Molecular Genetics, Microbiology and Virology最新文献

{"title":"Regulatory Non-Coding RNAs in Crops Health and Disease","authors":"Patrick Ferreira, Pedro A. Casquero, Altino Choupina","doi":"10.3103/s0891416823040080","DOIUrl":"https://doi.org/10.3103/s0891416823040080","url":null,"abstract":"<h3 data-test=\"abstract-sub-heading\">Abstract</h3><p>For many years it was thought that the function of RNA was limited to the process of producing proteins. In recent years, scientific discoveries have been proving the multiple roles of different RNAs in different regulatory mechanisms. These RNA’s are collectively called non-coding RNA’s (ncRNA’s). This review presents the latest advances on the different classes of non-coding RNA’s (ncRNA’s) from their function to mechanisms of action. Special emphasis is given to the long non-coding RNAs as new regulatory elements in eukaryote gene expression and in the processes of epigenetic regulation in plants. We believe that increasing studies of regulatory non-coding RNAs in plants will provide a better understanding of the different types of genes related to crop resistance.</p>","PeriodicalId":19005,"journal":{"name":"Molecular Genetics, Microbiology and Virology","volume":null,"pages":null},"PeriodicalIF":0.5,"publicationDate":"2024-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140167512","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Rare Mutations V453X and Y847X in the MYBPC3 Gene Do Not Lead to a Severe Form of Hypertrophic Cardiomyopathy in the Russian Population","authors":"A. L. Klass, N. S. Krylova, A. V. Lysenko, I. N. Vlasov, M. Yu. Maslova, G. I. Salagaev, E. A. Kovalevskaya, N. G. Poteshkina, M. I. Shadrina, P. A. Slominsky, E. V. Filatova","doi":"10.3103/s0891416823040043","DOIUrl":"https://doi.org/10.3103/s0891416823040043","url":null,"abstract":"<h3 data-test=\"abstract-sub-heading\">Abstract</h3><p>Mutations in the <i>MYBPC3</i> gene are currently believed to lead to the development of hypertrophic cardiomyopathy (HCM) in the majority of genetically determined cases. However, despite many years of research, both in worldwide and in Russia in particular, the genetic landscape of HCM is still insufficiently studied. Moreover, the insufficient study of genetically determined cases of HCM in the Russian population does not allow us to study the possible relation of the phenotypic characteristics of HCM patients with certain pathogenic variants of the genome of these patients. In this regard, the purpose of our work was to study the prevalence of rare pathogenic variants rs730880711 and rs397515974 of the <i>MYBPC3</i> gene in HCM patients from Russia and to assess the effect of these mutations on the severity of this disease. The sample included 180 patients with moderate HCM and 137 patients with severe HCM. Analysis of the genotypes of rs730880711 (NC_000011.10:g.47342928_47342929insG; V453X) and rs397515974 (NC_000011.10:g.47337452G&gt;C; Y847X) variants in the <i>MYBPC3</i> gene was carried out in genomic DNA samples isolated from peripheral blood by real-time PCR. The performed analysis of the prevalence of rare pathogenic variants rs730880711 and rs397515974 in the <i>MYBPC3</i> gene in patients with moderate and severe forms of HCM from Russia showed that the frequency of each mutation was 0.003. Both pathogenic variants were identified in individuals with the moderate disease. Thus, the indicated mutations are extremely rare in HCM patients from Russia and do not make a significant contribution to the development of this disease in the Russian population.</p>","PeriodicalId":19005,"journal":{"name":"Molecular Genetics, Microbiology and Virology","volume":null,"pages":null},"PeriodicalIF":0.5,"publicationDate":"2024-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140167586","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Molecular Identification of Leptospira interrogans and Its Treatment with Andrographis paniculata Extract","authors":"Abirami Madhaiyan, V. Muthumani, E. Angel Jemima, K. Rubika, V. Bharathi, N. Parameswari, V. Kowsalya Devi, K. Ramesh, P. Poongothai","doi":"10.3103/s0891416823040110","DOIUrl":"https://doi.org/10.3103/s0891416823040110","url":null,"abstract":"<p><b>Background</b>: Leptospirosis is a zoonotic disease that transmitted to humans by rodents and domestic animals that infected by <i>Leptospira</i> bacteria and considered as a endemic disease in South India and North-East India. <i>Andrographis paniculata</i> is an ancient herb with various therapeutic properties and has Andrographolide bioactive compound. <b>Purpose:</b> The aim of the present study is to isolate and molecular identification by ITS sequencing of the <i>Leptospira interrogans</i> from the soil sample of paddy field and to analyse the anti-leptospirosis activity of <i>Andrographis paniculata</i> extract in <i>in vitro</i>. <b>Methods:</b> <i>Andrographis paniculata</i> extract was extracted using methanol and screening of phytocompound was done by GC-MS analysis. <i>Leptospira</i> species was isolated from the soil sample and molecular identification was performed by 16s rRNA gene sequencing. BLAST and phylogenetic tree analysis confirmed the presence of <i>Leptospira interrogans.</i> Molecular Docking analysis was performed to study the interaction between the Andrographolide and Leptospira LRR protein. Anti-leptospirosis activities of <i>Andrographis paniculata</i> extracted using different solvents were determined by Minimum Inhibitory Concentration (MIC) methods. <b>Results:</b> The 16s rRNA sequencing and BLAST results confirmed the presence of <i>Leptospirosis interrogans</i>. The phylogenetic tree analysis showed the distance between the Leptospirosis species. Furthermore, the GCMS analysis of <i>Andrographis paniculata</i> methanolic extract showed the presence of phytocompound Andrographolide. Molecular docking analysis of Andrographolide with the LRR protein showed stable binding. The in vitro anti-leptospiral activity of different <i>Andrographis paniculata</i> extract showed the maximum inhibition at 500 μg/mL and the maximum MIC was observed at methanolic extract of <i>A. paniculata</i> at 31.2 μg/mL with 64.65 ± 7.33% of inhibition. <b>Conclusion:</b> From our study we concluded that <i>Andrographis paniculata</i> extracted using methanol has good anti-leptospirosis activity and Andrographolide will act as a good therapeutic agent against leptospirosis.</p>","PeriodicalId":19005,"journal":{"name":"Molecular Genetics, Microbiology and Virology","volume":null,"pages":null},"PeriodicalIF":0.5,"publicationDate":"2024-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140167465","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Association of Mutations in gyrA Gene with Resistance to Fluoroquinolones in Clinical Isolates of Multidrug-Resistant Mycobacterium tuberculosis","authors":"Hanieh Bagherifard, Mitra Salehi, Mona Ghazi","doi":"10.3103/s0891416823040092","DOIUrl":"https://doi.org/10.3103/s0891416823040092","url":null,"abstract":"<h3 data-test=\"abstract-sub-heading\">Abstract</h3><p>The purpose of the present study was to evaluate the association of mutations inside and outside quinolone-resistance determining region (QRDR) of gyrA gene with resistance to fluoroquinolones, particularly levofloxacin (LFX), moxifloxacin (MFX), ofloxacin (OFX), and ciprofloxacin (CIP). Therefore, a total of 255 clinical isolates of <i>Mycobacterium tuberculosis</i> were tested for drug susceptibility. Accordingly, 68 multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis strains were subjected to molecular analysis. Mutations were found in 25 (43.1%) of fluoroquinolone-resistant isolates including two rare mutations at codons 93 and 124. We then proceeded to predict the functional and structural impacts of the identified mutations on the protein via PredictSNP, PROVEAN, PoPMuSiC, and HoTMuSiC tools which revealed that they could be deleterious and/or destabilizing to GyrA. Our findings suggest that coupling genetic analysis with computational approaches could be of great value for unraveling molecular mechanisms involved in drug resistance.</p>","PeriodicalId":19005,"journal":{"name":"Molecular Genetics, Microbiology and Virology","volume":null,"pages":null},"PeriodicalIF":0.5,"publicationDate":"2024-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140167726","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The Cytotoxic Activity of Phenazine Compounds from Pseudomonas chlororaphis subsp. aurantiaca against the HeLa Cell Line","authors":"A. A. Zhyzneyskaya, A. A. Lukashevich, N. P. Maksimova, E. G. Veremeenko","doi":"10.3103/s0891416823040079","DOIUrl":"https://doi.org/10.3103/s0891416823040079","url":null,"abstract":"<p>In this investigation, the cytotoxic activity of phenazine compounds from different bacterial strains of <i>Pseudomonas chlororaphis</i> subsp. <i>aurantiaca</i> against the HeLa cervical adenocarcinoma cell line was studied. The cytotoxic concentrations of phenazines against HeLa cells were 300 μg/mL. After incubation with phenazines, cytological preparations of HeLa cells showing the presence of apoptotic bodies were obtained. The effect of phenazines on HeLa cells led to a change in the expression of their ABC transporter genes (<i>abcc1</i> and <i>abcg2</i>) and <i>tp53</i>. The activity of <i>tp53</i> increased almost 13-fold, while the expression of the <i>abcc1</i> and <i>abcg2</i> genes decreased. The activation of <i>tp53</i> is one of the probable causes of apoptotic death of HeLa cells in the presence of phenazine compounds from the bacterium <i>P. chlororaphis</i> subsp. <i>aurantiaca</i>.</p>","PeriodicalId":19005,"journal":{"name":"Molecular Genetics, Microbiology and Virology","volume":null,"pages":null},"PeriodicalIF":0.5,"publicationDate":"2024-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140167671","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cassette Construction and Co-Expression of NA and F Proteins of H1N1 Influenza Virus and Newcastle Disease Virus in Saccharomyces cerevisiae","authors":"A. Seyed Hamzehzadeh, A. Hesampour","doi":"10.3103/s0891416823040134","DOIUrl":"https://doi.org/10.3103/s0891416823040134","url":null,"abstract":"<p>The aim of this study is to optimize the design, construction, and expression of recombinant protein, NA-F, derived from pathogenic influenza and Newcastle viruses within the eukaryotic host <i>Saccharomyces cerevisiae</i>. The chimeric gene was constructed using a homologous recombination method, and the target genes were cloned onto the T&amp;A vector using PCR cloning techniques. Subsequently, the chimeric gene was expressed in <i>S. cerevisiae</i>, and its presence was confirmed through extracellular expression validation, Western blotting, and specific antibody detection. The recombinant protein displayed significant biological activity, demonstrating its functional characteristics and potential for use in immunization. The chimeric gene structure produced in this study holds promise as a candidate to replace existing vaccines worldwide, considering the high prevalence of influenza and Newcastle viruses in poultry and the need for a universal subunit vaccine. However, further research in animal and human models is essential to ensure safety before widespread application.</p>","PeriodicalId":19005,"journal":{"name":"Molecular Genetics, Microbiology and Virology","volume":null,"pages":null},"PeriodicalIF":0.5,"publicationDate":"2024-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140167488","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Phylogenetic Analysis and Frequency Estimation of HTLV-1 Virus in Hemophilic Patients Refer to Iranian Comprehensive Hemophilia Care Center (ICHCC)","authors":"Tina Taikandi, Navid Momenifar, Ali Namvar, Alireza Azizi Saraji, Mehdi Norouzi, Cobra Razavi Pashabayg","doi":"10.3103/s0891416823040109","DOIUrl":"https://doi.org/10.3103/s0891416823040109","url":null,"abstract":"<h3 data-test=\"abstract-sub-heading\">Abstract</h3><p>Human T-lymphotropic virus type 1 (HTLV-1) is associated with the development of malignant diseases, particularly adult T-cell leukemia/lymphoma (ATLL) and myelopathy/tropical spastic paraparesis (HAM/TSP). Hemophilia patients are at risk of acquiring blood–borne infections, making it critical to prevent the transmission of viral and other contaminants. The objective of this study was to determine the prevalence of HTLV-1 in Iranian hemophilia patients who were referred to the Iranian Comprehensive Hemophilia Care Center (ICHCC) and to analyze the results. A total of 320 blood samples were collected from hemophilia patients and screened using enzyme-linked immunosorbent assay (ELISA). Seropositive samples were confirmed by amplifying the long terminal repeat (LTR) region of HTLV-1 using nested PCR. The LTR region fragment was amplified, sequenced, and analyzed by MEGA 7 for phylogenetic analysis. Three out of four positive serological samples were confirmed using PCR, resulting in an HTLV-1 infection outbreak of 0.9%. Phylogenetic analysis of hemophilia patients infected with HTLV-1 revealed that the virus belongs to subtype a (Cosmopolitan) and subgroup A (Transcontinental). The findings suggest that hemophilia patients may be at high risk for HTLV-1 transmission. Furthermore, screening of blood and blood products can play a critical role in preventing the spread of the virus in endemic areas.</p>","PeriodicalId":19005,"journal":{"name":"Molecular Genetics, Microbiology and Virology","volume":null,"pages":null},"PeriodicalIF":0.5,"publicationDate":"2024-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140167490","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Historical Isolates of Semliki Forest and Chikungunya Viruses and Their Virological Characteristic, Whole-Genome Sequences, and Phylogeny","authors":"T. P. Mikryukova, E. V. Protopopova, A. S. Mezhevalova, R. B. Bayandin, Yu. A. Khoroshavin, A. N. Shvalov, O. S. Taranov, T. V. Tregubchak, V. A. Ternovoi, V. B. Loktev","doi":"10.3103/s0891416823040055","DOIUrl":"https://doi.org/10.3103/s0891416823040055","url":null,"abstract":"<p>The purpose of the study was to analyze alphavirus isolates collected in Uganda and Tanzania in the period preceding their global spread dating back to the middle of the last century. We supposed that analysis of their genomes could help to learn more about the specific features and the direction of molecular evolution of alphaviruses in the modern world. Archival samples of Chikungunya (CHIKV) and Semliki Forest (SFV) viruses were revived by cultivation in the Vero E6 cells. Isolates were identified by RT-PCR followed by sequencing. Whole genome sequences were obtained by NGS and used for phylogenetic analysis. The presence of two representatives of the <i>Alphavirus</i> genus, namely, CHIKV and SFV, was observed in the studied archival CHIKV sample. Only SFV was found in the archival sample from 1942. All isolates were capable of highly efficient replication in the C6/36, Vero E6, 293, and SPEV cell cultures showing the development of cytopathological effects and were able to produce pathomorphological changes typical of these alphaviruses in mice. Whole genome sequences have been obtained for these viruses and analyzed. Studied isolates clustered with the typical African CHIKV and SFV strains. These isolates may be attributed to the oldest known SFV and CHIKV strains dating back to 1942 and 1953 preserved in laboratory collections. The archival CHIKV isolate was genotyped as an ECSA variant, the modern representatives of which are associated with the global spread of CHIKV in recent decades. Semliki Forest and Chikungunya virus isolates were revived from archival laboratory samples presumably dating back to 1942 and 1953, and their virological characterization was carried out, followed by genotyping and phylogenetic analysis of their whole-genome sequences.</p>","PeriodicalId":19005,"journal":{"name":"Molecular Genetics, Microbiology and Virology","volume":null,"pages":null},"PeriodicalIF":0.5,"publicationDate":"2024-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140167521","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Monitoring the Spread of SARS-CoV-2 Gene Variants in Sverdlovsk and Chelyabinsk Oblasts and Perm Krai","authors":"A. E. Chernysheva, I. A. Korotkova, E. V. Bolgarova, A. Yu. Markaryan, A. V. Semenov","doi":"10.3103/s0891416823040031","DOIUrl":"https://doi.org/10.3103/s0891416823040031","url":null,"abstract":"<p>A genomic diversity study of SARS-CoV-2 was conducted in the context of lifting the coronavirus restrictions and reopening Russia’s national borders. SARS-CoV-2 genovariants in Sverdlovsk and Chelyabinsk oblasts and Perm krai were identified. A total of 1127 nasopharyngeal smears from patients with a new coronavirus infection treated in Sverdlovsk and Chelyabinsk oblasts and Perm krai were studied by Sanger sequencing. The paper presents the dynamics of new coronavirus strains and its several mutations for the period from May to December 2022 using descriptive statistics. The study period was notable for Omicron predominance (~97%) with a greater number of amino acid mutations compared with that for the Delta and Alpha variants. The Omicron BA.2 subvariant prevailed during the study period from May to September and from October to December 2022, then BA.2 was displaced by BA.4/BA.5 subvariants. Molecular similarity analysis for assessment of SARS-CoV-2 showed the presence of atypical mutations at positions E484K, G485R, and R408I in some samples, as well as an increase in the number of mutations in variants, especially in the receptor-binding domain RBD. According to the literature, amino acid substitutions led to an S protein conformational alteration which, in turn, increased the affinity of RBD binding to ACE2 and resulted in a milder disease and higher contagiousness compared with earlier SARS-CoV-2 variants.</p>","PeriodicalId":19005,"journal":{"name":"Molecular Genetics, Microbiology and Virology","volume":null,"pages":null},"PeriodicalIF":0.5,"publicationDate":"2024-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140167487","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comparative Analysis of Influenza Viruses Isolated from Early and Severe Cases in Epidemic Seasons before and during the COVID-19 Pandemic in Russia (2019–2023)","authors":"N. D. Boldyrev, A. S. Panova, N. P. Kolosova, A. V. Danilenko, S. V. Svyatchenko, G. S. Onkhonova, V. Yu. Marchenko, A. B. Ryzhikov","doi":"10.3103/s089141682304002x","DOIUrl":"https://doi.org/10.3103/s089141682304002x","url":null,"abstract":"<h3 data-test=\"abstract-sub-heading\">Abstract</h3><p>The influenza virus is one of the most dangerous causative agents of respiratory diseases, and its study is important for epidemiological control, especially in the case of cocirculation with SARS-CoV-2. Comparative analysis of influenza viruses isolated from early and severe cases in epidemic seasons before and during the COVID-19 pandemic in Russia. The article is based on the results of monitoring the circulation of seasonal influenza viruses obtained in 2019–2023. Samples from early and severe cases of influenza were studied using real-time PCR and whole-genome sequencing. Antigenic characterization of isolated viruses was carried out, and their sensitivity to antiviral drugs was studied. The flu season of 2019–2020 in Russia was the last epidemic season before the COVID-19 pandemic with the predominant cocirculation of influenza B and influenza A/H1 N1 pdm09 viruses. After the onset of the pandemic in the 2020–2021 season, the influenza virus was practically absent in Russia and was detected sporadically. Virus circulation resumed in the 2021–2022 season with dominance of A/H3N2 (clade 3C.2a1b.2a2) and continued in the 2022–2023 season with the dominance of A/Hi N1 pdm09 (clade 6B.1A.5a.2a) and the spread of influenza B/Victoria viruses (clade V1A.3a.2), which were antigenically different from the viruses circulating before the COVID-19 pandemic. Genetic analysis of the D222G/N mutations in the hemagglutinin of the A/H1N1pdm09 viruses, which are associated with increased disease severity, revealed an approximately equivalent selection of the D222G and D222N mutations in the 2019–2020 season and increased occurrence of the D222N variant in the 2022–2023 season. Cocirculation with SARS-CoV-2, the return of influenza circulation to epidemic levels, the emergence of new antigenic variants and pathogenicity factors emphasize the need to monitor and study influenza viruses for epidemiological analysis and prognosis, as well as for the development and application of effective measures to protect the population.</p>","PeriodicalId":19005,"journal":{"name":"Molecular Genetics, Microbiology and Virology","volume":null,"pages":null},"PeriodicalIF":0.5,"publicationDate":"2024-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140167492","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}