Mechanism of regulation of the Helicobacter pylori Cagß ATPase by CagZ.

The transport of the CagA effector into gastric epithelial cells by the Cag Type IV secretion system (Cag T4SS) of Helicobacter pylori (H. pylori) is critical for pathogenesis. CagA is recruited to Cag T4SS by the Cagß ATPase. CagZ, a unique protein in H. pylori, regulates Cagß-mediated CagA transport, but the underlying mechanisms remain unclear. Here we report the crystal structure of the cytosolic region of Cagß, showing a typical ring-like hexameric assembly. The central channel of the ring is narrow, suggesting that CagA must unfold for transport through the channel. Our structure of CagZ in complex with the all-alpha domain (AAD) of Cagß shows that CagZ adopts an overall U-shape and tightly embraces Cagß. This binding mode of CagZ is incompatible with the formation of the Cagß hexamer essential for the ATPase activity. CagZ therefore inhibits Cagß by trapping it in the monomeric state. Based on these findings, we propose a refined model for the transport of CagA by Cagß.

Sevoflurane protects against intracerebral hemorrhage via microRNA-133b/FOXO4/BCL2 axis.

The application of Sevoflurane (Sev) in neurological diseases has been documented. We herein clarified the role of Sev in intracerebral hemorrhage (ICH). Through bioinformatics analysis, ICH-related microRNA (miRNA) was collected with microRNA-133b (miR-133b) chosen for the study subject. Then, the related downstream gene Forkhead box O4 (FOXO4) was identified. For in vivo assays, an ICH mouse model was established by autologous blood injection. For in vitro assays, hippocampal neurons were extracted from mouse brain tissues, and erythrocyte lysates were employed to simulate in vitro hemorrhage. Interaction between miR-133b and FOXO4 as well as between FOXO4 and BCL2 were assayed. We found decreased miR-133b in the brain tissue of ICH mice and erythrocyte lysate-treated hippocampal neurons. Sev treatment attenuated ICH and hippocampal neuronal apoptosis in mice by upregulating miR-133b. miR-133b targeted FOXO4 expression, and inhibition of FOXO4 attenuated hippocampal neuronal apoptosis by increasing BCL2 expression. Sev attenuated ICH in mice by increasing BCL2 expression through regulation of miR-133b-mediated FOXO4 expression. The findings highlighted the protective effect of Sev on ICH mice through the regulation of miR-133b-mediated FOXO4 expression.

Structural Characteristics of Polysaccharide GP2a in Gardenia jasminoides and Its Immunomodulatory Effect on Macrophages.

Here, we elucidated the structural characteristics of a polysaccharide isolated from Gardenia jasminoides Ellis (labeled as GP2a) and its immunomodulatory activity. GP2a is an acidic polysaccharide with a molecular weight of 44.8 kDa, mostly comprising galacturonic acid. Methylation analysis revealed 4-GalpA (74.8%) to be the major sugar residue in GP2a. Nuclear magnetic resonance analysis indicated that its main chain comprised →4)-α-D-GalpA-6-OMe-(1→4)-α-D-GalpA-(1→ and →4)-α-D-GalpA-6-OMe-(1→2)-α-L-Rhap-(1→, with galactan and arabinans linked to the C-4 position of →2)-α-L-Rhap-(1→ residue as branched chains. Furthermore, GP2a showed no obvious toxicity to macrophages (RAW 264.7) while enhancing cell viability in a dose- and time-dependent manner. Compared with untreated cells, nitric oxide production and secretion of cytokines, such as tumor necrosis factor-α, interferon-γ, interleukin (IL)-1ß, IL-6, and granulocyte macrophage colony stimulating factor, in GP2a-treated cells significantly increased after 48 h. At 300 µg/mL GP2a concentration, there was no significant difference in the cytokine levels in GP2a- and lipopolysaccharide-treated cells (the positive control). In summary, GP2a is a pectic polysaccharide with homogalacturonan and rhamnogalacturonan-I structural regions in the main chain. Based on its immunomodulatory effects in vitro, GP2a may have potential uses in functional food and medicine.

Early immune response in large yellow croaker (Larimichthys crocea) after immunization with oral vaccine.

Mesoporous silica nanoparticles (MSNs) have been used in the field of biomedicine as antigen carriers and adjuvants for protective antigens. In the present study, an oral nanovaccine against Vibrio alginolyticus was prepared employing MSNs as carriers. The uptake of the dihydrolipoamide dehydrogenase (DLDH) antigens in the intestine of large yellow croaker was evaluated using an immunohistochemistry assay. Additionally, the effects of the nanovaccine on the early immune response in large yellow croaker were investigated via oral vaccination. The presence of the antigens was detected in the mucosa and lamina propria of the foregut, midgut, and hindgut of large yellow croaker at 3 h following oral immunization. The expression levels of cytokines (i.e., lysozyme, IFN-γ, IFITM, TNF-α, IL-1ß, IL-2, IL-4, IL-10, and IL-13) in the intestine, spleen, and head kidney tissues of large yellow croaker before and after the immune challenge were determined via RT-qPCR assay. The obtained results revealed that the expression levels of lysozyme, IFN-γ, IFITM, TNF-α, IL-1ß, IL-2, IL-4, IL-10, and IL-13 in the intestine and head kidney of the vaccinated large yellow croaker, as well as the expression of lysozyme, IL-1ß, and IL-10 in the spleen, exhibited time-dependent oscillation regulation patterns. Notably, the nanovaccine immunization could induce early (6 h) and high expression of IFN-γ in the spleen and kidney tissues after the bacterial infection. The current study supplements the available data on the early immune response to fish nanovaccines. It also provides a valuable theoretical basis for the future development of large yellow croaker oral vaccines.

KSRP modulates melanoma growth and efficacy of vemurafenib.

The majority of melanomas carry an oncogenic BRAF mutation (BRAFV600E), which results in constitutive kinase activity driving melanoma proliferation. While inhibitors of BRAFV600E (BRAFi) effectively lead to rapid tumor shrinkage, most patients treated with BRAFi develop acquired resistance. Identification of factors as regulators of melanoma growth and as potential sources of resistance is thus crucial for the design of improved therapies to treat advanced melanoma with more durable responses. Here, we show that KH-type splicing regulatory protein (KSRP) is critical for proliferation of melanoma cells without and with acquired resistance to vemurafenib. Silencing KSRP reduces cell proliferation and augments the growth suppressive effects of vemurafenib. We identify killin (KLLN), a p53-regulated DNA replication inhibitor, as a downstream effector of growth inhibition by KSRP silencing and demonstrate that KSRP promotes decay of KLLN mRNA through an RNA-protein interaction. Using heterologous mRNA reporters, we show that a U-rich element within the 3' untranslated region of KLLN is responsible for KSRP-dependent mRNA decay. These findings implicate that KSRP is an important regulator of melanoma cell growth in part through controlling KLLN mRNA stability.

Facile synthesis of Silicon quantum dot-Gadolinium: A potential fluorescent/T1-T2 multimodal imaging agent.

Highly stable and multifunctional fluorescent quantum dots are particularly attractive in practical applications. Here, a new kind of ultra-small-sized silicon quantum dot-gadolinium (SiQD-Gd) was successfully fabricated by a newly-designed facile hydrothermal growth and chelating method. The obtained SiQD-Gd exhibited outstanding water dispersibility, stability and good fluorescent property with the quantum yield of 11.6%. SiQD-Gd displayed a low cytotoxicity in normal cell lines (HELF, HEK293F) and tumor cell lines (H1299, A549). Meanwhile, SiQD-Gd showed excellent magnetic resonance response with r1 relaxation rate of 10.5 mmol L-1·s-1 and r2 relaxation rate of 47.5 mmol L-1·s-1, which are 2.5 and 7.4 times enhanced comparing to that of the commercial MR agent Magnevist. In vivo studies showed significant contrast enhancement effect of its T1- and T2-weighted MR imaging. In addition, in vivo fluorescent imaging for mice and zebrafish indicated its potential applications in fluorescent tracking. Thus, the excellent multimodal imaging capacity and biocompatibility of SiQD-Gd make it a potential imaging agent for clinic applications.

Chemical Synthesis, Versatile Structures and Functions of Tailorable Adjuvants for Optimizing Oral Vaccination.

Oral vaccines have become a recent focus because of their potential significance in disease prevention and therapy. In the development of oral vaccine-based therapeutics, synthetic materials with tailorable structures and versatile functions can act as antigen conveyers with adjuvant effects, reduce the time cost for vaccine optimization, and provide high security and enhanced immunity. This review presents an overview of the current status of tailoring synthetic adjuvants for oral vaccination, modification strategies for producing effectors with specific structures and functions, enhancement of immune-associated efficiencies, including the barrier-crossing capability to protect antigens in the gastrointestinal tract, coordination of the antigens penetrating mucosa and cell barriers, targeting of concentrated antigens to immune-associated cells, and direct stimulation of immune cells. Finally, we focus on prospective synthetic adjuvants that facilitate the use of oral vaccines via two approaches, namely, in vivo antigen expression and cancer immunotherapy.

Crystal Structure of Major Envelope Protein VP24 from White Spot Syndrome Virus.

White spot syndrome virus (WSSV) is one of the major and most serious pathogen in the shrimp industry. As one of the most abundant envelope protein, VP24 acts as a core protein interacting with other structure proteins and plays an important role in virus assembly and infection. Here, we have presented the crystal structure of VP24 from WSSV. In the structure, VP24 consists of a nine-stranded ß-barrel fold with mostly antiparallel ß-strands, and the loops extending out the ß-barrel at both N-terminus and C-terminus, which is distinct to those of the other two major envelope proteins VP28 and VP26. Structural comparison of VP24 with VP26 and VP28 reveals opposite electrostatic surface potential properties of them. These structural differences could provide insight into their differential functional mechanisms and roles for virus assembly and infection. Moreover, the structure reveals a trimeric assembly, suggesting a likely natural conformation of VP24 in viral envelope. Therefore, in addition to confirming the evolutionary relationship among the three abundant envelope proteins of WSSV, our structural studies also facilitate a better understanding of the molecular mechanism underlying special roles of VP24 in WSSV assembly and infection.

Envelope protein VP24 from White spot syndrome virus: expression, purification and crystallization.

White spot syndrome virus (WSSV) is a major shrimp pathogen known to infect penaeid shrimp and other crustaceans. VP24 is one of the major envelope proteins of WSSV. In order to facilitate purification, crystallization and structure determination, the predicted N-terminal transmembrane region of approximately 26 amino acids was truncated from VP24 and several mutants were prepared to increase the proportion of selenomethionine (SeMet) residues for subsequent structural determination using the SAD method. Truncated VP24, its mutants and the corresponding SeMet-labelled proteins were purified, and the native and SeMet proteins were crystallized by the hanging-drop vapour-diffusion method. Crystals of VP24 were obtained using a reservoir consisting of 0.1 M Tris-HCl pH 8.5, 2.75 M ammonium acetate with a drop volume ratio of two parts protein solution to one part reservoir solution. Notably, ATP was added as a critical additive to the drop with a final concentration of 10 mM. Crystals of SeMet-labelled VP24 mutant diffracted to 3.0 Šresolution and those of the native diffracted to 2.4 Šresolution; the crystals belonged to space group I213, with unit-cell parameters a = b = c = 140 Å.

A Versatile Monoclonal Antibody Specific Against Human DAB2IP.

Human DAB2 interaction protein (DAB2IP) is a member of Ras-GTPase activating protein family and functions as a tumor suppressor, implying it could serve as a prognostic biomarker in cancers. Here we generated a mouse monoclonal antibody, 2A4, directed against human DAB2IP. This antibody was identified as IgG1 and specifically recognizes DAB2IP in both its native and denatured forms. It will serve as a useful and versatile tool for further mechanistic study and development of the potential prognostic significance of DAB2IP.

Using inositol as a biocompatible ligand for efficient transgene expression.

Transgene transfection techniques using cationic polymers such as polyethylenimines (PEIs) and PEI derivatives as gene vectors have shown efficacy, although they also have shortcomings. PEIs have decent DNA-binding capability and good cell internalization performance, but they cannot deliver gene payloads very efficiently to cell nuclei. In this study, three hyperbranched polyglycerol-polyethylenimine (PG6-PEI) polymers conjugated with myo-inositol (INO) molecules were developed. The three resulting PG6-PEI-INO polymers have an increased number of INO ligands per molecule. PG6-PEI-INO 1 had only 14 carboxymethyl INO (CMINO) units per molecule. PG6-PEI-INO 2 had approximately 130 CMINO units per molecule. PG6-PEI-INO 3 had as high as 415 CMINO units approximately. Mixing PG6-PEI-INO polymers with DNA produced compact nanocomposites. We then performed localization studies using fluorescent microscopy. As the number of conjugated inositol ligands increased in PG6-PEI-INO polymers, there was a corresponding increase in accumulation of the polymers within 293T cell nuclei. Transfection performed with spherical 293T cells yielded 82% of EGFP-positive cells when using PG6-PEI-INO 3 as the vehicle. Studies further revealed that extracellular adenosine triphosphate (eATP) can inhibit the transgene efficiency of PG6-PEI-INO polymers, as compared with PEI and PG6-PEI that were not conjugated with inositol. Our work unveiled the possibility of using inositol as an effective ligand for transgene expression.

Crystal structure confirmation of JHP933 as a nucleotidyltransferase superfamily protein from Helicobacter pylori strain J99.

Helicobacter pylori is a well-known pathogen involved in the development of peptic ulcer, gastric adenocarcinoma and other forms of gastric cancer. Recently, there has been more considerable interest in strain-specific genes located in plasticity regions with great genetic variability. However, little is known about many of these genes. Studies suggested that certain genes in this region may play key roles in the pathogenesis of H. pylori-associated gastroduodenal diseases. JHP933, a conserved putative protein of unknown function, is encoded by the gene in plasticity region of H. pylori strain J99. Here we have determined the structure of JHP933. Our work demonstrates that JHP933 is a nucleotidyltransferase superfamily protein with a characteristic αßαßαßα topology. A superposition demonstrates overall structural homology of the JHP933 N-terminal fragment with lincosamide antibiotic adenylyltransferase LinA and identifies a possible substrate-binding cleft of JHP933. Furthermore, through structural comparison with LinA and LinB, we pinpoint conservative active site residues which may contribute to divalent ion coordination and substrate binding.

Inositol based non-viral vectors for transgene expression in human cervical carcinoma and hepatoma cell lines.

Myo-Inositol (INO) is a biomolecule with crucial functions in many aspects. In this study, hyperbranched copolymers for gene delivery were synthesized based on inositol and low molecular weight polyethylenimine. The capacity of INO-PEIs to load plasmid DNA and their biocompatibility was demonstrated. A tumor target ligand, folic acid (FA), which was widely used for drug delivery systems, was subsequently conjugated to INO-PEIs and resulted in INO-PEI-FA copolymers. The polymers were then evaluated on their activity to mediate transgene expression in mammalian cell lines. As indicated, INO-PEIs were able to mediate efficient transgene expression, which was particularly noticeable in carcinoma cell line HeLa. INO-PEI-FA further improved the efficiency in HepG2. Distribution of INO-PEI-FA polymers in non-carcinoma NIH 3T3 and carcinoma HeLa cell lines was discussed.

Binary gene vectors based on hyperbranched poly(l-lactide-co-polyglycerol) and polyethylenimine for prolonged transgene expression via co-assembly with DNA into fiber core-shell triplexes.

Hyper-branched PG6-PLA polymers based on hydrophilic hyperbranched polyglycerol (PG6) and the ester chain poly(l-lactide) (PLA) were synthesized and facilitated to develop a novel biocompatible release-controlled gene vector. The hyper-branched structure of PG6-PLA was verified by NMR, FT-IR and SEC-MALLS analysis. The co-assembly of PG6-PLA with high molecular weight polyethylenimine (PEI) of 25 kDa was discussed. The results of TEM, fluorescence tracking and size/zeta-potential analysis revealed that the PG6-PLA/PEI25k/DNA could co-assemble to generate a novel fiber core-shell conformation. In vitro cell experiment demonstrated that PG6-PLA significantly enhanced the ability of PEI25k to remain within cells and mediate luciferase and EGFP expression in the human embryonic kidney cell line 293T and human cervical carcinoma cell line HeLa, which was accompanied by improved cell biocompatibility and an extended period of transgene expression. Importantly, the binary vector PG6-PLA/PEI25k exhibited specific affinity to some tumour cell lines including HeLa and the HepG2 human hepatoma cell line. These results suggested that the novel gene delivery system based on fiber core-shell PG6-PLA/PEI25k/DNA can serve as a gene delivery system to mediate more efficient transgene expression.

The crystal structure of the putative peptide-binding fragment from the human Hsp40 protein Hdj1.

BACKGROUND: The mechanism by which Hsp40 and other molecular chaperones recognize and interact with non-native polypeptides is a fundamental question. How Hsp40 co-operates with Hsp70 to facilitate protein folding is not well understood. To investigate the mechanisms, we determined the crystal structure of the putative peptide-binding fragment of Hdj1, a human member of the type II Hsp40 family. RESULTS: The 2.7A structure reveals that Hdj1 forms a homodimer in the crystal by a crystallographic two-fold axis. The Hdj1 dimer has a U-shaped architecture and a large cleft is formed between the two elongated monomers. When compared with another Hsp40 Sis1 structure, the domain I of Hdj1 is rotated by 7.1 degree from the main body of the molecule, which makes the cleft between the two Hdj1 monomers smaller that that of Sis1. CONCLUSION: This structural observation indicates that the domain I of Hsp40 may possess significant flexibility. This flexibility may be important for Hsp40 to regulate the size of the cleft. We propose an "anchoring and docking" model for Hsp40 to utilize the flexibility of domain I to interact with non-native polypeptides and transfer them to Hsp70.

Crystal structure of yeast Sis1 peptide-binding fragment and Hsp70 Ssa1 C-terminal complex.

Heat shock protein (Hsp) 40 facilitates the critical role of Hsp70 in a number of cellular processes such as protein folding, assembly, degradation and translocation in vivo. Hsp40 and Hsp70 stay in close contact to achieve these diverse functions. The conserved C-terminal EEVD motif in Hsp70 has been shown to regulate Hsp40-Hsp70 interaction by an unknown mechanism. Here, we provide a structural basis for this regulation by determining the crystal structure of yeast Hsp40 Sis1 peptide-binding fragment complexed with the Hsp70 Ssa1 C-terminal. The Ssa1 extreme C-terminal eight residues, G634PTVEEVD641, form a beta-strand with the domain I of Sis1 peptide-binding fragment. Surprisingly, the Ssa1 C-terminal binds Sis1 at the site where Sis1 interacts with the non-native polypeptides. The negatively charged residues within the EEVD motif in Ssa1 C-terminal form extensive charge-charge interactions with the positively charged residues in Sis1. The structure-based mutagenesis data support the structural observations.

The crystal structure of the C-terminal fragment of yeast Hsp40 Ydj1 reveals novel dimerization motif for Hsp40.

The molecular chaperone Hsp40 functions as a dimer. The dimer formation is critical for Hsp40 molecular chaperone activity to facilitate Hsp70 to refold non-native polypeptides. We have determined the crystal structure of the C-terminal fragment of yeast Hsp40 Ydj1 that is responsible for Ydj1 dimerization by MAD method. The C-terminal fragment of Ydj1 comprises of the domain III of Ydj1 and the Ydj1 C-terminal dimerization motif. The crystal structure indicates that the dimerization motif of type I Hsp40 Ydj1 differs significantly from that of yeast type II Hsp40. The C terminus of type I Hsp40 Ydj1 from one monomer forms beta-strands with the domain III from the other monomer in the homo-dimer. The L372 from Ydj1 C terminus inserts its side-chain into a hydrophobic pocket on domain III. The modeled full-length Ydj1 dimer structure reveals that a large cleft is formed between the two monomers. The domain IIs of Ydj1 monomers that contain the zinc-finger motifs points directly against each other.

[Detection of serum total MMP-9 and its clinical significance for lung neoplasms].

BACKGROUND: To investigate the clinical value of serum total MMP-9 detection in lung malignancies. METHODS: Serum total MMP-9 levels were detected in 63 patients with lung malignancies by sandwich ELISA with monoclonal antibody against human total MMP-9. RESULTS: Serum total MMP-9 was (92.2± 74.39) µg/L in lung malignancy group, which was significantly higher than that of healthy control [(9.5± 6.74) µg/L](P < 0.05). In lung cancer group, there were significant differences in serum total MMP-9 level between patients with and without lymph node metastasis, and between progressive disease+death group and complete response+partial response group. Significantly negative correlation was observed between serum CA242 and serum total MMP-9 levels (P=0.021). CONCLUSIONS: Detection of serum total MMP-9 may be helpful to predict metastasis and treatment response of lung cancer.