There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. All authors have approved the final article. This work was supported by grants Palbociclib purchase from Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG – APQ01525) to L.M. Botion. E.G.M. was recipient of scholarship from Coordenadoria de Aperfeiçoamento do Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). “
“Eukaryotic antimicrobial peptides (AMPs) are a promising class of molecular tools that have

tremendous potential to be clinically relevant due to their activity against a wide spectrum of microorganisms. Over the last few decades, intense research has been focused on their biosynthesis and the mechanisms of their activity [6] and [16]. They are important components of the natural defenses of most living organisms and have been isolated from a wide variety of animals, plants and bacteria (http://www.bbem.univ.triest.it/-tossi/search.htm). Many of these AMPs are genetically

encoded, while others are secondary metabolites. Yet, both avenues generate peptides that display positive, negative or neutral net charge in physiological pH. The positive charge of the vast majority of AMPs allows the initial interaction with the negatively charged lipopolysacharides (LPS) in the outer membrane, and later, with negatively charged phospholipids in the inner membrane. LY294002 molecular weight However, there are exceptions such as the magainins and cecropins, which do not show any interaction with chiral centers in the membrane since the L and D enantiomers of these peptides have similar antimicrobial Selleckchem Neratinib activity. In contrast, apidaecin kills bacteria by a mechanism that involves stereospecificity. The selectivity of these peptides for bacterial membranes over eukaryotic membranes has been ascribed to the lack of cholesterol and cationic lipids in the bacterial membranes and the limited amounts of anionic lipids in the eukaryotic membranes.

The skin and skin mucus of several fish species have been shown to contain AMPs. A number of those show sequence similarity to AMPs isolated from other organisms. Pleurocidin (Plc), a α-helical cationic peptide isolated from the skin-secreted mucous of the winter flounder Pleuronectes americanus [6] and [7] is predicted as a 60 or 68 residue precursor prepropolypeptide that undergoes proteolytic cleavage of its amino-terminal signal and carboxy-terminal anionic propiece to form the active peptide [8] and [9]. The resulting active peptide consists of 25 amino acid residues and possesses a net positive charge at physiological pH. The activity has been extensively explored and has shown a broad spectrum of antimicrobial and hemolytic activity [14], [17], [40] and [42].

The intermediate washing steps with PBS-T and developing were as described earlier using AP substrate. Background was assessed by incubating the wells with the non-induced periplasmic extract. All assays were conducted in duplicate. We used a rapid dot-immunoblotting protocol. Volumes of 10 μl of sera samples from Toxoplasma sero-negative and sero-positive

patients selleck kinase inhibitor were spotted onto nitrocellulose membrane, allowed to air dry and then blocked with blotto. The membrane was incubated for 1 h at room temperature with a periplasmic preparation containing the SAG1–AP fusion protein. Specific immunocomplexes were detected by incubation for 20 min in the BCIP/NBT AP substrate buffer. The membrane was washed three times with PBS-T between each step. Background Gemcitabine price was assessed in the same conditions with the non-induced periplasmic extract. According to the primers used, sag1

coding gene fragment was PCR-amplified as 867 bp including SfiI/NotI clamp sequences (data not shown). After digestion with restriction enzymes, DNA fragment was ligated into the SfiI/NotI cloning site of the pLIP6-GN vector. The recombinant plasmid was transformed into the E. coli DH5α strain; rapid visual screening on BCIP containing agar plates allowed detection of recombinant clones and the corresponding plasmids were sequenced. As expected in all blue colonies, insertion of the sag1 gene between codons + 6 and Galactosylceramidase + 7 of AP gene restored the initial frame of the AP gene in the vector. In the retained plasmid, nucleotide and deduced amino acid sequences of sag1 were in agreement with GenBank database (accession no. X14080) (data not shown). The recombinant pLIP6-sag1–AP vector was subsequently used to transform E. coli XL1-blue strain. The colonies were grown in LB medium at 37 °C, and then induced with 0.5 mM IPTG at 28 °C overnight. Periplasmic fusion protein was extracted using cold osmotic shock. A protein band with an apparent molecular weight of 78 kDa

was detected after SDS-PAGE on homogenous 10% silver staining gel ( Fig. 1A, lane 2), in agreement with the SAG1–AP predicted molecular mass. This band was absent in the non-induced cell culture. The identity and the integrity of this band as the SAG1–AP conjugate were confirmed further by two Western blotting after SDS-PAGE. The first was revealed with the anti-bacterial AP MAb ( Fig. 1B) and detected the 78 kDa-recombinant protein in periplasmic and cytoplasmic fractions from induced recombinant bacteria tested. The second blot was revealed with the conformational anti-T. gondii SAG1 Mab ( Fig. 1C) and only the periplasmic SAG1–AP was detected. This means that the intact SAG1–AP fusion protein was released in soluble form into the bacterial periplasm, where the SAG1 antigen adopts a native-like structure. No visible degradation products are revealed using anti-SAG1, suggesting the stability of the fusion protein.

Of particular note is the presence selleck products of MC-RY (9) as the dominant microcystin, together with less common or unreported analogues such as MC-RA (10), MC-RL

(28) and MC-RF (13) and some of their [Dha7]- and [Asp3]-variants. As reported earlier (Miles et al., 2012), there was a marked effect from Arg-substitution on retention times for microcystins during LC–MS2 analysis (Table 1). Analogues containing two Arg groups (Arg2 and Arg4) eluted around 2 min (e.g. 3). For microcystins containing a single Arg, those containing an Arg4 group (e.g. 1) eluted at 3.3–4.1 min, whereas those containing an Arg2 group (e.g. 9) eluted at 4.8–6.4 min. Microcystins without Arg groups (e.g. 4) eluted at 7.6–10.5 min. Apart from [Mser7]-microcystins, all of the analogues in the present study reacted with mercaptoethanol and (MEMHEG), indicating the presence of Mdha or Dha (rather than Mdhb or Dhb) at position 7. The presence in African samples of MC-RY (9) and [Asp3]MC-RY (16) has recently been reported, based on mass spectral analyses (Miles et al., 2012; Okello et al., 2010a), although their identities were not confirmed by methods capable of discriminating structural and stereochemical isomers, such as NMR spectroscopy or amino

acid analysis. The MC-RY (9) used in the present study was therefore purified and its structure verified by one- and two-dimensional NMR spectroscopy, thus greatly strengthening interpretation of buy PF-02341066 its MS2 fragmentation patterns (which were largely consistent with those reported by Okello et al. (2010a)). This in turn strengthens the interpretation of the MS2 spectra (Fig. 4 and Supplementary data) leading to the tentative identification of the less common Arg2-containing analogues for which standards are not available, such as MC-RA (10), MC-RL (28) and MC-RF (13) and their [Dha7]-, [Asp3]-, and [Mser7]-congeners. Comparison of the MS2 spectrum of MC-RY (9) with that of MC-YR (2) (Fig. 4) cAMP reveals that while a number of prominent fragment ions (e.g. m/z

916, 911, 638, 620, 603, 602 and 375) are common to both compounds, several prominent fragment ions from 2 (e.g. m/z 728, 710, 682, 571, and, most notably, 599) are absent in the MS2 spectrum of 9. The MS2 spectrum of 9 also contained several fragment ions (m/z 865, 595, 368 and, most notably, 440) that were not present in the MS2 spectrum of 2. The fragment ion at m/z 440 is characteristic for Arg2-containing microcystins (weak fragment ions at both m/z 440 and 599 were present in the MS2 spectrum of MC-RR (3)), and appears to arise from amino acids 7 and 1–3 ( Supplementary data). Thus, [Asp3]MC-RY (16) and [Dha7]MC-RY (23) both showed prominent fragment ions at m/z 426, whereas [Asp3, Dha7]MC-RY ( Miles et al., 2012) and [Mser7]MC-RY (22) showed this fragment at m/z 412 and 458, respectively ( Supplementary data).

Because we did not have detailed information on serologic tests and histology of the small bowel to confirm CD, we used specific medical read codes instead to identify women with CD in the general

population. The method used to define CD has been validated previously in general practice databases,23 therefore we believe the ascertainment of CD in our study is likely to be good. Other recent studies also have made use of read codes in primary care data to identify cases of CD, reiterating that this method to identify a CD population is valid.36 and 37 When we further increased the specificity of our CD diagnosis by restricting our analysis only to cases with supporting evidence of a gluten-free prescription, our estimates remained broadly unchanged. Approximately selleck compound 30% of the women with CD did not have any record Doxorubicin in vitro of a gluten-free prescription in our study. Gluten-free prescriptions are considerably costly when prescribed on the UK National Health Service compared with similar products purchased directly.38

Therefore, women may end up purchasing gluten-free products directly, in which case there will be no primary care data recorded on these purchases. Our study also lacked data on compliance with a gluten-free diet. However, similar to most CD studies, we assumed that all women with diagnosed CD are broadly compliant with a gluten-free diet, which seems reasonable given previous evidence suggesting that complete nonadherence to a gluten-free diet is uncommon among patients with CD.39 We must acknowledge that approximately 1% of women in the

United Kingdom have serologic evidence of CD40 and therefore it is likely that there are women with undiagnosed CD among our general population comparison group. It therefore is possible that the presence of these women could have increased the rate of fertility problems in our comparison group if there was truly an increased risk of infertility Clostridium perfringens alpha toxin among women with undiagnosed CD as has been implied previously.10, 11, 14 and 41 However, against that hypothesis, our analysis of the women with undiagnosed CD showed that, if anything, their rates of clinically recorded fertility problems were even lower than in the women with diagnosed CD in almost all of the age groups we studied. Finally, there were communication delays between secondary and primary care.42 Although the exact time for this is unknown, there may be inaccuracies in the recording of the exact date of diagnosis of CD, which may have resulted in the misclassification of some diagnosed cases as being undiagnosed. Nevertheless, the rates of fertility problems in both diagnosed and undiagnosed CD were found to be very similar, and also were comparable with the rates in women without CD. Results from the limited studies assessing CD in women with fertility problems have been inconsistent.

A linear regression analysis between the single-plex and 3-plex assays is shown in Fig. 4B, yielding an R2 value ≥ 0.98 for all 3 biomarkers. To show the specificity of this metric, a linear regression between 3-plex measurements of GDF15 and p53 autoantibody,

in which no correlation is expected, yields an R2 value of 0.04. Finally, in a culmination of these efforts, the selleck kinase inhibitor full 3-plex assay was performed on 186 CRC and normal patient serum/plasma samples (59 normal and 127 CRC) (Fig. 5A). Using the aforementioned cutoff and scoring method, individually, CEA, GDF15 and the p53 TAA were 21%, 38% and 11% sensitive and 98%, 100% and 100% specific, respectively. Composite sensitivity and specificity of all 3 biomarkers in the multiplexed assay were 54% and 98%, respectively and biomarker overlap (or lack thereof) is shown in the Venn Diagram in Fig. 5B. Notably, while partial redundancy is observed, each biomarker detects several CRC patients that the other biomarkers do not (9, 11 and 29 unique patients for p53, CEA and GDF15, respectively). Here we demonstrate the novel adaptation of Illumina’s multiplexed, genomic, VeraCode™ micro-bead technology for high-throughput immunoassay and validation of two classes of serological biomarkers: autoantibodies to TAAs (see LBH589 Fig. 1)

and circulating non-antibody proteins, using colorectal cancer (CRC) as a model system. We have created a multiplexed “hybrid” assay for NADPH-cytochrome-c2 reductase the simultaneous detection of these two classes of serological biomarkers. To our knowledge, this is the first report of use of the VeraCode™ micro-beads

as a protein/immunoassay platform. The potential advantages of this assay include its requirement for only a small volume of blood, the ability to multiplex and perform this in a high-throughput manner, and the ability to add in new biomarkers to eventually achieve a higher level of sensitivity while maintaining a high specificity for CRC diagnosis. Our goal is to continue to add to and refine our 3-marker CRC panel, thereby creating an effective CRC diagnostic screening test, which would be predicted to have excellent compliance due to its non-invasive nature. This approach could be used as a targeted population-wide screening test (for people over 50), or could eventually replace the colonoscopy altogether, assuming that the appropriate level of sensitivity and specificity is achieved by the expansion of our CRC biomarker panel. Another use for this novel protein-based platform could be for the high-throughput clinical validation studies which are urgently needed for the constant stream of newly reported putative serological biomarkers.

The heat stimulus was applied with a constant water jet onto the centre of the receptive field. Data were captured and analysed by a CED 1401 interface coupled to a Pentium computer with Spike 2 software (Cambridge Electronic Design; PSTH and rate functions). Stable control responses to electrical and selected natural stimuli were established at 20 min intervals prior to drug administration; this was confirmed with at least 3 consistent responses (< 10%) to all measures. Means of these baseline responses were calculated and used as the ‘pre-drug’ controls from which drug effects on subsequent evoked responses were tested against. Ketanserin

(1, 10 and 100 μg/50 μl saline) was applied topically to the spinal cord in a cumulative manner or ritanserin (2 mg/kg) was administered subcutaneously

into Akt inhibitor the scruff of the neck. DOI (3.6 and 17.8 μg/50 μl saline) was applied topically to the spinal cord in a cumulative manner; a low dose of ketanserin (1 μg/50 μl/saline), which does not produce any effect on neuronal activity on its own, was then administered to the spinal cord. The two routes were used because spinal application of a drug will localise its pharmacological target to pre-or postsynaptic elements in the dorsal horn. We used sub cutaneous administration to assess the effects of systemic selleck exposure. The effect of each drug was followed over an hour per dose, with tests carried out at 10, 30 and 50 min time points post drug application. The nature of the drug injection and recording protocol meant that just one experiment, on one neurone, was performed per animal used. Data are presented as mean ± standard error of mean (SEM) unless otherwise stated. For all studies the maximal effect, compared with pre-drug baseline control, for each dose was selected, this varied and was seen at any of the time points tested i.e. 10, 30 and 50 min post drug

administration. However, in most cases the maximal change in response was observed at 30 or 50 min post drug application. Drug effects were then expressed as the mean maximal effect of the pre-drug control for each dose. Analyses were performed using Decitabine GraphPad Prism version 4 for Apple Macintosh OS 10.4, (GraphPad Software, USA), and for all data, a 95% confidence interval was used as a measure of statistical significance. All statistical analyses were performed on raw data using two-way analysis of variance with repeated measures (RM ANOVA) for responses to mechanical and thermal stimuli, and if significant, Bonferroni post hoc tests were performed. The effect of ketanserin and DOI effect on responses to electrical stimulation were assessed using a one-way RM ANOVA followed by Dunnett’s post hoc multiple comparisons test for significant values. The effect of ritanserin on electrical evoked responses was assessed using a paired Student’s t-test. This work was supported by the Wellcome Trust (R25878) and NIH (Y481862).

1% SDS) at constant voltage (200 V) for 30 min. Gels were stained with Oriole™ Fluorescent Gel Stain (Bio-Rad®) and viewed under UV light to determine the presence of protein bands. APRO© SP-2110 Broad Range ladder (APRO Life Science Institute Inc., Naruto city, Tokushima, Japan) was used to estimate molecular weight. Proteins on SDS–PAGE gels were transferred to a Sequi-Blot™ polyvinylidene fluoride (PVDF) membrane (Bio-Rad) using a semi-dry transfer cell as per

Hirano and Watanabe (1990). The transblotted membrane was blocked with Blocking One solution (Nacalai Tesque Inc., Kyoto, Japan) and applied with anti-A18 mutant endogenous termite cellulase rabbit serum (Tokuda et al., 2012) diluted 1:1000 in Solution 1 of the Can Get Signal® immunoreaction enhancer solution kit (Toyobo Co., Ltd, JAK inhibitor Osaka, Japan). Following washing with TPBS (phosphate-buffered saline with 0.01% Tween 20), the membrane was applied with goat anti-rabbit IgG-horseradish peroxidase conjugate (Santa Cruz Biotechnology Inc, CA, USA) followed by TPBS washing. Applications of blocking solution and both antisera were conducted with a Snap i.d.™ protein detection system (EMD Millipore, Sirolimus datasheet Billerica, MA, USA). Before vacuum-application of the antiserum solutions onto a PVDF membrane, the solution was incubated with agitation on the membrane for 10 min at room temperature.

Finally, presence of antigen on the membrane was visualized by incubation with 3,3′,5,5′-tetramethylbenzidine solution (T0565, Sigma). APRO SP-2110 Broad Range protein ladder was used as a negative control for the primary and secondary anti-sera. For a positive control for the second anti-serum, a protein ladder with IgG binding sites (MagicMark™ XP Western Protein Standard, Life Technologies) was employed. The volumes of the foregut and the midgut of a typical, full-grown, male E. calcarata averaged 3.0 and 777 mL, respectively. EG concentration of the

foregut and the first, second, and third sections of the midgut were 4.1, 20.9, 2.0, and 0 (not detected) units/mL, respectively ( Fig. 2). A unit is defined PFKL as the amount of enzyme that produces one μmole of reducing sugar per minute from CMC in the TZB method ( Calderón-Cortés et al., 2010). These findings support the hypothesis that digestive activity is concentrated in the anterior midgut, whose pleating and folding might slow down passage of food debris to increase digestibility. The role of the appendices of the posterior midgut is likely not enzyme production, as cellulase activity stops after the anterior midgut. The phasmid cellulase concentrations were relatively low compared with the exceptionally high midgut concentrations (>1000 units/mL) found in some termite species ( Tokuda et al., 2004 and Tokuda et al., 2005), but are still significant. An EG protein was isolated by hydrophobicity interaction chromatography using a HiTrap Phenyl FF (low sub) column. The protein was eluted as three separate peaks at different concentrations of ammonium sulfate (1, 0.

As a crop, maize was subjected to artificial selection during domestication [2], [3] and [4] with subsequent episodes of post-domestication selection or improvement [5] and innovative agronomic practices. Strong selection pressure directed at genes controlling traits of agronomic importance shapes genetic variation that is available to modern breeders as it affects genome-wide nucleotide diversity and patterns of linkage disequilibrium (LD) [6]. Thus, the variation can be optimized and the SB431542 ic50 direction of recombination enhanced via evolutionary

analyses using genomics information to exploit the variation acted on by artificial selection [6]. Human selection of maize has largely focused on grain since its early domestication. Therefore, a number of genes associated with maize ears, including those for kernel color (c1 [7] and y1 [8]), and kernel composition (bt2 and su1 [9], su1 [10], sh2 [11]), were analyzed for the effects of their association with selection [3]. The maize P locus is involved in the synthesis of a red flavonoid pigment in cobs, in

the kernel pericarp, and in other floral tissues [12]. Gene P1, encoding a Myb transcription factor [13], [14] and [15], confers different color phenotypes on pericarp and cob glumes through different epigenetic alleles or forms [14], [15], [16], [17], [18] and [19]. A sharp probability peak (highest, P = 10− 17) in a mapping study was found to coincide with the known location of P1 (Fengler K, personal communication in reference [20]). QTL mapping based on a number of populations developed selleck compound by crossing two functional, but distinct, P alleles has identified a QTL in bin 1.03 [21]. It had also been an important model for gene expression regulation since the early days of modern genetics [22]. Other findings suggested that the P locus is a complex locus with different copy-number variants in a tandem repeat pattern and regulated by methylation

[12] and [13]. Candidate gene association was conducted to verify Casein kinase 1 that P1 was associated with pericarp, cob, and silk pigmentation in 76 maize lines [23]. In addition, P1 was suggested to be tightly linked with a chromosomal region that is important for controlling yield in those source populations [24]. Later work demonstrated that selection for cob color had effects on several other traits including grain yield in different genetic backgrounds [25]. It was suggested that some maize color components were less preferred, by or more toxic, to caterpillars such as Helicoverpa zea (Boddie) and sap beetles such as Carpophilus lugubris [26], which are major pests of maize during kernel storage. All this information suggests that cob glume color might be a trait under selection or a result of selection during breeding and consumer preference during the post-domestication period.

For these assays, BSc2118 had to be i.p. administered Etoposide order for technical reasons, yielding no optimal inhibition of the 20S proteasome within the 24-hour animal groups. Nevertheless,

animals treated with BSc2118 at 30 mg/kg revealed a tendency to reduce the number of metastases as compared to controls (Figure 7C). In spite of a tendency of BSc2118 (30 mg/kg) to reduce angiogenesis, significant results were lacking ( Figure 7D; P = 0.06). Taken together, BSc2118 exerts local antitumor activity in a mouse melanoma model. Novel proteasome inhibitors are intensively developed and studied in order to find more specific and safer inhibitors with a broad spectrum of therapeutic applications [15], [33], [34] and [35]. Selleckchem PF-01367338 In this context, we studied for the first time the biodistribution of the novel proteasome inhibitor BSc2118 In Vivo followed by an analysis of its therapeutic potential and therapeutic safety in the context of malignant melanoma. For inhibitor tracking in living organisms, the fluorescent variant of BSc2118, BSc2118-FL, was synthesized. BSc2118-FL was cell-permeable, targets the proteasome specifically, co-localizes with the proteasome

and had a similar inhibition profile in comparison to its non-fluorescent variant. The bright fluorescence signal facilitated rapid and sensitive detection of proteasomes by fluorescence-based microscopy in living cells and in tissues. Because the proteasome inhibitor BSc2118 had a low toxicity, even the use of higher concentrations that allows monitoring of inhibitor biodistribution, was well tolerated in experimental models. The biodistribution and inhibition profile of proteasomes inhibited by BSc2118 in a mouse model was compared to bortezomib and was similar in equivalent concentrations. BSc2118 was given daily at maximal doses of 60 mg/kg

body weight for 7 days, which was well tolerated by mice with no signs of toxicity. Using this application schedule, no lethality was observed. Moreover as it was shown in a different publication, BSc2118 up to 60 mg/kg daily dose did not affect peripheral blood morphology in C57BL/6 mouse [36]. In contrast, bortezomib had to be given with at least a one-day break, whereas daily injection of 1 6-phosphogluconolactonase mg/kg body weight was lethal in most animals. As such, BSc2118 might serve as a potential, low toxic and well tolerated novel drug [30]. Therefore, we analyzed the potential for BSc2118 usage in different application forms to be considered for proteasome inhibition. These typically include anti-tumor effects based on cell cycle arrest and on inducing apoptosis [34] and [35]. Although Bortezomib was developed and approved for therapy of multiple myeloma and mantle cell lymphoma only, therapeutic potential for other tumors was investigated within the last years as well [37]. However, bortezomib was not effective in treatment of solid tumors until recently [38].

B cell crossmatching was performed for 80% of the patients; however a positive B cell crossmatch

was not considered an absolute contraindication to transplantation. Sera collected at the time of transplant were screened retrospectively for anti-HLA class I and/or class II antibodies using the Luminex Mixed Screen assay (OneLambda Inc.) and those with a positive screen were characterised for HLA class XAV-939 in vivo I and/or class II antibodies specificity using single antigen beads (LABScreen Single Antigen beads, OneLambda Inc.). Antibodies were considered to be positive if the normalised mean fluorescence intensity (MFI) value for a particular bead was greater than 500. HLA antibodies with an MFI > 500 directed against a donor HLA antigen were considered to be DSA. Transfusion history was recorded as never transfused (No-RBCT), transfused at any time prior to renal transplant but not after Epigenetic pathway inhibitor renal transplant surgery (Pre-RBCT), not transfused prior to transplant but transfused at the time of, or within 30 days of transplant surgery (Post-RBCT) and transfused both prior to and within 30 days of the transplant (Pre + Post-RBCT). Delayed

graft function (DGF) was defined as the need for dialysis within the first 48 h of transplantation. Graft loss was defined as the return to dialysis (i.e. death-censored) unless otherwise indicated. Sclareol All rejection episodes were proven by biopsy (BPAR) and the first BPAR was used to construct time to event analysis and where multiple rejections occurred, the highest reported grade was recorded. Time to AMR was recorded as a separate event to allow analysis by rejection type (AMR vs Non-AMR). Treatment of rejection was at the treating clinician’s discretion and was not mandated by protocol. Histological reporting of renal biopsies was undertaken by the local

histopathologists as part of routine clinical care and was initially made without information as to the presence or absence of DSA (due to varying laboratory testing and reporting changes over the period of study). The biopsy findings were graded according to the Banff classification 2003. AMR was defined as C4d positivity in PTC alone or in conjunction with transplant glomerulitis and/or peri-tubular capillaritis and/or arteritis, and also in the absence of C4d when transplant glomerulitis and peri-tubular capillaritis were detected. Statistical analyses were performed by using SPSSv18 (SPSS Inc., Chicago IL, USA). For categorical data Fisher’s exact test or Pearson’s chi-square tests were used. Parametric data were compared by ANOVA or t-test, and for non parametric data Mann–Whitney U test or Kruskal–Wallis one-way ANOVA was used. Comparisons of within group differences by z-test were made with Bonferroni adjustment reported at the p < 0.05 level.