Cancers are caused by accumulated DNA mutations. This recognition of the central role of mutations in cancer and recent advances in next‐generation sequencing, has initiated the massive screening of ...clinical samples and the identification of 1000s of cancer‐associated gene mutations. However, proteomic analysis of the expressed mutation products lags far behind genomic (transcriptomic) analysis. With comprehensive global proteomics analysis, only a small percentage of single nucleotide variants detected by DNA and RNA sequencing have been observed as single amino acid variants due to current technical limitations. Proteomic analysis of mutations is important with the potential to advance cancer biomarker development and the discovery of new therapeutic targets for more effective disease treatment. Targeted proteomics using selected reaction monitoring (also known as multiple reaction monitoring) and parallel reaction monitoring, has emerged as a powerful tool with significant advantages over global proteomics for analysis of protein mutations in terms of detection sensitivity, quantitation accuracy and overall practicality (e.g., reliable identification and the scale of quantification). Herein we review recent advances in the targeted proteomics technology for enhancing detection sensitivity and multiplexing capability and highlight its broad biomedical applications for analysis of protein mutations in human bodily fluids, tissues, and cell lines. Furthermore, we review recent applications of top‐down proteomics for analysis of protein mutations. Unlike the commonly used bottom‐up proteomics which requires digestion of proteins into peptides, top‐down proteomics directly analyzes intact proteins for more precise characterization of mutation isoforms. Finally, general perspectives on the potential of achieving both high sensitivity and high sample throughput for large‐scale targeted detection and quantification of important protein mutations are discussed.
Currently, no oral medications are available for type 1 diabetes (T1D). While our recent randomized placebo-controlled T1D trial revealed that oral verapamil had short-term beneficial effects, their ...duration and underlying mechanisms remained elusive. Now, our global T1D serum proteomics analysis identified chromogranin A (CHGA), a T1D-autoantigen, as the top protein altered by verapamil and as a potential therapeutic marker and revealed that verapamil normalizes serum CHGA levels and reverses T1D-induced elevations in circulating proinflammatory T-follicular-helper cell markers. RNA-sequencing further confirmed that verapamil regulates the thioredoxin system and promotes an anti-oxidative, anti-apoptotic and immunomodulatory gene expression profile in human islets. Moreover, continuous use of oral verapamil delayed T1D progression, promoted endogenous beta-cell function and lowered insulin requirements and serum CHGA levels for at least 2 years and these benefits were lost upon discontinuation. Thus, the current studies provide crucial mechanistic and clinical insight into the beneficial effects of verapamil in T1D.
Sensory neurons in the dorsal root ganglia (DRG) convey somatosensory and metabolic cues to the central nervous system and release substances from stimulated terminal endings in peripheral organs. ...Sex‐biased variations driven by the sex chromosome complement (XX and XY) have been implicated in the sensory–islet crosstalk. However, the molecular underpinnings of these male–female differences are not known. Here, we aim to characterize the molecular repertoire and the secretome profile of the lower thoracic spinal sensory neurons and to identify molecules with sex‐biased insulin sensing‐ and/or insulin secretion‐modulating activity that are encoded independently of circulating gonadal sex hormones. We used transcriptomics and proteomics to uncover differentially expressed genes and secreted molecules in lower thoracic T5‐12 DRG sensory neurons derived from sexually immature 3‐week‐old male and female C57BL/6J mice. Comparative transcriptome and proteome analyses revealed differential gene expression and protein secretion in DRG neurons in males and females. The transcriptome analysis identified, among others, higher insulin signaling/sensing capabilities in female DRG neurons; secretome screening uncovered several sex‐specific candidate molecules with potential regulatory functions in pancreatic β cells. Together, these data suggest a putative role of sensory interoception of insulin in the DRG–islet crosstalk with implications in sensory feedback loops in the regulation of β‐cell activity in a sex‐biased manner. Finally, we provide a valuable resource of molecular and secretory targets that can be leveraged for understanding insulin interoception and insulin secretion and inform the development of novel studies/approaches to fathom the role of the sensory–islet axis in the regulation of energy balance in males and females.
Transcriptome and proteome analyses of DRG neurons in sexually immature 3‐week‐old male and female mice revealed sex differences in cellular and molecular pathways relevant to the sensory neuron–islet crosstalk.
Prostate cancer (PCa) is the second leading cause of male cancer-related deaths in the United States. The pre-mature forms of prostate-specific antigen (PSA), proPSA, were shown to be associated with ...PCa. However, there is a technical challenge in the development of antibody-based immunoassays for specific recognition of each individual proPSA isoform. Herein, we report the development of highly specific, antibody-free, targeted mass spectrometry assays for simultaneous quantification of −2, −4, −5, and −7 proPSA isoforms in voided urine. The newly developed proPSA assays capitalize on Lys-C digestion to generate surrogate peptides with appropriate length (9–16 amino acids) along with long-gradient liquid chromatography separation. The assay utility of these isoform markers was evaluated in a cohort of 30 well-established clinical urine samples for distinguishing PCa patients from healthy controls. Under the 95% confidence interval, the combination of −2 and −4 proPSA isoforms yields the area under curve (AUC) of 0.86, and the AUC value for the combined all four isoforms was calculated to be 0.85. We have further verified −2proPSA, the dominant isoform, in an independent cohort of 34 clinical urine samples. Validation of proPSA isoforms in large-scale cohorts is needed to demonstrate their potential clinical utility.
Recent advances in sample preparation enable label-free mass spectrometry (MS)-based proteome profiling of small numbers of mammalian cells. However, specific devices are often required to downscale ...sample processing volume from the standard 50–200 μL to sub-μL for effective nanoproteomics, which greatly impedes the implementation of current nanoproteomics methods by the proteomics research community. Herein, we report a facile one-pot nanoproteomics method termed SOPs-MS (surfactant-assisted one-pot sample processing at the standard volume coupled with MS) for convenient robust proteome profiling of 50–1000 mammalian cells. Building upon our recent development of SOPs-MS for label-free single-cell proteomics at a low μL volume, we have systematically evaluated its processing volume at 10–200 μL using 100 human cells. The processing volume of 50 μL that is in the range of volume for standard proteomics sample preparation has been selected for easy sample handling with a benchtop micropipette. SOPs-MS allows for reliable label-free quantification of ∼1200–2700 protein groups from 50 to 1000 MCF10A cells. When applied to small subpopulations of mouse colon crypt cells, SOPs-MS has revealed protein signatures between distinct subpopulation cells with identification of ∼1500–2500 protein groups for each subpopulation. SOPs-MS may pave the way for routine deep proteome profiling of small numbers of cells and low-input samples.
A nonlinear constitutive model for a single lamina is proposed for the failure analysis of composite laminates. In the material model, both fiber and matrix are assumed to behave as elastic-plastic ...and the in-plane shear is assumed to behave nonlinearly with a variable shear parameter. The damage onset for individual lamina is detected by a mixed failure criterion, composed of the Tsai-Wu criterion and the maximum stress criterion. After damage takes place within the lamina, the fiber and in-plane shear are assumed to exhibit brittle behavior, and the matrix is assumed to exhibit degrading behavior. The proposed nonlinear constitutive model is tested against experimental data and good agreement is obtained. Then, numerical analyses are carried out to study the failure behavior of symmetric angle-ply composite laminates and symmetric cross-ply composite laminates subjected to biaxial loads. Finally, the conclusions obtained from the numerical analysis are given.
The enhanced precision and selectivity of liquid chromatography-tandem mass spectrometry (LC-MS/MS) makes it an attractive alternative to certain clinical immunoassays. Easily transferrable work ...flows could help facilitate harmonization and ensure high-quality patient care. We aimed to evaluate the interlaboratory comparability of antibody-free multiplexed insulin and C-peptide LC-MS/MS measurements.
The laboratories that comprise the Targeted Mass Spectrometry Assays for Diabetes and Obesity Research (TaMADOR) consortium verified the performance of a validated peptide-based assay (reproducibility, linearity, and lower limit of the measuring interval LLMI). An interlaboratory comparison study was then performed using shared calibrators, de-identified leftover laboratory samples, and reference materials.
During verification, the measurements were precise (2.7% to 3.7%CV), linear (4 to 15 ng/mL for C-peptide and 2 to 14 ng/mL for insulin), and sensitive (LLMI of 0.04 to 0.10 ng/mL for C-peptide and 0.03 ng/mL for insulin). Median imprecision across the 3 laboratories was 13.4% (inter-quartile range IQR 11.6%) for C-peptide and 22.2% (IQR 20.9%) for insulin using individual measurements, and 10.8% (IQR 8.7%) and 15.3% (IQR 14.9%) for C-peptide and insulin, respectively, when replicate measurements were averaged. Method comparison with the University of Missouri reference method for C-peptide demonstrated a robust linear correlation with a slope of 1.044 and r2 = 0.99.
Our results suggest that combined LC-MS/MS measurements of C-peptide and insulin are robust and adaptable and that standardization with a reference measurement procedure could allow accurate and precise measurements across sites, which could be important to diabetes research and help patient care in the future.
We characterized a prospective endometrial carcinoma (EC) cohort containing 138 tumors and 20 enriched normal tissues using 10 different omics platforms. Targeted quantitation of two peptides can ...predict antigen processing and presentation machinery activity, and may inform patient selection for immunotherapy. Association analysis between MYC activity and metformin treatment in both patients and cell lines suggests a potential role for metformin treatment in non-diabetic patients with elevated MYC activity. PIK3R1 in-frame indels are associated with elevated AKT phosphorylation and increased sensitivity to AKT inhibitors. CTNNB1 hotspot mutations are concentrated near phosphorylation sites mediating pS45-induced degradation of β-catenin, which may render Wnt-FZD antagonists ineffective. Deep learning accurately predicts EC subtypes and mutations from histopathology images, which may be useful for rapid diagnosis. Overall, this study identified molecular and imaging markers that can be further investigated to guide patient stratification for more precise treatment of EC.
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•APM status can be accurately predicted using a targeted proteomic assay•MYC activity is a potential biomarker for metformin treatment•PIK3R1 in-frame indels are associated with upregulated AKT1 phosphorylation•CTNNB1 exon 3 hotspot mutations block pS45 induced β-catenin degradation
Dou et al. report the characterization of a prospective endometrial carcinoma (EC) cohort using 10 different omics platforms. They identify potential molecular and imaging markers for guiding patient stratification for more precise treatment of EC.
Mobile devices (e.g., PDAs, smart phones and notebooks) currently have become the trend for personal use and are equipped with the capability of wireless communications. To authenticate the mobile ...user who transfers to the foreign area, the roaming authentication protocol is needed and usually requested for low costs to meet the requirements of lightweight communication devices. With the development of powerful functionalities on the mobile devices, the roaming payment service seems to be practical and with high commercial value. This paper proposes an efficient roaming payment protocol with low communication costs by using group signatures, inspired by the roaming authentication schemes proposed by Yang et al. and He et al. The major contribution of this paper is to integrate the PayWord-based micropayment scheme and the group signature scheme to enhance the effectiveness of the anonymous signatures and extend the fast authentication in roaming to perform lightweight payments by using hash chains.
碩士
高雄醫學大學
生物化學研究所碩士班
95
Diabetic nephropathy (DN) is a common complication of diabetic mellitus. Hyperplycemia, advanced glycation end-product (AGE), angiotensin II (Ang II) and transforming growth ...factor beta1 (TGF-β1) play important roles in DN. TGF-β1 is induced by hyperplycemia, AGE, Ang II, and reactive oxygen species (ROS). Additionally, our laboratory have shown that both EGF (epidermal growth factor) and EGF receptor (EGFR) are overexpressed in DN rats. Thus, we wished to understand the role of EGFR in an in vitro model of DN.
We found that TGF-β1 phosphorylated EGFR and up-regulated EGFR mRNA and protein expression in NRK-49F cells. TGF-β1 also induced FGF-2 and PAI-1 expression via inducing p-EGFR. Inhibition of EGFR (Iressa) or Smad2/3 (by SB-431542 and Smad2、Smad3 dominant negative plasmid) attenuated TGF-β1-induced FGF-2 and PAI-1 expression. Thus, TGF-β1 induced FGF-2 and PAI-1 expression via EGFR and Smad2/3. Inhibiting EGFR also attenuated cell cycle change. Moreover, TGF-β1-inducd proliferati
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