Transgenic overexpression of ITGB6 in intestinal epithelial cells exacerbates dextran sulfate sodium-induced colitis in mice

Integrins, as a large family of cell adhesion molecules, play a crucial role in maintaining intestinal homeostasis. In inflammatory bowel disease (IBD), homeostasis is disrupted. Integrin αvβ6, which is mainly regulated by the integrin β6 subunit gene (ITGB6), is a cell adhesion molecule that mediates cell-cell and cell-matrix interactions. However, the role of ITGB6 in the pathogenesis of IBD remains elusive. In this study, we found that ITGB6 was markedly upregulated in inflamed intestinal tissues from patients with IBD. Then, we generated an intestinal epithelial cell-specific ITGB6 transgenic mouse model. Conditional ITGB6 transgene expression exacerbated experimental colitis in mouse models of acute and chronic dextran sulphate sodium (DSS)-induced colitis. Survival analyses revealed that ITGB6 transgene expression correlated with poor prognosis in DSS-induced colitis. Furthermore, our data indicated that ITGB6 transgene expression increased macrophages infiltration, pro-inflammatory cytokines secretion, integrin ligands expression and Stat1 signalling pathway activation. Collectively, our findings revealed a previously unknown role of ITGB6 in IBD and highlighted the possibility of ITGB6 as a diagnostic marker and therapeutic target for IBD.     

Development and Evaluation of a Novel and Rapid Detection Assay for Botrytis cinerea Based on Loop-Mediated Isothermal Amplification

Botrytis cinerea is a devastating plant pathogen that causes grey mould disease. In this study, we developed a visual detection method of B. cinerea based on the Bcos5 sequence using loop-mediated isothermal amplification (LAMP) with hydroxynaphthol blue dye (HNB). The LAMP reaction was optimal at 63°C for 45 min. When HNB was added prior to amplification, samples with B. cinerea DNA developed a characteristic sky blue color after the reaction but those without DNA or with DNA of other plant pathogenic fungi did not. Results of HNB staining method were reconfirmed when LAMP products were subjected to gel electrophoresis. The detection limit of this LAMP assay for B. cinerea was 10⁻³ ng µL⁻¹ of genomic DNA per reaction, which was 10-fold more sensitive than conventional PCR (10⁻² ng µL⁻¹). Detection of the LAMP assay for inoculum of B. cinerea was possible in the inoculated tomato and strawberry petals. In the 191 diseased samples, 180 (94.2%) were confirmed as positive by LAMP, 172 (90.1%) positive by the tissue separation, while 147 (77.0%) positive by PCR. Because the LAMP assay performed well in aspects of sensitivity, specificity, repeatability, reliability, and visibility, it is suitable for rapid detection of B. cinerea in infected plant materials prior to storage and during transportation, such as cut flowers, fruits and vegetables.     

Inhibition of Tumor Vasculogenic Mimicry and Prolongation of Host Survival in Highly Aggressive Gallbladder Cancers by Norcantharidin via Blocking the Ephrin Type a Receptor 2/Focal Adhesion Kinase/Paxillin Signaling Pathway

Vasculogenic mimicry (VM) is a newly-defined tumor microcirculation pattern in highly aggressive malignant tumors. We recently reported tumor growth and VM formation of gallbladder cancers through the contribution of the ephrin type a receptor 2 (EphA2)/focal adhesion kinase (FAK)/Paxillin signaling pathways. In this study, we further investigated the anti-VM activity of norcantharidin (NCTD) as a VM inhibitor for gallbladder cancers and the underlying mechanisms. In vivo and in vitro experiments to determine the effects of NCTD on tumor growth, host survival, VM formation of GBC-SD nude mouse xenografts, and vasculogenic-like networks, malignant phenotypes i.e., proliferation, apoptosis, invasion and migration of GBC-SD cells. Expression of VM signaling-related markers EphA2, FAK and Paxillin in vivo and in vitro were examined by immunofluorescence, western blotting and real-time polymerase chain reaction (RT-PCR), respectively. The results showed that after treatment with NCTD, GBC-SD cells were unable to form VM structures when injecting into nude mouse, growth of the xenograft was inhibited and these observations were confirmed by facts that VM formation by three-dimensional (3-D) matrix, proliferation, apoptosis, invasion, migration of GBC-SD cells were affected; and survival time of the xenograft mice was prolonged. Furthermore, expression of EphA2, FAK and Paxillin proteins/mRNAs of the xenografts was downregulated. Thus, we concluded that NCTD has potential anti-VM activity against human gallbladder cancers; one of the underlying mechanisms may be via blocking the EphA2/FAK/Paxillin signaling pathway.     

Efficient biotransformation of cholesterol to androsta-1,4-diene-3,17-dione by a newly isolated actinomycete <Emphasis Type="Italic">Gordonia neofelifaecis</Emphasis>

A newly isolated actinomycete, Gordonia neofelifaecis (NRRL B-59395) from the faeces of Neofelis nebulosa, was used to selectively degrade the side-chain of cholesterol. The intermediates were purified and characterized. Quantitative analysis of the accumulated metabolites from cholesterol side-chain cleavage was conducted during the biotransformation. The results showed that the profile of accumulated intermediates was different from those of other reported microorganisms. Among the five metabolites, androsta-1,4-diene-3,17-dione (ADD) was the main product of the side-chain degradation, with a high conversion rate (87.2%), indicating its potential for industrial production of ADD. At the end of transformation, the substrate cholesterol was completely consumed. The effect of some factors on the bioconversion was also investigated. To our best knowledge, this is the first report regarding cholesterol side-chain cleavage using bacteria belonging to Gordonia.     

Calcium protects bacteria against cadmium stress via reducing nitric oxide production and increasing iron acquisition

Cadmium (Cd) is a common toxic heavy metal in the environment, and bacteria have evolved different strategies against Cd-toxicity. Here, we found that marine bacterium Bacillus sp. 98 could significantly alleviate Cd-toxicity by recruiting calcium (Ca) for reducing excessive intracellular nitric oxide (NO) and enhancing iron acquisition. To investigate the underlying mechanisms, mass spectrometry-based proteomic analysis was applied to Bacillus sp. 98 after treated with Cd supplemented with or without Ca. Compared with bacterial cells treated with Cd only, the proteomic results showed that the expression level of NO synthase was markedly down-regulated, while the expression levels of NO dioxygenase, which is responsible for converting NO to nitrate, and proteins associated with iron uptake were profoundly enhanced when Ca was supplemented. Consistently, bacterial intracellular NO amount was dramatically increased after Bacillus sp. 98 was treated with Cd, and reversed to a normal level when Ca or iron was supplemented. Notably, Ca also protected bacteria against stresses from other heavy metals including Cu, Cr, Mn, Ni and Zn, and this self-protection strategy was adopted as well in zebrafish, which encourages us to develop Ca-associated products against heavy metals toxicity in the future.     

Ion current rectification in a confined conical nanopore with high solution concentrations

Ion transport in a confined conical nanochannel with high solution concentrations was studied using molecular dynamics simulation. The simulation results indicated that the ion current rectification appeared at high solution concentrations, even without electrical double layer (EDL) overlapping, which was still influenced by both the solution concentration and surface charge properties as it would be in a low solution concentration. With solution concentrations increasing from 0.41 to 2.08 M, a maximum rectification ratio was obtained. This phenomenon was attributed to the competition between the axial binding energy gradient arising from the confined conical geometry intensifying the ion axial asymmetric concentration polarisation and the decreasing thickness of the EDL weakening the concentration polarisation.     

Characterization and functional analysis of a novel PGIP gene from Pyrus pyrifolia Nakai cv Huobali

Polygalacturonase-inhibiting proteins (PGIPs) are multifunctional proteins related to plant autoimmunity and belong to the plant extracellular leucine-rich repeat (eLRR) protein superfamily. PGIPs play a role in host defense in many plants. In the present study, a novel PGIP gene, PpPGIP was isolated from Pyrus pyrifolia Nakai cv Huobali. The nucleotide sequence of PpPGIP was highly homologous with PGIPs from other plant species and the protein encoded by PpPGIP has several conserved LRR domains. The putative protein PpPGIP was closely clustered with several PGIPs from horticultural plants on the phylogenetic tree. The constructed homology model of PpPGIP indicated that the main-chain conformation and the folding patterns of PpPGIP were highly similar to structural features of PvPGIP2 from Phaseolus vulgaris. The expression levels of PpPGIP in healthy tissue and organ of ‘Huobali’ were analyzed with RT-PCR, and PpPGIP accumulated a little in young leaves, but PpPGIP was expressed abundantly in the pericarp of ‘Huobali’ fruits. Furthermore, in order to verify the function of PpPGIP, the constitutive plant expression vector of PpPGIP was constructed and transferred into tobacco (Nicotiana tabacum L. cv Xanthi). The Southern blot and real-time PCR analyses demonstrated that the PpPGIP gene was integrated into the genome of the tobacco transformants and highly expressed in the transgenic lines. The antifungal activity of PpPGIP was detected in vitro plates, and the crude protein extract of transgenic tobacco plants inhibited the hyphal growth of Phomopsis sp., Alternaria sp., Penicillium sp., and Aspergillus niger in different degrees.