Objective To compare different blocking methods for eliminating autofluorescence of mouse liver frozen sections, so as to find the best method to reduce the interference to immunofluorescence positive signals and improve the accuracy of immunofluorescence. Methods Intrasplenic injection-liver colonization nude mice: Hepa1-6-GFP cells were intrasplenically injected into male athymic BALB/c nude mice to create liver colonization models. Liver tissues were frozen and continuously sectioned. Sections were blocked with AB reagent (A reagent: streptavidin reagent, B reagent: biotin reagent), blocking buffer, AB reagent＋blocking buffer, or acetone＋AB reagent＋blocking buffer, and then the autofluorescence of the frozen sections was detected. C57BL/6 mice: the liver tissues of C57BL/6 mice were frozen and continuously sectioned, and then the sections were blocked with AB reagent, blocking buffer, AB reagent＋blocking buffer, or acetone＋AB reagent＋blocking buffer. Liver macrophages (Kupffer cells) were labeled with F4/80, and then the autofluorescence of mouse liver frozen sections was detected. Results In the immunofluorescence staining of liver tissue frozen sections, all the above four blocking methods could reduce the autofluorescence of liver sections, and acetone＋AB reagent＋blocking buffer group had the best effect. Conclusion The combined buffers (acetone＋AB reagent＋blocking buffer) has the best effect in eliminating the autofluorescence of mouse liver frozen section.

Anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis (AAV) constitues a group of autoimmune diseases with poor prognosis. Inflammation and fibrinoid necrosis of the small vessels are the pathological features of the disease, and many organs and systems can be involved. Monocyte-macrophages are important to innate immune system. Monocyte-macrophage can respond rapidly to inflammation and participate in the progression of AAV. Recently, the role of monocyte-macrophage in AAV has been studied with great detail. This article reviews recent research progress of monocyte-macrophage in AAV so as to further understand the disease.

This experiment aimed to explore and research the process of preparing baicalein and wogonin through liquid fermentation with Bacillus natto. Active enzymes of produced by B. natto was used for the biological transformation of baclin and wogonoside, in order to increase the content of the haicalein and wogonin in the scutellaria. With the content of the baicalein and wogonin as evaluating indexes, the effects of carbon source, nitrogen source, the types and suitable concentration of inorganic salt, medium pH, granularities of medical materials, liquid volume in flask, shaking speed, liquid-to-solid ratio, fermentation time on the fermentation process were studied. The optimal process conditions for liquid fermentation of scutellaria were 1.0% of peptone, 0.05% of NaCl, pH at 6, the granularities of medical materials of the scutellaria screened through 40-mesh sifter, 33% of liquid, shaker incubator speed at 200 r x min(-1), liquid-to-solid ratio of 5:1, temperature at 37 degrees C, fermentation for 6 days, baclin's conversion rate at 97.6% and wogonoside's conversion rate at 97% in the scutellaria. According to the verification test, the process was stable and feasible, and could provide data reference for the industrial production.
Bacillus subtilis ; metabolism ; Biotransformation ; Fermentation ; Flavanones ; metabolism ; Flavonoids ; metabolism ; Glucosides ; metabolism ; Soy Foods ; microbiology

Enterocolitis, Necrotizing ; metabolism ; pathology ; Female ; Humans ; Immunohistochemistry ; Infant, Newborn ; Intestinal Mucosa ; metabolism ; pathology ; Male ; Platelet Activating Factor ; metabolism

PAK1 plays an important role in the development of tumors. It is of great significance to screen and develop new PAK1 inhibitors as targeted drugs for cancer treatment. The traditional PAK1 inhibitor screening method has the problems of high cost and low efficiency. Computer virtual screening can reduce the cost of finding active lead compounds and improve the screening efficiency. In this study, a kind of PAK1 candidate compound was screened by computer assisted virtual screening combined with Z′lyteTM high flux kinase screen. In vitro enzyme activity screening showed that compound 18(K788)had good PAK1 inhibitory activity(inhibition rate was 42. 7%). Furtherly by MTT detection, it was found that K788 had significant PAK1 positive tumor killing activity, which was even better than the positive drug IPA-3. Flow cytometry and Western Blot showed that K788 could activate caspase apoptosis pathway and induce apoptosis of colon cancer cell DLD-1 by inhibiting PAK1 expression and activation. K788 has great potential for clinical development and application, and can be used as a PAK1 target for further research.

This study was aimed to investigate whether endothelium-specific deletion of PTEN can affect hemangioblast development in the AGM region of mouse embryos. Based on Cre/loxP system, the Tie2CrePten(loxp/loxp) and Tie2CrePten(loxp/wt) mouse embryos were obtained. The genotype was identified by PCR. After treated with type I collagenase, the AGM region was dispersed into single-cell suspension, and then was cultured in blast colony-forming cell (BL-CFC) media. The number of BL-CFC was counted 4 or 5 days later. The hematopoietic capacity of BL-CFC was detected in methylcellulose culture system and the endothelial potential was assessed by tube-like structure formation on Matrigel. The results showed that the number of BL-CFC in AGM region of Tie2CrePten(loxp/loxp) mouse embryo decreased as compared with Tie2CrePten(loxp/wt) embryo. Whereas the hematopoietic capacity of mutant BL-CFC was enhanced, the endothelial potential, as evaluated by tube-like structure formation in vitro, was significantly reduced. It is concluded that the endothelial PTEN is capable of exerting regulatory functions on both the numbers and the dual potential of hemangioblast in mouse AGM region.
Animals ; Cell Differentiation ; Cells, Cultured ; Hemangioblasts ; Hematopoietic Stem Cells ; cytology ; Mice ; PTEN Phosphohydrolase ; genetics

OBJECTIVETo observe the antimicrobial activity of topical agents commonly used for burns on Acinetobacter baumannii (AB) in both free and biofilm states, and their synergistic effect with ambroxol on AB within biofilm.

METHODSEleven AB strains were isolated from wound excretion, respiratory tract, and blood of patients hospitalized in our hospital from August 2005 to April 2007. (1) The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of mafenide acetate and chlorhexidine acetate to free AB (including drug-resistant, drug-sensitive, and standard strains) were determined by dilution method. (2) AB was cultured with LB or TSB medium for 12, 24, and 48 h to form biofilm, and it was treated with above-mentioned two topical agents in MBC (mafenide group and chlorhexidine group) for 30 min. Biofilm not treated by topical agent was used as control group. The biofilm thickness was determined with confocal laser scanning microscope. The proportion of living bacteria in biofilm was calculated. AB biofilm in each topical agent group was mixed and inoculated into LB culture dish to observe the growth of bacteria. (3) AB was cultured with LB medium for 48 h to form biofilm, which was respectively treated by above-mentioned two topical agents in MBC (mafenide group and chlorhexidine group) and combination of each topical agent with 3.75 mg/mL ambroxol solution (ambroxol + mafenide group and ambroxol + chlorhexidine group) for 30 min. Biofilm not treated by topical agents was used as control group. Growth of bacteria in biofilm was detected with MTT method (denoted as absorbance value). Data were processed with one-way analysis of variance and LSD-t test.

RESULTS(1) MIC of mafenide acetate and chlorhexidine acetate for free AB was respectively 25.00 mg/mL and 0.03 mg/mL. MBC of both agents for free AB was the same as their MIC. (2) Among three groups, the thickness of biofilm of sensitive AB was thicker than that of drug-resistant bacteria at most of the time points. Compared with those in control group, biofilm thickness and proportion of living bacteria in biofilm were slightly decreased in mafenide and chlorhexidine groups. The growth of bacteria was abundant in each group. (3) Absorbance value of drug-resistant bacteria in control, mafenide, and chlorhexidine groups was respectively 0.776 ± 0.071, 0.625 ± 0.063, and 0.420 ± 0.068. Absorbance value of drug-resistant bacteria in ambroxol + mafenide group (0.174 ± 0.089) was significantly lower than that of control group (t = 11.823, P = 0.000) and mafenide group (t = 9.248, P < 0.01). Absorbance value of ambroxol + chlorhexidine group (0.178 ± 0.044) was significantly lower than that of control group (t = 16.009, P = 0.000) and chlorhexidine group (t = 6.681, P < 0.01).

CONCLUSIONSDrug-resistant AB forms biofilm readily, which prevents topical agents from killing the bacteria inside. Combined use of ambroxol with topical agents gives synergistic effect on killing AB in biofilm in the wound.

Acinetobacter baumannii ; drug effects ; isolation & purification ; Anti-Bacterial Agents ; pharmacology ; Biofilms ; drug effects ; Burns ; microbiology ; Chlorhexidine ; pharmacology ; Drug Resistance, Bacterial ; Humans ; Mafenide ; pharmacology ; Microbial Sensitivity Tests

OBJECTIVETo study the physiopathologic basis of Weikangfu Granule (WKFG) in treating precancerosis of gastric mucosa in patients of chronic gastritis with Pi-deficiency syndrome (CG-PDS).

METHODSOne hundred and fifteen patients of CG-PDS who suffered from intestinal metaplasia (IM) and atypical hyperplasia (ATHP) of gastric mucosa, were divided into two groups. The treated group (n = 61) was treated by WKFG with its ingredients modified according to the syndrome type of patients. The control group (n = 54) was treated with Weishu granule. The histopathological and subcellular ultrastructural changes were detected by optical microscope, screening electronic microscope, transmission electronic microscope and histochemical staining; the nuclear and mitochondrial ultrastructure of gastric mucosa were analyzed with energy dispersion X-ray analyser and image analysis system. And the changes of cAMP, lipid peroxide (LPO), superoxide dismutase (SOD) before and after treatment in the treated group were measured and compared with those of the health control group consisting of 15 volunteers.

RESULTSThe symptomatic and pathological therapeutic effect in the treated group were significantly superior to those in the control group (P < 0.05). The contents of Zn, Cu, cAMP, SOD and (3)H-TdR LCT in gastric mucosa of the treated group before treatment were all lower than those of the healthy control group, yet all these indexes markedly increased after treatment, while serum LPO level, which increased before treatment was lowered after treatment. All the changes showed statistical significance (P < 0.05 or P < 0.01).

CONCLUSIONWKFG can reverse IM and ATHP in patients of CG-PDS, and the effect may be realized by way of increasing the level of Zn, Cu, cAMP and SOD in gastric mucosa, promoting cell differentiation, enhancing cellular immunity and reducing oxygen free radicals and lipid peroxidation.

Adult ; Aged ; Antineoplastic Agents ; pharmacology ; therapeutic use ; Chronic Disease ; Copper ; analysis ; Cyclic AMP ; analysis ; Drugs, Chinese Herbal ; therapeutic use ; Gastric Mucosa ; chemistry ; pathology ; ultrastructure ; Gastritis, Atrophic ; pathology ; Humans ; Lipid Peroxides ; analysis ; Male ; Medicine, Chinese Traditional ; Middle Aged ; Precancerous Conditions ; pathology ; Stomach Neoplasms ; pathology ; Superoxide Dismutase ; analysis ; Syndrome ; Yang Deficiency ; complications ; Zinc ; analysis

Excessive fluid resuscitation in massive burn patients is a common phenomenon in burn management, and the reasons are mostly related with administering resuscitation of crystalloid alone and pursuing a goal-directed resuscitation with targeting normal hemodynamic parameters in the first 24 h post burn. Tissue edema caused by excessive fluid resuscitation is a vital factor that induces complications including respiratory compromise, abdominal compartment syndrome, and so on. Therefore, in order to control excessive fluid resuscitation and prevent its subsequent complications in massive burn patients, it is necessary to determine the optimal resuscitation regime, set appropriate resuscitation endpoints, and implement precise management of fluid resuscitation.
Fluid Therapy ; Humans ; Resuscitation

The fusion protein (F) gene of Newcastle disease virus was amplified by polymerase chain reaction (PCR) from the recombinant plasmid pVAX1-F, and subcloned into eukaryotic expression vector pmcDNA3. 1+. The F gene was identified by sequencing. The recombinant plasmid was transformed into attenuated Salmonella typhimurium SL7207, and the recombinant was designated as SL7207 (pmcDNA3. 1-F). In vitro and in vivo experiments showed that the plasmid stability of pmcDNA3. 1-F was apparently higher than that of pcDNA3. 1-F in SL7207. In order to compare the immune response induced by these two re combinant bacteria, BALB/c mice were immunized orally with them at the dosage of 2 x 10(9) CFU respectively. Both SL7207(pcDNA3. 1-F) and SL7207(pmcDNA3. 1-F) initiated F-specific serum and mucosal antibodies in immunized mice. Furthermore, 4-day-old SPF chickens were immunized with SL7207(pcDNA3. 1-F) and SL7207(pmcDNA3. 1-F) at the dosage of 5 x 10(9) CFU and boosted two weeks later with the same dosage. Humoral and intestinal mucosal immune responses were observed and their levels were significantly higher than that of negative and positive controls. The result of protective efficacy showed that the chickens immunized with SL7207(pmcDNA3. 1-F) had the protective rate of 70.0%, higher than that of the SL7207 (pcDNA3. 1-F) with 50.0%. In summary, the DNA vaccine delivered by attenuated Salmonella typhimurium has good immunogenicity. A novel mucosal DNA vaccine has been developed and could be useful for controlling the infection and epidemic of Newcastle disease in the poultry.
Animals ; Chickens ; Female ; Immunization ; Mice ; Mice, Inbred BALB C ; Newcastle disease virus ; immunology ; Plasmids ; Salmonella typhimurium ; genetics ; Vaccines, Attenuated ; immunology ; Vaccines, DNA ; immunology ; Viral Vaccines ; immunology