EGF-mediated signaling

EGF-mediated signaling Cisplatin FDA pathways are also known to play important roles in the organization of TJs, in which they regulate the expression and localization of claudin proteins. For instance, EGF was reported to induce the upregulation of claudins 1, 3 and 4, and the EGF-induced downregulation of claudin-2 increases the force of the intercellular barrier, as determined by an increased transepithelial electrical resistance (TER) in MDCK-II cells [14], [15]. However, using the same model (MDCK cells), other authors have reported that the downregulation of claudin-2 induced higher cell motility, even with increased TER [16]. Recently, the EGFR/ERK/c-Fos pathway was shown to up-regulate claudin-2, an increase that was correlated with increased intercellular permeability and cell migration in human lung adenocarcinoma cells [17], [18].

Little information is known about the molecular mechanisms underlying the alterations in claudin expression that are associated with colorectal tumorigenesis. We have shown that patients with colorectal cancer presented increased expression levels of claudins 1, 3 and 4, which altered the barrier function of TJs [19]. Recent studies have reported a controversial role for claudin-1 during colorectal carcinogenesis; increased claudin-1 expression was observed in hepatic metastatic lesions of colorectal cancer, but this expression was decreased in the lymph node metastases of colon carcinomas [20], [21]. Additionally, the ERK1/2 and PI3K pathways have been reported to mediate increases in EGF-induced claudin-2 expression in colon cancer cells; this event was accompanied by increases in proliferation, anchorage-independent growth and tumor growth in vivo [22].

Therefore, it is important to understand the molecular mechanisms that regulate the expression of other claudin family members and the implications of claudin overexpression in colorectal cancer progression. In the present study, we show that the EGF-mediated increased expression of claudin-3 is related to increased cell migration and the formation of anchorage-dependent and anchorage-independent colonies in human colorectal adenocarcinoma HT-29 cells. Furthermore, we show that these events were mediated by the ERK1/2 and PI3K-Akt pathways. We also demonstrated that the forced overexpression of claudin-3 in HT-29 cells by genetic manipulation increased the malignant potential, while the overexpression of claudin-1 decreased cell migration.

Most importantly, our results reveal that claudin-3 plays a role as tumor promoter when its expression is imbalanced and implicate the ERK1/2 and PI3K-Akt signaling pathways as modulators of claudin-3 upregulation-related tumor progression in colorectal GSK-3 cancer cells. Materials and Methods Materials Anti-claudin-1 (Cat. no. 51�C9000) and anti-claudin-3 (Cat. no. 34�C1700) rabbit polyclonal antibodies as well as an anti-��-tubulin mouse monoclonal antibody (Cat. no.

Hepatitis flare was defined as ALT >5 times

Hepatitis flare was defined as ALT >5 times Ceritinib FDA of the upper normal limit (2000 IU/mL, [18] at least 6 months after inclusion. Cirrhosis was defined by histological or ultrasonographic findings (two consecutive examinations 6 months apart for confirmation) along with clinical features of splenomegaly, esophageal varices, or ascites. [19] HCC was diagnosed either by histology/cytology or by typical dynamic imaging study (CT, MRI or angiography) plus a serum alpha-fetoprotein level >200 ng/mL. [20]. Statistics Continuous variables were expressed as median (25�C75th percentile) and categorical variables were expressed as number (percentage) as appropriate. The HBsAg, HBV-DNA and HBV-DNA/HBsAg levels were logarithmically transformed for analysis.

For those with HBsAg or HBV-DNA level below the detection limit (0.05 IU/mL for HBsAg and 20 IU/mL for HBV-DNA), an arbitrary value of 0.025 IU/mL for HBsAg and 10 IU/mL for HBV-DNA were assigned for analysis. Differences between subgroups were analyzed using one-way ANOVA tests or Student��s t test as appropriate. Categorical variables were compared by the Chi-square test as appropriate. The decline of HBsAg and HBV-DNA were calculated by the value at baseline �C value at EOF. Paired t test was used to compare the decline of HBsAg and HBV-DNA levels between the baseline and EOF. The annualized rate of HBsAg decline was computed by dividing the HBsAg decline by individual duration of follow-up. Nonparametric trend test was used for trend across ordered groups.

Receiver Operating Characteristic (ROC) analysis was used for a cut-off HBsAg level to predict HBsAg loss in the LVL group. The statistical analysis was performed by STATA (version 11.0; Stata Corp, College Station, TX, USA). All tests were 2-sided and a p value <0.05 was considered significant. Results Baseline and End-of-follow-up HBsAg and HBV-DNA Levels Differed Significantly between Active and Inactive Carriers The baseline and EOF data of our patients are shown in Table 1. The distribution of HBV genotypes was mostly B (126/166, 76%), followed by C (38/166, 23%), B+C (2/166, 1%) and was undetermined in 21 patients because of low viral-load. The median follow-up duration was 8 years. Two patients developed HCC during the longitudinal follow-up in the HVL and FVL groups, respectively.

Table 1 Characteristics of HBeAg-negative HBsAg carriers according to their hepatitis activities. The baseline characteristics were comparable in terms of gender, genotype, and percentage of liver cirrhosis among three groups [Table 1]. The baseline HBsAg and HBV-DNA levels were significantly lower in the LVL group compared with the GSK-3 FVL and HVL groups (HBsAg: 2.84, 3.43 and 3.37 log10 IU/mL, P<.001; HBV-DNA: 1.77, 3.43 and 4.78 log10 IU/mL, P<.001). Among them, HBsAg was quantified in 165 patients (LVL/FVL/HVL: 39/101/25) with available first-year sera. The median first-year HBsAg were 2.59, 3.29 and 3.

In that vein, the work of recovery-oriented professionals revolve

In that vein, the work of recovery-oriented professionals revolves around a logic of empowerment to stimulate personal growth Selinexor (KPT-330)? [43]. Craig [44, page 126] formulates the recovery-oriented task of the services as ��a matter of doing as much as possible to empower the individual. The aim is to have consumers assume more and more responsibility for themselves. Their particular responsibilities include developing goals, working with providers and others��for example, family and friends��to make plans for reaching these goals, taking on decision-making tasks, and engaging in self-care. In addition, responsibility is a factor in making choices and taking risks; full empowerment requires that consumers live with the consequences of their choices.

�� As Jacobson and Greenley [38, page 483] state, ��empowerment emerges from inside one’s self��although it may be facilitated by external conditions.��In the most favorable and far-reaching view, the individual approach to recovery suggests that people with mental health problems individually have to take ��personal responsibility through self-management, being responsible for your own well-being�� [1, page 268]. As Slade [2, page 703] asserts, ��the central shift in a recovery-oriented system, therefore, involves seeing an individual not as a patient��someone who is fundamentally different and therefore needs treatment before getting on with life��but as a person whose efforts to live the most fulfilling life possible are fundamentally similar to those of people without mental illness.

�� Nevertheless, although the recovery paradigm is heretical within the dominant biomedical model [33, 39], ��the fashionable concept of ��recovery’ can be a two-edged sword�� (Hopton, [12, pp. 65-66]). As Hopton [12, pp. 65-66] argues aptly, ��on one level, it represents a step away from the once prevalent idea that (��) only compliance to medication will prevent a relapse. Dacomitinib On the other hand, (sometimes) it also seems to have medical overtones.�� In clinical conceptualizations, for example, it is stated that recovery implies that it is possible to regain control of one’s life, to reintegrate socially and become independent [41], and to ��return to a normal or healthy state, free of the symptoms of illness, (��) being able to work, to go to college, to live in ordinary housing, have an active recreational life and find friendship and romance�� [44, page 125, our italics].

Signal transducer and activator of transcription

Signal transducer and activator of transcription selleck compound 3 (STAT3) is activated in response to growth factors, cytokines, and hormones that are known to play protective role after nerve injury and to mediate nerve regeneration [54]. COX-1 deficient mice show reduced neuroinflammatory and microglial responses to insults, through reduced activation of both Nf-��B and STAT3 [56]. PGE2 is known to regulate the activation of STAT3, and PGE2 inhibition alters a signal required for dendritic cell survival and development, leading to apoptosis [57]. Survivin, a regulator of cell survival, is regulated by COX-2-generated PGE2 in part trough a STAT3 pathway [58]. PGE2 also mediates survival of nonneural cells, such as myocytes, through a STAT3 pathway [59].

The phosphoinositide 3-kinase (PI3K) and the mitogen-activated protein kinase (MAPK) pathways are activated in injured neurons, and act as intracellular cascades that modulate regeneration associated genes. Indomethacin treatment in tumor cells has been shown to decrease expression of PI3K [60]. Latanoprost, the synthetic derivative of PGF2a, is able to promote retinal ganglion cell survival and promote neurite outgrowth through a PGF receptor-mediated modulation of the PI3K pathway [61]. PI3K inhibition leads to enhanced protsnoids production in activated microglia, and additionally, PI3K can regulate the expression of COX-2 in microglia as a response to neuroinflammation [62]. MAPK pathways are involved in neuroinflammation in many ways, including the enhancement of sensory neuron interleukin-6 production [63] and the regulation of interleukin-1-mediated COX-2 expression [64].

Latanoprost also uses a MAPK pathway in order to rescue nuroglial cells from apoptosis [65]. Specificity protein-1 (Sp1) is a transcription machinery theorized to act as a general regulator of many of the nerve injury-inducible transcription factors, including some discussed above [54]. COX inhibitors Entinostat are also known to alter Sp1 phosphorylation and lead to arrested cell growth in tumor cells [66], and PGE2 can stimulate cell growth through induction of Sp1 DNA-binding activity [67]. PGE2 enhances the phosphorylation, DNA binding, and transcriptional activity of Sp1, and it leads to enhanced neurotrophin production through a Sp1 pathway [68], suggesting a possible mechanism for PGE2-induced reinnervation. These finding illustrate the complex interactions between inflammatory and neuroregenerative signaling.

In the 200U group, PV was significantly reduced only at the six-m

In the 200U group, PV was significantly reduced only at the six-month evaluation. The impact of intraprostatic injection of 100U and 200U of BoNT-A in IPSS, Qmax , PVR, PSA, and prostate volume was comparable, as seen in Table 2.Complications and their management are depicted in Table 3. No severe Mdm2 complication was observed. Two (5.8%) patients had transient hematuria requiring bladder irrigation, two (5.8%) had short-term urinary retention, and two had acute prostatitis (5.8%). One patient who initially had mild improvement of symptoms developed urinary retention 5 months after 100U BoNT-A injection, requiring transurethral resection of the prostate. The complication rate did not differ between the groups (P = 0.921). When present, pain was usually mild and no patient needed narcotic analgesics.

Table 3Complications after intraprostatic botulinum toxin injection for BPH.4. DiscussionThe efficacy of BPH treatment is primarily determined by the magnitude of symptom relief as well as improvement of urinary flow rates. In the present study, both BoNT-A doses promoted significant improvement of symptoms and increased flow rates that continued throughout the followup period of six months.Maria et al. [15] pioneered BoNT-A injection as a BPH therapy in a double-blind, placebo-controlled trial with 30 men who no longer responded to oral medication and refused surgical treatment. A total of 13 (86.7%) patients in the treated group and 3 (20.0%) in the control group had symptomatic improvement at the 2-month follow-up.

Patients in the treatment group had significant improvement in the maximum urinary flow rate, post-void residual urine volume, and IPSS score. Furthermore, PSA levels and prostate volume decreased significantly.Further studies have documented that intraprostatic BoNT-A injection is an efficient therapy, capable to improve LUTS and Qmax , as well as to reduce PVR [17, 21]. Our study has also shown these benefits up to 6 months after treatment. Additionally, we recorded that both doses (100U and 200U) promoted similar effects. One patient treated with 100U of BoNT-A developed urinary retention five months after the injection. Baseline characteristics of this patient included an IPSS score of 23, maximum flow rate of 3mL/s, PVR of 135mL, and a prostate volume 88mL. These clinical features characterize a severe case of BPH, which might explain why he failed BoNT-A therapy.

The patient was treated with transurethral resection AV-951 of the prostate with a favorable outcome.The effect of BoNT-A injection on prostate volume is controversial. Experimental studies demonstrated generalized atrophy and apoptosis of glandular and stromal components of the prostate [22�C24]. Previous series have shown different rates of prostate volume reduction, ranging from 13 to 54% [15, 16, 19, 21].

3 1 2 Inputs Energy input should be considered as important as

3.1.2. Inputs Energy input should be considered as important as capital and labor. In the light of the majority of literature, this paper takes the number of employees every year as labor input and the energy consumption as energy input in subindustries, both of which can be inquired product info from NBSC [18]. Capital stock is one of the most important inputs, but NBSC does not provide details of the capital stock data; therefore, capital stock data need to be estimated. This paper estimates industry capital stock data with the method of perpetual inventory. Obviously, the calculation of capital stock of each year should be based on the capital stock of base year, depreciation rate, and constant price of investment. Following Chen’s method [15], this study gets capital stock of 1980 as capital stock of the base year.

The depreciation rates of subindustry are estimated with the data of depreciation value and fixed assets value from Chinese Statistical Yearbook and Chinese Industry Economy Statistical Yearbook published by NBSC [23]. This paper constructs the investment sequence data based on the difference of fixed assets and then converts them to constant price of 1990 by the investment price index of each year. After that, with the perpetual inventory method, capital stock data of various subindustries are calculated over the period from 1999 to 2009. 3.2. Environmental Efficiency and Its Components Based on SBM directional distance function and Luenberger productivity index, this paper measures the environmental efficiency and environmental TFP by the software package Excel Solver Prem Platform V5.

5 which is widely used in the present study. Environmental inefficiency values of every subindustry under the assumption of CRS and VRS are Drug_discovery measured, respectively, and the results are given in Table 1.Table 1Environmental inefficiency and its components of China’s industry. Since the environmental efficiency value under VRS assumption does not consider scale efficiency, the value under VRS assumption must be lower than CRS assumption, which is confirmed in Table 1. Under CRS assumption industrial production is in the conditions of optimal scale. However, many factors such as imperfect competition and externality may lead to nonoptimal scale. Therefore, when the value under CRS assumption is different from that under VRS assumption, the task is to analyze the efficiency under VRS assumption [24]. This study will focus on the analysis of environmental efficiency and its components under VRS assumption. The total value of environmental inefficiency of China’s industry is 60.8%. The main source of environmental inefficiency is the inefficiency of gross industrial output value (14.

0M in 50mM Tris buffer, pH 7 1 The enzymatic assays were perform

0M in 50mM Tris buffer, pH 7.1. The enzymatic assays were performed as described above. The influence of different cations was studied using KCl, NaCl, NH4Cl, MgCl2, and CaCl2 (all purchased from Merck KGaA, Darmstadt, Germany) in the range of 0.10 to 0.70M in the same conditions. 2.7. Inhibition of the Enzyme ActivityThe effect of inhibitors on the protease amydolytic activity was tested incubating the enzyme (50nM) with irreversible inhibitors as, N��-tosyl-Phe chloromethyl ketone (TPCK��50.0��M [17]), purchased from Fluka BioChemika (Saint Louis, MO, USA), L-trans-epoxysuccinyl-leucylamido-butane (E-64��5.0��M [18]) purchased from Merck KGaA (Darmstadt, Germany), phenylmethylsulphonyl fluoride (PMSF��0.50mM [19]), or N��-tosyl-Lys chloromethyl ketone (TLCK��50.

0��M [17]), both purchased from Sigma-Aldrich Chemical Company Inc. (Saint Louis, MO, USA), in 50mM Tris buffer pH 7.1, for 10min at 37��C. The activity was also tested in the presence of reversible inhibitors as CeKI (21.5 and 215nM [9]) purified by our group, benzamidine (0.50 and 4.0mM [20]) purchased from Sigma-Aldrich Chemical Company Inc., (Saint Louis, MO, USA), pepstatin A (1��M [21]) purchased from USB (Cleveland, OH, USA), aprotinin (92 and 307nM [22]), soybean trypsin inhibitor (SBTI: 49.8 and 498nM [23]), or ethylenediamine-tetraacetic acid (EDTA: 1.0 and 10.0mM [24]), all purchased from Calbiochem (Darmstadt, Germany), in 50mM Tris buffer, pH 7.1, for 10min at 37��C. After the preincubation, 0.40mM H-D-Pro-Phe-Arg-pNA was added, and the substrate hydrolysis was followed for 30min at 37��C at 405nm in the microplate reader.

3. Results and DiscussionIn the recent years, great efforts has been devoted to purification of plant proteases, which appear to be involved in several processes such as germination where specific degradation of cotyledon storage proteins occurs [25]. Also, they are thought to be useful toward understanding of several mechanisms at the subcellular level.This work focuses on the isolation and characterization of a protease of C. echinata seeds��CeSP. The crude protease was obtained by precipitation on 40% (NH4)2SO4. Purification to homogeneity was achieved by hydrophobic interaction and anion exchange chromatographies (Figures 1(a) and 1(b)) and gel filtration (Figure 1(c)). The details of the purification analysis are summarized in Table 1.

The specific activity of the purified enzyme was high (8,424U/mg) as compared to the activity in the crude extract (176U/mg). The total recovery of the activity was 8.2%. Besides, the enzyme was purified 47.8-fold.Figure 1Elution profile of CeSP purification. (a) Hydrophobic interaction chromatography, where a Hitrap GSK-3 Phenyl column was equilibrated with 50mM phosphate buffer, pH 7.0, 1.0M (NH4)2SO4, and proteins were eluted with (NH4)2SO4 (0.95, 0.25, …

Subsequently the bandwidth increases with increasing the cell tem

Subsequently the bandwidth increases with increasing the cell temperature. The bandwidth depends on the transverse relaxation time ��2, as evident from (4) and (6). An increase in the cell temperature increases the vapor pressure, which results in a larger number of atoms interacting with the light, leading to a longer relaxation time (smaller bandwidth). However, at high temperature, the number of collisions between atoms or with the walls of the cell increases significantly, leading to a shorter relaxation time and hence a larger bandwidth. This explains the existence of a critical temperature (around 45��) at which the intrinsic bandwidth is minimum. Figure 8Intrinsic bandwidth versus cell temperature for an input optical light power of 10��W, 15��W, and 20��W.

In the experiments, the measured maximum bandwidth was 175Hz, obtained at room temperature (23��C) with an input optical power of 10��W (blue curve in Figure 8), while the minimum bandwidth was 25Hz, obtained with an input optical power of 20��W at a temperature of 45��C (black curve in Figure 8).4.3. Low-Amplitude Magnetic Field MeasurementThe ultimate intrinsic sensitivity of the magnetometer can be calculated using (5). The best performance of the magnetometer was obtained for an input optical power of 20��W at cell temperature of 48��C; the ultimate intrinsic sensitivity was 327fT/Hz1/2 over a bandwidth of 26Hz. However, the external magnetic noise generated by power lines and surrounding equipment caused the actual ultimate sensitivity of the magnetometer to drop to 130pT/Hz1/2 over a bandwidth of 26Hz.

The magnetometer in its optimal configuration (input optical power of 20��W at cell temperature of 48��C) was then used to measure an applied small-signal sinusoidal magnetic field of amplitude 15pT oscillating at 25Hz, which was generated by a test coil placed at a distance of 6cm from the vapor cell. For this measurement, the uniform dc magnetic field was 13��T, corresponding to a Larmor frequency of 45.5kHz. The frequency of the rf magnetic field was then set at 45.5kHz resulting in a phase shift of ?90 degrees between the photodiode output and the driving rf signal, as predicted by (4), and verified experimentally by the result shown in Figure 7(c). When another 25Hz small-amplitude magnetic field was applied in addition to the dc and rf magnetic fields, the Larmor frequency changed and no more resonance occurred, Cilengitide causing the phase shift between the photodiode output and the driving rf signal to oscillate around ?90 degrees at 25Hz. This enabled the measurement of the new Larmor frequency and hence the calculation of the magnitude of the 25Hz small-amplitude magnetic field, which is proportional to the new Larmor frequency.

3 4 Avoiding Premature ConvergenceMost of the global optimizatio

3.4. Avoiding Premature ConvergenceMost of the global optimization methods suffer from premature convergence. One of the most used approaches to tackle this problem is to introduce diversity to the velocity sellckchem or the position of a particle. As mutation operators are to the genetic algorithm, so is introduction of diversity to PSO algorithms. The focus of this paper is to introduce the diversity by employing scout particles. The details of how the proposed DPSO algorithm circumvents premature convergence are described in Section 4.Garc��a-Villoria and Pastor [27] introduce the concept of diversity into the velocity updating function. The proposed dynamic diversity PSO (PSO-c3dyn) dynamically changes the diversity coefficients of all particles through iterations.

The more heterogeneity of the population will be, the less diversity will be introduced to the velocity updating function, and vice versa. Blackwell and Bentley [28] incorporate diversity into the population by preventing the homogeneous particles from clustering tightly to each other in the search space. They provide collision-avoiding swarms that reduce the attraction of the swarm center and increase the coverage of a swarm in the search space. Silva et al. [16] attempt to apply the diversity to both the velocity and the population by a predator particle and several scout particles. A predator particle is intended to balance the exploitation and exploration of the swarm, while scout particles are designed to implement different exploration strategies. The closer the predator particle will be to the best particle, the higher probability of perturbation will be.

4. DPSO with Scout ParticlesThis section details how to tackle the materials acquisition problem by discrete particle swarm optimization with scout particles. The representation of a particle and the initialization method for the studied problem are described in Section Brefeldin_A 4.1. Then, Section 4.2 elaborates on the details of preventing premature convergence by deploying scout particles. Section 4.3 redefines a constraints handling mechanism for solving the constrained optimization problem.4.1. Representation and InitializationThe solution of materials acquisition problem with n materials and m departments obtained by particle s at iteration t can be represented by an n �� m binary matrix, proposed by Wu et al. [29], as shown in (15). Each entry of the matrix (Pst)ij indicates whether material i is acquired by department j or not. Note that each entry of the matrix (Pst)ij corresponds to the decision variable (xij) that was mentioned in Section 2.1:Pst=[Ps(11)t?Ps(1m)t???Ps(n1)t?Ps(nm)t].

3 3 Genetic Relationships of 115 Sugarcane ParentsNine series fr

3.3. Genetic Relationships of 115 Sugarcane ParentsNine series from 115 accessions sorted by institution-based breeding programme are shown in Table 5. According to the information indicated in Table 1, we assigned them as the following nine series: GT series (13) from Guangxi Sugarcane Institute; YT series (13) from Guangzhou Institute of Sugarcane and Sugar Industry; YC series (10) from Hainan Sugarcane Breeding Station; FN series (7) from Sugarcane Research Institute of FAFU; MT series (7) from Sugarcane Research Institute, Fujian Academy of Agricultural Sciences; HoCP series (4) from Sugarcane Research Unit, Houma, Louisiana, United States Department of Agriculture, USA; CP series (10) from Sugarcane Experiment Station, Canal Point, Florida, United States Department of Agriculture, USA; and ��ROC�� series (13) from Taiwan Sugar Corporation.

The rest of the sugarcane parents included 37 accessions from several breeding institutions different from all the above eight and were termed as OTHER.Table 5Genetic diversity of sugarcane parents in 9 series released by different breeding institutions, estimated based on polymorphisms of 5 SSR loci.Genetic diversity parameters for the 5 microsatellite markers in the 9 sugarcane series were presented in Table 5, indicating that except the highest NPB value (62 stands for 70.45%) observed in OTHER series, the polymorphisms among eight determinate series were as follows: YT (50, 56.82%) > YC (49, 55.68%) > GT (47, 53.41%) > ��ROC�� (46, 52.27%) > MT (44, 50.00%) > FN (37, 42.05%) > CP (36, 40.91%) > HoCP (32, 36.36%).

Observed numbers of alleles (Na) were higher in OTHER (Na = 1.705) and YT (Na = 1.568) series compared to those of the remaining seven series. Moreover, effective numbers of alleles (Ne) were also higher in OTHER (Ne = 1.290) and YC (Ne = 1.290) series compared to those of the remaining seven determinate series. Except OTHER series (h = 0.177, I = 0.278), both the gene diversity (h) and the Shannon information index (I) were higher in YC (h = 0.178, I = 0.275) series but lower in CP series (h = 0.136, I = 0.181).The number of alleles based on 5 SSR loci in different series of GT, YT, YC, FN, MT, HoCP, CP, ��ROC,�� and OTHER was illustrated in Figure 1. A total of 1,395 alleles were detected for all the 115 testing sugarcane accessions with an average of 12.

The maximum number of alleles was 18 observed in YT93-159, while the minimum number was 7 in three accessions of GT90-55, YC96-48, and FN93-3608. Within the GT and FN series, the number of alleles both ranged from 7 to 15 with mean values of 11.8 and 11.1, respectively. In YT series, the number of alleles per locus ranged from 8 to 18 Dacomitinib with an average of 12.6. Within MT series, the number of alleles ranged from 8 to 14, and the average number was 11.3.