Utilizing chitooligosaccharides from shrimp waste biodegradation via recombinant chitinase A: a promising approach for emulsifying hydrocarbon and bioremediation.

BACKGROUND: Hydrocarbon pollution stemming from petrochemical activities is a significant global environmental concern. Bioremediation, employing microbial chitinase-based bioproducts to detoxify or remove contaminants, presents an intriguing solution for addressing hydrocarbon pollution. Chitooligosaccharides, a product of chitin degradation by chitinase enzymes, emerge as key components in this process. Utilizing chitinaceous wastes as a cost-effective substrate, microbial chitinase can be harnessed to produce Chitooligosaccharides. This investigation explores two strategies to enhance chitinase productivity, firstly, statistical optimization by the Plackett Burman design approach to  evaluating the influence of individual physical and chemical parameters on chitinase production, Followed by  response surface methodology (RSM) which delvs  into the interactions among these factors to optimize chitinase production. Second, to further boost chitinase production, we employed heterologous expression of the chitinase-encoding gene in E. coli BL21(DE3) using a suitable vector. Enhancing chitinase activity not only boosts productivity but also augments the production of Chitooligosaccharides, which are found to be used as emulsifiers. RESULTS: In this study, we focused on optimizing the production of chitinase A from S. marcescens using the Plackett Burman design and response surface methods. This approach led to achieving a maximum activity of 78.65 U/mL. Subsequently, we cloned and expressed the gene responsible for chitinase A in E. coli BL21(DE3). The gene sequence, named SmChiA, spans 1692 base pairs, encoding 563 amino acids with a molecular weight of approximately 58 kDa. This sequence has been deposited in the NCBI GenBank under the accession number "OR643436". The purified recombinant chitinase exhibited a remarkable activity of 228.085 U/mL, with optimal conditions at a pH of 5.5 and a temperature of 65 °C. This activity was 2.9 times higher than that of the optimized enzyme. We then employed the recombinant chitinase A to effectively hydrolyze shrimp waste, yielding chitooligosaccharides (COS) at a rate of 33% of the substrate. The structure of the COS was confirmed through NMR and mass spectrometry analyses. Moreover, the COS demonstrated its utility by forming stable emulsions with various hydrocarbons. Its emulsification index remained stable across a wide range of salinity, pH, and temperature conditions. We further observed that the COS facilitated the recovery of motor oil, burned motor oil, and aniline from polluted sand. Gravimetric assessment of residual hydrocarbons showed a correlation with FTIR analyses, indicating the efficacy of COS in remediation efforts. CONCLUSIONS: The recombinant chitinase holds significant promise for the biological conversion of chitinaceous wastes into chitooligosaccharides (COS), which proved its potential in bioremediation efforts targeting hydrocarbon-contaminated sand.

Optimization, gene cloning, expression, and molecular docking insights for enhanced cellulase enzyme production by Bacillus amyloliquefaciens strain elh1.

BACKGROUND: In this study, we isolated a cellulase-producing bacterium, Bacillus amyloliquefaciens strain elh, from rice peel. We employed two optimization methods to enhance the yield of cellulase. Firstly, we utilized a one-variable-at-a-time (OVAT) approach to evaluate the impact of individual physical and chemical parameters. Subsequently, we employed response surface methodology (RSM) to investigate the interactions among these factors. We heterologously expressed the cellulase encoding gene using a cloning vectorin E. coli DH5α. Moreover, we conducted in silico molecular docking analysis to analyze the interaction between cellulase and carboxymethyl cellulose as a substrate. RESULTS: The bacterial isolate eh1 exhibited an initial cellulase activity of 0.141 ± 0.077 U/ml when cultured in a specific medium, namely Basic Liquid Media (BLM), with rice peel as a substrate. This strain was identified as Bacillus amyloliquefaciens strain elh1 through 16S rRNA sequencing, assigned the accession number OR920278 in GenBank. The optimal incubation time was found to be 72 h of fermentation. Urea was identified as the most suitable nitrogen source, and dextrose as the optimal sugar, resulting in a production increase to 5.04 ± 0.120 U/ml. The peak activity of cellulase reached 14.04 ± 0.42 U/ml utilizing statistical optimization using Response Surface Methodology (RSM). This process comprised an initial screening utilizing the Plackett-Burman design and further refinement employing the BOX -Behnken Design. The gene responsible for cellulase production, egl, was effectively cloned and expressed in E. coli DH5α. The transformed cells exhibited a cellulase activity of 22.3 ± 0.24 U/ml. The egl gene sequence was deposited in GenBank with the accession number PP194445. In silico molecular docking revealed that the two hydroxyl groups of carboxymethyl cellulose bind to the residues of Glu169 inside the binding pocket of the CMCase. This interaction forms two hydrogen bonds, with an affinity score of -5.71. CONCLUSIONS: Optimization of cultural conditions significantly enhances the yield of cellulase enzyme when compared to unoptimized culturing conditions. Additionally, heterologous expression of egl gene showed that the recombinant form of the cellulase is active and that a valid expression system can contribute to a better yield of the enzyme.

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.

Spectrofluorimetric Approach for Quantification of Cyclizine in the Presence of its Toxic Impurities in Human Plasma; in silico Study and ADMET Calculations.

Cyclizine (CYZ); an antiemetic compound; is widely misused for its euphoric or hallucinatory effects, either by oral or intravenous routes. The concomitant abuse of CYZ among addicted adolescents contributes to neuromuscular disorders that are life-threatening. Consequently, with the company of 1-Methylpiperazine (MPZ) and diphenylmethanol (DPM, Benzhydrol) as pharmacopoeia-reported CYZ impurities, a novel spectrofluorimetric assay for the detection of CYZ, has been established either in human plasma samples or in its parenteral formulation. The native fluorescence of CYZ has been investigated under various conditions. Different parameters affecting relative fluorescence intensity of CYZ including diluting solvent, surfactant, plasma protein solvent, and pH were studied and optimized. The linearity obtained between the fluorescence intensity at emission wavelength 350 nm after excitation at 244 nm and the corresponding CYZ concentrations was in the range 10-1000 ng/mL for measurement of CYZ either in pure form or in human plasma samples, with a appropriate correlation coefficient (r = 0.9999) and 3.10 ng/mL as the limit of detection and 9.41 ng/mL as the limit of quantitation. The suggested procedure was created and validated in accordance with ICH guidelines for quantification of CYZ either in its pure form or its dosage form, and FDA guidelines for the assay of CYZ in human plasma. Finally, in silico study and ADMET predictions were conducted for the studied drug impurities to estimate their pharmacokinetic behaviors. The results showed that both CYZ impurities have higher cellular permeability and maximum tolerated doses, DPM has higher BBB and CNS permeability than MPZ, while MPZ exceeds DPM in total clearance and volume of distribution.

Voltammetric monitoring of linezolid, meropenem and theophylline in plasma.

Treatment of healthcare associated Pneumonia (HCAP) caused by Methicillin-resistant Staphylococcus aureus (MRSA) requires therapeutic protocols formed of linezolid (LIN) either alone or in combination with meropenem (MERO) and theophylline (THEO). The inter-individual pharmacokinetic variations require the development of reliable therapeutic drug monitoring (TDM) tools especially in immunocompromised patients. A sensitive square wave voltammetric sensor using multiwalled carbon nanotubes (MWCNTs) modified carbon paste electrode in Britton-Robinson buffer was developed and validated. Experimental parameters such as pH, percentage of MWCNTs, and pre-concentration time were optimized. The sensor was employed at pH 11.0 for the determination of LIN in plasma within a concentration range of 2.5 × 10-8 - 8.0 × 10-6 mol L-1without interference from co-administered medications. On the other hand, simultaneous monitoring of LIN, MERO and THEO in plasma was feasible at pH 3.0 over concentration ranges of 4.0 × 10-7- 9.0 × 10-5, 8.0 × 10-7- 9.0 × 10-5 and 8.0 × 10-7 - 9.0 × 10-5 mol L-1, respectively. The performance of the proposed sensor was validated and the applicability for TDM has been demonstrated in plasma of healthy volunteers.

Utilizing immobilized recombinant serine alkaline protease from Bacillus safensis lab418 in wound healing: Gene cloning, heterologous expression, optimization, and characterization.

Microbial proteases have proven their efficiency in various industrial applications; however, their application in accelerating the wound healing process has been inconsistent in previous studies. In this study, heterologous expression was used to obtain an over-yielding of the serine alkaline protease. The serine protease-encoding gene aprE was isolated from Bacillus safensis lab 418 and expressed in E. coli BL21 (DE3) using the pET28a (+) expression vector. The gene sequence was assigned the accession number OP610065 in the NCBI GenBank. The open reading frame of the recombinant protease (aprEsaf) was 383 amino acids, with a molecular weight of 35 kDa. The yield of aprEsaf increased to 300 U/mL compared with the native serine protease (SAFWD), with a maximum yield of 77.43 U/mL after optimization conditions. aprEsaf was immobilized on modified amine-functionalized films (MAFs). By comparing the biochemical characteristics of immobilized and free recombinant enzymes, the former exhibited distinctive biochemical characteristics: improved thermostability, alkaline stability over a wider pH range, and efficient reusability. The immobilized serine protease was effectively utilized to expedite wound healing. In conclusion, our study demonstrates the suitability of the immobilized recombinant serine protease for wound healing, suggesting that it is a viable alternative therapeutic agent for wound management.

Development of Different Separation Techniques for Impurity Profiling of Neostigmine Methylsulfate: A Comparative Study.

Different separation techniques have been employed to resolve mixtures of multicomponent preparations over the last few decades. They could be efficiently applied for impurity profiling of active drug substances. Impurity profiling has become a critical procedure in pharmaceutical companies to comply with numerous regulatory standards. Isolation and characterization of impurities are crucial for obtaining data that proves biological safety and efficacy. In this contribution, different HPLC, capillary electrophoresis (CE) and TLC-densitometric methods were developed for the determination of neostigmine methylsulfate (NEO) along with 3-hydroxyphenyltrimethyl ammonium methylsulfate (3-HPA) and 3-dimethylaminophenol (DAP) as its impurities in the presence of citric acid. The linearity for NEO was attained in the range of 5-120 µg/mL and 10-60 µg/mL for the HPLC and CE methods, respectively. Regarding the impurities, linearity was obtained over the range of 10-30 µg/mL for 3-HPA and 5-30 µg/mL for DAP in the two proposed methods. For the TLC method, NEO and DAP were determined within the range of 1-11 µg/band, whereas 3-HPA was assayed over the range of 2-12 µg/band. The suggested methods can be securely utilized for routine analysis of the cited components in quality control laboratories.

Functionalized SnO2 nanoparticles with gallic acid via green chemical approach for enhanced photocatalytic degradation of citalopram: synthesis, characterization and application to pharmaceutical wastewater treatment.

Eco-friendly stannic oxide nanoparticles functionalized with gallic acid (SnO2/GA NP) were synthesized and employed as a novel photocatalyst for the degradation of citalopram, a commonly prescribed antidepressant drug. SnO2/GA NP were characterized using high-resolution transmission electron microscopy, Fourier transform infrared spectroscopy, Brunauer-Emmett-Teller measurements and X-ray diffraction. A validated RP-HPLC assay was developed to monitor citalopram concentration in the presence of its degradation products. Full factorial design (24) was conducted to investigate the effect of irradiation time, pH, SnO2/GA NP loading and initial citalopram concentration on the efficiency of the photodegradation process. Citalopram initial concentration was found to be the most significant parameter followed by irradiation time and pH, respectively. At optimum conditions, 88.43 ± 0.7% degradation of citalopram (25.00 µg/mL) was obtained in 1 h using UV light (1.01 mW/cm2). Citalopram kinetics of degradation followed pseudo-first order rate with Kobs and t0.5 of - 0.037 min-1 and 18.73 min, respectively. The optimized protocol was successfully applied for treatment of water samples collected during different cleaning validation cycles of citalopram production lines. The reusability of SnO2/GA NP was studied for 3 cycles without significant loss in activity. This approach would provide a green and economic alternative for pharmaceutical wastewater treatment of organic pollutants.

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.

Comparative Chemometric Manipulations of UV-Spectrophotometric Data for the Efficient Resolution and Determination of Overlapping Signals of Cyclizine and Its Impurities in Its Pharmaceutical Preparations.

BACKGROUND: Cyclizine (CYZ), a commonly used antiemetic drug, has two pharmacopeial toxic impurities, 1-methylpiperazine (MPZ) and diphenylmethanol (DPM). When CYZ parenteral formulations are administered intravenously, both impurities are poisonous, toxic, and harmful to the human body. OBJECTIVE: Cyclizine was determined along with its hazardous impurities MPZ and DPM by green multivariate calibration using UV-spectroscopic data. METHODS: Three multivariate algorithms were used to resolve and quantify overlapped spectral signals: principal component regression (PCR), partial least squares (PLS), and synergistic intervals partial least squares (siPLS). A concentration set containing 16 distinct combinations of CYZ, MPZ, and DPM was randomly prepared, and the absorbance values of the concentration set were determined using the 376 point-wavelength set with an interval of 0.2 nm between 200 and 275 nm. RESULTS: Good linear correlations were established for CYZ, MPZ, and DPM in the concentration ranges of 5.00-25.0, 0.50-2.50, and 0.50-2.50 µg/mL, respectively. The ideal spectral range and associated combinations were chosen based on the lowest root mean error of prediction (RMSEP) and correlation coefficient values (r). The siPLS approach performed better than the PCR and PLS models. The combination of four subintervals, 1, 3, 4, and 7, demonstrated the greatest effect, with RMSEP values of 0.0272, 0.0053, and 0.0315 for CYZ, MPZ, and DPM, respectively, and correlation coefficients of 0.9991, 0.9999, and 0.9997, in order. Various assessment tools were used to evaluate and measure the greenness profile of the established methods. The proposed methods were validated using internal and external validation sets. CONCLUSIONS: The three methods were effectively used to determine CYZ in its pure form and parenteral formulations, as well as its toxic impurities. The acquired results were compared statistically to those obtained using the reported HPLC method. HIGHLIGHTS: Cyclizine and its toxic impurities can be determined spectrophotometrically by using the three developed chemometric models.

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.

Quality by design approach for green HPLC method development for simultaneous analysis of two thalassemia drugs in biological fluid with pharmacokinetic study.

This work implements a combined experimental approach of analytical quality-by-design (AQbD) and green analytical chemistry (GAC) to develop an HPLC method for simultaneous determination of the two thalassemia drugs, deferasirox (DFX) and deferiprone (DFP), in biological fluid for the first time. This integration was designed to maximize efficiency and minimize environmental impacts, as well as energy and solvent consumption. To accomplish this goal, an analytical quality-by-design approach was performed, beginning with quality risk assessment and scouting analysis, followed by Placket-Burman design screening for five chromatographic parameters. Critical method parameters were thoroughly recognized and then optimized by using a two levels-three factors custom experimental design to evaluate the optimum conditions that achieved the highest resolution with acceptable peak symmetry within the shortest run time. The desirability function was used to define the optimal chromatographic conditions, and the optimal separation was achieved using an XBridge® HPLC RP-C18 (4.6 × 250 mm, 5 µm) column with ethanol : acidic water at pH 3.0 adjusted by phosphoric acid in the ratio of (70 : 30, v/v) as the mobile phase at a flow rate of 1 mL min-1 with UV detection at 225 nm at a temperature of 25 °C. Linearity was obtained over the concentration range of 0.30-20.00 µg mL-1 and 0.20-20.00 µg mL-1 for DFX and DFP, respectively, using 20.00 µg mL-1 ibuprofen (IBF) as an internal standard. The established method's greenness profile was evaluated and measured using various assessment tools, and the developed method was green. For the validation of the developed method, FDA recommendations were followed, and all the results obtained met the acceptance criteria. The suggested method was successfully used to study the pharmacokinetic parameters of DFX and DFP in rat plasma. Due to the substantial increase in bioavailability of the two iron chelating drugs, the results from this study strongly recommend their co-administration.

Nanoparticle-enhanced in-line potentiometric ion sensor for point-of-care diagnostics for tropicamide abuse in biological fluid.

Point of care (POC), also identified as on-site testing, has evolved as a rapid and accurate technique for drug of abuse screening and analysis. The aim of this work is to detect tropicamide (TPC) abuse in biological fluids; we selected rat plasma as example. We developed a disposal miniaturized, portable, green, and budget-friendly POC solid-state electrochemical sensor based on potentiometric transduction. To attain that, an innovative microfabricated electrode modified with conducting polymer poly(3-octylthiophene) (POT) has been placed on sensitized printed circuit board (PCB). A two-stage optimization process was implemented to develop the fabricated electrode. The first stage of the optimization process depends on screening various ionophores in order to enhance the sensor selectivity towards tropicamide. Copper nanoparticles exhibited the highest selectivity towards TPC. The second stage was utilizing a polymer as an ion-to-electron transducer layer between the copper nanoparticles impregnated ion sensing membrane and the microfabricated solid-contact ion-selective electrode. This polymer was added to boost the stability of the potential drift (1.2 mV/h) due to the hydrophobic behavior of the POT, which precludes the formation of an aqueous layer at the Cu electrode/polymeric membrane interface and improve the limit of detection (1.1 × 10-8 M). Nernstian potentiometric response was accomplished for TPC with a slope of 58.46 ± 0.43 mV/decade and E0 ∼ 189.39 ± 2.12 over the concentration range 1.0 × 10-7 to 1.0 × 10-2 M. The suggested sensor's intrinsic figure of merits include a quick response time (13 ± 2 s) and long life time (45 days). The proposed sensor has been successfully employed in the selective determination of TPC in pharmaceutical formulations, and biological fluids. When the results were compared to those of the official approach, there was no statistically significant difference. The Eco-Scale tool assessed and measured the greenness profile of the established method.

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.

A local Talaromyces atroroseus TRP-NRC isolate: isolation, genetic improvement, and biotechnological approach combined with LC/HRESI-MS characterization, skin safety, and wool fabric dyeing ability of the produced red pigment mixture.

BACKGROUND: During the last decade, enormous research efforts have been directed at identifying potent microorganisms as sustainable green cell factories for eco-friendly pigments. Talaromyces atroroseus has recently been shown to excrete large amounts of azaphilone mycotoxin-free red pigment mixture comprising some known coloring components together with many uncharacterized metabolites. In this study, a new Talaromyces atroroseus isolate was identified via sequencing of the fragment of the nuclear ribosomal gene cluster containing internal transcribed spacers and 5.8S rRNA gene. The parameters that affected the level of pigment production were optimized in uncommon static conditions of culture and genetic improvement, via γ-irradiation, to improve pigment yield. Moreover, chemical characterization using LC/MS and skin safety test of the target pigment mixture were precisely conducted to maximize its benefits as a natural and safe red pigment for wool fabrics. RESULTS: Molecular identification via the sequencing of the ITS of the rDNA encoding gene cluster revealed that the fungal isolate TRP-NRC was T. atroroseus TRP-NRC (deposited in GenBank under accession number MW282329). In the static conditions of culture, pigment production was dramatically enhanced to 27.36 g/L in an optimum yeast malt peptone medium of 2% mannitol at pH 2-4.5 and 30 °C for 7 days of incubation. Under exposure to a 400-Gy γ-radiation dose, pigment yield was increased to a 3-fold level higher than that recorded for the wild type. Based on the inter-simple sequence repeats (ISSR), as a molecular marker tool, the wild-type T. atroroseus TRP-NRC strain and its mutants were discriminated. The UHPLC/HRESI-MS analytical tool characterized 60 metabolites, including many unknown molecules, at appropriate concentrations. It is worthy to note that four mitorubrin derivatives were identified for the first time in T. atroroseus, i.e., mitorubrinolamine acetate, dihydro-PP-O, mitorobrinal, and mitorubrinol. The range of irritation indexes (0-0.1) demonstrated an adequate skin safety after the direct local application of the pigment mixture. Finally, the pigment mixture exhibited a remarkably good dyeing ability in wool fabrics, with high-fastness properties. CONCLUSIONS: Because of its sustainable and economic production, the target red pigment mixture may be applied in the future in textile, food, cosmetics, or different pharmaceutical industries after extensive conventional safety and toxicity studies, which are currently under consideration.

Label-Free Potentiometric Ion Flux Immunosensor for Determination of Recombinant Human Myelin Basic Protein: Application to Downstream Purification from Transgenic Milk.

Recombinant human myelin basic protein (rhMBP) produced in the milk of transgenic cows was found exclusively associated with milk caseins. This hindered its direct determination without extensive sample pretreatment. Here, a label-free potentiometric immunosensor was developed and validated for the determination of rhMBP. An ion flux was generated under zero-current based on surface blocking of the polymeric membrane ion-selective electrode by anti-hMBP antibody and tetrabutylammonium bromide as a marker ion. The immunosensor was successfully employed in the quantitative determination of hMBP in the range of 0.10-20.00 µg/mL with a limit of detection of 50.00 ng/mL. The applicability of the passive ion flux immunosensor for determination of target analyte in complex matrices was investigated. Downstream purification of rhMBP from the milk of transgenic cows was achieved using cation exchange chromatography, immobilized metal affinity chromatography, and immunoaffinity chromatography. The specificity of the immunosensor along with matrix effect of milk proteins were demonstrated. Results obtained using the rhMBP immunosensor were further cross-validated using an orthogonal testing protocol assembled of RP-HPLC and SE-HPLC. It should be noted that the proposed ion flux immunosensor provided a feasible and specific tool for monitoring rhMBP concentration/purity, immunogenic activity, and stability. Such approach provides an attractive economic alternative to sophisticated biosensors required for in-process quality control of biopharmaceutical products.

Simultaneous determination of linezolid, meropenem and theophylline in plasma.

The data presented in this article are related to the research article entitled "Voltammeric monitoring of linezolid, meropenem and theophylline in plasma" (A.K. Attia, M.A. Al-Ghobashy, G.M. El-Sayed, S.M. Kamal, accepted in Anal. Biochem. 2018). This article describes a sensitive square wave voltammetric (SWV) method for simultaneous monitoring of linezolid (LIN), meropenem (MERO) and theophylline (THEO) in spiked plasma and in plasma of healthy volunteers.

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.

Utilization of 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) for kinetic spectrophotometric assay of befunolol hydrochloride in its pharmaceutical formulation.

A simple, accurate, precise, and sensitive kinetic spectrophotometric method for determination of befunolol hydrochloride is described. The method is based on the formation of a colored product with 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) in methanol at 70 degrees C for 45 min. The red-colored product was measured at 523 nm. The optimization of various experimental conditions is described; Beer's law was obeyed in the range 15.25 x 10(-6) to 122.04 x 10(-6) M. The results obtained showed good recoveries (100.1 +/- 0.80%). Application of the proposed method to a pharmaceutical formulation was successfully achieved. The determination of befunolol hydrochloride by fixed time, fixed concentration, and rate constant methods was feasible with the calibration equation obtained. However, the fixed time method proved to be more applicable.

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.