Effects and molecular mechanism of sugar transporter ESA_RS15745 on desiccation resistance, motility, and biofilm formation of Cronobacter sakazakii.

Cronobacter sakazakii, an important Gram-negative foodborne pathogen, can cause neonatal meningitis and sepsis with high rates of infection and death. Gene ESA_RS15745 encodes a sugar transporter protein, which is not only essential for osmotic pressure maintenance during bacterial growth and reproduction but also associated with their desiccation tolerance, motility, and biofilm formation. Here, a mutant strain of ESA_RS15745 (ΔESA_RS15745) and the complementation strain (cpESA_RS15745) were constructed using a suicide vector knockout and gene complementation. ΔESA_RS15745 was found to have a decrease in its ability to transport maltose and trehalose and resist desiccation, whereas an increase in the ability of motility and biofilm formation, implying that ESA_RS15745 may positively regulate sugar transport and desiccation tolerance and negatively regulate motility and biofilm formation. To further investigate the molecular mechanisms underlying the function of related genes, RNA-seq was performed to explore the differentially expressed genes in the mutants. RNA-seq results showed the upregulation of 114 genes (mainly including those regulating chemotaxis and flagellar motility) and the downregulation of 22 genes (mainly including those regulating sugar transport). qRT-PCR analysis supported the RNA-seq results and showed that ESA_RS15745 may influence the dehydration tolerance though decreasing the intracellular trehalose content and negatively regulate the motility though the chemotactic signaling pathway. In addition, the biofilm formation of C. sakazakii should also be speculated to negatively regulate by ESA_RS15745 by consuming the extracellular carbohydrates concentration and then downregulating the intracellular cyclic diguanosine monophosphate. This study offers a reference for comprehending the molecular mechanism of gene ESA_RS15745 in C. sakazakii.

An optimal medicinal and edible Chinese herbal formula attenuates particulate matter-induced lung injury through its anti-oxidative, anti-inflammatory and anti-apoptosis activities / 中草药·英文版

OBJECTIVE@#Identifying novel strategies to prevent particulate matter (PM)-induced lung injury is crucial for the reduction of the morbidity of chronic respiratory diseases. The combined intervention represented by herbal formulae for simultaneously targeting multiple pathological processes can provide a more beneficial effect than the single intervention. The aim of this paper is therefore to design a safe and effective medicinal and edible Chinese herbs (MECHs) formula against PM-induced lung injury.@*METHODS@#PM-induced oxidative stress, inflammatory response and apoptosis A549 cell model were used to screen anti-oxidant, anti-inflammatory and anti-apoptotic MECHs, respectively. A network pharmacology method was utilized to rationally design a novel herbal formula. Ultra performance liquid chromatography-mass spectrometer was utilized to assess the quality control of MECHs formula. The excretion of magnetic iron oxide nanospheres of the MECHs formula was estimated in zebrafish. The MECH formula against PM-induced lung injury was investigated with mice experiments.@*RESULTS@#Five selected herbs were rationally designed to form a new MECH formula, including Citri Exocarpium Rubrum (Juhong), Lablab Semen Album (Baibiandou), Atractylodis Macrocephalae Rhizoma (Baizhu), Mori Folium (Sangye) and Polygonati Odorati Rhizoma (Yuzhu). The formula effectively promoted the magnetic iron oxide nanospheres excretion in zebrafish. The mid/high dose formula significantly prevented PM-induced lung damage in mice by enhancing the activity of SOD and GSH-Px, reducing the MDA and ROS level and attenuating the upregulation of pro-inflammatory cytokine (IL-6, IL-8, IL-1β and TNF-α), down regulating the protein expression of NF-κB, STAT3 and Caspase-3.@*CONCLUSION@#Our findings suggest that the effective MECHs formula will become a novel strategy for preventing PM-induced lung injury and provide a paradigm for the development of functional foods using MECHs.

Construction, Screening and Antibody Structure Homology Modeling of Phage Single Chain Variable Fragment Library Against Ofloxacin / 分析化学

To construct a library of mouse single chain variable fragment (scFv) antibody against ofloxacin using phage display and recombinant antibody technique, specific anti-ofloxacin scFv was screened and 3D structure was homology modeling. Total RNA was extracted from hybridoma cell of ofloxacin mAb, and was used to amplify VH and VL gene by RT-PCR using random primer. Then they were linked by a DNA linker encoding (G1y4 Ser) 3 as VH-linker-VL sequence forming scFv by SOE(splicing by overlap extension) PCR. These fragments were inserted into phage T7 after double digestion and transformed with host bacteria BLT5403. 3 ×105 pfu / ml single chain antibody phage libraries were successfully constructed. Four positive phage scFv clones were screened by direct competitive ELISA after four times of enriched procedure in the order of adsorption-elution-amplificatio, 3D structure of specific scFv was homology modeling finally. This research lays a foundation for further massive expression of anti-ofloxacin scFv.

Molecular cloning, recombinant expression and characterization of lysozyme from Chinese shrimp Fenneropenaeus chinensis / 生物工程学报

Lysozyme hydrolyses bacterial cell walls and acts as a nonspecific innate immunity molecule against the invasion of bacterial pathogens. We cloned the cDNA of lysozyme from Fenneropenaeus chinensis and named Fc-lysozyme (FcLyz in short). The full length of the gene was of 709 bp, and the open reading frame (477 bp) encoded 158 amino acids. The predicted protein had a signal peptide (-1--18 residue) and molecular weight of the mature protein (residue 1-140) was of 16.2 kD. A Lyz 1 domain (residue 1-130) in the lysozyme was found by SMART analysis. The results of semiquantity RT-PCR showed that FcLyz was constitutively expressed in tested tissues in a low level in normal shrimp, and up-regulated in hemocytes, heart, hepatopancreas and gill of bacterial challenged shrimp. The DNA fragment of mature Fc-Lys was subcloned to pET-30a (+) expression vector, the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) and then induced by isopropylthio-beta-D-galactoside (IPTG). The antibacterial activity of the purified recombinant FcLys was analyzed and minimal inhibitory concentration (MIC) was assayed. The recombinant protein showed high antibacterial activity against some Gram-positive bacteria, and MIC reached 3.43 micromol/L, and relatively low activity against Gram-negative bacteria. All together, the Fc-Lys was regulated by pathogen infection and had antibacterial activity. This suggested that the FcLyz may be one of the important molecules against pathogens in innate immunity of the shrimp.