Tight junctions (TJs) are the outermost structures of intercellular junctions and are highly specialized membrane domains involved in many important cellular processes. However, most TJ proteins are four-time transmembrane proteins and are difficult to express in their correct soluble form, which limits their functional study and therapeutic application. Human occludin (OCLN) is a major component of TJs and an essential co-receptor for hepatitis C virus (HCV) cell entry. To explore expression strategy for recombinant TJ proteins possessing integrated and functional extracellular loops, OCLN was here used as a model molecule, and several prokaryotic fusion constructs were designed by docking OCLN extracellular loops (ECLs) to HIV-1 gp41 NHR and CHR six-helical bundle (6HV1); then their biophysical features and anti-HCV activity were evaluated. The proteins were successfully expressed and purified in E. coli, and the double-loop constructs (D1ECL1S+D2ECL2 as a representative) were found to have more potent HCV neutralizing activity than single-loop constructs at non-cytotoxic concentrations. Circular dichroism studies indicate that D1ECL1S+D2ECL2 adopt stable α-helical folds consistent with design. Thermal denaturation assay indicated that D1ECL1S+D2ECL2 is highly stable at 80°C (melting temperature, Tm, of 89.08 ± 2.0°C) and comparable in stability to the 6HV1 scaffold. Moreover, the time-of-addition experiment revealed that D1ECL1S+D2ECL2 predominantly functioned during the early stages of HCV entry. Taken together, these findings provide a novel strategy for recombinant TJ protein expression in vitro, which may shed light on functional and structural studies for TJs and may provide a new avenue to drug development.

Microsporidia are a diverse group of eukaryotic organisms, capable of causing parasitic infections in both vertebrates and invertebrates. During the germination process, there is an increase in the osmotic pressure of microsporidian spores. As part of this study, we cloned a homologous aquaporin gene in Nosema bombycis, and named it Nosema bombycis aquaporin (NbAQP). Sequence analysis revealed that the NbAQP contains an open reading frame with a length of 750 bp and encodes a polypeptide of 249 amino acids. Amino acid sequence homology was greater than 50% that of five aquaporins from other microsporidian species. Indirect immunofluorescence (IFA) and immunogold electron microscopy showed NbAQP to be located predominantly in the spore wall of N. bombycis spores. The results of qRT-PCR analysis revealed that NbAQP expression remained high 0 h after inoculation and decreased sharply to 24 h, increased gradually from 2 days and peaked at 6 days. After expression of NbAQP in Xenopus laevis oocytes, it was observed that NbAQP can promote rapid penetration of water into oocytes. The associated permeation rate was 2-3 times that of the water-injected and uninjected oocytes. Antibody blocking experiments showed that the inhibition rate of spore germination was approximately 28% after antibody blocking. The difference in germination rate between the control group and the NbAQP group was significant (P < 0.05). This study shows for the first time that N. bombycis contains functional water channel proteins and provides a platform suitable for further research into the mechanisms underlying the regulation of NbAQP protein expression. Further study of NbAQP and their inhibitors may have significance for prevention of microsporidiosis.

To exploit the biological and pharmacological properties of immunoglobulin constant domain Fc fragment and increase the killing efficacy of T cells, a single chain variable fragment specific to CD3 was fused with Fcab (Fc antigen binding), a mutant Fc fragment with specificity against Human epidermal growth factor receptor 2 (HER2) developed by F-star. The bispecific fusion named as FcabCD3 was expressed by transient transfection in HEK-293T cells and purified by affinity chromatography. Specific cytolytic activity of retargeted T cells to kill HER2 positive SKBR3 cell line was evaluated in vitro. FcabCD3 was able to retarget T cells to kill both Herceptin insensitive Colo205-luc cell line and HER2 low expression MDA-MB-231-luc cell line. Furthermore, FcabCD3 was effective in eliminating the Colo205 tumor established on BALB/c nu/nu mice.

Idiopathic pulmonary fibrosis (IPF) is a gradual lung disease with a survival of less than 5 years post-diagnosis for most patients. Poor molecular description of IPF has led to unsatisfactory interpretation of the pathogenesis of this disease, resulting in the lack of successful treatments. The objective of this study was to discover novel noninvasive biomarkers for the diagnosis of IPF. We employed a coupled isobaric tag for relative and absolute quantitation (iTRAQ)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach to examine protein expression in patients with IPF. A total of 97 differentially expressed proteins (38 upregulated proteins and 59 downregulated proteins) were identified in the serum of IPF patients. Using String software, a regulatory network containing 87 nodes and 244 edges was built, and the functional enrichment showed that differentially expressed proteins were predominantly involved in protein activation cascade, regulation of response to wounding and extracellular components. A set of three most significantly upregulated proteins (HBB, CRP and SERPINA1) and four most significantly downregulated proteins (APOA2, AHSG, KNG1 and AMBP) were selected for validation in an independent cohort of IPF and other lung diseases using ELISA test. The results confirmed the iTRAQ profiling results and AHSG, AMBP, CRP and KNG1 were found as specific IPF biomarkers. ROC analysis indicated the diagnosis potential of the validated biomarkers. The findings of this study will contribute in understanding the pathogenesis of IPF and facilitate the development of therapeutic targets.

In this study, a novel adamantanamine-paclitaxel (AD-PTX) incorporated oligochitosan- carboxymethyl-β-cyclodextrin (CSO-g-CM-β-CD) self-assembly macromolecular (CSO-g-CM-β-CD@AD-PTX) micelle was successfully prepared in water through sonication. The formed molecules were characterized by Fourier transform infrared spectroscopy, proton nuclear magnetic resonance (NMR) spectroscopy, two-dimensional NMR, elemental analysis, and liquid chromatography-mass spectrometry, while the correspondent micelles were characterized by dynamic light scattering and transmission electron microscopy. We showed that the macromolecular micelle contained a spherical core-shell structure with a diameter of 197.1 ± 3.3 nm and zeta potential of -19.1 ± 4.3 mV. The CSO-g-CM-β-CD@AD-PTX micelle exhibited a high drug-loading efficacy up to 31.3%, as well as a critical micelle concentration of 3.4 × 10-7 M, which indicated good stability. Additionally, the in vitro release profile of the CSO-g-CM-β-CD@AD-PTX micelle demonstrated a long-term release pattern, 63.1% of AD-PTX was released from the micelle during a 30-day period. Moreover, the CSO-g-CM-β-CD@AD-PTX micelle displayed cytotoxicity at a sub-μM scale similar to PTX in U87 MG cells, and CSO-g-CM-β-CD exhibited a good safety profile by not manifesting significant toxicity at concentrations up to 100 μM. These results indicated that β-CD-based inclusion complexation resulting in biodegradable self-assembled macromolecular micelles can be utilized as nanocarrier, and may provide a promising platform for drug delivery in the future medical applications.

Deep brain stimulation (DBS) of the nucleus accumbens (NAc) is a potential remedial therapy for drug craving and relapse, but the mechanism is poorly understood. We investigated changes in neurotransmitter levels during high frequency stimulation (HFS) of the unilateral NAc on morphine-induced rats. Sixty adult Wistar rats were randomized into five groups: the control group (administration of saline), the morphine-only group (systematic administration of morphine without electrode implantation), the morphine-sham-stimulation group (systematic administration of morphine with electrode implantation but not given stimulation), the morphine-stimulation group (systematic administration of morphine with electrode implantation and stimulation) and the saline-stimulation group (administration of saline with electrode implantation and stimulation). The stimulation electrode was stereotaxically implanted into the core of unilateral NAc and microdialysis probes were unilaterally lowered into the ipsilateral ventral tegmental area (VTA), NAc, and ventral pallidum (VP). Samples from microdialysis probes in the ipsilateral VTA, NAc, and VP were analyzed for glutamate (Glu) and γ-aminobutyric acid (GABA) by high-performance liquid chromatography (HPLC). The levels of Glu were increased in the ipsilateral NAc and VP of morphine-only group versus control group, whereas Glu levels were not significantly changed in the ipsilateral VTA. Furthermore, the levels of GABA decreased significantly in the ipsilateral NAc, VP, and VTA of morphine-only group when compared with control group. The profiles of increased Glu and reduced GABA in morphine-induced rats suggest that the presence of increased excitatory neurotransmission in these brain regions. The concentrations of the Glu significantly decreased while the levels of GABA increased in ipsilateral VTA, NAc, and VP in the morphine-stimulation group compared with the morphine-only group. No significant changes were seen in the morphine-sham stimulation group compared with the morphine-only group. These findings indicated that unilateral NAc stimulation inhibits the morphine-induced rats associated hyperactivation of excitatory neurotransmission in the mesocorticolimbic reward circuit.

Lipid production by oleaginous microorganisms is a promising route to produce raw material for the production of biodiesel. However, most of these organisms must be grown on sugars and agro-industrial wastes because they cannot directly utilize lignocellulosic substrates. We report the first comprehensive investigation of Mucor circinelloides, one of a few oleaginous fungi for which genome sequences are available, for its potential to assimilate cellulose and produce lipids. Our genomic analysis revealed the existence of genes encoding 13 endoglucanases (7 of them secretory), 3 β-D-glucosidases (2 of them secretory) and 243 other glycoside hydrolase (GH) proteins, but not genes for exoglucanases such as cellobiohydrolases (CBH) that are required for breakdown of cellulose to cellobiose. Analysis of the major PAGE gel bands of secretome proteins confirmed expression of two secretory endoglucanases and one β-D-glucosidase, along with a set of accessory cell wall-degrading enzymes and 11 proteins of unknown function. We found that M. circinelloides can grow on CMC (carboxymethyl cellulose) and cellobiose, confirming the enzymatic activities of endoglucanases and β-D-glucosidases, respectively. The data suggested that M. circinelloides could be made usable as a consolidated bioprocessing (CBP) strain by introducing a CBH (e.g. CBHI) into the microorganism. This proposal was validated by our demonstration that M. circinelloides growing on Avicel supplemented with CBHI produced about 33% of the lipid that was generated in glucose medium. Furthermore, fatty acid methyl ester (FAME) analysis showed that when growing on pre-saccharified Avicel substrates, it produced a higher proportion of C14 fatty acids, which has an interesting implication in that shorter fatty acid chains have characteristics that are ideal for use in jet fuel. This substrate-specific shift in FAME profile warrants further investigation.

Atrial fibrosis, as a hallmark of atrial structure remodeling, plays an important role in maintenance of chronic atrial fibrillation, but interrelationship of atrial fibrosis and atrial fibrillation is uncertain. Label-free proteomics can implement high throughput screening for finding and analyzing pivotal proteins related to the disease.. Therefore, we used label-free proteomics to explore and analyze differentially proteins in chronic atrial fibrillation patients with mitral valve disease.