, 1997; Fewell & Page Jr, 1999; Parrish, Viscido & Grünbaum, 2002; Theraulaz et al., 2003; Couzin, 2008). Importantly, emergence mechanisms require

only spatial proximity among individuals, leading to novel behaviors and patterns without underlying genetic changes in behavioral strategy as individuals interact with one another and their shared environment. If the defining features of eusociality are similarly self-organizing in nature, this would provide a mechanism for their appearance in a single step at the origin of group formation. Critically evaluating these alternative trajectories of social evolution is not straightforward, as the initial characteristics of extant social species, whether emergent or not, are likely to have long since been superseded by secondary adaptations

to social life. One approach to recovering what incipient groups may Lumacaftor have been like is to assemble artificial social groups in species that are normally solitary, but tolerant enough of conspecifics to persist in groups without fatal aggression and group dissolution. Because such individuals have no evolutionary history of social cooperation, their behaviors under experimental group formation should be a function of their intrinsic behavioral repertoires and any emergent properties resulting from interactions with the shared physical environment and/or other group members. As predicted by the emergent property hypothesis, artificially assembled groups Gefitinib nmr of insects that are normally solitary during the life stage being investigated

show pronounced division of labor in nonreproductive tasks such as nest construction and defense, suggesting that these can emerge from self-organizing processes (Fewell & Page Jr, 1999; Helms Cahan & Fewell, 2004; Jeanson, Kukuk & Fewell, 2005; Jeanson & Fewell, 2008; Holbrook et al., HSP90 2009). However, whether self-organization can also cause the emergence of division of labor in reproduction has scarcely been investigated, despite its centrality to the origin and elaboration of eusociality (Sakagami & Maeta, 1987). This has led authors to question whether emergent property scenarios have any applicability to the evolution of eusociality (Bourke, 2011; Duarte et al., 2011; Herre & Wcislo, 2011). In this study, we experimentally test whether self-organizing mechanisms can spontaneously generate reproductive division of labor by creating forced associations of colony-founding queens of the harvester ant species Pogonomyrmex barbatus. Although ants show highly derived eusocial structure during most of the life cycle, queens of many species found colonies alone, excavating the nest and rearing the first cohort of workers in complete social isolation. Because queens are strictly solitary during this period, they should be selected for a behavioral repertoire similar to that of a hypothetical solitary ground-nesting ant ancestor.

Informed consent was obtained from each patient and the study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in the a priori Internal Review Board’s approval. APAP history use was recorded and no subjects had taken APAP within a month of enrollment. Subjects were excluded if they had abnormal liver tests on screening or a history of chronic liver disease. Nine subjects AZD5363 cost were enrolled for 7 days each as inpatients in the General Clinical Research Center at the UNC Hospital. Human overdose subject descriptions have been reported.5 Subjects were placed on a defined liquid diet to assure uniform nutritional intake. The protein source was soy, the fat source was

safflower oil of known composition, and the carbohydrate source was cane or beet sugar. Other ingredients included Metamucil to provide fiber and vanilla. The overall macronutrient composition was 15% of total calories from protein, 30% from fat, and 55% from carbohydrate. Subject’s daily calorie intake, divided into five consistently timed meals per day, was based on the formula 35 kcal/kg actual body weight. On day 4 the subjects were fasting until 2 hours after receiving APAP. Weight was monitored daily and calories adjusted to maintain body weight. On the morning of the fourth day, six subjects received a single dose of 4 g of APAP administered as eight, 500-mg capsules, whereas three

received placebo pills. Blood was collected at 6 AM on each of the clinical days for ALT measurement. PB, 7.5 mL, was drawn into PAXgene (PreAnalytiX/Qiagen, Hilden, Germany) blood RNA Venetoclax ic50 collection tubes (3 tubes at 2.5 mL) immediately before the first dose and at 6, 18, 24, 48, 72, and 96 hours postdosing. Samples were mixed and allowed to remain at room temperature for 2 hours, then frozen at −20°C until RNA isolation. Blood was also collected at 6 AM on each of the clinical days for measurement of clinical chemistries SPTLC1 and complete blood counts (CBCs), performed by the UNC Hospital clinical laboratories. Serum was collected and frozen at −80°C predose

and at the following times postdose: 30 minutes, 60 minutes, 90 minutes, 2, 3, 4, 5, 6, 8, and 12 hours. Upon study completion, APAP and metabolites were assayed in the serum by high-performance liquid chromatography (HPLC).6 In order to measure APAP metabolite excretion, urine was also collected for 24 hours postdosing and stored at −20°C with ascorbic acid (1 g/L). RNA was isolated utilizing the PAXgene blood RNA isolation kit (PreAnalytiX/Qiagen) according to the manufacturer’s protocol, including the optional on-column DNase digestion. RNA quality was assessed with an Agilent Bioanalyzer (Palo Alto, CA) and only samples with intact 18S and 28S ribosomal RNA peaks were used for microarray analysis. Gene expression profiling was conducted using Agilent Human 1A(V2) oligo arrays with ≈20,000 genes represented.

[2] Thus, NAFLD is associated with an increased liver-related morbidity

and mortality and is emerging as a leading cause of liver transplantation.[3] In addition, patients with NAFLD exhibit an increased risk of developing both type 2 diabetes mellitus (T2DM) and cardiovascular disease.[4] For these reasons, timely and effective treatment of patients with NAFLD, and particularly those with NASH, is indicated to prevent metabolic consequences and eventually hamper the development of liver cirrhosis. However, current treatment options are limited to lifestyle changes, which are effective but difficult to achieve because of adherence issues.

Erismodegib nmr Although many pharmacological agents have been proposed to treat patients with NAFLD/NASH, the only drugs tested to date in large, randomized, controlled trials are pioglitazone and vitamin E, which have shown efficacy for treatment of NASH.[1] However, their therapeutic value is limited and several safety concerns have been raised recently. Therefore, the development of novel, pathophysiologically targeted, safe, and effective therapies is urgently needed. In this issue of HEPATOLOGY, Staels et al.[5] report promising preclinical data on the effects of a dual peroxisome proliferator-activated receptor (PPAR)-α/δ agonist Gefitinib supplier (GFT505) in rodent models of NAFLD/NASH and hepatic fibrosis, along with some clinical data on the effects of the

compound on liver function Dynein tests (LFTs) in humans. Before getting into details of their work, a few words on the role of PPARs in NAFLD/NASH are in order. PPARs are lipid-activated nuclear receptors highly conserved in mammals that, upon activation by the appropriate ligand, control complex networks of target genes involved in a myriad of processes, including energy homeostasis, inflammatory response, and lipid and carbohydrate metabolism.[6] Receptors of this family form heterodimers with the nuclear retinoid X receptor and are divided in three subtypes, each encoded by a different gene: PPAR-α (NRC11 1); PPAR-δ (NRC2, also named β/δ); and PPAR-γ (NRC3). Though PPAR-α and PPAR-γ have a relatively restricted tissue expression, being predominantly expressed in hepatocytes and adipocytes, respectively, PPAR-δ exhibits a more ubiquitous expression with particularly high abundance in muscle tissue and macrophages. Activation of different PPARs represents an important pharmacological target because of the multifaceted metabolic effects on lipid and carbohydrate metabolism and their effects on innate immunity and inflammatory responses.

96, 97 In selected patients with INCPH (e.g., abdominal discomfort or hypersplenism patients), these interventions can be regarded as effective therapeutic modalities. Based on the high prevalence of thrombophilia and incidence of portal vein thrombosis in INCPH patients, several investigators

have incriminated thrombosis of small intrahepatic portal veins as an important etiological factor in the development of this disorder.6, 30 Additionally, a trend toward poor prognosis has been reported in patients with INCPH who develop portal vein thrombosis.6 As a result, anticoagulation therapy has been proposed by Selleck Fludarabine several investigators to prevent disease progression and to maintain portal vein patency.6, 32, 98 However, considering the fact that gastrointestinal SAHA HDAC concentration bleeding is the main complication of INCPH and the uncertain role of thrombophilia in the pathogenesis, this treatment is still a matter of debate and cannot be generally implicated until more solid data are present. Nonetheless, we believe that anticoagulation therapy must be considered in patients

with underlying prothrombotic conditions and in patients who develop portal vein thrombosis. Generally, patients with isolated INCPH have a normal liver function and the complications of portal hypertension can be managed successfully with endoscopic therapy and shunting. However, several reports describing liver transplantation in patients with INCPH have been published. The reported indications requiring liver transplantation in these patients were medical unmanageable portal hypertension, hepatopulmonary syndrome, hepatic encephalopathy, and progressive Etofibrate hepatic failure.49, 63, 78 Recently, Karsinskas et al. described a small cohort of INCPH patients treated with liver transplantation.63 The main indication for liver transplantation was medically unmanageable severe portal hypertension; a minority was listed because of hepatic encephalopathy. Notwithstanding the fact that resistant bleeding in INCPH patients should be treated with portosystemic shunting before considering the option of

liver transplantation, only two patients underwent pretransplantation portosystemic shunting procedures (e.g., TIPS and mesocaval shunt). Presumably, the high frequency of cirrhosis misdiagnoses in these patients led to early referral for liver transplantation. To prevent unnecessary liver transplantation in these patients, early discrimination between cirrhosis and INCPH is extremely important. Based on small-sized cohorts (with limited follow-up), post-transplantation outcome in these patients is good and INCPH tends not to recur.63, 99, 100 Data on the etiology and management of INCPH are scarce, and currently applied diagnostic and therapeutic algorithms are based on personal experience or data from limited numbers of patients.

Prospective ascertainment of alcohol intake poses fewer problems concerning memory than retrospective ascertainment, but this advantage is offset by the problems involved in long-term studies of rare chronic diseases. In addition, several studies have found

that heavy drinkers report higher alcohol intakes retrospectively than prospectively,20-22 which suggests that people are more comfortable reporting past heavy drinking than current heavy drinking. Because intense pressure on patients to reduce their alcohol intake before HCV treatment seemed likely to foster denial, we chose to study patients who had already been treated to reduce denial, and we emphasized that patients’ data would be kept confidential, even from their care providers. Test-retest reliability of the CLDH was not reexamined in this study, but internal validity was good. Patients with a CD diagnosis or CD treatment reported consuming approximately twice as much alcohol before HCV treatment as patients without CD records. It is possible that patients who did not obtain an SVR might have minimized their alcohol intake if they thought it might jeopardize their future treatment. However,

successfully treated patients had little reason to exaggerate their drinking, and the high alcohol intakes reported both by patients who did and did not recover suggests that denial did not influence these findings. In conclusion, excellent P/R treatment completion rates and outcomes were not impaired by high pretreatment alcohol intakes or failure to abstain 6 months before treatment in patients of an integrated health care plan who were aggressively supported and closely monitored. These findings suggest that past heavy drinking and recent drinking represent low treatment risk in these patients. The fact that over 60% of

patients stopped drinking when HCV+ was diagnosed documents the before potential for immediate health benefits associated with case finding in this population. The authors thank Boris H. Ruebner, M.D. (University of California at Davis, Davis, CA), for confirming biopsy findings in our cohort. The authors also thank Lilli Remer of the Prevention Research Center for assistance in programming the computer-assisted interview used in this study and deriving measures from it, Fred Johnson, Ph.D., of the Prevention Research Center, for assistance in data management and analysis, John Edwards of Kaiser Permanente Chemical Dependency Services for IT assistance, and Sonia Menenberg, R.N., of Kaiser Permanente Chemical Dependency Services for supervising our interviewer. “
“Hepatolenticular degeneration, commonly known as Wilson disease, is an Opaganib manufacturer autosomal recessive inherited disease of abnormal copper metabolism, characterized by the accumulation of copper in the body due to decreased biliary excretion of copper from hepatocytes.

These data indicate that IL-32 affects several parameters of HCV pathology but itself might not have antiviral properties. Other viral infections such as influenza A virus infection also induce IL-32 expression.18, 42 Influenza virus induced cyclooxygenase (COX)-2-mediated prostaglandin E2 production was suppressed by overexpression of IL-32 but decreased by IL-32-specific siRNA, suggesting a feedback mechanism between IL-32 selleck and COX-2.18 A clear antiviral effect against influenza A virus for IL-32 has not been demonstrated in these studies. In conclusion, in patients with chronic HCV the presence of IL-32

is associated with severity of steatosis, ITF2357 hepatic inflammation, and liver fibrosis. IL-32 is expressed by hepatocytes and up-regulated upon stimulation with IL-1β or TNF-α as well as HCV infection. Although IL-32 lacks anti-HCV activity at least in a cell culture

system, our data suggest that viral infection stimulates expression of this cytokine, thus supporting a role for IL-32 in chronic HCV infection and related pathologies. “
“This review focuses on the hypothesis that biliary HCO secretion in humans serves to maintain an alkaline pH near the apical surface of hepatocytes and cholangiocytes to prevent the uncontrolled membrane permeation of protonated glycine-conjugated bile acids. Functional impairment of this biliary HCO umbrella or its regulation may lead to enhanced vulnerability of cholangiocytes

and periportal hepatocytes toward the attack of apolar hydrophobic bile acids. An intact interplay of hepatocellular and cholangiocellular adenosine triphosphate (ATP) selleckchem secretion, ATP/P2Y- and bile salt/TGR5-mediated Cl−/ HCO exchange and HCO secretion, and alkaline phosphatase–mediated ATP breakdown may guarantee a stable biliary HCO umbrella under physiological conditions. Genetic and acquired functional defects leading to destabilization of the biliary HCO umbrella may contribute to development and progression of various forms of fibrosing/sclerosing cholangitis. (HEPATOLOGY 2010) The pathogenesis of chronic cholestatic liver diseases such as primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC) and other fibrosing cholangiopathies remains enigmatic.1 Without adequate therapy, the prognosis is dismal and current treatment strategies may achieve stabilization but no resolution.1 Genetic factors contribute to the development of chronic cholestatic liver disease as indicated by sibling studies in PBC.2 An increased risk for first-degree relatives of PBC and PSC patients to develop the same disease also indicate a genetic background. Notably, various mutations of genes involved in bile formation present with a sclerosing/fibrosing cholangitis-like phenotype.

Though significant research has been carried out on the leptin induced NADPH oxidase in fibrogenesis, the molecular mechanisms that connect leptin-NA-DPH oxidase axis in upregulation of TGF p signaling has been unclear. Recently, there is an increased emphasis on non-coding RNAs in controlling NASH progression. For this we hypothesized that leptin mediated

upregulation of NADPH oxidase and its subsequent induction of mir21 H 89 purchase via Nf-κb activation causes increased TGF p signaling by inhibiting SMAD7. A high fat (60% kCal) diet fed chronic mouse model was used for inducing fatty liver and subsequent steatohepatitic lesions following administration of hepatotoxin bromodichloromethane. To prove the role of Leptin-NADPH oxidase-miR21 axis, mouse deficient in genes for leptin, p47 phox and mir21 were used. Results showed that wild type mice that had steatohepatiic lesions, had increased oxidative stress, increased p47 phox expression, augmented f-κb activation and increased mir21 levels. These mice showed increased TGF p,

INK 128 SMAD2/3 phosphorylation, COL1A1 and α-SMA expression with a concomitant decrease in both miRNA and protein levels of SMAD7 (inhibitor of TGF p signaling pathway and a regulatory SMAD that is a direct target of mir21). Mice that were deficient in leptin, leptin receptor or p47 phox had decreased Nf-κb and mir21 levels suggesting the role of these proteins in inducing NFkB mediated mir21. Further mir21 ko mice had decreased TGF b signaling, increased SMAD7 levels and decreased fibrogenesis as shown by a-SMA levels and picrosirius red staining. The increased markers for stellate cell activation, collagen deposition and fibrogenesis when compared to wild type mice were decreased in mice that were deficient in leptin and p47 phox genes, suggesting that leptin mediated NADPH oxidase plays a direct role in fibrogenesis via mir21-induced inhibition of SMAD7. Interestingly macrophage depletion by GdCl3 didn’t decrease TGF p signaling or kinetics of SMAD7

expression. Taken together the studies show the novel role of leptin-NADPH oxidase- mediated regulation of mir21 in NASH and identifies mir21 as a potential therapeutic target check details of fibrogenesis in NASH. Disclosures: Anna Mae Diehl – Consulting: Roche; Grant/Research Support: Gilead, Genfit The following people have nothing to disclose: Diptadip Dattaroy, Ratanesh K. Seth, Suvarthi Das, Sahar Pourhoseini, Mitzi Nagarkatti, Gregory A. Michelotti, Saurabh Chatterjee “
“Prohibitin 1 (PHB1) is a highly conserved, ubiquitously expressed protein that participates in diverse processes including mitochondrial chaperone, growth and apoptosis. The role of PHB1 in vivo is unclear and whether it is a tumor suppressor is controversial. Mice lacking methionine adenosyltransferase 1A (MAT1A) have reduced PHB1 expression, impaired mitochondrial function, and spontaneously develop hepatocellular carcinoma (HCC).

Double IF and co-immunoprecipitation were used to study protein-protein interactions. Results: In both an experimental liver metastasis mouse model and cancer

patients, colorectal cancer cells reaching liver sinusoids induced upregulation of VASP and α-SMA in HSCs as revealed by IF on liver biopsies. In a HSC/tumor coimplantation model, VASP knockdown HSCs significantly reduced tumor growth in mice as compared to control HSCs. In vitro, TGF-β1 stimulation resulted in myofibroblastic activation in more than 60% of HSCs as determined by α-SMA IF. Two different VASP shRNAs and a VASP siRNA significantly buy Erastin reduced this effect of TGF-β1 on HSC activation (P<0.05). The effect of VASP knockdown on HSC activation was also confirmed in LX2 cells. Biotinylation study and IF revealed that VASP knockdown reduced TβRII protein levels at the plasma membrane. Furthermore, check details VASP formed a trimeric protein complex with TβRII and Rab11, a Ras-like small GTPase and key regulator of recycling endosomes. VASP knockdown impaired Rab11 activity and Rab11 dependent targeting of TβRII to the plasma membrane thereby desensitizing HSCs to TGF-β1 stimulation. Conclusions: our study demonstrates a requirement of VASP for TGF-β

mediated HSC activation in the tumor micro-environment by regulating Rab11 dependent recycling of TβRII to the plasma membrane. VASP and its effector Rab11 in the tumor microenvironment thus present therapeutic targets for reducing tumor implantation and metastatic growth in the liver. Disclosures: The following people have nothing to disclose: Kangsheng Tu, Jiachu Li, Vijay Shah, Ningling Kang “
“The aim of this case–control study was to assess the efficacy and safety of dipeptidyl peptidase-4 inhibitor (sitagliptin)

selleck screening library for type 2 diabetes mellitus (T2DM) with non-alcoholic fatty liver disease (NAFLD). Twenty NAFLD patients with T2DM treated by sitagliptin were retrospectively enrolled as the sitagliptin group. These patients were given sitagliptin between January 2010 and July 2011. Another 20 NAFLD patients with T2DM treated only with diet and exercise for 48 weeks were selected as the control group. Serum levels of fasting plasma glucose (FPG), hemoglobin A1C (HbA1c), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured before and 12, 24, 36 and 48 weeks after the initiation of treatment. In the sitagliptin group, average HbA1c levels decreased approximately 0.7% at 48 weeks after the initiation of sitagliptin. Next, average FPG levels decreased approximately 15 mg/dL at 48 weeks after the initiation of sitagliptin. The serum levels of HbA1c and FPG in the sitagliptin group decreased with statistical significance compared to those in the control group (P < 0.05). All the patients could take sitagliptin of 50 mg/day without reduction necessitated by sitagliptin-related side-effects.

The balance/imbalance of selleck screening library an adipose tissue “mediator cocktail” may profoundly affect not only the situation in the adipose tissue but especially in important target organs such as the liver (Fig. 1). Adiponectin is an anti-inflammatory adipocytokine that signals through two receptors.54-56 Obesity is associated with hypoadiponectinemia, and adiponectin levels increase after weight loss.55

Adiponectin induces extracellular Ca2+ influx by adiponectin receptor 1, which is necessary for activation of adenosine monophosphate–activated protein kinase (AMPK) and Sirtuin 1 (Sirt1).57 Hepatocyte-specific deletion of Sirt1 leads not only to hepatic steatosis but also to ER stress and liver inflammation.58 Genetically obese leptin-deficient ob/ob mice exhibit a reversal of the diabetic phenotype with normalization of glucose and insulin levels upon transgenic overexpression of the full-length isoform of adiponectin, despite see more retaining the obese phenotype.59 This report convincingly demonstrates that, despite massive expansion of subcutaneous adipose tissue, high-level expression of adipose tissue adiponectin reduces liver fat content

and improves insulin resistance. Therefore, also in humans, a sufficient production of adiponectin might play a central role in establishing a balance where local and systemic/liver inflammation is prevented.60 In the

hierarchy of processes in the adipose tissue, soluble mediators such as adiponectin click here might be the “big players. Because adipocytes expand with triglycerides, leptin secretion increases proportionally.61 Hyperleptinemia reduces fat content in peripheral organs. Because leptin stimulates fatty acid oxidation, adipocytes would be oxidizing, rather than storing fat if the endogenous leptin they synthesize acts on them.62, 63 Such an autocrine/paracrine relationship between leptin and its secreting cell, the adipocyte, is prevented by a progressive decline of adipocyte leptin receptor expression. It is assumed that leptin’s capacity to oxidize lipids is fully operative in the liver, thereby minimizing ectopic lipid accumulation, at least temporarily. Whether such a mechanism is operative in NAFLD is not known. Expression of IL-6 and TNFα, two important proinflammatory cytokines, is profoundly increased in human fat cells from obese subjects and patients with insulin resistance.64 IL-6 serum levels are elevated in obese patients and weight loss results in decreased IL-6 serum levels.65, 66 Enhanced TNFα expression in adipose tissue of obese subjects decreases following weight loss.67 Insulin resistance is an important feature of NAFLD and is caused by a variety of factors, including soluble mediators derived from immune cells and/or adipose tissue.

Objective.— The objective of this study is to determine whether MAOI-A therapy should deter the use of 6 mg s.c. sumatriptan on pharmacokinetic grounds. Methods.— Summary pharmacokinetic data were taken from the literature and from GlaxoSmithKline (GSK) study C92-050. Half-times were converted into rate constants, which were then used in a parsimonious compartmental model (needing

only 3 simultaneous differential equations). Acceptance criteria for the model included observed plasma sumatriptan concentrations at Tmax, 1, 2, and 10 hours post-dose. A set of 1000 concentration Opaganib molecular weight measurements at a resolution of 36 seconds was generated. The model was then perturbed with elimination constants observed during concomitant moclobemide administration, creating a second set of concentration measurements. The 2 sets were then plotted, examined for their differences, and integrated for a second time to obtain and compare areas under the curve (AUCs). Results.— The greatest absolute difference between the 2 sets of measurements was 2.85 ng/mL at t = 2.95 hours. A 2-fold difference Selleck Selumetinib between the 2 sets occurred only after t = 5.96 hours, when the concentration

in the presence of the MAOI-A was 3.72 ng/mL (or <4% of Cmax). At t = 10 hours, the concentrations in both sets were <1 ng/mL (ie, below the lower limit of assay quantitation), and AUC0-10h was 97.4 and 117 ng.hour/mL in the absence this website and presence of the MAOI-A. Conclusions.— There are no pharmacokinetic grounds to deter co-administration of an MAOI-A and subcutaneous sumatriptan. The dominance of the distribution phase and completeness of absorption

of a 6 mg dose of s.c. sumatriptan explains the trivial effect size of the MAOI-A on plasma sumatriptan concentrations. Importantly, these findings should not be extrapolated to other routes of administration for sumatriptan. “
“(Headache 2010;50:99-168) Background.— It has been suggested that homocysteine (Hcy) and the 5′-10′-methylenetetrahydrofolate reductase (MTHFR) C677T variant are implicated in the pathogenesis of migraine. Homocysteine has the potential to damage endothelium and accelerate atherosclerosis. Genetic factors such as the MTHFR C677T polymorphism, and other polymorphisms in folate-related genes associated with high homocysteine levels, may contribute to increasing this vascular risk. Results.— We recruited 427 migraine patients (199 without aura [MO]; 228 with aura [MA]), and 310 controls in a neurologic clinic. Hcy levels and 6 polymorphisms corresponding to 6 folate-related genes, including the MTHFR C677T variant, were determined in all migraine participants and in a subset of 155 controls. We found higher sex-adjusted Hcy levels in MA (mean: 11.02 µM) than MO patients (9.86 µM; P = .005 for the difference). Hcy levels higher than 12.0 µM doubled the risk for MA (OR = 2.145; 95% confidence intervals [CI] = 1.3-3.4; P = .001), and those higher than 15.