Cardiomyocyte ßII spectrin plays a critical role in maintaining cardiac function by regulating mitochondrial respiratory function.

AIMS: ßII spectrin is a cytoskeletal protein known to be tightly linked to heart development and cardiovascular electrophysiology. However, the roles of ßII spectrin in cardiac contractile function and pathological post-myocardial infarction remodelling remain unclear. Here, we investigated whether and how ßII spectrin, the most common isoform of non-erythrocytic spectrin in cardiomyocytes, is involved in cardiac contractile function and ischaemia/reperfusion (I/R) injury. METHODS AND RESULTS: We observed that the levels of serum ßII spectrin breakdown products (ßII SBDPs) were significantly increased in patients with acute myocardial infarction (AMI). Concordantly, ßII spectrin was degraded into ßII SBDPs by calpain in mouse hearts after I/R injury. Using tamoxifen-inducible cardiac-specific ßII spectrin knockout mice, we found that deletion of ßII spectrin in the adult heart resulted in spontaneous development of cardiac contractile dysfunction, cardiac hypertrophy, and fibrosis at 5 weeks after tamoxifen treatment. Moreover, at 1 week after tamoxifen treatment, although spontaneous cardiac dysfunction in cardiac-specific ßII spectrin knockout mice had not developed, deletion of ßII spectrin in the heart exacerbated I/R-induced cardiomyocyte death and heart failure. Furthermore, restoration of ßII spectrin expression via adenoviral small activating RNA (saRNA) delivery into the heart reduced I/R injury. Immunoprecipitation coupled with mass spectrometry (IP-LC-MS/MS) analyses and functional studies revealed that ßII spectrin is indispensable for mitochondrial complex I activity and respiratory function. Mechanistically, ßII spectrin promotes translocation of NADH:ubiquinone oxidoreductase 75-kDa Fe-S protein 1 (NDUFS1) from the cytosol to mitochondria by crosslinking with actin filaments (F-actin) to maintain F-actin stability. CONCLUSION: ßII spectrin is an essential cytoskeletal element for preserving mitochondrial homeostasis and cardiac function. Defects in ßII spectrin exacerbate cardiac I/R injury.

Identification of a highly conserved neutralizing epitope within the RBD region of diverse SARS-CoV-2 variants.

The constant emergence of SARS-CoV-2 variants continues to impair the efficacy of existing neutralizing antibodies, especially XBB.1.5 and EG.5, which showed exceptional immune evasion properties. Here, we identify a highly conserved neutralizing epitope targeted by a broad-spectrum neutralizing antibody BA7535, which demonstrates high neutralization potency against not only previous variants, such as Alpha, Beta, Gamma, Delta and Omicron BA.1-BA.5, but also more recently emerged Omicron subvariants, including BF.7, CH.1.1, XBB.1, XBB.1.5, XBB.1.9.1, EG.5. Structural analysis of the Omicron Spike trimer with BA7535-Fab using cryo-EM indicates that BA7535 recognizes a highly conserved cryptic receptor-binding domain (RBD) epitope, avoiding most of the mutational hot spots in RBD. Furthermore, structural simulation based on the interaction of BA7535-Fab/RBD complexes dissects the broadly neutralizing effect of BA7535 against latest variants. Therapeutic and prophylactic treatment with BA7535 alone or in combination with BA7208 protected female mice from the circulating Omicron BA.5 and XBB.1 variant infection, suggesting the highly conserved neutralizing epitope serves as a potential target for developing highly potent therapeutic antibodies and vaccines.

Grpel2 alleviates myocardial ischemia/reperfusion injury by inhibiting MCU-mediated mitochondrial calcium overload.

Mitochondrial calcium ([Ca2+]m) overload is considered a major trigger of cardiomyocyte death during myocardial ischemia/reperfusion (I/R) injury. Grpel2 is located in mitochondria and facilitates the mtHSP70 protein folding cycle in oxidative stress. However, Grpel2 expression during I/R injury and its impact on I/R injury remain poorly understood. This study explored the role of Grpel2 in I/R injury and its underlying mechanism. Mice were intramyocardially injected with recombinant adenovirus vectors to knockdown cardiac Grpel2 expression, and a myocardial I/R model was established. We confirmed that cardiac Grpel2 is upregulated during I/R injury. Cardiac-specific Grpel2 knockdown exacerbates mitochondrial fission, cardiomyocyte death and cardiac contractile dysfunction induced by I/R injury. Moreover, our study revealed that Grpel2 knockdown increased both MCU expression and [Ca2+]m content. Excessive mitochondrial fission and apoptosis were rescued by Ru360, an inhibitor of MCU opening. In summary, our findings suggest that Grpel2 alleviates myocardial ischemia/reperfusion injury by inhibiting MCU-mediated mitochondrial calcium overload and provide new insights into the mechanism of MCU-mediated [Ca2+]m homeostasis during I/R injury.

Neuronatin gene expression levels affect foetal growth and development by regulating glucose transport in porcine placenta.

Imprinted genes play important regulatory roles in the growth and development of placentas and foetuses during pregnancy. In a previous study, we found that the imprinted gene Neuronatin (NNAT) is involved in foetal development; NNAT expression was significantly lower in the placentas of piglets that died neonatally compared to the placentas of surviving piglets. However, the function and mechanism of NNAT in regulating porcine placental development is still unknown. In this study, we collected the placentas of high- and low-weight foetuses at gestational day (GD 65, 90), (n = 4-5 litters/GD) to investigate the role of NNAT in regulating foetal growth and development. We found that the mRNA and protein levels of NNAT were significantly higher in the placentas of high-weight than low-weight foetuses. We then overexpressed NNAT in porcine placental trophoblast cell lines (pTr2) and demonstrated that NNAT activated the PI3K-AKT pathway, and further promoted the expression of glucose transporter 1 (GLUT1) and increased cellular calcium ion levels, which improved glucose transport in placental trophoblast cells in vitro. To conclude, our study suggests that NNAT expression impacts porcine foetal development by regulating placental glucose transport.

Establishment of Etv5 gene knockout mice as a recipient model for spermatogonial stem cell transplantation.

Spermatogonial stem cell (SSC) transplantation is an alternative reproductive method to achieve conservation and production of elite animals in livestock production. Creating a recipient animal without endogenous germ cells is important for effective SSC transplantation. However, natural mutants with depletion of SSCs are difficult to obtain, and drug ablation of endogenous germ cells is arduous to perform for practical use. In this study, we used mouse models to study the preparation of recipients with congenital germ cell ablation. We knocked out (KO) Ets-variant gene 5 (Etv5) in mice using the CRISPR/Cas9 system. The testicular weight of Etv5-/- mice was significantly lower than that of wild-type (WT) mice. The germ cell layer of the seminiferous tubules gradually receded with age in Etv5-/- mice. At 12 weeks of age, the tubules of Etv5-/- mice lacked almost all spermatogenic cells with a Sertoli cell-only phenotype, and sperm were completely absent in the epididymis. We subsequently transplanted allogeneic SSCs with enhanced green fluorescent protein (EGFP) into 3- (immature) or 7-week-old (mature) Etv5-/- mice. Partial restoration of germ cell layers in the seminiferous tubules and spermatogenesis was observed in all immature testes but not in mature adult testes at 2 months post-transplantation. The presence of heterologous genes Etv5 and EGFP in recipient testicular tissue and epididymal sperm by PCR indicated that sperm originated from the transplanted donor cells. Our study demonstrates that, although Etv5-/- mice could accommodate and support foreign germ cell transplantation, this process occurs in a quite low efficiency to support a full spermatogenesis of transplanted SSCs. However, using Etv5-/- mice as a recipient model for SSC transplantation is feasible, and still needs further investigation to establish an optimized transplantation process.

Identification and Expression Pattern of EZH2 in Pig Developing Fetuses.

The proper methylation status of histones is essential for appropriate cell lineage and organogenesis. EZH2, a methyltransferase catalyzing H3K27me3, has been abundantly studied in human and mouse embryonic development. The pig is an increasing important animal model for molecular study and pharmaceutical research. However, the transcript variant and temporal expression pattern of EZH2 in the middle and late porcine fetus are still unknown. Here, we identified the coding sequence of the EZH2 gene and characterized its expression pattern in fetal tissues of Duroc pigs at 65- and 90-day postcoitus (dpc). Our results showed that the coding sequence of EZH2 was 2241 bp, encoding 746 amino acids. There were 9 amino acid insertions and an amino acid substitution in this transcript compared with the validated reference sequence in NCBI. EZH2 was ubiquitously expressed in the fetal tissues of two time points with different expression levels. These results validated a different transcript in pigs and characterized its expression profile in fetal tissues of different gestation stages, which indicated that EZH2 played important roles during porcine embryonic development.

Increased Active Tumor Targeting by An αvß3-Targeting and Cell-Penetrating Bifunctional Peptide-Mediated Dendrimer-Based Conjugate.

PURPOSE: A bifunctional RGDTAT peptide-modified PEG-PAMAM dendrimer conjugate RGDTAT-PEG-PAMAM (RTPP) was established for the targeted treatment of αvß3-overexpressing tumor cells. METHODS: The RGDTAT peptide was synthesized and attached to PAMAM using PEG to construct the RTPP conjugate. The methotrexate (MTX) encapsulated RTPPM complex was prepared and characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM) and in vitro release. The targeting ability was then studied in cells and tumor-bearing nude mice using fluorescence microscopy, confocal fluorescence microscopy, flow cytometry, and in vivo imaging. The cytotoxicity and pharmacokinetics of the RTPPM complex was also evaluated in cells and rats. RESULTS: The successful synthesis of the RTPP conjugate was confirmed by 1H-NMR. DLS and TEM measurements revealed that the size was 37 nm and the complex had a spherical shape. RTPP and RTPPM were taken up by αvß3-overexpressing cells more efficiently than by αvß3-lowexpressing cells. The RTPP conjugate localized to the cell nucleus and accumulated in the tumor more efficiently than did the conjugates without RGDTAT. The pharmacokinetic study of the RTPPM complex showed sustained drug release. CONCLUSIONS: The bifunctional peptide-mediated dendrimer-based RTPP conjugate can serve as a promising nanocarrier for targeted drug delivery to improve anti-tumor activity.

Colon-Targeted Delivery of IgY Against Clostridium difficile Toxin A and B by Encapsulation in Chitosan-Ca Pectinate Microbeads.

This study investigated the use of a newly developed chitosan-Ca pectinate microbead formulation for the colon-targeted delivery of anti-A/B toxin immunoglobulin of egg yolk (IgY) to inhibit toxin binding to colon mucosa cells. The effect of the three components (pectinate, calcium chloride, and chitosan) used for the microbead production was examined with the aim of identifying the optimal levels to improve drug encapsulation efficiency, swelling ratio, and cumulative IgY release rate. The optimized IgY-loaded bead component was pectin 5% (w/v), CaCl2 3% (w/v), and chitosan 0.5% (w/v). Formulated beads were spherical with 1.2-mm diameter, and the drug loading was 45%. An in vitro release study revealed that chitosan-Ca pectinate microbeads inhibited IgY release in the upper gastrointestinal tract and significantly improved the site-specific release of IgY in the colon. An in vivo rat study demonstrated that 72.6% of biologically active IgY was released specifically in the colon. These results demonstrated that anti-A/B toxin IgY-loaded chitosan-Ca pectinate oral microbeads improved IgY release behavior in vivo, which could be used as an effective oral delivery platform for the biological treatment of Clostridium difficile infection (CDI).

Development of microbeads of chicken yolk antibodies against Clostridium difficile toxin A for colonic-specific delivery.

The incidence of Clostridium difficile infection has increased in Western world in the past 10 years, similar infection rates are also reported in developing countries such as China. Current antibiotics treatments have recurrence rates between 15% and 30%. IgY antibodies against toxin A of C. difficile could protect animal models from the challenge of lethal dose of C. difficile spores. However, IgY is sensitive to the low pH environment of the stomach and proteinases in the intestine. The objective of this study was to prepare colonic-specific delivery system of toxin A antigen-specific IgY to block the recognition of toxin A to the colon mucosa cells. Egg-laying hens were immunized with purified C. difficile toxin A C-terminal domain for 3 times, then egg IgY against the recombinant ToxA-C protein was purified from immunized egg yolk and frozen dried. IgY-loaded microbeads were prepared using mini fluid bed system; the loading efficiency was 21%. The pH and temperature stabilities of the microbeads were assayed. The IgY-loaded microbeads coated with 35% Eudragit S100 had colonic-specific IgY release specificity both in vitro and in vivo, the colonic-specific release of biological active IgY was 87.5% in the rat. Our study provides a new option for the biological treatment C. difficile infection.