Standards-Free Absolute Quantitation of Oxidizable Glycopeptides by Coulometric Mass Spectrometry.

Currently, glycopeptide quantitation is mainly based on relative quantitation due to absolute quantitation requiring isotope-labeled or standard glycopeptides which may not be commercially available or are very costly and time consuming to synthesize. To address this grand challenge, coulometric mass spectrometry (CMS), based on the combination of electrochemistry (EC) and mass spectrometry (MS), was utilized to quantify electrochemically active glycopeptides without the need of using standard materials. In this study, we studied tyrosine-containing glycopeptides, NYIVGQPSS(ß-GlcNAc)TGNL-OH and NYSVPSS(ß-GlcNAc)TGNL-OH, and successfully quantified them directly with CMS with a discrepancy of less than 5% between the CMS measured amount and the theoretical amount. Taking one step further, we applied this approach to quantify glycopeptides generated from the digestion of NIST mAb, a monoclonal antibody reference material. Through HILIC column separation, five N297 glycopeptides resulting from NIST mAb tryptic digestion were successfully separated and quantified by CMS for an absolute amount without the use of any standard materials. This study indicates the potential utility of CMS for quantitative proteomics research.

Serum proteins differentially expressed in gestational diabetes mellitus assessed using isobaric tag for relative and absolute quantitation proteomics.

BACKGROUND: As a well-known fact to the public, gestational diabetes mellitus (GDM) could bring serious risks for both pregnant women and infants. During this important investigation into the linkage between GDM patients and their altered expression in the serum, proteomics techniques were deployed to detect the differentially expressed proteins (DEPs) of in the serum of GDM patients to further explore its pathogenesis, and find out possible biomarkers to forecast GDM occurrence. AIM: To investigation serum proteins differentially expressed in GDM were assessed using isobaric tag for relative and absolute quantitation (iTRAQ) proteomics and bioinformatics analyses. METHODS: Subjects were divided into GDM and normal control groups according to the IADPSG diagnostic criteria. Serum samples were randomly selected from four cases in each group at 24-28 wk of gestation, and the blood samples were identified by applying iTRAQ technology combined with liquid chromatography-tandem mass spectrometry. Key proteins and signaling pathways associated with GDM were identified by bioinformatics analysis, and the expression of key proteins in serum from 12 wk to 16 wk of gestation was further verified using enzyme-linked immunosorbent assay (ELISA). RESULTS: Forty-seven proteins were significantly differentially expressed by analyzing the serum samples between the GDM gravidas as well as the healthy ones. Among them, 31 proteins were found to be upregulated notably and the rest 16 proteins were downregulated remarkably. Bioinformatic data report revealed abnormal expression of proteins associated with lipid metabolism, coagulation cascade activation, complement system and inflammatory response in the GDM group. ELISA results showed that the contents of RBP4, as well as ANGPTL8, increased in the serum of GDM gravidas compared with the healthy ones, and this change was found to initiate from 12 wk to 16 wk of gestation. CONCLUSION: GDM symptoms may involve abnormalities in lipid metabolism, coagulation cascade activation, complement system and inflammatory response. RBP4 and ANGPTL8 are expected to be early predictors of GDM.

[The Applications of qNMR in Drug Quality Control].

NMR is well known as one of the most important methods for elucidating the structure of organic compounds. Furthermore, it has recently been recognized as a powerful tool for quantitative analysis. The quantitative NMR (qNMR) has become an official analytical method described in detail in the Japanese Pharmacopoeia. And today, it is widely applied in drug development. The qNMR method offers many new advantages over traditional and conventional quantitative analysis methods. For example, this method requires only a few milligrams of the analyte and allows absolute quantitation of the analyte without using a qualified reference standard as a control sample. Then, it can be easily applied to most chemicals without expending significant time and resources on method development. In addition, residual solvent can be determined using qNMR methods. The peak area of an NMR spectrum is directly proportional to the number of protons contributing to the resonance. Based on this principle, the residual solvent can be determined by counting the signal corresponding to the residual solvent in the sample solution. We have applied qNMR as an alternative to GC. Thus, qNMR is an innovative and promising analytical technique that is expected to make significant progress in the future. Recently, the analytical research and quality control departments have been working together to expand this technology to a wide range of areas in the pharmaceutical industry.

Cathelicidin Antimicrobial Peptide Acts as a Tumor Suppressor in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is associated with high rates of metastasis and recurrence, and is one of the most common causes of cancer-associated death worldwide. This study examined the protein changes within circulating exosomes in patients with HCC against those in healthy people using isobaric tags for a relative or absolute quantitation (iTRAQ)-based quantitative proteomics analysis. The protein levels of von Willebrand factor (VWF), cathelicidin antimicrobial peptide (CAMP), and proteasome subunit beta type-2 (PSMB2) were altered in HCC. The increased levels of VWF and PSMB2 but decreased CAMP levels in the serum of patients with HCC were validated by enzyme-linked immunosorbent assays. The level of CAMP (the only cathelicidin found in humans) also decreased in the circulating exosomes and buffy coat of the HCC patients. The serum with reduced levels of CAMP protein in the HCC patients increased the cell proliferation of Huh-7 cells; this effect was reduced following the addition of CAMP protein. The depletion of CAMP proteins in the serum of healthy people enhances the cell proliferation of Huh-7 cells. In addition, supplementation with synthetic CAMP reduces cell proliferation in a dose-dependent manner and significantly delays G1-S transition in Huh-7 cells. This implies that CAMP may act as a tumor suppressor in HCC.

Grb2 Expression in Acute Spinal Cord Injury After Methylprednisolone Intrathecal Injection in Rats.

OBJECTIVE: This study aims to investigate the effect of methylprednisolone (MP) intrathecal injection on a rat model of acute spinal cord injury (ASCI). METHODS: Allen's frame was used to establish a rat model of ASCI. MP and normal saline were intrathecally injected to Sprague-Dawley rats at 0, 3, 6, 8, 12, and 24 hours after ASCI, and injured spinal cord tissues were sterilely extracted after 24 hours of treatment. Isobaric tags for relative and absolute quantitation (iTRAQ) were coupled with 2-dimensional liquid chromatography tandem mass spectrometry to separate and identify differentially expressed proteins. RESULTS: The expression of growth factor receptor-bound protein 2 (Grb2) was downregulated in the MP groups at 0 hours (iTRAQ ratio = 0.996), 3 hours (iTRAQ ratio = 0.737), 8 hours (iTRAQ ratio = 0.763), and 24 hours (iTRAQ ratio = 0.908) after injury compared with that in the control groups. No significant difference in Grb2 expression was observed between the control groups at 6 and 12 hours after ASCI. CONCLUSIONS: Standardized MP intrathecal injection after ASCI treatment reduces Grb2 activation in a rat ASCI model. Further studies should determine whether or not the same effect can be observed in human ASCIs.

iTRAQ-based differential proteomic analysis of high- and low-virulence strains of Spiroplasma eriocheiris.

Spiroplasma eriocheiris is one of the major pathogenic bacteria in crustaceans, featuring high infectivity, rapid transmission, and an absence of effective control strategies, resulting in significant economic losses to the aquaculture industry. Research into virulence-related factors provides an important perspective to clarify how Spiroplasma eriocheiris is pathogenic to shrimps and crabs. Therefore, in this study, isobaric tags for relative and absolute quantitation (iTRAQ) technology was utilized to undertake a differential proteomic analysis of high- and low-virulence Spiroplasma eriocheiris strains at different growth phases. A total of 868 differentially expressed proteins (DEPs) were obtained, of which 31 novel proteins were identified by proteogenomic analysis. There were 62, 61, 175, and 235 DEPs between the log phase (YD) and non-log phase (YFD) of the high-virulence strain, between the log phase (CD) and non-log phase (CFD) of the low-virulence strain, between YD and CD, and between CFD and YFD, respectively. All the DEPs were compared with virulence protein databases (MvirDB and VFDB), and 68 virulence proteins of Spiroplasma eriocheiris were identified, of which 12 were involved in a total of 21 metabolic pathways, including motility, chemotaxis, growth, metabolism and virulence of the bacteria. The results of this study form the basis for further research into the molecular mechanism of virulence and physiological differences between high- and low-virulence strains of Spiroplasma eriocheiris, and provide a scientific basis for a detailed understanding of its pathogenesis.

Lactobacillus gasseri LA39 promotes hepatic primary bile acid biosynthesis and intestinal secondary bile acid biotransformation. / æ ¼æ°ä¹³é ¸æ†èŒLA39ä¿ƒè¿›äº†è‚è„åˆçº§èƒ†æ±é ¸çš„ç”Ÿç‰©åˆæˆå’Œè‚ é“æ¬¡çº§èƒ†æ±é ¸çš„ç”Ÿç‰©è½¬åŒ–.

A growing body of evidence has linked the gut microbiota to liver metabolism. The manipulation of intestinal microflora has been considered as a promising avenue to promote liver health. However, the effects of Lactobacillus gasseri LA39, a potential probiotic, on liver metabolism remain unclear. Accumulating studies have investigated the proteomic profile for mining the host biological events affected by microbes, and used the germ-free (GF) mouse model to evaluate host-microbe interaction. Here, we explored the effects of L. gasseri LA39 gavage on the protein expression profiles of the liver of GF mice. Our results showed that a total of 128 proteins were upregulated, whereas a total of 123 proteins were downregulated by treatment with L. gasseri LA39. Further bioinformatics analyses suggested that the primary bile acid (BA) biosynthesis pathway in the liver was activated by L. gasseri LA39. Three differentially expressed proteins (cytochrome P450 family 27 subfamily A member 1 (CYP27A1), cytochrome P450 family 7 subfamily B member 1 (CYP7B1), and cytochrome P450 family 8 subfamily B member 1 (CYP8B1)) involved in the primary BA biosynthesis pathway were further validated by western blot assay. In addition, targeted metabolomic analyses demonstrated that serum and fecal ß|-muricholic acid (a primary BA), dehydrolithocholic acid (a secondary BA), and glycolithocholic acid-3-sulfate (a secondary BA) were significantly increased by L. gasseri LA39. Thus, our data revealed that L. gasseri LA39 activates the hepatic primary BA biosynthesis and promotes the intestinal secondary BA biotransformation. Based on these findings, we suggest that L. gasseri LA39 confers an important function in the gut‒liver axis through regulating BA metabolism.

Detecting the Neuraminidase R294K Mutation in Avian Influenza A (H7N9) Virus Using Reverse Transcription Droplet Digital PCR Method.

The R294K mutation in neuraminidase (NA) causes resistance to oseltamivir in the avian influenza virus H7N9. Reverse transcription droplet digital polymerase chain reaction (RT-dd PCR) is a novel technique for detecting single-nucleotide polymorphisms. This study aimed to develop an RT-dd PCR method for detecting the R294K mutation in H7N9. Primers and dual probes were designed using the H7N9 NA gene and the annealing temperature was optimized at 58.0 °C. The sensitivity of our RT-dd PCR method was not significantly different from that of RT-qPCR (p = 0.625), but it could specifically detect R294 and 294K in H7N9. Among 89 clinical samples, 2 showed the R294K mutation. These two strains were evaluated using a neuraminidase inhibition test, which revealed that their sensitivity to oseltamivir was greatly reduced. The sensitivity and specificity of RT-dd PCR were similar to those of RT-qPCR and its accuracy was comparable to that of NGS. The RT-dd PCR method had the advantages of absolute quantitation, eliminating the need for a calibration standard curve, and being simpler in both experimental operation and result interpretation than NGS. Therefore, this RT-dd PCR method can be used to quantitatively detect the R294K mutation in H7N9.

Alterations in the Proteome of Developing Neocortical Synaptosomes in the Absence of MET Signaling Revealed by Comparative Proteomics.

Alterations in the expression of genes encoding proteins involved in synapse formation, maturation, and function are a hallmark of many neurodevelopmental and psychiatric disorders. For example, there is reduced neocortical expression of the MET receptor tyrosine kinase (MET) transcript and protein in Autism Spectrum Disorder (ASD) and Rett syndrome. Preclinical in vivo and in vitro models manipulating MET signaling reveal that the receptor modulates excitatory synapse development and maturation in select forebrain circuits. The molecular adaptations underlying the altered synaptic development remain unknown. We performed a comparative mass spectrometry analysis of synaptosomes generated from the neocortex of wild type and Met null mice during the peak of synaptogenesis (postnatal day 14; data are available from ProteomeXchange with identifier PXD033204). The analyses revealed broad disruption of the developing synaptic proteome in the absence of MET, consistent with the localization of MET protein in pre- and postsynaptic compartments, including proteins associated with the neocortical synaptic MET interactome and those encoded by syndromic and ASD risk genes. In addition to an overrepresentation of altered proteins associated with the SNARE complex, multiple proteins in the ubiquitin-proteasome system and associated with the synaptic vesicle, as well as proteins that regulate actin filament organization and synaptic vesicle exocytosis/endocytosis, were disrupted. Taken together, the proteomic changes are consistent with structural and functional changes observed following alterations in MET signaling. We hypothesize that the molecular adaptations following Met deletion may reflect a general mechanism that produces circuit-specific molecular changes due to loss or reduction of synaptic signaling proteins.

AlacatDesigner─Computational Design of Peptide Concatamers for Protein Quantitation.

Protein quantitation via mass spectrometry relies on peptide proxies for the parent protein from which abundances are estimated. Owing to the variability in signal from individual peptides, accurate absolute quantitation usually relies on the addition of an external standard. Typically, this involves stable isotope-labeled peptides, delivered singly or as a concatenated recombinant protein. Consequently, the selection of the most appropriate surrogate peptides and the attendant design in recombinant proteins termed QconCATs are challenges for proteome science. QconCATs can now be built in a "a-la-carte" assembly method using synthetic biology: ALACATs. To assist their design, we present "AlacatDesigner", a tool that supports the peptide selection for recombinant protein standards based on the user's target protein. The user-customizable tool considers existing databases, occurrence in the literature, potential post-translational modifications, predicted miscleavage, predicted divergence of the peptide and protein quantifications, and ionization potential within the mass spectrometer. We show that peptide selections are enriched for good proteotypic and quantotypic candidates compared to empirical data. The software is freely available to use either via a web interface AlacatDesigner, downloaded as a Desktop application or imported as a Python package for the command line interface or in scripts.

The protective effects of Esculentoside A through AMPK in the triple transgenic mouse model of Alzheimer's disease.

BACKGROUND: Neurofibrillary tangles comprising hyperphosphorylated tau are vital factors associated with the pathogenesis of Alzheimer's disease (AD). The elimination or reduction of hyperphosphorylated and abnormally aggregated tau is a valuable measure in AD therapy. Esculentoside A (EsA), isolated from Phytolacca esculenta, exhibits pharmacotherapeutic efficacy in mice with amyloid beta-induced AD. However, whether EsA affects tau pathology and its specific mechanism of action in AD mice remains unclear. PURPOSE: To investigate the roles and mechanisms of EsA in cognitive decline and tau pathology in a triple transgenic AD (3 × Tg-AD) mouse model. METHODS: EsA (5 and 10 mg/kg) was administered via intraperitoneal injection to 8-month-old AD mice for eight consecutive weeks. Y-maze and novel object recognition tasks were used to evaluate the cognitive abilities of mice. Potential signaling pathways and targets in EsA-treated AD mice were assessed using quantitative proteomic analysis. The NFT levels and hippocampal synapse numbers were investigated using Gallyas-Braak silver staining and transmission electron microscopy, respectively. Western blotting and immunofluorescence assays were used to measure the expression of tau-associated proteins. RESULTS: EsA administration attenuated memory and recognition deficits and synaptic damage in AD mice. Isobaric tags for relative and absolute quantitation proteomic analysis of the mouse hippocampus revealed that EsA modulated the expression of some critical proteins, including brain-specific angiogenesis inhibitor 3, galectin-1, and Ras-related protein 24, whose biological roles are relevant to synaptic function and autophagy. Further research revealed that EsA upregulated AKT/GSK3ß activity, in turn, inhibited tau hyperphosphorylation and promoted autophagy to clear abnormally phosphorylated tau. In hippocampus-derived primary neurons, inhibiting AMP-activated protein kinase (AMPK) activity through dorsomorphin could eliminate the effect of EsA, as revealed by increased tau hyperphosphorylation, downregulated activity AKT/GSK3ß, and blocked autophagy. CONCLUSIONS: To our knowledge, this study is the first to demonstrate that EsA attenuates cognitive decline by targeting the pathways of both tau hyperphosphorylation and autophagic clearance in an AMPK-dependent manner and it shows a high reference value in AD pharmacotherapy research.

Effects of electroacupuncture on rats with cognitive impairment: An iTRAQ-based proteomics analysis.

OBJECTIVE: The study explores the effects of electroacupuncture (EA) at the governing vessel (GV) on proteomic changes in the hippocampus of rats with cognitive impairment. METHODS: Healthy male rats were randomly divided into 3 groups: sham, model and EA. Cognitive impairment was induced by left middle cerebral artery occlusion in the model and EA groups. Rats in the EA group were treated with EA at Shenting (GV24) and Baihui (GV20) for 7 d. Neurological deficit was scored using the Longa scale, the learning and memory ability was detected using the Morris water maze (MWM) test, and the proteomic profiling in the hippocampus was analyzed using protein-labeling technology based on the isobaric tag for relative and absolute quantitation (iTRAQ). The Western blot (WB) analysis was used to detect the proteins and validate the results of iTRAQ. RESULTS: Compared with the model group, the neurological deficit score was significantly reduced, and the escape latency in the MWM test was significantly shortened, while the number of platform crossings increased in the EA group. A total of 2872 proteins were identified by iTRAQ. Differentially expressed proteins (DEPs) were identified between different groups: 92 proteins were upregulated and 103 were downregulated in the model group compared with the sham group, while 142 proteins were upregulated and 126 were downregulated in the EA group compared with the model group. Most of the DEPs were involved in oxidative phosphorylation, glycolipid metabolism and synaptic transmission. Furthermore, we also verified 4 DEPs using WB technology. Although the WB results were not exactly the same as the iTRAQ results, the expression trends of the DEPs were consistent. The upregulation of heat-shock protein ß1 (Hspb1) was the highest in the EA group compared to the model group. CONCLUSION: EA can effect proteomic changes in the hippocampus of rats with cognitive impairment. Hspb1 may be involved in the molecular mechanism by which acupuncture improves cognitive impairment.

The new landscape of differentially expression proteins in placenta tissues of gestational diabetes based on iTRAQ proteomics.

INTRODUCTION: Gestational diabetes mellitus (GDM) refers to abnormal glucose tolerance that occurs or is firstly diagnosed during pregnancy. GDM is related to various adverse pregnancy outcomes, but GDM pathogeny has not been fully elucidated. Nevertheless, previous studies have observed that many proteins in the placentas of patients with GDM are dysregulated. The present study aimed to establish a novel differentially expressed protein (DEP) landscape of GDM and normal maternal placentas and to explore the possible connection between DEPs and GDM pathogenesis. This study provides new insights into the mechanism of GDM and should make an important contribution to the development of biomarkers. METHODS: The morphological characteristics of the placenta were observed on 30 GDM and normal maternal placental tissues stained with haematoxylin and eosin. Isobaric tags for relative and absolute quantitation (iTRAQ) was used in the proteomics screening of the DEPs of the normal and GDM maternal placentas. Bioinformatics analysis was performed on the DEPs, and parallel reaction monitoring (PRM) was performed to verify the DEPs. Finally, the quantitative analysis of iTRAQ and PRM was verified by immunohistochemical assay. RESULTS: A total of 68 DEPs in the GDM placenta were identified with iTRAQ proteomics experiment, comprising 21 up-regulated and 47 down-regulated DEPs. Bioinformatics analysis showed that the regulation of transport, catabolic process of non-coding RNA, cytoskeleton and cell binding were the most abundant Gene Ontology terms, and RNA degradation was an important pathway for significant enrichment. Protein-protein interaction network analysis showed that heterogeneous nuclear ribonucleoproteins A2/B1 (HNRNPA2B1), heterogeneous nuclear ribonucleoprotein A/B (HNRNPAB), heterogeneous nuclear ribonucleoprotein L (HNRNPL) and heterogeneous nuclear ribonucleoprotein A3 (HNRNPA3) were the cores of the up-regulated proteins. Band 3 anion transport protein (SLC4A1), spectrin beta chain erythrocytic (SPTB), ankyrin-1 (ANK1), spectrin beta chain non-erythrocytic 2 (SPTBN2), D-3-phosphoglycerate dehydrogenase (PHGDH) and exosome complex component RRP42 (EXOSC7) were the cores of the down-regulated proteins. These proteins are involved in the binding, splicing, processing, transport and degradation of RNA and in the formation and maintenance of the cytoskeleton. PRM verification results showed that seven proteins, namely, epiplakin (EPPK1), cold-inducible RNA-binding protein (CIRBP), HNRNPA2B1, HNRNPAB, HNRNPL, Ras-related protein Rab-21 (RAB21) and Ras-related protein Rab-3B (RAB3B), were up-regulated, whereas SPTB and SLC4A1 were down-regulated. The results of immunohistochemical assay also showed that the expression of five proteins, namely EPPK1, HNRNPA2B1, HNRNPAB, CIRBP and RAB21, were significantly higher in GDM placental tissues (P < 0.01). The GDM placentas showed changes in the morphological evaluation, including poor villous maturation, obvious increase in the number of syncytiotrophoblast nodules, thickening of the wall of dry villous arterioles with lumen stenosis, increased fibrinous exudation and excessive filling of villous interstitial vessels. DISCUSSION: Differentially expressed proteins related to a variety of biological processes in the GDM placenta were found. Fourteen proteins, namely, HNRNPA2B1, HNRNPAB, HNRNPL, HNRNPA3, EPPK1, CIRBP, RAB21, RAB3B, SLC4A1, SPTB, ANK1, SPTBN2, PHGDH and EXOSC7, which were differentially expressed in the placenta, may play an important role in regulating the occurrence and development of gestational diabetes through multi-channel and multi-link regulation.

Quantitative iTRAQ proteomics reveal the proteome profiles of bone marrow mesenchymal stem cells after cocultures with Schwann cells in vitro.

Background: Bone marrow mesenchymal stem cells (BMSCs) combined with Schwann cells (SCs) represent a better therapeutic cell transplantation strategy for treating spinal cord injury (SCI) than transplantation with BMSCs or SCs alone. In previous studies, we demonstrated that BMSCs are able to differentiate in neuron-like cells when cocultured with SCs. The detailed mechanism underlying SCI repair that occurs during the combined transplantation of BMSCs and SCs has not yet been studied. In this study, we adopted an isobaric tag for relative and absolute quantitation (iTRAQ)-based protein identification/quantification approach to examine the effects of the SC and BMSC coculture process on the BMSCs and then obtained and analyzed the differentially expressed proteins (DEPs) and their possible related pathways. Methods: This study included three groups based on the number of coculture days (i.e., 0, 3, and 7 days). Changes in BMSC protein expression levels were measured using the iTRAQ technique. A bioinformatics analysis of all the data was performed. Results: In total, 6,760 types of proteins were detected, corresponding to 5,181 data points with quantitative information. Of these, a total of 243 DEPs were identified, of which 169 proteins were upregulated and 74 proteins were downregulated. These DEPs were identified by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Intercellular adhesion molecule-1 (ICAM-1), integrin, and dioxygenase may play crucial roles in the repair of SCI. The data analysis indicates that the relevant biological processes may be regulated by lysosome function, cell adhesion molecules (CAMs), leukocyte transendothelial migration, and the phosphatidylinositol-3-kinase (PI3K) and peroxisome proliferator-activated receptor (PPAR) signaling pathways. Conclusions: The data provided in this study indicate that several molecular mechanisms and signaling pathways are involved in the BMSC and SC coculture process. This information may be useful for the further identification of specific targets and related mechanisms and guide new directions for SCI treatment.

Differential proteome response to H5N1 highly pathogenic avian influenza (HPAI) viruses infection in duck.

Ducks and wild aquatic birds are the natural reservoirs of avian influenza viruses. However, the host proteome response that causes disease in vivo by the H5N1 HPAI virus is still unclear. This study presented a comprehensive analysis of the proteome response in Muscovy duck lung tissue during 3 days of infection with either a highly virulent DK383 or an avirulent DK212. An unbiased strategy- isobaric tags for relative and absolute quantitation (iTRAQ) in conjunction with high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) was utilized to investigate the infection mechanism. Pathways derived from analysis of 292 significantly altered proteins may contribute to the high pathogenic nature and disease progression of H5N1 viruses. Global proteome profiles indicated improved correlation with the virus titers and gene expression patterns between the two strains of the H5N1 virus. DK383 replicated more efficiently and induced a stronger response specific to severe disease. While proteins involved in the immune response of neutrophils were increased markedly by DK383, DK212 evoked a distinct response characterized by an increase in proteins involved in the maturation of dendritic cells, adhesion of phagocytes, and immune response of macrophages. The differentially activated Akt/mTOR/p70S6K pathway might involve in the host response to H5N1 viruses. Therefore, systematically integrated with datasets from primary genomic and virus titer results, proteomic analyses may help reveal the potential pathogenesis.

Challenges to establish the diagnosis of aspergillosis in non-laboratory animals: looking for alternatives in veterinary medicine and demonstration of feasibility through two concrete examples in penguins and dolphins.

Aspergillosis remains difficult to diagnose in animals. Laboratory-based assays are far less developed than those for human medicine, and only few studies have been completed to validate their utility in routine veterinary diagnostics. To overcome the current limitations, veterinarians and researchers have to propose alternative methods including extrapolating from human diagnostic tools and using innovative technology. In the present overview, two specific examples were complementarily addressed in penguins and dolphins to illustrate how is challenging the diagnosis of aspergillosis in animals. Specific focus will be made on the novel application of simple testing in blood based on serological assays or protein electrophoresis and on the new information garnered from metabolomics/proteomics to discover potential new biomarkers. In conclusion, while the diagnostic approach of aspergillosis in veterinary medicine cannot be directly taken from options developed for human medicine, it can certainly serve as inspiration.

Predictive value of proteomic markers for advanced rectal cancer with neoadjuvant chemoradiotherapy.

BACKGROUND: Preoperative neoadjuvant chemoradiation (nCRT) has been the standard treatment for locally advanced rectal cancer. Serum biomarkers to stratify patients with respect to prognosis and response to nCRT are needed due to the diverse response to the therapy. METHODS: Thirteen paired pre- and post-nCRT sera from rectal cancer patients were analyzed by isobaric tags for relative and absolute quantitation (iTRAQ) method. Twenty-five proteins were selected for validation by parallel reaction monitoring (PRM) in ninety-one patients. RESULTS: Totally, 310 proteins were identified and quantified in sera samples. Reactome pathway analysis showed that the immune activation-related pathways were enriched in response to nCRT. Twenty-five proteins were selected for further validation. PRM result showed that the level of PZP was higher in pathological complete response (pCR) patients than non-pCR patients. The Random Forest algorithm identified a prediction model composed of 10 protein markers, which allowed discrimination between pCR patients and non-pCR patients (area under the curve (AUC) = 0.886 on testing set). Higher HEP2 and GELS or lower S10A8 in baseline sera were associated with better prognosis. Higher APOA1 in post nCRT sera was associated with better disease-free survival (DFS). CONCLUSIONS: We identified and confirmed a 10-protein panel for nCRT response prediction and four potential biomarkers HEP2, GELS, S10A8 and APOA1 for prognosis of rectal cancer based on iTRAQ-based comparative proteomics screening and PRM-based targeted proteomic validation.

Targeted proteomics using parallel reaction monitoring confirms salivary proteins indicative of metastatic triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype due to the absence of hormonal receptors. Our study aimed to identify and determine the effectiveness of salivary proteins as candidate markers for metastatic TNBC subtype using parallel reaction monitoring mass spectrometry (PRM-MS). Three salivary proteins (lipocalin-1, SMR3B, and plastin-2) that showed significant differential expression in label-free quantitation (LFQ) between TNBC (N = 6) and health subjects (HS; N = 6) were selected for further validation. The developed PRM assay was used to quantify peptides GLST and NNLE (lipocalin-1), VYAL and MINL (Plastin-2) and GPYP, and IPPP (SMR3B) on a different cohort of TNBC patients (N = 20) and HS (N = 20) for evaluating their discriminating performances. Quantitative validation using PRM correlated well with the LFQ results, and 5 peptides from three proteins showed a similar up-or down-regulation. Subsequently, these proteins were validated by Western blot analysis. Compared to one protein's performance as an individual marker, the five-signature panel with salivary GLST, VYAL, MINL, GPYP, and IPPP achieved better performance in differentiating aggressive TNBC and HS with sensitivity (80%) and specificity (95%). Targeted proteomic analysis of the prioritized proteins highlights a peptide-based signature in saliva as the potential predictor to distinguish between TNBC and HS. SIGNIFICANCE OF THE STUDY: This study was designed to identify and quantify potential markers in saliva from the triple-negative breast cancer (TNBC) patients using parallel reaction monitoring assay. Three salivary proteins, Lipocalin-1 (LCN-1), Submaxillary androgen-regulated protein 3B (SMR3B), and Plastin-2 (LCP-1) selected in the discovery-phase were further quantified by targeted proteomics and Western blots. The salivary proteins successfully differentiated TNBC patients from healthy subjects with a sensitivity (80%) and specificity (95%).

Validation of absolutely quantitated Ki67 and cyclinD1 protein levels for prognosis of Luminal-like breast cancer patients.

AIMS: To translate a clinical research finding into daily clinical practice requires well-controlled clinical trials. We have demonstrated the usage of absolute quantitation of Ki67 and cyclinD1 protein levels to improve prognosis of Luminal-like patients based on overall survival (OS) analysis of a cohort of 155 breast cancer specimens (cohort 1). However, this finding is considered the D level of evidence (LOE) to require subsequent validation before it may be used in daily clinical practice. To set the stage for future clinical trials, our findings were validated through OS analysis of an independent cohort (cohort 2) of 173 Luminal-like patients. METHODS: Both Ki67 and cyclinD1 levels were measured absolutely and quantitatively using the Quantitative Dot Blot (QDB) method in cohort 2. The proposed cutoffs for both biomarkers from cohort 1 were re-evaluated in cohort 2 and in the merged cohort of 1 and 2, respectively, through univariate, multivariate and Kaplan-Meier survival analysis. RESULTS: The proposed cutoffs of 2.31 nmol/g for Ki67 and 0.44 µmol/g for cyclinD1 were validated as effective cutoffs in cohort 2 and the merged cohort through OS analysis. The combined use of both biomarkers allowed us to identify patients with both biomarker levels below the cutoffs (59.3%) with10-year survival probability (SP) of 89%, in comparison to those above the cutoffs (8.3%) with 8 year SP of 28% through OS analysis in the merged cohort. CONCLUSIONS: This study validated our findings that absolute quantitation of Ki67 and cyclinD1 allows effective subtyping of luminal-like patients. It sets the stage for prospective or prospective-retrospective clinical studies.

Preliminary study on pathogenic mechanism of first Chinese family with PNKD.

Background: The first Chinese family with paroxysmal non-kinesigenic dystonia (PNKD) was confirmed to harbour a PNKD mutation. However, the pathogenic mechanism of the PNKD-causing gene mutation was unclear. Methods: Wild-type and mutant PNKD-L plasmids were prepared and transfected into the C6 cell line to study the distribution and stability of PNKD protein in C6 cells and its effect on the glutathione content. The blood and cerebrospinal fluid (CSF) of 3 PNKD patients and 3 healthy controls were collected. The differentially expressed proteins were identified using isobaric tags for relative and absolute quantitation. Furthermore, Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analyses were performed, and the protein-protein interaction network was constructed. Results: Wild-type PNKD protein was mainly distributed in the membranes, whereas mutant PNKD protein was distributed throughout the C6 cells. After transfection with mutant PNKD-L plasmid, the glutathione content decreased significantly in C6 cells; the stability of the mutant PNKD protein was significantly low. There were 172 and 163 differentially expressed proteins in CSF and plasma, respectively, of PNKD patients and healthy controls. For these proteins, blood microparticle and complex activation (classical pathway) were the common GO enrichment term, and complex and coordination cascade pathway were the common KEGG enrichment pathway. Recombinant mothers against decapentaplegic homolog 4 (SMAD4) was one of the differentially expressed proteins; it exhibited a relationship with the aforementioned enrichment GO terms and KEGG pathway. Conclusion: PNKD protein was mainly distributed in cell membranes. PNKD-L mutation affected subcellular localisation, PNKD protein stability, and glutathione content. SMAD4 was found to be a potential biomarker for PNKD diagnosis.