Genetic and Proteomic Evidence for Roles of Drosophila SUMO in Cell Cycle Control, Ras Signaling, and Early Pattern Formation

SUMO is a protein modifier that is vital for multicellular development. Here we present the first system-wide analysis, combining multiple approaches, to correlate the sumoylated proteome (SUMO-ome) in a multicellular organism with the developmental roles of SUMO. Using mass-spectrometry-based protein identification, we found over 140 largely novel SUMO conjugates in the early Drosophila embryo. Enriched functional groups include proteins involved in Ras signaling, cell cycle, and pattern formation. In support of the functional significance of these findings, sumo germline clone embryos exhibited phenotypes indicative of defects in these same three processes. Our cell culture and immunolocalization studies further substantiate roles for SUMO in Ras signaling and cell cycle regulation. For example, we found that SUMO is required for efficient Ras-mediated MAP kinase activation upstream or at the level of Ras activation. We further found that SUMO is dynamically localized during mitosis to the condensed chromosomes, and later also to the midbody. Polo kinase, a SUMO substrate found in our screen, partially colocalizes with SUMO at both sites. These studies show that SUMO coordinates multiple regulatory processes during oogenesis and early embryogenesis. In addition, our database of sumoylated proteins provides a valuable resource for those studying the roles of SUMO in development

Styrene-assisted maleic anhydride grafted poly(lactic acid) as an effective compatibilizer for wood flour/poly(lactic acid) bio-composites

This study aimed to evaluate the effect of styrene-assisted maleic anhydride-grafted poly(lactic acid) (PLA-g-St/MAH) on the interfacial properties of wood flour/poly(lactic acid) (PLA) bio-composites. PLA-g-St/MAH was synthesized by free-radical melt grafting using styrene as a comonomer and dicumyl peroxide as an initiator. The structure of PLA-g-St/MAH was characterized by Fourier transform infrared spectroscopy. Wood flour/PLA composites were prepared by compression molding using PLA-g-St/MAH as a compatibilizer. The effects of PLA-g-St/MAH on the rheological and mechanical properties, as well as on the fractured surface morphology of the composites were investigated. Results indicated that storage modulus, complex viscosity, equilibrium torque, and shear heat were significantly increased. The mechanical properties of the wood flour/PLA composites were also significantly increased after the addition of PLA-g-St/MAH. The maximum values were achieved at the loading rate of 3 wt % because of the improved interfacial adhesion between the wood flour and the PLA matrix

MiR-494-3p mediates oxaliplatin resistance of colorectal cancer cells via PTEN/AKT pathway

Purpose: To unravel the influence of miR-494-3p on the insensitivity of colorectal cancer (CRC) cells to oxaliplatin.Methods: The mRNA level of miR-494-3p in oxaliplatin-resistant HT-29 cells was evaluated with reverse transcript-polymerase chain reaction (RT-PCR). The cells were treated with miR-494-3p suppressor or mimic, and then apoptotic changes were determined flow cytometrically. Resistancerelated gene expressions were measured using RT-PCR and western blotting. In addition, in vivo mouse experiments were conducted.Results: MiR-494-3p expression in oxaliplatin-resistant HT-29 cells was much higher than that in parental HT-29 cells, accompanied by increased levels of MRP, P-gp, and AKT phosphorylation (p-AKT), and decreased phosphatase and tensin homolog (PTEN) (p < 0.001). The miR-494-3p mimic suppressed oxaliplatin-induced parental HT-29 cell apoptosis, while miR-494-3p inhibitor promoted oxaliplatin-resistant HT-29 cell apoptosis and decreased the levels of p-AKT, MRP and P-gp, while upregulating PTEN (p < 0.001). Furthermore, AKT inhibitor had similar effects as miR-494-3p inhibitor (p < 0.001). Experiments using nude mice demonstrated that inhibition of miR-494-3p accentuated the sensitivity of oxaliplatin-resistant HT-29 cells to oxaliplatin (p < 0.05).Conclusion: Suppression of miR-494-3p attenuates oxaliplatin insensitivity to CRC cells via a mechanism which may involve PTEN/AKT pathway. Therefore, miR-494-3p may be an effective target for overcoming drug resistance of CRC

Classification of Diabetic Foot Ulcers Using Class Knowledge Banks

Diabetic foot ulcers (DFUs) are one of the most common complications of diabetes. Identifying the presence of infection and ischemia in DFU is important for ulcer examination and treatment planning. Recently, the computerized classification of infection and ischaemia of DFU based on deep learning methods has shown promising performance. Most state-of-the-art DFU image classification methods employ deep neural networks, especially convolutional neural networks, to extract discriminative features, and predict class probabilities from the extracted features by fully connected neural networks. In the testing, the prediction depends on an individual input image and trained parameters, where knowledge in the training data is not explicitly utilized. To better utilize the knowledge in the training data, we propose class knowledge banks (CKBs) consisting of trainable units that can effectively extract and represent class knowledge. Each unit in a CKB is used to compute similarity with a representation extracted from an input image. The averaged similarity between units in the CKB and the representation can be regarded as the logit of the considered input. In this way, the prediction depends not only on input images and trained parameters in networks but the class knowledge extracted from the training data and stored in the CKBs. Experimental results show that the proposed method can effectively improve the performance of DFU infection and ischaemia classifications

3D-printed polycaprolactone-chitosan based drug delivery implants for personalized administration

Fused deposition molding (FDM) can complete most complex preparation of drug delivery implants to meet the personalized needs of patients. However, the drug activity has strict requirements on processing temperature and preparation method of filaments, the implant also has strict biocompatibility requirements for the materials. In this study, a drug delivery implant was prepared with good biocompatibility, controlled and efficient drug release using FDM printing for personalized administration. Drug-loaded filaments were developed for FDM process by hot-melt extrusion (HME). Polycaprolactone was used as a drug delivery carrier, and ibuprofen as the model drug. Notably, chitosan was dissolved to form controlled and efficient release channels. The printability, changes in physical and chemical properties during HME and FDM processes of the filament, and drug release behavior, mechanism and biocompatibility of the implants were investigated. The results showed that the filament tensile strength decreased with the increase of drug and chitosan content. No obvious degradation and chemical change occurred during the whole process. The drug release efficiency could reach\u3e99% and lasted for 120 h mainly via the diffusion - erosion mechanism. The viability of cells cultured for 24 h in 72 h, 100% implant extract was 75.3%

Pyrophosphate-Dependent ATP Formation from Acetyl Coenzyme A in Syntrophus aciditrophicus, a New Twist on ATP Formation.

UnlabelledSyntrophus aciditrophicus is a model syntrophic bacterium that degrades key intermediates in anaerobic decomposition, such as benzoate, cyclohexane-1-carboxylate, and certain fatty acids, to acetate when grown with hydrogen-/formate-consuming microorganisms. ATP formation coupled to acetate production is the main source for energy conservation by S. aciditrophicus However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome of S. aciditrophicus leaves it unclear as to how ATP is formed, as most fermentative bacteria rely on these two enzymes to synthesize ATP from acetyl coenzyme A (CoA) and phosphate. Here, we combine transcriptomic, proteomic, metabolite, and enzymatic approaches to show that S. aciditrophicus uses AMP-forming, acetyl-CoA synthetase (Acs1) for ATP synthesis from acetyl-CoA. acs1 mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, of S. aciditrophicus grown in pure culture and coculture. Cell extracts of S. aciditrophicus had low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified from S. aciditrophicus and recombinantly produced Acs1 catalyzed ATP and acetate formation from acetyl-CoA, AMP, and pyrophosphate. High pyrophosphate levels and a high AMP-to-ATP ratio (5.9 ± 1.4) in S. aciditrophicus cells support the operation of Acs1 in the acetate-forming direction. Thus, S. aciditrophicus has a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase.ImportanceBacteria use two enzymes, phosphate acetyltransferase and acetate kinase, to make ATP from acetyl-CoA, while acetate-forming archaea use a single enzyme, an ADP-forming, acetyl-CoA synthetase, to synthesize ATP and acetate from acetyl-CoA. Syntrophus aciditrophicus apparently relies on a different approach to conserve energy during acetyl-CoA metabolism, as its genome does not have homologs to the genes for phosphate acetyltransferase and acetate kinase. Here, we show that S. aciditrophicus uses an alternative approach, an AMP-forming, acetyl-CoA synthetase, to make ATP from acetyl-CoA. AMP-forming, acetyl-CoA synthetases were previously thought to function only in the activation of acetate to acetyl-CoA

The Reproducibility of Lists of Differentially Expressed Genes in Microarray Studies

Reproducibility is a fundamental requirement in scientific experiments and clinical contexts. Recent publications raise concerns about the reliability of microarray technology because of the apparent lack of agreement between lists of differentially expressed genes (DEGs). In this study we demonstrate that (1) such discordance may stem from ranking and selecting DEGs solely by statistical significance (P) derived from widely used simple t-tests; (2) when fold change (FC) is used as the ranking criterion, the lists become much more reproducible, especially when fewer genes are selected; and (3) the instability of short DEG lists based on P cutoffs is an expected mathematical consequence of the high variability of the t-values. We recommend the use of FC ranking plus a non-stringent P cutoff as a baseline practice in order to generate more reproducible DEG lists. The FC criterion enhances reproducibility while the P criterion balances sensitivity and specificity

Microstructure-sensitive estimation of small fatigue crack growth in bridge steel welds

A probabilistic finite element model is implemented to estimate microstructurally small fatigue crack growth in bridge steel welds. Simulations are based on a microstructure-sensitive crystal plasticity model to quantify fatigue indicator parameters (FIPs) at the slip system level and a fatigue model that relates FIPs to fatigue lives of individual grains. Microstructures from three weld zones, namely, fusion zone (FZ), heat affected zone (HAZ), and base metal (BM), are constructed based on their microstructural attributes such as grain morphology, size, and orientation. Statistical volume elements (SVEs) are generated and meshed independently for the three welding zones. Each grain within the SVEs is divided into several slip bands parallel to crystallographic planes. During the loading process, cracks nucleate at the slip bands (SBs) with the largest FIP next to the free surface. The crack extension path is assumed to be transgranular along SBs and the number of cycles required to crack the neighbor grain is calculated by the corresponding FIP-based crack growth rate equation. The simulation process is carried out using ABAQUS with a user defined subroutine UMAT for crystal plasticity. After the calibration of the constitutive model and irreversibility parameters, numerical simulations for small crack growth in three zones are presented. The crack length vs. the predicted fatigue resistance shows significant differences in the mean values and variability among the three weld zones

An Improved Protocol for N-Glycosylation Analysis of Gel-Separated Sialylated Glycoproteins by MALDI-TOF/TOF

Different glycoforms of some proteins have been identified as differential spots for certain diseases in 2-DE, indicating disease-related glycosylation changes. It is routine to determine the site-specific glycosylation of nonsialylated N-glycoproteins from a single gel spot, but some obstacles still exist in analyzing sialylated glycoproteins due to the lability and higher detection limit of acid glycans in MALDI-TOF/TOF analysis. Thus, we present an improved protocol here. Tryptic glycopeptides were separated and subjected to MALDI-TOF/TOF analysis, resulting in the identification of site-specific glycosylation of high-intensity glycopeptides. Sequential deglycosylation and desialylation were used to improve the identification of glycosylation sites and desialylated glycans. The site-specific glycosylation of large glycopeptides and low-intensity glycopeptides was deduced based on the masses of glycopeptides, deglycosylated peptides and desialylated glycans. By applying it to 2-DE separated human serum, the difference of N-glycosylation was successfully determined for α1-antitrypsin between different gel spots