Ependymal polarity defects coupled with disorganized ciliary beating drive abnormal cerebrospinal fluid flow and spine curvature in zebrafish.

Idiopathic scoliosis (IS) is the most common spinal deformity diagnosed in childhood or early adolescence, while the underlying pathogenesis of this serious condition remains largely unknown. Here, we report zebrafish ccdc57 mutants exhibiting scoliosis during late development, similar to that observed in human adolescent idiopathic scoliosis (AIS). Zebrafish ccdc57 mutants developed hydrocephalus due to cerebrospinal fluid (CSF) flow defects caused by uncoordinated cilia beating in ependymal cells. Mechanistically, Ccdc57 localizes to ciliary basal bodies and controls the planar polarity of ependymal cells through regulating the organization of microtubule networks and proper positioning of basal bodies. Interestingly, ependymal cell polarity defects were first observed in ccdc57 mutants at approximately 17 days postfertilization, the same time when scoliosis became apparent and prior to multiciliated ependymal cell maturation. We further showed that mutant spinal cord exhibited altered expression pattern of the Urotensin neuropeptides, in consistent with the curvature of the spine. Strikingly, human IS patients also displayed abnormal Urotensin signaling in paraspinal muscles. Altogether, our data suggest that ependymal polarity defects are one of the earliest sign of scoliosis in zebrafish and disclose the essential and conserved roles of Urotensin signaling during scoliosis progression.

Rassf7a promotes spinal cord regeneration and controls spindle orientation in neural progenitor cells.

Spinal cord injury (SCI) can cause long-lasting disability in mammals due to the lack of axonal regrowth together with the inability to reinitiate spinal neurogenesis at the injury site. Deciphering the mechanisms that regulate the proliferation and differentiation of neural progenitor cells is critical for understanding spinal neurogenesis after injury. Compared with mammals, zebrafish show a remarkable capability of spinal cord regeneration. Here, we show that Rassf7a, a member of the Ras-association domain family, promotes spinal cord regeneration after injury. Zebrafish larvae harboring a rassf7a mutation show spinal cord regeneration and spinal neurogenesis defects. Live imaging shows abnormal asymmetric neurogenic divisions and spindle orientation defects in mutant neural progenitor cells. In line with this, the expression of rassf7a is enriched in neural progenitor cells. Subcellular analysis shows that Rassf7a localizes to the centrosome and is essential for cell cycle progression. Our data indicate a role for Rassf7a in modulating spindle orientation and the proliferation of neural progenitor cells after spinal cord injury.

ZFYVE19 deficiency: a ciliopathy involving failure of cell division, with cell death.

BACKGROUND AND AIMS: Variants in ZFYVE19 underlie a disorder characterised by progressive portal fibrosis, portal hypertension and eventual liver decompensation. We aim to create an animal model to elucidate the pathogenic mechanism. METHODS: Zfyve19 knockout (Zfyve19-/- ) mice were generated and exposed to different liver toxins. Their livers were characterised at the tissue, cellular and molecular levels. Findings were compared with those in wild-type mice and in ZFYVE19-deficient patients. ZFYVE19 knockout and knockdown retinal pigment epithelial-1 cells and mouse embryonic fibroblasts were generated to study cell division and cell death. RESULTS: The Zfyve19-/- mice were normal overall, particularly with respect to hepatobiliary features. However, when challenged with α-naphthyl isothiocyanate, Zfyve19-/- mice developed changes resembling those in ZFYVE19-deficient patients, including elevated serum liver injury markers, increased numbers of bile duct profiles with abnormal cholangiocyte polarity and biliary fibrosis. Failure of cell division, centriole and cilia abnormalities, and increased cell death were observed in knockdown/knockout cells. Increased cell death and altered mRNA expression of cell death-related signalling pathways was demonstrated in livers from Zfyve19-/- mice and patients. Transforming growth factor-ß (TGF-ß) and Janus kinase-Signal Transducer and Activator of Transcription 3 (JAK-STAT3) signalling pathways were upregulated in vivo, as were chemokines such as C-X-C motif ligands 1, 10 and 12. CONCLUSIONS: Our findings demonstrated that ZFYVE19 deficiency is a ciliopathy with novel histological features. Failure of cell division with ciliary abnormalities and cell death activates macrophages and may thus lead to biliary fibrosis via TGF-ß pathway in the disease.

Novel compound heterozygous variants in ARL13B lead to Joubert syndrome.

Joubert syndrome (JBTS) is a systematic developmental disorder mainly characterized by a pathognomonic mid-hindbrain malformation. All known JBTS-associated genes encode proteins involved in the function of antenna-like cellular organelle, primary cilium, which plays essential roles in cellular signal transduction and development. Here, we identified four unreported variants in ARL13B in two patients with the classical features of JBTS. ARL13B is a member of the Ras GTPase family and functions in ciliogenesis and cilia-related signaling. The two missense variants in ARL13B harbored the substitutions of amino acids at evolutionarily conserved positions. Using model cell lines, we found that the accumulations of the missense variants in cilia were impaired and the variants showed attenuated functions in ciliogenesis or the trafficking of INPP5E. Overall, these findings expanded the ARL13B pathogenetic variant spectrum of JBTS.

Gradually Self-Strengthen DNA Supramolecular Hydrogels.

The dynamic mechanical strength of the extracellular matrix (ECM) has been demonstrated to play important role in determining the cell behavior. Growing evidences suggest that the gradual stiffening process of the matrix is particularly decisive during tissue development and wound healing. Herein, a novel strategy to prepare hydrogels with gradually enhanced mechanical strength is provided. Such hydrogels could maintain the dynamic properties at their initial states, such as self-healing and shear-thinning properties. With subsequent slow covalent crosslinking, the stability and mechanical properties would be gradually improved. This method is useful for sequence programmability and oxidation strategies, which has provided an alternated tool to study cell behavior during dynamic increase in mechanical strength of ECM.

Cell cycle arrest induced by trichoplein depletion is independent of cilia assembly.

Cilia assembly and centriole duplication are closely coordinated with cell cycle progression, and inhibition of cilia disassembly impedes cell cycle progression. The centrosomal protein trichoplein (TCHP) has been shown to promote cell cycle progression in the G1 -S phase by disassembling cilia. In this study, we showed that deletion of TCHP not only prevented the progression to the S phase but also resulted in cell cycle exit and entrance into G0 phase. Surprisingly, we found that loss of TCHP-induced G0 arrest could not be reversed by blocking the assembly of cilia. In cells without IFT20 or CEP164, two genes encoding key factors for ciliogenesis, depletion of TCHP still led to G0 arrest. Mechanistically, we also found that TCHP depletion-induced cell cycle arrest was not mediated through a centrosome surveillance mechanism, but inhibition of Rb or concomitant inhibition of both Rb and p53 signaling pathways was required to reverse the cell cycle phenotype. In conclusion, our study provides new insights into the function of TCHP in cell cycle progression.

Association of novel TMEM67 variants with mild phenotypes of high gamma-glutamyl transpeptidase cholestasis and congenital hepatic fibrosis.

TMEM67 (mecklin or MKS3) locates in the transition zone of cilia. Dysfunction of TMEM67 disrupts cilia-related signaling and leads to developmental defects of multiple organs in humans. Typical autosomal recessive TMEM67 defects cause partial overlapping phenotypes, including abnormalities in the brain, eyes, liver, kidneys, bones, and so forth. However, emerging reports of isolated nephronophthisis suggest the possibility of a broader phenotype spectrum. In this study, we analyzed the genetic data of cholestasis patients with no obvious extrahepatic involvement but with an unexplained high level of gamma-glutamyl transpeptidase (GGT). We identified five Han Chinese patients from three unrelated families with biallelic nonnull low-frequency TMEM67 variants. All variants were predicted pathogenic in silico, of which p. Arg820Ile and p. Leu144del were previously unreported. In vitro studies revealed that the protein levels of the TMEM67 variants were significantly decreased; however, their interaction with MKS1 remained unaffected. All the patients, aged 7-39 years old, had silently progressive cholestasis with elevated GGT but had normal bilirubin levels. Histological studies of liver biopsy of patients 1, 3, and 5 showed the presence of congenital hepatic fibrosis. We conclude that variants in TMEM67 are associated with a mild phenotype of unexplained, persistent, anicteric, and high GGT cholestasis without typical symptoms of TMEM67 defects; this possibility should be considered by physicians in gastroenterology and hepatology.

When Dynamic Diselenide Bonds Meet Dynamic Imine Bonds in Polymeric Materials.

In both natural and artificial functional systems, the cooperation between different dynamic interactions is of vital importance for realizing complicated functions. Dynamic covalent bonds are one kind of relatively stable dynamic interactions and have shown synergistic effect in natural systems such as functional proteins. However, synergistic interactions between different dynamic covalent bonds in polymeric materials are still unclear. Herein, polymeric materials containing diselenide and imine bonds are prepared, and then the synergistic effect between the two dynamic covalent bonds is quantitatively evaluated in typical processes of dynamic materials. The results reveal that dynamic covalent bonds show weak synergistic effect in the degradation process and have strong synergistic effect in stress relaxation process. Therefore, introducing multiple dynamic covalent bonds in polymeric materials can extensively enhance their dynamic properties.

Disrupted intraflagellar transport due to IFT74 variants causes Joubert syndrome.

PURPOSE: Ciliopathies are a group of disorders caused by defects of the cilia. Joubert syndrome (JBTS) is a recessive and pleiotropic ciliopathy that causes cerebellar vermis hypoplasia and psychomotor delay. Although the intraflagellar transport (IFT) complex serves as a key module to maintain the ciliary structure and regulate ciliary signaling, the function of IFT in JBTS remains largely unknown. We aimed to explore the impact of IFT dysfunction in JBTS. METHODS: Exome sequencing was performed to screen for pathogenic variants in IFT genes in a JBTS cohort. Animal model and patient-derived fibroblasts were used to evaluate the pathogenic effects of the variants. RESULTS: We identified IFT74 as a JBTS-associated gene in three unrelated families. All the affected individuals carried truncated variants and shared one missense variant (p.Q179E) found only in East Asians. The expression of the human p.Q179E-IFT74 variant displayed compromised rescue effects in zebrafish ift74 morphants. Attenuated ciliogenesis; altered distribution of IFT proteins and ciliary membrane proteins, including ARL13B, INPP5E, and GPR161; and disrupted hedgehog signaling were observed in patient fibroblasts with IFT74 variants. CONCLUSION: IFT74 is identified as a JBTS-related gene. Cellular and biochemical mechanisms are also provided.

Association Between Outdoor Activity and Insufficient Sleep in Chinese School-Aged Children.

BACKGROUND The aim of this study was to explore the relationship between outdoor activity (OA) and insufficient sleep duration (ISD) among Chinese children and to assess the potential age- and gender-specific effects. MATERIAL AND METHODS A national sample of 62 517 children aged 6-17 years were recruited from 7 provinces of China in the autumn of 2013. Children and their caregivers reported daily sleep duration (<7 h, 7-9 h and ≥9 h), daily OA time (<1 h, 1-2 h, and ≥2 h), and other behavior and demographic information by standardized questionnaire. ISD was defined as <9 h/day. Logistic regression analysis was used to evaluate the relationship between ISD and OA. RESULTS In total, 46 639 children (50.9% boys) completed this survey. The prevalence of ISD was 74.9% (73.7% in boys and 76.1% in girls). Stratified by age, compared with <1 h OA, the odds ratios for ISD were 0.67 (95% confidence interval: 0.62-0.72, aged 6-13 years) and 0.69 (0.49-0.98, 14-17 years) in the ≥2 h OA group; 0.91 (0.85-0.97, 6-13 years) and 0.73 (0.54-0.97, 14-17 years) in the 1-2 h OA group. Further stratified by gender, we found similar trends for both boys and girls in each age group, although some results became non-significant. CONCLUSIONS Adequate OA time per day is associated with decreased risks for ISD among children aged 6-17 years, but age and gender can modify the association.

Anaemia, iron deficiency, iron-deficiency anaemia and their associations with obesity among schoolchildren in Guangzhou, China.

OBJECTIVE: The association of Fe metabolism with obesity in children remains unclear. The present study aimed to assess the status of Fe metabolism parameters, the prevalence of anaemia, Fe deficiency (ID) and Fe-deficiency anaemia (IDA), and the associations of these variables with obesity in Chinese schoolchildren. DESIGN: A cross-sectional study conducted in 5295 schoolchildren aged 7-11 years in Guangzhou, China, 2014-2015. Full data of anthropometric and Fe metabolic parameters were collected to assess obesity, anaemia, ID and IDA. Logistic regression models were established to determine the possible associations of anaemia, ID and IDA with obesity. Two-tailed P values of <0·05 was considered statistically significant. SETTING: Guangzhou City, China. PARTICIPANTS: Schoolchildren aged 7-11 years (n 5295). RESULTS: In this sample, mean Hb concentration was 128·1 g/l and the prevalence of anaemia, ID and IDA was 6·6, 6·2 and 0·6 %, respectively. Of the participants, 14·0 % were overweight and 8·8 % were obese. Importantly, obesity was associated with lower anaemia risk (adjusted OR = 0·553; 95 % CI 0·316, 0·968) but higher ID risk (adjusted OR = 1·808; 95 % CI 1·146, 2·853) after adjustment for confounders. No significant relationship was found between obesity and IDA. CONCLUSIONS: Our results confirmed that anaemia and ID remain public health concerns among schoolchildren in Guangzhou, while IDA is remarkably less prevalent. Furthermore, obesity was associated with lower anaemia risk, but higher ID risk. More efforts should be made to prevent the onset of ID and obesity in the same individual, thus improving the health and fitness of children.

Association of sugar-sweetened beverage intake with risk of metabolic syndrome among children and adolescents in urban China.

OBJECTIVE: High sugar-sweetened beverage (SSB) intake has been shown to correlate with a higher risk for CVD and metabolic disorders, while the association between SSB intake and the risk of metabolic syndrome (MetS) remains unclear. The present study aimed to explore the association between SSB intake and MetS among children and adolescents in urban China. DESIGN: A cross-sectional study involving 7143 children and adolescents was conducted in urban China. MetS definition proposed by the International Diabetes Federation was adopted. Data on SSB intake, diet, physical activity and family environment factors were obtained through questionnaires. Logistic regression models with multivariable adjustment were adopted to analyse the association between SSB intake and the risk of MetS and its components. SETTING: Primary and secondary schools in three urban cities of China. PARTICIPANTS: Children and adolescents (n 5258) aged 7-18 years. RESULTS: Among the participants, 29·9 % of them had high SSB intake (at least 0·3 servings/d) and the overall MetS prevalence was 2·7 %. Participants with high SSB intake were at higher risk for MetS (OR = 1·60; 95 % CI 1·03, 2·54) and abdominal obesity (OR = 1·55; 95 % CI 1·28, 1·83) compared with their counterparts with no SSB intake (0 servings/d). CONCLUSIONS: High SSB intake is significantly associated with increased MetS and abdominal obesity risk among children and adolescents in urban China. These results suggest that strong policies focusing on controlling SSB intake might be effective in preventing MetS and abdominal obesity.

Physical Activity and Spatial Memory Are Minimally Affected by Moderate Growth Restriction in Preterm Piglets.

BACKGROUND: Preterm birth is associated with impaired brain functions, but it is unknown whether fetal growth restriction (GR) makes these deficits worse. Using piglets as a model for preterm infants, we hypothesized that moderate GR reduces growth rate, physical activity, and spatial memory in the first weeks after preterm birth. METHODS: Preterm pigs were delivered by caesarean section and fed until 19 days (n = 830 from 55 pregnant sows) and received intensive clinical care. GR pigs were classified as animals with the lowest 5-20% percentile birth weight within each litter and were compared with litter-mate controls (21-100% percentile birth weight). Basic motor skill development, physical activity, and morbidities (e.g., necrotizing enterocolitis) were recorded within the first week. Weight of internal organs and data from a T-maze spatial memory test were noted until 19 days. RESULTS: Moderate GR and control preterm pigs (birth weights 728 ± 140 and 1,019 ± 204 g, respectively) showed similar relative weights of internal organs (relative to body), except higher adrenal gland weights in GR pigs (+20-50%, p < 0.05). This was associated with a tendency to higher plasma cortisol (p < 0.05 on day 11). GR preterm pigs showed delayed ability to stand and walk (days 2-5, p < 0.01), but physical activity and proportion of correct choices in a T-maze test (70.3 vs. 71.6%) were similar. CONCLUSION: Moderate GR has limited effect on motor function and spatial memory in the early postnatal period of preterm pigs, despite some initial delays in basic motor skills. In the postnatal period, moderately growth-restricted preterm infants may adapt well with regards to organ growth and neurodevelopment.

Short sleep duration is associated with specific food intake increase among school-aged children in China: a national cross-sectional study.

BACKGROUND: The relationship between sleep duration and food intake is unclear. This study aims to examine the relationship among children aged 6-17 years in China. METHODS: The sample consisted of 70,519 children aged 6-17 years, which were randomly selected from 7 representative areas from China, from September to November, 2013. In the structured questionnaire, children reported daily sleep hours (less than 7 h, 7-9 h and more than 9 h), weekly food intake amount (including vegetables, fruit, sugar beverages and meat), physical activity and sedentary time. The relationship of sleep duration with vegetable, sugar beverage, fruit and meat intake was evaluated by multi-nominal logistic regression and multi-variable adjusted. RESULTS: A total of 62,517 children (51.6% boys) completed the study. Short sleep duration (SSD, < 7 h) was independently associated with increased sugar beverage intake (SBI, Odd Ratio, OR: 1.29, 95% CI: 1.19-1.40) but decreased vegetable (VI, OR: 0.94, 95% CI: 0.90-0.98) & fruit intake (FI, OR: 0.94, 95% CI: 0.88-0.99). Stratified by age and gender, SSD increased SBI for boys of both young (6-12 years) & older (13-17 years) groups and older girls (ORs: 1.25, 1.25, 1.49, 95% CI: 1.08-1.44, 1.04-1.50, 1.22-1.81, respectively), but decreased VI and FI for older girls (ORs: 0.84& 0.81, 95% CI: 0.74-0.96& 0.68-0.96, respectively). CONCLUSIONS: Among school-aged children in China, short sleep duration was associated with increased risks of more sugar beverage intake among those younger and boys but less vegetable & fruit intake among those older and girls. Longitudinal research is needed to clarify the causation in between.

Gender-dependent association between sleep duration and overweight incidence in CHINESE school children: a national follow-up study.

BACKGROUND: The relationship between sleep duration and overweight risk remains unexplored among Chinese children. This study aims to evaluate this association in a national investigation with school-aged population. METHODS: There were 18,302 normal weight children in this Chinese national study which conducted during 2013-2014 included in the research. Anthropometric measurements were performed both at baseline and after 6-9 month. Sleep duration, physical activity, food intake and social economic information were collected by self-report questionnaire. Overweight was defined according to the updated Chinese criterion. Cox regression was used to evaluate the relationships between sleep duration and overweight incidence with multivariable adjusted. RESULTS: In total, there were 443 new overweight cases recorded at the end of observation. Overweight incidence with greater than 9 h (long sleep duration, LSD), 7 to 9 h (middle sleep duration, MSD), and less than 7 h of sleep (short sleep duration, SSD) were 2.7, 3.1 and 3.3% respectively. Stratified by gender and compared with LSD, the hazard ratio (HR) of overweight for females with MSD was 1.60 (95% CI: 1.02-2.52). Stratified by age and gender, the HR in the group of MSD was 2.13 (1.20-3.77) in female aged 6-10 years and 0.24 (0.06-0.93) in female aged 15-17 years. CONCLUSION: The association between short sleep duration and overweight is age- and gender dependent. In group of small age and elder age, girls' adiposity states are independently associated with sleep duration. Sleep recommendation is a potential preventive action for overweight/obesity among girls.

The conserved ciliary protein Bug22 controls planar beating of Chlamydomonas flagella.

Eukaryotic flagella and cilia can exhibit planar and non-planar beating, and the mechanism controlling these beating patterns is not well understood. Chlamydomonas reinhardtii flagella beat in approximately the same plane with either an asymmetric ciliary-type or symmetric flagellar-type waveform. Each B-tubule of the number 1, 5 and 6 doublets of the flagellar axoneme possesses a beak-like structure. The number 5 and 6 beak structures are implicated in conversion of ciliary motion into flagellar motion. Here, we show that in a null mutant of Bug22, the asymmetric ciliary waveform is converted into a three-dimensional (non-planar) symmetric flagellar waveform. Bug22 is localized to approximately the proximal half to two-thirds of the flagellum, similar to localization of beak-like structures. However, as shown by immunogold labeling, Bug22 associates with axonemal microtubules without apparent preference for any particular doublets. Interestingly, bug22 mutants lack all beak-like structures. We propose that one function of Bug22 is to regulate the anchoring of the beak-like structures to the doublet microtubules and confine flagellar beating to a plane.

Activation loop phosphorylation of a protein kinase is a molecular marker of organelle size that dynamically reports flagellar length.

Specification of organelle size is crucial for cell function, yet we know little about the molecular mechanisms that report and regulate organelle growth and steady-state dimensions. The biflagellated green alga Chlamydomonas requires continuous-length feedback to integrate the multiple events that support flagellar assembly and disassembly and at the same time maintain the sensory and motility functions of the organelle. Although several length mutants have been characterized, the requisite molecular reporter of length has not been identified. Previously, we showed that depletion of Chlamydomonas aurora-like protein kinase CALK inhibited flagellar disassembly and that a gel-shift-associated phosphorylation of CALK marked half-length flagella during flagellar assembly. Here, we show that phosphorylation of CALK on T193, a consensus phosphorylation site on the activation loop required for kinase activity, is distinct from the gel-shift-associated phosphorylation and is triggered when flagellar shortening is induced, thereby implicating CALK protein kinase activity in the shortening arm of length control. Moreover, CALK phosphorylation on T193 is dynamically related to flagellar length. It is reduced in cells with short flagella, elevated in the long flagella mutant, lf4, and dynamically tracks length during both flagellar assembly and flagellar disassembly in WT, but not in lf4. Thus, phosphorylation of CALK in its activation loop is implicated in the disassembly arm of a length feedback mechanism and is a continuous and dynamic molecular marker of flagellar length during both assembly and disassembly.

Flagellar regeneration requires cytoplasmic microtubule depolymerization and kinesin-13.

In ciliated cells, two types of microtubules can be categorized: cytoplasmic and axonemal. It has been shown that axonemal tubulins come from a 'cytoplasmic pool' during cilia regeneration. However, the identity and regulation of this 'pool' is not understood. Previously, we have shown that Chlamydomonas kinesin-13 (CrKin13) is phosphorylated during flagellar regeneration, and required for proper flagellar assembly. In the present study, we show that CrKin13 regulates depolymerization of cytoplasmic microtubules to control flagellar regeneration. After flagellar loss and before flagellar regeneration, cytoplasmic microtubules were quickly depolymerized, which was evidenced by the appearance of sparse and shorter microtubule arrays and increased free tubulins in the cell body. Knockdown of CrKin13 expression by RNA interference inhibited depolymerization of cytoplasmic microtubules and impaired flagellar regeneration. In vitro assay showed that CrKin13 possessed microtubule depolymerization activity. CrKin13 underwent phosphorylation during microtubule depolymerization, and phosphorylation induced targeting of CrKin13 to microtubules. The phosphorylation of CrKin13 occurred at residues S100, T469 and S522 as determined by mass spectrometry. Abrogation of CrKin13 phosphorylation at S100 but not at other residues by inducing point mutation prevented CrKin13 targeting to microtubules. We propose that CrKin13 depolymerizes cytoplasmic microtubules to provide tubulin precursors for flagellar regeneration.

Physical activity level is impaired and diet dependent in preterm newborn pigs.

BACKGROUND: Preterm infants show delayed development of motor function after birth. This may relate to functional immaturity of many organs, including the gut and brain. Using pigs as model for preterm infants, we hypothesized that early initiation of enteral feeding stimulates both gut growth and neonatal physical activity. METHODS: In experiment 1, preterm and term pigs were fed parenteral nutrition (PN) or PN plus bovine colostrum (BC, 16-64 ml/kg/d enterally) for 5 d. In experiment 2, preterm pigs were fed PN+BC or PN+formula for 5 d. In experiment 3, preterm pigs were fed BC, formula, or human milk (HM) for 10 d. Incubator home cage activity (HCA) was quantified by continuous camera recordings. RESULTS: Preterm birth was associated with reduced intestinal weight and HCA (experiment 1), and BC or formula supplementation increased intestinal weights and HCA (experiments 1+2). Enteral BC and HM feeding increased HCA, intestinal weights, and necrotizing enteritis resistance, relative to formula (experiment 3). CONCLUSION: Preterm pigs show decreased physical activity, and the first enteral feeds diet dependently stimulate both gut growth and physical activity. The effects may arise from maturation of digestive, metabolic, and neurological functions, including gut serotonin production, by the first enteral feeds and milk bioactive factors.