Biology subjects

Shang, P.

Publications and source records attributed to Shang, P..

9 recordsLinked to original sources

bioRxiv2026

Characterization of HLA-restricted GAD65-specific CD8+ T cell responses in patients with GAD65 antibody-associated neurological disorders

BackgroundHigh-titer glutamic acid decarboxylase 65 (GAD65) antibodies are found in patients with GAD65 antibody-associated neurological disorders, including stiff-person syndrome (SPS), GAD65 cerebellar ataxia (CA), and GAD65 epilepsy. Given the intracellular localization of the antigenic target, a direct pathogenic role for GAD65 autoantibodies is unlikely. Instead, the autoantibody may be a biomarker for the existence of pathogenic anti-GAD65 autoreactive CD8+ T cells. MethodsPBMC-derived dendritic cells (DCs) from 20 GAD65 antibody-positive patients with neurological manifestations (SPS, n=10; CA, n=7; epilepsy, n=3) and 15 healthy controls were pulsed with full-length GAD65 protein, full-length GAD67 protein, overlapping 15-mer peptide pools spanning GAD65 and GAD67, individual GAD65 15-mer peptides, or GAD65 9-mer peptides selected from predicted class I binding hotspots. T cell activation was quantified by flow cytometry-based activation-induced marker (AIM) assays using CD69 upregulation. Class I and class II HLA haplotypes were determined by high resolution typing. NetMHCpan v4.2c was used to generate residue-level peptide:HLA binding density maps across GAD65, and candidate 9-mers were validated for HLA binding by peptide:MHC monomer affinity testing. GAD65-peptide-HLA-restricted CD8+ T cells were identified by co-staining with two separately assembled tetramers carrying the same peptide:HLA complex on different fluorophores (APC and BV421), with double-positive events scored as antigen-specific. HLA-restricted cytotoxicity was measured by coculture of patient CD8+ T cells with GAD65-expressing HEK-293T cells reconstituted with defined HLA class I alleles using AAV-delivered Cre-dependent HLA-2A-eGFP cassettes. ResultsCD8+ T cells from GAD65 antibody-positive patients showed increased activation in response to DCs pulsed with full-length GAD65 relative to healthy controls (P=0.0157, Welchs t-test), whereas responses to GAD67 did not differ significantly between groups. CD4+ T cells responded to both GAD65 (P=0.0004) and GAD67 (P=0.0051). Peptide pool screening of GAD65 identified discrete CD8+ and CD4+ immunogenic regions, with refinement using individual 15-mers localizing CD8+ activity to multiple subdomains within GAD65(205-300), GAD65(316-435), and GAD65(447-520). HLA class I haplotyping in 16 non-Hispanic White GAD65+ patients revealed enrichment of HLA-B*08:01 (3.0-fold) and HLA-B*40:01 (4.1-fold) relative to USA NMDP European Caucasian reference frequencies (both BH q < 0.05), with suggestive enrichment of HLA-C*03:04 (2.9-fold; q = 0.09). Additionally, 44% of patients carried the HLA-A*01:01, HLA-B*08:01, and HLA-C*07:01 8.1 ancestral haplotype, approximately four-fold higher than the expected population frequency. Dual-fluorophore tetramer staining identified CD8+ T cells in GAD65+ subjects that bound a subset of HLA-A*11:01- and HLA-B*08:01-restricted GAD65 9-mers, with the clearest disease-skewed signals localized to GAD65(213-221), GAD65(257-265), and GAD65(529-537). In an HLA-reconstituted target-cell killing assay, CD8+ T cells from an HLA-B*08:01-positive GAD65+ patient mediated antigen- and HLA-restricted depletion of GAD65-expressing HEK-293T cells, with HLA-restricted target loss also observed in single-donor experiments across additional HLA-A, -B, and -C contexts. ConclusionsPatients with GAD65 antibody-associated neurological disorders harbor circulating CD8+ T cells that recognize discrete HLA class I-restricted GAD65 peptides and that are capable of cytotoxicity against GAD65-expressing HLA-matched target cells. We characterize the immunogenetic and cellular features of class I-restricted CD8+ T cell responses in GAD65 autoimmunity, including overrepresentation of the 8.1 ancestral haplotype, complementing the historical focus on antibodies and CD4+ T cell help, and we provide a panel of validated GAD65 peptide:HLA tetramers for prospective isolation, clonotypic analysis, and longitudinal monitoring of candidate pathogenic CD8+ T cell populations across the GAD65 antibody-associated neurological disease spectrum.

immunology↗

bioRxiv2026

Complement Factor H (Y402H) polymorphism for age-related macular degeneration alters retinal lipids

Age-related macular degeneration (AMD), the leading cause of blindness in the elderly, is associated with multiple risk factors and involves death of the retinal pigment epithelium (RPE). We investigated how the Y402H polymorphism of Complement Factor H (CFH) and cigarette smoke extract (CSE), major AMD genetic and environmental risks, affect lipid metabolism in RPE differentiated from induced pluripotent stem cells (iPSC-RPE) that were derived from human donors genotyped for low-risk (LR) or high-risk (HR) CFH. Results from discovery-based (lipidomics, proteomics) and targeted (mitochondrial fatty acid oxidation (FAO)) assays found significant genotype-dependent differences under basal conditions include higher free fatty acids and cholesterol esters in HR cells. CSE induced differences in proteins regulating lipid handling, lipolysis, and inflammation. Lower FAO in HR cells was observed in multiple donors and pairs of parent/isogenic edited lines compared with LR lines. CSE induced lipid accumulation, lipid composition remodeling, and upregulation of proteins involved in lipid synthesis/hydrolysis, production of bioactive lipid mediators, and metabolism of ceramide and cholesterol. These results elucidate putative mechanisms driving pathology in RPE harboring CFH Y402H.

systems biology↗

bioRxiv2024

An intra-family conserved high-order RNA structure within the M ORF is important for arterivirus subgenomic RNA accumulation and infectious virus production

Synthesis of subgenomic RNAs is a strategy commonly used by polycistronic positive sense single-stranded RNA viruses to express 3'-proximal genes. Members of the order of Nidovirales, including coronaviruses and arteriviruses, use a unique discontinuous transcription strategy to synthesize subgenomic RNAs. In this study, in silico synonymous site conservation analysis and RNA structure folding predicted the existence of intra-family conserved high-order RNA structure within the M ORF of arteriviral genomes, which was further determined to be important for the transcription/accumulation of subgenomic RNAs and production of infectious viral particles. Mutations disrupting the stability of the RNA structures significantly decreased the accumulation of multiple subgenomic RNAs. In contrast, the impact of mutagenesis on full-length genomic RNA accumulation was limited. The degree to which wild-type levels of subgenomic RNA accumulation were maintained was found to correlate with the efficiency of infectious virus production. Moreover, the thermo-stability of stems within the high-order RNA structure is also well correlated with viral replication capacity and the maintenance of subgenomic RNA accumulation. This study is the first to report an intra-Arteriviridae conserved high-order RNA structure that is located in a protein-coding region and functions as an important cis-acting element to control the accumulation/transcription of arteriviral subgenomic RNAs. This work suggests a complex regulation mechanism between genome replication and discontinuous transcription in nidoviruses. IMPORTANCEArteriviruses are a group of RNA viruses that infect different animal species. They can cause diseases associated with respiratory/reproductive syndromes, abortion, or haemorrhagic fever. Among arteriviruses, porcine reproductive and respiratory syndrome virus (PRRSV) and equine arteritis virus (EAV) are economically important veterinary pathogens. The challenge in control of arterivirus infection reflects our limited knowledge of viral biology. In this study, we conducted a comprehensive bioinformatical analysis of arteriviral genomes and discovered intra-family conserved regions in the M ORF with a high-order RNA structure. The thermo-stability of the RNA structure influences sgRNA transcription/accumulation and correlates with the level of infectious virus production. Our studies provide a new insight on arterivirus replication mechanism, which may have implications in developing disease control and prevention strategies.

molecular biology↗

bioRxiv2023

Anti-tau antibodies targeting a conformation-dependent epitope selectively bind seeds

Neurodegenerative tauopathies are caused by transition of tau protein from a monomer to a toxic aggregate. They include Alzheimer disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and Pick disease (PiD). We have previously proposed that tau monomer exists in two conformational ensembles: an inert form (Mi), which does not self-assemble; and seed-competent form (Ms), which self-assembles and templates ordered assembly growth. We proposed that cis/trans isomerization of tau at P301, the site of dominant disease-associated S/L mis-sense mutations, might underlie the transition of wild-type tau to a seed-competent state. Consequently, we created monoclonal antibodies using non-natural antigens consisting of fluorinated proline (P*) at the analogous P270 in R1, biased towards the trans-configuration at either the R1/R2 (TENLKHQP*GGGKVQIINKK) or R1/R3 (TENLKHQP*GGGKVQIVYK) interfaces. Two antibodies, MD2.2 and MD3.1 efficiently immunoprecipitated soluble seeds from AD and PSP, but not CBD or PiD. They stained brain samples of AD, PSP, and PiD, but not CBD. They did not immunoprecipitate, or immunostain tau from control brain. Creation of potent anti-seed antibodies based on the trans-proline epitope implicates local unfolding around P301 in pathogenesis. MD2.2 and MD3.1 may also be useful for therapy and diagnosis. SummaryTau protein undergoes conformational change to self-assemble and trigger neurodegeneration. We have proposed local unfolding events centered on P301 cis/trans isomerization, which expose amyloidogenic sequences. We used a non-natural peptide with a trans-proline to generate monoclonal antibodies that distinguish tau seeds from native tau in human brain. In addition to being important therapeutic and diagnostic leads, the activity of these antibodies supports structural studies implicating local conformational change in tau that underlies disease initiation.

neuroscience↗

bioRxiv2023

Collateral RNA cleavage by CRISPR-Cas13 allows selective cell elimination

CRISPR-Cas13 exclusively targets RNA. In prokaryotic cells, Cas13 cleaves both target and non-target RNA indiscriminately upon activation by a specific target RNA, but in eukaryotic cells collateral cleavage activity has been limited. To investigate collateral cleavage by Cas13 in eukaryotic cells, we first compared various Cas13 orthologs and found that specifically LbuCas13a exhibits strong collateral RNA cleavage activity in human cells when delivered as ribonucleoprotein, independent of cell line and targeting both exogenous and endogenous transcripts. Collateral RNA cleavage started within 50 minutes of ribonucleoprotein delivery resulting in major alterations to the total RNA profile. In response to the collateral RNA cleavage, cells upregulated genes associated with stress and innate immune response, ultimately leading to apoptotic cell death. This enabled us to use LbuCas13a as a flexible and repeatable target-RNA-specific cell elimination tool. Finally, we used Nanopore sequencing to explore the identity of collaterally cleaved RNAs, the nucleotide position at which they are cleaved, and the temporal dynamics of collateral RNA cleavage. This revealed that LbuCas13a activation leads to rapid and global cleavage of cytoplasmic RNAs at specific nucleotide positions. In conclusion, we here report that LbuCas13a has high collateral activity in human cells and describe the temporal dynamics of the collateral RNA cleavage, the cellular responses ultimately leading to apoptosis, how this can be exploited as a cell elimination tool, and the collateral cleavage preferences of LbuCas13a.

molecular biology↗

bioRxiv2022

Modulating CRISPR-Cas genome editing using guide-complementary DNA oligonucleotides

CRISPR-Cas has revolutionized genome editing and has a great potential for applications, such as correcting human genetic disorders. To increase the safety of genome editing applications, CRISPR-Cas may benefit from strict control over Cas enzyme activity. Previously, anti-CRISPR proteins and designed oligonucleotides have been proposed to modulate CRISPR-Cas activity. Here we report on the potential of guide-complementary DNA oligonucleotides as controlled inhibitors of Cas9 ribonucleoprotein complexes. First, we show that DNA oligonucleotides down-regulate Cas9 activity in human cells, reducing both on and off-target cleavage. We then used in vitro assays to better understand how inhibition is achieved and under which conditions. Two factors were found to be important for robust inhibition: the length of the complementary region, and the presence of a PAM-loop on the inhibitor. We conclude that DNA oligonucleotides can be used to effectively inhibit Cas9 activity both ex vivo and in vitro.

molecular biology↗

bioRxiv2021

Ribosome profiling of porcine reproductive and respiratory syndrome virus reveals novel features of viral gene expression

Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus which causes significant economic losses to the swine industry worldwide. Here, we use ribosome profiling (RiboSeq) and parallel RNA sequencing (RNASeq) to characterise the transcriptome and translatome of both species of PRRSV and analyse the host response to infection. We quantified viral gene expression over a timecourse of infection, and calculated the efficiency of programmed ribosomal frameshifting (PRF) at both sites on the viral genome. At the nsp2 frameshift site (a rare example of protein-stimulated frameshifting), -2 PRF efficiency increases over time, likely facilitated by accumulation of the PRF- stimulatory viral protein (nsp1{beta}) during infection. This marks arteriviruses as the second example of temporally regulated PRF. Surprisingly, we also found PRF efficiency at the canonical ORF1ab frameshift site increases over time, in apparent contradiction of the common assumption that RNA structure-directed frameshift sites operate at a fixed efficiency. This has potential implications for the numerous other viruses with canonical PRF sites. Furthermore, we discovered several highly translated additional viral ORFs, the translation of which may be facilitated by multiple novel viral transcripts. For example, we found a 125-codon ORF overlapping nsp12, which is expressed as highly as nsp12 itself at late stages of replication, and is likely translated from novel subgenomic (sg) RNA transcripts that overlap the 3' end of ORF1b. Similar transcripts were discovered for both PRRSV-1 and PRRSV- 2, suggesting a potential conserved mechanism for temporal regulation of expression of the 3'-proximal region of ORF1b. In addition, we identified a highly translated, short upstream ORF (uORF) in the 5' UTR, the presence of which is highly conserved amongst PRRSV-2 isolates. This is the first application of RiboSeq to arterivirus-infected cells, and reveals new features which add to the complexity of gene expression programmes in this important family of nidoviruses.

pathology↗

bioRxiv2020

Neuron-intrinsic NF-κB Signaling Mediates Reovirus Virulence

Pathological effects of apoptosis associated with viral infections of the central nervous system are an important cause of morbidity and mortality. Reovirus is a neurotropic virus that causes apoptosis in neurons, leading to lethal encephalitis in newborn mice. Reovirus-induced encephalitis is diminished in mice with germline ablation of NF-{kappa}B subunit p50. It is not known whether the pro-apoptotic function of NF-{kappa}B is mediated by neuron-intrinsic processes, NF-{kappa}B-regulated cytokine production by inflammatory cells, or a combination of both. To determine the contribution of cell type-specific NF-{kappa}B signaling in reovirus-induced neuronal injury, we established mice that lack NF-{kappa}B p65 expression in neurons using the Cre/loxP recombination system. Following intracranial inoculation of reovirus, 50% of wild-type (WT) mice succumbed to infection, whereas more than 90% of mice lacking neural NF-{kappa}B p65 (Nsp65-/-) mice survived. While viral loads in brains of WT and Nsp65-/- were comparable, histological analysis revealed that reovirus antigen-positive areas in the brain of WT mice displayed enhanced cleaved caspase-3 immunoreactivity, a marker of apoptosis, compared with Nsp65-/- mice. These data suggest that neuron-intrinsic NF-{kappa}B-dependent factors are essential mediators of reovirus neurovirulence. RNA sequencing analysis of reovirus-infected cortices of WT and Nsp65-/- mice suggests that NF-{kappa}B activation in neurons upregulates genes involved in innate immunity, inflammation, and cell death following reovirus infection. A better understanding of the contribution of cell type-specific NF-{kappa}B-dependent signaling to viral neuropathogenesis could inform development of new therapeutics that target and protect highly vulnerable cell populations

microbiology↗

bioRxiv2018

A widespread decrease of chromatin accessibility in age-related macular degeneration

Age-related macular degeneration (AMD) is a leading cause of blindness in the elderly. The extent to which epigenetic changes regulate AMD progression is unclear. Here we globally profiled chromatin accessibility in the retina and retinal pigmented epithelium (RPE) from AMD patients and controls. Global decreases in chromatin accessibility occurr in RPE in early AMD, and in the retina with advanced disease, suggesting that dysfunction in RPE cells drives disease progression. Footprints of photoreceptor and RPE-specific transcription factors are enriched in differentially accessible regions (DARs). Genes associated with DARs show altered expression in AMD. Cigarette smoke treatment of RPE cells recapitulates epigenomic changes seen in AMD, providing an epigenetic link between the known risk factors for AMD and AMD pathology. Finally, overexpression of HDAC11 is partially responsible for the reduction in chromatin accessibility, identifying potential new targets for treatment of AMD.

genomics↗