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: Pneumonia-like primary pulmonary lymphoma (PPL) was commonly misdiagnosed as infectious pneumonia, leading to delayed treatment. The purpose of this study was to establish a computed tomography (CT)-based radiomics model to differentiate pneumonia-like PPL from infectious pneumonia. METHODS: In this retrospective study, 79 patients with pneumonia-like PPL and 176 patients with infectious pneumonia from 12 medical centers were enrolled. Patients from center 1 to center 7 were assigned to the training or validation cohort, and the remaining patients from other centers were used as the external test cohort. Radiomics features were extracted from CT images. A three-step procedure was applied for radiomics feature selection and radiomics signature building, including the inter- and intra-class correlation coefficients (ICCs), a one-way analysis of variance (ANOVA), and least absolute shrinkage and selection operator (LASSO). Univariate and multivariate analyses were used to identify the significant clinicoradiological variables and construct a clinical factor model. Two radiologists reviewed the CT images for the external test set. Performance of the radiomics model, clinical factor model, and each radiologist were assessed by receiver operating characteristic, and area under the curve (AUC) was compared. RESULTS: A total of 144 patients (44 with pneumonia-like PPL and 100 infectious pneumonia) were in the training cohort, 38 patients (12 with pneumonia-like PPL and 26 infectious pneumonia) were in the validation cohort, and 73 patients (23 with pneumonia-like PPL and 50 infectious pneumonia) were in the external test cohort. Twenty-three radiomics features were selected to build the radiomics model, which yielded AUCs of 0.95 (95% confidence interval [CI]: 0.94-0.99), 0.93 (95% CI: 0.85-0.98), and 0.94 (95% CI: 0.87-0.99) in the training, validation, and external test cohort, respectively. The AUCs for the two readers and clinical factor model were 0.74 (95% CI: 0.63-0.83), 0.72 (95% CI: 0.62-0.82), and 0.73 (95% CI: 0.62-0.84) in the external test cohort, respectively. The radiomics model outperformed both the readers' interpretation and clinical factor model ( P <0.05). CONCLUSIONS The CT-based radiomics model may provide an effective and non-invasive tool to differentiate pneumonia-like PPL from infectious pneumonia, which might provide assistance for clinicians in tailoring precise therapy.
Humans ; Retrospective Studies ; Pneumonia/diagnostic imaging* ; Analysis of Variance ; Tomography, X-Ray Computed ; Lymphoma/diagnostic imaging*

Objective:To investigate the value of multimodal MRI radiomics in the preoperative prediction of Fuhrman nuclear grade of clear cell renal cell carcinoma (ccRCC).Methods:A total of 129 patients with ccRCC confirmed by pathology from April 2011 to April 2021 in Third Affiliated Hospital of Soochow University were collected, and the imaging and clinicopathological data were retrospectively analyzed. All patients were divided into training set ( n=90) and validation set ( n=39) at the ratio of 7∶3 using random indicator method. According to the postoperative pathological results, Fuhrman grades Ⅰ and Ⅱ were included in the low grade group (96 cases, 65 cases in the training set and 31 cases in the validation set), and Fuhrman grades Ⅲ and Ⅳ were included in the high grade group (33 cases, 25 cases in the training set and 8 cases in the validation set). Two radiologists manually delineated regions of interest (ROI) on T 1WI, T 2WI, Dixon-water, Dixon-fat, susceptibility weighted imaging (SWI), blood oxygen level dependent (BOLD) images, and 396 texture features were extracted from each ROI. In the training set, intra-class correlation coefficient, Mann-Whitney U test, minimum redundancy maximum relevance and least absolute shrinkage and selection operator method were used to reduce the dimension of features to obtain the best texture features. The logistic regression was used to develop the multimodal radiomics model, and the receiver operating characteristic (ROC) curve was used to evaluate the effectiveness of the model in identifying high and low-grade ccRCC in training set and validation set. Results:Four SWI, one T 2WI and one BOLD texture features were selected for modeling. The areas under the ROC curve (95%CI) of the multimodal radiomics model for identifying high and low grade ccRCC in the training and validation sets were 0.859 (0.770-0.923) and 0.883 (0.740-0.964), with the specificity at 95.4% and 87.1%, the sensitivity at 68.0% and 87.5%, the accuracy at 87.8% and 87.2%, respectively. Conclusion:The multimodal MRI radiomics model based on T 2WI, SWI and BOLD images has high effectiveness in preoperative predicting Fuhrman nuclear grade of ccRCC.

Objective:To investigate the expression changes of lncRNAs and mRNAs in human umbilical vein endothelial cells(HUVEC) treated by tritiated water.Methods:HUVEC cells were divided into two groups, the control group cultured in DMEM medium, and the tritiated water exposure group cultured in a medium containing tritiated water with a final concentraion of 3.7×10 3 Bq/ml. After culture for 48 h, cells were collected for RNA extract.The differentially expressed lncRNAs and mRNAs were screened by high-through put chip technology and then analyzed. Results:Compared with the control group, 1 717 lncRNAs were significantly up-regulated and 3 994 lncRNAs significantly down-regulated, and 4 562 mRNAs were significantly up-regulated and 1 433 mRNAs down-regulated. Through co-expression analysis of differential mRNAs and lncRNAs, some key genes including SQSTM1, CXCL8, ITPR1, GADD45A, NF-kB1 and VDAC1 were obtained.Conclusions:Tritiated water exposure can induce multiple changes of mRNAs and lncRNAs in vascular endothelial cells, which may lead to toxic effects through signaling pathways including some key genes such as SQSTM1, CXCL8, and ITPR1.

The discovery of hydroxylases in the anticancer drug taxol biosynthesis pathway is a hotspot and difficulty in current research. In this study, a new hydroxylase gene TcCYP725A22 (GenBank accession number: MF448646.1) was used to construct a sub-cellular localization vector pCAMIBA1303-TcCYP725A22-EGFP to get the transient expression in onion epidermal cells. Laser confocal microscopy revealed that the protein encoded by this gene was localized in the cell membrane. Furthermore, the recombinant plant expression plasmid pBI121-TcCYP725A22 was constructed. After transient transformation to the Taxus chinensis mediated by Agrobacterium tumefaciens LBA4404, qRT-PCR and LC-MS were utilized to analyze the effects of TcCYP725A22 overexpression on the synthesis of taxol. The results showed that, in the TcCYP725A22 overexpressed cell line, expression levels of most defined hydroxylase genes for taxol biosynthesis were increased, and the yield of taxanes were also increased. It was concluded that the hydroxylase gene TcCYP725A22 is likely involved in the biosynthetic pathway of taxol.
Biosynthetic Pathways ; Mixed Function Oxygenases ; Paclitaxel ; Taxoids ; Taxus

Taxol is a secondary metabolite with prominent anti-tumor activity, but the yield cannot meet the growing clinical demand due to lower content in yew. Now, most enzyme genes involved in taxol biosynthesis have been cloned and identified, so that obtaining this drug by using synthetic biology method has become a hotspot in recent years. However, most hydroxylases involved in taxol biosynthetic pathway have not been explored. Here, we reviewed the progress on the biosynthesis pathway of taxol, especially concerning hydroxylase. The future research areas of taxol biosynthesis through synthetic biology were also discussed to provide basis for the discovery of uncharacterized hydroxylase genes and the mass taxol production by synthetic biology technology.
Biosynthetic Pathways ; Mixed Function Oxygenases ; metabolism ; Paclitaxel ; biosynthesis ; Synthetic Biology ; Taxus ; enzymology

OBJECTIVETo explore the effects of stress imposed on adenoid cystic carcinoma (ACC), therefore to clarify the molecular basis and mechanism of ACC's malignant phenotype under the elevated tumor interstitial fluid pressure.

METHODSACC cells were cultured under pressure (103.74 kPa), and were divided into four groups (3 h group, 6 h group, 12 h group, 24 h group) according the pressure time. Untreated ACC2 was as negative control group, untreated ACCM was as positive control group. The level of epidermal growth factor receptor (EGFR) was detected by semiquantitative analysis of immunochemistry. Matrix metalloproteinase 9 (MMP9) and EGFR mRNA expression were assessed by reverse transcriptase polymerase chain reaction. EGFR, phosphorylation epidermal growth factor receptor (P-EGFR), MMP9, keratinocyte growth factor (KGF) and phosphorylation extracellular signal-regulated kinase (P-ERK) protein expressions were assessed by Western blot.

RESULTSAs the extension of pressure time, the expression of EGFR, P-EGFR, MMP9, KGF, P-ERK in ACC2 gradually increased, which were positive correlation with pressure time, and were higher than that of negative control group.

CONCLUSIONUnder the stimulation of pressure, the mRNA and protein levels of adhesion molecules and metastatic relative molecules in ACC2 were sharply elevated.

OBJECTIVEThrough a simulation of interstitial fluid pressure (IFP), we developed an in vitro model to explore the change law of biological characteristics of adenoid cystic carcinoma (ACC) under different IFP.

METHODSA pressure cooker was refitted into a controllable pressure device. Cultured ACC-2 cells were subdivided into different groups, namely, negative control (untreated ACC-2) and experimental group (stressed for 3, 6, 12, 24 h under pressure of 7.551, 7.649, 7.747 kPa). CCK-8 and immunofluorescence of Ki67 were used to reflect proliferation ability. Transwell chamber assay was performed to observe the invasion ability of cells.

RESULTSThe proliferation ability was positively correlated with treatment time, and the peak value was obtained after the cells were subjected to 7.649 kPa of stress for 24 h. The invasion ability of ACC-2 cells was upregulated under stress.

CONCLUSIONWe successfully developed an in vitro model of IFP and found that high IFP can stimulate cell proliferation ability and upregulate invasion ability.

Objective Through a simulation of interstitial fluid pressure (IFP), we developed an in vitro model to explore the change law of biological characteristics of adenoid cystic carcinoma (ACC) under different IFP. Methods A pressure cooker was refitted into a controllable pressure device. Cultured ACC-2 cells were subdivided into different groups, namely, negative control (untreated ACC-2) and experimental group (stressed for 3, 6, 12, 24 h under pressure of 7.551, 7.649, 7.747 kPa). CCK-8 and immunofluorescence of Ki67 were used to reflect proliferation ability. Transwell chamber assay was performed to observe the invasion ability of cells. Results The proliferation ability was positively correlated with treatment time, and the peak value was obtained after the cells were subjected to 7.649 kPa of stress for 24 h. The invasion ability of ACC-2 cells was upregulated under stress. Conclusion We successfully developed an in vitro model of IFP and found that high IFP can stimulate cell proliferation ability and upregulate invasion ability.

Objective To explore the effects of stress imposed on adenoid cystic carcinoma (ACC), therefore to clarify the molecular basis and mechanism of ACC’s malignant phenotype under the elevated tumor interstitial fluid pressure. Methods ACC cells were cultured under pressure (103.74 kPa), and were divided into four groups (3 h group, 6 h group, 12 h group, 24 h group) according the pressure time. Untreated ACC2 was as negative control group, untreated ACCM was as positive control group. The level of epidermal growth factor receptor (EGFR) was detected by semiquantitative analysis of immunoche-mistry. Matrix metalloproteinase 9 (MMP9) and EGFR mRNA expression were assessed by reverse transcriptase polymerase chain reaction. EGFR, phosphorylation epidermal growth factor receptor (P-EGFR), MMP9, keratinocyte growth factor (KGF) and phosphorylation extracellular signal-regulated kinase (P-ERK) protein expressions were assessed by Western blot. Results As the extension of pressure time, the expression of EGFR, P-EGFR, MMP9, KGF, P-ERK in ACC2 gradually increased, which were positive correlation with pressure time, and were higher than that of negative control group. Conclusion Under the stimulation of pressure, the mRNA and protein levels of adhesion molecules and metastatic relative molecules in ACC2 were sharply elevated.