ZDHHC5-mediated NLRP3 palmitoylation promotes NLRP3-NEK7 interaction and inflammasome activation.

The nucleotide-binding domain (NBD), leucine-rich repeat (LRR), and pyrin domain (PYD)-containing protein 3 (NLRP3) inflammasome is a critical mediator of the innate immune response. How NLRP3 responds to stimuli and initiates the assembly of the NLRP3 inflammasome is not fully understood. Here, we found that a cellular metabolite, palmitate, facilitates NLRP3 activation by enhancing its S-palmitoylation, in synergy with lipopolysaccharide stimulation. NLRP3 is post-translationally palmitoylated by zinc-finger and aspartate-histidine-histidine-cysteine 5 (ZDHHC5) at the LRR domain, which promotes NLRP3 inflammasome assembly and activation. Silencing ZDHHC5 blocks NLRP3 oligomerization, NLRP3-NEK7 interaction, and formation of large intracellular ASC aggregates, leading to abrogation of caspase-1 activation, IL-1ß/18 release, and GSDMD cleavage, both in human cells and in mice. ABHD17A depalmitoylates NLRP3, and one human-heritable disease-associated mutation in NLRP3 was found to be associated with defective ABHD17A binding and hyper-palmitoylation. Furthermore, Zdhhc5-/- mice showed defective NLRP3 inflammasome activation in vivo. Taken together, our data reveal an endogenous mechanism of inflammasome assembly and activation and suggest NLRP3 palmitoylation as a potential target for the treatment of NLRP3 inflammasome-driven diseases.

Translationally controlled tumor protein interacts with TaCIPK23 to positively regulate wheat resistance to Puccinia triticina.

Hypersensitive response-programmed cell death (HR-PCD) regulated by Ca2+ signal is considered the major regulator of resistance against Puccinia triticina (Pt.) infection in wheat. In this study, the bread wheat variety Thatcher and its near-isogenic line with the leaf rust resistance locus Lr26 were infected with the Pt. race 260 to obtain the compatible and incompatible combinations, respectively. The expression of translationally controlled tumor protein (TaTCTP) was upregulated upon infection with Pt., through a Ca2+-dependent mechanism in the incompatible combination. The knockdown of TaTCTP markedly increased the area of dying cell and the number of Pt. haustorial mother cells (HMCs) at the infection sites, whereas plants overexpressing the gene exhibited enhanced resistance. The interaction between TaTCTP and calcineurin B-like protein-interacting protein kinase 23 (TaCIPK23) was also investigated, and the interaction was found occurred in the endoplasmic reticulum. TaCIPK23 phosphorylated TaTCTP in vitro. The expression of a phospho-mimic TaTCTP mutant in Nicotiana benthamiana promoted HR-like cell death. Silencing TaCIPK23 or TaCIPK23/TaTCTP co-silencing resulted in the same results as silencing TaTCTP. This suggested that TaTCTP is a novel phosphorylation target of TaCIPK23, and both participate in the resistance of wheat to Pt. in the same pathway.

TaCAMTA4 negatively regulates H2O2-dependent wheat leaf rust resistance by activating catalase 1 expression.

Leaf rust, caused by Puccinia triticina Erikss. (Pt), is a serious disease threatening wheat (Triticum aestivum L.) production worldwide. Hydrogen peroxide (H2O2) triggered by Pt infection in resistant wheat cultivars cause oxidative damage directly to biomolecules or is activated by calcium signaling and mediates the hypersensitive response. Calmodulin-binding transcriptional activator 4 (TaCAMTA4) has been reported to negatively regulate wheat resistance to Pt. In this study, we found that TaCAMTA4 was induced by Pt race 165 in its compatible host harboring the Pt resistant locus Lr26, TcLr26, and silencing of TaCAMTA4 increased local H2O2 accumulation and Pt resistance. Subcellular localization and autoactivation tests revealed that TaCAMTA4 is a nucleus-localized transcriptional activator. Furthermore, four DNA motifs recognized by TaCAMTA4 were identified by transcription factor-centered Y1H. Through analyzing the transcriptome database, four gene clusters were identified, each containing a different DNA motif on each promoter. Among them, the expression of catalase 1 (TaCAT1) with motif-1 was highly induced in the compatible interaction and was decreased when TaCAMTA4 was silenced. The results of EMSA, ChIP-qPCR, and RT-qPCR further showed that TaCAMTA4 directly bound motif-1 in the TaCAT1 promoter. Furthermore, silencing of TaCAT1 resulted in enhanced resistance to Pt and increased local H2O2 accumulation in wheat, which is consistent with that of TaCAMTA4. Since CAMTAs are Ca2+ sensors and catalases catalyze the decomposition of H2O2, we hypothesize that Ca2+ regulates the plant immune networks that are controlled by H2O2 and implicate a potential mechanism for Pt to suppress resistance by inducing the expression of the TaCAMTA4-TaCAT1 module, which consequently enhances H2O2 scavenging and attenuates H2O2-dependent resistance.

O-GlcNAcPRED-DL: Prediction of Protein O-GlcNAcylation Sites Based on an Ensemble Model of Deep Learning.

O-linked ß-N-acetylglucosamine (O-GlcNAc) is a post-translational modification (i.e., O-GlcNAcylation) on serine/threonine residues of proteins, regulating a plethora of physiological and pathological events. As a dynamic process, O-GlcNAc functions in a site-specific manner. However, the experimental identification of the O-GlcNAc sites remains challenging in many scenarios. Herein, by leveraging the recent progress in cataloguing experimentally identified O-GlcNAc sites and advanced deep learning approaches, we establish an ensemble model, O-GlcNAcPRED-DL, a deep learning-based tool, for the prediction of O-GlcNAc sites. In brief, to make a benchmark O-GlcNAc data set, we extracted the information on O-GlcNAc from the recently constructed database O-GlcNAcAtlas, which contains thousands of experimentally identified and curated O-GlcNAc sites on proteins from multiple species. To overcome the imbalance between positive and negative data sets, we selected five groups of negative data sets in humans and mice to construct an ensemble predictor based on connection of a convolutional neural network and bidirectional long short-term memory. By taking into account three types of sequence information, we constructed four network frameworks, with the systematically optimized parameters used for the models. The thorough comparison analysis on two independent data sets of humans and mice and six independent data sets from other species demonstrated remarkably increased sensitivity and accuracy of the O-GlcNAcPRED-DL models, outperforming other existing tools. Moreover, a user-friendly Web server for O-GlcNAcPRED-DL has been constructed, which is freely available at http://oglcnac.org/pred_dl.

In-Depth Endogenous Phosphopeptidomics of Serum with Zirconium(IV)-Grafted Mesoporous Silica Enrichment.

Detection of endogenous peptides, especially those with modifications (such as phosphorylation) in biofluids, can serve as an indicator of intracellular pathophysiology. Although great progress has been made in phosphoproteomics in recent years, endogenous phosphopeptidomics has largely lagged behind. One main hurdle in endogenous phosphopeptidomics analysis is the coexistence of proteins and highly abundant nonmodified peptides in complex matrices. In this study, we developed an approach using zirconium(IV)-grafted mesoporous beads to enrich phosphopeptides, followed by analysis with a high resolution nanoRPLC-MS/MS system. The bifunctional material was first tested with digests of standard phosphoproteins and HeLa cell lysates, with excellent enrichment performance achieved. Given the size exclusion nature, the beads were directly applied for endogenous phosphopeptidomic analysis of serum samples from pancreatic ductal adenocarcinoma (PDAC) patients and controls. In total, 329 endogenous phosphopeptides (containing 113 high confidence sites) were identified across samples, by far the largest endogenous phosphopeptide data set cataloged to date. In addition, the method was readily applied for phosphoproteomics of the same set of samples, with 172 phosphopeptides identified and significant changes in dozens of phosphopeptides observed. Given the simplicity and robustness of the proposed method, we envision that it can be readily used for comprehensive phosphorylation studies of serum and other biofluid samples.

Resistance to abemaciclib is associated with increased metastatic potential and lysosomal protein deregulation in breast cancer cells.

Cyclin dependent kinase 4 and 6 inhibitors such as abemaciclib are routinely used to treat metastatic estrogen receptor positive (ER+) breast cancer. However, adaptive mechanisms inhibit their effectiveness and allow for disease progression. Using ER+ breast cancer cell models, we show that acquired resistance to abemaciclib is accompanied by increase in metastatic potential. Mass spectrometry-based proteomics from abemaciclib sensitive and resistant cells showed that lysosomal proteins including CTSD (cathepsin D), cathepsin A and CD68 were significantly increased in resistant cells. Combination of abemaciclib and a lysosomal destabilizer, such as hydroxychloroquine (HCQ) or bafilomycin A1, resensitized resistant cells to abemaciclib. Also, combination of abemaciclib and HCQ decreased migration and invasive potential and increased lysosomal membrane permeability in resistant cells. Prosurvival B cell lymphoma 2 (BCL2) protein levels were elevated in resistant cells, and a triple treatment with abemaciclib, HCQ, and BCL2 inhibitor, venetoclax, significantly inhibited cell growth compared to treatment with abemaciclib and HCQ. Furthermore, resistant cells showed increased levels of Transcription Factor EB (TFEB), a master regulator of lysosomal-autophagy genes, and siRNA mediated knockdown of TFEB decreased invasion in resistant cells. TFEB was found to be mutated in a subset of invasive human breast cancer samples, and overall survival analysis in ER+, lymph node-positive breast cancer showed that increased TFEB expression correlated with decreased survival. Collectively, we show that acquired resistance to abemaciclib leads to increased metastatic potential and increased levels of protumorigenic lysosomal proteins. Therefore, the lysosomal pathway could be a therapeutic target in advanced ER+ breast cancer.

Demystifying the O-GlcNAc Code: A Systems View.

Post-translational modification with O-linked ß-N-acetylglucosamine (O-GlcNAc), a process referred to as O-GlcNAcylation, occurs on a vast variety of proteins. Mounting evidence in the past several decades has clearly demonstrated that O-GlcNAcylation is a unique and ubiquitous modification. Reminiscent of a code, protein O-GlcNAcylation functions as a crucial regulator of nearly all cellular processes studied. The primary aim of this review is to summarize the developments in our understanding of myriad protein substrates modified by O-GlcNAcylation from a systems perspective. Specifically, we provide a comprehensive survey of O-GlcNAcylation in multiple species studied, including eukaryotes (e.g., protists, fungi, plants, Caenorhabditis elegans, Drosophila melanogaster, murine, and human), prokaryotes, and some viruses. We evaluate features (e.g., structural properties and sequence motifs) of O-GlcNAc modification on proteins across species. Given that O-GlcNAcylation functions in a species-, tissue-/cell-, protein-, and site-specific manner, we discuss the functional roles of O-GlcNAcylation on human proteins. We focus particularly on several classes of relatively well-characterized human proteins (including transcription factors, protein kinases, protein phosphatases, and E3 ubiquitin-ligases), with representative O-GlcNAc site-specific functions presented. We hope the systems view of the great endeavor in the past 35 years will help demystify the O-GlcNAc code and lead to more fascinating studies in the years to come.

PEG-modified halloysite as a hydrophilic interaction and cation exchange mixed-mode sorbent for solid-phase extraction of biogenic amines in fish samples.

A novel type of PEG-modified halloysite was prepared and used as a hydrophilic interaction and cation exchange mixed-mode sorbent for solid-phase extraction of biogenic amines in fish samples. The eluates were analyzed by high-performance liquid chromatography-ultraviolet detection after the derivatization with benzoyl chloride. The developed sorbent was characterized by scanning electron microscopy, infrared spectroscopy, X-ray diffraction, zeta potential analyzer, and thermo-gravimetric analysis. After the optimization of various parameters influencing the extraction efficiency, the PEG-modified halloysite-based SPE method was evaluated. The adsorption capacities of putrescine, spermine, phenethylamine, and histamine were as high as 9.3, 8.5, 5.7, and 5.6 mg g-1, respectively. Satisfactory reproducibility of sorbent preparation was obtained with within-batch and batch-to-batch relative standard deviations (RSDs) lower than 3.9% and 8.6%, respectively. The biogenic amine spiking recoveries in fish samples ranged from 84.3 to 105.5% with good RSDs lower than 7.8%. Intra-day and inter-day precision, expressed as RSDs, were better than 8.8%. The limits of detection of histamine, putrescine, phenethylamine, and spermine were 9.4, 1.9, 0.5, and 0.9 µg L-1, respectively. This work provides a new hydrophilic interaction and cation exchange mixed-mode sorbent and is successfully applied to the extraction of trace biogenic amines from fish samples.

C18-modified halloysite as a novel sorbent in matrix solid-phase dispersion for the extraction of bisphenol A and diethylstilbestrol from human placenta.

In the current study, the C18-modified halloysite was fabricated via silylation reaction and subsequently used as sorbent in matrix solid-phase dispersion (MSPD) for the extraction of bisphenol A and diethylstilbestrol from human placenta, followed by high-performance liquid chromatography-tandem mass spectrometry analysis. The as-prepared sorbent was characterized by scanning electron microscopy, energy-dispersive spectrometry, Fourier transform infrared spectroscopy, X-ray diffraction, and thermo-gravimetric analysis. Varied parameters such as methanol concentration in wash solvent, pH and salt concentration in elution solvent, elution volume, and mass ratio of sample to sorbent were optimized. The adsorption capacities of bisphenol A and diethylstilbestrol on the developed C18-modified halloysite were 6.3 and 14.2 mg g-1, respectively, higher than those on the commercial C18 silica gel. Under the optimal condition, the average recoveries of bisphenol A and diethylstilbestrol by MSPD varied from 91.0 to 106.0%, and the relative standard deviations were less than 10.6% for human placenta samples. The limits of detection in the human placenta were 0.2 µg kg-1 for bisphenol A and diethylstilbestrol. The simple C18-modified halloysite-based MSPD method holds great potential for the determination of trace bisphenol A and diethylstilbestrol in the human placenta and other tissues of pregnant women with high sensitivity, accuracy, and reliability.

Coupling suspension trapping-based sample preparation and data-independent acquisition mass spectrometry for sensitive exosomal proteomic analysis.

It has been a challenge to analyze minute amounts of proteomic samples in a facile and robust manner. Herein, we developed a quantitative proteomics workflow by integrating suspension trapping (S-Trap)-based sample preparation and label-free data-independent acquisition (DIA) mass spectrometry and then applied it for the analysis of microgram and even nanogram amounts of exosome samples. S-Trap-based sample preparation outperformed the traditional in-solution digestion-based approach and the commonly used filter-aided sample preparation (FASP)-based approach with regard to the number of proteins and peptides identified. Moreover, S-Trap-based sample preparation coupled with DIA mass spectrometry also showed the highest reproducibility for protein quantification. In addition, this approach allowed for identification and quantification of exosome proteins with low starting amounts (down to 50 ~ 200 ng). Finally, the proposed method was successfully applied to label-free quantification of exosomal proteins extracted from MDA-MB-231 breast cancer cells and MCF-10A non-tumorigenic epithelial breast cells. Prospectively, we envision the integrated S-Trap sample preparation coupled with DIA quantification strategy as a promising alternative for highly efficient and sensitive analysis of trace amounts of proteomic samples (e.g., exosomal samples).

Hydrophilic-interaction-based magnetically assisted matrix solid-phase dispersion extraction of carbadox and olaquindox in feeds.

BACKGROUND: Carbadox and olaquindox have been banned from feeds since 1998 by the EU because of their mutagenic, photoallergic, and carcinogenic effects. Unfortunately, owing to their outstanding effect, they are frequently abused or misused in animal husbandry. There is an urgent need to develop a sensitive and reliable method for monitoring these drugs in animal feeds. RESULTS: This work reported a new method of hydrophilic-interaction-based magnetically assisted matrix solid-phase dispersion (MMSPD) extraction coupled with reversed-phase liquid chromatography-mass spectrometry for simultaneous determination of carbadox and olaquindox in animal feeds. 3-Trimethoxysilylpropyl methacrylate (γ-MAPS)-modified attapulgite (ATP) was crosslinked with γ-MAPS-modified iron(II,III) oxide (Fe3 O4 ), 1-vinyl-3-(butyl-4-sulfonate) imidazolium (VBSIm), acrylamide (AM), and N,N'-methylene-bis(acrylamide) (MBA) to synthesize ATP@Fe3 O4 @poly(VBSIm-AM-MBA) particles. The resultant particles were characterized by scanning electron microscopy, energy dispersive spectrometer, transmission electron microscopy, vibrating sample magnetometer, and Fourier transform infrared spectroscopy. Crosslinking of ATP into the magnetic particles has significantly increased the adsorption capacity of the particles. Under optimum conditions, the limits of detection (S/N = 3) were 0.3 µg kg-1 and 0.9 µg kg-1 for carbadox and olaquindox respectively. The intra-day and inter-day recoveries of the spiked targets in feed samples were in the range 83.5-98.3% with relative standard deviations of 1.0-8.3%. CONCLUSION: With a simplified procedure and a low amount of sample, the proposed hydrophilic-interaction-based MMSPD method is not only useful for the determination of carbadox and olaquindox in feeds but also holds great promise for the analysis of other polar targets in solid or semisolid matrices. © 2021 Society of Chemical Industry.

O-GlcNAcAtlas: A database of experimentally identified O-GlcNAc sites and proteins.

O-linked ß-N-acetylglucosamine (O-GlcNAc) is a post-translational modification (i.e., O-GlcNAcylation) on the serine/threonine residues of proteins. As a unique intracellular monosaccharide modification, protein O-GlcNAcylation plays important roles in almost all biochemical processes examined. Aberrant O-GlcNAcylation underlies the etiologies of a number of chronic diseases. With the tremendous improvement of techniques, thousands of proteins along with their O-GlcNAc sites have been reported. However, until now, there are few databases dedicated to accommodate the rapid accumulation of such information. Thus, O-GlcNAcAtlas is created to integrate all experimentally identified O-GlcNAc sites and proteins. O-GlcNAcAtlas consists of two datasets (Dataset-I and Dataset-II, for unambiguously identified sites and ambiguously identified sites, respectively), representing a total number of 4571 O-GlcNAc modified proteins from all species studied from 1984 to 31 Dec 2019. For each protein, comprehensive information (including species, sample type, gene symbol, modified peptides and/or modification sites, site mapping methods and literature references) is provided. To solve the heterogeneity among the data collected from different sources, the sequence identity of these reported O-GlcNAc peptides are mapped to the UniProtKB protein entries. To our knowledge, O-GlcNAcAtlas is a highly comprehensive and rigorously curated database encapsulating all O-GlcNAc sites and proteins identified in the past 35 years. We expect that O-GlcNAcAtlas will be a useful resource to facilitate O-GlcNAc studies and computational analyses of protein O-GlcNAcylation. The public version of the web interface to the O-GlcNAcAtlas can be found at http://oglcnac.org/.

Polyethyleneimine-functionalized Fe3O4/attapulgite particles for hydrophilic interaction-based magnetic dispersive solid-phase extraction of fluoroquinolones in chicken muscle.

Fluoroquinolone (FQ) residues in foods of animal origin may threaten public health but are challenging to determine because of their low contents and complex matrices. In this study, novel polyethyleneimine-functionalized Fe3O4/attapulgite magnetic particles were prepared by a simple co-mixing method and applied as hydrophilic sorbents for the magnetic dispersive solid-phase extraction (MSPE) of three FQs, i.e., ciprofloxacin, norfloxacin, and enrofloxacin, from chicken muscle samples. The preparation of the magnetic particles was of high reproducibility and the products could be reused many times with high adsorption capacity. The key experimental factors possibly influencing the extraction efficiencies, including sample solution, extraction time, sample loading volume, desorption solution, desorption time, and elution volume were investigated. Under optimum MSPE conditions, the analytes in chicken muscle samples were extracted and then determined by RPLC-MS/MS in MRM mode. Good linearity was obtained for the analytes with correlation coefficients ranged from 0.9975 to 0.9995. The limits of detection were in the range of 0.02-0.08 µg kg-1, and the recoveries of the spiked FQs in chicken muscle samples ranged from 83.9 to 98.7% with relative standard deviations of 1.3-6.8% (n = 3). Compared with the traditional MSPE methods based on hydrophobic mechanism, this hydrophilic interaction-based method significantly simplifies the sample pretreatment procedure and improves repeatability. This method is promising for accurate monitoring of FQs in foods of animal origin.

Novel synthesized attapulgite nanoparticles-based hydrophobic monolithic column for in-tube solid-phase microextraction of thiosildenafil, pseudovardenafil, and norneosildenafil in functional foods.

In this study, a novel method which involved in-tube solid-phase microextraction (SPME) using an attapulgite (ATP) nanoparticles-based hydrophobic monolithic column was successfully developed. It was coupled with high-performance liquid chromatography-ultraviolet detection for the determination of three phosphodiesterase-5 (PDE-5) inhibitors, including thiosildenafil, pseudovardenafil, and norneosildenafil, in functional foods. The monolithic column was prepared by one-step polymerization, using 3-trimethoxysilylpropyl methacrylate-modified ATP nanoparticles and 1-butyl-3-vinylimidazolium bromide (VBIMBr) as the functional monomers, and ethylene glycol dimethacrylate (EDMA) as the cross-linker. The obtained poly(ATP-VBIMBr-EDMA) monolith was characterized by scanning electron microscopy equipped with energy-dispersive analysis of X-ray, Fourier transform infrared spectroscopy, thermogravimetric analysis, and X-ray diffraction. The adsorption capacity, up to 2.00 µg/cm calculated by the Langmuir isotherm model, was about six times that of the poly(VBIMBr-EDMA) monolith. Crucial factors affecting the extraction efficiency, including sample solvent, elution solvent, flow rates of sampling loading and elution, sample loading volume, and elution volume, were investigated in details. Under the optimal in-tube SPME conditions, the proposed method showed good reproducibility with run-to-run, column-to-column, and batch-to-batch relative standard deviations less than 7.2%, and low limits of detection of 0.5-0.9 ng/mL in real samples. Thiosildenafil was detected in four types of functional foods with the contents of 1.30-4.78 µg/g. This newly proposed in-tube SPME method based on poly(ATP-VBIMBr-EDMA) monolith may provide a simple, efficient, and promising alternative to daily monitoring of PDE-5 inhibitors in functional foods.

OGT Protein Interaction Network (OGT-PIN): A Curated Database of Experimentally Identified Interaction Proteins of OGT.

Interactions between proteins are essential to any cellular process and constitute the basis for molecular networks that determine the functional state of a cell. With the technical advances in recent years, an astonishingly high number of protein-protein interactions has been revealed. However, the interactome of O-linked N-acetylglucosamine transferase (OGT), the sole enzyme adding the O-linked ß-N-acetylglucosamine (O-GlcNAc) onto its target proteins, has been largely undefined. To that end, we collated OGT interaction proteins experimentally identified in the past several decades. Rigorous curation of datasets from public repositories and O-GlcNAc-focused publications led to the identification of up to 929 high-stringency OGT interactors from multiple species studied (including Homo sapiens, Mus musculus, Rattus norvegicus, Drosophila melanogaster, Arabidopsis thaliana, and others). Among them, 784 human proteins were found to be interactors of human OGT. Moreover, these proteins spanned a very diverse range of functional classes (e.g., DNA repair, RNA metabolism, translational regulation, and cell cycle), with significant enrichment in regulating transcription and (co)translation. Our dataset demonstrates that OGT is likely a hub protein in cells. A webserver OGT-Protein Interaction Network (OGT-PIN) has also been created, which is freely accessible.

Echo decomposition of full-waveform LiDAR based on a digital implicit model and a particle swarm optimization.

In the waveform decomposition of full-waveform LiDAR, the Gaussian model (GSM) and the generalized Gaussian model (GGSM) are widely used to extract echoes from return waveforms. However, those models have explicit functions that follow specific distribution shapes, so they are suitable only for decomposing echo waveforms with similar shapes. This paper introduces a digital implicit model (DIM) and presents a universal decomposition method for the full-waveform LiDAR. In this method, the decomposition model is considered to be an implicit function, associated with a digital template waveform library, whose optimization is implemented by a modified particle swarm algorithm. The template waveform library is a customized fingerprint for any special full-waveform LiDAR, so the DIM can deal effectively with infinite echoes with arbitrary shapes. A full-waveform LiDAR system with asymmetric echo distribution is designed to compare the decomposition performances among the GSM, GGSM, and DIM. Experimental results show that, when decomposing the return waveform containing a single echo, the normalized sum of squares due to fitting error (SSE) of the DIM can be 60 times lower than the GSM and the GGSM. By comparing the estimation accuracies of the amplitude, the FWHM and the location of the echo component, the DIM has the best decomposition performance and the best ranging accuracy (subcentimeter level) among the three models; when decomposing the return waveform containing three overlapping echoes, the normalized SSE of the DIM can be 28 times lower than the GSM and 12 times lower than the GGSM. By comparing the estimation accuracies of the amplitude, FWHM, and location of echoes components, the DIM has the best decomposition performance and best ranging accuracy (centimeter level) among the three models.

Determination of vitamin A in blood serum based on solid-phase extraction using cetyltrimethyl ammonium bromide-modified attapulgite.

Cetyltrimethyl ammonium bromide-modified attapulgite was prepared and utilized as a novel sorbent in a simple solid-phase extraction method for the determination of vitamin A in blood serum. Several factors affecting extraction efficiency were systematically optimized, including the sampling solvent and its volume, as well as the elution solvent and its volume. Under the optimal solid-phase extraction conditions, the adsorption capacity of vitamin A was as high as 28 mg/g according to the Langmuir isotherm model. Based on the developed solid-phase extraction method, the level of vitamin A in 200 µL blood serum sample could be accurately determined by high-performance liquid chromatography. The recoveries of vitamin A spiked in 10% v/v methanol aqueous solutions were in the range of 86.9-92.8%, with the relative standard deviations not more than 8.1%. The method was applied to the determination of vitamin A in serum samples from 20 pregnant women. Compared with the previously reported solid-phase extraction methods for determination of vitamin A in serum, our developed cetyltrimethyl ammonium bromide-modified attapulgite-based solid-phase extraction method used lower serum volume, omitted extra steps (i.e. evaporation and re-dissolution), and eliminated internal standard. The results were promising for it to be used in routine monitoring during pregnancy.

Expression of heparanase is associated with breed-specific morphological characters of placental folded bilayer between Yorkshire and Meishan pigs.

The pig has a noninvasive epitheliochorial placenta, and trophoblast-endometrial epithelial bilayer development could impact on placental function. This work compared the morphological structures, the cell proliferation status as assessed by Ki67 staining, as well as the location and gene and protein expression of heparanase (HPSE) at the maternal-fetal interface between Yorkshire and Meishan pigs on Days 26, 50, and 95 of gestation. Histomorphometry showed that the widths of placental folds, endometrial stroma, and placental stroma in Meishan pigs were smaller than those in Yorkshire pigs during late gestation, while the complexity and the cell proliferation ability of the folded bilayer were greater in Meishan pigs in this period. The location and expression levels of HPSE mRNA and protein at the maternal-fetal interface were similar between the two breeds during early and midgestation. However, during late gestation, the mRNA and protein levels were higher in Meishan placentae. In addition, the HPSE mRNA was expressed by all the trophoblast cells, and the protein was located both at trophoblast and luminal epithelium cells in Meishan pigs during late gestation, while in Yorkshire pigs, the HPSE mRNA and protein were only identified in trophoblast cells located at the bottom and side of folds. The findings suggest that Meishan pigs may rely more upon the increase in the complexity of the folded bilayer within a reduced placenta to expand the exchange surface area and the HPSE may contribute to the development of the folded bilayer in pigs.

Developmental analysis of a Medicago truncatula smooth leaf margin1 mutant reveals context-dependent effects on compound leaf development.

Compound leaf development requires highly regulated cell proliferation, differentiation, and expansion patterns. We identified loss-of-function alleles at the SMOOTH LEAF MARGIN1 (SLM1) locus in Medicago truncatula, a model legume species with trifoliate adult leaves. SLM1 encodes an auxin efflux carrier protein and is the ortholog of Arabidopsis thaliana PIN-FORMED1 (PIN1). Auxin distribution is impaired in the slm1 mutant, resulting in pleiotropic phenotypes in different organs. The most striking change in slm1 is the increase in the number of terminal leaflets and a simultaneous reduction in the number of lateral leaflets, accompanied by reduced expression of SINGLE LEAFLET1 (SGL1), an ortholog of LEAFY. Characterization of the mutant indicates that distinct developmental domains exist in the formation of terminal and lateral leaflets. In contrast with the pinnate compound leaves in the wild type, the slm1 sgl1 double mutant shows nonpeltately palmate leaves, suggesting that the terminal leaflet primordium in M. truncatula has a unique developmental mechanism. Further investigations on the development of leaf serrations reveal different ontogenies between distal serration and marginal serration formation as well as between serration and leaflet formation. These data suggest that regulation of the elaboration of compound leaves and serrations is context dependent and tightly correlated with the auxin/SLM1 module in M. truncatula.

Integrating HexNAcQuest with Glycoproteomics Data Analysis Software to Distinguish HexNAc Isomers on Peptides.

Recently, HexNAcQuest was developed to help distinguish peptides modified by HexNAc isomers, more specifically O-linked ß-N-acetylglucosamine (O-GlcNAc) and O-linked α-N-acetylgalactosamine (O-GalNAc, Tn antigen). To facilitate its usage (particularly for datasets from glycoproteomics studies), herein we present a detailed protocol. It describes example cases and procedures for which users might need to use HexNAcQuest to distinguish these two modifications.