-
Molecular Brain Sep 2023Novelty-induced memory consolidation is a well-established phenomenon that depends on the activation of a locus coeruleus-hippocampal circuit. It is associated with the...
Novelty-induced memory consolidation is a well-established phenomenon that depends on the activation of a locus coeruleus-hippocampal circuit. It is associated with the expression of activity-dependent genes that may mediate initial or cellular memory consolidation. Several genes have been identified to date, however, to fully understand the mechanisms of memory consolidation, additional candidates must be identified. In this cross-species study, we used a contextual novelty-exploration paradigm to identify changes in gene expression in the dorsal hippocampus of both mice and rats. We found that changes in gene expression following contextual novelty varied between the two species, with 9 genes being upregulated in mice and 3 genes in rats. Comparison across species revealed that ArfGAP with a GTPase domain, an ankyrin repeat and PH domain 3 (Agap3) was the only gene being upregulated in both, suggesting a potentially conserved role for Agap3. AGAP3 is known to regulate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor trafficking in the synapse, which suggests that increased transcription of Agap3 may be involved in maintaining functional plasticity. While we identified several genes affected by contextual novelty exploration, we were unable to fully reverse these changes using SCH 23390, a dopamine D/D receptor antagonist. Further research on the role of AGAP3 in novelty-induced memory consolidation could lead to better understanding of this process and guide future research.
Topics: Animals; Mice; Rats; Dopamine; Glutamic Acid; Hippocampus; Locus Coeruleus; Memory Consolidation; Receptors, AMPA; GTPase-Activating Proteins
PubMed: 37749596
DOI: 10.1186/s13041-023-01056-4 -
BioRxiv : the Preprint Server For... Sep 2023General methods for spatiotemporal control of specific endogenous proteins would be broadly useful for probing protein function in living cells. Synthetic protein...
General methods for spatiotemporal control of specific endogenous proteins would be broadly useful for probing protein function in living cells. Synthetic protein binders that bind and inhibit endogenous protein targets can be obtained from nanobodies, designed ankyrin repeat proteins (DARPins), and other small protein scaffolds, but generalizable methods to control their binding activity are lacking. Here, we report robust single-chain photoswitchable DARPins (psDARPins) for bidirectional optical control of endogenous proteins. We created topological variants of the DARPin scaffold by computer-aided design so fusion of photodissociable dimeric Dronpa (pdDronpa) results in occlusion of target binding at baseline. Cyan light induces pdDronpa dissociation to expose the binding surface (paratope), while violet light restores pdDronpa dimerization and paratope caging. Since the DARPin redesign leaves the paratope intact, the approach was easily applied to existing DARPins for GFP, ERK, and Ras, as demonstrated by relocalizing GFP-family proteins and inhibiting endogenous ERK and Ras with optical control. Finally, a Ras-targeted psDARPin was used to determine that, following EGF-activation of EGFR, Ras is required for sustained EGFR to ERK signaling. In summary, psDARPins provide a generalizable strategy for precise spatiotemporal dissection of endogenous protein function.
PubMed: 37745504
DOI: 10.1101/2023.09.14.557687 -
Journal of Integrative Neuroscience Aug 2023To identify suitable reference genes for gene expression studies in rat dorsal root ganglia (DRG) neurons.
OBJECTIVE
To identify suitable reference genes for gene expression studies in rat dorsal root ganglia (DRG) neurons.
METHODS
The raw cycle threshold (Ct) values of 12 selected reference genes were obtained via quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) in neurons at different developmental stages or under different treatments. Two strategies were employed to screen the most stable reference genes: the genes were ranked according to the coefficient of biological variation and further validated using geNorm and NormFinder programs. The stable and unstable reference genes were subsequently used as internal controls to assess their effects on target gene expression.
RESULTS
All reference genes showed varying degrees of fluctuation in Ct values during the growth process of neurons or after different treatments. 18S ribosomal RNA () and β-actin () exhibited the most significant changes, while ubiquitin C (), hypoxanthine phosphoribosyl transferase (), and mitochondrial ribosomal protein L10 () showed relatively minor changes. The most stable and unstable genes obtained by different evaluation methods varied slightly. Overall, was found to be the most unstable reference gene, while was the relatively most stable reference gene. The use of unstable reference genes and ankyrin repeat domain 27 () as internal controls led to high variability within the control group, ultimately affecting the determination of target gene expression. In contrast, the stable reference gene had small inter-assay variation and high stability.
CONCLUSIONS
Our observations indicate that is a proper endogenous reference gene for qRT-PCR analysis in rat DRG neurons and thus provides a critical molecular basis for the genetic characterization in neurological disorders.
Topics: Animals; Rats; Ganglia, Spinal; Reverse Transcription; Neurons; Polymerase Chain Reaction
PubMed: 37735125
DOI: 10.31083/j.jin2205125 -
BMC Genomic Data Sep 2023Peucedanum praeruptorum Dunn, a traditional Chinese herbal medicine, contains coumarin and volatile oil components that have clinical application value. However, early...
BACKGROUND
Peucedanum praeruptorum Dunn, a traditional Chinese herbal medicine, contains coumarin and volatile oil components that have clinical application value. However, early bolting often occurs in the medicinal materials of Apiaceae plants. The rhizomes of the medicinal parts are gradually lignified after bolting, resulting in a sharp decrease in the content of coumarins. At present, the link between coumarin biosynthesis and early bolting in P. praeruptorum has not been elucidated.
RESULTS
Combining the genome sequencing and the previous transcriptome sequencing results, we reanalyzed the differential transcripts of P. praeruptorum before and after bolting. A total of 62,088 new transcripts were identified, of which 31,500 were unknown transcripts. Functional classification and annotation showed that many genes were involved in the regulation of transcription, defense response, and carbohydrate metabolic processes. The main domains are the pentatricopeptide repeat, protein kinase, RNA recognition motif, leucine-rich repeat, and ankyrin repeat domains, indicating their pivotal roles in protein modification and signal transduction. Gene structure analysis showed that skipped exon (SE) was the most dominant alternative splicing, followed by the alternative 3' splice site (A3SS) and the alternative 5' splice site (A5SS). Functional enrichment of differentially expressed genes showed that these differentially expressed genes mainly include transmembrane transporters, channel proteins, DNA-binding proteins, polysaccharide-binding proteins, etc. In addition, genes involved in peroxisome, hexose phosphate pathway, phosphatidylinositol signaling system, and inositol phosphate metabolism pathway were greatly enriched. A protein-protein interaction network analysis discoverd 1,457 pairs of proteins that interact with each other. The expression levels of six UbiA genes, three UGT genes, and four OMT genes were higher during the bolting stage. This observation suggests their potential involvement in the catalytic processes of prenylation, glycosylation, and methylation of coumarins, respectively. A total of 100 peroxidase (PRX) genes were identified being involved in lignin polymerization, but only nine PRX genes were highly expressed at the bolting stage. It is worth noting that 73 autophagy-related genes (ATGs) were first identified from the KEGG pathway-enriched genes. Some ATGs, such as BHQH00009837, BHQH00013830, and novel8944, had higher expression levels after bolting.
CONCLUSIONS
Comparative transcriptome analysis and large-scale genome screening provide guidance and new opinions for the identification of bolting-related genes in P. praeruptorum.
Topics: Transcriptome; Chromosome Mapping; Gene Expression Profiling; Exons; Apiaceae
PubMed: 37723451
DOI: 10.1186/s12863-023-01157-y -
Clinical and Translational Medicine Sep 2023Predictive biomarkers for oesophageal squamous cell carcinoma (ESCC) immunotherapy are lacking, and immunotherapy resistance remains to be addressed. The role of long...
BACKGROUND
Predictive biomarkers for oesophageal squamous cell carcinoma (ESCC) immunotherapy are lacking, and immunotherapy resistance remains to be addressed. The role of long noncoding RNA (lncRNA) in ESCC immune escape and immunotherapy resistance remains to be elucidated.
METHODS
The tumour-associated macrophage-upregulated lncRNAs and the exosomal lncRNAs highly expressed in ESCC immunotherapy nonresponders were identified by lncRNA sequencing and polymerase chain reaction assays. CRISPR-Cas9 was used to explore the functional roles of the lncRNA. RNA pull-down, MS2-tagged RNA affinity purification (MS2-TRAP) and RNA-binding protein immunoprecipitation (RIP) were performed to identify lncRNA-associated proteins and related mechanisms. In vivo, the humanized PBMC (hu-PBMC) mouse model was established to assess the therapeutic responses of specific lncRNA inhibitors and their combination with programmed cell death protein 1 (PD-1) monoclonal antibody (mAb). Single-cell sequencing, flow cytometry, and multiplex fluorescent immunohistochemistry were used to analyze immune cells infiltrating the tumour microenvironment.
RESULTS
We identified a lncRNA that is involved in tumour immune evasion and immunotherapy resistance. High LINC02096 (RIME) expression in plasma exosomes correlates with a reduced response to PD-1 mAb treatment and poor prognosis. Mechanistically, RIME binds to mixed lineage leukaemia protein-1 (MLL1) and prevents ankyrin repeat and SOCS box containing 2 (ASB2)-mediated MLL1 ubiquitination, improving the stability of MLL1. RIME-MLL1 increases H3K4me3 levels in the promoter regions of programmed death-ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase 1 (IDO-1), constitutively increasing the expression of PD-L1/IDO-1 in tumour cells and inhibiting CD8 T cells infiltration and activation. RIME depletion in huPBMC-NOG mice significantly represses tumour development and improves the effectiveness of PD-1 mAb treatment by activating T-cell-mediated antitumour immunity.
CONCLUSIONS
This study reveals that the RIME-MLL1-H3K4me3 axis plays a critical role in tumour immunosuppression. Moreover, RIME appears to be a potential prognostic biomarker for immunotherapy and developing drugs that target RIME may be a new therapeutic strategy that overcomes immunotherapy resistance and benefits patients with ESCC.
Topics: Animals; Mice; Antibodies, Monoclonal; B7-H1 Antigen; CD8-Positive T-Lymphocytes; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Leukocytes, Mononuclear; Myeloid-Lymphoid Leukemia Protein; Programmed Cell Death 1 Receptor; RNA, Long Noncoding; Tumor Microenvironment
PubMed: 37712124
DOI: 10.1002/ctm2.1410 -
Molecular Medicine Reports Oct 2023Chronic complications of diabetes increase mortality and disability of patients. It is crucial to find potential early biomarkers and provide novel therapeutic...
Chronic complications of diabetes increase mortality and disability of patients. It is crucial to find potential early biomarkers and provide novel therapeutic strategies for diabetic complications. Circular RNAs (circRNAs), covalently closed RNA molecules in eukaryotes, have high stability. Recent studies have confirmed that differentially expressed circRNAs have a vital role in diabetic complications. Certain circRNAs, such as circRNA ankyrin repeat domain 36, circRNA homeodomain‑interacting protein kinase 3 (circHIPK3) and circRNA WD repeat domain 77, are associated with inflammation, endothelial cell apoptosis and smooth muscle cell proliferation, leading to vascular endothelial dysfunction and atherosclerosis. CircRNA LDL receptor related protein 6, circRNA actin related protein 2, circ_0000064, circ‑0101383, circ_0123996, hsa_circ_0003928 and circ_0000285 mediate inflammation, apoptosis and autophagy of podocytes, mesangial cell hypertrophy and proliferation, as well as tubulointerstitial fibrosis, in diabetic nephropathy by regulating the expression of microRNAs and proteins. Circ_0005015, circRNA PWWP domain containing 2A, circRNA zinc finger protein 532, circRNA zinc finger protein 609, circRNA DNA methyltransferase 3β, circRNA collagen type I α2 chain and circHIPK3 widely affect multiple biological processes of diabetic retinopathy. Furthermore, circ_000203, circ_010567, circHIPK3, hsa_circ_0076631 and circRNA cerebellar degeneration‑related protein 1 antisense are involved in the pathology of diabetic cardiomyopathy. CircHIPK3 is the most well‑studied circRNA in the field of diabetic complications and is most likely to become a biological marker and therapeutic target for diabetic complications. The applications of circRNAs may be a promising treatment strategy for human diseases at the molecular level. The relationship between circRNAs and diabetic complications is summarized in the present study. Of note, circRNA‑targeted therapy and the role of circRNAs as biomarkers may potentially be used in diabetic complications in the future.
Topics: Humans; RNA, Circular; Diabetic Nephropathies; MicroRNAs; Biomarkers; Diabetic Retinopathy; Diabetes Mellitus
PubMed: 37681455
DOI: 10.3892/mmr.2023.13081 -
Virus Research Oct 2023Avipoxvirus 282E4 strain was extensively applied into recombinant vaccine vector to prevent other infectious diseases. However, little information on the genomic...
Avipoxvirus 282E4 strain was extensively applied into recombinant vaccine vector to prevent other infectious diseases. However, little information on the genomic background, functional and genetic evolutionary of the isolate 282E4 strain was clarified. The results showed that the linear genome of avipoxvirus 282E4 was 308,826 bp, containing 313 open reading frames (ORFs) and 12 new predicted ORFs. The 282E4 strain appears to encode two novel thymidine kinase proteins and two TGF-beta-like proteins that may be associated with the suppression of the host's antiviral response. Avipoxvirus 282E4 also encodes 57 ankyrin repeat proteins and 5 variola B22R-like proteins, which composed 7% of the avipoxvirus 282E4 genome. GO and KEGG analysis further revealed that 12 ORFs participate in viral transcription process, 7 ORFs may function during DNA repair, replication and biological synthesis, and ORF 208 is involved in the process of virus life cycle. Interestingly, phylogenetic analysis based on concatenated sequences p4b and DNA polymerase of avipoxviruses gene demonstrates that avipoxvirus 282E4 strain is divergent from known FWPV isolates and is similar to shearwater poxvirus (SWPV-1) that belongs to the CNPV-like virus. Sequencing avipoxvirus 282E4 is a significant step to judge the genetic position of avipoxviruses within the larger Poxviridae phylogenetic tree and provide a new insight into the genetic background of avipoxvirus 282E4 and interspecies transmission of poxviruses, meanwhile, explanation of gene function provides theoretical foundation for vaccine design with 282E4 strain as skeleton.
PubMed: 37678517
DOI: 10.1016/j.virusres.2023.199218 -
European Journal of Nuclear Medicine... Jul 2024Fluorescence-guided surgery (FGS) can play a key role in improving radical resection rates by assisting surgeons to gain adequate visualization of malignant tissue...
Preclinical evaluation of EpCAM-binding designed ankyrin repeat proteins (DARPins) as targeting moieties for bimodal near-infrared fluorescence and photoacoustic imaging of cancer.
PURPOSE
Fluorescence-guided surgery (FGS) can play a key role in improving radical resection rates by assisting surgeons to gain adequate visualization of malignant tissue intraoperatively. Designed ankyrin repeat proteins (DARPins) possess optimal pharmacokinetic and other properties for in vivo imaging. This study aims to evaluate the preclinical potential of epithelial cell adhesion molecule (EpCAM)-binding DARPins as targeting moieties for near-infrared fluorescence (NIRF) and photoacoustic (PA) imaging of cancer.
METHODS
EpCAM-binding DARPins Ac2, Ec4.1, and non-binding control DARPin Off7 were conjugated to IRDye 800CW and their binding efficacy was evaluated on EpCAM-positive HT-29 and EpCAM-negative COLO-320 human colon cancer cell lines. Thereafter, NIRF and PA imaging of all three conjugates were performed in HT-29_luc2 tumor-bearing mice. At 24 h post-injection, tumors and organs were resected and tracer biodistributions were analyzed.
RESULTS
Ac2-800CW and Ec4.1-800CW specifically bound to HT-29 cells, but not to COLO-320 cells. Next, 6 nmol and 24 h were established as the optimal in vivo dose and imaging time point for both DARPin tracers. At 24 h post-injection, mean tumor-to-background ratios of 2.60 ± 0.3 and 3.1 ± 0.3 were observed for Ac2-800CW and Ec4.1-800CW, respectively, allowing clear tumor delineation using the clinical Artemis NIRF imager. Biodistribution analyses in non-neoplastic tissue solely showed high fluorescence signal in the liver and kidney, which reflects the clearance of the DARPin tracers.
CONCLUSION
Our encouraging results show that EpCAM-binding DARPins are a promising class of targeting moieties for pan-carcinoma targeting, providing clear tumor delineation at 24 h post-injection. The work described provides the preclinical foundation for DARPin-based bimodal NIRF/PA imaging of cancer.
Topics: Humans; Animals; Mice; Photoacoustic Techniques; Ankyrin Repeat; Epithelial Cell Adhesion Molecule; Optical Imaging; Cell Line, Tumor; Tissue Distribution; HT29 Cells; Protein Binding; Infrared Rays; Female
PubMed: 37642704
DOI: 10.1007/s00259-023-06407-w -
International Journal of Molecular... Aug 2023Ankyrin repeat and single KH domain-containing protein 1 (ANKHD1) is a large, scaffolding protein composed of two stretches of ankyrin repeat domains that mediate... (Review)
Review
Ankyrin repeat and single KH domain-containing protein 1 (ANKHD1) is a large, scaffolding protein composed of two stretches of ankyrin repeat domains that mediate protein-protein interactions and a KH domain that mediates RNA or single-stranded DNA binding. ANKHD1 interacts with proteins in several crucial signalling pathways, including receptor tyrosine kinase, JAK/STAT, mechanosensitive Hippo (YAP/TAZ), and p21. Studies into the role of ANKHD1 in cancer cell lines demonstrate a crucial role in driving uncontrolled cellular proliferation and growth, enhanced tumorigenicity, cell cycle progression through the S phase, and increased epithelial-to-mesenchymal transition. Furthermore, at a clinical level, the increased expression of ANKHD1 has been associated with greater tumour infiltration, increased metastasis, and larger tumours. Elevated ANKHD1 resulted in poorer prognosis, more aggressive growth, and a decrease in patient survival in numerous cancer types. This review aims to gather the current knowledge about ANKHD1 and explore its molecular properties and functions, focusing on the protein's role in cancer at both a cellular and clinical level.
Topics: Humans; Neoplasms; Hyperplasia; Aggression; Ankyrin Repeat; Cell Division; RNA-Binding Proteins
PubMed: 37629022
DOI: 10.3390/ijms241612834 -
Cell Chemical Biology Dec 2023An emerging strategy for the therapeutic targeting of protein phosphatases involves the use of compounds that interfere with the binding of regulatory subunits or...
An emerging strategy for the therapeutic targeting of protein phosphatases involves the use of compounds that interfere with the binding of regulatory subunits or substrates. However, high-throughput screening strategies for such interfering molecules are scarce. Here, we report on the conversion of the NanoBiT split-luciferase system into a robust assay for the quantification of phosphatase subunit and substrate interactions in cell lysates. The assay is suitable to screen small-molecule libraries for interfering compounds. We designed and validated split-luciferase sensors for a broad range of PP1 and PP2A holoenzymes, including sensors that selectively report on weak interaction sites. To facilitate efficient hit triaging in large-scale screening campaigns, deselection procedures were developed to eliminate assay-interfering molecules with high fidelity. As a proof-of-principle, we successfully applied the split-luciferase screening tool to identify small-molecule disruptors of the interaction between the C-terminus of PP1β and the ankyrin-repeat domain of the myosin-phosphatase targeting subunit MYPT1.
Topics: Protein Phosphatase 1; Protein Binding; Myosin-Light-Chain Phosphatase; Phosphorylation
PubMed: 37625414
DOI: 10.1016/j.chembiol.2023.07.018