Pig Tissues Research Articles

Porcine transient receptor potential channel 1 promotes adipogenesis and lipid deposition

Adipose tissue, an important organ involved in energy metabolism and endocrine, is closely related to animal meat quality and human health. Transient receptor potential channel 1 (TRPC1), an ion transporter, is adipocytes’ major Ca2+ entry channel. However, its function in fat deposition is poorly understood, particularly in pigs, which are both an ideal model for human obesity research and a primary meat source for human diets. In the present investigation, our findings demonstrate a prominent expression of TRPC1 within the adipose tissue of pigs with a strong fat deposition ability. Functional analysis showed that TRPC1 promotes primary preadipocyte proliferation and adipogenic differentiation. In vivo, transgenic mice expressing porcine TRPC1 (Tg-pTRPC1) exhibited aggravated high-fat diet-induced obesity, hepatic steatosis, and insulin resistance. Moreover, TRPC1 may facilitate adipogenesis via activating PI3K/AKT and β-catenin signaling pathways. Our research underscores the pivotal role of porcine TRPC1 as a positive regulator in adipogenesis and lipid accumulation processes, providing a potential target for improving animal meat quality and treating obesity-related diseases in humans.

Insights and implications for transcriptomic analysis of heat stress-induced intestinal inflammation in pigs

BackgroundHeat stress (HS) can affect the physiology and metabolism of animals. HS-induced intestinal inflammation in pigs is a common disease, causing severe diarrhea, that can result in substantial economic losses to the pig industry, but the molecular mechanisms and pathogenicity of this disease are not fully understood. The objective of this study was to identify the differentially expressed genes (DEGs) and long noncoding RNAs (DELs) related to inflammation in the colon tissues of pigs under constant (1, 7, and 14 days) HS.ResultsLncRNA and targeted gene interaction networks were constructed. GO annotation and KEGG pathway analyses were subsequently performed to determine the functions of the DEGs and DELs. The results revealed 57, 212, and 54 DEGs and 87, 79, and 55 DELs in the CON/H01, CON/H07, and CON/H14 groups, respectively. KRT85, CLDN1, S100A12, TM7SF2, CCN1, NR4A1, and several lncRNAs may be involved in regulating the development of intestinal inflammation. GO analysis indicated that the DEGs and DELs were enriched in a series of biological processes involved in the innate immune response, RAGE receptor binding, and positive regulation of the ERK1 and ERK2 cascades. KEGG pathways related to inflammation, such as the tight junction (TJ) and MAPK signaling pathways, were enriched in DEGs and DELs.ConclusionsThis study have expanded the knowledge about colon inflammation-related genes and lncRNA biology in pigs under HS; analyzed the the lncRNA‒mRNA interaction for HS-induced intestinal inflammation. These results may provide some references for our understanding of the molecular mechanism of the intestinal response to HS in pig.

Porcine Airway Organoid-Derived Well-Differentiated Epithelial Cultures as a Tool for the Characterization of Swine Influenza a Virus Strains.

Swine influenza A viruses (IAVsw) are important causes of disease in pigs but also constitute a public health risk. IAVsw strains show remarkable differences in pathogenicity. We aimed to generate airway organoids from the porcine lower respiratory tract and use these to establish well-differentiated airway epithelial cell (WD-AEC) cultures grown at an air-liquid interface (ALI) for in vitro screening of IAVsw strain virulence. Epithelial cells were isolated from bronchus tissue of juvenile pigs, and airway organoids were cultured in an extracellular matrix in a culture medium containing human growth factors. Single-cell suspensions of these 3D organoids were seeded on Transwell filters and differentiated at ALI to form a pseudostratified epithelium containing ciliated cells, mucus-producing cells and tight junctions. Inoculation with a low dose of IAVsw in a low volume inoculum resulted in virus replication without requiring the addition of trypsin, and was quantified by the detection of viral genome loads in apical washes. Interestingly, inoculation of an H3N2 strain known to cause severe disease in pigs induced a greater reduction in trans-epithelial resistance and more damage to tight junctions than H1N2 or H1N1 strains associated with mild disease in pigs. We conclude that the porcine WD-AEC model is useful in assessing the virulence of IAVsw strains.

Study of the establishment of a guinea pig model of ocular accommodative spasm by carbachol eye drops.

To explore a method for establishing a model of ocular accommodative spasm in guinea pigs by using different concentrations of carbachol eye drops and different frequency of administration. Eighteen healthy guinea pigs were selected to establish models for both eyes. Guinea pigs were randomly divided into two groups. Group A was modeled with carbachol eye drops at a concentration of 2ml:0.1mg, and group B was modeled with carbachol eye drops at a concentration of 4ml:0.1mg. The two groups were further randomly divided into three groups according to the random number table method. Three groups of guinea pigs were established: In the A1, B1 group, the drug was administered one time/day; in the A2, B2 group, the drug was administered two times/day; and in the A3, B3 group, the drug was administered three times/day. Before modeling, retinoscopy optometry was performed on the guinea pigs without mydriasis and mydriasis, and a refractive state was recorded. On subsequent days, modeling was performed again, and carbachol eye drops were administered using the same administration times. Retinoscopy was performed in a dark room without mydriasis on days 1, 3, 7, 14 and 21, and the data were recorded. After optimizing the modeling method, the ciliary muscle tissue of the model guinea pig and normal guinea pig were obtained for hematoxylin and eosin (H&E) staining of paraffin sections to compare the morphological differences under a light microscope. (1) There was no statistical significance in the interaction of time*0 d and time*drug frequency between group A and group B (P > 0.05). However, the main effect of different dose times varied between group A and group B (P < 0.05), and the modeling effect of three drops was better. (2) The overall modeling methods of groups A and B were not affected by the time factor, but three doses of carbachol eye drops achieved the best modeling effect on the 7th day, and the diopter of guinea pigs stabilized on the 14th and 21st days. (3) To further optimize the medication, the A and B groups were given three drops. On the 7th day, the refractive data of guinea pigs were compared between the groups, and the refractive status of the two groups of guinea pigs before modeling was compared within the groups. There was no statistical difference between the groups (P > 0.05), and there was a statistical difference within the groups (P < 0.05). (4) In the H&E staining of paraffin sections, the color of the ciliary muscle of the guinea pig with spasm of accommodation was darker than that of the normal guinea pig, and the myofilaments were more closely arranged. (1) Carbachol has the effect of contracting the ciliary muscle; (2) Three drops of 2ml:0.1mg or 4ml:0.1mg of carbachol eye drops can be selected for seven days for modeling.

Dietary glycine supplementation enhances creatine availability in tissues of pigs with intrauterine growth restriction.

This study tested the hypothesis that dietary supplementation with glycine (Gly) enhances the synthesis and availability of creatine (Cr) in tissues of pigs with intrauterine growth restriction (IUGR). At weaning (21 d of age), IUGR pigs and litter mates with normal birth weights (NBW) were assigned randomly to one of two groups, namely, supplementation with 1% Gly or 1.19% L-alanine (isonitrogenous control) to a corn- and soybean meal-based diet. Blood, kidneys, liver, pancreas, jejunum, longissimus lumborum muscle (LLM), and gastrocnemius muscle (GM) were obtained from the pigs within 1wk after the feeding trial ended at 188 d of age to determine concentrations of guanidinoacetate (GAA), Cr, creatinine, and phosphocreatine (CrP). The organs were also analyzed for activities and mRNA levels for Cr-synthetic enzymes: L-arginine:glycine amidinotransferase (AGAT; forming GAA from Gly and L-arginine) and guanidinoacetate N-methyltransferase (GAMT; converting GAA and L-methionine into Cr). AGAT activity was present in the kidneys, liver, and pancreas, whereas GAMT activity was found in all the organs analyzed. AGAT and GAMT were most active per g of tissue in the kidneys and liver, respectively. Based on tissue mass, the kidneys had the greatest (P < 0.001) AGAT activity per whole organ, followed by the liver, while skeletal muscle had the greatest (P < 0.001) GAMT activity per whole organ, followed by the liver. Thus, the kidneys played a dominant role in forming GAA, whereas skeletal muscle and liver were the major sites for converting GAA into Cr. Dietary supplementation with 1% Gly enhanced AGAT activity in the kidneys and pancreas but reduced GAMT activity in the pancreas and small intestine, therefore directing GAA to the liver and skeletal muscle for Cr production. IUGR selectively reduced the concentration of Cr in LLM among all the organs studied. Except for the GM that had greater mRNA levels for GAMT in IUGR pigs, neither Gly nor IUGR affected mRNA levels for the selected genes in the tissues examined. Collectively, these novel results indicate that dietary Gly intake upregulates the Cr-synthetic pathway in swine.

The Spatial-Temporal Alternative Splicing Profile Reveals the Functional Diversity of FXR1 Isoforms in Myogenesis.

Alternative splicing (AS) is a fundamental mechanism contributing to proteome diversity, yet its comprehensive landscape and regulatory dynamics during skeletal muscle development remain largely unexplored. Here, the temporal AS profiles are investigated during myogenesis in five vertebrates, conducting comprehensive profiling across 27 developmental stages in skeletal muscle and encompassing ten tissues in adult pigs. The analysis reveals a pervasive and evolutionarily conserved pattern of alternative exon usage throughout myogenic differentiation, with hundreds of skipped exons (SEs) showing developmental regulation, particularly within skeletal muscle. Notably, this study identifies a muscle-specific SE (exon 15) within the Fxr1 gene, whose AS generates two dynamically expressed isoforms with distinct functions: the isoform without exon 15 (Fxr1E15 -) regulates myoblasts proliferation, while the isoform incorporating exon 15 (Fxr1E15+) promotes myogenic differentiation and fusion. Transcriptome analysis suggests that specifically knocking-down Fxr1E15+ isoform in myoblasts modulates differentiation by influencing gene expression and splicing of specific targets. The increased inclusion of exon 15 during differentiation is mediated by the binding of Rbm24 to the intron. Furthermore, in vivo experiments indicate that the Fxr1E15+ isoform facilitates muscle regeneration. Collectively, these findings provide a comprehensive resource for AS studies in skeletal muscle development, underscoring the diverse functions and regulatory mechanisms governing distinct Fxr1 isoforms in myogenesis.

A comprehensive atlas of pig RNA editome across 23 tissues reveals RNA editing affecting interaction mRNA-miRNAs.

RNA editing is a co-transcriptional/post-transcriptional modification that is mediated by the ADAR enzyme family. Profiling of RNA editing is very limited in pigs. In this study, we collated 3813 RNA-seq data from the public repositories across 23 tissues and carried out comprehensive profiling of RNA editing in pigs. In total, 127,927 A-to-I RNA-editing sites were detected. Our analysis showed that 98.2% of RNA-editing sites were located within repeat regions, primarily within the pig-specific SINE retrotransposon PRE-1/Pre0_SS elements. Subsequently, we focused on analyzing specific RNA-editing sites (SESs) in skeletal muscle tissues. Functional enrichment analyses suggested that they were enriched in signaling pathways associated with muscle cell differentiation, including DMD, MYOD1, and CAV1 genes. Furthermore, we discovered that RNA editing event in the 3'UTR of CFLAR mRNA influenced miR-708-5p binding in this region. In this study, the panoramic RNA-editing landscape of different tissues of pigs was systematically mapped, and RNA-editing sites and genes involved in muscle cell differentiation were identified. In summary, we identified modifications to pig RNA-editing sites and provided candidate targets for further validation.

Monoclonal Antibody against Porcine LAG3 Inhibits Porcine Reproductive and Respiratory Syndrome Virus Infection

Lymphocyte activation gene 3 (LAG3) is an inhibitory receptor and the interaction between fibrinogen-like protein 1 and LAG3 can inhibit the anti-tumor effect of T cells both in vivo and in vitro, which was regarded as a new immune evasion mechanism. Porcine reproductive and respiratory syndrome (PRRS), caused by PRRSV, is an infectious disease characterized by reproductive disorders in pregnant sows and gilts and respiratory problems in pigs of all ages, seriously impacting the pig industry worldwide. In this study, monoclonal antibodies (mAbs) against porcine LAG3 (pLAG3) were developed, and one mAb (1C2) showed good reactivity with pLAG3 on PHA-activated porcine peripheral blood lymphocytes. Epitope mapping showed the epitope recognized by mAb 1C2 was located at amino acid residues 214–435 of pLAG3. LAG3 expression in the tissues of PRRSV-infected pigs was detected, using mAb 1C2 as the primary antibody, and the results revealed that PRRSV infection caused a marked increase in LAG3 expression compared to the control group. Interference of LAG3 expression on PHA-activated lymphocytes promoted PRRSV replication in the co-culture system of monocyte-derived dendritic cells and lymphocytes, whereas overexpression of LAG3 or blocking of the LAG3 signal with mAb 1C2 inhibited PRRSV replication, indicating that PRRSV infection activates the LAG3-signaling pathway, suggesting that this pathway plays an important role in PRRSV pathogenesis. The results obtained lay the foundation for subsequent research on the role of LAG3 in PRRS and other diseases with persistent infection characteristics.

Identification of genomic regions associated with fatty acid metabolism across blood, liver, backfat and muscle in pigs

BackgroundThe composition and distribution of fatty acids (FA) are important factors determining the quality, flavor, and nutrient value of meat. In addition, FAs synthesized in the body participate in energy metabolism and are involved in different regulatory pathways in the form of signaling molecules or by acting as agonist or antagonist ligands of different nuclear receptors. Finally, synthesis and catabolism of FAs affect adaptive immunity by regulating lymphocyte metabolism. The present study performed genome-wide association studies using FA profiles of blood, liver, backfat and muscle from 432 commercial Duroc pigs.ResultsTwenty-five genomic regions located on 15 Sus scrofa chromosomes (SSC) were detected. Annotation of the quantitative trait locus (QTL) regions identified 49 lipid metabolism-related candidate genes. Among these QTLs, four were identified in more than one tissue. The ratio of C20:4n-6/C20:3n-6 was associated with the region on SSC2 at 7.56–14.26 Mb for backfat, liver, and muscle. Members of the fatty acid desaturase gene cluster (FADS1, FADS2, and FADS3) are the most promising candidate genes in this region. Two QTL regions on SSC14 (103.81–115.64 Mb and 100.91–128.14 Mb) were identified for FA desaturation in backfat and muscle. In addition, two separate regions on SSC9 at 0 – 14.55 Mb and on SSC12 at 0–1.91 Mb were both associated with the same multiple FA traits for backfat, with candidate genes involved in de novo FA synthesis and triacylglycerol (TAG) metabolism, such as DGAT2 and FASN. The ratio C20:0/C18:0 was associated with the region on SSC5 at 64.84–78.32 Mb for backfat. Furthermore, the association of the C16:0 content with the region at 118.92–123.95 Mb on SSC4 was blood specific. Finally, candidate genes involved in de novo lipogenesis regulate T cell differentiation and promote the generation of palmitoleate, an adipokine that alleviates inflammation.ConclusionsSeveral SNPs and candidate genes were associated with lipid metabolism in blood, liver, backfat, and muscle. These results contribute to elucidating the molecular mechanisms implicated in the determination of the FA profile in different pig tissues and can be useful in selection programs that aim to improve health and energy metabolism in pigs.

49 Crossing-talk between micronutrient and macronutrient metabolism: Implication in health and disease

Abstract Recent goals of swine ration formulation have evolved from preventing nutritional deficiency diseases and maximizing growth performance to optimizing immune function and disease resistance with least climate impact. Thus, certain micronutrients such as selenium (Se) are widely considered to be health beneficial at dietary concentrations above nutrient requirements. During the past decade, we have conducted a number of experiments to determine impacts and mechanisms of dietary Se concentrations (0.03 to 1 or 3 mg/kg) on metabolism of carbohydrate, lipid, and protein in pigs from growing to finishing. Strikingly, pigs fed high-Se diets developed hyperinsulinemia and insulin resistance. Those pigs also had increased lipid, protein, saturated fatty acids (myristic, palmitic, stearic, and behenic acids), and aspartic acid, glutamine, and alanine and decreased polyunsaturated fatty acids (dihomo-γ-linolenic, oleic, α-linolenic, and eicosatrienoic acids) in the liver and(or) adipose tissues. The high-Se diets increased and decreased amounts of 5 and 2 selenoproteins, respectively, in tissues of pigs. These effects were associated with down-regulated insulin signaling (INSR and AKT), glycolysis (glucokinase) and gluconeogenesis (PEPCK) and up-regulated lipogenesis (FOXO1, SREBP1, ACC and FAS), and protein synthesis (mTOR and RPS6/S6). In contrast, high dietary fat intakes up- or down-regulated expressions of all selenoproteins except for TXNRD2 and TXNRD3 and showed a strong effect on expressions of DIO2, GPX6, and SELENOK. These induced selenogenome expression changes were associated with increased serum malondialdehyde and plasma concentrations of tumor necrosis factor alpha and interleukin-6, along with risk of obesity and chronic inflammation. Altogether, metabolisms of micronutrients and macronutrients in pigs are coordinated through interdependent and mutually regulated crossing talks at tissue, cellular, biochemical, and signal levels.

FNDC5 IS THE KEY MOLECULE TO INHIBIT MUSCLE FIBER DEVELOPMENT IN TIBETAN PIGS

Differences in muscle fiber development between Tibetan and Yorkshire pigs determine their performance in terms of meat quality, taste, and food value. To investigate the molecular regulation of muscle fiber development by fibronectin type III domain-containing protein 5 (FNDC5), a preliminary study of FNDC5 gene expression in the longissimus dorsi muscle and Leg muscle tissues of Tibetan and Yorkshire Pigs was conducted using RT–qPCR and western blotting. Muscle tissues were sectioned and stained to observe the muscle fiber diameter and area under a microscope. The mRNA and protein expression levels of FNDC5, and the diameter and area of muscle fibers in the longissimus dorsi muscle and Leg muscle tissues of Tibetan pigs were significantly lower than those in Yorkshire Pigs. The results imply that FNDC5 negatively regulates muscle fiber diameter in pigs. The role of FNDC5 in pork quality has important implications for improving pork production efficiency, pork quality, and the sustainable development of the livestock industry. Keywords: Pig; FNDC5; Gene expression.

SP8.12 - Evaluating the pig tissue model for acute proctology simulation at General Surgery ST3 Bootcamp

Abstract Evaluating the pig tissue model for acute proctology simulation at General Surgery ST3 Bootcamp Background Acute proctology is an important skill for any general surgical trainee; however it is a skill which often is overlooked when it comes to training. Most, if not all teaching happens during patient procedures. Simulation in this area is underdeveloped and there is a lack of validated models for simulation. These models are also usually expensive and difficult to obtain. The aim of this study was to assess face validity of a low-cost model for teaching acute proctology during the ST3 general surgical bootcamp. Methods We created a model using a pig rectum and perineum. This provided two uses. Firstly, we were able to teach relevant clinical anatomy. Secondly, we were able to simulate EUA, rigid sigmoidoscopy, decompression of volvulus and haemorrhoidal banding. The models allowed for repeated use. Results Twenty-five trainees took part in this simulation. Overall, the trainees felt the rectal examination was realistic (76%), the sigmoidoscope moved realistically in the model (72%) as did the proctoscope (88%). In terms of ability, trainees felt the simulation improved their confidence in EUA (68%), rectal examination (72%) and banding (80%). A total of 80% of trainees thought the model was overall “Good or Excellent”. Conclusions Our study demonstrates that this low-cost, high-fidelity simulation model has a high face validity and may be explored for the use for training new general surgical registrars.

Visceral pleura mechanics: Characterization of human, pig, and rat lung material properties

Pulmonary air leaks are amongst the most common complications in lung surgery. Lung sealants are applied to the organ surface and need to synchronously stretch with the visceral pleura, the layer of tissue which encompasses the lung parenchymal tissue. These adhesives are commonly tested on pig and rat lungs, but applied to human lungs. However, the unknown mechanics of human lung visceral pleura undermines the clinical translatability of such animal-tested sealants and the absence of how pig and rat lung visceral pleura compare to human tissues is necessary to address. Here we quantify the biaxial planar tensile mechanics of visceral pleura from healthy transplant-eligible and smoker human lungs for the first time, and further compare the material behaviors to pig and rat lung visceral pleura. Initial and final stiffness moduli, maximum stress, low-to-high strain transition, and stress relaxation are analyzed and compared between and within groups, further considering regional and directional dependencies. Visceral pleura tissue from all species behave isotropically, and pig and human visceral pleura exhibits regional heterogeneity (i.e. upper versus lower lobe differences). We find that pig visceral pleura exhibits similar initial stiffness moduli and regional trends compared to human visceral pleura, suggesting pig tissue may serve as a viable animal model candidate for lung sealant testing. The outcomes and mechanical characterization of these scarce tissues enables future development of biomimetic lung sealants for improved surgical applications. Statement of significanceSurgical lung sealants must synchronously deform with the underlying tissue and with each breath to minimize post-operative air leaks, which remain the most frequent complications of pulmonary intervention. These adhesives are often tested on pig and rat lungs, but applied to humans; however, the material properties of human lung visceral pleura were previously unexplored. Here, for the first time, the mechanics of human visceral pleura tissue are investigated, further contrasting rarely acquired donated lungs from healthy and smoking individuals, and additionally, comparing biaxial planar material characterizations to animal models often employed for pulmonary sealant development. This fundamental material characterization addresses key hindrances in the advancement of biomimetic sealants and evaluates the translatability of animal model experiments for clinical applications.

Myosin Vb Traffics P-Glycoprotein to the Apical Membrane of Intestinal Epithelial Cells

The xenobiotic efflux pump P-glycoprotein is highly expressed on the apical membrane of the gastrointestinal tract, where it regulates the levels of intracellular substrates. P-glycoprotein is altered in disease, but the mechanisms that regulate the levels of P-glycoprotein are still being explored. The molecular motor myosin Vb (Myo5b) traffics diverse cargo to the apical membrane of intestinal epithelial cells. We hypothesized that Myo5b was responsible for the delivery of P-glycoprotein to the apical membrane of enterocytes. We used multiple murine models that lack functional Myo5b or the myosin binding partner Rab11a to analyze P-glycoprotein localization. Pig and human tissue were analyzed to determine P-glycoprotein localization in the setting of MYO5B mutations. Intestinal organoids were used to examine P-glycoprotein trafficking and to assay P-glycoprotein function when MYO5 is inhibited. In mice lacking Myo5b or the binding partner Rab11a, P-glycoprotein was improperly trafficked and had decreased presence in the brush border of enterocytes. Immunostaining of a pig model lacking functional Myo5b and human biopsies from a patient with an inactivating mutation in Myo5b also showed altered localization of intestinal P-glycoprotein. Human intestinal organoids expressing the motorless MYO5B tail domain had colocalization with P-glycoprotein, confirming that P-glycoprotein was trafficked by MYO5B in human enterocytes. Inhibition of MYO5 in human intestinal cell lines and organoids resulted in decreased P-glycoprotein capacity. Additionally, inhibition of MYO5 in human colon cancer cells diminished P-glycoprotein activity and increased cell death in response to a chemotherapeutic drug. Collectively, these data demonstrate that Myo5b is necessary for the apical delivery of P-glycoprotein.

Optimizing oilseed rape growth: Exploring the effect of foliar biostimulants on the interplay among metabolism, phenology, and yield.

The current agricultural system is in search of new strategies to achieve a more sustainable production while keeping or even increasing crop yield and quality. In this scenario, the application of biostimulants constitutes a potent solution. In the current study, the impact of a blue-green microalgal extract (MB) and a pig tissue hydrolysate (PTH) on rapeseed plants' development was characterized. Obtained results revealed a positive effect on yield parameters of plants treated with MB and, especially, PTH; this was associated to an improvement on the photosynthetic performance. Moreover, this study remarked the effects of biostimulants on plant phenology through their pivotal role in modulating developmental processes. More specifically, proteomic, metabolomic, and hormone content analyses revealed distinct alterations associated with the acceleration of phenology induced by biostimulant application. Additionally, some antioxidant enzymes and stress-related compounds were up-regulated upon MB and PTH treatments, indicating enhanced plant defense mechanisms in response to accelerated phenological transitions. Such findings highlight the intricate interplay between biostimulants and plant physiology, wherein biostimulants orchestrate rapid developmental changes, ultimately influencing growth dynamics. Altogether, the current study reveals that the application of both MB and PTH biostimulants promoted rapeseed plant phenology and productivity associated with an improvement in the photosynthetic machinery while boosting other physiological and molecular mechanisms.

Ssc-miR-101-3p inhibits hypoxia-induced apoptosis and inflammatory response in alveolar type-II epithelial cells of Tibetan pigs via targeting FOXO3

Tibetan pigs are a unique swine strain adapted to the hypoxic environment of the plateau regions in China. The unique mechanisms underlying the adaption by Tibetan pigs, however, are still elusive. Only few studies have investigated hypoxia-associated molecular regulation in the lung tissues of animals living in the plateau region of China. Our previous study reported that ssc-miR-101-3p expression significantly differed in the lung tissues of Tibetan pigs at different altitudes, suggesting that ssc-miR-101-3p plays an important role in the adaptation of Tibetan pigs to high altitude. To understand the underlying molecular mechanism, in this study, the target genes of ssc-miR-101-3p and their functions were analyzed via various methods including qRT-PCR and GO and KEGG pathway enrichment analyses. The action of ssc-miR-101-3p was investigated by culturing alveolar type-II epithelial cells (ATII) of Tibetan pigs under hypoxic conditions and transfecting ATII cells with vectors overexpressing or inhibiting ssc-miR-101-3p. Overexpression of ssc-miR-101-3p significantly increased the proliferation of ATII cells and decreased the expression of inflammatory and apoptotic factors. The target genes of ssc-miR-101-3p were significantly enriched in FOXO and PI3K-AKT signaling pathways required to mitigate lung injury. Further, FOXO3 was identified as a direct target of ssc-miR-101-3p. Interestingly, ssc-miR-101-3p overexpression reversed the damaging effect of FOXO3 in the ATII cells. In conclusion, ssc-miR-101-3p targeting FOXO3 could inhibit hypoxia-induced apoptosis and inflammatory response in ATII cells of Tibetan pigs. These results provided new insights into the molecular mechanisms elucidating the response of lung tissues of Tibetan pigs to hypoxic stress.

Revealing the lethal effects of Pasteurella multocida toxin on multiple organ systems.

Pasteurella multocida toxin (PMT) is one of the most important virulence factors of Pasteurella multocida type D. Pasteurella multocida infection has caused enormous economic losses in the pig farming industry. Although it is well known that this bacterial infection causes progressive atrophic rhinitis, its effects on other organ tissues in pigs are unclear. In this study, PMT was expressed and purified, and the cytotoxic effects of PMT on four types of swine cells, LLC-PK1, PAM, IPEC, and ST, were investigated. LLC-PK1 exhibited the highest sensitivity to the cytotoxic effects of PMT. Our studies revealed that a PMT concentration of 0.1 μg/kg can lead to weight loss, whereas a PMT concentration of 0.5 μg/kg can lead to death in mice. PMT causes damage to the intestines, kidneys, lungs, livers, and spleens of mice. Furthermore, PMT caused acute death in pigs at treatment concentrations greater than 5 μg/kg; at PMT concentration of 2.5 μg/kg, weight loss occurred until death. PMT mainly caused damage to the hearts, lungs, livers, spleens and kidneys of pigs. The organ coefficient showed that damage to the heart and kidneys was the most severe and caused the renal pelvis and renal pyramid to dissolve and become cavitated. Pathology revealed hemorrhage in the lungs, liver, and spleen, and the kidneys were swollen and vacuolated, which was consistent with the damaged target organs in the mice. In conclusion, these findings demonstrate that PMT is extremely toxic in vitro and in vivo, causing damage to various organs of the body, especially the kidneys and lungs. This study provides a theoretical basis for the in-depth exploration of the cytotoxic effects of PMT on target organs.

3D Humanized Bioprinted Tubulointerstitium Model to Emulate Renal Fibrosis In Vitro.

Chronic kidney disease (CKD) leads to a gradual loss of kidney function, with fibrosis as pathological endpoint, which is characterized by extracellular matrix (ECM) deposition and remodeling. Traditionally, in vivo models are used to study interstitial fibrosis, through histological characterization of biopsy tissue. However, ethical considerations and the 3Rs (replacement, reduction, and refinement) regulations emphasizes the need for humanized 3D in vitro models. This study introduces a bioprinted in vitro model which combines primary human cells and decellularized and partially digested extracellular matrix (ddECM). A protocol was established to decellularize kidney pig tissue and the ddECM was used to encapsulate human renal cells. To investigate fibrosis progression, cells were treated with transforming growth factor beta 1 (TGF-β1), and the mechanical properties of the ddECM hydrogel were modulated using vitamin B2 crosslinking. The bioprinting perfusable model replicates the renal tubulointerstitium. Results show an increased Young's modulus over time, together with the increase of ECM components and cell dedifferentiation toward myofibroblasts. Multiple fibrotic genes resulted upregulated, and the model closely resembled fibrotic human tissue in terms of collagen deposition. This 3D bioprinted model offers a more physiologically relevant platform for studying kidney fibrosis, potentially improving disease progression research and high-throughput drug screening.

Abstract We008: Hallmark Maturation of the Human Pluripotent Stem Cells-derived Cardiovascular Progenitors in Myocardial Infarcted Large Animal Model

For the goal of regenerative cardiology, cellular therapy may be used to restore injured heart muscle. The molecular mechanisms behind the engraftment and maturation of these transplanted cells, however, remain poorly understood. Thus, to obtain a comprehensive understanding of the complex transcriptional landscapes within their transplanted spatial context, we utilized spatial transcriptomics in this study. We have previously demonstrated the safety and effectiveness of transplanting our cardiovascular progenitors (CVPs) produced from laminin-221 into pig hearts that had suffered myocardial infarction (MI). Here, we undertake 10x spatial transcriptomics in chronic MI pig models at 1-, 4-, and 12 weeks post-transplantation to further our understanding of these cells in vivo. With confidence, we can distinguish between the transcripts from the pigs and the humans in the infarcted region. By using human-specific anti-Ku80 antibodies for immunohistochemical labeling of the pig tissue samples, this in silico prediction was verified. The CVPs' engraftment and longevity in the infracted region were shown by the long-term identification and staining of human cells at various time points. It is interesting to note that after 12 weeks, ribosomal and mitochondrial activity were higher than at 1 and 2 weeks. These results are consistent with the hallmarks of cardiac maturation, in which cells transit from immature fetal-like cells to mature cardiomyocytes (CMs) by switching their metabolic energy source from glucose to fatty acids. Also, by comparing the heart-specific genes, including MYH6 and MYH7, we were able to quantify the myosin isoform switches in the xenograft over time. Furthermore, to delineate the fate of the transplanted cells, we performed a ligand-receptor interaction analysis. Intriguing, we identified the midkine protein as a possible secreted human paracrine factor that enhances angiogenesis in the graft. In conclusion, we have shown the successful engraftment and maturation of human CVPs toward CMs using spatial transcriptomic technology. These data will be used to understand other aspects of regenerative cardiology such as the mechanism of VTs and in vivo cellular repair mechanisms. A shiny app atlas has been generated to share our transcriptomics data for all to explore gene expression of human transplanted cells in myocardial infarcted pig hearts.

Safety assessment of electrosurgical electrodes by using mini pig tissue

Electrosurgical electrodes are the main dissecting devices widely used for surgeries throughout the world. The present study aimed to evaluate the thermal injury and safety within animals' organs following a minimally invasive electrosurgery technique with electrosurgical electrode AE40-300 (LIPO) and AE20-80 (LIFT). To ensure the effective application of electrosurgery in a clinical environment, it is crucial to minimize heat-induced injury to nearby tissues. In this study, the skin, liver, kidney, and femoral muscle dissected from 9 minipigs were used in tissue thermal spread experiments. Thermal imaging area analysis, maximum temperature, and time to reach basal temperature were evaluated. Thermography results revealed that the surgical temperature was significantly lower in the minimally invasive electrosurgery with AE40-300 (LIPO) and AE20-80 (LIFT) compared to the predicate device. In addition, AE40-300 (LIPO) and AE20-80 (LIFT) created a relatively small thermal injury area and thermal diffusion. Our results indicated that the tested devices named AE40-300 (LIPO) and AE20-80 (LIFT) reduced excessive thermal injury and could be applied to clinical use safely.