Multiepitope glycan based laser assisted fluorescent nanocomposite with dual functionality for sensing and ablation of Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) infection is becoming a severe health hazard and needs early diagnosis with high specificity. However, the non-specific binding of a biosensor is a challenge to the current bacterial detection system. For the first time, we chemically synthesized a galactose tripod (GT) as a P. aeruginosa-specific ligand. We conjugated GT to a photothermally active fluorescent nanocomposite (Au@SiO2-TCPP). P. aeruginosa can be detected using Au@SiO2-TCPP-GT, and additionally ablated as well using synergistic photothermal and photodynamic therapy. Molecular dynamics and simulation studies suggested better binding of GT (binding energy = -6.6 kcal mol-1) with P. aeruginosa lectin than that of galactose monopod (GM) (binding energy = -5.9 kcal mol-1). Furthermore, a binding study was extended to target P. aeruginosa, which has a galactose-binding carbohydrate recognition domain receptor. The colorimetric assay confirmed a limit of detection (LOD) of 104 CFU mL-1. We also looked into the photosensitizing property of Au@SiO2-TCPP-GT, which is stimulated by laser light (630 nm) and causes photoablation of bacteria by the formation of singlet oxygen in the surrounding media. The cytocompatibility of Au@SiO2-TCPP-GT was confirmed using cytotoxicity assays on mammalian cell lines. Moreover, Au@SiO2-TCPP-GT also showed non-hemolytic activity. Considering the toxicity analysis and efficacy of the synthesized glycan nanocomposites, these can be utilized for the treatment of P. aeruginosa-infected wounds. Furthermore, the current glycan nanocomposites can be used for bacterial detection and ablation of P. aeruginosa in contaminated food and water samples as well.

Immobilization of α-transglucosidase on silica-coated magnetic nanoparticles and its application for production of isomaltooligosaccharide from the potato peel.

In this study, the production of isomaltooligosaccharide from potato peel starch was carried out in three steps: liquefaction, saccharification, and transglucosylation. Further, cloning α-transglucosidase gene from Aspergillus niger (GH31 family), transforming into E. coli BL21 (DE3), overexpressing and purifying the resulting protein for the production of α-transglucosidase. The generated α-transglucosidase was then bound with magnetic nanoparticles, which improved reusability up to 5 cycles with more than 60% activity. All the modifications were characterized using the following methods: Fourier transform infra-red analysis, Transmission Electron Microscopy, Field Emission Scanning Electron Microscopy, Energy Dispersive X-ray spectroscopy, X-Ray Diffraction Spectroscopy, Thermogravimetric Analysis, and Dynamic Light Scattering (DLS) analysis. Further, the optimum conditions for transglucosylation were determined by RSM as follows: enzyme-to-substrate ratio 6.9 U g-1, reaction time 9 h, temperature 45 °C, and pH 5.5 with a yield of 70 g l-1 (± 2.1). MALDI-TOF-MS analysis showed DP of the IMOs in ranges of 2-10. The detailed structural characterization of isomaltooligosaccharide by GC-MS and NMR suggested the α-(1 → 4) and α-(1 → 6)-D-Glcp residues as major constituents along with minor α-(1 → 2) and α-(1 → 3) -D-Glcp residues.

Mechanobactericidal, Gold Nanostar Hydrogel-Based Bandage for Bacteria-Infected Skin Wound Healing.

The emergence of multidrug resistant (MDR) microorganisms has led to the development of alternative approaches for providing relief from microbial attacks. The mechano-bactericidal action as a substitute for antimicrobials has become the focus of intensive research. In this work, nanostructure-conjugated hydrogel are explored as a flexible dressing against Staphylococcus aureus (S. aureus)-infected skin wounds. Herein gold nanostars (AuNst) with spike lengths reaching 120 nm are probed for antibacterial action. The bacterial killing of >95% is observed for Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli), while up to 60% for Gram-positive S. aureus. AuNst conjugated hydrogel (AuNst120@H) reduced >80% colonies of P. aeruginosa and E. coli. In comparison, around 35.4% reduction of colonies are obtained for S. aureus. The viability assay confirmed the presence of about 85% of living NIH-3T3 cells when grown with hydrogels. An animal wound model is also developed to assess the efficiency of AuNst120@H. A significant reduction in wound size is observed on the 10th day in AuNst120@H treated animals with fully formed epidermal layers, hair follicles, new blood vessels, and arrector muscles. These findings suggest that novel dressing materials can be developed with antimicrobial nanotextured surfaces.

Graphene oxide and fluorescent aptamer based novel biosensor for detection of 25-hydroxyvitamin D3.

For maintaining the healthy metabolic status, vitamin D is a beneficial metabolite stored majorly in its pre-activated form, 25-hydroxyvitamin D3 (25(OH)D3). Due to its important role in bone strengthening, the study was planned to quantify 25(OH)D3 levels in our blood. Quantification techniques for 25(OH)D3 are costly thus requiring a need for a low cost, and sensitive detection methods. In this work, an economic, and sensitive sensor for the detection of 25(OH)D3 was developed using aptamer and graphene oxide (GO). Aptamer is an oligonucleotide, sensitive towards its target, whereas, GO with 2D nanosheets provides excellent quenching surface. Aptamer labeled with fluorescein (5', 6-FAM) is adsorbed by π-π interaction on the GO sheets leading to quenching of the fluorescence due to Förster resonance energy transfer (FRET). However, in the presence of 25(OH)D3, a major portion of aptamer fluorescence remains unaltered, due to its association with 25(OH)D3. However, in the absence, aptamer fluorescence gets fully quenched. Fluorescence intensity quenching was monitored using fluorescence spectrophotometer and agarose gel based system. The limit of detection of 25(OH)D3 by this method was found to be 0.15 µg/mL whereas when GO-COOH was used, limit of detection was improved to 0.075 µg/mL. Therefore, this method could come up as a new sensing method in the field of vitamin D detection.

Liver Phosphoenolpyruvate Carboxykinase-1 Downregulation via siRNA-Functionalized Graphene Oxide Nanosheets Restores Glucose Homeostasis in a Type 2 Diabetes Mellitus In Vivo Model.

Metabolic disorders have been increasing at an alarming rate, and one such example of metabolic disorder is type 2 diabetes mellitus (T2DM). Unregulated gluconeogenesis in T2DM results in increased hepatic glucose output that causes fasting and postprandial hyperglycaemia. Extensive proofs have shown that the downregulation of the key rate-limiting enzyme phosphoenolpyruvate carboxykinase-1 (PCK-1) of gluconeogenesis improved glucose homeostasis in vivo. In the present study, we have synthesized and characterized liver-specific stearic acid conjugated octaarginine (StA-R8) functionalized 4arm-2K-PEGamineylated graphene oxide nanosheets (GPR8) for the delivery of siRNA against PCK-1 in T2DM C57BL/6 mice. We found that a single intravenous administration of siRNA (3 mg/kg BW) conjugated to GPR8 (GPR8:PCK-1siRNA(3 mg/kg BW) conjugate) in an optimized N/P ratio exploited as a therapeutic nanoformulation maintained glucose homeostasis for nearly 4 weeks in the T2DM mice. Efficient silencing of PCK-1 in T2DM liver tissue increased the phosphorylation of serine-256 of FOXO-1, thus showing a marked decrease in hepatic gluconeogenesis. Gluconeogenesis control and consequently glucose output from the liver furthermore partially enhanced liver and muscle insulin sensitivity results in the stimulation of the insulin/AKT-2 signaling pathway which indirectly restored glucose homeostasis in the treated T2DM group. Our therapeutic nanoformulation also improved glycogen storage in the liver and membrane translocation of GLUT4 in the muscle of the treated T2DM group. In conclusion, GPR8:PCK-1siRNA (3 mg/Kg BW) restored glucose homeostasis by controlling the hepatic glucose production and improved peripheral insulin sensitivity as a consequence of reduced hyperglycemia. Thus, the current approach offered an alternative strategy for the therapeutics for T2DM.

pH-Triggered, Synbiotic Hydrogel Beads for In Vivo Therapy of Iron Deficiency Anemia and Reduced Inflammatory Response.

Iron deficiency anemia (IDA) is the most common nutritional disorder worldwide nearly affecting two billion people. The efficacies of conventional oral iron supplements are mixed, intravenous iron administration acquaintances with finite but crucial risks. Usually, only 5-20% iron is absorbed in the duodenum while the remaining fraction reaches the colon, affecting the gut microbes and can significantly impact intestinal inflammatory responses. Therefore, administration of gut bacterial modulators such as probiotics, prebiotics, and any other dietary molecules that can stimulate healthy gut bacteria can enhance iron absorption without any adverse side effects. In this study, we have prepared an iron supplement to avoid the side effects of conventional oral iron supplements. The formulation includes co-encapsulation of iron with anti-inflammatory probiotic bacteria within alginate/starch hydrogels (B + I-Dex (H)), which has been demonstrated to be efficient in mitigating IDA in vivo. As intestinal pH increases, the pore size of hydrogel increases due to ionic interactions and thus releases the encapsulated bacteria and iron. The field emission scanning electron microscopy (FESEM) analysis confirmed the porous structure of hydrogel beads, and in vitro release studies showed a sustained release of iron and bacteria at intestinal pH. The hydrogel was found to be nontoxic and biocompatible in Caco2 cell lines. The formulation showed efficient in vitro and in vivo iron bioavailability in Fe depletion-repletion studies. B + I-Dex (H) was observed to generate less inflammatory response than FeSO4 or nonencapsulated iron dextran (I-Dex) in vivo. We entrust that this duly functional hydrogel formulation could be further utilized or modified for the development of oral therapeutics for IDA.

Organophosphonate functionalized Au/Si@Fe3O4: Versatile carrier for enzyme immobilization.

Magnetic nanoparticles with silica/gold (Au) shell were synthesized to be used as substrates for enzyme immobilization. Successful functionalization was followed by addition of phosphate linkers containing carboxyl end as free end for linking with enzyme. Effect of varied phospho-carboxyl chain lengths based linkers, 3-phosphonopropionic acid (3-PPA) and 16-phosphonopropionic acid (16-PHDA) on kinetic parameters of enzyme were studied. Immobilization of upto 670µg/mg of nanoparticles were observed. Enzyme bound with longer chain linker were observed to have Km (11.8mg/mL) and Vmax (1.59µmol/min/mg) which was found to be lower than 3-PPA. Reusability assay gave a minimum of 70% activity at 10th time of usage. Thus, an efficient, robust, cost effective and easily separable substrate for restraining the enzyme was prepared.

Efficacy of linalool to ameliorate uremia induced vascular calcification in wistar rats.

BACKGROUND: Uremia is the condition generally associated with the last stage of chronic kidney disease (CKD) due to highly reduced glomerular filtration rate. Mortality of the patients diagnosed with Uremia generally occurs due to cardiovascular involvement. This occurs due to the transdifferentiation of vascular smooth muscle cells (VSMCs) into osteogenic cells in hyperphosphatemic condition that is associated with kidney failure promoting extra-osseous calcification. PURPOSE: Linalool is an essential oil that has been recently studied for its procardiovascular effects, thus the aim of the study involved to identify its potential role on vascular calcification (VC). METHODS: Uremia was induced in male wistar rats, weighing 250-350 gm by giving adenine diet for 4 weeks followed by phosphate diet for next 4 weeks. Linalool was given orally at two different doses (100 mg/kg bodyweight and 150 mg/kg bodyweight) daily for 4 weeks with phosphate diet. RESULTS: Linalool being a moderate antioxidant probably scavenged superoxide radicals (in vitro analysis). Deposition of calcium was observed by alizarin and von-kossa stains in aorta of uremic rats whereas linalool co-administration prevents calcium deposition in aorta of uremic rats. Elevated mRNA expression of calcification markers, increased lipid peroxidation levels and increased levels of catalase and superoxide dismutase (SOD) was found in aorta of uremic animals. However, with supplementation of linalool reduction in the mRNA expression of calcification markers, lipid peroxidation and antioxidant enzymes were observed. CONCLUSION: Therefore it can be concluded that linalool could be a promising therapeutic candidate for exploring its clinical application in VC.

Multiple thermostable enzyme hydrolases on magnetic nanoparticles: An immobilized enzyme-mediated approach to saccharification through simultaneous xylanase, cellulase and amylolytic glucanotransferase action.

Microbe-derived enzymes such as xylanases, cellulases and amylases, are efficient at hydrolyzing plant biomass. Efforts to harness the functionalities of these enzymes towards applications in energy and fuel biosciences, and food and nutrition, continue apace in many laboratories. Given that enzymes derived from mesophile proteomes undergo facile denaturation and/or degradation at ambient temperatures, and require frequent replenishment during bioprocessing, it is desirable that they be replaced by structurally-stable enzymes capable of functioning efficiently and resisting denaturation and degradation, immobilized on solid media to further add to stability and facilitate recovery and reuse. Towards these objectives, we used synthetic magnetic nanoparticles (MNPs) and immobilized upon their surfaces three different structurally-stable hydrolases: a thermostable xylanase (BSX) derived from Bacillus sp. NG-27, a cellulase (RMCel12A) derived from Rhodothermus marinus, and an amylase-cum-glucanotransferase (PfuAmyGT) derived from Pyrococcus furiosus. The MNPs were activated with glutaraldehyde and BSX, RMCel12A, and PfuAmyGT, respectively, were covalently immobilized with efficiencies of ~92%, 45% and 93%. The enzymes and the MNPs were fully characterized before and after immobilization, and the immobilized enzymes were found to be active at 50 °C against synthetic substrates as well as pre-treated biomass derived from corn cob and rice husk. The enzyme-coupled MNPs displayed high stability upon storage properties, high operational stability as well as high reusability (retaining 69, 48, and 50% residual activity after 13 uses for BSX, RMCel12A and PfuAmyGT, respectively). Experiments were also conducted with MNPs loaded simultaneously with all three enzymes. Such immobilized enzyme combinations on MNPs can be used in the saccharification of plant biomass.

Xylanase immobilization on magnetite and magnetite core/shell nanocomposites using two different flexible alkyl length organophosphonates: Linker length and shell effect on enzyme catalytic activity.

Magnetite and magnetite core/shell (Fe3O4/SiO2) nanoparticles were synthesized and functionalized with two different alkyl chain length linkers that were 3-Phosphonopropionic acid (3-PPA) and 16-Phosphonohexadecanoic acid (16-PHDA). Xylanase (EC 3.2.1.8, endo-1,4-xylanase, endo-1), was immobilized on as synthesized bare and silica coated magnetite nanoparticles via well-known EDC coupling. Transmission Electron Microscopy (TEM), Field Emission Scanning Electron Microscopy (FESEM), X-Ray Diffraction Spectroscopy (XRD), X-ray Photoelectron Spectroscopy (XPS), Dynamic Light Scattering (DLS) and Thermogravimetric analysis (TGA) techniques were utilized to characterize all the modifications. The flexible linker chain length plays a vital role in the catalytic attributes of the immobilized enzyme. Result shows that long chain alkyl linker grafted magnetite and magnetite core/silica shell nanoparticles exhibited a superior performance in terms of lower Km, higher catalytic efficiency and better reusability. Furthermore, the immobilized xylanase shows improved tolerability performance at a wide range of pH and temperature. Silica-coated magnetite nanoparticles bound xylanase through 16-PHDA retained 90% of its initial activity after 10 consecutive cycles, further emphasize on the beneficial effect of linker chain length and inert silica coating.