Scientific Practicum for Students of the Specialty « Medical Biophysics »

According to the Russian Federation Government Decree on April 15, 2014, N. 295 «On approval of the Russian State Program of Development of Education for the 2013—2020 years» as amended on February 27, April 14, 2016, V. A. Negovsky Scientific Research Institute of General Reanimatology organized a specialized scientific Статья посвящена специализированному научному практикуму, организованному на базе лаборато# рии «Биофизики мембран клеток при критических состояниях» НИИ общей реаниматологи им. В. А. Не# говского для студентов, обучающихся по специальности «Медицинская биофизика» в Первом МГМУ им. И. М. Сеченова. В ходе работы студентами был освоен ряд методик: атомная силовая микроскопия, кали# брованная электропорация, спектрофотометрия. Студенты ознакомились с методологией научного экс# перимента и математической обработкой полученных результатов.

Практикум включал следующие темы: All research work within the Practicum was carried out on the basis of the Laboratory of bio physics of cells membranes in critical illness by pro fessor, Dr. Biol.Sci. A. M. Chernysh, the Head.
The objective of the Practicum -to teach stu dents to obtain research results independently, to explain them and to make appropriate conclusions.

Тhe authors set the following goals when creating the Practicum:
-To acquaint students with comprehensive meth ods of biophysics: atomic force microscopy, calibrated elec troporation; -To teach students to establish independently the necessary settings, and to employ sophisticated equipment in their research; -To teach students the methodology of scientific experiment; -To teach students to carry out mathematical pro cessing of study results and to submit the results for publi cation.
The Practicum included the following topics: 1. Atomic force microscopy: the idea of the method, the modes of operation of the atomic force microscope (AFM).
2. Methods of preparing and evaluating nanostruc ture of blood cell membranes.
3. AFM: defects of nanostructure of erythrocyte membranes during prolonged storage of donor packed red blood cells (PRBC).
4. AFM: defects of nanostructure erythrocyte mem branes under the action of toxins on the membrane of red blood cells (RBC) -Zn ions, hemin.
5. Calibrated electroporation method.Hidden dam age of erythrocyte membranes under the influence of mus cle relaxants (rocuronium Kabi).
6. Calibrated electroporation method.Hidden dam age to erythrocyte membranes under the action of ultravi olet (UV) radiation on human blood.
7. Spectrophotometry.Kinetics of hemoglobin derivatives under the action of sodium nitrite on human blood.
8. Methods of mathematical processing of the results.Using special laboratory programs created on the basis of Nonlinear Fitting software, OriginPro9 (USA).
All the experiments were performed in vitro.

Results and Discussion
The first theme « Atomic force microscopy, the idea of the method, the modes of operation of the atomic force microscope « was carried out with the whole group at once.Before the work the theoretical aspects of AFM(molecular interaction between the probe and the object of study, Lennard Jones inter actions depending on the distance between the object and the probe) were discussed.
During the work students independently estab lished the cantilever obtained diffraction of the laser beam on it, set the position of the laser beam on the photodiode, and used software to set the AFM.The most complicated operations including setting the cantilever resonance and obtaining high quality of the resonance curve at the AFM in a tapping mode.
The second theme was carried out individually by each student and was devoted to the methods of obtaining and evaluating the nanostructure of RBC membranes.Initially, it was necessary to form a monolayer of cells.It was performed by students with the aid of the V Sampler device (Austria).Then, the smears of cell monolayer was placed onto the AFM working table, andthe scale, speed and the number of scanning points were set.After imaging nanosurfaces of erythrocyte membranes and trans ferring the images to the program «FemtoScan» (Russia), the students quantified parameters of objects roughness by setting the markers.For this surface profiles constructed in any given direction.
In the course of the study, each student received an individual task to build their own image, determined profiled and estimated parameters of the erythrocyte membrane nanosurfaces [1].
The problem of evaluating the nanostructure defects in erythrocyte membrane during prolonged storage of PRBC was devoted to the work №3.По ходу выполнения работы каждый сту дент получал индивидуальное задание, строил operation of body tissue transplantation and it could further be contributed to the development of life threatening reactions.The quality of transfusion media is paramount in ensuring the effectiveness of transfusions and prevention of severe post transfu sion reactions [2].

Transfusion of blood components is considered as an
Main parameters of quality of donor PRBC are the morphology of cells and the structure of erythro cyte membranes [3,4].
The purpose of this work: to study the alter ations of RBC morphology and their membrane struc ture by AFM during long term storage of PRBC.
Bags were stored at 4°C for 30 days, in accor dance with WHO Recommendations.During the storage the morphology and nanostructure of ery throcyte membranes were evaluated as described previously [5].
During the period of storage of PRBC their morphology was transformed and their membranes were destructed [6].
Students compared nanostructure and mor phology of erythrocytes at 2, 8, 15 and 19 hours of storage of PRBC.Then they carried out the analysis of morphological changes at different periods of stor age [7][8][9].
As probes, the standard cantilevers fp N10 with an apex at an angle 22° and a radius of about 10 nm were used.The number of scan points: 512, field scanning: 10 10 μm, 1500 1500 nm, 800 800 nm and up to 150 150 nm.
Students employed various concentrations of zinc ions to obtain a wide range of damage of nanos tructure of erythrocyte membranes.Each student carried out an assessment of damages individually, according to specicicity of findings.
Hematin hydrochloride -hemin represented an another example of toixic agent the effect of which was studied.Hemin ise formed in a human body when hemoglobin enters the bloodstream and is undergone oxidation, for example, in the stomach.To prepare the working solution the chemically pure hemin (Sigma, USA) was used.50 mg of dry hemin was dissolved in solution containing 1 ml of NaOH solution and 5 ml of distilled water.The final con centration of hemin in the blood in the in vitro experiments was 1.8 mM.
The domains of different size (large and small one) were revealed on the membrane surface.A dis tinctive feature of the defects under the action of hemin was that all the domains were formed by grains of the same size.This makes it possible to study the mechanisms of formation of these domains in an individual blood cell [10].
In the next study the method of calibrated elec troporation was used [9,11,12].This method allows to determine the hidden damage of cell membranes, when the damage is not yet manifested at a physio logical level.The studied blood (3 ml) was placed in a quartz cuvette and was subjected to the effect of a high energy pulsed electric field.As a result of the exposure, the electrical breakdown of erythrocyte membranes arose.Then, the students developed the kinetic curves that determined the quality of the membrane.For these curves the rate constant was estimated for each patient's membranes.When the membrane of RBC in the patient's blood was normal, the rate constant was in a certain range.If the cells (membrane) were damaged, the constant rate was above the normal range.Thus, the value of the rate constant evaluated the extent of damage of the mem brane nanostructure.Students evaluated the effect of muscle relaxant Rocuronium bromide (Fresenius Kabi, USA) on erythrocyte membrane.In this exper iment, the erythrocyte suspension was administered in 1.0, 0.5, and 0.25 μl of drug per 1 mL of solution.The obtained kinetic curves are shown in Fig. 4.
Interesting scientific results were obtained dur ing the study course « Kinetics of hemoglobin deriva tives under the in vitro action of sodium nitrite on human blood «.The work was performed with the aid of a spectrophotometer linked to computer automatic Unico 2800 program (USA).The wavelengths were within ranges of 190-1100 nm at a step of 0.5 nm.
Spectrophotometry belongs to physico chemi cal methods of research based on the study of the absorption spectra in the ultraviolet (200-400 nm), visible (400-760 nm) and infrared (>760 nm) regions.Students study the dependence of optical density on the wavelength.This method is based on the law of Bouguer -Lambert -Beer -the weak ening of the beam of monochromatic light propagat ing in the light absorbing medium.Previously stu dents have investigated the absorption spectra of quartz and glass.Glass has a larger absorption value within the UV region (shorter than 300 nm).Therefore, for studies of spectra of biological sub stances it is necessary to use only quartz cuvettes.
Students studied the content of components of hemoglobin (oxy , deoxy -and methemoglobin) after the action of sodium nitrite.The first part of the experiments included 3 different concentrations of NaNO 2 added to the erythrocytes suspension.Evaluated spectra dependend on the concentrations of hemoglobin derivatives.The second stage -2 μl NaNO 2 was introduced into 2.4 ml hemoglobin solu tion.Time dependent kinetics of changes in concen trations of hemoglobin derivatives was recorded.This relationship is shown in Fig. 5.
Within 6 minutes concentrations of oxyhemo globin and methemoglobin were equal, and after 10 minutes the concentration of methemoglobin reached 90%.This method can be used to diagnose methemoglobinemia, detect poisoning by nitrates, and installations of biologically based standards for JDC and MAC values of nitrates in various food productss.
When performing the scientific Practicum the students have fulfilled all the objectives, mastered methods of atomic force microscopy, calibrated elec troporation [14] and spectrophotometry.The results ях спектров биологических веществ необходимо использовать только кварцевые кюветы.
Data obtained during the Practicum by AFM con firmed the presence of defects in nanostructures of red blood cells membranes during prolonged storage of donor PRBC, as well as the action of toxins on erythro cyte membrane.The results of applying of the calibrated electroporation, as seen from the kinetic curves (Fig. 7), showed the effectiveness of the method for detecting the hidden damages of erythrocytes membranes after action of muscle relaxant or ultraviolet radiation.Kinetics of hemoglobin derivatives concentrations, obtained by spectrophotometry, showed the increase of methemo globin under the influence of nitrate.
At the final lesson the results obtained by each student were discussed and the comparative analysis of these results was carried out.

Conclusion
Described Student's Practicum differed from the traditional methods of teaching because its main aim was to gain research experience through the obtaining scientific results.This paper contains examples of various results of the performed course.Each work required performing the number of inter dependent sequential operations and obtaining a whole complex of final evaluated variables.As exam ples, the concentration and time dependences of ori gin of the local membranes nanoscale defects, time and dose dependences of methemoglobin formation during UV irradiation of blood, a family of kinetic curves under the action of muscle relaxants and heavy metal ions on blood were determined during the course of education.Moreover, for each study individual initial experimental conditions were thor oughly set up.Therefore, each student performed the task by himself, independently, and gained personal experience to obtain the specific results.
On May 11, 2016 the Scientific Student Conference at the Department of Medical and Biological Physics of Sechenov First Moscow State Medical University was held, where all students pre sented the results of their works.
Students noted that techniques acquired dur ing Practicum and data obtained by employed meth ods exhibited broad perspectives for medical diag nostics.The conference was attended by representatives of the University administration and students of biophysics, biochemistry, bioengineering, biotechnology specialties and other related fields.The conference was very successful.
; 4 82 w w w .r e a n i m a t o l o g y .c o m DOI:10.15360/18139779 2016 4 79 88 Professional Education Рис. 2. Мембрана эритроцита в формате 3D и профиль поверхности.Fig. 2. Details of red blood cells membrane in 3D format and the profile of its surface.Note. a -the detailes of red blood cells membrane; b -image of a single red blood cell after the action of Zn ions; c -the detailes of red blood cells membrane hemin effect after; a1, b1, c1 -the profiles of the a, b, c surfaces of red blood cells.Примечание.a -фрагмент мембраны эритроцита; b -мембрана отдельного эритроцита после воздействия ионов Zn; c -фраг мент мембраны эритроцита после воздействия гемина; a1, b1, c1 -профили поверхностей a, b, c.Профессиональное образование w w w .r e a n i m a t o l o g y .c o m DOI:10.15360/18139779 2016 4 79 88
Practicum for students of the 4th year of the Sechenov First Moscow State Medical University.Students were trained in the specialty «Medical Biophysics»specialty code 30.05.02.The Practicum was carried out free of charge in accordance with the request of prof.T. M. Litvinova, the Vice Chancellor for Academic Affairs, I. M. Sechenov First Moscow State Medical University.Comprehensive support in the formulation of the Practicum was provided by the Professor V. V. Moroz, Corresponding Member of the Russian Academy of Sciences, Director of V. A. Negovsky Research Institute of General Reanimatology.
w w w .r e a n i m a t o l o g y .c o m