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ALS and the ice bucket challenge has been getting a lot of media coverage, with over $3 million being raised so far in Canada. What causes Amyotrophic lateral sclerosis (ALS) ?
Simple. Like most other human diseases it's Oxidative stress or excess free radicals, which C60 Hydrated Fullerenes, an ultra high, safe, enzymatic universal antioxidant from Ukraine neutralize. See Below
Front Cell Neurosci.... 2014 May 13;8:131. doi: 10.3389/fncel.2014.00131. eCollection 2014.
The role of oxidative stress in degeneration of the neuromuscular junction in amyotrophic lateral sclerosis. Pollari E1, Goldsteins G2, Bart G3, Koistinaho J2, Giniatullin R4.
Author information • 1Molecular Brain Research Laboratory, Department of Neurobiology, A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland Kuopio, Finland ; Experimental Neurology - Laboratory of Neurobiology, Department of Neurosciences, Vesalius Research Center, KULeuven - University of Leuven Leuven, Belgium. • 2Molecular Brain Research Laboratory, Department of Neurobiology, A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland Kuopio, Finland. • 3Cell Biology Laboratory, Department of Neurobiology, A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland Kuopio, Finland. • 4Cell Biology Laboratory, Department of Neurobiology, A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland Kuopio, Finland ; Laboratory of Neurobiology, Department of Physiology, Kazan Federal University Kazan, Russia.
Amyotrophic lateral sclerosis (ALS) is characterized by the progressive loss of motoneurons and degradation of the neuromuscular junctions (NMJ). Consistent with the dying-back hypothesis of motoneuron degeneration the decline in synaptic function initiates from the presynaptic terminals in ALS. Oxidative stress is a major contributory factor to ALS pathology and affects the presynaptic transmitter releasing machinery. Indeed, in ALS mouse models nerve terminals are sensitive to reactive oxygen species (ROS) suggesting that oxidative stress, along with compromised mitochondria and increased intracellular Ca(2+) amplifies the presynaptic decline in NMJ. This initial dysfunction is followed by a neurodegeneration induced by inflammatory agents and loss of trophic support. To develop effective therapeutic approaches against ALS, it is important to identify the mechanisms underlying the initial pathological events. Given the role of oxidative stress in ALS, targeted antioxidant treatments could be a promising therapeutic approach. However, the complex nature of ALS and failure of monotherapies suggest that an antioxidant therapy should be accompanied by anti-inflammatory interventions to enhance the restoration of the redox balance. KEYWORDS: ALS; ROS; neurodegeneration; neuromuscular junction; oxidative stress http://www.ncbi.nlm.nih.gov/pubmed/24860432
Meredith G. Dixon, MD1,2, Ilana J. Schafer, DVM1,2 (Author affiliations at end of text)
On March 21, 2014, the Guinea Ministry of Health reported the outbreak of an illness characterized by fever, severe diarrhea, vomiting, and a high case-fatality rate (59%) among 49 persons (1). Specimens from 15 of 20 persons tested at Institut Pasteur in Lyon, France, were positive for an Ebola virus by polymerase chain reaction (2). Viral sequencing identified Ebola virus (species Zaïre ebolavirus), one of five viruses in the genus Ebolavirus, as the cause (2). Cases of Ebola viral disease (EVD) were initially reported in three southeastern districts (Gueckedou, Macenta, and Kissidougou) of Guinea and in the capital city of Conakry. By March 30, cases had been reported in Foya district in neighboring Liberia (1), and in May, the first cases identified in Sierra Leone were reported. As of June 18, the outbreak was the largest EVD outbreak ever documented, with a combined total of 528 cases (including laboratory-confirmed, probable, and suspected cases) and 337 deaths (case-fatality rate = 64%) reported in the three countries. The largest previous outbreak occurred in Uganda during 2000–2001, when 425 cases were reported with 224 deaths (case-fatality rate = 53%) (3). The current outbreak also represents the first outbreak of EVD in West Africa (a single case caused by Taï Forest virus was reported in Côte d'Ivoire in 1994 ) and marks the first time that Ebola virus transmission has been reported in a capital city.
Bats provide vital ecologic services that humans benefit from, such as seed dispersal and pest control, and are a food source for some human populations. However, bats also are reservoirs for a number of high-consequence zoonoses, including paramyxoviruses, filoviruses, and lyssaviruses. The variety of viruses that bats harbor might be related to their evolutionary diversity, ability to fly large distances, long lifespans, and gregarious roosting behaviors. Every year a festival takes place in Idanre, Nigeria, in which males of all ages enter designated caves to capture bats; persons are forbidden from entering the caves outside of these festivities. Festival participants use a variety of techniques to capture bats, but protective equipment rarely is used, placing hunters at risk for bat scratches and bites. Many captured bats are prepared as food, but some are transported to markets in other parts of the country for sale as bushmeat. Bats also are presented to dignitaries in elaborate rituals. The health consequences of contact with these bats are unknown, but a number of viruses have been previously identified among Nigerian bats, including lyssaviruses, pegiviruses, and coronaviruses. Furthermore, the caves are home to Rousettus aegyptiacus bats, which are reservoirs for Marburg virus in other parts of Africa.
Cytotoxic and antiviral properties of fullerene C60 in the culture of animal cells
*State Scientific-Control Institute of Biotechnology and Strains of Microorganisms (Donetskaya St, 30,Kyiv-151, Ukraine);
**National Taras Shevchenko National University of Kyiv, Volodymyrska Str. 64, 01601 Kyiv, Ukraine
***The Institute of Veterinary Medicine of NAAS (Donetskaya St. 30, Kyiv-151, Ukraine)
One of the urgent problems of modern veterinary biotechnology is to solve the complex task that lies at the intersection of chemistry, physics, materials science, biology, veterinary medicine is focused design, synthesis and study of the functional properties of nanomaterials which characterized by high bioavailability and biocompatibility, low toxicity and high specific biological activity.
In our studies, was used C60 fullerene - fullerene in water-soluble form. This compound molecule is nearly spherical, with a diameter of 0.72 nm , the surface of which consists of 60 carbon atoms connected by single or double chemical called " links. Considered that C60- fullerenes are potential pharmaceutical compounds. However, along with a broad perspective on the use of such substances for the prevention and treatment of diseases, there are certain precautions, particularly with regard to the possible toxic effects on biological objects, including on cell.
Therefore, our research started with the determination of cytotoxic properties of C60 fullerene - on cell line BHK -21, which is continuous line origin from Syrian hamster and which is used in many medical and biological research.
In experiments used at least ten holes in plates with cell culture for each drug dilution in culture medium. The plates with cell culture incubated at +37˚ C with 5% СО2 for 96 hours.
Thus, we have found the maximum cytotoxic concentration of compound that was 0,0375 ±0,003 mg/ml (n=3,).
Determined the antiviral activity of C60 - fullerene, using as a biological model coronovirus (virus of transmissible gastroenteritis of swine). Coronoviruses affect both animals and humans, leading in many cases to a high degree of mortality. Investigation of antiviral activity of fullerene on transmissible gastroenteritis virus of swine in the system in vitro, n = 5 (each concentration: 0,15 , 0,075, 0,0375, 0,019, 0,009, 0,005 was tested in 10 holes).
We found that C60-fullerene reduced the infectious properties of the virus by 2.0 TCID 50/ml which is a significant result.
Therefore, preliminary data suggest recommend this compound for further preclinical and clinical studies.
Summary. Aim: To estimate the impact of C60 fullerene aqueous solution (C60FAS) on the rate of transplanted malignant tumor growth and metastasis. Methods: Lewis lung carcinoma was transplanted into С57Bl/6J male mice. Conventional methods for the evaluation of antitumor and antimetastatic effects have been used. Results: The C60FAS at low single therapeutic dose of 5 mg/kg inhibited the growth of transplanted malignant tumor (antitumor effect) and metastasis (antimetastatic effect): the maximum therapeutic effect was found to be of 76.5% for the tumor growth inhibition; the increase of animal life span by 22% was found; the metastasis inhibition index was estimated as 48%. Conclusion: It was found that water-soluble pristine С60 fullerenes efficiently inhibit the transplanted malignant tumor growth and metastasis
1Department of Neurology, Zhongshan Hospital; The State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai, 200032, China.
Oxidative stress plays a significant role in the pathogenesis of Alzheimer's disease (AD), a devastating disease of the elderly. The brain is more vulnerable than other organs to oxidative stress, and most of the components of neurons (lipids, proteins, and nucleic acids) can be oxidized in AD due to mitochondrial dysfunction, increased metal levels, inflammation, and β-amyloid (Aβ) peptides. Oxidative stress participates in the development of AD by promoting Aβ deposition, tau hyperphosphorylation, and the subsequent loss of synapses and neurons. The relationship between oxidative stress and AD suggests that oxidative stress is an essential part of the pathological process, and antioxidants may be useful for AD treatment.
Study demonstrates X-ray fluorescence spectroscopy is a non-destructive way to date artwork
Ancient Japanese gold leaf artists were truly masters of their craft. An analysis of six ancient Namban paper screens show that these artifacts are gilded with gold leaf that was hand-beaten to the nanometer scale. Study leader Sofia Pessanha of the Atomic Physics Center of the University of Lisbon in Portugal believes that the X-ray fluorescence technique her team used in the analysis could also be used to date other artworks without causing any damage to them. The results are published in Springer’s journal Applied Physics A: Material Science and Processing.
Gold leaf refers to a very thin sheet made from a combination of gold and other metals. It has almost no weight and can only be handled by specially designed tools. Even though the ancient Egyptians were probably the first to gild artwork with it, the Japanese have long been credited as being able to produce the thinnest gold leaf in the world. In Japanese traditional painting, decorating with gold leaf is named Kin-haku, and the finest examples of this craft are the Namban folding screens, or byobu. These were made during the late Momoyama (around 1573 to 1603) and early Edo (around 1603 to 1868) periods.
Pessanha’s team examined six screens that are currently either part of a museum collection or in a private collection in Portugal. Four screens belong to the Momoyama period, and two others were decorated during the early Edo period. The researchers used various X-ray fluorescence spectroscopy techniques to test the thickness and characteristics of the gold layers. The method is completely non-invasive, no samples needed to be taken, and therefore the artwork was not damaged in any way. Also, the apparatus needed to perform these tests is portable and can be done outside of a laboratory.
The gilding was evaluated by taking the attenuation or weakening of the different characteristic lines of gold leaf layers into account. The methodology was tested to be suitable for high grade gold alloys with a maximum of 5 percent influence of silver, which is considered negligible.
The two screens from the early Edo period were initially thought to be of the same age. However, Pessanha’s team found that gold leaf on a screen kept at Museu Oriente in Lisbon was thinner, hence was made more recently. This is in line with the continued development of the gold beating techniques carried out in an effort to obtain ever thinner gold leaf.
“This simple comparison allowed establishing a timeline between the manufacture of two pieces attributed to the same period, proving that X-ray fluorescence techniques can be an important asset in the dating of artworks,” says Pessanha.
Reference: Pessanha, S. et al (2014). Comparison of gold leaf thickness in Namban folding screens using X-ray fluorescence, Applied Physics A: Material Science and Processing. DOI 10.1007/s00339-014-8531-z
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CORAL GABLES, Fla. (June 25, 2014) — With the continuing need for very small devices in therapeutic applications, there is a growing demand for the development of nanoparticles that can transport and deliver drugs to target cells in the human body.
Recently, researchers created nanoparticles that under the right conditions, self-assemble – trapping complementary guest molecules within their structure. Like tiny submarines, these versatile nanocarriers can navigate in the watery environment surrounding cells and transport their guest molecules through the membrane of living cells to sequentially deliver their cargo.
Although the transport of molecules inside cells with nanoparticles has been previously achieved using various methods, researchers have developed nanoparticles capable of delivering and exchanging complementary molecules. For practical applications, these nanocarriers are highly desirable, explains Francisco Raymo, professor of chemistry in the University of Miami College of Arts and Sciences and lead investigator of this project.
"The ability to deliver distinct species inside cells independently and force them to interact, exclusively in the intracellular environment, can evolve into a valuable strategy to activate drugs inside cells," Raymo says.
The new nanocarriers are15 nanometers in diameter. They are supramolecular constructs made up of building blocks called amphiphilic polymers. These nanocarriers hold the guest molecules within the confines of their water-insoluble interior and use their water-soluble exterior to travel through an aqueous environment. As a result, these nanovehicles are ideal for transferring molecules that would otherwise be insoluble in water, across a liquid environment.
IMAGE: The sequential transport of donors and acceptors across cell membranes with independent and dynamic nanocarriers enables energy transfer exclusively in the intracellular space with concomitant fluorescence activation.
"Once inside a living cell, the particles mix and exchange their cargo. This interaction enables the energy transfer between the internalized molecules," says Raymo, director of the UM laboratory for molecular photonics. "If the complementary energy donors and acceptors are loaded separately and sequentially, the transfer of energy between them occurs exclusively within the intracellular space," he says. "As the energy transfer takes place, the acceptors emit a fluorescent signal that can be observed with a microscope."
Essential to this mechanism are the noncovalent bonds that loosely hold the supramolecular constructs together. These weak bonds exist between molecules with complementary shapes and electronic properties. They are responsible for the ability of the supramolecules to assemble spontaneously in liquid environments. Under the right conditions, the reversibility of these weak noncovalent contacts allows the supramolecular constructs to exchange their components as well as their cargo.
The experiments were conducted with cell cultures. It is not yet known if the nanoparticles can actually travel through the bloodstream.
"That would be the dream, but we have no evidence that they can actually do so," Raymo says. "However, this is the direction we are heading."
The next phase of this investigation involves demonstrating that this method can be used to do chemical reactions inside cells, instead of energy transfers.
"The size of these nanoparticles, their dynamic character and the fact that the reactions take place under normal biological conditions (at ambient temperature and neutral environment) makes these nanoparticles an ideal vehicle for the controlled activation of therapeutics, directly inside the cells," Raymo says.
IMAGE: The sequential transport of donors and acceptors across cell membranes with independent and dynamic nanocarriers enables energy transfer exclusively in the intracellular space with concomitant fluorescence activation.
The current study is titled "Intracellular guest exchange between dynamic supramolecular hosts." It's published in the Journal of the American Chemical Society. Other authors are John F. Callan, co-corresponding author of the study, from the School of Pharmacy and Pharmaceutical Sciences at the University of Ulster; Subramani Swaminathan and Janet Cusido from the UM's Laboratory for Molecular Photonics, Department of Chemistry in the College of Arts and Sciences; and Colin Fowley and Bridgeen McCuaghan, School of Pharmacy and Pharmaceutical Sciences at the University of Ulster.
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This work focuses on the interactions between molecules and in particular on "amphiphilic" molecules, which contain two distinct parts to them. Household detergent is a good example of a product that relies on interacting amphiphilic molecules. Detergent molecules comprise two distinct parts: one that prefers to form bonds with water (hydrophilic) and the other that likes oily substances (hydrophobic). Detergents are used for cleaning because when they are added to dirty water, they orient and assemble around oily dirt, forming small clusters that allow grease and dirt to be more easily removed from the water.
The newly reported method takes the concept of amphiphilic assembly one step further, and applies it to a completely new set of hydrophobic molecules, intriguingly with no water-loving part to them. These new "hydrophobic amphiphiles" still have different 'parts', but the assembly process relies on more subtle interactions.
The research was carried out by an international team of researchers led by Dr Martin Hollamby (Keele University, UK) and Dr Takashi Nakanishi (National Institute for Materials Science, Japan). Together they showed used neutron scattering techniques at the Institut Laue-Langevin (ILL) to investigate the arrangement of these clusters and showed that hydrophobic amphiphiles can still assemble into extended structures in much the same way as conventional amphiphiles.
One example is a molecule shaped like a football but with a long tail. The amphiphile has been tailor made from 'bucky balls' - football-shaped molecules made up of 60 carbon atoms (C60) which are chemically modified by attaching a much longer 'tail' made up of chains of carbon atoms, as found in a regular soap. The new detergents resemble "molecular tadpoles". When dissolved in solvents that interact with the tails, these molecules assemble to form a core of C60 spheres and a shell of carbon chains.
"Changing the chemistry of the chains can even lead to gels made of bundled C60 wires that have a measureable (photo)conductivity" explains Dr Martin Hollamby. "By adding pristine C60 in place of the solvent, we instead prepare a sheet-like material with totally different properties".
Small-angle neutron scattering data obtained on beamline D11 at the ILL was crucially used to prove the internal structure of these clusters.
"The light elements that makes up these 'molecular tadpoles' are easily located by neutrons" says Dr Isabelle Grillo, at the ILL. "Moreover, small angle neutron scattering which we use at the ILL allows to characterise the self-assembled systems from the nanometre scale to tenth of micrometres and is perfectly adapted to observe the coming together of the C60 footballs' into these beautiful core structures."
This flexibility is the remarkable thing about the new route towards self-organised structures. A great variety of different structures can be produced just by making small changes to the chemical structure and the additives (solvent or C¬60) used. This level of control over self-assembly in complex molecules such as C60 is unprecedented.
One area that could be significantly impacted by this new discovery is the field of 'molecular electronics'. These carbon-based electronics could provide a cheaper alternative to traditional silicon technology and allow for flexible handheld devices for many functions, including smartphones and tablets for watching TV.
Furthermore, the new molecular electronic components could lead to improved properties (e.g. higher efficiency, lower power consumption) simply by optimizing how the molecules interact with each other. In 2018 during the next World Cup in Russia you could be using football-shaped molecules to actually watch the football!
Excess Free Radicals in the body (called oxidative stress) triggers many diseases, but it's easy to prevent or in some cases reverse. To neutralize excess free radicals in the body, eat antioxidant foods daily, with high ORAC values (see ORAC table http://www.oracvalues.com/sort/orac-value ) and drink Hydrated Fullerene or C-60 water-the highest antioxidant in the world. For information contact me
The paths taken by a single cholinergic neuron as it branches through a thin section of the forebrain of a mouse
Nathans Lab, eLife
By studying laboratory mice, scientists at The Johns Hopkins University have succeeded in plotting the labyrinthine paths of some of the largest nerve cells in the mammalian brain: cholinergic neurons, the first cells to degenerate in people with Alzheimer’s disease.
“For us, this was like scaling Mount Everest,” says Jeremy Nathans, Ph.D., professor of molecular biology and genetics, neuroscience, and ophthalmology at the Johns Hopkins University School of Medicine. “This work reveals the amazing challenges that cholinergic neurons face every day. Each of these cells is like a city connected to its suburbs by a single, one-lane road, with all of the emergency services located only in the city. You can imagine how hard it would be in a crisis if all of the emergency vehicles had to get to the suburbs along that one road. We think something like this might be happening when cholinergic neurons trying to repair the damage done by Alzheimer’s disease.”
Each cholinergic neuron, Nathans explains, has roughly 1,000 branch points. If lined up end to end, one neuron’s branches would add up to approximately 15 times the length of the mouse brain. But all of the branches are connected by a single, extremely thin “pipeline” to one hub — the cell body — that provides for the needs of the branches. The challenge of moving material through this single pipeline could make it very difficult for cholinergic neurons to combat the challenges that come with a disorder like Alzheimer’s disease, he says. Now, by mapping the branches and pipelines, scientists will likely get a better fix on what happens when the neurons fail to meet the challenges.
Cholinergic neurons are among the largest neurons in the mammal brain. Named for their release of a chemical messenger called acetylcholine, they number only in the thousands in mouse brains, a tiny fraction of the 50 to 100 million total neurons. Their cell bodies are located at the base of the brain near its front end, but their branches extend throughout the cerebral cortex, the outermost, wrinkled layer of “grey matter” that is responsible for the mind’s most advanced intellectual functions. Therefore, although there are relatively few cholinergic neurons, they affect a very large part of the brain, Nathans says.
Due to the technical challenge of visualizing the complicated paths of hundreds of tiny branches from a single neuron tangled within millions of other neurons, the actual size and shape of individual cholinergic neurons — and the territory they cover — had been unknown until now, Nathans says. Using genetic engineering methods, the Nathans team programmed several cholinergic neurons per mouse to make a protein that could be seen with a colored chemical reaction. Critical to the success of the work was the ability to limit the number of cells making the protein — if all of the cholinergic neurons made the protein, it would have been impossible to distinguish individual branches.
Because microscopes cannot see through thick tissue, Nathans and his team preserved the mouse brains and then thinly sliced them to produce serial images. The branching path of each neuron was then painstakingly reconstructed from the serial images and analyzed. In adult mice, he says, the average length of the branches of a single cholinergic neuron, lined up end to end, is 31 cm (12 inches), varying from 11 to 49 cm (4 to 19 inches). The average length of a mouse brain is only 2 cm — a bit less than one inch. Although each cholinergic neuron, on average, contains approximately 1,000 branch points, they vary significantly in the extent of the territory that they cover.
The researchers used the same techniques to study the cholinergic neurons of mice with a rodent form of Alzheimer’s disease and found that the branches were fragmented. They also found clumps of material that may have been debris from the disintegrating branches.
Although the cholinergic neurons of human brains have not been individually traced, Nathans’ team was able to calculate that the average cholinergic neuron in the human brain has a total branch length of approximately 100 meters, a bit longer than a football field. “That is a really long pipeline, especially if one considers that the pipes have diameters of only 30 thousandths of a millimeter, far narrower than a human hair,” says Nathans.
He adds, “Although our study only defined a few simple, physical properties of these neurons, such as size and shape, it has equipped us to form and test better hypotheses about what goes wrong with them during disease.”
Other authors of the report include Hao Wu and John Williams of the Johns Hopkins University School of Medicine.
This work was supported by grants from the Human Frontier Science Program, the Howard Hughes Medical Institute and the Brain Science Institute of The Johns Hopkins University.
New Synthesis Method of Nickel-Carbon Heterofullerenes Presented
Heterofullerene molecula. Image courtesy of the authors of the research
Scientists from several British, Spanish and Russian research centers (MIPT, Institute for Spectroscopy RAS, Kurchatov Institute and Kintech Lab Ltd) have come up with a method of synthesizing a new type of nickel-carbon compound. The article titled Formation of nickel-carbon heterofullerenes under electron irradiation has been published by Dalton Transactions and is available as a pre-print at arxiv.org. The first author of the article is Alexander Sinitsa, an MIPT student, and the leading author is Andrey Popov (Institute for Spectroscopy RAS, 1989 MIPT graduate).
Heterofullerenes are hollow molecules with a nearly-spherical shape, which, unlike the typical fullerenes, contain atoms of elements other than carbon. Such compounds were synthesized quite a while ago, in 1991, but till now no heterofullerenes containing nickel, or any other transition metal, have been obtained. Yet, as the authors point out in their article, transition metals are now being studied as catalysts in the synthesis of carbon nanotubes and graphene.
“I’d like to emphasize that the majority of calculations have been performed by a student. Hopefully, students regularly visit the MIPT site and get inspired by their colleagues’ successes. If you are especially interested in the role of MIPT graduates in research, then I can tell you that Irina Lebedeva graduated from the Institute in 2008, and Andrey Knizhnik, perhaps in 1999, but I’m not exactly sure about the year. I’d also like to point out that Elena Bichoutskaia (a Saint Petersburg State University Faculty of Physics graduate) is a member of the Russian diaspora abroad, which is typical of international cooperation of Russian scientists,” Andrey Popov told the MIPT Press Service.
The synthesis of nickel heterofullerenes is supposed to be carried out under electron irradiation, which is used in high-resolution transmission electron microscopy (HRTEM) in order to obtain detailed snapshots showing, if needed, separate atoms. A number of previous experiments conducted by various research groups demonstrated that electronic irradiation can also be applied to synthesize a variety of nanostructures, e.g., one-layer carbon fullerene-filled nanotubes were transformed into two-layer ones.
Using the latest data obtained from the HRTEM images and the results of computer modelling by methods of molecular dynamics, the scientists have shown the potential possibility to transform graphene flakes with nickel cluster into nickel-carbon heterofullerene.
The scientists, though, are not sure about the practical application of such heterofullerenes. According to Andrey Popov, “these new-type molecules can reveal some interesting electronic, magnetic, and optic features, or it may be possible to combine them with some organic functional complexes of interest to biologists and physicians. They can also be used to create 3D organic-metallic structures to store hydrogen”.
In their work, the researchers developed and applied an authentic algorithm for modelling electron-nanostructure interactions. This allows taking into account both fast (just tens of picoseconds) and slow (lasting for full seconds) processes. The fast processes are associated with electron collisions, and the slow ones relate to molecular relaxation.
MIPT Press Service expresses gratitude to Andrey Popov for the invaluable help in preparing the material.