Closing dissertation fieldwork: Ecuador 2014

The Applied Biodiversity Sciences Perspectives Series is a student-directed collection of contributions from graduate student and faculty members of the integrative, NSF-IGERT Applied Biodiversity Sciences (ABS) program at Texas A&M University. The ABS Perspectives Series aims to highlight the application and practice of conservation science reflected in the experiences of ABS Scholars from both the social and biological sciences.Our online publication focuses on sharing our experiences with a diverse readership to raise awareness of biodiversity conservation issues and current research being undertaken at Texas A&M University. A foundational component of the ABS Program is to communicate within, across, and outside of our scientific disciplines. The ABS Perspectives Series is intended to communicate to the general public, the communities where our research takes place, fellow academics and practitioners, and institutions that provide logistics, infrastructure, and support the who, what, where, how, and why of Applied Biodiversity Science.Applied Biodiversity Science Program - Texas A&M Universit

MicroRNA markers for forensic body fluid identification obtained from microarray screening and quantitative RT-PCR confirmation

MicroRNAs (miRNAs) are non-protein coding molecules with important regulatory functions; many have tissue-specific expression patterns. Their very small size in principle makes them less prone to degradation processes, unlike messenger RNAs (mRNAs), which were previously proposed as molecular tools for forensic body fluid identification. To identify suitable miRNA markers for forensic body fluid identification, we first screened total RNA samples derived from saliva, semen, vaginal secretion, and venous and menstrual blood for the expression of 718 human miRNAs using a microarray platform. All body fluids could be easily distinguished from each other on the basis of complete array-based miRNA expression profiles. Results from quantitative reverse transcription PCR (RT-PCR; TaqMan) assays for microarray candidate markers confirmed strong over-expression in the targeting body fluid of several miRNAs for venous blood and several others for semen. However, no candidate markers from array experiments for other body fluids such as saliva, vaginal secretion, or menstrual blood could be confirmed by RT-PCR. Time-wise degradation of venous blood and semen stains for at least 1 year under lab conditions did not significantly affect the detection sensitivity of the identified miRNA markers. The detection limit of the TaqMan assays tested for selected venous blood and semen miRNA markers required only subpicogram amounts of total RNA per single RT-PCR test, which is considerably less than usually needed for reliable mRNA RT-PCR detection. We therefore propose the application of several stable miRNA markers for the forensic identification of blood stains and several others for semen stain identification, using commercially available TaqMan assays. Additional work remains necessary in search for suitable miRNA markers for other forensically relevant body fluids

Acute effects of continuous nasogastric tube-feeding on gastric function - comparison of a polymeric and a nonpolymeric formula

The acute effects of continuous intragastric administration of 1500 ml (4200 kJ/liter) of a polymeric and of a nonpolymeric formula on gastric function were studied in 15 healthy subjects. During 450 min 1500 ml, containing 6300 kJ (1500 kcal), was given through a nasogastric tube. At regular intervals the volume, the pH, the titratable acidity, and the pepsin activity of the gastric contents and the plasma gastrin concentration were determined. Maximal observed intragastric volumes occurred after 120 min (118 +/- 16 ml during polymeric formula, 212 +/- 37 ml during nonpolymeric one) and volumes subsequently halved (at 450 min 68 +/- 13 and 104 +/- 16 ml, respectively). During the administration of both polymeric and the nonpolymeric formula intragastric pH fell progressively to 3.15 and 2.67, respectively, at 450 min. Incremental plasma gastrin values increased between 120 and 450 min from 7 to 12 ng/liter during the polymeric formula. During the nonpolymeric one it stabilized after 120 min at 12 ng/liter. When the whole test periods were considered integrated, mean intragastric volumes tended to be larger during the nonpolymeric formula (153 +/- 23 ml) than during the polymeric formula (107 +/- 12 ml), but this difference was not statistically significant. Median integrated mean pH was lower during the nonpolymeric formula (2.89) compared with the polymeric one (3.26). Despite the limitation that the investigations were performed in healthy subjects only, it is concluded from this study that the risk of aspiration during continuous nasogastric tube feeding is probably greatest during the first few hours of administration because of the larger intragastric volumes. The findings further suggest that the risk of aspiration may be less with the polymeric formula because of the smaller intragastric volumes observed. However, studies in ill patients are necessary to confirm the clinical validity of our observations

Increased intestinal permeability during cytomegalovirus infection in renal transplant recipients

Cytomegalovirus (CMV) infections in renal transplant recipients can affect the gastrointestinal trace, but significant clinical manifestations are seldom seen. We hypothesize that subclinical involvement of the gastrointestinal tract may be quite frequent during CMV infection. In order to study this, we measured intestinal permeability by calculating the urinary lactulose mannitol (LM) excretion ratio after oral administration of lactulose and mannitol (normal <0.030) in patients with symptomatic and asymptomatic CMV infection. A total of 111 patients were enrolled in the study, 104 of whom were tested on postoperative day (POD) 10. Twenty-nine patients developed CMV infection, 12 of whom could be studied with the permeability test (median POD 40). Another nine patients without CMV infection were also studied at day 40 and served as controls. The LM ratio increased significantly during CMV infection compared to measurements before active infection (median 0.060 vs. 0.030, P <0.01) and was significantly higher during the infection than in the control group (median 0.007, P <0.01). No correlation could be found between the LM ratio and viral load, humoral response to the virus, or symptomatology of infection. We conclude that an increased intestinal permeability is found in a substantial number of patients with an active, albeit asymptomatic, CMV infection after renal transplantation. Pathophysiological mechanisms and clinical implications remain speculative but will be subject to further study

Dysplasia in fundic gland polyps is associated with nuclear beta-catenin expression and relatively high cell turnover rates

Background: Fundic gland polyps (FGPs) occur in both syndromic and sporadic form. Syndromic FGPs arise through mutations in the adenomatous polyposis coli (APC) gene, whereas sporadic FGPs are caused by beta-catenin gene mutations. Dysplasia in sporadic FGPs, found less often than in syndromic FGPs, was recently associated with APC rather than beta-catenin mutations. These data suggest different functional consequences of APC and beta-catenin mutations. To investigate this hypothesis, we examined proliferative activity, degree of apoptosis, beta-catenin expression and p53 expression in syndromic and sporadic FGPs. Methods: Syndromic FGPs (n = 9) from familial adenomatous polyposis (FAP) patients and sporadic FGPs ( n = 18) were studied. Proliferative activity, apoptotic cell death and expression of beta-catenin and p53 were examined by immunohistochemistry. In FGPs containing dysplasia, areas with and without dysplasia were compared. Results: Syndromic and sporadic FGPs without dysplasia exhibited similar proliferative activity, degree of apoptosis, beta-catenin and p53 expression. Dysplasia was observed more often in syndromic (4/9) than in sporadic FGPs (1/18). Within FGPs containing dysplasia, dysplastic areas showed abnormal nuclear beta-catenin staining in 3/5 cases and higher rates of cell proliferation and apoptosis than non-dysplastic areas. Overexpression of p53 was not observed. Conclusion: The finding of similar rates of proliferation and apoptosis in syndromic and sporadic FGPs does not support the hypothesis that APC and beta-catenin gene mutations have different functional consequences in FGPs. The association of dysplasia with relatively high cell turnover rates and nuclear expression of beta-catenin indicates activation of the Wnt-APC-beta-catenin pathway in dysplasia. The finding of dysplasia in some but not all syndromic FGPs suggests the involvement of other genes in addition to the APC gene in the development of dysplasia in FGPs

Independent induction of caspase-8 and cFLIP expression during colorectal carcinogenesis in sporadic and HNPCC adenomas and carcinomas

Background: TNF-Related Apoptosis Inducing Ligand (TRAIL) is a promising agent for the induction of apoptosis in neoplastic tissues. Important determinants of TRAIL sensitivity are two intracellular proteins of the TRAIL pathway, caspase-8 and its anti-apoptotic competitor cellular Flice-Like Inhibitory Protein (cFLIP). Methods: The aim of this Study was to investigate basic expression of caspase-8 and cFLIP in normal colorectal epithelium (n = 20), colorectal adenomas (n = 66) and colorectal carcinomas (n = 44) using immunohistochemistry performed on both sporadic and Hereditary Non-Polyposis Colorectal Cancer (HNPCC or Lynch syndrome)-associated adenomas and carcinomas. Results: Expression of both caspase-8 and cFLIP was similar in cases with sporadic and hereditary origin. Expression of caspase-8 in colorectal adenomas and carcinomas was increased when compared to normal colon tissue (P = 0.02). Nuclear, paranuclear as well as cytoplasmic localizations of caspase-8 were detected. Immunohistochemistry revealed an upregulation of cFLIP in colorectal carcinomas in comparison to normal epithelium and colorectal adenomas (P <0.001). A large variation in the caspase-8/cFLIP ratio was observed between the individual adenomas and carcinomas. Conclusion: Caspase-8 and cFLIP are upregulated during colorectal carcinogenesis. Upregulation of caspase-8 and/or downregulation of cFLIP maybe interesting approaches to maximize TRAIL sensitivity in colorectal neoplasms

The extrinsic apoptosis pathway and its prognostic impact in ovarian cancer

OBJECTIVE: Death ligand FasL, its agonistic receptor Fas, tumor necrosis factor related apoptosis inducing ligand (TRAIL) and its agonistic death receptors DR4 and DR5 are implied in carcinogenesis, tumor immune surveillance and response to chemotherapy. TRAIL receptor agonists are evaluated as anti-cancer agents. This study aimed to relate expression of death ligands/receptors and downstream initiator caspase 8 and its anti-apoptotic homologue FLICE like inhibitory protein (c-FLIP) in ovarian cancers to chemotherapy response and survival. METHODS: Fas, FasL, TRAIL, DR4, DR5, caspase 8 and c-FLIP were determined immunohistochemically on a tissue microarray containing 382 ovarian cancers. Protein expression profiles were correlated with clinicopathologic variables, chemotherapy response and survival. RESULTS: Most tumors expressed DR4, DR5, caspase 8 and c-FLIP. High c-FLIP expression was associated with expression of caspase 8 and both TRAIL receptors. TRAIL and Fas were associated with low tumor grade and better progression-free survival (HR 0.63, p=.018 and HR 0.54, p=.012), respectively, and Fas with disease-specific survival (HR 0.49, p=0.009) in univariate analysis. CONCLUSIONS: Fas and TRAIL loss is associated with dedifferentiation and worse prognosis. Expression of DR4, DR5, caspase 8 and c-FLIP by most ovarian cancers does not correlate with survival. High c-FLIP expression should be taken into account for death receptor targeted therapies

Cancer Stem Cells, Epithelial to Mesenchymal Markers, and Circulating Tumor Cells in Small Cell Lung Cancer

Background Small cell lung cancer (SCLC) has a poor prognosis, and even with localized (limited) disease, the 5-year survival has only been around 20%. Elevated levels of circulating tumor cells (CTCs) have been associated with a worse prognosis, and markers of cancer stem cells (CSCs) and epithelial to mesenchymal transition have been associated with increased chemoresistance and metastatic spread in SCLC. Patients and Methods The biopsy specimens of 38 SCLC patients were used for marker evaluation by immunohistochemistry. The markers for CSCs were CD44 and SOX2. The markers for epithelial to mesenchymal transition were E-cadherin, epithelial cell adhesion molecule, cytokeratins 8, 18, and 19, vimentin, and c-MET. Staining was scored as low (weak) or high (strong) intensity for SOX2, epithelial cell adhesion molecule, cytokeratins 8, 18, and 19, and c-MET and using the immunoreactive score for CD44, E-cadherin, and vimentin, expressed as low or high expression. Results High expression of c-MET (c-METH) and low expression of E-cadherin (E-cadL) showed a trend toward a better prognosis (P = .07 and P = .09, respectively). The combination of c-METH and E-cadL resulted in significantly better survival (P = .007). The tested markers were not associated with CTCs, although a trend was seen for c-METHE-cadL (P = .09) with low CTCs. The CSC markers SOX2 and CD44 were not associated with overall survival in this patient cohort. Conclusion SCLC with a mesenchymal-like phenotype (c-METHE-cadL) is associated with longer survival and showed a trend toward lower CTCs

Regulation of TRAIL receptor expression by β-catenin in colorectal tumours

Expression of the pro-apoptotic TRAIL receptors is regulated, at least in part, by beta-catenin. We show that beta-catenin co-localizes with DR4/5 in human and mouse colorectal tumours and that downregulation of beta-catenin in cell line models reduces TRAIL receptor expression and TRAIL sensitivity.Tumour-necrosis-factor-related apoptosis-inducing ligand (TRAIL) is being investigated as a targeted cancer therapeutic and the expression of its pro-apoptotic receptors, DR4 and DR5, increases during colorectal carcinogenesis. This study investigated the role of beta-catenin in the regulation of these receptors. In human colorectal adenoma and carcinoma cell lines, downregulation of beta-catenin resulted in lower total DR4 and DR5 protein levels. Similarly, cell membrane expression of DR4 and DR5 was reduced after downregulation of beta-catenin in colon carcinoma cells, whereas induction of beta-catenin in HeLa cells led to increased cell membrane expression of DR4 and DR5. Downregulation of beta-catenin decreased the recombinant human TRAIL sensitivity of human colon carcinoma cells. Activation of the transcription factor T-cell factor-4 (TCF-4) is an important function of beta-catenin. Dominant-negative TCF-4 overexpression, however, did not significantly affect TRAIL receptor expression or recombinant human TRAIL sensitivity. Human colorectal adenomas (N = 158) with aberrant (cytoplasmic and nuclear) beta-catenin expression had a higher percentage of immunohistochemical DR4 and DR5 staining per tumour (mean: 73 and 88%, respectively) than those with membranous beta-catenin staining only (mean: 50 and 70%, respectively, P <0.01 for both). Furthermore, aberrant beta-catenin staining co-localized with DR4 and DR5 expression in 92% of adenomas. In 53 human colorectal carcinomas, aberrant beta-catenin expression was present in most cases and DR4/5 expression was largely homogenous. Similarly, in adenomas from APC(min) mice, cytoplasmic beta-catenin staining co-localized with staining for the murine TRAIL death receptor. In conclusion, the gradual increase in TRAIL receptor expression during colorectal carcinogenesis is at least partially mediated through increased beta-catenin expression, independently of TCF-4-signalling