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.

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.

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.

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.

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.

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.

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.

Anti-proliferative effect of novel primary cetyl alcohol derived sophorolipids against human cervical cancer cells HeLa.

Sophorolipids (SLs) are glycolipid biosurfactants that have been shown to display anticancer activity. In the present study, we report anti-proliferative studies on purified forms of novel SLs synthesized using cetyl alcohol as the substrate (referred as SLCA) and their anticancer mechanism in human cervical cancer cells. Antiproliferative effect of column purified SLCA fractions (A, B, C, D, E and F) was examined in panel of human cancer cell lines as well as primary cells. Among these fractions, SLCA B and C significantly inhibited the survival of HeLa and HCT 116 cells without affecting the viability of normal human umbilical vein endothelial cells (HUVEC). The two fractions were identified as cetyl alcohol sophorolipids with non-hydroxylated tail differing in the degree of acetylation on sophorose head group. At an IC50 concentration SLCA B (16.32 µg ml-1) and SLCA C (14.14 µg ml-1) blocked the cell cycle progression of HeLa cells at G1/S phase in time-dependent manner. Moreover, SLCA B and SLCA C induced apoptosis in HeLa cells through an increase in intracellular Ca2+ leading to depolarization of mitochondrial membrane potential and increase in the caspase-3, -8 and -9 activity. All these findings suggest that these SLCAs could be explored for their chemopreventive potential in cervical cancer.

Diverse profiles of N-acyl-homoserine lactones in biofilm forming strains of Cronobacter sakazakii.

The present study investigates the role of quorum sensing (QS) molecules expressed by C. sakazakii in biofilm formation and extracellular polysaccharide expression. The QS signaling was detected using Chromobacterium violaceum 026 and Agrobacterium tumefaciens NTL4(pZLR4) based bioassay. Long chain N-acyl-homoserine lactones (AHLs) with C6- C18 chain length were identified using High Performance Liquid Chromatography and Liquid Chromatography-High Resolution Mass Spectrometry. A higher Specific Biofilm Formation (SBF) index (p < 0.05) with the presence of genes associated with cellulose biosynthesis (bcsA, bcsC and bcsG) was observed in the strains. AHLs and their mechanisms can serve as novel targets for developing technologies to eradicate and prevent biofilm formation by C. sakazakii.

Biosynthesis of Glycomonoterpenes to Attenuate Quorum Sensing Associated Virulence in Bacteria.

The acquisition of multidrug resistance in bacteria has become a bigger threat of late, mainly due to the bacterial signaling phenomenon, quorum sensing (QS). QS, among a population of bacteria, initiates the formation of biofilms and offers myriad advantages to bacteria. Burgeoning antibiotic resistance in biofilm-producing bacteria has motivated efforts toward finding new alternatives to these traditional antimicrobials. In the present study, we report the increased solubility and additional quorum quenching as well as biofilm disruption activity of glyco-derivatives of monoterpenes (citral and citronellal). Glycomonoterpenes of citral and citronellal were synthesized via conjugation of the monoterpenes with glucose by the non-pathogenic yeast Candida bombicola (ATCC 22214). Structural elucidation of newly synthesized glycomonoterpenes showed that one synthesized using citronellal contains three major lactonic forms with molecular weight 492.43, 473.47, and 330.39 Da whereas the one produced using citral has an acidic form with molecular weight 389.33 and 346.23 Da. The glycomonoterpenes were able to individually inhibit QS, mediated through various medium-chain and long-chain N-acyl homoserine lactones (AHLs). These new compounds are interesting additions to the known range of quorum sensing inhibitors (QSIs) and could be further explored for potential clinical applications.

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.

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.

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.

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.

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.

Biological activity of sophorolipids and their possible use as antiviral agents.

Sophorolipids are surface active glycolipids consisting of a hydrophilic sophorose unit and a hydrophobic portion composed of a fatty acid tail. Crude sophorolipid sample contains both acidic and lactonic forms of sophorolipid with different degrees of acetylation and varying lengths of the fatty acid chains depending on the substrates used in the production process. Carboxylic end in the acidic form of the fatty acid is free, whereas in the lactonic form, it is internally esterified. Sophorolipids show different physicochemical properties with wide range of applications for each structural compound. Lactonic form of sophorolipids shows surface tension reducing ability and biological activity, whereas the acidic form possesses better foam forming ability and higher solubility. Presence of acetyl groups gives hydrophilic nature to the sophorolipids which promotes its antiviral and cytokine-stimulating properties. The aim of this review is to explore and suggest the plausibility of sophorolipids as therapeutic and prophylactic agents for the treatment of viral diseases.

Pulsed Laser-Driven Molecular Self-assembly of Cephalexin: Aggregation-Induced Fluorescence and Its Utility as a Mercury Ion Sensor.

A fluorescent self-assembly of cephalexin is obtained by pulsed laser irradiation process. An intense fluorescence emission is found in the self-assembled form due to occurrence of a typical aggregation-induced emission in cephalexin molecules. It is observed that fluorescence quenching of the self-assembled fluorescent nanostructures occurs in the presence of extremely low Hg(++) ions concentrations (10(-7) m) as compared to other heavy metal ions e.g. Ferrous (Fe(++) ), Manganese (Mn(++) ), Magnesium (Mg(++) ), Cobalt (Co(++) ), Nickel (Ni(++) ) and Zinc (Zn(++) ) at the same concentrations.

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.