Molecular recognition of 2,4,6-trinitrotoluene by 6-aminohexanethiol and surface-enhanced Raman scattering sensor

2,4,6-trinitrotoluene (TNT) is one of the most commonly used nitro aromatic explosives in landmine, military and mining industry. This article demonstrates rapid and selective identification of TNT by surface-enhanced Raman spectroscopy (SERS) using 6-aminohexanethiol (AHT) as a new recognition molecule. First, Meisenheimer complex formation between AHT and TNT is confirmed by the development of pink colour and appearance of new band around 500 nm in UV-visible spectrum. Solution Raman spectroscopy study also supported the AHT:TNT complex formation by demonstrating changes in the vibrational stretching of AHT molecule between 2800-3000 cm−1. For surface enhanced Raman spectroscopy analysis, a self-assembled monolayer (SAM) of AHT is formed over the gold nanostructure (AuNS) SERS substrate in order to selectively capture TNT onto the surface. Electrochemical desorption and X-ray photoelectron studies are performed over AHT SAM modified surface to examine the presence of free amine groups with appropriate orientation for complex formation. Further, AHT and butanethiol (BT) mixed monolayer system is explored to improve the AHT:TNT complex formation efficiency. Using a 9:1 AHT:BT mixed monolayer, a very low detection limit (LOD) of 100 fM TNT was realized. The new method delivers high selectivity towards TNT over 2,4 DNT and picric acid. Finally, real sample analysis is demonstrated by the extraction and SERS detection of 302 pM of TNT from spiked.

Supramolecular selection in molecular alloys

Complexes of the type \[M(phen)3](PF6)2 (M = Ni(II), Fe(II), Ru(II) and phen = 1,10-phenanthroline) were found to co-crystallize to form molecular alloys (solid solutions of molecules) with general formula \[MAxMB1–x(phen)3](PF6)2·0.5H2O in which the relative concentrations of the metal complexes in the crystals closely match those in the crystallizing solution. Consequently, the composition of the co-crystals can be accurately predicted and controlled by modulating the relative concentrations of the metal complexes in the crystallizing solution. Although they are chemically and structurally similar, complexes of the type \[M(bipy)3](PF6)2 (M = Ni(II), Fe(II), Ru(II) and bipy = 2,2′-bipyridine) display markedly different behavior upon co-crystallization. In this case, the resulting co-crystals of general formula \[MAxMB1–x(bipy)3](PF6)2 have relative concentrations of the constituent complexes that are markedly different from the relative concentrations of the complexes initially present in the crystallizing solution. For example, when the nickel and iron complexes are co-crystallized from a solution containing a 50:50 ratio of each, the result is the formation of some crystals with a higher proportion of iron and others with a higher proportion of nickel. The relative concentrations of the metal complexes in the crystals can vary from those in the crystallizing solutions by as much as 15%. This result was observed for a range of combinations of metal complexes (Ni/Fe, Ni/Ru, and Fe/Ru) and a range of starting concentrations in the crystallizing solutions (90:10 through to 10:90 in 10% increments). To explain this remarkable result, we introduce the concept of “supramolecular selection”, which is a process driven by molecular recognition that leads to the partially selective aggregation of like molecules during crystallization.

Influences of molecular structure on co-crystallization of polypyridyl metal complexes.

Heteroleptic complexes of the type \[RuL2L′](PF6)2 (L, L′ = combinations of 1,10-phenanthroline (phen) and 2,2′-bipyridine (bipy)) were found to cocrystallize with \[Ni(phen)3](PF6)2 to produce cocrystals of \[Ni(phen)3]x\[RuL2L′]1–x(PF6)2. In this report we show that the ability of the complexes to cocrystallize is influenced by the number of common ligands between complexes in solution. Supramolecular selection is a phenomenon caused by molecular recognition through which cocrystals can grow from the same solution but contain different ratios of the molecular components. It was found that systems where L = phen displayed less supramolecular selection than systems where L = bipy. With increasing supramolecular selection, the composition of cocrystals was found to vary significantly from the initial relative concentration in the cocrystallizing solution, and therefore it was increasingly difficult to control the final composition of the resultant cocrystals. Consequently, modulation of concentration-dependent properties such as phase was also found to be less predictable with increasing supramolecular selection. Notwithstanding the complication afforded by the presence of supramolecular selection, our results reaffirm the robustness of the \[M(phen)3](PF6)2 structure because it was maintained even when ca. 90% of the complexes in the cocrystals were \[Ru(phen)(bipy)2](PF6)2, which in its pure form is not isomorphous with \[M(phen)3](PF6)2. Experiments between complexes without common ligands, i.e., \[Ru(bipy)3](PF6)2 cocrystallized with \[Ni(phen)3](PF6)2, were found to approach the limit to which molecular recognition processes can be confused into cocrystallizing different molecules to form single cocrystals. For these systems the result was the formation of block-shaped crystals skewered by a needle-shaped crystals.

Exploiting the 1,2,3-triazolium motif in anion-templated formation of a bromide-selective rotaxane host assembly

Stimulated by the efficacy of copper (I) catalysed Huisgen-type 1,3-dipolar cycloaddition of terminal alkynes and organic azides to generate 1,4-disubstituted 1,2,3-triazole derivatives, the importance of ‘click’ chemistry in the synthesis of organic and biological molecular systems is ever increasing.[1] The mild reaction conditions have also led to this reaction gaining favour in the construction of interlocked molecular architectures.[2-4] In the majority of cases however, the triazole group simply serves as a covalent linkage with no function in the resulting organic molecular framework. More recently a renewed interest has been shown in the transition metal coordination chemistry of triazole ligands.[3, 5, 6] In addition novel aryl macrocyclic and acyclic triazole based oligomers have been shown to recognise halide anions via cooperative triazole C5-H….anion hydrogen bonds.[7] In light of this it is surprising the potential anion binding affinity of the positively charged triazolium motif has not, with one notable exception,[8] been investigated. With the objective of manipulating the unique topological cavities of mechanically bonded molecules for anion recognition purposes, we have developed general methods of using anions to template the formation of interpenetrated and interlocked structures.[9-13] Herein we report the first examples of exploiting the 1,2,3-triazolium group in the anion templated formation of pseudorotaxane and rotaxane assemblies. In an unprecedented discovery the bromide anion is shown to be a superior templating reagent to chloride in the synthesis of a novel triazolium axle containing [2]rotaxane. Furthermore the resulting rotaxane interlocked host system exhibits the rare selectivity preference for bromide over chloride...

A Survey of the 2010 Quartz Crystal Microbalance Literature

In 2010 there has again been an increase in the number of papers published involving piezoelectric acoustic sensors, or quartz crystal microbalances (QCM), when compared to the last period reviewed 2006-2009. The average number of QCM publications per annum was 124 in the period 2001-2005, 223 in the period 2006-9, and 273 in 2010. There are trends towards increasing use of QCM in the study of protein adsorption to surfaces (93% increase), homeostasis (67% increase), protein-protein interactions (40% increase), and carbohydrates (43% increase). New commercial systems have been released that are driving the uptake of the technology for characterisation of binding specificities, affinities, kinetics and conformational changes associated with a molecular recognition event. This article highlights theoretical and practical aspects of the principals that underpin acoustic analysis, then reviews exemplary papers in key application areas involving small molecular weight ligands, carbohydrates, proteins, nucleic acids, viruses, bacteria, cells, and membrane interfaces.

Distamycin A affects the stability of NF-kappaB p50-DNA complexes in a sequence-dependent manner

The effect of two different DNA minor groove binding molecules, Hoechst 33258 and distamycin A, on the binding kinetics of NF-κB p50 to three different specific DNA sequences was studied at various salt concentrations. Distamycin A was shown to significantly increase the dissociation rate constant of p50 from the sequences PRDII (5′-GGGAAATTCC-3′) and Ig-κ B (5′-GGGACTTTCC-3′) but had a negligible effect on the dissociation from the palindromic target-κB binding site (5′-GGGAATTCCC-3′). By comparison, the effect of Hoechst 33258 on binding of p50 to each sequence was found to be minimal. The dissociation rates for the protein–DNA complexes increased at higher potassium chloride concentrations for the PRDII and Ig-κB binding motifs and this effect was magnified by distamycin A. In contrast, p50 bound to the palindromic target-κB site with a much higher intrinsic affinity and exhibited a significantly reduced salt dependence of binding over the ionic strength range studied, retaining a KD of less than 10 pM at 150 mM KCl. Our results demonstrate that the DNA binding kinetics of p50 and their salt dependence is strongly sequence-dependent and, in addition, that the binding of p50 to DNA can be influenced by the addition of minor groove-binding drugs in a sequence-dependent manner.

Mutations in the gene encoding IFT dynein complex component WDR34 cause jeune asphyxiating thoracic dystrophy

Bidirectional (anterograde and retrograde) motor-based intraflagellar transport (IFT) governs cargo transport and delivery processes that are essential for primary cilia growth and maintenance and for hedgehog signaling functions. The IFT dynein-2 motor complex that regulates ciliary retrograde protein transport contains a heavy chain dynein ATPase/motor subunit, DYNC2H1, along with other less well functionally defined subunits. Deficiency of IFT proteins, including DYNC2H1, underlies a spectrum of skeletal ciliopathies. Here, by using exome sequencing and a targeted next-generation sequencing panel, we identified a total of 11 mutations in WDR34 in 9 families with the clinical diagnosis of Jeune syndrome (asphyxiating thoracic dystrophy). WDR34 encodes a WD40 repeat-containing protein orthologous to Chlamydomonas FAP133, a dynein intermediate chain associated with the retrograde intraflagellar transport motor. Three-dimensional protein modeling suggests that the identified mutations all affect residues critical for WDR34 protein-protein interactions. We find that WDR34 concentrates around the centrioles and basal bodies in mammalian cells, also showing axonemal staining. WDR34 coimmunoprecipitates with the dynein-1 light chain DYNLL1 in vitro, and mining of proteomics data suggests that WDR34 could represent a previously unrecognized link between the cytoplasmic dynein-1 and IFT dynein-2 motors. Together, these data show that WDR34 is critical for ciliary functions essential to normal development and survival, most probably as a previously unrecognized component of the mammalian dynein-IFT machinery.

Unique and cross-reactive T cell epitope peptides of the major bahia grass pollen allergen, pas n 1

Background Bahia grass pollen (BaGP) is a major cause of allergic rhinitis. Subcutaneous allergen-specific immunotherapy is effective for grass pollen allergy, but is unsuitable for patients with moderate to severe asthma due to the risk of anaphylaxis. T cell-reactive but IgE nonreactive peptides provide a safer treatment option. This study aimed to identify and characterize dominant CD4+ T cell epitope peptides of the major BaGP allergen, Pas n 1. Methods Pas n 1-specific T cell lines generated from the peripheral blood of BaGP-allergic subjects were tested for proliferative and cytokine response to overlapping 20-mer Pas n 1 peptides. Cross-reactivity to homologous peptides from Lol p 1 and Cyn d 1 of Ryegrass and Bermuda grass pollen, respectively, was assessed using Pas n 1 peptide-specific T cell clones. MHC class II restriction of Pas n 1 peptide T cell recognition was determined by HLA blocking assays and peptide IgE reactivity tested by dot blotting. Results Three Pas n 1 peptides showed dominant T cell reactivity; 15 of 18 (83%) patients responded to one or more of these peptides. T cell clones specific for dominant Pas n 1 peptides showed evidence of species-specific T cell reactivity as well as cross-reactivity with other group 1 grass pollen allergens. The dominant Pas n 1 T cell epitope peptides showed HLA binding diversity and were non-IgE reactive. Conclusions The immunodominant T cell-reactive Pas n 1 peptides are candidates for safe immunotherapy for individuals, including those with asthma, who are allergic to Bahia and possibly other grass pollens.

Chemotherapy and zoledronate sensitize solid tumour cells to Vγ9Vδ2 T cell cytotoxicity

Combinations of cellular immune-based therapies with chemotherapy and other antitumour agents may be of significant clinical benefit in the treatment of many forms of cancer. Gamma delta (γδ) T cells are of particular interest for use in such combined therapies due to their potent antitumour cytotoxicity and relative ease of generation in vitro. Here, we demonstrate high levels of cytotoxicity against solid tumour-derived cell lines with combination treatment utilizing Vγ9Vδ2 T cells, chemotherapeutic agents and the bisphosphonate, zoledronate. Pre-treatment with low concentrations of chemotherapeutic agents or zoledronate sensitized tumour cells to rapid killing by Vγ9Vδ2 T cells with levels of cytotoxicity approaching 90%. In addition, zoledronate enhanced the chemotherapy-induced sensitization of tumour cells to Vγ9Vδ2 T cell cytotoxicity resulting in almost 100% lysis of tumour targets in some cases. Vγ9Vδ2 T cell cytotoxicity was mediated by perforin following TCR-dependent and isoprenoid-mediated recognition of tumour cells. Production of IFN-γ by Vγ9Vδ2 T cells was also induced after exposure to sensitized targets. We conclude that administration of Vγ9Vδ2 T cells at suitable intervals after chemotherapy and zoledronate may substantially increase antitumour activities in a range of malignancies.

Metabolite profiling and pathway analysis of acute hepatitis rats by UPLC-ESI MS combined with pattern recognition methods

BACKGROUND & AIMS Metabolomics is comprehensive analysis of low-molecular-weight endogenous metabolites in a biological sample. It could enable mapping of perturbations of early biochemical changes in diseases and hence provide an opportunity to develop predictive biomarkers that could provide valuable insights into the mechanisms of diseases. The aim of this study was to elucidate the changes in endogenous metabolites and to phenotype the metabolic profiling of d-galactosamine (GalN)-inducing acute hepatitis in rats by UPLC-ESI MS. METHODS The systemic biochemical actions of GalN administration (ip, 400 mg/kg) have been investigated in male wistar rats using conventional clinical chemistry, liver histopathology and metabolomic analysis of UPLC- ESI MS of urine. The urine was collected predose (-24 to 0 h) and 0-24, 24-48, 48-72, 72-96 h post-dose. Mass spectrometry of the urine was analysed visually and via conjunction with multivariate data analysis. RESULTS Results demonstrated that there was a time-dependent biochemical effect of GalN dosed on the levels of a range of low-molecular-weight metabolites in urine, which was correlated with developing phase of the GalN-inducing acute hepatitis. Urinary excretion of beta-hydroxybutanoic acid and citric acid was decreased following GalN dosing, whereas that of glycocholic acid, indole-3-acetic acid, sphinganine, n-acetyl-l-phenylalanine, cholic acid and creatinine excretion was increased, which suggests that several key metabolic pathways such as energy metabolism, lipid metabolism and amino acid metabolism were perturbed by GalN. CONCLUSION This metabolomic investigation demonstrates that this robust non-invasive tool offers insight into the metabolic states of diseases.

Molecular and cellular analysis of basement membrane invasion by human breast cancer cells in Matrigel-based in vitro assays

In vitro analyses of basement membrane invasiveness employing Matrigel (a murine tumor extract rich in basement membrane components) have been performed on human breast cancer model systems. Constitutive invasiveness of different human breast cancer (HBC) cell lines has been examined as well as regulation by steroid hormones, growth factors, and oncogenes. Carcinoma cells exhibiting a mesenchymal-like phenotype (vimentin expression, lack of cell border associated uvomorulin) show dramatically increased motility, invasiveness, and metastatic potential in nude mice. These findings support the hypothesis that epithelial to mesenchymal transition (EMT)-like events may be instrumental in the metastatic progression of human breast cancer. The MCF-7 subline MCF-7ADR appears to have undergone such a transition. The importance of such a transition may be reflected in the emergence of vimentin expression as an indicator of poor prognosis in HBC. Matrix degradation and laminin recognition are highlighted as potential targets for antimetastatic therapy, and analyses of laminin attachment and the matrix metalloproteinase (MMP) family in HBC cell lines are summarized. Matrigel-based assays have proved useful in the study of the molecular mechanisms of basement membrane invasiveness, their regulation in HBC cells, and their potential as targets for antimetastatic therapy.

Yhh1p/Cft1p directly links poly(A) site recognition and RNA polymerase II transcription termination

RNA polymerase II (pol II) transcription termination requires co‐transcriptional recognition of a functional polyadenylation signal, but the molecular mechanisms that transduce this signal to pol II remain unclear. We show that Yhh1p/Cft1p, the yeast homologue of the mammalian AAUAAA interacting protein CPSF 160, is an RNA‐binding protein and provide evidence that it participates in poly(A) site recognition. Interestingly, RNA binding is mediated by a central domain composed of predicted β‐propeller‐forming repeats, which occurs in proteins of diverse cellular functions. We also found that Yhh1p/Cft1p bound specifically to the phosphorylated C‐terminal domain (CTD) of pol II in vitro and in a two‐hybrid test in vivo. Furthermore, transcriptional run‐on analysis demonstrated that yhh1 mutants were defective in transcription termination, suggesting that Yhh1p/Cft1p functions in the coupling of transcription and 3′‐end formation. We propose that direct interactions of Yhh1p/Cft1p with both the RNA transcript and the CTD are required to communicate poly(A) site recognition to elongating pol II to initiate transcription termination.

Nanostructured hydroxyapatite surfaces-mediated adsorption alters recognition of BMP receptor IA and bioactivity of bone morphogenetic protein-2

Highly efficient loading of bone morphogenetic protein-2 (BMP-2) onto carriers with desirable performance is still a major challenge in the field of bone regeneration. Till now, the nanoscaled surface-induced changes of the structure and bioactivity of BMP-2 remains poorly understood. Here, the effect of nanoscaled surface on the adsorption and bioactivity of BMP-2 was investigated with a series of hydroxyapatite surfaces (HAPs): HAP crystal-coated surface (HAP), HAP crystal-coated polished surface (HAP-Pol), and sintered HAP crystal-coated surface (HAP-Sin). The adsorption dynamics of recombinant human BMP-2 (rhBMP-2) and the accessibility of the binding epitopes of adsorbed rhBMP-2 for BMP receptors (BMPRs) were examined by a quartz crystal microbalance with dissipation. Moreover, the bioactivity of adsorbed rhBMP-2 and the BMP-induced Smad signaling were investigated with C2C12 model cells. A noticeably high mass-uptake of rhBMP-2 and enhanced recognition of BMPR-IA to adsorbed rhBMP-2 were found on the HAP-Pol surface. For the rhBMP-2-adsorbed HAPs, both ALP activity and Smad signaling increased in the order of HAP-Sin < HAP < HAP-Pol. Furthermore, hybrid molecular dynamics and steered molecular dynamics simulations validated that BMP-2 tightly anchored on the HAP-Pol surface with a relative loosened conformation, but the HAP-Sin surface induced a compact conformation of BMP-2. In conclusion, the nanostructured HAPs can modulate the way of adsorption of rhBMP-2, and thus the recognition of BMPR-IA and the bioactivity of rhBMP-2. These findings can provide insightful suggestions for the future design and fabrication of rhBMP-2-based scaffolds/implants.

Molecular and pathological insights into Chlamydia pecorum-associated sporadic bovine encephalomyelitis (SBE) in Western Australia

Background Despite its global recognition as a ruminant pathogen, cases of Chlamydia pecorum infection in Australian livestock are poorly documented. In this report, a C. pecorum specific Multi Locus Sequence Analysis scheme was used to characterise the C. pecorum strains implicated in two cases of sporadic bovine encephalomyelitis confirmed by necropsy, histopathology and immunohistochemistry. This report provides the first molecular evidence for the presence of mixed infections of C. pecorum strains in Australian cattle. Case presentation Affected animals were two markedly depressed, dehydrated and blind calves, 12 and 16 weeks old. The calves were euthanized and necropsied. In one calf, a severe fibrinous polyserositis was noted with excess joint fluid in all joints whereas in the other, no significant lesions were seen. No gross abnormalities were noted in the brain of either calf. Histopathological lesions seen in both calves included: multifocal, severe, subacute meningoencephalitis with vasculitis, fibrinocellular thrombosis and malacia; diffuse, mild, acute interstitial pneumonia; and diffuse, subacute epicarditis, severe in the calf with gross serositis. Immunohistochemical labelling of chlamydial antigen in brain, spleen and lung from the two affected calves and brain from two archived cases, localised the antigen to the cytoplasm of endothelium, mesothelium and macrophages. C. pecorum specific qPCR, showed dissemination of the pathogen to multiple organs. Phylogenetic comparisons with other C. pecorum bovine strains from Australia, Europe and the USA revealed the presence of two genetically distinct sequence types (ST). The predominant ST detected in the brain, heart, lung and liver of both calves was identical to the C. pecorum ST previously described in cases of SBE. A second ST detected in an ileal tissue sample from one of the calves, clustered with previously typed faecal bovine isolates. Conclusion This report provides the first data to suggest that identical C. pecorum STs may be associated with SBE in geographically separated countries and that these may be distinct from those found in the gastrointestinal tract. This report provides a platform for further investigations into SBE and for understanding the genetic relationships that exist between C. pecorum strains detected in association with other infectious diseases in livestock.

Infection-induced IL-10 and Jak-Stat: A review of the molecular circuitry controlling immune hyperactivity in response to pathogenic microbes

Generation of effective immune responses against pathogenic microbes depends on a fine balance between pro- and anti-inflammatory responses. Interleukin-10 (IL-10) is essential in regulating this balance and has garnered renewed interest recently as a modulator of the response to infection at the JAK-STAT signaling axis of host responses. Here, we examine how IL-10 functions as the “master regulator” of immune responses through JAK-STAT, and provide a perspective from recent insights on bacterial, protozoan, and viral infection model systems. Pattern recognition and subsequent molecular events that drive activation of IL-10-associated JAK-STAT circuitry are reviewed and the implications for microbial pathogenesis are discussed.