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Maximizing the binding properties of thermoresponsive molecularly imprinted nanoparticles (MIN) was aimed to explore their feasibility as antibody substitutes in protein immunoprecipitation (IPP) with magnetic streptavidin beads (MSB). Thermoresponsive MIN targeting the cannabinoid CB1 receptor were produced by epitope imprinting through solid-phase synthesis. It was intended to determine how different variables influenced physicochemical features, binding behaviour and immunoprecipitation of the target recombinant glutathione S-transferase tagged fusion protein (GST-CTer). Such variables included the cross-linking degree of MIN, and variables like pH, temperature or the use of Tween-20 for binding and IPP experiments. The cross-linker (CL) amount influenced the coil-to-globule transition of thermoresponsive MIN, making the lower critical solution temperature (LCST) decrease from 37.2 °C using 5% of CL, to 29.0 °C using 25%, also suggesting higher plasticity on the former. Temperature influence on size was corroborated by dynamic light scattering, observing size reductions from 250–450 nm (RT) to 70–100 nm (> LCST) for MIN produced with 5–15% of CL. However, binding behaviour did not clearly improve for more than 10% CL. Further experiments revealed that temperature and pH control were critical for efficient binding and release, selecting 40 °C and pH 5 as appropriate. Following binding experiments, the GST-CTer-MIN complex was successfully immunoprecipitated using MSB, achieving an IPP efficiency of 11.48% over the initial input protein concentration, which was calculated after SDS-PAGE separation and Western blot analysis. The methodology may be exploited for selective protein extraction and quantification from complex tissue homogenates. [ABSTRACT FROM AUTHOR]

The role of long non-coding RNAs (lncRNAs) in malignant cell transformation remains elusive. We previously identified an enhancer-associated lncRNA, LINC01116 (named HOXDeRNA), as a transformative factor converting human astrocytes into glioma-like cells. Employing a combination of CRISPR editing, chromatin isolation by RNA purification coupled with sequencing (ChIRP-seq), in situ mapping RNA-genome interactions (iMARGI), chromatin immunoprecipitation sequencing (ChIP-seq), HiC, and RNA/DNA FISH, we found that HOXDeRNA directly binds to CpG islands within the promoters of 35 glioma-specific transcription factors (TFs) distributed throughout the genome, including key stem cell TFs SOX2, OLIG2, POU3F2, and ASCL1, liberating them from PRC2 repression. This process requires a distinct RNA quadruplex structure and other segments of HOXDeRNA, interacting with EZH2 and CpGs, respectively. Subsequent transformation activates multiple oncogenes (e.g., EGFR, miR-21, and WEE1), driven by the SOX2- and OLIG2-dependent glioma-specific super enhancers. These results help reconstruct the sequence of events underlying the process of astrocyte transformation, highlighting HOXDeRNA's central genome-wide activity and suggesting a shared RNA-dependent mechanism in otherwise heterogeneous and multifactorial gliomagenesis. [Display omitted] • HOXDeRNA transforms astrocytes into glioma-like stem cells • HOXDeRNA directly binds to PRC2-covered CpG islands in the promoters of glioma driver genes • HOXDeRNA removes EZH2 through its RNA quadruplex sequences Deforzh et al. demonstrate the ability of the lncRNA HOXDeRNA to transform astrocytes into glioma-like stem cells by sequestering and protecting key glioma genes from Polycomb2 repression in an RNA quadruplex-dependent manner. [ABSTRACT FROM AUTHOR]

DNA i-motif structures are formed in the nuclei of human cells and are believed to provide critical genomic regulation. While the existence, abundance, and distribution of i-motif structures in human cells has been demonstrated and studied by immunofluorescent staining, and more recently NMR and CUT&Tag, the abundance and distribution of such structures in human genomic DNA have remained unclear. Here we utilise high-affinity i-motif immunoprecipitation followed by sequencing to map i-motifs in the purified genomic DNA of human MCF7, U2OS and HEK293T cells. Validated by biolayer interferometry and circular dichroism spectroscopy, our approach aimed to identify DNA sequences capable of i-motif formation on a genome-wide scale, revealing that such sequences are widely distributed throughout the human genome and are common in genes upregulated in G0/G1 cell cycle phases. Our findings provide experimental evidence for the widespread formation of i-motif structures in human genomic DNA and a foundational resource for future studies of their genomic, structural, and molecular roles. Synopsis: i-motifs (iMs) are knot-like DNA structures structures formed in the nuclei of human cells and believed to provide critical genomic regulation. This study uses immunoprecipitation and next-generation sequencing to identify i-motif structures in human DNA on a genome-wide scale. DNA i-motif structures are common in human genomic DNA. ~53,000 iMs are observed among three human cells lines (MCF7, U2OS, HEK293T). iMs are widely distributed throughout the human genome and frequent in genes upregulated in G0/G1 phase. Genome-wide i-motif immunoprecipitation and next-generation sequencing shows that sequences capable of forming these knot-like DNA structures are widely distributed and common in G0/G1-expressed genes. [ABSTRACT FROM AUTHOR]

Background: the reference method for detection of myositis-specific and myositis-associated antibodies (MSAs and MAAs) is considered immunoprecipitation (IP), but it is routinely replaced by semi-automated methods, like lineblot (LB). Few data are available on the consistency with clinical diagnoses; thus, we aim at analysing these aspects. Methods: sixty-nine patients with idiopathic inflammatory myopathies (IIM) were studied via LB (Myositis Antigens Profile 3 EUROLINE, Euroimmun) and IP (RNA and protein antigens). The degree of concordance between methods was calculated using Cohen's coefficient. Results: a substantial concordance was found for anti-Ku and anti-PM/Scl and a moderate concordance was found for anti-Jo1 and anti–Mi-2, while a fair concordance was found for anti-EJ, anti-SRP, and anti-Ro52 antibodies. The concordance could not be calculated for anti-OJ, anti-PL-7, anti-PL-12, anti-NXP2, anti-TIF1ɣ, and anti-MDA5, because they were only detected with one method. Multiple MSAs were found only with LB in 2/69 sera. Anti-MDA5, TIF1ɣ, NXP2 (detected via IP), and anti-Jo1 in anti-synthetase syndrome (both LB and IP) had the best concordance with clinical diagnosis. Conclusions: LB and IP show substantial concordance for PM/Scl and Ku, and moderate concordance for Jo1 and Mi-2, with a good concordance with clinical diagnoses. IP shows a high performance for DM-associated MSAs. LB seems to be more sensitive in detecting anti-Ro52 antibodies, but it identified multiple MSAs, unlike IP. [ABSTRACT FROM AUTHOR]

Autophagy is essential for cell survival and cellular homeostasis under various stress conditions. Therefore, autophagy dysfunction is associated with the pathogenesis of various human diseases. We explored the regulatory role of RhoBTB3 in autophagy and its interaction with activating molecules in AMBRA1. RhoBTB3 deficiency was found to induce autophagy, while its overexpression inhibited autophagy induction. Through immunoprecipitation and mass spectrometry, AMBRA1 was identified as a substrate of RhoBTB3. The study revealed that RhoBTB3 regulates AMBRA1 stability by influencing its protein levels without affecting its mRNA levels. RhoBTB3 induced the ubiquitination of AMBRA1, leading to proteasome-mediated degradation, with the ubiquitination occurring at K45 on AMBRA1 through a K27-linked ubiquitin chain. The knockdown of AMBRA1 blocked RhoBTB3 knockdown-induced autophagy, indicating the dependency of autophagy on AMBRA1. Thus, RhoBTB3 negatively regulates autophagy by mediating AMBRA1 ubiquitination and degradation, suggesting RhoBTB3 as a potential therapeutic target for autophagy-related diseases. [ABSTRACT FROM AUTHOR]

Polyglutamine (polyQ)-mediated spinocerebellar ataxia (SCA), including SCA1, 2, 3, 6, 7, and 17, are caused by mutant genes with expanded CAG repeats, leading to the intracellular accumulation of aggregated proteins, the production of reactive oxygen species, and cell death. Among SCA, SCA3 is caused by a mutation in the ATXN3 (ataxin-3) gene. In a circumstance of polyQ aggregation, the autophagic pathway is induced to degrade the aggregated proteins, thereby suppressing downstream deleterious effects and promoting neuronal survival. In this study, we tested the effects of synthetic indole (NC009-1, -2, -3, -6) and coumarin (LM-022, -031) derivatives as chemical chaperones to assist mutant ATXN3-Q75 folding, as well as autophagy inducers to clear aggregated protein. Among the tested compounds, NC009-1, -2, and -6 and LM-031 interfered with Escherichia coli-derived ATXN3-Q75 aggregation in thioflavin T binding and filter trap assays. In SH-SY5Y cells expressing GFP-fused ATXN3-Q75, these compounds displayed aggregation-inhibitory and neurite growth-promoting potentials compared to untreated cells. Furthermore, these compounds activated autophagy by increasing the phosphatidylethanolamine-conjugated LC3 (microtubule associated protein 1 light chain 3)-II:cytosolic LC3-I ratio in these cells. A biochemical co-immunoprecipitation assay by using a mixture of HEK 293T cell lysates containing recombinant ATXN3-Q75-Venus-C-terminus (VC) or Venus-N-terminus (VN)-LC3 protein indicated that NC009-1 and -2 and LM-031 served as an autophagosome-tethering compound (ATTEC) to interact with ATXN3-Q75 and LC3, and the interaction was further confirmed by bimolecular fluorescence complementation analysis in cells co-expressing both ATXN3-Q75-VC and VN-LC3 proteins. The study results suggest the potential of NC009-1 and -2 and LM-031 as an ATTEC in treating SCA3 and, probably, other polyQ diseases. [ABSTRACT FROM AUTHOR]

Aims: Postpartum haemorrhage (PPH) is the leading cause of maternal mortality worldwide. To prevent PPH, the WHO recommends administration of oxytocin (OT) immediately after birth, i.e. during the third stage of labour (TSL). Previous studies demonstrate that methods to quantify OT in biological matrices, e.g. enzyme‐linked immunosorbent assays (ELISA), radioimmunoassays (RIA) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) lack the specificity and/or sensitivity to accurately quantify OT in plasma from women administered OT during TSL. This is due to increased metabolic clearance of OT in late‐stage pregnancy and at the time of childbirth, resulting in extremely low OT plasma concentrations. This study describes the development of an ultra‐sensitive bioanalytical method that overcomes the issues previously reported and enables accurate pharmacokinetic analyses of exogenously administered OT in TSL. Methods: A selective and sensitive assay to quantify OT in TSL plasma was developed. Immunoprecipitation (IP) was applied to selectively extract OT from the TSL plasma, thereby generating clean extracts compatible with nanoflow LC (nLC). nLC–MS/MS was chosen for its high sensitivity and ability to differentiate between OT and potentially co‐captured OT‐like immunoreactive products. Results: The presented methodology is accurate and precise, with a good linear fit between 100–10 000 fg mL−1 OT. TSL plasma samples from a clinical phase 1 study (NCT02999100) were analysed successfully, enabling OT quantification down to 100 fg mL−1. Conclusions: The presented IP–nLC–MS/MS method succeeded in overcoming the sensitivity challenge related to the assay of OT in TSL plasma and thereby revealing the PK profiles of OT in TSL plasma clinical study samples. [ABSTRACT FROM AUTHOR]

Formaldehyde (FA) is a chemical that facilitates crosslinking between DNA and proteins. It is widely used in various biochemical assays, such as chromosome conformation capture (3C) and Chromatin Immunoprecipitation (ChIP). While the concentration and temperature of FA treatment are recognized as crucial factors in crosslinking, their quantitative effects have largely remained unexplored. In this study, we employed 3C as a model system to systematically assess the impacts of these two factors on crosslinking. Our findings indicate that the strength of crosslinking significantly influences chromatin conformation detection at nearly all known structural levels. Specifically, a delicate balance between sensitivity and reliability is required when detecting higher-level structures, such as chromosome compartments. Conversely, intense crosslinking is preferred when targeting lower-level structures, such as topologically associated domains (TADs) or chromatin loops. Based on our data, we propose a conceptual molecular thermal motion model to elucidate the roles of these two factors in restricting FA crosslinking. Our results not only shed light on the previously overlooked confounding factor in FA crosslinking but also highlight the need for caution in new technology developments that rely on FA crosslinking. This methodological study examines the effects of temperature and formaldehyde concentration on the analysis of genome organization by chromosome conformation capture approaches and reveals that changes in these two key parameters can greatly affect the outcome and reproducibility of the analysis. [ABSTRACT FROM AUTHOR]

The six PCGF proteins (PCGF1-6) define the biochemical identity of Polycomb repressor complex 1 (PRC1) subcomplexes. While structural and functional studies of PRC1 subcomplexes have revealed their specialized roles in distinct aspects of epigenetic regulation, our understanding of the variation in the protein interaction networks of distinct PCGF subunits in different PRC1 complexes is incomplete. We carried out an affinity purification mass spectrometry (AP-MS) screening of three PCGF subunits, PCGF1 (NSPC1), PCGF2 (MEL18), and PCGF4 (BMI1), to define their interactome and potential cellular function in pluripotent human embryonal carcinoma cell "NT2". The bioinformatic analysis revealed that these interacting proteins cover a range of functional pathways, often involved in cell biology and chromatin regulation. We also found evidence of mutual regulation (at mRNA and protein level) between three distinct PCGF subunits. Furthermore, we confirmed that the disruption of these subunits results in reduced cell proliferation ability. We reveal an interplay between the compositional diversity of the distinct PCGF containing PRC1 complex and the potential role of PCGF proteins within the wider cellular network. [ABSTRACT FROM AUTHOR]

Summary Chromatin immunoprecipitation and sequencing (vs ChIP‐seq) is an essential tool for epigenetic and molecular genetic studies. Although being routinely used, ChIP‐seq is expensive, requires grams of plant materials, and is challenging for samples that enrich fatty acids such as seeds. Here, we developed an Ultrasensitive Plant ChIP‐seq (UP‐ChIP) method based on native ChIP‐seq combined with Tn5 tagmentation‐based library construction strategy. UP‐ChIP is generally applicable for profiling both histone modification and Pol II in a wide range of plant samples, such as a single Arabidopsis seedling, a few Arabidopsis seeds, and sorted nuclei. Compared with conventional ChIP‐seq, UP‐ChIP is much less labor intensive and only consumes 1 μg of antibody and 10 μl of Protein‐A/G conjugated beads for each IP and can work effectively with the amount of starting material down to a few milligrams. By performing UP‐ChIP in various conditions and genotypes, we showed that UP‐ChIP is highly reliable, sensitive, and quantitative for studying histone modifications. Detailed UP‐ChIP protocol is provided. We recommend UP‐ChIP as an alternative to traditional ChIP‐seq for profiling histone modifications and Pol II, offering the advantages of reduced labor intensity, decreased costs, and low‐sample input. [ABSTRACT FROM AUTHOR]

Members of the leucine rich repeat (LRR) and PDZ domain (LAP) protein family are essential for animal development and histogenesis. Densin-180, encoded by LRRC7, is the only LAP protein selectively expressed in neurons. Densin-180 is a postsynaptic scaffold at glutamatergic synapses, linking cytoskeletal elements with signalling proteins such as the α-subunit of Ca2+/calmodulin-dependent protein kinase II. We have previously observed an association between high impact variants in LRRC7 and Intellectual Disability; also three individual cases with variants in LRRC7 had been described. We identify here 33 individuals (one of them previously described) with a dominant neurodevelopmental disorder due to heterozygous missense or loss-of-function variants in LRRC7. The clinical spectrum involves intellectual disability, autism, ADHD, aggression and, in several cases, hyperphagia-associated obesity. A PDZ domain variant interferes with synaptic targeting of Densin-180 in primary cultured neurons. Using in vitro systems (two hybrid, BioID, coimmunoprecipitation of tagged proteins from 293T cells) we identified new candidate interaction partners for the LRR domain, including protein phosphatase 1 (PP1), and observed that variants in the LRR reduced binding to these proteins. We conclude that LRRC7 encodes a major determinant of intellectual development and behaviour. Here the authors identify 33 individuals with a dominant neurodevelopmental disorder due to heterozygous missense or loss-of-function variants in the gene encoding Densin-180, a scaffold protein present at postsynaptic sites in neurons of the central nervous system. [ABSTRACT FROM AUTHOR]

Abnormal expression of circRNAs has been observed in different types of carcinomas, and they play significant roles in the biology of these cancers. Nevertheless, the clinical relevance and functional mechanisms of the majority of circRNAs implicated in breast cancer progression remain unclear. The primary objective of our investigation is to uncover new circRNAs in breast cancer and elucidate the underlying mechanisms by which they exert their effects. The circRNA expression profile data for breast cancer and RNA-sequencing data were acquired from distinct public databases. Differentially expressed circRNAs and mRNA were identified through fold change filtering. The establishment of the competing endogenous RNAs (ceRNAs) network relied on the interplay between circular RNAs, miRNAs, and mRNAs. The hub genes were identified from the protein–protein interaction (PPI) regulatory network using the CytoHubba plugin in Cytoscape. Moreover, the expression levels and prognostic value of these hub genes in the PPI network were assessed using the GEPIA and Kaplan–Meier plotter databases. Fluorescence in situ hybridization (FISH) was used to identified the expression and intracellular localization of hsa_circ_0059665 by using the tissue microarray. Transwell analysis and CCK-8 analysis were performed to assess the invasion, migration, and proliferation abilities of breast cancer cells. Additionally, we investigated the interactions between hsa_circ_0059665 and miR-602 through various methods, including FISH, RNA-binding protein immunoprecipitation (RIP), and luciferase reporter assay. Rescue experiments were conducted to determine the potential regulatory role of hsa_circ_0059665 in breast cancer progression. A total of 252 differentially expressed circRNAs were identified. Among them, 246 circRNAs were up-regulated, while 6 circRNAs were down-regulated. Based on prediction and screening of circRNA-miRNA and miRNA-mRNA binding sites, we constructed a network consisting of circRNA-miRNA-mRNA interactions. In addition, we constructed a Protein–Protein Interaction (PPI) network and identified six hub genes. Moreover, the expression levels of these six hub genes in breast cancer tissues were found to be significantly lower. Furthermore, the survival analysis results revealed a significant correlation between low expression levels of KIT, FGF2, NTRK2, CAV1, LEP and poorer prognosis in breast cancer patients. The FISH experiment results indicated that hsa_circ_0059665 exhibits significant downregulation in breast cancer, and its decreased expression is linked to poor prognosis in breast cancer patients. Functional in vitro experiments revealed that overexpression of hsa_circ_0059665 can inhibit proliferation, migration and invasion abilities of breast cancer cells. Further molecular mechanism studies showed that hsa_circ_0059665 exerts its anticancer gene role by acting as a molecular sponge for miR-602. In our study, we constructed and analyzed a circRNA-related ceRNA regulatory network and found that hsa_circ_0059665 can act as a sponge for miR-602 and inhibit the proliferation, invasion and migration of breast cancer cells. [ABSTRACT FROM AUTHOR]

Background: Tumour dormancy, a resistance mechanism employed by cancer cells, is a significant challenge in cancer treatment, contributing to minimal residual disease (MRD) and potential relapse. Despite its clinical importance, the mechanisms underlying tumour dormancy and MRD remain unclear. In this study, we employed two syngeneic murine models of myeloid leukemia and melanoma to investigate the genetic, epigenetic, transcriptomic and protein signatures associated with tumour dormancy. We used a multiomics approach to elucidate the molecular mechanisms driving MRD and identify potential therapeutic targets. Results: We conducted an in-depth omics analysis encompassing whole-exome sequencing (WES), copy number variation (CNV) analysis, chromatin immunoprecipitation followed by sequencing (ChIP-seq), transcriptome and proteome investigations. WES analysis revealed a modest overlap of gene mutations between melanoma and leukemia dormancy models, with a significant number of mutated genes found exclusively in dormant cells. These exclusive genetic signatures suggest selective pressure during MRD, potentially conferring resistance to the microenvironment or therapies. CNV, histone marks and transcriptomic gene expression signatures combined with Gene Ontology (GO) enrichment analysis highlighted the potential functional roles of the mutated genes, providing insights into the pathways associated with MRD. In addition, we compared "murine MRD genes" profiles to the corresponding human disease through public datasets and highlighted common features according to disease progression. Proteomic analysis combined with multi-omics genetic investigations, revealed a dysregulated proteins signature in dormant cells with minimal genetic mechanism involvement. Pathway enrichment analysis revealed the metabolic, differentiation and cytoskeletal remodeling processes involved in MRD. Finally, we identified 11 common proteins differentially expressed in dormant cells from both pathologies. Conclusions: Our study underscores the complexity of tumour dormancy, implicating both genetic and nongenetic factors. By comparing genomic, transcriptomic, proteomic, and epigenomic datasets, our study provides a comprehensive understanding of the molecular landscape of minimal residual disease. These results provide a robust foundation for forthcoming investigations and offer potential avenues for the advancement of targeted MRD therapies in leukemia and melanoma patients, emphasizing the importance of considering both genetic and nongenetic factors in treatment strategies. [ABSTRACT FROM AUTHOR]

Objective: To screen the host interaction proteins of the severe fever with thrombocytopenia syndrome virus (SFTSV) nonstructural protein (NSs) by immunoprecipitation combined with mass spectrometry analysis, to discuss the functions, subcellular localization, and biological pathways of these interaction proteins, and to provide the basis for clarifying the replication and pathogenic mechanism of SFTSV. Methods: The eukaryotic expression vectors pSFTSV-NSs-Flag (experimental group) and Flag-CMV-3 (negative group) were transfected into the human embryonic kidney 293T cells, and contorl group (no treatment) was set up. The lysates of the cells in various groups were collected, and the expression and localization of SFTSV NSs in the host cells were verified by indirect immunofluorescence and Western blotting methods. The protein lysates were treated with protein A/G and immunoprecipitation was used to enrich host proteins binding to NSs. The captured interaction proteins were initially analyzed by silver staining and Coomassie brilliant blue staining to observe the differential protein bands in various groups; liquid chromatography-tandem mass spectrometry was used to obtain the information of protein sequences; the reliable proteins were retained and searched by UniProt database; Gene Ontology (GO) functional enrichment analysis, IPR, eukaryotic orthologous groups (KOGs) functional annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis, subcellular localization, and transcription factor (TF) functional annotation were used to determine the subcellular structure, gene functions, and biological processes of the interaction proteins. Results: The immunofluorescence results showed that the SFTSV NSs expressed a single specific band at relative molecular mass 33 000 and was localized in the cytoplasm in a granular inclusion body-like manner. The silver staining and Coomassie brilliant blue staining results showed there were significant differential protein bands between experimental group and negative group. The mass spectrometry results identified 46 potential interaction proteins. The GO functional enrichment analysis, KOGs functional annotation, and KEGG signaling pathway enrichment analysis results showed that the biological pathways related to viral translation, cellular metabolism, and protein transport were enriched with a considerable number of proteins. Eight annotated proteins had intermediate filament domains. The highest percentage of subcellular localization was cytoplasmic proteins, consistent with the NSs localization site. The TF functional annotation analysis results showed one protein from the NF-Y family. Conclusion: The interaction proteins play roles in assisting the proper protein folding, participating in the cribosome translation, and forming the cytoskeleton, which may be involved in antiviral replication. These proteins can be used as candidate proteins for further study on the replication mechanism of SFTSV. [ABSTRACT FROM AUTHOR]

The production of amylose is facilitated by granule-bound starch synthase (GBSS). Despite its importance, the specific protein interactions involving barley grain-bound starch synthase Ia (HvGBSSIa) remain poorly understood. To elucidate this, we engineered a pET-32a-HvGBSSIa prokaryotic expression vector for specific expression in E. coli Rosetta cells. A rabbit anti-HvGBSSIa polyclonal antibody was generated and employed to enrich HvGBSSIa-binding proteins from barley grains through immunoprecipitation. The isolated complexes were then resolved through SDS-PAGE, and the constituent proteins were identified using mass spectrometry coupled with database searches. Our results confirmed the successful preparation of a highly specific polyclonal antibody against HvGBSSI. Furthermore, differential expression of HvGBSSIa was assessed across various barley tissues and developmental stages of the grain, revealing peak expression at 25 days post-flowering. Proteins interacting with HvGBSSIa, including sucrose synthase and starch branching enzyme, were identified through co-immunoprecipitation. This study lays the groundwork for further detailed analyses of the HvGBSSIa protein complex in barley. [ABSTRACT FROM AUTHOR]

Abstract Heterozygous de novo mutations in the Activity-Dependent Neuroprotective Homeobox (ADNP) gene underlie Helsmoortel-Van der Aa syndrome (HVDAS). Most of these mutations are situated in the last exon and we previously demonstrated escape from nonsense-mediated decay by detecting mutant ADNP mRNA in patient blood. In this study, wild-type and ADNP mutants are investigated at the protein level and therefore optimal detection of the protein is required. Detection of ADNP by means of western blotting has been ambiguous with reported antibodies resulting in non-specific bands without unique ADNP signal. Validation of an N-terminal ADNP antibody (Aviva Systems) using a blocking peptide competition assay allowed to differentiate between specific and non-specific signals in different sample materials, resulting in a unique band signal around 150 kDa for ADNP, above its theoretical molecular weight of 124 kDa. Detection with different C-terminal antibodies confirmed the signals at an observed molecular weight of 150 kDa. Our antibody panel was subsequently tested by immunoblotting, comparing parental and homozygous CRISPR/Cas9 endonuclease-mediated Adnp knockout cell lines and showed disappearance of the 150 kDa signal, indicative for intact ADNP. By means of both a GFPSpark and Flag-tag N-terminally fused to a human ADNP expression vector, we detected wild-type ADNP together with mutant forms after introduction of patient mutations in E. coli expression systems by site-directed mutagenesis. Furthermore, we were also able to visualize endogenous ADNP with our C-terminal antibody panel in heterozygous cell lines carrying ADNP patient mutations, while the truncated ADNP mutants could only be detected with epitope-tag-specific antibodies, suggesting that addition of an epitope-tag possibly helps stabilizing the protein. However, western blotting of patient-derived hiPSCs, immortalized lymphoblastoid cell lines and post-mortem patient brain material failed to detect a native mutant ADNP protein. In addition, an N-terminal immunoprecipitation-competent ADNP antibody enriched truncating mutants in overexpression lysates, whereas implementation of the same method failed to enrich a possible native mutant protein in immortalized patient-derived lymphoblastoid cell lines. This study aims to shape awareness for critical assessment of mutant ADNP protein analysis in Helsmoortel-Van der Aa syndrome.

RNA-binding protein Fused-in Sarcoma (FUS) plays an essential role in various cellular processes. Mutations in the C-terminal domain region, where the nuclear localization signal (NLS) is located, causes the redistribution of FUS from the nucleus to the cytoplasm. In neurons, neurotoxic aggregates are formed as a result, contributing to neurogenerative diseases. Well-characterized anti-FUS antibodies would enable the reproducibility of FUS research, thereby benefiting the scientific community. In this study, we characterized ten FUS commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Rab10 is a small GTPase involved in cargo transport from the trans-Golgi network to the plasma membrane and endocytic recycling back to the cell membrane. It has garnered significant interest in neurodegenerative disease research, particularly due to its phosphorylation by the LRRK2 kinase. This relationship underscores the importance of Rab10 in cellular processes related to disease pathology, specifically Parkinson’s disease. The accessibility of renewable and selective antibodies against Rab10 would advance research efforts, enabling further understanding of its function and implications in disease. Here, we have characterized eight Rab10 commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

STING1 is an immune adaptor protein which promotes innate immune defense mechanisms against pathogens. Its role in modulating inflammation links STING1 to various pathologic conditions, positioning it as a key target for therapeutic interventions aimed at regulating immune responses. To advance our understanding of STING1-associated diseases, it is essential to make high-performing antibodies readily accessible to the scientific community. This study aims to improve reliability of STING1 research as we have characterized sixteen STING1 commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

SPARC-related modular calcium-binding protein 1, otherwise known as SMOC-1, is a secreted glycoprotein involved in various cell biological processes including cell-matrix interactions, osteoblast differentiation, embryonic development, and homeostasis. SMOC-1 was found to be elevated in asymptomatic Alzheimer’s disease (AD) patient cortex as well as being enriched in amyloid plaques and in AD patientcerebrospinal fluid, arguing for SMOC-1 as a promising biomarker for AD. Having access to high-quality SMOC-1 antibodies is crucial for the scientific community. It can ensure the consistency and reliability of SMOC-1 research, and further the exploration of its potential as both a therapeutic target or diagnostic marker.. In this study, we characterized seven SMOC-1 commercial antibodies for Western blot and immunoprecipitation, using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified successful antibodies in the tested applications and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Sphingosine 1-phosphate receptor 1 (S1PR1) is a G-coupled protein receptor that induces crucial biological processes when bound by sphingosine 1-phosphate. Here, we have characterized nine S1PR1 commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Casein kinase II subunit alpha (CSNK2A1), a serine/threonine kinase, phosphorylates multiple protein substrates and is involved in diverse cellular and biological processes. Implicated in various human diseases, high-performing antibodies would help evaluate its potential as a therapeutic target and benefit the scientific community. In this study, we have characterized ten CSNK2A1 commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Objectives Methods Results Conclusions A variety of myositis‐specific autoantibodies have been identified in sera from patients with idiopathic inflammatory myopathies, and they play a crucial role in tailoring personalized disease management. In particular, autoantibodies against 3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase (HMGCR) are now recognized as a key tool for diagnosing immune‐mediated necrotizing myopathy. The current gold standard for detecting anti‐HMGCR autoantibodies involves immunoprecipitation (IP) using radiolabeled proteins from cell extracts or purified proteins produced by in vitro transcription/translation (IVTT). Unfortunately, this radioisotope labeling is technically intricate and not suitable for routine laboratory use. To address this, we developed a novel assay called “Bio‐IVTT‐IP” for detecting anti‐HMGCR autoantibodies, which uses a nonradioactive biotinylated recombinant HMGCR protein produced by IVTT.We collected 14 clinical specimens containing anti‐HMGCR autoantibodies from patients with immune‐mediated necrotizing myopathy, which were validated using the gold standard IP assay, and a set of 35 control samples from patients with other autoimmune diseases, such as systemic lupus erythematosus.The Bio‐IVTT‐IP assay successfully identified 14 clinical samples positive for anti‐HMGCR. We confirmed that the performance of the Bio‐IVTT‐IP assay was completely consistent with that of the gold standard IP assay using radiolabeled proteins. Notably, no immunoprecipitates were found in control samples from patients with other autoimmune diseases.These findings show that the Bio‐IVTT‐IP assay can serve as a potential alternative to the gold standard IP assay for detecting anti‐HMGCR autoantibodies. It can be considered a practical immunodiagnostic tool for diagnosing immune‐mediated necrotizing myopathy, particularly owing to its accessibility in routine laboratory settings. [ABSTRACT FROM AUTHOR]

Background and Purpose: Methamphetamine (METH) use disorder has risen dramatically over the past decade, and there are currently no FDA‐approved medications due, in part, to gaps in our understanding of the pharmacological mechanisms related to METH action in the brain. Experimental Approach: Here, we investigated whether transient receptor potential ankyrin 1 (TRPA1) mediates each of several METH abuse‐related behaviours in rodents: self‐administration, drug‐primed reinstatement, acquisition of conditioned place preference, and hyperlocomotion. Additionally, METH‐induced molecular (i.e., neurotransmitter and protein) changes in the brain were compared between wild‐type and TRPA1 knock‐out mice. Finally, the relationship between TRPA1 and the dopamine transporter was investigated through immunoprecipitation and dopamine reuptake assays. Key Results: TRPA1 antagonism blunted METH self‐administration and drug‐primed reinstatement of METH‐seeking behaviour. Further, development of METH‐induced conditioned place preference and hyperlocomotion were inhibited by TRPA1 antagonist treatment, effects that were not observed in TRPA1 knock‐out mice. Similarly, molecular studies revealed METH‐induced increases in dopamine levels and expression of dopamine system‐related proteins in wild‐type, but not in TRPA1 knock‐out mice. Furthermore, pharmacological blockade of TRPA1 receptors reduced the interaction between TRPA1 and the dopamine transporter, thereby increasing dopamine reuptake activity by the transporter. Conclusion and Implications: This study demonstrates that TRPA1 is involved in the abuse‐related behavioural effects of METH, potentially through its modulatory role in METH‐induced activation of dopaminergic neurotransmission. Taken together, these data suggest that TRPA1 may be a novel therapeutic target for treating METH use disorder. [ABSTRACT FROM AUTHOR]

Objective: Rare study of the non-coding and regulatory regions of the genome limits our ability to decode the mechanisms of fatty liver hemorrhage syndrome (FLHS) in chickens. Methods: Herein, we constructed the high-fat diet-induced FLHS chicken model to investigate the genome-wide active enhancers and transcriptome by H3K27ac target chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-Seq) profiles of normal and FLHS liver tissues. Concurrently, an integrative analysis combining ChIP-seq with RNA-Seq and a comparative analysis with chicken FLHS, rat non-alcoholic fatty liver disease (NAFLD) and human NAFLD at the transcriptome level revealed the enhancer and super enhancer target genes and conservative genes involved in metabolic processes. Results: In total, 56 and 199 peak-genes were identified in upregulated peak-genes positively regulated by H3K27ac (Cor (peak-gene correlation) ≥0.5 and log2(FoldChange) ≥1) (PP) and downregulated peak-genes positively regulated by H3K27ac (Cor (peak-gene correlation) ≥0.5 and log2(FoldChange)≤-1) (PN), respectively; then we screened key regulatory targets mainly distributing in lipid metabolism (PCK1, APOA4, APOA1, INHBE) and apoptosis (KIT, NTRK2) together with MAPK and PPAR signaling pathway in FLHS. Intriguingly, PCK1 was also significantly covered in up-regulated super-enhancers (SEs), which further implied the vital role of PCK1 during the development of FLHS. Conclusion: Together, our studies have identified potential therapeutic biomarkers of PCK1 and elucidated novel insights into the pathogenesis of FLHS, especially for the epigenetic perspective. [ABSTRACT FROM AUTHOR]

Phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) is a de novo purine biosynthetic enzyme. It has been found to be overexpressed in various types of cancer and is related to cell proliferation, invasion, the epithelial–mesenchymal transition, and efficient tumor growth. In this study, we describe a rat monoclonal antibody (mAb) 6A10, which was generated as an antigen of human PAICS. This mAb was generated to interact with the N-terminal region of human PAICS and was found to recognize endogenous PAICS enzymes in several cancer cells. Our results also indicated that it can recognize monkey and dog PAICS, which possess the same amino acid sequence in the antigenic region as human PAICS, but it does not recognize rat and mouse PAICS. Furthermore, our data indicated that this mAb is suitable for immunoprecipitation and immunoblotting use for several cancer cell lines. We, therefore, anticipate that mAb 6A10 will be useful for functional analyses of human PAICS in several cancers and for diagnosis of malignant transformation. [ABSTRACT FROM AUTHOR]

Hypoxia is a critical factor contributing to a poor prognosis and challenging glioma therapy. Previous studies have indicated that hypoxia drives M2 polarization of macrophages and promotes cancer progression in various solid tumors. However, the more complex and diverse mechanisms underlying this process remain to be elucidated. Here, we aimed to examine the functions of hypoxia in gliomas and preliminarily investigate the underlying mechanisms of M2 macrophage polarization caused by hypoxia. We found that hypoxia significantly enhances the malignant phenotypes of U87 and U251 cells by regulating glycolysis. In addition, hypoxia mediated accumulation of the glycolysis product [lactic acid (LA)], which is subsequently absorbed by macrophages to induce its M2 polarization, and this process is reverted by both the glycolysis inhibitor and silenced monocarboxylate transporter (MCT-1) in macrophages, indicating that M2 macrophage polarization is associated with the promotion of glycolysis by hypoxia. Interestingly, we also found that hypoxia mediated LA accumulation in glioma cells upon uptake by macrophages upregulates H3K18La expression and promotes tumor necrosis factor superfamily member 9 (TNFSF9) expression in a histone-lactylation-dependent manner based on the results of chromatin immunoprecipitation sequencing (ChIP seq) enrichment analysis. Subsequent in vitro and in vivo experiments further indicated that TNFSF9 facilitated glioma progression. Mechanistically, hypoxia-mediated LA accumulation in glioma cells is taken up by macrophages and then induces its M2 macrophage polarization by regulating TNFSF9 expression via MCT-1/H3K18La signaling, thus facilitating the malignant progression of gliomas. NEW & NOTEWORTHY: Our study revealed that hypoxia induces the production of LA accumulation through glycolysis in glioma cells, which is subsequently absorbed by macrophages and leads to its M2 polarization via the MCT-1/H3K18La/TNFSF9 axis, ultimately significantly promoting the malignant progression of glioma cells. These findings are novel and noteworthy as they provide insights into the connection between energy metabolism and epigenetics in gliomas. [ABSTRACT FROM AUTHOR]

The genomes and annotated genes of allotetraploid cotton Gossypium hirsutum have been extensively studied in recent years. However, the expression, regulation, and evolution of intergenic genes (ITGs) have not been completely deciphered. In this study, we identified a novel set of actively expressed ITGs in G. hirsutum cotton, through transcriptome profiling based on deep sequencing data, as well as chromatin immunoprecipitation, followed by sequencing (ChIP-seq) of histone modifications and how the ITGs evolved. Totals of 17,567 and 8249 ITGs were identified in G. hirsutum and Gossypium arboreum, respectively. The expression of ITGs in G. hirsutum was significantly higher than that in G. arboreum. Moreover, longer exons were observed in G. hirsutum ITGs. Notably, 42.3% of the ITGs from G. hirsutum were generated by the long terminal repeat (LTR) insertions, while their proportion in genic genes was 19.9%. The H3K27ac and H3K4me3 modification proportions and intensities of ITGs were equivalent to genic genes. The H3K4me1 modifications were lower in ITGs. Additionally, evolution analyses revealed that the ITGs from G. hirsutum were mainly produced around 6.6 and 1.6 million years ago (Mya), later than the pegged time for genic genes, which is 7.0 Mya. The characterization of ITGs helps to elucidate the evolution of cotton genomes and shed more light on their biological functions in the transcriptional regulation of eukaryotic genes, along with the roles of histone modifications in speciation and diversification. [ABSTRACT FROM AUTHOR]

Pausing of RNA polymerase II (Pol II) at transcription start sites (TSSs) primes target genes for productive elongation. Coincidentally, DNA double-strand breaks (DSBs) enrich at highly transcribed and Pol II-paused genes, although their interplay remains undefined. Using androgen receptor (AR) signaling as a model, we have uncovered AR-interacting protein 4 (ARIP4) helicase as a driver of androgen-dependent transcription induction. Chromatin immunoprecipitation sequencing analysis revealed that ARIP4 preferentially co-occupies TSSs with paused Pol II. Moreover, we found that ARIP4 complexes with topoisomerase II beta and mediates transient DSB formation upon hormone stimulation. Accordingly, ARIP4 deficiency compromised release of paused Pol II and resulted in R-loop accumulation at a panel of highly transcribed AR target genes. Last, we showed that ARIP4 binds and unwinds R-loops in vitro and that its expression positively correlates with prostate cancer progression. We propose that androgen stimulation triggers ARIP4-mediated unwinding of R-loops at TSSs, enforcing Pol II pause release to effectively drive an androgen-dependent expression program. [ABSTRACT FROM AUTHOR]

Vitamin D plays an important pleiotropic role in maintaining global homeostasis of the human body. Its functions go far beyond skeletal health, playing a crucial role in a plethora of cellular functions, as well as in extraskeletal health, ensuring the proper functioning of multiple human organs, including the skin. Genes from the Grainyhead-like (GRHL) family code for transcription factors necessary for the development and maintenance of various epithelia. Even though they are involved in many processes regulated by vitamin D, a direct link between vitamin D-mediated cellular pathways and GRHL genes has never been described. We employed various bioinformatic methods, quantitative real-time PCR, chromatin immunoprecipitation, reporter gene assays, and calcitriol treatments to investigate this issue. We report that the vitamin D receptor (VDR) binds to a regulatory region of the Grainyhead-like 1 (GRHL1) gene and regulates its expression. Ectopic expression of VDR and treatment with calcitriol alters the expression of the GRHL1 gene. The evidence presented here indicates a role of VDR in the regulation of expression of GRHL1 and correspondingly a role of GRHL1 in mediating the actions of vitamin D. [ABSTRACT FROM AUTHOR]

Background: Megakaryocytic differentiation is a complicated process regulated by a series of transcription factors in a context- and stage-dependent manner. Recent studies have suggested that krüppel-like transcription factor 2 (KLF2) is involved in the control of embryonic erythroid precursor cell differentiation and maturation. However, the function and mechanism of KLF2 in regulating megakaryocytic differentiation remain unclear. Methods: The expression patterns of krüppel-like transcription factors (KLFs) during megakaryocytic differentiation were identified from public databases. Phorbol 12-myristate 13-acetate (PMA) treatment of the myeloid-erythroid-leukemic cell lines K562 and HEL were used as cellular megakaryocytic differentiation models. A lentiviral transduction system was utilized to achieve the goal of amplifying or reducing KLF2. The expression of KLF2 was examined using real-time PCR and western blot. The impact of KLF2 on the megakaryocytic differentiation of K562 cells was examined by flow cytometry, Giemsa staining, Phalloidin staining and western blot. RNA-sequencing (RNA-seq) and chromatin immunoprecipitation-sequencing (ChIP-seq) technologies were used to identify the KLF2-regulated targets. Results: KLF2 is increased in the maturation process of megakaryocytes. KLF2 overexpression accelerated the PMA-induced megakaryocytic differentiation, as reflected by an increased percentage of CD41/CD61 cells, an increased number of polyploid cells, and an elevated expression of P21 and P27. KLF2 knockdown exhibited the opposite results, indicating that KLF2 knockdown suppressed the megakaryocytic differentiation. Further, combination of the RNA-seq and ChIP-seq results suggested that chimerin 1 (CHN1) and potassium voltage-gated channel subfamily Q member 5 (KCNQ5) may be target genes regulated of KLF2. Both CHN1 and KCNQ5 knockdown could block the megakaryocytic differentiation to some content. Conclusion: This study implicated a regulatory role of KLF2 in megakaryocytic differentiation, which may suggest KLF2 as a target for illness with abnormal megakaryocytic differentiation. [ABSTRACT FROM AUTHOR]

Type II innate lymphoid cells (ILC2s) maintain homeostasis and barrier integrity in mucosal tissues. In both mice and humans, ILC2s poorly reconstitute after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Determining the mechanisms involved in their impaired reconstitution could improve transplant outcomes. By integrating single-cell chromatin and transcriptomic analyses of transplanted ILC2s, we identify a previously unreported population of converted ILC1-like cells in the mouse small intestine post-transplant. Exposure of ILC2s to proinflammatory cytokines resulted in a mixed ILC1-ILC2 phenotype but was able to convert only a small population of ILC2s to ILC1s, which were found post-transplant. Whereas ILC2s protected against acute graft-versus-host disease (aGVHD) mediated mortality, infusion of proinflammatory cytokine-exposed ILC2s accelerated aGvHD. Interestingly, murine ILC2 reconstitution post-HSCT is decreased in the presence of alloreactive T cells. Finally, peripheral blood cells from human patients with aGvHD have an altered ILC2-associated chromatin landscape compared to transplanted controls. These data demonstrate that following transplantation ILC2s convert to a pro-pathogenic population with an ILC1-like chromatin state and provide insights into the contribution of ILC plasticity to the impaired reconstitution of ILC2 cells, which is one of several potential mechanisms for the poor reconstitution of these important cells after allo-HSCT. Allogeneic hematopoietic stem cell transplantation restores the whole hematopoietic compartment of the recipient, but some cell types, such as Type II innate lymphoid cells (ILC2) are not reconstituted efficiently. Here authors show that ILC2s could be converted to ILC1-like cells in the mouse small intestine post-transplantation, which might contribute to the lower than physiological ILC2 numbers. [ABSTRACT FROM AUTHOR]

Background: Presbycusis, also referred to as age-related hearing loss (ARHL), is a condition that results from the cumulative effects of aging on an individual's auditory capabilities. Given the limited understanding of epigenetic mechanisms in ARHL, our research focuses on alterations in chromatin-accessible regions. Methods: We employed assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) in conjunction with unique identifier (UID) mRNA-seq between young and aging cochleae, and conducted integrated analysis as well as motif/TF-gene prediction. Additionally, the essential role of super-enhancers (SEs) in the development of ARHL was identified by comparative analysis to previous research. Meanwhile, an ARHL mouse model and an aging mimic hair cell (HC) model were established with a comprehensive identification of senescence phenotypes to access the role of SEs in ARHL progression. Results: The control cochlear tissue exhibited greater chromatin accessibility than cochlear tissue affected by ARHL. Furthermore, the levels of histone 3 lysine 27 acetylation were significantly depressed in both aging cochlea and aging mimic HEI-OC1 cells, highlighting the essential role of SEs in the development of ARHL. The potential senescence-associated super-enhancers (SASEs) of ARHL were identified, most of which exhibited decreased chromatin accessibility. The majority of genes related to the SASEs showed obvious decreases in mRNA expression level in aging HCs and was noticeably altered following treatment with JQ1 (a commonly used SE inhibitor). Conclusion: The chromatin accessibility in control cochlear tissue was higher than that in cochlear tissue affected by ARHL. Potential SEs involved in ARHL were identified, which might provide a basis for future therapeutics targeting SASEs related to ARHL. [ABSTRACT FROM AUTHOR]

Background: Hepatocellular carcinoma (HCC) ranks as the second leading cause of global cancer-related deaths and is characterized by a poor prognosis. Eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) have been proved to play important roles in various human cancers, whereas the deubiquitination of EEF1A1 was poorly understood. Methods: The binding and regulatory relationship between Ubiquitin carboxyl-terminal hydrolase L3 (UCHL3) and EEF1A1 was validated using clinical tissue samples, reverse transcription quantitative real-time fluorescence quantitative PCR (RT-qPCR), Western blotting, co-immunoprecipitation, and immunofluorescence, as well as ubiquitin detection and cyclohexamide tracking experiments. Finally, the impact of the UCHL3/EEF1A1 axis on HCC malignant behavior was analyzed through functional experiments and nude mouse models. Results: UCHL3 was found to have a high expression level in HCC tissues. Tissue samples from 60 HCC patients were used to evaluate the correlation between UCHL3 and EEF1A1. UCHL3 binds to EEF1A1 through the lysine site, which reduces the ubiquitination level of EEF1A1. Functional experiments and nude mouse models have demonstrated that the UCHL3/EEF1A1 axis promotes the migration, stemness, and drug resistance of HCC cells. Reducing the expression of EEF1A1 can reverse the effect of UCHL3 on the malignant behavior of HCC cells. Conclusion: Our findings revealed that UCHL3 binds and stabilizes EEF1A1 through deubiquitination. UCHL3 and EEF1A1 formed a functional axis in facilitating the malignant progression of HCC, proving new insights for the anti-tumor targeted therapy for HCC. [ABSTRACT FROM AUTHOR]

Macroautophagy, simply referred to below as autophagy, is an intracellular degradation system that is highly conserved in eukaryotes. Since the processes involved in autophagy are accompanied by membrane dynamics, RAB small GTPases, key regulators of membrane trafficking, are generally thought to regulate the membrane dynamics of autophagy. Although more than half of the mammalian RABs have been reported to be involved in canonical and selective autophagy, no consensus has been reached in regard to the role of RABs in mammalian autophagy. Here, we comprehensively analyzed a rab-knockout (KO) library of MDCK cells to reevaluate the requirement for each RAB isoform in basal and starvation-induced autophagy. The results revealed clear alteration of the MAP1LC3/LC3-II level in only four rab-KO cells (rab1-KO, rab2-KO, rab7a-KO, and rab14-KO cells) and identified RAB14 as a new regulator of autophagy, specifically at the autophagosome maturation step. The autophagy-defective phenotype of two of these rab-KO cells, rab2-KO and rab14-KO cells, was very mild, but double KO of rab2 and rab14 caused a severer autophagy-defective phenotype (greater LC3 accumulation than in single-KO cells, indicating an overlapping role of RAB2 and RAB14 during autophagosome maturation. We also found that RAB14 is phylogenetically similar to RAB2 and that it possesses the same properties as RAB2, i.e. autophagosome localization and interaction with the HOPS subunits VPS39 and VPS41. Our findings suggest that RAB2 and RAB14 overlappingly regulate the autophagosome maturation step through recruitment of the HOPS complex to the autophagosome.Abbreviation: AID2: auxin-inducible degron 2; ATG: autophagy related; BafA1: bafilomycin A1; CKO: conditional knockout; EBSS: Earle’s balanced salt solution; EEA1: early endosome antigen 1; HOPS: homotypic fusion and protein sorting; HRP: horseradish peroxidase; IP: immunoprecipitation; KD: knockdown; KO: knockout; LAMP2: lysosomal-associated membrane protein 2; MDCK: Madin-Darby canine kidney; mAb: monoclonal antibody; MEF: mouse embryonic fibroblast; MTORC1: mechanistic target of rapamycin kinase complex 1; 5-Ph-IAA: 5-phenyl-indole-3-acetic acid; pAb: polyclonal antibody; siRNA: small interfering RNA; SNARE: soluble NSF-attachment protein receptor; TF: transferrin; WT: wild-type. [ABSTRACT FROM AUTHOR]

Leaf adaxialeabaxial (adeabaxial) polarity is crucial for leaf morphology and function, but the genetic machinery governing this process remains unclear. To uncover critical genes involved in leaf adeabaxial patterning, we applied a combination of in silico prediction using machine learning (ML) and experimental analysis. A RandomForestmodel was trained using genes known to influence adeabaxial polarity as ground truth. Gene expression data from various tissues and conditions as well as promoter regulation data derived from transcription factor chromatin immunoprecipitation sequencing (ChIP-seq) was used as input, enabling the prediction of novel adeabaxial polarity-related genes and additional transcription factors. Parallel to this, available and newly-obtained transcriptome data enabled us to identify genes differentially expressed across leaf adeabaxial sides. Based on these analyses, we obtained a set of 111 novel genes which are involved in leaf adeabaxial specialization. To explore implications for vegetable crop breeding, we examined the conservation of expression patterns between Arabidopsis and Brassica rapa using single-cell transcriptomics. The results demonstrated the utility of our computational approach for predicting candidate genes in crop species. Our findings expand the understanding of the genetic networks governing leaf adeabaxial differentiation in agriculturally important vegetables, enhancing comprehension of natural variation impacting leaf morphology and development, with demonstrable breeding applications. [ABSTRACT FROM AUTHOR]

Background: A differential gene, triggering receptor expressed on myeloid cells 1 (TREM1), was identified in blood sequencing datasets from myocardial infarction patients and healthy controls. Myocardial fibrosis following myocardial infarction significantly contributes to cardiac dysfunction. Objectives: This study aimed to unveil the intrinsic regulatory mechanism of TREM1 in myocardial fibrosis. Methods: Mimicking pathology by angiotensin II (Ang II) treatment of human cardiac fibroblasts (HCFs), the impacts of TREM1 knockdown on its proliferation, migration, and secretion of the pro-fibrotic matrix were identified. Using the Human Transcription Factor Database (HumanTFDB) website, lysine-specific demethylase 5B (KDM5B) was found to bind to the TREM1 promoter, which was further validated through luciferase reporter and chromatin immunoprecipitation (ChIP). By promoting KDM5B overexpression, its effect on the regulation of TREM1 was examined. Results: TREM1 knockdown suppressed the proliferation, migration, and secretion of the pro-fibrotic matrix in HCFs upon Ang II treatment. KDM5B bound to the TREM1 promoter and upregulated its transcriptional expression. Furthermore, KDM5B overexpression reversed the regulation of the above cellular phenotypes by TREM1 knockdown. Conclusion: This study sheds light on the positive regulation of TREM1 by KDM5B, demonstrating their role in promoting myocardial fibrosis. This finding provides a theoretical foundation for understanding disease pathology and potentially advancing the development of new targeted therapies. [ABSTRACT FROM AUTHOR]

Primordial germ cells (PGCs) are the precursors of germ cells and play a crucial role in germline transmission. In chickens, PGCs can be cultured in vitro while maintaining their germline stem cell characteristics. The Deleted in Azoospermia-Like (DAZL) gene, which is highly expressed in PGCs, is essential for germ cell development. Here, through gene knockout experiments, we discovered that the loss of DAZL expression in chicken PGCs led to decreased proliferation and survival. By next employed techniques such as RIP-seq (RNA Binding Protein Immunoprecipitation) and Co-IP-MS/MS (Co-immunoprecipitation Mass Spectrometry), we identified genes directly regulated by DAZL or cooperating with DAZL at the transcriptomic and proteomic levels. DAZL was found to control genes related to germline development, pluripotency, and cell proliferation in PGCs. Additionally, we observed a significant overlap between RNAs and proteins that interact with both DAZL and DDX4, indicating their cooperation in the gene regulation network in chicken PGCs. Our research provides valuable insights into the function of the DAZL gene in germline cells. [ABSTRACT FROM AUTHOR]

[Display omitted] • ALKBH5 plays an important protective role during the post-MI repair phase. • The loss of ALKBH5 in fibroblasts leads to cardiac rupture and deteriorated cardiac function. • This study identities the ALKBH5 as hypoxia-related role in cardiac fibroblasts. • ALKBH5 regulates fibroblast activation via ErbB4 mRNA demethylation. • ALKBH5/ErbB4 are possible therapeutic targets to reduce the occurrence of cardiac rupture. N6-methyladenosine (m6A) methylation produces a marked effect on cardiovascular diseases. The m6A demethylase AlkB homolog 5 (ALKBH5), as an m6A "eraser", is responsible for decreased m6A modification. However, its role in cardiac fibroblasts during the post-myocardial infarction (MI) healing process remains elusive. To investigate the effect of ALKBH5 in cardiac fibroblasts during infarct repair. MI was mimicked by permanent left anterior descending artery ligation in global ALKBH5-knockout, ALKBH5-knockin, and fibroblast-specific ALKBH5-knockout mice to study the function of ALKBH5 during post-MI collagen repair. Methylated RNA immunoprecipitation sequencing was performed to explore potential ALKBH5 targets. Dramatic alterations in ALKBH5 expression were observed during the early stages post-MI and in hypoxic fibroblasts. Global ALKBH5 knockin reduced infarct size and ameliorated cardiac function after MI. The global and fibroblast-specific ALKBH5-knockout mice both exhibited low survival rates along with poor collagen repair, impaired cardiac function, and cardiac rupture. Both in vivo and in vitro ALKBH5 loss resulted in impaired fibroblast activation and decreased collagen deposition. Additionally, hypoxia, but not TGF-β1 or Ang II, upregulated ALKBH5 expression in myofibroblasts by HIF-1α-dependent transcriptional regulation. Mechanistically, ALKBH5 promoted the stability of ErbB4 mRNA and the degradation of ST14 mRNA via m6A demethylation. Fibroblast-specific ErbB4 overexpression ameliorated the impaired fibroblast-to-myofibroblast transformation and poor post-MI repair due to ALKBH5 knockout. Fibroblast ALKBH5 positively regulates post-MI healing by stabilization of ErbB4 mRNA in an m6A-dependent manner. ALKBH5/ErbB4 might be potential therapeutic targets for post-MI cardiac rupture. [ABSTRACT FROM AUTHOR]

[Display omitted] • The cotton miR319c- MYB33 module regulates trade-offs between plant growth and defense. • GhGAI1 interacts with GhMYB33 and affects its transcriptional activity. • GhMYB33 binds to the GhSPL9 promoter and activates its expression to increase plant height. • Under Verticillium dahliae infection, GhMYB33 expression is repressed to promote GhDFR1 transcription, increasing plant resistance. Sessile plants engage in trade-offs between growth and defense capacity in response to fluctuating environmental cues. MYB is an important transcription factor that plays many important roles in controlling plant growth and defense. However, the mechanism behind how it keeps a balance between these two physiological processes is still largely unknown. Our work focuses on the dissection of the molecular mechanism by which GhMYB33 regulates plant growth and defense. The CRISPR/Cas9 technique was used to generate mutants for deciphering GhMYB33 functions. Yeast two-hybrid, luciferase complementary imaging, and co-immunoprecipitation assays were used to prove that proteins interact with each other. We used the electrophoretic mobility shift assay, yeast one-hybrid, and luciferase activity assays to analyze GhMYB33 acting as a promoter. A β-glucuronidase fusion reporter and 5′ RNA ligase mediated amplification of cDNA ends analysis showed that ghr-miR319c directedly cleaved the GhMYB33 mRNA. Overexpressing miR319c-resistant GhMYB33 (rGhMYB33) promoted plant growth, accompanied by a significant decline in resistance against Verticillium dahliae. Conversely, its knockout mutant, ghmyb33, demonstrated growth restriction and concomitant augmentation of V. dahliae resistance. GhMYB33 was found to couple with the DELLA protein GhGAI1 and bind to the specific cis-elements of GhSPL9 and GhDFR1 promoters, thereby modulating internode elongation and plant resistance in V. dahliae infection. The ghr-miR319c was discovered to target and suppress GhMYB33 expression. The overexpression of ghr-miR319c led to enhanced plant resistance and a simultaneous reduction in plant height. Our findings demonstrate that GhMYB33 encodes a hub protein and controls the expression of GhSPL9 and GhDFR1, implicating a pivotal role for the miR319c- MYB33 module to regulate the trade-offs between plant growth and defense. [ABSTRACT FROM AUTHOR]

Background: During male gametogenesis of flowering plants, sperm cell lineage (microspores, generative cells, and sperm cells) differentiated from somatic cells and acquired different cell fates. Trimethylation of histone H3 on lysine 4 (H3K4me3) epigenetically contributes to this process, however, it remained unclear how H3K4me3 influences the gene expression in each cell type. Here, we conducted chromatin immunoprecipitation sequencing (ChIP-seq) to obtain a genome-wide landscape of H3K4me3 during sperm cell lineage development in tomato (Solanum lycopersicum). Results: We show that H3K4me3 peaks were mainly enriched in the promoter regions, and intergenic H3K4me3 peaks expanded as sperm cell lineage differentiated from somatic cells. H3K4me3 was generally positively associated with transcript abundance and served as a better indicator of gene expression in somatic and vegetative cells, compared to sperm cell lineage. H3K4me3 was mutually exclusive with DNA methylation at 3' proximal of the transcription start sites. The microspore maintained the H3K4me3 features of somatic cells, while generative cells and sperm cells shared an almost identical H3K4me3 pattern which differed from that of the vegetative cell. After microspore division, significant loss of H3K4me3 in genes related to brassinosteroid and cytokinin signaling was observed in generative cells and vegetative cells, respectively. Conclusions: Our results suggest the asymmetric division of the microspore significantly reshapes the genome-wide distribution of H3K4me3. Selective loss of H3K4me3 in genes related to hormone signaling may contribute to functional differentiation of sperm cell lineage. This work provides new resource data for the epigenetic studies of gametogenesis in plants. [ABSTRACT FROM AUTHOR]

Background: Mesaconitine (MA), a diester-diterpenoid alkaloid extracted from the medicinal herb Aconitum carmichaelii, is commonly used to treat various diseases. Previous studies have indicated the potent toxicity of aconitum despite its pharmacological activities, with limited understanding of its effects on the nervous system and the underlying mechanisms. Methods: HT22 cells and zebrafish were used to investigate the neurotoxic effects of MA both in vitro and in vivo, employing multi-omics techniques to explore the potential mechanisms of toxicity. Results: Our results demonstrated that treatment with MA induces neurotoxicity in zebrafish and HT22 cells. Subsequent analysis revealed that MA induced oxidative stress, as well as structural and functional damage to mitochondria in HT22 cells, accompanied by an upregulation of mRNA and protein expression related to autophagic and lysosomal pathways. Furthermore, methylated RNA immunoprecipitation sequencing (MeRIP-seq) showed a correlation between the expression of autophagy-related genes and N6-methyladenosine (m6A) modification following MA treatment. In addition, we identified METTL14 as a potential regulator of m6A methylation in HT22 cells after exposure to MA. Conclusion: Our study has contributed to a thorough mechanistic elucidation of the neurotoxic effects caused by MA, and has provided valuable insights for optimizing the rational utilization of traditional Chinese medicine formulations containing aconitum in clinical practice. [ABSTRACT FROM AUTHOR]

Background: Genome sequence variants affecting complex traits (quantitative trait loci, QTL) are enriched in functional regions of the genome, such as those marked by certain histone modifications. These variants are believed to influence gene expression. However, due to the linkage disequilibrium among nearby variants, pinpointing the precise location of QTL is challenging. We aimed to identify allele-specific binding (ASB) QTL (asbQTL) that cause variation in the level of histone modification, as measured by the height of peaks assayed by ChIP-seq (chromatin immunoprecipitation sequencing). We identified DNA sequences that predict the difference between alleles in ChIP-seq peak height in H3K4me3 and H3K27ac histone modifications in the mammary glands of cows. Results: We used a gapped k-mer support vector machine, a novel best linear unbiased prediction model, and a multiple linear regression model that combines the other two approaches to predict variant impacts on peak height. For each method, a subset of 1000 sites with the highest magnitude of predicted ASB was considered as candidate asbQTL. The accuracy of this prediction was measured by the proportion where the predicted direction matched the observed direction. Prediction accuracy ranged between 0.59 and 0.74, suggesting that these 1000 sites are enriched for asbQTL. Using independent data, we investigated functional enrichment in the candidate asbQTL set and three control groups, including non-causal ASB sites, non-ASB variants under a peak, and SNPs (single nucleotide polymorphisms) not under a peak. For H3K4me3, a higher proportion of the candidate asbQTL were confirmed as ASB when compared to the non-causal ASB sites (P < 0.01). However, these candidate asbQTL did not enrich for the other annotations, including expression QTL (eQTL), allele-specific expression QTL (aseQTL) and sites conserved across mammals (P > 0.05). Conclusions: We identified putatively causal sites for asbQTL using the DNA sequence surrounding these sites. Our results suggest that many sites influencing histone modifications may not directly affect gene expression. However, it is important to acknowledge that distinguishing between putative causal ASB sites and other non-causal ASB sites in high linkage disequilibrium with the causal sites regarding their impact on gene expression may be challenging due to limitations in statistical power. [ABSTRACT FROM AUTHOR]

Heterozygous de novo mutations in the Activity-Dependent Neuroprotective Homeobox (ADNP) gene underlie Helsmoortel-Van der Aa syndrome (HVDAS). Most of these mutations are situated in the last exon and we previously demonstrated escape from nonsense-mediated decay by detecting mutant ADNP mRNA in patient blood. In this study, wild-type and ADNP mutants are investigated at the protein level and therefore optimal detection of the protein is required. Detection of ADNP by means of western blotting has been ambiguous with reported antibodies resulting in non-specific bands without unique ADNP signal. Validation of an N-terminal ADNP antibody (Aviva Systems) using a blocking peptide competition assay allowed to differentiate between specific and non-specific signals in different sample materials, resulting in a unique band signal around 150 kDa for ADNP, above its theoretical molecular weight of 124 kDa. Detection with different C-terminal antibodies confirmed the signals at an observed molecular weight of 150 kDa. Our antibody panel was subsequently tested by immunoblotting, comparing parental and homozygous CRISPR/Cas9 endonuclease-mediated Adnp knockout cell lines and showed disappearance of the 150 kDa signal, indicative for intact ADNP. By means of both a GFPSpark and Flag-tag N-terminally fused to a human ADNP expression vector, we detected wild-type ADNP together with mutant forms after introduction of patient mutations in E. coli expression systems by site-directed mutagenesis. Furthermore, we were also able to visualize endogenous ADNP with our C-terminal antibody panel in heterozygous cell lines carrying ADNP patient mutations, while the truncated ADNP mutants could only be detected with epitope-tag-specific antibodies, suggesting that addition of an epitope-tag possibly helps stabilizing the protein. However, western blotting of patient-derived hiPSCs, immortalized lymphoblastoid cell lines and post-mortem patient brain material failed to detect a native mutant ADNP protein. In addition, an N-terminal immunoprecipitation-competent ADNP antibody enriched truncating mutants in overexpression lysates, whereas implementation of the same method failed to enrich a possible native mutant protein in immortalized patient-derived lymphoblastoid cell lines. This study aims to shape awareness for critical assessment of mutant ADNP protein analysis in Helsmoortel-Van der Aa syndrome. [ABSTRACT FROM AUTHOR]

Background: Telomeres consist of repetitive DNA sequences at the chromosome ends to protect chromosomal stability, and primarily maintained by telomerase or occasionally by alternative telomere lengthening of telomeres (ALT) through recombination-based mechanisms. Additional mechanisms that may regulate telomere maintenance remain to be explored. Simultaneous measurement of telomere length and transcriptome in the same human embryonic stem cell (hESC) revealed that mRNA expression levels of UBQLN1 exhibit linear relationship with telomere length. Methods: In this study, we first generated UBQLN1-deficient hESCs and compared with the wild-type (WT) hESCs the telomere length and molecular change at RNA and protein level by RNA-seq and proteomics. Then we identified the potential interacting proteins with UBQLN1 using immunoprecipitation-mass spectrometry (IP-MS). Furthermore, the potential mechanisms underlying the shortened telomeres in UBQLN1-deficient hESCs were analyzed. Results: We show that Ubiquilin1 (UBQLN1) is critical for telomere maintenance in human embryonic stem cells (hESCs) via promoting mitochondrial function. UBQLN1 deficiency leads to oxidative stress, loss of proteostasis, mitochondria dysfunction, DNA damage, and telomere attrition. Reducing oxidative damage and promoting mitochondria function by culture under hypoxia condition or supplementation with N-acetylcysteine partly attenuate the telomere attrition induced by UBQLN1 deficiency. Moreover, UBQLN1 deficiency/telomere shortening downregulates genes for neuro-ectoderm lineage differentiation. Conclusions: Altogether, UBQLN1 functions to scavenge ubiquitinated proteins, preventing their overloading mitochondria and elevated mitophagy. UBQLN1 maintains mitochondria and telomeres by regulating proteostasis and plays critical role in neuro-ectoderm differentiation. [ABSTRACT FROM AUTHOR]

Colon adenocarcinoma (COAD) is the second leading cause of cancer death, and there is still a lack of diagnostic biomarkers and therapeutic targets. In this study, bioinformatics analysis of the TCGA database was used to obtain RUNX1, a gene with prognostic value in COAD. RUNX1 plays an important role in many malignancies, and its molecular regulatory mechanisms in COAD remain to be fully understood. To explore the physiological role of RUNX1, we performed functional analyses, such as CCK-8, colony formation and migration assays. In addition, we investigated the underlying mechanisms using transcriptome sequencing and chromatin immunoprecipitation assays. RUNX1 is highly expressed in COAD patients and significantly correlates with survival. Silencing of RUNX1 significantly slowed down the proliferation and migratory capacity of COAD cells. Furthermore, we demonstrate that CDC20 and MCM2 may be target genes of RUNX1, and that RUNX1 may be physically linked to the deubiquitinating enzyme USP31, which mediates the upregulation of RUNX1 protein to promote transcriptional function. Our results may provide new insights into the mechanism of action of RUNX1 in COAD and reveal potential therapeutic targets for this disease. [ABSTRACT FROM AUTHOR]

The spatiotemporal regulation of inflammasome activation remains unclear. To examine the mechanism underlying the assembly and regulation of the inflammasome response, here we perform an immunoprecipitation-mass spectrometry analysis of apoptosis-associated speck-like protein containing a CARD (ASC) and identify NCF4/1/2 as ASC-binding proteins. Reduced NCF4 expression is associated with colorectal cancer development and decreased five-year survival rate in patients with colorectal cancer. NCF4 cooperates with NCF1 and NCF2 to promote NLRP3 and AIM2 inflammasome activation. Mechanistically, NCF4 phosphorylation and puncta distribution switches from the NADPH complex to the perinuclear region, mediating ASC oligomerization, speck formation and inflammasome activation. NCF4 functions as a sensor of ROS levels, to establish a balance between ROS production and inflammasome activation. NCF4 deficiency causes severe colorectal cancer in mice, increases transit-amplifying and precancerous cells, reduces the frequency and activation of CD8+ T and NK cells, and impairs the inflammasome-IL-18-IFN-γ axis during the early phase of colorectal tumorigenesis. Our study implicates NCF4 in determining the spatial positioning of inflammasome assembly and contributing to inflammasome-mediated anti-tumor responses. The NADPH oxidase 2 (NOX2) complex is a main reactive oxygen species (ROS) source during inflammation. Here the authors report that NCF4, a subunit of the NOX2 complex, acts a sensor of ROS levels and regulates NLRP3 and AIM2 inflammasome activation, associated with attenuated colorectal cancer progression. [ABSTRACT FROM AUTHOR]

SUMMARY: Juncus is the largest genus of Juncaceae and was considered holocentric for a long time. Recent findings, however, indicated that 11 species from different clades of the genus have monocentric chromosomes. Thus, the Juncus centromere organization and evolution need to be reassessed. We aimed to investigate the major repetitive DNA sequences of two accessions of Juncus effusus and its centromeric structure by employing whole‐genome analyses, fluorescent in situ hybridization, CENH3 immunodetection, and chromatin immunoprecipitation sequencing. We showed that the repetitive fraction of the small J. effusus genome (~270 Mbp/1C) is mainly composed of Class I and Class II transposable elements (TEs) and satellite DNAs. Three identified satellite DNA families were mainly (peri)centromeric, with two being associated with the centromeric protein CENH3, but not strictly centromeric. Two types of centromere organization were discerned in J. effusus: type 1 was characterized by a single CENH3 domain enriched with JefSAT1‐155 or JefSAT2‐180, whereas type 2 showed multiple CENH3 domains interrupted by other satellites, TEs or genes. Furthermore, while type 1 centromeres showed a higher degree of satellite identity along the array, type 2 centromeres had less homogenized arrays along the multiple CENH3 domains per chromosome. Although the analyses confirmed the monocentric organization of J. effusus chromosomes, our data indicate a more dynamic arrangement of J. effusus centromeres than observed for other plant species, suggesting it may constitute a transient state between mono‐ and holocentricity. [ABSTRACT FROM AUTHOR]

Background: While previous studies have primarily focused on Glucose transporter type 1 (GLUT1) related glucose metabolism signaling, we aim to discover if GLUT1 promotes tumor progression through a non-metabolic pathway. Methods: The RNA-seq and microarray data were comprehensively analyzed to evaluate the significance of GLUT1 expression in lung adenocarcinoma (LUAD). The cell proliferation, colony formation, invasion, and migration were used to test GLUT1 's oncogenic function. Co-immunoprecipitation and mass spectrum (MS) were used to uncover potential GLUT1 interacting proteins. RNA-seq, DIA-MS, western blot, and qRT-PCR to probe the change of gene and cell signaling pathways. Results: We found that GLUT1 is highly expressed in LUAD, and higher expression is related to poor patient survival. GLUT1 knockdown caused a decrease in cell proliferation, colony formation, migration, invasion, and induced apoptosis in LUAD cells. Mechanistically, GLUT1 directly interacted with phosphor-epidermal growth factor receptor (p-EGFR) and prevented EGFR protein degradation via ubiquitin-mediated proteolysis. The GLUT1 inhibitor WZB117 can increase the sensitivity of LUAD cells to EGFR-tyrosine kinase inhibitors (TKIs) Gefitinib. Conclusions: GLUT1 expression is higher in LUAD and plays an oncogenic role in lung cancer progression. Combining GLUT1 inhibitors and EGFR-TKIs could be a potential therapeutic option for LUAD treatment. [ABSTRACT FROM AUTHOR]

The ubiquitination-mediated protein degradation exerts a vital role in the progression of multiple tumors. NEDD4L, which belongs to the E3 ubiquitin ligase NEDD4 family, is related to tumor genesis, metastasis and drug resistance. However, the anti-tumor role of NEDD4L in esophageal carcinoma, and the potential specific recognition substrate remain unclear. Based on public esophageal carcinoma database and clinical sample data, it was discovered in this study that the expression of NEDD4L in esophageal carcinoma was apparently lower than that in atypical hyperplastic esophageal tissue and esophageal squamous epithelium. Besides, patients with high expression of NEDD4L in esophageal carcinoma tissue had longer progression-free survival than those with low expression. Experiments in vivo and in vitro also verified that NEDD4L suppressed the growth and metastasis of esophageal carcinoma. Based on co-immunoprecipitation and proteome analysis, the NEDD4L ubiquitination-degraded protein ITGB4 was obtained. In terms of the mechanism, the HECT domain of NEDD4L specifically bound to the Galx-β domain of ITGB4, which modified the K915 site of ITGB4 in an ubiquitination manner, and promoted the ubiquitination degradation of ITGB4, thus suppressing the malignant phenotype of esophageal carcinoma. [ABSTRACT FROM AUTHOR]