Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos.

Monkeys serve as important model species for studying human diseases and developing therapeutic strategies, yet the application of monkeys in biomedical researches has been significantly hindered by the difficulties in producing animals genetically modified at the desired target sites. Here, we first applied the CRISPR/Cas9 system, a versatile tool for editing the genes of different organisms, to target monkey genomes. By coinjection of Cas9 mRNA and sgRNAs into one-cell-stage embryos, we successfully achieve precise gene targeting in cynomolgus monkeys. We also show that this system enables simultaneous disruption of two target genes (Ppar-γ and Rag1) in one step, and no off-target mutagenesis was detected by comprehensive analysis. Thus, coinjection of one-cell-stage embryos with Cas9 mRNA and sgRNAs is an efficient and reliable approach for gene-modified cynomolgus monkey generation.

Mechanism and treatment of olfactory dysfunction caused by coronavirus disease 2019.

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since the start of the pandemic, olfactory dysfunction (OD) has been reported as a common symptom of COVID-19. In some asymptomatic carriers, OD is often the first and even the only symptom. At the same time, persistent OD is also a long-term sequela seen after COVID-19 that can have a serious impact on the quality of life of patients. However, the pathogenesis of post-COVID-19 OD is still unclear, and there is no specific treatment for its patients. The aim of this paper was to review the research on OD caused by SARS-CoV-2 infection and to summarize the mechanism of action, the pathogenesis, and current treatments.

Correction to: YTHDF2 reduction fuels inflammation and vascular abnormalization in hepatocellular carcinoma.

An amendment to this paper has been published and can be accessed via the original article.

Chemerin induces lipolysis through ERK1/2 pathway in intramuscular mature adipocytes of dairy bull calves.

The adipokine Chemerin has been reported to regulate differentiation and metabolism of adipocytes, but the mechanism underlying lipolysis is still largely unknown. The purpose of this study was to explore whether ERK1/2 pathway is involved in regulating Chemerin during bovine intramuscular mature adipocyte lipolysis. Intramuscular mature adipocytes of dairy bull calves were cultured in vitro and were treated with Chemerin or U0126, which is an inhibitor of ERK1/2 pathway. The results showed that TG content in cells was significantly decreased, glycerol and free fatty acid were significantly increased in cell culture media, and the expression of phosphorylated ERK1/2 in cells was increased in Chemerin-treated group, suggested that ERK1/2 pathway was involved in regulation of lipolysis by Chemerin. In addition, the expression of lipolytic-related critical factors ATGL, HSL, LPL, PPARα, UCP3, and CPT1 were upregulated, but the expression of adipogenic key factors, including PPARγ and C/EBPα were downregulated by Chemerin. Interestingly, all the effects of Chemerin on genes expression in intramuscular mature adipocytes or fat tissue were inhibited by U0126, showed that the function of Chemerin to promote adipose decomposition will be significantly weakened if the ERK1/2 pathway is suppressed, and confirmed that ERK1/2 pathway is involved in mediate Chemerin-enhanced lipolysis. In conclusion, the study demonstrated that Chemerin induce intramuscular mature adipocytes lipolysis through activation of the ERK1/2 pathway. Our research at least provide partial mechanisms of Chemerin on lipolysis and deposition of intramuscular fat tissue of dairy bull calves.

YTHDF2 reduction fuels inflammation and vascular abnormalization in hepatocellular carcinoma.

BACKGROUND: Dynamic N6-methyladenosine (m6A) modification was previously identified as a ubiquitous post-transcriptional regulation that affected mRNA homeostasis. However, the m6A-related epitranscriptomic alterations and functions remain elusive in human cancer. Here we aim to identify the profile and outcome of m6A-methylation in hepatocellular carcinoma (HCC). RESULTS: Using liquid chromatography-tandem mass spectrometry and m6A-immunoprecipitation in combination with high-throughput sequencing, we determined the m6A-mRNA levels in human HCC. Human HCC exhibited a characteristic gain of m6A modification in tandem with an increase of mRNA expression, owing to YTH domain family 2 (YTHDF2) reduction. The latter predicted poor classification and prognosis of HCC patients, and highly correlated with HCC m6A landscape. YTHDF2 silenced in human HCC cells or ablated in mouse hepatocytes provoked inflammation, vascular reconstruction and metastatic progression. Mechanistically, YTHDF2 processed the decay of m6A-containing interleukin 11 (IL11) and serpin family E member 2 (SERPINE2) mRNAs, which were responsible for the inflammation-mediated malignancy and disruption of vascular normalization. Reciprocally, YTHDF2 transcription succumbed to hypoxia-inducible factor-2α (HIF-2α). Administration of a HIF-2α antagonist (PT2385) restored YTHDF2-programed epigenetic machinery and repressed liver cancer. CONCLUSION: Our results have characterized the m6A-mRNA landscape in human HCC and revealed YTHDF2 as a molecular 'rheostat' in epitranscriptome and cancer progression.

CRISPR/Cas9-mediated Dax1 knockout in the monkey recapitulates human AHC-HH.

Mutations in the DAX1 locus cause X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH), which manifest with primary adrenal insufficiency and incomplete or absent sexual maturation, respectively. The associated defects in spermatogenesis can range from spermatogenic arrest to Sertoli cell only syndrome. Conclusions from Dax1 knockout mouse models provide only limited insight into AHC/HH disease mechanisms, because mouse models exhibit more extensive abnormalities in testicular development, including disorganized and incompletely formed testis cords with decreased number of peritubular myoid cells and male-to-female sex reversal. We previously reported successful clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome targeting in cynomolgus monkeys. Here, we describe a male fetal monkey in which targeted genome editing using CRISPR/Cas9 produced Dax1-null mutations in most somatic tissues and in the gonads. This DAX1-deficient monkey displayed defects in adrenal gland development and abnormal testis architecture with small cords, expanded blood vessels and extensive fibrosis. Sertoli cell formation was not affected. This phenotype strongly resembles findings in human patients with AHC-HH caused by mutations in DAX1. We further detected upregulation of Wnt/ß-catenin-VEGF signaling in the fetal Dax1-deficient testis, suggesting abnormal activation of signaling pathways in the absence of DAX1 as one mechanism of AHC-HH. Our study reveals novel insight into the role of DAX1 in HH and provides proof-of-principle for the generation of monkey models of human disease via CRISPR/Cas9-mediated gene targeting.

Efficient in vivo deletion of a large imprinted lncRNA by CRISPR/Cas9.

Recent genome-wide studies have revealed that the majority of the mouse genome is transcribed as non-coding RNAs (ncRNAs) and growing evidence supports the importance of ncRNAs in regulating gene expression and epigenetic processes. However, the low efficiency of conventional gene targeting strategies has hindered the functional study of ncRNAs in vivo, particularly in generating large fragment deletions of long non-coding RNAs (lncRNAs) with multiple expression variants. The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system has recently been applied as an efficient tool for engineering site-specific mutations of protein-coding genes in the genome. In this study, we explored the potential of using the CRISPR/Cas9 system to generate large genomic deletions of lncRNAs in mice. We developed an efficient one-step strategy to target the maternally expressed lncRNA, Rian, on chromosome 12 in mice. We showed that paired sgRNAs can precisely generate large deletions up to 23kb and the deletion efficiency can be further improved up to 33% by combining multiple sgRNAs. The deletion successfully abolished the expression of Rian from the maternally inherited allele, validating the biological relevance of the mutations in studying an imprinted locus. Mutation of Rian has differential effects on expression of nearby genes in different somatic tissues. Taken together, we have established a robust one-step method to engineer large deletions to knockout lncRNA genes with the CRISPR/Cas9 system. Our work will facilitate future functional studies of other lncRNAs in vivo.

A review of spatial profiling technologies for characterizing the tumor microenvironment in immuno-oncology.

Interpreting the mechanisms and principles that govern gene activity and how these genes work according to -their cellular distribution in organisms has profound implications for cancer research. The latest technological advancements, such as imaging-based approaches and next-generation single-cell sequencing technologies, have established a platform for spatial transcriptomics to systematically quantify the expression of all or most genes in the entire tumor microenvironment and explore an array of disease milieus, particularly in tumors. Spatial profiling technologies permit the study of transcriptional activity at the spatial or single-cell level. This multidimensional classification of the transcriptomic and proteomic signatures of tumors, especially the associated immune and stromal cells, facilitates evaluation of tumor heterogeneity, details of the evolutionary trajectory of each tumor, and multifaceted interactions between each tumor cell and its microenvironment. Therefore, spatial profiling technologies may provide abundant and high-resolution information required for the description of clinical-related features in immuno-oncology. From this perspective, the present review will highlight the importance of spatial transcriptomic and spatial proteomics analysis along with the joint use of other sequencing technologies and their implications in cancers and immune-oncology. In the near future, advances in spatial profiling technologies will undoubtedly expand our understanding of tumor biology and highlight possible precision therapeutic targets for cancer patients.

Research Progress of Olfactory Nerve Regeneration Mechanism and Olfactory Training.

The olfactory nerve (ON) is the only cranial nerve exposed to the external environment. Hence, it is susceptible to damage from head trauma, viral infection, inflammatory stimulation, and chemical toxins, which can lead to olfactory dysfunction. However, compared with all other cranial nerves, the ON is unique due to its inherent ability to regenerate. This characteristic provides a theoretical basis for treatment of olfactory dysfunction. Olfactory training (OT) is one of the main treatments for olfactory dysfunction. It is easy to apply and has few side-effects, and has been shown to be efficacious for patients with olfactory dysfunction of various causes. To further understand the application value of ON regeneration and OT on olfactory dysfunction, we review the research progress on the mechanism of ON regeneration and OT.

Long-term intraperitoneal injection of lipopolysaccharide induces high expression of Id2 in the brain of mice.

Lipopolysaccharide (LPS) from gram negative bacteria plays an important role in the pathophysiology of neurodegenerative diseases. Many evidences showed that LPS-induced neuroinflammation is related to upregulation of NF-kappaB. Here, we report that long-term treatment of lower dosage LPS mainly causes upregulation of Id2 protein. As an inhibitor of cell differentiation, Id2 plays an import role in adult olfactory neurogenesis. However, Id2 protein in brain acts as two edges in a sword, persist over-expression of Id2 in brain can induce neurodamages and may be related to neurodegeneration.

Sum frequency generation-compressive sensing microscope.

A new sum frequency generation imaging microscope using a novel sampling theory, compressive sensing (CS), has been developed for surface studies. CS differentiates itself from the conventional sampling methods by collecting fewer measurements than the traditional methods to reconstruct a high quality image. Pseudorandom patterns were applied to a light modulator and reflected the sum frequency (SF) signal generated from the sample into a photomultiplier tube detector. The image of the sample was reconstructed using sparsity preserving algorithms from the SF signal. The influences of the number of CS testing patterns applied and the number of SF pulses acquired for each pattern on the quality of the images was investigated and a comparison of the image quality with the traditional raster scan was made at varying resolutions for a gold patterned Si surface. Our results demonstrate the CS technique achieved 16 times the pixel density beyond the resolution where the raster scan strategy lost its ability to image the sample due to the dilution of the SF signal below the detection limit of the detector.

Intracellular XBP1-IL-24 axis dismantles cytotoxic unfolded protein response in the liver.

Endoplasmic reticulum (ER) stress-associated cell death is prevalent in various liver diseases. However, the determinant mechanism how hepatocytes survive unresolved stress was still unclear. Interleukin-24 (IL-24) was previously found to promote ER stress-mediated cell death, and yet its expression and function in the liver remained elusive. Here we identified an antiapoptotic role of IL-24, which transiently accumulated within ER-stressed hepatocytes in a X-box binding protein 1 (XBP1)-dependent manner. Disruption of IL-24 increased cell death in the CCL4- or APAP-challenged mouse liver or Tm-treated hepatocytes. In contrast, pharmaceutical blockade of eukaryotic initiation factor 2α (eIF2α) or genetical ablation of C/EBP homologous protein (CHOP) restored hepatocyte function in the absence of IL-24. In a clinical setting, patients with acute liver failure manifested a profound decrease of hepatic IL-24 expression, which was associated with disease progression. In conclusion, intrinsic hepatocyte IL-24 maintains ER homeostasis by restricting the eIF2α-CHOP pathway-mediated stress signal, which might be exploited as a bio-index for prognosis or therapeutic intervention in patients with liver injury.

Nucleofection with Plasmid DNA for CRISPR/Cas9-Mediated Inactivation of Programmed Cell Death Protein 1 in CD133-Specific CAR T Cells.

CRISPR/Cas9-mediated programmed cell death protein 1 (PD-1) disruption in chimeric antigen receptor (CAR) T cells could be an appealing choice to improve the therapeutic efficacy of CAR T cells in an immunosuppressive tumor microenvironment. In most of the reported cases, Cas9 was delivered into T cells by way of electroporation with RNA or protein. However, transient expression of Cas9 by transfection with a plasmid encoding its gene is apparently simpler, as it avoids the steps of in vitro transcription of DNA or protein production. This study tried nucleofection into human primary T cells of plasmids encoding both CRISPR/Cas9 for disrupting the PD-1 gene and the piggyBac transposon system for expressing CD133-specific CAR in one reaction. Based on drug selection, CD133-specific CAR T cells were obtained in which, on average, 91.5% of the PD-1 gene sites were disrupted, but almost no Cas9 gene expression was found in the final engineered CAR T cells. The PD-1-deficient CD133-specific CAR T cells showed similar levels of cytokine secretion and improved proliferation and cytotoxicity in vitro, and enhanced inhibition of tumor growth in an orthotopic mouse model of glioma, compared to conventional CD133-CAR T cells. The described method could be useful for the production of PD-1-deficient CAR T cells for cancer immunotherapy.

Deletion of miR-126a Promotes Hepatic Aging and Inflammation in a Mouse Model of Cholestasis.

MicroRNAs (miRNAs) act as regulators of aging at the tissue or organism level or as regulators of cellular senescence. Targeted deletion of miR-126 in mice causes partial embryonic lethality, but its biological function in the liver is still largely unknown. Here, we deleted miR-126a, using the CRISPR/Cas9 system in vitro and in vivo. miR-126a was reduced in the aging livers, and disruption of miR-126a in bone mesenchymal stem cells (BMSCs) induced age-associated telomere shortening, DNA damage responses, and proinflammatory cytokines. Moreover, disruption of miR-126a in mice caused hepatocyte senescence, inflammation, and metabolism deficiency. In addition, disruption of miR-126a via BMSC transplantation aggravated the severity of liver defects induced by cholestasis compared with that in the functional miR-126a BMSC group. Mechanistically, we identified versican (VCAN) as a novel direct miR-126a-5p target that induces telomere shortening, BMSC senescence, and nuclear factor κB (NF-κB) pathway activation. This study identified aging-related reduced expression of miR-126a and promotion of its target VCAN as a key mechanism in the regulation of hepatic metabolic function during aging and hepatic damage by inducing NF-κB pathway activation, DNA repair function disorder, and telomere attrition. The findings indicate that miR-126a may be a drug target for the treatment of hepatic failure.

Molecular characteristics of early-stage female germ cells revealed by RNA sequencing of low-input cells and analysis of genome-wide DNA methylation.

High-throughput stage-specific transcriptomics provides an unbiased approach for understanding the process of cell development. Here, we report transcriptome analysis of primordial germ cell, female germline stem cell (FGSC), germinal vesicle and mature oocyte by performing RNA sequencing of freshly isolated cells in mice. As expected, these stages and gene-expression profiles are consistent with developmental timing. Analysis of genome-wide DNA methylation during female germline development was used for confirmation. By pathway analysis and blocking experiments, we demonstrate PI3K-AKT pathway is critical for FGSC maintenance. We also identify functional modules with hub genes and lncRNAs, which represent candidates for regulating FGSC self-renewal and differentiation. Remarkably, we note alternative splicing patterns change dramatically during female germline development, with the highest occurring in FGSCs. These findings are invaluable resource for dissecting the molecular pathways and processes into oogenesis and will be wider applications for other types of stem cell research.

Runx3 Mediates Resistance to Intracellular Bacterial Infection by Promoting IL12 Signaling in Group 1 ILC and NCR+ILC3.

Innate lymphoid cells (ILCs) are the most recently identified family of the innate immune system and are hypothesized to modulate immune functions prior to the generation of adaptive immune responses. Subsets of ILCs reside in the mucosa and regulate immune responses to external pathogens; however, their role and the mechanism by which they protect against intracellular bacterial infection is not completely understood. In this report, using S. typhimurium and L. monocytogenes, we found that the levels of group 1 ILCs and NCR+ ILC3s were increased upon infection and that these increases were associated with Runt-related transcription factor 3 (Runx3) expression. Runx3 fl/fl PLZF-cre mice were much more sensitive to infection with the intracellular bacterial pathogens S. typhimurium and L. monocytogenes partially due to abnormal Group 1 ILC and NCR+ILC3 function. We also found that Runx3 directly binds to the Il12Rß2 promoter and intron 8 to accelerate the expression of Il12Rß2 and modulates IFNγ secretion triggered by the IL12/ STAT4 axis. Therefore, we demonstrate that Runx3 influences group 1 ILC- and NCR+ILC3-mediated immune protection against intracellular bacterial infections of both the gut and liver.

APOBEC3 induces mutations during repair of CRISPR-Cas9-generated DNA breaks.

The APOBEC-AID family of cytidine deaminase prefers single-stranded nucleic acids for cytidine-to-uridine deamination. Single-stranded nucleic acids are commonly involved in the DNA repair system for breaks generated by CRISPR-Cas9. Here, we show in human cells that APOBEC3 can trigger cytidine deamination of single-stranded oligodeoxynucleotides, which ultimately results in base substitution mutations in genomic DNA through homology-directed repair (HDR) of Cas9-generated double-strand breaks. In addition, the APOBEC3-catalyzed deamination in genomic single-stranded DNA formed during the repair of Cas9 nickase-generated single-strand breaks in human cells can be further processed to yield mutations mainly involving insertions or deletions (indels). Both APOBEC3-mediated deamination and DNA-repair proteins play important roles in the generation of these indels. Therefore, optimizing conditions for the repair of CRISPR-Cas9-generated DNA breaks, such as using double-stranded donors in HDR or temporarily suppressing endogenous APOBEC3s, can repress these unwanted mutations in genomic DNA.

Cyclin B2 can compensate for Cyclin B1 in oocyte meiosis I.

Mammalian oocytes are arrested at the prophase of the first meiotic division for months and even years, depending on species. Meiotic resumption of fully grown oocytes requires activation of M-phase-promoting factor (MPF), which is composed of Cyclin B1 and cyclin-dependent kinase 1 (CDK1). It has long been believed that Cyclin B1 synthesis/accumulation and its interaction with CDK1 is a prerequisite for MPF activation in oocytes. In this study, we revealed that oocyte meiotic resumption occurred in the absence of Cyclin B1. Ccnb1-null oocytes resumed meiosis and extruded the first polar body. Without Cyclin B1, CDK1 could be activated by up-regulated Cyclin B2. Ccnb1 and Ccnb2 double knockout permanently arrested the oocytes at the prophase of the first meiotic division. Oocyte-specific Ccnb1-null female mice were infertile due to failed MPF activity elevation and thus premature interphase-like stage entry in the second meiotic division. These results have revealed a hidden compensatory mechanism between Cyclin B1 and Cyclin B2 in regulating MPF and oocyte meiotic resumption.

[Comparison of sperm parameters between male adults at different altitudes].

OBJECTIVE: To investigate the influence of long-term anoxic exposure on the sperm function of male adults at different altitudes. METHODS: A total of 28 male adults that had stayed at the altitude of 5 340 m for 1-3 years were included as a high-altitude group (HAG), 34 at the mean altitude of 3 800 m for 2-5 years as a middle-altitude group (MAG) and 31 permanently at the altitude of 1 300 m as controls. Semen specimens were collected and the real-time semen analysis was performed by using computer-assisted semen analysis (CASA) system. RESULTS: The sperm density, VCL, VSL, VAP and LIN in the HAG were (51.12 +/- 14.61) x 10(6)/ ml, (48.17 +/- 13. 52) microm/s, (32.64 +/- 6.70) microm/s, (41.21 +/- 9.32) microm/s and 52.24 +/- 8.14, respectively, significantly lower than those of the control (P < 0.01 or P < 0.05). Compared with the control group, there was a progressive decrease in sperm concentration, sperm motility rate, VSL, VCL, LIN, VAP and ALH in the MAG. CONCLUSION: The higher the altitude, the more obvious was the negative effect of anoxic exposure on the sperm function of male adults.

Wisp2 disruption represses Cxcr4 expression and inhibits BMSCs homing to injured liver.

Liver regeneration/repair is a compensatory regrowth following acute liver failure, and bone marrow-derived mesenchyme stem cell (BMSC) transplantation is an effective therapy that promotes liver regeneration/repair. Wnt1 inducible signaling pathway protein 2 (Wisp2) is highly expressed in BMSCs, however, its function remains unclear. In this work, we used clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein -9 nuclease (CRISPR/Cas9) genome editing technology to knockdown Wisp2 in BMSCs, and these modified cells were then transplanted into rats which were induced by the 2-AAF/PH. By linking the expression of Cas9 to green fluorescent protein (GFP), we tracked BMSCs in the rats. Disruption of Wisp2 inhibited the homing of BMSCs to injured liver and aggravated liver damage as indicated by remarkably high levels of ALT and AST. Moreover, the key factor in BMSC transplantation, C-X-C chemokine receptor type 4 (Cxcr4), was down-regulated in the Wisp2 depleted BMSCs and had a lower expression in the livers of the corresponding rats. By tracing the GFP marker, more BMSCs were observed to differentiate into CD31 positive endothelial cells in the functional Wisp2 cells but less in the Wisp2 gene disrupted cells. In summary, Wisp2 promotes the homing of BMSCs through Cxcr4 related signaling during liver repair in rats.