Fertility in mammals requires appropriate communication within the hypothalamic-pituitary-gonadal axis and the GnRH receptor (GnRHR) is a central conduit for this communication. The GnRHR resides in ...discrete membrane rafts and raft occupancy is required for signaling by GnRH. The present studies use immunoprecipitation and mass spectrometry to define peptides present within the raft associated with the GnRHR and flotillin-1, a key raft marker. These studies revealed peptides from the F0F1 ATP synthase complex. The catalytic subunits of the F1 domain were validated by immunoprecipitation, flow cytometry, and cell surface biotinylation studies demonstrating that this complex was present at the plasma membrane associated with the GnRHR. The F1 catalytic domain faces the extracellular space and catalyzes ATP synthesis when presented with ADP in normal mouse pituitary explants and a gonadotrope cell line. Steady-state extracellular ATP accumulation was blunted by coadministration of inhibitory factor 1, limiting inorganic phosphate in the media, and by chronic stimulation of the GnRHR. Steady-state extracellular ATP accumulation was enhanced by pharmacological inhibition of ecto-nucleoside triphosphate diphosphohydrolases. Kisspeptin administration induced coincident GnRH and ATP release from the median eminence into the hypophyseal-portal vasculature in ovariectomized sheep. Elevated levels of extracellular ATP augmented GnRH-induced secretion of LH from pituitary cells in primary culture, which was blocked in media containing low inorganic phosphate supporting the importance of extracellular ATP levels to gonadotrope cell function. These studies indicate that gonadotropes have intrinsic ability to metabolize ATP in the extracellular space and extracellular ATP may serve as a modulator of GnRH-induced LH secretion.
•We introduce an “on-site laboratory”, the Taboga Field Laboratory, established outside the Taboga Forest Reserve in Costa Rica.•This manuscript is the first collaboration across three long-term ...field sites on the behavior and biology of wild white-faced capuchins (Cebus imitator).•We validate six steroid hormone assays for use on wild capuchin fecal samples (glucocorticoids, androgens, estrogens, and progestogens).
Hormone laboratories located “on-site” where field studies are being conducted have a number of advantages. On-site laboratories allow hormone analyses to proceed in near-real-time, minimize logistics of sample permits/shipping, contribute to in-country capacity-building, and (our focus here) facilitate cross-site collaboration through shared methods and a shared laboratory. Here we provide proof-of-concept that an on-site hormone laboratory (the Taboga Field Laboratory, located in the Taboga Forest Reserve, Costa Rica) can successfully run endocrine analyses in a remote location. Using fecal samples from wild white-faced capuchins (Cebus imitator) from three Costa Rican forests, we validate the extraction and analysis of four steroid hormones (glucocorticoids, testosterone, estradiol, progesterone) across six assays (DetectX® and ISWE, all from Arbor Assays). Additionally, as the first collaboration across three long-term, wild capuchin field sites (Lomas Barbudal, Santa Rosa, Taboga) involving local Costa Rican collaborators, this laboratory can serve as a future hub for collaborative exchange.
The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical ...relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation(1-16). The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and Müller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates (12,14-18). While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells (7,19-23). For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues (8,19-22,24-33). Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level (5,8,21,24,27-30,33-39). Xenopus laevis, a classic model system for the study of early neural development (19,27,29,31-32,40-42), serves as a particularly suitable system for retinal primary cell culture (10,38,43-45). Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction (25,38,43). In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products (10,24,44-45). However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).
Failure to recognize symptoms which signal cancer may delay contact with the medical care system, thus decreasing the chances of diagnosis at an early stage of disease. We investigated the ...determinants of cancer symptom recognition and delay in seeking medical care in a population-based sample of 625 newly diagnosed lung, breast and colorectal cancer patients. Although the majority (79.5%) of patients reported noticing symptoms prior to diagnosis, one quarter of these patients (24.7%) delayed longer than three months in seeking medical care. Contrary to the findings of research based on clinic samples, logistic regression analysis revealed that no demographic or social support factors were predictive of symptom recognition or delay, with the exception that older colorectal cancer patients were less likely to notice symptoms, but also less likely to delay. Lung and colorectal patients diagnosed with advanced disease were more likely to notice symptoms than patients with local disease. Results of a content analysis of patients' remarks indicate that breast cancer patients were significantly more likely than lung or colorectal cancer patients to attribute their symptoms to cancer (p < .001). Symptoms common to lung and colorectal cancer appear to be attributed to other, less serious causes. Given the lack of demographic predictors of symptom recognition and delay in seeking care, we suggest that education programs address risk groups for specific cancers, rather than the general public as a whole, grouping together all cancers and cancer symptomatology.
The Centers for Disease Control and Prevention (CDC) Quarantine Stations distribute select lifesaving drug products that are not commercially available or are in limited supply in the United States ...for emergency treatment of certain health conditions. Following a retrospective analysis of shipment records, the authors estimated an average of 6.66 hours saved per shipment when drug products were distributed from quarantine stations compared to a hypothetical centralized site from CDC headquarters in Atlanta, GA. This evaluation supports the continued use of a decentralized model which leverages CDC's regional presence and maximizes efficiency in the distribution of lifesaving drugs.
The Centers for Disease Control and Prevention (CDC) Quarantine Stations distribute select lifesaving drug products that are not commercially available or are in limited supply in the United States ...for emergency treatment of certain health conditions. Following a retrospective analysis of shipment records, the authors estimated an average of 6.66 hours saved per shipment when drug products were distributed from quarantine stations compared to a hypothetical centralized site from CDC headquarters in Atlanta, GA. This evaluation supports the continued use of a decentralized model which leverages CDC's regional presence and maximizes efficiency in the distribution of lifesaving drugs.
Summary Since first characterized in 1997, patients with hereditary mixed polyposis syndrome (HMPS) have been difficult to identify because of lack of well-established diagnostic criteria. Recently, ...HMPS was found to be caused by a duplication on chromosome 15 spanning the 3′ end of the SCG5 gene and a region upstream of the GREM1 locus. Clinical testing for the duplication is available; however, the clinical characteristics of hereditary mixed polyposis to support testing are ill defined. The clinicopathological findings of 10 HMPS patients with confirmed germline SCG5-GREM1 duplication were reviewed. Mean age at presentation was 33.3 years. Fifty-one colonoscopies yielded 207 polyp specimens, all of which were reexamined. Adenomas (n = 80) and a fairly unique polyp composed of a mixture of hyperplastic polyp and inflammatory polyp–type changes (n = 74) were the most common findings; however, other polyps, including hyperplastic (n = 28), mixed inflammatory polyp/adenoma (n = 8), inflammatory polyp (n = 7), prolapse-type polyp (n = 6), and lymphoid aggregates (n = 4), were encountered. None of the patients developed colorectal malignancy during surveillance, demonstrated extracolonic manifestations, or underwent colectomy on follow-up (mean, 26.2 years). SCG5-GREM1 duplication–associated polyposis is characterized by a few polyps per endoscopy with a mixture of phenotypes, most commonly adenoma and nondysplastic mixed hyperplastic/inflammatory polyps. Nine of 10 patients had at least 1 mixed hyperplastic-inflammatory polyp, which is the characteristic lesion of SCG5-GREM1 duplication–associated HMPS.
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