Stathmin is a prognostic marker in many cancers, including endometrial cancer. Preclinical studies, predominantly in breast cancer, have suggested that stathmin may additionally be a predictive marker for response to paclitaxel. We first evaluated the response to paclitaxel in endometrial cancer cell lines before and after stathmin knock-down. Subsequently we investigated the clinical response to paclitaxel containing chemotherapy in metastatic endometrial cancer in relation to stathmin protein level in tumors. Stathmin level was also determined in metastatic lesions, analyzing changes in biomarker status on disease progression. Knock-down of stathmin improved sensitivity to paclitaxel in endometrial carcinoma cell lines with both naturally higher and lower sensitivity to paclitaxel. In clinical samples, high stathmin level was demonstrated to be associated with poor response to paclitaxel containing chemotherapy and to reduced disease specific survival only in patients treated with such combination. Stathmin level increased significantly from primary to metastatic lesions. This study suggests, supported by both preclinical and clinical data, that stathmin could be a predictive biomarker for response to paclitaxel treatment in endometrial cancer. Re-assessment of stathmin level in metastatic lesions prior to treatment start may be relevant. Also, validation in a randomized clinical trial will be important. Stathmin1 (STMN1 hereafter indicated as ‘stathmin’) is an 18 kD cytosolic phosphoprotein, known to play an important role in the cell cycle. Stathmin is expressed in all tissues. It is a critical regulator of microtubule dynamics through its microtubule destabilizing properties, including both prevention of polymerization and active promotion of microtubule depolymerization A biomarker is defined as a 'characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes or pharmacologic responses to a specified therapeutic intervention Stathmin is upregulated in many solid cancers, including endometrial cancer KRAS mutational status indicating response to cetuximab and panitumumab in colorectal cancer Presently, few predictive markers are known in human cancers and even less are clinically applied. In endometrial cancer no clinically validated predictive markers are yet available Taxanes are a group of chemotherapeutic agents frequently used in the treatment of endometrial carcinoma. Preclinical studies in breast and prostate cancer and retinoblastoma Two endometrial cancer cell lines were selected due to the difference in their sensitivity profile to paclitaxel; Ishikawa  and Hec1B . The Cancer Cell Line Encyclopedia (CCLE) data confirms the difference in sensitivity Cells were cultured to 50–70% confluence prior to transfection by lentiviral transduction . A GIPZ lentiviral shRNA target gene set of 3  at MOI 2.5 was used. A non-silencing GIPZ lentiviral shRNAmir control (BV17110_2.80×10 8) was used as control. Cells were selected with puromycin (1 μg/ml) after transfection.Paclitaxel and carboplatin were purchased from Sigma.The cell lines were treated with paclitaxel in increasing concentrations (range 1–500 nM) for 24 h. As clinically taxanes are often combined with platinum derivates in endometrial cancer, we also treated cells with a combination of paclitaxel (in increasing concentrations (range 1–500 nM) and carboplatin  for 24 h to observe any synergistic treatment effects. Cells were subsequently either fixed in 2% formaldehyde for microscopic evaluation of apoptosis; used in a proliferation assay (MTS) or processed for immunoblotting. Experiments were at least performed in triplicate.ueous One Solution Cell Proliferation Assay (Promega) following instructions from the manufacturer. The absorbance was recorded at 490 nm using an ELISA plate reader (TECAN Magellan Sunrise).For assessment of apoptosis, at least 150 cells were counted in three different areas in 96-well plates. For proliferation assays, experiments were performed in triplicates in 96-well plates. Assays were performed with CellTiter 96® AQImmunoblots were performed according to a standard protocol. In short, cells were grown and treated in 6-well plates and harvested in lysisbuffer after 24 h paclitaxel treatment. Proteins (25 ug) were separated by SDS/PAGE and transferred to a nitrocellulose membrane . Stathmin and/or (cleaved) PARP were detected using cleaved PARP (Asp214) (D64E10) , diluted 1∶1000 and stathmin , diluted 1∶1000; β-actin served as a loading control (anti β-actin antibody (AC-15) (ab6276) AbCam), diluted 1∶10000. Alkaline phosphatase conjugated secondary antibodies were used (Anti-rabbit IgG (Sigma Aldrich A3687): Anti-mouse IgG (Sigma Aldrich A3562)) and chemoluminiscence substrate  for detection.Patients diagnosed with and treated for endometrial cancer at Haukeland University Hospital, Bergen, Norway, are after signing informed consent, prospectively and consecutively included in a database (population based setting) from 2001 onwards, preventing selection bias and ensuring optimal data collection for all patients, as previously reported TMA's were generated as previously described and validated in several studies 5 μm thick TMA sections were dewaxed with xylene/ethanol. Antigen retrieval was done by microwave in TRS pH6  for 20 minutes. Slides were blocked for peroxidase (Dako S2023) for 8 minutes and incubated for 60 minutes with stathmin , diluted 1∶50. EnVision+ system, HRP secondary antibody (Dako K4011) was used, followed by DAB+chromogen (DAKO K4011) as detection system. Slides were counterstained with hematoxylin .Blinded for patient characteristics and outcome, slides were scored by two authors (HMJW and JT) using standard light microscopy as previously described Statistical analyses were performed using PASW18 Statistics . Categorical variables were evaluated using the Pearson χ2-test or Fisher exact where applicable. Two-sided P-values of <0.05 were considered significant. Univariate analyses of time from primary treatment to death due to endometrial carcinoma  were carried out using the Kaplan-Meier method. The Cox proportional hazards method was used for a multivariate survival analysis .All patients have signed informed consent prior to inclusion in the study. The study has been approved by the Norwegian Data Inspectorate (961478-2), the Norwegian Social Science Data Services (15501) and the local Institutional Review Board .Response to paclitaxel varies between endometrial cancer cell lines Fluorescence microscopy showed a transfection rate of 70–80% at the start of experiments, with markedly reduced stathmin levels in the stathmin knock-down cell lines compared to the control knock-down and wild-type cell lines  and 3a.In both stathmin knock-down cell lines (Ishikawa and Hec1B), improved response to paclitaxel treatment was observed  and 3b. We then investigated patient tumor samples to see if a similar association between stathmin level and treatment response could be observed. Stathmin staining was predominantly cytoplasmic, as reported in literature Approaching from a different angle, in general, patients with high stathmin level showed a reduced disease specific survival, in line with stathmins role as a prognostic biomarker . HoweverThe percentage of patients with high stathmin level was significantly higher in metastases compared to primary lesions with pathologic (high) levels noted in 18% of the latter (n = 84 of 477 primary lesions with stathmin staining available) compared to 37% in metastatic samples (n = 29 of 79) .In the paired primary-metastasis samples, 35% of metastatic lesions showed high stathmin level. A discordance of 26% between metastatic lesions and their primaries was observed. In 16% there was a change to high level in metastases and in 10% to normal level.Stathmin protein level has been shown to be a prognostic marker of aggressive disease in many cancers, including endometrial cancer, where high stathmin level in primary tumor identifies patients at high risk for recurrent disease and lymph node metastases in vitro results in breast cancer, we show in endometrial cancer cell lines that, independent of the original stathmin level, sensitivity to paclitaxel increased and thereby apoptosis expedited after successful stathmin knock-down. This was shown by direct microscopic counting and in Ishikawa cells also substantiated by immunoblotting focusing on PARP cleavage. PARP cleavage is an established indicator of apoptosis, distinguishing it from other mechanisms of cell death, such as necrosis. The increased apoptotic body formation noted by microscopy in the stathmin knock-down cell lines fits with increased apoptosis In line with previous In our prospectively collected, retrospectively analyzed patient series, we also demonstrated a striking difference in response to paclitaxel containing chemotherapy comparing patients with normal to those with high stathmin level, also when correcting for the most important clinicopathological prognostic variables. Even when exploring such a large clinical series with endometrial cancer patients as ours, collected over more than 10 years, with adequate follow-up and RECIST in vitro breast cancer studies, in this study we were able to test and confirm the association in clinical samples from patients treated with the drug of interest; using data from a well-annotated prospectively collected patient series. Both the preclinical and clinical testing support that stathmin level influences sensitivity to paclitaxel. We have explored and excluded that this effect can be generalized to other chemotherapeutic agents such as carboplatin, also frequently used in endometrial cancer.Unlike previous studies looking at stathmin as a potential predictive marker, predominantly in Reporting recommendations for tumor marker prognostic studies (REMARK) guidelines have been developed with the aim to improve the methodological quality and reporting transparency in such studies Taxanes, originally isolated from the bark of the yew tree, belong to the family of anti-microtubule chemotherapeutic agents, with paclitaxel as their prototype. Simply put, taxanes bind to β -tubulin, causing microtubules to resist depolymerization, inhibiting cell cycle progression and promoting mitotic arrest and cell death We show a higher proportion of high stathmin level in metastatic (37%) compared with primary lesions (18%). Discrepancy in stathmin status was noted in a quarter of paired samples, paralleling findings in e.g. breast cancer where discrepancies between primary and metastatic lesions are shown in 14–55% and 0–40% for hormone receptors and HER2 respectively These results, including preclinical data and for the first time data from clinical samples, support that stathmin may be a predictive biomarker for the response to paclitaxel treatment in endometrial cancer. However, confirmatory studies, ideally from randomized clinical trials are needed. The biomarker discordance on tumor progression is in line with other studies on tumor biomarker heterogeneity and supports the need for repeated biopsy in metastatic disease.


Lactobacillus rhamnosus GG (LGG) feeding  effectively increased the LGG counts in the HGM Gn pig intestinal contents and significantly enhanced HRV-specific IFN-γ producing T cell responses to the AttHRV vaccine. Lower dose LGG  was ineffective. Neither doses of LGG significantly improved the protection rate, HRV-specific IgA and IgG antibody titers in serum, or IgA antibody titers in intestinal contents compared to the AttHRV vaccine alone, suggesting that an even higher dose of LGG is needed to overcome the influence of the microbiota to achieve the immunostimulatory effect in the HGM pigs. This study demonstrated that HGM Gn pig is an applicable animal model for studying immune responses to rotavirus vaccines and can be used for studying interventions  that may enhance the immunogenicity and protective efficacy of vaccines through improving the gut microbiota.This study aims to establish a human gut microbiota (HGM) transplanted gnotobiotic (Gn) pig model of human rotavirus (HRV) infection and diarrhea, and to verify the dose-effects of probiotics on HRV vaccine-induced immune responses. Our previous studies using the Gn pig model found that probiotics dose-dependently regulated both T cell and B cell immune responses induced by rotavirus vaccines. We generated the HGM transplanted neonatal Gn pigs through daily feeding of neonatal human fecal suspension to germ-free pigs for 3 days starting at 12 hours after birth. We found that attenuated HRV (AttHRV) vaccination conferred similar overall protection against rotavirus diarrhea and virus shedding in Gn pigs and HGM transplanted Gn pigs. HGM promoted the development of the neonatal immune system, as evidenced by the significantly enhanced IFN-γ producing T cell responses and reduction of regulatory T cells and their cytokine production in the AttHRV-vaccinated pigs. The higher dose  Lactobacillus acidophilus NCFM (LA) at the appropriate dose was effective in reducing rotavirus diarrhea as well as enhancing immunogenicity of oral rotavirus vaccines in Gn pigs 6 colony-forming-unit (CFU)/dose) significantly enhanced effector T cell responses and down-regulated regulatory T (Treg) cell responses; high dose LA  significantly down-regulated effector T cell responses and up-regulated Treg cell responses 6 CFU/dose) significantly enhanced rotavirus-specific antibody secreting cell (ASC) and memory B cell responses induced by rotavirus vaccines in Gn pigs Lactobacillus rhamnosus (LGG) strain also enhanced the immunogenicity of rotavirus vaccines in Gn pigs at the appropriate dose in our previous study (Manuscript in preparation). LGG, but not LA has been evaluated in several human clinical trials as a vaccine adjuvant Gnotobiotic (Gn) pig models have been extensively used in studies of human rotavirus (HRV) because of the similar susceptibility to the Wa strain G1P1A HRV infeBifidobacteria from human stool could not colonize the gut of HGM transplanted ex-germfree mice Interactions between probiotics and host immune responses can be influenced by gut microbiota, which is lacking in Gn pigs. A previous study of pigs showed that the gut microbiota composition in human gut microbiota (HGM) transplanted pigs resembles that from the human donor All animal experiments were performed in strict accordance with federal and university guidelines. Specifically, we adhered to the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and the American Veterinary Medical Association Guidelines on Euthanasia. The animal protocol was approved by the Institutional Animal Care and Use Committee at Virginia Tech (Protocol# 10-168-CVM and 13-187-CVM). Ethical Committee approval was received from Virginia Tech Institutional Review Board for the newborn human stool sample collection (IRB number 11-1049).Lactobacillus rhamnosus GG strain (ATCC 53103) was used in the current study and propagated in lactobacilli MRS broth . LGG inoculums were prepared and titrated as we previously described th passage in African green monkey kidney cells , was used as the AttHRV vaccine for inoculation of Gn pigs at a dose of 5×107 fluorescent focus-forming units (FFU) The cell culture-adapted human rotavirus Wa strain G1P1A, derivedth passage were used to challenge Gn pigs at a dose of 105 FFU. The median infectious dose (ID50) and median diarrhea dose (DD50) of the VirHRV in Gn pigs were determined as approximately 1 FFU The virulent HRV (VirHRV) Wa strain was passaged through Gn pigs and the pooled intestinal contents from the 27Human gut microbiota was obtained from stool samples of a healthy male infant born in the United States who was delivered by C-section, solely breast-fed, and did not exhibit any signs of digestive disorders or receive any medication prior to stool sample collection. A freshly passed stool was diluted 20-fold and homogenized in sterile and pre-reduced 0.1 M potassium phosphate buffer  containing 15% glycerol (v/v) to produce a 5% human fecal suspension, which was then aliquoted into 15 ml sterile tubes and injected with nitrogen for 1 min and stored at −80°C. After completing sample collection daily between 17 to 23 days of age, the fecal samples were then thawed, mixed and homogenized to make a large stock for the entire experiment. The stock was divided into 15 ml aliquots, injected with nitrogen, and stored at −80°C. In order to remove glycerol prior to feeding to piglets, the fecal samples were thawed and washed with at least ten-fold volumes of PBS, centrifuged at 2000 rpm/min for 10 min at 4°C, and then diluted to the original volume with PBS.Death of neonatal pigs caused by opportunistic bacteria infection after transplanting HGM from an apparently healthy 11 years old human donor has been reported previously 5 FFU VirHRV. After HGM inoculation from 0 to 12 of days of age and from postchallenge days (PCD) 0 to 7, pigs were examined daily for clinical signs (diarrhea) and fecal swabs were collected daily as we previously described Gn pigs were derived and maintained as we previously described Rectal swabs were collected for 7 days after VirHRV challenge to assess rotavirus diarrhea and shedding. Pigs with daily fecal scores of ≥2 were considered diarrheic. Fecal consistency was scored as follows: 0, normal; 1, pasty; 2, semiliquid; and 3, liquid. Daily scores were added up for each challenged pig from PCD 0 to 7 to get the cumulative fecal score and then the average cumulative fecal score for all the pigs in each group was calculated. Virus shedding was detected by ELISA and cell culture immunofluorescence (CCIF) assay in processed rectal swab fluids as described previously 2 SYBR® Green qPCR Mastermix . PCR cycling conditions were 5 min at 94°C, and then 40 cycles of 15 s at 94°C, 15 s at 60°C, and 30 s at 72°C. The reaction mixtures each contained 2 μl of the sample DNA and 18 μl of the master mix, which included the appropriate sense and antisense primers. The sense primer for LGG is 5′-CGCCCTTAACAGCAGTCTTCAAAT-3′ and the antisense primer is 5′-ACGCGCCCTCCGTATGCTTAAACC-3′Feces and LIC were collected and processed as described previously The frequencies of specific subsets of effector and regulatory T cells were determined by using intracellular cytokine staining and flow cytometry as we previously described Sera were collected at PID0, PID13, PID22, PID28 and PCD7, and the small intestinal contents (SIC) were collected at PID28 and PCD7 to measure rotavirus-specific IgA and IgG responses using indirect isotype-specific antibody ELISA as previously described Kruskal-Wallis rank sum test was performed to compare all data, except for proportions of virus shedding and diarrhea among treatment groups, which were compared using Fisher's exact test. Statistical significance was assessed at p<0.05. All statistical analysis was performed using SAS program 9.2 .Comparisons of clinical signs and fecal virus shedding of AttHRV-vaccinated pigs with or without HGM transplantation after VirHRV challenge are summarized in Two LGG dosing regimens were included to investigate the effectiveness of different LGG feeding regimens in modulating the LGG counts in the feces and intestine of the HGM transplanted pigs . AttHRV+To study interactions among HGM, neonatal immune system and rotavirus vaccine, and to evaluate the effect of HGM on the development of neonatal immune system in the context of rotavirus vaccination, we compared the total numbers and frequencies of effector  and TregConsistent with the enhanced Th1 type immune responses, HGM down-regulated or significantly down-regulated the frequencies of CD4+CD25−FoxP3+ Treg cells in all tissues of AttHRV-vaccinated pigs. HGM+AttHRV pigs had significantly lower numbers of Treg cells in spleen and blood at PID 28 and in ileum, IEL and blood at PCD 7 compared to AttHRV only pigs. Furthermore, HGM decreased or significantly decreased the frequencies of IL-10 or TGF-β producing Treg cells in all tissues (with one exception in IL-10 producing Treg cells in blood at PCD7) of AttHRV-vaccinated pigs.To investigate the dose effect of LGG as an adjuvant in enhancing the immunogenicity of the rotavirus vaccine, we measured the virus-specific effector T cell responses in AttHRV-vaccinated HGM pigs fed with or without the 9 and 14 doses of LGG at challenge (PID 28) and postchallenge (PCD 7) . The AttNo significant differences were observed in the frequencies of CD4+CD25−FoxP3+ Treg cells and the frequencies of their cytokine production in any tissues among the three AttHRV-vaccinated HGM pig groups with or without LGG feeding at PID 28 or PCD 7 (data not shown).In the HGM pigs, AttHRV vaccination with and without LGG induced significantly higher rotavirus-specific serum IgA responses at PID22, PID28 and PCD7 and serum IgG responses from PID12 to PCD7 compared to the unvaccinated pig groups . Of inteWhen analyzing the dynamics of antibody responses, pigs from all three AttHRV vaccinated groups had the highest rotavirus-specific serum IgA and IgG titers at PCD7, which were significantly higher than those examined at all previous time points . All AttKlebsiella oxytoca, Enterococcus faecalis, and Staphyloccus epidermdis in the pigs. Klebsiella oxytoca was considered the likely primary pathogen. These early euthanized pigs were excluded from this study.Although the multi-step safety testing was done to confirm the safety of the collected stool samples, 2–3 pigs in each litter became ill  during the experiments and had to be euthanized prior to the scheduled time point, despite treatment with 50% dextrose (8ml/dose) by mouth every 8 hrs. The mean mortality rate in the HGM pigs was 20.9% . The necGnotobiotic pigs offer distinct advantages for investigating enteric virus infections and vaccines and for dissecting immunomodulatory functions of probiotics. Gnotobiotic status prevents confounding factors from preexisting gut microflora and maternal antibodies that are present in conventionally reared animals or in humans. However, gnotobiotic animals also have some disadvantages. The major drawback is that the germ-free status causes underdeveloped intestinal lymphatic constituents and leads to the decreasing number of gut-associated lymphoid tissues L. acidophilus regulated T cell and B cell immune responses in a dose-dependent manner. We also found that the probiotic LGG dose-dependently regulated T cell and B cell immune responses in Gn pigs (manuscript in preparation). Due to competition between LGG and other bacterial species for colonizing the gut in HGM transplanted pigs, LGG doses higher than that used for Gn pigs were needed to effectively regulate immune responses in HGM pigs. We found that 14 LGG feedings (up to 109 CFU/dose), but not 9 feedings (up to 106 CFU/dose), enhanced virus-specific IFN-γ producing T cell responses in AttHRV vaccinated pigs transplanted with HGM; AttHRV+LGG14X pigs also had a trend for higher IgA titers than the AttHRV+LGG9X and AttHRV-only pigs at PID28, which suggests that an even higher dose of LGG may be needed to achieve the desired adjuvant effect in the HGM pigs 10 CFU, which was called “low dose” but higher than the highest dose  of LGG used in our studies. They found that 7×1010 CFU of LGG down regulated ileal IL-17A, enhanced ileal TGF-β1 and IL-10 mRNA but had no effect on IFN-γ, IL-12, and IL-4 mRNA expression in the small intestine Our previous studies Klebsiella pneumoniae present in the HGM from an apparently healthy human donor of 11 years of age Klebsiella oxytoca as the likely cause of the illness. To build safe inoculum pools of HGM from donor stools, a more comprehensive screening procedure is needed. In addition to the screening steps we performed in this study, high-depth and high-throughput sequencing analysis and species-specific PCR for detection of potential pathogenic bacterial species for pigs such as Klebsiella spp. should be performed.Because Gn pigs derived by hysterectomy (unsuckled) are devoid of maternal antibodies due to the impervious nature of the sow placenta to immunoglobulin, it is expected that Gn pigs are highly prone to opportunistic pathogen infections from transplanted HGM. One early study reported that in two litters of HGM pigs, 17 of 24 died due to the opportunistic pathogen L. acidophilus prior to rotavirus infection significantly increased the amount of rotavirus antigens and the virus titers L. acidophilus plus L. reuteri or inoculated with the AttHRV and fed with L. acidophilus, shed higher titers of the viruses in feces than the pigs not receiving lactobacilli feeding AttHRV vaccination with or without HGM transplantation conferred similar levels of protection in Gn pigs against diarrhea, virus shedding, and overall clinical signs upon VirHRV challenge, even though HGM pigs had significantly higher Th1 type effector T cell responses and lower Treg cell responses compared to Gn pigs. Furthermore, high dose LGG feeding increased the fecal and intestinal LGG counts, suggesting that sufficient doses of LGG fed to pigs with preexisting HGM and prior colonization by LGG were able to further enhance the fecal and intestinal counts of LGG. High dose LGG feeding also significantly enhanced the intestinal and circulating virus-specific effector T cell responses, but did not significantly enhance intestinal or serum virus-specific antibody responses in HGM pigs at the time of challenge (PID28) and did not significantly enhance the protective efficacy of the AttHRV vaccine. The magnitude of virus-specific serum and intestinal IgA antibody responses induced by rotavirus vaccines is an important indicator of protective efficacy of the vaccines In previously reported studies using HGM pigs, HGM from a healthy 3-month old baby In conclusion, both Gn pig and HGM Gn pig models are applicable models for the evaluation of rotavirus vaccines and therapeutics, but each model has its advantages and drawbacks. The Gn pigs have the advantage of being devoid of confounding factors from the gut microbiota, whereas HGM Gn pigs have the advantage of more closely mimicking real-world situations. This current study demonstrated that HGM Gn pig model is an appropriate model for studying HRV infection and vaccines and for evaluation of the immunomodulatory effects of probiotics. To the best of our knowledge, this is the first study to establish the neonatal HGM transplanted Gn pig model of HRV infection and immunity. The findings also have implications in using Gn animal models with HGM for studies of other pathogens and diseases.


The geographic distribution of genetic diversity and the population structure of tetraploid wheat landraces in the Mediterranean basin has received relatively little attention. This is complicated by the lack of consensus concerning the taxonomy of tetraploid wheats and by unresolved questions regarding the domestication and spread of naked wheats. These knowledge gaps hinder crop diversity conservation efforts and plant breeding programmes. We investigated genetic diversity and population structure in tetraploid wheats  using nuclear and chloroplast simple sequence repeats, functional variations and insertion site-based polymorphisms. Emmer and wild emmer constitute a genetically distinct population from durum and rivet, the latter seeming to share a common gene pool. Our population structure and genetic diversity data suggest a dynamic history of introduction and extinction of genotypes in the Mediterranean fields. Triticum aestivum L.). Although a ‘relic’ crop today, emmer wheat is used for bread making, animal feed and as a genetic resource for the improvement of durum and bread wheat varieties th century and its extinction was prevented only by inclusion of rivet accessions in germplasm bank collections Tetraploid wheats have played a critical role in human history. Durum is the primary wheat for pasta and semolina production and the second most cultivated wheat after bread wheat (th millennium BCE T. aestivum L.) has been demonstrated to have evolved from a domesticated tetraploid, most likely emmer, and the D-genome donor Aegilops tauschiiTetraploid wheats are genetically and morphologically diverse and their evolution under domestication has not been fully elucidated Triticum turgidum (for example in the Van Slageren system durum is T. turgidum subsp. durum). MacKey further classifies durum and rivet as convarieties of the subspecies T. turgidum subsp. turgidum (T. turgidum subsp. turgidum conv. durum). Conversely, Dorofeev and colleagues T. durum). Previous genetic studies based on expressed genes in the nuclear and chloroplast genomes have focused on the origin of the different genomes and the relationship between domesticated and wild wheats A rigorous taxonomical classification for wheat species is important for the conservation of wheat biodiversity We analysed four classes of genetic markers: nuclear simple-sequence repeats (nuSSRs), chloroplast SSRs (cpSSRs), insertion site-based polymorphisms (ISBPs) and functional markers in expressed genes to investigate within landrace and between landrace genetic diversity in the Mediterranean Basin. We addressed the following questions: 1) how genetically diverse are tetraploid wheat landraces?; 2) can the genetic diversity in neutral markers elucidate the taxonomy of tetraploid wheats?; 3) can the geographic distribution of genetic diversity reveal the evolution and history of tetraploid wheats in the Mediterranean basin?within landrace genetic diversity analysis, 75 individuals from seven tetraploid wheat landrace accessions from the Iberian Peninsula were analysed  preserved by cultivation in situ in Asturias, Spain. Population structure in the latter accessions was previously studied using chloroplast and nuclear SSRs For analysed . These abetween landrace genetic diversity analysis, 244 accessions were assayed  to ten (K = 10) clusters were tested using 1,000,000 MCMCs and 500,000 burn-in runs, with the admixture model. Twenty replicate runs were performed for each value of K. The best-fit model was determined by plotting the natural log probability of the data against KKQ-matrices from replicate runs in each value of K using the program CorrSieve 1.1 http://gmt.soest.hawaii.edu).We clustered landraces into populations based on the nuclear SSR data without any Genetic structure was further investigated on the 244 accessions by means of principal component analysis (PCA) based on microsatellite allele frequency data from the 29 nuSSR panel using the R packages ade4 CD genetic distance measure CD genetic distances were also calculated separately for the sets of microsatellites located in the A and B genomes. Finally, we grouped our landraces in eight a priori defined populations: emmer accessions, rivet accessions, and durum accessions grouped by regional geographical provenance i.e. Southwest Asia, Southeast Europe, Central Mediterranean, Southwest Europe, Northwest Africa and Northeast Africa  obtained by re-sampling the allelic frequency data. A majority-rule consensus tree was produced using the CONSENSUS routine in the PHYLIP package available in the Mobyle portal  and subsequently manipulated in Dendroscope version 2.5 Gene diversity, polymorphic information content (PIC) and allele richness for nuSSRs were calculated using PowerMarker t Africa . DC genewww.worldclim.org). These included altitude (r), mean annual temperature (s), maximum temperature of the warmest month (t), minimum temperature of the coldest month (u), mean annual precipitation (v), precipitation of the driest month (w) and precipitation of the wettest month (x). Environmental distances (ENVijD) between two locations i and j were calculated using equation 1  and the WorldClim database (Xgwm540) and 6 (Xgwm46) with a mean of 4 alleles per locus (Xgwm169) and 0.776 (Xgwm160 and Xgwm60) with a mean of 0.622  were detected in the 75 individual plants analysed with 15 nuSSRs. The number of alleles detected per locus ranged between 2  . The in ectively .T. aestivum outgroup). The number of alleles per locus ranged between 4 (WCt15) and 33 (Xgwm6) with a mean of 14.6 alleles per locus  were detected in the panel of tetraploid wheat accessions  followed by emmer (gene diversity of 0.708) and rivet (gene diversity of 0.682), while Northwest African durum was the least diverse (gene diversity of 0.546) , indicating that the phylogenetic information contained in the A and B genomes is different.Wild emmer was the most diverse of all ten f 0.546) . Among dr groups . Interesr groups . SoutheaT. aestivum landraces found well-supported population structure among wheats with a degree of admixture between clusters. In all models, most accessions were indicated to have received alleles from more than a single group. Analyses of ΔK, LnP(D) and Q-matrix correlations indicated that the solutions K = 2 and K = 6 were stable and well-supported , containing wild emmer, emmer and bread wheat, and Group II (red), including rivet and durum accessions  from Spain (Group Ib – yellow) from the remainder of Group I (Group Ia – dark green) (In the k green) . Group Ik green) . Group Ik green) . Group Ik green) . Group Ik green) . Group Ik green) . Table 4Q-matrices from the K = 6 model, we plotted clines of the six groups' allele frequencies on a map of the Mediterranean Basin (Using an Basin . Group IPCA corroborated the results obtained using STRUCTURE . The firT. aestivum) (T. aestivum var. Chinese Spring control), 2, 13 and 14 were the most frequent. Emmer and wild emmer had a high frequency of unique cp-haplotypes, indicating a higher diversity of maternal lineages in these forms. The frequencies of the four main cp-haplotypes in Southwest Europe, Northwest Africa and the Central Mediterranean were similar. Cp-haplotype 2 was very rare in emmer and rivet and its geographic distribution in durums was limited to accessions from Northwest Africa, Southwest Europe and Central Mediterranean (WCt22 (We identified 24 chloroplast haplotypes (cp-haplotypes) in our accession panel (excluding estivum) . Cp-haplerranean . 20 durun , Beladi Bouhi (Egypt) and Rubio o Rubial (Spain) with the ‘GS-105’ type deletion; Rubio (Spain) and Tri13912 (Libya) with the ‘GS-100’ type deletion; and Trigo Fuerte (Spain) where both deletions were detected. The presence of both mutations in this latter accession may be because bulks of 6 to 8 plants were used for DNA extractions and plants with the two types of mutations were included; at a phenotypic level all of the plants are expected to have the same photoperiod insensitivity response. However, these molecular results were not confirmed with the phenotypic analysis of their photoperiod response type. The accessions with the deletion were previously clustered in distinct groups by STRUCTURE.Almost all of the accessions had neither of the two VRN-A1 gene was obtained in 235 of the 244 accessions  in the intron 1 of the cessions . With onr = 0.211; p<0.001) was found between genetic and geographic distance  and in the Mediterranean European accessions . For the 53 Iberian durum accessions, there were no noteworthy correlations between CD and the logarithm of ENVijD or between CD and the individual environmental parameters, suggesting that none of them is having a substantial influence on microsatellite genetic diversity  was much higher than in emmer (3), durum (3) or rivet (2). Out of the 14 cp-haplotypes found in rivet and durum, 4 were also present in emmer accessions and 3 were present in wild emmer. Of the 7 cp-haplotypes found in emmer, 2 were also found in wild emmer. This suggests a scenario in which all three forms share a common maternal ancestral gene pool that would later become distinct between them due to different population histories. Zohary and Hopf Considering the strong genetic similarity between rivets and durums demonstrated in all our marker systems, it is likely that the two subspecies were originated from a common domesticated ancestor. Adaptation of plants to specific conditions as the species was introduced into Europe could have yielded landrace varieties with distinct morphological characteristics, such as the distinct head form in rivet or the latter's higher tolerance to frost and humidity in comparison with durum. These differences would be maintained by artificial selection giving rise to the agronomically distinct rivet and durum. These selective pressures were apparently not strong enough to create a distinct genetic pool between the two. Another possibility is that the distinction between rivet and durum is simply an artefact based on the criteria used by early botanists, focusing on differences rather than similarities between groups, whereas traditional farmers might have simply thought in terms of varieties with similar agronomic properties, like thresh-ability in situ and ex situ. In some of these Asturias accessions, Leigh and colleagues Xgwm169 could also be phylogenetically meaningful as they occurred only in Asturias accessions. Emmer landraces in Asturias can constitute relic varieties descending from the earliest cultivated forms that have been preserved from genetic erosion due to geographic isolation in mountainous terrain and small-scale farming Population structure was detected within emmer genotypes. Emmer accessions from Asturias were distinct from other emmers. These include accessions preserved both K = 6 model. It appears to be associated with cp-haplotype 2 which also shows an exclusive Western Mediterranean distribution  or natural selection . We investigated variation in our accession panel for two functional markers, one related to photoperiod response and another to vernalisation requirement. Only five accessions had a deletion in the Weak correlations between geographic and genetic distances suggest that a simple isolation by distance model does not apply to durum landraces in Iberia, North Africa or Mediterranean Europe. It is probable that durum wheat did not follow a linear and gradual route when it was introduced in Iberia, North Africa or Mediterranean Europe, or that subsequent historical processes have masked the primitive phylogeographic signal.The absence of correlations between genetic distances and environmental parameters for the Iberian durum suggests that natural selection has not significantly affected SSR allele distribution in Iberia. This is not to say that some plants would not have been selected due to their better performance under different environmental conditions, but simply that the genetic signal of this selection was not imprinted in the SSR markers analysed. Gene flow (by human-driven migration of genotypes) seems thus to be the major evolutionary force affecting the phylogeographic pattern of this species as revealed by nuSSR markers.Our study revealed a distinct population structure in tetraploid wheat accessions using even a small number of SSR markers. It is likely that a larger panel of SSRs would reveal further substructure in these wheat types. It remains to be seen if high-throughput screening of thousands of genome-wide SNPs in these accessions would confirm or falsify the population structure here reported. Moreover, using next-generation-sequencing technologies a large number of homologous genes related to climatic or physiological adaptations could be sequenced in landraces and wild types, revealing selective pressures occurring during wheat adaptation to distinct environments. Although caution would be advised when comparing these different marker systems, namely due to different ascertainment bias levels in SNPs and in SSRs in situ preserved varieties. An individual wheat landrace accession cannot always be seen as a more-or-less homogeneous group of individual plants with most alleles being fixed, as landraces are not static entities and episodes of introduction of new genotypes may occur throughout their history.In this paper we have demonstrated that tetraploid wheat accessions in the Mediterranean show population structure that may reflect early agricultural movements. This structure is corroborated by different genetic marker systems and adds strength to the taxonomic classification proposed to tetraploid wheats by MacKey Details S1PCR details.(DOC)Click here for additional data file.Figure S1Determination of the best STRUCTURE model for the panel of tetraploid wheats. The most meaningful values of K were determined using the LnP(D) (blue line) and the ΔK (red line) methods.(TIF)Click here for additional data file.Figure S2STRUCTURE results for domesticated tetraploid wheats (top and middle) and naked wheats (bottom). For the domesticated tetraploid wheats, models K = 3 and K = 6 are presented. For the naked wheats, model K = 4 is presented Groups were identified with the same letter and colour as their equivalent groups in the STRUCTURE runs using the complete panel of accessions.(TIF)Click here for additional data file.Figure S3Geographical distribution of population structure in domesticated tetraploid accessions. Wild emmer is excluded. Pie charts indicate the proportional membership of each landrace to each one of the different groups as determined by STRUCTURE. A) K = 3 model. B) K = 6 model.(TIF)Click here for additional data file.Figure S4Plot of ΔK and LnP(D) for 20 STRUCTURE runs with a panel of 215 tetraploid wheat accessions.(TIF)Click here for additional data file.Table S1Accession sourcing and genotype data. NSG-USDA: National Small Grains Research Facility, Idaho, USA; IPK: Institute für Pflanzengenetik und Kulturpflanzenforschung, Gatersleben, Germany; CRF-INIA: Centro de Recursos Fitogenéticos, Madrid, Spain; ENMP-INIA: Estação Nacional de Melhoramento de Plantas, Elvas, Portugal.(XLS)Click here for additional data file.Table S2Markers used to characterise genetic diversity in 235 tetraploid wheat and 9 bread wheat accessions. For each marker chromosome location (Chr.), PCR annealing temperature (Tm), Dye used for labelling primers, number of alleles detected (Allele Richness), gene diversity and polymorphic information content (PIC) are provided. “Touchdown” PCRs are denoted by “t*” under annealing temperature.(XLS)Click here for additional data file.Table S3Genetic markers used to characterise the within landrace genetic diversity in seven tetraploid wheat accessions. For each marker measures of genetic diversity are provided. MAF: frequency of the most frequent allele; PIC: polymorphic information content.(XLS)Click here for additional data file.Table S4Genetic marker systems used in the characterisation of tetraploid wheat landraces. For each system the percentage of null alleles detected, average frequency of the most frequent allele (MAF), average number of alleles and average gene diversity detected and polymorphic information content (PIC) is presented.(XLS)Click here for additional data file.Table S5Chloroplast haplotypes identified in tetraploid wheat accessions combining the alleles of five chloroplast SSR markers.(XLS)Click here for additional data file.Table S6ISBP haplotypes detected in tetraploid wheat accessions. The haplotype found in the T. aestivum var. Chinese Spring control was labelled “I”. Numbers correspond to the number of accessions where haplotypes were detected.(XLS)Click here for additional data file.Table S7Haplotypes identified in tetraploid wheat accessions using four ISBP markers.(XLS)Click here for additional data file.


Borago officinalis L.) seed oil has been used as a treatment for various degenerative diseases. Many useful properties of this oil are attributed to its high gamma linolenic acid content . The purpose of this study was to demonstrate the safety and suitability of the use of borage seed oil, along with one of its active components, GLA, with respect to DNA integrity, and to establish possible in vivo toxic and in vitro cytotoxic effects. In order to measure these properties, five types of assays were carried out: toxicity, genotoxicity, antigenotoxicity, cytotoxicity (using the promyelocytic leukaemia HL60 cell line), and life span . Results showed that i) Borage seed oil is not toxic to D. melanogaster at physiological concentrations below 125 µl/ml and the studies on GLA indicated non-toxicity at the lowest concentration analyzed ii) Borage seed oil and GLA are DNA safe (non-genotoxic) and antimutagenic compared to hydrogen peroxide, thereby confirming its antioxidant capacity; iii) Borage seed oil and GLA exhibited cytotoxic activity in low doses  iv) Low doses of borage seed oil (0.19%) increased the health span of D. melanogaster; and v) GLA significantly decreased the life span of D. melanogaster.Borage (D. melanogaster, increases the health span and exerts cytotoxic activity towards promyelocytic HL60 cells.Based on the antimutagenic and cytotoxic effects along with the ability to increase the health span, we propose supplementation with borage seed oil rather than GLA, because it protects DNA by modulating oxidative genetic damage in  Borago officinalis L.) is an annual plant used from ancient times for culinary and medicinal purposes. Recently, interest in borage has been renewed because its seeds are considered as one of the best sources of gamma-linolenic ). This unusual fatty acid is an intermediate of indispensable compounds in the body such as prostaglandin E1 and its derivatives Many recently developed remedial therapeutic and preventive medicines include the use of traditional plant-based preparations. Borage  and cytotoxicity (using the promyelocytic leukaemia HL60 cell line).The purpose of this study was to demonstrate the safety and suitability in the use of borage seed oil along with one of its active components (GLA) with respect to DNA integrity and to establish possible Commercial borage seed oil obtained by first cold pressure of seeds from organic cultivation was provided by Biolasi . Organic farming methods for borage seed production in combination with cold-pressing methods allow the maximum content and recovery of antioxidants in the seed oil.cis-6,9,12-octadecatrienoic acid), grade analytical standard  was purchased from Sigma ,Gamma linolenic acid . Fatty acid methyl esters of the borage seed oil were prepared according to Garcés and Mancha The triglycerides of the borage seed oil were separated by HPLC according to the COI T.20 Doc 25 method 2 column). Results were adjusted for the average specific gravity of borage seed oil and expressed in mg/kg.The tocopherol isomers in borage seed oil were determined in accordance with the IUPAC method 2432 Drosophila SMART test Drosophila melanogaster strains carrying visible wing genetic markers were used: the flare (flr) strain 3/ln (3LR) TM3, Bdsflr and the multiple wing-hair (mwh) strain mwh/mwh. The marker multiple wing hairs  is a recessive mutation that is viable in homozygous flies, producing multiple hairs per cell instead of the wild type single-hair trichome 3, 3_38.3flr) is a recessive mutation which produces individual wing hairs that are malformed. The 3flr allele is retained via a balancer chromosome carrying multiple inversions and a dominant marker which is homozygous lethal  Analysis of the genotoxic and antigenotoxic activities of borage seed oil and GLA, was evaluated by the flr3/TM3, sBd strain and mwh/mwh males. After 8 h egg-laying, 72±3 h old larvae were washed in distilled water, transferred to fresh vials with 0.85 g Drosophila Instant Medium  which were wetted with 4 ml after of put solutions of increasing concentrations of the borage seed oil and GLA. Test groups consisting of 100 larvae each were: (i) negative control (distilled water); (ii) mutagenic positive control (hydrogen peroxide 0.12 M); (iii) 5 vials with increasing concentrations of borage seed oil ; and (iv) 5 vials with increasing concentrations of GLA . Taking into account the average daily food intake of D. melanogaster (1 mg/day) and the average body weight of D. melanogaster individuals (1 mg), the concentration range for both borage seed oil and GLA were calculate to fit them into the recommended fat daily intake for humans . Emerging adults of all groups were counted and stored in 70% ethanol to be analysed. Procedures are: 1) Toxicity assays evaluated x100. 2) Genotoxicity assays evaluated mutation rate diagnosis of different groups and 3) Antigenotoxicity testing, combined treatments were performed as previously described by Anter et al., 2011 Crosses were performed with optimally fertile virgin females of the 3mwh/flr) were mounted on slides with Faurés solution. Both dorsal and ventral surfaces of the wings containing 22,000 cells were screened under a photonic microscope at 400× magnification for the occurrence of individual spots (mwh or flr phenotype) or twin spots (mwh clone adjacent to flr clone). Small individual spots with one or two cells exhibiting the mwh phenotype corresponded to gene mutation and somatic recombination between the two marker genes occurring during the last mitotic rounds in the imaginal discs of the larvae. Large individual spots with three or more cells showing mwh or flr phenotypes corresponded to mutational events occurring earlier during larvae development. Twin spots with two juxtaposed clones corresponded uniquely to recombination events between the 3flr gene and the centromere.Marker-heterozygous genotypes  and passed every 2 days.The human promyelocytic leukaemia cell line HL60 was obtained from Dr. José M. Villalba Montoro  5 cells/ml) and treated for 72 h with increasing concentrations of borage seed oil and GLA. Cell viability was determined by the Trypan Blue dye exclusion test . Cells were counted by adding an aliquot of 10 µl of the culture to 10 µl of the Trypan Blue dye. The mix was counted under a light inverted microscope using a Neubauer chamber . Aliquots were taken after 24, 36, 48, 60 and 72 h incubation. After each incubation period, a growth curve was established and IC50 values (concentration of tested compound causing 50% inhibition of cell growth) were estimated. Curves are expressed as the percentage of survival with respect to controls at 72 h of growth.For the assessment of cell viability, HL60 cells were placed in 12 well culture plates  using the Log-Rank (Mantel-Cox) method.Viability curves of leukaemia cells were plotted as mean viability ± standard error of three independent replicas for each compound and concentration.The data given in cis-9 octadecenoic), linoleic  and gamma-linolenic acid , were the major fatty acids of the oil, representing the 18,5%, 36,6% and 21,1% of the oil respectively. Borage seed oil contained a very high proportion of unsaturated fatty acids to saturated fatty acids , and these data are in agreement with those reported in previous studies Unsaturated oleic . The concentrations 62.5 µl/ml to 125 µl/ml decreased survival considerably in a dose-dependent manner. This range of oil concentration in the medium  is similar to that used with other vegetable oils in a previous work Toxicity assays showed that borage seed oil is not toxic to However, the data on GLA indicated non-toxicity at the lowest concentration resulting in a survival rate of 99%, although the next higher concentration tested showed a 66% decrease in survival and higher concentrations showed a strong dose-dependent effect.In summary, taking the average toxicities into account  a general pattern of responses was observed indicating that treatments with borage seed oil and GLA, resulted in strongly reduced survival rates when combined with hydrogen peroxide .D. melanogaster. The genotoxicant hydrogen peroxide resulted mutagenic as expected 50) of 1 µl/ml and 0.087 mM, respectively. The concentrations of GLA used in the cytotoxicity experiments corresponded to the content of GLA of each oil dose. The cytotoxic effect of borage seed oil could almost entirely be due to the GLA content of these concentrations, deducible from the similarity of the curve shapes , compared to the control group (55 days). We compared the survival curves ≥ 75%, of living flies using water as control with the remaining substances. This part of the life span is considered as the health span of a curve, characterized by low and more or less constant age-specific mortality rate values D. melanogaster, but decreased the mean survival time significantly.When compared to acute toxicity treatments, lower concentrations were applied to larvae for longevity tests, as these treatments were chronic. Total lifespan curves are plotted in in vivo, in the eukaryotic system D. melanogaster because more than half of its genes are known to have mammalian homologues D. melanogaster provides an excellent in vivo genetic model system for toxicity studies Drosophila has been used to model human conditions such as neurodegenerative and infectious diseases Drosophila and higher mammals have similar dose–response relationship with many compounds such as monofunctional alkylating agents Pleiotropic activities with beneficial effects have been attributed to borage seed oil for the treatment of several disorders such as acute respiratory distress syndrome, rheumatoid arthritis, diabetic neuropathy, menopause and atopic dermatitis D. melanogaster, acting as a desmutagenic agents against hydrogen peroxide by scavenging the reactive oxygen species originated by the model genotoxicant used. The similarity in the antigenotoxic inhibitory potencies of GLA (38.1%) and borage seed oil (38.2%) as well as the high content of GLA in borage seed oil (21.1%) indicates that GLA may account for the antigenotoxic ability of borage seed oil.More than 200 effects of hydrogen peroxide as a genotoxin have been previously described Additionally, our results show that both substances tested have a cytotoxic effect on HL-60 tumour cells. Specific mechanisms inducing cytotoxicity have been observed for GLA in other studies: i) GLA induces apoptosis in this cell line checked by DNA fragmentation hallmark The cytotoxic effect of polyunsaturated fatty acids particularly arachidonic acid and the eicosanoids generated from it, is long known, being characterized as apoptotic and associated with oxidative stress. Chen et al., (1998) show that pretreatment with 0.03 mM arachidonic acid causes significant toxicity to the human hepatoma HepG2 cells (E9 cells) that over express human CYP2E1. The PUFA toxicity is associated with increased lipid peroxidation and can be diminished by antioxidants that prevent lipid peroxidation  showed no significant toxicity to the E9 cells even at concentrations (0.05 mM) at which arachidonic acid is highly cytotoxic Similarly, Pompeia et al., (2002) demonstrated the cytotoxic effect of arachidonic acid in leukocytes . Use this fatty acid was also demonstrated to have an apoptotic effect at low concentrations (10–400 µM) and a necrotic effect at high concentrations (400–1600 µM) D. melanogaster strains in order to perform life span trials with specific chronic diets in controlled environments D. melanogaster. This animal model is an excellent system to investigate longevity-promoting properties of compounds and nutraceutical extracts. Drosophila has a short life span, can be cultured with simple diets, and represents a rich genetic resource with a fully sequenced genome. The life span of this insect is relatively short and adults seem to show many of the cell senescence features seen in mammals D. melanogaster (Drosophila strains used in this work is not extended-life mutants. Average life span data of D. melanogaster vary widely and are strongly dependent on rearing conditions. The average control life span (between 33 and 80 days) found in previous works Anti-ageing and anti-degenerative assays can be carried out using different nogaster . No posinogaster  The DrosD. melanogaster are in agreement with the toxicity results for this fatty acid  borage seed oil and GLA are DNA safe (non-genotoxic) and antimutagenic compared to hydrogen peroxide, thereby confirming their antioxidant capacity; iii) borage seed oil and GLA exhibited cytotoxic activity in low doses  iv) Low doses of borage seed oil (0.19%) increased the health span of D. melanogaster; and v) GLA significantly decreased the life span of D. melanogaster.Based on the findings of the present study, we conclude that i) borage seed oil is non- toxic to in vivo oxidative genetic damage in D. melanogaster, increases the health span and exerts in vitro cytotoxic activity towards promyelocytic HL60 cells.Based on the antimutagenic and cytotoxic effects along with the ability to increase the health span, we propose the use of borage seed oil, rather than GLA, as a substance with antimutagenic/anticarcinogenic properties because it protects DNA by modulating the


Pongo pygmaeus morio)  in the Lower Kinabatangan Wildlife Sanctuary of Sabah, Malaysian Borneo, we predicted that i) fecal glucocorticoid metabolite concentrations would be elevated on the day after tourist visitation  compared to samples taken before or during tourist visitation in wild, habituated orangutans, and ii) that samples collected from habituated animals would have lower fecal glucocorticoid metabolites than unhabituated animals not used for tourism. Among the habituated animals used for tourism, fecal glucocorticoid metabolite levels were significantly elevated in samples collected the day after tourist visitation (indicative of elevated cortisol production on the previous day during tourist visitation). Fecal glucocorticoid metabolite levels were also lower in the habituated animals compared to their age-matched unhabituated counterparts. We conclude that the habituated animals used for this singular ecotourism project are not chronically stressed, unlike other species/populations with documented permanent alterations in stress responses. Animal temperament, species, the presence of coping/escape mechanisms, social confounders, and variation in amount of tourism may explain differences among previous experiments. Acute alterations in glucocorticoid measures in wildlife exposed to tourism must be interpreted conservatively. While permanently altered stress responses can be detrimental, preliminary results in these wild habituated orangutans suggest that low levels of predictable disturbance can likely result in low physiological impact on these animals.Nature-based tourism can generate important revenue to support conservation of biodiversity. However, constant exposure to tourists and subsequent chronic activation of stress responses can produce pathological effects, including impaired cognition, growth, reproduction, and immunity in the same animals we are interested in protecting. Utilizing fecal samples (N = 53) from 2 wild habituated orangutans ( Tourism generates more than 9% of the global gross domestic product Rapid, unmonitored development of nature-based tourism projects can also lead to degradation of habitats and deleterious effects on animal well-being, negatively impacting the very species we wish to conserve Tourists also likely serve as a source of infection, particularly to nonhuman primates who are genetically closely related to humans and share a number of common infections. A significant proportion of visitors to primate rehabilitation centers are not adequately vaccinated and are likely infectious, creating unnecessary risk of pathogen transmission to wildlife because they are largely unaware of the impacts they may directly have on animal health, despite their interests in environmental protection Anthropogenic disturbances may also affect adversely animal physiology. Stressors are noxious stimuli that trigger responses to maintain homeostasis. Normal activation of stress responses  is important for survival via alteration of metabolism and behaviors. Chronic exposure to stressors can lead to ‘allostatic overload’ Canis lupus), pine martens (Martes martes), elk (Cervus elaphus), and wood grouse  Field endocrinology has recently been used to assess alterations of the hypothalamic-pituitary-adrenal axis in endangered and economically valuable wildlife species Pongo pygmaeus ssp.) are endangered The present study proposed to evaluate the effects of tourist exposure on fGM levels of wild habituated orangutans. Fecal steroids Ethical approvals were obtained from the Institutional Animal Care and Use Committee of Indiana University (protocol #08-047) and by the Scientific Research Review Committee of Cleveland Metroparks Zoo (project #CS2005-030). Permission to conduct this research was granted by the Sabah Wildlife Department. As fecal samples were collected noninvasively from the captive and wild orangutan populations, all research complied with all recommendations of the 2006 Weatherall report for the use of non-human primates in research.Pongo pygmaeus pygmaeus) at Cleveland Metroparks Zoo. Using a wooden applicator stick, first-morning samples were collected between 7 and 8 am (immediately following defecation) off of a clean enclosure floor, uncontaminated with water or urine. Three samples were collected from each animal on non-consecutive days. Immediately following defecation, a portion of the fecal mass was stored in a closed centrifuge tube at enclosure temperature. A portion of each sample was removed from the tube and processed (see below) precisely every 30 minutes for up to 6 hours . Solid-phase extraction for this portion of the project took place in a laboratory at Cleveland Metroparks Zoo; sample solvolysis and enzyme immunoassay took place in the Evolutionary Physiology and Ecology Laboratory at Indiana University.Under field conditions, it is not usually possible to process fecal samples immediately following defecation or collection. Therefore, we determined the effects of time on hormone degradation using samples from one male and one female adult orangutan  when they were encountered during area surveys in the Kinabatangan (see below). Like the Cleveland samples, these samples (presumably first morning voids before 8 am) were collected immediately following defecation and processed in a similar fashion described above (a solid-phase extraction every 30 minutes for up to 6 hours) so as to determine the effects of time on hormone degradation.Pongo pygmaeus morio), and 17 samples from her 11-year old sub-adult male offspring, Etin. These wild animals have been monitored semi-continuously since 1998 as part of the Kinabatangan Orangutan Conservation Programme (KOCP) near the village of Sukau, in the Malaysian state of Sabah (north Borneo)  female orangutan ( Borneo)  and 2. TIn 2001, KOCP, the Sabah Wildlife Department and the Danish International Development Agency formed the Red Ape Encounters (RAE) community-based ecotourism program in the village of Sukau in the Lower Kinabatangan Wildlife Sanctuary, an area acknowledged as a prominent tourism destination in Sabah's National Ecotourism Plan To evaluate changes in fecal cortisol levels associated with tourist visitations, our goal was to obtain fecal samples from both Jenny and Etin i) 24 hours before tourist visitation , ii) during tourist visitation (to measure the effects of the previous day's exposure to our two field assistants) and iii) 24 hours after tourist visitation (to measure the effects of the previous day's exposure to the tourists). But obtaining samples for each animal and time point is difficult because of the relatively few tourists that presently utilize RAE, and because these animals are completely wild and utilize a very large territory, coming and going as they please. It is very difficult to stay with a single animal for more than a few days at a time, leading to a compromised sampling schedule.Samples were collected in association with 25 tourist visitation events. Of the 53 samples (36 from Jenny and 17 from Etin), 17 were obtained on the day before tourist visitation, 24 were obtained during tourist visitation, and 12 were obtained the day after tourist visitation. Samples were collected by 8 am each day  to minimize any diurnal effects. Animal identification and behaviors , animal physical condition, height of animal in canopy, tourist behaviors , number of tourists present, distance between animal and tourists, date, time of animal defecation and sample extraction were recorded. Given the nature of fecal hormone secretion, elevated fGM levels detected in the samples obtained the day after tourist visitation would be indicative of elevated stress response on the previous day (during the tourist visitation), assuming a gut retention time of approximately 24 hours for a large-bodied primate with a high fibrous diet Fecal samples were also obtained from 4 previously unidentified (and unhabituated) orangutans  when they were encountered during area surveys, expeditions and line transects in the Kinabatangan. Once these animals were located, they were followed every day that was possible. Ten fecal samples were obtained from the 2 unknown females , and 16 samples from the 2 unknown males .Solid-phase extraction for this portion of the project took place in the KOCP laboratory in Sukau; sample solvolysis and enzyme immunoassay took place in the Evolutionary Physiology and Ecology Laboratory at Indiana University.Each sample was thoroughly mixed within its collection tube. 0.2 grams (g) of feces  was weighed and mixed with 2.5 milliliters (ml) of distilled water and 2.5 ml of 95% ethanol in a new tube. The tube was then vortexed for exactly 5 minutes (min) . The tube was then centrifuged at 3300 revolutions per minute (rpm) for 10 min, and the fecal extract (supernatant) decanted into a clean tube. An Alltech Prevail C18 Maxi-Clean reversed-phase non-polar silica solid-phase extraction cartridge (500 milligrams [mg] bed weight) was primed with 2 ml of distilled water. Using a syringe, 2 ml of the fecal extract was slowly loaded into the cartridge, followed by another 2 ml of distilled water. Both ends of the cartridge were capped, the cartridge was labeled and placed into a zip-lock bag containing a silica gel packet. Cartridges were stored at ambient temperature in the dark for two weeks before being shipped to the laboratory and frozen at −20 degrees Celsius (°C). This method of adhering the fecal analytes onto a sorbet matrix in the field requires the availability of organic solvents and electricity, but also allows for shipping diagnostic, non-infectious samples at ambient temperature, eliminating the need for shipping alcohol, dry ice or liquid nitrogen. This method was also preferred in the present study because of unpredictable freezer/electricity availability in the forest.2SO4 with 43.06 ml water], 100 µl of a saturated sodium chloride solution [31.13 g made up to 100 ml water], and 4.5 ml of ethyl acetate, mixed with sample, incubated overnight in a water bath [40°C]) to hydrolyze conjugated steroids. The next morning, 2.5 ml of water were added, the tubes vortexed for 5 min, and then centrifuged for 3 min at 1000 rpm. The ethyl acetate fraction was transferred into clean tubes, evaporated until dryness and rehydrated with 1 ml of 100% ethanol.Using a vacuum manifold, the cartridges were washed using 1 ml of 20% methanol and eluted using 2 ml of 100% methanol. The eluted samples were evaporated to dryness using an Organomation Multi-Vap nitrogen evaporator . Samples were rehydrated with 1 ml of 100% ethanol and vortexed. Samples underwent solvolysis  added to each well, sealed and frozen until use.Cortisol antibody and cortisol-horseradish peroxidase (HRP) were obtained from Coralie Munro at the University of California-Davis. 200 µl of antibody were mixed with 1.8 ml carbonate buffer (2 Sigma carbonate-bicarbonate capsules with 200 ml water). 124 µl of this solution were mixed with 198 ml of carbonate buffer to yield a 1∶16,000 antibody dilution. 100 µl of this was pipetted into each well of a Nunc Maxisorb plate. The plates were incubated inside a humidity chamber in a refrigerator overnight, after which they were washed (4 cycles with 10 second soaks on a Bio-Tek ELx-405 plate washer) with buffer , blotted, 150 µl buffer (10.842 g NaHEight standard concentrations (5–1000 picograms [pg] per well) of cortisol were created by dilution of a stock solution (10 mg of cortisol in 100 ml 100% ethanol). 900 µl of unknowns were transferred into clean tubes, and these along with standard and controls (62.5 pg and 17 pg) were evaporated to dryness. 300 µl of cortisol-HRP conjugate solution (17 ml buffer with 11.3 µl HRP solution [5 µl HRP with 495 µl buffer]) were added to each tube, vortexed, and then transferred to a deep-well plate. 200 µl of the cortisol-HRP conjugate solution were added to the zero concentration well, and 300 µl buffer to the non-specific binding well.2O2 solution [0.5 ml H2O2 with 7.5 ml water], and 24.67 ml citrate buffer ) were added to each well. The plate was resealed and rotated for 5 min, and incubated in the humidity chamber for 25 min at room temperature. 100 µl stop solution (10 ml of water with 20 µl ethylenediaminetetraacetic acid (EDTA) solution [41.623 mg EDTA made up to 100 ml water] and 10 ml hydrofluoric acid solution [5.048 ml of 49% hydrofluoric acid with 1.2 NaOH [5 normal]) were added to each well. Absorbance  of each well was read at 415 nanometers (nm) (reference wavelength of 570 nm) using a Bio-Tek Synergy 2 plate reader. The average optical density of the non-specific binding wells was subtracted from the average optical density of all other wells. Percent binding was plotted against the standard concentrations using a log-logit transformation. Based on the dilutions used above, the value of each unknown was transformed from pg per well to nanograms (ng) of glucocorticoid metabolite per gram of feces by multiplying by 0.042, with separate dilution factors for standards and controls. A detailed protocol can be made available upon request. This assay was adapted from previous methods used by Dr. Toni Ziegler and Dan Wittwer at the Wisconsin National Primate Research Center for other nonhuman primate species Antibody-coated plates were removed from the freezer and allowed to reach room temperature within a humidity chamber, after which the buffer was dumped and the plates blotted. 100 µl of from each well in the deep-well plate were then pipetted in duplicate to the corresponding wells on the antibody-coated plate. The plate was sealed and incubated for 2 hours (5 min on a plate rotator and then 155 min in the humidity chapter at room temperature). The plate was washed with buffer as before, and 100 µl of substrate-citrate buffer solution  for statistical analysis. Intra-assay coefficients of variation were assessed using the mean coefficients of variation of control duplicates. Intra-assay coefficients of variation were less than 8%. Interassay coefficients of variation were assessed using the mean coefficients of variation of control (high and low) duplicates in five separate assays. Interassay coefficients of variation were less than 10%.Continuous measures were summarized by mean, median, and standard deviation. For the fecal degradation samples , average cortisol values for each time point (every 30 minutes between 0 and 6 hours) were plotted, and a first-order auto correlation was used to evaluate change over time. For the Cleveland samples, a repeated measures analysis of variance was run on their sequential samples (3 samples collected on non-consecutive days) to evaluate stability in fGM across days.Samples from the wild habituated animals (Jenny and Etin) collected before (N = 17), during (N = 24) and after (N = 12) tourist visitation were compared using analysis of co-variance . Samples from the 4 unhabituated animals collected at initial encounter (N = 19) and after exposure to researchers (N = 7) were compared using analysis of co-variance. Samples from the wild, habituated animals were compared with those of the unhabituated animals  as well as the captive Cleveland samples using non-parametric Mann-Whitney U tests. Level of significance  was set at 0.05. Because of low sample size (and subsequent power less than 80%), borderline significant results are interpreted conservatively to avoid conclusions based on false negatives.Pongo pygmaeus pygmaeus) at Cleveland Metroparks Zoo as well as two wild orangutans (Pongo pygmaeus morio) in Borneo. Under field conditions, it is not usually possible to process fecal samples immediately following defecation or collection. Therefore, we determined the effects of time on hormone degradation using samples from one male and one female adult orangutan . We contend that results from other studies dependent upon samples collected randomly without witnessing defecation, and those not extracted (or frozen) within 3 hours of defecation, are limited. The time between defecation and extraction should be standardized for a given research population, ideally with all samples and cohorts extracted at approximately the same time since defecation. Samples extracted immediately can yield very different results from those extracted after 3 hours.Increased variation in fGM over time likely reflects bacterial metabolism, conjugate cleavage and other physiological processes Of the 25 different tourist visitations that were monitored, mean number of tourists at each visitation was 6.2 (SD 5.1) with a mean length of visit of 143 min (SD 111). These variables were unassociated with fGM levels in either animal (results not shown). The two habituated animals exhibited no behavioral indicators of stress  and animal physical condition appeared excellent. Height of animal in canopy, tourist behaviors , number of tourists present, and distance between animals and tourists did not vary significantly to allow further analysis.In comparing Jenny and Etin's  samples obtained the day before, the day of and the day after tourist visitation (ANCOVA controlling for time between defecation and sample extraction), fGM levels were significantly elevated in samples collected the day after tourist visitation (indicative of elevated cortisol production on the previous day: during tourist visitation) .For the unknown animals, fGM levels in samples obtained after previous contact with researchers were numerically higher but not statistically significantly different from their initial samples  see .In comparing the habituated animals' (Jenny and Etin's) samples obtained the day before tourist visitation  with the samples obtained from the 2 unhabituated animals upon initial contact or no contact with humans in the 2 preceding days (N = 19), fGM levels were lower in the habituated animals . However, fGM levels in the habituated animals' samples the day after tourist visitation  were not statistically different  from samples obtained from the 2 unhabituated animals the day after exposure to the researchers (N = 7).Alouatta pigra) in Belize regularly visited by tourists have significantly higher fGM values than other study groups not visited by tourists Macaca sylvanus) in Morocco only following ‘aggressive’ human behavior towards the animals (and not other tourist behaviors), although self-scratching in males (used here as a proxy for anxiety) was related directly to rates of all tourist-macaque interactions fGM levels following an anthropogenic stressor provide valuable biometric data that complement previous work illustrating behavioral alterations in wild populations of nonhuman primates in response to habituation and tourism Spheniscus magellanicus) in Argentina exposed to tourism exhibit blunted corticosterone responses following capture and restraint as well as blunted responses following exogenous treatment with adrenocorticotropin hormone Eudyptula minor) at tourist sites exhibit higher glucocorticoid concentrations than animals at non-tourist sites Megadyptes antipodes) exhibit significantly higher corticosterone responses to capture and restraint Amblyrhynchus cristatus) in the Galápagos islands exhibit reduced stress responses to capture and restraint compared to more isolated animals Rupicapra pyrenaica pyrenaica) Crocuta crocuta) The present study, although not without limitations, utilizes a more powerful study design  to identify apparently normal fGM responses to tourist exposure in wild orangutans, with fGM levels increasing in response to tourist visitation, and then returning to a lower level. fGM levels were also lower in habituated than in nonhabituated animals. We therefore conclude that these habituated animals utilized for tourism in Sabah are not chronically stressed, unlike other animals with documented permanent alterations in stress responses. For example, habituated wild adult Magellanic penguins . Those with PTSD  typically display lower baseline (tonic) cortisol levels compared to healthy controls Other studies have paradoxically observed increased baseline cortisol levels in those with PTSD Similarly, animal temperament may influence the outcome of anthropogenic disturbance, with some animals responding more negatively to human disturbance Opisthocomus hoatzin) than undisturbed groups Megadyptes antipodes) exposed to tourism exhibit significantly lower breeding success than those in other populations Tourism seems to negatively affect fitness of some wildlife populations. Survival is lower in tourist-exposed groups of Amazonian hoatzin juveniles  could be utilized to ensure the balance between animal well-being and economic opportunities. Furthermore, animal usage must be kept minimal , and the habituation processes gradual. The costs associated with tourism development must be carefully considered before new groups of animals are habituated. And most importantly, consumer behavior and visitor management must change. The public must be informed better about the risks they may pose to wildlife.Low sample sizes (for reasons explained above) prevent us from concluding if acute variation in cortisol in these orangutans correlates with survival or reproduction probabilities. It is unknown if there are changes in neurotransmitter or endogenous opioid levels or receptor functions in these animals. An adrenocorticotropic hormone (ACTH)-challenge as well as a ‘capture-and-restraint’ test would certainly yield useful information


Survival of patients with lung cancer could be significantly prolonged should the disease be diagnosed early. Growing evidence indicates that the immune response in the form of autoantibodies to developing cancer is present before clinical presentation. We used a phage-displayed antibody library to select for recombinant scFvs that specifically bind to lung cancer-associated IgM autoantibodies. We selected for scFv recombinant antibodies reactive with circulating IgM autoantibodies found in the serum of patients with early stage lung adenocarcinoma but not matched controls. Discriminatory performance of 6 selected scFvs was validated in an independent set of serum from stage 1 adenocarcinoma and matching control groups using two independent novel methods developed for this application. The panel of 6 selected scFvs predicted cancer based on seroreactivity value with sensitivity of 0.8 and specificity of 0.87. Receiver Operative Characteristic curve (ROC) for combined 6 scFv has an AUC of 0.88  as determined by fluorometric microvolume assay technology (FMAT) The ROC curve generated using a homogeneous bridging Mesa Scale Discovery (MSD) assay had an AUC of 0.72 . The panel of all 6 antibodies demonstrated better discriminative power than any single scFv alone. The scFv panel also demonstrated the association between a high score - based on seroreactivity - with poor survival. Selected scFvs were able to recognize lung cancer associated IgM autoantibodies in patient serum as early as 21 months before the clinical presentation of disease. The panel of antibodies discovered represents a potential unique non-invasive molecular tool to detect an immune response specific to lung adenocarcinoma at an early stage of disease. Non-small cell lung cancer (NSCLC) is the most prevalent form of lung cancer and, if not detected early, has a low cure rate. Survival rate improves dramatically from 13% when diagnosed at stage IIIA to 70% when detected at stage IA A promising approach to the early detection of cancer is to look for the immune response to the developing cancer. Solid tumors produce unique tumor associated antigens (TAA) that are recognized by the immune system resulting in the production of autoantibodies against them. Growing evidence indicates that humoral immune response in the form of autoantibodies is present in cancer patients before clinical demonstration of disease Antibodies represent some of the most abundant proteins in human serum and can be present in serum at concentrations that supersedes that of their respective antigens. The amino acids present in an antibody’s heavy and light chain variable (V-region) or idiotypic regions interact with and bind to an antigenic site, and are unique to the antibody. Antibodies known as anti-idiotypic antibodies can, like an antigen, also interact with some or all of the same amino acids that make up the idiotype of an antibody.IgM antibodies are the first class of antibodies produced during a humoral immune response, and represent the most logical class of antibodies to detect as early indicators of disease. Antibody class switch from IgM to IgA, IgE or IgG is dependent upon antigen presentation by MHC molecules present on T-cells and the antibody producing B-cells. Typically, MHC molecules present peptide fragments, but do not usually present carbohydrate or other non-amino acid fragments. If an IgM antibody is produced to a site on a self- or “cancer antigen” that has been glycosylated in response to tumor formation, then it is unlikely that other classes of antibodies will be made to the exact same site if MHC presentation does not occur. We have tested the hypothesis that IgM antibodies produced against proteins up-regulated or modified in response to tumor formation would be present and more readily detected than their respective antigens; and, as such, would represent useful biomarkers as indicators of early stage lung cancer.in vitro method to select for peptides and antibodies to target molecules The goal of this study was to use phage-display to select for single chain fragment variable (scFv) antibodies for use in assays to detect cancer-associated IgM autoantibodies as biomarkers of early stage NSCLC. Since antibodies of identical antigen-binding site specificities have similar or identical idiotypic amino acid sequences, then any IgM antibodies that undergo IgM to IgA, IgE or IgG class switch - without undergoing significant amino acid change due to V-region somatic mutations - would also be detected by the same scFv, if the scfv were specific for the idiotype of an antibody.Antibody fragments, as Fab or scFv, have been among the first proteins to be displayed on the surface of a filamentous virus or bacteriophage using a procedure initially described in 1990 by McCafferty et al. Recognizing that the heterogeneity of NSCLC may determine the diversity of disease-related humoral immune response, we have restricted ourselves to the discovery of autoantibody present in adenocarcinoma - the most common type of lung cancer. To select for scFv binding to cancer patient IgM that could be early disease biomarker, sera from only stage I adenocarcinoma patients were used.A phage-displayed scFv antibody library was used to select scFv specific for lung cancer IgM autoantibodies as biomakers of early stage I adenocarcinoma. The procedure is summarized in The 6 remaining scFv were further characterized by FMAT analysis using an independent set of 15 lung cancer and 15 non-cancer serum samples. Three of the 6 scfv  showed significant difference between cancer and negative control groups . The scFEach IgA, IgG and IgE antibody has, at minimum, 2 identical sets of idiotypes or heavy and light chains, while IgM antibodies have 10. Therefore, mono-specific scFv stemming from the same bacterial clone can bind, at minimum, 2 sites on the same “autoantibody”. As such, a mono-specific, monoclonal scFv can conceivably be used in the same assay to both capture and detect the same “autoantibody” to enhance assay specificity. We developed and utilized an electrochemiluminescent homogenous assay to confirm results obtained using the FMAT homogenous assay. An aliquot of a single scFv (e.g. B6) was biotinylated and used to capture autoantibodies from serum samples. Another aliquot of the same scFv (i.e. B6) was conjugated with an MSD sulfo-Tag and used to detect autoantibodies captured by the biotinylated (i.e. B6) scFv see . HomogenFor validation of 6 scFvs in homogenous bridging assay all 43 samples were used. The discriminatory capacity of selected scFvs was evaluated for individual clones as well as for their combination. Validation was done with the independent set of 43 samples: 22 early adenocarcinoma samples (19 of stage I and 3 of stage IIB), and 21 matched controls . These 2Although survival was not our primary outcome, we speculated based on our observation that seroreactivity of selected scFvs could be associated with prognosis. We used the panel of all 6 scFv to build a survival prediction model to compare the equality of survival curves for the low and high scFv score groups . The panel demonstrated the associating of high score with poor survival . HoweverWe took advantage of the exquisite sensitivity of the immune system to detect lung cancer specific antibodies at an early stage in tumor progression and develop a new autoantibody testing platform. Autoantibody to tumor-associated antigens was found in the circulation of patients in the preclinical setting We decided to focus this research project on stage 1 NSCLCs because prompt management of malignant pulmonary nodules presenting at stage I  could have 5 year survival rate up to 80%, and because NSCLC is the most prevalent in heterogeneous populations of lung cancer.We used large phage-display recombinant scFv antibody library to select for lung cancer specific circulating autoantibodies. There are several advantages in using this approach: 1) the binding strength of the phage-displayed recombinant antibodies due to their larger size is greater than that for a phage-displayed peptide, resulting in better specificity, selectivity and potency of antigen binding; 2) the ability to use the E-tagged scFv antibodies to affinity-purify and conjugate lung cancer-specific antibody/antigen for subsequent characterization; 3) the affinity-purified recombinant antibodies can be labeled with biotin, dyes, enzymes, or conjugated to beads, and be used in a variety of inexpensive immunoassay formats.We successfully selected for scFv recombinant antibodies reactive with circulating IgM autoantibodies obtained from adenocarcinoma patient but not normal human samples. In a validation set of patients matched by age, gender and smoking status, 6 scFv discriminated serum samples from stage I adenocarcinoma and no-cancer control at very significant statistical level.A novel homogeneous bridging scFv assay was developed using MSD technology. This technology has demonstrated more sensitivity and broad dynamic range and it requires significantly less volume of serum sample than ELISA, which is commonly used in similar serological studies. The ability to multiplexing is also benefit over standard ELISA. In contrast to other multiplex platforms, such as Luminex, the MSD technology is advantageous because it does not require chemical conjugation of antigens to solid phase, therefore minimizing the potential impact on immunogenicity of tested antibody. This platform can be used in multi-array format for the high-throughput screening. To our best knowledge, we were the first to develop this type of assay using scFv for the autoantibody detection.We were able to detect high titers of IgM lung cancer associated autoantibodies in the serum of patients 2 year before clinical presentation. This is what makes this approach promising. If such observation is confirmed, it would be of great interest to identify the proteins that changes so early at disease onset and these changes are so dramatic that organism starts to consider these proteins as “non-self” and develops autoantibody against them. We believe that these autoantibodies can be detected and measured in a simple immunoassay and their presence or concentration can be correlated with occurrence of disease.The identification of panels of antibodies that are specific for particular cancer type may yield a molecular signature for non-invasive early diagnosis. While surgical resection is the most effective therapy in NSCLC, 75% of patients with lung cancer present with unresectable disease. A set of serological biomarkers may offer cost-effective ways for early detection of lung cancer, and by detecting NSCLC early in its course patients could be provided a better chance for a cure. Additional validation of our results is essential and would be particularly well suited in independent cohorts of high-risk individuals for lung cancer development. Upon follow up, we may be able to detect lung cancer earlier in patients found to have chest CT-detected lung nodules or ground glass opacities. Further investigations towards the identification of the targets may help us gain insight in the nature of the signal and the biology of the early disease process.The Vanderbilt University and Nashville Veterans Affairs Medical Center Institutional Review Boards approved the study and all patients provided written informed consent. Copies of all written consent forms are available under IRB Protocol 030763.Blood was collected from patients diagnosed with lung cancer and from non-cancerous patients. All cancer samples were collected prior to resection - at the time of diagnosis. Patients’ characteristics are presented in 9 or 2.9 billion members) phage-displayed ScFv (single-chain fragment variable) recombinant antibody library. The library has been used to obtain antibodies to different recombinantproteins E. coli.Modifications of previously published protocols E. coli TG1 cells. 4) Infected TG1 cells were plated onto 2xYT agar plates containing ampicillin and 2% glucose (2xYTAG). Colonies stemming from this 1st round of phage antibody selection were M13KO7 helper virus rescued to produce phage-displayed scFv and then PEG precipitated. 5) Human IgM antibodies pooled from 8 normal human serum samples were immobilized onto anti-human IgG agarose beads as described in step 2. 6) Beads from step bearing normal human IgM were used to cross-absorb PEG precipitated phage-displayed scFv to remove scFv cross-reactive with normal and adenocarcinoma IgM antibodies. 7) Nunc Maxisorb tubes were coated with goat anti-human IgM μ-chain specific antibody (Sigma cat#I0759) at 5 µg/ml in PBS and blocked with PBS-T. 8) Unbound phage-displayed scFv from step 6 were cross-absorbed against goat anti-human IgM in Maxisorb tubes (step 7) to remove scFv reactive with goat anti-human IgM antibodies.9) Eight Nunc Maxiasorb tubes coated with goat anti-human IgM antibodies were used to separately capture IgM from 10 µl of each of 8 different human adenocarcinoma serum samples. 10) Unbound phage-displayed scFv were applied to tubes from step 8 to select for scFv specific for adenocarcinoma IgM. 11) Bacterial colonies stemming from the 2nd round of phage selection (step 10) were pooled and frozen down as glycerol stocks for use in subsequent assays to identify colonies producing scFv that bind to adenocarcinoma but not normal human IgM. It should be noted here that eight 1st and 2nd round phage selections were performed on 8 individual stage I adenocarcinoma serum samples, with all selections for each patient sample being kept separate.Phage-displayed scFv specific for adenocarcinoma IgM were selected using IgM from human samples bound to beads. The following 11 steps were used to obtain scFv specific for andenocarcinoma IgM: 1) The phage-displayed scFv library was cross-absorbed against goat anti-human IgM (μ-chain) specific antibodies coupled to agarose beads (Sigma cat# A9935) to remove scFv reactive with goat antibodies. 2) Ten to 20 µl of serum from stage I adenocarcinoma patients was applied to 100 µl of goat anti-human IgM agarose beads to capture IgM from serum. Beads bearing human IgM were washed with PBS containing 0.1% Tween-20 (PBS-T) to remove non-bound human serum components. 3) The cross-absorbed phage-displayed scFv from step 1 was applied to beads from step 2 and incubated overnight at 4–8°C. Beads were washed with PBS-T to remove unbound phage-displayed antibodies, after which phage on beads were used to infect nd round phage scFv selections were plated onto 2xYTAG agar plates to obtain individual colonies. Individual colonies were picked from 2xYTAG agar plates into 384 well microtiter plates containing 2xYTAI (ampicillin and 1 mM IPTG) to induce E. coli scFv expression. A 384 well pin replicator was used to transfer bacteria from 384 well 2xYTAI to prepare 2xYTAG agar master replica plates. Cells in 2xYTAI microtiter plates were centrifuged to pellet cells. The supernatant was removed and cells were suspended in 40 µl of TES  and 60 µl of 1/5th X TES (1 volume of TES plus 4 volumes of distilled water) then incubated on ice for at least 1 hour to obtain scFv in E. coli periplasmic extract.Cells from frozen glycerol stocks stemming from the 2The Anti-E tag monoclonal antibody conjugated to Alexa 647 was used for FMAT assays (Fluorometric Microvolume Assay Technology) to detect E-tagged scFv bound to adenocarcinoma IgM bound on beads. Briefly, the Anti-E tag monoclonal antibody was conjugated to Alexa 647 (Invitrogen cat# 20006) at a ratio of 2.25 dye molecules per antibody. Unincorporated dye was removed from the dye-labeled antibody using a NAP-10 column  equilibrated with PBS.E. coli periplasmic extract were transferred from 384 well culture plates to corresponding wells in 384 well assay plates. Assay plates were incubated for 4 hours at room temperature, or overnight at 4–8°C in the dark. Assay plates were then read using an FMAT 8100 plate reader (Applied Biosystems).Streptavidin-coated microtiter beads (Spherotech cat# SVP-100-4) at a concentration of 5 µg/µl were coupled with 0.2 µl biotinylated goat anti-human IgM (Sigma cat# B1265) per 1 µl of beads, washed with PBS-T to remove unbound anti-human IgM, then blocked with PBS-T. Approximately 0.6 µl of human serum was added per 1 µl of beads to immobilize normal or adenocarcinoma human serum IgM onto beads. Beads were washed with PBS-T to remove unbound IgM. Alexa 647 conjugated Anti-E tag monoclonal antibody was added to beads to a final concentration of 0.38 µg/ml of beads. Fifty microliters of beads containing Alexa 647 conjugated to Anti-E tag monoclonal antibody were dispensed to individual microtiter wells (∼0.138 µl of beads/well) of a 384 well Corning (cat#3912) microtiter assay plate. Fifteen µl of scFv in www.mesoscale.com) electrochemiluminescent assay platform was developed to detect circulating autoantibodies. Purified scFv were biotinylated with biotinamidocaproate-NHS (Sigma cat# B2643) at a ratio of 2 biotins : 1 scFv. Purified scFv were also conjugated to the MSD Sulfo-tag (MSD cat# R91AN-1) at a ratio of 18 tags : 1 scFv for 4 hours at room temperature, in the dark. MSD Sulfo-tag conjugated scFv were used to detect autoantibodies captured by biotinylated scFv immobilized onto wells of streptavidin-coated microtiter plates. Unincorporated biotin or Sulfo-tags present in scFv conjugation reactions were removed with desalting columns (Pierce cat# 89883).An MSD pre-blocked with MSD blocking buffer (MSD cat.# R93AA-2), incubated for 2 hrs at RT on a shaking platform, washed with PBS-T and emptied, after which MSD read buffer (MSD cat.# R92TC-3) was added. An MSD Sector Imager 2400 plate reader was used to quantify electrochemiluminescence assay signals.To assess the primary hypothesis that these 6 selected scFvs autoantibody candidate biomarkers, individually or jointly, have the ability to predict the patient status , we applied logistic regression model. Univariate analysis was applied by only using one of the candidate autoantibody in the logistic model. Multivariable analysis was carried out using backward selection as well as weighted compound score method. The patient classification prediction performance was assessed by a widely used measurement of diagnostic discrimination: receiver operating characteristic (ROC) curves with corresponding area under the curve (AUC). Bootstrapping techniques were used to validate the models and to obtain a bias-corrected estimate of the model’s diagnostic accuracy. In addition, we tested the secondary hypothesis that these 6 scFvs are potential prognostic biomarkers that can predict patients’ survival by applying Cox proportional hazards models. Kaplan-Meier method was also applied to estimate the overall survival time. The results are shown in the results and discussion section.