Understanding vertebrate embryonic development under conditions present in outer space

The ability of humans to survive, thrive, and sustain life in outer space requires that human embryos develop normally under conditions such as microgravity. We are using the zebrafish model system due to the fact that they share similarities with humans during development. The larval zebrafish will be used to assess embryonic and neural development under simulated microgravity conditions, with specific focus on neural development which has not been well studied. Before performing these experiments, it is critical that histological procedures, including cryosectioning and immunohistochemistry, are up and running in our lab for stages of early zebrafish development. This includes immunohistochemistry for the neuronal marker HuC/D (commonly used to label neurons in zebrafish), and RNA-binding proteins Rbfox1l and Rbfox2 (label neuronal populations). Cryosectioning of 7 day post-fertilization (dpf) larval zebrafish was performed, followed by immunohistochemistry for HuC/D in conjunction with Rbfox labeling, and samples will be imaged on a compound fluorescent microscope to determine whether the protocol is working. Our work aims to better understand vertebrate embryonic development under conditions present in outer space

Understanding vertebrate embryonic development under conditions present in outer space

It is currently unknown whether humans can survive and thrive in outer space, which includes human embryonic development. In this study we investigate vertebrate embryonic development under conditions present in outer space including microgravity and an altered day-and-night cycle using zebrafish and chicken model systems. Zebrafish and chicken model systems are widely used in developmental biology research given their similarity as vertebrates to humans. We aim to analyze the development of brain, muscle and other tissues under conditions of microgravity in both zebrafish and chicken embryos. Additionally, we will analyze zebrafish development under an altered day-and-night cycle (16 sunrises and 16 sunsets per day), and determine whether adult zebrafish can survive and reproduce under these conditions. Zebrafish and chicken embryos will be placed onto a clinostat, which is a device used to simulate a microgravity environment. Embryos will be harvested between 2 and 14 days of incubation on the clinostat, and markers of cell proliferation, death, and differentiation will be analyzed on tissue sections of the brain, skeletal muscle, and other tissues. We expect that our results may allow us to better understand embryonic development under conditions present in outer space, which may shed light on this process in humans

Role of RNA-binding proteins Rbfox1l and Rbfox2 in neuronal development and behavior in zebrafish

Rbfox proteins are RNA-binding proteins that play a significant role in the alternative splicing of neuronal transcripts in the central nervous system (CNS). Rbfox proteins are required for proper brain development and function. In humans, RBFOX1 has been implicated in a variety of neurological disorders, including autism, anxiety, epilepsy, and schizophrenia. Rbfox2 is involved in cerebellar development in mammals. The zebrafish is used as a model system for studies in neurobiology given their neuroanatomical conservation with mammals, and remarkable capability to regenerate parts of their CNS. Rbfox1l (Rbfox1-like) and Rbfox2 have been identified in neurons of the adult zebrafish brain. Rbfox1l was found in a restricted population of dorsal telencephalic neurons, and Rbfox2 was found broadly throughout the brain. Both genes have been found in Purkinje cells of the cerebellum. Utilizing antibody staining on zebrafish brain tissue sections, we will analyze expression of Rbfox1l and Rbfox2 at larval stages and stages leading up to adulthood. Furthermore, we will use rbfox1l and rbfox2 mutant zebrafish (in collaboration with Ohio State University) to better understand the role of rbfox1l in behavior and determine whether rbfox2 is necessary for the regeneration of the cerebellum. Understanding the role of the Rbfox proteins in neural development, regeneration, and behavior may lead to a substantial advancement in the research field and health care

Role of RNA-binding proteins Rbfox1l and Rbfox2 in neuronal development and behavior in zebrafish

Rbfox proteins are RNA-binding proteins that play a significant role in the alternative splicing of neuronal transcripts in the central nervous system (CNS). Rbfox proteins are required for proper brain development and function. In humans, RBFOX1 has been implicated in a variety of neurological disorders, including autism, anxiety, epilepsy, and schizophrenia. Rbfox2 is involved in cerebellar development in mammals. The zebrafish is used as a model system for studies in neurobiology given their neuroanatomical conservation with mammals, and remarkable capability to regenerate parts of their CNS. Rbfox1l (Rbfox1-like) and Rbfox2 have been identified in neurons of the adult zebrafish brain. Rbfox1l was found in a restricted population of dorsal telencephalic neurons, and Rbfox2 was found broadly throughout the brain. Both genes have been found in Purkinje cells of the cerebellum. Utilizing antibody staining on zebrafish brain tissue sections, we will analyze expression of Rbfox1l and Rbfox2 at larval stages and stages leading up to adulthood. Furthermore, we will use rbfox1l and rbfox2 mutant zebrafish (in collaboration with Ohio State University) to better understand the role of rbfox1l in behavior and determine whether rbfox2 is necessary for the regeneration of the cerebellum. Understanding the role of the Rbfox proteins in neural development, regeneration, and behavior may lead to a substantial advancement in the research field and health care

Spatial and temporal evaluation of soil erosion in Turkey under climate change scenarios using the Pan-European Soil Erosion Risk Assessment (PESERA) model

The impacts of climate change on soil erosion are mainly caused by the changes in the amount and intensity of rainfall and rising temperature. The combination of rainfall and temperature change is likely to be accompanied by negative or positive variations in agricultural and forest management. Turkey contains vast fertile plains, high mountain chains and semi-arid lands, with a climate that ranges from marine to continental and therefore is susceptible to soil erosion under climate change, particularly on high gradients and in semi-arid areas. This study aims to model the soil erosion risk under climate change scenarios in Turkey using the Pan-European Soil Erosion Assessment (PESERA) model, predicting the likely effects of land use/cover and climate change on sediment transport and soil erosion in the country. For this purpose, PESERA was applied to estimate the monthly and annual soil loss for 12 land use/cover types in Turkey. The model inputs included 128 variables derived from soil, climate, land use/cover and topography data. The total soil loss from the land surface is speculated to be approximately 285.5 million tonnes per year. According to the IPCC 5th Assessment Report of four climate change scenarios, the total soil losses were predicted as 308.9, 323.5, 320.3 and 355.3 million tonnes for RCP2.6, RCP4.5, RCP6.0 and RCP8.5 scenarios respectively from 2060 to 2080. The predicted amounts of fertile soil loss from agricultural land in a year were predicted to be 55.5 million tonnes at present, and 62.7, 59.9, 61.7 and 58.1 under RCP2.6, RCP4.5, RCP6.0 and RCP8.5 respectively. This confirms that approximately 30% of the total erosion occurs over the agricultural lands. In this respect, degraded forests, scrub and arable lands were subjected to the highest erosion rate (68%) of the total, whereas, fruit trees and berry plantations reflected the lowest erosion rates. Low soil organic carbon, sparse vegetation cover and variable climatic conditions significantly enhanced the erosion of the cultivated lands by primarily removing the potential food for organisms. Finally, process-based models offer a valuable resource for decision-makers when improving environmental management schemes and also decrease uncertainty when considering risks

Next-generation sequencing reveals deep intronic cryptic ABCC8 and HADH splicing founder mutations causing hyperinsulinism by pseudoexon activation

Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc.Next-generation sequencing (NGS) enables analysis of the human genome on a scale previously unachievable by Sanger sequencing. Exome sequencing of the coding regions and conserved splice sites has been very successful in the identification of disease-causing mutations, and targeting of these regions has extended clinical diagnostic testing from analysis of fewer than ten genes per phenotype to more than 100. Noncoding mutations have been less extensively studied despite evidence from mRNA analysis for the existence of deep intronic mutations in >20 genes. We investigated individuals with hyperinsulinaemic hypoglycaemia and biochemical or genetic evidence to suggest noncoding mutations by using NGS to analyze the entire genomic regions of ABCC8 (117 kb) and HADH (94 kb) from overlapping ~10 kb PCR amplicons. Two deep intronic mutations, c.1333-1013A>G in ABCC8 and c.636+471G>T HADH, were identified. Both are predicted to create a cryptic splice donor site and an out-of-frame pseudoexon. Sequence analysis of mRNA from affected individuals' fibroblasts or lymphoblastoid cells confirmed mutant transcripts with pseudoexon inclusion and premature termination codons. Testing of additional individuals showed that these are founder mutations in the Irish and Turkish populations, accounting for 14% of focal hyperinsulinism cases and 32% of subjects with HADH mutations in our cohort. The identification of deep intronic mutations has previously focused on the detection of aberrant mRNA transcripts in a subset of disorders for which RNA is readily obtained from the target tissue or ectopically expressed at sufficient levels. Our approach of using NGS to analyze the entire genomic DNA sequence is applicable to any disease

A feature extraction software tool for agricultural object-based image analysis

A software application for automatic descriptive feature extraction from image-objects, FETEX 2.0, is presented and described in this paper. The input data include a multispectral high resolution digital image and a vector file in shapefile format containing the polygons or objects, usually extracted from a geospatial database. The design of the available descriptive features or attributes has been mainly focused on the description of agricultural parcels, providing a variety of information: spectral information from the different image bands; textural descriptors of the distribution of the intensity values based on the grey level co-occurrence matrix, the wavelet transform and a factor of edgeness; structural features describing the spatial arrangement of the elements inside the objects, based on the semivariogram curve and the Hough transform; and several descriptors of the object shape. The output file is a table that can be produced in four alternative formats, containing a vector of features for every object processed. This table of numeric values describing the objects from different points of view can be externally used as input data for any classification software. Additionally, several types of graphs and images describing the feature extraction procedure are produced, useful for interpretation and understanding the process. A test of the processing times is included, as well as an application of the program in a real parcel-based classification problem, providing some results and analyzing the applicability, the future improvement of the methodologies, and the use of additional types of data sets. This software is intended to be a dynamic tool, integrating further data and feature extraction algorithms for the progressive improvement of land use/land cover database classification and agricultural database updating processes. © 2011 Elsevier B.V.The authors appreciate the financial support provided by the Spanish Ministerio de Ciencia e Innovacion and the FEDER in the framework of the Project CGL2009-14220 and CGL2010-19591/BTE, the Spanish Institut Geografico Nacional (IGN), Institut Cartografico Valenciano (ICV), Institut Murciano de Investigacion y Desarrollo Agrario y Alimentario (IMIDA) and Banco de Terras de Galicia (Bantegal).Ruiz Fernández, LÁ.; Recio Recio, JA.; Fernández-Sarría, A.; Hermosilla, T. (2011). A feature extraction software tool for agricultural object-based image analysis. Computers and Electronics in Agriculture. 76(2):284-296. https://doi.org/10.1016/j.compag.2011.02.007S28429676

Quantitative Pathology: Historical Background, Clinical Research and Application of Nuclear Morphometry and DNA Image Cytometry

Quantitative analysis of histo- and cytochemical components such as DNA, RNA or chromatin pattern on one hand (cytometry) and the quantitative analysis of geometric non-chemical cell and tissue components (morphometry and sterology) on the other, have developed somewhat independently. Today, many different techniques, such as morphometry, sterology, and static image and flow cytometry are well established and routinely used in diagnostic quantitative pathology. The potential significance of these techniques in the individualization of care in cancer patients include the objective distinction between benign, borderline and malignant lesions, objective grading of invasive tumours, prediction of prognosis, and therapy response

One Step Nucleic Acid Amplification (OSNA) - a new method for lymph node staging in colorectal carcinomas

<p>Abstract</p> <p>Background</p> <p>Accurate histopathological evaluation of resected lymph nodes (LN) is essential for the reliable staging of colorectal carcinomas (CRC). With conventional sectioning and staining techniques usually only parts of the LN are examined which might lead to incorrect tumor staging. A molecular method called OSNA (One Step Nucleic Acid Amplification) may be suitable to determine the metastatic status of the complete LN and therefore improve staging.</p> <p>Methods</p> <p>OSNA is based on a short homogenisation step and subsequent automated amplification of cytokeratin 19 (CK19) mRNA directly from the sample lysate, with result available in 30-40 minutes. In this study 184 frozen LN from 184 patients with CRC were investigated by both OSNA and histology (Haematoxylin & Eosin staining and CK19 immunohistochemistry), with half of the LN used for each method. Samples with discordant results were further analysed by RT-PCR for CK19 and carcinoembryonic antigen (CEA).</p> <p>Results</p> <p>The concordance rate between histology and OSNA was 95.7%. Three LN were histology+/OSNA- and 5 LN histology-/OSNA+. RT-PCR supported the OSNA result in 3 discordant cases, suggesting that metastases were exclusively located in either the tissue analysed by OSNA or the tissue used for histology. If these samples were excluded the concordance was 97.2%, the sensitivity 94.9%, and the specificity 97.9%. Three patients (3%) staged as UICC I or II by routine histopathology were upstaged as LN positive by OSNA. One of these patients developed distant metastases (DMS) during follow up.</p> <p>Conclusion</p> <p>OSNA is a new and reliable method for molecular staging of lymphatic metastases in CRC and enables the examination of whole LN. It can be applied as a rapid diagnostic tool to estimate tumour involvement in LN during the staging of CRC.</p