Search Results (413)

In order to explore the main regulatory genes and related pathways of growth traits, transcriptome sequencing was performed on the hypothalamus, pituitary, and liver tissues of 12-month-old greater amberjack (Seriola dumerili) with different growth rates. In total, 504 (118 up- and 386 down-regulated), 556 (283 up- and 273 down-regulated), and 699 (224 up- and 475 down-regulated) differentially expressed genes (DEGs) were identified in the hypothalamus, pituitary, and liver tissues, respectively. GO and KEGG pathway analyses revealed significant differences in the expression of several genes involved in growth, metabolism, and immune-related pathways. The mRNA expression levels of genes related to growth (ghrh, ghra, igf1), cell proliferation (fgf19, fgfr4, mapk8b, map2k4b, and map4k3), and lipid metabolism (acsl5, dgat2, lipeb, cyp7a1, and fabp10a) were up-regulated in the fast-growing (FG) group, while the cartl and sst1.1 were down-regulated. Conversely, genes associated with glycolysis (fbp1a, pklr, pgm2), citrate cycle (aclya, idh1), and immune-related pathways (irf1b, cxcl10, tnfb, lysg, ifi44, mapk11, and mapk12b) were up-regulated in the slow-growing (SG) group. These findings indicate that the FG exhibited greater lipid metabolism capacity and cell proliferation ability, while the SG expended additional energy to cope with environmental stress, with hindered growth during immune response. This study enhances our understanding of the genetic mechanisms underlying differences in growth rates and provides essential gene resources for future growth-related molecular breeding programs in greater amberjack. Full article

CRISPR-Cas9 technology has opened new perspectives in genome editing of clonally, asexually propagated and polyploid plants by enabling multiple allelic gene edits. Traditional Agrobacterium- and particle bombardment-mediated transformations, which rely on integration of gene-editing transgene cassettes, have been efficiently applied to several plants; however, concerns about the acceptability of resultant edited transgenic genotypes make these methods less attractive for vegetatively propagated crops. We leveraged and optimized the CRISPR-Cas9/sgRNA-RNPs system for delivery into protoplasts of the hexaploid sweetpotato cultivar PI-318846, targeting eukaryotic translation initiation factor isoform 4E genes to enhance resistance to SPFMV potyviruses. To evaluate the efficiency of pre-assembled Cas9/sgRNA-RNP in sweetpotato transfection, single guide RNAs were designed to target putative host susceptibility genes: IbeIF4E, IbeIF(iso)4E, and IbCBP. Freshly isolated leaf protoplasts were subjected to CRISPR-CAS9-RNP PEG-mediated transfection under different parameters. Sweetpotato regenerants screened using PCR-RE-T7 assay, sequencing, and Inference CRISPR Edit analyses of target-site amplicons revealed the most efficient editing conditions utilizing 25% PEG with a 3:1 (15 µg:45 µg) ratio of Cas9/sgRNA-RNP for 25 min and 48 h incubation period. Different allelic InDels were obtained with editing efficiencies of 10–20% in regenerated plantlets, demonstrating that PEG-mediated CRISPR-RNP transfection system is key for advancing DNA-free editing tools in polyploid and vegetatively propagated crops. Full article

Potyvirus genomes are expressed as a single large open reading frame, which is translated into a polyprotein that is post-translationally cleaved by three virus-encoded proteases into 10 functional proteins. Several of these potyviral proteins, including nuclear inclusion protein b (NIb), are multifunctional. Here, using the classic GFP silencing in Nicotiana benthamiana gfp-transgenic plants, we show that potato virus Y (PVY) NIb, in addition to its canonical role as the viral RNA-dependent RNA polymerase (RdRP), functions as a suppressor of RNA silencing. Mutational analyses reveal a previously unreported NIb nuclear localization signal (NLS) consisting of a triple-lysine motif. NIb suppression of RNA silencing activity was lost when the NLS was mutated, suggesting that nuclear localization is required for NIb suppression of RNA silencing activity. Analysis of sequenced GFP siRNAs revealed three reproducible hotspot regions at ≈175 nt, ≈320–330 nt, and a broader 3′-proximal region spanning ≈560–700 nt that contains multiple local maxima. These data show differences in the positional distribution of siRNAs between samples expressing NIb and those expressing NIb^Del3×2, the NIb null mutant that does not suppress RNA silencing. However, the positional distribution of GFP-derived small RNAs across the transgene differed modestly between NIb and NIb^Del3×2, while both treatments showed the same three reproducible hotspot regions. Furthermore, NIb was found to interact with four key RNA silencing pathway proteins—AGO4, HSP70, HSP90, and SGS3. Except for HSP90, each of these proteins showed degradation products that were absent in NIb mutants that did not suppress RNA silencing. These findings support a role for NIb in countering host defense during virus infection. Full article

Since the COVID-19 pandemic, wastewater monitoring has become an additional tool in the surveillance of infectious diseases. Many EU countries put wastewater surveillance systems (WSS) in place to track SARS-CoV-2 and its variants and other pathogens, such as the influenza virus or Respiratory syncytial virus (RSV). In Germany, several research and pilot projects funded by the EU, the Federal Ministry of Education and Research, the Federal Ministry of Health, and projects at Federal State level have been launched in the last four years. In Germany, wastewater monitoring was not implemented as a public health tool before the COVID-19 pandemic, but in September 2022, it has been legally determined in the German infection protection act (Infektionsschutzgesetz, IfSG). As Germany is a federal state, competencies in epidemic management partly belong to the 16 federal states (“Länder”). In the federal states, the local health authorities at the county (“Kreise”) level also have specific risk management and communication competencies. Furthermore, WSS has been incorporated into the revised Urban Wastewater Treatment Directive (EU) 2024/3019. For this reason, the federal states and local health authorities play a pivotal role in successfully implementing wastewater monitoring as a supplementary component of disease surveillance in Germany. Between November 2021 and August 2022, the federal state of Thuringia, Germany, supported a pilot study to implement a surveillance system for SARS-CoV-2-RNA in wastewater of 23 wastewater treatment plants in 17 counties in Thuringia. Here, we describe the study design and the system behind the logistics and the planning, and we provide an overview of the options for involving the public health service. Furthermore, the possibilities for IT concepts and approaches to innovative AI solutions are shown. We also aim to explore the feasibility and potential barriers to further implementing wastewater surveillance as a supplementary public health tool in Thuringia. Full article

GmSRC7 is a broad-spectrum antiviral R gene from soybean, but its downstream and functionally related genes remain unclear. Virus-induced gene silencing (VIGS) assays in Nicotiana benthamiana (Nb) showed that suppression of several gene families—WRKY transcription factors, chaperones, ethylene pathway components, MAPK cascade elements, salicylic acid (SA) signaling genes, calcium-dependent protein kinases, nuclear migration proteins, RNA replication-related genes, and immune regulators—consistently weakened GmSRC7-mediated resistance to Soybean Mosaic Virus (SMV) and Tobacco Mosaic Virus (TMV). Targeted silencing of four regulatory genes—NbEDS1, NbARF1, NbSGT1, and NbCOI1—markedly enhanced GmSRC7-mediated resistance to SMV and TMV in our experiments. Silencing the serine/threonine kinase gene NbPBS1 increased GmSRC7-conferred resistance to SMV but did not significantly alter its resistance to TMV. Transient expression assays showed that NbARF1, NbSGT1, and NbCOI1 antagonize GmSRC7-mediated defense against SMV and TMV, whereas NbPBS1 specifically suppresses anti-SMV activity without affecting TMV resistance. Transient overexpression of SA-degrading enzymes (AtS3H, AtS5H, and NahG) significantly reduced GmSRC7-conferred resistance to SMV, indicating that SA is essential for this R protein-mediated defense. Genes were also grouped by immune pathways and function: co-expression of chaperone family genes inhibited GmSRC7 activity against SMV and TMV, while co-expression of WRKY family genes enhanced anti-SMV activity of GmSRC7. Finally, transient silencing of soybean genes GmEDS1, GmSGT1-1, GmSGT1-2, GmJAR1, and GmSGS3 compromised GmSRC7-mediated resistance to SMV. Full article

Cells are continuously exposed to physiological and environmental stressors that disrupt homeostasis, triggering adaptive mechanisms such as the integrated stress response (ISR). A central feature of ISR is the selective translation of activating transcription factor 4 (ATF4), which orchestrates gene programs essential for metabolic adaptation and survival. Stress-induced acute ATF4 expression occurs in diverse mammalian cell types and is typically protective; however, chronic activation contributes to pathologies including cancer and neurodegeneration. Canonical ISR (c-ISR) is initiated by phosphorylation of eIF2α in response to stressors such as endoplasmic reticulum or mitochondrial dysfunction, hypoxia, nutrient deprivation, and infections. This modification suppresses global protein synthesis while promoting ATF4 translation through upstream open reading frames (uORFs) in its 5′UTR. Recently, an alternative pathway, split ISR (s-ISR), enabling ATF4 translation independently of eIF2α phosphorylation, was identified in mice, suggesting ISR adaptability, though its relevance in humans remains unclear. Under normal conditions, cap-dependent translation predominates, mediated by the eIF4F complex and requiring the activity of eIF2B at its initial steps. During translational stress, eIF2α phosphorylation inhibits eIF2B activity, resulting in the formation of stalled initiation complexes, which can aggregate into stress granules (SGs). SGs sequester mRNAs and translation initiation factors, further repressing global translation, while ATF4 mRNA largely escapes sequestration, enabling selective translation. This partitioning highlights a finely tuned regulatory mechanism balancing ATF4 expression during stress. Recent advances reveal that, beyond cis-regulatory uORFs, trans-acting factors such as translation initiation factors and associated RNA-binding proteins critically influence ATF4 translation. Understanding these mechanisms provides insight into ISR plasticity and its implications for development, aging, and disease. Full article

Stress granules (SGs) are dynamic, membraneless organelles that form in response to stress and play pivotal roles in translational control, RNA metabolism, and cell survival. In cancer, SGs are increasingly recognized as central mediators of therapy resistance, enabling malignant cells to evade apoptosis, reprogram metabolism, and modulate immune responses. Understanding the mechanistic and clinical insights into SG kinetics in healthy versus cancer cells holds significant potential for targeting them in precision oncology. This review integrates current knowledge on how chemotherapeutic agents, oncogenic signaling pathways, and tumor microenvironmental stressors promote SG formation, as well as evidence of altered SG kinetics across tumor types. We further highlight how the upregulation of SG components within the tumor microenvironment shapes cancer cell behavior and adaptability, and how crosstalk between SGs and other biomolecular condensates could contribute to resistance. Finally, we discuss emerging therapeutic strategies targeting SGs, including kinase inhibitors and modulators of SG dynamics, and propose that SGs represent tractable vulnerabilities in precision oncology. By bridging mechanistic insights with clinical implications, this review positions SGs as a promising frontier in overcoming cancer therapy resistance. Full article

Portunus trituberculatus is an economically important marine crustacean in East Asia’s aquaculture industry. Nevertheless, precise genome modification has not yet been established. In this study, we evaluated the applicability of the CRISPR-Cas9 gene editing system in P. trituberculatus using electroporation for efficient delivery of the Cas9-sgRNA complex into zygotes. We systematically investigated electroporation parameters, including buffer composition, voltage, capacitance, and pulse times. Our results showed that artificial seawater was a superior buffer to phosphate-buffered saline (PBS) and identified an effective electroporation condition of 600 V, 1 μF capacitance, and two pulses, resulting in approximately 72.7% fluorescent zygotes. Under these electroporated conditions, we detected gene indels and putative insertion events at the targeted locus of myostatin (mstn) gene. These results demonstrate the feasibility of Cas9-based genome editing in P. trituberculatus and provide a proof-of-concept for functional genomics studies and future genetic improvement of this species. Full article

The presence of aberrant double-stranded DNA (dsDNA) in the cytoplasm of cells is sensed by unique pattern recognition receptors (PRRs) to trigger innate immune response. The cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) signaling pathway is activated by the presence of non-self or mislocalized self-dsDNA from nucleus or mitochondria released in response to DNA damage or cellular stress in the cytoplasm. Activation of cGAS leads to the synthesis of the second messenger cyclic GMP–AMP (cGAMP), which binds and activates STING, triggering downstream signaling cascades that result in the production of type I interferons (IFNs) and proinflammatory cytokines. Here, we show that diverse immunostimulatory dsDNA ligands and chemotherapy agents like Doxorubicin and Taxol trigger the integrated stress response (ISR) by activating endoplasmic reticulum (ER) stress kinase, protein kinase RNA-like ER kinase (PERK), in addition to the canonical IFN pathways. PERK-mediated phosphorylation and inactivation of the alpha subunit of eukaryotic translation initiation factor-2 (eIF2α) result in the formation of stress granules (SGs). SG formation by dsDNA was significantly reduced in PERK knockout cells or by inhibiting PERK activity. Transcriptional induction of IFNβ and cytokines, ISR signaling, and SG formation by dsDNA was dampened in cells lacking PERK activity, STING, or key stress-granule nucleating protein, Ras-GAP SH3 domain-binding protein 1 (G3BP1), demonstrating an important role of the signal transduction pathway mediated by STING and SG assembly. Lastly, STING regulates reactive oxygen species (ROS) production in response to DNA damage, highlighting the crosstalk between DNA sensing and oxidative stress pathways. Together, our data identify STING–PERK–G3BP1 signaling axis that couples cytosolic DNA sensing to stress response pathways in maintaining cellular homeostasis. Full article

Background: Diabetic nephropathy (DN) is largely driven by transforming growth factor-β1 (TGF-β1)-mediated fibrosis. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) ribonucleoprotein (RNP) complexes offer precise gene disruption, yet effective non-viral delivery remains a challenge. This study developed cationic lipid-based hybrid nanostructured lipid carriers (NLCs) for intracellular delivery of TGFB1-targeting RNP as an early-stage platform for DN gene modulation. Methods: A single-guide RNA (sgRNA) targeting human TGFB1 was assembled with Cas9 protein (1:1 and 1:2 molar ratios). Hybrid NLCs comprising squalene, glyceryl trimyristate, and the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were formulated via optimized emulsification–sonication to achieve sub-100 nm particles. Physicochemical properties, including polydispersity index (PDI), were assessed via dynamic light scattering (DLS), while silencing efficacy in HEK293T cells was quantified using quantitative reverse transcription PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). Results: Optimized NLCs achieved hydrodynamic diameters of 65–99 nm (PDI < 0.5) with successful RNP complexation. The 1:2 Cas9:sgRNA formulation produced the strongest gene-editing response, reducing TGFB1 mRNA by 67% (p < 0.01) compared with 39% for the 1:1 ratio. This translated to a significant reduction in TGF-β1 protein (p < 0.05) within 24 h. Conclusions: DOTAP-based hybrid NLCs enable efficient delivery of CRISPR–Cas9 RNP and achieve significant suppression of TGFB1 expression at both transcriptional and protein levels. These findings establish a promising non-viral platform for upstream modulation of profibrotic signaling in DN and support further evaluation in kidney-derived cells and in vivo renal models. Full article

Type-2 Diabetes Mellitus (T2DM) is related to cardiac arrhythmias. The stellate ganglion (SG), part of the sympathetic nervous system, regulates heart function. Within the SG, satellite glial cells (SGCs) have gap junction channels (Cx43). Increased Cx43 permeability induces SGC depolarization and activates the PKC-MAPK14-ADAM17 signaling pathway, releasing some endogenous factors that stimulate nearby cardiac postganglionic sympathetic neurons (CPSN). This study investigated the activation of the PKC-MAPK14-ADAM17 signaling pathway in T2DM SGs and SGCs as a novel mechanism of sympathetic overactivation. A total of 56 Sprague-Dawley rats were randomly assigned to sham and T2DM groups, and T2DM was induced using a high-fat diet combined with low-dose streptozotocin. Real-time RT-PCR, Western blot, and ELISA quantified mRNA/protein expression and enzymatic activity. The patch clamp technique assessed neuronal voltage-gated Ca²⁺ currents and action potentials, while electrophysiological recording measured cardiac sympathetic nerve activity (CSNA). T2DM rats exhibited marked upregulation of MAPK14, PKC-α, and ADAM17 mRNA/protein in the SG, alongside elevated enzymatic activities of PKC and ADAM17. T2DM also increased Ca²⁺ currents and neuronal excitability in the CPSN and induced the elevation of the CSNA. Upregulated PKC-MAPK-ADAM17 signaling in the SG might contribute to cardiac sympathetic overactivation in T2DM rats by enhancing the cell excitability of the CPSN. Full article

Ganoderma sichuanense is a widely used medicinal and edible fungus. Genomic studies have revealed substantial genetic variation among its different strains, indicating that a genetic transformation system optimized for one genotype may not be effective in others. However, no study has systematically evaluated the efficiency of a genetic transformation system across diverse genotypes, which has potentially limited functional genetic studies in this species. In this study, we first evaluated eight wild and cultivated monokaryotic strains with different genotypes based on their hygromycin B resistance and green fluorescent protein (GFP) expression efficiency. Three strains (CCMJ1500101, CCMJ1509001, and CCMJ1507802) were identified as capable of stable foreign gene expression, achieving transformation efficiencies of 20.0–66.7% via PEG-mediated protoplast transformation. Subsequently, a CRISPR/Cas9 system incorporating seven key elements to enhance editing efficiency was constructed and applied to these three strains using the ura3 gene as a test target. Gene editing efficiencies varied significantly among genotypes, ranging from 14.3% to 75.0%, confirming the system’s high efficacy and genotype dependence. Importantly, to rigorously assess the robustness and versatility of the established transformation platform, we further validated its broad applicability in the best-performing strain, CCMJ1500101, by successfully editing five functional genes involved in growth, development, and metabolism. Notably, gene inversion events were detected for the first time in edited transformants of Ganoderma, providing new clues for understanding non-homologous end joining (NHEJ) repair in this species. This study establishes a robust dual-sgRNA CRISPR/Cas9 platform for G. sichuanense and provides valuable strain resources to facilitate future gene functional studies and genetic improvement. Full article

Background/Objectives: The Notch–ADAM17 pathway is a fundamental signaling mechanism where ADAM17, a disintegrin and metalloprotease, cleaves the Notch receptor after the Notch receptor binds to a ligand. Crosstalk between Notch and ADAM17 is often altered in pathological situations. Alterations in Notch2 expression, in particular, appears to be correlated with the onset of various autoimmune diseases. In primary Sjögren’s disease (pSjD), an autoimmune disorder characterized by chronic inflammation, the role of ADAM17 has been extensively explored, but a correlation with Notch2 has not yet been evaluated. Methods: To analyze the gene and protein expression of Notch2 in pSjD and a possible correlation with ADAM17 expression and with the patient’s inflammatory grade, we employed an integrated co-detection protocol to analyze salivary gland tissue sections by combining in situ hybridization (ISH) with immunohistochemistry (IHC). Results: combined ISH/IHC allows us to demonstrate an increased expression of Notch2 mRNA and protein in pSjD salivary glands (SGs) biopsies, which appears correlated with an increased expression of ADAM17, both in acinar and duct cells and in infiltrating lymphocytes. Notch2/ADAM17 expression is higher in biopsies of pSjD SGs characterized by a high degree of inflammation. Conclusions: this work demonstrates the correlated expression in pSjD SGs of ADAM17, which plays multiple roles in the pathogenesis of SjD, and Notch2, widely considered a key player in various inflammatory mechanisms, offering a starting point for future therapeutic interventions to investigate. Full article

GC-rich sequences affect DNA replication, recombination and repair, as well as RNA transcription in vivo. Such sequences may also impede site-directed mutagenesis in vitro. P3a site-directed mutagenesis is a highly efficient method, but it has not been tested with plasmids possessing GC-rich sequences. Here we report that it is very efficient with a BRPF3 expression vector but unsuccessful with that for KAT2B. Because two GC-rich regions located within the synthetic CAG promoter and the KAT2B coding region may form guanine (G)-quadruplexes and hinder plasmid denaturation during PCR, we developed P3b site-specific mutagenesis, achieving an average efficiency of 97.5% in engineering ten KAT2B mutants. Importantly, deletion mutagenesis revealed that either of the two GC-rich regions is sufficient for rendering the plasmid incompatible with P3a mutagenesis. Consistent with this, only P3b mutagenesis worked efficiently with several widely used sgRNA/Cas9 expression vectors, which contain the CAG promoter, and with an expression vector for CDK13, which possesses an intrinsically disordered domain encoded by a GC-rich DNA fragment. Thus, this study highlights serious challenges posed by GC-rich sequences to site-directed mutagenesis and provides an effective remedy to address such challenges. The findings support that G-quadruplex formation is one mechanism whereby such sequences impede regular PCR-based mutagenesis methods. Full article

Cytosine base editors (CBEs) enable precise C-to-T (G-to-A) conversions in genomic DNA, offering significant potential for specific gene editing. This study compared the prototypical Base Editor 3 (BE3) and a modified variant, BE3-Y130F, which utilizes an hA3A deaminase with the Y130F mutation, focusing on their editing efficiency and editing window characteristics using bovine zygotes. Following in vitro fertilization (IVF), sgRNA and Cas9 mRNA were injected as a targeting efficiency control, which resulted in 100% editing with no wild-type sequence. Then, either BE3 or BE3-Y130F mRNA, synthesized via in vitro transcription, and an sgRNA targeting exon 4 of the MYO7A gene was injected into zygotes. Genomic DNA was extracted from both blastocysts and developmentally arrested embryos, and Sanger sequencing was performed to evaluate C-to-T conversion efficiency and editing window. Both BE3 and BE3-Y130F achieved 100% C-to-T conversion efficiency at the primary target cytosine. BE3 displayed a defined editing window, primarily affecting cytosines at positions 7 and 8, indicating a predictable profile. In contrast, BE3-Y130F maintained high efficiency but had a less clearly defined editing window, resulting in incomplete editing and a remaining cytosine on the target sequence. Full article