Gene Cloning, Heterologous Expression, and In Silico Analysis of Chitinase B from Serratia marcescens for Biocontrol of Spodoptera frugiperda Larvae Infesting Maize Crops.

Spodoptera frugiperda, the fall armyworm (FAW), is a highly invasive polyphagous insect pest that is considered a source of severe economic losses to agricultural production. Currently, the majority of chemical insecticides pose tremendous threats to humans and animals besides insect resistance. Thus, there is an urgent need to develop new pest management strategies with more specificity, efficiency, and sustainability. Chitin-degrading enzymes, including chitinases, are promising agents which may contribute to FAW control. Chitinase-producing microorganisms are reported normally in bacteria and fungi. In the present study, Serratia marcescens was successfully isolated and identified from the larvae of Spodoptera frugiperda. The bacterial strain NRC408 displayed the highest chitinase enzyme activity of 250 units per milligram of protein. Subsequently, the chitinase gene was cloned and heterologously expressed in E. coli BL21 (DE3). Recombinant chitinase B was overproduced to 2.5-fold, driven by the T7 expression system. Recombinant chitinase B was evaluated for its efficacy as an insecticidal bioagent against S. frugiperda larvae, which induced significant alteration in subsequent developmental stages and conspicuous malformations. Additionally, our study highlights that in silico analyses of the anticipated protein encoded by the chitinase gene (ChiB) offered improved predictions for enzyme binding and catalytic activity. The effectiveness of (ChiB) against S. frugiperda was evaluated in laboratory and controlled field conditions. The results indicated significant mortality, disturbed development, different induced malformations, and a reduction in larval populations. Thus, the current study consequently recommends chitinase B for the first time to control FAW.

Green chromatographic methods for determination of co-formulated lidocaine hydrochloride and miconazole nitrate along with an endocrine disruptor preservative and potential impurity.

Recently, green analytical chemistry (GAC) is a key issue towards the idea of sustainability, the analytical community is focused on developing analytical methods that incorporate green chemistry principles to minimize adverse impacts on the environment and humans. Herein, we present 2 sustainable, selective, and validated chromatographic methods. Initially, lidocaine hydrochloride (LDC) and miconazole nitrate (MIC) with two preservatives; methyl paraben (MTP) and saccharin sodium (SAC) were chromatographed via TLC-densitometric method which employed ethyl acetate: methanol: formic acid (9:1:0.1, by volume) as the mobile phase with UV detection at 220.0 nm, good correlation was obtained in the range of 0.3-3.0 µg/band for MIC and LDC. Following that, RP-HPLC was successfully applied for separating quinary mixture of LDC, MIC, MTP, SAC along with LDC impurity; dimethyl aniline (DMA) using C18 column, and a gradient green mobile phase composed of methanol and phosphate buffer (pH 6.0) in different ratios with a flow rate 1.5 mL/min and UV detection at 210.0 nm, linearity ranges from 1.00 to 100.00 µg/mL for MIC, 2.00-100.00 µg/mL for LDC and 1.00--20.00 µg/mL for MTP and DMA. No records to date regarding the determination of the two drugs, besides MTP and DMA. The proposed methods were validated according to the ICH guidelines and applied successfully to the analysis of the compounds. The methods' results were statistically compared to those obtained by applying the reported one, indicating no significant difference regarding both accuracy and precision. The methods' greenness profiles have been assessed and compared with those of the reported method using different assessment tools.

Safe production of Aspergillus terreus xylanase from Ricinus communis: gene identification, molecular docking, characterization, production of xylooligosaccharides, and its biological activities.

BACKGROUND: The production of industrial enzymes such as xylanase using sufficient cost-effective substrates from potent microorganisms is considered economically feasible. Studies have reported castor cake (Ricinus communis) as the most potent and inexpensive alternative carbon source for production of xylanase C by using Aspergillus terreus (A. terreus). RESULTS: A. terreus strain RGS Eg-NRC, a local isolate from agro-wastes, was first identified by sequencing the internal transcribed spacer region of a nuclear DNA encoding gene cluster deposited in GenBank (accession number MW282328). Before optimization of xylanase production, A. terreus produced 20.23 U/g of xylanase after 7 days using castor cake as a substrate in a solid-state fermentation (SSF) system that was employed to achieve ricin detoxification and stimulate xylanase production. Physicochemical parameters for the production of xylanase were optimized by using a one-variable-at-a-time approach and two statistical methods (two-level Plackett-Burman design and central composite design, CCD). The maximum xylanase yield after optimization was increased by 12.1-fold (245 U/g). A 60-70% saturation of ammonium sulfate resulted in partially purified xylanase with a specific activity of 3.9 IU/mg protein. At 60 °C and pH 6, the partially purified xylanase had the highest activity, and the activation energy (Ea) was 23.919 kJmol. Subsequently, antioxidant capacity and cytotoxicity tests in normal Ehrlich ascites carcinoma human cells demonstrated xylooligosaccharides produced by the xylanase degradation of xylan as a potent antioxidant and moderate antitumor agent. Further investigations with sodium dodecyl sulfate polyacrylamide gel electrophoresis then determined the molecular weight of partially purified xylanase C to be 36 kDa. Based on the conserved regions, observations revealed that xylanase C belonged to the glycosyl hydrolase family 10. Next, the xylanase-encoding gene (xynC), which has an open reading frame of 981 bp and encodes a protein with 326 amino acids, was isolated, sequenced, and submitted to the NCBI GenBank database (accession number LC595779.1). Molecular docking analysis finally revealed that Glu156, Glu262, and Lys75 residues were involved in the substrate-binding and protein-ligand interaction site of modeled xylanase, with a binding affinity of -8.7 kcal. mol-1. CONCLUSION: The high production of safe and efficient xylanase could be achieved using economical materials such as Ricinus communis.

Different Approaches in Manipulating Ratio Spectra for Analyzing Amlodipine Besylate and Irbesartan Combination.

BACKGROUND: Hypertension is a key risk factor for ischemic heart disease and atherosclerosis. Most patients require a combination of antihypertensive medications to accomplish their therapeutic goals. Antihypertensive medicines such as calcium channel blockers and angiotensin receptor blockers are indicated for patients whose high blood pressure cannot be controlled with monotherapy. The combination of amlodipine besylate (AML) with irbesartan (IRB) is an example of this synergistic activity in lowering blood pressure. OBJECTIVE: In this regard, the goal of the research is to develop sensitive spectrophotometric methods for the simultaneous determination of amlodipine besylate and irbesartan. METHODS: Three simple ratio spectra-manipulating spectrophotometric methods namely, ratio difference, mean centering of ratio spectra, and derivative ratio, were developed for the simultaneous assay of the cited mixture. RESULTS: Linear correlations were attained over the concentration range of 1-35 µg/mL and 2-35 µg/mL for amlodipine besylate and irbesartan, respectively. The methods were validated according to the International Conference on Harmonization guidelines with good results. CONCLUSION: The methods developed were successfully applied for the assay of the cited drugs in their marketed formulation. They could be efficiently used for routine analysis of the mentioned drugs in QC laboratories. HIGHLIGHTS: The proposed approaches do not require expensive solvents or complex instruments. They could be used in routine laboratory tests where time and cost are crucial.

Determination of voriconazole and co-administered drugs in plasma of pediatric cancer patients using UPLC-MS/MS: A key step towards personalized therapeutics.

Untreated invasive aspergillosis results in high mortality rate in pediatric cancer patients. Voriconazole (VORI), the first line of treatment, requires strict dose monitoring because of its narrow therapeutic index and individual variation in plasma concentration levels. Commonly co-administered drugs; either Esomeprazole (ESO) or Ondansetron (OND) have reported drug-drug interaction with VORI that should adversely alter therapeutic outcomes of the latter. Although VORI, ESO and OND are co-administered to pediatric cancer patients, the combined effect of ESO and OND on the plasma concentration levels of VORI has not been fully explored. In this study, an accurate, reliable and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated for simultaneous determination of VORI, ESO, and OND in ultra-low sample volumes (25 µL) of plasma of pediatric cancer patients. Based on the physicochemical properties of the studied drugs and internal standard, liquid-liquid extraction was successfully adopted with methyl t-butyl ether. Consistent and reproducible recovery of the three drugs and the internal standard were calculated using plasma and matrix matched samples (RE% > 72.97%, RSD < 8.29%). Chromatographic separation was carried out using UPLC with C18 column and a mobile phase of acetonitrile:water:methanol (70:25:5 V/V/V) at 0.3 mL/min. Mass spectrometric determination at positive electrospray ionization in the MRM mode was employed. The analysis was achieved within 4 min over a linear concentration range of 1.00-200.00 ng/mL for the three drugs. The assay validity was assessed as per the Food and Drug Administration guidelines for bioanalytical method validation, and satisfactory results were obtained. The accuracy and precision were within the acceptable limits for the three drugs in both quality control and incurred plasma samples. Matrix effect and process efficiency were investigated in neat solvent, post-extraction matrix, and plasma. Correlation of the plasma concentration levels of the three drugs revealed differences from the reported drug-drug interactions. This confirmed the need for simultaneous determination of VORI and co-administered drugs in order to achieve optimal therapeutic outcomes. To achieve this, analysis results of this study, genetic polymorphisms in CYP2C19 and clinical data will be used to establish one model incorporating all possible factors that might lead to variation in therapeutic outcomes.

Bcl-2 protein expression in egyptian acute myeloid leukemia.

OBJECTIVE: The primary cause of treatment failure in acute myeloid leukemia (AML) is the emergence of both resistant disease and early relapse. The bcl-2 gene encodes a 26-kDa protein that promotes cell survival by blocking programmed cell death (apoptosis). In the present study, bcl-2 protein expression was evaluated in newly diagnosed AML patients and correlated with the induction of remission and overall survival (OS), in an attempt to define patients who might benefit from modified therapeutic strategies. PATIENTS AND METHODS: Pretreatment cellular bcl-2 protein expression was measured in bone marrow samples obtained from 68 patients of newly diagnosed acute myeloid leukemia and 10 healthy controls by western blotting. RESULTS: The mean bcl-2 protein expression was significantly higher in patients (0.686+/-0.592) compared to controls (0.313+/-0.016) (p=0.002). The overall survival for patients with mean bcl-2 expression of less, and more than or equal to 0.315, was 67% and 56%, respectively, with no significant difference between the two groups (p=0.86). CONCLUSION: Even though we did not observe a significant difference in overall survival between patients with high and low levels of bcl-2, modulation of this protein might still be considered as an option for enhancing the effectiveness of conventional chemotherapy. KEY WORDS: Acute myeloid leukemia (AML) - Bcl-2 - prognosis - Western blot.

Intercellular adhesion molecule-1(ICAM-1), CD44s expression and serum level of sICAM-1 in disseminated non-Hodgkin's lymphoma: correlation with overall survival.

BACKGROUND AND PURPOSE: Uncontrolled growth, invasion and distant spread are the characteristics of neoplastic cells. Changes in adhesion molecule expression, such as loss of expression, de novo expression or functional alterations, can be observed in each of these steps. The aim of the present work was to study the expression of two adhesion molecules [standard CD44 (CD44s) and intercellular adhesion molecule-1 (ICAM-1)] and the soluble form of ICAM-1(sICAM-1) in adult disseminated non-Hodgkin's lymphoma (D-NHL) and to correlate the findings with overall survival. PATIENTS AND METHODS: The expression of CD44s and ICAM-1 adhesion molecules and the level of sICAM-1 were studied in 34 cases of (D-NHL). They included: 13 cases of lymphoblastic lymphoma (LBL), 5 cases of Burkitt's lymphoma (BL), one case of diffuse large cell lymphoma (DLCL), 14 cases of small lymphocytic lymphoma (SLL), one case of mantle cell lymphoma (MCL). Ten apparently healthy individuals were taken as a control group. CD44s expression was evaluated by direct immunofluoresence, ICAM-1 by immunoperoxidase on cyto-preps and serum level of sICAM-1 by ELISA. RESULTS: ICAM-1 was positive in 6/34 cases (17.6%) of D-NHL. ICAM-1 was expressed in 2/15 (13.3%) of low-grade NHL and 4/19 (21%) of high-grade lymphoma with no significant difference between the two groups (p=0.6). CD44s was expressed in 13/34 cases (38.2%) of D-NHL; in 4/15 (26.6%) of low grade NHL and 9/19 (47.3%) of high grade lymphoma with no significant difference between the two groups (p=0.2). The serum levels of sICAM-1 were elevated in all patients with D-NHL compared to healthy controls with a statistically significant difference (p <0.001), (median 1160 ng/ml, range from 150 to 2500 ng/ml, and 375 ng/ml, range from 270 to 620 ng/ml, respectively). The median of sICAM-1 in patients with high-grade D-NHL was significantly (p=0.006) lower than that of those with low-grade D-NHL (median 890 ng/ml, range from 150 to 1540 ng/ml and 1440 ng/ml, range from 380 to 2500 ng/ml respectively). The median follow-up duration for all patients was 18 months. No statistical significant difference was achieved (p=0.8) on comparing overall survival pattern between D-NHL lymphoma patients with positive or negative expression of ICAM-1 or sCD44 expression. Also, no statistically significant difference (p=0.9) was found between patients with sICAM (above and below 600 ng/ml) at 18 months from diagnosis. CONCLUSION: There is a marked heterogeneity in cell adhesion molecule (CAM) expression in NHL and this may correlate with degree of differentiation of malignant lymphocytes. However the exact significance of these findings will require functional studies to determine the role of these CAMs in each subtype of NHL.