However, this peptide has a poor eukaryotic cell penetrating
<

However, this peptide has a poor eukaryotic cell penetrating

potential and, as a result, it requires the assistance this website of other cell penetrating peptides for effective translocation in micromolar concentrations. In an effort to improve the cell penetrating potential of KLA, we have created a library in which pairs of residues on its hydrophobic face are replaced by Cys. The double Cys mutants were then transformed to bundle dimers by oxidatively generating two intermolecular disulfide bonds. We envisioned that once transported into cells, the disulfide bonds would undergo reductive cleavage to generate the monomeric peptides. The results of these studies showed that one of the mutant peptides, dimer B, has a high cell penetrating ability that corresponds to 100% of fluorescence positive cells at 250 nM. Even though dimer B induces

disruption of the mitochondrial potential and cytochrome c release followed by caspase activation at submicromolar concentrations, it displays an LD50 of 1.6 mu M under serum conditions using HeLa cells. Taken together, the results demonstrate that the strategy involving formation of bundle dimeric peptides is viable for the design of apoptosis inducing KLA www.selleckchem.com/products/LY2603618-IC-83.html peptide that translocate into cells at submicromolar concentrations.”
“Objective: To evaluate the efficacy, safety, and tolerability of the selective serotonin norepinephrine inhibitor duloxetine in children and adolescents with

generalized anxiety disorder (GAD). Method: Youth aged 7 through 17 years with a primary diagnosis of GAD were treated with flexibly dosed duloxetine (30-120 mg daily, n = 135) or placebo (n = 137) for 10 weeks, followed by open-label duloxetine (30-120mg selleck screening library daily) for 18 weeks. Efficacy measures included the Pediatric Anxiety Rating Scale (PARS), Clinical Global Impression-Severity (CGI-Severity) scale, and Children’s Global Assessment Scale (CGAS). Safety measures included the Columbia-Suicide Severity Rating Scale (C-SSRS) as well as vital signs and electrocardiographic and laboratory monitoring. Results: On the primary efficacy measure (PARS severity for GAD), mean improvement from baseline to 10 weeks was statistically significantly greater for duloxetine (-9.7) compared with placebo (-7.1, p smaller than .001, Cohen’s d: 0.5). Symptomatic response (50% improvement on the PARS severity for GAD), remission (PARS severity for GAD smaller than = 8), and functional remission (CGAS bigger than 70) rates for the duloxetine group (59%, 50%, 37%, respectively) were statistically significantly greater than for the placebo group (42%, 34%, 24%, respectively, p smaller than =.05) during acute treatment.

Results Recently, we presented the in vitro based Genomic Allerge

Results Recently, we presented the in vitro based Genomic Allergen Rapid Detection (GARD) assay as a novel testing strategy for classification of skin sensitizing chemicals based on measurement of a genomic biomarker signature. We have expanded the applicability domain of the GARD assay to classify also respiratory sensitizers by identifying a separate biomarker signature containing 389 differentially regulated genes for respiratory sensitizers in comparison to non-respiratory sensitizers. By using an independent data set in combination with supervised machine learning, we validated the assay, showing that the identified genomic

mTOR inhibitor biomarker is able to accurately classify respiratory sensitizers. Conclusions We have identified a genomic biomarker signature for classification of respiratory sensitizers. Combining this newly identified biomarker signature with our previously identified biomarker signature for classification of skin sensitizers, we have developed a novel in vitro testing strategy with a potent ability to predict both skin and respiratory sensitization in the

same this website sample.”
“The pathologic classification of rhabdomyosarcoma (RMS) into embryonal or alveolar subtype is an important prognostic factor guiding the therapeutic protocol chosen for an individual patient. Unfortunately, this classification is not always straightforward, and the diagnostic criteria are controversial in a subset of cases. Ancillary studies are used to aid in the classification, but their potential use as independent prognostic factors is rarely studied. The aim of this study is to identify immunohistochemical markers of potential prognostic significance in pediatric RMS and to correlate their expression with PAX-3/FKHR and PAX-7/FKHR fusion status. A single tissue microarray containing 71 paraffin-embedded pediatric RMSs was immunostained with Fosbretabulin antibodies against p53, bcl-2, Ki-67, CD44, myogenin, and MyoD1. The tissue microarray and whole paraffin blocks were studied for PAX-3/FKHR and

PAX-7/FKHR gene fusions by fluorescence in situ hybridization and reverse transcription-polymerise chain reaction. Clinical follow-up data were available for each patient. Immunohistochemical staining results and translocation status were correlated with recurrence-free interval (RFI) and overall survival (OS) using the Kaplan-Meier method, the log-rank test, and Cox proportional hazard regression. The minimum clinical follow-up interval was 24 months (median follow-up = 57 mo). On univariable analysis, immunohistochemical expression of myogenin, bcl-2, and identification of a gene fusion were associated with decreased 5-year RFI and 10-year OS (myogenin RFI P = 0.0028, OS P = 0.0021; bcl-2 RFI P = 0.037, OS P = 0.032; gene fusion RFI P = 0.0001, OS P = 0.0058).

We aimed to investigate an alternative subdivision of FGD, taking

We aimed to investigate an alternative subdivision of FGD, taking into account gastric sensorimotor function, anxiety & depression and ‘somatization’, besides gastroduodenal symptoms. Methods Gastroduodenal symptom data were available for 857 consecutive FGD patients (Rome II criteria). In a subsample (n = 259), additional data were obtained on gastric sensitivity, anxiety, depression and ‘somatization’. Two separate cluster analyses were performed. In analysis 1, clustering was based on individual gastroduodenal symptom scores. In analysis 2, gastric sensitivity,

anxiety & depression and ‘somatization’, besides total gastroduodenal symptoms score, were used for clustering. Key Results Analysis 1 identified four clusters, largely supporting the Rome III classification, with

early satiation, I-BET-762 in vitro pain and nausea/vomiting clusters, besides a limited severity cluster (R(2) = 0.32). Analysis 2 suggested a five-cluster solution (R(2) = 0.48). Anxiety, depression and ‘somatization’ GSI-IX in vitro were the most important variables separating the clusters. ‘Primary somatization’ (with low psychiatric symptom levels) as well as ‘secondary somatization’ (with high anxiety & depression scores) subgroups were identified, besides three other subgroups characterized by psychiatric/gastroduodenal symptoms, mild anxiety symptoms and limited overall severity, respectively. Conclusions & Inferences We propose an alternative to the current subgrouping in FGD that is exclusively based on gastroduodenal symptoms. This may have consequences

for future classification of FGD, as well as broader relevance towards the debate on subgrouping click here ‘functional somatic syndromes’.”
“Objective: This study aimed to examine the extent to which illness perceptions and coping strategies among women diagnosed with breast cancer explain psychological distress at diagnosis and at 6 months post diagnosis relative to demographic and illness-related variables.\n\nMethods: Women were recruited to the study shortly after diagnosis. A total of 90 women completed study materials (Illness Perception Questionnaire-Revised, the Cancer Coping Questionnaire and the Hospital Anxiety and Depression Scale) at time 1. The same questionnaires were sent approximately 6 months later to those who had consented at time 1, and completed questionnaires were returned by 72 women.\n\nResults: Cluster analysis was used to identify groups of respondents who reported a similar profile of illness perception scores. Regression analysis demonstrated that one of these clusters was more likely to experience psychological distress than the other both at diagnosis and at 6 months post diagnosis.

Using samples of EHR data between January 1, 2007, and March 31,

Using samples of EHR data between January 1, 2007, and March 31, 2011, from two health systems, the accuracy of the MediClass processor for identifying these counseling elements was evaluated in postpartum visits of 600 women with gestational diabetes mellitus (GDM) compared

to manual chart review as the gold standard. Data were analyzed in 2013.\n\nResults: Mean sensitivity and specificity for each of the 5As compared to Quizartinib Angiogenesis inhibitor the gold standard was at or above 85%, with the exception of sensitivity for Assist, which was 40% and 60% for each of the two health systems. The automated method identified many valid Assist cases not identified in the gold standard.\n\nConclusions: The MediClass processor has performance capability sufficiently similar to human abstractors to permit automated assessment of counseling for weight loss in postpartum encounter records. (C) 2014 American Journal of Preventive Medicine”
“The Epoxomicin in vitro purpose of this study was to determine the late decline in viability of fat cells over time for fat tissue stored at -15 degrees C and -70 degrees C after harvest from abdominal liposuction. A total of 16 females were recruited for this study.

The viability of fat cell specimens was measured after freezing for 1, 3, 7, 14, 28, and 56 days. A number of viable mature adipocytes were evaluated by fluorescence microscopy after staining with fluorescein diacetate and propidium iodide. The glycerol-3-phosphate dehydrogenase activity was measured in lipoaspirates before digestion and the XTT reduction assay was performed. In addition, the Dinaciclib nmr XTT reduction assay was also performed on isolated lipocytes and preadipocytes.\n\nThe viability of mature adipocytes was very low for both the -15 degrees C and -70 degrees C samples after 1 day of freezing (13.3% +/- 7.4% and 12.6% +/- 6.3%, respectively). There was no statistically significant

difference between the samples stored at the 2 temperatures. The GPDH activity of the lipoaspirates frozen, for 1 day, at -15 degrees C and -70 degrees C was 25.1% +/- 10% and 28.7% +/- 11%, respectively. For the XTT test, the fractional enzyme activity of the lipoaspirates frozen, for 1 day, at -15 degrees C and -70 degrees C was 30.0% +/- 10.9% and 36.1% +/- 12.3%, respectively. In addition, the adipocytes had low activity from day one: 15.4% +/- 7.2% at -15 degrees C and 11.5% +/- 5.6% at -70 degrees C. Furthermore, the preadipocytes had a low activity of 8.0% +/- 6.0% at -15 degrees C and 8.6% +/- 3.8% at -70 degrees C. At 8 weeks, there were few viable mature adipocytes and the activity of the cells was very low by XTT and GPDH testing.

There is a spectrum of proliferation and cellular atypia in these

There is a spectrum of proliferation and cellular atypia in these tumors with benign serous cystadenoma, borderline tumors, and low grade or type I serous carcinoma at the lower

end and type 11 or high-grade serous papillary cystadenocarcinoma at the higher end. Fascin is a globular actin cross-linking protein involved in cell motility that has been shown to be upregulated in many human neoplasms and associated with the aggressiveness of malignancy. The aim of this Study was to investigate fascin expression in serous tumors of ovary and to evaluate its relationship https://www.selleckchem.com/products/mi-503.html with the aggressiveness of tumor. Sections from a total of 66 serous tumors of ovary were collected including 26 serous carcinomas, 20 borderline serous tumors. and 20 benign serous cystadenomas. Ten benign ovaries with inclusion cysts were used as controls. Sections were immunostained with fascin. Fascin expression was significantly increased in borderline (13/20. 65%) and malignant serous tumors (22/26, 84%) compared with benign serous cystadenoma (0/20) (P<0.001). There was increased quantitative expression of fascin in carcinoma compared with borderline tumors (diffuse versus patchy). Fascin expression also correlated well with 3-deazaneplanocin A cost the tumor grade in serous carcinoma cases with 8/12 (66%) of grade I/II tumors staining positive compared with all 14 (100%) of grade Ill

tumors showing fascin expression (P<0.05). Our findings Suggest that upregulation of fascin plays a role in increasing Vorinostat nmr aggressiveness of serous ovarian tumors and Could potentially be a molecular therapeutic target and it prognostic marker.”
“Objectives: To highlight a case series of 3 cases of focal cortical dysplasia that were unrecognized

for many years though the patients were seen by various neurologists and received the appropriate neuroimaging studies, and to retrospectively characterize the clinical elements, neuroimaging, electroencephalography, and pathologic findings in these cases.\n\nDesign: Retrospective descriptive study.\n\nSetting: Tertiary urban and suburban neurology and epilepsy outpatient and inpatient clinic settings and hospitals.\n\nPatients: We analyze retrospectively 3 patients in whom magnetic resonance images were previously deemed as normal, who, in fact, exhibited subtle gyral abnormalities and who underwent focal surgical resections of these regions after invasive electroencephalography monitoring or electrocorticography and were cured of their epilepsy.\n\nMain Outcome Measures: Clinical semiology and neuroimaging findings.\n\nResults: Focal cortical dysplasias may present with subtle gyral abnormalities. These gyral abnormalities may guide invasive electroencephalography or electrocorticography and may delineate seizure onsets with precision. Resection of these areas in 3 such patients resulted in excellent surgical outcomes.

HYPK has the ability of reducing rate of

HYPK has the ability of reducing rate of 4-Hydroxytamoxifen concentration aggregate formation and subsequent toxicity caused by mutant Huntingtin. Further investigation revealed that HYPK is involved in diverse cellular processes and required for normal functioning of cells. In this study we observed that hyperthermia increases HYPK expression in human and mouse cells in culture. Expression of exogenous Heat Shock Factor 1 (HSF1), upon heat treatment could

induce HYPK expression, whereas HSF1 knockdown reduced endogenous as well as heat-induced HYPK expression. Putative HSF1-binding site present in the promoter of human HYPK gene was identified and validated by reporter assay. Chromatin immunoprecipitation revealed in vivo interaction of HSF1 and RNA polymerase II with HYPK promoter sequence. Additionally, acetylation of histone H4, a known epigenetic buy Nutlin-3 marker of inducible HSF1 binding, was observed in response to heat shock in HYPK gene promoter. Overexpression of HYPK inhibited cells from lethal heat-induced death whereas knockdown of HYPK made the cells susceptible to lethal heat shock-induced death. Apart from

elevated temperature, HYPK was also upregulated by hypoxia and proteasome inhibition, two other forms of cellular stress. We concluded that chaperone-like protein HYPK is induced by cellular stress and under transcriptional regulation of HSF1.”
“The generation of transgenic cell lines is acquired by facilitating the uptake and integration of DNA. Unfortunately, most of the systems generating VX770 stable expression systems are cost and time-consuming and transient expression is optimized to generate milligram amounts of the recombinant protein. Therefore we improved and compared two transfection systems, one based on cationic liposomes consisting of DOTAP/DOPE and the second one on polyethylenimine (PEI). Both systems have been used as chemically defined transfection systems in combination with serum-free cultivated host cell line. At first we had determined the toxicity and ideal ratio of DNA to PEI followed by determination of the optimal transfection

conditions in order to achieve maximum transfection efficiency. We then directly compared DOTAP/DOPE and PEI in transient transfection experiments using enhanced green fluorescence protein (EGFP) and a human monoclonal antibody, mAb 2F5, as a model protein. The results which were achieved in case of EGFP were more than 15% transfectants at a viability of 85%. Despite the fact that expression of the mAb was found negligible we used both techniques to generate stable mAb 2F5 expressing cell lines that underwent several cycles of screening and amplification with methotrexate, and resulted in cell lines with similar volumetric production titers. These experiments serve to demonstrate the potential of stable cell lines even in case where the transient systems did not show satisfying results.

Results We identified 28 906 new episodes of medication use,

\n\nResults. We identified 28 906 new episodes of medication use, including TNF-alpha antagonists (8%), MTX alone (15%) and glucocorticoids alone (57%). Compared with MTX initiation, TNF-alpha antagonist initiation did not significantly increase the

risk of hospitalizations for pneumonia [adjusted hazard ratio (aHR) 1.61; 95% CI 0.85, 3.03] or any infection (aHR 1.31; 95% CI 0.78, 2.19). selleck screening library Initiation of LEF, SSZ or HCQ did not increase serious infections, compared with MTX. Both initiation and concurrent glucocorticoid use were associated with a dose-dependent increase in serious infections. Sensitivity analyses showed consistent results.\n\nConclusions. Compared with initiation of MTX alone, initiation of TNF-alpha antagonists was not associated with a large increase in the risk of serious infections. Glucocorticoid use was associated with a dose-dependent increase in the risk of these infections.”
“The aim of the report is to document

two cases of biopsy-proven temporal arteritis, resulting in major visual loss that recovered almost completely after treatment with intravenous methylprednisolone and intravenous heparin infusion. An 80-year-old Caucasian lady presented with vision of perception of light (PL) due to a central retinal artery occlusion. Following systematic treatment with anterior chamber paracentesis, intravenous heparin, and intravenous steroids, vision improved to 6/5. A definitive diagnosis was made following temporal artery biopsy. A 77-year-old Caucasian lady presented with treated polymyalgia rheumatica, which on further history and examination was confirmed to be temporal click here arteritis. Prompt treatment of her arteritic anterior ischaemic optic neuropathy with intravenous methylprednisolone and intravenous heparin infusion resulted in improvement of visual acuity from 6/12 to 6/5 with Rocilinostat a sustained improvement in visual fields and in the funduscopically visible optic nerve ischaemic changes. The authors conclude that near-complete resolution of major visual loss in these two patients with biopsy-proven temporal arteritis suggests

that the appropriate use of a therapeutic intravenous methylprednisolone and heparin protocol could represent a way forward in the management of this potentially blinding disease. This would need to be confirmed in large prospective, randomised controlled studies.”
“Climate change models for California’s Sierra Nevada predict greater inter-annual variability in precipitation over the next 50 years. These increases in precipitation variability coupled with increases in nitrogen deposition from fossil fuel consumption are likely to result in increased productivity levels and significant increases in forest understory fuel loads. Higher understory plant biomass contributes to fuel connectivity and may increase future fire size and severity in the Sierra Nevada.

Cultural variables made a significant contribution to explaining

Cultural variables made a significant contribution to explaining the variance in EP scores. Harsh parenting was significantly associated with increased levels of EP in both age

groups, and supportive parenting was a mental health protective factor for younger children. Immigrant family human and social capital, according to which immigrants are selected for admission to Canada, play a relatively small role in determining children’s mental health. These effects are overshadowed by resettlement contingencies and cultural influences. Concentrating on trying to find a formula to select the “right” immigrants while neglecting settlement and culture is likely to pay limited dividends for ensuring the mental health of children.”
“The Nutlin-3 ic50 protein LC3 is indispensible for the cellular recycling process of autophagy and plays critical roles during cargo recruitment, autophagosome biogenesis, and completion. Here, we report that LC3 is phosphorylated at threonine 50 (Thr(50)) by the mammalian Sterile-20 kinases STK3 and STK4. Loss of phosphorylation at this site blocks autophagy by impairing fusion of autophagosomeswith lysosomes, and compromises the ability of cells to clear intracellular bacteria, an established cargo for autophagy. Strikingly, mutation of LC3 mimicking constitutive phosphorylation at Thr(50) reverses the autophagy block in STK3/STK4-deficient

cells and restores Selleckchem GM6001 their capacity to clear bacteria. Loss of STK3/STK4 impairs autophagy in diverse species, indicating that these kinases are conserved autophagy regulators. We conclude that phosphorylation of LC3 by STK3/STK4 is an essential step in the autophagy process. Since several

pathological conditions, including bacterial infections, display aberrant autophagy, we propose that pharmacological agents targeting this regulatory circuit hold therapeutic potential.”
“We developed a new layout optimization method that incorporates dynamic Selleckchem QNZ fatigue and static failure criteria under constant and proportional mechanical loads. Although fatigue failure is one of the most important design considerations to ensure the safety of a mechanical structure, it has rarely been considered in terms of topology optirhization due to several major theoretical and numerical difficulties. In addition to the numerical and theoretical issues associated with static failure criteria, we found that the local mode issue and the non-differentiability of the fatigue failure criteria needed to be addressed prior to performing fatigue constraint topology optimization. By resolving the inherent and newly-found issues associated with fatigue criteria, we were able to perform successful topology optimization with dynamic fatigue criteria under constant and proportional mechanical loads. (C) 2014 Elsevier Inc. All rights reserved.

Results: A steady increase in the number of cases was detecte

\n\nResults: A steady increase in the number of cases was detected, from 24 in 1998, peaking at 82 cases in 2005, followed by a gradual decline (though still high) with 68 cases in 2006 and 51 cases in 2007. Male/female ratio was 1.35/1 (p > 0.05). The occupational exposure to asbestos was 13.9%. Residential exposure plays a major role in two regions, Helwan and Shoubra (27.3% and 20.6% respectively), while in Upper and Lower Egypt the level was 12.7% and 17.5% respectively.

Kaplan-Meier survival for sex, residence and the pathological types epithelioid, biphasic and sarcomatoid was insignificant. The median survival MLN4924 inhibitor for different grades and treatment modalities was significant (P < 0.001).\n\nConclusions: There was a steady increase in the incidence of MPM from 1998 to 2005 followed by a decline during 2006-2007. Mesothelioma in Egypt is mainly concentrated in areas of high environmental pollution. The decline within the last 2 years may be attributed to recent strict industrial preventive measures. However, a better environmental control programme would benefit Egypt.”
“In this study, we propose a reliable measurement method for the effective transverse piezoelectric coefficient for thin

films especially on anisotropic substrate. This coefficient for piezoelectric Pb(Zr, TGF beta inhibitor Ti)O-3 (PZT) thin films was calculated by measuring the electric field-induced tip displacement of unimorph cantilevers composed of PZT thin films and Si substrates. We evaluated the reliability of the proposed measurement method by comparing it with numerical analysis and confirmed that the relative error of the piezoelectric coefficient (e(31,f)) was less than 1%. We prepared 16 different unimorph cantilevers composed of identical PZT films on different Si beam check details geometries that had various substrate thicknesses and cantilever widths. Although the effective transverse piezoelectric coefficient e(31,f) of PZT thin

films ranged from – 6.5 to – 14 C/m(2) as a function of the applied voltage, the difference among the 16 samples with an applied voltage of 25V was within 10%. These results demonstrate that the proposed measurement method has sufficient reliability and can be used to evaluate the effective transverse piezoelectric coefficient e(31,f) of thin films. (C) 2013 American Institute of Physics. [http://0-dx.doi.org.brum.beds.ac.uk/10.1063/1.4789347]“
“Background: In Japan, hospital infection control (IC) programs are frequently underresourced, and their improvement is considered a pressing issue.\n\nMethods: In 2005, we conducted a questionnaire survey of 638 teaching hospitals (most with 300 or more beds) and 882 nonteaching hospitals (most with fewer than 300 beds) in Japan. We analyzed associations among resources, infrastructures, activities, and performance related to IC.\n\nResults: A total of 423 teaching hospitals (66.3%) and 377 nonteaching hospitals (50.

Copurification experiments performed on infected cell extracts in

Copurification experiments performed on infected cell extracts indicate that the avian-derived polymerase is assembled and interacts physically with the cellular RNA polymerase II at least as efficiently as does the human-derived polymerase in human as well as in avian cells. Restricted growth of the avian AZD1208 cell line isolate in human cells correlates with low levels of the core proteins in infected cell extracts and with poor association of the NP with the polymerase compared to what is observed for the human isolate. The NP-polymerase association is restored by a Glu-to-Lys substitution at residue 627 of PB2. Overall, our data point to viral and cellular factors regulating the NP-polymerase

interaction as key determinants of influenza A virus host range. Recombinant viruses expressing a tagged polymerase should prove useful for further

studies of the molecular interactions between viral polymerase and host factors during the infection cycle.”
“Objective To determine the clinical effectiveness of real time continuous glucose monitoring compared with self monitoring of blood glucose in type 1 diabetes.\n\nDesign Meta-analysis of randomised controlled trials.\n\nData sources Cochrane database for randomised controlled trials, Ovid Medline, Embase, Google Scholar, lists of papers supplied by manufacturers of continuous glucose monitors, and cited literature in retrieved articles.\n\nStudies reviewed Randomised controlled trials of two FG 4592 or more months’ duration in men and non-pregnant women with type 1 diabetes that compared real time continuous glucose monitoring with self monitoring

of blood glucose and where insulin delivery was the same in both arms.\n\nAnalysis Two step meta-analysis of individual patient U0126 cost data with the primary outcome of final glycated haemoglobin (HbA(1c)) percentage and area under the curve of hypoglycaemia (glucose concentration <3.9 mmol/L) during either treatment, followed by one step metaregression exploring patient level determinants of HbA(1c) and hypoglycaemia.\n\nResults Six trials were identified, consisting of 449 patients randomised to continuous glucose monitoring and 443 to self monitoring of blood glucose. The overall mean difference in HbA(1c) for continuous glucose monitoring versus self monitoring of blood glucose was -0.30% (95% confidence interval -0.43% to -0.17%) (-3.0, -4.3 to -1.7 mmol/mol). A best fit regression model of determinants of final HbA(1c) showed that for every one day increase of sensor usage per week the effect of continuous glucose monitoring versus self monitoring of blood glucose increased by 0.150% (95% credibility interval -0.194% to -0.106%) (1.5, -1.9 to -1.1 mmol/mol) and every 1% (10 mmol/mol) increase in baseline HbA(1c) increased the effect by 0.126% (-0.257% to 0.0007%) (1.3, -2.6 to 0.0 mmol/mol). The model estimates that, for example, a patient using the sensor continuously would experience a reduction in HbA(1c) of about 0.