Effects of Catecholamine Stress Hormones Norepinephrine and Epinephrine on Growth, Antimicrobial Susceptibility, Biofilm Formation, and Gene Expressions of Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is a significant contributor to diarrhea. To determine whether ETEC-catecholamine hormone interactions contribute to the development of diarrhea, we tested the effects of catecholamine hormones acting on ETEC in vitro. The results showed that in the presence of norepinephrine (NE) and epinephrine (Epi), the growth of 9 out of 10 ETEC isolates was promoted, the MICs of more than 60% of the isolates to 6 antibiotics significantly increased, and the biofilm formation ability of 10 ETEC isolates was also promoted. In addition, NE and Epi also significantly upregulated the expression of the virulence genes feaG, estA, estB, and elt. Transcriptome analysis revealed that the expression of 290 genes was affected by NE. These data demonstrated that catecholamine hormones may augment the diarrhea caused by ETEC.

The Synergistic Activity of Rhamnolipid Combined with Linezolid against Linezolid-Resistant Enterococcus faecium.

Enterococci resistance is increasing sharply, which poses a serious threat to public health. Rhamnolipids are a kind of amphiphilic compound used for its bioactivities, while the combination of nontraditional drugs to restore linezolid activity is an attractive strategy to treat infections caused by these pathogens. This study aimed to investigate the activity of linezolid in combination with the rhamnolipids against Enterococcus faecium. Here, we determined that the rhamnolipids could enhance the efficacy of linezolid against enterococci infections by a checkerboard MIC assay, a time-kill assay, a combined disk test, an anti-biofilm assay, molecular simulation dynamics, and mouse infection models. We identified that the combination of rhamnolipids and linezolid restored the linezolid sensitivity. Anti-biofilm experiments show that our new scheme can effectively inhibit biofilm generation. The mouse infection model demonstrated that the combination therapy significantly reduced the bacterial load in the feces, colons, and kidneys following subcutaneous administration. This study showed that rhamnolipids could play a synergistic role with linezolid against Enterococcus. Our combined agents could be appealing candidates for developing new combinatorial agents to restore antibiotic efficacy in the treatment of linezolid-resistant Enterococcus infections.

Effect of Structure and Intramolecular Distances on Photoswitchable Magnetic Resonance Imaging Contrast Agents.

Light-activated sensors are of great interest for biological applications but are limited by the depth of penetration of light. We have been interested in transducing light activation to a magnetic signal that can be detected through noninvasive imaging by magnetic resonance imaging (MRI). We have previously developed agents incorporating spiropyran derivatives as the sensing moiety and characterized features that influence photoswitching; however, we found the MRI response to be unpredictable. In this work, we delve deeper into the potential mechanisms for the observed MRI responses in an effort to better understand the structural effects on controlling magnetic properties. A series of light-activatable MRI contrast agents were synthesized and characterized to assess the effect of spiropyran positioning on contrast agent functions and properties. These compounds are based on the same spiropyran skeleton, also named 1',3',3'-trimethyl-6-nitrospiro[chromene-2,2-indoline], which is linked with an MRI contrast agent, gadolinium-1,4,7,10-tetraazacyclododecane-1,4,7-triacetate (DO3A). We investigated the photo-to-magnetic conversion properties of these novel compounds by adjusting linker lengths over a range from three to seven methylene groups. The primary results indicated that the contrast agent with a five-carbon linker (25) showed the highest light-sensing ability after irradiation with visible light. The results will aid in the design of future spiropyran-based MRI sensors.

Recombinant ferritin nanoparticles can induce dendritic cell maturation through TLR4/NF-κB pathway.

OBJECTIVE: Immune response initiation and regulation require activation of dendritic cells (DCs). However, the mechanism by which ferritin, a carrier for immunogen, induces DCs maturation remains unclear. RESULTS: Recombinant ferritin nanoparticle (RFNp), were prepared through the baculovirus expression vector system, formed spherical and hollow cage-liked proteins with a diameter of approximately 12.17 ± 0.87 nm. They induced bone marrow-derived DC (BMDC) maturation via surface molecules up-regulation of (MHC II, CD80, CD86 and CD40), increased pro-inflammatory cytokines production (IL-6, IL-12, TNF-α, and IFN-γ), and decreased antigen capturing capacity. They positively regulated IκBα and NF-κB (p65) phosphorylation, and facilitate NF-κB (p65) translocation into mature BMDCs nuclei. Following pre-treatment of RFNp-treated BMDCs with TLR4 and NF-κB (p65) inhibitors, respectively, surface molecule expression, pro-inflammatory cytokines production, and IκBα and NF-κB (p65) activities were suppressed. RFNp-treated BMDCs can also facilitate T-cell proliferation and differentiation into Th1 and Th2. CONCLUSION: RFNps induced DCs maturation lends the potential application of RFNps as carrier platforms in DC-based vaccine.

Expression, purification, and characterisation of recombinant ferritin in insect cells using the baculovirus expression system.

OBJECTIVE: Ferritin is an attractive vector for the delivery of drug molecules and antigen proteins because of its unique structural and biochemical features. In this study, recombinant ferritin from Helicobacter pylori was expressed in the soluble form employing the baculovirus expression system. RESULTS: The optimum conditions for producing recombinant ferritin comprised MOI 5 of rBV-ferritin for 96 h of infection. The recombinant ferritin was purified by Ni Sepharose™ 6 Fast Flow, with a purity and yield of 92.5% and 11.25 mg/L, respectively. In addition, the recombinant ferritin showed a multimeric structure under non-denaturing conditions, as well as self-assembled spherical cage architecture with a diameter of approximately 12 nm. Dot-ELISA results suggested that the His-tag at the N-terminus likely existed on the surface of the recombinant ferritin. CONCLUSION: Recombinant ferritin was produced by the baculovirus expression system, which has the potential to display exogenous proteins, and may aid in the delivery of drugs for disease prevention and treatment.

Copper nitrate-mediated synthesis of 3-aryl isoxazolines and isoxazoles from olefinic azlactones.

A copper nitrate-mediated [2 + 2 + 1] cycloaddition reaction was developed for the expedient synthesis of pharmacologically interesting 3-aryl substituted isoxazolines and isoxazoles through C[double bond, length as m-dash]C bond cleavage. Copper nitrate is employed as a reaction promoter and precursor of nitrile oxides. The given approach features a new mode of cycloaddition from olefinic azlactones, copper nitrate and unsaturated compounds with wide substrate scope, good functional group tolerance and operational simplicity.

Comparative liver transcriptome analysis in ducklings infected with duck hepatitis A virus 3 (DHAV-3) at 12 and 48 hours post-infection through RNA-seq.

Duck hepatitis A virus 3 (DHAV-3), the only member of the novel genus Avihepatovirus, in the family Picornaviridae, can cause significant economic losses for duck farms in China. Reports on the pathogenicity and the antiviral molecular mechanisms of the lethal DHAV-3 strain in ducklings are inadequate and remain poorly understood. We conducted global gene expression profiling and screened differentially expressed genes (DEG) of duckling liver tissues infected with lethal DHAV-3. There were 1643 DEG and 8979 DEG when compared with mock ducklings at 12 hours post-infection (hpi) and at 48 hpi, respectively. Gene pathway analysis of DEG highlighted mainly biological processes involved in metabolic pathways, host immune responses, and viral invasion. The results may provide valuable information for us to explore the pathogenicity of the virulent DHAV-3 strain and to improve our understanding of host-virus interactions.

Copper nitrate: a privileged reagent for organic synthesis.

Copper has been explored as an ideal candidate for replacing noble metals in organic synthesis, especially for practical large scale preparation. Recent decades have witnessed the renaissance and improvement of copper-catalyzed and copper-mediated organic reactions. Copper nitrate is a common inorganic copper salt which has been proved to be a ubiquitous reactant in organic synthesis due to its commercial availability, stability, inexpensiveness and environmentally benign nature. Copper nitrate could be used as a nitration reagent, oxidant, catalyst or promoter, and Lewis acid as well. Remarkably, great attention has been devoted to the efficient transformation of copper nitrate into functionalized or complicated compounds through various reaction types including cyclization, C-H activation, difunctionalization, nitration, rearrangement and asymmetric synthesis with chiral ligands. Further modification of copper nitrate, such as solid-supported copper nitrate or copper nitrate complexes, extends its applications in organic synthesis. The present review highlights recent advances of copper nitrate in organic synthesis, along with the mechanisms.

Transition Metal-Participated Synthesis and Utilization of N-containing Heterocycles: Exploring for Nitrogen Sources.

This account aims to describe our recent efforts on the synthesis and utilization of N-containing heterocycles, where transition metals participate in the synthesis. A variety of nitrogen sources, including amines, amides, hydrazones, pyrimidines, isocyanides, and copper nitrate, have been disclosed for the synthesis of diverse bioactive and pharmacologically interesting N-containing heterocycles under the participation of transition metals. The well-known nitrogen sources, such as amines and amides, were used for the construction of indoles, isatins, and quinolones. Dihydrophthalazines, isoquinolines, indazoles, and pyrazoles were obtained from hydrazones, while various pyrimidine-containing heterocycles were afforded through regioselective C-H functionalizations using pyrimidine as the directing group. Recent research has focused on the chemistry of isocyanides to achieve several kinds of heterocyclic compounds with high efficiency under the catalysis of transition metals (Pd, Rh, Mn, Cu), through oxidative cyanation reactions, sequential isocyanide insertions into C-H, N-H, or O-H bonds, and tandem radical annulation. More recently, an efficient route to isoxazolines has been reported using copper nitrate as a novel nitrogen source.

Copper-catalyzed aerobic cascade cycloamination and acyloxylation: a direct approach to 4-acyloxy-1H-pyrazoles.

A novel direct transformation of hydrazones to acyloxylated pyrazoles by copper-catalyzed regioselective olefinic C(sp(2))-H bond cycloamination and acyloxylation was performed under mild conditions, which combines the formation of the pyrazole skeleton and installation of an acyloxyl group in a single step, using facile carboxylic acids as the acyloxylation reagents.

Copper Nitrate Mediated Regioselective [2+2+1] Cyclization of Alkynes with Alkenes: A Cascade Approach to Δ(2)-Isoxazolines.

An efficient method for the regioselective synthesis of pharmacologically relevant polysubstituted Δ(2)-isoxazolines is based on the copper-mediated direct transformation of simple terminal alkynes and alkenes. The overall process involves the formation of four chemical bonds with inexpensive and readily available copper nitrate trihydrate as a novel precursor of nitrile oxides. The reaction can be easily handled and proceeds under mild conditions.

Molecular cloning and characterization of the α-chain gene of goose immunoglobulin heavy chain.

A novel gene encoding the α-chain of goose immunoglobulin heavy-chain (Igα) was cloned by reverse transcription-PCR. The cDNA had 1,760 bp and encompassed a partial V-D-J region of the heavy chain, a constant region (Cα) and 3'-untranslated region of α-chain. The Cα gene contains four constant region domains (CH1-CH4). Phylogenetic analysis indicated that goose IgCα has a close genetic relationship with duck, ostrich and chicken IgCα. Three-dimensional modeling and glycosylation analysis revealed the goose Igα is consistent with the characterization of immunoglobulin. Western blotting suggested the goose IgCα has the same antigenicity to natural IgA. In general, the identification of goose immunoglobulin not only provides insights into the evolution of the Ig heavy-chain gene family, but may also benefit future studies of the avian immune system.

The US2 protein is involved in the penetration and cell-to-cell spreading of DEV in vitro.

Previous studies have shown that the unique short 2 (US2) protein contributes to the infection of several herpesviruses. However, little is known about whether the US2 protein of duck enteritis virus (DEV) could also contribute to the DEV infection in susceptible cells. This study is aimed at investigating the role of US2 protein in the DEV infection in vitro. The US2 gene was amplified from DEV and cloned into pET32a (+). The expression of recombinant US2 protein was induced in Escherichia coli. After purification, the recombinant US2 protein was immunized into rabbits to generate anti-US2 sera. The immunoreactivity of anti-US2 sera was determined by Western blot and immunofluorescent assays. The US2 protein was detected in the cytoplasm and plasma membrane of DEV-infected duck embryo fibroblast (DEF) cells by immunofluorescent assay using the anti-US2 serum. When DEV infected DEF cells in vitro, pre-treatment with anti-US2 serum did not affect the attachment of virus, but significantly reduced the penetration and cell-to-cell spreading of DEV in DEF cells. Our data suggest that the US2 protein is involved in the penetration and cell-to-cell spreading during the DEV infection of susceptible cells in vitro.

A novel bacterium-like particles platform displaying antigens by new anchoring proteins induces efficacious immune responses.

Bacterium-like particles (BLP) are the peptidoglycan skeleton particles of lactic acid bacteria, which have high safety, mucosal delivery efficiency, and adjuvant effect. It has been widely used in recent years in the development of vaccines. Existing anchoring proteins for BLP surfaces are few in number, so screening and characterization of new anchoring proteins are necessary. In this research, we created the OACD (C-terminal domain of Escherichia coli outer membrane protein A) to serve as an anchoring protein on the surface of BLP produced by the immunomodulatory bacteria Levilactobacillus brevis 23017. We used red fluorescent protein (RFP) to demonstrate the novel surface display system's effectiveness, stability, and ability to be adapted to a wide range of lactic acid bacteria. Furthermore, this study employed this surface display method to develop a novel vaccine (called COB17) by using the multi-epitope antigen of Clostridium perfringens as the model antigen. The vaccine can induce more than 50% protection rate against C. perfringens type A challenge in mice immunized with a single dose and has been tested through three routes. The vaccine yields protection rates of 75% for subcutaneous, 50% for intranasal, and 75% for oral immunization. Additionally, it elicits a strong mucosal immune response, markedly increasing levels of specific IgG, high-affinity IgG, specific IgA, and SIgA antibodies. Additionally, we used protein anchors (PA) and OACD simultaneous to show several antigens on the BLP surface. The discovery of novel BLP anchoring proteins may expand the possibilities for creating mucosal immunity subunit vaccines. Additionally, it may work in concert with PA to provide concepts for the creation of multivalent or multiple vaccines that may be used in clinical practice to treat complex illnesses.

Complete genome sequence of a bovine viral diarrhea virus 2 from commercial fetal bovine serum.

We isolated a bovine viral diarrhea virus (BVDV) from commercial fetal bovine serum and designated it HLJ-10. The complete genome is 12,284 nucleotides (nt); the open reading frame is 11,694 nt, coding 3,898 amino acids. Phylogenetic analysis indicated that this strain belongs to BVDV group 2.

Characterization of the optimized C2 domain of protein G: finding its additional chicken IgY-binding ability.

The C2 domain of streptococcal protein G is a small (55 residue) peptide with immunoglobulin-binding activity. Following codon optimization, the gene was divided into four oligonucleotide fragments and amplified by overlap PCR. The recombinant plasmid pET30a-C2 was transformed into Escherichia coli Rosetta (DE3) PLysS for expression. After purification by Ni-NTA, the fusion protein was identified by western-blotting, Dot-ELISA and ELISA. His-tagged C2 bound to human, rabbit, cattle, pig, goat, mouse or guinea pig IgG had no affinity for goose, duck, wild duck, wild turkey and red-crowned crane IgY. Its affinity for chicken IgY, however, was comparable to that of guinea pig IgG. The C2 domain may therefore provide an ideal material for the purification and detection of immunoglobulin G from various mammals.

Subcutaneous Streptococcus dysgalactiae GAPDH vaccine in mice induces a proficient innate immune response.

BACKGROUND: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) on the surface of Streptococcus dysgalactiae, coded with gapC, is a glycolytic enzyme that was reported to be a moonlighting protein and virulence factor. OBJECTIVE: This study assessed GAPDH as a potential immunization candidate protein to prevent streptococcus infections. METHODS: Mice were vaccinated subcutaneously with recombinant GAPDH and challenged with S. dysgalactiae in vivo. They were then evaluated using histological methods. rGAPDH of mouse bone marrow-derived dendritic cells (BMDCs) was evaluated using immunoblotting, reverse transcription quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay methods. RESULTS: Vaccination with rGAPDH improved the survival rates and decreased the bacterial burdens in the mammary glands compared to the control group. The mechanism by which rGAPDH vaccination protects against S. dysgalactiae was investigated. In vitro experiments showed that rGAPDH boosted the generation of interleukin-10 and tumor necrosis factor-α. Treatment of BMDCs with TAK-242, a toll-like receptor 4 inhibitor, or C29, a toll-like receptor 2 inhibitor, reduced cytokines substantially, suggesting that rGAPDH may be a potential ligand for both TLR2 and TLR4. Subsequent investigations showed that rGAPDH may activate the phosphorylation of MAPKs and nuclear factor-κB. CONCLUSIONS: GAPDH is a promising immunization candidate protein for targeting virulence and enhancing immune-mediated protection. Further investigations are warranted to understand the mechanisms underlying the activation of BMDCs by rGAPDH in a TLR2- and TLR4-dependent manner and the regulation of inflammatory cytokines contributing to mastitis pathogenesis.

Direct Nitration of Vinylcycles with Copper Nitrate.

A direct nitration of vinylcyclopropanes is disclosed with Cu(NO3)2 and KI in a regio- and stereoselective manner to afford nitroalkenes efficiently, where the cyclopropane skeleton was retained. The given method could be extended to other vinylcycles as well as biomolecule derivatives with wide substrate scope, good functionality tolerance, and efficient synthesis modularity. Further transformations illustrated the obtained products as versatile building blocks in organic synthesis. The proposed ionic pathway could account for the untouched small ring and the effect of KI during the reaction.

Identification of a First-in-Class Small-Molecule Inhibitor of the EIF4E-RBM38 Complex That Enhances Wild-type TP53 Protein Translation for Tumor Growth Suppression.

EIF4E, an mRNA cap-binding protein, is necessary for cap-dependent translation. Overexpression of EIF4E is known to promote cancer development by preferentially translating a group of oncogenic mRNAs. Thus, 4EGI-1, a disruptor of EIF4E-EIF4G1 interaction, was developed to inhibit oncoprotein expression for cancer therapy. Interestingly, RBM38, an RNA-binding protein, interacts with EIF4E on TP53 mRNA, prevents EIF4E from binding to TP53 mRNA cap, and inhibits TP53 expression. Thus, Pep8, an eight amino acid peptide derived from RBM38, was developed to disrupt the EIF4E-RBM38 complex, leading to increased TP53 expression and decreased tumor cell growth. Herein, we have developed a first-in-class small-molecule compound 094, which interacts with EIF4E via the same pocket as does Pep8, dissociates RBM38 from EIF4E, and enhances TP53 translation in RBM38- and EIF4E-dependent manners. Structure-activity relationship studies identified that both the fluorobenzene and ethyl benzamide are necessary for compound 094 to interact with EIF4E. Furthermore, we showed that compound 094 is capable of suppressing three-dimensional tumor spheroid growth in RBM38- and TP53-dependent manners. In addition, we found that compound 094 cooperates with the chemotherapeutic agent doxorubicin and EIF4E inhibitor 4EGI-1 to suppress tumor cell growth. Collectively, we showed that two distinct approaches can be used together to target EIF4E for cancer therapy by enhancing wild-type TP53 expression (094) and by suppressing oncoprotein expression (4EGI-1).

Biological Activity of Optimized Codon Bovine Type III Interferon Expressed in Pichia pastoris.

Type III interferons (IFN-λs) exhibit potent antiviral activity and immunomodulatory effects in specific cells. Nucleotide fragments of the bovine ifn-λ (boifn-λ) gene were synthetized after codon optimization. The boifn-λ gene was then amplified by splicing using overlap extension PCR (SOE PCR), resulting in the serendipitous acquisition of the mutated boIFN-λ3V18M. The recombinant plasmid pPICZαA-boIFN-λ3/λ3V18M was constructed, and the corresponding proteins were expressed in Pichia pastoris with a high-level extracellular soluble form. Dominant expression strains of boIFN-λ3/λ3V18M were selected by Western blot and ELISA and cultured on a large scale, and the recombinant proteins purified by ammonium sulfate precipitation and ion exchange chromatography yielded 1.5g/L and 0.3 g/L, with 85% and 92% purity, respectively. The antiviral activity of boIFN-λ3/λ3V18M exceeded 106 U/mg, and they were neutralized with IFN-λ3 polyclonal antibodies, were susceptible to trypsin, and retained stability within defined pH and temperature ranges. Furthermore, boIFN-λ3/λ3V18M exerted antiproliferative effects on MDBK cells without cytotoxicity at 104 U/mL. Overall, boIFN-λ3 and boIFN-λ3V18M did not differ substantially in biological activity, except for reduced glycosylation of the latter. The development of boIFN-λ3 and comparative evaluation with the mutant provide theoretical insights into the antiviral mechanisms of boIFN-λs and provide material for therapeutic development.