Expression patterns of intermediate filament proteins desmin and lamin A in the developing conduction system of early human embryonic hearts.

Since embryonic heart development is a complex process and acquisition of human embryonic specimens is challenging, the mechanism by which the embryonic conduction system develops remains unclear. Herein, we attempt to gain insights into this developmental process through immunohistochemical staining and 3D reconstructions. Expression analysis of T-box transcription factor 3, cytoskeleton desmin, and nucleoskeleton lamin A protein in human embryos in Carnegie stages 11-20 showed that desmin is preferentially expressed in the myocardium of the central conduction system compared with the peripheral conduction system, and is co-expressed with T-box transcription factor 3 in the central conduction system. Further, lamin A was first expressed in the embryonic ventricular trabeculations, where the terminal ramifications of the peripheral conduction system develop, and extended progressively to all parts of the central conduction system. The uncoupled spatiotemporal distribution pattern of lamin A and desmin indicated that the association of cytoskeleton desmin and nucleoskeleton lamin A may be a late event in human embryonic heart development. Compared with model animals, our data provide a direct morphological basis for understanding the arrhythmogenesis caused by mutations in human DES and LMNA genes.

MCU inhibition protects against intestinal ischemia‒reperfusion by inhibiting Drp1-dependent mitochondrial fission.

Intestinal ischemia‒reperfusion (IIR) injury is a common complication of surgery, but clear molecular insights and valuable therapeutic targets are lacking. Mitochondrial calcium overload is an early sign of various diseases and is considered a vital factor in ischemia‒reperfusion injury. The mitochondrial calcium uniporter (MCU), which is located on the inner mitochondrial membrane, is the primary mediator of calcium ion entry into the mitochondria. However, the specific mechanism of MCU in IIR injury remains to be clarified. In this study, we generated an IIR model using C57BL/6 mice and Caco-2 cells and found increases in the calcium levels and MCU expression following IIR injury. The specific inhibition of MCU markedly attenuated IIR injury. Moreover, MCU knockdown alleviates mitochondrial dysfunction by reducing oxidative stress and apoptosis. Mechanistically, MCU knockdown substantially reduced the translocation of Drp1 and thus its binding to Fis1 receptors, resulting in decreased mitochondrial fission. Taken together, our findings demonstrated that MCU is a novel upstream regulator of Drp1 in ischemia‒reperfusion and represents a predictive and therapeutic target for IIR.

Screening and evaluation of human single-chain fragment variable antibody against hepatitis B virus surface antigen.

BACKGROUND: Phage display technology has become a vital tool in studies aimed at identifying molecules binding to a specific target. It enables the rapid generation and selection of high affinity, fully human antibody product candidates to essentially any disease target appropriate for antibody therapy. In this study, we prepared the recombinant single-chain fragment variable (ScFv) antibody to hepatitis B virus surface antigen (HBsAg) by the phage display technology for obtaining a virus-targeting mediator. METHODS: mRNA was isolated from B-lymphocytes from a healthy volunteer and converted into cDNA. The fragment variables of heavy and light chain were amplified separately and assembled into ScFv DNA with a specially constructed DNA linker by polymerase chain reaction. The ScFv DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E.coli TG1. The transformed cells were infected with M13K07 helper phage to form a human recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by bacterial colony count and restriction analysis. After two rounds of panning with HBsAg, the phage clones displaying ScFv of the antibody were selected by enzyme-linked immunosorbant assay (ELISA) from the enriched phage clones. The antigen binding affinity of the positive clone was detected by competition ELISA. HB2151 E.coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the anti-HBsAg ScFv. ELISA assay was used to detect the antigen binding affinity of the soluble anti-HBsAg ScFv. Finally, the relative molecular mass of soluble anti-HBsAg ScFv was measured by SDS-PAGE. RESULTS: The variable heavy (VH) and variable light (VL) and ScFv DNAs were about 340 bp, 320 bp and 750 bp, respectively. The volume of the library was up to 2 x 10(6) and 8 of 10 random clones were recombinants. Two phage clones could strongly compete with the original HBsAb for binding to HBsAg. Within 2 strong positive phage clones, the soluble anti-HBsAg ScFv from one clone was found to have the binding activity with HBsAg. SDS-PAGE showed that the relative molecular weight of soluble anti-HBsAg ScFv was 32 kDa. CONCLUSION: The anti-HBsAg ScFv successfully produced by phage antibody technology may be useful for broadening the scope of application of the antibody.