Standardisation and comparison of methods employed for microbial cell surface hydrophobicity and charge determination

Whilst there are a number of methods available to characterise the cell surface hydrophobicity (CSH) and cell surface charge (CSC) of microorganisms, there is still debate concerning the correlation of results between individual methods. In this study, the techniques of bacterial adherence to hydrocarbons (BATH) and hydrophobic interaction chromatography (HTC) were used to measure CSH. Electrostatic interaction chromatography (ESIC) and zeta potential (ZP) measurements were used to determine CSC. To allow meaningful comparisons between the BATH and HIC tests, between ESIC and ZP and also between CSH and CSC, the buffer systems employed in each test were standardised (phosphate buffered saline, pH 7.3, 0.01 mM). Isolates of Staphylococcus epidermidis derived from microbial biofilm were used as the test organism in this study. The isolates examined exhibited primarily medium to high CSH and a highly negative CSC. Good correlation of CSH measurement was observed between the BATH and HIC tests (r = 0.89). Good correlation was observed between ESIC (anionic exchange column) and ZP measurements. No correlations were observed between isolate CSC and either increased or decreased CSH. It is recommended that whenever comparisons of various methods to determine either CSC or CSH (by partitioning methods), the buffer systems should remain constant throughout to achieve consistency of results.

Do Foreign Greenfields Outperform Foreign Acquisitions or Vice Versa? An Institutional Perspective.

Prior studies of the comparative performance of greenfields and acquisitions have advanced competing arguments, with some arguing that greenfields should outperform acquisitions because acquisitions are costlier to integrate, and others that acquisitions should outperform greenfields because greenfields suffer from a liability of newness. Moreover, while the costs of integration and the liability of newness are at their greatest during a subsidiary's first years, prior studies have tested their competing arguments on samples containing older subsidiaries. We extend these prior studies by (1) developing an institutional theory-based framework that simultaneously considers the costs of integration and the liability of newness, (2) recognizing that both types of costs vary with the level of subsidiary integration, and (3) focusing on the stage of their life during which subsidiaries predominantly incur these costs. To measure subsidiary performance, we ask managers of Dutch multinationals how their ex ante performance expectations compare to the subsidiary's ex post performance during its first two years. Analysing a sample of 191 foreign subsidiaries and controlling for entry mode self-selection and other factors, we find that acquisitions outperform greenfields at low and intermediate levels of subsidiary integration, but that greenfields outperform acquisitions at higher integration levels. [ABSTRACT FROM AUTHOR]

Functional analysis of the glycero-manno-heptose 7-phosphate kinase domain from the bifunctional HldE protein, which is involved in ADP-L-glycero-D-manno-heptose biosynthesis

The core oligosaccharide component of the lipopolysaccharide can be subdivided into inner and outer core regions. In Escherichia coli, the inner core consists of two 3-deoxy-d-manno-octulosonic acid and three glycero-manno-heptose residues. The HldE protein participates in the biosynthesis of ADP-glycero-manno-heptose precursors used in the assembly of the inner core. HldE comprises two functional domains: an N-terminal region with homology to the ribokinase superfamily (HldE1 domain) and a C-terminal region with homology to the cytidylyltransferase superfamily (HldE2 domain). We have employed the structure of the E. coli ribokinase as a template to model the HldE1 domain and predict critical amino acids required for enzyme activity. Mutation of these residues renders the protein inactive as determined in vivo by functional complementation analysis. However, these mutations did not affect the secondary or tertiary structure of purified HldE1, as judged by fluorescence spectroscopy and circular dichroism. Furthermore, in vivo coexpression of wild-type, chromosomally encoded HldE and mutant HldE1 proteins with amino acid substitutions in the predicted ATP binding site caused a dominant negative phenotype as revealed by increased bacterial sensitivity to novobiocin. Copurification experiments demonstrated that HldE and HldE1 form a complex in vivo. Gel filtration chromatography resulted in the detection of a dimer as the predominant form of the native HldE1 protein. Altogether, our data support the notions that the HldE functional unit is a dimer and that structural components present in each HldE1 monomer are required for enzymatic activity.

Apparent tolerance of turkey vultures (Cathartes aura) to the non-steroidal anti-inflammatory drug diclofenac

The nonsteroidal anti-inflammatory drug diclofenac is extremely toxic to Old World Gyps vultures (median lethal dose -0.1-0.2 mg/kg), evoking visceral gout, renal necrosis, and mortality within a few days of exposure. Unintentional secondary poisoning of vultures that fed upon carcasses of diclofenac-treated livestock decimated populations in the Indian subcontinent. Because of the widespread use of diclofenac and other cyclooxygenase-2 inhibiting drugs, a toxicological study was undertaken in turkey vultures (Cathartes aura) as an initial step in examining sensitivity of New World scavenging birds. Two trials were conducted entailing oral gavage of diclofenac at doses ranging from 0.08 to 25 mg/kg body weight. Birds were observed for 7 d, blood samples were collected for plasma chemistry (predose and 12, 24, and 48 h and 7 d postdose), and select individuals were necropsied. Diclofenac failed to evoke overt signs of toxicity, visceral gout, renal necrosis, or elevate plasma uric acid at concentrations greater than 100 times the estimated median lethal dose reported for Gyps vultures. For turkey vultures receiving 8 or 25 mg/kg, the plasma half-life of diclofenac was estimated to be 6 h, and it was apparently cleared after several days as no residues were detectable in liver or kidney at necropsy. Differential sensitivity among avian species is a hallmark of cyclooxygenase-2 inhibitors, and despite the tolerance of turkey vultures to diclofenac, additional studies in related scavenging species seem warranted.

Distinct CCK-2 Receptor Conformations Associated with β-Arrestin-2 Recruitment or Phospholipase-C Activation Revealed by a Biased Antagonist

Seven-transmembrane receptors (7TMRs), also termed G protein-coupled receptors (GPCRs), form the largest class of cell surface membrane receptors, involving several hundred members in the human genome. Near 30% of marketed pharmacological agents target 7TMRs. 7TMRs adopt multiple conformations upon agonist binding. Biased agonists, in contrast to non-biased agonists, are believed to stabilize conformations preferentially activating either G-protein- or ß-arrestin-dependent signalling pathways. However, proof that cognate conformations of receptors display structural differences within their binding site where biased agonism initiates, are still lacking. Here, we show that a non-biased agonist, cholecystokinin (CCK) induces conformational states of the CCK2R activating Gq-protein-dependent pathway (CCK2RG) or recruiting ß-arrestin2 (CCK2Rß) that are pharmacologically and structurally distinct. Two structurally unrelated antagonists competitively inhibited both pathways. A third ligand (GV150,013X), acted as a high affinity competitive antagonist on CCK2RG but was nearly inefficient as inhibitor of CCK2Rß. Several structural elements on both GV150,013X and in CCK2R binding cavity, which hinder binding of GV150,013X only to the CCK2Rß were identified. At last, proximity between two conserved amino acids from transmembrane helices 3 and 7 interacting through sulphur-aromatic interaction was shown to be crucial for selective stabilization of the CCK2Rß state. These data establish structural evidences for distinct conformations of a 7TMR associated with ß-arrestin-2 recruitment or G-protein coupling and validate relevance of the design of biased ligands able to selectively target each functional conformation of 7TMRs.

Functional expression of KCNQ (Kv7) channels in guinea-pig bladder smooth muscle and their contribution to spontaneous activity

Background and Purpose: The aim of the study was to determine whether KCNQ channels are functionally expressed in bladder smooth muscle cells (SMC) and to investigate their physiological significance in bladder contractility. 

Experimental Approach: KCNQ channels were examined at the genetic, protein, cellular and tissue level in guinea pig bladder smooth muscle using RT-PCR, immunofluorescence, patch-clamp electrophysiology, calcium imaging, detrusor strip myography, and a panel of KCNQ activators and inhibitors. 

Key Results: KCNQ subtypes 1-5 are expressed in bladder detrusor smooth muscle. Detrusor strips typically displayed TTX-insensitive myogenic spontaneous contractions that were increased in amplitude by the KCNQ channel inhibitors XE991, linopirdine or chromanol 293B. Contractility was inhibited by the KCNQ channel activators flupirtine or meclofenamic acid (MFA). The frequency of Ca2+-oscillations in SMC contained within bladder tissue sheets was increased by XE991. Outward currents in dispersed bladder SMC, recorded under conditions where BK and KATP currents were minimal, were significantly reduced by XE991, linopirdine, or chromanol, and enhanced by flupirtine or MFA. XE991 depolarized the cell membrane and could evoke transient depolarizations in quiescent cells. Flupirtine (20M) hyperpolarized the cell membrane with a simultaneous cessation of any spontaneous electrical activity. 

Conclusions and Implications: These novel findings reveal the role of KCNQ currents in the regulation of the resting membrane potential of detrusor SMC and their important physiological function in the control of spontaneous contractility in the guinea pig bladder.

Meta-analysis of randomised controlled trials comparing latanoprost with timolol in the treatment of patients with open angle glaucoma or ocular hypertension

Aim - To evaluate the comparative efficacy and tolerance of latanoprost versus timolol through a meta-analysis of randomised controlled trials (RCTs). Methods - Systematic retrieval of RCTs of latanoprost versus timolol to allow pooling of results from head to head comparison studies. Quality of trials was assessed based on randomisation, masking, and withdrawal. Sensitivity analyses were used to estimate the effects of quality of study on outcomes. The data sources were Medline, Embase, Scientific Citation Index, Merck Glaucoma, and Pharmacia and Upjohn ophthalmology databases. There were 1256 patients with open angle glaucoma or ocular hypertension reported in 11 trials of latanoprost versus timolol. The main outcome measures were (i) percentage intraocular pressure (IOP) reduction for efficacy; (ii) relative risk, risk difference, and number needed to harm for side effects such as hyperaemia, conjunctivitis, increased pigmentation, hypotension, and bradycardia expressed as dichotomous outcomes; and (iii) reduction in systemic blood pressure and heart rate as side effects. Results - Both 0.005% latanoprost once daily and 0.5% timolol twice daily reduced IOP. The percentage reductions in IOP from baseline (mean (SE)) produced by latanoprost and timolol were 30.2 (2.3) and 26.9 (3.4) at 3 months. The difference in IOP reduction between the two treatments were 5.0 (95% confidence intervals 2.8, 7.3). However, latanoprost caused iris pigmentation in more patients than timolol (relative risk = 8.01, 95% confidence intervals 1.87, 34.30). The 2 year risk with latanoprost reached 18% (51/277). Hyperaemia was also more often observed with latanoprost (relative risk = 2.20, 95% confidence intervals 1.33, 3.64). Timolol caused a significant reduction in heart rate of 4 beats/minute (95% confidence interval 2, 6). Conclusion - This meta-analysis suggests that latanoprost is more effective than timolol in lowering IOP. However, it often causes iris pigmentation. While current evidence suggests that this pigmentation is benign, careful lifetime evaluation of patients is still justified.

Complementary Therapy: perceptions of older people with lung or colorectal cancer

Aim: To explore how older people with lung and colorectal cancer
view registered complementary therapy (CT) services in Northern
Ireland. Background: A literature review highlighted gaps around
information, access, and communication between patients and health
professionals regarding CT services. Methods: Using structured
interviews, a survey of 68 patients in one hospital and one hospice was
conducted in Belfast, Northern Ireland. Results: All respondents felt
that CT services should be better promoted and more easily
accessible to older people with cancer. Some patients were concerned
about the lack of written information provided regarding CT services,
which they believed led to poorer uptake and uncertainty regarding
the potential benefits. Others were concerned that engaging in or
disclosing CT usage might negatively affect existing relationships with
medical professionals. Conclusion: Patients should be offered high
quality written information on CT services to enable choice, improve
knowledge, and promote wider access. Increased physician education
may facilitate provision of such information.

No evidence for linkage or association of neuregulin-1 (NRG1) with disease in the Irish study of high-density schizophrenia families (ISHDSF)

The neuregulin-1 gene (NRG1) at chromosome 8p21-22 has been implicated as a schizophrenia susceptibility gene in Icelandic, Scottish, Irish and mixed UK populations. The shared ancestry between these populations led us to investigate the NRG1 polymorphisms and appropriate marker haplotypes for linkage and/or association to schizophrenia in the Irish study of high-density schizophrenia families (ISHDSF). Neither single-point nor multi-point linkage analysis of NRG1 markers gave evidence for linkage independent of our pre-existing findings telomeric on 8p. Analysis of linkage disequilibrium (LD) across the 252 kb interval encompassing the 7 marker core Icelandic/Scottish NRG1 haplotype revealed two separate regions of modest LD, comprising markers SNP8NRG255133, SNP8NRG249130 and SNP8NRG243177 (telomeric) and microsatellites 478B14-428, 420M9-1395, D8S1810 and 420M9-116I12 (centromeric). From single marker analysis by TRANSMIT and FBAT we found no evidence for association with schizophrenia for any marker. Haplotype analysis for the three SNPs in LD region 1 and, separately, the four microsatellites in LD region 2 (analyzed in overlapping 2-marker windows), showed no evidence for overtransmission of specific haplotypes to affected individuals. We therefore conclude that if NRG1 does contain susceptibility alleles for schizophrenia, they impact quite weakly on risk in the ISHDSF.

Newly Acquired Fear of Falling Leads to Altered Eye Movement Patterns and Reduced Stepping Safety: A Case Study

This opportune case study describes visual and stepping behaviours of an 87 year old female (P8), both prior to, and following two falls. Before falling, when asked to walk along a path containing two stepping guides positioned before and after an obstacle, P8 generally visually fixated the first stepping guide until after foot contact inside it. However, after falling P8 consistently looked away from the stepping guide before completing the step into it in order to fixate the upcoming obstacle in her path. The timing of gaze redirection away from the target (in relation to foot contact inside it) correlated with absolute stepping error. No differences in eyesight, cognitive function, or balance were found between pre- and post-fall recordings. However, P8 did report large increases in fall-related anxiety and reduced balance confidence, supporting previously suggested links between anxiety/increased fear or falling and maladaptive visual/stepping behaviours. The results represent a novel insight into how psychological and related behavioural factors can change in older adults following a fall, and provide a possible partial rationalisation for why recent fallers are more likely to fall again in the following 12 months. These findings highlight novel possibilities for falls prevention and rehabilitation.

An inherited duplication at the gene p21 Protein-Activated Kinase 7 (PAK7) is a risk factor for psychosis

Identifying rare, highly penetrant risk mutations may be an important step in dissecting the molecular etiology of schizophrenia. We conducted a gene-based analysis of large (>100kb), rare copy number variants (CNVs) in the Wellcome Trust Case Control Consortium 2 (WTCCC2) schizophrenia sample of 1,564 cases and 1,748 controls all from Ireland, and further extended the analysis to include an additional 5,196 UK controls. We found association with duplications at chr20p12.2 (P=0.007) and evidence of replication in large independent European schizophrenia (P=0.052) and UK bipolar disorder case-control cohorts (P=0.047). A combined analysis of Irish/UK subjects including additional psychosis cases (schizophrenia and bipolar disorder) identified 22 carriers in 11,707 cases and 10 carriers in 21,204 controls (meta-analysis CMH P value=2x10(-4) (odds ratio (OR)=11.3, 95% CI=3.7, ∞)). Nineteen of the 22 cases and 8 of the 10 controls carried duplications starting at 9.68Mb with similar breakpoints across samples. By haplotype analysis and sequencing we identified a tandem ∼149kb duplication overlapping the gene p21 Protein-Activated Kinase 7 (PAK7, also called PAK5) which was in linkage disequilibrium with local haplotypes (P=2.5x10(-21)), indicative of a single ancestral duplication event. We confirmed the breakpoints in 8/8 carriers tested and found co-segregation of the duplication with illness in two additional family members of one of the affected probands. We demonstrate that PAK7 is developmentally co-expressed with another known psychosis risk gene (DISC1) suggesting a potential molecular mechanism involving aberrant synapse development and plasticity.

Adolescent ecstasy use and depression: cause and effect, or two outcomes of home environment?

Background: This study assessed the association between adolescent ecstasy use and depressive symptoms in adolescence. Methods: The Belfast Youth Development Study surveyed a cohort annually from age 11 to 16 years. Gender, Strengths and Difficulties Questionnaire emotional subscale, living arrangements, parental affluence, parent and peer attachment, tobacco, alcohol, cannabis and ecstasy use were investigated as predictors of Short Mood and Feelings Questionnaire (SMFQ) outcome. Results: Of 5371 respondents, 301 (5.6%) had an SMFQ > 15, and 1620 (30.2) had missing data for SMFQ. Around 8% of the cohort had used ecstasy by the end of follow-up. Of the non-drug users, ∼2% showed symptoms of depression, compared with 6% of those who had used alcohol, 6% of cannabis users, 6% of ecstasy users and 7% of frequent ecstasy users. Without adjustment, ecstasy users showed around a 4-fold increased odds of depressive symptoms compared with non-drug users [odds ratio (OR) = 0.26; 95% confidence interval (CI) = 0.10, 0.68]. Further adjustment for living arrangements, peer and parental attachment attenuated the association to under a 3-fold increase (OR = 0.37; 95% CI = 0.15, 0.94). There were no differences by frequency of use. Conclusions: Ecstasy use during adolescence may be associated with poorer mental health; however, this association can be explained by the confounding social influence of family dynamics. These findings could be used to aid effective evidence-based drug policies, which concentrate criminal justice and public health resources on reducing harm.

Tailoring ionic liquid catalysts: structure, acidity and catalytic activity of protonic ionic liquids based on anionic clusters, [(HSO4)(H2SO4)x]− (x = 0, 1, or 2)

Aiming at inexpensive Brønsted-acidic ionic liquids, suitable for industrial-scale catalysis, a family of protonic ionic liquids based on nitrogen bases and sulfuric acid has been developed. Variation of the molar ratio of sulfuric acid, χH2SO4, was used to tune acidity. The liquid structure was studied using 1H NMR and IR spectroscopies, revealing the existence of hydrogen-bonded clusters, [(HSO4)(H2SO4)]−, for χH2SO4 > 0.50. Acidity, quantified by Gutmann Acceptor Number (AN), was found to be closely related to the liquid structure. The ionic liquids were employed as acid catalysts in a model reaction; Fischer esterification of acetic acid with 1-butanol. The reaction rate depended on two factors; for χH2SO4 > 0.50, the key parameter was acidity (expressed as AN value), while for χH2SO4 > 0.50 it was the mass transport (solubility of starting materials in the ionic liquid phase). Building on this insight, the ionic liquid catalyst and reaction conditions have been chosen. Conversion values of over 95% were achieved under exceptionally mild conditions, and using an inexpensive ionic liquid, which could be recycled up to eight times without diminution in conversion or selectivity. It has been demonstrated how structural studies can underpin rational design and development of an ionic liquid catalyst, and in turn lead to a both greener and economically viable process.