Biosensing with the singular phase of an ultrathin metal-dielectric nanophotonic cavity.

The concept of point of darkness has received much attention for biosensing based on phase-sensitive detection and perfect absorption of light. The maximum phase change is possible at the point of darkness where the reflection is almost zero. To date, this has been experimentally realized using different material systems through the concept of topological darkness. However, complex nanopatterning techniques are required to realize topological darkness. Here, we report an approach to realize perfect absorption and extreme phase singularity using a simple metal-dielectric multilayer thin-film stack. The multilayer stack works on the principle of an asymmetric Fabry-Perot cavity and shows an abrupt phase change at the reflectionless point due to the presence of a highly absorbing ultrathin film of germanium in the stack. In the proof-of-concept phase-sensitive biosensing experiments, we functionalize the film surface with an ultrathin layer of biotin-thiol to capture streptavidin at a low concentration of 1 pM.

Bacterial cyclic ß-(1,2)-glucans sequester iron to protect against iron-induced toxicity.

Cellular iron homeostasis is critical for survival and growth. Bacteria employ a variety of strategies to sequester iron from the environment and to store intracellular iron surplus that can be utilized in iron-restricted conditions while also limiting the potential for the production of iron-induced reactive oxygen species (ROS). Here, we report that membrane-derived oligosaccharide (mdo) glucan, an intrinsic component of Gram-negative bacteria, sequesters the ferrous form of iron. Iron-binding, uptake, and localization experiments indicated that both secreted and periplasmic ß-(1,2)-glucans bind iron specifically and promote growth under iron-restricted conditions. Xanthomonas campestris and Escherichia coli mutants blocked in the production of ß-(1,2)-glucan accumulate low amounts of intracellular iron under iron-restricted conditions, whereas they exhibit elevated ROS production and sensitivity under iron-replete conditions. Our results reveal a critical role of glucan in intracellular iron homeostasis conserved in Gram-negative bacteria.

Polymeric Nanomaterials Based on the Buckybowl Motif: Synthesis through Ring-Opening Metathesis Polymerization and Energy Storage Applications.

Ring-opening metathesis polymerization (ROMP) of buckybowl corannulene-based oxa-norbornadiene monomer is shown to give rise to polymeric nanomaterials with an average pore size of about 1.4 nm and a surface area of 49.2 m2/g. Application in supercapacitor devices show that the corannulene-based nanomaterials exhibit a specific capacitance of 134 F·g-1 (1.0 V voltage window) in a three-electrode cell configuration. Moreover, the electrode assembled from these materials in a symmetric configuration (1.6 V voltage window) exhibits long-term cyclability of 90% capacitance retention after undergoing 10000 cycles. This work demonstrates that ROMP is a valuable method in synthesizing nanostructured corannulene polymers, and that materials based on the nonplanar polycyclic aromatic motif represents an attractive active component for fabrication of devices targeted at electrochemical energy storage applications.

Ca2+ and ßγ-crystallins: An affair that did not last?

BACKGROUND: During the last three decades, lens ß- and γ-crystallins have found a huge number of kin from numerous taxonomical sources. Most of these proteins from invertebrates and microbes have been demonstrated or predicted to bind Ca2+ involving a distinct double-clamp motif, which is largely degenerated in lens homologues. SCOPE OF REVIEW: The various aspects of transformation of ßγ-crystallins from a quintessential Ca2+-binding protein into a primarily structural molecule have been reviewed. MAJOR CONCLUSIONS: In lens members of ßγ-crystallins, the residues involved in Ca2+ binding have diverged considerably from the classical consensus with consequent reduction in their Ca2+-binding properties. This evolutionary change is congenial to their new role as robust constituents of lens. The exact functions of the residual affinity for Ca2+ are yet to be established. GENERAL SIGNIFICANCE: This review highlights the significance of reduction in Ca2+-binding ability of the ßγ-crystallins for lens physiology and why this residual affinity may be functionally important. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

Synthesis and evaluation of new diaryl ether and quinoline hybrids as potential antiplasmodial and antimicrobial agents.

Synthesis and bioevaluation of new diaryl ether hybridized quinoline derivatives as antiplasmodial, antibacterial and antifungal agents is reported. It was encouraging to discover that several compounds displayed 2-3 folds better efficacy than chloroquine in chloroquine-resistant K1 strain of Plasmodium falciparum. Further, a few members of the library displayed good antibacterial efficacy against gram positive strains of bacteria but none of the compounds displayed any significant antifungal activity.

Microbial ßγ-crystallins.

ßγ-Crystallins have emerged as a superfamily of structurally homologous proteins with representatives across the domains of life. A major portion of this superfamily is constituted by members from microorganisms. This superfamily has also been recognized as a novel group of Ca(2+)-binding proteins with huge diversity. The ßγ domain shows variable properties in Ca(2+) binding, stability and association with other domains. The various members present a series of evolutionary adaptations culminating in great diversity in properties and functions. Most of the predicted ßγ-crystallins are yet to be characterized experimentally. In this review, we outline the distinctive features of microbial ßγ-crystallins and their position in the ßγ-crystallin superfamily.

Ca2+-binding motif of ßγ-crystallins.

ßγ-Crystallin-type double clamp (N/D)(N/D)XX(S/T)S motif is an established but sparsely investigated motif for Ca(2+) binding. A ßγ-crystallin domain is formed of two Greek key motifs, accommodating two Ca(2+)-binding sites. ßγ-Crystallins make a separate class of Ca(2+)-binding proteins (CaBP), apparently a major group of CaBP in bacteria. Paralleling the diversity in ßγ-crystallin domains, these motifs also show great diversity, both in structure and in function. Although the expression of some of them has been associated with stress, virulence, and adhesion, the functional implications of Ca(2+) binding to ßγ-crystallins in mediating biological processes are yet to be elucidated.

Disability for function: loss of Ca(2+)-binding is obligatory for fitness of mammalian ßγ-crystallins.

Vertebrate ßγ-crystallins belonging to the ßγ-crystallin superfamily lack functional Ca(2+)-binding sites, while their microbial homologues do not; for example, three out of four sites in lens γ-crystallins are disabled. Such loss of Ca(2+)-binding function in non-lens ßγ-crystallins from mammals (e.g., AIM1 and Crybg3) raises the possibility of a trade-off in the evolutionary extinction of Ca(2+)-binding. We test this hypothesis by reconstructing ancestral Ca(2+)-binding motifs (transforming disabled motifs into the canonical ones) in the lens γB-crystallin by introducing minimal sets of mutations. Upon incorporation of serine at the fifth position in the N/D-N/D-X-X-S/T(5)-S motif, which endowed a domain with microbial characteristics, a decreased domain stability was observed. Ca(2+) further destabilized the N-terminal domain (NTD) and its serine mutants profoundly, while the incorporation of a C-terminal domain (CTD) nullified this destabilization. On the other hand, Ca(2+)-induced destabilization of the CTD was not rescued by the introduction of an NTD. Of note, only one out of four sites is functional in the NTD of γB-crystallins responsible for weak Ca(2+) binding, but the deleterious effects of Ca(2+) are overcome by introduction of a CTD. The rationale for the onset of cataracts by certain mutations, such as R77S, which have not been clarified by structural means, could be explained by this work. The findings presented here shed light on the evolutionary innovations in terms of the functional loss of Ca(2+)-binding and acquisition of a bilobed domain, besides imparting additional advantages (e.g., protection from light) required for specialized functions.

Bauhinia variegata leaf extracts exhibit considerable antibacterial, antioxidant, and anticancer activities.

The present study reports the phytochemical profiling, antimicrobial, antioxidant, and anticancer activities of Bauhinia variegata leaf extracts. The reducing sugar, anthraquinone, and saponins were observed in polar extracts, while terpenoids and alkaloids were present in nonpolar and ethanol extracts. Total flavonoid contents in various extracts were found in the range of 11-222.67 mg QE/g. In disc diffusion assays, petroleum ether and chloroform fractions exhibited considerable inhibition against Klebsiella pneumoniae. Several other extracts also showed antibacterial activity against pathogenic strains of E. coli, Proteus spp. and Pseudomonas spp. Minimum bactericidal concentration (MBC) values of potential extracts were found between 3.5 and 28.40 mg/mL. The lowest MBC (3.5 mg/mL) was recorded for ethanol extract against Pseudomonas spp. The antioxidant activity of the extracts was compared with standard antioxidants. Dose dependent response was observed in reducing power of extracts. Polar extracts demonstrated appreciable metal ion chelating activity at lower concentrations (10-40 µg/mL). Many extracts showed significant antioxidant response in beta carotene bleaching assay. AQ fraction of B. variegata showed pronounced cytotoxic effect against DU-145, HOP-62, IGR-OV-1, MCF-7, and THP-1 human cancer cell lines with 90-99% cell growth inhibitory activity. Ethyl acetate fraction also produced considerable cytotoxicity against MCF-7 and THP-1 cell lines. The study demonstrates notable antibacterial, antioxidant, and anticancer activities in B. variegata leaf extracts.

Thiourea and guanidine derivatives as antimalarial and antimicrobial agents.

Thiourea and guanidine substructural units are of significant importance as the compounds containing these core units either in the open or the cyclic form are known to display an array of pharmacological properties. This brief review assimilates the literature on the medicinal significance of thiourea and guanidine derivatives with respect to antimalarial and antimicrobial activities.

Antioxidant mediated protective effect of Parthenium hysterophorus against oxidative damage using in vitro models.

BACKGROUND: Parthenium hysterophorus L. (Asteraceae) is a common weed occurring throughout the globe. In traditional medicine its decoction has been used for treatment of many infectious and degenerative diseases. This work was therefore designed to assess the phytochemical constitution of P. hysterophorus flower and root extracts and to evaluate their reducing power, radical scavenging activity as well as protective efficacy against membrane lipid damage. METHODS: Dried flower and root samples were sequentially extracted with non-polar and polar solvents using Soxhlet apparatus. The phytochemical screening was done using standard chemical methods and thin layer chromatography. Total phenolic content was determined spectrophotometrically. Reducing power and hydroxyl radical scavenging activity assays were used to measure antioxidant activity. Protection against membrane damage was evaluated by inhibition of lipid peroxidation (TBARS assay) in rat kidney homogenate. RESULTS: Flavonoids, terpenoids, alkaloids and cardiac glycosides were present in all the extract. The total phenol contents in flower and root extracts were found to be in the range 86.69-320.17 mg propyl gallate equivalent (PGE)/g and 55.47-253.84 mg PGE/g, respectively. Comparatively better reducing power was observed in hexane fractions of flower (0.405) and root (0.282). Benzene extract of flower and ethyl acetate fraction of root accounted for appreciable hydroxyl radical scavenging activity (75-77%). Maximum protection against membrane lipid peroxidative damage among flower and root extracts was provided by ethanol (55.26%) and ethyl acetate (48.95%) fractions, respectively. Total phenolic content showed positive correlations with reducing power and lipid peroxidation inhibition (LPOI) % in floral extracts as well as with hydroxyl radical scavenging activity and LPOI % in root extracts. CONCLUSION: Study established that phytochemicals present in P. hysterophorus extracts have considerable antioxidant potential as well as lipo-protective activity against membrane damage.

Association properties and unfolding of a ßγ-crystallin domain of a Vibrio-specific protein.

The ßγ-crystallin superfamily possesses a large number of versatile members, of which only a few members other than lens ßγ-crystallins have been studied. Understanding the non-crystallin functions as well as origin of crystallin-like properties of such proteins is possible by exploring novel members from diverse sources. We describe a novel ßγ-crystallin domain with S-type (Spherulin 3a type) Greek key motifs in protein vibrillin from a pathogenic bacterium Vibrio cholerae. This domain is a part of a large Vibrio-specific protein prevalent in Vibrio species (found in at least fourteen different strains sequenced so far). The domain contains two canonical N/D-N/D-X-X-S/T-S Ca(2+)-binding motifs, and bind Ca(2+). Unlike spherulin 3a and other microbial homologues studied so far, ßγ-crystallin domain of vibrillin self-associates forming oligomers of various sizes including dimers. The fractionated dimers readily form octamers in concentration-dependent manner, suggesting an association between these two major forms. The domain associates/dissociates forming dimers at the cost of monomeric populations in the presence of Ca(2+). No such effect of Ca(2+) has been observed in oligomeric species. The equilibrium unfolding of both forms follows a similar pattern, with the formation of an unfolding intermediate at sub-molar concentrations of denaturant. These properties exhibited by this ßγ-crystallin domain are not shown by any other domain studied so far, demonstrating the diversity in domain properties.

Scientific validation of the medicinal efficacy of Tinospora cordifolia.

Present communication reports the scientific evaluation of Tinospora cordifolia for its medicinal efficacy which includes phytochemical screening, antimicrobial, antioxidant, and anticancer activities of the plant. Secondary metabolites including anthraquinones, terpenoids, and saponins were present in many extracts in addition to phenolics. Total phenol contents in various extracts were found in the range of 8.75-52.50 catechol equivalent per gram (CE/g). In disc diffusion assays, polar extracts exhibited considerable inhibition against Klebsiella pneumoniae. Several other extracts also showed antibacterial activity against pathogenic strains of E. coli, Pseudomonas spp., and Proteus spp. Minimum bactericidal concentration (MBC) values of potential extracts were found between 1.29 and 22.73 mg/mL. The lowest MBC (1.29 mg/mL) was recorded for acetone and ethyl acetate extracts against K. pneumoniae and Pseudomonas spp., respectively. The antioxidant activity of the extracts was comparable to that of standard antioxidants and concentration-dependent response was shown in reducing power assay. Aqueous extracts demonstrated substantial metal ion chelating activity (67-95%) at lower concentrations (10-40 µ g/mL). Other extracts also exhibited considerable metal chelating response. Most of the extracts revealed considerable inhibition of MCF-7 cancer cell line. The study established remarkable antibacterial, antioxidant, and anticancer potential in T. cordifolia stem extracts.

Decoding the molecular design principles underlying Ca(2+) binding to ßγ-crystallin motifs.

Numerous proteins belonging to the recently expanded ßγ-crystallin superfamily bind Ca(2+) at the double-clamp N/D-N/D-X(1)-X(2)-S/T-S motif. However, there have been no attempts to understand the intricacies involving Ca(2+) binding, such as the determinants of Ca(2+)-binding affinity and their contributions to gain in stability. This work is an in-depth analysis of understanding the modes and determinants of Ca(2+) binding to ßγ-crystallin motifs. We have performed extensive naturally occurring substitutions from related proteins on the ßγ-crystallin domains of flavollin, a low-affinity Ca(2+)-binding protein, and clostrillin, a moderate-affinity protein. We monitored the consequences of these modifications on Ca(2)(+) binding by isothermal titration calorimetry, thermal stability and conformational and crystal structure analyses. We demonstrate that Ca(2)(+) binding to the two sites of a ßγ-domain is interdependent and that the presence of Arg at the fifth position disables a site. A change from Thr to Ser, or vice versa, influences Ca(2+)-binding affinity, highlighting the basis of diversity found in these domains. A subtle change in the first site has a greater influence on Ca(2)(+) binding than a similar alteration in the second site. Thus, the second site is more variable in nature. Replacing an acidic or hydrophobic residue in a binding site alters the Ca(2+)-binding properties drastically. While it appears from their binding site sequence that these domains have evolved randomly, our examination illustrates the subtlety in the design of these modules. Decoding such design schemes would aid in our understanding of the functional themes underlying differential Ca(2)(+) binding and in predicting these in emerging sequence information.

Evolutionary remodeling of ßγ-crystallins for domain stability at cost of Ca2+ binding.

The topologically similar ßγ-crystallins that are prevalent in all kingdoms of life have evolved for high innate domain stability to perform their specialized functions. The evolution of stability and its control in ßγ-crystallins that possess either a canonical (mostly from microorganisms) or degenerate (principally found in vertebrate homologues) Ca2+-binding motif is not known. Using equilibrium unfolding of ßγ-crystallin domains (26 wild-type domains and their mutants) in apo- and holo-forms, we demonstrate the presence of a stability gradient across these members, which is attained by the choice of residues in the (N/D)(N/D)XX(S/T)S Ca2+-binding motif. The occurrence of a polar, hydrophobic, or Ser residue at the 1st, 3rd, or 5th position of the motif is likely linked to a higher domain stability. Partial conversion of a microbe-type domain (with a canonical Ca2+-binding motif) to a vertebrate-type domain (with a degenerate Ca2+-binding motif) by mutating serine to arginine/lysine disables the Ca2+-binding but significantly augments its stability. Conversely, stability is compromised when arginine (in a vertebrate-type disabled domain) is replaced by serine (as a microbe type). Our results suggest that such conversions were acquired as a strategy for desired stability in vertebrate members at the cost of Ca2+-binding. In a physiological context, we demonstrate that a mutation such as an arginine to serine (R77S) mutation in this motif of γ-crystallin (partial conversion to microbe-type), implicated in cataracts, decreases the domain stability. Thus, this motif acts as a "central tuning knob" for innate as well as Ca2+-induced gain in stability, incorporating a stability gradient across ßγ-crystallin members critical for their specialized functions.

The betagamma-crystallin superfamily contains a universal motif for binding calcium.

The betagamma-crystallin superfamily consists of evolutionarily related proteins with domain topology similar to lens beta- and gamma-crystallins, formed from duplicated Greek key motifs. Ca(2+) binding was found in a few betagamma-crystallin members earlier, although its prevalence and diversity as inherent molecular properties among members of the superfamily are not well studied. To increase our understanding of Ca(2+) binding in various betagamma-crystallins, we undertook comprehensive structural and Ca(2+)-binding studies of seven members of the superfamily from bacteria, archaea, and vertebrates, including determination of high-resolution crystal structures of three proteins. Our structural observations show that the determinants of Ca(2+) coordination remain conserved in the form of an N/D-N/D-#-I-S/T-S motif in all domains. However, binding of Ca(2+) elicits varied physicochemical responses, ranging from passive sequestration to active stabilization. The motif in this superfamily is modified in some members like lens crystallins where Ca(2+)-binding abilities are partly or completely compromised. We show that reduction or loss of Ca(2+) binding in members of the superfamily, particularly in vertebrates, is due to the selective presence of unfavorable amino acids (largely Arg) at key Ca(2+)-ligation positions and that engineering of the canonical Ca(2+)-binding residues can confer binding activity on an otherwise inactive domain. Through this work, we demonstrate that betagamma-crystallins with the N/D-N/D-#-I-S/T-S motif form an extensive set of Ca(2+)-binding proteins prevalent in all of the three kingdoms of life.

Inhibitory activity of Indian spice plant Cinnamomum zeylanicum extracts against Alternaria solani and Curvularia lunata, the pathogenic dematiaceous moulds.

BACKGROUND: Dematiaceous moulds are pathogenic microorganisms and act as etiological agents of mycoses with different degrees of severity in humans and animals. These moulds also cause loss of food crops and storage food products. The information regarding antimicrobial efficacy of the plant preparations on these moulds is scanty. The present study reveals phytochemical characterization and the effect of bark and leaf extracts of Indian spice plant, Cinnamomum zeylanicum (Cz), against the growth of two species of dematiaceous moulds, Alternaria solani and Curvularia lunata. METHODS: Cz bark and leaf samples were sequentially extracted in different solvents using Soxhlet apparatus. Phytochemical analyses of extracts were done as per standard protocols. The antifungal bioassay of extracts was done by hanging drop technique. The inhibition of fungal spore germination was monitored under influence of three different concentrations of extracts. RESULTS: The lowest test concentration (50 microg/ml) of extracts of Cz bark prepared into acetone and that of Cz leaf into petroleum ether and ethanol exhibited complete inhibition (100%) of spore germination in both the moulds. At 100 microg/ml concentration all the extracts showed about 50 to 100% inhibition. However, the treatment of the spores of the two fungal species with highest concentration (500 microg/ml) of bark and leaf extracts in all the solvents showed 100% fungicidal activity as it completely arrested the germination of spores. Relatively lower activity of aqueous extracts at 50 and 100 microg/ml concentrations suggests that the antifungal ingredients present in Cz bark and leaf are more soluble in organic solvents than water. CONCLUSION: The results demonstrated that the Cz bark and leaves contain certain fungicidal constituents exhibiting potential antimould activity against A. solani and C. lunata.

Three-dimensional domain swapping in nitrollin, a single-domain betagamma-crystallin from Nitrosospira multiformis, controls protein conformation and stability but not dimerization.

The betagamma-crystallin superfamily has a well-characterized protein fold, with several members found in both prokaryotic and eukaryotic worlds. A majority of them contain two betagamma-crystallin domains. A few examples, such as ciona crystallin and spherulin 3a exist that represent the eukaryotic single-domain proteins of this superfamily. This study reports the high-resolution crystal structure of a single-domain betagamma-crystallin protein, nitrollin, from the ammonium-oxidizing soil bacterium Nitrosospira multiformis. The structure retains the characteristic betagamma-crystallin fold despite a very low sequence identity. The protein exhibits a unique case of homodimerization in betagamma-crystallins by employing its N-terminal extension to undergo three-dimensional (3D) domain swapping with its partner. Removal of the swapped strand results in partial loss of structure and stability but not dimerization per se as determined using gel filtration and equilibrium unfolding studies. Overall, nitrollin represents a distinct single-domain prokaryotic member that has evolved a specialized mode of dimerization hitherto unknown in the realm of betagamma-crystallins.

Ethylene induced cotton leaf abscission is associated with higher expression of cellulase (GhCel1) and increased activities of ethylene biosynthesis enzymes in abscission zone.

Ethylene induced cotton (Gossypium hirsutum var RST-39) leaf abscission has been characterized by measuring the activities of ACC synthase (ACS, E.C. 4.4.1.14), ACC oxidase (ACO, E.C. 1.14.17.4) and cellulase (E.C. 3.2.1.4). In addition, a leaf abscission specific cDNA (GhCel1) has been cloned from cotton, which belongs to the alpha(2) subgroup of cellulases that possess a C-terminus carbohydrate-binding domain. Measurement of enzyme activity in the abscission zones of cotton leaf explants exposed to ethylene for 48h compared to non-treated controls indicated a more than 5-fold increase in the activity of ACS, 1.2-fold increase in the activity of ACO and about 2.7-fold increase in the activity of cellulase in the ethylene treated explants. This increase was accompanied by a substantial decrease in the force required to separate the petiole from the stem (break strength) and an increased accumulation of cellulase transcript in the abscission zone. Treatment of explants with 1-Methylcyclopropene (1-MCP) prior to ethylene resulted in significant inhibition of enzyme activities and transcript accumulation. It is concluded that ethylene response of cotton leaf abscission leads to higher cellulase expression and increased activities of ethylene biosynthesis enzymes in the abscission zone.