In infancy, males are at higher risk of dying than females. Birthweight and gestational age are potential confounders or mediators but are also familial and correlated, posing epidemiological challenges that can be addressed by studying male-female twin pairs.We studied 28 558 male-female twin pairs born in Brazil between 2012 and 2016, by linking their birth and death records. Using a co-twin control study matched for gestational age and familial factors, we applied logistic regression with random effects (to account for paired data) to study the association between male sex and infant death, adjusting for: birthweight, within- and between-pair effects of birthweight, birth order and gestational age, including interactions. The main outcome was infant mortality (0–365 days) stratified by neonatal (early and late) and postneonatal deaths.P = 0.001] and after  adjusting for birthweight and birth order. When adjusting for birthweight within-pair difference and mean separately, the OR attenuated to 1.40 , with evidence of familial confounding . We found evidence of interaction (P = 0.001) between male sex and gestational age for early neonatal death.Males were 100 g heavier and more at risk of infant death than their female co-twins before [odds ratio (OR)  = 1.28, 95% confidence interval (CI): 1.11–1.49, After matching for gestational age and familial factors by design and controlling for birthweight and birth order, males remain at greater risk of infant death than their female co-twins. Birthweight’s role as a confounder can be partially explained by familial factors. Key MessagesMale twins are about 40% more likely to die in the first year of life than their female co-twins, despite being born 100g heavier, on average.The association between male sex and infant mortality remains after adjusting for familial confounders.Term births might increase the relative risk of neonatal death for males compared with female co-twins, although the absolute risk is decreased for both males and females.Infant mortality, defined as deaths within the first year of life, remains a substantial public health problem globally. In 2019 alone, there were nearly four million infant deaths globally, and around 63% of those were neonatal deaths (within the first 28 days of life).Studying sex differences in early life mortality is challenging, not least because birthweight and gestational age, which are likely to be potential confounders or mediators, are familial and correlated with each other.A co-twin control study designRandom-effects models can be used to estimate the association between male sex and infant mortality while controlling for unmeasured familial factors and measured covariates that differ between twins in a pair. This is important because birthweight is influenced by maternal and genetic factors.We have designed a matched opposite-sex co-twin control study to investigate the association between male sex and infant mortality while matching for gestational age, and unmeasured maternal and familial factors, and adjusting for birthweight and birth order as measured covariates. We applied deterministic data linkage to create a whole-of-population prospective cohort of male-female twin pairs by linking their birth and death records available from national health administrative databases in Brazil, and then studied this cohort to address our study objectives.The University of Melbourne's Human Research Ethics Committee (HREC) waived the need for ethics approval for this study, due to the use of unidentifiable secondary data freely available in the public domain.We used population-complete databases publicly available from the Brazilian population health databases system called Datasus.P = 0.093). This linkage process was entirely deterministic, performed by a straightforward linkage using the birth record number from twins in SINASC to all death records in SIM within 1 year of birth over 2012–17 with this unique identifier. We included all death records (not only twins) to avoid missing deaths that were not coded for a ‘double’ pregnancy due to potential coding errors. The birth record number was not present in 31% of death records. Given our interest in relative differences between males and females, we conducted a sensitivity analysis and found no evidence that proportions of males and females in death records of twins with the birth identifier were different from those records without the identifier , parity at birth, gestational age (weeks), preterm ,Birth outcomes were congenital anomalies detected at birth and Apgar scores at the first and fifth minutes (Apgar1 and Apgar5) scored from 1 to 10 and then categorised as low if <7. Following WHO guidelines,First, we calculated descriptive statistics for maternal characteristics of the twin pairs  and testSecond, we investigated the association between male sex and the outcomes by estimating odds ratios (OR) for males  in: Model 1, an unadjusted random-effects model using maximum likelihood estimation; Model 2, which was the same as Model 1 but adjusted for birth order and birthweight; and Model 3, the same as Model 1 but adjusted for birth order and within- and between-pair differences of birthweight separately, by including both the within-pair absolute difference and the within-pair mean of birthweight in the models .Twins were matched in each design. The difference between the models is the level of confounding adjustment, going from the more generic crude Model 1 to the more specific Model 3 which accounted for the familial aggregation in birthweight as a confounder. We used a likelihood ratio test (LR Test) to compare Models 2 and 3 to assess evidence of familial confounding.To study interactions, we then fitted Model 4, which modelled the association between male sex and outcomes adjusting for birth order, birthweight pair difference, birthweight pair mean, gestational age and an interactive term between male sex and low birthweight, with ORs presented separately for twins with low (<2500 g) and normal (≥2500) birthweight. Model 5 adjusted for the same variables, but the interactive term was for male sex by preterm births, with separate results for preterm (<37 weeks) and term (≥37 weeks) births.We studied interactions of male sex with birthweight pair mean and male sex with gestational age as continuous variables while adjusting for covariates for all mortality outcomes  Statement.All analyses were conducted using Stata/SE 16.0, using the P <0.001). There was no evidence of sex differences in Apgar1 and Apgar5 scores (The mean age of the twins' mothers was approximately 30 years . The mea5 scores . There w5 scores .P <0.001), increasing from 1.28  in the unadjusted model. The aOR for the association between male sex and neonatal mortality (0–28 days) was 1.59  from the adjusted Model 2. This positive association was mainly driven by early neonatal deaths. For those, the adjusted OR was 1.72 . We found evidence of a sex association for postneonatal death  but not for late neonatal death  from the adjusted Model 2.There was a consistent increase in ORs from those estimated in the unadjusted Model 1 to Model 2 adjusted for birth order and birthweight in a single variable for all outcomes, suggesting that birthweight is a negative confounder of the sex associations . After aP <0.001), favouring Model 3 for all mortality outcomes except postneonatal death. However, the confidence intervals of estimates of the sex association overlapped between these models, with better precision in Model 3.P <0.001) to 1.43  between Models 2 and 3, suggesting that the effect of birthweight as a negative confounder can be partially explained by familial confounding. For infant death, an increase of 100 g in the birthweight pair mean was a stronger protective factor  than being 100g heavier than their co-twin .The aOR for early neonatal deaths attenuated from 1.72  and early neonatal death (P = 0.033) . In Model 4, we found evidence of interaction between male sex and low birthweight for low Apgar1 (P = 0.003) and postneonatal death (P = 0.008).We found some evidence of an interaction between male sex and preterm status for neonatal death , and male sex and birthweight pair mean (P = 0.001), both as continuous variables  for all outcomes but postneonatal death . We did We investigated the roles of birthweight, gestational age and familial confounding in the association between male sex and infant mortality by applying a twin design to study male-female twin pairs. Our results show that males were at substantially higher risk than females for low Apgar5, congenital anomalies and infant, neonatal, early neonatal and postneonatal deaths after adjusting for birth order and within- and between-pair estimates of birthweight separately. This was despite males being heavier at birth by 100g on average.,,The overall proportion of infant mortality for boys compared with girls was greater than previously reported for the non-twin population,For the first time, we found that the within- and between-pair differences in birthweight were independent predictors of infant mortality when fitted separately (Model 4). Nonetheless, the attenuation from the OR of 1.60 (95% CI: 1.39–1.83) to 1.40 (95% CI: 1.21–1.61) provides an estimate of the magnitude of familial (negative) confounding related to birthweight in the association between sex and infant mortality. Our evidence that familial confounding is more important for early neonatal than later deaths has not been previously reported in the literature. Our study found that male sex predicts infant death independently of birth order and within- and between-pair differences in birthweight .We found strong evidence of an interaction between male sex and gestational age for early neonatal deaths. This is important, given the previously reported longer gestational age for opposite-sex twin pairs compared with same-sex twin pairsOur study had some limitations related to contingencies in the data linkage process. The sensitivity analysis  revealedGiven that around 30% of the mortality records did not have a unique identifier (birth number) available for linkage with birth records, we might have underestimated absolute mortality numbers. Differences in the quality of mortality records in Brazil depending on death location have been reported.,Our findings have clear implications for the Brazilian public health care setting, which is primarily guided by low birthweight and prematurity guidelines to determine eligibility for primary care interventions. For example, the ‘kangaroo method’,Our study showed that boys are at particularly higher risk of death in the first 7 days of life compared with their female co-twins. We showed that the familial aggregation of birthweight should be properly considered when adjusting for this variable, by demonstrating the application of the ‘within-between’ method for the first time in twin pairs of the opposite sex. Our findings also advance the understanding of the interplay between birthweight, gestational age and familial confounding in the association between sex and infant mortality.IJE online.L.C.F., P.H.F. and K.J.S. were funded by the National Health and Medical Research Council’s (NHMRC) Australian Centre of Excellence in Twin Research (APP1079102). M.E.B. is a Newton International Fellow Alumnus  and holds grants from Google.org, NVIDIA, Bill & Melinda Gates Foundation, MRC (UK) and Wellcome Trust (UK). G.S.D. is employed by Genetic Technologies Ltd. M.H. was funded by an NHMRC Investigator Grant (APP1193838). J.L.H. is an NHMRC Senior Principal Research Fellow (APP1137349). None of the funding sources had any role in the preparation of this manuscript.L.C.F. co-designed the study and the analytical strategy, conducted the statistical analysis and drafted the manuscript. M.E.B. and E.M. co-designed the data linkage strategy, acquired the data and revised the manuscript. G.S.D., M.H. and P.H.F. co-designed the study, helped interpret findings and revised the manuscript. K.J.S. and J.L.H. co-designed the study and the analytical strategy, guided the statistical analysis, helped interpret findings and revised the manuscript. All authors approved the final version of this manuscript.None declared.dyab242_Supplementary_DataClick here for additional data file.


The creation of electromagnetic energy may be realised by engineering a device with a method of transduction, which allows an external energy source, such as mechanical, chemical, nuclear, etc., to be impressed into the electromagnetic system through a mechanism that enables the separation of opposite polarity charges. For example, a voltage generator, such as a triboelectric nanogenerator, enables the separation of charges through the transduction of mechanical energy, creating an active physical dipole in the static case, or an active Hertzian dipole in the time-dependent case. The net result is the creation of a static or time-dependent permanent polarisation, respectively, without an applied electric field and with a non-zero vector curl. This system is the dual of a magnetic solenoid or permanent magnet excited by a circulating electrical current or fictitious bound current, respectively, which supplies a magnetomotive force described by a magnetic vector potential and a magnetic geometric phase proportional to the enclosed magnetic flux. Thus, the active electric dipole voltage generator has been described macroscopically by a circulating fictitious magnetic current boundary source and exhibits an electric vector potential with an electric geometric phase proportional to the enclosed electric flux density. This macroscopic description of an active dipole is a semi-classical average description of some underlying microscopic physics, which exhibits emergent nonconservative behaviour not found in classical closed-system laws of electrodynamics. We show that the electromotive force produced by an active dipole in general has both electric scalar and vector potential components to account for the magnitude of the electromotive force it produces. Independent of the electromagnetic gauge, we show that Faraday’s and Ampere’s law may be derived from the time rate of change of the magnetic and dual electric geometric phases. Finally, we analyse an active cylindrical dipole in terms of scalar and vector potential and confirm that the electromotive force produced, and hence potential difference across the terminals is a combination of vector and scalar potential difference depending on the aspect ratio (AR) of the dipole. For long thin active dipoles (AR approaches 0), the electric field is suppressed inside, and the voltage is determined mainly by the electric vector potential. For large flat active dipoles (AR approaches infinity), the electric flux density is suppressed inside, and the voltage is mainly determined by the scalar potential. Classically, a permanent polarisation consists of equal and opposite charges, ve force ,2,3,4, aThe ideal oscillating time dependent active conducting dipole is commonly known as a Hertzian dipole, and in the quasi static limit, otential . In contotential ,2,3,4,5.otential ,9,10,11.In the case that the medium is an insulator, a macroscopic active bound charge dipole is known as an electret  and exhiFurthermore, related to this, modern polarisation theory introduced in the 1990s ,26,27 haIn this work, we use the fact that a permanent vector polarisation, generated without an electric field has both a non-zero curl and divergence. For the curl of the polarisation to be nonzero, an energy input is required to separate the bound charge; this describes a permanent electret or energy harvesting material ,35,36,37For a dipole, some standard text book example assumes point charges, which are unphysical, a better approximation is to assume ideal surface charges, σei , so qei=shown in P→ei=ϵE→eMaxwell’s equations for an ideal voltage generator with impressed bound or free charge 15) back g either  or 14)  16)∂(∇→∂(∇→(16)∂ituting,  and (15)ituting,  and (15)Thus, we have successfully calculated the potentials in terms of the impressed sources, The magnetic Aharonov–Bohm (AB) effect is a phenomenon where a charged particle’s wave function is affected by the magnetic vector potential, ing zero . Underlying zero  apparenting zero  and not ing zero ,68, conding zero ,70, fluiing zero , and so ing zero ,73,74,75ing zero . Like thing zero ; (2) Theing zero ,78, dualing zero ,80,81 oring zero  due to aSince we have defined a macroscopic polarisation with respect to a 3D electric vector potential Equation , D→T=ϵE→e system ,80,81. Ie system ,82, due e system , or the e system ,27,29,80S, in which the magnetic flux flows, with the first term on the RHS the static contribution to the AB geometric phase, while the second term adds the time dependent term. For the static case, if we consider n Cooper pairs , Equation , we can quation . Thus, S, in which the electric flux flows. Thus, in analogy, the first term on the RHS gives the static dual geometric phase, while the second gives the general time dependent term. For the static case, the geometric phase depends on the enclosed electric flux, Analogous to this, the dual electric phase Equation  becomesquation  spin gauge field —in a simFirst, we consider the time rate of change of Equation , combini becomesE=∮PE→T·dNext, we consider the time rate of change of Equation  and, com becomesF=1μ0∮PB→L is the axial length, and V, and vector, (1)The electric scalar potential,(2)The electric vector potential,(3)The electric field vector ((4)The electric flux density 38) give In this section, we analyze the electronic properties of a static (or quai-static) cylindrical active electronic dipole of varying aspect ratios (ae)σeiϕ^  will be Some interesting points come out of these simulations, and the potential difference , and no work is required to keep the dipole energised.A macroscopic, time-independent, active magnetic dipole can in principle exist without loss as a persistent DC current in a superconducting wire loop or coil not requiring any extra energy or power input. For this situation, all parts of Faraday’s law in Equation  are zeroEquation . Thus, oThe electromagnetic dual of the active macroscopic magnetic dipole (or permanent magnet) is the active macroscopic electric dipole. This type of dipole is a permanent dipole, such as a macroscopic electret, polar molecule, or atomic system with a dipole moment determined by the first-order linear Stark effect. This description does not include instantaneous or induced dipoles, which are not permanent. However, for the macroscopic electric dipole (or an electret) to exist, an emf must be generated to force separation of charges, unlike the magnetic dipole, this charge separation requires an impressed force per unit charge from an external energy source. For example, a solar cell contains a p-n junction, where an array of bound dipoles existed in the depletion region and, when photons enter this region, the dipoles are polarized to essentially form an electret and an electromotive force. Conversely, once an electret is polarized, the natural tendency is for the active electric dipole to discharge or decay and emit a photon , which mAn interesting point in understanding the physics of an emf generator is to understand the microscopic description, which will be a different description for each type of generator, which inevitably involves quantum mechanics ,22,23,24


To investigate the incidence and related factors of extrauterine growth retardation (EUGR) and “true EUGR” in very preterm infants (VPI) from different regions of China.Clinical data of VPI were prospectively collected from 28 hospitals in seven different regions of China from September 2019 to December 2020. The infants were divided into a small for gestational age (SGA) group or non-SGA group at birth, with non-SGA infants at 36 weeks of gestation or at discharge being further divided into a EUGR group or a non-EUGR group. Infants in the EUGR and non-SGA group were defined as “true EUGR.” The general information of VPI, such as maternal complications during pregnancy, use of enteral nutrition and parenteral nutrition, and complications during hospitalization were compared between the groups.Among the 2,514 VPI included in this study, 47.3, 41.5, and 33.3% of VPI were below the 10th percentile, and 22.6, 22.4, and 16.0% of VPI were below the 3rd percentile for weight, height, and head circumference at 36 weeks of gestation or at discharge, respectively, by the percentile on the 2013 Fenton curve. The incidences of EUGR and “true EUGR” evaluated by weight were 47.3 and 44.5%, respectively. Univariate analysis showed that there were statistically significant differences in the aspects of perinatal and nutritional characteristics, treatment, and complications between the groups. Multivariate analysis showed that in non-SGA infants, the cumulative caloric intake during the first week was a protective factor for “true EUGR,” while days to reach total enteral nutrition, late initiation of human milk fortifier, and moderate to severe bronchopulmonary dysplasia were independent risk factors for “true EUGR.”More attention should be paid to the nutritional management of VPI to prevent “true EUGR.” Cumulative caloric intake should be ensured and increased during the first week, total enteral nutrition should be achieved as early as possible, human milk fortifier should be added early, and moderate to severe bronchopulmonary dysplasia should be prevented. These strategies are very important for reducing the incidence of “true EUGR” in VPI. The prevalence of very preterm infants (VPI) is increasing all over the world . DespiteFigueras-Aloy et al.  have sugIn recent years, increasing attention has been paid to the prevention and treatment of nutritional deficiency and EUGR in preterm infants in China , 11, butParticipants for this study were recruited from 28 tertiary hospitals located in seven different regions of China  and the hospitals included 13 general hospitals, 11 children’s hospitals, and four maternal and childcare hospitals.Participants were divided into the SGA group and non-SGA group at birth, and the non-SGA infants at the corrected GA of 36 weeks (36 weeks PCA) or at discharge (when before 36 weeks PCA) were further divided into the EUGR group and non-EUGR group, according to whether their body weight, length, and head circumference (HC) were below the 10th percentile on the Fenton curve.Inclusion criteria were as follows: (1) GA < 32 w; (2) Length of hospital stay > 2 weeks; (3) Hospital admission within 24 h after birth.Exclusion criteria were as follows: (1) Congenital malformation or inherited metabolic disease; (2) Hospital stay < 2 weeks; (3) Death during hospitalization, interruption of treatment, or automatic discharge.Discharge criteria were as follows: (1) The primary disease was cured, and the vital signs were stable [infants with bronchopulmonary dysplasia (BPD) were allowed to be discharged with oxygen]; (2) milk volume had reached total enteral feeding; (3) Weight was more than 1,800–2,000 g; (4) Corrected GA was ≥ 36 weeks.This study was conducted by the Nutrition Professional Committee of Neonatologists Branch of Chinese Medical Doctor Association, and it was registered in the Chinese Clinical Trial Registry (Registration number: ChiCTR1900023418). The research protocol was approved by the Ethics Committee of Women and Children’s Hospital affiliated to Xiamen University/Xiamen Maternal and Child Health Hospital (Batch number kY-2019-016).This was a multicenter prospective study, and the study period was from September 2019 to December 2020.Using a unified questionnaire, data on the maternal complications during pregnancy, general clinical data of preterm infants, nutritional status during hospitalization, complications, and major treatments were collected. Perinatal data included GA at birth, birth weight (BW), sex, delivery mode, single or multiple births, 1-min Apgar score, and full course of prenatal glucocorticoid use. Maternal complications during pregnancy included gestational hypertension, gestational diabetes, thyroid disease, and connective tissue disease. Growth and nutrition related indicators included maximum weight loss, days to regain BW, growth velocity (GV) after regaining BW, start of enteral nutrition (EN), age at reaching total EN, duration of parenteral nutrition (PN), cumulative fasting days, calories accumulated during the first week of hospitalization, days to reach the target total calorie intake and oral calorie intake, accumulative doses of amino acid and fat emulsions during the first week of hospitalization and throughout hospitalization, breast milk volume after addition of human milk fortifier (HMF), and days needed for full fortification. The main treatments included duration of invasive and non-invasive mechanical ventilation, duration of oxygen use, cumulative duration of antibiotic use, and total length of hospital stay. Primary complications during hospitalization included neonatal respiratory distress syndrome (RDS), moderate to severe BPD, early-onset sepsis (EOS), late-onset sepsis (LOS), necrotizing enterocolitis (NEC) ≥ stage 2, intraventricular hemorrhage (IVH) grade III-IV, periventricular leukomalacia (PVL), hemodynamically significant patent ductus arteriosus (hsPDA), retinopathy of prematurity (ROP), feeding intolerance (FI), metabolic bone disease of prematurity (MBDP), and PN associated cholestasis (PNAC).The data entry personnel of each unit were uniformly trained, and they strictly fulfilled the requirements of the research program. The EpiData database was established in this study, data of the case report form were recorded in double pairs, all participating units collected and uploaded the nutrition data of preterm infants in time, and the database was locked after verification. The team leader maintained close contact with all participating units at any time point, checked the case records, and solved the possible problems in time.(1) The term “SGA” referred to BW below the 10th percentile of the average BW of infants of the same sex and GA; the term ‘appropriate for gestational age’ was defined as BW within the 10th to 90th percentile of the BW of infants of the same sex and age; and the term “large for gestational age” referred to BW above the 90th percentile of the BW for infants of the same sex and age. (2) Evaluation criteria for EUGR: With reference to Fenton 2013 , EUGR waAccording to the literature, the incidence of EUGR was about 60% and the allowable error (δ) was 2%. If we considered α = 0.05, the required sample size was 2,305 and the required total sample size was 2,400. The average sample size for each collaborating unit was expected to be 90.t-test, while Mann–Whitney U test was used for non-normal distributions. Univariate analysis was performed for the factors that may affect the clinical outcome of infants. To determine the effect of the prenatal and postnatal variables on the outcome of true EUGR, binomial logistic regression was used to arrive at odds ratios (OR) and 95% confidence intervals (CI). In the multivariate model, true EUGR (yes or no) was considered as the dependent variable, and nutritional factors and complications related variables were analyzed as independent variables. The Hosmer–Lemeshow test was conducted to determine the model’s goodness of fit. All statistical tests were two-tailed, and P-values ≤ 0.05 were considered statistically significant.All data were analyzed with SPSS 23.0 for Windows . Chi-square test was used to assess the association between categorical variables unless the cell frequency was ≤ 5, in which case the Fisher’s exact test was used. Distribution of continuous variables was assessed using the Shapiro–Wilk test. Normally distributed continuous variables were analyzed using independent sample A total of 2,800 preterm infants with GA < 32 weeks were admitted during the study period. A total of 184 preterm infants, aged < 24 weeks at birth, died during hospitalization, or had severe congenital developmental abnormalities, inherited metabolic diseases, chromosomal abnormalities, or underwent surgical procedures, were excluded. A total of 55 infants were excluded from the study as the corrected age was less than 36 weeks, their weight was less than 1,800 g at discharge, or the hospital stay was less than 2 weeks. A total of 47 infants were excluded because of missing data on weight, height, or HC. Finally, 2,514 VPI were enrolled in the study. The flow chart of the included infants is shown in The infants were divided into the SGA and non-SGA groups at birth, and non-SGA infants at 36 weeks of gestation or at discharge were further divided into the EUGR group and non-EUGR group. According to weight, the incidence of EUGR was 98.5, 44.5, and 47.3% in SGA infants, non-SGA infants, and all 2,514 infants, respectively; and the incidences of EUGR and “true EUGR” evaluated by weight were 47.3 and 44.5%, respectively. According to body length, the incidence of EUGR was 76.7, 36.6, and 41.5% in SGA infants, non-SGA infants, and all infants. When evaluated by HC, the incidence of EUGR was 62.4, 29.8, and 33.2% in SGA infants, non-SGA infants, and all infants, respectively. The incidence of EUGR in SGA and non-SGA infants, and the specific proportions of infants who had values less than P3 and P10 at discharge in each group are shown in P < 0.05, data not shown). Preterm infants with GA < 28 weeks had the highest incidence of EUGR , and those with GA 31-31 + 6 weeks had the lowest incidence of EUGR . P < 0.05, data not shown). Preterm infants with BW < 1,000 g had the highest incidence of EUGR , and those with BW 2,000–2,500 g had the lowest incidence of EUGR .P < 0.05). The incidence of gestational diabetes was higher in the non-SGA group and non-EUGR group when compared with the SGA group and EUGR group, respectively (P < 0.05). There was a tendency that the incidence of full course of prenatal steroids was higher in the SGA group (P = 0.072), and at discharge, this incidence was higher in the non-EUGR group (P < 0.05).Comparisons of obstetrical and neonatal characteristics of VPI are shown in P < 0.05). For weight, length, HC, the corresponding Z score, and the change in Z score, all except two indicators were significant between the SGA and non-SGA groups at birth, and between the EUGR and non-EUGR groups belonging to the non-SGA group at discharge (P < 0.05). Changes in weight Z score and HC at discharge were not significantly different between the SGA and non-SGA groups at birth . However, they became significant between the EUGR and non-EUGR groups belonging to the non-SGA group at discharge (P < 0.05).P < 0.05). The cumulative dose of amino acid and fat emulsions during the first week (W1) and during hospitalization was higher in the SGA group and EUGR group (P < 0.05). When comparing the indicators for the start of EN, cumulative calories during W1, breast milk volume after addition of HMF, and days taken for full fortification, the differences were not significant between the SGA and non-SGA groups at birth, but they became significant between the EUGR and non-EUGR groups belonging to the non-SGA group at discharge (P < 0.05).Comparisons of nutrition-related characteristics between the groups are shown in P < 0.05). Nosocomial infection, EOS, brain injury, IVH, and PVL were not significant. With respect to LOS, NEC ≥ stage 2, PDA, hsPDA, ROP, and ROP requiring intervention, the differences were not significant between the SGA and non-SGA groups at birth, but they became significant between the EUGR and non-EUGR groups belonging to the non-SGA group at discharge (P < 0.05). The results are presented in The results of univariate analysis of complications during hospitalization showed that there were statistically significant differences in the incidences of RDS, BPD, moderate to severe BPD, postnatal corticosteroid use, FI, PNAC, MBDP, anemia, and transfusion between the groups  . Recent studies in China have shown that VPI account for 13.1% of all preterm infants and EPI account for 1.1% of all preterm infants. The success rate of treatment of VPI in China is 93.3% , and theIn this study, the incidence of EUGR evaluated by weight, length, and HC at discharge in the SGA and non-SGA groups was statistically different. The incidences of “true EUGR” and severe “true EUGR” evaluated by weight were 43.5 and 18.9% at discharge, respectively. A national multicenter study performed in 2009 in China showed that among 696 singleton preterm infants, the incidence of EUGR in the non-SGA group was 47.4% . Lima etIn this study, the variables independently related to the presence of “true EUGR” were low BW, days to reach total EN, late initiation of HMF, and moderate to severe BPD. Lima et al.  highlighIncreasing the energy intake in the first week of postnatal life is associated with postnatal weight gain and reduced risk of EUGR and BPD , 23. TheEarly attainment and maintenance of adequate EN can promote growth and weight recovery in preterm infants, and it can reduce the short-term and long-term complications , 26. A lIn this study, multivariate logistic regression analysis showed that late initiation of HMF was an independent risk factor for “true EUGR.” Early addition of HMF can increase protein intake within 4 weeks after birth and reduce early protein accumulation deficiency, and it does not increase the chances of complications, such as NEC or FI , 29. ForMultivariate logistic regression analysis showed that moderate to severe BPD was an independent risk factor for “true EUGR.” Several studies have supported the claim that moderate to severe BPD was associated with more severe EUGR , 32. LonDomestic and foreign guidelines have been revised and updated in the last 10 years, with the management of EN and PN becoming more active and standardized. Most of the previous multicenter studies have focused on VLBWI , 33, andOver the past 10 years, enteral nutrition and parenteral nutrition of VPI have made great progress, and the incidence of “true EUGR” has decreased significantly. However, it should be emphasized that perinatal care should continue to be strengthened to further reduce the occurrence of SGA. Cumulative caloric intake should be ensured and increased during the first week of hospitalization. More attention should be paid toward days to reach total enteral nutrition, initiation of HMF, and moderate to severe BPD to reduce the risk of “true EUGR.”The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Women and Children’s Hospital, School of Medicine, Xiamen University (KY-2019-016). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.X-ZL, X-MT, and CC: conceptualization, writing - review and editing, and project administration. WS, Y-MC, RZ, and ZZ: methodology and data curation. WS, X-ZY, and Y-PQ: software. LM, RC, HW, and D-MC: validation. WS and FW: formal analysis. FW, JM, LL, Y-MC, RZ, ZZ, X-ZY, Y-PQ, LM, RC, HW, and D-MC: investigation and resources. WS: writing—original draft preparation. FW, JM, and LL: visualization and supervision. All authors have read and agreed to the published version of the manuscript.


What is the relative prevalence of congenital heart defects among boys born with hypospadias compared with boys born without hypospadias?In this large multistate birth defects registry cohort study of data from population-based birth defect surveillance programs on all male infants born in 11 US states, boys born with hypospadias were 6 times more likely than boys without hypospadias to have any major congenital heart defect.This study suggests that screenings for additional birth defects among boys born with hypospadias may be warranted; in addition, molecular studies are needed to uncover the shared etiology of co-occurring defects in different developmental fields. Hypospadias is a common birth defect of the male urinary tract that may be isolated or may co-occur with other structural malformations, including congenital heart defects (CHDs). The risk for co-occurring CHDs among boys with hypospadias remains unknown, which limits screening and genetic testing strategies.To characterize the risk of major CHDs among boys born with hypospadias.This retrospective cohort study used data from population-based birth defect surveillance programs on all male infants born in 11 US states from January 1, 1995, to December 31, 2014. Statistical analysis was performed from September 2, 2020, to March 25, 2022.Hypospadias.Demographic and diagnostic data were obtained from 2 active state-based birth defect surveillance programs for primary analyses, the Texas Birth Defects Registry and the Arkansas Reproductive Health Monitoring System, with validation among 9 additional states in the National Birth Defects Prevention Network (NBDPN). Birth defect diagnoses were identified using the British Pediatric Association coding for hypospadias (exposure) and major CHDs (primary outcomes). Maternal covariates and birth year were also abstracted from the vital records. Poisson regression was used to estimate adjusted prevalence ratios and 95% CIs for major CHDs within Texas and Arkansas and combined using inverse variance–weighted meta-analysis. Findings were validated using the NBDPN.Among 3.7 million pregnancies in Texas and Arkansas, 1485 boys had hypospadias and a co-occurring CHD. Boys with hypospadias were 5.8 times  more likely to have a co-occurring CHD compared with boys without hypospadias. Associations were observed for every specific CHD analyzed among boys with hypospadias, occurred outside of chromosomal anomalies, and were validated in the NBDPN. An estimated 7.024%  of boys with hypospadias in Texas and 5.503%  of boys with hypospadias in Arkansas have a co-occurring CHD. In addition, hypospadias severity and maternal race and ethnicity were independently associated with the likelihood for hypospadias to co-occur with a CHD; boys in Texas with third-degree  hypospadias were 2.7 times  more likely than boys with first-degree hypospadias to have a co-occurring CHD, with consistent estimates in Arkansas , and boys with hypospadias born to Hispanic mothers in Texas were 1.5 times  more likely to have a co-occurring CHD than boys with hypospadias born to non-Hispanic White mothers.In this cohort study, boys with hypospadias had a higher prevalence of CHDs than boys without hypospadias. These findings support the need for consideration of additional CHD screening programs for boys born with hypospadias. This cohort study uses data from the Texas Birth Defects Registry and Arkansas Reproductive Health Monitoring System to characterize the risk of major congenital heart defects among boys born with hypospadias. Advancements in early detection and intervention have improved CHD outcomes,18 yet unrecognized CHD suggests that targeted screening could further reduce morbidity and mortality.Hypospadias and congenital heart defects (CHDs) are 2 of the most prevalent birth defects in the United States.21 Case reports highlight key hypotheses in the shared etiology of hypospadias and CHDs ,29 yet the exact mechanisms remain unknown. Similarly, birth defects may cluster in nonspecific patterns . A recent study by Ludorf et al21 used an agnostic approach to characterize complex patterns of defects co-occurring with hypospadias using a large population-based birth defects registry. Congenital heart defects accounted for more than half of the most frequent defects co-occurring with hypospadias, including ostium secundum–type atrial septal defect (390 [18.7%] of 2084 cases of nonisolated hypospadias) and ventricular septal defect (290 [13.9%] of 2084 cases of nonisolated hypospadias), which lays the foundational work that hypospadias and CHDs specifically cluster.To our knowledge, few studies to date have evaluated the co-occurrence of hypospadias and CHDs.21 in the Texas Birth Defects Registry (TBDR), we extended these findings in independent birth defect surveillance programs. We performed a retrospective cohort study of hypospadias and CHDs among pregnancies spanning 2 decades in comprehensive birth defect surveillance programs, leveraging the strength of each program. The prevalence of CHDs in association with hypospadias was estimated between 2 contiguous states with active surveillance for major birth defects, which allows for precise estimates of CHD prevalence. Statistical validation of observed associations used data from 9 additional states throughout the US with both active and passive surveillance programs. We therefore estimated and validated the prevalence of specific CHDs that co-occur with hypospadias, leveraging data from the TBDR, the Arkansas Reproductive Health Monitoring System (ARHMS), and the National Birth Defects Prevention Network.Based on mounting evidence for the co-occurrence of hypospadias and CHDs, with advancements in CHD screening through echocardiograms, clinical examinations, and genetic sequencing, we sought to estimate the prevalence of specific CHDs among boys with hypospadias. From the specific clustering of hypospadias and CHDs established by Ludorf et alSTROBE) reporting guideline for cohort studies.This study was approved by the institutional review boards of the Texas Department of State Health Services, the University of Texas Health Science Center, and the University of Arkansas for Medical Sciences. Parental consent for all registries was waived by their respective governing bodies, based on the deidentified nature of data. We followed the Strengthening the Reporting of Observational Studies in Epidemiology  conducts active surveillance of live births, stillbirths, and fetal deaths to identify infants with birth defects delivered by women residing in Texas.31 Information on birth defects is obtained through medical record abstraction at more than 40 delivering hospitals, prenatal diagnostic clinics, and specialty clinics or hospitals. Birth defects reported in the ARHMS are diagnosed in the first 2 years of life; analyses included resident births from January 1, 1995, to December 31, 2013.The ARHMS  is an active, statewide surveillance program for birth defects conducted at Arkansas Children’s Hospital.32 Each surveillance program uses birth certificates to link to the index case and obtain information on demographic characteristics and births in the catchment area. Birth defects in the NBDPN are diagnosed within the first 3 years of life. Pooled individual-level data from births (1999-2007) in 9 states  were available for these analyses.The National Birth Defects Prevention Network  is a network of active and passive programs from 43 birth defect surveillance registries in the US.33 Birth defects were coded by the TBDR and the ARHMS using 6-digit British Pediatric Association (BPA) codes for major birth defects. Registries in the NBDPN used BPA codes or International Classification of Diseases, Ninth Revision (ICD-9) codes, with ICD-9 codes centrally linked to relevant BPA codes prior to analyses. From these data, we selected individuals with isolated or any co-occurring birth defects to form the following 3 groups: (1) boys with hypospadias without a CHD, (2) boys with a CHD without hypospadias, and (3) boys with co-occurring hypospadias and CHD. Hypospadias cases included all severities with or without chordee . Congenital heart defect cases, including critical CHDs, were grouped by major defect based on the first 4 digits of the BPA code; specific CHD codes considered minor or incompletely ascertained by the registries were excluded from analyses  and birth year . Regression models using NBDPN data were additionally adjusted for state of birth. Within each of the 3 data sets, regression analyses were restricted to CHDs that occurred among at least 3 boys who also had hypospadias. Regression results are reported as adjusted prevalence ratios (PRs) and 95% CIs. To generate combined PR estimates, we performed fixed-effects inverse variance–weighted meta-analysis to report overall PRs as estimated in Texas and Arkansas. Prevalence ratios across all states in the NBDPN served as validation of our primary findings from meta-analysis of the single-state registry analyses. Heterogeneity was assessed using the I2 statistic. To assess whether known syndromes were associated with the co-occurrence of hypospadias and CHDs, we performed sensitivity analyses using Texas data by excluding cases with any chromosomal syndrome (BPA codes 758.0-758.9). Analyses were conducted in R, version 3.5.2  and Stata, version 16 (StataCorp LLC). All P values were from 2-sided tests and results were deemed statistically significant at P < .05.Statistical analysis was performed from September 2, 2020, to March 25, 2022. All analyses were restricted to boys. Descriptive analysis using counts and percentages was performed for demographic variables that included maternal race and ethnicity, age, and educational level. Poisson regression was used to compare the prevalence of each CHD phenotype (outcome) between boys born with hypospadias and boys born without hypospadias (exposure).We also performed analyses only among boys with hypospadias in Texas and Arkansas to characterize CHD prevalence. First, we estimated the overall prevalence and 95% CIs for any type of CHD among boys with hypospadias within each of the single-state registries. We then used logistic regression to assess the association of hypospadias severity and maternal race and ethnicity with the co-occurrence of CHD, after adjusting for birth year. Hypospadias severity was coded as first, second, or third degree, excluding hypospadias of unspecified severity from logistic regression analyses.Among 3.7 million pregnancies in Texas and Arkansas, we identified 1485 boys who had hypospadias and a co-occurring CHD . In bothWe found that boys with hypospadias in Texas and Arkansas were 5.8 times  more likely to have any type of co-occurring major CHD compared with boys born without hypospadias . Our anaI2 < 60%), with substantial heterogeneity for other major, noncritical CHDs (heterogeneity I2 > 80%). For example, despite being a relatively common CHD, ventricular septal defects were 6.2 times  more prevalent among boys with hypospadias in Texas and Arkansas, but 26.3 times  more prevalent among boys with hypospadias in the NBDPN (heterogeneity I2 = 99.4%).We found that prevalence across all CHDs was elevated among boys with hypospadias in the NBDPN, consistent with our findings in Texas and Arkansas , but estimated PRs did not substantially differ in magnitude after these exclusions  and 5.503% of boys with hypospadias in Arkansas  have a co-occurring CHD . We perfWe analyzed 11 population-based birth defects registries that comprised more than 8.1 million pregnancies spanning 2 decades and found consistent evidence that boys with hypospadias are substantially more likely than boys without hypospadias to have a co-occurring major CHD. In general, boys with hypospadias have a 6-fold or greater risk for co-occurrence of any major CHD, with further elevated prevalence across specific CHDs. We found that boys with hypospadias have critical CHDs that require surgical intervention in the first years of life and have improved prognosis with earlier detection. In addition, our analyses suggest that differences in both hypospadias severity and maternal race and ethnicity could be associated with the likelihood for hypospadias to co-occur with a major CHD. Together, these findings suggest that, for a boy with a diagnosis of any form of hypospadias, there may be a benefit associated with screening for additional structural defects.35 In addition, to our knowledge, there are currently no recommended additional screenings for boys with a diagnosis of hypospadias, regardless of hypospadias severity.36 However, it is increasingly recognized that boys with hypospadias could experience a benefit associated with clinical sequencing and genetic counseling,38 and the American College of Medical Genomics and Genetics now recommends sequencing for children with a diagnosis of a congenital anomaly prior to 1 year of age.39 Sequencing of individuals with multiple congenital anomalies, particularly those across body systems, has a high likelihood to yield a molecular diagnosis.41 Together, these findings suggest that additional clinical and genetic evaluations of boys with hypospadias may identify subclinical birth defects and improve molecular diagnoses for boys with multiple congenital anomalies.Screenings through prenatal sonography and newborn pulse oximetry are widely implemented to detect critical CHDs. Despite the success of these screening programs, these tests have imperfect sensitivity and positive predictive values for critical CHDs and often fail to detect noncritical CHDs.42 Hypospadias is known to frequently co-occur with other anomalies in the genitourinary system, likely owing to the shared developmental origins of the kidneys and urogenital tract.43 However, evidence is emerging that hypospadias frequently co-occurs with defects in other developmental fields. Agnostic analyses of co-occurring defects with hypospadias in the TBDR have established that hypospadias specifically clusters with CHDs beyond nonspecific birth defect clustering.21 Building on those findings using an expanded set of registry data, we found that 5.5% to 7.0% of boys with hypospadias had a major CHD, which is consistent with smaller hospital-based reports of developmental anomalies among boys treated for hypospadias (19 of 356 [5.3%] with CHD).43Typically, defects that emerge in the same developmental fields are suspected to result from disturbances in shared, complex embryologic pathways.44 In addition, this study found that 30% of hospital-based patients with CHD exhibited kidney defects on imaging results, which together support the shared etiology between these systems. Genetic studies of children with CHDs revealed a complex architecture that includes single-gene disorders and polygenic inheritance.53 By contrast, less is known about the genetic etiology of hypospadias, but evidence is also emerging of a complex architecture.56 Approximately 70% of hypospadias cases occur outside of known genetic syndromes and have incompletely characterized origins.57 Many genetic syndromes that include hypospadias also feature CHDs, including Mowat-Wilson syndrome (ZEB2 [OMIM 605802]),58 Opitz G/BBB syndrome (MID1 [OMIM 300552]),59 and Wolf-Hirschhorn syndrome .60The shared developmental etiology of urogenital and cardiac anomalies is now being investigated in molecular studies. In animal studies of single-gene CHDs, 29.8% of the variant lines exhibited renal anomalies.63 We found that boys with hypospadias born to Hispanic mothers were more likely to have a co-occurring CHD than boys with hypospadias born to non-Hispanic White mothers. We additionally identified a higher prevalence of CHDs among boys with hypospadias in Texas, which may reflect the high proportion of Hispanic individuals living in Texas compared with Arkansas. Disparities in outcomes for select CHDs disproportionately affect Hispanic individuals,64 so it is important to identify vulnerable populations that could benefit from enhanced screening and surveillance of co-occurring conditions.Risk for hypospadias and CHDs independently differs by maternal race or ethnicity.Our study has some strengths. We performed independent analyses of 3 birth defect databases comprising 11 states. Prevalence of specific CHDs in association with hypospadias was estimated in 2 states with active surveillance programs, which (1) minimizes information bias owing to differences in surveillance programs, (2) is a powerful approach to identify rare co-occurring birth defects, and (3) minimizes sampling bias owing to the large population-based samples. In addition, a large proportion of cases in Texas (60%) and Arkansas (30%) are reviewed by a clinical geneticist. We also used a large, multistate network of surveillance programs to statistically validate findings in our primary analyses.65 which makes it difficult to make inferences about the observed associations relative to associations between other birth defects. That is, we expect that many pairwise combinations of birth defects will have an increased association even without shared etiology, but the magnitude of association at which shared etiology is suggested remains unknown. In addition, birth defect registries are limited to major defects reportable to each registry. Specific CHDs that are considered minor defects  are therefore not included in these registry analyses, so we cannot assess from these analyses whether prevalence of minor CHDs was also elevated with hypospadias. Likewise, minor and noncritical CHDs may go undiagnosed early in life, yet they may become symptomatic as the individual ages, so it is important to evaluate whether these defects are also more likely to co-occur with hypospadias. We found that atrial septal defects and ventricular septal defects—which may go undiagnosed—are more prevalent with hypospadias but had estimates that differed between the surveillance systems. These CHDs may be incorrectly captured through birth defect registries, so it is difficult to know whether the prevalence estimates reported for atrial septal defects and ventricular septal defects are accurate.However, our study should be considered in light of certain limitations. Our approach did not account for the known tendency of birth defects to cluster nonspecifically,The high prevalence of co-occurring CHDs in this cohort study suggests that hypospadias may not be an isolated defect and that additional birth defect screening among boys born with hypospadias is warranted. The ability to characterize molecular alterations associated with the development of both hypospadias and CHDs may lead to targeted prevention strategies and improved counseling for these relatively common and medically significant birth defects.


Fruits along with vegetables are crucial for a balanced diet. These not only have delicious flavors but are also reported to decrease the risk of contracting various chronic diseases. Fruit by-products are produced in huge quantity during industrial processing and constitute a serious issue because they may pose a harmful risk to the environment. The proposal of employing fruit by-products, particularly fruit peels, has gradually attained popularity because scientists found that in many instances peels displayed better biological and pharmacological applications than other sections of the fruit. The aim of this review is to highlight the importance of fruit peel extracts and natural products obtained in food industries along with their other potential biological applications. Approximately 89 million tons of food waste is produced in the European Union, and this figure is anticipated to increase by a factor of 40 in the future . Fruits It has been reported that large amounts of secondary metabolites are present in fruit and vegetable wastes and these waste materials have been studied for phenolic molecules, dietary fibers and other biologically active metabolites by extraction ,2. ScienC. reticulata and C. unshiu are used under the trade name “Chimpi” in Japan as crude drugs. Similarly, C. aurantium dried peels are employed as the popular folkloric drug “Touhi.”  ether (135), tricin 4′-O-[threo-β-guaiacyl-(7″-O-methyl-9″-O-acetyl)-glyceryl] ether (136) and quercetin (137) , quercetin 5,4′-dimethyl ether (139), isorhamnetin-3-O-glucoside (140) and isorhamnetin (141) were produced by Opuntia ficus-indica peels and flavone 139 demonstrated significant antibacterial effects towards Streptococcus pneumoniae, Stenotrophomonas maltophilia, Moraxella catarrhalis, Klebsiella pneumoniae, Pseudomonas aeruginosa and Legionella pneumophila. In addition, flavone 140 illustrated good antibacterial potential towards Streptococcus pneumoniae, Stenotrophomonas maltophilia, Moraxella catarrhalis, Klebsiella pneumoniae and Pseudomonas aeruginosa [The pear (in (137) . The flaectively . Astragaruginosa . O-rhamnopyranoside (142), acacetin-7-O-neohesperidoside (143) and acacetin-7-C-neohesperidoside (144) were reported from fruit peels of Fortunella japonica [145), vitexin (146), orientin (147), vitexin-2″-O-rhamnoside (148) and vitexin-4″-O-glucoside (149) were reported from Benincasae exocarpium fruit peels and screened for antidiabetic effects. Flavones 145–149 inhibit AGE better than AMG  with IC50: 20.2, 19.5, 9.6, 29.5 and 14.5 μM, respectively. In addition, flavones 145–149 inhibit the α-glucosidase enzyme better than the standard acarbose  with IC50: 48.1, 52.2, 36.1, 12.8 and 65.3 μM, respectively [Kaempferide-3-anoside 1, acacetiectively . 150) and (−)-pinoresinol (151)  and sesamolin (153) were produced by Strychnos nuxblanda fruit peels. Notably, metabolites 152 and 153 exhibited α-glucosidase with IC50: 447 and 215 μM, respectively, while the activities of these same lignans were higher than acarbose . On the other hand, these compounds were not active as acetylcholinesterase enzymes [154) and medioresinol (155) were isolated from Annona squamosa fruit peels and both compounds illustrated anti-inflammatory effects with IC50: 45.4 and 6.2 µM, respectively [Two lignans named syringaresinol (ol (151)  were repectively . Sesamin enzymes . Furofur156)  [156), punicalin (157) and ellagic acid (158) were isolated from pomegranate fruit peels. Tannins 156–158 prevent protease-mediated effects in vitro through acting on HCV NS3/4A protease directly (with IC50: < 0.1 µM for tannins 156 and 157 and compound 158 has IC50: 1.0 µM). Notably, these compounds are all reported to be associated with no toxic effects side effects ex vivo and were quite safe at 5000 mg/kg when given acutely in BALB/c mice. Pharmacokinetics data indicated that compounds 156–158 are readily bioavailable [Punicalagin (156)  was isolll (90%) . Punicalvailable . 156 and 157 possess galloyl residues that provide further evidence for their inhibitory effects against NS3/4A protease. Literature revealed that tannins can form complexes with metal ions as well as target macromolecular polysaccharides and proteins . Hence, 156–158 demonstrated biological effects towards RNA replication of HCV and the exact mechanism of this activity is yet to be investigated. Since compounds 156–158 inhibit NS3 protease, thus affecting HCV polyprotein proteolytic processing which in turn leads to a decreased active viral RNA-dependent RNA polymerase level. This eventually causes a decreasing level of HCV RNA [158 results in apoptosis in the cell line of human prostatic cancer by decreasing antiapoptotic factors including HuR (human antigen R), HO-1 (Heme oxygenase-1) and SIRT1 (silent information regulator). It also decreases apoptotic markers including IL-6 (interleukin-6) and TGF-β (transforming growth factor -β) in cell line of prostatic cancer [158 also mediates the apoptotic process in cancer cells of the pancreas through inhibiting NF-kB and the mitochondrial depolarization process [Further study revealed that compounds  HCV RNA . Of notec cancer . Compoun process  and demo process .159) and rhodomyrtone (160)  [Myrciarone A (ne (160)  are prod5 µg/mL) . 160) towards B. cereus and M. luteus (MIC: 0.78 µg/mL) were 8-fold higher than kanamycin (MIC: 6.25 µg/mL). The antimicrobial effects of molecule 160 against S. mutans (MIC: 1.56 µg/mL) were equal to the standard kanamycin, whereas the same compound was 2-fold more active towards S. aureus (MIC: 0.78 µg/mL) and S. epidermidis. Myrciarone A (159) displayed the same level of activities against S. epidermidis and S. mutans with MIC: 3.13 µg/mL and its effects were 2-fold less than kanamycin (MIC: 1.56 µg/mL). Metabolites 159 and 160 were unfortunately not active towards Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Candida albicans and Saccharomyces cerevisiae [The activities of rhodomyrtone (revisiae . Lansium domesticum produced onocerane triterpenoids named lansiosides A-C (161–163) (164) and all were effectively shown to decrease leukotriene D4-mediated contraction of guinea pig ileum [165), lansionic acid (166), 3β-hydroxyonocera-8(26),14-dien-21-one (167), compound 168 and lansiolic acid (169) were isolated from the fruit peels of Lansium parasiticum. Lamesticumin G (165) demonstrated α-glucosidase inhibition with IC50: 2.27 mM [170), lansiosides B (171) and C (172) were reported from the fruit peels of Lansium parasiticum. Triterpenoids 165 (SC50: 14.5 mM) and 171 (SC50: 13.7 mM) exerted moderate antioxidant effects, whereas triterpenoid 172 possessed weaker antioxidant effects (SC50: 23.6 mM) while compounds 170–172 were not active in α-glucosidase screening [The fruit peel of 161–163)  along wiig ileum . In anot 2.27 mM . In anotcreening . Lansium domesticum fruit peels resulted in the isolation of an onoceranoid named onoceradienedione (173)  which possessed cytotoxic effects towards epithelial breast cancer (T47D) with IC50: 15.6 μg/mL [L. domesticum fruit peels and reported finding onoceranoid triterpenoids 175–177 all of which displayed moderate toxicity towards Artemia salina. The molecule described as α,γ-onoceradienedione (178) was produced by Lansium domesticum fruit peels and possessed moderate effects towards C. albicans and A. niger [Phytochemical investigation of ne (173)  which diectively . In anot.6 μg/mL . Tanaka .6 μg/mL  also invA. niger .179), taraxerone (180) and taraxerol (181)  which was demonstrated to inhibit cathepsin D with IC50: 8.3 µg/mL [183) was isolated from C. reticulata fruit peels [184–188 [189–192 [Musa sapientum fruit peels (banana). Lupenone (193), β-amyrin (194) and α-amyrin (195) were obtained from fruit peels of Fortunella japonica [Lupeol (ol (181)  were repectively . Fruit p.3 µg/mL . Friedelit peels . Cycloar[184–188  and 189–[189–192  were projaponica .Wisteria floribunda produced β-sitosterol (196) and β-sitosterol glucopyranoside (197)  were produced by the fruit peels of Solanum melongena [199 was isolated from C. reticulata fruit peels [197 and 198 demonstrated cytotoxic effects towards HCT116 (colon cancer), HEP2 (larynx cancer), MCF7 (breast cancer), HEPG2 (liver cancer) and HELA (cervix cancer) with IC50 ranging from 2.2 to 13.4 µM [200–202 were reported from the fruit peel of Annona squamosal and illustrated anti-inflammatory effects with IC50: 5.0, 5.2 and 5.4 µM, respectively [Fruit peels of de (197)  and exerectively . The steelongena , whereasit peels . Steroid 13.4 µM . In anotectively . 203) (Punica granatum fruit peel [204 and 205 produced by the fruit peels of C. medica had their structures established via extensive spectroscopic techniques [Pepstatin A (203)  was repouit peel . This peuit peel . Two cycchniques .206)  was isolated from the peels of Citrus hystrix fruits and possesses butyrylcholinesterase inhibition activity with IC50: 376 µM [The tetranortriterpenoid, kokosanolide D (206)  was repoit peels . BiphenyR method . Citruso: 376 µM . 209) (Strychnos nuxblanda fruit peels produced tyrosol (210) which exhibited α-glucosidase effects with an IC50: 295 μM [211 and 212 were isolated from C. reticulata fruit peels [213–216 were isolated from Pyrus pyrifolia fruit peels (pear) and 216 displayed higher antioxidant effects than caffeic acid (standard) [Passiflora edulis fruit peels produce edulilic acid (209)  that exh: 295 μM . Compounit peels . Metaboltandard) . 217  peels produced orobol (221), daidzein (222) and possessed antioxidant effects with IC50: 29.1 and 88.5 μg/mL, respectively [Fortunella japonica fruit peels produced α-tocopherol (223) [Pyrus pyrifolia (pear) fruit peels produced trans-chlorogenic acid (224), cis-chlorogenic acid (225) and 3,5-dicaffeoylquinic acid (226) [Benzaldehyde 217  was prodticulate  while diuit peel  and illuectively . The peaectively . The Forol (223)  while Pyid (226) .227–232  essential oil (OPEO) with chitosan film increased the shelf life of fresh shrimps (Parapenaeus longirostris) to 15 days [Edible coatings are applied as thin layers on the food surface and results in longer food shelf life, retention of food characteristics, properties and functionality at low cost. Shin et al.  develope 15 days . Escherichia coli and Bacillus sp. and the addition of this essential oil with an edible coating  significantly decreases the degradation of fresh strawberry and tofu [Escherichia coli, as well as anticancer and antioxidant effects [Lactobacillus casei, L. acidophilus and L. paracasei [In another study, the lemon peel essential oil possessed potent antimicrobial effects against and tofu . In addiand tofu  of orangand tofu . Probiot effects . It was aracasei . In addiaracasei  and oranaracasei . Fruit peels, which form a significant portion as a food processing by-product, have not yet been employed as a useful resource for many health supportive products. It is abundantly clear from this review that the potential possibilities to utilize these bioactive components in the food chain are available to the pharmaceutical industry. One of the possible factors for such an unused resource is an erroneous general misconception that fruit peels are unhealthy and considered an undesired waste. The current review has undoubtedly established that fruit peels are indeed a very rich resource of biologically active natural compounds and will have significant benefits for human and animal health. Investing in the fruit peel processing industry, food processors may expand their usability and flexibility to include a fruit-peel-based novel products business venture to establish a profitable existing enterprise. Producing healthy foods with natural ingredients or with fruit peels could bring so many new advantages via the reduction or elimination of food preservatives, artificial additives and replacing them with cheap natural ingredients. In addition, fruit and vegetable peels are being widely employed as food additives and in the modern era of health-conscious young people, such a venture will add to their repertoire of food buying possibilities.


The bacterial presence, abundance, and composition were analyzed by endpoint PCR of full-length 16S rRNA and the V4 amplicon sequence variants (ASVs). The results demonstrate that bacterial prevalence and abundance were both significantly increased in stillbirth, even after adjusting for maternal age, race, body mass index, number of pregnancies, gestational age, and multiple gestations. Composition of bacterial communities in the cord blood also differed significantly. Using a group of 25 ASVs differentially abundant between the two groups, a Random Forest classification model achieved an accuracy score of 0.76 differentiating stillbirth and live birth, with Group B Streptococcus as the most enriched species in stillbirth. Positive PCR was also significantly associated with early preterm birth. A group of oral anaerobes, including Actinomyces, Campylobacter, Fusobacterium, Peptostreptococcus, Porphyromonas, and Prevotella, were enriched in live early preterm birth, suggesting possible oral origin of infection. Our ASV-based microbiome analysis revealed specific candidate pathogens associated with infections in stillbirth and early preterm birth. The cord blood microbial signatures may be markers of adverse pregnancy outcomes. Our study will help identify possible mechanism of infection and improve our ability to prevent stillbirth and early preterm birth.Stillbirths account for half of all perinatal mortality, but the underlying cause of a significant portion of the cases remains unknown. We set out to test the potential role and extent of microbial infection in stillbirth through a case-control analysis of fetal cord blood collected from the multisite Stillbirth Collaborative Research Network. Cases ( Fusobacterium nucleatum, a Gram-negative anaerobe that is difficult to detect by routine culture yet known to be associated with adverse pregnancy outcomes such as preterm birth, stillbirth, and early-onset neonatal sepsis , antibiotics given prior to delivery, or fetal sex. The live birth group had higher rates of preterm labor and spontaneous labor than the stillbirth group. The stillborn infants were more likely to be born at an earlier gestational age, weighed less at birth, and had higher rates of meconium in the amniotic fluid .Both clinical and histologic chorioamnionitis were noted to be more prevalent in stillbirths, but the differences did not reach significance . After aEnd-point PCR amplification of the full-length 16S rRNA gene was performed to examine the presence of bacterial DNA. Detection of the 16S rRNA gene  was significantly higher in stillbirths than live births . After aWhen the subjects were further divided according to gestational age into early preterm birth (<32 weeks of gestation), preterm birth (32–36 weeks), and term birth (>37 weeks), the PCR+ rate was significantly higher among stillbirths than live births in each gestational age group . FurtherTo quantify the bacterial abundance and to characterize the microbial community in the umbilical cord blood, the V4 regions of 16S rRNA were sequenced on the Illumina MiSeq platform. In addition to the cord blood samples of stillbirths (cases) and live births (controls), we also included 12 blank buffer controls. A total of 4,651 amplicon sequence variants (ASVs) were identified after denoising by DADA2 . To remoThe total ASV read counts were significantly higher in stillbirths compared to live births . The difTo compare the microbial composition between samples, we downsized each sample to 1,000 reads, which corresponded to 1,875 reliable microbial ASVs from 39 cases (stillbirth) and 66 control (live birth) samples in the subsequent analysis.P = 0.039) (P = 0.0017), and a PERMANOVA test also showed a significant difference . A significant but smaller difference between the case and control groups was observed based on unweighted UniFrac distance as well (P = 0.021) and a significant PERMANOVA test result . At the ASV level, we found 30 ASVs with both different relative abundance (Mann-Whitney test) and prevalence (Fisher’s exact test) between the case and control groups , has been reported as a high-risk factor in stillbirth  . There w= 0.039) . Princip as well , as evidl groups . While al groups . To expll groups . Notably 24 ASVs . S. agalillbirth . The secal death . Eight aal death . These f10.1128/mbio.02036-22.1FIG S1n = 39) and controls (n = 66). Boxes show the medians and the interquartile ranges (IQRs), the whiskers denote the lowest and highest values that were within 1.5 times the IQR from the first and third quartiles, and outliers are shown as individual points. Download FIG S1, PDF file, 0.03 MB.Comparison of (a) Shannon index and (b) ASV number between stillbirth (case) and live birth (control). Mann-Whitney test (two-sided) was applied to compare cases  and control (n = 66). *, P < 0.05; **, P < 0.01. Boxes show the medians and the interquartile ranges (IQRs), and the whiskers denote the lowest and highest values that were within 1.5 times the IQR from the first and third quartiles. Download FIG S2, PDF file, 0.03 MB.Different microbial composition of umbilical cord blood between stillbirth (case) and live birth (control) based on unweighted UniFrac distance. (a) Principal coordinates analysis (PCoA) plot based on unweighted UnFirac distance. (b) PC1 of PCoA plot. (c) PC2 of PCoA plot. Mann-Whitney test (two-sided) was applied to compare case  (P = 0.026 compared to 32–36 weeks) (P = 0.033) , with both a lower Shannon index (7 weeks)  and a lo6 weeks) . In the 6 weeks)  and b. A= 0.033) . These rP = 0.011, R2 = 0.035). PCoA plot showed that the microbial structure in the stillbirth group changed along PC1 (<32 weeks versus ≥37 weeks: P = 0.010), while in live birth it changed along PC2  (P = 0.048) and ≥37 weeks (P = 0.021)  . Signifi= 0.021) . TherefoDifferences between stillbirth and live birth were detected at the ASV level at different gestational stages . Among t10.1128/mbio.02036-22.3FIG S3FIG S3, PDF file, 0.04 MB.Microbiota-based model to predict stillbirth (case) and live birth (control) in <32 weeks gestational age. Classification model based on Random Forest algorithm by using the abundance of the 3 ASVs. (a) Number of variables and mean squared error of the corresponding model. (b) The Random Forest model assigns a mean error rate or feature-importance score to each feature; this value indicates the extent to which each predictor contributes to the accuracy of the model. This model achieves an accuracy of 0.74. Download Copyright © 2022 Vander Haar et al.2022Vander Haar et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.02036-22.4FIG S4FIG S4, PDF file, 0.04 MB.Microbiota-based model to predict stillbirth (case) and live birth (control) in ≥37 weeks gestational age. Classification model based on Random Forest algorithm by using the abundance of the 24 ASVs. (a) Number of variables and mean squared error of the corresponding model. (b) The Random Forest model assigns a mean error rate or feature-importance score to each feature; this value indicates the extent to which each predictor contributes to the accuracy of the model. This model achieves an accuracy of 0.78. Download Copyright © 2022 Vander Haar et al.2022Vander Haar et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Fusobacterium, with Fusobacterium nucleatum subsp. animalis ATCC 51191 identified as the best hit for three of them based on NCBI BLASTN. This is consistent with a previous report that subsp. animalis was the most prevalent F. nucleatum detected in intrauterine infection  and false negative  results. As contamination is a serious concern in analyzing utero samples , we condThe current findings indicate that the bacterial infection rate in stillbirth may be severely underestimated in previous studies. Both the bacterial DNA prevalence and abundance were significantly increased in stillbirth than in live birth, indicating enhanced severity of infection in stillbirth. Among them, GBS was the major contributor. As GBS colonizes the maternal genital track, it likely invades into the intrauterine cavity via an ascending mechanism, although hematogenous infection cannot be ruled out. Although previous meta-analysis identified GBS as an important risk predictor of stillbirth, it was only attributed to 1% of stillbirths worldwide and 4% in Africa . Our resF. nucleatum was detected as the most prevalent species in early preterm birth, consistent with the report that intrauterine infection of F. nucleatum is inversely related to gestational age  , conducted from March 2006 to September 2008, with follow-up through December 2009, at 59 hospitals associated with five clinical sites . The casn = 101) . The resn = 101) .g for 15 min. The pellets were used for DNA extraction using the QIAamp BiOstic Bacteremia DNA Kit (Qiagen). The full-length 16S rRNA gene was amplified by PCR using universal primers A17F (GTTTGATCCTGGCTCAG) and 1512R (TACCTTGTTACGACTT), which can detect the majority of eubacteria, as previously described  and R Version 3.6.0 . Alpha- and beta-diversity metrics were calculated using QIIME2  upon request.


In China, it is common for pregnant women with a high load of hepatitis B virus (HBV) to take nucleos(t)ide analogue (NA) to prevent maternal-to-child transmission of HBV. However, the impact of NA intervention on virological and biochemical parameters in pregnant and postpartum women and the safety of drug cessation remain unclear. A prospective observational cohort was established in this study to analyze the clinical characteristics of hepatitis flares in pregnant and postpartum chronic HBV carriers, with or without NA intervention.Pregnant women who were chronic HBV carriers were enrolled in this study and divided into an NA intervention group and a non-intervention group according to their preferences. Liver function, HBV DNA level, and HBV serological markers were regularly measured during pregnancy and at approximately 6 weeks, 12 weeks, 24 weeks, 36 weeks, and 48 weeks postpartum.A total of 417 patients were enrolled, including 303 in the NA intervention group and 114 in the non-intervention group. The incidence rates of postpartum hepatitis flares in both groups were higher than that of during pregnancy . The second trimester was the peak of the incidence of flares during pregnancy and the incidence peak of postpartum flares was about 6 weeks postpartum. A total of 98% (145/148) of postpartum flares occurred within 24 weeks postpartum. After drug cessation, the incidence rate of flares was 34.1% (44/129).In pregnant chronic HBV carriers, a certain proportion of hepatitis flares occurred during pregnancy and postpartum regardless of whether NA intervention was used, and the incidence of postpartum flares (44.6%) was significantly higher than that (12.8%) of during pregnancy. The flare incidence peaked at approximately 6 weeks postpartum, which may be the time period suitable for treatment. Since 98% of postpartum flares occurred within 24 weeks postpartum, the follow-up after drug cessation should be at least 24 weeks postpartum. In China, most patients with chronic hepatitis B virus (HBV) infection acquired the infection at a young age, mother-to-child transmission as the main transmission route, accounting for approximately 40% of all HBV infections . Among p6 IU/ml, no history of antiviral therapy, and normal alanine aminotransferase (ALT) levels in at least two tests within 2 years before pregnancy. The exclusion criteria were as follows: hepatitis A, C, D, or E infection; other chronic liver diseases; human immunodeficiency virus infection; cirrhosis or hepatocellular carcinoma; and intrahepatic cholestasis of pregnancy and a history of miscarriage.The subjects of the study were chronic HBV carriers visited in Beijing You’an Hospital Affiliated to Capital Medical University(from November 2015 to July 2018), who were planning for pregnancy or pregnant (less than 8 weeks of gestation). The inclusion criteria were as follows: age 18-40 years, hepatitis B surface antigen (HBsAg) positivity for more than 6 months, HBeAg hepatitis B e antigen (HBeAg) positivity, HBV DNA ≥ 2 × 10A prospective, open-cohort design was adopted for this clinical study. The patients were given the choice between joining either the non-intervention group (group A) or the NA intervention group (group B) during pregnancy. Medications used for intervention included telbivudine (LDT) and tenofovir disoproxil (TDF), which were started at 24 weeks to 28 weeks of gestation at a dose of 600 mg/day for LDT and 300 mg/day for TDF. To select LDT or TDF was based on patients’ willingnesses. All patients were informed about this study and signed informed consent forms before enrollment .The team members of the joint research group of the Department of Obstetrics and the Department of Hepatology followed up the patients regularly from pregnancy to 48 weeks postpartum. Patients were followed up once every 4-6 weeks during pregnancy. The first postpartum follow-up was approximately 6 weeks postpartum (6 ± 2 weeks), and afterwards the patients were followed up approximately every 12 weeks until the completion of the 48-week follow-up. HBV DNA, HBV serological markers, liver function and renal function should be tested during each follow-up. Abnormal liver function was defined as ALT > upper limit of normal , including mildly abnormal liver function (ULN < ALT < 2 × ULN), hepatitis flare (ALT ≥ 2 × ULN), and hepatitis exacerbation . Patients with abnormal liver function test results were re-examined within 2 weeks, and any liver injury-inducing factors, such as alcohol, fat and drugs, had to be excluded by a clinicians’ assessment.HBV DNA was detected using the Roche Cobas/Taqman Real-Time fluorescence quantitative polymerase chain reaction (PCR) system , and the low limit of detection was 20 IU/mL. The HBV serological markers were detected using the Roche E601 automatic chemiluminescence analyzer, with lower detection limit of 0.05 IU/ml for HBsAg and 1 COI for HBeAg. An OLMPUS-AU5400 biochemistry analyzer was used to detect biochemical parameters such as liver function parameters. HBV was genotyped with the HBV genotyping reagent kit (fluorescence PCR) .SPSS 25.0  software was used for the statistical analysis. Continuous variables were presented as the mean ± standard deviation or median (interquartile range). Categorical variables were expressed as frequencies and percentages. Continuous variables were assessed using Student’s t test or Ranksum test. Categorical variables were compared with the Chi-squared test or Fisher’s exact test. A two-sided P value of < 0.05 indicated statistical significance.A total of 471 HBsAg-positive women who were pregnant or were planning for pregnancy were screened in this study Figure 1There were no significant differences in age, the baseline HBV DNA, HBeAg, and ALT levels, and the genotypes of patients between group A and group B Table 1.The rate of abnormal liver function during pregnancy was 20.9% (87/415), including 34 (8.2%) with mildly abnormal liver function, 53 (12.8%) with hepatitis flares, and 8 (1.9%) with hepatitis exacerbation. Among the 53 cases of hepatitis flares during pregnancy, 46 happened in non-intervention state, an incidence rate of 11.1% (46/415). Seven patients in group B had hepatitis flares during NA intervention, an incidence rate of 2.3% (7/302). The incidence rate of hepatitis flares in non-intervention state during pregnancy was significantly higher than that in intervention state, the difference was statistically significant . There was no significant difference in the incidence of hepatitis flares with or without NA intervention during pregnancy, with 17.7% (20/113) and 10.9% (33/302) in group A and B, respectively Table 2.In terms of the time of hepatitis flares, the second trimester was more common, with 65.0% (13/20) of patients with hepatitis flares during pregnancy in group A experienced hepatitis flares and 54.5% (18/33) of patients with hepatitis flares during pregnancy in group B experienced hepatitis flares in the second trimester.After 48 weeks of postpartum follow-up, 30 patients were lost to follow-up. The 53 patients with hepatitis flares during pregnancy were not included in the analysis of postpartum hepatitis flares. Therefore, 332 patients (85 in group A and 247 in group B) were included for the overall postpartum analysis, including 132 (39.7%) with mild abnormal liver function, 148 (44.6%) with hepatitis flares. The incidence rate of hepatitis flares during the postpartum period was significantly higher than that during pregnancy . The other 138 patients did not stop NA and the incidence of hepatitis flares in these patients was 33.3% (46/138).The difference was not significant (P=0.893).th week and the 48th week of the 48-week follow-up period, accounting for 2.0% of all postpartum hepatitis flare cases  was 113, including patients with drug cessation and patients without drug cessation, and the median value of HBV DNA during hepatitis flares was 4.4 log10 IU/ml, significantly lower than that in group A (P < 0.001). In group B, the HBV DNA levels at the onset of hepatitis flares were compared between patients with drug cessation and patients without drug cessation. The former had 67 cases of hepatitis flares and the median value of HBV DNA was higher, 5.6 log10 IU/ml; the latter had 46 cases of hepatitis flares and the median value of HBV DNA was lower, 3.3 log10 IU/ml, the difference was significant (P < 0.001).Within the 48 weeks of the postpartum follow-up, the cumulative number of patients with postpartum hepatitis flares in non-intervention group (group A) was 35, and the median value of HBV DNA during hepatitis flares was 7.6 logAccording to the recommendations of the recent guidelines for the management of hepatitis B –10, NAs In this study, among the 332 patients who were followed up to 48 weeks postpartum and included in the postpartum analysis, the incidence rate was 39.7% for mildly abnormal liver function, 44.6% for hepatitis flares, and 4.8% for hepatitis exacerbation. In the non-intervention group and the intervention group, mildly abnormal liver function and hepatitis flares were 36.5% and 40.9%, 41.2% and 45.7%, respectively, and the differences were not significant. But the proportion of hepatitis exacerbation in the non-intervention group (9.4%) was higher than that in the intervention group (3.2%), the difference was significant. Pan et al. . Hepatitis flares could not be avoided even during NA intervention, and the incidence rates of hepatitis flares were 2.3% in pregnant women during NA intervention and 33.3% in postpartum women. Bzowej . The resRegarding the time of onset of hepatitis flares during pregnancy, it had been reported  that aboRegarding the safe time for drug cessation, 92.3% 385/417) of patients completed the 48-week follow-up, and 98% of postpartum hepatitis flares (145/148) occurred within 24 weeks of postpartum, only 2% after 24 weeks. A retrospective study  showed t of patieIn addition, the HBV DNA load varied significantly among the patients with hepatitis flares in the intervention group in this study. Virological rebound occurred first in patients who stopped taking NA postpartum, and the HBV DNA level increased (5.6 log10 IU/ml) and then could cause hepatitis flares, which was in line with the general pattern of hepatitis flares, namely, high viral load and high ALT level. However, the hepatitis flare in patients who did not stop NA was different from the above. Even when the HBV DNA load was lower (3.3 log10 IU/ml), the ALT level was still elevated. The pattern of hepatitis flares in patients who did not stop NA was characterized by high ALT level but low viral load. The difference between the pattern of hepatitis flare before drug cessation and that after drug cessation was related to the different status of drug administration. In general, when the HBV DNA load is low, it usually does not trigger hepatitis flares. If the ALT level increases when the viral load declines, it is more likely to indicate the enhancement of immune function and the transition from immune tolerance to immune clearance period. In addition, other liver injury–inducing factors, such as fatty liver and drug-induced hepatitis, were excluded in this study. Pregnancy was generally considered to be in a relatively immunosuppressive state  to avoidIn summary, the status of immune tolerance in pregnant women who were chronic HBV carriers was not always unchanged. Regardless of whether NA intervention was applied, a certain proportion of hepatitis flares occurred during pregnancy and postpartum period, and the incidence rate of hepatitis flares during postpartum(44.6%) was significantly higher than that during pregnancy (12.8%). The peak of postpartum hepatitis flare was in 6 weeks. Since 98% of the postpartum hepatitis flares occurred within 24 weeks postpartum, patients should be followed up for at least 24 weeks postpartum to determine whether they can safely stop the treatment.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by The ethics committee of Beijing Youan Hospital, Capital Medical University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.XC, XW, and AS conceived and designed the protocol and study. AS, XL and SR collected the data. XW analyzed the data and drafted the manuscript. YZ performed the follow up of patients. JL, SZ, and LM guided statistical analysis. XC contributed to the interpretation of the results and critical revision of the manuscript for important intellectual content and approved the final version of the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the Capital Clinical Diagnostic Techniques and the Translational Application Projects (Z211100002921059), Chinese National Natural Science Foundation (81900537), Beijing Hospitals Authority Clinical medicine Development of special funding support (XMLX202125), and the Capital Health Research and Development Projects (2020–1–2181).The authors thank Yunxia Zhu, the director of Obstetrics and Gynecology, Beijing Youan Hospital, Capital Medical University, for supporting the enrollment and follow-up of patients.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The handling editor declared a shared parent affiliation with the authors at the time of review.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cytokine storm is a condition in which the immune system produces an excessive number of inflammatory signals, which can result in organ failure and death. It is also known as cytokine release syndrome, CRS, or simply cytokine storm, and it has received a lot of attention recently because of the COVID-19 pandemic. It appears to be one of the reasons why some people experience life-threatening symptoms from COVID-19, a medical condition induced by SARS-CoV-2 infection. In situations where natural substances can be exploited as therapeutics to reduce cytokine storm, the drug development process has come to the rescue. In the present study, we tested the potentiality of Andrographolide, labdane diterpenoid targeting several key cytokines that are secreted as a result of cytokine storm. We used molecular docking analyses, molecular dynamics simulations, and pharmacokinetic properties to test the stability of the complexes. The compound’s binding energy with some cytokines was over −6.5 Kcal/mol. Furthermore, a post-molecular dynamics (MD) study revealed that Andrographolide was extremely stable with these cytokines. The compound’s pharmacokinetic measurements demonstrated excellent properties in terms of adsorption, distribution, metabolism, and excretion. Our research revealed that this compound may be effective in lowering cytokine storm and treating severe symptoms. Cytokines are small proteins secreted by cells that have a specific effect on cell contacts and communication . They arAccording to studies, cytokine storm is linked to the advancement of SARS-CoV-2 infection; a higher level of cytokine storm is linked to a more severe disease development . In patiAndrographolide is a labdane diterpenoid isolated from Andrographis paniculate, a medicinal herb. It has anti-inflammatory, anti-allergic, anti-platelet aggregation, antineoplastic, anti-HIV, and hepatoprotective qualities . AndrogrThe drug design and discovery driven by advances in powerful cheminformatics and various in silico tools culminated in the revolution of the ‘bioinformatics era’ . The in Taking advantage of these techniques, we selected Andrographolide to identify its potential efficacy against eighteen cytokines in this study. Their classification, main sources, receptor, target cell and major function are listed in http://accelrys.com/products/discovery-studio/, accessed on 20 June 2022) [Eighteen cytokines named IL-2, IL-3, IL-4, IL-5, IL-6, IL-13, IFN-γ, IL-8, IL-10, IL-12, IL-17, G-GSF, GM-CSF, IP10, M-CSF, MiP1α, MiP1β, and TNF-α were selected for this study. The crystal structures of the cytokines were collected from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB) . All thehttps://sts.bioe.uic.edu/castp/, accessed on 20 June 2022) and were enclosed within the suitable parameters of grid box dimensions. AutoDock Vina (ADV) [Molecular docking was performed using PyRX , an openna (ADV) , implemena (ADV) ,22+ and Cl− ions, which were added while 0.15 mol/L salt concentrations were set in all the systems. Using the steepest descent method, all the solvated systems were subjected to energy minimization for 5000 steps. Afterwards, the NVT  series, NPT  series, and the production run were conducted in the MD simulation [Molecular dynamics simulation of the four protein-ligand complexes was performed using GROMACS 2021.1 the University of Groningen, city of Groningen, Northern Netherlands  version mulation . The NVTmulation  program http://www.swissadme.ch, accessed on 20 June 2022) was utilized to evaluate the pharmacokinetic properties of Andrographolide.The SwissADME  online tAutoDock Vina predicted nine possible binding positions for each compound as output. The best position was chosen for each compound based on the lowest docking energy. The docking energy score of all the cytokines with Andrographolide  ranged fThe dynamic movements of atoms and conformational variations of Cα atoms of the protein–ligand complexes were calculated by RMSD to detect their stability. It is observed that except for the IL5, all the complexes exhibit lower RMSD (<1.0 nm) with no major fluctuations, indicating their greater stability shown in p (skin permeation) was in a good range and it also had efficacy to cross the blood–brain barrier. A PAINS alert confirmed the absence of any catechol moieties in the compound and synthetic accessibility values were also found to be low.The ADME properties of Andrographolide were predicted using a Swiss ADME server . In manyApart from traditional methods, computer-assisted drug design (CADD) approaches are now used efficiently in the drug discovery and development process. Molecular docking and molecular dynamics simulations are commonly used to perform the virtual screening of compounds to examine how ligands inhibit a target and confirm their stability towards specific target proteins, among numerous approaches to CADD that are recognized as promising techniques. Additionally, the rising development of robust computational techniques allows for the assessment of the pharmacological properties of drugs prior to engaging in an experimental trial. As a result, in this study, rigorous CADD methodologies were used to examine the drug capabilities of Andrographolide against a range of cytokines.Considering the significance of cytokines and chemokines in immune system mediation maintaining anti-viral immunity, we tested Andrographolide’s efficacy against different cytokines by looking at both its binding affinity and ADMET characteristics. Andrographolide was tested against seventeen cytokines for this purpose. When binding with IL3, the compound had the maximum binding affinity of −7.5 kcal/mol and interacted with six amino acid residues in the cytokines’ active site. We further investigated their stability with Andrographolide, conducting MD simulation for a 100 ns time period, even though they had strong binding affinity with other cytokines as well.The compound was further used for molecular docking analysis to determine their binding affinities towards different cytokines. The result of the analysis indicated good binding energies of most cytokines ranging between −5.3 and −7.5 kcal/mol, better than the minimum binding energies of MiP1α , IP10 , IL-8 , and IL-2  . The comThe stability of Andrographolide with the respected cytokines was confirmed by the MD simulation findings of the top 11 complexes. The RMSD plot demonstrated that all of the complexes are stable, and the RMSD values did not fluctuate significantly over time. The complexes did not show significant variability and were within an acceptable range, according to the RMSF analysis. The radius of gyration (Rg) data revealed that each complex had a substantially comparable compactness characteristic. The SASA data indicated that the volume of the complexes had dropped somewhat. Throughout the simulation, a significant number of hydrogen bonds were identified in all the complexes, which once again explained their conformational stability. As a result, Andrographolide can bind to these cytokines, interfering with their action. Thus, by inhibiting these cytokines, several functions such as the production of granulocyte and monocyte, and the proliferation of B and T cells can be altered, thereby reducing the cytokine storm.Andrographolide has a high gastrointestinal absorption potency, with a skin permeability coefficient (Log Kp) of −6.90, according to its ADME characteristics. It does not break Lipinski’s rule of five; therefore, it might readily be mimicked by other natural cytokine ligands. Furthermore, the permeability of the blood–brain barrier membrane revealed that the compound is unable to cross the blood–brain barrier. Pan-assay interference chemicals (PAINS) are chemical compounds that react nonspecifically with a variety of biological targets, resulting in false positive results in high-throughput screens. It does not have any catechol groups, according to the PAINS filter. It has a low synthetic accessibility score, indicating that the compound can considerably more easily be produced in experimental laboratories.There may be some limitations in this study because it was conducted via in silico analysis. Despite the positive results with the cytokines, the compound has yet to be tested in an animal model. To assure its therapeutic efficacy, sufficient experimental validations are required.Our study proposed and tested a new compound, Andrographolide, against several crucial cytokines involved in cytokine storm. Binding of the compound to the target cytokines may eventually create an alteration in entering in their action and producing a lesser amount. The compound showed better results in both binding with the proteins in efficient oral bioavailability and less toxicity. Considering binding affinities, interactions with target proteins, and ADME profile, we have analyzed and validated the stability of protein–ligand complexes using an MD simulation approach and found intriguing results for all of them. The results of this study might be valuable references for further experimental research in designing new potent cytokine regulators. Hence, it can be concluded, after further in vitro and in vivo experiments, that Andrographolide can be used as a therapeutic against cytokine storm.