Alterations in Self-Aggregating Neuropeptides in Cerebrospinal Fluid of Patients with Parkinsonian Disorders.

BACKGROUND: Parkinson's disease (PD), progressive supranuclear palsy (PSP), and multiple system atrophy (MSA) present with similar movement disorder symptoms but distinct protein aggregates upon pathological examination. OBJECTIVE: Discovery and validation of candidate biomarkers in parkinsonian disorders for differential diagnosis of subgroup molecular etiologies. METHODS: Untargeted liquid chromatography (LC)-mass spectrometry (MS) proteomics was used for discovery profiling in cerebral spinal fluid (CSF) followed by LC-MS/MS based multiple reaction monitoring for validation of candidates. We compared clinical variation within the parkinsonian cohort including PD subgroups exhibiting tremor dominance (TD) or postural instability gait disturbance and those with detectable leukocytes in CSF. RESULTS: We have identified candidate peptide biomarkers and validated related proteins with targeted quantitative multiplexed assays. Dopamine-drug naïve patients at first diagnosis exhibit reduced levels of signaling neuropeptides, chaperones, and processing proteases for packaging of self-aggregating peptides into dense core vesicles. Distinct patterns of biomarkers were detected in the parkinsonian disorders but were not robust enough to offer a differential diagnosis. Different biomarker changes were detected in male and female patients with PD. Subgroup specific candidate biomarkers were identified for TD PD and PD patients with leukocytes detected in CSF. CONCLUSION: PD, MSA, and PSP exhibit overlapping as well as distinct protein biomarkers that suggest specific molecular etiologies. This indicates common sensitivity of certain populations of selectively vulnerable neurons in the brain, and distinct therapeutic targets for PD subgroups. Our report validates a decrease in CSF levels of self-aggregating neuropeptides in parkinsonian disorders and supports the role of native amyloidogenic proteins in etiologies of neurodegenerative diseases.

Targeted Multiple Reaction Monitoring Analysis of CSF Identifies UCHL1 and GPNMB as Candidate Biomarkers for ALS.

The neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD) share some common molecular deficits including disruption of protein homeostasis leading to disease-specific protein aggregation. While insoluble protein aggregates are the defining pathological confirmation of diagnosis, patient stratification based on early molecular etiologies may identify distinct subgroups within a clinical diagnosis that would respond differently in therapeutic development programs. We are developing targeted multiple reaction monitoring (MRM) mass spectrometry methods to rigorously quantify CSF proteins from known disease genes involved in lysosomal, ubiquitin-proteasomal, and autophagy pathways. Analysis of CSF from 21 PD, 21 ALS, and 25 control patients, rigorously matched for gender, age, and age of sample, revealed significant changes in peptide levels between PD, ALS, and control. In patients with PD, levels of two peptides for chromogranin B (CHGB, secretogranin 1) were significantly reduced. In CSF of patients with ALS, levels of two peptides from ubiquitin carboxy-terminal hydrolase like protein 1 (UCHL1) and one peptide each for glycoprotein non-metastatic melanoma protein B (GPNMB) and cathepsin D (CTSD) were all increased. Analysis of patients with ALS separated into two groups based on length of survival after CSF sampling revealed that the increases in GPNMB and UCHL1 were specific for short-lived ALS patients. While analysis of additional cohorts is required to validate these candidate biomarkers, this study suggests methods for stratification of ALS patients for clinical trials and identifies targets for drug efficacy measurements during therapeutic development.

Nitrogen diagnosis based on dynamic characteristics of rice leaf image.

Digital image processing is widely used in the non-destructive diagnosis of plant nutrition. Previous plant nitrogen diagnostic studies have mostly focused on characteristics of the rice canopy or leaves at some specific points in time, with the long sampling intervals unable to provide detailed and specific "dynamic features." According to plant growth mechanisms, the dynamic changing rate in leaf shape and color differ between different nitrogen supplements. Therefore, the objective of this study was to diagnose nitrogen stress levels by analyzing the dynamic characteristics of rice leaves. Scanning technology was implemented to collect rice leaf images every 3 days, with the characteristics of the leaves from different leaf positions extracted utilizing MATLAB. Newly developed shape characteristics such as etiolation area (EA) and etiolation degree (ED), in addition to shape (area, perimeter) and color characteristics (green, normalized red index, etc.), were used to quantify the process of leaf change. These characteristics allowed sensitive indices to be established for further model validation. Our results indicate that the changing rates in dynamic characteristics, in particular the shape characteristics of the first incomplete leaf (FIL) and the characteristics of the 3rd leaf (leaf color and etiolation indices), expressed obvious distinctions among different nitrogen treatments. Consequently, we achieved acceptable diagnostic accuracy (training accuracy 77.3%, validation accuracy 64.4%) by using the FIL at six days after leaf emergence, and the new shape characteristics developed in this article (ED and EA) also showed good performance in nitrogen diagnosis. Based on the aforementioned results, dynamic analysis is valuable not only in further studies but also in practice.

Proteomic approach to reveal the regulatory function of aconitase AcnA in oxidative stress response in the antibiotic producer Streptomyces viridochromogenes Tü494.

The aconitase AcnA from the phosphinothricin tripeptide producing strain Streptomyces viridochromogenes Tü494 is a bifunctional protein: under iron-sufficiency conditions AcnA functions as an enzyme of the tricarboxylic acid cycle, whereas under iron depletion it is a regulator of iron metabolism and oxidative stress response. As a member of the family of iron regulatory proteins (IRP), AcnA binds to characteristic iron responsive element (IRE) binding motifs and post-transcriptionally controls the expression of respective target genes. A S. viridochromogenes aconitase mutant (MacnA) has previously been shown to be highly sensitive to oxidative stress. In the present paper, we performed a comparative proteomic approach with the S. viridochromogenes wild-type and the MacnA mutant strain under oxidative stress conditions to identify proteins that are under control of the AcnA-mediated regulation. We identified up to 90 differentially expressed proteins in both strains. In silico analysis of the corresponding gene sequences revealed the presence of IRE motifs on some of the respective target mRNAs. From this proteome study we have in vivo evidences for a direct AcnA-mediated regulation upon oxidative stress.

Development of a MALDI-TOF MS strategy for the high-throughput analysis of biomarkers: on-target aptamer immobilization and laser-accelerated proteolysis.

An aptamer-based strategy was developed for the high-throughput analysis of protein biomarkers, such as lysozyme, by on-target MALDI-TOF MS. The aptamers were immobilized on the target plate through formation of covalent bonds with a stable and porous gold layer. An infrared laser was subsequently applied for fast proteolysis. High sensitivities were observed both in standard solutions and human urine.

Developing a strong anion exchange/RP (SAX/RP) 2D LC system for high-abundance proteins depletion in human plasma.

Human plasma is dominated by high-abundance proteins which severely impede the detection of low-abundance proteins. Unfortunately, now there is no efficient method for large-scale depletion of high-abundance proteins in human plasma. In this study, we developed a new strategy, strong anion exchange (SAX)/RP 2D LC system, which has potential for large-scale depletion of high-abundance proteins in human plasma. Separation gradients of the system were optimized to ensure an extensive separation of plasma proteins. Plasma was fractionated into 67 fractions by SAX. All these fractions were subjected a thorough separation by the 2D RPLC and 66 peaks with high UV absorption (>20 mAU) at 215 nm were collected. Proteins in these peaks were identified by LC-MS/MS analysis. Results showed that 83 proteins could be identified in these peaks, 68 among them were reported to be high- or middle-abundance proteins in plasma. All these proteins had definite retention times and were mapped in the 2D SAX-RP system, which resulted in accurate depletion of high-abundance proteins with ease. Our studies provide a convenient and effective method for large-scale depletion of high-abundance proteins and in-depth research in human plasma proteomics.

Neurocognitive and clinical dysfunction in adult Chinese, nonpsychotic relatives of patients with schizophrenia: Findings from the Changsha study and evidence for schizotaxia.

Many first-degree relatives of patients with schizophrenia demonstrate deficits in neurocognitive, social, clinical and other dimensions, in the absence of psychosis. Based on a reformulation of Meehl's concept of "schizotaxia" as a clinically meaningful syndrome reflecting liability to schizophrenia, we proposed research criteria in relatives focused on negative symptoms and neurocognitive deficits. Here we assess validity of the syndrome in a sample of Chinese adult relatives by assessing measures of concurrent validity, and by using cluster analysis to test the hypothesis that relatives could be grouped into distinct schizotaxic and non-schizotaxic subgroups based on our diagnostic criteria. Thirty community comparison subjects (CCS) and 189 relatives were evaluated with measures of clinical, cognitive, medical and social function at the Mental Health Institute, Second Xiangya Hospital of Central South University, Changsha (Hunan, China), as part of a larger study to identify and ameliorate symptoms of schizotaxia. Using modified research criteria based on negative symptoms and neurocognitive deficits, 103 relatives did not meet criteria for schizotaxia, and 86 did. The cluster analysis confirmed a two-group solution that corresponded to our non-schizotaxic and schizotaxic groups, but it increased the non-schizotaxic group to 135, and reduced the schizotaxic group to 53. Both schizotaxic groups, but especially the cluster-derived group, showed significant impairment in a variety of independent (i.e. non-criterion related) measures of clinical and social function. These findings provide additional validity for a liability syndrome, and for its utility as an intervention target for strategies aimed at ameliorating both its core and its associated symptoms.

Are neurocognitive, clinical and social dysfunctions in schizotaxia reversible pharmacologically?: Results from the Changsha study.

The Changsha study identifies adult, non-psychotic relatives of patients with schizophrenia who show deficits in neurocognitive, social, clinical and other dimensions, and who meet provisional criteria for a liability syndrome for schizophrenia ('schizotaxia'). In this study, we investigated whether negative symptoms, neurocognitive deficits, or other measures of clinical and social function in subjects who met our research criteria for schizotaxia were amenable to pharmacological remediation with a low dose (2.0 mg) of risperidone, a second generation antipsychotic medication. One hundred eighty nine relatives were assessed at the Mental Health Institute, Second Xiangya Hospital of Central South University, Changsha (Hunan Province, China), between 12/06 - 12/08. Eighty six of these individuals met modified criteria for schizotaxia, and 36 agreed to enter a 6-week, double-blind, placebo-controlled protocol. ANCOVAs using age and gender as covariates showed significant improvement in the risperidone group (n=20) on neurocognitive function (Wisconsin Card Sorting Test Total Errors and Perseverative Errors) and on a self-report measure of social function (Social Adjustment Scale), compared to the placebo-control group (n=16). Effect sizes were small to medium. Notably, risperidone effect sizes were larger (medium to large) in a subset of subjects (risperidone=15; placebo=10) whose membership in the schizotaxic group was supported empirically by cluster analysis. Negative symptoms did not change significantly in either analysis. The results are generally consistent with previous open-label investigations of risperidone administration in subjects with schizotaxia, and provide evidence that some neurocognitive and clinical problems are amenable to remediation in non-psychotic relatives of people with schizophrenia.

An aptamer based on-plate microarray for high-throughput insulin detection by MALDI-TOF MS.

An aptamer microarray was directly fabricated on a MALDI target plate for high-throughput insulin detection. High sensitivities were observed both in standard solutions (5 ng mL(-1), 0.86 nM) and serum sample (20 ng mL(-1), 3.4 nM). This method shows great promise in the field of biomarker detection.

Highly sensitive thrombin detection by matrix assisted laser desorption ionization-time of flight mass spectrometry with aptamer functionalized core-shell Fe3O4@C@Au magnetic microspheres.

Here, we describe a sensitive and specific method for thrombin detection with aptamer functionalized core-shell Fe(3)O(4)@C@Au magnetic microspheres (Au-MMPs). Firstly, Au-MMPs were synthesized through surface adsorption of gold nanoparticles onto PDDA functionalized Fe(3)O(4)@C magnetic microspheres. Then, the as-synthesized Au-MMPs were developed as new substrate for immobilization of thrombin binding aptamer (TBA) through easy formation of Au-S bond. After that, the prepared aptamer functionalized Au-MMPs (TBA@Au-MMPs) were used as effective magnetic absorbent to extract trace level of thrombin from dilute solutions. Finally, enriched thrombin was digested by trypsin and analyzed by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Taking advantage of the efficient affinity extraction ability of our TBA@Au-MMPs and the sensitive mass readout of MALDI-TOF, highly sensitive detection of thrombin was achieved. The limit of detection was as low as 18 fmol, corresponding to 0.36 nM thrombin in 50 µL original solution. Linear relation was observed within a concentration range from 0.5 nM to 10nM with linear correlation R(2)=0.998. Other proteins including human serum albumin (HSA), Ig G, transferrin, oval albumin (OVA) and fetal calf serum did not interfere with thrombin detection. This simple method holds great potential for analyzing, sensing, purification and preconcentration of proteins in biological fluids.

Rapid and efficient proteolysis through laser-assisted immobilized enzyme reactors.

In this report, laser radiation (808nm) for the first time was employed to enhance the efficiency of proteolysis through immobilized enzyme reactor (IMER). IMER based monolithic support was prepared in the fused-silica capillary via a simple two-step procedure including acryloylation on trypsin surface and in situ aqueous polymerization/immobilization. The feasibility and high efficiency of the laser-assisted IMER were demonstrated by the digestion of bovine serum albumin (BSA), cytochrome c (Cyt-c) and ß-casein. The digestion process was achieved in 60s. The peptides were identified by MALDI-TOF-MS, yielding the sequence coverage of 33% for BSA, 73% for Cyt-c and 22% for ß-casein. The comparisons between the in-solution digestion and on IMER reaction with/without laser assistance were made. To further confirm its efficiency in proteome analysis, the laser-assisted IMER was also applied to the analysis of one fraction of human serum sample through two-dimensional (2-D) separation of strong anion exchange/reversed-phase liquid chromatography (SAX/RPLC). After a database search, 49 unique peptides corresponding to 5 proteins were identified. The results showed that the laser-assisted IMER provides a promising platform for the high-throughput protein identification.

[Construction of a two-dimensional liquid chromatography separation system for high abundance proteins depletion in human plasma].

High abundance proteins existing in human plasma severely impede the detection of low abundance proteins. This is one of the most difficult problems encountered in plasma proteomics research. We developed a two-dimensional liquid chromatography system with strong anion exchange chromatography-reversed-phase liquid chromatography (SAX-RPLC) for the extensive separation of plasma proteins and selective depletion of high abundance proteins. TSKgel SuperQ-5PW was selected as the first dimensional separation column for crude human plasma fractionation and Jupiter C4 column was selected as the second dimensional separation column. Separation gradients of the two-dimensional liquid chromatography system were optimized to ensure an extensive separation of plasma proteins. Ten peaks with high signal intensities ( >20 mAU) at 215 nm during the second dimensional separation were collected and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). As a result, 32 proteins, all of which were reported to be high abundance proteins in plasma, including human serum albumin (HSA), immunoglobulin G (IgG) and so on were successfully identified. This system provides an effective method for future depletion of more high abundance proteins and in-depth research in human plasma proteomics.

[Cloning, expression and identification of membrane protein Tetraspanin 2-A of Schistosoma japonicum].

OBJECTIVE: To clone and express a membrane protein (Tetraspanin 2) gene of Schistosoma japonicum (SjTsp2). METHODS: A pair of primers was designed to amplify the SjTsp2 gene which was subcloned into prokaryotic plasmid pET28a(+). The recombinant plasmid was transformed into E. coli BL21(D3) and followed by expression of the protein induced by IPTG. The protein was purified by affinity chromatography and used to immunize BALB/c mice. Dilution of antibody against SjTsp2 was determined by ELISA. The protein was also identified by Western blotting. RESULTS: Big loop of SjTsp2-A, 228 bp, was amplified in vitro by PCR. Its deduced amino sequence shared 52% similarity with SmTsp2. The soluble recombinant SjTsp2-A was expressed in the experiment and high dilution antibody against the recombinant (1:32,000 in maximum) was produced in immunized mice. SjTsp2-A reacted positively with sera of acute and chronic schistosomiasis patients but not with sera from healthy persons by Western blotting. CONCLUSION: SjTsp2 has been expressed and shows certain antigenicity.

Schistosoma japonicum: inhibition of Mago nashi gene expression by shRNA-mediated RNA interference.

RNA interference (RNAi) mediated by short interfering RNA (siRNA) is a powerful reverse genetics tool and holds enormous therapeutic potential for various diseases, including parasite infections. siRNAs bind their complementary mRNA and lead to degradation of their specific mRNA targets. RNAi has been widely used for functional analysis of specific genes in various cells and organisms. In this paper, we tested the potential of silencing the expression of the Mago nashi gene in Schistosoma japonicum by siRNAs derived from shRNA expressed by mammalian Pol III promoter H1. Schistosomula, transformed from cercariae by mechanical shearing of the tails, were electroporated with Mago nashi shRNA expression vector. Aliquots of parasites were harvested at days 1, 3, and 5 after electroporation, respectively. Levels of Mago nashi mRNA and protein were determined by RT-PCR and Western blotting analysis. The results showed that shRNA expressed from mammalian Pol III promoter H1 specifically reduced the levels of Mago nashi mRNA and proteins in S. japonicum. Changes in testicular lobes were apparent when parasites were introduced into mammalian hosts. Thus, vector-mediated gene silencing is applicable to S. japonicum, which provides a means for the functional analysis of genes in this organism.

[Immunoscreening of schistosomulum cDNA library of Schistosoma japonicum and preliminary identification of membrane-associated protein abundant in tegument].

OBJECTIVE: To obtain genes encoding the novel molecules for diagnosis of schistosomiasis. METHODS: Juvenile S. japonicum cDNA library was immunoscreened to obtain positive clones. By DNA sequencing and sequence analysis, the target gene was amplified by PCR and subcloned into prokaryotic plasmid pET28a. The recombinant plasmid was transformed into E. coli BL21 followed by expression of the protein induced by IPTG. The protein was identified by Western blotting. RESULTS: 34 positive clones were obtained, 24 of which were chosen to be sequenced, 13 of which were Sj22600 gene. The protein were recognized with sera of infected rabbits and patients with acute and chronic schistosomiasis by Western blotting. CONCLUSION: The gene coding for Sj22600 membrane protein was screened with high frequency in the cDNA library. The E. coli BL21 transformed with the recombinant plasmid can express the fusion protein, which shows immunoactivity.