Immediate adequacy assessment (IADA) during fine-needle aspiration (FNA) is not universal and the optimal number of passes has not been well determined. The aim of this study was to evaluate the nondiagnostic rates (NDR) with and without the IADA forthyroid aspirates. Subsequent cytological and surgical follow-up were reviewed for non-diagnostic cases. In addition, we evaluated the number of passes performed in each FNA to determine the optimal number. Retrospective analysis of NDR was performed on 883 thyroid FNA specimens retrieved through a Computer SNOMED Search from our files between January 2001 to December 2003. For FNAs with IADA, one Diff-Quick and one fixed smear for each pass were prepared, and the needle was rinsed in CytoLyt solution for a ThinPrep and/or a cell-block. FNAs without IADAwere received in CytoLyt solution, from which a ThinPrep and a cell-block were prepared for each case. Of the total 883 cases, 443 were performed with IADA, of which 417 cases were diagnostic. The remaining 440 cases were performed without IADA, of which 300 cases were diagnostic.NDR for IADA was 5.9% (26 cases-group-I)compared to 31.8% (140 cases-group-II)without IADA. In group-I, 5 cases were followed-up by repeat FNA, 10 cases by surgical resection, and 11 cases received no tissue follow-up. In group-II, 23 cases were followed-up by repeat FNA, 36 by surgical resection, and 82 cases received no tissue follow-up. Interestingly, follow-up in group-I did not reveal any missed malignancy, while that in group-II resulted in a malignant diagnosis in 13.8% (8 cases). We also found that the optimal number of passes with least NDR was 4-6 passes.NDR was 25% for < 3 passes, 11% for 4 passes, 5.2% for 5 passes, 1.4% for 6 passes, and 2.5% for 7 passesor more. IADA significantly reduces the NDR and increases the sample adequacy for diagnosis. Optimal number of passes is 4-6 passes, and additional passes did not improve the diagnostic rate. Our study also emphasizes the significance of repeat FNA or histological follow-up for nondiagnostic cases, especially for those without IADA.

Although it is a member of the amidohydrolase superfamily, LigW catalyzes the nonoxidative decarboxylation of 5-carboxyvanillate to form vanillate in the metabolic pathway for bacterial lignin degradation. We now show that membrane inlet mass spectrometry (MIMS) can be used to measure transient CO2 concentrations in real time, thereby permitting us to establish that C−C bond cleavage proceeds to give CO2 rather than HCO3 − as the initial product in the LigW-catalyzed reaction. Thus, incubation of LigW at pH 7.0 with the substrate 5-carboxyvanillate results in an initial burst of CO2 formation that gradually decreases to an equilibrium value as CO2 is nonenzymatically hydrated to HCO3 − . The burst of CO2 is completely eliminated with the simultaneous addition of substrate and excess carbonic anhydrase to the enzyme, demonstrating that CO2 is the initial reaction product. This finding is fully consistent with the results of density functional theory calculations, which also provide support for a mechanism in which protonation of the C5 carbon takes place prior to C−C bond cleavage. The calculated barrier of 16.8 kcal/mol for the rate-limiting step, the formation of the C5-protonated intermediate, compares well with the observed kcat value of 27 s − 1 for Sphingomonas paucimobilis LigW, which corresponds to an energy barrier of ∼16 kcal/mol. The MIMS-based strategy is superior to alternate methods of establishing whether CO2 or HCO3 − is the initial reaction product, such as the use of pH-dependent dyes to monitor very small changes in solution pH. Moreover, the MIMS-based assay is generally applicable to studies of all enzymes that produce and/or consume small-molecule, neutral gases. KEYWORDS: decarboxylase, membrane inlet mass spectrometry, reaction mechanism, cluster approach, density functional theory, quantum chemistry ■ INTRODUCTION5-Carboxyvanillate decarboxylase (LigW) catalyzes the formation of vanillate (3-methoxy-4-hydroxybenzoate) via the nonoxidative decarboxylation reaction shown in Scheme 1a. LigW is an integral component within the biochemical degradation pathway of lignin from plant biomass by bacteria. Although LigW is a member of the amidohydrolase superfamily (AHS), this enzyme is located within a subset from cog2159 that catalyzes decarboxylation reactions rather than the much more common hydrolysis of carboxylate and phosphate esters performed by other AHS members.1 Recently, we interrogated the chemical reaction mechanism of LigW using the enzymes from Sphingomonas paucimobilis and Novosphingobium aromaticivorans by high-resolution X-ray crystallography, mutation of active site residues, substrate−activity relationships, and product isotope eff ects. 2 These studies led to the conclusion that LigW catalyzes the decarboxylation of 5-carboxyvanillate (5-CV) via a reaction intermediate formed by protonation of the substrate at C5 prior to carbon−carbon bond cleavage, as shown in Scheme 1b.Experimental support for the existence of the C5-protonated intermediate was provided by the 1.0...

Galangin, a natural flavonol, has attracted much attention for its potential anti-inflammatory properties. However, its role in the regulation of airway remodelling in asthma has not been explored. The present study aimed to elucidate the effects of galangin on chronic inflammation and airway remodelling and to investigate the underlying mechanisms both in vivo and in vitro. Ovalbumin (OVA)-sensitised mice were administered with galangin 30 min before challenge. Our results showed that severe inflammatory responses and airway remodelling occurred in OVA-induced mice. Treatment with galangin markedly attenuated the leakage of inflammatory cells into bronchoalveolar lavage fluid (BALF) and decreased the level of OVA-specific IgE in serum. Galangin significantly inhibited goblet cell hyperplasia, collagen deposition and α-SMA expression. Lowered level of TGF-β1 and suppressed expression of VEGF and MMP-9 were observed in BALF or lung tissue, implying that galangin has an optimal anti-remodelling effect in vivo. Consistently, the TGF-β1-induced proliferation of airway smooth muscle cells was reduced by galangin in vitro, which might be due to the alleviation of ROS levels and inhibition of MAPK pathway. Taken together, the present findings highlight a novel role for galangin as a promising anti-remodelling agent in asthma, which likely involves the TGF-β1-ROS-MAPK pathway.

ObjectiveThe objective of this survey was to study the association between Helicobacter pylori infection and the severity of coronary atherosclerosis.MethodsThe study population consisted of 961 consecutive patients (711 males and 250 females) who underwent coronary angiography for suspected or known coronary atherosclerosis. The patients' body mass index, blood pressure, the blood lipid, blood glucose, leukocyte count (109/L), neutrophil count (109/L), and Helicobacter pylori-specific IgG antibodies were performed. Coronary angiograms were scored according to vessel score and Gensini's score.ResultsA significant association between H. pylori infection and coronary atherosclerosis as well as its severity was not find in this cross section study (p = 0.858). And, the level distribution of vessel score (p = 0.906) and Gensini's score (p = 0.905) were similar in the seropositivity group and seronegativity group of Helicobacter pylori infection. However, the level of fasting high-density lipoprotein cholesterol (mmol/L) (p = 0.013) was significantly lower in the seropositivity group than that in the seronegativity group of Helicobacter pylori infection.ConclusionsIn conclusion, in the present study, a significantly correlation between Helicobacter pylori seropositivity and angiographically evaluated severity of atherosclerosis was not find. And, the present study showed a good correlation between Helicobacter pylori infection and decreased HDL cholesterol. However, the exact mechanisms need further study.

Expression of human asparagine synthetase (ASNS) promotes metastatic progression and tumor cell invasiveness in colorectal and breast cancer, presumably by altering cellular levels of L-asparagine. Human ASNS is therefore emerging as a bona fide drug target for cancer therapy. Here we show that a slow-onset, tight binding inhibitor, which exhibits nanomolar affinity for human ASNS in vitro, exhibits excellent selectivity at 10 μM concentration in HCT-116 cell lysates with almost no off-target binding. The high-resolution (1.85 Å) crystal structure of human ASNS has enabled us to identify a cluster of negatively charged side chains in the synthetase domain that plays a key role in inhibitor binding. Comparing this structure with those of evolutionarily related AMP-forming enzymes provides insights into intermolecular interactions that give rise to the observed binding selectivity. Our findings demonstrate the feasibility of developing second generation human ASNS inhibitors as lead compounds for the discovery of drugs against metastasis.

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Oxalate decarboxylase (OxDC) catalyzes the disproportionation of oxalic acid monoanion into CO2 and formate. The enzyme has long been hypothesized to utilize dioxygen to form mononuclear Mn(III) or Mn(IV) in the catalytic site during turnover. Recombinant OxDC, however, contains only tightly bound Mn(II), and direct spectroscopic detection of the metal in higher oxidation states under optimal catalytic conditions (pH 4.2) has not yet been reported. Using parallel mode electron paramagnetic resonance spectroscopy, we now show that substantial amounts of Mn(III) are indeed formed in OxDC, but only in the presence of oxalate and dioxygen under acidic conditions. These observations provide the first direct support for proposals in which Mn(III) removes an electron from the substrate to yield a radical intermediate in which the barrier to C-C bond cleavage is significantly decreased. Thus, OxDC joins a small list of enzymes capable of stabilizing and controlling the reactivity of the powerful oxidizing species Mn(III).

Pyrroloquinoline quinone (PQQ) is an important redox active quinocofactor produced by a wide variety of bacteria. A key step in PQQ biosynthesis is a carbon−carbon cross-link reaction between glutamate and tyrosine side chains within the ribosomally synthesized peptide substrate PqqA. This reaction is catalyzed by the radical SAM enzyme PqqE. Previous X-ray crystallographic and spectroscopic studies suggested that PqqE, like the other members of the SPASM domain family, contains two auxiliary Fe−S clusters (AuxI and AuxII) in addition to the radical SAM [4Fe−4S] cluster. However, a clear assignment of the electron paramagnetic resonance (EPR) signal of each Fe−S cluster was hindered by the isolation of a His 6tagged PqqE variant with an altered AuxI cluster. In this work, we are able to isolate soluble PqqE variants by using a less disruptive strep-tactin chromatographic approach. We have unambiguously identified the EPR signatures for four forms of Fe−S clusters present in PqqE through the use of multifrequency EPR spectroscopy: the RS [4Fe−4S] cluster, the AuxII [4Fe−4S] cluster, and two different clusters ([4Fe−4S] and [2Fe−2S]) bound in the AuxI site. The RS [4Fe−4S] cluster, the AuxII [4Fe−4S] cluster, and the [2Fe−2S] cluster form in the AuxI site can all be reduced by sodium dithionite, with g tensors of their reduced form determined as [2.040, 1.927, 1.897], [2. 059, 1.940, 1.903], and [2.004, 1.958, 1.904], respectively. The AuxI [4Fe−4S] cluster that is determined on the basis of its relaxation profile can be reduced only by using low-potential reductants such as Ti(III) citrate or Eu(II)-DTPA to give rise to a g 1 = 2.104 signal. Identification of the EPR signature for each cluster paves the way for further investigations of SPASM domain radical SAM enzymes.