Long-Term In Vitro Culture Alters Gene Expression Pattern of Genes Involved in Ontological Groups Representing Cellular Processes.

The oviduct provides an optimal environment for the final preparation, transport, and survival of gametes, the fertilization process, and early embryonic development. Most of the studies on reproduction are based on in vitro cell culture models because of the cell's accessibility. It creates opportunities to explore the complexity of directly linked processes between cells. Previous studies showed a significant expression of genes responsible for cell differentiation, maturation, and development during long-term porcine oviduct epithelial cells (POECs) in vitro culture. This study aimed at establishing the transcriptomic profile and comprehensive characteristics of porcine oviduct epithelial cell in vitro cultures, to compare changes in gene expression over time and deliver information about the expression pattern of genes highlighted in specific GO groups. The oviduct cells were collected after 7, 15, and 30 days of in vitro cultivation. The transcriptomic profile of gene expression was compared to the control group (cells collected after the first day). The expression of COL1A2 and LOX was enhanced, while FGFBP1, SERPINB2, and OVGP1 were downregulated at all selected intervals of cell culture in comparison to the 24-h control (p-value < 0.05). Adding new detailed information to the reproductive biology field about the diversified transcriptome profile in POECs may create new future possibilities in infertility treatments, including assisted reproductive technique (ART) programmes, and may be a valuable tool to investigate the potential role of oviduct cells in post-ovulation events.

In vitro culture of reptile PGCS to preserve endangered species.

Primordial germ cells (PGCs), are the source of gametes in vertebrates. There are similarities in the development of PGCs of reptiles with avian and mammalian species PGCs development. PGCs culture has been performed for avian and mammalian species but there is no report for reptilian PGCs culture. In vitro culture of PGCs is needed to produce transgenic animals, preservation of endangered animals and for studies on cell behaviour and research on fertility. Reptiles are traded as exotic pets and a source of food and they are valuable for their skin and they are useful as model for medical research. Transgenic reptile has been suggested to be useful for pet industry and medical research. In this research different aspects of PGCs development was compared in three main classes of vertebrates including mammalian, avian and reptilian species. It is proposed that a discussion on similarities between reptilian PGCs development with avian and mammalian species helps to find clues for studies of reptilian PGCs development details and finding an efficient protocol for in vitro culture of reptilian PG.

New Gene Markers of Exosomal Regulation Are Involved in Porcine Granulosa Cell Adhesion, Migration, and Proliferation.

Exosomal regulation is intimately involved in key cellular processes, such as migration, proliferation, and adhesion. By participating in the regulation of basic mechanisms, extracellular vesicles are important in intercellular signaling and the functioning of the mammalian reproductive system. The complexity of intercellular interactions in the ovarian follicle is also based on multilevel intercellular signaling, including the mechanisms involving cadherins, integrins, and the extracellular matrix. The processes in the ovary leading to the formation of a fertilization-ready oocyte are extremely complex at the molecular level and depend on the oocyte's ongoing relationship with granulosa cells. An analysis of gene expression from material obtained from a primary in vitro culture of porcine granulosa cells was employed using microarray technology. Genes with the highest expression (LIPG, HSD3B1, CLIP4, LOX, ANKRD1, FMOD, SHAS2, TAGLN, ITGA8, MXRA5, and NEXN) and the lowest expression levels (DAPL1, HSD17B1, SNX31, FST, NEBL, CXCL10, RGS2, MAL2, IHH, and TRIB2) were selected for further analysis. The gene expression results obtained from the microarrays were validated using quantitative RT-qPCR. Exosomes may play important roles regarding intercellular signaling between granulosa cells. Therefore, exosomes may have significant applications in regenerative medicine, targeted therapy, and assisted reproduction technologies.

Expression Profile of New Marker Genes Involved in Differentiation of Human Wharton's Jelly-Derived Mesenchymal Stem Cells into Chondrocytes, Osteoblasts, Adipocytes and Neural-like Cells.

Wharton's jelly (WJ) contains mesenchymal stem cells (MSCs) exhibiting broad immunomodulatory properties and differentiation capacity, which makes them a promising tool for cellular therapies. Although the osteogenic, chondrogenic and adipogenic differentiation is a gold standard for proper identification of MSCs, it is important to elucidate the exact molecular mechanisms governing these processes to develop safe and efficient cellular therapies. Umbilical cords were collected from healthy, full-term deliveries, for subsequent MSCs (WJ-MSCs) isolation. WJ-MSCs were cultivated in vitro for osteogenic, chondrogenic, adipogenic and neurogenic differentiation. The RNA samples were isolated and the transcript levels were evaluated using NovaSeq platform, which led to the identification of differentially expressed genes. Expression of H19 and SLPI was enhanced in adipocytes, chondrocytes and osteoblasts, and NPPB was decreased in all analyzed groups compared to the control. KISS1 was down-regulated in adipocytes, chondrocytes, and neural-like cells compared to the control. The most of identified genes were already implicated in differentiation of MSCs; however, some genes (PROK1, OCA2) have not yet been associated with initiating final cell fate. The current results indicate that both osteo- and adipo-induced WJ-MSCs share many similarities regarding the most overexpressed genes, while the neuro-induced WJ-MSCs are quite distinctive from the other three groups. Overall, this study provides an insight into the transcriptomic changes occurring during the differentiation of WJ-MSCs and enables the identification of novel markers involved in this process, which may serve as a reference for further research exploring the role of these genes in physiology of WJ-MSCs and in regenerative medicine.

Apoptosis Related Human Wharton's Jelly-Derived Stem Cells Differentiation into Osteoblasts, Chondrocytes, Adipocytes and Neural-like Cells-Complete Transcriptomic Assays.

Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) exhibit multilineage differentiation potential, adhere to plastic, and express a specific set of surface markers-CD105, CD73, CD90. Although there are relatively well-established differentiation protocols for WJ-MSCs, the exact molecular mechanisms involved in their in vitro long-term culture and differentiation remain to be elucidated. In this study, the cells were isolated from Wharton's jelly of umbilical cords obtained from healthy full-term deliveries, cultivated in vitro, and differentiated towards osteogenic, chondrogenic, adipogenic and neurogenic lineages. RNA samples were isolated after the differentiation regimen and analyzed using an RNA sequencing (RNAseq) assay, which led to the identification of differentially expressed genes belonging to apoptosis-related ontological groups. ZBTB16 and FOXO1 were upregulated in all differentiated groups as compared to controls, while TGFA was downregulated in all groups. In addition, several possible novel marker genes associated with the differentiation of WJ-MSCs were identified (e.g., SEPTIN4, ITPR1, CNR1, BEX2, CD14, EDNRB). The results of this study provide an insight into the molecular mechanisms involved in the long-term culture in vitro and four-lineage differentiation of WJ-MSCs, which is crucial to utilize WJ-MSCs in regenerative medicine.

Diagnostic Test Accuracy of Artificial Intelligence in Detecting Periapical Periodontitis on Two-Dimensional Radiographs: A Retrospective Study and Literature Review.

This study aims to evaluate the diagnostic accuracy of artificial intelligence in detecting apical pathosis on periapical radiographs. A total of twenty anonymized periapical radiographs were retrieved from the database of Poznan University of Medical Sciences. These radiographs displayed a sequence of 60 visible teeth. The evaluation of the radiographs was conducted using two methods (manual and automatic), and the results obtained from each technique were afterward compared. For the ground-truth method, one oral and maxillofacial radiology expert with more than ten years of experience and one trainee in oral and maxillofacial radiology evaluated the radiographs by classifying teeth as healthy and unhealthy. A tooth was considered unhealthy when periapical periodontitis related to this tooth had been detected on the radiograph. At the same time, a tooth was classified as healthy when no periapical radiolucency was detected on the periapical radiographs. Then, the same radiographs were evaluated by artificial intelligence, Diagnocat (Diagnocat Ltd., San Francisco, CA, USA). Diagnocat (Diagnocat Ltd., San Francisco, CA, USA) correctly identified periapical lesions on periapical radiographs with a sensitivity of 92.30% and identified healthy teeth with a specificity of 97.87%. The recorded accuracy and F1 score were 96.66% and 0.92, respectively. The artificial intelligence algorithm misdiagnosed one unhealthy tooth (false negative) and over-diagnosed one healthy tooth (false positive) compared to the ground-truth results. Diagnocat (Diagnocat Ltd., San Francisco, CA, USA) showed an optimum accuracy for detecting periapical periodontitis on periapical radiographs. However, more research is needed to assess the diagnostic accuracy of artificial intelligence-based algorithms in dentistry.

The pathogenic roles of lncRNA-Taurine upregulated 1 (TUG1) in colorectal cancer.

Colorectal cancer (CRC) is a gastrointestinal tumor that develops from the colon, rectum, or appendix. The prognosis of CRC patients especially those with metastatic lesions remains unsatisfactory. Although various conventional methods have been used for the treatment of patients with CRC, the early detection and identification of molecular mechanisms associated with CRC is necessary. The scientific literature reports that altered expression of long non-coding RNAs (lncRNAs) contributed to the pathogenesis of CRC cells. LncRNA TUG1 was reported to target various miRNAs and signaling pathways to mediate CRC cell proliferation, migration, and metastasis. Therefore, TUG1 might be a potent predictive/prognostic biomarker for diagnosis of CRC.

The Potential of a Protein Model Synthesized Absent of Methionine.

Methionine is an amino acid long thought to be essential, but only in the case of protein synthesis initiation. In more recent years, methionine has been found to play an important role in antioxidant defense, stability, and modulation of cell and protein activity. Though these findings have expanded the previously held sentiment of methionine having a singular purpose within cells and proteins, the essential nature of methionine can still be challenged. Many of the features that give methionine its newfound functions are shared by the other sulfur-containing amino acid: cysteine. While the antioxidant, stabilizing, and cell/protein modulatory functions of cysteine have already been well established, recent findings have shown a similar hydrophobicity to methionine which suggests cysteine may be able to replace methionine in all functions outside of protein synthesis initiation with little effect on cell and protein function. Furthermore, a number of novel mechanisms for alternative initiation of protein synthesis have been identified that suggest a potential to bypass the traditional methionine-dependent initiation during times of stress. In this review, these findings are discussed with a number of examples that demonstrate a potential model for synthesizing a protein in the absence of methionine.

A highly efficient hybrid peptide ameliorates intestinal inflammation and mucosal barrier damage by neutralizing lipopolysaccharides and antagonizing the lipopolysaccharide-receptor interaction.

Intestinal inflammatory disorders, such as inflammatory bowel disease, are major contributors to mortality and morbidity in humans and animals worldwide. While some native peptides have great potential as therapeutic agents against intestinal inflammation, potential cytotoxicity, anti-inciting action, and suppression of anti-inflammatory activity may limit their development as anti-inflammatory agents. Peptide hybridization is an effective approach for the design and engineering of novel functional peptides because hybrid peptides combine the advantages and benefits of various native peptides. In the present study, a novel hybrid anti-inflammatory peptide that combines the active center of Cecropin A (C) and the core functional region of LL-37 (L) was designed [C-L peptide; C (1-8)-L (17-30)] through in silico analysis to reduce cytotoxicity and improve the anti-inflammatory activity of the parental peptides. The resulting C-L peptide exhibited lower cytotoxicity than either C or L peptides alone. C-L also exerted a protective effect against lipopolysaccharide (LPS)-induced inflammatory responses in RAW264.7 macrophages and in the intestines of a mouse model. The hybrid peptide exhibited increased anti-inflammatory activity compared to the parental peptides. C-L plays a role in protecting intestinal tissue from damage, LPS-induced weight loss, and leukocyte infiltration. In addition, C-L reduces the expression levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), IL-1ß, and interferon-gamma (IFN-γ), as well as reduces cell apoptosis. It also reduced mucosal barrier damage caused by LPS. The anti-inflammatory effects of the hybrid peptide were mainly attributed to its LPS-neutralizing activity and antagonizing the activation of LPS-induced Toll-like receptor 4-myeloid differentiation factor 2 (TLR4/MD2). The peptide also affected the TLR4-(nuclear factor κB) signaling pathway, modulating the inflammatory response upon LPS stimulation. Collectively, these findings suggest that the newly designed peptide, C-L, could be developed into a novel anti-inflammatory agent for animals or humans.

Expression Profile of New Marker Genes Involved in Differentiation of Canine Adipose-Derived Stem Cells into Osteoblasts.

Next-generation sequencing (RNAseq) analysis of gene expression changes during the long-term in vitro culture and osteogenic differentiation of ASCs remains to be important, as the analysis provides important clues toward employing stem cells as a therapeutic intervention. In this study, the cells were isolated from adipose tissue obtained during routine surgical procedures and subjected to 14-day in vitro culture and differentiation. The mRNA transcript levels were evaluated using the Illumina platform, resulting in the detection of 19,856 gene transcripts. The most differentially expressed genes (fold change >|2|, adjusted p value < 0.05), between day 1, day 14 and differentiated cell cultures were extracted and subjected to bioinformatical analysis based on the R programming language. The results of this study provide molecular insight into the processes that occur during long-term in vitro culture and osteogenic differentiation of ASCs, allowing the re-evaluation of the roles of some genes in MSC progression towards a range of lineages. The results improve the knowledge of the molecular mechanisms associated with long-term in vitro culture and differentiation of ASCs, as well as providing a point of reference for potential in vivo and clinical studies regarding these cells' application in regenerative medicine.

Expression Profile of Genes Encoding Proteins Involved in Regulation of Vasculature Development and Heart Muscle Morphogenesis-A Transcriptomic Approach Based on a Porcine Model.

Despite significant advances in treatment of acute coronary syndromes (ACS) many subjects still develop heart failure due to significantly reduced ejection fraction. Currently, there are no commonly available treatment strategies that replace the infarcted/dysfunctional myocardium. Therefore, understanding the mechanisms that control the regeneration of the heart muscle is important. The development of new coronary vessels plays a pivotal role in cardiac regeneration. Employing microarray expression assays and RT-qPCR validation expression pattern of genes in long-term primary cultured cells isolated form the right atrial appendage (RAA) and right atrium (RA) was evaluated. After using DAVID software, it indicated the analysis expression profiles of genes involved in ontological groups such as: "angiogenesis", "blood vessel morphogenesis", "circulatory system development", "regulation of vasculature development", and "vasculature development" associated with the process of creation new blood vessels. The performed transcriptomic comparative analysis between two different compartments of the heart muscle allowed us to indicate the presence of differences in the expression of key transcripts depending on the cell source. Increases in culture intervals significantly increased expression of SFRP2, PRRX1 genes and some other genes involved in inflammatory process, such as: CCL2, IL6, and ROBO1. Moreover, the right atrial appendage gene encoding lysyl oxidase (LOX) showed much higher expression compared to the pre-cultivation state.

New Gene Markers Expressed in Porcine Oviductal Epithelial Cells Cultured Primary In Vitro Are Involved in Ontological Groups Representing Physiological Processes of Porcine Oocytes.

Changes that occur within oviducts after fertilization are dependent on post-ovulation events, including oocyte-oviduct interactions. Although general processes are well-defined, the molecular basis are poorly understood. Recently, new marker genes involved in 'cell development', 'cell growth', 'cell differentiation' and 'cell maturation' processes have been identified in porcine oocytes. The aim of the study was to assess the expression profile of genes in primary in vitro cultured oviductal epithelial cells (OECs), clustered in Gene Ontology groups which enveloped markers also identified in porcine oocytes. OECs (from 45 gilts) were surgically removed and cultured in vitro for ≤ 30 days, and then subjected to molecular analyses. The transcriptomic and proteomic profiles of cells cultured during 7, 15 and 30 days were investigated. Additionally, morphological/histochemical analyzes were performed. The results of genes expression profiles were validated after using RT-qPCR. The results showed a significant upregulation of UNC45B, NOX4, VLDLR, ITGB3, FMOD, SGCE, COL1A2, LOX, LIPG, THY1 and downregulation of SERPINB2, CD274, TXNIP, CELA1, DDX60, CRABP2, SLC5A1, IDO1, ANPEP, FST. Detailed knowledge of the molecular pathways occurring in the OECs and the gametes that contact them may contribute both to developments of basic science of physiology, and new possibilities in advanced biotechnology of assisted reproduction.

Genes regulating hormone stimulus and response to protein signaling revealed differential expression pattern during porcine oocyte in vitro maturation, confirmed by lipid concentration.

Genes influencing oocyte maturation may be valuable for predicting their developmental potential, as well as discerning the mechanistic pathways regulating oocyte development. In the presented research microarray gene expression analysis of immature and in vitro matured porcine oocytes was performed. Two groups of oocytes were compared in the study: before (3 × n = 50) and after in vitro maturation (3 × n = 50). The selection of viable oocytes was performed using the brilliant cresyl blue (BCB) test. Furthermore, microarrays and RT-qPCR was used to analyze the transcriptome of the oocytes before and after IVM. The study focused on the genes undergoing differential expression in two gene-ontology groups: "Cellular response to hormone stimulus" and "Cellular response to unfolded protein", which contain genes that may directly or indirectly be involved in signal transduction during oocyte maturation. Examination of all the genes of interest showed a lower level of their expression after IVM. From the total number of genes in these gene ontologies ten of the highest change in expression were identified: FOS, ID2, BTG2, CYR61, ESR1, AR, TACR3, CCND2, EGR2 and TGFBR3. The successful maturation of the oocytes was additionally confirmed with the use of lipid droplet assay. The genes were briefly described and related to the literature sources, to investigate their potential roles in the process of oocyte maturation. The results of the study may serve as a basic molecular reference for further research aimed at improving the methods of oocyte in vitro maturation, which plays an important role in the procedures of assisted reproduction.

Transcriptomic analysis of expression of genes regulating cell cycle progression in porcine ovarian granulosa cells during short-term in vitro primary culture.

The primary function of ovarian granulosa cells (GCs) is the support of oocytes during maturation and development. Molecular analyses of granulosa cell-associated processes, leading to improvement of understanding of the cell cycle events during the formation of ovarian follicles (folliculogenesis), may be key to improve the in vitro fertilization procedures. Primary in vitro culture of porcine GCs was employed to examine the changes in the transcriptomic profile of genes belonging to "cell cycle", "cell division", "cell cycle process", "cell cycle phase transition", "cell cycle G1/S phase transition", "cell cycle G2/M phase transition" and "cell cycle checkpoint" ontology groups. During the analysis, microarrays were employed to study the transcriptome of GCs, analyzing the total RNA of cells from specific periods of in vitro cultures. This research was based on material obtained from 40 landrace gilts of similar weight, age and the same living conditions. RNA was isolated at specific timeframes: before the culture was established (0 h) and after 48 h, 96 h and 144 h in vitro. Out of 133 differentially expressed genes, we chose the 10 most up-regulated (SFRP2, PDPN, PDE3A, FGFR2, PLK2, THBS1, ETS1, LIF, ANXA1, TGFB1) and the 10 most downregulated (IGF1, NCAPD2, CABLES1, H1FOO, NEK2, PPAT, TXNIP, NUP210, RGS2 and CCNE2). Some of these genes known to play key roles in the regulation of correct cell cycle passage (up-regulated SFRP2, PDE3A, PLK2, LIF and down-regulated CCNE2, TXNIP, NEK2). The data obtained provide a potential reference for studies on the process of mammalian folliculogenesis, as well as suggests possible new genetic markers for cell cycle progress in in vitro cultured porcine granulosa cells.

Skeletal Muscle and the Effects of Ammonia Toxicity in Fish, Mammalian, and Avian Species: A Comparative Review Based on Molecular Research.

Typically, mammalian and avian models have been used to examine the effects of ammonia on skeletal muscle. Hyperammonemia causes sarcopenia or muscle wasting, in mammals and has been linked to sarcopenia in liver disease patients. Avian models of skeletal muscle have responded positively to hyperammonemia, differing from the mammalian response. Fish skeletal muscle has not been examined as extensively as mammalian and avian muscle. Fish skeletal muscle shares similarities with avian and mammalian muscle but has notable differences in growth, fiber distribution, and response to the environment. The wide array of body sizes and locomotion needs of fish also leads to greater diversity in muscle fiber distribution and growth between different fish species. The response of fish muscle to high levels of ammonia is important for aquaculture and quality food production but has not been extensively studied to date. Understanding the differences between fish, mammalian and avian species' myogenic response to hyperammonemia could lead to new therapies for muscle wasting due to a greater understanding of the mechanisms behind skeletal muscle regulation and how ammonia effects these mechanisms. This paper provides an overview of fish skeletal muscle and ammonia excretion and toxicity in fish, as well as a comparison to avian and mammalian species.

The Role of the Adipokines in the Most Common Gestational Complications.

Adipocytokines are hormonally active molecules that are believed to play a key role in the regulation of crucial biological processes in the human body. Numerous experimental studies established significant alterations in the adipokine secretion patterns throughout pregnancy. The exact etiology of various gestational complications, such as gestational diabetes, preeclampsia, and fetal growth abnormalities, needs to be fully elucidated. The discovery of adipokines raised questions about their potential contribution to the molecular pathophysiology of those diseases. Multiple studies analyzed their local mRNA expression and circulating protein levels. However, most studies report conflicting results. Several adipokines such as leptin, resistin, irisin, apelin, chemerin, and omentin were proposed as potential novel early markers of heterogeneous gestational complications. The inclusion of the adipokines in the standard predictive multifactorial models could improve their prognostic values. Nonetheless, their independent diagnostic value is mostly insufficient to be implemented into standard clinical practice. Routine assessments of adipokine levels during pregnancy are not recommended in the management of both normal and complicated pregnancies. Based on the animal models (e.g., apelin and its receptors in the rodent preeclampsia models), future implementation of adipokines and their receptors as new therapeutic targets appears promising but requires further validation in humans.

The Proliferation and Differentiation of Adipose-Derived Stem Cells in Neovascularization and Angiogenesis.

Neovascularization and angiogenesis are vital processes in the repair of damaged tissue, creating new blood vessel networks and increasing oxygen and nutrient supply for regeneration. The importance of Adipose-derived Mesenchymal Stem Cells (ASCs) contained in the adipose tissue surrounding blood vessel networks to these processes remains unknown and the exact mechanisms responsible for directing adipogenic cell fate remain to be discovered. As adipose tissue contains a heterogenous population of partially differentiated cells of adipocyte lineage; tissue repair, angiogenesis and neovascularization may be closely linked to the function of ASCs in a complex relationship. This review aims to investigate the link between ASCs and angiogenesis/neovascularization, with references to current studies. The molecular mechanisms of these processes, as well as ASC differentiation and proliferation are described in detail. ASCs may differentiate into endothelial cells during neovascularization; however, recent clinical trials have suggested that ASCs may also stimulate angiogenesis and neovascularization indirectly through the release of paracrine factors.

Glutamine synthetase in avian muscle contributes to a positive myogenic response to ammonia compared with mammalian muscle.

In mammalian models of cirrhosis, plasma ammonia concentration increases, having numerous adverse effects, including sarcopenia. The objective of this study was to identify differences between avian and mammalian myogenic response to applied ammonia and glutamine. Primary chicken breast and thigh, primary rat, and C2C12 myotubes were treated with ammonium acetate (AA, 10 mM) or glutamine (10 mM) for 24 h and compared with sodium acetate (10 mM) and untreated controls. Myostatin mRNA was significantly higher in C2C12 and rat myotubes treated with AA compared with glutamine and controls (P < 0.01), whereas myostatin was unchanged in chicken myotubes. AA-treated C2C12 myotubes had significantly higher glutamine synthetase (GS) mRNA expression compared with controls, but GS protein expression was unchanged. In contrast, GS mRNA expression was unchanged in thigh myotubes, but GS protein expression was significantly higher in AA-treated thigh myotubes (P < 0.05). In both breast and thigh myotubes, intracellular glutamine concentration was significantly increased in AA- and glutamine-treated myotubes compared with controls but was only increased in glutamine-treated C2C12 and rat myotubes (P < 0.05). Glutamine concentration was significantly higher in all treatment media collected from avian myotube cultures compared with both C2C12 and rat media (P < 0.01). Myotube diameter was significantly larger in avian myotubes after treatment with both AA and glutamine (P < 0.05). C2C12 and rat myotubes had a significantly smaller myotube diameter after AA treatment (P < 0.001). Altogether, these data support species differences in skeletal muscle ammonia metabolism and suggest that glutamine synthesis is a mechanism of ammonia utilization in avian muscle.

Differential ammonia metabolism and toxicity between avian and mammalian species, and effect of ammonia on skeletal muscle: A comparative review.

Comparative aspects of ammonia toxicity, specific to liver and skeletal muscle and skeletal muscle metabolism between avian and mammalian species are discussed in the context of models for liver disease and subsequent skeletal muscle wasting. The purpose of this review is to present species differences in ammonia metabolism and to specifically highlight observed differences in skeletal muscle response to excess ammonia in avian species. Ammonia, which is produced during protein catabolism and is an essential component of nucleic acid and protein biosynthesis, is detoxified mainly in the liver. While the liver is consistent as the main organ responsible for ammonia detoxification, there are evolutionary differences in ammonia metabolism and nitrogen excretory products between avian and mammalian species. In patients with liver disease and all mammalian models, inadequate ammonia detoxification and successive increased circulating ammonia concentration, termed hyperammonemia, leads to severe skeletal muscle atrophy, increased apoptosis and reduced protein synthesis, altogether having deleterious effects on muscle size and strength. Previously, an avian embryonic model, designed to determine the effects of increased circulating ammonia on muscle development, revealed that ammonia elicits a positive myogenic response. Specifically, induced hyperammonemia in avian embryos resulted in a reduction in myostatin, a well-known inhibitor of muscle growth, expression, whereas myostatin expression is significantly increased in mammalian models of hyperammonemia. These interesting findings imply that species differences in ammonia metabolism allow avians to utilize ammonia for growth. Understanding the intrinsic physiological mechanisms that allow for ammonia to be utilized for growth has potential to reveal novel approaches to muscle growth in avian species and will provide new targets for preventing muscle degeneration in mammalian species.

Z-band and M-band titin splicing and regulation by RNA binding motif 20 in striated muscles.

Titin (TTN) has multifunctional roles in sarcomere assembly, mechanosignaling transduction, and muscle stiffness. TTN splicing generates variable protein sizes with different functions. Therefore, understanding TTN splicing is important to develop a novel treatment for TTN-based diseases. The I-band TTN splicing regulated by RNA binding motif 20 (RBM20) has been extensively studied. However, the Z- and M-band splicing and regulation remain poorly understood. Herein, we aimed to define the Z- and M-band splicing in striated muscles and determined whether RBM20 regulates the Z- and M-band splicing. We discovered four new Z-band TTN splicing variants, and one of them dominates in mouse, rat, sheep, and human hearts. But only one form can be detected in frog and chicken hearts. In skeletal muscles, three new Z repeats (Zr) were detected, and Zr4 to 6 exclusion dominates in the fast muscles, whereas Zr4 skipping dominates in the slow muscle. No developmental changes were detected in the Z-band. In the M-band, two new variants were discovered with alternative 3' splice site in exon363 (Mex5) and alternative 5' splice site in intron 362. However, only the sheep heart expresses two new variants rather than other species. Skeletal muscles express three M-band variants with altered ratios of Mex5 inclusion to Mex5 exclusion. Finally, we revealed that RBM20 does not regulate the Z- and M-band splicing in the heart, but does in skeletal muscles. Taken together, we characterized the Z- and M-band splicing and provided the first evidence of the role of RBM20 in the Z- and M-band TTN splicing.