Single-cell analyses reveal distinct expression patterns and roles of long non-coding RNAs during hESC differentiation into pancreatic progenitors.

BACKGROUND: Deep understanding the differentiation process of human embryonic stem cells (hESCs) is essential for developing cell-based therapeutic strategy. Substantial efforts have been made to investigate protein-coding genes, yet it remains lacking comprehensive characterization of long non-coding RNAs (lncRNAs) during this process. METHODS: hESCs were passaged every 5-6 days and had maintained stable karyotype even until the 50th generation. Pancreatic progenitor specification of in vitro differentiation from hESCs was performed and modified. The nuclei were stained with 4,6-Diamidino-2-phenylindole (DAPI). Droplet-based platform (10X Genomics) was applied to generate the single-cell RNA sequencing (scRNA-seq) data. The quality of the filtered read pairs was evaluated by using FastQC. Batch effects were removed using the size factor method. Dimension reduction and unsupervised clustering analyses were performed using Seurat R package. The Monocle 2 and MetaCell algorithms were used to order single cells on a pseudotime course and partition the scRNA-seq data into metacells, respectively. Co-expression network was constructed using WGCNA. Module- and hub-based methods were adopted to predict the functions of lncRNAs. RESULTS: A total of 77,382 cells during the differentiation process of hESCs toward pancreatic progenitors were sequenced. According to the single-cell map, the cells from different time points were authenticated to constitute a relatively homogeneous population, in which a total of 7382 lncRNAs could be detected. Through further analyzing the time course data, conserved and specific expression features of lncRNAs during hESC differentiation were revealed. Based upon pseudotime analysis, 52 pseudotime-associated lncRNAs that grouped into three distinct expression patterns were identified. We also implemented MetaCell algorithm and network-based methods to explore the functional mechanisms of these lncRNAs. Totally, 464 lncRNAs, including 49 pseudotime-associated lncRNAs were functionally annotated by either module-based or hub-based methods. Most importantly, we demonstrated that the lncRNA HOTAIRM1, which co-localized and co-expressed with several HOX genes, may play crucial role in the generation of pancreatic progenitors through regulation of exocytosis and retinoic acid receptor signaling pathway. CONCLUSIONS: Our single-cell analyses provide valuable data resources for biological researchers and novel insights into hESC differentiation processes, which will guide future endeavors to further elucidate the roles of lncRNAs.

Ginsenosides from stems and leaves of ginseng prevent ethanol-induced lipid accumulation in human L02 hepatocytes.

OBJECTIVE: To investigate the effect of ginsenosides from stems and leaves of ginseng on ethanol-induced lipid deposition in human L02 hepatocytes. METHODS: L02 cells were exposed to ethanol for 36 h and treated with or without ginsenosides. The viability of L02 cells was evaluated by methylthiazolyldiphenyl-tetrazolium bromide assay and the triglyceride (TG) content was detected. Lipid droplets were determined by oil red O staining. Intracellular reactive oxygen species (ROS) production and the mitochondrial membrane potential were tested by flow cytometry. The ATP level was measured by reverse phase high performance liquid chromatography. The expression of cytochrome p450 2E1 (CYP2E1) and peroxisome proliferator-activated receptor α (PPARα) was detected by reverse transcriptase-polymerase chain reaction and Western blotting, respectively. RESULTS: Ethanol exposure resulted in the increase of TG level, lipid accumulation and ROS generation, and the decrease of mitochondrial membrane potential and ATP production in the cells. However, ginsenosides significantly reduced TG content (9.69±0.22 µg/mg protein vs. 4.93±0.49 µg/mg protein, P<0.01), and ROS formation (7254.8±385.7 vs. 5825.2±375.9, P<0.01). Meanwhile, improvements in mitochondrial membrane potential (10655.33±331.34 vs. 11129.52±262.35, P<0.05) and ATP level (1.20±0.18 nmol/mg protein vs. 2.53±0.25 nmol/mg protein, P<0.01) were observed by treatment with ginsenosides. Furthermore, ginsenosides could down-regulate CYP2E1 expression (P<0.01) and upregulate PPARα expression (P<0.01) in ethanol-treated cells. CONCLUSIONS: Ginsenosides could prevent ethanol-induced hepatocyte steatosis in vitro related to the inhibition of oxidative stress and the improvement of mitochondrial function. In addition, the modulation of CYP2E1 and PPARα expression may also play an important role in the protective effect of ginsenosides against lipid accumulation.

Risk factors associated with clinical prognosis of cervical cancer treated with laparoscopic surgery / 中国内镜杂志

Objective To investigate the risk factors associated with clinical prognosis of cervical cancer treated with laparoscopic surgery. Methods 80 patients with cervical cancer were recruited in the study, who underwent radical surgery with laparoscopic surgery from March 2013 to December 2016. The patients were followed up after surgery, and the overall survival and disease free survival was analyzed with tumor recurrence and death as the terminal events. And the risk factors associated with clinical prognosis were identified by using Cox regression analysis. Results The patients were followed up from 12 months to 46 months, and the median period was 39 months. There were 16 recurrences and 6 deaths during the period of follow-up, yielding a disease-free survival of (41.85 ± 1.06) year and an overall survival of (44.86 ± 0.74) year. Cox regression analysis demonstrated that tumor size, clinical stage, lymph node metastasis and vascular invasion were independent risk factors associated with clinical prognosis of cervical cancer treated with laparoscopic surgery. Conclusion Tumor size, clinical stage, lymph node metastasis and vascular invasion were independent risk factors associated with clinical prognosis of cervical cancer treated with laparoscopic surgery.

Risk factors associated with clinical prognosis of cervical cancer treated with laparoscopic surgery / 中国内镜杂志

Objective To investigate the risk factors associated with clinical prognosis of cervical cancer treated with laparoscopic surgery. Methods 80 patients with cervical cancer were recruited in the study, who underwent radical surgery with laparoscopic surgery from March 2013 to December 2016. The patients were followed up after surgery, and the overall survival and disease free survival was analyzed with tumor recurrence and death as the terminal events. And the risk factors associated with clinical prognosis were identified by using Cox regression analysis. Results The patients were followed up from 12 months to 46 months, and the median period was 39 months. There were 16 recurrences and 6 deaths during the period of follow-up, yielding a disease-free survival of (41.85 ± 1.06) year and an overall survival of (44.86 ± 0.74) year. Cox regression analysis demonstrated that tumor size, clinical stage, lymph node metastasis and vascular invasion were independent risk factors associated with clinical prognosis of cervical cancer treated with laparoscopic surgery. Conclusion Tumor size, clinical stage, lymph node metastasis and vascular invasion were independent risk factors associated with clinical prognosis of cervical cancer treated with laparoscopic surgery.

Berberine inhibits norepinephrine-induced apoptosis in neonatal rat cardiomyocytes via inhibiting ROS-TNF-α-caspase signaling pathway.

OBJECTIVE: To determine the effect of berberine (Ber) on norepinephrine (NE)-induced apoptosis in neonatal rat cardiomyocytes. METHODS: The cultured neonatal rat cardiomyocytes were treated with NE in the presence or absence of Ber. The activity of lactate dehydrogenase (LDH) in the culture medium was examined, and apoptosis of cardiomyocytes was assessed by Hoechst 33258, isothiocyanate (FITC)-conjugated annexin-V, and propidine iodide (PI) staining. In addition, the activities of caspases-2 and-3 were measured by a fluorescent assay kit. The level of secreted tumor necrosis factor α (TNF-α) and production of intracellular reactive oxygen species (ROS) were also determined. RESULTS: NE at a concentration of 50 µ mol/L induced an obvious increase in the activity of LDH in the culture medium (P<0.05), which was inhibited by coincubation with 0.5, 1.0, or 2.0 µ mol/L Ber (P<0.05). Ber also significantly attenuated NE-induced apoptosis in a dose-dependent manner (P<0.01). Moreover, Ber at a dose of 2 µ mol/L markedly decreased the ROS and TNF-α productions (P <0.05) and inhibited the activation of caspases-2 and -3 in cardiomyocytes exposed to NE (P<0.05)h. CONCLUSION: The present study suggested that Ber could reduce NE-induced apoptosis in neonatal rat cardiomyocytes through inhibiting the ROS-TNF-α-caspase signaling pathway.

Berberine protects against lipopolysaccharide-induced intestinal injury in mice via alpha 2 adrenoceptor-independent mechanisms.

AIM: To investigate the mechanisms responsible for the protective action of berberine (Ber) against gut damage in endotoxemic mice. METHODS: Male BALB/c mice were administered intragastrically with distilled water (0.1 mL/10 g), Ber (50 mg/kg) alone, yohimbine (2 mg/kg) alone, or Ber (50 mg/kg) in combination with yohimbine (2 mg/kg) for 3 d. On the third day, lipopolysaccharide (LPS, 18 mg/kg) or normal saline was intraperitoneally injected one hour after the intragastric administration. Following the treatment, intestinal injury in the ileum was histopathologically accessed; enterocyte apoptosis was examined using TUNEL method; Toll-like receptor 4 (TLR4) mRNA expression was measured using RT-PCR assay; inhibitor protein-κBα (I-κBα) phosphorylation and myeloperoxidase content were examined using Western blloting. The macrophage inflammatory protein-2 (MIP-2) production was measured using ELISA assay. RESULTS: Mice challenged with LPS caused extensive ileum injury, including a significantly increased injury score, decreased intestinal villus height, reduced gut mucosal weight and increased intestinal permeability. Furthermore, LPS significantly induced enterocyte apoptosis, increased TLR4 mRNA expression, I-κBα phosphorylation, MIP-2 production and myeloperoxidase content in the ileum. Pretreatment with Ber significantly alleviated all the alterations in the ileum in the endotoxemic mice. Pretreatment with the α2-adrenoceptor antagonist yohimbine did not block the protective action of Ber against LPS-induced intestinal injury. In addition, treatment with yohimbine alone did not prevent LPS-induced intestinal injury. CONCLUSION: Pretreatment with Ber provides significant protection against LPS-induced intestinal injury in mice, via reducing enterocyte apoptosis, inhibiting the TLR4-nuclear factor κB-MIP-2 pathway and decreasing neutrophil infiltration that are independent of α2-adrenoceptors.

Pretreatment with berberine and yohimbine protects against LPS-induced myocardial dysfunction via inhibition of cardiac I-[kappa]B[alpha] phosphorylation and apoptosis in mice.

Myocardial dysfunction is a common complication in sepsis and significantly contributes to the mortality of patients with septic shock. Our previous study demonstrated that pretreatment with berberine (Ber) protected against the lethality induced by LPS, which was enhanced by yohimbine, an [alpha]2-adrenergic receptor antagonist, and Ber combined with yohimbine also improved survival in mice subjected to cecal ligation and puncture. However, no studies have examined whether Ber and yohimbine reduce LPS-induced myocardial dysfunction. Here, we report that pretreatment with Ber, Ber combined with yohimbine, or yohimbine significantly reduced LPS-induced cardiac dysfunction in mice. LPS-provoked cardiac apoptosis, I-[kappa]B[alpha] phosphorylation, IL-1[beta], TNF-[alpha], and NO production were attenuated by pretreatment with Ber and/or yohimbine, whereas cardiac Toll-like receptor 4 mRNA expression, malondialdehyde content, and superoxide dismutase activity were not affected. These data demonstrate for the first time that pretreatment with Ber and/or yohimbine prevents LPS-induced myocardial dysfunction in mice through inhibiting myocardial apoptosis, cardiac I-[kappa]B[alpha] phosphorylation, and TNF-[alpha], IL-1[beta], and NO production, suggesting that activation of [alpha]2-adrenergic receptor in vivo may be responsible at least in part for LPS-induced cardiac dysfunction, and Ber in combination with yohimbine may be a potential agent for preventing cardiac dysfunction during sepsis.

[The effect of gastrodine on hepatitic cell line L02 injury induced by ethanol].

AIM: To explore the the effect of gastrodine on hepatitic cell line L02 injury induced by ethanol. METHODS: The growing L02 cells were treated with different concentrations of alcohol. For screening the proper concentration of alcohol, the proliferation of the cells were measured by MTT colorimetry. The level of reactive oxygen species (ROS) and change of mitochondrial membrane potential in cells were tested by flow cytometry. Reversed-phase high-performance liquid chromatography (RP-HPLC) was applied to assay ATP content in cells. RESULTS: The cells were injured when treated with 25 mL/L alcohol for 36 h. Compared with the control group , the ROS level in cells markedly increased while mitochondrial membrane potential and ATP content in cells significantly decreased (P<0.01). Gastrodine obviously inhibited cell damage, reduced ROS level, and increased mitochondrial membrane potential and ATP content in cells. CONCLUSION: Above results suggest that gastrodine may suppress cellular injury and lipid peroxidation induced by ethanol, improve mitochondrial function and raise ATP level, which play an important role in protecting hepatocytes.

[Construction of T7 phage display library from the anther of Honglian hybrid line of rice].

Phage display is a powerful method to study protein-protein interactions. In order to study the molecular mechanism of cytoplasmic male sterility and fertility restoration in Honglian rice, the mRNA was isolated with PolyA Tract mRNA Isolation Kit from the anther of F1 hybrid rice and the double strand (ds) cDNA was synthesized by reverse transcription. Then the directional EcoRI /Hind III linkers were ligated into the ends of ds cDNA and the ds cDNA was further digested with EcoR I and Hind, which resulted in ds cDNA with EcoR I and Hind III ends. The digested ds cDNA fragments longer than 300 bp in length were fractionated with Mini Column, then ligated into the T7 Select 10-3b vertor with EcoR I and Hind III ends. After packaging in vitro, the T7 Select 10-3b vertor was transformed into BL T5403 to construct the T7 phage display library. Analysis showed that the library contained 1.03 x 106 clones per microliter, and approximately 100% of the clones in library was recombinant. The titer of the amplied library was 2.14 x 1012 pfu/mL, and the insert length of the recombinants over 300 bp was about 97%.

[Silencing HSV1 gD expression in cultured cells by RNA interference].

To explore the anti-HSV-1 effect of silencing gD gene expression by RNA interference, five 21-nucleotide duplex small interfering RNAs(siRNAs) targeting the HSV1 gD sequence were designed and the gD-EGFP fusion gene expression vector was constructed, then co-transfected into Vero cell, and screened the effective siRNA through analyzing the intensity of the EGFP fluorescence. Finally, the anti-HSV1 effect was confirmed by plaque reduction assay, real-time PCR and daughter virus titration of HSV1 infected Vero cells transfected with siRNAs. The study demonstrated that siRNAs could effectively and specifically inhibit gD gene expression in HSV1-infected cells, but only had a little effect on HSV1 infection, so taking gD as the target of siRNA against HSV1 needs further study.

[Cluster analysis applied in the epidemiological stratification analysis].

OBJECTIVE: To establish a new method on stratification analysis when the stratification limits of confounding factors was not clear or contradictory. METHOD: Data on a study of diabetes mellitus in Guangdong province collected in the year of 1997 and 1998 was analyzed using cluster-stratification analysis. RESULTS: The efficiency of stratification analysis was improved and the confounding bias was effectively controlled with information bias avoided when the clusters-stratification analysis was applied. CONCLUSION: The problem was logically solved using cluster analysis as an assistant stratification means.