PhthisisBioMed Artificial Medical Intelligence: Software for Automated Analysis of Digital Chest X-ray/Fluorograms.

The scope of diagnostic medical examinations increases from year to year causing a reasonable desire to develop and implement new technologies to diagnostics and medical data analysis. Artificial intelligence (AI) algorithms became one of the most promising solutions to this problem and proved themselves in the course of mass practical application. During the three-year Moscow experiment started in 2020, the possibility was achieved to develop methodologies of AI use and to successfully implement it into the regional level healthcare system. In this article, the authors share their experience in developing a medical AI service using the example of PhthisisBioMed AI service and the results of its application in real clinical activities environment. This AI service has shown its quality and reliability confirmed by technological monitoring. Clinical trials of PhthisisBioMed AI service were conducted on a specially prepared verified data set (n=1536) considering epidemiological indicators of the thoracic organs major diseases prevalence. The mean sensitivity of the service was 0.975 (95% CI: 0.966-0.984). PhthisisBioMed medical AI service is registered as a medical device (medical device registration certificate No.RZN 2022/17406 dated May 31, 2022) and is actively used in the Russian Federation as a diagnostic tool to reduce the burden on radiologists and to accelerate the process of medical report obtaining.

Methodology for Conducting Post-Marketing Surveillance of Software as a Medical Device Based on Artificial Intelligence Technologies.

The aim of the study was to develop a methodology for conducting post-registration clinical monitoring of software as a medical device based on artificial intelligence technologies (SaMD-AI). Materials and Methods: The methodology of post-registration clinical monitoring is based on the requirements of regulatory legal acts issued by the Board of the Eurasian Economic Commission. To comply with these requirements, the monitoring involves submission of the review of adverse events reports, the review of developers' routine reports on the safety and efficiency of SaMD-AI, and the assessment of the system for collecting and analyzing developers' post-registration data on the safety and efficiency of medical devices. The methodology was developed with regard to the recommendations of the International Medical Device Regulators Forum and the documents issued by the Food and Drug Administration (USA). Field-testing of this methodology was carried out using SaMD-AI designed for diagnostic imaging. Results: The post-registration monitoring of SaMD-AI consists of three key stages: collecting user feedback, technical monitoring and clinical validation. Technical monitoring involves routine evaluation of SaMD-AI output data quality to detect and remove flaws in a timely manner, and to secure the product stability. Major outcomes include an ordered list of technical flaws in SaMD-AI and their classification using evidence from diagnostic imaging studies. The application of this methodology resulted in a gradual reduction in the number of studies with flaws due to timely improvements in artificial intelligence algorithms: the number of flaws decreased to 5% in various aspects during subsequent testing. Clinical validation confirmed that SaMD-AI is capable of producing clinically meaningful outputs related to its intended use within the functionality determined by the developer. The testing procedure and the baseline testing framework were established during the field testing. Conclusion: The developed methodology will ensure the safety and efficiency of SaMD-AI taking into account its specifics as intangible medical devices. The methodology presented in this paper can be used by SaMD-AI developers to plan and carry out the post-registration clinical monitoring.

Chest MRI of patients with COVID-19.

During the pandemic of novel coronavirus infection (COVID-19), computed tomography (CT) showed its effectiveness in diagnosis of coronavirus infection. However, ionizing radiation during CT studies causes concern for patients who require dynamic observation, as well as for examination of children and young people. For this retrospective study, we included 15 suspected for COVID-19 patients who were hospitalized in April 2020, Russia. There were 4 adults with positive polymerase chain reaction (PCR) test for COVID-19. All patients underwent magnetic resonance imaging (MRI) examinations using MR-LUND PROTOCOL: Single-shot Fast Spin Echo (SSFSE), LAVA 3D and IDEAL 3D, Echo-planar imaging (EPI) diffusion-weighted imaging (DWI) and Fast Spin Echo (FSE) T2 weighted imaging (T2WI). On T2WI changes were identified in 9 (60,0%) patients, on DWI - in 5 (33,3%) patients. In 5 (33,3%) patients lesions of the parenchyma were visualized on T2WI and DWI simultaneously. At the same time, 4 (26.7%) patients had changes in lung tissue only on T2WI. (P(McNemar) = 0,125; OR = 0,00 (95%); kappa = 0,500). In those patients who had CT scan, the changes were comparable to MRI. The results showed that in case of CT is not available, it is advisable to conduct a chest MRI for patients with suspected or confirmed COVID-19. Considering that T2WI is a fluid-sensitive sequence, if imaging for the lung infiltration is required, we can recommend the abbreviated MRI protocol consisting of T2 and T1 WI. These data may be applicable for interpreting other studies, such as thoracic spine MRI, detecting signs of viral pneumonia of asymptomatic patients. MRI can detect features of viral pneumonia.

[Artificial intelligence for diagnosis of vertebral compression fractures using a morphometric analysis model, based on convolutional neural networks].

BACKGROUND: Pathological low-energy (LE) vertebral compression fractures (VFs) are common complications of osteoporosis and predictors of subsequent LE fractures. In 84% of cases, VFs are not reported on chest CT (CCT), which calls for the development of an artificial intelligence-based (AI) assistant that would help radiology specialists to improve the diagnosis of osteoporosis complications and prevent new LE fractures. AIMS: To develop an AI model for automated diagnosis of compression fractures of the thoracic spine based on chest CT images. MATERIALS AND METHODS: Between September 2019 and May 2020 the authors performed a retrospective sampling study of ССТ images. The 160 of results were selected and anonymized. The data was labeled by seven readers. Using the morphometric analysis, the investigators received the following metric data: ventral, medial and dorsal dimensions. This was followed by a semiquantitative assessment of VFs degree. The data was used to develop the Comprise-G AI mode based on CNN, which subsequently measured the size of the vertebral bodies and then calculates the compression degree. The model was evaluated with the ROC curve analysis and by calculating sensitivity and specificity values. RESULTS: Formed data consist of 160 patients (a training group - 100 patients; a test group - 60 patients). The total of 2,066 vertebrae was annotated. When detecting Grade 2 and 3 maximum VFs in patients the Comprise-G model demonstrated sensitivity - 90,7%, specificity - 90,7%, AUC ROC - 0.974 on the 5-FOLD cross-validation data of the training dataset; on the test data - sensitivity - 83,2%, specificity - 90,0%, AUC ROC - 0.956; in vertebrae demonstrated sensitivity - 91,5%, specificity - 95,2%, AUC ROC - 0.981 on the cross-validation data; for the test data sensitivity - 79,3%, specificity - 98,7%, AUC ROC - 0.978. CONCLUSIONS: The Comprise-G model demonstrated high diagnostic capabilities in detecting the VFs on CCT images and can be recommended for further validation.

Metagenomic Analysis of the Dynamic Changes in the Gut Microbiome of the Participants of the MARS-500 Experiment, Simulating Long Term Space Flight.

A metagenomic analysis of the dynamic changes of the composition of the intestinal microbiome of five participants of the MARS-500 experiment was performed. DNA samples were isolated from the feces of the participants taken just before the experiment, upon 14, 30, 210, 363 and 510 days of isolation in the experimental module, and two weeks upon completion of the experiment. The taxonomic composition of the microbiome was analyzed by pyrosequencing of 16S rRNA gene fragments. Both the taxonomic and functional gene content of the microbiome of one participant were analyzed by whole metagenome sequencing using the SOLiD technique. Each participant had a specific microbiome that could be assigned to one of three recognized enterotypes. Two participants had enterotype I microbiomes characterized by the prevalence of Bacteroides, while the microbiomes of two others, assigned to type II, were dominated by Prevotella. One participant had a microbiome of mixed type. It was found that (1) changes in the taxonimic composition of the microbiomes occurred in the course of the experiment, but the enterotypes remained the same; (2) significant changes in the compositions of the microbiomes occurred just 14-30 days after the beginning of the experiment, presumably indicating the influence of stress factors in the first stage of the experiment; (3) a tendency toward a reversion of the microbiomes to their initial composition was observed two weeks after the end of the experiment, but complete recovery was not achieved. The metagenomic analysis of the microbiome of one of the participants showed that in spite of variations in the taxonomic compositions of microbiomes, the "functional" genetic composition was much more stable for most of the functional gene categories. Probably in the course of the experiment the taxonomic composition of the gut microbiome was adaptively changed to reflect the individual response to the experimental conditions. A new, balanced taxonomic composition of the microbiome was formed to ensure a stable gene content of the community as a whole without negative consequences for the health of the participants.

An expression system for regulated protein production in Synechocystis sp. PCC 6803 and its application for construction of a conditional knockout of the ferrochelatase enzyme.

Synechocystis sp. PCC 6803 is a model organism for the study of photosynthetic processes. Methods to genetically manipulate this bacterium are essential to investigate these processes and to evaluate potential biotechnological applications. We developed a vector for controllable expression of proteins using a platform for stable integration of the expression cassette into the genome. The respective gene is translationally fused to the promoter of the petJ gene encoding cytochrome c(553) that is repressed by copper. Maximal expression from this promoter is achieved under copper depletion, whereas normal copper concentrations in standard medium lead to low expression rates. We show here the application of this system for construction of a conditional knockout mutant for the ferrochelatase, which is an essential enzyme in heme biosynthesis. Using different amounts of copper in the medium we were able to control the amount of ferrochelatase in the cell resulting in a varying expression of the phenotype.

[Classification of domestic probiotic cultures of Lactobacillus genus].

AIM: To study strains of bacteria from Lactobacillus genus using combination of microbiological and molecular biological methods in order to define more accurately their systematic position and biochemical characteristics. MATERIALS AND METHODS: Thirteen cultures of Lactobacillus bacteria isolated from stool of healthy persons were studied: L. plantarum CS 396, L. plantarum 8-PA-3, L. plantarum 421-2, L. fermentum 90-TC-4, L. delbrueckii gKNM 101, L. delbrueckii gKNM 526, L. acidophilus Er 317/402 NARINE, L. acidophilus 100 ash, L. acidophilus NK-1, L. acidophilus NNIE, L. acidophilus K3sh24, L. brevis gKNM 23 11, L. casei gKNM 577. Their enzymatic activity relative to 50 sugars was studied using API-50 system. Structure of proximal region of 16S rRNA gene was studied also. RESULTS: According to results of 16S rRNA gene sequence analysis strains were divided on 2 groups: 1) L. casei gKNM 577, L. plantarum 8-PA-3, L. plantarum CS 396, which species belonging corresponded to stated description. Comparison of nucleotide sequence of 16S rRNA gene of group 2 strains with nucleotide sequences database revealed that cultures NK-1, Er315/402 NARINE, 100 ash, NNIE identified early as L. acidophilus belong to species L. helveticus; L. brevis gKNM 23 and L. acidophilus K3sh24--to group L. casei/paracasei, L. delbrueckii gKNM 101 and L. fermentum 90-TC-4--to L. plantarum, L. delbrueckii gKNM 526--to L. fermentum, and L. plantarum 421-2--to L. rhamnosus. CONCLUSION: Obtained data allowed to perform taxonomic reclassification of species belonging of studied probiotic cultures of lactobacilli according to modem level of systematic of bacteria.

[Revised classification of native probiotic strains of Lactobacillus used in Russian Federation].

Thirteen strains of industrial bacterial cultures of the genus Lactobacillus (from a collection of Gabrichevsky Research Institute of Epidemiology and Microbiology) were studied. These strains were used for decades in Russian Federation for food and drug production, as ferments for lactic acid products, for production of probiotics, biologically active and veterinary preparations. Complex analysis of data on cultures obtained using microbiological and molecular-genetic methods was conducted for the first time. Biochemical characteristics of these cultures were studied and the sequence of the proximal region of 16S ribosomal RNA gene was determined. The employment of the test system API-50CHL was shown to broaden the opportunities of a more accurate biochemical identification of bacteria belonging to the genus Lactobacillus, in comparison with the set ANAEROTEST-23. According to the results obtained in a comparative analysis of nucleotide sequences of 16S rRNA gene, all strains examined show 97-99% homology of the proximal region of this gene with that of the type representatives of studied species. These data allowed taxonomic reclassification of the species position of cultures with consideration of the more advanced level of systematics. Nucleotide sequences of gene fragments of examined lactobacilli strains were recorded in NCBI database (accession numbers of deposits GU560031, GU560032, GU560033, GU560034, GU560035, GU560036, GU560037, GU560038, GU560039, GU560040, GU560041, GU560042, GU560043).

[Genetic diversity of the genus Lactobacillus bacteria from the human gastrointestinal microbiome].

The species and strain genetic diversity of bacterial cultures belonging to the genus Lactobacillus, which were isolated from the gastrointestinal microbiome of the human population living in the former Soviet Union in the years 1960-1980, was studied. The bacteria demonstrated probiotic characteristics. Phylogenetic analysis of sequences of the gene coding for 16S rRNA detected earlier by us, showed that the gene found in bacteria isolated from the intestinal content of healthy adults and represented by species L. plantarum, L. helveticus, L. casei/paracasei, L. rhamnosus, and L. fermentum has high homology (97-100%) with this gene in type representatives of the species. The genotypic and strain diversity of cultures was studied using RAPD-PCR and nonspecific primers. This method with the use of the ERIC-1 primer gave reliable and reproducible results as compared that using with M13 and MSP primers and allowed the identification of examined bacteria belonging to the genus Lactobacillus at the level of species and certification at the strain level.

[The prqA and mvrA genes encoding carrier proteins control resistance to methyl viologen in the cyanobacterium Synechocystis sp. PCC6803]. / Geny prqA i mvrA, kodiruiushchie belki-transportery, kontroliruiut ustoichivost' k metilviologenu u tsianobakterii Synechocystis sp. PCC6803.

Derivatives with insertional inactivation of prqA and mvrA genes were obtained and studied in the Synechocystis sp. PCC6803 wild-type strain and in the mutant Prq20 resistant to methyl viologen (MV). It was shown that the formation of resistance to MV is associated with the operation of two systems: constitutive and inducible. The prqA gene encoding drug efflux proteins controls the constitutive system of cell resistance to MV. Derepression of the prqA gene is the main reason for an enhanced MV resistance in the Prq20 mutant with impaired repressor function of the PrqR protein. The mvrA gene encoding the transmembrane protein from the family of transporters of sugar and other compounds controls the inducible MV resistance. It is assumed that the MvrA protein is required for efficient elimination from cells of toxic substances formed upon oxidative stress or participates in the repair of membranes destroyed by oxidants. The data obtained demonstrated for the first time that transport systems are involved in the formation of MV resistance in photosynthetic organisms.

[On the involvement of the regulatory gene prqR in the development of resistance to methyl viologen in cyanobacterium Synechocystis sp. PCC6803]. / Ob uchastii reguliartornogo gena prqR v razvitii ustoichivosti k metilviologenu u tsianobakterii Synechocystis sp. PCC6803.

The role of the prqR gene in the regulation of the adaptive response of the cyanobacterium Synechocystis sp. PCC6803 to the oxidative stress induced with methyl viologen (MV) was studied. For this, transcription activity of prqR and the genes, which may be involved in the control of resistance to MV, was determined by means of Northern blot hybridization in wild-type cells and in the MV-resistant Prq20 mutant with a mutation located in the DNA-binding domain of the PrqR protein. It was ascertained that the prqR gene is a component of the prqR-prqA operon and down regulates its transcription. In cells of the wild-type strain containing MV, the autorepressor activity of the PrqR protein enhances and transcription of mvrA and sodB genes encoding an respectively assumed transporter protein and iron-containing superoxide dismutase increases. The prqR gene may be involved in the negative, indirect control of transcription of these genes. The Prq20 mutant is characterized by an MV-independent derepression of the prqR-prqA operon and by a slightly increased transcription of mvrA and sodB genes not stimulated by MV. Nevertheless, the expression of mvrA and sodB genes was lower than in wild-type cells after the MV treatment. On the strength of this evidence, it is assumed that the main mechanism underlying for the resistance to MV in the Prq20 mutant is derepression of the prqA gene, the product of which is homologous to multidrug transporters, drug efflux proteins.

[Trans- and cis-acting autorepressors of the prqR gene in Synechocystis cyanobacteria sp. PCC6803]. / Trans- i tsis-deistvuiushchaia avtorepressiia gena prqR u tsianobakterii Synechocystis sp. PCC 6803.

Genetic analysis of the allele interactions was carried out with the use of recombinant plasmids and reporter genes to study the autorepressor function of prqR, which negatively regulates the prqR-prqA operon and the response to oxidative stress inductor methyl viologen (MV) in cyanobacterium Synechocystis sp. PCC 6803. The wild-type (WT) prqR showed negative autoregulation and suppressed in trans the derepressed mutant alleles. Frameshift mutation C134fs, which was introduced in prqR by site-directed mutagenesis, impaired the autoregulation, implicating the C-terminal domain in transcriptional repression by PrqR. Missense mutation C134S altering the only redox-sensitive Cys of PrqR, had no effect on prqR expression, indicating that oxidation and consequent disulfide bridging of two PrqR molecules was not responsible for MV-induced autorepression of prqR. Analysis of the prqR-prqA deletion derivatives lacking the promoter and most of prqR revealed weak uncontrollable expression of reporter cat, testifying to the existence of a constitutive promoter in prqA responsible for MV resistance. The interaction of the WT and mutant prqR alleles in Synechocystis cells revealed a cis-dominant character of the alteration of prqR autoregulation. Stimulation of in cis autorepression of prqR was assumed to contribute to the induction of systems protecting cyanobacteria from oxidative stress.

[clpP2 gene encoding peptidase in cyanobacteria Synechocystis sp. PCC 6803 controls the sensitivity of cells to photoinhibition]. / Gen clpP2, kodiruiushchii peptidazu u tsianobakterii Synechocystis sp. PCC 6803, kontroliruet chuvstvitel'nost' kletok k fotoingibirovaniiu.

A homozygous insertion mutant with the inactivated clpP2 gene, which encodes the proteolytic subunit of ATP-dependent peptidase, was obtained in the unicellular cyanobacterium Synechocystis sp. PCC 6803. The mutant cannot grow under photoautotrophic conditions, but cells grown under heterotrophic conditions in a glucose-containing medium have active photosystems I and II (PS I and PS II). The loss of capacity for photoautotrophic growth is determined by a high sensitivity of mutant cells to the inactivating effect of light. Their incubation under light with an intensity above 10 microE m-2 s-1 inhibits cell growth in culture and causes degradation of photosynthetic pigments. It is proposed that the ClpP2 peptidase is involved in the protection of Synechocystis 6803 cells from photoinhibition.

Regulation of nitrogenase in the photosynthetic bacterium Rhodobacter sphaeroides containing draTG and nifHDK genes from Rhodobacter capsulatus.

The photosynthetic bacteria Rhodobacter capsulatus and Rhodospirillum rubrum regulate their nitrogenase activity by the reversible ADP-ribosylation of nitrogenase Fe-protein in response to ammonium addition or darkness. This regulation is mediated by two enzymes, dinitrogenase reductase ADP-ribosyl transferase (DRAT) and dinitrogenase reductase activating glycohydrolase (DRAG). Recently, we demonstrated that another photosynthetic bacterium, Rhodobacter sphaeroides, appears to have no draTG genes, and no evidence of Fe-protein ADP-ribosylation was found in this bacterium under a variety of growth and incubation conditions. Here we show that four different strains of Rba. sphaeroides are incapable of modifying Fe-protein, whereas four out of five Rba. capsulatus strains possess this ability. Introduction of Rba. capsulatus draTG and nifHDK (structural genes for nitrogenase proteins) into Rba. sphaeroides had no effect on in vivo nitrogenase activity and on nitrogenase switch-off by ammonium. However, transfer of draTG from Rba. capsulatus was sufficient to confer on Rba. sphaeroides the ability to reversibly modify the nitrogenase Fe-protein in response to either ammonium addition or darkness. These data suggest that Rba. sphaeroides, which lacks DRAT and DRAG, possesses all the elements necessary for the transduction of signals generated by ammonium or darkness to these proteins.

[Analysis of amino acid sequences of Rhodobacter sphaeroides glutamine synthetase]. / Analiz aminokislotnoi posledovatel'nosti glutaminsintetazy Rhodobacter sphaeroides.

A comparative analysis of the amino acid sequence of glutamine synthetase (GS) of the photosynthetic purple bacterium Rhodobacter sphaeroides revealed that the enzyme is typical for first type procaryotic GSs and structurally resembles GSs of enteric bacteria. The data obtained indicate that the complex phenotype of purple bacterial mutants at the glnA gene coding for GS may be conditioned by specific regulation of nitrogen metabolism in bacterial cells rather than by structural-and-functional peculiarities of GS.

[Cloning and identification of the gene controlling nitrogen metabolism in the photosynthetic purple bacteria Rhodobacter sphaeroides]. / Klonirovanie i identifikatsiia gena, kontroliruiushchego azotistyi metabolizm u fotosinteziruiushchei purpurnoi bakterii Rhodobacter sphaeroides.

A recombinant plasmid has been selected from the genomic library of Rhodobacter sphaeroides that restores the properties of the wild type strain in the mutant Drn121. The latter possesses the derepressed synthesis of nitrogenase when grown in the light, inability of nitrogen fixation in the dark and growth on potassium nitrate as a single source of nitrogen, disruption of ammonium ions and methylamine transportation, decreased activity of glutamine synthetase. The gene complementing the drn121 mutation is localized within the EcoRI-HindIII fragment of Rhodobacter sphaeroides chromosome 2.25 kb in size. Analysis of the fragment nucleotide sequence has revealed the fragments with a high level of homology to regulatory genes ntrB (the 3'-end) and ntrC of Rhodobacter capsulatus. The plasmid pRCN102, containing the nifR3-ntrB-ntrC operon of Rhodobacter capsulatus, is able to complement the drn121 mutation while its derivatives having inactivated ntrN or ntrC genes are not. Hence, in Rhodobacter sphaeroides mutant Drn121 the mutation is localized in ntrC gene the product of which is involved not only in nitrogen fixation but also in nitrogen metabolism on the whole.

[Expression of the nif and ntr genes of Klebsiella pneumoniae in Rhodobacter sphaeroides cells]. / Issledovanie ékspressii nif- i ntr-genov Klebsiella pneumoniae v kletkakh Rhodobacter sphaeroides.

The genes glnA, ntr, nif or their promoters from Klebsiella pneumoniae cloned on the vectors, based on the plasmid RSF1010, were introduced into Rhodobacter sphaeroides cells. It was found that K. pneumoniae genes glnA, nifB, nifE, nifL and nifH are not expressed in R. sphaeroides. Neither was the glnA gene from cyanobacterium Anabaena 7120 expressed in R. sphaeroides. No functional activity of K. pneumoniae product of ntrA gene which is expressed from its own promoter, and the product of the gene nifA which is expressed from the constitutive promoter of the kanamycin resistance gene of the transposon Tn903, was detected. The implications of these findings are discussed.

[Use of the plasmid R89S replicon for constructing integration vectors]. / Ispol'zovanie replikona plazmidy R89S dlia konstruirovaniia vektorov integratsii.

It has been shown that the plasmid R89S derivatives can be used as integrative vectors for bacteria in which the plasmid is unable to replicate autonomously. The chromosomal and plasmid fragments of phototrophic bacterium Rhodobacter sphaeroides have been cloned in plasmid pVZ365, a SmRKmR-derivative of R89S. The obtained recombinant plasmids were mobilized into R. sphaeroides cells by the I pcP-group conjugative plasmid R751. The frequencies of the SmR-transconjugants formation are 3.7.10(-5) to 5.6.10(-3) per recipient cell. The formation of the SmR-transconjugants has not been revealed in case of the plasmid pVZ365 mobilization. The recombinant molecules containing R. sphaeroides plasmid fragments have been shown to integrate into endogenous plasmids and form cointegrates with them.

A new IncQ plasmid R89S: properties and genetic organization.

The new small (8.18 kb) streptomycin-resistant multicopy plasmid R89S of the Q group incompatibility is described. In contrast to other IncQ plasmids, replication of R89S is dependent on DNA polymerase 1 and proceeds in the absence of de novo protein synthesis. According to our data up to now, the host spectrum of the plasmid R89S is limited to Enterobacteriaceae. A genetic map of the plasmid R89S has been prepared through the construction of deletion and insertion derivatives. Phenotypic analysis of these derivatives has identified the location of genes encoding resistance to streptomycin, and the region essential for mobilization of R89S. The origin of vegetative replication has been located within a 0.7-kb fragment. Another region highly homologous to oriV of the plasmid RSF1010, but not functioning as an origin of replication, was localized. Two regions involved in the expression of incompatibility have also been identified. The data from the restriction analyses, DNA-DNA hybridization, and genetic experiments enable us to assume that the plasmid R89S is a naturally occurring recombinant between part of an IncQ plasmid and another narrow host range replicon of unknown incompatibility group.