Production of a monoclonal antibody against aflatoxin M1 and its application for detection of aflatoxin M1 in fortified milk

AbstractAflatoxin M1 (AFM1) is a toxic metabolite of the fungal product aflatoxin found in milk. For food safety concern, maximum residual limits of AFM1 in milk and dairy products have been differently enforced in many countries. A suitable detection method is required to screen a large number of product samples for the AFM1 contamination. In this study, monoclonal antibodies (MAbs) against AFM1 were generated using a conventional somatic cell fusion technique. After screening, five MAbs (AFM1-1, AFM1-3, AFM1-9, AFM1-11, and AFM1-17) were obtained that showed cross-reactivity with aflatoxin B1 (AFB1) and aflatoxin G1 (AFG1) but with no other tested compounds. An indirect competitive enzyme-linked immunosorbent assay (ELISA) using a partially purified MAb and antigen-coated plates yielded the best sensitivity with the 50% inhibition concentration (IC50) and the limit of detection (LOD) values of 0.13 ng/mL and 0.04 ng/mL, respectively. This indirect competitive ELISA was used to quantify the amount of fortified AFM1 in raw milk. The precision and accuracy in terms of % coefficient of variation (CV) and % recovery of the detection was investigated for both intra- (n = 6) and inter- (n = 12) variation assays. The % CV was found in the range of 3.50–15.8% and 1.32–7.98%, respectively, while the % recovery was in the range of 92–104% and 100–103%, respectively. In addition, the indirect ELISA was also used to detect AFM1 fortified in processed milk samples. The % CV and % recovery values were in the ranges of 0.1–33.0% and 91–109%, respectively. Comparison analysis between the indirect ELISA and high performance liquid chromatography was also performed and showed a good correlation with the R2 of 0.992 for the concentration of 0.2–5.0 ng/mL. These results indicated that the developed MAb and ELISA could be used for detection of AFM1 in milk samples

The role of notch signaling in regulating nuclear hormone receptor activity in mammalian T lymphocytes and Drosophila

Regulation of programmed cell death is crucial for proper development and maintaining homeostasis of organisms. This thesis work attempts to elucidate the relationships between two evolutionarily well-conserved pathways, i.e. nuclear hormone receptor and Notch signaling pathway, using programmed cell death in T lymphocytes and Drosophila melanogaster as model systems. During metamorphosis of insects, all larval tissues undergo apoptotic cell death, a process triggered by steroid hormone ecdysone. In larval tissues, ecdysone pulses directly regulate expression of cell death genes. In this thesis work, the effect of Notch signaling on ecdysone receptor (EcR) activity was examined in larval salivary gland undergoing apoptosis. Aberrant expression of activated Notch or mutations in Suppressor of Hairless (Su(H)), a downstream effector protein of Notch, resulted in inhibition of cell death in the larval salivary glands. This inhibitory effect was due to the inability of EcR to induce ecdysone responsive genes during prepupal ecdysone pulse. Genetic interaction experiments revealed an allele-specific genetic interaction between Su(H) and EcR. These results suggest the cross talk between Notch signaling and EcR in Drosophila. There are two signalings involved in apoptosis of thymocytes during thymic development, steroid hormone, glucocorticoid, and orphan nuclear receptor Nur77. Previously, aberrant expression of Notch was shown to inhibit apoptosis induced by both signalings in T cell hybridomas. In this thesis work, the role for nuclear hormone receptor in regulating Notch activity was examined. Glucocorticoid treatment of thymocytes resulted in rapid down-modulation of Notch I proteins. This event was inhibited by caspase and proteasome inhibitors, suggesting that glucocorticoid-induced down-modulation of Notch protein is mediated through caspase and the proteasome pathways. Furthermore, thymocytes from transgenic mice expressing a Notch antisense construct, showed an increased in susceptibility to glucocorticoid-induced apoptosis. Lastly, upregulation of Notch proteins, and activation of Notch signaling were observed in T lymphocytes upon stimulation. This observation suggests a novel role of Notch signaling in regulating functions of peripheral T cells. The work described in this thesis provides an insight into the link between Notch signaling and nuclear hormone receptors

Notch Signaling in Macrophages in the Context of Cancer Immunity

Macrophages play both tumor-suppressing and tumor-promoting roles depending on the microenvironment. Tumor-associated macrophages (TAMs) are often associated with poor prognosis in most, but not all cancer. Understanding how macrophages become TAMs and how TAMs interact with tumor cells and shape the outcome of cancer is one of the key areas of interest in cancer therapy research. Notch signaling is involved in macrophage activation and its effector functions. Notch signaling has been indicated to play roles in the regulation of macrophage activation in pro-inflammatory and wound-healing processes. Recent evidence points to the involvement of canonical Notch signaling in the differentiation of TAMs in a breast cancer model. On the other hand, hyperactivation of Notch signaling specifically in macrophages in tumors mass has been shown to suppress tumor growth in an animal model of cancer. Investigations into how Notch signaling is regulated in TAMs and translates into pro- or anti-tumor functions are still largely in their infancy. Therefore, in this review, we summarize the current understanding of the conflicting roles of Notch signaling in regulating the effector function of macrophages and the involvement of Notch signaling in TAM differentiation and function. Furthermore, how Notch signaling in TAMs affects the tumor microenvironment is reviewed. Finally, the direct or indirect cross-talk among TAMs, tumor cells and other cells in the tumor microenvironment via Notch signaling is discussed along with the possibility of its clinical application. Investigations into Notch signaling in macrophages may lead to a more effective way for immune intervention in the treatment of cancer in the future

(alpha )-Mangostin and apigenin induced the necrotic death of BT474 breast cancer cells with autophagy and inflammation

Objective: To find new compounds in order to overcome the mainstay of metastatic breast cancer due to the adverse side effects from, and increasing resistance to, current chemotherapeutic agents. Methods: α-Mangostin and apigenin were reported in comparison to doxorubicin, a chemotherapeutic drug. Ductal carcinoma (BT474) cell line and non-tumorigenic epithelial tissue from mammary gland (MCF-10A) were used. Cell viability assessment was calculated by the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Cell morphology was investigated by light microscopy. By flow cytometry analysis, programmed cell death was observed using annexin V and propidium iodide staining while cell-cycle arrest was observed using propidium iodide staining. Change in transcriptional expression was evaluated by real-time quantitative reverse transcription PCR. Results: In 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the result revealed α-mangostin and apigenin were more cytotoxic to BT474 cells. Longer exposure times to α-mangostin and apigenin caused more floating cells and a lower density of adhered cells with more vacuoles present in the colonies in BT474 only. α-Mangostin and apigenin caused necrosis in BT474 cells in a 24 h exposure, but a small amount of early apoptotic cells could also be detected at 24, 48 and 72 h exposure, whereas doxorubicin caused early apoptosis to BT474 cells at 24 h. Transcript expression and activity analysis supported caspase-3 was involved in the death of BT474 cells treated by all compounds. Moreover, α-mangostin and apigenin arrested the cell-cycle at the G1-phase, but at the G2/M-phase by doxorubicin. All three compounds induced a change in transcript expression levels of inflammation-associated, proto-oncogene, autophagy-associated and apoptosis-associated genes. Conclusions: α-Mangostin and apigenin are worth investigating as potential new sources of chemotherapeutic agents for breast cancer treatment

Biological activities and safety of Thanaka (Hesperethusa crenulata) stem bark

Ethnopharmacological relevance The stem bark powder of Hesperethusa crenulata or Thanaka has been used on the face by Myanmar women for more than a thousand years as a skin care regiment. Aim of the study The aim of the current study was to both verify the safety and evaluate some biological activities of the Thanaka bark. Materials and methods Maceration of the Thanaka bark powder resulted in hexane, dichloromethane, ethyl acetate, methanol, 85% ethanol and water extracts. For the safety evaluation, cytotoxicity and genotoxicity of each extract were tested. Antibacterial, tyrosinase inhibition, antioxidant and anti-inflammatory activities were evaluated for each extract. Results and conclusions Extracts from Thanaka bark showed strong anti-inflammatory, significant antioxidation, mild tyrosinase inhibition and slight antibacterial activities. All extracts and the original bark powder showed no detectable genotoxicity while very low cytotoxicity with IC50 value of more than 12 mg/ml was detected in the water extract. Thus, the use of the Thanaka bark in the form of a watery paste as a skin care regiment is not only safe but also beneficial to skin

Clusters of Carbon Nanospheres Derived from Graphene Oxide

Stable and water-dispersible cluster of carbon nanospheres (CCNs) can be synthesized from graphite or graphene nanoplatelets via sonication assisted chemical exfoliation/oxidation. Separation of CCNs with a diameter of ∼130 nm from other products in the reaction including the graphene oxide sheets (GOShs) and other smaller carbon-based particles, could be done by centrifugation. Structural analysis of these CCNs by UV–vis absorption, fluorescent, IR, Raman, and X-ray photoelectron spectroscopic, combustion elemental, and electron diffraction analyses, indicates an extended π–π conjugation network of sp²-hybridized carbon atoms together with COOH, CO, and OH functionalities. The spherical morphology of the CCNs is established by scanning electron microscopic analysis, while transmission electron microscopy indicates that the obtained CCNs are an aggregate of 5 nm carbon spheres (giant fullerenes) and GOShs. Dispersing the CCNs in curcumin solution results in curcumin adsorption onto the CCNs at 18.5 ± 1.4% (wt curcumin/wt loaded CCNs). The curcumin-loaded CCNs are taken up into human embryonic kidney (HEK293T) cells with detachment of the curcumin from the CCNs once inside the cells, such that curcumin is detected in the nucleus while the CCN carriers are only found outside the nucleus. The use of CCNs as the carrier gives a significantly better transportation of curcumin into the nucleus of the cells compared to curcumin alone. CCNs show no significant in vitro cytotoxicity to the HEK293T and human epidermoid cervical carcinoma cell lines at up to 3 μg/mL