The major goal of microbial ecology is to study the structure and function of complex micro-bial communities. New molecular biological techniques have been successfully applied to analyze mi-crobial community structure. However they do not provide information on the physiologic properties of the detected microorganisms. A new tool for structure-function analyses in microbial ecology, micro-autoradiography combined with fluorescence in situ hybridization (MAR-FISH) can be used to simul-taneously examine the phylogenetic identity and the specific activity of microorganisms within a com-plex microbial community at a single-cell level. This article reviews the principle, experimental steps of MAR-FISH technique. The application of this technique in study of the environmental microbial com-munity and function is also summarized.

Objective:To investigate the protective effect and potential mechanism of cordycepin on renal proximal tubular cells injury induced by lipopolysaccharide (LPS).Methods:Renal proximal tubular cells NRK-52E were incubated on a cell culture plated at a density of 1×10 5/mL for experiment, then divided into control group (Ctrl group), LPS group (cells were stimulated with 1 mg/L LPS), 10 μmol/L or 20 μmol/L cordycep in intervention groups (LPS+C 10 group and LPS+C 20 group). Cell viability was measured using cell counting kit-8 (CCK-8) reagent. The level of intracellular reactive oxygen species (ROS) was detected by 2',7'-dichlorofluorescin diacetate (DCFH-DA) staining. The protein expressions of inflammatory factors intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), interleukin-1β (IL-1β), and nuclear factor-κB (NF-κB) were detected by Western blotting. Results:Compared with the Ctrl group, LPS significantly inhibited NRK-52E cell viability, increased intracellular ROS, and up-regulated the expressions of ICAM-1, VCAM-1, IL-1β and NF-κB. Compared with LPS group, after treated with 10 μmol/L or 20 μmol/L cordycepin, NRK-52E cell viability was significantly increased (Ctrl group as 1: 0.717±0.017, 0.916±0.036 vs. 0.554±0.046) and intracellular ROS level was significantly decreased (Ctrl group as 1: 1.527±0.165, 1.098±0.168 vs. 2.543±0.127), meanwhile the expressions of ICAM-1, VCAM-1, IL-1β and NF-κB were significantly down-regulated [Ctrl group as 1, ICAM-1/GAPDH: 2.364±0.097, 1.561±0.074 vs. 3.101±0.121; VCAM-1/GAPDH: 2.866±0.135, 1.920±0.098 vs. 4.170±0.119; IL-1β/GAPDH: 2.358±0.107, 1.563±0.179 vs. 3.301±0.210; phosphorylation NF-κB p65 (NF-κB p-p65)/GAPDH: 2.559±0.166, 1.596±0.148 vs. 3.183±0.098], the differences were statistically significant (all P < 0.05). Compared with the LPS+C 10 group, the cell activity of LPS+C 20 group was more significant (0.916±0.036 vs. 0.717±0.017, P < 0.01), and the expressions of ICAM-1, VCAM-1, IL-1β, NF-κB were down-regulated more significantly (ICAM-1/GAPDH: 1.561±0.074 vs. 2.364±0.097, VCAM-1/GAPDH: 1.920±0.098 vs. 2.866±0.135, IL-1β/GAPDH: 1.563±0.179 vs. 2.358±0.107, NF-κB p-p65/GAPDH: 1.596±0.148 vs. 2.559±0.166, all P < 0.05).Conclusion:Cordycepin could significantly increase the survival rate of NRK-52E cells, reduce intracellular ROS level, and inhibit inflammation, and the anti-inflammation effect can be related with NF-κB pathway.

Objective To prepare monoclonal antibody(McAb) against human tissue kallikrein (HK) and develop an ELISA kit allows for the in vitro quantitative determination of human tissue kallikrein in urine. Methods To generate a monoclonal antibody specific for TK, the synthetic TK peptide consisting of 12 amine acids(12P), was fused to keyhole limpet hemocyanin(KLH) and used for immunization. Using hybridoma screening, monoclonal secreting cell lines were identified and used to generate ascites in BALB/c mouse. Antibody was purified by affinity column chromatography. 12% SDS-PAGE and Western blot were used to visualize the purified antibody. This kit employs indirect competitive ELISA technique and BiotinAvidin System. 12P was fused to bovine serum albumin(BSA) and has been pre-coated onto a microplate at first. Standards and samples were added to the appropriate microplate wells with a biotin-conjugated McAb croplate well. A TMB substrate solution is added to each well. The enzyme-substrate reaction is terminating by the addition of a sulphuric acid solution and the color change is measured spectrotometrically at a wavelength of 450 nm. The concentration of tissue kallikrein in the samples is then determined by comparing the O.D. of the samples to the standard curve. Results 8 hybridoma cell lines secreting mAbs special to HK,SDS-PAGE and Western blot demonstrated successful preparing and purification of McAb( 100% ). The linearity of this ELISA kit is demonstrated(r =0. 990). The range of detection of the assay is 0.008 μg/ml to 0. 5 μg/ml. The assay remained stable, with no change in the values measured, over five cycles of freezing and thawing. Conclusion 8 McAbs against HK have been prepared successfully and possess high titer and specificity. The development of an ELISA kit for detecting HK can meet the needs of detection of HK in urine samples.

4-Hydroxycoumarins ; blood ; Animals ; Chromatography, High Pressure Liquid ; methods

Acupuncture Therapy ; Adult ; Aged ; Diplopia ; therapy ; Female ; Humans ; Male ; Medicine, Chinese Traditional ; Middle Aged

Background Many researches confirmed that basic fibroblast growth factor (bFGF)plays an important role on the proliferation and differentiation of retinal progenitor cells,but its low intraocular permeability limits its clinical application.To explore an effective approach to enhance the intraocular permeability of bFGF has an important significance for the treatment of retinopathy. Objective This study was to investigate the effect of azone on bFGF intraocular permeability after its topical administration. Methods Eighteen New Zealand white rabbits were randomly divided into four groups on random number table method.Distilled water( blank control group),5％ bFGF eyedrops(5％ bFGF group ),0.4％ azone+5％ bFGF eyedrops (0.4％ azone + 5％ bFGF group ) and 0.4％ azone+ 10％ bFGF eyedrops (0.4％ azone + 10％ bFGF group)were topically administered in different groups at 5- minute interval for 3 times.Aqueous and vitreous fluid were extracted 30 minutes after administration of eyedrops,and those in the 0.4％ azone + 5％ bFGF group were obtained 30,60 and 120 minutes after administration.bFGF concentration in the aqueous and vitreous fluid was quantified with ELISA. Results The bFGF levels(A value)in aqueous and vitreous fluid were 0.1007±0.0100 and 0.1340±0.0100 after topical administration of the 5％ bFGF eyedrops,those in blank control group were 0.1363 ±0.0100 and 0.1130±0.0100,respectively,and those in the 0.4％ azone+5％ bFGF group and 0.4％ azone+10％ bFGF group were significantly higher than the 5％ bFGF group ( both P=0.000).However,no significant difference was found in bFGF levels between 0.4％ azone+5％ bFGF group and 0.4％ azone + 10％ bFGF groups in both aqueous and vitreous fluid ( P =0.985,0.098 ).A value of bFGF in aqueous was gradually increased with prolong of time in the 0.4％ azone+5％ bFGF group,with the values 0.9413±0.0300 at 30 minutes,0.3865±0.0300 at 60 minutes,and 0.2550±0.0300 at 120 minutes,showing a positive linear correlation between bFGF level and time( R2 =0.736,P =0.003 ),but no significant correlation was seen in vitreous sample(R2=0.196,P=0.233). Conclusions Azone can improve the intraocular penetration of bFGF eyedrops.Increasing the concentration of bFGF in eyedrop from 5％ to 10％ dose not change its intraocular distribution.The highest content of the bFGF in aqueous is at 30 minutes following the administration of 0.4％ azone+5％ bFGF eyedrops.

At present, majority of the small molecular drugs used in clinics target proteins, they exert the efficacy through the binding to specific sites on the target protein. However, the "druggable" protein targets account for a small portion of the total number of proteins, and "non-druggable" proteins account for 80%, because of not having suitable drug binding sites. In the central rule, RNA is located in the upstream of proteins and controls the transcription of proteins. The research of small molecule drugs targeting RNA can solve the problem of protein "undruggable proteins" in some extent. This review summarizes the representative research achievements of small molecular drugs targeting RNA in recent years, and the screening methods applied to this field, with the focuses on the latest progress of small molecular drugs targeting novel coronavirus RNA.

In order to identify Aucklandiae Radix, Vladimiriae Radix, Inulae Radix, Aristolochiae Radix and Kadsurae Radix using ITS2 barcodes, genomic DNA from sixty samples was extracted and the ITS2 (internal transcribed spacer) regions were amplified and sequenced. The genetic distances were computed using MEGA 5.0 in accordance with the kimura 2-parameter (K2P) model and the neighbor-joining (NJ) phylogenetic tree was constructed. The results indicated that for Aucklandiae Radix (Aucklandia lappa), Vladimiriae Radix (Vladimiria souliei and V. souliei var. cinerea), Inulae Radix (Inula helenium), Aristolochiae Radix (Aristolochia debilis) and Kadsurae Radix (Kadsura longipedunculata), the intra-specific variation was smaller than inter-specific one. There are 162 variable sites among 272 bp after alignment of all ITS2 sequence haplotypes. For each species, the intra-specific genetic distances were also smaller than inter-specific one. Furthermore, the NJ tree strongly supported that Aucklandiae Radix, Vladimiriae Radix, Inulae Radix, Aristolochiae Radix and Kadsurae Radix can be differentiated. At the same time, V. souliei (Dolomiaea souliei) and V. souliei var. cinerea( D. souliei var. cinerea) belonging to Vladimiriae Radix were clearly identified. In conclusion, ITS2 barcode could be used to identify Aucklandiae Radix, Vladimiriae Radix, Inulae Radix, Aristolochiae Radix and Kadsurae Radix. Our study may provide a scientific foundation for clinical safe use of the traditional Chinese medicines.
Aristolochia ; classification ; genetics ; Base Sequence ; DNA Barcoding, Taxonomic ; methods ; DNA, Plant ; genetics ; DNA, Ribosomal Spacer ; genetics ; Drugs, Chinese Herbal ; chemistry ; classification ; Molecular Sequence Data ; Phylogeny ; Plants, Medicinal ; classification ; genetics ; Quality Control

Objective To study the therapeutic effects of human umbilical cord mesenchymal stem cells (hUCMSCs) on acute liver failure ( ALF) induced by D-galactosamine (D-gal) and lipopolysaccharide (LPS) in rats. Methods The ALF model was obtained through intraperitoneal injection of D-gal(300 mg/kg)and LPS (20μg/kg)in Wister rats. The hUCMSCs were transplanted after intoxication. All rats were divided into four groups, and each group received either hUCMSCs or 0.9% NaCl solution through intraperitoneal or tail-intravenous injection. To evaluate the liver function of each group, the levels of alanine aminotransferase (ALT), total bilirubin (TBil) and serum albumin (Alb) were measured on the day of hUCMSCs transplantation and the following 1, 2, 3, 5 and 7 days. All rats were then sacrificed to examine the liver histology at day 7. Analyses were done by using Fisher's exact test, unpaired t test and Mann-Whitney U test. Results There were no significant differences of survival rates among four groups (Fisher's exact test, both P = 1. 00). The levels of ALT, TBil and Alb in group receiving hUCMSCs intraperitoneally were (804. 9 ± 88. 0) U/L,(17. 4±2. 7) μmol/L and (20. 9±0. 8) g/L, respectively after 2 days of injection, whereas in the corresponding control group, those were (1294. 3± 171. 4) U/L, (32. 3±5. 5) μmol/L and (16. 1±0. 9) g/L, respectively, which indicated that hUCMSCs transplantation significantly improved the liver function (t = 2. 640, P =0.020;t=2.529, P = 0. 025;t= - 3. 833, P = 0. 002). Both of hUCMSCs-transplanted groups showed no significant differences. Liver histological data showed that transplantation of hUSMSCs through either intraperitoneal or tail-intravenous injection alleviated liver damage (U=4. 500, P = 0. 005;U=4. 500, P = 0. 008) and the mitotic index also increased in hUCMSCs-transplanted groups (U=4. 000, P = 0. 005; U=5. 500, P = 0. 013). Conclusions The levels of ALT, TBil and Alb can rapidly normalize in ALF rats after injected with hUCMSCs either intraperitoneally or tail-intravenously. hUCMSCs application raises the mitotic index, enhances hepatocellular regeneration and improves histological status.

Objective To investigate the relationship between arsenic in drinking water and skin lesions in endemic arsenism area in Shanyin County of Shanxi Province,in order to provide epidemiologic data for further arsenism research.Methods One hundred and eighty-nine endemic arsenism patients and 59 controls were randomly selected in 17 endemic amenism countries in Shanyin County of Shanxi Province.The content of arsenic in drinking water which wa8 collected indoom was half-quantitatively screened by a kit made by Chinese Center for Disease Control and Prevention,then quantitatively determined by HPLC-ICP-MS.Patients of endemic arsenism were diagnosed by "The Standard of Diagnosis for Endemic Amenism"(WS/T 211-2001).Results There were 64.9% (87/134)samples above the arsenic level(50μg/L)of drinking water and the median value of arsenic in drinking water was 91.43 μg/L in 134 water samples.The OR(95%CI)value between arsenic in drinking water and hyperkeratosis,hyperpigmentation,depigmentation was 2.46(1.22-4.94),3.34(1.50～7.44)and 2.86(1.50-5.46),respectively.The prevalence of hyperkeratosis,hyperpigmentation and depigmentation increased,as the arsenic in drinking water increased(≤10,≤50,≤200,＞200μg/L),especially in＞200μg/L group(OR=6.15,13.96,11.41,P＜0.05).The arsenic level in drinking water of Ⅲ degree of depigmentation patients(318.300μg/L)was higher(P＜0.05)than that of 0,Ⅰ and Ⅱ degree groups(86.670,131.800,1 10.590μg/L,P＜0.05).Conclusions Shanyin County is a medial arsenic pollution area. Arsenic in drinking water is considered as a risk factor of skin lesion. The degree of skin lesions increased,as the arsenic in drinking water increased.