export from the ER. Our revised manuscript highlights this new message. In addition, we have added a table that summarizes our current understanding of the SNARE proteins localized at the ER-Golgi interface. This will help the readers to understand the complexity of this process and appreciate our findings.1) The data are convincing regarding the phenotypes, but the possibility remains that this could be an indirect effect, despite the authors attempt to demonstrate otherwise. […] An alternative explanation is that PCVII is an especially sensitive cargo, and its incorporation into COPII vesicles is hindered by general trafficking perturbations that do not affect most other cargos. Furthermore, how do the authors know that membranes are the limiting component for PCVII vesicle formation upon SLY1 knockdown? Might the recycling ERGIC membranes contain specific protein factors that might be important for this process? These are very tricky issues to address, but we offer a suggestion for how the authors might make their case stronger:Although the authors identify SLY1 as an interactor of TANGO1, this interaction is not explored functionally. Can the authors show that the interaction is important for PCVII to exit the ER? .The reviewer lists a number of different issues in this point that we address separately.A) As stated in the response above, it is impossible for us to map the binding interaction between TANGO1 and SLY1. We have changed the Title, Abstract, Introduction, and the Discussion extensively to state more clearly the facts.B) Effect on specific cargo: a V-SNARE for example. We address this in the discussion.C) Is PC VII somehow more susceptible to our experimental perturbations? It cannot be because of our data that the export of PC I, which is roughly the same size is not affected by either the loss of TANGO1, SLY1 or STX18. This is now stated more clearly in the text.2) The authors conclude with a speculative model suggesting that Sly1 helps fuse ERGIC derived membranes to build collagen VII carriers. Equally possible is a model in which the SNAREs drive fusion of the carriers with the target membranes and cannot form without packaging of the SNAREs. This should be discussed as well. Moreover, the authors should summarize what is known about all ER localized SNARE proteins, perhaps in a table, to highlight gaps in our understanding. The authors find that the SM protein SLY1 interacts with TANGO1, and find that SLY1 and STX18 are important for ER export of the large cargo PC VII. The key experimental results in this paper are that SLY1 knockdown dramatically affects the ER exit of PCVII and overexpressed ssHRP, but not ER exit of other cargos like PCI.We state this in the text but also highlight that we favor an alternative model. A table with the known v-and t-SNAREs at the ER-Golgi interface is now included in the paper.3) If SLY1 is directly involved in PCVII ER export, one might expect that levels of SLY1 would be highest in those tissues or cells responsible for secreting PCVII. Is there any evidence in the literature  that SLY1 is particularly abundant in such tissues?No, there is no evidence in the literature that SLY1 or TANGO1 levels are particularly higher in tissues that export PC VII.4) One reviewer took issue with the authors' claim that SLY1 and STX18 act at a “post-COPII assembly step”. The evidence for this claim is based on the fact that SEC31 still forms punctae on the same ER compartments where PCVII is accumulating in SLY1-knockdown cells . However, there is no reason to think that these SEC31 punctae represent stalled PCVII vesicle buds. These SEC31 punctae might be unaffected ERES through which other cargos are continuing to exit normally (because the authors show that other cargos are not affected by SLY1-knockdown). SLY1-knockdown may have no affect at all on the observed SEC31 punctae, and may have actually prevented the formation of PCVII-nucleated SEC31 punctae (which would remain unseen). If SLY1 acts downstream of COPII assembly, one would expect to see a proliferation of SEC31 punctae in the SLY1-knockdown cells, corresponding to stalled PCVII buds. Please modify the text accordingly.We have changed the subheading to “SLY1 knockdown does not affect Sec13/Sec31 localization to the ER exit sites“. It is also important to note that we do not state that these are stalled PC VII buds. All we say is that SLY1 depletion does not affect the connection made by TANGO1 between PC VII and the COPII coats of the exit site. We have deleted our statement that SLY1 acts post COPII coat assembly from the discussion to alleviate the issue raised by the reviewer.


Background. This study was done to assess the maternal and sociodemographic factors associated with low birth weight (LBW) babies. Methods. An unmatched case control study was done involving 159 cases (mothers having LBW singleton babies) and 159 controls . Results. More than 50% of LBW babies were from the mothers with height ≤145 cm while only 9.43% of NBW babies were from the mothers with that height. Finally, after multivariate logistic regression analysis, maternal height, time of first antenatal care (ANC) visit, number of ANC visits, iron supplementation, calcium supplementation, maternal education, any illness during pregnancy, and hypertension were found as the significant predictors of LBW. However, maternal blood group AB, normal maternal Body Mass Index (BMI), mother's age of 30 or more years, and starting ANC visit earlier were found to be protective for LBW. Conclusion. Study findings suggest that selectively targeted interventions such as delay age at first pregnancy, improving maternal education and nutrition, and iron and calcium supplementation can prevent LBW in Nepal. Low birth weight (LBW) has been defined by WHO as weight at birth of less than 2.5 kg . By inteAcross the world, neonatal mortality is 20 times more likely for LBW babies compared to NBW babies >2.5 kg) . It is n kg . It The incidence of LBW is estimated to be 16% worldwide, 19% in the least developed and developing countries, and 7% in the developed countries. The incidence of LBW is 31% in South Asia followed by East and North Africa (15%), Sub-Saharan Africa (14%), and East Asia and Pacific (7%). Asia accounts for 75% of worldwide LBW followed by Africa (20%) and Latin America (5%). In Nepal, the LBW prevalence is relatively high, ranging from 14 to 32%, as documented from various hospital and community based studies [ Pahad (hills) and 12% in Terai (plain land areas) [Only 36% of children born in Nepal were weighed at birth. Among them, 12% are of low birth weight. The percentage of children with LBW varies from 15% in mountains to 13% ind areas) .This study was done to assess the maternal and sociodemographic factors associated with LBW babies, an important indicator of maternal and newborn health in Nepal.A hospital based unmatched case control study was carried out at two hospitals: B.P. Koirala Institute of Health Sciences (BPKIHS), Dharan, and Koshi Zonal Hospital (KZH), Biratnagar, in east Nepal. Both hospitals are referral hospitals in the region. BPKIHS is 700 bedded teaching hospital while KZH is 300 bedded hospital. The high uptake of services at these hospitals is probably owing to their low cost. After admission, the majority of births take place within 2 days. The hospital stay is usually at least 1 day after delivery unless the mother or infant experiences problems or the mode of delivery was surgical.The study data were collected between September 2011 and February 2012 by using interview technique. Eligible mothers were interviewed face to face within 24 hours after delivery. Abstraction of ANC cards and medical records of mothers were also made and anthropometric measurements were taken after interview.Mothers delivering live born singleton term baby with birth weight less than 2500 gm were taken as cases, while mothers delivering live born singleton term baby with birth weight 2500 gm or more were taken as controls. The mothers delivering babies of more than 4 kilograms or babies with congenital anomalies or twins or preterm babies were excluded from the study.Sample size was estimated using software Epi Info 7.0 version and cross-checked using software nMaster 2.0 version. The sample size estimation was done taking 80% power, 5% alpha error, and 2 as anticipated odds ratio. One hundred and fifty-nine cases and the same number of controls were included in the study. Questionnaire was translated into local language and pretested before data collection. Mothers were interviewed by researchers only.Ethical clearance was obtained from the Institutional Ethical Review Board of BPKIHS. Before starting the interview, verbal individual consent for participation was taken after the study's aim, methods, benefits, and the potential discomfort were adequately explained. Subjects were informed that they were free to abstain or to withdraw from participation at any time. Questions were asked in a way that did not hurt their dignity. The respondents were assured that the answers they give would remain private, anonymous, and confidential.The questionnaire contained the variables on maternal factors  and calcium (500 mg) supplementation, and interpregnancy interval), sociodemographic factors , and diseases during pregnancy .The filled questionnaires were checked and rechecked for their completeness at the end of the day during data collection. They were coded before the data entry. Data were entered into the MS Excel 2007 version and were analyzed using PASW Statistical software 18.0. Crude and adjusted odds ratios were used to investigate the factors affecting incidence of LBW, by bivariate and multivariate logistic regression, respectively. Bivariate associations between independent variables and low birth weight were analyzed using simple logistic regression, and crude odds ratios and confidence intervals were calculated. Chi-square analysis was used to test possible bivariate associations between independent variables and low birth weight.Based on the bivariate analysis and a priori information, a multivariate logistic regression model was constructed to examine the relationship between variables and low birth weight, while also considering possible covariate effects. Entry of the exposure variable was forced, followed by stepwise entry of potentially relevant covariates. The Hosmer-Lemeshow test was used to identify and exclude variables that caused poor fit in the model. Independent associations between variables were characterized by adjusted odds ratios and 95% confidence intervals.A total sample of 318 mothers comprising 159 cases and 159 controls were included in the study. Out of 318 subjects, 70.75 percent were from rural area while 29.25 percent were from urban area. More than seventy percent of both the case (70.44%) and the control (71.07%) groups were from rural area. Majority of the subjects were Hindu (94.02%). The rest were Muslims (5.03%) and Christians (0.95%). Majority of the cases (92.45%) and controls (95.60) were Hindus. Although they were very few in number, all Hill occupational castes  were cases. Muslims were three times more in percentage in case group (7.55%) than control (2.51%) group while Hill natives were three times more in the control (23.27%) group than in the case (7.55%) group.Only 24.85% of total respondents belonged to nuclear family, while the rest belonged to joint (64.46%) and extended family (10.69%). Only 10.03% of the total controls were illiterate, while 24.53% of the cases were illiterate.More than three-fourths of the mothers were housewives while the rest worked outside home. Only 3.14% of the mothers were involved in business while 7.55% of them were laborers.Majority (46.86%) of the respondent mothers were of 20–24 years. Teenage mothers constituted 19.18% of total mothers. More than one-fifth (22.01%) of the cases were born by teenage mothers, while teenage mothers gave birth to 16.36% of the controls.Nearly two-thirds (63.52%) of the respondents were below poverty level. Based on modified Kuppuswamy's socioeconomic scale, 44.34% of total mothers were from the upper lower class while 4.41% of them were also from lower class. All the mothers from the lower class gave birth to cases.p < 0.05). There was no significant difference in the maternal age between cases and controls. However, the mothers of LBW infants had significantly lower weight than the controls (p < 0.05). The mothers who had their first ANC in the third trimester of pregnancy were threefold  more likely to give birth to LBW neonates than the mothers who had their first ANC in the first trimester. The chance of having LBW babies was also significantly higher in the mothers who had their first ANC in second trimester .The chances of delivering LBW babies were found to be increased as the mothers started the first ANC visit lately. The association between LBW neonates and the time of the first ANC visit was found to be significantly associated (p < 0.001). The mothers who had 1 to 2 ANC visits were 16-fold  more prone to have LBW neonates than the mothers who had more than 4 ANC visits in total. The chance of delivering LBW neonates by the mothers who had total ANC visits of 3-4 times was also higher .LBW was found to be significantly associated with the total number of ANC visits (p < 0.001). The mothers who were supplemented with iron for 90 or fewer days were nearly threefold more prone to have LBW babies than the mothers having iron supplementation for more than 90 days.Iron supplementation was found to be significantly associated with LBW  had not taken calcium supplement, while less than one-third of the controls (30.19%) had not taken the calcium supplement. In control group, the proportion of having calcium supplement for 90 days or more was found more than in the case group. It was more than double the proportion in the controls (40.25%) compared to the cases (18.24%). The mothers having no calcium supplement were more likely to have LBW babies than mothers having it for 90 days or more . The mothers having iron supplement for 90 or fewer days were also more prone to deliver LBW babies .p < 0.05). Maternal blood group AB has some protective effect against delivering low birth weight babies .The association between maternal blood group and LBW babies was also found to be significantly associated (p = 0.902), religion (p = 0.236), paternal education (p = 0.213), paternal occupation (p = 0.251), socioeconomic status based on modified Kuppuswamy scale (p = 0.48), and per-capita income (p = 0.162) were found to be insignificant. But caste (p < 0.001), type of family (p = 0.013), maternal education (p = 0.001), maternal occupation (p = 0.037), and type of house (p = 0.009) were found to be significantly associated with LBW. The odds ratio was found significant for Muslims  and major Hill castes . Illiterate mothers were more at risk to have LBW babies than mothers who were educated up to SLC or more . Laborer mothers were three times more common to deliver LBW babies than housewives . Mothers having katcha  house were twofold more likely to have LBW neonates than mothers having pucca houses  .Maternal residence (p < 0.05). Mothers having any illness during pregnancy were at more than twofold risk to have LBW babies compared to mothers having no illness . Mothers having fever during pregnancy, night blindness, and pain in abdomen during pregnancy were not significantly associated with LBW babies. But hypertension was found to be significantly associated with low birth weight babies. Mothers having hypertension during pregnancy were four times more likely to deliver LBW neonates than mothers having no hypertension . Anaemic mothers were also more likely to deliver LBW babies .Any illness during pregnancy was significantly associated with LBW , and house type, were included in the multivariate regression analysis. Maternal height, time of first ANC visit, total number of ANC visits, iron supplementation, calcium supplementation, maternal education, any illness during pregnancy, and hypertension are the significant predictors of LBW .Maternal blood group AB, normal maternal BMI, mother's age of 30 years or more, and starting ANC visit earlier were found to be significantly protective for LBW from multivariate regression analysis. Likewise, maternal blood group AB and normal maternal BMI had also protective effect on LBW after adjustment for other variables .The variables maternal weight, iron supplementation, anaemia, and poverty level were found to be significantly associated with LBW during univariate logistic analysis; however, those were found to be insignificant after being adjusted for other variables. It means odds ratios were significant, but adjusted odds ratios were not significant.In this study, maternal age had no significant association with LBW which is consistent with studies conducted by Mavalankar et al.  in Indiap < 0.001) with the birth weight of the baby which is consistent with some studies [Maternal postpartum weight in this study was significantly associated  was not found to be significantly associated with LBW babies in this study which is in accordance with the study by Ojha and Malla [The relationship between a low maternal BMI and LBW has been known for several decades. Some studies , 18 alsond Malla .p < 0.05) in this study which is in agreement with Roy et al. [Interpregnancy interval was found to be significantly associated with LBW babies p < 0.0 in this y et al.  but is iy et al. .The time of the first ANC visit was found to be significantly associated with LBW in this study and it was in accordance with the studies by Kercher  but in cSingh et al.  support Intake of iron supplements during pregnancy was found to have a protective effect with respect to LBW in this study which is consistent with Rizvi et al. . MaternaMaternal education was found to be significantly associated with LBW babies in this study as in some previous studies , but it Maternal occupation was found to be significantly associated with LBW babies in our study which is in accordance with some studies , 23 but LBW babies were more common in mothers having katcha houses than mothers having pucca houses in our study. Rizvi et al.  suggest There was significant negative association between LBW and maternal blood group AB in our study which is in accordance with the findings of Fedrick and Adelstein .Exposure variables, maternal weight, height, age, time of first ANC visit, total ANC visits, iron supplementation, calcium supplementation, maternal blood group, maternal education, any illness during pregnancy, hypertension, anaemia, BMI, poverty level (per-capita income), and house type, were included in the multivariate regression analysis.Out of those exposure variables, maternal height, time of first ANC visit, total number of ANC visits, iron supplementation, calcium supplementation, maternal education, any illness during pregnancy, and hypertension were found to be the significant predictors of LBW.Maternal blood group AB, normal maternal BMI, mother's age of 30 years or more, and starting ANC visit earlier were some variables which were found to be significant but with protective effect for LBW.Study findings suggest that selectively targeted interventions such as delay age at first pregnancy, improving maternal education and nutrition, and iron and calcium supplementation can prevent LBW in Nepal.It is suggested to conduct more comprehensive studies to assess maternal blood groups and different diseases during pregnancy as associated factors for low birth weight babies.