• Genomics & Informatics
• /
• 제20권3호
• /
• pp.30.1-30.9
• /
• 2022

Hereditary spastic paraplegia is a not common inherited neurological disorder with heterogeneous clinical expressions. ALDH18A1 (located on 10q24.1) gene-related spastic paraplegias (SPG9A and SPG9B) are rare metabolic disorders caused by dominant and recessive mutations that have been found recently. Autosomal recessive hereditary spastic paraplegia is a common and clinical type of familial spastic paraplegia linked to the SPG11 locus (locates on 15q21.1). There are different symptoms of spastic paraplegia, such as muscle atrophy, moderate mental retardation, short stature, balance problem, and lower limb weakness. Our first proband involves a 45 years old man and our second proband involves a 20 years old woman both are affected by spastic paraplegia disease. Genomic DNA was extracted from the peripheral blood of the patients, their parents, and their siblings using a filter-based methodology and quantified and used for molecular analysis and sequencing. Sequencing libraries were generated using Agilent SureSelect Human All ExonV7 kit, and the qualified libraries are fed into NovaSeq 6000 Illumina sequencers. Sanger sequencing was performed by an ABI prism 3730 sequencer. Here, for the first time, we report two cases, the first one which contains likely pathogenic NM_002860: c.475C>T: p.R159X mutation of the ALDH18A1 and the second one has likely pathogenic NM_001160227.2: c.5454dupA: p.Glu1819Argfs Ter11 mutation of the SPG11 gene and also was identified by the whole-exome sequencing and confirmed by Sanger sequencing. Our aim with this study was to confirm that these two novel variants are direct causes of spastic paraplegia.

• Genomics & Informatics
• /
• 제18권4호
• /
• pp.40.1-40.8
• /
• 2020

Avian influenza (AIV) outbreaks can induce fatal human pulmonary infections in addition to economic losses to the poultry industry. In this study, we aimed to develop a rapid and sensitive point-of-care AIV test using loop-mediated isothermal amplification (LAMP) technology. We designed three sets of reverse transcription LAMP (RT-LAMP) primers targeting the matrix (M) and hemagglutinin (HA) genes of the H5 and H9 subtypes. RT-LAMP targeting the universal M gene was designed to screen for the presence of AIV and RT-LAMP assays targeting H5-HA and H9-HA were designed to discriminate between the H5 and H9 subtypes. All three RT-LAMP assays showed specific amplification results without nonspecific reactions. In terms of sensitivity, the detection limits of our RT-LAMP assays were 100 to 1,000 RNA copies per reaction, which were 10 times more sensitive than the detection limits of the reference reverse-transcription polymerase chain reaction (RT-PCR) (1,000 to 10,000 RNA copies per reaction). The reaction time of our RT-LAMP assays was less than 30 min, which was approximately four times quicker than that of conventional RT-PCR. Altogether, these assays successfully detected the existence of AIV and discriminated between the H5 or H9 subtypes with higher sensitivity and less time than the conventional RT-PCR assay.

• Journal of Veterinary Science
• /
• 제21권4호
• /
• pp.56.1-56.13
• /
• 2020

Background: The live bird market (LBM) plays an important role in the dynamic evolution of the avian influenza H5N1 virus. Objectives: The main objective of this study was to monitor the genetic diversity of the H5N1 viruses in LBMs in Indonesia. Methods: Therefore, the disease surveillance was conducted in the area of Banten, West Java, Central Java, East Java, and Jakarta Province, Indonesia from 2014 to 2019. Subsequently, the genetic characterization of the H5N1 viruses was performed by sequencing all 8 segments of the viral genome. Results: As a result, the H5N1 viruses were detected in most of LBMs in both bird' cloacal and environmental samples, in which about 35% of all samples were positive for influenza A and, subsequently, about 52% of these samples were positive for H5 subtyping. Based on the genetic analyses of 14 viruses isolated from LBMs, genetic diversities of the H5N1 viruses were identified including clades 2.1.3 and 2.3.2 as typical predominant groups as well as reassortant viruses between these 2 clades. Conclusions: As a consequence, zoonotic transmission to humans in the market could be occurred from the exposure of infected birds and/or contaminated environments. Moreover, new virus variants could emerge from the LBM environment. Therefore, improving pandemic preparedness raised great concerns related to the zoonotic aspect of new influenza variants because of its high adaptivity and efficiency for human infection.

• BMB Reports
• /
• 제55권10호
• /
• pp.488-493
• /
• 2022

The specific pair of heat shock protein 70 (Hsp70) and Hsp40 constitutes an essential molecular chaperone system involved in numerous cellular processes, including the proper folding/refolding and transport of proteins. Hsp40 family members are characterized by the presence of a conserved J-domain (JD) that functions as a co-chaperone of Hsp70. Tumorous imaginal disc 1 (Tid1) is a tumor suppressor protein belonging to the DNAJA3 subfamily of Hsp40 and functions as a co-chaperone of the mitochondrial Hsp70, mortalin. In this work, we performed nuclear magnetic resonance spectroscopy to determine the solution structure of JD and its interaction with the glycine/phenylalanine-rich region (GF-motif) of human Tid1. Notably, Tid1-JD, whose conformation was consistent with that of the DNAJB1 JD, appeared to stably interact with its subsequent GF-motif region. Collectively with our sequence analysis, the present results demonstrate that the functional and regulatory mode of Tid1 resembles that of the DNAJB1 subfamily members rather than DNAJA1 or DNAJA2 subfamily proteins. Therefore, it is suggested that an allosteric interaction between mortalin and Tid1 is involved in the mitochondrial Hsp70/Hsp40 chaperone system.

• Journal of Microbiology and Biotechnology
• /
• 제32권11호
• /
• pp.1416-1426
• /
• 2022

The need to discover new types of antimicrobial agents has grown since the emergence of antibiotic-resistant pathogens that threaten human health. The world's oceans, comprising complex niches of biodiversity, are a promising environment from which to extract new antibiotics-like compounds. In this study, we newly isolated Pseudomonas sp. NIBR-H-19 from the gut of the sea roach Ligia exotica and present both phenotypes and genomic information consisting of 6,184,379 bp in a single chromosome possessing a total of 5,644 protein-coding genes. Genomic analysis of the isolated species revealed that numerous genes involved in antimicrobial secondary metabolites are predicted throughout the whole genome. Moreover, our analysis showed that among twenty-five pathogenic bacteria, the growth of three pathogens, including Staphylococcus aureus, Streptococcus hominis and Rhodococcus equi, was significantly inhibited by the culture of Pseudomonas sp. NIBR-H-19. The characterization of marine microorganisms with biochemical assays and genomics tools will help uncover the biosynthesis and action mechanism of antimicrobial metabolites for development as antagonistic probiotics against fish pathogens in an aquatic culture system.

• Clinical and Experimental Reproductive Medicine
• /
• 제50권3호
• /
• pp.177-184
• /
• 2023

Objective: Reconstructed oocytes after polar body genome transfer constitute a potential therapeutic option for patients with a history of embryo fragmentation and advanced maternal age. However, the rescue of genetic material from the first polar body (PB1) through introduction into the donor cytoplasm is not yet ready for clinical application. Methods: Eighty-five oocytes were obtained following in vitro maturation (IVM) and divided into two groups: PB1 nuclear transfer (PB1NT; n=54) and control (n=31). Following enucleation and PB1 genomic transfer, PB1 fusion was assessed. Subsequently, all fused oocytes underwent intracytoplasmic sperm injection (ICSI) and were cultured in an incubator under a time-lapse monitoring system to evaluate fertilization, embryonic morphokinetic parameters, and cleavage patterns. Results: Following enucleation and fusion, 77.14% of oocytes survived, and 92.59% of polar bodies (PBs) fused. However, the normal fertilization rate was lower in the PB1NT group than in the control group (56.41% vs. 92%, p=0.002). No significant differences were observed in embryo kinetics between the groups, but a significant difference was detected in embryo developmental arrest after the four-cell stage, along with abnormal cleavage division in the PB1NT group. This was followed by significant between-group differences in the implantation potential rate and euploidy status. Most embryos in the PB1NT group had at least one abnormal cleavage division (93.3%, p=0.001). Conclusion: Fresh PB1NT oocytes successfully produced normal zygotes following PB fusion and ICSI in IVM oocytes. However, this was accompanied by low efficiency in developing into cleavage embryos, along with an increase in abnormal cleavage patterns.

• Genomics & Informatics
• /
• 제21권1호
• /
• pp.7.1-7.11
• /
• 2023

The gram-positive bacterium Listeria monocytogenes is an important foodborne intracellular pathogen that is widespread in the environment. The functions of hypothetical proteins (HP) from various pathogenic bacteria have been successfully annotated using a variety of bioinformatics strategies. In this study, a HP Imo0888 (NP_464414.1) from the Listeria monocytogenes EGD-e strain was annotated using several bioinformatics tools. Various techniques, including CELLO, PSORTb, and SOSUIGramN, identified the candidate protein as cytoplasmic. Domain and motif analysis revealed that the target protein is a PemK/MazF-like toxin protein of the type II toxin-antitoxin system (TAS) which was consistent with BLASTp analysis. Through secondary structure analysis, we found the random coil to be the most frequent. The Alpha Fold 2 Protein Structure Prediction Database was used to determine the three-dimensional (3D) structure of the HP using the template structure of a type II TAS PemK/MazF family toxin protein (DB ID_AFDB: A0A4B9HQB9) with 99.1% sequence identity. Various quality evaluation tools, such as PROCHECK, ERRAT, Verify 3D, and QMEAN were used to validate the 3D structure. Following the YASARA energy minimization method, the target protein's 3D structure became more stable. The active site of the developed 3D structure was determined by the CASTp server. Most pathogens that harbor TAS create a crucial risk to human health. Our aim to annotate the HP Imo088 found in Listeria could offer a chance to understand bacterial pathogenicity and identify a number of potential targets for drug development.

• Journal of Microbiology and Biotechnology
• /
• 제33권9호
• /
• pp.1228-1237
• /
• 2023

The CRISPR-Cas system has emerged as the most efficient genome editing technique for a wide range of cells. Delivery of the Cas9-sgRNA ribonucleoprotein complex (Cas9 RNP) has gained popularity. The objective of this study was to develop a quantitative polymerase chain reaction (qPCR)-based assay to quantify the double-strand break reaction mediated by Cas9 RNP. To accomplish this, the dextransucrase gene (dsr) from Leuconostoc citreum was selected as the target DNA. The Cas9 protein was produced using recombinant Escherichia coli BL21, and two sgRNAs were synthesized through in vitro transcription to facilitate binding with the dsr gene. Under optimized in vitro conditions, the 2.6 kb dsr DNA was specifically cleaved into 1.1 and 1.5 kb fragments by both Cas9-sgRNA365 and Cas9-sgRNA433. By monitoring changes in dsr concentration using qPCR, the endonuclease activities of the two Cas9 RNPs were measured, and their efficiencies were compared. Specifically, the specific activities of dsr365RNP and dsr433RNP were 28.74 and 34.48 (unit/㎍ RNP), respectively. The versatility of this method was also verified using different target genes, uracil phosphoribosyl transferase (upp) gene, of Bifidobacterium bifidum and specific sgRNAs. The assay method was also utilized to determine the impact of high electrical field on Cas9 RNP activity during an efficient electroporation process. Overall, the results demonstrated that the qPCR-based method is an effective tool for measuring the endonuclease activity of Cas9 RNP.

• Genomics & Informatics
• /
• 제21권3호
• /
• pp.38.1-38.8
• /
• 2023

Non-small cell lung cancer (NSCLC) is an important cause of cancer-associated deaths worldwide. Therefore, the exact molecular mechanisms of NSCLC are unidentified. The present investigation aims to identify the miRNAs with predictive value in NSCLC. The two datasets were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed miRNAs (DEmiRNA) and mRNAs (DEmRNA) were selected from the normalized data. Next, miRNA-mRNA interactions were determined. Then, co-expression network analysis was completed using the WGCNA package in R software. The co-expression network between DEmiRNAs and DEmRNAs was calculated to prioritize the miRNAs. Next, the enrichment analysis was performed for DEmiRNA and DEmRNA. Finally, the drug-gene interaction network was constructed by importing the gene list to dgidb database. A total of 3,033 differentially expressed genes and 58 DEmiRNA were recognized from two datasets. The co-expression network analysis was utilized to build a gene co- expression network. Next, four modules were selected based on the Z_summary score. In the next step, a bipartite miRNA-gene network was constructed and hub miRNAs (let-7a-2-3p, let-7d-5p, let-7b-5p, let-7a-5p, and let-7b-3p) were selected. Finally, a drug-gene network was constructed while SUNITINIB, MEDROXYPROGESTERONE ACETATE, DOFETILIDE, HALOPERIDOL, and CALCITRIOL drugs were recognized as a beneficial drug in NSCLC. The hub miRNAs and repurposed drugs may act a vital role in NSCLC progression and treatment, respectively; however, these results must validate in further clinical and experimental assessments.