New 4,5‐seco‐20(10→5)‐abeo‐Abietane Diterpenoids with Anti‐Inflammatory Activity from Isodon lophanthoides var. graciliflorus (Benth.) H.Hara

Three new 4,5‐seco‐20(10→5)‐abeo‐abietane diterpenoids, 16‐hydroxysalvilenone ( 1 ), 15‐hydroxysalprionin ( 2 ), and 11β,15‐dihydroxysalprionin‐12‐one ( 3 ), and nine known abietane diterpenoids, 4 – 12 , along with one known sempervirane diterpenoid, hispidanol A ( 13 ), were isolated from the aerial parts of Isodon lophanthoides var. graciliflorus. The structures of compounds 1 – 3 were determined on the basis of spectroscopic methods including extensive analysis of NMR and mass spectroscopic data. All diterpenoids were tested for their TNF‐α inhibitory effects on LPS‐induced RAW264.7 cells. Compound 9 (16‐acetoxyhorminone) was the most potent with an IC₅₀ value of 3.97±0.70 μm .     

Virus‐inspired and mimetic designs in non‐viral gene delivery

Virus‐inspired mimics for nucleic acid transportation have attracted much attention in the past decade, especially the derivative microenvironment stimuli‐responsive designs. In the present mini‐review, the smart designs of gene carriers that overcome biological barriers and realize an efficient delivery are categorized with respect to the different “triggers” provided by tumor cells, including pH, redox potentials, ATP, enzymes and reactive oxygen species. Some dual/multi‐responsive gene vectors have also been introduced that show a more precise and efficient delivery in the complicated environment of human body. In addition, inspired by the special recognition mechanisms and components of viruses, improvements in the design of carriers relating to targeting/penetration properties, as well as chemical component evolution, are also addressed.     

Bending of the BST‐2 coiled‐coil during viral budding

BST‐2/tetherin is a human extracellular transmembrane protein that serves as a host defense factor against HIV‐1 and other viruses by inhibiting viral spreading. Structurally, BST‐2 is a homo‐dimeric coiled‐coil that is connected to the host cell membrane by N and C terminal transmembrane anchors. The C‐terminal membrane anchor of BST‐2 is inserted into the budding virus while the N‐terminal membrane anchor remains in the host cell membrane creating a viral tether. The structural mechanism of viral budding and tethering as mediated by BST‐2 is not clear. To more fully describe the mechanism of viral tethering, we created a model of BST‐2 embedded in a membrane and used steered molecular dynamics to simulate the transition from the host cell membrane associated form to the cell‐virus membrane bridging form. We observed that BST‐2 did not transition as a rigid structure, but instead bent at positions with a reduced interface between the helices of the coiled‐coil. The simulations for the human BST‐2 were then compared with simulations on the mouse homolog, which has no apparent weak spots. We observed that the mouse homolog spread the bending across the ectodomain, rather than breaking at discrete points as observed with the human homolog. These simulations support previous biochemical and cellular work suggesting some flexibility in the coiled‐coil is necessary for viral tethering, while also highlighting how subtle changes in protein sequence can influence the dynamics and stability of proteins with overall similar structure.     

Higher viral load and genetic diversity of HIV‐1 in seminal compartments than in blood of seven Chinese men who have sex with men and have early HIV‐1 infection

To date, there have been no reports characterizing HIV‐1 in the semen of Chinese men who have sex with men (MSM) with early infection. In this study, genetic diversity and viral load of HIV‐1 in the seminal compartments and blood of Chinese MSM with early HIV‐1 infection were examined. Viral load and genetic diversity of HIV‐1 in paired samples of semen and blood were analyzed in seven MSM with early HIV‐1 infection. HIV‐1 RNA and DNA were quantitated by real‐time PCR assays. Through sequencing the C2‐V5 region of the HIV‐1 env gene, the HIV‐1 genotype and genetic diversity based on V3 loop amino acid sequences were determined by using Geno2pheno and PSSM programs co‐receptor usage. It was found that there was more HIV‐1 RNA in seminal plasma than in blood plasma and total, and more 2‐LTR circular and integrated HIV‐1 DNA in seminal cells than in peripheral blood mononuclear cells from all seven patients with early HIV‐infection. There was also greater HIV‐1 genetic diversity in seminal than in blood compartments. HIV‐1 in plasma displayed higher genetic diversity than in cells from the blood and semen. In addition, V3 loop central motifs, which present some key neutralizing antibody epitopes, varied between blood and semen. Thus, virological characteristics in semen may be more representative when evaluating risk of transmission in persons with early HIV infection.

     

Cladosporactone A,a Unique Polyketide with 7‐Methylisochromen‐3‐one Skeleton from the Deep‐Sea‐Derived Fungus Cladosporium cladosporioides

A unique polyketide cladosporactone A along with eight known compounds were isolated from the deep‐sea‐derived Cladosporium cladosporioides. The structure of cladosporactone A was established by spectroscopic analyses, and the absolute configuration was clarified by the theoretical ECD calculation. Cladosporactone A is the first member of polyketide with the 7‐methylisochromen‐3‐one skeleton.     

Quantitation of viral load using real-time amplification techniques

Real-time PCR amplification techniques are currently used to determine the viral load in clinical samples for an increasing number of targets. Real-time PCR reduces the time necessary to generate results after amplification. In-house developed PCR and nucleic acid sequence-based amplification (NASBA)-based systems combined with several detection strategies are being employed in a clinical diagnostic setting. The importance of these assays in disease management is still in an exploration phase. Although these technologies have the implicit capability of accurately measuring DNA and RNA in clinical samples, issues related to standardization and quality control must be resolved to enable routine implementation of these technologies in molecular diagnostics.     

Non‐viral gene delivery for cancer immunotherapy

Recent decades have witnessed the revolutionary development of cancer immunotherapies, which boost cancer‐specific immune responses for long‐term tumor regression. However, immunotherapy still has limitations, including off‐target side effects, long processing times and limited patient responses. These disadvantages of current immunotherapy are being addressed by improving our understanding of the immune system, as well as by establishing combinational approaches. Advanced biomaterials and gene delivery systems overcome some of these delivery issues, harnessing adverse effects and amplifying immunomodulatory effects, and are superior to standard formulations with respect to eliciting antitumor immunity. Nucleic acid‐based nanostructures have diverse functions, ranging from gene expression and gene regulation to pro‐inflammatory effects, as well as the ability to specifically bind different molecules. A brief overview is provided of the recent advances in the non‐viral gene delivery methods that are being used to activate cancer‐specific immune responses. Furthermore, the tumor microenvironment‐responsive synergistic strategies that modulate the immune response by targeting various signaling pathways are discussed. Nanoparticle‐based non‐viral gene delivery strategies have great potential to be implemented in the clinic for cancer immunotherapy.     

LincRNA‐EPS impairs host antiviral immunity by antagonizing viral RNA–PKR interaction

LincRNA‐EPS is an important regulator in inflammation. However, the role of lincRNA‐EPS in the host response against viral infection is unexplored. Here, we show that lincRNA‐EPS is downregulated in macrophages infected with different viruses including VSV, SeV, and HSV‐1. Overexpression of lincRNA‐EPS facilitates viral infection, while deficiency of lincRNA‐EPS protects the host against viral infection in vitro and in vivo. LincRNA‐EPS ^−/− macrophages show elevated expression of antiviral interferon‐stimulated genes (ISGs) such as Mx1, Oas2, and Ifit2 at both basal and inducible levels. However, IFN‐β, the key upstream inducer of these ISGs, is downregulated in lincRNA‐EPS ^−/− macrophages compared with control cells. RNA pulldown and mass spectrometry results indicate that lincRNA‐EPS binds to PKR and antagonizes the viral RNA–PKR interaction. PKR activates STAT1 and induces antiviral ISGs independent of IFN‐I induction. LincRNA‐EPS inhibits PKR‐STAT1‐ISGs signaling and thus facilitates viral infection. Our study outlines an alternative antiviral pathway, with downregulation of lincRNA‐EPS promoting the induction of PKR‐STAT1‐dependent ISGs, and reveals a potential therapeutic target for viral infectious diseases.     

Predictive modeling of non‐viral gene transfer

In non‐viral gene delivery, the variance of transgenic expression stems from the low number of plasmids successfully transferred. Here, we experimentally determine Lipofectamine‐ and PEI‐mediated exogenous gene expression distributions from single cell time‐lapse analysis. Broad Poisson‐like distributions of steady state expression are observed for both transfection agents, when used with synchronized cell lines. At the same time, co‐transfection analysis with YFP‐ and CFP‐coding plasmids shows that multiple plasmids are simultaneously expressed, suggesting that plasmids are delivered in correlated units (complexes). We present a mathematical model of transfection, where a stochastic, two‐step process is assumed, with the first being the low‐probability entry step of complexes into the nucleus, followed by the subsequent release and activation of a small number of plasmids from a delivered complex. This conceptually simple model consistently predicts the observed fraction of transfected cells, the cotransfection ratio and the expression level distribution. It yields the number of efficient plasmids per complex and elucidates the origin of the associated noise, consequently providing a platform for evaluating and improving non‐viral vectors. Biotechnol. Bioeng. 2010. 105: 805–813. © 2009 Wiley Periodicals, Inc.     

Multi‐targeting of viral RNAs with synthetic trans‐acting small interfering RNAs enhances plant antiviral resistance

RNA interference (RNAi)‐based tools are used in multiple organisms to induce antiviral resistance through the sequence‐specific degradation of target RNAs by complementary small RNAs. In plants, highly specific antiviral RNAi‐based tools include artificial microRNAs (amiRNAs) and synthetic trans‐acting small interfering RNAs (syn‐tasiRNAs). syn‐tasiRNAs have emerged as a promising antiviral tool allowing for the multi‐targeting of viral RNAs through the simultaneous expression of several syn‐tasiRNAs from a single precursor. Here, we compared in tomato plants the effects of an amiRNA construct expressing a single amiRNA and a syn‐tasiRNA construct expressing four different syn‐tasiRNAs against Tomato spotted wilt virus (TSWV), an economically important pathogen affecting tomato crops worldwide. Most of the syn‐tasiRNA lines were resistant to TSWV, whereas the majority of the amiRNA lines were susceptible and accumulated viral progenies with mutations in the amiRNA target site. Only the two amiRNA lines with higher amiRNA accumulation were resistant, whereas resistance in syn‐tasiRNA lines was not exclusive of lines with high syn‐tasiRNA accumulation. Collectively, these results suggest that syn‐tasiRNAs induce enhanced antiviral resistance because of the combined silencing effect of each individual syn‐tasiRNA, which minimizes the possibility that the virus simultaneously mutates all different target sites to fully escape each syn‐tasiRNA.     

Granzyme B‐expressing neutrophils correlate with bacterial load in granulomas from Mycobacterium tuberculosis‐infected cynomolgus macaques

The role of neutrophils in tuberculosis (TB), and whether neutrophils express granzyme B (grzB), a pro‐apoptotic enzyme associated with cytotoxic T cells, is controversial. We examined neutrophils in peripheral blood (PB) and lung granulomas of Mycobacterium tuberculosis‐infected cynomolgus macaques and humans to determine whether mycobacterial products or pro‐inflammatory factors induce neutrophil grzB expression. We found large numbers of grzB‐expressing neutrophils in macaque and human granulomas and these cells contained more grzB+ granules than T cells. Higher neutrophil, but not T cell, grzB expression correlated with increased bacterial load. Although unstimulated PB neutrophils lacked grzB expression, grzB expression increased upon exposure to M. tuberculosis bacilli, M. tuberculosis culture filtrate protein or lipopolysaccharide from Escherichia coli. Perforin is required for granzyme‐mediated cytotoxicity by T cells, but was not observed in PB or granuloma neutrophils. Nonetheless, stimulated PB neutrophils secreted grzB as determined by enzyme‐linked immunospot assays. Purified grzB was not bactericidal or bacteriostatic, suggesting secreted neutrophil grzB acts on extracellular targets, potentially enhancing neutrophil migration through extracellular matrix and regulating apoptosis or activation in other cell types. These data indicate mycobacterial products and the pro‐inflammatory environment of granulomas up‐regulates neutrophil grzB expression and suggests a previously unappreciated aspect of neutrophil biology in TB.     

Tick‐borne viruses: Current trends in large‐scale viral surveillance

Ticks are ectoparasites that transmit pathogens, such as tick‐borne viruses, to their hosts. Tick‐borne viruses are diverse: they can be categorized into two orders, nine families, and at least 12 genera. Almost 25% of these viruses are infectious to humans and some are a serious threat to public health. The global rise in tick‐borne virus diseases has been linked to climate change which has reduced tick mortality in the winter and extended their active period. The spread of tick‐borne viral diseases to humans has received significant interest due to the increased threat to human life; epidemiological monitoring of tick‐borne viruses using molecular, immunological, and environmental methods is now a priority. Nevertheless, many tick‐borne diseases remain undiagnosed, which poses a challenge to public administration and health care officials. This review discusses three major tick‐borne RNA viruses that cause serious infection in humans: severe fever with thrombocytopenia syndrome (SFTS) virus, tick‐borne encephalitis (TBE), and Crimean–Congo hemorrhagic fever (CCHF) virus. Specifically, we discuss the epidemiological monitoring, vector control measures, molecular diagnostics, vaccines, and environmental determinants related to these viruses. Furthermore, we review the current surveillance of these tick‐borne viruses with a specific focus on diagnostic approaches that employ molecular interventions such as viral nucleic acid isolation, PCR‐based diagnostics, and high‐throughput sequencing technologies.     

Synthesis of Catechin‐Rare Earth Complex with Efficient and Broad‐Spectrum Anti‐Biofilm Activity

Biofilm is the crucial reason of clinical infections. Herein, green tea based polyphenol (catechin) and rare earth (RE) metal ions were employed for the preparation of catechin–RE complexes with significant anti‐biofilm properties. The complexes were characterized by FT‐IR, Raman spectroscopy, X‐ray photoelectron spectroscopy (XPS) and dynamic light scattering (DLS), which suggested that catechin coordinated with RE³⁺ through its ortho phenolic hydroxy groups. The prepared catechin‐RE showed significant effects in anti‐biofilm growth against P. aeruginosa (Gram‐negative bacteria), S. sciuri (Gram‐positive bacteria), and A. niger (fungi), which significantly exceeded the utilization of catechin or RE³⁺. Morphological observations indicated that catechin supplied cell affinity to transfer RE³⁺ and helped to damage cell membrane, which act as a carrier to exert cytotoxicity of RE³⁺ to realize anti‐biofilm. Differential gene expression analysis described gene expression changes induced by catechin‐RE, including 56, 272 and 2160 downregulated genes for P. aeruginosa, S. sciuri and A. niger, respectively, which suggested critical changes in cellular metabolism, growth and other processes. These results illustrate the outstanding superiority of catechin‐RE complexes in anti‐infection aspect, i. e., the green tea based rare earth complexes are promising candidates for anti‐biofilm applications to address serious challenges in the prevention of multiple infections.