FAS/FASL Expression Profile as a Prognostic Marker in Squamous Cell Carcinoma of the Oral Cavity

FAS/FASL altered expression may cause tumor protecting immunomodulation, with a direct impact on patient prognosis. FAS expression was studied in 60 squamous cell carcinomas of the oral cavity. FAS expression did not show a significant association with tumor histopathological characteristics, but was significantly associated with lymph node positivity. FAS expression was significantly associated with disease specific death and negative FAS expression was an independent risk factor, increasing risk 4 times when compared to positive expression. When FAS and FASL expression results were combined, we were able to define high, intermediate and low risk profiles. Disease-free and disease-specific survival were significantly correlated with FAS/FASL expression profiles. The high risk category was an independent marker for earlier disease relapse and disease-specific death, with approximately 4- and 6-fold increased risk, respectively, when compared to the low risk profile. Risk profiles based on FAS/FASL expression showed that high risk was significantly associated with increased disease relapse and death, as well as shorter disease-free or disease-specific survival. This categorization, added to patient clinical data, may facilitate the choice of therapy, minimizing treatment failure and increasing disease control.     

Molecular survey and characterization of Theileria annulata and Ehrlichia ruminantium in cattle from Northwest China

Theileriosis and ehrlichiosis are two important tick-borne diseases affecting cattle farming in China. However, limited information is available regarding prevalence and molecular characterization of Theileria annulata and Ehrlichia ruminantium in cattle in Xinjiang Uygur Autonomous Region (XUAR), northwestern China. In this study, a total of 176 blood samples of cattle from three rural areas of XUAR were collected in June 2017 and were tested by nested-PCR. A total of 34 (19.3%) samples were found to be infected with one or two pathogens. The overall prevalence rates of T. annulata and E. ruminantium were 18.2% and 1.7%, respectively. Phylogenetic analyses revealed that the E. ruminantium isolates from XUAR were located in the same clade but diverged from the isolates from African countries using pCS20 gene while T. annulata isolates from XUAR revealed differences in the genotypes using Tams1 sequences. To our knowledge, this is the first report of E. ruminantium infection in cattle in China. It also provides the first genetic characterization of T. annulata in cattle in XUAR. The current findings are important for understanding the distribution of agents of theileriosis and ehrlichiosis and in designing measures for the prevention and control of tick-borne diseases in cattle, other animals, and humans.     

Comparison of M.I.C.E. and Etest with CLSI Agar Dilution for Antimicrobial Susceptibility Testing against Oxacillin-Resistant Staphylococcus spp

Objective

The main objective of this study was to comparatively evaluate the performance of M.I.C.E. and Etest methodologies to that of agar dilution for determining the antimicrobial susceptibility profile of oxacillin-resistant Staphylococcus spp.

Methods

A total of 100 oxacillin-resistant Staphylococcus spp. isolates were collected from hospitalized patients at a teaching hospital. Antimicrobial susceptibility testing for vancomycin, teicoplanin and linezolid was performed using the reference CLSI agar dilution method (2009), Etest and M.I.C.E. methodologies. The MIC values were interpreted according to CLSI susceptibility breakpoints and compared by regression analysis.

Results

In general, the essential agreement (±1-log₂) between M.I.C.E. and CLSI agar dilution was 93.0%, 84.0% and 77.0% for linezolid, teicoplanin and vancomycin, respectively. Essential agreement rates between M.I.C.E. and Etest were excellent (>90.0%) for all antibiotics tested. Both strips (M.I.C.E. and Etest) yielded two very major errors for linezolid. Unacceptable minor rates were observed for teicoplanin against CoNS and for vancomycin against S. aureus.

Conclusions

According to our results, linezolid and teicoplanin MICs against all staphylococci and S. aureus, respectively, were more accurately predicted by M.I.C.E. strips. However, the Etest showed better performance than M.I.C.E. for predicting vancomycin MICs against all staphylococci. Thus, microbiologists must be aware of the different performance of commercially available gradient strips against staphylococci.     

Inflammation and Cancer: Role of Annexin A1 and FPR2/ALX in Proliferation and Metastasis in Human Laryngeal Squamous Cell Carcinoma

The anti-inflammatory protein annexin A1 (ANXA1) has been associated with cancer progression and metastasis, suggesting its role in regulating tumor cell proliferation. We investigated the mechanism of ANXA1 interaction with formylated peptide receptor 2 (FPR2/ALX) in control, peritumoral and tumor larynx tissue samples from 20 patients, to quantitate the neutrophils and mast cells, and to evaluate the protein expression and co-localization of ANXA1/FPR2 in these inflammatory cells and laryngeal squamous cells by immunocytochemistry. In addition, we performed in vitro experiments to further investigate the functional role of ANXA1/FPR2 in the proliferation and metastasis of Hep-2 cells, a cell line from larynx epidermoid carcinoma, after treatment with ANXA1_2–26 (annexin A1 N-terminal-derived peptide), Boc2 (antagonist of FPR) and/or dexamethasone. Under these treatments, the level of Hep-2 cell proliferation, pro-inflammatory cytokines, ANXA1/FPR2 co-localization, and the prostaglandin signalling were analyzed using ELISA, immunocytochemistry and real-time PCR. An influx of neutrophils and degranulated mast cells was detected in tumor samples. In these inflammatory cells of peritumoral and tumor samples, ANXA1/FPR2 expression was markedly exacerbated, however, in laryngeal carcinoma cells, this expression was down-regulated. ANXA1_2–26 treatment reduced the proliferation of the Hep-2 cells, an effect that was blocked by Boc2, and up-regulated ANXA1/FPR2 expression. ANXA1_2–26 treatment also reduced the levels of pro-inflammatory cytokines and affected the expression of metalloproteinases and EP receptors, which are involved in the prostaglandin signalling. Overall, this study identified potential roles for the molecular mechanism of the ANXA1/FPR2 interaction in laryngeal cancer, including its relationship with the prostaglandin pathway, providing promising starting points for future research. ANXA1 may contribute to the regulation of tumor growth and metastasis through paracrine mechanisms that are mediated by FPR2/ALX. These data may lead to new biological targets for therapeutic intervention in human laryngeal cancer.     

Expression, purification, and circular dichroism analysis of human CDK9

The human cyclin-dependent kinase 9 (CDK9) protein was expressed in E. coli BL21 using the pET23a vector at 30 degrees C. Several milligrams of protein were purified from soluble fraction using ionic exchange and ATP-affinity chromatography. The structural quality of recombinant CDK9 and the estimation of its secondary structure were obtained by circular dichroism. Structural models of CDK9 presented 26% of helices in agreement with the spectra by circular dichroism analysis. This is the first report on human CDK9 expression in Escherichia coli and structure analysis and provides the first step for the development of CDK9 inhibitors.     

Preliminary functional characterization, cloning and primary sequence of Fastuosain, a cysteine peptidase isolated from fruits of Bromelia fastuosa

The present work reports the characterization of Fastuosain, a novel cysteine protease of 25kDa, purified from the unripe fruits of Bromelia fastuosa, a wild South American Bromeliaceae. Proteolytic activity, measured using casein and synthetic substrates, was dependent on the presence of thiol reagents, having maximum activity at pH 7.0. The present work reports cDNA cloning of Fastuosain; cDNA was amplified by PCR using specific primers. The product was 1096pb long. Mature fastuosain has 217 residues, and with the proregion has a total length of 324 residues. Its primary sequence showed high homology with ananain(74%), stem bromelain (66%) and papain (44%).