scholarly journals Design of a 3D printed, motorized, uniaxial cell stretcher for microscopic and biochemical analysis of mechanotransduction

Biology Open ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. bio057778
Author(s):  
Noor A. Al-Maslamani ◽  
Abdulghani A. Khilan ◽  
Henning F. Horn
Keyword(s):  
Biochemical Analysis ◽  
Cellular Responses ◽  
Control Parameters ◽  
Cell Nuclei ◽  
Static Stretch ◽  
Cyclic Stretching ◽  
3D Printed ◽  
Biochemical Analyses ◽  
Cell Stretcher ◽  
Proof Of Principle

ABSTRACTCells respond to mechanical cues from their environment through a process of mechanosensing and mechanotransduction. Cell stretching devices are important tools to study the molecular pathways responsible for cellular responses to mechanobiological processes. We describe the development and testing of a uniaxial cell stretcher that has applications for microscopic as well as biochemical analyses. By combining simple fabrication techniques with adjustable control parameters, the stretcher is designed to fit a variety of experimental needs. The stretcher can be used for static and cyclic stretching. As a proof of principle, we visualize stretch induced deformation of cell nuclei via incremental static stretch, and changes in IEX1 expression via cyclic stretching. This stretcher is easily modified to meet experimental needs, inexpensive to build, and should be readily accessible for most laboratories with access to 3D printing.

Differential effects of static and cyclic stretching during elastase digestion on the mechanical properties of extracellular matrices

2007 ◽  
Vol 103 (3) ◽  
pp. 803-811 ◽  
Author(s):  
Rajiv Jesudason ◽  
Lauren Black ◽  
Arnab Majumdar ◽  
Phillip Stone ◽  
Bela Suki
Keyword(s):  
Mechanical Properties ◽  
Enzyme Activity ◽  
Failure Stress ◽  
Cyclic Stretch ◽  
Mechanical Forces ◽  
Peak Strain ◽  
Static Stretch ◽  
Cyclic Stretching ◽  
Rate Dependent ◽  
Physiological Processes

Enzyme activity plays an essential role in many physiological processes and diseases such as pulmonary emphysema. While the lung is constantly exposed to cyclic stretching, the effects of stretch on the mechanical properties of the extracellular matrix (ECM) during digestion have not been determined. We measured the mechanical and failure properties of elastin-rich ECM sheets loaded with static or cyclic uniaxial stretch (40% peak strain) during elastase digestion. Quasistatic stress-strain measurements were taken during 30 min of digestion. The incremental stiffness of the sheets decreased exponentially with time during digestion. However, digestion in the presence of static stretch resulted in an accelerated stiffness decrease, with a time constant that was nearly 3× smaller (7.1 min) than during digestion alone (18.4 min). These results were supported by simulations that used a nonlinear spring network model. The reduction in stiffness was larger during static than cyclic stretch, and the latter also depended on the frequency. Stretching at 20 cycles/min decreased stiffness less than stretching at 5 cycles/min, suggesting a rate-dependent coupling between mechanical forces and enzyme activity. Furthermore, pure digestion reduced the failure stress of the sheets from 88 ± 21 kPa in control to 29 ± 15 kPa ( P < 0.05), while static and cyclic stretch resulted in a failure stress of 7 ± 5 kPa ( P < 0.05). We conclude that not only the presence but the dynamic nature of mechanical forces have a significant impact on enzyme activity, hence the deterioration of the functional properties of the ECM during exposure to enzymes.

scholarly journals Biochemical Analysis of a β-d-Xylosidase and a Bifunctional Xylanase-Ferulic Acid Esterase from a Xylanolytic Gene Cluster in Prevotella ruminicola 23

2009 ◽  
Vol 191 (10) ◽  
pp. 3328-3338 ◽  
Author(s):  
Dylan Dodd ◽  
Svetlana A. Kocherginskaya ◽  
M. Ashley Spies ◽  
Kyle E. Beery ◽  
Charles A. Abbas ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Ferulic Acid ◽  
Biochemical Analysis ◽  
Directed Mutagenesis ◽  
Ferulic Acid Esterase ◽  
Prevotella Ruminicola ◽  
Obligate Anaerobic ◽  
Catalytic Sites ◽  
Cellular Machinery ◽  
Biochemical Analyses

ABSTRACT Prevotella ruminicola 23 is an obligate anaerobic bacterium in the phylum Bacteroidetes that contributes to hemicellulose utilization within the bovine rumen. To gain insight into the cellular machinery that this organism elaborates to degrade the hemicellulosic polymer xylan, we identified and cloned a gene predicted to encode a bifunctional xylanase-ferulic acid esterase (xyn10D-fae1A) and expressed the recombinant protein in Escherichia coli. Biochemical analysis of purified Xyn10D-Fae1A revealed that this protein possesses both endo-β-1,4-xylanase and ferulic acid esterase activities. A putative glycoside hydrolase (GH) family 3 β-d-glucosidase gene, with a novel PA14-like insertion sequence, was identified two genes downstream of xyn10D-fae1A. Biochemical analyses of the purified recombinant protein revealed that the putative β-d-glucosidase has activity for pNP-β-d-xylopyranoside, pNP-α-l-arabinofuranoside, and xylo-oligosaccharides; thus, the gene was designated xyl3A. When incubated in combination with Xyn10D-Fae1A, Xyl3A improved the release of xylose monomers from a hemicellulosic xylan substrate, suggesting that these two enzymes function synergistically to depolymerize xylan. Directed mutagenesis studies of Xyn10D-Fae1A mapped the catalytic sites for the two enzymatic functionalities to distinct regions within the polypeptide sequence. When a mutation was introduced into the putative catalytic site for the xylanase domain (E280S), the ferulic acid esterase activity increased threefold, which suggests that the two catalytic domains for Xyn10D-Fae1A are functionally coupled. Directed mutagenesis of conserved residues for Xyl3A resulted in attenuation of activity, which supports the assignment of Xyl3A as a GH family 3 β-d-xylosidase.

scholarly journals A 3D-printed hand-powered centrifuge for molecular biology

2019 ◽  
Author(s):  
Gaurav Byagathvalli ◽  
Aaron F. Pomerantz ◽  
Soham Sinha ◽  
Janet Standeven ◽  
M. Saad Bhamla
Keyword(s):  
Molecular Biology ◽  
Low Cost ◽  
Power Source ◽  
Medical Diagnostics ◽  
Resource Limited Settings ◽  
Resource Limited ◽  
External Power Source ◽  
External Power ◽  
3D Printed ◽  
Proof Of Principle

The centrifuge is an essential tool for many aspects of research and medical diagnostics. However, conventional centrifuges are often inaccessible outside of conventional laboratory settings, such as remote field sites, require a constant external power source, and can be prohibitively costly in resource-limited settings and STEM-focused programs. Here we present the 3D-Fuge, a 3D-printed hand-powered centrifuge, as a novel alternative to standard benchtop centrifuges. Based on the design principles of a paper-based centrifuge, this 3D-printed instrument increases the volume capacity to 2 mL and can reach hand-powered centrifugation speeds up to 6,000 rpm. The 3D-Fuge devices presented here are capable of centrifugation of a wide variety of different solutions such as spinning down samples for biomarker applications and performing nucleotide extractions as part of a portable molecular lab setup. We introduce the design and proof-of-principle trials that demonstrate the utility of low-cost 3D printed centrifuges for use in remote and educational settings.

scholarly journals Experimental investigation on flexural properties of FDM-processed PET-G specimen using response surface methodology

2021 ◽  
Vol 349 ◽  
pp. 01008
Author(s):  
Nikolaos A. Fountas ◽  
Ioannis Papantoniou ◽  
John D. Kechagias ◽  
Dimitrios E. Manolakos ◽  
Nikolaos M. Vaxevanidis
Keyword(s):  
Flexural Strength ◽  
Response Surface ◽  
Fused Deposition Modeling ◽  
Performance Metrics ◽  
Optimal Parameter ◽  
Strong Dependence ◽  
Control Parameters ◽  
Fused Deposition ◽  
Deposition Modeling ◽  
3D Printed

The properties of fused deposition modeling (FDM) products exhibit strong dependence on process parameters which may be improved by setting suitable levels for parameters related to FDM. Anisotropic and brittle nature of 3D-printed components makes it essential to investigate the effect of FDM control parameters to different performance metrics related to resistance for improving strength of functional parts. In this work the flexural strength of polyethylene terephthalate glycol (PET-G) is examined under by altering the levels of different 3D-printing parameters such as layer height, infill density, deposition angle, printing speed and printing temperature. A response surface experiment was established having 27 experimental runs to obtain the results for flexural strength (MPa) and to further investigate the effect of each control parameter on the response by studying the results using statistical analysis. The experiments were conducted as per the ASTM D790 standard. The regression model generated for flexural strength adequately explains the variation of FDM control parameters on flexural strength and thus, it can be implemented to find optimal parameter settings with the use of either an intelligent algorithm, or neural network.

scholarly journals Biochemical composition of the hoof capsule of buffaloes and its influence on hoof quality

2017 ◽  
Vol 69 (1) ◽  
pp. 57-64 ◽  
Author(s):  
B.M. Assis ◽  
V.A.S. Vulcani ◽  
L.A.F. Silva ◽  
M. Dias ◽  
A. Pancotti ◽  
...  
Keyword(s):  
Organic Matter ◽  
Body Mass ◽  
Biochemical Composition ◽  
Biochemical Parameters ◽  
Crude Protein ◽  
Biochemical Analysis ◽  
Mineral Matter ◽  
Ether Extract ◽  
Dorsal Wall ◽  
Biochemical Analyses

ABSTRACT The purpose of this study was to establish the biochemical parameters of the abaxial wall, dorsal wall and sole of the hoof of the medial thoracic, lateral, and medial pelvic digits of buffalos. The hoof samples were subjected to destructive biochemical analyses to identify the dry material (DM), mineral matter (MM), organic matter (OM), crude protein (CP) and ether extract (EE) contents. Sulfur (S), calcium (Ca), potassium (K), phosphorus (P), zinc (Zn) and copper (Cu) levels were determined based on nondestructive biochemical analyses. The parameters of dry material, mineral matter, organic matter, crude protein and ether extract of hoof capsule of the digits of buffalos can be determined by means of both destructive and nondestructive biochemical analysis. In addition, this study revealed that the highest concentrations of DM, CP and minerals such as, K, Zn and Cu are concentrated in the digits that bear the greatest body mass weight, suggesting that there is a positive correlation between the aforementioned parameters and the strength and growth of the hoof capsule in the digits. As for the element S, this study demonstrated that its highest concentration is located in the lateral digits of the pelvic members.

scholarly journals Toxicological and Biochemical Analyses of an Autopsy Case Involving Oral Overdose of Multiple Antidiabetic and Antihypertensive Drugs

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Tomoya Ikeda ◽  
Naoto Tani ◽  
Shigeki Oritani ◽  
Alissa Shida ◽  
Yayoi Aoki ◽  
...  
Keyword(s):  
Biochemical Analysis ◽  
Antihypertensive Drugs ◽  
Serum Insulin ◽  
Reduction Rate ◽  
Pericardial Fluid ◽  
Oral Antidiabetics ◽  
Fatal Overdose ◽  
Biochemical Profiles ◽  
Biochemical Analyses ◽  
Lifestyle Related Diseases

Oral antidiabetics can cause fatal hypoglycemia; although they can be chemically identified and quantified, biochemical investigations are important for assessing the biological consequences of an overdose. Such cases of overdose involving oral antidiabetics may involve other drugs for treating lifestyle-related diseases, particularly antihypertensives. Here, we report a toxicological and biochemical investigation of drugs and biochemical profiles in a fatal overdose involving multiple oral antidiabetics and antihypertensives. A 55-year-old woman died about 2 days after the ingestion of around 110 tablets of antidiabetics and antihypertensives that had been prescribed for her husband. A forensic autopsy and histological analysis demonstrated no evident pathology as the cause of death. A toxicological analysis suggested hypoglycemia and an overdose of antihypertensives as well as the retention of antidiabetics and diuretics in the pericardial fluid. A relatively low pericardial amlodipine concentration was observed, which may have been the result of its long half-life (slower distribution and reduction rate) and/or possible affinity with the myocardium. In addition, a biochemical analysis indicated hypoglycemia, without increased serum insulin and C-peptide, but with increased glucagon levels, as the possible influence of glibenclamide overdose. These observations suggest the usefulness of a combination of toxicological and biochemical analyses in postmortem investigations involving a fatal overdose of such drugs.

scholarly journals Cloning of a human phosphoinositide 3-kinase with a C2 domain that displays reduced sensitivity to the inhibitor wortmannin

1997 ◽  
Vol 326 (1) ◽  
pp. 139-147 ◽  
Author(s):  
Jan DOMIN ◽  
Françoise PAGES ◽  
Stefano VOLINIA ◽  
Susan E. RITTENHOUSE ◽  
Marketa J. ZVELEBIL ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Biochemical Analysis ◽  
Cellular Responses ◽  
C2 Domain ◽  
Substrate Specificities ◽  
Sequence Heterogeneity ◽  
Phosphoinositide 3 Kinase ◽  
Lipid Substrate ◽  
K Activity

The generation of phosphatidylinositide 3-phosphates has been observed in a variety of cellular responses. The enzymes that mediate synthesis are the phosphoinositide 3-kinases (PI3-Ks) that form a family of structurally diverse enzymes with distinct substrate specificities. In this paper, we describe the cloning of a novel human PI3-K, namely PI3-K-C2α, which contains a C-terminal C2 domain. This enzyme can be assigned to the class II PI3-Ks, which was defined by characterization of the Drosophila 68D enzyme and includes the recently described murine enzymes m-cpk and p170. Despite the overall similarity in the amino acid sequence of the murine and human enzymes, which suggests that they are encoded by closely related genes, these molecules show marked sequence heterogeneity at their N-termini. Biochemical analysis of recombinant PI3-K-C2α demonstrates a restricted lipid substrate specificity. As reported for other members of this class, the enzyme only phosphorylates PtdIns and PtdIns4P when the lipids are presented alone. However, when lipids were presented together with phosphatidylserine acting as a carrier, phosphorylation of PtdIns(4,5)P2 was also observed. The catalytic activity of PI3-K-C2α is refractory to concentrations of wortmannin and LY294002 which inhibit the PI3-K activity of other family members. The comparative insensitivity of PI3-K-C2α to these inhibitors suggests that their use should be re-evaluated in the study of PI3-Ks.

3D-printed individual labware in biosciences by rapid prototyping: A proof of principle

2014 ◽  
Vol 15 (1) ◽  
pp. 51-56 ◽  
Author(s):  
Tim H. Lücking ◽  
Franziska Sambale ◽  
Sascha Beutel ◽  
Thomas Scheper
Keyword(s):  
Rapid Prototyping ◽  
3D Printed ◽  
Proof Of Principle

scholarly journals Matrisome Properties of Scaffolds Direct Fibroblasts in Idiopathic Pulmonary Fibrosis

2019 ◽  
Vol 20 (16) ◽  
pp. 4013 ◽  
Author(s):  
Linda Elowsson Rendin ◽  
Anna Löfdahl ◽  
Emma Åhrman ◽  
Catharina Müller ◽  
Thomas Notermans ◽  
...  
Keyword(s):  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Cellular Responses ◽  
Biomechanical Properties ◽  
Static Stretch ◽  
Cell Matrix ◽  
Associated Proteins ◽  
Approaches To Study ◽  
Specific Proteins ◽  
Cell Matrix Interactions

In idiopathic pulmonary fibrosis (IPF) structural properties of the extracellular matrix (ECM) are altered and influence cellular responses through cell-matrix interactions. Scaffolds (decellularized tissue) derived from subpleural healthy and IPF lungs were examined regarding biomechanical properties and ECM composition of proteins (the matrisome). Scaffolds were repopulated with healthy fibroblasts cultured under static stretch with heavy isotope amino acids (SILAC), to examine newly synthesized proteins over time. IPF scaffolds were characterized by increased tissue density, stiffness, ultimate force, and differential expressions of matrisome proteins compared to healthy scaffolds. Collagens, proteoglycans, and ECM glycoproteins were increased in IPF scaffolds, however while specific basement membrane (BM) proteins such as laminins and collagen IV were decreased, nidogen-2 was also increased. Findings were confirmed with histology, clearly showing a disorganized BM. Fibroblasts produced scaffold-specific proteins mimicking preexisting scaffold composition, where 11 out of 20 BM proteins were differentially expressed, along with increased periostin and proteoglycans production. We demonstrate how matrisome changes affect fibroblast activity using novel approaches to study temporal differences, where IPF scaffolds support a disorganized BM and upregulation of disease-associated proteins. These matrix-directed cellular responses emphasize the IPF matrisome and specifically the BM components as important factors for disease progression.

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