Partial Functional Rescue of Helicoverpa armigera Single Nucleocapsid Nucleopolyhedrovirus Infectivity by Replacement of F Protein with GP64 from Autographa californica Multicapsid Nucleopolyhedrovirus

Two distinct envelope fusion proteins (EFPs) (GP64 and F) have been identified in members of the Baculoviridae family of viruses. F proteins are found in group II nucleopolyhedroviruses (NPVs) of alphabaculoviruses and in beta- and deltabaculoviruses, while GP64 occurs only in group I NPVs of alphabaculoviruses. It was proposed that an ancestral baculovirus acquired the gp64 gene that conferred a selective advantage and allowed it to evolve into group I NPVs. The F protein is a functional analogue of GP64, as evidenced from the rescue of gp64-null Autographa californica multicapsid nucleopolyhedrovirus (MNPV) (AcMNPV) by F proteins from group II NPVs or from betabaculoviruses. However, GP64 failed to rescue an F-null Spodoptera exigua MNPV (SeMNPV) (group II NPV). Here, we report the successful generation of an infectious gp64-rescued group II NPV of Helicoverpa armigera (vHaBacΔF-gp64). Viral growth curve assays and quantitative real-time PCR (Q-PCR), however, showed substantially decreased infectivity of vHaBacΔF-gp64 compared to the HaF rescue control virus vHaBacΔF-HaF. Electron microscopy further showed that most vHaBacΔF-gp64 budded viruses (BV) in the cell culture supernatant lacked envelope components and contained morphologically aberrant nucleocapsids, suggesting the improper BV envelopment or budding of vHaBacΔF-gp64. Bioassays using pseudotyped viruses with a reintroduced polyhedrin gene showed that GP64-pseudotyped Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) significantly delayed the mortality of infected H. armigera larvae.The envelope fusion protein (EFP) of budded viruses (BV) (30) of baculoviruses is critical for virus entry (attachment and fusion) and egress (assembly and budding) (7, 13, 21). Two types of BV EFPs have been identified in the Baculoviridae family of viruses. The F proteins are similar in structure, but they are very diverse in their amino acid sequences (20 to 40% identity). They are widespread within the baculovirus family (group II NPVs of the alphabaculoviruses and in beta- and deltabaculoviruses) (23) and are thought to be carried by ancestral members (26). In contrast, the baculovirus GP64 homologs are all closely related EFPs (>74% sequence identity) and found only in group I NPVs of the alphabaculoviruses (23). It has been suggested that a gp64 gene was acquired relatively recently by an ancestral virus of the group II NPV, thereby giving these viruses a selective advantage and obviating the need of the envelope fusion function of the F protein (23). A nonfusogenic F homolog (F-like protein), however, is maintained in the genome of group I NPVs, functioning as a virulence factor (9, 17, 24, 32).GP64 and F proteins play similar roles during the baculovirus infection processes, such as virus-cell receptor attachment, membrane fusion, and efficient budding. However, there are striking differences between the receptor usage of GP64 and F proteins as well. These two types of proteins are very different in structure, mode of action, and receptor exploitation. The crystal structure reveals that GP64 belongs to class III viral fusion proteins, with its fusion loop located in the internal region of the protein, and proteolytic cleavage is not required for activation of fusion activity (10). F proteins by contrast share common features of class I viral fusion proteins (12). The proteolytic cleavage of the F precursor (F₀) by a furin-like protease generates an N-terminal F₂ fragment and a C-teminal F₁ fragment. This cleavage is essential for exposing the N-terminal fusion peptide of F₁ and for activating F fusogenicity (8, 36). Although the nature of the baculovirus host cell receptors is still enigmatic, it has been reported that Autographa californica multicapsid nucleopolyhedrovirus (MNPV) (AcMNPV)) and Orgyia pseudotsugata MNPV (OpMNPV), both using GP64 as their EFPs, exploit the same insect cell receptor, while Lymantria dispar MNPV (LdMNPV) with an F protein as the EFP utilizes a cell receptor different from that used by AcMNPV (7, 37). Additionally, in the case of SeMNPV, using competition assays, it was confirmed that the baculovirus F protein and GP64 recognized distinct receptors to gain entry into cultured insect cells (34).Pseudotyping viral nucleocapsid with heterologous EFPs to form pseudotype virions is a valuable approach to studying the structure, function, and specificity of heterologous EFPs. It has been a successful strategy to expand or alter viral host range, i.e., in gene delivery (3). For example, vesicular stomatitis virus G (VSV-G)-pseudotyped lentivirus and AcMNPV gp64-pseudotyped HIV-1 exhibit high virus titers and wider tropism (5, 14, 38); the gp64-pseudotyped human respiratory syncytial virus (HRSV) lacking its own glycoproteins is of high and stable infectivity (22); furthermore, pseudotyped lentiviruses with modified fusion proteins of GP64 with targeting peptides (i.e., hepatitis B virus PreS1 peptide, involved in viral attachment) or with the decay accelerating factor (DAF) facilitate the targeting to specific cell types or confer stability against serum inactivation, respectively (6, 19). For the Baculoviridae, a series of pseudotyping studies have investigated the functional analogy between GP64 and F proteins. F proteins of group II NPVs (SeMNPV, LdMNPV, and Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus [HearNPV]) can substitute for GP64 in gp64-null AcMNPV viruses (15, 16). Recent studies indicated that many granulovirus (GV) F proteins, but not F protein from Plutella xylostella GV (PxGV), can rescue a gp64-null AcMNPV (16, 39). These results demonstrated that baculovirus F proteins are functional analogues to GP64. Since it was postulated that GP64 was captured by a baculovirus during evolution (24), one would expect the functional incorporation of GP64 into the BV of an F-null group II NPV. However, the reverse substitution of a group II NPV (SeMNPV) F protein by GP64 failed to produce infectious progeny viruses (35).In this paper, we show that AcMNPV gp64 could be inserted into an F-null HearNPV genome and produce infectious progeny virus upon transfection of insect cells. The infectivity of the pseudotyped virus, however, was greatly impaired, and large amounts of morphologically defective BV were produced. Bioassay experiments indicated that the infectivity of GP64-pseudotyped F-null HearNPV for insect larvae was not reduced, but that the time to death was significantly delayed. These results demonstrate that GP64 alone can only partially complement HearNPV F protein function.     

Identification and Characterization of Bmi-1-responding Element within the Human p16 Promoter

Bmi-1, the first functionally identified polycomb gene family member, plays critical roles in cell cycle regulation, cell immortalization, and cell senescence. Bmi-1 is involved in the development and progression of carcinomas and is a potent target for cancer therapy. One important pathway regulated by Bmi-1 is that involving two cyclin-dependent kinase inhibitors, p16^Ink4a and p19^Arf, as Bmi-1 represses the INK4a locus on which they are encoded. A close correlation between the up-regulation of Bmi-1 and down-regulation of p16 has been demonstrated in various tumors; however, how Bmi-1 regulates p16 expression is not clear. In this study, we revealed that Bmi-1 regulates the expression of p16 by binding directly to the Bmi-1-responding element (BRE) within the p16 promoter. The BRE resided at bp −821 to −732 upstream of the p16 ATG codon. BRE alone was sufficient to allow Bmi-1-mediated regulation of the CMV promoter. Bmi-1 typically functions by forming a complex with Ring2; however, regulation of p16 was independent of Ring2. Chromatin immunoprecipitation sequencing of Bmi-1-precipitated chromatin DNA revealed that 1536 genes were targeted by Bmi-1, including genes involved in tissue-specific differentiation, cell cycle, and apoptosis. By analyzing the binding sequences of these genes, we found two highly conserved Bmi-1-binding motifs, which were required for Bmi-1-mediated p16 promoter regulation. Taken together, our results revealed the molecular mechanism of Bmi-1-mediated regulation of the p16 gene, thus providing further insights into the functions of Bmi-1 as well as a sensitive high-throughput platform with which to screen Bmi-1-targeted small molecules for cancer therapy.     

The Legs at odd angles (Loa) Mutation in Cytoplasmic Dynein Ameliorates Mitochondrial Function in SOD1G93A Mouse Model for Motor Neuron Disease

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal late-onset neurodegenerative disease. Familial cases of ALS (FALS) constitute ∼10% of all ALS cases, and mutant superoxide dismutase 1 (SOD1) is found in 15–20% of FALS. SOD1 mutations confer a toxic gain of unknown function to the protein that specifically targets the motor neurons in the cortex and the spinal cord. We have previously shown that the autosomal dominant Legs at odd angles (Loa) mutation in cytoplasmic dynein heavy chain (Dync1h1) delays disease onset and extends the life span of transgenic mice harboring human mutant SOD1^G93A. In this study we provide evidence that despite the lack of direct interactions between mutant SOD1 and either mutant or wild-type cytoplasmic dynein, the Loa mutation confers significant reductions in the amount of mutant SOD1 protein in the mitochondrial matrix. Moreover, we show that the Loa mutation ameliorates defects in mitochondrial respiration and membrane potential observed in SOD1^G93A motor neuron mitochondria. These data suggest that the Loa mutation reduces the vulnerability of mitochondria to the toxic effects of mutant SOD1, leading to improved mitochondrial function in SOD1^G93A motor neurons.     

A Comprehensive Proteomic Analysis of the Type III Secretome of Citrobacter rodentium

Enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium belong to the family of attaching and effacing (A/E) bacterial pathogens. They intimately attach to host intestinal epithelial cells, trigger the effacement of intestinal microvilli, and cause diarrheal disease. Central to their pathogenesis is a type III secretion system (T3SS) encoded by a pathogenicity island called the locus of enterocyte effacement (LEE). The T3SS is used to inject both LEE- and non-LEE-encoded effector proteins into the host cell, where these effectors modulate host signaling pathways and immune responses. Identifying the effectors and elucidating their functions are central to understanding the molecular pathogenesis of these pathogens. Here we analyzed the type III secretome of C. rodentium using the highly sensitive and quantitative SILAC (stable isotope labeling with amino acids in cell culture)-based mass spectrometry. This approach not only confirmed nearly all known secreted proteins and effectors previously identified by conventional biochemical and proteomic techniques, but also identified several new secreted proteins. The T3SS-dependent secretion of these new proteins was validated, and five of them were translocated into cultured cells, representing new or additional effectors. Deletion mutants for genes encoding these effectors were generated in C. rodentium and tested in a murine infection model. This study comprehensively characterizes the type III secretome of C. rodentium, expands the repertoire of type III secreted proteins and effectors for the A/E pathogens, and demonstrates the simplicity and sensitivity of using SILAC-based quantitative proteomics as a tool for identifying substrates for protein secretion systems.     

Site-Directed Mutagenesis on Human Cystathionine-γ-Lyase Reveals Insights into the Modulation of H2S Production

In recent years, increased interest has been directed towards hydrogen sulfide (H₂S) as the third gasotransmitter and its role in various diseases. Cystathionine-γ-lyase (CSE) is one of the enzymes responsible for the endogenous production of H₂S in mammals. With the aid of the crystal structures of human CSE and site-directed mutagenesis studies, we have identified several amino acid residues in CSE that are actively involved in the catalysis of H₂S production. Contrary to reports suggesting that Tyr114 is required for substrate binding, our results reveal a significant increase in the production of H₂S upon mutation of Tyr114 to phenylalanine. This is attributed to an increased rate of pyridoxal 5′-phosphate (PLP) regeneration due to weakened π-stacking interactions between Phe114 and PLP. Thr189 is also identified as a crucial residue where hydrogen bonding to Asp187 keeps the latter in an optimal position for hydrogen bonding to the pyridoxal nitrogen of PLP. Furthermore, mutation of Glu339 to lysine, alanine or tyrosine reveals the importance of the hydrophobicity of the 339th amino acid in determining the specificity of the enzyme for the catalysis of α,γ-elimination or α,β-elimination reaction. Our study also shows that the rate of H₂S production is increased with increasing exogenous PLP concentration, hence supporting our hypothesis that apo-CSE is formed during the catalysis of H₂S production. Taken together, these findings suggest novel routes towards the design of activators or inhibitors that modulate the production of H₂S; these modulators may also serve as lead compounds in the development of drugs or mechanistic probes in the study of various H₂S-related diseases.     

Metal-organic open frameworks with one-dimension channels assembled with pyridine 2,6-dicarboxylic acid and N-containing auxiliary ligands: Syntheses, crystal structures, and physical characterization

Four new complexes, {[Mn(imH)₂(pdc)]·H₂O}_n (1), [Zn₂(pdc)₂(H₂O)₅]·2H₂O (2), [Zn(imH)₂(pdc)]·H₂O (3), {[Zn₂(pdc)₂(bpy)(H₂O)₂]·5H₂O}_n (4) [imH = imidazole pdc = pyridine 2,6-dicarboxylate, bpy = 4,4′-bipyridine] have been synthesized under hydrothermal conditions and structurally characterized by elemental analysis, IR, PXRD, single-crystal X-ray diffraction and thermogravimetric analyses. All the four complexes display a three-dimensional (3D) open framework with one-dimensional (1D) channels that are filled with lattice water molecules. Particularly, in 4, the lattice water molecules form an infinite water chain. Both 1 and 4 consist of 1D polymeric chains. While 2 contains a dinuclear Zn(II) unit, and 3 is a mononuclear complex. Further, the result of thermal analysis of 1 and 2 shows the robustness of the overall supramolecular three-dimensional architecture. Complexes 1, 3, and 4 exhibit strong fluorescent emissions in the solid state at room temperature and could be significant in the field of photoactive materials.     

Synthesis and characterization of chitosan/ZnO nanoparticle composite membranes

Novel chitosan/ZnO nanoparticle (CS/nano-ZnO) composite membranes were prepared via the method of sol-cast transformation and studied by UV-vis absorption spectroscopy (UV-vis), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy dispersive X-ray fluorescence spectrometry (EDX). The characterization revealed that ZnO nanoparticles dispersed homogeneously within the chitosan matrix. The mechanical and antibacterial properties of the product were investigated. The results showed that the ZnO content had an effect on the mechanical properties of CS/nano-ZnO composite membranes, and that the antibacterial activities of CS membranes for Bacillus subtilis, Escherichia coli, and Staphylococcus aureus were enhanced by the incorporation of ZnO. Further, CS/nano-ZnO composite membranes with 6-10 wt % ZnO exhibited high antibacterial activities.     

Regulatory effect of vasoactive intestinal peptide on the balance of Treg and Th17 in collagen-induced arthritis

Vasoactive intestinal peptide (VIP) is a well-known anti-inflammatory neuropeptide. The capacity of VIP can be exhibited through inhibiting inflammatory responses, shifting the Th1/Th2 balance in favor of anti-inflammatory Th2 immunity and inducing regulatory T cells (Tregs) with suppressive activity. In addition to pro-inflammatory Th1 response, Th17 are also believed to play important roles in the pathogenesis of rheumatoid arthritis (RA). In this study, we used collagen-induced arthritis (CIA) model in Wistar rats to investigate the role of VIP in the balance of CD4⁺ CD25⁺ Tregs and Th17 on RA. Data presented here showed that administration of VIP decreased incidence and severity of CIA. Disease suppression was associated with the upregulation of CD4⁺ CD25⁺ Tregs, downregulation of Th17- and Th1-type response and influence on the RANK/RANKL/OPG system. The results provide novel evidence that the therapeutic effects of VIP on CIA rats were associated with the balance of CD4⁺ CD25⁺ Tregs and Th17.     

Confounding from cryptic relatedness in haplotype-based association studies

Cryptic relatedness was suggested to be an important source of confounding in population-based association studies (PBAS). The impact of cryptic relatedness on the performance of haplotype phase inference and haplotype-based association tests is not clear. In this study, we used the Hapmap genetic data to simulate a set of related samples. We evaluated the accuracy of haplotype phase inferred by PHASE 2.1 and calculated the power, type I error rates, accuracy and positive prediction value (PPV) of haplotype frequency-based association tests (HFAT) and haplotype similarity-based association tests (HSAT) under various scenarios, considering relatedness levels, disease models and sample sizes. Cryptic relatedness appeared to slightly increase the accuracy of haplotype phase inference. We observed significant negative effect of cryptic relatedness on the performance of HFAT and HSAT. Ignoring cryptic relatedness may increase spurious association results in haplotype-based PBAS.