Cyanidin-3-O-glucoside Regulates the Expression of Ucp1 in Brown Adipose Tissue by Activating Prdm16 Gene

(1) Background: Brown adipose tissue (BAT) burns energy to produce heat. Cyanidin-3-O-glucoside (C3G) can then enhance the thermogenic ability of BAT in vivo. However, the mechanism by which C3G regulates Ucp1 protein expression remains unclear. (2) Methods: In this study, C3H10T12 brown adipose cells and db/db mice and mice with high-fat, high-fructose, diet-induced obesity were used as the model to explore the effect of C3G on the expression of the Ucp1 gene. Furthermore, the 293T cell line was used for an in vitro cell transgene, a double luciferase reporting system, and yeast single hybridization to explore the mechanism of C3G in regulating Ucp1 protein. (3) Results: we identified that, under the influence of C3G, Prdm16 directly binds to the −500 to −150 bp promoter region of Ucp1 to activate its transcription and, thus, facilitate BAT programming. (4) Conclusions: This study clarified the mechanism by which C3G regulates the expression of the Ucp1 gene of brown fat to a certain extent.

Fully Human Antibody Immunoglobulin from Transchromosomic Bovines is Potent Against SARS-CoV-2 Variant Pseudoviruses

SAB-185 is a fully human polyclonal anti-SARS-CoV-2 immunoglobulin produced from the plasma of transchromosomic bovines that are hyperimmunized with recombinant SARS-CoV-2 Wuhan-Hu-1 Spike protein. SAB-185 is being evaluated for efficacy in an adaptive phase 2/3 clinical trial. The World Health Organization (WHO) has identified multiple Variants-of-Concern and Variants-of-Interest (VOC/VOI) that have mutations in their Spike protein that appear to increase transmissibility and/or reduce the effectiveness of therapeutics and vaccines, among other parameters of concern. SAB-185 was evaluated using a lentiviral-based pseudovirus assay performed in a BSL2 environment that incorporates a stable 293T cell line expressing human angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). The results indicate that SAB-185 retained neutralization potency against multiple SARS-CoV-2 pseudovirus variants, including the Delta, Kappa and Lambda variants, that are supplanting other VOC/VOI in many countries and regions around the world.

Mutants of human ACE2 differentially promote SARS-CoV and SARS-CoV-2 spike mediated infection

SARS-CoV and SARS-CoV-2 encode spike proteins that bind human ACE2 on the cell surface to enter target cells during infection. A small fraction of humans encode variants of ACE2, thus altering the biochemical properties at the protein interaction interface. These and other ACE2 coding mutants can reveal how the spike proteins of each virus may differentially engage the ACE2 protein surface during infection. We created an engineered HEK 293T cell line for facile stable transgenic modification, and expressed the major human ACE2 allele or 28 of its missense mutants, 24 of which are possible through single nucleotide changes from the human reference sequence. Infection with SARS-CoV or SARS-CoV-2 spike pseudotyped lentiviruses revealed that high ACE2 cell-surface expression could mask the effects of impaired binding during infection. Drastically reducing ACE2 cell surface expression revealed a range of infection efficiencies across the panel of mutants. Our infection results revealed a non-linear relationship between soluble SARS-CoV-2 RBD binding to ACE2 and pseudovirus infection, supporting a major role for binding avidity during entry. While ACE2 mutants D355N, R357A, and R357T abrogated entry by both SARS-CoV and SARS-CoV-2 spike proteins, the Y41A mutant inhibited SARS-CoV entry much more than SARS-CoV-2, suggesting differential utilization of the ACE2 side-chains within the largely overlapping interaction surfaces utilized by the two CoV spike proteins. These effects correlated well with cytopathic effects observed during SARS-CoV-2 replication in ACE2-mutant cells. The panel of ACE2 mutants also revealed altered ACE2 surface dependencies by the N501Y spike variant, including a near-complete utilization of the K353D ACE2 variant, despite decreased infection mediated by the parental SARS-CoV-2 spike. Our results clarify the relationship between ACE2 abundance, binding, and infection, for various SARS-like coronavirus spike proteins and their mutants, and inform our understanding for how changes to ACE2 sequence may correspond with different susceptibilities to infection.

IFI16 induces inflammation in Hepatitis B Virus-Associated Glomerulonephritis by Regulating the Caspase-1 /IL-1ß pathway

Aims & backgroundIFI16 plays an important role in innate immunity against invasive microbial infection by sensing double-stranded DNA viruses due to caspase-1-dependent inflammasome activation and subsequent maturation and secretion of IL-1β. However, the role of IFI16 in regulating the immune response to viruses in Hepatitis B Virus-Associated Glomerulonephritis(HBV-GN), especially in sensing the hepatitis B virus (HBV), has not been determined. In this study,, we investigated the inflammatory role of IFI16 in HBV-GN.MethodsA total of 75 kidney tissues including 50 HBV-GN and 25 chronic glomerulonephritis (CCN) were collected to determine expression of IFI16, Caspase-1, and IL-1𝛽 by immunohistochemistry (IHC), and then the correlation between them was analyzed. In vitro, the overexpression or knockdown of IFI16 in regulating the immune response to HBV infection in the human glomerular mesangial (HGM) cell line and HEK-293T cell line. Quantitative Real-time PCR and western blotting were used to determine the expression of IFI16, Caspase-1 and IL-1β. The role effect of IFI16 in vivo was further investigated.ResultsIFI16 expression in HBV-GN biopsies (80.0%) was significantly higher than in CGN (24.0%) and was positively correlated with caspase-1 and IL-1𝛽 expression in HBV-GN. In vitro, over expression of IFI16 increased caspase-1 and IL-1𝛽 expression in HBV-infected HGM and HEK-293T cell lines, whereas knockdown of IFI16 mRNA by siRNA resulted in downregulation of the caspase-1 and IL-1𝛽 expression in both cell lines.ConclusionsThe elevation of IFI16 during HBV infection or replication may contribute to renal damage due to inflammation, thus providing a putative therapeutic target and a new avenue for studying the pathogenesis of HBV-GN.

Expression of Secreted Neutrophil Gelatinase-Associated Lipocalin in 293T Cell Using the Inducible Dual-Function System

Neutrophil gelatinase-associated lipocalin (NGAL) has emerged as a promising biomarker for the early prediction of acute kidney injury (AKI). The production of recombinant NGAL is considered to be necessary for the development of a detection method. This study intended to express the recombinant NGAL protein in 293T cell under the Tet-On inducible system and human serum albumin signal sequence (HSA-SS). The transfection efficiency and protein modulation were assessed by detecting the expression of the enhanced green fluorescent protein (EGFP) and secreted NGAL protein. Both proteins were detected only in the presence of a doxycycline (Dox) inducer. Cell toxicity was not found under any conditions. Moreover, a higher level of soluble NGAL protein in the supernatant secreted by HSA-SS compared with a native signal peptide (Nat-SS) was observed. In summary, this work successfully optimized the conditions for induction of NGAL expression. This system will provide as an efficient strategy to produce other recombinant proteins secreted from a mammalian cell.

Generation and utilization of a HEK-293T murine GM-CSF expressing cell line

Macrophages and dendritic cells (DCs) are innate immune cells that play a key role in defense against pathogens. In vitro cultures of bone marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs) are well-established and valuable methods for immunological studies. Typically, commercially available recombinant GM-CSF is utilized to generate BMDCs and is also used to culture alveolar macrophages. We have generated a new HEK-293T cell line expressing murine GM-CSF that secretes high levels of GM-CSF (~180 ng/ml) into complete media as an alternative to commercial GM-CSF. Differentiation of dendritic cells and expression of various markers were kinetically assessed using the GM-CSF HEK293T cell line, termed supGM-CSF and compared directly to purified commercial GMCSF. After 7–9 days of cell culture the supGM-CSF yielded twice as many viable cells compared to the commercial purified GM-CSF. In addition to differentiating BMDCs, the supGM-CSF can be utilized to culture functionally active alveolar macrophages. Collectively, our results show that supernatant from our GM-CSF HEK293T cell line supports the differentiation of mouse BMDCs or alveolar macrophage culturing, providing an economical alternative to purified GM-CSF.

Establishment of a well-characterized SARS-CoV-2 lentiviral pseudovirus neutralization assay using 293T cells with stable expression of ACE2 and TMPRSS2

Pseudoviruses are useful surrogates for highly pathogenic viruses because of their safety, genetic stability, and scalability for screening assays. Many different pseudovirus platforms exist, each with different advantages and limitations. Here we report our efforts to optimize and characterize an HIV-based lentiviral pseudovirus assay for screening neutralizing antibodies for SARS-CoV-2 using a stable 293T cell line expressing human angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). We assessed different target cells, established conditions that generate readouts over at least a two-log range, and confirmed consistent neutralization titers over a range of pseudovirus input. Using reference sera and plasma panels, we evaluated assay precision and showed that our neutralization titers correlate well with results reported in other assays. Overall, our lentiviral assay is relatively simple, scalable, and suitable for a variety of SARS-CoV-2 entry and neutralization screening assays.

Determining the pathogenicity of MUT gene variant by mini-gene splicing assay

ObjectiveTo perform gene mutation splicing analysis on 12 suspected pathogenic MMUT/MUT gene mutation sites to verify the pathogenicity of the mutation.MethodsWild-type and mutant minigenes were inserted into pcMINI vector, and total 5 wild-type recombinant vectors and 12 mutant recombinant vectors were constructed. The total RNA in 293T cells was extracted after the recombinant vectors were transfected into 293T cell line. Then PCR products were detected by agarose gel electrophoresis and analyzed by sequencing.ResultsRT-PCR and sequencing results showed that among 12 mutations, 9 mutations (c.419T>C, 469G>T, c.470T>A, c.626dupC, c.693C>G, c.976A>G, c.1009T>C, c.1777G>T and c.1874A>C) did not affect the gene splicing, and the other 3 mutations (c.454C>T, c.421G>A and c.2125-3C>G) all affected mRNA splicing.ConclusionIn recent case reports of MMUT/MUT gene mutation sites, variant of uncertain significance (VUS) variation is very common. In this study, the pathogenicity of three mutation sites is confirmed by the mini-gene method.

Establishment of a well-characterized SARS-CoV-2 lentiviral pseudovirus neutralization assay using 293T cells with stable expression of ACE2 and TMPRSS2

Pseudoviruses are useful surrogates for highly pathogenic viruses because of their safety, genetic stability, and scalability for screening assays. Many different pseudovirus platforms exist, each with different advantages and limitations. Here we report our efforts to optimize and characterize an HIV-based lentiviral pseudovirus assay for screening neutralizing antibodies for SARS-CoV-2 using a stable 293T cell line expressing human angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). We assessed different target cells, established conditions that generate readouts over at least a two-log range, and confirmed consistent neutralization titers over a range of pseudovirus input. Using reference sera and plasma panels, we evaluated assay precision and showed that our neutralization titers correlate well with results reported in other assays. Overall, our lentiviral assay is relatively simple, scalable, and suitable for a variety of SARS-CoV-2 entry and neutralization screening assays.

Crispra: A Promising Tool for β-Thalassemia Treatment

Induction of hemoglobin γ expression is a reliable strategy to treat β-thalassemia. Gene editing using CRISPR/Cas9 technology has been widely used. However, application in vivo is limited due to the uncertainty on genomic cleavages of Cas9. In contrast, CRISPR/Cas9-based gene activation (CRISPRa) can only locate genomic locus but not interrupt sequence. Here, we use SAM system of CRISPRa to locate and activate HBG1 and HBG2, exploring the great potential of CRISPRa for β-thalassemia treatment. WWe designed 8 single-guide RNAs (sgRNAs) online and cloned into vector SAM V2, which fused dCas9 and VP64. To test the over-expression efficiency, vector containing sgRNA and MPH (fused HSF1, p65 and MS2) were transfected into 293T cell. After 72h transfection, 293T cells were collected. Q-PCR data showed that two sgRNAs were excellent on activating HBG expression with over 1000-fold increase. WTo test the activating function in hematological cell and the persistence of hemoglobin γexpression, two screened sgRNA were transfected into NB4 cells using lentivirus system. We harvested NB4 cells at different time-point (3 day, 1 week and 2 weeks), and implemented q-PCR assay. HBG expression were increased 50-hold and 1000-hold, respectively. However, the expression were reducing over time and the intrinsic mechanism is unknown. WThis study set out to increase HBG without interrupt genome using CRISPRa system. This study has found two sgRNA to activate the expression of HBG in 293T cell and NB4 cell. Further research is required to vertify the efficiency of sgRNA in hematopoietic stem cells and prolong the expression time. Figure Disclosures No relevant conflicts of interest to declare.