Relationship between immune cells and diabetic nephropathy: a Mendelian randomization study.

OBJECTIVE: Although previous studies have suggested potential correlations between immunocytes and diabetic nephropathy (DN), the causal correlations between them remain unclarified. In this study, we employed Mendelian randomization (MR) to analyze the potential causative correlations between immune 731 cells and DN. METHODS: We used the Genome-Wide Association Studies (GWAS) database to aggregate signatures of immune cells and DN from European and East Asian populations. Single-nucleotide polymorphisms (SNPs) were used as instrumental variables. MR analysis was conducted using Mendelian randomization-Egger (MR-Egger) regression and the random-effects inverse-variance weighted (IVW) method. RESULTS: A total of 3,571 SNPs were included as instrumental variables. The MR-Egger regression model indicated no genetic pleiotropy (P = 0.6284). The results of the IVW method indicated a statistically significant causal relationship between immune cell HLA-DR on CD14-CD16- (P = 0.029), CD45RA-CD28-CD8 + T cell% T cells (P = 0.0278), CD11c on myeloid dendritic cells (P = 0.0352), HLA-DR on CD14+ monocytes (P < 0.001), CD27 on CD24 + CD27 + B cells (P = 0.0334), CD27 on IgD + CD24 + B cells (P = 0.0137), CD4 on CD39 + CD4 + T cells (P = 0.0347), CD28 on CD39 + CD4 + T cells (P = 0.0414), CD39 on CD39 + CD4 + T cells (P = 0.0426), and DN. Additionally, there was no heterogeneity in SNPs related HLA-DR on CD14-CD16-cells and DN(I2 = 32%, Cochran's Q = 2.9476, P = 0.2291). Moreover, leave-one-out analysis showed a causal correlation between HLA-DR on CD14-CD16- cells and DN. CONCLUSION: Higher expression of immune cell HLA-DR on CD14-CD16- cells may indicate a lower risk of DN.

Ileocecal involvement in intestinal Behçet's disease and Crohn's disease: comparison of clinicopathological and immunophenotypic features.

OBJECTIVES: Intestinal Behçet's disease (BD) predominantly affects the ileocecal region and is currently diagnosed based on endoscopic features and clinical manifestations. It is difficult to distinguish between intestinal BD and Crohn's disease (CD) due to similar patient populations, gastrointestinal involvement, extraintestinal manifestations, and long-term recurrent course. In this study we aimed to compare the clinicopathological and immunophenotypic features of intestinal BD to CD. METHODS: The medical and pathological records of 29 cases of intestinal BD and 120 cases of CD diagnosed at Sir Run Run Shaw Hospital were retrospectively analyzed. Immunohistochemistry for CD3, CD20, FOXP3, myeloperoxidase, and quantitative analysis of the infiltrating inflammatory cells was conducted. RESULTS: Intestinal BD with ileocecal ulcer had a higher incidence of abdominal pain and a higher erythrocyte sedimentation rate than CD, while chronic diarrhea was more common in CD. Excessive neutrophils in the mucosal lamina propria, neutrophilic exudate on the ulcer surface, and prominent lymphocytic infiltration in ulcer tissues were statistically more frequent in intestinal BD than in CD. The numbers of FOXP3+ T cells, CD3+ T cells, and CD20+ B cells in biopsy tissue from intestinal BD were significantly higher than CD, but the ratio of FOXP3+ T cells to CD3+ T cells was not statistically different. CONCLUSION: Besides the typical clinical and endoscopic findings, diagnostic biopsies from the ileocecal region in intestinal BD show some histological and immunophenotypic features that are different from CD, which may be useful in distinguishing these two entities.

Characterization and Function of Glycans on the Spike Proteins of SARS-CoV-2 Variants of Concern.

SARS-CoV-2 variants of concern (VOCs) pose a great challenge to viral prevention and treatment owing to spike (S) protein mutations, which enhance their infectivity and capacity for immune evasion. However, whether these S protein mutations affect glycosylation patterns and thereby influence infectivity and immunogenicity remains unclear. In this study, four VOC S proteins-S-Alpha, S-Beta, S-Delta, and S-Omicron-were expressed and purified. Lectin microarrays were performed to characterize their glycosylation patterns. Several glycans were differentially expressed among the four VOC S proteins. Furthermore, the functional examination of glycans differentially expressed on S-Omicron revealed a higher expression of fucose-containing glycans, which modestly increased the binding of S-Omicron to angiotensin converting enzyme 2 (ACE2). A higher abundance of sialic acid and galactose-containing glycan was observed on S-Omicron, which significantly reduced its sensitivity against broad S protein-neutralizing antibodies. These findings contribute to the further understanding of SARS-CoV-2 infection mechanisms and provide novel glycan targets for emerging and future variants of SARS-CoV-2. IMPORTANCE Though glycosylation sites of SARS-CoV-2 S protein remain highly conserved, we confirmed that mutations in the Spike gene affect the S protein glycan expression pattern in different variants. More importantly, we found that glycans were differentially expressed on the S protein of the Omicron variant, enabling different forms of receptor binding and neutralization resistance. This study improves our understanding of SARS-CoV-2 glycomics and glycobiology and provides novel therapeutic and preventive strategies for SARS-CoV-2 VOCs.

Near-Infrared Luciferase Complementation Assay with Enhanced Bioluminescence for Studying Protein-Protein Interactions and Drug Evaluation Under Physiological Conditions.

Identification of protein-protein interactions (PPIs) that occur in various cellular processes helps to reveal their potential molecular mechanisms, and there is still an urgent need to develop the assays to explore PPIs in living subjects. Here, we reported a near-infrared split luciferase complementation assay (SLCA) with enhanced bioluminescence produced by cleaving a luciferase, Akaluc, for exploring and visualizing PPIs in living cells and live mice. Compared with the previously developed and widely used red SLCA based on split firefly luciferase (Fluc-SLCA), the signal intensities for PPI recognition in living cells and live mice of the Akaluc-SLCA increased by ∼3.79-fold and ∼18.06-fold in the measured condition, respectively. Additionally, the interactions between the nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and cellular RNA processing proteins were identified, and the drug evaluation assays were also performed in living cells using Akaluc-SLCA. This study provides a new tool in the near-infrared region for the identification of PPIs in living cells and in vivo and new information for the understanding and treatment of SARS-CoV-2.

Synthesis of Dual-Emitting CdZnSe/Mn:ZnS Quantum Dots for Sensing the pH Change in Live Cells.

Intracellular pH is an important regulator of cell function, and its subtle changes may greatly affect cell activities and cause diseases. Reliable imaging of intracellular pH remains a huge challenge. Dual-emitting Mn2+-doped quantum dots (QDs) can be directly used as a ratiometric fluorescent probe without further modification, but they displayed low performance in terms of photoluminescence, stability, and intensity ratio regulation. Here, we report intrinsic dual-emitting CdZnSe/Mn:ZnS QDs with high photoluminescence efficiency, good stability, and biocompatibility. The emission intensity ratio was selectively regulated by Mn2+ doping. Because of aggregation-induced quenching of QDs, the exciton emission of CdZnSe/Mn:ZnS QDs (471 nm) was sensitive to pH, while the Mn2+-doped emission (606 nm) was passive to pH, which was probably due to little self-quenching in Mn2+-doped emission caused by weak Mn-Mn coupling interaction. Dual-emitting CdZnSe/Mn:ZnS QDs exhibited excellent pH-responsiveness in the range of pH 4.0 to 12.0 and were used for pH imaging in live HeLa cells. When the pH value of HeLa cells changed from 5.0 to 9.0, the emission changed from red to blue. Furthermore, these dual-emitting CdZnSe/Mn:ZnS QDs can provide a versatile platform for biosensors and biological imaging.

Aridity modifies the responses of plant stoichiometry to global warming and nitrogen deposition in semi-arid steppes.

Global warming and nitrogen (N) deposition are known to unbalance the stoichiometry of carbon (C), N, and phosphorus (P) in terrestrial plants, but it is unclear how water availability regulates their effects along a natural aridity gradient. Here, we conducted manipulative experiments to determine the effects of experimental warming (WT) and N addition (NT) on plant stoichiometry in desert, typical, and meadow steppes with decreasing aridity. WT elevated air temperatures by 1.2-2.9 °C using open-top chambers. WT increased forb C:N ratio and thus its N use efficiency and competitiveness in desert steppes, whereas WT reduced forb C:N and C:P ratios in typical and meadow steppes. Plant N:P ratio, which reflects nutrient limitation, was reduced by WT in desert steppes but not for typical or meadow steppes. NT reduced plant C:N ratios and increased N:P ratios in all three steppes. NT reduced forb C:P ratios in desert and typical steppes, but it enhanced grass C:P ratio in meadow steppes, indicating an enhancement of P use efficiency and competitiveness of grasses in wet steppes. WT and NT had synergetic effects on grass C:N and C:P ratios in all three steppes, which helps to increase grasses' productivity. Under WT or NT, the changes in community C:N ratio were positively correlated with increasing aridity, indicating that aridity increases plants' N use efficiency. However, aridity negatively affected the changes in N:P ratios under NT but not WT, which suggests that aridity mitigates P limitation induced by N deposition. Our results imply that warming could shift the dominant functional group into forbs in dry steppes due to altered stoichiometry, whereas grasses become dominated plants in wet steppes under increasing N deposition. We suggest that global changes might break the stoichiometric balance of plants and water availability could strongly modify such processes in semi-arid steppes.

The smallest near-infrared fluorescence complementation system for imaging protein-protein and RNA-protein interactions.

Bimolecular fluorescence complementation (BiFC) and its derivative molecular biosensor systems provide effective tools for visualizing biomolecular interactions. The introduction of red and near-infrared fluorescence emission proteins has expanded the spectrum of signal generating modules, enabling BiFC for in vivo imaging. However, the large size of the signal module of BiFC can hinder the interaction between proteins under investigation. In this study, we constructed the near-infrared BiFC and TriFC systems by splitting miRFP670nano, the smallest cyanobacteriochrome-evolved phytochrome available. The miRFP670nano-BiFC sensor system identified and enabled visualization of protein-protein interactions in living cells and live mice, and afforded a faster maturation rate and higher photostability and cellular stability when compared with those of reported near-infrared BiFC systems. We used the miRFP670nano-BiFC sensor system to identify interactions between the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and cellular stress granule proteins in living cells and found that the N protein downregulated the expression level of granule protein G3BP1. With the advantages of small size and long wavelength emission of the signal module, the proposed molecular biosensor system should be suitable for various applications in cell imaging studies.

The intraviral protein-protein interaction of SARS-CoV-2 reveals the key role of N protein in virus-like particle assembly.

Intraviral protein-protein interactions (PPIs) of SARS-CoV-2 in host cells may provide useful information for deep understanding of virology of SARS-CoV-2. In this study, 22 of 55 interactions of the structural and accessory proteins of SARS-CoV-2 were identified by biomolecular fluorescence complementation (BiFC) assay. The nucleocapsid (N) protein was found to have the most interactions among the structural and accessory proteins of SARS-CoV-2, and also specifically interacted with the putative packaging signal (PS) of SARS-CoV-2. We also demonstrated that the PS core containing PS576 RNA bears a functional PS, important for the assembly of the viral RNA into virus like particles (VLPs), and the packaging of SARS-CoV-2 RNA was N dependent.

SARS-CoV-2 spike protein receptor-binding domain N-glycans facilitate viral internalization in respiratory epithelial cells.

N-glycosylation plays an important role in the pathogenesis of viral infections. However, the role of SARS-CoV-2 RBD N-glycosylation in viral entry remains elusive. In this study, we expressed and purified N331 and N343 N-glycosite mutants of SARS-CoV-2 RBD. We found that de-glycosylation at N331 and N343 drastically reduces the RBD binding to ACE2. More importantly, based on qualitative and quantitative virology research methods, we show that the mutation of RBD N-glycosites interfered with SARS-CoV-2 internalization rather than attachment potentially by decreasing RBD binding to the receptors. Also, the double N-glycosites mutant (N331 + N343) showed significantly increased sensitivity against the designated RBD neutralizing antibodies. Taken together, these results suggest that N-glycosylation of SARS-CoV-2 RBD is not only critical for viral internalization into respiratory epithelial cells but also shields the virus from neutralization. It may provide new insights into the biological process of early-stage SARS-CoV-2 infection with potential therapeutic implications.

Pavement 3D Data Denoising Algorithm Based on Cell Meshing Ellipsoid Detection.

As a new measuring technique, laser 3D scanning technique has advantages of rapidity, safety, and accuracy. However, the measured result of laser scanning always contains some noise points due to the measuring principle and the scanning environment. These noise points will result in the precision loss during the 3D reconstruction. The commonly used denoising algorithms ignore the strong planarity feature of the pavement, and thus might mistakenly eliminate ground points. This study proposes an ellipsoid detection algorithm to emphasize the planarity feature of the pavement during the 3D scanned data denoising process. By counting neighbors within the ellipsoid neighborhood of each point, the threshold of each point can be calculated to distinguish if it is the ground point or the noise point. Meanwhile, to narrow down the detection space and to reduce the processing time, the proposed algorithm divides the cloud point into cells. The result proves that this denoising algorithm can identify and eliminate the scattered noise points and the foreign body noise points very well, providing precise data for later 3D reconstruction of the scanned pavement.

The cardiac translational landscape reveals that micropeptides are new players involved in cardiomyocyte hypertrophy.

Hypertrophic growth of cardiomyocytes is one of the major compensatory responses in the heart after physiological or pathological stimulation. Protein synthesis enhancement, which is mediated by the translation of messenger RNAs, is one of the main features of cardiomyocyte hypertrophy. Although the transcriptome shift caused by cardiac hypertrophy induced by different stimuli has been extensively investigated, translatome dynamics in this cellular process has been less studied. Here, we generated a nucleotide-resolution translatome as well as transcriptome data from isolated primary cardiomyocytes undergoing hypertrophy. More than 10,000 open reading frames (ORFs) were detected from the deep sequencing of ribosome-protected fragments (Ribo-seq), which orchestrated the shift of the translatome in hypertrophied cardiomyocytes. Our data suggest that rather than increase the translational rate of ribosomes, the increased efficiency of protein synthesis in cardiomyocyte hypertrophy was attributable to an increased quantity of ribosomes. In addition, more than 100 uncharacterized short ORFs (sORFs) were detected in long noncoding RNA genes from Ribo-seq with potential of micropeptide coding. In a random test of 15 candidates, the coding potential of 11 sORFs was experimentally supported. Three micropeptides were identified to regulate cardiomyocyte hypertrophy by modulating the activities of oxidative phosphorylation, the calcium signaling pathway, and the mitogen-activated protein kinase (MAPK) pathway. Our study provides a genome-wide overview of the translational controls behind cardiomyocyte hypertrophy and demonstrates an unrecognized role of micropeptides in cardiomyocyte biology.

A tandem near-infrared fluorescence complementation system with enhanced fluorescence for imaging protein-protein interactions in vivo.

Bimolecular fluorescence complementation (BiFC) is an effective tool for visualizing protein-protein interactions (PPIs). However, a BiFC system with long wavelength and high fluorescence intensity is yet to be developed for in vivo imaging. In this study, we constructed a tandem near-infrared BiFC (tBiFC) system by splitting a near-infrared phytochrome, IFP2.0. This system allows the identification and visualization of PPIs in live cells and living mice. The photophysical properties of the complementary fluorescence of the tBiFC system were similar to those of its parent protein IFP2.0, but the intensity was twice that of a single-copy IFP2.0-based BiFC system. Compared with previously reported near infrared BiFC systems-iRFP-BiFC and IFP1.4-BiFC-the signal intensity of the tBiFC system increased by ~1.48- and ~400-fold for weak PPIs in living cells, and ~1.51- and ~8-fold for strong PPIs, respectively. When applied to imaging PPIs in live mice, the complementary fluorescence intensity of the tBiFC system was also significantly higher than that of the other near-infrared BiFC systems. The use of this bright phytochrome in a tandem arrangement constitutes a powerful tool for imaging PPIs in the near infrared region.

Aridity stimulates responses of root production and turnover to warming but suppresses the responses to nitrogen addition in temperate grasslands of northern China.

Global warming and nitrogen (N) deposition are known to affect root dynamics in grasslands. However, previous studies were based only on a single ecosystem type, so it is unclear how warming and N addition affect root traits (root biomass, root-shoot ratio, root production and turnover) along the aridity gradient. In this study, we conducted an experiment to determine the effects of warming and N addition on root traits in desert, typical, and meadow grasslands in northern China, where the aridity gradually decreases from west to east across the region. Warming increased root-shoot ratio in dry year due to decline in soil water, but had a downward trend in root production and turnover in all three grasslands. N addition decreased root-shoot ratio in humid year due to increase in soil N, whereas did not significantly affect root production in any grasslands and increased root turnover in desert and meadow grasslands rather than typical grassland. Warming combined with N addition had negatively additive effects on root turnover in typical and meadow grasslands rather than desert grassland. N addition-induced changes in root biomass and root-shoot ratio were negatively affected by aridity in dry year. Aridity positively affected responses of root production and turnover to warming but negatively affected those responses to N addition. However, root-shoot ratio, root production and turnover under warming combined with N addition were not affected by aridity. Our results suggest that warming suppresses root carbon (C) input but N addition may exacerbate it in temperate grasslands, and warming combined with N addition suppresses it only in wet grasslands. Aridity promotes root C input under warming but suppresses it under N addition. However, aridity may little affect soil C and nutrient dynamics under global warming combined with N deposition in temperate grasslands in the future.

Small Molecule Epigenetic Modulators in Pure Chemical Cell Fate Conversion.

Although innovative technologies for somatic cell reprogramming and transdifferentiation provide new strategies for the research of translational medicine, including disease modeling, drug screening, artificial organ development, and cell therapy, recipient safety remains a concern due to the use of exogenous transcription factors during induction. To resolve this problem, new induction approaches containing clinically applicable small molecules have been explored. Small molecule epigenetic modulators such as DNA methylation writer inhibitors, histone methylation writer inhibitors, histone acylation reader inhibitors, and histone acetylation eraser inhibitors could overcome epigenetic barriers during cell fate conversion. In the past few years, significant progress has been made in reprogramming and transdifferentiation of somatic cells with small molecule approaches. In the present review, we systematically discuss recent achievements of pure chemical reprogramming and transdifferentiation.

Nonlinear Optimization of Orthotropic Steel Deck System Based on Response Surface Methodology.

The steel bridge deck system, directly subjected to the vehicle load, is an important component to be considered in the optimization design of the bridges. Due to its complex structure, the design parameters are coupled with each other, and many fatigue details in the system result in time-consuming calculation during structure optimization. In view of this, a nonlinear optimization method based on the response surface methodology (RSM) is proposed in this study to simplify the design process and to reduce the amount of calculations during optimization. The optimization design of the steel bridge deck system with two-layer pavement on the top of the steel deck plate is taken as an example, the influence of eight structural parameters is considered. The Box-Behnken design is used to construct a sample space in which the eight structural parameters can be distributed evenly to reduce the calculation workload. The finite element method is used to model the mechanical responses of the steel bridge deck system. From the regression analysis by the RSM, the explicit relationships between the fatigue details and the design parameters can be obtained, based on which the nonlinear optimization design of the bridge deck system is conducted. The influence of constraint functions, objective functions, and optimization algorithms is also analyzed. The method proposed in this study is capable of considering the influence of different structural parameters and different optimization objectives according to the actual needs, which will effectively simplify the optimization design of the steel bridge deck system.

An EDTA-resistant pyrazinamidase from non-pathogen Pseudonocardia carboxydivorans.

OBJECTIVES: To characterize a pyrazinamidase from non-pathogen Pseudonocardia carboxydivorans. RESULTS: A pyrazinamidase gene pncA encoding a 23-kDa protein PncA-Pse from P. carboxydivorans was over-expressed in Escherichia coli and characterized. This PncA-Pse can convert both pyrazinamide and nicotinamide efficiently with the optimal pH and temperature of pH 8.5 and 45 °C, respectively. Although ferrous iron and manganese were detected in PncA-Pse, the enzymatic activity is not affected by EDTA with the final concentration of 10 mM. Moreover, the enzymatic activity was not significantly affected with the addition of several metal ions, respectively. Based on the structure modeling, the 61st histidine which is associated with the metal binding, was mutated into alanine to get mutant H61A. No activity, iron and manganese were detected for H61A, which implies that PncA-Pse is a metal enzyme with resistance of the metal ion chelator EDTA, which is different from the previous reports. CONCLUSION: This is the first characterized pyrazinamidase from the genus Pseudonocardia, a non-pathogen.

Direct Reprogramming of Mouse Fibroblasts to Neural Stem Cells by Small Molecules.

Although it is possible to generate neural stem cells (NSC) from somatic cells by reprogramming technologies with transcription factors, clinical utilization of patient-specific NSC for the treatment of human diseases remains elusive. The risk hurdles are associated with viral transduction vectors induced mutagenesis, tumor formation from undifferentiated stem cells, and transcription factors-induced genomic instability. Here we describe a viral vector-free and more efficient method to induce mouse fibroblasts into NSC using small molecules. The small molecule-induced neural stem (SMINS) cells closely resemble NSC in morphology, gene expression patterns, self-renewal, excitability, and multipotency. Furthermore, the SMINS cells are able to differentiate into astrocytes, functional neurons, and oligodendrocytes in vitro and in vivo. Thus, we have established a novel way to efficiently induce neural stem cells (iNSC) from fibroblasts using only small molecules without altering the genome. Such chemical induction removes the risks associated with current techniques such as the use of viral vectors or the induction of oncogenic factors. This technique may, therefore, enable NSC to be utilized in various applications within clinical medicine.

Antagonistic effect of protein extracts from Streptococcus sanguinis on pathogenic bacteria and fungi of the oral cavity.

An antibacterial substance from Streptococcus sanguinis (S. sanguinis) is known to have an inhibitory effect on putative periodontal pathogens, but its inhibitory effect on pathogens of oral candidiasis is unknown. In this study, intracellular and exocrine proteins were extracted from S. sanguinis. The antagonistic effect of the protein extracts on Prevotella intermedia (P. intermedia) and Porphyromonas gingivalis (P. gingivalis) was detected by a well-plate technique, and the effects of the protein extracts on biofilms formed by these bacteria were evaluated by confocal laser scanning microscopy. The antagonistic effect of the protein extracts on pathogenic fungi was investigated using Candida albicans (C. albicans) and Candida tropicalis (C. tropicalis). The growth curves of C. albicans and C. tropicalis were determined from ultraviolet absorption measurements, their morphological changes following treatment were observed by optical microscopy and scanning electron microscopy, and the effects of the protein extracts on the thickness of their biofilms and the distribution of dead/live bacteria within the biofilms were detected by confocal laser scanning microscopy. The results showed significant inhibitory effects of the intracellular proteins extracted from S. sanguinis on pathogenic bacteria (P. intermedia and P. gingivalis), fungi (C. albicans and C. tropicalis) and the biofilms formed by them. Furthermore, the growth curves and morphology of C. albicans and C. tropicalis were altered following treatment with the intracellular proteins, resulting in disc-like depressions in the surfaces of the fungal spores and mycelia. By contrast, the exocrine proteins demonstrated no significant inhibitory effect on the pathogenic bacteria, fungi and the biofilms formed by them. Thus, it may be concluded that intracellular proteins of S. sanguinis have antibacterial activity and exert an antagonistic effect on certain pathogenic bacteria and fungi of the oral cavity.

The intracellular domain of sortilin interacts with amyloid precursor protein and regulates its lysosomal and lipid raft trafficking.

The processing of Amyloid precursor protein (APP) is multifaceted, comprising of protein transport, internalization and sequential proteolysis. However, the exact mechanism of APP intracellular trafficking and distribution remains unclear. To determine the interaction between sortilin and APP and the effect of sortilin on APP trafficking and processing, we studied the binding site and its function by mapping experiments, colocalization, coimmunoprecipitation and sucrose gradient fractionation. We identified for the first time that sortilin interacts with APP at both N- and C-terminal regions. The sortilin-FLVHRY (residues 787-792) and APP-NPTYKFFE (residues 759-766) motifs are crucial for the C-terminal interaction. We also found that lack of the FLVHRY motif reduces APP lysosomal targeting and increases APP distribution in lipid rafts in co-transfected HEK293 cells. These results are consistent with our in vivo data where sortilin knockout mice showed a decrease of APP lysosomal distribution and an increase of APP in lipid rafts. We further confirmed that overexpression of sortilin-FLVHRY mutants failed to rescue the lysosomal degradation of APP. Thus, our data suggests that sortilin is implicated in APP lysosomal and lipid raft targeting via its carboxyl-terminal F/YXXXXF/Y motif. Our study provides new molecular insights into APP trafficking and processing.

An optimal method of DNA silver staining in polyacrylamide gels.

DNA silver staining has widely been used to detect DNA fragments in polyacrylamide gels with high sensitivity. We developed an optimal method for DNA silver staining on polyacrylamide gels. The novel procedure can be completed within 10 min instead of over 20 min with the conventional methods. The sensitivity is significantly improved by the silver-ion sensitizer (Eriochrome black T (EBT)) and the minimum of 0.11 and 1.75 ng of DNA amount can be detected in denaturing and nondenaturing polyacrylamide gel, respectively. Compared with the conventional silver staining methods, the improved optimal method can save time and display high sensitivity, color uniformity, and long storage time of the staining gels.