However, there are numerous molecules that have been categorized as costimulatory based solely on their ability to generate a second signal when ligated with CYC202 datasheet antibodies (Leitner et al., 2010). Recombinant proteins representing the extracellular domains of costimulatory ligands are valuable and widely used tools to study T cell activation processes. However, their generation is time consuming and costly and they might differ from

their membrane resident natural counterparts regarding their capability to modulate T cell responses. We have developed a simple cellular system to assess the role of costimulatory ligands in the activation of human T cells. This system, which we have designated T cell stimulator cells, is based on the murine thymoma cell line Bw5417 that expresses membrane-bound anti-human CD3 single chain antibody fragments at high or low densities. Upon retroviral expression of BGJ398 molecular weight human costimulatory ligands on these cells their contribution to the activation of human T cells can readily be determined. In this study we

describe this system in detail and demonstrate that T cell stimulator cells are an efficient and versatile tool to study various aspects of human T cell costimulatory processes. 293T cells and the mouse thymoma cell line Bw5147 (short designation within this work Bw) were cultured as described (Pfistershammer et al., 2006 and Pfistershammer et al., 2008). The ethical review board of the General Hospital and the Medical University of Vienna approved the human studies performed within this work and informed consent was obtained from the donors. PBMC were isolated from heparinised whole blood of healthy volunteer donors by standard density centrifugation with Ficoll-Paque (Amersham Bioscience, Roosendaal, Netherlands). Human T cells were obtained through depletion of CD11b, CD14, CD16, CD19, CD33 and MHC-class II bearing cells with the respective mAbs by MACS (Miltenyi Biotech,

Bergisch Gladbach, Germany). The mAbs to CD11b (VIM12), CD14 (VIM13), CD33 (4D3), HSP90 MHC-class II (1/47), CD80 (7-480), CD58 (1-456) and the non-binding control antibody VIAP (calf intestine alkaline phosphatase specific) were produced at our institute. The mAbs to CD14 (MEM-18) was purchased from An der Grub (Kaumberg, Austria), CD19 mAb (BU12) from Ancell (Bayport, MN), and 41BB-L and CD150/SLAM (A12) from Biolegend (San Diego, CA). Goat anti-human TL1A/TNFSF15 antibodies were obtained from R&D (Minneapolis, MN). FACS analysis was performed as described previously (Pfistershammer et al., 2006). Briefly, binding of primary antibodies was detected with PE-conjugated goat anti-mouse IgG-Fcγ specific Abs or donkey anti-goat IgG (H + L) (both Jackson ImmunoResearch, West Grove, PA).

26 The effect of exercise or time-control (ie, no exercise) protocols on vascular reactivity was analyzed by means of a repeated-measures ANOVA, followed by the Fisher post hoc test in case selleckchem of significant F values. Vascular reactivity was compared between groups using analysis of covariance (ANCOVA) models, where all subjects’ characteristics were considered as covariates. At first, a model was used to compare the baseline vascular reactivity

between groups. Then, a different model was used to compare groups throughout time (ie, ANCOVA main factors: group [wild vs polymorphic] and time [baseline vs 10 minutes vs 60 minutes vs 120 minutes]). In the ANCOVA of the haplotypes, the haplotype containing only wild-type alleles (H1) was separately compared with each of the haplotypes containing polymorphic alleles (ie, H1 vs H2, H1 vs H3, H1 vs H4).26 Greenhouse–Geisser correction was used to correct P values from ANCOVA main effects due to deviation from the sphericity assumption. In case of significant F values, Cohen’s d effect size was calculated. Results are presented as mean ± standard error of the mean. Statistical significance was considered for P ≤ .05 based on 2-tailed comparisons. All analyses were performed using STATISTICA version 8.0 software

(StatSoft Inc, Tulsa, Okla). The characteristics of the entire sample were as follows: age 32 ± 1 years, BMI 25.0 ± 0.3 m/kg, total cholesterol 176 ± 3 mg/dL, HDL 55 ± 1 mg/dL, LDL 104 ± 2 mg/dL, triglycerides 89 ± 3 mg/dL, glycemia 85 ± 1 mg/dL, VO2peak 31.1 ± 0.7 mL/kg/min, Veliparib nmr SBP 114 ± 1 mm Hg, and DBP 73 ± 1 mm Hg. Vascular reactivity increased 10 minutes after exercise in the entire sample (main effect P < 0.01; baseline: 218 ± 11% vs 10 minutes: 284 ± 15%, P = 0.001), remained increased at 60 minutes (239 ± 12%, P = 0.02 vs baseline), and returned to baseline at 120 minutes (210

± 10%, P = 0.83 vs baseline). In the control protocol, there was no change in vascular reactivity (main U0126 nmr effect P = 0.96; baseline = 227 ± 24%, 10 minutes = 228 ± 36%, 60 minutes = 237 ± 31%, 120 minutes = 237 ± 29%). The distribution of genotype frequencies for the polymorphisms studied showed no deviation from Hardy–Weinberg’s equilibrium (locus −786, P = 0.93; intron 4, P = 1.00; locus 894, P = 0.70). Two subjects had the 4b4c genotype, and 1 subject had the 4c4c genotype. Because of the low frequency of the c allele, subjects with the 4b4c genotype were grouped with those with the 4b4a genotype, whereas the subject with the 4c4c genotype was grouped with those with the 4a4a genotype. Table I shows partial correlations among eNOS gene polymorphisms and vascular reactivity according to dominant, recessive, and additive models.

Disruption of calcium homeostasis

and free radicals generation are among the detrimental effects associated with MeHg-induced toxicity (Limke et al., 2003 and Ikeda et al., 1999). In this scenario, mitochondria play a crucial role, as these organelles can act as a buffer against cytosolic calcium and can mediate (RS) formation in cells (Norenberg and Rama-Rao, 2007 and Chacko et al., 2009). It has been shown that mitochondrial dysfunctions induced by MeHg include the failure of energy metabolism, the disruption of calcium homeostasis and the dissipation of the mitochondrial membrane potential, effects which lead to a mitochondrial burst of reactive oxygen species (ROS) production (Kim and Sharma, 2003, Kang et al., 2006 and Dreiem and Seegal, 2007). ROS are important mediators of damage to cell structures, including lipids and membranes, as well as proteins and nucleic

acids (Poli et al., 2004). The detrimental effects of ROS TGF-beta inhibition are balanced by the antioxidant action of non-enzymatic antioxidants in addition to antioxidant enzymes (Poli et al., 2004). However, in vivo and in vitro experimental observations have shown that the toxic effects of MeHg are accompanied by a significant deficit of antioxidant defenses, such as the depletion of GSH and the inhibition ABT 199 of GSH peroxidase activity ( Farina et al., 2004, Chang and Tsai, 2008, Stringari et al., 2008 and Farina et al., 2009). Thus, oxidative stress has been implicated

in a number of events involved in MeHg-induced cytotoxicity ( Roos et al., 2009). Based on the evidence presented above, it is reasonable to assume that Met, acting as competitive inhibitor of MeHg–Cys transport through system L could prevent or reduce MeHg-induced cytotoxicity. To date, there have been no studies on the efficacy of Met to attenuate mitochondrial MeHg uptake and mitochondrial function. The experimental model employed, namely hepatic cells, possess a particular propensity to accumulate appreciable quantities of Hg after exposure to MeHg (de Freitas et al., 2009). Specifically, we Methamphetamine have examined, for the first time, the effects of Met pre-treatment on Hg uptake, RS formation, oxygen consumption and cellular viability in both liver slices and mitochondria isolated from these slices, after exposure to MeHg or the MeHg–Cys complex. MeHgCl and l-Cysteine chloride were obtained from Aldrich (St. Louis, MO). All other chemicals were of analytical reagent grade and were purchased from Merck (Rio de Janeiro, Brazil). Adult male Wistar rats from our own breeding colony (200–250 g) were maintained in Plexiglas cages with food and water ad libitum, in a temperature-controlled room (22–25 °C) and on a 12 h-light/dark cycle with lights on at 7:00 a.m. Animals were handled and treated according to the guidelines set forth by the Committee on Care and Use of Experimental Animal Resources of the Federal University of Santa Maria, Brazil.

, 2004 and Graves et al., 2007). Here we examined whether skilled readers differed in their use of semantic information in reading aloud, and whether such individual differences map onto structural neural differences in connectivity of the reading network. We found considerable variation across individuals in the influence of semantics, and this

variation corresponded specifically to differences in the degree of structural connectivity between regions connecting areas that process semantic information with areas that process phonological information. These findings have implications for cognitive models of reading, and suggest that there are different ways to be a skilled Obeticholic Acid in vivo reader. This work was supported by National Institutes

of Health grants from the National Institute of Neurological Disorders and Stroke (Grant Number R01 NS033576 to J.R.B.) and the Eunice Kennedy Shriver National Institute of Child Health and Human Development (Grant Number K99/R00 HD065839 to W.W.G.). “
“Language is a human-specific trait used for communication. Existence of familial language impairments offers the possibility Natural Product Library ic50 of using genetics to study language. Indeed, genetic variants, such as mutations or single nucleotide polymorphisms (SNPs), in candidate genes for speech/language impairments have been identified using molecular biological approaches in patients with inherited language disorders, or association studies in clinical cohorts (Falcaro et al., 2008, Francks et al., 2004, Monaco, 2007, Newbury and Monaco, 2010, Newbury et al., 2009, Newbury et al., 2011, SLI Consortium (SLIC), 2002 and SLI Consortium (SLIC), 2004). Speech is a possible external interface for language and consists of articulation, vocalization Tau-protein kinase and Fluency. Vocalization is the sound produced by animals includes human using lung and vocal tract. A mutation in the forkhead box P2 (FOXP2) gene is present in affected KE family members. Approximately half the members of this family have speech disorders, including verbal and orofacial dyspraxia ( Belton et al., 2003, Fisher et al., 1998, Lai et al., 2001, Liegeois et al., 2003, Vargha-Khadem

et al., 2005 and Vargha-Khadem et al., 1995). It has also been reported that FOXP1, a molecule that directly interacts with FOXP2, is associated with language impairments ( Carr et al., 2010 and Hamdan et al., 2010; Horn et al., 2010, Palumbo et al., 2013, Pariani et al., 2009 and Vernes et al., 2009). Specific language impairments (SLI) are found in children with delayed or disordered language development for no apparent reason. Candidate genes for SLI have been reported, and include contactin associated protein-like 2 (CNTNAP2) and c-Maf inducing protein (CMIP) ( Newbury and Monaco, 2010, SLI Consortium (SLIC), 2002 and Vernes et al., 2008). Furthermore, both FOXP1 and CNTNAP2 are known to interact with FOXP2, and both FOXP1 and FOXP2 are known to regulate CNTNAP2 ( Horn et al.

These RG7204 datasheet trials should also include an assessment of safety. For the most part, the new BLA references the original BLA, including the non-clinical, chemistry, manufacturing and controls data. Continuous post-licensure surveillance is used to confirm the safety of vaccines in the general population, including people with a variety of health backgrounds. Post-licensure studies of safety and effectiveness of vaccines are now considered increasingly important and are often based on national

immunisation programmes and safety surveillance. Due to the nature of surveillance methods, such as patient registers and call-backs, post-licensure data may not appear in the

literature until 5–10 years after a vaccine has been granted a licence. It is important to assess the background incidence in non-vaccinees of rare conditions and AI disorders that might be possibly diagnosed in temporal association with vaccination (Table 5.2). The background incidence is required to determine whether temporal associations with vaccination are in line with the natural expected incidence rate or if there is an increased incidence see more that may suggest a causal link with the vaccine, as described in the rotavirus case study (see case study 3). Vaccine pharmacovigilance is defined by the Council for International Organizations of Medical Sciences as ‘the science and activities relating to the

detection, assessment, understanding, prevention and communication of AEs following immunisation, or of any other vaccine- or immunisation-related issues’. This covers many activities such as continuous benefit–cost assessment, risk management or communication activities to improve vaccine safety. Pharmacovigilance activities include the collection, analysis and reporting of AEs following authorisation. These reports are received from different sources, with the most frequent being healthcare professionals. Reporting to the competent authorities Methamphetamine can be expedited or periodic. The expedited reporting of serious unexpected suspected adverse events (SUSARs) to regulatory authorities should be done no later than 15 days from their receipt. In Europe, life-threatening or fatal events must be reported within 7 days. Periodic safety reporting, which in Europe takes the form of a periodic safety update report (PSUR), should be submitted to regulatory authorities at 6-monthly intervals until a full 2 years of marketing experience has been completed, then one is due every year for the following 2 years and every 3 years thereafter. Examples of assessments, requirements and timings in vaccine pharmacovigilance are summarised in Figure 5.7. Case study 2.

Unlike CdCl2 or CdS, Cd(CH3)2 is a volatile compound (bp 105.5 °C), which readily reacts with water to yield cadmium hydroxide but does not oxidize spontaneously in air. In fact, there is a gradation in stability among the Group 12 methyl derivatives, with Cd(CH3)2 ranking in an intermediate position between dimethylmercury, quite stable and dimethylzinc, Metformin cost very reactive toward oxygen and water [122]. Indicative of its stability, Cd(CH3)2 toxicity could be assessed, including through animal inhalation

studies, and a maximum 8-h work-place exposure has been set at 1 μg/m3[123]. While CH3 is the most abundant alkyl radical generated in the high temperature zone, homologue radicals with higher carbon content are also present that could react in the same way. In fact many other radicals present

in smoke could be expected to react with Cd(0) but very little information is available on such reactions. Thus, the following discussion is focused on Cd(CH3)2, since its reactivity is well documented and it is epitomical when discussing the consequences of the transitory formation of a volatile and reactive cadmium derivative. It should however be understood that Cd(CH3)2 may not be the main cadmium volatile intermediate that is actually formed in smoke. Cd(CH3)2 could certainly move to the filter during a puff, and exit the cigarette with mainstream smoke. Because of its reactivity, Cd(CH3)2 will deposit onto the unburnt tobacco downstream with Buparlisib mouse a high efficiency; yet, elements captured on the unburnt tobacco Racecadotril during a puff can be mobilized in subsequent puffs, so that this capture

is not incompatible with the observed cadmium transfer to mainstream smoke (only 5–10%). The consequence of this high capture is a yield per puff that increases with puff number, which has indeed been observed [78]. Moreover, in such a case it is expected that a higher smoke flow rate through the tobacco rod would decrease the retention of gas-phase cadmium since it is diffusion-controlled. This was also observed. Compared to the ISO yields, cadmium yield was found to be more increased under HCI than nicotine was, whereas lead yield remains to a constant ratio to nicotine (Table 6 and Table 8). Specifically, a high and flow rate-sensitive capture of cadmium by the tobacco filler was evidenced by studies where the deposited cadmium was separately assessed in the unburnt tobacco and in the filter plug after machine-smoking the cigarettes using both ISO conditions and undefined “heavy” puffing conditions [82]. The fact that elements captured on the unburnt tobacco during a puff can be mobilized by subsequent heating also increases the possibility of transfer to sidestream smoke. Hot gases can diffuse out of a smoldering cigarette as sidestream emission, the temperature of this gas stream is about 350 °C [116]. Cadmium can diffuse out as CdCl2, which would be gaseous.

“
“Toxins from animal venoms with cytolytic activity play an important role in offensive and defensive actions in different organisms. In general, these roles are achieved by enzymatic cell lysis by phospholipases A2 and C.

However, a wide variety of cytolytic proteins and peptides lacking enzymatic activity have been isolated from reptilian, amphibian, insect, cnidaria, microbial and mammalian origins (Bernheimer and Rudy, 1986, Brinkman and Burnell, 2008, Frazão et al., 2012 and Kini and Evans, 1989). Differently from phospholipases, whose hemolytic activity is due to their ability to destroy cell membranes, most of those non-enzymatic proteins and peptides lyses cells by forming discrete transmembrane pores. Small osmoticants PI3K inhibitor can move in or out of the cell through those pores, while larger molecules such as proteins cannot. Thus the cell interior becomes hyperosmotic, attracting a net influx of water, which results in a sustained cell swelling and may

result in subsequent lysis (Menestrina et al., 1994). Pore-forming toxins interact to either lipids or proteins in the external cell membrane. It has been demonstrated that some toxins interact with erythrocyte membrane glycoproteins, such as glycophorin or band 3 (Garland and Buckley, 1988). Cytolytic activity on erythrocytes has been described for selleck inhibitor numerous animal venoms, including fish venoms, which exhibit high in vitro species-specific hemolytic activity. Hemolytic effect has been demonstrated in Pterois volitans, Pterois antennata ( Kiriake and

Shiomi, 2011), Scorpaena guttata ( Carlson et al., 1971), Scorpaena plumieri ( Andrich et al., 2010 and Carrijo et al., 2005), Synanceja verrucosa ( Garnier et al., 1995), Thalassophryne natterei ( Lopes-Ferreira et al., 1998 and Lopes-Ferreira et al., 2001) and Trachinus draco fish venoms ( Chhatwal and Dreyer, 1992). The hemolytic action of these venoms is very specific for rabbit erythrocytes. Erythrocytes from human, pig and chicken are resistant to hemolysis and weak hemolytic activity Glutathione peroxidase is observed on mice and cattle erythrocytes ( Chhatwal and Dreyer, 1992 and Kreger, 1991). Because fish venoms lack phospholipase A2 activity, this hemolytic action on erythrocytes can be seen as a direct hemolysis ( Khoo et al., 1992). Chhatwal and Dreyer (1992) suggested that the hemolytic activity of the T. draco venom is preceded by the binding of the hemolytic component to a protein receptor on the surface of erythrocytes. Recently, a new cytolytic toxin, referred to as Sp-CTx has been purified from the venom of the scorpionfish S. plumieri by our group ( Andrich et al., 2010).

In a

secondary step, EHMT2 is recruited to the Slc22a2 and Slc22a3 promoters and is required to maintain repression of these genes [ 35••]. The repressed genes then Obeticholic Acid attract PRC1 and PRC2 to catalyse the H2A119u1 and H3K27me3 modifications causing chromatin compaction and the formation of a repressive compartment ( Figure 2b bottom). This compaction brings the Airn macro ncRNA, the Slc22a2 and Slc22a3 promoters and EHMT2 in close physical proximity that can be detected by sensitive techniques like TRAP and RNA immunoprecipitation. This model is supported by the formation of a repressive compartment on the paternal chromosome containing Airn ncRNA, a contracted Igf2r cluster, PRC1 and PRC2 and the repressive H2A119u1, H3K27me3 and H3K9me3 modifications [ 29••]. Recent reports have highlighted the importance of long ncRNAs in disease. this website Overexpression of the lincRNA HOTAIR in breast and colorectal cancers is associated with increased PRC2 activity and an altered H3K27me3 distribution, and correlates with metastasis

and a poor prognosis [ 42 and 43•]. The prostate cancer associated long ncRNA, PCAT-1, is correlated with aggressive prostate cancer, and appears to have a prostate specific role in regulating cell proliferation [ 44•]. The many long ncRNAs that have been recently discovered are likely to play a role in gene regulation and misregulation in disease, demonstrating the need for well-characterised model systems to understand their different mechanisms of action. Understanding the mechanism of imprinted macro ncRNA action may reveal new drug targets and enable improved therapy for diseases where macro ncRNAs play a role. Papers of particular interest, published within the period of review, have been highlighted as: • of special interest This work was funded by: FWF ‘RNA Regulation of the Transcriptome’ (SFB-F43), FWF ‘DK RNA Biology’ (W1207-BO9) and GEN-AU III ‘Epigenetic Control Of Cell Identity’ (GZ200.141/1-VI/12009). We thank Tomasz Kulinski

for comments on the manuscript. “
“In the published version of the paper, there is an error in the Abstract. Line 6 of the abstract showed “control group (n = 117)”, the clonidine correct information is “control group (n = 17)”. “
“The author regrets that in the above article, “channelepsy” was lacking in the keywords list. The correct list of keywords is as below: SCN1A; Nav1.1; Na+ channel; channelepsy; Epilepsy; SMEI; GEFS+; Seizure. “
“If you wear glasses or contact lenses, you are already enjoying the benefits of personalized medicine. Eye-care specialists can precisely diagnose your degree of nearsightedness or farsightedness and prescribe corrective measures tailored specifically to your individual needs, including, for example, spectacles, lenses or laser eye surgery, to restore 20/20 vision.

While this model revealed distance to active gas wells as exhibiting a negative control on methane concentrations, this does not indicate that gas wells are definitively causing higher methane concentrations; since these gas wells are inherently

producing from methane-rich strata this may indicate that methane concentrations are higher in close proximity to these particular formations, but it is not possible to discern the cause of the relationship without further investigation. Seliciclib Sulfate was also found to be negatively correlated to methane in this model, providing further evidence for some biologically driven methane production. This follows thermodynamic principles given that sulfate reduction yields more energy than methanogenesis; thus methane is produced when sulfate concentrations are reduced ( Schlesinger, 1997).

The three most significant variables in the model (p < 0.001) – hardness, sodium, and barium – together could explain 77% of the observed variation in dissolved methane. We acknowledge check details that including both sodium and hardness could introduce some multicollinearity into the model since sodium and hardness (as the sum of magnesium and calcium) tend to be negatively correlated; however, we find that removing either sodium or hardness from the model strongly reduces its predictive power, indicating that they are both contributing to it. These results are informative for better understanding the drivers of observed methane patterns. Sodium was positively correlated with methane concentrations

and hardness was negatively correlated with methane. This is consistent with previously described geochemical patterns that indicated that methane likely resulted from bedrock-groundwater interactions and lengthy residence times. The positive correlation between barium and methane concentrations also indicates that there is a geologic relationship with methane patterns. While barium can be present Telomerase due to human activities, including use in gas well drilling mud, it also is naturally present in geologic formations. Barium has been found in western New York to be primarily sourced from the mineral barite (BaSO4) ( Moore and Staubitz, 1984), which may also be present in formations underlying this study region. Using measured environmental variables, regression models for methane were developed with high explanatory power. While these models were developed using data from Chenango County, New York, they could have similar predictive power in nearby areas of New York and Pennsylvania with similar shale-dominated bedrock geology. With other studies in New York observing some higher methane concentrations than here (Kappel and Nystrom, 2012 and Heisig and Scott, 2013), it will be important to refine this model to try to better capture these patterns. In the future, it would also be beneficial to work toward creating improved regression models based on more easily quantified parameters (e.g.

Quality control (QC) samples were prepared using blank saliva (Innovative Research, Novi, MI, USA) which was analysed both as a blank, and spiked Y-27632 clinical trial with 10 μg/L lead. For “Device” QCs, 1 mL of saliva was sampled from a plastic beaker using the StatSure sampling device. The device was stored overnight at −20 °C and then prepared as

the samples were. For “Fresh” QCs, 1 mL spiked saliva was added to 1 mL ultrapure water (to replicate the volume of buffer in the device) and mixed. This mixture was then analysed as the device contents were. “Fresh” and “Device” QCs (blank and 10 μg/L spike) were analysed at the beginning and end of the analysis and after every 20 samples. An external CRM, Lyphochek Urine Metals Control level Lapatinib molecular weight 1 (no saliva CRM material is commercially available), lot 69151 (Bio-Rad Laboratories Ltd., Hemel Hempstead, UK) was prepared as the “Fresh” QC

was, and also analysed at the beginning and end of the analysis and after every 20 samples. The diluted blood and saliva samples were analysed using a Thermo X7 Series 2 ICP-MS instrument (Thermo-Fisher Scientific, Hemel Hempstead, UK). The instrument was tuned on a daily basis to ensure optimisation. The instrument was set up with direct nebulisation in normal mode with optimised conditions. Extraction voltage was typically – 100 V, Rf Power 1400 W, focus voltage 12.0 V and nebuliser gas flow rate (using a Burgener Miramist nebuliser (Burgener Research International, Kingston-upon-Thames, UK)) 0.83 L/min. Dwell times were 50 ms for 208Pb for blood analysis and 100 ms for 208Pb for saliva analysis,

both methods had a dwell of 10 ms for 195Pt. 3 replicates per sample were carried out. For blood analysis there were 100 sweeps per replicate, for saliva analysis, 50 sweeps per replicate. An additional investigation was carried out, to investigate whether any contamination of the sample could occur from the StatSure sampling device, and if so, whether the freezing/thawing process had any effect. Four sample Fenbendazole types were prepared using blank saliva: • A) 1 mL of refrigerated blank saliva – prepared as the “Fresh” QC above. Ten of each sample type were prepared and analysed using the same ICP-MS method as specified above. The individual components of the sampling device were also investigated, in order to elucidate from which part of the device any possible contamination originated. Samples were prepared from the buffer contained within the device, the paddle with which the saliva is collected, and the outer tube. From each device, the buffer was decanted into a 5 mL screw-cap polypropylene tube. The outer tube component was then rinsed thoroughly with ultrapure water and dried, before adding 2 mL ultrapure water and capping the tube. The head of the paddle component was cut from its stick and placed in a screw-cap 5 mL polypropylene tube. All tubes were vortex-mixed for 10 s and then rolled for 1 h before being stored overnight at −20 °C.