Inter-species somatic cell nuclear transfer (iSCNT) is significant in the study of biological problems such as embryonic genome activation and the mitochondrial function of embryos. Here, we used ...iSCNT as a model to determine whether abnormal embryo genome activation was caused by mitochondrial dysfunction. First, we found the ovine-bovine iSCNT embryos were developmentally blocked at the 8-cell stage. The reactive oxygen species level, mitochondrial membrane potential, and ATP level in ovine-bovine cloned embryos were significantly different from both bovine-bovine and IVF 8-cell stage embryos. RNA sequencing and q-PCR analysis revealed that mitochondrial transport, mitochondrial translational initiation, mitochondrial large ribosomal subunit, and mitochondrial outer membrane genes were abnormally expressed in the ovine-bovine embryos, and the mitochondrial outer membrane and mitochondrial ribosome large subunit genes, mitochondrial fusion gene 1, and ATPase Na+/K+ transporting subunit beta 3 gene were expressed at lower levels in the ovine-bovine cloned embryos. Furthermore, we found that overexpression and knockdown of Mfn1 significantly affected mitochondrial fusion and subsequent biological functions such as production of ATP, mitochondrial membrane potential, reactive oxygen species and gene expressions in cloned embryos. These findings enhance our understanding of the mechanism by which the Mfn1 gene regulates embryonic development and embryonic genome activation events.
Cationic nanocarrier mediated intracellular therapeutic agent delivery acts as a double-edged sword: the carriers promote cellular uptake, but interact nonspecifically and strongly with negatively ...charged endogenic proteins and cell membranes, which results in aggregates and high cytotoxicity. The present study was aimed at exploring zwitterionic polyaspartamide derivative nanoparticles for efficient intracellular delivery with low cytotoxicity. Poly(aspartic acid) partially grafted tetraethylenepentamine (PASP-pg-TEPA) with different isoelectric points (IEPs) was synthesized. The PASP-pg-TEPA formed zwitterionic nanoparticles with an irregular core and a well-defined shell structure in aqueous medium. Their particle size decreased from about 300 to 80 nm with an increase of the IEP from 7.5 to 9.1. The surface charge of the PASP-pg-TEPA nanoparticles could be tuned from positive to negative with a change of the pH of the medium. The nanoparticles with an IEP above 8.5 exhibited good stability under simulated physiological conditions. It was noted that the zwitterionic PASP-pg-TEPA nanoparticles displayed highly efficient cellular uptake in HeLa cells (approximately 99%) in serum-containing medium and did not adversely affect the cell viability at concentrations up to 1 mg/mL. Furthermore, thermodynamic analysis using isothermal titration calorimetry provided direct evidence that these zwitterionic nanoparticles had low binding affinities for serum protein. Therefore, the zwitterionic PASP-pg-TEPA nanoparticles could overcome limitations of cationic nanocarriers and achieve efficient intracellular delivery with low cytotoxicity.
Myostatin (MSTN) is a major negative regulator of skeletal muscle mass and causes a variety of metabolic changes. However, the effect of MSTN knockout on bile acid metabolism has rarely been ...reported. In this study, the physiological and biochemical alterations of serum in MSTN
and wild type (WT) cattle were investigated. There were no significant changes in liver and kidney biochemical indexes. However, compared with the WT cattle, lactate dehydrogenase, total bile acid (TBA), cholesterol, and high-density lipoprotein (HDL) in the MSTN
cattle were significantly increased, and glucose, low-density lipoprotein (LDL), and triglycerides (TG) were significantly decreased, indicating that MSTN knockout affected glucose and lipid metabolism and total bile acids content. Targeted metabolomic analysis of the bile acids and their derivatives was performed on serum samples and found that bile acids were significantly increased in the MSTN
cattle compared with the WT cattle. As the only bile acid synthesis organ in the body, we performed metabolomic analysis on the liver to study the effect of MSTN knockout on hepatic metabolism. Metabolic pathway enrichment analysis of differential metabolites showed significant enrichment of the primary bile acid biosynthesis and bile secretion pathway in the MSTN
cattle. Targeted metabolomics data further showed that MSTN knockout significantly increased bile acid content in the liver, which may have resulted from enhanced bile acid synthesis due to the expression of bile acid synthesis genes, cholesterol 7 alpha-hydroxylase (CYP7A1) and sterol 27-hydroxylase (CYP27A1), and upregulation in the liver of the MSTN
cattle. These results indicate that MSTN knockout does not adversely affect bovine fitness but regulates bile acid metabolism via enhanced bile acid synthesis. This further suggests a role of MSTN in regulating metabolism.
(1) Background: Myostatin (MSTN) is a protein that regulates skeletal muscle development and plays a crucial role in maintaining animal body composition and muscle structure. The loss-of-function ...mutation of MSTN gene can induce the muscle hypertrophic phenotype. (2) Methods: Growth indexes and blood parameters of the cattle of different months were analyzed via multiple linear regression. (3) Results: Compared with the control group, the body shape parameters of F2 cattle were improved, especially the body weight, cross height, and hip height, representing significant development of hindquarters, and the coat color of the F2 generation returned to the yellow of Luxi cattle. As adults, MSTN gene-edited bulls have a tall, wide acromion and a deep, wide chest. Both the forequarters and hindquarters are double-muscled with clear muscle masses. The multiple linear regression demonstrates that MSTN gene-edited hybrid beef cattle gained weight due to the higher height of the hindquarters. Significant differences in blood glucose, calcium, and low-density lipoprotein. Serum insulin levels decreased significantly at 24 months of age. MSTN gene editing improves the adaptability of cattle. (4) Conclusions: Our findings suggest that breeding with MSTN gene-edited Luxi bulls can improve the growth and performance of hybrid cattle, with potential benefits for both farmers and consumers.
Background
Cornelia de Lange syndrome (CdLS) is a multisystem genetic disorder, and cases caused by variants in the structural maintenance of chromosomes protein 3 (SMC3) gene are uncommon. Here, we ...report two cases of CdLS associated with novel pathogenic variants in SMC3 from two Chinese families.
Methods
Clinical presentations of two patients with CdLS were evaluated, and specimens from the patients and other family members were collected for Trio‐based whole‐exome sequencing. Pyrosequencing, chip‐based digital PCR, minigene splicing assay, and in silico analysis were carried out to elucidate the impact of novel variants.
Results
Novel heterozygous variants in SMC3 were identified in each proband. One harbored a novel splicing and mosaic variant (c.2535+1G>A) in SMC3. The mutated allele G>A conversion was approximately 23.1% by digital PCR, which indicated that 46.2% of peripheral blood cells had this variant. Additionally, in vitro minigene splicing analysis validated that the c.2535+1G>A variant led to an exon skipping in messenger RNA splicing. The other carried a heterozygous variant (c.435C>A), which was predicted to be pathogenic as well as significantly altered in local electrical potential. The former showed multiple abnormalities and marked clinical severity, and the latter mainly exhibited a speech developmental disorder and slightly facial anomalies.
Conclusion
Both patients were clinically diagnosed with Cornelia de Lange syndrome 3 (CdLS3). The newly identified SMC3 gene variants can expand the understanding of CdLS3 and provide reliable evidence for genetic counseling to the affected family.
Identification of two novel potential pathogenic variants in SMC3 in two clinically heterogeneous children with CdLS.
Chinese Yellow Cattle, an ancient and domesticated breed for draft service, provide unique animal genetic resources with excellent genetic features, including crude feed tolerance, good stress ...resistance, strong adaptability, and tender meat quality; however, their production performance and meat yield are significantly inferior. Herein, the myostatin gene (
), a negative regulator of skeletal muscle development, was knocked out by CRISPR/Cas9 technology. Eight
gene-edited bull calves (MT) were born, and six of them are well-developed. Compared with the control cattle (WT), the growth trait indexes of MT cattle were generally increased, and the hindquarters especially were significantly improved. The biochemical indexes and the semen characteristics demonstrated that MT bulls were healthy and fertile. Consistent with our conjecture, the wobble and beating of MT bull spermatozoa were significantly higher than that of WT. Nine sperm motility-related proteins and nineteen mitochondrial-related proteins were identified by up-regulation in MT bull spermatozoa using FLQ proteomic technique and act to govern sperm flagellum assembly, organization, and beating and provide sufficient energy for sperm motility. The current study confirmed that the
gene-edited Chinese Yellow cattle have improved growth traits and normal fertility, which can be used for beef cattle production and breeding.
Myostatin (MSTN) is a negative regulator of skeletal muscle genesis during development. MSTN mutation leads to increased lean meat production and reduced fat deposition in livestock. However, the ...mechanism by which MSTN promotes myogenesis by regulating metabolism is not clear. In this study, we compared the metabolomics of the livers of wild-type (WT) and MSTN mutation cattle (MT), and found changes in the content and proportion of fatty acids and bile acids in MT cattle. The differential metabolites were enriched in sterol synthesis and primary bile acid synthesis. We further analyzed the expression of genes involved in the regulation of lipid and bile acid metabolism, and found that the loss of MSTN may alter lipid synthesis and bile acid metabolism. This study provides new basic data for MSTN mutations in beef cattle breeding.
Short interfering RNAs (siRNAs) as chemotherapeutic RNAi agents hold great promise for a significant improvement in cancer therapy. Despite the promise, effective transport of siRNA with minimal side ...effects remains a challenge. The common problem associated with the low delivery efficiencies of current polycation-based gene delivery systems is their low stability in the presence of salt and serum. In the present study we developed the polyglutamate derivatives (PGS) polyelectrolyte brushes for NF-κB p65 siRNA delivery. The PGS polyelectrolyte brushes/siRNA polyplex was colloidally stable (150 nm diameter) in physiological saline (150 mM NaCl), likely due to the osmotic brushes of PGS. The size-controlled siRNA/PGS polyplex also showed the serum resistance resulting in their efficient cellular uptake was not negatively influenced by the presence of serum. The endothermic profile of ITC, their low values of Gibbs free energy and binding constants K b under salt conditions provided the direct evidence that PGS polyelectrolyte brushes had a much lower binding affinity for serum proteins, compared with PEI 25KDa. PGS polyelectrolyte brushes delivering NF-κB p65 siRNA achieved efficient down-regulation of NF-κB p65 protein in HeLa cells. The NF-κB p65 down-regulation mediated by PGS polyelectrolyte brushes was more significant than PEI 25KDa and comparable to Lipofectamine 2000. Furthermore, the combination treatment with PGS polyelectrolyte brushes/NF-κB p65 siRNA polyplex and doxorubicin demonstrated synergistic apoptotic and cytotoxic effects on HeLa cancer cells. The high stability in physiological saline and salt-induced serum resistance of PGS polyelectrolyte brushes/siRNA polyplex has potential applications together with standard chemotherapies such as doxorubicin to be a viable method to improve the clinical outcomes in cancer therapies.
The transfection efficiency of cationic polymers decreases dramatically in the presence of serum, which hampers the
in vivo application of these polymers for gene delivery. Due to its shielding ...effect of poly(α-glutamic acid) (PGA) from negatively charged serum proteins, it was introduced into DNA polyplexes to overcome the serum inhibitory effect. In the present studies, the transfection efficiency of DNA/PEI/PGA terplex system was compared to PEI 25
kDa and Lipofectamine 2000 in the presence of serum. The successful formation of DNA/PEI/PGA terplexes was confirmed by their near-neutral surface charge. Interaction between components in the terplex system demonstrated that PGA was competing with DNA to combine with PEI. PEI/PGA combined carriers were not cytotoxic at a C/N ratio higher than 0.3. The
in vitro transfection efficiency of DNA/PEI/PGA terplexes was not significantly different from those of DNA/PEI25
kDa in serum-free medium. Importantly, in serum-containing medium, the DNA terplexes at their optimal C/N ratios maintained the same level of transfection efficiency as that of serum-free medium, even though the transfection efficiency of PEI 25
kDa and Lipofectamine 2000 was significantly decreased under serum-containing conditions. CLSM results confirmed that the cellular import of pDNA delivered by PEI/PGA combined carriers was more efficient than PEI 25
kDa alone under serum-containing conditions. Therefore, PGA could be used as a versatile serum-resistant reagent to overcome the serum inhibitory effect of polycations for gene delivery.
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