Identification of potential serum markers for nasopharyngeal carcinoma from a xenografted mouse model using Cy-dye labeling combined with three-dimensional fractionation

Nasopharyngeal carcinoma (NPC), one of the most common cancers in Southeast Asia, is commonly diagnosed late due to its deep location and vague symptoms. To identify biomarkers for improving NPC diagnosis, we established a proteomic platform for detecting aberrant serum proteins in nude mice bearing NPC xenografts. We first removed the three most abundant proteins from serum samples of tumor-bearing and control mice, and then labeled the samples with different fluorescent cyanine (Cy) dyes. The labeled serum proteins were then mixed equally and fractionated with ion-exchange chromatography followed by SDS-PAGE. Differentially expressed proteins were identified by in-gel tryptic digestion and MALDI-TOF MS. We identified peroxiredoxin 2 (Prx-II) and carbonic anhydrase 2 (CA-II) as being elevated in the xenograft mouse model compared to controls. Western blot analysis confirmed up-regulation of Prx-II and CA-II in plasma from five NPC patients, and ELISA showed that plasma Prx-II levels were significantly higher in NPC patients (n = 84) versus healthy controls (n = 90) (3.03 +/- 4.47 versus 1.90 +/- 2.74 microg/mL, p = 0.047). In conclusion, Cy dye labeling combined with three-dimensional fractionation is a feasible strategy for identifying differentially expressed serum proteins in an NPC xenograft model, and Prx-II may represent a potential NPC biomarker.     

基于蛋白组学的脓毒症补体凝血级联通路及标志物KLKB1研究

摘要 目的：通过蛋白质组学方法鉴定脓毒症关键通路及诊断标志物。方法：选取2019年1月至12月西南医科大学附属医院急诊科收治的56例脓毒症患者（脓毒症组）,另取同期50名健康体检志愿者（对照组）。采用随机抽样法分别选取两组中12名脓毒症患者和8名健康体检志愿者,利用非数据依赖模式（DIA）进行血清蛋白数据采集,将数据上传至iDEP在线平台分析脓毒症患者外周血中差异表达蛋白,进一步对这些差异蛋白进行生物信息学分析,包括主成分分析（PCA）、基因本体富集分析（GO）、通路富集分析和蛋白-蛋白相互作用网络（PPI）分析,进而筛选出脓毒症关键蛋白。采用酶联免疫吸附试验（ELISA）对脓毒症组、对照组进行关键蛋白表达验证分析。采用受试者工作特征（ROC）曲线分析关键蛋白对脓毒症的诊断效能。结果：蛋白质组学分析共鉴定出690个蛋白,筛选出171个差异表达蛋白（DEPs）,其中39个蛋白显著下调,132个蛋白显著上调。DEPs富集的核心通路为补体和凝血级联通路。该条通路中的血清激肽释放酶 1（KLKB1）在脓毒症组的表达水平为（121.80±55.63 ng/mL）,显著高于对照组的（68.30±57.11 ng/mL）,差异具有统计学意义（t=4.881,P=0.000）。根据ELISA结果进行脓毒症诊断ROC曲线分析得出,KLKB1蛋白诊断脓毒症的 AUC（95%CI）为0.759（0.594～0.923）。结论：补体和凝血级联通路为脓毒症免疫途径的重要通路,KLKB1具有较好的脓毒症诊断特性,可能是脓毒症潜在的诊断生物标志物。     

Plasma Peptide Biomarker Discovery for Amyotrophic Lateral Sclerosis by MALDI –TOF Mass Spectrometry Profiling

The diagnostic of Amyotrophic lateral sclerosis (ALS) remains based on clinical and neurophysiological observations. The actual delay between the onset of the symptoms and diagnosis is about 1 year, preventing early inclusion of patients into clinical trials and early care of the disease. Therefore, finding biomarkers with high sensitivity and specificity remains urgent. In our study, we looked for peptide biomarkers in plasma samples using reverse phase magnetic beads (C18 and C8) and MALDI-TOF mass spectrometry analysis. From a set of ALS patients (n=30) and healthy age-matched controls (n=30), C18- or C8-SVM-based models for ALS diagnostic were constructed on the base of the minimum of the most discriminant peaks. These two SVM-based models end up in excellent separations between the 2 groups of patients (recognition capability overall classes > 97%) and classify blinded samples (10 ALS and 10 healthy age-matched controls) with very high sensitivities and specificities (>90%). Some of these discriminant peaks have been identified by Mass Spectrometry (MS) analyses and correspond to (or are fragments of) major plasma proteins, partly linked to the blood coagulation.     

Identification of putative serum glycoprotein biomarkers for human lung adenocarcinoma by multilectin affinity chromatography and LC-MS/MS

Glycoproteins in human serum play fundamental roles in many biological processes, and also have clinical value as biomarkers for disease progression and treatment. In this study, we isolated glycoproteins from the sera of three healthy individuals and three lung adenocarcinoma patients using multilectin affinity chromatography. The recovered glycoproteins were subjected to treatment with peptide-N-glycosidase F (PNGase F) and in-gel digestion by trypsin. Tryptic peptides were analyzed by nano-LC coupled to ESI-MS/MS and the MS/MS spectra were processed by Bioworks 3.2 and an in-house bioinformatics tool, ProtAn. Approximately 90% of the proteins identified contained more than one potential glycosylation site. Comparison of the serum glycoproteome of healthy and adenocarcinoma individuals revealed 38 cancer-selective proteins. Among them, 60% have previously been reported as low abundance proteins in human sera. We identified several cancer-selective proteins that have been previously characterized as potential indicators of lung cancer in serum or plasma, including haptoglobin (HP), inter-alpha-trypsin inhibitor heavy chain 4 (ITI-H4), complement C3 precursor, and leucine-rich alpha-2-glycoprotein. In addition, plasma kallikrein (KLKB1) and inter-alpha-trypsin inhibitor heavy chain 3 (ITI-H3) were identified as being potentially elevated in the lung cancer group, and were validated by Western blot analysis. Furthermore, approximately 18 kDa plasma kallirein protein fragment was detected at high levels in 25 out of 28 adenocarcinoma patients, while one of the eight normal individuals showed moderate positive. The results suggest that KLKB1 represents a potential candidate serum biomarker of lung cancer.     

A differential proteomics study of cerebrospinal fluid from individuals with Niemann-Pick disease,Type C1

Niemann-Pick, type C1 (NPC1) is a fatal, neurodegenerative disease, which belongs to the family of lysosomal diseases. In NPC1, endo/lysosomal accumulation of unesterified cholesterol and sphingolipids arise from improper intracellular trafficking resulting in multi-organ dysfunction. With the proximity between the brain and cerebrospinal fluid (CSF), performing differential proteomics provides a means to shed light to changes occurring in the brain. In this study, CSF samples obtained from NPC1 individuals and unaffected controls were used for protein biomarker identification. A subset of these individuals with NPC1 are being treated with miglustat, a glycosphingolipid synthesis inhibitor. Of the 300 identified proteins, 71 proteins were altered in individuals with NPC1 compared to controls including cathepsin D, and members of the complement family. Included are a report of 10 potential markers for monitoring therapeutic treatment. We observed that pro-neuropeptide Y (NPY) was significantly increased in NPC1 individuals relative to healthy controls; however, individuals treated with miglustat displayed levels comparable to healthy controls. In further investigation, NPY levels in a NPC1 mouse model corroborated our findings. We posit that NPY could be a potential therapeutic target for NPC1 due to its multiple roles in the central nervous system such as attenuating neuroinflammation and reducing excitotoxicity.     

Plasma biomarkers for detecting Hodgkin's lymphoma in HIV patients

The lifespan of people with human immunodeficiency virus (HIV) infection has increased as a result of effective antiretroviral therapy, and the incidences of the AIDS-defining cancers, non-Hodgkin''s lymphoma and Kaposi sarcoma, have declined. Even so, HIV-infected individuals are now at greater risk of other cancers, including Hodgkin''s lymphoma (HL). To identify candidate biomarkers for the early detection of HL, we undertook an accurate mass and elution time tag proteomics analysis of individual plasma samples from either HIV-infected patients without HL (controls; n = 14) and from HIV-infected patient samples with HL (n = 22). This analysis identified 60 proteins that were statistically (p<0.05) altered and at least 1.5-fold different between the two groups. At least three of these proteins have previously been reported to be altered in the blood of HL patients that were not known to be HIV positive, suggesting that these markers may be broadly useful for detecting HL. Ingenuity Pathway Analysis software identified “inflammatory response” and “cancer” as the top two biological functions associated with these proteins. Overall, this study validated three plasma proteins as candidate biomarkers for detecting HL, and identified 57 novel candidate biomarkers that remain to be validated. The relationship of these novel candidate biomarkers with cancer and inflammation suggests that they are truly associated with HL and therefore may be useful for the early detection of this cancer in susceptible populations.     

基于脂质组学技术研究急性缺血性脑卒中临床诊断标志物

目的:急性缺血性脑卒中(Acute ischemic stroke, AIS)是由于血流减少导致的脑功能突然丧失。由于AIS发病机制是异质性和多因素的,我们建立全面的脂质组学方法来阐明AIS进程相关的脂质变化和复杂的脂质代谢网络。方法:选取26例AIS患者血液标本和27例健康志愿者血清作为研究对象,进行总脂抽提,通过基于LC-MS策略的非靶向脂质组学方法进行规模性、整体性的脂质组学分析。结果:对AIS患者和健康志愿者血浆进行大规模脂质定性定量分析,通过Progenesis~? QI软件分析Xevo~? G2-XS QTOF质谱系统MSE采集的子离子数据,精确定量到1054个脂质特征差异,准确定性得到368个脂质分子,多变量统计分析中差异脂质组成能将AIS患者和健康志愿者区分开来,通路富集分析图显示差异脂质主要参与甘油磷脂代谢的紊乱。结论:AIS患者血浆脂质组成与健康志愿者存在显著差异,差异表达的脂质可能与AIS发生有关。这些发现有助于开发新的诊断标志物和AIS治疗靶点。     

Resistin,Elastase, and Lactoferrin as Potential Plasma Biomarkers of Pediatric Inflammatory Bowel Disease Based on Comprehensive Proteomic Screens

Inflammatory bowel disease (IBD) is an immune-mediated chronic inflammation of the intestine, which can present in the form of ulcerative colitis (UC) or as Crohn’s disease (CD). Biomarkers are needed for reliable diagnosis and disease monitoring in IBD, especially in pediatric patients. Plasma samples from a pediatric IBD cohort were interrogated using an aptamer-based screen of 1322 proteins. The elevated biomarkers identified using the aptamer screen were further validated by ELISA using an independent cohort of 76 pediatric plasma samples, drawn from 30 CD, 30 UC, and 16 healthy controls. Of the 1322 proteins screened in plasma from IBD patients, 129 proteins were significantly elevated when compared with healthy controls. Of these 15 proteins had a fold change greater than 2 and 28 proteins had a fold change >1.5. Neutrophil and extracellular vesicle signatures were detected among the elevated plasma biomarkers. When seven of these proteins were validated by ELISA, resistin was the only protein that was significantly higher in both UC and CD (p < 0.01), with receiver operating characteristic area under the curve value of 0.82 and 0.77, respectively, and the only protein that exhibited high sensitivity and specificity for both CD and UC. The next most discriminatory plasma proteins were elastase and lactoferrin, particularly for UC, with receiver operating characteristic area under the curve values of 0.74 and 0.69, respectively. We have identified circulating resistin, elastase, and lactoferrin as potential plasma biomarkers of IBD in pediatric patients using two independent diagnostic platforms and two independent patient cohorts.     

Serum protein profiling to identify biomarkers for small renal cell carcinoma

Diagnostic biomarkers for early detection of renal cell carcinoma (RCC) are in great need. In the present study, we compared the serum protein profiles of patients with small RCC to those of healthy individuals to identify the differentially expressed proteins with potential to serve as biomarkers. Serum samples were collected from 10 patients with small RCC and 10 healthy individuals. The serum protein expression profiles were analyzed by two-dimensional (2-D) gel electrophoresis. Twenty-seven proteins with differences in expression levels between RCC patients and healthy volunteers were identified. Of these, 19 were expressed at different levels and eight were expressed in serum from the RCC group, but not from the control group. Six differentially expressed proteins identified by using mass spectrometry included coagulation factor XIII B, complement C3 and its precursor, misato homolog 1 (isoform CRA_b), hemopexin, and alpha-1-B-glycoprotein. Some of these serum proteins are known regulators of tumor progression in human malignancies. In conclusion, we successfully applied 2-D gel electrophoresis and identified six serum proteins differentially expressed between patients with small RCC and healthy volunteers. These proteins may provide novel biomarkers for early detection and diagnosis of human RCC.     

The ovarian cancer-derived secretory/releasing proteome: A repertoire of tumor markers

Ovarian cancer is the most lethal gynecological malignancy worldwide, and early detection of this disease using serum or plasma biomarkers may improve its clinical outcome. In the present study, a large scale protein database derived from ovarian cancer was created to enable tumor marker discovery. First, primary organ cultures were established with the tumor tissues and corresponding normal tissues obtained from six ovarian cancer patients, and the serum-free conditioned medium (CM) samples were collected for proteomic analysis. The total proteins from the CM sample were separated by SDS-PAGE, digested with trypsin and then analyzed by LC-MS/MS. Combining data from the tumor tissues and the normal tissues, 1129 proteins were identified in total, of which those categorized as "extracellular proteins" and "plasma membrane proteins" accounted for 21.4% and 16.9%, respectively. For validation, three secretory proteins (NID1, TIMP2, and VCAN) involved in "organ development"-associated subnetwork, showed significant differences between their levels in the circulating plasma samples from ovarian cancer patients and healthy women. In conclusion, this ovarian cancer-derived protein database provides a credible repertoire of potential biomarkers in blood for this malignant disease, and deserves mining further.     

Coagulation Changes during Presyncope and Recovery

Orthostatic stress activates the coagulation system. The extent of coagulation activation with full orthostatic load leading to presyncope is unknown. We examined in 7 healthy males whether presyncope, using a combination of head up tilt (HUT) and lower body negative pressure (LBNP), leads to coagulation changes as well as in the return to baseline during recovery. Coagulation responses (whole blood thrombelastometry, whole blood platelet aggregation, endogenous thrombin potential, markers of endothelial activation and thrombin generation), blood cell counts and plasma mass density (for volume changes) were measured before, during, and 20 min after the orthostatic stress. Maximum orthostatic load led to a 25% plasma volume loss. Blood cell counts, prothrombin levels, thrombin peak, endogenous thrombin potential, and tissue factor pathway inhibitor levels increased during the protocol, commensurable with hemoconcentration. The markers of endothelial activation (tissue factor, tissue plasminogen activator), and thrombin generation (F1+2, prothrombin fragments 1 and 2, and TAT, thrombin-antithrombin complex) increased to an extent far beyond the hemoconcentration effect. During recovery, the markers of endothelial activation returned to initial supine values, but F1+2 and TAT remained elevated, suggestive of increased coagulability. Our findings of increased coagulability at 20 min of recovery from presyncope may have greater clinical significance than short-term procoagulant changes observed during standing. While our experiments were conducted in healthy subjects, the observed hypercoagulability during graded orthostatic challenge, at presyncope and in recovery may be an important risk factor particularly for patients already at high risk for thromboembolic events (e.g. those with coronary heart disease, atherosclerosis or hypertensives).     

Lack of Plasma Kallikrein-Kinin System Cascade in Teleosts

The kallikrein-kinin system (KKS) consists of two major cascades in mammals: “plasma KKS” consisting of high molecular-weight (HMW) kininogen (KNG), plasma kallikrein (KLKB1), and bradykinin (BK); and “tissue KKS” consisting of low molecular-weight (LMW) KNG, tissue kallikreins (KLKs), and [Lys⁰]-BK. Some components of the KKS have been identified in the fishes, but systematic analyses have not been performed, thus this study aims to define the KKS components in teleosts and pave a way for future physiological and evolutionary studies. Through a combination of genomics, molecular, and biochemical methods, we showed that the entire plasma KKS cascade is absent in teleosts. Instead of two KNGs as found in mammals, a single molecular weight KNG was found in various teleosts, which is homologous to the mammalian LMW KNG. Results of molecular phylogenetic and synteny analyses indicated that the all current teleost genomes lack KLKB1, and its unique protein structure, four apple domains and one trypsin domain, could not be identified in any genome or nucleotide databases. We identified some KLK-like proteins in teleost genomes by synteny and conserved domain analyses, which could be the orthologs of tetrapod KLKs. A radioimmunoassay system was established to measure the teleost BK and we found that [Arg⁰]-BK is the major circulating form instead of BK, which supports that the teleost KKS is similar to the mammalian tissue KKS. Coincidently, coelacanths are the earliest vertebrate that possess both HMW KNG and KLKB1, which implies that the plasma KKS could have evolved in the early lobe-finned fish and descended to the tetrapod lineage. The co-evolution of HMW KNG and KLKB1 in lobe-finned fish and early tetrapods may mark the emergence of the plasma KKS and a contact activation system in blood coagulation, while teleosts may have retained a single KKS cascade.     

Label-free quantitative proteomic analysis reveals dysfunction of complement pathway in peripheral blood of schizophrenia patients: evidence for the immune hypothesis of schizophrenia

Schizophrenia is a complex mental disease caused by a combination of serial alterations in genetic and environmental factors. Although the brain is usually considered as the most relevant organ in schizophrenia, accumulated evidence suggests that peripheral tissues also contribute to this disease. In particular, abnormalities of the immune system have been identified in the peripheral blood of schizophrenia patients. To screen the serum proteomic signature of schizophrenia patients, we conducted shotgun proteomic analysis on serum samples of schizophrenia patients and healthy controls. High-abundance proteins were eliminated by immunoaffinity before LC-MS/MS analysis. The multivariate statistical test partial least squares-discriminant analysis (PLS-DA) was applied to build models for screening out variable importance in the projection (VIP) and 27 proteins were identified as being responsible for discriminating between the proteomic profiles of schizophrenia patients and healthy controls. Pathway analysis based on these 27 proteins revealed that complement and coagulation cascades was the most significant pathway. ELISA-based activity analyses indicated that the alternative complement pathway was suppressed in schizophrenia patients. Ingenuity pathways analysis was used to conduct the interaction network of 27 proteins. The network exhibited common features such as, nervous system development and function, humoral immune response and inflammatory response, and highlighted some proteins with important roles in the immune system, such as hub nodes. Our findings indicate dysregulation of the alternative complement pathway in schizophrenia patients. The protein interaction network enhances the interpretation of proteomic data and provides evidence that the immune system may contribute to schizophrenia.     

Investigation of plasma biomarkers in HIV-1/HCV mono- and coinfected individuals by multiplex iTRAQ quantitative proteomics

The analysis of plasma samples from HIV-1/HCV mono- and coinfected individuals by quantitative proteomics is an efficient strategy to investigate changes in protein abundances and to characterize the proteins that are the effectors of cellular functions involved in viral pathogenesis. In this study, the infected and healthy plasma samples (in triplicate) were treated with ProteoMiner beads to equalize protein concentrations and subjected to 4-plex iTRAQ labeling and liquid chromatography/mass spectrometry (LC-MS/MS) analysis. A total of 70 proteins were identified with high confidence in the triplicate analysis of plasma proteins and 65% of the proteins were found to be common among the three replicates. Apolipoproteins and complement proteins are the two major classes of proteins that exhibited differential regulation. The results of quantitative analysis revealed that APOA2, APOC2, APOE, C3, HRG proteins were upregulated in the plasma of all the three HIV-1 mono-, HCV mono-, and coinfected patient samples compared to healthy control samples. Ingenuity pathway analysis (IPA) of the upregulated proteins revealed that they are implicated in the hepatic lipid metabolism, inflammation, and acute-phase response signaling pathways. Thus, we identified several differentially regulated proteins in HIV-1/HCV mono and coinfected plasma samples that may be potential biomarkers for liver disease.     

microRNAs in Circulation Are Altered in Response to Influenza A Virus Infection in Humans

Changes in microRNA expression have been detected in vitro in influenza infected cells, yet little is known about them in patients. microRNA profiling was performed on whole blood of H1N1 patients to identify signature microRNAs to better understand the gene regulation involved and possibly improve diagnosis. Total RNA extracted from blood samples of influenza infected patients and healthy controls were subjected to microRNA microarray. Expression profiles of circulating microRNAs were altered and distinctly different in influenza patients. Expression of highly dysregulated microRNAs were validated using quantitative PCR. Fourteen highly dysregulated miRNAs, identified from the blood of influenza infected patients, provided a clear distinction between infected and healthy individuals. Of these, expression of miR-1260, -26a, -335*, -576-3p, -628-3p and -664 were consistently dysregulated in both whole blood and H1N1 infected cells. Potential host and viral gene targets were identified and the impact of microRNA dysregulation on the host proteome was studied. Consequences of their altered expression were extrapolated to changes in the host proteome expression. These highly dysregulated microRNAs may have crucial roles in influenza pathogenesis and are potential biomarkers of influenza.     

A wide range of protein isoforms in serum and plasma uncovered by a quantitative intact protein analysis system

We have implemented an orthogonal 3-D intact protein analysis system (IPAS) to quantitatively profile protein differences between human serum and plasma. Reference specimens consisting of pooled Caucasian-American serum, citrate-anticoagulated plasma, and EDTA-anticoagulated plasma were each depleted of six highly abundant proteins, concentrated, and labeled with a different Cy dye (Cy5, Cy3, or Cy2). A mixture consisting of each of the labeled samples was subjected to three dimensions of separation based on charge, hydrophobicity, and molecular mass. Differences in the abundance of proteins between each of the three samples were determined. More than 5000 bands were found to have greater than two-fold difference in intensity between any pair of labeled specimens by quantitative imaging. As expected, some of the differences in band intensities between serum and plasma were attributable to proteins related to coagulation. Interestingly, many proteins were identified in multiple fractions, each exhibiting different pI, hydrophobicity, or molecular mass. This is likely reflective of the expression of different protein isoforms or specific protein cleavage products, as illustrated by complement component 3 precursor and clusterin. IPAS provides a high resolution, high sensitivity, and quantitative approach for the analysis of serum and plasma proteins, and allows assessment of PTMs as a potential source of biomarkers.     

Genomic structure of the human plasma prekallikrein gene, identification of allelic variants, and analysis in end-stage renal disease

Kallikreins are serine proteases that catalyze the release of kinins and other vasoactive peptides. Previously, we have studied one tissue-specific (H. Yu et al., 1996, J. Am. Soc. Nephrol. 7: 2559-2564) and one plasma-specific (H. Yu et al., 1998, Hypertension 31: 906-911) human kallikrein gene in end-stage renal disease (ESRD). Short sequence repeat polymorphisms for the human plasma kallikrein gene (KLKB1; previously known as KLK3) on chromosome 4 were associated with ESRD in an African American study population. This study of KLKB1 in ESRD has been extended by determining the genomic structure of KLKB1 and searching for allelic variants that may be associated with ESRD. Exon-spanning PCR primer sets were identified by serial testing of primer pairs designed from KLKB1 cDNA sequence and DNA sequencing of PCR products. Like the rat plasma kallikrein gene and the closely related human factor XI gene, the human KLKB1 gene contains 15 exons and 14 introns. The longest intron, F, is almost 12 kb long. The total length of the gene is approximately 30 kb. Sequence of the 5'-proximal promoter region of KLKB1 was obtained by shotgun cloning of genomic fragments from a bacterial artificial clone containing the KLKB1 gene, followed by screening of the clones using exon 1-specific probes. Primers flanking the exons and 5'-proximal promoter region were used to screen for allelic variants in the genomic DNA from ESRD patients and controls using the single-strand conformation polymorphism technique. We identified 12 allelic variants in the 5'-proximal promoter and 7 exons. Of note were a common polymorphism (30% of the population) at position 521 of KLKB1 cDNA, which leads to the replacement of asparagine with a serine at position 124 in the heavy chain of the A2 domain of the protein. In addition, an A716C polymorphism in exon 7 resulting in the amino acid change H189P in the A3 domain of the heavy chain was observed in 5 patients belonging to 3 ESRD families. A third polymorphism in the coding sequence was a C699A shift that caused an amino acid change, H183Q. This allele was observed in 8 cases from 6 ESRD families but was not found in any control DNAs. Individually or combined, the allelic variants observed are not statistically associated with ESRD, though in several cases (e.g., H183Q) the small number of people in the population carrying these alleles limits our ability to statistically test for significant association with ESRD. Two new CA/GT repeat polymorphic markers, designated KLK3f and KLK3g, that have heterozygosities of 0.65 and 0.84, respectively, were identified within introns M and N. Analysis using the relative predispositional effect technique indicated that the frequencies of alleles 4 and 8 of KLK3f and allele 8 of KLK3g were significantly different between controls and ESRD cases. They accounted for 0.226, 0.096, and 0.313, respectively, in the probands of 166 ESRD families compared to 0.172, 0.066, and 0.244 in 139 healthy race-matched controls (allele P and total P < 0.05 for all three alleles). Therefore, although polymorphisms in the coding and 5'-proximal promoter of KLKB1 show no statistically significant association with ESRD in African Americans, there is still evidence for association of this part of chromosome 4 with ESRD. This observation suggests that other sequences within or near KLKB1, or another gene nearby, may contribute to ESRD susceptibility.     

Quantitative Proteomic Analysis of Niemann-Pick Disease,Type C1 Cerebellum Identifies Protein Biomarkers and Provides Pathological Insight

Niemann-Pick disease, type C1 (NPC1) is a fatal, neurodegenerative disorder for which there is no definitive therapy. In NPC1, a pathological cascade including neuroinflammation, oxidative stress and neuronal apoptosis likely contribute to the clinical phenotype. While the genetic cause of NPC1 is known, we sought to gain a further understanding into the pathophysiology by identifying differentially expressed proteins in Npc1 mutant mouse cerebella. Using two-dimensional gel electrophoresis and mass spectrometry, 77 differentially expressed proteins were identified in Npc1 mutant mice cerebella compared to controls. These include proteins involved in glucose metabolism, detoxification/oxidative stress and Alzheimer disease-related proteins. Furthermore, members of the fatty acid binding protein family, including FABP3, FABP5 and FABP7, were found to have altered expression in the Npc1 mutant cerebellum relative to control. Translating our findings from the murine model to patients, we confirm altered expression of glutathione s-transferase α, superoxide dismutase, and FABP3 in cerebrospinal fluid of NPC1 patients relative to pediatric controls. A subset of NPC1 patients on miglustat, a glycosphingolipid synthesis inhibitor, showed significantly decreased levels of FABP3 compared to patients not on miglustat therapy. This study provides an initial report of dysregulated proteins in NPC1 which will assist with further investigation of NPC1 pathology and facilitate implementation of therapeutic trials.