Transcriptional Profiling Research Articles

Single-cell transcriptome analysis reveals characteristic transcription factors in polydactyly.

The study aims to provide guidance on the identification of multiple-digit malformations as potential biomarkers and therapeutic targets. Single-cell RNA sequencing (scRNA-seq) data of four multiple-finger malformation samples were downloaded from the GEO public database. Fibroblasts and keratinocytes were divided into cellular subpopulations and the transcription factors of different subpopulations were analyzed. The regulatory network of transcription factors and their target genes were constructed to analyze the functionality of regulons. Examination of the transcriptional profile data from 11,806 single cells uncovered significant associations between regulons and cell function in polydactyly. Specifically, the analysis highlighted the involvement of HOX family members and GLI2 transcription factors, including HOXD13, MSX2, LHX2, EMX2, LEF1, CREB3L2, and LHX2, in the polydactyly process within fibroblast cells. Furthermore, it sheds light on the roles of HES2 and GLIS1 in the formation and development of keratinocytes. Significant presence of transcription factors, especially HOXD13, MSX2, and LHX2, may be strongly related to the development of polydactyly.

Exacerbating effects of circadian rhythm disruption on the systemic lupus erythematosus

ObjectiveCircadian rhythm disruption (CRD) has been associated with inflammation and immune disorders, but its role in SLE progression is unclear. We aimed to investigate the impact of circadian rhythms on...

1242 Metabologenomic Characterization Uncovers Heterogeneity Amongst IDH Mutant Gliomas

INTRODUCTION: Mutations in isocitrate dehydrogenase (IDH) enzymes are recognised to drive the molecular footprint of diffuse gliomas through the accumulation of oncometabolite R-2-hydroxyglutarate which drives the widespread restructuring of the DNA methylome; however, beyond R-2-hydroxyglutarate, a comprehensive metabologenomic characterization of IDH-mutant gliomas has yet to be performed. METHODS: Glioma samples from a cohort of 154 patients underwent multiplatform molecular analysis, including metabolomic studies, genome-wide DNA methylation profiling and bulk RNA sequencing. A integrative analysis was performed, including hierarchical clustering and principal component analysis of identified metabolites, differentially methylated probes, copy number alteration and bulk mRNA data. Survival analysis as well as multivariable hazard ratios for clinical variables were calculated by fitting Cox Proportional Hazards Models. RESULTS: We discovered a group of IDH-mutant gliomas whose metabolic profile highly resembled IDH wildtype tumors. Notably, these IDH-mutant gliomas with dysregulated metabolism were distinguished from their IDH-mutant counterparts by significantly shorter overall survival (median OS 118.9 months vs 173.6 months, p=0.048). The IDH-mutant tumors with dysregulated metabolism harbored distinct epigenetic alterations that converged to drive proliferative and stem-like transcriptional profiles. The prognostic relevance of dysregulated metabolism complements, but was not wholly explained by canonically recognized prognostic classifications in IDH-mutant gliomas including 1p/19q codeletion, glioma CpG Island Hypermethylator (GCIMP) status and CDKN2A homozygous deletion. CONCLUSIONS: Utilizing a cross-platform analysis we have uncovered a novel subtyping of IDH-mutant gliomas with dysregulated cellular metabolism with similar survival to IDH-wildtype tumors. The metabolic profile provides unique information on glioma phenotypes, which may facilitate a more comprehensive understanding of glioma biology, and provide a window to target novel dependencies.

Evolutionary dynamics of the cytoskeletal profilin gene family in Brassica juncea L. reveal its roles in silique development and stress resilience

Essential to plant adaptation, cell wall (CW) integrity is maintained by CW-biosynthesis genes. Cytoskeletal actin-(de)polymerizing, phospholipid-binding profilin (PRF) proteins play important roles in maintaining cellular homeostasis across kingdoms. However, evolutionary selection of PRF genes and their systematic characterization in family Brassicaceae, especially in Brassica juncea remain unexplored. Here, a comprehensive analysis of genome-wide identification of BjPRFs, their phylogenetic association, genomic localization, gene structure, and transcriptional profiling were performed in an evolutionary framework. Identification of 23 BjPRFs in B. juncea indicated an evolutionary conservation within Brassicaceae. The BjPRFs evolved through paralogous and orthologous gene formation in Brassica genomes. Evolutionary divergence of BjPRFs indicated purifying selection, with nonsynonymous (dN)/synonymous (dS) value of 0.090 for orthologous gene-pairs. Hybrid homology-modeling identified evolutionary distinct and conserved domains in BjPRFs which suggested that these proteins evolved following the divergence of monocot and eudicot plants. RNA-seq profiles of BjPRFs revealed their functional evolution in spatiotemporal manner during plant-development and stress-conditions in diploid/amphidiploid Brassica species. Real-Time PCR experiments in seedling, vegetative, floral and silique tissues of B. juncea suggested their essential roles in systematic plant development. These observations underscore the expansion of BjPRFs in B. juncea, and offer valuable evolutionary insights for exploring cellular mechanisms, and stress resilience.

Transcriptional profiling reveals fundamental differences in iPS-derived CD34+ Cells versus adult circulating CD34+

CD34+ cells hold significant promise in regenerative medicine and the treatment of various vascular degenerative diseases primarily because of their ability to regenerate and differentiate into various cell types. These cells can be derived from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), or adult stem cells like blood and bone marrow, and can differentiate into endothelial, hematopoietic, myeloid, and other cell types. However, their characteristics vary based on the source, making detailed definitions essential for specific therapeutic applications. In this study, it was aimed to compare these two different CD34-positive cell populations by full genome transcriptional profiling (RNAseq). To this end, we first optimized a CD34+ cell differentiation protocol and found that Vascular Endothelial Growth Factor (VEGF) is critical for the transition of cells from mesodermal precursors to CD34+ cells. Additionally, principal component analysis (PCA) of RNAseq data showed that blood-derived CD34+ cells clustered far from iPS-derived CD34+ cells which illustrates these populations are fundamentally different. This data will be useful to better define these cell populations and facilitate the translation of regenerative approaches in this field as well as providing potentially novel diagnostic tools.

In silico analysis of the key molecules of SARS-CoV-2: ACE2 and TMPRSS2 in head and neck cancer

It has been reported that cancer patients are more susceptible to SARS-CoV-2 infection due to their immunocompromised state. Although ACE2 and TMPRSS2 are essential for SARS-CoV-2 virus entry, the significant role of these molecules in HNSC is still unknown. Thus, the study aimed to delineate the fundamental role these molecules in HNSC using a systems biology approach. The transcriptional profile of ACE2 and TMPRSS2 in HNSC was evaluated using the TIMER and UALCAN databases. The association between the mRNA levels of ACE2 and TMPRSS2 was assessed using GEPIA2. cBioPortal was used to assess the genetic alterations in the ACE2 and TMPRSS2 protein sequences involved in HNSC. The gene associated with COVID-19 and HNSC was identified using the CTD database. Finally, the STRING and PANTHER online platforms were used to construct a protein-protein interaction (PPI) network and to determine the functional role of genes retrieved from the CTD database, respectively. ACE2 expression was found to be unaltered in HNSC and uninfected HNSC datasets compared to normal and HPV-infected HNSC datasets, respectively. However, the TMPRSS2 levels in HNSC and HPV-infected HNSC were significantly (P ≤ 0.001) downregulated and upregulated compared to healthy controls and uninfected HNSC, respectively. A significant (P ≤ 0.014) but weak positive correlation was noted between ACE2 and TMPRSS2 in HNSC. The PPI network also showed significant involvement of COVID-19 and HNSC-related genes in catalysis, binding activity, and several cancers signaling pathways. Based on the in-silico analysis, it could be concluded that ACE2 and TMPRSS2, the key molecules of SARS-CoV-2, might be involved in HNSC. However, the long-term effects of SARS-CoV-2 infection, if any, on HNSC patients in clinical settings require further study.

Transcriptional Responses of Different Brain Cell Types to Oxygen Decline.

Brain hypoxia is associated with a wide range of physiological and clinical conditions. Although oxygen is an essential constituent of maintaining brain functions, our understanding of how specific brain cell types globally respond and adapt to decreasing oxygen conditions is incomplete. In this study, we exposed mouse primary neurons, astrocytes, and microglia to normoxia and two hypoxic conditions and obtained genome-wide transcriptional profiles of the treated cells. Analysis of differentially expressed genes under conditions of reduced oxygen revealed a canonical hypoxic response shared among different brain cell types. In addition, we observed a higher sensitivity of neurons to oxygen decline, and dissected cell type-specific biological processes affected by hypoxia. Importantly, this study establishes novel gene modules associated with brain cells responding to oxygen deprivation and reveals a state of profound stress incurred by hypoxia.

The key role of myostatin b in somatic growth in fishes derived from distant hybridization.

The basic mechanism of heterosis has not been systematically and completely characterized. In previous studies, we obtained three economically important fishes that exhibit rapid growth, WR (WCC ♀ × RCC ♂), WR-II (WR ♀ × WCC ♂), and WR-III (WR-II ♀ × 4nAU ♂), through distant hybridization. However, the mechanism underlying this rapid growth remains unclear. In this study, we found that WR, WR-II, and WR-III showed muscle hypertrophy and higher muscle protein and fat contents compared with their parent species (RCC and WCC). Candidate genes responsible for this rapid growth were then obtained through an analysis of 12 muscle transcriptomes. Notably, the mRNA level of mstnb (myostatin b), which is a negative regulator of myogenesis, was significantly reduced in WR, WR-II, and WR-III compared with the parent species. To verify the function of mstnb, a mstnb-deficient mutant RCC line was generated using the CRISPR-Cas9 technique. The average body weight of mstnb-deficient RCC at 12 months of age was significantly increased by 29.57% compared with that in wild-type siblings. Moreover, the area and number of muscle fibers were significantly increased in mstnb-deficient RCC, indicating hypertrophy and hyperplasia. Furthermore, the muscle protein and fat contents were significantly increased in mstnb-deficient RCC. The molecular regulatory mechanism of mstnb was then revealed by transcription profiling, which showed that genes related to myogenesis (myod, myog, and myf5), protein synthesis (PI3K-AKT-mTOR), and lipogenesis (pparγ and fabp3) were highly activated in hybrid fishes and mstnb-deficient RCC. This study revealed that low expression or deficiency of mstnb regulates somatic growth by promoting myogenesis, protein synthesis, and lipogenesis in hybrid fishes and mstnb-deficient RCC, which provides evidence for the molecular mechanism of heterosis via distant hybridization.

Select EZH2 inhibitors enhance viral mimicry effects of DNMT inhibition through a mechanism involving NFAT:AP-1 signaling.

DNA methyltransferase inhibitor (DNMTi) efficacy in solid tumors is limited. Colon cancer cells exposed to DNMTi accumulate lysine-27 trimethylation on histone H3 (H3K27me3). We propose this Enhancer of Zeste Homolog 2 (EZH2)-dependent repressive modification limits DNMTi efficacy. Here, we show that low-dose DNMTi treatment sensitizes colon cancer cells to select EZH2 inhibitors (EZH2is). Integrative epigenomic analysis reveals that DNMTi-induced H3K27me3 accumulates at genomic regions poised with EZH2. Notably, combined EZH2i and DNMTi alters the epigenomic landscape to transcriptionally up-regulate the calcium-induced nuclear factor of activated T cells (NFAT):activating protein 1 (AP-1) signaling pathway. Blocking this pathway limits transcriptional activating effects of these drugs, including transposable element and innate immune response gene expression involved in viral defense. Analysis of primary human colon cancer specimens reveals positive correlations between DNMTi-, innate immune response-, and calcium signaling-associated transcription profiles. Collectively, we show that compensatory EZH2 activity limits DNMTi efficacy in colon cancer and link NFAT:AP-1 signaling to epigenetic therapy-induced viral mimicry.

Characterization of Th17 tissue-resident memory cells in non-inflamed intestinal tissue of Crohn's disease patients

Crohn's disease (CD) is a chronic inflammatory disorder affecting the bowel wall. Tissue-resident memory T (Trm) cells are implicated in CD, yet their characteristics remain unclear. We aimed to investigate the transcriptional profiles and functional characteristics of Trm cells in the small bowel of CD and their interactions with immune cells. Seven patients with CD and four with ulcerative colitis as controls were included. Single-cell RNA sequencing and paired T cell receptor sequencing assessed T cell subsets and transcriptional signatures in lamina propria (LP) and submucosa/muscularis propria-enriched fractions (SM/MP) from small bowel tissue samples. We detected 58,123 T cells grouped into 16 populations, including the CD4+ Trm cells with a Th17 signature and CD8+ Trm clusters. In CD, CD4+ Trm cells with a Th17 signature, termed Th17 Trm, showed significantly increased proportions within both the LP and SM/MP areas. The Th17 Trm cluster demonstrated heightened expression of tissue-residency marker genes (ITGAE, ITGA1, and CXCR6) along with elevated levels of IL17A, IL22, CCR6, and CCL20. The clonal expansion of Th17 Trm cells in CD was accompanied by enhanced transmural dynamic potential, as indicated by significantly higher migration scores. CD-prominent Th17 Trm cells displayed an increased interferon gamma (IFNγ)-related signature possibly linked with STAT1 activation, inducing chemokines (i.e., CXCL10, CXCL8, and CXCL9) in myeloid cells. Our findings underscored the elevated Th17 Trm cells throughout the small bowel in CD, contributing to disease pathogenesis through IFNγ induction and subsequent chemokine production in myeloid cells.

Transcriptome-Wide Identification and Expression Analysis of Genes Encoding Defense-Related Peptides of Filipendula ulmaria in Response to Bipolaris sorokiniana Infection.

Peptides play an essential role in plant development and immunity. Filipendula ulmaria, belonging to the Rosaceae family, is a medicinal plant which exhibits valuable pharmacological properties. F. ulmaria extracts in vitro inhibit the growth of a variety of plant and human pathogens. The role of peptides in defense against pathogens in F. ulmaria remains unknown. The objective of this study was to explore the repertoire of antimicrobial (AMPs) and defense-related signaling peptide genes expressed by F. ulmaria in response to infection with Bipolaris sorokiniana using RNA-seq. Transcriptomes of healthy and infected plants at two time points were sequenced on the Illumina HiSeq500 platform and de novo assembled. A total of 84 peptide genes encoding novel putative AMPs and signaling peptides were predicted in F. ulmaria transcriptomes. They belong to known, as well as new, peptide families. Transcriptional profiling in response to infection disclosed complex expression patterns of peptide genes and identified both up- and down-regulated genes in each family. Among the differentially expressed genes, the vast majority were down-regulated, suggesting suppression of the immune response by the fungus. The expression of 13 peptide genes was up-regulated, indicating their possible involvement in triggering defense response. After functional studies, the encoded peptides can be used in the development of novel biofungicides and resistance inducers.

Mesenchymal stem cells alleviate mouse liver fibrosis by inhibiting pathogenic function of intrahepatic B cells.

The immunomodulatory characteristics of mesenchymal stem cells (MSCs) make them a promising therapeutic approach for liver fibrosis (LF). Here, we postulated that MSCs could potentially suppress the pro-fibrotic activity of intrahepatic B cells, thereby inhibiting LF progression. Administration of MSCs significantly ameliorated LF as indicated by reduced myofibroblast activation, collagen deposition, and inflammation. The treatment efficacy of MSCs can be attributed to decreased infiltration, activation, and pro-inflammatory cytokine production of intrahepatic B cells. Single-cell RNA sequencing revealed a distinct intrahepatic B cell atlas, and a subtype of naive B cells (B-II) was identified, which were markedly abundant in fibrotic liver, displaying mature features with elevated expression of several proliferative and inflammatory genes. Transcriptional profiling of total B cells revealed that intrahepatic B cells displayed activation, proliferation, and pro-inflammatory gene profile during LF. Fibrosis was attenuated in mice ablated with B cells (μMT) or in vivo treatment with anti-CD20. Moreover, fibrosis was recapitulated in μMT after adoptive transfer of B cells, which in turn could be rescued by MSC injection, validating the pathogenic function of B cells and the efficacy of MSCs on B cell-promoted LF progression. Mechanistically, MSCs could inhibit the proliferation and cytokine production of intrahepatic B cells through exosomes, regulating the Mitogen-activated protein kinase and Nuclear factor kappa B signaling pathways. Intrahepatic B cells serve as a target of MSCs, play an important role in the process of MSC-induced amelioration of LF, and may provide new clues for revealing the novel mechanisms of MSC action.

Exploring the PpEXPs Family in Peach: Insights into Their Role in Fruit Texture Development through Identification and Transcriptional Analysis

Expansins (EXPs) loosen plant cell walls and are involved in diverse developmental processes through modifying cell-walls; however, little is known about the role of PpEXPs in peach fruit. In this study, 26 PpEXP genes were identified in the peach genome and grouped into four subfamilies, with 20 PpEXPAs, three PpEXPBs, one PpEXPLA and two PpEXPLBs. The 26 PpEXPs were mapped on eight chromosomes. The primary mode of gene duplication of the PpEXPs was dispersed gene duplication (DSD, 50%). Notably, cis-elements involved in light responsiveness and MeJA-responsiveness were detected in the promoter regions of all PpEXPs, while ethylene responsive elements were observed in 12 PpEXPs. Transcript profiling of PpEXPs in the peach fruit varieties of MF (melting), NMF (non-melting) and SH (stony hard) at different stages showed that PpEXPs displayed distinct expression patterns. Among the 26 PpEXPs, 15 PpEXPs were expressed in the fruit. Combining the expressing patterns of PpEXPs in fruits with different flesh textures, PpEXPA7, PpEXPA13 and PpEXPA15 were selected as candidate genes, as they were highly consistent with the patterns of previous reported key genes (PpPGM, PpPGF and PpYUC11) in regard to peach fruit texture. The genes with different expression patterns between MF and NMF were divided into 16 modules, of which one module, with pink and midnightblue, negatively correlated with the phenotype of fruit firmness and was identified as PpEXPA1 and PpEXPA7, while the other module was identified as PpERF in the pink module, which might potentially effect fruit texture development by regulating PpEXPs. These results provide a foundation for the functional characterization of PpEXPs in peach.

Evidence that Xrn1 is in complex with Gcn1, and is required for full levels of eIF2α phosphorylation.

The protein kinase Gcn2 and its effector protein Gcn1 are part of the general amino acid control signalling (GAAC) pathway best known in yeast for its function in maintaining amino acid homeostasis. Under amino acid limitation, Gcn2 becomes activated, subsequently increasing the levels of phosphorylated eIF2α (eIF2α-P). This leads to the increased translation of transcriptional regulators, such as Gcn4 in yeast and ATF4 in mammals, and subsequent re-programming of the cell's gene transcription profile, thereby allowing cells to cope with starvation. Xrn1 is involved in RNA decay, quality control and processing. We found that Xrn1 co-precipitates Gcn1 and Gcn2, suggesting that these three proteins are in the same complex. Growth under starvation conditions was dependent on Xrn1 but not on Xrn1-ribosome association, and this correlated with reduced eIF2α-P levels. Constitutively active Gcn2 leads to a growth defect due to eIF2α-hyperphosphorylation, and we found that this phenotype was independent of Xrn1, suggesting that xrn1 deletion does not enhance eIF2α de-phosphorylation. Our study provides evidence that Xrn1 is required for efficient Gcn2 activation, directly or indirectly. Thus, we have uncovered a potential new link between RNA metabolism and the GAAC.

Influence of Two Hexose Transporters on Substrate Affinity and Pathogenicity in Magnaporthe oryzae.

Hexose transporters (HXT) play a crucial role in the pathogenicity of Magnaporthe oryzae, serving not only as key facilitators for acquiring and transporting sugar nutrients to support pathogen development, but also as sugar sensors which receive transduction signals. The objective of this study is to investigate the impact of MoHXT1-3 on rice pathogenicity and hexose affinity. MoHXT1-3 deletion mutants were generated using CRISPR/Cas9 technology, and their affinity for hexose was evaluated through yeast complementation assays and electrophysiological experiments in Xenopus oocytes. The results suggest that MoHXT1 does not contribute to melanin formation or hexose transportation processes. Conversely, MoHXT2, despite displaying lower affinity towards the hexoses tested in comparison to MoHXT3, is likely to have a more substantial impact on pathogenicity. The analysis of the transcription profiles demonstrated that the deletion of MoHXT2 caused a decrease in the expression of MoHXT3, whereas the knockout of MoHXT3 resulted in an upregulation of MoHXT2 transcription. It is noteworthy that the MoHXT2M145K variant displayed an incapacity to transport hexoses. This investigation into the functional differences in hexose transporters in Magnaporthe oryzae provides insights into potential advances in new strategies to target hexose transporters to combat rice blast by blocking carbon nutrient supply.

Autophagy-enhancing ATG16L1 polymorphism is associated with improved clinical outcome and T-cell immunity in chronic HIV-1 infection

Chronic HIV-1 infection is characterized by T-cell dysregulation that is partly restored by antiretroviral therapy. Autophagy is a critical regulator of T-cell function. Here, we demonstrate a protective role for autophagy in HIV-1 disease pathogenesis. Targeted analysis of genetic variation in core autophagy gene ATG16L1 reveals the previously unidentified rs6861 polymorphism, which correlates functionally with enhanced autophagy and clinically with improved survival of untreated HIV-1-infected individuals. T-cells carrying ATG16L1 rs6861(TT) genotype display improved antiviral immunity, evidenced by increased proliferation, revamped immune responsiveness, and suppressed exhaustion/immunosenescence features. In-depth flow-cytometric and transcriptional profiling reveal T-helper-cell-signatures unique to rs6861(TT) individuals with enriched regulation of pro-inflammatory networks and skewing towards immunoregulatory phenotype. Therapeutic enhancement of autophagy recapitulates the rs6861(TT)-associated T-cell traits in non-carriers. These data underscore the in vivo relevance of autophagy for longer-lasting T-cell-mediated HIV-1 control, with implications towards development of host-directed antivirals targeting autophagy to restore immune function in chronic HIV-1 infection.

Transcriptome analysis of the bipotential gonads of hermaphroditic bivalve species Tridacna crocea unravels the potential genes responsible for spermatogenesis

Sex is an integral part of the evolution of species and often takes the form of intersex and sexual change in mollusks. As a major member of the deep-sea coral reef ecosystem, Tridacna crocea has complex gonadal development mechanisms and reproductive strategies, and the regulation model of spermatogenesis needs to be further explored. In this study, gonadal transcripts of T. crocea in resting, spermatogonia and sperm stage were evaluated. According to the statistics of bioinformatics analysis, a total of 10,260 DEGs were identified, including 3021 genes shared by three developmental stages. Through dynamic expression analysis, we identified the expression patterns and functional classification of genes that regulate spermatogenesis. These candidate genes were involved in regulatory mechanisms such as nucleotide binding, cell cycle, sexual reproduction and DNA replication. Subsequently, based on functional screening, PPI analysis was performed on the target gene cluster to learn the network relationships among candidate genes and other genes closely related to candidate genes. The results showed that in addition to candidate genes (zmcm6, gins2, mcm9, blm, mcm4, psmc3ip, haus, ccnb3, spice, rad1, rad17, ncapg, bub1b, lin9, tipin, and cks), several genes that have been shown to be associated with gonadal development were present in the network (such as fanci, aurka, kif20a and nek2), suggesting the reliability of the results. Finally, the expression levels of candidate genes between individuals at different developmental stages were compared with transcriptional expression profiles, which further proved that these candidate genes were related to T. crocea gonad development traits. These results make up for the lack of research on T. crocea gametogenesis, spermatogenesis and gonad development, and provide a reference for the research on Tridacna reproductive mechanism.

The impact of canonical Wnt transcriptional repressors TLE3 and TLE4 on postsynaptic transcription at the neuromuscular junction.

Here, we investigated the role of the canonical Wnt signaling pathway transcriptional regulators at the neuromuscular junction. Upon applying a denervation paradigm, the transcription levels of Ctnnb1, Tcf7l1, Tle1, Tle2, Tle3, and Tle4 were significantly downregulated. A significant decrease in canonical Wnt signaling activity was observed using the denervation paradigm in Axin2-lacZ reporter mice. Alterations in the transcriptional profile of the myogenic lineage in response to agrin (AGRN) suggested that TLE3 and TLE4, family members of groucho transducin-like enhancer of split 3 (TLE3), transcriptional repressors known to antagonize T cell factor/lymphoid enhancer factor (TCF)-mediated target gene activation, could be important regulators of canonical Wnt signaling activity at the postsynapse. Knockouts of these genes using CRISPR/Cas9 gene editing in primary skeletal muscle stem cells, called satellite cells, led to decreased AGRN-dependent acetylcholine receptor (CHRN) clustering and reduced synaptic gene transcription upon differentiation of these cells. Overall, our findings demonstrate that TLE3 and TLE4 participate in diminishing canonical Wnt signaling activity, supporting transcription of synaptic genes and CHRN clustering at the neuromuscular junction.

Radiofrequency radiation reshapes tumor immune microenvironment into antitumor phenotype in pulmonary metastatic melanoma by inducing active transformation of tumor-infiltrating CD8+ T and NK cells

Immunosuppression by the tumor microenvironment is a pivotal factor contributing to tumor progression and immunotherapy resistance. Priming the tumor immune microenvironment (TIME) has emerged as a promising strategy for improving the efficacy of cancer immunotherapy. In this study we investigated the effects of noninvasive radiofrequency radiation (RFR) exposure on tumor progression and TIME phenotype, as well as the antitumor potential of PD-1 blockage in a model of pulmonary metastatic melanoma (PMM). Mouse model of PMM was established by tail vein injection of B16F10 cells. From day 3 after injection, the mice were exposed to RFR at an average specific absorption rate of 9.7 W/kg for 1 h per day for 14 days. After RFR exposure, lung tissues were harvested and RNAs were extracted for transcriptome sequencing; PMM-infiltrating immune cells were isolated for single-cell RNA-seq analysis. We showed that RFR exposure significantly impeded PMM progression accompanied by remodeled TIME of PMM via altering the proportion and transcription profile of tumor-infiltrating immune cells. RFR exposure increased the activation and cytotoxicity signatures of tumor-infiltrating CD8+ T cells, particularly in the early activation subset with upregulated genes associated with T cell cytotoxicity. The PD-1 checkpoint pathway was upregulated by RFR exposure in CD8+ T cells. RFR exposure also augmented NK cell subsets with increased cytotoxic characteristics in PMM. RFR exposure enhanced the effector function of tumor-infiltrating CD8+ T cells and NK cells, evidenced by increased expression of cytotoxic molecules. RFR-induced inhibition of PMM growth was mediated by RFR-activated CD8+ T cells and NK cells. We conclude that noninvasive RFR exposure induces antitumor remodeling of the TIME, leading to inhibition of tumor progression, which provides a promising novel strategy for TIME priming and potential combination with cancer immunotherapy.

Transcriptional Profiling of BpWRKY49 Reveals Its Role as a Master Regulator in Stress Signaling Pathways in Birch (Betula platyphylla)

The WRKY family of transcription factors (TFs) is one of the most diverse families in plants, playing crucial roles in various plant growth and stress response processes. Asian white birch (Betula platyphylla) is a globally distributed tree species that holds ecological, medical, and economic significance. However, the regulatory mechanisms of WRKY TFs in birch remain poorly understood. Herein, we cloned and characterized the BpWRKY49 gene from birch. Through bioinformatics analyses, we revealed the potential involvement of BpWRKY49 in both biotic and abiotic stress responses. In addition, BpWRKY49 was found to be localized in the nucleus and exhibited transcriptional activity in yeast. Transactivation assays further confirmed that BpWRKY49 exhibited transcriptional activity at its C-terminal end. Notably, our binding specificity assays demonstrated the specific interaction of BpWRKY49 with the W-box cis element in vitro. Furthermore, tissue-specific expression analysis demonstrated that BpWRKY49 exhibited the highest expression level in the roots. Real-time quantitative PCR (RT-qPCR) analysis of birch plants subjected to salt and drought treatments revealed that BpWRKY49 displayed significant 30-fold and 10-fold upregulations under salt and drought stress conditions, respectively. DAP-seq analysis of BpWRKY49 identified a total of 21,832 peaks, with 3477 occurring in the promoter region of genes. Gene ontology (GO) enrichment analysis highlighted prominent terms related to defense against biotic stress, followed by terms associated with abiotic stress and development. Y1H assays of three genes provided evidence for the binding ability of BpWRKY49 to the promoters of BpPUB21, BpBTL15, and BpHIP47 in vitro. Collectively, our findings strongly suggest that BpWRKY49 possesses diverse functions and may activate multiple genes to contribute to various biological processes, including salt stress tolerance, in birch.