With a sharp increase in the cases of multi-drug resistant (MDR) bacteria all over the world, there is a huge demand to develop a new generation of antibiotic agents to fight them. As an alternative to the traditional drug discovery route, we have designed an effective antibacterial agent by modifying an existing commercial antibiotic, kanamycin, conjugated on the surface of gold nanoparticles (AuNPs). In this study, we report a single-step synthesis of kanamycin-capped AuNPs (Kan-AuNPs) utilizing the combined reducing and capping properties of kanamycin. While Kan-AuNPs have increased toxicity to a primate cell line (Vero 76), antibacterial assays showed dose-dependent broad spectrum activity of Kan-AuNPs against both Gram-positive and Gram-negative bacteria, including Kanamycin resistant bacteria. Further, a significant reduction in the minimum inhibitory concentration (MIC) of Kan-AuNPs was observed when compared to free kanamycin against all the bacterial strains tested. Mechanistic studies using transmission electron microscopy and fluorescence microscopy indicated that at least part of Kan-AuNPs increased efficacy may be through disrupting the bacterial envelope, resulting in the leakage of cytoplasmic content and the death of bacterial cells. Results of this study provide critical information about a novel method for the development of antibiotic capped AuNPs as potent next-generation antibacterial agents.

BackgroundPhloridzin, an antidiabetic and antineoplastic agent usually found in fruit trees, is a dihydrochalcone constituent that has a clinical/pharmaceutical significance as a sodium-glucose linked transport 2 (SGLT2) inhibitor. While the aglycone metabolite of phloridzin, phloretin, displays a reduced capacity of SGLT2 inhibition, this nutraceutical displays enhanced antineoplastic activity in comparison to phloridzin.PurposeThe objective of this study was to develop gold nanoparticle (AuNP) mediated delivery of phloridzin and phloretin and explore their anticancer mechanism through conjugation of the dihydrochalcones and the AuNP cores.MethodsPhloridzin and phloretin conjugated AuNPs (Phl-AuNP and Pht-AuNP) were synthesized in single-step, rapid, biofriendly processes. The synthesized AuNPs morphology was characterized via transmission electron microscopy and ultraviolet-visible spectroscopy. The presence of phloridzin or phloretin was confirmed using scanning electron microscopy-energy dispersive x-ray spectroscopy. The percentage of organic component (phloridzin/phloretin) onto AuNPs surface was characterized using thermogravimetric analysis. Assessment of the antineoplastic potency of the dihydrochalcones conjugated AuNPs against cancerous cell lines (HeLa) was accomplished through monitoring via flow cytometry.ResultsThe functionalized AuNPs were synthesized via a single-step method that relied only upon the redox potential of the conjugate itself and required no toxic chemicals. The synthesized Phl-AuNPs were found to be in the size range of 15±5 nm, whereas the Pht-AuNP were found to be 8±3 nm, placing both conjugated AuNPs well within the size range necessary for successful pharmaceutical applications. These assays demonstrate a significant increase in the cancerous cell toxicities as a result of the conjugation of the drugs to AuNPs, as indicated by the 17.45-fold increase in the efficacy of Pht-AuNPs over pure phloretin, and the 4.49-fold increase in efficacy of Phl-AuNP over pure phloridzin.ConclusionWe report a simple, biofriendly process using the reducing and capping potential of the dihydrochalcones, phloridzin and phloretin, to synthesize stable AuNPs that have promising futures as potential antineoplastic agents.

The high sensitivity and functional group selectivity of surface-enhanced Raman scattering (SERS) make it an attractive method for enzyme sensing, but there is currently a severe lack of enzyme substrates that release SERS reporter molecules with favorable detection properties. We find that 2-mercaptopyridine-3-carbonitrile ( o-MPN) and 2-mercaptopyridine-5-carbonitrile ( p-MPN) are highly effective as SERS reporter molecules that can be captured by silver or gold nanoparticles to give intense SERS spectra, each with a distinctive nitrile peak at 2230 cm–1. p-MPN is a more sensitive reporter and can be detected at low nanomolar concentrations. An assay validation study synthesized two novel substrate molecules, Glc-o-MPN and Glc-p-MPN, and showed that they can be cleaved efficiently by β-glucosidase (K m = 228 and 162 μM, respectively), an enzyme with broad industrial and biomedical utility. Moreover, SERS detection of the released reporters ( o-MPN or p-MPN) enabled sensing of β-glucosidase activity and β-glucosidase inhibition. Comparative experiments using a crude almond flour extract showed that the presence of β-glucosidase activity could be confirmed by SERS detection in a much shorter time period (>10 time shorter) than by UV–vis absorption detection. It is likely that a wide range of enzyme assays and diagnostic tests can be developed using 2-mercaptopyridine-carbonitriles as SERS reporter molecules.

prone to aggregation. In this study, to overcome this issue and chemically regulate disulfide-coupled peptide and protein folding, a series of chemical reagents was examined in the refolding of hepcidin and prohepcidin, as a model peptide and protein, and their folding recoveries were estimated. Hepcidin consists of 25 amino acid residues and four intra-molecular disulfide bonds, which are absolutely required for its biological activity [2], not only for iron homeostasis, but also for anti-microbial activity. To investigate structurefunction relationships, hepcidin was chemically synthesized. However, the yield of synthesized hepcidin was quite low under the typical folding conditions. The major problem in the disulfide-coupled folding of hepcidin is that it undergoes aggregation during its folding reaction [3]. To solve this problem, several types of redox reagents and solvents were examined to improve the folding efficiency of hepcidin [3]. However, all of the reagents resulted in quite low yields for the disulfide-coupled folding of hepcidin. Therefore, to regulate the folding reaction of hepcidin and its precursor protein, we estimated the folding conditions, such as pH values, salt concentrations, and a variety of redox reagents. The results will be discussed in this paper.[1] Chakravarthi, S.; Jessop, C.E.; Bulleid, N.J. EMBO reports 2006, 7, 271-275. [2] Hocquellet, A.; le Senechal, C.; Garbay, B. Peptides 2012, 36, 303-307. [3] Zhang, J.; Diamond, S.; Arvedson, T.; Sasu, BJ.; Miranda, LP. Biopolymers 2010, 94, 257-264. Human cystatin C (HCC) is a cysteine protease inhibitor. This protein in pathological conditions, forms dimers via a ''domain swapping'' mechanism. HCC is also associated with two types of amyloid deposition diseases -hereditary amyloid angiopathy (related to the Leu68Gln mutation) and wild-type cystatin C co-precipitation. The aim of our studies was the characterisation of the self-assembling properties of native and mutated (at positions 57 or 68) forms of human cystatin C in solution. The structure, overall conformation and secondary structure changes in solution were studied by Fourier transformed infrared spectroscopy (FTIR), circular dichroism spectroscopy (CD), dynamic light scattering (DLS) and time resolved small angle scattering of synchrotron radiation (TR-SAXS). SAXS data for native and mutated HCC were subjected to analysis by using SVD and MCR-ALS methods as well as the low resolution structure determination. Besides the monomeric forms of human cystatin C, also dimers and higher oligomers were formed even after short (50-ms) exposure on synchrotron radiation. In addition we observed for first time, formation of domain swapped dimers of human cystatin C induced by irradiation. The spectroscopic studies confirmed conformational changes. Light chain (AL) amyloidosis is a protein misfolding disease where immunoglobulin light chains sample partially folded states that lead to misfolding and amyloid formation, resulting in organ dysfunction and death. In vivo, amyloid deposits...

Polysaccharide lyases (PLs) are enzymes that our commonly used by microorganisms to degrade polyuronides. These enzymes are produced by bacteria to aid in reducing the high molecular weight of the host's extracellular matrix to enhance its virulence. PLs can also be used to degrade polysaccharides as a source of carbon for the pathogen. Our focus is on the alginate lyase of Stenotrophomonas maltophilia (Smlt1473), which has been shown recently to be active against alginate, hyaluronic acid (HA) and poly-D-glucuronic acid at varying pH values (J. Biol. Chem. 289: 312 ( 2014)). The active site appears to have regions specific to binding these three sugars and an active catalytic site. Our objective is to understand how pH influences binding of a HA and if this involves structural changes in the protein. Docking of HA to a homology-based structure of Smlt1473 (based on the crystal structure of alginate lyase A1-III) was used to provide initial starting conformations for our molecular dynamics (MD) simulations. ProPKa was used to determine the ionization state of residues at our three simulated pHs (5, 7 and 9). MD simulations at neutral pH confirm the binding residues seen in experiment. However, at the acidic and basic condtions, the homology-based model of the binding pocket opens. At ph¼5, there is an increase in the exposure of HA-specific residues in the vicinity of W171 suggesting this exposure is the cause for Smlt1473 increased activity (J. Biol. Chem. 289: 312 ( 2014)). Therefore, Smlt1473's structure appears to strongly depend on pH and is used to control its activity toward different sugars.