Actin polymerization inhibition by targeting ARPC2 affects intestinal stem cell homeostasis.

Background: The rapid turnover of the intestinal epithelium is driven by the proliferation and differentiation of intestinal stem cells (ISCs). The dynamics of the F-actin cytoskeleton are critical for maintaining intercellular force and the signal transduction network. However, it remains unclear how direct interference with actin polymerization impacts ISC homeostasis. This study aims to reveal the regulatory effects of the F-actin cytoskeleton on the homeostasis of intestinal epithelium, as well as the potential risks of benproperine (BPP) as an anti-tumor drug. Methods: Phalloidin fluorescence staining was utilized to test F-actin polymerization. Flow cytometry and IHC staining were employed to discriminate different types of intestinal epithelial cells. Cell proliferation was assessed through bromo-deoxyuridine (BrdU) and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays. The proliferation and differentiation of intestinal stem cells were replicated in vitro through organoid culture. Epithelial migration was evaluated through BrdU pulse labeling and chasing in mice. Results: The F-actin content was observed to significantly increase as crypt cells migrated into the villus region. Additionally, actin polymerization in secretory cells, especially in Paneth cells (PCs), was much higher than that in neighboring ISCs. Treatment with the newly identified actin-related protein 2/3 complex subunit 2 (ARPC2) inhibitor BPP led to a dose-dependent increase or inhibition of intestinal organoid growth in vitro and crypt cell proliferation in vivo. Compared with the vehicle group, BPP treatment decreased the expression of Lgr5 ISC feature genes in vivo and in organoid culture. Meanwhile, PC differentiation derived from ISCs and progenitors was decreased by inhibition of F-actin polymerization. Mechanistically, BPP-induced actin polymerization inhibition may activate the Yes1-associated transcriptional regulator pathway, which affects ISC proliferation and differentiation. Accordingly, BPP treatment affected intestinal epithelial cell migration in a dose-dependent manner. Conclusion: Our findings indicate that the regulation of cytoskeleton reorganization can affect ISC homeostasis. In addition, inhibiting ARPC2 with the Food and Drug Administration-approved drug BPP represents a novel approach to influencing the turnover of intestinal epithelial cells.

Tannins amount determines whether tannase-containing bacteria are probiotic or pathogenic in IBD.

The role of dietary tannin in inflammatory bowel disease (IBD) is still not clear. Therefore, we aim to study the effect of TA in the progression of IBD. Dextran sulphate sodium (DSS)-induced model was used to mimic IBD. Metagenomics and metabolomics were performed to study the alteration of intestinal microbiota and metabolites. NCM460 and THP-1 cells were used for in vitro study. The amount of TA was associated with the outcomes of DSS-induced IBD as evidenced by in vivo and in vitro studies. Metabolomic and metagenomic analyses revealed that TA-induced enrichment of microbial metabolite gallic acid (GA) was responsible for the action of TA. Mechanistically, protective dose of GA promoted colonic mucus secretion to suppress bacterial infection and that it ameliorated DSS-induced epithelial damage by inhibiting p53 signaling, whereas toxic dose of GA directly caused epithelial damage by promoting cell cycle arrest. Therapeutic experiment showed protective dose of GA-promoted recovery of DSS-induced colonic inflammation. The role of tannase-containing bacteria can be transformed under different conditions in IBD progression.

Production of gene-edited pigs harboring orthologous human mutations via double cutting by CRISPR/Cas9 with long single-stranded DNAs as homology-directed repair templates by zygote injection.

Precise gene editing of model organisms is required for accurately modeling human diseases and deciphering gene functions. In this study, we used a pair of guide RNAs (sgRNAs), which in vitro transcribed along with other CRISPR RNA components, to generate two cleavage sites flanking pig GJB2 (pGJB2) CDS. By using long single-stranded DNAs (lssDNA) as homology-directed repair (HDR) templates, we efficiently obtained two gene-edited pigs, of which GJB2 CDS replaced with CDSs containing human GJB2 c.235delC mutation and orthologous human p.V37I mutation, respectively. These mutations were commonly observed in patients with hearing loss. Genetic analysis of the two gene-edited pigs showed that the HDR-derived gene-editing efficiency were as high as 80% (4/5) and 50% (2/4), respectively. While no mutation was observed in the group of single cutting with one sgRNA covering the 235th nucleotide C in pGJB2 CDS, using a short single-stranded oligo DNA containing c.235delC mutation as HDR template. Extra experiments proved that the intended mutations were successfully transmitted to offspring or extensively integrated into various tissues including gonad of founder pigs. Our work indicated that the new "double cutting with lssDNA template" gene editing method can expand sgRNA selection scope and avoids direct cutting of gene CDS. Additionally, can introduce precise mutations into mammalian genomic sites, especially those with unavailable proper protospacer sequence or being resistant to gene editing. Moreover, this method can be performed with CRISPR RNA reagents instead of CRISPR ribonucleoproteins applied in previous reports.

Metabolite identification in fecal microbiota transplantation mouse livers and combined proteomics with chronic unpredictive mild stress mouse livers.

Major depressive disorder (MDD) is a common mood disorder. Gut microbiota may be involved in the pathogenesis of depression via the microbe-gut-brain axis. Liver is vulnerable to exposure of bacterial products translocated from the gut via the portal vein and may be involved in the axis. In this study, germ-free mice underwent fecal microbiota transplantation from MDD patients and healthy controls. Behavioral tests verified the depression model. Metabolomics using gas chromatography-mass spectrometry, nuclear magnetic resonance, and liquid chromatography-mass spectrometry determined the influence of microbes on liver metabolism. With multivariate statistical analysis, 191 metabolites were distinguishable in MDD mice from control (CON) mice. Compared with CON mice, MDD mice showed lower levels for 106 metabolites and higher levels for 85 metabolites. These metabolites are associated with lipid and energy metabolism and oxidative stress. Combined analyses of significantly changed proteins in livers from another depression model induced by chronic unpredictive mild stress returned a high score for the Lipid Metabolism, Free Radical Scavenging, and Molecule Transports network, and canonical pathways were involved in energy metabolism and tryptophan degradation. The two mouse models of depression suggest that changes in liver metabolism might be involved in the pathogenesis of MDD. Conjoint analyses of fecal, serum, liver, and hippocampal metabolites from fecal microbiota transplantation mice suggested that aminoacyl-tRNA biosynthesis significantly changed and fecal metabolites showed a close relationship with the liver. These findings may help determine the biological mechanisms of depression and provide evidence about "depression microbes" impacting on liver metabolism.

Pilot study of large-scale production of mutant pigs by ENU mutagenesis.

N-ethyl-N-nitrosourea (ENU) mutagenesis is a powerful tool to generate mutants on a large scale efficiently, and to discover genes with novel functions at the whole-genome level in Caenorhabditis elegans, flies, zebrafish and mice, but it has never been tried in large model animals. We describe a successful systematic three-generation ENU mutagenesis screening in pigs with the establishment of the Chinese Swine Mutagenesis Consortium. A total of 6,770 G1 and 6,800 G3 pigs were screened, 36 dominant and 91 recessive novel pig families with various phenotypes were established. The causative mutations in 10 mutant families were further mapped. As examples, the mutation of SOX10 (R109W) in pig causes inner ear malfunctions and mimics human Mondini dysplasia, and upregulated expression of FBXO32 is associated with congenital splay legs. This study demonstrates the feasibility of artificial random mutagenesis in pigs and opens an avenue for generating a reservoir of mutants for agricultural production and biomedical research.

Normal Electrocardiogram of Bama Miniature Pigs (Sus scrofa domestica).

This study determined the normal ECG patterns and values for Bama miniature pigs. Standard limb-lead ECG were recorded from 120 clinically healthy, unanesthetized piglets (age, 2 to 4 mo). The values for the ECG parameters (mean ± 1 SD) were: heart rate, 125.56 ± 18.80 bpm; P amplitude, 0.11 ± 0.03 mV; QRS amplitude, 0.63 ± 0.31 mV; P duration, 43.99 ± 5.98 ms; QRS complex, 55.27 ± 7.02 ms; RR interval, 487.55 ± 77.32 ms; PR interval, 90.72 ± 11.94 ms; QT interval, 244.72 ± 25.27 ms; and mean electrical axis, 22.2 ± 80.3°. The P waves were predominantly positive in leads I and II and in the augmented unipolar limb aVF lead; by comparison, the QRS patterns were less uniform. The T waves were slightly positive in leads II, III, and aVF. The determination and publication of the normal ECG patterns and values of Bama minipigs facilitates understanding of the electrocardiographic changes that arise under experimental conditions.

Expression of Bama Minipig and Human CYP3A Enzymes: Comparison of the Catalytic Characteristics with Each Other and Their Liver Microsomes.

Minipigs represent a good animal model because of the physiologic and anatomic similarities they share with humans. Three cytochrome P450 (CYP) 3A isozymes, CYP3A22, CYP3A29, and CYP3A46, have recently been reported to be expressed in Bama minipigs, which have limited data relating to their metabolic characteristics. In the present study, Bama minipig CYP3A22, CYP3A29, and CYP3A46 were recombinantly expressed and their metabolic manners were compared with those of human CYP3A4 and CYP3A5 and also human and Bama minipig liver microsomes. The results indicated Bama minipigs and human CYP3A enzymes showed similar metabolic kinetics and metabolite profiles using testosterone, midazolam, and nifedipine as substrates. However, the differences in amino acid sequences change the elimination velocity and metabolic preference of CYP3A enzymes to their substrates. It was demonstrated that CYP3A29, CYP3A4, and CYP3A5 were the most active enzymes for all reactions, whereas CYP3A46 was the least active enzyme. Substrate-dependent metabolism characteristics between human and Bama minipig CYP3A isoenzymes exist.

Transcriptome profiling identifies differentially expressed genes in postnatal developing pituitary gland of miniature pig.

In recent years, Tibetan pig and Bama pig are popularly used as animal models for medical researches. However, little genomic information is available for the two breeds, particularly regarding gene expression pattern at the whole-transcriptome level. In this study, we characterized the pituitary transcriptome profile along their postnatal developmental stages within and between the two breeds in order to illustrate the differential dynamics and functions of differentially expressed genes. We obtained a total of ∼300 million 80-bp paired-end reads, detected 15 715 previously annotated genes. Most of the genes (90.33%) were shared between the two breeds with the main functions in metabolic process. Four hormone genes (GH, PRL, LHB, and FSHB) were detected in all samples with extremely high levels of expression. Functional differences between the three developmental stages (infancy, puberty and adulthood) in each breed were dominantly presented by the gene expressions at the first stage. That is, Bama pig was over-represented in the genes involved in the cellular process, while Tibetan pig was over-represented in the genes represented by the reproductive process. The identified SNPs indicated that the divergence between the miniature pig breeds and the large pig (Duroc) were greater than that between the two miniature pig breeds. This study substantially expands our knowledge concerning the genes transcribed in the pig pituitary gland and provides an overview of pituitary transcriptome dynamics throughout the period of postnatal development.

Constitutive expression of CYP3A mRNA in Bama miniature pig tissues.

The pig, particularly the miniature pig (minipig), is becoming an important animal model due to its physiological and anatomical similarities to humans. Bama minipigs (Sus scrofa domestica), a Chinese natural minipig breed, are a promising animal model. The pig is a useful model for drug metabolism and pharmacological studies due to the similar properties of cytochrome P450 (CYP)3A between pigs and humans. However, a detailed investigation regarding the abundance and expression of CYP3A in porcine tissues, particularly in minipig tissues, has not been performed. The present study investigated constitutive expression of CYP3A mRNA in Bama minipig tissues using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). We found that the expression of CYP3A mRNAs relative to the endogenous control, ß-actin (ACTB), was lower than when compared to the expression of the endogenous control, TATA-binding protein (TBP), except for the expression of CYP3A29 mRNA in the spleen, adrenal gland, testis, and epididymis, and CYP3A46 in the spleen. Expression levels of all three CYP3As were highest in the liver amongst all the tissues tested, and the order of relative mRNA expression level of the three CYP3As was different between other tissues. We also analyzed the relative expression of the three CYP3A mRNAs in each tissue. CYP3A46 had the highest expression in all extrahepatic tissues, whereas CYP3A22 had the highest expression in the liver, and CYP3A29 had the lowest expression in all tissues except in the duodenum, where it had higher expression than CYP3A22. Because CYP3A22 and CYP3A46 were the most highly expressed isoforms, it could follow that they are probably important functional CYP3A genes for Bama miniature pig. Our present work will broaden the understanding of the physiological functions of CYP3As in the Bama minipig and promote its application in drug metabolism and pharmacological studies. Our results also indicate that the breed, age, and castration status of the pig should be considered when using the pig as an animal model in pharmacological applications.

A study on optimizing the cryopreservation methods for Bama miniature pig semen.

In this study, the cryopreservation methods for Bama miniature pig semen were investigated and optimized. First, using an orthogonal experimental design, the semen freezing-thawing procedure for Bama miniature boars was optimized based on analysis of the effects of concentrations of LDL (LC, parameter A), trehalose (TC, parameter B) and glycerol (GC, parameter C), the equilibration time at 15°C (ET, parameter D), and the thawing method (TM, parameter E) on sperm motility. The results showed that the effects of the parameters could be arranged as A>C>B>D>E. The LDL concentration and final glycerol concentration had exceedingly significant effects on the motility of thawed spermatozoa (P<0.01), and the effects of the trehalose concentration, equilibration time at 15°C, and the thawing method were not significant (P>0.05). Scheme 2 (A(3)B(4)C(2)D(3)E(1)) gave a motility of 52.26% after thawing. Then, using sperm motility, acrosome integrity, plasma membrane integrity, and DNA injury rate as indicators, four combinations, on the basis of scheme 2, were designed to analyze the protective effects of different combinations of LDL, glycerol, and trehalose; the results showed that combination of 9% LDL, 200 mM trehalose, and 2% glycerol (i.e., combination 4) demonstrated significantly better protective effects than the other combinations (P<0.05), further verifying that scheme 2 was the best for cryopreservation of Bama miniature boar semen. In this way, a method with favorable performance was established for cryopreservation of semen of Bama miniature boars.

Lentiviral transgenic microRNA-based shRNA suppressed mouse cytochromosome P450 3A (CYP3A) expression in a dose-dependent and inheritable manner.

Cytochomosome P450 enzymes (CYP) are heme-containing monooxygenases responsible for oxidative metabolism of many exogenous and endogenous compounds including drugs. The species difference of CYP limits the extent to which data obtained from animals can be translated to humans in pharmacodynamics or pharmacokinetics studies. Transgenic expression of human CYP in animals lacking or with largely reduced endogenous CYP counterparts is recognized as an ideal strategy to correct CYP species difference. CYP3A is the most abundant CYP subfamily both in human and mammals. In this study, we designed a microRNA-based shRNA (miR-shRNA) simultaneously targeting four members of mouse CYP3A subfamily (CYP3A11, CYP3A16, CYP3A41 and CYP3A44), and transgenic mice expressing the designed miR-shRNA were generated by lentiviral transgenesis. Results showed that the CYP3A expression level in transgenic mice was markedly reduced compared to that in wild type or unrelated miR-shRNA transgenic mice, and was inversely correlated to the miR-shRNA expression level. The CYP3A expression levels in transgenic offspring of different generations were also remarkably lower compared to those of controls, and moreover the inhibition rate of CYP3A expression remained comparable over generations. The ratio of the targeted CYP3A transcriptional levels was comparable between knockdown and control mice of the same gender as detected by RT-PCR DGGE analysis. These data suggested that transgenic miR-shRNA suppressed CYP3A expression in a dose-dependent and inheritable manner, and transcriptional levels of the targeted CYP3As were suppressed to a similar extent. The observed knockdown efficacy was further confirmed by enzymatic activity analysis, and data showed that CYP3A activities in transgenic mice were markedly reduced compared to those in wild-type or unrelated miR-shRNA transgenic controls (1.11±0.71 vs 5.85±1.74, 5.9±2.4; P<0.01). This work laid down a foundation to further knock down the remaining murine CYP3As or CYPs of other subfamilies, and a basis to generate CYP knockdown animals of other species.