L-asparaginase from Dickeya chrysanthemi: expression, purification and cytotoxicity assessment.

Microbial L-asparaginases are aminohydrolases that hydrolyze L-asparagine to L-aspartate. They are used to treat acute lymphoblastic leukemia and Hodgkin's lymphomas and in food industries. Increasing demand for L-ASNases is therefore needed. In the current study, the recombinant L-ASNase from Dickeya chrysanthemi (DcL-ASNase) was cloned into pET28a (+) expression vector and expressed in Escherichia coli as a 6His-tagged fusion protein and purified using Ni2+ chelated Sepharose chromatography resin, yielding a highly purified enzyme. Kinetics analysis allowed the determination of its substrate specificity and the physicochemical parameters that affect enzyme activity. The enzyme showed operational stability at 37 °C and 45 °C. The immunogenicity of the purified DcL-ASNase was evaluated by measuring the IgG and IgM levels in rats after injection. The cytotoxicity DcL-ASNase in selected cancer cell lines and peripheral blood monocytes was determined. The results showed that the enzyme induces pleiotropic effects, including significant morphological changes and the formation of apoptotic bodies. No cytotoxic effects were observed in peripheral blood monocytes at the same concentrations. In addition, gene expression analysis by RT-PCR of apoptotic biomarkers (Bax, survivin, and Ki-67) allowed the study of the apoptotic mechanism induced by DcL-ASNase on THP-1 cells.

Combination versus monotherapy as definitive treatment for Pseudomonas aeruginosa bacteraemia: a multicentre retrospective observational cohort study.

BACKGROUND: Pseudomonas aeruginosa bacteraemia is a common and serious infection. No consensus exists regarding whether definitive combination therapy is superior to monotherapy. We aimed to evaluate the impact of combination therapy on mortality. METHODS: This was a multicentre retrospective study (nine countries, 25 centres), including 1277 patients with P. aeruginosa bacteraemia during 2009-15. We evaluated the association between ß-lactam plus aminoglycoside or quinolone combination therapy versus ß-lactam monotherapy and mortality. The primary outcome was 30 day all-cause mortality. Univariate and multivariate Cox regression analyses were conducted, introducing combination as a time-dependent variable. Propensity score was conducted to adjust for confounding for choosing combination therapy over monotherapy. RESULTS: Of 1119 patients included, 843 received definitive monotherapy and 276 received combination therapy (59% aminoglycoside and 41% quinolone). Mortality at 30 days was 16.9% (189/1119) and was similar between combination (45/276; 16.3%) and monotherapy (144/843; 17.1%) groups (P = 0.765). In multivariate Cox regression, combination therapy was not associated with reduced mortality (HR 0.98, 95% CI 0.64-1.53). No advantage in terms of clinical failure, microbiological failure or recurrent/persistent bacteraemia was demonstrated using combination therapy. Likewise, adverse events and resistance development were similar for the two regimens. CONCLUSIONS: In this retrospective cohort, no mortality advantage was demonstrated using combination therapy over monotherapy for P. aeruginosa bacteraemia. Combination therapy did not improve clinical or microbiological failure rates, nor affect adverse events or resistance development. Our finding of no benefit with combination therapy needs confirmation in well-designed randomized controlled trials.

Pseudomonas aeruginosa recombinant L-asparaginase: Large scale production, purification, and cytotoxicity on THP-1, MDA-MB-231, A549, Caco2 and HCT-116 cell lines.

In previous studies Pseudomonas aeruginosal-ASNase complete coding sequence gene, 984 bp (GenBank accession number KU161101.2) was isolated by PCR, cloned into pET28a(+) vector, expressed in E. coli DE3(BL21) pLysS, purified to apparent homogeneity and biochemically characterized. In the present work we highlight large scale production, affinity purification of the recombinant enzyme, effect of osmolytes on the stability of the l-ASNase and cytotoxicity on different cancer cell lines. Successful overexpression was achieved in E. coli as a 6-His-Tag fusion protein after 18 h of induction with lactose at a concentration of 2 g/L in fermentation medium and at 37 °C. The recombinant enzyme was purified to homogeneity using Ni2+ chelated Fast Flow Sepharose resin with 19758.8 specific activity and 10.28 purification fold. With respect to the effect of osmolytes on the stability of the purified enzyme, the majority of the tested osmolytes namely 5% maltose, 5% mannitol, 30% glycerol and 5% BSA were found to increase the stability of the recombinant l-ASNase as compared to the free enzyme. Triple negative breast cancer cell line, MDA-MB-231 treated with recombinant l-ASNase showed significant morphological changes and the IC50 of the purified enzyme was found to be 3.1 IU. Human leukemia cell line, THP-1 treated with l-ASNase showed apoptotic bodies and morphological changes with IC50 of the purified enzyme 1.75 IU. Moreover, the purified recombinant l-ASNase was found to induced cytotoxic effects on colorectal adenocarcinoma cell line, Caco-2 with IC50 of 68.28 IU. Results of apoptosis assay on THP-1 cells revealed that the purified l-ASNase induced early and late apoptosis at 14.16% and 7.56 respectively as compared to the control untreated cells.

Ceftazidime, Carbapenems, or Piperacillin-tazobactam as Single Definitive Therapy for Pseudomonas aeruginosa Bloodstream Infection: A Multisite Retrospective Study.

BACKGROUND: The optimal antibiotic regimen for Pseudomonas aeruginosa bacteremia is controversial. Although ß-lactam monotherapy is common, data to guide the choice between antibiotics are scarce. We aimed to compare ceftazidime, carbapenems, and piperacillin-tazobactam as definitive monotherapy. METHODS: A multinational retrospective study (9 countries, 25 centers) including 767 hospitalized patients with P. aeruginosa bacteremia treated with ß-lactam monotherapy during 2009-2015. The primary outcome was 30-day all-cause mortality. Univariate and multivariate, including propensity-adjusted, analyses were conducted introducing monotherapy type as an independent variable. RESULTS: Thirty-day mortality was 37/213 (17.4%), 42/210 (20%), and 55/344 (16%) in the ceftazidime, carbapenem, and piperacillin-tazobactam groups, respectively. Type of monotherapy was not significantly associated with mortality in either univariate, multivariate, or propensity-adjusted analyses (odds ratio [OR], 1.14; 95% confidence interval [CI], 0.52-2.46, for ceftazidime; OR, 1.3; 95% CI, 0.67-2.51, for piperacillin-tazobactam, with carbapenems as reference in propensity adjusted multivariate analysis; 542 patients). No significant difference between antibiotics was demonstrated for clinical failure, microbiological failure, or adverse events. Isolation of P. aeruginosa with new resistance to antipseudomonal drugs was significantly more frequent with carbapenems (36/206 [17.5%]) versus ceftazidime (25/201 [12.4%]) and piperacillin-tazobactam (28/332 [8.4%] (P = .007). CONCLUSIONS: No significant difference in mortality, clinical, and microbiological outcomes or adverse events was demonstrated between ceftazidime, carbapenems, and piperacillin-tazobactam as definitive treatment of P. aeruginosa bacteremia. Higher rates of resistant P. aeruginosa after patients were treated with carbapenems, along with the general preference for carbapenem-sparing regimens, suggests using ceftazidime or piperacillin-tazobactam for treating susceptible infection.

Is It Time for Systematic Voriconazole Pharmacogenomic Investigation for Central Nervous System Aspergillosis?

Voriconazole is the standard treatment for invasive aspergillosis but requires therapeutic drug monitoring to optimize therapy. We report two cases of central nervous system aspergillosis treated with voriconazole. Because of low trough plasma concentrations, we identified gain-of-function mutations in CYP2C19 that were partially responsible for the therapeutic failure of voriconazole. We suggest that systematic voriconazole pharmacogenomic investigation of cerebral aspergillosis be performed to avoid effective therapy delay in this life-threatening disease.

Predicting Chronic Kidney Disease Using Hybrid Machine Learning Based on Apache Spark.

Chronic kidney disease (CKD) has become a widespread disease among people. It is related to various serious risks like cardiovascular disease, heightened risk, and end-stage renal disease, which can be feasibly avoidable by early detection and treatment of people in danger of this disease. The machine learning algorithm is a source of significant assistance for medical scientists to diagnose the disease accurately in its outset stage. Recently, Big Data platforms are integrated with machine learning algorithms to add value to healthcare. Therefore, this paper proposes hybrid machine learning techniques that include feature selection methods and machine learning classification algorithms based on big data platforms (Apache Spark) that were used to detect chronic kidney disease (CKD). The feature selection techniques, namely, Relief-F and chi-squared feature selection method, were applied to select the important features. Six machine learning classification algorithms were used in this research: decision tree (DT), logistic regression (LR), Naive Bayes (NB), Random Forest (RF), support vector machine (SVM), and Gradient-Boosted Trees (GBT Classifier) as ensemble learning algorithms. Four methods of evaluation, namely, accuracy, precision, recall, and F1-measure, were applied to validate the results. For each algorithm, the results of cross-validation and the testing results have been computed based on full features, the features selected by Relief-F, and the features selected by chi-squared feature selection method. The results showed that SVM, DT, and GBT Classifiers with the selected features had achieved the best performance at 100% accuracy. Overall, Relief-F's selected features are better than full features and the features selected by chi-square.

Highly efficient Pyrococcus furiosus recombinant L-asparaginase with no glutaminase activity: Expression, purification, functional characterization, and cytotoxicity on THP-1, A549 and Caco-2 cell lines.

L-Asparaginase (L-ASNase EC 3.5.1.1) is considered as an important biopharmaceutical drug enzyme in the treatment of childhood acute lymphoblastic leukemia (ALL). In the present study, Pyrococcus furiosus L-ASNase gene was cloned into pET26b (+), expressed in E. coli BL21(DE3) pLysS, and purified to homogeneity using Ni2+ chelated Fast Flow Sepharose resin with 5.7 purification fold and 23.9% recovery. The purified enzyme exhibited a molecular weight of ~33,660 Da on SDS-PAGE and showed maximal activity at 50 °C and pH 8.0. It retained 98.3% and 60.7% initial activity after 60 min at 37 °C and 50 °C, respectively. The recombinant enzyme showed highest substrate specificity towards L-ASNase substrate, while no detectable specificity was observed for l-glutamine, urea, and acrylamide at 10 mM concentration. The Km and Vmax of the purified recombinant enzyme as calculated using Lineweaver-Burk plot were determined to be 1.623 mM and 105 µmol min-1 mg-1, respectively. Human leukemia cell line THP-1 treated with recombinant L-ASNase showed significant morphological changes, and the IC50 of the purified enzyme was found to be 0.8 IU. Moreover, the purified recombinant L-ASNase induced cytotoxic effects on lung adenocarcinoma A549 and colorectal adenocarcinoma Caco-2 cell lines with IC50 of 1.78 IU and 30 IU, respectively.

The Arabian camel, Camelus dromedarius interferon epsilon: Functional expression, in vitro refolding, purification and cytotoxicity on breast cancer cell lines.

The current study highlights, for the first time, cloning, overexpression and purification of the novel interferon epsilon (IFNƐ), from the Arabian camel Camelus dromedaries. The study then assesses the cytotoxicity of IFNε against two human breast cancer cell lines MDA-MB-231 and MCF-7. Full-length cDNA encoding interferon epsilon (IFNε) was isolated and cloned from the liver of the Arabian camel, C. dromedarius using reverse transcription-polymerase chain reaction. The sequence analysis of the camel IFNε cDNA showed a 582-bp open reading frame encoding a protein of 193 amino acids with an estimated molecular weight of 21.230 kDa. A BLAST search analysis revealed that the C. dromedarius IFNε shared high sequence identity with the IFN genes of other species, such as Camelus ferus, Vicugna pacos, and Homo sapiens. Expression of the camel IFNε cDNA in Escherichia coli gave a fusion protein band of 24.97 kDa after induction with either isopropyl ß-D-1-thiogalactopyranoside or lactose for 5 h. Recombinant IFNε protein was overexpressed in the form of inclusion bodies that were easily solubilized and refolded using SDS and KCl. The solubilized inclusion bodies were purified to apparent homogeneity using nickel affinity chromatography. We examined the effect of IFNε on two breast cancer cell lines MDA-MB-231 and MCF-7. In both cell lines, IFNε inhibited cell survival in a dose dependent manner as observed by MTT assay, morphological changes and apoptosis assay. Caspase-3 expression level was found to be increased in MDA-MB-231 treated cells as compared to untreated cells.

Tumor shape pulmonary mucormycosis associated with sinonasal aspergillosis in a diabetic patient.

Mucormycosis is a rare and life-threatening fungal infection of the Mucorales order occurring mainly in immunosuppressed patients. The most common forms are rhinocerebral but pulmonary or disseminated forms may occur. We report the case of a 61-year-old patient in whom pulmonary mucormycosis was diagnosed during his first-ever episode of diabetic ketoacidosis. While receiving liposomal amphotericin B, a sinusal aspergillosis due to Aspergillus fumigatus occurred. Evolution was slowly favorable under antifungal tritherapy by liposomal amphotericin B, posaconazole and caspofungin.

Screening for human papillomavirus (HPV) in Egyptian women by the second-generation hybrid capture (HC II) test.

BACKGROUND: HPV infection is the main cause of cervical cancer and cervical intraepithelial neoplasia worldwide. The second-generation HC II test is a liquid hybridization assay designed to detect 18 HPV types. The aim of the present study was to detect the rate of HPV infection and its various genotypes among Egyptian women. MATERIAL/METHODS: We evaluated 166 Egyptian women. They were classified according to cytology into those with normal cytology, chronic nonspecific cervicitis, and squamous intraepithelial lesions (SILs). RESULTS: The overall prevalence of HPV DNA in the studied groups was 15.06% (25/166). Among the 25 HPV-positive women, 16 (64%) were infected with high-risk HPV types, 4 (16%) with low risk HPV types, while 5 (20%) had both types. Twenty-one (84%) of the infected women harbored at least one high-risk HPV type, while 9 (36%) harbored at least one low-risk HPV type. Values of HPV viral load for low-risk HPV infection showed no significant difference in the normal and chronic nonspecific cervicitis groups. But when HPV viral load of high-risk HPV infection was compared in the normal, chronic nonspecific cervicitis, and SIL groups, a significant difference was found. The same was detected between chronic nonspecific cervicitis and SIL and between normal cytology and SIL, suggesting an association between viral load and risk of SIL and, accordingly, cancer. CONCLUSIONS: It may be concluded that HPV testing using the HC II assay is a useful tool when combined with cytology in the diagnosis of high-risk HPV viral types in apparently normal tissues.