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Tumorigenesis and metastasis are highly dependent on the interactions between the tumor and the surrounding microenvironment. In 3D matrix, the fibrous structure of the extracellular matrix (ECM) undergoes dynamic remodeling during tumor progression. In particular, during the late stage of tumor development, the fibers become more aggregated and oriented. However, it remains unclear how cancer cells respond to the organizational change of ECM fibers and exhibit distinct morphology and behavior. Here, we used electrospinning technology to fabricate biomimetic ECM with distinct fiber arrangements, which mimic the structural characteristics of normal or tumor tissues and found that aligned and oriented nanofibers induce cytoskeletal rearrangement to promote directed migration of cancer cells. Mechanistically, caveolin-1(Cav-1)-expressing cancer cells grown on aligned fibers exhibit increased integrin β1 internalization and actin polymerization, which promoted stress fiber formation, focal adhesion dynamics and YAP activity, thereby accelerating the directional cell migration. In general, the linear fibrous structure of the ECM provides convenient tracks on which tumor cells can invade and migrate. Moreover, histological data from both mice and patients with tumors indicates that tumor tissue exhibits a greater abundance of isotropic ECM fibers compared to normal tissue. And Cav-1 downregulation can suppress cancer cells muscle invasion through the inhibition of YAP-dependent mechanotransduction. Taken together, our findings revealed the Cav-1 is indispensable for the cellular response to topological change of ECM, and that the Cav-1/YAP axis is an attractive target for inhibiting cancer cell directional migration which induced by linearization of ECM fibers.

Abstract Introduction Exercise enhances one's health and competitiveness. A strong physical fitness status can pave the way for a promising future. This study presents the time-based trends in physical fitness indicators—including height, weight, BMI, lung capacity, dash, long-distance running, and standing long jump—among medical undergraduates during their university years. Additionally, we analyzed the impact of students' physical fitness on their career paths. Method We conducted a retrospective database study by collecting physical fitness test data and career paths information for 634 medical students from a university in southwestern China. These students graduated in 2022. The career paths included pursuits in further studies, employment, and unemployment. To detect differences in these aspects, we used the t-test and Chi-square test. Results Our study indicates a significant declining trend in the physical fitness of medical students during their university years. The changes observed between the first and fourth tests are as follows: Weight (kg): 58.52 ± 10.48 to 60.73 ± 12.07, P

Abstract Background Kaposiform hemangioendothelioma (KHE) is a rare intermediate vascular tumor with unclear pathogenesis. Recently, three dimensional (3D) cell spheroids and organoids have played an indispensable role in the study of many diseases, such as infantile hemangioma and non-involuting congenital hemangiomas. However, few research on KHE are based on the 3D model. This study aims to evaluate the 3D superiority, the similarity with KHE and the ability of drug evaluation of EOMA spheroids as an in vitro 3D KHE model. Results After two days, relatively uniform morphology and high viability of EOMA spheroids were generated by the rotating cell culture system (RCCS). Through transcriptome analysis, compared with 2D EOMA cells, focal adhesion-related genes such as Itgb4, Flt1, VEGFC, TNXB, LAMA3, VWF, and VEGFD were upregulated in EOMA spheroids. Meanwhile, the EOMA spheroids injected into the subcutaneous showed more obvious KMP than 2D EOMA cells. Furthermore, EOMA spheroids possessed the similar characteristics to the KHE tissues and subcutaneous tumors, such as diagnostic markers (CD31 and LYVE-1), cell proliferation (Ki67), hypoxia (HIF-1α) and cell adhesion (E-cadherin and N-cadherin). Based on the EOMA spheroid model, we discovered that sirolimus, the first-line drug for treating KHE, could inhibit EOMA cell proliferation and downregulate the VEGFC expression. Through the extra addition of VEGFC, the effect of sirolimus on EOMA spheroid could be weakened. Conclusion With a high degree of similarity of the KHE, 3D EOMA spheroids generated by the RCCS can be used as a in vitro model for basic researches of KHE, generating subcutaneous tumors and drug screening.

Introduction: Gene-edited pigs have become prominent models for studying human disease mechanisms, gene therapy, and xenotransplantation. CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-associated 9 (CRISPR/Cas9) technology is a widely employed tool for generating gene-edited pigs. Nevertheless, delivering CRISPR/Cas9 to pre-implantation embryos has traditionally posed challenges due to its reliance on intricate micromanipulation equipment and specialized techniques, resulting in high costs and time-consuming procedures. This study aims to introduce a novel one-step approach for generating genetically modified pigs by transducing CRISPR/Cas9 components into pre-implantation porcine embryos through oviductal injection of recombinant adeno-associated viruses (rAAV).Methods: We first used rAAV-1, rAAV-6, rAAV-8, rAAV-9 expressing EGFP to screen for rAAV serotypes that efficiently target porcine embryos, and then, to achieve efficient expression of CRISPR/Cas9 in vivo for a short period, we packaged sgRNAs targeting the GHR genes to self-complementary adeno-associated virus (scAAV), and Cas9 proteins to single-stranded adeno-associated virus (ssAAV). The efficiency of porcine embryos -based editing was then validated in vitro. The feasibility of this one-step method to produce gene-edited pigs using rAAV-CRISPR/Cas9 oviductal injection into sows within 24 h of conception was then validated.Results: Our research firstly establishes the efficient delivery of CRISPR/Cas9 to pig zygotes, both in vivo and in vitro, using rAAV6. Successful gene editing in pigs was achieved through oviductal injection of rAAV-CRISPR/Cas9.Conclusion: This method circumvents the intricate procedures involved in in vitro embryo manipulation and embryo transfers, providing a straightforward and cost-effective approach for the production of gene-edited pigs.

Abstract Xenotransplantation is considered a solution for the shortage of organs, and pigs play an indispensable role as donors in xenotransplantation. The biosecurity of pigs, especially the zoonotic viruses carried by pigs, has attracted attention. This review introduces several viruses, including porcine endogenous retroviruses that are integrated into the pig genome in a DNA form, herpesviruses that have been proven to clearly affect recipient survival time in previous xenotransplant surgeries, the zoonotic hepatitis E virus, and the widely distributed porcine circoviruses. The detail virus information, such as structure, caused diseases, transmission pathways, and epidemiology was introduced in the current review. Diagnostic and control measures for these viruses, including detection sites and methods, vaccines, RNA interference, antiviral pigs, farm biosecurity, and drugs, are discussed. The challenges faced, including those posed by other viruses and newly emerged viruses, and the challenges brought by the modes of transmission of the viruses are also summarized.

Multicellular three-dimensional (3D) in vitro models, such as cell spheroids and organoids, can significantly improve the viability, histomorphology, genotype stability, function and drug metabolism of cells [1–3]. In general, several culture methods of 3D models, including the hanging drop, microwell-mesh and hydrogel encapsulating methods, have difficulty building a standard mode and controlling the size and arrangement of cell spheroids or organoids, which could severely affect the authenticity and repeatability of experimental results [4]. Another key factor in 3D in vitro models is the extracellular matrix (ECM), which can determine cell viability, proliferation, differentiation, function, migration and organization [5]. In this study, micropattern array chips combined with decellularized ECM (dECM) not only provide tissue-specific ECM but also control the size and arrangement of 3D models. • Methods have been established to demonstrate the use of dECM as a bioink to generate dECM-coated micropattern array chips by microcontact printing. • The micropattern can limit cell growth and migration, and cells spontaneously assemble into cell spheroids with uniform size and orderly arrangement.

In the world’s first pig-to-human cardiac cytomegalovirus (PCMV), xenotransplant and elevated levels of porcine key factors contributing to patient mortality were considered. This has renewed attention on PCMV, a virus widely prevalent in pigs. Currently, there are no effective drugs or vaccines targeting PCMV, and its high detection difficulty poses challenges for prevention and control research. In this study, antiviral small hairpin RNA (shRNA) was selected and inserted into the Rosa26 and miR-17-92 loci of pigs via a CRISPR/Cas9-mediated knock-in strategy. Further in vitro viral challenge experiments demonstrated that these genetically edited pig cells could effectively limit PCMV replication. Through this process, we constructed a PCMV-infected cell model, validated partial viral interference sites, enhanced gene knock-in efficiency, performed gene editing at two different gene loci, and ultimately demonstrated that RNA interference (RNAi) technology combined with CRISPR/Cas9 has the potential to generate pig cells with enhanced antiviral infection capabilities. This opens up possibilities for the future production of pig populations with antiviral functionalities.

Globally, gastric cancer is the third leading cause of death from tumors. Prevention and individualized treatment are considered to be the best options for reducing the mortality rate of gastric cancer. Artificial intelligence (AI) technology has been widely used in the field of gastric cancer, including diagnosis, prognosis, and image analysis. Eligible papers were identified from PubMed and IEEE up to April 13, 2022. Through the comparison of these articles, the application status of AI technology in the diagnosis of gastric cancer was summarized, including application types, application scenarios, advantages and limitations. This review presents the current state and role of AI in the diagnosis of gastric cancer based on four aspects: 1) accurate sampling from early diagnosis (endoscopy), 2) digital pathological diagnosis, 3) molecules and genes, and 4) clinical big data analysis and prognosis prediction. AI plays a very important role in facilitating the diagnosis of gastric cancer; however, it also has shortcomings such as interpretability. The purpose of this review is to provide assistance to researchers working in this domain.

Aims: This study aims to evaluate ECM-coated micropattern arrays derived from decellularization of native porcine lungs as a novel three-dimensional cell culture platform. Methods: ECM derived from decellularization of native porcine lungs was exploited to prepare hydrogels. Then, dECM-coated micropattern arrays were fabricated at four different diameters (50, 100, 150 and 200 ​μm) using polydimethylsiloxane (PDMS). Two lung cancer cell lines, A549 and H1299, were tested on a dECM-coated micropattern array as a novel culture platform for cell adhesion, distribution, proliferation, viability, phenotype expression, and drug screening to evaluate the cytotoxicity of paclitaxel, doxorubicin and cisplatin. Results: The ECM derived from decellularization of native porcine lungs supported cell adhesion, distribution, viability and proliferation better than collagen I and Matrigel as the coated matrix on the surface. Moreover, the optimal diameter of the micropattern arrays was 100–150 ​μm, as determined by measuring the morphology, viability, proliferation and phenotype of the cancer cell spheroids. Cell spheroids of A549 and H1299 on dECM-coated micropattern arrays showed chemoresistance to anticancer drugs compared to that of the monolayer. The different distributions of HIF-1α, MCL-1 (in the center) and Ki-67 and MRP2 (in the periphery) of the spheroids demonstrated the good establishment of basal-lateral polarity and explained the chemoresistance phenomenon of spheroids. Conclusions: This novel three-dimensional cell culture platform is stable and reliable for anticancer drug testing. Drug screening in dECM-coated micropattern arrays provides a powerful alternative to existing methods for drug testing and metabolic profiling in the drug discovery process.

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems can precisely manipulate DNA sequences to change the characteristics of cells and organs, which has potential in the mechanistic research on genes and the treatment of diseases. However, clinical applications are restricted by the lack of safe, targeted and effective delivery vectors. Extracellular vesicles (EVs) are an attractive delivery platform for CRISPR/Cas9. Compared with viral and other vectors, EVs present several advantages, including safety, protection, capacity, penetrating ability, targeting ability and potential for modification. Consequently, EVs are profitably used to deliver the CRISPR/Cas9 in vivo. In this review, the advantages and disadvantages of the delivery form and vectors of the CRISPR/Cas9 are concluded. The favorable traits of EVs as vectors, such as the innate characteristics, physiological and pathological functions, safety and targeting ability of EVs, are summarized. Furthermore, in terms of the delivery of the CRISPR/Cas9 by EVs, EV sources and isolation strategies, the delivery form and loading methods of the CRISPR/Cas9 and applications have been concluded and discussed. Finally, this review provides future directions of EVs as vectors of the CRISPR/Cas9 system in clinical applications, such as the safety, capacity, consistent quality, yield and targeting ability of EVs.

Immunohistochemistry (IHC) plays an important role in evaluating the status of ER, PR, Ki-67 and human epidermal growth factor receptor 2 (HER-2) during diagnosis of breast cancer. Although some existing automated approaches can solve the high time-consumption and inter-/intra-observer variability drawbacks to a certain extent, most of them are can’t analyze both nuclear staining and cell membrane staining using the same method. This is attributed to the difference in localization of the positive signal of immunohistochemical staining in different biological markers. The present study proposes a novel automated image analysis model for scoring and grading of ER, PR, Ki-67 and HER-2 immunohistochemical images based on whole tissue sections in breast cancer. The scoring results of the trained model and manual interpretation of ER, PR, Ki-67 and HER-2 were then finally analyzed and compared. Experimental results show that the F1-measure was 0.8450, 0.8533 and 0.7962 for nuclear recognition of Ki-67, ER/PR and HER-2 respectively. For stain grading of Ki-67, ER/PR and HER-2, the F1-measure was 0.9776, 0.8306 and 0.9573 respectively. The scoring consistency of ER/PR, Ki-67 and HER-2 between our model and expert interpretation was 0.9279, 0.9712 and 0.8046 respectively. Our results demonstrate that artificial intelligence technology is a feasible and accurate method for accurate quantitative immunohistochemical analysis that can solve the drawbacks of low repeatability and time consumption brought by manual counting. The main contribution of our proposed model is that it can recognize both nuclear staining and cell membrane staining and grade the staining intensity as a sequential learning task.

Abstract Background The scoring of Ki-67 is highly relevant for the diagnosis, classification, prognosis, and treatment in breast invasive ductal carcinoma (IDC). Traditional scoring method of Ki-67 staining followed by manual counting, is time-consumption and inter−/intra observer variability, which may limit its clinical value. Although more and more algorithms and individual platforms have been developed for the assessment of Ki-67 stained images to improve its accuracy level, most of them lack of accurate registration of immunohistochemical (IHC) images and their matched hematoxylin-eosin (HE) images, or did not accurately labelled each positive and negative cell with Ki-67 staining based on whole tissue sections (WTS). In view of this, we introduce an accurate image registration method and an automatic identification and counting software of Ki-67 based on WTS by deep learning. Methods We marked 1017 breast IDC whole slide imaging (WSI), established a research workflow based on the (i) identification of IDC area, (ii) registration of HE and IHC slides from the same anatomical region, and (iii) counting of positive Ki-67 staining. Results The accuracy, sensitivity, and specificity levels of identifying breast IDC regions were 89.44, 85.05, and 95.23%, respectively, and the contiguous HE and Ki-67 stained slides perfectly registered. We counted and labelled each cell of 10 Ki-67 slides as standard for testing on WTS, the accuracy by automatic calculation of Ki-67 positive rate in attained IDC was 90.2%. In the human-machine competition of Ki-67 scoring, the average time of 1 slide was 2.3 min with 1 GPU by using this software, and the accuracy was 99.4%, which was over 90% of the results provided by participating doctors. Conclusions Our study demonstrates the enormous potential of automated quantitative analysis of Ki-67 staining and HE images recognition and registration based on WTS, and the automated scoring of Ki67 can thus successfully address issues of consistency, reproducibility and accuracy. We will provide those labelled images as an open-free platform for researchers to assess the performance of computer algorithms for automated Ki-67 scoring on IHC stained slides.

Quantitative analysis of immunohistochemically stained breast cancer specimens by cell counting is important for prognosis and treatment planning. This paper presents a robust, accurate, and novel method to label immunopositive and immunonegative cells automatically. During preprocessing, we developed an adaptive method to correct the colour aberration caused by imaging conditions. Next, a pixel-level segmentation was performed on preprocessed images using a support vector machine with a radial basis function kernel in HSV colour space. The segmentation result was processed by mathematical morphology operations to correct error-segmented regions and extract the marker for each cell. Validation studies showed that the automated cell-counting method had divergences varying from –5.05% to 3.99% compared with manual counting by a pathologist, indicating considerable agreement of the present automated cell counting method with manual counting. Thus, this method can free pathologists from laborious work and can potentially improve the accuracy and the reproducibility of diagnosis.

Infantile hemangioma (IH) is the most prevalent type of vascular tumor in infants. The pathophysiology of IH is unknown. The tissue structure and physiology of two-dimensional cell cultures differ greatly from those in vivo, and spontaneous regression often occurs during tumor formation in nude mice and has severely limited research into the pathogenesis and development of IH. By decellularizing porcine aorta, we attempted to obtain vascular-specific extracellular matrix as the bioink for fabricating micropattern arrays of varying diameters via microcontact printing. We then constructed IH-derived CD31+ hemangioma endothelial cell three-dimensional microtumor models. The vascular-specific and decellularized extracellular matrix was suitable for the growth of infantile hemangioma-derived endothelial cells. The KEGG signaling pathway analysis revealed enrichment primarily in stem cell pluripotency, RAS, and PI3KAkt compared to the two-dimensional cell model according to RNA sequencing. Propranolol, the first-line medication for IH, was also used to test the model’s applicability. We also found that metformin had some impact on the condition. The three-dimensional microtumor models of CD31+ hemangioma endothelial cells were more robust and efficient experimental models for IH mechanistic exploration and drug screening.

Long-term culture of hepatocyte spheroids with high ammonia clearance is valuable for therapeutic applications, especially the bioartificial liver. However, the optimal conditions are not well studied. We hypothesized that liver urea cycle enzymes can be induced by high protein diet and maintain on a higher expression level in rat hepatocyte spheroids by serum-free medium (SFM) culture and coculture with mesenchymal stromal cells (MSCs). Rats were feed normal protein diet (NPD) or high protein diet (HPD) for 7 days before liver digestion and isolation of hepatocytes. Hepatocyte spheroids were formed and maintained in a rocked suspension culture with or without MSCs in SFM or 10% serum-containing medium (SCM). Spheroid viability, kinetics of spheroid formation, hepatic functions, gene expression, and biochemical activities of rat hepatocyte spheroids were tested over 14 days of culture. We observed that urea cycle enzymes of hepatocyte spheroids can be induced by high protein diet. SFM and MSCs enhanced ammonia clearance and ureagenesis and stabilized integrity of hepatocyte spheroids compared to control conditions over 14 days. Hepatocytes from high protein diet-fed rats formed spheroids and maintained a high level of ammonia detoxification for over 14 days in a novel SFM. Hepatic functionality and spheroid integrity were further stabilized by coculture of hepatocytes with MSCs in the spheroid microenvironment. These findings have direct application to development of the spheroid reservoir bioartificial liver.

Innovative cell-based therapies, including hepatic tissue engineering following hepatocyte transplantation, are considered as theoretical alternatives to liver transplant or for partial replacement of liver function in patients. However, recent progress in hepatic tissue engineering has been hampered by low initial hepatocyte engraftment and insufficient blood supply in vivo. We developed an intact 3D scaffold of an extracellular matrix (ECM) derived from a decellularized liver lobe, with layer-by-layer (LbL) heparin deposition to avoid thrombosis, which we repopulated with hepatocytes and successfully implanted as a tissue-engineered liver (TEL) into the portal system. The TEL provided sufficient volume for transplantation of cell numbers representing up to 10% of whole-liver equivalents and was perfused by portal vein blood. Treatment of extended hepatectomized rats with a TEL improved liver function and prolonged survival; mean lifespan was extended from 16 to 72 h. At 72 h postoperation, the TEL sustained functional and viable hepatocytes. In conclusion, we propose the TEL as a state-of-the-art substitute for whole-liver transplantation and as a proof of concept for the technology that will eventually allow for the transplantation of a reconstituted liver.

C33H28N4O2, monoclinic, P21/c (no. 14), a = 10.263(8) Å, b = 23.48(2) Å, c = 12.28(1) Å, β = 114.498(9)°, V = 2692.9 Å3, Z = 4, Rgt(F) = 0.0451, wRref(F2) = 0.1189, T = 296 K.

Aging is an irresistible natural law of the progressive decline of body molecules, organs, and overall function with the passage of time, resulting in eventual death. World Health Organization data show that aging is correlated with a wide range of common chronic diseases in the elderly, and is an essential driver of many diseases. Panax Ginseng C.A Meyer is an ancient herbal medicine, which has an effect of "long service, light weight, and longevity" recorded in the ancient Chinese medicine book "Compendium of Materia Medica." Ginsenoside Rg2, the main active ingredient of ginseng, also exerts a marked effect on the treatment of liver injury. However, it remains unclear whether Rg2 has the potential to ameliorate aging-induced liver injury. Hence, exploring the hepatoprotective properties of Rg2 and its possible molecular mechanism by Senescence Accelerate Mouse Prone 8 (SAMP8) and gut microbiota. Our study demonstrated that Rg2 can inhibit pyroptosis and apoptosis through caspase 8, and regulate the gut-liver axis to alleviate liver inflammation by changing the composition of gut microbiota, thus improving aging-induced liver injury. These findings provide theoretical support for the pharmacological effects of ginsenosides in delaying aging-induced liver injury. [ABSTRACT FROM AUTHOR]