p; 50 mg/kg; Cristalia, Brazil). Heparin (1000 IU; Cristalia, Brazil) was injected into the left cardiac ventricle, then the animals were transcardially perfused through the left ventricle using a peristaltic pump (Control Company, Brazil, 20 mL/min) with 400 mL of 0.9% saline solution, followed by 400 mL of a fixative solution 4% GDC-0449 purchase paraformaldehyde (Synth, Brazil) in 0.1 M phosphate buffer, pH 7.4 (PB). The brains were removed from the skulls, post-fixed in the same solution at room temperature for 4 h and cryoprotected by immersion in a 15% and 30% sucrose (Synth, Brazil) solution in PB at 4 °C until they sank. After these procedures, the brains were quickly frozen in isopentane (Merck, Germany) cooled in liquid nitrogen and kept in a

freezer (−70 °C) for further analyses. Coronal sections (50 μm) from VTA and SNpc were obtained from each brain using a cryostat (CM1850, Leica, Germany) at −20 °C and collected in a PB saline (PBS), pH 7.4. These areas were identified using Paxinos and Watson’s Atlas (1998). The free-floating sections were pre-treated with 3% hydrogen peroxide for

30 min, carefully washed and treated with 2% bovine serum albumin (Inlab, Brazil) in PBS containing 0.4% Triton X-100 (PBS-Tx) for 30 min and incubated with monoclonal TH antibody (Sigma Chemical Co., USA) raised in mice, diluted 1:2000 in PBS-Tx for 48 h at 4 °C. Sections were again washed in PBS-Tx and incubated in an anti-mouse antibody conjugated with peroxidase (Sigma Chemical Co., Selleck AZD6244 USA) diluted 1:200 in PBS-Tx for 2 h at room temperature. The reaction was revealed in a medium containing 0.06% 3,3′-diaminobenzidine (DAB, Sigma Chemical Co., USA) dissolved in PBS for 10 min and

then 1 μL of 3% H2O2/mL was added to the DAB medium for an additional 10 min. Finally, the sections were rinsed in PBS, dehydrated in ethanol, cleared with xylene and covered with Entellan (Merck, Germany) and coverslips. Control sections were prepared omitting the primary antibody by replacing it with PBS. Semi-quantitative densitometric analysis was used to measure the intensity of the TH immunoreaction using a Nikon Optiphot-2 microscope (40×, Japan) coupled to a Micrometrics camera (Accu Scope, only USA) and Image Pro Plus Software 6.0 (Media Cybernetics, USA). The digitized images obtained from the selected areas were converted to an 8-bit gray scale (0–255 gray levels). All lighting conditions and magnifications were held constant. Picture elements (pixels) employed to measure optical density were obtained from squares measuring 9680 μm2 (area of interest, AOI) overlaid on the gray scale image. Both left and right sides of each brain were used. For each rat, 10 measures were taken from the VTA and 10 measures each from the medial, lateral and intermediary regions of the SNpc. The results shown for the SNpc were the total mean value from the three studied regions. Background staining subtraction and correction were done in accordance with our previous published protocol (Xavier et al., 2005).

Protein kinases (EC 2.7.10/EC 2.7.11) which phosphorylate the hydroxyl group present on serine, threonine, or tyrosine residues represent an enzyme family for which many assays have been developed due to their central role in controlling signaling pathways (Glickman et al., 2004). Measurement of substrate depletion by detecting the remaining ATP in a kinase assay using firefly luciferase (EC 1.13.12.7) is an example

of a generic assay format for protein kinases (Koresawa and Okabe, 2004 and Singh et al., 2004). The use of a bioluminescent signal for ATP levels results in an increase in luminescence when the kinase is inhibited. Drawbacks of the see more ATP depletion method include the need to run the assay at high substrate turnover which often requires larger amounts of enzyme, the assay is performed using check details single endpoint, and the assay requires the presence of a luciferase coupling enzyme. This latter point requires that appropriate counter-screens against luciferase alone are performed (Auld et al., 2008). Additionally, as the system measures substrate depletion, the standard steady-state assumption that So~St does not apply, thus confounding MoI studies. The assay is best performed using 50% conversion of substrate where the signal:background ratio is ~2-fold, a range often yielding acceptable assay performance for luminescent assays, and the expected shift in IC50 from ideal initial rate conditions is expected to be <2-fold

( Wu et al., 2003). A more desirable method to measure enzyme activity is by detecting product formation. Recently, generic methods for kinase assays have been developed that detect the ADP product. These include both a non-antibody based system that employs coupling enzymes with a fluorescent readout (DiscoveRx, ADP Quest™; λex=530 nm, λem=590 nm) ( Charter et al., 2006) and a system that uses an ADP specific antibody and red-shifted FP by employing an Alexa® 633-labeled ADP (BellBrooks Lab, Transcreener™; λex=612 nm,

λem=670 nm) ( Huss et al., 2007 and Kleman-Leyer et al., 2009). The red-shifted fluorescence limits fluorescent Cyclin-dependent kinase 3 interference by compounds and the ratiometric nature of FP aides in minimizing artifacts due to liquid handling. The BellBrook system has also been adapted to a TR-FRET format employing terbium labeled ADP-antibody and fluorescein labeled ADP ( Klink et al., 2008). The TR-FRET format of this assay limits interference by faster decaying background fluorescence due to compounds or buffer components ( Comley, 2006). Indeed, with any fluorescent-based assay, a consideration of interference by fluorescent compounds ( Simeonov et al., 2008) or by the inner-filter effect ( Palmier and Van Doren, 2007) needs to be considered. Low-molecular weight (LMW) compounds present in typical chemical libraries can show a good-deal of blue fluorescent, therefore using red-shifted fluorophores for detection can reduce interference by compound fluorescence ( Simeonov et al., 2008).

All of these processes interact in a complex way. Nonetheless, in experimentally well controlled tasks, some of these variables can be varied, whereas others can be kept constant. The experimental

variation of attentional processes is a typical characteristic of tasks that are used to investigate the P1. Spatial cuing paradigms are a good example. According to our hypotheses, two different processes, T and S are of primary importance in this type of tasks. In type 1 tasks, T is experimentally manipulated by instructing subjects to attend to the left or right hemifield. In type 2 tasks, T is varied by the cue and its validity. T establishes a top–down control process that operates to increase SNR in task relevant networks. In contrast, S is a process that blocks information Nintedanib concentration processing in interfering networks. Thus, attentional benefits – associated with the influence of T – and attentional costs – associated with the influence of S – are both due to an increase in inhibition which leads to an increase in P1 amplitude. The difference between T and S is seen in different inhibitory processes that operate in task relevant vs. interfering networks (cf. Fig. 5A). Attentional processes are not the only class of cognitive processes that affect the P1 component. Processing complexity (C) during early stimulus Z-VAD-FMK categorization is another important

cognitive process that shapes the P1. As an example, orthographic neighborhood size (N), and word length may be considered variables that directly affect C. A pop-out color target search may be considered an example affecting D, the focused Rucaparib search for a complex target lacking pop-out features may be considered an example affecting primarily T, whereas the processing of a distractor item may be considered an example for S. In this section we apply the proposed theory particularly to those findings which are difficult to interpret in terms of stimulus evoked activity

or on the basis of an enhancement hypothesis. An overview over the findings reviewed in Section 2 and their interpretation on the basis of the P1 inhibition timing hypothesis are presented in Fig. 5B. The central prediction of the proposed theory rests on inhibition and on the idea that suppression of task irrelevant and potentially competing information and or neural structures leads to a particularly large increase in the P1 amplitude. Under controlled conditions this suppression related increase will be at least as large or larger than for task relevant processes where inhibition is used to increase the SNR. As a first example let us consider the finding of a large ipsilateral P1 amplitude. We assume that the increased ipsilateral P1 reflects inhibition of task irrelevant and potentially competing processes.

, 2012a, Brodie and Waterhouse, 2012 and Lewis et al., 2009). Satellite imagery effectively captures these events and their associated flood plumes migrating up to 50 km offshore as far as the midshelf coral reefs (Bainbridge et al., 2012). A wide spectrum of pesticides this website have been detected in waters of the GBR, but herbicides are often more water soluble and mobile than contemporary insecticides and fungicides, and as a consequence, are more frequently detected in the river mouths and GBR lagoon (Brodie et al., 2012b, Davis et al., 2011 and Lewis et al., 2009). The photosystem II herbicides have

been the primary group detected in GBR waters; however, glyphosate (CAS number 1071-83-6) is the most widely used herbicide in Australia, in the GBR catchments and elsewhere, with approximately 15,000 tonnes applied annually to control agricultural, urban and roadside weeds (Beeton et al., 2006 and Radcliffe, 2002). The popularity of glyphosate has increased steadily since its introduction in the mid 1970s as it exhibits: (i) relatively low toxicity to non-target organisms (Borggaard and Gimsing, 2008 and Duke and Powles, 2008); (ii) apparent rapid microbial

degradation to a major metabolite aminophosphonic selleck kinase inhibitor acid (AMPA) (Giesy et al., 2000) and (iii) strong adsorption to soils and sediments potentially limiting runoff in surface water (Duke and Powles, 2008, Pérez et al., 2012 and Solomon and Thompson, 2003). Glyphosate has not often been included in regular monitoring programs as the stand-alone analytical methods are often cost-prohibitive, resulting in a long term deficiency in global datasets (Barceló and Hennion, 2003). However, glyphosate has been regularly detected in a diversity of waterbodies when samples were analysed (see Table 1). For example, glyphosate and AMPA were detected in 36% and 69% of water samples respectively, following extensive sampling of aquatic ecosystems

in the Midwestern United States (Battaglin et al., 2005 and Scribner et al., 2003). Concentrations measured in field studies in Australia have Cyclin-dependent kinase 3 been reported as high as 54 μg L−1 (Davis et al., 2011). A similar concentration (40.8 μg L−1) was measured in Canada (Struger et al., 2008), while field dissipation studies found concentrations as high as 1700 μg L−1 (Mensink and Janssen, 1994 and NHMRC, 2011). Glyphosate exhibits a relatively low toxicity to non-target marine organisms, with the LC50s of glyphosate (lethal concentration which affects half of the sample population) in the 10–1000 mg L−1 range. However, recent research suggests that low μg L−1 concentrations can affect natural coastal microbial communities (Stachowski-Haberkorn et al., 2008).

Neuroimaging is typically limited to patients with recent falls or head trauma, use of anticoagulation, focal neurologic signs, or fever without other explanation.3 The prescribing practitioner may use antipsychotics at the lowest effective dose for the shortest possible duration to treat patients who are severely agitated or distressed, and are threatening substantial harm to self and/or others. In all cases, treatment with antipsychotics should be employed only if behavioral interventions have failed

or are not possible, and ongoing use should be evaluated daily with in-person examination of patients. The evidence for pharmacologic treatment of postoperative PI3K inhibitor delirium with antipsychotic medications is difficult to interpret because of the heterogeneity in the drugs studied, dosages administered, patient populations, and outcomes examined.87, 88 and 89 The potential benefit of antipsychotics is decreased STA-9090 delirium severity, although results of clinical trials are not consistent. The potential harms associated with antipsychotic medication

are numerous.62, 63, 64, 65, 66 and 90There is no evidence of benefit from treatment of antipsychotics in patients without agitation. The use of antipsychotics should be reserved for short-term management of acute agitation in the setting of possible substantial harm, ie, for treatment of postoperative delirium in older surgical patients with behavior such as agitation that substantially threatens the patient’s safety or the safety of others. No current evidence

supports the routine use of Dimethyl sulfoxide benzodiazepines in the treatment of delirium. There is substantial evidence that benzodiazepines promote delirium.91 However, benzodiazepines remain the recommended treatment of alcohol withdrawal.92 Developing a set of national guidelines for postoperative delirium care is the first step in the translational discovery to delivery cycle. This translational cycle is considered inefficient and expensive.93, 94 and 95 New, emerging “implementation science” may help in speeding the translational cycle by understanding the barriers and facilitators of implementing evidence-based knowledge such as the current guideline on postoperative delirium care into the real world of health care practice. Thus, it is important to translate the current guideline set into locally sensitive implementation tools that can be easily adapted by local quality improvement offices within each health care system. Successful postoperative management of delirium for older adults requires knowledge of approaches for screening, diagnosis, risk factor assessment, and nonpharmacologic and pharmacologic interventions aimed to prevent and treat delirium. The recommendation statements within provide a framework to allow hospital systems and health care professionals to implement actionable, evidence-based measures to address the highly morbid problem of delirium in perioperative patients.

In a recent study, the widths of lateral ventricles (frontal horns) were monitored with TCS in 37 patients with intraventricular hemorrhage [46]. The authors reported a cut-off value for increase of lateral ventricular width of 5.5 mm that yielded high sensitivity (100%) and specificity (83%) in combination with a 100% negative predictive value for reopening of the external ventricular

or lumbar drainage. In conclusion, TCS can be regarded as a reliable tool for monitoring the midline shift, as well as the ventricular width in patients with acute supratentorial brain lesions who have adequate acoustic bone windows (>80% of patients). In many neurological and neurosurgical departments with appropriate expertise in neurosonology, TCS is already today routinely used for Etoposide clinical trial this purpose. Becker et al. [47] were the first to describe the TCS finding of SN hyperechogenicity in PD patients (Fig. 2). In the past decade, this finding has been confirmed by a number of independent groups MG-132 ic50 [23], [24], [25], [27], [28],

[48], [49], [50], [51], [52], [53] and [54]. This TCS finding, present in about 90% of PD patients at cross-section is independent from PD duration and severity [55] and [56], and was found to be stable in a 5-year follow-up study of PD patients [57]. Also there was no correlation found between the degree of SN hyperechogenicity and the striatal uptake of N-omega-fluoropropyl-2beta-carbomethoxy-3beta-4-[(123)I]iodophenyl-nortropane (FP-CIT) on SPECT, which is thought to represent a correlate for the degeneration of presynaptic dopaminergic neurons in PD [58]. These

findings indicate that SN hyperechogenicity is not a correlate of the progressive degeneration of SN neurons. However, a close correlation between SN echogenicity Megestrol Acetate and tissue iron content has been shown in post-mortem studies of human brains [59], suggesting that SN hyperechogenicity in PD is at least in part, caused by an elevated iron content of the SN. Also in a number of other neurodegenerative disorders TCS was demonstrated to detect accumulation of trace metals (iron, copper, manganese) in the basal ganglia with higher sensitivity than MRI supporting the idea that TCS can display trace metal accumulation in deep brain structures [59], [60], [61] and [62]. On the other hand, increased iron content alone cannot be the only explanation for SN hyperechogenicity since iron accumulates over time in the SN of PD patients, and other iron-rich brain structures, such as red nucleus or globus pallidus internus normally show no increased echogenicity on TCS [2]. Therefore, additional factors, such as abnormal iron–protein bindings were proposed to contribute to SN hyperechogenicity [59].

, 2010) and a common R2* for all peaks were used in the modeling. The R2* parameter can be thought of as the peak width in frequency domain and can be used to detect liver iron deposition (positive correlation). In the present study, the R2* parameter was used as an additional Protein Tyrosine Kinase inhibitor biomarker of liver status. The liver fat content and R2* from the entire liver was analyzed by manual identification of the volume of interest and by fitting of a Gaussian function to the liver fat fraction and R2* histograms (see Fig. 1f and g). The center of the Gaussian function was used to sample robust estimates of liver fat content and

R2*. At termination blood was collected from the abdominal aorta in EDTA-treated tubes (Greiner bio-one, Frickenhausen, Germany) and centrifuged for 10 min to prepare plasma. Aliquotes were stored at −70 °C pending

biochemical analyses of the following circulating markers: triglycerides, cholesterol, and apolipoprotein A-I (apo A-I). The liver and the left perirenal fat pad (see Fig. 2) were dissected and weighed. The liver weight was used click here to calculate the liver somatic index (LSI, liver weight × 100/body weight). The analysis of cholesterol and triglycerides was a standard laboratory technique and was performed on an Architect C 8000 analyzer (Abbott Laboratories, Abbott Park, IL, USA) and reported using SI units. Analysis of protein apo A-I: Prior to western blot 1 μl of plasma from rats of all groups (W; n = 12, F; n = 12, BPA 0.025 mg/L; n = 11, BPA 0.25 mg/L; n = 8 and BPA 2.5 mg/L; n = 9) were separated on SDS-polyacrylamide gradient gels (T = 5–20%,

C = 1.5%) with stacking gels (T = 5%, C = 1.5%) for 1 h (180 V, 60 mA) in electrode buffer (0.15% (w/v) Tris, Succinyl-CoA 0.72% (w/v) glycine, 0.05% (w/v) SDS) using a Mini Protean II electrophoresis cell (Bio Rad). Samples were diluted in sample cocktail (4% (w/v) SDS, 200 mM DTT, 20% (w/v) sucrose) and boiled for 3 min. Plasma proteins separated by SDS PAGE were transferred to a PVDF membrane. After blocking 1 h (5% milk in TBS) and incubation over night with primary antibodies 1:1000 (2% milk in TTBS) against apo A-I (rabbit anti rat apoA-I, polyclonal, Ab 20453, Abcam, UK), the membrane was incubated for 1 h with goat anti-rabbit HRP-conjugated secondary antibodies 1:40 000 (2% milk in TTBS). Proteins were visualized using an ECL plus western blotting detection system. Gel images were evaluated using Image Lab 3.0.1 (Bio Rad, Hercules, CA) and apo A-I levels were determined as intensity/mm2. Differences between the fructose control group and the three BPA plus fructose exposed groups were evaluated by factorial ANOVA. When the three BPA groups were analyzed vs the fructose control group one by one, a Bonferroni adjustment for 3 tests was used and p < 0.0167 considered significant (p = 0.05/3 = 0.0167). In the secondary analysis, when the water control group was compared with the fructose control group p < 0.05 was considered as significant.

As all cell lines respond to NVP-AUY922, the increase in Hsp70 is very significant and occurs rapidly. In the HCUVA-CC-34 primary culture however, EGFR depletion, ERK1/2 phosphorylation, and Hsp70

up-regulation are not very dramatic, which explain the moderate effects of this drug in anchorage-dependent and anchorage-independent growth assays. Experiments are Ivacaftor solubility dmso underway to try to identify a possible mechanism of resistance of HCUVA-CC-34 and other colorectal cellular models to NVP-AUY922. Since all our cellular models, apart from the exception just mentioned, were sensitive to NVP-AUY922, we sought to find markers of sensitivity/resistance to 17-AAG. In fact, phospho-kinase arrays were performed in 17-AAG–sensitive as well as in 17-AAG–resistant cell lines with the intention to find putative markers. However, we could not clearly associate differences found between cell lines to resistance to this drug. As it has been suggested that ABC transporters may play a role in resistance to Hsp90 inhibitors, we analyzed Mdr-1, MRP1, and BRCP1 protein levels

in these cell lines and found that none of the 17-AAG–resistant pancreatic and colorectal carcinoma cell lines expressed these transporters, Dabrafenib with the exception of Caco-2 cells that express very low levels of BRCP1. However, many of the 17-AAG–sensitive cell lines express some of these ABC transporters (Figure 7). Therefore, we can rule out the role of these ABC transporters

in 17-AAG resistance. In addition to Pgp (Mdr-1), it has been suggested in several reports that NQO1/DT-diaphorase is necessary for benzoquinone ansamycin function. This enzyme is able to metabolize quinones to the corresponding hydroquinones, which are more stable and bind Hsp90 with greater affinity. We have found that the 17-AAG–resistant pancreatic carcinoma PANC-1 and CFPAC-1 cells lack NQO1 protein and activity (Figure 8), confirming the results previously reported by Siegel et al. [39]. The 17-AAG–resistant Caco-2 cells also lack NQO1 protein and enzymatic activity. However, LoVo cells, which are also devoid of NQO1 enzyme (Figure 8), are still responsive to 17-AAG, as demonstrated especially in soft Diflunisal agar assays and cell cycle analyses (Figure 2 and Figure 3). We speculate that other reductases, albeit with less potency, may be able to reduce 17-AAG to 17-AAGH2 in these cells. Another possibility is that although less potent, the nonreduced benzoquinones may also have an activity and be able to exert the same effects as their reduced counterparts at higher concentrations. When we blocked NQO1 activity in 17-AAG–sensitive cell lines with ES936, these cells were still growth inhibited by 17-AAG (Figure 9).

There is no conflict of interest related to the submitted manuscript. This research protocol was reviewed and approved by the Institutional Ethics Committees from University of Taubaté (2008/0098) and Guarulhos University (09/2005). “
“The authors regret that the units of Table 3 were incorrect. It should be as below The authors would like to apologise for any inconvenience caused. “
“Orthodontic tooth movement (OTM) occurs through remodelling of alveolar bone after mechanical stimuli. The orthodontic forces generate and propagate signalling cascades through all paradental selleck tissue cells, triggering important changes in the homeostatic periodontal environment.1 and 2

The orthodontic loading leads to a focal tissue injury and, consequently, an aseptic inflammatory

find more response characterised by the release of several important inflammatory mediators on periodontal tissues,2 and 3 such as the cytokine interleukin-1 (IL-1).4 IL-1 is directly involved in bone resorption by taking part in the survival, fusion and activation of osteoclasts and it exerts its activities by binding to two types of receptors, IL-1-RI and IL-1-RII.5 Whilst the latter has no described signalling properties and acts as a “decoy” target for IL-1, the former develops pro-inflammatory functions, such as cell recruitment and release of other cytokines, which also are involved in bone resorption.6 However, IL-1 functions are physiologically ID-8 controlled by the naturally occurring interleukin-1 receptor antagonist (IL-1Ra), which competitively blocks the interactions of IL-1 with its receptors and inhibits its activity.7 and 8 IL-1Ra has long been studied in clinical and experimental surveys as a physiological and therapeutic target in inflammatory conditions related to bone resorption, such as rheumatoid arthritis9 and 10 and periodontal disease.11 and 12 These studies reported that administration of exogenous IL-1Ra may be a useful strategy to control bone resorption, mainly for its anti-inflammatory properties related to the antagonism of IL-1.9, 10,

11 and 13 However, only a few studies have investigated the effect of IL-1Ra on OTM, showing a positive correlation between decreased IL-1Ra gingival expression and faster OTM in humans.14, 15, 16 and 17 Despite these findings, there is a lack of evidence describing the effects of IL-1Ra therapy on bone remodelling after mechanical loading. Therefore, the aim of this study was to investigate the effects of IL-1Ra administration on OTM in a mouse model. Thirty five ten-week-old wild-type mice (WT) (C57BL6/J) were used in this study. For histomorphometric analysis, 10 mice with orthodontic appliance were used. In this set of experiments, the left side of maxillae (without orthodontic appliance) was used as control.

, 2005), our research did not find a significant association between the

CRP gene and the metabolic syndrome. However, we showed an interaction between CRP rs1205 and affective status on the risk of the metabolic syndrome. Our finding of adolescent emotional problems being associated with elevated risk for the metabolic syndrome only in rs1205 CC homozygotes may be linked to their higher CRP levels. According the study by Halder et al., C allele carriers had a higher mean CRP level than the TT genotype ( Halder et al., 2010). Consistently with this finding, we showed that depressive symptoms were associated with higher risk Z-VAD-FMK clinical trial of the metabolic syndrome only in CC homozygotes, possibly through higher level of inflammation. The same study also reported interaction effect between three-marker haplotype (A–G–T, rs1417938–rs1800947–rs1205) and depressive symptoms on the higher level of CRP ( Halder et al., 2010). Unfortunately, our results are not directly comparable with these findings, since we do not have the information on the two other SNPs. It is possible that this three-marker haplotype, with T allele of rs1205, captures another functional significant variant within CRP gene. Our findings are in line with

the following hypothesis explaining the association Veliparib in vitro between depression and the metabolic syndrome: that depression dysregulates immune system pathways in ways that promote inflammation and through inflammation lead to higher risk of the metabolic syndrome. Recent studies have shown that early life trauma, with or without clinical depression, is associated with clinically significant levels of inflammation in adulthood (Danese

et al., 2007 and Pace et al., 2006). Stress system activation might promote inflammation process through several mechanisms: through activation of the sympathetic nervous system, through vagal withdrawal or through the development of glucocorticoid resistance associated with increased cytokine production (Raison et al., 2006). Thus, HPA axis hyperactivity and autonomic nervous system dysfunction could be one Clomifene plausible mechanism that explains how emotional problems in adolescence affect the metabolic syndrome in adulthood via the inflammation process (Kop and Gottdiener, 2005). In conclusion, we find that adolescent emotional problems are associated with the metabolic syndrome 40 years later, in women but not in men, although this sex difference was not statistically significant. We also show that a CRP polymorphism modifies the association between adolescent affective status and the metabolic syndrome. This suggests that inflammatory system genes could provide a link between depression and the metabolic syndrome but through more complex interactions than simple associations. Funding organisations had no role in design and conduct of the study or in preparation of the manuscript. The authors have no conflict of interests to disclosure.