Cell isolation and Cell Culture

Regardless of the species to be studied, growing large populations of isolated cells for biochemical analysis and microscopic observation is helpful. This is straightforward for the unicellular organisms such as fungi or bacteria, which can be grown suspended in a nutrient medium. These organisms can also be grown on the surface of gelled agar in a petri dish. When single cells are dispersed widely on an agar surface, each multiplies to form a macroscopic colony, all descendents of a single cell. This family of cells is called a clone.
For multicellular organisms, it is often possible to isolate single live cells by dissociating a tissue with proteolytic enzymes and media that weaken adhesions between the cells. Tissues provide the most realistic source of material. Several approaches are used to separate the different cell types from solid tissue or liquid tissue. For solid tissue, the first step in isolating individual cells is to get nixed cell suspension through proteolytic enzymes digestion to disrupt the extracellular matrix and cell-cell junctions. After that, a flow cytometer, also known as fluorescence activated cell sorter (FACs), can be used to isolate target cells from the mixed cell suspension. For isolating cells, an antibody coupled to a fluorescent dye is used to specifically bind to the surface of specific cells, then the labeled cells are chosen from the unlabeled ones using the flow cytometer. The flow cytometer can sort 20 thousand cells each second, the purity of selected cell can reach to 95%. Immunomagnetic separation (IMS) is another useful tool that can efficiently isolate viable and functional cells. In one approach, cells are incubated with immunomagnetic beads precoating antibodies which will bind to antigens present on the surface of cells, thus the se bead-attached cells will be captured by a magnet placed on the side of the test tube. Compared with FACs, this technique is easy to work and its procedure is very rapid. In additio......

Spherical Assemblies Formed by Regular Polygons of Subunits

Geometric constraints limit the ways that identical subunits can be arranged on a closed spherical surface with equivalent or nearly equivalent contacts between the subunits. By far, the most favored arrangement is based on a net of equilateral triangles. On a plane surface, these triangles will pack hexagonally with six fold vertices. Since the time of Plato, it has been appreciated that introducing vertices surrounded by three, four, or five triangles will cause such a network of triangles to pucker and, given an appropriate number of puckers, to close up into a complete shell. Four threefold vertices make a tetrahedron, six fourfold vertices make an octahedron, and 12 fivefold vertices make an icosahedron. Remarkably, no other ways of arranging triangles will complete a shell. In addition to threefold, fourfold, or fivefold vertices that introduce puckers, a closed polygon can contain additional triangular faces and sixfold vertices to expand the volume. The sixfold vertices can be placed symmetrically with respect to the fivefold vertices to produce a spherical shell or asymmetrically to form an elongated structure.
Most closed macromolecular assemblies in biology are polygons with fivefold vertices. An important reason for this is that most structures require some sixfold vertices to provide sufficient internal volume. This favors fivefold vertices for the puckers, as they require much less distortion of the subunits located on the triangular faces of the hexagonal plane sheet than do threefold or fourfold vertices. Further, the distortion in the contacts between the triangles is minimized if the fivefold vertices are in equivalent positions. Closed icosahedral shells can be assembled from any type of asymmetrical subunit given two provisions: (1) The subunit must be able to form bonds with like subunits in a triangular network; and (2) these subunits must be able to accommodate the distortion required to form both fivefold and sixfold vertices. Both fi......

Proteins

Proteins constitute most of a cell's dry mass. They are not only the cell's building blocks but also execute nearly all the cell's functions. Proteins with catalytic activity, called enzymes, speed up many chemical reactions. Proteins embedded in the plasma membrane form channels and pumps that control the passage of small molecules into and out of the cell. Other proteins carry messages from one cell to another, or act as signal integrator that relay sets of signals inward from the plasma membrane to the cell nucleus. Contractile proteins are responsible for movement, and other specialized proteins act as antibodies, toxins and hormones. This section presents some basic concepts about protein structure that help to explain how proteins function in cells. More extensive converage of this topic is available in biochemistry textbooks and specialized books on protein chemistry.
Proteins consist of one or more linear polymers called polypeptides, which consist of various combinations of 20 different amino acids linked together by peptide bonds. When linked in polypeptides, amino acids are referred to as residues. The sequence of amino acids in each type of polypeptide is unique. It is specified by the gene encoding the protein and is read out precisely during protein synthesis. The polypeptides of proteins with more than one chain are usually synthesized separately. However, in some cases, a single chain is divided into pieces by cleavage after synthesis.
Polypeptides range widely in length. Small peptide hormones, such as oxytocin, consist of as few as nine residues, while the giant structural protein titin has more than 25,000 residues. Most cellular proteins fall in the range of 100 to 1000 residues. Without stabilization by disulfide bonds or bound metal ions, about 40 residues are required for a polypeptide to adopt a stable three-dimensional structure in water.

Xenotransplantation-Specific modified gene select for genetically edited pigs

Pig organs are very close to human organs in structure, size and physiological function, and are the main research objects of xenotransplantation.However, due to the natural alpha-galactosyl antibody (Gal) of the human carrier, cross-species xenotransplantation can cause severe hyperacute rejection.The antibody binds to the vascular endothelial cells of the graft and activates the complement system, causing vascular endothelial edema and microvascular thrombosis, which in turn causes necrosis of the transplanted organ.
On Apr 15, 2019 , the David K. C. Cooper team of the Alabama University published a review article entitled "Justification of specific genetic modifications inpigs for clinical organ xenotransplantation" at Xenotransplantation. A systematic review of genetic modification schemes that have been found to promote xenotransplantation has been conducted.This review article covers the expression or knockout of nine genes : knock out of pig GGTA1, CMAH and β4GALNT2, expression of human CD46,CD55, TBM, ERCP, CD47, and HO-1.
The authors say that based on current technology, donor pigs for xenotransplantation must be “TKO pigs”, which knock out pig endogenous genes: GGAT1, CMAH and β4GalNT2. These three genes, which are abundantly expressed in pig organs and tissues, are the three major antigens that cause xenograft immune rejection. The "TKO pig" produced by CRISPR/Cas9 technology can greatly reduce its immunogenicity.
In addition, genetically edited pigs expressing human proteins can also significantly reduce xenotransplant rejection, vascular endothelial edema and microvascular thrombosis. Combinatorial expression can provide better inhibition, for example, human complement regulatory proteins (CD46, CD55), human thrombomodulin (TBM, EPCR). Related experiments showed that the "TKO pig" expressing human CD55 had a heterologous kidney transplantation survival time of up to 90 days, while the "TKO pig" that did not expres......

Origin and Evolution of Mitochondria

Overwhelming molecular evidence has established that eukaryotes acquired mitochondria when an alpha-proteobacterium became an endosymbiont. Modern-day alpha-proteobacteriainclude pathogenic Rickettsias. When the two formerly independent cells established a stable, endosymbiotic relationship, the Bacterium contributed molecular machinery for ATP synthesis by oxidative phosphorylation. The host cell might have supplied organic substrates to fuel ATP synthesis. Together, they had a reliable energy supply for processes such as biosynthesis, regulation of the internal ionic environment, and cellular motility. Given that some primitive eukaryotes lack full-fledged mitochondria, the singular event that created mitochondria was believed to have occurred well after eukaryotes branched from prokaryotes.

An alternative idea is that the recipient of theα- proteobacterium was an archaean cell rather than a eukaryote. If so, this union could have created not only the mitochondrion but also the first eukaryote! This parsimonious hypothesis is consistent with some but not all of the available data, so it is currently impossible to rule out other scenarios.

The mitochondrial progenitor brought along its own genome and biosynthetic machinery, but over many years of evolution, most bacterial genes either moved to the host cell nucleus or were lost. Like their bacterial ancestors, mitochondria are enclosed by two membranes, with the inner membrane equipped for synthesis of ATP. Mitochondria maintain a few genes for mitochondrial components and the capacity to synthesize proteins. Nuclear genes encode most mitochondrial proteins, which are synthesized in the cytoplasm and imported into the organelle. The transfer of bacterial genes to the nucleus sealed the dependence of the organelle on its eukaryotic host.

Even though acquisition of mitochondria might have been the earliest event in eukaryotic evolution, some eukaryotes lack fully functional mitoc......