Post cross-linked ROS-responsive poly(ß-amino ester)-plasmid polyplex NPs for gene therapy of EBV-associated nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is one of the most common tumors in South China and Southeast Asia and is thought to be associated with Epstein-Barr virus (EBV) infection. Downregulation of latent membrane protein 1 (LMP1) encoded by EBV can reduce the expression of NF-κB and PI3K, induce apoptosis, and inhibit the growth of EBV-related NPC. For targeted cleavage of the Lmp1 oncogene via the CRISPR/Cas9 gene editing system, a post cross-linked ROS-responsive poly(ß-amino ester) (PBAE) polymeric vector was developed for the delivery of CRISPR/Cas9 plasmids both in vitro and in vivo. After composition optimization, the resultant polymer-plasmid polyplex nanoparticles (NPs) showed a diameter of ∼230 nm and a zeta potential of 22.3 mV with good stability. Compared with the non-cross-linked system, the cross-linked NPs exhibited efficient and quick cell uptake, higher transfection efficiency in EBV-positive C666-1 cells (53.5% vs. 40.6%), more efficient gene editing ability against the Mucin2 model gene (Muc2) (17.9% vs. 15.4%) and Lmp1 (8.5% vs. 5.6%), and lower intracellular reactive oxygen species (ROS) levels. The NPs achieved good tumor penetration and tumor growth inhibition in the C666-1 xenograft tumor model via Lmp1 cleavage, indicating their potential for gene therapy of EBV-related NPC.

Cyclophosphamide reduces gene transcriptional activity and embryo in vitro development by inhibiting NF-κB expression through decreasing AcH4K12.

Cyclophosphamide (CTX), a widely used chemotherapeutic agent for cancer treatment, has been associated with long-term toxicity and detrimental effects on oocytes and ovaries, resulting in female reproductive dysfunction. This study aimed to investigate the potential impact of CTX on in vitro maturation (IVM) injury of porcine oocytes and subsequent embryonic development, as well as its effects on epigenetic modification and gene activation during early embryonic development. The results demonstrated that CTX treatment caused aberrant spindle structure and mitochondrial dysfunction during oocyte maturation, inducing DNA damage and early apoptosis, which consequently disrupted meiotic maturation. Indeed, CTX significantly reduced the in vitro developmental capacity of porcine embryos, and induced DNA damage and apoptosis in in vitro fertilization (IVF) blastocysts. Importantly, CTX induced abnormal histone modification of AcH4K12 in early porcine embryos. Moreover, addition of LBH589 before zygotic genome activation (ZGA) effectively increased AcH4K12 levels and restored the protein expression of NF-κB, which can effectively enhance the in vitro developmental potential of IVF embryos. The DNA damage and apoptosis induced by CTX compromised the quality of the blastocysts, which were recovered by supplementation with LBH589. This restoration was accompanied by down-regulation of BAX mRNA expression and up-regulation of BCL2, POU5F1, SOX2 and SOD1 mRNA expression. These findings indicated that CTX caused abnormal histone modification of AcH4K12 in early porcine embryos and reduced the protein expression of NF-κB, a key regulator of early embryo development, which may block subsequent ZGA processes.

Research advances in peptide‒drug conjugates.

Peptide‒drug conjugates (PDCs) are drug delivery systems consisting of a drug covalently coupled to a multifunctional peptide via a cleavable linker. As an emerging prodrug strategy, PDCs not only preserve the function and bioactivity of the peptides but also release the drugs responsively with the cleavable property of the linkers. Given the ability to significantly improve the circulation stability and targeting of drugs in vivo and reduce the toxic side effects of drugs, PDCs have already been extensively applied in drug delivery. Herein, we review the types and mechanisms of peptides, linkers and drugs used to construct PDCs, and summarize the clinical applications and challenges of PDC drugs.

Ginsenoside Rg1 activates brown adipose tissue to counteract obesity in high-fat diet-fed mice by regulating gut microbes and bile acid composition.

Obesity is a global health problem strongly linked to gut microbes and their metabolites. In this study, ginsenoside Rg1 (Rg1) reduced lipid droplet size and hepatic lipid accumulation by activating uncoupling protein 1 expression in brown adipose tissue (BAT), which in turn inhibited high-fat diet (HFD)-induced weight gain in mice. Furthermore, the intestinal flora of mice was altered, the abundance of Lachnoclostridium, Streptococcus, Lactococcus, Enterococcus and Erysipelatoclostridium was upregulated, and the concentrations of fecal bile acids were altered, with cholic acid and taurocholic acid concentrations being significantly increased. In addition, the beneficial effects of Rg1 were eliminated in mice treated with a combination of antibiotics. In conclusion, these results suggest that Rg1 activates BAT to counteract obesity by regulating gut microbes and bile acid composition in HFD-fed mice.

Fecal transplant from myostatin deletion pigs positively impacts the gut-muscle axis.

The host genome may influence the composition of the intestinal microbiota, and the intestinal microbiota has a significant effect on muscle growth and development. In this study, we found that the deletion of the myostatin (MSTN) gene positively regulates the expression of the intestinal tight junction-related genes TJP1 and OCLN through the myosin light-chain kinase/myosin light chain pathway. The intestinal structure of MSTN-/- pigs differed from wild-type, including by the presence of a thicker muscularis and longer plicae. Together, these changes affect the structure of intestinal microbiota. Mice transplanted with the intestinal microbiota of MSTN-/- pigs had myofibers with larger cross-sectional areas and higher fast-twitch glycolytic muscle mass. Microbes responsible for the production of short-chain fatty acids (SCFAs) were enriched in both the MSTN-/- pigs and recipient mice, and SCFAs levels were elevated in the colon contents. We also demonstrated that valeric acid stimulates type IIb myofiber growth by activating the Akt/mTOR pathway via G protein-coupled receptor 43 and ameliorates dexamethasone-induced muscle atrophy. This is the first study to identify the MSTN gene-gut microbiota-SCFA axis and its regulatory role in fast-twitch glycolytic muscle growth.

Dynamic changes in intestinal microbiota and metabolite composition of pre-weaned beef calves.

Gut microbes and their metabolites are essential for maintaining host health and production. The intestinal microflora of pre-weaned calves gradually tends to mature with growth and development and has high plasticity, but few studies have explored the dynamic changes of intestinal microbiota and metabolites in pre-weaned beef calves. In this study, we tracked the dynamics of faecal microbiota in 13 new-born calves by 16S rRNA gene sequencing and analysed changes in faecal amino acid levels using metabolomics. Calves were divided into the relatively high average daily gain group (HA) and the relatively low average daily gain group (LA) for comparison. The results demonstrated that the alpha diversity of the faecal microbiota increased with calf growth and development. The abundance of Porphyromonadaceae bacterium DJF B175 increased in the HA group, while that of Lactobacillus reuteri decreased. The results of the LEfSe analysis showed that the microbiota of faeces of HA calves at eight weeks of age was enriched with P. bacterium DJF B175, while Escherichia coli and L. reuteri were enriched in the microbiota of faeces of LA calves. Besides, the total amino acid concentration decreased significantly in the eighth week compared with that in the first week (P < 0.05). Overall, even under the same management conditions, microorganisms and their metabolites interact to play different dynamic regulatory roles. Our results provide new insights into changes in the gut microbiota and metabolites of pre-weaned calves.

Effects of myostatin gene knockout on porcine extraocular muscles.

Myostatin (MSTN), a negative regulator of skeletal muscle mass, is not well known in extraocular muscles (EOMs). EOMs are specialized skeletal muscles. Hence, in this study, the effect of MSTN on the superior rectus (SR) and superior oblique (SO) of 2-month-old MSTN knockout (MSTN-/-) and wild-type (WT) pigs of the same genotype was investigated. SR (P < 0.01) and SO (P < 0.001) fiber cross-sectional areas of MSTN-/- pigs were significantly larger than those of WT pigs. Compared with WT pigs, MSTN-/- SO displayed a decrease in type I fibers (WT: 27.24%, MSTN-/-: 10.32%, P < 0.001). Type IIb fibers were higher in MSTN-/- pigs than in WT pigs (WT: 30.38%, MSTN-/-: 62.24%, P < 0.001). The trend in SR was the same as that in SO, although the trend in SO was greater than that in SR. The expression of myogenic differentiation factor (MyoD) and myogenic (MyoG) showed a significant increase in MSTN-/- SO (about 2.5-fold and 2-fold, respectively at the gene expression level, about 1.5-fold at the protein level) compared with WT pigs. MSTN plays an important role in the development of EOMs and regulates the muscle fiber type by modulating the gene expression of MyoD and MyoG in pigs.

Combined Biomimetic MOF-RVG15 Nanoformulation Efficient Over BBB for Effective Anti-Glioblastoma in Mice Model.

Introduction: The blood-brain barrier (BBB) is a key obstacle to the delivery of drugs into the brain. Therefore, it is essential to develop an advanced drug delivery nanoplatform to solve this problem. We previously screened a small rabies virus glycoprotein 15 (RVG15) peptide with 15 amino acids and observed that most of the RVG15-modified nanoparticles entered the brain within 1 h of administration. The high BBB penetrability gives RVG15 great potential for brain-targeted drug delivery systems. Moreover, a multifunctional integrated nanoplatform with a high drug-loading capacity, tunable functionality, and controlled drug release is crucial for tumor treatment. Zeolitic imidazolate framework (ZIF-8) is a promising nanodrug delivery system. Methods: Inspired by the biomimetic concept, we designed RVG15-coated biomimetic ZIF-8 nanoparticles (RVG15-PEG@DTX@ZIF-8) for docetaxel (DTX) delivery to achieve efficient glioblastoma elimination in mice. This bionic nanotherapeutic system was prepared by one-pot encapsulation, followed by coating with RVG15-PEG conjugates. The size, morphology, stability, drug-loading capacity, and release of RVG15-PEG@DTX@ZIF-8 were thoroughly investigated. Additionally, we performed in vitro evaluation, cell uptake capacity, BBB penetration, and anti-migratory ability. We also conducted an in vivo evaluation of the biodistribution and anti-glioma efficacy of this bionic nanotherapeutic system in a mouse mode. Results: In vitro studies showed that, this bionic nanotherapeutic system exhibited excellent targeting efficiency and safety in HBMECs and C6 cells and high efficiency in crossing the BBB. Furthermore, the nanoparticles cause rapid DTX accumulation in the brain, allowing deeper penetration into glioma tumors. In vivo antitumor assay results indicated that RVG15-PEG@DTX@ZIF-8 significantly inhibited glioma growth and metastasis, thereby improving the survival of tumor-bearing mice. Conclusion: Our study demonstrates that our bionic nanotherapeutic system using RVG15 peptides is a promising and powerful tool for crossing the BBB and treating glioblastoma.

Gastrodia elata Blume extract improves high-fat diet-induced type 2 diabetes by regulating gut microbiota and bile acid profile.

In this study, we aimed to characterize the anti-type 2 diabetes (T2D) effects of Gastrodia elata Blume extract (GEBE) and determine whether these are mediated through modification of the gut microbiota and bile acids. Mice were fed a high-fat diet (HFD), with or without GEBE, and we found that GEBE significantly ameliorated the HFD-induced hyperglycemia, insulin resistance, and inflammation by upregulating glucose transporter 4 (GLUT4) and inhibiting the toll-like receptor 4-nuclear factor kappa-B signaling pathway in white adipose tissue (WAT). In addition, we found that GEBE increased the abundance of Faecalibaculum and Lactobacillus, and altered the serum bile acid concentrations, with a significant increase in deoxycholic acid. The administration of combined antibiotics to mice to eliminate their intestinal microbiota caused a loss of the protective effects of GEBE. Taken together, these findings suggest that GEBE ameliorates T2D by increasing GLUT4 expression in WAT, remodeling the gut microbiota, and modifying serum bile acid concentrations.

miR-455-3p Is Negatively Regulated by Myostatin in Skeletal Muscle and Promotes Myoblast Differentiation.

Myostatin (MSTN) is a growth and differentiation factor that regulates proliferation and differentiation of myoblasts, which in turn controls skeletal muscle growth. It may regulate myoblast differentiation by influencing miRNA expression, and the present study aimed to clarify its precise mechanism of action. Here, we found that MSTN-/- pigs showed an overgrowth of skeletal muscle and upregulated miR-455-3p level. Intervention of MSTN expression using siMSTN in C2C12 myoblasts also showed that siMSTN significantly increased the expression of miR-455-3p. It was found that miR-455-3p directly targeted the 3'-untranslated region of Smad2 by dual-luciferase assay. qRT-PCR, Western blotting, and immunofluorescence analyses indicated that miR-455-3p overexpression or Smad2 silencing in C2C12 myoblasts significantly promoted myoblast differentiation. Furthermore, siMSTN significantly increased the expression of GATA3. The levels of miR-455-3p were considerably reduced in C2C12 myoblasts following GATA3 knockdown. Consistently, GATA3 knockdown also reduced the enhanced miR-455-3p expression caused by siMSTN. Finally, we illustrated that GATA3 has a role in myoblast differentiation regulation. Taken together, we identified the expression profiles of miRNAs in MSTN-/- pigs and found that miR-455-3p positively regulates myoblast differentiation. In addition, we revealed that MSTN acts through the GATA3/miR-455-3p/Smad2 cascade to regulate muscle development.

Positive growth of smooth muscle in uterine horns of myostatin homozygous mutant gilt.

Current studies on myostatin (MSTN), a well-known negative regulator of skeletal muscle, studies mainly focus on the its effects on skeletal muscle.However, its effects on smooth muscle are less studied, especially in the uterine horns. To identify the role of MSTN in uterine horn smooth muscle, this study used 6-8-month-old homozygous MSTN mutant (MSTN-/-) gilts in anoestrum as animal models. Histochemical and immunofluorescence staining, western blotting, and RT-qPCR were performed. The results showed that the uteri of the MSTN-/- gilts were morphologically normal, and the uterine horn smooth muscle content was increased (MSTN-/-: 75.19%, Wild type: 51.52%, P < 0.01). In vivo immunofluorescence staining showed that the expression of the uterine horn smooth muscle-specific marker proteins, namely α-smooth muscle actin (ACTA2) and calponin, increased after MSTN knockout (1.41- and 1.21-fold, respectively, P < 0.05). Increased gene expression was also seen in MSTN-/- gilts in vivo for ACTA2 (approximately 2-fold), smooth muscle myosin heavy chain (7.14-fold), myocardin (9.32-fold), and serum response factor (2.17-fold). Protein expression of smooth muscle-specific markers was increased (1.51-fold for ACTA2, 1.57-fold for calponin, P<0.05). MSTN knockout promoted proliferation of the smooth muscle cell and the gene expression of c-kit, a peristaltic marker (2.43-fold, P < 0.05). The results of the in vitro experiments were consistent with those of the in vivo experiments. The present study indicates that MSTN knockout can increase the smooth muscle content of uterine horns, thus providing potential therapeutic targets for pregnancy disorders caused by increased smooth muscle content.

Esophageal striated muscle hypertrophy and muscle fiber type transformation in MSTN knockout pigs.

Myostatin (MSTN) is a member of the transforming growth factor-ß superfamily that inhibits skeletal muscle growth and development. The esophagus is composed of skeletal muscle and smooth muscle, but the effect of MSTN on esophagus striated muscle (ESM) is unknown. The present study investigated the role of MSTN in ESM using MSTN mutant pigs through histological, gene and protein expression analysis in ESM of MSTN knockout (MSTN-/-) pigs and their wild type (WT) littermates. Hematoxylin-eosin staining showed that the fiber cross-sectional areas in ESM of MSTN-/- pigs were significantly larger than WT pigs (P < 0.05). Immunofluorescence staining showed that the percentage of type I muscle fibers in MSTN-/- pigs were significantly lower than WT pigs (P < 0.01) and type IIA muscle fibers in MSTN-/- pigs were significantly higher than WT pigs (21% higher, P < 0.01). However, type IIB muscle fibers were not detected in the ESM of MSTN-/- or WT pigs indicating that muscle fiber types in pig ESM was composed of type I and IIA. The mRNA levels of myogenic regulatory factors (MRFs) including myogenic differentiation (MyoD), myogenin (MyoG), myogenic factor 5 (Myf5) and myogenic regulatory factor 4 (MRF4) in ESM of MSTN-/- pigs showed a significant increase (P < 0.05 at least) when compared to WT pigs while mRNA level of myocyte enhancer factor 2C (MEF2C) displayed a decrease (P < 0.001). Protein expression of myosin heavy chain I (MHC-I) in MSTN-/- ESM was decreased and myosin heavy chain IIA (MHC-IIA) was increased (P < 0.01, P < 0.05). These findings indicate that MSTN plays an important role in esophageal striated muscle development and regulates muscle fiber types.

miR-320 regulates myogenesis by targeting growth factor receptor-bound protein-2 and ameliorates myotubes atrophy.

Loss of muscle mass can lead to diseases such as sarcopenia, diabetes, and obesity, which can worsen the quality of life and increase the incidence of disease. Therefore, understanding the mechanism underlying skeletal muscle differentiation is vital to prevent muscle diseases. We previously found that microRNA-320 (miR-320) is highly expressed in the lean muscle-type pigs, but its regulatory role in myogenesis remains unclear. The bioinformatics prediction indicated that miR-320 could bind to the 3 'untranslated region of growth factor receptor-bound protein-2 (Grb2). We hypothesized that miR-320 targets Grb2 to regulate myoblasts differentiation. To verify this, we transfected miR-320 mimic and inhibitor into C2C12 myoblasts to assess the role of miR-320 during myoblasts differentiation. We used real-time qPCR, luciferase reporter assays, and western blotting to confirm that miR-320 directly targets Grb2 to promote myoblasts differentiation. Moreover, by using a dexamethasone-induced atrophic model of myotubes, we discovered that miR-320 promotes the repair of damaged myotubes. Our findings expand understanding of miRNAs and genes related to regulating skeletal muscle differentiation, and provide insight into underlying therapeutic strategies for muscle diseases.