Gut Microbial Compositions in Four Age Groups of Tibetan Minipigs

In this study, the gut microbiota was characterized in four age strata of Tibetan minipigs. Results indicated that the fecal bacteria of 7-, 28-, 56-, and 180-day-old minipigs did not significantly differ in terms of phylogenetic diversity (i.e., PD whole tree) or the Shannon index (both, p > 0.05). Findings of a principal coordinate analysis demonstrated that fecal bacteria of 180-day-old minipigs were discernable from those of the other three age groups. From ages seven to 56 days, the abundance of Bacteroidetes or Firmicutes appeared to vary. Regarding genera, the populations of Bacteroides and Akkermansia decreased with increasing age

Use of the 2A Peptide for Generation of Multi-Transgenic Pigs through a Single Round of Nuclear Transfer

Multiple genetic modifications in pigs can essentially benefit research on agriculture, human disease and xenotransplantation. Most multi-transgenic pigs have been produced by complex and time-consuming breeding programs using multiple single-transgenic pigs. This study explored the feasibility of producing multi-transgenic pigs using the viral 2A peptide in the light of previous research indicating that it can be utilized for multi-gene transfer in gene therapy and somatic cell reprogramming. A 2A peptide-based double-promoter expression vector that mediated the expression of four fluorescent proteins was constructed and transfected into primary porcine fetal fibroblasts. Cell colonies (54.3%) formed under G418 selection co-expressed the four fluorescent proteins at uniformly high levels. The reconstructed embryos, which were obtained by somatic cell nuclear transfer and confirmed to express the four fluorescent proteins evenly, were transplanted into seven recipient gilts. Eleven piglets were delivered by two gilts, and seven of them co-expressed the four fluorescent proteins at equivalently high levels in various tissues. The fluorescence intensities were directly observed at the nose, hoof and tongue using goggles. The results suggest that the strategy of combining the 2A peptide and double promoters efficiently mediates the co-expression of the four fluorescent proteins in pigs and is hence a promising methodology to generate multi-transgenic pigs by a single nuclear transfer

Improved Biocompatibility of Novel Biodegradable Scaffold Composed of Poly-L-lactic Acid and Amorphous Calcium Phosphate Nanoparticles in Porcine Coronary Artery

Using poly-L-lactic acid for implantable biodegradable scaffold has potential biocompatibility issue due to its acidic degradation byproducts. We have previously reported that the addition of amorphous calcium phosphate improved poly-L-lactic acid coating biocompatibility. In the present study, poly-L-lactic acid and poly-L-lactic acid/amorphous calcium phosphate scaffolds were implanted in pig coronary arteries for 28 days. At the follow-up angiographic evaluation, no case of stent thrombosis was observed, and the arteries that were stented with the copolymer scaffold had significantly less inflammation and nuclear factor-κB expression and a greater degree of reendothelialization. The serum levels of vascular endothelial growth factor and nitric oxide, as well the expression of endothelial nitric oxide synthase and platelet-endothelial cell adhesion molecule-1, were also significantly higher. In conclusion, the addition of amorphous calcium phosphate to biodegradable poly-L-lactic acid scaffold minimizes the inflammatory response, promotes the growth of endothelial cells, and accelerates the reendothelialization of the stented coronary arteries

KLF4 Promotes Angiogenesis by Activating VEGF Signaling in Human Retinal Microvascular Endothelial Cells

<div><p>The transcription factor Krüppel-like factor 4 (KLF4) has been implicated in regulating cell proliferation, migration and differentiation in a variety of human cells and is one of four factors required for the induction of pluripotent stem cell reprogramming. However, its role has not been addressed in ocular neovascular diseases. This study investigated the role of KLF4 in angiogenesis and underlying molecular mechanisms in human retinal microvascular endothelial cells (HRMECs). The functional role of KLF4 in HRMECs was determined following lentiviral vector mediated inducible expression and shRNA knockdown of KLF4. Inducible expression of KLF4 promotes cell proliferation, migration and tube formation. In contrast, silencing KLF4 inhibits cell proliferation, migration, tube formation and induces apoptosis in HRMECs. KLF4 promotes angiogenesis by transcriptionally activating VEGF expression, thus activating the VEGF signaling pathway in HRMECs.</p></div

KLF4 promotes VEGF-induced tube formation and enhances angiogenesis in vivo.

<p><b>A.B.</b> Tube formation assays were performed in KLF4 expressing and knockdown HRMECs, respectively. The angiogenic effect of KLF4 on VEGF induced tube formation was determined by counting nodes and sprouts of tube-like structures from at least three different fields of three independent experiments and normalized to vehicle treated control cells. Significance was compared between KLF4 expressing and control cells with or without VEGF treatment (*p<0.05, **p<0.01, ***p<0.001). Images were presented from one representative experiment. C. Sections of plugs were stained using CD31 antibody and microvessels were counted from 4 sections of each plug and averaged from total 3 plugs. Significance of CD31 positive vessels were compared between sections of KLF4 expressing and control plugs (*p<0.05).</p

Schematic diagram of KLF4 mediated VEGF signaling pathway.

<p>KLF4 binds to the VEGF promoter and induces VEGF expression, subsequently phosphorylates VEGFR2 and activates downstream ERK1/2 and AKT to promote cell proliferation, migration and angiogenesis.</p