黑果枸杞不同组织内生细菌群落多样性

【目的】黑果枸杞是我国荒漠区特有的药用盐生植物,本研究分析了黑果枸杞不同组织中内生细菌群落多样性特征及分布规律。【方法】应用Illumina MiSeq高通量测序技术对黑果枸杞内生细菌的16S rRNAV5-V7区域序列进行测定,并分析群落组成、多样性及功能等生物学信息。【结果】黑果枸杞不同组织内生细菌群落多样性及功能均有较大的差异。花、叶、果、茎和根产生的OTUs分别是182、173、119、187和254,群落多样性表现为根花果、茎叶。从门水平上看,变形菌门是优势菌门,在不同组织中均有分布,花、叶、果、茎和根中的相对丰度分别为87.66%、41.51%、81.76%、97.67%和61.85%。在属水平上显示内生细菌的分布表现出器官差异性。花部能够准确分类的优势菌属为沙雷氏菌属和不动杆菌属,相对丰度分别为11.57%和8.55%。叶部为红球菌属和慢生根瘤菌属,相对丰度分别为29.68%和5.53%。果实中为泛菌属、红球菌属和沙雷氏菌属,相对丰度分别为23.12%、5.52%和4.29%。茎部为沙雷氏菌属和假单胞菌属,相对丰度分别为12.03%和17.71%。根部为盐单胞菌属、Fodinicurvata和Lipingzhangella,相对丰度分别为24.18%、5.16%和4.86%。在不同组织中分布较广的盐单胞菌、沙雷氏菌、不动杆菌、红球菌、泛菌等菌属均具有较高耐盐性和促生、生防、降解有机污染物及抗氧化等功能。PICRUSt功能预测分析显示,黑果枸杞组织中内生细菌功能中涉及丰富的多糖、萜类和酮类、酶及维他命等次生代谢产物的生物合成。【结论】黑果枸杞内生细菌具有丰富的群落和功能多样性,拥有多种益生功能性状,也含有多个与人和植物体代谢相关的功能信息。不同组织优势菌属和功能信息各有不同,其中根部的内生细菌物种最丰富,花部和茎部参与各种代谢调控的细菌丰度最高。此外,不同组织中还含有大量未知种属的微生物类群,这些都为内生细菌功能利用和挖掘新的有益微生物资源提供广阔的发展空间。     

Dual stimuli-responsive Fe<Subscript>3</Subscript>O<Subscript>4</Subscript> graft poly(acrylic acid)-<Emphasis Type="Italic">block</Emphasis>-poly(2-methacryloyloxyethyl ferrocenecarboxylate) copolymer micromicelles: surface RAFT synthesis,self-assembly and drug release applications

Background

Stimuli-responsive polymer materials are a new kind of intelligent materials based on the concept of bionics, which exhibits more significant changes in physicochemical properties upon triggered by tiny environment stimuli, hence providing a good carrier platform for antitumor drug delivery.

Results

Dual stimuli-responsive Fe₃O₄ graft poly(acrylic acid)-block-poly(2-methacryloyloxyethyl ferrocenecarboxylate) block copolymers (Fe₃O₄-g-PAA-b-PMAEFC) were engineered and synthesized through a two-step sequential reversible addition-fragmentation chain transfer polymerization route. The characterization was performed by FTIR, ¹H NMR, SEC, XRD and TGA techniques. The self-assembly behavior in aqueous solution upon triggered by pH, magnetic and redox stimuli was investigated via zeta potentials, vibration sample magnetometer, cyclic voltammetry, fluorescent spectrometry, dynamic light scattering, XPS, TEM and SEM measurements. The experimental results indicated that the Fe₃O₄-g-PAA-b-PMAEFC copolymer materials could spontaneously assemble into hybrid magnetic copolymer micromicelles with core–shell structure, and exhibited superparamagnetism, redox and pH stimuli-responsive features. The hybrid copolymer micromicelles were stable and nontoxic, and could entrap hydrophobic anticancer drug, which was in turn swiftly and effectively delivered from the drug-loaded micromicelles at special microenvironments such as acidic pH and high reactive oxygen species.

Conclusion

This class of stimuli-responsive copolymer materials is expected to find wide applications in medical science and biology, etc., especially in drug delivery system.

     

Adhesion contact kinetics of HepG2 cells during Hepatitis B virus replication: Involvement of SH3-binding motif in HBX

It has been shown that Hepatitis B virus (HBV) replication directly alters the expression of key cytoskeleton-associated proteins which play key roles in mechanochemical signal transduction. Nevertheless, little is known on the correlation between HBV replication and the subsequent adhesion mechanism of HBV-replicating cells. In this study, it is demonstrated that the lag time of adhesion contact evolution of HepG2 cells with HBV replication is significantly increased by two times compared to that of normal HepG2 cell on collagen coated substrate. During the initial 20 min of cell seeding, only diffuse forms of vinculin was detected in HBV replicating cells while vinculin-associated focal complexes were found in normal and control cells. Similar delay in cell adhesion in HBV-replicating cells was observed in cells transfected with HBX, the smallest HBV protein, suggesting its involvement in this cellular process. In addition, a proline rich region found in many SH3 binding proteins was identified in HBX. HBX was found to interact with the focal adhesion protein, vinexin-beta, through the SH3 binding. Furthermore, HepG2 cells with HBV replication showed evidence of cell rounding up, possibly resulting from cytoskeletal reorganizations associated with interaction between HBX and vinexin-beta. Taken together, our results suggest that HBX is involved in the cytoskeletal reorganization in response to HBV replication.     

Reversal of diabetic nephropathy by a ketogenic diet

Intensive insulin therapy and protein restriction delay the development of nephropathy in a variety of conditions, but few interventions are known to reverse nephropathy. Having recently observed that the ketone 3-beta-hydroxybutyric acid (3-OHB) reduces molecular responses to glucose, we hypothesized that a ketogenic diet, which produces prolonged elevation of 3-OHB, may reverse pathological processes caused by diabetes. To address this hypothesis, we assessed if prolonged maintenance on a ketogenic diet would reverse nephropathy produced by diabetes. In mouse models for both Type 1 (Akita) and Type 2 (db/db) diabetes, diabetic nephropathy (as indicated by albuminuria) was allowed to develop, then half the mice were switched to a ketogenic diet. After 8 weeks on the diet, mice were sacrificed to assess gene expression and histology. Diabetic nephropathy, as indicated by albumin/creatinine ratios as well as expression of stress-induced genes, was completely reversed by 2 months maintenance on a ketogenic diet. However, histological evidence of nephropathy was only partly reversed. These studies demonstrate that diabetic nephropathy can be reversed by a relatively simple dietary intervention. Whether reduced glucose metabolism mediates the protective effects of the ketogenic diet remains to be determined.     

Newcastle disease virus-vectored rabies vaccine is safe, highly immunogenic, and provides long-lasting protection in dogs and cats

Effective, safe, and affordable rabies vaccines are still being sought. Newcastle disease virus (NDV), an avian paramyxovirus, has shown promise as a vaccine vector for mammals. Here, we generated a recombinant avirulent NDV La Sota strain expressing the rabies virus glycoprotein (RVG) and evaluated its potential to serve as a vaccine against rabies. The recombinant virus, rL-RVG, retained its high-growth property in chicken eggs, with titers of up to 10^9.8 50% egg infective doses (EID₅₀)/ml of allantoic fluid. RVG expression enabled rL-RVG to spread from cell to cell in a rabies virus-like manner, and RVG was incorporated on the surface of the rL-RVG viral particle. RVG incorporation did not alter the trypsin-dependent infectivity of the NDV vector in mammalian cells. rL-RVG and La Sota NDV showed similar levels of sensitivity to a neutralization antibody against NDV and similar levels of resistance to a neutralization antibody against rabies virus. Animal studies demonstrated that rL-RVG is safe in several species, including cats and dogs, when administered as multiple high doses of recombinant vaccine. Intramuscular vaccination with rL-RVG induced a substantial rabies virus neutralization antibody response and provided complete protection from challenge with circulating rabies virus strains. Most importantly, rL-RVG induced strong and long-lasting protective neutralization antibody responses to rabies virus in dogs and cats. A low vaccine dose of 10^8.3 EID₅₀ completely protected dogs from challenge with a circulating strain of rabies virus for more than a year. This is the first study to demonstrate that immunization with an NDV-vectored vaccine can induce long-lasting, systemic protective immunity against rabies.     

Protocorm-like body (PLB) formation and plant regeneration from the callus culture of <Emphasis Type="Italic">Dendrobium candidum</Emphasis> Wall <Emphasis Type="Italic">ex</Emphasis> Lindl.

An efficient system was established for a higher frequency of protocorm-like body (PLB) formation from the callus of Dendrobium candidum Wall ex Lindl. The calluses were induced from longitudinally bisected segments of protocorms and subcultured two times every 40d on Murashige and Skoog medium with macronutrients at half strength, micronutrients at full strength, 2% sucrose, and with 8.8μM 6-Benzylaminopurine. PLB formation was achieved when calluses were transferred onto the same basal medium without any plant growth regulators. PLBs developed into intact plantlets about 2cm in height and with four roots when on basal medium with 2.7μM 1-naphthaleneacetic acid. Plantlets were transplanted into vermiculite with a 95% survival rate in a greenhouse. Histological observations proved that globular somatic embryos could be produced from the inside and surface of the embryogenic callus during PLB formation.     

miR-300 regulates cellular radiosensitivity through targeting p53 and apaf1 in human lung cancer cells

microRNAs (miRNAs) play a crucial role in mediation of the cellular sensitivity to ionizing radiation (IR). Previous studies revealed that miR-300 was involved in the cellular response to IR or chemotherapy drug. However, whether miR-300 could regulate the DNA damage responses induced by extrinsic genotoxic stress in human lung cancer and the underlying mechanism remain unknown. In this study, the expression of miR-300 was examined in lung cancer cells treated with IR, and the effects of miR-300 on DNA damage repair, cell cycle arrest, apoptosis and senescence induced by IR were investigated. It was found that IR induced upregulation of endogenous miR-300, and ectopic expression of miR-300 by transfected with miR-300 mimics not only greatly enhanced the cellular DNA damage repair ability but also substantially abrogated the G2 cell cycle arrest and apoptosis induced by IR. Bioinformatic analysis predicted that p53 and apaf1 were potential targets of miR-300, and the luciferase reporter assay showed that miR-300 significantly suppressed the luciferase activity through binding to the 3′-UTR of p53 or apaf1 mRNA. In addition, overexpression of miR-300 significantly reduced p53/apaf1 and/or IR-induced p53/apaf1 protein expression levels. Flow cytomertry analysis and colony formation assay showed that miR-300 desensitized lung cancer cells to IR by suppressing p53-dependent G2 cell cycle arrest, apoptosis and senescence. These data demonstrate that miR-300 regulates the cellular sensitivity to IR through targeting p53 and apaf1 in lung cancer cells.     

Plasmodium chabaudi AS: Distinct CD4+CD25+Foxp3+ regulatory T cell responses during infection in DBA/2 and BALB/c mice

Malaria infections display variation patterns of clinical course and outcome. Although CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells play an essential role in immune homeostasis, the immune regulatory roles involved in malaria infection remains to be elucidated. Herein, we compared the disparity in Treg cells response during the course of blood stage Plasmodium chabaudi chabaudi AS (P. c chabaudi AS) infection in DBA/2 and BALB/c mice. BALB/c mice initiated a Th1/Th2 profile respond to P. c chabaudi AS infection, but DBA/2 mice failed to control P. c chabaudi AS infection and almost of them died post-peak parasitemia. At the peak parasitemia, we found that higher proportion of Treg cells with elevated Foxp3 expression in DBA/2 than in BALB/c mice. We used anti-CD25 mAb to deplete Treg cells and found that the survival time and rate were prolonged in DBA/2 mice treated with anti-CD25 mAb. Treatment with anti-CD25 mAb in vivo led to enhanced pro-inflammation responses and Foxp3 expression decline on Treg cells. In contrast, after DBA/2 was treatment with anti-IL-10R mAb, IL-10R blockade in vivo caused excessive pro-inflammation responses and Foxp3 expression loss on CD4⁺CD25⁺ T cells. Earlier death was found in all of DBA/2 mice with anti-IL-10R mAb. It suggested that IL-2 and IL-10 signal involved in maintaining Foxp3 expression on Treg cells. In all, the moderate suppressive activity of Treg cells may facilitate resistance to P. c chabaudi AS infection.     

GEF-H1/RhoA signalling pathway mediates lipopolysaccharide-induced intercellular adhesion molecular-1 expression in endothelial cells via activation of p38 and NF-κB

The purpose of study is to investigate the effects of GEF-H1/RhoA pathway in regulating intercellular adhesion molecule-1 (ICAM-1) expression in lipopolysaccharide (LPS)-activated endothelial cells. Exposure of human umbilical vein endothelial cells (HUVECs) to LPS induced GEF-H1 and ICAM-1 expression in dose- and time-dependent up-regulating manners. Pretreatment with Clostridium difficile toxin B-10463 (TcdB-10463), an inhibitor of Rho activity, reduced LPS-related phosphorylation of p65 at Ser 536 in a dose-dependent manner. Inhibition of TLR4 expression significantly blocked LPS-induced RhoA activity, NF-κB transactivation, GEF-H1 and ICAM-1 expression. Coimmunoprecipitation assay indicated that LPS-activated TLR4 and GEF-H1 formed a signalling complex, suggesting that LPS, acting through TLR4, stimulates GEF-H1 expression and RhoA activity, and thereby induces NF-κB transactivation and ICAM-1 gene expression. However, GEF-H1/RhoA regulates LPS-induced NF-κB transactivation and ICAM-1 expression in a MyD88-independent pathway because inhibition of MyD88 expression could not block LPS-induced RhoA activity. Furthermore, pretreatment with Y-27632, an inhibitor of ROCK, significantly reduced LPS-induced p38, ERK1/2 and p65 phosphorylation, indicating that ROCK acts as an upstream effector of p38 and ERK1/2 to promote LPS-induced NF-κB transactivation and ICAM-1 expression. What is more, the p38 inhibitor (SB203580) but not ERK1/2 inhibitor (PD98059) blocked LPS-induce NF-κB transactivation and ICAM-1 expression, which demonstrates that RhoA mediates LPS-induced NF-κB transactivation and ICAM-1 expression dominantly through p38 but not ERK1/2 activation. In summary, our data suggest that LPS-induced ICAM-1 synthesis in HUVECs is regulated by GEF-H1/RhoA-dependent signaling pathway via activation of p38 and NF-κB.