Obesity-associated microbiomes instigate visceral adipose tissue inflammation by recruitment of distinct neutrophils.

Neutrophils are increasingly implicated in chronic inflammation and metabolic disorders. Here, we show that visceral adipose tissue (VAT) from individuals with obesity contains more neutrophils than in those without obesity and is associated with a distinct bacterial community. Exploring the mechanism, we gavaged microbiome-depleted mice with stool from patients with and without obesity during high-fat or normal diet administration. Only mice receiving high-fat diet and stool from subjects with obesity show enrichment of VAT neutrophils, suggesting donor microbiome and recipient diet determine VAT neutrophilia. A rise in pro-inflammatory CD4+ Th1 cells and a drop in immunoregulatory T cells in VAT only follows if there is a transient spike in neutrophils. Human VAT neutrophils exhibit a distinct gene expression pattern that is found in different human tissues, including tumors. VAT neutrophils and bacteria may be a novel therapeutic target for treating inflammatory-driven complications of obesity, including insulin resistance and colon cancer.

Interferon gamma mediates the reduction of adipose tissue regulatory T cells in human obesity.

Decreased adipose tissue regulatory T cells contribute to insulin resistance in obese mice, however, little is known about the mechanisms regulating adipose tissue regulatory T cells numbers in humans. Here we obtain adipose tissue from obese and lean volunteers. Regulatory T cell abundance is lower in obese vs. lean visceral and subcutaneous adipose tissue and associates with reduced insulin sensitivity and altered adipocyte metabolic gene expression. Regulatory T cells numbers decline following high-fat diet induction in lean volunteers. We see alteration in major histocompatibility complex II pathway in adipocytes from obese patients and after high fat ingestion, which increases T helper 1 cell numbers and decreases regulatory T cell differentiation. We also observe increased expression of inhibitory co-receptors including programmed cell death protein 1 and OX40 in visceral adipose tissue regulatory T cells from patients with obesity. In human obesity, these global effects of interferon gamma to reduce regulatory T cells and diminish their function appear to instigate adipose inflammation and suppress adipocyte metabolism, leading to insulin resistance.

New 19F NMR methodology reveals structures of molecules in complex mixtures of fluorinated compounds.

Although the number of natural fluorinated compounds is very small, fluorinated pharmaceuticals and agrochemicals are numerous. 19F NMR spectroscopy has a great potential for the structure elucidation of fluorinated organic molecules, starting with their production by chemical or chemoenzymatic reactions, through monitoring their structural integrity, to their biotic and abiotic transformation and ultimate degradation in the environment. Additionally, choosing to incorporate 19F into any organic molecule opens a convenient route to study reaction mechanisms and kinetics. Addressing limitations of the existing 19F NMR techniques, we have developed methodology that uses 19F as a powerful spectroscopic spy to study mixtures of fluorinated molecules. The proposed 19F-centred NMR analysis utilises the substantial resolution and sensitivity of 19F to obtain a large number of NMR parameters, which enable structure determination of fluorinated compounds without the need for their separation or the use of standards. Here we illustrate the 19F-centred structure determination process and demonstrate its power by successfully elucidating the structures of chloramination disinfectant by-products of a single mono-fluorinated phenolic compound, which would have been impossible otherwise. This novel NMR approach for the structure elucidation of molecules in complex mixtures represents a major contribution towards the analysis of chemical and biological processes involving fluorinated compounds.

19F-centred NMR analysis of mono-fluorinated compounds.

Addressing limitations of the existing NMR techniques for the structure determination of mono-fluorinated compounds, we have developed methodology that uses 19F as the focal point of this process. The proposed 19F-centred NMR analysis consists of a complementary set of broadband, phase-sensitive NMR experiments that utilise the substantial sensitivity of 19F and its far reaching couplings with 1H and 13C to obtain a large number of NMR parameters. The assembled 1H, 13C and 19F chemical shifts, values of J HF, J HH, and J FC coupling constants and the size of 13C induced 19F isotopic shifts constitute a rich source of information that enables structure elucidation of fluorinated moieties and even complete structures of molecules. Here we introduce the methodology, provide a detailed description of each NMR experiment and illustrate their interpretation using 3-fluoro-3-deoxy-d-glucose. This novel approach performs particularly well in the structure elucidation of fluorinated compounds embedded in complex mixtures, eliminating the need for compound separation or use of standards to confirm the structures. It represents a major contribution towards the analysis of fluorinated agrochemicals and (radio)pharmaceuticals at any point during their lifetime, including preparation, use, biotransformation and biodegradation in the environment. The developed methodology can also assist with the investigations of the stability of fluoroorganics and their pharmacokinetics. Studies of reaction mechanisms using fluorinated molecules as convenient reporters of these processes, will also benefit.

Molecular level study of hot water extracted green tea buried in soils - a proxy for labile soil organic matter.

Understanding the composition of soil organic matter (SOM) is vital to our understanding of how soils form, evolve and respond to external stimuli. The shear complexity of SOM, an inseparable mixture of thousands of compounds hinders the determination of structure-function relationships required to explore these processes on a molecular level. Litter bags and soil hot water extracts (HWE) have frequently been used to study the transformation of labile SOM, however these are still too complex to examine beyond compound classes. In this work, a much simpler mixture, HWE buried green tea, was investigated by Nuclear Magnetic Resonance (NMR) spectroscopy and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR-MS), as a proxy for labile SOM. Changes induced by the burial over 90 days in a grassland, woodland and two peatland sites, one damaged by drainage and one undergoing restoration by drain-blocking, were analysed. Major differences between the extracts were observed on the level of compound classes, molecular formulae and specific molecules. The causes of these differences are discussed with reference to abiotic and biotic processes. Despite the vastly different detection limits of NMR and MS, chemometric analysis of the data yielded identical separation of the samples. These findings provide a basis for the molecular level interrogation of labile SOM and C-cycling processes in soils.

Zebrafish-Based Discovery of Antiseizure Compounds from the Red Sea: Pseurotin A2 and Azaspirofuran A.

In search for novel antiseizure drugs (ASDs), the European FP7-funded PharmaSea project used zebrafish embryos and larvae as a drug discovery platform to screen marine natural products to identify promising antiseizure hits in vivo for further development. Within the framework of this project, seven known heterospirocyclic γ-lactams, namely, pseurotin A, pseurotin A2, pseurotin F1, 11- O-methylpseurotin A, pseurotin D, azaspirofuran A, and azaspirofuran B, were isolated from the bioactive marine fungus Aspergillus fumigatus, and their antiseizure activity was evaluated in the larval zebrafish pentylenetetrazole (PTZ) seizure model. Pseurotin A2 and azaspirofuran A were identified as antiseizure hits, while their close chemical analogues were inactive. Besides, electrophysiological analysis from the zebrafish midbrain demonstrated that pseurotin A2 and azaspirofuran A also ameliorate PTZ-induced epileptiform discharges. Next, to determine whether these findings translate to mammalians, both compounds were analyzed in the mouse 6 Hz (44 mA) psychomotor seizure model. They lowered the seizure duration dose-dependently, thereby confirming their antiseizure properties and suggesting activity against drug-resistant seizures. Finally, in a thorough ADMET assessment, pseurotin A2 and azaspirofuran A were found to be drug-like. Based on the prominent antiseizure activity in both species and the drug-likeness, we propose pseurotin A2 and azaspirofuran A as lead compounds that are worth further investigation for the treatment of epileptic seizures. This study not only provides the first evidence of antiseizure activity of pseurotins and azaspirofurans, but also demonstrates the value of the zebrafish model in (marine) natural product drug discovery in general, and for ASD discovery in particular.

IL-23R-activated STAT3/STAT4 is essential for Th1/Th17-mediated CNS autoimmunity.

The factors that promote the differentiation of pathogenic T cells in autoimmune diseases are poorly defined. Use of genetically modified mice has provided insight into molecules necessary for the development of autoimmunity, but the sum of the data has led to contradictory observations based on what is currently known about specific molecules in specific signaling pathways. To define the minimum signals required for development of encephalitogenic T cells that cause CNS autoimmunity, myelin-specific T cells were differentiated with various cytokine cocktails, and pathogenicity was determined by transfer into mice. IL-6+IL-23 or IL-12+IL-23 generated encephalitogenic T cells and recapitulated the essential cytokine signals provided by antigen-presenting cells, and both IL-6 and IL-12 induced IL-23 receptor expression on both mouse and human naive T cells. IL-23 signaled through both STAT3 and STAT4, and disruption in STAT4 signaling impaired CNS autoimmunity independent of IL-12. These data explain why IL-12-deficient mice develop CNS autoimmunity, while STAT4-deficient mice are resistant. CD4+ memory T cells from multiple sclerosis patients had significantly higher levels of p-STAT3/p-STAT4, and p-STAT3/p-STAT4 heterodimers were observed upon IL-23 signaling, suggesting that p-STAT3/p-STAT4 induced by IL-23 signaling orchestrate the generation of pathogenic T cells in CNS autoimmunity, regardless of Th1 or Th17 phenotype.

Does adding noradrenaline reuptake inhibition to selective serotonin reuptake inhibition improve efficacy in patients with depression? A systematic review of meta-analyses and large randomised pragmatic trials.

Selective serotonin reuptake inhibitors (SSRIs) are recommended as first-line pharmacological treatment for depression and are the most commonly prescribed class of antidepressants. However, there is substantial evidence that noradrenaline has a role in the pathogenesis and treatment of depression. This review aims to examine the evidence of including noradrenaline reuptake inhibition with serotonin reuptake inhibition with respect to increasing efficacy in the treatment of depression. Evidence from meta-analysis of randomised controlled trials (RCTs) and randomised pragmatic trials was found in support of greater efficacy of the serotonin noradrenaline reuptake inhibitors (SNRIs), venlafaxine and duloxetine, in moderate to severe depression compared to SSRIs but no evidence was found for superiority of milnacipran. There is sufficient current evidence that demonstrates an increase in efficacy, when noradrenaline reuptake is added to serotonin (5-HT) reuptake, to suggest that patients with severe depression or those who have failed to reach remission with a SSRI may benefit from treatment with a SNRI. However, as these conclusions are drawn from the evidence derived from meta-analyses and pragmatic trials, large adequately powered RCTs using optimal dosing regimens and clinically relevant outcome measures in severe depression and SSRI treatment failures are still required to confirm these findings.

Comparison of a classical Th1 bacteria versus a Th17 bacteria as adjuvant in the induction of experimental autoimmune encephalomyelitis.

The relative contribution of myelin-specific Th1 and Th17 cells in the pathology of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), is controversial. IL-12, the key cytokine necessary for the differentiation of Th1 cells, has been found to be dispensable for EAE induction; while the related cytokine associated with Th17 cells, IL-23, is a critical factor for inducing EAE. Since EAE is induced by immunization with myelin proteins in CFA which contains M. tuberculosis that generates a prototypical Th1-mediated immune response, we sought to determine if replacing the M. tuberculosis in the adjuvant with a bacterium that induces an IL-23-dependent Th17 cell response during infection would induce EAE with a different phenotype. C. rodentium, a bacterium that requires IL-23 for protective immunity, was used as the adjuvant in EAE and compared to CFA. Mice immunized with C. rodentium adjuvant (CRA) developed classical signs of EAE, similar to CFA-immunized mice, but disease was less severe with a later onset and slower progression than CFA. Surprisingly, the peripheral cytokine profile revealed similar numbers of Th1 and Th17 cells for both CFA and CRA-immunized mice; however, the number of Th1 and Th17 cells was significantly reduced in the CNS of CRA-immunized mice. The development of EAE in CRA-immunized mice was associated with epitope spreading. The unique clinical course of CRA immunizations helps serve as a useful alternative model for studying EAE pathogenesis and potential therapeutics for MS.

T-bet is essential for encephalitogenicity of both Th1 and Th17 cells.

The extent to which myelin-specific Th1 and Th17 cells contribute to the pathogenesis of experimental autoimmune encephalomyelitis (EAE) is controversial. Combinations of interleukin (IL)-1beta, IL-6, and IL-23 with transforming growth factor beta were used to differentiate myelin-specific T cell receptor transgenic T cells into Th17 cells, none of which could induce EAE, whereas Th1 cells consistently transferred disease. However, IL-6 was found to promote the differentiation of encephalitogenic Th17 cells. Further analysis of myelin-specific T cells that were encephalitogenic in spontaneous EAE and actively induced EAE demonstrated that T-bet expression was critical for pathogenicity, regardless of cytokine expression by the encephalitogenic T cells. These data suggest that encephalitogenicity of myelin-specific T cells appears to be mediated by a pathway dependent on T-bet and not necessarily pathway-specific end products, such as interferon gamma and IL-17.

Venlafaxine extended release versus citalopram in patients with depression unresponsive to a selective serotonin reuptake inhibitor.

Findings from the National Institute of Mental Health's Sequenced Treatment Alternatives to Relieve Depression trial indicate that approximately 50% of patients with major depressive disorder do not experience a treatment response after adequate first-line treatment with a selective serotonin reuptake inhibitor (SSRI). This study was designed to test the hypothesis that, after treatment failure with an SSRI, switching to venlafaxine extended release (ER) would offer advantages over switching to another SSRI, citalopram. The objectives of this trial were to compare the efficacy and safety of venlafaxine ER and citalopram in the treatment of moderate-to-severe depression in patients who did not experience a treatment response to an SSRI other than citalopram and to investigate the effects of severity of depression by categorizing treatment groups according to baseline severity. This was a 12-week, double-blind, randomized, parallel-group, multicenter study. Participants were adult outpatients who, following 8 weeks of monotherapy with an adequate dosing regimen of an SSRI other than citalopram and had not responded, met the diagnostic criteria for depression as described in the Diagnostic and statistical manual of mental disorders, fourth edition, and had a score > or =20 on the 21-item Hamilton Rating Scale for Depression (HAM-D21). After a 7-day screening period, patients were randomly assigned to receive venlafaxine ER 75 mg/day or citalopram 20 mg/day for the first 2 weeks. Doses could be increased every 2 weeks through week 6. Treatment lasted 12 weeks and was followed by a 1-week tapering period. Maximum dosages were venlafaxine ER 300 mg/day or citalopram 60 mg/day. The primary end point was the final on-therapy total HAM-D21 score. To investigate the treatment effects of the severity of depression on efficacy, a subgroup analysis was performed for baseline HAM-D21 total score < or =31 and >31. The analyses for HAM-D21, Montgomery-Asberg Depression Rating Scale (MADRS), Clinical Global Impressions - Severity (CGI-S), and Clinical Global Impressions - Improvement scores were based on intent-to-treat (ITT) population, for both the primary analysis and subgroup analysis according to baseline HAM-D21 total scores < or =31 or >31. Safety assessments included the recording of adverse events (AEs). A total of 406 patients (200 venlafaxine ER, 206 citalopram) were randomly assigned and 396 patients were included in the ITT population (194 venlafaxine ER, 202 citalopram). Treatment groups were similar in terms of demographics and baseline psychiatric assessments. Two hundred and eighty-four patients (137 venlafaxine ER, 147 citalopram) were present in the ITT population with a baseline HAM-D21 total score < or =31 and 112 patients (57 venlafaxine ER, 55 citalopram) in the >31 group. In the primary analysis, there was no statistical difference between groups. The group with a baseline HAM-D21 total score of 20-31 did not differ significantly in any efficacy parameters. In the group with a baseline HAM-D21 total score >31, the venlafaxine ER group differed significantly from the citalopram group on the primary end point HAM-D21 total score (P=0.0121). The secondary end point CGI-S score was statistically significant (P=0.0359), although the MADRS total score (P=0.0930) was not. AEs were reported by 57.8 and 63.4% of venlafaxine ER and citalopram patients, respectively. Overall discontinuation rates were 24.5% for venlafaxine ER and 20.9% for citalopram. Discontinuation rates owing to an AE as a primary or secondary reason were 5.5% for venlafaxine ER and 5.3% for citalopram. Overall, venlafaxine ER and citalopram showed similar efficacy in patients who had an inadequate response to an SSRI. In the subset of more severely depressed patients, venlafaxine ER was significantly more effective on a number of efficacy measures. Patients who remain severely depressed following treatment with an SSRI may gain more benefit from the dual-action drug venlafaxine, rather than switching to another SSRI.

Innovative reflecting interview: effect on high-utilizing patients with medically unexplained symptoms.

This pilot study was conducted to determine the effect of an innovative reflecting interview on the health care utilization, physical health, mental function, and health care satisfaction of high-utilizing primary care patients with medically unexplained physical symptoms. Twenty-four high-utilizing patients met study selection criteria and were randomly assigned to a no-intervention control group or a reflecting interview intervention group. Outcomes were measured at 4 weeks, 6 months, and 1 year after the date of study enrollment. Results indicated that high-utilizing patients with medically unexplained physical symptoms who participated in a reflecting interview had reduced total health care costs, primarily through the reduction of hospitalization or inpatient expenses, despite a modest increase in outpatient primary care clinic visits. These data suggest that participation in a reflecting interview and regular visits with a primary care clinician can decrease health care utilization without adversely affecting patient satisfaction.

Validity of index of learning styles scores: multitrait-multimethod comparison with three cognitive/learning style instruments.

Cognitive and learning styles research is limited by the lack of evidence supporting valid interpretations of style assessment scores. We sought evidence to support the validity of scores from 4 instruments: the Index of Learning Styles (ILS); the Learning Style Inventory (LSI); the Cognitive Styles Analysis (CSA), and the Learning Style Type Indicator (LSTI). The ILS assesses 4 domains: sensing-intuitive (SensInt), active-reflective (ActRefl), sequential-global (SeqGlob) and visual-verbal (VisVerb), each of which parallel a similar domain in at least 1 of the other instruments. We administered the ILS, LSI and CSA to family medicine and internal medicine residents and Year 1 and 3 medical students and applied the multitrait-multimethod matrix to evaluate convergence and discrimination. After 3 months participants repeated the ILS and completed the LSTI. A total of 89 residents and medical students participated. Multitrait-multimethod analysis showed evidence of both convergence and discrimination for ActRefl (ILS, LSI and LSTI) and SensInt (ILS and LSTI) scores. ILS SeqGlob and SensInt scores showed unanticipated correlation. No other domains met the criteria for convergence or discrimination. Test-retest reliabilities for ILS scores were 0.856 for SensInt, 0.809 for ActRefl, 0.703 for SeqGlob and 0.684 for VisVerb. Cronbach's alpha values were > or = 0.810 for LSI and 0.237-0.758 for LSTI. At least 9 participants misinterpreted the LSI instructions. These data support the validity of ILS active-reflective and sensing-intuitive scores, LSI active-reflective scores and LSTI sensing-intuitive scores for determining learning styles in this population. Cognitive style and learning style scores may not be interchangeable, even for constructs with similar definitions.

Investigation of the pathway for inter-copper electron transfer in peptidylglycine alpha-amidating monooxygenase.

Peptidylglycine alpha-amidating monooxygenase catalyzes the oxidative cleavage of glycine extended peptides at their terminus. In the course of the reaction, there is a requisite long-range electron transfer between the two copper centers (CuH and CuM) located in the hydroxylating domain. This communication presents data that argue against the participation of the extended peptide backbone of substrate in the long-range electron transfer. We propose that electron transfer occurs via the bulk solvent that separates CuH from CuM.

Detection of gas leakage from landfills using infrared thermography--applicability and limitations.

Although landfill gas emission can be greatly reduced by extraction and converting gas to energy, in practice not all gas can be collected and some leaks can still occur. Management of landfill gas can be improved if leaks can be detected and rectified effectively. This paper provides a brief review of methods available for detecting landfill leakage, with a focus on infrared thermography. It then describes a study which was conducted to test if an infrared camera can be used to detect gas leaks accurately by identifying them as anomalies. It examined the applicability and limitations of the technique by investigating fundamental factors such as weather conditions, ground conditions and distance of sensor from source. The paper also describes a test case conducted to reinforce the findings. It concluded that unless all the fundamental factors are clearly understood and addressed, the technique currently can only be used as a screening tool rather than as a precise tool to detect landfill gas leakages. For this reason, it would be difficult to use the technique as a basis for modelling gas emission from landfills.

A double-blind, randomised, placebo controlled study of venlafaxine XL in patients with generalised anxiety disorder in primary care.

BACKGROUND: Generalised anxiety disorder (GAD) is one of the commonest anxiety disorders and is treated almost entirely in primary care. Most recent studies performed in GAD have excluded depression for regulatory reasons. As GAD is usually a co-morbid disease, often co-existing with depression, the results from recent studies have only limited relevance to the naturalistic population. This study was set up to investigate venlafaxine XL in a more naturalistic population of patients with GAD. AIM: To assess the efficacy of venlafaxine XL in patients with generalised anxiety disorder with and without co-morbid depression. DESIGN OF STUDY: Double-blind, randomised, placebo controlled, parallel-group, 24-week study. SETTING: Primary care in the UK. METHODS: Patients enrolled in the study were over 18 years old, met DSM-IV criteria for GAD, and had a score of 20 or more on the Hamilton Anxiety Scale (HAM-A). A score of more than 23 on the Montgomery Asberg Depression Rating Scale (MADRS) excluded patients. Eligible patients were randomised to receive 75 mg of venlafaxine or a matching placebo. After 2 weeks the dose could be doubled if the physician considered the response to be poor. The study duration was 24 weeks. RESULTS: 244 patients were enrolled, with 122 randomised to the placebo and 122 to venlafaxine. Baseline characteristics were similar for both groups, each having a mean total HAM-A score at baseline of 28. The difference from the placebo group at 24 weeks on the total HAM-A score was 2.1 points (95% 0 to 4.2), which was statistically significant (P = 0.05). Remission rates at week 24 were 27.9% for the venlafaxine XL group and 18.9% for placebo group (P = 0.11). CONCLUSION: Venlafaxine was efficacious in the treatment of patients with GAD with and without depression over a 24-week period.