ABSTRACT
MICB*002:06 differs from MICB*002:01:01 by one nucleotide change at nucleotide 33 in exon 1 from C to T.
Subject(s)
Exons , Histocompatibility Antigens Class I , Humans , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/immunology , Alleles , Base Sequence , Sequence Analysis, DNA/methods , Histocompatibility Testing , Sequence Alignment , CodonABSTRACT
Glioblastoma (GBM) is a lethal brain cancer with no effective treatment; understanding how GBM cells respond to tumor microenvironment remains challenging as conventional cell cultures lack proper cytoarchitecture while in vivo animal models present complexity all at once. Developing a culture system to bridge the gap is thus crucial. Here, we employed a multicellular approach using human glia and vascular cells to optimize a 3-dimensional (3D) brain vascular niche model that enabled not only long-term culture of patient derived GBM cells but also recapitulation of key features of GBM heterogeneity, in particular invasion behavior and vascular association. Comparative transcriptomics of identical patient derived GBM cells in 3D and in vivo xenotransplants models revealed that glia-vascular contact induced genes concerning neural/glia development, synaptic regulation, as well as immune suppression. This gene signature displayed region specific enrichment in the leading edge and microvascular proliferation zones in human GBM and predicted poor prognosis. Gene variance analysis also uncovered histone demethylation and xylosyltransferase activity as main themes for gene adaption of GBM cells in vivo . Furthermore, our 3D model also demonstrated the capacity to provide a quiescence and a protective niche against chemotherapy. In summary, an advanced 3D brain vascular model can bridge the gap between 2D cultures and in vivo models in capturing key features of GBM heterogeneity and unveil previously unrecognized influence of glia-vascular contact for transcriptional adaption in GBM cells featuring neural/synaptic interaction and immunosuppression.
ABSTRACT
Communication between glial cells has a profound impact on the pathophysiology of Alzheimer's disease (AD). We reveal here that reactive astrocytes control cell distancing in peri-plaque glial nets, which restricts microglial access to amyloid deposits. This process is governed by guidance receptor Plexin-B1 (PLXNB1), a network hub gene in individuals with late-onset AD that is upregulated in plaque-associated astrocytes. Plexin-B1 deletion in a mouse AD model led to reduced number of reactive astrocytes and microglia in peri-plaque glial nets, but higher coverage of plaques by glial processes, along with transcriptional changes signifying reduced neuroinflammation. Additionally, a reduced footprint of glial nets was associated with overall lower plaque burden, a shift toward dense-core-type plaques and reduced neuritic dystrophy. Altogether, our study demonstrates that Plexin-B1 regulates peri-plaque glial net activation in AD. Relaxing glial spacing by targeting guidance receptors may present an alternative strategy to increase plaque compaction and reduce neuroinflammation in AD.
Subject(s)
Alzheimer Disease , Nerve Tissue Proteins , Neuroglia , Plaque, Amyloid , Animals , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Plaque, Amyloid/metabolism , Plaque, Amyloid/pathology , Mice , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/genetics , Neuroglia/metabolism , Receptors, Cell Surface/metabolism , Receptors, Cell Surface/genetics , Humans , Astrocytes/metabolism , Mice, Transgenic , Microglia/metabolism , Amyloid beta-Peptides/metabolism , Male , Mice, Knockout , FemaleABSTRACT
Compared with HLA-DRB1*09:01:02:05, the alleles HLA-DRB1*09:57 and HLA-DRB1*09:58 each show one nucleotide change, respectively.
Subject(s)
Alleles , Asian People , Base Sequence , Exons , HLA-DRB1 Chains , Humans , HLA-DRB1 Chains/genetics , Asian People/genetics , Histocompatibility Testing , China , Sequence Analysis, DNA/methods , Sequence Alignment , Codon , East Asian PeopleABSTRACT
HLA-A*31:01:53 differs from HLA-A*31:01:02:01 by one nucleotide change at nucleotide 900 in exon 5 from G to A.
Subject(s)
Alleles , Asian People , Exons , HLA-A Antigens , High-Throughput Nucleotide Sequencing , Humans , Asian People/genetics , Base Sequence , Codon , East Asian People , Histocompatibility Testing/methods , HLA-A Antigens/genetics , Polymorphism, Single Nucleotide , Sequence Analysis, DNA/methodsABSTRACT
Background: Infection is the main cause of death for patients after allogeneic hematopoietic stem cell transplantation (HSCT). However, pathogen profiles still have not been reported in detail due to their heterogeneity caused by geographic region. Objective: To evaluate the performance of metagenomic next-generation sequencing (mNGS) and summarize regional pathogen profiles of infected patients after HSCT. Methods: From February 2021 to August 2022, 64 patients, admitted to the Department of Hematology of The First Hospital of Jilin University for HSCT and diagnosed as suspected infections, were retrospectively enrolled. Results: A total of 38 patients were diagnosed as having infections, including bloodstream (n =17), pulmonary (n =16), central nervous system (CNS) (n =4), and chest (n =1) infections. Human betaherpesvirus 5 (CMV) was the most common pathogen in both bloodstream (n =10) and pulmonary (n =8) infections, while CNS (n =2) and chest (n =1) infections were mainly caused by Human gammaherpesvirus 4 (EBV). For bloodstream infection, Mycobacterium tuberculosis complex (n =3), Staphylococcus epidermidis (n =1), and Candida tropicalis (n =1) were also diagnosed as causative pathogens. Furthermore, mNGS combined with conventional tests can identify more causative pathogens with high sensitivity of 82.9% (95% CI 70.4-95.3%), and the total coincidence rate can reach up to 76.7% (95% CI 64.1-89.4%). Conclusions: Our findings emphasized the importance of mNGS in diagnosing, managing, and ruling out infections, and an era of more rapid, independent, and impartial diagnosis of infections after HSCT can be expected.
Subject(s)
Hematopoietic Stem Cell Transplantation , Humans , Retrospective Studies , China , Hematopoietic Stem Cell Transplantation/adverse effects , High-Throughput Nucleotide Sequencing , Candida tropicalis , Herpesvirus 4, Human , Metagenomics , Sensitivity and SpecificityABSTRACT
OBJECTIVE: To investigate the accuracy of next-generation sequencing technology (NGS) in detecting the polymorphisms of HLA-DRB1, DQB1, DQA1, DRB3, DRB4, DRB5, DPA1 and DPB1 alleles in randomly-selected unrelated healthy individuals from Shenzhen Han population, investigate the potential reason for HLA-DRB1 allele dropout in routine NGS, and establish an internal quality control system. METHODS: NGS-based HLA class II genotyping was performed on 1 012 samples using the MiSeqDxTM platform. The suspected missed alleles indicated by the quality control software and HLA-DRB1 homozygotes were confirmed by PCR-SSOP or PCR-SBT methods. RESULTS: A total of 139 alleles were detected, including HLA-DRB1(45), DRB3(7), DRB4(5), DRB5(7), DQA1(17), DQB1(21), DPA1(10) and DPB1(27). HLA-DRB1*09:01(17.09%),15:01(10.72%); DRB3*02:02(25.99%),03:01(10.18%); DRB4*01:03(36.46%); DRB5*01:01(15.42%); DQA1*01:02(20.01%),03:02(17.19%); DQB1*03:01(19.47%),03:03(17.98%), 05:02(11.66%), 06:01(10.67%); DPA1*02:02(54.45%), 01:03(31.18%) and DPB1*05:01(39.13%), 02:01(16.90%) alleles were the most common alleles in Shenzhen Han population (frequencies >10%). There was no statistical difference between the gene frequencies of HLA-DRB1 and DQB1 loci in our study. The HLA Common and Well-Documented Alleles in China (CWD2.4) (χ2=12.68, P >0.05). 94 cases of HLA-DRB1 homozygous samples detected by NGS were retested by PCR-SSOP or SBT method, and one case of allele dropout at HLA-DRB1 locus was found. SBT method confirmed that the allele of DRB1*04:03 was missed. The laboratory internal quality control system was established. Two cases of new alleles were detected and named by WHO Nomenclature Committee for Factors of the HLA System. CONCLUSION: The HLA genotyping results based on NGS showed a significantly lower ambiguity rate. The HLA class II alleles exhibit genetic polymorphism in the Han population of unrelated healthy individuals in Shenzhen. The independent method based on NGS in clinical histocompatibility testing has limitations and requires internal quality control strategies to avoid allele-dropout events.
Subject(s)
East Asian People , Genotype , High-Throughput Nucleotide Sequencing , Histocompatibility Antigens Class II , Humans , Alleles , Gene Frequency , Polymorphism, Genetic , East Asian People/genetics , Histocompatibility Antigens Class II/geneticsABSTRACT
Glioblastoma (GBM) is the most common primary malignant cancer of the central nervous system. Insufficient oxygenation (hypoxia) has been linked to GBM invasion and aggression, leading to poor patient outcomes. Hypoxia induces gene expression for cellular adaptations. However, GBM is characterized by high intertumoral (molecular subtypes) and intratumoral heterogeneity (cell states), and it is not well understood to what extent hypoxia triggers patient-specific gene responses and cellular diversity in GBM. Here, we surveyed eight patient-derived GBM stem cell lines for invasion phenotypes in 3D culture, which identified two GBM lines showing increased invasiveness in response to hypoxia. RNA-seq analysis of the two patient GBM lines revealed a set of shared hypoxia response genes concerning glucose metabolism, angiogenesis, and autophagy, but also a large set of patient-specific hypoxia-induced genes featuring cell migration and anti-inflammation, highlighting intertumoral diversity of hypoxia responses in GBM. We further applied the Shared GBM Hypoxia gene signature to single cell RNA-seq datasets of glioma patients, which showed that hypoxic cells displayed a shift towards mesenchymal-like (MES) and astrocyte-like (AC) states. Interestingly, in response to hypoxia, tumor cells in IDH-mutant gliomas displayed a strong shift to the AC state, whereas tumor cells in IDH-wildtype gliomas mainly shifted to the MES state. This distinct hypoxia response of IDH-mutant gliomas may contribute to its more favorable prognosis. Our transcriptomic studies provide a basis for future approaches to better understand the diversity of hypoxic niches in gliomas.
Subject(s)
Brain Neoplasms , Glioblastoma , Glioma , Humans , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Glioma/pathology , Glioblastoma/pathology , Hypoxia/genetics , Hypoxia/metabolism , Cell Line, Tumor , Gene Expression Profiling , Neoplastic Stem Cells/metabolism , Cell Hypoxia/geneticsABSTRACT
Double minutes (DMs), extrachromosomal gene fragments found within certain tumors, have been noted to carry onco- and drug resistance genes contributing to tumor pathogenesis and progression. After screening for SUMO-related molecule expression within various tumor sample and cell line databases, we found that SUMO-conjugating enzyme UBC9 has been associated with genome instability and tumor cell DM counts, which was confirmed both in vitro and in vivo. Karyotyping determined DM counts post-UBC9 knockdown or SUMOylation inhibitor 2-D08, while RT-qPCR and Western blot were used to measure DM-carried gene expression in vitro. In vivo, fluorescence in situ hybridization (FISH) identified micronucleus (MN) expulsion. Western blot and immunofluorescence staining were then used to determine DNA damage extent, and a reporter plasmid system was constructed to detect changes in homologous recombination (HR) and non-homologous end joining (NHEJ) pathways. Our research has shown that UBC9 inhibition is able to attenuate DM formation and lower DM-carried gene expression, in turn reducing tumor growth and malignant phenotype, via MN efflux of DMs and lowering NHEJ activity to increase DNA damage. These findings thus reveal a relationship between heightened UBC9 activity, increased DM counts, and tumor progression, providing a potential approach for targeted therapies, via UBC9 inhibition.
Subject(s)
Chromosome Aberrations , DNA Damage , Humans , Cell Nucleus , In Situ Hybridization, FluorescenceABSTRACT
Glioblastoma (GBM) is a malignant brain tumor with uncontrolled invasive growth. Here, we demonstrate how GBM cells usurp guidance receptor Plexin-B2 to gain biomechanical plasticity for polarized migration through confined space. Using live-cell imaging to track GBM cells negotiating microchannels, we reveal active endocytosis at cell front and filamentous actin assembly at rear to propel GBM cells through constrictions. These two processes are interconnected and governed by Plexin-B2 that orchestrates cortical actin and membrane tension, shown by biomechanical assays. Molecular dynamics simulations predict that balanced membrane and actin tension are required for optimal migratory velocity and consistency. Furthermore, Plexin-B2 mechanosensitive function requires a bendable extracellular ring structure and affects membrane internalization, permeability, phospholipid composition, as well as inner membrane surface charge. Together, our studies unveil a key element of membrane tension and mechanoelectrical coupling via Plexin-B2 that enables GBM cells to adapt to physical constraints and achieve polarized confined migration.
ABSTRACT
Developing a simple, reliable, and sensitive hepatitis C virus (HCV) genetic sensing platform is of great significance for diagnosing diseases and selecting appropriate antiviral treatments. Herein, a tandem nucleic acid amplification strategy for sensitive detection of HCV genotype 1b (HCV-1b) was developed by stringing the catalytic hairpin assembly (CHA) and the triggered DNAzyme amplifier. The hairpin reactants were initiated by the target to produce lots of triggering double-stranded DNA sequences which can efficiently activate the subsequent blocked DNAzyme. Thereby, the continuous cleavage of substrate was realized, resulting in the fluorescence signal amplification. The DNA-based isothermal CHA-DNAzyme (CDz) sensing platform was successfully applied for sensitive detection of HCV-1b with the limit of detection (84 pM) and showed good selectivity. Moreover, the practical detection of target DNA in the complex biologic matrix indicated that the developing strategy had good potential for early HCV infection diagnosis.
Subject(s)
Biosensing Techniques , DNA, Catalytic , Hepatitis C , Humans , DNA, Catalytic/genetics , Hepacivirus/genetics , Feedback , Biosensing Techniques/methods , DNA/genetics , Hepatitis C/diagnosis , Genotype , Limit of DetectionABSTRACT
OBJECTIVE: To delineate a deletional mutation of the HLA-B gene in a Chinese pedigree. METHODS: A female patient with acute myeloid leukemia who had visited Liuzhou People's Hospital in April 2022 was selected as the study subject. Routine human leukocyte antigen (HLA) was determined by using PCR-sequence specific oligonucleotide polymorphism (PCR-SSOP) and PCR-sequence-based typing (PCR-SBT) methods. Next generation sequencing (NGS) was used to validate the candidate variant in the HLA-B gene. RESULTS: The PCR-SBT and SSOP results for the HLA-B locus were inconsistent for the patient and her daughter. The SSOP results of the two individuals were HLA-B*35:01, 40:02 and HLA-B*35:01, 40:01, respectively. However, the PCR-SBT results has indicated a mismatch with the nearest HLA-B*35:01 at exon 4. NGS results showed that the HLA-B*35:01 had a 9 bp deletion in the intron 5. The patient's husband was HLA-B*40:01, 58:01, which was normal. CONCLUSION: The variant in intron 5 of the HLA-B gene in this pedigree has mapped to a primer-binding region for the SBT reagent, which has affected the accuracy of PCR-SBT results.
Subject(s)
HLA Antigens , HLA-B Antigens , Humans , Female , Alleles , Pedigree , HLA Antigens/genetics , HLA-B Antigens/genetics , China , Histocompatibility Testing/methods , Sequence Analysis, DNA/methodsABSTRACT
HLA-A*30:211 differs from HLA-A*30:01:01:01 by one nucleotide change at nucleotide 344 in exon 3 from G to C.
Subject(s)
High-Throughput Nucleotide Sequencing , Nucleotides , Humans , Alleles , China , HLA-A Antigens/geneticsABSTRACT
HLA-A*02:1103 differs from HLA-A*02:01:01:01 by one nucleotide change at nucleotide 811 in exon 4 from G to A.
Subject(s)
HLA-A Antigens , High-Throughput Nucleotide Sequencing , Humans , Alleles , HLA-A Antigens/genetics , Nucleotides , ChinaABSTRACT
Injured neurons sense environmental cues to balance neural protection and axon regeneration, but the mechanisms are unclear. Here, we unveil aryl hydrocarbon receptor (AhR), a ligand-activated bHLH-PAS transcription factor, as molecular sensor and key regulator of acute stress response at the expense of axon regeneration. We demonstrate responsiveness of DRG sensory neurons to ligand-mediated AhR signaling, which functions to inhibit axon regeneration. Ahr deletion mimics the conditioning lesion in priming DRG to initiate axonogenesis gene programs; upon peripheral axotomy, Ahr ablation suppresses inflammation and stress signaling while augmenting pro-growth pathways. Moreover, comparative transcriptomics revealed signaling interactions between AhR and HIF-1α, two structurally related bHLH-PAS α units that share the dimerization partner Arnt/HIF-1ß. Functional assays showed that the growth advantage of AhR-deficient DRG neurons requires HIF-1α; but in the absence of Arnt, DRG neurons can still mount a regenerative response. We further unveil a link between bHLH-PAS transcription factors and DNA hydroxymethylation in response to peripheral axotomy, while neuronal single cell RNA-seq analysis revealed a link of the AhR regulon to RNA polymerase III regulation and integrated stress response (ISR). Altogether, AhR activation favors stress coping and inflammation at the expense of axon regeneration; targeting AhR can enhance nerve repair.
ABSTRACT
Axon regeneration of dorsal root ganglia (DRG) neurons after peripheral axotomy involves reconfiguration of gene regulatory circuits to establish regenerative gene programs. However, the underlying mechanisms remain unclear. Here, through an unbiased survey, we show that the binding motif of Bmal1, a central transcription factor of the circadian clock, is enriched in differentially hydroxymethylated regions (DhMRs) of mouse DRG after peripheral lesion. By applying conditional deletion of Bmal1 in neurons, in vitro and in vivo neurite outgrowth assays, as well as transcriptomic profiling, we demonstrate that Bmal1 inhibits axon regeneration, in part through a functional link with the epigenetic factor Tet3. Mechanistically, we reveal that Bmal1 acts as a gatekeeper of neuroepigenetic responses to axonal injury by limiting Tet3 expression and restricting 5hmC modifications. Bmal1-regulated genes not only concern axon growth, but also stress responses and energy homeostasis. Furthermore, we uncover an epigenetic rhythm of diurnal oscillation of Tet3 and 5hmC levels in DRG neurons, corresponding to time-of-day effect on axon growth potential. Collectively, our studies demonstrate that targeting Bmal1 enhances axon regeneration.
Subject(s)
Circadian Clocks , Dioxygenases , Epigenesis, Genetic , Nerve Regeneration , Sensory Receptor Cells , Animals , Mice , Axons , Circadian Clocks/genetics , Nerve Regeneration/geneticsABSTRACT
HLA-B*13:179 differs from HLA-B*13:99 by one nucleotide substitution at position 829(A>G) in exon 4.
Subject(s)
East Asian People , High-Throughput Nucleotide Sequencing , Humans , Alleles , Asian People/genetics , HLA-B Antigens/geneticsABSTRACT
Glioblastoma (GBM), a highly lethal brain cancer, is notorious for immunosuppression, but the mechanisms remain unclear. Here, we documented a temporospatial patterning of tumor-associated myeloid cells (TAMs) corresponding to vascular changes during GBM progression. As tumor vessels transitioned from the initial dense regular network to later scant and engorged vasculature, TAMs shifted away from perivascular regions and trafficked to vascular-poor areas. This process was heavily influenced by the immunocompetence state of the host. Utilizing a sensitive fluorescent UnaG reporter to track tumor hypoxia, coupled with single-cell transcriptomics, we revealed that hypoxic niches attracted and sequestered TAMs and cytotoxic T lymphocytes (CTLs), where they were reprogrammed toward an immunosuppressive state. Mechanistically, we identified chemokine CCL8 and cytokine IL-1ß as two hypoxic-niche factors critical for TAM trafficking and co-evolution of hypoxic zones into pseudopalisading patterns. Therefore, perturbation of TAM patterning in hypoxic zones may improve tumor control.
Subject(s)
Glioblastoma , T-Lymphocytes, Cytotoxic , Humans , Tumor-Associated Macrophages , Macrophages , Immunosuppression Therapy , Glioblastoma/pathology , Tumor MicroenvironmentABSTRACT
B*15:664 differs from B*15:02:01:01 by one nucleotide change at nucleotide 755 in exon 4 from C to G.
Subject(s)
HLA-B Antigens , Humans , Alleles , East Asian People/genetics , High-Throughput Nucleotide Sequencing , HLA-B Antigens/genetics , NucleotidesABSTRACT
Plasmonic nanomaterials have attracted great attention in the field of catalysis and sensing for their outstanding electrical and optical properties. Here, a representative type of nonstoichiometric Cu2-xSe nanoparticles with typical near-infrared (NIR) localized surface plasma resonance (LSPR) properties originating from their copper deficiency was applied to catalyze the oxidation of colorless TMB into their blue product in the presence of H2O2, indicating they had good peroxidase-like activity. However, glutathione (GSH) inhibited the catalytic oxidation of TMB, as it can consume the reactive oxygen species. Meanwhile, it can induce the reduction of Cu(II) in Cu2-xSe, resulting in a decrease in the degree of copper deficiency, which can lead to a reduction in the LSPR. Therefore, the catalytic ability and photothermal responses of Cu2-xSe were decreased. Thus, in our work, a colorimetric/photothermal dual-readout array was developed for the detection of GSH. The linear calibration for GSH concentration was in the range of 1-50 µM with the LOD as 0.13 µM and 50-800 µM with the LOD as 39.27 µM. To evaluate the practicability of the assay, tomatoes and cucumbers were selected as real samples, and good recoveries indicated that the developed assay had great potential in real applications.