Downstream Genes Research Articles

Differential Localization and Functional Roles of mGluR6 Paralogs in Zebrafish Retina.

To define the location of mglur6 paralogs in the outer zebrafish retina and delineate their contribution to retina light responses across the visual spectrum. In situ hybridization and immunolocalization with custom-made antibodies were used to localize mglur6 transcripts, proteins, and additional components of the mGluR6 signaling complex. Gene editing was used to generate knockout mutants that were analyzed with white light and spectral electroretinography. Both mglur6 paralogs colocalized with known downstream pathway genes, such as trpm1a, nyctalopin, and gnaoβ. All rod photoreceptors contacted mGluR6-positive cells, while cone connectivity presented a more complex situation with no red cones and only a few UV and blue-sensitive cones connecting to mGluR6a-positive bipolar cells. All cone subtypes contacted mGluR6b-positive cells with markedly fewer red-sensitive cones. Retinas of knockout animals displayed no morphologic alterations. While ERG responses were unaffected in mglur6a knockout animals, mglur6b mutants displayed decreased responses over all spectral wavelengths. We demonstrated that mGlurR6 signalplex components are similar in the zebrafish and the mammalian retina. Despite mglur6b knockout animals having significantly impaired ERG b-wave responses, a residual b-wave persists, even in double knockouts, suggesting additional pathway components yet to be identified.

IFI27 inhibits HIV-1 replication by degrading Gag protein through the ubiquitin-proteasome pathway.

Type I interferon (IFN-I) and its downstream genes play a profound role in HIV infection. In this study, we found that an IFN-inducible gene, IFI27, was upregulated in HIV-1 infection, which in turn efficiently suppressed HIV-1 replication, specially degraded the viral gag protein, including p24 and p55 subunits. Notably, the anti-HIV-1 activity of IFI27 in Old World monkeys surpassed that in New World monkeys, and IFI27 has a higher potentially inhibitory effect on HIV-1 than simian immunodeficiency virus (SIV). Our initial observations showed that NPM-IFI27, the IFI27 variant in northern pig-tailed macaque (Macaca leonina, NPM), exhibited a strong anti-HIV-1 activity. Further investigation demonstrated that NPM-IFI27 degraded p24 and p55 via the ubiquitin-proteasome pathway, with NPM-IFI27-37-115 interacting with the p24-N domain, and the NPM-IFI27-76-122 domain was closely associated with K48 ubiquitin recruitment. Additionally, Skp2 was identified as the probable E3 ubiquitin ligase responsible for the degradation of p24 and p55. Similarly, human IFI27 (Hu-IFI27) showed a mechanism similar to NPM-IFI27 in HIV-1 inhibition. These findings underscore the pivotal role of NPM-IFI27 in HIV-1 infection and provide a potential strategy for clinical anti-HIV-1 therapy.IMPORTANCEHIV-1 infection can trigger the production of IFN-I, which subsequently activates the expression of various IFN-stimulated genes (ISGs) to antagonize the virus. Therefore, discovering novel host antiviral agents for HIV-1 treatment is crucial. Our previous study revealed that IFI27 can influence HIV-1 replication. In this study, we observed that the NPM-IFI27 complex specifically inhibited HIV-1 by targeting its Gag protein. Further exploration demonstrated that IFI27 interacted with the HIV-1 p24 and p55 proteins, leading to their degradation through the ubiquitin-proteasome pathway. Notably, the NPM-IFI27-37-122 variant exhibited potent anti-HIV-1 activity, comparable to that of SAMHD1. These findings highlight the critical role and inhibitory mechanism of NPM-IFI27 in HIV-1 infection, providing a potential strategy for clinical antiviral therapy.

Isotschimgine promotes lifespan, healthspan and neuroprotection of Caenorhabditis elegans via the activation of nuclear hormone receptors.

Isotschimgine (ITG) is a bornane-type monoterpenoid derivative naturally occurring in genus Ferula plants and propolis. Its effects on aging and the underlying mechanisms are not yet well understood. This study employed Caenorhabditis elegans (C. elegans) as a model organism to evaluate the potential of ITG in extending lifespan, enhancing healthspan, and promoting neuroprotection, while exploring the underlying mechanisms involved. The results showed that ITG extended the lifespan and healthspan of C. elegans, significantly enhanced stress resistance and detoxification functions. Studies on mutants and qPCR data indicated that ITG-mediated lifespan extension was modulated by the insulin/IGF-1 signaling pathway and nuclear hormone receptors. Furthermore, ITG markedly increased stress-responsive genes, including daf-16 and its downstream genes sod-3 and hsp-16.2, as well as NHR downstream detoxification-related genes cyp35a1, cyp35b3, cyp35c1, gst-4, pgp-3 and pgp-13. Additionally, ITG alleviated β-amyloid-induced paralysis and behavioral dysfunction in transgenic C. elegans strains. The neuroprotective efficacy of ITG was weakened by RNAi knockdown of nuclear hormone receptors daf-12 and nhr-8. Overall, our study identifies ITG as a potential compound for promoting longevity and neuroprotection, mediated through nuclear hormone receptors.

Overexpression of SLAP2 inhibits triple-negative breast cancer progression by promoting macrophage M1-type polarization

Breast cancer is the most common malignant tumor in women, and triple-negative breast cancer (TNBC) is a specific subtype of breast cancer characterized by high invasiveness, high metastatic potential, ease of recurrence, and poor prognosis. Src-like adaptor protein 2 (SLAP2), which can be involved in the regulation of multiple signaling pathways, may be a key target for TNBC. The aim of this study was to investigate the effect of overexpression of SLAP2 on TNBC and to explore the underlying mechanisms. First, we constructed and transfected SLAP2 overexpressing lentivirus based on MDA-MB-231 human TNBC cell line, screened for differential downstream target genes in combination with mRNA high-throughput sequencing (RNA-Seq), and predicted their functions and enriched pathways in conjunction with bioinformatics analysis. The effects of SLAP2 overexpression on macrophage polarization, as well as on tumor proliferation and apoptosis, were assessed by tail vein injection of a stable transfection line of 4T1 cells transfected with SLAP2 overexpressing lentivirus. The effect of SLAP2 on macrophage polarization was assessed by inducing M1/M2 polarization and transfecting SLAP2 overexpressing lentivirus. Meanwhile, a transwell co-culture system was constructed between differently treated macrophages and 4T1 cells to assess the effect of SLAP2 overexpression on the malignant behavior of the cells via macrophage polarization. Overexpression of SLAP2 revealed 179 genes up-regulated and 74 genes down-regulated by mRNA high-throughput sequencing, and the enriched functions and pathways of differential genes were mainly related to immunity response. In vivo experiments revealed that overexpression of SLAP2 inhibited the growth of tumor in nude mice, decreased the expression of ki67 in tumor tissues, and increased the rate of apoptosis in tumor tissues. Meanwhile, we found that overexpression of SLAP2 promoted macrophage polarization toward M1 type and inhibited M2 type polarization in tumors. In vitro experiments further verified its effect on M1/M2 polarization by transfecting SLAP2 overexpressing lentivirus. By transwell co-culture system, we further demonstrated that overexpression of SLAP2 inhibits cell proliferation and invasion, promotes apoptosis, up-regulates the expression of Bax in cells, and down-regulates the expression of Bcl-2 in cells by promoting macrophage M1-type polarization. Overexpression of SLAP2 inhibits TNBC progression by promoting macrophage M1-type polarization.

Epithelial membrane protein 1 in human cancer: a potential diagnostic biomarker and therapeutic target.

Epithelial membrane protein 1 (EMP1) is a member of the small hydrophobic membrane protein subfamily. EMP1 is aberrantly expressed in various tumor tissues and governs multiple cellular behaviors (e.g., proliferation, differentiation, and migration). The resultant regulation of the cancer pathway is responsible for the metastasis of cancer cells and determines the risk of malignant tumor progression. This review provides an updated overview of EMP1 as either an oncogene or a tumor suppressor contingent on the cancer type and summarizes its upstream regulators and downstream target genes. This systematic review summarizes our current understanding of the role of EMP1 in malignant tumor development, including critical functional mechanisms and implications for its potential use as the biomarker and therapeutic target.

Overexpression of wheat C2H2 zinc finger protein transcription factor TaZAT8-5B enhances drought tolerance and root growth in Arabidopsis thaliana.

TaZAT8-5B, a C2H2 zinc finger protein transcription factor, positively regulates drought tolerance in transgenic Arabidopsis. It promotes root growth under drought stress via the Aux/IAA-ARF module in the auxin signaling pathway. C2H2 zinc finger proteins (C2H2-ZFPs) represent the largest but relatively unexplored family of transcription factors in plants. This is particularly evident in wheat, where the functions of only a few C2H2-ZFP genes have been confirmed. In this study, we identified a novel C2H2-ZFP gene, TaZAT8-5B. This gene shows high expression in roots and flowers and is significantly induced by heat, drought, and salt stress. Under drought stress, overexpressing TaZAT8-5B in Arabidopsis resulted in increased proline content and superoxide dismutase (SOD) activity in leaves. It also led to reduced stomatal aperture and water loss, while inducing the expression of P5CS1, RD29A, and DREB1A. Consequently, it alleviated drought stress-induced malondialdehyde (MDA) accumulation and improved drought tolerance. Additionally, TaZAT8-5B promoted lateral root initiation under mannitol stress and enhanced both lateral and primary root growth under long-term drought stress. Moreover, TaZAT8-5B was induced by indole-3-acetic acid (IAA). Overexpressing TaZAT8-5B under drought stress significantly inhibited the expression of auxin signaling negative regulatory genes IAA12 and IAA14. Conversely, downstream genes (ARF7, LBD16, LBD18, and CDKA1) of IAA14 and IAA12 were upregulated in TaZAT8-5B overexpressing plants compared to wild-type (WT) plants. These findings suggest that TaZAT8-5B regulates root growth and development under drought stress via the Aux/IAA-ARF module in the auxin signaling pathway. In summary, this study elucidates the role of TaZAT8-5B in enhancing drought tolerance and its involvement in root growth and development through the auxin signaling pathway. These findings offer new insights into the functional analysis of homologous genes of TaZAT8-5B, particularly in Gramineae species.

Proteomic profiling reveals CEACAM6 function in driving gallbladder cancer aggressiveness through integrin receptor, PRKCD and AKT/ERK signaling

Gallbladder cancer (GBC) presents as an aggressive malignancy with poor patient outcome. Like other epithelial cancers, the mechanisms of GBC cancer progression remain vague and efforts in finding targeted therapies fall below expectations. This study combined proteomic analysis of formalin-fixed paraffin-embedded (FFPE) GBC samples, functional and molecular characterization of potential oncogenes and identification of potential therapeutic strategies for GBC. We identified Carcinoembryonic Antigen-related Cell Adhesion Molecule 6 (CEACAM6) as one of the significantly most upregulated proteins in GBC. CEACAM6 overexpression has been observed in other cancer entities but the molecular function remains unclear. Our functional analyses in vitro and in vivo mouse models revealed that CEACAM6 supported the initial steps of cancer progression and metastasis by decreasing cell adhesion and promoting migration and invasion of GBC cells. Conversely, CEACAM6 knockdown abolished GBC aggressiveness by increasing cell adhesion while reducing cell migration, cell proliferation, and colony formation. BirA-BioID followed by mass-spectrometry revealed Integrin Beta-1 (ITGB1) and Protein Kinase C Delta (PRKCD) as direct molecular and functional partners of CEACAM6 supporting GBC cell migration. ERK and AKT signaling and their downstream target genes were regulated by CEACAM6 and thus the treatment with AKT inhibitor capivasertib or ERK inhibitor ulixertinib mitigated the CEACAM6-induced migration. These findings demonstrate that CEACAM6 is crucially involved in gallbladder cancer progression by promoting migration and inhibiting cell adhesion through ERK and AKT signaling providing specific options for treatment of CEACAM6-positive cancers.

Quinic acid protects against the development of Huntington’s disease in Caenorhabditis elegans model

BackgroundQuinic acid (QA), a cyclitol and cyclohexanecarboxylic acid, is a natural product that is present and can be isolated from edible herbals like tea, coffee and several fruits and vegetables. It was previously reported that QA exerted antioxidant and neuroprotective activity against dementia. However, it was not tested for its neuroprotective potential against Huntington’s disease (HD). Since aging related disorders are greatly linked to oxidative stress conditions, we focused on testing the oxidative stress resistant activity and protective effect of QA against the development of HD by using the multicellular Caenorhabditis elegans (C. elegans) worm model.MethodsFirstly, QA was tested for its oxidative stress resistant properties. In survival assay, wild type and mutant skn-1 and daf-16 worms were exposed to oxidative stress conditions by using H2O2. Activation of SKN-1 pathway and expression of its downstream genes gcs-1 and gst-4 were also tested. Secondly, the effect of QA was evaluated on HD by testing its ability to decrease the formation of polyQ150 aggregates. Furthermore, its effect on the accumulation of polyglutamine (polyQ35 and polyQ40 aggregates) was tested.ResultsHere we report that QA could improve the survival of C. elegans after exposure to oxidative stress caused by H2O2 while also exerting antioxidant effects through the activation of SKN-1/Nrf2 pathway. Moreover, QA could be a potential candidate to protect against HD due to its effects on decreasing the formation of polyQ150, polyQ35 and polyQ40 aggregates.ConclusionsThis study highlights the importance of QA as a natural compound in defending against oxidative stress and the development of neurodegenerative diseases like HD.

The circRNA circSCAF8 promotes tumor growth and metastasis of gastric cancer via miR-1293/TIMP1signaling.

SR-like CTD-associated factor 8 (SCAF8) can regulate transcriptional termination, but the function of circSCAF8 remains unclear. In our study, we observed a significant increase in circSCAF8 expression in gastric cancer, particularly in tissues with lymph node metastasis. The Kaplan-Meier curve revealed that high circSCAF8 expression was associated with a low overall survival time in gastric cancer patients. Moreover, circSCAF8 shRNA effectively decreased gastric cancer proliferation, invasion, and migration in vitro. Additionally, using bioluminescence imaging (BLI) technology in vivo, we found that circSCAF8 shRNA viruses inhibited the growth of xenograft tumors and gastric cancer lung metastasis. RNA immunoprecipitation (RIP) and circRNA pulldown assays confirmed the direct binding of circSCAF8 to miR-1293, but circSCAF8 could not regulate the expression of miR-1293 in gastric cancer. Interestingly, circSCAF8 regulated the downstream gene tissue inhibitor of metalloproteinases 1 (TIMP1) of miR-1293, and this observation was further verified in gastric cancer tissues. Moreover, we confirmed that miR-1293 directly suppressed TIMP1 expression. Subsequent rescue experiments revealed that TIMP1 overexpression reversed the impact of circSCAF8 shRNA viruses on gastric cancer. In conclusion, circSCAF8 expression was elevated in gastric cancer, and circSCAF8 shRNA viruses inhibited gastric cancer growth and metastasis by upregulating TIMP1 expression via miR-1293.

Multifunctional extracellular vesicles and edaravone-loaded scaffolds for kidney tissue regeneration by activating GDNF/RET pathway

With the severity of chronic kidney disease worldwide, strategies to recover renal function via tissue regeneration provide alternatives to kidney replacement therapy. To exclude side effects from direct cell transplantation, extracellular vesicles (EVs) are great substitutes representing paracrine cell signaling. To build three-dimensional structures for implantation into the 5/6 nephrectomy model by incorporating bioactive materials, including multifunctional EVs (mEVs), porous PMEZE/mEV scaffolds were developed in combination with edaravone (EDV; E) and mEV based on PMEZ scaffolds with PLGA (P), MH-RA (M), ECM (E), ZnO-ALA (Z). The oxygen free radical scavenger EDV was incorporated to induce tubular regeneration. mEVs were engineered to serve regenerative activities with a combination of two EVs from SDF-1α overexpressed tonsil-derived mesenchymal stem cells (sEVs) and intermediate mesoderm (IM) cells during differentiation into kidney progenitor cells (dEVs). mEVs displayed beneficial effects on regeneration by facilitating migration and inducing differentiation of surrounding stem cells, and EDV improved kidney function by regulating the GDNF/RET pathway and their downstream genes. The promotion of MSC recruitment was confirmed with sEV particles number dependently, and the regulation of the GDNF/RET pathway by the effect of EDV and its enhanced effect by mEVs were elucidated using in vitro analysis. The regeneration of tubules was additionally demonstrated through the increased expression of aquaporin-1 (AQP-1) and cadherin-16 (CDH16) for proximal tubules, and calbindin and PAX2 for distal tubules in the renal defect model. With these, structural regeneration and functional recovery were achieved with kidney regeneration in the 5/6 nephrectomy mice model.Graphical abstract

Advances in the study of artemisinin and its derivatives for the treatment of rheumatic skeletal disorders, autoimmune inflammatory diseases, and autoimmune disorders: a comprehensive review.

Artemisinin and its derivatives are widely recognized as first-line treatments for malaria worldwide. Recent studies have demonstrated that artemisinin-based antimalarial drugs, such as artesunate, dihydroartemisinin, and artemether, not only possess excellent antimalarial properties but also exhibit antitumor, antifungal, and immunomodulatory effects. Researchers globally have synthesized artemisinin derivatives like SM735, SM905, and SM934, which offer advantages such as low toxicity, high bioavailability, and potential immunosuppressive properties. These compounds induce immunosuppression by inhibiting the activation of pathogenic T cells, suppressing B cell activation and antibody production, and enhancing the differentiation of regulatory T cells. This review summarized the mechanisms by which artemisinin and its analogs modulate excessive inflammation and immune responses in rheumatic and skeletal diseases, autoimmune inflammatory diseases, and autoimmune disorders, through pathways including TNF, Toll-like receptors, IL-6, RANKL, MAPK, PI3K/AKT/mTOR, JAK/STAT, and NRF2/GPX4. Notably, in the context of the NF-κB pathway, artemisinin not only inhibits NF-κB expression by disrupting upstream cascades and/or directly binding to NF-κB but also downregulates multiple downstream genes controlled by NF-κB, including inflammatory chemokines and their receptors. These downstream targets regulate various immune cell functions, apoptosis, proliferation, signal transduction, and antioxidant responses, ultimately intervening in systemic autoimmune diseases and autoimmune responses in organs such as the kidneys, nervous system, skin, liver, and biliary system by modulating immune dysregulation and inflammatory responses. Ongoing multicenter randomized clinical trials are investigating the effects of these compounds on rheumatic, inflammatory, and autoimmune diseases, with the aim of translating promising preclinical data into clinical applications.

The expression of integron arrays is shaped by the translation rate of cassettes

Integrons are key elements in the rise and spread of multidrug resistance in Gram-negative bacteria. These genetic platforms capture cassettes containing promoterless genes and stockpile them in arrays of variable length. In the current integron model, expression of cassettes is granted by the Pc promoter in the platform and is assumed to decrease as a function of its distance. Here we explored this model using a large collection of 136 antibiotic resistance cassettes and show the effect of distance is in fact negligible. Instead, cassettes have a strong impact in the expression of downstream genes because their translation rate affects the stability of the whole polycistronic mRNA molecule. Hence, cassettes with reduced translation rates decrease the expression and resistance phenotype of cassettes downstream. Our data puts forward an integron model in which expression is contingent on the translation of cassettes upstream, rather than on the distance to the Pc.

Non-electrophilic NRF2 activators promote wound healing in human keratinocytes and diabetic mice and demonstrate selective downstream gene targeting

The transcription factor NRF2 plays an important role in many biological processes and is a promising therapeutic target for many disease states. NRF2 is highly expressed in the skin and is known to play a critical role in diabetic wound healing, a serious disease process for which treatment options are limited. However, many existing NRF2 activators display off-target effects due to their electrophilic mechanism, underscoring the need for alternative approaches. In this work, we investigated two recently described non-electrophilic NRF2 activators, ADJ-310 and PRL-295, and demonstrated their efficacy in vitro and in vivo in human keratinocytes and Leprdb/db diabetic mice. We also compared the downstream targets of PRL-295 to those of the widely used electrophilic NRF2 activator CDDO-Me by RNA sequencing. Both ADJ-310 and PRL-295 maintained human keratinocyte cell viability at increasing concentrations and maintained or improved cell proliferation over time. Both compounds also increased cell migration, improving in vitro wound closure. ADJ-310 and PRL-295 enhanced the oxidative stress response in vitro, and RNA-sequencing data showed that PRL-295 activated NRF2 with a narrower transcriptomic effect than CDDO-Me. In vivo, both ADJ-310 and PRL-295 improved wound healing in Leprdb/db diabetic mice and upregulated known downstream NRF2 target genes in treated tissue. These results highlight the non-electrophilic compounds ADJ-310 and PRL-295 as effective, innovative tools for investigating the function of NRF2. These compounds directly address the need for alternative NRF2 activators and offer a new approach to studying the role of NRF2 in human disease and its potential as a therapeutic across multiple disease states.

Discovery of a novel methionine biosynthetic route via O-phospho-l-homoserine.

Methionine (Met), a sulfur-containing amino acid, is essential for the underlying biological processes in living organisms. In addition to its importance as a starting building block for peptide chain elongation in protein biosynthesis, Met is a direct precursor of S-adenosyl-l-methionine, an indispensable methyl donor molecule in primary and secondary metabolism. Streptomyces bacteria are well known to produce diverse secondary metabolites, but many strains lack canonical Met pathway genes for l-homocysteine, a direct precursor of Met in bacteria, plants, and archaea. Here, we report the identification of a novel gene (metM) responsible for the Met biosynthesis in Streptomyces strains and demonstrate the catalytic function of the gene product, MetM. We further identified the metO gene, a downstream gene of metM, and showed that it encodes a sulfur-carrier protein (SCP). In in vitro analysis, MetO was found to play an important role in a sulfur donor by forming a thiocarboxylated SCP. Together with MetO (thiocarboxylate), MetM directly converted O-phospho-l-homoserine to l-homocysteine. O-Phospho-l-homoserine is also known as an intermediate for threonine biosynthesis in bacteria and plants, and MetM shares sequence homology with threonine synthase. Our findings thus revealed that MetM seizes O-phospho-l-homoserine from the threonine biosynthetic pathway and uses it as an intermediate of the Met biosynthesis to generate the sulfur-containing amino acid. Importantly, this MetM/MetO pathway is highly conserved in Streptomyces bacteria and distributed in other bacteria and archaea.IMPORTANCEMethionine (Met) is a sulfur-containing proteinogenic amino acid. Moreover, Met is a direct precursor of S-adenosyl-l-methionine, an indispensable molecule for expanding the structural diversity of natural products. Because Met and its derivatives benefit humans, the knowledge of Met biosynthesis is important as a basis for improving their fermentation. Streptomyces bacteria are well known to produce diverse and valuable natural products, but many strains lack canonical Met pathway genes. Here, we identified a novel l-homocysteine synthase (MetM) in Streptomyces and demonstrated that it converts O-phospho-L-homoserine to l-homocysteine using a thiocarboxylated sulfur-carrier protein as a sulfur donor. Since the metM is distributed in other bacteria and archaea, our pioneering study contributes to understanding Met biosynthesis in these organisms.

Overexpression of the RAV Transcription Factor OsAAT1 Confers Enhanced Arsenic Tolerance by Modulating Auxin Hemostasis in Rice.

Characterization of arsenic (As)-responsive genes is fundamental to solving the issue of As contamination in rice. Herein, we establish the involvement of an RAV transcription factor OsAAT1 (Arsenic Accumulation and Tolerance 1) in regulating As response in rice. The expression of OsAAT1 is significantly higher in roots and stems of rice seedlings and is clearly upregulated by higher concentrations of arsenite [As(III)]. Compared with wild-type (WT) plants, OsAAT1-overexpressed transgenic lines (OE-OsAAT1) exhibit tolerance, while OsAAT1-knockout mutants (Osaat1) are sensitive to As(III) stress. Notably, the application of exogenous 1-naphthylacetic acid (NAA) greatly enhances the As tolerance of WT and transgenic lines, with stronger effects on OE-OsAAT1. The change in OsAAT1 expression leads to the alteration of As and auxin accumulation in transgenic plants by regulating the expression of OsLsi1, OsLsi2, OsCRL4, and OsAUX1 genes. Moreover, overexpression of OsAAT1 accelerates ROS scavenging and phytochelatins (PCs) synthesis, especially with the addition of exogenous NAA. OsAAT1 localizes in the nucleus and works as a transcriptional suppressor. OsGH3-12, belonging to the auxin-responsive GH3 gene family, is the downstream target gene of OsAAT1, whose expression is extensively downregulated by As(III). These findings provide new insights into As response via auxin signaling pathway in rice.

Stat stimulates histone H3K4 methylation via KDM5 inhibition in adult stem cells of budding tunicates.

The branchial epithelium is one of the main tissues in which histone H3K4 trimethylation (H3K4me3) occurs in the budding tunicate, Polyandrocarpa misakiensis. It contains proliferating and undifferentiated cell aggregates at the bottom of each pharyngeal cleft, providing the nest for the adult stem cell niche. We examined the sustainable mechanism enabling epigenetic histone methylation in adult stem cells. Histone H3K4 demethylase (PmisKdm5) was not co-expressed in vivo with the transcription factor, signal transduction and activator of transcription (PmisStat) in the same cells. PmisStat mRNA, when electroporated into zooids, suppressed the gene expression of PmisKdm5 and facilitated the trimethylation of H3K4. A STAT5 inhibitor blocked the nuclear localization of PmisStat. It stimulated PmisKdm5 gene expression irrespective of PmisStat mRNA. The KDM5 inhibitor, CPI-455, stimulated H3K4me3 similarly to PmisStat mRNA. PmisStat mRNA and CPI-455 both induced the gene expression of PmisAp2 and PmisSp8, which were recently identified as budding/regeneration-related genes. When zooid tissues were treated with both CPI-455 and the STAT5 inhibitor, CPI-455 overwhelmed the effects of the STAT inhibitor on PmisAp2 and PmisSp8. PmisStat is involved in epigenetic histone methylation at H3K4 through the inhibition of PmisKdm5. H3K4me3 affects downstream gene expression more directly and strongly than PmisStat.

Identification of Candidate Genes Associated with Flesh Firmness by Combining QTL Mapping and Transcriptome Profiling in Pyrus pyrifolia.

Flesh firmness is an important quality of pear fruits. Breeding cultivars with suitably low flesh firmness is one of the popular pear breeding goals. At present, SNP markers related to pear flesh firmness and genes affecting flesh firmness are still uncertain. In this study, a QTL analysis was performed, and the result showed that the position of 139.857 cM in lineage group 14 (LG14) had the highest average logarithm of odds (3.41) over two years. This newly discovered locus was identified as a flesh firmness-related QTL (qFirmness-LG14). The 'C/T' SNP was found in corresponding Marker1512129. The 'C' genotype is the high-firmness genotype, which is a dominant trait. The average firmness of fruits with genotype C is 21.4% higher than genotype without the C genotype. Transcriptome profiling was obtained between 'Zaoshengxinshui' and 'Qiushui' at five time points. Three candidate genes in the interval of qFirmness-LG14 might affect firmness. A gene of xyloglucan endotransglucosylase 1 (PpXTH1) was upregulated in 'Qiushui' at all five time points. Two transcription factors (PpHY5 and PpERF113) were upregulated in 'Zaoshengxinshui', which might be negative regulatory genes for high flesh firmness. The transcriptome results also isolated a large number of cell wall-related genes (e.g., Pectate lyase, Pectin acetylesterase, Pectin methylesterase, and 4-coumarate-CoA ligase) and transcription factors (e.g., ERF, WRKY). These genes are all potential upstream and downstream genes related to flesh firmness. In conclusion, this study provides valuable insights into the QTLs and molecular mechanisms associated with fruit firmness in Pyrus pyrifolia.

Analyzing super-enhancer temporal dynamics reveals potential critical enhancers and their gene regulatory networks underlying skeletal muscle development.

Super-enhancers (SEs) govern the expression of genes defining cell identity. However, the dynamic landscape of SEs and their critical constituent enhancers involved in skeletal muscle development remains unclear. In this study, using pig as a model, we employed CUT&Tag to profile the enhancer-associated histone modification marker H3K27ac in skeletal muscle across two prenatal and three postnatal stages and investigated how SEs influence skeletal muscle development. We identified three SE families with distinct temporal dynamics: continuous (Con, 397), transient (TS, 434), and de novo (DN, 756). These SE families are associated with different temporal gene expression trajectories, biological functions, and DNA methylation levels. Notably, several lines of evidence suggest a potential prominent role of Con SEs in regulating porcine muscle development and meat traits. To pinpoint key cis-regulatory units in Con SEs, we developed an integrative approach that leverages information from eRNA annotation, GWAS signals and high-throughput capture STARR-seq experiments. Within Con SEs, we identified 20 candidate critical enhancers with meat and carcass-associated DNA variations that affect enhancer activity and inferred their upstream TFs and downstream target genes. As a proof of concept, we experimentally validated the role of one such enhancer and its potential target gene during myogenesis. Our findings reveal the dynamic regulatory features of SEs in skeletal muscle development and provide a general integrative framework for identifying critical enhancers underlying the formation of complex traits.

METTL3 affects the biological function of lung adenocarcinoma through the FGF2/PI3K/AKT /mTOR pathway.

This study aims to investigate the role of the m6A regulatory factor METTL3 in LUAD. By examining the expression of METTL3 in LUAD and conducting cellular functional experiments, the biological functions of METTL3 were discussed. mRNA-seq and MeRIP-qPCR were used to identify downstream target genes and pathways. The expression level of METTL3 in LUAD is lower than that in the control group. The downregulation of METTL3 promoted the proliferation, migration, and invasion of LUAD cells, while overexpression of METTL3 results in the opposite effects. Furthermore, we found that FGF2 was negatively regulated by METTL3. Inhibiting FGF2 reversed the tumor-promoting effects caused by METTL3 downregulation in LUAD cells. Silencing METTL3 enhanced the stability of FGF2 mRNA. Silencing FGF2 resulted in reduced activity of the PI3K/AKT/mTOR signaling pathway in METTL3 knockdown LUAD cells. In summary, our findings unveil an intricate signaling network involving METTL3/FGF2/PI3K/AKT/mTOR in LUAD and provide valuable insights into the molecular mechanisms underlying tumor progression, thus holding significant implications for targeted therapy and advancing LUAD research.

Inhibiting p38α and β MAPK affects testis development in the marsupial tammar wallaby.

The MAPK genes are critical for gonadal differentiation in eutherian mammals but their role in marsupial mammals is unknown. Characterisation and phylogenetic analyses of the tammar wallaby MAPK genes shows these genes are highly conserved with their orthologues in mammalian and non-mammalian species. We cultured sexually indifferent tammar gonads in the presence of p38α and β MAPK inhibitor, SB202190. SB202190 downregulated SOX9 and AMH levels in XY treated gonads when compared to controls, similar to the effects of oestrogen on the MAPK pathway in males. In contrast, XX gonads treated with the SB202190 inhibitor showed no change in mRNA expression between the control and treated gonads for any of the markers tested. This study confirms that components of the MAPK pathway drive testis differentiation via the key downstream genes SOX9 and AMH in marsupials as is observed in eutherian mammals.