HPLC Experiments Research Articles

A novel AIE-based mitochondria-targeting fluorescent probe for monitoring of the fluctuation of endogenous hypochlorous acid in ferroptosis models.

Ferroptosis is a way of cell death mainly due to the imbalance between the production and degradation of lipid reactive oxygen species, which is closely associated with various diseases. Endogenous hypochlorous acid (HOCl) mainly produced in mitochondria is regarded as an important signal molecule of ferroptosis. Therefore, monitoring the fluctuation of endogenous HOCl is beneficial to better understand and treat ferroptosis-related diseases. Inspired by the promising aggregation-induced emission (AIE) properties of tetraphenylethene (TPE), herein, we rationally constructed a novel AIE-based fluorescent probe, namely QTrPEP, for HOCl with nice mitochondria-targeting ability and high sensitivity and selectivity. Probe QTrPEP consisted of phenylborate ester and the AIE fluorophore of quinoline-conjugated triphenylethylene (QTrPE). HOCl can brighten the strong fluorescence through a specific HOCl-triggered cleavage of the phenylborate ester bond and release ofQTrPE, which has been demonstrated by MS, HPLC, and DLS experiments. In addition, combining QTrPE-doped test strips with a smartphone-based measurement demonstrated the excellent performance of the probe to sense HOCl. The obtained favorable optical properties and negligible cytotoxicity allowed the use of this probe for tracking of HOCl in three different cells. In particular, this work represents the first AIE-based mitochondria-targeting fluorescent probe for monitoring the fluctuation of HOCl in ferroptosis.

HPLC, NMR Based Characterization, Antioxidant and Anticancer Activities of Chemical Constituents from Therapeutically Active Fungal Endophytes.

Fungi generate different metabolites some of which are intrinsically bioactive and could therefore serve as templates for drug development. In the current study, six endophytic fungi namely Aspergillus flavus, Aspergillus tubigenesis, Aspergillus oryzae, Penicillium oxalicum, Aspergillus niger, and Aspergillus brasiliensis were isolated and identified from the medicinal plant, Silybum marianum. These endophytic fungi were identified through intra transcribed sequence (ITS) gene sequencing. The bioactive potentials of fungal extracts were investigated using several bioassays such as antibacterial activity by well-diffusion, MIC, MBC, anti-biofilm, antioxidant, and haemolysis. The Pseudomonas aeruginosa PAO1 was used to determine the antibiofilm activity. The ethyl acetate extract of Aspergillus flavus showed strong to moderate efficacy against Staphylococcus aureus, Escherichia coli, P. aeruginosa, and Bacillus spizizenii. Aspergillus flavus and Aspergillus brasiliensis exhibited significant antibiofilm activity with IC50 at 4.02 and 3.63 mg/ml, while A. flavus exhibited maximum antioxidant activity of 50.8%. Based on HPLC, LC-MS, and NMR experiments kojic acid (1) and carbamic acid (methylene-4, 1-phenylene) bis-dimethyl ester (2) were identified from A. flavus. Kojic acid exhibited DPPH free radical scavenging activity with an IC50 value of 99.3 μg/ml and moderate activity against ovarian teratocarcinoma (CH1), colon carcinoma (SW480), and non-small cell lung cancer (A549) cell lines. These findings suggest that endophytic fungi are able to produce promising bioactive compounds which deserve further investigation.

Activation of a silent lactose utilization pathway in an evolved Listeria monocytogenes F2365 outbreak isolate

Listeria monocytogenes, a widespread food-borne pathogen, utilizes diverse growth substrates including mono- and di-saccharides via PEP-phosphotransferase (PTS) systems. We evaluated a collection of L. monocytogenes isolates of different origins for their ability to utilize lactose, a disaccharide composed of galactose and glucose and the main carbon source in milk and dairy products. Notably, the dairy-associated outbreak strain F2365 could not utilize lactose efficiently, conceivably due to a frameshift mutation (lacR887del) resulting in a truncated LacR. Transcriptional activator LacR is involved in the expression of two PTS systems, encoded by the lpo operon lmo1718-1720 in combination with lmo2708 and the lmo2683-2685 operon, and linked to lactose and/or cellobiose metabolism in L. monocytogenes. Via experimental evolution of the ancestral strain F2365, an evolved isolate F2365 EV was obtained which showed enhanced growth and metabolism of lactose. Using the lactose-positive model strain L. monocytogenes EGDe as a control, HPLC experiments showed that EGDe and F2365 EV could consume lactose and utilize the glucose moiety, while the galactose moiety was exported from the cells. Genome sequencing of F2365 EV found the original lacR887del mutation was still present but an additional point mutation lmo2766C415T had occurred, resulting in an amino acid substitution in the putative regulator Lmo2766. The lmo2766 gene is located next to operon lmo2761-2765 with putative PTS genes in the genome. Notably, comparative RNAseq analysis confirmed that the lmo2761-2765 operon was strongly upregulated in F2365 EV in the presence of lactose but not in EGDe and F2365. Conversely, the LacR-regulated lpo operon, lmo2708, and lmo2683-2685 operon were only upregulated in EGDe. Additional growth and HPLC experiments, using mutants constructed in lactose-positive L. monocytogenes EGDe, showed reduced growth of the EGDe lacR887del mutant with no utilization of lactose, while the double mutant EGDe lacR887dellmo2766C415T showed enhanced growth and lactose utilization. Hence, these results demonstrate that an amino acid substitution in the Lmo2766 regulator activates a previously silent lactose utilization pathway encoded by PTS operon lmo2761-2765, facilitating the growth and metabolism of L. monocytogenes with lactose as a substrate. This finding enhances our understanding of the metabolic capabilities and adaptability of L. monocytogenes, offering a broader view of the lactose utilization capacity of this pathogen.

Two Fluorescent Probes for Recognition of Acetylcholinesterase: Design, Synthesis, and Comparative Evaluation.

In this study, two "on-off" probes (BF2-cur-Ben and BF2-cur-But) recognizing acetylcholinesterase (AChE) were designed and synthesized. The obtained probes can achieve recognition of AChE with good selectivity and pH-independence with a linear range of 0.5~7 U/mL and 0.5~25 U/mL respectively. BF2-cur-Ben has a lower limit of detection (LOD) (0.031 U/mL), higher enzyme affinity (Km = 16 ± 1.6 μM), and higher inhibitor sensitivity. A responsive mechanism of the probes for AChE was proposed based on HPLC and mass spectra (MS) experiments, as well as calculations. In molecular simulation, BF2-cur-Ben forms more hydrogen bonds (seven, while BF2-cur-But has only four) and thus has a more stable enzyme affinity, which is mirrored by the results of the comparison of Km values. These two probes could enable recognition of intracellular AChE and probe BF2-cur-Ben has superior cell membrane penetration due to its higher log p value. These probes can monitor the overexpression of AChE during apoptosis of lung cancer cells. The ability of BF2-cur-Ben to monitor AChE in vivo was confirmed by a zebrafish experiment.

Statistical Methods in the Study of Protein Binding and Its Relationship to Drug Bioavailability in Breast Milk.

Protein binding (PB) is indicated as the factor most severely limiting distribution in the organism, reducing the bioavailability of the drug, but also minimizing the penetration of xenobiotics into the fetus or the body of a breastfed child. Therefore, PB is an important aspect to be analyzed and monitored in the design of new drug substances. In this paper, several statistical analyses have been introduced to find the relationship between protein binding and the amount of drug in breast milk and to select molecular descriptors responsible for both pharmacokinetic phenomena. Along with descriptors related to the physicochemical properties of drugs, chromatographic descriptors from TLC and HPLC experiments were also used. Both methods used modification of the stationary phase, using bovine serum albumin (BSA) in TLC and human serum albumin (HSA) in HPLC. The use of the chromatographic data in the protein binding study was found to be positive —the most effective application of normal-phase TLC and HPLCHSA data was found. Statistical analyses also confirmed the prognostic value of affinity chromatography data and protein binding itself as the most important parameters in predicting drug excretion into breast milk.

A Metabolic Network Mediating the Cycling of Succinate, a Product of ROS Detoxification into α-Ketoglutarate, an Antioxidant.

Sulfur is an essential element for life. However, the soil microbe Pseudomonas (P.) fluorescens can survive in a low sulfur environment. When cultured in a sulfur-deficient medium, the bacterium reprograms its metabolic pathways to produce α-ketoglutarate (KG) and regenerate this keto-acid from succinate, a by-product of ROS detoxification. Succinate semialdehyde dehydrogenase (SSADH) and KG decarboxylase (KGDC) work in partnership to synthesize KG. This process is further aided by the increased activity of the enzymes glutamate decarboxylase (GDC) and γ-amino-butyrate transaminase (GABAT). The pool of succinate semialdehyde (SSA) generated is further channeled towards the formation of the antioxidant. Spectrophotometric analyses, HPLC experiments and electrophoretic studies with intact cells and cell-free extracts (CFE) pointed to the metabolites (succinate, SSA, GABA) and enzymes (SSADH, GDC, KGDC) contributing to this KG-forming metabolic machinery. Real-time polymerase chain reaction (RT-qPCR) revealed significant increase in transcripts of such enzymes as SSADH, GDC and KGDC. The findings of this study highlight a novel pathway involving keto-acids in ROS scavenging. The cycling of succinate into KG provides an efficient means of combatting an oxidative environment. Considering the central role of KG in biological processes, this metabolic network may be operative in other living systems.

Photo-electrochemical and ozonation process to degrade ciprofloxacin in synthetic municipal wastewater, using C, N-codoped TiO2 with high visible-light absorption

Carbon, nitrogen-codoped TiO2 nanoroughness surfaces (CN-TiNRS) are successfully synthesized through electrochemical anodization at 50, 70 and 90 V (50 V-CN-TiNRS, 70 V-CN-TiNRS, and 90 V-CN-TiNRS, respectively), with the aim of shifting their absorption capacities to the visible-light region. The analysis by X-ray diffraction (XRD) and scanning electron microscopy (SEM) indicate that a TiO2 film with a major proportion of crystalline anatase and nanoroughness morphology is formed on the entire surface of all photoelectrodes. Carbon and nitrogen elements are detected by X-ray photoelectron spectroscopy (XPS) analysis for all samples, obtaining similar weight percentages. The best photoelectrochemical performance is reached for the 70 V-CN-TiNRS electrode subjected to Xe lamp radiation. Accordingly, this photocatalyst is chosen to perform all the ciprofloxacin (CIP) degradation tests in distilled water, involving the following methods: photocatalysis (PC), photoelectrocatalysis (PEC), Ozone (O3), photocatalytic ozonation (PCO) and photoelectrocatalytic ozonation (PECO). Degradation tests are additionally conducted in synthetic municipal wastewater (SWW) using PECO, to analyze its oxidation capacity in a real scenario (i.e. organic composition matrix). All techniques revealed that the highest CIP removal occurs with PECO, decreasing TOC by almost 86.79% (95% degradation by HPLC) after 90 min of treatment, thus, exploiting the intrinsic advantages of single PEC and O3. The radical trapping technique reveals that superoxide radicals (O2-•), holes (h+) and hydroxyl radicals (OH•) are responsible of the CIP abatement in the PECO. Reaction mechanisms are proposed for the production of these oxidants, while the CIP degradation pathway is rationalized using HPLC experiments. A toxicity test of the treated water is carried out after the process with PECO by means of germination with sativa lettuce seeds.

Perovskite oxides as electrocatalyst for glycerol oxidation

• LaNiO 3 and LaCoO 3 present catalytic activity towards glycerol electrooxidation. • At relatively high potentials, LaNiO 3 and LaCoO 3 are more active than metal oxide. • Substitution of the transition metal can tune the perovskite activity for GEOR. • LaCoO 3 presents high resistance to surface poisoning. • The products of glycerol electrooxidation are mainly glycolate and formate. Glycerol is a co-product from Biodiesel production with high abundance and low market price. Its transformation into valuable products or its use as an alternative source of energy has driven the search for selective catalysts. Herein we show first findings of perovskites as electrocatalysts for glycerol electrooxidation reaction (GEOR). Alternatively to Pt and Pd based catalyst, perovskite oxides (LaNiO 3 and LaCoO 3 ) demonstrated high catalytic performance with faradaic currents of ca. 18 mA mg −1 at potentials higher than 1.6 V vs. RHE. LaCoO 3 exhibited resistance to poisoning during GEOR. Based on in situ FTIR and online HPLC experiments, GEOR on these perovskites yields formate and glycolate as final products, with no evidence of CO 2 formation. Our results indicate that perovskites can be considered as alternative electrocatalysts for the selective oxidation of glycerol, opening the door for a new class of catalysts for polyols oxidation with efficiency in terms of current densities and poisoning resistance.

Triphenylethylene analogues: Design, synthesis and evaluation of antitumor activity and topoisomerase inhibitors

To structurally relate anticancer drug tamoxifen used in the treatment of breast cancer, a sequence of compounds is designed and synthesized as potential drug candidates. McMurry coupling reaction is used as the key synthetic step in the preparation of these analogues and the ratios of E/Z-isomers are determined on the basis of NMR and HPLC experiments. The new compounds are found to be cytotoxic in the micromolar range with 60 human tumor cell lines at one dose and five dose concentration levels. Detailed studies on the most active compounds 11-13 show these compounds are capable to inhibit the growth of cancer cells. Finally, with the aim to correlate the antiproliferative activity with an intracellular target(s), the effect on relaxation activity of DNA topoisomerase-II is assayed. The relevance of interaction of most active compounds with topoisomerase-II is demonstrated which is also supported by docking studies.

An organic monolithic capillary column functionalized with human serum albumin and its application for the nano – chromatography study of its binding with universal cancer peptides and its impact on immunogenicity

Sixty picomoles of human serum albumin (HSA) was immobilized on a glycidylmethacrylate (GMA) capillary column previously developed by our group to study its binding with two universal cancer peptides (UCPs). Zonal HPLC experiments demonstrated that UCP2 and UCP4 have 1:1 interaction at the well-known site I of HSA with similar affinities in the micromolar range. At pH 7.4 UCP2 and UCP4 bound to HSA site I with negative ΔH values increasing with increasing temperature leading to positive heat capacity changes (UCP2: ΔC p=1.06 kJ/mol/K; UCP4: ΔC p=0.53 kJ/mol/K). The sign and magnitude of these thermodynamic data were explained by the burial of the polar groups of the peptide inside the hydrophobic binding pocket of HSA leading to coil helix transition upon the binding. The strong linear coefficient (r 2 = 0.995) for the plot comparing the immunogenicity index (II) between a series of four UCPs against the enthalpy gain confirmed a high degree of similarity for the mechanism of immunogenicity of these short peptides. The positive slope (+2.59) indicated that at physiological pH (7.4), the helix gain upon UCPs/HSA binding promoted the immunogenicity of the peptide vaccine.

Genome-scale metabolic models of Microbacterium species isolated from a high altitude desert environment

The Atacama Desert is the most arid desert on Earth, focus of important research activities related to microbial biodiversity studies. In this context, metabolic characterization of arid soil bacteria is crucial to understand their survival strategies under extreme environmental stress. We investigated whether strain-specific features of two Microbacterium species were involved in the metabolic ability to tolerate/adapt to local variations within an extreme desert environment. Using an integrative systems biology approach we have carried out construction and comparison of genome-scale metabolic models (GEMs) of two Microbacterium sp., CGR1 and CGR2, previously isolated from physicochemically contrasting soil sites in the Atacama Desert. Despite CGR1 and CGR2 belong to different phylogenetic clades, metabolic pathways and attributes are highly conserved in both strains. However, comparison of the GEMs showed significant differences in the connectivity of specific metabolites related to pH tolerance and CO2 production. The latter is most likely required to handle acidic stress through decarboxylation reactions. We observed greater GEM connectivity within Microbacterium sp. CGR1 compared to CGR2, which is correlated with the capacity of CGR1 to tolerate a wider pH tolerance range. Both metabolic models predict the synthesis of pigment metabolites (β-carotene), observation validated by HPLC experiments. Our study provides a valuable resource to further investigate global metabolic adaptations of bacterial species to grow in soils with different abiotic factors within an extreme environment.

Quantitative structure retention relationship modeling as potential tool in chromatographic determination of stability constants and thermodynamic parameters of β-cyclodextrin complexation process

When cyclodextrins (CDs) are used in chromatography analytes’ retention time is decreased with an increase in concentration of CD in the mobile phase. Thus complex stability constants can be determined from the change in retention time of the ligand molecule upon complexation. Since the preceding approach implies extensive and time-consuming HPLC experiments, the goal of this research was to investigate the possibility of using in silico prediction tools instead. Quantitative structure–retention relationship (QSRR) model previously developed to explain the retention behavior of risperidone, olanzapine and their structurally related impurities in β-CD modified HPLC system was applied to predict retention factor under different chromatographic conditions within the examined domains. Predicted retention factors were further used for calculation of stability constants and important thermodynamic parameters, namely standard Gibbs free energy, standard molar enthalpy and entropy, contributing to inclusion phenomenon. Unexpected prolonged retention with an increase in β-CD concentration was observed, in contrast to the employed chromatographic theory used for the calculation of the stability constants. Consequently, it led to failure in stability constants and thermodynamic parameters calculation for almost all analytes when acetonitrile content was 20% (v/v) across the investigated pH range. Moreover, ionization of investigated analytes and free stationary phase silanol groups are pH dependent, leading to minimization of secondary interactions if free silanol groups are non-ionized at pH lower than 3. In order to prove accuracy of predicted retention factors, HPLC verification experiments were performed and good agreement between predicted and experimental values was obtained, confirming the applicability of proposed in-silico tool. However, the obtained results opened some novel questions and revealed that chromatographic method is not overall applicable in calculation of stability constants and thermodynamic parameters indicating the complexity of β-CD modified systems.

Catecholaminergic hippocampal activation is necessary for object recognition memory persistence induced by one-single physical exercise session

Previously we demonstrated that a single physical exercise session promotes the persistence of object recognition (OR) memory and this effect involves the activation of the noradrenergic system. Here, using adult male Wistar rats (3 months old) we confirm that an aerobic single physical exercise session (30 min of treadmill running at an intensity of 60–70 % of indirect VO2 max.) after OR learning promotes memory persistence. We also demonstrate that this effect involves the dopaminergic system, since it is blocked when a D1-family receptor antagonist (SCH-23390, 1μg/μl) is infused into the hippocampus after the physical exercise session. Additionally, through HPLC experiments we demonstrate that a physical exercise session increases the hippocampal dopamine levels. Taken together, our results demonstrate that acute post-learning physical exercise is able to promote the persistence of OR memory, inducing the release of dopamine in hippocampus, which is necessary for the modulation of memory persistence. This work brings new evidences on the benefit of a single physical exercise session to memory, as well as suggests that catecholaminergic mechanisms are behind this effect.

Electrochemical degradation of triclosan in aqueous solution. A study of the performance of an electro-Fenton reactor

The electro-Fenton degradation of Triclosan in aqueous solution was studied using a cylindrical reactor in which polarized carbon cloth electrodes and a cation exchange resin were employed. Using a factorial design of experiments approach, the effect of four variables (considering two levels for each one), was measured on four response parameters that reflect the electrooxidation efficiency of the electrochemical reactor. The results revealed that in all cases triclosan degradation was very efficient (above 95%) and that while there is a reasonable effect of all variables and their interactions, the one with the strongest influence on the process is the nature and magnitude of the ionic strength of the electrolytic solution. In this way, while the presence of a buffer species in this solution can keep the pH in a value that affects the generation of •OH radicals from the Fenton mixture, a high ionic strength solution can promote the elimination of Fe ionic species from the reactor by decreasing resin Fe retention due to competition effects of other ions for the binding sites of the substrate. HPLC experiments of the effluent solutions, also revealed that the degradation by-products of triclosan were dependent on the nature and ionic strength of the electrolytic solution in the electro-Fenton process under study. Finally, comparison of the different operation modes, also suggested that electro-adsorption of Fe cationic species in the negatively polarized cathode surface, is the main factor that controls Fe ion retention within the reactor.

Effect of ligand and bridge substitution on chiral recognition of 1-phenylethylammonium cation by an anionic binuclear Ni(II) complex

Chiral recognition is an important phenomenon. Identifying factors those influences recognition and resolution have significance in multiple areas. Earlier, we had reported recognition and resolution (30% ee) of 1-phenylethylammonium cation using an anionic binuclear Ni(II) complex of a l-histidine derived ligand. We have now synthesized its l-methionine analogue and also substituted the bridging acetate to benzoate. Substitution of amino acid and subsequent reaction with racemic 1-phenylethylamine resulted in isolation of crystals of both possible diastereoisomers (1 and 2) as a conglomerate. This is different from l-histidine analogue which showed pseudo racemate formation. HPLC experiments on the bulk of the crystals showed 30% ee. On the other hand, substitution of both the amino acid and bridging ligand resulted in isolation of crystals of single diastereoisomer (3). HPLC experiment on the bulk of the crystals showed an improved 58% ee. Thus, changes in amino acid changed the mechanism of crystal formation without improving the enantiomeric excess (ee) but the changes in bridge almost doubled the % ee. To understand the reasons, the complexes 1–3 were structurally characterized and compared with previously reported l-histidine analogue. Comparison showed, the absence of strong imidazole-carboxylate H-bonding in the present system narrowed the solubility difference between diastereomers, which may have resulted in the formation of conglomerate. On the other hand, changing the bridging acetate to benzoate increase the number of inter molecular non-covalent interactions which may have influenced the improved chiral enhancement.

Persistent increase in ecto‑5′‑nucleotidase activity from encephala of adult zebrafish exposed to ethanol during early development

Prenatal alcohol exposure causes alterations to the brain and can lead to numerous cognitive and behavioral outcomes. Long-lasting effects of early ethanol exposure have been registered in glutamatergic and dopaminergic systems. The purinergic system has been registered as an additional target of ethanol exposure. The objective of this research was to evaluate if the ecto‑5′‑nucleotidase and adenosine deaminase activities and gene expression of adult zebrafish exposed to 1% ethanol during early development could be part of the long-lasting targets of ethanol. Zebrafish embryos were exposed to 1% ethanol in two distinct developmental phases: gastrula/segmentation (5–24 h post-fertilization) or pharyngula (24–48 h post-fertilization). At the end of three months, after checking for morphological outcomes, the evaluation of enzymatic activity and gene expression was performed. Exposure to ethanol did not promote gross morphological defects; however, a significant decrease in the body length was observed (17% in the gastrula and 22% in the pharyngula stage, p < 0.0001). Ethanol exposure during the gastrula/segmentation stage promoted an increase in ecto‑5′‑nucleotidase activity (39.5%) when compared to the control/saline group (p < 0.0001). The ecto‑5′‑nucleotidase gene expression and the deamination of adenosine exerted by ecto and cytosolic adenosine deaminase were not affected by exposure to ethanol in both developmental stages. HPLC experiments did not identify differences in adenosine concentration on the whole encephala of adult animals exposed to ethanol during the gastrula stage or on control animals (p > 0.05). Although the mechanism underlying these findings requires further investigation, these results indicate that ethanol exposure during restricted periods of brain development can have long-term consequences on ecto‑5′‑nucleotidase activity, which could have an impact on subtle sequels of ethanol early exposure.

Stereochemical Stability and Absolute Configuration of Atropisomeric Alkylthioporphyrazines by Dynamic NMR and HPLC Studies and Computational Analysis of HPLC‐ECD Recorded Spectra

The conformational and stereodynamic properties of a pyrene‐substituted ethylthioporphyrazine (PzPy), promising as a dye for organic optoelectronic applications and light‐harvesting systems, have been investigated by DFT computations and variable‐temperature NMR and HPLC. In PzPy, rotation around the pyrene–macrocycle bond is hindered and the molecule displays atropisomerism, existing as a pair of enantiomers, thus representing the first chiral alkyl‐porphyrazine to be reported in the literature. The rotational barrier (ΔG‡ ≈ 22.0 kcal mol–1), measured by variable‐temperature HPLC experiments, allows chromatographic separation of the enantiomers on a chiral stationary phase below 0 °C, whereas racemization spontaneously occurs at room temperature. The absolute configurations of the eluted enantiomers were also determined by computational analysis of their electronic circular dichroism spectra. The influence of alkyl chain length and metal complexation on the molecular stereodynamic properties have also been investigated by studying its novel octylthio derivative (PzPy‐octyl) and PdII complex (PdPzPy), respectively. The latter also is the first example of a PdII–alkylthioporphyrazine complex. Given that PzPy has been used in the preparation of hybrid materials with nanocarbons for optoelectronic applications, the presence of an axial chirality element may play a key role in its interaction with semiconducting chiral nanotubes.

Removal of antibiotics from water and waste milk by ozonation: kinetics, byproducts, and antimicrobial activity

The use of antibiotics in the dairy farming for curing and growth promotion results in the production of massive quantities of non-recyclable wastewater by the conventional purification techniques. Additionally, waste milk is produced during the drug withholding periods, which is not suitable for human or animal consumption and cause huge economic loss as well as present serious environmental waste. This study was designed to investigate the decomposition of various antibiotic compounds in un-buffered aqueous solutions and milk samples by ozonation process. Commonly administered broad-spectrum antibiotics such as amoxicillin, doxycycline, ciprofloxacin, and sulphadiazine were selected as model examples in the current investigation. Gradual exposure of these antibiotics to increasing ozone gas concentration induced increasing removal percentages of the antibiotics in spiked water and milk samples. The removal reached 95% across all the tested treated antibiotics with ozone dose as low as 75 mg L−1. It was noted that the removal of antibiotics in milk samples is more efficient with faster rate constants. This was attributed to the self-buffering characteristic of milk that maintains the neutral pH, keeping the amine groups un-protonated and more reactive towards the electrophilic attack by the molecular ozone. 1H NMR as well as HPLC experiments support the near complete removal of antibiotics and indicated the break down to simpler and more soluble fragments of acidic nature. Bacterial growth experiments, conducted with E. coli, and milk ageing experiments provided clear evidences that the resulting decomposition byproducts lack both toxicity effect and antimicrobial activity. This study provides a viable route to remove hazardous materials, which contribute to a growing issue of antibiotic resistance of pathogenic bacteria.

Investigating the Kinetics, Mechanism, and Reaction Pathway of a Biodesulfurizing Enzyme from Rhodococcus erythropolis, Dibenzothiophene Monooxygenase

Fossil fuel combustion is a major source of sulfur dioxide, dangerous to respiratory health and the environment. Sulfur removal from fossil fuels eases these effects, however, the current method, hydrodesulfurization, requires costly high temperatures and pressures. The bacterial species Rhodococcus erythropolis uses a four‐step enzymatic process, known as biodesulfurization, to remove sulfur in fuel compounds. The first enzyme in the pathway, dibenzothiophene monooxygenase (DszC), uses reduced flavin and molecular oxygen to generate a flavin intermediate that oxygenates its substrate, dibenzothiophene, to dibenzothiophene sulfoxide, then dibenzothiophene sulfone. Our objectives are to understand DszC's enzymatic mechanism and steps of the catalytic pathway to engineer DszC to accept various organosulfur compounds and increase catalytic efficiency. Towards our goals, we have purified active DszC; determined steady‐state kinetic parameters of DszC as a function of dibenzothiophene, dibenzothiophene sulfoxide, and flavin substrates; performed steady‐state experiments to describe DszC's enzymatic mechanism; and used transient‐state kinetics to ascertain the effects of dibenzothiophene and age times on flavin binding. Using affinity chromatography, we purified DszC at 0.5 gram/liter of culture in its native tetrameric state. Reverse‐phase HPLC experiments showed DszC had faster product turnover using dibenzothiophene sulfoxide as substrate versus dibenzothiophene (11.8 × 10−3 s−1 vs. 4.84 × 10−3 s−1). HPLC assays also demonstrated that having equimolar flavin and DszC resulted in a faster turnover rate than using excess flavin (4.84 × 10−3 s−1 vs. 1.49 × 10−3 s−1). Varying flavin and dibenzothiophene concentrations under steady‐state conditions indicated DszC likely forms a ternary complex with the flavin intermediate and dibenzothiophene to facilitate oxygenation. Using stopped‐flow spectrophotometry, we observed fast flavin binding (&lt;1 second) and that having equimolar dibenzothiophene and DszC increased intermediate formation rates. Dibenzothiophene pre‐incubation with DszC, however, decreased intermediate formation rates, suggesting reduced flavin is the initial substrate to bind to DszC. These results provided us kinetic and mechanistic insights towards understanding the full catalytic pathway of DszC to support our goals of engineering a robust and efficient DszC enzyme for industrial applications.Support or Funding InformationNational Institute of Health (SCORE 5SC2AI109500)Research Corporation CCSA 22672MARC/RISE Program (NIH Grant Number 2R25GM063787‐10)CSUPERB New Investigator GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Development of a promising drug delivery for formononetin: Cyclodextrin-modified single-walled carbon nanotubes

The design of hydroxypropyl-β-cyclodextrin (HP-β-CD) modified carboxylated single-walled carbon nanotubes (CD-SWCNTs) composition was to improve the biocompatibility and reduce the toxicity of carbon nanotubes as the delivery of anticancer drug formononetin (FMN). According to the result of fourier-transform infrared spectrometry (FTIR), HP-β-CD was grafted to carboxylated single-walled carbon nanotubes (SWCNTs-COOH) successfully. The entrapment efficiency and loading capacity of the CD-SWCNTs loading with FMN (CD-SWCNTs-FMN) were evaluated by HPLC experiments, which could reach to (88.66 ± 3.13) % and (8.43 ± 1.11) % respectively. The samples were characterized by x-ray diffractometry (XRD), differential scanning calorimetry (DSC), laser particle size analysis and scanning electron microscopy (SEM). Moreover, the research on drug release kinetics demonstrated a slow and sustained release. The in vitro cytotoxicity assay revealed that the antitumor activity of CD-SWCNTs-FMN is stronger than that of the free FMN. In conclusion, CD-SWCNTs synthesized in this study were prospective sustained-release and drug-targeting system for antitumor drugs.