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BACKGROUND: The intricate biological mechanism underlying lung adenocarcinoma (LUAD), characterized by a deficiency of distinctive biomarkers, remain elusive. The presence of Long non-coding RNAs (lncRNAs) have been established to play a role in carcinogenesis. Nevertheless, the regulatory effects and mechanisms of lncRNA CYTOR in LUAD have yet to be elucidated. METHODS: In this study, RT-qPCR and Western blot were adopted to examine gene mRNA and protein expression, respectively. Cell proliferation was evaluated by CCK-8 assays. Transwell was performed to assay cell migration and invasion. The function of CYTOR in vivo was investigated through a xenograft animal model. RESULTS: We observed an apparent upregulation of CYTOR in LUAD. Silencing CYTOR significantly reduced proliferation, migration, and invasion capabilities of LUAD cells. Mechanism analysis indicated that CYTOR targeted the miR-503-5p/PCSK9 axis. Additionally, inhibiting of miR-503-5p partially reversed the inhibitory effects of CYTOR silencing on the malignant progression of LUAD cells. Animal experiments revealed that CYTOR/miR-503-5p/PCSK9 curbed tumor formation of nude mice in vivo. CONCLUSION: These findings demonstrated that lncRNA CYTOR acted as an oncogene in LUAD, regulating tumor malignant progression through the miR-503-5p/PCSK9 axis. This study unveiled a new regulation mechanism of LUAD progression, offering potential therapeutic targets for LUAD.
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The quality of Ti alloy casing is crucial for the safe and stable operation of aero engines. However, the fluctuation of key process parameters during the investment casting process of titanium alloy casings has a significant influence on the volume and number of porosity defects, and this influence cannot be effectively suppressed at present. Therefore, this paper proposes a strategy to control the influence of process parameters on shrinkage volume and number. This study constructed multiple regression prediction models and neural network prediction models of porosity volume and number for a ZTC4 casing by simulating the gravity investment casting process. The results show that the multiple regression prediction model and neural network prediction model of shrinkage cavity total volume have an accuracy of over 99%. The accuracy of the neural network prediction model is higher than that of the multiple regression model, and the neural network model realizes the accurate prediction of shrinkage defect volume and defect number through pouring temperature, pouring time, and mold shell temperature. The sensitivity degree of casing defects to key process parameters, from high to low, is as follows: pouring temperature, pouring time, and mold temperature. Further optimizing the key process parameter window reduces the influence of process parameter fluctuation on the volume and number of porosity defects in casing castings. This study provides a reference for actual production control process parameters to reduce shrinkage cavity and loose defects.
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The improved wear and corrosion resistance of gray cast iron (GCI) with enhanced mechanical properties is a proven stepping stone towards the longevity of its versatile industrial applications. In this article, we have tailored the microstructural properties of GCI by alloying it with titanium (Ti) and tungsten (W) additives, which resulted in improved mechanical, wear, and corrosion resistance. The results also show the nucleation of the B-, D-, and E-type graphite flakes with the A-type graphite flake in the alloyed GCI microstructure. Additionally, the alloyed microstructure demonstrated that the ratio of the pearlite volume percentage to the ferrite volume percentage was improved from 67/33 to 87/13, whereas a reduction in the maximum graphite length and average grain size from 356 ± 31 µm to 297 ± 16 µm and 378 ± 18 µm to 349 ± 19 µm was detected. Consequently, it improved the mechanical properties and wear and corrosion resistance of alloyed GCI. A significant improvement in Brinell hardness, yield strength, and tensile strength of the modified microstructure from 213 ± 7 BHN to 272 ± 8 BHN, 260 ± 3 MPa to 310 ± 2 MPa, and 346 ± 12 MPa to 375 ± 7 MPa was achieved, respectively. The substantial reduction in the wear rate of alloyed GCI from 8.49 × 10-3 mm3/N.m to 1.59 × 10-3 mm3/N.m resulted in the upgradation of the surface roughness quality from 297.625 nm to 192.553 nm. Due to the increase in the corrosion potential from -0.5832 V to -0.4813 V, the impedance of the alloyed GCI was increased from 1545 Ohm·cm2 to 2290 Ohm·cm2. On the basis of the achieved experimental results, it is suggested that the reliability of alloyed GCI based on experimentally validated microstructural compositions can be ensured during the operation of plants and components in a severe wear and corrosive environment. It can be predicted that the proposed alloyed GCI components are capable of preventing the premature failure of high-tech components susceptible to a wear and corrosion environment.
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LGALS12, also known as galectin12, belongs to the galectin family with ß-galactoside-binding activity. We previously reported that LGALS12 is an important regulator of adipogenesis in porcine adipocytes in vitro, but its value in pig breeding needed to be explored in vivo. In this study, we used CRISPR/Cas9 to construct porcine fetal fibroblasts (PFFs) with a 43 bp deletion in LGALS12 exon 2. Using these PFFs as donor cells, a LGALS12 knockout pig model was generated via somatic cell nuclear transfer. Primary cultures of porcine intramuscular (IM) and subcutaneous (SC) adipocytes were established using cells from LGALS12 knockout pigs and wild-type pigs. A comparison of these cells proved that LGALS12 deficiency suppresses cell proliferation via the RAS-p38MAPK pathway and promotes lipolysis via the PKA pathway in both IM and SC adipocytes. In addition, we observed AKT activation only in IM adipocytes and suppression of the Wnt/ß-catenin only in SC adipocytes. Our findings suggest that LGALS12 deficiency affects the adipogenesis of IM and SC adipocytes through different mechanisms.
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Adipocitos , Sistemas CRISPR-Cas , Porcinos , Animales , Técnicas de Inactivación de Genes , Adipocitos/metabolismo , Adipogénesis/genética , Proliferación CelularRESUMEN
Lung adenocarcinoma is one of the most common cancers in the world, and accurate diagnosis of lung nodules is an important factor in reducing its mortality. In the diagnosis of pulmonary nodules, artificial intelligence (AI) assisted diagnosis technology has been rapidly developed, so testing its effectiveness is conducive to promoting its important role in clinical practice. This paper introduces the background of early lung adenocarcinoma and lung nodule AI medical imaging, and then makes academic research on early lung adenocarcinoma and AI medical imaging, and finally summarizes the biological information. In the experimental part, the relationship analysis of 4 driver genes in group X and group Y showed that there were more abnormal invasive lung adenocarcinoma genes, and the maximum uptake value and uptake function of metabolic value were also higher. However, there was no significant correlation between mutations in the four driver genes and metabolic values, and the average accuracy of AI-based medical images was 3.88% higher than that of traditional images.
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This study reports new phenomena of the interstitial fluid (ISF) microflow along perivascular and adventitial clearances (PAC) around neurovascular bundles. The fluorescent tracing was used to observe the ISF flow along the PAC of neurovascular bundles in 8-10 week old BALB/c mice. The new results include: (1) the topologic structure of the PAC around the neurovascular bundles is revealed; (2) the heart-orientated ISF flow along the PAC is observed; (3) the double-belt ISF flow along the venous adventitial clearance of the PAC is recorded; (4) the waterfall-like ISF flow induced by the small branching vessel or torn fascia along the PAC is discovered. Based on the above new phenomena, this paper approached the following objectives: (1) the kinematic laws of the ISF flow along the PAC around neurovascular bundles are set up; (2) the applicability of the hypothesis on the PAC and its subspaces by numerical simulations are examined. The findings of this paper not only enriched the image of the ISF flow through the body but also explained the kernel structure of the ISF flow (i.e., the PAC). It helps to lay the foundation for the kinematics and dynamics of the ISF flow along the PAC around neurovascular bundles.
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The TiAl47Cr2Nb2 alloy fatigue specimens were prepared by investment casting, and three kinds of surface processes were applied to fatigue specimens. These three processes were sand-blasting (SB), sand-blasting and shot-peening (SBSP) and sand-blasting and mechanical grinding (SBMG). The surface condition evolutions before and after thermal exposure at 700 °C for 24 h were investigated. The fatigue performances of specimens after thermal exposure were evaluated. The results show that the surface roughness Ra after SB, SBSP and SBMG processes were 3.14, 2.35 and 0.04 µm, respectively. After thermal exposure, they almost remained unchanged for all three processes. The SB process caused work hardening in the near-surface region and the work hardening reached saturation after the SB process. Due to the mechanical grinding (MG) process removing the uncertain thick hardening layer, the maximum hardness after SBMG process was noticeably lower than those after SB and SBSP processes. After thermal exposure, the maximum hardness after SB, SBSP and SBMG processes significantly recovered. The SBMG specimens had the highest fatigue limit of 350 MPa. This is attributed to the SBMG specimens having very smooth surfaces and some work hardening remaining near their surface layers.
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Objective: To observe the efficacy of complete video-assisted thoracoscopic surgery (CVATS) and traditional open surgery (TOS) in the treatment of elderly patients with non-small cell lung cancer (NSCLC) and their influence on cardiopulmonary function. Methods: A total of 120 elderly patients with primary NSCLC who were treated surgically in our hospital from January 2018 to January 2021 were selected and divided into the study group and the control group according to the different surgical procedures, 60 patients in each group. CVATS was used in the observation group and TOS in the control group. The surgical indexes and cardiopulmonary function indexes were observed and compared between the two groups. The serum C-reactive protein (CRP) level and visual analog scale's (VAS) score of the patients at different time points were detected. The incidence of postoperative complications was compared between the two groups. Results: The perioperative indexes such as operation time were significantly different between the two groups (p < 0.05), but the number of lymph node dissection was not significantly different (p > 0.05). The serum CRP level and VAS score of the observation group were significantly lower than those of the control group on the 1st, 3rd, and 7th postoperative days (p < 0.05). There were significant differences in cardiopulmonary function between the two groups on the 7th postoperative day (p < 0.05). The incidence of adverse reactions in the observation group was significantly lower than that in the control group (p > 0.05). Conclusion: CVATS is effective in the treatment of NSCLC. Compared with TOS therapy, CVATS has less damage to cardiopulmonary function and fewer complications, which is conducive to the rehabilitation of elderly patients. It is a safe and reliable scheme for the treatment of elderly patients with NSCLC.
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In this paper, the self-similar functional circuit models of arteries are proposed for bioinspired hemodynamic materials design. Based on the mechanical-electrical analogous method, the circuit model can be utilized to mimic the blood flow of arteries. The theoretical mechanism to quantitatively simulate realistic blood flow is developed by establishing a fractal circuit network with an infinite number of electrical components. We have found that the fractal admittance operator obtained from the minimum repeating unit of the fractal circuit can simply and directly determine the blood-flow regulation mechanism. Furthermore, according to the operator algebra, the fractal admittance operator on the aorta can be represented by Gaussian-type convolution kernel function. Similarly, the arteriolar operator can be described by Bessel-type function. Moreover, by the self-similar assembly pattern of the proposed model, biomimetic materials which contain self-similar circuits can be designed to mimic physiological or pathological states of blood flow. Studies show that the self-similar functional circuit model can efficiently describe the blood flow and provide an available and convenient structural theoretical revelation for the preparation of in vitro hemodynamic bionic materials.
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Algoritmos , Arterias/fisiología , Materiales Biocompatibles/química , Fractales , Hemodinámica , Modelos Cardiovasculares , Biomimética , Velocidad del Flujo Sanguíneo , HumanosRESUMEN
Through the continuity of the DREIDING force field, we propose, for the first time, the finite-deformation plate theory for the single-layer hexagonal boron nitride (h-BN) to clarify the atomic source of the structure against deformations. Divergent from the classical Föppl-von Karman plate theory, our new theory shows that h-BN's two in-plane mechanical parameters are independent of two out-of-plane mechanical parameters. The new theory reveals the relationships between the h-BN's elastic rigidities and the atomic force field: (1) two in-plane elastic rigidities come from the bond stretching and the bond angle bending; (2) the bending rigidity comes from the inversion angle and the dihedral angle torsion; (3) the Gaussian rigidity only comes from the dihedral angle torsion. Mechanical parameters obtained by our theory align with atomic calculations. The new theory proves that two four-body terms in the DREIDING force field are necessary to model the h-BN's mechanical properties. Overall, our theory establishes a foundation to apply the classical plate theory on the h-BN, and the approach in this paper is heuristic in modelling the mechanical properties of the other two-dimensional nanostructures.
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Interstitial fluid (ISF) flow through vascular adventitia has been discovered recently. However, its kinetic pattern was unclear. We used histological and topographical identification to observe ISF flow along venous vessels in rabbits. By magnetic resonance imaging (MRI) in live subjects, the inherent pathways of ISF flow from the ankle dermis through the legs, abdomen, and thorax were enhanced by paramagnetic contrast. By fluorescence stereomicroscopy and layer-by-layer dissection after the rabbits were sacrificed, the perivascular and adventitial connective tissues (PACTs) along the saphenous veins and inferior vena cava were found to be stained by sodium fluorescein from the ankle dermis, which coincided with the findings by MRI. The direction of ISF transport in a venous PACT pathway was the same as that of venous blood flow. By confocal microscopy and histological analysis, the stained PACT pathways were verified to be the fibrous connective tissues, consisting of longitudinally assembled fibers. Real-time observations by fluorescence stereomicroscopy revealed at least two types of spaces for ISF flow: one along adventitial fibers and another one between the vascular adventitia and its covering fascia. Using nanoparticles and surfactants, a PACT pathway was found to be accessible by a nanoparticle of <100 nm and contained two parts: a transport channel and an absorptive part. The calculated velocity of continuous ISF flow along fibers of the PACT pathway was 3.6â15.6 mm/s. These data revealed that a PACT pathway was a "slit-shaped" porous biomaterial, comprising a longitudinal transport channel and an absorptive part for imbibition. The use of surfactants suggested that interfacial tension might play an essential role in layers of continuous ISF flow along vascular vessels. A hypothetical "gel pump" is proposed based on interfacial tension and interactions to regulate ISF flow. These experimental findings may inspire future studies to explore the physiological and pathophysiological functions of vascular ISF or interfacial fluid flow among interstitial connective tissues throughout the body.
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Adventicia/fisiología , Líquido Extracelular/fisiología , Animales , Transporte Biológico , Vasos Linfáticos/fisiología , Imagen por Resonancia Magnética , ConejosRESUMEN
Although various phenomena have confirmed that surface geometry has an impact on surface energy at micro/nano scales, determining the surface energy on micro/nano curved surfaces remains a challenge. In this paper, based on Lennard-Jones (L-J) pair potential, we study the geometrical effect on surface energy with the homogenization hypothesis. The surface energy is expressed as a function of local principle curvatures. The accuracy of curvature-based surface energy is confirmed by comparing surface energy on flat surface with experimental results. Furthermore, the surface energy for spherical geometry is investigated and verified by the numerical experiment with errors within 5%. The results show that (i) the surface energy will decrease on a convex surface and increase on a concave surface with the increasing of scales, and tend to the value on flat surface; (ii) the effect of curvatures will be obvious and exceed 5% when spherical radius becomes smaller than 5 nm; (iii) the surface energy varies with curvatures on sinusoidal surfaces, and the normalized surface energy relates with the ratio of wave height to wavelength. The curvature-based surface energy offers new insights into the geometrical and scales effect at micro/nano scales, which provides a theoretical direction for designing NEMS/MEMS.
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This study aimed to explore the influences of microRNA-195 (miRNA-195)/Rap2C/MAPK in the proliferation and apoptosis of small cell lung cancer (SCLC) cells. QRT-PCR analysis were executed to evaluate miRNA-195 expression in lung cancer tissues and SCLC cells, and the western blot was implemented to monitor Rap2C protein level and uncovered whether the MAPK signaling pathway in lung cancer tissues and SCLC cells was activated. The CCK-8 experiment was performed to detect cell proliferation ability, and the flow cytometry was utilized to examine cell apoptosis level. Luciferase reporter gene system was executed to disclose the interaction between miRNA-195 and Rap2C. Subcutaneous implantation mouse models of SCLC cells were constructed to detect cell proliferation in vivo, and Kaplan-Meier method calculated patient survival. The expression of Rap2C was higher in lung cancer tissues and SCLC cells than in normal tissues and cells, while the expression of miRNA-195 was lower in lung cancer tissues and SCLC cells than in normal tissues and cells. miRNA-195 lower expression predicted showed reduced overall survival in lung cancer patients. Further loss of function and enhancement experiments revealed that miRNA-195 overexpression could significantly inhibit SCLC cell proliferation and promote cell apoptosis by upregulation of Bax and down-regulation of bcl-2; Luciferase reporter assay demonstrated that miRNA-195 could bind to Rap2C mRNA and inhibit its expression, Rap2C overexpression also related to the poorer prognosis of lung patients. Knockdown of Rap2C suppressed cell proliferation and expedited apoptosis. In addition, overexpression of Rap2C reversed miRNA-195-induced apoptosis and proliferation inhibition. Furthermore, miRNA195 prohibited the activation of MAPK signaling pathway by down-regulating Rap2C. These consequences indicated that miRNA-195 promotes the apoptosis and inhibits the proliferation of small cell lung cancer (SCLC) cells via inhibiting Rap2C protein-dependent MAPK signal transduction.
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Apoptosis/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Sistema de Señalización de MAP Quinasas , MicroARNs/genética , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Proteínas ras/metabolismo , Adulto , Anciano , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Genes Reporteros , Humanos , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Interferencia de ARN , Carcinoma Pulmonar de Células Pequeñas/patología , Carga Tumoral , Proteínas ras/genéticaRESUMEN
The regulation of porcine subcutaneous (SC) and intramuscular (IM) fat deposition significantly affects pork quality and the lean meat percentage of the carcass, respectively. The adipokine C1q/tumor necrosis factor-related protein 6 (CTRP6), plays a significant role in regulating animal fat deposition. The purpose of this study was to understand the effects of CTRP6 gene knockdown in IM and SC adipocytes by RNA-seq analysis. A total of 1830 and 2936 differentially expressed genes (DEGs) were identified in SC and IM adipocytes, respectively. 844 were down- and 2092 were upregulated in SC adipocytes, while 648 were down- and 1182 were upregulated in IM adipocytes. Furthermore, 1778 DEGs were detected only in SC adipocytes, 672 DEGs only in IM adipocytes, and 1158 DEGs in both types of adipocytes. GO analysis indicated that DEGs involved in adipocyte differentiation were significantly enriched in both SC and IM adipocytes following treatment with CTRP6-siRNA. Moreover, KEGG pathway enrichment analysis revealed differences of metabolic regulation between IM and SC adipocytes. With CTRP6-silencing, the signaling pathways related to Ras and arachidonic acid metabolism were significantly enriched in IM adipocytes, while four other signaling pathways, encompassing the TNF, MAPK, p53 and adipokine pathway were specifically enriched in SC adipocytes. Interestingly, the effect of CTRP6-siRNA treatment was attenuated by the specific Ras activator ML-097 in IM adipocytes, while the specific p53 activator SJ-172550 had the corresponding effect in SC adipocytes. Altogether, we suggest that CTRP6 may be a differential regulator of the development and metabolism of IM and SC adipose tissues.
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Adipoquinas/metabolismo , Grasa Subcutánea/metabolismo , Adipocitos/metabolismo , Adipoquinas/deficiencia , Adipoquinas/genética , Animales , Masculino , Transducción de Señal , PorcinosRESUMEN
Fluid in interstitial spaces accounts for ~20% of an adult body weight and flows diffusively for a short range. Does it circulate around the body like vascular circulations? This bold conjecture has been debated for decades. As a conventional physiological concept, interstitial space is a micron-sized space between cells and vasculature. Fluid in interstitial spaces is thought to be entrapped within interstitial matrix. However, our serial data have further defined a second space in interstitium that is a nanosized interfacial transport zone on a solid surface. Within this fine space, fluid along a solid fibre can be transported under a driving power and identically, interstitial fluid transport can be visualized by tracking the oriented fibres. Since 2006, our data from volunteers and cadavers have revealed a long-distance extravascular pathway for interstitial fluid flow, comprising at least four types of anatomic distributions. The framework of each extravascular pathway contains the longitudinally assembled and oriented fibres, working as a fibrorail for fluid flow. Interestingly, our data showed that the movement of fluid in a fibrous pathway is in response to a dynamic driving source and named as dynamotaxis. By analysis of previous studies and our experimental results, a hypothesis of interstitial fluid circulatory system is proposed.
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Transporte Biológico Activo/fisiología , Transporte Biológico/fisiología , Tejido Conectivo/metabolismo , Líquido Extracelular/metabolismo , Animales , Matriz Extracelular/metabolismo , HumanosRESUMEN
OBJECTIVE: Interstitial fluid in extracellular matrices may not be totally fixed but partially flow through long-distance oriented fibrous connective tissues via physical mechanisms. We hypothesized there is a long-distance interstitial fluid transport network beyond vascular circulations. MATERIALS AND METHODS: We first used 20 volunteers to determine hypodermic entrant points to visualize long-distance extravascular pathway by MRI. We then investigated the extravascular pathways initiating from the point of thumb in cadavers by chest compressor. The distributions and structures of long-distance pathways from extremity ending to associated visceral structures were identified. RESULTS: Using fluorescent tracer, the pathways from right thumb to right atrium wall near chest were visualized in seven of 10 subjects. The cutaneous pathways were found in dermic, hypodermic and fascial tissues of hand and forearm. The perivascular pathways were along the veins of arm, axillary sheath, superior vena cava and into the superficial tissues on right atrium. Histological and micro-CT data showed these pathways were neither blood nor lymphatic vessels but long-distance oriented fibrous matrices, which contained the longitudinally assembled micro-scale fibres consistently from thumb to superficial tissues on right atrium. CONCLUSIONS: These data revealed the structural framework of the fibrous extracellular matrices in oriented fibrous connective tissues was of the long-distance assembled fibres throughout human body. Along fibres, interstitial fluid can systemically transport by certain driving-transfer mechanisms beyond vascular circulations.
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Tejido Conectivo/metabolismo , Matriz Extracelular/metabolismo , Puntos de Acupuntura , Adulto , Cadáver , Tejido Conectivo/química , Tejido Conectivo/patología , Medios de Contraste/química , Medios de Contraste/metabolismo , Femenino , Fluoresceína/química , Fluoresceína/metabolismo , Pie/diagnóstico por imagen , Mano/diagnóstico por imagen , Humanos , Vasos Linfáticos/anatomía & histología , Vasos Linfáticos/diagnóstico por imagen , Imagen por Resonancia Magnética , Masculino , Microscopía Confocal , Puntos Cuánticos/química , Puntos Cuánticos/metabolismo , Adulto JovenRESUMEN
BACKGROUND: Livestock production aims to provide meats of high and consistent eating quality. Insufficient intramuscular (IM) fat and excessive subcutaneous (SC) fat are paramount pork quality challenges. IM fat and SC fat, which are modulated by the adipogenesis of IM and SC adipocytes, play key roles in pork quality. Galectin-12 (LGALS12) was proven to be an important regulator of fat deposition in porcine. However, the current knowledge of the transcriptome-wide role of LGALS12 in adipocytes is still limited. This study was aimed to discover the different regulatory mechanisms of LGALS12 in porcine IM and SC adipocyte. RESULTS: The siRNA-mediated knockdown of the expression of LGALS12 identified 1075 and 3016 differentially expressed genes (DEGs) in IM and SC adipocytes, respectively. Among these, 585 were up- and 490 were downregulated in the IM adipocytes, while 2186 were up- and 830 were downregulated in the SC adipocytes. Moreover, 418 DGEs were observed only in the IM adipocytes, 2359 DGEs only in the SC adipocytes, and 657 DGEs in both types of adipocytes. According to Gene Ontology (GO) analysis, DEGs in both IM and SC adipocytes were mainly enriched in categories related to lipids or fat cell differentiation. Pathway analysis of the DEGs revealed 88 changed signaling pathways in the IM adipocytes and 86 in the SC adipocytes. The signaling pathways present in only one type of adipocyte were identified from among the top 50 signaling pathways in each type of adipocyte. Four signaling pathways, encompassing PI3K-AKT, cardiac muscle contraction, fatty acid metabolism and Ras, were significantly enriched in the IM adipocytes. On the other hand, four different signaling pathways, encompassing TNF, WNT, cGMP-PKG and NF-kappa B, were greatly enriched in the SC ones. The pathway changes were confirmed by chemical inhibition assays. CONCLUSIONS: Our data reveals that LGALS12 knockdown alters the expression of numerous genes involved in key biological processes in the development of adipocytes. These observations provide a global view of the role of LGALS12 in porcine IM and SC adipocytes; thus, improving our understanding of the regulatory mechanisms by which this gene acts in fat development.
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Adipocitos/metabolismo , Galectinas/genética , Perfilación de la Expresión Génica , Músculos/citología , Grasa Subcutánea/citología , Porcinos/metabolismo , Animales , Galectinas/deficiencia , Regulación de la Expresión Génica/genética , Ontología de Genes , Silenciador del Gen , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Melanocortin 3 receptor (MC3R), a rhodopsin-like G protein-coupled receptor, is an important regulator of metabolism. Although MC3R knock-out (KO) mice and rats were generated in earlier studies, the function of MC3R remains elusive. Since pig models have many advantages over rodents in metabolism research, we generated an MC3R-KO pig using a CRSPR/Cas9-based system combined with somatic cell nuclear transfer (SCNT) technology. METHOD: Four CRSPR/Cas9 target vectors were constructed and then their cleavage efficiency was tested in porcine fetal fibroblasts (PFFs). The pX330-sgRNA1 and pX330-sgRNA4 vectors were used to co-transfect PFFs to obtain positive colonies. PCR screening and sequencing were conducted to identify the genotype of the colonies. The biallelically modified colonies and wild-type control colonies were used simultaneously as donor cells for SCNT. A total of 1203 reconstructed embryos were transferred into 6 surrogates, of which one became pregnant. The genotypes of the resulting piglets were determined by PCR and sequencing, and off-target effects in the MC3R KO piglets were detected by sequencing. Then, offspring were obtained through breeding and six male KO pigs were used for the growth performance analysis. RESULTS: Four vectors were constructed successfully, and their cleavage efficiencies were 27.96, 44.89, 32.72 and 38.86%, respectively. A total of 21 mutant colonies, including 11 MC3R-/- and 10 MC3R+/- clones, were obtained, corresponding to a gene targeting efficiency of 29.17%, with 15.28% biallelic mutations. A total of 6 piglets were born, and only two MC3R KO piglets were generated, one with malformations and a healthy one. No off-target effects were detected by sequencing in the healthy mutant. Six male MC3R KO pigs were obtained in the F2 generation and their body weight and body fat were both increased compared to wild-type full siblings. CONCLUSION: A MC3R KO pig strain was generated using the CRSIPR/Cas9-based system, which makes it possible to study the biological function of MC3R in a non-rodent model.
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Sistemas CRISPR-Cas/genética , Técnicas de Inactivación de Genes , Técnicas de Transferencia Nuclear , Receptor de Melanocortina Tipo 3/deficiencia , Adiposidad , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Peso Corporal , Feto/citología , Fibroblastos/metabolismo , Marcación de Gen , Vectores Genéticos/metabolismo , ARN Guía de Kinetoplastida/metabolismo , Receptor de Melanocortina Tipo 3/metabolismo , PorcinosRESUMEN
Biological nanochannels control the movements of different ions through cell membranes depending on not only those channels' static inherent configurations, structures, inner surface's physicochemical properties but also their dynamic shape changes, which are required in various essential functions of life processes. Inspired by ion channels, many artificial nanochannel-based membranes for nanofluidics and biosensing applications have been developed to regulate ionic transport behaviors by using the functional molecular modifications at the inner surface of nanochannel to achieve a stimuli-responsive layer. Here, the concept of a dynamic nanochannel system is further developed, which is a new way to regulate ion transport in nanochannels by using the dynamic change in the curvature of channels to adjust ionic rectification in real time. The dynamic curvature nanochannel-based membrane displays the advanced features of the anomalous effect of voltage, concentration, and ionic size for applying simultaneous control over the curvature-tunable asymmetric and reversible ionic rectification switching properties. This dynamic approach can be used to build smart nanochannel-based systems, which have strong implications for flexible nanofluidics, ionic rectifiers, and power generators.