Isolation and characterization of two human transcription factor IIH (TFIIH)-related complexes: ERCC2/CAK and TFIIH.

Transcription factor IIH (TFIIH) is a multisubunit protein complex essential for both the initiation of RNA polymerase class II (pol II)-catalyzed transcription and nucleotide excision repair of DNA. Recent studies have shown that TFIIH copurifies with the cyclin-dependent kinase (cdk)-activating kinase complex (CAK) that includes cdk7, cyclin H, and p36/MAT1. Here we report the isolation of two TFIIH-related complexes: TFIIH* and ERCC2/CAK. TFIIH* consists of a subset of the TFIIH complex proteins including ERCC3 (XPB), p62, p44, p41, and p34 but is devoid of detectable levels of ERCC2 (XPD) and CAK. ERCC2/CAK was isolated as a complex that exhibits CAK activity that cosediments with the three CAK components (cdk7, cyclin H, and p36/MAT1) as well as the ERCC2 (XPD) protein. TFIIH* can support pol II-catalyzed transcription in vitro with lower efficiency compared with TFIIH. This TFIIH*-dependent transcription reaction was stimulated by ERCC2/CAK. The ERCC2/CAK and TFIIH* complexes are each active in DNA repair as shown by their ability to complement extracts prepared from ERCC2 (XPD)- and ERCC3 (XPB)-deficient cells, respectively, in supporting the excision of DNA containing a cholesterol lesion. These data suggest that TFIIH* and ERCC2/CAK interact to form the TFIIH holoenzyme capable of efficiently assembling the pol II transcription initiation complex and directly participating in excision repair reactions.

Common origin for surface reconstruction and the formation of chains of metal atoms

During the fracture of nanocontacts gold spontaneously forms freely suspended chains of atoms, which is not observed for the isoelectronic noble metals Ag and Cu. Au also differs from Ag and Cu in forming reconstructions at its low-index surfaces. Using mechanically controllable break junctions we show that all the 5d metals that show similar reconstructions (Ir, Pt, and Au) also form chains of atoms, while both properties are absent in the 4d neighbor elements (Rh, Pd, and Ag), indicating a common origin for these two phenomena. A competition between s and d bonding is proposed as an explanation.

Embracing ligands. Synthesis, characterisation and the correlation between 59Co NMR and ligand field parameters of Co(III) complexes with a new class of nitrogen-thioether multidentate ligand

The syntheses of the hexadentate ligands 2,2,10,10-tetra(methyleneamine)-4,8-dithiaundecane (PrN(4)S(2)amp), 2,2,11,11-tetra(methyleneamine)-4,9-dithiadodecane (BuN(4)S(2)amp), and 1,2-bis(4,4-methyleneamine)-2-thiapentyl)benzene (XyN(4)S(2)amp) are reported and the complexes [Co(RN(4)S(2)amp)](3+) (R = Pr, Bu, Xy) characterised by single crystal X-ray study. The low-temperature (11 K) absorption spectra have been measured in Nafion films. From the observed positions of both spin-allowed (1)A(1g) --> T-1(1g) and (1)A(1g) --> T-1(2g) and spin forbidden (1)A(1g) --> T-3(1g) and (1)A(1g) --> T-3(2g) bands, octahedral ligand-field parameters (10D(q), B and C) have been determined. DFT calculations suggest that significant interaction between the d-d and CT excitations occurs for the complexes. The calculations offer an explanation for the observed deviations from linearity of the relationship between Co-59 magnetogyric ratio and beta(DeltaE)(-1) (beta = the nephelauxetic ratio; DeltaE the energy of the (1)A(1g) --> T-1(1g) transition) for a series of amine and mixed amine/thioether donor complexes.

Polymer-phospholipid complexes for the solubilisation and delivery of drugs to the eye

Aim: Topical application of ophthalmic drugs is very inefficient; contact lenses used as drug delivery devices could minimize the drug loss and side effects. Styrene-maleic acid copolymers (PSMA) can form polymer-phospholipid complexes with dipalmitoyl phosphatidylcholine (DMPC) in the form of nanometric vesicles, which can easily solubilise hydrophobic drugs. They can be dispersed on very thin contact lens coatings to immobilize the drug on their surface. Methods: Two types of complexes stable at different pH values (5 and 7 respectively) where synthesized and loaded with drugs of different hydrophilicities during their formation process. The drug release was studied in vitro and compared to the free drug. Results: The mean sizes of the complexes obtained by light scattering were 50 nm and 450 nm respectively with low polydispersities. However, they were affected by the drugs load and release. An increase was observed in the duration of the release in the case of hydrophobic drugs, from days to weeks, avoiding initial “burst” and with a lesser amount of total drug released due to the interaction of the drug with the phospholipid core. The size and charge of the different drugs and the complexes nature also affected the release profile. Conclusions: Polymer-phospholipid complexes in the form of nanoparticles can be used to solubilise and release hydrophobic drugs in a controlled way. The drug load and release can be optimised to reach therapeutic values in the eye.

Synthesis and characterization of some Mn(II) and Mn(III) complexes of N,N′-o-phenylenebis(salicylideneimine)(LH2) and N,N′-o-phenylenebis(5-bromosalicylideneimine)(L′H2). Crystal structures of [Mn(L)(H2O)(ClO4)], [Mn(L)(NCS)] and an infinite linear chain of [Mn(L)(OAc)]

A series of manganese(II) [Mn(L)] and manganese(III) [Mn(L)(X)] (X = ClO4, OAc, NCS, N3, Cl, Br and I) complexes have been synthesized from Schiff base ligands N,N′-o- phenylenebis(salicylideneimine)(LH2) and N,N′-o-phenylenebis(5- bromosalicylideneimine)(L′H2) obtained by condensation of salicylaldehyde or 5-Br salicylaldehyde with o-phenylene-diamine. The complexes have been characterized by the combination of IR, UV-Vis spectroscopy, magnetic measurements and electrochemical studies. Three manganese(III) complexes 3 [Mn(L)(ClO4)(H2O)], 5 [Mn(L)(OAc)] and 13 [Mn(L)(NCS)] have been characterized by X-ray crystallography. The X-ray structures show that the manganese(III) is hexa-coordinated in 3, it is penta-coordinated in 13, while in 5 there is an infinite chain where the MnL moieties are connected by acetate ions acting as bridging bidentate ligand. The cyclic voltammograms of all the manganese(III) complexes exhibit two reversible/quasi-reversible/ irreversible responses assignable to Mn(III)/Mn(II) and Mn(IV)/Mn(III) couples. It was observed that the ligand L′H2 containing the 5-bromosal moiety always stabilizes the lower oxidation states compared to the corresponding unsubstituted LH2. Cyclic voltammograms of the manganese(II) complexes (1 and 2) exhibit a quasi-reversible Mn(III)/Mn(II) couple at E1/2 -0.08 V for 1 and 0.054 V for 2. © 2005 Elsevier B.V. All rights reserved.

Interrogation of a sonogashira cross-coupling of 8-bromoguanosine with phenylacetylene on amberlite:evidence for Pd/Cu ion binding and propagation of Pd/Cu nanoparticles

The reactivity of Amberlite (IRA-67) base "heterogeneous" resin in Sonogashira cross-coupling of 8-bromoguanosine 1 with phenylacetylene 3 to give 2 has been examined. Both 1 and 2 coordinate to Pd and Cu ions, which explains why at equivalent catalyst loadings, the homogeneous reaction employing triethylamine base is poor yielding. X-ray photo-electron spectroscopy (XPS) has been used to probe and quantify the active nitrogen base sites of the Amberlite resin, and postreaction Pd and Cu species. The Pd2Cl3(PPh)2 precatalyst and CuI cocatalyst degrade to give Amberlite-supported metal nanoparticles (average size ∼2.7 nm). The guanosine product 2 formed using the Amberlite Pd/Cu catalyst system is of higher purity than reactions using a homogeneous Pd precatalyst, a prerequisite for use in biological applications. Copyright © Taylor and Francis Group, LLC.

Structure, energetics and reactions of metal cation complexes of dipeptides in the gas phase

Structure, energetics and reactions of ions in the gas phase can be revealed by mass spectrometry techniques coupled to ions activation methods. Ions can gain enough energy for dissociation by absorbing IR light photons introduced by an IR laser to the mass spectrometer. Also collisions with a neutral molecule can increase the internal energy of ions and provide the dissociation threshold energy. Infrared multiple photon dissociation (IRMPD) or sustained off-resonance irradiation collision-induced dissociation (SORI-CID) methods are combined with Fourier Transform Ion Cyclotron Resonance (FT-ICR) mass spectrometers where ions can be held at low pressures for a long time. The outcome of ion activation techniques especially when it is compared to the computational methods results is of great importance since it provides useful information about the structure, thermochemistry and reactivity of ions of interest. In this work structure, energetics and reactivity of metal cation complexes with dipeptides are investigated. Effect of metal cation size and charge as well as microsolvation on the structure of these complexes has been studied. Structures of bare and hydrated Na and Ca complexes with isomeric dipeptides AlaGly and GlyAla are characterized by means of IRMPD spectroscopy and computational methods. At the second step unimolecular dissociation reactions of singly charged and doubly charged multimetallic complexes of alkaline earth metal cations with GlyGly are examined by CID method. Also structural features of these complexes are revealed by comparing their IRMPD spectra with calculated IR spectra of possible structures. At last the unimolecular dissociation reactions of Mn complexes are studied. IRMPD spectroscopy along with computational methods is also employed for structural elucidation of Mn complexes. In addition the ion-molecule reactions of Mn complexes with CO and water are explored in the low pressures obtained in the ICR cell.

Elevated Micronucleus Frequency In Patients With Type 2 Diabetes, Dyslipidemia And Periodontitis.

The over-production of reactive oxygen species (ROS) can cause oxidative damage to a large number of molecules, including DNA, and has been associated with the pathogenesis of several disorders, such as diabetes mellitus (DM), dyslipidemia and periodontitis (PD). We hypothesise that the presence of these diseases could proportionally increase the DNA damage. The aim of this study was to assess the micronucleus frequency (MNF), as a biomarker for DNA damage, in individuals with type 2 DM, dyslipidemia and PD. One hundred and fifty patients were divided into five groups based upon diabetic, dyslipidemic and periodontal status (Group 1 - poor controlled DM with dyslipidemia and PD; Group 2 - well-controlled DM with dyslipidemia and PD; Group 3 - without DM with dyslipidemia and PD; Group 4 - without DM, without dyslipidemia and with PD; and Group 5 - without DM, dyslipidemia and PD). Blood analyses were carried out for fasting plasma glucose, HbA1c and lipid profile. Periodontal examinations were performed, and venous blood was collected and processed for micronucleus (MN) assay. The frequency of micronuclei was evaluated by cell culture cytokinesis-block MN assay. The general characteristics of each group were described by the mean and standard deviation and the data were submitted to the Mann-Whitney, Kruskal-Wallis, Multiple Logistic Regression and Spearman tests. The Groups 1, 2 and 3 were similarly dyslipidemic presenting increased levels of total cholesterol, low density lipoprotein cholesterol and triglycerides. Periodontal tissue destruction and local inflammation were significantly more severe in diabetics, particularly in Group 1. Frequency of bi-nucleated cells with MN and MNF, as well as nucleoplasmic bridges, were significantly higher for poor controlled diabetics with dyslipidemia and PD in comparison with those systemically healthy, even after adjusting for age, and considering Bonferroni's correction. Elevated frequency of micronuclei was found in patients affected by type 2 diabetes, dyslipidemia and PD. This result suggests that these three pathologies occurring simultaneously promote an additional role to produce DNA impairment. In addition, the micronuclei assay was useful as a biomarker for DNA damage in individuals with chronic degenerative diseases.

Pharmacokinetic And Pharmacodynamic Evaluation Of A Nanotechnological Topical Formulation Of Lidocaine/prilocaine (nanorap) In Healthy Volunteers.

Nanorap is a new nanotechnological formulation for topical anesthesia composed of lidocaine (2.5%) and prilocaine (2.5%). The present study evaluated the pharmacokinetics (PK) of Nanorap. For the determination of lidocaine and prilocaine in human plasma a new method using high-performance liquid-chromatography coupled to tandem mass spectrometry was developed. Nanorap pharmacodynamic (PD) and its physical proprieties were also evaluated. Nanorap was administered by topical application of 2g to healthy volunteers and blood samples were collected for the PK analysis. The drugs were extracted from plasma by liquid-liquid extraction with ether/hexane (80/20, v/v). The chromatography separation was performed on a Genesis C18 analytical column 4 µm (100 x 2.1 mm i.d.) with a mobile phase of methanol/acetonitrile/water (40/30/30, for lidocaine, and 50/30/20, for prilocaine, v/v/v) + 2 mM of ammonium acetate and ropivacaine as internal standard. The drugs were quantified using a mass spectrometer with an electrospray source in the ESI positive mode (ES+) configured for multiple reaction monitoring. The PD of Nanorap was evaluated with the use of a visual analogue scale. Nanorap was characterized by cryofracture. The chromatography run time was 5.5 min for lidocaine and 3.3 min for prilocaine and the lower limit of quantification was 0.05 ng/mL for both drugs. Mean Cmax was 6.62 and 1.72 ng/mL for lidocaine and prilocaine, respectively. Median Tmax was 6.5 hours for both drugs. Nanocapsules had a mean size of 88nm and mean drug association of 92.5% and 89% for lidocaine and prilocaine, respectively. The PD study showed that Nanorap has a sufficient analgesic effect (>30% reduction in pain) after 10 minutes of application. A new simple, selective and sensitive method for determination of lidocaine and prilocaine in human plasma was developed. Nanorap generated safe plasma levels of the drugs and satisfactory analgesic effect.

Transcutaneous Tibial Nerve Stimulation In The Treatment Of Lower Urinary Tract Symptoms And Its Impact On Health-related Quality Of Life In Patients With Parkinson Disease: A Randomized Controlled Trial.

A randomized controlled trial study was performed to evaluate the efficacy of transcutaneous tibial nerve stimulation (TTNS) and sham TTNS, in patients with Parkinson disease (PD) with lower urinary tract symptoms (LUTS). Randomized controlled trial. Thirteen patients with a diagnosis of PD and bothersome LUTS were randomly allocated to one of the following groups: Group I: TTNS group (n = 8) and group II: Sham group (n = 5). Both groups attended twice a week during 5 weeks; each session lasted 30 minutes. Eight patients received TTNS treatment and 5 subjects allocated to group II were managed with sham surface electrodes that delivered no electrical stimulation. Assessments were performed before and after the treatment; they included a 3-day bladder diary, Overactive Bladder Questionnaire (OAB-V8), and the International Consultation on Incontinence Quality of Life Questionnaire Short Form (ICIQ-SF), and urodynamic evaluation. Following 5 weeks of treatment, patients allocated to TTNS demonstrated statistically significant reductions in the number of urgency episodes (P = .004) and reductions in nocturia episodes (P < .01). Participants allocated to active treatment also showed better results after treatment in the OAB-V8 and ICIQ-SF scores (P < .01, respectively). Urodynamic testing revealed that patients in the active treatment group showed improvements in intravesical volume at strong desire to void (P < .05) and volume at urgency (P < .01) when compared to subjects in the sham treatment group. These findings suggest that TTNS is effective in the treatment of LUTS in patients with PD, reducing urgency and nocturia episodes and improving urodynamic parameters as well as symptom scores measured by the OAB-V8 and health-related quality-of-life scores measured by the ICIQ-SF.

Sim-xl: A Powerful And User-friendly Tool For Peptide Cross-linking Analysis.

Chemical cross-linking has emerged as a powerful approach for the structural characterization of proteins and protein complexes. However, the correct identification of covalently linked (cross-linked or XL) peptides analyzed by tandem mass spectrometry is still an open challenge. Here we present SIM-XL, a software tool that can analyze data generated through commonly used cross-linkers (e.g., BS3/DSS). Our software introduces a new paradigm for search-space reduction, which ultimately accounts for its increase in speed and sensitivity. Moreover, our search engine is the first to capitalize on reporter ions for selecting tandem mass spectra derived from cross-linked peptides. It also makes available a 2D interaction map and a spectrum-annotation tool unmatched by any of its kind. We show SIM-XL to be more sensitive and faster than a competing tool when analyzing a data set obtained from the human HSP90. The software is freely available for academic use at http://patternlabforproteomics.org/sim-xl. A video demonstrating the tool is available at http://patternlabforproteomics.org/sim-xl/video. SIM-XL is the first tool to support XL data in the mzIdentML format; all data are thus available from the ProteomeXchange consortium (identifier PXD001677).

Relationship between periodontitis and rheumatoid arthritis and the effect of non-surgical periodontal treatment

This study analyzed the association of periodontal disease (PD) and rheumatoid arthritis (RA). Seventy-five 35-60-year-old patients were assigned to 5 groups according to the presence (+) or not (-) of PD and RA and the treatment received (TR+) or not (TR-) for PD. Group 3 uses total prosthesis (TP). Clinical and laboratory evaluations were performed at baseline, 3 and 6 months of follow-up by probing pocket depth, bleeding on probing and plaque index for PD, HAQ, DAS28, SF-36 and laboratory: AAG, ESR, CRP for RA. Statistically significant differences for PD after 3 (p=0.0055) and after 6 months (p=0.0066) were obtained in Group 1 (RA+PD+TR+) and 2(RA+PD+TR-); significant reduction in the % of BOP after 6 months (p=0.0128) and significant reduction in the % of Pl after 3 (p=0.0128) and 6 months (p=0.0002) in Group 1. Statistically significant differences between Groups 1 and 3 (RA+TP) for DAS28 at baseline and after 3 months were observed, but not after 6 months. No other parameters for RA were significantly affected. The relationship between RA and PD disease activities is not clear, but the importance of periodontal treatment in the control of inflammation to avoid tooth extraction is evident.

Theory of nitride oxide adsorption on transition metal (111) surfaces: a first-principles investigation

In this work, we report a density functional theory study of nitric oxide (NO) adsorption on close-packed transition metal (TM) Rh(111), Ir(111), Pd(111) and Pt(111) surfaces in terms of adsorption sites, binding mechanism and charge transfer at a coverage of Theta(NO) = 0.25, 0.50, 0.75 monolayer (ML). Based on our study, an unified picture for the interaction between NO and TM(111) and site preference is established, and valuable insights are obtained. At low coverage (0.25 ML), we find that the interaction of NO/TM(111) is determined by an electron donation and back-donation process via the interplay between NO 5 sigma/2 pi* and TM d-bands. The extent of the donation and back-donation depends critically on the coordination number (adsorption sites) and TM d-band filling, and plays an essential role for NO adsorption on TM surfaces. DFT calculations shows that for TMs with high d-band filling such as Pd and Pt, hollow-site NO is energetically the most favorable, and top-site NO prefers to tilt away from the normal direction. While for TMs with low d-band filling (Rh and Ir), top-site NO perpendicular to the surfaces is energetically most favorable. Electronic structure analysis show that irrespective of the TM and adsorption site, there is a net charge transfer from the substrate to the adsorbate due to overwhelming back-donation from the TM substrate to the adsorbed NO molecules. The adsorption-induced change of the work function with respect to bare surfaces and dipole moment is however site dependent, and the work function increases for hollow-site NO, but decreases for top-site NO, because of differences in the charge redistribution. The interplay between the energetics, lateral interaction and charge transfer, which is element dependent, rationalizes the structural evolution of NO adsorption on TM(111) surfaces in the submonolayer regime.

Preslaughter mortality of broilers in relation to lairage and season in a subtropical climate

The preslaughter handling and transport of broilers are stressful operations that might affect welfare and meat quality and could increase numbers of deaths before slaughter. However, the influence of thermal factors during transportation and lairage at slaughterhouses is complex in subtropical regions, where increasing temperature and high RH are the major concerns regarding animal survival before slaughter. In this study we assessed the influence of a controlled lairage environment on preslaughter mortality rates of broiler chickens that were transported during different seasons of the year and had varying lairage times in the subtropical climate. Preslaughter data from 13,937 broiler flocks were recorded daily during 2006 in a commercial slaughterhouse in southeastern Brazil. The main factors that influenced daily mortality rate were mean dry bulb temperature and RH, lairage time, daily periods, density of broilers per crate, season of the year, stocking density per lorry, transport time, and distance between farms and slaughterhouse. A holding area at the slaughterhouse with environmental control was assessed. Using a double GLM for mean and dispersion modeling, the seasons were found to have significant effects (P < 0.05) on average mortality rates. The highest incidence was observed in summer (0.42%), followed by spring (0.39%), winter (0.28%), and autumn (0.23%). A decrease of preslaughter mortality of broilers during summer (P < 0.05) was observed when the lairage time was increased, mainly after 1 h of exposure to a controlled environment. Thus, lairage for 3 to 4 h in a controlled lairage environment during the summer and spring is necessary to reduce the thermal load of broiler chickens.

Enterotoxigenic and genetic profiles of Bacillus cereus strains of food origin in Brazil

In Brazil. the incidence of Bacillus cereus outbreaks is unknown, and there is little information about B. cereus occurrence in food. In addition, data on toxin production and genetic characterization of the B. cereus isolates cannot be found. This pathogen causes two distinct types of toxin-mediated foodborne illnesses known as diarrheal and emetic syndromes. Diarrheal syndrome has been linked to three different enterotoxins: two protein complexes, hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE); and an enterotoxic protein, cytotoxin K (cytK). Emetic syndrome is related to cereulide, a toxin encoded by the ces gene. In this study, NHE and HBL production capacities of 155 strains of B. cereus isolated from Brazilian food products were evaluated with an immunoassay. Strains were also tested for the presence of the genes of the HBL and NHE complexes, cytK, cytK-1, cytK-2, and ces, using PCR. HBL was detected in 105 (67.7%) strains and NHE in 154 (99.4%) strains. All the strains harbored at least one gene of the NHE complex, while 96.1% of them were positive for at least one of those of the HBL complex. Genes cytK1 and ces were not detected. All strains showed toxigenic capacity and could represent a risk for consumers if good practices are not followed. This is the first report on toxigenic and genetic profiles of B. cereus strains isolated in Brazil.