Liquid Chromatography Time-of-flight Mass Spectrometry Research Articles

Optimization of Metabolite Extraction Protocols for Untargeted Metabolite Profiling of Mycoparasitic Scytalidium parasiticum using LC-TOF-MS

Basal stem rot disease of oil palm caused by Ganoderma boninense is one of the most devastating diseases in oil palm plantation resulting in low yield, loss of palm stands and shorter replanting cycle. To-date, there is no effective treatment for Ganoderma infected palms. Control measures, either chemical or cultural approaches, show varying degrees of effectiveness. The application of biological control agents which is environmental-friendly could be an attractive solution to overcome the problem. Earlier, we had isolated a mycoparasite, Scytalidium parasiticum, from the basidiomata of Ganoderma boninense. In vitro assay and nursery experiment showed that this fungus could suppress Ganoderma infection and reduce disease severity. However, metabolites which might contribute to the antagonistic or mycoparasitic effect remain unknown. In the current study, optimization of fungal sample processing, extraction, and analytical procedures were conducted to obtain metabolites from the maize substrate colonized by mycoparasitic ascomycetous Scytalidium parasiticum. This technique capable of producing sexual spores in sac-like organs. Untargeted metabolomics profiling was carried out by using Liquid Chromatography Time of Flight Mass Spectrometry (LC-ToF-MS). We found that S. parasiticum in both liquid- and solid-state cultivation gave higher metabolite when extracted with 60% methanol with 1% formic acid in combination with homogenisation methods such as ultrasonication and grinding. The findings from this study are useful for optimisation of metabolite extraction from other fungi-Ganoderma-plant interactions.

Elucidation and Characterization of New Chlorinated By-Products after Electrochemical Degradation of Hydrochlorothiazide Using Graphite–Poly Vinyl Chloride Electrode

This paper describes an electrochemical treatment process of hydrochlorothiazide (HDZ) under different conditions such as initial concentration, sodium chloride and applied voltage. In this present study, HDZ was treated by electrochemical oxidation process using graphite-PVC composite electrode as anode and Platinum (Pt) as cathode. All results were analyzed using liquid chromatography-time of flight/mass spectrometry (LC-TOF/MS). It was found that at high applied voltages, and high amounts of NaCl, the electrochemical treatment process was more efficient. The removal% of HDZ was 92% at 5 V after 60 min. From the obtained results, the electrochemical oxidation process of HDZ followed pseudo first order with rate constant values ranged between 0.0009 and 0.0502 min−1, depending on the experimental conditions. Energy consumption was also considered in this study, it was ranged between 0.9058 and 5.56 Wh/mg using 0.5, 0.3 and 0.1 g NaCl within interval times of (10, 20, 30, 40, 50, 60, 70, and 80 min). Five chlorinated and one non-chlorinated by-products were formed and analyzed in negative ionization (NI) mode during the electrochemical process. Due to the strong oxidizing potential of the chlorine (Cl2) and hypochlorite ion (ClO−), HDZ and its by-products were removed after 140 min. Furthermore, a novel synthesis of chlorothiaizde as one of the new by-products was reported in this present study. Toxicity was impacted by the formation of the by-products, especially at 20 min. The inhibition percentage (I%) of E. coli bacteria was decreased to be the lowest value after 140 min.

Aflatoxin B1 degradation by salt tolerant Tetragenococcus halophilus CGMCC 3792

This study explores aflatoxin B1 (AFB1) degradation by salt tolerant Tetragenococcus halophilus CGMCC 3792 (T. halophilus CGMCC 3792). Six non-toxic degradation products of AFB1 were identified by liquid chromatography/time-of-flight mass spectrometry (LC/TOF-MS), including m/z 243.06 (C14H10O4), 361.09 (C18H16O8), 229.09 (C14H12O3), 277.14 (C16H20O4), 217.12 (C14H16O2), 221.15 (C14H20O2). Two pathways were proposed based on molecular formulas and MS/MS spectra, and the final degradation product was m/z 221.15 (C14H20O2). The degradation ratio of active cell component (66%) and intracellular component (57%) was significantly higher than extracellular component (14%). AFB1 degradation ratio of intracellular component, initially at around 60%, was decreased to 32% after proteinase K treatment, and to 7% after heating, to 9% after proteinase K plus SDS treatment, and to 16% after TFA treatment. It suggests that the AFB1 removal mainly resulted from enzyme biodegradation. The degradation ratio was 92% in AFB1 polluted soy sauce mash. The high degradation ratio of AFB1 by T. halophilus CGMCC 3792 indicates its great potential for application in oriental fermentation condiment process.

Isolation and identification of novel antioxidant and antimicrobial oligopeptides from enzymatically hydrolyzed anchovy fish meal

Abstract Natural Antioxidants and antimicrobials are preferred to prolong food shelf life, therefore we isolated and identified novel natural antioxidant and antimicrobial oligopeptides from anchovy fish meal hydrolysate. Firstly, the oligopeptide mixture was prepared by two-step hydrolysis using alkaline and neutral proteases step by step for enriching oligopeptides. Then, four novel oligopeptide fractions were purified using reversed phase-high performance liquid chromatography, and their sequences were determined using high performance liquid chromatography-time of flight-mass spectrometry. Meanwhile, antioxidant and antimicrobial activities of the fractions were evaluated. Thr-Pro-Ser-Ala-Gly-Lys exhibited the highest hydroxyl scavenging activity, lipid peroxidation inhibition, ferrous ion chelating activity as well as Escherichia coli growth inhibition. Thr-Pro-Ser-Asn-Leu-Gly-Gly-Lys showed the highest superoxide scavenging activity, lipid peroxidation inhibition, and Staphylococcus aureus growth inhibition. Leu-Glu and Leu-Glu-Glu displayed similarly high inhibition activity on 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid. Almost no suppression effects on Aspergillus flavus and Candida albicans growth were observed for all isolated fractions. These results indicate that the oligopeptide mixture derived from anchovy fish meal could serve as a suitable source of antioxidant and antimicrobial oligopeptides.

Photolysis of atorvastatin in aquatic environment: Influencing factors, products, and pathways

Atorvastatin (ATV), a second generation cholesterol-lowering drug, is detected frequently in natural water because of its extensive use and incomplete removal from wastewater. In this study, the photochemical behavior of ATV under simulated solar irradiation was systematically investigated in order to assess the potential of photolysis as its transformation pathway in aquatic environment. The quantum yield of ATV direct photolysis was determined to be 0.0041. Among various water components investigated, including pH, Suwannee River Fulvic Acid (SRFA), Fe3+, HCO3−, SO42− and NO3−, the major factors contributing to the indirect photolysis of ATV were SRFA and NO3−, and the co-existence of SRFA and NO3− showed no interaction in synthetic water containing the above water components. The results were further verified in natural water samples. Singlet oxygen (1O2) played dominant role in the indirect photolysis of ATV, and the contributions of 1O2 and ·OH to the photolysis of ATV in the solution with optimum combination of water components were calculated to be 67.14% and 0.66%, respectively. Nine phototransformation intermediates were identified by liquid chromatography - time-of-flight - mass spectrometry (LC-TOF-MS), and the degradation pathways were speculated as hydroxyl addition, pyrrole-ring open and debenzamide reactions. In addition, the evolution of products in the degradation process showed that the ring-opened product P416 and hydroxylation product P575 still remained at a certain level after two days of photodegradation, which may accumulate and cause additional ecological risks. This study provides significant information for understanding the risk and fate of ATV in aquatic environment.

Transportation of Different Therapeutic Classes of Pharmaceuticals to the Surface Water, Sewage Treatment Plant, and Hospital Samples, Malaysia

All pharmaceuticals are separated chromatographically using the liquid chromatography-time of flight/mass spectrometry (LC-ToF/MS) on a 5 µm, 2.1 mm × 250 mm, C18 column at 0.3 mL/min. The recovery is investigated at two spiking levels, 10 and 1 ng/mL; the mean recovery is higher than 77, 84, and 93% in sewage treatment plants (STP) influent, STP effluent, and surface water, respectively. The limit of quantification (LOQ) averages 29, 16, 7, and 2 ng/L in STP influent, STP effluent, surface water, and drinking water, respectively. The matrix effect is also evaluated in STP influent and effluent. It is observed that sulfamethoxazole, prednisolone, ketoprofen, and glibenclamide are highly impacted compared to other compounds, −99, −110, 77, and 91%, respectively. The results show that six out of nine pharmaceuticals, namely atenolol, acetaminophen, theophylline, caffeine, metoprolol, and sulfamethoxazole are detected in STP influent, STP effluent, and surface water. However, the means of concentration are 561, 3305, 1805, 3900, 78, and 308 ng/L for atenolol, acetaminophen, theophylline, caffeine, metoprolol, and sulfamethoxazole, respectively, in STP influent. Caffeine and acetaminophen are detected with the highest concentration, reaching up to 8700 and 4919 ng/L, respectively, in STP influent.

Ultrasound-assisted extraction of hemicellulose and phenolic compounds from bamboo bast fiber powder.

Ultrasound-assisted extraction of hemicellulose and phenolic compounds from bamboo bast fibre powder was investigated. The effect of ultrasonic probe depth and power input parameters on the type and amount of products extracted was assessed. The results of input energy and radical formation correlated with the calculated values for the anti-nodal point (λ/4; 16.85 mm, maximum amplitude) of the ultrasonic wave in aqueous medium. Ultrasonic treatment at optimum probe depth of 15 mm improve 2.6-fold the extraction efficiencies of hemicellulose and phenolic lignin compounds from bamboo bast fibre powder. LC-Ms-Tof (liquid chromatography-mass spectrometry-time of flight) analysis indicated that ultrasound led to the extraction of coniferyl alcohol, sinapyl alcohol, vanillic acid, cellobiose, in contrast to boiling water extraction only. At optimized conditions, ultrasound caused the formation of radicals confirmed by the presence of (+)-pinoresinol which resulted from the radical coupling of coniferyl alcohol. Ultrasounds revealed to be an efficient methodology for the extraction of hemicellulosic and phenolic compounds from woody bamboo without the addition of harmful solvents.

Mechanism and kinetics of degrading aflatoxin B1 by salt tolerant Candida versatilis CGMCC 3790

Four products were identified by liquid chromatography/time-of-flight mass spectrometry (LC/TOF-MS) for the degradation of aflatoxin B1 (AFB1) is by salt tolerant Candida versatilis CGMCC 3790 (C. versatilis CGMCC 3790), includingⅡ(C14H10O4), Ⅲ (C14H12O3), Ⅳ (C13H12O2), Ⅴ (C11H10O4), which were not toxic. Based on these products, it is speculated that AFB1 degradation has two pathways. The degradation ratio of active cell component (69.40%) and intracellular component (64.99%) was significantly higher than extracellular component (29.61%), suggesting that the AFB1 removal mainly resulted from biodegradation. The optimal degradation conditions of AFB1 (20 ng/mL) were: incubated at pH 5.0, 25 °C for 60 min in liquid medium system. The degradation ratio was ranged from 41.23%∼100% at 10.26∼130.44 ng/g in an actual system. This is the first report revealing that a salt tolerant yeast could effectively degrade AFB1. Therefore, Candida versatilis CGMCC 3790 might be an excellent candidate for bioremediation and detoxification for oriental fermentation condiment process.

Determination of theobromine and caffeine in some Malaysian beverages by liquid chromatography-time-offlight mass spectrometry

Purpose: To determine the concentration of theobromine (TB) and caffeine (CF) in tea and other beverages using liquid chromatography-time of flight-mass spectrometry (LC-TOF-MS).Methods: The extract of caffeine and theobromine from tea and other beverages was filtered by 0.45 μm nylon micro-syringe and then injected into a LC-ToF-MS system. Theobromine and caffeine were separated using Thermo Scientific C18-column (length 250 mm, width 2.1 mm and diameter 5 μm). Acetonitrile-methanol (ACN – MeOH, 3:1 v/v) was used as mobile phase B, while mobile phase A was 0.1 % FA in DIW. The volume injected was 30 μL at a rate of 0.3 mL/min.Results: Good linearity was obtained in the range of 0.3 – 400 and 0.2 – 200 mg/L for theobromine and caffeine, respectively (regression coefficient (R2) > 0.970). The limits of detection were 0.15 and 0.05 μg/mL for theobromine and caffeine, respectively. The highest concentrations of caffeine and theobromine determined in tea samples were 159.1 and 255.8 mg/L, respectively.Conclusion: Theobromine and caffeine have been successfully analysed in tea, coffee and soft drinks. LC-TOF-MS is an accurate and promising instrument for the determination of the studied compounds in beverages.Keywords: Theobromine, Caffeine, Tea, Coffee, LC-TOF/MS

Metabolomic profiling identifies distinct phenotypes for ASS1 positive and negative GBM

BackgroundTumour cells have a high demand for arginine. However, a subset of glioblastomas has a defect in the arginine biosynthetic pathway due to epigenetic silencing of the rate limiting enzyme argininosuccinate synthetase (ASS1). These tumours are auxotrophic for arginine and susceptible to the arginine degrading enzyme, pegylated arginine deiminase (ADI-PEG20). Moreover, ASS1 deficient GBM have a worse prognosis compared to ASS1 positive tumours. Since altered tumour metabolism is one of the hallmarks of cancer we were interested to determine if these two subtypes exhibited different metabolic profiles that could allow for their non-invasive detection as well as unveil additional novel therapeutic opportunities.MethodsWe looked for basal metabolic differences using one and two-dimensional gas chromatography-time-of-flight mass spectrometry (1D/2D GC-TOFMS) followed by targeted analysis of 29 amino acids using liquid chromatography-time-of-flight mass spectrometry (LC-TOFMS). We also looked for differences upon arginine deprivation in a single ASS1 negative and positive cell line (SNB19 and U87 respectively). The acquired data was evaluated by chemometric based bioinformatic methods.ResultsOrthogonal partial least squares-discriminant analysis (OPLS-DA) of both the 1D and 2D GC-TOFMS data revealed significant systematic difference in metabolites between the two subgroups with ASS1 positive cells generally exhibiting an overall elevation of identified metabolites, including those involved in the arginine biosynthetic pathway. Pathway and network analysis of the metabolite profile show that ASS1 negative cells have altered arginine and citrulline metabolism as well as altered amino acid metabolism. As expected, we observed significant metabolite perturbations in ASS negative cells in response to ADI-PEG20 treatment.ConclusionsThis study has highlighted significant differences in the metabolome of ASS1 negative and positive GBM which warrants further study to determine their diagnostic and therapeutic potential for the treatment of this devastating disease.

The Effects of Glaucoma Drainage Devices on Oxygen Tension, Glycolytic Metabolites, and Metabolomics Profile of Aqueous Humor in the Rabbit.

PurposeGlaucoma drainage device (GDD) implantation can lead to corneal decompensation. We evaluated changes over time in oxygen tension and in the metabolic environment of the aqueous humor after GDD implantation in the rabbit eye.MethodsAhmed Glaucoma Valves were implanted in the left eyes of eight male New Zealand white rabbits. Right eyes were used as a control. Oxygen tension was measured immediately before surgery and at 1 and 2 months postoperation. Aqueous humor was collected from the surgical and control eyes at 1, 2, and 5 months postoperation. Aqueous humor samples collected at 1 and 5 months postoperation were selected for broad-spectrum metabolomics analysis using ultra-performance liquid chromatography-time of flight-mass spectrometry (UPLC TOF-MS). Multivariate analysis methods were used to identify metabolite profiles that separated the surgical and control eye at 1 and 5 months.ResultsThere was a significant decrease in oxygen tension in aqueous humor of the surgical eyes (9 mm Hg, 95% confidence interval [CI]: −14.7 to −3.5). Differences in the metabolic profiles between the surgical and control eye at 1 and 5 months were observed, as were differences for the surgical eye at 1 and 5 months. In addition, a metabolite profile was identified that differentiated the surgical eyes at 1 and 5 months.ConclusionChanges in the oxygen tension and metabolic intermediates occur within the aqueous humor as early as 1 month after GDD implantation.Translational RelevanceCorneal decompensation following GDD implantation could be secondary to disruption of the normal aqueous circulation, resulting in hypoxia and an altered metabolic profile. Alterations to the GDD design might minimize aqueous disruption and prevent corneal decompensation.

Lowering the Bar for Mass Spectrometry: A Comparison between Immunoassay and Rapid Time-of-Flight for Presumptive Screening of Drugs in Urine.

Immunoassay-based techniques and creatinine quantification have historically been the methods of choice for urine drug screening. Positive presumptive drug screen results are reflexed to more specific, confirmatory testing using gas or liquid chromatography coupled to mass spectrometry. False positives and false negatives with immunoassay techniques are common problems that have substantial down-stream consequences for patient care, laboratory operations, and total costs. The final workflow included rapid enzymatic hydrolysis, rapid liquid chromatographic methods, and time-of-flight mass spectrometry for detection. In total, 84 drugs and metabolites were included and reported qualitatively using 11 isotopically labeled internal standards selected to represent compound classes, retention time, and expected abundances to control for method inefficiencies and matrix suppression/enhancement. The method performance validation included 420 individual urine specimens. Of the 420 samples screened by immunoassay, 117 failed to confirm by mass spectrometry and were immunoassay false positives. None of these 117 samples screened positive on the liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS) assay. The LC-TOF-MS method failed to detect 1 sample in each of the following classes: buprenorphine, ethanol markers, and opiates owing to concentrations below the established cutoffs. Out of 579 samples, 275 (47.4%) screened positive by LC-TOF-MS for nicotine and at least 2 of its metabolites. Quantitative creatinine comparison to an existing Jaffe method yielded a slope of 0.91 and a correlation coefficient of 0.96. We investigated whether immunoassay-based drug screening and creatinine quantification could be sufficiently replaced by a rapid LC-TOF-MS screen with higher specificity and accuracy than existing methods. The LC-LC-TOF-MS method is a sensitive and more specific way to screen for drugs, providing creatinine quantification and potential novel specimen validity testing with the inclusion of nicotine metabolites.

Full Factorial Experimental Design for Carbamazepine Removal Using Electrochemical Process: a Case Study of Scheming the Pathway Degradation

Carbamazepine is an antiepileptic drug which is considered one of the persistent compounds detected in Malaysian aquatic environment. In this present study, a full factorial experimental design was applied for the analysis of effect NaCl amount, initial concentration of carbamazepine, applied voltage and treatment time on the electrochemical removal percentage of carbamazepine. However, applied voltage was found as the most significant factor with p = 0.00 lower than 95% confidence level. The investigation of effect NaCl and Na2SO4 on the pathway degradation for carbamazepine was provided using liquid chromatography-time of flight/mass spectrometry (LC-TOF/MS). The results showed that new by-products were identified such chloro-epoxy carbamazepine, and other by-products, were detected in the presence of NaCl and Na2SO4 electrolytes. High inhibition percentage of Escherichia coli (E. coli) bacteria was observed at 24.0 h incubation time for both NaCl and Na2SO4 electrolytes after 80 and 20 min of treatment, respectively.

Antioxidant activity of an anatolian herbal tea—Origanum minutiflorum: isolation and characterization of its secondary metabolites

ABSTRACTOriganum species are significant aromatic and medicinal plants used in food and pharmaceutical industries. Isolation of bioactive compounds was executed on n-butanol extract to yield the compounds responsible for the activities. Tricosan-1-ol (1), (8E,16E)-tetracosa-8,16-diene-1,24-diol (2), azepan-2-one (3), 3,4-dihydroxybenzoic acid (4), apigenin (5), eriodictyol (6), globoidnan-A (7), luteolin (8), rosmarinic acid (9), apigenin-7-O-glucuronide (10), and vicenin-2 (11) were isolated by chromatographic methods (column chromatography and semi-preparative High Performance Liquid Chromatography (HPLC) and structures were elucidated on the basis of spectroscopic techniques including 1D/2D nuclear magnetic resonance (NMR) and Liquid chromatography/Time-of-flight/Mass spectrometry (LC-TOF/MS). The isolated compounds and extracts were applied for antioxidant assays including 1,1-diphenyl-2-picrylhydrazyl (DPPH•) scavenging, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS•+) scavenging, reducing power, and cuprac techniques. 3,4-Dihydroxy benzoic acid (4), eriodictyol (6), luteolin (8), and rosmarinic acid (9) revealed the considerable antioxidant activities.

Novel contaminants identified in fish kills in the Red River watershed, 2011-2013.

Provisional molecular weights and chemical formulas were assigned to 4 significant previously unidentified contaminants present during active fish kills in the Red River region of Oklahoma. The provisional identifications of these contaminants were determined using high-resolution liquid chromatography-time-of-flight mass spectrometry (LC-TOFMS), LC-Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICRMS), and LC-ion trap mass spectrometry (LC-ITMS). Environmental water samples were extracted using a solid-phase extraction (SPE) method, and sediment samples were extracted using a modified sonication liquid extraction method. During screening of the samples, 2 major unknown chromatographic peaks were detected at m/z 624.3 and m/z 639.3. The peak at m/z 639.3 was firmly identified, through the use of an authentic standard, as a porphyrin, specifically chlorin-e6-trimethyl ester, with m/z 639.31735 (M + H)+ and molecular formula C37 H43 N4 O6 . The other major peak, at m/z 624.3 (M + H)+ , was identified as an amide-containing porphyrin. It was discovered that the amide compound was an artifact created during the SPE process by reaction of ammonium hydroxide at 1 of 3 potential reaction sites on chlorin-e6-trimethyl ester. Other unique nontargeted chemicals were also detected and the importance of their identification is discussed. Environ Toxicol Chem 2018;37:336-344. Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Non-Targeted Secondary Metabolite Profile Study for Deciphering the Cosmeceutical Potential of Red Marine Macro Alga Jania rubens—An LCMS-Based Approach

This study aims to unveil the cosmeceutical traits of Jania rubens by highlighting its mineral composition, antioxidant potential, and presence of bioactive molecules using non-targeted metabolite profiling. This study showed that among minerals, (macro), Ca (14790.33 + 1.46 mg/100 g dry weight (DW)) and in (micro) Fe (84.93 + 0.89 mg/100 g DW) was the highest. A total of 23 putative metabolites in the +ESI (Electrospray Ionization) mode of LCMS-TOF (Liquid Chromatography Mass Spectrometry-Time of Flight) were detected. Two anthocyanins—malonylshisonin and 4′′′-demalonylsalvianin (m/z 825.19; anti-aging, antioxidant, anticancer properties) were detected. Two flavonoids, viz, medicocarpin and agecorynin C, 4′-O-methylglucoliquiritigenin—a flavonoid-7-O-glycoside, and 5,6,7,8,3′,4′,5′-heptamethoxyflavone, a polymethoxygenated flavone (m/z 415.15), were detected. Maclurin 3-C-(2″,3″,6″-trigalloylglucoside) (m/z 863.15) (antioxidant, antimicrobial and anticancer traits) and theaflavonin (m/z 919.18), belonging to the class of theaflavins (whitening and anti-wrinkle agent), were obtained. Pharmacologically active metabolites like berberrubin (m/z 305.1; antitumor activity), icaceine (m/z 358.24; anticonvulsant properties), agnuside (m/z 449.15; constituent for treatment of premenstrual syndrome), γ-coniceine (m/z 108.12; formulations to treat breast cancer), eremopetasitenin B2, and eremosulphoxinolide A (m/z 447.18; therapeutic effect of allergy and asthma) were observed. 6-O-Methylarmillaridin (m/z 445.18) (antimicrobial and antifungal) and simmondsin 2-ferulate, (m/z 534.21) (insecticidal, antifungal and antifeedant) were detected. Aromatic lignans, viz, 8-Acetoxy-4′-methoxypinoresinol, sesartemin, and cubebinone (m/z 413.16), in addition to an aromatic terpene glycoside, tsangane L3 glucoside (m/z 357.23), were detected. Zizybeoside I, benzyl gentiobioside, and trichocarposide were also detected. The determination of antioxidant potential was performed through assays such as like DPPH (2,2-diphenyl-1-picrylhydrazyl), FRAP (Ferric Ion Reducing Antioxidant Power), ABTS (2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid)), and total antioxidants. Therefore, this study progresses the probability for the inclusion of J. rubens as an ingredient in modern day cosmetic formulations.

A Novel and Native Microcystin-Degrading Bacterium of Sphingopyxis sp. Isolated from Lake Taihu.

A native, highly efficient microcystin-LR (MC-LR)-degrading bacterium named a7 was isolated from Lake Taihu and identified as Sphingopyxis sp. by 16S rDNA sequence analysis. The strain a7 could totally degrade MC-LR at a rate of 3.33 mg/(L·h), as detected by high-performance liquid chromatography (HPLC). The mlrA, mlrC, and mlrD genes were detected in the strain a7 by sequence analysis. Tetrapeptide and Adda—which are the middle metabolites of MC-LR—were analyzed via liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS) during degradation. These metabolites were degraded completely, which suggested that the native Sphingopyxis sp. a7 was highly efficient in MC-LR degradation under bench conditions. Thus, strain a7 exhibited a significant potential application for bioremediation in water bodies contaminated by MC-LR produced by harmful cyanobacterial blooms.

Enzymatic Systems for Cellulose Acetate Degradation

Cellulose acetate (CA)-based materials, like cigarette filters, contribute to landscape pollution challenging municipal authorities and manufacturers. This study investigates the potential of enzymes to degrade CA and to be potentially incorporated into the respective materials, enhancing biodegradation. Deacetylation studies based on Liquid Chromatography-Mass Spectrometry-Time of Flight (LC-MS-TOF), High Performance Liquid Chromatography (HPLC), and spectrophotometric analysis showed that the tested esterases were able to deacetylate the plasticizer triacetin (glycerol triacetate) and glucose pentaacetate (cellulose acetate model compound). The most effective esterases for deacetylation belong to the enzyme family 2 (AXE55, AXE 53, GAE), they deacetylated CA with a degree of acetylation of up to 1.8. A combination of esterases and cellulases showed synergistic effects, the absolute glucose recovery for CA 1.8 was increased from 15% to 28% when an enzymatic deacetylation was performed. Lytic polysaccharide monooxygenase (LPMO), and cellobiohydrolase were able to cleave cellulose acetates with a degree of acetylation of up to 1.4, whereas chitinase showed no activity. In general, the degree of substitution, chain length, and acetyl group distribution were found to affect CA degradation. This study shows that, for a successful enzyme-based deacetylation system, a cocktail of enzymes, which will randomly cleave and generate shorter CA fragments, is the most suitable.

Fluorescent ligand fishing combination with in-situ imaging and characterizing to screen Hsp 90 inhibitors from Curcuma longa L. based on InP/ZnS quantum dots embedded mesoporous nanoparticles

Although ligand fishing has been shown to be an efficient technique for the identification of bioactive components from complex mixtures such as natural products, it cannot be applied to biomedical image processing. Herein, a specific fluorescent ligand fishing combined with in situ imaging approach is presented for the identification of heat shock protein 90 (Hsp 90) inhibitors from complex matrixes, Curcuma longa L., using N-terminus immobilized Hsp 90α functionalized InP/ZnS quantum dots embedded mesoporous nanoparticles (i.e. Hsp 90α (NT)-FQDNs) as extraction sorbents and fluorescent tracer. The fished ligands were identified by liquid chromatography time-of-flight/mass spectrometry (LC-TOF/MS) and gas chromatography-mass spectrometry (GC-MS). Moreover, in situ imaging by confocal laser scanning microscopy (CLSM) was applied for evaluating the effect of fished-ligands on bioactivity-induced apoptosis morphologically in HeLa cells. MTT assay verified the bioactivity of the ligands and molecular docking results further provided convincing information to verify the feasible binding mode between ligands and protein. Twelve ligands as potential Hsp 90 inhibitors were ultimately fished and identified from Curcuma longa L. crude extracts. The proposed approach based on Hsp 90α functionalized nanocomposites is superior in the combination of highly specific screening efficiency and concurrent visual in situ imaging, which could have great promise for the development of other plant-derived Hsp 90 inhibitors, and providing a rapid and reliable platform for discovering biologically active molecules in natural products.

Synthesis, characterization and toxicological evaluation of pH-sensitive polyelectrolyte Nanogels

Nanogels are polymeric nanoparticles that have similar characteristic to hydrogels, but have the size in nano range. The pH-sensitive nanogel have gained much interest in the field of pharmaceutical nanotechnology as they have potential to be used as nanocarriers in drug delivery system. The aim of the present study was to synthesize pH-sensitive polyelectrolyte MMA/IA nanogels using free radical polymerization containing methyl methacrylate (MMA), itaconic acid (IA), and a crosslinker ethylene glycol dimethacrylate (EGDMA). In the synthesis of nanogels four parameters i.e. ethanol/water ratios (v/v), dilution volume using ethanol/water (v/v), crosslinker EGDMA concentration, and monomers MMA/IA ratios were optimized. Their effect on particle size, PdI, zeta potential and swelling ratio were evaluated. The swelling behaviour of the nanogels was studied by measuring swelling ratio using gravimetric method. The optimized nanogels were characterized by proton nuclear magnetic resonance (1H NMR), Fourier transform infrared spectroscopy (FTIR), liquid chromatography/time-of-flight/mass spectrometry (LC-TOF-MS), X-ray powder diffraction (X-RD) and transmission electron microscopy (TEM). Polyelectrolyte characteristic was confirmed by measuring isoelectric point using aqueous electrophoresis. The in-vitro and in-vivo toxicity studies were performed by MTT assays using Caco-2 cells and Limit test using female Sprague Dawly rats, respectively. The nanogels were amorphous in nature, exhibited pH-responsive property and polyelectrolyte characteristics, which showed an isoelectric point at pH 2.78. They had an average particle size <250 nm, narrow size distribution (PdI < 0.3), and negative zeta potential. The in-vitro MTT assays indicated that the nanogels had no sign of cytotoxicity. The in vivo Limit test showed that the LD50 was greater than 2000 mg/kg body weight. The necrospy, histopathology and hematological studies also revealed no sign of toxicity. These findings suggested that the MMA/IA nanogels are pH-sensitive, non-toxic and have potential to form a polyelectrolyte complex with oppositely charged of macromolecular drugs.