Quantification Quality Control Emerges as a Crucial Factor to Enhance Single-Cell Proteomics Data Analysis.

Mass spectrometry (MS)-based single-cell proteomics (SCP) provides us the opportunity to unbiasedly explore biological variability within cells without the limitation of antibody availability. This field is rapidly developed with the main focuses on instrument advancement, sample preparation refinement, and signal boosting methods; however, the optimal data processing and analysis are rarely investigated which holds an arduous challenge because of the high proportion of missing values and batch effect. Here, we introduced a quantification quality control to intensify the identification of differentially expressed proteins (DEPs) by considering both within and across SCP data. Combining quantification quality control with isobaric matching between runs (IMBR) and PSM-level normalization, an additional 12% and 19% of proteins and peptides, with more than 90% of proteins/peptides containing valid values, were quantified. Clearly, quantification quality control was able to reduce quantification variations and q-values with the more apparent cell type separations. In addition, we found that PSM-level normalization performed similar to other protein-level normalizations but kept the original data profiles without the additional requirement of data manipulation. In proof of concept of our refined pipeline, six uniquely identified DEPs exhibiting varied fold-changes and playing critical roles for melanoma and monocyte functionalities were selected for validation using immunoblotting. Five out of six validated DEPs showed an identical trend with the SCP dataset, emphasizing the feasibility of combining the IMBR, cell quality control, and PSM-level normalization in SCP analysis, which is beneficial for future SCP studies.

Circ_0000877 accelerates proliferation and immune escape of non-small cell lung cancer cells by regulating microRNA-637/E2F2 axis.

BACKGROUND: Recently, circular RNA (circRNA) has become a vital targeted therapy gene for non-small-cell lung cancer (NSCLC) cells. CircRNA_0000877 (Circ_0000877) has been researched in diffuse large B-cell lymphoma (DLBCL). However, whether circ_0000877 regulated NSCLC cell progression is still poorly investigated. The research attempted to investigate the influence of circ_0000877 in NSCLC. METHODS: Circ_0000877 levels in NSCLC tissues and cell lines were determined applying RT-qPCR. Cell functions were evaluated by CCK-8, EdU, flow cytometry, ELISA, and western blot. Gene interactions were predicted by Cirular RNA interactome database and Target Scan website and certified by dual-luciferase reporter, RIP, and RNA pull-down assays. Finally, mice experimental model was established to explore the effects of circ_0000877 on tumor growth in vivo. RESULTS: The elevated trend of circ_0000877 expression was discovered in NSCLC tissues compared to para-carcinoma tissues. The clinicopathological data uncovered that up-regulated circ_0000877 was linked to tumor size, differentiation, and TNM stages of NSCLC patients. Knockdown of circ_0000877 inhibited the proliferation, triggered apoptosis, and prohibited immune escape in NSCLC cells. It was certified that miR-637 was directly interacted with circ_0000877 and targeted by E2F2. Overexpressed E2F2 strongly overturned the functions of circ_0000877 knockdown in NSCLC cells. Mice experimental data demonstrated that circ_0000877 knockdown suppressed tumor growth in vivo. CONCLUSION: The research demonstrated that circ_0000877 exhibited the promotive effect on NSCLC cells proliferation and immune escape by regulating miR-637/E2F2 axis.

Effects of ceramic thickness, ceramic translucency, and light transmission on light-cured bulk-fill resin composites as luting cement of lithium disilicate based-ceramics.

PURPOSE: To assess the effects of ceramic thickness, ceramic translucency, and light transmission on restorative composites used as luting cement for lithium disilicate-based ceramics. METHODS: Four luting types of cement were tested (n=8); a dual-cured resin cement (Multilink N), a light-cured conventional flowable composite (Tetric N-Flow), and two light-cured bulk-fill flowable composites (Tetric N-Flow Bulk Fill and X-tra base). The 20 s- or 40 s-light (1000 mW/cm2) was transmitted through 1- or 2-mm-thick high- or low-translucency (HT- or LT-) ceramic discs (IPS e.Max press) to reach the 1-mm-thick luting cement. Light transmitted to cement without ceramic served as a control. Vickers hardness number (VHN), flexural strength (FS), fractography, and degree of conversion (DC) were evaluated. One-way and multi-way analysis of variance was conducted to determine the effects of factors on VHN and FS. RESULTS: Ceramic thickness, light transmission time, and cement type significantly affected the VHN of the luting cement (P < .000). Only Multilink N (LT- and HT-1mm) and Tetric N-Flow (HT-1mm) reached 90% VHN of corresponding control by 20 s-light transmissions, but Tetric N-Flow exhibited lowest VHN and approximately 1/3-1/2 VHN of Multilink N (P < 0.05). X-tra base expressed superior physicochemical properties to Tetric N-Flow Bulk Fill (P < 0.05) and reached >90% VHN of control in all conditions with 40 s-light transmissions except for LT-2 mm. DC, FS, and fractography supported these findings. CONCLUSIONS: The light-cured bulk-fill composite served as a luting cement for lithium-disilicate-based ceramics in a product-dependent manner. Light transmission time is crucial to ensure sufficient luting cement polymerization.

Canonical Wnt Signaling Promotes Formation of Somatic Permeability Barrier for Proper Germ Cell Differentiation.

Morphogen-mediated signaling is critical for proper organ development and stem cell function, and well-characterized mechanisms spatiotemporally limit the expression of ligands, receptors, and ligand-binding cell-surface glypicans. Here, we show that in the developing Drosophila ovary, canonical Wnt signaling promotes the formation of somatic escort cells (ECs) and their protrusions, which establish a physical permeability barrier to define morphogen territories for proper germ cell differentiation. The protrusions shield germ cells from Dpp and Wingless morphogens produced by the germline stem cell (GSC) niche and normally only received by GSCs. Genetic disruption of EC protrusions allows GSC progeny to also receive Dpp and Wingless, which subsequently disrupt germ cell differentiation. Our results reveal a role for canonical Wnt signaling in specifying the ovarian somatic cells necessary for germ cell differentiation. Additionally, we demonstrate the morphogen-limiting function of this physical permeability barrier, which may be a common mechanism in other organs across species.

Long-Term Rehabilitation Utilization Pattern Among Stroke Patients Under the National Health Insurance Program.

OBJECTIVE: The aim of this study was to understand the frequency of patients receiving rehabilitation services at various periods after stroke and the possible medical barriers to receiving rehabilitation. DESIGN: A retrospective cohort study was conducted using a nationally representative sample in Taiwan. A total of 14,600 stroke patients between 2005 and 2011 were included. Utilization of physical therapy or occupational therapy at different periods after stroke onset was the outcome variable. Individual and geographic characteristics were investigated to determine their effect on patients' probability of receiving rehabilitation. RESULTS: More severe stroke or more comorbid diseases increased the odds of receiving physical therapy and occupational therapy; older age was associated with decreased odds. Notably, sex and stroke type influenced the odds of rehabilitation only in the early period. Copayment exemption lowered the odds of rehabilitation in the first 6 mos but increased the odds in later periods. Rural and suburban patients had significantly lower odds of receiving physical therapy and occupational therapy, as did patients living in areas with fewer rehabilitation therapists. CONCLUSIONS: Besides personal factors, geographic factors such as urban-rural gaps and number of therapists were significantly associated with the utilization of post-stroke rehabilitation care. Furthermore, the influence of certain factors, such as sex, stroke type, and copayment exemption type, changed over time.

Color-complexity enabled exhaustive color-dots identification and spatial patterns testing in images.

Our computational developments and analyses on experimental images are designed to evaluate the effectiveness of chemical spraying via unmanned aerial vehicle (UAV). Our evaluations are in accord with the two perspectives of color-complexity: color variety within a color system and color distributional geometry on an image. First, by working within RGB and HSV color systems, we develop a new color-identification algorithm relying on highly associative relations among three color-coordinates to lead us to exhaustively identify all targeted color-pixels. A color-dot is then identified as one isolated network of connected color-pixel. All identified color-dots vary in shapes and sizes within each image. Such a pixel-based computing algorithm is shown robustly and efficiently accommodating heterogeneity due to shaded regions and lighting conditions. Secondly, all color-dots with varying sizes are categorized into three categories. Since the number of small color-dot is rather large, we spatially divide the entire image into a 2D lattice of rectangular. As such, each rectangle becomes a collective of color-dots of various sizes and is classified with respect to its color-dots intensity. We progressively construct a series of minimum spanning trees (MST) as multiscale 2D distributional spatial geometries in a decreasing-intensity fashion. We extract the distributions of distances among connected rectangle-nodes in the observed MST and simulated MSTs generated under the spatial uniformness assumption. We devise a new algorithm for testing 2D spatial uniformness based on a Hierarchical clustering tree upon all involving MSTs. This new tree-based p-value evaluation has the capacity to become exact.

Role of Pneumococcal NanC in the Severe Disease of Streptococcus pneumoniae Superinfection with Influenza.

Bacterial superinfection aggravates the disease of influenza. Streptococcus pneumoniae is the most common bacterial pathogen. Synergistic virulence has been demonstrated between influenza neuraminidase and pneumococcal NanA and NanB. NanC, the other pneumococcal neuraminidase infrequently present in clinical isolates, is not well characterized. In this study, we report that superinfection with a NanC-negative pneumococcus strain suppresses anti-influenza immunity and impairs viral clearance with higher TGF-ß activation in mice. Bacterial load in the lungs also increases as the host immunity is suppressed. NanC-positive isogenic mutant reverses wild type S. pneumoniae-mediated immune suppression and facilitates virus clearance. However, it causes more severe disease as the augmented inflammation causes collateral damage. Both virus-mediated damage and immune response-mediated inflammation are important for pathogenesis of severe influenza. Inflammation may be more critical than virus-mediated damage in influenza with bacterial superinfection.

Protective Evaluation of Compounds Extracted from Root of Rhodiola rosea L. against Methylglyoxal-Induced Toxicity in a Neuronal Cell Line.

Rhodiola rosea L. (R. rosea) is one of the most beneficial medicinal plants and it is studied as an adaptogen. This study aims to evaluate the neuroprotective activity of compounds extracted from the root of R. rosea against methylglyoxal (MG)-induced apoptosis in neuro-2A (N2A) cells. The root of R. rosea was extracted with ethanol and partitioned with water, ethyl acetate, and n-butanol fractions to evaluate acetylcholinesterase (AChE) inhibitory activity and neuroprotective activity. The ethyl acetate fraction exhibited the highest values of AChE inhibitory activity (49.2% ± 3%) and cell viability (50.7% ± 4.8%) for neuroprotection. The structure identification of the most potential fraction (ethyl acetate fraction) revealed 15 compounds, consisting of three tannins, five flavonoids, and seven phenolics by infrared spectroscopy, nuclear magnetic resonance, and mass spectroscopy. All compounds were evaluated for their neuroprotective activity. Salidroside had the most potential neuroprotective activity. Gallic acid and methyl gallate had potential cytotoxicity in N2A cells. This study showed that R. rosea might have potential neuroprotective activities.

Effect of aloin on viral neuraminidase and hemagglutinin-specific T cell immunity in acute influenza.

BACKGROUND: Millions of people are infected by the influenza virus worldwide every year. Current selections of anti-influenza agents are limited and their effectiveness and drug resistance are still of concern. PURPOSE: Investigation on in vitro and in vivo effect of aloin from Aloe vera leaves against influenza virus infection. METHODS: In vitro antiviral property of aloin was measured by plaque reduction assay in which MDCK cells were infected with oseltamivir-sensitive A(H1N1)pdm09, oseltamivir-resistant A(H1N1)pdm09, H1N1 or H3N2 influenza A or with influenza B viruses in the presence of aloin. In vivo activity was tested in H1N1 influenza virus infected mice. Aloin-mediated inhibition of influenza neuraminidase activity was tested by MUNANA assay. Aloin treatment-mediated modulation of anti-influenza immunity was tested by the study of hemagglutinin-specific T cells in vivo. RESULTS: Aloin significantly reduced in vitro infection by all the tested strains of influenza viruses, including oseltamivir-resistant A(H1N1)pdm09 influenza viruses, with an average IC50 value 91.83 ± 18.97 µM. In H1N1 influenza virus infected mice, aloin treatment (intraperitoneal, once daily for 5 days) reduced virus load in the lungs and attenuated body weight loss and mortality. Adjuvant aloin treatment also improved the outcome with delayed oseltamivir treatment. Aloin inhibited viral neuraminidase and impeded neuraminidase-mediated TGF-ß activation. Viral neuraminidase mediated immune suppression with TGF-ß was constrained and influenza hemagglutinin-specific T cell immunity was increased. There was more infiltration of hemagglutinin-specific CD4+ and CD8+ T cells in the lungs and their production of effector cytokines IFN-γ and TNF-α was boosted. CONCLUSION: Aloin from Aloe vera leaves is a potent anti-influenza compound that inhibits viral neuraminidase activity, even of the oseltamivir-resistant influenza virus. With suppression of this virus machinery, aloin boosts host immunity with augmented hemagglutinin-specific T cell response to the infection. In addition, in the context of compromised benefit with delayed oseltamivir treatment, adjuvant aloin treatment ameliorates the disease and improves survival. Taken together, aloin has the potential to be further evaluated for clinical applications in human influenza.

Management of Giant Retinal Tear with microincision vitrectomy and metallic retinal tacks fixation-a case report.

BACKGROUND: Giant retinal tear is usually challenging among retinal detachment with recurrent rate up to 45%. Here we presented a case of giant retinal tear being treated by microincision vitrectomy and retinal tacks fixation. CASE PRESENTATION: A 53-year-old male presented to our hospital with blurred vision of his right eye for one week with floaters and obscured sensation over nasal visual field. Ocular examination showed a 120 degree giant tear with large inverted flap and retinal detachment of his right eye. The BCVA was only naming digit. Under the impression of giant retinal tear with retinal detachment, 23-gauge pars plana vitrectomy were performed using Constellation high speed vitrectomy system and Topcon non-contact wide angle viewing system. During surgery, the vitreous was removed and perfluorocarbon liquids (PFCL) was injected to help unfolding the large inverted retinal flap. Three retinal tacks were applied to help fixating the large inverted retinal flap. Then, fluid-gas exchange, endolaser photocoagulation and intraocular silicone oil tamponade were performed as well. Initial reattachment of his right retina was achieved and his best corrected visual acuity improved to 0.3 of his right eye postoperatively. There was no recurrent retinal detachment during follow up period of 19 months. CONCLUSIONS: Primary microincision vitrectomy using wide-angle viewing system with intraoperative perfluorocarbon liquids (PFCL) assistant, retinal tacks fixation and intraocular silicone oil tamponade appears to be safe and feasible for managing giant retinal tear with retinal detachment.

Ultrasound-assisted emulsification microextraction for rapid determination of unmetabolized synthetic polycyclic and nitro-aromatic musks in human urine.

An effective method to rapidly determine the presence of seven unmetabolized synthetic musks in human urine samples is developed. The target musks are five synthetic polycyclic musks (i.e., celestolide (ADBI), phantolide (AHMI), traseolide (ATII), galaxolide (HHCB), tonalide (AHTN)), and two nitro-aromatic musks (i.e., musk xylene (MX) and musk ketone (MK)). The method involved an ultrasound-assisted emulsification microextraction (USAEME) coupled with gas chromatography-mass spectrometry (GC-MS). The factors that affect USAEME efficiency were optimized in detail, and the optimized procedure involved the rapid injection of 50 µL of carbon tetrachloride into 1.0 mL of urine sample (contained 0.1-g of sodium chloride) in a conical bottom glass tube. After 1.0 min ultrasonication and 3 min centrifugation (at 7000 rpm), the sedimented extract 10 µL was directly injected into the GC-MS system. The limits of quantitation (LOQs) varied from 0.1 to 0.5 ng/mL. The precisions for both repeatability and reproducibility were <8%. The trueness varied from 79 to 96% with the RSD ranging from 2 to 8%. The total concentrations of the seven unmetabolized target musks in collected human urine samples were in the range from 0.93 to 3.74 ng/mL. HHCB and AHTN were detected in all the collected samples, and the daily excretion doses were evaluated.

Alkanethiolate-Capped Palladium Nanoparticles for Regio- and Stereoselective Hydrogenation of Allenes.

Colloidal Pd nanoparticles capped with octanethiolate ligands have previously shown an excellent selectivity toward the mono-hydrogenation of both isolated and conjugated dienes to internal alkenes. This paper reports an efficient stereoselective mono-hydrogenation of cumulated dienes (allenes) to either Z or E olefinic isomers, depending on the substitution pattern around C=C bonds. Kinetic studies indicate that the reaction progresses through the hydrogenation of less hindered C=C bonds to produce internal Z olefinic isomers. In the cases of di-substitued olefinic products, this initial hydrogenation step is followed by the subsequent isomerization of Z to E isomers. In contrast, the slow isomerization of Z to E isomers for tri-substituted olefinic products results in the preservation of Z stereochemistry. The high selectivity of Pd nanoparticles averting an additional hydrogenation is steered from the controlled electronic and geometric properties of the Pd surface, which are the result of thiolate-induced partial poisoning and surface crowding, respectively. The high activity of colloidal Pd nanoparticle catalysts allows the reactions to be completed at room temperature and atmospheric pressure.

Alkanethiolate-capped palladium nanoparticles for selective catalytic hydrogenation of dienes and trienes.

Selective hydrogenation of dienes and trienes is an important process in the pharmaceutical and chemical industries. Our group previously reported that the thiosulfate protocol using a sodium S-alkylthiosulfate ligand could generate catalytically active Pd nanoparticles (PdNP) capped with a lower density of alkanethio-late ligands. This homogeneously soluble PdNP catalyst offers several advantages such as little contamination via Pd leaching and easy separation and recycling. In addition, the high activity of PdNP allows the reactions to be completed under mild conditions, at room temperature and atmospheric pressure. Herein, a PdNP catalyst capped with octanethiolate ligands (C8 PdNP) is investigated for the selective hydrogenation of conjugated dienes into monoenes. The strong influence of the thiolate ligands on the chemical and electronic properties of the Pd surface is confirmed by mechanistic studies and highly selective catalysis results. The studies also suggest two major routes for the conjugated diene hydrogenation: the 1,2-addition and 1,4-addition of hydrogen. The selectivity between two mono-hydrogenation products is controlled by the steric interaction of substrates and the thermodynamic stability of products. The catalytic hydrogenation of trienes also results in the almost quantitative formation of mono-hydrogenation products, the isolated dienes, from both ocimene and myrcene.