Assessing motor-related phenotypes of Caenorhabditis elegans with the wide field-of-view nematode tracking platform

Caenorhabditis elegans is a valuable model organism in biomedical research that has led to major discoveries in the fields of neurodegeneration, cancer and aging. Because movement phenotypes are commonly used and represent strong indicators of C. elegans fitness, there is an increasing need to replace manual assessments of worm motility with automated measurements to increase throughput and minimize observer biases. Here, we provide a protocol for the implementation of the improved wide field-of-view nematode tracking platform (WF-NTP), which enables the simultaneous analysis of hundreds of worms with respect to multiple behavioral parameters. The protocol takes only a few hours to complete, excluding the time spent culturing C. elegans, and includes (i) experimental design and preparation of samples, (ii) data recording, (iii) software management with appropriate parameter choices and (iv) post-experimental data analysis. We compare the WF-NTP with other existing worm trackers, including those having high spatial resolution. The main benefits of WF-NTP relate to the high number of worms that can be assessed at the same time on a whole-plate basis and the number of phenotypes that can be screened for simultaneously

Non-Overlapping Functions for Pyk2 and FAK in Osteoblasts during Fluid Shear Stress-Induced Mechanotransduction

Mechanotransduction, the process by which cells convert external mechanical stimuli such as fluid shear stress (FSS) into biochemical changes, plays a critical role in maintenance of the skeleton. We have proposed that mechanical stimulation by FSS across the surfaces of bone cells results in formation of unique signaling complexes called mechanosomes that are launched from sites of adhesion with the extracellular matrix and with other bone cells [1]. Deformation of adhesion complexes at the cell membrane ultimately results in alteration of target gene expression. Recently, we reported that focal adhesion kinase (FAK) functions as a part of a mechanosome complex that is required for FSS-induced mechanotransduction in bone cells. This study extends this work to examine the role of a second member of the FAK family of non-receptor protein tyrosine kinases, proline-rich tyrosine kinase 2 (Pyk2), and determine its role during osteoblast mechanotransduction. We use osteoblasts harvested from mice as our model system in this study and compared the contributions of Pyk2 and FAK during FSS induced mechanotransduction in osteoblasts. We exposed Pyk2+/+ and Pyk2−/− primary calvarial osteoblasts to short period of oscillatory fluid flow and analyzed downstream activation of ERK1/2, and expression of c-fos, cyclooxygenase-2 and osteopontin. Unlike FAK, Pyk2 was not required for fluid flow-induced mechanotransduction as there was no significant difference in the response of Pyk2+/+ and Pyk2−/− osteoblasts to short periods of fluid flow (FF). In contrast, and as predicted, FAK−/− osteoblasts were unable to respond to FF. These data indicate that FAK and Pyk2 have distinct, non-redundant functions in launching mechanical signals during osteoblast mechanotransduction. Additionally, we compared two methods of generating FF in both cell types, oscillatory pump method and another orbital platform method. We determined that both methods of generating FF induced similar responses in both primary calvarial osteoblasts and immortalized calvarial osteoblasts

The disruption of proteostasis in neurodegenerative diseases

Cells count on surveillance systems to monitor and protect the cellular proteome which, besides being highly heterogeneous, is constantly being challenged by intrinsic and environmental factors. In this context, the proteostasis network (PN) is essential to achieve a stable and functional proteome. Disruption of the PN is associated with aging and can lead to and/or potentiate the occurrence of many neurodegenerative diseases (ND). This not only emphasizes the importance of the PN in health span and aging but also how its modulation can be a potential target for intervention and treatment of human diseases.info:eu-repo/semantics/publishedVersio

Enhanced Longevity by Ibuprofen, Conserved in Multiple Species, Occurs in Yeast through Inhibition of Tryptophan Import

The common non-steroidal anti-inflammatory drug ibuprofen has been associated with a reduced risk of some age-related pathologies. However, a general pro-longevity role for ibuprofen and its mechanistic basis remains unclear. Here we show that ibuprofen increased the lifespan of Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster, indicative of conserved eukaryotic longevity effects. Studies in yeast indicate that ibuprofen destabilizes the Tat2p permease and inhibits tryptophan uptake. Loss of Tat2p increased replicative lifespan (RLS), but ibuprofen did not increase RLS when Tat2p was stabilized or in an already long-lived strain background impaired for aromatic amino acid uptake. Concomitant with lifespan extension, ibuprofen moderately reduced cell size at birth, leading to a delay in the G1 phase of the cell cycle. Similar changes in cell cycle progression were evident in a large dataset of replicatively long-lived yeast deletion strains. These results point to fundamental cell cycle signatures linked with longevity, implicate aromatic amino acid import in aging and identify a largely safe drug that extends lifespan across different kingdoms of life.The open access fee for this work was funded through the Texas A&M University Open Access to Knowledge (OAK) Fund

C. elegans as a Model for Synucleinopathies and Other Neurodegenerative Diseases:Tools and Techniques

Caenorhabditis elegans is widely used to investigate biological processes related to health and disease. Multiple C. elegans models for human neurodegenerative diseases do exist, including those expressing human α-synuclein. Even though these models do not feature all pathological and molecular hallmarks of the disease they mimic, they allow for the identification and dissection of molecular pathways that are involved. In line with this, genetic screens have yielded multiple modifiers of proteotoxicity in C. elegans models for neurodegenerative diseases. Here, we describe a set of common screening approaches and tools that can be used to study synucleinopathies and other neurodegenerative diseases in C. elegans. RNA interference and mutagenesis screens can be used to find genes that affect proteotoxicity, while relatively simple molecular, cellular (fractionation studies), metabolic (respiration studies), and behavioral (thrashing and crawling) readouts can be used to study the effects of disease proteins and modifiers more closely.</p

Assessing and Modulating Kynurenine Pathway Dynamics in Huntington's Disease: Focus on Kynurenine 3-Monooxygenase.

The link between disturbances in kynurenine pathway (KP) metabolism and Huntington's disease (HD) pathogenesis has been explored for a number of years. Several novel genetic and pharmacological tools have recently been developed to modulate key regulatory steps in the KP such as the reaction catalyzed by the enzyme kynurenine 3-monooxygenase (KMO). This insight has offered new options for exploring the mechanistic link between this metabolic pathway and HD, and provided novel opportunities for the development of candidate drug-like compounds. Here, we present an overview of the field, focusing on some novel approaches for interrogating the pathway experimentally

Tryptophan metabolism along the kynurenine and serotonin pathways reveals substantial differences in colon and rectal cancer

Introduction: Induction of tryptophan (TRP) catabolism is an adaptation mechanism to restrict excessive acute immune response in tissues. In the tumour microenvironment, TRP catabolism’s dysregulation plays an important role in local antitumour immune response suppression. Aim: We investigated changes in the plasma concentrations of TRP and its metabolites in a cohort of colorectal cancer (CRC) patients at different tumour stages and in subjects at risk of developing CRC. TRP metabolites were assessed along kynurenine and serotonin pathways, and the activity of involved enzymes and their tissue expression were monitored. Method: Plasmatic levels of tryptophan metabolites were quantified in 80 patients’ plasma samples by means of High-Pressure Liquid Chromatography coupled to UltraViolet/Fluorescence Detectors (HPLC-UV/FD), after a simple dilution step. Tissue IDO1 gene expression during to the adenoma-carcinoma sequence and samples were obtained from formalin-fixed and paraffin-embedded (FFPE) normal colon and tumour tissues from a subset of patients (n = 21). Results: Altered TRP concentrations were detected in plasma samples concomitant to pre-cancerous lesion and persisted during the adenoma-carcinoma transition. Moreover, the anatomical site of cancer lesions (colon or rectum) strongly influences the TRP metabolic profiles. Colon cancer patients exhibited increased TRP catabolism with respect to those affected by rectal cancer, suggesting that TRP’s metabolism alterations play an important role in the onset and progression of colon cancer, but not in those of rectal cancer

Molecular assembly of the aerolysin pore reveals a swirling membrane-insertion mechanism

Aerolysin is the founding member of a superfamily of β-pore–forming toxins whose pore structure is unknown. We have combined X-ray crystallography, cryo-EM, molecular dynamics and computational modeling to determine the structures of aerolysin mutants in their monomeric and heptameric forms, trapped at various stages of the pore formation process. A dynamic modeling approach based on swarm intelligence was applied, whereby the intrinsic flexibility of aerolysin extracted from new X-ray structures was used to fully exploit the cryo-EM spatial restraints. Using this integrated strategy, we obtained a radically new arrangement of the prepore conformation and a near-atomistic structure of the aerolysin pore, which is fully consistent with all of the biochemical data available so far. Upon transition from the prepore to pore, the aerolysin heptamer shows a unique concerted swirling movement, accompanied by a vertical collapse of the complex, ultimately leading to the insertion of a transmembrane β-barrel

The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus

We discovered that the hepatitis C virus (HCV) envelope glycoprotein E2 binds to human hepatoma cell lines independently of the previously proposed HCV receptor CD81. Comparative binding studies using recombinant E2 from the most prevalent 1a and 1b genotypes revealed that E2 recognition by hepatoma cells is independent from the viral isolate, while E2–CD81 interaction is isolate specific. Binding of soluble E2 to human hepatoma cells was impaired by deletion of the hypervariable region 1 (HVR1), but the wild-type phenotype was recovered by introducing a compensatory mutation reported previously to rescue infectivity of an HVR1-deleted HCV infectious clone. We have identified the receptor responsible for E2 binding to human hepatic cells as the human scavenger receptor class B type I (SR-BI). E2–SR-BI interaction is very selective since neither mouse SR-BI nor the closely related human scavenger receptor CD36, were able to bind E2. Finally, E2 recognition by SR-BI was competed out in an isolate-specific manner both on the hepatoma cell line and on the human SR-BI-transfected cell line by an anti-HVR1 monoclonal antibody