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This question arises because I saw that monocytes and leukocytes are commonly called 'mononuclear cells' in the scientific literature. The implication of course being that other immune sub-types are multi-nuclear!
I know of granulocytes (e.g. neutrophils) that are classed as 'polymorphonuclear' because their nuclei are segmented, and can alter their shapes, and muscle cells that fuse together to form one long cell (muscle fiber) with multiple nuclei. What other examples of multi-nucleated cells are there?
I am also interested in the advantages gained by cells having multiple (or segmented) nuclei?
Muscle cells are the only cells I know of that are polynuclear. With respect to monocytes, a concise review of their nomenclature can be found in this paper by L Ziegler-Heitbrock, P Ancuta, S Crowe, et al. (Blood, 2010). Apparently it has had quite a complex and confused biochemical characterization, but the article states the name indeed derived from its single lobed, mononuclear morphology. This is in distinction to other phagocytes which have multi-lobular nuclei (polymorphonuclear cells).
With respect to advantages, a multinucleated cell makes sense when the speed of intracellular signalling is important (e.g., calcium diffusion). It may also be useful in the case of cells when the cell needs to coordinate the synthesis of large amounts of protein.
Another example of multinucleated cells is osteoclasts, which are specialized derivatives of macrophages that degrade bone matrix. They form by fusion of mononucleated progenitors and can accumulate many nuclei in a single large cell. In cell culture with mouse macrophages, it's common to obtain individual osteoclasts with 50-100 or more nuclei each.
- Multinucleate cell angiohistiocytoma (MCAH)
- muscle fiber
- Types of giant cells A. Physiological giant cells: • Osteoclast • Megakaryocytes • Striated muscle • Syncytiotrophoblast B. Pathological giant cells: • Langhan's giant cell • Foreign body giant cell • Touton giant cell • Tumor giant cell • Warthin-Finkeldey giant cell
- Liver cells
- Skeletal muscle
- Some cardiac muscles
Cloning of Genes: Methods and Advantages | Biochemistry
In this article we will discuss about: 1. Methods of Cloning, 2. Cloning of a Specific Gene, 3. Cloning of a Specific Gene, 4. Expression of Cloned Genes, 5. Cloning of Genes in Eucaryotic Cells and 6. Precautions to be taken during Experiments of Genetic Recombination.
Methods of Cloning:
In the past few years, an increasing number of laboratories have undertaken experiments of genetic recombination in vitro. These experiments consist in incorporating heterologous DNA in a stable manner in a bacterial cell (generally the strain of E.coli K12), through a vector which can be, either a plasmid, or a phage such as phage , i.e. genetic material capable of existing independently of the bacterial chromosome and of replicating autonomously.
For example, if a plasmid is used (i.e. a circular double-stranded covalently closed DNA molecule present in the bacterial cell independently of the chromosome), the principle of the experiments will consist in isolating the plasmid from the bacterial cell, splitting this DNA molecule by means of a very specific enzyme (a restriction endonuclease), joining the cleaved plasmid DNA to the heterologous DNA to be cloned and finally re-introducing — thanks to the transformation process — in the bacterial cell, the plasmid carrying the heterologous DNA.
Among the most frequently used plasmids, one can mention those made in vitro using the DNA recombination techniques, and which often belong to the Col series, because they contain genes coding for the production of a colicine (an extracellular protein, having antibiotic properties, produced by E.coli).
The plasmids used for the cloning of genes generally have the following proper­ties: their size is comparatively small they possess at least one single site sensitive to a restriction endoculease they contain one (or several) gene(s) which allow the selection of the cells transformed by the plasmid they are present at the concentration of about 20 molecules per cell, but in presence of chloromphenicol their replication leads to the accumulation of 1 000 to 2 000 circular plasmid DNA molecules per cell they can be easily isolated from bacterial cells and can also be easily reintroduced in them.
The diagram of fig. 6-51 shows the main steps allowing the cloning of heterologous genes in a bacterial cell. Owing to its large size, the bacterial DNA is very sensitive to shearing forces, with the result that it is cut into fragments during the extraction of DNAs, while the plasmid DNA much smaller in size, present in the form of circular (covalently closed) double- stranded super-coiled molecules, remains intact and can be easily separated from the bacterial DNA by centrifugation in a density gradient, in presence of a molecule which intercalates between the bases of the DNA, ethidium bromide.
The physical constraints existing in a covalently closed circular DNA, molecule are such that the quantity of ethidium bromide bound will be limited. These constraints do not exist in a linear molecule and the quantity or ethidium bromide bound per unit DNA length will be much greater.
Compared to the density of the circular complexes, the density of the linear complexes will therefore be sufficiently lowered to permit their separation during an isopicnic sedimentation (the decrease in density of the DNA is proportional to the quantity of ethidium bromide bound per unit length).
To cleave the plasmid DNA and the heterologous DNA one can use a restriction endonuclease producing cohesive ends which on the one hand, will subsequently facilitate the joining of the heterologous DNA fragment with the plasmid by means of polynucleotide ligase, and on the other hand will permit — with the help of the same restriction enzyme — the excision of the inserted DNA from the plasmid (if, for example, one wants to study the structure of the DNA fragment inserted).
But one can also use other restriction enzymes (which do not produce cohesive ends, e.g. Hae III), and achieve cyclization after addition of poly dA at the 3′ ends of the heterologous DNA fragments and of poly dT at the 3′ end of the cleaved plasmid DNA, which permits pairing of poly dA and poly dT segments, and then covalent joining of the fragment and the plasmid with the help of polynucleotide ligase this method offers the advantage of avoiding the cyclization of the plasmid without insertion of foreign DNA, with the result that practically all the transformed bacterial cells, selected using genetic marker of the plasmid, will contain a hybrid plasmid (i.e. having inserted heterologous DNA).
In general, in optimal conditions, one E.coli cell out of 10 5 or 10 6 will be transformed by the plasmid. It must be noted that there is no minimal size for the heterologous DNA inserted in a plasmid, and that this size can reach at least 40 x 10 6 daltons, i.e. about 60 000 base pairs (= 60 kb).
Moreover, the insertion of foreign DNA in plasmids of the Col series does not affect the number of plasmid molecules which can be present in the cell, so that the heterologous DNA inserted will be present also at the concentration of several copies in the transformed bacterial cell. Lastly, under the effect of chloram­phenicol one can have an amplification such that the circular DNA of the hybrid plasmid represents 40 to 50% of the totality of the DNA of the cell.
The DNA of the bacteriophage (47 000 base pairs = 47 kb) is often used as cloning vector because it offers some advantages over the bacterial plasmids. About 15 kb of the central part of the genome of this phage are not essential for its survival and can be replaced by heterologous DNA to be cloned.
One can carry out in vitro the encapsidation of the recombinant DNA obtained and thus considerably increase the infection efficiency of this DNA. The encapsidation mechanism of the phage is such that only DNA molecules having a final size of about 47 kb will be encapsidated, which brings about a spontaneous selection of DNA molecules, having inserted a heterologous fragment of about 15 kb.
Cosmids are small plasmids which contain an encapsidation signal (the “cos” site of phage ), an origin of replication and a gene of resistance to an antibiotic.
They therefore combine the advantages of the bacteriophage (ef­ficiency of infection of the encapsidated form) and those of bacterial plasmids (autonomous replication and ease of selection). Furthermore, one can insert in cosmids, fragments of heterologous DNA of larger size (up to 40 kb).
These two vectors of cloning (phage and cosmids) are often used for constructing genomic “banks” or “libraries” which are collections of recom­binant phages or plasmids that together comprise the totality of the sequences of a given genome. It is clear that the greater the size of genomic DNA fragments inserted, the smaller the number of clones needed to cover the totality of the genome: this is the special advantage of cosmids.
Cloning of a Specific Gene:
The problem is to select from a vast population of transformed cells, a cell which contains a hybrid plasmid carrying a particular heterologous gene. This is a serious problem, especially if the starting heterologous DNA is complex and if the fragments generated by the restriction enzyme are numerous. It is clear that either there must be a phenotypical property associated with the gene cloned, or one must have an appropriate probe.
If one clones a gene coding for an enzyme for example, one can transform a bacterial mutant lacking this enzyme and thus select the appropriate trans­formed cells on the basis of the restoration of the corresponding enzymatic activity.
It was thus possible to clone the genes of the tryptophan operon, or the gene of the ligase of E.coli, starting from DNA fragments resulting from the hydrolysis of the whole chromosome of E.coli. But if one wants to clone a eucaryotic gene, it is often preferable to clone first the DNA copy (cDNA) of the mRNA corresponding to the gene sought.
The size of these cDNAs is indeed generally much smaller than that of their genomic counterpart due to the absence of introns. One therefore constructs a cDNA “library” by inserting, in a plasmid or in a vector derived from bacteriophage (e.g., gt 10), cDNA copies of the entire population of cytoplasmic mRNAs extracted from the cells expressing the gene concerned.
These copies are made with the help of the reverse transcriptase which synthesizes the cDNA strands complementary to the mRNAs starting from oligo dT primers previously hybridized to the poly A tails of mRNAs.
After alkaline hydrolysis of the RNAs, double-stranded cDNA molecules are then obtained by action of DNA polymerase I of E.coli which makes strands complementary to those synthesized during the first step. The synthesis is “self-primed” at the 3′ end of the single-stranded cDNA which folds back upon itself (see fig. 6-31a).
A digestion by nuclease S1 (specific of single stranded nucleic acids) finally eliminates the loops at this end and gives perfectly double-stranded cDNAs. A cDNA library is said to be complete if it contains at least one bacterial clone for each starting mRNA.
There are several methods to identify the clones sought. If the cDNAs have been inserted downstream a bacterial promoter (as in the case of gt 11), one can analyse (or “screen”) the “library” by the expression of the cDNAs. This is done by immunological identification of the producer clones antibodies specific of the corresponding protein will react selectively with the relevant clones which will thus be labeled.
The success of this “screening” technique however depends on several factors: the cDNA must be inserted in the correct orientation with respect to the bacterial promoter of the vector, in order to permit the transcription of its coding strand the translation of the messenger thus produced must take place in the correct reading frame lastly, the tertiary structure adopted by the synthesized polypeptide must reconstitute the an­tigenic determinants (epitopes) recognized by the antibodies.
A second ap­proach, independent of these prerequisites, is based on recent developments in the micro-sequencing techniques of proteins and in the production of synthetic oligodeoxynucleotides. With the knowledge of the partial amino acid sequence of the protein one can, on the basis of the genetic code, deduce (taking into consideration the fact that the code is degenerate) the corresponding nucleotide sequence and carry out its chemical synthesis.
These oligonucleotides, labeled by 32 P, are then used as radioactive probes to identify, by hybridization with the complementary sequences, the bacterial clones con­taining the cDNA sought.
The cDNAs thus isolated can in their turn serve as probes to look for the corresponding genes in a genomic “bank”.
Expression of Cloned Genes:
These hybrid plasmids represent not only an abundant source of inserted heterologous DNA, but also allow the production – by the bacterial cell – of large quantities of products specified by the genes inserted, particularly when the cloned genes are from the same bacterium or another procaryote: for example, the cloning of the tryptophan operon of E.coli in a plasmid of the Col series has allowed the induced synthesis of such large quantities of the five enzymes of this operon that they represented 40% of the total proteins of the cell.
But when genes of eucaryotes are cloned in E.coli plasmids, it is observed that the synthesis — by the bacterium — of the products corresponding to these genes is very small, or even nil, most probably because of differences between the systems controlling transcription and translation in procaryotes and eucaryotes (for example in the initiation and termination of transcription and translation) or because of the absence of intron splicing systems in procaryotes.
Some of these problems can be solved by the insertion of a cDNA (DNA complementary to the mature mRNA) directly downstream a functional procaryotic promoter in the plasmid. In this manner one has ob­tained in bacteria, the synthesis of proteins of different origins: animal (growth hormone, somatostatin, insulin, interferon), plant (large sub-unit of ribulose 1, 5 bisphosphate carboxylase) or viral (antigen of the hepatitis B virus, or of the foot and mouth disease virus).
It must be emphasized however that the biologi­cal activity of some of these proteins can depend on subsequent modifications (specific proteolytic cleavages in the case of precursors or glycosylation) which the procaryotic host cannot perform.
Cloning of Genes in Eucaryotic Cells:
At the beginning, the cloning of genes was mostly carried out using E.coli, but the principles on which this process is based are also applicable to animal and plant cells. Viruses or plasmids capable of introducing genes in the chromosomes of eucaryotic cells were needed it was therefore envisaged to use viruses derived from Simian virus 40 (SV 40).
As far as the plant kingdom is concerned, it was shown that a fragment of the DNA (T-DNA) of a plasmid (Ti plasmid) of the bacterium Agrobacterium tumefaciens (responsible for the formation of a tumour called Crown-Gall) could be transferred to plant cell chromosomes.
Moreover, one could insert, in this T-DNA, genes of bacterial origin (e.g., gene of resistance to an antibiotic) or plant origin (e.g., gene of the small sub-unit of ribulose 1,5 bisphosphate carboxylase) and thus obtain their transfer to a tobacco protoplast since in various species (including tobacco) one can regenerate an entire plant from a protoplast, one could observe, starting from transformed protoplasts, the presence of the gene inserted in diverse tissues of the transformed plant, observe its expression and note that this expression is controlled by factors (such as light) which exert their regulatory effects at the transcriptional level in plants.
Some recent techniques enable the direct introduction of DNA into plant or animal cells without the help of any plasmid or viral vector (direct transfer of genes).
Advantages of the Cloning of Genes:
Cloning of genes provided for the first time, large quantities of specific DNA fragments, in other words genes, especially of higher organisms. These DNA fragments can be used as probe and permit, by hybridization, the detection and dosage of the corresponding mRNAs in cells which are at different stages of their development or placed in diverse physiological conditions.
On the other hand one can, thanks to the cloning of genes, undertake the study of the structure of genes and chromosomes of eucaryotes, and the study of the control mechanisms of gene expression in higher organisms.
But, beside the great advances that may be expected in the domain of fundamental knowledge in molecular biology, the cloning of genes can lead to extremely important applications.
In the field of agronomy, one can envisage transferring to plants:
1. A gene responsible for resistance to a herbicide (isolated for example from a microorganism) thus allowing the protection of the cultivated plant when weeds are eliminated by the herbicide
2. A gene responsible for resistance to a pathogen (e.g., fungus, virus, insect). It has been possible to obtain plants protected from infection by a virus (e.g., Tobacco Mosaic Virus) by introducing into these plants the gene coding for the coat protein of this virus, or plants protected from attack by insects by introducing into these plants the gene of a bacterial toxin having insecticide properties
3. Genes improving the growth of plants, for example, enabling a more efficient photosynthesis, or permitting atmospheric nitrogen fixation (although, in the microorganisms studied, this process implies the coordinated expression of 17 genes)
4. Genes improving the nutritional value of plants, for example, providing seed storage proteins richer in some essential amino acids, like lysine.
This assumes that the gene is not only introduced in the plant, but also expressed in the right tissue (e.g., the seed) and at the right time.
In medicine, the production of therapeutically important substances, by (bacterial, animal, human) cells in culture after introduction of the correspond­ing gene, is a major application of genetic engineering. Among substances which are already being produced or under study, one may cite: insulin (used in the treatment of diabetes), growth hormone (used against some forms of dwarfism), interferons (anti-viral action), the plasminogen activator (active against thromboses, because plasmin is an enzyme which degrades the fibrin of clots), α-anti-trypsin (active against emphysema, because it inhibits elastase which digests the elastin of the pulmonary conjunctive tissue), the blood factors VIII and IX (against hemophilia A and B, respectively), various viral antigens, like those of hepatitis B or foot and mouth disease (for the preparation of vaccines), etc.
It must be noted that the advantage lies not only in the production of large quantities of human proteins, which will not cause allergies (unlike for example, pig insulin used till now), but also in the fact that the products are free from contaminants, especially of viral origin, present particularly in substances prepared from human blood, particularly when it becomes necessary to pool the blood of many donors to prepare appreciable quantities of these products (for example in the case of antihemophilic factors), which increases risks (several hemophilic patients were thus contaminated by the hepatitis or Aids virus).
Genetic engineering techniques have also led to major advances in the diagnosis of hereditary diseases. After action of a restriction enzyme on the DNA of a patient, one visualizes the DNA fragments produced at the genetic locus of the disease by hybridization with an appropriate radioactive probe (DNA segment or synthetic oligonucleotide corresponding to this locus or to a portion of it).
The comparison of the size of DNA fragments thus revealed, with those produced in the same manner from a healthy subject permits the detection of anomalies if any: deletions or even, if the hybridization conditions are sufficiently selective (or “stringent”), point mutations.
When the gene responsible for the disease in question is not known, one may utilize the existence in the genome, of zones whose sequence may vary from one in­dividual to another (polymorphic zones or mutation hot points). These zones, characterized by the appearance or disappearance of cleavage sites for a given restriction enzyme, therefore present a restriction fragment length polymor­phism (RFLP).
Thus, if one has a probe which can reveal such a polymorphism near the presumed locus of the disease, one can associate the appearance of the disease in an individual with a restriction profile characteristic of the DNA of this individual. It is then possible, by analysis of the RFLP of the DNA of parents and descendants of this individual, to follow the segregation of the gene responsible for the disease, with the help of this probe.
It has also been envisaged to introduce the intact gene, into animal or human cells deprived of a particular enzymatic activity because of a mutation in the corresponding gene, and thus restore the activity lacking in these cells.
Important applications of genetic engineering are also possible:
i. In the field of environment, for example for the biodegradation of toxic products, or for microbiological extraction of various metals from low grade ore.
ii. In the field of energy, for example, for the production of methane and methanol, or for the recovery of crude oil not easily accessible by conventional extraction methods.
Precautions to be taken during Experiments of Genetic Recombination:
It has been said that living species could preserve their identity for a multi­tude of generations thanks to the presence of natural barriers, and that cloning of genes, by violating these barriers, could create new forms of life, dangerous to man and his environment.
Experiments on genetic recombination in vitro and cloning of genes, as well as the possible risks which could endanger humanity, have been the object of many controversies. Scientists carrying out such experiments in the United States as also in Europe have decided to lay down a number of precautionary measures.
While we will not discuss them here, we will just mention that such measures concern on the one hand, the laboratories where the experiments are carried out and on the other hand the plasmids and bacteria used. As regards the laboratories, one must naturally work in conditions which exclude risks of dissemination as in the case of experiments with pathogenic bacteria and viruses.
As far as the plasmids are concerned, one must preferably use plasmids lacking genes which permit bacterial conjugation this will reduce the risk of dissemination of hybrid plasmid molecules in our environment.
What Are Advantages of Multicellular Organisms?
Multicellular organisms enjoy several distinct benefits, including a larger size and greater complexity than unicellular organisms. Multicellular organisms include many types of plants and animals while the class of unicellular organisms forms primarily from microorganisms, amoeba and bacteria.
Multicellular organisms, as the name implies, have many types of cells, while unicellular organisms contain just one cell each. Both kinds of organisms reproduce through meiosis or mitosis. Multicellular organisms generally form the higher tiers in the web of life. These living beings, including plants and animals, have a more complex internal layout than unicellular creatures. They rely on specialized cells and cell departments for carrying out specific tasks, such as breaking down food, sending electrical messages and other duties necessary for supporting life. This distinction, called cell differentiation, lets multicellular organisms engage in more complex physical and cognitive tasks than unicellular organisms.
In addition to having specialized cells, multicellular organisms have separate organ systems to perform specific tasks. These systems, such as the cardiovascular, digestive and respiratory systems, perform life processes necessary for survival. Digestive systems, for instance, deliver nutrients and energy to organs in the digestive tract, letting them process and digest food. These organ systems also facilitate communications between different types of systems, such as the circulatory and nervous systems.
Morphologic and Structural Effects
Infection of permissive cells with virus leads to productive infection and often results in cell death (cytocidal, cytolytic infection). The first effects of the replication of cytocidal viruses to be described were the morphologic changes known as cytopathic effects. Cultured cells that are infected by most viruses undergo morphologic changes, which can be observed easily in unfixed, unstained cells by a light microscope. Some viruses cause characteristic cytopathic effects thus, observation of the cytopathic effect is an important tool for virologists concerned with isolating and identifying viruses from infected animals or humans (Fig. 44-1).
Development and progression of viral cytopathology. Human embryo skin muscle cells were infected with human cytomegalovirus and stained at selected times to demonstrate (A) uninfected cells, (B) late virus cytopathic effects (nuclear inclusions, cell (more. )
Many types of cytopathic effects occur. Often the first sign of viral infections is rounding of the cells. In some diseased tissues, intracellular structures called inclusion bodies appear in the nucleus and/or cytoplasm of infected cells. Inclusion bodies were first identified by light microscopy in smears and stained sections of infected tissues. Their composition can often be clarified by electron microscopy. In an adenovirus infection, for example, crystalline arrays of adenovirus capsids accumulate in the nucleus to form an inclusion body.
Inclusions may alternatively be host cell structures altered by the virus. For example, in reovirus-infected cells, virions associate with the microtubules, giving rise to a crescent-shaped perinuclear inclusion. Infection of cells by other viruses causes specific alterations in the cytoskeleton of cells. For example, extensive changes in cellular intermediate filaments in relation to formation of viral inclusions may be observed after cytomegalovirus infection (Fig. 44-4). Some characteristics of inclusion bodies produced by various viruses are listed in Table 44-2.
Alteration of cytoskeleton organization by virus infection. Normal cells have networks of microtubules, and intermediate filaments throughout the cytoplasm. Infection with reovirus causes a perinuclear aggregation of microtubules, and infection with cytomegalovirus (more. )
Viral Inclusion Bodies in Some Human Diseases.
A particularly striking cytopathic effect of some viral infections is the formation of syncytia, or polykaryocytes, which are large cytoplasmic masses that contain many nuclei (poly, many karyon, nucleus) and are usually produced by fusion of infected cells (Fig. 44-2). The mechanism of cell fusion during viral infection probably results from the interaction between viral gene products and host cell membranes. Cell fusion may be a mechanism by which virus spreads from infected to uninfected cells.
Formation of multinucleated cells. The figure represents the cytopathology of measles virus-induced syncytia.
Effects on Cell Physiology
Research into the pathogenesis of virus infections suggests a close correlation between cellular physiologic responses and the replication of some viruses (Fig. 44-3). In other words, the physiological state of living cells has a significant effect on the outcome of the virus infection, since the host cell provides the synthetic machinery, key regulatory molecules, and precursors for the newly synthesized viral proteins and nucleic acids. The optimal intracellular environment for virus replication develops through events that begin to take place with attachment of virus to the cell membrane. Binding of virus to the cell membrane receptor(s) may be followed by cascades of events that are associated with biochemical, physiological and morphological changes in the cells. The virus receptor is a cell membrane component that participates in virus binding, facilitates viral infection, and is a determinant of virus host range, as well as tissue tropism. Some viruses recognize more than one cellular receptor (e.g., HIV, adenoviruses) and the binding is a multistage process (see Table 44-3). Multiple receptors may act together either to modulate each other's activity or to contribute complementary functions.
Relationship of morphological, physiological, and biochemical cellular effects to the replication of human cytomegalovirus. Examples for biochemical and physiological cellular responses: a formation of secondary messengers, Ca 2+ influx, activation of protein (more. )
Proposed Cell Membrane Receptors for Viruses.
Other virus-associated alterations in cell physiology are related to insertion of viral proteins or other changes in the cell membrane. One example is the leaky cell membrane that appears after infection with picornaviruses or Sindbis virus the change in intracellular ion concentrations that results from the leaky membrane may favor translation of the more salt-stable (e.g., Na + or K + ) viral mRNA over cellular, mRNA. These and other effects may be maintained or modified by immediate early and/or early viral gene products (e.g., changes in transcription and protein levels of cell cycle regulatory molecules). Figure 44-3 demonstrates the coordination of cellular physiologic responses with the replication of a herpesvirus (human cytomegalovirus).
Effects on Cell Biochemistry
Virus binding to the cell membrane in concert with immediate early (e.g., IE proteins of herpesviruses), early non-structural proteins (e.g., E-6, E-7 of HPVs) or virion components (e.g., ICP0, ICP4, VP16 of herpes simplex virus, penton protein of adenoviruses) may mediate a series of biochemical changes that optimize the intracellular milieu for use of cellular synthetic machinery, low molecular weight precursors for productive virus replication or to achieve latency, chronic, slow or transforming infection. For example, studies of transcriptional regulation of viral genes and post-transcriptional modification of gene products (splicing, polyadenylation of RNA) demonstrate that the nature of the basic biochemical processes for virus replication are similar to the mechanisms used to regulate expression of cellular genes. Viruses have sequence motifs in their nucleic acid for binding of known transcriptional regulators of cellular origin. Thus, promoter regions of regulatory and structural proteins for many viruses (Table 44-4) contain contiguous binding sites for a large array of identifiable mammalian cellular transcription factors (e.g., NFκ B, Sp1, CRE/B, AP-1, Oct-1, NF-1). These cellular transcription factors in concert with regulatory viral proteins are involved in activation or repression of viral and cellular genes to develop latent, persistent, transforming virus infections, as well as to produce progeny virus. Most cellular transcription factors must be activated prior to binding to their specific recognition (consensus) sequences. The biochemical events may include phosphorylation, dephosphorylation, disassociation (from inhibitory subunit) and dimerization. These activation processes can be accomplished as a result of the cascade of events initiated by the virus and cell receptor interaction. Events associated with these cascades may include, for example, formation of secondary messengers (phosphatidyl inositols, diacylglycerols, cAMP, cGMP, etc.), activation of protein kinases, and ion (e.g., Ca 2+ ) influxes.
Cellular Transcription Factors that are Involved in Regulation of Viral Gene Expression.
To maintain cell activation processes, viruses have evolved unique mechanisms to regulate these cellular processes, adapting their proteins to interact with cellular proteins. Examples include the association of early virus gene products (e.g., E-6, E-7 of papillomaviruses IE proteins of herpesviruses SV40 T antigen) with the Rb tumor suppresser protein which results in liberation of the E2F transcription factor that is required for modification (activation/inhibition) of cellular biochemical pathways, for synthesis of viral DNA, or initiation of cellular apoptotic processes (programmed cell death).
In some cases the virus directly incorporates cellular biochemical regulatory strategies by triggering the cells to overproduce and excrete regulatory molecules (e.g., transforming growth factors, tumor necrosis factors, interleukins), which may activate in an autocrine fashion cellular biochemical cascades involved in virus (e.g., HIV, herpesviruses, papillomaviruses) replication, maintenance of or reactivation from a latent state, or maintaining a transformed phenotype. On the other hand, these soluble cellular regulatory molecules may inhibit biochemical reactions of immune cells in a paracrine manner to compromise elimination of infected cells.
Inhibition of cellular macromolecule synthesis may result from virus infection and provide an advantage for synthesis of virus proteins and nucleic acids in the absence of competing synthesis of cellular products. This inhibition occurs in characteristic ways. In poliovirus or herpes simplex infections, for example, selective inhibition of host protein synthesis occurs prior to the maximal synthesis of viral proteins. In some cases, viral products inhibit both protein and nucleic acid synthesis. Purified adenovirus penton fibers significantly decrease the synthesis of host protein, RNA, and DNA. Total inhibition of host macromolecular synthesis also may occur when excess viral products accumulate in the cell late in the viral replicative cycle. Some picornaviruses specify a protein that causes cell damage independent of the viral proteins that inhibit cell macromolecular synthesis. Cellular mRNA may be degraded. For example, in influenza virus and herpes simplex virus infections, cellular mRNA stops binding with ribosomes to form polyribosomes only virus-specific mRNA is bound, giving viral mRNAs a selective advantage. Cell DNA synthesis is inhibited in most cytolytic virus infections. This may be achieved by virus-induced apoptosis or by a decrease in cellular protein synthesis. Reoviruses and some herpesviruses may be exceptions in that they cause a decrease in cell DNA synthesis before a substantial decline in cellular protein synthesis occurs. Direct degradation of host DNA is seen in vaccinia virus infections due to a virion-associated DNase.
Chromosome damage may be caused directly by the virus particle or indirectly by events occurring during synthesis of new viral macromolecules (RNA, DNA, protein). The chromosome damage (Fig. 44-5) may or may not be faithfully repaired, and in either case, it may or may not be compatible with survival of the infected cell. When the cell survives, the virus genome may persist within the cell, possibly leading to continued instability of cellular genomic material or to altered expression of cellular genes (e.g., cellular oncogenes). Virus-induced genomic instability appears to be associated with accumulation of mutations and related to the process of cell immortalization and oncogenic transformation.
Chromosomal aberrations resulting from cytomegalovirus infection of human peripheral blood lymphocytes.
The biologic consequences of virus infection results from the aforementioned biochemical, physiological, structural, morphological and genetic changes. In productive infections virus-induced biological modifications of the cell may be closely related to the efficiency of virus replication or to the recognition of these cells by the immune system. For cells that are persistently infected, the cellular changes caused by the virus could lead to disease (e.g., subacute sclerosing panencephalitis after measles infection), cellular genetic damage (e.g., hepatitis B virus), immortalization (e.g., Epstein-Barr virus), or malignant transformation (e.g., HTLV-1, HTLV-2, hepatitis B virus). The wide variety of these effects of virus infection points to the complex interaction between the viruses and their host cell.
Relation of Cellular Effects to Viral Pathogenesis
Although most of the events that damage or modify the host cell during lytic infection are difficult to separate from viral replication, the effects are not always linked directly to the production of progeny virions. For example, changes in cell size, shape, and physiologic parameters may occur before progeny virions or even many virus proteins, are produced. These alterations in cell structure and function may be important aspects of the pathogenesis of a number of viral infections (see Ch. 45). For example, through their cellular effects many viruses (e.g., rotaviruses, caliciviruses, Norwalk viruses) induce gastrointestinal symptoms (ranging from mild alteration in absorption of ions to severe watery diarrhea). Cytocidal viral infections (e.g., herpesviruses, togaviruses, flaviviruses, bunyaviruses) of the central nervous system are related to necrosis, inflammation or phagocytosis by supporting cells. Rubella virus infections are associated with demyelination without neural degeneration. The long-term effects of persistent virus infections (see below) may also be related to such progressive diseases as atherosclerosis and demyelination in multiple sclerosis.
Organization of the Nucleus and Its DNA
Like most other cellular organelles, the nucleus is surrounded by a membrane called the nuclear envelope. This membranous covering consists of two adjacent lipid bilayers with a thin fluid space in between them. Spanning these two bilayers are nuclear pores. A nuclear pore is a tiny passageway for the passage of proteins, RNA, and solutes between the nucleus and the cytoplasm. Proteins called pore complexes lining the nuclear pores regulate the passage of materials into and out of the nucleus. Inside the nuclear envelope is a gel-like nucleoplasm with solutes that include the building blocks of nucleic acids. There also can be a dark-staining mass often visible under a simple light microscope, called anucleolus (plural = nucleoli). The nucleolus is a region of the nucleus that is responsible for manufacturing the RNA necessary for construction of ribosomes. Once synthesized, newly made ribosomal subunits exit the cell’s nucleus through the nuclear pores. The genetic instructions that are used to build and maintain an organism are arranged in an orderly manner in strands of DNA. Within the nucleus are threads of chromatin composed of DNA and associated proteins (Figure 3.22). Along the chromatin threads, the DNA is wrapped around a set of histone proteins. Anucleosome is a single, wrapped DNA-histone complex. Multiple nucleosomes along the entire molecule of DNA appear like a beaded necklace, in which the string is the DNA and the beads are the associated histones. When a cell is in the process of division, the chromatin condenses into chromosomes, so that the DNA can be safely transported to the “daughter cells.” The chromosome is composed of DNA and proteins it is the condensed form of chromatin. It is estimated that humans have almost 22,000 genes distributed on 46 chromosomes.
18 Questions on the Function of the Cell Nucleus
The mains elements of the nucleus are chromatin (made of DNA molecules), the nucleolus, the karyolymph, or nucleoplasm, and the nuclear membrane (or karyotheca).
More Bite-Sized Q&As Below
2. Do all eukaryotic cells have only one nucleus?
Some eukaryotic cells do not contain a nucleus and others contain more than one. For example, osteoclasts, the cells responsible for resorption of bone matrix, are multinucleate cells, meaning they have more than one nucleus. Striated muscle fibers are also multinucleate. Red blood cells are an example of enucleated (no nucleus) specialized cells.
Chromatin, Heterochromatin and Euchromatin
3. What substances is chromatin made of?
Chromatin is made of DNA molecules bound to proteins called histones.
Cell Nucleus Review - Image Diversity: chromatin
4. What are heterochromatin and euchromatin?
Chromatin is uncondensed nuclear DNA, the typical DNA morphology during interphase (the phase of the cell cycle in which the cell does not divide). During this phase of the cell cycle, which is the chromatin can be found as heterochromatin, more condensed portion of DNA molecules and which appears darker under electron microscopy, and as euchromatin, which is less condensed and composes the lighter portions of DNA molecules.
Since it is less condensed, euchromatin is the biologically active part of the DNA, that is, the region that contains active genes to transcribe into RNA. Heterochromatin makes up the inactive portions of the DNA molecule.
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5. What is the relationship between the concepts of chromatin and chromosomes? Are euchromatin and heterochromatin a part of chromosomes?
Every filament of chromatin is a complete DNA molecule (a complete double helix), or rather, a complete chromosome. A DNA molecule may contain euchromatin and heterochromatin portions and, as a result, both are a part of chromosomes.
6. During the phase when the cell is not dividing (interphase), is there activity within the cell nucleus?
During interphase, there is intense metabolic activity in the cell nucleus: DNA is duplicating, euchromatin is being transcripted and RNA is produced.
7. How are the concepts of chromosomes, chromatin and chromatids related? During which phase of the cell cycle does DNA replicate?
Chromatin is a set of filamentous DNA molecules dispersed in the karyoplasm, made up of euchromatin and heterochromatin portions. Each chromatin filament is a complete chromosome (a DNA molecule, or double helix). The chromatin of the human somatic cell is formed by 46 DNA molecules (22 homologous chromosomes and 1 pair of sex chromosomes).
During interphase, the cell prepares itself for division and the duplication of DNA molecules occurs. The duplication of every DNA molecule forms two identical DNA double helices bound by a structure called the centromere. During this phase, each identical chromosome of these pairs is called a chromatid. It is also during interphase that chromatids begin to condense, taking on the thicker and shorter shape typical of chromosome illustrations. Therefore, the phase of the cell cycle during which DNA duplicates is interphase.
Some biology textbooks refer to a chromosome as a unique filament of chromatin as well as the condensed structure made of two identical chromatids after DNA duplication. The pair of identical chromatids bound in the centromere is always made up of two copies of the same chromosome, therefore, they are two identical chromosomes (and not only one).
8. What structure maintains the binding of identical chromatids?
The structure that maintains the binding of identical chromatids is the centromere.
9. What is the name given to region of the chromosome where the centromere is located? How are chromosomes classified in relation to the position of their centromere?
The region of the chromosome where the centromere is located is called primary constriction. Under a microscopic view, this region is narrower (a stricture) than most parts of the chromosome.
Depending on the position of the primary constriction, chromosomes are classified as telocentric, acrocentric, submetacentric or metacentric.
10. What are the primary and secondary constrictions of a chromosome? What is the other name given to the secondary constriction?
Primary constriction is the narrower region of a condensed chromosome where the centromere, the structure that binds identical chromatids, is located. The secondary constriction is a region similar to the primary constriction, narrower than the normal thickness of the chromosome, and which is normally related to genes that coordinate the formation of the nucleolus and which control ribosomal RNA (rRNA) synthesis. For this reason, secondaryonstrictions (there can be one or more in a chromosome) are called nucleolus organizer regions (NOR).
11. What are homologous chromosomes? Which human cells do not have homologous chromosomes?
Chromosomes contain genes (genetic information in the form of nucleotide sequences) that control protein synthesis, thus regulating and controlling cell activities. In the nuclei of somatic cells of diploid beings, every chromosome has its corresponding homologous chromosome, both of which contain alleles of the same genes related to the same functions. This occurs because one chromosome of one pair comes from the father and the other comes from the mother of an individual. Chromosomes that form a pair with alleles of the same genes are called homologous chromosomes. In humans, there are 22 pairs of homologous chromosomes plus one pair of sex chromosomes (sex chromosomes are partially homologous).
The only human cells that do not have homologous chromosomes are gametes, as during meiosis, the homologous chromosomes are separated.
Karyotypes and Genomes
12. What is the difference between karyotype and a genome?
A genome is the set of DNA molecules that characterizes each living being or each species. This concept includes the specific nucleotide sequence of the DNA molecules of each individual or species. A karyotype is the set of chromosomes of a given individual or species, and focuses on the number of pairs of chromosomes as well as their morphology.
13. Can two normal individuals of the same species with sexual reproduction have identical genomes and identical karyotypes? How is the human karyotype usually represented?
Except for clones (individuals created from nucleus transplantation, like Dolly the sheep) and monozygotic twins, it is very improbable that the genomes of two individuals of the same species generated by sexual reproduction will be identical. Nevertheless, the karyotypes of two normal individuals of the same species and of the same sex are always identical. The normal human karyotype is represented by the formula 44+XX for women and 44+XY for men.
Alosomes and Ploidy
14. What is the other name given to sex chromosomes? What is the function of sex chromosomes?
Sex chromosomes are also called allosomes (other chromosomes that are not sex chromosomes are called autosomes).
Sex chromosomes take their name from the fact that they have genes that determine the sex (male or female) of an individual. Sex chromosomes also contain genes related to other biological functions.
15. How many chromosomes does a normal human haploid cell have? How many chromosomes does a normal human diploid cell have? How many sex chromosomes do each of them contain?
The human haploid cell is the gamete (egg cell and sperm cell). The human gamete has 22 autosomes and 1 allosome, i.e., 23 chromosomes. The diploid cell is the somatic cell and it has 44 autosomes and 2 allosomes, i.e., 46 chromosomes.
Gametes have one sex chromosome and somatic cells have two sex chromosomes.
16. Do phylogenetically close species have cells with similar chromosome counts?
The number of chromosomes typical of each species is similar for phylogenetically close species (for example, orangutans, gorillas, chimpanzees and humans). However, it is not impossible for evolutionarily distant species, such as rats and oats, to have similar karyotypes and the same total number of chromosomes.
Even if they present equal number of chromosomes, evolutionarily distant species have radically different characteristics, since the quantity and the sequence of nucleotides that make up their DNA molecules are quite different.
The Nucleolus and the Nuclear Membrane
17. What is the nucleolus?
The nucleolus is a small and optically dense region in the interior of the cell nucleus. It is made of ribosomic RNA (rRNA) and proteins. One nucleus can have one or more nucleolus.
18. What structures make up the nuclear membrane?
Eukaryotic cells have a nucleus that is enclosed by two juxtaposed membranes that are a continuation of the membrane of the endoplasmic reticulum. The nuclear membrane, or karyotheca, contains pores through which substances pass. In addition, its external surface contains ribosomes.
Now that you have finished studying Cell Nucleus, these are your options:
Examples of symbiosis
As will be clearer through the examples, symbiosis relationships are very important in the environment , as they enable many species to survive. That is why we believe that symbiosis works as an enhancer of the evolution of these species, which manage to improve their way of life by establishing relationships with other organisms and species.
The examples are very http://kamagrawiki.org numerous and varied. Next, we present some examples of symbiosis in ecology and biology so that, in this way, the importance that these types of relationship suppose for the survival of these organisms becomes clearer.
- Ants and aphids: some species of ants, such as the black ant ( Lasius niger ), protect herds of aphids that in turn provide them with food and molasses, a sugary substance they produce rich in carbohydrates. In the main image of this article, we can see this same example.
- Ants and acacias: other species of ants such as Pseudomyrmex ferruginea protect acacias from other parasites or herbivores. In return, the tree provides shelter and food.
- Crocodiles and plovers: it is by all known the great power that the crocodiles possess in the jaws. These present no less than 80 teeth, which replace 2 or 3 times a year and the remains of food can cause serious problems such as infections. Thus arises the relationship with the Egyptian plovers. They obtain their food by cleaning the remains they find between the teeth of the crocodiles and these thus avoid oral problems allowing them to move inside their mouths.
- Sharks and remoras: this is the clearest case of commensalism. Surely you have seen other sharks under the sharks. They adhere to the sharks and obtain from them protection and food from the remains of food that do not ingest them. For sharks, the presence of remora is practically indifferent.
- Goby fish and blind prawn: the shrimp, despite its lack of vision, digs the burrow that keeps it clean and allows the fish to share so that it acts as their guide for the search for food and, in addition, warns of the dangers that they lurk through movements of its tail that create vibrations that the shrimp is capable of detecting, at which time both can hide in the burrow.
- The clownfish and the anemone: these fish make their whole lives inside the anemones, which are very poisonous. They establish a mutualistic relationship in which the clownfish attracts other predatory fish that, when they come in contact with the anemone, are paralyzed and serve as food, the remains of which are used by the clownfish.
- Lichens: are the symbiotic association between a fungus and algae. The fungus protects the algae from dehydration and provides a structure to grow on, and the algae make carbohydrates that the fungus can use as food. There is a great variety of lichens since they are very resistant and capable of colonizing very diverse environments.
- Mycorrhizae: Mycorrhizae are fungi that establish symbiotic relationships with multiple plant species of vascular plants. How? The roots of these plants secrete useful substances for these fungi and these, in turn, make materials found in the soil as minerals and other materials in decomposition are more assimilable by plants.
- Intestinal flora and microbiota: in our intestine, as in many other parts of our body, there is a large number of bacteria and other microorganisms that live in symbiosis with our cells and that are of great importance to our health to such an extent that variations in this microbiota can cause alterations in our body.
Now that you know well what is the symbiosis in ecology and biology and have seen several examples, you may also be interested in knowing with this other Green Ecology article the interspecific relationships: types and examples
The difference between specialised and unspecialised cells.
What cell differentiation is.
A specialised cell is when a cell has certain features that make it very good at its job.
Cell differentiation is when an unspecialised cell becomes specialised.
Before you were born, you started as just a bunch of cells! These cells were stem cells. Your DNA had told certain cells to change their appearance and contents, to become specialised! When specialised cells start to form, then things like skin and bones can start forming - making you!
2D Versus 3D Cell Cultures: Advantages and Disadvantages
/>2D cell cultures have been used since the early 1900s. In recent years, 3D cell culture techniques have received much attention from scientists, as these might provide more accurate models of tissues.
Here, we will explore the differences between the two types of cell cultures and explain why 3D cell culture systems are becoming so popular.
A (Very) Brief History of 3D Cell Cultures
3D cell cultures have been around for longer than you might think. Ross Granville Harrison (1870&ndash1959) adapted the hanging drop method from bacteriology to carry out the first tissue culture. This method allowed for further studies in embryology as well as experimental improvements in oncology, virology, genetics and a number of other fields.
The importance of the tissue microenvironment and 3D culturing techniques for cancer research was first proposed in the early 1980s by Mina Bissell, a lead researcher at Lawrence Berkeley National Laboratory. Since then, Mina has set up her own lab and continues to develop 3D techniques, especially in cancer discovery and treatment.
The 1990s were a quiet time for 3D cell cultures. This all started to change over the last five to 10 years. We&rsquove seen venture capitalists, angel investors and pharmaceuticals pouring lots of money into 3D cell culture technologies, research and production.
The reason? The enormous potential for 3D cell culture in drug development, which represents a big business opportunity. In our bodies, cells don&rsquot grow in 2D, and it&rsquos precisely the human body that we should model to develop better therapies against cancer and other diseases.
2D Cell Culture Systems
2D cell culture systems grow cells on flat dishes, typically made of plastic. The cells are put onto coated surfaces where they adhere and spread. Unfortunately, 2D cell cultures do have some inherent flaws:
- They aren&rsquot representative of real cell environments &mdash Growing on a flat surface isn&rsquot a good way to understand how cells grow and function in a human body, where they surrounded by other cells in three dimensions.
- Lack of predictivity &mdash 2D cell testing isn&rsquot always predictive, which increases the cost and failure rate of new drug discovery and clinical trials. Pharmaceutical companies spend hundreds of millions on failed drug development every year.
- Issues caused by the growth media and expansion of cells &mdash As cells grow in a standard 2D culture, they consume growth media and exude waste. This can result in toxic waste products, dead cells, nutrition depletion and damage of the environment the cells are in.
Why 2D Cultures Are Still Used in the Majority of Cell Research
Despite these disadvantages, 2D cell cultures are still used for the majority of cell cultures. Reasons include:
- It&rsquos inexpensive &mdash Economies of scale mean 2D cell culture systems and technologies are less expensive than some other systems.
- It&rsquos well established &mdash 2D cell cultures have been used since the early 1900s, gaining widespread acceptance in the 1940s and 1950s.
- There&rsquos a lot of comparative literature &mdash It&rsquos easier to compare current results vs. previous results and studies.
- It&rsquos what most researchers and scientists understand &mdash Everyone involved with lab work has been taught 2D cell culture technology.
- Easier cell observation and measurement &mdash 2D cell cultures are typically easier to analyze than some 3D cell culture systems.
3D Cell Culture Techniques
The reason for the growth in 3D cell culture is it&rsquos simply a better way of representing human tissue outside the body. 2D cell cultures only exist in two dimensions. That&rsquos not an accurate representation of how cells grow or how they are affected by disease and injury.
Although 2D cell cultures are still used for most research, the 3D cell culture industry is starting to catch up, especially in cancer treatment and stem cell research. Reasons for the increasing acceptance and use of 3D cell cultures include:
- More relevant cell models &mdash Much better biomimetic tissue models make 3D cell cultures more physiologically relevant and predictive than 2D cultures. 3D plate cultures also show a higher degree of structural complexity and retain a &ldquosteady state&rdquo (homeostasis) for longer.
- Interaction between different types of cells &mdash Creation of complex systems linked together by microfluidics means that 3D tissue systems can better model how different types of cells interact.
- Integration of flow &mdash Fluid flow, for example blood flow, interstitial fluid flow and urine flow, is crucially important for the functioning of all tissues. Cells respond to flow through differentiation and metabolic adaptation
- Establishment of barrier tissues &mdash Epithelia typically separate organ compartments, interact with the environment and protect the organism from the environment. Their proper functioning is crucial for survival and barrier malfunction plays a role in many diseases. Representation of barrier tissues is greatly enhanced in 3D culture.
- Better simulation of conditions in a living organism &mdash Microfluidics continuously provide nutrients to where they&rsquore needed, meaning cells and organs grow in a more realistic way.
- Reduces use of animal models &mdash Animal models aren&rsquot a reliable way to predict how drug treatments will affect humans. Screening drugs against human organs grown on chips is a much more reliable method.
- More realistic way to grow and treat tumor cells &mdash 3D cell culture systems are good simulators of diseased tissue, including cancer tumors. They can exhibit similar growth and treatment patterns.
3D Cell Culture Techniques: Issues and Solutions
Some 3D cell culture systems do still have problems, but at Mimetas, we've come up with solutions.
- Throughput &mdash Many 3D culture techniques are cumbersome and time-consuming, rendering them unsuitable for drug development screening and research. The OrganoPlate ® is based on a standard 384 well plate and provide the throughput needed for large-scale experimentation and screening.
- Challenges in microscopy and measurement &mdash Due to the larger size of 3D cell cultures compared to 2D cell cultures, some types of measurement and microscopic analysis can be difficult. Mimetas has created technology specifically designed to allow for easy microscopy results, assays and readouts across all of its 3D plates.
- Getting oxygen and other essential nutrients to the right place &mdash For larger cultures, it can be a challenge to distribute nutrients to where they need to be in the cell culture. Enhanced pump-free OrganoPlates® vascularization in organ-on-a-chip devices, which Mimetas is developing and architecture are helping to deal with this.
- Certain tissue models may contain undesirable elements &mdash In rare cases, 3D cultures created from specific tissue types (e.g. basement membrane extracts) can contain unwanted components such as growth factors or viruses.
A Vital Part of 3D Cell Culture: The 3D Scaffold
There are two main types of 3D cell culture systems, a scaffold system, and a scaffold-free system. Scaffold-free systems typically use techniques such as &ldquohanging drop templates,&rdquo &ldquomagnetic levitation,&rdquo and &ldquomagnetic 3D bioprinting.&rdquo Although there&rsquos been some development in these fields, most of the focus is on regular 3D scaffold systems.
A 3D scaffold provides a way for cells to grow in three dimensions on a 3D plate. These are typically provided through something called hydrogel, an extracellular matrix (ECM) in which cells can survive, grow and proliferate. These ECMs normally have tiny pores that allow the passage of nutrients and gasses to give the cells the environment they need to thrive. Well-developed and selected ECMs also provide essential cues to cells, rendering them crucial for the establishment of physiologically relevant 3D tissue cultures.
This means 3D cell cultures with ECMs can be accurately grown and measured, providing an ideal environment for drug discovery and development and other types of research.
The Evolution of 3D Cell Culture Systems
3D cell cultures, systems, plates, and techniques have come a huge way over the last 10 years. Still, we&rsquove barely started. As physicians, scientists, and pharmaceutical companies learn the potential of 3D tissue culture, we&rsquoll see more rapid innovation and wide-ranging applications.
At MIMETAS, we&rsquore proud to be at the forefront, making personalized medicine and affordable drug screening a reality for everyone with OrganoPlates and OrganoPlate-based applications.
To build predictive models for malaria parasite multiplication we need to understand the dynamic relation between cycle time, cell volume and nuclear numbers. Therefore, we need to quantify those very basic cellular parameters in a time-resolved manner. The description of blood-stage development has been dominated by the useful, but limited, separation in ring, trophozoite and schizont stages. The more gradual change of key biophysical parameters, however, is best assessed by dynamic single cell analysis i.e. 4D live cell imaging, a technology that still has potential for a broader application in the malaria field (Gruring etਊl., 2011 De Niz etਊl., 2016). By verifying the dependency of merozoite number on schizogony duration we can establish whether timing plays a determinant role. This assumes that we also assess the variability in nuclear division rates. A strict dependency on the concentration of specific extrinsic factors would support a counting mechanism. In this context it will be important to verify whether progeny number generated by a parasite is an inheritable, possibly epigenetic, trait. Investigating this requires tracking progeny number from one to the next generation. Whether this number correlates with final cell size is another relevant parameter. Finally, all these parameters should be linked to parasite multiplication rates to predict how they might correlate with disease severity. Knowing that multiplication rates can increase throughout culture adaptation, we further need to assess the validity of those cellular parameters in samples that have more recently been isolated from patients (Stewart etਊl., 2020). Ultimately, we advocate for the systematic assessment of merozoite number in Plasmodium mutants that develop a growth phenotype, so that we can expand our list of candidate proteins implicated in defining progeny numbers and advance our understanding of malaria parasites proliferation mechanisms.