We found evidence of bacterial DNA in the majority of meconium samples in our study. The bacterial DNA structure  of meconium differed significantly from that of the transitional stool microbiota. There was a significant reduction in bacterial alpha diversity  and change in bacterial composition  in the transition from meconium to stool. However, changes in predicted microbiota metabolic function from meconium to transitional stool were only observed in vaginally-delivered neonates. Within sample comparisons showed that delivery mode was significantly associated with bacterial structure, composition and predicted microbiota metabolic function in transitional-stool samples, but not in meconium samples. Specifically, compared to vaginally delivered neonates, the transitional stool of C-section delivered neonates had lower proportions of the genera Bacteroides, Parabacteroides and Clostridium. These differences led to C-section neonates having lower predicted abundance of microbial genes related to metabolism of amino and nucleotide sugars, and higher abundance of genes related to fatty-acid metabolism, amino-acid degradation and xenobiotics biodegradation. In summary, microbiota diversity was reduced in the transition from meconium to stool, and the association of delivery mode with microbiota structure, composition and predicted metabolic function was not observed until the passing of the transitional stool after meconium.Cesarean (C-section) delivery, recently shown to cause excess weight gain in mice, perturbs human neonatal gut microbiota development due to the lack of natural mother-to-newborn transfer of microbes. Neonates excrete first the in-utero intestinal content (referred to as meconium) hours after birth, followed by intestinal contents reflective of extra-uterine exposure (referred to as transition stool) 2 to 3 days after birth. It is not clear when the effect of C-section on the neonatal gut microbiota emerges. We examined bacterial DNA in carefully-collected meconium, and the subsequent transitional stool, from 59 neonates [13 born by scheduled C-section and 46 born by vaginal delivery] in a private hospital in Brazil. Bacterial DNA was extracted, and the V4 region of the 16S  C-section delivery is a common surgical procedure intended to increase the chances of successful delivery and to protect the health of the mother. Yet this highly overused intervention ,2,3,4 haStaphylococcus and Corynebacterium) [Neonates delivered vaginally are exposed to maternal vaginal microbiota in labor, and additionally to maternal intestinal microbiota at birth ,11,12,13cterium) . Recent cterium) ,18. Yet,Findings of bacterial DNA in the placenta, amniotic fluid, and newborn meconium have given rise to the hypothesis that the human fetus may be exposed to microbiota in utero through ingestion of amniotic fluid ,19,20,21Trained staff invited women to participate in the study if they were scheduled for a vaginal delivery or an elective C-section delivery between 38 and 42 weeks of gestation (confirmed by an ultrasound taken before the 20th week of pregnancy) in the participating private hospital in Porto Alegre, Brazil. Women were not eligible for the study if they were administered oral antibiotics in the third trimester of pregnancy; if they had diabetes, hypertensive disorders, autoimmune disorders, or HIV/AIDS during pregnancy; if they had smoked during pregnancy; or if they were on a restrictive diet during pregnancy. For vaginal births, we further only included women whose water broke less than 12 h before delivery. All vaginal deliveries were by spontaneous vaginal birth and no instruments were used. Women also had to consent to allowing study staff collect meconium or stool from their neonate, and to completing a postnatal questionnaire. The Research and Ethics Committee of the Hospital de Clinicas (protocol No. 11/0388) and the Hospital Mae de Deus (protocol No. 524/11) in Porto Alegre, Brazil, approved the study protocols and consent.From medical records, we ascertained information on mode of delivery, gravidity, parity, history of urinary tract infection during pregnancy, antibiotic use during pregnancy, birth weight, labor length, head circumference, placenta weight, sex and race. Other clinical information was derived from a questionnaire administered after birth before discharge.Of the 89 mother-child pairs who consented and enrolled in the original study, we were able to collect 78 meconium samples . From the 78 meconium samples, 59 (75.6%) had detectable bacterial DNA. Transitional stool samples (collected 2–3 days after birth) with detectable bacterial DNA for analysis were available for 50 of 59 of these neonates. All specimens were collected from diapers with sterile spatulas, transferred to sterile tubes, which were kept at 4 °C for <6 h, and then frozen at −80 °C until DNA extraction.® DNA Stool mini kit (QIAGEN) according to the manufacturer’s instructions. After DNA extraction , we concentrated DNA samples to 20 µL using 3 M Na Acetate precipitation and kept it stored at −20 °C. We determined the DNA concentration of prepared samples using the Quant-iT PicoGreen dsDNA reagent and kit  based on the manufacturer’s instructions. We amplified the V4 region of the 16S ribosomal RNA (rRNA) gene by polymerase chain reaction (PCR) using the Illumina-adapted universal primers 515F/806R [Total DNA was extracted using QIAamp15F/806R . PCR wasWe analyzed 16S rRNA gene sequence data using the Quantitative Insights Into Microbial Ecology (QIIME) software package (v1.8) . Using qt tests with 10,000 Monte Carlo permutations for significant test of microbial diversity.We rarefied to 1634 reads per sample to calculate microbial diversity. We calculated alpha diversity using the phylogenetic distance and the detected number of species metrics. Principle coordinate analysis (PCoA) with weighted Unifrac distance metric were used to evaluate beta diversity  between C-section and vaginally-delivered neonatal fecal samples . We perfrRNA gene dataset [We used linear discriminant analysis (LDA) effect size (LEfSe)  to ident dataset , with Ky dataset . An LDA Of the 78 neonates that provided meconium samples, 59 (75.6%) had bacterial DNA present in their meconium. There were no significant differences in the clinical characteristics of mothers or neonates who had meconium with vs. without detectable bacterial DNA. Of the 59 neonates with meconium bacterial DNA, 50 also provided a subsequent transitional stool sample with bacterial DNA. We analyzed the meconium and transitional stool from these neonates for the current study. Demographic, clinical, and anthropometric characteristics of the 59 neonates  who had meconium with detectable bacterial DNA (and their mothers) are presented in p < 0.005; Observed species, p < 0.05).We obtained 604,570 sequences  from the 59 meconium samples, and 472,165 sequences from the 50 transitional stools. There was an average of 9878 reads per sample, binned into 5939 Operating Taxonomic Units OTUs; . Alpha dp = 0.1047). It did, however, vary in the transitional stools (p < 0.0001). There were also inter-individual differences in microbiota .Microbial community structure (beta diversity) did not differ significantly by delivery mode in the meconium  in transitional stool of vaginally born neonates, is also consistent with findings in mice [Lactobacilli are facultative anaerobes that ferment milk lactose and casein and produce lactate [Our findings on the transition in microbiome composition and structure from meconium to transitional stool are consistent with previous work reporting loss of alpha diversity postnatally in mice  and huma in mice . Lactoba lactate ,35,36. L lactate  commonlyPrevious investigations on the impact of delivery mode on meconium bacterial composition have been mixed, with some studies having found differences ,16,38, aBifidobacterium strains by delivery mode in 12 vaginally-delivered neonates and 5 C-section neonates, but only at 3 days after birth [Our amplicon-sequencing results compliment previous studies ,11,12 ther birth ,45, chorer birth , cord bler birth , fetal mer birth , and theer birth , some iner birth . Howeverer birth .The detection of bacterial DNA in the meconium samples in our study does not clearly support nor refute the in-utero colonization hypothesis, as meconium may reflect not only prenatal but also peri- and postnatal exposures . About 7In summary, we found evidence of bacterial DNA in most meconium samples and we observed that bacterial diversity in the neonatal intestinal tract decreases in the first days of life, from the passing of meconium to transitional stool. Moreover, the initial transition in neonatal intestinal bacterial structure, composition and predicted metagenome function appears to be impacted by delivery mode. Our findings also add to the literature that vaginal birth-acquired microbes are important in the normal development of the early neonatal gut microbiota development and also their metabolic function, since C-section delivered neonates fail to show transitional changes in predicted bacterial metagenomic functions. Future research should determine whether the differential transition of these pioneering microbial communities and their metagenomic functions are on the pathway to the metabolic and immune-mediated diseases that have been associated with C-section delivery.


Non-ruptured arteriovenous malformations (AVMs) rarely cause tract disruption. Few studies have described how ruptured AVMs influence white matter (WM) tract morphology. We reviewed consecutive AVM cases treated at a tertiary care hospital where diffusion tensor imaging (DTI) tractography was obtained preoperatively. DTI was performed using the Synaptive Plan . Quality control was performed by clinical application specialist. Perinidal fractional anisotropy (FA) value of corticospinal tracts (CST) was obtained. A reference FA value was obtained from the corresponding area on the contralateral side. Images were evaluated by a consultant neuroradiologist. Radiological findings were correlated with clinical findings. White matter morphology was described by a consultant neuroradiologist.All three cases included in the study had a history of haemorrhage in the past. Two patients had disruption of CST and presented with a significant neurological deficit. In one patient FA value of CST around the nidus was comparable to the contralateral side and did not show any neurological deficit. DTI integrated neuronavigation was used to plan the trajectory and complete resection of the AVM with excellent postoperative recovery. Diffusion tensor imaging (DTI) is a non-invasive imaging modality that determines the spatial distribution of the diffusion of water molecules. It provides a three-dimensional image of the white matter (WM) tracts. The extent of white matter damage can be measured by fractional anisotropy (FA) across the tracts through DTI tractography .FA measures the preferential direction of the diffusion of water molecules . The infArteriovenous malformation is the congenitally localized anomalous accumulation of dilated arteries and veins which lack a capillary network subsequently creating a direct channel between the cerebral arteries and veins, risking rupture . Few stuThe purpose of this study was to describe the morphology of white tracts on the basis of DTI. We then described the clinical correlation of morphological information with the clinical picture. We utilized DTI to evaluate the integrity and spatial relationship of the tracts to the lesion and chose a safe surgical corridor in three patients by using it for neuronavigation.Technical details DTI studies were performed using the Synaptive DTI protocol  on Siemens© 3 Tesla machine . The tractography was generated using the BrightMatter™ Plan . The protocol required a minimum of 20 gradient directions preferred +1 baseline B0 scan. B0 is an image of the anatomy that takes into account tissue signals and contrasts in the absence of diffusion gradients. B value of 1,000 was preferred. Slices of 2 mm thickness were obtained with gap/spacing 0.0 mm, square matrix 128 x 128. A DTI scan was completed prior to contrast injections. All the images were evaluated for their quality using a quality control (QC) algorithm. The QC was performed by the Synaptive ImageDrive™  by a Synaptive clinical application specialist. The protocol was used as part of the BrightMatter™ Guide (navigation) and BrightMatter™ Drive (robotic arm) technology . FA values were calculated by selecting a region of interest (ROI) close to the nidus in corticospinal tracts (CST) on FA map. Corresponding FA value was obtained from the identical area on the uninvolved side by estimating left-right asymmetry. Selection of ROI and calculation of FA value was performed by a consultant neuroradiologist. Selected cases were studied in detail using the hospital electronic database and medical records. Demographic, clinical, and radiological variables were recorded and described. A correlation between FA values, white matter integrity, and neurological deficits was made. Surgical resection was aided by neuronavigation with DTI and CT angiograms merged by advanced options on the BrightMatter™ Guide for all the cases.Case 1A 12-year-old girl presented with left-sided hemiparesis following a right frontoparietal hemorrhage two months prior. On examination, she was fully awake, alert, and oriented. She had pyramidal weakness in her left upper and lower limbs of grade 4/5. Magnetic resonance imaging (MRI) of the brain with contrast demonstrated a right parietal intraparenchymal hemorrhage with maturation changes suggested by a hemosiderin rim. The lesion appeared hyperintense on T1 and T2 images with central diffusion restriction. It measured 65 x 48 x 37 mm in the anteroposterior, craniocaudal, and transverse dimensions   was found.On DTI tractography, fibers of the posterior corona radiata and the posterior part of the medial limb of the superior longitudinal fasciculus were disrupted with abnormal color hues on a colored map Figure . An FA vIn this report, we have presented three cases of AVMs with previous rupture. We utilized classifications system introduced by Witwer, et al. to describe the morphology of WM tracts . Two of Our results are congruent with the aforementioned studies, showing the quantitative analyses to be more reflective of the symptomatology of the patients than the appearance of the tracts. The FA value of Patient 1 was around three times lower compared to the normal contralateral, with Patient 3 having an FA value two times lower than its contralateral unaffected side, and both patients had neurological deficits. Patient 2 had no neurological symptoms and did not appear to have a significantly low FA value in comparison to the normal side.The lack of visualization of the corticospinal WM tracts has been shown to not necessarily be correlated with associated symptoms, particularly in hemorrhage . In our The surgical resection of AVM follows the “all or nothing” principle. Microsurgical resection is relatively superior to stereotactic radiosurgery and endovascular embolization wherever possible . DTI, esThe study has a limited number of cases; however, it provides an important perspective on the role of DTI in the management of AVM. We have utilized a standardized and well-described nomenclature to describe WM tract morphology. This is the first study to describe the use of the Synaptive DTI protocol and Guide in the understanding of WM. We recommend more prospective studies on the topic for improved understanding of the topic.Ruptured AVMs can result in non-visualization and disruption of tracts around the nidus. Tract morphology around the nidus correlates with the neurological deficits in a patient and may be used to guide surgical planning, prognosis, and outcomes.


Preterm birth and small for gestational age (SGA) are strong indicators of neonatal adverse outcomes. With the growing importance of preterm SGA infants, we aim to evaluate the prevalence and risk factors for preterm SGA in China.We analyzed the data of parents and infants from a population-based cohort research of the free National Pre-pregnancy Checkups Project (NPCP) in rural China. Only singleton live births that occurred between 24 weeks +0 days and 36 weeks +6 days of pregnancy were included in this study. SGA was defined as birth weight less than the 10th percentile of the reference birth-weight-for-gestational-age population. A multiple logistic regression model was built using the statistically significant variables from the 371 variables in the questionnaire.A total of 11,474 singleton, preterm, live-birth infants were included. Of the total infants, 317 (2.77%) were preterm SGA infants. A higher risk of preterm SGA infants was observed among mothers who were on oral contraceptives , mothers who had syphilis , and mothers with a high eosinophil percentage . Maternal intake of folic acid at least 3 months before pregnancy  and paternal intake of egg and meat  were protective factors. Compared with North China, the incidence of preterm SGA infants was higher in South China.Preterm SGA infants were associated with both maternal and paternal factors. Gestational age and birth weight are two of the most important factors for evaluating the prognosis of infants. Small for gestational age (SGA) infants may show a decrease in their growth due to intrauterine growth restriction. Limitations in fetal growth affect the development of the cardiovascular system or other organs, which can have life-long effects on an individual . PretermA population-based retrospective cohort study was performed on 248,501 couples and their children who were part of the free National Pre-pregnancy Checkups Project (NPCP) in 220 pilot counties in 30 provinces in China between January 2010 and December 2012. The project was implemented by the Chinese National Health and Family Planning Commission and Ministry of Finance with aim of preventing birth defects in China, it is the largest pregnancy retrospective cohort study of the preconception stage in China. It covered all volunteer couples who planned to conceive within the next 6 months. The clinical data were collected during the preconception medical examination. Information on socioeconomic background, reproductive history and history of illness, lifestyle, and dietary habits was carefully collected through face-to-face interviews by qualified nurses. Physical examinations and biochemical studies were also carried out by medical staff at the same time .SGA was defined by a 1995 WHO expert committee as infants with body weight below the 10th percentile of a birth-weight-for-gestational-age, using the gender-specific reference population with the local growth standards of Li Zhu et al. [The inclusion and exclusion criteria are shown in Fig. A structured questionnaire was constructed by well-trained investigators; the questionnaire included 371 variables from the National Free Preconception Health Examination Project , 11. As The questionnaire involves 19 aspects including baseline characters of couples, physical examination, laboratory examination family history of couples etc. We chose the variables with a high data integrity over 80%. Among these variables, 38 variables were statistically significant exposures in univariate analysis including education level of the parents, maternal preconception intake of narcotics and paternal second-hand smoking, maternal intake of eggs and meat, the beginning time of maternal intake of folic acid, paternal intake of eggs and meat and paternal intake of vegetables, tense maternal and paternal relationship with relatives and co-workers, paternal exposure to heavy metals, organic solutes and vibrations, maternal syphilis and Candida infection, maternal drug use, pet exposure and influenza virus infection during pregnancy, maternal medical history of diabetes mellitus (DM), maternal medical history of hepatitis B, maternal oral contraceptive use, maternal family history of neonatal death, paternal hepatitis B vaccination and paternal family history of DM, maternal height (meter), maternal weight (kilogram), maternal BMI (kg/m2), maternal red blood cell count (109/L), maternal eosinophil percentage, maternal blood glucose level (mmol/L) and paternal height. It also included the presence of maternal HBe antibodies, maternal rubella virus IgG antibodies, maternal CMV IgG antibodies, maternal toxoplasma IgG antibodies, paternal HBs antibodies. The folic acid supplementation, the preconception habits and socioeconomic status of the parents were based on self-report. The location was classified as North or South region of China by the Qinling Mountain-Huaihe River Line.U test for analysis of continuous variables with a skewed distribution as all of continuous variables were of skewed distribution in this cohorts examined by Kolmogorov-Smirnov test. The continuous skewed variables were expressed in the form of mid-values . When a variable was found to be significant at the 0.1 level, it was entered into the multivariate model. Stepwise logistic regression was used to examine the correlation between risk factors and preterm SGA. In logistic regression, p < 0.05 was considered statistical significance. The results were presented using the OR and 95% CI values. The analyses were performed with SPSS .All risk factor variables were first examined by univariate analysis to assess the importance of each of them on preterm SGA. We used chi-square test for analysis of categorical variables, and the Mann-Whitney A total of 248,501 infants were recruited in our database, of which 12,164 were preterm infants (5.63%). The preterm neonatal mortality was 1.69% in our study. The mean weight of 11,474 preterm singleton live-birth infants was 3104.87 ± 636.03 g, 6307 infants of them were male (54.97%). Among them, 317 (2.77%) were preterm singleton live-birth SGA infants. The mean weight of them was 1778.26 ± 438.58 g, 189 of them were male (59.62%). 11,157 of them were preterm singleton live-birth non-SGA infants, the mean weight was 3141.73 ± 595.51 g, 6118 of them were male (54.84%).The following tables show the risk factors in preterm SGA deliveries. Table Paternal exposure to heavy metals, organic solutes and vibrations were associated with a higher incidence of preterm SGA. Parental infections were also identified as important risk factors. Syphilis, Candida infection, rubella virus infection, CMV infection, toxoplasma infection were associated with higher rate of preterm SGA. Hepatitis B is common in China. Positive maternal HBe antibodies is associated with higher prevalence of preterm SGA. Moreover, maternal family history of hepatitis B was associated with higher rate in preterm SGA infants while paternal hepatitis B vaccination was associated with lower rate in preterm SGA infants.Mothers who were taking medications, came into contact with pets or had influenza virus infection were more likely to have preterm SGA infants. With regard to the medical history of the parents, maternal family history of neonatal death were associated with a higher rate of preterm SGA.The Qinling Mountain-Huaihe River Line is an important demarcation line of climate, hydrology, and topography in China . The NorAs expected, the parental weight, height and BMI were associated with preterm SGA as shown in Table Table Birth weight and gestational age are considered as strong predictors of short-term and long-term prognosis of infants. Given the growing attention paid to preterm SGA infants, our study attempted to determine the incidence of the preterm SGA infants and the risk factors associated with delivering preterm SGA infants.A major strength of this study is its large sample size and the large number of variables analyzed. To the best of our knowledge, this is the most extensive multi-center study in China to evaluate the risk factors associated with preterm SGA infants. The large number of variables allows us to analyze more risk factors than previous studies on preterm SGA infants. The effect of paternal factors on preterm SGA infants, for example, the maternal eosinophil percentage has rarely been reported before.This database has several unique features. Compared with earlier study, the mortality rate of preterm infants in our study (5.63%) was lower than the average rate reported for eastern Asia (7.2% (5.4–9.0)) . With ecIn our study, we discovered a gender-based difference in the incidence of preterm SGA infants in China; 59.62% of preterm SGA infants were male. It has been reported that boys are more likely to be born before term in a different of populations . A possiInsufficient periconceptional folic acid intake is associated with a number of birth defects that may also be related to genetic and environmental factors before conception or during early pregnancy . Recent Oral contraceptives use is one of the most popular reversible methods of contraception. However, the adverse effects of oral contraceptives on fetal development are unclear. Previous studies have reported the association of oral contraceptive use and preterm birth and low birth weight , 25. It Eosinophils have been shown to be a significant cellular infiltrate of the placenta and uterus, including the infiltration and degranulation of eosinophils in the cervix of pregnant humans . The rolDespite being easily detectable and treatable during pregnancy, syphilis remains an important cause of adverse pregnancy outcomes . SyphiliOur study showed that diet containing egg and meat of the father, which reflected the paternal nutritional status, particularly protein intake, was significantly associated with lower incidence of preterm SGA infants. Animal studies have demonstrated that all stages of gamete maturation and preimplantation embryo development are influenced directly by parental nutrition and hormonal status . MoreoveThe primary limitation of our study is that several risk factors such as the beginning time of maternal folic acid intake, the paternal intake of egg and meat, and the use of oral contraceptive were based on self-report of the parents. More quantitative variables are needed in our questionnaire. With the large number of subjects, it is difficult to assure the completeness of data. This study identified several factors that are associated with preterm SGA, due to diverse culture and social economic status of these subjects, some confounding factors might be overlooked.Our results show that preterm SGA infants were associated with both maternal and paternal factors. Maternal use of oral contraceptives, maternal syphilis infection, maternal higher eosinophil percentage, maternal folic acid intake less than 3 months before pregnancy and paternal low protein diet were associated with preterm SGA.


Preterm birth is the leading cause of death among children <5 years of age. Accurate determination of prematurity is necessary to provide appropriate neonatal care and guide preventive measures. To estimate the most accurate method to identify infants at risk for adverse outcomes, we assessed the validity of two widely available methods—last menstrual period (LMP) and the New Ballard (NB) neonatal assessment—against ultrasound in determining gestational age and preterm birth in highland Guatemala.Pregnant women (n = 188) were recruited with a gestational age <20 weeks and followed until delivery. Ultrasound was performed by trained physicians and LMP was collected during recruitment. NB was performed on infants within 96 hours of birth by trained study nurses. LMP and NB accuracy at determining gestational age and identifying prematurity was assessed by comparing them to ultrasound.By ultrasound, infant mean gestational age at birth was 38.3 weeks (SD = 1.6) with 16% born at less than 37 gestation. LMP was more accurate than NB (mean difference of +0.13 weeks for LMP and +0.61 weeks for NB). However, LMP and NB estimates had low agreement with ultrasound-determined gestational age (Lin’s concordance<0.48 for both methods) and preterm birth (κ<0.29 for both methods). By LMP, 18% were judged premature compared with 6% by NB. LMP underestimated gestational age among women presenting later to prenatal care . Gestational age for preterm infants was overestimated by nearly one week using LMP and nearly two weeks using NB. New Ballard neuromuscular measurements were more predictive of preterm birth than those measuring physical criteria.In an indigenous population in highland Guatemala, LMP overestimated prematurity by 2% and NB underestimated prematurity by 10% compared with ultrasound estimates. New, simple and accurate methods are needed to identify preterm birth in resource-limited settings worldwide. This buf total) . ChildhoIn Guatemala, there are an estimated thirty-six thousand preterm births (8%) per year , which aUltrasound during the first trimester of pregnancy is considered the most accurate method for determining gestational age . Howeverst or 15th of month, or rounding to zero or five) [Using the known date of last menstrual period (LMP) can be highly accurate. Estimates have been shown to be within days of those from ultrasound, even in low- and middle-income countries –16. Addior five) , 16 and or five) , 11. As or five) , 13, 15.Other methods for determining gestational age use neonatal physical and neuromuscular measurements to determine developmental maturity. These include the 21-item Dubowitz , 12-itemThe purpose of this exploratory study is to compare gestational age and preterm estimates from ultrasound, LMP and Ballard under real field conditions and investigate factors influencing accuracy to determine the best method for use in the highlands of rural Guatemala.Embarazo Seguro Bebé Sano  Study, investigating several potential environmental, nutritional and infectious risk factors for disease during pregnancy and infancy [Asociación de Comadronas del Área Mam and Pies del Occidente from May 2013 to February 2014. Medical chart reviews and interviews of the pregnant women were used to determine eligibility. Women were eligible if they were non-smokers between the ages of 18 and 40 years old, had a low-risk, singleton pregnancy less than 20 weeks gestation by LMP or ultrasound and not greater than 24 weeks by the other method, lived and planned to stay in the area for at least six months post-delivery and had access to a mobile phone. Low risk pregnancies were defined as maternal blood pressure less than139/89 mmHg and no protein, glucose or ketones in the urine at the baseline clinical exam.This methodological study was part of a larger prospective cohort study completed between 2013 and 2015, the  infancy , 28. StuA home visit was made within one week after enrollment to assess baseline household and maternal characteristics. Weekly phone calls and cell phone text messages were conducted after baseline to assess pregnancy status until the mother delivered her infant. Mothers were instructed to contact the study personnel if they went into labor. Study personnel assessed newborns either at the hospital or in the home, depending on place of delivery and timing of the assessment.Three methods were used to calculate the gestational age at birth: fetal ultrasound, New Ballard and LMP. Preterm births were those infants having a gestational age at birth less than 37 weeks. As defined by the WHO, moderate to late preterm births are those between 32 and 37 weeks, very preterm between 28 and 32 weeks, and extremely preterm less than 28 weeks .th day of the month was used. We determined the estimated delivery date (LMP + 280 days). The difference between the estimated delivery date by LMP and actual delivery date was then used to determine gestational age in weeks:LMP was self-reported during the baseline clinical exam. If the exact date of the month could not be recalled, the 15Project nurses assessed newborn gestational age using the New Ballard (NB) maturational assessment. The New Ballard Score measures 6 physical and 6 neuromuscular criteria; the total score ranges from -10 to 50. Nurses were trained over 2 days; didactic training including videos of the New Ballard assessment was followed by hands-on assessments of newborns in the postpartum ward of the hospital.NB scores were calculated for infants examined within 96 hours of birth in 156 (83%) of the live births. Typically the New Ballard gives gestational age in 2 week increments. To provide gestational ages in smaller increments, gestational age (in weeks) was determined from the NB score by a regression equation from published scores and estimated gestational ages (0.4*NB score + 24) .All mothers had an ultrasound taken as part of the screening for recruitment eligibility. To standardize measures among those performing ultrasounds, study physicians received didactic and hands-on training on ultrasound use before the study began, complemented by follow-up in-person trainings during the course of the study. Consistent quality measurements were required before physicians were permitted to perform ultrasounds for the study. For quality control, 33% of study images were assessed by a board-certified obstetrician and ultrasounds were repeated if they were found to be of low quality. When multiple ultrasounds were performed, the earliest set of high quality images was used. Before 14 weeks gestational age, crown-rump length was used to determine gestational age . After 1For the calculation of small for gestational age, newborn weight was measured to the nearest 5 grams using a Seca 334 digital infant scale. Weight was measured in triplicate and clothing weight was subtracted from infant weight. Measurements within 50 g of each other were used to determine the average. If either the birthweight or clothing weight were implausible (<1400 grams or >5000 grams for birthweight and <100 g or >1000 g for clothing) or taken later than two weeks after birth, the recorded birthweight from the hospital medical record was used, if available (n = 25), or excluded from analysis, if unavailable. Only weights taken within 2 weeks of birth (n = 184) were used in the estimation of small-for-gestational age.All statistical analyses were performed in Stata 13. Descriptive statistics  were used to describe the data.Lin’s concordance correlations  and BlanCohen’s kappa statistic  was usedth weight centile for gestational age as determined by the INTERGROWTH-21st Newborn Size Application Tool using standards derived from births in eight ethnically distinct countries (http://intergrowth21.ndog.ox.ac.uk/).Small-for-gestational age was defined as an infant being below the 10This study was approved by human subjects committees at Universidad del Valle de Guatemala, Emory University, and the Guatemalan Ministry of Public Health and Social Welfare. This study was reviewed in accordance with CDC human subjects protection policy and was determined to be human subjects research, but CDC involvement did not constitute engagement in human subjects research. All participants signed a written consent that was read to them in Spanish and, if they were unable to read or write, a witness also signed to ensure comprehension by the participant.In total, 637 women were assessed for eligibility with 221 meeting all inclusion criteria and consenting to participate in the study . The meaOverall, the distribution of gestational ages as determined by NB was shifted toward higher gestational ages compared to ultrasound  by an avThe accuracy of gestational age estimates by the New Ballard was dependent on trimester of ultrasound and whether the infant was preterm or SGA. The mean difference in gestational age estimates between New Ballard and ultrasound was larger among those having a first trimester ultrasound, when ultrasonography is most accurate, compared to those with second trimester ultrasounds (+1.10 weeks vs. +0.47 weeks in second trimester) and among preterm infants . New BalLMP performed better than the New Ballard at determining gestational age. While the distribution of gestational age estimates by LMP was more disperse , it overst trimester ultrasounds) but underestimated gestational age at birth for later ultrasounds. Mother’s age and educational attainment had no statistically significant effect on the accuracy of the LMP estimation  in the study population, whereas LMP slightly overestimated preterm birth (17.8%) compared to ultrasound (16.0%) . LMP hadBecause the New Ballard is a composite of six neurological and six physical criteria, we investigated whether some measures of the New Ballard scale performed better than others. While the physical and neuromuscular subscores had similar correlations with gestational age, more individual neuromuscular (4/6) than physical scores (3/6) were significantly correlated with ultrasound-determined gestational age . SimilarLMP performed better than methods assessing newborn characteristics using the New Ballard; gestational age as determined by LMP had a higher agreement with ultrasound determined gestational age and preterm birth. This finding is despite 18% of women being able to recall only the month of their LMP in the present study. The performance of LMP reported here was similar to that previously reported. LMP, on average, overestimated gestational age by ~1 day which is in the range of studies performed in other low- and middle-income countries–from an underestimation of 1 day to an overestimation of 4 days , 13, 15.While overall LMP appeared quite accurate, our present study demonstrated large and potentially clinically relevant variation in accuracy among individual participants with nearly half disagreeing by more than a week and wide 95% limits of agreement. This variation is greater than that seen in Brazil , BangladThe accuracy of LMP was dependent on whether the infant was preterm or not; LMP consistently overestimated gestational age among preterm births more than term infants. However, another study using LMP found gestational age was underestimated in preterm infants compared to term infants . That stThe wide variation in accuracy of gestational age estimates based on LMP led to a slight reduction in the proportion of preterm birth as compared to ultrasound and, more importantly, a low kappa statistic, sensitivity and specificity. LMP has previously been shown to lead to slight increases , 11, 14 We found, as other studies have , 24, thaMost infants on whom the New Ballard was performed were between 35 and 39 weeks of gestational age at birth (62%) with most preterm births between 35 and 37 weeks (79%). Because physical characteristics and muscle tone are easier to differentiate at the extremes (very premature or post-mature), the New Ballard is less accurate between 32 and 41 weeks gestational age compared to 28 to 31 and greater than 42 weeks . The ranGiven the degree of accuracy of the New Ballard within this study, its long training and test procedure and need for standardization, a shorter and more accurate alternative is desirable. One alternative is the 6-item Capurro, five of which were measured within this study as part of the New Ballard. Gestational age estimates by Capurro can be performed by trained birth attendants and have been shown to be highly correlated with those from New Ballard . HoweverA new alternative method can adapt the New Ballard or Dubowitz methods to find and use only those elements most highly correlated with gestational age, as in the development of the Capurro , or adjuIn Guatemala, few women have formal antenatal care during pregnancy and, thus, there may be a selection bias among study participants. The small sample size in this study limited the ability to look at differences in sensitivity and specificity for preterm birth among only those births with first trimester ultrasound and LMP reports. Additionally, results might not be generalizable to other contexts due to sample size. However, they fit with the broad trends from similar studies in low and middle-income countries , 13, 24.Preventing childhood mortality is essential for enhancing health security. Preterm birth is a major contributor to childhood mortality worldwide and identification of premature newborns is necessary for proper allocation of care and prevention of mortality. While prenatal ultrasound is the most accurate method for determining gestational age if performed early in pregnancy, it is frequently not available. LMP and New Ballard are widely available methods that have been shown to be quite accurate under ideal conditions. However, as presented here they suffered from wide variations in accuracy and had systematic biases based on varying newborn and maternal characteristics. Using these methods would have led to inaccurate estimations for rates of both preterm birth and small for gestational age. Given suboptimal performance, there is a need for simple, novel methods to accurately determine gestational age and preterm birth in low and middle-income countries where the burden is greatest, ultrasound is not readily available and women present late to antenatal care.S1 Dataset(CSV)Click here for additional data file.S1 FigRed dashed line indicates the mean difference between methods and blue lines indicate the upper and lower limits of agreement.(PPTX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


The birth of a child is a vital event that needs to be registered but this is not always the case as an estimated 40 million births go unregistered annually. Birth registration safeguards the basic rights of children and gives them an identity, citizenship/nationality and legal protection against violence, abuse and human rights violations. It is therefore necessary that all births are registered and even more critical that the registration of a birth is followed by the issuance of a birth certificate. But sadly, birth registration in many African countries continues to remain below acceptable international standards and not all registered births are certified. This paper examined birth registration and certification in Ghana. Differentials in the characteristics of children and mothers of children whose births are registered and certified, children whose births are registered but not certified and children whose births are not registered were examined.This paper analysed data from the 2014 Ghana Demographic and Health Survey drawing on variables from the household and children’s data files. Descriptive analytical tools  and multinomial logistic regression analysis were used to examine differentials in birth registration status among an analytical sample of 3880 (weighted) children aged 0–4 years.The birth of about every 1 in 4 (28.89%) children in Ghana have never been registered. Birth registration and certification was lowest among children born to young mothers (15–19 years), children whose mothers have no formal education, mothers who reside in rural areas and mothers in the poorest wealth quintile. Additionally, home births and births that were not assisted by a medical professional were observed to have the lowest proportion of registered and certified births. Furthermore, the birth of children who are less than a year old was significantly more likely not to be registered or issued with a birth certificate.Efforts aimed at improving birth registration and certification in Ghana need to target groups of children and mothers with low levels of registration and certification particularly children who are born at home, children born to young mothers and children whose mothers are poor and or reside in rural areas. But eveWhile it is necessary to ensure that all births are registered, the registration process is not complete if a birth certificate is not issued following registration. Without a birth certificate, the legal protection, identity and rights of the child is not guaranteed even though the birth of the child is registered. Furthermore, a birth certificate serves as proof of a child’s age and a potential means of protection against child labour, child marriage, child trafficking, sexual exploitation and forceful conscription into the armed forces . With thThe importance of birth registration has become a topical development issue particularly in regions such as Southern Asia and sub-Sharan Africa where just about half of children under the age of five have been registered . In GhanIn spite of these relatively high levels of birth registration, there is little empirical evidence on barriers and drivers of birth registration in Ghana and other developing country context . Amo-AdjThe laws of Ghana as mandated by the Registration of Births and Deaths Act of 1965 (Act 301) requires that all births (and deaths) that occur in Ghana are registered . Birth rIn spite of this success story , an estiVital event registration in Ghana started in 1888 in the then Gold Coast . HoweverThe provisions for civil registration have also changed as the various ordinances/acts were amended. In 1888, the Cemeteries Ordinance was only concerned with the registration of deaths. Birth registration was later introduced after the 1912 amendment . The 196The Births and Deaths Registry operates a centralised system with the national head office in Accra being the central co-ordination and administrative office . There aThis study draws on data from the 2014 Ghana Demographic and Health Survey (GDHS). The GDHS is a nationally representative sample survey which has been conducted every 5 years since 1988. The most recent round of the survey (2014 GDHS) was, however, conducted 6 years after the 2008 GDHS. The GDHS serves a number of policy and programmatic purposes. Policy makers for instance, use these surveys to monitor progress in population and health indicators. The surveys are also used to inform policy decisions such as allocation of limited resources to health services. Respondents for the 2014 GDHS were selected using a two-stage sampling technique. The first stage of sampling involved the selection of a total of 427 clusters (sample points) consisting of Enumerations Areas (EAs) delineated for the 2010 Population and Housing Census. The second stage of sampling involved the systematic selection of 30 households from the selected EAs [Does (NAME) have a birth certificate? Responses to this question included four categories of: (1) has certificate, (2) registered, (3) neither, and (4) don’t know. The “has certificate” category denotes children whose birth has been registered and who have a birth certificate while the “registered” category denotes children whose birth is reported to have been registered but do not have a birth certificate. The “neither” category represents those children whose birth has not been registered and who do not have a birth certificate. For the purpose of this study, the “don’t know” category was excluded from the analysis. The dependent variable; birth registration status therefore includes three categories of; (1) neither registered nor has a birth certificate, (2) registered without a birth certificate and (3) registered and has a birth certificate.The dependent variable in this study is birth registration status. The dependent variable was generated from the question that asked if children aged 0–4 years on the household roster have a birth certificate: The independent variables considered in this study were grouped under four main categories; 1) child’s characteristics, which included age  and sex , (2) mothers characteristics, which included age , level of education , marital status  and wealth status , (3) geographic location which included place of residence  and region of residence  and (4) delivery characteristics which include place of delivery  and assistance at delivery , 2 = other child’s Descriptive statistical tools including frequencies and cross tabulations were used to examine the characteristics of children and their mothers by birth registration status. The dependent variable for this study is nominal with three categories, hence, a multinomial logistic regression model was used to examine the factors associated birth registration status.Descriptive results in Table The results in Table Regarding the characteristics of mothers and the registration status of their children, the results show that a higher proportion of children born to young mothers (15–19 years) were neither registered nor had a birth certificate 53.2%) whereas among women aged 30–44 years, about six out of ten of their most recent births were registered with a birth certificate. Births that were neither registered nor had birth certificates were more common among women with primary education (38%) and lowest among mothers with higher level of education (13%). Furthermore, the results show that the proportion of births registered with certificates increases with increasing level of education, from 46% among mothers with no education to 82% among mothers with higher level of education. Birth registration with certification was highest among currently married women (57%) and lowest among never married women (48%). On the other hand, the highest proportion of births that have not been registered nor issued a birth certificate was observed among never married women while the lowest proportion was observed among currently married women (Table % whereasThe distribution by place of residence shows a higher proportion of births which were neither registered nor issued a certificate (36%), and births which were registered without the issuance of a certificate (19%) being common in rural areas while births that were registered with a birth certificate were higher in urban areas (68%). The regional variations showed the Volta region recording the highest proportion (50%) of births that were not registered nor issued a birth certificate whereas just about one-fifth of births in the Greater Accra (19%), Ashanti (19%), and Central (20%) regions were neither registered nor issued a certificate. The results with regards to household wealth status show that the proportion of births that were neither registered nor issued a certificate decreased with increasing wealth status from approximately 41% among the poorest to 12% among the richest.And as is to be expected, a higher proportion of births that occurred at home were not registered nor issued a birth certificate. On the other hand, the highest proportion of births registered with the issuance of a birth certificate was observed for births occurring in private health facilities (69%), followed by public health facilities (61%) and home births (37%). Similarly, registration of births with the issuance of a birth certificate was highest among children whose birth was assisted by medical professionals (62%) compared to births that were assisted by TBAs (38%), other people (34%) or no one (39%).P < 0.05) compared to mothers with no education.The results of the multivariate logistic regression analysis shown in Table The regional results show that whereas children in the Volta region were more likely to neither be registered nor have a certificate compared to children in the Greater Accra region, births to mothers in Upper East and Upper West were less likely to neither be registered nor have a certificate. Additionally, births to mothers in Central, Brong Ahafo, Northern and Upper West regions were more likely to be registered without the issuance of a birth certificate compared to births in the Greater Accra region. With respect to wealth quintile, the risk of births not being registered nor having a birth certificate was observed to decrease with increasing wealth. However, there was no significant relationship between wealth quintile and births being registered without the issuance of a birth certificate. Additionally, place of residence did not show a statistically significant association with birth registration status. So was the case for mothers’ marital status and delivery characteristics (place of delivery and assistance at delivery).This study examined the socio-demographic characteristics of children and their mothers that influence the birth registration status of children aged 0–4 years. The findings indicate that the sex of a child does not determine whether or not the child’s birth is registered as has been reported in earlier studies conducted in Ghana, South Africa and India , 20, 21.The findings of the present study indicate that beyond the demographic characteristics of children, the socio-economic status of mothers’ plays an influential role on the birth registration status of their children. For instance, the chances of births being neither registered nor issued a birth certificate decreases with increasing mother’s age . Additionally, mothers with a higher level of education were significantly less likely to have their births registered without the issuance of birth certificates. Similarly, in countries such as Peru, Haiti, India, Brazil and Honduras, children born to illiterate parents have been found to be less likely to have a birth certificate compared to children born to literate parents . FurtherIn addition to the aforementioned socio-demographic variations in birth registration status, the results show spatial variations in birth registration status along a north south divide. The results show lower levels of birth registration in the southern part of Ghana compared to the northern part as has been found in other studies conducted in Ghana . This noThe findings of this study demonstrate that the characteristics of children and the socio-demographic characteristics of their parents/families particularly their mothers influence birth registration status. Unlike previous studies that focused on only birth registration in specific communities, the present study uses the most recent nationally representative data to examine birth registration as well as certification in Ghana. The findings are thus nationally representative and generalizable to the whole country. The study however has a few limitations that are worth mentioning. Firstly, there is the possibility that the proportion of births that are registered with a birth certificate could be over-reported as not all the certificates were seen by the interviewer. Secondly, the reporting of birth registration status may not be accurate as this information was asked at the household level and the required information may not have been provided by the child’s mother or parents. The findings of this study thus need to be interpreted in light of these potential limitations.The findings of this study indicate that while about every 1 in 2 children aged less than 5 years in Ghana are registered with a birth certificate, another 1 in 6 are registered but without a birth certificate. More importantly, the birth of about 1 in 4 children aged less than 5 years in Ghana have never been registered nor do they have a birth certificate. Furthermore, the findings demonstrate that there is a propensity for late rather than early registration. Additionally, while some births are reported to have been registered, these births were not issued with a birth certificate. It is important to educate the Ghanaian population on the importance of birth registration and more importantly that parents/guardians ensure that they obtain a birth certificate following the registration of a birth. Policy interventions aimed at improving birth registration and certification in Ghana need to target groups of children and mothers with low levels of registration and certification particularly children who are born at home, children born to young mothers and children whose mothers are poor and or reside in rural areas.


Bees and flowering plants have a long-standing and remarkable co-evolutionary history. Flowers and bees evolved traits that enable pollination, a process that is as important to plants as it is for pollinating insects. From the sensory ecological viewpoint, bee–flower interactions rely on senses such as vision, olfaction, humidity sensing, and touch. Recently, another sensory modality has been unveiled; the detection of the weak electrostatic field that arises between a flower and a bee. Here, we present our latest understanding of how these electric interactions arise and how they contribute to pollination and electroreception. Finite-element modelling and experimental evidence offer new insights into how these interactions are organised and how they can be further studied. Focussing on pollen transfer, we deconstruct some of the salient features of the three ingredients that enable electrostatic interactions, namely the atmospheric electric field, the capacity of bees to accumulate positive charge, and the propensity of plants to be relatively negatively charged. This article also aims at highlighting areas in need of further investigation, where more research is required to better understand the mechanisms of electrostatic interactions and aerial electroreception. Animal pollinators detect and select flowers by their colours, shapes, patterns, fragrant volatiles Raguso , and, inElectroreception, defined as the ability of an organism to detect external electric forces, has long been known in animals living in aquatic, electrically conductive environments, for example, in fish Kalmijn , in amphBombus terrestris  is the term given to the vertical electric field that exists between the earth and the upper atmosphere . This increase in voltage continues towards a maximum of around 300 kV at an altitude of 30–50 km, above which the potential begins to decrease and invert  should have a distinct pattern of electrical contrast from a petunia (Petunia sp.) for example, and likewise, a raceme will have a different electrical contrast to an umbel inflorescence. Features with sharp edges or points, both within a flower  or on the overall plant , should have a stronger electric field purely due to their geometry.An aesthetic if not quantitative way of visualising the presence of an electric field around a flower can be achieved by dusting natural flowers with electrostatically charged coloured powder  allows the partial specification of a physical situation using empirical data, which can then be used to calculate many difficult or impossible to measure features of that situation. For aerial electroreception, these models stand in where direct measurement of the electric field is unavailable, providing a description of how the morphology and material properties of flowers and bees interact to induce forces on one another, and on charged objects in their vicinity. In Fig. In this model Fig. , the colWith currently available instrumentation, it is not possible to directly measure the computed quantities with as good a spatial or temporal resolution as that provided by finite-element analysis. A further benefit conferred by the finite-element analysis is that it allows experimenters to quickly explore the wide parameter space that exists within the specification of the model, changing and tweaking many aspects of it and comparing the results of thousands of simulated experiments. This can be extended from 2D into 3D, and from stationary to dynamic, time-domain, and frequency-domain studies. Ultimately, a dynamic representation of the electrical scene involving flying bees, growing plants, and an ever-changing atmospheric potential gradient could be constructed.During the bee’s final approach to the flower, electrostatic forces between these two bodies grow rapidly larger Fig. . This haBrassica napus) show the stigma and outer nectaries to have lower impedance paths to earth than the petals, sepals, styles, and anthers , or attached to the ventral side of the bee (where they have a positive charge of between 0 and +40 fC). All grains had a mass (m) between 1 and 5 ng. There were three forces included in the model that influence the motion of individual grains: gravitational forces (G*m in the downward direction), electric forces (using Coulomb’s law), and viscous drag (in low-Reynolds number fluid). Pollen acceleration is modelled as the sum of the forces on the grain divided by the grain’s mass.t = 0 ms), there is a repulsive electrostatic force (green arrow) acting on the positive pollen grain in the direction normal to the surface of the bee . The grain reaches terminal velocity almost instantaneously as viscous drag forces in the opposite direction to the velocity of the grain, quickly balance the other forces. Fifty milliseconds later, the pollen grain has moved closer to the stigma due to electric forces and gravity. Here, at point 2, the drag force perfectly opposes these other two forces, and the grain moves at terminal velocity. At this point, the grain is following a trajectory that would take it below and past the stigma (points 3 and 4), but as it approaches the stigma, the floral electric field rapidly increases  accelerates the grain towards the stigma where it finally makes contact and is no longer simulated. The second grain’s journey is similar but in the opposite direction. Here, the initial force from the plant is again repulsive (negative pollen and negative plant), followed by attractive forces from the opposite surface (bee). Gravity plays a minor role in determining the trajectory of the pollen grains and simulation with many (2000) grains with varied initial location, charge or mass shows that almost all, if free to move, will reach either the protruding sexual organs of the flower or the hairy surface of the bee (Fig. The results of the model show pollen grains of masses up to and including the mass of maize pollen undergo significant electrical forces, with the strong electric field between flower and bee more influential than gravity Fig. . Pollen bee Fig. c, as prebee Fig. , Corbet bee Fig. , and Vakith many  grains wbee Fig. .bona fide part of the multiple co-evolutionary adaptations between plants and their pollinators. For example, Armbruster (This simulation highlights the non-random and organised directed trajectory of pollen grains under electrostatic forces. Considering the picture generated by both empirical and modelled data, some questions naturally arise. Are plants morphologically and physiologically adapted to increase or take advantage of these electrostatic effects? Do some flower species actively generate or control the structure and dynamics of the electric field surrounding them? Are floral electric fields adapted, much like colours and fragrances are, to signal resource provisioning to bees, or their readiness for pollination? Given that bees can perceive detail within the floral electric field (Clarke et al. mbruster  suggestsAs a recently defined field, there is still a vast amount that is unknown in the electrical ecology of pollination, each from the perspectives of the bee the flower and the electric field. Finite-element models are proving a powerful tool in describing electrical interactions between the bee and the flower, but we need empirical data to confirm their predictions under a broader range of conditions. In many cases, the influence of electric fields may be strongly reduced by environmental factors not simulated in our models. High humidity, for example, leads to the formation of moist films on surfaces which prevent charge build up, and a dense rainforest canopy acts like a Faraday cage, negating the influence of the atmospheric electric field at ground level, possibly preventing the formation of floral electric fields altogether. How do these different environmental conditions affect the foraging behaviour of pollinators? At the scale of the pollen grain, even small humidity gradients in the floral headspace (von Arx et al. The tripartite interactions between bees, flowers, and the electric field that exist all around them, reveal thus far underappreciated physical and sensory ecologies. These interactions are likely to be diverse and maybe ubiquitous among insects since all are subject to the laws that govern the force of electromagnetism. The electrical ecology of bees is not unique. Every flying pollinator of comparable size to a bee is subject to the same electrostatic forces from flowers and comparable forces from conspecifics and other species. Antennae and mechanosensory hairs are widespread in arthropods (Casas and Dangles


Hydatic cyst of seminal vesicles is very rarely seen. We report a case who complained of the inability to void, which developed progressively with dysuria, frequency, nocturia, and tenesmus, due to a giant retrovesical hydatid cyst that displaced the bladder and rectosigmoid region.


An effective method to monitor heavy metal stress in crops is of critical importance to assure agricultural production and food security. Phenology, as a sensitive indicator of environmental change, can respond to heavy metal stress in crops and remote sensing is an effective method to detect plant phenological changes. This study focused on identifying the rice phenological differences under varied heavy metal stress using EVI (enhanced vegetation index) time-series, which was obtained from HJ-1A/B CCD images and fitted with asymmetric Gaussian model functions. We extracted three phenological periods using first derivative analysis: the tillering period, heading period, and maturation period; and constructed two kinds of metrics with phenological characteristics: date-intervals and time-integrated EVI, to explore the rice phenological differences under mild and severe stress levels. Results indicated that under severe stress the values of the metrics for presenting rice phenological differences in the experimental areas of heavy metal stress were smaller than the ones under mild stress. This finding represents a new method for monitoring heavy metal contamination through rice phenology. With rapid development of the industry, rice paddies in some areas have been subjected to heavy metal pollution. According to the related statistics, more than 12 million tons of grains are contaminated in China every year . Heavy mAt present, most research on heavy metals in rice are carried out from the aspects of chlorophyll, leaf area index, cell structure  ,8,9, or Compared with methods based on traditional ground remote sensing and model simulation, extracting the rice phenological information through satellite remote sensing to monitor heavy metal stress is more convenient and direct. Knowledge of crop calendars and phenology is often a key element in vegetation interpretation. Since the 1970s, the potential of multi-temporal satellite observations to provide information on the phenological development of natural vegetation and crops have been recognized by many researchers ,23,24. PThe objective of this study was to explore the potential of evaluating heavy metal stress based on rice phenology extracted by remote sensing technology. In order to verify the effectiveness of using phenological characteristics to monitor heavy metal stress in rice, we compared and analyzed the phenological differences under mild and severe heavy metal stress by two kinds of metrics with phenological characteristics for proving that the specific phenological characteristics are effective indicators for monitoring heavy metal stress in rice. The findings are expected to assist in monitoring the heavy metal contamination of rice and measure the degree of regional heavy metal pollution from the perspective of phenology.Environmental Quality Standard for Soils in China [The city of Zhuzhou, in the Hunan Province, China, is an important commodity grain producing area and, simultaneously, is an old industrial base. From the industrial activity, the Xiangjiang River and its tributaries were contaminated. The main heavy metals in this area are cadmium (Cd), plumbum (Pb) and arsenic (As). Four rice fields adjacent to the Xiangjiang watershed in Zhuzhou were selected as experimental fields , and eacl sample  and variin China , the depin China . In thesIn this study, 16 CCD images from the small sun-synchronous satellites, HJ-1A/B, which are used for monitoring and forecasting environmental and natural disasters, were adopted as the remote sensing data covering four experimentation areas in the entire growing season of rice paddies (dated from 13 June to 29 September 2013). The HJ-1A/B satellite can cover a ground swath 700 km wide, and two CCD sensors were loaded on satellites which formed a constellation to constitute an observation network that covered China and its surrounding areas on a two-day-repeat cycle. The CCD sensors captured ground features with a 30 m pixel resolution at a nadir angle in the visible bands. These CCD images consisted of the following four channels: band 1: 0.43–0.52 μm (blue waveband), band 2: 0.52–0.6 μm (green waveband), band 3: 0.63–0.69 μm (red waveband), and band 4: 0.76–0.90 μm (near-infrared waveband). Wang et al.  integratThe time is expressed as the day of the year (DOY) in this paper. Among the 16 images , there were two images in June (DOY168 and DOY181), four images in July , five images in August , and five images in September . All of the 30 m multispectral CCD images were processed prior to use, and the specific process can be summarized as: (1) radiometric-calibration was performed for each band, and the four bands of each scenes were packed; (2) the CCD images were clipped to obtain a subset The extraction of vegetation phenological information by remote sensing are mostly based on the vegetation indices time-series curves, that is, the vegetation indices of time-series using remote sensing images are analyzed by smoothing and parameter analysis of the vegetation indices to extract phenological information while reflecting seasonal characteristics of vegetation community for temporal-spatial analysis ,34,35,36NDVI is the most widely used vegetation index for monitoring vegetation phenology. EVI, the enhanced vegetation index based on NDVI, is applied increasing widely. To a certain extent, the two VIs can reflect the comprehensive situation of the land cover types from pixels in corresponding regions. The periodic increase and decrease of the VIs value of the region is a typical performance of the vegetation growth cycle, which can be used to determine the vegetation growth and overall situation. Additionally, the differences of VIs among different regions can reflect the vegetation, soil type, precipitation, topography and land use, and so on . Thus, tectively .Due to the presence of cloud, shadow, and other factors, the time-series EVI data still had a lot of noise, so they need to be filtered and reconstructed before application . In the Many researchers performed the studies about the fidelity performance and the capability of keeping main characteristics on the three algorithms for vegetation phenology detection. Wang, Xin, and Shu  used EVIAfter the original data were smoothed, the temporal profiles of EVI were obtained. Next, according to the characteristics of EVI time-series curves, the specific phenological periods were extracted. The rice growth period was from transplanting time to harvest time. Remote sensing in rice using optical satellite sensors exploited the phenological characteristics of the rice life-cycle. About one to two weeks after transplanting, the rice turned into the tillering period. In the tillering period, due to the development of rice root and leaf systems, EVI rapidly increased until the heading period when the vegetative development of rice reached its maximum, and rice growth becomes a descending stage where EVI decreases gradually, due to the nutrients transferred into the seeds. During the maturation period, due to the loss of most of the chlorophyll, the leaves rapidly withered, and the vegetation index was minimized in the harvest period . As ment0) [max) was used to estimate the active tillering period; and (3) similarly, at the end of the maturation stage, EVI declines rapidly due to leaf senescence [min) to detect the maturity period (mid-September). According to the characteristics of rice growth stages described above, the identification of rice growth and development period can be detected through the vegetation index of remote sensing image time-series.Based on the analysis method of first derivative, we selected the appropriate indicators to identify the rice growth stage: (1) due to a high level of LAI and biomass in the heading period (mid-August), this period will have the maximum EVI corresponding to the value of FD (FD0) ,47; (2) 0) . Thus, tnescence . Hence, As the four experimental areas were from different places, there were differences in the specific transplanting period (mostly in mid-June) and the definition of phenological periods by traditional methods, therefore, we could not identify the delay and advance for the specific phenological period. To detect the impact of heavy metals in soil on rice phenology, we selected the date-intervals, which meant the lengths of time between different phenological phases and eliminated the impact of different planting time, as the heavy metal stress metrics of rice phenology to extract the same differences under varied heavy metal stress levels. The specific metrics are calculated as follows :(3)L1=DOTo further analyze the effect of heavy metal concentration on rice phenology, the differences between L1 and L2 under same heavy metal stress were taken as a measure, that is, the variation tendency of two adjacent phenological intervals (between the tillering period and heading period and between the heading period and maturity period) under different heavy metal stresses was analyzed, and the formula is as follows:The dates of the tillering, heading, and maturation periods can be extracted by remote sensing data using the phenology monitoring algorithms, as the EVI time-series reflect changes in the morphological and physiological condition of the rice during the rice growth cycle . The timIn this study, approximately 50 pixels from CCD images were extracted in each of the four experimental areas to fit the time-series of the EVI and extract phenological periods by pixel, before averaging the pixel information in the same experimental area. max, FD0, and FDmin) of smoothed EVI temporal profiles, three phenological periods  in the four experiment areas were obtained and averaged, respectively. According to first derivative (FD) analysis  were selected as the metrics to present the phenological differences under stress. Different concentrations of heavy metals had distinct effects on the rice different growth stages. As shown in Different characteristics of rice growth status presented different responses under heavy metal stress. Based on the time-series curves constructed by the EVI values obtained from the remote sensing images, the trend and abundance of curves can reflect the size of rice growth season. This study presents a new methodology for monitoring heavy metal stress in crops based on crop phenology with remote sensing technology. The methodology was identified through extracting rice phenological differences under mild and severe heavy metal stress levels by the EVI time-series, which was obtained from HJ-1A/B CCD images and fitted with asymmetric Gaussian model functions (AG). With first derivative (FD) analysis, three phenological periods  were detected, and then two kinds of metrics with phenological characteristic: date-intervals and time-integrated EVI were constructed and compared in the four experimental areas. The results indicated that the phenology is an effective indicator for monitoring heavy metal stress in rice.It is critical that phenological differences under different heavy metals stress are measured. In this study, we selected the EVI time-series, which were composed of CCD images, to define three key phenological periods. CCD images from HJ-1A/B have high temporal-spatial resolution were obtained, but due to the monsoon climate, the cloud-free remote sensing data during the growth phase of the paddy rice was limited. Compared with other remote sensing data commonly used for monitoring vegetation phenology, CCD data represents the value of the day unlike AVHRR and MODIS remote sensing data of usually eight or 16 days of synthetic products, which means any one of the images were compounded by the optimal algorithm between eight days or 16 days. Thus, we can obtain better and closer to the true value of smoothed curves of the EVI time-series that also contributed to detect phenological differences under the two heavy metal stress levels. Some studies found that the crop growth under heavy metal stress becomes slower comparing with non-contaminated conditions ,51,52,53This study provides a new way to monitor heavy metal pollution in agriculture through remote sensing technology. Compared to traditional ground-based methods used to monitor heavy metal stress, remote sensing is a simple and convenient resource. Furthermore, the analysis of phenology presented here concerned the effects of temperature, climate, rainfall, and so on. In this study, the results of the metrics’ analyses clearly indicate that heavy metal stress is also an important factor that affects phenological changes. At present, in the case of serious heavy metal pollution, when investigating the phenological changes of terrestrial ecosystems, it is necessary to consider the impact of heavy metal pollution in heavy metal contaminated areas. However, it is necessary to declare that the relationship between the phenological differences and stress metrics found in this study are specific to the selected phenological periods studied , and the results might differ for other phenological periods monitored, other types of data used, other smoothed functions, other measures of phenological period estimation, and other stress metrics. Thus, when the metrics are applied to other areas or other crops, another comparative analysis should be undertaken. Furthermore, the values of the metrics presented in this study ought to be proved. In conclusion, the generality of this finding in other areas and crops require further research.Our results are limited by the selected phenological extraction method’s sensitivity and fidelity and the metrics used, but under the severe stress level, the length of time between the tillering period and the maturation period was around 49 days, and the integral areas of the EVI time-series during the tillering and maturation periods was about 32. These were identified via the statistical properties, and a comparative analysis of the metrics is an important indicator of monitoring phenological differences under mild and severe stress levels to fully investigate heavy metal stress levels of rice paddies based on extracting phenological periods from EVI time-series curves by remote sensing data and the fitting method. Meanwhile, it should be pointed out that, in this study, two kinds of metrics were qualitatively analyzed, and the performance of the metrics could be further tested through detailed quantification using measured data and more detailed procedures. The results in this paper could be further verified by collecting field observation data. In addition, more rice phenology periods, like the transplanting periods and panicle development, should be extracted to analyze the phenological differences by means of ground-based spectrometers.max, FD0 and FDmin) of smoothed EVI temporal profiles. Two kinds of metrics with phenological characteristics, date-intervals and time-integrated EVI, were constructed to detect rice phenological differences under varied heavy metal stress. The comparison of the two kinds of metrics indicated that the values of the metrics for presenting rice phenological differences under severe stress in the four experimental areas were smaller than the ones under mild stress. It has been proved that it is feasible to use remote sensing technology to monitor the rice phenological variations caused under different heavy metal stress, and measured the rice phenological differences with the two kinds of metrics under mild and severe heavy metal stress. This conclusion strongly supported the theory that phenology is one of the sensitive indicators of environmental change. Thus, phenology could be a useful indicator for heavy metal stress in rice plants.In this study, we monitored rice phenological differences under mild and severe heavy metal stress levels using remote sensing technology for exploring the potential of evaluating heavy metal stress based on rice phenology by remote sensing. We used the EVI time-series smoothed with AG function to represent the status of plant growth. Three phenological periods  were detected through first derivative (FD) analysis (FD


The wfsl1 displayed white fine stripe leaves since tillering stage and abnormal chloroplast structure. Map-based cloning and Bioinformatic analysis indicated that WFSL1 on chromosome 1 contains an “A” to “T” substitution in protein coding region, and encodes a putative metal-dependent phosphohydrolase with HD domain at the N-terminus. WFSL1 was targeted to the chloroplasts and had higher expression in mature leaves and sheaths. RNA-seq analysis revealed that chloroplast development and photosynthesis genes were significantly affected in wfsl1 plants. Levels of WFSL1 and chloroplast encoded proteins were decreased in wfsl1 mutants via western blot analysis. Compared with WT, wfsl1 exhibits lower Chl content and defective in biogenesis of chloroplast ribosomes, which resulted in reduced grain yield. Taken together, our results show that WFSL1 is critical for chloroplast development, ribosome biogenesis, and light energy utilization, finally affects grain yield.Plant leaves are a crucial organ associated closely with chloroplast development, photosynthesis rate and crop productivity. In this study, a  Chloroplast development is regulated by genes encoded of nuclear and plastids, but for the limited coding capacity of plastids, chloroplast development is mainly under nuclear control. The coordination of gene expression through nuclear and plastids is essential for chloroplasts biogenesis in plant2. The transcription of nuclear and chloroplast genes mainly depends on two RNA polymerase. One is the bacterial-type nuclear-encoded RNA polymerase (NEP). NEP transcribes plastid genes involved RpoA, B, C that are necessary for the development of plastidic genetic systems in the early stage of chloroplast development. The other bacterial-type plastid-encoded RNA polymerase (PEP) is response for the photosynthesis genes  development at mature stage4. Owing to defects in PEP activity, the formation of thylakoid membrane and photosynthesis was repressed, suggesting it is an important role of PEP in chloroplast development5. Proteins encoded by the plastid genome are synthesized by plastidic prokaryotic type 70 S ribosome that are composed of 30 S and 50 S subunits9. Deficiency in content of 70 S ribosome results in stunted chloroplast development. Arabidopsis konck-down mutant RH22 accumulated precursors 23 S rRNA that displayed virescent phenotype. RH22 affected ribosome assembly in rRNA metabolism10. ObgC participated in 70 S ribosome assembly. The knock down and RNAi of ObgC result in chlorotic phenotype in rice8. Thus, 70 S ribosome biogenesis is essential for chloroplast development in higher plants.Chloroplast, the center for leaf cell metabolism which plays an important role in light reception and carbon sequestration in higher plants11. Immutans (im) and var2 are two types variegation mutants in Arabidopsis. The im mutant is induced by a nuclear recessive gene and the extent of variegation can be modulated by light and temperature. IM protein is a plastid homologue of the mitochondrial alternative oxidase and functions as a redox component of the phytoene desaturation pathway11. Mutations in the VAR2 locus cause variegation due to loss of a chloroplast thylakoid membrane protein that is similar to the FtsH family of AAA protein. FtsH functions in a number of diverse membrane-associated events and VAR2 protein functions in thylakoid membrane biogenesis12. Both im and var2 mutants provide an excellent system to understand the molecular mechanism of nuclear-plastid interactions in Arabidopsis. In rice, many genes were also associated with nuclear-plastid interaction. V1 encodes a chloroplast-localized protein NUS1 regulating chloroplast RNA metabolism16. V2 encodes a plastids/mitochondria Guanylate kinase (pt/mtGK) which catalyzes GMP to GDP in guanylate biosynthesis and affects chloroplast development. It suggests that pt/mtGK is an important part for chloroplast development17. RNRL1 and RNRS1 encode the large and small subunits of rice ribonucleotide reductase, respectively. They are necessary for the DNA replication in chloroplast18. OsDVR plays certain roles in nuclear-cytoplasmic signal transduction by which nucleus directly regulates cytoplasm20. Leaf-color mutations are a crucial for dissection of regulation mechanism of with chloroplast development, photosynthesis and key agronomic traits.Plant variegations are characterized by the presence of white sectors and green sectors. The white sectors contain defective chloroplasts and the green sectors contain normal chloroplastswhitef inestripeleaf (wfsl1) mutant in rice. The phenotype of mutant not very likely with im and var2 mutants, the green and white sectors longitudinal distributed on the blade of mutants. It’s interesting that mutant phenotype mainly first occurs on the newly grown leaves and sheath at tillering stage, continued to the mature stage. Our study discovers that map-based cloning identifies WFSL1 on chromosome 1 encoding an HD domain containing gene. The WFSL1 protein is localized to chloroplast and was decreased in wfsl1 mutant leaves. The wfsl1 mutant was defective in biogenesis of chloroplast ribosomes, and affected photosynthesis rate and grain yield. Our results suggest that WFSL1 is critical for chloroplast development and regulating chloroplast ribosome biogenesis.Here, we isolated a wfsl1 mutant was isolated from an M2 population of the japonica rice Zhonghua11 (ZH11) after Ethyl methanesulfonate mutagenesis, and was designated according to the chromosome location and white fine stripe leaf phenotype. At seedlings wfsl1 showed normal phenotype , suggesting that the phenotype of wfsl1 mutant is controlled by a single recessive gene . The predicted region was sequenced to detect the mutation and discovered that the target ORF (LOC_Os01g01920) had an “A” to “T” substitution in protein coding region . The overexpression plants exhibited normal green leaves and relative expression increased significantly compared with wild type and mutants  is responsible for the phenotype of wfsl1 mutant.The e 1 Fig. . Then eie 1 Fig. . The locion Fig. . The genion Fig. . Gene prion Fig. . To confves Fig. , and theves Fig. . Besidesves Fig. . And a 1nts Fig. . Thus, tWFSL1 cDNA was amplified from the cDNA library of wild type. The WFSL1 cDNA was ligated to GFP sequence, and injected into rice protoplasts. Its expression was under the control of 35 S promoter. The GFP fluorescence in transformed protoplasts was examined using confocal fluorescence microscope, which showed that WFSL1 was located to chloroplast , mature leaves and mature roots (heading stage), stems, sheaths and panicles, respectively. WFSL1 was highly expressed in sheaths and mature leaves, but low expression in young leaves, roots, stems and panicles  was used as control27 , Rubisco activase (RCA) were all decreased in wfsl1. while the expression levels of nuclear-encoded genes RpoA, RpoB, RpoC1, RpoC2 increased in wfsl1. These results suggest wfsl1 is defective in biogenesis of plastid-encoded genes and affects chloroplast development.qRT-PCR was used to examine the tissue-specific expression pattern of les Fig. . Next, wage Fig. . The relwfsl1 mutation on gene expression. More than 40 million reliable clean reads were obtained from wild type and wfsl1. About 355 up regulated genes while 536 repressed genes in wfsl1  and its chloroplasts were defective in tillering stage  ppGpp hydrolase activity of SpoT protein in E.coli, which supports that the protein containing HD-domain is a phosphohydrolase33. Cyclic nucleotide Phosphodiesterase (PDEs) has a HD domain, and is regulated by Zn2+ coordination. Other metals, such as Mn2+, Co2+ and Mg2+, also have catalytic activity34. RelA and SpoT regulate ppGpp levels in E.coli35. In SpoT protein, HD domain has ppGppase activity33, and the His-Asp doublet is involved in the hydrolysis for ppGpp36. Arabidopsis has four RelA/SpoT homologues AtRSH1, AtRSH2, AtRSH3 and AtCRSH. They are all targeted to plastids and expressed in green tissues and flowers which have important functions in chloroplast development and reproduction via ppGpp synthetase activity38. The transcripts encoding the RC and LHC subunits of PSI and PSII, the small and large subunits of Rubisco was decreased in OX:RSH2 and OX:RSH3 plants. Plastid-encoded genes were affected in OX:RSH2 and OX:RSH3 plants39.We cloned nts Fig. . WFSL1 h38. Go term analysis found that genes of photosynthesis, photosystem II assembly, and plastid were highly expressed in wfsl1  after Ethyl methanesulfonate mutagenesis. The japonica rice ZH11 and wfsl1 mutant were grown in the paddy fields of Zhejiang  and Hainan  provinces in China under local growing conditions.The wfsl1 were collected and weighted. Then, the leaves were soaked in an acetone and ethanol mixture solution at 26 °C in dark for 24 h. The content of chl a, chl b and carotenoids were calculated as described previously45.The leaves from different stages of wild type and wfsl1 were selected at tillering stages and cut into small pieces. They were fixed by 2.5% glutaraldehyde (PH 7.2) and vacuumed until fully sinking to the bottom. Subsequently, samples were successively washed three times with 0.2 mol/L sodium cacodylate buffer for 30 min, fixed in 10% osmic acid for 1 h, distilled three times with deionized water for 45 min, dehydrated with ethanol, treated with acetone and embedded in epoxy resins and polymerized at 70 °C. The samples were then cut into about 500–800 Ǻ thick with a slicer and stained by the mixture of uranyl acetate dihydrate and lead citrate. The sections were washed with deionized water and visualized using HITACHI Transmission Electron Microscope (HT7700).The leaves of wild type and wfsl1 and the indica cultivars TN1, 93–11, SH527 (Shuhui527) to analyze whether a dominant/recessive single/multiple gene control(s) the wfsl1 phenotype. The segregation population of F2 was examined by χ2 test . A total of 117 pairs of rice chromosome markers were used for primary mapping, and the sequence-tagged-site (STS) markers for fine mapping were developed based on the gap difference between the contig sequences of the japonica cultivar Nipponbare and the indica cultivar 93–11 (http://ensemblgenomes.org). WFSL1 was ultimately mapped to a 50.5 kb region on chromosome 1. The predicted region was PCR-amplified and sequenced to detect the mutation.To map the genomic location of E-value cut-off of 0.001. Using the Clustal Omega  to identify motif that are conserved in the alignment sequence.Using blastp program to search the protein sequence database at the NCBI with an WFSL1 sequence, the upstream and downstream sequences was amplified by two pairs of PCR primers: 5′-ggatcccttgggtgtgccgtcgatgtgagc-3′, 5′-tatccacaacagtgaaaggatatgtggttaac-3′ and 5′-gcattgtgtcattcaggctgcgggatctaaaac-3′, 5′-ccaagcttcggtggtaggaggactcccgttgg-3′. The 11,296 bp PCR product was inserted into the binary vector pCAMBIA1300 (containing CaMV 35 S promoter) to construct the pCAMBIA1300-WFSL1 vector. This vector was then introduced into the wfsl1 mutants by Agrobacterium-mediated transformation using the Agrobacterium tumefacien EHA105. For the WFSL1 overexpression construct, a 1,743-bp fragment of full-length cDNA was amplified by two pairs of PCR primers: 5′-GGGGTACCATGAAACATCCCTCCCGCATTAAATTGGC-3′ and 5′-GCTCTAGATCAGTTGTAGGTTCTCGAAGGCTTCTG-3′ from the cDNA library of wild type and inserted into the binary overexpression vector pCAMBIA1300S (pCAMBIA1300 containing 35 S promoter) into pCAM-BIA1300S-WFSL1. The vector was transformed into wfsl1 mutants to generate overexpression plants.A 11,296 bp genomic DNA fragment containing WFSL1 were: 5′-gctctagaatgaaacatccctcccgcattaaattggc-3′ and 5′-cgggatcccggttgtaggttctcgaaggcttctg-3′. The pCAMBIA1300-WFSL1-GFP vector, as well as the control were transformed into rice protoplasts according to the protocol described previously46. The transformed rice protoplasts cells were incubated for 16 h at 28 °C under dark conditions. The GFP fluorescence in transformed protoplasts was examined by confocal fluorescence microscope .To investigate the subcellular location of WFSL1, a 1,743-bp fragment of full-length cDNA was amplified by PCR and ligated into pCAMBIA1300-GFP (containing 35 s promoter and GFP reporter protein) vector to generate the pCAMBIA1300-WFSL1-GFP construct. The PCR primers for wfsl1 mutants using a Total RNA Extraction Kit  according to the manufacturer’s instructions. The complementary DNA was synthesized using a ReverTra Ace qPCR-RT Kit . RT-PCR was run in Applied Biosystems 7900HT Real-time System using 2 × SYBR Green PCR Master Mix (Applied Biosystems). The RT-PCR program was as follows: initial denaturation at 95 °C for 10 min, followed by 40 cycles at 95 °C for 10 s and 60 °C for 1 min. The analysis of each sample was based on three technical replicates and biological replicates. The relative expression level of each transcript was compared with that of UBQ5 and quantified with the 2C−ΔΔT method47. The primers for the genes are listed . A total of 45 million reads genes from wild type and 40 million from wfsl1 were obtained. The significance of differentially expressed genes (DEGs) were using log2 (fold change) > 1 and q values < 0.05. Gene ontology analysis was performed on GOseq48. Pathway enrichment analysis was using the Kyoto Encyclopedia of Genes and Genomes database49.Total RNA was extracted from wild type and 50 isolated from wild type and wfsl1 at seedling and tillering stage. The tissues were ground in liquid nitrogen and thawed in extraction buffer  for 15 min on ice. The supernatant was collected by centrifugation at 12,000 g for 10 min at 4 °C. Total proteins were separated by SDS-PAGE gels (8%), transferred to the polyvinylidene difluoride (PVDF) membranes , blotted with different primary antibodies, detected with ECL prime . Anti-WFSL1 antibody was obtained from Shanghai Youke Biotechnology (http://www.youke-ab.cn/) and other antibodies such as anti-PsaA , anti-AtpB , anti-RCA  and anti-RpoA  from Beijing Protein Innovation (http://www.proteomics.org.cn/) and anti-PsbA  from Agrisera.Total proteins extraction was performed as previously describedSupplementary information