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In order to overcome the challenges of insufficient restriction enzyme sites, and construct a fusion-expression vector with flexible fusion direction, we designed an LB cloning system based on the type IIS and type IIT restriction enzymes LguⅠ and BbvCⅠ. The LB cloning system is constructed by inserting the LB fragment (GCTCTTCCTCAGC) into the multiple cloning site region of the broad-host plasmid pBBR1MCS-3 using PCR. The LB fragment contains partially overlapped recognition sites of LguⅠ and BbvCⅠ. Therefore, the same non-palindromic sequence will be generated by these two restriction endonucleases digestion. This feature can be used to quickly and flexibly insert multiple genes into the expression vector in a stepwise and directed way. In order to verify the efficacy of the cloning system, two glycosyltransferase genes welB and welK of Sphingomonas sp. WG were consecutively fused to the LB cloning vector, and the recombinant plasmid was transferred into Sphingomonas sp. WG by triparental mating. The results showed that gene fusion expression has little effect on sphingan titer, but enhanced the viscosity of sphingan. The viscosity of the sphingan produced by recombinant strain Sphingomonas sp. WG/pBBR1MCS-3-LB-welKB was 24.7% higher than that of the wild strain after fermentation for 84 h, which would be beneficial for its application. In conclusion, the application of LB cloning system were verified using Sphingomonas sp. WG. The LB cloning system may provide an efficient tool for fusion expression of target genes.
Subject(s)
Base Sequence , Cloning, Molecular , Fermentation , Plasmids/genetics , Sphingomonas/metabolismABSTRACT
Background: The objectives of the present study were to clinically evaluate the effectiveness of chitosan mouthwash on Plaque formation and to evaluate the antimicrobial activity against salivary Streptococcus mutans (SM) and Lactobacillus (LB).Methods: A total of 20 participants with decay-missing-filled index ?4 and simplified oral hygiene index score >1.3 were recruited for the study after taking informed consent. All the participants were provided a bottle of chitosan mouthwash in addition to their usual oral hygiene procedure. Baseline salivary SM and LB levels were determined, using chair-side CRT� bacteria test prior giving chitosan mouthwash and after 14 days, during which children are advised to use 10 ml of mouthwash for 60 seconds two times daily. Plaque index score recorded using Silness and Loe plaque index. Paired t-test (or corresponding non-parametric) and percentage comparison method using cross tables were used for statistical analysis.Results: The baseline plaque index score of 0.758 and after 14 days of mouthwash use, it reduced to 0.434. The mean difference in the plaque index showed significant reduction in the plaque score (i.e., with the difference of 0.324). The results showed the antiplaque effects with a short duration of 14 days use of chitosan (CH) mouthwash. The antibacterial activity demonstrated a range of inhibitory effect on salivary SM and LB.Conclusions: CH showed an evident strong effect against salivary SM and LB levels and controlling the plaque biofilm formation. So water soluble CH can be used in new formulations for oral applications not only as antimicrobial agent but also for plaque biofilm control.
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Objective To investigate the effects of 3 kinds of broth media,including tryptose soya broth(TSB),LB and M-H,on the biofilm formation of Staphylococcus epidermidis (S.epidermidis).Methods The effects of TSB,LB and M-H broth media on the biofilm formation of S.epidermidis were investigated by the construction of bacterial biofilm in 96-well and 6-well microplates and the crystal violet straining for the semi-quantitative analysis and microscopic observation of the bacterial biofilm.The effects of TSB,LB and M-H broth media on the expression of adhesion-related genes in S.epidermidis were determined by the extraction of bacterial total RNA,reverse transcription and real-time PCR.Results Compared with LB (0.149 ± 0.047) and M-H (0.323 ± 0.003) media,TSB medium (2.954 ± 0.287) could significantly promote the biofilm formation of S.epidermidis (TSB vs LB,t =16.706,P ＜ 0.01;TSB vs M-H,t =15.877,P ＜ 0.01).Compared with LB medium,TSB medium could significantly enhance the expression of icaA gene (t =9.667,P＜0.01) but inhibit icaR gene (t =13.283,P＜0.01).Conclusion Compared with LB and M-H broth media,TSB medium may significantly improve the biofilm formation and the expression of adhesion-related gene icaA of S.epidermidis.
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Resumen:La Entomología Forense es una ciencia que implica el estudio de las especies antropofágicas que practican un ciclo vital en el cadáver. Contribuye en la determinación del Intervalo posmortem. Los insectos de importancia forense que se estudiaron pertenecen a la orden Dípteros y Coleópteros. En el estudio realizado en el municipio de Pucarani, ubicado en la provinciaLos Andes del Departamento La Paz a 3852 m.s.n.m. entre otoño e invierno, se obtuvo dos ejemplares de cerdo S. scrofa, en dos ambientes: abierto (cerdo A) y cerrado: (cerdo B). Se identificó 339 especímenes entre dípteros y coleópteros. No se encontraron diferencias significativas en la sucesión de entomofauna entre ambos ambientes, sin embargo predominó la familia Fannide sp. en el lugar cerrado por ser un ambiente húmedo. Los dípteros de mayor importancia son: Sarconesia Chlorogaster, Clorobrachycoma splendida, que pertenecen a la familia Calliphoridae, que se mantuvo en constante actividaden su estadio larval y su forma adulta en los cuatro estados de descomposición a temperaturas de 14 - 16°C, humedad relativa de 30%; no se encontró a Calliphora nigribasis en la descomposición avanzada; Sarconesiopsis magellánica solo se presenta en la etapa Fresco. Entre los Coleópteros: Oxelitrum apicale apareció desde la etapa hinchado. La sucesión de entomofauna cadavérica y su particularidad en cada etapa de descomposición, es el elemento que nos ayudaría a determinar el IPM, que dependerá de las condiciones climáticas a gran altitud como Bolivia.
Abstract:Forensic entomology is a science that involves the study of the cannibalistic species that plays a vital cycle in the body. It helps to determinate the postmortem interval. Insects with forensic importance that were studied belong to the order Diptera and Coleoptera. In the study conducted in the Municipality of Pucarani, located in the province of Los Andes La Paz Department at 3852 meters between autumn and winter, two copies of pork S. scrofa was obtained in two environments: open (pork A), closed (pig B). 339 specimens between Diptera and Coleoptera were identified. No significant differences were found in the entomofauna sequence between the two environments, however the Fannide sp. In the place closed for being a humid environment. The most important dipterans are: Sarconesia Chlorogaster, Clorobrachycoma splendida, belonging to the family Calliphoridae, which remained in constant activity in its larval stage and its adult form in the four decaying states at temperatures of 14 - 16 ° C, humidity Relative to 30%; Calliphora nigribasis was not found in advanced decomposition; Magellanic sarconesiopsis only occurs in the Fresco stage. Among the Coleoptera: Oxelitrum apicale appeared from the swollen stage. The succession of cadaveric entomofauna and its particularity in each stage of decomposition is the element that would help us determine the MPI, which will depend on high altitude climatic conditions like Bolivia.
Subject(s)
Animals , Swine , Coleoptera , Bolivia , Cadaver , Forensic Sciences , Diptera , Entomology , InsectaABSTRACT
OBJECTIVES: This study was aimed at evaluating the clinical usability of a new caries activity test (Cariview(R)), which was based on the acidogenic potential of plaque by evaluating it's correlation with the DMFT index and comparing the results of Cariview(R) with those of previous caries activity tests in an adult group. METHODS: Teeth of 74 subjects were examined, and the DMFT index was calculated according to World Health Organization (WHO) criteria to determine the past caries experience. Caries activity tests were performed according to manufacturer's instructions. For Cariview(R) test, plaque samples were collected from the tooth surface by using a sterile cotton swab and incubated at 37degrees C for 48 h; an indicator was added to observe the color changes according to the plaque acidogenicity. The subjects were classified into three groups (Low, Moderate, High) according to the level of caries risk. The data were assessed using one-way ANOVA to compare the caries experiences of risk groups according to the caries activity test results, and the relationships between the caries activity tests and the DMFT index were evaluated. RESULTS: The overall mean DMFT index was 5.70+/-4.42. There was a significant difference in the DMFT index among the caries risk groups (Low, Moderate, High) according to the Cariview(R) test result (P=0.036). Further, there was a positive correlation between the Cariview(R) score and the DMFT index (r=0.23, P=0.047) and between the Cariview(R) score and the previous caries activity tests score (P<0.01). CONCLUSIONS: The new caries activity test (Cariview(R)) can be easily used at dental clinics and enables precise caries risk assessment.
Subject(s)
Adult , Humans , Dental Clinics , Dental Plaque , Risk Assessment , Tooth , World Health OrganizationABSTRACT
A new kind of biosensor for immunology was developed by ultrasonic technique and LB membrane. A double delay-line resonator was made by using ST-cut quartz crystal with working frequency of 149.7 MHz. Then a layer of LB membrane was covered on it. When anti-IgM antibody of various concentrations was added to it, the sensor can be used to detect IgM antigen. The biosensor was highly sensitive, small and light. The experimental results showed that the working frequency change of the sensor was proportional to the concentration of antibody with its dilution ratio between 1:10000 and 1:100. It was also first observed that the frequency curve of the sensor resulting from the reaction of IgM antigen and antibody undulated in the experiment.
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A new kind of biosensor for immunology was developed by ultrasonic technique and LB membrane. A double delay-line resonator was made by using ST-cut quartz crystal with working frequency of 149.7 MHz. Then a layer of LB membrane was covered on it. When anti-IgM antibody of various concentrations was added to it, the sensor can be used to detect IgM antigen. The biosensor was highly sensitive, small and light. The experimental results showed that the working frequency change of the sensor was proportional to the concentration of antibody with its dilution ratio between 1:10000 and 1:100. It was also first observed that the frequency curve of the sensor resulting from the reaction of IgM antigen and antibody undulated in the experiment.
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BACKGROUND: The expression of adhesion molecules contribute to development of systemic diseases. Vascular cell adhesion molecule-l(VCAM-1) is an endothelial cell membrane glycoprotein that has been implicated in leukocyte/endothelial cell interactions in inflammation. OBJECTIVE: The aim of this study was to characterize the surface expression and regulation of VCAM-1 on two different endothelial cells. METHOD: We examined the effects of the expression of VCAM-1 in two different endothelial cells, isolated from human umbilical cords and human glomerulus. Expression of VCAM-1 was measured by enzyme-linked immunosorbent assay(ELISA) and flow cytometry. RESULTS: In human umbilical cord endothelial cells(HUVECs), both interleukin-l B(IL-lB) and tumor necrosis factor-a (TNF-a) increased VCAM-1 expression. VCAM-1 expression increased by TNF-a was higher than that increased by IL-lB. In human glomerular endothelial cells(HGECs), IL-lB and TNF-a markedly increased VCAM-1 expression. Conclusion. The regulation of VCAM-1 appears to be somewhat different in HGECs compared with HUVECs. These differences between the responsiveness of the two cells may possibly indicate inherent differences in endothelial cell derived from different vascular beds.