Background: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a lethal threat.Increasing Severe fever with thrombocytopenia syndrome (SFTS) risk in Asia and the United States stems from the spread of natural host, Haemaphysalis longicornis. Rapid and accurate SFTSV molecular diagnosis is crucial for treatment decisions, reducing fatality risk. Methods: Blood samples from 17 suspected SFTS patients at Chuncheon Sacred Heart Hospital (September-December 2022) were collected. SFTSV was diagnosed using two reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays from Gangwon Institute of Health and Environment (RT-qPCR/GIHE) and Asan Medical Center (RT-qPCR/AMC). To address RT-qPCR disparities, amplicon-based MinION sequencing traced SFTSV genomic sequences in clinical samples. Results: In two samples (N39 and N50), SFTSV was detected in both RT-qPCR/GIHE and RTqPCR/AMC. Among 11 samples, RT-qPCR/AMC exclusively detected SFTSV. In four samples, both assays yielded negative results. Amplicon-based MinION sequencing enabled nearly whole-genome sequencing of SFTSV in samples N39 and N50. Among 11 discordant samples, five contained significant SFTSV reads, aligning with the RT-qPCR/AMC findings. However, another six samples showed insufficient viral reads in accordance with the negativity observed in RT-qPCR/GIHE. The phylogenetic pattern of SFTSV demonstrated N39 formed a genetic lineage with genotype A in all segments. SFTSV N50 grouped with the B-1 sub-genotype for L segment and B-2 sub-genotype for the M and S segments, indicating genetic reassortment. Conclusion The study demonstrates the robust sensitivity of amplicon-based MinION sequencing for the direct detection of SFTSV in clinical samples containing ultralow copies of viral genomes. Next-generation sequencing holds potential in resolving SFTSV diagnosis discrepancies, enhancing understanding of diagnostic capacity, and risk assessment for emerging SFTSV.

Background: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a lethal threat.Increasing Severe fever with thrombocytopenia syndrome (SFTS) risk in Asia and the United States stems from the spread of natural host, Haemaphysalis longicornis. Rapid and accurate SFTSV molecular diagnosis is crucial for treatment decisions, reducing fatality risk. Methods: Blood samples from 17 suspected SFTS patients at Chuncheon Sacred Heart Hospital (September-December 2022) were collected. SFTSV was diagnosed using two reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays from Gangwon Institute of Health and Environment (RT-qPCR/GIHE) and Asan Medical Center (RT-qPCR/AMC). To address RT-qPCR disparities, amplicon-based MinION sequencing traced SFTSV genomic sequences in clinical samples. Results: In two samples (N39 and N50), SFTSV was detected in both RT-qPCR/GIHE and RTqPCR/AMC. Among 11 samples, RT-qPCR/AMC exclusively detected SFTSV. In four samples, both assays yielded negative results. Amplicon-based MinION sequencing enabled nearly whole-genome sequencing of SFTSV in samples N39 and N50. Among 11 discordant samples, five contained significant SFTSV reads, aligning with the RT-qPCR/AMC findings. However, another six samples showed insufficient viral reads in accordance with the negativity observed in RT-qPCR/GIHE. The phylogenetic pattern of SFTSV demonstrated N39 formed a genetic lineage with genotype A in all segments. SFTSV N50 grouped with the B-1 sub-genotype for L segment and B-2 sub-genotype for the M and S segments, indicating genetic reassortment. Conclusion The study demonstrates the robust sensitivity of amplicon-based MinION sequencing for the direct detection of SFTSV in clinical samples containing ultralow copies of viral genomes. Next-generation sequencing holds potential in resolving SFTSV diagnosis discrepancies, enhancing understanding of diagnostic capacity, and risk assessment for emerging SFTSV.

Background: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a lethal threat.Increasing Severe fever with thrombocytopenia syndrome (SFTS) risk in Asia and the United States stems from the spread of natural host, Haemaphysalis longicornis. Rapid and accurate SFTSV molecular diagnosis is crucial for treatment decisions, reducing fatality risk. Methods: Blood samples from 17 suspected SFTS patients at Chuncheon Sacred Heart Hospital (September-December 2022) were collected. SFTSV was diagnosed using two reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays from Gangwon Institute of Health and Environment (RT-qPCR/GIHE) and Asan Medical Center (RT-qPCR/AMC). To address RT-qPCR disparities, amplicon-based MinION sequencing traced SFTSV genomic sequences in clinical samples. Results: In two samples (N39 and N50), SFTSV was detected in both RT-qPCR/GIHE and RTqPCR/AMC. Among 11 samples, RT-qPCR/AMC exclusively detected SFTSV. In four samples, both assays yielded negative results. Amplicon-based MinION sequencing enabled nearly whole-genome sequencing of SFTSV in samples N39 and N50. Among 11 discordant samples, five contained significant SFTSV reads, aligning with the RT-qPCR/AMC findings. However, another six samples showed insufficient viral reads in accordance with the negativity observed in RT-qPCR/GIHE. The phylogenetic pattern of SFTSV demonstrated N39 formed a genetic lineage with genotype A in all segments. SFTSV N50 grouped with the B-1 sub-genotype for L segment and B-2 sub-genotype for the M and S segments, indicating genetic reassortment. Conclusion The study demonstrates the robust sensitivity of amplicon-based MinION sequencing for the direct detection of SFTSV in clinical samples containing ultralow copies of viral genomes. Next-generation sequencing holds potential in resolving SFTSV diagnosis discrepancies, enhancing understanding of diagnostic capacity, and risk assessment for emerging SFTSV.

Background: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a lethal threat.Increasing Severe fever with thrombocytopenia syndrome (SFTS) risk in Asia and the United States stems from the spread of natural host, Haemaphysalis longicornis. Rapid and accurate SFTSV molecular diagnosis is crucial for treatment decisions, reducing fatality risk. Methods: Blood samples from 17 suspected SFTS patients at Chuncheon Sacred Heart Hospital (September-December 2022) were collected. SFTSV was diagnosed using two reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays from Gangwon Institute of Health and Environment (RT-qPCR/GIHE) and Asan Medical Center (RT-qPCR/AMC). To address RT-qPCR disparities, amplicon-based MinION sequencing traced SFTSV genomic sequences in clinical samples. Results: In two samples (N39 and N50), SFTSV was detected in both RT-qPCR/GIHE and RTqPCR/AMC. Among 11 samples, RT-qPCR/AMC exclusively detected SFTSV. In four samples, both assays yielded negative results. Amplicon-based MinION sequencing enabled nearly whole-genome sequencing of SFTSV in samples N39 and N50. Among 11 discordant samples, five contained significant SFTSV reads, aligning with the RT-qPCR/AMC findings. However, another six samples showed insufficient viral reads in accordance with the negativity observed in RT-qPCR/GIHE. The phylogenetic pattern of SFTSV demonstrated N39 formed a genetic lineage with genotype A in all segments. SFTSV N50 grouped with the B-1 sub-genotype for L segment and B-2 sub-genotype for the M and S segments, indicating genetic reassortment. Conclusion The study demonstrates the robust sensitivity of amplicon-based MinION sequencing for the direct detection of SFTSV in clinical samples containing ultralow copies of viral genomes. Next-generation sequencing holds potential in resolving SFTSV diagnosis discrepancies, enhancing understanding of diagnostic capacity, and risk assessment for emerging SFTSV.

This study aimed to establish an organoid culture model using small intestine tissues from male and female C57BL/6 mice and to compare it with rat organoid cultures derived from frozen tissues. Crypts were isolated from the small intestines of eight-week-old male and female mice and cultured in 3D extracellular matrix with Wnt, R-spondin, and Noggin. In addition, small intestine tissues from sixteen-week-old F344 rats were preserved in a storage solution immediately post-sacrifice and stored at –80°C before being transferred to a nitrogen tank. Upon thawing, crypts from frozen rat tissues failed to develop into organoids due to structural damage, suggesting the need for fresh tissues or optimized preservation methods. In contrast, mouse-derived organoids showed viability for 7 days, with distinct morphological changes and clear differentiation by Day 7. Quantitative real-time PCR analysis revealed that Lgr5, a stem cell marker, showed significantly higher expression in organoids than in tissues, confirming the successful establishment of the organoid culture. Among epithelial markers, the antimicrobial enzyme Lyz1 was more highly expressed in organoids, while Muc2, a key goblet cell marker, was more highly expressed in male tissues. The enterocyte marker Alp exhibited higher expression in male organoids compared to females, with no sex differences in tissues. These findings highlight sex-specific differences in gene expression related to small intestine differentiation and demonstrate the challenges in organoid culture from frozen rat tissues. The results suggest the importance of immediate tissue processing or improved preservation methods for successful organoid cultures.

This study aimed to establish an organoid culture model using small intestine tissues from male and female C57BL/6 mice and to compare it with rat organoid cultures derived from frozen tissues. Crypts were isolated from the small intestines of eight-week-old male and female mice and cultured in 3D extracellular matrix with Wnt, R-spondin, and Noggin. In addition, small intestine tissues from sixteen-week-old F344 rats were preserved in a storage solution immediately post-sacrifice and stored at –80°C before being transferred to a nitrogen tank. Upon thawing, crypts from frozen rat tissues failed to develop into organoids due to structural damage, suggesting the need for fresh tissues or optimized preservation methods. In contrast, mouse-derived organoids showed viability for 7 days, with distinct morphological changes and clear differentiation by Day 7. Quantitative real-time PCR analysis revealed that Lgr5, a stem cell marker, showed significantly higher expression in organoids than in tissues, confirming the successful establishment of the organoid culture. Among epithelial markers, the antimicrobial enzyme Lyz1 was more highly expressed in organoids, while Muc2, a key goblet cell marker, was more highly expressed in male tissues. The enterocyte marker Alp exhibited higher expression in male organoids compared to females, with no sex differences in tissues. These findings highlight sex-specific differences in gene expression related to small intestine differentiation and demonstrate the challenges in organoid culture from frozen rat tissues. The results suggest the importance of immediate tissue processing or improved preservation methods for successful organoid cultures.

This study aimed to establish an organoid culture model using small intestine tissues from male and female C57BL/6 mice and to compare it with rat organoid cultures derived from frozen tissues. Crypts were isolated from the small intestines of eight-week-old male and female mice and cultured in 3D extracellular matrix with Wnt, R-spondin, and Noggin. In addition, small intestine tissues from sixteen-week-old F344 rats were preserved in a storage solution immediately post-sacrifice and stored at –80°C before being transferred to a nitrogen tank. Upon thawing, crypts from frozen rat tissues failed to develop into organoids due to structural damage, suggesting the need for fresh tissues or optimized preservation methods. In contrast, mouse-derived organoids showed viability for 7 days, with distinct morphological changes and clear differentiation by Day 7. Quantitative real-time PCR analysis revealed that Lgr5, a stem cell marker, showed significantly higher expression in organoids than in tissues, confirming the successful establishment of the organoid culture. Among epithelial markers, the antimicrobial enzyme Lyz1 was more highly expressed in organoids, while Muc2, a key goblet cell marker, was more highly expressed in male tissues. The enterocyte marker Alp exhibited higher expression in male organoids compared to females, with no sex differences in tissues. These findings highlight sex-specific differences in gene expression related to small intestine differentiation and demonstrate the challenges in organoid culture from frozen rat tissues. The results suggest the importance of immediate tissue processing or improved preservation methods for successful organoid cultures.

Primary cilia function as critical sensory organelles that mediate multiple signaling pathways, including the Hedgehog (Hh) pathway, which is essential for organ patterning and morphogenesis. Disruptions in Hh signaling have been implicated in supernumerary tooth formation and molar fusion in mutant mice. Cilk1, a highly conserved serine/threonine-protein kinase localized within primary cilia, plays a critical role in ciliary transport. Loss of Cilk1 results in severe ciliopathy phenotypes, including polydactyly, edema, and cleft palate. However, the role of Cilk1 in tooth development remains unexplored. In this study, we investigated the role of Cilk1 in tooth development. Cilk1 was found to be expressed in both the epithelial and mesenchymal compartments of developing molars. Cilk1 deficiency resulted in altered ciliary dynamics, characterized by reduced frequency and increased length, accompanied by downregulation of Hh target genes, such as Ptch1 and Sostdc1, leading to the formation of diastemal supernumerary teeth. Furthermore, in Cilk1^-/-;PCS1-MRCS1^△/△ mice, which exhibit a compounded suppression of Hh signaling, we uncovered a novel phenomenon: diastemal supernumerary teeth can be larger than first molars. Based on these findings, we propose a progressive model linking Hh signaling levels to sequential changes in tooth patterning: initially inducing diastemal supernumerary teeth, then enlarging them, and ultimately leading to molar fusion. This study reveals a previously unrecognized role of Cilk1 in controlling tooth morphology via Hh signaling and highlights how Hh signaling levels shape tooth patterning in a gradient-dependent manner.
Animals ; Hedgehog Proteins/physiology* ; Mice ; Signal Transduction/physiology* ; Tooth, Supernumerary ; Molar ; Cilia/physiology* ; Odontogenesis/physiology* ; Patched-1 Receptor ; Protein Serine-Threonine Kinases/physiology* ; Mice, Knockout ; Adaptor Proteins, Signal Transducing

A standardized protocol for the isolation of cell-free DNA (cfDNA) from cerebrospinal fluid (CSF) is lacking. Therefore, we established a cfDNA isolation protocol optimized for clinical CSF specimens, integrating acceptable modifications and using artificial CSF generated from remnant CSF spiked with reference cell-free tumor DNA (ctDNA). We compared the isolation yields of in vitro diagnostic (IVD)-certified column-based (CB) and magnetic beadbased (MB) isolation. Furthermore, we modified both methods, including pre- and postelution steps. To confirm ctDNA integrity and quantify the variant allele frequency after isolation, we performed droplet digital PCR (ddPCR) targeting IDH1 R132C in the reference ctDNA. MB isolation had a higher yield than CB isolation (P < 0.0001), and post-isolation vacuum increased the final concentration in both methods, with little effect on cfDNA integrity. Our study provides a protocol to maximize CSF-ctDNA concentrations in IVD testing and future studies.

Lupus is characterized by the autoantibodies against nuclear Ags, underscoring the importance of identifying the B cell subsets driving autoimmunity. Our research focused on the mitochondrial activity and CXCR4 expression in CD11c + B cells from lupus patients after ex vivo stimulation with a TLR9 agonist, CpG-oligodeoxyribonucleotide (ODN). We also evaluated the response of CD11c + B cells in ODN-injected mice. Post-ex vivo ODN stimulation, we observed an increase in the proportion of CD11chi cells, with elevated mitochondrial activity and CXCR4 expression in CD11c + B cells from lupus patients. In vivo experiments showed similar patterns, with TLR9 stimulation enhancing mitochondrial and CXCR4 activities in CD11chi B cells, leading to the generation of anti-dsDNA plasmablasts. The CXCR4 inhibitor AMD3100 and the mitochondrial complex I inhibitor IM156 significantly reduced the proportion of CD11c + B cells and autoreactive plasmablasts. These results underscore the pivotal roles of mitochondria and CXCR4 in the production of autoreactive plasmablasts.