Probiotics

The notion of probiotics has recently developed and most pharmacists have not been trained in these new food supplements.

From birth, our gastrointestinal tract is colonized by many microorganisms that constitute the digestive microbiota. This complex and diversified ecosystem, unique to each individual, contributes to the proper functioning of the intestine through the many activities it carries out. However, the balance of the microbiota is sensitive and its rupture occurs in the pathophysiology of various intestinal disorders, hence the idea of positively modulating a microbiota unbalanced by the administration of probiotics.

The term “probiotic” means “for life” and refers to living microorganisms that, when ingested in appropriate amounts, produce a benefit to the health of the host that goes beyond basic nutritional functions.

Probiotics are often lactic acid bacteria (lactobacilli and bifidobacteria) or yeasts introduced into the diet in the form of fermented milk products or food supplements.

These microorganisms strengthen the intestinal and vaginal flora. Their presence makes it possible to fight against the proliferation of pathogenic bacteria.

Several clinical studies have already demonstrated the efficacy of certain probiotics in the treatment of systemic and infectious diseases such as acute diarrhea and Crohn’s disease.

Other studies have suggested a potential application for the treatment of urogenital infections, colon cancer, atopic dermatitis and allergic diseases including food allergy such as lactose intolerance.

History

The definition of probiotics has evolved over time according to researchers, scientific knowledge and technological advances.

In the 20th century the Nobel Prize winner, Elie Metchnikoff, observed that a surprising number of people in Bulgaria lived for more than 100 years. This longevity could not be explained by the advances in modern medicine, because Bulgaria, one of the poorest countries in Europe at that time, did not benefit from such advances. Dr. Metchnikoff found that Bulgarians consume large quantities of yogurt, he associated the increase in longevity observed with the consumption of living microorganisms from fermented dairy products. Although Metchnikoff saw germs as rather harmful to human health, he considered it beneficial to replace bacteria in the gastrointestinal tract with yogurt, including the Bulgarian bacillus. He then explained the better beneficial effect of this bacteria by the absence of alcohol production (harmful to longevity), compared to bacteria present in other fermented milk such as kefir or kumys. In addition, he assumed that the lactic acid produced, as well as other unidentified factors, would act synergistically to inhibit the growth of putrefaction bacteria in the colon.

At the same time, in 1906, the French pediatrician Henry Tissier observed that the stools of children with diarrhea contained a small number of bifidobacteria compared to the stools of healthy children. He then suggested that these bacteria be administered to diarrheal patients to help them restore a healthy intestinal microbiota.

Metchnikoff and Tissier are therefore the first to put forward the idea of administering exogenous microorganisms to compensate for a possible dysfunction in our intestinal ecosystem. The concept of “probiotics” was born.

Nevertheless, it was not until 1954 that the term probiotics was introduced into the literature by Ferdinand Vergin in a paper entitled “Anti-und Probiotika”. This term derived from the Greek “pro bios”, which literally means “for life” as opposed to the harmful effects of antibiotics

In 1965, Lilly and Stilwell, in the journal Science, defined probiotics as substances produced by microorganisms capable of stimulating the growth of other microorganisms.

In 1989, Fuller highlighted the microbial nature of probiotics by redefining the term as a “living microbial nutritional supplement that has a positive effect on the host animal by improving its intestinal balance”.

In 1992, Havenaar and Huis in’t Velt further refined the term to “a viable culture composed of one or a mixture of bacteria that, when applied to animals or humans, has a beneficial effect on the host by improving the properties of native flora. ».

In 1998, Guarner and Schaafsmaa specified that probiotics are “living microorganisms, which, when consumed in adequate amounts, have a beneficial effect on the health of the host”.

In 2002, the World Health Organization (WHO) and the Food and Agriculture Organization of the United Nations (FAO) formalized the definition of probiotics to avoid any drift.

Probiotics are therefore defined as “living organisms that, when ingested in sufficient quantities, have a beneficial effect on the health of the host”.

History, therefore, underlines that the current definition could still evolve, as there are still many fields of research to better understand and understand the action of probiotics.

Regulation

The conditions and marketing authorization of probiotics are defined according to their drug or food application. Most probiotics are functional foods or are used as food supplements. These “healthy foods” are at the border between the drug and the traditional food and are governed by food legislation.

Probiotic foods

The global market of probiotic foods has been growing rapidly since the early 2000s, particularly in Europe. This dynamic is supported in particular by the link between food and health benefits.

Probiotics used as food supplements, as well as functional foods, are considered as food and are governed by the relevant legislation. They are different from dietary foods that are intended for a particular food and require a specific formulation or manufacturing process to differentiate them from the common food, and from medicinal products.




Transposition

Aside from homologous recombination, the overall stability of the genome is interrupted by mobile sequences called transposable elements or transposons. There are different classes of distinct DNA sequences that are able to transport themselves to other locations within the genome. This process utilizes recombination but does not result in an exchange. Rather, a transposon moves directly from one site of the genome to another without an intermediary such as plasmid or phage DNA. This results in rearrangements that create new sequences and change the functions of target sequences. Transposons may be a major source of evolutionary changes in the genome. In some cases they cause disease when inserted into a functioning gene. Three examples are presented below: insertion sequences (IS), transposons (Tn), and retroelements transposing via an RNA intermediate.

A. Insertion sequences (IS) and transposons (Tn)

A characteristic feature of IS transposition is the presence of a pair of short direct repeats of target DNA at either end. The IS itself carries inverted repeats of about 9–13 bp at both ends and depending on the particular class consists of about 750–1500 bp, which contain a single long coding region for transposase (the enzyme responsible for transposition of mobile sequences). Target selection is either random or at particular sites. The presence of inverted terminal repeats and the short direct repeats of host DNA result in a characteristic structure (1). Transposons carry in addition a central region with genetic markers unrelated to transposition, e.g., antibiotic resistance (2). They are flanked either by direct repeats (same direction) or by inverted repeats (opposite direction, 3).

 Insertion sequences (IS) and transposons (Tn)
Insertion sequences (IS) and transposons (Tn)

B. Replicative and nonreplicative transposition

With replicative transposition, the original transposon remains in place and creates a new copy of itself, which inserts into a recipient site elsewhere. Thus, this mechanism leads to an increase in the number of copies of the transposon in the genome. This type involves two enzymatic activities: a transposase acting on the ends of the original transposon and resolvase acting on the duplicated copies. In nonreplicative transposition, the transposing element itself moves as a physical entity directly to another site. The donor site is either repaired (in eukaryotes) or may be destroyed (in bacteria) if more than one copy of the chromosome is present.

C. Transposition of retroelements

Retrotransposition requires synthesis of an RNA copy of the inserted retroelement. Retroviruses including the human immunodeficiency virus and RNA tumor viruses are important retroelements. The first step in retrotransposition is the synthesis of an RNA copy of the inserted retroelement, followed by reverse transcription up to the polyadenylation sequence in the 3′ long terminal repeat (3’LTR). Three important classes of mammalian transposons that undergo or have undergone retrotransposition through an RNA intermediary are shown. Endogenous retroviruses , are sequences that resemble retroviruses but cannot infect new cells and are restricted to one genome. Non viral retrotransposons , lack LTRs and usually other parts of retroviruses. Both types contain reverse transcriptase and are therefore capable of independent transposition. Processed pseudogenes, or retropseudogenes lack reverse transcriptase and cannot transpose independently. They contain two groups: low copy number of processed pseudogenes transcribed by RNA polymerase II and high copy number of mammalian SINE sequences, such as human Alu and the mouse B1 repeat families.




DNA Amplification by Polymerase Chain Reaction (PCR)

The introduction of cell-free methods for multiplying DNA fragments of defined origin (DNA amplification) in 1985 ushered in a new era in molecular genetics (the principle of PCR is contained in earlier publications). This fundamental technology has spread dramatically with the development of automated equipment used in basic and applied research.

Polymerase chain reaction (PCR)

PCR is a cell-free, rapid, and sensitive method for cloning DNA fragments. A standard reaction and a wide variety of PCR-based methods have been developed to assay for polymorphisms and mutations. Standard PCR is an in vitro procedure for amplifying defined target DNA sequences, even from very small amounts of material or material of ancient origin. Selective amplification requires some prior information about DNA sequences flanking the target DNA. Based on this information, two oligonucleotide primers of about 15–25 base pairs length are designed. The primers are complementary to sequences outside the 3′ ends of the target site and bind specifically to these. PCR is a chain reaction because newly synthesized DNA strands act as templates for further DNA synthesis for about 25–35 subsequent cycles. Theoretically each cycle doubles the amount of DNA amplified. At the end, at least 10^5 copies of the specific target sequence are present. This can be visualized as a distinct band of a specific size after gel electrophoresis. Each cycle, involving three precisely time-controlled and temperature-controlled reactions in automated thermal cyclers, takes about 1–5min. The three steps in each cycle are (1) denaturation of double-stranded DNA, at about 93–95°C for human DNA, (2) primer annealing at about 50–70°C depending on the expected melting temperature of the duplex DNA, and (3) DNA synthesis using heat-stable DNA polymerase (from microorganisms living in hot springs, such as Thermophilus aquaticus, Taq polymerase), typically at about 70–75°C. At each subsequent cycle the template (shown in blue) and the DNA newly synthesized during the preceding cycle (shown in red) act as templates for another round of synthesis. The first cycle results in newly synthesized DNA of varied lengths (shown with an arrow) at the 3′ ends because synthesis is continued beyond the target sequences. The same happens during subsequent cycles, but the variable strands are rapidly outnumbered by new DNA of fixed length at both ends because synthesis cannot proceed past the terminus of the primer at the opposite template DNA.

cDNA amplification and RT-PCR

A partially known amino acid sequence of a polypeptide can be used to obtain the sequence information required for PCR. From its mRNA one can derive cDNA, and determine the sequence of the sense and the antisense strand to prepare appropriate oligonucleotide primers (1). When different RNAs are available in small amounts, rapid PCR based methods are employed to amplify cDNA from different exons of a gene. cDNA is obtained by reverse transcriptase from mRNA, which is then removed by alkaline hydrolysis (2). After a complementary new DNA strand has been synthesized, the DNA can be amplified by PCR (3). Reverse transcriptase PCR (RT-PCR) can be used when the known exon sequences are widely separated within a gene. With rapid amplification of cDNA ends (RACE-PCR), the 5? and 3? end sequences can be isolated from cDNA.

Polymerase chain reaction (PCR)
Polymerase chain reaction (PCR)




Recombination

Recombination lends the genome flexibility. Without genetic recombination, the genes on each individual chromosome would remain fixed in their particular position. Changes could occur by mutation only, which would be hazardous. Recombination provides the means to achieve extensive restructuring, eliminate unfavorable mutation, maintain and spread favorable mutations, and endow each individual with a unique set of genetic information. This greatly enhances the evolutionary potential of the genome. Recombination must occur between precisely corresponding sequences (homologous recombination) to ensure that not one base pair is lost or added. The newly combined (recombined) stretches of DNA must retain their original structure in order to function properly. Two types of recombination can be distinguished: (1) generalized or homologous recombination, which in eukaryotes occurs at meiosis and (2) site-specific recombination. A third process, transposition, utilizes recombination to insert one DNA sequence into another without regard to sequence homology. Here we consider homologous recombination, a complex biochemical reaction between two duplexes of DNA. The necessary enzymes, which can involve any pair of homologous sequences, are not considered. Two general models can be distinguished, recombination initiated from a single-strand DNA break and recombination initiated from a double-strand break.

A. Recombination initiated by single-strand breaks

This model assumes that the process starts with breaks at corresponding positions of one of the strands of homologous DNA (same sequences of different parental origin) (1). A nick is made by a single-strand-breaking enzyme (endonuclease) in each molecule at the corresponding site (2), but see below. This allows the free ends of one nicked strand to join with the free ends of the other nicked strand, from the other molecule, to form single-strand exchanges between the two duplex molecules at the recombination joint (3). The recombination joint moves along the duplex (branch migration) (4). This is an important feature because it ensures that sufficient distance for the second nick is present in each of the other strands (5). After the two other strands have joined and gaps have been sealed (6), a reciprocal recombinant molecule is generated (7). Recombination involving DNA duplexes requires topological changes, i.e., either the molecules must be free to rotate or the restraint must be relieved in some other way. This model has an unresolved difficulty: How is it assured that the single-strand nicks shown in step 2 occur at precisely the same position in the two double helix DNA molecules?

B. Recombination initiated by double-strand breaks

The current model for recombination is based on initial double-strand breaks in one of the two homologous DNA molecules (1). Both strands are cleaved by an endonuclease, and the break is enlarged to a gap by an exonuclease that removes the new 5′ ends of the strands at the break and leaves 3′ single-stranded ends (2). One free 3′ end recombines with a homologous strand of the other molecule (3). This generates a D loop consisting of a displaced strand from the “donor” duplex. The D loop is extended by repair synthesis until the entire gap of the recipient molecule is closed (4). This displaced strand anneals to the single-stranded complementary homologous sequences of the recipient strand and closes the gap (5). DNA repair synthesis from the other 3′ end closes the remaining gap (6). The integrity of the two molecules is restored by two rounds of single strand repair synthesis. In contrast to the single-strand exchange model, the double strand breaks result in hetero-duplex DNA in the entire region that has undergone recombination. An apparent disadvantage is the temporary loss of information in the gaps after the initial cleavage. However, the ability to retrieve this information by resynthesis from the other duplex avoids permanent loss.




Chemical bounds

Some Types of Chemical Bonds close to 99% of the weight of a living cell is composed of just four elements: carbon (C), hydrogen (H), nitrogen (N), and oxygen (O). Almost 50% of the atoms are hydrogen atoms; about 25% are carbon, and 25% oxygen. Apart from water (about 70% of the weight of the cell) almost all components are carbon compounds. Carbon, a small atom with four electrons in its outer shell, can form four strong covalent bonds withotheratoms.But most importantly,carbon atoms can combine with each other to build chains and rings, and thus large complex molecules with specific biological properties.

A. Compounds of hydrogen (H), oxygen (O), and carbon (C)

Four simple combinations of these atoms occur frequently in biologically important molecules: hydroxyl (—OH; alcohols), methyl (—CH 3 ), carboxyl (—COOH), and carbonyl (C=O; aldehydes and ketones) groups. They impart to the molecules characteristic chemical properties, including possibilities to form compounds.

B. Acids and esters

Many biological substances contain a carbon– oxygen bond with weak acidic or basic (alkaline) properties. The degree of acidity is expressed by the pH value, which indicates the concentration of H + ions in a solution, ranging from 10 –1 mol/L (pH 1, strongly acidic) to 10 –14 mol/L (pH 14, strongly alkaline). Pure water contains 10 –7 moles H + per liter (pH 7.0). An ester is formed when an acid reacts with an alcohol. Esters are frequently found in lipids and phosphate compounds.

C. Carbon–nitrogen bonds (C—N)

C—N bonds occur in many biologically important molecules: in amino groups, amines, and amides, especially in proteins. Of paramount significance are the amino acids, which are the subunits of proteins. All proteins have a specific role in the functioning of an organism.

D. Phosphate compounds

Ionized phosphate compounds play an essential biological role. HPO 4 2– is a stable inorganic phosphate ion from ionized phosphoric acid. A phosphate ion and a free hydroxyl group can form a phosphate ester. Phosphate compounds playan important role in energy-rich molecules and numerous macromolecules because they can store energy.

E. Sulfur compounds

Sulfur often serves to bind biological molecules together, especially when two sulfhydryl groups(—SH)reacttoformadisulfidebridge(— S—S—). Sulfur is a component of two amino acids (cysteine and methionine) and of some polysaccharides and sugars. Disulfide bridges play an important role in many complex molecules, serving to stabilize and maintain particular three-dimensional structures.




DNA Polymorphism

Genetic polymorphism is the existence of variants with respect to a gene locus (alleles), a chromosome structure(e.g., sizeofcentromeric heterochromatin), a gene product (variants in enzymatic activity or binding affinity), or a phenotype. The term DNA polymorphism refers to a wide range of variations in nucleotide base composition, length of nucleotide repeats, or single nucleotide variants. DNA polymorphisms are important as genetic markers to identify and distinguish alleles at agene locus and to determine their parental origin.

A. Single nucleotide polymorphism (SNP)

These allelic variants differ in a single nucleotide at a specific position. At least one in a thousand DNA bases differs among individuals (1). The detection of SNPs does not require gel electrophoresis. This facilitates large-scale detection. A SNP can be visualized in a Southern blot as a restriction fragment length polymorphism (RFLP) if the difference in the two alleles corresponds to a difference in the recognition site of a restriction enzyme (see Southern blot, p. 62). B. Simple sequence length polymorphism (SSLP) These allelic variants differ in the number of tandemly repeated short nucleotide sequences in noncoding DNA. Short tandem repeats (STRs) consist of units of 1,2,3,or 4 base pairs repeated from 3 to about 10 times. Typical short tandem repeats are CA repeats in the 5′ to 3′ strand, i.e., alternating CG and AT base pairs in the double strand. Each allele is defined by the number of CA repeats, e.g., 3 and 5, as shown (1). These are also called microsatellites. The size differences due to the number of repeats are determined by PCR. Variable number of tandem repeats (VNTR), also called minisatellites, consist of repeat units of 20–200 base pairs (2). C. Detection of SNP by oligonucleotide hybridization analysis Oligonucleotides, short stretches of about 20 nucleotides with a complementary sequence to the single-stranded DNA to be examined, will hybridize completely only if perfectly matched. If there is a difference of even one base, such as due to an SNP, the resulting mismatch can be detected because the DNA hybrid is unstable and gives no signal.

D. Detection of STRs by PCR

Short tandem repeats (STRs) can be detected by the polymerase chain reaction (PCR). The allelic regions of a stretch of DNA are amplified; the resulting DNA fragments of different sizes are subjected to electrophoresis; and their sizes are determined.

E. CEPH families

CEPH family
CEPH family

An important step in gene identification is the analysis of large families by linkage analysis of polymorphic marker loci on a specific chromosomal region near a locus of interest. Large families are of particular value. DNA from such families has been collected by the Centre pour l’Étude du Polymorphisme Humain (CEPH) in Paris, now called the Centre Jean Dausset, after the founder. Immortalized cell lines are stored from each family. A CEPH family consists of four grandparents, the two parents, and eight children. If four alleles are present at a given locus they are designated A, B, C, and D. Starting with the grandparents, the inheritance of each allele through the parents to the grandchildren can be traced (shown here as a schematic pattern in a Southern blot). Of the four grandparents shown, three are heterozygous (AB, CD, BC) and one is homozygous (CC). Since the parents are heterozygous for different alleles (AD the father and BC the mother), all eight children are heterozygous (BD, AB, AC, or CD).




Mutation Due to Different Base Modifications

Mutations can result from chemical or physical events that lead to base modification. When they affect the base-pairing pattern, they interfere with replication or transcription. Chemical
substancees able to induce such changes are called mutagens. Mutagens cause mutations in different ways. Spontaneous oxidation, hydrolysis, uncontrolled methylation, alkylation, and ultraviolet irradiation result in alterations that modify nucleotide bases. DNA-reactive chemicals change nucleotide bases into different chemical structures or remove a base.

A. Deamination and methylation

Cytosine, adenine, and guanine contain an amino group. When this is removed (deamination), a modified base with a different basepairing pattern results. Nitrous acid typically removes the amino group. This also occurs spontaneously at a rate of 100 bases per genome per day (Alberts et al., 1994, p. 245). Deamination of cytosine removes the amino group in position 4 (1). The resulting molecule is uracil (2). This pairs with adenine rather than guanine. Normally this change is efficiently repaired by uracil-DNA glycosylase. Deamination at the RNA level occurs in RNA editing (see Expression of genes). Methylation of the carbon atom in position 5 of cytosine results in 5 methylcytosine, containing a methyl group in position 5 (3). Deamination of 5-methylcytosine will result in a change to thymine, containing an oxygen in position 4 instead of an amino group (4). This mutation will not be corrected because thymine is a natural base. Adenine (5) can be deaminated in position 6 to form hypoxanthine, which contains an oxygen in this position instead of an amino group (6), and which pairs with cytosine instead of thymine. The resulting change after DNA replication is a cytosine instead of a thymine in the mutant strand.

B. Depurination

About 5000 purine bases (adenine and guanine) are lost per day from DNA in each cell (depurination) owing to thermal fluctuations. Depurination of DNA involves hydrolytic cleavage of the N-glycosyl linkage of deoxyribose to the guanine nitrogen in position 9.This leaves a depurinated sugar. The loss of a base pair will lead to a deletion after the next replication if not repaired in time (see DNA repair).

C. Alkylation

Alkylation is the introduction of a methyl or an ethyl group into a molecule. The alkylation of guanine involves the replacement of the hydrogen bond to the oxygen atom in position 6 by a methyl group, to form 6-methylguanine. This can no longer pair with cytosine. Instead, it will pair with thymine. Thus, after the next replication the opposite cytosine (C) is replaced by a thymine (T) in the mutant daughter molecule. Important alkylating agents are ethylnitroso urea (ENU), ethylmethane sulfonate (EMS), dimethylnitrosamine, and N-methyl-N-nitroN-nitrosoguanidine.

D. Nucleotide base analogue

Base analogs are purines or pyrimidines that are similar enough to the regular nucleotide DNA bases to be incorporated into the new strand during replication. 5-Bromodeoxyuridine (5BrdU) is an analog of thymine. It contains a bromine atom instead of the methyl group in position 5.Thus, it can be incorporated into the new DNA strand during replication. However, the presence of the bromine atom causes ambiguous and often wrong base pairing. E. UV-light-induced thymine dimers Ultraviolet irradiation at 260 nm wavelength induces covalent bonds between adjacent thymine residues at carbon positions 5 and 6. If located within a gene, this will interfere with replication and transcription unless repaired. Another important type of UV-induced change is a photoproduct consisting of a covalent bond between the carbons in positions 4 and 6 of two adjacent nucleotides, the 4–6 photoproduct.




Eukaryotic gene structure

Eukaryotic genes consist of coding and noncoding segments of DNA, called exons and introns, respectively.At first glance it seems to be an unnecessary burden to carry DNA without obvious functions within a gene. However, it has been recognized that this has great evolutionary advantages. When parts of different genes are rearranged on new chromosomal sites during evolution, new genes may be constructed from parts of previously existing genes.

Exons and introns

In 1977, it was unexpectedly found that the DNA of a eukaryotic gene is longer than its corresponding mRNA. The reason is that certain sections of the initially formed primary RNA transcript are removed before translation occurs. Electron micrographs show that DNA and its corresponding transcript (RNA) are of different lengths (1). When mRNA and its complementary single-stranded DNA are hybridized, loops of single-stranded DNA arise because mRNA hybridizes only with certain sections of the single stranded DNA. In (2), seven loops (A to G) and eight hybridizing sections are shown (1 to 7 and the leading section L). Of the total 7700 DNA base pairs of this gene (3), only 1825 hybridize with mRNA. A hybridizing segment is called an exon. An initially transcribed DNA section that is subsequently removed from the primary transcript is an intron. The size and arrangement of exons and introns are characteristic for every eukaryotic gene (exon/intron structure). (Electron micrograph from Watson et al., 1987).

Intervening DNA sequences (introns)

In prokaryotes, DNA is colinear with mRNA and contains no introns (1). In eukaryotes, mature mRNA is complementary to only certain sections of DNA because the latter contains introns (2). (Figure adapted from Stryer, 1995).

Basic eukaryotic gene structure

Basic eukaryotic gene structure
Basic eukaryotic gene structure

Exons and introns are numbered in the 5′ to 3′ direction of the coding strand. Both exons and introns are transcribed into a precursor RNA (primary transcript).The first and the last exons usually contain sequences that are not translated. These are called the 5′ untranslated region (5′ UTR) of exon 1 and the 3′ UTR at the 3′ end of the last exon. The non coding segments (introns) are removed from the primary transcript and the exons on either side are connected by a process called splicing. Splicing must be very precise to avoid an undesirable change of the correct reading frame. Introns almost always start with the nucleotides GT in the 5′ to 3′ strand (GU in RNA) and end with AG. The sequences at the 5′ end of the intron beginning with GT are called splice donor site and at the 3′ end, ending with AG,are called the splice acceptor site. Mature mRNA is modified at the 5? end by adding a stabilizing structure called a “cap” and by adding many adenines at the 3’end (polyadenylation).

Splicing pathway in GU–AG introns

Splicing pathway in GU – AG introns
Splicing pathway in GU – AG introns

RNA splicing is a complex process mediated by a large RNA-containing protein called a spliceosome. This consists of five types of small nuclear RNA molecules (snRNA) and more than 50 proteins (small nuclear riboprotein particles). The basic mechanism of splicing schematically involves autocatalytic cleavage at the 5’end of the intron resulting in lariat formation. This is an intermediate circular structure formed by connecting the 5′ terminus (UG) to a base (A) within the intron. This site is called the branch site. In the next stage, cleavage at the 3′ site releases the intron in lariat form. At the same time the right exon is ligated (spliced) to the left exon. The lariat is debranched to yield a linear intron and this is rapidly degraded. The branch site identifies the 3′ end for precise cleavage at the splice acceptor site. It lies 18–40 nucleotides upstream (in 5′ direction) of the 3′ splice site. (Figure adapted from Strachan and Read, 1999)




The Strange world of Bionanomachines

Very remarkably, many of nanomachines will always perform their functions automatically, after they are isolated and purified, provided the environment is not too hard. They must not be sequestered inside the cells in the pure state. Each of them constitutes a self-sufficient molecular machine. Natural digestive enzymes such as pepsin and lysozyme are so hard that they can be added to the laundry detergent to help dissociate partial stains. Amylases are used on an industrial scale to convert pulverulent starch into corn syrup. The entire field of genetic engineering and biotechnology is made possible by a collection of DNA and manipulative nanomachines, now commercially available. Generally the natural bionanomachines are remarkably robust. This chapter explores the bionanomachines made by living cells. They are different from machines in our world.

Bionanomachines are also chosen by their very specific tasks in a given environment, and are also subject to unfamiliar forces exerted by this environment. It is imperative to keep these notions of difference in mind before embarking on the biotechnology study.

A strange world

Natural biomolecules have often unbelievable shapes, unlike designs arranged in bread grids and tractors. They perform their functions in a strange environment, where the thermal movements, the nervous movements constantly influenced by pushing and attracting forces on their constituents. They are linked by a complex set of links and external forces. The bionanomachines are almost immune to the laws of gravity and inertia which dominate our machines.

Gravity and inertia are negligible at the nano scale

Physical properties such as friction, tensile strength, adhesion, and resistance to disintegration are comparable in magnitude to the forces exerted by inertia and gravity. This balance changes, however, when we move to larger or smaller objects. As we move to larger objects, the graduation laws shift the balance. Mass increases with the cube of size of an object, and properties such as force and friction increase linearly with the square of the waist.

The graduation laws also go in the opposite direction, the grains of micrometer-sized sands, or the cells, act on each other differently from macroscopic objects. In fact, if we consider a bacterial cell that swims in a volume of water by its ciliature, it moves slowly, and stops at a given moment as does the submarine in the ocean. However, if one considers the inertial forces relative to the viscosity of the surrounding water, the cell would stop less than the diameter of an atom!

Gravity is neglected at the nano scale, so molecules dissociated in water or propagated in the air are in continuous motion, the forces of intermolecular attraction are more important than the force of gravity. Flies take advantage of these forces and can crawl up a wall, even the water droplets may remain adhered to the ceiling as a result of these forces.

Nanomachines can show the granularity of atoms

Objects on the nanoscale scale are constructed by discrete combinations of atoms, which act on each other by specific atom-atom interactions. They must consist of a fixed number of atoms (rotary engine at the nanometer scale). Similar engines already existing, such as adenosine triphosphate synthetase, the bacterial flagellar engine.

Due to atomic granularity, typical continuous representations are not used in technology because of physical properties; Such as viscosity and friction, are not defined for discrete atomic groups. Instead, various atomic substitutive properties are used, quantum mechanics in fact provides a deep understanding of atomic properties, but fortunately most of the properties of atoms can be understood qualitatively by using simple rules: 1. The links 2. Steric repulsion between unconnected atoms, electrostatic interactions, hydrogen bonds that allow the understanding of most aspects, structures, and molecular interactions.

Generally, biomolecules are considered as articulated chains of atoms that interact precisely with one another.

Heat flux is a significant force at the nano scale

Molecular nanotechnology seeks to create the environment of the “machine phase” with different organized nanomachines, in order to form objects on the micro or macroscopic scale. On the other hand, natural ionanomachinery takes a different approach because it requires the introduction of nanomachinery inside the cell. The various parts act upon each other by induced movements and diffusion.

Biomachines work in a chaotic environment, they are continually bombarded by molecules of water. They will disperse but always held in place. Biomachines can establish specific interactions with other biomachines, adapting together to the state of rest, and separating in the event of operation. If two closely matched molecules have a good complementarity of chemical groups, they will interact for long periods, but if the interactions are weak, they will establish temporary interactions before moving on to the next.

The careful design of the bionanomachines and the rigorous regulation of the forces of their interactions give them the character of being able to form stable molecular beams that can remain years or build biological Detectors capable of detecting molecular traces. Bionanomachines require water as an environment. The shape and function of bionanomolecules are dominated by two things: the chemistry of their component atoms and the irregular properties of the surrounding water molecules. The energy of these interactions is different from what is meant in the macroscopic world, the water molecules interact strongly with each other by hydrogen bonds. They do not separate and do not interact with other molecules unless they have something to offer. Bionanomolecules, which have an important value in terms of electron richness, nitrogen (N) and Oxygen (O), interact favorably with water molecules, and therefore have good solubility in water. The hydrophobicity of biomolecules strongly influences their function and shape. The geometry of the molecular chain alone, creates a large number of conformations, one would thus rarely find simple structures. Once placed in water, the biomolecules rapidly respond to the environment, folding into a conformation, so that the hydrophobic regions are stacked inwards, the surface being decorated by the H 2 O-hydrophilic molecules . For proteins: the chain is most often forced into a compact globule, for DNA the pairs of bases are sequestered without alteration inside, leaving the phosphates heavily loaded on the surface, for lipids, hydrophobic groups ( In the cell membranes) are directed inside the lamellae as well as the hydrophilic groups are directed outwards. If carefully constructed, one would obtain a unique structure with an appropriate conformation in order to perform its task properly.




Impact of Particle Irradiation on the Immune System: From the Clinic to Mars

Despite the generalized use of photon-based radiation (i.e., gamma rays and X-rays) to treat different cancer types, particle radiotherapy (i.e., protons and carbon ions) is becoming a popular, and more effective tool to treat specific tumors due to the improved physical properties and biological effectiveness. Current scientific evidence indicates that conventional radiation therapy affects the tumor immunological profile in a particular manner, which in turn, might induce beneficial effects both at local and systemic (i.e., abscopal effects) levels. The interaction between radiotherapy and the immune system is being explored to combine immune and radiation (including particles) treatments, which in many cases have a greater clinical effect than any of the therapies alone. Contrary to localized, clinical irradiation, astronauts are exposed to whole body, chronic cosmic radiation, where protons and heavy ions are an important component. The effects of this extreme environment during long periods of time, e.g., a potential mission to Mars, will have an impact on the immune system that could jeopardize the health of the astronauts, hence the success of the mission. To this background, the purpose of this mini review is to briefly present the current knowledge in local and systemic immune alterations triggered by particle irradiation and to propose new lines of future research. Immune effects induced by particle radiation relevant to clinical applications will be covered, together with examples of combined radiotherapy and immunotherapy. Then, the focus will move to outer space, where the immune system alterations induced by cosmic radiation during spaceflight will be discussed.

Keywords :  protons, carbon ions, immunotherapy, space flight, cosmic radiation, immune response, cancer therapy.

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