Antibacterial cotton fabrics with in situ generated silver and copper bimetallic nanoparticles using red sanders powder extract as reducing agent

Using aqueous extraction of red sanders powder as a reducing agent, silver and copper bimetallic nanoparticles were in situ generated in cotton fabrics. Silver and copper nanoparticles were also generated separately for comparison. The resulted nanocomposite cotton fabrics (NCFs) were characterized by Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), X-ray diffraction (XRD), thermogravimetric analysis (TGA), and antibacterial tests. SEM analysis indicated the generation of more number of nanoparticles when bimetallic source solutions were used. Further, the size range of the generated bimetallic nanoparticles was found to be lower than when individual metal nanoparticles were generated in NCFs. XRD analysis confirmed the in situ generation of silver and copper nanoparticles when equimolar bimetallic salt source solutions were utilized. The NCFs with bimetallic nanoparticles exhibited higher antibacterial activity against both Gram-negative and Gram-positive bacteria and hence can be considered for applications as antibacterial bed and dressing materials.     

Investigation of Sequence Clipping and Structural Heterogeneity of an HIV Broadly Neutralizing Antibody by a Comprehensive LC-MS Analysis

CAP256 is one of the highly potent, broadly neutralizing monoclonal antibodies (bNAb) designed for HIV-1 therapy. During the process development of one of the constructs, an unexpected product-related impurity was observed via microfluidics gel electrophoresis. A panel of complementary LC-MS analyses was applied for the comprehensive characterization of CAP256 which included the analysis of the intact and reduced protein, the middle-up approach, and a set of complementary peptide mapping techniques and verification of the disulfide bonds. The designed workflow allowed to identify a clip within a protruding acidic loop in the CDR-H3 region of the heavy chain, which can lead to the decrease of bNAb potency. This characterization explained the origin of the additional species reflected by the reducing gel profile. An intra-loop disulfide bond linking the two fragments was identified, which explained why the non-reducing capillary electrophoresis (CE) profile was not affected. The extensive characterization of CAP256 post-translational modifications was performed to investigate a possible cause of CE profile complexity and to illustrate other structural details related to this molecule’s biological function. Two sites of the engineered Tyr sulfation were verified in the antigen-binding loop, and pyroglutamate formation was used as a tool for monitoring the extent of antibody clipping. Overall, the comprehensive LC-MS study was crucial to (1) identify the impurity as sequence clipping, (2) pinpoint the clipping location and justify its susceptibility relative to the molecular structure, (3) lead to an upstream process optimization to mitigate product quality risk, and (4) ultimately re-engineer the sequence to be clip-resistant.

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Quantitation of methylated hemoglobin adducts in a signature peptide from rat blood by liquid chromatography/negative electrospray ionization tandem mass spectrometry

Hemoglobin adducts are often used as biomarkers for exposure to reactive chemicals in toxicology studies. Therefore, fast, sensitive, accurate, and reproducible methods for quantifying these protein adducts are key to evaluate test material dosimetry. A methodology has been developed for the quantitation of methylated hemoglobin adducts isolated from rats exposed to the model alkylating agent: methyl methane sulfonate (MMS). After 4 days of MMS exposure by oral gavage, hemoglobin was isolated from rat blood and digested with trypsin. The tryptic digestion solution was used for the adducted hemoglobin signature peptide quantitation via liquid chromatography/negative tandem mass spectrometry (LC/ESI-MS/MS). The limit of quantitation (LOQ) for the methylated hemoglobin beta chain N-terminal signature peptide (MeVHLTDAEK) was 1.95 ng/mL (5.9 pmol/mg globin). The calibration curves were linear over a concentration range of 1.95 to 625 ng/mL, with a correlation coefficient R2 >0.998, accuracy of 85.8 to 119.3%, and precision of 0.9 to 19.4%.     

Quantitation of lower levels of the DNA adduct of thymidylyl(3'-5')thymidine methyl phosphotriester by liquid chromatography/negative atmospheric pressure chemical ionization tandem mass spectrometry

A sensitive method has been developed for the direct quantitation of the methyl phosphotriester DNA adduct of thymidyl(3'-5')thymidine (dTp(Me)dT) from enzymatic DNA hydrolysates prepared from cultured cells treated with low levels of N-methyl-N-nitrosourea (MNU) and methyl methane sulfonate (MMS), by rapid and selective liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry (LC/APCI-MS/MS). The lower limit of quantitation was 0.1 ng/mL (6.4 adducts per 10(8) nucleotides). Linearity of the calibration curve was greater than 0.999 from 0.1 to 50 ng/mL. Intra-day precision for three levels of quality control samples ranged from 4.27 to 15.62%. Interday precision ranged from 2.46 to 11.95%. Using this method, the levels of dTp(Me)dT in DNA enzymatic hydrolysates obtained from a series of incubations of mouse lymphoma cells with low doses of MNU (50 microM) were quantified.     

Quantitation of DNA adduct of thymidylyl(3'-5')thymidine methyl phosphotriester by liquid chromatography/negative electrospray tandem mass spectrometry

A rapid and selective method based on liquid chromatography/electrospray tandem mass spectrometry (LC/ESI-MS/MS) has been developed for the direct quantitation of a methyl phosphotriester DNA adduct, thymidyl (3'-5') thymidine [dTp(Me)dT] from enzymatic hydrolysates of DNA (either in vitro DNA or in cell culture) treated with MNU (N-methyl-N-nitrosourea) or MMS (methyl methane sulfonate). The lower limit of quantitation was 2 ng/mL. Linearity of the calibration curve was greater than 0.999 from 2 to 1000 ng/mL. Intraday precision for four levels of quality controls ranged from 2.8 to 20.1%, and interday precision ranged from 2.9 to 5.6%. This method was used to quantify the levels of dTp(Me)dT in enzymatic hydrolysates of DNA obtained from a series of incubations of salmon testis DNA or mouse lymphoma cells with either MNU or MMS.     

An efficient approach for the synthesis of novel methyl sulfones in acetic acid medium and evaluation of antimicrobial activity

A series of nine methyl sulphones ( 3a –3 i ) starting from the aldehydes ( 1a–1i ) were synthesized in two consecutive steps. In the first step, preparation of allyl alcohols ( 2a–2i ) from their corresponding aldehydes by the reaction of sodium borohydride in methanol at room temperature is reported. Finally, methyl sulphones are synthesized by condensing sodium methyl sulfinates with allyl alcohols in the presence of BF ₃ .Et ₂ O in acetic acid medium at room temperature for about 2–3 h. The reaction conditions are simple, yields are high (85%–95%), and the products were obtained with good purity. All the synthesized compounds were characterized by their ¹ H, ¹³ C NMR, and mass spectral analysis. All the title compounds were screened for antimicrobial activity. Among the compounds tested, the compound 3f has inhibited both Gram positive and Gram negative bacteria effectively and compound 3i has shown potent antifungal activity. These promising components may help to develop more potent drugs in the near future for the treatment of bacterial and fungal infections.     

Betulinic acid binding to human serum albumin: a study of protein conformation and binding affinity

Betulinic acid (BA) has anti cancer and anti-HIV activity and has been proved to be therapeutically effective against cancerous and HIV-infected cells. Human serum albumin (HSA) is the predominant protein in the blood. Most drugs that bind to HSA will be transported to other parts of the body. Using micro TOF-Q mass spectrometry, we have shown, for the first time that BA isolated from a plant (Tephrosia calophylla) binds to HSA. The binding constant of BA to HSA was calculated from fluorescence data and found to be K(BA)=1.685+/-0.01 x 10(6) M(-1), indicating a strong binding affinity. The secondary structure of the HSA-BA complex was determined by circular dichroism. The results indicate that the HSA in this complex is partially unfolded. Further, binding of BA at nanomolar concentrations of BA to free HSA was detected using micro TOF-Q mass spectrometry. The study revealed a mass increase from 65199 Da (free HSA) to 65643 Da (HSA+drug), where the additional mass of 444 Da was due to bound BA. Based on the results of this study, it is suggested that micro TOF-Q mass spectrometry is useful technique for drug binding studies.     

Differential adduction of proteins vs. deoxynucleosides by methyl methanesulfonate and 1-methyl-1-nitrosourea in vitro

The reactions of two model mutagenic and carcinogenic alkylating agents, N-methyl-N-nitrosourea (MNU) and methyl methanesulfonate (MMS), with proteins and deoxynucleosides in vitro, were investigated. The protein work used an approach involving trypsin digestion and high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS). This technique permitted identification of the specific location of protein adduction by both MNU and MMS with commercial apomyoglobin and human hemoglobin, under physiological conditions. MNU treatment resulted in predominantly carbamoylation adducts on the proteins, but in contrast only methylated protein adducts were found following treatment with MMS. Further analyses, using TurboSequest, and the Scoring Algorithm for Spectral Analysis (SALSA), revealed that MNU carbamoylation was specific for modification of either the N-terminal valine or the free amino group in lysine residues of apomyglobin and human hemoglobin. However, MMS methylation modified the N-terminal valine and histidine residues of the proteins. Despite their clear differences in protein modifications, MNU and MMS formed qualitatively the same methylated deoxynucleoside adduct profiles with all four deoxynucleosides in vitro under physiological conditions. In light of their different biological potencies, where MMS is considered a 'super clastogen' while MNU is a 'super mutagen', these differences in reaction products with proteins vs. deoxynucleosides may indicate that these two model alkylating agents work via different mechanisms to produce their mutagenic and carcinogenic effects.     

Quantitation of glutathione by liquid chromatography/positive electrospray ionization tandem mass spectrometry

Glutathione (GSH) is a tripeptide composed of glutamate, cysteine, and glycine. It is present in practically all cells and has several important roles, such as preventing the oxidation of the sulfhydryl groups of proteins within a cell. Evidence for GSH deficiency or depletion has been found in a variety of diseases and toxicity-related studies, including diabetes and induction of oxidative stress to form reactive oxygen species which cause DNA, lipid, and protein oxidations. A simple, selective, and sensitive analytical method for measuring low levels of GSH in biological fluids would therefore be desirable to conduct GSH deficiency or depletion-related mechanistic toxicity studies. Here a method for both low- and high-level quantitation of GSH from cultured cells and rat liver tissues via liquid chromatography/positive electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) has been developed. The lower limit of quantitation (LOQ) of the method was 5 ng/mL. The method is linear over a wide dynamic concentration range of 5.0 to 5000.0 ng/mL, with a correlation coefficient R2 > 0.99. The intra-day assay precision relative standard deviation (RSD) values for all quality control (QC) samples were < or =16.31%, with accuracy values ranging from 94.13 to 97.80%. The inter-day assay precision RSD values for all QC samples were < or =15.94%, with accuracy values ranging from 94.51 to 100.29%. With this method, low levels of GSH from diethyl maleate (DEM)-treated mouse lymphoma cells, and GSH in rat liver tissues, were quantified.