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*

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 explore the difference of the vessel and plaque characteristics, myocardial perfusion and cardiac function between patients with ischemia with non-obstructive coronary artery disease (INOCA) and obstructive coronary artery disease (CAD).Methods:From July 2021 to June 2022, 101 patients with angina were referred to dynamic computed tomography myocardial perfusion (CTP) and coronary computed tomography angiography (CCTA) and retrospectively included in our hospital. Based on the results of CTP and CCTA, patients were divided into INOCA (27 cases), moderate obstructive CAD (26 cases) and severe obstructive CAD (48 cases). The anatomical coronary artery stenosis, plaque characteristics and myocardial perfusion features of all patients were analyzed. Furthermore, left ventricular global longitudinal strain (GLS), global circumferential strain (GCS), and global radial strain (GRS) were obtained on full-phase reconstruction CCTA image by using Medis Suite 3.2 postprocessing software. Multigroup analysis used one way ANOVA or Kruskal Wallis H test. Results:Patients with INOCA were younger than patients with moderate and severe obstructive CAD ( P<0.001). INOCA patients (7.4%, 2/27) had lower rate of positive remodeling than both moderate (57.7%, 15/26, P<0.001) and severe obstructive CAD patients (33.3%, 16/48, P=0.017). The percentage of ischemic myocardium volume in patients with INOCA were similar with those in patients with severe CAD (all P>0.05), but significantly higher than those in patients with moderate CAD (all P<0.05). No significant difference in terms of GLS was detected between patients with INOCA [-17.4% (-21.6%, -11.6%)] and severe CAD [-17.6% (-21.9%, -14.8%), P=0.536], however, patients both with INOCA and severe CAD also had higher GLS than patients with moderate obstructive CAD [-22.3% (-29.8%, -19.0%), all P<0.05]. Conclusions:Based on"one-stop-shop"CTP combined with CCTA imaging, early cardiac functional changes including abnormal myocardial perfusion and myocardial strain in INOCA patients were similar to those in patients with severe obstructive CAD and more severe than those in patients with moderate obstructive CAD.

Objective:To evaluate the effect of hyperthermia on the apoptosis and the expression levels of cysteine-containing aspartate-specific protease-3 (Caspase-3) and phosphorylated protein kinase B (p-AKT) in laryngeal squamous cell carcinoma cells.Methods:A prospective study was conducted on 30 patients with laryngeal squamous cell carcinoma who were treated at the First Affiliated Hospital of Zhengzhou University from October, 2021 to October, 2022. Three times of hyperthermia were performed with a time interval of 24 h. The tumor tissue samples were collected from 30 patients before and after hyperthermia and divided into before hyperthermia group (group A ) and after hyperthermia group (group B). Self-control study mode was adopted for comrparative analysis. The cell apoptosis was detected by TUNEL assay. The expression levels of Caspase-3 and p-AKT in the tissues were detected by immunohistochemistry. Positive cell ratio and immunohistochemistry (IHC) score were recorded. Comparison between two groups was performed by paired t-test. The correlation between the degree of apoptosis and the changes of Caspase-3 and p-AKT molecules was assessed by Pearson correlation analysis. Results:No evident adverse reactions were observed in 30 patients after hyperthermia. The apoptosis index of laryngeal squamous cell carcinoma cells in group A was 2.37%±1.33%, and 4.27%±3.93% in group B ( P=0.006). In group A, the ratio of Caspase-3 positive cells in tumor tissues was 62.31%±19.49% and 80.79%±17.15% in group B ( P=0.001). The ratio of p-AKT positive cells in group A was 31.26%±19.30%, and 26.26%±15.86% in group B ( P=0.023). There was a positive correlation between the degree of apoptosis and the changes of Caspase-3 molecule ( r=0.544, P=0.002), but a negative correlation was noted between the degree of apoptosis and the changes of p-AKT molecule ( r=-0.434, P=0.017). Conclusion:Hyperthermia can promote the apoptosis of tumor cells in laryngeal squamous cell carcinoma, which may be related to Caspase-3 dependent apoptosis, and the inhibition of AKT phosphorylation is also involved in this process.

Objective:To investigate the effect of Mediterranean diet on blood glucose control and cardiovascular risk factors in patients with type 2 diabetes.Methods:As to December 2021, the PubMed, Cochrance Central Register of Controlled Trials and Cochrance Database, Cochranc Library, Embase, China National Knowledge Infrastructure and Wanfang Medical Network system were searched for clinical randomized controlled trials(RCTs) of Mediterranean diet in patients with type 2 diabetes to conduct Meta-analysis The main observation index were cardiovascular risk factors, and the mean difference and its 95% confidence interval were used to estimate the effect size.Results:There were six RCTs, and 1181 patients met the inclusion criteria and entered the Meta-analysis. Compared with the control group, the intervention group can significantly reduce the level of systolic blood pressure ( MD=-1.20, 95% CI-2.21 to -0.19) and diastolic blood pressure ( MD=-4.17, 95% CI-7.12 to -1.22) in patients with type 2 diabetes mellitus, but there were no significant difference in the level of TC ( MD=2.92, 95% CI-0.84 to-6.67), HDL ( MD=2.33, 95% CI-0.27 to -4.92) and LDL ( MD=-2.34, 95% CI-5.67 to -0.99) between the two groups (all P>0.05). Conclusions:The meta-analysis provided evidence the Mediterranean diet showed the beneficial improvements in blood pressure glycemic control, but the effect of Mediterranean diet on lipid profile was not significant, which needed further verification.

Esophageal cancer is a malignant tumor of the digestive system that has a high incidence in China. The traditional treatment methods include surgery, radiotherapy, and chemotherapy, but the long-term efficacy is not good and the side effects are obvious. As a traditional physical therapy, hyperthermia has no significant toxic and side effects. Studies have shown that hyperthermia can increase the sensitivity of esophageal cancer to radiotherapy and chemotherapy, and its combined use in the treatment of esophageal cancer can prolong the survival and improve the quality of life. In addition, the innovation of materials and technologies brings new breakthroughs to tumor hyperthermia.

Objective:To initially investigate whether simultaneous radiochemotherapy with hyperthermia can prolong the survival of glioblastoma (GBM) patients.Methods:Clinical data of 61 GBM patients undergoing surgery in our hospital from September 2016 to June 2019 were retrospectively analyzed. According to different treatment methods, all patients were divided into the control group ( n=34) and observation group ( n=27). In the control group, three-dimensional radiotherapy with a dose of 60 Gy combined with temoazolamine chemotherapy was delivered. In the observation group, simultaneous radiochemotherapy with 15-20 cycles of hyperthermia at 40-41℃ was supplemented. The survival time was calculated by Kaplan-Meier method, and the survival time was compared with log-rank test between two groups. Results:The median progression-free survival in the observation group was significantly longer than that in the control group (14.33 months vs.9.94 months, P<0.05). The median overall survival in the observation group was also remarkably higher than that in the control group (18 months vs. 14 months, P<0.05). Conclusions:Simultaneous radiochemotherapy with hyperthermia is innovatively applied to treat GBM after surgical resection. Preliminary findings demonstrate that compared with chemoradiotherapy, simultaneous radiochemotherapy with hyperthermia can prolong the survival time of GBM patients.

Objective:To observe the molecular mechanism of Hemoporfin-mediated photodynamic therapy on vascular endothelial cells.Methods:Human umbilical vein endothelial cells (HUVEC) were cultured in vitro and divided into four groups: PDT group(Hemoporfin concentration: 5 μg/ml, light fluences: 4 J/cm 2), drug group (only Hemporfin: 5 μg/ml), light group(only irradiation by 4J/cm 2 light), and blank control (no drug, no light). The cell viability and proliferation were detected by cck-8 cytotoxicity test and Brdu testafter different treatments as mentioned above. Expression levels of VEGF-A/MAPK/ERK pathway related molecules in the cells before and after photodynamic treatment were detected by real-time quantitative PCR, Western blot and immunofluorescence staining. Results:Compared with the black control group, the cell viability[(0.45±0.08)vs(1.02±0.11), t=12.02, P<0.05] and cell proliferation level [(0.42±0.02)vs(1.00±0.01), t=31.20, P<0.05]were significantly decreased in PDT group.The mRNA expression levels, including Ras[(0.62±0.02)vs(1.05±0.03), t=10.35, P<0.05], c-Raf [(0.72±0.04)vs(1.00±0.05), t=7.35, P<0.05], Mek[(0.73±0.12)vs(1.15±0.04), t=7.74, P<0.05], Erk [(0.56±0.11)vs(1.02±0.03), t=5.56, P<0.05], VEGF-Α [(0.34±0.04)vs(1.02±0.07), t=7.59, P<0.05], and VEGFR2[(0.54±0.05)vs(1.00±0.03), t=5.34, P<0.05] were significantly decreased. The proteinphosphorylation level of c-Raf[(0.44±0.02)vs(1.02±0.05), t=46.7, P<0.05], Mek[(0.72±0.05)vs(1.05±0.04), t=5.35, P<0.05], Erk[(0.62±0.15)vs(1.03±0.03), t=8.58, P<0.05] and the proteinexpression level of VEGF-A[(0.64±0.03)vs(1.03±0.04), t=21.65, P<0.05] were significantly down-regulated in PDT group compared with the black control group. Compared with the blank control group, there were no significant differences expression between the drug group and the light group at cell activity, molecular proliferation level and molecular expressions. Conclusions:HMME-PDT inhibits the activity and proliferation of vascular endothelial cells by inhibiting the expression of the VEGF-A/MAPK/ERK pathway to achieve the purpose of inhibiting vascular hyperplasia and repair.

Objective:To observe the molecular mechanism of Hemoporfin-mediated photodynamic therapy on vascular endothelial cells.Methods:Human umbilical vein endothelial cells (HUVEC) were cultured in vitro and divided into four groups: PDT group(Hemoporfin concentration: 5 μg/ml, light fluences: 4 J/cm 2), drug group (only Hemporfin: 5 μg/ml), light group(only irradiation by 4J/cm 2 light), and blank control (no drug, no light). The cell viability and proliferation were detected by cck-8 cytotoxicity test and Brdu testafter different treatments as mentioned above. Expression levels of VEGF-A/MAPK/ERK pathway related molecules in the cells before and after photodynamic treatment were detected by real-time quantitative PCR, Western blot and immunofluorescence staining. Results:Compared with the black control group, the cell viability[(0.45±0.08)vs(1.02±0.11), t=12.02, P<0.05] and cell proliferation level [(0.42±0.02)vs(1.00±0.01), t=31.20, P<0.05]were significantly decreased in PDT group.The mRNA expression levels, including Ras[(0.62±0.02)vs(1.05±0.03), t=10.35, P<0.05], c-Raf [(0.72±0.04)vs(1.00±0.05), t=7.35, P<0.05], Mek[(0.73±0.12)vs(1.15±0.04), t=7.74, P<0.05], Erk [(0.56±0.11)vs(1.02±0.03), t=5.56, P<0.05], VEGF-Α [(0.34±0.04)vs(1.02±0.07), t=7.59, P<0.05], and VEGFR2[(0.54±0.05)vs(1.00±0.03), t=5.34, P<0.05] were significantly decreased. The proteinphosphorylation level of c-Raf[(0.44±0.02)vs(1.02±0.05), t=46.7, P<0.05], Mek[(0.72±0.05)vs(1.05±0.04), t=5.35, P<0.05], Erk[(0.62±0.15)vs(1.03±0.03), t=8.58, P<0.05] and the proteinexpression level of VEGF-A[(0.64±0.03)vs(1.03±0.04), t=21.65, P<0.05] were significantly down-regulated in PDT group compared with the black control group. Compared with the blank control group, there were no significant differences expression between the drug group and the light group at cell activity, molecular proliferation level and molecular expressions. Conclusions:HMME-PDT inhibits the activity and proliferation of vascular endothelial cells by inhibiting the expression of the VEGF-A/MAPK/ERK pathway to achieve the purpose of inhibiting vascular hyperplasia and repair.

Objective To investigate the inhibition mechanism of tetramethylpyrazine combined with cisplatin on angiogenesis in Lewis lung cancer mice and to observe the mechanism of Arresten on angiogenesis in lung cancer. Methods The model of Lewis lung adenocarcinoma mouse xenograft was established in this work, and 40 mice were randomly divided into 4 groups: 0.9% sodium chloride solution group(NS group), tetramethylpyrazine group(TMP group), cisplatin group(DDP group), tetramethylpyrazine plus cisplatin group(TMP + DDP group), 10 mice in each group.Mice in NS group were given 0.2 mL of 0.9% sodium chloride solution, mice in DDP group were given 0.2 mL of 2 mg.kg-1 of cisplatin, mice in TMP group were given 0.2 mL of 100 mg.kg-1 of tetramethylpyrazine, mice in TMP+DDP group were given 2 mg.kg-1 of cisplatin and 100 mg.kg-1 of tetramethylpyrazine, each 0.1 mL .Tumor size was measured every day to calculate the tumor volume.The mice were sacrificed to stripp the subcutaneous tumor after continuous medication. The expressions of Arresten, integrin α1β1 and VEGF were determinated by immunhistochemistry and Western blotting. Results The tumor growth of NS group was the fastest and TMP+DDP group was the slowest. Compared with NS group, the expression of Arresten in the other three groups was increased( P<0.01) , and the TMP+DDP group exhibited the highest expression;at the same time, integrin α1β1 , VEGF in the other three groups was decreased(P<0.01), and the TMP+DDP group exhibited the lowest expression.The expression of integrinα1β1 and VEGF was negatively related to Arresten, and the expression of integrin α1β1 was positively correlated with VEGF. Conclusion TMP can inhibited the growth of Lewis lung carcinoma and angiogenesis. Moreover, in combination with cisplatin, TMP can also improved the effect of chemotherapy and then the survival state of mice. The mechanism of action, which TMP suppress tumor angiogenesis may be through improving Arresten and inhibiting integrin α1β1 and VEGF. And the action mechanism of Arresten may be implemented by inhibiting the expression of VEGF by incorporation with integrinα1β1 or by itself to inhibit the expression of VEGF.