Expression, Purification and Crystallization of Asrij, A Novel Scaffold Transmembrane Protein.

Asrij/OCIAD1 is a scaffold transmembrane protein belonging to the Ovarian Carcinoma Immunoreactive Antigen Domain containing protein family. In Drosophila and mouse models, Asrij localizes at the endosomal and mitochondrial membrane and is shown to regulate the stemness of hematopoietic stem cells. Interaction of Asrij with ADP Ribosylation Factor 1 (Arf1) is shown to be crucial for hematopoietic niche function and prohemocyte maintenance. Here, we report the heterologous expression, standardization of detergents and purification methodologies for crystallization of Asrij/OCIAD1. To probe the activity of bacterially expressed Asrij, we developed a protein complementation assay and conclusively show that Asrij and Arf1 physically interact. Further, we find that sophorolipids improve the solubility and monodispersibility of Asrij. Hence, we propose that sophorolipids could be novel additives for stabilization of membrane proteins. To our knowledge, this is the first study detailing methodology for the production and crystallization of a heterologously expressed scaffold membrane protein and will be widely applicable to understand membrane protein structure and function.

Red fluorescent ultra-small gold nanoclusters functionalized with signal molecules to probe specificity in quorum sensing receptors in gram-negative bacteria.

Ultra-small (size < 2 nm) gold nanoclusters (AuNCs) are used as fluorescent probes which have excellent applications in bioimaging and sensing due to their emission in visible and NIR spectral region. Here, this property is exploited for understanding the quorum sensing phenomenon in bacteria which is regulated by signal molecules which are specific to various species. AuNCs are then functionalized with the signal molecules, Acyl Homoserine Lactones (AHL) of varying carbon chain length, C-6, C-8, and C-12 without 3rd C modification, to sense different strains of gram-negative bacteria i.e., Escherichia coli, Cronobacter sakazakii and Pseudomonas aeruginosa. In the concentration employed, selectivity to a limited extent is observed between the three Gram-negative bacteria tested. E. coli showed emission with all the AHL conjugates and P. aeruginosa did not interact with any of the three conjugates, whereas C. sakazakii showed specificity to C-8AHL. This is probably due to selectivity for cognate AHL molecules of appropriate concentrations.

Silk Fibroin-Sodium Dodecyl Sulfate Gelation: Molecular, Structural, and Rheological Insights.

A gelling agent is necessary to accelerate sol to gel transition in an aqueous solution of silk fibroin (SF), which otherwise takes several days to complete. In this paper, we investigate the mechanism of gelation of Bombyx mori SF by a model anionic surfactant, sodium dodecyl sulfate (SDS). Even though interactions between SDS and proteins have been extensively investigated, most of these studies have focused on globular proteins, which undergo denaturation. The interaction with a fibrous protein such as SF is different and results in an altered secondary structure leading to gelation. In this work, the concentration-dependent gelation process of the SF-SDS system is examined using rheology, SANS, FTIR, and NMR. We observed preferential binding of SDS to specific regions on the SF chain, which aids structural changes favoring ß-sheet formation. We propose a mechanism for the accelerated sol-gel transition in the SF-SDS system.

Self-Exfoliated Guanidinium-Based Ionic Covalent Organic Nanosheets (iCONs).

Covalent organic nanosheets (CONs) have emerged as functional two-dimensional materials for versatile applications. Although π-π stacking between layers, hydrolytic instability, possible restacking prevents their exfoliation on to few thin layered CONs from crystalline porous polymers. We anticipated rational designing of a structure by intrinsic ionic linker could be the solution to produce self-exfoliated CONs without external stimuli. In an attempt to address this issue, we have synthesized three self-exfoliated guanidinium halide based ionic covalent organic nanosheets (iCONs) with antimicrobial property. Self-exfoliation phenomenon has been supported by molecular dynamics (MD) simulation as well. Intrinsic ionic guanidinium unit plays the pivotal role for both self-exfoliation and antibacterial property against both Gram-positive and Gram-negative bacteria. Using such iCONs, we have devised a mixed matrix membrane which could be useful for antimicrobial coatings with plausible medical benefits.

Inhibition of quorum-sensing-mediated biofilm formation in Cronobacter sakazakii strains.

The present study investigated plant extracts for their anti-quorum-sensing (QS) potential to inhibit the biofilm formation in Cronobacter sakazakii strains. The bioassay based on loss of pigment production by Chromobacterium violaceum 026 and Agrobacterium tumefaciens NTL4(pZLR4) was used for initial screening of the extracts. Further, the effect of extracts on the inhibition of QS-mediated biofilm in C. sakazakii isolates was evaluated using standard crystal violet assay. The effect on biofilm texture was studied using SYTO9 staining and light and scanning electron microscopy. Among the tested extracts, Piper nigrum and Cinnamomum verum at 100 ppm resulted in 78 and 68 % reduction in the production of violacein as well as blue-green colour in both biosensor strains. A higher inhibitory activity (>50 %) on biofilm formation in C. sakazakii was observed for Pip. nigrum and Cin. verum, whereas the other extracts possessed moderate (25-50 %) and minimal (<25 %) inhibitory activities. Further, the fluorescent and scanning electron microscopic images indicated a major disruption in the architecture of biofilms of tested strains by Pip. nigrum. This study points to the possibility of using Pip. nigrum and Cin. verum as inhibitor of QS-mediated biofilm formation by C. sakazakii that could be further explored for novel bioactive molecules to limit the emerging infections of C. sakazakii.

Silk Fibroin-Sophorolipid Gelation: Deciphering the Underlying Mechanism.

Silk fibroin (SF) protein, produced by silkworm Bombyx mori, is a promising biomaterial, while sophorolipid (SL) is an amphiphilic functional biosurfactant synthesized by nonpathogenic yeast Candida bombicola. SL is a mixture of two forms, acidic (ASL) and lactonic (LSL), which when added to SF results in accelerated gelation of silk fibroin. LSL is known to have multiple biological functionalities and hence hydrogels of these green molecules have promising applications in the biomedical sector. In this work, SANS, NMR, and rheology are employed to examine the assembling properties of individual and mixed SLs and their interactions with SF to understand the mechanism that leads to rapid gelation. SANS and NMR studies show that ASL assembles to form charged micelles, while LSL forms micellar assemblies and aggregates of a mass fractal nature. ASL and LSL together form larger mixed micelles, all of which interact differently with SF. It is shown that preferential binding of LSL to SF causes rapid unfolding of the SF chain leading to the formation of intermolecular beta sheets, which trigger fast gelation. Based on the observations, a mechanism for gelation of SF in the presence of different sophorolipids is proposed.

Purification and partial characterization of novel penicillin V acylase from Acinetobacter sp. AP24 isolated from Loktak Lake, an Indo-Burma biodiversity hotspot.

Members of the bacterial genus Acinetobacter have attracted great attention over the past few decades, on account of their various biotechnological applications and clinical implications. In this study, we are reporting the first experimental penicillin V acylase (PVA) activity from this genus. Penicillin acylases are pharmaceutically important enzymes widely used in the synthesis of semisynthetic beta-lactam antibiotics. The bacterium, identified as Acinetobacter sp. AP24, was isolated from the water of Loktak Lake (Manipur, India), an Indo-Burma biodiversity hotspot. PVA production was increased threefold in an optimized medium with 0.2% sodium glutamate and 1% glucose as nitrogen and carbon sources respectively, after 24 hr of fermentation at 28°C and pH 7.0 with shaking at 180 rpm. The enzyme was purified to homogeneity by cation-exchange chromatography using SP-sepharose resin. The PVA is a homotetramer with subunit molecular mass of 34 kD. The enzyme was highly specific toward penicillin V with optimal hydrolytic activity at 40°C and pH 7.5. The enzyme was stable from pH 5.0 to 9.0 at 25 °C for 2 hr. The enzyme retained 75% activity after 1 hr of incubation at 40°C at pH 7.5.

Evaluating the anticancer activity and nanoparticulate nature of homeopathic preparations of Terminalia chebula.

BACKGROUND: Breast cancer is the most common cancer diagnosed among women and is the second leading cause of cancer death. Homeopathic medicines are part of the alternative medicines that are given as a supportive therapy in breast cancer. The objective of this study was to investigate the anticancer activity of commercially available homeopathic preparations of Terminalia chebula (TC) and evaluate their nanoparticulate nature. METHODS: Mother tincture (MT) and other homeopathic preparations (3X, 6C and 30C) of TC were tested for their effect on the viability of breast cancer (MDAMB231 and MCF7) and non-cancerous (HEK 293) cell lines by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell growth assay was performed to analyze the effect of the different potencies on the growth kinetics of breast cancer cells. MT and 6C were evaluated for the presence of nanoparticles by using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). RESULTS: MT decreased the viability of breast cancer (MDAMB231 and MCF7) and non-cancerous (HEK 293) cells. However, the other potencies (3X, 6C and 30C) decreased the viability of only breast cancer cells without affecting the viability of the non-cancerous cells. All the potencies, MT, 3X, 6C and 30C, reduced growth kinetics of breast cancer cells, more specifically at 1:10 dilution at 24, 48 and 72 h. Under SEM, MT appeared as a mesh-like structure whereas under TEM, it showed presence of nanoclusters. On the other hand, 6C potency contained 20 nm sized nanoparticles. CONCLUSION: The current study reports the anticancer activity of homeopathic preparations of TC against breast cancer and reveals their nanoparticulate nature. These preliminary results warrant further mechanistic studies at both in vitro and in vivo levels to evaluate the potential of TC as nanomedicine in breast cancer.

A new class of bacterial quorum sensing antagonists: glycomonoterpenols synthesized using linalool and alpha terpineol.

With increasing burden of antibiotic resistant microorganism search for newer drug targets and potent drug molecules is a never ending scenario. Quorum sensing (QS), the phenomenon of bacterial cross-talk, is one such target that has captured the attention of many and has been touted as the future of new age antimicrobials. Quorum sensing has the potential to regulate a plethora of bacterial virulence phenotypes and search of molecules with powerful quorum sensing inhibitory (QSI) capacity are underway. Monoterpene alcohols like linalool and alpha terpineol have been shown to possess antimicrobial and anti-biofilm activity. However in this article we attempt to bring forth a new class of compounds, glycomonoterpenols, derived from monoterpenoids alcohols. These glycomonoterpenols have been synthesized using Candida bombicola ATCC 22214 by feeding the cells with linalool and alpha terpineol respectively as substrates in 10% glucose, production medium. The advantage of these molecules over their parent compound is their additional surfactant like property, increased solubility and enhanced QSI potential. A variety of gram-negative bacteria capable of elaborating quorum sensing mediated phenotypes have been selected and both these glycoterpenoid derivatives have been shown to possess strong anti-QS activity.

Novel glycolipids synthesized using plant essential oils and their application in quorum sensing inhibition and as antibiofilm agents.

Essential oils (EOs) form an important part of traditional medicine so their anti-microbial and, in the recent past, antiquorum sensing activity has been well studied. However it is likely that due to their hydrophobic nature and reduced solubility in aqueous environments full potential of their activity cannot be realized. hence it is only rational to formulate a process to make these molecules more polar in nature. The present paper reports synthesis of sophorolipids using 12 different essential oils as substrates, thus providing surfactant-like properties to these EOs. The synthesis protocol makes the use of Candida bombicola ATCC 22214 as producer organism. The production process required 7 days of incubation at 28°C and 180 rpm. Preliminary characterization of the synthesized essential oil sophorolipids (EOSLs) was performed using thin layer chromatography (TLC) and Fourier transform infrared spectroscopy (FTIR). Additionally, essential oils that were incapable of mediating quorum sensing inhibition (QSI) on their own became potent quorum sensing inhibitors upon conversion into their corresponding EOSLs. Antibiofilm potential of these EOSLs was also demonstrated using V. cholerae as test organism. Use of essential oils as substrates for glycolipid synthesis has not been attempted previously, and hence this is the first report.

Effect of pH on the hydrolytic kinetics of gamma-glutamyl transferase from Bacillus subtilis.

The effect of pH on the steady state kinetics of gamma-glutamyl transferase (GGT) from Bacillus subtilis was examined using glutamyl-(3-carboxyl)-4-nitroanilide as the chromogenic reporter substrate. The enzyme was active in the pH range 7.0-11.0 with the optimum activity at pH 11.0. We noticed a pH dependent transformation in the nature of substrate consumption kinetics. The substrate saturation curves were hyperbolic in the pH range 7.0-9.0 but changed into sigmoid form at pH 10.0 and 11.0. Hill's coefficients were >1. We also analysed the effect of pH on the structure of the enzyme. The circular dichroism spectra of the enzyme sample at pH 9.0 and 11.0 were coincidental in both far and near UV regions indicating conservation of the secondary and tertiary structures, respectively. The molecular weight of the enzyme sample was the same in both pH 7.0 and 11.0 indicating conservation of the quaternary structure. These results show that the kinetic transformation does not involve significant conformational changes. Cooperative binding of multiple substrate molecules may not be the basis for the sigmoid kinetics as only one substrate binding site has been noticed in the reported crystal structures of B. subtilis GGT.

Crystallization and X-ray structure analysis of a thermostable penicillin G acylase from Alcaligenes faecalis.

The enzyme penicillin G acylase (EC 3.5.1.11) catalyzes amide-bond cleavage in benzylpenicillin (penicillin G) to yield 6-aminopenicillanic acid, an intermediate chemical used in the production of semisynthetic penicillins. A thermostable penicillin G acylase from Alcaligenes faecalis (AfPGA) has been crystallized using the hanging-drop vapour-diffusion method in two different space groups: C222(1), with unit-cell parameters a = 72.9, b = 86.0, c = 260.2 , and P4(1)2(1)2, with unit-cell parameters a = b = 85.6, c = 298.8 . Data were collected at 293 and the structure was determined using the molecular-replacement method. Like other penicillin acylases, AfPGA belongs to the N-terminal nucleophilic hydrolase superfamily, has undergone post-translational processing and has a serine as the N-terminal residue of the ß-chain. A disulfide bridge has been identified in the structure that was not found in the other two known penicillin G cylase structures. The presence of the disulfide bridge is perceived to be one factor that confers higher stability to this enzyme.

Structural determination and chemical esterification of the sophorolipids produced by Candida bombicola grown on glucose and α-linolenic acid.

The extracellular surface-active glycolipids produced by the yeast, Candida bombicola when grown on glucose and α-linolenic acid, were analyzed by HPLC with electro-spray ionization (ESI-MS) and collision-induced dissociation mass spectrometry. The analysis confirmed that the sophorolipid (SL) mixture contained three different forms of C18:3 SL molecules: free acid, lactone and a diacetylated lactone, which has not been reported previously. Also a minor amount of diacetylated lactone form of C18:1 SL was detected. Further, the SL mixture was subjected to chemical esterification reaction with sodium methoxide. The reaction product was analyzed with ESI-MS and confirmed to be the single homogenous esterified product containing C18:3 moieties in its fatty acid chain.

Myristic Acid Derived Sophorolipid: Efficient Synthesis and Enhanced Antibacterial Activity.

Microbial glycolipids are one of the most interesting alternatives to chemical-based surfactants as they exhibit improved biodegradability and less toxicity. However, their potential has been limited because of specificity of the yeast toward fatty acids having a carbon 16 or carbon 18 chain. This study focuses on sophorolipid (SL) production by the yeast Starmerella bombicola using myristic acid, a medium-chain carbon-14 fatty acid that has not been used as a substrate for SL production. The production was optimized for inoculum size and lipophilic substrate concentration. Furthermore, we also studied the effect of medium-chain fatty acid on yeast cell growth and optimized the process for excellent yield. The myristic acid SL (MASL) so synthesized consisted of mono- and diacetylated forms with preferential glycosylation at the methyl end group, as determined by high-resolution mass spectrometry. Individual congeners of the crude mixture were separated using dry column chromatography and then structurally characterized by mass spectrometry. The synthesized MASL was also shown to have promising surface tension, lowering abilities with a low CMC of 14 mg/L. The SL derived from myristic acid exhibited superior antibacterial activity as compared to SL derived from oleic acid. MASL was also found to be more potent against Gram-positive organisms as compared to Gram-negative organisms. This work, therefore, demonstrates successful synthesis of myristic acid-derived SL and its superior antibacterial activity, establishing a promising future for this biosurfactant.

A curcumin-sophorolipid nanocomplex inhibits Candida albicans filamentation and biofilm development.

Candida albicans is an opportunistic fungal pathogen that is highly resistant to contemporary antifungals, due to their biofilm lifestyle. The ability of C. albicans to invade human tissues is due to its filamentation. Therefore, inhibition of biofilms and filamentation of the yeast are high value targets to develop the next-generation antifungals. Curcumin (CU) is a natural polyphenol with excellent pharmacological attributes, but limitations such as poor solubility, acid, and enzyme tolerance have impeded its practical utility. Sophorolipids (SL) are biologically-derived surfactants that serve as efficient carriers of hydrophobic molecules such as curcumin into biofilms. Here, we synthesised a curcumin-sophorolipid nanocomplex (CUSL), and comprehensively evaluated its effects on C. albicans biofilms and filamentation. Our results demonstrated that sub-inhibitory concentration of CUSL (9.37 µg/mL) significantly inhibited fungal adhesion to substrates, and subsequent biofilm development, maturation, and filamentation. This effect was associated with significant downregulation of a select group of biofilm, adhesins, and hyphal regulatory genes. In conclusion, the curcumin-sophorolipid nanocomplex is a potent inhibitor of the two major virulence attributes of C. albicans, biofilm formation and filamentation, thus highlighting its promise as a putative anti-fungal agent with biofilm penetrative potential.

Lauric Acid Sophorolipid: Accelerating the Gelation of Silk Fibroin.

Silk fibroin (SF) hydrogels find wide applications in tissue engineering. However, their scope has been limited due to the long gelation time in ambient conditions. This paper shows the reduction in gelation time of silk fibroin to minutes upon doping with a newly synthesized lauric acid sophorolipid (LASL). LASL comprises a fatty acid, lauric acid (with a 12-carbon aliphatic chain), that is derivatized by glucose molecules using a non-pathogenic yeast Candida bombicola. LASL was characterized using spectroscopic (Fourier transform infrared spectroscopy) and chromatographic (high-performance liquid chromatography, thin-layer chromatography, and high-resolution mass spectrometry) methods. This gelation of SF is comparable to the effect of an anionic surfactant, sodium dodecyl sulfate (SDS). The microstructure of SF-LASL hydrogels was investigated by small-angle neutron scattering (SANS) measurements and exhibited the beads-on-a-necklace model. The rheological properties of these hydrogels show similarity to SF-SDS hydrogels, therefore presenting a greener alternative for tissue engineering applications.

Structural and enzymatic analysis of a dimeric cholylglycine hydrolase like acylase active on N-acyl homoserine lactones.

The prevalence of substrate cross-reactivity between AHL acylases and ß-lactam acylases provides a glimpse of probable links between quorum sensing and antibiotic resistance in bacteria. Both these enzyme classes belong to the N-terminal nucleophile (Ntn)-hydrolase superfamily. Penicillin V acylases alongside bile salt hydrolases constitute the cholylglycine hydrolase (CGH) group of the Ntn-hydrolase superfamily. Here we report the ability of two acylases, Slac1 and Slac2, from the marine bacterium Shewanella loihica-PV4 to hydrolyze AHLs. Three-dimensional structure of Slac1reveals the conservation of the Ntn hydrolase fold and CGH active site, making it a unique CGH exclusively active on AHLs. Slac1homologs phylogenetically cluster separate from reported CGHs and AHL acylases, thereby representing a functionally distinct sub-class of CGH that might have evolved as an adaptation to the marine environment. We hypothesize that Slac1 could provide the structural framework for understanding this subclass, and further our understanding of the evolutionary link between AHL acylases and ß-lactam acylases.

Characterization of the smallest dimeric bile salt hydrolase from a thermophile Brevibacillus sp.

A thermophilic microorganism producing bile salt hydrolase was isolated from hot water springs, Pali, Maharashtra, India. This microorganism was identified as Brevibacillus sp. by 16S rDNA sequencing. Bile salt hydrolase (BSH) was purified to homogeneity from this thermophilic source using Q-sepharose chromatography and its enzymatic properties were characterized. The subunit molecular mass of the purified enzyme was estimated to be 28 kDa by SDS-PAGE and, 28.2 kDa by MALDI-TOF analysis. The native molecular mass was estimated to be 56 kDa by gel filtration chromatography, indicating the protein to be a homodimer. The pH and temperature optimum for the enzyme catalysis were 9.0 and 60 degrees C, respectively. Even though BSH from Brevibacillus sp. hydrolyzed all of the six major human bile salts, the enzyme preferred glycine conjugated substrates with apparent K(M) and k(cat) values of 3.08 microM and 6.32 x 10(2) s(-1), respectively, for glycodeoxycholic acid. The NH(2)-terminal sequence of the purified enzyme was determined and it did not show any homology with other bacterial bile salt hydrolases. To our knowledge, this is the first report describing the purification of BSH to homogeneity from a thermophilic source.

Glycomonoterpene-Functionalized Crack-Resistant Biocompatible Silk Fibroin Coatings for Biomedical Implants.

Surface coatings for biomedical implants have been used to prevent premature failure of the implant due to bacterial biofilm formation and foreign body reaction. Delamination, cracking, crazing, etc. are frequent problems associated with coatings when implants are subjected to mechanical deformation either during surgical handling or during use. We demonstrate here a novel process that results in the formation of a coating that is stable under mechanical stresses in tensile, torsion, and bending modes. The coating process involves a combination of two conventional coating processes, namely, dip coating and electrospinning. Polydimethylsiloxane was selected as the substrate owing to its wide use in biomedical implants. Silk fibroin, a natural biocompatible protein polymer obtained from the Bombyx mori silkworm, was used for demonstrating the process of coating. The coating was also further functionalized using a green biomolecule , glycomonoterpene prepared using citronellal and glucose. These functional compounds are being touted as the next-generation antibiofilm molecules on account of quorum sensing inhibitory activity. We have demonstrated that the quorum-quenching activity of the biomolecule is retained during the processing steps and that the coatings exhibited an excellent antibiofilm activity against common infection-causing bacteria, Pseudomonas aeruginosa and Staphylococcus epidermidis. These silk fibroin-glycomonoterpene coatings can be used for implants in biomedical applications such as breast implants and catheter tubings.

Photophysical studies on curcumin-sophorolipid nanostructures: applications in quorum quenching and imaging.

Sophorolipid biosurfactants are biodegradable, less toxic and FDA approved. The purified acidic form of sophorolipid is stimuli-responsive with self-assembling properties and used for solubilizing hydrophobic drugs. This study encapsulated curcumin (CU) with acidic sophorolipid (ASL) micelles and analysed using photophysical studies like UV-visible spectroscopy, photoluminescence (PL) spectroscopy and time-correlated single photon counting (TCSPC). TEM images have revealed ellipsoid micelles of approximately 100 nm size and were confirmed by dynamic light scattering. The bacterial fluorescence uptake studies showed the uptake of formed CUASL nanostructures into both Gram-positive and Gram-negative bacteria. They also showed quorum quenching activity against Pseudomonas aeruginosa. The results have demonstrated this system has potential theranostic applications.