Mechanistic Investigation on the Antibacterial Activity of Biogenic Silver Nanoparticles Prepared Using Root Extract of Sarsaparilla and Demonstrated their In Vivo Efficacy in Zebrafish Model.

Antibiotic success rates are decreasing as drug-resistant bacteria become more prevalent, prompting the development of new therapeutic drugs. Herein, we demonstrated the antimicrobial activity of sarsaparilla root extract fabricated silver nanoparticles (sAgNPs). The UV-Visible spectra revealed that the surface Plasmon resonance maxima of sAgNPs were at 415 nm. Transmission electron microscopy confirms that the particles are spherical with size of 12-35 nm. The minimum inhibitory concentration (MIC) of sAgNPs against Escherichia coli, uropathogenic Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus was 62.5, 62.5, 62.5, 62.5, 125 and 125 µM, respectively. At 1X MIC, sAgNPs induces excess reactive oxygen species (ROS) production and disturbs the bacteria membrane intergity, causing cytoplamic membrane depolarization. Interestingly, antibacterial activity of sAgNPs was considerably reduced in the presence of an antioxidant, N-acetyl cysteine, suggesting that ROS-induced membrane damage is a plausible cause of cell death. In contrast to many studies that only report the in vitro activity of NPs, we determined the in vivo antibacterial efficacy using the zebrafish model. It was found that sAgNPs protect fish from infection by inhibiting bacterial growth and eliminating them from the fish. In addition, the catalytic potential of sAgNPs for wastewater decontamination was demonstrated by degrading organic pollutants such as methyl orange, congo red, reactive black, and acid blue. The pollutants degraded in less than 10 min, and the reaction follows pseudo-first-order kinetics. As a proof of concept, the catalytic potential of sAgNPs in degrading mixed dyes to satisfy industrial wastewater treatment needs was established. In summary, sAgNPs have the potential to act as nanocatalysts and nano-drugs, addressing key challenges in medical and environmental research.

Activity of lipid-loaded lectin against co-infection of Candida albicans and Staphylococcus aureus using the zebrafish model.

AIM: Nosocomial infection caused by mixed species of methicillin-resistant Staphylococcus aureus (MRSA) and Candida albicans (CA) is difficult to manage with existing antimicrobials, particularly in the presence of mixed-species biofilm. This study evaluates the activity of cationic lipid, specifically functionalized with lectin, against mixed biofilms of MRSA and CA and their effectiveness in vivo using the zebrafish model. METHODS AND RESULTS: The present study demonstrates for the first time the antimicrobial activity of 2-((N-[2-hydroxyethyl]palmitamido)methyl)-1-methylpyridin-1-ium iodide (cN16E) against MRSA and mixed species of MRSA + CA. The cN16E functionalized with Butea monosperma seed lectin (BMSL) showed a lower minimum inhibitory concentration (MIC) as compared with cN16E. BMSL-cN16E (BcN16E) exhibited strong membrane-damaging activity at a lower concentration than cN16E. Crystal violet assay showed that BcN16E inhibits mixed-species biofilm at the concentration of 15.63 µM, which is four-fold lower than the MIC. Especially, BcN16E was found to be effective in disturbing mature mixed biofilm at 31.25 µM, which is two-fold lower than the MIC, suggesting true antibiofilm activity without pressurizing the microorganisms. The treatment with BcN16E significantly reduced the exopolysaccharide synthesis (> 78%), cell surface hydrophobicity (> 70 %), hyphae formation, staphyloxanthin biosynthesis (> 41 %), and antioxidant enzyme and hemolysin activity (> 70 %). Notably, BcN16E was efficient in reducing the in vivo colonization of bacterial and fungal burden in the blood and muscle tissues of zebrafish. CONCLUSION: Antimicrobial and antibiofilm efficacy of BcN16E against MRSA, and mixed species of MRSA + CA were demonstrated. Importantly, BcN16E treatment rescued Zebrafish coinfected with mixed species of MRSA + CA. Significance and Impact of the study: The results highlight that antimicrobial loaded on lectin provides an additional advantage to recognize microorganism surface glycans and maximize drug delivery to treat polymicrobial infections caused by MRSA and CA.

Revealing the Significance of the Glycan Binding Property of Butea monosperma Seed Lectin for Enhancing the Antibiofilm Activity of Silver Nanoparticles against Uropathogenic Escherichia coli.

The incompetence of conventional antibiotics against bacteria residing in biofilms demands newer therapeutic intervention. In this study, we demonstrated that the interaction between silver nanoparticles (AgNPs) and Butea monosperma seed lectin (BMSL) forms efficient surface-functionalized AgNPs with excellent antibiofilm competency against uropathogenic Escherichia coli (UPEC). The minimum biofilm inhibitory concentration (MBIC) of AgNPs and the BMSL-AgNP conjugate (BAgNP) against UPEC was 75 and 9.37 µM, respectively. The eight-fold reduction in the MBIC of AgNPs was attributed to lectin functionalization. The chemical modification of serine amino acids affects the hemagglutination activity of BMSL but not its interaction with the AgNPs. At the same time, AgNPs surface-functionalized with modified BMSL display poor antibiofilm activity. Molecular docking studies revealed that BMSL binds to galactose with a free energy of -5.72 kcal/mol, whereas the serine residue-modified BMSL showed the lowest free energy values, suggesting incompetence for binding galactose. These results showcase that the sugar binding site of BMSL aids in the adhesion of AgNPs to the biofilm matrix and disturbs the formation of the biofilm, which was confirmed by light microscopy using crystal violet staining. At 37.5 µM, BAgNPs also have the capability to eradicate preformed biofilm. As a proof of concept, UPEC biofilm prevention and eradication were demonstrated on a urinary catheter. A scanning electron microscopy study showed that BAgNPs prevent bacterial colonization and thereby curtail biofilm growth. In addition to antibiofilm activity, BAgNPs exert antibacterial activity at 18.75 µM, which is four-fold lower than the MIC of AgNPs. A mechanistic study revealed that BAgNPs affect the integrity of the bacterial outer membrane and generate an imbalance in the antioxidant defense, which induces cell death. The results highlight that lectin functionalization can be extended to other nanoparticles and different antibiotics to enhance their efficacy against drug-resistant bacteria.

Nitrated Fatty Acids Modulate the Physical Properties of Model Membranes and the Structure of Transmembrane Proteins.

Nitrated fatty acids (NO2 -FAs) act as anti-inflammatory signal mediators, albeit the molecular mechanisms behind NO2 -FAs' influence on diverse metabolic and signaling pathways in inflamed tissues are essentially elusive. Here, we combine fluorescence measurements with surface-specific sum frequency generation vibrational spectroscopy and coarse-grained computer simulations to demonstrate that NO2 -FAs alter lipid organization by accumulation at the membrane-water interface. As the function of membrane proteins strongly depends on both, protein structure as well as membrane properties, we consecutively follow the structural dynamics of an integral membrane protein in presence of NO2 -FAs. Based on our results, we suggest a molecular mechanism of the NO2 -FA in vivo activity: Driven by the NO2 -FA-induced lipid layer reorganization, the structure and function of membrane-associated (signaling) proteins is indirectly affected.

C-H oxidation and chelation of a dipyrromethane mediated rapid colorimetric naked-eye Cu(ii) chemosensor.

Copper(ii) ion mediated C-H oxidation of dipyrromethanes (DPMs) to the corresponding dipyrrins followed by complexation invoked the selective sensing of copper(ii) ions in aqueous solutions. On the addition of copper, the colour of the DPM solution instantaneously changes from yellow to pink with the detection limit of 0.104 µM measured by absorption spectroscopy, whereas visible colour changes could be observed by the naked eye for concentrations as low as 3 µM.

Correction: Platinum nanoparticles inhibit bacteria proliferation and rescue zebrafish from bacterial infection.

[This corrects the article DOI: 10.1039/C6RA03732A.].

Hybrid of Metapenaeus dobsoni lectin and platinum nanoparticles exert antimicrobial and immunostimulatory effects to reduce bacterial bioburden in infected Nile tilapia.

A novel antibacterial immunostimulant using Platinum nanoparticles (PtNPs) and lectin from Metapenaeus dobsoni (Md-Lec) was developed. The Md-Lec and PtNPs (Pt-lec) hybrid formed through non-covalent interaction exhibits antimicrobial activity against fish specific pathogens by affecting membrane integrity and producing excess reactive oxygen species. The therapeutic efficacy of Pt-lec was demonstrated through rescuing Aeromonas hydrophila infected Nile Tilapia. Pt-lec prevents the infection spreading and reduces the bacterial bioburden in less than 12 h, and as a result of this the fish were restored to normalcy. To assess immunostimulation, we studied the expression of three different immune related genes, namely LEC, Myd88 and COX-2 in the gills, liver, spleen and kidney of fish under various experimental conditions. Our results showed that Pt-lec treatment appeared to be better when compared to lectin alone in enhancing the expression of Myd88 and COX-2, but LEC was not as expected. These results suggest that Pt-lec has the ability to protect Nile Tilapia against bacterial infection by restricting bacterial bioburden through their direct effects on the bacterial membrane and indirectly through their effects on host immune-related gene expression. This hybrid could have potential "green" application in fish farming in rescuing infected animals when compared to widely and unregulated antibiotics.

Heme binding constricts the conformational dynamics of the cytochrome b(559)' heme binding cavity.

Cytochrome b(559)' is a transmembrane protein formed by homodimerization of the 44-residue PsbF polypeptide and noncovalent binding of a heme cofactor. The PsbF polypeptide can dimerize in the absence and presence of heme. To monitor structural alterations associated with binding of heme to the apo-cytochrome, we analyzed the apo- and holo-cytochrome structure by electron paramagnetic resonance spectroscopy. Spin labeling of amino acids located close to the heme binding domain of the cytochrome revealed that the structure of the heme binding domain is unconstrained in the absence of heme. Heme binding restricts the conformational dynamics of the heme binding domain, resulting in the structurally more constricted holo-cytochrome structure.

The membrane environment modulates self-association of the human GpA TM domain--implications for membrane protein folding and transmembrane signaling.

The influence of lipid bilayer properties on a defined and sequence-specific transmembrane helix-helix interaction is not well characterized yet. To study the potential impact of changing bilayer properties on a sequence-specific transmembrane helix-helix interaction, we have traced the association of fluorescent-labeled glycophorin A transmembrane peptides by fluorescence spectroscopy in model membranes with varying lipid compositions. The observed changes of the glycophorin A dimerization propensities in different lipid bilayers suggest that the lipid bilayer thickness severely influences the monomer-dimer equilibrium of this transmembrane domain, and dimerization was most efficient under hydrophobic matching conditions. Moreover, cholesterol considerably promotes self-association of transmembrane helices in model membranes by affecting the lipid acyl chain ordering. In general, the order of the lipid acyl chains appears to be an important factor involved in determining the strength and stability of transmembrane helix-helix interactions. As discussed, the described influences of membrane properties on transmembrane helix-helix interactions are highly important for understanding the mechanism of transmembrane protein folding and functioning as well as for gaining a deeper insight into the regulation of signal transduction via membrane integral proteins by bilayer properties.

31P NMR and AFM studies on the destabilization of cell and model membranes by the major bovine seminal plasma protein, PDC-109.

The effect of PDC-109 binding to dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylglycerol (DPPG) multilamellar vesicles (MLVs) and supported membranes was investigated by (31)P NMR spectroscopy and atomic force microscopy. Additionally, the effect of cholesterol on the binding of PDC-109 to phosphatidylcholine (PC) membranes was studied. Binding of PDC-109 to MLVs of DMPC and DPPG induced the formation of an isotropic signal in their (31)P NMR spectra, which increased with increasing protein/lipid ratio and temperature, consistent with protein-induced disruption of the MLVs and the formation of small unilamellar vesicles or micelles but not inverse hexagonal or cubic phases. Incorporation of cholesterol in the DMPC MLVs afforded a partial stabilization of the lamellar structure, consistent with previous reports of membrane stabilization by cholesterol. AFM results are consistent with the above findings and show that addition of PDC-109 leads to a complete breakdown of PC membranes. The fraction of isotropic signal in (31)P NMR spectra of DPPG in the presence of PDC-109 was less than that of DMPC under similar conditions, suggesting a significantly higher affinity of the protein for PC. Confocal microscopic studies showed that addition of PDC-109 to human erythrocytes results in a disruption of the plasma membrane and release of hemoglobin into the solution, which was dependent on the protein concentration and incubation time.

Unfolding a transmembrane helix dimer: A FRET study in mixed micelles.

The exact nature of membrane protein folding and assembly is not understood in detail yet. Addition of SDS to a membrane protein dissolved in mild, non-polar detergent results in formation of mixed micelles and in subsequent denaturation of higher ordered membrane protein structures. The exact nature of this denaturation event is, however, enigmatic, and separation of an individual helix pair in mixed micelles has also not been reported yet. Here we followed unfolding of the human glycophorin A transmembrane helix dimer in mixed micelles by fluorescence spectroscopy. Energy transfer between differently labelled glycophorin A transmembrane helices decreased with increasing SDS mole fractions albeit without modifying the helicity of the peptides. The energetics and kinetics of the dimer dissociation in mixed micelles is analyzed and discussed, and the experimental data demonstrate that mixed micelles can be used as a general method to investigate unfolding of alpha-helical membrane proteins.

Targeting the glycan of receptor binding domain with jacalin as a novel approach to develop a treatment against COVID-19.

In silico analysis revealed that a lectin, jacalin from jackfruit seeds, recognizes a glycosylated region of the receptor-binding domain (RBD) of SARS-CoV2. Jacalin binding induces conformational changes in RBD and significantly affects its interaction with human angiotensin-converting enzyme 2. The result may open up exploration of lectin-based strategies against COVID-19.

Maximizing the direct recovery and stabilization of cellulolytic enzymes from Trichoderma harzanium BPGF1 fermented broth using carboxymethyl inulin nanoparticles.

The carboxymethylated inulin (CMI) nanoparticles prepared by the salt out method was demonstrated to harvest cellulolytic enzymes (Ez) directly from the clarified fermented broth of Trichoderma harzanium BPGF1. The formation of CMI nanoparticles and entrapment of Ez in CMI was confirmed by scanning electron microscopy and Fourier transform infrared spectroscopy, respectively. A factorial design was developed to maximize enzymes recovery directly from the fermented broth. A maximum of 71.68 ± 8.61% cellulolytic enzymes was recovered using 20 mg/L inulin, 2 M sodium chloroacetate at 80 °C for 2 h. The resultant CMIEz nanohybrid displayed excellent activity in broad pH and temperature. Moreover, CMIEz was reusable for >30 cycles without losing efficiency. The real-time application of CMIEz was demonstrated by hydrolyzing acid pretreated corncob. High-pressure liquid chromatography revealed that the hydrolyzed corncob contained cellobiose, glucose, galactose, xylose, mannose, and arabinose. The results highlight that carbohydrate nanoparticles was useful in engulfing enzymes directly from the fermentation broth.

A residue-level investigation of the equilibrium unfolding of the C2A domain of synaptotagmin 1.

Fibroblast growth factor (FGF) 1 is known to be released in response to cellular stress conditions through formation of a multi-protein complex with synaptotagmin 1 and S100A13. In this study, we characterized the denaturant-induced unfolding of synaptotagmin 1, C2A domain in a residue-specific manner by NMR spectroscopy. The amide protons of 30 residues distributed throughout the 3D structure of the whole protein could be followed in a series of (1)H-(15)N HSQC spectra recorded from 0 to 8 M urea under equilibrium conditions. The midpoint for the urea-induced unfolding obtained from NMR coincides with those obtained from steady state fluorescence and CD spectroscopy, revealing that the protein unfolds via a two-state mechanism without accumulating stable intermediates. The thermodynamic parameter obtained from the denaturation curve illustrates the cooperative unfolding of the C2A domain. The implications of C2A domain folding in relation to the release of FGF-1 from the multi-protein complex were discussed.

Simple admixture of 4-nitrobenzaldehyde and 2,4-dimethylpyrrole for efficient colorimetric sensing of copper(II) ions.

An easily accessible chemo-probe based on physical mixture of 2,4-dimethylpyrrole and 4-nitrobenzaldehyde has been developed. Based on NMR spectroscopic analysis, catalyst free formation of dipyrromethane was observed in the physical mixture of chemo-probe. The probe is utilized in effective colorimetric sensing of copper(II) ions present in environmental solutions by instantaneous appearance of red colour, even in the co-existence of various metal ions. The lowest detection limit of 2.51 µM for this chemo-probe towards copper(II) sensing is significantly lower than the WHO prescribed level (<30 µM of copper(II) ions) in potable water. The sensing mechanism is explained via rapid formation of bis(dipyrrinato)copper(II) complex, as confirmed by Jobs plot, UV-Vis spectroscopy and IR spectroscopy.

Remarkable Effect of Jacalin in Diminishing the Protein Corona Interference in the Antibacterial Activity of Pectin-Capped Copper Sulfide Nanoparticles.

Herein, we report a new strategy based on jacalin functionalization to diminish the impact of biological fluids in the antibacterial applications of nanoparticles (NPs). Precoating pectin-capped copper sulfide NPs (pCuS) with bovine serum albumin produced a protein corona, which affects the antibacterial activity of pCuS. It was found that the minimum inhibitory concentration (MIC) increases fourfold because of the formation of the protein corona. Interestingly, the pCuS functionalized with jacalin enhance the targeting capabilities through bacterial cell surface glycan recognition with no interference from the protein corona. The MIC of pCuS decreases 16-fold on functionalization with jacalin. Mechanistic studies indicated that the pCuS functionalized with jacalin impede the protein corona interference and induce bacterial cell death by impairing the GSH/reactive oxygen species balance and disrupting the bacteria cell membrane. As a proof of concept, we used a bacteria-infected zebrafish animal model to demonstrate the interference of biological fluids in the antibacterial activity of NPs. Infected zebrafish treated with 1× MIC of pCuS failed to recover from the infection, but 4× MIC rescues the fish. The requirement of a high dose of NPs to treat the infection confirms the interference of biological fluids in nanotherapeutic applications. At the same time, the jacalin-pCuS complex rescues the infected fish at 16-fold lesser MIC. The results obtained from this study suggest that jacalin-mediated NP targeting may have broad implications in the development of future nanomedicine.

Evaluation of the toxicities of silver and silver sulfide nanoparticles against Gram-positive and Gram-negative bacteria.

In this study, the endogenous lipid signalling molecules, N-myristoylethanolamine, were explored as a capping agent to synthesise stable silver nanoparticles (AgNPs) and Ag sulphide NPs (Ag2S NPs). Sulphidation of the AgNPs abolishes the surface plasmon resonance (SPR) maximum of AgNPs at 415 nm with concomitant changes in the SPR, indicating the formation of Ag2S NPs. Transmission electron microscopy revealed that the AgNPs and Ag2S NPs are spherical in shape with a size of 5-30 and 8-30 nm, respectively. AgNPs and Ag2S NPs exhibit antimicrobial activity against Gram-positive and Gram-negative bacteria. The minimum inhibitory concentrations (MIC) of 25 and 50 µM for AgNPs and Ag2S NPs, respectively, were determined from resazurin microtitre plate assay. At or above MIC, both AgNPs and Ag2S NPs decrease the cell viability through the mechanism of membrane damage and generation of excess reactive oxygen species.

Preparation of self-assembled platinum nanoclusters to combat Salmonella typhi infection and inhibit biofilm formation.

In this work, phytoprotein functionalized platinum nanoparticles (PtNCs) were synthesized using the proteins from fresh green spinach leaves. Transmission electron microscopy showed that PtNCs were spherical shape with size ∼5 nm, which self assembled into spherical platinum nanoclustures (PtNCs) with size within the range of 100-250 nm. The presence of elemental platinum was confirmed by EDX analysis. FTIR studies confirm that the PtNCs were stabilized by the protein. As prepared PtNCs inhibits the growth of the food borne pathogen, Salmonella typhi with minimum inhibitory concentration (MIC) of 12.5 µM. Light microscopy evidenced that the PtNCs can damage the established biofilms. Antibacterial mechanistic study revealed that PtNCs damages the S. typhi membranes, which was confirmed by scanning electron microscopy and further by fluorescence microscopy using acridine orange/propidium iodide dual staining assay. Besides membrane damage, PtNCs also triggered the intracellular ROS-mediated oxidative damage over the antioxidant defense and kills S. typhi. The hemolytic test showed low cytotoxicity of PtNCs at 100 µM (four times higher the MIC). Finally, the therapeutic efficacy of PtNCs was validated in S. typhi infected zebrafish animal model and the obtained results are discussed.

Facile synthesis of gold nanoparticles using carbon dots for electrochemical detection of neurotransmitter, dopamine in human serum and as a chemocatalyst for nitroaromatic reduction.

Herein, the authors reported a carbon dots mediated synthesis of gold nanoparticles (AuNPs) at room temperature. Transmission electron microscopy revealed that the AuNPs are spherical in shape with a size of 10 nm. As-prepared AuNPs was immobilised on carbon paste electrode and subjected to electrochemical sensing of an important neurotransmitter dopamine. Differential pulse voltammetry studies revealed sensitive and selective determination of dopamine in the presence of commonly interfering ascorbic acid and uric acid. The linear detection range was 10-600 µM and the limit of detection was 0.7 ± 0.18 µM. The practical application was demonstrated by measuring dopamine in human blood serum and urine samples. The catalytic activity of AuNPs was evaluated by sodium borohydride mediated reduction of nitroaromatic compounds. The reduction kinetics was found to be pseudo-first-order kinetics. All the tested nitroaromatics reduced to corresponding amines in <10 min.

Immobilization of levan-xylanase nanohybrid on an alginate bead improves xylanase stability at wide pH and temperature.

Despite the sustainable availability, levan, a fructose based natural polysaccharide has not received significant attention in the development of enzyme immobilization technology. Herein, we prepared levan-xylanase (LXy) nanohybrid and characterized by scanning electron microscopy, particle size analyzer and zeta potential. To prevent the enzyme leakage from the nanohybrid, LXy was immobilized onto an alginate beads (NaAlg). Immobilization yield was optimized using a statistical method, central composite design. A maximum immobilization yield of 95.3% was achieved at 2.13% (w/v) of sodium alginate, 2.14% (w/v) of calcium chloride, 64min of curation time and 1.4mm bead size. Immobilized LXy retains nearly 80% of the enzyme activity at a wide range of temperature (20-90°C) and pH (3-10). Immobilization of LXy onto NaAlg increases the activation energy from 28.50Jmol-1K-1 to 39.38Jmol-1K-1. Collectively, this result implies that LXy immobilized onto NaAlg increases the enzyme stability and retains its activity.