Lactobacillus strains of dairy origin were evaluated for some functional properties relevant to their use as probiotics. A preliminary subtractive screening based on the abilities to inhibit the growth of microbial pathogens and hydrolyze conjugated bile salts was applied, and six strains were selected for further characterization including survival under gastrointestinal environmental conditions, adhesion to gut epithelial tissue, enzymatic activity, and some safety properties. All selected strains maintained elevated cell numbers under conditions simulating passage through the human gastrointestinal tract, well comparable to the values obtained for the probiotic strain Lactobacillus rhamnosus GG, and were able to adhere to Caco-2 cells to various extents (from 3 to 20%). All strains exhibited high aminopeptidase, and absent or very low proteolytic and strong β-galactosidase activities; none was found to be haemolytic or to produce biogenic amines and all were susceptible to tetracycline, chloramphenicol, erythromycin, ampicillin, and amoxicillin/clavulanic acid. Our results indicate that the Lactobacillus strains analyzed could be considered appropriate probiotic candidates, due to resistance to GIT simulated conditions, antimicrobial activity, adhesion to Caco-2 cell-line, and absence of undesirable properties. They could be used as adjunct cultures for contributing to the quality and health related functional properties of dairy products.Twenty-three  Lactic acid bacteria (LAB) are widespread in nature and are one of the major microbial groups involved in the fermentation of different types of food. They represent the dominant microorganisms in milk and milk products where they play important roles during both manufacture and ripening. LAB possess a large number of metabolic properties that are responsible for the organoleptic characteristics of the final product as well as its preservation and microbial safety . They arAs a result of increasing awareness of the close interrelationship between health and diet, special attention is presently given to functional properties of LAB associated with traditional fermented products. These foods could represent alternative sources of novel probiotic candidates with physiological and functional properties for potential biotechnological use in the manufacturing of functional products in which the probiotic cultures are more active and protected from the gastrointestinal stress.Among food ecosystem, cheese has some intrinsic chemical and physical characteristics  that make it suitable for delivery of viable probiotic microorganisms into the human intestine . Raw mil Lactobacillus strains have been extensively exploited for their probiotic properties [Among LAB,operties  and are operties .Lactobacilli are generally regarded as safe (GRAS) due to their long history of safe use and their presence in the intestinal microbiota of humans, and several species have received the qualified presumption of safety (QPS) status [) status , 8. Lactobacillus plantarum, L. paracasei, and L. brevis, which constitute the majority of NSLAB found in most ripened cheese varieties, may play a role as health promoters, beyond their technological function [Recently an increasing interest was paid to the possibility that strains belonging to species such asfunction .To perform their probiotic action these bacteria must arrive at the intestinal tract alive. This requires their survival during food processing, product maturation, and shelf-life and, after consumption, their resistance to the acidic conditions of the stomach as well as to bile salts in the small intestine . Lactobacillus strains of dairy origin were investigated for some functional properties relevant to their use as probiotic cultures. Antagonism toward microbial pathogens, ability to deconjugate bile salts, survival in the gastrointestinal tract, adhesion capability to intestinal epithelial cells, enzymatic activities, and some safety features such as hemolytic activity, antimicrobial resistance, and production of biogenic amines were in vitro analyzed.As a part of a larger study involving the selection and characterization of new probiotic candidates, 23 autochthonous Lactobacillus strains  isolated from raw milk and artisanal ewes' cheeses were included in this study. The strains were identified on the basis of phenotypic tests and genetic analysis based on polymerase chain reaction amplification using species-specific primers derived from 16S rRNA sequences, as previously reported [ Listeria monocytogenes ATCC 7644, Escherichia coli ATCC 35150, Enterococcus faecalis ATCC 29212, Salmonella Typhimurium ATCC 14028, Staphylococcus aureus ATCC 25923, and Candida albicans ATCC 10231 were used as indicators. All indicator strains were stored on nutrient broth (Microbiol) plus 20% (v/v) glycerol at −20°C. Before use, they were subcultured twice in appropriate medium.A total of 23reported . The str Lactobacilli were spotted onto the surface of MRS agar (1.2% (w/v) agar—0.2% (w/v) glucose) plates, which were then incubated anaerobically for 24 h at 37°C. The indicator strains were inoculated into 7 mL of soft agar medium (nutrient broth containing 0.7% w/v agar) to a final concentration of approximately 107 colony forming unit (cfu)/mL; then the soft media were poured on the plates. After 24 h of incubation at the optimal growth temperature and atmosphere for the indicator strains, inhibition halos were measured. The width of the clear zone (R) was calculated as follows: R = (dInhib − dSpot)/2, where dInhib is the diameter of the zone without pathogen growth and dSpot is the diameter of the spot.The strains were screened for antimicrobial compounds production against the indicator strains using an agar spot method . OvernigμL of cultures grown overnight in MRS broth on the surface of MRS agar plates supplemented with 0.5% (w/v) sodium salt taurodeoxycholic acid  or 0.2% (w/v) glycodeoxycholic acid  and 0.37 g/L of CaCl2 [ Enterococcus faecalis ATCC 14433 was used as BSH-positive strain.Bile salt hydrolase (BSH) activity was screened by spotting in duplicate 10 of CaCl2 . Plates  in vitro model simulating gastric and pancreatic juices , as reported by Vizoso Pinto et al. [8 cfu/mL in 10 mL of simulated gastric juice  and incubated at 37°C in a shaking water bath  to simulate peristalsis. After 90 min, 17.5 mL of synthetic duodenum juice , adjusted to pH 7.4 with 5 M HCl, and 4 mL of 10% (w/v) oxgall (Sigma) were added to the cell suspensions to simulate passage into the upper intestinal tract [Transit tolerance in the upper GIT was assessed using ano et al. . The stral tract . After 0μg/mL streptomycin). Cells were maintained in T-75 culture flasks at 37°C in a 5% CO2 atmosphere. For adhesion assay, the Caco-2 cells were seeded at a concentration of 105 cells/well in 6-well tissue culture plates  to obtain confluence and cultured for 20 days prior to use in adhesion assay. The cell culture medium was changed on alternate days and replaced by fresh DMEM supplemented with 2% (w/v) FBS and without antibiotic at least 1 h before the adhesion assay. A 1 mL aliquot of Lactobacillus suspension  was added to each well of the tissue culture plate and incubated at 37°C in 5% CO2 atmosphere for 3 h. Afterwards, the cells were washed three times with 1 mL of PBS in order to remove nonadherent bacteria and lysed by addition of Triton X 100 (0.05% solution) for 10 min; then appropriate dilutions were plated on MRS agar. Adhesion was expressed as the percentage of bacteria adhered to Caco-2 cells compared to the initial amount of bacteria.The human colonic carcinoma cell line Caco-2  was routinely cultured in Dulbecco's modified Eagle's minimal essential medium DMEM (Sigma), supplemented with 10% (w/v) foetal bovine serum , 1% (w/v) nonessential amino acids solution (Sigma), and antibiotic solution  as described by the manufacturer. The results were graded from 0 to 5 by comparing the colour developed within 5 min with the API-ZYM colour reaction chart. The results were expressed in nanomoles of hydrolysed substrate, from the intensity of the reactions obtained, in the range 0 (no activity) to 5 (40 or more nanomoles liberated), following the manufacturer's instructions.Enzymatic activities of Lactobacillus strains for the production of biogenic amines. Briefly, the test strains were subcultured twice at 24 h intervals in MRS broth containing 1% of each precursor amino acid: tyrosine disodium salt, l-histidine monohydrochloride, l-ornithine monohydrochloride, and lysine monohydrochloride (Sigma), and 0.005% pyridoxal-5-phosphate (Sigma) as a codecarboxylase factor. All strains were then streaked in duplicate on decarboxylase medium plates each containing only one of the abovementioned amino acids and bromocresol purple as pH indicator and incubated for 4 days in anaerobic conditions at 37°C. Decarboxylase medium without amino acids was used as control. A colour change from brown to purple in the medium indicated an increase in pH and was considered a positive result.The method of Bover-Cid and Holzapfel  was usedμg), amoxicillin/clavulanic acid , vancomycin , teicoplanin  , tetracycline , streptomycin , kanamycin , gentamicin , chloramphenicol , erythromycin  (inhibitors of protein synthesis), ciprofloxacin , and rifampicin  (inhibitors of nucleic acids). A suspension from fresh overnight cultures with a density of McFarland 0.5 in buffered saline was plated on MRS agar plates; then antibiotic discs were dispensed onto the plates. After incubation at 37°C for 24 h in anaerobiosis, the diameters of the bacterial free-zone were measured and results expressed in terms of resistance according to the interpretative criteria issued by the CLSI [Antibiotic susceptibilitytesting was carried out by disc diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines  but Muelthe CLSI .β-haemolysis), a partial hydrolysis and greenish zone (α-haemolysis), or no reaction (γ-haemolysis).Haemolytic activity was determined by streaking the strains on Columbia Blood (Microbiol) agar plates supplemented with 5% defibrinated sheep blood after 48 h of incubation at 37°C. The haemolytic reaction was recorded by observation of a clear zone of hydrolysis around the colonies . One-way ANOVA followed by Bonferroni test was used to determine significant differences of viability of the L. monocytogenes, S. aureus, and E. coli O157:H7 with clear inhibition zones of more than 6 mm in agar-spot plates. A lower activity was detected against E. faecalis, while one strain showed an inhibition zone of 4 mm against C. albicans ATCC 10231. Such broad antagonistic activity of Lactobacillus towards different pathogens has been reported [ L. plantarum isolated from cheese showed no activity against selected indicator pathogens [A preliminary subtractive screening based on the abilities to inhibit the growth of microbial pathogens and hydrolyse conjugated bile salts was applied to the strains. As shown in reported , 20; howathogens .LAB are known to produce many different substances with antimicrobial activity, including the major metabolic end products such as organic acids, hydrogen peroxide, ethanol, and bacteriocins . When thL. plantarum 11/20966, 4/16868, 19/20711 and L. paracasei 62LP39, 1A6 M) demonstrated also the ability to hydrolyze both sodium glycodeoxycholate (GDCA) and sodium taurodeoxycholate (TDCA), and four were able to deconjugate only GDCA, as indicated by the BSH test on agar plates. Some authors have shown that in Lactobacilli the resistance to bile salts toxicity is not related to hydrolase activity [BSH activity is a relevant property for probiotic strains to survive the toxicity of conjugated bile salts in the duodenum . In our activity , 24–26. activity . Lactobacillus, Bifidobacterium, and Enterococcus isolated from the gastrointestinal tract (GIT): it has been reported for L. plantarum [ L. paracasei strains [Bifidobacteria and Lactobacilli) are not capable of dehydroxylating deconjugated bile salts [BSH activity is frequently observed in specific groups of bacteria likelantarum , 28 but  strains , 29. Pre strains  or negat strains . Howeverle salts , 33 and le salts . On the le salts . C. albicans ATCC 10231 were selected for further functional characterization including in vitro tests to assess their survival under gastrointestinal environmental conditions and adhesion to gut epithelial tissue and to determine the enzymatic activity and some safety properties.Among the BSH positive strains, five showing the ability to ferment the trisaccharide raffinose, known for its prebiotic activity, and one with a high antagonistic activity against L. rhamnosus GG was included in the study for comparison purpose. All strains retained high viability during simulated gastric juice (pH 3.0) transit for 90 min, and no significant reduction (P > 0.05) was found for any of the strains after exposure to artificial duodenum juice (pH 8.0). The strains L. paracasei 62LP39 and L. plantarum 11/20966 and 4/16868 presented the best survival rates values with more than 98% surviving cells.The survival responses of the strains, after 90 and 180 min of exposure to different gastrointestinal conditions, are shown in  L. rhamnosus GG. The survival rates were above 90% after 3 h despite the high bile concentration (more than 1%) used in the test. These results are consistent with those previously reported for other potentially probiotic strains belonging to L. paracasei [ L. plantarum [Exposure to gastric and intestinal fluids is the main environmental stress that decreases viability of ingested probiotics . All selaracasei  and L. plantarum  and corr Lactobacillus strains is reported in  L. rhamnosus GG (P < 0.05). L. paracasei 62LP39 was the most adhesive strain since approximately 20% of the added bacteria were bound to Caco-2 cells, followed by L. brevis 1C3 M (14%) and L. plantarum 11/20966 (10.5%). A lower adhesion rate was observed for the strain L. paracasei 1A6 M (3%). The percentage of adhesion of L. rhamnosus GG, used as positive control, was similar to those obtained with the same strain in other studies [ L. plantarum strains have been shown to adhere more efficiently to Caco-2 cells than L. paracasei or L. brevis [ L. paracasei strains were effective in reducing adhesion of Salmonella to Caco-2 cells in competitive assays, in spite of their low adherence ability [The adhesion ability of studies , 38 but . brevis , 38, alt. brevis  obtained ability –42. in vitro studies suggested that probiotic adhesion may interfere with the adherence of pathogens, exerting a barrier against pathogen colonization through competitive exclusion mechanisms [Severalchanisms , 40.β-glucosaminidase activities were absent or very low. All strains exhibited strong β-galactosidase and moderate-to-high α-galactosidase activities, which are responsible for the hydrolysis of lactose and raffinose, respectively. None of the strains showed β-glucuronidase or α-mannosidase activity.Enzymatic characterization of the selected strains was carried out in order to evaluate their potential for using as adjunct cultures in the manufacturing of probiotic cheese and is reported in  L. plantarum [β-galactosidase in 14 L. paracasei isolates and Herreros et al. [ L. brevis. Strains with low proteinase, high peptidase, and low esterase-lipase activities may be useful in reducing bitterness and improving body and texture defects [β-galactosidase activity is an important feature for strains to be used as probiotics, since it may be useful in improving lactose tolerance in the gut [β-glucuronidase activity is an important trait, as well, since its negative role has been suggested in increasing the risk of carcinogenesis [Overall, the enzymatic profiles of our strains are similar to those reported by other authors particularly for the specieslantarum –44. Mathlantarum  could nos et al.  found a  defects , 44. β-g the gut . The lacogenesis .In order for a strain to be used as probiotic culture, it should be evaluated for the presence of virulence determinants to determine what potential risks might be involved in its use. Biogenic amines are produced by LAB during the process of fermentation of foods and beverages by amino acid decarboxylation. Bover-Cid and Holzapfel  suggeste Lactobacillus strains were assayed for their resistance to 12 antibiotics using a disc diffusion method on MRS agar plates under anaerobic conditions, and the results are reported in The L. plantarum and L. paracasei strains [Our results are in substantial agreement with those reported for strains , 29, alt strains , 48. Lactobacilli [ Lactobacilli and it has been attributed to the synthesis of modified cell wall peptidoglycan precursors that end in a depsipeptide d-alanine-d-lactate instead of the dipeptide d-alanine-d-alanine, the target for vancomycin activity [ Lactobacillus strains is not of the transmissible type. The strains tested in our work do not seem to represent a source for transfer of genes encoding resistance, since they were phenotypically susceptible to tetracycline, erythromycin, and chloramphenicol, but the absence of the genes needs to be confirmed genotypically. Comunian et al. [The resistance observed against some antibiotics tested suggests that our strains would not be affected by therapies using these antibiotics and might help maintain the natural balance of intestinal microflora during antibiotic treatments . Resistaobacilli , 51. In activity . Moreoveactivity  showed tn et al.  recentlyFinally, none of the strains was found to be haemolytic (data not shown), in agreement with previous studies . Lactobacillus strains  could be considered appropriate probiotic candidates, due to resistance to GIT simulated conditions, antimicrobial properties, adhesion to human intestinal epithelial cell-line (Caco-2), and absence of undesirable properties. They could be used as adjunct cultures for contributing to the quality and health related functional properties of dairy products; however, additional studies are required to confirm in vivo this findings as well as to assess the strains stability to manufacturing processes.In conclusion, our results indicate that some


The intensity and quality of pain and anxiety were evaluated using the visual analogue scale, short form and modified-McGill pain questionnaire (SFM-MPQ) and the Spielberger situational anxiety level inventory (STAII) scale, respectively. Patients in all treatment groups had significantly lower pain intensity and anxiety compared with the control group immediately, 5, 10 and 15 min after CTR. There was no statistically significant difference in the SFM-MPQ total scores between the intervention groups. With respect to anxiety score, there was a significantly reduced anxiety level immediately after CTR in the aromatherapy and cold-aromatherapy combination groups versus the cold application group. The present results suggested the efficacy of cold application and aromatherapy with lavender oil in reducing pain and anxiety associated with post-CABG CTR.Post-surgical chest tube removal (CTR) is associated with a significant pain and discomfort for patients. Current treatment strategies for reducing CTR-associated pain and anxiety are limited and partially efficacious. To determine the effects of cold application, inhalation of lavender essential oil, and their combination on pain and anxiety during CTR was investigated. This randomized controlled open-label trial was conducted with 80 patients in the cardiac surgery intensive care unit who had a chest tube for duration of at least 24 hours after coronary artery bypass grafting (CABG). Patients were randomized ( Abbreviations: BMI: Body mass index; CABG: Coronary artery bypass grafting; CTR: Chest tube removal; GC: Gas chromatography; ICU: Intensive care unit; MS: Mass spectrometry; RI: Retention index; SFM-MPQ: Short form and modified-McGill pain questionnaire; STAII: Spielberger situational anxiety level inventory; VAS: Visual analogue scaleInsertion of chest tube following coronary artery bypass grafting (CABG) is a frequent practice aimed at maintaining hemodynamic stability and cardiopulmonary function. Chest tube facilitates drainage of the air, blood and other fluids from the pleural, pericardial or mediastinal cavity and prevents pleural effusion, pneumothorax and hemothorax. Chest tubes are typically removed 24-48 hours after surgery, or when the excess air, blood, or fluid has been properly drained. The tube adheres to the surrounding tissues, and its removal and separation of adjoined tissues is painful for the patient .Cold applications are commonly used as a non-pharmacological method for pain relief. Cold slows down tissue metabolism and nerve conduction velocity locally and also has vasoconstrictive, anti-inflammatory, anti-spasmodic and analgesic effects. The analgesic effects of cold treatment can also be explained by the Melzack and Wall's gate control theory. According to this theory, the stimulus caused by CTR activates fibers within the parietal pleura, chest muscles, and chest tube insertion site. It is believed that activation of these pain transmitting fibers results in a release of various excitatory neurotransmitters. Application of cold may lead to a reduction or reversal of the pain impulse via activating descending inhibitory neurons that block ascending nociceptive nerves originating from the substantia gelatinosa. Reduction of anxiety is another mechanism that can lead to a decreased perception of pain , is a commonly used agent in aromatherapy . Lavendal., 2012; Morris,al., 2012; Sasanneal., 2012; Bagherial., 2012). The anal., 2012; Sasanneal., 2012). It hasal., 2012; Olapoural., 2012; Bagherial., 2012). These al., 2012; Babashaal., 2012).Owing to the contradictory results on the efficacy of conventional pharmacological methods in relieving CTR-associated pain and discomfort, the present research was conducted to examine the effects of cold application, aromatherapy with lavender oil, and combination of both of these methods on the severity of pain and anxiety during CTR. 2 (2 ml/ min); and detector temperature 280 °C.Lavender oil was provided by the Giah Essence Pharmaceutical Company (Gorgan-Iran). The plant material was collected by Dr. H. Soleimani from the Tooskestan region  located at the Southeast Gorgan, Golestan province, Iran. The plant was identified by Mrs. Souzani and a voucher sample (no. 13144) was identified and deposited in the herbarium of the School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran. Chemical composition of the essential oil was analyzed using Gas Chromatography (GC) and GC-Mass Spectrometry (GC-MS) methods. GC analysis was performed using a Varian CP-3800 apparatus equipped with a FID detector and fused silica column . The operating conditions were oven temperature 50 °C (5 min), 50 °C-250 °C (3 °C /min), 250 °C (10 min); injector temperature 260 °C; split ratio 1:5; carrier gas NGC-MS analyses were performed using an Agilent 5975 apparatus with a HP-5ms column  interfaced with a quadruple mass detector and a computer equipped with Wiley 7n.l library. The operating conditions were oven temperature 50 C (5 min), 50 C-250 C (3 C/min), 250 C (10 min); injector temperature 250 °C; injection volume 0.1 µL; split ration, 1:50; carrier gas Helium at 1.1 mL/min; ionization potential 70 eV; ionization current 150 µA; ion source temperature 250 °C; and mass range 35-465 mui  and the oil on a CP-Sil 8CB column. Identification of individual compounds was made by comparison of their mass spectra and retention indices (RI) with those of authentic samples and those reported in the literature . Quanti2, full consciousness, ability to report pain, experiencing cardiac surgery and CTR for the first time, and having normal vital signs. Exclusion criteria were receiving mechanical ventilation support, continuous infusion of sedatives and analgesics or opioid analgesics during less than 4 hours before intervention, presence of psychiatric disease or any disease that could affect the research question , difficulty in breathing, nausea, vomiting, history of hypersensitivity or dissatisfaction during aromatherapy or cold application, and history of drug addiction or smoking. Eighty ICU-hospitalized patients who had a chest tube for at least 24 hours after cardiothoracic surgery were included in this study. Other inclusion criteria were age > 18 yrs, body mass index (BMI) < 30 kg/ mThis study was a randomized comparative trial with a factorial design that was conducted between December 2014 and March 2015 in the cardiac surgery intensive care unit (ICU) of the Imam Reza Hospital, Mashhad, Iran. Approval for the study was obtained from the Ethics Committee of the Mashhad University of Medical Sciences . Written informed consent was obtained from each participant prior to the study commencement.Eligible subjects were randomized (using random number tables) in an open-label fashion (using a random number table) to one of the following four groups:Group I: applied cold with cooling gel pack .Group II: inhaled lavender essential oil .Group III: applied cold with cooling gel pack and inhaled lavender essential oil .Group IV: did not receive either cold application or lavender oil inhalation . Pain intensity was measured using a vertical visual analog scale (VAS) from 0 to 10 . Numerous studies have used this scale to measure pain severity in CABG patients  was used to evaluate pain quality during CTR. Reliability and validity of the MPQ was previously established by Melzack (1975). The vaOn the first postoperative morning, patients' readiness for CTR was determined by a physician according to the standard criteria. Patients were instructed by the researcher to use VAS, SFM-MPQ and STAII for rating pain intensity, quality and anxiety level. In addition, some information about cold application and inhalation of lavender essential oil was presented to the participants. At 6 am CTR day, an ICU nurse administered 1 gr acetaminophen intravenously to all study subjects. Cold packs were kept in freezer for at least 2 h and then were covered with gauze or special pocket (according to the manufacturer's leaflet) and applied to the area surrounding the chest tube until the skin temperature reached 13 °C.The researcher asked the patients to mark the pain intensity, quality and situational anxiety they felt with the chest tube in place. Pain intensity was measured 10 minutes before, and immediately, 5, 10, and 15 minutes after CTR. Pain quality and situational anxiety of the patients were measured 10 minutes before and immediately after CTR.All patients were reassured that analgesic drugs would be administered as needed. All chest tubes were removed by the same ICU nurse to ensure the reliability of the results. The procedures for the application of cold and aromatherapy are as follows.In the cold application group, cooling gel packs  were used to reduce the body temperature around the chest tube during undressing. The bandage was removed from the chest area and body temperature was measured and recorded by a non-contact infrared thermometer . Then, a single layer of sterile gauze pad was placed around the skin area of pericardial tube insertion  and cooling packs (14×18) were twisted in gauze and placed on top of it.Cold application was terminated when the skin temperature reached 13 °C. After removal of cooling packs, the nurse removed the pericardial tube within 1-2 minutes. The intensity and quality of pain experienced by the patient and the anxiety level were measured and recorded at the time points described above. In the aromatherapy group, 1-2 drops of aromatherapy blend containing lavender essential oil were poured on cotton and fixed at a 10 cm distance from the patients' nose. The patient was asked to breathe slowly for 20 minutes and then pain and anxiety were measured as described above.SPSS software version 11.5 was used to analyze the recoded data. Descriptive statistics were used to summarize demographic characteristics of the study participants. Normality of data were checked using Kolmogorov-Smirnov and Shapiro-Wilk tests. Baseline parameters of the study groups were compared using one-way ANOVA, Chi-square and Fisher's exact tests were used. To compare the efficacy measures among the study groups, one-way ANOVA, Kruskal-Wallis, and repeated measures ANOVA tests were used. Bivariate correlations between anxiety and pain scores before and immediately after CTR were performed using Pearson's correlation coefficient. In all analyses, a two-sided P value of <0.05 was considered as statistically significant.Ninety-three compounds were identified in the lavender oil, representing 99.0 % of the total oil composition Table 1. The maiThe mean age of participants was 54.3 ± 10.9 yrs. The study population consisted of 56.2 % men and 43.8 % women, and distribution of genders among the study groups was similar (P=0.77). All subjects had two chest tubes  after CABG.Mean tube remaining time was 45.9±1.3 hrs, and there was no significant difference among the study groups regarding this parameter (P=0.17). Likewise, the groups were comparable with respect to demographic characteristics including marital status (P=0.23), education (P=0.86), and residential status  (P=0.91), the worst pain experienced in the past (P=0.58) and its description (P=0.33). Baseline characteristics of the study groups are summarized in Table 2P=0.55). Repeated-measures analysis revealed a significant increase in pain severity immediately after CTR, followed by a continuous decreasing trend until the last assessed time point in all study groups (P<0.001). Post-hoc multiple comparisons revealed significantly reduced pain intensity in all intervention groups compared with the control group immediately after CTR, as well as at 5, 10 and 15 min post-CTR time points. However, there was no significant difference among cold application, aromatherapy and cold-aroma groups.The study flow diagram is show in Figure 1P<0.001 [cold and cold-aroma groups] and P=0.020 [aroma group]); however, there was no statistically significant difference between the interventions.The words selected by most of the subjects to describe the present pain endured during CTR were “discomforting” (reported by 37.5 %), “distressing” (22.5 %), and “horrible” (21.2 %). Also, the words selected by subjects to describe pain during CTR, according to the SF-MPQ sclae were “fearful” (70 %), “shooting” (51.2 %), “tender” (51.2 %), “sickening” (48.8 %), “hot or burning” (46.2 %) and “sharp” (30 %). The total SFM-MPQ score in cold, aroma, cold-aroma and control groups were 19.5±5.8, 21.9±7.2, 17.1±9.9 and 29.1±5.5, respectively. All interventions were associated with a reduced total SFM-MPQ score compared with the control group (P<0.001). Post-hoc multiple comparisons revealed a significant difference between the aroma group with cold (P<0.009) and control groups (P<0.007), and also between the cold-aroma group with cold (P<0.001) and control groups (P<0.001).Table 4Bivariate correlations using Pearson's correlation coefficients did not suggest any significant correlation between anxiety and pain scores either before or immediately after CTR. Likewise, there was no significant correlation between age, sleep duration, length of chest tube in the body, duration of chest tube presence in the body and fatigue score with either pain or anxiety score immediately after CTR.Findings of the present study showed that all three types of interventions  could reduce pain and anxiety associated with CTR. However, there was no significant difference among the applied interventions, and combination therapy did not further improve the analgesic and anti-anxiety effects of each intervention. Although numerous studies have shown the efficacy of non-pharmacological methods in reducing pain, to the best of authors' knowledge, this is the first study comparing the effects of cold application and lavender aromatherapy in reducing the pain associated with CTR. In order to eliminate the confounding effect of surgery type and chest tube type on the study outcomes, CTR-associated pain was assessed in subjects undergoing pericardial tube removal following CABG. L. angustifolia. The volatile oil of the herb had 19 compounds representing 90.4 % of the oil composition, and the main components were linalool (39.5 %), terpinen-4-ol (7.3 %) and linalyl acetate (32.4 %). Recently, Tayarani-Najaran et al.  studiedal. (2014) identifIn previous studies, lavender oil was shown to be useful in reducing the pain associated with migraine, cesarean section, and needle insertion into a fistula in hemodialysis patients . In thiThe MPQ words selected most frequently by subjects in this study were “fearful”, “shooting”, “tender”, “terrible” and “stinging”. In earlier studies on CTR, the frequency of selection of “terrible” from the MPQ varied considerably. Sauls (2002) found tIn the literature, there has been only a few studies related to anxiety associated with CTR. Mimnaugh et al. (1999) also reAs a limitation, this study was performed on a relatively small number of subjects and was limited to patients who underwent CABG surgery. Whilst this inclusion criteria increase the homogeneity of the study population, it will make generalizability of findings difficult. Moreover, the subjective nature of efficacy measures might have introduced bias into the results, though the effect of such a potential bias could be minimized by the randomized design of the study. Finally, this study was performed in an open-label fashion because the nature of interventions made it impossible to design appropriate placebo.In conclusion, the findings of the present randomized controlled trial suggested that cold application, aromatherapy, and cold application in combination with lavender oil inhalation could be used as non-pharmacologic interventions to relieve pain associated with CTR. Given the simplicity and inexpensive nature of such approaches, their application in clinical practice could be suggested to the nursing staff. However, before routine application, confirmation of the present findings in larger scale trials, and in CTR-associated pain of non-CABG subjects is recommended. This study was financially supported by the Research Council at the Mashhad University of Medical Sciences, Mashhad, Iran.The authors have no competing interests to disclose.


Podocyte injury is an important mechanism of diabetic nephropathy (DN). Accumulating evidence suggests that nephrin expression is decreased in podocyte in DN. Moreover, it has been demonstrated that tongxinluo (TXL) can ameliorate renal structure disruption and dysfunction in DN. However, the effect of TXL on podocyte injury in DN and its molecular mechanism is unclear. In order to explore the effect of TXL on podocyte injury and its molecular mechanism in DN, our in vivo and in vitro studies were performed. Our results showed that TXL increased nephrin expression in diabetic rats and in high glucose cultured podocyte. Meanwhile, TXL decreased ICN1 (the intracellular domain of notch), HES1, and snail expression in podocyte in vivo and in vitro. More importantly, we found that TXL protected podocyte from injury in DN. The results demonstrated that TXL inhibited the activation of notch1/snail pathway and increased nephrin expression, which may be a mechanism of protecting effect on podocyte injury in DN. Diabetic nephropathy (DN) has become the leading cause of end stage renal disease. As we know, microalbuminuria is an early clinical hallmark of DN. The presence of albuminuria indicates the injury of the glomerular filtration barrier (GFB), of which podocyte is an important component . AccumulNephrin is a signature molecule of podocyte . NephrinEvidence is available that notch pathway is a common pathway of podocyte injury . Waters  Panax ginseng and Paeonia lactiflora Pallas. TXL has been widely used in clinical practice in China over recent years. It has been demonstrated that TXL is beneficial for heart disease and cerebral disease [TXL, which is a new kind of Chinese herbal compound, consists of a group of herbal medicines, such as disease , 15. Man disease . Moreove disease . Liu et  disease . Moreove disease , 20. Mor disease . HoweverIn the present study, TXL decreased ICN1, HES1, and snail expression and increased nephrin expression in vivo and in vitro. More importantly, TXL had protecting effect on podocyte injury in diabetic rats and in high glucose cultured podocyte. Taken together, our results showed that TXL inhibited the activation of notch1/snail pathway and increased nephrin expression, which may be a mechanism of protecting effect on podocyte injury. Chinese Pharmacopoeia (2005). The species, origin, harvest time, medicinal parts, and concoction methods for each component were strictly standardized. The components of TXL were ground to superfine powder with a diameter ⩽10 μm by a Micronizer. Moreover, the security of TXL has been approved by the State Food and Drug Administration in China. In our in vivo study, TXL superfine powder was dissolved in aquadistillate and intragastrically administered at 60 mg/kg for DN rats each day. In our in vitro study, the TXL superfine powder was dissolved in serum-free DMEM/low glucose medium and sonicated for 1 hour. Then the TXL solution was centrifuged at 3,500 rpm for 10 min and the supernatant was filtrated by a 0.22-μm micropore filter. In order to calculate practical volume of dissolved TXL superfine powder, the precipitate was heated and dried. The final concentration of TXL superfine powder solution is 2000 μg/mL [TXL superfine powder was provided by Shijiazhuang Yiling pharmaceutical Co. . The herbal drugs of TXL were seriously authenticated and standardized through their marker compounds according to then = 12) and DN with TXL group . The rats injected with vehicle were as normal control group . The rats of TXL group were treated with TXL solution  and the rats of NC group and DN group were treated with an equal volume of vehicle . All rats were provided with free access to food and water throughout the experiment. The rats of the three groups were observed for 12 weeks. At the end of 0, 4, 8, and 12 weeks, serum glucose and urinary albumin excretion (UAE) were detected. At the end of 12 weeks after STZ, all rats were sacrificed and renal cortex was collected for electron microscope observation and purposed experiments.The experiments were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at Capital Medical University. Male Sprague-Dawley rats, aged 8 weeks and weighing 180–200 g, were purchased from the Chinese Academy of Medical Sciences . All rats were maintained in a conventional environment with a regular 12 h light/dark cycle and 24 ± 1°C temperature. The diabetic rats were induced by intraperitoneally injecting STZ at 60 mg/kg. A total of 12 rats were injected with an equal volume of vehicle  as control. Forty-eight hours after injection, tail vein blood was collected and serum glucose was detected in all rats. The diabetic rat model was considered to be successful when its serum glucose ≥16.7 mmol/L. The diabetic rats were randomly divided into two groups: diabetic nephropathy group  for proliferation. In order to induce differentiation, podocytes were grown under nonpermissive conditions at 37°C in DMEM/low glucose medium without recombinant IFN-γ. When they grew to about 80% confluence, podocytes were maintained in serum-free conditions for 24 h. To detect the optimum concentration of TXL, podocytes were treated with different concentrations of TXL  for 24 h. The podocytes were divided into three groups: normal control group , high glucose group , and TXL group (DMEM containing 30 mmol/L glucose + 100 μg/mL). The podocytes of three groups were treated for 24 h and then were used for purposed experiments.The conditionally immortalized mouse podocyte line was obtained from the National Platform of Experimental Cell Resources for Sci-Tech. Cell was cultured under permissive conditions at 33°C in DMEM/low glucose (Hyclone) medium supplemented with 10% fetal bovine serum (Excell) and recombinant IFN-MTT Assay. Cells were cultured in a 96-well plate at 5000 cells/well. After incubation with serum-free DMEM, podocytes were treated with different concentrations of TXL  for 24 h. After that, cells were cultured with MTT solution at a concentration of 0.5 mg/mL for 4 h. The MTT solution was removed and DMSO was added to dissolve the purple crystals of formazan. The optical density (OD) was detected by a spectrophotometer at 570 nm.Renal cortexes were fixed with 2% glutaraldehyde in 0.1 mol/L phosphate buffer at 4°C for 120 min. They were then sectioned into ultrathin slices and were double stained with 4% uranyl acetate and lead citrate. The ultrathin slices were observed by a Hitachi 7100 transmission electron microscope .µg per lane) were separated by electrophoresis through 10% SDS-PAGE and then transferred to polyvinylidene difluoride membranes. After blocking with 5% nonfat dry milk in PBS + 0.05% Tween 20, the membranes were treated with primary antibody, washed, and then incubated with peroxidase secondary antibody. Antibodies and dilutions included the following: rabbit monoclonal to nephrin antibody , goat polyclonal to snail antibody , rabbit polyclonal to notch1-cleaved-val1744 antibody (1 : 2000 Abcam), and mice monoclonal to GAPDH antibody (1 : 20000). The blots were visualized with LumiGLO reagent and peroxide, followed by exposure to X-ray film. Western blot analyses were performed at least in triplicate.Renal cortexes and treated cells were collected and lysed as described previously . Equal aμm) were used to perform immunohistochemical staining for nephrin and snail. After antigen retrieval, the sections were incubated with the primary antibodies of nephrin  and snail . Then renal tissues were incubated with species-specific secondary antibodies and diaminobenzidine. After that, the sections were counterstained with hematoxylin and observed with fluorescent microscope. The semiquantitative analyses for immunohistochemistry study were performed using Image-Pro Plus 6.0 software. Twenty high-power microscope fields of each group were randomly selected, and their optical density (OD) was detected. Mean optical density (MOD) of each group was calculated and then used for statistical analysis.Renal tissue sections . Antibodies and dilutions were as follows: rabbit polyclonal to notch1-cleaved-val1744 antibody  and goat polyclonal to snail antibody .Cells were cultured on the cover glasses in 24-well plates. When grown to about 80% confluence, cells were fixed with 4% paraformaldehyde for 30 min. After that, cells were incubated with Hoechst 33258 for 30 min at room temperature. Cells were observed under fluorescent microscope. Apoptotic cells were identified by nuclear condensation and/or fragmentation. The number of apoptotic cells was counted in 10 random fields per group.Cells were cultured on the cover glasses in 24-well plates. When grown to about 80% confluence, cells were fixed with 4% paraformaldehyde for 30 min. After that, cells were incubated with phalloidin for 30 min at room temperature. After washing in PBS, cells were counterstained with DAPI. Cells were observed under a confocal microscope .−ΔΔCt method. The sequences of primers are the following: mice nephrin: forward primer, 5′-CCCAACACTGGAAGAGGTGT-3′, reverse primer, 5′-CTGGTCGTAGATTCCCCTTG-3′; rat HES1: forward primer, 5′-CAAACCAAAGACAGCCTCTG-3′, reverse primer, 5′-ATGCCGGGAGCTATC-TTTCT-3′; mice HES1: forward primer, 5′-CACGACACCGGACAAACCA-3′, reverse primer, 5′-GCCGGGAGCTATCTTTCTTAAGTG-3′; mice snail: forward primer, 5′-AGCCCAACTATAGCGAGCTG-3′, reverse primer, 5′-CCAGGAGAGA-GTCCCAGATG-3′. All RT-PCRs were performed in triplicate and the data were presented as mean ± SD.Total RNA was isolated using the TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Then RNA was reverse transcribed into cDNAs by the SuperScript RT kit (Invitrogen). Relative mRNA levels were examined using SYBR Green real-time quantitative reverse transcription-PCR (qRT-PCR) (Applied Biosystems) and were calculated by the 2t-test (for comparison of 2 groups). P < 0.05 was considered statistically significant.Data were presented as mean ± SEM. Statistical analyses were performed by one-way ANOVA followed by the Bonferroni multiple comparison test (for comparison of more than 2 groups) or Student's μg/mL has no cytotoxicity on podocyte. However, cell viability was significantly decreased when it was incubated with TXL at 200 μg/mL. To determine the optimum concentration of TXL, nephrin protein expression was detected by western blot in cultured podocyte treated with different concentrations of TXL . Our results showed that HG repressed nephrin expression and TXL increased nephrin expression in high glucose cultured podocyte. More importantly, we found that TXL at 100 μg/mL had the optimum effect on nephrin expression in high glucose cultured podocyte . Moreover, nephrin mRNA transcript level was also significantly decreased in high glucose cultured podocyte (P < 0.05). TXL reversed decreased nephrin protein expression and mRNA transcript level induced by high glucose (P < 0.05)  .γ-secretase, with consequent release of ICN1 that translocates into the nucleus. HES1, a downstream target gene of ICN1, is an important component of notch pathway [P < 0.05). TXL markedly decreased ICN1 protein expression induced by HG (P < 0.05). Moreover, high glucose significantly increased HES1 mRNA expression (P < 0.05). TXL markedly decreased HES1 mRNA expression induced by HG (P < 0.05)  .P < 0.05). TXL markedly inhibited snail protein and mRNA expression induced by HG (P < 0.05)  .P < 0.05). Administration of TXL significantly decreased the number of apoptotic cells induced by high glucose (P < 0.05). In NC group, podocyte cytoskeleton was observed as parallel bundles of stress fibers. In HG group, intracellular actin stress fibers were abolished and placed by cortical actin web, resulting in a polygonal cellular shape. Moreover, TXL resumed intracellular actin stress fibers and maintained the normal cellular shape of high glucose cultured podocyte (Decreased nephrin expression has an intensive relationship with podocyte apoptosis and podocyte cytoskeleton change. The change of podocyte cytoskeleton can lead to podocyte foot process effacement and proteinuria. Herein, UAE, podocyte foot process, podocyte cytoskeleton, and podocyte apoptosis were detected in our in vivo and in vitro study. In our in vivo study, UAE was increased and podocyte foot process became fused and flattened in DN group. TXL significantly decreased UAE and alleviated podocyte foot effacement in diabetic rats. Our in vitro study showed that apoptotic cells were significantly increased in HG group compared with NC group (podocyte .Accumulate evidence has shown that podocyte loss and injury are an important mechanism of DN –4. NephrEvidence is available that notch pathway is a common pathway of podocyte injury . It has As we know, UAE is increased and podocyte foot becomes effaceable in DN. Moreover, previous studies have demonstrated that decreased nephrin expression can lead to podocyte apoptosis and podocyte cytoskeleton change –29. Our In conclusion, we speculated that TXL might have a pleiotropic effect on notch/snail pathway and nephrin expression both indirectly and directly, which may be a mechanism of protected effect on podocyte injury in DN. However, our study has some limitations and pitfalls. Although TXL has a pleiotropic effect on podocyte injury, the main therapeutic target of TXL is still unclear. Thus, further studies need to be performed.


Escherichia coli, Staphylococcus aureus, Candida albicans and Aspergillus fumigatus) and blood collected from patients with clinical symptoms of sepsis. The developed method is based on nested-multiplex real-time PCR .The study describes the application of the PCR method for the simultaneous detection of DNA of Gram-negative bacteria, Gram-positive bacteria, yeast fungi and filamentous fungi in blood and, thus, a whole range of microbial etiological agents that may cause sepsis. Material for the study was sterile blood inoculated with four species of microorganisms  allow to detect bacteria in whole blood samples, (2) are much more sensitive than culture method, (3) allow differentiation of the main groups of microorganisms within a few hours. Given the nonspecific clinical symptoms of sepsis, especially in its early stages, and the need for rapid implementation of appropriate therapy, microbiological and laboratory testing is of importance. The key role in diagnostics is determining the etiological agent of infection. Until now, the so-called diagnostic “gold standard” is still blood cultures performed in specialized media, preferably in automated culture systems. An important advantage of blood cultures is their low cost of testing. However, the long period of waiting for the results, in relation to the need for rapid implementation of appropriate antibiotic therapy, is undoubtedly a disadvantage of this method. The downside is also its low sensitivity – positive blood culture results, despite the presence of clinical signs of sepsis, are obtained in less than 50% of cases ,2. The sConsequently, the aim of the study was to develop an alternative nested, multiplex, real time PCR (qPCR) method serving to detect the presence of microbes in blood in order to diagnose sepsis.Four external primer sequences have been designed. Their sequences are presented in Table 2 (DNAGdansk) – 50 mM; Perpetual Taq Polymerase 2,5 U/μl (EURx). DNA amplification was carried out under the following thermal conditions for amplification I: 95°C for 5 min  30 cycles and for amplification II: 95°C for 5 min  40 cycles.The annealing temperature of the primers (amplification I) was determined to be 46.0°C and for amplification II – 65.0°C Table . AfterwaThe indication of sensitivity was performed separately for amplification II  and in the nested system, i.e. in successive amplifications I and II. The obtained results were compared in Table 102 blood samples from patients with clinical symptoms of sepsis were examined with the use of the developed method in the nested multiplex system and of BacT/ALERT® 3D (bioMérieux) blood culture. The application of the developed method for microbial detection allowed to increase the percentage of positive results from 18.6% as for culture to 69.6% in the case of nested-multiplex qPCR Table . The elaMolecular diagnostics of microbial etiological agents of sepsis is currently at an initial stage and is limited more to scientific research than to diagnostic practice. Only few kits for the detection of microorganisms that cause sepsis are available on the market: SeptiFast (Roche) and SeptiTest (Molzym), but in no way do they satisfy the needs of molecular sepsis diagnostics ,9. The S2 CFU/ml to 5.4 × 103 CFU/ml depending on the group of microorganisms , but without typing specific species. This information is very useful to the physician when selecting the appropriate treatment before he receives the final identification from microbiological laboratory.Bacillus sp. (ATCC 51912), Enterobacter aerogenes (ATCC 29009), Enterococcus faecalis (ATCC 33186), Escherichia coli (ATCC 25922), Haemophilus influenzae (DSM 4690), Neisseria meningitidis (ATCC 53414), Proteus mirabilis (DSM 4479), Pseudomonas aeruginosa (DSM 13626), Serratia marcescens (DSM 50904), Staphylococcus aureus (ATCC 33497), Staphylococcus epidermidis (ATCC 35983), Staphylococcus haemolyticus (DSM 20263), Streptococcus agalactiae (DSM 2134), Streptococcus pneumoniae (ATCC 49619), Streptococcus pyogenes (DSM 20565), Streptococcus salivarius (DSM 20617), fungi – Aspergillus fumigatus (ATCC 14110), Candida albicans (ATCC 10231), Candida glabrata (DSM 11950), Candida parapsilosis (DSM 5784), Candida tropicalis (ATCC 20115).Several strains were used in the research: bacteria – The research was granted approval by the local Bioethics Committee of the Jagiellonian University (KBET/94/B/2009). Written informed consent was obtained from participants before their enrollment in the study.3E (BectonDickinson) test tubes.Blood was collected from volunteers, who had no clinical symptoms of sepsis and no inflammatory markers . Additionally, 102 blood samples were taken from patients with clinical symptoms of sepsis, hospitalized in the John Paul II Hospital in Krakow. Blood samples were drawn into 2-ml Vacutainer KThe blood culture was carried out in the John Paul II Hospital in Krakow in the Microbiology Department using BacT/ALERT® 3D apparatus (bioMérieux).The bacterial and fungal DNA was isolated with the application of a specialized kit for DNA extraction . The isolation was carried out in accordance with the manufacturer’s report.E. coli, S. aureus, C. albicans, A. fumigatus) in order to obtain a gradient of their number from 105 CFU/ml to 100 CFU/ml for each one of them. DNA isolation was carried out according to the method described by Gosiewski et al. with the employment of a ready-to-use Blood Mini (A&A Biotechnology) kit [With the aim of determining the sensitivity of the PCR method, microbial DNA was isolated from 1.5-ml blood samples, collected from volunteers, which were simultaneously inoculated with four model microbial reference strains (ogy) kit . The samA260 and A280. It was performed in a NanoDrop machine (Thermo Scientific).The concentration and purity of total DNA isolates in the samples were measured spectrophotometrically at wavelengths of All the processes of DNA amplification were performed with the use of the real-time PCR method (qPCR) in a CFX96 thermal cycler (BioRad) by employing species-specific starters and TaqMan probes. The sequences of oligonucleotides utilized in the research and amplification procedures are presented in Table In each amplification reaction was used DNA isolated from the sterile human blood samples derived from healthy volunteers was used, serving as a PCR negative control.Additionally, in every sample of DNA isolated from blood, β-actin gene detection was performed in order to check whether qPCR inhibition takes place; SYBR®Green JumpStart Taq ReadyMix (Sigma) was used for that purpose  Table 11.http://www.ncbi.nlm.nih.gov/blast) provided in the public domain by the National Center for Biotechnology: bacteria – Bacillus thuringiensis (KC153529), Enterobacter aerogenes (AB844449), Enterococcus faecalis (KC150142), Escherichia sp. (KF453959), Haemophilus influenzae (AB377170), Neisseria meningitidis (AJ239312), Proteus mirabilis (KC150143), Pseudomonas sp. (JQ613981), Serratia marcescens (KC130920), Staphylococcus aureus (CP000736.1), Staphylococcus epidermidis (CP000029), Staphylococcus haemolyticus (EF522132), Stenotrophomonas maltophilia (AB008509), Streptococcus agalactiae (AB002480), Streptococcus pneumoniae (CP000410.1), Streptococcus pyogenes (AB002521), Streptococcus salivarius (NR042776); fungi – Ascomycota sp. (JX869355), Aspergillus fumigatus (HQ871898), Aspergillus sp. (KC120773), Candida albicans (JN941105), Candida glabrata (AY083231), Candida parapsilosis (DQ218328), Candida tropicalis (EU034726), Candida tunisiensis (JQ612155).16S rDNA and 18S rDNA sequences of the following organisms were obtained from GenBank (http://www.thermoscientificbio.com/webtools/multipleprimer/) software in order to check whether they form dimers or if they hybridize with one another. The primer set and probes were described in Table The universal external primers EXT_BAC_F and EXT_BAC_R (for bacteria detection) and EXT_FUN_F and EXT_FUN_R (for fungi detection) were designed by aligning in the conservative region of 16S rDNA (bacteria) or 18S rDNA (fungi), yielding products of approximately 610 bp and 440 bp. Selected sequences were aligned with 16S rDNA and 18S rDNA regions with the use of ChromasPro ver 1.7 (Technelysium Pty Ltd) software. The designed primers were later tested using Multiple Primer Analyzer .The standardization of the method was carried out with the use of DNA samples isolated from blood  simultaneously inoculated with four model reference microbial strains  and the amplification method II with internal primers and TaqMan probes Table . OptimizE. coli, S. aureus, C. albicans, A. fumigatus) in the same blood sample, so as to obtain a gradient of their number from 105 CFU/ml to 100 CFU/ml - as regards the resulting gradient, we prepared 5 samples for each of the points representing a specific number of microorganisms. Later, DNA was isolated with the use of the methodology described above. The indication of sensitivity was performed separately for amplification II  and in the nested system, i.e. in subsequent amplifications I and II. The obtained results were compared in Table The evaluation of the PCR method sensitivity consisted in simultaneously inoculating the blood samples taken from healthy volunteers with four reference strains  (Table Amplification sensitivity was defined as the relation of the Ct) Table .P value of <0.05 was taken as statistically significant.The relationship between the proportion positive from each replicate of 5 and the corresponding log concentrations of the four reference strains was examined using probit regression analysis (Gretl software ver. 1.9.4.). Using the probit model, the Nested qPCR and qPCR tests were compared. A The authors declared that they have no competing interests.TG was involved in protocol development, researched literature and conceived the study and wrote the first draft of the manuscript; DJ-B – patient recruitment and data analysis; AS – DNA isolation and PCR; MBW – optimization of the DNA amplification reaction; MB – data analysis. All authors read and approved the final manuscript.


Metformin is a widely used anti-diabetic drug with potential anti-tumor activity. However, little is known about its global metabolic and transcriptional impacts on tumor cells. In current study, we performed a metabolic profiling on human-derived colon cancer LoVo cells treated by 10 mM metformin for 8, 24 and 48 h. An obvious time-dependent metabolic alteration was observed from 8 to 48 h, prior to the reduction of cell viability. A total of 47, 45 and 66 differential metabolites were identified between control and metformin-treated cells at three time points. Most of the metabolites were up-regulated at 8 h, but down-regulated at 24 and 48 h by metformin. These metabolites were mainly involved in carbohydrates, lipids, amino acids, vitamins and nucleotides metabolism pathways. Meanwhile, the transcirptomic profile revealed 134 and 3061 differentially expressed genes at 8 and 24 h by metformin. In addition to the cancer signaling pathways, expression of genes involved in cell energy metabolism pathways was significantly altered, which were further validated with genes in glucose metabolism pathway. Altogether, our current data indicate that metformin suppressed the proliferation of LoVo cells, which may be due to the modulation on cell energy metabolism at both metabolic and transcriptional levels in a time-dependent way. Colorectal cancer is one of the leading causes of tumor-associated death worldwide, and a higher risk of colorectal cancer is observed in patients with type 2 diabetes1The alteration in cell energy metabolism is a hallmark of tumor cells, which are more dependent on aerobic glycolysis to generate ATP for cell growth. Metformin is a potent activator of AMP-activated protein kinase (AMPK), which plays a crucial role in modulating cell energy metabolism and insulin sensitivity. The anti-tumor property of metformin is proposed in either AMPK-dependent or –independent way58Metabolomics holds the advantages of revealing the comprehensive metabolic alterations in a biological system either alone or in combination with other omics approaches. In breast cancer cells, metabolomic fingerprint indicates that metformin treatment results in significant accumulation of 5-formimino-tetrahydrofolate, and the supplementation of hypoxanthine for purine salvage pathway greatly attenuates the anti-tumor effect of metforminIn the present study, we performed a combined metabolomic and transcriptomic study on the global effects of metformin with different culture time on a human-derived colon cancer LoVo cells. Our results indicate metformin treatment exerts transparent impacts on LoVo cells both at transcriptional and metabolic levels earlier than the appearance of cell viability reduction. The metabolomic data indicate that most of the cellular metabolites are depleted during the culture period from 8 to 48 h in control LoVo cells, whereas metformin treatment accelerates the depletion of cellular metabolites at 24 and 48 h, except for at 8 h. Meanwhile, the transcriptomic results indicate that metformin treatment resulted in over 130 and 3000 differentially expressed genes at 8 and 24 h, respectively. The combined metabolic and transcriptional results suggest the cell energy metabolism pathway is the main target of metformin.2 in 10% FBS DMEM supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin. The metabolic profiling was performed on cells treated with or without 10 mM metformin  for 8, 24 and 48 h, respectively, while the transcriptomic profiling was conducted on cells after 8 and 24 h of metformin treatment.Human-derived colon cancer LoVo cells (CCL-229) from ATCC were routinely cultured in 10 cm dishes at 37 °C in a humidified atmosphere of 5% CO4 cells/well in 10% FBS DMEM . After 24-h culture, cells were treated with Metformin at different concentrations from 0, 0.5, 1, 2.5, 5, and 10 mM in 10% FBS DMEM for 24 h. Then, cell viability was determined using the cell counting kit-8 according to the instructions.Cells were seeded in 96-well plates at 2 × 107 cells/each on ice. Remove the culture medium and wash with 5 ml of ice-cold isotonic saline  mixed solvent which has been refrigerated at −20 °C completely. Vortex for 30 s and sonicate for 5 min in ice water. Centrifuge at 13,200 rpm for 15 min at 4 °C. Transfer 175 μl of supernatant to a GC and LC sampling vial respectively for the following GC/TOFMS and LC/TOFMS analysis. The sample residue was extracted with 350 μl of methanol again. Vortex for 30 s, and sonicate for 5 min. Centrifuge at 13,200 rpm for 15 min at 4 °C, and transfer 175 μl of supernatant to the GC and LC sampling vials respectively. Then, each sample was added with internal standards . For GC/TOFMS analysis, the metabolite extraction was vacuum dried at room temperature, and the residue was then chemically derivatized with BSTFA, while the LC/TOFMS was performed directly with the solvent extraction. The protocols for GC/TOFMS and LC/TOFMS were described previously13The cellular metabolic profiling was carried out on GC/TOFMS  and LC/TOFMS (Agilent Corporation) after the two-step extraction of cellular metabolites with mixed solvent. The cellular metabolite extraction process was described as following: Place the 10 cm dishes containing about 1 × 10http://www.ncbi.nlm.nih.gov/geo) with the accession number GSE67342.After the time course experiments, cells were collected and total RNA was extracted using TRIzol reagent , followed by purification on an RNeasy column  and quantified by UV absorption . RNA quality was evaluated by RNA 6000 Nano LabChip  on an Agilent 2100 Bioanalyzer. Following quantification, 1 μg of each total RNA sample was amplified, labeled and hybridized according to the standard Affymetrix protocols by Expression Analysis, Inc. . The platform was used the Affymetrix HG-U133_Plus_2.0 Array. Expression values were calculated using Affymetrix GeneChip analysis software MAS 5.0. CEL files have been deposited to the Gene Expression Omnibus database  in LoVo cells treated with or without metformin for 8 and 24 h, which are independently different with those used for transcriptomic profiling. Briefly, the total RNA was extracted with RNeasy Mini Kit  and 1 μg of total RNA was subjected to first strand cDNA synthesis with RT2 HT First Strand Kit  according to the manufacturer’s instructions. The synthesized cDNA samples were analyzed with RT2 Profiler PCR Arrays in triplicate on ABI7500 system (ABI).Following the analysis of transcriptomic data, commercial RTFor GC/TOFMS data analysis, the sample information, peak retention time, and peak area (quant mass) were included in the final data set. All those known artificial peaks, such as peaks caused by noise, column bleed, and the BSTFA derivatization procedure, were removed from the data set. The resulting data were normalized to the internal standard prior to statistical analysis. The normalized data were mean centered and unit variance scaled during chemometric data analysis in the SIMCA-p 13.0 Software package . The unsupervised multivariate statistic, principal component analysis (PCA) was first used to compare the metabolic profiles between groups. Differential variables were then selected with the criteria of variable importance in the projection (VIP > 1) in the partial least-squares-discriminant analysis (PLS-DA) model and p < 0.05 in a Student’s t-test. Compound identification was performed by comparing the mass fragments of interesting variables with NIST 05 standard mass spectral databases in NIST MS search 2.0  software at a similarity score of greater than 70%. The identified differential metabolites were then validated by using available reference compounds. The corresponding fold change shows how these selected differential metabolites varied between groups.http://metlin.scripps.edu), which runs in the statistical package R (v.2.12.1) (http://www.r-project.org), to pick, align, and quantify features . The software was used with default settings as described (http://metlin.scripps.edu) except for xset (bw = 5) and rector . The created .tsv file was opened using Excel software and saved as .xls file. The resulting data sheet was normalized to the internal standard and used for further analysis. Metabolite annotation was performed by comparing the accurate mass (m/z) and retention time (Rt) of reference standards in our in-house library and the accurate mass of compounds obtained from the web-based resources such as the Human Metabolome Database (http://www.hmdb.ca/) and The METLIN Metabolite Database (http://metlin.scripps.edu/).For LC/TOFMS data analysis, The resulting .d files were then centroided, deisotoped, and converted to mzData xml files using the MassHunter Qualitative Analysis Program (vB.03.01) (Agilent). Following the conversion, the xml files were analyzed using the open source XCMS package (v1.24.1) (http://david.abcc.ncifcrf.gov/)http://www.paintomics.org/cgi-bin/main2.cgi)http://www.genome.jp/kegg/) databaseFor the microarray data analysis, only those probes with “perfect value” present in >2 samples (60%) in each group were applied in further analysis. Principal component analysis (PCA) was used to summarize gene expression profiles between groups. The SAM  method to evaluate statistical significance of changes in gene expression, and took the two-class unpaired analysis at false discovery rate (FDR) <0.01 and adopted a cutoff of 1.2-fold changesTo determine an optimal concentration of metformin for suppressing proliferation of LoVo cells, we first treated LoVo cells with a series of concentrations of metformin from 0.5 to 10 mM for 48 h, and found that the proliferation of LoVo cells were significantly suppressed by metformin in a dose-dependent way . Then, wThe metabolic profile was evaluated between control and metformin treated cells at three different time points using unsupervised statistics, PCA on the basis of 158 identified cellular metabolites from GC/TOFMS and LC/TOFMS. The PCA loading plots showed that the cell samples of con 8 h and con 24 h almost clustered together, but were clearly separated from those of con 48 h , suggestTo further investigate the metabolic impacts of metformin on LoVo cells, the supervised PLS-DA models were constructed between control and metformin-treated groups at different time points. The differential metabolites between groups were determined based on the criteria of VIP > 1 in the PLS-DA model and P < 0.05 in Student’s t-test. We identified 47, 45 and 66 differential metabolites between control and metformin-treated cells at 8, 24 and 48 h, respectively  in glucose metabolism pathways was consistent with the transcriptomic profiling. In glycolysis and gluconeogenesis pathway, a lot of genes were upregulated with culture time in control cells, but most of them were downregulated by metformin treatment, especially at 24 h . In glucTCA cycle is the key process for tumor cells to generate ATP and intermediates for macromolecule biosynthesis . In this study, we found that about half of observed genes in TCA cycle was downregulated at con 24 h, and most of them was further reduced by metformin treatment . At metaOur current study demonstrated that metformin treatment resulted in a comprehensive metabolic and transcriptional alteration on LoVo cells, which are prior to the phenotypic changes in cell viability. There are about 50 differential metabolites between metformin and control cells at 8, and 24 hours, whereas 66 at 48 hours. Meanwhile, more than 130 and 3000 differently expressed genes are observed in metformin treated cells at 8 and 24 hours comparing to corresponding control cells, respectively. In addition, the subsequent analysis of genes in glucose metabolism pathways confirms the time-dependent transcriptional modulation of metformin on LoVo cells.LoVo cells are derived from a colorectal cancer patient who is classified as Duke’s type C and grade IV. Since different types of cancer cells usually present different genetic and metabolic phenotypes leading to variable responses towards identical treatment, in current study, the inhibitory effect of metformin was also validated in another two colorectal cancer cells, Caco-2 and HT-29, in addition to LoVo cells. The results showed consistent reduction in cell viability by metformin treatment at different concentrations . However, a more satisfactory time- and dose-dependent suppressive effect was only observed in metformin-treated LoVo cells in our study. Meanwhile, the antitumor effect of metformin has been investigated in several other colorectal cancer cells such as HCT116, HT-29, and SW6201920The dysregulation of energy metabolism is the common pathophysiological basis for both metabolic diseases and tumorsTumor cells are usually characterized with aerobic glycolysis (Warburg effect) in glucose metabolism leading to overproduction of lactate from pyruvate even though in the presence of sufficient oxygen25The genes responsible for regulation of gluconeogenesis were differently modulated by metformin treatment. Phosphoenolpyruvate carboxykinases (PCKs), fructose-1,6-bisphosphatase 1,2 , and pyruvate carboxylase (PC) are the main control for gluconeogenesis. We observed that the expression of FBP1, FBP2, PC and PCK2  genes was significantly suppressed by metformin at 24 h, except for the obvious up-regulation of PCK1 (soluble). PCK1 (soluble) and PCK2  encode a cytosolic or mitochondrial enzyme catalyzing the formation of phosphoenolpyruvate from oxaloacetate along with GTP. In current study, the paradoxical modulation on these two genes by metformin suggested that metformin could differently regulate the expression of cytosolic or mitochondrial isoforms of particular genes. Since metformin can inhibit protein synthesis and gluconeogenesis in cells by activating AMPK2, and provides the link between glycolysis and TCA cycle. The activity of PDH is inhibited by pyruvate dehydrogenase kinase 4 (PDK4) leading to the suppression of pyruvate conversion to acetyl-CoAIn tumor cells, TCA cycle is also an important source of biosynthetic precursors, in addition to the production of ATPIn addition to the glucose-derived energy supply, glutamine/glutamate also fuels tumor cell through conversion toα-ketoglutarate in TCA cycle. In previous report, metformin has been observed to reduce levels of both glutamine and glutamate in breast cancer stem cells, and no defect in glutamine transport across the cell membraneIn summary, our current study indicates that metformin treatment produces comprehensive metabolic and transcriptional alteration on LoVo cells, which is time-dependent and prior to the occurrence of cell viability reduction. Moreover, the tumor cell energy metabolism pathway is one of the main targets for metformin activity.How to cite this article: He, J. et al. Metformin suppressed the proliferation of LoVo cells and induced a time-dependent metabolic and transcriptional alteration. Sci. Rep.5, 17423; doi: 10.1038/srep17423 (2015).


Overall, group B and group C  patients had similar renal function and were not significantly associated with an increased risk of CIN . During a 2.33-year period (median), multivariate analysis demonstrated that attempted PCI for CTOs was independently associated with lower mortality . Attempted PCI for CTOs improved the long-term prognosis in patients with high-risk renal insufficiency and did not increase the risk of CIN.We investigated whether attempted percutaneous coronary intervention (PCI) for chronic total occlusion (CTO) would improve the prognosis in patients with renal insufficiency at high risk of contrast-induced nephropathy (CIN). We analyzed 2,330 consecutive patients with renal insufficiency with or without CTOs who underwent coronary angiography or PCI from prospectively collected data. The long-term death and risk of CIN were evaluated among three groups: patients without CTOs (group A, n With great advances in expertise and technologies, successful percutaneous coronary intervention (PCI) for chronic total occlusion (CTO) was shown to be associated with reduced long-term mortality in a recent studyIn patients with renal insufficiency undergoing PCI, the incidence of contrast-induced nephropathy (CIN), which is associated with prolonged hospitalization and an increased risk of death and healthcare costs, is as high as 30%4567Thus, we investigated whether attempted PCI for CTOs will improve prognosis in patients with renal insufficiency at high risk of CIN.P <.001; 13.8% vs. 9.7%, P = 0.013). Patients who underwent attempted CTO-PCI  had slightly better renal function than those that did not undergo attempted CTO-PCI, but it was not significantly different . Furthermore, patients who underwent attempted PCI for CTOs showed a significantly lower rate of previous coronary artery bypass grafting (CABG: P <0.001) and higher usage of statins than those who did not undergo attempted PCI for CTOs (P = 0.039)  were included in the final analysis . There w= 0.039) . However= 0.039) .P < 0.001). There were a higher percentage of patients with a V/CrCl (ratio of contrast volume to creatinine clearance) of >2.62 who underwent attempted PCI for CTOs . Peri-procedural hydration volume was not significantly higher in group C than in group B , and the same was found for the high hydration volume . In addition, in 96% of cases, an antegrade approach was followed, and in 4%, a retrograde approach was followed. However, the hydration volume was not significantly different among the three groups.The angiographic and procedural characteristics are presented in Overall, CIN developed in 7.4% of patients . There was no difference in the prevalence of CIN among groups. For the different renal insufficiency categories, the prevalence of CIN was 5.8%, 8.5%, and 17.4% among the low-risk, moderate-risk, and high-risk categories, respectively. Furthermore, the incidence of renal replacement therapy, in-hospital mortality, and other adverse events were not higher in patients who underwent attempted PCI for CTOs than in the other groups .25%/0.5 compared to un-attempted PCI for CTOs  (0.5 (Attempted PCI for CTOs was not significantly associated with an increased risk of CIN= 0.758) , which w= 0.758)  and for 58)  (Patients who underwent CTO-PCI were not significantly associated with an increased risk of CINdisease) .25%/0.5  (0.5)  , which w3) (0.5) . Multiva(25%/0.5 .P = 0.414) were not significantly associated with an increased risk of CIN25%/0.5 . Particularly, patients with CIN who underwent attempted PCI for CTOs did not show a significantly higher risk of death than those without CIN (P = 0.396).In this cohort, the median follow-up was 2.33 years (interquartile range: 1.8–3.2 years). Kaplan-Meier curves for the clinical endpoints are shown in 25%/0.5, multivariate Cox regression analysis showed that attempted PCI for CTOs (group C vs. group B) was significantly associated with a reduced risk of death . The independent risk factors of death included CIN25%/0.5 and LVEF <40% , and it still improved the long-term prognosis without increasing the risk of CIN in patients with renal insufficiency.Patients who underwent attempted PCI for CTOs presented with slightly better renal function, but a higher contrast medium volume and higher percentage of V/CrCl of >2.62 than those that did not undergo attempted PCI for CTOs. Regardless of the contrast media insult during the procedure (PCI or no-PCI), the contrast volume of attempted PCI was more than un-attempted PCI for CTOs in this study. Additionally, a higher volume of contrast medium is associated with an increased risk of CIN due to hypoxia in the renal medulla and a direct toxic effect on renal cells111214816619et al.In Aguiar-Souto’s study (9), the incidence of CIN following PCI for CTO was as high as 6.16%, and Yu-Sheng Our study was consistent with a previous study that demonstrated that attempts to achieve recanalization of CTO in patients with CKD may not increase the risk of CIN; however, it lacked sufficient power to evaluate the long-term outcome due to a relatively small study population29The present study has several limitations. First, although the present study was prospective, it was observational in nature; thus, it cannot ascribe causality. Since it was also conducted at a single center among a relatively small patient population, it may not be representative of the wider population of patients who undergo PCI for CTOs. Second, measurements of the peak SCr levels may have been missed because of the variation in the measurement times, which were not standardized. This can potentially cause an underestimation of the true incidence of nephropathy in our study population. Third, there is not a standardized protocol for oral or intravenous periprocedural hydration. Finally, since data on oral hydration was missing, we were unable to exclude the effect of the whole hydration strategy on the risk of CIN in patients with renal insufficiency.In conclusion, attempted PCI for CTOs still improved the long-term prognosis without increasing the risk of CIN in high-risk patients with renal insufficiency. When CIN risk stratification and not satisfaction, an individual contrast dose limit, and periprocedural hydration were also used to protect patient’s renal function, and attempted PCI for CTOs was considered in patients with renal insufficiency. = 83), no use of low-osmolarity contrast agents (n = 130), creatinine clearance (CrCl) ≥90 mL/min or an emergency coronary angiography (n = 943), cardiovascular surgery or endovascular repair (n = 382), end-stage renal disease or renal replacement (n = 7), missing preoperative or postoperative creatinine value (n = 61), malignancy (n = 3), and no use of isotonic saline for hydration (n = 18).In this prospective, observational, single-center study, we reviewed 3,957 consecutive patients who underwent coronary angiography or PCI between January 2010 and October 2012 according to the institutional protocol at our institution. As described in our previous study < 90 mL/min) were retrospectively analyzed, of whom 501 were diagnosed with CTO. Among patients with CTO, 142 mainly underwent PCI of no occluded vessels, 359 underwent PCI for CTO. CTO was defined as thrombolysis in myocardial infraction grade 0 for >3 months with the presence of typical angina or reversible myocardial ischemia on a stress studyFinally, 2,330 patients with renal insufficiency CrCl < 0 mL/min Follow-up events were carefully monitored and were recorded by trained nurses through office visits and telephone interviews at 1, 6, 12, and 24 months after coronary angiography. The mean follow-up time was 2.45 ± 0.82 years . All experiments were performed in accordance with the approved protocols, guidelines, and regulations. This study was approved by the Guangdong General Hospital institutional ethics research committee, and all patients provided written informed consent.Coronary angiography was performed according to general clinical practice using general guide catheters, guidewires, balloon catheters, and stents through the femoral or radial approach. The contrast dose was left to the discretion of the interventional cardiologist. All patients received 370 mg I/mL of nonionic, low-osmolality contrast agents, either lopamiron or ultravist. Subjects were treated according to the American Heart Association/American College of Cardiology Foundation guidelinesThe success criteria of PCI for CTOs was defined as a final diameter stenosis <30% with a TIMI flow grade 3 of the CTO vessel without death, or Q-wave myocardial infarction, or emergency coronary surgery. A Q-wave myocardial infarction was defined as new Q waves in two or more contiguous leads in addition to creatine kinase-myocardial band elevation. Creatine kinase-myocardial band fraction was routinely assessed 12 h after PCI in all patients or at least three times every 6h in patients with recurrent chest painThe CrCl was calculated by applying the Cockcroft-Gault formula to the serum creatinine concentration25%/0.5 was defined as an increase in the serum creatinine level of >0.5 mg/dL (≥44.2 μmol/L) or of ≥25% from the baseline within 48–72 h of contrast exposure0.5 was defined as an increase in the serum creatinine level of >0.5 mg/dL (≥44.2 μmol/L) from baseline within 48–72 h of contrast exposure.The primary endpoint was long-term mortality; the second endpoint was long-term major adverse clinical events . We also observed patients’ in-hospital clinical outcomes, including renal replacement therapy, acute heart failure, re-AMI, the use of an intra-aortic balloon pump (IABP), arrhythmia, stroke, bleeding and death. CINP value <0.05 was considered statistically significant. All data analyses were performed using SAS, version 9.4 (SAS Institute).First, all the sample patients were divided into two groups: patients without CTOs and patients with CTOs. Then patients with CTOs were further divided into patients who did not undergo attempted PCI for CTOs and those who underwent attempted PCI for CTOs. Continuous variables are presented as mean ± standard deviation or median with interquartile range if they fit a normal or non-normal distribution, respectively. Comparisons between two groups were performed using Student’s t-test or the Wilcoxon rank-sum test. Pearson’s chi-square or Fisher’s exact tests were used, as appropriate, for categorical data, and the data are expressed as percentages. The incidence of CIN was compared among three groups using unadjusted and adjusted logistic regression analyses. Multivariable logistic regression analyses were also performed. Univariable analyses of mortality were performed using the log-rank test, and multivariable Cox regression analyses were performed. The data were analyzed on an available case basis, and missing data were not imputed. A two-tailed How to cite this article: Liu, Y. et al. Percutaneous coronary intervention for chronic total occlusion improved prognosis in patients with renal insufficiency at high risk of contrast-induced nephropathy. Sci. Rep.6, 21426; doi: 10.1038/srep21426 (2016).


Diffuse amyloid goiter (AG) is an entity characterized by the deposition of amyloid in the thyroid gland. AG may be associated with either primary or secondary amyloidosis. Secondary amyloidosis is rarely caused by inflammatory bowel diseases. Secondary amyloidosis is relatively more common in the patients with Crohn's disease, whereas it is highly rare in patients with ulcerative colitis. Diffuse amyloid goiter caused by ulcerative colitis is also a rare condition. In the presence of amyloid in the thyroid gland, medullary thyroid cancer should be kept in mind in the differential diagnosis. Imaging techniques and biochemical tests are not very helpful in the diagnosis of secondary amyloid goiter and the definitive diagnosis is established based on the histopathologic analysis and histochemical staining techniques. In this report, we present a 35-year-old male patient with diffuse amyloid goiter caused by secondary amyloidosis associated with ulcerative colitis. The operational definition of amyloid, which has been adopted by the community of pathologists, is that the fibers are unbranched, usually extracellular, and found in vivo; in addition, the fibers bind the dye Congo Red and then show green birefringence when viewed between crossed polarizers [β peptide (Aβ) with Alzheimer's disease (AD), islet amyloid polypeptide (IAPP) with diabetes type 2, and prion protein (PrP) with the spongiform encephalopathies [Amyloidosis is a general term describing a group of diseases characterized by the deposition of an insoluble, proteinaceous, amorphous, and eosinophilic material, called amyloid, in the extracellular matrix of some organs and tissues . Many prlarizers . By thisopathies .Primary amyloidosis is characterized by the deposition of amyloid L (AL), which is the major fibrillary protein originating from the light chain components of immunoglobulins, whereas secondary amyloidosis is characterized by the deposition of amyloid A (AA), which constitutes a portion of the acute-phase serum amyloid A (SAA) protein produced by the liver during inflammation. Amyloid goiter is clinically defined as the deposition of amyloid in the thyroid gland in such quantities as to produce an enlargement in the gland. To date, an approximate total of 85 cases of amyloid goiter have been reported in the literature. Deposition of amyloid in the thyroid gland may coexist with systemic amyloidosis, with medullary thyroid carcinoma, and less commonly with primary amyloidosis involving the thyroid gland. Systemic amyloidosis may be accompanied by the involvement of the thyroid gland alone . The proOur patient presented with diffuse intrathyroidal thyroid amyloidosis that developed on the basis of secondary amyloidosis caused by ulcerative colitis.3, hemoglobin: 12.6 g/dL, platelet: 159,000/L, C-reactive protein (CRP): 25 mg/L , glucose: 90, urea: 47, creatinine: 1.07 mg/dL (0.84–1.25), aspartate transaminase (AST): 26 U/L (8–40), alanine transaminase (ALT): 43 U/L (5–35), sodium: 139 mmol/L (136–146), potassium: 4.7 mmol/L (2.5–4.5), triglyceride: 342 mg/dL (35–150), HDL: 35 mg/dL (40–60), total cholesterol: 302 mg/dL (120–200), and sedimentation: 76/h. The cardiothoracic ratio was increased on the PA-AC graph. Pericardial effusion was detected during the consultations with the relevant departments. During the investigation of the etiology of the pericardial effusion, nephrotic-range proteinuria (3.6 gr/day) was detected. Subcutaneous biopsy was performed on suspicion of amyloidosis in the etiology of proteinuria but no sign of amyloidosis was found in the immunohistochemical analysis. Duodenal and rectal mucosal biopsies were performed but amyloid was detected only in the rectal mucosa. The presence of amyloid was confirmed by the appearance of Congo Red and apple-green birefringence and immunohistochemical staining patterns. Thyroid function tests were as follows: Ft3: 2.14 pg/mL , Ft4: 1.01 ng/dL (0.61–1.12), TSH: 0.29 μIU/mL (0.34–4.2), anti-thyroid peroxidase (Anti-TPO): 28 IU/mL (0–35), and antithyroglobulin (AntiTG): 110 IU/mL (0–115). On ultrasonography, both thyroid parenchymata were heterogeneous, whereas a 12 × 8 mm heterogeneous, degenerated, mixed nodule was detected in the isthmus and a 13 × 8 mm cystic nodule was detected in the inferior segment of the right lobe. Thyroid scintigraphy revealed low thyroid activity and increased background activity in the thyroid area. Fine needle aspiration of the thyroid gland revealed a 12 × 8 mm mixed nodule with benign cytological results in the isthmus and an 18 mm cystic nodule at the junction of the left lobe with the isthmus. Thyroid scintigraphy was normal. In the follow-up period, total thyroidectomy was performed due to euthyroidism. Pathological analysis revealed amyloid deposition in the entire thyroid gland and deposition of amorphous eosinophilic material between the follicular structures. Amyloid was histochemically stained with PAS and Congo Red stains and was immunohistochemically classified as AA  is a rare thyroid lesion although it was described a long time ago . AG may Fine needle aspiration biopsy is a simple, sensitive, and safe method used in the diagnosis of thyroid diseases. Nevertheless, in patients with amyloid goiter, the definitive diagnosis relies on the histological analysis of the thyroid gland that is excised. Moreover, histochemical staining techniques including Congo Red, thioflavin T, and crystal violet stains are used for confirming the presence of amyloid. Among these, Congo Red is the most commonly used staining method, which imparts an apple-green birefringence under polarized light and is accepted as a pathognomonic property of amyloid. AA can be differentiated from other amyloid proteins via immunohistochemical analysis . In our In conclusion, amyloid goiter should be kept in mind in the patients suspected of primary or secondary amyloidosis, particularly in the patients presenting with a rapidly growing goiter and obstructive symptoms.