The cultures were then removed and the plates were washed twice with distilled water. The number of the adherent cells in an area of 1 mm2 was counted under an optic microscope (Olympus CKX41) to estimate the primary attachment ability of the bacteria in different mediums. The culture of S. aureus NCTC8325 was started

by diluting the overnight culture to an OD600 nm=0.05 and was then allowed to grow at 37 °C (200 r.p.m.) to exponential phase (OD600 nm=0.6). The cultures were then treated with 5 mM dithiothreitol, 10 mM cysteine or 20 mM BME, respectively, for 30 min. Staphylococcus aureus cells were predigested with digestion buffer containing 40 U mL−1 lysostaphin, 10 mg mL−1 lysozyme and 10% (v/v) glycerol. RNA extraction was performed using the SV Total RNA Isolation System (Promega). Residue DNA in extracted RNA was removed by treatment with 10 U of DNaseI (Takara) at 37 °C for an hour. Purified total RNA were qualified and quantified Selleckchem ERK inhibitor by a DU730 Nucleic LGK974 Acid/Protein Analyzer (Beckman Coulter). Reverse transcription of ica was carried out following the technical manual of ImProm-II Reverse Transcription System (Promega) with primer PicaD-R (TCACGATTCTCTTCCTCTC). Q-PCR was performed using StepOne Real-time System (Applied Biosystems) with primers PicaD-F (ATGGTCAAGCCCAGACAG) and PicaD-R. The crude extracellular matrix was prepared as described previously (Sadovskaya et al., 2005). Briefly, S. aureus

cells were grown in the respective medium at 37 °C with moderate shaking (50 r.p.m.) to reach an OD600 nm of 2.0. Bacterial cells were collected by centrifugation (3000 g), resuspended in phosphate-buffered saline (pH 7.4) and sonicated immediately on ice to extract the cell-associated extracellular material. Bacterial cells and insoluble material were removed by centrifugation (10 000 g). Then, the Elson–Morgan assay was performed to measure the amount of PIA in the supernatant after acidolysis as described previously

(Morgan & Elson, 1934). The cultures of S. aureus NCTC8325 were started by diluting the overnight culture to an OD600 nm=0.05 in TSB or TSB supplemented with 5 mM dithiothreitol and incubating at 37 °C (200 r.p.m.) for an additional 6 h. Total protein was purified Methane monooxygenase by trichloroacetic acid/ice-cold acetone precipitation as described previously (Resch et al., 2006). Protein sample (500 μg) was loaded in each 17-cm gradient gel strip in the pH range of 4–7 and separated by isoelectrofocusing (IEF) using a Protean IEF cell (Bio-Rad). Second-dimension electrophoresis was carried out on a sodium dodecyl sulfate polyacrylamide gel. The gels were stained with Coomassie G-250 and then scanned on a flatbed scanner. Images were analyzed with imagemaster 2d-platium 5.0 (Amersham), setting the threshold at 3.0-fold. Differential protein dots of interest were marked and 21 of them were excised manually and digested with trypsin (Worthington). The digests were analyzed by HPLC-ES-MS (MDLC-LTQ, Amersham).

In the present study, we investigated the electrophysiological and morphological properties of GABAergic neurons in SGI by whole-cell patch-clamp recordings and intracellular find more staining using biocytin in GAD67-GFP knock-in mice (PND17-22), in which GABAergic neurons specifically express GFP fluorescence. The most common firing properties among these GABAergic neurons (n = 231) were fast spiking (58%), followed by burst spiking (29%), late spiking (8%) and, the least common, regular spiking (2%) and rapid

spike inactivation (3%). Morphological analysis of axonal trajectories of intracellularly-labeled GABAergic neurons revealed three major subclasses: (i) intralaminar interneurons, which were further divided check details into two subclasses, local and horizontal interneurons; (ii) interlaminar interneurons; and (iii) commissural and tectofugal neurons. These results reveal distinct subsets of GABAergic neurons including neurons that mediate local and long-range inhibition in the SC, neurons that potentially modulate visual and other sensory inputs to the SC, and neurons that project to nuclei outside the SC. “
“Repetitive transcranial magnetic stimulation paradigms such as continuous theta burst stimulation (cTBS) induce long-term potentiation- and long-term depression-like plasticity

in the human motor

cortex. However, responses to cTBS are highly variable and may depend on the activity of the cortex at the time of stimulation. We investigated whether power in different Thalidomide electroencephalogram (EEG) frequency bands predicted the response to subsequent cTBS, and conversely whether cTBS had after-effects on the EEG. cTBS may utilize similar mechanisms of plasticity to motor learning; thus, we conducted a parallel set of experiments to test whether ongoing electroencephalography could predict performance of a visuomotor training task, and whether training itself had effects on the EEG. Motor evoked potentials (MEPs) provided an index of cortical excitability pre- and post-intervention. The EEG was recorded over the motor cortex pre- and post-intervention, and power spectra were computed. cTBS reduced MEP amplitudes; however, baseline power in the delta, theta, alpha or beta frequencies did not predict responses to cTBS or learning of the visuomotor training task. cTBS had no effect on delta, theta, alpha or beta power. In contrast, there was an increase in alpha power following visuomotor training that was positively correlated with changes in MEP amplitude post-training. The results suggest that the EEG is not a useful state-marker for predicting responses to plasticity-inducing paradigms.

0), 140 mM choline GSK126 price Cl, 5 mM MgCl2, 1 mM dithiothreitol and 10% v/v glycerol]. They were passed through a French press (Avanti Products)

to disrupt the cells. Membrane fractions containing the inside-out vesicles were collected by ultracentrifugation. The fluorescence assays of cation/proton antiport activities were conducted on the membrane vesicles using acridine orange, a ΔpH probe which may be used to infer the transmembrane pH gradient. To assess antiport activity, 66 μg of membrane vesicle protein was mixed into 2 mL of assay buffer (10 mM Bis-Tris propane, 140 mM choline Cl, 5 mM MgCl2, 1 μM acridine orange). The assay was initiated by adding Tris-succinate to a final concentration of 5 mM. This induced a quenching of fluorescence intensity as succinate metabolism caused the respiratory chain to establish a pH gradient of ‘acid in’ across the vesicle membrane. Then, for evaluating cation/proton antiport activities, 1 mM of cation (Na+, Li+, K+, or Ca2+) was added to the assay buffer. Finally, 10 mM NH4Cl was added into the assay buffer to re-establish a baseline by collapsing the pH gradient. We have estimated the cation/proton antiport activity as the % dequenching by the observed changes in the acridine orange fluorescence intensity after cation addition. Measurements were conducted PARP inhibitor using a Hitachi High-Technologies

model F-4500 fluorescence spectrophotometer. Genomic analysis showed that the Tr-mrp genes cluster was located on a large plasmid of 0.92 Mb. The cluster is composed of seven separate genes Pyruvate dehydrogenase lipoamide kinase isozyme 1 encoding putative hydrophobic proteins as seen in the mrp operon from Bacillus (Fig. 1). The sodium sensitive phenotype of E. coli KNabc can be complemented by the heterogenous expression of Na+-efflux systems including Mrp, as previously described (Yang et al., 2006; Swartz et al., 2007). As shown in Fig. 2a, expression of Bp-Mrp strongly restored the sodium tolerance of E. coli KNabc. However,

no such recovery of the growth was observed in E. coli KNabc transformed with Tr-Mrp, indicating it was unlikely that Tr-Mrp conferred Na+-efflux in E. coli. In addition, its expression led to a decreased growth level of the transformants even in LBK medium without added NaCl. Cation/proton antiport activities of Tr-Mrp were measured in the inside-out vesicles prepared from E. coli KNabc transformants. The inside-out vesicles expressing Bp-Mrp, which has been characterized previously, exhibited the obvious pH-dependent Na+/H+ antiport activity (Fig. 2b) (Swartz et al., 2007). On the other hand, no Na+/H+ antiport activity was observed in the inside-out vesicles containing Tr-Mrp (Fig. 2b). However, the vesicles containing Tr-Mrp exhibited Ca2+-stimulated dequenching, which was detectable in the broad pH range of 7.0–9.0 (Fig. 3a). As no significant dequenching was observed by added CaCl2 to the vesicles containing Bp-Mrp or the empty vector (Fig.

Thus, 660 000 cases

of sepsis occur in the United States each year, and combined with the high mortality, it ranks as a leading cause of death. Staphylococci, including methicillin-resistant Staphylococcus aureus, have become the most frequently isolated bacteria in nosocomial infections, giving rise, according to some reports, to more than 50% of the cases (Bearman & Wenzel, 2005). Severe sepsis occurs when sepsis (the combined events of infection and the systemic inflammatory response syndrome, i.e. SIRS) is complicated by organ dysfunction, hypotension or hypoperfusion; septic shock is characterized by persistent arterial hypotension despite adequate fluid resuscitation (Bone et al., 1992; Levy et al., 2003). The Sequential Organ Failure Assessment (SOFA) score, which evaluates the respiratory, blood clotting, hepatic, cardiovascular, central nervous and renal systems, has been advocated as a simple (bedside) method to monitor organ CDK inhibitor dysfunction and guiding supportive therapy of critically ill patients, including those with sepsis (Vincent et al., 1996, 1998; Afessa et al., 2007). Several papers have demonstrated that the SOFA scoring system predicts mortality, which increases with an increasing

score and the number of failing organs. Dysfunction or failure of the cardiovascular system, followed by the renal and central nervous systems, had the single most serious impact on severity in patients in intensive care (Moreno et al., 1999), as also evidenced in septic shock patients, who suffer the highest mortality (Vincent et al., 2006). The mean Protein Tyrosine Kinase inhibitor time to reach the maximum SOFA scores was the highest for the liver; dysfunction of the liver, however, did not contribute to an increased risk of death (Moreno et al., 1999). The liver thus seems to be an organ on which the sum of ailments converges, making failure a late and secondary event. In a previous study (Nielsen et al., 2009b), we showed that an intravenous inoculation of S. aureus

in pigs induces acute pyaemia, with the formation of microabscesses in various organs 4– 6 h after the challenge. However, no 3-mercaptopyruvate sulfurtransferase systemic inflammatory response, and thus sepsis, was induced, probably due to the short duration of the experiment. The aim of the present study was to further characterize the pig as a model for human S. aureus-induced sepsis and severe sepsis. Therefore, the inoculated pigs in the present study were kept for up to 48 h, inoculation was repeated in some pigs and the study included the evaluation of the possible late event of liver dysfunction or failure. Details on the experimental animals, the bacterial inocula and the design of the study have been published previously (Jensen et al., 2010). Briefly, 12 clinically healthy female specific pathogen-free (SPF) Yorkshire–Landrace crossbreed 8-week-old pigs with a body weight (BW) of 20–25 kg were used. The pigs, which remained clinically healthy during the acclimatization period of 7 days, were randomly assigned to three groups.

We did not observe

an association between low BMI and baseline BMD values, although we did find an association between low BMI and subsequent decline in hip BMD. However, most patients were normal weight (BMI between 20 and 25), which Bioactive Compound Library mouse may have diminished the influence of BMI. Of interest, Aukrust et al. found markedly decreased levels of bone formation markers and increased levels of bone resorption markers in untreated patients with advanced HIV infection and also found indications of normalization of the bone remodelling process during HAART [15]. The decrease in BMD observed during the initial 24 to 48 weeks of therapy could also partly be due to an ongoing BMD loss in untreated HIV-infected individuals, which do not reverse immediately after initiation of HAART. However, bone loss of the magnitude we observed in the 24–48-week period after HAART initiation

could not have taken place selleck chemicals during the often many years of asymptomatic HIV infection without producing more pronounced osteopenia than observed at baseline in this and other studies [25]. In our study, the factors associated with a low baseline BMD were different from the factors associated with bone loss after HAART initiation; most notably, there was no association between low baseline CD4 cell count and low baseline BMD. It is important to note that the between-patient variability and statistical power concerning baseline BMD in the cross-sectional analysis are different from those in the analyses concerning percentage change in BMD from baseline to 24 weeks, but different processes may also drive the bone loss before and PJ34 HCl after HAART initiation. Data on the effect of different drug classes on

BMD have not been consistent. While some observational studies found that PIs increased the risk of BMD decline, others did not confirm these results. In particular, studies that controlled for HIV-related and traditional risk factors for osteoporosis did not find an independent effect of PIs [26,27]. A randomized French study (n=71) of three different class-sparing strategies found a more pronounced decrease in lumbar spine BMD in the two PI-containing arms compared with the PI-sparing arm; however, no differences were found at the hip [6]. In contrast, recent results from a Dutch study including 50 patients indicated a role of zidovudine/lamivudine, as patients randomized to zidovudine/lamivudine and lopinavir/ritonavir had more pronounced bone loss than patients randomized to lopinavir/ritonavir and nevirapine [17].

We did not observe

an association between low BMI and baseline BMD values, although we did find an association between low BMI and subsequent decline in hip BMD. However, most patients were normal weight (BMI between 20 and 25), which GSK2118436 mw may have diminished the influence of BMI. Of interest, Aukrust et al. found markedly decreased levels of bone formation markers and increased levels of bone resorption markers in untreated patients with advanced HIV infection and also found indications of normalization of the bone remodelling process during HAART [15]. The decrease in BMD observed during the initial 24 to 48 weeks of therapy could also partly be due to an ongoing BMD loss in untreated HIV-infected individuals, which do not reverse immediately after initiation of HAART. However, bone loss of the magnitude we observed in the 24–48-week period after HAART initiation

could not have taken place Trametinib price during the often many years of asymptomatic HIV infection without producing more pronounced osteopenia than observed at baseline in this and other studies [25]. In our study, the factors associated with a low baseline BMD were different from the factors associated with bone loss after HAART initiation; most notably, there was no association between low baseline CD4 cell count and low baseline BMD. It is important to note that the between-patient variability and statistical power concerning baseline BMD in the cross-sectional analysis are different from those in the analyses concerning percentage change in BMD from baseline to 24 weeks, but different processes may also drive the bone loss before and PLEKHB2 after HAART initiation. Data on the effect of different drug classes on

BMD have not been consistent. While some observational studies found that PIs increased the risk of BMD decline, others did not confirm these results. In particular, studies that controlled for HIV-related and traditional risk factors for osteoporosis did not find an independent effect of PIs [26,27]. A randomized French study (n=71) of three different class-sparing strategies found a more pronounced decrease in lumbar spine BMD in the two PI-containing arms compared with the PI-sparing arm; however, no differences were found at the hip [6]. In contrast, recent results from a Dutch study including 50 patients indicated a role of zidovudine/lamivudine, as patients randomized to zidovudine/lamivudine and lopinavir/ritonavir had more pronounced bone loss than patients randomized to lopinavir/ritonavir and nevirapine [17].

, 2012; Wacongne et al., 2012). In the present study, we found that the omission in the random sequence caused greater

brain activity than that in the group sequence. This result could be explained Fulvestrant mouse by the predictability of omission. The omission in the group sequence only occurred after the first ‘L’ tone or the last ‘S’ tones, so the subjects could easily predict the position of omission. However, the omission in the random sequence occurred randomly and was less predictable than the group sequence. Thus, the prediction error for the omission in the random sequence should be greater than that in the group sequence, leading to greater brain activity for the omission in the random sequence. An alternative explanation would be that we have different brain mechanisms for perceptual grouping, depending on whether the subjects ignore or attend to the stimuli. Bregman (1990) also suggested the existence of two different forms of perceptual grouping, namely pre-attentive and attentive. Although the predictive coding theory considered the pre-attentive perceptual grouping, several studies have shown evidence that attention modulates regularity processing, including deviance detection, feature binding, and stream segregation (Cusack et al., 2004; Takegata et al., 2005; Haroush et al., 2010).

Our results may be interpreted as resulting from this kind of attentive isocitrate dehydrogenase inhibitor processing. However, the design of the present experiment is not suitable to evaluate this idea and further investigation will be conducted in the future. The MEG measurement suggests that

the right IPL and left STG are part of the network for perceptual grouping in musicians and non-musicians, respectively. The contribution of these areas in perceptual grouping has also been found in studies of auditory stream segregation. When A and B tones are rapidly presented as ‘ABA_ABA_…’, it can be heard as either a single ‘ABA_’ sequence or two different streams of A and B tones. When a subject hears the sequence as two streams, activity in the left STG and right IPL is increased, compared with hearing it as one stream Amobarbital (Deike et al., 2004, 2010; Cusack, 2005; Rahne et al., 2008). Our findings are compatible with these results, but we found that the activated areas were different between musicians and non-musicians. The STG includes the auditory cortex and, in particular, the left hemisphere appears to be specialised for temporal feature processing, whereas the right hemisphere is specialised for spectral processing (Samson et al., 2001; Peretz & Zatorre, 2003; Vuust et al., 2005). Because the omission of a tone works as a kind of temporal deviation, the observed difference in the left STG in non-musicians might be caused by such left hemisphere dominance in temporal feature processing. Conversely, the difference in the right IPL in musicians is more difficult to interpret.

Furthermore, apnoea-related reductions in airflow lead to hypoxia and hypercapnia. A range of neurocognitive check details impairments have been associated with OSA, including

decreases in memory, attention and executive function (Campana et al., 2010). These symptoms negatively impact the daily life of patients, with reports of difficulty accomplishing routine work tasks (Ulfberg et al., 1996), and increased risk of motor vehicle (Tregear et al., 2009) and occupational (Ulfberg et al., 2000) accidents. Although the pathophysiology of these cognitive deficits remains largely unknown, several studies have shown alterations in brain structure and function in patients with OSA. For example, patients with OSA show decreased grey matter in various brain regions (Joo et al., 2010b; Morrell et al., 2010; Torelli

et al., 2011), and differences in neural activation of sensorimotor and autonomic brain regions during respiratory challenges (Zimmerman & Aloia, 2006). Furthermore, studies using transcranial magnetic stimulation (TMS) have shown increased motor thresholds (Joo et al., 2010a) and prolonged cortical silent periods (CSPs) in patients with OSA (Civardi et al., 2004; Grippo et al., 2005), reflecting cortical hypoexcitability (see Civardi et al., 2009). These changes may result from diminished corticospinal fibre integrity in patients (Macey et al., 2008), and are presumed to be a consequence of chronic intermittent hypoxaemia and sleep fragmentation (Morrell et al., 2003; Ohga et al., 2003). Plasticity of cortical circuits is an important component of the http://www.selleckchem.com/products/SB-431542.html brain’s ability to adapt, learn and recover from injury. It is also known to be a fundamental process in memory function, which has been shown to be defective in OSA (Jackson et al., 2011). The application of repetitive trains of TMS (rTMS) is commonly used to non-invasively induce plasticity of neural circuits within the

human motor cortex. A recently developed protocol known as continuous theta burst stimulation Thiamet G (cTBS) uses a specific pattern of rTMS that can suppress motor-evoked potentials (MEPs) for up to 1 h (Huang et al., 2005), and is thought to induce long-term depression (LTD)-like synaptic changes (Cardenas-Morales et al., 2010) within cortical circuits (Di Lazzaro et al., 2005). The primary aim of this study was to examine motor cortex plasticity in patients with moderate-to-severe OSA using cTBS. As mouse models of OSA have shown impaired hippocampal plasticity (Xie et al., 2010) and sleep fragmentation could affect processes that promote plasticity (Diekelmann & Born, 2010), we hypothesised that cortical plasticity would be reduced in patients with OSA. Furthermore, as increased intracortical inhibition (ICI) can reduce neuroplastic capacity of cortical circuits (Ziemann et al., 2001), a secondary aim of this study was to quantify baseline ICI in patients with OSA compared with controls. Based on previous observations of increased CSP in OSA (Civardi et al.

Therefore, a high baseline weight and 80 mg of d4T daily are directly correlated and difficult to untangle in analysis. In light of

this, it is important to consider that cases with SHLA were more likely to have a baseline weight of ≥60 kg but were at even greater risk if their baseline weight was ≥75 kg in multivariate regression. These findings are consistent with those of a smaller cohort study in the same setting [17]. The rapid increase in risk with increasing weight cannot be explained by dose escalation at 60 kg alone, and suggests a biological phenomenon peculiar to women with high BMIs. Obesity and rapid weight gain are closely linked to both insulin resistance and nonalcoholic fatty liver disease selleck kinase inhibitor (NAFLD) [25,26]. Once NAFLD is present, other factors including oxidative stress and mitochondrial dysfunction (which has been shown to be caused by NRTI drugs [27,28]) may cause progression from NAFLD to nonalcoholic steatohepatitis (NASH; inflammation of and damage to the liver) [25,26,28,29]. In the setting of this study, there is a high prevalence selleck chemical of obesity [30] and metabolic syndrome in African women [31], which could result in many patients having or being predisposed to NAFLD or NASH at the start of ART. Rapid weight gain on ART and the mitochondrial toxicity caused

by NRTIs are likely to exacerbate this. As lactate is cleared predominantly by the liver and kidneys [22], a metabolically dysfunctional fatty liver may be unable to clear excess lactate, potentially contributing to SHLA [25,32]. The clinical utility of

low-grade increases in ALT serving as an early marker for progressive NAFLD warrants further exploration. The well-recognized major symptoms of SHLA (abdominal pain and vomiting) were frequently observed early manifestations of SHLA. These associations were expected, given the a priori anticipated association, and because they are amongst the symptoms that prompt clinicians to measure lactates. Less frequently described early symptoms were poor appetite and weight loss. An important finding was the independent Thalidomide association of symptoms of peripheral neuropathy with development of SHLA. This was probably attributable to their shared underlying pathogenesis of NRTI mitochondrial toxicity. Symptoms of peripheral neuropathy should be a further prompt for clinicians to assess for SHLA. This study has a number of strengths. The universal use of d4T, combined with the matching on duration of therapy, provided a unique opportunity to explore other associations in greater depth. The concentration of 71 cases in a single service setting enabled the collection of clinical follow-up data that facilitated the exploration of early signs of progressive disease. The incompleteness of some clinical data was, however, an important limitation in this study.

, 1990; Timenetsky et al., 2006). Here, we show Torin 1 clinical trial for the first time that

contamination of SH-SY5Y cells by a strain of M. hyorhinis results in increased levels of calpastatin. The mycoplasma-infected cells exhibit lower calpain activation and diminished calpain-promoted proteolysis, compared with the noninfected (clean) cells. These findings have implications for studies on mycoplasma-contaminated cultured cells, and may be relevant to the role of mycoplasmas in some diseases. For the detection of mycoplasma contamination of the SH-SY5Y cell cultures, the EZ-PCR Mycoplasma Test Kit (Biological Industries, Israel) was used according to the manufacturer’s instructions. For the specific identification of the contaminating Mycoplasma species, PCR was performed using the primers 1623F – ACACCATGGGAG(C/T)TGGTAAT and 1623R – CTTC(A/T)TCGACTT(C/T)CAGACCCAAGGCAT, designed to amplify the variable spacer between the conserved 23s and 16s rRNA mycoplasma genes. The PCR product was isolated, sequenced and analyzed using the program blastn

of the National Center for Biotechnology Information with the nucleotide collection database (nr). The contaminating Mycoplasma species was grown in a modified Chanock medium supplemented with 10% heat-inactivated fetal calf serum (FCS) (Biological Industries), as described previously (Yavlovich et al., 2004). The medium was inoculated with 1–5% of a frozen culture and incubated at 37 °C for 48–96 h. Cells were harvested at the late find more exponential phase of growth (pH 5.9–6.2) by centrifugation at 12 000 g for 15 min, washed and suspended in a solution containing 250 mM NaCl and 10 mM Tris-HCl (pH 7.4). Mycoplasma-free SH-SY5Y cells (obtained from Dr Talia Han, Kaplan Medical Center, Rehovot, Israel) were grown in RPMI-1640

supplemented with 2 mM l-glutamine, 10% FCS, 100 IU mL−1 penicillin and 100 μg mL−1 streptomycin (pen-strep solution) [growth medium (GM)] in 25-cm2 plastic culture flasks. Cells were induced to differentiate, by plating 1–2 × 106 cells in 60-mm Petri dishes, and cultured for 7 days in Dulbecco’s modified Eagle’s medium, Prostatic acid phosphatase supplemented with 2 mM l-glutamine, 10% FCS and pen-strep solution [differentiation medium (DM)], in the presence of 20 μM all-trans retinoic acid (Sigma, St. Louis, MO). The DM and retinoic acid were replenished every 48 h during the differentiation. Cultures were routinely checked (every 3–4 weeks) by PCR, as described above, to ensure that they were uncontaminated (clean). Clean SH-SY5Y cells, cultured in GM, were infected with the mycoplasma (isolated from the original contaminated SH-SY5Y cells), at a multiplicity of infection of 50. The infected cells were differentiated under the same conditions described above for clean cells. To study the effects of Ca2+ on differentiated clean and infected cells, CaCl2 (Sigma) (100 mM stock solution in double-distilled water) and ionomycin (Calbiochem, La Jolla, CA) (0.