Anti-diabetic effects of linarin from Chrysanthemi Indici Flos via AMPK activation / 中草药·英文版

Objective: The purpose of this study is to investigate the anti-diabetic effects of linarin, a flavonoid extracted from Chrysanthemi Indici Flos (CIF), and its potential mechanisms. Methods: The effects of linarin on cell viability and glucose consumption in HepG2 cells were measured. Meanwhile, monosodium glutamate (MSG) mouse model was constructed to monitor the changes of insulin tolerance, glucose tolerance, triglyceride and cholesterol. The protein expression levels of p-AMPK, p-ACC, PEPCK and p-GS were detected by Western blot. Results: Linarin could increase the relative glucose consumption of HepG2 cells, improve insulin tolerance and glucose tolerance, and decrease the levels of triglyceride and cholesterol of MSG mice. Simultaneously, the expression levels of p-AMPK and p-ACC in HepG2 cells and the liver tissue of MSG mice were increased, while the expression levels of PEPCK and p-GS were decreased after treatment with linarin. Conclusion: Insulin resistance could be ameliorated by linarin in type 2 diabetes, and its mechanism may be related to AMPK signaling pathway.

Crystal clear: visualizing the intervention mechanism of the PD-1/PD-L1 interaction by two cancer therapeutic monoclonal antibodies

Antibody-based PD-1/PD-L1 blockade therapies have taken center stage in immunotherapies for cancer, with multiple clinical successes. PD-1 signaling plays pivotal roles in tumor-driven T-cell dysfunction. In contrast to prior approaches to generate or boost tumor-specific T-cell responses, antibody-based PD-1/PD-L1 blockade targets tumor-induced T-cell defects and restores pre-existing T-cell function to modulate antitumor immunity. In this review, the fundamental knowledge on the expression regulations and inhibitory functions of PD-1 and the present understanding of antibody-based PD-1/PD-L1 blockade therapies are briefly summarized. We then focus on the recent breakthrough work concerning the structural basis of the PD-1/PD-Ls interaction and how therapeutic antibodies, pembrolizumab targeting PD-1 and avelumab targeting PD-L1, compete with the binding of PD-1/PD-L1 to interrupt the PD-1/PD-L1 interaction. We believe that this structural information will benefit the design and improvement of therapeutic antibodies targeting PD-1 signaling.

The receptors and entry of measles virus: a review / 生物工程学报

Measles virus is an enveloped virus with a non-segmented negative-sense RNA genome. Two envelope glycoproteins on the viral surface, namely hemagglutinin (H) and membrane fusion protein (F), are responsible for the virus entry into susceptible host cells. The specific interaction between H and its cellular receptors is a key step in successful virus infection, determining the infectivity and tissue tropism of the measles virus. Thus far, three H receptors have been identified, including the complement regulatory molecule CD46, the signaling lymphocyte activation molecule (SLAM) and the cell adhesion molecule Nectin-4. Here, we reviewed our molecular understanding on the recognition mechanism of these receptors by the viral H protein, aiming to promote future studies on antiviral drug design and measles virus-based oncolytic therapy.

Expression, crystallization and crystallographic study of the 1st IgV domain of human CD96 / 生物工程学报

CD96 (Tactile) is an adhesion receptor expressed mainly on activated T cells, NK cells. As a family member of the immunoglobulin-like cell receptor, CD96 consists of three immunoglobulin-like domains (V1, V2/C and C) in the extracellular region. Recent studies have shown that the 1st IgV domain of CD96 (CD96V1) plays an essential role in cell adhesion and NK cell-mediated killing. In this study, the 1st IgV domain of human CD96 (hCD96V1) was cloned and expressed in Escherichia coli (BL21). The soluble protein was obtained by refolding of the hCD96V1 inclusion bodies. From analytical ultracentrifugation, we could predict that CD96 V1 maily exists as dimer with approximate molecular weight of 26.9 kDa. The protein was then successfully crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to 1.9 angstrom resolution and belonged to space group P21, with unit-cell parameters a = 35.1, b = 69.5, c = 49.6A, alpha=gamma=90 degrees, beta=105.4 degrees.

Crystal structures of the two membrane-proximal Ig-like domains (D3D4) of LILRB1/B2: alternative models for their involvement in peptide-HLA binding

Leukocyte immunoglobulin-like receptors (LILRs), also called CD85s, ILTs, or LIRs, are important mediators of immune activation and tolerance that contain tandem immunoglobulin (Ig)-like folds. There are 11 (in addition to two pseudogenes) LILRs in total, two with two Ig-like domains (D1D2) and the remaining nine with four Ig-like domains (D1D2D3D4). Thus far, the structural features of the D1D2 domains of LILR proteins are well defined, but no structures for the D3D4 domains have been reported. This is a very important field to be studied as it relates to the unknown functions of the D3D4 domains, as well as their relative orientation to the D1D2 domains on the cell surface. Here, we report the crystal structures of the D3D4 domains of both LILRB1 and LILRB2. The two Ig-like domains of both LILRB1-D3D4 and LILRB2-D3D4 are arranged at an acute angle (∼60°) to form a bent structure, resembling the structures of natural killer inhibitory receptors. Based on these two D3D4 domain structures and previously reported D1D2/HLA I complex structures, two alternative models of full-length (four Ig-like domains) LILR molecules bound to HLA I are proposed.

Effect of antibody CR6261 V(H) and scFv expression on influenza virus infection / 生物工程学报

The emerging CR6261 antibody could neutralize several subtype influenza virus with high affinity, whose VH domain binds to the HA protein conserved domain. Therefore, it has drawn much attention as a potential broad-spectrum therapeutic antibody against influenza virus. In this study, we constructed the eukaryotic expression vectors pCR6261V(H), pCR6261V(H)-GFP and pCR6261scFv and screened the monoclonal cell lines that could stably express CR6261V(H), CR6261V(H)-GFP and CR6261scFv on the cell membrane. After influenza virus infecting the stable cell lines, the titers of viruses were tested by hemagglutination inhibition test. The result shows that the titers of viruses in CR6261scFv and CR6261V(H)-GFP stable expression cell lines decreased and there was no obvious discrimination between the CR6261V(H) expression cell line and the negative control, suggesting that CR6261V(H) and CR6261scFv expressing on the cell membrane could partly inhibit the virus infection. Though the effective inhibition strategy is undergoing, our research will provide new clues for the breeding of anti influenza transgenic animals.

X-ray diffraction analysis of glycoprotein D from herpes simplex virus type 2 / 生物工程学报

Glycoprotein D (gD) of Herpes simplex virus type 2 (HSV-2) is a key factor mediating the entry of HSV-2 into host cells. In order to explain the mechanism underlying the gD-mediated receptor-binding and viral entry, we performed a structural study on HSV-2 gD. The ectodomain of the gD protein encompassing residues 1 to 285 was expressed by baculovirus-infected insect cells as a secreted soluble protein with a C-terminal hexa-his tag. The protein was then purified by affinity and size-exclusion chromatography. The purified protein was successfully crystallized using the hanging-drop vapor-diffusion at 18 degrees C in a condition consisting of 0.1 mol/L Hepes pH 7.2, 5% (V/V) 2-methyl-2,4-pentanediol (MPD) and 10% PEG 10 000. The crystals diffracted to 1.8 angstroms resolution and belonged to space group P21, with unit-cell parameters alpha = 63.6, b = 55.4, c = 65.3 angstroms, beta = 96.3 degrees.

hNUDT16: a universal decapping enzyme for small nucleolar RNA and cytoplasmic mRNA

Human NUDT16 (hNUDT16) is a decapping enzyme initially identified as the human homolog to the Xenopus laevis X29. As a metalloenzyme, hNUDT16 relies on divalent cations for its cap-hydrolysis activity to remove m⁷GDP and m²²⁷GDP from RNAs. Metal also determines substrate specificity of the enzyme. So far, only U8 small nucleolar RNA (snoRNA) has been identified as the substrate of hNUDT16 in the presence of Mg²(+). Here we demonstrate that besides U8, hNUDT16 can also actively cleave the m⁷GDP cap from mRNAs in the presence of Mg²(+) or Mn²(+). We further show that hNUDT16 does not preferentially recognize U8 or mRNA substrates by our cross-inhibition and quantitative decapping assays. In addition, our mutagenesis analysis identifies several key residues involved in hydrolysis and confirms the key role of the REXXEE motif in catalysis. Finally an investigation into the subcellular localization of hNUDT16 revealed its abundance in both cytoplasm and nucleus. These findings extend the substrate spectrum of hNUDT16 beyond snoRNAs to also include mRNA, demonstrating the pleiotropic decapping activity of hNUDT16.

Transdermal delivery of Gentiana macrophylla complex components system under micro-needle conditions / 药学学报

The purpose of this study is to investigate the transdermal delivery characteristics of Gentiana macrophylla complex components system through different parts of the skin under micro-needles conditions. Two-chamber diffusion cells were used, different parts of isolated skin and micro-needle pretreated isolated mouse skin were applied separately, high performance liquid chromatography (HPLC) similarity evaluation methods were used to evaluate transdermal delivery characteristics of Gentiana macrophylla complex components system on receiving pool and the permeation rate and penetration amount of Gentiopicroside at different parts of mouse skin. In the 24 h, the similarity between receiving fluid which was on passive transdermal delivery and micro-needle transdermal delivery conditions and original fluid were ranged from 83.0% to 98.9%; By the micro-needle pretreatment with different parts of the mouse skin, the time that Gentiana macrophylla complex components system though abdominal skin to the receiving fluid which reached 90% similarity compared with that of original fluid was 4 h, which was 18 h at back skin and 12 h at neck skin separately. Micro-needles can be used as the ideal ingredients for traditional Chinese medicine complex transdermal delivery; transdermal absorption time delay could be greatly reduced and its bioavailability was improved. The permeation rate and similarity to original liquid of Chinese medicine complex components increased significantly in the abdominal skin relative to the neck and back skin under micro-needle conditions.

Crystal structure of the swine-origin A (H1N1)-2009 influenza A virus hemagglutinin (HA) reveals similar antigenicity to that of the 1918 pandemic virus

Influenza virus is the causative agent of the seasonal and occasional pandemic flu. The current H1N1 influenza pandemic, announced by the WHO in June 2009, is highly contagious and responsible for global economic losses and fatalities. Although the H1N1 gene segments have three origins in terms of host species, the virus has been named swine-origin influenza virus (S-OIV) due to a predominant swine origin. 2009 S-OIV has been shown to highly resemble the 1918 pandemic virus in many aspects. Hemagglutinin is responsible for the host range and receptor binding of the virus and is therefore a primary indicator for the potential of infection. Primary sequence analysis of the 2009 S-OIV hemagglutinin (HA) reveals its closest relationship to that of the 1918 pandemic influenza virus, however, analysis at the structural level is necessary to critically assess the functional significance. In this report, we report the crystal structure of soluble hemagglutinin H1 (09H1) at 2.9 Å, illustrating that the 09H1 is very similar to the 1918 pandemic HA (18H1) in overall structure and the structural modules, including the five defined antiboby (Ab)-binding epitopes. Our results provide an explanation as to why sera from the survivors of the 1918 pandemics can neutralize the 2009 S-OIV, and people born around the 1918 are resistant to the current pandemic, yet younger generations are more susceptible to the 2009 pandemic.

Interaction of Hsp40 with influenza virus M2 protein: implications for PKR signaling pathway

Influenza virus contains three integral membrane proteins: haemagglutinin, neuraminidase, and matrix protein (M1 and M2). Among them, M2 protein functions as an ion channel, important for virus uncoating in endosomes of virus-infected cells and essential for virus replication. In an effort to explore potential new functions of M2 in the virus life cycle, we used yeast two-hybrid system to search for M2-associated cellular proteins. One of the positive clones was identified as human Hsp40/Hdj1, a DnaJ/Hsp40 family protein. Here, we report that both BM2 (M2 of influenza B virus) and A/M2 (M2 of influenza A virus) interacted with Hsp40 in vitro and in vivo. The region of M2-Hsp40 interaction has been mapped to the CTD1 domain of Hsp40. Hsp40 has been reported to be a regulator of PKR signaling pathway by interacting with p58(IPK) that is a cellular inhibitor of PKR. PKR is a crucial component of the host defense response against virus infection. We therefore attempted to understand the relationship among M2, Hsp40 and p58(IPK) by further experimentation. The results demonstrated that both A/M2 and BM2 are able to bind to p58(IPK) in vitro and in vivo and enhance PKR autophosphorylation probably via forming a stable complex with Hsp40 and P58(IPK), and consequently induce cell death. These results suggest that influenza virus M2 protein is involved in p58(IPK) mediated PKR regulation during influenza virus infection, therefore affecting infected-cell life cycle and virus replication.

Construction and identification of recombinant lentiviral vector of hNoc4L gene / 生物工程学报

Formation and nuclear export of pre-ribosomes requires many nucleolar complexes, hNoc4L which contains a conserved Noc doman is a homolog of nucleolar complex associated 4 (S. cerevisiae), but its function is completely unclear. Here, we successfully got the recombinant lentiviral vector p113.7-EF1-hNoc4L-Flag by replacing the U6 promoter in p113.7 with EF1alpha promoter, and then inserted hNoc4L to down-stream of the EF1alpha prompter. We determined the transduction efficiency in different mammalian cell lines based on lentiviral packaging system. Subsequently, we analyzed the immunogenicity of the recombinant lentivirus and stable expression of hNoc4L in RAW264.7 cells. The results showed that the recombinant lentivirus characterized a high transduction efficiency, long-term expression and low immunogenicity. Therefore, we pave the way for further identification of the biological activity of hNoc4L protein during ribosome biogenesis in mammalian.

Effects of methylprednisolone on cytokines in patients after successful cardiopulmonary resuscitation / 中华急诊医学杂志

Objective To investigate the effects of methylprednisolone on the release of cytokines in patients after successful cardiopulmonary resuscitation(CPR).Method Thirty patients after CPR with restoration of spontaneous circulation(ROSC)were randomly divided into two groups:group A(methylprednisolone group,n=14)and group B(control group,n=16)during the period from May 2005 through May 2007.The patients of group were treated with methylprednisolone 3 mg/kg by intravenously twice a day after ROSC.The levels of serum tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-6(IL-6),interleukin-8(IL-8),interleukin-10 (IL-10)were measured by enzyme-linked immunosorbent assay(ELISA)before CPR,and 24,48,72 hours and 7 days after ROSC.The data were analyzed studentis t test and chi-gquare test.A P value less than 0.05 indicated significant difference.Results There was on significant difference in the mean time from cardiac arrest to return of spontaneous circulation,and the levels of serum cytokines between the two groups before CPR(P＞0.05).In comparisorl with group B,the levels of serum TNF-α,IL-1β,IL-6,IL-8 decreased markedly at 24 and 48 hour after ROSC in group A(P＜0.05-0.01),and the levels of serum IL-8 decreased markedly at 72 hours after ROSC in groupA(P＜0.05).Thelevels of serum TNF-α,IL-1β,IL-6,IL-8 were not of significant differences between the two groups at 7 hys after ROSC(P＞0.05).There was no significant difference in the levels of serum IL-10between the two groups at different time points after ROSC(P＞0.05).Conclusions Methylprednisolone plays a role of preventive effects on patients with ROSC after PCR through decreasing the levels of serum TNF-α,IL-1β,IL-6,IL-8.