EGFR functional polymorphisms, a (CA)n repeat in intron 1, and a single nucleotide polymorphism, R497K, may affect EGFR expression and breast cancer clinical profile.The epidermal growth factor receptor (EGFR) is differently expressed in breast cancer, and its presence may favor cancer progression. We hypothesized that two (CA)n and R497K polymorphisms, and the associations between (CA)n polymorphism and EGFR transcript levels (n = 129), or between either polymorphism and histopathological features (n = 505) were evaluated. The REMARK criteria of tumor marker evaluation were followed.The study population consisted of 508 Brazilian women with unilateral breast cancer, and no distant metastases. Patients were genotyped for the (CA)16 , which was set as the cut-off length to define the Short allele. Variant (CA)n genotypes had no significant effect in tumoral EGFR mRNA levels, but patients with two (CA)n Long alleles showed lower chances of being negative for progesterone receptor . The evaluation of R497K polymorphism indicated a frequency of 0.21 (95% CI = 0.19 – 0.24) for the variant (Lys) allele. Patients with variant R497K genotypes presented lower proportion of worse lymph node status (pN2 or pN3) when compared to the reference genotype Arg/Arg , which resulted in lower tumor staging , and lower estimated recurrence risk . The combined presence of both EGFR polymorphisms n) resulted in lower TNM status  and lower ERR . When tumors were stratified according to biological classification, the favorable effects of variant EGFR polymorphisms were preserved for luminal A tumors, but not for other subtypes.(CA)n lengths ranged from 14 to 24 repeats, comprehending 11 alleles and 37 genotypes. The most frequent allele was EGFR polymorphisms may lead to better prognosis in breast cancer, especially in patients with luminal A tumors.The data suggest that the presence of the variant forms of  Breast cancer is the most frequent type of cancer in women both in the developed and the developing world . It is aThe epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase (TK) receptor of the ErbB family, whose activation leads to mitogenic signaling . EGFR isEGFR gene, located at 7p12.3-p.1, contains multiple polymorphisms [myc, fos, and jun[The orphisms , two of orphisms , and app, and jun.EGFR polymorphisms among Brazilian breast cancer patients, and to evaluate their impact on breast cancer prognosis, exploring the effects of (CA)n polymorphism on EGFR transcript levels, and the associations of both polymorphisms with histopathological features and prognostic estimates.In the present work, we aimed to describe the frequency of these two The study population consisted of a prospective cohort of Brazilian women with first diagnosis of unilateral breast cancer and no distant metastases, admitted at the Brazilian National Cancer Institute (INCA) during the period from February 2009 to April 2011, and who were assigned for tumor resection as their first therapeutic approach. The recruitment occurred before surgery, but the inclusion was only completed after diagnosis confirmation by histopathological evaluation of the resected tumor. The study protocol was approved by the Ethics Committee of the Brazilian National Cancer Institute (INCA #129/08), and all patients gave written consent to participate. The REMARK guidelines (REporting recommendations for tumor MARKer prognostic studies) were followed .The histopathological evaluation of resected tumors was performed following institutional routine procedures, and all individual data were obtained from electronic medical records. The histopathological characterization was based on the TNM classification by the American Joint Committee on Cancer  and on tet al. [The data on hormone receptors, i.e. Estrogen Receptor (ER), and Progesterone Receptor (PR), and on the Human Epidermal growth factor Receptor 2 (HER2) status were used for biological classification of the tumors, as proposed by Huober et al. . The Estet al. , with thR497K (rs11543848) or by capillary electrophoresis for the (CA)n repeat polymorphism in intron 1 (rs72554020). The PCR amplifications were performed with the following primers : 5′-AGGTCTGCCATGCCTTGT-3′ (sense) and 5′-CAACGCAAGGGGATTAAAGA-3′ (antisense) for R497K; or 5′-TTCTCCTCAAAACCCGGAGAC-3′ labeled with 6-FAM™ (sense) and 5′-GTCACGAAGCCAGACTCGCT-3′ for (CA)n repeat (antisense). The R497K PCR products (5 μL) were digested with 5U of BstN1 restriction enzyme  at 60°C for 3 hours, and the digestion products were resolved on 2% agarose gel and stained with ethidium bromide for visualization under UV light. The digestion of the homozygous G alleles (Arginine) produced two fragments (100 bp and 56 bp), whereas the homozygous A alleles (Lysine) remained intact (156 bp). The method was validated by direct sequencing of four samples of each genotype.Peripheral blood samples (3 mL) were collected from the subjects, and DNA was extracted using the Blood Genomic Prep Mini Spin Kit , following the procedures recommended by the manufacturer. The genotyping analyses were performed using PCR-RFLP for the SNP The (CA)n repeat PCR products (0.5 μl) were denatured at 95°C for 3 min in the presence of 0.5 μl of the GeneScan™ 400HD ROX molecular weight standard  and 9.0 μl of Hi-Di™ Formamide , refrigerated to 4°C for 2 min, and then submitted to separation by capillary electrophoresis in ABI Prism® 3130 Genetic Analyzer, using POP7™ polymer . The analyses were performed using the GeneMapper® Software v.3.7 . The PCR products identified as homozygous, i.e. those presenting a single retention time at the capillary electrophoresis, were submitted to direct sequencing, using the BigDye® Terminator Kit , in order to establish a correspondence between each retention time and the respective number of CA repeats .2, and stored at the Brazilian National Bank of Tumors (BNT-INCA). Frozen sections of breast specimens (with approximately 2 mm) were used for RNA isolation, which was performed using the RNeasy Mini Kit , following the manufacturer’s instructions. The RNA samples were stored in RNAse-free distilled water at -80°C, and the corresponding cDNA was synthesized using 2 μg of RNA, with High Capacity cDNA Reverse Transcription Kit , according to the manufacturer’s instructions.Fresh specimens of breast tumors were dissected by clinical pathologists after tumor resection, frozen in liquid NEGFR transcripts was performed using quantitative real-time RT-PCR (TaqMan) assays, in an ABI PRISM 7500 Sequence Detector System . Each reaction contained: cDNA templates (approximately 40 ng), 10 μl of reaction mix containing 5 μl Taqman® Gene Expression Master Mix, and Taqman® probes, which were as follows: EGFR Hs01076078_m1 (with FAM), PPIA 4326316E (with VIC) . The thermal cycling conditions comprised an initial denaturation step at 95°C for 10 min, followed by 40 cycles of 95°C denaturation for 15 sec, and annealing at 60°C for 1 min. The experiments were carried out in 96-well plates, including a nontemplate control, and a reference control, consisting of cDNA obtained from a commercial Human Mammary Gland (HMG) total RNA . The relative quantification of EGFR mRNA was calculated as the average 2-ΔΔCt, where ΔΔCT = ΔCTEGFR - ΔCTHMG, and ΔCTEGFR = CtEGFR - CtPPIA, and ΔCTHMG = CtHMG - CtPPIA. All data were generated in triplicates and expressed as median +/- SD with the 25–75 percentiles.The relative quantification of EGFR genotypes were evaluated by the Chi-square or Fisher’s exact tests. In the cases of significant associations between EGFR genotypes and independent histopathological variables, the odds ratios (OR) and their respective 95% confidence intervals (95% CI) were tested for linear-by-linear associations, with calculation of trend significances (Ptrend), and definition of phenotypic inheritance models. The odds ratios between EGFR phenotypic groups and histopathological categorical features were adjusted for all other independent clinical variables (ORadjusted) using multiple regression analyses. The comparison of the relative quantities of EGFR mRNA as a function of histopathological features or EGFR genotypes was performed with the GraphPad Prism 5.0 software , using the non-parametric Mann–Whitney U-test for comparison of two groups, or the Kruskal-Wallis test for comparison of multiple groups. All other statistical analyses were conducted using SPSS 13.0 for Windows . The threshold for significance was set at P < 0.05.A descriptive study of the cohort was conducted, presenting measures of central tendency and dispersion for continuous variables, or relative frequencies for each categorical variable. Allelic and genotypic frequencies were derived by gene counting. The histopathological features were dichotomized for better and worse prognostic values, and their associations with EGFR polymorphisms for the 508 patients evaluated. The median age was 59 years old, ranging from 27 to 92.A total of 576 patients were recruited when admitted for surgery, and 528 had the diagnosis and inclusion criteria confirmed after pathological evaluation of their resected tumor. Blood samples were available for 511 cases, and 508 of them had good DNA quality for genotyping assays. Table R497K polymorphism was obtained for 505 patients, whereas the characterization of the number of (CA)n repeats by electrophoresis was conclusive in 477 cases  was 0.21 (95% CI = 0.19-0.24).The genotyping of the es Table . The fre(CA)16 , which was taken as the cut-off length to group Short alleles. All the other variant alleles, with more than 16 (CA) repeats were considered as Long alleles. Thus, the genotypic distribution used for further analyses was: Short/Short (reference homozygous genotype), Short/Long (heterozygous) and Long/Long (variant homozygous genotype).The evaluation of the (CA)n lengths indicated a range of 14 to 24 repeats n genotypes or prognostic categories of breast tumors were explored n genotypes . As a consequence of these two associations, patients with higher ERR presented higher EGFR mRNA expression levels n genotypes according to prognostic categories. The distribution of R497K genotypes was statistically different as a function of the lymph node status, whereas the distribution of (CA)n genotypes was statistically different as a function of the PR status.Table R497K genotypes and lymph node status, or between (CA)n genotypes and the PR status is further explored in Figure Arg/Lys presented lower proportion of the worse lymph node status (pN2 or pN3), when compared to the reference homozygous genotype Arg/Arg , whereas among patients with the homozygous variant genotype Lys/Lys (n = 21), there was only 1 case of pN2 or pN3 . These results indicate that the magnitude of the association between R497K polymorphism and lymph node status depends on the number of variant Lys alleles (Ptrend = 0.001). Similarly, Figure (CA)n alleles on the proportion of negative PR status. The results indicate an apparently progressive effect of the number of long (CA)n alleles (Ptrend = 0.008). Thus, patients with the Short/Long genotype showed a slightly lower proportion of negative PR status when compared to the reference Short/Short genotype , and a significant protective effect was observed for the variant Long/Long genotype .The association between pN2 or pN, when coR497K polymorphism and lymph node status and of recessiveness for (CA)n Long allele and PR status. Thus, the genotypes Arg/Lys and Lys/Lys were grouped for evaluation of their impact on lymph node status, whereas the (CA)n Long/Long genotype was evaluated in comparison with the combined Short/Short and Short/Long genotypes for its effect on the PR status. The two EGFR polymorphisms were also evaluated in a combined analysis in order to investigate a possible interaction between them on the distribution of breast cancer prognostic features n polymorphism, the association between (CA)n Long/Long genotype and PR negative status also remained significant after adjustment for other independent individual prognostic variables , but did not affect TNM status or the ERR. When the two EGFR polymorphisms are present, there is lower TNM status  and lower ERR .The results indicate a significantly protective effect of the EGFR polymorphisms and better lymph node status occurs for tumors classified as luminal A, but not for the other biological subtypes.The stratification of breast tumors according to their biological classification indicates that the association between combined variant EGFR functional polymorphisms in the Brazilian population was not known before the current study. Our data indicate a frequency of 0.21 (95% CI = 0.19 – 0.24) for the 497 K , and of 0.43 (95% CI = 0.40 – 0.46) for the (CA)16. These results are similar to the frequencies reported for Europeans and North-Americans (including African-Americans), either for R497K polymorphisms [(CA)n[Lys allele [(CA)n alleles [The distribution of the two orphisms  or (CA)nms [(CA)n,21. Asias allele ,23, and  alleles ,21,24,25(CA)n polymorphism in gene transcriptional activity in vivo is the vast distribution of the number of (CA) repeats, with various possible heterozygous genotypes, and no clear model on how the two alleles interact for the final cell phenotype. Amador et al. [EGFR mRNA in head and neck cancer cell lines. Buerger et al. [et al. [(CA)20 and other long alleles, had lower amounts of EGFR protein than tumors from German patients, who have a predominance of (CA)16 and other short alleles. Other authors, however, found no correlation between the length of the (CA)n region and the relative quantification of EGFR mRNA [One difficulty of evaluating the effects of r et al.  considerr et al. , studyin [et al.  showed tGFR mRNA  or EGFR GFR mRNA .EGFR mRNA, with no apparent inverse correlation between the number of (CA) repeats, considering either the smaller allele or the combined length within each genotype (data not shown). In order to investigate a possible effect of somatic mutations on the tumoral (CA)n genotype, we evaluated a set of 40 tumor samples. The number of CA repeats was preserved in relation to genomic DNA in all cases (data not shown). Although we did not extend such analyses to all patients, it appears that mutational events, such as loss of heterozigosity, are not affecting the EGFR locus of breast tumors. Nevertheless, an accurate characterization of the impact of EGFR polymorphisms on the gene transcriptional activity in vivo would ideally include quantification of gene amplification in the tumors [EGFR polymorphisms (-216G/T or rs712829 and -191C/A or rs712830), located in the promoter region, which might have functional impact on EGFR transcriptional activity [EGFR expression [Our data confirm the great dispersion of (CA) lengths and indicate great variability on the expression of e tumors . In addipression .EGFR polymorphisms on histopathological and molecular characteristics of breast cancer indicated significant association between R497K variant genotypes and better lymph node status, corroborating the findings of Kallel et al. [Long/Long (CA)n genotypes and positive PR status. These two associations seem protective in relation to breast cancer evolution, since a greater number of affected lymph nodes increases the risk of systemic metastasis [The evaluation of the impact of l et al. , and bettastasis , and thetastasis .Lys) and better lymph node status appear to corroborate the notion of reduced signaling with the variant EGFR isoform [With regards to the molecular mechanisms underlying lymph node metastases, EGFR appears to activate integrins  and meta isoform , leadingEGFR polymorphisms and lymph node metastases and negative PR status appear to corroborate the role of EGFR in breast cancer pathogenesis.The interaction between the EGFR activity and the PR status might occur via a cross-talk mechanism between steroid and growth factor receptors , resultiLong/Long (CA)n genotypes and Lys R497K alleles appears to favor better prognostic estimates in breast cancer. Other studies involving different types of cancer also point to an interaction between the two EGFR polymorphisms, with a combined protective effect in relation to disease progression. Zhang et al. [Arg alleles and <20 CA repeats. Bandrés et al. [Arg allele and both (CA)n repeats ≤ 16 presented higher risk of death. Press et al. [Arg/Arg genotype and two short alleles (< 20 CA repeats) had shorter overall survival than men with the Lys/Lys or Arg/Lys variant genotypes and any long allele (≥ 20 CA repeats).The combined presence of g et al. , evaluats et al. , studyins et al. , studyinEGFR polymorphisms are characteristic of luminal A tumors. This apparently selective effect of EGFR polymorphisms might be due to the lower genomic instability of luminal A tumors in relation to other subtypes, which present more aggressive phenotypes due to superposed molecular alterations [EGFR polymorphisms with prognostic features at diagnosis cannot be considered as actually predictive of disease progression or therapy response,The stratification of breast tumors according to their biological subtypes suggests that the apparently protective effects of erations . NeverthEGFR polymorphisms as independent prognostic factors, especially in early-stage luminal A tumors, as they might contribute to identify patients at higher risk of progression. We propose that EGFR genotyping should be further evaluated for their prognostic value in prospective studies of breast cancer survival.In conclusion, the current results indicate a potential benefit of The study was conducted following the international precepts of ethics in research and of good clinical practice. The authors complied with the Brazilian regulation of clinical research. The protocol was approved by the Ethics Committee of the Brazilian National Cancer Institute (INCA #129/08), and all patients gave written consent to participate.The authors declare that they have no competing interests.(CA)n polymorphism. MAC coordinated the expression assays, collaborated with data interpretation and revised the manuscript. RV-J conceived, designed, and coordinated the study, analyzed the data, wrote and revised the manuscript. All authors read and approved the final manuscript.MSL recruited patients, collected clinical information, set the genotyping and expression assays, characterized genotypes and haplotypes, performed statistical analyses, generated tables and figures, and drafted the manuscript. LCG recruited patients, collected clinical information, and helped with genotyping assays. DNP set and performed the expression assays, and helped revising the manuscript. JSF-V collected and revised histopathological data, and helped revising the manuscript. VI-do-B recruited patients, collected clinical information, and helped with the statistical analyses. SK conceived the epidemiological design of the cohort. RSMN coordinated the genotyping of The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/190/prepub


The aim of this study was to investigate effects of endurance and resistance training (ET and RT) on CGRP and AChRs at slow and fast twitch muscles and sciatic nerve in rats. Twenty-five male rats were randomly assigned into three groups including sedentary (SED), endurance training (ET), and resistance training (RT). Animals of ET exercised for 12 weeks, five times/week, and 60 min/day at 30 m/min. Animals of RT were housed in metal cage with 2 m high wire-mesh tower, with water bottles set at the top. 48 h after the last session of training protocol, animals were anaesthetized. The right sciatic nerves were removed; then, Soleus (SOL) and Tibialis anterior (TA) muscles were excised and immediately snap frozen in liquid nitrogen. All frozen tissues were stored at −80°C. Results showed that, after both ET and RT, CGRP content as well as AChR content of SOL and TA muscles significantly increased. But there was no significant difference among groups at sciatic nerve' CGRP content. In conclusion, data demonstrate that ET and RT lead to changes of CGRP and AChR content of ST and FT muscles. The changes indicate to the importance of neuromuscular activity. Calcitonin gene-related peptide (CGRP), generated from the calcitonin gene –3, is diα enhances the expression of the acetylcholine receptor  (AChR)α-subunit mRNA in skeletal muscles [α but not CGRPβ is upregulated by axotomy or blockade of neuronal activity, suggesting that this particular peptide plays a role in motoneuron regeneration [α acts as an anterograde “trophic” agent that controls the synthesis and function of muscle AChRs via cAMP-mediated pathways at the neuromuscular junction (NMJ) [α plays a key role in the trophic control of AChE molecular forms in adult NMJs [In the peripheral nervous system, CGRP coexists with ACh in motoneurons , and stu muscles , and pro muscles . Other sneration , 14. Theon (NMJ) , 15. Itson (NMJ)  and incron (NMJ) –19 and ion (NMJ) , 21. Theon (NMJ) . Fernandult NMJs .Studies have shown that motoneurons supplying fast-twitch muscles  show higher levels of CGRP staining than do motoneurons innervating muscles of slow-twitch (ST) fiber  –20, 23. It has been postulated for many years that an increase in neuromuscular activity has resulted in an increased motoneuronal CGRP. Homonko and Theriault showed an increased CGRP in the motor neurons of medial gastrocnemius muscles in rats 72 hours after eccentric downhill running. Their results may indicate a preferential response of FG fibers after unaccustomed exercise, resulting in synaptic reorganization . In addiHowever, there is no evidence of RT-induced CGRP and AChRs possible changes in muscles and sciatic nerve. Thus, we have investigated the content of CGRP of slow and fast twitch muscles and the sciatic nerve, following ET and RT protocols.n = 8), endurance training (ET) (n = 8), and resistance training (RT) (n = 9). Ethics Committee of TMU approved the experimental protocol, and their Guidelines for Care and Use of Laboratory Animals were followed.Twenty-five Wistar rats , purchased from Pasture Institute  and maintained in the Animal House, School of Medical Sciences of Tarbiat Modares University (TMU). The animals were housed four per cage with volume of 46 L. The light-dark cycle was 12 h. Temperature was 22 ± 1.4°C, and humidity was 55.6 ± 4.0%. Animals were fed with a pellet rodent diet and had free access to water. Animals were randomly assigned into three groups including: sedentary (SED)  and tibialis anterior  [2 and dry ice then divided into two parts which were used to examine CGRP and AChR content.48 h after the last training session, animals were anaesthetized with a mixture of Ketamine (75 mg/kg-1) and Xylazine (5 mg/kg-1) which was administered intraperitoneally. The right sciatic nerves were surgically removed and frozen in liquid nitrogen. The  muscle) , 31 wereSOL and TA muscle samples and sciatic nerve samples were washed with ice-cold PBS, homogenized 1 : 10 in 10 mM PBS, pH 7.4 at 4°C, and centrifuged (20000 rpm/45 min), and CGRP content was measured. Then, CGRP content was determined using available commercial enzyme immunoassay method  according to kit manufacture's instruction. The assay sensitivity was 5 pg/mL, and the intra-assay coefficient of variation was 7.9%.Levels of AChR in the homogenized  and centrifuged (20000 rpm/45 min) muscle samples were determined using Rat nicotinic acetylcholine receptor ELISA kit  according to the manufacturer's instructions.t-test and Two-way ANOVA. P values <0.05 were considered significant.Results are expressed as means ± Std. E. Variables were analyzed by unpaired As presented in Tables P = 0.001 and P = 0.017, resp.).As Figures P = 0.001) as well as AT muscles (P = 0.013). CGRP content was increased significantly at SOL muscle by ET (P = 0.036), but no significant increase following ET at TA muscle was found (P = 0.287). However, no significant difference was observed between ET and RT at SOL muscle CGRP (P = 0.212), and there was also no significant difference between ET and RT regarding AT muscle CGRP content (P = 0.283). As data suggests there is no significant change in CGRP content at SOL and TA muscles in SED as unpaired t-test analysis showed (P = 0.984).Results showed that RT leads to a significant increase in CGRP content of SOL . Thereby, there was a difference, but not significant, in AChR content between SED and ET at SOL muscle (P = 0.116) (P = 0.010)  . In addi= 0.010) .P = 0.001 and P = 0.005, resp.)  .As data analysis suggests, there were no significant changes between AChR content at SOL and AT muscles of SED, ET, and RT groups .The results of our study demonstrate that both ET and RT protocols have been capable of significantly altering CGRP and AChR contents of ST and FT muscles and probably CGRP of the sciatic nerve.In this study, no differences of CGRP and AChR content between ST and FT muscles of SED were observed. These results show that muscle fiber type, probably, is not the main criteria for the basal level of CGRP as well as AChR. There are, however, contradictory documents related to the fiber type-dependent differences among muscles and related motor units. For example, Osterlund et al. found that the overall distribution of CGRP immunoreactivity was similar when comparing motoneurons innervating SOL, TA, and lateral gastrocnemius . Later, In general, both ET and RT protocols lead to change CGRP and AChR content of ST and FT muscles. Reports have documented that both ET and RT stimulate morphological remolding of NMJ. For example, weight lifting has affected morphological profiles in NMJ architecture , 35. IncIn accordance with other reports, in present study CGRP that is a neurotrophic and neuromodulator factor at NMJ , 32 was As described, neuromuscular activity has contribution in NMJ remodeling, and it may induce unstable NMJ. Therefore, in the present study, CGRP content probably is changed at NMJ area. Eisen et al. were the first who showed the effect of long-time ET protocol on motoneuronal CGRP in cell bodies of SOL and neighbouring motoneurons in endurance-trained rats which was significantly increased (90%) . For exaMuscle CGRP content was measured after eccentric exercise by Jonhagen et al. They observed increased concentrations of CGRP after eccentric exercise together with increased experience of pain. Thus, they believed that CGRP may be involved in the regulation of DOMS after heavy exercise while simultaneously stimulating tissue regeneration . More reAlthough there is no available document investigating CGRP content change through RT, the findings of the present research might be explained by the well-known effect of RT on NMJ, and its mediators such as CGRP. Our data showed that RT has affected both ST and FT muscles. These results show that CGRP perhaps depends on the neuromuscular activity of muscle in adult animals. Although muscle CGRP was increased significantly at both ST and FT muscles by RT, the signaling mechanism is unknown.It should be noted that contraction type probably has a key role in CGRP changes. Since eccentric contractions lead to FT fiber activity , and NMJIt is well known that spinal motoneuron cell bodies synthesize CGRP which is conveyed to nerve terminals by axonal transport  where stThere is a few research investigating CGRP content of sciatic nerve; however, it should be noted that in most of previous studies there has been a ligature on sciatic nerve by which it has been possible to show the accumulation of CGRP, as Forsgren et al. have suggested that content of CGRP sciatic nerve measured 4 h following application of a ligature . We atteα and δ AChR subunits were increased in 10–70 day electrostimulated muscle specimens [Our data showed all training protocols of this study significantly increased the muscle AChR content. Although possible morphological changes of NMJ were not measured in this study, findings showed that probably this aspect of NMJ remodeling is affected through the training protocols. The increased AChRs content is in coordination with other findings of the present study as increased level of muscle CGRP and, most likely, the facilitated transportation of this peptide. It is believed that exercise-induced MEP distribution has a direct relation to the number of AChRs . It has pecimens . The enhpecimens , 35.Moreover, it is suggested that low-intensity running decreases postsynaptic development, while high-intensity running increases it . TherefoAChR content also was increased through RT of the present study as many of studies that reported RT are known to have effects at NMJ , 36. DenCGRP has been found to colocalize with AChE and AChRs in all of the individual MEPs examined . Followiα-subunit mRNA, thereby promoting the synthesis of AChR [In our study, data analysis showed that RT significantly increased both CGRP and AChR content at ST and FT muscles. Although we did not measure factors such as cAMP, it could be speculated that exercise-induced CGRP increase probably has been involved in AChR increase. It should be noted that investigations have shown that treatment with exogenous CGRP elevates muscle intracellular cAMP activity , upregul of AChR , enhance of AChR , increas of AChR , and pro of AChR .In conclusion, present data demonstrate that both ET and RT lead to changes of CGRP and AChR content of ST and FT muscles. The changes were independent of muscle fiber type that indicates to the importance of neuromuscular activity in the form of ET and RT affecting the fundamental components of neuromuscular adaptations.


Objective. We investigated the relationship between superior mesenteric artery blood flow volume (SMA BFV) and autonomic nerve activity in acupuncture stimulation of lower limb points through heart rate variability (HRV) evaluations. Methods. Twenty-six healthy volunteers underwent crossover applications of bilateral manual acupuncture stimulation at ST36 or LR3 or no stimulation. Heart rate, blood pressure, cardiac index, systemic vascular resistance index, SMA BFV, and HRV at rest and 30 min after the intervention were analyzed. Results. SMA BFV showed a significant increase after ST36 stimulation ; very low frequency (VLF), high frequency (HF), low frequency (LF), and LF/HF were significantly greater than those at rest . Changes in HF and LF also differed significantly from those resulting from LR3 stimulation . Conclusion. Increased vagus nerve activity after ST36 stimulation resulted in increased SMA BFV. This partly explains the mechanism of acupuncture-induced BFV changes. In traditional medicine in East Asia, acupuncture therapy is achieved through acupoints, which are reactive points on the surface of the body. Each acupoint is thought to achieve specific and sometimes unique effects on human organ systems . HoweverAcupuncture stimulation of ST36 is considered to have beneficial effects on gastrointestinal symptoms and has been used since ancient times . We haveNoninvasive methods are preferable to elucidate mechanisms by which the autonomic nervous system controls physiological phenomena such as increases in blood flow volume. In research and clinical practice, the spectral analysis of heart rate variability (HRV) has gained popularity and has gained wide popularity as a noninvasive monitor of the autonomic response . SpectraIn the present study, we evaluated the involvement of autonomic nerve activity and changes in SMA BFV resulting from acupuncture stimulation of acupoints using a spectral analysis of HRV.Crossover trials of stimulation at ST36 and LR3 and nonstimulation (CTL) were conducted . The subBefore each trial, resting measurements were obtained for each subject after 10 min of rest in the supine position at a room temperature of 25-26°C. Thereafter, acupuncture stimulation was performed by bilateral insertion of a number 1 needle  at ST36 or LR3 to a depth of 1 cm over 18 sec, twirling the needle for 18 sec, and leaving the needle for 15 min. In the CTL group, the subject was observed for the same duration without acupuncture stimulation. An acupuncturist with over 5 years of acupuncture experience administered the acupuncture stimulation. ST36 is located on the lower leg, 3 units below the lateral “eye” of the knee and approximately 1 finger width lateral to the tibia [In each trial, heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), cardiac index (CI), systemic vascular resistance index (SVRI), SMA BFV, and power of each frequency domain calculated from HRV (very low frequency (VLF), low frequency (LF), high frequency (HF), and LF/HF) at rest (before) and 30 min after stimulation were extracted for analysis. The details of the extracted items were as follows.2) generally reflects functions such as thermoregulation and renin-angiotensin system activity.VLF: very low frequency domain (≤0.04 Hz) power (ms2) generally reflects both the sympathetic and parasympathetic nervous systems. LF: low frequency domain (0.04–0.15 Hz) power (ms2) generally reflects the parasympathetic nervous system.HF: high frequency domain (0.15–0.40 Hz) power . The data were extracted from the record for 3 min at rest and at 30 min after the intervention to avoid the period of the SMA BFV measurement using ultrasonographic diagnostic equipment. The data were subjected to a spectral analysis using LabChart analysis software  . The Tasα10; Hitachi Aloka Co. Ltd., Tokyo, Japan). SMA BFV was measured within 2-3 cm from the origin of the artery using ultrasonographic diagnostic equipment .CI was calculated from intrathoracic impedance measured using 4 dual sensors of a BioZ ICG Module, Dash 3000 .t-test. A 2-sample t-test was used for intergroup comparisons. The difference was considered significant when the P value was less than 0.05. For the statistical analysis, values of HR, SBP, CI, SVRI, SMA BFV, and the power of each HRV frequency domain  at rest and 30 min after stimulation in each group were represented by the percentage change (%) relative to the resting value. The values before and after the intervention were compared by a paired This study was conducted after approval by the Ethics Committee of Tohoku University School of Medicine. All subjects provided written consent regarding the contents of the experiments. Tables P = 0.045, P = 0.006, P = 0.048, and P = 0.039, resp.), SBP significantly decreased (P = 0.037), and SMA BFV significantly increased (P = 0.007)  .P = 0.014) and LF/HF significantly increased (P = 0.013), while SMA BFV showed no significant change  and CI significantly decreased (P < 0.001), while SMA BFV showed no significant change  was observed in the change in SMA BFV as well as in LF and HF   .In the present study, acupuncture stimulation at ST36 resulted in an increased SMA BFV together with significantly increased VLF, LF, HF, LF/HF, and significantly decreased SBP values. The intergroup comparison between the 2 acupoint stimulations showed significant differences in LF and HF.The SMA supplies blood widely to the small and large intestines, and its blood flow is regulated by the peritoneal vagus and abdominal sympathetic nerves . HF in HLR3 stimulation caused no significant change in SMA BFV. In addition, when HR, SBP, CI, and SVRI showed no significant change, HF significantly decreased, LF/HF significantly increased, and LF showed no significant change. LR3 stimulation might have suppressed parasympathetic nerve (cardiac vagus nerve) activity while exerting no influence on sympathetic nerve activity.In CTL, while no significant change was seen in SMA BFV, CI significantly decreased, SVRI significantly increased, and HR and SBP showed no significant changes. While VLF significantly increased, HF, LF, and LF/HF showed no significant changes. Although there is a report stating that an increase of VLF indicates an enhancement of parasympathetic nerve activity , the incIn the comparison between ST36 and LR3 stimulations in the present experimental system, SMA BFV as well as HF and LF in HRV showed significant differences. These 2 acupoints are present in the same segment, that is, on the same afferent fiber. Mori et al. performed acupuncture stimulation at 2 acupoints in the same segment (afferent fiber) and indicated that the reaction of the autonomic nervous system may vary because of differences in the muscle (muscular segment), even though the input is made on the same afferent fiber . Zhao etWatanabe et al. speculated that increased SMA BFV after ST36 stimulation resulted from the suppression of the sympathetic nervous system through stimulation of the parasympathetic nervous system and spinal reflex . In the β and Ach receptors [HR effects due to sympathetic nerve activity have distinctly different characteristics from those due to vagus nerve activity. HRV does not exceed 0.15 Hz with sympathetic nerve transmission, whereas HRV up to around 1 Hz occurs with cardiac vagus nerve transmission because of differences in intracellular communication mechanisms downstream of eceptors . To evaleceptors , 18. In eceptors . FurtherThe Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology define 2–5 min as the extraction time required for a spectral analysis of HRV . NotablyBecause ultrasonographic diagnostic equipment is used to measure SMA BFV, respiration needs to be controlled for a short time during the measurement. We preliminarily investigated the presence or absence of the influence of respiratory control on HRV. We investigated the change in HRV after ST36 stimulation in a protocol without SMA BFV measurement (respiratory control) and compared it with that with SMA BFV measurement. As a result, no influence of respiratory control on HRV was seen. Thus, when examining multiple organs, we need to preliminarily check possible interference among examination procedures because multiple examinations need to be conducted simultaneously.When SMA BFV increased after ST36 stimulation, HF and LF in HRV showed a significant increase and differed from those observed after LR3 stimulation. This indicated that an increase of vagus nerve activity most likely is involved in an increase of SMA BFV induced by ST36 stimulation. This result also demonstrated acupoint specificity.


There was no significant difference in genotypic distribution and allelic frequencies of the BDNF gene C270T polimorphism between the PE and control groups. However, the BDNF gene TT genotype was more frequently seen in the epileptic children . Interestingly, in the epilepsy group, both two children with TT genotype have posttraumatic epilepsy. The data indicate a possible association with the 270T genotype of the BDNF gene with a posttraumatic epilepsy. To draw any conclusion, further studies using larger sample sizes should be carried out in various ethnic populations in childhood epilepsies.Brain-derived factor (BDNF) is a member of neurotrophin family and is localized and upregulated in areas implicated in epileptogenesis. Several lines of evidence make the BDNF gene a plausible candidate gene for predisposition to epilepsy. In this study, we tested that BDNF might be involved in the etiology of childhood PE. To assess whether BDNF gene C270T polimorphism could be implicated in vulnerability to PE, we conducted a case-control association analysis  in Turkish children. Epileptic children were divided into two groups: 1—idiopathic ( Epilepsy is a condition or a group of conditions involving transient disturbances in cerebral function caused by abnormal neuronal discharges and characterized by recurrent seizures. Epilepsy is divided into idiopathic, cryptogenic, and symptomatic forms. Among them, idiopathic epilepsy means that the primary etiology of epilepsy is believed to be genetic, and no any underlying pathological disturbance exists . Recent in vivo and in vitro and exogenous BDNF induces spontaneous seizures in rats [P = 0.003). As indicated by Lohoff et al. [Brain-derived neurotrophic factor (BDNF), a small dimeric protein and a member of the neurotrophic factor, is expressed widely throughout in the developing brain. Various animal experiments as well as clinical studies have shown that BDNF regulates neuronal morphology, synaptogenesis, and neuroprotective effects in diverse areas of the CNS during development . Several in rats , 6. Furt in rats , 8 and i in rats , 10. Var in rats  recently in rats  reportedf et al. , the C/Tf et al. . Becausef et al. –20 than In our previous study , we inven = 100) comprised unrelated neurologically normal individuals. Diagnoses were carried out from seizure symptomatology , and those who showed focal EEG abnormalities or focal lesions on imaging studies but did not have corresponding seizure symptoms suggestive of focal origin were excluded. Study group was divided into two categories: those with PE and MRI evidence (symptomatic epilepsy) (n = 27), those with PE essentially normal MRI findings (idiopathic epilepsy) (n = 85).This study included all outpatients of the Dr. Behçet Uz Child Disease and Pediatric Surgery Training and Research Hospital-Pediatric Neurology Polyclinic from 2008 to December 2010 who gave their informed consent. The patient group consisted of 112 children, 62.5% males. The control group ; 5x of Tuneup solution; 25 nM of each primer; 1 U of Taq polymerase . The amplification protocol conditions selected were as follows: initial denaturation at 95°C for 3 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 45 s, extension at 72°C for 45 s, and a final extension at 72°C for 7 min. PCR products were purified using PureHelix PCR Purification Kit according to the manufacturer's instructions  and subjected to automatic sequence analysis  by BigDye terminator reaction according to the supplier's instructions  using BDNF-F: 5′-CAG AGG AGC CAG CCC GCT GCG-3′ primer. The obtained sequences were analyzed using BioEdit software, version 7.0.5.3 (http://www.mbio.ncsu.edu/bioedit/bioedit.html).Genomic DNA was extracted from peripheral whole blood using NucleoSpin Blood kit according to the manufacturer's instructions . The C270T polymorphism of intron 1 of the 5_-UTR sequence was first reported by Kunugi et al. (GenBank accession no. X60202) . The genχ2 test. A value of P < 0.05 was considered statistically significant.Statistical analysis was performed by using PE group consists of 112 children with ages ranging from 2 to 17 years and an average age of 10.4 (SD: 3.18) years, those with 85 (75.9%) children in the idiopathic epilepsy group and 27 (24.1%) children in the symptomatic group. One hundred control children  with mean age 10.8 (SD: 3.15) were enrolled in the study.P = 0.748). Comparison of each group by sex and BDNF 270T allele frequencies is presented in BDNF 270T allele was found to be in 15 (13%) epileptic and 11 (11%) control children. The BDNF 270T allele variation was not significantly different between the two groups (P = 0.379). Interestingly, in the epilepsy group, both two children with TT genotype have posttraumatic epilepsy. Further, for the genotype frequencies of BDNF gene polymorphism, all two patient subgroups (idiopathic/symptomatic epilepsy), epileptic children with mental retardation and cerebral palsy showed similar distribution of the 270C/T allele . There was no statisticaly significant difference between the epilepsy and control groups for allele frequency of BDNF gene polymorphism (P = 0.738).The determination of allelic frequencies of the BDNF gene for idiopathic and symptomatic epilepsy patients is presented in Various mechanisms and associated genes have been shown to role in the epileptogenesis, and currently there are many problems in the treatment of epilepsy a need to new solutions. A genetic contribution to the development of PE as a sequel of acquired focal brain disorder has been suggested for a long time. Rimoin and Metrakos  concludeIn our study, we did not find any significant difference between the PE and control groups for the BDNF C270T variation. To our knowledge, this was the first study which investigates this polymorphism in the childhood epilepsy. Kanemoto et al.  detectedIn search of a polymorphism in the BDNF gene an associated study by Kunugi et al.  found thin vivo. Thus, transgenic mice with decreased BDNF have a higher seizure threshold, and overexpression of BDNF facilitates seizures [γ-aminobutyric acid (GABA)ergic transmission, phosphorilation of N-methyl-d-aspartate (NMDA) receptors, and actions at sodium channels [Various studies have shown that BDNF increased neuronal excitability and is the ability to potentiate glutamatergic synaptic transmission. BDNF is highly localized and upregulated in areas implicated in epileptogenesis. Indeed, it is perhaps no coincidence that BDNF-immunoreactive fibers innervate the regions most vulnerable in temporal lobe epilepsy . BDNF mRseizures . Particuseizures . Both thchannels –32. ThesMost of the epilepsy-associated genes identified so far encode ion channels. Interestingly, those proteins encoded by the nonion channel genes have been suggested to interact with ion channels. This is conceivable from a pathophysiological point of view, as ion channels provide the basis for both the electrophysiological excitability of neuronal cell membranes and the communication between neurons. Also, most anticonvulsant drugs in clinical use today modulate different types of ion channels . GeneticThe major hypotheses for the functional effects of insult-induced neurotrophin changes are protection against neuronal damage and stimulation of sprouting and synaptic reorganization; therefore, a BDNF gene polymorphism is an ideal candidate for a genetic predisposition that regulates development of epileptogenesis following insult to the brain . The BDNThere was an idiopathic PE dominance, in our patient population. Some PEs such as Rolandic epilepsy have long been known to have a strong genetic contribution; however, the strictly age-dependent nature as well as family history associated with that condition presents a unique clinical picture suggestive of a close kinship with idiopathic generalized epilepsies. Also, many sporadic PE cases cannot be explained as a single-gene disease, because they seem as a polygenic or multifactorial. Nevertheless, genetic and acquired risk factors are frequent in PE patients . In summ


The possible use of municipal solid waste compost (MSWC) in the production of marigold and basil seedlings examined. Six medium prepared from commercial peat (CP) and MSWC . There was not any plant growth when MSWC used alone (100%). The addition of MSWC in low content (15% and 30%) improved seed emergence for marigold and basil respectively, while greater content revealed opposed impacts. Mean emergence time delayed as MSWC content increased into substrates. Addition of MSWC  into CP reduced (from 34 to 64%) plant height, leaf number and stem diameter as a consequence reduced plant fresh weight (plant biomass) for both species. The number of lateral stems decreased (up to 81%) in basil when MSWC added into substrate mixtures. Chlorophyll b content decreased (up to 58%) in substrates with MSWC content greater than 15% or 30% while similar reduction observed in content of Chlorophyll a and total carotenoids for basil with MSWC > 60%. However, Chlorophyll a and total carotenoids content increased as MSWC content increased for marigold. K and Na leaf content increased but P equivalent decreased as MSWC content increased. Nursery-produced basil and marigold seedlings grown in 15% MSWC; displayed quality indices similar to those recorded for conventional mixtures of peat and may act as component substitute. Transplants, compared with direct sowing, are a more reliable method of ensuring the proper establishment of a range of commercial horticultural crops with great economic value. The production of container-grown flowers and aromatic plants is a highly competitive business; uniform and rapid seed emergence is essential prerequisites to increase yield, quality, and profits in crops. Use of good crop substrates is therefore critical .It is well known that peat, a wide used substrate, is a nonrenewable resource, and diminishing availability is prompting price increases. The extensive use of peat as a substrate has led growers to consider its replacement in the medium to long term  with altMunicipal solid waste compost (MSWC) as an organic soil additive when applied in field trials suggested that it can be used in agricultural production, improving soil physicochemical properties, increasing water retention as well as supply with considerable amount of essential nutrients , 9. HoweA previous study showed that the growth and development of nursery-produced tomato seedlings using a peat and MSWC mixture was similar to that obtained with the standard peat mixture . The preTagetes erecta L. cv Erecta) and basil (Ocimum basilicum L. cv sweet basil) were purchased from Agrospito company  and Agrimore , respectively.Seeds of marigold , based in Chania. The compost used was made from the organic fraction of selectively collected urban waste. Following electromagnetic separation, manual sorting and use of an 80 mm trommel screen to remove as many bulking agents as possible, organic material was arranged in piles of 5 m wide of 2.5 m high of 45 m long, which were regularly turned and watered over a 140-day period to ensure appropriate composting conditions (turned windrow system). This material was then passed through a densimetric table and a 15 mm trommel screen to remove the largest particles. The composting procedure lasted for 5-6 months. The 60% of compost consisted of particles with <4 mm size. Marigold and basil seeds were used for nursery tests. A mix of commercial compost peat , perlite , and MSWC were used to create six treatments which were (% v/v) (1) peat : MSWC (100 : 0) as control, (2) peat : MSWC (85 : 15), (3) peat : MSWC (70 : 30), (4) peat : MSWC (55 : 45), (5) peat : MSWC (40 : 60), and (6) peat : MSWC (0 : 100). In each substrate medium was added 10% of perlite. Perlite physicochemical properties were reported in previous studies .3 well capacity)) on top of the surface of the media. The experiment was carried out in a completely randomized design in an unheated glasshouse with a north-south orientation at the School of Agricultural Technology at Heraklion, Crete, Greece, located at the latitude of 35′ 35°N, longitude 24′ 02°E, and 8 m altitude : 93.5 ± 1.9 max, 74.8 ± 4.1 min) with alternate-day watering by mist system . Over the growth-period in the nursery, no fertilizer was applied; seedling nutritional requirements were thus met entirely by the substrates. Daily observations were recorded for seed germination (seeds recorded as emerged when the hypocotyls appeared above the surface of substrate medium). After 15 days seedlings were thinned to single plant, maintaining 4-5 cm distance among seedlings. Mean germination time (MGT) was calculated as follows, according to Labouriau [Seeds were sown  of chlorophyll a (Chla), chlorophyll b (Chlb), and total carotenoids (Car) determined. Leaf elemental analysis for K, Na (photometric), P (spectrophotometric), and N (Kjeldahl) was determined.After 48 days, seedling growth was assessed by harvesting six individuals/treatment. Seedlings were harvested above substrate, the leaf number and height (cm) per seedling, measured from substrate surface, stem diameter (mm) measured below the cotyledon node, number of stems (for basil), number of flowers (for marigold), upper fresh weight (g), total dry matter content (%), leaf fluoresces, content . Significant differences between mean values were determined using Duncan's Multiple Range test following One-Way ANOVA. Various correlations were also calculated. Statistical analyses were performed using SPSS .The main physicochemical characteristics of compost (dry weight: dwt) were pH, 7.7; electrical conductivity (EC), 18.2 mS/cm; ashes, 50.6% dwt; organic matter, 49.4% dwt; Carbon, 27.5% dwt; N, 1.9% dwt; and ratio C/N, 7.2, under low limits for heavy metal content. The C/N ratio is widely used as an indicator of the maturity and stability of organic matter. The low values recorded here for the C/N ratio in MSWC suggest that composts were stable and mature. Davidson et al.  reportedThe first germination was observed after two and six days of sowing for marigold and basil, respectively, while the first true-leaf was emerged after five and nine days, respectively. The ratios of mix when peat and MSWC were used affected seed germination/emergence . MarigolThe ratios of mix when CP and/or MSWC were used affected seed MGT . For marAdding MSWC into the substrate in low content benefits the seed germination/emergence, possible due to the fact that MSWC provided nutritional value as organic material and/or improved substrate medium properties. The stimulation of several presowing treatments  of seedAlthough there is no single, ideal growth medium for nursery-produced horticultural crops , most grSeedling growth failed when 100% MSWC was used as substrate due to seed emergence failure. The highest values for all growth parameters were recorded in substrate without MSWC (control), which in most cases differed significantly from the other substrates Tables . AnalyseIn marigold seedlings, significant increases in Chla and total carotenoids were observed in substrates with >60% MSWC and >45% MSWC, respectively, while Chlb decreased when the content of MSWC was >45% . In basiLeaf elemental content for marigold revealed that K and Na increase  with the addition of MSWC into the substrate, being in agreement with melon seedling production with the same MSWC , while PMSW compost was found to be an ideal component of mixed-peat substrates for marigold and basil seedlings, provided that it accounts for less than 15% of the mixture. These proportions reduce the negative effects of high pH and EC on seedling growth and provide a seedling comparable to that obtained using standard peat-based mixtures, being in agreement with previous study in melon seedling production . Thus, tThere is a growing public concern about the environmental impact of industrial development and population expansion in recent decades. Improved methods of selective waste collection and compost processing will enable increasingly widespread use of this renewable organic compost, as an alternative to high-quality sphagnum peat, which—because they are nonrenewable—are less available and more expensive for growers.


The methanol extracts of Mesua beccariana showed its potency towards the proliferation of B-lymphoma cell (Raji). In addition, only the nonpolar to semipolar extracts (hexane to ethyl acetate) of the three Mesua species indicated cytotoxic effects on the tested panel of human cancer cell lines. Antioxidant assays were evaluated using DPPH scavenging radical assay and Folin-Ciocalteu method. The methanol extracts of M. beccariana and M. ferrea showed high antioxidant activities with low EC50 values of 12.70 and 9.77 μg/mL, respectively, which are comparable to that of ascorbic acid (EC50 = 5.62 μg/mL). Antibacterial tests were carried out using four Gram positive and four Gram negative bacteria on Mesua beccariana extracts. All the extracts showed negative results in the inhibition of Gram negative bacteria. Nevertheless, methanol extracts showed some activities against Gram positive bacteria which are Bacillus cereus, methicillin-sensitive Staphylococcus aureus (MSSA), and methicillin-resistant Staphylococcus aureus (MRSA), while the hexane extract also contributed some activities towards Bacillus cereus.The  Mesua species belong to the Clusiaceae family and consist of many health-promoting bioactive compounds [Mesua ferrea leaf and fruit extracts showed both methanolic extracts to exhibit good antibacterial activity towards Staphylococcus aureus [Mesua species have shown that these plants exhibit many biological activities such as antioxidant [Mesua beccariana, Mesua ferrea, and Mesua congestiflora against a panel of human cancer cell lines and DPPH scavenging agent. Furthermore, antimicrobial assay was also carried out using four Gram positive and four Gram negative bacteria on the Mesua beccariana extracts. Preliminary screening results of cytotoxic and antioxidant activities for Mesua beccariana, Mesua ferrea, and Mesua congestiflora will be reported. We have reported a novel cyclodione coumarin together with other known compounds from Mesua beccariana in a previous paper [Traditional medicines for human diseases have been widely used in many parts of the world. Herbal plants are usually the primary source of medicine in many developing countries. Natural product compounds from plants provide biologically active compounds, many of which have been developed as new lead chemicals for pharmaceuticals . Only a ompounds –10. Reces aureus . Previouioxidant –14, hepaioxidant , antiartioxidant , immunomioxidant , antibacioxidant , antiaceioxidant , and larioxidant  activitiioxidant , 20–22. us paper .Mesua beccariana (Baill.) Kosterm., root bark of Mesua ferrea Linn., and roots of Mesua congestiflora were collected from the Sri Aman district in Sarawak, Malaysia. All the plant materials were identified by Associate Professor Dr. Rusea Go, Department of Biology, Faculty of Science, Universiti Putra Malaysia and deposited in the herbarium of the Department of Chemistry, Faculty of Science, Universiti Putra Malaysia. The voucher specimen numbers for M. beccariana, M. ferrea, and M. congestiflora are RG211, RG2694, and RG203, respectively.The stem bark of 2 humidified incubator at 37°C (ESCO Celculture CO2 Incubator with model number CCL-170B-8). Cell stocks were placed in a −86°C  ultralow temperature freezer (Scancool SCL 50 P) and preserved in a liquid nitrogen tank (Taylor-Wharton LS300).All the apparatus used for cell culture were sterilized and decontaminated using Hirayama HICLAVE HVE-50. Cell culture handling was carried out in an ESCO Class II BSC Biosafety Cabinet. The healthy cells were spun down, adhered together, and separated from unhealthy and dead cells by using Thermo Scientific Sorvall ST 16R centrifuge machine. All cultures were incubated in 5% COThe aerial parts of the plant samples were extracted using the conventional extraction method in which solvent was added and removed in batches. The dried and milled samples were soaked in nonpolar (hexane), medium polar  and polar (methanol) solvents in a polarity increasing order. The solvent was decanted and replaced with new solvent after 48 hours.Mesua beccariana stem bark, Mesua ferrea roots, and Mesua congestiflora stem bark were extracted successively with n-hexane (Hex), dichloromethane (DCM), ethyl acetate (EA), and methanol (MeOH). The extracts were dried under reduced pressure using a rotary evaporator. Mesua species.The air-dried and powdered 2 T-flask and maintained at 37°C in 5% CO2 humidified incubator. The MTT assay is based on the protocol illustrated by Mosmann [μg/mL were prepared by a serial dilution method. An aliquot of 100 μL of each substock with different concentrations was added to each well together with 100 μL of selected cells to give concentrations of 100.00, 50.00, 25.00, 12.50, 6.25, and 3.13 μg/mL and made up to a final volume of 200 μL in each well. 200 μL of cells with no extracts (untreated cell control—positive control) and 200 μL of medium only (blank medium—negative control) were prepared in the same plate. Each of the samples and controls were prepared in triplicates.Preliminary screening tests for cytotoxic activity against a panel of human cancer cell lines on the crude extracts and quercetin (standard) were undertaken by using MTT -2,5-diphenyltetra zolium bromide) assay. The nine tested cancer cell lines were Raji (human B lymphocyte), SNU-1 (human gastric carcinoma), K562 (human erythroleukemia cells), LS-174T , HeLa , SK-MEL-28 , NCI-H23 (human lung adenocarcinoma), IMR-32 (human neuroblastoma), and Hep-G2 (human hepatocellular liver carcinoma). The Raji, SNU-1, K562, HeLa, Hep-G2, and NCI-H23 cells were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), while the LS-174T, SK-MEL-28, and IMR-32 were maintained in MEM supplemented with 10% FBS. All the cell lines were cultured in 75 cm Mosmann  and was 2 humidifier incubator. After 72 hours, 20 μL of MTT solution was added to all the wells and incubated for 3 hours in a 5% CO2 humidifier incubator. The plate was then spun at 3000 rpm for 10 minutes. 160 μL of supernatant from each well was discarded and then added with 160 μL of DMSO to dissolve the purple formazan crystals. The plate was then incubated for 72 hours at 37°C in 5% COA is the absorbance of sample, B is the absorbance of negative control, and C is the absorbance of positive control.The absorbance of each well was determined using a microplate reader at 550 nm. The average absorbance of each crude extract was calculated, and the average value was used to determine the percentage of cell viability by using the following formula:50) values were obtained from the plotted graph. Further dilutions will only be performed on the extracts with IC50 values less than 3.13 μg/mL. Three independent experiments were conducted to assure the accuracy of the results. Quercetin was used as standard drug throughout the cytotoxicity experiments.A graph of percentage of cell viability versus concentration was plotted for each extract. The half maximal inhibitory concentration  were prepared in the same plate. Each sample and the control were prepared in triplicates. The plate was then incubated in a dark room at room temperature for 30 minutes. The absorbance of each well was recorded after 30 minutes using a microplate reader at 517 nm. The average absorbance of each crude extract was calculated, and the average value was used to determine the percentage of total radical scavenging activity by using the following formula:A is the absorbance of blank, and B is the absorbance of sample.The DPPH  radical scavenging assay was used to evaluate the antioxidant property of the sample. The assay was performed in 96-well flat bottom plates. The DPPH solution was prepared in a stock solution of 5 mg/2 mL in ethanol (EtOH) and wrapped with aluminium foil. The varying concentrations of 3.13, 6.25, 12.50, 25.00, 50.00, and 100.00 50) values were obtained from the plotted graphs. Three independent experiments were conducted to ensure the precision of the results. Ascorbic acid (vitamin C) was used as the standard drug.A graph of percentage of total radical scavenging activity versus concentration was plotted for each extracts. The half maximal effective concentration  and subsequently neutralized by sodium bicarbonate, NaHCOy = 0.0026x + 0.0402, where y is absorbance and x is gallic acid content in μg/mL. This equation was used for each extract to determine their percentage of gallic acid content. The total phenolic contents of crude extracts were expressed as gallic acid equivalent . A standard curve was constructed using the equation μL of each solution with different concentrations of gallic acid was added into each well together with 750 μL of FC reagent (previously diluted 10fold with distilled water). A blank control solution (EtOH without extracts) was prepared in the same plate. Consequently, 750 μL of 60 g/L (60 g in 1 L distilled water) of NaHCO3 was added into each well and left in the dark for 90 minutes. The absorbance of each extracts was measured at 725 nm. The experiment was executed in triplicate for accuracy. All the previous steps were repeated by different extracts with only one concentration which was 500 μg/mL.An aliquot of 100 8 coliform units (cfu)/mL by diluting each suspension in 10 mL of sterile distilled water to the appropriate density. Nutrient agar (NA) and Mueller Hinton agar (MHA) were prepared according to the manufacturer's instructions. The medium was cooled to 50°C after autoclaving, before approximately 25.0 to 30.0 mL of the medium was poured into 15 × 100 mm plastic petri dishes at a uniform depth of 4 mm. Each type of microorganism (from the stock culture) was streaked onto a noninhibitory NA plate to obtain isolated colonies. After an overnight incubation (16–20 hours) at 37°C, one single colony was selected and inoculated with a loop, transferred into 10.0 mL of sterile nutrient broth (NB), and incubated in a shaking incubator at 37°C for 16–20 hours. The density of bacteria was standardized using McFarland 0.5 turbidity standard to 1 × 10μg or vancomycin 30 μg) and negative controls (blank disc impregnated with methanol) were used in the antibacterial test. The plates were left at 4°C for an hour to allow for the diffusion of the extracts before they were incubated for 16–20 hours at 37°C, and the diameter of the inhibition zones was then measured [The antibacterial assay was performed using disc diffusion (Kirby-Bauer) method as described by Aksoy et al. The standardized bacterial suspensions were swabbed on MHA surface using sterile cotton wool. For initial screening, 1 mg of DCM : MeOH extract was loaded onto each Whatman No. 1 filter paper discs  and were impregnated. The positive , a crude extract can be considered active if it possesses an IC20 μg/mL , 27. FroM. ferrea displayed moderate to strong antiproliferative effects against all the cancer cell lines except for the Raji cells. The IC50 values were in the range of 8.85 to 43.75 μg/mL. However, the DCM extract barely gave any activity towards the HeLa cells, while the ethyl acetate and methanol extract, were inactive towards the proliferation of the tested cell lines.On the other hand, the hexane and dichloromethane extracts of Mesua congestiflora indicated mild cytotoxicities towards SNU-1, K562, and NCI-H23 cells and weak cytotoxic activity towards the rest of the cell lines. Both the ethyl acetate and methanol extracts were not cytotoxic towards all the investigated cells.Additionally, the hexane extract of Mesua beccariana as shown in 50 values ranging from 2.00 to 31.25 μg/mL. In our previous paper, we reported eleven xanthones from the three plant species, and three of which were very cytotoxic towards all the nine cancer cell lines tested. The structure-activity relationship (SAR) study revealed that the diprenyl, dipyrano, and prenylated pyrano substituent groups of the xanthone derivatives contributed towards the cytotoxicities [In conclusion, only nonpolar to semipolar extracts contribute to the cytotoxic effects on the panel of human cancer cell lines except for the polar extract of xicities . Moreovexicities , 21.Mesua beccariana, Mesua ferrea, and Mesua congestiflora along with ascorbic acid (standard drug) at different concentrations were evaluated for their scavenging potentials on the DPPH free radical.All the extracts of Mesua beccariana and Mesua ferrea exhibited significant scavenging activity with EC50 values of 12.70 and 9.77 μg/mL which are comparable with ascorbic acid (EC50 = 5.62 μg/mL). The ethyl acetate (EA) extract of Mesua beccariana and Mesua congestiflora as well as the dichloromethane (DCM) extract of Mesua ferrea reflected mild scavenging capability, while the dichloromethane extract of Mesua beccariana gave weak scavenging effect. Besides this, the hexane extract of all three Mesua species, the EA extract of Mesua ferrea, and MeOH extract of Mesua congestiflora were seen to be inactive towards DPPH scavenging agent.Several semipolar to polar extracts possess medium to strong scavenging activity against DPPH radical where the methanol (MeOH) extracts of 50 values for the extracts of three Mesua species towards DPPH free radicals are summarized in  The ECMesua beccariana and Mesua ferrea exhibited the highest phenolic contents among the extracts with values of 363.82 and 441.33 μg in gallic acid equivalent (GAE), whereas the ethyl acetate extracts of both species also possess moderate phenolic contents with values of 145.26 and 206.67 μg in GAE. Inversely, the ethyl acetate extract of Mesua congestiflora gave a higher phenolic content compared to its methanol extract with values of 369.26 and 273.87 μg in GAE, respectively. The hexane and dichloromethane extracts of the three Mesua species contributed low to moderate phenolic contents with GAE values less than 130 μg of gallic acid per mg of extract. The total phenolic content (TPC) analysis was carried out on all the crude extracts by using Folin-Ciocalteu method. The TPC values give information on the phenolic contents of the crude extracts and their antioxidant activities. The methanolic extracts of Mesua beccariana were screened against both Gram positive and Gram negative bacteria. Streptomycin and vancomycin were used as positive control standards in the assay. The four types of Gram positive bacteria used were Bacillus cereus, Micrococcus luteus, methicillin-sensitive Staphylococcus aureus (MSSA), and methicillin-resistant Staphylococcus aureus (MRSA), while four types of Gram negative bacteria including Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, and Enterobacter aerogenes were tested in this experiment. All the crude extracts showed negative results in the inhibition of Gram negative bacteria. The methanol extracts, however, showed some activity against B. cereus, MSSA, and MRSA. In addition, the hexane extract also gave weak activity against B. cereus bacterium. Mesua beccariana.The extracts of Mesua species could be a good source for the development of lead compounds.Both DPPH radical assay and total phenolic content indicate that semipolar to polar extracts had high antioxidant properties, while non-polar extracts exhibited good cytotoxic activity. The pharmacognosy investigation showed adverse effects of crude extracts suggesting that the three


Background: Although cyclosporine (CsA) and calcium channel blockers (CCBs) parallel to each other may provoke gingival enlargement (GE), there are few considerations about combined effects of CsA and CCBs on gingival tissues.Objectives: This study aimed to determine prevalence of GE among renal transplant recipients and to compare its occurrence in patients who received only CsA and those who were on CsA and amlodipine.Patients and Methods: We conducted a prospective randomized case-control trial including 213 renal transplant recipients between February 2010 and August 2010. They were randomly divided into two groups including control group  and trial group . Buccal, lingual, and inter-proximal membranes at last 12 anterior teeth were assessed for GE and packet depth (PD) using Gingival Index of McGaw and others, and Packet Index of Turesky–Gilmore–Glickman, respectively.Results: Marked GE was observed in 26 patients (25.7%) in trial group and only in 4 individuals (3.6%) in control group (P = 0.000). In logistic regression analysis, obese , older , and female  recipients as well as who received high dose amlodipine  were at risk for marked GE.Conclusions: There is a strong correlation between GE, in particular marked GE, and combination therapy with CsA and amlodipine in transplant patients compared to those treated by CsA alone. We suggest CsA dose reduction may restrain this adverse effect. Cyclosporine (CsA) has been widely prescribed to prevent graft rejection after kidney transplantation . HoweverDrug induced GE is a clinical condition characterized by an increase in the size of gingival tissues leading to an alteration in gingival contour . It coulAlthough CsA and CCB parallel to each other may provoke GE, there are few considerations about combined effects of CsA and CCB on gingival tissues. Therefore, this study aimed to determine prevalence of GE among renal transplant recipients and to compare its occurrence in patients who received only CsA and those who were managed by CsA and amlodipine.We conducted a prospective randomized case-control trial including 213 renal transplant recipients to evaluate correlation between amlodipine and GE. Living and deceased kidney transplants recipients were both included. Eligible subjects had at least six maxillary and six mandibular anterior teeth with stable renal function and maintained on triple immunosuppressive therapy . Subjects were excluded if they exhibited poor oral hygiene, had a history of kidney transplantation less than 3 months, managed by anticonvulsant drugs or oral contraceptives, suffered from major illnesses, or were pregnant. They were randomly divided into two groups of control  and trial . The current study protocol was approved by the local Ethics Committee of Baqiyatallah University of Medical Sciences and an informed consent was obtained from all patients.The clinical data collected for all patients included age at transplantation and at diagnosis, gender, body mass index (weight/hieght2), CsA dose, GE, PD, and amlodipine dose and treatment duration . Biochemical data such as plasma creatinine concentration, and C0 (through) and C2 (2 hour post dose) blood levels of CsA were also assessed.Marked PD and marked GE were defined as PD more than 1.5 and GE more than 2.5.Immunosuppression was maintained in all patients based on CsA plus mycophenolate mofetil, and prednisolone. The amount of cyclosporine given to transplant patients was mostly tailored by blood levels of drug . CsA monitoring using its blood levels was periodically performed at different times and the dose was adjusted if necessary. Cyclosporine was targeted at 150-250 ng/mL through blood level during 3 months and tapered subsequently to 100-150 ng/mL by 1 year, while we performed C2 target levels 800 to 1000 ng/mL in month one to three after transplantation, and C2 target levels 400 to 600 ng/mL thereafter. Control patients were only maintained on CsA microemulsion but trial patients while continued to receive CsA, amlodipine was added to control their hypertension at initial dose of 2.5 mg twice daily with subsequent dose adjustment.During February 2010 to August 2010, all patients were examined for periodontal status by an expert periodontist who was blinded to both control and trial groups. Buccal, lingual, and inter-proximal membranes at last 12 anterior teeth were assessed for GE and packet depth (PD) using Gingival Index of McGaw and others (12), and Packet Index of Turesky–Gilmore–Glickman (13), respectively.Statistical analysis was performed using SPSS version 17.0 for Windows. All quantitative variables have been expressed as mean ± SD and the qualitative variables have been shown by percentage. Continuous data were compared by Student’s t-test and Mann-Withney, and categorical data were analyzed by Chi-square or Fisher’s exact test. Univariate and multivariate analysis were performed to evaluate correlation between GE and PD with confounding factors using a logistic model. Independent variables with P values ≤ 0.2 in univariate analysis were entered into a multivariate logistic regression model. P values less than 0.05 were considered statistically significant.No significant differences were observed between two groups in terms of gender, age at transplantation, age at diagnosis of GE, BMI, and renal allograft function . The meaAs a whole, no significant differences were observed between two groups unless about GE which was higher in trial group , and C0 and C2 levels which were more significant in control group (P = 0.02 and P = 0.001); however, the doses of CsA in both groups were not significantly different . FrequenThe correlation between marked GE and marked PD with CsA blood levels in trial and control groups were summarized in The mean amount of GE and PD were 0.72 ± 0.6 (0-2.7) and 2.7 ± 0.9 (0.33-4.8), respectively; whereas marked GE and marked PD were observed in 14% (n = 30) and in 63% (n = 134) of all patients, respectively. In addition, marked GE was seen in 26 (25.7%) patients of trial group but in only 4 (3.6%) individuals in control group (P = 0.000). Furthermore, marked PD was observed in 61 (60.4%) cases in trial group and in 73 (65.2%) subjects in control group (P = 0.4). We also found a marked PD in older recipients . Higher doses of amlodipine was significantly associated with marked GE .At univariate analysis in trial group, PD enhanced with increasing dose of amlodipine (0.008); also GE was correlated with high dose of amlodipine (P = 0.000) and greater BMI (P = 0.01) . FurtherAlthough marked GE was significantly correlated with higher doses of amlodipine (P = 0.000), this correlation was modest in lower levels of CsA . Higher Obese , older , and female  recipients as well as who received high doses of amlodipine  were at risk for marked GE .The older age , high C0 level , and high dose of amlodipine  were risk factors for marked PD .Drug induced GE is a well-documented complication in renal transplant recipients. The highest prevalence of GE is reported among those treated with CsA and CCBs (76%) , consistAdvance reports have revealed that more severe gingival changes developed in patients who received a combination of CsA and CCB than those who treated only by CsA , 15, 16.Drug induced GE should be treated based on the medication being used and clinical presentation of each individual. First of all, we have to consider the possibility of ceasing or replacing the drug. Simple withdrawal of offending agent is usually not a practical decision. However, its replacement with another medication might be a practical solution . CsA disIn our transplant center, we monitor recipients with CsA blood levels and its side effects. In the presence of CsA side effects, dose reduction is the major approach. According to Rodwan et al. study in 2003  CsA dosaHowever, the prevalence of GE in our patients was lower than that in report of James et al.  showing The fact that marked PD was more dominant in all recipients and in control group than marked GE, motivate a theory that possibly PD – compared to GE - was more influenced by CsA This theory might be supported by the evidence that in trial group which –compared to control group - CsA was prescribed in lower dose, occurrence of PD was less dominant. In contrary, lower prevalence of GE in all recipients and control group, may support this theory that amlodipine potentially induce dose dependent GE more frequent than PD. It seems that the extent of PD depends on other contributing factors, too.There are so many potential risk factors such as genetic susceptibility, oral hygiene, and demographic, pharmacologic, and periodontal variables which contribute GE and PD -28. In RWe suggest co-management of hypertension by other drugs such as aldosterone receptor blockers or CCBs with lower side effects like diltiazem that did not induce GE . Prescri


Neurologic impairments in female heterozygotes for X-linked Adrenoleukodystrophy (X-ALD) are poorly understood. Our aims were to describe the neurological and neurophysiological manifestations of a cohort of X-ALD heterozygotes, and to correlate them with age, disease duration, mutations, X-inactivation and serum concentrations of a marker of neuronal damage, neuron-specific enolase (NSE).All 45 heterozygotes identified in our region, with previous VLCFA and molecular diagnosis, were invited to be evaluated through myelopathy scales JOA and SSPROM, nerve conduction studies and somatosensory evoked responses. X inactivation pattern was tested by HUMARA methylation assay. Serum NSE was measured by eletrochemiluminescense.Thirty three heterozygote women were recruited: 29 (87%) were symptomatic. Symptomatic and asymptomatic women presented different m ± sd ages (43.9 ± 10.2 versus 24.3 ± 4.6), JOA (14.5 ± 1.7 versus 16.6 ± 0.2) and SSPROM (86.6 ± 7.9 versus 98.4 ± 1.1) scores (p < 0.05). Both JOA (r = −0.68) and SSPROM (r = −0.65) correlated with age, irrespectively of the disease status . Delayed latencies in the central ascending conduction studies on the lower limbs were present in 72% of all heterozygotes, and correlated with SSPROM . NSE values were higher in heterozygote than in control women . Mutation severity and inactivation patterns were not associated with neurologic status.Neurologic manifestations, clearly related to age, were quite common in the present cohort. JOA and SSPROM scales were able to discriminate the asymptomatic from the symptomatic heterozygotes. Both scales might be useful tools to follow disease progression, in future studies. ABCD1 gene that codes for ALD protein , where d = digested, u = undigested sample, and the two allele peaks (1 and 2) are compared [Genomic DNA was obtained from peripheral leukocytes by the salting out procedure . X-inactcompared . Inactivcompared .Patient characteristics are given as mean ± SD and range, when applicable. Categorical variables were represented by absolute and relative frequencies and were compared through Fisher exact test. The majority of continuous variables did not show a normal distribution on the Shapiro-Wilk test, and therefore were tested by Mann-Whitney U, Spearman correlation and, when applicable, by stepwise linear regression. Statistical significance was defined as p < 0.05. All statistical tests were performed in PASW 18.3.Forty five heterozygote women (belonging to 25 families) had been detected by molecular studies in our institution, from 2008 to 2013. Thirty three heterozygotes  gave their consent and were included in this study. The another 27% (12 women) were lost: six were not found by two phone calls and letter, two refused to participate in the study and another four fulfilled exclusion criteria. The mean ± sd ages of the 33 participants and of the 12 non-participants, in 2013, were of 41.2 ± 11.9 and 40.9 ± 15, respectively . Twenty-nine of the 33 participants classified themselves as symptomatic and four, as asymptomatic, after learning about X-ALD symptoms in females. Their general characteristics are present in Table Symptomatic were significantly older and presented worse JOA and SSPROM scores and higher disability scores than asymptomatic women. Significant differences in SSPROM scores were due to a high proportion of symptomatic females (27/29) with spasticity/hyperreflexia; and to motor disabilities Table . The autAmong symptomatic women, although SSPROM correlated with age and DD, only DD was maintained on linear regression . When all heterozygote women were analyzed together, the association with age was confirmed  . When all heterozygotes were analyzed together, JOA correlated with age  , among symptomatic women, were 3.7 ± 2.6. This score did not correlate with age or any other parameters.Symptomatic classification produced more associations with independent parameters than the disability classification, in this cohort. Due to that, it was maintained as the stratification criteria between the present heterozygote women, when necessary.Twenty-two out of 33 heterozygotes (20 symptomatic and 2 asymptomatic women) performed neurophysiologic studies. The others denied for personal reasons. Nineteen (including one asymptomatic heterozygote) presented at least one altered proof. Results are presented in Table All m-NCV and most of s-NCV were normal, and there were no correlations between NCVs and symptomatic status, age, AO, DD, disability status, JOA, SSPROM, disease severity, inactivation studies or plasma NSE concentrations (data not shown). Twelve out of 20 symptomatic heterozygotes showed reduced s-NCV of the median nerve, measured on the elbow. This finding was unrelated to any variable under study (data not shown).No evidence of generalized axonal damage was found in any of the women studied, since the majority of CMAPs and SNAPs amplitudes were between the normal range. The amplitude of fibular superficial nerve SNAPs correlated with AO among the heterozygotes .Twenty-five heterozygote females  were tested for SSE. Eighteen (17 symptomatic) presented delayed latencies in the central ascending conduction studies starting on the lower limbs  Table . LatenciABCD1 gene are depicted on Additional file The mutations detected on Twenty heterozygotes were informative in the technique applied to determine the inactivation pattern. Six had a skewed inactivation pattern of 70:30 or more. Skewed inactivation was not associated to symptomatic state , disability, SSPROM, JOA, disease severity  or AO . A curious trend to relate larger skewing with age was also seen .The medium ± sd values of NSE were higher in heterozygotes than in controls .VLCFA had been determined at the time of diagnosis of the present 33 heterozygotes, sometimes several years before the present evaluation. Their mean ± sd ages at the collection were of 30 ± 11.3 (range: 7 to 43) years-old. In the case of symptomatic women, for whom the symptom onset has been determined as year zero, blood collections were done at a −3.8 ± 4.5 range: -12 to 3) years from the start of their symptoms. Correlations of VLCFA levels with age at the time of blood collection and with time from disease start were then tested. There were direct correlations between age and both C26:0 and VLCFA discriminant factor .Neurological impairment was better evaluated by clinical scales than by neurophysiology. Both JOA and SSPROM scales were able to discriminate symptomatic from asymptomatic heterozygotes, with almost no overlap, correlated very well with DD in the symptomatic group, and with age in the overall group.The lack of any substantial alterations on peripheral neurophysiology reported here corroborates the findings of Schmidt and Cols . One intAfter clinical scores, the SSER of lower limbs have shown the most prevalent abnormalities in heterozygote women. Similar results have been seen by others ,27-30 SSX chromosome inactivation has been studied as a modifying factor that could influence clinical presentation in women with X linked disorders. Relating a skewed inactivation in peripheral leukocytes to a neuronal disorder, as in the heterozygote women with X-ALD, is a complex hypothesis. If this association would exist, at least two possible mechanisms should be invoked: a good mirroring between neurons and leukocytes, where a skewed X inactivation in neurons would be related to differences in clinical presentation; and/or an intercellular mechanism, operating from skewed cells other than neurons. Differently from some authors  but simiVLCFA also increased with age, at least in this cohort, whose blood collections had been done between 7 and 43 years of age. Other authors have observed a similar finding in this age group, with decreasing VLCFA concentrations in women older than 50 years old . AlthougNSE is a cytoplasmatic glycolytic enzyme found in neurons and cells with neuroendocrine differentiation. Since NSE is not physiologically secreted, an increase of its serum concentrations can be associated with structural damage to neuronal cells . NSE conAccording to former studies, around 50% of X-ALD heterozygotes showed some degree of neurologic involvement . Our resEmotional issues should also be remembered. Male X-ALD is a catastrophic disease for families. A mother or a female caregiver of an affected boy usually lives in very stressful conditions, where priorities are put in hemizygote men rather than in her well-being. Maybe the actual numbers of symptomatic heterozygote females had been previously underestimated, due to a hasty report of these women about their healthy states. The hypothesis that the majority of heterozygote females will present a neurologic impairment is not negligible. Moreover, the associations between JOA and SSPROM with age might suggest that the symptomatic status is a matter of time and that, as heterozygote females grow older, they get symptomatic.With the prominent involvement of pyramidal tracts, X-ALD presenting in symptomatic heterozygotes and as male AMN, can be seen as one form of complicated spastic paraplegia (SPG) . TherapeNeurologic impairments are frequent and are clearly related to age and to the CNS involvement, in heterozygote females. SSER of lower limbs and plasma NSE concentrations were altered in the majority of heterozygote females. Since they did not differentiate between symptomatic and asymptomatic heterozygotes, nor were related to age or disease duration, we cannot postulate them as good candidates as biomarkers of disease progression. VLFCA and skewing inactivation were also unable to separate symptomatic from asymptomatic heterozygotes. Both increased with age, but dissociated from the increasing neurological symptoms. Among the number of potential biomarkers of disease progression, JOA and SSPROM presented the most robust associations with disease duration and age. Besides that, SSPROM correlated with SSER of lower limbs. We suggest that JOA and SSPROM might be valuable instruments in future natural history studies in heterozygotes for X-ALD.AMN: Adrenomyeloneuropathy; AO: Age at onset; C22:0: Docosanoic acid; C24:0: Tetracosanoic acid; C26:0: Hexacosanoic acid; CALD: Cerebral form of ALD; CMAP: Compound muscle action potential; DD: Disease duration; HUMARA: Human androgen-receptor locus; JOA: Japanese Orthopaedic Association scale of myelopathy; m-NCV: Motor conduction velocity; NSE: Neuron-specific enolase; SNAP: Sensory nerve action potential; SPG: Spastic paraplegia; SPRS: Spastic Paraplegia Rating Scale; SSER: Somatosensory evoked responses; SSPROM: Severity Score System for Progressive Myelopathy; VLCFA: Very long chain fatty acids; X-ALD: X-linked Adrenoleukodystrophy.The authors declare that they have no competing interests.CTH conceived the study, participated in the design of the study, carried out the recruitment, interviews and clinical studies, and helped to draft the manuscript. PS carried out the SSER studies and participated in the analysis. PS and VTF carried out peripheral neurophysiology studies and participated in the analysis of the data. DMC performed VLCFA analyses and helped in the coordination of data. CRV performed VLCFA analyses and helped in the acquisition of funding. VT performed NSE analyses and participated in the analysis of data. LVP performed NSE studies and helped in the acquisition of funding. FSP carried out the molecular genetic studies and participated in the analysis. UM participated in the molecular analysis and in the coordination of data. LBJ conceived the study, participated in the design and coordination of the study, performed the statistical analysis, and drafted the manuscript. All authors read and approved the final manuscript.Mutations found at ABCD1 gene in the present X-ALD heterozygote women, and the distribution of ages at examination and at onset of symptoms.Click here for fileAssociations between age and (6A) C26:00 plasma levels and (6B) VLCFA discriminant factor 3,805(C24:0/C22:0) + 5,296(C26:0/C22:0) + 5,15(C26:0).Click here for file