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*

The study was designed to investigate the peripheral anti-inflammatory effect of nicotinamide riboside(NR)in experimental autoimmune encephalomyelitis(EAE)mice and its mechanisms.Female C57BL/6 mice were induced by MOG35-55 to prepare EAE model,which then randomly divided into EAE model group and NR treatment group.Mice in EAE model group were given normal saline at a dose of 200 μl/d and mice in NR treatment group were given NR at a dose of 500 mg/kg(200 μl/d)by intragastric administration.Clinical score and body weight of mice in each group were observed and recorded.After mice were sacrificed on the 28th day after immunization,frozen sections of spleen and spinal cord were prepared and proteins of spinal cord were extracted.HE staining was used to detect peripheral inflammatory cells infiltrating spinal cord;immunofluorescence staining was used to detect the number of CD4+T cells and CD68+macrophages in spinal cord of mice;Western blot was used to detect the expression of IFN-γ and IL-1β in spinal cord of mice;immunofluorescence staining was used to detect the number of ROCK-Ⅰ+cells,TLR4+cells,p-NF-κB+cells,TNF-α+cells,IL-1β+cells,IFN-γ+cells,IL-6+cells,IL-10+cells,IL-17+cells,iNOS+cells and Arg-1+cells in spleen of mice.Data showed that compared with EAE model group,NR significantly delayed the onset time of EAE mice(P<0.05),decreased clinical score(P<0.05 or P<0.01),alleviated weight loss,prevented peripheral inflammatory cells from infiltrating spinal cord,decreased the number of CD4+T cells and CD68+macrophages in spinal cord(P<0.01),down-regulated the expression of IFN-γ and IL-1β of spinal cord(P<0.05),inhibited the expression of ROCK-Ⅰ,TLR4 and p-NF-κB in spleen of mice(P<0.01),reduced the secretion of IFN-γ,iNOS,IL-6 and other pro-inflammatory factors in spleen(P<0.05 or P<0.01),and increased the secretion of anti-inflammatory factors Arg-1 and IL-10 in spleen(P<0.05 or P<0.01).In conclusion,NR can effectively alleviate the clinical symptoms of EAE mice and significantly reduce inflammatory response of peripheral and central nervous system,and its mechanism may be related to the inhibition of Rho/ROCK signaling pathway and TLR4/NF-κB signaling pathway in spleen of EAE mice.

Objective:To explore mechanism of Fasudil reducing Aβ1-42 induced BV2 cell injury based on NLRP3 inflamma-some.Methods:BV2 cells were divided into:normal control group,Aβ stimulation group,Aβ+Fasudil intervention group,Aβ+MCC950(NLRP3 inhibitor)intervention group.Cell morphology was observed under microscope.Cell activity was determined of by CCK8.NO release was measured by Griess.NLRP3,caspase 1 and IL-18 expressions were detected by immunofluorescence staining.NLRP3,ASC,caspase 1,IL-1β and IL-18 expressions were detected by Western blot.Results:Compared with normal control group,BV2 cells in Aβ stimulation group were activated and showed amoeba-like shape,cell activity was decreased,NO production was increased,NLRP3,ASC,caspase 1,IL-1β and IL-18 expressions were increased.Fasudil intervention and MCC950 intervention inhibited cell injury induced by Aβ1-42 in which BV2 cell morphology tended to be normal,cell activity was increased,while produc-tion of NO was reduced,and NLRP3,ASC,caspase 1,IL-1β and IL-18 expressions were down-regulated,there was no significant difference between Fasudil intervention group and MCC950 intervention group.Conclusion:Fasudil may alleviate Aβ1-42 induced BV2 cell injury and inflammatory reaction by inhibiting NLRP3 inflammasome activation.

Objective:To explore the pupil size distribution of the Chinese myopic population under different mesopic conditions, and to analyze the possible influencing factors.Methods:A cross-sectional study was conducted.Two hundred and fourteen myopic patients (428 eyes) who underwent refractive surgery in Tianjin Eye Hospital from December 2018 to April 2019 were randomly selected.The patients were 17 to 45 years old, with an average age of (22.62±4.88) years old.The patients were divided into astigmatism <-1.5 D group (372 eyes) and astigmatism ≥-1.5 D group (56 eyes) according to their astigmatism measurements.The low mesopic pupil size (LMPS) (0.2 lx) was measured with the infrared Colvard pupillometer, and the high mesopic pupil size (HMPS) (6-12 lx) was obtained through the anterior Pentacam segment analyzer.The pupil size was compared between both eyes, different sexes and different astigmatism measurements.The relationship between pupil size and possible influencing factors, such as age, sex, spherical equivalent, spherical diopter, cylinder diopter, axis, mean keratometry(Km), and central cornea thickness was analyzed.This study adhered to the Declaration of Helsinki.The study protocol was approved by the Ethics Committee of Tianjin Eye Hospital (No.201912). Written informed consent was obtained from each subject or their guardians.Results:The pupil sizes measured by the Colvard pupillometer and Pentacam were (6.806±0.776)mm and (3.312±0.540)mm, respectively.The pupil size of male subjects was (6.692±0.754)mm, which was larger than (6.668±0.792)mm of females, showing a statistically significant difference ( t=2.935, P=0.004). Under the high mesopic condition, the pupil size of astigmatism ≥-1.5 D group was lower than that of astigmatism <-1.5 D group, with a statistically significant difference ( t=2.611, P=0.009). Under the low mesopic condition, pupil size was negatively correlated with age and Km ( r=-0.213, -0.210; both at P<0.001). Under the high mesopic condition, pupil size was weakly positively correlated with cylinder power ( r=0.124, P=0.010) and was weakly negatively correlated with Km ( r=-0.142, P=0.003). The multiple linear regression analysis revealed that the LMPS=0.659×HMPS-0.019×age-0.084×Km+ 8.662.About 28% of pupil size under low mesopic conditions could be predicted by Pentacam.LMPS of ≤7 mm could be better predicted when the results were below 3.6 mm. Conclusions:Age and corneal curvature are influencing factors of mesopic pupil size.Older people with steep curvature have a smaller pupil.At high mesopic conditions, astigmatism affects pupil size.Pentacam measurements can predict LMPS to some degree but are not a substitute for dark-adapted pupil diameter.

Objective:To investigate the effect of Superfine Cordyceps militarisv Powder on the immune functions of the mice,and to provide basis for improving the utilizable ratio of Cordyceps militaris.Methods:A total of 40 mice were randomly divided into negative control group,low dose (0.166 5 g·kg-1),middle dose (0.333 3 g·kg-1),and high dose (0.999 9 g·kg-1) of Superfine Cordyceps militaris Powder groups,and there were 10 mice in each group.The mice in different doses of Superfine Cordyceps militaris Powder groups were administered with the Superfine Cordyceps militaris Powder for 30 d by intragastric infusion respectively,whereas,the mice in negative control group were administered with the same volume of water for 30 d by intragastric infusion.The body weights,the spleen indexes and thymus indexes of the mice in various groups were measured;the lymphocyte transformation abilities of the mice in various groups were observed by lymphocyte transformation experiment;the levels of delayed-type hypersensitivity reaction of the mice were examined with toe incrassation;the number of antibody forming cells,phagocytic rate and phagocytic index of chicken erythrocytes phagocytized by peritoneal macrophages of the mice were detected.Results:Compared with negative control group,the body weights,the spleen indexes a nd thymus indexes of the mice in different doses of Superfine Cordyceps militaris Powder groups had no significant differences (P>0.05).The lymphocyte transformation abilities of the mice in middle and high doses of Superfine Cordyceps militarisPowder groups were higher than that in negative control group(P<0.05).The levels of delayed-type hypersensitivity reaction of the mice in middle and high doses of Superfine Cordyceps militaris Powder groups were higher than those in low dose of Superfine Cordyceps militaris powder group and negative control group(P<0.05).The numbers of antibody forming cells of the mice in low,middle and high doses of Superfine Cordyceps militaris Powder groups were higher than that in negative control group(P<0.05).The phagocytic rates and phagocytic indexes of chicken erythrocytes phagocytized by peritoneal macrophage of the mice in low,middle and high doses of Superfine Cordyceps militaris Powder groups were higher than that in negative control group(P<0.05).Conclusion:Superfine Cordyceps militaris Powder can strengthen the immune functions of the mice,and the recommended doses are 0.333 3 and 0.999 9 g·kg-1.