Nucleation temperature‐controlled synthesis and in vitro toxicity evaluation of l‐cysteine‐capped Mn:ZnS quantum dots for intracellular imaging

Quantum dots (QDs), one of the fastest developing and most exciting fluorescent materials, have attracted increasing interest in bioimaging and biomedical applications. The long‐term stability and emission in the visible region of QDs have proved their applicability as a significant fluorophore in cell labelling. In this study, an attempt has been made to explore the efficacy of l ‐cysteine as a capping agent for Mn‐doped ZnS QD for intracellular imaging. A room temperature nucleation strategy was adopted to prepare non‐toxic, water‐dispersible and biocompatible Mn:ZnS QDs. Aqueous and room temperature QDs with l ‐cysteine as a capping agent were found to be non‐toxic even at a concentration of 1500 µg/mL and have wide applications in intracellular imaging. Copyright © 2015 John Wiley & Sons, Ltd.     

Thermoluminescence of mercaptoethanol‐capped ZnS:Mn nanoparticles

The thermoluminescence (TL) of nanoparticles has become a matter of keen interest in recent times but is rarely reported. This article reports the synthesis of ZnS:Mn nanocrystals using a chemical route, with mercaptoethanol (ME) as the capping agent. The particle sizes for the nanocrystals were measured by X‐ray diffraction (XRD) and also by studying transmission electron microscopy (TEM) patterns. The particle sizes of the synthesized samples were found to be between 1 and 3 nm. For samples with different concentrations of the capping agent, it was found that the TL intensity of the ZnS:Mn nanoparticles increased as the particle size decreased. A shift in the peak position of the TL glow curve was also seen with decreasing particle size. The TL intensity was found to be maximal for samples with 1.2% of Mn. A change in the peak position was not found for samples with different concentrations of Mn. The half‐width glow peak curve method was used to determine the trap‐depth. The frequency factor of the synthesized samples was also calculated. The stability of the charge carriers in the traps increases with decreasing nanoparticle size. The higher stability may be attributed to the higher surface/volume ratio and also to the increase in the trap‐depth with decreasing particle size. Copyright © 2014 John Wiley & Sons, Ltd.     

Enhanced dissolution of the substrate d,l‐2‐amino‐Δ2‐thiazoline‐4‐carboxylic acid and enzymatic production of l‐cysteine at high concentrations

l ‐Cysteine is widely used as a precursor in the pharmaceutical, cosmetic, food, and feed additive industries. It has been industrially produced from hydrolysis of human and animal hairs, which is limited for industrial production. At the same time, chemical hydrolysis causes the formation of intractable waste material. Thus, environmentally friendly methods have been developed. A big obstacle of currently available methods is the low substrate solubility leading to poor l ‐cysteine yield. Here, a method for improving the low solubility of the substrate d ,l ‐2‐amino‐Δ²‐thiazoline‐4‐carboxylic acid (d ,l ‐ATC) is presented and the enzymatic reaction at high concentration levels was optimized. The substrate was dissolved in large amounts in aqueous solutions by pH control using salts. d ,l ‐ATC solubility increased with an increasing solution pH due to its enhanced hydrophilicity, which can be achieved by a shift to dissociated carboxylic group (–COO^?). The highest d ,l ‐ATC solubility of 610 mM was obtained at pH 10.5. The maximum l ‐cysteine yield of 250 mM was attained at pH 9.1, which lies between the optimum values for high substrate solubility and reaction rate. The product yield could be increased by more than 10 times compared to those in previous reports, which is industrially meaningful.     

Dual doped biocompatible multicolor luminescent carbon dots for bio labeling,UV‐active marker and fluorescent polymer composite

We report on metal–non‐metal doped carbon dots with very high photoluminescent properties in solution. Magnesium doping to tamarind extract associated with nitrogen‐doping is for the first time reported here which also produce very high quantum yield. Our aim is to develop such dual doped carbon dots which can also serve living cell imaging with easy permeation towards cells and show non‐cytotoxic attributes. More importantly, the chemical signatures of the carbon dots unveiled in this work can support their easy solubilization into water; even in sub‐ambient temperature. The cytotoxicity assay proves the almost negligible cytotoxic effect against human cell lines. Moreover, the use of carbon dots in UV‐active marker and polymer composites are also performed which gave clear distinguishable features of fluorescent nanoparticles. Hitherto, the carbon dots can be commercially prepared without adopting any rigorous methods and also can be used as non‐photo‐bleachable biomarkers of living cells.     

Labeling of BSA and imaging of mouse T‐lymphocyte as well as mouse spleen tissue by l‐glutathione capped CdTe quantum dots

l ‐glutathione capped highly fluorescent CdTe quantum dots (QDs) were prepared by an aqueous approach and used as fluorescent labels to link albumin bovine serum (BSA) and rat anti‐mouse CD4, which was expressed on mouse T‐lymphocyte and mouse spleen tissue. The sharp and narrow emission peaks showed that the as‐prepared QDs have desirable dispersibility, uniformity and good fluorescence properties. Both CdTe–BSA and CdTe–CD4 conjugates showed an enhancement of fluorescence intensity over that of bare CdTe QDs. The experimental result of gel electrophoresis confirmed the successful conjugation of CdTe–BSA and CdTe–CD4. The fluorescent microscopic images of CdTe–CD4 labeled mouse T‐lymphocyte cells and mouse spleen tissue were compared with that obtained from fluorescein isothiocyanate labeling. It was demonstrated that the CdTe QDs‐based probe exhibited much better photostability and fluorescence intensity than fluorescein isothiocyanate, showing a good application potential in the immuno‐labeling of cells and tissues. Copyright © 2009 John Wiley & Sons, Ltd.     

Biofabrication of ZnS:Mn luminescent nanocrystals using histidine,hexahistidine, and His-tagged proteins: A comparison study

The ubiquitous hexahistidine purification tag has been used to conjugate proteins to the shell of CdSe:ZnS quantum dots (QDs) due to its affinity for surface-exposed Zn²⁺ ions but little attention has been paid to the potential of His-tagged proteins for mineralizing luminescent ZnS nanocrystals. Here, we compare the ability of free histidine, a His tag peptide, His-tagged thioredoxin (TrxA, a monomeric protein), and N- and C-terminally His-tagged versions of Hsp31 (a homodimeric protein) to support the synthesis of Mn-doped ZnS nanocrystals from aqueous precursors under mild conditions of pH (8.2) and temperature (37 °C). We find that: (1) it is possible to produce poor quality QDs when histidine is used at high (8 mM) concentration; (2) an increase in local histidine concentration through repetition of the amino acid as a His tag decreases the amount of needed reagent ≈10-fold and improves optical properties; (3) fusion of the same His tag to TrxA allows for ZnS:Mn QDs mineralization at micromolar concentrations; and (4) doubling the local hexahistidine concentration by exploiting Hsp31 dimerization further improves nanocrystal luminescence with the brightest particles obtained when His tags are spatially co-localized at the Hsp31 N-termini. Although hexahistidine tracts are not as efficient as combinatorially selected ZnS binding peptides at QD synthesis, it should be possible to use the large number of available His-tagged proteins and the synthesis approach described herein to produce luminescent nanoparticles whose protein shell carries a broad range of functions.     

Extraction and long‐term storage of S‐layer proteins and flagella from Lysinibacillus sphaericus NCTC 9602: Building blocks for nanotechnology

Self‐assembling surface layer (SL) proteins of bacteria have been widely studied, in particular their use as molecularly defined, 2D coatings of technical surfaces. An important prerequisite is the availability of a sufficient amount of protein. However, a detailed and optimized protocol for the complete SL extraction is so far not available. Here, we describe the complete purification and reassembly procedure of an SL protein of Lysinibacillus sphaericus NCTC 9602, starting from the cultivation of cells, the preparation and purification of SL proteins up to the long‐term storage and in vitro self‐assembly of the proteins. All crucial steps of the procedure are assessed by different microscopic techniques, such as light microscopy, atomic force microscopy, and scanning electron microscopy as well as by SDS‐PAGE as a biochemical method. We demonstrate that storage of the protein in the presence of sodium azide or upon lyophilization allows the preservation of the self‐assembly properties for at least 9 years. Additionally, we describe a method allowing the extraction of intact flagella with lengths in the range up to 4 μm. Flagella may have applications in bio‐nanotechnology, for example as templates for metallic nanowires.     

Assessment of poly‐l‐lysine dendrigrafts for virus concentration in water: use of MS2 bacteriophage as proof of concept

Aims

Virus detection has often been difficult due to a low concentration in water. In this study, we developed a new procedure based on concentration of virus particles on an innovative support: poly‐l ‐lysine dendrigrafts (DGL), coupled with directed nucleic acid extraction and real‐time PCR quantification.

Methods and Results

This method was evaluated using the bacteriophage MS2 as a model virus. This virus exhibited the size and structural properties of human pathogenic enteric viruses and has often been used to assess new supports of concentration. Moreover, this bacteriophage is also a faecal contamination indicator. In this study, many water filtration conditions were tested (volume of water, concentration, etc.), and more than 80% of bacteriophage were recovered after filtration on polymer, in most conditions. We demonstrated that the method was linear (slope = 0·99 ± 0·04 and Y intercept when x = ?0·02 ± 0·28), valid (as manipulators, tested concentrations, volumes of sample and batch of polymer did not have any influence on concentration) and sensitive (allowing to concentrate up to 16 600‐fold 1 l of sample and to detect and quantify down to 750 GC l^?1 and 7500 GC l^?1, respectively).

Conclusions

To conclude, this support exhibits high interest to retain viruses and to allow to detect low concentration of virus in water.

Significance and Impact of the Study

This study gives valuable advance in the methods of concentration and diagnosis of virus in water.     

A Green and Sustainable Approach: Celebrating the 30th Anniversary of the Asymmetric l‐Menthol Process

Takasago has been devoted to producing l‐menthol since 1954, and our long history of manufacturing this important aroma chemical is reviewed here. The current asymmetric catalytic process had its 30th anniversary in 2013. Our l‐menthol process is considered carbon‐neutral, and, therefore, ‘green’ and sustainable. It uses renewable myrcene obtained from gum rosin as a starting material. In addition, the Rh‐BINAP (=2,2′‐bis(diphenylphosphino)‐1,1′‐binaphthyl) catalytic system is highly efficient. This pathway not only leads l‐menthol, but a variety of 100% biobased aroma chemical products as well. By measuring the ¹⁴C levels in a material, one can determine the percentage of carbon that is biobased. This biobased assay, described as the ratio plant‐derived C/fossil‐derived C, can clarify how renewable a product really is. This will be highlighted for several of Takasago's key aroma chemicals.     

Purification,Characterization and Antibacterial Activity of l‐amino Acid Oxidase from Cerastes cerastes

Antibiotic resistance presents a real problem in which new antibacterial molecules from natural secretions could be beneficial in the development of new drugs. In this study, Cerastes cerastes venom was investigated for its antibacterial activity against Gram‐positive and Gram‐negative bacteria. The antibacterial activity was evaluated by measuring the halo inhibition and minimum inhibitory concentration (MIC). An l ‐amino acid oxidase (CcLAAO) was purified from this venom using three chromatographic steps; its homogeneity (60 kDa) was confirmed by SDS‐PAGE. LC–MS/MS analysis of CcLAAO showed similarities with other LAAO enzymes from Echis ocellatus and Viridovipera stejnegeri venoms. CcLAAO presents an antibacterial activity against three bacterial strains (Staphylococcus aureus, Methicillin‐resistant S. aureus, and Pseudomonas aeruginosa) with MIC values of 10, 10, and 20 μg/mL, respectively. However, no effect was observed against Escherichia coli and yeast strains. Kinetic parameters of CcLAAO evaluated on l ‐leucine at pH 8.0 and 20°C were K_m = 0.06 mmol and V_max = 164 mmol/min.     

Highly luminescent nitrogen‐doped carbon dots for simultaneous determination of chlortetracycline and sulfasalazine

Here, we have presented a green and facile strategy to fabricate nitrogen‐doped carbon dots (N‐CDs) and their applications for determination of chlortetracycline (CTC) and sulfasalazine (SSZ). The fluorescent N‐CDs, prepared by one‐step hydrothermal reaction of citric acid and l ‐arginine, manifested numerous excellent features containing strong blue fluorescence, good water‐solubility, narrow size distribution, and a high fluorescence quantum yield (QY) of 38.8%. Based on the fluorescence quenching effects, the as‐synthesized N‐CDs as a fluorescent nanosensor exhibited superior analytical performances for quantifying CTC and SSZ. The linear range for CTC was calculated to be from 0.85 to 20.38 μg ml^?1 with a low detection limit of 0.078 μg ml^?1. Meanwhile, the linear range for SSZ was estimated to be from 0.34 to 6.76 μg ml^?1 with a low detection limit of 0.032 μg ml^?1. Therefore, the N‐CDs hold admirable application potential for constructing a fluorescent sensor for pharmaceutical analysis.     

Sodium Butyrate Ameliorates l‐Arginine‐Induced Pancreatitis and Associated Fibrosis in Wistar Rat: Role of Inflammation and Nitrosative Stress

Several reports indicated that histone deacetylases (HDACs) play a crucial role in inflammation and fibrogenesis. Sodium butyrate (SB) is a short‐chain fatty acid having HDAC inhibition potential. The present study aimed to evaluate the protective effect of SB against l ‐arginine (l ‐Arg)‐induced pancreatic fibrosis in Wistar rats. Pancreatic fibrosis was induced by twice intraperitoneal (i.p.) injections of 20% l ‐Arg (250 mg/100 g) at 2‐h interval on day 1, 4, 7, and 10, whereas SB (800 mg/kg/day) was administrated for 10 days. At the end of the study, biochemical estimations, histological alterations, DNA damage, and the expression of various proteins were evaluated. Posttreatment of SB decreased l ‐Arg‐induced oxidative and nitrosative stress, DNA damage, histological alterations, and fibrosis. Interestingly, posttreatment of SB significantly decreased the expression of α‐smooth muscle actin, interleukin‐1β, inducible nitric oxide synthase, and 3‐nitrotyrosine. The present study demonstrated that posttreatment of SB alleviates l ‐Arg‐induced pancreatic damage and fibrosis in rat.     

Purification,immobilization, and biochemical characterization of l‐arginine deiminase from thermophilic Aspergillus fumigatus KJ434941: Anticancer activity in vitro

l ‐Arginine deiminase (ADI) has a powerful anticancer activity against various tumors, via arginine depletion, arresting the cell cycle at G₁ phase. However, the current clinically tried bacterial ADI displayed a higher antigenicity and lower thermal stability. Thus, our objective was to purify and characterize this enzyme from thermophilic fungi, to explore its catalytic and antigenic properties for therapeutic uses. ADI was purified from thermophilic Aspergillus fumigatus KJ434941 to its electrophoretic homogeneity by 5.1‐fold, with molecular subunit 50 kDa. The purified ADI was PEGylated and covalently immobilized on dextran to explore its catalytic properties. The specific activity of free ADI, PEG‐ADI, and Dex‐ADI was 26.7, 21.5, and 18.0 U/mg, respectively. At 50°C, PEG‐ADI displays twofold resistance to thermal denaturation (t_1/2 13.9 h), than free ADI (t_1/2 6.9 h), while at 70°C, the thermal stability of PEG‐ADI was increased by 1.7‐fold, with similar stability to Dex‐ADI with the free one. Kinetically, free ADI had the higher catalytic affinity to arginine, followed by PEG‐ADI and Dex‐ADI. Upon proteolysis for 30 min, the residual activity of native ADI, PEG‐ADI, and Dex‐AD was 8.0, 32.0, and 20.0% for proteinase K and 10.0, 52.0, and 90.0% for acid protease, respectively. The anticancer activity of the ADIs was assessed against HCT, HEP‐G2, and MCF7, in vitro. The free and PEG‐ADI exhibits a similar cytotoxic efficacy for the tested cells, lower than Dex‐ADI. The free ADI had IC₅₀ value 22.0, 16.6, and 13.9 U/mL, while Dex‐ADI had 3.98, 5.18, and 4.43 U/mL for HCT, MCF7, and HEPG‐2, respectively. The in vitro anticancer activity of ADI against HCT, MCF7, and HEPG‐2 was increased by five‐, three‐, and threefold upon covalent modification by dextran. The biochemical and hematological parameters of the experimented animals were not affected by ADIs dosing, with no signs of anti‐ADI immunoglobulins in vivo. The in vivo half‐life time of free ADI, PEG‐ADI, and Dex‐ADI was 29.7, 91.1, 59.6 h, respectively. The present findings explored a novel thermostable, less antigenic ADI from thermophilic A. fumigatus, with further molecular and crystallographic analyses, this enzyme will be a powerful candidate for clinical trials. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:396–405, 2015     

Synthesis,binding ability,and cell cytotoxicity of fluorescent probes for l‐arginine detection based on naphthalene derivatives: Experiment and theory

Inspired by biological related parts, Schiff base derivatives and functional groups of chemical modification can provide efficient detection method of amino acids. Therefore, we have designed and prepared 4 compounds based on Schiff base derivatives involving ─NO₂, ─OH, and naphthyl group. Results indicated that compound 4 containing 2 nitro groups showed strong sensitivity and high selectivity for arginine (Arg) among normal 18 kinds of standard amino acids (alanine, valine, leucine, isoleucine, methionine, aspartic acid, glutamic acid, arginine, glycine, serine, asparagine, phenylalanine, histidine, tryptophan, proline, lysine, glutamine, and cysteine). Theoretical investigation also approved the strong binding ability of compound 4 for Arg. In addition, compound 4 displayed high combining ability of Arg and low cytotoxicity of MCF‐7 cell in the 0 to 150 μg mL^?1 of concentration range; it can be used for Arg in vivo detection of fluorescent probe.     

Aminooxy‐naphthylpropionic acid and its derivatives are inhibitors of auxin biosynthesis targeting l‐tryptophan aminotransferase: structure–activity relationships

We previously reported l ‐α‐aminooxy‐phenylpropionic acid (AOPP) to be an inhibitor of auxin biosynthesis, but its precise molecular target was not identified. In this study we found that AOPP targets TRYPTOPHAN AMINOTRANSFERASE of ARABIDOPSIS 1 (TAA1). We then synthesized 14 novel compounds derived from AOPP to study the structure–activity relationships of TAA1 inhibitors in vitro. The aminooxy and carboxy groups of the compounds were essential for inhibition of TAA1 in vitro. Docking simulation analysis revealed that the inhibitory activity of the compounds was correlated with their binding energy with TAA1. These active compounds reduced the endogenous indole‐3‐acetic acid (IAA) content upon application to Arabidopsis seedlings. Among the compounds, we selected 2‐(aminooxy)‐3‐(naphthalen‐2‐yl)propanoic acid (KOK1169/AONP) and analyzed its activities in vitro and in vivo. Arabidopsis seedlings treated with KOK1169 showed typical auxin‐deficient phenotypes, which were reversed by exogenous IAA. In vitro and in vivo experiments indicated that KOK1169 is more specific for TAA1 than other enzymes, such as phenylalanine ammonia‐lyase. We further tested 41 novel compounds with aminooxy and carboxy groups to which we added protection groups to increase their calculated hydrophobicity. Most of these compounds decreased the endogenous auxin level to a greater degree than the original compounds, and resulted in a maximum reduction of about 90% in the endogenous IAA level in Arabidopsis seedlings. We conclude that the newly developed compounds constitute a class of inhibitors of TAA1. We designated them ‘pyruvamine’.