An ultrasensitive, homogeneous fluorescence quenching immunoassay integrating separation and detection of aflatoxin M1 based on magnetic graphene composites.

A homogeneous fluorescence quenching immunoassay is described for simultaneous separation and detection of aflatoxin M1 (AFM1) in milk. The novel assay relies on monoclonal antibody (mAb) functionalized Fe3O4 decorated reduced-graphene oxide (rGO-Fe3O4-mAb) as both capture probe and energy acceptor, combined with tetramethylrhodamine cadaverine-labeled aflatoxin B1 (AFB1-TRCA) as the energy donor. In the assay, AFB1-TRCA binds to rGO-Fe3O4-mAb in the absence of AFM1, quenching the fluorescence of TRCA by resonance energy transfer. Significantly, the immunoassay integrates sample preparation and detection into a single step, by using magnetic graphene composites to avoid washing and centrifugation steps, and the assay can be completed within 10 min. Under optimized conditions, the visual and quantitative detection limits of the assay for AFM1 were 50 and 3.8 ng L-1, respectively, which were significantly lower than those obtained by fluorescence polarization immunoassay using the same immunoreagents. Owing to its operation and highly sensitivity, the proposed assay provides a powerful tool for the detection of AFM1.

Knockout of pprM Decreases Resistance to Desiccation and Oxidation in Deinococcus radiodurans.

Deinococcus radiodurans has attracted a great interest in the past decades due to its extraordinary resistance to ionizing radiation and highly efficient DNA repair system. Recent studies indicated that pprM is a putative pleiotropic gene in D. radiodurans and plays an important role in radioresistance and antioxidation, but its underlying mechanisms are poorly elucidated. In this study, pprM mutation was generated to investigate resistance to desiccation and oxidative stress. The result showed that the survival of pprM mutant under desiccation was markedly retarded compared to the wild strain from day 7-28. Furthermore, knockout of pprM increases the intercellular accumulation of ROS and the sensibility to H2O2 stress in the bacterial growth inhibition assay. The absorbance spectrum experiment for detecting the carotenoid showed that deinoxanthin, a carotenoid that peculiarly exists in Deinococcus, was reduced in the pprM mutant in the pprM mutant. Quantitative real time PCR showed decreased expression of three genes viz. CrtI (DR0861, 50%),CrtB (DR0862, 40%) and CrtO (DR0093, 50%), which are involved in deinoxanthin synthesis, and of Dps (DNA protection during starving) gene (DRB0092) relevant to ion combining and DNA protection in cells. Our results suggest that pprM may affect antioxidative ability of D. radiodurans by regulating the synthesis of deinoxanthin and the concentration of metal ions. This may provide new clues for the treatment of antioxidants.

RNA-Binding Domain is Necessary for PprM Function in Response to the Extreme Environmental Stress in Deinococcus radiodurans.

Deinococcus radiodurans was considered as one of the most radiation-resistant organisms on Earth because of its strong resistance to the damaging factors of both DNA and protein, including ionizing radiation, ultraviolet radiation, oxidants, and desiccation. PprM, as a bacterial cold shock protein homolog, was involved in the radiation resistance and oxidative stress response of D. radiodurans, but its potential mechanisms are poorly expounded. In this study, we found that PprM was highly conserved with the RNA-binding domain in Deinococcus genus through performing phylogenic analysis. Moreover, the paper presents the analysis on the tolerance of environmental stresses both in the wild-type and the pprM/pprM RBD mutant strains, demonstrating that pprM and RNA-binding domain disruptant strain were with higher sensitivity than the wild-type strain to cold stress, mitomycin C, UV radiation, and hydrogen peroxide. In the following step, the recombinant PprM was purified, with the finding that PprM was bound to the 5'-untranslated region of its own mRNA by gel mobility shift assay in vitro. With all these findings taken into consideration, it was suggested that PprM act as a cold shock protein and its RNA-binding domain may be involved in reaction to the extreme environmental stress in D. radiodurans.

Removal of B-cell epitopes for decreasing immunogenicity in recombinant immunotoxin against B-cell malignancies.

Recombinant immunotoxin HA22, composed of an anti- CD22 Fv fragment fused to PE38, a truncated portion of Pseudomonas Exotoxin A (PE), has been developed for targeted treatment of various B-cell malignancies. As a foreign, internalized macromolecule, PE38 often induces lysosomal degradation and neutralizing antibodies to limit the efficacy of treating B-cell malignancies. The region of PE38 containing lysosomal protease cleavage sites deleted, leaving only furin processing site. The resulting immunotoxin HA22-LR (lysosome resistant) retains excellent biologic activity and removes immunogenic epitopes as an additional benefit. Another approach for avoiding immunogenicity is to identify B-cell epitopes and remove them by mutagenesis. Previously, to determine B-cell epitopes on PE38, murine Ab as a model, 7 major mouse-specific B-cell epitope groups with 13 subgroups were identified and located through a series of point mutations. Two new mutants, HA22-8X and HA22-LR-8X, were prepared, containing 8 epitope-silencing mutations which greatly reduced immunogenicity in mice. Later, by phage-display assay, human Fvs against PE toxin were isolated and human-specific B-cell epitopes were located by alanine scanning mutagenesis. HA22-LR as a scaffold, HA22-LR-LO10 with 7 point mutations was constructed, has low reactivity with human antisera, yet has high cytotoxic and antitumor activity. In this review, theoretical aspects and experimental evidence for the removal of B-cell epitope is discussed.

Kinetics model of DNA double-strand break repair in eukaryotes.

This manuscript outlines the kinetics of two main repair pathways of DNA double-strand break (DSB) in eukaryotes: non-homologous end joining (NHEJ) and homologous recombination repair (HRR). In this review, we discuss the precise study of recruitment kinetics of repair proteins based on the latest technologies in the past two decades. Then we simulate the theoretical description of the DNA repair process by mathematical models. In our study, the consecutive reactions chain (CRC) model and continuous-time random walk (CTRW) model have been unified by us, so that we can obtain the function of the number of intermediates with time in the same framework of equations, overcome the incompatibility between the two models. On this basis, we propose a data fitting workflow using these both models. Finally, we give an overview of different real-time quantitative methods and the new mechanism complexity that can be found from the corresponding dynamic models.

Antioxidative system of Deinococcus radiodurans.

Deinococcus radiodurans is famous for its extreme resistance to various stresses such as ionizing radiation (IR), desiccation and oxidative stress. The underlying mechanism of exceptional resistance of this robust bacterium still remained unclear. However, the antioxidative system of D. radiodurans has been considered to be the determinant factor for its unparalleled resistance and protects the proteome during stress, then the DNA repair system and metabolic system exert their functions to restore the cell to normal physiological state. The antioxidative system not only equipped with the common reactive oxygen species (ROS) scavenging enzymes (e.g., catalase and superoxide dismutase) but also armed with a variety of non-enzyme antioxidants (e.g., carotenoids and manganese species). And the small manganese complexes play an important role in the antioxidative system of D. radiodurans. Recent studies have characterized several regulators (e.g., PprI and PprM) in D. radiodurans, which play critical roles in the protection of the bacteria from various stresses. In this review, we offer a panorama of the progress regarding the characteristics of the antioxidative system in D. radiodurans and its application in the future.

Endoplasmic reticulum stress and destruction of pancreatic ß cells in type 1 diabetes.

Type 1 diabetes (T1D) results from dysfunction of pancreatic islets ß cells. Recent studies supported that endoplasmic reticulum (ER) stress takes an important role in pancreatic ß cell excessive loss, resulting in T1D. Here, we aimed to review the relationship between ER stress and T1D. Additionally, we also reviewed the potential mechanisms underlying ER stress mediated T1D. Studies have shown that severe ER stress is directly involved in the pancreatic ß cells destruction and pathogenesis of T1D. ER stress plays a key part in pancreatic ß cells and T1D, which will help in developing new effective therapeutics for T1D.

Gene regulation for the extreme resistance to ionizing radiation of Deinococcus radiodurans.

Deinococcus radiodurans is a model microorganism used for studies on DNA repair and antioxidation due to its extraordinary tolerance to ionizing radiation and other DNA-damaging agents. Various transcriptome analyses have revealed that hundreds of genes are induced and that many other genes are repressed during recovery of D. radiodurans following irradiation, suggesting that gene regulation is of great importance for the extreme resistance of this microorganism to ionizing radiation. In this article, we focus on some reported strategies that are employed by D. radiodurans to regulate the genes implicated in its extreme tolerance to ionizing radiation for a comprehensive understanding of the reasons this bacterium can survive such extraordinary stress. We expect this review to shed light on potential radioprotective agents and applications for use in a range of fields.

Use of recombinant Lactobacillus sakei for the prevention and treatment of radiation-induced enteritis.

Radiation-induced enteritis is one of the most common complications in patients under radiotherapy at abdominal or pelvic cavity. Up to 50% of patients treated with pelvic radiotherapy has been reported radiation-induced acute enteritis, and half of them developed chronic enteritis. Overproduction of free radicals, activation of inflammatory pathways and vascular endothelial dysfunction were considered as the primary mechanisms of radiation-induced enteritis. Because probiotics have been demonstrated as a promising potential candidate for treating intestinal diseases, it may be a safer and more effective radioprotector for the enteritis compared to conventional chemical agents with inherent toxicities. Here, we propose that a recombinant Lactobacillus sakei would decrease the complications or symptoms significantly through against different pathogenic mechanisms simultaneously. Therefore, application of higher radiation dose for tumor control would be feasible when co-treating with recombinant Lactobacillus sakei.

[Compound K suppresses myeloid-derived suppressor cells in a mouse model bearing CT26 colorectal cancer xenograft].

OBJECTIVE: To investigate the effect of ginseng-derived compound K (C-K) on apoptosis, immunosuppressive activity, and pro-inflammatory cytokine production of myeloid-derived suppressor cells (MDSCs) from mice bearing colorectal cancer xenograft. METHODS: Flow-sorted bone marrow MDSCs from Balb/c mice bearing CT26 tumor xenograft were treated with either C-K or PBS for 96 h and examined for apoptosis with Annexin V/7-AAD, Cox-2 and Arg-1 expressions using qRT-PCR, and supernatant IL-1ß, IL-6, and IL-17 levels with ELISA. C-K- or PBS-treated MDSCs were subcutaneously implanted along with CT26 tumor cells in WT Balb/c mice, and the tumor size and morphology were evaluated 21 days later. RESULTS: C-K treatment significantly increased the percentages of early and late apoptotic MDSCs in vitro (P<0.01 and P<0.05, respectively), decreased the expressions of immunosuppression-related genes Cox-2 (P<0.05) and Arg-1 (P<0.01), and suppressed the production of IL-1ß (P<0.05), IL-6 (P<0.01), and IL-17 (P<0.05) by the MDSCs . Compared with PBS-pre-treated cells, C-K-pretreated MDSCs showed significantly attenuated activity in promoting CT26 tumor growth in mice (P<0.01). CONCLUSION: C-K can suppress the immunosuppresive effect of MDSCs to inhibit tumor cell proliferation in mice, which suggests a new strategy of tumor therapy by targeting MDSCs.

Development of a lateral flow immunoassay (LFA) strip for the rapid detection of 1-aminohydantoin in meat samples.

UNLABELLED: Due to the potential toxic effects of the nitrofuran family of antibiotics, their use in animals in the food industry has raised health concerns. This study was aimed to develop a lateral flow assay (LFA) based on competitive format for the detection of 1-aminohydantoin (AHD) in meat samples. The assay could be completed in 1 min and detected AHDs derivates (CPAHD) at 3 ng/mL, equivalent to 1.40 ng/mL of AHD, which was much lower than that reported in the literature by similar method. The antibody showed no cross-reactivity with a panel of more than 10 nitrofuran analogs except for nitrofurantoin at a high concentration. The test strip was stable at room temperature for up to 8 wk or at 37 °C for 4 wk. Parallel analyses of meat samples with LFA and enzyme-linked immunosorbent assay (ELISA) obtained data in good agreement. This developed gold nanoparticle based LFA had a good specificity, sensitivity, stability, and reliability. It was potentially suitable for on-the-spot large-scale screening of meat samples, and even more other applications. PRACTICAL APPLICATION: Nitrofurantoin is one of antibiotics of the nitrofuran family, which has been used not only to prevent and treat diseases, but also to promote growth in animals. However, concerning the carcinogenicity of the metabolite of nitrofurantoin (AHD), a new fast and convenient method for monitoring AHD should be established. We describe the development of a new test assay for rapid screening of meat samples.