Influence of dietary patterns and physical activity on bone mineral content and density, osteoporosis among children with stimulant use.

Aim: To examine the relationship between dietary patterns (DPs) and physical activity (PA) on bone mineral content (BMC), bone mineral density (BMD), and osteoporosis in children with stimulant use. Methods: A cross-sectional study collected information on participants from the National Health and Nutrition Examination Survey (NHANES) via multistage stratified sampling. The baseline variables included the following: age, gender, the dietary approaches to stop hypertension (DASH) score, the Mediterranean diet (MD) score, and the Alternative Healthy Eating Index-2010 (AHEI-2010). The univariate and multivariate linear-regression analyses were carried out to explore the statistical correlation between the DPs and PA on BMC and BMD in children with stimulant use or non-stimulant use. In addition, we also investigated the association between DPs and PA on osteoporosis via logistic regression analyses. Results: A total of 6,294 participants were eligibly enrolled in this study eventually. After adjusting age, gender, body mass index (BMI), race, family income, serum 25-hydroxyvitamin D, and serum cotinine, the multivariate linear-regression analysis showed that the MD was positively associated with total femur BMD, total femur BMC, femoral neck BMD, and femoral neck BMC among stimulant use group; high PA was associated with total femur BMD, total femur BMC, femoral neck BMD, femoral neck BMC, lumbar spine BMD, lumbar spine BMC and osteoporosis in stimulant use group. Conclusion: Improved adherence to MD, DASH, AHEI-2010 or increased physical activity may increase BMD, BMC and reduce the risk of osteoporosis; children with stimulant use should improve their adherence to the MD and do more PA compared with children without stimulant use.

Identifying Rare Genetic Variants of Immune Mediators as Risk Factors for Autism Spectrum Disorder.

Autism spectrum disorder (ASD) affects more than 1% of children, and there is no viable pharmacotherapeutic agent to treat the core symptoms of ASD. Studies have shown that children with ASD show changes in their levels of immune response molecules. Our previous studies have shown that ASD is more common in children with folate receptor autoantibodies. We also found that children with ASD have abnormal gut immune function, which was characterized by a significant increase in the content of immunoglobulin A and an increase in gut-microbiota-associated epitope diversity. These studies suggest that the immune mechanism plays an important role in the occurrence of ASD. The present study aims to systematically assess gene mutations in immune mediators in patients with ASD. We collected genetic samples from 72 children with ASD (2−12 years old) and 107 healthy controls without ASD (20−78 years old). We used our previously-designed immune gene panel, which can capture cytokine and receptor genes, the coding regions of MHC genes, and genes of innate immunity. Target region sequencing (500×) and bioinformatics analytical methods were used to identify variants in immune response genes associated with patients with ASD. A total of 4 rare variants were found to be associated with ASD, including HLA-B: p.A93G, HLA-DQB1: p.S229N, LILRB2: p.R322H, and LILRB2: c.956-4C>T. These variants were present in 44.44% (32/72) of the ASD patients and were detected in 3.74% (4/107) of the healthy controls. We expect these genetic variants will serve as new targets for the clinical genetic assessment of ASD, and our findings suggest that immune abnormalities in children with ASD may have a genetic basis.

Oxymatrine attenuates oxidized low­density lipoprotein­induced HUVEC injury by inhibiting NLRP3 inflammasome­mediated pyroptosis via the activation of the SIRT1/Nrf2 signaling pathway.

Oxymatrine, a quinolizidine alkaloid isolated from the traditional Chinese herb Sophora flavescens Aiton, has been demonstrated to exert anti­inflammatory and atherosclerotic effects, but the molecular mechanism has yet to be elucidated. Accumulating evidence indicates an important role of NLR family pyrin domain containing 3 (NLRP3) inflammasome­mediated pyroptosis in the pathogenesis of atherosclerosis. The present study was undertaken to investigate whether oxymatrine attenuates oxidized low­density lipoprotein (ox­LDL)­induced human umbilical vein endothelial cell (HUVEC) injury, an in vitro cell model of atherosclerosis, by inhibiting NLRP3 inflammasome­mediated pyroptosis, and elucidate the role of the sirtuin (SIRT)1/nuclear factor­erythroid 2­related factor 2 (Nrf2) signaling pathway in this process. Cell viability and cytotoxicity were detected by CCK­8 assay and a lactate dehydrogenase (LDH) assay kit. Cell apoptosis was detected by flow cytometry. Reactive oxygen species (ROS) generation was detected using a ROS assay kit. The malondialdehyde (MDA) content, mitochondrial membrane potential (MMP) level, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH­Px) activities were determined using commercial kits. The inflammatory cytokines levels were measured by ELISA and protein expression was monitored by western blot analysis. The results revealed that oxymatrine alleviated ox­LDL­induced cytotoxicity and apoptosis. Concurrently, oxymatrine inhibited ox­LDL­induced NLRP3 inflammasome­mediated pyroptosis in HUVECs, as evidenced by the significant decreases in the expression of NLRP3, apoptosis­associated speck­like protein containing a C­terminal caspase recruitment domain (ASC), cleaved caspase­1, interleukin (IL)­1ß and IL­18 in HUVECs. In addition, NLRP3 siRNA transfection efficiently suppressed ox­LDL­induced pyroptosis and HUVEC injury. Furthermore, oxymatrine promoted SIRT1/Nrf2 signaling pathway activation in HUVECs subjected to ox­LDL treatment, and SIRT1 deficiency induced by SIRT1 siRNA transfection abolished the protective effect of oxymatrine against ox­LDL­induced injury. SIRT1 siRNA also mitigated the oxymatrine­induced decreases in ROS generation and MDA content, and the increases in MMP as well as the activities of SOD, CAT and GSH­Px in HUVECs. Moreover, SIRT1 siRNA transfection blocked the inhibitory effect of oxymatrine on NLRP3 inflammasome­mediated pyroptosis in ox­LDL­treated HUVECs. Collectively, these results indicated that oxymatrine may attenuate ox­LDL­induced HUVEC injury by inhibiting NLRP3 inflammasome­mediated pyroptosis via activating the SIRT1/Nrf2 signaling pathway.

Neuroprotective gain of Apelin/APJ system.

Apelin is an endogenous ligand of G protein-coupled receptor APJ. In recent years, many studies have shown that the apelin/APJ system has neuroprotective properties, such as anti-inflammatory, anti-oxidative stress, anti-apoptosis, and regulating autophagy, blocking excitatory toxicity. Apelin/APJ system has been proven to play a role in various neurological diseases and may be a promising therapeutic target for nervous system diseases. In this paper, the neuroprotective properties of the apelin/APJ system and its role in neurologic disorders are reviewed. Further understanding of the pathophysiological effect and mechanism of the apelin/APJ system in the nervous system will help develop new therapeutic interventions for various neurological diseases.

Virulence factor-related gut microbiota genes and immunoglobulin A levels as novel markers for machine learning-based classification of autism spectrum disorder.

Autism spectrum disorder (ASD) is a neurodevelopmental condition for which early identification and intervention is crucial for optimum prognosis. Our previous work showed gut Immunoglobulin A (IgA) to be significantly elevated in the gut lumen of children with ASD compared to typically developing (TD) children. Gut microbiota variations have been reported in ASD, yet not much is known about virulence factor-related gut microbiota (VFGM) genes. Upon determining the VFGM genes distinguishing ASD from TD, this study is the first to utilize VFGM genes and IgA levels for a machine learning-based classification of ASD. Sequence comparisons were performed of metagenome datasets from children with ASD (n = 43) and TD children (n = 31) against genes in the virulence factor database. VFGM gene composition was associated with ASD phenotype. VFGM gene diversity was higher in children with ASD and positively correlated with IgA content. As Group B streptococcus (GBS) genes account for the highest proportion of 24 different VFGMs between ASD and TD and positively correlate with gut IgA, GBS genes were used in combination with IgA and VFGMs diversity to distinguish ASD from TD. Given that VFGM diversity, increases in IgA, and ASD-enriched VFGM genes were independent of sex and gastrointestinal symptoms, a classification method utilizing them will not pertain only to a specific subgroup of ASD. By introducing the classification value of VFGM genes and considering that VFs can be isolated in pregnant women and newborns, these findings provide a novel machine learning-based early risk identification method for ASD.

DeepBindPoc: a deep learning method to rank ligand binding pockets using molecular vector representation.

Accurate identification of ligand-binding pockets in a protein is important for structure-based drug design. In recent years, several deep learning models were developed to learn important physical-chemical and spatial information to predict ligand-binding pockets in a protein. However, ranking the native ligand binding pockets from a pool of predicted pockets is still a hard task for computational molecular biologists using a single web-based tool. Hence, we believe, by using closer to real application data set as training and by providing ligand information, an enhanced model to identify accurate pockets can be obtained. In this article, we propose a new deep learning method called DeepBindPoc for identifying and ranking ligand-binding pockets in proteins. The model is built by using information about the binding pocket and associated ligand. We take advantage of the mol2vec tool to represent both the given ligand and pocket as vectors to construct a densely fully connected layer model. During the training, important features for pocket-ligand binding are automatically extracted and high-level information is preserved appropriately. DeepBindPoc demonstrated a strong complementary advantage for the detection of native-like pockets when combined with traditional popular methods, such as fpocket and P2Rank. The proposed method is extensively tested and validated with standard procedures on multiple datasets, including a dataset with G-protein Coupled receptors. The systematic testing and validation of our method suggest that DeepBindPoc is a valuable tool to rank near-native pockets for theoretically modeled protein with unknown experimental active site but have known ligand. The DeepBindPoc model described in this article is available at GitHub (https://github.com/haiping1010/DeepBindPoc) and the webserver is available at (http://cbblab.siat.ac.cn/DeepBindPoc/index.php).

Aberrant white matter properties of the callosal tracts implicated in girls with attention-deficit/hyperactivity disorder.

Aberrant microstructure of the callosal tracts has been found in boys with attention-deficit/hyperactivity disorder (ADHD). However, it is unclear whether the previously identified white matter (WM) alterations in boys with ADHD are also present in girls with ADHD. Thus, we applied diffusion tensor imaging (DTI) to investigate WM alterations in the callosal tracts in girls with ADHD. In this study, twenty-four adolescent girls (fourteen ADHD patients and ten typically developed girls) were recruited for high-resolution DTI. Automated fiber quantification of the callosum forceps major and the callosum forceps minor was then conducted. Diffusion parameters, including fractional anisotropy (FA), mean diffusivity (MD), radial diffusivity (RD) and axial diffusivity (AD), were calculated to investigate the microstructural integrity of the two callosal tracts. We also investigated correlations between diffusion properties and clinical measurements, including scores on Conners' Parent Rating Scale, the Stroop Color-Word Test, the Wisconsin Card Sorting Test and the Continuous Performance Test, in ADHD patients and typically developed girls. Compared to typically developed adolescent girls, girls with ADHD had reduced FA values at nodes 59-70 and increased RD values at nodes 60-68 along the callosum forceps major. Lower FA values correlated with higher Hyperactivity-Impulsivity scores and lower control quotients, while higher RD values correlated with lower control quotients. This study revealed the disruption of interhemispheric connectivity, particularly across the right side of the occipital CC tract, which might be involved in visual processes in girls with ADHD. These findings enhanced current knowledge about the neuropathological basis of female ADHD.

Alterations in Gut Glutamate Metabolism Associated with Changes in Gut Microbiota Composition in Children with Autism Spectrum Disorder.

Changes in the gut microenvironment may influence the pathogenesis of autism spectrum disorders (ASD). Here, we investigated the composition of the gut microbiota and metabolites in children with ASD. Ninety-two children with ASD and 42 age-matched children exhibiting typical development (TD) were enrolled in the two-stage study. In the discovery stage, shotgun metagenomic sequencing and liquid chromatography-mass spectrometry (LC-MS) were performed simultaneously on fecal samples obtained from 43 children in the ASD group and 31 children in the TD group. Systematic bioinformatic analyses were performed to identify gut metabolites associated with altered gut microbiota composition. At the validation stage, differential metabolites were tested using LC-MS with an additional 49 and 11 children in the ASD and TD groups, respectively. Altered glutamate metabolites were found in the ASD group, along with a decline in 2-keto-glutaramic acid and an abundance of microbiota associated with glutamate metabolism. These changes in glutamate metabolism were correlated with lower levels of the highly abundant bacteria Bacteroides vulgatus and higher levels of the potentially harmful Eggerthella lenta and Clostridium botulinum. Lower gut cortisol levels have also been identified in the ASD group and associated with changes in gut microbiota glutamate metabolism. Finally, gut 2-keto-glutaramic acid was validated as a potential biomarker for ASD. The significant changes in the gut microenvironment in children with ASD may provide new insight into the cause of ASD and aid in the search for diagnostic and therapeutic approaches. IMPORTANCE Multiple lines of evidence suggest that the gut microbiota may play an important role in the pathogenesis of ASD, but the specific mechanism is still unclear. Through a comprehensive gut metagenomic and metabolome study of children with ASD, alterations in gut metabolite composition were found in children with ASD, and these alterations were linked to changes in gut microbiota composition. This may give us a deeper understanding of the role of gut microbiota in the pathogenesis of ASD.

Association of serum bilirubin in newborns affected by jaundice with gut microbiota dysbiosis.

BACKGROUND AND AIMS: Breast milk jaundice (BMJ) is common and benign, but neonatal cholestasis (NC) is rare and not benign, so early differentiation between NC and non-NC jaundice is important and may facilitate diagnosis and treatment. Gut microbiota plays an important role in enterohepatic circulation, which in turn plays an important role in the secretion of bilirubin. We aimed to determine the composition of gut microbiota in patients with NC and BMJ, and to identify the gut microbiota composition associated with NC and BMJ. METHODS: Data on age, gender, delivery, feeding mode, serum total bilirubin, direct bilirubin, and liver function were collected for NC patients, BMJ patients and healthy controls, respectively. Shotgun metagenomic sequencing and metagenome-wide association were performed. RESULTS: Forty NC patients, 16 patients affected by BMJ, and 14 healthy controls (CON) without jaundice were enrolled. A significant increase in species richness, especially Bacteroides, was found in NC patients. The abundances of potentially pathogenic species and KEGG orthologies (KOs) of virulence factor genes were positively correlated with serum bilirubin level. The abundances of nine species of Bifidobacterium and three KOs of galactose metabolism were significantly decreased in the jaundice group (NC and BMJ) and were negatively correlated with serum bilirubin level. CONCLUSIONS: The gut microbiota in NC patients is characterized by a significant increase in species richness, possibly due to the proliferation of potentially pathogenic species. Additionally, the gut microbiota in jaundice patients is characterized by a decreased abundance of Bifidobacterium. Decreased Bifidobacterium has been associated with elevated bilirubin and abnormal gut microbiota galactose metabolic pathway. Further, ten bacteria species were identified as potential biomarker of jaundice. KEY POINTS: Question Is there any alteration of gut microbiotain neonatal cholestasis patients? Does gut microbiota have any involvement in the occurrence of neonatal cholestasis or breast milk jaundice? Findings The alteration of gut microbiota in neonatal cholestasis patients mainly manifested as a significant increase in species richness and an increased abundance of potentially pathogenic species, while the main manifestation in jaundice patients was a significant decrease in Bifidobacterium which may be involved in the metabolism of bilirubin through the galactose metabolic pathway. Meaning The results suggest that an imbalance of gut microbiota exist in neonatal cholestasis and breast milk jaundice patients, primarily in the form of a substantial reduction in the abundance of Bifidobacterium, suggesting the possibility of intervention treatment for neonatal cholestasis and breast milk jaundice by supplementing probiotics.

Alteration of gut microbiota-associated epitopes in children with autism spectrum disorders.

BACKGROUND: Autism spectrum disorder (ASD) affects 1% of children and has no cure. Gastrointestinal (GI) problems are common in children with ASD, and although gut microbiota is known to play an important role in ASD through the gut-brain axis, the specific mechanism is unknown. Recent evidence suggests that gut microbiota may participate in the pathogenesis of ASD through immune- and inflammation-mediated pathways. Here, we identified potentially immunogenic epitopes derived from gut microbiota in stool samples from ASD children with and without GI problems and typically developing (TD) children. METHODS: Candidate gut microbiota-associated epitopes (MEs) were identified by blast shotgun metagenomic sequencing of fecal samples from 43 ASD children (19 with and 24 without GI involvement) and 31 sex- and age-matched typically developing (TD) children. Potentially immunogenic epitopes were screened against a predictive human Immune Epitope Database. The composition and abundance of candidate MEs were compared between the three groups of children. RESULTS: MEs identified in ASD children with GI problems were significantly more diverse than those in TD children. ME composition could discriminate between the three groups of children. We identified 34 MEs that were significantly more or less abundant in ASD children than TD children, most (29/34) of the differences in MEs were reduced in ASD and associated with abnormal gut IgA level and altered gut microbiota composition, these MEs were limited effected by clinical factors such as age, gender, and GI problems, of which eleven MEs were pathogenic microorganisms peptides with strong T or B cell response, nine MEs showed high homology to peptides from human self proteins associated with autoimmune disease occurrence, eliciting immune attack against hematopoietic stem cells and inhibition antigen binding. We also found that the abundance of five MEs were increased in ASD, including three human self proteins, gap junction alpha-1 (GJA1), paired box protein Pax-3 (PAX3) and eyes absent homolog 1 isoform 4 (EYA1) which associated with cancer, and a ME with homology to a Listeriolysin O peptide from the pathogenic bacterium Listeria monocytogenes was significantly increased in ASD children compared with TD children. CONCLUSIONS: Our findings demonstrate the abnormal of MEs composition in the gut of children with ASD, moreover, the abnormality in MEs composition was associated with abnormal gut IgA levels and altered gut microbiota composition, this abnormality also suggests that there may be abnormalities in intestinal immunity in children with ASD; In all, thirty-four MEs identified were potential biomarker of ASD, and alterations in MEs may contribute to abnormalities in gut immunity and/or homeostasis in ASD children. Further study of the MEs identified here may advance our understanding of the pathogenesis of ASD.

Mass spectrometry identification of potential biomarker proteins in the 150-kD electrophoretic band in patients with schizophrenia.

BACKGROUND: Diagnosing schizophrenia is primarily based on the presentation of defined signs and symptoms, none of which is pathognomonic for this group of syndromes. However, few significant genome-wide associations between schizophrenia and individual have detected. Protein profiling of candidate serum biomarkers in schizophrenia is therefore an area of great interest. METHODS: In the present study, we used a combination of 7% polyethylene glycol (PEG) enrichment of immune complexes and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to separate abnormal band, then analyse the band with liquid chromatography mass spectrometry (LC-MS). RESULTS: There is a special 150-kD electrophoretic band in patients with schizophrenia, bipolar disorder, or depression relative to healthy controls (each 30 samples). Analysis of the band using LC-MS resulted in the identification of 11 serum proteins whose abundance was altered between patients and controls. Among them, 8 proteins (CFH, CFB, cDNA FLJ75416, zinc finger protein 729, isoform 2 of nidogen-1, diaphanous-1, cDNA FLJ77762, and cDNA FLJ58411) were up regulated, while one protein (isoform 1 of collagen alpha-1 (II) was down regulated in patients with schizophrenia, but only zinc finger protein 729 has statistics significance (P < .05). No differences were noted with regard to thrombospondin-1 or collagen alpha-2 (I) among the 3 groups. These proteins take part in several biological functions such as focal adhesion, complement cascades, ECM-receptor interaction, and Staphylococcus aureus infection. CONCLUSIONS: The 150-kD electrophoretic band or zinc finger protein 729 may become biomarkers in patients with schizophrenia. In the future increasing sample size and function research of zinc finger protein 729 should be executed continuously.

[A multi-parameter resting-state functional magnetic resonance imaging study of brain intrinsic activity in attention deficit hyperactivity disorder children].

A great number of studies have demonstrated functional abnormalities in children with attention-deficit/hyperactivity disorder (ADHD), although conflicting results have also been reported. And few studies analyzed homotopic functional connectivity between hemispheres. In this study, resting-state functional magnetic resonance imaging (MRI) data were recorded from 45 medication-naïve ADHD children and 26 healthy controls. The regional homogeneity (ReHo), degree centrality (DC) and voxel-mirrored homotopic connectivity (VMHC) values were compared between the two groups to depict the intrinsic brain activities. We found that ADHD children exhibited significantly lower ReHo and DC values in the right middle frontal gyrus and the two values correlated with each other; moreover, lower VMHC values were found in the bilateral occipital lobes of ADHD children, which was negatively related with anxiety scores of Conners' Parent Rating Scale (CPRS-R) and positively related with completed categories of Wisconsin Card Sorting Test (WCST). Our results might suggest that less spontaneous neuronal activities of the right middle frontal gyrus and the bilateral occipital lobes in ADHD children.

A comparison of blood metal levels in autism spectrum disorder and unaffected children in Shenzhen of China and factors involved in bioaccumulation of metals.

The present study compared blood plasma metals in children with autism spectrum disorder (ASD) with those in unaffected children in Shenzhen (China). Factors associated with the metal bioaccumulation were further investigated. Thirty-four blood samples of children with ASD were collected in a local hospital (Shenzhen Children's Hospital), while those of 38 unaffected children were from a local large public kindergarten, during March to April in 2016. Metal analysis was carried out by inductively coupled plasma-optical emission spectrometry. The results showed that children with ASD had higher (P < 0.01, 0.05) Pb (ASD 31.9 µg/L, unaffected children 18.6 µg/L), Hg (3.83, and 1.09 µg/L), and Cd (0.70 and 0.26 µg/L) than unaffected children, while essential elements Zn (ASD 4552.0 µg/L, unaffected children 5118.6 µg/L), Se (61.7 and 90.6 µg/L), and Mn (13.5 and 21.4 µg/L) showed an opposite pattern. Moreover, the children exposed to passive smoking had higher (P < 0.05) Cd (passive smoking 1.08 µg/L; non-passive smoking 0.22 µg/L) than those without the exposure. Positive associations were found between levels of Hg or Pb and seafood consumption as well as body mass index (BMI). More future work is needed in order to clarify the association between metal exposure and ASD occurrence in China.

High prevalence of serum folate receptor autoantibodies in children with autism spectrum disorders.

BACKGROUND: Supplementation of folic acid by pregnant mothers is thought to lower the risk of autism spectrum disorders (ASDs) in the offspring. Folic acid is taken up by cells via receptors with high affinity for folate and reduced folic acid derivatives. However, this is blocked by the presence of folate receptor autoantibodies (FRAA). Cerebral FRAA have been detected with high frequency in children with ASDs, suggesting the existence of a link between folic acid uptake and disease aetiology. METHODS: We investigated the frequency of FRAA in serum samples from 40 children with ASDs and 42 gender- and age-matched children with typical development (TD). Serum FRAA concentrations were measured by enzyme-linked immunosorbent assay. RESULTS: We found a significant difference in the frequency of serum FRAA in the two study cohorts. Serum FRAA were present in 77.5% (31/40) of children with ASDs compared with 54.8% (23/42) of TD children (p = 0.03746, Fischer's exact test). Thus, serum FRAA are more prevalent in children with ASDs than in TD children. CONCLUSIONS: Our data suggest that children with ASDs may have defects in folic acid absorption that play a role in the onset of ASDs.

Increased stool immunoglobulin A level in children with autism spectrum disorders.

BACKGROUND: There are currently no effective treatments for the core symptoms of autism spectrum disorders (ASDs). However, alleviating gastrointestinal (GI) problems, which are prevalent in ASD patients, can significantly improve the core symptoms of autism. Previous studies have associated GI disorders in ASD patients with abnormal gut microbiota, although few disease-related microorganisms have been identified. Considering that the gut microbiome affects the intestinal immune system and the patient's behavior, and that immunoglobulin A (IgA) is the main antibody secreted by intestinal immune cells, we investigated stool IgA content as a means of understanding the gut immune status of ASD patients. The IgA level in gut can be used as factor to know the Gene x Environment interactions and diagnose of ASDs. METHODS: We enrolled 43 ASD patients and 31 gender- and age-matched healthy children. Stool IgA content was measured by enzyme-linked immunosorbent assay. RESULTS: We found that IgA levels were significantly higher in stool samples from ASD patients than from healthy children (p<0.05, Student's t test). CONCLUSIONS: This finding may suggest the presence of gut immune abnormalities in ASD patients. Further studies with larger patient and control cohorts will be necessary to determine whether stool IgA levels can be used as a biomarker for ASDs.

Diversity of Gut Microbiota Metabolic Pathways in 10 Pairs of Chinese Infant Twins.

Early colonization of gut microbiota in human gut is a complex process. It remains unclear when gut microbiota colonization occurs and how it proceeds. In order to study gut microbiota composition in human early life, the present study recruited 10 healthy pairs of twins, including five monozygotic (MZ) and five dizygotic (DZ) twin pairs, whose age ranged from 0 to 6 years old. 20 fecal samples from these twins were processed by shotgun metagenomic sequencing, and their averaged data outputs were generated as 2G per sample. We used MEGAN5 to perform taxonomic and functional annotation of the metagenomic data, and systematically analyzed those 20 samples, including Jaccard index similarity, principle component, clustering, and correlation analyses. Our findings indicated that within our study group: 1) MZ-twins share more microbes than DZ twins or non-twin pairs, 2) gut microbiota distribution is relatively stable at metabolic pathways level, 3) age represents the strongest factor that can account for variation in gut microbiota, and 4) a clear metabolic pathway shift can be observed, which speculatively occurs around the age of 1 year old. This research will serve as a base for future studies of gut microbiota-related disease research.