Transcriptional Analysis Research Articles

Molecular characterization of mixed-histology endometrial carcinoma provides prognostic and therapeutic value over morphologic findings

We performed molecular analysis of a single-institution cohort of clinically diagnosed mixed-histology endometrial carcinoma (MEC). A gynecologic pathologist confirmed that 72 cases met diagnostic criteria for MEC based on WHO 2020 guidelines, and these were molecularly classified using both a DNA-based and histologic approach. Tumors were classified as: POLE-mutated (13.9%), microsatellite instability (MSI)-high/mismatch repair deficient (MMRd) (26.4%), TP53/p53 abnormal (p53abnl) (48.6%), no specific molecular profile (NSMP) (11.1%). Recurrence risk significantly differed based upon molecular class, but not histology. 44% of MEC cases had a HER2 IHC score of 2–3+, and this was not limited to p53abnl tumors. Transcriptional analysis demonstrated 93 differentially expressed genes between p53abnl and NSMP tumors, including many associated with the innate immune response and DNA damage repair. While p53abnl and NSMP tumors have similarly poor outcomes, transcriptome analysis revealed biologic differences that could impact targeted therapeutics in this high-risk group.

Early metabolic changes in the brain of Alzheimer's disease rats are driven by GLAST+ cells.

Glucose metabolic dysfunction is a hallmark of Alzheimer's disease (AD) pathology and is used to diagnose the disease or predict imminent cognitive decline. The main method to measure brain metabolism in vivo is positron emission tomography with 2-Deoxy-2-[18F]fluoroglucose ([18F]FDG-PET). The cellular origin of changes in the [18F]FDG-PET signal in AD is controversial. We addressed this by combining [18F]FDG-PET with subsequent cell-sorting and γ-counting of [18F]FDG-accumulation in sorted cell populations. 7-month-old male TgF344-AD rats and wild-type controls (n = 24/group) received sham or ceftriaxone (200 mg/kg) injection prior to [18F]FDG-PET imaging to increase glutamate uptake and glucose utilisation. The same animals were injected again one week later, and radiolabelled brains were dissected, with hippocampi taken for magnetically-activated cell sorting of radioligand-treated tissues (MACS-RTT). Radioactivity in sorted cell populations was measured to quantify cell-specific [18F]FDG uptake. Transcriptional analyses of metabolic enzymes/transporters were also performed. Hypometabolism in the frontal association cortex of TgF344-AD rats was identified using [18F]FDG-PET, whereas hypermetabolism was identified in the hippocampus using MACS-RTT. Hypermetabolism was primarily driven by GLAST+ cells. This was supported by transcriptional analyses which showed alteration to metabolic apparatus, including upregulation of hexokinase 2 and altered expression of glucose/lactate transporters. See Figure 1 for summary.

The lived experience of migrant Syrian mothers’ interaction with the neonatal screening program

BackgroundThe aim of this study was to evaluate the knowledge, attitudes, and practices of migrant Syrian mothers regarding the neonatal screening programs (NSP) using in-depth interviews.MethodsWomen were selected from the Migrant Health Center (MHC), which is the primary health care institution in Istanbul. A translator who spoke Arabic, the native language of participants, used an open-ended, semi-structed interview form to guide face-to-face interviews with participants. Questions were about sociodemographic information, opinions about NSP’s heel prick testing, diseases the test screens for, reasons for agreeing to the heel prick test, and challenges faced accessing health care services. The interviews were audio recorded. The translator then transcribed the audio recordings in Arabic and translated them into Turkish. The framework method for analysis of qualitative data was used to analyze transcripts of the interviews.ResultsForty-one migrant women were interviewed. Four themes were identified from analysis of transcripts of the interviews, regarding (1) knowledge of the NSP, (2) attitudes about the NSP, (3) practices that create barriers to accessing health care services, (4) practices to overcome barriers to accessing health care services. Most participants had no knowledge of the diseases the NSP tests for. A few participants had heard of congenital hypothyroidism (CH), congenital adrenal hyperplasia (CAH), and spinal muscular atrophy (SMA), but they were not aware of the symptoms and effects of these diseases. Some participants thought the heel prick test benefited their child’s health as it enables early diagnosis and treatment of diseases. All participants agree to heal prick testing after the birth of their most recent child. Participants noted the information and recommendations health care professionals provided influenced their decision to agree to the heel prick test. Most participants experienced language barriers when accessing health care services.ConclusionsMigrant women expressed a positive attitude toward the NSP. Future health care interventions regarding migrants should aim to reduce the negative experiences they encounter accessing health care, such as language barriers.

Long and Accurate: How HiFi Sequencing is Transforming Genomics.

Recent developments in PacBio high-fidelity (HiFi) sequencing technologies have transformed genomic research, with circular consensus sequencing now achieving 99.9% accuracy for long (up to 25 kb) single-molecule reads. This method circumvents biases intrinsic to amplification-based approaches, enabling thorough analysis of complex genomic regions [including tandem repeats, segmental duplications, ribosomal DNA (rDNA) arrays, and centromeres] as well as direct detection of base modifications, furnishing both sequence and epigenetic data concurrently. This has streamlined a number of tasks including genome assembly, variant detection, and full-length transcript analysis. This review provides a comprehensive overview of the applications and challenges of HiFi sequencing across various fields, including genomics, transcriptomics, and epigenetics. By delineating the evolving landscape of HiFi sequencing in multi-omics research, we highlight its potential to deepen our understanding of genetic mechanisms and to advance precision medicine.

RE teachers’ ways of balancing children’s existential concerns and the curriculum: mirrored through established RE approaches

Abstract The relationship between learner and curriculum is foundational in education. In this study the purpose is to investigate how Swedish middle-school RE teachers balance children’s existential concerns and the curriculum content in their teaching, as well as how they describe current curricular goals and pupils’ questions, and to explore how these ways of balancing positions can be understood in light of how well-known RE approaches balance the child and the curriculum. In the analysis of the interview transcripts of the eleven middle-school teachers focussing on what the teachers express as important goals for RE, the findings were placed on a continuum between a focus on the child’s experiences and on the curriculum. The result also shows that the teachers recurrently describe pupils’ concerns and questions as relatively absent. The responses vary between “questions are lacking” and “questions are present”, with a middle position of “some interactionally created questions are present”. The child and the curriculum can never in practice be separated since they are integrated in the learning individual: in education, a particular individual is learning something specific. However, revisiting these approaches in combination with concrete examples of teachers’ experiences has brought our understanding further, and has also drawn attention to the need for awareness concerning the varying character of this relationship, as a tension, one between whose poles the teaching constantly moves or as one that falls apart in two different poles that do not necessarily need to be seen in the light of each other.

Case report: Deciphering the clinical significance of a novel partial BRCA1 exon 10 duplication in a patient with triple-negative breast cancer

Pathogenic/likely pathogenic germline variants in the BRCA1 and BRCA2 genes are associated with an increased risk of developing cancer, particularly breast and/or ovarian tumors. The identification and correct classification of these variants is crucial to find individuals with an increased risk of cancer and to support physicians in their clinical and therapeutic decisions. In addition, the status of BRCA1 and BRCA2 variants is important for appropriate management of patients’ family members. Here, we describe the case of a woman who developed triple-negative breast cancer at the age of 49 years. NGS analysis of BRCA1 and BRCA2 genes revealed the presence of a new partial BRCA1 exon 10 duplication of 2.012 bp. The identified duplication comprises 395 nucleotides from the final portion of intron 9 and 1617 nucleotides from the beginning of exon 10. Using specific primers, we were able to identify the breakpoint at the DNA level and characterize the alteration as a tandem duplication leading to the formation of a premature stop codon after 10 residues. RNA analysis allowed to confirm the production of an altered mRNA showing the duplicated sequence. In this way, we were able to assign a clinical significance to the new alteration and classify it as a pathogenic variant. Although new ClinGen ENIGMA guidelines have been produced to provide tools for the accurate interpretation of variants in the BRCA1 and BRCA2 genes, defining the clinical significance of copy number variants, particularly duplications, remains a challenging goal that requires complex approaches to accurately determine the role of such variants. Other investigations, such as the detection of breakpoints by RNA analysis, are often essential to classify the identified alteration. Our study suggests that RNA transcript analysis is an ideal methodology to support the accurate classification of variants and clarify their effects.

Deciphering the Interplay of the PD-L1/MALT1/miR-200a Axis During Lung Cancer Development.

Lung cancer remains a leading cause of cancer-related mortality worldwide. Our study investigates the involvement of the PD-L1/MALAT1/miR-200a-3p axis in lung tumor progression using a murine model of lung carcinogenesis. Lung tumors were induced in rats, which were divided into groups and sacrificed at different stages of tumor development. A histopathological examination was performed to assess tumor progression. Immunohistochemistry was applied to evaluate the expression of Ki-67 and programmed death-ligand 1 (PD-L1). The level of carcinoembryonic antigen (CEA) and expression analysis of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), miR-200a-3p, and zinc finger E-box binding homeobox 1 (ZEB1) were evaluated for each stage of induction. Immunohistochemical analysis demonstrated a progressive upregulation of the proliferative marker Ki-67 and the immune checkpoint protein PD-L1 during the induction process, indicative of enhanced tumor proliferation and immune evasion. Additionally, CEA levels revealed a progressive increase across induction stages, with a significant increase in advanced tumor stages, highlighting its clinical relevance as a biomarker for lung cancer progression. Expression analysis revealed dynamic upregulation of MALAT1 and downregulation of miR-200a during lung tumor induction, which correlated with advanced tumor stages and elevated PD-L1 expression, suggesting that the negative correlation between MALAT1 and miR-200a is involved in the development of lung tumors. ZEB1 expression exhibited a notable increase in the advanced stages of induction, consistent with its association with aggressive lung cancer. Our findings underscore the interplay between molecular pathways involved in lung tumor development and the potential diagnostic and therapeutic implications of the PD-L1/MALAT1/miR-200a-3p axis.

Temáticas en los pódcast españoles conducidos por mujeres: de la política y la familia al trabajo y las emociones

The success of podcasts created by women is evident in the Spanish offer, as evidenced by the awards for the programs Deforme Semanal Ideal Total and Estirando el chicle. Despite this, the literature on this phenomenon is scarce, so it is necessary to study the narrative of these products, which are increasingly consumed by the audience. This proposal quantitatively analyzes a selection of the main Spanish conversational native podcasts hosted by women, in order to distinguish the topics covered. A large-scale content analysis of YouTube transcripts of the programs Deforme Semanal, Dulces y Saladas, Estirando el chicle, Keep It Cutre, Nos Tienen Contentas and Saldremos Mejores was carried out. The sample is comprised of 143,319 lines of text, subjected to a word frequency analysis to explore the most common subjects. The results reveal the predominance of a set of categories: politics, family and relationships, work, emotions and feelings, feminism, sexuality and sexual orientation, leisure and culture. This diversity of topics shows that this is not a homogeneous phenomenon, but rather that it has a diverse content that moves away from the label of a niche product.

Zinc-limited Mycobacterium tuberculosis stimulate distinct responses in macrophages compared with standard zinc-replete bacteria.

Tuberculosis (TB) is notoriously difficult to treat, likely due to the complex host-pathogen interactions driven by pathogen heterogeneity. An understudied area of TB pathogenesis is host responses to Mycobacterium tuberculosis bacteria (Mtb) that are limited in zinc ions. This distinct population resides in necrotic granulomas and sputum and could be the key player in tuberculosis pathogenicity. In this study, we tested the hypothesis that macrophages differentiate between Mtb grown under zinc limitation or in the standard zinc-replete medium. Using several macrophage infection models, such as murine RAW 264.7 and murine bone marrow-derived macrophages (BMDMs), as well as human THP-1-derived macrophages, we show that macrophages infected with zinc-limited Mtb have increased bacterial burden compared with macrophages infected with zinc-replete Mtb. We further demonstrate that macrophage infection with zinc-limited Mtb trigger higher production of reactive oxygen species (ROS) and cause more macrophage death. Furthermore, the increased ROS production is linked to the increased phagocytosis of zinc-limited Mtb, whereas cell death is not. Finally, transcriptional analysis of RAW 264.7 macrophages demonstrates that macrophages have more robust pro-inflammatory responses when infected with zinc-limited Mtb than zinc-replete Mtb. Together, our findings suggest that Mtb's access to zinc affects their interaction with macrophages and that zinc-limited Mtb may be influencing TB progression. Therefore, zinc availability in bacterial growth medium should be considered in TB drug and vaccine developments.

FRET-Based Sensor Zebrafish Reveal Muscle Cells Do Not Undergo Apoptosis in Starvation or Natural Aging-Induced Muscle Atrophy.

Muscle atrophy occurs during natural aging and under disease conditions. Muscle cell apoptosis is considered one of the main causes of muscle atrophy, while several recent studies argued that muscle cells do not die during muscle atrophy. Here, sensor zebrafish are generated to visualize muscle cell apoptosis and the engulfment of dead muscle cells by macrophages. Using these sensor zebrafish, starvation, and natural aging-induced muscle atrophy models are established. The data showed that the diameters of muscle cells decreased in both models; however, muscle cell apoptosis is not found in the process of muscle atrophy. In starvation-induced muscle atrophy, it also showed that the number of nuclei in muscle cells remained constant, and there is no increase in the number of macrophages in muscle tissues, both of which further confirmed that muscle cells do not die. In both models, transcriptional analysis showed that the apoptosis pathway is down-regulated, and autophagy and protein degradation pathways are up-regulated. All these data indicated that although there is a great reduction of muscle mass during starvation or aging-induced muscle atrophy, muscle cells do not die by apoptosis. These findings provide new insights into muscle atrophy and can benefit the treatments for muscle atrophy-related diseases.

Identification of the BBX gene family in blueberry at different chromosome ploidy levels and fruit development and response under stress

BackgroundBlueberry (Vaccinium spp.) fruits are rich in flavonoids such as anthocyanins and have a high nutritional value. The zinc finger protein transcription factor B-box (BBX) plays important roles in plant growth and development, hormone response, abiotic stress, and anthocyanin accumulation. However, studies on the BBX family in blueberry are lacking.ResultsIn total, 83 VcBBX and 24 VdBBX genes were identified in tetraploid and diploid blueberry, respectively. A correlation was observed between the number of BBX genes in blueberry and chromosome ploidy. Gene loss and specific replication during blueberry evolution may lead to an imbalance of quantitative relationship between VcBBX and VdBBX genes. The analysis of transcriptome and quantitative reverse transcription–polymerase chain reaction data revealed that the expression pattern of BBX genes depended on the developmental stage of blueberry fruit. Gibberellin inhibited the expression of most VcBBX genes. Abscisic acid promoted the expression of some members of the BBX family. The expression levels of VcBBX15b4, VcBBX21a1, and VcBBX30a in blueberry leaves were significantly downregulated under blue light treatment, whereas that of VcBBX15c3 was significantly upregulated under red light treatment.ConclusionIn total, 83 VcBBX and 24 VdBBX genes were identified in 2 types of blueberries. Fruit development and transcription profiles under different stresses were analyzed. These findings will support further investigation of how BBX genes are involved in regulating hormone treatment and light stress during the growth and development of blueberry.

The Feasibility and Comparability of Using Artificial Intelligence for Qualitative Data Analysis in Equity-Focused Research

In this essay, we explored the feasibility of utilizing artificial intelligence (AI) for qualitative data analysis in equity-focused research. Specifically, we compare thematic analyses of interview transcripts conducted by human coders with those performed by GPT-3 using a zero-shot chain-of-thought prompting strategy. Our results suggest that the AI model, when provided with suitable prompts, can proficiently perform thematic analysis, demonstrating considerable comparability with human coders. Despite potential biases inherent in its training data, the model was able to analyze and interpret the data through social justice perspectives. We discuss the applications of integrating AI into qualitative research, provide code snippets illustrating the use of GPT models, and highlight unresolved questions to encourage further dialogue in the field.

Exploring biomarkers and molecular mechanisms of Type 2 diabetes mellitus promotes colorectal cancer progression based on transcriptomics

Type 2 diabetes mellitus (T2DM) has been confirmed as an independent risk factor for colorectal cancer (CRC) in many studies. However, the mechanisms behind T2DM’s role in the progression of CRC remain unclear. This study aims to explore the potential biomarkers and molecular mechanisms involved in T2DM-promoted CRC progression. The limma package was used to identify differentially expressed genes in tumor tissue from CRC patients with or without T2DM. The key biological processes were screened by gene ontology and gene set enrichment analysis. A diagnostic model for co-morbidities was constructed by logistic regression model with least absolute shrinkage and selection operator (Lasso) regularization method. The diagnostic performance was assessed by supplementing external datasets to draw ROC curves on the diagnostic model. The diagnostic model was further screened for key genes by prognostic analysis. The relationship of key genes with immune cells and other cells was evaluated by immune infiltration algorithm and single-cell transcription analysis. Drug prediction was performed by cMAP and the obtained drugs were molecularly docked with the key genes. The differentially expressed genes of T2DM-promoted CRC progression were mainly enriched to O-linked glycosylation-related processes. The diagnostic model constructed based on Lasso logistic regression had good diagnostic performance (AUC > 0.8). COX11 was the key gene for co-morbidities: in tumor tissues, COX11 expression was significantly higher than that in normal colon tissues. However, COX11 gene expression was significantly lower in patients with comorbidities than in patients without T2DM in tumor tissue. External datasets confirmed from both mRNA and protein expression levels that low COX11 expression was significantly associated with poor CRC prognosis. Immune infiltration analysis suggested that its expression related to the proportion of M2 macrophages. Single-cell transcriptome analysis revealed a close association of COX11 expression with endothelial cells and macrophages. The top4 drugs predicted bound well to COX11. Our study revealed that the pathogenesis of T2DM-promoted CRC progression related to O-linked glycosylation. We constructed a diagnostic model for T2DM-CRC co-morbidity. Meanwhile, we identified COX11 as a potential immune-related molecular marker closely associated with T2DM-promoted CRC progression. These mechanisms and molecular markers may provide new ideas for further studies of T2DM-promoted CRC progression and contribute to drug discovery for the treatment of co-morbidities.

Alternating inverse modulation of xylem K+/NO3 - loading by HY5 and PIF facilitates diurnal regulation of root-to-shoot water and nutrient transport.

Diurnal light-dark cycles regulate nutrient uptake and transport; however, the underlying molecular mechanisms remain largely unknown. Transcription factor MYB59 and ion transporter NPF7.3 participate in root-to-shoot K+/NO3 - translocation in Arabidopsis. In this study, transcriptional analyses and western blotting experiments revealed the diurnal expression of the MYB59-NPF7.3 module. ChIP-qPCR and EMSA showed that transcription factors HY5 and PIF directly bind to the MYB59 promoter. Phenotype analyses and ion content measurement indicated that HY5 and PIF antagonistically control root-to-shoot K+/NO3 - translocation through the MYB59-NPF7.3 module. We found HY5 proteins accumulate in roots and repress MYB59 transcription during daytime, while PIF proteins promote MYB59 transcription in the dark. The expression levels of the NPF7.3 transcript and protein are gradually decreased during daytime, but increased at night. The enhancement of K+/NO3 - loading into the xylem mediated by NPF7.3 could increase root pressure at night, which maintained the root-to-shoot water/nutrient translocation. This study reveals a synergistic mechanism between light signaling and nutrient transport in plants, and defines a diurnal molecular switch of driving forces for root-to-shoot water/nutrient translocation.

The essential role of the hickory StMADS11 subfamily in flower organogenesis and flowering time in Arabidopsis.

The StMADS11 subfamily genes play a crucial role in regulating flowering time, flower development, and bud dormancy in plants. These genes exhibit functional differences between annual and perennial woody plants. In hickory (Carya cathayensis Sarg.), the specific roles of these genes in flowering regulation have not been elucidated. In this study, we identified five StMADS11 subfamily genes in the hickory genome, designated as CcSVP-like, CcAGL24-like1, CcAGL24-like2, CcJOINTLESS-like1, and CcJOINTLESS-like2, based on their clustering characteristics. Sequence analyses revealed distinct structural features in this subfamily, including differences in intron length, C domain, and conserved motifs. Transcript analysis indicated high expression levels of these genes in female flower buds, along with a notable seasonal expression pattern. Overexpression studies on Arabidopsis have demonstrated that the StMADS11 subfamily genes lead to various floral organ and pod anomalies. Specifically, overexpression of CcSVP-like resulted in delayed flowering, while overexpression of CcAGL24-like1, CcAGL24-like2, CcJOINTLESS-like1, and CcJOINTLESS-like2 promoted flowering. Protein interaction studies have shown that the StMADS11 subfamily proteins bind to the CcFUL-like protein. Notably, CcFUL-like, CcSVP-like, CcJOINTLESS-like1, and CcJOINTLESS-like2 proteins were able to bind to the CcSOC1-like promoter and suppress its expression. Our findings elucidate the distinct roles of the StMADS11 subfamily genes in flower development and timing, contribute to developing the current understanding of flowering regulation in hickory, and offer a foundation for further studies in perennial woody plants.

Protective effects and bioinformatic analysis of narciclasine on vascular aging via cross-talk between inflammation and metabolism through inhibiting skeletal muscle-specific ceramide synthase 1.

The senescence of smooth muscle is one of the independent risk factors in atherosclerosis progression in which the vascular inflammation plays an important role on vascular dysfunction. This study is designed to explore the novel vascular aging biomarkers and screen the potential molecular interventional targets through bioinformatic analysis. Transcriptional analysis was conducted based on the GSE16487 open access database, which included 15 human vascular tissue samples from two groups: young group (≤ 60 years old, n = 8) and aged group (≥ 75 years old, n = 7). There were 275 differential expression genes (119 upregulated and 156 downregulated genes) with minimum 1.5-fold change between two groups. 9 genes were mainly participated in inflammation-related signaling pathways, in which narciclasine was validated as the most effective candidate for modulation the ceramide synthesis. In vitro and animal study demonstrated that narciclasine reversed vascular aging by inhibiting skeletal muscle-specific ceramide synthase 1 (CerS1), reducing the ceramide level derived from CerS1, and improving fat deposition and circulating glycolipid metabolism. Narciclasine attenuates vascular aging and modulates the cross-talk between inflammation and metabolism via inhibiting skeletal muscle-specific ceramide synthase 1.

Improving consumer trust in digital health: A mixed methods study involving people living with chronic kidney disease.

To explore preferences, experience and trust in digital health in people living with chronic kidney disease (CKD), and tailor these findings towards solutions that may enhance uptake of digital health services. Mixed methods study, with cross-sectional survey and individual interviews with adults living with CKD attending specialist appointments at an Australian metropolitan hospital. Descriptive statistics and Wilcoxon matched-pairs test were used for survey responses and thematic analysis of interview transcripts, both reported on a theme-by-theme basis provided an overall understanding of trust in digital healthcare. Digital health is changing the way health services are provided, and our results demonstrate that despite limited familiarity, participants are open to learn and adapt to existing digital models of care. Limited exposure to technology may undermine trust in digital health, and telehealth can promote improvements in health literacy. Having the choice in healthcare modalities can promote trust, which can arise from trustful relationships with clinicians who demonstrate genuine interest in patient care. Participants expressed more concerns about sharing identity data than health data online and worry about fragmented information among providers. They preferred public health services due to distrust generated by the perceived risk of private sector data commercialisation. Building trust requires increasing awareness of digital health benefits, promoting positive experiences, improving digital literacy and ensuring interoperability and transparency in digital healthcare systems. People with CKD want to learn and benefit from digital health. Choice and open disclosure on data management and purpose are paramount to building trust.

Harnessing the spatial and transcriptional regulation of monoterpenoid indole alkaloid metabolism in Alstonia scholaris leads to the identification of broad geissoschizine cyclase activities

Monoterpene indole alkaloids (MIAs) are valuable metabolites produced in numerous medicinal plants from the Apocynaceae family such as Alstonia scholaris, which synthesizes strictamine, a MIA displaying neuropharmacological properties of a potential importance. To get insights into the MIA metabolism in A. scholaris, we studied here both the spatial and transcriptional regulations of MIA genes by performing a robust transcriptomics analysis of the main plant organs, leaf epidermis but also by sequencing RNA from leaves transiently overexpressing the master transcriptional regulator MYC2. These transcriptomic studies notably demonstrated that the first steps of the MIA pathway are successively distributed in the internal phloem associated parenchyma and epidermis, and that MYC2 exert a remarkable transcriptional effect by modulating the expression of around 1000 genes. By combining these distinct datasets, we initiated the search for MIA-related genes encoding CYP71, based on the similarity of expression compared to already known MIA genes. Transient expression of these candidates in Nicotiana benthamiana leaves and yeast notably led to the identification of a related isoform of rhazimal synthase (RHS) capable of converting the MIA precursor geissoschizine into akuammicine, strictamine and 16-epi-pleiocarpamine. Investigating its catalytic mechanism revealed that strictamine results from rhazimal deformylation and that a similar mechanism may also explain 16-epi-pleiocarpamine synthesis. This prompted us to rename these enzymes geissoschizine cyclase due to their capacity of cyclizing geissoschizine into three different MIA scaffolds and to form both C-C and C-N bonds. This identification thus illustrates the potential of integrating spatial and transcriptional regulation analysis for MIA gene identification.

Interferon regulatory factor 2 of red-spotted grouper (Epinephelus akaara): Insights into its transcriptional profiling, antiviral potential, and function in macrophage polarization.

Interferon regulatory factor 2 (IRF2) is a member of the IRF family that is specifically involved in diverse immune responses via interferon (IFN)/IRF-dependent signaling pathways. In this study, IRF2 of Epinephelus akaara (EAIRF2) was identified and characterized by evaluating its structural and functional properties. EAIRF2 showed the highest homology with IRF2 of Epinephelus coioides and clustered with teleosts in the phylogenetic tree. The highest level of EAIRF2 mRNA was found in the blood under normal physiological conditions. In the immune challenge experiment, significant transcriptional modulation of EAIRF2 upon lipopolysaccharide (LPS), polyinosinic: polycytidylic acid (poly I:C), and nervous necrosis virus (NNV) challenge were observed. The subcellular localization assay confirmed the role of EAIRF2 as a transcription factor by revealing its specific nuclear localization. To elucidate its functional implications in antiviral defense, EAIRF2 was overexpressed in fathead minnow cells, which were subsequently infected with viral hemorrhagic septicemia virus (VHSV). Notably, cells overexpressing EAIRF2 exhibited a significant reduction in the transcription of VHSV genes. Concurrently, the genes associated with the IFN/IRF signaling pathway were upregulated. Furthermore, the Hoechst and propidium iodide dual staining assay, water-soluble tetrazolium-1 (WST-1) assay, and transcriptional analysis of B-cell lymphoma 2-associated X protein (Bax)/B-cell lymphoma 2 (Bcl-2) indicated that EAIRF2 possesses anti-apoptotic properties during viral infection and poly I:C treatment. Additionally, EAIRF2 overexpression in murine macrophages induced M1 polarization and augmented relative marker gene expression. Collectively, these findings suggest that EAIRF2 is a pivotal immune-related gene, specifically implicated in the IFN/IRF-mediated antiviral defense mechanism, apoptotic signaling pathway, and activation of macrophage-mediated immune responses in Epinephelus akaara. The finding of this study enhances our understanding of IRF2's function in teleost immunity and presents potential avenues for developing therapeutic strategies against viral infections and other immune-related conditions in aquaculture species.

Characterization of tumor necrosis factor receptor-associated factor 2 (TRAF2) in red-spotted grouper (Epinephelus akaara): In vivo and in vitro investigation of its role in the regulation of antiviral immunity and cell death.

Tumor necrosis factor receptor-associated factor 2 (TRAF2) is a key adaptor molecule in tumor necrosis factor receptor signaling complexes, facilitating downstream immune-related signaling cascades. This study aimed to elucidate its function in teleost fish by characterizing the TRAF2 homolog of the red-spotted grouper (Epinephelus akaara, EaTraf2). The open reading frame of EaTraf2 encodes a putative protein of 520 amino acids, containing several characteristic domains of TRAF2. These structural features of EaTraf2 are conserved across diverse organisms, with a relatively higher sequence identity to TRAF2 orthologs from other bony fish. Transcriptional analysis demonstrated that EaTraf2 was ubiquitously expressed in all examined tissues, with the highest level observed in blood. Upon immune challenge, EaTraf2 expression significantly increased in the early stages of stimulation in both blood and spleen. Subcellular localization analysis revealed that EaTraf2 is predominantly localized in the cytoplasm. Overexpression of EaTraf2 in fathead minnow (FHM) cells resulted in elevated levels of interferon and inflammation-associated genes following viral hemorrhagic septicemia virus (VHSV) infection, along with reduced viral gene expression. This provided compelling evidence that EaTraf2 possesses antiviral properties. Furthermore, EaTraf2 demonstrated the ability to promote cell death induced by oxidative stress. Additionally, luciferase reporter assays revealed that EaTraf2 activates the NF-κB signaling pathway upon poly(I:C) stimulation and the Jun N-terminal kinase (JNK) signaling pathway in response to H2O2 treatment. Overall, our study elucidated the role of EaTraf2 in regulating innate immune responses and mediating stress-induced cell death. These findings enhance our understanding of TRAF2 in fish and may contribute to improved health management strategies in finfish aquaculture.