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 identify the biomarkers for early rheumatoid arthritis(RA)diagnosis and explore the possible immune regulatory mechanisms.Methods The differentially expressed genesin RA were screened and functionally annotated using the limma,RRA,batch correction,and clusterProfiler.The protein-protein interaction network was retrieved from the STRING database,and Cytoscape 3.8.0 and GeneMANIA were used to select the key genes and predicting their interaction mechanisms.ROC curves was used to validate the accuracy of diagnostic models based on the key genes.The disease-specific immune cells were selected via machine learning,and their correlation with the key genes were analyzed using Corrplot package.Biological functions of the key genes were explored using GSEA method.The expression of STAT1 was investigated in the synovial tissue of rats with collagen-induced arthritis(CIA).Results We identified 9 core key genes in RA(CD3G,CD8A,SYK,LCK,IL2RG,STAT1,CCR5,ITGB2,and ITGAL),which regulate synovial inflammation primarily through cytokines-related pathways.ROC curve analysis showed a high predictive accuracy of the 9 core genes,among which STAT1 had the highest AUC(0.909).Correlation analysis revealed strong correlations of CD3G,ITGAL,LCK,CD8A,and STAT1 with disease-specific immune cells,and STAT1 showed the strongest correlation with M1-type macrophages(R=0.68,P=2.9e-08).The synovial tissues of the ankle joints of CIA rats showed high expressions of STAT1 and p-STAT1 with significant differential expression of STAT1 between the nucleus and the cytoplasm of the synovial fibroblasts.The protein expressions of p-STAT1 and STAT1 in the cell nuclei were significantly reduced after treatment.Conclusion CD3G,CD8A,SYK,LCK,IL2RG,STAT1,CCR5,ITGB2,and ITGAL may serve as biomarkers for early diagnosis of RA.Gene-immune cell pathways such as CD3G/CD8A/LCK-γδ T cells,ITGAL-Tfh cells,and STAT1-M1-type macrophages may be closely related with the development of RA.

Objective To identify the biomarkers for early rheumatoid arthritis(RA)diagnosis and explore the possible immune regulatory mechanisms.Methods The differentially expressed genesin RA were screened and functionally annotated using the limma,RRA,batch correction,and clusterProfiler.The protein-protein interaction network was retrieved from the STRING database,and Cytoscape 3.8.0 and GeneMANIA were used to select the key genes and predicting their interaction mechanisms.ROC curves was used to validate the accuracy of diagnostic models based on the key genes.The disease-specific immune cells were selected via machine learning,and their correlation with the key genes were analyzed using Corrplot package.Biological functions of the key genes were explored using GSEA method.The expression of STAT1 was investigated in the synovial tissue of rats with collagen-induced arthritis(CIA).Results We identified 9 core key genes in RA(CD3G,CD8A,SYK,LCK,IL2RG,STAT1,CCR5,ITGB2,and ITGAL),which regulate synovial inflammation primarily through cytokines-related pathways.ROC curve analysis showed a high predictive accuracy of the 9 core genes,among which STAT1 had the highest AUC(0.909).Correlation analysis revealed strong correlations of CD3G,ITGAL,LCK,CD8A,and STAT1 with disease-specific immune cells,and STAT1 showed the strongest correlation with M1-type macrophages(R=0.68,P=2.9e-08).The synovial tissues of the ankle joints of CIA rats showed high expressions of STAT1 and p-STAT1 with significant differential expression of STAT1 between the nucleus and the cytoplasm of the synovial fibroblasts.The protein expressions of p-STAT1 and STAT1 in the cell nuclei were significantly reduced after treatment.Conclusion CD3G,CD8A,SYK,LCK,IL2RG,STAT1,CCR5,ITGB2,and ITGAL may serve as biomarkers for early diagnosis of RA.Gene-immune cell pathways such as CD3G/CD8A/LCK-γδ T cells,ITGAL-Tfh cells,and STAT1-M1-type macrophages may be closely related with the development of RA.

Pulmonary fibrosis(PF)is a chronic progressive end-stage lung disease.However,the mechanisms un-derlying the progression of this disease remain elusive.Presently,clinically employed drugs are scarce for the treatment of PF.Hence,there is an urgent need for developing novel drugs to address such diseases.Our study found for the first time that a natural source of Prismatomeris connata Y.Z.Ruan(Huang Gen,HG)ethyl acetate extract(HG-2)had a significant anti-PF effect by inhibiting the expression of the transforming growth factor beta 1/suppressor of mothers against decapentaplegic(TGF-β1/Smad)pathway.Network pharmacological analysis suggested that HG-2 had effects on tyrosine kinase phosphorylation,cellular response to reactive oxygen species,and extracellular matrix(ECM)disassembly.Moreover,mass spec-trometry imaging(MSI)was used to visualize the heterogeneous distribution of endogenous metabolites in lung tissue and reveal the anti-PF metabolic mechanism of HG-2,which was related to arginine biosyn-thesis and alanine,asparate and glutamate metabolism,the downregulation of arachidonic acid meta-bolism,and the upregulation of glycerophospholipid metabolism.In conclusion,we elaborated on the relationship between metabolite distribution and the progression of PF,constructed the regulatory metabolic network of HG-2,and discovered the multi-target therapeutic effect of HG-2,which might be conducive to the development of new drugs for PF.

ObjectiveTo explore the possible peripheral analgesic mechanism of electroacupuncture （EA） at promimal and distal acupoints in treatment of myofascial pain syndrome （MPS）. MethodsTwenty-four SD rats were randomly divided into blank group， model group， proximal group， and distal group， with six rats in each group. MPS model was prepared by “strike combined with centrifugal exercise” in all groups except for the blank group. After modeling， the rats in the proximal group received EA at the local myofascial trigger points （MTrPs）， namely the Ashi points， with dilatational waves of frequency of 2/100 HZ and voltage of 2-4 V， current intensity depending on a slight trembling of the left lower limbs， once a day， 15min each time，for 14 days. The rats in the distal group received EA at “Yanglingquan” （GB 34） and “Yinlingquan” （SP 9）， with the same operations as the proximal group. The rats in the blank group and the model group were only grasped and hedged， without other interventions. After intervention， the paw withdrawl mechanical threshold （PWMT） was measured， and variability between the left and right hind paws was calculated. Musculoskeletal ultrasound imaging and electromyography monitoring were performed on the left lower extremity vastus medialis. The morphological changes of vastus medialis muscle of the left lower extremity were observed by HE staining. The positive expression of substance P （SP）， calcitonin gene-related peptide （CGRP）， CD68 and CD206 in muscle tissue was detected by immunohistochemistry. Abdominal aortic serum interleukin-1β （IL-1β）， interleukin-6 （IL-6）， tumor necrosis factor-α （TNF-α）， interleukin-10 （IL-10）， and interleukin-8 （interleukin-8） were detected by ELISA. ResultsCompared to those in the blank group， the fibers of the vastus medial muscle of the rats in the model group were broken and distorted with thickness in variation， and the myofascia was broken， with fibrillation potential， enlarged muscle cells， inward moved nucleus， and widened muscle space； the variability of PWMT between the left and right hind paws significantly increased， as well as the levels of SP， CGRP， CD68， and CD206 in the vastus medialis muscle （P＜0.01）， and the serum IL-8 and TNF-αlevels were significantly elevated （P＜0.05 or P＜0.01）. Compared to those in the model group， the muscle fibers in the proximal and distal group were complete in shape and arranged in an orderly manner， with continued non-broken myofascia， regular shape of muscle cells， and significantly reduced level of IL-8 （P＜0.01）； the amplitude and frequency of spontaneous discharge in the proximal group significantly decreased， as well as the variability of PWMT between the left and right hind paws， and the levels of SP， CGRP， and CD68 in the vastus medialis muscle， while the CD206 level increased significantly （P＜0.05 or P＜0.01 ）； there was complex discharges in the distal group， with significantly decreased level of CD68 in the vastus medialis muscle and increased level of CD206 （P＜0.01）. Compared to the proximal group， the level of IL-8 in the distal group was significantly higher （P＜0.05）. ConclusionsEA at proximal acupoints can significantly improve the pain threshold and local muscle tissue morpho-logy in rats， and its mechanism may be related to reducing the levels of pain-causing substances and related inflammatory factors and promoting the polarization of macrophages. The analgesic effect of EA at distal acupoints is not obvious， and the mechanism is still unclear.

Objective:To explore the curative effect of surgical treatment for Crohn′s disease (CD), to investigate the timing of surgical intervention and the choice of surgical methods.Methods:From January 1, 2016 to August 31, 2020, at Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, the clinical data of 123 patients with CD and receiving surgical treatment were retrospectively analyzed, which included the type of lesion, the location of lesion, clinical manifestation, surgical method, preoperative inflammatory and nutritional indicators, postoperative recovery of digestive tract function, and the development and treatment of postoperative complications. CD was diagnosed according to Consensus opinion on diagnosis and treatment of inflammatory bowel disease ( Beijing 2018). Patient was classitied according to the Montreal Classification. Postoperative complications were graded according to the Clavien-Dindo Criteria. Mann-Whitney U test was used for statistical analysis. Results:Among 123 patients, according to the Montreal classification, two cases (1.6%) were diagnosed at ≤16 years old (type A1), 66 cases (53.7%) were diagnosed at 17 to 40 years old (type A2), and 55 cases (44.7%) were diagnosed at >40 years old (type A3). The lesions were 52 cases (42.3%) of terminal ileum (L1) type, 20 cases (16.3%) of colon (L2) type, and 51 cases (41.5%) of ileocolon (L3) type. Four cases (3.2%) were non-stenosis and non-penetrating (B1) type, 87 cases (70.7%) were stenosis (B2) type, and 32 cases (26.0%) were penetrating (B3) type. Eighteen patients (14.6%) underwent emergency surgery due to complete intestinal obstruction (10 cases), gastrointestinal perforation (five cases), gastrointestinal bleeding (two cases), and rectovesical fistula complicated with septic shock (one case). One hundred and five patients (85.4%) received selective surgery due to poor conservative treatment effects. 51 cases (41.5%) underwent traditional open surgery and 72 cases (58.5%) underwent laparoscopic surgery. Nineteen patients (15.4%) received temporary or permanent ostomy. The preoperative C reactive protein level of patients with emergency surgery was higher than that of patients undergoing selective surgery ((39.23±24.13) mg/L vs. (11.48±2.68) mg/L), while the levels of plasma albumin (ALB) and pre-ALB were lower than those of patients receiving selective surgery ((29.90±10.60) g/L vs. (38.38±8.30) g/L, (146.00±125.49) mg/L vs. (209.06±61.19) mg/L), and the differences were statistically significant ( Z=9.603, 8.754 and 7.111, all P<0.01). During the follow-up, a total of 23 cases (18.7%) developed postoperative complications, including one case of postoperative intra-abdominal hemorrhage and underwent re-operation (Clavien-Dindo grade Ⅲ complication); four cases of anastomotic leakage after operation; six cases of postoperative paralytic ileus; 11 cases of surgical site infection, all of which were Clavien-Dindo grade Ⅱ complications, and one case of deep venous thrombosis of lower extremity. No patient with severe intraoperative complication was observed, and no patients died during the operation or hospitalization. The postoperative exhaust time of patients was (3.2±1.4) d, the time of open fluid diet was (5.8±0.8) d, the length of hospital stay was (18.0±14.1) d, and the length of postoperative hospital stay was (11.2±8.8) d. Conclusions:The concept of multidisciplinary collaboration should be emphasized in the treatment of CD. Surgical treatment can effectively control the complications and improve the quality of life of patients, but the timing of operation and the choice of surgical methods should be decided prudently after perioperative treatment, multi-disciplinary participated and regulation of the internal environment. The standardized and targeted treatments for the surgical difficulties of inflammatory bowel disease should be conducted.

Objective:To investigate the clinical efficacy of gastrojejunal bypass surgery combined with radical gastrectomy following conversion therapy for gastric cancer with outlet obstruction.Methods:The retrospective and descriptive study was conducted. The clinicopatho-logical data of 10 initially unresectable gastric cancer patients with outlet obstruction who were admitted to Ruijin Hospital of Shanghai Jiao Tong University School of Medicine from October 2019 to July 2020 were collected. There were 8 males and 2 females, aged from 41 to 59 years, with a median age of 53 years. Patients underwent 'sandwich therapy' of gastrojejunal bypass surgery combined with gastrectomy following conversion therapy. Observation indicators: (1) gastrojejunal bypass surgery and postoperative situations; (2) conversion therapy and complications; (3) radical gastrectomy and postoperative situations; (4) follow-up. Follow-up using outpatient examinations or telephone interview was conducted to detect postoperative complications, progress-free survival, tumor recurrence and metastasis up to March 2019. Measurement data with normal distribution were represented as Mean± SD, and measurement data with skewed distribution were represented as M(range). Count data were described as absolute numbers. Results:(1) Gastrojejunal bypass surgery and postoperative situations: 10 patients received modified gastrojejunal bypass surgery combined with No.4sb lymph node dissection, without intraoperative serious complications, conversion to laparotomy or death. The operation time, volume of intraoperative blood loss, time to postoperative first flatus, time to liquid diet intake were 73 minutes(range, 60-87 minutes), 33 mL(range,20-110 mL), 3 days(range, 2-6 days), 4 days(range, 4-9 days). One patient had post-operative Clavien-Dindo grade Ⅱ complication of anastomotic bleeding, and was improved after transfusion of blood products. (2) Conversion therapy and complications: of 10 patients, 9 cases received 4 cycles of FLOT regimen. One of the 9 cases was suspended chemotherapy due to Clavien-Dindo grade Ⅱ anastomotic edema after 2 cycles of FLOT regimen. Of 10 patients, there were 6 cases with partial response and 4 cases with stable disease. Of 6 patients with partial response, 4 cases with preoperative cT4b stage were down stage to T4a stage, showing the relationship of tumor with transverse mesentery and pancreatic capsule clearer than the first exploration, 2 cases with preoperative lymph nodes fusion had shrank obviously. Of 4 patients with stable disease, 3 cases were negative for lymph nodes shranking, and the rest 1 case with tumor peritoneal metastasis diagnosed by initial laparoscopy can not be evaluated by imaging examination after chemotherapy. Two of 10 patients had Clavien-Dindo grade I complication of elevated blood glucose during the chemotherapy, which were improved after insulin therapy. (3) Radical gastrectomy and post-operative situations: 10 patients underwent radical resection after conversion therapy. Of 4 cases with stable disease, 3 cases with preoperative lymph nodes fusion showed obvious space between lymph nodes and surrounding tissues at resurgical exploration and received radical resection, 1 case with peritoneal metastasis showed abdominal wall nodelus and omental tuberosity as fibrous scars at resurgical exploration and received radical resection. The operation time, volume of intra-operative blood loss, time to postoperative first flatus, time to initial liquid diet intake, duration of total hospital stay, duration of postoperative hospital stay of 10 patients were 148 minutes(range, 95-195 minutes), 108 mL(range, 100-180 mL), 3 days(range, 2-7 days), 4 days(range, 3-9 days), 11 days(range, 10-21 days), 8 days(range, 7-16 days). Two of 10 patients had perioperative complications. Results of pathological examination of 10 patients showed the number of dissected lymph nodes as 25±6. There were 1 case of stage T1, 5 cases of stage T3, 4 cases of stage T4a. There were 1 case of stage N0, 2 cases of stage N1, 3 cases of stage N2, 4 cases of stage N3. There were 3 cases of tumor regression grade 1a, 1 case of grade 1b, 4 cases of grade 2, 2 cases of grade 3. (4) Follow-up: 10 patients were followed up for 3.9-13.0 months, with a median follow-up time of 6.0 months. The median progression-free survival time of 10 patients was 6.0 months. During the follow-up, 1 case underwent postoperative Clavien-Dindo grade Ⅱ complication of delayed gastric emptying and was improved after symptomatic treatment.Conclusion:The gastrojejunal bypass surgery combined with gastrectomy following conversion therapy for gastric cancer with outlet obstruction is safe and effective.

Objective: To investigate the surgical options for splenic lymph node dissection in patients with advanced gastric cancer undergoing radical total gastrectomy, and to evaluate the sentinel effect of No. 4s lymph node on splenic lymph node metastasis. Methods: A prospective, single-center, randomized and controlled study was carried out (Trial registration, No.NCT02980861). Enrollment criteria: (1) >18 years old and <65 years old; (2) gastric adenocarcinoma locating in the proximal or corpus; (3) preoperative clinical staging as cT2-4aN0-3M0; (4) D2 radical total gastrectomy feasible judged before operation; (5) physical ability score 0 to 1; (6) I to III of ASA classification. Pregnant or lactating women, patients with severe mental illness or previous history of upper abdominal surgery, those suffered from other malignant tumors in the past 5 years, or heart and lung system diseases judged to affect surgery before operation, those receiving preoperative chemotherapy, radiotherapy or targeted therapies, and distant metastases being found during surgery were excluded. According to above criteria, 222 patients at The First Medical Center of Chinese PLA General Hospital from December 2016 to December 2017 were enrolled prospectively and were randomly divided into the laparoscopic splenic hilar lymph node dissection group (laparoscopic group, n=114) and the open splenic hilar lymph node dissection group (open group, n=108). The result of rapid frozen immunohistochemistry of harvested No.4s lymph nodes was used to evaluate the sensitivity and specificity of sentinel effect on splenic hilar lymph node metastasis. The surgical parameters, postoperative recovery parameters, and complication rates were compared between the two groups. Results: There were 80 males and 34 females in the lapascopic group with a mean age of (56.1±10.2) years, and 69 males and 39 females in the open group with a mean age of (58.4±10.9) years. There were no significant differences in baseline data between the two groups (all P>0.05). Total blood loss was less in the laparoscopic group [(96.3±82.4) ml vs. (116.6±101.9) ml, t=1.124, P<0.001], and the amount of bleeding from the splenic hilar lymph nodes dissected was also less than that in the open group [(25.3±17.8) ml vs. (59.5±36.4) ml, t=1.172, P<0.001]. However, the operation time, the time of splenic hilar lymph node, the number of lymph node dissected and number of splenic hilar lymph node dissected were not significantly different between the two groups (all P>0.05). As compared to the open group, the laparoscopic group had shorter time to the first flatus [(1.3±1.2) days vs. (1.6±1.5) days, t=1.665, P=0.021], shorter time to fluid diet [(4.6±1.4) days vs. (4.9 ± 1.6) days, t=1.436, P=0.007], shorter time to remove nasogastric tube [(3.9±2.6) days vs. (4.3±2.4) days, t=0.687, P<0.001] and shorter hospital stay [(10.3±6.6) days vs. (12.1±7.2) days, t=0.697, P<0.001]. Complication rate was 14.0% (16/114) and _12.0% (₁3/108) in the laparoscopic group and the open group, respectively, without significant difference (χ²=6.723, P=0.331). The sensitivity of the No. 4s lymph node for the prediction of splenic hilar lymph node metastasis reached 89.5%, and the specificity reached 99.6%. Conclusions Laparoscopic technique is safe and feasible in the treatment of splenic hilar lymph node dissection in advanced gastric cancer. The No.4s lymph node examination has good sentinel effect on predicting the metastasis of splenic hilar lymph nodes.

With the worldwide adoption of minimally invasive surgery, innovation again becomes the theme. In the past hundred years, molecular biology technology, minimally invasive surgery technology, pharmaceutical research and therapies have been constantly innovated to promote the development of medicine. As the subject of medical innovation, the innovative idea from medical staff is also very important. For surgeons, the invention and improvement of a new operation or surgical instrument directly depends on curiosity and exploration. Whether a novel idea or technology can be transformed and used in clinical practice depends on its commercial prospects and the adjustment of macro health policies. The protection of intellectual property and appropriate distribution of interests are the key to ensure sustainable innovation. We should put scientific and technological innovation and achievement transformation in the important position of the "healthy China" strategy, take scientific and technological innovation as the basis and achievements transformation as the means, promote the development of China's health protection, implement the healthy China strategy, and make our own contribution to provide people with all-round health services.

With the worldwide adoption of minimally invasive surgery, innovation again becomes the theme. In the past hundred years, molecular biology technology, minimally invasive surgery technology, pharmaceutical research and therapies have been constantly innovated to promote the development of medicine. As the subject of medical innovation, the innovative idea from medical staff is also very important. For surgeons, the invention and improvement of a new operation or surgical instrument directly depends on curiosity and exploration. Whether a novel idea or technology can be transformed and used in clinical practice depends on its commercial prospects and the adjustment of macro health policies. The protection of intellectual property and appropriate distribution of interests are the key to ensure sustainable innovation. We should put scientific and technological innovation and achievement transformation in the important position of the "healthy China" strategy, take scientific and technological innovation as the basis and achievements transformation as the means, promote the development of China's health protection, implement the healthy China strategy, and make our own contribution to provide people with all-round health services.