scholarly journals A standardized method to purify cardiomyocytes from individual mouse hearts of any age

2021 ◽  
Author(s):  
Amy M Nicks ◽  
Sara R Holman ◽  
Andrea Y Chan ◽  
Michael Tsang ◽  
Paul E Young ◽  
...  
Keyword(s):  
Heart Development ◽  
Enzymatic Digestion ◽  
High Yield ◽  
Coronary Perfusion ◽  
Isolation And Purification ◽  
Immunomagnetic Bead ◽  
Rapid Isolation ◽  
Neonatal Cardiomyocytes ◽  
Standardized Method

Rationale: Primary cardiomyocytes are invaluable for understanding postnatal heart development and elucidating disease mechanisms in genetic and pharmacological models, however, a method to obtain freshly purified cardiomyocytes at any postnatal age, without using different age-dependent isolation procedures and cell culture, is lacking. Objective: To develop a standardized method that allows rapid isolation and purification of cardiomyocytes in high yield and viability from individual neonatal, infant, and adult mice. Methods and Results: Hearts of C57BL/6J mice were cannulated using a novel in situ aortic cannulation procedure optimized to allow cannulation of even the very small vessel of neonates (postnatal day 0-2, P0-2). Hearts were then subjected to Langendorff retrograde perfusion and enzymatic digestion. Cardiomyocytes were isolated after subsequent tissue disaggregation and filtration, in high yield (1.56-2.2x106 cardiomyocytes/heart) and viability (~70-100%). The larger size of infant (P10 and P13) and adult (P70), but not neonatal, cardiomyocytes relative to non-myocytes, allowed enrichment by differential centrifugation. Cardiomyocytes from all ages were further purified by immunomagnetic bead-based depletion of non-myocytes. Together, these procedures resulted in the isolation of highly purified cardiomyocytes (~94%) within 1 hour, enabling experiments using individual replicates. For example, RNA-sequencing of cardiomyocytes purified from one P2 male and female heart per litter (n=4 litters) showed distinct clustering by litters and sex differences for nine differentially expressed genes (FDR<0.005). In situ fixation via coronary perfusion, performed immediately after tissue digestion, preserved the cytoarchitecture of isolated cardiomyocytes (yielding ~94% rod-shaped cardiomyocytes at all ages), allowing capture of spindle-shaped neonatal cells undergoing mitosis, as well as enabling accurate quantitation of cardiomyocyte area and nucleation state. Conclusion: The procedures developed here provide a universal protocol for the rapid isolation and purification of high-quality cardiomyocytes from hearts of any postnatal age, even those of neonates, thereby enabling direct comparisons between individual hearts.

scholarly journals HIGH-YIELD PREPARATION OF ISOLATED RAT LIVER PARENCHYMAL CELLS

1969 ◽  
Vol 43 (3) ◽  
pp. 506-520 ◽  
Author(s):  
M. N. Berry ◽  
D. S. Friend
Keyword(s):  
Gap Junctions ◽  
Rat Liver ◽  
Structural Integrity ◽  
Cell Injury ◽  
High Yield ◽  
Isolated Cells ◽  
Nylon Mesh ◽  
Parenchymal Cells

A new technique employing continuous recirculating perfusion of the rat liver in situ, shaking of the liver in buffer in vitro, and filtration of the tissue through nylon mesh, results in the conversion of about 50% of the liver into intact, isolated parenchymal cells. The perfusion media consist of: (a) calcium-free Hanks' solution containing 0.05% collagenase and 0.10% hyaluronidase, and (b) magnesium and calcium-free Hanks' solution containing 2 mM ethylenediaminetetraacetate. Biochemical and morphologic studies indicate that the isolated cells are viable. They respire in a medium containing calcium ions, synthesize glucose from lactate, are impermeable to inulin, do not stain with trypan blue, and retain their structural integrity. Electron microscopy of biopsies taken during and after perfusion reveals that desmosomes are quickly cleaved. Hemidesmosome-containing areas of the cell membrane invaginate and appear to pinch off and migrate centrally. Tight and gap junctions, however, persist on the intact, isolated cells, retaining small segments of cytoplasm from formerly apposing parenchymal cells. Cells which do not retain tight and gap junctions display swelling of Golgi vacuoles and vacuoles in the peripheral cytoplasm. Cytoplasmic vacuolization in a small percentage of cells and potassium loss are the only indications of cell injury detected. By other parameters measured, the isolated cells are comparable to normal hepatic parenchymal cells in situ in appearance and function.

1-Carba-arachno-pentaborane(10) Derivatives

1997 ◽  
Vol 62 (8) ◽  
pp. 1254-1262 ◽  
Author(s):  
Bernd Wrackmeyer ◽  
Hans-Jörg Schanz
Keyword(s):  
High Yield ◽  
Exchange Reactions ◽  
Nmr Data ◽  
Substituted 1 ◽  
C Nmr

The formation of organo substituted 1-carba-arachno-pentaborane(10) derivatives is shown to proceed in high yield via in situ generated 1,1,1-tris(diethylboryl)propane (2) from diethyl(propyn-1-yl)borane (1) by hydroboration with an excess of diethylborane (hydride bath). In the hydride bath, exchange reactions between 2 and other geminal bis(diethylboryl)alkanes take place until the carbaborane skeleton is formed. If tris(diethylboryl)methane is used under the same conditions, the corresponding 1-carba-arachno-pentaborane(10) derivatives 11 and 12 are formed in mixture with other unknown boranes or carboranes. 11B and 13C NMR data are presented to allow for straightforward identification of the 1-carba-arachno-pentaboranes(10).

Application of Epoxy Resin for Improvement of the Banana Stem-Acoustic Panel

2021 ◽  
Vol 15 ◽  
Author(s):  
Mohd Azril Riduan ◽  
Mohd Jumain Jalil ◽  
Intan Suhada Azmi ◽  
Afifudin Habulat ◽  
Danial Nuruddin Azlan Raofuddin ◽  
...  
Keyword(s):  
Oleic Acid ◽  
Kinetic Modelling ◽  
Low Cost ◽  
Cost Effective ◽  
Performic Acid ◽  
High Yield ◽  
Renewable Materials ◽  
Runge Kutta Method ◽  
To Come

Background: Greener epoxidation by using vegetable oil to create an eco-friendly epoxide is being studied because it is a more cost-effective and environmentally friendly commodity that is safer than non-renewable materials. The aim of this research is to come up with low-cost solutions for banana trunk acoustic panels with kinetic modelling of epoxy-based palm oil. Method: In this study, the epoxidation of palm oleic acid was carried out by in situ performic acid to produce epoxidized palm oleic acid. Results: Banana trunk acoustic panel was successfully innovated based on the performance when the epoxy was applied. Lastly, a mathematical model was developed by using the numerical integration of the 4th order Runge-Kutta method, and the results showed that there is a good agreement between the simulation and experimental data, which validates the kinetic model. Conclusion: Overall, the peracid mechanism was effective in producing a high yield of epoxy from palm oleic acid that is useful for the improvement of acoustic panels based on the banana trunk.

Abstract 124: Function of the Cytoskeletal Proteins Alpha-catenins in Myocardial Growth Control

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Jifen Li ◽  
Sarah Carrante ◽  
Roslyn Yi ◽  
Frans van Roy ◽  
Glenn L. Radice
Keyword(s):  
Heart Development ◽  
Growth Control ◽  
Cytoskeletal Proteins ◽  
Neonatal Rat ◽  
Nuclear Accumulation ◽  
Mouse Heart ◽  
Cardiomyocyte Proliferation ◽  
Rat Cardiomyocytes ◽  
Neonatal Cardiomyocytes

Introduction: Mammalian heart possesses regenerative potential immediately after birth and lost by one week of age. The mechanisms that govern neonatal cardiomyocyte proliferation and regenerative capacity are poorly understood. Recent reports indicate that Yap-Tead transcriptional complex is necessary and sufficient for cardiomyocyte proliferation. During postnatal development, N-cadherin/catenin adhesion complex becomes concentrated at termini of cardiomyocytes facilitating maturation of a specialized intercellular junction structure, the intercalated disc (ICD). This process coincides with the time cardiomyocytes exit cell cycle soon after birth. Hypothesis: We hypothesize that coincident with maturation of ICD α-catenins sequester transcriptional coactivator Yap in cytosol thus preventing activation of genes critical for cardiomyocyte proliferation. Methods: We deleted αE-catenin / αT-catenin genes (α-cat DKO) in perinatal mouse heart and knockdown (KD) α-catenins in neonatal rat cardiomyocytes to study functional impact of α-catenins ablation on ICD maturation. Results: We previously demonstrated that adult α-cat DKO mice exhibited decrease in scar size and improved function post myocardial infarction. In present study, we investigated function of α-catenins during postnatal heart development. We found increase in the number of Yap-positive nuclei (58.7% in DKO vs. 35.8 % in WT, n=13, p<0.001) and PCNA (53.9% in DKO vs. 47.8%, n=8, p<0.05) at postnatal day 1 and day 7 of α-cat DKO heart, respectively. Loss of α-catenins resulted in reduction in N-cadherin at ICD at day 14. We observed an increase number of mononucleated myocytes and decrease number of binucleated myocytes in α-cat DKO compared to controls. Using siRNA KD, we were able to replicate α-cat DKO proliferative phenotype in vitro. The number of BrdU-positive cells was decreased in α-cat KD after interfering with Yap expression (2.91% in α-cat KD vs. 2.02% in α-cat/Yap KD, n>2500 cells, p<0.05), suggesting α-catenins regulate cell proliferation through Yap in neonatal cardiomyocytes. Conclusion: Our results suggest that maturation of ICD regulates α-catenin-Yap interactions in cytosol, thus preventing Yap nuclear accumulation and cardiomyocyte proliferation.

scholarly journals New Biochemical Insights to Unravel the Pathogenesis of Alzheimer's Lesions

1991 ◽  
Vol 18 (S3) ◽  
pp. 408-410 ◽  
Author(s):  
Alex E. Roher ◽  
Kenneth C. Palmer ◽  
John Capodilupo ◽  
Arun R. Wakade ◽  
Melvyn J. Ball
Keyword(s):  
Amyloid Plaque ◽  
Extracellular Proteins ◽  
High Yield ◽  
Chemical Fractionation ◽  
Core Proteins ◽  
Β Amyloid ◽  
Neuritic Sprouting

ABSTRACT:Purification of amyloid plaque core proteins (APCP) from Alzheimer's disease brains to complete homogeneity and in high yield permitted its chemical fractionation and characterization of its components. APCP is mainly made of β-amyloid (βA) and an assortment of glycoproteins (accounting for 20%) rich in carbohydrates compatible with N-and O-linked saccharides. When added to tissue culture of sympathetic and sensory neurons APCP and βA inhibited neuritic sprouting, a reversible phenomenon at low doses. Higher concentrations of both substances kill the neurons in culture. APCP is significantly more toxic than βA, suggesting the minor components may play an important role in increasing the toxicity of βA. If the observed toxic effects of APCP in situ are occurring in vivo during the course of AD, then the accumulation of these extracellular proteins could be largely responsible for some of the neuronal death observed in this neuropathology.

In situ electrogeneration of o-benzoquinone and high yield reaction with benzenethiols in a microflow system

2012 ◽  
Vol 48 (22) ◽  
pp. 2806 ◽  
Author(s):  
Tsuneo Kashiwagi ◽  
Fumihiro Amemiya ◽  
Toshio Fuchigami ◽  
Mahito Atobe
Keyword(s):  
High Yield

scholarly journals Microstructure and mechanical properties of slowly cooled Cu47.5Zr47.5Al5

2007 ◽  
Vol 22 (2) ◽  
pp. 326-333 ◽  
Author(s):  
J. Das ◽  
S. Pauly ◽  
C. Duhamel ◽  
B.C. Wei ◽  
J. Eckert
Keyword(s):  
Mechanical Properties ◽  
Fracture Strain ◽  
Volume Fraction ◽  
Spherical Shape ◽  
High Yield ◽  
High Yield Strength ◽  
Scanning Calorimetry ◽  
Arc Melting ◽  
Martensite Matrix

Cu47.5Zr47.5Al5 was prepared by arc melting and solidified in situ by suction casting into 2–5-mm-diameter rods under various cooling rates (200–2000 K/s). The microstructure was investigated along the length of the rods by electron microscopy, differential scanning calorimetry and mechanical properties were investigated under compression. The microstructure of differently prepared specimens consists of macroscopic spherical shape chemically inhomogeneous regions together with a low volume fraction of randomly distributed CuZr B2 phase embedded in a 2–7 nm size clustered “glassy-martensite” matrix. The as-cast specimens show high yield strength (1721 MPa), pronounced work-hardening behavior up to 2116 MPa and large fracture strain up to 12.1–15.1%. The fracture strain decreases with increasing casting diameter. The presence of chemical inhomogenities and nanoscale “glassy-martensite” features are beneficial for improving the inherent ductility of the metallic glass.

scholarly journals Agro morphological characterization of cashew trees (Anacardium occidental L.), in improvement for the high yield and high quality of raw nuts in Burkina Faso

2021 ◽  
Vol 14 (9) ◽  
pp. 3188-3199
Author(s):  
Windpouiré Vianney Tarpaga ◽  
Larbouga Bourgou ◽  
Moussa Guira ◽  
Albert Rouamba
Keyword(s):  
Burkina Faso ◽  
Core Collection ◽  
Morphological Diversity ◽  
Morphological Characterization ◽  
High Yield ◽  
Morphological Description ◽  
Average Weight ◽  
In Situ Collection

The cashew sector has taken a great economic interest, due to the strong global demand for raw nuts. However, in Burkina Faso, the productivity of orchards still remains low, a consequence of a lack of breeding program on the species. In order to increase yields and improve the quality of the raw nut, efficient clones must be developed. Surveys were carried out and a primary in situ collection was made from which a core collection of 15 trees was extracted. An agro-morphological description of the selected trees and an analysis of the structuration of the variability within them were carried out. The study revealed appreciable agro-morphological diversity through shape, size and color of apple, type of inflorescence, fruiting period and the health of trees. A structure of diversity based on agronomic criteria led to identify 5 groups of trees, characterized by an average weight of the nut between 6.64 and 8.32 g, a kernel rate between 28.10 and 31.26% and a yield per tree between 34.27 and 104.42 kg. Performing trees will be erected as heads of clones for the production of grafted plants. La filière anacarde a pris un grand intérêt économique, suite à la forte demande mondiale en noix brutes. Cependant, au Burkina Faso, la productivité des vergers demeure encore faible, conséquence d’une absence de sélection variétale du matériel végétal. En vue d’accroître les rendements et d’améliorer la qualité de la noix brute, une recherche de clones performants était indispensable. Des prospections ont été conduites et une collection primaire in situ a été constituée dont une core collection de 15 arbres a été extraite. Une description agro-morphologique des arbres sélectionnés et une analyse de la structuration de la variabilité en son sein ont été conduites. L’étude a révélé une diversité agro morphologique appréciable à travers la forme, le calibre et la couleur de la pomme, le type d’inflorescence, la période de fructification et l’état sanitaire des arbres. Une structuration de la diversité basée sur les critères agronomiques a permis d’identifier 5 groupes d’arbres, caractérisés par un poids moyen de la noix compris entre 6,64 et 8,32 g, un taux d’amande compris entre 28,10 et 31,26% et un rendement par arbre compris entre 34,27 et 104,42 kg. Les arbres les plus performants constitueront des têtes de clones pour la production de plants par greffage.

scholarly journals Selective benzylic C–H monooxygenation mediated by iodine oxides

2019 ◽  
Vol 15 ◽  
pp. 602-609
Author(s):  
Kelsey B LaMartina ◽  
Haley K Kuck ◽  
Linda S Oglesbee ◽  
Asma Al-Odaini ◽  
Nicholas C Boaz
Keyword(s):  
Selective Oxidation ◽  
High Yield ◽  
Hypervalent Iodine ◽  
Iodine Species ◽  
Mechanistic Analysis ◽  
Substrate Tolerance ◽  
Iodine Oxides ◽  
Moderate Yield

A method for the selective monooxdiation of secondary benzylic C–H bonds is described using an N-oxyl catalyst and a hypervalent iodine species as a terminal oxidant. Combinations of ammonium iodate and catalytic N-hydroxyphthalimide (NHPI) were shown to be effective in the selective oxidation of n-butylbenzene directly to 1-phenylbutyl acetate in high yield (86%). This method shows moderate substrate tolerance in the oxygenation of substrates containing secondary benzylic C–H bonds, yielding the corresponding benzylic acetates in good to moderate yield. Tertiary benzylic C–H bonds were shown to be unreactive under similar conditions, despite the weaker C–H bond. A preliminary mechanistic analysis suggests that this NHPI-iodate system is functioning by a radical-based mechanism where iodine generated in situ captures formed benzylic radicals. The benzylic iodide intermediate then solvolyzes to yield the product ester.

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