scholarly journals A Novel Method Facilitating the Simple and Low-Cost Preparation of Human Osteochondral Slice Explants for Large-Scale Native Tissue Analysis

2021 ◽  
Vol 22 (12) ◽  
pp. 6394
Author(s):  
Jacob Spinnen ◽  
Lennard K. Shopperly ◽  
Carsten Rendenbach ◽  
Anja A. Kühl ◽  
Ufuk Sentürk ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Viability ◽  
Large Scale ◽  
Marker Gene ◽  
Cell Swelling ◽  
Tissue Cell ◽  
Joint Research ◽  
Sample Number ◽  
Tnf Α ◽  
Novel Method

For in vitro modeling of human joints, osteochondral explants represent an acceptable compromise between conventional cell culture and animal models. However, the scarcity of native human joint tissue poses a challenge for experiments requiring high numbers of samples and makes the method rather unsuitable for toxicity analyses and dosing studies. To scale their application, we developed a novel method that allows the preparation of up to 100 explant cultures from a single human sample with a simple setup. Explants were cultured for 21 days, stimulated with TNF-α or TGF-β3, and analyzed for cell viability, gene expression and histological changes. Tissue cell viability remained stable at >90% for three weeks. Proteoglycan levels and gene expression of COL2A1, ACAN and COMP were maintained for 14 days before decreasing. TNF-α and TGF-β3 caused dose-dependent changes in cartilage marker gene expression as early as 7 days. Histologically, cultures under TNF-α stimulation showed a 32% reduction in proteoglycans, detachment of collagen fibers and cell swelling after 7 days. In conclusion, thin osteochondral slice cultures behaved analogously to conventional punch explants despite cell stress exerted during fabrication. In pharmacological testing, both the shorter diffusion distance and the lack of need for serum in the culture suggest a positive effect on sensitivity. The ease of fabrication and the scalability of the sample number make this manufacturing method a promising platform for large-scale preclinical testing in joint research.

Cis/transgene optimization: systematic discovery of some key gene expression elements integrating bioinformatics and computational biology

2016 ◽  
Author(s):  
Saeid Kadkhodaei ◽  
Farahnaz S. Golestan Hashemi ◽  
Morvarid Akhavan Rezaei ◽  
Sahar Abbasiliasi ◽  
Tan Joo Shun ◽  
...  
Keyword(s):  
Gene Expression ◽  
Large Scale ◽  
Computational Analysis ◽  
Production Systems ◽  
Marker Gene ◽  
Optimization Procedure ◽  
Expression Vectors ◽  
Secretion Systems ◽  
Specific Expression ◽  
Gene Optimization

In recombinant protein production, quantity and quality are the major challenges particularly for large scale and high-throughput production systems. The present study mainly focused on computational analysis and in silico systematic discovery of some key functional gene expression elements in microalgae Dunaliella salina as a case study which there is no or poor information in this regard. Among the key factors, we took a shot at matrix attachment regions (MARs), translation initiation sites (TIS), signal peptide (SP) sequences, gene optimization and transformation system. Computational analysis of MARs sequences provided enough information about the structure of these sequences and led us to design an artificial MAR sequence considering the essential motifs and underlain rules. As the consensus TIS, we revealed that A-3, G-6C-5C-4 and G+1C+2G+3 arrange the specific context in this microalgae which help in locating the ribosome at the correct reading frame. Bioinformatics studies unveiled the sequence of MASTRAPLLALLALLCAGSARA with the highest signal score as the specific SP for secretion systems. A multi-criteria optimization procedure was performed to redesign the coding sequence of the BAR selectable marker gene. The optimized version of the gene mainly covered the host codon preference, the less structured mRNA and exposure of TIS. As the intragenic factors, we selected an efficient promoter, a 5ˈ-UTR and an intron from the closely related species (Chlamydomonas Sp.) to construct the specific expression vectors. The expression cassettes containing optimized genetic elements could be delivered into the microalgae cells and conferred the resistance to the transformants for at least 90 generations. The findings indicated that the MARs flanking the expression cassette along with the optimized expression elements particularly codon adaptation could potentially improve transformation efficiency and stability. The findings can be efficiently deployed as an empirical model for systematic discovery of the key expression elements and optimization of the cis/transgenes.

scholarly journals The Effects of Antioxidant Supplements on the Inflammatory Gene Expression of Osteoarthritis-Like Chondrocytes

2020 ◽  
Vol 11 (1) ◽  
pp. 239
Author(s):  
Chen-Ying Su ◽  
Yongxiang Luo ◽  
Chi-Hau Fang ◽  
Hsu-Wei Fang
Keyword(s):  
Gene Expression ◽  
Hydrogen Peroxide ◽  
Vitamin E ◽  
Cell Viability ◽  
Combined Treatment ◽  
Inflammatory Gene Expression ◽  
Inflammatory Gene ◽  
Tnf Α ◽  
Osteoarthritis Treatment ◽  
Antioxidant Supplements

Osteoarthritis patients often experience pain and inflammation in joints. To understand whether antioxidant supplements could reduce inflammation and could potentially be applied for osteoarthritis treatment, vitamin E and resveratrol were investigated in this study. Hydrogen peroxide was used for inducing inflammation in porcine chondrocytes, and the lumen density was measured for the level of oxygen-derived free radicals. In total, 5, 50, 100, and 200 μM of vitamin E and resveratrol were analyzed for cell viability and the effect of lumen density. In addition, the expressions of IL-1β, TNF-α, MMP-1, MMP-13, and COL2A1 were investigated when treating chondrocytes with 100 μM vitamin E, 5 μM resveratrol, or their combination before or after hydrogen peroxide induction. Inducing chondrocytes with hydrogen peroxide for 30 min increased the level of lumen density sufficiently and stimulated the expression of inflammatory genes. All the concentrations of vitamin E and resveratrol showed good cell viability. Single treatment with either vitamin E or resveratrol reduced inflammatory gene expression, and combined treatment increased the expression of collagen type II. The results suggested that post-treatment with both vitamin E and resveratrol has a better effect on the reduction in inflammation in osteoarthritis-like chondrocytes.

scholarly journals An NMRA-Like Protein Regulates Gene Expression in Phytophthora capsici to Drive the Infection Cycle on Tomato

2018 ◽  
Vol 31 (6) ◽  
pp. 665-677 ◽  
Author(s):  
Jasmine Pham ◽  
Remco Stam ◽  
Victor Martinez Heredia ◽  
Michael Csukai ◽  
Edgar Huitema
Keyword(s):  
Gene Expression ◽  
Crop Production ◽  
Large Scale ◽  
Phytophthora Capsici ◽  
Marker Gene ◽  
Infection Cycle ◽  
Distinct Evolutionary Lineage ◽  
Phytophthora Spp ◽  
Transcriptional Changes

Phytophthora spp. cause devastating disease epidemics on important crop plants and pose a grave threat to global crop production. Critically, Phytophthora pathogens represent a distinct evolutionary lineage in which pathogenicity has been acquired independently. Therefore, there is an urgent need to understand and disrupt the processes that drive infection if we aspire to defeat oomycete pathogens in the field. One area that has received little attention thus far in this respect is the regulation of Phytophthora gene expression during infection. Here, we characterize PcNMRAL1 (Phyca11_505845), a homolog of the Aspergillus nidulans nitrogen metabolite repression regulator NMRA and demonstrate a role for this protein in progression of the Phytophthora capsici infection cycle. PcNmrAL1 is coexpressed with the biotrophic marker gene PcHmp1 (haustorial membrane protein 1) and, when overexpressed, extends the biotrophic infection stage. Microarray analyses revealed that PcNmrAL1 overexpression in P. capsici leads to large-scale transcriptional changes during infection and in vitro. Importantly, detailed analysis reveals that PcNmrAL1 overexpression induces biotrophy-associated genes while repressing those associated with necrotrophy. In addition to factors controlling transcription, translation, and nitrogen metabolism, PcNMRAL1 helps regulate the expression of a considerable effector repertoire in P. capsici. Our data suggests that PcNMRAL1 is a transcriptional regulator that mediates the biotrophy to necrotrophy transition. PcNMRAL1 represents a novel factor that may drive the Phytophthora disease cycle on crops. This study provides the first insight into mechanisms that regulate infection-related processes in Phytophthora spp. and provides a platform for further studies aimed at disabling pathogenesis and preventing crop losses.

scholarly journals A Cell-Based Platform for the Investigation of Immunoproteasome Subunit β5i Expression and Biology of β5i-Containing Proteasomes

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 3049
Author(s):  
Alexander Burov ◽  
Sergei Funikov ◽  
Elmira Vagapova ◽  
Alexandra Dalina ◽  
Alexander Rezvykh ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Large Scale ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Tnf Α ◽  
Anti Cancer ◽  
A Cell ◽  
Established Cell

The degradation of most intracellular proteins is a dynamic and tightly regulated process performed by proteasomes. To date, different forms of proteasomes have been identified. Currently the role of non-constitutive proteasomes (immunoproteasomes (iPs) and intermediate proteasomes (intPs)) has attracted special attention. Here, using a CRISPR-Cas9 nickase technology, four cell lines: histiocytic lymphoma, colorectal adenocarcinoma, cervix adenocarcinoma, and hepatocarcinoma were modified to express proteasomes with mCherry-tagged β5i subunit, which is a catalytic subunit of iPs and intPs. Importantly, the expression of the chimeric gene in modified cells is under the control of endogenous regulatory mechanisms and is increased following IFN-γ and/or TNF-α stimulation. Fluorescent proteasomes retain catalytic activity and are distributed within the nucleus and cytoplasm. RNAseq reveals marginal differences in gene expression profiles between the modified and wild-type cell lines. Predominant metabolic pathways and patterns of expressed receptors were identified for each cell line. Using established cell lines, we demonstrated that anti-cancer drugs Ruxolitinib, Vincristine and Gefitinib stimulated the expression of β5i-containing proteasomes, which might affect disease prognosis. Taken together, obtained cell lines can be used as a platform for real-time studies of immunoproteasome gene expression, localization of iPs and intPs, interaction of non-constitutive proteasomes with other proteins, proteasome trafficking and many other aspects of proteasome biology in living cells. Moreover, the established platform might be especially useful for fast and large-scale experiments intended to evaluate the effects of different conditions including treatment with various drugs and compounds on the proteasome pool.

scholarly journals Osteochondral Live Slices as Physiological in-vitro Model of the Human Joint for High-throughput Applications

2020 ◽  
Author(s):  
Jacob Spinnen ◽  
Carsten Rendenbach ◽  
Anja Kühl ◽  
Ufuk Sentürk ◽  
Daniel Kendoff ◽  
...  
Keyword(s):  
High Throughput ◽  
Cartilage Oligomeric Matrix Protein ◽  
Matrix Protein ◽  
Laser Scanning Microscopy ◽  
Cell Swelling ◽  
Tissue Cell ◽  
Tnf Α ◽  
Human Joint

Abstract Background: Current advances in musculoskeletal research yielded numerous new insights and therapy options for diseases of the joints but preclinical in-vitro testing is currently limited to self-manufactured 2D and 3D cell cultures only remotely mimicking the in-vivo situation in regards of tissue composition and cell configuration. While in-vivo animal models are a lot more lifelike, they are inherently connected with ethical concerns, translation to the human situation, logistic efforts and high costs. Here we explored the use of native human life slice cultures from explanted tibial plateaus upon their feasibility to serve as a highly lifelike and easy-to-handle model of the human joint which can be obtained in masses from few tissue samples.Methods: Osteochondral slices (1cm x 1cm x 500µm) were prepared with a special microtome from 23 human tibial plateaus and subsequently cultivated in hanging inserts over the course of 7 or 21 days. Short-term cultivated slices were stimulated with either 800 pM/1.2 nM TNF-α or 800 pM TGF-β3. During cultivation, viability of tissue cells was assessed via laser microscopy / resazurin assay. After cultivation, gene expression of cartilage ECM Marker proteins was quantified with RT-PCR. TNF-α stimulated slices and their controls were stained with Safranin-O and analyzed via histomorphometry to quantify tissue proteoglycan content. Results: Tissue cell viability remained >90% over the three weeks. Laser scanning microscopy revealed highly conserved spatial alignment of cells inside the cartilage. Incubation of the slice cultures with TNF-α showed a significant, dose dependent decrease in mRNA expression of cartilage proteins collagen II, cartilage oligomeric matrix protein and aggrecan while incubation with TGF-β3 caused a significant increase of early bone formation proteins collagen I and also cartilage oligomeric matrix protein. Histologically, TNF-α incubation caused a 32% reduction of proteoglycans, detachment of collagen fibers and cell swelling.Conclusion: In summary, native osteochondral slice cultures provide a stable and manipulable, physiological model of human joint bone and cartilage biology. The setup is easy to produce and handle, scalable, and contributes to 3R principles in biomedical research, suggesting itself as a platform for preclinical testing especially regarding high-throughput applications.

scholarly journals The Effect of Acute and Chronic Thermotherapy on Type 2 Diabetic Skeletal Muscle Gene Expression and Inflammatory Markers

Biomedicines ◽  
2021 ◽  
Vol 9 (9) ◽  
pp. 1276
Author(s):  
Louay Bachnak ◽  
Jean Sparks ◽  
Daniel E. Newmire ◽  
Xavier F. Gonzales ◽  
Felix O. Omoruyi
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Cell Viability ◽  
Cell Lines ◽  
Muscle Cell ◽  
Skeletal Muscle Cell ◽  
Muscle Gene Expression ◽  
Tnf Α ◽  
Muscle Gene

Background: Type 2 diabetes (T2D) is a chronic illness associated with resistance to or defective insulin secretion. This study investigates the effects of thermotherapy on cell viability, gene expression and inflammation in skeletal muscle cell lines. Methods: Healthy and T2D human skeletal muscle cell lines (HSMM and D-HSMM, respectively) were subjected to acute or chronic thermo-therapy (AT or CT, respectively). CT consisted of a 30 min exposure to 40 °C, three times a week for three weeks; AT was a one-time exposure. Results: A significant decrease in D-HSMM cell viability percentage followed AT; however, no significant change occurred in CT. HSMM yielded the highest elevations of genes following CT. In D-HSMM, both treatments yielded gene upregulation. Both treatments significantly down-regulated IL-1β, IL-6, IL-10 and TNF-α in HSMM. AT significantly decreased IL-1β, IL-6 and upregulated IL-10 and TNF-α levels in D-HSMM, while CT yielded a reduction in IL-4, TNF-α and an upregulation of IL-6 and IL-10. Conclusions: An increase in gene expression indicates actin activity and cellular responses, suggesting an increase in transcriptional regulation. The upregulation of IL-6 and IL-10 in D-HSMM negatively correlated with a decrease in TNF-α and IL-1β, indicating improved adverse inflammatory effects associated with the disease.

scholarly journals A Benchmark of the Marker Gene Tool: MGFM

2017 ◽  
Vol 3 (1) ◽  
pp. 55
Author(s):  
Khadija El Amrani ◽  
Nancy Mah ◽  
Andreas Kurtz
Keyword(s):  
Gene Expression ◽  
Microarray Data ◽  
Molecular Mechanisms ◽  
Marker Gene ◽  
Genomic Research ◽  
Marker Genes ◽  
Tissue Cell ◽  
Specific Gene ◽  
Data Set ◽  
Tissue Specific

Large amounts of microarray experimental data are available in public repositories. Although a variety of tools have been developed to make use of these data, the number of tools that detect marker genes is limited. Identification of marker genes associated with a specific tissue/cell type is a fundamental challenge in genetic and genomic research. In addition to other genes, marker genes are of great importance for understanding the gene function, the molecular mechanisms underlying complex diseases, and may lead to the development of new drug targets. We have previously developed a Bioconductor R package (MGFM) for marker gene detection from microarray data. The tool is freely available from the Bioconductor web site (https://www.bioconductor.org/packages/release/bioc/html/MGFM.html), and it is also provided as an online application integrated into the CellFinder platform (http://cellfinder.org/analysis/marker). In this work, we applied our tool to a public microarray data set from the NCBI’s Gene Expression Omnibus public repository encompassing samples for 12 human tissues. We compared the set of predicted marker genes to a set of tissue-specific genes obtained from the Tissue-specific Gene Expression and Regulation (TiGER) database. Furthermore, we tested the performance of the tool using two normalization methods, RMA and YuGene. YuGene performed slightly better than RMA. Our tool identified 38,4 % or 37,9 % of the TiGER derived tissue-specific genes using YuGene or RMA, respectively.

Molecular Epidemiology and Biomarkers in Etiologic Cancer Research: The New in Light of the Old

2020 ◽  
Author(s):  
Lungwani Muungo
Keyword(s):  
Gene Expression ◽  
Cancer Research ◽  
Molecular Epidemiology ◽  
Exposure Assessment ◽  
Large Scale ◽  
First Generation ◽  
Cancer Epidemiology ◽  
Policy Changes ◽  
The Past ◽  
New Generation

The purpose of this review is to evaluate progress inmolecular epidemiology over the past 24 years in canceretiology and prevention to draw lessons for futureresearch incorporating the new generation of biomarkers.Molecular epidemiology was introduced inthe study of cancer in the early 1980s, with theexpectation that it would help overcome some majorlimitations of epidemiology and facilitate cancerprevention. The expectation was that biomarkerswould improve exposure assessment, document earlychanges preceding disease, and identify subgroupsin the population with greater susceptibility to cancer,thereby increasing the ability of epidemiologic studiesto identify causes and elucidate mechanisms incarcinogenesis. The first generation of biomarkers hasindeed contributed to our understanding of riskandsusceptibility related largely to genotoxic carcinogens.Consequently, interventions and policy changes havebeen mounted to reduce riskfrom several importantenvironmental carcinogens. Several new and promisingbiomarkers are now becoming available for epidemiologicstudies, thanks to the development of highthroughputtechnologies and theoretical advances inbiology. These include toxicogenomics, alterations ingene methylation and gene expression, proteomics, andmetabonomics, which allow large-scale studies, includingdiscovery-oriented as well as hypothesis-testinginvestigations. However, most of these newer biomarkershave not been adequately validated, and theirrole in the causal paradigm is not clear. There is a needfor their systematic validation using principles andcriteria established over the past several decades inmolecular cancer epidemiology.

Sign in / Sign up

Export Citation Format

Share Document