Supramolecular citrate poly allylamine hydrochloride nanoparticles for citrate delivery and calcium oxalate nanocrystal dissolution.

HYPOTHESIS: Renal calculi (kidney stones) are mainly made by calcium oxalate and can cause different complications including malfunction of the kidney. The most important urinary stone inhibitors are citrate molecules. Unfortunately, the amount of citrate reaching the kidney after oral ingestion is low. We hypothesized that nanoparticles of polyallylamine hydrochloride (CIT-PAH) carrying citrate ions could simultaneously deliver citrates while PAH would complex oxalate triggering dissolution and removal of CaOx nanocrystals. EXPERIMENTS: We successfully prepared nanoparticles of citrate ions with polyallylamine hydrochloride (CIT-PAH), PAH with oxalate (OX-PAH) and characterize them by Small Angle X ray Scattering (SAXS), Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS) and NMR. Dissolution of CaOx nanocrystals in presence of CIT-PAH have been followed with Wide Angle Xray Scattering (WAXS), DLS and Confocal Raman Microscopy. Raman spectroscopy was used to study the dissolution of crystals in synthetic urine samples. The release of citrate from CIT-PAH was followed by diffusion NMR. Molecular dynamics (MD) simulations were carried out to study the interaction of CIT and OX ions with PAH. FINDINGS: CIT-PAH nanoparticles dissolves CaOx nanocrystals as shown by NMR, DLS, TEM and WAXS in water and by Raman spectroscopy in artificial human urine. WAXS and Raman show that the crystal structure of CaOx disappears in the presence of CIT-PAH. DLS shows that the time required for CaOX dissolution will depend on the concentration of CIT-PAH NPs. NMR proves that citrate ions are released from the CIT PAH NPs during CaOX dissolution, MD simulations showed that oxalates exhibit a stronger interaction for PAH than citrate, explaining the removal of oxalate ions and replacement of the citrate in the polymer nanoparticles.

Fabrication and Characterization of a Polydopamine-Modified Bacterial Cellulose and Sugarcane Filter Cake-Derived Hydroxyapatite Composite Scaffold.

The search for environmentally friendly and sustainable sources of raw materials has been ongoing for quite a while, and currently, the utilization and applications of agro-industrial biomass residues in biomedicine are being researched. In this study, a polydopamine (PDA)-modified bacterial cellulose (BC) and hydroxyapatite (HA) composite scaffold was fabricated using the freeze-drying method. The as-prepared hydroxyapatite was synthesized via the chemical precipitation method using sugarcane filter cake as a calcium source, as reported in a previous study. X-ray diffraction analysis revealed a carbonated phase of the prepared hydroxyapatite, similar to that of the natural bone mineral. Wide-angle X-ray scattering analysis revealed the successful fabrication of BC/HA composite scaffolds, while X-ray photoelectron spectroscopy suggested that PDA was deposited on the surface of the BC/HA composite scaffolds. In vitro cell viability assays indicated that BC/HA and PDA-modified composite scaffolds did not induce cytotoxicity and were biocompatible with MC3T3-E1 preosteoblasts. PDA-modified composite scaffolds showed enhanced protein adsorption capacity in vitro compared to the unmodified scaffolds. On a concluding note, these results demonstrate that agro-industrial biomass residues have the potential to be used in biomedical applications and that PDA-modified BC/HA composite scaffolds are a promising biomaterial for bone tissue engineering.

Tissue-specific expression of senescence biomarkers in spontaneously hypertensive rats: evidence of premature aging in hypertension.

Background: Cellular senescence is an age-related physiological process that contributes to tissue dysfunction and accelerated onset of chronic metabolic diseases including hypertension. Indeed, elevation of blood pressure in hypertension coincides with premature vascular aging and dysfunction. In addition, onsets of metabolic disturbance and osteopenia in patients with hypertension have also been reported. It is possible that hypertension enhances premature aging and causes progressive loss of function in multiple organs. However, the landscape of cellular senescence in critical tissues affected by hypertension remains elusive. Materials and Methods: Heart, liver, bone, hypothalamus, and kidney were collected from spontaneously hypertensive rats (SHR) and age- and sex-matched normotensive Wistar rats (WT) at 6, 12, 24 and 36 weeks of age (n = 10 animals/group). Changes in mRNA levels of senescence biomarkers namely cyclin-dependent kinase (CDK) inhibitors (CDKIs), i.e., Cdkn2a (encoding p16Ink4a) and Cdkn1a (encoding p21cip1) as well as senescence-associated secretory phenotypes (SASPs), i.e., Timp1, Mmp12, Il6 and Cxcl1, were determined. Additionally, bone collagen alignment and hydroxy apatite crystal dimensions were determined by synchrotron radiation small- and wide-angle X-ray scattering (SAXS/WAXS) techniques. Results: Real-time PCR revealed that transcript levels of genes encoding CDKIs and SASPs in the heart and liver were upregulated in SHR from 6 to 36 weeks of age. Expression of Timp1 and Cxcl1 was increased in bone tissues isolated from 36-week-old SHR. In contrast, we found that expression levels of Timp1 and Il6 mRNA were decreased in hypothalamus and kidney of SHR in all age groups. Simultaneous SAXS/WAXS analysis also revealed misalignment of bone collagen fibers in SHR as compared to WT. Conclusion: Premature aging was identified in an organ directly affected by high blood pressure (i.e., heart) and those with known functional defects in SHR (i.e., liver and bone). Cellular senescence was not evident in organs with autoregulation of blood pressure (i.e., brain and kidney). Our study suggested that cellular senescence is induced by persistently elevated blood pressure and in part, leading to organ dysfunction. Therefore, interventions that can both lower blood pressure and prevent cellular senescence should provide therapeutic benefits for treatment of cardiovascular and metabolic consequences.

Biophysical Characterization of p51 and p66 Monomers of HIV-1 Reverse Transcriptase with Their Inhibitors.

Human immunodeficiency virus (HIV)-1 reverse transcriptase (HIV-1 RT) is responsible for the transcription of viral RNA genomes into DNA genomes and has become an important target for the treatment of acquired immune deficiency syndrome (AIDS). This study used biophysical techniques to characterize the HIV-1 RT structure, monomer forms, and the non-nucleoside reverse transcriptase inhibitors (NNRTIs) bound forms. Inactive p66W401A and p51W401A were selected as models to study the HIV-1 RT monomer structures. Nuclear magnetic resonance (NMR) spectroscopy revealed that the unliganded forms of p66W401A protein and p51W401A protein had similar conformation to each other in solution. The complexes of p66W401A or p51W401A with inhibitors showed similar conformations to p66 in the RT heterodimer bound to the NNRTIs. Furthermore, the results of paramagnetic relaxation enhancement (PRE)-assisted NMR revealed that the unliganded forms of the p66W401A and p51W401A conformations were different from the unliganded heterodimer, characterized by a greater distance between the fingers and thumb subdomains. Small-angle X-ray scattering (SAXS) experiments confirmed that p66W401A and p51W401A can bind with inhibitors, similar to the p66/p51 heterodimer. The findings of this study increase the structural knowledge base of HIV-1 RT monomers, which may be helpful in the future design of potent viral inhibitors.

Structural and binding studies of a new chitin-active AA10 lytic polysaccharide monooxygenase from the marine bacterium Vibrio campbellii.

Vibrio spp. play a crucial role in the global recycling of the highly abundant recalcitrant biopolymer chitin in marine ecosystems through their ability to secrete chitin-degrading enzymes to efficiently hydrolyse chitinous materials and use them as their major carbon source. In this study, the first crystal structures of a complete four-domain chitin-active AA10 lytic polysaccharide monooxygenase from the chitinolytic bacterium Vibrio campbellii type strain ATCC BAA-1116 are reported. The crystal structures of apo and copper-bound VhLPMO10A were resolved as homodimers with four distinct domains: an N-terminal AA10 catalytic (CatD) domain connected to a GlcNAc-binding (GbpA_2) domain, followed by a module X domain and a C-terminal carbohydrate-binding module (CBM73). Size-exclusion chromatography and small-angle X-ray scattering analysis confirmed that VhLPMO10A exists as a monomer in solution. The active site of VhLPMO10A is located on the surface of the CatD domain, with three conserved residues (His1, His98 and Phe170) forming the copper(II)-binding site. Metal-binding studies using synchrotron X-ray absorption spectroscopy and X-ray fluorescence, together with electron paramagnetic resonance spectroscopy, gave consistently strong copper(II) signals in the protein samples, confirming that VhLPMO10A is a copper-dependent enzyme. ITC binding data showed that VhLPMO10A could bind various divalent cations but bound most strongly to copper(II) ions, with a Kd of 0.1 ± 0.01 µM. In contrast, a Kd of 1.9 nM was estimated for copper(I) ions from redox-potential measurements. The presence of ascorbic acid is essential for H2O2 production in the reaction catalysed by VhLPMO10A. MALDI-TOF MS identified VhLPMO10A as a C1-specific LPMO, generating oxidized chitooligosaccharide products with different degrees of polymerization (DP2ox-DP8ox). This new member of the chitin-active AA10 LPMOs could serve as a powerful biocatalyst in biofuel production from chitin biomass.

Structural interpretations of a flexible cold-active AMS8 lipase by combining small-angle X-ray scattering and molecular dynamics simulation (SAXS-MD).

Determining structure of highly flexible protein with multiple conformations can be challenging. This paper aims to combine molecular dynamics (MD) and small angle X-ray diffraction (SAX) techniques as a solution to overcome issues related to protein conformation in hardly crystallized protein. Based on prior studies, a cold-active lipase AMS8 was simulated in solvents showing stability in its N-terminal and high flexibility in its C-terminal. However, MD in its own algorithm could not explain the basis of macromolecule conformational transitions or changes related to protein through folding. Hence, by combining SAXS with MD, it is possible to understand the structure of flexible AMS8 lipase in natural space. Based on the findings, SAXS ab-initio model of AMS8 lipase was identified as a monomeric protein in which the optimized model of cold-active lipase AMS8 derived from SAXS data was found to be aligned with AMS8 homology model under series of MD timeframe.

A flap motif in human serine hydroxymethyltransferase is important for structural stabilization, ligand binding, and control of product release.

Human cytosolic serine hydroxymethyltransferase (hcSHMT) is a promising target for anticancer chemotherapy and contains a flexible "flap motif" whose function is yet unknown. Here, using size-exclusion chromatography, analytical ultracentrifugation, small-angle X-ray scattering (SAXS), molecular dynamics (MD) simulations, and ligand-binding and enzyme-kinetic analyses, we studied the functional roles of the flap motif by comparing WT hcSHMT with a flap-deleted variant (hcSHMT/Δflap). We found that deletion of the flap results in a mixture of apo-dimers and holo-tetramers, whereas the WT was mostly in the tetrameric form. MD simulations indicated that the flap stabilizes structural compactness and thereby enhances oligomerization. The hcSHMT/Δflap variant exhibited different catalytic properties in (6S)-tetrahydrofolate (THF)-dependent reactions compared with the WT but had similar activity in THF-independent aldol cleavage of ß-hydroxyamino acid. hcSHMT/Δflap was less sensitive to THF inhibition than the WT (Ki of 0.65 and 0.27 mm THF at pH 7.5, respectively), and the THF dissociation constant of the WT was also 3-fold lower than that of hcSHMT/Δflap, indicating that the flap is important for THF binding. hcSHMT/Δflap did not display the burst kinetics observed in the WT. These results indicate that, upon removal of the flap, product release is no longer the rate-limiting step, implying that the flap is important for controlling product release. The findings reported here improve our understanding of the functional roles of the flap motif in hcSHMT and provide fundamental insight into how a flexible loop can be involved in controlling the enzymatic reactions of hcSHMT and other enzymes.

Crystal structure of the flavin reductase of Acinetobacter baumannii p-hydroxyphenylacetate 3-hydroxylase (HPAH) and identification of amino acid residues underlying its regulation by aromatic ligands.

The first step in the degradation of p-hydroxyphenylacetic acid (HPA) is catalyzed by the two-component enzyme p-hydroxyphenylacetate 3-hydroxylase (HPAH). The two components of Acinetobacter baumannii HPAH are known as C1 and C2, respectively. C1 is a flavin reductase that uses NADH to generate reduced flavin mononucleotide (FMNH-), which is used by C2 in the hydroxylation of HPA. Interestingly, although HPA is not directly involved in the reaction catalyzed by C1, the presence of HPA dramatically increases the FMN reduction rate. Amino acid sequence analysis revealed that C1 contains two domains: an N-terminal flavin reductase domain, and a C-terminal MarR domain. Although MarR proteins typically function as transcription regulators, the MarR domain of C1 was found to play an auto-inhibitory role. Here, we report a crystal structure of C1 and small-angle X-ray scattering (SAXS) studies that revealed that C1 undergoes a substantial conformational change in the presence of HPA, concomitant with the increase in the rate of flavin reduction. Amino acid residues that are important for HPA binding and regulation of C1 activity were identified by site-directed mutagenesis. Amino acid sequence similarity analysis revealed several as yet uncharacterized flavin reductases with N- or C-terminal fusions.

A Coarse-Grained Model for Free and Template-Bound Porphyrin Nanorings.

Coarse-grained simulation models are developed to study both template-bound and free porphyrin nanoring systems. Key interactions are modeled with relatively simple (and physically motivated) energy functions which allow for relatively facile transfer both between different ring sizes and between the template-bound and free nanoring systems. The effects of varying the model parameters on the respective radii of gyration are determined. The effects of including different templates on the ring structure are investigated both in terms of the detailed geometry of the template and the interaction strength between the template and the metal centers in the nanorings. The role of the template-nanoring interaction strength in controlling potential "caterpillar track" rotational motion is discussed. The relationship of the model to experimental small-angle X-ray, exchange spectroscopy, and electron spin resonance results is discussed.

Template-directed synthesis of linear porphyrin oligomers: classical, Vernier and mutual Vernier.

Three different types of template-directed syntheses of linear porphyrin oligomers are presented. In the classical approach the product has the same number of binding sites as the template, whereas in Vernier reactions the product has the lowest common multiple of the numbers of binding sites in the template and the building block. Mutual Vernier templating is like Vernier templating except that both strands of the Vernier complex undergo coupling simultaneously, so that it becomes impossible to say which is the 'template' and which is the 'building block'. The template-directed synthesis of monodisperse linear oligomers is more difficult than that of cyclic oligomers, because the products of linear templating have reactive ends. All three types of templating are demonstrated here, and used to prepare a nickel(ii) porphyrin dodecamer with 4-pyridyl substituents on all twelve porphyrin units. The stabilities and cooperativities of the double-strand complexes involved in these reactions were investigated by UV-vis-NIR titration. The four-rung ladder duplex has a stability constant of about 2 × 1018 M-1 in dichloromethane at 298 K.

Size-Independent Energy Transfer in Biomimetic Nanoring Complexes.

Supramolecular antenna-ring complexes are of great interest due to their presence in natural light-harvesting complexes. While such systems are known to provide benefits through robust and efficient energy funneling, the relationship between molecular structure, strain (governed by nuclear coordinates and motion), and energy dynamics (arising from electronic behavior) is highly complex. We present a synthetic antenna-nanoring system based on a series of conjugated porphyrin chromophores ideally suited to explore such effects. By systematically varying the size of the acceptor nanoring, we reveal the interplay between antenna-nanoring binding, local strain, and energy dynamics on the picosecond time scale. Binding of the antenna unit creates a local strain in the nanoring, and this strain was measured as a function of the size of the nanoring, by UV-vis-NIR titration, providing information on the conformational flexibility of the system. Strikingly, the energy-transfer rate is independent of nanoring size, indicating the existence of strain-localized acceptor states, spread over about six porphyrin units, arising from the noncovalent antenna-nanoring association.

Ultrafast energy transfer in biomimetic multistrand nanorings.

We report the synthesis of LH2-like supramolecular double- and triple-stranded complexes based upon porphyrin nanorings. Energy transfer from the antenna dimers to the π-conjugated nanoring occurs on a subpicosecond time scale, rivaling transfer rates in natural light-harvesting systems. The presence of a second nanoring acceptor doubles the transfer rate, providing strong evidence for multidirectional energy funneling. The behavior of these systems is particularly intriguing because the local nature of the interaction may allow energy transfer into states that are, for cyclic nanorings, symmetry-forbidden in the far field. These complexes are versatile synthetic models for natural light-harvesting systems.

The first microbeam synchrotron X-ray fluorescence beamline at the Siam Photon Laboratory.

The first microbeam synchrotron X-ray fluorescence (µ-SXRF) beamline using continuous synchrotron radiation from Siam Photon Source has been constructed and commissioned as of August 2011. Utilizing an X-ray capillary half-lens allows synchrotron radiation from a 1.4 T bending magnet of the 1.2 GeV electron storage ring to be focused from a few millimeters-sized beam to a micrometer-sized beam. This beamline was originally designed for deep X-ray lithography (DXL) and was one of the first two operational beamlines at this facility. A modification has been carried out to the beamline in order to additionally enable µ-SXRF and synchrotron X-ray powder diffraction (SXPD). Modifications included the installation of a new chamber housing a Si(111) crystal to extract 8 keV synchrotron radiation from the white X-ray beam (for SXPD), a fixed aperture and three gate valves. Two end-stations incorporating optics and detectors for µ-SXRF and SXPD have then been installed immediately upstream of the DXL station, with the three techniques sharing available beam time. The µ-SXRF station utilizes a polycapillary half-lens for X-ray focusing. This optic focuses X-ray white beam from 5 mm × 2 mm (H × V) at the entrance of the lens down to a diameter of 100 µm FWHM measured at a sample position 22 mm (lens focal point) downstream of the lens exit. The end-station also incorporates an XYZ motorized sample holder with 25 mm travel per axis, a 5× ZEISS microscope objective with 5 mm × 5 mm field of view coupled to a CCD camera looking to the sample, and an AMPTEK single-element Si (PIN) solid-state detector for fluorescence detection. A graphic user interface data acquisition program using the LabVIEW platform has also been developed in-house to generate a series of single-column data which are compatible with available XRF data-processing software. Finally, to test the performance of the µ-SXRF beamline, an elemental surface profile has been obtained for a piece of ancient pottery from the Ban Chiang archaeological site, a UNESCO heritage site. It was found that the newly constructed µ-SXRF technique was able to clearly distinguish the distribution of different elements on the specimen.