The role of extracellular vesicles in animal reproduction and diseases

Extracellular vesicles(EVs)are nanosized membrane-enclosed compartments that serve as messengers in cell-to-cell communication,both in normal physiology and in pathological conditions.EVs can transfer functional proteins and genetic information to alter the phenotype and function of recipient cells,which undergo different changes that positively affect their structural and functional integrity.Biological fluids are enriched with several subpopulations of EVs,including exosomes,microvesicles(MVs),and apoptotic bodies carrying several cargoes,such as lipids,proteins,and nucleic acids.EVs associated with the reproductive system are actively involved in the regulation of different physiological events,including gamete maturation,fertilization,and embryo and fetal development.EVs can influence follicle development,oocyte maturation,embryo production,and endometrial-conceptus communication.EVs loaded with cargoes are used to diagnose various diseases,including pregnancy disorders;however,these are dependent on the type of cell of origin and pathological characteristics.EV-derived microRNAs(miRNAs)and proteins in the placenta regulate inflammatory responses and trophoblast invasion through intercellular delivery in the placental microenvironment.This review presents evidence regarding the types of extracellular vesicles,and general aspects of isolation,purification,and characterization of EVs,particularly from various types of embryos.Further,we discuss EVs as mediators and messengers in reproductive biology,the effects of EVs on placentation and pregnancy disorders,the role of EVs in animal reproduction,in the male reproductive system,and mother and embryo cross-communication.In addition,we emphasize the role of microRNAs in embryo implantation and the role of EVs in reproductive and therapeutic medicine.Finally,we discuss the future perspectives of EVs in reproductive biology.

Amylase intrapancreatic infusion delays insulin release during an intravenous glucose tolerance test,proof of acini–islet–acinar interactions

BACKGROUND The possible existence of an acini–islet–acinar(AIA)reflex,involving mutual amylase and insulin interactions,was investigated in the current acute experiment on pigs.AIM To confirm the existence of an AIA reflex and justify the placement of the exocrine and endocrine pancreatic components within the same organ.METHODS The study was performed on six pigs under general anesthesia.An intravenous glucose tolerance test was performed,with a bolus infusion of 50%glucose to the jugular vein,while amylase(5000 U/kg)or vehicle intrapancreatic infusions were administered via the pancreaticoduodenalis cranialis artery during 30 min with a 1 mL/min flow rate.RESULTS The amylase infusion to pancreatic arterial circulation inhibited and delayed the insulin release peak which is usually associated with the highest value of blood glucose and is typically observed at 15 min after glucose infusion,for>1 h.The intrapancreatic infusion of the vehicle(saline)did not have any effect on the time frame of insulin release.Infusion of 1%bovine serum albumin changed the insulin release curve dramatically and prolonged the high range of insulin secretion,far beyond the glucose peak.CONCLUSION Intrapancreatic arterial infusion of amylase interrupted the integrated glucose–insulin interactions.This confirms an AIA reflex and justifies placement of the exocrine and endocrine pancreatic components within the same organ.

Current trends in three-dimensional visualization and real-time navigation as well as robot-assisted technologies in hepatobiliary surgery

With the continuous development of digital medicine,minimally invasive precision and safety have become the primary development trends in hepatobiliary surgery.Due to the specificity and complexity of hepatobiliary surgery,traditional preoperative imaging techniques such as computed tomography and magnetic resonance imaging cannot meet the need for identification of fine anatomical regions.Imaging-based three-dimensional(3D)reconstruction,virtual simulation of surgery and 3D printing optimize the surgical plan through preoperative assessment,improving the controllability and safety of intraoperative operations,and in difficult-to-reach areas of the posterior and superior liver,assistive robots reproduce the surgeon’s natural movements with stable cameras,reducing natural vibrations.Electromagnetic navigation in abdominal surgery solves the problem of conventional surgery still relying on direct visual observation or preoperative image assessment.We summarize and compare these recent trends in digital medical solutions for the future development and refinement of digital medicine in hepatobiliary surgery.

Double knock-in pig models with elements of binary Tet-On and phiC31 integrase systems for controllable and switchable gene expression

Inducible expression systems are indispensable for precise regulation and in-depth analysis of biological process.Binary Tet-On system has been widely employed to regulate transgenic expression by doxycycline.Previous pig models with tetracycline regulatory elements were generated through random integration.This process often resulted in uncertain expression and unpredictable phenotypes,thus hindering their applications.Here,by precise knock-in of binary Tet-On 3G elements into Rosa26 and Hipp11 locus,respectively,a double knock-in reporter pig model was generated.We characterized excellent properties of this system for controllable transgenic expression both in vitro and in vivo.Two att P sites were arranged to flank the td Tomato to switch reporter gene.Single or multiple gene replacement was efficiently and faithfully achieved in fetal fibroblasts and nuclear transfer embryos.To display the flexible application of this system,we generated a pig strain with Dox-inducing h KRASexpression through phiC31 integrase-mediated cassette exchange.After eight months of Dox administration,squamous cell carcinoma developed in the nose,mouth,and scrotum,which indicated this pig strain could serve as an ideal large animal model to study tumorigenesis.Overall,the established pig models with controllable and switchable transgene expression system will provide a facilitating platform for transgenic and biomedical research.

Hepatitis B virus infection modeling using multi-cellular organoids derived from human induced pluripotent stem cells

Chronic infection with hepatitis B virus(HBV)remains a global health concern despite the availability of vaccines.To date,the development of effective treatments has been severely hampered by the lack of reliable,reproducible,and scalable in vitro modeling systems that precisely recapitulate the virus life cycle and represent virus-host interactions.With the progressive understanding of liver organogenesis mechanisms,the development of human induced pluripotent stem cell(iPSC)-derived hepatic sources and stromal cellular compositions provides novel strategies for personalized modeling and treatment of liver disease.Further,advancements in three-dimensional culture of self-organized liver-like organoids considerably promote in vitro modeling of intact human liver tissue,in terms of both hepatic function and other physiological characteristics.Combined with our experiences in the investigation of HBV infections using liver organoids,we have summarized the advances in modeling reported thus far and discussed the limitations and ongoing challenges in the application of liver organoids,particularly those with multi-cellular components derived from human iPSCs.This review provides general guidelines for establishing clinical-grade iPSC-derived multi-cellular organoids in modeling personalized hepatitis virus infection and other liver diseases,as well as drug testing and transplantation therapy.

In vivo evaluation of guide-free Cas9-induced safety risks in a pig model

The CRISPR/Cas9 system has shown great potential for treating human genetic diseases through gene therapy.However,there are concerns about the safety of this system,specifically related to the use of guide-free Cas9.Previous studies have shown that guidefree Cas9 can induce genomic instability in vitro.However,the in vivo safety risks associated with guide-free Cas9 have not been evaluated,which is necessary for the development of gene therapy in clinical settings.In this study,we used doxycycline-inducible Cas9-expressing pigs to evaluate the safety risks of guide-free Cas9 in vivo.Our findings demonstrated that expression of guide-free Cas9 could induce genomic damages and transcriptome changes in vivo.The severity of the genomic damages and transcriptome changes were correlate with the expression levels of Cas9 protein.Moreover,prolonged expression of Cas9 in pigs led to abnormal phenotypes,including a significant decrease in body weight,which may be attributable to genomic damage-induced nutritional absorption and metabolic dysfunction.Furthermore,we observed an increase in whole-genome and tumor driver gene mutations in pigs with long-term Cas9 expression,raising the risk of tumor occurrence.Our in vivo evaluation of guide-free Cas9 in pigs highlights the necessity of considering and monitoring the detrimental effects of Cas9 alone as genome editing via the CRISPR/Cas9 system is implemented in clinical gene therapy.This research emphasizes the importance of further study and implementation of safety measures to ensure the successful and safe application of the CRiSPR/Cas9 system in clinical practice.

Multiple gene modifications of pigs for overcoming obstacles of xenotransplantation

Xenotransplantation,involving animal organ transplantation into humans to address the human organ shortage,has been studied since the 17th century.Early attempts to obtain organs from animals such as goats,dogs,and non-human primates proved unsuccessful.In the 1990s,scientists agreed that pigs were the most suitable donor animals for xenotransplantation.However,immune rejection between pig and human has hindered the application.To overcome these challenges,researchers developed genetically modified pigs that deactivate xenoreactive antigen genes and express human protective genes.These advances extended xenograft survival from days to years in non-human primates,resulting in the first human heart xenotransplant trial.Using genetically engineered pigs for the organ shortage is promising.This review provides an overview of potential incompatibilities of immunogenicity and functional proteins related to xenotransplantation between humans and pigs.Furthermore,it elucidates possible approaches for multiplex gene modification to breed better-humanized pigs for clinical xenotransplantation.

Generation of pigs with humanized type Ⅱ collagen by precise human COL2A1 gene knock-in

Osteoarthritis(OA)is the most common chronic disease,characterized by progressive cartilage breakdown,subchondral bone sclerosis,and aberrant bone outgrowth(Yucesoy et al.,2015;Hussain et al.,2016).OA is one of the leading causes of cartilage damage.Patients with severe cartilage damage require transplantation of articular cartilage to improve their quality of life.Type Ⅱ collagen is a major component of articular cartilage and intervertebral discs and plays an important role in the structure and strength of connective tissues that support muscles and joints(Byers,1994).

Base editors:development and applications in biomedicine

Base editor(BE)is a gene-editing tool developed by combining the CRISPR/Cas system with an individual deaminase,enabling precise single-base substitution in DNA or RNA without generating a DNA double-strand break(DSB)or requiring donor DNA templates in living cells.Base editors offer more precise and secure genome-editing effects than other conventional artificial nuclease systems,such as CRISPR/Cas9,as the DSB induced by Cas9 will cause severe damage to the genome.Thus,base editors have important applications in the field of biomedicine,including gene function investigation,directed protein evolution,genetic lineage tracing,disease modeling,and gene therapy.Since the development of the two main base editors,cytosine base editors(CBEs)and adenine base editors(ABEs),scientists have developed more than 100 optimized base editors with improved editing efficiency,precision,specificity,targeting scope,and capacity to be delivered in vivo,greatly enhancing their application potential in biomedicine.Here,we review the recent development of base editors,summarize their applications in the biomedical field,and discuss future perspectives and challenges for therapeutic applications.