The treated cells were harvested and washed with PBS containing 1

The treated cells were harvested and washed with PBS containing 1% bovine serum albumin. Cells were incubated with anti-DR4 or anti-DR5 antibody for 30 min

at 4°C in the dark. After incubation, cells were washed twice and reacted with PE-labeled secondary antibody for 30 min at 4°C in the dark. Isotype-matched nonbinding antibodies (Iso) were the negative control cells. Samples were measured by flow cytometry. Analysis of the cell cycle was performed by staining with PI. Cells were seeded into a 100-mm dish, which contained selleck products 1 × 106 cells per plate. After 24 h, the media were changed to RPMI 1640 medium supplemented with indicated concentrations of Rg5. After 48 h of incubation, the cells were trypsinized and washed with ice-cold PBS, fixed with ice-cold 90% ethanol, and then incubated at −20°C until analysis. For cell cycle analysis, the cells were resuspended in 300 mL of PBS containing 30 μL RNase A solution (10 mg/mL; Sigma-Aldrich) and 1.5 μL PI solution (1 mg/mL; Molecular Probes). After incubation at 37°C for 30 min, cells were determined using the FACSCanto II Flow Cytometer (BD

Biosciences). The cell cycle distribution was analyzed by FlowJo software (Tree Star, Inc., Ashland, OR, USA). Cells were plated at 0.3 × 106 cells in six-well plates. After treatment, the cells were fixed in DMSO/methanol (1:4) solution for 12 h at 4°C, stained with 4′,6-diamidino-2-phenylindole LY294002 in vivo (DAPI) for 20 min, and observed by fluorescence microscopy. Statistical significance was performed by Turkey’s multiple comparison tests (Sigma Plot version 10.0; Systat Software, San Jose, CA). All experiments were repeated at least three times. Data were analyzed by one-way analysis of variance (ANOVA), and each value was presented as the mean ± the standard deviation. The yield of ginsenosides from ginseng hairy root (i.e., fine root) was higher than the yield from the main root [2], and the saponin GPX6 content of FBG was higher

than that of BG [23]. First of all, the HPLC results showed Rg5 was the main constituent among the ginsenosides in FBG (Fig. 1A). Rg5 was separated from FBG BF using column chromatography (silica gel, ODS) (Figs. 1B, 1C), and the chemical structure was confirmed by spectroscopic methods [e.g., NMR, mass spectroscopy (MS)] (Fig. 2). The effects of FBG EE and FBG BF on cell viability were evaluated in MCF-7 and MDA-MB-453 breast cancer cell lines by MTT assay. The results showed that EE reduced MCF-7 cell viability after 48 h of treatment and it decreased cell viability of MDA-MB-453 cells after 72 h (Figs. 3A, 3B). Increased cell viability was detected in MCF-7 cells when it was treated with 50 μg/mL (at 24 h, 48 h, and 72 h) and 100 μg/mL (24 h) of BF, but at higher concentrations (150 μg/mL and 200 μg/mL) the cell viability was decreased in a dose-dependent manner (Figs. 3C, 3D). As Figs.

2010),

these two flows have quite different dimensional a

2010),

these two flows have quite different dimensional and non-dimensional dynamic parameters. The Słupsk Furrow gravity current has a larger width W   (25 km vs. 10 km) and thickness H   (34 m vs. 11 m) and a smaller mean downstream interfacial slope Sx′   (1.5 × 10−4 vs. 5.0 × 10−4), bulk buoyancy B   (0.034 m s−2 vs. 0.07 m s−2), friction velocity u* (0.015 m s−1 vs. 0.02 ms−1), bulk flow velocity U   (0.3 m s−1 vs. 0.5 ms−1), Froude number Fr (0.27 vs. 0.54) and Ekman number Ek=(u′*2U−1f−1H−1)2(2.7×10−2vs.≈1). Though Ek ≪ l in the case of Słupsk Furrow, both this website gravity currents can be regarded as frictionally controlled, because the Ekman depth δE = 0.4u*/f exceeds H ( Umlauf & Arneborg 2009a). That is why in both cases the transverse structure of the gravity current is characterized by the presence of a thin interfacial jet directed to the right of the down-channel flow. Note that in the case when the Ekman layer thickness is much smaller than the channelized gravity flow itself, the transverse velocity structure does

not display a thin interfacial jet but a secondary flow field consisting of frictionally induced Ekman transports across the channel in the benthic and interfacial boundary layers and a return flow in the interior ( Cossu et al. 2010). The small value of the Froude number in the Słupsk Furrow gravity current relative to that of the Arkona Basin (Fr = 0.27 vs. Fr = 0.54) implies a reduced amount Megestrol Acetate of entrainment in the former case. To estimate the entrainment of surrounding waters to a gravity 5-FU datasheet current, one can use a new empirical parameterization suggested by Cenedese & Adduce (2010) based on laboratory and field measurements equation(4) E=Min+A Frα1+ACinf(FR+FR0)α,Cinf=1Max+1Reβ,where E = we/U is the entrainment ratio, we is the entrainment velocity, Re = U H/v is the Reynolds number, v ≈ 1.3×10−6 m2 s−1 is the kinematic molecular viscosity of water, and Min = 4 × 10−5, Max = 1, A = 3.4 × 10−3, Fr0 = 0.51, α = 7.18 and β = 0.5 are empirical constants based on the limited oceanographic and laboratory data available. Substituting the above parameters of gravity flows into

equation (4) one obtains E = 4.03 × 10−5 ≈ Min for the simulated gravity flow in the Słupsk Furrow and E = 8.0 × 10−5 for the Arkona Basin gravity current. Therefore, the entrainment in the Słupsk Furrow is twice as small as that of the Arkona Basin. Note that the last estimate (E = 8.0 × 10−5) is close to the observed value E = 6.6 × 10−5 ( Arneborg et al. 2007). The simulation of the same flow using MIKE 3 yielded results almost identical to those of POM (cf. Figures 4 and 6). The only difference worth mentioning is an inverted, hydrostatically unstable salinity/density stratification in BBL simulated with MIKE 3 instead of the vertically uniform stratification simulated with POM. This difference can be interpreted as follows.

The proliferation phase starts immediately after microneedling an

The proliferation phase starts immediately after microneedling and may reach its peak after 2 months. At present it is not known how epidermal and dermal stem cells are affected by microneedling. New type III collagen fibers integrate into the existing skin matrix without any trace of fibrotic tissue (Compare Figure 10 and Figure 11). An interesting fact is that the new collagen formation is deposited from a depth of 0.6 mm upwards and towards the basal membrane, in most cases when needles

with a length of 1.5 mm are used.8 Skin improvement is evident 3–4 weeks after a microneedle session.9 However, collagen maturation needs time, especially to transform into the more elastic collagen type I. Former atrophic scars show a relatively early improvement that is evident around 2–3 weeks post-needling. As mentioned earlier, the degradation of hypertrophic scars, especially

burn scars, may need many months for a visible improvement. Permanent or lasting find more erythema after thermal exposure responds very well to microneedling. It is assumed that the contraction capabilities of the burned vessel proteins are damaged by heat exposure. MMPs degrade the perforated endothelial cells and stimulate angiogenesis for new capillaries. We would like to emphasize that in contrast to ablative procedures, post-op infections after microneedling are very unlikely due to the rapid closure of the SC within a maximum of 15 minutes. Bal et al10 have not reported any negative side effects in BKM120 manufacturer their reports. Microneedling

is a fascinating and intriguing new procedure for skin improvement based on induced cell proliferation by electrical signals. We speculate that reduction of hyperpigmentation may be influenced by expression of MMPs, however, research is needed to verify the mechanism(s) involved. Very good results have been obtained after microneedling of flourishing acne. Acne is triggered by androgens that stimulate increased proliferation of keratinocytes that block the ducts of sebaceous glands. After one or two treatments the hyper proliferation of keratinocytes may be down-regulated. Thus it can only be speculated that MMPs, induced by microneedles, somehow balance or equilibrate cell proliferation. “
“A major effect of climate change is a present and continuing increase in sea level, caused mainly by thermal expansion of seawater and the addition of water to the oceans Interleukin-3 receptor from melted land ice (e.g. Meehl et al., 2007, as reported in the Fourth Assessment Report (AR4) of the Intergovernmental Panel on Climate Change (IPCC)). Over the last two decades, the rate of global-average sea-level rise was about 3.2 mm yr−1 (Church and White, 2011). At the time of AR4 in 2007, sea level was projected to rise at a maximum rate of about 10 mm yr−1 and to a maximum level of about 0.8 m (relative to 1990) by the last decade of the 21st century, in the absence of significant mitigation of greenhouse-gas emissions (Meehl et al., 2007, Table 10.

The concept of knee and hip OA as different diseases is supported

The concept of knee and hip OA as different diseases is supported C59 wnt clinical trial by the fact that hip OA appears to be more heritable than knee OA [18], and genetic studies indicate little genetic correlation between the two disorders [19]. The role of specific risk factors for OA at these two joint

sites is also thought to differ; for example, the relationship between obesity and OA is reported to be stronger at the knee compared with the hip [15], [20] and [21], and knee OA is more prevalent in females than males [14]. We therefore wished to establish whether any relationship between HBM and OA of the knee is similar to that previously observed at the hip. The aim of this study was to investigate radiographic knee OA in our HBM population, determining i) whether HBM is associated with an increased prevalence of radiographic knee OA, ii) the phenotype of knee OA in HBM compared with controls in terms of individual

radiographic features, and iii) the role of potential mediators such as BMI. We hypothesized that, in line with Nivolumab our previous findings and evidence from general population studies, HBM would be associated with a bone-forming phenotype of radiographic knee OA. HBM cases were recruited as part of the UK-based HBM study, a multi-centre observational study of adults with unexplained HBM. Index cases were initially identified by screening DXA databases for T and/or Z-scores ≥ + 4. All DXA images were inspected by trained clinicians in order to exclude scans with artefactual elevation of DXA BMD, resulting in 49.4% of scans being excluded due to degenerative disease/osteoarthritis/scoliosis, and a further 15.5% for other reasons including surgical/malignant/Pagetic artefacts etc.

Then, in order to identify generalised HBM, the HBM index case definition was refined to either a) L1 Z-score ≥ + 3.2 plus total hip Z-score ≥ + 1.2 or b) total hip Z-score ≥ + 3.2 plus L1 Z-score ≥ + 1.2. A + 3.2 threshold was consistent with the only published precedent for identifying HBM using DXA [22]. L1 Z-score was used to avoid misclassifying individuals with lower lumbar OA as having HBM [9] and [23]. Z rather than T-score limited age bias. Further HBM cases were identified through DXA assessment of the relatives and spouses Pomalidomide of index cases. In first-degree relatives, HBM was defined as a summed L1 Z-score plus total hip Z-score ≥ + 3.2. 41% of relatives screened were affected and combined with HBM index cases, with remaining unaffected first-degree relatives/spouses forming a family control group. Full details of this DXA database screening and recruitment have been previously reported [9]. Assessments, including a structured interview and clinical examination, were identical in both HBM cases and controls, and AP weight-bearing knee X-rays were performed in all participants according to local protocols at each centre.

Second, we evaluated the difference

in other gastrointest

Second, we evaluated the difference

in other gastrointestinal and constitutional toxicity observed between treatments when dogs were fed and fasted. No significant difference between the incidence and scores of appetite, diarrhea, or activity was evident between treatments when first dose or paired data were analyzed (Table 2). Similarly, no differences in the incidence or scores of neutropenia or thrombocytopenia were detected (Table 2). Lastly, there was no significant difference anti-PD-1 antibody in IGF-1 concentration between when dogs were fed or fasted before treatment (Table 2 and Figure 2). To the authors’ knowledge, this study is the first randomized prospective clinical evaluation of the effects of Alectinib fasting on the incidence of CINV in cancer-bearing patients. Here, we reported our findings assessing the impact of fasting for 18 hours before and 6 hours after doxorubicin chemotherapy in cancer-bearing dogs. Our data suggest that fasting for 24 hours significantly reduces the incidence of vomiting in dogs treated with doxorubicin but did not appear to affect nausea or other potential adverse effects commonly seen in doxorubicin-treated cancer-bearing dogs. The effect of fasting on the modulation of digestive tract cellular proliferation has long been known [10] and [11]. Theoretically, by blocking

gastrointestinal cells in the G1

phase with fasting, these cells should be less sensitive to the effects of doxorubicin, which is preferentially toxic to cells in the S phase [8] and [9]. In addition to the effects of fasting on the cell cycle, it also appears that protection is elicited in part by other mechanisms that likely alter gene expression [18]. In one study, protection of mice from doxorubicin toxicity by fasting before treatment appeared to be mediated by a reduction in circulating IGF-1 levels such that administration of IGF-1 abolished the protective effect of fasting [18]. Furthermore, mouse embryonic fibroblasts grown to confluence in vitro and then treated with Org 27569 doxorubicin were found to be protected from cell killing by IGF-1 receptor deletion compared to cells that overexpressed IGF-1 receptor [18]. In this case, the proliferation rate was kept relatively constant by the confluence of the cells in culture and therefore cytoprotection appeared to be independent of the cell cycle. Supporting the notion that fasting before chemotherapy might result in reduced clinical toxicity are several studies in mice illustrating that cellular stress resistance is elicited by fasting [13] and [18]. In one report, etoposide administered at 80 mg/kg killed 43% of control mice compared to 6% of mice that were fasted for 48 hours before administration [13].

This difference was significant between intention ET and postural

This difference was significant between intention ET and postural ET groups (Kruskall–Wallis test H=2.84, P<0.05) but not between cerebellar

tremor and either essential tremor group (P>0.05). In combination with the analysis of coherence, these results demonstrate that postural ET is Inhibitor Library cost different from intention ET at the level of spike×EMG interaction. The results show that the physiology of postural ET is different from that of cerebellar tremor, as demonstrated in 7/9 physiological variables which were significantly different from cerebellar tremor. These 7 variables included: peak frequency in the tremor frequency range, firing rates (in Vim and Vop separately), incidence of sensory cells, firing rates of sensory cells, SNR, and SP600125 order coherence (in Vim only). Vop coherence, and phase lead were not different. Intention ET was not different from cerebellar tremor in any of these variables. Based on these results postural ET had more physiological differences from cerebellar tremor than intention ET had from cerebellar tremor (7/9 vs. 0/9, P<0.05, Fisher). These results suggest that postural ET is different from cerebellar tremor while intention ET is not. Postural ET had similar numbers of physiological differences from cerebellar

tremor and from intention ET (7/9 vs. 5/9, P=1, Fisher), which demonstrates again that intention ET is not apparently different from cerebellar tremor. We next examined the result of a cerebellar lesion in a patient with intention ET since the lesion should increase tremor due to a cerebellar disruption but decrease tremor due to a pacemaker in the cerebellum and related structures. Patient 4 (Table 1) with intention ET began to have tremor in the right upper extremity which then spread to the left. The patient had no family history of tremor and did not know the effect of alcohol upon the tremor. Propranolol and primidone have been tried without benefit. Tremor was demonstrated with posture and intention, both graded at 4/4 bilaterally on the Fahn clinical rating scale (Fahn et al., 1988). There

was no head tremor and the remainder of the neurological examination was within normal limits. The preoperative Tolmetin MRI scan was within normal limits. Fifteen years after the onset of tremor, the patient underwent an uncomplicated left thalamotomy which resulted in a small lesion, as shown in Fig. 1A (Lenz et al., 1994b). At follow-up 2 months later, the patient had a substantial improvement in activities of daily living, with a tremor rating of 1/4 in the right upper extremity with posture only. Twenty years after the onset of tremor, the patient had a total knee replacement and one week thereafter developed an acute onset of true vertigo and imbalance leading to falls, but no other symptoms or signs. These symptoms resolved and physical therapy was completed as planned.

I felt both excited and nervous at the prospect What was most am

I felt both excited and nervous at the prospect. What was most amazing to me was his incredible humility; he was known to physiotherapists from all around the world, yet had time for me. I remember watching him examine a patient with foot pain and he spent ages hunting around to try and reproduce this guys pain; I can’t quite

remember whether he ever did manage to, what struck me was the enormous effort and dedication in trying to help him. After this visit I returned to the UK and kept in contact by phone and at various conferences here and in Australia. At an IFOMT conference in Cambridge he publically requested that therapists stop using the term ‘Maitland mobilisations’, saying that mobilisations are mobilisations and are not related to a person. After writing the foreword to a book http://www.selleckchem.com/products/Dapagliflozin.html on examination and assessment, he said that the sooner he died and let things move on, the better. He felt he was somehow holding things back. Again his humility astonished me. While our paths crossed infrequently, Geoff left a lasting impression on me that I will always treasure. God bless you Geoff. “
“Figure options Download full-size image Download high-quality image (39 K) Download as PowerPoint slide Geoff Maitland passed away peacefully on Friday 22 January 2010 almost one year after the death selleck products of his dear wife Anne. It is, therefore, a poignant time for the whole of the Physiotherapy World

to stop and reflect upon the achievements and legacy of a man who has done as much as anyone to shape and define the Physiotherapy profession as it is today. Geoff and Anne were inseparable. Both of them possessed an unshakable

Christian faith and a strong Duty of Care. Anne, invariably, would be present at his lectures, seminars and workshops. She would give him honest feedback Mannose-binding protein-associated serine protease on his performance and tell him how he could improve. He would add to this with his own self-criticism. From the outset, they developed a robust internal moderation system to ensure quality control and quality assurance of his work. A quote by Dr D.A. Brewerton in the foreword to Maitland’s 1st edition of Peripheral Manipulation [1970] sums up Geoff Maitland’s approach to his work as a Physiotherapist. “Geoffrey Maitland is well aware of the limitations of our knowledge and he is always modest in describing his results. Undoubtedly he is putting forward his own views with humility, hoping to promote discussion so that others can improve on his own suggestions. Geoff was a great listener and a great communicator. He placed a great emphasis on the art and skill of listening [as opposed to just hearing]. He would hang on every word his patients would say so that he did not miss the subtle hints from the language or its tone that would help him understand, in depth, what the individual was experiencing. He would use every facet of “the bodies capacity to inform” both verbal and non-verbal.

The sustained ability of practices to “offer more” by incorporati

The sustained ability of practices to “offer more” by incorporating aspects associated with DMPs into regular practice and by expanding activities beyond the care setting and into the community is important

in this regard as is the focus on patient-led communication. The study has several limitations. First and most importantly, this study did not include control groups corresponding to all the different patient groups. Although we found that physical quality of life declined over the 1-year period, we do not know whether this reduction Ion Channel Ligand Library mw was smaller compared with chronically ill patients not enrolled in DMPs. Worsening of the disease, poor medication adherence or an unhealthy diet may also explain declines

in quality of life. Ku-0059436 Future research should investigate the role of other health behaviors. Secondly, we included only patients’ and project managers’ reported perceptions, and did not report the effects of DMP implementation on patients’ objective health outcomes. Thirdly, respondents who completed questionnaires at T0 and T1 were on average older and more physically active than were those who completed only one questionnaire, which may have resulted in non-response bias. Physical activity may also be higher compared to patients not responding at all, which limits generalizability of our study findings. Finally, non-response bias at T0 may have affected our findings. We did however test the final full tetracosactide model on imputed data which showed similar results. DMPs based on the CCM appear to improve

physical activity among chronically ill patients over time. Furthermore, this research showed that smoking and (changes in) physical activity were important for the physical quality of life of these patients. To improve health behavior among chronically ill patients healthcare providers are advised to: • Focus on supporting patients to make healthier lifestyle choices by listening to the needs and desires of patients, for example through motivational interviewing or regular meetings with dieticians and specialized nurses; This research was supported by a grant provided by the Netherlands Organization for Health Research and Development (ZonMw, project no. 300030201). The views expressed in the paper are those of the authors. The authors declare that they have no competing interests and confirm all patient/personal identifiers have been removed or disguised so the patient/person(s) described are not identifiable and cannot be identified through the details of the story. The authors are thankful to all healthcare workers, patients and project managers that participated in the research.

All patients with post-operative hypertension, i e blood pressur

All patients with post-operative hypertension, i.e. blood pressure (BP) >160 mmHg systolic (absolute), >20% above the pre-operative

BP, or BP risen above the individual restriction in patients with an intra-operative Vmean increase >100%, underwent strict individualized BP control during the early post-operative period with intravenous labetalol (first choice) or clonidine (second choice). CHS was diagnosed if the patient developed headache, confusion, seizures, intracranial hemorrhage or focal neurological deficits in the presence of post-operative cerebral hyperperfusion (defined as >100% increase of the pre-operative Vmean) after a symptom-free interval. Of the 560 patients undergoing CEA during the time of the study, 72 (13%) received both intra- and post-operative TCD monitoring and were included for the present analysis. See Table 1 for patient characteristics. The majority of patients were symptomatic (86%). About a third of the

PR-171 molecular weight patients required the use of an intra-luminal shunt because of either EEG asymmetry or a decrease of >60% of Vmean measured by TCD. Twelve patients (17%) had an intra-operative Vmean increase >100%. Post-operatively, Vmean increase >100% was found in the 13 patients (18%). During all TCD measurements no significant selleck screening library increase in BP was found after declamping compared to the pre-clamping systolic BP or when the post-operative measurement was compared to the pre-operative systolic BP. Of all 72 patients, 19 patients (26%) developed post-operative hypertension and 5 patients (7%) suffered from CHS. All patients with CHS had hypertension during the post-operative phase. The

overall 30-day rate of death/stroke was 1%. Of 12 patients with an intra-operative increase of Vmean > 100%, 2 patients developed CHS. On the other hand, in 60 patients who had an intra-operative increase less than 100%, 3 patients suffered from CHS. This results in a PPV of 17% (2/12) and NPV of 95% (57/60) in the prediction of CHS ( Table 2). With respect to the post-operative TCD measurements 5 of the 13 patients with at least a doubling of post-operative Vmean 17-DMAG (Alvespimycin) HCl developed CHS. In the subgroup of 59 patients with a post-operative increase of less than 100% CHS did not occur. This results in a PPV of 38% (5/13) and a NPV of 100% (59/59) for the development of CHS. In the present retrospective study, as previously published, an increase in Vmean measured with post-operative TCD is superior in predicting the development of CHS to the commonly used increase in Vmean measured three minutes after declamping versus pre-clamping value [12]. The PPV of the post-operative measurement in the prediction of CHS is more than two times higher than the PPV of the intra-operative measurement (38% and 17% respectively). Moreover, absence of doubling of the Vmean at the post-operative measurement completely excluded the development of CHS (NPV 95% vs. 100% for the intra-operative and post-operative measurements, respectively).

6 and 7) As showed in Table 2, the immunizer and potential donor

6 and 7). As showed in Table 2, the immunizer and potential donors share the same beta subunit in the HLA DQ molecule (DQB1*03:01), however combined to different alpha subunits. Such beta subunit presents an

only potentially immunogenic eplet: 45EV. Nevertheless, as the MFI value of the HLA heterodimer DQA1*02:01–DQB1*03:01 of the potential donors is 921, the immunological risk is low for class II HLA. Contrariwise, we were able to detect a strong reactivity against A*68:02, representing a high immunological risk for antibody-mediated rejection. As there is no agreement upon current CDC assay with the flow cytometry crossmatch and SPA results, we believe that the recipient has a mixture of antibodies with a prevalence of non-fixing complement isotypes, Trichostatin A or the titles of the fixing complement antibodies present in the current serum were not enough to activate the classic pathway of the complement system. Thus, the potential donors’ allele A*68:02 is considered one of the unacceptable mismatches for this recipient. As the calculated PRA was 91%, this case exemplifies the importance of using the Acceptable Mismatch approach. The implementation of the EpHLA program this website will allow a simple and automated analysis

of antibody data using the HLAMatchmaker algorithm and prevent the many laborious manual steps used in the current analyses. Based on the HLA types of the recipient/donor pair and the SPA result, it is possible to generate reports automatically which will support the transplantation team to define the risk of developing antibody-mediated rejection. The automated analysis is important to increase the efficiency in generating results with at least the same degree of accuracy [19]. Automation will certainly decrease the incidence of administrative errors and facilitate the information management within the organization [20]. In conclusion, the EpHLA program integrates SPA results and the HLAMatchmaker algorithm in

selleck inhibitor an automated histocompatibility analysis. The program will certainly benefit the donor selection and risk assessment for HLA sensitized recipients. The work was supported by the Immunogenetics and Molecular Biology Laboratory from UFPI. Thanks to CNPq for the scholarship granted to Herton Luiz Alves Sales Filho. The authors also acknowledge João Batista de Oliveira Silva Jr. for his corrections of the English version in preparation of the manuscript. “
“Since the introduction of potent immunosuppressants such as calcineurin inhibitors and improved immunological matching, the risk of acute transplant rejection has been reduced considerably. However, despite a wide range of immunosuppressive agents, severe episodes of rejection still occur and chronic allograft rejection still poses a significant problem [1] and [2].