Identification of hub genes associated with Helicobacter pylori infection and type 2 diabetes mellitus:A pilot bioinformatics study

BACKGROUND Helicobacter pylori(H.pylori)infection is related to various extragastric diseases including type 2 diabetes mellitus(T2DM).However,the possible mechanisms connecting H.pylori infection and T2DM remain unknown.AIM To explore potential molecular connections between H.pylori infection and T2DM.METHODS We extracted gene expression arrays from three online datasets(GSE60427,GSE27411 and GSE115601).Differentially expressed genes(DEGs)commonly present in patients with H.pylori infection and T2DM were identified.Hub genes were validated using human gastric biopsy samples.Correlations between hub genes and immune cell infiltration,miRNAs,and transcription factors(TFs)were further analyzed.RESULTS A total of 67 DEGs were commonly presented in patients with H.pylori infection and T2DM.Five significantly upregulated hub genes,including TLR4,ITGAM,C5AR1,FCER1G,and FCGR2A,were finally identified,all of which are closely related to immune cell infiltration.The gene-miRNA analysis detected 13 miRNAs with at least two gene cross-links.TF-gene interaction networks showed that TLR4 was coregulated by 26 TFs,the largest number of TFs among the 5 hub genes.CONCLUSION We identified five hub genes that may have molecular connections between H.pylori infection and T2DM.This study provides new insights into the pathogenesis of H.pylori-induced onset of T2DM.

Pathogenesis of chronic enteropathy associated with the SLCO2A1 gene:Hypotheses and conundrums

Chronic enteropathy associated with the SLCO2A1 gene(CEAS)is a complex gastroenterological condition characterized by multiple ulcers in the small intestine with chronic bleeding and protein loss.This review explores the potential mechanisms underlying the pathogenesis of CEAS,focusing on the role of SLCO2A1-encoded prostaglandin transporter OATP2A1 and its impact on prostaglandin E2(PGE2)levels.Studies have suggested that elevated PGE2 levels contribute to mucosal damage,inflammation,and disruption of the intestinal barrier.The effects of PGE2 on macrophage activation and Maxi-Cl channel functionality,as well as its interaction with nonsteroidal anti-inflammatory drugs play crucial roles in the progression of CEAS.Understanding the balance between its protective and pro-inflammatory effects and the complex interactions within the gastrointestinal tract can shed light on potential therapeutic targets for CEAS and guide the development of novel,targeted therapies.

Trifunctional Cu-Mesh/Cu_(2)O@FeO Nanoarrays for Highly Efficient Degradation of Antibiotic, Inactivation of Antibiotic-Resistant Bacteria, and Damage of Antibiotics Resistance Genes

Trifunctional Cu-mesh/Cu_(2)O@FeO nanoarrays heterostructure is designed and fabricated by integrating CuCu_(2)O@FeO nanoarrays onto Cu-mesh(CM)via an in situ growth and phase transformation process.It is successfully applied to efficiently mitigate the antibiotic pollution,including degradation of antibiotics,inactivation of antibiotic-resistant bacteria(ARB),and damage of antibiotics resistance genes(ARGs).Under visible-light irradiation,CM/CuCu_(2)O@FeO nanoarrays exhibit a superior degradation efficiency on antibiotics(e.g.,up to 99%in 25 min for tetracycline hydrochloride,TC),due to the generated reactive oxygen species(ROS),especially the dominant·O^(2−).It can fully inactivate E.coli(HB101)with initial number of~108 CFU mL^(−1) in 10 min,which is mainly attributed to the synergistic effects of 1D nanostructure,dissolved metal ions,and generated ROS.Meanwhile,it is able to damage ARGs after 180 min of photodegradation,including tetA(vs TC)of 3.3 log 10,aphA(vs kanamycin sulfate,KAN)of 3.4 log 10,and tnpA(vs ampicillin,AMP)of 4.4 log 10,respectively.This work explores a green way for treating antibiotic pollution under visible light.

The relationship of Imp2 and DR3 genes with susceptibility to type Ⅰ diabetes mellitus in south China Han population

AIN To study the relationship of Imp2 and DR3genes with type Ⅰ diabetes mellitus.NETHODS Imp2 genotypes and DR3 wereidentified in 68 patients with type Ⅰ diabetesmellitus(Ⅰ-DM)and 71 healthy controls.Then,Ⅰ-DM patients and controls were respectivelyallocated into DR3-positive and DR3-negativegroups.The frequencies of Imp2 and DR3 genein random subjects,and Imp2 genotypes in DR3-matched subjects were compared between Ⅰ-DMpatients and controls.At the same time,Ⅰ-DMpatients were divided into 3 groups based on theonset age of diabetics:group A≤14 years,group B 15-30 years and group C≥31 years.RESULTS The frequency of DR3 in Ⅰ-DMpatients was significantly higher than that incontrols(47% vs 21%,P【0.005),and it wassignificantly higher in group A than that in groupB+C(70% vs 36%,x^2=7.07,P【0.01).Therewas a significant difference among groups withdifferent onset age of diabetics(x^2=8.19,rp=0.33,P【0.05).In random subjects,thefrequency of Imp2.R/R in Ⅰ-DM patients waslower(43% vs 61%,P【0.05)and Imp2.R/Hhigher(53% vs 28%,P【0.05)than that incontrols,and there was no significant differenceamong groups with different onset age ofdiabetics.In DR3-positive subjects,thefrequency of Imp2.R/R in Ⅰ-DM patients waslower(47% vs 87%,P【0.05)and Imp2-R/H higher(47% vs 13%,P【0.05)than that incontrols.In DR3-negative subjects,thefrequency of Imp2.R/H in Ⅰ-DM patients washigher than that in controls(58% vs 32%,P【0.01),but the frequency of Imp2-R/R and Imp2H/H was not significantly different betweenthese two groups.CONCLUSION DR3 gene may be one of thesusceptible genes of Ⅰ-DM,and significantlyrelated to the onset age of diabetics,and thepersons with DR3 may have an younger onsetage of diabeteS.The Imp2-R/R may be theprotective genotype of Ⅰ-DM,and Imp2-R/H thesusceptible genotype.These were not affectedby DR3 gene.Imp-2 genotypes were not relatedwith the onset age of diabetics.

Detections of mefA, ermB, and mphA Macrolides Resistant Genes in Bacteria Isolated from Covid-19 Patients from Selected Health Facilities in Ibadan, Nigeria

Background: COVID-19 is a disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Epidemiological data indicated that bacterial complications in COVID-19 would decrease clearance rate of the infecting agent and increase mortality rate. Macrolides such as Azithromycin are usually administered to COVID-19 patients as palliative treatments. Currently, a considerable number of bacterial strains have developed resistance to various antibiotics, especially macrolides. Resistance is reported to be due to possession of mefA, ermB, and mphA genes by Gram positive and Gram negative bacteria. Therefore, this study determined antibiotic resistance patterns and identify mefA, ermB and mphA macrolide-resistant genes in bacterial pathogens isolated from COVID-19 cases in Ibadan, Nigeria. Methods: 400 Nasopharyngeal samples were collected from symptomatic cases before antibiotic medication;structured questionnaires were administered to collect socio-demographic data of participants. Samples were cultured on Blood, Chocolate, MacConkey and Mannitol salt agar at 37°C for 48 hrs. Bacterial identification was performed using VITEK 2.0 ID cards and API 20E for Gram positive and negative bacteria respectively. Antibiotic Susceptibility Testing was performed using Kirby Bauer disc diffusion methods and VITEK 2.0 AST card kits. DNA of multidrug resistant bacterial isolates was extracted;resistant genes were determined using a polymerase chain reaction with specific primers. Amplified genes were detected using agarose gel electrophoresis. Results: 240 (60%) had bacterial growth and 97 (22.2%) yielded no growth. From the 240 bacterial isolates, 38 (15.83%) were multi-drug resistant including resistance to macrolides (Azithromycin) 20 (52.63%) of which were positive for either mefA or ermB, and none (0.0%) possess mphA gene;14 (36.8%) isolates had mefA gene, 10 (26.3%) isolates carried ermB gene. Conclusion: Multi-drug bacterial resistance including macrolides and quinolones was detected. Only mefA and ermB genes were detected in the bacterial isolates, especially in Gram positive organisms. The detection of mefA and ermB genes in the MDR bacterial isolates raised concern on the use of azithromycin as palliative treatment for COVID-19 symptomatic patients.

Identification of key genes and biological pathways in lung adenocarcinoma by integrated bioinformatics analysis

BACKGROUND The objectives of this study were to identify hub genes and biological pathways involved in lung adenocarcinoma(LUAD)via bioinformatics analysis,and investigate potential therapeutic targets.AIM To determine reliable prognostic biomarkers for early diagnosis and treatment of LUAD.METHODS To identify potential therapeutic targets for LUAD,two microarray datasets derived from the Gene Expression Omnibus(GEO)database were analyzed,GSE3116959 and GSE118370.Differentially expressed genes(DEGs)in LUAD and normal tissues were identified using the GEO2R tool.The Hiplot database was then used to generate a volcanic map of the DEGs.Weighted gene co-expression network analysis was conducted to cluster the genes in GSE116959 and GSE-118370 into different modules,and identify immune genes shared between them.A protein-protein interaction network was established using the Search Tool for the Retrieval of Interacting Genes database,then the CytoNCA and CytoHubba components of Cytoscape software were used to visualize the genes.Hub genes with high scores and co-expression were identified,and the Database for Annotation,Visualization and Integrated Discovery was used to perform enrichment analysis of these genes.The diagnostic and prognostic values of the hub genes were calculated using receiver operating characteristic curves and Kaplan-Meier survival analysis,and gene-set enrichment analysis was conducted.The University of Alabama at Birmingham Cancer data analysis portal was used to analyze relationships between the hub genes and normal specimens,as well as their expression during tumor progression.Lastly,validation of protein expression was conducted on the identified hub genes via the Human Protein Atlas database.RESULTS Three hub genes with high connectivity were identified;cellular retinoic acid binding protein 2(CRABP2),matrix metallopeptidase 12(MMP12),and DNA topoisomerase II alpha(TOP2A).High expression of these genes was associated with a poor LUAD prognosis,and the genes exhibited high diagnostic value.CONCLUSION Expression levels of CRABP2,MMP12,and TOP2A in LUAD were higher than those in normal lung tissue.This observation has diagnostic value,and is linked to poor LUAD prognosis.These genes may be biomarkers and therapeutic targets in LUAD,but further research is warranted to investigate their usefulness in these respects.

THE EXPRESSIONS OF HBV X GENE AND ets-2, IGF-Ⅰ, c-myc AND N-ras ONCOGENES IN HUMAN HEPATOCELLULAR CARCINOMA AND TUMOR-ADJACENT TISSUES

The expressions of HBV X gene and ets-2, IGF-I, c-myc and N-ras were studied in 7 pairs of human primary hepatocellular carcinoma (PHC) and tumor-adjacent tissues, using RNA hybridization and im-munoblot methods. The results showed that specific 17 and 28 kD HBV X gene products (HBxAg) were existed in a portion of PHC and tumor-adjacent tissues. The 17 kD HBxAg was detected in the sera of 3 patients who also had 17 kD HBxAg in their liver tissues. Multiple expressions of oncogenes such as ets-2, c-myc and N-ras were observed in PHC and tumor-adjacent tissues that had HBxAg expressed, indicating HBxAg might function as a transactivator in the course of intracellular proto-oncogene activation. It is also observed that in some tumor-adjacnet tissues the expressions of ets-2, c-myc and N-ras were higher than those in corresponding PHC. The relationship of HBxAg to the expression of est-2, IGF-Ⅱ, c-myc and their possible roles in the carcinogenesis of PHC are discussed.

Expression of Genes Associated with Nickel Resistance in White Spruce (<i>Picea glauca</i>) under Nickel Stress: Analysis of AT2G16800 and <i>NRAMP</i>Genes

Heavy metals such nickel (Ni) can cause toxicity by 1) displacing essential components in the biomolecules, 2) blocking the functional group of molecules, or 3) modifying enzymes, proteins, the plasma membrane, and membrane transporters. The main objective of the present study was to investigate the effect of nickel (Ni) on gene expression of nitrate on gene expression with a focus on the genes coding for the high affinity Ni transporter family protein AT2G16800, and natural resistance-associated macrophage protein (NRAMP). Ni toxicity was assessed by treating seedlings with an aqueous solution of nickel nitrate salt [Ni(NO3)2] at the concentrations of 150 mg, 800 mg, and 1600 mg of nickel per 1 kg of dry soil. RT-qPCR was used to measure the expression of AT2G16800, and NRAMP genes in samples treated with nickel nitrates and controls. The results revealed that P. glauca is resistant to Ni based on lack of plant damage at all nickel concentrations. Ni has no effect on the expression of the AT2G16800 gene in needles or roots. However, it induced an upregulation of the NRAMP genes in roots at all the doses tested (150 mg/kg, 800 mg/kg, and 1600 mg/kg). On the other hand, Ni has no effect on the expression of the NRAMP gene in needle but the lowest dose of potassium (150 mg/kg) upregulated this gene in needle tissues.

Cloning and Expression Analysis of <i>RrRUP</i>2 Gene Related to Photomorphogenesis Biosynthesis in <i>Rosa rugosa</i>

Plants have evolved and perfected a series of light receptors to feel the light at different bands and regulate the expression, modification and interaction of related genes in plants through signal transduction. So far, many photoreceptors have been identified in plants, UVR8 has recently been identified as a receptor for UV-B light. This paper cloned a WD40 gene related to UVR8 protein subunit, named RrRUP2, based on the Rosa rugose transcriptome data, using Rosa rugose “Zi zhi” as experimental materials. The full length of cDNA of the gene was obtained by RT-PCR and RACE methods. The total length of this gene is 1173 bp, and it encodes 390 amino acids. After bioinformatics analysis, the molecular formula C3415H5659N1173O1434S313 was predicted;the relative molecular weight was 96129.27 Da;the theoretical isoelectric point PI value was 5.00;and its instability index was 47.06. The total average hydrophobic index was 0.750. In the secondary structure of RrRUP2 protein, there are 10 α-helix, 45 β-helix, 181 Random coil, and 154 Extended strand. Gene Bank Blast results showed that the amino acid sequence encoded by RrRUP2 was more than 90% homologous with the RUP2 protein of Rosa chinensis, Fragaria, Malus, Pyrus, Prunus, Juglans, Arabidopsis and Tobacco, so it can be inferred that the RrRUP2 gene is a WD repeat-containing protein. Regarding to fluorescence quantitative expression analysis of RrRUP2, we find its experssion pattern is corresponded with the accumulation of anthocyanins.

Correlation research of Runt-related transcription factor 2 with proliferation genes, tumor suppressor genes and angiogenesis molecules in colon cancer lesions

Objective: To investigate the correlation of Runt-related transcription factor 2 (RunX2) with proliferation genes, tumor suppressor genes and angiogenesis molecules in colon cancer lesions. Methods: A total of 90 patients with primary colon cancer were enrolled in colon cancer group, 68 patients with benign colon polyps were enrolled in colon polyps group, the differences in the expression levels of RunX2, proliferation genes, tumor suppressor genes and angiogenesis molecules in the two groups of lesions were compared, and Pearson test was further used to evaluate the correlation of RunX2 expression level with proliferation gene, tumor suppressor gene and angiogenesis molecule expression levels in colon cancer tissues. Results: RunX2 mRNA expression level in the lesions of colon cancer group was higher than that of colon polyps group. Proliferation genes GTPBP4, HOXB7, ZNF331, ADAM17 and HSP60 mRNA expression levels in the lesions of colon cancer group were higher than those of colon polyps group;tumor suppressor genes ATF3, FOXN3, OTUD1 and NDRG2 mRNA expression levels were lower than those of colon polyps group;angiogenesis molecules Musashi 1, NF-κB, RegⅣ and STAT3 mRNA expression levels were higher than those of colon polyps group. RunX2 mRNA expression level in the colon cancer lesions was directly correlated with the expression levels of the above proliferation genes, tumor suppressor genes and angiogenesis molecules. Conclusion: RunX2 expression is abnormally high in colon cancer lesions, the specific expression level is positively correlated with cancer cell proliferation activity and angiogenesis activity, and it is an important molecular target that can lead to the occurrence and development of colon cancer.

The Role of HER2/Neu and BRCA1 Genes in the Diagnosis of Breast Cancer among Sudanese Women

Background: Knowledge of HER2/Neu and BRCA1 Genes might be helpful for development of strategies for decreasing the burden of risk of breast cancer. Therefore, the aim of this study to detect the role of HER2/Neu and BRCA1 Genes expression in diagnosis of breast cancer in Sudanese women. Methodology: A total of 100 tissue samples obtained from patients with breast cancer in addition to 50 tissue samples obtained from patients with benign breast lesions, were detected the expression of HER2/Neu and BRCA1 Genes by Polymerase Chain Reaction (PCR). Results: The prevalence of HER2/Neu and BRCA1 Genes, among cases was 6%, and 10% respectively. Conclusion: HER2/Neu and BRCA1 Genes have a considerable contribution to etiology of breast cancer in Sudan that requires further consideration.

Role of p53 suppression in the pathogenesis of hepatocellular carcinoma

In the world,hepatocellular carcinoma(HCC)is among the top 10 most prevalent malignancies.HCC formation has indeed been linked to numerous etiological factors,including alcohol usage,hepatitis viruses and liver cirrhosis.Among the most prevalent defects in a wide range of tumours,notably HCC,is the silencing of the p53 tumour suppressor gene.The control of the cell cycle and the preservation of gene function are both critically important functions of p53.In order to pinpoint the core mechanisms of HCC and find more efficient treatments,molecular research employing HCC tissues has been the main focus.Stimulated p53 triggers necessary reactions that achieve cell cycle arrest,genetic stability,DNA repair and the elimination of DNA-damaged cells’responses to biological stressors(like oncogenes or DNA damage).To the contrary hand,the oncogene protein of the murine double minute 2(MDM2)is a significant biological inhibitor of p53.MDM2 causes p53 protein degradation,which in turn adversely controls p53 function.Despite carrying wt-p53,the majority of HCCs show abnormalities in the p53-expressed apoptotic pathway.High p53 in-vivo expression might have two clinical impacts on HCC:(1)Increased levels of exogenous p53 protein cause tumour cells to undergo apoptosis by preventing cell growth through a number of biological pathways;and(2)Exogenous p53 makes HCC susceptible to various anticancer drugs.This review describes the functions and primary mechanisms of p53 in pathological mechanism,chemoresistance and therapeutic mechanisms of HCC.

Growth arrest-specific gene 2 suppresses hepatocarcinogenesis by intervention of cell cycle and p53-dependent apoptosis

BACKGROUND Growth arrest-specific gene 2(GAS2)plays a role in modulating in reversible growth arrest cell cycle,apoptosis,and cell survival.GAS2 protein is universally expressed in most normal tissues,particularly in the liver,but is depleted in some tumor tissues.However,the functional mechanisms of GAS2 in hepatocellular carcinoma(HCC)are not fully defined.AIM To investigate the function and mechanism of GAS2 in HCC.METHODS GAS2 expression in clinic liver and HCC specimens was analyzed by real-time PCR and western blotting.Cell proliferation was analyzed by counting,MTS,and colony formation assays.Cell cycle analysis was performed by flow cytometry.Cell apoptosis was investigated by Annexin V apoptosis assay and western blotting.RESULTS GAS2 protein expression was lower in HCC than in normal tissues.Overexpression of GAS2 inhibited the proliferation of HCC cells with wide-type p53,while knockdown of GAS2 promoted the proliferation of hepatocytes(P<0.05).Furthermore,GAS2 overexpression impeded the G1-to-S cell cycle transition and arrested more G1 cells,particularly the elevation of sub G1(P<0.01).Apoptosis induced by GAS2 was dependent on p53,which was increased by etoposide addition.The expression of p53 and apoptosis markers was further enhanced when GAS2 was upregulated,but became diminished upon downregulation of GAS2.In the clinic specimen,GAS2 was downregulated in more than 60%of HCCs.The average fold changes of GAS2 expression in tumor tissues were significantly lower than those in paired non-tumor tissues(P<0.05).CONCLUSION GAS2 plays a vital role in HCC cell proliferation and apoptosis,possibly by regulating the cell cycle and p53-dependent apoptosis pathway.

Development of highly glyphosate-tolerant tobacco by coexpression of glyphosate acetyltransferase gat and EPSPS G2-aroA genes

The widely used herbicide glyphosate targets 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS).Glyphosate acetyltransferase(GAT)effectively detoxifies glyphosate by N-acetylation.With the aim of identifying a new strategy for development of glyphosate-tolerant crops,the plant expression vector pG2-GAT harboring gat and G2-aroA(encoding EPSPS)has been transformed into tobacco(Nicotiana tabacum)to develop novel plants with higher tolerance to glyphosate.Results from Southern and Western blotting analyses indicated that the target genes were integrated into tobacco chromosomes and expressed effectively at the protein level.Glyphosate tolerance was compared among transgenic tobacco plants containing gat,G2-aroA,or both genes.Plants containing both gat and G2-aroA genes were the most glyphosate-tolerant.This study has shown that a combination of different strategies may result in higher tolerance in transgenic crops,providing a new approach for development of glyphosate-tolerant crops.

Genetic diversity of the msp-1,msp-2,and glurp genes of Plasmodium falciparum isolates along the Thai-Myanmar borders

Objective:To study the genetic diversity at the msp—1,msp—2,and glurp genes of Plasmodium falciparum(P.falciparum) isolates from 3 endemic areas in Thailand:Tak.Kanchanaburi and Ranong provinces.Methods:A total of 144 P.falciparum isolates collected prior to Irealmenl during January.2012 to June,2013 were genotyped.DNA was extracted:allele frequency and diversity of msp-1.msp-2,and glurp genes were investigated by nested polymerase chain reaction.Results:P.falciparum isolates in this study had high rate of multiple genotypes infection(96.5%)with an overall mean multiplicity of infection of 3.21.The distribution of allelic families of msp-1was significantly different among isolales from Tak.kanchanahnri.and Ranong but not for the msp-2.K1 and MAD20 were the predominant allelic families at the msp-1 gene,whereas alleles belonging to 3D7 were more frequent at the msp-2 gene.The glurp gene had the least diverse alleles.Population structure of P.falciparum isolates from Tak and Ranong was quite similar as revealed by the presence of similar proportions of MAD20 and K1 alleles at msp-1 loci.3D7 and FC27 alleles at msp-2 loci as well as comparable mean MOI.Isolates from Kanchanaburi had different structures;the most prevalent alleles were K1 and RO33.Conclusions:The present study shows that P.falciparum isolates from Tak and Ranong provinces had similar allelic pattern of msp—1 and msp—2 and diversity but different from Kanchanaburi isolates.These allelic variant profiles are valuable baseline data for future epidemiological study of malaria transmission and for continued monitoring of polymorphisms associated with antimalarial drug resistance in these areas.

Sequence and phylogenetic analysis of hemagglutinin genes of H9N2 influenza viruses isolated from chicken in China from 2013 to 2015

H9N2 avian influenza virus（AIV） infection is a major problem in poultry industry worldwide. In this study, molecular characterizations and phylogenetic relationships of hemagglutinin（HA） gene sequences of H9N2 AIV of 5 Chinese isolates in 2014 recently available in Gen Bank, 3 widely used vaccine strains, and 52 novel isolates in China from 2013 to 2015 were analyzed. The homology analysis showed that the nucleotide sequences of HA gene of these recent Chinese H9N2 AIV isolates shared homologies from 94.1 to 99.9%. Phylogenetic analysis showed that all isolates belonged to AIV lineage h9.4.2.5. Fifty-six out of the 57 recent Chinese H9N2 AIV isolates had the motifs PSRSSR↓GLF at the cleavage sites within the HA protein, while one isolate PWH01 harbored LSRSSR↓GLF. Remarkably, all of the recent Chinese H9N2 AIV strains had the Q216 L substitution in the receptor binding site, which indicated that they had potential to infect humans. Most of recent Chinese H9N2 AIV isolates lost the potential N-linked glycosylation site at residues 200–202 compared with vaccine strains. This present study demonstrated that AIV lineage h9.4.2.5 was more predominant in China than other lineages as it harbored all the H9N2 AIV isolated between 2013 and 2015. Also we showed the importance of continuous surveillance of emerging H9N2 AIV in China and update of vaccine formulation accordingly in order to prevent and control H9N2 AIV.

Functionality of a bicistronic construction containing HEXA and HEXB genes encoding β-hexosaminidase A for cell-mediated therapy of GM2 gangliosidoses

Tay-Sachs disease and Sandhoff disease are severe hereditary neurodegenerative disorders caused by a deficiency ofβ-hexosaminidase A(HexA)enzyme,which results in the accumulation of GM2 gangliosides in the nervous system cells.In this work,we analyzed the efficacy and safety of cell-mediated gene therapy for Sandhoff disease and Sandhoff disease using a bicistronic lentiviral vector encoding cDNA of HexAα-andβ-subunit genes separated by the nucleotide sequence of a P2A peptide(HEXA-HEXB).The functionality of the bicistronic construct containing the HEXA-HEXB genetic cassette was analyzed in a culture of HEK293T cells and human umbilical cord blood mononuclear cells(hUCBMCs).Our results showed that the enzymatic activity of HexA in the conditioned medium harvested from genetically modified HEK293T-HEXA-HEXB and hUCBMCs-HEXA-HEXB was increased by 23 and 8 times,respectively,compared with the conditioned medium of native cells.Western blot analysis showed that hUCBMCs-HEXA-HEXB secreted both completely separated HEXA and HEXB proteins,and an uncleaved protein containing HEXA+HEXB linked by the P2A peptide.Intravenous injection of genetically modified hUCBMCs-HEXA-HEXB to laboratory Wistar rats was carried out,and the HexA enzymatic activity in the blood plasma of experimental animals,as well as the number of live cells of immune system organs(spleen,thymus,bone marrow,lymph nodes)were determined.A significant increase in the enzymatic activity of HexA in the blood plasma of laboratory rats on days 6 and 9(by 2.5 and 3 times,respectively)after the administration of hUCBMCsHEXA-HEXB was shown.At the same time,the number of live cells in the studied organs remained unchanged.Thus,the functionality of the bicistronic genetic construct encoding cDNA of the HEXA and HEXB genes separated by the nucleotide sequence of the P2A peptide was shown in vitro and in vivo.We hypothesize that due to the natural ability of hUCBMCs to overcome biological barriers,such a strategy can restore the activity of the missing enzyme in the central nervous system of patients with GM2 gangliosidoses.Based on the obtained data,it can be concluded that intravenous administration of hUCBMCs with HexA overexpression is a promising method of the therapy for GM2 gangliosidoses.The animal protocol was approved by the Animal Ethics Committee of the Kazan Federal University(No.23)on June 30,2020.

Identification and Expression Analysis of Abscisic Acid Signal Transduction Genes in Hemp Seeds

Abscisic acid(ABA)is involved in regulating diverse biological processes,but its signal transduction genes and roles in hemp seed germination are not well known.Here,the ABA signaling pathway members,PYL,PP2C and SnRK2 gene families,were identified from the hemp reference genome,including 7 CsPYL(pyrab-actin resistance1-like,ABA receptor),8 CsPP2CA(group A protein phosphatase 2c),and 7 CsSnRK2(sucrose nonfermenting1-related protein kinase 2).The content of ABA in hemp seeds in germination stage is lower than that in non-germination stage.Exogenous ABA(1 or 10μM)treatment had a significant regulatory effect on the selected PYL,PP2C,SnRK2 gene families.CsAHG3 and CsHAI1 were most significantly affected by exogenous ABA treatment.Yeast two-hybrid experiments were performed to reveal that CsPYL5,CsSnRK2.2,and CsSnRK2.3 could interact with CsPP2CA7 and demonstrate that this interaction was ABA-independent.Our results indicated that CsPYL5,CsSnRK2.2,CsSnRK2.3 and CsPP2CA7 might involve in the ABA signaling transduction pathway of hemp seeds during the hemp seed germination stages.This study suggested that novel genetic views can be brought into investigation of ABA signaling pathway in hemp seeds and lay the foundation for further exploration of the mechanism of hemp seed germination.

Study of pathogenic genes in a pedigree with familial dilated cardiomyopathy

BACKGROUND Dilated cardiomyopathy(DCM)is a genetically heterogeneous cardiac disorder characterized by left ventricular dilation and contractile dysfunction.The substantial genetic heterogeneity evident in patients with DCM contributes to variable disease severity and complicates overall prognosis,which can be very poor.AIM To identify pathogenic genes in DCM through pedigree analysis.METHODS Our research team identified a patient with DCM in the clinic.Through invest-igation,we found that the family of this patient has a typical DCM pedigree.High-throughput sequencing technology,next-generation sequencing,was used to sequence the whole exomes of seven samples in the pedigree.RESULTS A novel and potentially pathogenic gene mutation-ANK2p.F3067L-was discovered.The mutation was completely consistent with the clinical information for this DCM pedigree.Sanger sequencing was used to further verify the locus of the mutation in pedigree samples.These results were consistent with those of high-throughput sequencing.CONCLUSIONS ANK2p.F3067L is considered a novel and potentially pathogenic gene mutation in DCM.

Liver expression of Nrf2-related genes in different liver diseases

BACKGROUND： The KEAP1-Nrf2 antioxidant signaling pathway is important in protecting liver from various insults. However,little is known about the expression of Nrf2-related genes in human liver in different diseases.METHODS： This study utilized normal donor liver tissues（n=35）, samples from patients with hepatocellular carcinoma（HCC, n=24）, HBV-related cirrhosis（n=27）, alcoholic cirrhosis（n=5） and end-stage liver disease（n=13）. All of the liver tissues were from the Oriental Liver Transplant Center, Beijing,China. The expressions of Nrf2 and Nrf2-related genes, including its negative regulator Kelch-like ECH-associated protein 1（KEAP1）, its targeted gene NAD（P）H-quinone oxidoreductase 1（NQO1）, glutamate-cysteine ligase catalytic subunit（GCLC） and modified subunit（GCLM）, heme oxygenase 1（HO-1） and peroxiredoxin-1（PRDX1） were evaluated. RESULTS： The expression of Nrf2 was decreased in HCC, increased in alcoholic cirrhosis and end-stage liver disease. The expression of KEAP1 was increased in all of the liver samples.The most notable finding was the increased expression of NQO1 in HCC（18-fold）, alcoholic cirrhosis（6-fold）, endstage liver disease（5-fold） and HBV-related cirrhosis（3-fold）.Peri-HCC also had 4-fold higher NQO1 m RNA as compared to the normal livers. GCLC m RNA levels were lower only in HCC, as compared to the normal livers and peri-HCC tissues.GCLM m RNA levels were higher in HBV-related cirrhosis and end-stage liver disease. HO-1 m RNA levels were increased in all liver tissues except for HCC. Peri-HCC had higher PRDX1 m RNA levels compared with HCC and normal livers.CONCLUSION： Nrf2 and Nrf2-related genes are aberrantly expressed in the liver in different diseases and the increase of NQO1 was the most notable finding, especially in HCC.