Design and Fabrication of Silicon Nanowire-Based Biosensors with Integration of Critical Factors: Toward Ultrasensitive Specific Detection of Biomolecules.

As critical factors affecting the sensing performance of silicon nanowire (SiNW) biosensors, the structure, functional interface, and detection target were analyzed and designed to improve sensing performance. For an improved understanding of the dependence of sensor structure on sensitivity, a simple theoretical analysis was proposed to predict the sensitivity of biosensors with different SiNW types, widths, and doping concentrations. Based on the theoretical analysis, a biosensor integrating optimized critical factors was designed and fabricated. Optimizations focusing on the following aspects are considered: (1) employing n-type SiNW and controlling the impurity doping concentration of SiNW at approximately 2 × 1016-6 × 1016 atoms/cm3 to obtain a suitable charge density, (2) minimizing the SiNW width to 16.0 nm to increase the surface area-to-volume ratio, (3) using a native oxide layer on SiNW as a gate insulator to transport the captured charge molecules closer to the SiNW surface, (4) modifying the SiNW surface by 2-aminoethylphosphonic acid coupling to form a high-density self-assembled monolayer for enhancing the stability bound molecules, and (5) functionalizing the SiNW with ovalbumin molecules for specifically capturing the target immunoglobulin G (IgG) molecules. The sensing performance was evaluated by detecting IgG with concentrations ranging from 6 aM to 600 nM and control experiments. The SiNW biosensor revealed ultrahigh sensitivity and specific detection of target IgG with a measured limit of detection of 6 aM. The integration of the critical SiNW biosensor factors provides a significant possibility of a rapid and ultrasensitive diagnosis of diseases at their early stages.

Identification of ceramide 2-aminoethylphosphonate molecular species from different aquatic products by NPLC/Q-Exactive-MS.

Ceramide 2-aminoethylphosphonate (CAEP) is a type of phosphonosphingolipids with potential trophic activity. In this work, complicated CAEP species from different aquatic products were comprehensively identified and semi-quantified by utilizing normal phase liquid chromatography/Q-Exactive mass spectrometry (NPLC/Q-Exactive-MS). We elucidated the fragment schemes of CAEP molecules and found the presence of methylated CAEP (Me-CAEP) species. Remarkably, quantitative results revealed that Loligo chinensis had the highest CAEP content of 4.9 ±â€¯0.4 mg/g dry weight and the most complex molecular species composition, whereas Asterias amurenis had the lowest CAEP content of 1.9 ±â€¯0.6 mg/g dry weight. The most common molecule was CAEP (d19:3-16:0). Additionally, statistical analysis revealed that five aquatic products can be effectively distinguished from their CAEP species; thus, CAEP molecules can play an important role in identifying processed products from aquatic products.

Chirality-Driven Mode of Binding of α-Aminophosphonic Acid-Based Allosteric Inhibitors of the Human Farnesyl Pyrophosphate Synthase (hFPPS).

Thienopyrimidine-based allosteric inhibitors of the human farnesyl pyrophosphate synthase (hFPPS), characterized by a chiral α-aminophosphonic acid moiety, were synthesized as enantiomerically enriched pairs, and their binding mode was investigated by X-ray crystallography. A general consensus in the binding orientation of all (R)- and (S)-enantiomers was revealed. This finding is a prerequisite for establishing a reliable structure-activity relationship (SAR) model.

A New Microbial Pathway for Organophosphonate Degradation Catalyzed by Two Previously Misannotated Non-Heme-Iron Oxygenases.

The assignment of biochemical functions to hypothetical proteins is challenged by functional diversification within many protein structural superfamilies. This diversification, which is particularly common for metalloenzymes, renders functional annotations that are founded solely on sequence and domain similarities unreliable and often erroneous. Definitive biochemical characterization to delineate functional subgroups within these superfamilies will aid in improving bioinformatic approaches for functional annotation. We describe here the structural and functional characterization of two non-heme-iron oxygenases, TmpA and TmpB, which are encoded by a genomically clustered pair of genes found in more than 350 species of bacteria. TmpA and TmpB are functional homologues of a pair of enzymes (PhnY and PhnZ) that degrade 2-aminoethylphosphonate but instead act on its naturally occurring, quaternary ammonium analogue, 2-(trimethylammonio)ethylphosphonate (TMAEP). TmpA, an iron(II)- and 2-(oxo)glutarate-dependent oxygenase misannotated as a γ-butyrobetaine (γbb) hydroxylase, shows no activity toward γbb but efficiently hydroxylates TMAEP. The product, ( R)-1-hydroxy-2-(trimethylammonio)ethylphosphonate [( R)-OH-TMAEP], then serves as the substrate for the second enzyme, TmpB. By contrast to its purported phosphohydrolytic activity, TmpB is an HD-domain oxygenase that uses a mixed-valent diiron cofactor to enact oxidative cleavage of the C-P bond of its substrate, yielding glycine betaine and phosphate. The high specificities of TmpA and TmpB for their N-trimethylated substrates suggest that they have evolved specifically to degrade TMAEP, which was not previously known to be subject to microbial catabolism. This study thus adds to the growing list of known pathways through which microbes break down organophosphonates to harvest phosphorus, carbon, and nitrogen in nutrient-limited niches.

Sphingoid bases of dietary ceramide 2-aminoethylphosphonate, a marine sphingolipid, absorb into lymph in rats.

Various functions of dietary sphingolipids have been reported; however, little is known about marine sphingolipids. Ceramide 2-aminoethylphosphonate (CAEP), an abundant sphingolipid in marine mollusks, frequently has a unique triene type of sphingoid base [2-amino-9-methyl-4,8,10-octadecatriene-1,3-diol (d19:3)]. We previously reported that dietary CAEP prepared from the skin of squid was digested in the intestinal mucosa of mice via ceramides to yield free sphingoid bases. How dietary CAEP is then used in the body remains unclear. Here, we investigated the absorption of dietary CAEP using a lipid absorption assay on the lymph collected from rats with thoracic duct cannulation. Our results reveal that sphingoid bases derived from CAEP, including d16:1, d18:1, and d19:3, were detected in the lymph after administration of CAEP. Lymphatic recovery of d19:3 was lower than that of other sphingoid bases. A large fraction of the absorbed sphingoid bases was present as complex sphingolipids, whereas a smaller portion was present in the free form. Fatty acids in ceramide moieties found in the lymph were partially different from dietary CAEP, which indicates that sphingoid bases derived from CAEP could be, at least in part, resynthesized into complex sphingolipids. Future studies should elucidate the metabolism of sphingoid bases derived from CAEP.

Synthesis and biological analysis of novel glycoside derivatives of l-AEP, as targeted antibacterial agents.

To develop targeted methods for treating bacterial infections, the feasibility of using glycoside derivatives of the antibacterial compound l-R-aminoethylphosphonic acid (l-AEP) has been investigated. These derivatives are hypothesized to be taken up by bacterial cells via carbohydrate uptake mechanisms, and then hydrolyzed in situ by bacterial borne glycosidase enzymes, to selectively afford l-AEP. Therefore the synthesis and analysis of ten glycoside derivatives of l-AEP, for selective targeting of specific bacteria, is reported. The ability of these derivatives to inhibit the growth of a panel of Gram-negative bacteria in two different media is discussed. ß-Glycosides (12a) and (12b) that contained l-AEP linked to glucose or galactose via a carbamate linkage inhibited growth of a range of organisms with the best MICs being <0.75mg/ml; for most species the inhibition was closely related to the hydrolysis of the equivalent chromogenic glycosides. This suggests that for (12a) and (12b), release of l-AEP was indeed dependent upon the presence of the respective glycosidase enzyme.

Combined C-H functionalization/O-H insertion reaction to form tertiary ß-alkoxy substituted ß-aminophosphonates catalyzed by [Cu(MeCN)4]PF6.

The copper-catalyzed C-H functionalization/O-H insertion reaction of α-diazophosphonates with alcohols has been developed with iodine as an additive. In order to understand this reaction, we present here a possible mechanism for the combined reaction. This process provides straightforward access to tertiary ß-alkoxy substituted ß-aminophosphonate derivatives with moderate to good yields.

Probing a coral genome for components of the photoprotective scytonemin biosynthetic pathway and the 2-aminoethylphosphonate pathway.

Genome sequences of the reef-building coral, Acropora digitifera, have been decoded. Acropora inhabits an environment with intense ultraviolet exposure and hosts the photosynthetic endosymbiont, Symbiodinium. Acropora homologs of all four genes necessary for biosynthesis of the photoprotective cyanobacterial compound, shinorine, are present. Among metazoans, these genes are found only in anthozoans. To gain further evolutionary insights into biosynthesis of photoprotective compounds and associated coral proteins, we surveyed the Acropora genome for 18 clustered genes involved in cyanobacterial synthesis of the anti-UV compound, scytonemin, even though it had not previously been detected in corals. We identified candidates for only 6 of the 18 genes, including tyrP, scyA, and scyB. Therefore, it does not appear that Acropora digitifera can synthesize scytonemin independently. On the other hand, molecular phylogenetic analysis showed that one tyrosinase gene is an ortholog of vertebrate tyrosinase genes and that the coral homologs, scyA and scyB, are similar to bacterial metabolic genes, phosphonopyruvate (ppyr) decarboxylase and glutamate dehydrogenase (GDH), respectively. Further genomic searches for ppyr gene-related biosynthetic components indicate that the coral possesses a metabolic pathway similar to the bacterial 2-aminoethylphosphonate (AEP) biosynthetic pathway. The results suggest that de novo synthesis of carbon-phosphorus compounds is performed in corals.

(AEDPH3)·(BtaH): a novel supramolecular plaster with formaldehyde adsorption and formaldehyde/ultraviolet ray-induced luminescence switching performance.

A novel supramolecular plaster, (AEDPH(3))·(BtaH) (1), is synthesised and characterized. The supramolecular plaster is easy to synthesise and process, and displays good mechanical properties. It can adsorb and eliminate formaldehyde (HCHO) with high efficiency and exhibits very interesting HCHO/ultraviolet ray-induced luminescence switching.

Binding affinity of surface functionalized gold nanoparticles to hydroxyapatite.

Gold nanoparticles (Au NPs) have been investigated for a number of biomedical applications, including drug and gene delivery vehicles, thermal ablation therapy, diagnostic sensors, and imaging contrast agents. Surface functionalization with molecular groups exhibiting calcium affinity can enable targeted delivery of Au NPs to calcified tissue, including damaged bone tissue. Therefore, the objective of this study was to investigate the binding affinity of functionalized Au NPs for targeted delivery to bone mineral, using hydroxyapatite (HA) crystals as a synthetic analog in vitro. Au NPs were synthesized to a mean particle size of 10-15 nm and surface functionalized with either L-glutamic acid, 2-aminoethylphosphonic acid, or alendronate, which exhibit a primary amine for binding gold opposite carboxylate, phosphonate, or bisphosphonate groups, respectively, for targeting calcium. Bisphosphonate functionalized Au NPs exhibited the most rapid binding kinetics and greatest binding affinity to HA, followed by glutamic acid and phosphonic acid. All functional groups reached complete binding after 24 h. Equilibrium binding constants in de-ionized water, determined by nonlinear regression of Langmuir isotherms, were 3.40, 0.69, and 0.25 mg/L for bisphosphonate, carboxylate, and phosphonate functionalized Au NPs, respectively. Functionalized Au NPs exhibited lower overall binding in fetal bovine serum compared to de-ionized water, but relative differences between functional groups were similar.

Structural analysis of O-glycans of mucin from jellyfish (Aurelia aurita) containing 2-aminoethylphosphonate.

The structure of O-glycan in qniumucin (Q-mucin), which is a novel mucin extracted from jellyfish, was analyzed by a combination of NMR and ESI-MS/MS. A previously unidentified monosaccharide involved in the glycan chains was determined to be N-acetylgalactosamine (GalNAc) substituted by 2-aminoethylphosphonate (AEP) at the C-6. The O-glycans in Q-mucin from Aurelia aurita were proved to be mainly composed of three monosaccharides: GalNAc, AEP-(O-->6)-GalNAc, and P-6-GalNAc. To the best of our knowledge, this is the first example of an O-glycan structure of glycoproteins containing AEP. This exceptionally simple structure of Q-mucin and its potential use in material science and technology are revealed.

In vitro and in vivo anti-angiogenic activities and inhibition of hormone-dependent and -independent breast cancer cells by ceramide methylaminoethylphosphonate.

Ceramide methylaminoethylphosphonate (CMAEPn) was isolated from eastern oyster ( Crassostrea virginica ) and screened against in vitro and in vivo angiogenesis and against MCF-7 and MDA-MB-435s breast cancer cell lines. In vitro angiogenesis was evaluated by the vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cell (HUVEC) tube formation assay. MCF-7 and MDA-MB-435s cell viability was evaluated by the CellTiter 96 AQ(ueous) One Solution Cell Proliferation assay. Apoptosis was evaluated by the caspase-9 assay, autophagy by acridine orange staining and beclin-1 level. Our study indicates that CMAEPn at 50 microM inhibited VEGF-induced tube formation by HUVEC. The viability of MCF-7 and MDA-MB-435s breast cancer cells exposed to 125 microM CMAEPn for 48 h was reduced to 76 and 85%, respectively. The viability of MCF-7 and MDA-MB-435s cells exposed to 250 microM CMAEPn for 48 h under the same conditions was reduced to 38 and 45%, respectively. CMAEPn at 125 microM inhibited VEGF-induced MDA-MB-435s cell migration and invasion. CMAEPn at 125 microM also decreased VEGF, EGF levels in the conditioned media, PI3K, IkappaB phosphorylation and degradation in the cytoplasmic extracts, and NFkappaB nuclear translocation. Both acridine orange staining and beclin-1 indicated autophagic cell death in MCF-7 and MDA-MB-435s cells, respectively. In vivo, CMAEPn at 30 mg/kg body weight inhibited bFGF-induced angiogenesis and caused a 57% reduction in hemoglobin levels in the matrigel plug assay within 7 days. This is the first report on CMAEPn-inhibited angiogenesis both in vitro and in vivo.

Lipid classes and fatty acid composition of the tropical nudibranch mollusks Chromodoris sp. and Phyllidia coelestis.

Two nudibranch mollusks, Chromodoris sp. and Phyllidia coelestis, collected from tropical waters of the Northwestern Pacific, were analyzed for lipids. The aim of this study was to fill the gap in knowledge of lipid biochemistry of mollusks. Phospholipids (PL) were the dominating lipid class followed by sterols (13%). Neutral lipids were not detected in Chromodoris sp. By contrast, P. coelestis contained TAG, diacylglyceryl ether, long chain alcohol and esters of sterols. Among PL, PC was predominant (about 50%); PE, PS and CAEP were almost in equal proportions. Sixty five FA were identified as methyl esters and N-acyl pyrrolidides by GC-MS. The sea slugs exhibited a wide diversity of FA. The common marine n-3 PUFA, 20:5n-6 and 22:6n-3, constituted 0.6-1.3% of the total FA, whereas n-6 PUFA, 22:4n-6, 20:4n-6, and 18:2n-6, were the main (25%). Among monounsaturated FA, 7-21:1 was the main (up to 6.2%). The non-methylene-interrupted (NMI) FA were found (9.4 and 12.4%), including the known 5,11-20:2, 5,13-20:2, 7,13-22:2, 7,15-22:2 and a novel isomer 7,13-21:2 (up to 3.9%). The pathway of its biosynthesis was suggested. A series of very long chain FA (VLC FA), with the main 5,9-25:2 and 5,9-26:2, were identified. High level of VLC FA (8.7 and 11.7%) in sea slugs is apparently the result of predation on sponges. Another unique feature concerned a high abundance of various odd and branched FA (16.7 and 34%), which could have originated from the dietary origin or symbiotic bacteria. This is the first report on lipid and FA composition of nudibranchs.

Glyco- and sphingophosphonolipids from the medusa Phyllorhiza punctata: NMR and ESI-MS/MS fingerprints.

The medusa Phyllorhiza punctata has been found in Brazilian waters where it is an exotic species, having arrived in ballasts from the Indo-Pacific Ocean in the general region of North Australia and Indonesia. Fatty acids of the intact animal and its component umbrella, oral arms, and mucus were identified. Two different groups of glycolipids and a sphingolipid were isolated by silica-gel column chromatography and characterized using GC-MS, ESI-MS, 1D, 2D (13)C, (1)H and (31)P NMR spectroscopy. They were sulfoquinovosyldiacylglycerol (SQDG), monogalactosyldiacylglycerol (MGDG), and ceramide aminoethylphosphonate (CAEP). The CAEP long chain base (LCB) and its polar head group (PHG) formed by partial hydrolysis, were analyzed by ESI-MS/MS. The probable origin of MGDG and SQDG in the jellyfish is the result of an endosymbiotic association with a microalga of the Dinoflagellate group, since these lipids are commonly found in photosynthetic membranes.

Inhibition of rabbit brain 4-aminobutyrate transaminase by some taurine analogues: a kinetic analysis.

The use of the antiepileptic drug, 4-aminobutyrate transaminase (GABA-T) inhibitor vigabatrin (VIGA), has been recently cautioned because it is associated to irreversible field defects from damage of the retina. Since novel GABA-T inhibitors might prove useful in epilepsy or other CNS pathologies as VIGA substitutes, the aim of the present investigation was to characterize the biochemical properties of some taurine analogues (TA) previously shown to act as GABA-T inhibitors. These include (+/-)piperidine-3-sulfonic acid (PSA), 2-aminoethylphosphonic acid (AEP), (+/-)2-acetylaminocyclohexane sulfonic acid (ATAHS) and 2-aminobenzenesulfonate (ANSA). Kinetic analysis of the activity of partially purified rabbit brain GABA-T in the presence of VIGA and TA showed that PSA and AEP caused a linear, mixed-type inhibition (Ki values 364 and 1010 microM, respectively), whereas VIGA, ANSA and ATAHS behaved like competitive inhibitors (Ki values 320, 434 and 598 microM, respectively). Among the compounds studied, only VIGA exerted a time-dependent, irreversible inhibition of the enzyme, with Ki and k(inact) values of 773 microM and 0.14 min(-1), respectively. Furthermore, the ability of VIGA and TA to enhance GABA-ergic transmission was assessed in rabbit brain cortical slices by NMR quantitative analysis. The results demonstrate that VIGA as well as all TA promoted a significant increase of GABA content. In conclusion, PSA, ANSA and ATAHS, reversible GABA-T inhibitors with Ki values close to that of VIGA, represent a new class of compounds, susceptible of therapeutic exploitation in many disorders associated with low levels of GABA in brain tissues.

High performance carbon-supported catalysts for fuel cells via phosphonation.

Carbon-supported catalysts were phosphonated using 2-aminoethylphosphonic acid, and the resulting catalysts with largely enhanced proton conductivity performed substantially better than the untreated counterparts in proton-exchange membrane fuel cells.

Degradation pathway of the phosphonate ciliatine: crystal structure of 2-aminoethylphosphonate transaminase.

Phosphonates allow certain organisms to thrive in otherwise hostile environments, and 2-aminoethylphosphonate (AEP) is a precursor of many cellular phosphonates. AEP transaminase (AEPT) is an enzyme essential to phosphonate synthesis and degradation pathways. The crystal structure of AEP transaminase was determined by multiwavelength anomalous diffraction of 66 selenium atoms. The refined structure at 2.2 A resolution revealed an overall fold and active site location similar to those of the dimeric, two-domain structure of type I aminotransferases. The active site contains a cofactor, pyridoxal 5'-phosphate (PLP), and the product phosphonoacetaldehyde. Comparison with other type I aminotransferase structures shows that the PLP-protein interactions are conserved. Modeling of bound substrates and products reveals the structural basis for AEP recognition and the stereospecificity of proton elimination at the alpha-carbon and indicates conformational changes along the reaction pathway.

Reaction of alanine racemase with 1-aminoethylphosphonic acid forms a stable external aldimine.

(R)-1-Aminoethylphosphonic acid (L-Ala-P), a synthetic L-alanine analogue, has antibacterial activity and is a time-dependent inactivator of all purified Gram-positive bacterial alanine racemases that have been tested. L-Ala-P forms an external aldimine with the bound pyridoxal 5'-phosphate (PLP) cofactor, but is neither racemized nor efficiently hydrolyzed. To understand the structural basis of the inactivation of the enzyme by L-Ala-P, we determined the crystal structure of the complex between L-Ala-P and alanine racemase at 1.6 A resolution. The cofactor derivative in the inhibited structure tilts outward from the protein approximately 20 degrees relative to the internal aldimine. The phosphonate oxygens are within hydrogen bonding distance of four amino acid residues and two water molecules in the active site of the enzyme. L-Ala-P is an effective inhibitor of alanine racemase because, upon formation of the external aldimine, the phosphonate group interacts with putative catalytic residues, thereby rendering them unavailable for catalysis. Furthermore, this aldimine appears to be inappropriately aligned for efficient Calpha proton abstraction. The combination of these effects leads to a stable aldimine derivative and potent inactivation of alanine racemase by this compound.

Aromatic L-amino acid decarboxylase: conformational change in the flexible region around Arg334 is required during the transaldimination process.

Aromatic L-amino acid decarboxylase (AADC) catalytic mechanism has been proposed to proceed through two consecutive intermediates (i.e., Michaelis complex and the external aldimine). Limited proteolysis of AADC that preferentially digested at the C-terminal side of Arg334 was slightly retarded in the presence of dihydroxyphenyl acetate that formed a stable Michaelis complex. On the contrary, AADC was scarcely digested in the presence of L-dopa methyl ester that formed a stable external aldimine. Similar protection by the substrate analogs was observed in the chemical modification experiment. From these results, we concluded that the region around Arg334 must be exposed and flexible in the unliganded state, and forming the Michaelis complex generated a subtle conformational change, then underwent marked conformational change during the subsequent transaldimination process prerequisite to forming the external aldimine. For further analyses, we constructed a mutant gene encoding in tandem the two peptides of AADC cleaved at the Asn327-Met328 bond inside the putative flexible region. The gene product, fragmentary AADC, was still active with L-dopa as substrate, but its k(cat) value was decreased 57-fold, and the Km value was increased 9-fold compared with those of the wild-type AADC. The absorption spectra of the fragmentary AADC in the presence of L-dopa methyl ester showed shift in the equilibrium of the transaldimination from the external aldimine to the Michaelis complex. Tryptic digestion of the fragmentary AADC removed seven amino acid residues, Met328-Arg334, and resulted in complete inactivation. Susceptibility of the fragmentary enzyme to trypsin was not changed by L-dopa methyl ester revealing the loss of appropriate conformational change in the flexible region induced by substrate binding. From these results we propose that the conformational change in the flexible region is required during the transaldimination process.