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International Journal of Molecular... May 2024In aquaculture, viral diseases pose a significant threat and can lead to substantial economic losses. The primary defense against viral invasion is the innate immune...
In aquaculture, viral diseases pose a significant threat and can lead to substantial economic losses. The primary defense against viral invasion is the innate immune system, with interferons (IFNs) playing a crucial role in mediating the immune response. With advancements in molecular biology, the role of non-coding RNA (ncRNA), particularly microRNAs (miRNAs), in gene expression has gained increasing attention. While the function of miRNAs in regulating the host immune response has been extensively studied, research on their immunomodulatory effects in teleost fish, including silver carp (), is limited. Therefore, this research aimed to investigate the immunomodulatory role of microRNA-30b-5p (miR-30b-5p) in the antiviral immune response of silver carp () by targeting cytokine receptor family B5 (CRFB5) via the JAK/STAT signaling pathway. In this study, silver carp were stimulated with polyinosinic-polycytidylic acid (poly (I:C)), resulting in the identification of an up-regulated miRNA (miR-30b-5p). Through a dual luciferase assay, it was demonstrated that CRFB5, a receptor shared by fish type I interferon, is a novel target of miR-30b-5p. Furthermore, it was found that miR-30b-5p can suppress post-transcriptional CRFB5 expression. Importantly, this study revealed for the first time that miR-30b-5p negatively regulates the JAK/STAT signaling pathway, thereby mediating the antiviral immune response in silver carp by targeting CRFB5 and maintaining immune system stability. These findings not only contribute to the understanding of how miRNAs act as negative feedback regulators in teleost fish antiviral immunity but also suggest their potential therapeutic measures to prevent an excessive immune response.
Topics: Animals; MicroRNAs; Carps; Poly I-C; Signal Transduction; Janus Kinases; STAT Transcription Factors; Fish Proteins; Fish Diseases; Immunity, Innate; Gene Expression Regulation
PubMed: 38891899
DOI: 10.3390/ijms25115712 -
International Journal of Molecular... May 2024Dysferlin is a large transmembrane protein involved in critical cellular processes including membrane repair and vesicle fusion. Mutations in the dysferlin gene () can... (Review)
Review
Dysferlin is a large transmembrane protein involved in critical cellular processes including membrane repair and vesicle fusion. Mutations in the dysferlin gene () can result in rare forms of muscular dystrophy; Miyoshi myopathy; limb girdle muscular dystrophy type 2B (LGMD2B); and distal myopathy. These conditions are collectively known as dysferlinopathies and are caused by more than 600 mutations that have been identified across the gene to date. In this review, we discuss the key molecular and clinical features of LGMD2B, the causative gene , and the associated dysferlin protein structure. We also provide an update on current approaches to LGMD2B diagnosis and advances in drug development, including splice switching antisense oligonucleotides. We give a brief update on clinical trials involving adeno-associated viral gene therapy and the current progress on CRISPR/Cas9 mediated therapy for LGMD2B, and then conclude by discussing the prospects of antisense oligomer-based intervention to treat selected mutations causing dysferlinopathies.
Topics: Humans; Muscular Dystrophies, Limb-Girdle; Dysferlin; Genetic Therapy; Mutation; Oligonucleotides, Antisense; Animals
PubMed: 38891760
DOI: 10.3390/ijms25115572 -
Cells May 2024Respiratory viruses cause airway inflammation, resulting in epithelial injury and repair. miRNAs, including miR-149-5p, regulate different pathological conditions. We...
Respiratory viruses cause airway inflammation, resulting in epithelial injury and repair. miRNAs, including miR-149-5p, regulate different pathological conditions. We aimed to determine how miR-149-5p functions in regulating pro-inflammatory IL-6 and p63, key regulators of airway epithelial wound repair, in response to viral proteins in bronchial (BEAS-2B) and alveolar (A549) epithelial cells. BEAS-2B or A549 cells were incubated with poly (I:C, 0.5 µg/mL) for 48 h or SARS-CoV-2 spike protein-1 or 2 subunit (S1 or S2, 1 μg/mL) for 24 h. miR-149-5p was suppressed in BEAS-2B challenged with poly (I:C), correlating with IL-6 and p63 upregulation. miR-149-5p was down-regulated in A549 stimulated with poly (I:C); IL-6 expression increased, but p63 protein levels were undetectable. miR-149-5p remained unchanged in cells exposed to S1 or S2, while S1 transfection increased IL-6 expression in BEAS-2B cells. Ectopic over-expression of miR-149-5p in BEAS-2B cells suppressed and mRNA levels and inhibited poly (I:C)-induced and mRNA expressions. miR-149-5p directly suppressed mRNA in BEAS-2B cells. Hence, BEAS-2B cells respond differently to poly (I:C), S1 or S2 compared to A549 cells. Thus, miR-149-5p dysregulation may be involved in poly (I:C)-stimulated but not S1- or S2-stimulated increased IL-6 production and p63 expression in BEAS-2B cells.
Topics: Humans; MicroRNAs; Interleukin-6; A549 Cells; Epithelial Cells; Poly I-C; SARS-CoV-2; COVID-19; Tumor Suppressor Proteins; Transcription Factors; Gene Expression Regulation
PubMed: 38891051
DOI: 10.3390/cells13110919 -
Applied Microbiology and Biotechnology Jun 2024Mushroom poisoning contributes significantly to global foodborne diseases and related fatalities. Amanita mushrooms frequently cause such poisonings; however,...
Mushroom poisoning contributes significantly to global foodborne diseases and related fatalities. Amanita mushrooms frequently cause such poisonings; however, identifying these toxic species is challenging due to the unavailability of fresh and intact samples. It is often necessary to analyze residues, vomitus, or stomach extracts to obtain DNA sequences for the identification of species responsible for causing food poisoning. This usually proves challenging to obtain usable DNA sequences that can be analyzed using conventional molecular biology techniques. Therefore, this study aimed to develop a DNA mini-barcoding method for the identification of Amanita species. Following the evaluation and optimization of universal primers for DNA mini-barcoding in Amanita mushrooms, we found that the internal transcribed spacer (ITS) gene sequence primer ITS-a was the most suitable DNA barcode primer for identifying Amanita species. Forty-three Amanita samples were subsequently amplified and sequenced. The sequences obtained were analyzed for intra- and inter-species genetic distances, and a phylogenetic tree was constructed. The findings indicated that the designed primers had strong universality among the Amanita samples and could accurately identify the target gene fragment with a length of 290 bp. Notably, the DNA mini-barcode accurately identified the 43 Amanita samples, demonstrating high consistency with the conventional DNA barcode. Furthermore, it effectively identified DNA from digested samples. In summary, this DNA mini-barcode is a promising tool for detecting accidental ingestion of toxic Amanita mushrooms. It may be used as an optimal barcode for species identification and traceability in events of Amanita-induced mushroom poisoning. KEY POINTS: • Development of a DNA mini-barcoding method for Amanita species identification without fresh samples. • The ITS-a primer set was optimized for robust universality in Amanita samples. • The mini-barcode is suitable for screening toxic mushroom species in mushroom poisoning cases.
Topics: Mushroom Poisoning; Amanita; DNA Barcoding, Taxonomic; Phylogeny; DNA, Fungal; DNA Primers; DNA, Ribosomal Spacer; Sequence Analysis, DNA; Humans
PubMed: 38884656
DOI: 10.1007/s00253-024-13219-x -
Experimental Biology and Medicine... 2024Rheumatoid fibroblast-like synoviocytes (RFLS) have an important role in the inflammatory pathogenesis of rheumatoid arthritis (RA). Toll-like receptor 3 (TLR3) is...
Rheumatoid fibroblast-like synoviocytes (RFLS) have an important role in the inflammatory pathogenesis of rheumatoid arthritis (RA). Toll-like receptor 3 (TLR3) is upregulated in RFLS; its activation leads to the production of interferon-β (IFN-β), a type I IFN. IFN-stimulated gene 56 (ISG56) is induced by IFN and is involved in innate immune responses; however, its role in RA remains unknown. Therefore, the purpose of this study was to investigate the role of TLR3-induced ISG56 in human RFLS. RFLS were treated with polyinosinic-polycytidylic acid (poly I:C), which served as a TLR3 ligand. ISG56, melanoma differentiation-associated gene 5 (MDA5), and C-X-C motif chemokine ligand 10 (CXCL10) expression were measured using quantitative reverse transcription-polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay. Using immunohistochemistry, we found that ISG56 was expressed in synovial tissues of patients with RA and osteoarthritis. Under poly I:C treatment, ISG56 was upregulated in RFLS. In addition, we found that the type I IFN-neutralizing antibody mixture suppressed ISG56 expression. ISG56 knockdown decreased CXCL10 expression and MDA5 knockdown decreased ISG56 expression. In addition, we found that ISG56 was strongly expressed in the synovial cells of patients with RA. TLR3 signaling induced ISG56 expression in RFLS and type I IFN was involved in ISG56 expression. ISG56 was also found to be associated with CXCL10 expression, suggesting that ISG56 may be involved in TLR3/type I IFN/CXCL10 axis, and play a role in RA synovial inflammation.
Topics: Humans; Toll-Like Receptor 3; Arthritis, Rheumatoid; Poly I-C; Signal Transduction; Synoviocytes; Chemokine CXCL10; Interferon-Induced Helicase, IFIH1; Cells, Cultured; Synovial Membrane; Adaptor Proteins, Vesicular Transport; RNA-Binding Proteins; Adaptor Proteins, Signal Transducing; Apoptosis Regulatory Proteins
PubMed: 38881847
DOI: 10.3389/ebm.2024.10122 -
Journal of Microbiology and... Jun 2024The accurate and rapid detection of methicillin-resistant (MRSA) holds significant clinical importance. This work presents a new method for detecting...
The accurate and rapid detection of methicillin-resistant (MRSA) holds significant clinical importance. This work presents a new method for detecting methicillin-resistant () in clinical samples. The method uses an aptamer-based colorimetric assay that combines a recognizing probe to identify the target and split DNAzyme to amplify the signal, resulting in a highly sensitive and direct analysis of methicillin-resistance. The identification of the PBP2a protein on the membrane of in clinical samples leads to the allosterism of the recognizing probe, and thus provides a template for the proximity ligation of split DNAzyme. The proximity ligation of split DNAzyme forms an intact DNAzyme to identify the loop section in the L probe and generates a nicking site to release the loop sequence ("3" and "4" fragments). The "3" and "4" fragments forms an intact sequence to induce the catalytic hairpin assembly, exposing the G-rich section. The released the G-rich sequence of LR probe induces the formation of G-quadruplex-hemin DNAzyme as a colorimetric signal readout. The absorption intensity demonstrated a strong linear association with the logarithm of the concentration across a wide range of 5 orders of magnitude dynamic range under the optimized experimental parameters. The limit of detection was calculated to be 23 CFU/ml and the method showed high selectivity for MRSA.
Topics: DNA, Catalytic; Colorimetry; Methicillin-Resistant Staphylococcus aureus; Aptamers, Nucleotide; Staphylococcal Infections; Humans; Biosensing Techniques; Bacterial Proteins; Staphylococcus aureus; Sensitivity and Specificity; Methicillin Resistance; Penicillin-Binding Proteins
PubMed: 38881169
DOI: 10.4014/jmb.2404.04012 -
Parasites & Vectors Jun 2024The Anopheles dirus complex plays a significant role as a malaria vector in the Greater Mekong Subregion (GMS), with varying degrees of vector competence among species....
BACKGROUND
The Anopheles dirus complex plays a significant role as a malaria vector in the Greater Mekong Subregion (GMS), with varying degrees of vector competence among species. Accurate identification of sibling species in this complex is essential for understanding malaria transmission dynamics and deploying effective vector control measures. However, the original molecular identification assay, Dirus allele-specific polymerase chain reaction (AS-PCR), targeting the ITS2 region, has pronounced nonspecific amplifications leading to ambiguous results and misidentification of the sibling species. This study investigates the underlying causes of these inconsistencies and develops new primers to accurately identify species within the Anopheles dirus complex.
METHODS
The AS-PCR reaction and thermal cycling conditions were modified to improve specificity for An. dirus member species identification. In silico analyses with Benchling and Primer-BLAST were conducted to identify problematic primers and design a new set for Dirus complex species identification PCR (DiCSIP). DiCSIP was then validated with laboratory and field samples of the An. dirus complex.
RESULTS
Despite several optimizations by reducing primer concentration, decreasing thermal cycling time, and increasing annealing temperature, the Dirus AS-PCR continued to produce inaccurate identifications for Anopheles dirus, Anopheles scanloni, and Anopheles nemophilous. Subsequently, in silico analyses pinpointed problematic primers with high Guanine-Cytosine (GC) content and multiple off-target binding sites. Through a series of in silico analyses and laboratory validation, a new set of primers for Dirus complex species identification PCR (DiCSIP) has been developed. DiCSIP primers improve specificity, operational range, and sensitivity to identify five complex member species in the GMS accurately. Validation with laboratory and field An. dirus complex specimens demonstrated that DiCSIP could correctly identify all samples while the original Dirus AS-PCR misidentified An. dirus as other species when used with different thermocyclers.
CONCLUSIONS
The DiCSIP assay offers a significant improvement in An. dirus complex identification, addressing challenges in specificity and efficiency of the previous ITS2-based assay. This new primer set provides a valuable tool for accurate entomological surveys, supporting effective vector control strategies to reduce transmission and prevent malaria re-introducing in the GMS.
Topics: Anopheles; Animals; Polymerase Chain Reaction; DNA Primers; Mosquito Vectors; Malaria; Asia, Southeastern; Sensitivity and Specificity
PubMed: 38880909
DOI: 10.1186/s13071-024-06321-6 -
Journal of Medical Case Reports Jun 2024Homozygous mutations in the APOA5 gene constitute a rare cause of monogenic hypertriglyceridemia, or familial chylomicronemia syndrome (FCS). We searched PubMed and...
BACKGROUND
Homozygous mutations in the APOA5 gene constitute a rare cause of monogenic hypertriglyceridemia, or familial chylomicronemia syndrome (FCS). We searched PubMed and identified 16 cases of homozygous mutations in the APOA5 gene. Severe hypertriglyceridemia related to monogenic mutations in triglyceride-regulating genes can cause recurrent acute pancreatitis. Standard therapeutic approaches for managing this condition typically include dietary interventions, fibrates, and omega-3-fatty acids. A novel therapeutic approach, antisense oligonucleotide volanesorsen is approved for use in patients with FCS.
CASE PRESENTATION
We report a case of a 25-years old Afghani male presenting with acute pancreatitis due to severe hypertriglyceridemia up to 29.8 mmol/L caused by homozygosity in APOA5 (c.427delC, p.Arg143Alafs*57). A low-fat diet enriched with medium-chain TG (MCT) oil and fibrate therapy did not prevent recurrent relapses, and volanesorsen was initiated. Volanesorsen resulted in almost normalized triglyceride levels. No further relapses of acute pancreatitis occurred. Patient reported an improve life quality due to alleviated chronic abdominal pain and headaches.
CONCLUSIONS
Our case reports a rare yet potentially life-threatening condition-monogenic hypertriglyceridemia-induced acute pancreatitis. The implementation of the antisense drug volanesorsen resulted in improved triglyceride levels, alleviated symptoms, and enhanced the quality of life.
Topics: Humans; Male; Adult; Pancreatitis; Apolipoprotein A-V; Hypertriglyceridemia; Recurrence; Homozygote; Mutation; Oligonucleotides; Hyperlipoproteinemia Type I; Diet, Fat-Restricted; Triglycerides
PubMed: 38872171
DOI: 10.1186/s13256-024-04532-0 -
PloS One 2024Quantitative transcription regulation studies in vivo and in vitro often make use of reporter proteins. Here we show that using Broccoli aptamers, quantitative study of...
Quantitative transcription regulation studies in vivo and in vitro often make use of reporter proteins. Here we show that using Broccoli aptamers, quantitative study of transcription in various regulatory scenarios is possible without a translational step. To explore the method we studied several regulatory scenarios that we analyzed using thermodynamic occupancy-based models, and found excellent agreement with previous studies. In the next step we show that non-coding DNA can have a dramatic effect on the level of transcription, similar to the influence of the lac repressor with a strong affinity to operator sites. Finally, we point out the limitations of the method in terms of delay times coupled to the folding of the aptamer. We conclude that the Broccoli aptamer is suitable for quantitative transcription measurements.
Topics: Aptamers, Nucleotide; Transcription, Genetic; Brassica; Thermodynamics; Gene Expression Regulation
PubMed: 38870160
DOI: 10.1371/journal.pone.0304677 -
BMC Genomics Jun 2024Reverse transcription quantitative PCR (RT-qPCR) with intercalating dyes is one of the main techniques to assess gene expression levels used in basic and applied...
BACKGROUND
Reverse transcription quantitative PCR (RT-qPCR) with intercalating dyes is one of the main techniques to assess gene expression levels used in basic and applied research as well as in diagnostics. However, primer design for RT-qPCR can be complex due to the high demands on primer quality. Primers are best placed on exon junctions, should avoid polymorphic regions, be specific to the target transcripts and also prevent genomic amplification accurately, among others. Current software tools manage to meet all the necessary criteria only insufficiently. Here, we present ExonSurfer, a novel, user-friendly web-tool for qPCR primer design.
RESULTS
ExonSurfer combines the different steps of the primer design process, encompassing target selection, specificity and self-complementarity assessment, and the avoidance of issues arising from polymorphisms. Amplification of potentially contaminating genomic DNA is avoided by designing primers on exon-exon junctions, moreover, a genomic alignment is performed to filter the primers accordingly and inform the user of any predicted interaction. In order to test the whole performance of the application, we designed primer pairs for 26 targets and checked both primer efficiency, amplicon melting temperature and length and confirmed the targeted amplicon by Sanger sequencing. Most of the tested primers accurately and selectively amplified the corresponding targets.
CONCLUSION
ExonSurfer offers a comprehensive end-to-end primer design, guaranteeing transcript-specific amplification. The user interface is intuitive, providing essential specificity and amplicon details. The tool can also be used by command line and the source code is available. Overall, we expect ExonSurfer to facilitate RT-qPCR set-up for researchers in many fields.
Topics: Exons; Software; DNA Primers; Internet; Humans; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 38867172
DOI: 10.1186/s12864-024-10456-2