Sequential gene expression analysis of myelodysplastic syndrome transformation identifies HOXB3 and HOXB7 as the novel targets for mesenchymal cells in disease.

BACKGROUND: Myelodysplastic syndrome (MDS) is known to arise through the pathogenic bone marrow mesenchymal stem cells (MSC) by interacting with hematopoietic stem cells (HSC). However, due to the strong heterogeneity of MDS patients, it is difficult to find common targets in studies with limited sample sizes. This study aimed to describe sequential molecular changes and identify biomarkers in MSC of MDS transformation. METHODS: Multidimensional data from three publicly available microarray and TCGA datasets were analyzed. MDS-MSC was further isolated and cultured in vitro to determine the potential diagnostic and prognostic value of the identified biomarkers. RESULTS: We demonstrated that normal MSCs presented greater molecular homogeneity than MDS-MSC. Biological process (embryonic skeletal system morphogenesis and angiogenesis) and pathways (p53 and MAPK) were enriched according to the differential gene expression. Furthermore, we identified HOXB3 and HOXB7 as potential causative genes gradually upregulated during the normal-MDS-AML transition. Blocking the HOXB3 and HOXB7 in MSCs could enhance the cell proliferation and differentiation, inhibit cell apoptosis and restore the function that supports hematopoietic differentiation in HSCs. CONCLUSION: Our comprehensive study of gene expression profiling has identified dysregulated genes and biological processes in MSCs during MDS. HOXB3 and HOXB7 are proposed as novel surrogate targets for therapeutic and diagnostic applications in MDS.

Effect of peptidoglycan amidase MSMEG_6281 on fatty acid metabolism in Mycobacterium smegmatis.

Mycobacterium smegmatis MSMEG_6281, a peptidoglycan (PG) amidase, is essential in maintaining cell wall integrity. To address the potential roles during the MSMEG_6281-mediated biological process, we compared proteomes from wild-type M.smegmatis and MSMEG_6281 gene knockout strain (M.sm-ΔM_6281) using LC-MS/MS analysis. Peptide analysis revealed that 851 proteins were differentially produced with at least 1.2-fold changes, including some proteins involved in fatty acid metabolism such as acyl-CoA synthase, acyl-CoA dehydrogenase, MCE-family proteins, ATP-binding cassette (ABC) transporters, and MmpL4. Some proteins related to fatty acid degradation were enriched through protein-protein interaction analysis. Therefore, proteomic data showed that a lack of MSMEG_6281 affected fatty acid metabolism. Mycobacteria can produce diverse lipid molecules ranging from single fatty acids to highly complex mycolic acids, and mycobacterial surface-exposed lipids may impact biofilm formation. In this study, we also assessed the effects of MSMEG_6281 on biofilm phenotype using semi-quantitative and morphology analysis methods. These results found that M.sm-ΔM_6281 exhibited a delayed biofilm phenotype compared to that of the wild-type M.smegmatis, and the changes were recovered when PG amidase was rescued in a ΔM_6281::Rv3717 strain. Our results demonstrated that MSMEG_6281 impacts fatty acid metabolism and further interferes with biofilm formation. These results provide a clue to study the effects of PG amidase on mycobacterial pathogenicity.

Structural Change in Microbiota by a Probiotic Cocktail Enhances the Gut Barrier and Reduces Cancer via TLR2 Signaling in a Rat Model of Colon Cancer.

BACKGROUND: Structural change in the gut microbiota is implicated in cancer. The beneficial modulation of the microbiota composition with probiotics and prebiotics prevents diseases. AIM: We investigated the effect of oligofructose-maltodextrin-enriched Lactobacillus acidophilus, Bifidobacteria bifidum, and Bifidobacteria infantum (LBB), on the gut microbiota composition and progression of colorectal cancer. METHODS: Sprague Dawley rats were acclimatized, given ampicillin (75 mg/kg), and treated as follows; GCO: normal control; GPR: LBB only; GPC: LBB+ 1,2-dimethylhydrazine dihydrochloride (DMH); and GCA: DMH only (cancer control). 16S V4 Pyrosequencing for gut microbiota analysis, tumor studies, and the expression of MUC2, ZO-1, occludin, TLR2, TLR4, caspase 3, COX-2, and ß-catenin were conducted at the end of experiment. RESULTS: Probiotic LBB treatment altered the gut microbiota. The relative abundance of genera Pseudomonas, Congregibacter, Clostridium, Candidactus spp., Phaeobacter, Escherichia, Helicobacter, and HTCC was decreased (P < 0.05), but the genus Lactobacillus increased (P < 0.05), in LBB treatment than in cancer control. The altered gut microbiota was associated with decreased tumor incidence (80 % in GPC vs. 100 % in GCA, P = 0.0001), tumor volume (GPC 84.23 (42.75-188.4) mm(3) vs. GCA 243 (175.5-344.5) mm(3), P < 0.0001) and tumor multiplicity/count (GPC 2.92 ± 0.26 vs. GCA 6.27 ± 0.41; P < 0.0001). The expression of MUC2, ZO-1, occludin, and TLR2 was increased, but expression of TLR4, caspase 3, Cox-2, and ß-catenin was decreased by LBB treatment than in cancer control GCA (P < 0.05). CONCLUSION: Administration of LBB modulates the gut microbiota and reduces colon cancer development by decreasing tumor incidence, multiplicity/count, and volume via enhanced TLR2-improved gut mucosa epithelial barrier integrity and suppression of apoptosis and inflammation.

Transcriptional profiling and biological pathway analysis of human equivalence PCB exposure in vitro: indicator of disease and disorder development in humans.

BACKGROUND AND AIMS: Our earlier gene-expression studies with a Slovak PCBs-exposed population have revealed possible disease and disorder development in accordance with epidemiological studies. The present investigation aimed to develop an in vitro model system that can provide an indication of disrupted biological pathways associated with developing future diseases, well in advance of the clinical manifestations that may take years to appear in the actual human exposure scenario. METHODS: We used human Primary Blood Mononuclear Cells (PBMC) and exposed them to a mixture of human equivalence levels of PCBs (PCB-118, -138, -153, -170, -180) as found in the PCBs-exposed Slovak population. The microarray studies of global gene expression were conducted on the Affymetrix platform using Human Genome U133 Plus 2.0 Array along with Ingenuity Pathway Analysis (IPA) to associate the affected genes with their mechanistic pathways. High-throughput qRT-PCR Taqman Low Density Array (TLDA) was done to further validate the selected 6 differentially expressed genes of our interest, viz., ARNT, CYP2D6, LEPR, LRP12, RRAD, TP53, with a small population validation sample (n=71). RESULTS: Overall, we revealed a discreet gene expression profile in the experimental model that resembled the diseases and disorders observed in PCBs-exposed population studies. The disease pathways included endocrine system disorders, genetic disorders, metabolic diseases, developmental disorders, and cancers, strongly consistent with the evidence from epidemiological studies. INTERPRETATION: These gene finger prints could lead to the identification of populations and subgroups at high risk for disease, and can pose as early disease biomarkers well ahead of time, before the actual disease becomes visible.

Functional identification of MSMEG_6402 protein from Mycobacterium smegmatis in decaprenylphosphoryl-D-arabinose biosynthesis.

The arabinogalactan (AG) of the mycobacterial cell wall consists of an arabinan region, a galactan region and a disaccharide linker. Decaprenylphosphoryl-D-arabinose (DPA) is the donor for arabinofuran residues, which are formed from phosphoribose diphosphate (PRPP) and decaprenyl phosphate (DP). DP is sequentially catalyzed by a three-step process that involves a transferase, a phosphatase and an epimerase. Rv3807c is a putative phospholipid phosphatase that might generate the intermediate product of decaprenyl-phosphoryl-ribose (DPR) in DPA biosynthesis. Mycobacterium smegmatis MSMEG_6402 is a homolog gene of Mycobacterium tuberculosis Rv3807c and was substituted for the functional identification of Rv3807c. Previously, we generated a conditional MSMEG_6402 gene knockout strain (M. sm-ΔM_6402) that exhibited significantly affected cell wall structure. To understand the function of MSMEG_6402 in DPA biosynthesis, this gene was amplified and expressed, and the resulting protein was identified and purified using a His-tagged approach. A MSMEG_6402 enzymatic reaction system with PRPP and DP as substrates was utilized, and the reaction products were separated using thin layer chromatography (TLC). The results revealed a specific lipid-linked sugar band that appeared in the reaction with the addition of MSMEG_6402. Furthermore, ESI-MS detection was utilized in this study, and the results revealed that the enzymatic reaction products involving MSMEG_6402 included DPPR and a sodium ion adduct of DPR. Additionally, the phosphatase activity of MSMEG_6402 was also determined through phosphate group detection using the colorimetric method. Based on our results together with the results of previous studies, including the functional identification and bioinformatics analysis of M. tuberculosis Rv3807c, we propose that MSMEG_6402, as a phosphatase, has an intimate relationship with DPA biosynthesis.

Discovery and characterization of the flavonoids in Cortex Mori Radicis as naturally occurring inhibitors against intestinal nitroreductases.

Gut bacterial nitroreductases are found to be heavily related with the intestinal toxicity of nitroaromatic compounds in food or medicine, which can be converted into mutagenic and enterotoxic nitroso or N-hydroxyl intermediates. Thus, inhibiting the gut microbe-encoded nitroreductases has become an attractive method to reduce the mutagen metabolites in colon and prevent intestinal diseases. In this study, the inhibitory effects of sixteen constituents in Cortex Mori Radicis on two kinds of gut bacterial nitroreductases (EcNfsA and EcNfsB) were evaluated with nitrofurazone (NFZ) as substrate and NADPH as electron donor. The results clearly demonstrated that four flavonoids including kuwanon G, kuwanon A, sanggenol A and kuwanon C showed dual inhibition on both EcNfsA and EcNfsB mediated NFZ reduction; morusin, morin, and sanggenone C were strong inhibitors towards EcNfsA; kuwanon H and kuwanon E exhibited effective inhibition on EcNfsB. Further inhibition kinetic analysis and molecular docking simulations displayed that all inhibitors above suppressed both EcNfsA and EcNfsB activities in competitive manners, except non-competitive inhibition of morin on EcNfsA and non-competitive inhibition of kuwanon C on EcNfsB, respectively. Taking together, these findings revealed that most flavonoids in Cortex Mori Radicis presented effective inhibition on gut microbial nitroreductases, suggesting that Cortex Mori Radicis might be a promising candidate for ameliorating nitroreductases mediated intestinal mutagenicity.

Effect of crude polysaccharide from seaweed, Dictyopteris divaricata (CDDP) on gut microbiota restoration and anti-diabetic activity in streptozotocin (STZ)-induced T1DM mice.

Type-1 Diabetes Mellitus (T1DM) is regarded as a multifunctional, immune-related disease which causes massive destruction of islet ß-cells in pancreas resulting in hyperglycemic, hypoinsulinemia and hyperlipidimic conditions. The aim of the present study, was to investigate the hypothesis that streptozotocin (STZ)-induced T1DM in Balb/c mice when treated with crude polysaccharide from seaweed, Dictyopteris divaricata (CDDP) depicts improvement in diabetes-related symptoms. Treatment with CDDP resulted in decreased body weight loss, improved food consumption and water intake disbalances. The CDDP effectively improved fasting blood glucose, oral glucose tolerance (OGTT), serum insulin, insulin secretion, rejuvenation of ß-cells mass, serum lipid profile and pro-inflammatory cytokines levels. Additionally, treatment with CDDP increased the population of beneficial bacteria such as Firmicutes, Bacteroidetes and Lactobacillus at phylum, family and genus levels by 16S rRNA sequencing. Furthermore, immunohistological examination confirmed that CDDP reduces the inflammation and restored the structural morphology of colon and upraised the levels of insulin receptor substrate-1 (IRS-1), Mucin-2 (MUC-2) and tight-junction proteins (TJs) whereby maintaining the gut structures and barrier permeability. Thus, the above presented data, highlights the safe and therapeutic effects of crude polysaccharide (CDDP) from D. divaricata in the treatment and restoration of T1DM disorders and can be used as a food supplement alternative to diabetes medicine.

The effect of MSMEG_6402 gene disruption on the cell wall structure of Mycobacterium smegmatis.

Arabinogalactan (AG) of mycobacterial cell wall consists of arabinan region, galactan region and disaccharide linker. The arabinan is composed of D-arabinofuranose residues, and decaprenyphosphoryl-D-arabinose (DPA) is the donor of the D-arabinofuranose residues. DPA is formed from phosphoribose diphosphate (PRPP) in a four-step process catalyzed by transferase, phosphatase and epimerase, respectively. Mycobacterium tuberculosis Rv3806c has been identified as PRPP: decaprenyl-phosphate 5-phosphoribosyltransferase, and heteromeric Rv3790/Rv3791 has epimerase activity. Rv3807c is putative phospholipid phosphatase. However, there is no direct biochemical evidence since expression of Rv3807c has been unsuccessful. Mycobacterium smegmatis MSMEG_6402 is ortholog of Rv3807c. To investigate the function of MSMEG_6402 on AG biosynthesis, a conditional MSMEG_6402 gene knock out (M. sm-ΔM_6402) strain was constructed through homologous recombination technique. The morphological and compositional changes of cell wall were examined in the M. sm-ΔM_6402 strain. The M. sm-ΔM_6402 strain grew at non-permissive temperature slower than that at permissive temperature, indicating that MSMEG_6402 is non-essential for growth of M. smegmatis. The change of cell shape and detectable bulging on the cell surface of M. sm-ΔM_6402 strain were observed by scanning electron microscopy, and curled as well as deformed cell wall of M. sm-ΔM_6402 strain was revealed by transmission electron microscopy. Analysis of sugar composition in the cell wall by HPLC indicated that the ratio of arabinofuran to galactofuran in M. sm-ΔM_6402 strain was changed to 1.7:1 comparing with 2:1 in the wild type. It demonstrates that the lacking MSMEG_6402 interferes the biosynthesis of arabinan. Analyzing 5' P-DPR and DPR from both M. sm-ΔM_6402 strain and wild type M. smegmatis is undergoing in this lab.

The novel responses of ethambutol against Mycobacterium smegmatis mc²155 Revealed by proteomics analysis.

Ethambutol (EMB), one of the effective anti-mycobacterial drugs, inhibits the biosynthesis of mycobacterium cell wall. To elucidate the molecular mechanism of EMB against tuberculosis (TB), Mycobacterium smegmatis mc²155 was employed as a model of mycobacterial system in this study. We compared the protein profiles on M. smegmatis mc²155 treated by EMB and untreated using fluorescence difference two-dimensional gel electrophoresis (2-D DIGE). A total of 40 differential protein spots were selected and 22 proteins were identified by HPLC-nano ESI-MS/MS analysis, including 16 over-expressed proteins and 6 under-expressed proteins. These proteins mainly affected energy metabolism, as well as synthesis and modification of macromolecules. The expressions of correspondent genes were confirmed by RT-PCR. This investigation provided some clues for searching potential drug targets.

Small extracellular vesicle-bound vascular endothelial growth factor secreted by carcinoma-associated fibroblasts promotes angiogenesis in a bevacizumab-resistant manner.

The blood vessel growth inhibitor bevacizumab targets vascular endothelial growth factor (VEGF), a crucial regulator of angiogenesis. Recently, small extracellular vesicles (sEVs) have been demonstrated to be important vehicles in the transport of growth factors to target cells. In this study, we isolated primary carcinoma-associated fibroblasts (CAFs) from four human oral squamous cell carcinoma (OSCC) specimens. Compared with other non-extracellular vesicle components, CAF-derived sEVs were found to be the main regulators of angiogenesis. The ability of CAF sEVs to activate VEGF receptor 2 (VEGFR2) signaling in human umbilical vein endothelial cells (HUVEC) was dependent on the association between sEVs and VEGF. In addition, sEV-bound VEGF secreted by CAFs further activated VEGFR2 signaling in HUVEC in a bevacizumab-resistant manner. VEGF was found to interact with heparan sulfate proteoglycans on the CAF sEV surface and could be released by heparinase I/III. The bioactivity of the dissociated VEGF was retained in vitro and in vivo and could be neutralized by bevacizumab. These findings suggest that the combined use of heparinase and bevacizumab might inhibit angiogenesis in patients with high levels of sEV-bound VEGF.

Pancreatic lipase inhibitory constituents from Fructus Psoraleae.

Pancreatic lipase (PL), a crucial enzyme in the digestive system of mammals, has been proven as a therapeutic target to prevent and treat obesity. The purpose of this study is to evaluate and characterize the PL inhibition activities of the major constituents from Fructus Psoraleae (FP), one of the most frequently used Chinese herbs with lipid-lowering activity. To this end, a total of eleven major constituents isolated from Fructus Psoraleae have been obtained and their inhibition potentials against PL have been assayed by a fluorescence-based assay. Among all tested compounds, isobavachalcone, bavachalcone and corylifol A displayed strong inhibition on PL (IC50 < 10 µmol·L-1). Inhibition kinetic analyses demonstrated that isobavachalcone, bavachalcone and corylifol A acted as mixed inhibitors against PL-mediated 4-methylumbelliferyl oleate (4-MUO) hydrolysis, with the Ki values of 1.61, 3.77 and 10.16 µmol·L-1, respectively. Furthermore, docking simulations indicated that two chalcones (isobavachalcone and bavachalcone) could interact with the key residues located in the catalytic cavity of PL via hydrogen binding and hydrophobic interactions. Collectively, these finding provided solid evidence to support that Fructus Psoraleae contained bioactive compounds with lipid-lowering effects via targeting PL, and also suggested that the chalcones in Fructus Psoraleae could be used as ideal leading compounds to develop novel PL inhibitors.

Inhibition of pancreatic lipase by the constituents in St. John's Wort: In vitro and in silico investigations.

Herbal medicines are frequently used for the prevention and treatment of obesity and obesity-related disorders. Our preliminary screening showed that St. John's Wort (SJW) displayed potent inhibition on pancreatic lipase (PL), a key hydrolase responsible for lipid digestion and absorption in mammals. Herein, the inhibition potentials and inhibitory mechanism of SJW extract and its major constituents on PL were fully investigated by a set of in vitro and in silico studies. The results clearly demonstrated that the naphthodianthrones, biflavones and most of flavonoids in SJW displayed strong to moderate inhibition on PL. Among all tested natural compounds, two naphthodianthrones (hypericin and pseudohypericin) and one biflavone (I3,II8-biapigenin) isolated from SJW exhibited potent PL inhibition activity, with the IC50 values of <1 µM. Inhibition kinetics analyses showed that hypericin, pseudohypericin and I3,II8-biapigenin inhibited PL via a mixed manner, while molecular dynamics simulations revealed that three newly identified PL inhibitors could bind on PL at both the catalytic cavity and the interface between colipase and the C-terminal domain of PL. Collectively, our findings suggested that part of major constituents in SJW displayed potent PL inhibition activities, which could be used as lead compounds for the development of novel PL inhibitors.

[Identification of biomarkers for early detection in gastric cancer and its clinical biological significance.].

OBJECTIVE: To systematically understand the cellular and molecular mechanism of gastric cancer (GC) development and to discover early diagnosis and predictive biomarkers, which will be used for early diagnosis and novel treatment targets. METHODS: 70 mer 22 K-oligonucleotide microarrays and bioinformatic analysis were conducted to recognize gene expression profiles in GC and normal appearing tissue (NAT). The control group was collected from non-tumor patients including 20 specimen mixture as a common reference (CR) and 5 individuals as additional control. Our results showed that 837 different expression genes (DEGs) were identified in GC while 570 DEGs were in NATs by Bayesian analysis (P<0.001, Fold change>2.0) as compared respectively with CR. An interesting finding is that we identified 67 over-expressed genes in both GC and NAT tissues, and these gene expression alterations could not be detected by comparison of GC with NATs, which were normally used in routine experiment design. Most of these genes were involved in the control of cell proliferation, metabolism and differentiation. RESULTS: These differential expressed genes were confirmed at mRNA and protein levels in primary tumors using RT-PCR and immunohistochemistry (IHC). The results showed that three genes, EGR1, CYR61 and ADAMTS1 were over expressed in both GC and NATs at mRNA level. These results were consistent with oligo microarray data. Another interesting finding is that these three genes were also over-expressed in intestinal metaplasia (IM) and dysplasia (DYS), which indicated that these three genes might be potential biomakers for early detection of GC. CONCLUSION: Through the systematic analysis of gene expression profiles in GC tissues, NAT and CR normal tissues, we identified a group of genes over-expressed both in GC and precancerous lesions, which might be potential biomarkers for early GC diagnosis.

Dynamic changes of bacterial communities and nitrite character during northeastern Chinese sauerkraut fermentation.

Northeastern Chinese sauerkraut is a well-known traditional fermented vegetable in China. Incomplete identification of the microorganisms' (bacteria in spontaneous fermentation) diversity and accumulation of nitrite make it difficult to normalize the fermentation process and product qualities of northeastern Chinese sauerkraut. Conventional culturing and polymerase chain reaction-denaturing gradient gel electrophoresis methods were combined to describe microbial structure and diversity. Lactobacillus, Leuconostoc, Enterobacter, Accumulibacter, Thermotoga, Pseudomonas, Clostridium, Rahnella and Citrobacter were predominant microorganisms in different fermentation periods. The pH value and nitrite concentration presented a certain relevance to the amount of lactic acid bacteria. Lactobacillus and Leuconostoc had the ability to decrease nitrite by inhibiting nitrate-reducing bacteria such as Enterobacter. Therefore, Northeastern Chinese sauerkraut should not be eaten until 4 weeks of fermentation for the safety and quality of fermented foods. Northeastern Chinese sauerkraut is rich in lactic acid bacteria, which demonstrate its ability as an excellent probiotic for applications in functional foods.

Detection of CD22 Expression in Living Cancer Cells by Semiconductor Quantum Dots.

CD22 is an important drug target for the treatment of autoimmune diseases and B cell-derived malignancies. In this study, N-acetylneuraminic acid functionalized quantum dots nanoconjugate was synthesized and used for targeting and fluorescence imaging of CD22 on living cells. The nanoprobe was prepared by conjugating N-acetylneuraminic acid (NANA) on the carboxyl groups modified CdSe/ZnS quantum dots (COOH-QDs) via NHS/EDC mediated esterification. The NANA-QDs nanoprobe showed excellent size distribution, very low cytotoxicity and super fluorescent properties for biological imaging applications. The specificity of NANA-QDs nanoparticles for CD22 on living cancer cells was validated by cellular uptake inhibition assays, colocalization of the immunofluorescence staining with both anti-CD22 antibody and NANA-QDs nanoparticles. Furthermore, CD22 mediated endocytosis of NANA-QDs nanoparticles was investigated by cellular internalization kinetics in Daudi cells at multiple time points. The newly developed NANA-QDs based assay was successfully used to determine the expression levels of CD22 on various cancer cells, which were highly consistent with the results determined by immunofluorescence staining assay and western blotting. All these findings demonstrated that NANA-QDs nanoparticles system was a practical fluorescent nanoprobe for bioimaging of CD22, which held great promise in a wide variety of biomedical applications of CD22 related studies.

Integration of statistical inference methods and a novel control measure to improve sensitivity and specificity of data analysis in expression profiling studies.

Statistical methods have proven invaluable tools for enhancing the quality of microarray analysis. In this study, we used different methods such as significance analysis of microarrays (SAM) and Bayesian analysis of gene expression levels (BAGEL), to analyze the same set of raw data in an attempt to maximize the chance of identifying genes whose expression were significantly altered in gastric cancers. In addition, we examined the utility of an additional set of reference in controlling the variances and enhancing the quality of the results. Our results showed that BAGEL has the advantage of detecting small yet statistically significant differences, which might be of biological significance. Furthermore, introducing an additional control into the BAGEL, we were able to minimize the influence of the variances and significantly reduce number of potential false positive hits. BAGEL incorporates a novel control significantly improve the sensitivity and specificity of gene expression profiling analysis.

Homology modeling and identification of amino acids involved in the catalytic process of Mycobacterium tuberculosis serine acetyltransferase.

Serine acetyltransferase (CysE) belongs to the hexapeptide acetyltransferase family and is involved in the biosynthesis of L­cysteine in microorganisms. Mycobacterium tuberculosis CysE is regarded as a potential target for anti­tuberculosis (TB) drugs; however, the structure and active sites of M. tuberculosis CysE remain unknown. The present study aimed to predict the secondary structure and to construct a 3D model for M. tuberculosis CysE using bioinformatics analysis. To determine the essential amino acids that are associated with CysE enzymatic activity, amino acid sequences from several microorganisms were compared, and a consensus sequence was identified. Subsequently, site­directed mutagenesis was used to generate mutant M. tuberculosis CysE proteins. Enzyme assays demonstrated that D67A, H82A and H117A mutants abolished ~75% activity of M. tuberculosis CysE. Prediction of the protein structure and identification of the active amino acids for M. tuberculosis CysE is essential for designing inhibitors, which may aid the discovery of effective anti­TB drugs.

[Relations of transforming growth factor-beta1 expression to differentiation and prognosis of advanced gastric cancer].

OBJECTIVE: To clarify the correlation of transforming growth factor-beta1 (TGF-beta1) expression with the differentiation and prognosis of advanced gastric cancer (GC). METHODS: Whole genome expression chip hybridization, was used to detect the expression of TGF-beta1 and TGF-betaR1 in 20 specimens of intestinal-type GC and para-cancer tissues. RT-PCR and immunohistochemistry (IHC) analysis were used to detect the mRNA and protein expression of TGF-beta1 and TGF-betaR1 in 30 specimens of intestinal-type GC tissue and para-cancer tissues. The mixture of gastric mucosa tissues from 20 non-tumor patients was used as common reference. RESULTS: The expression level of TGF-beta1 and TGF-betaR-1 genes was higher in the GC tissues than in the para-cancer tissues. However, the expression of Smad gene family was not significantly different between the GC tissues and para-tumor normal tissues. TGF-beta1 gene expression and TGF-betaR1 gene expression were higher in the GC tissues. RT-PCR showed that both TGF-beta1 and TGF-betaR-1 genes were highly expressed in the mRNA level in 21 of the 30 CC patients IHC showed that TGF-beta1 protein was expressed mainly in the cytoplasm. 32 of the 90 specimens of GC tissue were highly positive in TGF-beta1 protein (64%), in comparison with the positive rate of 5% (1/20) in the para-cancer normal tissues. The TGF-beta1 protein expression rate of the highly and moderately differentiated GC tissues was 59% (59%, 23/39), significantly higher than that of the lowly differentiated GC tissues (18%, 9/51, P < 0.01). IHC showed that the TGF-beta R-I rate was 57% (42/74) in the well differentiated specimens, particularly 68% (26/38) in the highly differentiated specimens, and was 44% in the poorly differentiated GC (6/20, P < 0.05). Log rank test showed that the prognosis of the patients positive in TGF-beta1 was significantly better than those negative in TGF-beta1 (P = 0.0058). However, the survival rate did not differ significantly according to TGF-beta R-I expression (P = 0.8453). CONCLUSION: TGF-beta1 expression is significantly correlated with the differentiation degree of GC. Moreover, positive expression of TGF-beta1 is a favorable prognostic factor in advanced GC. Expression of TGF-beta1 may be an important preoperative prognostic variable for advanced GC.

Effects of epigallocatechin gallate on the cell-wall structure of Mycobacterial smegmatis mc²155.

Epigallocatechin gallate (EGCG) is the main component of green tea extracts that inhibits the growth of Mycobacterial smegmatis mc(2)155, and the mechanism is not clear. This study showed the effects of EGCG on the growth of mc(2)155. The content and the structure of EGCG in LB medium with mc(2)155 were identified by HPLC and LC/MS. Transmission electron microscopy was utilised to identify the cell envelope structure. As a result, the optional inhibition concentration was determined to be 20 µg mL(-1). Most of EGCG was transferred into its isomeride in LB medium, but the inhibition effects against mc(2)155 had yet been maintained. The changes of cell envelope structure were showed after EGCG treatment for 18 h. The cell wall appeared to have a less electron-translucent zone, turn rougher and thicker. The results show that EGCG impacts the integrity of mycobacterial cell wall and is likely be a better prophylactic agent against tuberculosis.