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F1000Research 2022Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic disorders caused by mutations in the gene. The full mutation spectrum of the gene in...
BACKGROUND
Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic disorders caused by mutations in the gene. The full mutation spectrum of the gene in Indonesian patients is currently unknown. Mutation-specific therapies are currently being developed, such as exon skipping or stop codon read-through therapy. This study was conducted with the aim of identifying the mutation spectrum of the gene in Indonesia to guide future development and application of feasible therapeutic strategies.
METHODS
This study is a cross sectional study that enrolled 43 male patients with a clinical suspicion of or BMD. Multiplex ligation-dependent probe amplification (MLPA) reaction was performed to screen for the common mutations in the gene.
RESULTS
Out of 43 subjects, deletions accounted for 69.77% (n=30) cases, while duplications were found in 11.63% (n=5) cases. One novel duplication spanning exons 2 to 62 was identified. Deletion mutations clustered around the distal (66.67%) and proximal (26.67%) hot spot regions of the gene while duplication mutations were observed solely at the proximal region. Two false positive cases of single exon deletion detected through MLPA were attributed to sequence mutations affecting primer ligation sites, confirming the need to validate all single exon deletions when using this screening method. Analysis of available maternal DNA samples showed that the rate of de novo mutations (48.15%) appears higher than expected in this population. Out of 31 patients who were classified as DMD based on clinical and genotype characterizations, 60.47% (n=26) of cases were suitable for exon skipping therapy.
CONCLUSION
This is the first comprehensive study showing the feasibility of implementing the MLPA method for routine screening of patients in Indonesia. This is also the first study showing the potential applicability of exon skipping therapy in the majority of cases in the country.
Topics: Humans; Male; Cross-Sectional Studies; Dystrophin; Gene Deletion; Indonesia; Muscular Dystrophy, Duchenne; Mutation
PubMed: 38009102
DOI: 10.12688/f1000research.73476.3 -
Cellular and Molecular Life Sciences :... Oct 2023Deep mutational scanning (DMS) makes it possible to perform massively parallel quantification of the relationship between genetic variants and phenotypes of interest....
Deep mutational scanning (DMS) makes it possible to perform massively parallel quantification of the relationship between genetic variants and phenotypes of interest. However, the difficulties in introducing large variant libraries into mammalian cells greatly hinder DMS under physiological states. Here, we developed two novel strategies for DMS library construction in mammalian cells, namely 'piggyBac-in vitro ligation' and 'piggyBac-in vitro ligation-PCR'. For the first strategy, we took the 'in vitro ligation' approach to prepare high-diversity linear dsDNAs, and integrate them into the mammalian genome with a piggyBac transposon system. For the second strategy, we further added a PCR step using the in vitro ligation dsDNAs as templates, for the construction of high-content genome-integrated libraries via large-scale transfection. Both strategies could successfully establish genome-integrated EGFP-chromophore-randomized libraries in HEK293T cells and enrich the green fluorescence-chromophore amino-acid sequences. And we further identified a novel transcriptional activator peptide with the 'piggyBac-in vitro ligation-PCR' strategy. Our novel strategies greatly facilitate the construction of large variant DMS library in mammalian cells, and may have great application potential in the future.
Topics: Animals; Humans; DNA Transposable Elements; HEK293 Cells; Transfection; Mutation; Genomics; Mammals
PubMed: 37815732
DOI: 10.1007/s00018-023-04976-5 -
Biomedicine & Pharmacotherapy =... Jul 2024Therapeutic proteins provided new opportunities for patients and high sales volumes. However, they are formulated for extracellular targets. The lipophilic barrier of... (Review)
Review
Therapeutic proteins provided new opportunities for patients and high sales volumes. However, they are formulated for extracellular targets. The lipophilic barrier of the plasma membrane renders the vast array of intracellular targets out of reach. Peptide-based delivery systems, namely cell-penetrating peptides (CPPs), have few safety concerns, and low immunogenicity, with control over administered doses. This study investigates CPP-based protein delivery systems by classifying them into CPP-protein "covalent conjugation" and CPP: protein "non-covalent complexation" categories. Covalent conjugates ensure the proximity of the CPP to the cargo, which can improve cellular uptake and endosomal escape. We will discuss various aspects of covalent conjugates through non-cleavable (stable) or cleavable bonds. Non-cleavable CPP-protein conjugates are produced by recombinant DNA technology to express the complete fusion protein in a host cell or by chemical ligation of CPP and protein, which ensures stability during the delivery process. CPP-protein cleavable bonds are classified into pH-sensitive and redox-sensitive bonds, enzyme-cleavable bonds, and physical stimuli cleavable linkers (light radiation, ultrasonic waves, and thermo-responsive). We have highlighted the key characteristics of non-covalent complexes through electrostatic and hydrophobic interactions to preserve the conformational integrity of the CPP and cargo. CPP-mediated protein delivery by non-covalent complexation, such as zippers, CPP adaptor methods, and avidin-biotin technology, are featured. Conclusively, non-covalent complexation methods are appropriate when a high number of CPP or protein samples are to be screened. In contrast, when the high biological activity of the protein is critical in the intracellular compartment, conjugation protocols are preferred.
Topics: Cell-Penetrating Peptides; Humans; Drug Delivery Systems; Animals; Proteins
PubMed: 38852512
DOI: 10.1016/j.biopha.2024.116910 -
Scientific Reports Nov 2023Activation-induced cytidine deaminase (AID) is the key mediator of antibody diversification in activated B-cells by the process of somatic hypermutation (SHM) and class...
Activation-induced cytidine deaminase (AID) is the key mediator of antibody diversification in activated B-cells by the process of somatic hypermutation (SHM) and class switch recombination (CSR). Targeting AID to the Ig genes requires transcription (initiation and elongation), enhancers, and its interaction with numerous factors. Furthermore, the HIRA chaperon complex, a regulator of chromatin architecture, is indispensable for SHM. The HIRA chaperon complex consists of UBN1, ASF1a, HIRA, and CABIN1 that deposit H3.3 onto the DNA, the SHM hallmark. We explored whether UBN1 interacts with AID using computational and in-vitro experiments. Interestingly, our in-silico studies, such as molecular docking and molecular dynamics simulation results, predict that AID interacts with UBN1. Subsequently, co-immunoprecipitation and pull-down experiments established interactions between UBN1 and AID inside B-cells. Additionally, a double immunofluorescence assay confirmed that AID and UBN1 were co-localized in the human and chicken B-cell lines. Moreover, proximity ligation assay studies validated that AID interacts with UBN1. Ours is the first report on the interaction of genome mutator enzyme AID with UBN1. Nevertheless, the fate of interaction between UBN1 and AID is yet to be explored in the context of SHM or CSR.
Topics: Humans; Chromatin; Cytidine Deaminase; Immunoglobulin Class Switching; Immunoglobulins; Molecular Chaperones; Molecular Docking Simulation; Nuclear Proteins; Somatic Hypermutation, Immunoglobulin; Transcription Factors
PubMed: 37949972
DOI: 10.1038/s41598-023-46448-7 -
Heliyon Sep 2023The protective effects of electroacupuncture (EA) preconditioning against myocardial ischemia-reperfusion injury (MIRI) have been reported. However, the underlying...
BACKGROUND
The protective effects of electroacupuncture (EA) preconditioning against myocardial ischemia-reperfusion injury (MIRI) have been reported. However, the underlying mechanism remains unclear. Recent research has indicated that the dynamic inflammatory response following MIRI plays an essential role in the progression of myocardial injury. This study aimed to investigate the myocardial protective effects of EA preconditioning on MIRI in rats and to explore the relevant mechanism from the perspective of dynamic inflammatory response.
METHODS
A MIRI model was employed, and the rats were subjected to EA on Neiguan for four days prior to modeling. The myocardial protective effect of EA preconditioning was evaluated by echocardiography, Evans blue and triphenyltetrazolium chloride staining. Real-time polymerase chain reaction, Western blot, hematoxylin & eosin staining, and immunohistochemistry were utilized to detect the content of mitochondrial DNA, NOD receptor family protein 3 (NLRP3) inflammasome activation, neutrophil recruitment and macrophage infiltration in blood samples and myocardium below the ligation.
RESULTS
We found that EA preconditioning could accelerate the recovery of left ventricle function after MIRI and reduce the myocardial infarction area, thereby protecting the myocardium against MIRI. Furthermore, EA preconditioning was observed to ameliorate mitochondrial impairment, reduce the level of plasma mitochondrial DNA, modulate NLRP3 inflammasome activation, attenuate neutrophil infiltration, and promote the polarization of M1 macrophages towards M2 macrophages in the myocardium after MIRI.
CONCLUSION
EA preconditioning could reduce plasma mtDNA, suppress overactivation of the NLRP3 inflammasome, facilitate the transition from the acute pro-inflammatory phase to the anti-inflammatory reparative phase after MIRI, and ultimately confer cardioprotective benefits.
PubMed: 37809701
DOI: 10.1016/j.heliyon.2023.e19396 -
Exploration (Beijing, China) Oct 2023Gliomas are histologically and genetically heterogeneous tumors. However, classical histopathological typing often ignores the high heterogeneity of tumors and thus...
Gliomas are histologically and genetically heterogeneous tumors. However, classical histopathological typing often ignores the high heterogeneity of tumors and thus cannot meet the requirements of precise pathological diagnosis. Here, proximity-anchored in situ spectral coding amplification (ProxISCA) is proposed for multiplexed imaging of RNA mutations, enabling visual typing of brain gliomas with different pathological grades at the single-cell and tissue levels. The ligation-based padlock probe can discriminate one-nucleotide variations, and the design of proximity primers enables the anchoring of amplicons on target RNA, thus improving localization accuracy. The DNA module-based spectral coding strategy can dramatically improve the multiplexing capacity for imaging RNA mutations through one-time labelling, with low cost and simple operation. One-target-one-amplicon amplification confers ProxISCA the ability to quantify RNA mutation copy number with single-molecule resolution. Based on this approach, it is found that gliomas with higher malignant grades express more genes with high correlation at the cellular and tissue levels and show greater cellular heterogeneity. ProxISCA provides a tool for glioma research and precise diagnosis, which can reveal the relationship between cellular heterogeneity and glioma occurrence or development and assist in pathological prognosis.
PubMed: 37933281
DOI: 10.1002/EXP.20220175 -
Journal of Personalized Medicine Aug 2023Chronic lymphocytic leukemia (CLL) is known for its wide-ranging clinical and genetic diversity. The study aimed to assess the associations between copy number...
Chronic lymphocytic leukemia (CLL) is known for its wide-ranging clinical and genetic diversity. The study aimed to assess the associations between copy number variations (CNVs) and various biological and clinical features, as well as the survival rates of CLL patients and to evaluate the effectiveness of the multiplex ligation-dependent probe amplification (MLPA) technique in CLL patients.DNA was extracted from 110 patients, and MLPA was performed. Mutations in , , and were also analyzed. A total of 52 patients showed at least one CNV, 26 had at least one somatic mutation, and 10 presented both, CNVs, and somatic mutations. The most commonly identified CNVs were del(114.3), del(11q22.3), and dup(12q23.2). Other CNVs identified included del(17p13.1), del(14q32.33), dup(10q23.31), and del(19p13.2). One patient was identified with concomitant trisomy 12, 13, and 19. and mutations were found in 13 patients each, either alone or in combination with other mutations or CNVs, while mutation was identified in one patient. Forty-two patients had normal results. Associations between the investigated CNVs and gene mutations and patients' overall survival were found. The presence of and mutations or the combination of mutation and CNVs significantly influenced the survival of patients with CLL. Both mutations are frequently associated with different CNVs. Del(13q) is associated with the longest survival rate, while the shortest survival is found in patients with del(17p). Even if MLPA has constraints, it may be used as the primary routine analysis in patients with CLL.
PubMed: 37623489
DOI: 10.3390/jpm13081239 -
Advanced Science (Weinheim,... Sep 2023Digital PCR (dPCR) is an important tool for precise nucleic acid quantification in clinical setting, but the limited multiplexing capability restricts its applications...
Digital PCR (dPCR) is an important tool for precise nucleic acid quantification in clinical setting, but the limited multiplexing capability restricts its applications for quantitative gene panel profiling. Here, this work describes melt-encoded-tags for expanded optical readout in digital PCR (METEOR-dPCR), a simple two-step assay that enables simultaneous quantification of a large panel of arbitrary genes in a dPCR platform. Target genes are quantitatively converted into DNA tags with unique melting temperatures through a ligation approach. These tags are then counted and distinguished by their melt-curve profiles on a dPCR platform. A multiplexing capacity of M^N, where M is the number of resolvable melting temperature and N is the number of fluorescence channel, can be achieved. This work validates METEOR-dPCR with simultaneous DNA copy number profiling of 60 targets using dPCR in cancer cells, and demonstrates its sensitivity for estimating tumor fraction in mixed tumor and normal DNA samples. The rapid, quantitative, and highly multiplexed METEOR-dPCR assay will have wide appeal for many clinical applications.
Topics: Polymerase Chain Reaction; DNA
PubMed: 37485651
DOI: 10.1002/advs.202301630 -
Cells Oct 2023Nucleolar and Spindle-Associated Protein 1 (NuSAP1) is an important mitotic regulator, implicated in control of mitotic microtubule stability and chromosome segregation....
Nucleolar and Spindle-Associated Protein 1 (NuSAP1) is an important mitotic regulator, implicated in control of mitotic microtubule stability and chromosome segregation. NuSAP1 regulates these processes by interacting with several protein partners. Its abundance, activity and interactions are therefore tightly regulated during mitosis. Protein conjugation with SUMO (Small Ubiquitin-like MOdifier peptide) is a reversible post-translational modification that modulates rapid changes in the structure, interaction(s) and localization of proteins. NuSAP1 was previously found to interact with RANBP2, a nucleoporin with SUMO ligase and SUMO-stabilizing activity, but how this interaction affects NuSAP1 activity has remained elusive. Here, we show that NuSAP1 interacts with RANBP2 and forms proximity ligation products with SUMO2/3 peptides in a RANBP2-dependent manner at key mitotic sites. A bioinformatic search identified two putative SUMO consensus sites in NuSAP1, within the DNA-binding and the microtubule-binding domains, respectively. Site-specific mutagenesis, and mitotic phenotyping in cell lines expressing each NuSAP1 mutant version, revealed selective roles of each individual site in control of NuSAP1 localization and in generation of specific mitotic defects and distinct fates in daughter cells. These results identify therefore two new regulatory sites for NuSAP1 functions and implicate RANBP2 in control of NuSAP1 activity.
Topics: Humans; Sumoylation; Consensus; HeLa Cells; Microtubule-Associated Proteins; Microtubules
PubMed: 37947624
DOI: 10.3390/cells12212545 -
Plant Disease Nov 2023Trichosanthis fructus is one of the most common medicinal plants in China. In September 2022, T. fructus fruit showed black necrotic spots and surface irregularities,...
Trichosanthis fructus is one of the most common medicinal plants in China. In September 2022, T. fructus fruit showed black necrotic spots and surface irregularities, with water-soaked lesions (Fig 1). The affected T. fructus fruit (five weeks after blossom) were located in a field in Huai'an Municipality, Jiangsu Province (33.85°N, 119.00°E). The incidence was approximately 50%, causing great losses in fruit production. To isolate the causal agent, two symptomatic fruit from different plants were surface-disinfested with 75% (v/v) ethanol for 1 min, washed three times with sterile distilled water, and cultured on Nutrient agar (NA) plates at 28°C for 24 h. The obtained colonies were light yellow and transferred to fresh NA plates using the conventional repetitive streaking technique to obtain pure cultures. The purified bacterial cells were rod shaped, 1.64 to 2.47 μm long (n = 45), and 0.58 to 0.74 μm wide (n = 45) (Figure S2). Three isolates were used for further characterization. Biochemical tests indicated that the three isolates were Gram negative. DNA was extracted from the three bacterial isolates and used to amplify the16S rRNA (27F/1492R primers) and partial gyrB (UP1/Up2r primers) genes (Marchesi et al. 1998; Yamamoto and Harayama 1995). PCR products were purified using the DNA Clean-up Kit (CW2301, CWBIO), ligated into the PMD-19 vector (6013, Takara), and sequenced by Beijing Tsingke Biotech. The obtained 16S rRNA (GenBank accessions: OQ923996-OQ923998) and gyrB sequences (OR140942-OR140944) showed the best match, over 99%and 98% identity with 100% coverage to the K. cowanii type strain JCM 10956 (CP019445.1). To fulfill Koch's postulates, pathogenicity tests were conducted on healthy T. fructus fruit. T. fructus fruit showed no wounds or lesions, and were surface disinfected with 75% alcohol. The three isolates were grown in nutrient broth at 200 rpm in 28 oC for 24 h and re-suspended in sterilized ddH2O to OD600 = 0.6~1.0 (108~109cfu/mL). Five μL of bacterial suspension was inoculated into the healthy fruit surface with a sterile knife. For the control experiment, the same volume of sterilized ddH2O was used. In each treatment, four healthy T. fructus fruit were treated. All samples were incubated at 25°C and 75% humidity in a plant incubator (Bluepard, MGC-350BP-2). After 12 days, bacterial fruit blotch symptoms were observed in all the inoculated fruit. The pathogen was recovered from the infected fruit, and its identity was confirmed by 16S rRNA/gyrB sequencing and morphological analysis. To further investigate the pathogenicity, four-week-old T. fructus plant leaves were inoculated with the above three isolated suspension (OD600=0.6~1.0) using the leaf cutting method (Kauffman et al. 1973). Sterilized ddH2O was used as mock control. After 10 days, bacterial blight symptoms were observed in all inoculated leaves. To our knowledge, this is the first report of K. cowanii causing fruit blotch on T. fructus worldwide. This species has been previously associated with acute cholecystitis in humans (Berinson et al. 2020; Petrzik et al. 2021), but it was recently identified as the causal agent of bacterial wilt on patchouli, bacterial blight on soybean, and stalk rot in foxtail millet (Han et al. 2023; Krawczyk and Borodynko-Filas 2020; Zhang et al. 2022). China is the largest producer of T. fructus. This report reveals that K. cowanii has a greater host range than was known. This report will help to better understand the pathogens that affects T. fructus production in China.
PubMed: 37971893
DOI: 10.1094/PDIS-10-23-2048-PDN