Soluble ACE2-mediated cell entry of SARS-CoV-2 via interaction with proteins related to the renin-angiotensin system

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Comparison of expression of three different sub-cellular targeted GFPs in transgenic Valencia sweet orange by confocal laser scanning microscopy

The green fluorescent protein (GFP) has become an ideal visual marker to monitor and quantify the expression of the transgene. It can be targeted to specific subcellular locations, including the endoplasmic reticulum, mitochondria, actin cytoskeleton and nuclei through the addition of signal peptides. Our previous work has resulted in transgenic citrus plants expressing cytoplasmic targeted GFP (Cy-GFP) or endoplasmic reticulum targeted GFP (Er-GFP) gene. To evaluate the localization of three different subcellular targeted GFP, i.e., Cy-GFP, Er-GFP and mitochondria targeted GFP (Mt-GFP) in citrus tissues and to utilize cell lines containing Mt-GFP for basic research in cell fusion, the plasmid pBI-mgfp4-coxIV encoding the Mt-GFP gene was successfully transferred into embryogenic callus of Valencia sweet orange (Citrus sinensis (L.) Osbeck) via Agrobacterium tumefaciens-mediated transformation. Furthermore, we compared the specific expression of these three different subcellular localized GFP constructs in cells of different mature leaf tissues (upper epidermis, palisade parenchyma, spongy parenchyma and lower epidermis) by a confocal laser scanning microscope (CLSM). Cytoplasmic-localized GFP expression was observed throughout the cytoplasm but appeared to accumulate within the nucleoplasm. The Er-GFP occurred within a layer very close to the cell wall. In addition, a stable fluorescence on the ER network throughout the guard cells was detected. Interestingly, the Mt-GFP specifically expressed in the guard cells to particles of about 1–2 μm within the cytoplasm in this case. To verify that the fluorescent particles observable in the guard cells are indeed mitochondria, we co-localize the Mt-GFP fusion protein with a mitochondrial-specific dye in citrus protoplasts. These results demonstrate that the subcellular distribution of the three subcellular targeted GFP is very distinct in citrus leaf cells and the cell lines containing Mt-GFP gene can be further used in citrus basic cell fusion research.     

Immunization with a vaccine combining herpes simplex virus 2 (HSV-2) glycoprotein C (gC) and gD subunits improves the protection of dorsal root ganglia in mice and reduces the frequency of recurrent vaginal shedding of HSV-2 DNA in guinea pigs compared to immunization with gD alone

Attempts to develop a vaccine to prevent genital herpes simplex virus 2 (HSV-2) disease have been only marginally successful, suggesting that novel strategies are needed. Immunization with HSV-2 glycoprotein C (gC-2) and gD-2 was evaluated in mice and guinea pigs to determine whether adding gC-2 to a gD-2 subunit vaccine would improve protection by producing antibodies that block gC-2 immune evasion from complement. Antibodies produced by gC-2 immunization blocked the interaction between gC-2 and complement C3b, and passive transfer of gC-2 antibody protected complement-intact mice but not C3 knockout mice against HSV-2 challenge, indicating that gC-2 antibody is effective, at least in part, because it prevents HSV-2 evasion from complement. Immunization with gC-2 also produced neutralizing antibodies that were active in the absence of complement; however, the neutralizing titers were higher when complement was present, with the highest titers in animals immunized with both antigens. Animals immunized with the gC-2-plus-gD-2 combination had robust CD4+ T-cell responses to each immunogen. Multiple disease parameters were evaluated in mice and guinea pigs immunized with gC-2 alone, gD-2 alone, or both antigens. In general, gD-2 outperformed gC-2; however, the gC-2-plus-gD-2 combination outperformed gD-2 alone, particularly in protecting dorsal root ganglia in mice and reducing recurrent vaginal shedding of HSV-2 DNA in guinea pigs. Therefore, the gC-2 subunit antigen enhances a gD-2 subunit vaccine by stimulating a CD4+ T-cell response, by producing neutralizing antibodies that are effective in the absence and presence of complement, and by blocking immune evasion domains that inhibit complement activation.     

Additive mixed effect model for clustered failure time data

We propose an additive mixed effect model to analyze clustered failure time data. The proposed model assumes an additive structure and includes a random effect as an additional component. Our model imitates the commonly used mixed effect models in repeated measurement analysis but under the context of hazards regression; our model can also be considered as a parallel development of the gamma-frailty model in additive model structures. We develop estimating equations for parameter estimation and propose a way of assessing the distribution of the latent random effect in the presence of large clusters. We establish the asymptotic properties of the proposed estimator. The small sample performance of our method is demonstrated via a large number of simulation studies. Finally, we apply the proposed model to analyze data from a diabetic study and a treatment trial for congestive heart failure.     

(+)-Borneol suppresses conditioned fear recall and anxiety-like behaviors in mice

Fear- and anxiety-related psychiatric disorders have been one of the major chronic diseases afflicting patients for decades, and new compounds for treating such disorders remain to be developed. (+)-Borneol, a bicyclic monoterpene found in several species of Artemisia and Dipterocarpaceae, is widely used for anxiety, pain and anesthesia in Chinese medicine. Meanwhile, it can potentiate GABA (γ-aminobutyric acid) activity directly in recombinant GABAA receptors. The present study was to investigate the effects of (+)-Borneol on both contextual and cued fear recall. Interestingly, microinjection of (+)-Borneol into the dorsal hippocampus inhibited 24 h and 7 d contextual fear, whereas its infusion into ventral hippocampus only reduced 24 h cued fear responses. Moreover, microinjection of (+)-Borneol into dorsal but not ventral hippocampus suppressed anxiety-like behaviors in the open field test, light/dark exploration and the elevated plus maze test. As selective GABA_A receptor antagonist bicuculline reversed the effect of (+)-Borneol on contextual fear paradigm and the drug potentiated GABA-evoked currents in acute hippocampus slices, modulation of the GABAergic neurotransmission may explain the effects of (+)-Borneol. Our findings suggest that (+)-Borneol can serve as a new therapeutic in fear- and anxiety-related disorders.     

Dexamethasone suppresses osteogenesis of osteoblast via the PI3K/Akt signaling pathway in vitro and in vivo

The hypofunction of osteoblasts induced by glucocorticoids (GCs) has been identified as a major contributing factor for GC-induced osteoporosis (GIO). However, the biological mechanism underlying the effect of GC in osteoblasts are not fully elucidated. Recent studies implicated an important role of phosphoinositide 3-kinase (PI3K)/protein kinase B(Akt) signaling pathway in the regulation of bone growth. We propose that the PI3K/Akt signaling may be implicated in the process of GC-induced osteogenic inhibition in osteoblasts. In this study, primary osteoblasts were used in vitro and in rats in vivo to evaluate the biological significance of the PI3K/Akt pathway in GC-induced bone loss. In vivo, dexamethasone (Dex)-treated rats had low bone mineral density and decreased expression levels of alkaline phosphatase (ALP), osteocalcin (OCN), and phosphorylated Akt (p-Akt) in bone tissue. In vitro study shows that Dex over the dose of 10^–8 M remarkably inhibited cellular osteogenesis, as represented by decreased cell viability, lessened ALP activity, and suppressed osteogenic protein expressions including ALP and OCN. Meanwhile, a dramatic downregulation in the PI3K/Akt pathway phosphorylation was also observed in Dex-treated osteoblasts. These changes were marked rescued by treatment with a PI3K agonist 740Y-P. Moreover, downregulation of ALP and OCN expressions by LY294002 can mimic the suppressive effects of Dex. These data together reveal that the suppressed PI3K/Akt pathway is involved in the regulatory action of Dex on osteogenesis.     

大鼠正中神经一级传入纤维在脊髓胶状质定位投射的定量分析——FRAP法

本文用抗氟化物酸性磷酸酶(FRAP)法和显微测量,对大鼠正中神经一级传入纤维在脊髓胶状质(SG)的定位投射进行了定量分析.大鼠正中神经向SG的纵向投射主要为C_5～T_1.C_5～T_1各节段SG水平向眉毛状反应带所测均值(mm)分别为0.888、0.935、0.957、0.905和 0.776,而正中神经向C_5～T_1各节段SG水平向投射所测均值(mm)分别在0～0.204、0～0.303、0～0.409、0～0.432和0～0.336的范围,这显示了正中神经投射区均位于SG的内侧带和部分中间带.