Up-Regulation of MTHFD2 is Associated With Clinicopathological Characteistics and Poor Survival in Ovarian Cancer, Possibly By Regulating MOB1A Signaling

Background: MTHFD2 is a folate-coupled metabolic enzyme, which has been proved to participant in the metabolic reprogramming and tumor cell-sustaining proliferative capacity. However, the function of MTHFD2 in the development of ovarian cancer and its potential molecular mechanisms is still unclear. Materials and methods: The expression, various mutations, prognosis, and related network signaling pathways of MTHFD2 were analyzed using bioinformatics-related websites, including Oncomine, GEPIA, UCSC, cBioPortal, KM Plotter, TISIDB and TIMER. The prognostic value of MTHFD2 expression was validated by our own ovarian cancer samples using RT-qPCR. The migration ad invasion of ovarian cancer cells were further analyzed by CCK-8 and transwell assay. The Western-blot assay was performed to explore the protein levels of MTHFD2 and MOB1A. Results: We obtained the following important results. (1) MTHFD2 expression was markedly up-regulated in ovarian cancer than normal samples. (2) Among patients with ovarian cancer, those with higher MTHFD2 expression was associated with lower survival rate. (3) The major mutation type of MTHFD2 in ovarian cancer samples was missense mutation. (4) MTHFD2 knockdown inhibited proliferation, migration, invasion, as well as the expression of MOB1A in vitro. Conclusion: MTHFD2, as a NAD+-dependent enzyme, accelerated tumor progression by up-regulating MBO1A, suggesting that this protein may be an independent prognostic factor and a potential therapeutic target for future ovarian cancer treatments.

Prevalence of resistance mutations associated with integrase inhibitors in therapy-naive HIV-positive patients in Hungary

Widespread introduction of HIV integrase inhibitors into clinical care may result in appearance of drug resistance mutations affecting treatment outcome. The aim of our study was to monitor the resistance patterns of integrase inhibitors beside protease and reverse transcriptase inhibitors in newly diagnosed therapy-naive HIV-positive patients in Hungary between 2017 and 2019.Genotype-based resistance testing of HIV integrase, protease and reverse transcriptase was performed by amplification and Sanger population sequencing from plasma samples. Drug resistance mutations were identified by the algorithm of Stanford HIV Drug Resistance Database.Potentially transmitted, non-polymorphic integrase major mutation was detected in 1 out of 249 samples, while accessory mutations were observed in further 31 patients (12.4%). The overall prevalence of transmitted drug resistance (TDR) mutations related to protease and reverse transcriptase inhibitors was 5.8% (10/173) between the end of 2017 and 2019. Nucleoside reverse transcriptase inhibitor associated resistance mutations were the most frequent indicators of TDR (6/173; 3.5%), followed by resistance mutations associated with protease (3/173; 1.7%) and non-nucleoside reverse transcriptase inhibitors (2/173, 1.2%).The first detection of integrase major mutation and the changing patterns of other resistance mutations in Hungarian untreated HIV-positive population indicate the necessity of continuous molecular surveillance of Hungarian HIV epidemic.

Krabbe’s Disease mutation spectrum in Russian Federation

Болезнь Краббе (БКр) - редкое наследственное аутосомно-рецессивное заболевание, входящее в группу лизосомных болезней накопления. Заболевание обусловлено мутациями в гене GALC, приводящими к дефициту фермента галактозилцерамидазы. Частота БКр оценивается как 1:100 000 живых новорожденных, хотя в некоторых странах регистрируется более высокая частота заболевания. Точная частота БКр в Российской Федерации и в ее регионах неизвестна. Мажорной мутацией, приводящей к развитию БКр, является крупная делеция затрагивающая 11-17 экзоны гена GALC c.1161+6532_polyA+9kbdel (IVS10del30kb). Доля этой мутации в европейской популяции оценивается примерно в 50% от всех мутаций. Для изучения спектра и частот мутаций гена GALC на территории РФ были обследованы пациенты из разных регионов. Частая делеция составила 54% от общего числа выявленных мутаций, что сопоставимо с данными по европейской популяции. Однако в Чеченской Республике данная мутация встречалась гораздо чаще, чем в других регионах. Среди 950 исследованных образцов было выявлено 7 гетерозиготных носителей частой мутации. Учитывая вклад других мутаций, расчётная частота БКр в республике составила 1:51237, что превышает таковую в европейской популяции. Дополнительный анализ всех найденных мутаций гена GALC позволил выявить вариант c.578T>C, p.Ile193Thr, аллельная частота которого составила 8%. Данная замена впервые была описана у пациентов из нашей выборки и встречается только при поздней инфантильной форме заболевания у русских пациентов. Krabbe’s disease (KD) is a rare inherited autosomal recessive lysosomal storage disease. KD is caused by mutations in the GALC gene leading to deficiency of galactosylceramidase. KD oссurs in 1 per 100 000 newborns, although some countries have a higher incidence rate. The exact KD incidence in Russia is unknown. A major mutation leading to the KD development is a large deletion affecting exons 11-17 of the GALC gene с.1161+6532_polyA+9kbdel (IVS10del30kb). This mutation occurs in 50% of KD cases in the European population. Patients from different regions were studied to analyze the mutation spectrum and the incidence in the Russian population. The incidence rate of the large deletion in our study equals 54%, that is comparable with European population. However, in the Chechen Republic this mutation is much more common than in other regions. 950 samples were studied, 7 heterozygous carriers of frequent mutation were identified. Thus, the estimated KD incidence rate is 1:51237 considering other mutations, and it is higher than that in the European population. Additional analysis of all detected GALC mutations revealed a genetic variant c.578T>C, p.Ile193Thr with allelic frequency measured up 8%. This substitution was described in our selection for the first time and presented only in Russian patients with late infantile form of the disease.

Identification of Ferroptosis Genes In Immune Infiltration and Prognosis In Thyroid Papillary Carcinoma Using Network Analysis

Background: Papillary thyroid carcinoma (PTC) is the most common thyroid cancer. While many patients survive, a portion of PTC cases display high aggressiveness and even develop into refractory differentiated thyroid carcinoma. This may be alleviated by developing a novel model to predict the risk of recurrence. Ferroptosis is an iron-dependent form of regulated cell death (RCD) driven by lethal accumulation of lipid peroxides, is regulated by a set of genes and shows a variety of metabolic changes. To elucidate whether ferroptosis occurs in PTC, we analysed the gene expression profiles of the disease and established a new model for the correlation. Methods: The thyroid carcinoma (THCA) datasets were downloaded from The Cancer Genome Atlas (TCGA), UCSC Xena and MisgDB, and included 502 tumour samples and 56 normal samples. A total of 60 ferroptosis related genes were summarised from MisgDB database. Gene set enrichment analysis (GSEA) and Gene set variation analysis (GSVA) were used to analyse pathways potentially involving PTC subtypes. Single sample GSEA (ssGSEA) algorithm was used to analyse the proportion of 28 kinds of immune cells in the tumour immune infiltration microenvironment in THCA and the hclust algorithm was used to conduct immune typing according to the proportion of immune cells. Spearman correlation analysis was performed on the ferroptosis gene expression and the correlation between immune infiltrating cells proportion. We established the WGCNA to identify genes modules that are highly correlated with the microenvironment of immune invasion. DEseq2 algorithm was further used for differential analysis of sequencing data to analyse the functions and pathways potentially involving hub genes. GO and KEGG enrichment analysis was performed using Clusterprofiler to explore the clinical efficacy of hub genes. Univariate Cox analysis was performed for hub genes combined with clinical prognostic data, and the results was included for lasso regression and constructed the risk regression model. ROC curve and survival curve were used for evaluating the model. Univariate Cox analysis and multivariate Cox analysis were performed in combination with the clinical data of THCA and the risk score value, the clinical efficacy of the model was further evaluated.Results: We identified two subtypes in PTC, using Braf as the major mutation of subtype C1 and NRAS as the major mutation of subtype C2. The proportion of cluster 1 was significantly higher than cluster 2 in ferroptosis signature genes that are positively associated. Subsequent analyses of TME immune cells infiltration indicated cluster 1 was remarkably richer than cluster 2. The risk score of THCA in good performance evaluated by ROC curve and survival curve, in conjunction with univariate Cox analysis and multivariate Cox analysis results based on the clinical data showed that the risk score of the proposed model could be used as an independent prognostic indicator to predict the prognosis of patients with papillary thyroid cancer.Conclusions: Our study found seven crucial genes, including Ac008063.2, Apoe, Bcl3, Acap3, Alox5ap, Atxn2l and B2m, and regulation of apoptosis by parathyroid hormone-related proteins significantly associated with ferroptosis and immune cells in PTC, and we constructed the risk score model which can be used as an independent prognostic index to predict the prognosis of patients with PTC.

Structural and Thermodynamic Analysis of the Resistance Development to Pimodivir (VX-787), the Clinical Inhibitor of Cap Binding to PB2 Subunit of Influenza A Polymerase

Influenza A virus (IAV) encodes a polymerase composed of three subunits: PA, with endonuclease activity, PB1 with polymerase activity and PB2 with host RNA five-prime cap binding site. Their cooperation and stepwise activation include a process called cap-snatching, which is a crucial step in the IAV life cycle. Reproduction of IAV can be blocked by disrupting the interaction between the PB2 domain and the five-prime cap. An inhibitor of this interaction called pimodivir (VX-787) recently entered the third phase of clinical trial; however, several mutations in PB2 that cause resistance to pimodivir were observed. First major mutation, F404Y, causing resistance was identified during preclinical testing, next the mutation M431I was identified in patients during the second phase of clinical trials. The mutation H357N was identified during testing of IAV strains at Centers for Disease Control and Prevention. We set out to provide a structural and thermodynamic analysis of the interactions between cap-binding domain of PB2 wild-type and PB2 variants bearing these mutations and pimodivir. Here we present four crystal structures of PB2-WT, PB2-F404Y, PB2-M431I and PB2-H357N in complex with pimodivir. We have thermodynamically analysed all PB2 variants and proposed the effect of these mutations on thermodynamic parameters of these interactions and pimodivir resistance development. These data will contribute to understanding the effect of these missense mutations to the resistance development and help to design next generation inhibitors.

Clinical and genetic characteristics and orthopedic manifestations of the Saul–Wilson syndrome in two Russian patients

Background. SaulWilson syndrome (SWS, microcephalic osteodysplastic dysplasia) is a rare genetic variant of skeletal dysplasia and is determined based on the modern classification for thin bone dysplasias. To date, 16 patients with SWS from different countries have been identified. Clinical cases. We presented the first description of the clinical and genetic characteristics of two Russian patients with SWS and compared them with published data. The main clinical manifestations of SWS are characterized by a combination of nanism and pathology of long tubular bones, spine, and eyes. Changes in the phenotype of patients in different age groups were analyzed. Discussion. In the analysis of the clinical manifestations of the observed patients and patients described in the literature, typical dysmorphic features of the face and radiographic data help in the diagnosis of SWS upon clinical examination. In the majority of the described patients, the nucleotide substitution c.1546GA is the major mutation in the gene responsible for SWS, which leads to the replacement of the amino acid Gly516Arg in the protein molecule. Conclusion. Based on the identified specific features of the phenotype of patients with SWS and the presence of a major mutation in the COG4 gene, a priority analysis of gene mutations is necessary. Orthopedic manifestations of SWS can lead to life-threatening conditions (cervical spine instability) and motor limitations (progressive osteoarthritis) and thus should be monitored dynamically.

Clinical course of cystic fibrosis in children

Annotation. This study is devoted to evaluation of clinical course of cystic fibrosis (CF) in children. The study involved 84 children who were diagnosed with cystic fibrosis. All participants were asked about complaints, life history, physical examination was made, and the results of objective testing and instrumental examination were assessed. The statistical system “IBM SPSS Statistics” version 12 (20) was used using parametric and non-parametric methods of descriptive statistics. Student's criterion was used to assess the significance of the difference between the independent samples, and the Fisher's factor was used for the percentage data. It was shown that 86.91% of subjects had severe or moderate course of CF. Major mutation of CFRT gene F508del/F508del was found in 45.2% children. Common complaints were productive cough (92.9%; р<0.01), failure to thrive (61.9%; р<0.01) and shortness of breath during exercise (40.5%; р<0.01). Results of bacteriologic test of sputum showed significant prevalence of St. aureus (54.0%; р<0.01) and Ps. aeruginosa (38.10%; р<0.01). Patients with severe course of CF had lower rates of FVC, FEV1, PEF, FEF 25%, FEF 50%, FEF 75% than children with moderate and mild course. At the age of 3 years, the diagnosis of cystic fibrosis was established in time by only 76.92% of respondents, which aims to further efforts to improve the quality of its diagnosis.

The Cystic Fibrosis Patient Registry as a guarantee of effective disease management

The aim of the study was to analyze the data of Ukrainian cystic fibrosis (CF) patients entered to the European Cystic Fibrosis Society Patient Registry (ECFSPR) during 2017–2018 years, and compare with the parameters entered in previous years. Materials and methods. Using the computer program ecfstracker.eu, the clinical, paraclinical and molecular genetic data of CF patients were analyzed and their statistical analysis was performed. Research results. Among 210 CF patients 49 alleles and 68 different genotypes of the CF transmembrane conductance regulator gene were identified. It was found that 57% of CF patients were homozygous for the F508del major mutation, and 31% — heterozygous. The ratio of the proportion of СF patients between children and adults (over 18 years) was 78% and 22%, with an increasing in the proportion of adult patients compared to previous years. Conclusions. The entering data CF patients to the ECFSPR opens new opportunities for a comprehensive analysis of the disease at both on the regional (the center) and on the continental levels. Studying the experience and management of the disease based on the experience of 35 European countries (over 40,000 patients) will not only help to compare the data of Ukrainian patients, but also borrow the best monitoring and treatment schemes from European countries, which help to organize and optimize medical care in Ukraine. No conflict of interest was declared by the authors. Key words: The European Cystic Fibrosis Society Patient Registry, gene, genotype, DNA, molecular genetic research, cystic fibrosis, mutation, CFTR.

Evolution of the Resistance of Botrytis cinerea to Carbendazim and the Current Efficacy of Carbendazim Against Gray Mold After Long-Term Discontinuation

Gray mold caused by Botrytis cinerea is a fungal disease that critically threatens agricultural production, and carbendazim was the first fungicide used to control B. cinerea. However, B. cinerea developed serious resistance to carbendazim, and this fungicide has thus rarely been used in the past decade in China. Due to the extended discontinuation of carbendazim use, the evolution of the resistance of B. cinerea to carbendazim in recent years is unclear, and whether carbendazim can effectively control gray mold is largely unknown. Therefore, this study determined the sensitivity of 407 B. cinerea isolates collected from 2014 to 2018 to carbendazim and the ability of carbendazim to control gray mold in the field. The results showed that the frequency of B. cinerea isolates resistant to carbendazim remained above 95%. Three different mutation types responsible for the resistance of B. cinerea to carbendazim were identified at codon 198 in the β-tubulin gene sequence: E198V (changed from GAG to GTG), E198A (changed from GAG to GCG), and E198K (changed from GAG to AAG). Over the last 5 years, E198V was the major mutation. However, an analysis of its evolution revealed that the percentage of the E198V mutation declined after 2017 to 56.5% in 2018. In addition, the proportion of isolates with the E198K mutation decreased over time, and no isolates with this mutation were found in either 2017 or 2018. The proportion of the E198A mutation increased over the 5-year test period to reach 43.5% in 2018. Furthermore, three greenhouse experiments demonstrated that carbendazim has lost its ability to control gray mold. We attribute the above findings to our results showing that the carbendazim-resistant isolates had no fitness penalties compared with the carbendazim-sensitive isolates for sporulation and mycelial growth. In particular, the E198A mutant isolates exhibited a strong ability to sporulate, suggesting that the E198A mutation might become dominant in the future. Interestingly, the results showed that carbendazim-sensitive isolates could be easily controlled by four conventional fungicides, namely boscalid, procymidone, iprodione, and pyrimethanil, with mean EC50 values of 0.71 ± 0.2 mg liter−1, 1.33 ± 0.39 mg liter−1, 0.59 ± 0.33 mg liter−1, and 6.02 ± 3.02 mg liter−1, respectively. In conclusion, carbendazim has lost its application value and is ineffective for the control of gray mold.

The genomic variation landscape of globally-circulating clades of SARS-CoV-2 defines a genetic barcoding scheme

ABSTRACTWe describe fifteen major mutation events from 2,058 high-quality SARS-CoV-2 genomes deposited up to March 31st, 2020. These events define five major clades (G, I, S, D and V) of globally-circulating viral populations, representing 85.7% of all sequenced cases, which we can identify using a 10 nucleotide genetic classifier or barcode. We applied this barcode to 4,000 additional genomes deposited between March 31st and April 15th and classified successfully 95.6% of the clades demonstrating the utility of this approach. An analysis of amino acid variation in SARS-CoV-2 ORFs provided evidence of substitution events in the viral proteins involved in both host-entry and genome replication. The systematic monitoring of dynamic changes in the SARS-CoV-2 genomes of circulating virus populations over time can guide therapeutic and prophylactic strategies to manage and contain the virus and, also, with available efficacious antivirals and vaccines, aid in the monitoring of circulating genetic diversity as we proceed towards elimination of the agent. The barcode will add the necessary genetic resolution to facilitate tracking and monitoring of infection clusters to distinguish imported and indigenous cases and thereby aid public health measures seeking to interrupt transmission chains without the requirement for real-time complete genomes sequencing.