Changes in fibrin clot properties in patients after Roux-en-Y gastric bypass surgery.

Background: Obesity is a complex condition associated with prothrombotic fibrin networks that are resistant to fibrinolysis. Altered fibrin clot properties enhance cardiovascular risk and associate with a poorer prognosis following acute ischemic events. Bariatric surgery is commonly employed to improve cardiometabolic outcomes in individuals with obesity. However, the effects of this surgical intervention on fibrin clot properties have not been comprehensively studied. Objectives: To examine fibrin clot and lysis parameters in Roux-en-Y gastric bypass (RYGB) patients before and after surgery. Methods: The fibrin clot properties of 32 individuals living with obesity before and 9 months after RYGB surgery were determined using turbidimetric analysis. Correlation and regression analyses were used to identify relationships between clot properties and anthropomorphic and clinical measures. Results: RYGB surgery resulted in a significant reduction in adiposity-associated anthropometric measures as well as improvements in glycemia and lipid profile. Clot maximum absorbance was reduced from 0.43 ± 0.11 at baseline to 0.29 ± 0.10 at 9 months postsurgery (P < .0001), while fibrin clot lysis time failed to show a difference. The change in maximum absorbance was not caused by alterations in fibrinogen levels, while plasminogen activator inhibitor-1 concentration was significantly increased after surgery from 10,560 ± 6681 pg/mL to 15,290 ± 6559 pg/mL (P = .009). Correlation and regression analyses indicated that maximum absorbance was influenced by markers of adiposity as well as glycated hemoglobin and high-sensitivity C-reactive protein concentrations. Conclusion: RYGB surgery led to a decrease in the maximum absorbance of the fibrin clot. Values of maximum absorbance were associated with measures of glycemic control and inflammation. In contrast to previous reports, fibrin clot lysis time was not affected after surgery.

The most polyphagous insect herbivore? Host plant associations of the Meadow spittlebug, Philaenus spumarius (L.).

A comprehensive list of all known host plant species utilised by the Meadow Spittlebug (Philaenus spumarius (L.)) is presented, compiled from published and unpublished sources. P. spumarius feeds on 1311 host plants in 631 genera and 117 families. This appears, by a large margin, to be the greatest number of host species exploited by any herbivorous insect. The Asteraceae (222 species) and Rosaceae (110) together account for 25% of all host species. The Fabaceae (76) and Poaceae (73), are nearly tied for third and fourth place and these four families, combined with the Lamiaceae (62), Apiaceae (50), Brassicaceae (43) and Caprifoliaceae (34), comprise about half of all host species. Hosts are concentrated among herbaceous dicots but range from ferns and grasses to shrubs and trees. Philaenus spumarius is an "extreme polyphage", which appears to have evolved from a monophage ancestor in the past 3.7 to 7.9 million years. It is also the primary European vector of the emerging plant pathogen Xylella fastidiosa. Its vast host range suggests that it has the potential to spread X. fastidiosa among multiple hosts in any environment in which both the spittlebug and bacterium are present. Fully 47.9% of all known hosts were recorded in the Xylella-inspired BRIGIT citizen science P. spumarius host survey, including 358 hosts new to the documentary record, 27.3% of the 1311 total. This is a strong demonstration of the power of organized amateur observers to contribute to scientific knowledge.

Total plasma magnesium, zinc, copper and selenium concentrations in obese patients before and after bariatric surgery.

Obesity enhances the risk of type-2 diabetes, cardiovascular disease and inflammatory conditions and often leads to metal dyshomeostasis, which contributes to the negative health aspects associated with the disease. In severe cases, bariatric surgery can be recommended to achieve sustained weight loss and improvement in health. Here, magnesium, zinc, copper and selenium concentrations were examined in 24 obese patients (7 males; 17 females) before and 9 months after undergoing Roux-en-Y gastric bypass surgery. All patients lost weight over this period, with the mean BMI reducing from 51.2±7.1 kg/m2 to 37.2±5.5 kg/m2. Moreover, whole-blood glycated haemoglobin (HbA1c), as a marker of average glycaemia, was also measured and a correlative analysis of this parameter with metal concentrations performed. Significant alterations in the plasma concentrations of magnesium, zinc (both increased by 13.2% and 25.2% respectively) and copper (decreased by 7.9%) were observed over this period (plasma selenium concentration was unchanged), with BMI values correlating with plasma magnesium (p = 0.004) and zinc (p = 0.022) concentrations. At 9 months post-surgery, an increase in mean zinc/copper ratio was observed (0.86±0.29 compared to 0.63±0.14 pre-surgery). Comparison of whole-blood HbA1c concentrations pre- and post-surgery revealed a reduction from 6.50±1.28% pre-surgery to 5.51±0.49% post-surgery. Differences in plasma HbA1c and magnesium at either pre- and post-surgery correlated significantly, as did HbA1c and magnesium levels when pre- and post-surgery values were analysed together. Collectively, this work reveals that bariatric surgery, in conjunction with lifestyle/dietary changes, lead to improvements in the nutritional status of magnesium, zinc and copper. Furthermore, the observed improvements in magnesium and zinc were associated with weight loss and in the case of magnesium, to better glycaemic control.

Periarticular metal hypersensitivity complications of hip bearings containing cobalt-chromium.

Hip joints with bearings composed of cobalt-chromium alloy (metal-on-metal bearings) have been one of the most widely used implants in joint replacement arthroplasty. Unfortunately, these implants can contribute to a complication called aseptic lymphocyte-dominated vasculitis-associated lesion (ALVAL), a type IV metal hypersensitivity response around the joint. Consistent with such bearings, increased metal debris can be found in the surrounding fluids and in remote tissues and organs, due to wear and corrosion. It is hypothesized that metal ions released from the prosthesis (including Co2+) can potentially form haptens with proteins such as serum albumin in synovial fluid that in turn elicit ALVAL. Generally, elevated cobalt and chromium levels in synovial fluids may indicate implant failure. However, such measurements cannot be used as a reliable tool to predict the onset of ALVAL. To detect ALVAL, some diagnostic tests, questionnaires and imaging techniques have been used clinically with some success, but a standardized approach is lacking. At present, guidelines for implant usage and patient management are ambiguous and inconsistent across health care authorities. To reduce and better manage the development of ALVAL, further research into the precise molecular mechanism(s) by which ALVAL develops is urgently needed. Identification of diagnostic and prognostic biomarkers for ALVAL is required, as are more standardized guidelines for surgery and patient management.

Changes in plasma free fatty acids in obese patients before and after bariatric surgery highlight alterations in lipid metabolism.

Obesity is a complex disease that increases an individual's risk of developing other diseases and health-related problems. A common feature is dyslipidemia characterized by increased levels of plasma lipids, which include non-esterified fatty acids (NEFAs). The role of NEFAs in obesity-related morbidity is interesting as NEFAs constitute a reservoir of metabolic energy, are principal components of cell membranes and are precursors for signalling molecules. Bariatric surgery promotes sustained weight loss in severely obese patients, reducing the incidence and severity of co-morbidities. In this study we measure changes in circulating NEFA species in plasma samples taken from 25 obese individuals before and 9 months after Roux-en-Y gastric bypass surgery. The mean weight of the cohort reduced by 29.2% from 149.0 ± 25.1 kg pre-surgery to 105.5 ± 19.8 kg post-surgery and the BMI by 28.2% from 51.8 ± 6.3 kg/m2 pre-surgery to 37.2 ± 5.4 kg/m2. Mean glycated haemoglobin (HbA1c) reduced from 6.5 ± 1.3 to 5.5 ± 0.5%, consistent with the intervention leading to improved glycaemic control, particularly in those who were dysglycemic prior to surgery. Total and LDL cholesterol concentrations were markedly reduced following surgery. Concentrations of seven NEFAs were found to decrease 9 months after surgery compared to pre-surgery levels: myristate, palmitoleate, palmitate, linoleate, oleate, stearate and arachidonate. Bariatric surgery led to increased lipogenesis and elongase activity and decreased stearoyl-CoA desaturase 1 activity. This study therefore highlights metabolic changes that take place following gastric bypass surgery in severely obese patients.

Strategies for Therapeutic Amelioration of Aberrant Plasma Zn2+ Handling in Thrombotic Disease: Targeting Fatty Acid/Serum Albumin-Mediated Effects.

The initiation, maintenance and regulation of blood coagulation is inexorably linked to the actions of Zn2+ in blood plasma. Zn2+ interacts with a variety of haemostatic proteins in the bloodstream including fibrinogen, histidine-rich glycoprotein (HRG) and high molecular weight kininogen (HMWK) to regulate haemostasis. The availability of Zn2+ to bind such proteins is controlled by human serum albumin (HSA), which binds 70-85% of plasma Zn2+ under basal conditions. HSA also binds and transports non-esterified fatty acids (NEFAs). Upon NEFA binding, there is a change in the structure of HSA which leads to a reduction in its affinity for Zn2+. This enables other plasma proteins to better compete for binding of Zn2+. In diseases where elevated plasma NEFA concentrations are a feature, such as obesity and diabetes, there is a concurrent increase in hypercoagulability. Evidence indicates that NEFA-induced perturbation of Zn2+-binding by HSA may contribute to the thrombotic complications frequently observed in these pathophysiological conditions. This review highlights potential interventions, both pharmaceutical and non-pharmaceutical that may be employed to combat this dysregulation. Lifestyle and dietary changes have been shown to reduce plasma NEFA concentrations. Furthermore, drugs that influence NEFA levels such as statins and fibrates may be useful in this context. In severely obese patients, more invasive therapies such as bariatric surgery may be useful. Finally, other potential treatments such as chelation therapies, use of cholesteryl transfer protein (CETP) inhibitors, lipase inhibitors, fatty acid inhibitors and other treatments are highlighted, which with additional research and appropriate clinical trials, could prove useful in the treatment and management of thrombotic disease through amelioration of plasma Zn2+ dysregulation in high-risk individuals.

Identification of plasma proteins associated with oesophageal cancer chemotherapeutic treatment outcomes using SWATH-MS.

Oesophageal adenocarcinoma (OAC) is an aggressive cancer with a five-year survival of <15%. Current chemotherapeutic strategies only benefit a minority (20-30%) of patients and there are no methods available to differentiate between responders and non-responders. We performed quantitative proteomics using Sequential Window Acquisition of all THeoretical fragment-ion spectra-Mass Spectrometry (SWATH-MS) on albumin/IgG-depleted and non-depleted plasma samples from 23 patients with locally advanced OAC prior to treatment. Individuals were grouped based on tumour regression (TRG) score (TRG1/2/3 vs TRG4/5) after chemotherapy, and differentially abundant proteins were compared. Protein depletion of highly abundant proteins led to the identification of around twice as many proteins. SWATH-MS revealed significant quantitative differences in the abundance of several proteins between the two groups. These included complement c1q subunit proteins, C1QA, C1QB and C1QC, which were of higher abundance in the low TRG group. Of those that were found to be of higher abundance in the high TRG group, glutathione S-transferase pi (GSTP1) exhibited the lowest p-value and highest classification accuracy and Cohen's kappa value. Concentrations of these proteins were further examined using ELISA-based assays. This study provides quantitative information relating to differences in the plasma proteome that underpin response to chemotherapeutic treatment in oesophageal cancers. SIGNIFICANCE: Oesophageal cancers, including oesophageal adenocarcinoma (OAC) and oesophageal gastric junction cancer (OGJ), are one of the leading causes of cancer mortality worldwide. Curative therapy consists of surgery, either alone or in combination with adjuvant or neoadjuvant chemotherapy or radiation, or combination chemoradiotherapy regimens. There are currently no clinico-pathological means of predicting which patients will benefit from chemotherapeutic treatments. There is therefore an urgent need to improve oesophageal cancer disease management and treatment strategies. This work compared proteomic differences in OAC patients who responded well to chemotherapy as compared to those who did not, using quantitative proteomics prior to treatment commencement. SWATH-MS analysis of plasma (with and without albumin/IgG-depletion) from OAC patients prior to chemotherapy was performed. This approach was adopted to determine whether depletion offered a significant improvement in peptide coverage. Resultant datasets demonstrated that depletion increased peptide coverage significantly. Additionally, there was good quantitative agreement between commonly observed peptides. Data analysis was performed by adopting both univariate as well as multivariate analysis strategies. Differentially abundant proteins were identified between treatment response groups based on tumour regression grade. Such proteins included complement C1q sub-components and GSTP1. This study provides a platform for further work, utilising larger sample sets across different treatment regimens for oesophageal cancer, that will aid the development of 'treatment response prediction assays' for stratification of OAC patients prior to chemotherapy.

The influence of HLA genotype on the development of metal hypersensitivity following joint replacement.

Background: Over five million joint replacements are performed across the world each year. Cobalt chrome (CoCr) components are used in most of these procedures. Some patients develop delayed-type hypersensitivity (DTH) responses to CoCr implants, resulting in tissue damage and revision surgery. DTH is unpredictable and genetic links have yet to be definitively established. Methods: At a single site, we carried out an initial investigation to identify HLA alleles associated with development of DTH following metal-on-metal hip arthroplasty. We then recruited patients from other centres to train and validate an algorithm incorporating patient age, gender, HLA genotype, and blood metal concentrations to predict the development of DTH. Accuracy of the modelling was assessed using performance metrics including time-dependent receiver operator curves. Results: Using next-generation sequencing, here we determine the HLA genotypes of 606 patients. 176 of these patients had experienced failure of their prostheses; the remaining 430 remain asymptomatic at a mean follow up of twelve years. We demonstrate that the development of DTH is associated with patient age, gender, the magnitude of metal exposure, and the presence of certain HLA class II alleles. We show that the predictive algorithm developed from this investigation performs to an accuracy suitable for clinical use, with weighted mean survival probability errors of 1.8% and 3.1% for pre-operative and post-operative models respectively. Conclusions: The development of DTH following joint replacement appears to be determined by the interaction between implant wear and a patient's genotype. The algorithm described in this paper may improve implant selection and help direct patient surveillance following surgery. Further consideration should be given towards understanding patient-specific responses to different biomaterials.

Levothyroxine Treatment and the Risk of Cardiac Arrhythmias - Focus on the Patient Submitted to Thyroid Surgery.

Levothyroxine (LT4) is used to treat frequently encountered endocrinopathies such as thyroid diseases. It is regularly used in clinical (overt) hypothyroidism cases and subclinical (latent) hypothyroidism cases in the last decade. Suppressive LT4 therapy is also part of the medical regimen used to manage thyroid malignancies after a thyroidectomy. LT4 treatment possesses dual effects: substituting new-onset thyroid hormone deficiency and suppressing the local and distant malignancy spreading in cancer. It is the practice to administer LT4 in less-than-high suppressive doses for growth control of thyroid nodules and goiter, even in patients with preserved thyroid function. Despite its approved safety for clinical use, LT4 can sometimes induce side-effects, more often recorded with patients under treatment with LT4 suppressive doses than in unintentionally LT4-overdosed patients. Cardiac arrhythmias and the deterioration of osteoporosis are the most frequently documented side-effects of LT4 therapy. It also lowers the threshold for the onset or aggravation of cardiac arrhythmias for patients with pre-existing heart diseases. To improve the quality of life in LT4-substituted patients, clinicians often prescribe higher doses of LT4 to reach low normal TSH levels to achieve cellular euthyroidism. In such circumstances, the risk of cardiac arrhythmias, particularly atrial fibrillation, increases, and the combined use of LT4 and triiodothyronine further complicates such risk. This review summarizes the relevant available data related to LT4 suppressive treatment and the associated risk of cardiac arrhythmia.

Prognostic features of the tumour microenvironment in oesophageal adenocarcinoma.

Oesophageal adenocarcinoma (OAC) is a disease with an incredibly poor survival rate and a complex makeup. The growth and spread of OAC tumours are profoundly influenced by their surrounding microenvironment and the properties of the tumour itself. Constant crosstalk between the tumour and its microenvironment is key to the survival of the tumour and ultimately the death of the patient. The tumour microenvironment (TME) is composed of a complex milieu of cell types including cancer associated fibroblasts (CAFs) which make up the tumour stroma, endothelial cells which line blood and lymphatic vessels and infiltrating immune cell populations. These various cell types and the tumour constantly communicate through environmental cues including fluctuations in pH, hypoxia and the release of mitogens such as cytokines, chemokines and growth factors, many of which help promote malignant progression. Eventually clusters of tumour cells such as tumour buds break away and spread through the lymphatic system to nearby lymph nodes or enter the circulation forming secondary metastasis. Collectively, these factors need to be considered when assessing and treating patients clinically. This review aims to summarise the ways in which these various factors are currently assessed and how they relate to patient treatment and outcome at an individual level.

Leptin and Obesity: Role and Clinical Implication.

The peptide hormone leptin regulates food intake, body mass, and reproductive function and plays a role in fetal growth, proinflammatory immune responses, angiogenesis and lipolysis. Leptin is a product of the obese (ob) gene and, following synthesis and secretion from fat cells in white adipose tissue, binds to and activates its cognate receptor, the leptin receptor (LEP-R). LEP-R distribution facilitates leptin's pleiotropic effects, playing a crucial role in regulating body mass via a negative feedback mechanism between adipose tissue and the hypothalamus. Leptin resistance is characterized by reduced satiety, over-consumption of nutrients, and increased total body mass. Often this leads to obesity, which reduces the effectiveness of using exogenous leptin as a therapeutic agent. Thus, combining leptin therapies with leptin sensitizers may help overcome such resistance and, consequently, obesity. This review examines recent data obtained from human and animal studies related to leptin, its role in obesity, and its usefulness in obesity treatment.

Differential outcomes of novel plant-herbivore associations between an invading planthopper and native and invasive Spartina cordgrass species.

Non-native plants may benefit, briefly or permanently, from natural enemy release in their invaded range, or may form novel interactions with native enemy species. Likewise, newly arrived herbivores may develop novel associations with native plants or, where their hosts have arrived ahead of them, re-establish interactions that existed previously in their ancestral ranges. Predicting outcomes from this diversity of novel and re-established interactions between plants and their herbivores presents a major challenge for invasion biology. We report on interactions between the recently arrived invasive planthopper Prokelisia marginata, and the multi-ploidy Spartina complex of four native and introduced species in Britain, each representing a different level of shared evolutionary history with the herbivore. As predicted, S. alterniflora, the ancestral host, was least impacted by planthopper herbivory, with the previously unexposed native S. maritima, a nationally threatened species, suffering the greatest impacts on leaf length gain, new leaf growth and relative water content. Contrary to expectations, glasshouse trials showed P. marginata to preferentially oviposit on the invasive allododecaploid S. anglica, on which it achieved earlier egg hatch, faster nymphal development, larger female body size and greatest final population size. We suggest P. marginata is in the process of rapid adaptation to maximise its performance on what is now the most abundant and widespread host in Britain. The diversity of novel and re-established interactions of the herbivore with this multi-ploidy complex makes this a highly valuable system for the study of the evolutionary ecology of plant-insect interactions and their influence on invasion dynamics.

Sub-Micromolar Pulse Dipolar EPR Spectroscopy Reveals Increasing CuII -labelling of Double-Histidine Motifs with Lower Temperature.

Electron paramagnetic resonance (EPR) distance measurements are making increasingly important contributions to the studies of biomolecules by providing highly accurate geometric constraints. Combining double-histidine motifs with CuII spin labels can further increase the precision of distance measurements. It is also useful for proteins containing essential cysteines that can interfere with thiol-specific labelling. However, the non-covalent CuII coordination approach is vulnerable to low binding-affinity. Herein, dissociation constants (KD ) are investigated directly from the modulation depths of relaxation-induced dipolar modulation enhancement (RIDME) EPR experiments. This reveals low- to sub-µm CuII KD s under EPR distance measurement conditions at cryogenic temperatures. We show the feasibility of exploiting the double-histidine motif for EPR applications even at sub-µm protein concentrations in orthogonally labelled CuII -nitroxide systems using a commercial Q-band EPR instrument.

Influence of zinc on glycosaminoglycan neutralisation during coagulation.

Heparan sulfate (HS), dermatan sulfate (DS) and heparin are glycosaminoglycans (GAGs) that serve as key natural and pharmacological anticoagulants. During normal clotting such agents require to be inactivated or neutralised. Several proteins have been reported to facilitate their neutralisation, which reside in platelet α-granules and are released following platelet activation. These include histidine-rich-glycoprotein (HRG), fibrinogen and high-molecular-weight kininogen (HMWK). Zinc ions (Zn2+) are also present in α-granules at a high concentration and participate in the propagation of coagulation by influencing the binding of neutralising proteins to GAGs. Zn2+ in many cases increases the affinity of these proteins to GAGs, and is thus an important regulator of GAG neutralisation and haemostasis. Binding of Zn2+ to HRG, HMWK and fibrinogen is mediated predominantly through coordination to histidine residues but the mechanisms by which Zn2+ increases the affinity of the proteins for GAGs are not yet completely clear. Here we will review current knowledge of how Zn2+ binds to and influences the neutralisation of GAGs and describe the importance of this process in both normal and pathogenic clotting.

Changes in cardiac Na+/K+-ATPase expression and activity in female rats fed a high-fat diet.

The aim of this study was to investigate whether the presence of endogenous estradiol alters the effects of a high-fat (HF) diet on activity/expression of the cardiac Na+/K+-ATPase, via PI3K/IRS and RhoA/ROCK signalling cascades in female rats. For this study, female Wistar rats (8 weeks old, 150-200 g) were fed a standard diet or a HF diet (balanced diet for laboratory rats enriched with 42% fat) for 10 weeks. The results show that rats fed a HF diet exhibited a decrease in phosphorylation of the α1 subunit of Na+/K+-ATPase by 30% (p < 0.05), expression of total α1 subunit of Na+/K+-ATPase by 31% (p < 0.05), and association of IRS1 with p85 subunit of PI3K by 42% (p < 0.05), while the levels of cardiac RhoA and ROCK2 were significantly increased by 84% (p < 0.01) and 62% (p < 0.05), respectively. Our results suggest that a HF diet alters cardiac Na+/K+-ATPase expression via molecular mechanisms involving RhoA/ROCK and IRS-1/PI3K signalling in female rats.

17ß-Estradiol protects against the effects of a high fat diet on cardiac glucose, lipid and nitric oxide metabolism in rats.

The aim of this study was to investigate the in vivo effects of 17ß-estradiol (E2) on myocardial metabolism and inducible nitric oxide synthase (iNOS) expression/activity in obese rats. Male Wistar rats were fed with a normal or a high fat (HF) diet (42% fat) for 10 weeks. Half of the HF fed rats were treated with a single dose of E2 while the other half were placebo-treated. 24 h after treatment animals were sacrificed. E2 reduced cardiac free fatty acid (FFA) (p < 0.05), L-arginine (p < 0.01), iNOS mRNA (p < 0.01), and protein (p < 0.05) levels and translocation of the FFA transporter (CD36) (p < 0.01) to the plasma membrane (PM) in HF fed rats. In contrast, Akt phosphorylation at Thr308 (p < 0.05) and translocation of the glucose transporter GLUT4 (p < 0.05) to the PM increased after E2 treatment in HF rats. Our results indicate that E2 acts via the PI3K/Akt signalling pathway to partially protect myocardial metabolism by attenuating the detrimental effects of increased iNOS expression/activity in HF fed rats.

Influence of a High-Fat Diet on Cardiac iNOS in Female Rats.

BACKGROUND: Overexpression of inducible nitric oxide synthase (iNOS) is a key link between high-fat (HF) diet induced obesity and cardiovascular disease. Oestradiol has cardioprotective effects that may be mediated through reduction of iNOS activity/expression. METHODS: In the present study, female Wistar rats were fed a standard diet or a HF diet (42% fat) for 10 weeks. iNOS gene and protein expressions were measured in heart tissue. HF-fed rats exhibited a significant increase in cardiac iNOS mRNA by 695% (p<.05), iNOS protein level by 248% (p<0.01), without changes in nitrate/nitrite levels. Expression of CD36 protein in plasma membranes was increased by 37% (p<0.05), while the concentration of free fatty acids (FFA) was reduced by 25% (p<0.01) in HF-fed rats. Expression of the p50 subunit of nuclear factor-kB (NFkB-p50) in heart was increased by 77% (p<0.01) in HF-fed rats. Expression of protein kinase B (Akt) and extracellular signalregulated kinases 1/2 (ERK1/2) were unchanged between the groups. There was a significant increase in the ratio of phospho-Akt/total Akt but not for phospho-ERK1/2/total ERK1/2 in HF-fed rats. Estrogen receptor-α levels (by 50%; p<0.05) and serum oestradiol concentrations (by 35%; p<0.05) were shown to be significantly reduced in HF-fed rats. RESULTS AND CONCLUSION: Our results revealed that a HF diet led to increased iNOS expression, most likely via a mechanism involving Akt and NFκB-p50 proteins. Decreased levels of oestradiol and ERα protein in the HF-fed group, in combination with increased iNOS levels are consistent with the hypothesis that oestradiol has a cardioprotective effect through its ability to regulate iNOS expression.

A high fat diet induces sex-specific differences in hepatic lipid metabolism and nitrite/nitrate in rats.

Men and women differ substantially with regard to the severity of insulin resistance (IR) but the underlying mechanism(s) of how this occurs is poorly characterized. We investigated whether a high fat (HF) diet resulted in sex-specific differences in nitrite/nitrate production and lipid metabolism and whether these variances may contribute to altered obesity-induced IR. Male and female Wistar rats were fed a standard laboratory diet or a HF diet for 10 weeks. The level of plasma nitrite/nitrate, as well as free fatty acid (FFA), in both plasma and liver lysates were assessed. The levels of inducible nitric oxide (NO) synthase (iNOS), p65 subunit of NFκB, total and phosphorylated forms of Akt, mTOR and PDK-1 in lysates, and the levels of glucose transporter 2 (Glut-2) and fatty acid translocase/cluster of differentiation 36 (FAT/CD36) in plasma membrane fractions of liver were assessed. HF-fed male rats exhibited a significant increase in plasma nitrite/nitrate, and hepatic FFA and FAT/CD36 levels compared with controls. They also displayed a relative decrease in iNOS and Glut-2 levels in the liver. Phosphorylation of Akt (at Ser(473) and Thr(308)), mTOR and PDK-1 was also reduced. HF-fed female rats exhibited increased levels of NFκB-p65 in liver compared with controls, while levels of Glut-2, FAT/CD36 and Akt phosphorylation at Thr(308) and PDK-1 were decreased. Our results reveal that altered lipid and glucose metabolism in obesity, lead to altered iNOS expression and nitrite/nitrate production. It is likely that this mechanism contributes to sex-specific differences in the development of IR.

Phospholipase C-η2 interacts with nuclear and cytoplasmic LIMK-1 during retinoic acid-stimulated neurite growth.

Neurite growth is central to the formation and differentiation of functional neurons, and recently, an essential role for phospholipase C-η2 (PLCη2) in neuritogenesis was revealed. Here we investigate the function of PLCη2 in neuritogenesis using Neuro2A cells, which upon stimulation with retinoic acid differentiate and form neurites. We first investigated the role of the PLCη2 calcium-binding EF-hand domain, a domain that is known to be required for PLCη2 activation. To do this, we quantified neurite outgrowth in Neuro2A cells, stably overexpressing wild-type PLCη2 and D256A (EF-hand) and H460Q (active site) PLCη2 mutants. Retinoic acid-induced neuritogenesis was highly dependent on PLCη2 activity, with the H460Q mutant exhibiting a strong dominant-negative effect. Expression of the D256A mutant had little effect on neurite growth relative to the control, suggesting that calcium-directed activation of PLCη2 is not essential to this process. We next investigated which cellular compartments contain endogenous PLCη2 by comparing immunoelectron microscopy signals over control and knockdown cell lines. When signals were analyzed to reveal specific labeling for PLCη2, it was found to be localized predominantly over the nucleus and cytosol. Furthermore in these compartments (and also in growing neurites), a proximity ligand assay revealed that PLCη2 specifically interacts with LIMK-1 in Neuro2A cells. Taken together, these data emphasize the importance of the PLCη2 EF-hand domain and articulation of PLCη2 with LIMK-1 in regulating neuritogenesis.

In vivo effects of 17ß-estradiol on cardiac Na(+)/K(+)-ATPase expression and activity in rat heart.

In this study the in vivo effects of estradiol in regulating Na(+)/K(+)-ATPase function in rat heart was studied. Adult male Wistar rats were treated with estradiol (40µg/kg, i.p.) and after 24h the animals were sacrificed and the heart excised. Following estradiol administration, cardiac Na(+)/K(+)-ATPase activity, expression of the α1 subunit, and phosphorylation of the α1 subunit were significantly increased. These animals also had significantly decreased levels of digoxin-like immunoreactive factor(s). Na(+) levels were also significantly reduced but to a level that was still within the normal physiological range, highlighting the ability of the Na(+)/K(+)-ATPase to balance the ionic composition following treatment with estradiol. Estradiol treated rats also showed increased phosphorylation of protein kinase B (Akt), and extracellular-signal-regulated kinase 1/2 (ERK1/2). We therefore suggest a role for Akt and/or ERK1/2 in estradiol-mediated regulation of cardiac Na(+)/K(+)-ATPase expression and activity in rat heart.