Multiple Developmental Stages Research Articles

Multi-omic characterization of allele-specific regulatory variation in hybrid pigs

Hybrid mapping is a powerful approach to efficiently identify and characterize genes regulated through mechanisms in cis. In this study, using reciprocal crosses of the phenotypically divergent Duroc and Lulai pig breeds, we perform a comprehensive multi-omic characterization of regulatory variation across the brain, liver, muscle, and placenta through four developmental stages. We produce one of the largest multi-omic datasets in pigs to date, including 16 whole genome sequenced individuals, as well as 48 whole genome bisulfite sequencing, 168 ATAC-Seq and 168 RNA-Seq samples. We develop a read count-based method to reliably assess allele-specific methylation, chromatin accessibility, and RNA expression. We show that tissue specificity was much stronger than developmental stage specificity in all of DNA methylation, chromatin accessibility, and gene expression. We identify 573 genes showing allele specific expression, including those influenced by parent-of-origin as well as allele genotype effects. We integrate methylation, chromatin accessibility, and gene expression data to show that allele specific expression can be explained in great part by allele specific methylation and/or chromatin accessibility. This study provides a comprehensive characterization of regulatory variation across multiple tissues and developmental stages in pigs.

P-127 The RNA m6A landscape of mouse oocytes and preimplantation embryos

Abstract Study question Despite the significance of N6-methyladenosine (m6A) in gene regulation, the requirement for large amounts of RNA has hindered m6A profiling in mammalian early embryos. Summary answer We apply picoMeRIP-seq to map m6A in mouse oocytes and preimplantation embryos. We define the landscape of m6A during the maternal-to-zygotic transition. What is known already N6-methyladenosine (m6A), the most prevalent internal modification in eukaryotic messenger RNA (mRNA), plays key regulatory roles in many biological processes (for example, RNA stability, splicing, transport and translation) and is involved in a variety of physiological processes (for example, cell differentiation and reprogramming, embryonic development and stress responses). Study design, size, duration We recently developed an m6A mapping assay for low RNA input, low-input methyl RNA immunoprecipitation and sequencing (picoMeRIP-seq), which enables mapping of the m6A methylome using a low number of cells. In this article, we applied picoMeRIP-seq to multiple developmental stages of mouse oocytes and early embryos to profile their m6A landscapes. Participants/materials, setting, methods We use mouse as study materials.Using picoMeRIP-seq, we generated transcriptome-wide m6A maps for six stages (GV, MII, zygote, two-cell, eight-cell and blastocyst), with two biological replicates per stage and 49–110 oocytes/embryos per replicate. Main results and the role of chance We define the landscape of m6A during the maternal-to-zygotic transition, including stage-specifically expressed transcription factors essential for cell fate determination. Both the maternally inherited transcripts to be degraded post fertilization and the zygotically activated genes during zygotic genome activation are widely marked by m6A. In contrast to m6A-marked zygotic ally-activated genes, m6A-marked maternally inherited transcripts have a higher tendency to be targeted by microRNAs. Moreover, RNAs derived from retrotransposons, such as MTA that is maternally expressed and MERVL that is transcriptionally activated at the two-cell stage, are largely marked by m6A. Our results provide a foundation for future studies exploring the regulatory roles of m6A in mammalian early embryonic development. Limitations, reasons for caution The effect of m6A on the turnover of retrotransposon-derived transcripts abundant in oocytes and early embryos needs to be further addressed in future studies. Wider implications of the findings Our results provide a foundation for future studies exploring the regulatory roles of m6A in mammalian early embryonic development. Trial registration number not applicable

Genome-Wide Identification of the WUSCHEL-Related Homeobox (WOX) Gene Family in Three Cymbidium Species and Expression Patterns in C. goeringii

Numerous members of the WOX gene family play pivotal roles during the processes of growth and development in many plants, as has been demonstrated. Cymbidium goeringii, Cymbidium ensifolium, and Cymbidium sinense are ornamental plants with a fascinating floral morphology that are economically important in China. However, there is limited knowledge about the members of the WOX gene family and their functions in these three Cymbidium species. Hence, the WOX genes in three Cymbidium species were identified on the ground of the genomes data of C. goeringii, C. ensifolium, and C. sinense in this study. These identified WOX genes were further studied for their physicochemical properties, evolutionary relationship, gene structure, protein structure, and cis-acting elements of promoters, as well as the expression pattern of the WOX genes in different tissues of C. goeringii. The findings revealed that eight WOX genes in C. goeringii, twelve WOX genes in C. ensifolium, and nine WOX genes were identified. These WOX genes were further subdivided into WUS, ancient, and intermediate clades. The length of the coding region ranged from 149 to 335 aa, and it was predicted that all WOX genes would be located on the cell nucleus. The promoter cis-acting elements primarily comprised stress response, phytohormone response, plant growth and development, and transcription factor elements. Furthermore, both the transcriptomic data and RT-qPCR analysis showed that most WOX genes may be involved in multiple developmental stages of C. goeringii. To sum up, these results may serve as a theoretical foundation for further study of the function analysis of WOX genes in orchids.

Developmental priorities shift with ontogeny during the early life stages of the American lobster Homarus americanus H. Milne Edwards, 1837 (Decapoda: Astacidea: Nephropidae)

Abstract The American lobster (Homarus americanusMilne Edwards, 1837) is an ecologically and economically valuable invertebrate in the Northwest Atlantic. Its geographic range is shifting northward due to ocean warming. While extensive research on the thermal tolerance of this species has been performed on adults and postlarvae, there have been few studies focused on its multiple early developmental stages. We applied transcriptomics to investigate transcriptional changes in laboratory-reared American lobster developmental stages I through V. Changes in gene expression were contextualized in the ontogenetic shifts in distribution that these different life history stages experience, with highly active stage IV exhibiting increased cellular metabolism and shell-building processes. We identified differential expression of transcripts related to thermal and UV stress in planktonic stages I-IV compared to benthic stage V, which suggests innate molecular defenses against these stressors. Together, these findings further our understanding of crustacean development in the context of climate change and can be used to inform population distribution modeling efforts. They also provide evidence for the need to investigate the potential trade-offs associated with responding to a changing environment on a stage-by-stage basis.

Single Jar for Collecting and Rearing Mosquito Life Stages: An Innovative Prototype.

Mosquitoes vectors are the key threat that spread viruses, bacteria, nematodes, protozoans, and other infections responsible for the transmission of serious public health ailments including dengue, yellow fever, malaria, chikungunya, etc. To rear and cultivate mosquitoes at multiple stages of development (egg, larva, pupa, and adult) to be employed in various biomedical research, an innovative tool mosquito larva adult 2-in-1 rearing jar was designed and developed. The eggs/larvae or adults collected from the field or laboratory cultures can be released into the lower larvae or upper adult-rearing chamber so that they can find a suitable breeding place to oviposit or metamorphose into wrigglers and tumblers attaining the adult stage. Aedes, Anopheles, and Culex are three significant genera (Diptera: Culicidae) that were reared in experiments using this jar (Method I) in contrast to other conventional instruments available (Method II). Results of one-way ANOVA showed that the prototype was compatible and competitive for rearing as the data was non-significant when compared in both groups. However, throughout the trials, it was discovered that the prototype had a greater record of mean percentage of adult emergence (98.33), which supports the novelty of the instrument. This unique device eliminates the need for multiple tools, causes minimum bodily damage to stages during handling, can be used in both the lab and the field, is inexpensive, lightweight, portable, and requires a single manpower to operate.

Targeting the Plasmodium falciparum UCHL3 ubiquitin hydrolase using chemically constrained peptides

The ubiquitin-proteasome system is essential to all eukaryotes and has been shown to be critical to parasite survival as well, including Plasmodium falciparum, the causative agent of the deadliest form of malarial disease. Despite the central role of the ubiquitin-proteasome pathway to parasite viability across its entire life-cycle, specific inhibitors targeting the individual enzymes mediating ubiquitin attachment and removal do not currently exist. The ability to disrupt P. falciparum growth at multiple developmental stages is particularly attractive as this could potentially prevent both disease pathology, caused by asexually dividing parasites, as well as transmission which is mediated by sexually differentiated parasites. The deubiquitinating enzyme PfUCHL3 is an essential protein, transcribed across both human and mosquito developmental stages. PfUCHL3 is considered hard to drug by conventional methods given the high level of homology of its active site to human UCHL3 as well as to other UCH domain enzymes. Here, we apply the RaPID mRNA display technology and identify constrained peptides capable of binding to PfUCHL3 with nanomolar affinities. The two lead peptides were found to selectively inhibit the deubiquitinase activity of PfUCHL3 versus HsUCHL3. NMR spectroscopy revealed that the peptides do not act by binding to the active site but instead block binding of the ubiquitin substrate. We demonstrate that this approach can be used to target essential protein-protein interactions within the Plasmodium ubiquitin pathway, enabling the application of chemically constrained peptides as a novel class of antimalarial therapeutics.

A Timeline of Cardiac Maturation

Background: Much is known about the differences between adult and immature fetal cardiac myocytes. For example, the switch from glycolysis to oxidative phosphorylation, the increase in sarcomere distance and cell size, along with changes in mitochondrial structure and organization are all known to occur at some point between late embryonic development and adulthood. However, the precise timing of when these changes take place is ill-defined. Here, we report in high temporal resolution when hallmarks of maturation develop to provide a timeline and the order of events that are necessary for structural and physiological cardiomyocyte maturation. Methods and Results: Using murine samples from embryonic day 15 all the way to adulthood, we performed characterization of basic organelles, contractile machinery, and extracellular matrix composition, all shown to be necessary for cardiac maturation. We observed clear differences in when maturation hallmarks became established. Features that increased continuously include growth of the size of the heart and cardiomyocytes. Accompanying cell size changes, an increase in endothelial cell count was observed, although cardiomyocyte growth continued after the endothelial cell population reached a plateau. Seahorse assays revealed that mitochondrial respiration increased continuously over time for Complex 1 and 2 until about postnatal day 15, with complex 4 being relatively stable throughout maturation. Lipid droplets appeared at a higher frequency in ventricular tissue starting at postnatal day 5, coinciding with an increase in sarcomere length. T-tubules began to appear at postnatal day 7 alongside changes in calcium handling genes. Mitochondrial size was found to increase rapidly at postnatal day 19. Discussion: Overall, we found that multiple stages of development seem important to target for enhancing maturation as not all changes occur in a linear fashion. Ultimately, knowledge of when maturational hallmarks develop might inform strategies to enhance maturation of human induced pluripotent stem cell derived cardiomyocytes. T32 Minnesota Muscle Training Grant; Summer’s Wish; Regenerative Medicine Minnesota; Stem Cell Institute Predoctoral Infuse Award. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Genome-wide identification of the monovalent cation/proton antiporter (CPA) superfamily and expression analysis in tomato

The cation/proton antiporter (CPA) superfamily, crucial for regulating Na+, K+, and pH equilibrium in plant cells, plays an essential role in plant growth and stress adaptation. However, limited research has been conducted on the CPA gene superfamily in tomatoes. In this study, a total of 31 SlCPA members were identified in the tomato genome, comprising 18 cation/H+ exchangers (SlCHXs), 7 Na+/H+ exchangers (SlNHXs), and 6 K+ efflux antiporters (SlKEAs). A comprehensive analysis encompassing their chromosomal locations, gene structures, phylogenetic relationships, collinearity, tissue-specific expression profiles, and responses to abiotic stress was undertaken. Collinearity and phylogenetic analyses unveiled two groups, represented by Solyc08g081810, Solyc01g008190, Solyc12g056160, and Solyc06g060630, Solyc08g078880, suggesting potential redundancy resulting from duplication events. Tissue-specific expression analysis revealed SlNHXs and SlKEAs expression at multiple developmental stages, while SlCHXs were predominantly expressed during early flower and fruit development, indicating their likely involvement in these processes. Salt treatment notably induced the expression of SlSOS1 (Solyc01g005020) and Solyc04g056600, implicating their role in responding to salt stress. Under drought stress, the expression of Solyc01g005020 and Solyc08g081820 was also significantly induced, supported by promoter element analyses indicating the presence of drought response-related cis-elements like ABRE and MYB binding sites. This study establishes a foundation for further elucidating the gene functions of individual members within the tomato SlCPA family.

Exploration of the *ST Mall Acquisition Event Based on the Theory of Corporate Life Cycle

*ST Mall, a publicly listed company that has gone through multiple development stages, is currently facing severe financial difficulties and industry challenges. Through financial data analysis, it was found that *ST Mall is dealing with issues such as high debts, tight cash flow, and declining profitability. Especially in the recession stage, the financial condition of *ST Mall continues to deteriorate, leading to a significant drop in stock prices. In addition, they are also facing challenges like high market saturation and severe customer loss. The financial events of 2021 have had a profound impact on the company, bringing new financial risks and challenges. To address the current difficulties, the mall urgently needs to take measures to reduce financial risks, improve operational efficiency, closely monitor industry trends, and ensure that the company can effectively deal with future challenges.

Variable expression of MECP2, CDKL5, and FMR1 in the human brain: Implications for gene restorative therapies

MECP2, CDKL5, and FMR1 are three X-linked neurodevelopmental genes associated with Rett, CDKL5-, and fragile-X syndrome, respectively. These syndromes are characterized by distinct constellations of severe cognitive and neurobehavioral anomalies, reflecting the broad but unique expression patterns of each of the genes in the brain. As these disorders are not thought to be neurodegenerative and may be reversible, a major goal has been to restore expression of the functional proteins in the patient's brain. Strategies have included gene therapy, gene editing, and selective Xi-reactivation methodologies. However, tissue penetration and overall delivery to various regions of the brain remain challenging for each strategy. Thus, gaining insights into how much restoration would be required and what regions/cell types in the brain must be targeted for meaningful physiological improvement would be valuable. As a step toward addressing these questions, here we perform a meta-analysis of single-cell transcriptomics data from the human brain across multiple developmental stages, in various brain regions, and in multiple donors. We observe a substantial degree of expression variability for MECP2, CDKL5, and FMR1 not only across cell types but also between donors. The wide range of expression may help define a therapeutic window, with the low end delineating a minimum level required to restore physiological function and the high end informing toxicology margin. Finally, the inter-cellular and inter-individual variability enable identification of co-varying genes and will facilitate future identification of biomarkers.

Knockdown of best1 Gene in Zebrafish Caused Abnormal Neuronal and Skeletal Development - A Subtype of Craniovertebral Junction Malformation?

To investigate the developmental defects caused by knockdown of best1 gene in zebrafish as a model for a subtype of craniovertebral junction (CVJ) malformation. Two antisense morpholinos (MOs) were designed targeting zebrafish best1 to block translation (ATG-MO) or to disrupt splicing (I3E4-MO). MOs were microinjected into fertilized one-cell embryos. Efficacy of splicing MO was confirmed by reverse transcription-polymerase chain reaction. Phenotypes were analyzed and quantified by microscopy at multiple developmental stages. Neuronal outgrowth was assessed in transgenic zebrafish expressing green fluorescent protein in neurons. Skeletal ossification was visualized by Calcein staining. Knockdown of best1 resulted in zebrafish embryos with shorter body length, curved axis, low survival rate, microcephaly, reduced eye size, smaller head and brain, impaired neuronal outgrowth, and reduced ossification of craniofacial and vertebral bone. Best1 gene plays critical roles in ophthalmologic, neurological and skeletal development in zebrafish. A patient with a premature stop codon in BEST1 gene exhibited similar phenotypes, implying a subtype of CVJ malformation.

Application of Forward Chaining and Rule-Based Reasoning Methods to Design an Expert System for Pregnant Women Disease Diagnosis in a Private Hospital

This research aims to discover how to create and implement an expert system for prenatal disease diagnosis. To prepare this study, the author employed several data collection techniques, including observation, interviews, and a literature review. The system to be designed was developed using an object-oriented paradigm and a Rapid Application Development (RAD) technique. Multiple development stages are involved in creating RAD, including requirement planning, workshop design, and implementation. Based on the study findings, it was discovered that the PHP programming language, MySQL database management system, Apache web server, UML tool, and RAD system development methodology were used to create an expert system for identifying illnesses in expectant mothers. The forward chaining method is used for this expert system, where the first stages are to create an inference tree and set rules. This system likewise computes the consistency test using the Bayes theorem method. With an 80% accuracy rate, SISPADIBU can be used as a diagnostic tool for disorders related to pregnancy. SISPADIBU can use the user-entered symptoms to present the diagnosis results. According to the survey results, 80% of users said that SISPADIBU was highly user-friendly, that the layout display was simple to use, and that it was constructive in educating users about pregnancy disorders.

Nurturing positive mental health and wellbeing in educational settings - the PRICES model.

This article presents a comprehensive framework for the implementation of social and emotional learning (SEL) programs as a means of health promotion in educational settings, to positively impact student, school, and adult mental health and wellbeing across education. It emphasizes the profound impact of education on life trajectories and success definitions. Drawing inspiration from the Greek myth of Icarus and Maya Angelou's poem "Still I Rise," it explores contrasting notions of success and their consequences. Schools play a pivotal role in shaping students' wellbeing across multiple dimensions and developmental stages. Because of this, holistic mental health and wellbeing promotion that takes a whole-school approach is critical. The paper introduces the PRICES framework (Preparation and Access, Restoration, Integration, Connection and Community, Educator Support, Strengths-Based Cultivation and Student Voice) as a comprehensive method for implementing SEL programs in educational systems. Each component of the PRICES framework is discussed in detail, emphasizing its role in fostering positive health promotion within schools. Examples of implementation plans that operationalize this model through a co-development process focusing on the Social, Emotional, and Ethical (SEE) Learning program are also presented. The PRICES model encourages a shift towards a more holistic approach to education, nurturing social and emotional development alongside academic achievement. By prioritizing wellbeing, fostering a sense of community, and integrating evidence based SEL interventions, schools can positively impact the mental health and overall flourishing of students and educators, contributing to thriving communities.

Histone H3.3 variant plays a critical role on zygote-to-oocyst development in malaria parasites

The Plasmodium life cycle involves differentiation into multiple morphologically distinct forms, a process regulated by developmental stage-specific gene expression. Histone proteins are involved in epigenetic regulation in eukaryotes, and the histone variant H3.3 plays a key role in the regulation of gene expression and maintenance of genomic integrity during embryonic development in mice. However, the function of H3.3 through multiple developmental stages in Plasmodium remains unknown. To examine the function of H3.3, h3.3-deficient mutants (Δh3.3) were generated in P. berghei. The deletion of h3.3 was not lethal in blood stage parasites, although it had a minor effect of the growth rate in blood stage; however, the in vitro ookinete conversion rate was significantly reduced, and the production of the degenerated form was increased. Regarding the mosquito stage development of Δh3.3, oocysts number was significantly reduced, and no sporozoite production was observed. The h3.3 gene complemented mutant have normal development in mosquito stage producing mature oocysts and salivary glands contained sporozoites, and interestingly, the majority of H3.3 protein was detected in female gametocytes. However, Δh3.3 male and female gametocyte production levels were comparable to the wild-type levels. Transcriptome analysis of Δh3.3 male and female gametocytes revealed the upregulation of several male-specific genes in female gametocytes, suggesting that H3.3 functions as a transcription repressor of male-specific genes to maintain sexual identity in female gametocytes. This study provides new insights into the molecular biology of histone variants H3.3 which plays a critical role on zygote-to-oocyst development in primitive unicellular eukaryotes.

Accumulation patterns of flavonoids during multiple development stages of tea seedlings

Accumulation patterns of flavonoids during multiple development stages of tea seedlings

Silencing the rhythm gene AgCLK-1 reduced feeding of Aphis gossypii

The cotton aphid Aphis gossypii Glover is an important cotton pest, and means of controlling this insect is a primary research focus. Although biological rhythm is an important mechanism that regulates numerous insect processes and activities, its role in cotton aphid has not been elucidated. In the present study, four highly-expressed circadian rhythm genes were selected from the cotton aphid genome database and their physicochemical properties and protein structures were analyzed. These genes were in the Takeout, Timeless, and Timeless interacting-related families, and the corresponding proteins contained highly-conserved Swis and TIMELESS domains. Gene expression analysis at multiple developmental stages revealed differing expression patterns between the four genes. AgCLK-1 had the highest relative expression of the four, especially during the nymph period. Silencing AgCLK-1 caused a significant refusal of the cotton aphids to feed at 1, 3, and 5 d of treatment. These results demonstrated that AgCLK-1 played a key role in regulating the feeding behavior of cotton aphid. This new functional understanding provides novel insights into cotton aphid biology and suggests new targeting strategies for agricultural pest control.

Toltrazuril alkalizer-modifying solid dispersions against Toxoplasma gondii: A pharmacotechnical strategy to improve the efficacy of the drug

Toxoplasma gondii is a zoonotic protozoan that can parasitize nucleated cells of all warm-blooded animals, and seriously harm human and livestock. Toltrazuril (TOL) has insecticidal activity against parasites of the phylum Apicomplexan at multiple development stages, but the clinical application is limited by its poor water solubility. To improve the dissolution of TOL, nine ternary solid dispersions (SD) were prepared with PEG6000 as the carrier and various alkalizers as the pH modifier. Compared with the binary SD, all ternary SDs had improved TOL dissolution although dissolution rates differed. The complete dissolution was achieved for the Ca(OH)2-SD, associated with a gradual release of the alkalizer and adequate pH regulation of the microenvironment. DSC, PXRD and FTIR analyses indicated that TOL in the Ca(OH)2-SD was present in an amorphous form and had a strong hydrogen bond with Ca(OH)2. Within the drug concentration of 100 μg/mL, Ca(OH)2-SD was proved to have no damage to host cells by in vitro cytotoxicity analysis, and its anti-T. gondii efficacy was significantly higher than that of TOL and binary SD. The in vivo efficacy of Ca(OH)2-SD against T. gondii in mice further confirmed that Ca(OH)2-SD could be used as a new strategy to prevent T. gondii from killing mice and treat toxoplasmosis. In conclusion, Ca(OH)2-SD is expected to eventually turn into a clinical candidate for toxoplasmosis treatment in the future.

Genome-Wide Analysis of Amino Acid Transporter Gene Family Revealed That the Allele Unique to the Aus Variety Is Associated with Amino Acid Permease 17 (OsAAP17) Amplifies Both the Tiller Count and Yield in Indica Rice (Oryza sativa L.)

Amino acid transporters (AATs) play a crucial role in facilitating the movement of amino acids across cellular membranes, which is vital for the growth and development of plants. Amino acid permease (AAP), which belongs to the AAT family, has been the subject of extensive functional research in plants. Although its importance is recognized, a comprehensive grasp of this family’s dynamics in indica rice remains lacking. In this investigation, a total of 27 AAP genes were identified in the genome of indica rice. Further, the phylogenetic analysis unveiled that the 69 AAP genes from both the model species and other plant species could be classified into 16 distinct subfamilies. The analysis of chromosomal mapping revealed an uneven distribution of the 27 OsAAP genes across the 12 rice chromosomes. Notably, the OsAAP family displayed a total of 10 duplicated gene pairs, along with the identification of numerous conserved motifs. The examination of cis-elements within OsAAP genes unveiled that their promoters contain cis-elements related to phytohormones, plant growth and development, as well as stress responses. Additionally, transcriptome profiling demonstrated that a substantial portion of these genes exhibited responsiveness to various hormones, with their activation spanning multiple tissues and developmental stages in rice. The study identified miRNAs with a specific affinity for OsAAP genes. Out of the 27 OsAAP genes investigated, seventeen were discovered to be targeted by a total of forty-three miRNAs. Furthermore, the aus allele of OsAAP3 that we named OsAAP17 was validated for its effect on productive tillers and yield, and seventeen genetic variants of OsAAP17 were found to be associated with a culm number in indica rice. In addition, indica rice varieties were monomorphic, while aus genotypes displayed polymorphism for OsAAP17 gene-specific in/dels. Moreover, in Season II (rabi season), it was found that the aus allele of OsAAP17 increased the number of productive tillers and the single plant yield by 22.55% and 9.67%, respectively, in a recombinant inbred population created by crossing N22 and JR 201. Remarkably, this enhancement was more pronounced during the dry cultivation season, highlighting the influence of environmental factors in the regulation of tiller numbers mediated by OsAAP17. The discoveries presented here lay a strong foundation for further exploration into the roles of OsAAP family genes across a range of developmental processes. Therefore, the identified allelic variations in the utilization of OsAAP17 has the potential to enhance rice crop production via molecular breeding in the changing climate scenario.

Characterisation, evolution and expression analysis of the interferon regulatory factor (IRF) family from olive flounder (Paralichthys olivaceus) in response to Edwardsiella tarda infection and temperature stress

Interferon regulatory factor (IRF) family involves in the transcriptional regulation of type I Interferons (IFNs) and IFN-stimulated genes (ISGs) and plays a critical role in cytokine signaling and immune response. However, systematic identification of the IRF gene family in teleost has been rarely reported. In this study, twelve IRF members, named PoIRF1, PoIRF2, PoIRF3, PoIRF4a, PoIRF4b, PoIRF5, PoIRF6, PoIRF7, PoIRF8, PoIRF9, PoIRF10 and PoIRF11, were identified from genome-wide data of olive flounder (Paralichthys olivaceus). Phylogenetic analysis indicated that PoIRFs could be classified into four clades, including IRF1 subfamily (PoIRF1, PoIRF11), IRF3 subfamily (PoIRF3, PoIRF7), IRF4 subfamily (PoIRF4a, PoIRF8, PoIRF9, PoIRF10) and IRF5 subfamily (PoIRF5, PoIRF6). They were evolutionarily related to their counterparts in other fish. Gene structure and motif analysis showed that PoIRFs protein sequences were highly conserved. Under normal physiological conditions, all PoIRFs were generally expressed in multiple developmental stages and healthy tissues. After E. tarda attack and temperature stress, twelve PoIRFs showed significant and different changes in mRNA levels. The expression of PoIRF1, PoIRF3, PoIRF4a, PoIRF5, PoIRF7, PoIRF8, PoIRF9, PoIRF10 and PoIRF11 could be markedly induced by E. tarda, indicating that they played a key role in the process of antibacterial immunity. Besides, temperature stress could significantly stimulate the expression of PoIRF3, PoIRF5, PoIRF6 and PoIRF7, indicating that they could transmit signals rapidly when the temperature changes. In conclusion, this study reported the molecular properties and expression analysis of PoIRFs, and explored their role in immune response, which laid a favorable foundation for further studies on the evolution and functional characteristics of the IRF family in teleost fish.

Zebrafish as a high throughput model for organ preservation and transplantation research.

Despite decades of effort, the preservation of complex organs for transplantation remains a significant barrier that exacerbates the organ shortage crisis. Progress in organ preservation research is significantly hindered by suboptimal research tools that force investigators to sacrifice translatability over throughput. For instance, simple model systems, such as single cell monolayers or co-cultures, lack native tissue structure and functional assessment, while mammalian whole organs are complex systems with confounding variables not compatible with high-throughput experimentation. In response, diverse fields and industries have bridged this experimental gap through the development of rich and robust resources for the use of zebrafish as a model organism. Through this study, we aim to demonstrate the value zebrafish pose for the fields of solid organ preservation and transplantation, especially with respect to experimental transplantation efforts. A wide array of methods were customized and validated for preservation-specific experimentation utilizing zebrafish, including the development of assays at multiple developmental stages (larvae and adult), methods for loading and unloading preservation agents, and the development of viability scores to quantify functional outcomes. Using this platform, the largest and most comprehensive screen of cryoprotectant agents (CPAs) was performed to determine their toxicity and efficiency at preserving complex organ systems using a high subzero approach called partial freezing (i.e., storage in the frozen state at -10°C). As a result, adult zebrafish cardiac function was successfully preserved after 5 days of partial freezing storage. In combination, the methods and techniques developed have the potential to drive and accelerate research in the fields of solid organ preservation and transplantation.