Characterization of two β-galactosidases LacZ and WspA1 from Nostoc flagelliforme with focus on the latter’s central active region

The identification and characterization of new β-galactosidases will provide diverse candidate enzymes for use in food processing industry. In this study, two β-galactosidases, Nf-LacZ and WspA1, from the terrestrial cyanobacterium Nostoc flagelliforme were heterologously expressed in Escherichia coli, followed by purification and biochemical characterization. Nf-LacZ was characterized to have an optimum activity at 40\ua0\ub0C and pH 6.5, different from that (45\ua0\ub0C and pH 8.0) of WspA1. Two enzymes had a similar Michaelis constant (Km = 0.5\ua0mmol/liter) against the substrate o-nitrophenyl-β-D-galactopyranoside. Their activities could be inhibited by galactostatin bisulfite, with IC50 values of 0.59\ua0\ub5M for Nf-LacZ and 1.18\ua0\ub5M for WspA1, respectively. Gel filtration analysis suggested that the active form of WspA1 was a dimer, while Nf-LacZ was functional as a larger multimer. WspA1 was further characterized by the truncation test, and its minimum central region was found to be from residues 188 to 301, having both the glycosyl hydrolytic and transgalactosylation activities. Finally, transgenic analysis with the GFP reporter protein found that the N-terminus of WspA1 (35 aa) might play a special role in the export of WspA1 from cells. In summary, this study characterized two cyanobacterial β-galactosidases for potential applications in food industry

Storax Protected Oxygen-Glucose Deprivation/Reoxygenation Induced Primary Astrocyte Injury by Inhibiting NF-κB Activation in vitro

Stroke is the second leading cause of death and the leading cause of long-term disability in the world. There is an urgent unmet need to develop a range of neuroprotective strategies to restrain the damage that occurs in the hours and days following a stroke. Storax, a natural resin extracted from injuring Liquidambar orientalis Mill, has been used to treat acute stroke in traditional Chinese medicine for many centuries. Storax has demonstrated the neuroprotective effects in cerebrovascular diseases. However, the neuroprotective mechanisms activated by storax in ischemia/reperfusion-injured astrocytes have not been elucidated. In this study, we established an oxygen-glucose deprivation/reoxygenation (OGD/R)-induced astrocytes injury model to investigate the effects of storax on OGD/R-induced astrocytes injury and potential mechanisms. Experimental results showed that storax alleviated expression of inflammatory cytokines and protected primary cortical astrocytes injured by OGD/R. Furthermore, storax could inhibit NF-κB activation in injured astrocytes by OGD/R and inhibition of NF-κB with Bay-11-7082 obscured the neuroprotective effects of storax. In conclusion, storax alleviated expression of inflammatory cytokines and protected primary cortical astrocytes injured by OGD/R, which was partially mediated by NF-κB signaling pathway activation

Cucurbitacin B induces neurogenesis in PC12 cells and protects memory in APP/PS1 mice

Cucurbitacin B (CuB) isolated from Cucumis melo by using a PC12 cell bioassay system exhibited significant nerve growth factor (NGF)-mimic or NGF-enhancing activity in PC12 arid primary neuron cells. It was also demonstrated pro-neurogenesis effects in ICR and APP/PSI mice and improved memory deficit of APP/PSI mice. Its possible mechanism includes significant induction of the phosphorylation of glucocorticoid receptor (GR), protein kinase C (PKC), phospholipase C (PLC) and inhibition of cofilin. ChemProteoBase profiling, binding assay and cellular thermal shift assay (CETSA) were used to determine the target protein. Results revealed that CuB could affect actin dynamics as an actin inhibitor but did not bind with GR. The protein level of cofilin in PC12 cells after treating 0.3 mu M and different temperatures was significantly higher than that of control group. Other neurotrophic signalling pathways, such as TrkA/TrkB, were analysed with specific inhibitors and Western blot. The inhibitors of TrkA, PLC, PKC, Ras, Raf and ERK1/2 significantly decreased the percentage of PC12 cells with neurite outgrowth and shortened the length of neurite outgrowth induced by CuB. CuB significantly induced the phosphorylation of TrkA, ERK and CREB. The phosphorylation of these proteins was obviously decreased by their specific inhibitors. These results suggest that cofilin is a candidate target protein of CuB in PC12 cells and that the GR/PLC/PKC and TrkA/Ras/Raf/ERK signalling pathways play important roles in the neuroprotective effect of CuB.11Ysciescopu

Energy Route Multi-Objective Optimization of Wireless Power Transfer Network: An Improved Cross-Entropy Method

This paper identifies the Wireless Power Transfer Network (WPTN) as an ideal model for long-distance Wireless Power Transfer (WPT) in a certain region with multiple electrical equipment. The schematic circuit and design of each power node and the process of power transmission between the two power nodes are elaborated. The Improved Cross-Entropy (ICE) method is proposed as an algorithm to solve for optimal energy route. Non-dominated sorting is introduced for optimization. A demonstration of the optimization result of a 30-nodes WPTN system based on the proposed algorithm proves ICE method to be efficacious and efficiency

Decreased Peripheral BDNF Levels and Cognitive Impairment in Late-Life Schizophrenia

Objectives: There are relatively few studies on mechanisms of cognitive deficits in late-life schizophrenia (LLS). Brain-derived neurotrophic factor (BDNF), as an important neuroplastic molecule, has been reported to be involved in neurocognitive impairment in schizophrenia. This study aimed to examine whether peripheral BDNF levels were associated with cognitive deficits in LLS, which has not been explored yet.Methods: Forty-eight LLS patients and 45 age-matched elderly controls were recruited. We measured all participants on the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) for cognition and serum BDNF levels. Psychopathological symptoms in patients were assessed by the Positive and Negative Syndrome Scale (PANSS).Results: The levels of BDNF in LLS patients were significantly lower than those in healthy controls (8.80 ± 2.30 vs. 12.63 ± 5.08 ng/ml, p < 0.001). The cognitive performance of LLS patients was worse than that of the controls on RBANS total score and scores of immediate memory, attention, language, and delayed memory (all p ≤ 0.005). BDNF was positively associated with attention in LLS patients (r = 0.338, p = 0.019).Conclusion: Our findings suggest that older patients with schizophrenia exhibit lower BDNF levels and more cognitive deficits than older controls, supporting the accelerated aging hypothesis of schizophrenia. Moreover, decreased BDNF is related to attention deficits, indicating that BDNF might be a candidate biomarker of cognitive impairments in LLS patients

Production, purification and characterization of polyclonal antibody against the truncated gK of the duck enteritis virus

Duck virus enteritis (DVE) is an acute, contagious herpesvirus infection of ducks, geese, and swans, which has produced significant economic losses in domestic and wild waterfowl. With the purpose of decreasing economic losses in the commercial duck industry, studying the unknown glycoprotein K (gK) of DEV may be a new method for preferably preventing and curing this disease. So this is the first time to product and purify the rabbit anti-tgK polyclonal antibody. Through the western blot and ELISA assay, the truncated glycoprotein K (tgK) has good antigenicity, also the antibody possesses high specificity and affinity. Meanwhile the rabbit anti-tgK polyclonal antibody has the potential to produce subunit vaccines and the functions of neutralizing DEV and anti-DEV infection because of its neutralization titer. Indirect immunofluorescent microscopy using the purified rabbit anti-tgK polyclonal antibody as diagnostic antibody was susceptive to detect a small quantity of antigen in tissues or cells. This approach also provides effective experimental technology for epidemiological investigation and retrospective diagnose of the preservative paraffin blocks

Expression and characterization of duck enteritis virus gI gene

<p>Abstract</p> <p>Background</p> <p>At present, alphaherpesviruses gI gene and its encoding protein have been extensively studied. It is likely that gI protein and its homolog play similar roles in virions direct cell-to-cell spread of alphaherpesviruses. But, little is known about the characteristics of DEV gI gene. In this study, we expressed and presented the basic properties of the DEV gI protein.</p> <p>Results</p> <p>The special 1221-bp fragment containing complete open reading frame(ORF) of duck enteritis virus(DEV) gI gene was extracted from plasmid pMD18-T-gI, and then cloned into prokaryotic expression vector pET-32a(+), resulting in pET-32a(+)-gI. After being confirmed by PCR, restriction endonuclease digestion and sequencing, pET-32a(+)-gI was transformed into <it>E.coli </it>BL21(DE3) competent cells for overexpression. DEV gI gene was successfully expressed by the addition of isopropyl-β-D-thiogalactopyranoside(IPTG). SDS-PAGE showed that the recombinant protein His6-tagged gI molecular weight was about 61 kDa. Subsequently, the expressed product was applied to generate specific antibody against gI protein. The specificity of the rabbit immuneserum was confirmed by its ability to react with the recombinant protein His6-tagged gI. In addition, real time-PCR was used to determine the the levels of the mRNA transcripts of gI gene, the results showed that the DEV gI gene was transcribed most abundantly during the late phase of infection. Furthermore, indirect immunofluorescence(IIF) was established to study the gI protein expression and localization in DEV-infected duck embryo fibroblasts (DEFs), the results confirmed that the protein was expressed and located in the cytoplasm of the infected cells, intensively.</p> <p>Conclusions</p> <p>The recombinant prokaryotic expression vector of DEV gI gene was constructed successfully. The gI protein was successfully expressed by <it>E.coli </it>BL21(DE3) and maintained its antigenicity very well. The basic information of the transcription and intracellular localization of gI gene were presented, that would be helpful to assess the possible role of DEV gI gene. The research will provide useful clues for further functional analysis of DEV gI gene.</p

RSL3 Drives Ferroptosis Through GPX4 Inactivation and ROS Production in Colorectal Cancer

Ferroptosis is an iron-dependent, oxidative cell death, and is characterized by iron-dependent accumulation of reactive oxygen species (ROS) within the cell. It has been implicated in various human diseases, including cancer. Recently, ferroptosis, as a non-apoptotic form of cell death, is emerging in specific cancer types; however, its relevance in colorectal cancer (CRC) is unexplored and remains unclear. Here, we showed that ferroptosis inducer RSL3 initiated cell death and ROS accumulation in HCT116, LoVo, and HT29 CRC cells over a 24 h time course. Furthermore, we found that ROS levels and transferrin expression were elevated in CRC cells treated with RSL3 accompanied by a decrease in the expression of glutathione peroxidase 4 (GPX4), indicating an iron-dependent cell death, ferroptosis. Overexpression GPX4 resulted in decreased cell death after RSL3 treatment. Therefore, RSL3 was able to induce ferroptosis on three different CRC cell lines in vitro in a dose- and time-dependent manner, which was due to increased ROS and an increase in the cellular labile iron pool. Moreover, this effect was able to be reversed by overexpression of GPX4. Taken together, our results suggest that the induction of ferroptosis contributed to RSL3-induced cell death in CRC cells and ferroptosis may be a pervasive and dynamic form of cell death for cancer treatment