[Stochastic Packaging of Cas Proteins into Exosomes].

CRISPR/Cas systems are perspective molecular tools for targeted manipulation with genetic materials, such as gene editing, regulation of gene transcription, modification of epigenome etc. While CRISPR/Cas systems proved to be highly effective for correcting genetic disorders and treating infectious diseases and cancers in experimental settings, clinical translation of these results is hampered by the lack of efficient CRISPR/Cas delivery vehicles. Modern synthetic nanovehicles based on organic and inorganic polymers have many disadvantages, including toxicity issues, the lack of targeted delivery, and complex and expensive production pipelines. In turn, exosomes are secreted biological nanoparticles that exhibit high biocompatibility, physico-chemical stability, and the ability to cross biological barriers. Early clinical trials found no toxicity associated with exosome injections. In the recent years, exosomes have been considered as perspective delivery vehicles for CRISPR/Cas systems in vivo. The aim of this study was to analyze the efficacy of CRISPR/Cas stochastic packaging into exosomes for several human cell lines. Here, we show that Cas9 protein is effectively localized into the compartment of intracellular exosome biogenesis, but stochastic packaging of Cas9 into exosomes turns to be very low (~1%). As such, stochastic packaging of Cas9 protein is very ineffective and cannot be used for gene editing purposes. Developing novel tools and technologies for loading CRISPR/Cas systems into exosomes is needed.

Neurotropic enteroviruses (Picornaviridae: Enterovirus): predominant types, basis of neurovirulence.

Enteroviruses are one of the most common causative agents of infectious diseases of the central nervous system. They are characterized by genetic variability, the ability to infect a wide range of cells, including brain microglial cells and astrocytes, and persist in the central nervous system tissue, causing delayed and chronic diseases. The review presents data on the basis of neurovirulence of non-polio enteroviruses and the most common pathogens causing enteroviral neuroinfections.

[Antiviral Activity of CRISPR/Cas9 Ribonucleoprotein Complexes on a Hepatitis B Virus Model In Vivo].

Chronic hepatitis B (CHB) is caused by hepatitis B virus (HBV) infection. This disease is a key issue for global health. Modern methods of therapy do not completely eliminate HBV from infected cells and do not cure chronic infection. The CRISPR/Cas9 systems of site-specific nucleases can effectively cleave do not target DNA including viral genomes. The cleavage of the major form of the HBV genome, i.e., covalently closed circular DNA (cccDNA), leads to a robust reduction in viral replication and degradation or mutational inactivation of cccDNA. CRISPR/Cas9-based approaches are one of the most promising ways to achieve a 'sterilizing' cure of CHB, i.e., complete elimination of the virus from the body. Here, the HBV mouse model in vivo has been used to analyze the antiviral activity of the high-specific Cas9 protein and sgRNA targeting HBV genome. We have found that a single injection of short-lived ribonucleoprotein complexes of CRISPR/Cas9 results in a ~10-fold reduction in HBV DNA levels in the serum and liver of mice as early as 48 h after the start of the experiment. The remaining HBV DNAs have been found to harbor rare indel mutations. Developing new antivirals for treating CHB based on CRISPR/Cas9 ribonucleoprotein complexes could substantially reduce the duration of CHB therapy and, potentially, achieve complete elimination of viral infection.

[HBx Protein Potentiates Hepatitis B Virus Reactivation].

Hepatitis B virus (HBV) can cause chronic hepatitis B, one of the most prevalent infectious diseases in the world. Global estimates suggest that over 2 billion people are affected by HBV, with over 250 million people developing chronic infection. Upon treatment of comorbidities, patients with chronic infection may develop an abrupt increase of viral replication-HBV reactivation-leading to liver decompensation and, in some cases, death. HBV reactivation occurs mostly due to suppression of antiviral immune response and activation of intracellular pro-viral signaling. Defining the mechanisms of HBV reactivation is necessary for the rational use of drugs and reduction of mortality rates in patients with chronic infection. In this study, for the first time we analyzed the effects of HBx protein on HBV reactivation, described reactivation of HBV from the transcriptionally inactivated state at the methylated recombinant HBV genome model, and investigated HBV reactivation upon treatment with genotoxic agents (doxorubicin and hydrogen peroxide) and targeted drug therapies (sunitinib and bortezomib). We report that both wild-type HBx protein and, to a greater extent, the mutant form of HBx protein lacking the nuclear exportation signal, potentiate viral replication and promote HBV reactivation. For the first time, we demonstrate that HBV can reactivate from the transcriptionally inactive state. Doxorubicin and hydrogen peroxide induce HBV reactivation at models of both transcriptionally active and transcriptionally silenced viral genome. Sunitinib weakly reactivates HBV, while bortezomib does not affect HBV replication in vitro.

Genetic Association between Alzheimer's Disease Risk Variant of the PICALM Gene and Auditory Event-Related Potentials in Aging.

Aging and genetic predisposition are major risk factors in age-related neurodegenerative disorders. The most common neurodegenerative disorder is Alzheimer's disease (AD). Genome-wide association studies (GWAS) have identified statistically significant association of the PICALM rs3851179 polymorphism with AD. The PICALM G allele increases the risk of AD, while the A allele has a protective effect. We examined the association of the PICALM rs3851179 polymorphism with parameters of the P3 component of auditory event-related potentials (ERPs) in 87 non-demented volunteers (age, 19-77 years) subdivided into two cohorts younger and older than 50 years of age. We found statistically significant association between the AD risk variant PICALM GG and increase in the P3 latency in subjects over 50 years old. The age-dependent increase in the P3 latency was more pronounced in the PICALM GG carriers than in the carriers of the PICALM AA and PICALM AG genotypes. The observed PICALM-associated changes in the neurophysiological processes indicate a decline in the information processing speed with aging due, probably, to neuronal dysfunction and subclinical neurodegeneration of the neuronal networks in the hippocampus and the frontal and parietal cortical areas. Such changes were less pronounced in the carriers of the PICALM gene A allele, which might explain the protective effect of this allele in the cognitive decline and AD development.

The role of nelarabine in the treatment of T-cell acute lymphoblastic leukemia: literature review and own experience.

AIM: The analysis of experience of nelarabine use in refractory/relapsed T-cell acute lymphoblastic leukemia (T-ALL) depending on the immunophenotype and the line of therapy. MATERIALS AND METHODS: All the patients with relapsed or refractory T-ALL aged from 0 to 18 years who received treatment with nelarabine as a part of the therapeutic element R6 were included in the study. For all patients a detailed immunological analysis of leukemia cells with discrimination of immunological variants TI, TII, TIII or TIV was performed. Patients administered with nelarabine as a first therapeutic element were referred to the first-line therapy group, other patients were referred to the second-line therapy group. Nelarabine was ad- ministered as intravenous infusion at a dose of 650 mg/m2, on days 1-5. Allogeneic hematopoietic stem cells transplantation (allo-HSCT) was considered for all patients. RESULTS: From 2009 to 2017, 54 patients with refractory/relapsed T-ALL were treated with nelarabine. Five-year event-free survival (EFS) and overall survival (OS) was 28% for all patients, cumulative risk of relapse (CIR) was 27%. EFS was significantly higher in nelarabine first-line therapy group in comparison with second-line therapy group (34±8% vs 8±8%, p=0,05). In patients after allo-HSCT EFS, OS and CIR were 51±10%, 50±10% and 39,1±9,5% accordingly. The best results were achieved in patients with TI immunophenotype. No toxicity-related mortality as well as severe neurologic complications or discontinuation of therapy associated with use of nelarabine were reported. CONCLUSION: The use of nelarabine is an effective strategy for the treatment of relapsed and refractory T-ALL. The best treatment outcomes were obtained in patients with TI immunophenotype and in the first-line therapy group. Optimal dosage regimens can be established dur- ing controlled clinical trials.

[Age-related characteristics of the efficacy of different glucocorticosteroids in the therapy of acute lymphoblastic leukemia].

AIM: To determine predictors for decision-making on a differential approach to choosing glucocorticosteroids (GCS) for children and adolescents with acute lymphoblastic leukemia (ALL). SUBJECTS AND METHODS: The analysis covered 1064 primary patients aged to 1 to 18 years with ALL who had been registered at the clinics of Russia and Belorussia in April 2002 to November 2006. Before induction therapy, the patients were randomized into a dexamethasone (DEXA) 6 mg/m2 group (n=539) and a methylprednisolone (MePRED) 60 mg/m2 one (n=525). RESULTS: The entire group showed no statistically significant differences in survival rates between the patients receiving DEXA or MePRED. However, an analysis of age groups revealed the benefits of DEXA in children younger than 14 years (the event-free survival (EFS) was 76±2 and 71±2%, respectively (p=0.048); the overall survival (OS) was 81±2 and 77±2%, respectively (p=0.046); therapy-induced mortality was 6.4% (DEXA) andl 1.1% (MePRED) (p=0.01 4); the rate of isolated extramedullary relapses was 1.5% (DEXA) and 4.4% (MePRED) (p=0.009). At the same time, EFS and OS in 14-to-18-year-old adolescents were statistically significantly higher than in those who used MePRED (EFS, 65±6 and 52±6%, respectively (p=0.087); OS, 72±6 and 61±6%, respectively; (p=0.l 7). CONCLUSION: The findings suggest that it is possible that the choice of a GCS for ALL therapy must be also based on a patient's age. There is a need for further studies of this matter in prospective randomized multicenter trials in children and adolescents.

[Prevalence, features of circulation, and diversity of human parechoviruses in Nizhny Novgorod].

A total of 5230 specimens from children with gastroenteritis collected in Nizhny Novgorod in 2006-2010 were screened for human parechoviruses (HPeV). HPeV were observed every year with mean frequency of 6.16%. The majority of HpeV (65.83%) was detected in children younger than 3 years. The typing of 71 detected HPeV with the use of partial sequencing of the VP3-VP1 region revealed the presence of HPeV1 (91.55%), HpeV6 (5.63%), HPeV3 (3.08%), HPeV4 (1.54%). HPeV1B was predominant among HPeV1, HPeV1A was identified rarely. Six stains of HPEV1 formed separate phylogenetic cluster, had sequence gomology with HPEV1A or HPeV1B not more than 88% and could be characterized as members of a separate genotype HPeV1.

[Electrophysiological markers of middle cerebral artery blood flow velocity in healthy subjects].

AIM: to determine electrophysiological markers of middle cerebral artery blood flow velocity (BFV). PATIENTS AND METHODS: transcranial Doppler registration of middle cerebral artery BFV and direct current (DC) potentials recording from surface of head were performed in 30 healthy volunteers. Analysis of correlation between the BFV and DC potentials was used. RESULTS: significant correlation between BFV and DC potential characteristics was observed. The highest correlation was found between BFV in middle cerebral artery and the difference of DC potentials between central and temporal areas of head (r = 0,55; p = 0,003). These areas coincide with the location of middle cerebral artery and the correlation observed may be connected with streaming potential generated by the blood flow in middle cerebral artery. If electrode placement did not coincide with blood current, DC potentials and BFV were not correlated. CONCLUSIONS: it is assumed that electrical field created BFV in middle cerebral artery may contribute to the generation of DC potentials registered from the head.

[Two patterns of Ct-brain perfusion and Dc-potentials of the brain evoked cognitive task in discirculatory encephalopathy patients].

In patients with discirculatory encephalopathy the influence of verbal fluency test on the characteristics of cerebral perfusion, DC-potentials of the brain, as well as on blood pressure and heart rate was investigated. Two patterns of responses to the verbal fluency test were observed. The first one is the process of generalized activation, manifested by the reduction of the TTP (time to peak) parameters of brain perfusion, the rise of the DC-potentials in all areas of brain and the modulation of blood pressure and heart rate. The second process, directly connected with cognitive processing, was manifested by the shifts of local characteristics of brain perfusion and DC-potentials in the frontal, temporal and central cortex, especially in the left hemisphere. Correlations were found between the characteristics of cerebral perfusion and DC-potentials on the one hand and the number of words during the verbal fluency test performance on the other hand.

[Association between changes in characteristics of local cerebral blood flow and slow brain electrical activity in patients with dyscirculatory encephalopathy].

Associated changes in the characteristics of local cerebral blood flow (CBF) and slow brain electrical activity were studied in 40 patients with dyscirculatory encephalopathy. CBF and CBV (cerebral blood volume) values for the frontal and temporal cortex and basal ganglia positively correlated with the constant potential values in the central lead influenced by the blood flow rate in the upper sagittal sinus.

[Molecular genetic echovirus 9 variants identified in patients with aseptic meningitis in Russia in 2007-2009].

In 2009 echovirus 9 caused a higher seasonal incidence of enterovirus infection (EVI) and its several outbreaks in a number of regions of Russia. Analysis of the partial VP1 coding region differentiated 4 phylogenetic lineages of echoviruses 9 variants identified in patients with aseptic meningitis and EVI in 2007-2009. One variant of echovirus 9 was most commonly encountered in 2009. Echoviruses 9 identified in different areas, which had a high (98.2-100%) homology of nucleotide sequences of the partial VP1 coding region, varied in the amino acid sequences within the B-C loop.

[Efficiency of the ALL-MB-2002 protocol in children with acute lymphoblastic leukemia].

AIM: To evaluate the efficiency of the original ALL-MB-2002 protocol within the multicenter study of treatment of acute lymphoblastic leukemia (ALL) in children. SUBJECTS AND METHODS: A total of 1873 primary patients with ALL aged 1 to 18 years, of whom 1544 patients were enrolled in this study, were notified at 36 clinics of Russia and Belarus from April 15, 2002, to January 1, 2008. RESULTS: With the median observation of 4.12 years, 7-year event-free survival (EFS) was 73 +/- 13%; overall survival (OS) 78 +/- 2%; relapse-free survival 82 +/- 1%. The rates of EFS and OS were equal and amounted to 76 +/- 2 and 80 +/- 2% in the standard-risk group (SRG) and intermediate-risk group (ImRG), respectively. In the high-risk group (HRG) patients, EFS and OS were as high as 30 +/- 6 and 37 +/- 6%, respectively. The frequency of relapses with central nervous system lesion was as much as 4.7% in all the patients, 6-year cumulative risk for isolated neurorecurrences being 2.5% in the SRG patients. Adolescents, patients with the baseline leukocytosis (more than 100 x 10(9)/l), and those with a splenic size of over 4 cm or more from the costal arch margin had substantially worse survival rates. A poor early response to therapy (on induction days 8 and 15) was also associated with its lower efficiency. CONCLUSION: Despite a considerable rise in the number of centers and a slight increase in the intensity of therapy, the results of the new ALL-MB-2002 protocol are as minimum equivalents obtained in the use of the previous ALL-MB-91 protocol. A significant improvement in the overall results of therapy and a reduction in the cumulative risk for isolated neurorecurrences were noted in the ImRG patients.

[Interhemisphere asymmetry of regulation of the local brain blood flow in patients with dyscirculatory encephalopathy].

Patients with dyscirculatory encephalopathy (DE) were examined to elucidate interhemisphere relationships by recording brain constant potential (CP) and perfusion CT. Electrophysiological characteristics were shown to relate to local brain blood flow. Dispersion of intrehemisphere CP level in DE patients proved more pronounced than in healthy subjects. Interhemisphere CP difference in temporal region significantly (p < 0.001) correlated with the time needed to reach maximum concentration of contrast agent in the frontal and temporal cortex, basal ganglia, and thalamus. Left temporal CP level positively correlated with contrast agent concentration in the above brain structures of both hemispheres and negatively with that in the right hemisphere. It is concluded that intertemporal relationships may influence vegetative nervous system that in turn changes cardiovascular function.

[Nucleotide sequence analysis of the NSP4 gene from group A rotaviruses isolated in Nizhni Novgorod].

The complete nucleotide sequences of the NSP4 gene from Russian isolates of Group A rotaviruses with different G[P] types obtained in Nizhni Novgorod have been first ascertained. They belong to their affiliation to three NSP4 (A, B, and C) of the 5 known genotypes. Eleven strains had NSP4 genotype. Phylogenetic analysis based on the complete nucleotide sequences of the NSP4 gene revealed the sequence homology of G1P[8] viruses with the strains circulating in Scandinavia and that of the G3 type viruses with the strains from South-Eastern Asia. The rotavirus strains G1, G3, G4, and P[8] demonstrated the homology with porcine rotavirus strains, while the strains G3P[6], G3P[9], and G2P[4] did the homology with bovine rotavirus strains. There was an unusual strain of human rotaviruses G3P[9] genetically related to feline rotaviruses.

EEG alterations in non-demented individuals related to apolipoprotein E genotype and to risk of Alzheimer disease.

Identification of preclinical markers is required for early diagnosis of Alzheimer's disease (AD) and cognitive dysfunction in advancing age. Quantitative EEG was examined in 145 individuals with AD, their unaffected relatives and unrelated individuals. The AD patients and their relatives were stratified by ApoE genotype. The resting EEG parameters were severely changed in AD patients, and in patients carrying the ApoE epsilon4 allele the decrease in alpha power was higher than in epsilon4 non-carriers. The resting EEG parameters were indistinguishable in AD relatives with different ApoE genotypes and similar to EEG pattern in common population. Under hyperventilation the presence of the epsilon4 allele in AD relatives was associated with the manifestation of synchronous high-voltage delta-, theta-activity and sharp-waves, pronounced decrease in alpha and increase in delta and theta relative powers. The data suggest that neurophysiological endophenotype of non-demented individuals at genetic risk for AD, characterized by increased excitability and dysfunction of deep brain and alpha rhythm-generating structures, may be revealed decades before the first clinical symptoms of presumable dementia.

[Respiratory infection in patients with bronchial asthma. Clinicolaboratory characteristics].

AIM: To study clinicolaboratory signs of respiratory infection in patients with bronchial asthma (BA). MATERIAL AND METHODS: Respiratory infection and its influence on BA were studied in 108 BA patients (66 female and 42 male, mean age 51.0 +/- 15.2 years) with symptoms of bronchitis and non-nosocomial (n = 71) and nosocomial (n = 37) pneumonia. The patients referred for medical care and started antibacterial therapy (ABT) after 4.7 +/- 2.8 days of respiratory infection (RI). Before assignment to the trial BA exacerbation ran for 9.8 +/- 6.0 days. RESULTS: RI preceded BA exacerbation in 62% patients with pneumonia. BA exacerbation preceded RI in 66% patients with bronchitis. RI ran with moderate and severe intoxication. Severity of BA exacerbation correlated with severity of RI Antibacterial treatment was uneffective and the drugs were changed for others in 18% bronchitis and 16% pneumonia patients. Laboratory signs of inflammation were more obvious in pneumonias than in bronchitis and did not depend on the disease severity. External respiration function at RI onset was significantly affected in all the patients. CONCLUSION: Respiratory infections aggravate the course of BA and damage external respiration function.

[The dynamics of the direct current potentials of the brain during sustained attention in 10-11-year-old children].

The dynamics of direct current potentials of the brain was studied in 10-11-year-old children during sustained attention to successive presentation of series of Shulte tables. Children were examined twice: before and after the series of training to fast reading. A gradual increase in the level of direct current potentials during sustained attention was observed. The increase was more pronounced in children with excessive than in children with moderate reactions to the loading. After the series of training to fast reading, the increase in the level of direct current potentials was reduced in both groups. This aftertraining neurophysiological phenomenon was combined with a transformation of psychophysiological characteristics: a decrease in the time of viewing of Shulte tables and increase in the speed of reading. It is suggested that the shifts of direct current potentials reflects the dynamics of intensity of the cerebral energy metabolism.