Cancer patients not admissible for adjuvant chemotherapy are generally at high risk of considerably inferior prognosis. The aim of this retrospective study was to evaluate poorer survival without administration of oral adjuvant chemotherapy of stage III colon cancer patients in clinical settings.Between April 2007 and September 2011, 259 patients with stage III colon cancer who underwent curative surgery were retrospectively assigned to the adjuvant chemotherapy group of 171 patients (66%) and the surgery alone group of 88 patients. Oral fluorouracil (5-FU) derivatives used in adjuvant chemotherapy, such as oral uracil and tegafur plus leucovorin (UFT/LV) or capecitabine, were the most commonly used.P = 0.0001). The chemotherapy group was associated with a dramatic improvement in survival for stage IIIB  and stage IIIC  patients. There was a significant difference in the overall recurrence rate between groups . Multivariate analysis identified adjuvant therapy as an independent predictive factor of reduced recurrence (hazard ratio (HR): 3.231; P = 0.004) and improved RFS .The 3-year relapse-free survival (RFS) rates were 74.9% for all cases, 58.3% for the surgery alone group, and 83.4% for the adjuvant chemotherapy group (In clinical settings, adjuvant therapy was the only significant prognostic factor of survival. Since many patients prefer not to receive chemotherapy, it is critical to inform stage III colon cancer patients that chemotherapy raises their chances of survival by three-fold compared with curative surgery alone. Adjuvant chemotherapy after curative resection for stage III colon cancer patients has been widely recommended as a standard treatment to prolong disease-free survival (DFS) and overall survival (OS) since the early 1990s . The NatP = 0.12). The 3-year OS rates were 81.3% with capecitabine and 77.6% with 5-FU/LV, giving an absolute difference of 3.7%  between treatment groups. Since oral capecitabine offers equivalent clinical benefits in terms of efficacy, safety, convenience, and treatment cost, it can replace intravenous 5-FU/LV in the adjuvant treatment of stage III colon cancer.The Xeloda® in Adjuvant Colon Cancer Therapy (X-ACT) trial was undertaken to compare the efficacy and tolerability of oral capecitabine with 5-FU/LV (intravenous bolus) as adjuvant therapy for patients with stage III colon cancer ,11. The Generally, patients not receiving adjuvant chemotherapy have considerably inferior prognosis. The Japanese Society for Cancer of the Colon and Rectum (JSCCR) reported recurrence rates of 30.8% for stage III colon cancer . The preThe limitations of the study are that it has retrospective clinical observations, a lack of patient randomization, and a dependence on the information available in the patient files. Which is more benefit or more harm associated with various treatment strategies is decided depending on the situation of each patients in clinical settings. Therefore, the aim of this study was to demonstrate the poor prognosis without oral adjuvant chemotherapy for stage III colon cancer patients in clinical settings.Between April 2007 and September 2011, 259 patients with stage III colon cancer who underwent curative surgery were retrospectively assigned to the adjuvant chemotherapy group (n = 171) or the surgery alone group (n = 88). The database contained detailed information on patient characteristics, operative findings, histology, laboratory findings, and adjuvant therapies. The follow-up survival data were collected retrospectively through medical record analyses. The exclusion criteria were as follows: rectal cancer; malignancy other than colon cancer; and patients who did not receive adjuvant chemotherapy or did not have any information about the chemotherapy. Cancer was staged using the American Joint Committee on Cancer (AJCC) 7th edition TNM classification: colon and rectum .All patients started receiving adjuvant ≥8 weeks after curative surgery. They were required to have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 to 2, to have signed informed consent, and be aged between 20 and 80 years old. The JSCCR recommends four adjuvant therapy regimens for stage III colorectal cancer: intravenous 5-FU/LV, UFT/LV, capecitabine, or FOLFOX . In Japan, given the expected benefits and possible risks of toxicity, a consensus has not been reached as to whether adjuvant regimens containing oxaliplatin should be given to stage III patients. Several oral 5-FU derivatives are available, such as oral UFT/LV or capecitabine, and they are preferred for their convenience. Consequently, these were the most commonly used regimens in this study .2/day, and LV was administered at a dose of 75 mg/day. The daily doses of UFT/LV were divided into three doses administered every 8 h with water. Patients were instructed to avoid food from 1 h before to 1 h after each dose. Capecitabine is an oral fluoropyrimidine that generates 5-FU preferentially in tumor tissue through a three-step enzymatic cascade [2 twice a day for 14 days, followed by 7 days of rest. Standard care included a total of eight cycles. The amount of drug received was based on reported pill counts and patient declarations at the end of each cycle.The UFT regimen is a preparation of tegafur-uracil in a molar ratio of 1:4. Tegafur is the 5-FU prodrug converted to 5-FU in the liver. LV is used to modulate 5-FU biochemically, and has been widely adopted for the treatment of advanced colorectal cancer ,15. The  cascade . The adjThe patients were scheduled for follow-up visits every 3 months during the first 3 years, every 6 months during the next 2 years, and annually thereafter. Each visit included a physical examination and computed tomography (CT) scans of the chest, abdomen, and pelvis. Colonoscopies were performed during the first, third, and fifth year of follow-up. The median follow-up period was 41 months (range: 3 to 73 months).P <0.05) to identify the independent factors of recurrence. Survival rates were calculated by the Kaplan–Meier method and compared by the log-rank test. Stepwise forward Cox regression model was conducted for parameters found to be significantly associated with survival by the log-rank test (P <0.05) in order to identify the independent factors of survival. Values of P <0.05 were considered significant in all analyses. This analysis was conducted using the intention-to-treat theory.Statistical analysis was performed using SPSS version 21.0 software . The chi-squared test or Fisher’s exact probability test were used to compare recurrence rates. Logistic regression analysis was conducted on the parameters found to be significantly associated with recurrence by chi-squared tests or Fisher’s exact probability test (P <0.0001). The causes for adjuvant chemotherapy rejection were older patients (>80 years), high risk of severe comorbidities or postoperative complication, and self-judgment of refusal.The two groups were similar with respect to gender, tumor location, depth of invasion, tumor differentiation and lymphovascular invasion, and TNM sub-classification (IIIA/IIIB/IIIC) Table . HoweverP <0.0001).The UFT/LV regimen was performed in 104 patients (61%), capecitabine in 59 patients (34%), and other types in eight patients . The overall compliance was 77%, as 40 patients discontinued the therapy without evidence of a protocol event. The most common symptoms of drug toxicity were severe nausea and diarrhea (3.5%); the risks of severe hepatitis, neutropenia, and hand foot syndrome (5%) only with capecitabine. The completion rate of adjuvant chemotherapy was significantly less in patients with UFT/LV (72%) than with capecitabine  and RFS . Furthermore, chemotherapy did not affect the 3-year OS of stage IIIA and IIIC patients, and the 3-year RFS of stage IIIA patients.The Kaplan–Meier estimates of 3-year OS and 3-year RFS survival are presented in Figure P = 0.001), aside from oncological factors (depth of tumor invasion and TNM stage).Univariate and multivariate analyses were conducted for each clinicopathological factor Table . Based oCancer recurrence occurred in 62 patients (recurrence rate (rec rate), 23.9%) during the follow-up period. The risk of recurrence during the first 3 years was two-fold higher for surgery alone  than with adjuvant chemotherapy . The overall median time to recurrence after initial resection for colon cancer was 10.5 months. In the surgery only group, the recurrence sites included the liver , lung , dissemination , local site , and lymph node metastasis . In the adjuvant chemotherapy group, the recurrence sites were the liver , lung , dissemination , local sites , and lymph node metastasis .P = 0.004) apart from oncological factors (depth of tumor invasion and TNM stage).Multivariate analysis was performed to identify independent predictive factors of recurrence Table . ChemothRecently, practice-based research  which got rid of the distance of a clinician and the researcher tends to be regarded as important . It is aThe 3-year RFS rate of the surgery alone group was significantly lower than for the chemotherapy group. In addition, chemotherapy was associated with a two-fold lower cancer recurrence rate during the first 3 years for stage III colorectal cancer patients, with 18% and 35%, respectively. Both stage IIIB and IIIC patients benefited from chemotherapy with a dramatic improvement in survival rate over surgery alone. Multivariate analysis only identified chemotherapy as an independent predictive factor for improvements in prognosis and reduction in cancer recurrence. Therefore, patients unable or unwilling to receive chemotherapy have a poor oncological prognosis. It is critical to provide this information to the patients unable to receive adjuvant chemotherapy for stage IIIB and IIIC. When recurrence occurs in stage IIIB and IIIC colon cancer patients, the oncologic surgeon who makes the decision of surgery alone treatment will provide the best supportive care.The present study emphasizes the needless of chemotherapy in cases of stage IIIA colon cancer. Whereas both groups had an excellent outcome, chemotherapy after surgery raised the 3-year RFS from 87.4% to 100%. While the sample sizes were too small to reach statistical significance, it is likely that chemotherapy could be omitted for certain types of stage IIIA colon cancer with low risk of recurrence, such as node positive T1/T2 patients. Adjuvant administration must be decided based on the status of each patient with stage IIIA colon cancer. While the benefits of 100% 3-year RFS is important for patients, it must be weighed against the adverse effects.We must determine how to improve the treatment of patients with poor performance status, severe comorbidities, and older patients who are not normally eligible for chemotherapy . The oldAlthough other reports have documented treatment outcomes in stage III colon cancer, the present study analyzed a rare subset of colon cancer patients  treated only with oral adjuvant chemotherapy at a single institution. Receiving adjuvant chemotherapy was the only prognostic factor for an improved treatment outcome in a clinical setting. Patients that are uncertain about the benefits of adjuvant chemotherapy should be informed of the poor survival rates for stage IIIB and IIIC colon cancer, as well as the excellent survival rates for stage IIIA colon cancer without adjuvant chemotherapy. Unfortunately, stage IIIB and IIIC colon cancer patients who cannot undergo adjuvant chemotherapy because of severe comorbidities, an advanced age, or by choice, need to be informed of the inferior prognosis without treatment.5-FU: Fluorouracil; AJCC: American Joint Committee on Cancer; ASA: American Society of Anesthesiologists physical status classification system; CEA: Carcinoembryonic antigen; CI: Confidence interval; CT: Computed tomography; DFS: Disease-free survival; ECOG: Eastern Cooperative Oncology Group; HR: Hazard ratio; JSCCR: Japanese Society for Cancer of the Colon and Rectum; LV: Leucovorin; NSABP: National Surgical Adjuvant Breast and Bowel Project; OS: Overall survival; rec rate: Recurrence rate; RFS: Relapse-free survival; TNM: Tumor, node, metastasis; UFT: Uracil and tegafur; X-ACT: Xeloda in Adjuvant Colon Cancer Therapy.The authors declare that they have no competing interests.JT acted as guarantor of the integrity of the study, conceived the study concept, designed the study, defined the intellectual content, performed the literature research, undertook clinical and experimental studies, performed data acquisition, data analysis, and statistical analysis, and prepared and edited the manuscript. SY reviewed the manuscript. All authors read and approved the final manuscript.


P<0.001) than in ANT. Versican was majorly expressed in the stroma surrounding tumor epithelium and minorly some areas of tumor epithelium. The Versican expression level was higher in GC than in ANT (P=0.004), but no significant difference was observed between ANT and IN (P=0.517). The Versican mRNA and protein levels were consistent in GC. High Versican mRNA and protein expression correlated with greater tumor invasion depth . Univariate and multivariate analysis revealed that patients with high Versican mRNA expression exhibited poor disease-specific survival (P<0.001). In vitro experiments showed that Versican overexpression promoted cell proliferation and invasion. Our data indicate that Versican may be a novel prognostic indicator in GC and may be a potential target for clinical diagnosis.Gastric cancer (GC) is the leading malignancy in the digestive system. Versican is a ubiquitous component of the extracellular matrix and has a role in tumor progression. We aim to examine the expression of Versican in GC and the relationship between Versican levels and patient survival. We detected the mRNA expression of Versican in tumorous pairs and adjacent normal tissues (ANTs) of 78 GC patients by quantitative real-time polymerase chain reaction. The protein expression of Versican in 101 cases of matched GC and ANT, as well as in 27 intraepithelial neoplastic (IN) samples, was evaluated by immunohistochemistry. We analyzed the correlation between Versican levels and clinical outcomes. Finally, we performed CCK-8 cell counting assay and transwell assay in GC cell lines. Versican mRNA expression was significantly greater in tumor tissues ( Using intracellular signaling pathways, PG and growth factor signals are transmitted and alter the cellular response,5 thus, PGs are directly implicated in a large variety of human diseases. Several studies indicated that significant PG content changes occur in the tumor stroma of epithelial neoplasms, thereby supporting tumor growth and invasion. Versican, a member of the aggregating chondroitin sulfate PGs family,6 is accumulated predominantly in the tumor stroma,7 providing hygroscopic properties to create a loose and hydrated matrix that is necessary to support key events in development and disease. Through direct or indirect interactions with cells and molecules, Versican has significant roles in modulating cell proliferation, differentiation, adhesion and migration, all of which are features of cancer invasion and metastasis.8 Thus, Versican may serve a wide range of functions in the invasion and metastasis of tumor cells.The extracellular matrix (ECM), composed of proteoglycans (PGs), glycoproteins and collagens, is a highly organized structure with many physiological and pathological roles.12 pharyngeal,13 ovarian cancer14 and cervical cancer.15 Increased accumulation of structurally modified Versican is reportedly related to the progression of laryngeal cancer.16 However, the significance of Versican has been less extensively studied in gastric adenocarcinoma. The aim of our study was to investigate Versican expression in gastric adenocarcinoma and to determine its relationship with clinicopathologic factors, with a special emphasis on its prognostic significance.Previous studies have shown that Versican can be detected in many malignancies including melanoma, non-small cell lung cancers, breast tumors,P<0.001; The first goal of our study was to investigate whether Versican is detectable and altered in gastric adenocarcinoma tissues compared with adjacent normal ones. So we performed quantitative real-time (RT) polymerase chain reaction with RNAs isolated from gastric adenocarcinoma tissues to detect the expression levels of Versican. It turned out that Versican expression was significantly greater in tumor tissues compared with adjacent normal tissues  or ANT  and 27 intraepithelial neoplastic (IN) samples. First, we observed that the staining of Versican was in nucleus or both nucleus and cytoplasm. The staining areas majorly covered the stromal cells characteristically surrounding the epithelial lesions, and also covered some part of the epithelium of cancer areas . The staP=0.000; .Furthermore, we chose 50 cases with both results of RT–quantitative polymerase chain reaction (qPCR) in tumor tissues and immunochemistry in paraffin sections to examine the correlation between mRNA level and protein expression of Versican. As shown in figure, the mRNA levels of Versican in these 50 cases were highly correlated with protein expression levels in immunochemistry . Taken t18 At both the mRNA and protein levels, elevated Versican expression was correlated with greater tumor invasion than lower expression . There was no significant correlation between Versican expression and other clinicopathologic features, such as age, gender, tumor size, tumor location, histologic grade, lymphatic metastasis, peritoneal metastasis, vascular invasion, nervous invasion or tumor stage . Univariate analysis of DSS revealed that the relative level of Versican expression , nervous invasion (P=0.024), lymphatic metastasis (P=0.004), peritoneal metastasis (P=0.027) and tumor stage (P<0.001) were prognostic indicators , Versican expression was an independent prognostic indicator for DSS in GC patients  curves were plotted according to Versican mRNA expression levels using the Kaplan–Meier method. During follow-up, 20 patients died of gastric adenocarcinoma. As presented in P<0.001; , tumor ddicators . The oth P>0.05; . MultivaP=0.002; .in vitro. We detected the mRNA level of Versican in normal GES1 and five gastric carcinoma cell lines by RT–qPCR first  and C-(G3)terminal globular domains, which collectively interact with a large number of ECM and cell surface structural components.8 Nevertheless, it is likely that target genes differ between specific tissues and cell types, and the specific target genes controlled by Versican in GC remain unknown. In future work, high-throughput techniques such as RNA sequencing and microarray analysis should be applied to obtain a global view of the changes of downstream molecules, thereby elucidating the regulation mechanisms of Versican.Neoplastic remodeling of the ECM by modulating stromal cell secretion of macromolecules, such as Versican, is a well-recognized phenomenon and may be one mechanism by which tumor cells control their microenvironment to facilitate local invasion and metastasis.Our results show that Versican expression is significantly increased in GC tissues. Greater Versican expression levels were detected in more invasive tumors. In addition, the upregulation of Versican expression was associated with poor GC prognosis. These findings suggested that Versican may be a novel prognostic indicator in GC and may be a specific and accessible biomarker as well as a potential new target for GC clinical diagnosis.29 Patient follow-up was performed every 2–3 months during the first postoperative year and 3–6 months thereafter, until 30 July 2015. All patients completed the follow-up. The DSS was defined as the length of time between the surgery and death from the cancer. During the follow-up, 20 patients died of disease. This study was approved by the research ethics committee of Fudan University Shanghai Cancer Center, and the participants provided informed consent for the use of their tissues in this study.This study collected all 78 tissues of patients with gastric adenocarcinoma stored in tissue bank of Shanghai Cancer Center from 2009 to 2014. All the patients analyzed in this study underwent resection of primary gastric adenocarcinoma at Fudan University Shanghai Cancer Center from 2009 to 2014. All the cases were histopathologically confirmed as gastric adenocarcinoma according to WHO classifications of Tumors of Digestive System 2010 version. None of the patients underwent pre-operative treatment. All the resected tissue samples were immediately frozen in liquid nitrogen and stored at −80 °C until RNA extraction in the tissue bank. The data collected from all subjects included age, gender, DSS and clinicopathological features . The clinical stage was evaluated using the TNM classification system.20 Relative gene expression was calculated using the comparative cycle threshold (CT) (2−ΔΔCT) method, with beta-actin as the endogenous control to normalize the data.30 The following primers were used in this study: Versican, 5′-GATGTGTATTGTTATGTGGATCA-3′ (forward) and 5′-CATCAAATCTGCTATCAGGG-3′ (reverse); and beta-actin, 5′-TCCTCTCCCAAGTCCACACA-3′ (forward) and 5′-GCACGAAGGCTCATCATTCA-3′ (reverse).Total RNA was extracted from the tumorous and ANTs using Trizol  following the manufacturer's protocol. RT and qPCR kits were used to evaluate the Versican expression from tissue samples. The RT and qPCR reactions were performed as previously described.2O2 in methanol for 20 min to block endogenous peroxidase activity. Antigen retrieval was performed , and the sections were rinsed in phosphate-buffered saline. Subsequently, the sections were stained for Versican (overnight) using a 1:200 dilution of rabbit polyclonal anti-human Versican  in phosphate-buffered saline containing 1% bovine serum albumin. Subsequently, the slides were incubated with Powervision-Poly-HRP-goat anti-mouse/rat/rabbit IgG , and immune complexes were visualized with diamino-benzidine.A total of 101 GC sample pairs and 27 IN samples were studied. Slides and paraffin blocks were selected from the archives of the Pathology Department of Fudan University Shanghai Cancer Center. Immunohistochemical analysis was performed using 4-μm paraffin sections mounted on aminopropyl-triethoxysilane-coated slides. Sections were deparaffinized, rehydrated and treated with 0.3% H31 The combined scores were then divided into two groups by the middle value: category 0  and category 1 . Human lung carcinoma tissue and normal gastric tissue are the positive and negative controls, respectively.31The expression of Versican was scored by two pathologists independently of the study. Only staining for nucleus (regardless of cytoplasmic staining) was regarded as positive. The expression was scored according to previous studies, which divided the staining intensity into none (0), mild (1), moderate (2) and intense (3) at low magnification (x200) and scored the percentage of positive tumor cells as 0% , 1–5% , 6–25% , 26–50% , 51–75%  and 76–100% .The full-length versican sequence was amplified by PCR from complementary DNA of NIH: AGS cells and then subcloned into pcDNA3.1 (+) vector .2 in a humidified atmosphere.Cell lines involved including human GES cell line, GC cell lines AGS, HGC-27, MKN-45, MGC-803, and NCI-N87 were cultured in Dulbecco's modified Eagle's medium  or RMPI-1640 (Gibco) respectively, supplemented with 10% fetal bovine serum (Gibco), 50 U/ml penicillin and 50 μg/ml streptomycin (Gibco). All cell lines were maintained at 37 °C, 5% CO3 cells per well were seeded onto 96-well plated in 100 μl complete culture medium. The cells were cultured for 0, 24, 48, 72, 96 h before adding 10 μl CCK-8 (5 mg/ml) into the culture medium in each well. After a 2-h incubation at 37 °C, optical density at 450 nm was measured with an automatic microplate reader .Cell proliferation was evaluated using CCK-8 . In all, 2 × 104 cells in 100 μl serum-free medium were loaded into the upper inserts, and 500 μl culture medium containing 10% fetal bovine serum were loaded into the lower chamber as chemo-attractant. After a 24-h incubation at 37 °C, the cells that had migrated through the filters were fixed with ethanol and stained with 0.1% crystal violet. Photographs were taken at 200 × under microscope  and the number of invaded cells was counted at 400 × .The transwell chambers  were used for cell invasion assay. A total of 4 × 10t-test, χ2 test or Wilcoxon test, as appropriate. DSS rates were calculated using the Kaplan–Meier method, with the log-rank test applied for comparison. Variables with a value of P<0.05 in univariate analysis were used in a subsequent multivariate analysis, based on the Cox proportional hazards model. Two-sided P-values were calculated, and a probability level of 0.05 indicated statistical significance. Statistical analyses of immunohistochemistry staining evaluation were performed using the Kruskal–Wallis one-way analysis of variance test and Mann–Whitney U-test.All statistical analyses were performed using SPSS 20.0 software . The significance of between-group differences was estimated using Student's


Lepidoptera varies greatly in different development stages. To date, the effects of key genes in the melanin metabolism pathway on larval and adult body color are distinct, yet the effects on pupal pigmentation remains unclear. In the silkworm, Bombyx mori, the black pupa (bp) mutant is only specifically melanized at the pupal stage. Using positional cloning, we found that a mutation in the Aspartate decarboxylase gene (BmADC) is causative in the bp mutant. In the bp mutant, a SINE-like transposon with a length of 493 bp was detected ~2.2 kb upstream of the transcriptional start site of BmADC. This insertion causes a sharp reduction in BmADC transcript levels in bp mutants, leading to deficiency of β-alanine and N-β-alanyl dopamine (NBAD), but accumulation of dopamine. Following injection of β-alanine into bp mutants, the color pattern was reverted that of the wild-type silkworms. Additionally, melanic pupae resulting from knock-down of BmADC in the wild-type strain were obtained. These findings show that BmADC plays a crucial role in melanin metabolism and in the pigmentation pattern of the silkworm pupal stage. Finally, this study contributes to a better understanding of pupa pigmentation patterns in Lepidoptera.The pigmentation pattern of  TH (Tyrosine Hydroxylase gene) and DDC (Dopa Decarboxylase gene), can catalyze the synthesis of Dopamine and result in Dopamine accumulation, which allows melanin to be created in insects8910111314151718202122232425black mutant of Tribolium castaneum is a type of β-alanine deficiency melanic mutant that resulted from the marked down-regulation of Aspartate decarboxylase (ADC), which can catalyze aspartic acid to β-alanineADC genes  have been identified in several Lepidoptera insects; however, no direct phenotypic evidence has been reported to indicate that ADC participates in pigmentation28293031The melanin metabolism pathway is involved in insect cuticle pigmentation and plays an important role in ecological adaption, such as in escaping predation, mimicry, sexual selection, signaling, and thermo-regulation12356Lepidoptera13141628353637There are more than 100 body color mutants that have been reported in silkworms33Bmebony is abolished during this period, Dopamine will accumulate excessively because it cannot be converted to NBAD, which will makes the pupae adopt a melanism phenotype, which is the cause of the sooty (26-0.0) mutantmelanism (18-41.5) is another body color mutant in which melanism is expressed in the late pupal–moth stage1314331314black pupa (bp), showed melanization specifically in the pupal stage33bp (11-42.5) is independent of the sooty and melanism loci, and we speculate that there is other unknown pivotal gene that determines pupa pigmentation. Using positional cloning, gene expression analysis, RNAi, and biochemical and physiological analysis, we identified the Bombyx mori gene Aspartate gecarboxylase (BmADC) that is responsible for the mutant phenotype. BmADC shows low expression in the pupa stage of the bp mutant, which reduces β-alanine content. NBAD synthesis is hindered, which subsequently leads to an accumulation of excess dopamine, and eventually results in the melanism phenotype. To the best of our knowledge, the bp mutant is the first phenotype that shows the role of ADC in melanin metabolism in Lepidoptera. Our study should provide a theoretical basis to understand the role that ADC plays in this pigmentation model of Lepidoptera insect pupae metamorphosis, and also represents an important contribution to body pigmentation research.In the pupae metamorphosis period of silkworms, normal pupae exhibit an amber body color. If the function of bp mutant strain 16-100 (bp/bp) and three wild-type strains, Dazao (+bp/+bp), C108 (+bp/+bp), and N4 (+bp/+bp), were obtained from the silkworm gene bank at Southwest University and were reared on fresh mulberry leaves under a 12 hr/12 hr light/dark photoperiod at 24 °C.The β-alanine (A9920) and Dopamine (H8502) were purchased from Sigma. NBAD standards were provided by Professor Michael R. Kanost and Dr. Neal T. Dittmer . The β-alanine and aspartate standards used for quantitative analysis were provided by the Institute of Animal Nutrition, Sichuan Agricultural University .bp/+bp) and 16–100 (bp/bp) were selected for genetic mapping. F1 offspring were produced from a cross between a female C108 and a male bp. 20 BC1F, and progeny from the cross (C108 × 16–100)♀ × 16-100♂ were used for linkage analysis, while 562 BC1M progeny from the cross 16-100♀ × (C108 × 16-100)♂ were used for recombination analysis. Polymorphic PCR markers  and melanic body color (homozygosis on bp locus) were genotyped. Primers used for genotyping are listed in Two silkworm strains, C108 (+BmADC gene was obtained by rapid amplification of cDNA ends (RACE) using the GeneRacer kit (Invitrogen) according to the manufacturer’s protocol. The primers used for full-length cloning of BmADC are listed in BmADC cDNA in Dazao and 16–100 were KM523624 and KM523625, respectively. All sequence determinations were performed on three biological replicates per sample.Total RNA was extracted from Dazao and 16–100 at 6 h of pupation. The full-length cDNA sequence of the http://www.ncbi.nlm.nih.gov/), flybase (http://flybase.org/), bettlebase (http://beetlebase.org/), silkDB (http://www.silkdb.org/silkdb/), MonarchBase (http://monarchbase.umassmed.edu/), and Manduca base (http://agripestbase.org/manduca/). Additionally, the amino acid decarboxylase with a Pyridoxal Phosphate domain in Drosophila melanogaster, B. mori, and Tribolium castaneum were also searched using the BlastP and tBlastN programs. Using the online Muscle program (http://www.ebi.ac.uk/Tools/msa/muscle/) with defult settings, the amino acid sequences were aligned. Subsequently, the neighbor-joining method in MEGA4The ADC homology was searched by BlastP at the NCBI website (BmADC gene between Dazao (wild-type) and 16–100 (bp mutant). The PCR products were cloned into a PMD19-T vector (Takara) and sequenced . The characteristics of each sequence were analyzed using BmTEdb (http://gene.cqu.edu.cn/BmTEdb/) and genomatix (http://www.genomatix.de/). RT-PCR was performed to analyze the expression patterns of BmADC in the wild-type strains. Total RNA was extracted and purified from the silkworm whole body for several developmental stages (from the wandering stage to eclosion of adults) using TRIzol (Invitrogen) according to the manufacuturer’s protocol, and was subjected to cDNA synthesis using oligo (dT) primer and MLV reverse transcriptase provided in the Promega P1300 kit . Primers designed for RT-PCR are listed in BmActin3 gene was used as an internal control.Primers were designed primers based on the silkworm 9x assembly genome database to detect differences in the upstream sequences of the transcriptional start site for the BmADC in both the Dazao and 16–100 strains using the ABI Prism 7000 sequence detection system  with a SYBR Premix Ex-Taq kit (Takara) according to the manufacturer’s protocol. The primers designed for qRT-PCR are listed in B. mori gene Eukaryotic translation initiation factor 4A (microarray probe ID: sw22934) was used as an internal control. All assessments were performed on three biological replicates per sample.Quantitative RT-PCR was performed to measure the expression levels of BmADC gene. The dsBmADC and dsRed  targeting oligos were synthesized using the T7 RiboMAXTM Express RNAi System (Promega). Then, dsRNA was diluted to 12 μg/μl, and a dose of 120 μg was administered to each individual. The injection time was selected according to the temporal expression patterns of the BmADC gene 48 h after wandering. Subsequently, observation and qRT-PCR analysis of gene expression were carried out at day 1 of pupation, and the tissues for gene expression analysis after RNAi were selected from the parts with a distinct phenotype difference between the interference individuals and control individuals . The non-diapause strain N4 (wild-type) was selected for RNAi. The statistical analyses of the RNAi experiments are listed in RNAi was performed to validate the function of the Pupae were selected at 0 h of pupation of Dazao and 16–100 for amino acids and catecholamine content analysis . The free amino acids were extracted as follows: samples were homogenized with 1 ml 0.1 M HCl in centrifuge tubes, then were dissociated from the homogenate for 15 min using ultrasonication followed by centrifugation at 12,000 rpm and 4 °C. The supernatants (600 μl) were transferred to new tubes containing 600 μl 10% sulfosalicylic acid, and were centrifuged one more time at 12,000 rpm and 4 °C for 15 min. The supernatants were transferred and filtered through 0.22 μm membranes. The free amino acids were quantified according to the Le Boucher methodbp mutant strain 16–100 was selected for β-alanine  injection at 60 h after wandering, and the dose was 400 μg per pupa. Mutant pupae injected with 0.75% saline were used as controls. After injection, a pupae phenotype was observed and the expression of BmDDC and Bmebony were measured at 6 h of pupation. The efficiency of β-alanine treatment is listed in The bp mutant also begins at 3 h of pupation, yet melanism also occurs , and the Pfam database (http://pfam.xfam.org/)  . Additioilkworms . This geilkworms . In the BmADC in wild-type (Dazao) and bp mutant B. mori were cloned. In the wild-type animals, the transcript was 1848 bp (without poly A) and contained four exons, encoding a protein of 511 amino acids (bp mutant (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) online analysis , the expression of BmADC was reduced remarkably , no difference between the new moth cuticle was detected in this stage for the bp mutant and wild-type silkworms (data not shown). To explore the reason for these findings, we investigated the expression pattern of the NBAD synthetase gene, Bmebony, as well as Bm-iaaNAT, from the pupal stage to the moth stage in the Dazao strain. We found that the expression of Bmebony was significantly down-regulated in the late pupal (P8) and moth (M1) stages, indicating that NBAD synthesis in this stage is less active than in the early stage  are the respective critical rate-limiting enzymes among the two mentioned pathways17252945pression . We specmelanism . Howevermelanism , Fig. S4ow level . Thus, ientation . Additioth stage . Indeed,ly stage . This fized moth . Bm-iaaNh stages . AlthougBmADC is also sensitive to temperature. The expressions levels of BmADC under the high and low temperature are both down-regulated compared with the expression levels at room temperature  were analysed in wild-type and bp mutant silkworms via the genomatrix online program, and 11 heat shocking factor binding sites were detected that might be involved in the regulation of BmADC expression , which further suggests that BmADC participates in the pigmentation pattern at the early pupal stage . To explain this discrepancy, the possible explanation may be that the higher temperature can induce a much higher activity of BmADC to catalyze the synthesis of β-alanine 495051In addition to the specifically high expression in the early stage of pupation, the expression of e 24 °C; . Approxipression . The lowal stage . SimultaADC gene on insect body color have only been reported in Drosophila and Tribolium22ADC gene can also affect pupa pigmentation, and this is the first report confirming that this gene could be involved in insect pigmentation patterns in Lepidoptera. Additionally, the pigmentation pattern in the early pupal stage of many other moths, such as Manduca sexta or Biston betularia, are similar to that in silkworms, which indicates that ADC might participate in early pupal stage pigmentation and play a role similar to BmADC in these species. Overall, not only can our research uncover the molecular mechanism of the silkworm pupal-specific melanism mutant bp, which is involved in replenishing the silkworm melanin metabolism pathway, but it also provides a reference for body color pattern research in other insects, especially Lepidoptera.To date, the effects of the How to cite this article: Dai, F. et al. Aspartate Decarboxylase is Required for a Normal Pupa Pigmentation Pattern in the Silkworm, Bombyx mori. Sci. Rep.5, 10885; doi: 10.1038/srep10885 (2015).


This was associated with greater numbers of CXCR4+ progenitors in sputum from COPD. Increased migrational responsiveness of progenitor cells may promote lung-homing of VEPC in COPD which may disrupt maintenance and repair of the airways and contribute to COPD disease pathogenesis.Chronic obstructive pulmonary disease (COPD) is characterized by fixed airflow limitation and progressive decline of lung function and punctuated by occasional exacerbations. The disease pathogenesis may involve activation of the bone marrow stimulating mobilization and lung-homing of progenitor cells. We investigated the hypothesis that lower circulating numbers of vascular endothelial progenitor cells (VEPCs) are a consequence of increased lung-sequestration in COPD. Nonatopic, current or ex-smokers with diagnosed COPD and nonatopic, nonsmoking normal controls were enrolled. Blood and induced sputum extracted primitive hemopoietic progenitors (HPCs) and VEPC were enumerated by flow cytometry. Migration and adhesive responses to fibronectin were assessed. In sputum, VEPC numbers were significantly greater in COPD compared to normal controls. In blood, VEPCs were significantly lower in COPD versus normal controls. There were no differences in HPC levels between the two groups in either compartment. Functionally, there was a greater migrational responsiveness of progenitors from COPD subjects to stromal cell-derived factor-1alpha (SDF-1 Chronic obstructive pulmonary disease (COPD) is characterized by fixed airflow limitation and progressive decline of lung function punctuated by frequent exacerbations , 2. PulmStudies have shown that circulating levels of VEPC in COPD are lower at baseline compared to normal subjects , increasα), in order to understand the factors promoting lung-homing of progenitors in COPD.Both vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) are proangiogenic factors that have different action profiles , 12. VEGWe investigated the hypothesis that diminished circulating numbers of vascular endothelial progenitor cells (VEPCs) in subjects with COPD are a consequence of increased lung-sequestration. The novel findings of this study are that reduced number of circulating VEPCs but not HPCs in COPD is associated with increased sputum levels. We found that progenitors from COPD subjects had a greater migrational response than normal subjects, suggesting that increased homing of VEPC from the blood to the airways may be an important component to the pathogenesis of COPD.n = 9) subjects (18–75 years of age) were nonatopic, current or ex-smokers (at least 15 pack year smoking history), with chronic bronchitis, a FEV1 < 70% predicted and prebronchodilator FEV1/FVC < 0.70, and a postbronchodilator  of FEV1/FVC < 0.70. Subjects were clinically stable at the time of the study without exacerbations or oral steroid treatment for 4 months prior to study. As shown in n = 8) (18–75 years of age) were nonatopic, nonsmokers, with FEV1/FVC > 0.70. The study protocol was approved by Hamilton Integrated Research Ethics Board (#07-2914), and all subjects provided written informed consent.COPD .Subjects attended the clinic for medical history, skin-prick testing to assess allergy to common aeroallergen extracts, spirometry before and after salbutamol to assess FEVSputum (SP) samples were induced using 0.9% normal saline and mucous plugs were selected and dispersed with dithiothreitol, as previously described . Cytospiα, IL-3Rα, or GMCSFRα, chemoattractant marker CXCR4, and adhesion markers β1 integrins VLA4 (CD49d) and VLA5 (CD49e) and β2 integrin Mac-1 (CD11b) which were conjugated to PerCP. PB and SP extracted cells were suspended in FACS buffer, immunostained with isotype or specific mAbs , then washed with NaN3, and fixed in PBS plus 1% paraformaldehyde. Cells were acquired using a Becton Dickinson FACScan flow cytometer equipped with 3-colour argon ion laser. Analyses of FACS data were performed using a four-step gating strategy as previously described [low/SSClow/CD45dim/CD34high events and VEPCs were identified as FSClow/SSClow/CD45dim/CD34high/CD133high events.Enumeration and phenotypic analyses of peripheral blood (PB) and SP HPC were assessed by flow cytometry using monoclonal antibodies (mAbs) to lineage markers: CD45-FITC, CD34-PE, and CD133, IL-5Rescribed . HPCs we+ cells using magnetic beads as previously described [Blood was drawn into heparin (1000 units/mL) and layered onto 60% Percoll, for density gradient fractionation to isolate low-density mononuclear cells (MNC). The interface cells were washed with McCoy's 5A and subject to adherence on plastic to deplete macrophages. Nonadherent MNC fraction was subjected to positive selection of CD34escribed .+ cells was assessed by intracellular filamentous (F) compared to globular (G) actin ratio [+ cells were washed and resuspended in IMDM with 0.25% BSA (1 × 106 cells/mL) and prewarmed for 10 minutes at 37°C, followed by coincubation with SDF-1α (10 ng/mL), HGF (50 ng/mL), VEGF (50 ng/mL), or diluent at various time-points . Cells were fixed for 15 minutes, washed, and then permeabilized for 5 minutes. This was followed by incubation for 20 minutes with 1 U/mL of Alexa Phalloidin (detecting F-actin) and Texas Red DNAse I (detecting G-actin), resuspended in PBS and acquired FACScan flow cytometer. F : G ratios were then calculated for each cell sample as previously described [Migrational responsiveness of PB CD34in ratio . Brieflyescribed .+ cells were incubated in fibronectin-coated plates for 45 minutes at 37°C  and exposed to SDF-1α , HGF , VEGF , or diluent for 30 minutes at 37°C. Adherent cells were then recovered with cell dissociation buffer and Iscove's modified Dulbecco's medium plus 10% FBS and then stained with antibodies (CD45 and CD34 mAb) for flow cytometry enumeration and analysis as previously described [Adhesion assays were performed as previously described . Brieflyescribed .U test) and ANOVA was used within group analyses. Significance was set to a p value of <0.05.Between groups analyses were performed using nonparametric tests (Mann-Whitney dimCD34+ cells) in COPD subjects versus normal subjects,  (dimCD34+CD133+ cells) in PB from COPD versus normal subjects    . Further < 0.05) . The abs < 0.05) .6 WBC, p < 0.05) (There were no significant differences in the expression of the lineage-commitment markers between the two groups as shown in  < 0.05) . In cont < 0.05) .6 WBC, resp.)    . In cont= 0.038) . Similar < 0.05) . There wα compared with HPC from normal subjects   .COPD is characterized by structural and functional changes in the pulmonary parenchyma, central and peripheral respiratory tract, and pulmonary circulation. Evidence suggests that HPCs and VEPCs play important roles in angiogenesis, working in concert to maintain and repair the capillary endothelium. HPCs migrate to areas of injury, where they produce growth factors that induce blood vessel sprouting and endothelial proliferation . VEPCs pVEGF is a potent chemoattractant and survival factor for both HPC and VEPC, mediated through VEGF receptors , 39. Mouα compared to HPC from normal subjects. These data support our hypothesis that decreased numbers of circulating HPCs in COPD may be due to increased lung-homing. This is in part supported by reports of increased numbers of CD45+CD133+ cells on endothelial surfaces and intimal layers of the pulmonary arteries in smokers with COPD [In transmigration assays, blood-derived HPC from subjects with COPD had greater migrational responsiveness to SDF1-dimCD34+ cells between COPD and healthy controls [α, IL-5Rα, and GM-CSFRα. These receptors can enumerate HPC that have the potential to differentiate into eosinophil/basophil-CFU or granulocyte/macrophage-CFU, which have been shown to contribute to the pathogenesis of asthma [Notably, although we found marked differences in VEPCs between COPD and normal subjects, we found no difference in total absolute numbers of HPCs. This is consistent with Janssen et al. who found no difference in circulating CD45controls . We concf asthma , 47. How+ or CD133+ progenitor cells [Notably, we found no difference in the migrational response of HPC to HGF or VEGF between the two groups. It is interesting in cells from COPD subjects there was no increased responsiveness to VEGF, given that this is a potent chemoattractant, yet we saw an increase in VEPC within the airways. Kim et al. demonstrated that, in COPD, circulating VEPC showed significantly lower migratory capacity at baseline and in response to VEGF than those from normal nonsmoking controls . Howeveror cells . We concα, HGF, and VEGF, there was significant adhesion to fibronectin, compared to the diluent. However, there was no difference in the magnitude of the adhesive response between the two groups. We have previously shown that adhesion of progenitors to fibronectin is mediated by either VLA4 or VLA5 to the same extent with no additive effect [We were able to demonstrate that, in the presence of optimal concentrations of SDF-1e effect . This maThis study had some notable limitations. Firstly, 8 of 9 of the COPD subject had no cardiovascular risk factors aside from smoking or metabolic diseases. Although this is not representative of the common COPD population, the absence of comorbidities and cardiovascular risk factors allowed us to avoid potential confounding variables on progenitor cell numbers within the circulation and airways , 50–54. Secondly, our study is limited by the lack of healthy nonatopic current or ex-smoker controls. We investigated healthy nonatopic nonsmoker individuals for the control group of this study. The effect of cigarette smoking on progenitor cells is controversial. Some studies have reported an inverse correlation between the numbers of cigarettes smoked and frequency of VEPCs, which increase rapidly following smoking cessation , 59. VEPdimCD34+, which further complicates VEPC standardisation [dimCD34+CD133+. In the future, there is a need to identify more specific phenotypes for VEPCs, to allow for better profiling of distinct cell populations.Lastly, there is no clear consensus exact phenotypic profile for identifying VEPC. Palange and Liu et al. identified EPCs as cells positive for CD34 and CD133, and, similar to our findings, they found decreased circulating numbers in COPD , 44. Othdisation . Using oWe propose two explanations as to the role of progenitor cells in the airways that may contribute to the pathogenesis of COPD. Firstly, upon infiltration to the lungs, the activity of progenitor cells may be dysfunctional, thereby preventing the repair and maintenance of pulmonary vasculature homeostasis, which could in turn promote COPD. Secondly, recruited progenitor cells to the airways may have increased activity, which could lead to elevated inflammation and exacerbated endothelial cell proliferation of the vascular wall, causing pulmonary vessel remodelling and pulmonary hypertension, contributing to the pathogenesis of COPD.In summary, the findings of this study suggest that in COPD there is increased migrational and adhesive responsiveness of blood-derived progenitor cells and that increased homing to the airways may explain the reduced circulating levels of VEPC and increased numbers in the airways in these subjects. We propose that increased infiltration of VEPC to the airways in COPD may in part contribute to the pathogenesis of this disease although this requires further investigation.


Drug induced myocardial infarction is a known entity with different forms of steroids linked to coronary artery disease (CAD) either through promoting its traditional risk factors, inducing coronary spasm, or by other unidentified mechanisms. Dexamethasone is known to promote an atherogenic and hypercoagulable state. We report a case of a 75-year-old woman who had ST elevation myocardial infarction (STEMI) associated with dexamethasone use just 4 days following an angiogram showing minor luminal irregularities. ST elevation myocardial infarction is a substantial health problem with estimated 30-day mortality between 2.5 and 10% [Our patient is a 75-year-old African-American woman with a history of diabetes mellitus, hypertension, hyperlipidemia, and four prior cerebrovascular accidents with mild right sided residual weakness and ventral hernia status after surgical repair. She has no prior history of smoking, alcohol use, illicit drug use, or family history of premature coronary artery disease. She initially presented with shortness of breath, rhinorrhea, and cough productive of yellowish sputum and tested positive for influenza A for which she was started on oseltamivir. On her second day of hospitalization she reported mild retrosternal chest pain accompanied with a Troponin I elevation up to 0.3 ng/mL with an unchanged EKG showing right bundle branch block (RBBB) . She recOn the following day, she underwent a coronary angiogram revealing only minor luminal irregularities and no significant CAD . Two dayOne day later, she complained of severe retrosternal chest pain with a blood pressure of 90/65 mmHg, heart rate of 95 bpm, and oxygen saturation of 95% on room air. Cardiovascular exam was unremarkable revealing regular heart rate and normal S1 and S2 with no appreciated murmurs, rubs, or gallop. She had no JVD and clear lungs on auscultation. Distal pulses were well felt without appreciated lower extremity edema. The EKG however showed ST elevation in the anterior leads, V2 to V5 . BedsideBlood work revealed a platelet count of 461 k/cumm, hemoglobin of 10.5 mg/dL, and white blood cell count of 9 k/cumm. Homocysteine level was mildly elevated . High sensitivity CRP was elevated at 40.9 mg/L. Factor VIII activity , factor XI activity , and thrombin antithrombin complex  were all increased while factor VII activity was normal. Fibrinogen level and protein C and S activity were normal. Cardiolipin antibody, JAK2 V617F mutation, and Factor V Leiden were all negative.Besides impacting traditional risk factors for CAD such as hypertension (HTN), glucose intolerance, obesity, and hyperlipidemia (HLD), glucocorticoids influence vascular functions, atherogenesis, and remodeling following ischemia or intravascular injury. This influence is mediated by both glucocorticoids and mineralocorticoid receptors and is modified by 11b-hydroxysteroid dehydrogenase enzyme local metabolism of glucocorticoids .Dexamethasone was shown to promote an atherogenic and hypercoagulable state through different mechanisms. In high doses, it increases Von Willebrand Factor (VWF) which is a prothrombotic marker and increases the platelet activator P-selectin. Moreover, short term use of dexamethasone is associated with increased levels of Fibrinogen, VII, VIII, and XI , 6.In 2014 Okumura et al. reported 3 cases of steroid induced coronary spasm . This miInterestingly, Okumura hypothesized that corticosteroid-induced vasospastic angina might be underestimated due to being mistakenly confused with its known side effect of epigastric discomfort. Wei et al. reported that, among patients using high dose glucocorticoids (>7.5 mg/day), the relative risk for cardiovascular events occurrence was 2.56 .In 2007, Varas-Lorenzo et al. performed a cohort study with nested case-control analysis to estimate the risk of acute myocardial infarction (AMI) associated with the use of oral corticosteroids by dose and duration. The study included 4795 hospitalized cases of AMI or CAD deaths and randomly sampled 20,000 controls and frequency matched by sex, age, and calendar year.The adjusted OR for AMI in current users of oral corticosteroids compared to nonusers was 1.42 (95% CI: 1.17–1.72). The risk during the first 30 days of use  was greater than with longer duration . The risk was more pronounced  among users of prednisolone equivalent doses >10 mg/day. The study concluded that there is a small increased risk of AMI with oral corticosteroid use with a greater risk observed among users of high corticosteroid dose .In 2013, Coloma et al. identified 163 drugs reported to cause acute MI in various case reports, out of which 39 had a more probable association with MI including betamethasone, prednisolone, and dexamethasone  3.2 , OR 1.9 , and IRR 5.4 ). Of note, 285 excess cases representing patients on dexamethasone who had an AMI however with other more plausible causes were identified . We are Different forms of steroids have been linked to CAD either through promoting its traditional risk factors or by other unidentified mechanisms. In most reported cases of steroid-associated AMI, the temporal relation was the main factor suggesting causality. We believe that, with the wide use of corticosteroids for a variety of indications, physicians should be aware of this possible association. Cautious use should be considered particularly in patients with other risk factors for CAD until there is a larger and more convincing pool of evidence.


In summary 36 ablations were performed and 25 of them were successful . Fluoroscopy time was 16 ± 14.4 minutes. Electrophysiological studies in patients with pulmonary hypertension are feasible and safe. Ablation procedures are as effective in these patients as in non-PAH patients with atrial flutter and atrioventricular nodal reentrant tachycardia and should be performed likewise. The prognostic relevance of ventricular stimulations and inducible ventricular tachycardias in these patients is still unclear and requires further investigation. Few studies have investigated patients with pulmonary hypertension and arrhythmias. Data on electrophysiological studies in these patients are rare. In a retrospective dual-centre design, we analysed data from patients with indications for electrophysiological study. Fifty-five patients with pulmonary hypertension were included . Clinical data, 6-minute walk distance, laboratory values, and echocardiography were collected/performed. Nonsustained ventricular tachycardia was the most frequent indication ( Pulmonary hypertension (PH) is defined as mean pulmonary arterial pressure ≥25 mm Hg at rest and a pulmonary capillary wedge pressure ≤15 mm Hg in patients with precapillary forms and ≥15 mm Hg in postcapillary PH [Five different pathophysiological underlying mechanisms leading to PH are identified and accordingly the current classification distinguishes five groups of PH .Supraventricular arrhythmias are frequent in these patients as well as cases of sudden deaths. The prognostic implications, especially of documented ventricular arrhythmias, are uncertain. Few studies have investigated patients with PH and arrhythmias –5. SupraIn general data on electrophysiological studies (EPS) in patients with PH are rare and studies investigating results of EPS in patients with PH for arrhythmias other than atrial flutter and AVNRT are lacking.Our study retrospectively analysed the indications and results of EPS in patients with PH at two experienced PAH centres.We retrospectively investigated all consecutive patients with PAH that had undergone electrophysiological study in two centres  in the last fourteen years (2000–2013). Medical records of all included patients were screened for relevant data, including the following: demographic data, aetiology of PH, comorbidities, indications for EPS, procedure data and results from EPS, and additional data from echocardiography, right heart catheterisation, and clinical presentation were collected. Data collected within the six months prior to EPS were considered.Depending on the expected/documented arrhythmia, the appropriate catheter setup was chosen. In patients with supraventricular tachycardias, we used a 4-catheter approach (high right atrium (HRA), coronary sinus (CS), His bundle electrogram (HBE), and RV-apex) with the aim of inducing and mapping the tachycardia. At least dual AV-Node physiology and two echo beats must be shown if no tachycardia was inducible but documentation showed the typical pattern. Ablation of AVNRT was performed by 4 mm nonirrigated (50 W/50°C). Success was defined as noninducibility and slow-pathway ablation. In patients with atrial flutter, we used a two-catheter approach with duodecapolar catheter (HRA->coronary sinus); ablation catheters were either 8 mm nonirrigated (50 W/50°C) or 4 mm irrigated tip (40 W/43°C). Success was defined as bidirectional block of the cavotricuspid annulus (>150 ms).Nonsustained ventricular tachycardia during Holter-ECG monitoring was assessed, relevant and leading to EPS when causing clinical symptoms. Ventricular stimulation in patients with nonsustained ventricular tachycardia in Holter-ECG monitoring was performed from RV-apex and RV-outflow tracts, BD 500 and 400 ms, and coupling of three extrastimuli with coupling intervals of a minimum of 200 ms.In supraventricular tachycardias other than typical atrial flutter or AVNRT, as well as in patients with monomorphic ventricular extrasystoly, a 3D mapping system  was used to locate the origin (LAT and propagation map).The following diameters and valves were measured.Left atrial diameter , right atrial area , left ventricle diameter , right ventricle diameter , left ventricular ejection fraction , tricuspid annular plane systolic excursion (TAPSE), Tei-Index, systolic pulmonary artery pressure (sPAP), tissue Doppler of the free wall of the right ventricle (S′), stenosis, and insufficiency of valves –13.Right heart catheterization was performed via the right jugular vein. The hemodynamic measurements included right atrial pressure (RAP), mean pulmonary pressure (mPAP), pulmonary capillary wedge pressure (PCWP), pulmonary vascular resistance (PVR), and cardiac output (thermodilution).t-tests or chi-square tests as appropriate. P values of <0.05 were considered statistically significant. Statistical calculations were performed using SPSS, version 21 .Patient data are presented as absolute numbers (mean or median) and standard deviations (SDs) or interquartile ranges (IQRs). Comparisons between groups were done using The association of arrhythmias with cardiac diameter and measurements of right heart catheterization was assessed with Pearson's correlation coefficient.Fifty-five patients were included, fourteen patients with pulmonary arterial hypertension (group 1), 23 patients with PH associated with left heart disease (group 2), 4 patients with PH associated with lung disease (group 3), 8 patients with chronic thromboembolic PH , 2 patients with miscellaneous PH (group 5), and 4 patients with exercised-induced PH .n = 10) were older  than patients without CHD (n = 41).Mean age was 65 ± 13 years (range 30–94). Patients walked 334 ± 98.5 m in six minutes. Ten patients (18.2%) had anamnesis of coronary heart disease (CHD) with myocardial infarction in 5 patients (data available for 51 patients) and 37 (67.3%) patients with arterial hypertension. Patients with CHD , a bidirectional isthmus block could not be achieved. In one patient ablation was complicated by a third degree heart block necessitating cardiac pacemaker implantation.The lowest success of ablation occurred in patients with atrial tachycardia without feasibility of ablation because of multifocal tachycardia and in other cases placement of catheter was not successful because of right atrial enlargement.Atypical “incisional” atrial flutter , the diagnosis was confirmed and slow pathway ablation was performed successfully in all patients.In 15 patients a programmed ventricular stimulation protocol was performed because of nonsustained ventricular tachycardia (nsVT) during Holter-ECG monitoring. VT or VF could not be induced during EPS in any of these patients.n = 2) and atrial tachycardia (n = 1). In patients with an ICD indications for ablation were a slow VT (n = 1) and an HBE ablation in terms of rate control in one patient suffering from permanent rapidly conducted (>100/min) atrial fibrillation, despite medical therapy.Indications for EPS in patients with cardiac pacemakers were nonsustained VT (n = 1), nsVT (n = 1), syncope (n = 1), slow VT (n = 1), and “incisional” reentry tachycardia (n = 1).Patients with history of myocardial infarction suffered from the following arrhythmias: atrial flutter . In patients with atrial flutter, fluoroscopy time was 21.9 ± 14.6 minutes, 7.3 ± 10.6 minutes in patients with nsVT, 28.5 ± 15.9 minutes in patients with atrial tachycardia, and 7.3 ± 5.9 minutes in patients with AVNRT.Fluoroscopy time was 16 ± 14.4 minutes for all patients, 19.8 ± 17.4 minutes for patients of group 1, 18.4 ± 14.9 minutes for patients of group 2, and 15.2 ± 13.2 minutes for patients of group 4. No differences showed statistical significance  placement of catheter was not successful. The indications and results of EPS are shown in n = 4, 28%) and 2 , and most of the patients with AVNRT suffered from PH of group 1. Incidence of coronary heart disease was different in patients of the three PH groups  suffered from ventricular arrhythmias such as nonsustained VTs and slow VTs detected by Holter-ECG monitoring. The vast majority of patients with atrial tachycardia suffered from PH of groups 1 (n = 4), miscellaneous PH (n = 2), exercised-induced PH (n = 4) atrial flutter , and nsVT  were the most common arrhythmias leading to EPS. Other indications for EPS were syncope (group 3: 1 patient) and atrial tachycardia (exercised-induced PH: 1 patient). None of the patients of these groups had anamnesis of coronary heart disease. In one patient, data relating to coronary disease were not available.In patients with pulmonary hypertension associated with lung disease .Taking CHD into consideration, more patients without CHD (non-CHD) showed normal- and middle-grate reduction of LV-EF , LV (0.138), RA (0.209), RV (0.018), sPAP (0.007), mPAP (0.35), PVR (0.276), TAPSE (0.171), LV-EF (0.271), LVH (0.034), or PCWP (0.12), The present study is the first to analyse indications and results of EPS in patients with different kinds of pulmonary hypertension over a long period in centres with high expertise for pulmonary hypertension and diagnosis and therapy of cardiac arrhythmias. The study revealed that the incidence of EPS in these patients is low, approximately 2.2% (55 patients underwent EPS/2500 patients with pulmonary hypertension). The low number of EPS in these patients reflects the lower incidence of arrhythmias in our collective in contrast to other studies. In accordance to studies of Luesebrink, Bradfield, Ruiz-Cano, and Showkathali EPS were successful (85%) in patients with atrial flutter –9.Impossibility of placement of catheter because of right atrial enlargement was also described by Bradfield et al. .In our study atrial flutter was the second-most frequent arrhythmia in all patients and most frequent arrhythmia in patients without CHD. The results are in accordance with other studies, which describe a high incidence of atrial flutter in their cohorts –5, 7–9.It is noteworthy that in nearly half of the patients who underwent EPS, the indication was nonsustained VT revealed by Holter-ECG monitoring. The comparably high number of nsVT in our study could be achieved because patients underwent Holter-ECG monitoring over a minimum period of 24 hours. It is remarkable that nsVTs were present in 11 patients who were not suffering from coronary heart disease. A third of the patients with coronary heart disease of groups 1 and 2 and no patient from group 4 had nsVTs. More patients with CHD showed (middle- and high-grade) reductions in LV-EF known as a risk factor for ventricular arrhythmias. These findings indicate that, in patients with PH, mechanisms other than ischemia/coronary heart disease seem to be responsible for ventricular tachycardias. Umar et al. revealed in their study on rats with PH induced by subcutaneous injection of monocrotaline early after depolarisations (EADs) from right ventricular epicardial surface triggering VT . VT was The majority of patients (57%) with atrial tachycardia showed multifocal complex substrates; ablation could only be performed successfully in two individuals. Studies in patients with pulmonary hypertension and atrial tachycardias are lacking.The treatment of AVNRT was feasible; the results were identical (100% success) to Ruiz-Cano's study .Our study revealed that the numbers of EPS in patients with PH have been increasing since 2000. Potential reasons include an improvement of ablation techniques and studies demonstrate that EPS and ablations are possible with high rates of success, especially in patients with atrial flutter and AVNRT –9.Fluoroscopy time did not differ between groups 1, 2, and 4 but was longer in patients with atrial arrhythmias in contrast to nsVTs and AVNRTs. We found longer fluoroscopy time than in Luesebrink's study  but shorter than in patients of Bradfield's study (44 ± 20 minutes). It can be assumed that enlargement of the right atrium/ventricle is the reason for the differences between the fluoroscopy times in the studies, whereas Bradfield and Luesebrink do not give information about the diameter of the right atrium/ventricle .In our study we did not reveal correlations between the types of arrhythmias, left and right cardiac diameters, LVH, LV-EF, TAPSE, sPAP, mPAP, PVR, and PCWP.EPS in patients with pulmonary hypertension is feasible and safe. Ablation procedures are effective in patients with atrial flutter and AVNRT. Atrial tachycardias are often multifocal without possibility for ablation. The prognostic relevance of ventricular stimulations and inducible VTs in these patients is unclear. In patients with PH and atrial fibrillation, studies are lacking.


Ethanol abuse is linked to several acute and chronic injuries that can lead to healthproblems. Ethanol addiction is one of the most severe diseases linked to the abuse ofthis drug. Symptoms of ethanol addiction include compulsive substance intake andwithdrawal syndrome. Stress exposure has an important role in addictive behavior formany drugs of abuse (including ethanol), but the consequences of stress and ethanolin the organism when these factors are concomitant results in a complex interaction.We investigated the effects of concomitant, chronic administration of ethanol andstress exposure on the withdrawal and consumption of, as well as the preference for,ethanol in mice. Male Swiss mice  were exposed to an ethanolliquid diet as the only source of food for 15 days. In the final 5 days, they wereexposed to forced swimming stress. Twelve hours after removal of the ethanol liquiddiet, animals were evaluated for ethanol withdrawal by measuring anxiety-relatedbehaviors and locomotor activity. Twenty-four hours after evaluation of ethanolwithdrawal, they were evaluated for voluntary consumption of ethanol in a“three-bottle choice” paradigm. Mice exposed to chronic consumption of ethanol haddecreased locomotor activity during withdrawal. Contrary to our expectations, aconcomitant forced swimming stress did not aggravate ethanol withdrawal.Nevertheless, simultaneous ethanol administration and stress exposure increasedvoluntary consumption of ethanol, mainly solutions containing high concentrations ofethanol. These results showed that stressful situations during ethanol intake mayaggravate specific addiction-related behaviors. Alcohol addiction can be conceptualized as a set of cognitive, behavioral andphysiologic symptoms that indicate a loss of control of substance use by individuals,along with continued use of the substance despite adverse consequences. Symptoms ofethanol addiction include compulsive substance intake and withdrawal syndrome  .Several animal models have been developed to study alcohol addiction. In general, theyinvolve forced exposure of animals to alcohol for prolonged periods. A useful model toinduce behavioral and neurologic changes related to addiction is forced consumption ofethanol as the only source of food in a liquid diet for several weeks. Rodents exposedto this regimen show behavioral and neural alterations related to ethanol use .Changes in patterns of ethanol consumption are also features of alcoholism. Animalstreated chronically with ethanol previously show increased consumption of ethanol in a“two-bottle choice” paradigm and in operant procedures 45.Individual risk factors for the development of abuse and addiction of alcohol areincompletely understood. Nevertheless, acute and chronic stress have an important rolein motivation for the abuse of addictive drugs . For exaIn general, chronic administration of ethanol or stress exposure enhances addiction-likebehaviors. However, if these two factors are present simultaneously, the interactionbecomes complex. Alcohol has anxiety-reducing properties and can relieve stress but actssimultaneously as a stressor and activates stress-response systems . AlcoholWe investigated the effects of concomitant, chronic administration of ethanol and stressexposure upon the withdrawal and consumption of, as well as the preferences for, ethanolin mice.The experimental protocol was approved by the Ethics Committee for Animal Utilizationof Universidade Federal de Uberlândia (CEUA 120/11). Experiments were conductedaccording to the principles of the Brazilian College of Animal Experimentation, whichare based on the Guidelines for the Care and Use of Laboratory Animals .ad libitum(except during theliquid-diet procedure). Experiments were carried out during the light phase of thecycle, and animals were tested randomly across this time period (8–10 mice pergroup).Male Swiss mice  were transferredto our animal facility ≥5 days before the start of experimentation and housed ingroups of 4-5 per cage. The room was maintained at 23±2°C on a 12-h light-dark cyclewith access to water and food The procedure for chronic administration of ethanol was adapted from experiments withintake of a forced liquid diet undertaken by Bonassoli et al. . The liqad libitum.Bottles were weighed before and after exposure to mice to evaluate diet consumptionper animal group; any remaining food was removed after 24-h exposure. One bottle ofdrinking water was available simultaneously with the liquid diet. Mice were kept ingroups with no restricted volumes of the diet to reduce the stress related toisolation or food restriction.Animals allocated in plastic cages  had free access to bottles containing a solution composed ofSustagen M¯  at 28.5 g/100 mL and no chow. The liquid diet was the only source of food available to mice; it wasprepared fresh every day and presented to animals at the same time (12 pm). Thisliquid diet provided all necessary nutrients for rodents and was presented in alarger volume than normally eaten to ensure consumption Liquid diet was administered for 15 days. It was distributed in three cycles of 5days, with a 2-day interval without the diet between each diet cycle. This procedure mimics a kindling mechanism in alcoholwithdrawal in which repeated experiences of withdrawal intensify its symptoms . Groups Over 5 consecutive days, mice from the stress group were placed in a cylindrical 5-Lcontainer [22 (height)×17 cm (diameter)] filled with 3.5 L of water, from which micecould not escape. On days 1, 3, and 5, mice remained in the water for a single trialof 15 min. On days 2 and 4, mice underwent three trials of 6 min with intervals of 5min. After each swimming session, mice were towel-dried and returned to their homecages. Stress procedures were undertaken at different schedules (9 am to 5 pm). Thecontrol group comprised mice not exposed to stress.Ethanol withdrawal was evaluated by behavioral analyses in the elevated plus maze(EPM) and open field (OF) for 12 h and 14 h, respectively, after final exposure tothe liquid diet. All behavioral tests were conducted between 9 am to 12 pm.The EPM comprises a plus-shaped wooden apparatus elevated 38.5 cm above floor levelwith two open arms [30 cm (length)×5 cm (width)×0.25 cm (height)] and two closed arms[30 cm (length)×5 cm (width)×15 cm (height)] connected by a common central platform[5 cm (length)×5 cm (width)].Mice were placed in the central platform of the EPM facing an open arm and had theirbehavior video-recorded by a camera fixed to the roof for 5 min for analyses of the:number of entries (arm entry = 4 paws in the arm) in closed arms; percentage ofentries in open arms [×100]; percentage of time spent in open arms [×100)]; number of head dips (exploratory movement of the head over theside of the maze); stretched attend postures . Behavioralanalyses were carried out using the X-Plo-Rat 2005 software .The OF is a circular arena , 30 cm in diameter, surrounded bytransparent walls  with its circular floor divided by lines into fourcentral and eight peripheral quadrants of equivalent area.Mice were placed in the middle of the apparatus and the percentage of time spent inthe center ×100), time spent in each central quadrant  and number of quadrants crossed was quantifiedover 5 min for assessment of peripheral, central and total locomotion using theOpenFLD software .Voluntary consumption of ethanol was evaluated using the “three-bottle choice”paradigm 24 h after evaluation of ethanol withdrawal as described above.ad libitumduring this experiment. Bottles were presented to miceat the same time each day (12 pm).Every other day, for 9 days, mice housed individually in home cages [19 (width)×30(length)×13 cm (height)] were exposed to three bottles simultaneously: one containingfresh water, one containing ethanol 4% in fresh water (v/v) and another containingethanol 8% in fresh water (v/v). Every day the positions of the bottles wereinterchanged, with no preference given to the side of placement. Only fresh water wasavailable during the days that consumption was not measured. Food was availableviabottles placed in empty cages. Amount of liquid lost in theseempty cages was subtracted from the respective solution exposed to mice. Values ofethanol consumption are reported relative to the body weight of mice (g/kg), inpreference to ethanol [×100] and in preference to bottles containing 4% or 8% of ethanol [×100]. Amount of liquid lost by evaporation or leakage was measuredStatistical analyses were done using the Statistica software .Data are reported as means±SE of 8-10 animals per group. Results of EPM and OFexperiments were analyzed by two-way analysis of variance (ANOVA) considering thefactors “chronic ethanol” (ethanol×vehicle) and “stress” (stress×control). Voluntaryconsumption of ethanol and body weight were analyzed by three-way ANOVA consideringthe factors “chronic ethanol”, “stress” and “time”. If ANOVA showed significantdifferences (P≤0.05), a planned comparison test between the groups of interest wasundertaken.5,160=7.68, P≤0.05).Body weight of mice in groups did not differ from each other throughout the experimentalprocedure . Two-wayNo significant differences were found for mouse behavior in EPM . All gro1,32=9.72,P≤0.05) in peripheral locomotion but not for the stress factor or interaction, showing adecrease in peripheral locomotion in those groups  in total locomotion but not for the stress factoror interaction, showing a decrease in total locomotion in those groups . Mice from ethanol-treated groupsspent more time in the central part of the apparatus than vehicle-treated groups .In terms of OF behaviors , two-waye groups . Two-waye groups . The perd groups . No diffd groups . Animalsd groups , and two1,28=13.63, P≤0.05) and the interaction between ethanol, stress and timefactors .On days 1 and 3, no significant differences were found between experimental groupsrelating to total consumption of ethanol (P>0.05). On day 5, the ethanol/controlgroup and ethanol/stress group consumed more ethanol compared with the vehicle/controlgroup and vehicle/stress group (P≤0.05). On day 7, the ethanol/control group andethanol/stress group consumed more ethanol only when compared with the vehicle/stressgroup (P≤0.05). On day 9, the ethanol/stress group consumed more ethanol when comparedwith all other groups (P≤0.05).1,29=12.15, P≤0.05) and theinteraction between ethanol, stress and time factors .The total ethanol preference was changed by treatment. Three-way ANOVA showedsignificant differences for the ethanol factor . On day 5, the total ethanolpreference of the ethanol/stress group was significantly greater than the ethanolpreference of the vehicle/control group and vehicle/stress group (P≤0.05). On this day,the ethanol/control group had higher preference scores only when compared with thevehicle/control group (P≤0.05). On day 7, the ethanol/stress group had greaterpreference for ethanol solutions when compared with all other groups (P≤0.05). On day 9,the only difference found in total ethanol preference was between the ethanol/stressgroup and vehicle/stress group, in which the ethanol/stress group had a higherpreference for ethanol solutions.1,29=6.85, P≤0.05) and interaction between ethanol, stress and timefactors . On day 1, the ethanol/control group consumedmore 4% ethanol solution only when compared with the ethanol/stress group (P≤0.05). Onday 3, consumption of 4% ethanol solution of the ethanol/control group was greater thanall other groups (P≤0.05). On day 5, the ethanol/control group consumed more 4% ethanolsolution compared with the vehicle/control group and vehicle/stress group (P≤0.05). Ondays 7 and 9, the ethanol/stress group consumed more 4% ethanol solution when comparedwith the vehicle/stress group (P≤0.05).The ethanol/control group showed increased consumption of 4% ethanol solution. Three-wayANOVA for consumption of 4% ethanol solution showed significant effects for the ethanolfactor . On day 1, the ethanol/control group hada higher preference for 4% ethanol solution when compared with the ethanol/stress group(P≤0.05). On day 3, the ethanol/control group had the greatest scores for preference of4% ethanol solution (P≤0.05). On day 5, the preference of the ethanol/control group for4% ethanol solution was higher only when compared with the vehicle/control group(P≤0.05). On day 7, the ethanol/stress group had higher preference for 4% ethanolsolution than groups that did not receive ethanol  (P≤0.05). No differences between groups were found for preferenceof 4% ethanol solution on day 9.Preference for 4% ethanol solution was altered in the ethanol/control group. Three-wayANOVA for preference for the 4% ethanol solution showed a significant effect of theethanol factor  and a trend ofsignificance for the interactions between stress and ethanol factors. On day 1, the ethanol/stress group consumedsignificantly more 8% ethanol solution than the vehicle/control group and vehicle/stressgroup (P≤0.05). On day 3, the ethanol/stress group consumed more 8% ethanol solutiononly when compared with the vehicle/control group (P≤0.05). On day 5, the ethanol/stressgroup showed increased consumption of 8% ethanol solution compared with all experimentalgroups (P≤0.05). On days 7 and 9, the ethanol/stress group consumed more 8% ethanolsolution compared with the ethanol/control group and vehicle/stress group (P≤0.05).Animals in the ethanol/stress group consumed more 8% ethanol solution than animals inall other groups. Three-way ANOVA for consumption of 8% ethanol solution showed asignificant effect of the ethanol factor  and a trend of significance for the stress factor. No changes were found in preference for 8% ethanolsolution on day 1. On day 3, the preference for 8% ethanol solution of theethanol/stress group was greater than the preference for 8% ethanol solution of thevehicle/control group and vehicle/stress group (P≤0.05). On days 5 and 7, theethanol/stress group had higher preference for 8% ethanol solution than all other groups(P≤0.05).Preference for the 8% ethanol solution was also greater in animals of the ethanol/stressgroup. Three-way ANOVA showed a significant effect of the ethanol factor. However, no differences werefound between groups during the experiment on voluntary consumption of ethanol (data notshown).Three-way ANOVA for fluid intake (water + ethanol 4% + ethanol 8%) showed a significanteffect of the time factor .Ethanol withdrawal in animal models can be assessed through several behavioral andphysiologic alterations after cessation of ethanol exposure. Protocols can varyaccording to the animal species, strains, time after cessation of ethanol consumption,and test apparatus employed . DespiteSigns of ethanol withdrawal syndrome start to appear when levels of alcohol in bloodapproach zero. The type, duration and severity of these signs are dependent on theamount of ethanol administered and duration of treatment. In general, anxiety related toethanol withdrawal appears 6-15 h after drug discontinuation . Our expIn the OF apparatus, ethanol-treated mice showed decreased locomotor activity. Indeed,alterations in locomotor activity in the OF are, in general, used to indicate ethanolwithdrawal. In rats, removal of ethanol could result in hyper-locomotion andhypo-locomotion whereas, in mice, hypo-locomotion is seen more commonly thanhyper-locomotion ,222324Time spent at the center of the OF was increased in ethanol-treated animals. Despite useof this behavior in the measurement of anxiety in animal models , in our Psychomotor stimulation induced by drugs of abuse reflects activation of thedopaminergic mesolimbic pathway induced by these substances. This property seen inanimal models is shared by all drugs known to cause addiction in humans . SimilarExposure to stressful situations results in changes in locomotor activity andanxiety-like behavior in mice and rats. For example, chronic restraint stress increaseslocomotor activity  and coulConcomitant exposure to stress and ethanol results in complex interactions. For example,stress and ethanol alone cause impairment of memory function in object recognition testsand the y-maze apparatus. Concomitant stress/ethanol exposure reverses these effects.Alcohol also blocks stress-induced increases in anxiety-like behaviors in the plus mazetest. However, alcohol cannot block weight loss and increases in plasmaticcorticosterone levels induced by stress . Inthe Data from experiments on voluntary consumption of ethanol showed that pre-exposure toethanol caused an increase in the consumption of, and preference for, ethanol. Thisincrease was observed only for 4% ethanol solution. Scholars have shown increasedconsumption of ethanol or self-administration of ethanol after forced and chronicpre-exposure to ethanol in rats and mice 353637A large body of work examining the impact of acute or sub-chronic stress on ethanolconsumption has been conducted on rodents. Several stress procedures have been employedand, despite a general observation that stress is associated with increased drinking ofalcohol, the literature reveals equivocal findings: increased, decreased, or no changein alcohol consumption . Humans Animal housing during testing of voluntary consumption of ethanol was probably not asignificant stressor that would increase ethanol intake in mice because the effect ofisolation stress in mice is significant only if isolation starts during early ages andnot during adulthood . NeverthThe increase in ethanol intake in mice co-exposed to stress and ethanol in our studydoes not seem to be related to an attempt to alleviate the symptoms of ethanolwithdrawal. First, the ethanol/stress group did not show higher levels of ethanolwithdrawal compared with the ethanol/control group. Second, increased consumption of,and preference for, ethanol was long-lasting (continuing to appear on the last day ofvoluntary consumption of ethanol), whereas withdrawal  was short-lived.Hence, concomitant exposure to ethanol and stress may result in long-lasting neurologicadaptations that aggravate ethanol addiction. The relationship between stress andethanol drinking is complex because of many ethanol-related factors , along with stress-related factors  that interact with several biologic variables  .In summary, chronic pre-exposure to ethanol led to a decrease in locomotor activityrelated to withdrawal of this substance, an increase in voluntary intake of ethanol, andpreference for low-concentration ethanol solutions. Concomitant stress did not aggravateethanol withdrawal, but potentiated the intake of and preference for ethanol, therebychanging patterns of voluntary consumption towards highly concentrated ethanolsolutions.