Parallel, redundant circuit organization for homeostatic control of feeding behavior.

Neural circuits for essential natural behaviors are shaped by selective pressure to coordinate reliable execution of flexible goal-directed actions. However, the structural and functional organization of survival-oriented circuits is poorly understood due to exceptionally complex neuroanatomy. This is exemplified by AGRP neurons, which are a molecularly defined population that is sufficient to rapidly coordinate voracious food seeking and consumption behaviors. Here, we use cell-type-specific techniques for neural circuit manipulation and projection-specific anatomical analysis to examine the organization of this critical homeostatic circuit that regulates feeding. We show that AGRP neuronal circuits use a segregated, parallel, and redundant output configuration. AGRP neuron axon projections that target different brain regions originate from distinct subpopulations, several of which are sufficient to independently evoke feeding. The concerted anatomical and functional analysis of AGRP neuron projection populations reveals a constellation of core forebrain nodes, which are part of an extended circuit that mediates feeding behavior.

Histone methyltransferase MLL4 protects against pressure overload-induced heart failure via a THBS4-mediated protection in ER stress.

Pressure overload-induced pathological cardiac hypertrophy eventually leads to heart failure (HF). Unfortunately, lack of effective targeted therapies for HF remains a challenge in clinical management. Mixed-lineage leukemia 4 (MLL4) is a member of the SET family of histone methyltransferase enzymes, which possesses histone H3 lysine 4 (H3K4)-specific methyltransferase activity. However, whether and how MLL4 regulates cardiac function is not reported in adult HF. Here we report that MLL4 is required for endoplasmic reticulum (ER) stress homeostasis of cardiomyocytes and protective against pressure overload-induced cardiac hypertrophy and HF. We observed that MLL4 is increased in the heart tissue of HF mouse model and HF patients. The cardiomyocyte-specific deletion of Mll4 (Mll4-cKO) in mice leads to aggravated ER stress and cardiac dysfunction following pressure overloading. MLL4 knockdown neonatal rat cardiomyocytes (NRCMs) also display accelerated decompensated ER stress and hypertrophy induced by phenylephrine (PE). The combined analysis of Cleavage Under Targets and Tagmentation sequencing (CUT&Tag-seq) and RNA sequencing (RNA-seq) data reveals that, silencing of Mll4 alters the chromatin landscape for H3K4me1 modification and gene expression patterns in NRCMs. Interestingly, the deficiency of MLL4 results in a marked reduction of H3K4me1 and H3K27ac occupations on Thrombospondin-4 (Thbs4) gene loci, as well as Thbs4 gene expression. Mechanistically, MLL4 acts as a transcriptional activator of Thbs4 through mono-methylation of H3K4 and further regulates THBS4-dependent ER stress response, ultimately plays a role in HF. Our study indicates that pharmacologically targeting MLL4 and ER stress might be a valid therapeutic approach to protect against cardiac hypertrophy and HF.

Early cartilage lesion and 5-year incident joint surgery in knee osteoarthritis patients: a retrospective cohort study.

OBJECTIVE: to investigate the association between cartilage lesion-related features observed in knee osteoarthritis (OA) patients' first MRI examination and incident knee surgery within 5 years. Additionally, to assess the predictive value of these features for the incident knee surgery. METHODS: We identified patients diagnosed with knee OA and treated at our institution between January 2015 and January 2018, and retrieved their baseline clinical data and first MRI examination films from the information system. Next, we proceeded to determine joint space narrowing grade, cartilage lesion size grade, cartilage full-thickness loss grade and cartilage lesion sum score for the medial and lateral compartments, respectively. Generalized linear regression models examined the association of these features with 5-year incident knee surgery. Positive and negative predictive values (PPVs and NPVs) were determined referring to 5-year incident knee surgery. RESULTS: Totally, 878 participants (knees) were found eligible to form the study population. Within the 5 years, surgery was performed on 61 knees. None of the cartilage-related features had been found significantly associated with incident surgery. The results were similar for medial and lateral compartments. The PPVs were low for all the features. CONCLUSIONS: Among symptomatic clinically diagnosed OA knees, cartilage lesions observed in the first MRI examinations were not found to be associated with the occurrence of joint surgery within a 5-year period. All these cartilage-related features appear to have no additional value in predicting 5-year incident joint surgery.

ERK/CREB and p38 MAPK/MMP14 Signaling Pathway Influences Spermatogenesis through Regulating the Expression of Junctional Proteins in Eriocheir sinensis Testis.

The hemolymph-testis barrier (HTB) is a reproduction barrier in Crustacea, guaranteeing the safe and smooth process of spermatogenesis, which is similar to the blood-testis barrier (BTB) in mammals. The MAPK signaling pathway plays an essential role in spermatogenesis and maintenance of the BTB. However, only a few studies have focused on the influence of MAPK on crustacean reproduction. In the present study, we knocked down and inhibited MAPK in Eriocheir sinensis. Increased defects in spermatogenesis were observed, concurrently with a damaged HTB. Further research revealed that es-MMP14 functions downstream of ERK and p38 MAPK and degrades junctional proteins (Pinin and ZO-1); es-CREB functions in the ERK cascade as a transcription factor of ZO-1. In addition, when es-MMP14 and es-CREB were deleted, the defects in HTB and spermatogenesis aligned with abnormalities in the MAPK. However, JNK impacts the integrity of the HTB by changing the distribution of intercellular junctions. In summary, the MAPK signaling pathway maintains HTB integrity and spermatogenesis through es-MMP14 and es-CREB, which provides insights into the evolution of gene function during barrier evolution.

Type I/type III IFN and related factors regulate JEV infection and BBB endothelial integrity.

BACKGROUND: Japanese encephalitis virus (JEV) remains a predominant cause of Japanese encephalitis (JE) globally. Its infection is usually accompanied by disrupted blood‒brain barrier (BBB) integrity and central nervous system (CNS) inflammation in a poorly understood pathogenesis. Productive JEV infection in brain microvascular endothelial cells (BMECs) is considered the initial event of the virus in penetrating the BBB. Type I/III IFN and related factors have been described as negative regulators in CNS inflammation, whereas their role in JE remains ambiguous. METHODS: RNA-sequencing profiling (RNA-seq), real-time quantitative PCR, enzyme-linked immunosorbent assay, and Western blotting analysis were performed to analyze the gene and protein expression changes between mock- and JEV-infected hBMECs. Bioinformatic tools were used to cluster altered signaling pathway members during JEV infection. The shRNA-mediated immune factor-knockdown hBMECs and the in vitro transwell BBB model were utilized to explore the interrelation between immune factors, as well as between immune factors and BBB endothelial integrity. RESULTS: RNA-Seq data of JEV-infected hBMECs identified 417, 1256, and 2748 differentially expressed genes (DEGs) at 12, 36, and 72 h post-infection (hpi), respectively. The altered genes clustered into distinct pathways in gene ontology (GO) terms and KEGG pathway enrichment analysis, including host antiviral immune defense and endothelial cell leakage. Further investigation revealed that pattern-recognition receptors (PRRs, including TLR3, RIG-I, and MDA5) sensed JEV and initiated IRF/IFN signaling. IFNs triggered the expression of interferon-induced proteins with tetratricopeptide repeats (IFITs) via the JAK/STAT pathway. Distinct PRRs exert different functions in barrier homeostasis, while treatment with IFN (IFN-ß and IFN-λ1) in hBMECs stabilizes the endothelial barrier by alleviating exogenous destruction. Despite the complex interrelationship, IFITs are considered nonessential in the IFN-mediated maintenance of hBMEC barrier integrity. CONCLUSIONS: This research provided the first comprehensive description of the molecular mechanisms of host‒pathogen interplay in hBMECs responding to JEV invasion, in which type I/III IFN and related factors strongly correlated with regulating the hBMEC barrier and restricting JEV infection. This might help with developing an attractive therapeutic strategy in JE.

Single-cell RNA sequencing reveals small extracellular vesicles derived from malignant cells that contribute to angiogenesis in human breast cancers.

BACKGROUND: Breast cancer is the most common cancer affecting women across the world. Tumor endothelial cells (TECs) and malignant cells are the major constituents of the tumor microenvironment (TME), but their origin and role in shaping disease initiation, progression, and treatment responses remain unclear due to significant heterogeneity. METHODS: Tissue samples were collected from eight patients presenting with breast cancer. Single-cell RNA sequencing (scRNA-seq) analysis was employed to investigate the presence of distinct cell subsets in the tumor microenvironment. InferCNV was used to identify cancer cells. Pseudotime trajectory analysis revealed the dynamic process of breast cancer angiogenesis. We validated the function of small extracellular vesicles (sEVs)-derived protein phosphatase 1 regulatory inhibitor subunit 1B (PPP1R1B) in vitro experiments. RESULTS: We performed single-cell transcriptomics analysis of the factors associated with breast cancer angiogenesis and identified twelve subclusters of endothelial cells involved in the tumor microenvironment. We also identified the role of TECs in tumor angiogenesis and confirmed their participation in different stages of angiogenesis, including communication with other cell types via sEVs. Overall, the research uncovered the TECs heterogeneity and the expression levels of genes at different stages of tumor angiogenesis. CONCLUSIONS: This study showed sEVs derived from breast cancer malignant cells promote blood vessel formation by activating endothelial cells through the transfer of PPP1R1B. This provides a new direction for the development of anti-angiogenic therapies for human breast cancer.

mTORC1/rpS6 and mTORC2/PKC regulate spermatogenesis through Arp3-mediated actin microfilament organization in Eriocheir sinensis.

Mammalian target of rapamycin (mTOR) is a crucial signaling protein regulating a range of cellular events. Numerous studies have reported that the mTOR pathway is related to spermatogenesis in mammals. However, its functions and underlying mechanisms in crustaceans remain largely unknown. mTOR exists as two multimeric functional complexes termed mTOR complex 1 (mTORC1) and mTORC2. Herein, we first cloned ribosomal protein S6 (rpS6, a downstream molecule of mTORC1) and protein kinase C (PKC, a downstream effector of mTORC2) from the testis of Eriocheir sinensis. The dynamic localization of rpS6 and PKC suggested that both proteins may be essential for spermatogenesis. rpS6/PKC knockdown and Torin1 treatment led to defects in spermatogenesis, including germ cell loss, retention of mature sperm and empty lumen formation. In addition, the integrity of the testis barrier (similar to the blood-testis barrier in mammals) was disrupted in the rpS6/PKC knockdown and Torin1 treatment groups, accompanied by changing in expression and distribution of junction proteins. Further study demonstrated that these findings may result from the disorganization of filamentous actin (F-actin) networks, which were mediated by the expression of actin-related protein 3 (Arp3) rather than epidermal growth factor receptor pathway substrate 8 (Eps8). In summary, our study illustrated that mTORC1/rpS6 and mTORC2/PKC regulated spermatogenesis via Arp3-mediated actin microfilament organization in E. sinensis.

Two New Protolimonoids from the Chinese Mangrove Xylocarpus granatum Koenig.

Two new compounds including one apotirucallane protolimonoid, xylogranatriterpin A (1), and one glabretal protolimonoid, xylocarpusin A (2), along with three known related compounds were isolated from the twigs and leaves of the Chinese mangrove Xylocarpus granatum. The apotirucallane xylogranatriterpin A (1) bears an unprecedented 24-ketal carbon connecting ring E with an epoxide ring. The structures of new compounds were elucidated by extensive spectroscopic analysis and by comparison of the spectroscopic data with those reported in the literatures. Plausible biosynthetic pathway to xylogranatriterpin A (1) was also proposed. None of them showed cytotoxic, neuroprotective, or protein tyrosine phosphatase 1B (PTP1B) inhibitory activity.

mTORC1/C2 regulate spermatogenesis in Eriocheir sinensis via alterations in the actin filament network and cell junctions.

Spermatogenesis is a finely regulated process of germ cell proliferation and differentiation that leads to the production of sperm in seminiferous tubules. Although the mammalian target of rapamycin (mTOR) signaling pathway is crucial for spermatogenesis in mammals, its functions and molecular mechanisms in spermatogenesis remain largely unknown in nonmammalian species, particularly in Crustacea. In this study, we first identified es-Raptor (the core component of mTOR complex 1) and es-Rictor (the core component of mTOR complex 2) from the testis of Eriocheir sinensis. Dynamic localization of es-Raptor and es-Rictor implied that these proteins were indispensable for the spermatogenesis of E. sinensis. Furthermore, es-Raptor and es-Rictor knockdown results showed that the mature sperm failed to be released, causing almost empty lumens in the testis. We investigated the reasons for these effects and found that the actin-based cytoskeleton was disrupted in the knockdown groups. In addition, the integrity of the testis barrier (similar to the blood-testis barrier in mammals) was impaired and affected the expression of cell junction proteins. Further study revealed that es-Raptor and es-Rictor may regulate spermatogenesis via both mTORC1- and mTORC2-dependent mechanisms that involve es-rpS6 and es-Akt/es-PKC, respectively. Moreover, to explore the testis barrier in E. sinensis, we established a cadmium chloride (CdCl2)-induced testis barrier damage model as a positive control. Morphological and immunofluorescence results were similar to those of the es-Raptor and es-Rictor knockdown groups. Altogether, es-Raptor and es-Rictor were important for spermatogenesis through maintenance of the actin filament network and cell junctions in E. sinensis.

Follicle-stimulating hormone signaling in Sertoli cells: a licence to the early stages of spermatogenesis.

Follicle-stimulating hormone signaling is essential for the initiation and early stages of spermatogenesis. Follicle-stimulating hormone receptor is exclusively expressed in Sertoli cells. As the only type of somatic cell in the seminiferous tubule, Sertoli cells regulate spermatogenesis not only by controlling their own number and function but also through paracrine actions to nourish germ cells surrounded by Sertoli cells. After follicle-stimulating hormone binds to its receptor and activates the follicle-stimulating hormone signaling pathway, follicle-stimulating hormone signaling will establish a normal Sertoli cell number and promote their differentiation. Spermatogonia pool maintenance, spermatogonia differentiation and their entry into meiosis are also positively regulated by follicle-stimulating hormone signaling. In addition, follicle-stimulating hormone signaling regulates germ cell survival and limits their apoptosis. Our review summarizes the aforementioned functions of follicle-stimulating hormone signaling in Sertoli cells. We also describe the clinical potential of follicle-stimulating hormone treatment in male patients with infertility. Furthermore, our review may be helpful for developing better therapies for treating patients with dysfunctional follicle-stimulating hormone signaling in Sertoli cells.

Systematic analysis of the expression and prognostic value of ITPR1 and correlation with tumor infiltrating immune cells in breast cancer.

BACKGROUND: ITPR1 is a key gene for autophagy, but its biological function is still unclear, and there are few studies on the correlation between ITPR1 gene expression and the occurrence and development of breast cancer. METHODS: Analyze the expression of ITPR1 through online databases such as Oncomine and TIMER. Kaplan-Meier plotter and other databases were used to evaluate the impact of ITPR1 on clinical prognosis. The expression of ITPR1 in analysis of 145 cases of breast cancer and 30 cases of adjacent normal tissue was detected by Immunohistochemistry. Statistical analysis was used to evaluate the clinical relevance and prognostic significance of abnormally expressed proteins. And the Western Blot was used to detect the expression of ITPR1 between breast cancer tissues and cells. The TIMER database studied the relationship between ITPR1 and cancer immune infiltration. And used the ROC plotter database to predict the response of ITPR1 to chemotherapy, endocrine therapy and anti-HER2 therapy in patients with breast cancer. RESULTS: Compared with normal breast samples, ITPR1 was significantly lower in patients with breast cancer. And the increased expression of ITPR1 mRNA was closely related to longer overall survival (OS), distant metastasis free survival (DMFS), disease specific survival (DSS) and relapse free survival (RFS) in breast cancer. And the expression level of ITPR1 was higher in patients treated with chemotherapy than untreated patients. In addition, the expression of ITPR1 was positively correlated with related gene markers of immune cells in different types of breast cancer, especially with BRCA basal tissue breast cancer. CONCLUSION: ITPR1 was lower expressed in breast cancer. The higher expression of ITPR1 suggested favorable prognosis for patients. ITPR1 was related to the level of immune infiltration, especially in BRCA-Basal patients. All research results indicated that ITPR1 might affect breast cancer prognosis and participate in immune regulation. In short, ITPR1 might be a potential target for breast cancer therapy.

Quality of life in pregnancy after percutaneous closure of atrial septal defect guided by transthoracic echocardiography.

PURPOSE: We evaluated quality of life (QoL) in pregnant women who underwent transthoracic echocardiography-guided percutaneous closure of atrial septal defect (ASD). METHODS: A total of 45 pregnant women underwent transthoracic echocardiography-guided percutaneous closure of ASD. We assessed QoL using the 36-Item Short Form Survey (SF-36) and compared results between pre- and post-procedure patients, as well as between those with ASD and healthy women in their second and third trimesters of pregnancy. RESULTS: All patients showed improved right ventricular function and were classified as Class I, post-procedure. Mean SF-36 scores of the post-procedure group were better on all sub-scales than those of the pre-procedure group (p < 0.05), with the exception of role-emotional and mental health. Mean SF-36 scores for the pre-procedure group were also lower on all sub-scales than those of healthy pregnant controls (p < 0.05), with the exception of role physical, role emotional, and mental health. There was no difference between the post-procedure group and healthy pregnant controls. In a subgroup analysis, scores were better in some dimensions (social functioning and role emotional) for post-procedure patients in the 31-40 years of age group and the group on their second or third pregnancies than those of the 20-30 years of age group and the group on their first pregnancies (p < 0.05). CONCLUSION: After closure of ASD, QoL in pregnant women was improved. In a subgroup analysis, the younger women and those on their first pregnancy performed more poorly in some dimensions (social functioning and role emotional); this suggested that these groups should receive more proactive intervention.

Limited data was available on the general quality of life (QoL) in pregnant women with atrial septal defect (ASD), even though the condition could produce anxiety over health of the pregnancy and fetus. The percutaneous closure procedure was available for ASD during pregnancy; however, pregnant women were often concerned that the required X-rays would harm the fetus. A safe and effective procedure, percutaneous closure of ASD guided by transthoracic echocardiography, was widely used for this condition. This study used the 36-Item Short Form Survey (SF-36) to assess QoL in pregnant women with ASD pre- and post-procedure and compared the results to those of healthy pregnant women at a similar stage of pregnancy. Post-procedure QoL in pregnant women with ASD was improved; however, the younger women and those on their first pregnancy performed more poorly in some dimensions (social functioning and role emotional). Our results suggested that these groups should receive more proactive intervention.

Bridging the cervicothoracic junction during posterior cervical laminectomy and fusion for the treatment of multilevel cervical ossification of the posterior longitudinal ligament: a retrospective case series.

BACKGROUND: The purpose of this study was to investigate the surgical efficacy of crossing the cervicothoracic junction during posterior cervical laminectomy and fusion for the treatment of multilevel cervical ossification of the posterior longitudinal ligament (OPLL). METHODS: From October 2009 to October 2017, 46 consecutive patients with multilevel cervical OPLL underwent posterior cervical laminectomy and crossing the cervicothoracic junction fusion were obtained in the study. Their medical records were retrospectively collected. Cervical lordosis and cervical sagittal balance were used to assess radiographic outcomes. Japanese Orthopedic Association (JOA), axial symptom, C5 root palsy, blood loss, and operation time were used to assess clinical outcomes. The mean follow-up period was 20.7 ± 8.3 months. RESULTS: The operation time was 205.2 ± 39.8 min and the intraoperative blood loss was 352.2 ± 143.7 ml. Analysis of the final follow-up data showed significant differences in JOA score (P < 0.01), C2-C7 lordosis angle (P < 0.01), and C2-C7 SVA (P < 0.01). CT confirmed that grafted bone was completely fused in all patients and progression of OPLL was observed in two patients (4.3%) at final follow-up. No adjacent segment disease (ASD) or instrument failure occurred in any patients. CONCLUSIONS: Cervical laminectomy and crossing the cervicothoracic junction fusion are effective and safe methods to treat multilevel cervical OPLL. Randomized controlled studies compared constructs ending at cervical vertebrae or thoracic vertebrae are needed to confirm these results.

Neurons for hunger and thirst transmit a negative-valence teaching signal.

Homeostasis is a biological principle for regulation of essential physiological parameters within a set range. Behavioural responses due to deviation from homeostasis are critical for survival, but motivational processes engaged by physiological need states are incompletely understood. We examined motivational characteristics of two separate neuron populations that regulate energy and fluid homeostasis by using cell-type-specific activity manipulations in mice. We found that starvation-sensitive AGRP neurons exhibit properties consistent with a negative-valence teaching signal. Mice avoided activation of AGRP neurons, indicating that AGRP neuron activity has negative valence. AGRP neuron inhibition conditioned preference for flavours and places. Correspondingly, deep-brain calcium imaging revealed that AGRP neuron activity rapidly reduced in response to food-related cues. Complementary experiments activating thirst-promoting neurons also conditioned avoidance. Therefore, these need-sensing neurons condition preference for environmental cues associated with nutrient or water ingestion, which is learned through reduction of negative-valence signals during restoration of homeostasis.

Synthesis, Characterization, DNA/HSA Interactions, and Anticancer Activity of Two Novel Copper(II) Complexes with 4-Chloro-3-Nitrobenzoic Acid Ligand.

The purpose of this study was to identify new metal-based anticancer drugs; to this end, we synthesized two new copper(II) complexes, namely [Cu(ncba)4(phen)] (1) and [Cu(ncba)4(bpy)] (2), comprised 4-chloro-3-nitrobenzoic acid as the main ligand. The single-crystal XRD approach was employed to determine the copper(II) complex structures. Binding between these complexes and calf thymus DNA (CT-DNA) and human serum albumin (HSA) was explored by electronic absorption, fluorescence spectroscopy, and viscometry. Both complexes intercalatively bound CT-DNA and statically and spontaneously quenched DNA/HSA fluorescence. A CCK-8 assay revealed that complex 1 and complex 2 had substantial antiproliferative influences against human cancer cell lines. Moreover, complex 1 had greater antitumor efficacy than the positive control cisplatin. Flow cytometry assessment of the cell cycle demonstrated that these complexes arrested the HepG2 cell cycle and caused the accumulation of G0/G1-phase cells. The mechanism of cell death was elucidated by flow cytometry-based apoptosis assays. Western blotting revealed that both copper(II) complexes induced apoptosis by regulating the expression of the Bcl-2(Bcl-2, B cell lymphoma 2) protein family.

[Role of Contrast-enhanced Ultrasound in Distinguishing between Benign and Malignant Thyroid Nodules with Calcification].

Objective To explore the roles of conventional ultrasound and contrast-enhanced ultrasound in distinguishing between benign and malignant thyroid nodules with calcification. Methods A total of 102 solid thyroid nodules with calcification in 76 patients were evaluated by conventional ultrasound alone and conventional ultrasound combined with contrast-enhanced ultrasound.The features obtained through conventional ultrasound alone and that combined with contrast-enhanced ultrasound were scored,and the diagnostic performance of the two methods was analyzed based on the final pathological results. Results The distribution of microcalcification(P<0.0001),aspect ratio>1(P=0.039),unclear boundary(P=0.027),ring enhancement around nodules(P=0.000),and the degree(P=0.000)and uniformity(P=0.001)of enhancement for the non-calcified part were statistically different between benign and malignant nodules.Compared with conventional ultrasound alone,conventional ultrasound combined with contrast-enhanced ultrasound significantly improved the area under the curve(0.841 vs.0.701,P<0.001)and specificity(97.06% vs. 44.12%,P=0.007),without significant change in sensitivity(67.55% vs. 73.53%,P=0.727). Conclusions The combination with contrast-enhanced ultrasound can improve the performance of conventional ultrasound in the differential diagnosis of benign and malignant thyroid solid nodules with calcification.Eight malignant signs include solid,hypoechoic,microcalcification,aspect ratio,and blurred edges detected by conventional ultrasound,and non-circular enhancement around nodules,low enhancement and non-uniform enhancement of solid parts detected by contrast-enhanced ultrasound.A solid thyroid nodule with calcification presenting five or more malignant signs highly suggests malignancy.

[Progress on abnormal development of cloned pigs generated by somatic cell transfer nuclear].

Cloning, also known as somatic cell nuclear transfer (SCNT), is an asexual reproduction technique that reprograms differentiated cells to the totipotent state, and generates offspring with a genotype identical to the donor cells. Pig cloning technique holds great promise for propagating excellent breeding boars, generating genetically modified pigs, protecting rare and endangered pigs and studying the mechanisms of somatic cell nucleus reprogramming. However, cloned pigs suffer from various developmental defects, including low birth rate, low birth weight, and high stillbirth occurrence, neonatal mortality and congenital malformations, which severely hamper their applications. Errors in epigenetic reprogramming of donor nucleus are considered as the main causes of low cloning efficiency and abnormal embryonic development in cloned embryos and animals. However, most studies to correct the errors in epigenetic reprogramming of cloned pig embryos have not substantially improved the birth and survival rates of cloned pigs. In this review, we summarize the abnormal phenotypes, causes of abnormal development of cloned pigs and effective methods for improving pig cloning efficiency, thereby providing a reference for the future research to improve the development and survival rates of cloned pig embryos and cloned pigs.

[Recent developments in enhancing the efficiency of CRISPR/Cas9- mediated knock-in in animals].

Gene-editing technology can artificially modify genetic material of targeted loci by precise insertion, deletion, or replacement in the genomic DNA. In recent years, with the developments of zinc-finger endonuclease (ZFN), transcription activator-like effector nuclease (TALEN), clustered regularly interspaced short palindromic repeats/CRISPR- associated protein 9 (CRISPR/Cas9) technologies, such precise modifications of the animal genomes have become possible. Although gene-editing tools, such as CRISPR/Cas9, can efficiently generate double-strand breaks (DSBs) in mammalian cells, the homology-directed repair (HDR) mediated knock-in (KI) efficiency is extremely low. In this review, we briefly describe the current development of gene-editing tools and summarize the recent strategies to enhance the CRISPR/Cas9- mediated KI efficiency, which will provide a reference for the generation of human disease models, research on gene therapy and livestock genetic improvement.

[Advances in assay for transposase-accessible chromatin with high-throughput sequencing].

Assay for transposase accessible chromatin with high-throughput sequencing (ATAC-seq) was developed in 2013. It has the advantages of more convenient operation and higher efficiency for DNA recovery than DNase I hypersensitive site sequencing (DNase-seq) and micrococcal nuclease sequencing (MNase-seq). ATAC-seq currently is the most popular technique of genome-wide mapping for chromatin accessibility. It provides information on binding regions of transcription factors and nucleosome localization on the chromatin. Thus, ATAC-seq is of great significance for studying the epigenetics and molecular mechanisms in chromatin structure. In this review, we compare the advantages and disadvantages of multiple techniques for profiling chromatin accessibility, and summarize the principles, main process, development and applications of ATAC-seq. We hope this review will provide a reference for study of genome-wide mapping for chromatin accessibility, identification of cis-regulatory elements, and dissection of the epigenetic and genetic regulatory networks using the ATAC-seq technology in eukaryotes.

Transcriptome heterogeneity of porcine ear fibroblast and its potential influence on embryo development in nuclear transplantation.

There is heterogeneity among donor cells of the same source. Many studies have shown that donor cell affects the efficiency of somatic cell nuclear transfer (SCNT). However, the potential influence of donor cell heterogeneity on the efficiency of nuclear transplantation were rarely analyzed at the single-cell level. In this study, single-cell transcriptome sequencing was performed on 52 porcine ear fibroblasts randomly selected from the same source to compare their gene expression patterns. The results showed that 48 cells had similar gene expression patterns, whereas 4 cells (D11_1, D12_1, DW61_2, DW99_2) had significantly different gene expression patterns from those of other cells. There were no two cells with identical gene expression patterns. The gene expression patterns of D11_1, D12_1, DW61_2 and DW99_2 were analyzed, using the 48 cells with similar gene expression patterns as controls. Firstly, we used the R language statistics to select the differentially expressed genes in the 4 single cells, and identified the top 50 most significant differentially expressed genes. Then GO enrichment analysis and KEGG pathway analysis were performed on the differentially expressed genes. Enrichment analysis revealed that the main molecular functions of the differentially expressed genes included energy metabolism, protein metabolism and cell response to stimulation. The main pathways from KEGG enrichment were related to cell cycle, cell metabolism, and DNA replication. Finally, based on the above results and in consideration with the SCNT research progress, we discussed the potential effects of differential gene expression patterns of the 4 single cells on the embryonic development efficiency of nuclear transplantation. This study revealed transcriptional heterogeneity of porcine ear tissue fibroblasts and provided an effective method to analyze elite donor cells, thereby providing new ideas on improving the cloning efficiency of SCNT.