Contamination-free visual detection of CaMV35S promoter amplicon using CRISPR/Cas12a coupled with a designed reaction vessel: Rapid, specific and sensitive.

An ultrafast and convenient method for visually detecting CaMV35S promoter amplicon (amplified products) was established by using CRISPR/Cas12a system coupled with a designed reaction vessel. Genetically modified (GM) soybean (Roundup Ready®) powders containing CaMV35S promoter were employed as detection targets, which were amplified by loop-mediated isothermal amplification (LAMP). The CRISPR/Cas12a system directly mixed with amplified products at 37 °C for 5 min and detection results could be clearly identified by the naked eye under UV light (254 nm). A designed reaction vessel was employed to make operation easier and could effectively prevent contamination at the source. The CRISPR/Cas12a detection system was optimized in our study and the concentration of magnesium ions was proved to be important for the work of CRISPR/Cas12a system. The optimized concentration range of magnesium ions was between 10 mM and 12 mM. Besides, the activated Bst DNA polymerase also had little effects on CRISPR/Cas12a system. The developed method could significantly distinguish the specific and non-specific amplification. And as low as 0.05% transgenic contents in soybean powders could be detected. It would have the potential to be complementary to instrument-based ultrahigh sensitive method and provide a new solution for on-site rapid detection.

[Tea polyphenols delays human glomerular mesangial cells senescence induced by high glucose via regulating STAT3/miR-126/telomere signaling pathway activation].

The aim of this paper was to explore the effects and possible mechanisms in vitro of tea polyphenols (TP) delaying human glomerular mesangial cells (HGMCs) senescence induced by high glucose (HG). HGMCs were cultured in vitro and divided into the normal group (N, 5.5 mmol·L⁻¹ glucose), the mannitol group(MNT, 5.5 mmol·L⁻¹ glucose plus 24.5 mmol·L⁻¹ mannitol), the high dose of D-glucose group (HG, 30 mmol·L⁻¹ glucose), the low dose of TP group (L-TP, 30 mmol·L⁻¹ glucose plus 5 mg·L⁻¹ TP) and the high dose of TP group (H-TP, 30 mmol·L⁻¹ glucose plus 20 mg·L⁻¹ TP), which were cultured in 5% CO2 at 37 °C, respectively. Firstly, the effects of TP on the cell morphology of HGMCs were observed after 72 h-intervention. Secondly, the cell cycle, the positive rate of senescence-associated-ß-galactosidase (SA-ß-gal) staining and the telomere length were detected, respectively. Finally, the protein expressions of p53, p21 and Rb in the p53-p21-Rb signaling pathway were investigated, respectively. And the expressions of p-STAT3 and miR-126 were examined severally. The results indicated that HG not only arrested the cell cycle in G1 phase but also increased the positive rate of SA-ß-gal staining, and shortened the telomere length. HG led to the protein over-expressions of p53, p21 and Rb and HGMCs senescence by activating the p53-p21-Rb signaling pathway. In addition, L-TP delayed HGMCs senescence by improving the cell cycle G1 arrest, reducing SA-ß-gal staining positive rate and lengthening the telomere length. L-TP reduced the protein over-expressions of p53, P21 and Rb induced by HG and inhibited the telomere-p53-p21-Rb signaling pathway. Moreover, the expression of p-STAT3 was increased and the expression of miR-126 was decreased in HGMCs induced by HG. L-TP reduced the expression of p-STAT3 and increased the expression of miR-126 in HGMCs. In conclusion, HG could induce HGMCs senescence by activating the telomere-p53-p21-Rb signaling pathway in vitro. L-TP could delay HGMCs senescence through regulating STAT3/miR-126 expressions and inhibiting the telomere-p53-p21-Rb signaling pathway activation. These findings could provide the effective interventions in clinic for preventing and treating renal cell senescence in diabetic kidney disease.

Efficacy of early treatment on 52 patients with preneoplastic hepatitis B virus-associated hepatocellular carcinoma by compound Phyllanthus Urinaria L.

OBJECTIVE: To observe the change in the number of antibodies of preneoplastic hepatocellular carcinoma (HCC) using early treatment by Compound Phyllanthus Urinaria L. (CPUL) on patients with preneoplastic hepatitis B virus (HBV)-associated HCC. METHODS: A total of 102 cirrhosis patients with regenerative or dysplastic nodules whose sera were tested positive for at least one of these six proteins (five up-regulated genes URG4, URG7, URG11, URG12 and URG19, and one down-regulated gene DRG2) were assigned randomly to two groups using continual random codes by SPSS software. Fifty-two patients were in the treatment group and 50 patients were in the control group. CPUL was used in the treatment group for 3 years, while the control group did not receive any treatment. The changes in HBV-DNA level, number of antibodies, and hepatocarcinogenesis occurred were observed. Patients who did not develop HCC were followed up for another 2 years. RESULTS: HBV-DNA levels decreased ⩾2log in 22.2% (10/45) of patients in the treatment group in contrast to only 5.0% (2/40) of patients in the control group (P=0.0228). The number of antibodies that were tested positive in the treatment group (1.08±1.01) was significantly lower compared with the control group (2.11±1.12) after 24 months of drug treatment (P<0.01). Both the positive rates of anti-URG11 (33/52) and anti-URG19 (31/52) were over 60% at baseline in the two groups, and were decreased to 48.1% (25/52) and 46.2% (24/52) respectively at 36 months of drug treatment, while the rates increased to 68.0% (34/50) and 66.0% (33/50) respectively (P=0.0417, P=0.0436) in the control group. The positive rate of anti-DRG2 was increased to 55.8% (29/52) at 36 months of drug treatment, while in the control group was decreased to 36.0% (18/50, P=0.0452). Among the 102 patients who developed HCC, 2 were in the treatment group and 9 were in the control group, meaning that a significant difference between the two groups (P=0.0212). In 11 patients who developed HCC, anti-URG11 and anti-URG19 were always positive, while anti-DRG2 was negative. Patients newly developing HCC were 6 (20.0%) in the control group, and only one (2.5%) in the treatment group (P=0.0441) during 2-year follow-up after the end of the treatment. CONCLUSIONS: Anti-URG11, anti-URG19 and anti-DRG2 could be used as early markers in the prediction of the therapeutic efficacy of CPUL in treating preneoplastic HCC. CPUL is useful in preventing or delaying the development of HBV-associated cirrhosis to HCC.

Efficacy of ursodeoxycholic acid combined with Tongdan Decoction () on immunological indices and histopathological changes in primary biliary cirrhosis patients.

OBJECTIVE: To observe the efficacy of ursodeoxycholic acid (UDCA) combined with Tongdan: Decoction () on immunological indices and histopathological changes in patients with primary biliary cirrhosis (PBC) of IIor III histological stage. METHODS: Sixty PBC patients were assigned randomly and equally: to the control group treated with UDCA alone and the treatment group treated with UDCA combined with Tongdan Decoction. The immunological indices and histopathological changes were detected before and after 24-week treatment, and the follow-up lasted for 1-3 years. RESULTS: After 24-week treatment, CD4(+)CD28(-) in the peripheral blood was lowered and CD4(+)CD25(+) was increased in both groups, and better effect was shown in the treatment group (P<0.01). The levels of IgM, IgG, and IgA decreased markedly after 96-week treatment in the treatment group (P< 0.05, P< 0.01), while in the control group, only the latter two showed significant decrease after 148 week (all P<0.05). At the end of the 3-year follow-up, the medians of histopathological

[Effects of ligustrazine injection on high glucose-induced type I collagen, matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 expressions in human peritoneal mesothelial cells in vitro].

OBJECTIVE: To investigate the effects of ligustrazine injection on type I collagen, matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) expressions in human peritoneal mesothelial cells (HPMCs) cultured in high glucose conditions. METHODS: HPMCs were isolated from human omenta by trypsin digestion method and subcultured. Then, the HPMCs were divided into normal control group, high glucose group and high glucose plus low-, medium- and high-dose ligustrazine (10, 20 and 40 mg/L ligustrazine respectively) groups. Semi-quantitative reverse transcription-polymerase chain reaction was used to detect the expressions of type I collagen, MMP-1 and TIMP-1 mRNAs in HPMCs. Proteins of type I collagen, MMP-1 and TIMP-1 in culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA). Cell protein concentration was measured by trace bicinchoninic acid method to correct the ELISA assay results. RESULTS: Ligustrazine injection could significantly decrease high glucose-induced type I collagen and TIMP-1 expressions in a dose-dependent manner both in protein and gene levels (P<0.05, P<0.01). In addition, medium- and high-dose ligustrazine injection could significantly increase MMP-1 expression which was inhibited by high glucose concentrations (P<0.05). CONCLUSION: Ligustrazine injection does not only decrease type I collagen synthesis, but also promote its degradation by modulating unbalanced MMP-1/TIMP-1 expression in HPMCs cultured in high glucose conditions.

[Combined therapy of shehuang paste and colonic dialysis with Chinese medicines for treatment of refractory cirrhotic ascites complicated with azotemia].

OBJECTIVE: To observe the clinical efficacy of combined therapy of Shehuang Paste (SHP) with colonic dialysis in treating patients with refractory cirrhotic ascites complicated with azotemia. METHODS: Adopting a multi-centered, randomized, double blinded and 1:1 parallel controlled trial, 120 patients were equally randomized into 2 groups, the control group was treated by conventional basic therapy (umbilical application of placebo paste and colonic dialysis with normal saline), and the treatment group by, besides the same basic therapy, umbilical application of SHP once a day and colonic dialysis with herbal medicine once every other day. The course was 1 month for both groups. Changes of ascites volume, renal function, serum and urinary levels of Na+ and K+, blood vasoactive substance, and portal dynamics in patients before and after treatment were observed. RESULTS: The total effective rate for ascites was 71.7% (43/60 cases) in the treatment group and 18.3% (11/60 cases) in the control group, showing significant difference between groups (P < 0.01). Significant difference of blood creatinine, urea nitrogen, serum Na+ levels, and urinary Na+/K+ ratio were shown in the treatment group (P < 0.01) before and after treatment, and between groups after treatment (P < 0.05, P < 0.01). Portal vein blood flow was significantly lowered in the treatment group after treatment (P < 0.01), which showed significant difference as compared with that in the control group (P < 0.01). Besides, levels of atrial natriuretic peptide, renin, angiotensin, nitric oxide, and aldosterone decreased and endotoxemia improved remarkably in the treatment group (P < 0.01). One-year follow-up showed that the ascites eliminating rate and the incidence of hepato-renal syndrome in the treatment group was 38.3% (23/60 cases) and 23.3% (14/60 cases) respectively, while in the control group 0 and 41.7% (25/60 cases) respectively, all showed statistical difference between groups (all P < 0.05). CONCLUSION: Combined therapy of SHP and colonic dialysis with herbal medicine could effectively eliminate the ascites, improve the hemodynamic condition of portal and splenic veins, reduce the content of vasoactive substance and noxious substances like ammonia and endotoxin in blood, and lower the incidence of hepato-renal syndrome.

Effects of Shehuang Paste on hemodynamics, endotoxin, nitric oxide and endothelin-1 in patients with refractory cirrhotic ascites.

OBJECTIVE: To explore the influence of Shehuang Paste (SHP) to the hemodynamics, endotoxin, nitric oxide (NO), and endothelin-1 (ET-1) in patients with refractory cirrhotic ascites. METHODS: Fifty-nine cases of refractory cirrhotic ascites were randomly assigned to two groups, 32 cases in the treatment group and 27 cases in the control group. The basic treatment was the same for both groups, including liver protecting medicines, diuretics and supportive drugs, but SHP navel sticking was applied for the treatment group additionally once a day. A course of one month of treatment was applied and the general efficacy on ascites was observed by the end of the therapeutic course. Before and after the treatment, examinations by limulus lysate chromogenic test was conducted to measure plasma endotoxin content; colorimetry to measure plasma content of NO indirectly, radioimmunoassay to measure plasma ET-1 content; and color Doppler ultrasonography to measure the blood flow of portal vein and splenic vein. The relationship between the blood flow of portal vein and splenic vein and endotoxin, NO and ET-1 in the treatment group was analyzed as well. RESULTS: The total effective rate on ascites was 84.4% in the treatment group, and 48. 1% in the control group, with significant difference shown between them (P<0.01). In the treatment group the blood flow of portal vein and splenic vein, contents of endotoxin, NO and ET-1 all got significantly reduced after treatment ( P<0.05 or P<0.01); while these indexes in the control group were not significantly changed ( P 0.05). Moreover, it was found that in the treatment group, the blood flow of portal vein and splenic vein had a positive correlation to the levels of NO, ET-1, and endotoxin, either before or after treatment. CONCLUSION: Application of SHP navel sticking could clearly reduce the blood flow of portal vein and splenic vein, and lower the content of endotoxin, NO and ET-1. The blood flow of portal vein and splenic vein in the treatment group showed a positive correlation with the contents of endotoxin, NO and ET-1. liver cirrhosis, refractory ascites, vasoactive substance, hemodynamics