Analyzing Collagen Alpha 1(XI) Using a Zebrafish Model System

Zebrafish (Danio rerio) are progressively becoming more popular as a model organism for research. The use of zebrafish is advantageous for several reasons. Zebrafish exhibit high fecundity, and are easy to maintain in large numbers. The embryos produced are transparent, and have fewer cells than many other vertebrates. The embryos develop from a single cell to a small fish within 24 hours, which allows the research process to advance quickly. Also, the existence of the embryo outside of the mother allows for direct access. Using zebrafish as a model organism, the aim of this project is to determine the function of Collagen a1(XI) during early development. To begin with, it is imperative that the zebrafish are kept healthy. Hence, we control the water quality and nutrition of the zebrafish. The healthy zebrafish are then able to breed. The embryos are collected and their RNA is extracted for RT-PCR. Then anti-sense ribo-probes are synthesized for in situ hybridization to determine when Collagen a1(XI) is being expressed during embryonic development. To study the function of Collagen a(XI), we microinject anti-sense mopholinos into 1-2 cell stage embryos to diminish its expression. Combining these techniques, we are able to determine the function of Collagen a1(XI) by comparing the phenotypes of the morphants to those of controls

Cell Adherence and Its Effect on Collagen Expression in Immortalized Rat Chondrocytes (IRC) in Culture

Chondrocytes are cells found in cartilage, and are responsible for the production and secretion of all of the molecules of the extracellular matrix (ECM). Chondrocytes do not survive in the laboratory unless they have been modified in a way that “immortalizes” them. Immortalized chondrocytes serve as a valuable model system in which biomedical research can be carried out without relying on live animal models. Here, we present information on the characterization of the immortalized rat chondrocyte cell line with respect to extracellular matrix production, proliferation rate, optimal culture conditions, the prevelance of apoptotic events, and calcium homeostasis. This work is significant because it will allow us to use this cell line for future studies into the cellular and molecular mechanisms of stress-response of chondrocytes during skeletal development, cancer progression, osteoarthritis, tissue engineering and tissue regeneration. Such studies will depend upon the application of cytochemistry and immunocytochemistry, which are laboratory technique widely used to detect small molecules and proteins within a cultured cell. Antibodies are used to detect the specific proteins via antigen- antibody affinity complex. Fluorescent molecules can be used to visualize the proteins under a microscope. Studies have shown that stress factors on the endoplasmic reticulum such as nutrient depletion, mechanical stress or oxidative stress lead to a decrease in protein expression. Depending on the stress inducer, the cells undergo apoptosis in certain cases. We would like to test how calcium homeostasis is altered and further, how the alteration of calcium homeostasis in chondrocytes affects the expression of collagen II

The folic acid metabolism gene mel-32/Shmt is required for normal cell cycle lengths in Caenorhabditis elegans

Neural tube defects are common and serious birth defects in which the brain and/or spinal cord are exposed outside the body. Supplementation of foods with folic acid, an essential vitamin, is linked to a lower risk of neural tube defects; however, the mechanisms by which folic acid influence neural tube defect risk are unclear. Our research seeks to identify the basic cellular roles of known folic acid metabolism genes during morphogenesis using the roundworm Caenorhabditis elegans (C. elegans) as a simple model system. Here, we used live imaging to characterize defects in embryonic development when mel-32 is depleted. mel-32 is an essential folic acid metabolism gene in C. elegans and a homolog to the mammalian enzyme serine hydroxymethyltransferase (Shmt). Disruption of mel-32 resulted in a doubling or tripling of cell cycle lengths and a lack of directed cell movement during embryogenesis. However, the order of cell divisions, as determined by lineage analysis, is unchanged compared to wild type embryos. These results suggest that mel32/Shmt is required for normal cell cycle lengths in C. elegans

The Expression Patterns of Minor Fibrillar Collagens During Development in Zebrafish

Minor fibrillar collagens are recognized as the organizers and nucleators during collagen fibrillogenesis but likely serve additional functions. The minor fibrillar collagens include collagens type V and type XI. Mutations of collagen type V and XI can cause Ehlers Danlos, Stickler\u27s, and Marshall\u27s syndromes in human. We have characterized the spatiotemporal expression patterns of Col11a1, Col11a2, Col5a1 as well as Col5a3 in zebrafish embryos by in situ hybridization. Col5a1 is expressed in developing somites, neural crest, the head mesenchyme, developing cranial cartilage, pharyngeal arches and vertebrae. Col5a3 is detected in the notochord, mesenchyme cells in the eyes and lens. Both Col11a1 and Col11a2 have similar expression patterns, including notochord, otic vesicle, and developing cranial cartilages. Zebrafish may therefore serve as a valuable vertebrate model system for the study of diseases associated with collagens type V and XI mutations

Gonadal Development and Sexual Dimorphism of Gobiomorus dormitor from the Estuarine System of Tecolutla, Veracruz, Mexico

The bigmouth sleeper, Gobiomorus dormitor, is a benthic, euryhaline species, and is very abundant in river mouths, coastal lagoons, and sites away from marine influence from south Florida to Dutch Guyana. There are few studies of its life history, ecology, and abundance, particularly within Mexican waters. Nine trips to Tecolutla estuary, Veracruz, Mexico, were taken between October 1995 and May 1998 to estimate the gonadal development and sexual dimorphism of G. dormitor. A total of 94 individuals ranging from 15–260 mm SL and 0.05–181 g were captured. Seventy-two specimens were adults (60 females, 12 males) and 22 were juveniles that did not show external sexual dimorphism. Both juvenile and adult stages of G. dormitor were captured year-round in seagrass beds and adjacent shallow, muddy or sandy areas. This study has shown that G. dormitor are resident and undergo sexual maturation in the Tecolutla estuary. Histological evidence suggests both males and females undergo gonadal recrudescence in the estuary and have an extended reproductive season from May through November. However, it is unclear if the species actually spawns in the estuary, since females in the final stages of oocyte maturation were not captured. Additional research on the reproductive biology and ecology of this under-studied species is necessary to determine its role in tropical estuaries in the southern Gulf of Mexico. Information learned from areas in the center of its distribution may aid in conserving the species at the periphery of its range in Florida, where it is considered threatened

Depletion of Beclin-1 Due to Proteolytic Cleavage by Caspases in the Alzheimer\u27s Disease Brain

The Beclin-1 protein is essential for the initiation of autophagy and recent studies suggest this function may be compromised in Alzheimer’s disease (AD). In addition, in vitro studies have supported a loss of function of Beclin-1 due to proteolytic modification by caspases. In the present study we examined whether caspase-cleavage of Beclin-1 occurs in the AD brain by designing a site-directed caspase-cleavage antibody based upon a known cleavage site within the protein at position D149. We confirmed that Beclin-1 is an excellent substrate for caspase-3 and demonstrate cleavage led to the formation of a 35 kDa C-terminal fragment labeled by our novel antibody following Western blot analysis. Application of this antibody termed Beclin-1 caspase-cleavage product antibody or BeclinCCP in frontal cortex tissue sections revealed strong immunolabeling within astrocytes that localized with plaque-regions and along blood vessels in all AD cases examined. In addition, weaker, more variable BeclinCCP labeling was also observed within neurofibrillary tangles that co-localized with the early tau conformational marker, MC-1 as well as the late tangle marker, PHF-1. Collectively, these data support a depletion of Beclin-1 in AD following caspase-cleavage

Mind your Ps: A probabilistic model to aid the interpretation of molecular epidemiology data

Background: Assessing relatedness of pathogen sequences in clinical samples is a core goal in molecular epidemiology. Tools for Bayesian analysis of phylogeny, such as the BEAST software package, have been typically used in the analysis of sequence/time data in public health. However, they are computationally-, time-, and knowledge-intensive, demanding resources that many laboratories do not have available or cannot allocate frequently. Methods: To evaluate a faster and simpler alternative method to support the routine interpretation of sequence data for epidemiology, we obtained sequences for two regions in the measles virus genome, N-450 and MF-NCR, from patient samples of genotypes B3, D4 and D8 taken between 2011 and 2017 in the UK and Romania. A mathematical model incorporating time, possible shared ancestry and the Poisson distribution describing the number of expected substitutions at a given time point was developed to exclude epidemiological relatedness between pairs of sequences. The model was validated against the commonly used Bayesian phylogenetic method using an independent dataset collected in 2017–19. Findings: We demonstrate that our model, using time and sequence information to predict whether two samples may be related within a given time frame, minimises the risk of erroneous exclusion of relatedness. An easy-to-use implementation in the form of a guide and spreadsheet is provided for convenient application. Interpretation: The proposed model only requires a previously calculated substitution rate for the locus and pathogen of interest. It allows for an informed but quick decision on the likelihood of relatedness between two samples within a time frame, without the need for phylogenetic reconstruction, thus facilitating rapid epidemiological interpretation of sequence data.This work was funded by the United Kingdom Health Security Agency (UKHSA). The World Health Orga- nization European Regional Office funded Aurora Fernández-García and Mihaela Lazar training visits to UKHSAS

A Validated Software Application to Measure Fiber Organization in Soft Tissue

The mechanical behavior of soft connective tissue is governed by a dense network of fibrillar proteins in the extracellular matrix. Characterization of this fibrous network requires the accurate extraction of descriptive structural parameters from imaging data, including fiber dispersion and mean fiber orientation. Common methods to quantify fiber parameters include fast Fourier transforms (FFT) and structure tensors, however, information is limited on the accuracy of these methods. In this study, we compared these two methods using test images of fiber networks with varying topology. The FFT method with a band-pass filter was the most accurate, with an error of 0.71 ± 0.43 degrees in measuring mean fiber orientation and an error of 7.4 ± 3.0% in measuring fiber dispersion in the test images. The accuracy of the structure tensor method was approximately 4 times worse than the FFT bandpass method when measuring fiber dispersion. A free software application, FiberFit, was then developed that utilizes an FFT band-pass filter to fit fiber orientations to a semicircular von Mises distribution. FiberFit was used to measure collagen fibril organization in confocal images of bovine ligament at magnifications of 63x and 20x. Grayscale conversion prior to FFT analysis gave the most accurate results, with errors of 3.3 ± 3.1 degrees for mean fiber orientation and 13.3 ± 8.2% for fiber dispersion when measuring confocal images at 63x. By developing and validating a software application that facilitates the automated analysis of fiber organization, this study can help advance a mechanistic understanding of collagen networks and help clarify the mechanobiology of soft tissue remodeling and repair

Structural Differences Observed in Arboviruses of the Alphavirus and Flavivirus Genera

Arthropod borne viruses have developed a complex life cycle adapted to alternate between insect and vertebrate hosts. These arthropod-borne viruses belong mainly to the families Togaviridae, Flaviviridae, and Bunyaviridae. This group of viruses contains many pathogens that cause febrile, hemorrhagic, and encephalitic disease or arthritic symptoms which can be persistent. It has been appreciated for many years that these viruses were evolutionarily adapted to function in the highly divergent cellular environments of both insect and mammalian phyla. These viruses are hybrid in nature, containing viral-encoded RNA and proteins which are glycosylated by the host and encapsulate viral nucleocapsids in the context of a host-derived membrane. From a structural perspective, these virus particles are macromolecular machines adapted in design to assemble into a packaging and delivery system for the virus genome and, only when associated with the conditions appropriate for a productive infection, to disassemble and deliver the RNA cargo. It was initially assumed that the structures of the virus from both hosts were equivalent. New evidence that alphaviruses and flaviviruses can exist in more than one conformation postenvelopment will be discussed in this review. The data are limited but should refocus the field of structural biology on the metastable nature of these viruses