VESTIBULAR DYSFUNCTIONS IN CHRONIC BRAIN ISCHEMIA IN THE POST COVID PERIOD.

OBJECTIVE: The aim: The aim of the study is the clinical-pathogenetic reasoning of vestibular dysfunctions (VD) development against the background of chronic brain ischemia in the presence of degenerative changes in the cervical spine (CS) in the post COVID period. PATIENTS AND METHODS: Materials and methods: 82 patients, in the conditions of the clinical base of the Odessa National Medical University in 2019-2021 were examined. Group I with VD against the background of chronic brain ischemia (CBI) at the compensated phase; Group II with VD against the background of CBI at the subcom¬pensated phase (33 men; 49 women), aged from 18 to 55 years. The control group (CG) consisted of 20 patients of the corresponding gender and age. The condition of the state of the autonomic nervous system, vestibular functions, cervical spine, cerebral arteries and emotional condition were examined. RESULTS: Results: Vestibulo-ataxic disorders were higher compared to CG and increased along with the degree of brain damage. An important aspect of the development of VD is autonomic dysfunction against the background of pathological autonomic characteristics with predominant parasympathetic orientation of autonomic tone, especially in the case of insufficiency of autonomic recativity (AR) and pathological autonomic support of activity. Such changes significantly increased in the presence of subcompensation of CBI. The correlation between psychoemotional disorders and changes in autonomic characteristics with VD against the background of CBI with initial regularities depending on the degree of brain damage was defined. The progression of CBI is facilitated by coronavirus infection and manifested in autonomic and psychoemotional dysfunctions. A characteristic hemodynamic feature in groups with compensated and subcompensated CBI is the presence of reduced perfusion in basilar (BA) and vertebral (VA) arteries. Changes in cerebral vascular reactivity with a decrease in cerebrovascular reactivity indicators were characteristic of the subcompensated phase of CBI. Hyperactivity to rotational functional loads in both clinical groups has a high correlation with the presence of stair descent and, to a lesser extent, isolated instability in CS. CONCLUSION: Conclusions: 1. The occurrence of VD is facilitated by the presence of autonomic dysfunction and degenerative-dystrophic changes in the CS, especially in case of subcompensation of CBI. 2. Psychoemotional changes were a characteristic feature of patients with VD against the background of CBI and had certain regularities depending on the phase of CBI. 3. Suffered coronavirus infection contributes to the progression of VD and further decompensation of CBI due to direct damage to the autonomic and vascular systems of the brain. 4. Changes in cerebral hemodynamics in the form of reduced perfusion in BA and VA, a decrease in cerebrovascular reactivity, and an increase in reactivity to rotational functional load were determined in patients with VD against the background of subcompensated CBI.

STATE OF AUTONOMIC REGULATION AND CEREBROVASCULAR REACTIVITY IN PATIENTS WITH HEADACHE WITH ARTERIAL HYPERTENSION.

OBJECTIVE: The aim: A comprehensive assessment of the state of the autonomic nervous system and cerebrovascular reactivity of the cerebral blood flow in the patients with the headache accompanied by arterial hypertension (AH) and somatoform autonomic dysfunction (SAD). PATIENTS AND METHODS: Materials and methods: We conducted the clinical, autonomic regulation and Doppler sonography examination of 124 young patients (18-45 years old), including 51 men and 73 women in the conditions of the clinical base of the Kharkiv Medical Academy of Postgraduate Education in 2018-2021. All patients with cephalgias were divided into three groups: with AH stage II (Group I - 41 patients), AH stage I (Group II - 40 patients), SAD (Group III - 43 patients). The control group consisted of 50 patients of the corresponding gender and age. RESULTS: Results: The intensity of cephalalgia in patients with SAD was maximum. The autonomic tone (AT) was changed in 68.5%examined patients. It had a pronounced shift towards sympathicotonia. According to the visual analogue scale the maximum intensity of cephalgias was against the sympathicotonia. In the groups with organic lesions of the cerebral vessels the latent period delay was registered with the progression of the organic pathology. The regularity was revealed - the shortening of the evoked skin sympathetic potentials latency with the severity of cephalalgia, which can be interpreted as an increase in ergotropic effects with the realisation of the pain syndrome. The obtained data on the state of the AT indicate the depletion of the ergotropic processes with the progression of cerebral ischemia with a known increase in parasympathicotonia. In the patients of SAD group the CrCO2 and KrFNTvalues were significantly increased, in AH stage I group they slightly exceeded the standard values , in AH stage II group they were reduced. The reactivity to the orthostatic loads and functional metabolic test in all groups exceeded the control values. CONCLUSION: Conclusions: 1. The cephalgic syndrome is one of the main symptoms of the autonomic dysfunction and arterial hypertension; the frequency and intensity of the headache increases with the hyperreactivity of the sympathetic system. 2. The SSP data indicate that the sympathetic activity triggers and maintains the pain syndrome, and can also be realized in the form of arterial hypertension. 3. The dysfunction of the central link of the GSR indicates the instability of the autonomous regulation, the work of the limbic-reticular complex, which is clinically manifested by the changes in the cerebral vascular tone. 4. The cerebrovascular hyperreactivity as a sign of the search for the optimal sanogenetic variant of the cerebral hemodynamics in patients with SAD and AH stage I occurs predominantly due to the vasodilatory component. 5. In the patients with AH stage II the vasoconstrictor reactions are observed with the depletion of the vasodilation reserves, which is a marker of the autoregulation failure.

FEATURES OF HEMODYNAMICS IN HEAD MAGISTRAL AND CEREBRAL ARTERIES IN THE PATIENTS WITH MIGRAINE.

OBJECTIVE: The aim of the study was to Doppler sonography study of the structural and functional state of head magistral arteries (HMA) and cerebral arteries in the patients with various forms of migraine. PATIENTS AND METHODS: Materials and methods: We conducted the clinical Doppler examination of 124 young patients (18-45 years old), including 55 men and 69 women in the conditions of the clinical base of the Kharkiv Medical Academy of Postgraduate Education in 2017-2019. The criteria for involvement of patients in the study were: migraine without aura (group 1 - 63 patients), migraine with aura (group 2 - 61 patients) The control group consisted of 45 patients of the corresponding gender and age. The condition of HMA and cerebral arteries was studied using the ultrasound device. RESULTS: Results: The presence of extravasal compressions of vertebral arteries (VA) is typical for the patients with migraine, as well as for some cases of the hypoplasia of the VA in the group of the patients with migraine with aura. In the patients with migraine with aura, there was a decrease in the velocity values in the extracranial VA segments. The velocity values in the external carotid arteries (ECA) were slightly reduced in both groups. The most significant were the changes in the hemodynamics in the middle cerebral arteries (MCA), which were manifested by the pattern of the excessive perfusion in the patients of the migraine without aura and the pattern of the hampered perfusion in the MCA in the patients of the migraine with aura. CONCLUSION: Conclusions: 1. In the patients with migraine with aura, a decrease in the velocity values in the extracranial segments of the VA was observed, in some cases combined with the hypoplasia of the vertebral artery, the hampered perfusion in middle cerebral arteries. 2. The excessive perfusion in middle cerebral arteries is the most critical hemodynamic pattern in the patients with migraine without aura. 3. The extravasal compression of vertebral arteries, combined with the hyperperfusion in posterior cerebral arteries, is a typical hemodynamic pattern both in the group of the patients with migraine with aura, and in the group of the patients with migraine without aura.

FEATURES OF CEREBROVASCULAR REACTIVITY IN PATIENTS OF YOUNG AGE WITH MIGRAINE.

OBJECTIVE: The aim of the study was to Doppler sonography study of cerebrovascular reactivity in young patients with migraine and comparison of autoregulation patterns between groups of migraine patients with aura and migraine without aura. PATIENTS AND METHODS: Materials and methods: We conducted the clinical Doppler examination of 124 young patients (18-45 years old), including 55 men and 69 women in the conditions of the clinical base of the Kharkiv Medical Academy of Postgraduate Education in 2017-2019. The criteria for involvement of patients in the study were: migraine without aura (group 1-63 patients), migraine with aura (group 2-61 patients) The control group consisted of 45 patients of the corresponding gender and age. The indicators of CVR were studied using the ultrasound device. RESULTS: Results: A hyperreactive response to tests with CO2 and photoreactivity were more pronounced in the patients with migraine without aura. The patients with migraine with aura showed hyperreactivity in the test with O2, which was an indicator of the tendency to hyperconstriction. The hyperreactivity in the functional nitroglycerin test and the carotid compression test shows the state of the myogenic mechanism of changes in vascular tone. Insignificant hyperreactivity to orthostatic load detected in both groups shows the interest of the neurogenic regulatory link. CONCLUSION: Conclusions: 1. The most important hemodynamic patterns in the patients with migraine are excessive perfusion with migraine without aura and difficult perfusion with migraine with aura. 2. For the patients with migraine without aura, a characteristic criterion for autoregulation is the pattern of hyperreactivity hypercapnic load and photostimulation. 3. The most important difference in the autoregulatory response in the patients with migraine with aura compared with migraine without aura is the hyperresponse to hyperventilation load, compression carotid and nitroglycerin tests.

Higher degree of glycation of hemoglobin S compared to hemoglobin A measured by mass spectrometry: Potential impact on HbA1c testing.

BACKGROUND: Glycated hemoglobin (GHb), reported as HbA1c, is used as marker of long-term glycemia for diabetic patients. HbA1c results from boronate affinity methods are generally considered to be unaffected by most hemoglobin variants; this assumes comparable glycation of variant and non-variant (HbAA) hemoglobins. In this report, glycation of HbA beta chain (ßA) and HbS beta chain (ßS) for the most common Hb variant trait (HbAS) are examined. METHODS: We analyzed 41 blood samples from subjects with HbAS, both with and without diabetes. Using LC-MS, ratios of glycated HbS to glycated HbA were determined by comparison of areas under the curves from extracted ion chromatograms. RESULTS: Glycation of ßS chains was significantly higher (p<0.001) than ßA chains; this difference was consistent across subjects. Total (α+ß) glycated HbAS was theoretically estimated to be ~5% higher than glycated HbAA. CONCLUSION: This novel mass-spectrometric approach described allows for relative quantification of glycated forms of ßS and ßA. Although ßS glycation was significantly higher than that of ßA, the difference in total glycation of HbAS versus HbAA was smaller and unlikely to impact clinical interpretation of boronate affinity HbA1c results. These data support the continued use of boronate affinity to measure HbA1c in patients with HbAS.

Nanopore Event-Transduction Signal Stabilization for Wide pH Range under Extreme Chaotrope Conditions.

Operation of an α-hemolysin nanopore transduction detector is found to be surprisingly robust over a critical range of pH (6-9), including physiological pH = 7.4 and polymerase chain reaction (PCR) pH = 8.4, and extreme chaotrope concentration, including 5 M urea. The engineered transducer molecule that is captured in the standard α-hemolysin nanopore detector, to transform it into a transduction detector, appears to play a central role in this stabilization process by stabilizing the channel against gating during its capture. This enables the nanopore transduction detector to operate as a single molecule "nanoscope" in a wide range of conditions, where tracking on molecular state is possible in a variety of different environmental conditions. In the case of streptavidin biosensing, results are shown for detector operation when in the presence of extreme (5 M) urea concentration. Complications involving degenerate states are encountered at higher chaotrope concentrations, but since the degeneracy is only of order two, this is easily absorbed into the classification task as in prior work. This allows useful detector operation over a wide range of conditions relevant to biochemistry, biomedical engineering, and biotechnology.

Two-step ion-exchange chromatographic purification combined with reversed-phase chromatography to isolate C-peptide for mass spectrometric analysis.

A liquid chromatography with mass spectrometry on-line platform that includes the orthogonal techniques of ion exchange and reversed phase chromatography is applied for C-peptide analysis. Additional improvement is achieved by the subsequent application of cation- and anion-exchange purification steps that allow for isolating components that have their isoelectric points in a narrow pH range before final reversed-phase mass spectrometry analysis. The utility of this approach for isolating fractions in the desired "pI window" for profiling complex mixtures is discussed.

Isotope dilution assay in peptide quantification: the challenge of microheterogeneity of internal standard.

Isotope dilution analysis allows quantitation of elements and different compounds in complex mixtures. The quantitation is based on a known amount of reference material (internal standard, IS) added to a sample that makes the result critically dependent on the value assigned to the standard. In the case of peptides, IS concentration is determined by nitrogen and amino acid analysis while purity is normally assessed by methods such as chromatography or electrophoresis that might not be able to detect many possible amino acid modifications, either naturally occurring or chemically induced. Microheterogeneity of the IS, if it is not accounted for when assigning a reference value to the standard, results in highly overestimated values in target analyte quantitation. In this viewpoint article, we illustrate the problem of internal standard microheterogeneity by analyzing synthetic human C-peptide labeled analogs.

Human C-peptide Quantitation by LC-MS Isotope-Dilution Assay in Serum or Urine Samples.

In this communication we report a simple and efficient approach to C-peptide quantitation using isotope dilution mass-spectrometry analysis. The method facilitates quantitation of C-peptide levels at least one order of magnitude lower compared to concentration levels achieved with an IDA method reported previously. The improvement was due to more intensive sample preparation procedure that, in turn, makes it possible to increase the sample load without a corresponding increase in matrix effects. We also show the results of a comparison study with a second laboratory using a similar previously reported method for C-peptide quantitation.

IEF-based multidimensional applications in proteomics: toward higher resolution.

This review focuses on the alternative isoelectrofocusing methods and IEF-related techniques in protein analysis and characterization. The main emphasis is placed on the evaluation of the resolving power of new approaches, as well as potential advantages of IEF-related techniques for multidimensional analysis. In particular, the inverse 2D gel electrophoresis where the Mw separation stage precedes IEF mode is considered.

Use of cation exchange chromatography for human C-peptide isotope dilution - mass spectrometric assay.

An application of ion exchange chromatography for C-peptide analysis is described here. At the stage of C-peptide isolation, a strong cation exchanger (SP HP or MonoS) was used to purify the analyte from ballast proteins and peptides. The conditions of ion-exchange chromatographic separations were optimized using theoretical modeling of the net surface electric charge of the peptide as a function of pH. The purified and concentrated sample was further subjected to LC-MS/MS. In order to improve the reliability of analysis, two fragment ions were monitored simultaneously both for native C-peptide and internal standard, isotopically labeled C-peptides analogues (fragments with m/z of 927.7 and 147.2). Using ion-exchange chromatography, it became possible to process larger sample volumes, important for testing patients with very low C peptide levels, compared to currently used solid phase extraction methods.

Pattern recognition-informed feedback for nanopore detector cheminformatics.

Pattern recognition-informed (PRI) feedback using channel current cheminformatics (CCC) software is shown to be possible in "real-time" experimental efforts. The accuracy of the PRI classification is shown to inherit the high accuracy of our offline classifier: 99.9% accuracy in distinguishing between terminal base pairs of two DNA hairpins. The pattern recognition software consists of hidden Markov model (HMM) feature extraction software, and support vector machine (SVM) classification/ clustering software that is optimized for data acquired on a nanopore channel detection system. For general nanopore detection, the distributed HMM and SVM processing used here provides a processing speedup that allows pattern recognition to complete within the time frame of the signal acquisition - where the sampling is halted if the blockade signal is identified as not in the desired subset of events (or once recognized as nondiagnostic in general). We demonstrate that Nanopore Detection with PRI offers significant advantage when applied to data acquisition on antibody-antigen system, or other complex biomolecular mixtures, due to the reduction in wasted observation time on eventually rejected "junk" (nondiagnostic) signals.

Protein fractionation according to molecular size in constant pH media with immobilized charges in colinear porosity gradient.

A new method of protein electrophoresis is described here. Electrophoretic separation is performed in gel media with uniform concentration of immobilized charges, combined with porosity gradient directed against protein movement. Successful separation becomes possible due to the effect of strong sample zone compression; the latter effect is connected with complex conductivity profile dynamics in a gel system containing immobilized charges. Immobilized buffers combined with porosity gradient provide an opportunity of protein discrimination based on molecular size, while in the case of uniform gel concentration the separation is based on mobility differences and strongly affected by non-uniform electric field strength profile. The proposed method does not require ionic detergent for protein separation according to their molecular weight.

Nanopore-based kinetics analysis of individual antibody-channel and antibody-antigen interactions.

BACKGROUND: The UNO/RIC Nanopore Detector provides a new way to study the binding and conformational changes of individual antibodies. Many critical questions regarding antibody function are still unresolved, questions that can be approached in a new way with the nanopore detector. RESULTS: We present evidence that different forms of channel blockade can be associated with the same antibody, we associate these different blockades with different orientations of "capture" of an antibody in the detector's nanometer-scale channel. We directly detect the presence of antibodies via reductions in channel current. Changes to blockade patterns upon addition of antigen suggest indirect detection of antibody/antigen binding. Similarly, DNA-hairpin anchored antibodies have been studied, where the DNA linkage is to the carboxy-terminus at the base of the antibody's Fc region, with significantly fewer types of (lengthy) capture blockades than was observed for free (un-bound) IgG antibody. The introduction of chaotropic agents and its effects on protein-protein interactions have also been observed. CONCLUSION: Nanopore-based approaches may eventually provide a direct analysis of the complex conformational "negotiations" that occur upon binding between proteins.

On the possibility of applying noncovalent dyes for protein labeling in isoelectric focusing.

Noncovalent fluorescent dyes are widely used for protein quantification and postcolumn detection in electrophoretic separations and recently some attempts to separate the precolumn labeled proteins using isoelectric focusing (IEF) have been made. In the present study, the possibility of applying the technique of protein labeling with noncovalent dyes for IEF is investigated. We found that fluorescent signal emitted by NanoOrange dye increases essentially in presence of carrier ampholyte (CA) components, which makes problematic a reliable protein detection in CA environment. Since in an isoelectric focusing mode the CA species are present in much greater concentration than the concentrations of fractionated proteins, the method of protein labeling with NanoOrange is not suitable for precolumn labeling and cannot be used for CA-IEF, at least without more detailed study of the dye-protein interaction mechanism.

Conductivity properties of carrier ampholyte pH gradients in isoelectric focusing.

The conductivity properties of natural pH gradient created by carrier ampholytes were studied during the process of isoelectric focusing (IEF). IEF was performed in capillaries (10-30 mm long) or in microchips with the same channel length. A 10-30x reduction of the conductivity of the separation medium was observed during the establishment of pH gradient. Results obtained using different IEF voltages indicate that there is a nonlinear relationship between the conductivity of an established pH gradient and the applied electric field. Our theoretical analysis using a simplified model generated values that reasonably agree with the experimental data. In addition, we found that above a certain electric field ( approximately 300 V/cm), resolution does not increase with the applied voltage as predicated; we observed band-broadening and gel breakdown. The approach presented in this work can be used for optimization of the IEF separation and judicious selection of IEF conditions.

Buffer composition changes in background electrolyte during electrophoretic run in capillary zone electrophoresis.

The electrophoretic behaviors of different analytes in capillary zone electrophoresis were studied by the Whole Column Imaging Detection (WCID). For capillary zone electrophoresis (CZE) in conventional buffer systems, non-constant sample plug movement characterized by progressive decrease of peak migration velocity was observed. The appropriate velocity decrease was correlated with a degree of ionization of the analyzed ion, thus the effect observed could be explained by fast buffer composition change resulting in the development of a non-linear pH gradient. To visualize the appropriate pH gradient, the concentration profile of initially uniformly distributed amphoteric substances was also monitored. The evolution of the concentration profile exhibited very complex dynamics. In addition, it was found that the nature of the electrode solutions strongly affect changes in the background electrolyte. In the case of traditional background electrolytes with an acid-base pair for electrode solutions a non-uniform ampholyte concentration developed quickly, leading finally to a quasi-stationary profile similar to those typical of IEF. Possible approaches to suppress a negative impact of the background electrolyte composition changes during electrophoretic run on CE-separation are presented herein. In particular it was observed that zwitterionic buffers are able to withstand prolonged electrolysis much better compared to traditional buffers.

CE in a nonuniform capillary modulated by a cylindrical insert, and zone-narrowing effects during sample injection.

The electrophoretic behavior of an analyte in a capillary consisting of two parts of different cross section has been investigated. Modulation of the cross-sectional area of the separation channel has been achieved by inserting a cylindrical fiber different distances into the capillary. It was shown that the zone injected into the capillary part with smaller cross section could be moved using electromigration into the wider part of the capillary with zone compression. As we observed, the zone narrowed longitudinally in the wide part of the capillary in accordance with the ratio of the electric field strength in the two parts of the capillary. The concentration of plug introduced into the capillary by electroinjection can be increased by use of low-conductivity sample buffer. Efficient introduction of extracted analytes desorbed from an SPME fiber to the separation channel was achieved using this approach. Thermoinduced effects caused by temperature increase in the narrow part of the capillary and their influence on sample stacking are analyzed. Possible applications of the effect observed to the sample introduction optimization are also discussed in this study.

Heterogeneity of protein labeling with a fluorogenic reagent, 3-(2-furoyl)quinoline-2-carboxaldehyde.

Fluorogenic reagents are used for protein labeling when high-sensitivity fluorescence detection is required. Similar to traditional labeling with activated fluorescent dyes, such as fluorescein isothiocyanate, a fluorogenic reaction is expected to change the physical-chemical properties of proteins. Knowledge of these changes may be essential for efficient separation and identification of labeled proteins. Here we studied the effect of labeling of myoglobin with a fluorogenic reagent on the acid-base properties of the protein. The fluorogenic reagent used was 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ). In slab-gel isoelectric focusing, we found that the labeling reaction generated at least six species with pI values lower than that of non-labeled myoglobin. These species can be identified as products of progressive labeling of myoglobin with one to six FQ molecules. The same series of FQ-labeled species were observed when the reaction products were analyzed by capillary zone electrophoresis. The comparison of experimental and theoretical pI values allowed us to elucidate the labeling pattern--the number of FQ molecules corresponding to each labeled product detected by isoelectric focusing.