Search

With the convergent global emergence of SARS-CoV-2 variants of concern (VOC), a precise comparison study of viral fitness and transmission characteristics is necessary for the prediction of dominant VOCs and the development of suitable countermeasures. While airway temperature plays important roles in the fitness and transmissibility of respiratory tract viruses, it has not been well studied with SARS-CoV-2. Here we demonstrate that natural temperature differences between the upper (33°C) and lower (37°C) respiratory tract have profound effects on SARS-CoV-2 replication and transmission. Specifically, SARS-COV-2 variants containing the P323L or P323L/G671S mutation in the NSP12 RNA-dependent RNA polymerase (RdRp) exhibited enhanced RdRp enzymatic activity at 33°C compared to 37°C and high transmissibility in ferrets. MicroScale Thermophoresis demonstrated that the NSP12 P323L or P323L/G671S mutation stabilized the NSP12-NSP7-NSP8 complex interaction. Furthermore, reverse genetics-derived SARS-CoV-2 variants containing the NSP12 P323L or P323L/G671S mutation displayed enhanced replication at 33°C, and high transmission in ferrets. This suggests that the evolutionarily forced NSP12 P323L and P323L/G671S mutations of recent SARS-CoV-2 VOC strains are associated with increases of the RdRp complex stability and enzymatic activity, promoting the high transmissibility.

Alternative cancer models that are close to humans are required to create more valuable preclinical results during oncology studies. Here, a new onco-pig model via developing a CRISPR-Cas9-based Conditional Polycistronic gene expression Cassette (CRI-CPC) system to control the tumor inducing simian virus 40 large T antigen (SV40LT) and oncogenic HRAS

Although the human brain would be an ideal model for studying human neuropathology, it is difficult to perform in vitro culture of human brain cells from genetically engineered healthy or diseased brain tissue. Therefore, a suitable model for studying the molecular mechanisms responsible for neurological diseases that can appropriately mimic the human brain is needed. Somatic cell nuclear transfer (SCNT) was performed using an established porcine Yucatan EGFP cell line and whole seeding was performed using SCNT blastocysts. Two Yucatan EGFP porcine embryonic stem-like cell (pESLC) lines were established. These pESLC lines were then used to establish an in vitro neuro-organoids. Aggregates were cultured in vitro until 61 or 102 days after neural induction, neural patterning, and neural expansion. The neuro-organoids were sampled at each step and the expression of the dopaminergic neuronal marker (TH) and mature neuronal marker (MAP2) was confirmed by reverse transcription-PCR. Expression of the neural stem cell marker (PAX6), neural precursor markers (S100 and SOX2), and early neural markers (MAP2 and Nestin) were confirmed by immunofluorescence staining. In conclusion, we successfully established neuro-organoids derived from pESLCs in vitro. This protocol can be used as a tool to develop in vitro models for drug development, patient-specific chemotherapy, and human central nervous system disease studies.

Recent advances in somatic cell nuclear transfer (SCNT) in canines facilitate the production of canine transgenic models. Owing to the importance of stable and strong promoter activity in transgenic animals, we tested human elongation factor 1α (hEF1α) and cytomegalovirus (CMV) promoter sequences in SCNT transgenic dogs. After transfection, transgenic donor fibroblasts with the hEF1α-enhanced green fluorescence protein (EGFP) transgene were successfully isolated using fluorescence-activated cell sorting (FACS). We obtained four puppies, after SCNT, and identified three puppies as being transgenic using PCR analysis. Unexpectedly, EGFP regulated by hEF1α promoter was not observed at the organismal and cellular levels in these transgenic dogs. EGFP expression was rescued by the inhibition of DNA methyltransferases, implying that the hEF1α promoter is silenced by DNA methylation. Next, donor cells with CMV-EGFP transgene were successfully established and SCNT was performed. Three puppies of six born puppies were confirmed to be transgenic. Unlike hEF1α-regulated EGFP, CMV-regulated EGFP was strongly detectable at both the organismal and cellular levels in all transgenic dogs, even after 19 months. In conclusion, our study suggests that the CMV promoter is more suitable, than the hEF1α promoter, for stable transgene expression in SCNT-derived transgenic canine model.

Transgenic (TG) pigs are important in biomedical research and are used in disease modeling, pharmaceutical toxicity testing, and regenerative medicine. In this study, we constructed two vector systems by using the promoter of the pig glial fibrillary acidic protein (pGFAP) gene, which is an astrocyte cell marker. We established donor TG fibroblasts with pGFAP-CreERT2/LCMV-EGFPLoxP and evaluated the effect of the transgenes on TG-somatic cell nuclear transfer (SCNT) embryo development. Cleavage rates were not significantly different between control and transgene-donor groups. Embryo transfer was performed thrice just before ovulation of the surrogate sows. One sow delivered 5 TG piglets at 115 days after pregnancy. Polymerase chain reaction (PCR) analysis with genomic DNA isolated from skin tissues of TG pigs revealed that all 5 TG pigs had the transgenes. EGFP expression in all organs tested was confirmed by immunofluorescence staining and PCR. Real-time PCR analysis showed that pGFAP promoter-driven Cre fused to the mutated human ligand-binding domain of the estrogen receptor (CreERT2) mRNA was highly expressed in the cerebrum. Semi-nested PCR analysis revealed that CreERT2-mediated recombination was induced in cerebrum and cerebellum but not in skin. Thus, we successfully generated a TG pig with a 4-hydroxytamoxifen (TM)-inducible pGFAP-CreERT2/EGFPLoxP recombination system via SCNT.

Figure S1. Representative images of isolated canine fibroblasts with an extended cellular life span and morphologies targeted after transfection of CRISPR/Cas9 vectors of each TP53 gRNA#30, #39, and #51. Figure S2. Surveyor assay to identify indel mutations in on-target TP53 or off-target in candidate gene loci in TP53KO#30 and TP53KO#39 cells. Table S1. Possible off-target genes of #30 and #39 TP53 gRNA. Table S2. Primer sets for Surveyor assay. (PDF 280â kb)

Comparing the coding and regulatory sequences of genes in different species provides information on whether proteins translated from genes have conserved functions or gene expressions are regulated by analogical mechanisms. Herein, we compared the coding and regulatory sequences of glial fibrillary acidic protein (GFAP) from humans, mice, and pigs. The GFAP gene encodes a class III intermediate filament protein expressed specifically in astrocytes of the central nervous system. On comparing the mRNA, regulatory region (promoter), and protein sequences of GFAP gene in silico, we found that GFAP mRNA 3'-untranslated region (3'-UTR), promoter, and amino acid sequences showed higher similarities between humans and pigs than between humans and mice. In addition, the promoter-luciferase reporter gene assay revealed that the pig GFAP promoter functioned in human astrocytes. Notably, the 1.8-kb promoter fragment upstream from transcription initiation site showed strongest transcriptional activity compared to 5.2-kb DNA fragment or other regions of GFAP promoter. We also found that pig GFAP mRNA and promoter activity increased in pig fibroblasts by human IL-1β treatment. Taken together, these results suggest that the regulatory mechanisms and functions of pig genes might be more similar to those of humans than mice, indicating that pigs, particularly miniature pigs, are a useful model for studying human biological and pathological events.

Ethnopharmacological relevance Korean Red ginseng extract (RG) is one of the most widely used traditional health functional food in Asia, which invigorates immunity and vital energy. RG have been suggested to inhibit proliferation, invasion, and inflammation in several cancer cell lines. Correspondingly, clinical studies have raised the possibility that RG could augment therapeutic efficacy in cancer patients. However, little is known about the anti-cancer effects of RG in glioblastoma (GBM), the most common and aggressive brain tumor for which effective therapeutic regimens need to be developed. Aim of this study: Here, we assessed the in vivo and in vitro anti-cancer properties of RG in a patient-derived xenograft mouse model and GBM stem cell (GSC) line. Materials and methods We evaluated the anti-cancer effects of RG in patient-derived GBM xenograft mice with and without combined concurrent chemo- and radiation therapy (CCRT). Furthermore, we verified the in vitro effects of RG on the proliferation, cell death, and stem cell-like self-renewal capacity of cancer cells. Finally, we investigated the signaling pathway affected by RG, via which its anti-cancer effects were mediated. Results When combined with CCRT, RG impeded GBM progression by reducing cancer cell proliferation and ionized calcium-binding adapter molecule 1 (IBA1)-positive immune cell recruitment. The anti-cancer effects of RG were mediated by Rg3 and Rh2 ginsenosides. Rg3 promoted cell death while Rh2 did not. Furthermore, both Rg3 and Rh2 reduced cell viability and self-renewal capacity of GSCs by inhibiting Wnt/β-catenin signaling. Conclusion Therefore, our observations imply that RG could be applied to the GBM patients in parallel with CCRT to enhance therapeutic efficacy.

OCT4, also known as POU5F1 (POU domain class 5 transcription factor 1), is a transcription factor that acts as a master regulator of pluripotency in embryonic stem cells and is one of the reprogramming factors required for generating induced pluripotent stem cells. The human OCT4 encodes three isoforms, OCT4A, OCT4B, and OCT4B1, which are generated by alternative splicing. Currently, the functions and expression patterns of OCT4B remain largely unknown in malignancies, especially in human glioblastomas. Here, we demonstrated the function of OCT4B in human glioblastomas. Among the isoform of OCT4B, OCT4B-190 (OCT4B19kDa) was highly expressed in human glioblastoma stem cells and glioblastoma cells and was mainly detected in the cytoplasm rather than the nucleus. Overexpression of OCT4B19kDa promoted colony formation of glioblastoma cells when grown in soft agar culture conditions. Clinical data analysis revealed that patients with gliomas that expressed OCT4B at high levels had a poorer prognosis than patients with gliomas that expressed OCT4B at low levels. Thus, OCT4B19kDa may play a crucial role in regulating cancer cell survival and adaption in a rigid environment.

Background Somatic cell nuclear transfer (SCNT) is a useful biotechnological tool for transgenic animal production using genetically modified somatic cells (GMSCs). However, there are several limitations preventing successful transgenic animal generation by SCNT, such as obtaining proper somatic donor cells with a sufficiently long life span and proliferative capacity for generating GMSCs. Here, we established simian virus 40 large T antigen (SV40LT)-mediated lifespan-extended canine fibroblast cells (SV40LT-K9 cells) and evaluated their potential as nuclei donors for SCNT, based on cellular integrity and SCNT embryo development. Results SV40LT did not cause canine cell transformation, based on cell morphology and proliferation rate. No anchorage-independent growth in vitro and tumorigenicity in vivo were observed. After SCNT with SV40LT-K9 cells, embryos were transferred into surrogate dogs. All dogs failed to become pregnant. Most embryos did not proceed past the 8-cell stage and only one surrogate showed an implantation trace in its oviduct, indicating that the cells rarely developed into blastocysts. Because of the absence of an in vitro maturation method for canine embryos, we performed identical experiments using porcine fibroblast cells. Similarly, SV40LT did not transform porcine fibroblast cells (SV40LT-Pig cells). During in vitro development of SV40LT-Pig cell-driven SCNT embryos, their blastocyst formation rate was clearly lower than those of normal cells. Karyotyping analysis revealed that both SV40LT-K9 and SV40LT-Pig cells had aberrant chromosomal statuses. Conclusions Although lifespan-extended canine and porcine cells via SV40LT exhibit no apparent transforming changes, they are inappropriate for use as nuclei donors for SCNT because of their aneuploidy.

Glioblastoma multiforme (GBM) is one of the most aggressive and lethal human brain tumors, and the median survival of patients with GBM is only 14 months. Glioblastoma stem cells (GSCs) are regarded as a main cause of GBM recurrence, because of their self-renewal and drug resistance properties. Therefore, targeting GSCs is an important therapeutic strategy for GBM. In this study, we show the effects of BRM270, a compound from natural plant extracts, on GSCs in vitro and GBM recurrence in vivo. BRM270 induced apoptotic cell death and inhibited cell growth and “stemness” both in vitro and in vivo. Combining BRM270 treatment with concurrent chemoradiotherapy (CCRT) dramatically increased mice survival and tumor growth inhibition. Taken together, our results suggested that BRM270 synergizes with CCRT as a therapeutic agent to target GSCs.

Most cancer-related signaling pathways sustain their active or inactive status via genetic mutations or various regulatory mechanisms. Previously, we demonstrated that platelet-derived growth factor (PDGF) activates Notch signaling through nitric oxide (NO)-signaling-driven activation of inhibitor of differentiation 4 (ID4) in glioblastoma (GBM) stem cells (GSCs) and endothelial cells in the vascular niche of GBM, leading to maintenance of GSC traits and GBM progression. Here, we determined that the PDGF-NO-ID4-signaling axis is constantly activated through a positive regulatory circuit. ID4 expression significantly increased PDGF subunit B expression in both in vitro cultures and in vivo tumor xenografts and regulated NO synthase 2 (NOS2) expression and NO production by activating PDGF signaling, as well as that of its receptor (PDGFR). Additionally, ectopic expression of PDGFRα, NOS2, or ID4 activated the PDGF-NO-ID4-signaling circuit and enhanced the self-renewal of GBM cell lines. These results suggested that the positive regulatory circuit associated with PDGF-NO-ID4 signaling plays a pivotal role in regulating the self-renewal and tumor-initiating capacity of GSCs and might provide a promising therapeutic target for GBM.

Most of the cancers are still incurable human diseases. According to recent findings, especially targeting cancer stem cells (CSCs) is the most promising therapeutic strategy. CSCs take charge of a cancer hierarchy, harboring stem cell-like properties involving self-renewal and aberrant differentiation potential. Most of all, the presence of CSCs is closely associated with tumorigenesis and therapeutic resistance. Despite the numerous efforts to target CSCs, current anti-cancer therapies are still impeded by CSC-derived cancer malignancies; increased metastases, tumor recurrence, and even acquired resistance against the anti-CSC therapies developed in experimental models. One of the most forceful underlying reasons is a "cancer heterogeneity" due to "CSC plasticity". A comprehensive understanding of CSC-derived heterogeneity will provide novel insights into the establishment of efficient targeting strategies to eliminate CSCs. Here, we introduce findings on mechanisms of CSC reprogramming and CSC plasticity, which give rise to phenotypically varied CSCs. Also, we suggest concepts to improve CSC-targeted therapy in order to overcome therapeutic resistance caused by CSC plasticity and heterogeneity. [BMB Reports 2017; 50(3): 117-125].

Pigs are one of the most suitable alternative laboratory models than other animals, because they have similar cardiovascular, renal and gastrointestinal organs with those of human. However, in the case of genetically engineered animals, early development of embryos is inhibited by expression of foreign genes, there are many cases of miscarriage or birth early mortality. To overcome these problems, we constructed pig glial fibrillary acidic protein (GFAP) promoter-Cre recombinase fused to a mutated ligand-binding domain of the human oestrogen receptor (CreERT2) and enhanced green fluorescent protein (EGFP)-LoxP transgenes for tamoxifen(TM)-inducible CreERT2-mediated recombination. We then established donor transgenic pig fibroblasts with pGFAP-CreERT2; LCMV-EGFPLoxP transgenes for somatic cell nuclear transfer (SCNT). We produced the SCNT embryos using a Cloud male #5 pGFAP-CreERT2+LCMV-EGFPLoxP donor cell line that was verified in vitro. It was transferred into a surrogate mother and then 5 pGFAP-CreERT2; EGFPLoxP TG piglets were born. By immunofluorescence staining and semi-nested PCR analysis, it was proved that CreER-mediated astrocytic-specific recombination system was operated in some cerebral astrocytic cells after TM-administration to TG pig #4. Additionally, we obtained brain magnetic resonance imaging (MRI) images using 3T-tesla MRI. Brain compartment volume (total brain, grey matter, white matter, cerebellum, brainstem, lateral ventricle, thalamus, midbrain, pons, medulla oblongata, hypophysis) was no significant differences between normal pig and pGFAP-CreERT2; EGFPLoxP transgenic (TG) pig. In summary, we verified the pGFAP promoter-driven CreERT2-LoxP recombination system in TG pig generated by SCNT depending on the TM administration. We suggest that this technology will be a useful tool for studying physiology of astrocytes and generating TG pig model of neurological disease such as Huntington’s disease, Alzheimer’s disease and brain tumour.

Transgenic pigs are currently believed to be an important model for biomedical research, including for disease models, pharmaceutical toxicity testing, and regenerative medicine. However, production efficiency of animal disease models using somatic cell NT (SCNT) is very low. One of the main reasons is probably characteristics of the transgene. In this study, we introduce SV40LT oncogene into the fibroblast cells in order to establish immortalized transgenic cell line for producing the pig model of human brain cancer. We evaluated the effect of SV40LT oncogene on transgenic SCNT embryo development. As a results, the cleavage rates (73.8 ± 4.0 and 48.6 ± 2.4 in the normal and SV40LT group, respectively; P