Expression of Toll-like receptors in the cerebellum during pathogenesis of prion disease.

Prion diseases, such as scrapie, entail the accumulation of disease-specific prion protein (PrPSc) within the brain. Toll-like receptors (TLRs) are crucial components of the pattern recognition system. They recognize pathogen-associated molecular patterns (PAMPs) and play a central role in orchestrating host innate immune responses. The expression levels of Toll-like receptors (TLRs) in the central nervous system (CNS) were not well-defined. To establish a model of prion diseases in BALB/C mice, the 22L strain was employed. The features of the 22L strain were analyzed, and the cerebellum exhibited severe pathological changes. TLR1-13 levels in the cerebellum were measured using quantitative polymerase chain reaction (qPCR) at time points of 60, 90, 120, and the final end point (145 days post-infection). During the pathogenesis, the expression levels of Toll-like receptors (TLRs) 1, 2, 7, 8, and 9 increased in a time-dependent manner. This trend mirrored the expression patterns of PrPSc (the pathological isoform of the prion protein) and glial fibrillary acidic protein. Notably, at the end point, TLR1-13 levels were significantly elevated. Protein level of TLR7 and TLR9 showed increasing at the end point of the 22L-infected mice. A deeper understanding of the increased Toll-like receptors (TLRs) in prion diseases could shed light on their role in initiating immune responses at various stages during pathogenesis. This insight is particularly relevant when considering TLRs as potential therapeutic targets for prion diseases.

Repetitive DNA sequence detection and its role in the human genome.

Repetitive DNA sequences playing critical roles in driving evolution, inducing variation, and regulating gene expression. In this review, we summarized the definition, arrangement, and structural characteristics of repeats. Besides, we introduced diverse biological functions of repeats and reviewed existing methods for automatic repeat detection, classification, and masking. Finally, we analyzed the type, structure, and regulation of repeats in the human genome and their role in the induction of complex diseases. We believe that this review will facilitate a comprehensive understanding of repeats and provide guidance for repeat annotation and in-depth exploration of its association with human diseases.

Adipose-derived stem cell exosome NFIC improves diabetic foot ulcers by regulating miR-204-3p/HIPK2.

BACKGROUND: Diabetic foot ulcers (DFU) are a serious complication of diabetes that lead to significant morbidity and mortality. Recent studies reported that exosomes secreted by human adipose tissue-derived mesenchymal stem cells (ADSCs) might alleviate DFU development. However, the molecular mechanism of ADSCs-derived exosomes in DFU is far from being addressed. METHODS: Human umbilical vein endothelial cells (HUVECs) were induced by high-glucose (HG), which were treated with exosomes derived from nuclear factor I/C (NFIC)-modified ADSCs. MicroRNA-204-3p (miR-204-3p), homeodomain-interacting protein kinase 2 (HIPK2), and NFIC were determined using real-time quantitative polymerase chain reaction. Cell proliferation, apoptosis, migration, and angiogenesis were assessed using cell counting kit-8, 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, wound healing, and tube formation assays. Binding between miR-204-3p and NFIC or HIPK2 was predicted using bioinformatics tools and validated using a dual-luciferase reporter assay. HIPK2, NFIC, CD81, and CD63 protein levels were measured using western blot. Exosomes were identified by a transmission electron microscope and nanoparticle tracking analysis. RESULTS: miR-204-3p and NFIC were reduced, and HIPK2 was enhanced in DFU patients and HG-treated HUVECs. miR-204-3p overexpression might abolish HG-mediated HUVEC proliferation, apoptosis, migration, and angiogenesis in vitro. Furthermore, HIPK2 acted as a target of miR-204-3p. Meanwhile, NFIC was an upstream transcription factor that might bind to the miR-204-3p promoter and improve its expression. NFIC-exosome from ADSCs might regulate HG-triggered HUVEC injury through miR-204-3p-dependent inhibition of HIPK2. CONCLUSION: Exosomal NFIC silencing-loaded ADSC sheet modulates miR-204-3p/HIPK2 axis to suppress HG-induced HUVEC proliferation, migration, and angiogenesis, providing a stem cell-based treatment strategy for DFU.

LCAT: an isoform-sensitive error correction for transcriptome sequencing long reads.

As the carrier of genetic information, RNA carries the information from genes to proteins. Transcriptome sequencing technology is an important way to obtain transcriptome sequences, and it is also the basis for transcriptome research. With the development of third-generation sequencing, long reads can cover full-length transcripts and reflect the composition of different isoforms. However, the high error rate of third-generation sequencing affects the accuracy of long reads and downstream analysis. The current error correction methods seldom consider the existence of different isoforms in RNA, which makes the diversity of isoforms a serious loss. Here, we introduce LCAT (long-read error correction algorithm for transcriptome sequencing data), a wrapper algorithm of MECAT, to reduce the loss of isoform diversity while keeping MECAT's error correction performance. The experimental results show that LCAT can not only improve the quality of transcriptome sequencing long reads but also retain the diversity of isoforms.

An efficient classification algorithm for NGS data based on text similarity.

With the advancement of high-throughput sequencing technologies, the amount of available sequencing data is growing at a pace that has now begun to greatly challenge the data processing and storage capacities of modern computer systems. Removing redundancy from such data by clustering could be crucial for reducing memory, disk space and running time consumption. In addition, it also has good performance on reducing dataset noise in some analysis applications. In this study, we propose a high-performance short sequence classification algorithm (HSC) for next generation sequencing (NGS) data based on efficient hash function and text similarity. First, HSC converts all reads into k-mers, then it forms a unique k-mer set by merging the duplicated and reverse complementary elements. Second, all unique k-mers are stored in a hash table, where the k-mer string is stored in the key field, and the ID of the reads containing the k-mer are stored in the value field. Third, each hash unit is transformed into a short text consisting of reads. Fourth, texts that satisfy the similarity threshold are combined into a long text, the merge operation is executed iteratively until there is no text that satisfies the merge condition. Finally, the long text is transformed into a cluster consisting of reads. We tested HSC using five real datasets. The experimental results showed that HSC cluster 100 million short reads within 2 hours, and it has excellent performance in reducing memory consumption. Compared to existing methods, HSC is much faster than other tools, it can easily handle tens of millions of sequences. In addition, when HSC is used as a preprocessing tool to produce assembly data, the memory and time consumption of the assembler is greatly reduced. It can help the assembler to achieve better assemblies in terms of N50, NA50 and genome fraction.

A new mutation site in the AIRE gene causes autoimmune polyendocrine syndrome type 1.

Autoimmune polyendocrine syndrome type 1 (APS-1, OMIM 2403000) is a rare autosomal recessive disease that is caused by autoimmune regulator (AIRE). The main symptoms of APS-1 are chronic mucocutaneous candidiasis, autoimmune adrenocortical insufficiency (Addison's disease) and hypoparathyroidism. We collected APS-1 cases and analysed them. The AIRE genes of the patient and his family members were sequenced to identify whether the APS-1 patient had an AIRE mutation. We discovered a mutation site (c.206A>C) that had never before been reported in the AIRE gene located in exon 2 of the AIRE gene. This homogyzous mutation caused a substitution of the 69th amino acid of the AIRE protein from glutamine to proline (p.Q69P). A yeast two-hybrid assay, which was used to analyse the homodimerization properties of the mutant AIRE protein, showed that the mutant AIRE protein could not interact with the normal AIRE protein. Flow cytometry and RT-qPCR analyses indicated that the new mutation site could decrease the expression levels of the AIRE, glutamic acid decarboxylase 65 (GAD65) and tryptophan hydroxylase-1 (TPH1) proteins to affect central immune tolerance. In conclusion, our research has shown that the new mutation site (c.206A>C) may influence the homodimerization and expression levels and other aspects of the AIRE protein. It may also impact the expression levels of tissue-restricted antigens (TRAs), leading to a series of autoimmune diseases.

Macrophages overexpressing Aire induce CD4+Foxp3+ T cells.

Aire plays an important role in central immune tolerance by regulating the transcription of thousands of genes. However, the role of Aire in the peripheral immune system is poorly understood. Regulatory T (Treg) cells are considered essential for the maintenance of peripheral tolerance, but the effect of Aire on Treg cells in the peripheral immune system is currently unknown. In this study, we investigated the effects of macrophages overexpressing Aire on CD4+Foxp3+ Treg cells by co-culturing Aire-overexpressing RAW264.7 cells or their supernatant with splenocytes. The results show that macrophages overexpressing Aire enhanced the expression of Foxp3 mRNA and induced different subsets of Treg cells in splenocytes through cell-cell contact or a co-culture supernatants. TGF-ß is a key molecule in the increases of CD4+CD45RA+Foxp3hi T cell and activating Treg (aTreg) levels observed following cell­supernatant co-culturing. Subsets of Treg cells were induced by Aire-overexpressing macrophages, and the manipulation of Treg cells by the targeting of Aire may provide a method for the treatment of inflammatory or autoimmune diseases.

Overexpressing autoimmune regulator regulates the expression of toll-like receptors by interacting with their promoters in RAW264.7 cells.

Autoimmune regulator (Aire) is a transcriptional activator that regulates the ectopic expression of many tissue-restricted antigens in medullary thymic epithelial cells, and that has an important role in the negative selection of autoreactive T cells. However, the roles of Aire expression in peripheral lymphoid tissues and hematopoietic cells, especially monocytes/macrophages, remain poorly understood. In this study, we found that the mRNA and protein expression levels of toll-like receptor (TLR)1, TLR3, and TLR8 were notably up-regulated in a mouse macrophage-like cell line (RAW264.7) stably expressing Aire, while the expression of TLR2, TLR4, TLR5, TLR6, TLR7, and TLR9 were not significantly changed. In addition, the mRNA expression of TLR3 and TLR8 were significantly increased in primary peritoneal macrophages transiently transfected with Aire. Using chromatin immunoprecipitation and a luciferase activity assay, we also found that Aire interacted with the TLR1, TLR3, and TLR8 promoters and increased the luciferase transcriptional activity of these promoters in RAW264.7 cells. Moreover, after stimulation by Pam(3)CSK(4), a TLR1 ligand, and poly(I:C), a TLR3 ligand, we found that the mRNA expression levels of IL-1α, TNFα, iNOS, and IFNα were increased in RAW264.7 cells stably expressing Aire. Together, these data suggest that Aire has a crucial role in the recognition of pathogenic microorganisms and peripheral immune tolerance in antigen-presenting cells (APCs) by regulating the expression of TLRs.