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.




Drugs Identification in Urine, Bile and Gastric Contents using Thin Layer Chromatography in Multiple Screening Systems

A method of simultaneous identification of 25 molecules in human urine, bile and gastric contents using liquid-liquid extraction followed by thin layer chromatography (TLC) using multiple screening systems is described. The analytes were extracted at 25°C under isocratic conditions using chloroform after acidification with 1 to 2 drops of HCl 6 N for 10 mL of the biological sample, and dichloromethane after alkalization with 1 to 2 drops of NaOH 10 N for 10 mL of the biological sample. Employing LLE, the best conditions were achieved with double extraction of 10 mL of the biological sample, pH=9.5 for alkaline extraction and pH=2 for acid extraction. The organic extractums were filtered and dehydrated using anhydrous sodium thiosulfate powder and concentrated after evaporation of the organic solvents at 65°C. The extraction residues were solubilized in 500 µl of methanol and spotted with the molecules of reference onto four TLC plates (10 cm × 10 cm). The TLC plates were put into twin-through development chambers previously incubated 30 minutes for saturation namely TA (methanol:ammoniac 5% (50:0.750, v/v)), TD (chloroform:acetone (40:10, v/v)), TE (Ethyl acetate:methanol:ammoniac (42.3:5:2.5, v/v/v)), TB (cyclohexan:toluene:diethylamine (37.5:7.5:5, v/v/v)). The mobile phase migrates by capillarity through the stationary phase, driving at different speeds the molecules to be separated. The migration time (several minutes) depends on various parameters. When the solvent front has moved through a distance considered as sufficient (a few centimetres), the TLC plates were removed and dried, then exposed to ultraviolet light, the retardation factors Rf of each visible spot was measured. Some chemical processes might also be used to reveal spots. The total number of substances present in the biological sample was determined by counting the number of spots found on each TLC plate, the biggest number among the four counted values is considered as the default number of the present substances. A mathematical formula was applied to guess all possible matches according to a data table of Rf profiles of standards already calculated by the same method. The validation parameters obtained in LLE were linearly range of 50-1000 µg mL-1 biological fluid (r≥0.9815). This method has shown its suitable applicability in order to rapidly identify a wide verity of substances of toxicological interest present in the biological samples. Moreover, it’s inexpensive and could be suggested in various routine drug screening processes, especially for toxicological/forensic analysis.

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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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Simple flow cytometry protocol to determine simultaneously live, dead and apoptotic stallion spermatozoa

A simple flow cytometry protocol to determine simultaneously live, dead and apoptotic stallion spermatozoa in fresh and frozen thawed samples.

Flow cytometry is increasingly used in clinical andrology in stallions (Love, 2016; Munoz et al., 2016). Numerous protocols have been developed in the last decade allowing the simultaneous assessment of multiple parameters in the spermatozoa. Developments in flow cytometry have allowed for an enhanced understanding of the biology of spermatozoa, particularly in the fields of clinical spermatology and sperm biotechnology. One of the aspects recently discovered with the aid of flow cytometry, relate to the dependence of the stallion spermatozoa on oxidative phosphorylation to obtain energy for motility and sperm house-keeping functions (Gibb et al., 2014; Pena et al., 2015; Plaza Davila et al., 2015; Davila et al., 2016).

This dependence also results in a greater production of the superoxide anion (O2 −●), that if deregulation of redox homeostasis occurs, leads to oxidative stress, accelerated senescence of the spermatozoa and ultimately death of the cell (Aitken et al., 2012b; Morillo Rodriguez et al., 2012; Gallardo Bolanos et al., 2014; Martin Munoz et al., 2015; Munoz et al., 2016; Ortega-Ferrusola et al., 2017). This accelerated sperm senescence has been attributed to changes resembling apoptosis and the term spermptosis was proposed to refer to this biological phenomenon (Gallardo Bolanos et al., 2014; Ortega-Ferrusola et al., 2017).

A similar form of cell death after cryopreservation has also been described in somatic cells and is termed cryopreservation induced delayed onset cell death (Baust et al., 2001; Baust et al., 2016). These changes have been described in relation to cryopreservation but also may be attributable to stallion subfertility. Due to the importance of these changes, numerous assays have been developed for the identification of cell states using flow cytometry (Martinez-Pastor et al., 2010; Pena et al., 2016b; Ortega-Ferrusola et al., 2017). Many of these assays require the use of three different probes, usually the H-42 probe, to identify spermatozoa and eliminate non-sperm debris from the analysis. If simpler protocols, with minimal spectral overlap, can be developed the assessment of apoptotic changes could be facilitated.

The aim of this study was to develop a simple protocol to assess apoptotic changes, using a combination of two probes with minimal spectral overlap (H-42 and Eth-1). The use of this combination of probes was validated by comparing with use of a classical, and well proven assay for caspase 3 activation (Moran et al., 2008; Ortega-Ferrusola et al., 2008; Ortega Ferrusola et al., 2009; Gallardo Bolanos et al., 2014).

Material and methods

Reagents and media

The H-42 (Excitation: 350 nm, Emission: 461 nm) probe (Ref: H3570); Cell Event Caspase-3/7 Green (Excitation, 502 nm; Emission: 530 nm) (Ref: C10423) and Eth-1 (Excitation, 528 nm; Emission, 617 nm) (Ref E1169) were purchased from Fisher Scientific Spain (Madrid, Spain). All other chemicals were purchased from SIGMA (Madrid, Spain).

Semen collection and processing

Three ejaculates per horse were collected from seven fertile stallions. The samples were obtained on a routine basis (three collections/week) during the 2016 breeding season. Stallions were maintained according to institutional and European regulations (Law 6/2913 June 11th and European Directive 2010/63/EU). Ejaculates were obtained and processed (fresh and frozen/thawed) using protocols that have been previously published in detail (Rodriguez et al., 2011; Morillo Rodriguez et al., 2012; Martin Munoz et al., 2015; Ortega-Ferrusola et al., 2017).

Staining for determination of live and dead cells and caspase 3 and 7 activity

This protocol has been developed in the laboratory where the present research was conducted and has been extensively described in previous publications (Gallardo Bolanos et al., 2014; Martin Munoz et al., 2015; Munoz et al., 2016; Ortega-Ferrusola et al., 2017). In brief, stock solutions of CellEvent (2 mM in DMSO), Eth-1 (1.167 mM in DMSO), and Hoechst 33342 (1.62 mM in water) were prepared. Spermatozoa (5 × 106 /mL) in 1 mL of PBS were stained with 1 μL of CellEvent, and 0.3 μL of Hoechst 33342 and incubated 25′ in the dark at r.t. Then 0.3 μL of Eth-1 was added to each sample and after incubation for 5 additional minutes the samples were immediately evaluated using the flow cytometer. Representative cytograms of the assay and gating strategy are shown in Fig. 1.

Flow cytometry

Flow cytometry analyses were conducted in a MACSQuant Analyzer 10 (Miltenyi Biotech), equipped with three lasers emitting at 405, 488, and 635 nm and 10 photomultiplier tubes (PMTs): V1 (excitation 405 nm, emission 450/50 nm), V2 (excitation 405 nm, emission 525/50 nm), B1 (excitation 488 nm, emission 525/50 nm), B2 (excitation 488 nm, emission 585/40 nm), B3 (excitation 488 nm, emission 655–730 nm (655LP + split 730)), B4 (excitation 499 nm, emission 750 LP), R1 (excitation 635 nm, emission 655–730 nm (655LP + split 730)), and R2 (excitation 635 nm, emission filter 750 LP). The system was controlled using MACS Quantify software. The quadrants or regions used to quantify the frequency of each sperm subpopulation depended on the particular assay. Forward and sideways light scatter was recorded for a total of 50,000 events per sample. Gating the sperm population after Hoechst 33342 staining eliminated non-sperm events. The instrument was calibrated daily using specific calibration beads provided by the manufacturer. A compensation overlap was performed before each experiment. The data were analyzed using Flowjo V 10.2 Software (Ashland, OR, USA). Unstained and singlestained controls were used to determine compensations and positive and negative events, as well as to set regions of interest as described in previous publications (Pena et al., 2003; Gallardo Bolanos et al., 2014; Martin Munoz et al., 2015).

Statistical analyses

The normality of the data was assessed using the Kolmogorov-Smirnoff test. Because the data were normally distributed, the results were analyzed using an ANOVA followed by use of a Tukey post-hoc test for pair-wise comparisons (using SPSS 19.0 software for Mac). Differences were considered significant when P < 0.05. The results are displayed as the mean ± SEM. Bland and Altman Plots, a method agreement analysis technique, of the two staining protocols were also constructed. The differences between paired measurements of the same samples were calculated, and the mean of the differences (d) were used to estimate the average bias of one method relative to the other (Nagy et al., 2003; Pena et al., 2005). The 95% limit of agreement was calculated as d ± 2 SD, where SD is the standard deviation of the differences between paired measurements. These calculations were performed using Microsoft Excel for Mac OS X and SPSS for Mac.

Figure 1. Representative cytograms of the assays reported in the present study. Stallion spermatozoa were obtained and processed as described in material and methods. a) Dot plot showing H33342 fluorescence the region gated in a1 corresponds to spermatozoa and this region is further used set the rest of populations of interest. In a two population of H-42 positive spermatozoa can be easily distinguished. H-42 Dim and H-42 Bright b) Events gated in a1 are now plotted against H-43 and Eth-1 fluorescence showing three populations, Live (H-42 +), apoptotic (H-42 +/Eth-1 +) and dead spermatozoa (Eth-1 +). c) Representative dot plot confronting caspase 3 (X axis) positive vs. ethidium homodimer fluorescence (Y axis); three subpopulations are evident live spermatozoa, negative for caspase 3 and for Eth-1, Apoptotic spermatozoa, positive for caspase 3 and negative for ethidium homodimer, and dead spermatozoa, positive for ethidium homodimer. d) Representative cytograms showing relation between H-42 and Caspase 3 positivity. Three subpopulations can be detected, live spermatozoa (H-42 positive events), early apoptotic (Caspase 3 dim) and late apoptotic (Caspase bright events).

Experimental design

Semen was obtained and processed for cryopreservation or kept as fresh samples. Split samples from each kind of semen were stained with H-42, Eth-1, and CellEvent for caspase 3 detection. Comparisons were made of the same samples constructing 2D dot plots for each combination of probes assessed.

Conclusion

The bimodal distribution of H-42 positive spermatozoa (faint and bright) can be used to detect live and dead spermatozoa, while the addition of Eth-1 improves the assay allowing also the detection of apoptotic spermatozoa in the same fashion as specific assays detecting active caspases 3 and 7. Additionally, this assay offers the advantages of minimal spectral overlap and easy detection of non-sperm particles.




Measuring Apoptosis by Flow Cytometry

Mitochondria are double-membraned organelles believed to have been integrated into modern eukaryotes via symbiosis of proteobacteria into an anaerobic pre-eukaryotic (host) cell 1.5–2 billion years ago. According to modern thinking (pioneered by Mitchell;), an essential role of mitochondria is to produce ATP via oxidative phosphorylation (OXPHOS). In this process, the chemical energy stored in nutrients (carbohydrates, fats, etc.) is converted to an electrochemical gradient across the inner mitochondrial membrane via the electron transport chain (ETC) complexes. This electrochemical gradient acts as a store of energy. ATP synthase uses this stored energy to convert ADP to ATP. This bioenergetic picture of the role of mitochondria is now widely accepted. A second role of mitochondria is in the so-called intrinsic apoptosis pathway. This pathway converges (figuratively and literally) at the membrane of the mitochondria. Upon certain cell death signals [such as reactive oxygen species (ROS), DNA damage, etc.], the outer membrane of mitochondria becomes permeable enough to release the soluble hemeprotein cytochrome C (CytC), as well as Smac/Diablo, endonuclease G, and other intermembrane space proteins, which irreversibly activate downstream caspases to carry out the apoptosis process.

Apoptosis is a programmed mode of cell death that is accompanied by numerous morphological as well as biochemical changes to the cellular architecture. This results not only in cell death but also in the effi- cient removal of apoptotic cells by phagocytes. Apoptotic cells display a range of common characteristics that include cell shrinkage, plasma membrane blebbing, cell detachment, nuclear condensation, DNA fragmentation, externalization of phosphatidylserine (PS) and activation of caspases. In contrast, necrotic cell death is characterised by rapid plasma membrane, organelle swelling and plasma membrane rupture with none of the features of apoptosis. Apart from severe physical stresses, necrotic cell death often betrays the activities of viral infection and the activities of bacterial toxins. While necrotic cell death is characterized by the release of endogenous ‘danger signals’ and subsequent inflammation, apoptosis is largely tolergenic. Therefore, care must be taken when assessing whether cells are dying via apoptosis or necrosis. Here, we highlight a number of assays, utilizing flow cytometry, to determine whether cells have undergone apoptosis or alternative modes of cell death.

Detection of fragmented DNA by flow cytometry as a measure of apoptotic cell death

Intranucleosomal DNA fragmentation is a major hallmark of apoptosis. DNA fragmentation may be assessed by flow cytometry. Analysis of a cell population’s replication state (cell cycle profile) can be readily achieved with the fluorescent dye Propidium iodide (PI), which binds stoichiometrically to nucleic acids resulting in a fluorescence emission proportional to the DNA content of the cell. The rationale behind the approach is as follows: quiescent and G1 cells have two chromosome copies, while cells undergoing mitosis G2/M have double the amount of DNA and so will have double the fluorescence intensity of G1 cells. Cells in S phase will have a fluorescent signal between G1 and G2/M, because these cells are synthesizing DNA on their way to G2/M (Fig. 3A).

Figure 3. Measure of DNA fragmentation during apoptosis by flow cytometry. Jurkat cells were treated with 200 ng/ml anti-Fas (CH-11). Cells were harvested at indicated timepoints (A–E) and analysed by flow cytometry. (F) Gating strategy to discriminate cells aggregates from single cells.

Due to the generation of lowmolecular weight DNA fragments during apoptosis, cells undergoing apoptosis can be readily identified on DNA content histograms as cells with fractional hypodiploid or ‘‘sub-G1’’ content (Fig. 3B–E). Cellular DNA content is measured using a fluorescent dye after cell fixation with ethanol. Cell fixation does not retain small nuclear fragments in apoptotic cells. These low molecular weight DNA fragments leak out during subsequent wash steps. As a result, apoptotic cells contain a fractional DNA content relative to viable cells that can be readily distinguished by flow cytometry.

Methodology

The following protocol is tailored towards suspension cells, however, if using adherent cells remember to harvest the supernatant (late apoptotic cells become detached and float in the medium) in addition to the adherent/semi adherent cells on the plate and proceed as outlined below:

1.Apoptosis was induced in 2 10^6 Jurkat cells by incubation with 200 ng/ml anti-Fas antibody (CH-11) for 1–4 h. Cells are harvested at the desired time points and centrifuged at 400g for 5 min. Cells are washed with PBS pH 7.2 and centrifuged at 400g for 1 min.

2.Resuspend cells in 1 ml ice-cold 70% ethanol and incubate for at least 1 h at -20 °C to fix (cells can be stored for up to 6 months at -20 °C).

3.Centrifuge cells at 2500g for 5 min (a higher centrifuge speed is required as fixed cells become buoyant and may fail to pellet or stick along the side of the eppendorf). Aspirate off the ethanol without disturbing the cell pellet and resuspend with 1 ml phosphate-citrate wash buffer (200 mM Na2HPO4, 100 mM citric acid) followed by centrifugation at 2500g for 1 min.

4.To stain nuclei, prepare PBS pH 7.2 containing propidium iodide 10 lg/ml and RNase A 100 lg/ml (included to degrade RNA and to prevent PI staining of RNA) and incubate with cells for 30 min.

5.Samples are ready for analysis by flow cytometry (no need to wash out PI/RNase but this can be done if desired).

Setting correct parameters for cell cycle analysis by flow cytometry

A few considerations must be taken into account when using flow cytometry for cell cycle analysis:

A) Ensure that the fluorescence channel 2 (FL2) is set at linear (LIN) scale. It is harder to distinguish the differential fluorescence between G0/G1 and G2/M peaks on a logarithmic (LOG) scale. LIN amplification allows for clear separation between G0/G1 and G2/M peaks.

B) A common problem to control for during cell cycle analysis is aggregation of cells. For example cells can stick together and pass through the flow cytometer’s laser intercept simultaneously. In either case, two cells in G0/G1 that are stuck together or pass through the laser intercept at the same time will have a fluorescence signature equivalent to a cell in G2/ M. Therefore, the number of events recorded as G2/M will be artificially high. A way to exclude these events is by excluding non-single cell events from the analysis using scatter properties (FSC/SSC).

C) To discriminate between cellular aggregation and single cells, select a plot with FL2-A parameter as the y-axis and FL2-W as the x-axis (Fig. 3F). Single cells (G0/G1 or G2/M) will have pulse width values (FL2-W) that are similar, however aggregates will have larger pulse width values (due to increased cell width). In the example (Fig. 3F), single cells have been gated (R1-single cells) and the FL2-A histograms (Fig. 3A–E) have been formatted to display only events within this region (R1-single-cells).

Reference : ScienceDirect Measuring apoptosis by microscopy and flow cytometry




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.




Replication Cycle of Viruses

With all their different genomic structures, forms, and sizes, viruses basically have a relatively simple replication cycle. While only the genome of a bacteriophage enters a bacterium, the complete virus (genome and capsid) enters a eukaryotic cell.

General sequence of the replication cycle of a virus in a cell

The replication cycle of a virus consists of five principal consecutive steps: (1) entrance into the cell and release of the genome (uncoating), (2) transcription of the viral genes and(3) translation of the mRNAs to form viral proteins, (4) replication of the viral genome, (5) assembly of new viral particles in the cell and release of the complete virions from the host cell (6).

General sequence of the replication cycle of a virus in a cell
General sequence of the replication cycle of a virus in a cell

Uptake of a virus by endocytosis

Besides fusion of the lipid membrane of membrane-enclosedviruseswiththecellmembrane of the host cell, the most frequent mechanism for a virion to enter a cell is by a special form of endocytosis. The virus attaches to the cell membrane by using cell surface structures (receptors), which serve othe rimportant functions for the cell, e.g., for the uptake of macromolecules. Like these, the virus is taken into the cytoplasm by a special mechanism, receptor-mediated endocytosis (coated pits, coated vesicles). Within the cell, the virus-containing vesicle fuses with other cellular vesicles (e.g., primary lysosomes). The viral coat is extensively degraded in the endocytotic vesicle, and the viral core (genome, associated with viruscoded proteins) is released into the cytoplasm or nucleus, depending on the viral type. Replication and expression of the viral genome follow. Whether a cell can be infected by a virion depends on a specific interaction between the virus and a cellular receptor. Some viruses, such as the paramyxoviruses (e.g., mumps and Sendai virus), enter the cell by direct fusion of the viral and cellular membranes, mediated by a viral coat glycoprotein (F or fusion protein).

Uptake of a virus by endocytosis
Uptake of a virus by endocytosis

Transcription and replication of a virus

The first viral genes to be expressed after the virus has entered the cell are the early genes of the viral genome. Gene products of these early viral genes regulate transcription of the remaining viral genes and are involved in replicating the viral genome. Synthesis of the capsid proteins begins later (late genes), at the same time as genome replication, when new virions are formed from the genome and capsids (assembly). The virions (nucleocapsids = genome plus capsid) are then released from the cell by one of several mechanisms, depending on the type of virus.

Transcription and replication of a virus
Transcription and replication of a virus

Release of a virus by budding

The release of a virus coated by a lipid membrane occurs by budding. First, molecules of a viral-coded glycoprotein are built into the cell membrane, to which the virus capsid or virus core (containing the viral genome) attaches. Attachment of the genome leads to increased budding of that region of the cell membrane. Eventually, the entire virion is surrounded by a lipid membrane envelope of cellular origin containing viral proteins and is released. Virions can be expelled from the cell continuously and in great numbers without the death of the virus-producing cell. (Figures from J. D. Watson et al., 1987).

Release of a virus by budding
Release of a virus by budding




Viruses

Viruses are important pathogens in plants and animals, including man. The complete infectious viral particle is called a virion. Its genome carries a limited amount of genetic information, and it can replicate only in host cells. From analysis of the structure and expression of viral genes, fundamental biological processes such as DNA replication, transcription regulation, mRNA modification (RNA splicing, RNA capping, RNA polyadenylation), reverse transcription of RNA to DNA, viral genome integration into eukaryotic DNA, tumor induction by viruses, and cell surface proteins have been recognized and elucidated. The extracellular form of a virus particle includes a protein coat (capsid), which encloses the genome of DNA or RNA. The capsids contain multiple units of one or a few different protein molecules coded for by the virus genome. Capsids usually have an almost spherical, icosahedral (20 plane surfaces), or occasionally a helical structure. Some viral capsids are surrounded by a lipid membrane envelope.

Classification of viruses

Viruses can be classified on the basis of the structure of their viral coat, their type of genome, and their organ or tissue specificity. The genome of a virus may be enclosed simply in a virus-coded protein coat (capsid) or in the capsid plus an additional phospholipid membrane,which is of cellular origin. The genome of a virus may consist of single-stranded DNA(e.g., parvovirus), double-stranded DNA (e.g., papovavirus, adenovirus, herpesvirus, and poxvirus), single-stranded RNA (e.g., picornavirus, togavirus, myxovirus, rhabdovirus), or double stranded RNA (e.g., reovirus). Viruses with genomes of single-stranded RNA are classified according to whether their genome is a positive (plusRNA) or negative (minusRNA) RNAstrand. Only an RNA plus strand can serve as a template for translation (5′ to 3′ orientation).

Replication and transcription of viruses

Since viral genomes differ, the mechanisms for replicating their genetic material also differ. Viruses must pack all their genetic information into a small genome; thus, one transcription unit (gene) of a viral genome is often used to produce several mRNAs by alternative splicing, and each mRNA codes for a different protein. In some RNA viruses, initially large precursor proteins are formed from the mRNA and subsequently split into several smaller functional proteins. An RNA plus strand can be used directly for protein synthesis. An RNA minus strand cannot be used directly; an RNA plus strand must be formed from the RNA minus strand by a transcriptase before translation is possible. RNA viruses contain a transcriptase to replicate their RNA genomes. RNA viruses in which DNA is formed as an intermediate step (retroviruses) contain a reverse transcriptase. This can form DNA from RNA. The DNA intermediate step in the replication of retroviruses becomes integrated into the host cell. Several RNA viruses have segmented genomes. They consist of individual pieces of RNA genome, each of which codes for one or more proteins (e.g., influenza virus). The exchange of individual pieces of RNA genome of different viral serotypes plays an important role in the formation of new viral strains (e.g., influenza strains). (Figure after Watson et al., 1987).

Replication and transcription of viruses
Replication and transcription of viruses




DNA Transfer between Cells

Transfer of DNA occurs not only by fusion of gametes in sexual reproduction but also between other cells of prokaryotic and eukaryotic organisms (conjugation of bacteria, transduction between bacteriophages and bacteria, transformation by plasmids in bacteria, transfection in cultures of eukaryotic cells). Cells altered genetically by taking up DNA are said to be transformed. The term transformation is used in different contexts and refers to the result, not the mechanism.

A. Transduction by viruses

In 1952, N. Zinder and J. Lederberg described a new type of recombination between two strains of bacteria. Bacteria previously unable to produce lactose (lac – ) acquired the ability to produce lactose after being infected with phages that had replicated in bacteria containing a gene for producing lactose (lac + ). A small segment of DNA from a bacterial chromosome had been transferred by a phage to another bacterium (transduction). General transduction (insertion of phage DNA into the bacterial genome at any unspecified location) is distinguished from special transduction (insertion at a particular location). Genes regularly transduced together (cotransduction) were used to determine the positions of neighboring genes on the bacterial chromosome (mapping of genes in bacteria).

Transduction by viruses
Transduction by viruses

B. Transformation by plasmids

Plasmids are small, autonomously replicating, circular DNA molecules separate from the chromosome in a bacterial cell. Since they of ten contain genes for antibiotic resistance (e.g., ampicillin), their incorporation into a sensitive cell renders the cell resistant to the antibiotic (transformation). Only these bacteria can grow in culture medium containing the antibiotic (selective medium).

Transformation by plasmids
Transformation by plasmids

C. Multiplication of a DNA segment in transformed bacteria

Plasmids are well suited as vectors for the transfer of DNA. A selective medium is used so that only those bacteria that have incorporated a recombinant plasmid containing the DNA to be investigated can grow.

Multiplication of a DNA segment in transformed bacteria
Multiplication of a DNA segment in transformed bacteria

D. Transfection by DNA

The transfer of DNA between eukaryotic cells in culture (transfection) can be used to examine the transmission of certain genetic traits (transfection assay). Left, a DNA transfer experiment is shown in a culture of mouse fibroblasts; right, in a culture of human tumor cells (Weinberg, 1985, 1987). The mouse fibroblast culture (see p. 122) is altered by the chemical carcinogen methylcholanthrene (left). DNA from these cells is precipitated with calcium phosphate, extracted, and then taken up by a normal culture (transfection). About 2 weeks later, cells appear that have lost contact inhibition (transformed cells). When these cells are injected into mice that lack a functional immune system (naked mice), tumors develop. DNA from cultured human tumor cells (right) also can transform normal cells after several transfer cycles. The DNA segment must be of limited size (e.g., a gene), since long DNA segments do not remain intact after repeated cycles of extraction and precipitation. Detailed studies of cancer-causing genes (oncogenes) in eukaryotic cells were first carried out using transfection (see p. 90). (Figures in A–C adapted from Watson et al. 1987, D from Weinberg 1987).

Transfection by DNA
Transfection by DNA




Bacteriophages

The discovery of bacterial viruses (bacteriophages or phages) in 1941 opened a new era in the study of the genetics of prokaryotic organisms. Although they were disappointing in the original hope that they could be used to fight bacterial infections, phages served during the 1950s as vehicles for genetic analysis of bacteria. Unlike viruses that infect plant or animal cells, phages can relatively easily be analyzed in their host cells. Names associated with phage analysis are Max Delbrück, Salvador Luria, and Alfred D. Hershey (the “phage group,” see: Cairns et al., 1966).

A. Attachment of a bacteriophage

Phages consist of DNA, a coat (coat protein) for protection, and a means of attachment (terminal filaments). Like other viruses, phages are basically nothing more than packaged DNA. One or more bacteriophages attach to a receptor on the surface of the outer cell membrane of a bacterium. The figure shows how an attached phage inserts its DNA into a bacterium. Numerous different phages are known,e.g., for Escherichia coli and Salmonella (phages T1, T2, P1, F1, lambda, T4, T7, phiX174 and others).

B. Lytic and lysogenic cycles of a bacteriophage

Phages do not reproduce by cell division like bacteria, but by intracellular formation and assembly of the different components. This begins with the attachment of a phage particle to a specific receptor on the surface of a sensitive bacterium. Different phages use different receptors, thus giving rise to specificity of interaction (restriction). The invading phage DNA contains the information for production of coat proteins for new phages and factors for DNA replication and transcription. Translation is provided for by cell enzymes. The phage DNA and phage protein synthesized in the cell are assembled into new phage particles. Finally, the cell disintegrates (lysis) and hundreds of phage particles are released. With attachment of a new phage to a new cell, the procedure is repeated (lytic cycle). Phage reproduction does not always occur after invasion of the cell. Occasionally, phage DNA is integrated into the bacterial chromosome and replicated with it (lysogenic cycle). Phage DNA that has been integrated into the bacterial chromosome is designated a prophage. Bacteria containing prophages are designated lysogenic bacteria; the corresponding phages are termed lysogenic phages. The change from a lysogenic to a lytic cycle is rare. It requires induction by external influences and complex genetic mechanisms.

C. Insertion of a lambda phage into the bacterial chromosome by crossing-over

A phage can be inserted into a bacterial chromosome by different mechanisms. With the lambda phage ( λ ), insertion results from crossing-over between the E. coli chromosome and the lambda chromosome. First, the lambda chromosome forms a ring. Then it attaches to a homologous section of the bacterial chromosome. Both the bacterial and the lambda chromosome are opened by a break and attach to each other. Since the homologies between the two chromosomes are limited to very small regions, phage DNA is seldom integrated. The phage is released (and the lytic cycle is induced) by there verse procedure.(Figures adapted from Watson et al., 1987).