Many studies have assessed perspectives of medical students toward institutional diversity, but few of them have attempted to map changes in diversity climate over time.This study aims to investigate changes in diversity climate at a Jesuit medical institution over a 12-year period.In 1999, 334 medical students completed an anonymous self-administered online survey, and 12 years later, 406 students completed a comparable survey in 2011. Chi-square tests assessed the differences in percent responses to questions of the two surveys, related to three identities: gender, race, and sexual orientation.p<0.001); ‘in the practice of medicine’ based on gender (p<0.001), race/ethnicity (p=0.60), and sexual orientation (p=0.43); as well as in the medical school curriculum, including course text content, professor’s delivery and student–faculty interaction (p<0.001) across the three identities. There was a statistically significant decrease in experienced or witnessed events related to gender bias (p<0.001) from 1999 to 2011; however, reported events of bias based on race/ethnicity (p=0.69) and sexual orientation (p=0.58) only showed small decreases.The 1999 versus 2011 samples were 46% versus 49% female, 61% versus 61% Caucasian, and 41% vs. 39% aged 25 years or older. Findings suggested improvements in medical students’ perceptions surrounding equality ‘in general’ across the three identities (It may be postulated that the improvement in students’ self-perceptions of equality and diversity over the past 12 years may have been influenced by a generational acceptance of cultural diversity and, the inclusion of diversity training courses within the medical curriculum. Diversity training related to race and sexual orientation should be expanded, including a follow-up survey to assess the effectiveness of any intervention. The Liaison Committee on Medical Education (LCME) defines an appropriate institutional setting for medical education as an ‘environment characterized by, and supportive of, diversity and inclusion’ . DiversiInstitutional leadership can help define this essential atmosphere of cultural competency with ‘creation of curricula and environments’ that supports critical dialogue on the potentially contentious issues of gender, race/ethnicity, and sexual orientation . TherefoThere have been many studies gauging perceptions and attitudes of medical students toward diversity and cultural competency, with or without interventions –9. Very Cura Personalis, an Ignatian principle adapted for healthcare. Translated as ‘care of the whole person’, Cura Personalis advocates individualized attention to other’s needs, ‘distinct respect for unique circumstances’, and a suitable appreciation for ‘singular gifts and insights’ .The 1999 vs. 2011 sample demographic compositions were as follows: 46% (153/334) vs. 49% (199/406) female, 61% (204/334) vs. 61% (247/406) Caucasian, and 41% (137/334) vs. 38% (156/406) were older than 25 years. The two samples were not significantly different with respect to gender, age, student status, and race .p<0.001); race/ethnicity ; and sexual orientation   a–c.p<0.001), race , and sexual orientation , with gender equality demonstrating a statistically significant improvement ; gender equality in student–faculty interaction and professor’s delivery demonstrated the greatest positive increase of 33.5 and 27.9%, respectively  from 1999 to 2011 (p=0.69) and sexual orientation  only showed small decreases that were not statistically significant  to facilitating an environment conducive for excellence in medicine. Three previous questionnaire-based studies demonstrated that gender-based discrimination (GD) and SH were prevalent in undergraduate medical education as well as among medical school faculty –15. A 20US national-level surveys indicate that small shifts in attitudes and perspectives on racial equality have occurred over the past decade, reflecting results of our study. A 2013 Pew Research survey, assessing diversity and inclusion within society-at-large, indicated that 35% of Blacks, 20% of Hispanics, and 10% of Whites say that they have experienced discrimination because of their race and ethnicity over the past year . MoreovePew survey indicated that 92% of America’s LGBT  adults say ‘society has become more accepting of them in the past decade’ and an equal number expect it to ‘grow even more accepting in the decade ahead’  a generational acceptance of cultural diversity as reflected in the society-at-large facilitated by the use of the Internet and social networks; and 2) the inclusion of diversity training and health disparities courses within the medical curriculum. Diversity training related to race and sexual orientation should be expanded, including a follow-up survey to assess the effectiveness of any intervention.


Mycobacterium tuberculosis, i.e., IS6110, MPB64, and protein b, was performed on intraocular fluid samples of 25 subjects. Nine had presumed tubercular uveitis, six had intraocular inflammation secondary to a nontubercular etiology (disease controls), and ten had no evidence of intraocular inflammation . As described previously, response to antitubercular therapy was considered as the gold standard.The objective of this study was to report the use of multi-targeted polymerase chain reaction (PCR) in the diagnosis of presumed tubercular uveitis. Multi-targeted PCR using three targets specific for Multi-targeted PCR was positive in seven out of nine patients with presumed tubercular uveitis and negative in all normal and disease controls. The sensitivity and specificity were 77.77% and 100%, respectively. For the diagnosis of presumed tubercular uveitis, multi-targeted PCR had a positive predictive value of 100% and a negative predictive value of 88.88%.Multi-targeted PCR can be a valuable tool for diagnosing presumed tubercular uveitis. Mycobacterium tuberculosis (MTB)[IS6110 and MPB64 in patients with osteoarticular tuberculosis (TB) and IS6110, MPB64, and protein b in patients with TB meningitis (TBM) resulted in enhanced sensitivity of 82.5% and 86.63%, respectively[IS6110, MPB64, and protein b, for establishing a diagnosis of TB and hence conducted the study to assess the role of this novel multi-targeted PCR in the diagnosis of presumed tubercular uveitis.Establishing a definitive diagnosis of presumed tubercular uveitis is a challenge as the intraocular specimen is seldom available for histopathological or microbiological evaluation. A low bacterial count amounting to a negative acid-fast bacillus smear examination and a delay of about 4 to 6 weeks in confirming diagnosis by bacterial culture further limit the utility of these tests in uveitis patients of presumed tubercular etiology,2. The nis (MTB)-11. Howeectively,13. We sFrom a total of 25 patients, 18 vitreous fluid samples, 6 aqueous fluid samples, and 1 iris biopsy were taken for the study. These 25 patients were divided into three groups:1. Group 1 included nine patients with presumed tubercular uveitis based on clinical signs and corroborative evidence[(a) Signs of active uveitis such as cells/flare in the anterior chamber, with or without granulomatous keratic precipitates or iris nodules, iris granuloma, vitreous cells, snowballs, snowbanking, retinal vasculitis with/without perivascular choroiditis scars with/without vitreous hemorrhage or tractional retinal detachment, serpiginous-like choroiditis, choroidal granuloma, or neuroretinitis.(b) A positive TST according to the CDC recommendations  All known causes of infectious uveitis or known noninfectious uveitis syndromes ruled out. Patients already receiving ATT prior to initiating our study were excluded.2. Group 2 included six patients with evidence of intraocular inflammation. These served as positive disease controls and had the following inclusion criteria:(a) Evidence of active inflammation in the anterior and/or posterior segment.(b) A documented negative TST (less than 10 mm of induration) at 48 to 72 h and/or negative QuantiFERON-TB Gold test, and a negative chest X-ray for TB.(c) No evidence of extraocular manifestation of TB.(d) Never received ATT.3. Group 3 included ten patients without any evidence of inflammation in the eye who underwent intraocular surgery for noninflammatory ocular conditions such as cataract surgery or vitreous surgery for retinal detachment, macular hole, etc. This group served as the normal controls. No additional tests were done for this group of patients.Depending upon the severity of intraocular inflammation in the anterior or posterior segment in groups 1 and 2, aqueous or vitreous fluid samples were obtained for the PCR analysis. In group 3, aqueous or vitreous samples were collected depending upon the intraocular surgery being performed (cataract or vitreous surgery). Hence, of nine patients in group 1, aqueous sample was collected from one patient (chronic anterior uveitis with complicated cataract), vitreous from seven (serpiginous-like choroiditis with scleritis (one), retinal vasculitis (three), choroidal granuloma (one), panuveitis (one), intermediate uveitis (one)), and iris biopsy from one patient (iris granuloma). In group 2, all six patients underwent vitreous fluid sample analysis. In group 3, aqueous and vitreous fluid samples were obtained from five patients each. All samples were subjected to MPCR.Treponema pallidum hemagglutination (TPHA) test. When required, as after a negative response to ATT, patient no. 8  underwent pars plana vitrectomy with internal tamponade. No additional laboratory tests were done. Patient no. 9, after a failed response to ATT and corticosteroids, underwent brain MRI on clinically suspecting primary intraocular lymphoma (PIOL), followed by a chorioretinal biopsy for cytological examination that confirmed the diagnosis of PIOL. In group 2 patients, the investigations were ordered as follows: TST, chest X-ray, and TPHA test in intermediate uveitis (one), which were negative; smear and culture examination of the vitreous sample in postoperative (one) and posttraumatic (one) endophthalmitis; smear and culture evaluation of vitreous, blood, and urine samples in presumed endogenous endophthalmitis (one); and serology for Toxoplasma antibodies in toxoplasmic retinochoroiditis (one). The remaining patient with phacoantigenic uveitis did not require investigations specific to uveitis.Group 1 patients underwent baseline uveitis investigations that included TST and/or QFT-TB Gold test, chest X-ray, hemogram, erythrocyte sedimentation rate, and Patients in group 3 were included only to serve as normal (disease-free) controls. These patients underwent a detailed ocular examination as a routine protocol prior to surgery. Since there was no evidence of any intraocular inflammation, uveitis-related investigations were not considered in these patients. All patients in group 1 were reviewed by a consultant internist. None in groups 2 and 3 required his consultation.Prior to enrolment, all patients had undergone a detailed ocular examination including best-corrected visual acuity, intraocular pressure, and slit lamp biomicroscopy. Ancillary tests like fundus photography, fundus fluorescein angiography, spectral domain optical coherence tomography, and ultrasound B scan were performed as and when required. Systemic evaluation was done by a consultant internist (AS) with special interest in systemic inflammatory diseases. This study has been approved by the Institute Ethics Committee.An informed consent was taken from all the study participants. The vitreous humor, aqueous fluid, and/or iris biopsy were collected from the eye with active uveitis (groups 1 and 2) under strict aseptic precautions and transferred to the laboratory immediately.DNA was extracted from the samples using DNeasy Tissue Kit  according to the manufacturer's instructions. The extracted DNA specimens were stored at −20°C until used.IS6110, protein antigen b, and MPB64 in the MTB complex, and the expected band size was 123 bp for IS6110, 419 bp for protein b, and 240 bp for MPB64. The sequences of primers used for protein b PCR were Pab f and Pab r: 5-ACC ACC GAG CGG TTC GCC TGA-3 and 5-GAT CTG CGG GTC GTC CCA GGT-3, respectively. Primers used for IS6110 were IS1: CCTGCGAGCGTAGGCGT-30 and IS2: 5-CTCGTCCAGCG CCGCTTCGG-3. Primers used for MPB64 were MPB1: 5-TCC GCT GCC AGT CGT CTT CC-30 and MPB2: 50-GTC CTC GCG AGT CTA GGC CA-3. The following components were added to the Eppendorf tube (for a 50-μl reaction): PCR buffer (10X), dNTPs , primer IS1 (10 pm/μl), IS2 (10 pm/μl), P1 (10 pm/μl), P2 (10 pm/μl), MPB1 (10 pm/μl), MPB2 (10 pm/μl), Taq polymerase (5 U/μl), DNA template, and water. DNA amplification was performed for 40 cycles following an initial denaturation step at 94°C for 5 min in a thermocycler using the following program: denaturation at 94°C for 1 min, annealing at 65°C for 1.5 min, extension at 72°C for 1.5 min, and final extension at 72°C for 10 min.Multi-targeted PCR was carried out as described previously. To checIS6110, 419 bp for protein b, and 240 bp for MPB64 using a molecular weight marker of 100-bp ladder. The samples showing the presence of these bands under ultraviolet transillumination were considered positive.The amplified product was stored at 4°C until detection on 1.5% agarose gel stained with ethidium bromide. The stained gel was examined under UV light to look for bands 123 bp for Mycobacterium species, such as M. avium, M. fortuitum, or M. kansasii (data not shown). Sensitivity was estimated by serial dilutions of MTB DNA. The MPCR detected 10 fg, which is equivalent to two mycobacterial genomes.In agreement with Eisenach et al., muti-taTo avoid contamination during DNA extraction and amplification, strict precautions were taken, including separate areas for DNA extraction, reagent preparation, amplification and product detection, and regular meticulous cleaning of surfaces with 10% hypochlorite. In addition, all the reagents were aliquoted upon arrival in the laboratory. Positive and negative controls were included with each set of reaction. The positive control was DNA extracted from H37RV, whereas the negative control was PCR-grade water. To demonstrate the presence of inhibitors in multi-targeted PCR, all negative samples were spiked with positive control DNA, and no inhibitors were detected on spiked samples as all were positive with spiked DNA.a = disease, b = no disease; negative test: c = disease, d = no disease):The sensitivity, specificity, positive predictive value, and negative predictive value were calculated using standard formulae as follows (positive test: a / (a + c).• Sensitivity = d / (b + d).• Specificity = a / (a + b).• Positive predictive value = d / (c + d).• Negative predictive value = Group 1 comprised of nine patients with six males and three females. The mean age was 42.5 ± 9.8 years (range 30 to 50 years). Group 2 (six positive controls) had five males and one female. The mean age was 42 ± 20.8 years (range 4 to 60 years). Group 3 (ten negative controls) had five males and five females. The mean age was 47.9 ± 19.8 years (range 5 to 70 years).The clinical diagnosis in group 1 included vasculitis with or without vitreous hemorrhage/tractional retinal detachment (three), panuveitis (one), serpiginous-like choroiditis (one), choroidal granuloma (one), intermediate uveitis (one), chronic anterior uveitis (one), and iris granuloma (one). Table IS6110, protein b, and MPB64) were present in two cases, and two bands (MPB64 and IS6110) were present in two cases, and a single band each for MPB64 and IS6110 was present in two cases and one case, respectively . Since multi-targeted PCR was negative in groups 2 and 3 subjects, the specificity was 100%. Overall sensitivity of multi-targeted PCR, MPB64, IS6110, and protein b PCR were 77.77%, 66.66%, 55.55%, and 22.22%, respectively (Table Of nine patients with presumed tubercular uveitis group 1), seven (77.77%) were positive by multi-targeted PCR . All patients were started on ATT with corticosteroids . Six of these patients were quiescent at the final visit. One developed recurrence of anterior segment inflammation  during follow-up. Of the other two cases that were negative by multi-targeted PCR, one (patient no. 8) presented with right eye retinal vasculitis with rhegmatogenous retinal detachment and left eye intermediate uveitis. The TST was positive. The patient underwent pars plana vitrectomy with silicon oil tamponade in the right eye and received ATT with oral corticosteroids. The left eye showed persistent inflammation despite treatment at 19 months of follow-up. The other case (patient no. 9) presented with diffuse retinal vasculitis in the right eye and had a positive TST but did not respond to oral corticosteroids and ATT. The patient developed discrete hypopigmented lesions in the fundus that were below the retinal pigment epithelium as revealed by spectral domain optical coherence tomography. Following a negative vitreous biopsy report and other relevant investigations, the patient underwent a repeat vitreous surgery for obtaining retinal and subretinal biopsy that revealed an intraocular B-cell lymphoma.The clinical diagnosis of presumed tubercular uveitis is challenging due to protean manifestations, especially in an endemic country and BCG-vaccinated population. The chronic, recurrent nature of the disease demands an early and accurate diagnosis since a highly effective ATT is available for treating these patients. The NAAT have emerged as important tools for a rapid and an accurate diagnosis of TB. A meta-analysis of NAAT used in the diagnosis of TB concluded that commercial tests yielded results with high specificity but low sensitivity especially in smear-negative cases, while heterogeneity and low diagnostic accuracy were a concern with in-house PCR tests.Mycobacterium genome have been targeted[IS6110) for the amplification and detection of tuberculosis[IS6110 is absent in 10% to 40% of MTB isolates especially in geographically endemic areas like India, the likelihood of false-negative tests is increased[IS6110, MPB64, and protein antigen b (Pab). To check the internal reproducibility of the test, it was repeated three times and put up by two people independently.For the diagnosis of MTB, a large number of different sequences of the targeted-17. Mostrculosis. Since Increased. A low sncreased,20 and encreased,13,21. IIS6110, MPB64, and Pab specific for MTB. IS6110 was chosen because of multiple copy numbers of IS6110 (6 to 24) in the MTB genome, making it an attractive target for PCR amplification[MBP64 and protein b primers had shown good sensitivity for detection of MTB in our previous experience for the diagnosis of TBM patients[In the present study, we evaluated MPCR using three different targets, i.e., fication. MBP64 apatients.IS6110. The sensitivity of PCR is reported between 37% and 58.82%[IS6110 was 55.55%, which is comparable to that of previously reported studies[MPB64 was 66.66%, and the specificity was 100%. An important finding was the presence of two cases, which were only MPB64 positive but IS6110 negative. In the absence of MPB64 primers, these cases would have tested negative for MTB. Similarly, one case that was only IS6110 positive would have tested MTB negative by using only MPB64 primers. MPB64 as a target for diagnosis of tubercular uveitis has been evaluated in 23 patients suspected of Eales' disease and 27 control group subjects by Madhavan et al. and had shown a sensitivity of 47.8% and a specificity of 88.8%[MPB64 is 10,000 times more sensitive than IS6110 for the diagnosis of tubercular uveitis.For the diagnosis of tubercular uveitis, most of the previous studies had used only of 88.8%. The autProtein b PCR was positive in two out of nine patients in group 1. Protein b is a 38-kDa protein which is specific for MTB complex. This genome has not been evaluated for diagnosis of tubercular uveitis previously. It did not enhance the sensitivity of MPCR in the present study but had a good specificity for diagnosis of TBM in a previous study[us study and needMultiple gene amplification studies for the diagnosis of tubercular uveitis have not been reported, although a few studies have evaluated multiple targets as separate uniplex PCRs in other extrapulmonary conditions. These have not been studied as multi-targeted PCR and had reported increase in sensitivity by using two targets together. This muMycobacterium infection within the eye but does not differentiate the clinical picture from an immune response to mycobacterial antigens being loaded from the retinal pigment epithelium (RPE) cells, a possible sanctuary for MTB, or from somewhere else in the body. Demonstration of several acid-fast bacteria localized within the necrotic RPE cells in an earlier report by Rao et al. supports the possibility of tubercular uveitis as an immune response to these sequestered bacteria within the RPE cells[Presence of TB DNA in the ocular fluid suggests the presence of PE cells. DNA-basWe have earlier reported the use of quantitative PCR that allowed a fast detection and quantification of the mycobacterial load in the tested specimens of presumed tubercular uveitis. HoweverEarly diagnosis and prompt treatment of presumed tubercular uveitis is required for better patient outcome. Novel multi-targeted PCR using three primers has shown a high sensitivity and specificity for diagnosing presumed tubercular uveitis.In resource-limited countries that are endemic for tuberculosis, multi-targeted PCR has a high potential to be used as a diagnostic modality. However, more studies with a large sample size are required to corroborate its utility in a rapid and accurate diagnosis of presumed tubercular uveitis.The authors declare that they have no competing interests.KS participated in designing the study, carried out the PCR tests, performed the statistical analysis, and wrote the manuscript. VG participated in the patient recruitment and clinical sample collection. RB participated in the patient recruitment, clinical sample collection, patient treatment follow-up, and manuscript writing. AS participated in the study design, clinical diagnosis, statistical analysis, and manuscript writing. MS participated in carrying out the microbiological investigations. AG participated in the overall designing of the study, patient recruitment, clinical sample collection, clinical diagnosis, treatment and follow-up, manuscript writing, and overall supervision of the research project. All authors read and approved the final manuscript.


RYR3) encodes an intracellular calcium channel that mediates the efflux of Ca2+ from intracellular stores. Two single-nucleotide polymorphisms (SNPs) in the RYR3 gene have been shown to associate with stroke (rs877087) and carotid intima-media thickness (rs2229116) in two independent genome-wide association studies (GWAS) in Caucasian. We investigated the effect of these two SNPs as well as the 31.1 kilobases spanning region on atherosclerosis in Japanese population.The Ryanodine receptor 3 gene  and pathological atherosclerosis index (PAI) (n = 1262), which is a macroscopic examination of the luminal surfaces of 8 systemic arteries in consecutive autopsy samples. 4 tag SNPs in the 31.1 Kb region, rs877087 and rs2229116 were associated with PAI (OR = 2.07 [1.04-4.12] (95% CI), p = 0.038; and OR = 1.38 [1.02-1.86], p = 0.035, respectively). rs2229116 was also associated with common carotid atherosclerosis . The risk allele of rs2229116 was opposite from the original report. The haplotype block of this 31.1 Kb region was different between Caucasian and Japanese. Haplotype analysis revealed that only TAGG haplotype was associated with PAI  and atherosclerosis of common carotid artery .rs877087 and rs2229116 of RYR3 gene are associated with atherosclerosis severity in Japanese. The functional difference caused by rs2229116 needs to be investigated. Atherosclerosis is currently thought to be an inflammatory disease with deposits of cholesterol and inflammatory cells in the arterial wall -3. In thRYR3 gene [RYR3 gene is located on chromosome 15q14-q15, and encode a large intracellular homotetrameric protein (> 2MDa) that comprises 4780 amino acids [rs877087 polymorphism, which is an intron SNP, was associated with stroke in a Caucasian population from 4 large cohorts  [rs2229116, which is an Ile to Val non-synonymous SNP, was associated with common carotid intima-media thickness (cIMT), a clinical parameter for subclinical atherosclerosis [rs877087 and rs2229116 on RYR3 gene is separated by 31.1 Kb, we hypothesize that this region might confer susceptibility to atherosclerosis, and thus studied this region in detail using tag SNPs and haplotypes.Recently, two independent genome-wide association studies (GWAS) of atherosclerotic diseases in Caucasians pointed to two SNPs, closely resided in the 31.1 Kb (intron 14 ~ exon 19) region of the YR3 gene ,12. RYR3no acids -15; RYR3no acids ,17. The no acids . In huma 19,602) . The rs2clerosis . This wahttp://www.tmghig.jp/jg-snp/english/E_top.html) which contains abundant pathological information on atherosclerosis [A total of 1536 consecutive autopsy cases of aged patients registered in the JG-SNP database were examined  was analyzed by the Taqman assay (Applied BioSystems) using specific primers and probes on a Light-Cycler 480 (Roche), according to previously described protocols [The rotocols . The pathttp://pngu.mgh.harvard.edu/purcell/plink/) [Statistical analyses were performed with the IBM SPSS 19.0.0 statistical software. The associations of genotypes with atherosclerosis severity were tested by multiple logistic regression analyses with adjustment for conventional risk factors. All continuous and discrete variables of atherosclerotic severity were categorized by the cut-point of 75% severity, and divided subjects into Ath(+) group with top 25% severity, and Ath(−) group with severity lower than that, as we did previously . The con/plink/) .rs877087 and rs2229116 is 31.1 Kb apart and the D’ and r2 values of this susceptibility region in Japanese and Caucasian were shown in Figure rs2132207 and rs658750, both are intron SNPs) between rs877087 and rs2229116, were selected by using Haploview software . Genotype and haplotype data of the four RYR3 polymorphisms are summarized in Table rs877087 and rs2229116 genotypes were performed using Fisher’s exact test, and followed by logistic regression analysis in dominate or recessive models, adjusted by gender, history of hyperlipidemia, hypertension, diabetes mellitus, drinking and smoking  after adjustment for conventional risk factors. Also, rs2229116 A allele present significant associations with PAI  and atherosclerosis in common carotid arteries  after adjustment. The two tag SNPs, rs2132207 and rs658750, are not significantly associated with PAI and atherosclerosis in common carotid arteries  and from linear regression test (p = 0.010), indicating that there is significant linear relationship between degree of atherosclerosis and the rs2229116 polymorphism. Also, in PAI, the same trend was seen with regard to the genotype but it was not statistically significant (p = 0.060) (Table The tendency from the means of atherosclerosis degree in three genotypes of es Table . In commt p = 0.00 and frop = 0.032) and observed in the TAAA haplotype group in PAI . Also, a negative beta value was observed in TAGG haplotype group in common carotid arteries  , and in PAI   in several SNP sites, including the 2 tag SNPs (rs2132207 and rs658750), suggesting that the haplotypes within this region may be different between CEU and JPT  and build haplotypes  itself may be the primary determinant for the observed associations  at the 731st amino acid site of RYR3. However, the functional consequence has not yet been investigated. RYR3 have a major role in calcium signaling in the vasculation and thus is a good candidate gene in atherosclerosis pathogenesis.The Along with the function of RYR3 in vascular cells, recent studies have found that RYR3 have function in T cells, which is involved in the inflammation process of atherosclerotic lesions ,28. RYR3http://www.mhlw.go.jp/english/database/db-hw/lifetb10/index.html) were as follows; 25.9% men vs. 16.5% women by malignant neoplasms, 15.2% men vs. 20.3% women by heart diseases, 10.4% men vs. 12.1% women by cerebrovascular diseases, and 15.3% men vs. 12.4% women by pneumonia. Almost all death rates for major diseases at 75 years of age in Tokyo were consistent with our autopsy data, except for the frequency of malignant neoplasms is a little higher and that of cerebrovascular diseases is lower in our autopsy subjects. Since the national health insurance covers the whole population of Japan and in Japan most death occurred in hospitals, the selection bias from socioeconomic difference of the subjects seems minimal. Altogether, the subjects in our study did not differ greatly from the standard elderly residents in Tokyo, and may represent the general population, including young residents, in Japan rather than a selected hospitalized population.Our study has some limitations. The subjects of the present study were consecutive autopsy cases of patients in a geriatric hospital. Therefore, the selection bias and whether the subjects can represent the demographics of Japan should be evaluated. The autopsy rate of the hospital was kept around 40% and those who died suddenly outside the hospital and medicolegal cases were not autopsied for this study. The leading mortality probability at the age of 75 years according the census data in ‘Abridged Life Tables For Japan 2010’ by the Ministry of Health, Labor and Welfare of Japan  and atherosclerosis severity >75%.Click here for fileRelationship between RYR3 polymorphisms and atherosclerosis severity >75% in age subgroups.Click here for fileThe LD (D’) heatmap of the 31.1 Kb region in CEU.Click here for fileThe LD (D’) heatmap of the 31.1 Kb region in JPT.Click here for file


Although the outcomes of pancreatic cancer have been improved by gemcitabine, the changes in its characteristics and long-term outcomes within the gemcitabine era remain unclear. This study was conducted to identify clinical characteristics of pancreatic cancer patients within the gemcitabine era.A retrospective chart review was performed at 10 centers for 1,248 consecutive patients who were ever considered to have a diagnosis of pancreatic cancer between 2001 and 2010. Data collected included demographics, diagnosis date, clinical stage, treatment, and outcome; 1,082 patients met the inclusion criteria and were analyzed further. The chi-square test, Student’s t-test, and Mann–Whitney U-test were used for statistical analysis. Outcomes were analyzed using the Kaplan-Meier method and Cox proportional hazards regression. Differences in survival analyses were determined using the log-rank test.The distribution of clinical stages was: I, 2.2%; II, 3.4%; III, 13%; IVa, 27%; and IVb, 55%. Chemotherapy alone was administered to 42% of patients and 17% underwent resection. The 1-, 3-, and 5-year survival rates were 39%, 13%, and 6.9%, respectively. The median survival time was 257 days, but differed considerably among treatments and clinical stages. Demographics, distribution of clinical stage, and cause of death did not differ between groups A  and B . However, group B included more patients who underwent chemotherapy (P < 0.0001) and fewer treated with best supportive care (P = 0.0004), mirroring improvements in this group’s long-term outcomes (P = 0.0063). Finally, factors associated with long-term outcomes derived from multivariate analysis were clinical stage (P < 0.0001), location of the tumor (P = 0.0294) and treatments  (P < 0.0001).Long-term outcomes in pancreatic cancer has improved even within the gemcitabine era, suggesting the importance of offering chemotherapy to patients previously only considered for best supportive care. Most patients are still diagnosed at an advanced stage, making clinical strategy development for diagnosing pancreatic cancer at earlier stages essential. Pancreatic cancer (PC) has one of the worst prognoses among cancers worldwide , and the number of patients with PC is increasing globally . For exaIn this context, the Ehime Pancreato-Cholangiology (EPOCH) Study Group was established. This group conducted a retrospective study to identify the clinical characteristics of PC in Japan within the GEM era (after 2001).A retrospective chart review was conducted and included 1,248 consecutive patients who were ever considered to have a diagnosis of PC at the gastroenterology clinic at Ehime University Hospital or one of its 9 affiliated hospitals (EPOCH Study Group), between January 2001 and December 2010. As of August 2011, data collected included demographics (age and sex), date of diagnosis, tumor location, clinical stage (JPS TNM classification ), treatmThe chi-square test, Student’s t-test, and Mann–Whitney U-test were used for statistical analysis, where appropriate. Outcomes were analyzed using the Kaplan-Meier method and Cox proportional hazards regression. Differences in survival analyses were determined using the log-rank test. Two-tailed significance was defined in all analyses as a P-value < 0.05. All statistical analyses were performed using a statistical software package . Data were stored in a secure database and patients were numerically coded to anonymize data. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the local ethics committee at the Ehime University Graduate School of Medicine.The mean ± standard deviation for the age of male subjects included in the cohort was 69 ± 11 years  and 74 ± 11 years  for female subjects. The female subjects were significantly older than the male subjects (P < 0.0001). The distribution of clinical stages was as follows: Stage I , II , III , IVa , and IVb . This distribution did not change significantly over time: Stage I, 0–3.8%; Stage II, 0–6%; Stage III, 6.6–19%; Stage IVa, 23–33%; and Stage IVb, 50–59%  underwent chemotherapy alone  and the number of patients receiving chemotherapy increased over time , 685 patients died. The cause of death was identified in 629 patients (92%) and was related to PC in 95.1% (n = 598). Only 4.9% (n = 31) of deaths were unrelated to PC . Overall, the 1-, 3-, and 5-year survival rates were 39%, 13%, and 6.9%, respectively. The median survival time (MST) of the entire cohort was 257 days but differed significantly among clinical stages  did not show any significant differences in demographics (age and sex), distribution of clinical stage, and cause of death (PC-related vs. PC-unrelated). Although the resection rate did not differ between Group A and Group B , Group B (n = 148) included more patients with Stage III disease (46% vs. 36% in group A) and fewer with Stage IVa disease . Group B also included more patients treated with chemotherapy and fewer with BSC than Group A  , earlier stages , tumor location , treatments , and the period diagnosed as PC  (Table This multicenter retrospective study performed in gastroenterology clinics (EPOCH Study Group) in Japan, reports findings for 1,082 patients with PC. The results of this study support previously reported findings in that (1) a vast majority of patients with PC are still diagnosed at advanced stages; (2) patient prognosis in PC remains extremely poor; (3) tumors located in the pancreatic head are encountered most often at diagnosis; (4) tumors located in the tail are more often diagnosed at Stage IVb; (5) patients with PC involving the pancreatic head have a better prognosis than those with tumors in the body or tail; and (6) very few patients with PC may expect long-term survival, unless diagnosed at an earlier stage and treated with surgical resection. More importantly, this study was the first to observe and identify changes in the clinical characteristics of PC in a large number of patients within the GEM era.Overall, this study showed an improvement in long-term outcomes within the GEM era, and the associated factors for were clinical stage, tumor location and treatments (surgery and chemotherapy). The former finding is likely to be related to more patients being given an opportunity to undergo chemotherapy (the majority with a GEM-based regimen as first-line treatment) and fewer patients choosing BSC in the second half of the study period. Although not investigated, this was presumably due to physician’s skill up in chemotherapy rather than the change of patient characteristics, and indeed the referral pattern did not change during the study period. The improvement in MST differed by only 51 days between Group A (2001–2005) and Group B (2006–2010). However, considering that MST in Group A was as short as 229 days and PC is associated with one of the worst prognoses among cancers worldwide, it is important to appreciate the role of chemotherapy in the treatment of PC. Significant improvement in long-term outcomes was not seen over time (Group A vs. Group B) for chemotherapy . However, patients who were given chemotherapy had significantly better long-term outcomes than those who chose BSC in the 2 groups. Significant improvement in long-term outcomes was also not seen over time for surgical resection . This may be due to a small number of patients for surgical resection in our study population among gastroenterology clinics. Notably, more than 20% of patients with PC still chose BSC. In general, quality of life (QOL) is one of the most important factors when patients are considering chemotherapy as a treatment option, but this study did not investigate QOL data. Therefore, this study cannot evaluate changes in QOL with either chemotherapy or BSC. Nevertheless, a linear increase in the number of patients choosing chemotherapy and a concomitant decrease in patients choosing BSC were seen  in Stage I and 11 patients (30%) in Stage II chose BSC, which led to a poorer prognosis associated with these stages. This observation was likely due to referral patterns in this retrospective study conducted among gastroenterology clinics . The results of this study could not help identify the precise reasons in all patients in Stages I and II who did not undergo resection or chemotherapy. Previous studies have clearly shown that surgical resection improves prognosis among patients with locally advanced PC ,7. HowevThe strengths of this study include a large number of consecutive patients, the inclusion of gastroenterology clinics located in teaching, academic, and community hospitals, and an observation period limited to the GEM era. All of these strengths support generalization of these findings as they represent the current state of routine healthcare service. Also our patients were much older than the previous national study  . This maAlthough a significant improvement in the long-term outcomes of PC was achieved over the decade included in this study, it is important to stress that the clinical stage at diagnosis did not change over the same period Figure a. As repThis study showed that long-term outcomes in pancreatic cancer have improved even within the GEM era. These results suggest the importance of offering chemotherapy to patients previously only considered for BSC. Nevertheless, the vast majority of patients are still diagnosed at an advanced stage, making an effective clinical strategy for the diagnosis of PC at earlier stages a crucial requirement.PC: Pancreatic cancer; GEM: Gemcitabine; JPS: Japan pancreas society; BSC: Best supportive care; MST: Median survival time; QOL: Quality of life.The authors declare that they have no competing interests.Guarantor of the article: Teru Kumagi, MD, PhD. Specific author contributions: Conception and design of the study, generation, assembly, analysis of data, interpretation of data and statistical analysis: TKur, TKum, MK, HY, NA, TM MH; data collection from each center: TKur, TKum, TY, HS, MN, YI, NI, NS, SI, SO, HT, HU, YT, JM, YO; and drafting of the paper: TKur, TKum, YH, MO. Approval of the final draft submitted was undertaken by all authors. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-230X/13/134/prepub


Purpose. To assess the efficacy and safety of intraprostatic injection of two botulinum neurotoxin type A (BoNT-A) doses for the treatment of benign prostatic hyperplasia (BPH). Materials and Methods. Men with symptomatic BPH who failed medical treatment were randomized to receive 100 U or 200 U of BoNT-A into the prostate. The International Prostatic Symptom Score (IPSS), maximum flow rate (Qmax), post-void residual volume (PVR), PSA levels and prostate volume before injection and after 3 and 6 months were evaluated. Adverse events were compared between the groups. Results. Thirty four patients were evaluated, including 17 in the BoNT-A 100 U group and 17 in the BoNT-A 200 U group. Baseline characteristics were similar in both groups. Both doses produced significant improvements in IPSS, Qmax and PVR after 3 and 6 months and both doses promoted comparable effects. Prostate volume was affected by 200 U BoNT-A injection only after 6 months of treatment. PSA levels were significantly affected in the 100 U group only after 6 months of treatment. In the 200 U group, PSA levels were significantly decreased after 3 and 6 months. The complication rate was similar in both groups. Conclusions. Efficacy and safety of both BoNT-A doses are similar for BPH treatment in the short term followup. Benign prostatic hyperplasia (BPH) is a nonmalignant enlargement of the prostate that affects men in the adulthood . The purα-reductase inhibitors and alpha-adrenergic antagonists, is the most common treatment for BPH [Several therapeutic options are available, including watchful waiting, pharmacological therapy, and surgical procedures –4. Pharm for BPH , 5–8. Ho for BPH , 4. When for BPH , 9. Furt for BPH . Consequently, there has been much interest in alternative treatments for BPH and during the last decade, botulinum neurotoxin type A (BoNT-A) has been used to treat LUTS from different etiologies, such as striated sphincter dyssynergia , 11, refmax) of no more than 12 mL/s, and an enlarged prostate gland on digital rectal examination. Exclusion criteria were previous surgery for BPH, urethral stenosis, urinary tract infection, prostate or bladder cancer, pelvic surgery or radiotherapy, neurological diseases, use of any bladder or prostate medications including alpha-blockers, 5-alpha reductase inhibitors or antimuscarinics for the past three months and BPH-associated complications requiring surgical treatment including urinary retention, bladder stone, and bilateral hydronefrosis. This study was approved by the Local Ethics Committee and all participants gave written informed consent. Over a period of 2 years, men with symptomatic BPH were invited to participate in this prospective study. Inclusion criteria were age above 50 years, persistent moderate to severe LUTS as determined by International Prostatic Symptom Score (IPSS) >8 after medical therapy with at least one alpha-adrenergic antagonist, peak urinary flow rate , transrectal prostatic and transabdominal urinary tract sonography, free uroflowmetry, and measurement of post-void residual volume (PVR).Just before the injection procedure, patients were randomized to receive either 100 U or 200 U of BoNT-A. Injection procedure: with the patient lying on the lithotomy position and under cardiovascular monitoring, local anesthesia was performed with 20 mL of lldocaine 2% gel injected transurethraily and waited for 10-minutes. The injections were performed using a 22Fr rigid cystoscope  and a 23-gauge needle . The two different BoNT-A  doses (100 U or 200 U) were reconstituted with saline 0.9% to a total volume of 5 ml. All patients received five injections of 1 mL of the BoNT-A solution, including two injections in each lateral lobe  and one injection in the medium lobe. The injection depth was 7–10 mm. After the procedure, patients remained under observation until they were able to void spontaneously without hematuria. Oral levofloxacin (500 mg once a day) was administered for five days. Evaluations were performed 3 and 6 months after treatment and included a clinical assessment of LUTS with the IPSS score as well as measurement of peak urinary flow rate, postvoid residual volume, serum PSA levels, and prostate volume. The primary endpoint was improvement of IPSS scores. t-test. Fisher's test was used for categorical variables. A sample size of 17 in each group has 80% power to detect a difference between means of 3.00 (units in the IPSS score), at a two-tailed significance level of 0.05 or less. Data were processed using commercially available statistical software .Data were expressed as means ± standard deviation (SD) and range or absolute values and fractions. Intergroup changes from baseline of continuous variables were analyzed with analysis of variance for repeated measurements. Intragroup comparisons were performed using the Student's paired max, PVR, PSA, and PV . When present, pain was usually mild and no patient needed narcotic analgesics. Complications and their management are depicted in The efficacy of BPH treatment is primarily determined by the magnitude of symptom relief as well as improvement of urinary flow rates. In the present study, both BoNT-A doses promoted significant improvement of symptoms and increased flow rates that continued throughout the followup period of six months.Maria et al.  pioneeremax, as well as to reduce PVR [Further studies have documented that intraprostatic BoNT-A injection is an efficient therapy, capable to improve LUTS and Qduce PVR , 21. OurOne patient treated with 100 U of BoNT-A developed urinary retention five months after the injection. Baseline characteristics of this patient included an IPSS score of 23, maximum flow rate of 3 mL/s, PVR of 135 mL, and a prostate volume 88 mL. These clinical features characterize a severe case of BPH, which might explain why he failed BoNT-A therapy. The patient was treated with transurethral resection of the prostate with a favorable outcome.max. These data suggest that BoNT-A may act on the dynamic obstructive component of BPH. The neurotoxin was originally thought to act only by inhibiting acetylcholine release at the presynaptic neuromuscular junction [The effect of BoNT-A injection on prostate volume is controversial. Experimental studies demonstrated generalized atrophy and apoptosis of glandular and stromal components of the prostate –24. Prevjunction . Presentjunction . Furtherjunction . This isjunction . Different doses (range from 100 to 300 U) have been studied in several series, but there is a lack of consistency in some studies –21. SomeThe procedure has been performed by transperineal, transrectal and transurethral approaches. We chose the transurethral route because the vast majority of urologists are trained on cystoscopic procedures and also because it permits direct vision and injection in the transition zone of the prostate. Kuo  firstly Complications were uncommon in our series and seemed to be related to the urethral manipulation rather than a direct result of BoNT-A injection. Gross hematuria, although conservatively managed, was observed in 2 (5.8%) patients. Transient urinary retention was observed in 2 (5.8%) patients, and treated conservatively with an indwelling catheter for 5 days. Two patients (5.8%) developed acute prostatitis. As mentioned, these adverse effects seem to be associated with the route of administration of BoNT-A, since they are potential complications of any procedure requiring urethral instrumentation. Series that adopted the transperineal or transrectal approach did not report such complications –27. Given the vascular nature of the prostate, systemic absorption of the toxin could occur. The doses used in intraprostatic injection of BoNT-A are well below the presumed fatal dose. To our knowledge, no systemic complications have been reported after intraprostatic injections, even with 300 U of BoNT-A .Kuo injected 200 U of BoNT-A in the prostate of ten poor surgical candidates, with BPH and urinary retention or large postvoid residual volume. All patients improved spontaneous voiding after treatment. Both voiding pressure and postvoid residual volume were significantly decreased after treatment. Total prostate volume was significantly reduced and maximal flow rate was significantly increased after treatment . Our stuTransurethral intraprostatic BoNT-A injection is a simple and safe therapy for men with symptomatic BPH. Efficacy and safety of 100 U and 200 U BoNT-A doses are similar.


Microsomes are derived mostly from endoplasmic reticulum and are an ideal target to investigate compound metabolism, membrane-bound enzyme functions, lipid-protein interactions, and drug-drug interactions. To better understand the molecular mechanisms of the liver and its diseases, mouse liver microsomes were isolated and enriched with differential centrifugation and sucrose gradient centrifugation, and microsome membrane proteins were further extracted from isolated microsomal fractions by the carbonate method. The enriched microsome proteins were arrayed with two-dimensional gel electrophoresis (2DE) and carbonate-extracted microsome membrane proteins with one-dimensional gel electrophoresis (1DE). A total of 183 2DE-arrayed proteins and 99 1DE-separated proteins were identified with tandem mass spectrometry. A total of 259 nonredundant microsomal proteins were obtained and represent the proteomic profile of mouse liver microsomes, including 62 definite microsome membrane proteins. The comprehensive bioinformatics analyses revealed the functional categories of those microsome proteins and provided clues into biological functions of the liver. The systematic analyses of the proteomic profile of mouse liver microsomes not only reveal essential, valuable information about the biological function of the liver, but they also provide important reference data to analyze liver disease-related microsome proteins for biomarker discovery and mechanism clarification of liver disease. The liver, a vital organ, has a wide range of physiological functions and plays a major role in metabolism, biosynthesis, and chemical neutralizing. Liver diseases, such as viral hepatitis and liver cancer, pose a worldwide public health challenge. The Human Liver Proteome Project (HLPP) was launched in 2002 to better understand molecular liver functions and diseases, and liver proteome expression profile is one of the major parts of HLPP . BecauseMicrosomes are composed primarily of closed sacs of membrane called vesicles that are derived mostly from endoplasmic reticulum (ER). As for liver, in addition to components of the protein secretary pathway, microsomes contain a multitude of proteins that are involved in lipid/lipoprotein biosynthesis and drug metabolism. The liver microsome is an ideal way to study the metabolism of compounds, the functional properties of membrane-bound enzymes, lipid-protein interactions, and drug-drug interactions , 5. The Two-dimensional gel electrophoresis (2DE) is one of the most widespread techniques for the proteomic profiling of soluble proteins and visualizes isoforms and posttranslational modifications in a proteome , 7. MembAlthough many of microsome proteins have been studied, many more remain to be isolated and characterized. With the improvement of current methodologies and experimental techniques, more proteomic data will be obtained. Also, biological interpretation of proteomic data and extracting biological knowledge are essential to further understanding liver function.2CO3-treated microsomes were separated by 1DE. Moreover, bioinformatics analysis of microsome proteomic data was performed to discover biological roles of the proteins. The results showed that the combination of 1DE and 2DE was more efficient for analyzing microsomes. Bioinformatics analysis can provide a valuable molecular basis to interpret the mechanisms underlying microsome biological functions and give insight into the biological function of the liver at the level of microsomes.In our study, 2DE was first used to array the isolated microsome proteins of the liver. Because of the low performance of 2DE in separating membrane proteins  and the  Male C57 mice (9 weeks old) were purchased from the Experimental Animal Center of Central South University . The mice were starved overnight for liver subcellular fractionation. All experiments were performed with the approval of the institutional ethics committee on animal research.3PO4 buffer (pH 7.1), and the density of sucrose was adjusted to 43.7%. The fraction was placed in one type-70i rotor centrifuge tube; overlayered sequentially with 7 mL, 5 mL, 5 mL, and 6 mL of 38.7%, 36.0%, 33.0%, and 29.0% sucrose, respectively, and centrifuged . The upper four layers of the sucrose gradient were discarded by aspiration, and the bottom layer (43.7%) was diluted with two volumes of cold distilled water and centrifuged  in a type-70i rotor to get the “heavy” microsomes. The pellets, light and heavy microsomes, were suspended in 3 mL of 0.25 M sucrose (pH 7.0) and combined. The mixture was diluted to 14 mL with 0.25 M sucrose containing CsCl with its final concentration of 0.015 M. The suspension was layered into an equal volume of 1.3 M sucrose/0.015 M CsCl and then centrifuged  in an SW 55Ti rotor. The rough microsomes were in the pink sediment, and the smooth microsomes were at the interface. The smooth microsomes were diluted with an equal volume of 0.25 M sucrose (pH 7.0) and centrifuged  in an SW 55i rotor. Microsome apparatus-rich fractions were prepared from mice livers with differential centrifugation and sucrose gradient centrifugation as described . Mice li) were prepared and stained with uranyl acetate and lead citrate then examined with a transmission electron microscope . For Western blotting analysis, the microsome fractions were lysed  in lysis buffer . The protein samples (50 μg) were subjected to electrophoresis on SDS-PAGE with 12% gel and transferred to PVDF membrane (Millipore). The PVDF membranes with proteins were immunoblotted with antibodies to endoplasmin (ER marker), OxPhos complex IV subunit I , catalase , and cadherin (cytoplasmic marker), respectively.Electron microscopy and Western blotting were used to detect and validate the purity of prepared microsomes. For electron microscope analysis, the prepared microsomes were fixed with 2.5% glutaraldehyde for 24 h and 2% OsO4 for 2 h, dehydrated with alcohol , and processed into epoxy resin. Thin sections (500 Åμg) were diluted to 450 μL with rehydration solution  pH3–10 NL IPG buffer, and trace bromophenol blue) and applied to IPG strips  for rehydration . Proteins were focused successively  to give a total of 68 kVh on an IPGphor. After equilibration, SDS-PAGE was performed with 12% gel on Ettan DALT II system. Then, the blue silver staining method was used to visualize the protein spots on the 2DE gels [ 2DE was performed as described by the manufacturer (Amersham Biosciences). Protein samples  at room temperature. The proteins were reduced with 10 mM dithiothreitol (DDT)  and alkylated with 50 mM iodoacetamide in 100 mM NH4HCO3 . The gel pieces that contained proteins were dried and then incubated in the digestion solution . The tryptic peptides were extracted with 50% ACN/2.5% TFA and then dried using a Speed-Vac. The proteins contained in the 2D gel spots and 1D gel bands were subjected to in-gel digestion with trypsin. Gel spots or bands were excised and destained with 100 mM NHμm ID × 150 mm, 100 Å pore size, 3 mm particle size) at a column flow rate of 300 nL/min. The ACN gradient  started at 5% B and ended at 70% B in 45 min. Mass spectrometry (MS) and MS/MS data were acquired using a Micromass quadrupole time of flight Micromass spectrometer (Waters). Database searches were carried out with the MASCOT server by using a decoy database . A mass tolerance of 0.3 Da for both parent (MS) and fragmented (MS/MS) ions, allowance for up to one trypsin miscleavage, variable amino acid modifications consisting of methionine oxidation and cysteine carbamidomethylation were used. MS/MS ion score threshold was determined to produce a false-positive rate less than 5% for a significant hit (P < 0.05). The false-positive rate was calculated with 2* reverse/(reverse + forward)/100. In the current study, the MS/MS ion score threshold was 23 and a false-positive rate was approximately 3.1%. For all the proteins that were identified with only one peptide, each MS/MS spectrum was checked manually.The tryptic peptide mixture was fractionated with reverse-phase (RP) high-performance liquid chromatography (HPLC) by using an Ultimate nano-HPLC system (Dionex). Peptide samples were purified and concentrated with a C18-PepMap precolumn and then separated on an analytical C18-PepMap column , was used to map target proteins to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The pathway visualization was based on the pathway mapping service provided in KEGG. Protein annotations were obtained primarily from UniProt 7.0 including accession, entry name, comments such as function, catalytic activity, subcellular location, and similarity. The Cytoscape plugin, Biological Networks Gene Ontology (BinGO), was used to find statistically overrepresented GO categories of the protein dataset. An online tool, WebGestalt  in Supplementary Material available online at doi:10.1155/2012/832569). Western blotting analyses showed that, with the standard immunoblotting protocol, the ER marker endoplasmin was enriched in the isolated microsome fractions without the contamination marker  being detected ). The results demonstrated an optimized preparation of microsomes. n = 10 gels). A representative 2DE map of microsome proteins was shown (n = 41) from ER and Golgi, 11% of proteins (n = 20) from mitochondria and other membranes, 50% of proteins (n = 91) from cytosolic and other soluble proteins, 8% of secreted proteins (n = 15), and 9% of proteins without unambiguous location (n = 16).The 2DE reference maps display  514 ± 83  protein spots , (b) putative membrane proteins , and (c) other proteins . Those identified proteins were categorized according to the reported annotation in the UniProt database (http://www.uniprot.org/) and predictions for transmembrane regions (http://www.cbs.dtu.dk/services/TMHMM/). Of the 99 proteins, 59 (60%) were described as “membrane-associated” proteins (category (a) and (b)), including ER-characteristic proteins .The Nastaining . A totalstaining . Those pstaining . Those mstaining : (a) prohttp://us.expasy.org/tools/protparam.html) is an index to evaluate the hydrophobic status of a protein, indicates a hydrophobic protein, and suggests a membrane association. In the current study, 69 (70%) of the 99 proteins identified from 1DE had a GRAVY > −0.4 , a score indicating the probability for membrane association. Moreover, some alkaline proteins with PI values close to or greater than 10 were separated by 1DE , but they could not be detected in a conventional 2DE map.Hydrophobicity is an important characteristic of a membrane protein. The grand average of hydropathy (GRAVY) scores (>−0.4)  were identified in the microsome fraction through the strategy of combining 2DE with 1DE protein-separation technologies followed by ESI-Q-TOF MS/MS. The microsome consisted of a complex network of continuous membranes including ER, ER-Golgi intermediate complex—also referred to as the vesiculotubular clusters or pre-Golgi intermediates—and the Golgi apparatus [Among the 2DE dataset  was compared to a reference set of complete mouse proteome (IPI mouse) that was provided by Biological Networks Gene Ontology. The analysis was done with a hypergeometric test, and all significant (P < 0.01) GO terms were selected after correcting for a multiple term testing with a Benjamini and Hochberg false discovery rate. The analysis was performed separately for molecular function, cellular component, and biological process categories, and x-fold enrichment for every overrepresented term in three GO categories was calculated . The results showed that the terms were related to mostly catalytic activity in terms of molecular function, including metabolism-related oxidoreductase, hydrolase, and dehydrogenase. Similarly, terms belonging to the cellular component namespace include mitochondrion, ER, and ribosome. Finally, terms from the biological process namespace included metabolic process, localization, transport, and translation. All of the information suggested the main functions and compositions of microsome.Biological Networks Gene Ontology  and Cytohttp://bioinfo.vanderbilt.edu/webgestalt/) [http://www.genome.jp/kegg/). In addition, an enrichment score was obtained of the frequency of occurrence of a specific protein (or gene) within any given experimental subset with respect to a species-specific background set. Thus, an enrichment factor (the observed frequency in input set/the expected frequency in background set) was created with a statistical value that indicated that the protein (or gene) was specifically overrepresented in the input dataset. In this current study, all the proteins except 81 (n = 259 − 81 = 178) were linked to a total of 99 biological pathways in the KEGG, including metabolic pathway, glycolysis/gluconeogenesis, metabolism of xenobiotics by cytochrome P450, and PPAR signaling pathway. Among those pathways, 34 significantly (P < 0.01) enriched biological processes analyzed by WebGestalt were obtained  . This toobtained . Those bn = 9) that played a key role in every pathway of metabolism of xenobiotics were included in our dataset  were related to the well-known function and composition of the microsome . Enzyme  dataset . Thirtee dataset  and are  dataset . These pPI value range limited by the IPG strip and the high dependence on sample preparation, some problems exist for the available 2DE protocols to resolve membrane-associated proteins [Proteome analysis of the cell membrane-bound organelles is a daunting task mainly because of (a) isolation of membrane that is free from nonconstituents and (b) solubilization of membrane proteins in a manner amenable to isoelectric focusing . 2DE is proteins , 22. Thehttp://us.expasy.org/tools/protparam.html). A calculated GRAVY score of up to –0.4 indicates a hydrophobic protein, suggesting a membrane association [PI values close to or greater than 10 were separated by 1DE; they could not be detected in conventional 2DE map. Only 23 proteins were found to be consistent between 2DE and 1DE datasets with 6 proteins classified as membrane proteins . It appears to be as effective as the low detergent procedure in selectively releasing microsomal content. In the current study, some proteins that were identified from Na2CO3-extracted fraction were classified as membrane associated mainly based on published reports, even though their predicted transmembrane domains (TMDs) did not suggest a membrane origin. The observations point out the fact that structure alone may not be the deciding factor, as far as the association of proteins with cell membrane is concerned. First, the proteins may be bound to the membrane simply to perform their functional obligations. Consequently, they could become part of complexes involving membrane proteins and may not depart from them easily under the conditions of sample preparation. For example, many enzymes were identified in the extracted membrane fraction, such as Cis-retinol androgen dehydrogenase 1 (short-chain dehydrogenase family). It is anchored to the ER membrane facing the cytoplasm by an N-terminal signaling sequence of 22 residues and takes part in the membrane-associated retinoid metabolism [Microsome-sealed vesicles could be converted into flat membrane sheets with cisternal contents that were released effectively with the treatment solution  was used to identify GO-category significant enrichment with all the identified proteins. BiNGO is a plugin for Cytoscape, which is an open source bioinformatics software platform to visualize and integrate molecular interaction networks. BinGO maps the predominant functional themes of a given gene set on the GO hierarchy. Of the 259 target proteins and direct partners analyzed, 182 target proteins linked to one or more GO terms. GO-term enrichment analysis revealed that the most highly represented GO terms in the cellular GO category component were organelles such as ER, mitochondrial, and organelle membrane. An analysis of the proteins that were identified according to their potential roles in biological processes indicated that the proteins were mainly involved in metabolic process, localization, transport, and translation. All the results were highly statistically significant.Two datasets from 1DE and 2DE are part of the complete protein composition of microsomes. A bioinformatics analysis of the two datasets combined offers more information. For an overview of the proteomic data and comprehending their biological importance, biological networks GO (BinGO) (P < 0.01) compared to statistical expectations. Pathways that are involved in benzoate degradation, metabolism of xenobiotic, glutamate metabolism, and cysteine metabolism were among the most enriched biologically. This finding was consistent with the fact that microsomes were used to investigate the metabolism of compounds and to examine drug-drug interaction by in vitro studies.The KEGG pathway database integrates current knowledge on molecular interaction networks in biological processes. To gain a broad understanding of our dataset, WebGestalt  was used to map the identified proteins to KEGG pathways. The results showed that 112 of the total proteins were associated with one or more KEGG pathways. Meanwhile, 97 of 112 target proteins (87%) fell into 34 KEGG pathways; they were specifically enriched  membrane proteins. A total of 259 nonredundant proteins were identified including 62 (62/259 = 24%) membrane proteins. Compared to the documented data [n = 259 proteins) of mouse liver microsome proteome relative to the documented data  that were derived from 1DE and 2DE strategies.Supplemental figure 5: is significant enrichment of GO terms for mouse liver microsome proteins Supplemental Table 1: MS/MS identification of 2DE-arrayed proteins from mouse liver microsomal preparations.2CO3-extracted mouse liver microsomal membrane preparationsSupplemental Table 2: MS/MS identification of 1DE-separated proteins from NaClick here for additional data file.Click here for additional data file.Click here for additional data file.


The antioxidant activity of essential oil and oleoresins was evaluated against linseed oil system at 200 ppm concentration by peroxide value, thiobarbituric acid value, ferric thiocyanate, ferrous ion chelating activity, and 1,1-diphenyl-2-picrylhydrazyl radical scavenging methods. The essential oil and ethyl acetate oleoresin were found to be better than synthetic antioxidants. The total phenol contents  in black cumin essential oil, ethyl acetate, ethanol, and n-hexane oleoresins were calculated as 11.47 ± 0.05, 10.88 ± 0.9, 9.68 ± 0.06, and 8.33 ± 0.01, respectively, by Folin-Ciocalteau method. The essential oil showed up to 90% zone inhibition against Fusarium moniliforme in inverted petri plate method. Using agar well diffusion method for evaluating antibacterial activity, the essential oil was found to be highly effective against Gram-positive bacteria.Gas chromatography-mass spectrometry (GC-MS) analysis revealed the major components in black cumin essential oils which were thymoquinone (37.6%) followed by p-cymene (31.2%),  Preservation of food degradation, mainly by oxidation processes or by microorganism activity, during production, storage, and marketing is an important issue in the food industry. There is currently a large interest in substituting synthetic food preservatives and synthetic antioxidants for substance that can be marketed as natural. Synthetic antioxidants such as gallates, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and tert-butyl hydroquinone (TBHQ) were the first preservatives designed for widespread industrial use. However, some physical properties of BHA and BHT, such as their high volatility and instability at elevated temperatures, strict legislation on the use of synthetic food additives, and consumer preferences, have shifted the attention of manufacturers from synthetic to natural antioxidant . It is wNigella sativa L.) belonging to family Ranunculaceae is a spice that has been used for decades for both culinary and medicinal purposes. It is also used as a natural remedy for asthma, hypertension, diabetes, inflammation, cough, bronchitis, headache, eczema, fever, dizziness, and influenza [Black cumin  of black cumin seeds.The seeds of black cumin were purchased from the local market of Gorakhpur, Uttar Pradesh, India. A voucher specimen was deposited at the herbarium of the Faculty of Science, DDU Gorakhpur University.Thiobarbituric acid (TBA), 1,1′-diphenyl-2-picrylhydrazyl radical (DPPH), and linoleic acid are of Acros ; butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and propyl gallate (PG) are of S D Fine Chemicals Ltd., Mumbai, India. Folin-Ciocalteu reagent and gallic acid were from Qualigens Chemicals Ltd., Mumbai, India, and Qualikems Chemicals Ltd., New Delhi, India, respectively. Tween 20 and ferrozine were from Merck Pvt. Ltd., Mumbai, India. Ampicillin was purchased from Ranbaxy Fine Chemicals (New Delhi), India. Crude linseed oil was obtained from local oil mill in Gorakhpur. All solvents used were of analytical grade.Powdered seeds of black cumin (250 g) were subjected to hydrodistillation in Clevenger apparatus for 3 h according to the method recommended by European Pharmacopoeia, . A volatOleoresins were obtained by extracting 30 g of powdered spice with 300 mL of various solvents  for 3 h in Soxhlet extractor. Evaporation of the solvents at reduced pressure gave viscous extracts. The oleoresins were stored in freezer until further use.μm). GC-MS interphase, ion source, and selective mass detector temperatures were maintained at 280°C, 230°C, and 150°C, respectively. Carrier gas used was helium with a flow rate of 1.0 mL min−1. The oven temperature was programmed as follows.Analysis of the volatile oils and oleoresins was run on a Hewlett Packard (6890) GC-Ms system coupled to a quadruple mass spectrometer (model HP 5973) with a capillary column of HP-5MS  was measured according to the method described by Decker and Welch [2 . After incubation the reaction was initiated by addition of ferrozine (5 mM and 0.2 mL) for 10 min at room temperature, and then the absorbance was measured at 562 nm in a spectrophotometer. A lower absorbance indicates a higher chelating power. The chelating activity of the extract on Fe2+ was compared with that of EDTA that was used as positive control. Chelating activity was calculated using the following formula:The chelating activity of the aqueous and ethanolic extract on ferrous ions  versus concentration in The DPPH assay constitutes a quick and low cost method, which has frequently been used for the evaluation of the antioxidative potential of various natural products, . Due to μg mL−1) was prepared by dissolving 100 mg of gallic acid in 100 mL of ethanol. Various dilutions of standard gallic acid were prepared from this stock solution. Calibration curve  and 4.0 mL of sodium carbonate solution (75 g L−1). The absorbance was measured after 30 min at 20°C at 765 nm.TPC were determined using the Folin-Ciocalteu reagent method described by Singleton and Rossi . Gallic on curve  was plot−1, was taken to assess the antioxidant activity of black cumin oil and its oleoresins. This oil is most frequently used edible oil in central Europe and is rather unstable because of the presence of substantial amount of linoleic acid. The antioxidant activity of volatile oil and extract was examined by comparing the activity of known antioxidants such as PG, BHT, and BHA by the following peroxide value and thiobarbituric acid value methods.For present investigation, crude linseed oil, having initial peroxide value 5.2 meq kg−1) were measured in every seven days and test was replicated for three times. A control sample was prepared under similar condition without any additive. The effects of oil and oleoresins in term of peroxidation at 90°C are shown in For measuring the peroxide value (PV), a modified oven test was used . The antA is absorbance of the test sample, B is absorbance of the reagent blank, and M is mass of the sample.TBA value of different samples was determined according to the method previously reported . About 12 in 3.5% HCl; the absorbance of red colour was measured at 500 nm in Hitachi-U-2000 spectrophotometer . The control and standard were subjected to the same procedure except for the control, where there was no addition of sample and for the standard 1 mL of sample were replaced with 1 mg of PG, BHT, and BHA. These steps are repeated every 48 h until the control sample reached its maximum. Low absorbance value indicates the efficiency of the test samples to inhibit lipid oxidation. The results were reported as incubation time versus absorbance in Antioxidant activity of black cumin oil and its oleoresins was compared to synthetic standards according to the ferric thiocyanate method in linoleic acid emulsion . The reaAspergillusflavus (1884), Aspergillus niger (2479), Fusarium moniliforme (1893), Fusarium graminearum (2088), and Penicillium viridicatum (2007), were tested by the inverted petri plate [dc and dt are the average diameters of the mycelial colony of the control and treated sets, respectively.The antifungal activity of the essential oil and extract against various pathogenic fungi, ri plate  and poisri plate . All theStaphylococcus aureus (3103), Bacillus cereus (430), and Bacillus subtilis (1790), and two Gram negative bacteria, Escherichia coli (1672) and Pseudomonas aeruginosa (1942). All the bacterial strains were procured from the Microbial Type Culture Collection (MTCC), Institute of Microbial Technology , and their reference numbers are given in parentheses. The bacterial cultures were grown on nutrient agar medium and stored at 4°C. In order to prepare a bacterial strain for test, initially one loopful of bacterial culture was transferred from slant to nutrient broth solution (10 mL) and was stored at 37°C for 24 h. The control plate without the addition of essential oil or extract containing DMSO was also maintained under the same conditions. After incubating for 24 h at 37°C, all plates were examined for any zones of growth inhibition and the diameters of these zones were measured in millimeters.The essential oil and extract were individually tested against a panel of microorganisms using agar well diffusion method . Three GP < 0.05 was considered to be significant.For the essential oil or oleoresin, three samples were prepared for assays of every antioxidant and antimicrobial attribute. The data are presented as mean (standard deviation of three determinations (data are not shown). Statistical analyses were performed using a one-way analysis of variance . A probaα-thujene (5.6%), thymohydroquinone (3.4%), longifolene (2.0%), and carvacrol (1.4%). Burits and Bucar [Careful and detailed interpretations of the experimental GC-MS data  were carried out which permitted identification of a large number of components in essential oil and oleoresins Tables  and 2.  nd Bucar  charactend Bucar  who repohujene 5.%, thymohFrom 2+) chelating effect of black cumin oil and its different oleoresins is presented in μg mL−1 for black cumin oil and EDTA was found to be 74.56% and 87.90%, respectively, whereas the oleoresins were less effective in metal chelation and their metal chelating activity ranges from 22.1 to 44.5%.The ferrous ion  become less significant. Ethanol and n-hexane oleoresins showed moderate scavenging activity.DPPH μg mL−1 . At 20 μy = 0.0101x + 0.0178, R2 = 0.982  but lower than PG whereas the ethanol and n-hexane oleoresin showed moderate inhibition at 0.02% concentration as compared to the other additives. From the above results, it should be confirmed that formation of primary oxidation species, peroxides, was quite similar with the secondary oxidation products and the changes of both oxidation characteristics are in a good correlation.Simultaneously with the measurements of PV, changes in secondary product such as malondialdehyde, the compound used as an indicator of lipid peroxidation was measured by TBA values , were alP < 0.05) between the control and the tested essential oil and oleoresins. As can be seen in P < 0.05) different from the control.The FTC method was used to measure the amount of peroxides at the primary stage of linoleic acid peroxidation . Since tAntioxidant activities of essential oils and oleoresins may be related to the diverse compounds present in them including terpenes, sesquiterpenes, and phenolic acids, which act in various ways such as inhibition of peroxidation, scavenging the radicals, and chelating the metal ions. The main constituents of black cumin oil were thymoquinone (37.6%) and p-cymene 31.4%) with minor amounts of longifolene, carvacrol, and thymohydroquinone which were responsible for the antioxidant activity of black cumin oil , 28–31. .4% with F. moniliforme and P. viridicatum. It was also found to be highly effective in controlling the growth of Aspergillus species and F. graminearum where (50%) and (65%) zone inhibition was observed, respectively. For other tested fungi, the essential oil exerted less activity. However using the same method, the oleoresins have revealed less activity except for F. moniliforme, in which only up to 40% mycelial zone inhibition was obtained. Moreover using food poison technique (F. graminearum at 10 μL. The volatile oil showed strong antifungal activity against all tested Aspergillus species in the food poison method. Ethyl acetate and ethanol oleoresin showed up to 30% zone inhibition at 10 μL dose. The n-hexane oleoresin showed very feeble inhibition zone in both techniques. The lower antimicrobial efficacy of the oleoresins is due to their low volatility [Using inverted petri plate technique , the volechnique , the vollatility .B. subtilis, B. cereus, and S. aureus and showed complete zone of inhibition at 3000 ppm concentration whereas in oleoresins more than 20 mm inhibition was obtained for Gram-positive bacteria. In addition, more than 20 and 25 mm zone inhibition was obtained for P. aeruginosa and E. coli. The results obtained using agar well diffusion method were well correlated with the earlier reported work [B. cereus, B. subtilis, S. aureus, S. epidermis, E. coli, and P. aeruginosa.The antibacterial investigations were undertaken using agar well diffusion method . Using tted work , 30, 35 Nigella sativa seeds. Oleoresins have high concentration of unsaturated fatty acids along with thymohydroquinone in small amount which is responsible for its moderate antimicrobial effects. Long chain fatty acids like linoleic acid and oleic acid were previously reported to possess antibacterial and antifungal activity [p-Cymene is not an efficient antimicrobial compound when used alone, but it potentiate the activity of compounds like carvacrol [The results obtained in antimicrobial investigations of black cumin oil and oleoresins were in good agreement with the previous reported work . Thymoquactivity –39. p-Cyarvacrol . The antarvacrol . The difSeeds of black cumin seem to possess magical properties and have been worked out extensively. This study revealed that black cumin essential oil and its oleoresins constitute a good alternative source of essential fatty acids compared with common vegetable oil. The present results showed that essential oil and oleoresins of black cumin exhibited higher antioxidant activity than synthetic antioxidants. These findings could be used to prepare multipurpose products for pharmaceutical applications and its usage as dietary source of antioxidant should be considered largely for alleviating and ameliorating diseases.


Background. To investigate the link and mechanisms between intestine and lung in the ulcerative colitis (UC) rat model. Materials and Methods. We used the UC rat model by immunological sensitization combined with local 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) in 50% ethanol enema, observed dynamically animal general state and body weight, examined the histological and functional changes in the colon, lung, liver, and kidney tissues, and detected microvascular endothelium response towards inflammation characterized with the expression of iNOS, TXB2, P-selectin, ICAM-1, and vascular endothelial growth factor A (VEGF-A) in the colon and lung tissue. Results. Pulmonary function results suggested ventilator disorder, and pathological findings showed interstitial pneumonia. There were no significant changes in the liver and kidney function and histopathology. The colon and lung tissue iNOS, TXB2, P-selectin, ICAM-1, and VEGF-A expression of the model rats was significantly higher than the normal rats at both time points. Conclusions. Our study is the first to demonstrate the close association between the large intestine and lung in the immune-TNBS-ethanol-induced UC rat model. Different organs and tissues with the same embryonic origin may share the same pathological specificities in a disease. The present study provided a new way of thinking for pathological changes in clinical complex diseases manifested with multiorgan damage. Inflammatory bowel disease (IBD) is a term that describes a group of inflammatory diseases of the gastrointestinal tract. Ulcerative colitis (UC) is one of the most common forms of IBD . UC is cUC also may have many extraintestinal involvements during the process of disease. The pulmonary abnormality may represent one of the extraintestinal disorders. Turner-Warwick  describeγ, TNF-α, and IL-12) and Th2 cytokines  -10 knockouts), and other models that are used for various research purposes –23. Althd IL-13) –26. It qd IL-13) . Here, wAlthough the links between large intestine and lung have been clearly identified in the clinical literature, there have been few basic research studies that have investigated the mechanisms of the inflammatory cross-talk involved. It has now been clearly established that the microvascular changes associated with angiogenesis are key contributors to the tissue injury and remodeling process that inevitably accompanies chronic inflammation. Vascular endothelial growth factor A (VEGF-A) is a fundamental mediator of IBD by promoting intestinal angiogenesis and inflammation, and targeting VEGF-A is a promising new therapeutic strategy to dampen inflammation . In thisAll chemicals were purchased from Sigma laboratories  unless stated otherwise.24 h (n = 10); model24 h (n = 15)—immune-TNBS-ethanol-induced UC rats, for 24 hours; normal4 w (n = 10); model24 w (n = 15)—immune-TNBS-ethanol-induced UC rats, for 4 weeks.Male Wistar rats weighing 200 ± 10 g were purchased from the Academy of Military Medical Laboratory Animal Centre . Ten male New Zealand rabbits weighing about 3 kg were purchased from Haidian Thriving Animal Farm . The animals were housed at SPF Animal Laboratory of Dongzhimen Hospital  with free access to food and water and were kept in a regulated environment (22 ± 2°C) under a 12-hour light/dark cycle (light on at 8:00 am). All animal procedures were performed strictly within national regulations and guidelines and approved by the Animal Experimentation Committee at Beijing University of Chinese Medicine. The rats were randomly divided into four groups as follows: normalAntigen preparation: ten rabbits were sacrificed by air embolism and immediately removed the colon. The colon was rinsed with sterile saline and scraped the mucosa. The mucosa was mixed with equal saline and homogenized 30 times with a tight Dounce homogenizer . Samples were further disrupted by intermittent sonication (six 30 s pulse with a 1 min cooling period in between) and then centrifuged at 3,000 rpm  for 30 min at 4°C. The supernatant was then aliquoted and stored at −20°C. The protein concentration was measured using bicinchoninic acid (BCA) assay . −1, i.p.), and then a medical-grade polyurethane cannula for enteral feeding  was inserted into the anus, and the tip was advanced to 8 cm proximal to the anal verge. TNBS dissolved in 50% ethanol was instilled rapidly into the colon through the cannula at 100 mg·kg−1 and the animals were maintained in a head-down position for a few minutes to prevent leakage of the intracolonic instillation. Rats were killed at day 1 (24 hours after TNBS-ethanol enema) and day 28 (4 weeks after TNBS-ethanol enema)  .Paraffin-embedded sections were stained with hematoxylin-eosin (HE) and masson-trichrome (MT) to evaluate general morphology and collagen formation. The tissue of 8 cm colon , lung, liver, and kidney were, respectively, removed, and washed in saline. HE and MT staining were done as previously described , 29. To At the end of the experiment, rats were deprived of food overnight and anesthetized with 10% chloral hydrate. The abdomen was sprayed with 70% ethanol and cut open. The viscera were displaced to one side, and the tissues covering the dorsal aorta were reflected with forceps. The abdominal aorta was punctured with a 2 mL syringe, and the blood was collected into a 1.5 mL tube. 1 mL of blood was collected from each rat. The blood was allowed to clot for about 3 hours at room temperature and then centrifuged at 3,000 rpm for 15 min at 4°C. The serum was removed, aliquoted, and stored at −80°C. The serum concentration of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine (Cr) was detected by clinical laboratory of Dongzhimen Hospital affiliated with Beijing University of Chinese Medicine .−1, i.p.) to surgical depth in a whole body box, and a tracheal cannula  was inserted. The parameters for the pulmonary function tests included the resistance of airways at inspiration , resistance of airways at expiration , lung compliance , maximal voluntary ventilation , forced vital capacity , forced expiratory volume in 0.2 second , ration of FEV0.2 and FVC in % , peak expiratory flow , forced midexpiratory flow rate . Following the pulmonary function tests, the animals were kept warm, allowed to recover from the anesthetic, and were extubated. They were housed in a laminar hood in standard rat cages with sterile paper pellets as bedding and were allowed free access to food and water.Pulmonary function tests were conducted on five rats from each group at Respiratory Foundation Lab of Chaoyang Hospital . Animals were anesthetized with 12% urethane , and band intensities were quantified using Image J software .To determine the levels of ICAM-1 and VEGF-A in the colon and lung tissue, western blotting was used as previously described , 30. TheAt the end of the experiment, rats were anesthetized with 10% chloral hydrate and immediately removed the colon and the lung and then rinsed with sterile saline. 100 mg tissue was mixed with 9 volumes of saline and homogenized. Samples were further disrupted by intermittent sonication and then centrifuged at 3,000 rpm for 30 min at 4°C. The supernatant was then aliquoted and stored at −20°C. Samples were detected by Radioimmunoassay Institute of Science and Technology Development Centre affiliated with People's Liberation Army General Hospital .The mRNA expression for iNOS was determined by RT-PCR . The seqP < 0.05. Statistical analysis was performed with SPSS software 13.0.Quantitative data were expressed as mean ± SD. The significance of differences between groups was assessed using one-way ANOVA, post hoc comparisons being made using the nonparametric Dunn multiple comparison test. In all tests, the criterion for statistical significance was The changes in general state and weight can partially reflect the disease state and recovery of UC rats. Normal rats were agile and active, glossy hair, active foraging, and natural breathing. Feces were hard, granular ellipsoid, dark brown, and without mucus blood. Inflammation became evident in animals received rabbit colonic mucosal antigen emulsion injection. The paws appeared red and swelled. Groin injecting appeared nodules, sporadical suppuration, and ulceration. Some rats had ocular hyperemia. Sickness was also shown as reduced activity, drowsiness, loss of appetite, slowing response, and emaciation. Hair color was greenish yellow withered. Feces gradually became softer, bigger, and more watery. After TNBS-ethanol enema, the feces appeared mucopurulent bloody, loose, or even water on the night of enema. Three days later, it became constant diarrhea mixed with blood, of gradual onset. Respiratory symptoms were also displayed in some animals with appeared breathless and asthmatic wheeze . PatholoP < 0.01). While all animals showed a tendency of weight loss, animals in the TNBS-ethanol enema group weighted significantly less (P < 0.01)  . Histological examination of tissue sections of the colon, lung, liver, and kidney were performed using HE and MT staining as described previously. As the results of HE staining shown in The normal histology of the lung is shown in MT staining was used to label collagen fibers and collagen deposition. Differing from HE staining, the MT staining is able to differentiate clearly the important morphological keys for the colon and lung tissue of UC assessment such as haemoglobin, cellulose and muscle fiber (red color), cytoplasm and adipose cells (light red or pink), cell nuclei (blue black), elastic fiber (brown to dark brown), and collagen fiber which stained green in color . The intP > 0.05) at 24 hours and 4 weeks after TNBS-ethanol enema , while no significant changes were seen in other parameters , especially 24 hours (P < 0.001). In the lung tissue, the iNOS concentration was also increased in the model group (P < 0.001). Changes in iNOS concentration seem more substantial at early time point than that at 4 weeks (P < 0.05).Endothelial dysfunction: intrinsic alterations in the inflamed and remodeled microcirculation that underlie vascular dysfunction seem to play a fundamental role in deregulated inflammation, which characterizes UC. The expression of iNOS in the lung and colon tissue of animals was measured by immunochemistry and RT-PCR analysis . There w2, 6-Keto-PGFα1, and P-selectin in the lung and colon tissue of UC rats was measured by radioimmunoassay , while the 6-Keto-PGFα1 expression of model was significantly lower than normal animals (P < 0.05).Platelet activation: the intimate adherence of platelets to the endothelium is one of the characteristics of UC, and it is well established that platelets behave aberrantly in UC. Expression of TXBunoassay . ResultsP < 0.001), within 24 hours. Concentration of ICAM-1 in the lung continuously increased within 4 weeks compared to that at 24 hours (P < 0.05).Leukocyte-endothelium interaction: the inflamed endothelium displays enhanced leukocyte binding capacity and upregulation of adhesion molecules. ICAM-1 expression was measured with immunochemistry and western blotting . ICAM-1 P < 0.05).Angiogenesis: the enhancement of angiogenesis in UC highlights neovascularization is a major contributor to the initiation and perpetuation of chronic intestinal inflammation. The expression of VEGF-A in the lung and colon tissue of UC rats was measured by immunochemistry and western blotting . ResultsA large number of epidemiological investigations , 33, casIt is now clear that the abnormalities underlying UC pathogenesis are not only restricted to those mediated by classic immune cells, such as lymphocytes, macrophages, and dendritic cells but also involve nonimmune cells . In part2, and 6-Keto-PGFα1 are also localized to the intestinal or pulmonary microcirculation. 6-Keto-PGFα1 is thought to possess anti-inflammatory, immunosuppressive, and immunomodulatory actions. TXB2 enhances leucocyte recruitment and causes focal mucosal ischemia and injury through platelet activation and aggregation [Patients suffering from exacerbations of UC have an increase in circulating platelet number . Both leregation . Our findings confirmed that immune-TNBS-ethanol can induce platelet activation, thromboxane synthesis, and blood hypercoagulability in the colon and lung tissues, resulting in the microcirculation thrombosis and microcirculatory dysfunction. One of the most novel aspects that directly implicate endothelial participation in inflammation is the process of angiogenesis . It is nIn conclusion, this study provides evidence in support of immune-TNBS-ethanol-induced UC rat model as a useful tool to study possible association between the intestine and lung in ulcerative colitis. We identified a specific correlation in abnormalities in the large intestine and lung but not other organs in this model. Microvascular endothelium response towards inflammation by endothelial dysfunction, platelet activation, leukocyte-endothelium interaction, and angiogenesis may reflect a close relation in the large intestine and lung of this model. Our results suggest that different organs and tissues with the same embryonic origin may share the same pathological specificities in a disease. The present study provided a new way of thinking for pathological changes in clinical complex diseases manifested with multiorgan damage.