ANOX: A robust computational model for predicting the antioxidant proteins based on multiple features.

As an indispensable component of various living organisms, the antioxidant proteins have been studied for anti-aging and prevention of various diseases, such as altitude sickness, coronary heart disease, and even cancer. However, the traditional experimental methods for identifying the antioxidant proteins are very expensive and time-consuming. Thus, to address the challenge, a new predictor, named ANOX, was developed in this study. Multiple features, such as frequency matrix features (FRE), amino acid and dipeptide composition (AADP), evolutionary difference formula features (EEDP), k-separated bigrams (KSB), and PSI-PRED secondary structure (PRED), were extracted to generate the original feature space. To find the optimized feature subset, the Max-Relevance-Max-Distance (MRMD) algorithm was implemented for feature ranking and our model received the best performance with the top 1170 features. Rigorous tests were performed to evaluate the performance of ANOX, and the results showed that ANOX achieved a major improvement in the prediction accuracy of the antioxidant proteins (AUC:0.930 and 0.935 using 5-fold cross-validation or the jackknife test) compared to the state-of-the-art predictor AOPs-SVM (AUC:0.869 and 0.885). The dataset used in this study and the source code of ANOX are all available at https://github.com/NWAFU-LiuLab/ANOX.

m7GPredictor: An improved machine learning-based model for predicting internal m7G modifications using sequence properties.

As one of the most important post-transcriptional modifications, the N7-methylguanosine (m7G) plays a key role in many RNA processing events. The accurate identification of m7G is crucial for elucidating its biological significance and future application in the medical field. In this study, a machine learning-based model was developed for the prediction of internal m7G sites, and five different feature extraction methods (Pseudo dinucleotide composition, Pseudo k-tuple composition, K monomeric units, Ksnpf frequency, and Nucleotide chemical property) were used in the feature extraction. The Random Forest algorithm was used to find the optimized feature subset and the SVM-based predictor achieved the best performance by taking the top 240 features for model training. With different performance assessment methods, 10-fold cross validation, Jackknife test, and independent test, m7GPredictor achieved competitive performance compared with the state-of-the-art predictor iRNA-m7G. The predictor developed in this study can offer useful information for elucidating the mechanism of internal m7G sites and related experimental validations. The dataset used in this study and the source code of m7Gpredictor is all available at https://github.com/NWAFU-LiuLab/m7Gpredictor.

Identification and profiling of microRNAs responsive to cadmium toxicity in hepatopancreas of the freshwater crab Sinopotamon henanense.

BACKGROUND: Cadmium (Cd) is a ubiquitous environmental toxicant for aquatic animals. The freshwater crab, Sinopotamon henanense (S. henanense), is a useful model for monitoring Cd exposure since it is widely distributed in sediments whereby it tends to accumulate several toxicants, including Cd. In the recent years, the toxic effects of Cd in the hepatopancreas of S. henanense have been demonstrated by a series of biochemical analysis and ultrastructural observations as well as the deep sequencing approaches and gene expression profile analysis. However, the post-transcriptional regulatory network underlying Cd toxicity in S.henanense is still largely unknown. RESULTS: The miRNA transcriptional profile of the hepatopancreas of S. henanense was used to investigate the expression levels of miRNAs in response to Cd toxicity. In total, 464 known miRNAs and 191 novel miRNAs were identified. Among these 656 miRNAs, 126 known miRNAs could be matched with the miRNAs of Portunus trituberculatus, Eriocheir sinensis and Scylla paramamosain. Furthermore, a total of 24 conserved miRNAs were detected in these four crab species. Fifty-one differentially expressed miRNAs were identified in the Cd-exposed group, with 31 up-regulated and 20 down-regulated. Eight of the differentially expressed miRNAs were randomly selected and verified by the quantitative real-time PCR (qRT-PCR), and there was a general consistency (87.25%) between the qRT-PCR and miRNA transcriptome data. A total of 5258 target genes were screened by bioinformatics prediction. GO term analysis showed that, 17 GO terms were significantly enriched, which were mainly related to the regulation of oxidoreductase activity. KEGG pathway analysis showed that 18 pathways were significantly enriched, which were mainly associated with the biosynthesis, modification and degradation of proteins. CONCLUSION: In response to Cd toxicity, in the hepatopancreas of S. henanense, the expressions of significant amount of miRNAs were altered, which may be an adaptation to resist the oxidative stress induced by Cd. These results provide a basis for further studies of miRNA-mediated functional adaptation of the animal to combat Cd toxicity.

Effects of polysialic acid on sensory innervation of the cornea.

Sensory trigeminal growth cones innervate the cornea in a coordinated fashion during embryonic development. Polysialic acid (polySia) is known for its important roles during nerve development and regeneration. The purpose of this work is to determine whether polySia, present in developing eyefronts and on the surface of sensory nerves, may provide guidance cues to nerves during corneal innervation. Expression and localization of polySia in embryonic day (E)5-14 chick eyefronts and E9 trigeminal ganglia were identified using Western blotting and immunostaining. Effects of polySia removal on trigeminal nerve growth behavior were determined in vivo, using exogenous endoneuraminidase (endoN) treatments to remove polySia substrates during chick cornea development, and in vitro, using neuronal explant cultures. PolySia substrates, made by the physical adsorption of colominic acid to a surface coated with poly-d-lysine (PDL), were used as a model to investigate functions of the polySia expressed in axonal environments. PolySia was localized within developing eyefronts and on trigeminal sensory nerves. Distributions of PolySia in corneas and pericorneal regions are developmentally regulated. PolySia removal caused defasciculation of the limbal nerve trunk in vivo from E7 to E10. Removal of polySia on trigeminal neurites inhibited neurite outgrowth and caused axon defasciculation, but did not affect Neural Cell Adhesion Molecule (NCAM) expression or Schwann cell migration in vitro. PolySia substrates in vitro inhibited outgrowth of trigeminal neurites and promoted their fasciculation. In conclusion, polySia is localized on corneal nerves and in their targeting environment during early developing stages of chick embryos. PolySias promote fasciculation of trigeminal axons in vivo and in vitro, whereas, in contrast, their removal promotes defasciculation.

Substrate Stiffness Regulates Cholesterol Efflux in Smooth Muscle Cells.

The infiltration and deposition of cholesterol in the arterial wall play an important role in the initiation and development of atherosclerosis. Smooth muscle cells (SMCs) are the major cell type in the intima. Upon exposure to cholesterol, SMCs may undergo a phenotype switching into foam cells. Meanwhile, the pathological processes of the blood vessel such as cholesterol deposition and calcification induce the changes in the substrate stiffness around SMCs. However, whether substrate stiffness affects the cholesterol accumulation in SMCs and the formation of foam cells is not well-understood. In this study, SMCs were cultured on the substrates with different stiffnesses ranging from 1 to 100 kPa and treated with cholesterol. We found that cholesterol accumulation in SMCs was higher on 1 and 100 kPa substrates than that on intermediate stiffness at 40 kPa; consistently, total cholesterol (TC) content on 1 and 100 kPa substrates was also higher. As a result, the accumulation of cholesterol increased the expression of macrophage marker CD68 and downregulated SMC contractile marker smooth muscle α-actin (ACTA2). Furthermore, the mRNA and protein expression level of cholesterol efflux gene ATP-binding cassette transporter A1 (ABCA1) was much higher on 40 kPa substrate. With the treatment of a liver X receptor (LXR) agonist GW3965, the expression of ABCA1 increased and cholesterol loading decreased, showing an additive effect with substrate stiffness. In contrast, inhibition of LXR decreased ABCA1 gene expression and increased cholesterol accumulation in SMCs. Consistently, when ABCA1 gene was knockdown, the cholesterol accumulation was increased in SMCs on all substrates with different stiffness. These results revealed that substrate stiffness played an important role on SMCs cholesterol accumulation by regulating the ABCA1 expression. Our findings on the effects of substrate stiffness on cholesterol efflux unravel a new mechanism of biophysical regulation of cholesterol metabolism and SMC phenotype, and provide a rational basis for the development of novel therapies.

Analyte transport past a nanofluidic intermediate electrode junction in a microfluidic device.

A glass microfluidic device is presented in which a microchannel is split into two regions with different electric fields by a nanochannel intermediate electrode junction formed by dielectric breakdown. The objective is to sink current through the nanochannel junction without sample loss or broadening of the band as it passes the junction. This type of performance is desired in many microfluidic applications, including the coupling of microchannel/CE with ESI-MS, electrochemical detection, and electric field gradient focusing. The voltage offsets in this study are suitable for microchannel/CE-ESI-MS. Imaging of the transport of model anions and cations through the junction indicates that the junction exhibits nanofluidic behavior and the mean depth of the nanochannel is estimated to be approximately 105 nm. The ion permselectivity of the nanochannel induces concentration polarization and enriched and depleted concentration polarization zones form on opposite sides of the nanochannel, altering the current and electric field distributions along the main microchannel. Anion transport efficiency past the junction was high, 96.0%, and varied little over the pH range of 4.0-8.0. In contrast, cation transport is much lower, and decreases from 72 to 11% from pH 4.0 to 8.0. Band broadening increases with increasing pH less than 70% over the pH range of 4.0-8.0. It is anticipated that this characterization will aid in the understanding and optimization of such junctions made from permselective membranes and porous glass.

Online coupling of reverse-phase and hydrophilic interaction liquid chromatography for protein and glycoprotein characterization.

We have developed a novel system for coupling reverse-phase (RP) and hydrophilic interaction liquid chromatography (HILIC) online in a micro-flow scheme. In this approach, the inherent solvent incompatibility between RP and HILIC is overcome through the use of constant-pressure online solvent mixing, which allows our system to perform efficient separations of both hydrophilic and hydrophobic compounds for mass spectrometry-based proteomics applications. When analyzing the tryptic digests of bovine serum albumin, ribonuclease B, and horseradish peroxidase, we observed near-identical coverage of peptides and glycopeptides when using online RP-HILIC--with only a single sample injection event--as we did from two separate RP and HILIC analyses. The coupled system was also capable of concurrently characterizing the peptide and glycan portions of deglycosylated glycoproteins from one injection event, as confirmed, for example, through our detection of 23 novel glycans from turkey ovalbumin. Finally, we validated the applicability of using RP-HILIC for the analysis of highly complex biological samples (mouse chondrocyte lysate, deglycosylated human serum). The enhanced coverage and efficiency of online RP-HILIC makes it a viable technique for the comprehensive separation of components displaying dramatically different hydrophobicities, such as peptides, glycopeptides, and glycans.

Endothelial Cell Morphology Regulates Inflammatory Cells Through MicroRNA Transferred by Extracellular Vesicles.

Vascular inflammation plays an important role in the pathogenesis and the development of cardiovascular diseases such as arteriosclerosis and restenosis, and the dysfunction of endothelial cells (ECs) may result in the activation of monocytes and other inflammatory cells. ECs exhibit an elongated morphology in the straight part of arteries but a cobblestone shape near the pro-atherogenic region such as branch bifurcation. Although the effects of hemodynamic forces on ECs have been widely studied, it is not clear whether the EC morphology affects its own function and thus the inflammatory response of monocytes. Here we showed that elongated ECs cultured on poly-(dimethyl siloxane) membrane surface with microgrooves significantly suppressed the activation of the monocytes in co-culture, in comparison to ECs with a cobblestone shape. The transfer of EC-conditioned medium to monocytes had the same effect, suggesting that soluble factors were involved in EC-monocyte communication. Further investigation demonstrated that elongated ECs upregulated the expression of anti-inflammatory microRNAs, especially miR-10a. Moreover, miR-10a was found in the extracellular vesicles (EVs) released by ECs and transferred to monocytes, and the inhibition of EV secretion from ECs repressed the upregulation of miR-10a. Consistently, the inhibition of miR-10a expression in ECs reduced their anti-inflammatory effect on monocytes. These results reveal that the EC morphology can regulate inflammatory response through EVs, which provides a basis for the design and the optimization of biomaterials for vascular tissue engineering.

Transgenic expression of cytotoxic T-lymphocyte-associated antigen 4-immunoglobulin prolongs xenogeneic skin graft survival without extensive immunosuppression in rat burn wounds.

BACKGROUND: We sought to establish a transgenic animal line skin-specifically overexpressing cytotoxic T-lymphocyte-associated antigen 4-immunoglobulin (CTLA4Ig) as a reproducible source of xenogeneic skin grafts with extended survival for wound coverage. We tested this strategy in mice based on a previously established transgenic mouse line that stably and skin-specifically expresses CTLA4Ig for lifetimes and generations. METHODS: CTLA4Ig expression was examined by immunohistochemical assay, and its bio-activity was tested by mixed lymphocyte reaction. The survival of transgenic mouse skin grafted onto rat burn wounds was observed. The impact of transgenic skin grafting on recipient immunity was evaluated by inspecting the survival of the wild-type skin grafted along with transgenic skin onto a separate wound on the same rat. The circulatory CTLA4Ig protein in recipient was detected by sandwich enzyme-linked immunosorbent assay, and its impact on recipient lymphocyte response against donor antigen was tested by mixed lymphocyte reaction. RESULTS: The transgenic CTLA4Ig protein suppressed lymphocyte proliferation in vitro, and the transgenic skin graft survival was remarkably prolonged compared with the wild-type skin derived from the same mouse strain. The survival of the wild-type skin grafted along with transgenic skin exhibited no significant difference from that grafted alone. Circulatory CTLA4Ig protein was detected in recipients, however, no significantly reduced recipient lymphocyte response against donor antigen was observed. CONCLUSION: transgenic expression of CTLA4Ig may be a potential and safe method to prolong xenogenic skin graft survival in burn wounds, and transgenic animal lines can be established as a reproducible source of skin grafts with extended survival for wound coverage.

Resistance of corneal RFUVA­cross-linked collagens and small leucine-rich proteoglycans to degradation by matrix metalloproteinases.

PURPOSE: Extracellular matrix metalloproteinases (MMPs) are thought to play a crucial role in corneal degradation associated with the pathological progression of keratoconus. Currently, corneal cross-linking by riboflavin and ultraviolet A (RFUVA) has received significant attention for treatment of keratoconus. However, the extent to which MMPs digest cross-linked collagen and small leucine-rich proteoglycans (SLRPs) remains unknown. In this study, the resistance of RFUVA-cross-linked collagens and SLRPs to MMPs has been investigated. METHODS: To investigate the ability of MMPs to digest cross-linked collagen and SLRPs, a model reaction system using purified collagen type I, type IV, and nonglycosylated, commercially available recombinant SLRPs, keratocan, lumican, mimecan, decorin, and biglycan in solution in vitro has been compared using reactions inside an intact bovine cornea, ex vivo. RESULTS: Our data demonstrate that corneal cross-linked collagen type I and type IV are resistant to cleavage by MMP-1, MMP-2, MMP-9, and MMP-13, whereas non-cross-linked collagen I, IV, and natively glycosylated SLRPs are susceptible to degradation by MMPs. In addition, both cross-linked SLRPs themselves and cross-linked polymers of SLRPs and collagen appear able to resist degradation. These results suggest that the interactions between SLRPs and collagen caused by RFUVA protect both SLRPs and collagen fibrils from cleavage by MMPs. CONCLUSIONS: A novel approach for understanding the biochemical mechanism whereby RFUVA cross-linking stops keratoconus progression has been achieved.

Expression and localization of neural cell adhesion molecule and polysialic acid during chick corneal development.

PURPOSE: To assay for expression and localization of neural cell adhesion molecule (NCAM) and polysialic acid (polySia) in the chick cornea during embryonic and postnatal development. METHODS: Real time quantitative PCR and Western blot analyses were used to determine NCAM expression and polysiaylation in embryonic, hatchling, and adult chick corneas. Immunofluorescence staining for NCAM and polySia was conducted on cryosections of embryonic and adult corneas, whole embryonic corneas, and trigeminal neurons. RESULTS: NCAM and ST8SiaII mRNA transcripts peaked by embryonic day (E)9, remained steady between E10 and E14 and slowly decreased thereafter during embryonic development. Both gene transcripts showed > 190-fold decline in the adult chick cornea compared with E9. In contrast, ST8SiaIV expression gradually decreased 26.5-fold from E6 to E19, increased thereafter, and rose to the early embryonic level in the adult cornea. Western blot analysis revealed NCAM was polysialylated and its expression developmentally changed. Other polysiaylated proteins aside from NCAM were also detected by Western blot analysis. Five NCAM isoforms including NCAM-120, NCAM-180 and three soluble NCAM isoforms with low molecular weights (87-96 kDa) were present in chick corneas, with NCAM-120 being the predominate isoform. NCAM was localized to the epithelium, stroma, and stromal extracellular matrix (ECM) of the embryonic cornea. In stroma, NCAM expression shifted from anterior to posterior stroma during embryonic development and eventually became undetectable in 20-week-old adult cornea. Additionally, both NCAM and polySia were detected on embryonic corneal and pericorneal nerves. CONCLUSIONS: NCAM and polySia are expressed and developmentally regulated in chick corneas. Both membrane-associated and soluble NCAM isoforms are expressed in chick corneas. The distributions of NCAM and polySia in cornea and on corneal nerves suggest their potential functions in corneal innervation.

The viability change of pigskin in vitro.

BACKGROUND: It is widely recognised that take of grafts is strongly influenced by tissue viability. Although porcine skin is currently the most widely used xenograft, the viability change of pigskin in vitro has not been extensively studied. The purpose of this study was to assess the change of the viability of Bama miniature pigskin after harvest and cryopreservation, and to set up a guideline for pigskin preservation and storage that would allow the skin to retain the highest viability after treatment and still be used in the clinical applications. METHODS: Harvested pigskin grafts were divided into five groups: normal saline medium/4 degrees C (group 1), Dulbecco's minimum essential medium (DMEM)/4 degrees C (group 2), normal saline medium/25 degrees C (group 3), DMEM/25 degrees C (group 4) and cryopreserved (group 5). In our experiment, the viability was investigated by 3-(4,5)-dimethylthiazol-2,5-diphenyl tetrasolium bromide (MTT) salt assay. We also evaluated the transplantation performance of preserved skin in different conditions by using a rat recipient model, in which primary take was evaluated by gross observation and predetermined histological criteria after 7 days. RESULTS: Skin stored at 4 degrees C showed a very slow viability decrease with time. The sample showed a viability decrease of about 70% after 3 days in normal saline and 4 days in DMEM medium. Nevertheless, skin stored in DMEM at 25 degrees C underwent a viability increase during the first 4h and then decreased gradually to about 70% after 20 h, while the viability declined very quickly for skin grafts stored in normal saline medium at 25 degrees C, and maintained the same viability only within 6h of preservation. On the other hand, cryopreserved skin has been shown to maintain a level of skin metabolism equal to 77% of the fresh sample when measured immediately after thawing, and the viability remained about 70% after 6h at 25 degrees C and 2 days at 4 degrees C in DMEM. The graft performance of skin specimens with 70% viability of fresh skin stored in different conditions has not shown statistical significance compared with fresh pigskin. CONCLUSIONS: Based on these results, we suggest that the conservation period of fresh pigskin should not exceed 72 or 96 h when stored in normal saline or DMEM at 4 degrees C, and should not exceed 6 or 18 h when stored in normal saline or DMEM at 25 degrees C. Cryopreserved pigskin should be stored in DMEM for a maximum period of 48 h at 4 degrees C and 6h at 25 degrees C after thawing.

A sheath-flow nanoelectrospray interface of microchip electrophoresis MS for glycoprotein and glycopeptide analysis.

Microchip was coupled with MS through a stable, sensitive, and controllable sheath-flow nanoelectrospray (nES) interface for glycoprotein and glycopeptide analysis. The nano-ESI (nESI) was made with a delivery capillary, a commercial nES capillary, and a stainless steel (SS) tube which were connected together through a tee unit. High voltage for nES was applied on the SS tube and the commercial nES capillary was used as nES emitter. The delivery capillary was attached to the microchannel for delivering liquid from microchip to the nESI source. The flow rate of sheath liquid was optimized to be 100-200 nL/min which largely reduced the sample dilution. The detection limit of peptides on this microchip/MS platform was at femtomole level. Glycoprotein and glycopeptides were also successfully analyzed on the platform. All the glycoforms and glycopeptides of ribonuclease B (RNase B) were identified with this method. Some structures of the glycopeptides from RNase B were further characterized with MS/MS on the microchip, coupled with a quadrupole IT-MS.

Analysis of chicken and turkey ovalbumins by microchip electrophoresis combined with exoglycosidase digestion.

The polypeptide and carbohydrate patterns of two glycoproteins, chicken ovalbumin (CO) and turkey ovalbumin (TO), were analyzed by microchip electrophoresis (ME), following digestion with proteases and exoglycosidases. Glycopeptides derived from ovalbumin were obtained by digestion with Pronase, followed by dialysis, and then separated by ME. Using CO as model, the method was developed to deduce the structure of glycans from glycoproteins by comparing the electropherograms of glycopeptides with and without digestion of exolycosidases. Applying the same approach, the structure of oligosaccharides linked to TO was determined. TO was found to contain high-mannose type oligosaccharides and oligosaccharides with terminal N-acetylglucosamine residues. The complete primary analysis of CO and TO by ME described in this paper provides a basis for an analysis of glycoproteins with an integrated microfluidic chip.

Integrated lectin affinity microfluidic chip for glycoform separation.

Lectin affinity chromatography was miniaturized into a microfluidic format, which results in improvement of performance, as compared to the conventional method. A lectin affinity monolith column was prepared in the microchannel of a microfluidic chip. The porous monolith was fabricated by UV-initiated polymerization of ethylene dimethacrylate (EDMA) and glycidyl methacrylate (GMA) in the presence of porogeneities, followed by immobilization of pisum sativum agglutinin (PSA) on the monolith matrix. Using electroosmosis as the driven force, lectin affinity chromatographies of three kinds of glycoprotein, turkey ovalbumin (TO), chicken ovalbumin (CO), and ovomucoid (OM), were carried out on the microfluidic system. All the glycoproteins were successfully separated into several fractions with different affinities toward the immobilized PSA. The integrated system reduces the time required for the lectin affinity chromatography reaction to approximately 3%, thus, the overall analysis time from 4 h to 400 s. Only 300 pg of glycoprotein is required for the whole separation process. Moreover, troublesome operations for lectin affinity chromatography are simplified.

[Direct UV detection of glucose and its derivatives in capillary zone electrophoresis].

UV labeling detection has been commonly used to determine the association constants between lectins and saccharides, but the interaction is always between the labeled carbohydrates, rather than the truly underivatized carbohydrates, and lectins. In order to directly detect saccharides during the study on the interaction of glucose and its derivatives with lectins (e.g., concanavalin A), a capillary zone electrophoretic method with detection at a wavelength of 195 nm has been developed. The influences of various separation conditions including buffer concentration, pH and voltage were investigated. By using an uncoated silica capillary (50 microns i.d., 375 microns o.d., 48.5 cm of total length, and 44.0 cm to the detector) and 50 mmol/L Na2HPO(4)-50 mmol/L NaH2PO4 solution (near to the physiological pH of 7.4) as buffer, the underivatized sugars, including glucosamine, N-acetylglucosamine, glucose, and sodium gluconate, were sufficiently separated within 11 min at an applied voltage of 10 kV. On-column UV monitoring allowed the detection of these compounds at less than 4 mmol/L level, and quantification by the peak area method allowed reproducible determination of them at least at their respective concentration ranges. The method is characterized by its simplicity, rapidity, and reproducibility, and should be useful for the analysis of the interaction of glucose and its derivatives with lectins.