Seven-colour multiplex immunochemistry/immunofluorescence and whole slide imaging of frozen sections.

Multiplex Immunochemistry/Immunofluorescence (mIHC/IF) aims to visualise multiple biomarkers in a single tissue section and is especially powerful when used on slide scanners coupled with digital analysis tools. mIHC/IF is commonly employed in immuno-oncology to characterise features of the tumour microenvironment (TME) and correlate them with clinical parameters to guide prognostication and therapy. However, mIHC/IF can be applied to a wide range of organisms in any physiological or disease context. Recent innovation has extended the number of markers that can be detected using slide scanners well beyond the 3-4 markers typically reported in traditional fluorescence microscopy. However, these methods often require sequential antibody staining and stripping, and are not compatible with frozen tissue sections. Using fluorophore-conjugated antibodies, we have established a simple mIHC/IF imaging workflow that enables simultaneous staining and detection of seven markers in a single section of frozen tissue. Coupled with automated whole slide imaging and digital quantification, our data efficiently revealed the tumour-immune complexity in metastatic melanoma. Computational image analysis quantified the immune and stromal cell populations present in the TME as well as their spatial interactions. This imaging workflow can also be performed with an indirect labelling panel consisting of primary and secondary antibodies. Our new methods, combined with digital quantification, will provide a valuable tool for high-quality mIHC/IF assays in immuno-oncology research and other translational studies, especially in circumstances where frozen sections are required for detection of particular markers, or for applications where frozen sections may be preferred, such as spatial transcriptomics.

Spacesuit Center of Gravity Assessments for Partial Gravity EVA Simulation in an Underwater Environment.

OBJECTIVE: The objective is to analytically determine the expected CG and build hardware to measure and verify the suited subject's CG for lunar extravehicular activity (EVA) training in an underwater environment. BACKGROUND: For lunar EVAs, it is necessary for astronauts to train with a spacesuit in a simulated partial gravity environment. NASA's Neutral Buoyancy Laboratory (NBL) can provide these conditions by producing negative buoyancy for a submerged suited subject. However, it is critical that the center of gravity (CG) for the human-spacesuit system to be accurate for conditions expected during planetary EVAs. METHODS: An underwater force-transducer system and individualized human-spacesuit model was created to provide real-time measurement of CG, including recommendations for weight placement locations and quantity of weight needed on the spacesuit to achieve a realistic lunar spacesuit CG. This method was tested with four suited subjects. RESULTS: Across tested weighout configurations, it was observed that an aft and high CG location will have large postural differences when compared to low and fore CG locations, highlighting the importance of having a proper CG. The system had an accuracy of ±5lbs of the total lunar weight and within ± 15 cm for fore-aft and left-right CG directions of the model predictions. CONCLUSION: The developed method offers analytical verification of the suited subject's CG and improves simulation quality of lunar EVAs. Future suit design can also benefit by recommending hardware changes to create ideal CG locations that improve balance and mobility. APPLICATION: The developed methodology can be used to verify a proper CG location in future planetary EVA simulations such as different reduced gravity training analogs (e.g. active cable offloading systems).

Evidence That GRIN2A Mutations in Melanoma Correlate with Decreased Survival.

Previous whole-exome sequencing has demonstrated that melanoma tumors harbor mutations in the GRIN2A gene. GRIN2A encodes the regulatory GluN2A subunit of the glutamate-gated N-methyl-d-aspartate receptor (NMDAR), involvement of which in melanoma remains undefined. Here, we sequenced coding exons of GRIN2A in 19 low-passage melanoma cell lines derived from patients with metastatic melanoma. Potential mutation impact was evaluated in silico, including within the GluN2A crystal structure, and clinical correlations were sought. We found that of 19 metastatic melanoma tumors, four (21%) carried five missense mutations in the evolutionarily conserved domains of GRIN2A; two were previously reported. Melanoma cells that carried these mutations were treatment-naïve. Sorting intolerant from tolerant analysis predicted that S349F, G762E, and P1132L would disrupt protein function. When modeled into the crystal structure of GluN2A, G762E was seen to potentially alter GluN1-GluN2A interactions and ligand binding, implying disruption to NMDAR functionality. Patients whose tumors carried non-synonymous GRIN2A mutations had faster disease progression and shorter overall survival (P < 0.05). This was in contrast to the BRAF V600E mutation, found in 58% of tumors but showing no correlation with clinical outcome (P = 0.963). Although numbers of patients in this study are small, and firm conclusions about the association between GRIN2A mutations and poor clinical outcome cannot be drawn, our results highlight the high prevalence of GRIN2A mutations in metastatic melanoma and suggest for the first time that mutated NMDARs impact melanoma progression.

Conservation partnerships between Zoos and Aquariums, federal and state agencies, and nongovernmental organizations.

Partnerships are essential for the success of conservation organizations as they strive to achieve the ultimate goal of restoring and preserving biodiversity. Now is a particularly crucial time to develop partnerships owing to increasing financial constraints on all organizations and the urgent need for species recovery and habitat preservation. This study identified characteristics of successful conservation partnerships between Association of Zoos and Aquariums (AZA) accredited institutions and related facilities, US and international governmental agencies, and nongovernmental organizations. One hundred and five AZA accredited zoos and aquariums or related facilities participated in the preliminary survey. Staff at 75 of those zoos and aquariums were interviewed by telephone for a follow-up survey. Respondents were asked which characteristics most contributed to the success of their past and current conservation partnerships. Data were analyzed in two ways: descriptive statistics and principal component analysis. Descriptive statistics showed that effective leadership, clear and consistent communication, and trust between partners were the top three characteristics that led to partnership success. Ineffective leadership by those in charge, lack of clear, consistent communication between partners, and unreliable or insufficient sources of funding were the top three characteristics that inhibited partnership success. Using principal component analysis, the variables for each question on the questionnaire were reduced to a smaller subset of categories. Structure, personalities, process, and commitment were the four principal components of successful conservation partnerships. The three principal components that inhibited conservation partnerships were: communication, partnership personnel, and partner inequality. Results gained from this research are sure to increase the probability of success both for conservation partnerships that have already been established and for those that may develop in the future. Zoo Biol 26:471-486, 2007. (c) 2007 Wiley-Liss, Inc.

Estimation of radiation-induced interphase cell death in cultures of human tumor material and in cell lines.

A short-term assay method able to estimate the radiation response of human cancer tissue samples would be of great advantage to the individualization of radiotherapy in cancer patients. However, the effect of radiation on [3H]thymidine incorporation by proliferating cells reflects a composite of cell cycle arrest and induced cell death pathways. Here we consider whether it is feasible to correct for cell cycle effects based on comparison of the effects of radiation and the mitotic inhibitor paclitaxel on [3H]thymidine incorporation. Sixty-two short-term (7-day) cultures of human tumor tissue from 61 patients with melanoma, gynecological cancer, brain cancer, and head and neck cancer, as well as 18 5-day cultures of low passage human tumor cell lines, were irradiated at doses from 2 to 9 Gy, or exposed to paclitaxel (200 nM). [3H]Thymidine incorporation was measured at the end of the incubation. Cell cycle times could be estimated from the paclitaxel data and were 2.7 to 18.6 days for melanomas, 2.5 to >40 days for carcinomas, 3.9 to 39 days for brain tumors, and 1.1 to 3.8 days for cell lines. The effects of radiation on [3H]thymidine incorporation varied widely (0-97% and 0-99% inhibition for 2 and 9 Gy, respectively), and in 23 of the clinical samples, but in none of the cell lines, radiation caused significantly greater inhibition of [3H]thymidine incorporation than paclitaxel (p < 0.05). We argue that that these differences reflect radiation-induced cell loss from G1 phase and/or S phase. Responses of short-term cultures of clinical tumor material to radiation, with appropriate correction for cell cycle effects, might have the potential to provide information on radiation-induced cell death in individual patients.

Devemos adicionar fluoretos as aguas de abastecimento publico?

The Scientific Monthly (4), October 1954 (English)