Microbial communities such as those residing in the human gut are highly diverse and complex, and many with important implications for health and diseases. The effects and functions of these microbial communities are determined not only by their species compositions and diversities but also by the dynamic intra- and inter-cellular states at the transcriptional level. Powerful and scalable technologies capable of acquiring single-microbe-resolution RNA sequencing information in order to achieve a comprehensive understanding of complex microbial communities together with their hosts are therefore utterly needed. Here we report the development and utilization of a droplet-based smRNA-seq (single-microbe RNA sequencing) method capable of identifying large species varieties in human samples, which we name smRandom-seq2. Together with a triple-module computational pipeline designed for the bacteria and bacteriophage sequencing data by smRandom-seq2 in four human gut samples, we established a single-cell level bacterial transcriptional landscape of human gut microbiome, which included 29,742 single microbes and 329 unique species. Distinct adaptive response states among species in Prevotella and Roseburia genera and intrinsic adaptive strategy heterogeneity in Phascolarctobacterium succinatutens were uncovered. Additionally, we identified hundreds of novel host-phage transcriptional activity associations in the human gut microbiome. Our results indicated that smRandom-seq2 is a high-throughput and high-resolution smRNA-seq technique that is highly adaptable to complex microbial communities in real-world situations and promises new perspectives in the understanding of human microbiomes.
Humans ; Gastrointestinal Microbiome/genetics* ; Bacteriophages/physiology* ; High-Throughput Nucleotide Sequencing ; Sequence Analysis, RNA/methods* ; Bacteria/virology*

Objective:To investigate the effects of repetitive transcranial magnetic stimulation(rTMS) on the core symptoms, brain functional connectivity and activation in children with attention deficit hyperactivity disorder (ADHD) using functional near-infrared spectroscopy (fNIRS).Methods:From September 2022 to March 2024, a total of 35 children with ADHD were selected as research subjects and they were randomly divided into observation group ( n=17) and control group ( n=18). The control group received conventional rehabilitation therapy, while the observation group received rTMS therapy in addition to the conventional therapy. Both groups were treated every other day, with each course of treatment lasting four weeks, and a total of three courses of treatment were administered consecutively. The clinical symptoms of the children with ADHD were assessed using Swanson, Nolan, and Pelham-Ⅳ(SNAP-Ⅳ) before and after treatment. fNIRS was used to detect the relative concentration changes of oxyhemoglobin (HbO 2) and deoxyhemoglobin (HbR) in the prefrontal cortex under resting-state and Go/Nogo task conditions before and after treatment. Statistical analysis was performed using SPSS 26.0 software. Paired sample t-test were used for within-group comparisons, and independent sample t-test were used for between-group comparisons. Results:(1) After treatment, the scores for inattention, hyperactivity/impulsivity and oppositional defiant behavior in the two groups were significantly lower than before treatment ( t=3.51-18.86, all P<0.05). The scores for inattention, hyperactivity/impulsivity and oppositional defiant behavior in the observation group were significantly lower than those in the control group ( t=2.21, 2.03, 2.39, all P<0.05). (2) After treatment, the functional connectivity strength between all regions of interest in both groups was significantly higher than before treatment ( t=3.53-37.90, all P<0.05). The functional connectivity strength of the left dorsolateral prefrontal cortex (0.25±0.03, 0.21±0.03), right dorsolateral prefrontal cortex (0.12±0.02, 0.09±0.02), left medial prefrontal cortex (0.13±0.02, 0.10±0.01) and right medial prefrontal cortex (0.31±0.04, 0.24±0.06) in the observation group was significantly higher than those in the control group (all P<0.05). (3) In the Go/Nogo task, after treatment, the average HbO 2 concentrations in the left dorsolateral prefrontal cortex, right dorsolateral prefrontal cortex, left medial prefrontal cortex, right medial prefrontal cortex, left temporal lobe, and right temporal lobe in both groups were all higher than before treatment (all P<0.05). After treatment, the average HbO 2 concentrations in the left dorsolateral prefrontal cortex, right dorsolateral prefrontal cortex, left medial prefrontal cortex and right medial prefrontal cortex of the observation group were significantly higher than those of the control group (all P<0.05). Conclusion:rTMS therapy can improve the core symptoms of children with ADHD, which may be related to the strength of brain functional connectivity and activation of ADHD brain function by rTMS.

Objective To analyze the symptomatic improvement effects of vitamin D in children with autism spectrum disorder(ASD)based on the microbiota-gut-brain axis.Methods Seventy-two children with ASD were randomly divided into an observation group and a control group,with 36 cases in each group.Three cases dropped out in the control group.The observation group received 1200 IU/day of vitamin D supplementation in addition to conventional rehabilitation training,while the control group received only conventional rehabilitation training.The intervention lasted for 12 weeks.Assessments were conducted before and after the intervention using the childhood autism rating scale(CARS),autism behavior checklist(ABC),and repetitive behavior scale-revised(RBS-R).Resting-state functional connectivity of the brain was measured using near-infrared functional imaging,and serum levels of 25(OH)D3,inflammatory cytokines,and gut microbiota were analyzed.The differences in these indicators before and after the intervention were compared between the two groups to evaluate clinical efficacy.Results The between-group differences in pre-and post-intervention changes showed that the observation group had significantly greater improvements than the control group in the following measures:CARS scores(-5.92±1.40 vs.-2.55±1.43),RBS-R scores(-5.99±1.01 vs.-3.10±1.47),resting-state brain functional connectivity(0.19±0.15 vs.0.10±0.18),serum 25(OH)D3 levels[(34.89±8.18)ng/mL vs.(0.68±6.73)ng/mL],serum interleukin-6(IL-6)levels[(-6.60±6.07)pg/mL vs.(-0.74±9.45)pg/mL],IL-1β levels[(-2.56±1.33)pg/mL vs.(-0.04±2.13)pg/mL],and tumor necrosis factor-α(TNF-α)levels[(-4.09±3.85)pg/mL vs.(0.21±4.05)pg/mL](P<0.05).Post-intervention,significant differences in gut microbial β-diversity were observed between the two groups(R2=0.030,P=0.040,Adonis).LEfSe analysis revealed that the observation group exhibited enrichment in Clostridia(LDA=4.747,P=0.003),Clostridiales(LDA=4.747,P=0.003),Clostridiaceae(LDA=3.476,P=0.001),Lachnospiraceae(LDA=4.709,P=0.004),Odoribacteraceae(LDA=3.458,P=0.027),Odoribacter(LDA=3.458,P=0.027),Burkholderiales(LDA=3.339,P=0.038),Firmicutes(LDA=4.764,P=0.003),and Betaproteobacteria(LDA=3.338,P=0.037).Conclusion Vitamin D supplementation can modulate gut microbial diversity in children with ASD,significantly influence the abundance of specific gut microbiota,reduce systemic inflammatory cytokines,enhance brain functional connectivity,and alleviate clinical symptoms of ASD.

Objective:To investigate the effects of repetitive transcranial magnetic stimulation(rTMS) on the core symptoms, brain functional connectivity and activation in children with attention deficit hyperactivity disorder (ADHD) using functional near-infrared spectroscopy (fNIRS).Methods:From September 2022 to March 2024, a total of 35 children with ADHD were selected as research subjects and they were randomly divided into observation group ( n=17) and control group ( n=18). The control group received conventional rehabilitation therapy, while the observation group received rTMS therapy in addition to the conventional therapy. Both groups were treated every other day, with each course of treatment lasting four weeks, and a total of three courses of treatment were administered consecutively. The clinical symptoms of the children with ADHD were assessed using Swanson, Nolan, and Pelham-Ⅳ(SNAP-Ⅳ) before and after treatment. fNIRS was used to detect the relative concentration changes of oxyhemoglobin (HbO 2) and deoxyhemoglobin (HbR) in the prefrontal cortex under resting-state and Go/Nogo task conditions before and after treatment. Statistical analysis was performed using SPSS 26.0 software. Paired sample t-test were used for within-group comparisons, and independent sample t-test were used for between-group comparisons. Results:(1) After treatment, the scores for inattention, hyperactivity/impulsivity and oppositional defiant behavior in the two groups were significantly lower than before treatment ( t=3.51-18.86, all P<0.05). The scores for inattention, hyperactivity/impulsivity and oppositional defiant behavior in the observation group were significantly lower than those in the control group ( t=2.21, 2.03, 2.39, all P<0.05). (2) After treatment, the functional connectivity strength between all regions of interest in both groups was significantly higher than before treatment ( t=3.53-37.90, all P<0.05). The functional connectivity strength of the left dorsolateral prefrontal cortex (0.25±0.03, 0.21±0.03), right dorsolateral prefrontal cortex (0.12±0.02, 0.09±0.02), left medial prefrontal cortex (0.13±0.02, 0.10±0.01) and right medial prefrontal cortex (0.31±0.04, 0.24±0.06) in the observation group was significantly higher than those in the control group (all P<0.05). (3) In the Go/Nogo task, after treatment, the average HbO 2 concentrations in the left dorsolateral prefrontal cortex, right dorsolateral prefrontal cortex, left medial prefrontal cortex, right medial prefrontal cortex, left temporal lobe, and right temporal lobe in both groups were all higher than before treatment (all P<0.05). After treatment, the average HbO 2 concentrations in the left dorsolateral prefrontal cortex, right dorsolateral prefrontal cortex, left medial prefrontal cortex and right medial prefrontal cortex of the observation group were significantly higher than those of the control group (all P<0.05). Conclusion:rTMS therapy can improve the core symptoms of children with ADHD, which may be related to the strength of brain functional connectivity and activation of ADHD brain function by rTMS.

Objective To analyze the symptomatic improvement effects of vitamin D in children with autism spectrum disorder(ASD)based on the microbiota-gut-brain axis.Methods Seventy-two children with ASD were randomly divided into an observation group and a control group,with 36 cases in each group.Three cases dropped out in the control group.The observation group received 1200 IU/day of vitamin D supplementation in addition to conventional rehabilitation training,while the control group received only conventional rehabilitation training.The intervention lasted for 12 weeks.Assessments were conducted before and after the intervention using the childhood autism rating scale(CARS),autism behavior checklist(ABC),and repetitive behavior scale-revised(RBS-R).Resting-state functional connectivity of the brain was measured using near-infrared functional imaging,and serum levels of 25(OH)D3,inflammatory cytokines,and gut microbiota were analyzed.The differences in these indicators before and after the intervention were compared between the two groups to evaluate clinical efficacy.Results The between-group differences in pre-and post-intervention changes showed that the observation group had significantly greater improvements than the control group in the following measures:CARS scores(-5.92±1.40 vs.-2.55±1.43),RBS-R scores(-5.99±1.01 vs.-3.10±1.47),resting-state brain functional connectivity(0.19±0.15 vs.0.10±0.18),serum 25(OH)D3 levels[(34.89±8.18)ng/mL vs.(0.68±6.73)ng/mL],serum interleukin-6(IL-6)levels[(-6.60±6.07)pg/mL vs.(-0.74±9.45)pg/mL],IL-1β levels[(-2.56±1.33)pg/mL vs.(-0.04±2.13)pg/mL],and tumor necrosis factor-α(TNF-α)levels[(-4.09±3.85)pg/mL vs.(0.21±4.05)pg/mL](P<0.05).Post-intervention,significant differences in gut microbial β-diversity were observed between the two groups(R2=0.030,P=0.040,Adonis).LEfSe analysis revealed that the observation group exhibited enrichment in Clostridia(LDA=4.747,P=0.003),Clostridiales(LDA=4.747,P=0.003),Clostridiaceae(LDA=3.476,P=0.001),Lachnospiraceae(LDA=4.709,P=0.004),Odoribacteraceae(LDA=3.458,P=0.027),Odoribacter(LDA=3.458,P=0.027),Burkholderiales(LDA=3.339,P=0.038),Firmicutes(LDA=4.764,P=0.003),and Betaproteobacteria(LDA=3.338,P=0.037).Conclusion Vitamin D supplementation can modulate gut microbial diversity in children with ASD,significantly influence the abundance of specific gut microbiota,reduce systemic inflammatory cytokines,enhance brain functional connectivity,and alleviate clinical symptoms of ASD.

Objective To investigate the effect of dioscin on uric acid(UA)-induced oxidative stress injury of human renal tubular epithelial cells(HK-2)and its molecular mechanism.Methods HK-2 cells were cultured and divided into four groups:blank group(normal group),model group(uric acid-stimulation modeling),condition control group(UA+DMSO)and dioscin group(UA+dioscin).Oxidative stress injury model was induced by UA in HK-2 cells.Cells viability was detected by CCK-8.ROS level was detected by flow cytometry.Real-time PCR was used to detect the expressions of glycogen synthase kinase 3β(GSK3β),nuclear factor erythroid 2-related factor 2(Nrf2)and heme oxygenase 1(HO-1)at mRNA level,and Western Blot was used to detect the expressions of phosphorylated glycogen synthesis kinase 3β(p-GSK3β),GSK3β,Nrf2 and HO-1 at protein level.Results After stimulation by UA,HK-2 cells viability was obviously decreased,and ROS level was significantly increased(all P＜0.001).When treated with dioscin,HK-2 cells viability was obviously increased,and the ROS level of HK-2 cells was significantly decreased(all P＜0.001).The expressions of Nrf2 and HO-1 decreased at the protein and mRNA levels after stimulation with UA.But the expressions of Nrf2 and HO-1 significantly increased after treated with dioscin(all P＜0.001).Compared with the blank group,the p-GSK3β/GSK3β ratio in the model group decreased significantly at the protein level,but the p-GSK3β/GSK3β ratio increased after treated with dioscin(all P＜0.001).Conclusion Dioscin can alleviate UA-induced oxidative stress injury in HK-2 cells.The mechanism might be that dioscin can promote phosphorylation of GSK3β,and activate Nrf2/HO-1 pathway.

BACKGROUND:The increase in multi-drug resistant bacterial infections has become a major problem in modern healthcare due to the development of bacterial resistance to antibiotics and the development of new antibacterial alternative drug materials is of great importance. OBJECTIVE:To synthesize and perform a series of characterization of a CeO2 nanoenzyme to investigate its biocompatibility and antibacterial properties against Escherichia coli. METHODS:CeO2 nanoenzymes were synthesized using a hydrothermal method.The morphology,product composition,and chemical composition were analyzed using characterization methods such as X-ray diffraction,X-ray photoelectron spectroscopy,Fourier infrared analysis,Raman spectroscopy,scanning electron microscopy,and transmission electron microscopy.The peroxide-mimetic enzyme activity of CeO2 nanoenzymes was characterized using TMB color development assay.The toxic effect of CeO2 nanoenzymes at different concentrations(10,25,and 50 μg/mL)on mouse fibroblast L929 cells was evaluated using the CCK-8 assay.The antibacterial properties of CeO2 nanoenzymes against Escherichia coli under different conditions were evaluated using the plate coating method.Changes in intra-bacterial reactive oxygen species after treatment with different conditions were detected using a reactive oxygen species detection kit. RESULTS AND CONCLUSION:(1)The morphology of the synthesized CeO2 nanoparticles was rod-shaped,with Ce3+ accounting for 29.87%of the total Ce3+/Ce4+ and an average grain size of 7.4 nm.In a slightly acidic environment containing TMB and pH=5.5,CeO2 nanoenzymes mixed with H2O2 showed excellent peroxidase activity,but did not show peroxidase simulated activity at pH=7.4.(2)There was no statistically significant difference in the toxic effects of CeO2 nanoparticles at various mass concentrations on mouse fibroblast L929 cells.(3)In a slightly acidic environment at pH 5.5,Escherichia coli was inhibited to a certain extent in the presence of CeO2 nanoenzyme alone at a concentration of 10 μg/mL,with a decrease in CFU results of about 0.5 log(P＜0.01);in a slightly acidic environment containing 50 μmol/L H2O2,CeO2 nanoenzyme showed excellent antibacterial effects against Escherichia coli,with a decrease in Escherichia coli CFU results of by about 1.5 log(P＜0.001).After CeO2 nanoenzymes interacted with Escherichia coli,the level of reactive oxygen species in Escherichia coli increased(P＜0.05);after CeO2 nanoenzymes interacted with Escherichia coli together with H2O2,the level of reactive oxygen species in Escherichia coli increased significantly(P＜0.001).(4)The results show that the CeO2 nanoenzymes have good biocompatibility,are inherently antibacterial,and can exhibit peroxidase activity in a slightly acidic environment containing low concentrations of H2O2,and generate reactive oxygen species to kill bacteria,thus showing excellent antibacterial effects.

Objective To investigate the expression levels of serum G protein-coupled receptor-associated sorting protein 1(GASP-1),lipocalin-2(LCN2)and targeting protein for Xenopus kinesin-like protein 2(TPX2)in lung cancer patients,and their correlations with tumor-node-metastasis classification(TNM)pathological stage and prognosis.Methods A total of 92 patients with lung cancer who were admitted to General Hospital of Southern Theatre Command from April 2019 to May 2022 were selected as observation group.Meanwhile,92 healthy subjects were selected as control group.Peripheral venous blood samples of the two groups were collected to detect serum GASP-1,LCN2 and TPX2 levels.Serum GASP-1,LCN2 and TPX2 levels of lung cancer patients with different TNM stages were analyzed.According to the prognosis,the patients were divided into good prognosis group and poor prognosis group.Serum GASP-1,LCN2 and TPX2 levels were compared between the two groups.The correlations of serum GASP-1,LCN2 and TPX2 levels with TNM stage and prognosis in patients with lung cancer were investigated.Results The serum GASP-1,LCN2 and TPX2 levels in the observation group were significantly higher than those in the control group(P＜0.05).The serum levels of GASP-1,LCN2 and TPX2 in patients with TNM stage Ⅲ and Ⅳ were significantly higher than those in patients with TNM stage Ⅰand Ⅱ(P＜0.05).The levels of serum GASP-1,LCN2 and TPX2 in patients with TNM stage Ⅳ were significantly higher than those in patients with TNM stage Ⅲ(P＜0.05).Serum GASP-1,LCN2 and TPX2 levels in the good prognosis group were significantly lower than those in the poor prognosis group(P＜0.05).Serum GASP-1,LCN2 and TPX2 levels were positively correlated with TNM stage before treatment(P＜0.05),but were negatively correlated with the prognosis after treatment(P＜0.05).Conclusion Serum levels of GASP-1,LCN2 and TPX2 may be related to the TNM pathological stages of lung cancer patients.The detection of the above serum indicators after treatment can reflect the prognosis of patients to a certain extent,and provide reference for clinical practice.

Objective To explore the features of frontotemporal lobes'resting-state functional connectivity(rsFC)in preschool children with autism spectrum disorders(ASD)based on functional near-infrared spectroscopy(fNIRS)and to explore the possible neurological markers for early identification of ASD.Methods Sixty-three preschool ASD children and 72 typical development(TD)children were enrolled.Selected bilateral dorsolateral prefrontal cortex(DLPFC),bilateral premotor cortex(PMC),and bilateral temporal lobe(TL)cortex as the regions of interest(ROI).Changes of Oxyhemoglobin in the 6 ROIs in resting-state were measured by using functional near-infrared spectroscopy(fNIRS).Compared the frontotemporal rsFC strength and calculate the laterality index(LI)between two groups.Results Compared with the TD group,rsFC strength was significantly lower in the ASD group(P<0.05),and the differences existed mainly within the left ROIs(0.21±0.11 vs.0.32±0.18),right ROIs(0.16±0.16 vs.0.30±0.14),bilateral DLPFCs(0.20±0.14 vs.0.39±0.17;0.15±0.13 vs.0.36±0.13),bilateral TLs(0.15±0.14 vs.0.28±0.17;0.14±0.15 vs.0.31±0.17),and between the 10 groups of ROIs-ROIs(including right DLPFC-left DLPFC,right DLPFC-right PMC,right DLPFC-left PMC,right DLPFC-right TL,right DLPFC-left TL,left DLPFC-right PMC,left DLPFC-left PMC,left DLPFC-right TL,left DLPFC-left TL,right TL-left TL).There were a significant differences in the rsFC's laterality index of DLPFC and whole-brain between the two groups(t=2.002,P=0.047;t=3.003,P=0.003),and the ASD group showed left-lateralized connectivity.Conclusion Frontotemporal lobe's resting-state functional connectivity is abnormal in preschool children with ASD,characterized by low short-range functional connectivity of bilateral DLPFCs and TLs,low long-range functional connectivity associated with DLPFCs,and left-lateralized connectivity.

Objective:To identify the pathogenic gene mutations in a family with Leber congenital amaurosis (LCA).Methods:In October 2018, 1 patient and 3 normal family members from a LCA family was enrolled in this retrospective study. Detailed medical history of proband was obtained and fixation test, cycloplegic refraction, slit-lamp, fundus color photography and full-field ERG were performed. And other family members underwent BCVA, refraction slit-lamp, fundus biomicroscopy with the slit lamp, fundus color photography and full-field ERG. The family was investigated with a specific hereditary eye disease enrichment panel which contained 441 known pathogenic genes and based on targeted exome capture technology first to indentify the potential pathogenic genes and mutations. Then the potential pathogenic mutations were conformed by Sanger sequencing. Finally, the results were analyzed via bioinformatics analysis.Results:The proband showed no trace object from childhood, but had obvious photophobia and nystagmus. No positive changes were found in the anterior segment, vitreous and retina in both eyes. Both cone and rod system function decreased significantly in full-field ERG in both eyes. Gene tests showed the proband carried both RPGRIP1 c.1635dupA and c.3565C> T, which composited a heterozygous mutation. Bioinformatics analysis showed RPGRIP1 c.1635dupA was a pathogenic mutation, and RPGRIP1 c.3565C> T which was a novel potential pathogenic mutation in LCA.Conclusion:The compound heterozygous mutation, c.1635dupA and c.3565C> T in RPGRIP1 may be responsible for the pathogenesis in this Chinese Han LCA pedigree.