Ultrathin metal-organic framework nanosheets (Cu-TCPP)-based isothermal nucleic acid amplification for food allergen detection

The rapid and accurate detection of peanuts and soybeans allergen is important to the food safety. In this study, Cu-TCPP nanosheet, a kind of ultra-thin metal-organic framework(MOF)was synthesized and applied in loop-mediated isothermal amplification(named Cu-TCPP@LAMP), which can inhibit the non-specific amplification by absorbing and precise temperature releasing of single primer. As thus, Cu-TCPP@LAMP can achieve high sensitivity and specific amplification of the target gene. As a result, peanut and soybean allergens genes contained in food were successfully detected with a favorable detection sensitivity(5 ng/μL for peanuts and 10 ng/μL for soybeans)and reliable repeatability(The coefficient of variation was 3.38% for peanuts and 3.33% for soybeans). Moreover, the established method was utilized for detection of several commercial products, and had a high consistency with the standard method. Apart from food allergens, this novel assay can be widely used in other areas, such as pathogen detection, tumor nucleic acid detection and so on.

DCK confers sensitivity of DCTD-positive cancer cells to oxidized methylcytidines

Dear Editor,Cytidine analogs,such as decitabine(DAC)and cytarabine(ara-C),have been widely used in the clinical treatment for several cancer types,including myelodysplastic syndrome and acute myeloid leukemia(AML;Appelbaum et al.1999;Saba 2007).However,drug resistance causing treatment failure and disease relapse is an unresolved problem to date.Certain cancer cells rely on the salvage enzymes cytidine deaminase(CDA)and dCMP deaminase(DCTD)to inactivate these cytidine derivative drugs by deamination(Jamieson et al.1987;Ebrahem et al.2012).It is imperative to develop new categories of chemotherapeutic nucleosides to overcome the drug resistance caused by such increased cellular deamination activity.

Response of peptide intensity to concentration in ESI–MS-based proteome

High-throughput quantification with label-free methods has received considerable attention in electrospray ionization(ESI)-mass spectrometry(MS),but the manner by which MS signals respond to peptide concentration remains unclear in proteomics.We developed a new mathematical formula to describe the intrinsic log-log relationship between the MS intensity response and peptide concentration in an analytical ESI process.Experimental results showed that the calibration curve is fairly fit to the log-log formula with a linear dynamic range of approximate four to five orders of magnitude.However,we found that the ionization of analytical peptides can be severely suppressed by coexisting matrix peptides,such that the calibration curve can be poorly leveled off on both ends.Our study suggests that the interferences from coexisting matrix peptides should be reduced in the ESI process to use the log-log calibration curve successfully for the high-throughput quantification.

Facile Synthesis of Boronic Acid-Functionalized Magnetic Mesoporous Silica Nanocomposites for Highly Specific Enrichment of Glycopeptides

In the work, aminophenylboronic acid （APB）-functionalized magnetic mesoporous silica, which holds the attractive features of high magnetic responsivity and large surface area, was developed to enrich glycopeptides. At first, magnetic mesoporous silica nanocomposites were prepared. And then, the nanocomposites were functioned with glycidoxypropyltrimethoxysilane （GLYMO） for boronic acid immobilization. Due to that the boronic acid group on the surface of magnetic mesoporous silica nanocomposites can form tight yet reversible covalent bond with glycopeptides containing cis-1,2-diols groups, the magnetic mesoporous silica nanocomposites were successfully applied to selective enrichment of glycopeptides. APB functionalized magnetic mesoporous silica was also demonstrated to have high selectivity for the glycopeptides in the presence of a 10-fold excess bovine serum albumin （BSA） over horseradish peroxidase （HRP） in the tryptic digest. We also find that magnetic rnesoporous silica has better sensitivity in HRP digest compared with that of commercial aminophenylboronic acid-functionalized magnetic nanoparticles beads. The limit of detection for glycopeptides from glycoprotein HRP is about 0.01 ng/μL.

Continuous Detection of pH-responsive Drug Delivery System in Cells in situ by Confocal Laser Scanning Microscopy

Mesoporous silica nanoparticles （MSN） were coated by pH-responsive polymer chitosan-poly （methacrylic acid） （CS-PMAA）. This nano drug delivery system showed good application prospects and the polymer-coated microspheres were promising site-specific anticancer drug delivery carriers in biomedical field. A continuous detection of pH-responsive drug delivery system in cells in situ, utilizing MSN/CS-PMAA composite microspheres, was pro- posed. Two kinds of different cell lines, tumor cell line （Hela） and normal somatic cells （293T）, were used to inves- tigate the behaviours of the drug loaded system in the cells. Conclusions could be drawn from the fluorescent im- ages obtained by confocal laser scanning microscopy （CLSM）, modified drug-loaded microspheres （MSN/CS-PMAA） were ingested into cells more easily, the uptake of DOX@FITC-MSN/CS-PMAA by HeLa/293T cells were performed at pH 7.4/pH 6.8, DOX was released during the ingestion process, fluorescence intensity de- creased with time because of efflux transport and photo-bleaching. Fluoresence detection by flow cytometry was performed as comparison. The continuous fluorescent observation in situ could be widely used in the pH-responsive releasing process of drug delivery system in the cells.

OGP: A Repository of Experimentally Characterized O-glycoproteins to Facilitate Studies on O-glycosylation

Numerous studies on cancers, biopharmaceuticals, and clinical trials have necessitated comprehensive and precise analysis of protein O-glycosylation. However, the lack of updated and convenient databases deters the storage of and reference to emerging O-glycoprotein data. To resolve this issue, an O-glycoprotein repository named OGP was established in this work.It was constructed with a collection of O-glycoprotein data from different sources. OGP contains 9354 O-glycosylation sites and 11,633 site-specific O-glycans mapping to 2133 O-glycoproteins, and it is the largest O-glycoprotein repository thus far.Based on the recorded O-glycosylation sites, an O-glycosylation site prediction tool was developed. Moreover, an OGP-based website is already available(http://www.oglyp.org/). The website comprises four specially designed and user-friendly modules:statistical analysis, database search, site prediction, and data submission. The first version of OGP repository and the website allow users to obtain various O-glycoprotein-related information, such as protein accession Nos., O-glycosylation sites,O-glycopeptide sequences, site-specific O-glycan structures, experimental methods, and potential O-glycosylation sites.O-glycosylation data mining can be performed efficiently on this website, which will greatly facilitate related studies. In addition, the database is accessible from OGP website(http://www.oglyp.org/download.php).

Enhanced Ionization of Phosphatidylcholines during MALDI Mass Spectrometry Using DCTB as Matrix

Phosphatidylcholines （PCs） are the most abundant phospholipids constituents of the cellular membrane, which exhibit a variety of biological functions. The distinct critical roles in cellular function make their analysis quite de- manding. Although MALDI-MS is one of the most powerful tools for biomolecules identification, it is still limited to phospholipids studies. The ionization of phosphatidylcholines is insufficient, and signals of PCs are frequently confused and suppressed by matrix clusters occurring in the same mass range. As an alternative matrix, T-2-（3-（4-t-butyl-phenyl）-2-methyl-2-propenylidene） malononitrile （DCTB） was introduced to overcome these problems in our study. Specifically, the signal intensity of phosphatidylcholines from soybean was enhanced more than several ten-folds using DCTB during positive ion MALDI-TOFMS. Peak overlaps caused by the wide distri- butions of series fatty acid residues from phospholipids were separated by introducing cesium cation. The occurred mass shift of 131.90 Da between [M＋Cs]＋ and [M＋H]~ （＂M＂ represents the molecular weight of the corre- sponding neutral hosphatidylcholine） was approved to be helpful in the assignments of ambiguous peaks of PCs from soybean. For real sample analysis, employing cesium chloride as an auxiliary reagent successfully facilitated the profiling and characterizing of PCs extracted from mouse lung and egg yolk followed by analysis with MALDI-TOFMS using DCTB as matrix.

Asymmetric synthesis of emericellamide B

Asymmetric total synthesis of emericellamide B（9.4%, 17 longest linear steps） is detailed in this report. In this synthetic route, the highly methylated（2R,3R,4S,6S）-3-hydroxy-2,4,6-trimethyldodecanoic acid（HTMD） unit was effectively prepared through the asymmetric methylation, Wittig and Horner–Wadsworth–Emmons reaction. Moreover, pentafluorophenyl diphenylphophinate（FDPP） proved to be an effective condensation reagent for the macrolactamization between C14 and C18.

GTF2H4 regulates partial EndMT via NF-κB activation through NCOA3 phosphorylation in ischemic diseases

Partial endothelial-to-mesenchymal transition(EndMT)is an intermediate phenotype observed in endothelial cells(ECs)undergoing a transition toward a mesenchymal state to support neovascularization during(patho)physiological angiogenesis.Here,we investigated the occurrence of partial EndMT in ECs under hypoxic/ischemic conditions and identified general transcription factor IIH subunit 4(GTF2H4)as a positive regulator of this process.In addition,we discovered that GTF2H4 collaborates with its target protein excision repair cross-complementation group 3(ERCC3)to co-regulate partial EndMT.Furthermore,by using phosphorylation proteomics and site-directed mutagenesis,we demonstrated that GTF2H4 was involved in the phosphorylation of receptor coactivator 3(NCOA3)at serine 1330,which promoted the interaction between NCOA3 and p65,resulting in the transcriptional activation of NF-κB and the NF-kB/Snail signaling axis during partial EndMT.In vivo experiments confirmed that GTF2H4 significantly promoted partial EndMT and angiogenesis after ischemic injury.Collectively,our findings reveal that targeting GTF2H4 is promising for tissue repair and offers potential opportunities for treating hypoxic/ischemic diseases.