A stable colorimetric biosensor for highly selective detection of malathion residue in food based on aptamer-regulated laccase-mimic activity.

Malathion causes a serious threat to human health due to its widespread use in the environment. Herein, a novel and stable smartphone-integrated colorimetric biosensor for malathion detection is firstly established based on aptamer-enhanced laccase-mimicking activity. The results indicate that the M17-F aptamer can increase the affinity of Ag2O nanoparticles to the substrate 2,4-dichlorophenol and enhance their laccase-mimicking activity. Thus, abundant semiquinone radicals are produced in the catalytic system, which are combined with chromogenic agent to generate dark red products. The corresponding RGB values for the colour change of the solution can be easily obtained using smartphones, which is used for the rapid detection of malathion. The established biosensor for malathion has a limit of detection as low as 5.85 nmol·L-1, and displays good selectivity for other competitive pesticides. Moreover, further studies have verified the applicability of the biosensor in actual samples, indicating that it may have the potential for application in malathion detection in food.

Recent Thymic Emigrants Require RBPJ-Dependent Notch Signaling to Transition into Functionally Mature Naive T Cells.

Recent thymic emigrant (RTE) cells are nascent T cells that continue their post-thymic maturation in the periphery and dominate T cell immune responses in early life and in adults having undergone lymphodepletion regimens. However, the events that govern their maturation and their functionality as they transition to mature naive T cells have not been clearly defined. Using RBPJind mice, we were able to identify different stages of RTE maturation and interrogate their immune function using a T cell transfer model of colitis. As CD45RBlo RTE cells mature, they transition through a CD45RBint immature naive T (INT) cell population that is more immunocompetent but shows a bias toward IL-17 production at the expense of IFN-γ. Additionally, the levels of IFN-γ and IL-17 produced in INT cells are highly dependent on whether Notch signals are received during INT cell maturation or during their effector function. IL-17 production by INT cells showed a total requirement for Notch signaling. Loss of Notch signaling at any stage of INT cells resulted in an impaired colitogenic effect of INT cells. RNA sequencing of INT cells that had matured in the absence of Notch signals showed a reduced inflammatory profile compared with Notch-responsive INT cells. Overall, we have elucidated a previously unknown INT cell stage, revealed its intrinsic bias toward IL-17 production, and demonstrated a role for Notch signaling in INT cell peripheral maturation and effector function in the context of a T cell transfer model of colitis.

DL4-µbeads induce T cell lineage differentiation from stem cells in a stromal cell-free system.

T cells are pivotal effectors of the immune system and can be harnessed as therapeutics for regenerative medicine and cancer immunotherapy. An unmet challenge in the field is the development of a clinically relevant system that is readily scalable to generate large numbers of T-lineage cells from hematopoietic stem/progenitor cells (HSPCs). Here, we report a stromal cell-free, microbead-based approach that supports the efficient in vitro development of both human progenitor T (proT) cells and T-lineage cells from CD34+cells sourced from cord blood, GCSF-mobilized peripheral blood, and pluripotent stem cells (PSCs). DL4-µbeads, along with lymphopoietic cytokines, induce an ordered sequence of differentiation from CD34+ cells to CD34+CD7+CD5+ proT cells to CD3+αß T cells. Single-cell RNA sequencing of human PSC-derived proT cells reveals a transcriptional profile similar to the earliest thymocytes found in the embryonic and fetal thymus. Furthermore, the adoptive transfer of CD34+CD7+ proT cells into immunodeficient mice demonstrates efficient thymic engraftment and functional maturation of peripheral T cells. DL4-µbeads provide a simple and robust platform to both study human T cell development and facilitate the development of engineered T cell therapies from renewable sources.

High-Oxygen Submersion Fetal Thymus Organ Cultures Enable FOXN1-Dependent and -Independent Support of T Lymphopoiesis.

T cell development is effectively supported in fetal thymus organ cultures (FTOCs), which places thymus lobes atop an air-liquid interface (ALI) culture system. The direct exposure to air is critical for its success, as fetal thymus lobes placed in low oxygen submersion (LOS)-FTOCs fail to support thymocyte development. However, submersion cultures performed in the presence of high concentration of ambient oxygen (60~80%) allow for normal thymocyte development, but the underlying mechanism for this rescue has remained elusive. Here, we show that FOXN1 expression in thymic epithelial cells (TECs) from LOS-FTOCs was greatly reduced compared to conventional ALI-FTOCs. Consequently, the expression of important FOXN1 target genes, including Dll4 and Ccl25, in TECs was extinguished. The loss of DLL4 and CCL25 interrupted thymocyte differentiation and led to CD4+CD8+ cells exiting the lobes, respectively. High oxygen submersion (HOS)-FTOCs restored the expression of FOXN1 and its target genes, as well as maintained high levels of MHCII expression in TECs. In addition, HOS-FTOCs promoted the self-renewal of CD4-CD8-CD44-CD25+ cells, allowing for the continuous generation of later stage thymocytes. Forced FOXN1 expression in TECs rescued thymocyte developmental progression, but not cellularity, in LOS-FTOCs. Given that oxidative stress has been reported to accelerate the onset of age-associated thymic involution, we postulate that regulation of FOXN1 by oxygen and antioxidants may underpin this biological process.

Application of DNA barcoding and metabarcoding for species identification in salmon products.

Mislabelling is a significant manifestation of food fraud. Traditional Sanger sequencing technology is the gold standard for seafood species identification. However, this method is not suitable for analysing processed samples that may contain more than one species. This study tested the feasibility of next-generation sequencing in identifying mixed salmon products. Salmon samples containing up to eight species were amplified using 16S rRNA mini-barcode primers, and sequenced on an Illumina HiSeq2500 platform. All species were accurately identified, and mixtures as low as 1% (w/w) could be detected. Furthermore, this study conducted a market survey of 32 products labelled as salmon. For pure and mixed fish products, Sanger and next-generation sequencing techniques were respectively used for species identification, and for NGS results, we also used real-time PCR method to cross-validate the mixed products to further verify the accuracy of the DNA metabarcoding technology established in this study. DNA barcoding and metabarcoding of commercial salmon food products revealed the presence of mislabelling in 16 of 32 (50%) samples. The developed DNA barcoding and metabarcoding methods are useful for the identification of salmon species in food and can be used for quality control of various types of salmon products.

A 2020 View of Thymus Stromal Cells in T Cell Development.

The thymus is an intricate primary lymphoid organ, wherein bone marrow-derived lymphoid progenitor cells are induced to develop into functionally competent T cells that express a diverse TCR repertoire, which is selected to allow for the recognition of foreign Ags while avoiding self-reactivity or autoimmunity. Thymus stromal cells, which can include all non-T lineage cells, such as thymic epithelial cells, endothelial cells, mesenchymal/fibroblast cells, dendritic cells, and B cells, provide signals that are essential for thymocyte development as well as for the homeostasis of the thymic stroma itself. In this brief review, we focus on the key roles played by thymic stromal cells during early stages of T cell development, such as promoting the homing of thymic-seeding progenitors, inducing T lineage differentiation, and supporting thymocyte survival and proliferation. We also discuss recent advances on the transcriptional regulation that govern thymic epithelial cell function as well as the cellular and molecular changes that are associated with thymic involution and regeneration.

Rapid Classification and Quantification of Camellia (Camellia oleifera Abel.) Oil Blended with Rapeseed Oil Using FTIR-ATR Spectroscopy.

Currently, the authentication of camellia oil (CAO) has become very important due to the possible adulteration of CAO with cheaper vegetable oils such as rapeseed oil (RSO). Therefore, we report a Fourier transform infrared (FTIR) spectroscopic method for detecting the authenticity of CAO and quantifying the blended levels of RSO. In this study, two characteristic spectral bands (1119 cm-1 and 1096 cm-1) were selected and used for monitoring the purity of CAO. In combination with principal component analysis (PCA), linear discriminant analysis (LDA), and partial least squares regression (PLSR) analysis, qualitative and quantitative methods for the detection of camellia oil adulteration were proposed. The results showed that the calculated I1119/I1096 intensity ratio facilitated an initial check for pure CAO and six other edible oils. PCA was used on the optimized spectral region of 1800-650 cm-1. We observed the classification of CAO and RSO as well as discrimination of CAO with RSO adulterants. LDA was utilized to classify CAO from RSO. We could differentiate and classify RSO adulterants up to 1% v/v. In the quantitative PLSR models, the plots of actual values versus predicted values exhibited high linearity. Root mean square error of calibration (RMSEC) and root mean square error of cross validation (RMSECV) values of the PLSR models were 1.4518%-3.3164% v/v and 1.7196%-3.8136% v/v, respectively. This method was successfully applied in the classification and quantification of CAO adulteration with RSO.

DNA barcoding and mini-barcoding in authenticating processed animal-derived food: A case study involving the Chinese market.

This study used DNA barcoding and DNA mini-barcoding to test a variety of animal-derived food products sold in the Chinese market for potential mislabeling. Samples (52) including meat, poultry, and fish purchased from retail and online sources were examined. Regions of cytochrome C oxidase I (COI) gene (~650 bp) and 16S rRNA (~220 bp) were used as full- and mini-barcode markers, respectively. Approximately 94% (49 of 52) of the samples generated barcode sequences. The failure rate for full COI full-barcodes was 44%, but we obtained the 16S rRNA mini-barcode from 87% of the COI-failed cases. Overall, the survey revealed that 23% (12 of 52) of animal-derived products were mislabeled and, in most cases, contain undeclared species. Thus, regulatory measures and continuous monitoring for mislabeling of animal-derived products should be conducted.

Authentication of Edible Bird's Nest (EBN) and its adulterants by integration of shotgun proteomics and scheduled multiple reaction monitoring (MRM) based on tandem mass spectrometry.

Edible bird's nest (EBN) has been traditionally regarded as a kind of medicinal and healthy food in Asia. However, economically motivated adulteration (EMA) has been an issue in the EBN supply chain. To develop an accurate high-throughput approach for detecting EBN and its adulterants (exemplified by porcine skin, swim bladder, white fungus, and egg white), shotgun proteomics was applied for discovery of specific peptides that were subsequently converted into scheduled multiple reaction monitoring (MRM) transitions. Totally, 28 specific peptides were verified as unique to EBN and its adulterants by tandem mass spectrometry. Subsequently, 9 quantitative MRM-transitions of peptides from adulterants and 2 internal standard references from EBN were screened for the quantitative analysis of the adulterants, which allowed detection of adulterants in EBN matrix in the range of 1-80%. These results suggested that integration of shotgun proteomics and scheduled MRM had potential for the authentication of EBN and its adulterants.

[Effect of High Hydrostatic Pressure Combined with Temperature on the Antigenicity and Conformation of Tropomyosin Purified from Shrimp].

Foodborne shrimp allergy events have occurred in recent years. To illustrate the mechanism of high hydrostatic pressure technology to change the allergenicity of shrimp, the major allergen tropomyosin was separated and purified from Litopenaeus vannamei, and indentified with matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The effect of temperature factor under high hydrostatic pressure was measured with indirect ELISA method, CD and fluorescence spectrum. The results showed that the antigenicity of TM protein had an increase after being heated at 35 or 45 ℃ when treated at 300 MPa for 15 minutes, while the antigenicity decreased at 55, 65, and 75 ℃. With the increase of heat temperature, the secondary structure of TM also changed. The mutual transformation happened between the alpha-helix and beta-sheet, beta-turn, and the random coil. The tertiary structure of TM was observed dynamic changes from the extended state to the folded state, and then re-extended state to re-folded state. These results suggested that high hydrostatic pressure combined with temperature could influence the antigenicity of TM by the change of conformation which would be useful as theoretical guidance on developing new methods or technologies for producing hypoallergenic shrimp products.

A Real-Time PCR Method Targeting Camel Ingredient for Food Authentication.

The special nutritious value of camel showed high potential for market exploitation. In this paper, a real-time PCR method targeting camel ingredient in camel meat and milk is reported as an approach to fight against adulteration. To understand the impact of processing procedures on the amplifiability of cytb gene, four kinds of processed camel meat were investigated, and the rate of DNA breakage was explored. The method was able to detect 5 fg/µL camel DNA and highly processed food containing 0.01% camel meat with a high confidence level.

Species-specific identification of seven vegetable oils based on suspension bead array.

Species adulteration of vegetable oils has become a main form of adulteration in vegetable oils, severely violating consumer rights and causing disorder in the market. A reliable method of species authentication of vegetable oils is desirable. This paper reports a novel method for identification of seven species of vegetable oils based on suspension bead array. One pair of universal primers and seven species-specific probes were designed targeting rbcl gene of the chloroplast. Each probe was coupled to a unique color-coded microsphere. Biotinylated PCR amplicons of seven oils were hybridized to the complementary probes on microsphere sets. Bound amplicons were detected fluorometrically using a reporter dye, streptavidin-R-phycoeryt hrin (SA-PE). A sample could be analyzed less than 1 h after PCR amplification. With the exception of olive probe, all probes showed no cross-reactivity with other species. Absolute detection limit of the seven probes ranged from 0.01 ng/µL to 0.0001 ng/µL. Detection limit in DNA mixture was from 10% to 5%. Detection of vegetable oils validated the effectiveness of the method. The suspension bead array as a rapid, sensitive, and high-throughput technology has potential to identify more species of vegetable oils with increased species of probes.

Targeting γ-secretase in breast cancer.

γ-secretase complexes are multisubunit protease complexes that perform the intramembrane cleavage of more than 60 type-I transmembrane proteins, including Notch receptors. Since dysregulated Notch signaling has been implicated in the tumorigenesis and progression of breast cancer, small molecule γ-secretase inhibitors (GSIs) are being tested for their therapeutic potential in breast cancer treatment in several clinical trials. Here, the structure of γ-secretase complex and the development of GSIs are briefly reviewed, the roles of Notch and several other γ-secretase substrates in breast cancer are discussed, and the difference between γ-secretase inhibition and Notch inhibition, as well as the side effects associated with GSIs, are described. A better understanding of molecular mechanisms that affect the responsiveness of breast cancer to GSI might help to develop strategies to enhance the antitumor activity and, at the same time, alleviate the side effects of GSI.

Notch signaling as a therapeutic target for breast cancer treatment?

Aberrant Notch signaling can induce mammary gland carcinoma in transgenic mice, and high expressions of Notch receptors and ligands have been linked to poor clinical outcomes in human patients with breast cancer. This suggests that inhibition of Notch signaling may be beneficial for breast cancer treatment. In this review, we critically evaluate the evidence that supports or challenges the hypothesis that inhibition of Notch signaling would be advantageous in breast cancer management. We find that there are many remaining uncertainties that must be addressed experimentally if we are to exploit inhibition of Notch signaling as a treatment approach in breast cancer. Nonetheless, Notch inhibition, in combination with other therapies, is a promising avenue for future management of breast cancer. Furthermore, since aberrant Notch4 activity can induce mammary gland carcinoma in the absence of RBPjκ, a better understanding of the components of RBPjκ-independent oncogenic Notch signaling pathways and their contribution to Notch-induced tumorigenesis would facilitate the deployment of Notch inhibition strategies for effective treatment of breast cancer.

Simultaneous detection of eight food allergens using optical thin-film biosensor chips.

Food allergies are important food safety issues nowadays. To maintain the safety of people who experience allergic reactions, labeling is required in many countries and efficient and reliable detection methods are necessary. This paper reports a novel method for the rapid identification of food allergens through the use of a silicon-based optical thin-film biosensor chip with which color change results can be perceived by the naked eye without any extra equipment. The whole system can detect eight food allergens including soybean, wheat, peanut, cashew, shrimp, fish, beef, and chicken simultaneously. Sensitive and specific detection of the absolute detection limit of this method was 0.5 pg of cashew DNA, and the practical detection limit of 0.001%. The biosensor chip detection time was about 30 min after PCR amplification. The assay is proposed as a sensitive, specific, high-throughput, and ready-to-use analytical tool to detect the presence or confirm the absence of eight food allergens.

Characterization and comparison of protein complexes initiated by the intracellular domain of individual Notch paralogs.

The Notch signaling pathway is essential for embryonic development, organogenesis, and tissue homeostasis. Aberrant Notch signaling is associated with several types of cancers. The active form of Notch receptor is its intracellular domain (NICD), which is released from the cell membrane by serial proteolytic cleavages following ligand binding. Dose-dependent effects of NICD on cellular phenotypes have been observed under several conditions although the underlying mechanisms have not been well studied. Moreover, there are four mammalian Notch paralogs that have redundant as well as unique functions. The molecular basis for this variability is also not well understood. In this study, we used size exclusion chromatography to examine the overall distribution of NICD among NICD-containing protein complexes under conditions of increasing NICD abundance. We found that the assembly of NICD protein complexes was dose-dependent and that the abundance of the canonical complex was limited by, MAML, one of the proteins involved in the formation of canonical NICD transactivation complex, which became saturated with increasing NICD abundance. In addition, N4ICD showed a unique elution profile among the four NICDs. These results help to explain the dose-dependent and paralog-specific activities of NICD. These results are informative for the development of new reagents to block Notch signaling for therapeutic benefit.

The cytotoxicity of gamma-secretase inhibitor I to breast cancer cells is mediated by proteasome inhibition, not by gamma-secretase inhibition.

INTRODUCTION: Notch is a family of transmembrane protein receptors whose activation requires proteolytic cleavage by gamma-secretase. Since aberrant Notch signaling can induce mammary carcinomas in transgenic mice and high expression levels of Notch receptors and ligands correlates with overall poor clinical outcomes, inhibiting gamma-secretase with small molecules may be a promising approach for breast cancer treatment. Consistent with this hypothesis, two recent papers reported that gamma-secretase inhibitor I (GSI I), Z-LLNle-CHO, is toxic to breast cancer cells both in vitro and in vivo. In this study, we compared the activity and cytotoxicity of Z-LLNle-CHO to that of two highly specific GSIs, DAPT and L-685,458 and three structurally unrelated proteasome inhibitors, MG132, lactacystin, and bortezomib in order to study the mechanism underlying the cytotoxicity of Z-LLNle-CHO in breast cancer cells. METHODS: Three estrogen receptor (ER) positive cell lines, MCF-7, BT474, and T47D, and three ER negative cell lines, SKBR3, MDA-MB-231, and MDA-MB-468, were used in this study. Both SKBR3 and BT474 cells also overexpress HER2/neu. Cytotoxicity was measured by using an MTS cell viability/proliferation assay. Inhibition of gamma-secretase activity was measured by both immunoblotting and immunofluorescent microscopy in order to detect active Notch1 intracellular domain. Proteasome inhibition was determined by using a cell-based proteasome activity assay kit, by immunoblotting to detect accumulation of polyubiquitylated protein, and by immunofluorescent microscopy to detect redistribution of cellular ubiquitin. RESULTS: We found that blocking gamma-secretase activity by DAPT and L-685,458 had no effect on the survival and proliferation of a panel of six breast cancer cell lines while Z-LLNle-CHO could cause cell death even at concentrations that inhibited gamma-secretase activity less efficiently. Furthermore, we observed that Z-LLNle-CHO could inhibit proteasome activity and the relative cellular sensitivity of these six breast cancer cell lines to Z-LLNle-CHO was the same as observed for three proteasome inhibitors. Finally, we found that the cell killing effect of Z-LLNle-CHO could be reversed by a chemical that restored the proteasome activity. CONCLUSIONS: We conclude that the cytotoxicity of Z-LLNle-CHO in breast cancer cells is mediated by proteasome inhibition, not by gamma-secretase inhibition.

Quantitative analysis reveals asynchronous and more than DSB-associated histone H2AX phosphorylation after exposure to ionizing radiation.

Rapid phosphorylation of histone H2AX after exposure of cells to ionizing radiation occurs at DSB sites and extends to a region including as much as 30 Mbp of chromatin to form visible microscopic structures called gamma-H2AX foci. Although the kinetics of total cellular histone H2AX phosphorylation after irradiation has been characterized, we still know little about the phosphorylation kinetics of individual gamma-H2AX foci. In addition, there are hundreds of smaller gamma-H2AX foci that are not associated with DNA double-strand breaks. We refer to these sites as DSB-unrelated gamma-H2AX foci. By using indirect immunofluorescence microscopy, deconvolution and three-dimensional image analysis, we established an objective method to quantitatively analyze each gamma-H2AX focus as well as to discriminate DSB-related gamma-H2AX foci from DSB-unrelated gamma-H2AX foci. Using this method, we found that histone H2AX phosphorylation at different DSB sites was asynchronous after exposure to ionizing radiation. This may reflect the heterogeneous characteristic of free DNA ends that are generated under these conditions. In addition, we found that increased histone H2AX phosphorylation also occurred outside of DSB sites after exposure to ionizing radiation. The function of this DSB-unassociated phosphorylation is not known.