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Psychosis is a severe mental condition that is characterized by a loss of contact with reality and is typically associated with hallucinations and delusional beliefs. There are numerous psychiatric conditions that present with psychotic symptoms, most importantly schizophrenia, bipolar affective disorder, and some forms of severe depression referred to as psychotic depression. The pathological mechanisms resulting in psychotic symptoms are not understood, nor is it understood whether the various psychotic illnesses are the result of similar biochemical disturbances. The identification of biological markers (so-called biomarkers) of psychosis is a fundamental step towards a better understanding of the pathogenesis of psychosis and holds the potential for more objective testing methods.
Methods and Findings
Surface-enhanced laser desorption ionization mass spectrometry was employed to profile proteins and peptides in a total of 179 cerebrospinal fluid samples (58 schizophrenia patients, 16 patients with depression, five patients with obsessive-compulsive disorder, ten patients with Alzheimer disease, and 90 controls). Our results show a highly significant differential distribution of samples from healthy volunteers away from drug-naïve patients with first-onset paranoid schizophrenia. The key alterations were the up-regulation of a 40-amino acid VGF-derived peptide, the down-regulation of transthyretin at ~4 kDa, and a peptide cluster at ~6,800–7,300 Da (which is likely to be influenced by the doubly charged ions of the transthyretin protein cluster). These schizophrenia-specific protein/peptide changes were replicated in an independent sample set. Both experiments achieved a specificity of 95% and a sensitivity of 80% or 88% in the initial study and in a subsequent validation study, respectively.
Our results suggest that the application of modern proteomics techniques, particularly mass spectrometric approaches, holds the potential to advance the understanding of the biochemical basis of psychiatric disorders and may in turn allow for the development of diagnostics and improved therapeutics. Further studies are required to validate the clinical effectiveness and disease specificity of the identified biomarkers.
Citation: Huang JT-J, Leweke FM, Oxley D, Wang L, Harris N, Koethe D, et al. (2006) Disease Biomarkers in Cerebrospinal Fluid of Patients with First-Onset Psychosis. PLoS Med 3(11): e428. https://doi.org/10.1371/journal.pmed.0030428
Academic Editor: John McGrath, University of Queensland, Australia
Received: March 6, 2006; Accepted: August 14, 2006; Published: November 7, 2006
Copyright: © 2006 Huang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by the Stanley Medical Research Institute. Further thanks to the Henry Smith Charity and the Biotechnology and Biological Sciences Research Council for financial support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: ANOVA, analysis of variance; CSF, cerebrospinal fluid; ELISA, enzyme-linked immunosorbent assay; m/z, , mass-to-charge ratio; OCD, obsessive-compulsive disorder; PCA, principal component analysis; PLS, partial least-squares; PLS-DA, partial least-squares discriminate analysis; SELDI, surface-enhanced laser desorption ionization
Editors' Summary
Psychosis is an abnormal mental state characterized by loss of contact with reality, often associated with hallucinations, delusions, personality changes, and disorganized thinking. Psychotic symptoms occur in several psychiatric disorders, including schizophrenia, bipolar disorder, and psychotic depression. It is not clear what the underlying biological abnormalities in the brain are, and whether they are the same for the different psychotic illnesses. The hope is that recent advances in brain imaging and systematic characterization of genetic activity and protein composition in the brain might help to shed light on mental diseases, eventually leading to better diagnosis, treatment, and possibly even prevention.
Why Was This Study Done?
This study was carried out in order to search for biomarkers for psychosis and schizophrenia by comparing the protein composition in the cerebrospinal fluid (the clear body fluid that surrounds the brain and the spinal cord) of patients with different psychotic disorders and normal individuals who served as controls.
What Did the Researchers Do and Find?
The researchers used a technique called surface-enhanced laser desorption ionization mass spectrometry, which allows a comprehensive analysis of the protein composition of a particular sample, on a total of 179 cerebrospinal fluid samples. The samples came from 90 individuals without mental illness who served as controls, 58 people with schizophrenia who were very recently diagnosed and had not yet taken any medication, 16 patients with depression, five patients with obsessive-compulsive disorder, and ten patients with Alzheimer disease. All of the patients gave their informed consent to participate in the study. The researchers found that samples from treatment-naïve schizophrenic patients had a number of characteristic changes compared with samples from control individuals, and that those changes were not found in the patients with other mental illnesses. The researchers then wanted to test whether they would see the same pattern in a separate set of patients with schizophrenia versus controls, which turned out to be the case. Two of the changes in the cerebrospinal fluid that were associated with schizophrenia, namely higher levels of parts of a protein called VGF and lower levels of a protein called transthyretin, were also found in post-mortem brain samples of patients with schizophrenia compared with samples from controls. Lower levels of transthyretin were also found in serum (blood) of first-onset drug naïve schizophrenia patients.
What Do These Findings Mean?
These results suggest that this approach has the potential to find biomarkers for psychosis and, possibly, schizophrenia that might help in the understanding of the molecular basis for these conditions. If shown, in future studies, to be directly involved in causing the disease symptoms, they would be important targets for treatment and prevention efforts, and might also be useful for diagnostic purposes. Overall, there are promising examples, such as this study, suggesting that new molecular techniques can yield fresh insights into psychiatric illnesses such as schizophrenia and other psychotic disorders. Additional studies are needed to confirm the findings presented here and to address many open questions, and would seem well justified given these results.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030428.
- MedlinePlus entries on psychosis and schizophrenia
- The National Alliance for Research on Schizophrenia and Depression
- The National Alliance for the Mentally Ill
- The Schizophrenia Society of Canada
- Wikipedia entries on psychosis and schizophrenia (note that Wikipedia is an online encyclopedia that anyone can edit)
Schizophrenia is the most devastating and enduring psychotic disorder affecting as many as 1% of the population worldwide, with a similar prevalence between the sexes and throughout diverse cultures and geographic areas [1,2]. To date, the etiology of schizophrenia remains elusive. The disorder is almost certainly the result of a complex interaction between numerous predisposing genes and environmental factors. Many studies have focused on the identification of schizophrenia "risk genes"/genetic polymorphisms, and although such "risk genes" may play a role in a small number of schizophrenia cases, the great majority of patients do not exhibit any apparent polymorphisms [3]. An alternative approach to genetic studies is to screen for disease markers (biomarkers). Biomarkers or biomarker signatures are indicators of a disease state that are usually linked to an ongoing pathophysiology and thus may also provide information and insights into the underlying molecular mechanisms of a given disease. Typical examples include diabetes and cardiovascular disorders, where increased glucose and low-density lipoprotein levels, respectively, are hallmark biomarkers for the diseases. Both cardiovascular disorders and diabetes (particularly Type II diabetes) are very similar to schizophrenia in that they are a result of genetic and environmental factors. Thus, even if the etiologies for schizophrenia and other psychotic disorders remain unknown, a biomarker or biomarker signature that accurately identifies the clinical syndrome would allow for improved diagnosis, prognosis, and disease monitoring as well as the development of novel therapeutic approaches.
Surface-enhanced laser desorption ionization (SELDI) mass spectrometry is a powerful tool for identifying a characteristic "fingerprint" of proteins and peptides in body fluids and tissues for a given condition, e.g., drug treatments and diseases (for review, see [4]). This technology utilizes modified surfaces to capture proteins/peptides, while a time-of-flight mass spectrometer is used to quantitate and measure the molecular weights of compounds ranging from small molecules and peptides of less than 1,000 Da up to proteins of 500 kDa. Quantifiable differences in protein/peptide patterns can be statistically evaluated using automated computer programs that represent each protein/peptide measured in the biofluid as a coordinate in multi-dimensional space. This approach has been most successful in the field of clinical biomarker discovery (for example, see [5–7]) as it can be used as a diagnostic tool without needing to know the biomarker's identity. The SELDI system also has the capability to run hundreds of samples in a single experiment. In addition, all the signals from SELDI mass spectrometry are derived from native proteins/peptides (unlike some other proteomics technologies requiring protease digestion), thus directly reflecting the underling physiology of a given condition.
In this study, we undertook an extensive protein/peptide–profiling analysis of cerebrospinal fluid (CSF), investigating a total of 179 CSF samples (90 controls, 58 first-onset, drug-naïve schizophrenia patients, 16 depression patients, five patients with obsessive-compulsive disorder [OCD], and ten patients with Alzheimer disease) using SELDI mass spectrometry in combination with computerized pattern-recognition analysis. We found highly significant and reproducible differences in samples obtained from first-onset, drug-naïve patients with a diagnosis of paranoid schizophrenia as compared to age-matched controls. However, as no samples from patients with non-schizophrenia psychosis were included in the analysis (with the exception of three samples from patients with psychotic depression), no clear conclusions can be drawn with regards to the schizophrenia-specificity of the biomarkers as compared to a first-onset psychosis signature.
Materials and Methods
Clinical Samples
The Ethical Committee of the Medical Faculty of the University of Cologne reviewed and approved the protocol of this study and the procedures for sample collection and analysis. All study participants gave their written informed consent. All clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. CSF and serum samples were collected from drug-naïve patients diagnosed with first-episode paranoid schizophrenia or brief psychotic disorder due to duration of illness (DSM-IV 295.30 [n = 54] or 298.8 [n = 4], total n = 58), from 16 patients diagnosed with major depressive disorder (DSM-IV 296.22–4; 296.33–4), from five patients with OCD (DSM-IV 300.3), and from demographically matched healthy volunteers (n = 80) (Tables 1 and 2). Fresh-frozen prefrontal cortex tissue (Brodmann area 9) from gray matter of eight schizophrenia and eight well-matched control individuals was obtained from the Neuropathology Consortium of the Stanley Brain Collection (Stanley Medical Research Institute, http://www.stanleyresearch.org). A detailed history of antipsychotic drug use of included patients is listed in Tables S1 and S2.
Demographic Details of Subjects in the First CSF SELDI Experiment
Demographic Details of Subjects in the CSF Validation Sample Set
Preparation of CSF Samples for SELDI Analysis
An amount (5 μl) of each CSF sample was applied to protein chips with different chemical properties at various pH conditions. The best condition was chosen at pH 9.0 on strong anion exchanger Q10 chips, based on number and separation of peaks resolved. Briefly, the array spots were pre-activated twice with binding buffer (100 mM Tris-HCl [pH 9.0]) at room temperature for 10 min on a shaker (frequency = 600 rpm). An amount (5 μl) of binding buffer was added to each spot prior to the addition of 5 μl of CSF sample. The protein chips were incubated on a shaker for 60 min at room temperature, then washed twice with binding buffer and once with H2O, and air-dried. The chips were then sequentially treated twice with 0.6 μl of a 100% saturated sinapinic acid (3,5-dimethoxy-4-hydroxycinnamic acid) in 50% acetonitrile and 0.5% trifluoroacetic acid. The chips were analyzed with the Ciphergen ProteinChip Reader (Ciphergen ProteinChip System Series 4000, Ciphergen Biosystems, http://www.ciphergen.com). Each sample was analyzed twice to confirm reproducibility in identifying the differentially expressed proteins and to ensure stability of instrument performance.
SELDI–Time-of-Flight Mass Spectrometer Analysis
The arrays were analyzed with the Ciphergen ProteinChip System Series 4000 (Ciphergen Biosystems). Mass spectra of proteins were generated by using an average of 254 laser shots at a laser intensity of 1,800 arbitrary units. For data acquisition, the detection size ranged between 2.5 and 200 kDa. The laser was focused at 10 kDa. The mass-to-charge ratio (m/z) of each of the proteins captured on the array surface was determined relative to external calibration standards (Ciphergen Biosystems): bovine insulin (5,733.6 Da), human ubiquitin (8,564.8 Da), bovine cytochrome c (12,230.9 Da), bovine superoxide dismutase (15,591.4 Da), horseradish peroxidase (43,240 Da), and BSA (66,410 Da). The data were analyzed with ProteinChip data analysis software version 3.0 and Ciphergen Express Software 3.0 (Ciphergen Biosystems). Matrix attenuation was set at 2,500 Da, and shot sequence was set with one warming shot at 2,000 nJ followed by four shots at 1,800 nJ. The Ciphergen Express Software 3.0 was used to compile all spectra and to autodetect quantified mass peaks within a mass range of between 2,500 and 200,000 Da, and the signal-to-noise threshold was set at 5. Peak labeling was completed using second-pass peak selection with 0.2% of the mass window, and estimated peaks were added. The peak information of all spectra was exported for further statistical analysis.
Peptide and Protein Identification
Typically, 10-μl CSF samples from both the control and schizophrenia groups were applied to Q10 protein chips at pH 9.0 (50 mM Tris-HCl). Proteins/peptides bound to the chip were eluted with 5 μl of elution buffer (30% acetonitrile, 50 mM sodium acetate [pH 3.0]) and desalted using a C18 Ziptip (Millipore, Billerica, Massachusetts, United States) according to the manufacturer's instructions. The peptides were eluted with 0.1% formic acid/50% aqueous acetonitrile (2 μl) and were further examined by MALDI mass spectrometry for the confirmation of the enriched 3,959-Da peak in the CSF sample of the patients with schizophrenia. A CSF sample with a high 3.959 peptide level was also loaded onto a C18 nano-column for online LC-ESI-MS/MS on a QSTAR quadrupole-TOF mass spectrometer (Applied Biosystems, Foster City, California, United States) for de novo sequencing.
For identification of protein biomarkers, CSF proteins were purified from pooled CSF by a combination of anion exchange chromatography (HyperD, Ciphergen Biosystems) followed by SDS-PAGE. CSF protein (~1 mg) was diluted in two volumes of buffer (50 mM Tris-HCl [pH 9]) and applied to the HyperD column equilibrated with the same buffer. The bound protein was then eluted by step gradients using pH 7.0, 6.0, 5.0, 4.0, and 3.0 buffers (Expression Difference Mapping Kit, Ciphergen Biosystems). The fractions were concentrated using Centricon YM10 (Millipore) to ~20 μl, followed by SDS-PAGE analysis (NuPAGE Bis-Tris gel, 4%–12%, Invitrogen, Carlsbad, California, United States). The band expected to correspond to the SELDI peak was cut from the gel. Approximately one third of the excised gel band was used for passive elution of the protein followed by SELDI analysis to confirm that the correct protein had been isolated. The remaining two thirds of the gel band was in-gel digested with trypsin (1:50; Promega, Madison, Wisconsin, United States) overnight at room temperature. The resulting peptide mixtures were then analyzed by LC-ESI-MS/MS (QSTAR, Applied Biosystems), and the protein was identified by database searching using Mascot software (Matrix Science, London, United Kingdom).
S-cysteinylated or S-glutathionylated isoforms of proteins were confirmed by comparing the spectra before and after on-chip reduction using β-mecaptoethanol. In brief, CSF protein and peptide binding was performed as described above and, in the final step, each spot was washed with 100 μl of 1 mM HEPES [pH 7.5]. The proteins and peptides on the chips were then reduced with 1/40 β-mecaptoethanol (1 μl) for 30 min at room temperature. Water (1 ml) was added onto each spot and evaporated. This procedure was repeated twice. Matrix was then added, and data were acquired using ProteinChip Reader (Ciphergen ProteinChip System Series 4000).
Quantitative analysis of transthyretin in human serum samples was carried out by enzyme-linked immunosorbent assay (ELISA). Samples were defrosted from −80 °C and mixed on a benchtop vortexer for 10 min before experimental work commenced. All samples were assayed blind to the clinical conditions. The identities of all subjects were kept blind by code numbers until all biochemical analyses had been completed.
Controls and patient-derived human serum samples were diluted 1,000 times with PBS (pH 7.4) (Sigma, St. Louis, Missouri, United States), and 100 μl of each diluted sample was loaded onto ELISA Maxisorb plates (Nunc, http://www.nuncbrand.com) together with transthyretin standards (Sigma) before being incubated for 1 h. All samples were tested in duplicate. After washing with washing buffer (0.03% Tween 20 in PBS), the plates were blocked with 100 μl of 5% skimmed-milk powder for 60 min. Transthyretin antibody (100 μl) (DakoCytomation, Glostrup, Denmark, 1:500 diluted in 2.5% skimmed-milk powder) was incubated in 96-well plates for 60 min. The plates were washed four times with washing buffer followed by the addition of 100 μl of secondary antibody (anti-rabbit HPP-linked IgG [Cell Signaling Technology, Beverly, Massachusetts, United States] 1:2000) to each well and incubated for 60 min. After washing with washing buffer three times, 100 μl of substrate (TMB One solution, Promega) was added into each well, and the mixture was incubated at room temperature for 10 min. The plate was read at 450 nm (Model 680, Bio-Rad, Hercules, California, United States).
Western Blot Analysis
The preparation of human brain samples for Western blot analysis and the details of performing Western blotting were as described previously [8]. In brief, equivalent amounts of protein (30 μg per sample) were resolved by electrophoresis on 10% polyacrylamide gels and transferred onto nitrocellulose, which was then incubated with primary antibody (anti-transthyretin [DakoCytomation] 1:1,000) or anti-VGF (Santa Cruz Biotechnology [Santa Cruz, California, United States] 1:500) in 3% milk–PBS overnight at 4 °C, followed by incubation of a secondary antibody (HRP-conjugated anti-rabbit secondary antibody [Cell Signaling Technology, 1:2500]) at room temperature for 1 h. Enhanced chemiluminescence (LumiGlu, Cell Signaling Technology) was used to detect signals from the blot. Consistency of protein loading and transfer was determined by Ponseau S staining.
Statistical Analysis
Multivariate statistical analysis techniques including principal component analysis (PCA), partial least-squares discriminate analysis (PLS-DA), and partial least-squares (PLS) were employed to summarize the data output from Ciphergen Express. Holdout cross-validation was performed three times so that the sensitivity and specificity of the PLS model could be estimated. In each of the three rounds of holdout cross-validation, one third of the samples were randomly selected to form the validation data and the remaining samples were used as the training data. All multivariate analyses were performed using SIMCA-P+ 10 (Umetrics, http://www.umetrics.com). Sensitivity is defined as the proportion of true positives that it detects of all the positives, and specificity is defined as the proportion of true negatives that it detects of all the negatives. Where appropriate, a t-test was performed using the Statistical Package for Social Scientists (SPSS/PC+; SPSS, Chicago, Illinois, United States). Correlation analysis was performed using Matlab 7.0.1 (Mathwork, Natick, Massachusetts, United States). Two-way analysis of variance (ANOVA) was performed using the "ANOVA" function from the CAR package in R (R project, http://cran.r-project.org). Type III sums of squares was used as this study had an unbalanced experimental design.
Alterations of CSF Protein/Peptide Profiles in First-Onset, Drug-Naïve, Paranoid Schizophrenia Patients
In a first set of experiments, we examined protein/peptide profiles of CSF samples from 41 first-onset, drug-naïve, paranoid schizophrenia patients and 40 demographically matched healthy volunteers using SELDI mass spectrometry. CSF proteins and peptides were profiled using Q10 (strong anion exchanger) chips at pH 9.0. An example of the CSF protein/peptide profile of a healthy volunteer is shown in Figure 1A. Approximately 75 peaks can be readily detected with a signal-to-noise ratio of >5 under this binding condition. Plots of PLS-DA scores based on SELDI spectra of CSF samples showed a clear differentiation between healthy volunteers and drug-naïve patients with first-onset, paranoid schizophrenia (Figure 1B and 1C). Similar results were found using PCA (unpublished data). The loading coefficients indicated that the peak with an m/z of 3,959 contributed the most to the separation of the two groups (Figure 1D).
(A) Typical CSF protein/peptide spectrum using an anion exchanger chip (Q10; 50 mM Tris-HCl [pH 9.0]) showing the m/z range of 2,500–15,000 from a healthy volunteer is shown.
(B) The peak intensity of protein/peptide peaks from SELDI spectra were analyzed using PCA and PLS-DA models. A 3D PLS-DA scores plot indicates clusters of healthy volunteers (in blue) and untreated, drug-naïve schizophrenia patients (in red).
(C) and (D) PLS-DA scores and loadings plots. The scores plot is similar to (B) but only the first two components were used to discriminate between healthy controls and patients. The loading plot as shown in (D) indicates the key protein/peptide peaks contributing the most towards the separation as shown in (C). The 3,959-Da VGF23–62 peptide was found to predominantly contribute to the separation between the two groups.
Further analyses showed a 2.8-fold elevation of an m/z = 3,959 peptide in first-onset, drug-naïve, paranoid schizophrenia patients as compared to the demographically matched control group (p = 10−8, t-test; Figure 2). In addition, the loading plot also showed significant reductions in peaks of m/z = 23,490 and clusters of peaks between m/z = 13,600–14,000 and m/z = 6,800–7,300 (~20% decreases, p < 0.01, t-test), all of which also contributed to the separation between classes. We have identified the proteins and peptides at m/z = 3,959 and in the cluster of m/z = 13,600–14,000. The m/z = 6,800–7,300 biomarkers are likely to be influenced by the doubly charged ion signals of the m/z = 13,600–14,000 protein cluster and may only reflect the altered levels in m/z = 13,600–14,000 (Figure S1). The sensitivity and specificity of this model based on holdout cross validation was 80% and 95%, respectively (Table 3; see Materials and Methods). These alterations were not affected by most demographic variables including age, gender, alcohol, and illicit drug use, although we identified a correlation with smoking (Table S3). However, a disease-associated increase in VGF levels remains significant after controlling for smoking effects using a two-way ANOVA (Table S3.5).
Forty healthy volunteers and 41 untreated paranoid schizophrenia CSF samples were included in this study. A clear up-regulation of the peak at 3,959 Da was observed (as shown in [A], with five representative spectra from each group). Figure 2B reveals the relative intensity of a 3,959-Da peak in healthy volunteers and first-onset, drug-naïve schizophrenia patients. Figure 2C shows a Western blot analysis of VGF protein in the prefrontal cortex of age-matched controls and patients with schizophrenia. Eight schizophrenia and eight control brains (prefrontal cortex, age matched) were randomly selected, and Western analysis was performed using a polyclonal antibody against the C-terminal region of the VGF protein. VGF was found up-regulated in four patients with schizophrenia (out of eight patients), while no signal was detected in the control patients. This experiment was repeated twice on the same samples under identical conditions, except that a different batch antibody from the same company was used. Similar results were obtained. For demographic details, see Table 4.
Sensitivity and Specificity of PLS Models Calculated from the Two Independent Experiments
Demographic Details of Subjects Investigated for VGF Expression by Western Blot Analysis of Post-Mortem Brain
Identification of the 3,959-Da Peptide as a VGF Fragment
The 3,959-Da peptide was purified using identical conditions as employed in the profiling experiment followed by C18-Ziptip purification (see Figure S2). The eluted proteins and peptides from Ziptips were subjected to MALDI to confirm the mass, and the peak was identified as a 40-amino acid peptide using de novo sequencing (Figure S2). This peptide sequence was mapped to amino acids 23–62 of the native VGF protein (in bold underlined, Figure 3A), a neurosecretory protein known to regulate metabolism [9] and synaptic plasticity [10], and is immediately next to a predicted secretory signal peptide. Interestingly, another VGF-derived peptide with an m/z of 3,690 in CSF that did not appear to differ between control and schizophrenia patients was identified (p = 0.85, t-test). This peptide showed the same peptide sequence as the 40-amino acid VGF peptide except that three amino acids were absent at the first N-terminal (Figure 3B; de novo sequencing data not shown). This indicates that the 40-amino acid VGF peptide with the "APP" sequence in the amino terminus may have specific functions and/or may be linked to the pathophysiology of schizophrenia.
The 3,959-Da "schizophrenia" peptide was mapped to amino acids 23–62 of the native VGF protein (in bold; underlined), immediately next to a predicted secretory signal peptide (using InterProScan: http://www.ebi.ac.uk/cgi-bin/iprscan).
Another native VGF-derived peptide did not show differential expression between healthy volunteers and patients with schizophrenia (as shown in Figure 2A). This peptide has an identical sequence except that it is three amino acids shorter at the N-terminus compared to the "disease-specific" VGF peptide.
Increased VGF Protein Expression in Post-Mortem Brain Tissue from Patients with Schizophrenia
To examine whether VGF protein expression is also altered in schizophrenic post-mortem brain tissue, we performed Western blot analysis on gray matter in prefrontal cortex tissue from eight patients with schizophrenia and eight demographically matched control subjects. Western blot analysis using an antibody recognizing the C-terminal sequences of VGF showed a strong expression in four out of eight patients while, in control brains, the level of VGF was below the detection level (Figure 2C).
Identification of the 13.6–14-kDa Protein Cluster as Transthyretin
The protein cluster between 13.6 and 14.1 kDa contains four peaks (Figure 4A and 4B), three of which were consistently down-regulated in CSF from first-onset, drug-naïve schizophrenia patients (p < 0.01; Figure 4B, lower panel). Studies have suggested that these peaks may be from S-cysteinylated derivatives of transthyretin protein [11,12], a thyroid hormone–binding protein that transports thyroxine from the bloodstream to the brain. On-chip reduction of CSF peptide/protein performed using β-mercaptoethanol at room temperature showed that the three peaks m/z = 13,741, 13,875, and 13,923 were reduced to a single peak (Figure 4C), confirming that they are derived from the same protein. To determine the protein's identity, a pair of CSF samples from a healthy volunteer and a patient with schizophrenia were applied to an anion exchanger column (HyperD, Ciphergen Biosystems) and eluted with pH 9–pH 3 buffers. A major band (~13–15 kDa) was eluted in the pH 3 fraction (Figure 4D, left panel). The band was confirmed to be the peak cluster at around 13.6–14 kDa in the SELDI spectrum by eluting the protein from the band and running on an NP20 chip to match the mass (Figure 4E). This protein was then digested with trypsin and sequenced using LC-MS/MS. The protein was identified as transthyretin (Figure 4D, right panel).
Examples of CSF spectra from healthy volunteers and patients with schizophrenia within 6–17 kDa are shown in (A). The peak cluster indicated (red cursor) is enlarged in (B). Statistical details of each sub-peak are listed in the table presented just below (B). On-chip reduction of CSF peptide/protein with β-mecaptoethanol at room temperature showed that the three peaks 13,741, 13,875, and 13,923 Da were reduced into a single peak (C), suggesting that they are different S-cysteinylated derivatives of the same protein. To identify the protein, CSF samples from a healthy volunteer and a patient with schizophrenia were applied to an anion exchanger column (HyperD, Ciphergen Biosystems) and eluted with pH 9–pH 3 buffers. A major ~14–15-kDa band was eluted in the pH 3 fraction (D, left panel). The protein was identified as transthyretin using LC-MS/MS (D, right panel) and the sequence coverage is shown in red. Note, that all the peptides shown have an E-score of < 0.05, indicating a high probability for the assigned protein identity. In addition, the band was confirmed to be the peak cluster at around 13.5–14 kDa in the spectra by eluting the protein(s) from the gel and SELDI analysis (E).
Down-Regulation of Transthyretin in Serum Samples from the Same Subjects as well as Prefrontal Cortex Tissue from Patients with Schizophrenia
It has been estimated that 3% of transthyretin in the ventricular CSF and 10% of the transthyretin in lumbar CSF are derived from blood [13]. To evaluate the contribution of blood transthyretin to the changes found in CSF in patients with schizophrenia, we also investigated serum transthyretin levels taken from the same individuals (at the same time as the CSF was collected) on whom we had reported in this study (for demographics, see Tables 1 and 2) using ELISA. We found a moderate, but significant, decrease in transthyretin levels in sera from patients with schizophrenia compared to controls (15% decrease, p = 0.0007, t-test) (Figure 5A). However, we found no correlation between CSF and serum transthyretin from the same individuals, suggesting that the transthyretin level is regulated independently in CSF and serum (Figure 5B). Interestingly, we found an ~40% down-regulation of transthyretin levels in post-mortem prefrontal cortex from patients with schizophrenia compared with controls using Western blot (Figure 5C).
The serum samples from the same patients whose CSF protein profiles were measured in Figures 1 and 6 were included in this study. Figure 5A shows serum tranythyretin levels in patients with schizophrenia significantly decreased by ~15% compared to control subjects. Data are shown in mean ± standard deviation, and the asterisk denotes p = 0.0007 (t-test). Figure 5B indicates no correlation between serum transthyretin and CSF SELDI signals from one of the transthyretin isoforms (m/z = 13,741) in the second sample set (for demographics, see Table 2). Similar results were found when comparing with signals from other isoforms in CSF (unpublished data). Figure 5C shows an ~40% decrease of transthyretin expression in prefrontal cortex of five patients with schizophrenia and in five control subjects. For demographic details, see Table 5.
The demographic details of this sample set are listed in Table 2. The PLS-DA scores plot shows a separation between patients (in red) and healthy volunteers (in blue). The loadings plot indicates similar key variables (e.g., the 3,959-Da VGF23–62 peptide [in red] and transthyretin protein signals between 13,600 and 14,000 [in blue]) identified in the first experiment (see Figure 1).
Demographic Details of Subjects Investigated for Transthyretin Expression by Western Blot Analysis of Post-Mortem Brain
Validation of Protein/Peptide Biomarkers in an Independent Sample Set
We validated our biomarker model in Figure 1 using an independent sample set comprising a further 17 first-onset, drug-naïve schizophrenia patients and 40 demographically matched healthy volunteers. These samples were run using identical conditions as in the previously described experiment. PLS-DA scores and loadings plots showed a very similar result as found in Figure 1 with predominant changes in m/z = 3,959, the cluster of 6,800–7,300-Da peptides (likely influenced by the doubly charged ions from transthyretin proteins), and the cluster of 13,600–14,000 proteins (Figure 6). This suggests that these identified alterations in CSF proteins and peptides are a consistent finding and thus may reflect genuinely the early pathophysiology of schizophrenia. The sensitivity and specificity of this model were 88% and 95%, respectively (Table 3). As with the results from the first study, we did not find an influence of most demographic variables on these alterations, with the exception of a potential effect of cannabis use (urine-positive drug screen) on CSF transthyretin levels (p = 0.08; Table S3). However, as for the smoking effect, disease-associated changes in transthyretin levels remain significant after controlling for cannabis use using a two-way ANOVA (Table S3.6–S3.8).
Disease Specificity of the Schizophrenia Biomarker Panel
As many psychiatric disorders and neurological diseases share similar symptoms with schizophrenia, it is crucial to test the disease specificity of these protein/peptide biomarkers. We therefore examined CSF samples of three additional psychiatric/neurological disorders, namely depression, OCD, and Alzheimer disease. CSF samples from 16 patients with depression and five patients with OCD, together with CSF samples from 40 healthy volunteers, were analyzed with SELDI-MS using the condition described previously. We found a partial separation between controls and patients with depression (Figure 7A); the key variables contributing to the separation were the above-mentioned VGF23–62 peptide and a secretogranin II peptide 529–566 (Figure 7B; sequencing data not shown). No difference was found between controls and the five patients with OCD (unpublished data). These results indicate that the VGF peptide alone may not be a specific marker for a given psychiatric disease (possibly due to overlapping disease processes, which are not least implied by the fact that patients with a family history of affective disorder have an increased risk for developing schizophrenia, and vice versa). Our data thus suggest that disease specificity may be achieved with a panel of biomarkers (Table S4).
CSF samples from 16 patients with depression (green triangles), together with 40 healthy volunteers (blue dots), were analyzed with SELDI-MS using Q10 anion exchanger protein chips at pH 9 (50 mM Tris-HCl).
(A) The scores plot shows a partial separation between controls and patients with schizophrenia.
(B) The loading plot indicates VGF23–62 and a depression-specific secretogranin II 529–566 peptide to be the key protein/peptide peaks contributing the most towards the group separation.
As three out of 16 depression patients presented with psychotic symptoms, we analyzed the psychotic and non-psychotic subgroups separately to examine whether the identified biomarkers are specific for schizophrenia or merely for acute psychosis. No differences could be observed between these two subgroups, and the psychotic subgroup did not co-cluster with schizophrenia patients, suggesting that VGF23–62 and transthyretin peptide/protein changes are biomarkers for schizophrenia rather than acute psychosis (Figure S3). However, as the sample size of patients with psychotic depression was very small, the specificity of the biomarker signature will need to be confirmed on a larger set of samples from patients with psychotic depression, bipolar affective psychosis, and other neuropsychiatric illnesses that present with psychotic states.
In addition, we also analyzed CSF samples from ten patients with Alzheimer disease and from ten age-matched and sex-matched controls using identical experimental conditions. No significant differences between the two groups were identified, although some clear alterations were found using other experimental conditions (unpublished data).
Initial analysis of SELDI spectra of a total of 81 CSF samples (41 from patients with schizophrenia, and 40 from controls) showed a differential distribution of samples from drug-naïve patients with first-onset paranoid schizophrenia away from healthy volunteer samples (Figure 1B–1D). The protein/peptide profile of CSF was found to be characteristically altered in paranoid schizophrenia patients, with the key alterations being the prominent up-regulation of a 40-amino acid VGF peptide and the down-regulation of transthyretin at ~14 kDa along with a peptide cluster at ~6,800–7,300 Da that appeared co-regulated with the transthyretin cluster. These schizophrenia-specific protein/peptide changes were replicated/validated in an independent sample set (n = 58) using identical conditions (Figure 6). Both experiments achieved an astonishingly high specificity (rate of true negative) of 95% and a sensitivity of 80% or 88% in the initial study and in the subsequent validation study, respectively (Table 3). This means that virtually no control samples clustered with the schizophrenia group (Figure 1B and 1C). For a high diagnostic validity and consequent therapeutic interventions, an accurate identification of those individuals who truly have the disease is most critical. Correlation analyses suggest that these key alterations are not due to sex, gender, alcohol, smoking, or illicit drug use.
We also investigated the disease specificity of this biomarker panel with a smaller number of CSF samples from patients with depression (including three patients with acute psychotic depression), OCD, and Alzheimer disease. Interestingly, depression and schizophrenia share an increase in the VGF23–62 peptide expression, while transthyretin protein levels were not found to be significantly altered in depression (although we observed a trend towards down-regulation). Indeed, depression samples were most prominently characterized by a distinct decrease of a secretogranin II 529–566 peptide. No difference was found between controls and OCD patients (unpublished data). Additionally, we did not find any significant difference between control and Alzheimer disease CSF samples regarding the schizophrenia biomarker panel (unpublished data).
One of the key findings of this study is the up-regulation of a 40-amino acid VGF23–62 peptide in CSF of first-onset, drug-naïve schizophrenia patients and, to a lesser degree, in patients with depression (Figure 7). Interestingly, a 3.69-kDa VGF26–62 peptide with an identical sequence to the VGF23–62 peptide, except for the absence of the first three amino acids at the amino terminus, did not appear to be differentially expressed in CSF from schizophrenia or depression patients compared to healthy volunteers. However, this VGF26–62 peptide was reported to be decreased in frontotemporal dementia [11]. In addition, a down-regulation of a peptide of 4.82 kDa corresponding to a further VGF peptide (containing amino acids 378–398) was found to be changing in amyotrophic lateral sclerosis [14] and Alzheimer disease [15], highlighting the importance of this protein in neurological/psychiatric disorders. The identified 40-amino acid VGF23–62 peptide may have a distinct function in the schizophrenia/depression disease process, may reflect a disease-associated protease/peptidase dysfunction, and/or may imply the presence of pathological processes in neurons since VGF is selectively expressed in neurons in brain, particularly in the hypothalamus (for review see [16,17]). At this point in time, the biological function of this 40-amino acid VGF23–62 peptide is unknown; however, the full-length protein has been linked to synaptic plasticity [10] as well as penile erection through the activation of paraventricular oxytocinergic neurons [18], and the circadian clock [19]. Future studies are required to explore the possible functions of this and other disease-associated VGF peptides and their respective peptidases.
The increased level of the 40-amino acid VGF peptide may indicate an increase of native (full-length) VGF protein and/or the processing of the protein in the brain. In addition, our data showed that VGF expression was increased in half of the schizophrenia post-mortem brains (Figure 2C). As post-mortem samples were collected from chronic patients who had been chronically treated with antipsychotic drugs, we cannot exclude the possibility of medication effects. However, in view of our CSF results from drug-naïve schizophrenia patients (Figure 1), it is likely that the observed changes in VGF protein/peptide expression are a disease-related phenomenon. Studies have shown that VGF is a secreted polypeptide that is selectively expressed by neurons and by several endocrine and neuroendocrine tissues (for review [16,17]), and is transported in dense core vesicles with the VGF-derived processed peptides being released via a regulated secretory pathway [16,17]. Knockout of the VGF gene in mice generated a lean, hypermetabolic, hyperactive phenotype, demonstrating that VGF may regulate energy balance [9]. The observation of an increase of the VGF peptide in CSF from patients with schizophrenia, therefore, may point to a hypometabolic state in the schizophrenia brain. Whether or not this is linked to "hypofrontality", a consistent finding showing decreased metabolic activity in the prefrontal cortex during cognitive activation in patients with schizophrenia [20,21], requires further examination.
Our "metabonomics" data derived from the identical samples used in the current study indicates that there is an increase of CSF glucose level in first-onset, drug-naïve schizophrenia patients which was not observed in sera [22]. This further suggests that the schizophrenia brain may be in a hyperglycemic state, possibly linked to deficiencies in glucose metabolism since we found the majority of glycolysis enzymes down-regulated in prefrontal cortex in a study on post-mortem schizophrenia brain [23]. Interestingly, knockout of the VGF gene increases insulin sensitivity in animal models [24], suggesting that an increase of VGF may theoretically decrease insulin sensitivity in the brain. This may explain the increase in glucose levels in CSF from first-onset, drug-naïve schizophrenia patients.
In this study, we also observed a moderate, but consistent, decrease in the transthyretin proteins in CSF from first-onset schizophrenia patients. Transthyretin is a thyroid hormone–binding protein that transports thyroxine from the bloodstream to the brain [25]. This protein is expressed at a high rate in the brain choroid plexus, from which it is released into the CSF [25]. In peripheral tissues, it is expressed primarily in liver [13]. Only an estimated 3% of transthyretin in the ventricular CSF and an estimated 10% of the transthyretin in lumbar CSF are derived from blood [13]. Our ELISA results on the serum samples collected from the identical individuals whose CSF were investigated in this study showed that there is an ~15% decrease of transthyretin levels in serum (p = 0.0007, t-test; Figure 5A). However, there was no correlation between the level of serum transthyretin and SELDI signals from CSF transthyretin, suggesting that liver-derived tranthyretin may not contribute to the down-regulation in CSF (Figure 5B). Experiments perfusing isolated sheep brains showed that all newly synthesized transthyretin was secreted from the choroid plexus towards the ventricles. The synthesis of this protein is required for the transport of thyroxine [26]. Thus, the reduced level of transthyretin in CSF suggests a lower thyroxine transport in brains of patients with schizophrenia. Indeed, our result showing a down-regulation in transthyretin protein in the post-mortem brain tissue from patients with schizophrenia (Figure 5C) further supports this notion
It is noteworthy that thyroid dysfunction is relatively common in patients with schizophrenia [27,28], and indeed with other psychiatric disorders [29], possibly genetically linked to the disorders. In addition, in patients with severe forms of both hypo- and hyper-thyroidism, psychotic symptoms may occur, and the clinical picture frequently resembles that of schizophrenia [30], which may imply that an increase in central nervous system thyroxine function may be linked. Interestingly, long-term administration of clozapine has been shown to induce transthyretin expression in rat hippocampus and cerebral cortex [31], implying that clozapine enhances central nervous system thyroxine function, supporting the clinical relevance of transthyretin in the early pathophysiology of schizophrenia. Transthyretin has also been found to be differentially expressed in CSF of several other neurological and psychiatric diseases. Sullivan et al. found a 9.6% down-regulation of transthyretin levels (p = 0.05, t-test) in CSF from patients with [32]. This is in keeping with our observation of a trend towards down-regulation of transthyretin signals (Figure 7A). In frontotemporal dementia, Hansson et al. observed increased expression of two isoforms of transthyretin in CSF using 2D electrophoresis. Taken together, our results stress the importance of transthyretin in the pathophysiology of psychiatric and neurological diseases.
In summary, our results suggest that the application of modern proteomics techniques, particularly mass spectrometric approaches, holds the potential to advance the understanding of the biochemical basis of psychiatric disorders and may in turn allow for the development of diagnostics and better therapeutics. Further studies are required to validate the clinical utility and disease specificity of the identified biomarkers.
Limitations and Clinical Potential of the Findings
One of the key limitations of the present study is that no (or very few) samples from patients with non-schizophrenia psychosis were investigated. The inclusion of samples from patients with psychosis in the context of bipolar disorder would be particularly desirable. However, it represents a great challenge to collect CSF from patients with acute mania (as the patient must give informed consent and must lie still for safe CSF collection).
A larger number of patient samples, ideally collected in different hospital settings, and the inclusion of samples from patients with "prodromal" schizophrenia (most patients with schizophrenia present with vague mental disturbances for months or years prior to developing overt psychosis) as well as patients with chronic schizophrenia, would increase the confidence in the findings and would have prognostic utility. The responsiveness of the biomarker signature to drug treatment would also be of great interest, as this could be used as a surrogate end point in drug-discovery studies and could help to differentiate drug-responders from non-responders and generally may allow for the development of personalized treatment approaches.
The main limitation of the findings with regards to translation into clinical practice is that, in most countries (with the exception of Germany and some Scandinavian countries), CSF is not routinely collected in patients with first-onset psychosis. A CSF-based diagnostic assay would therefore require a sea-change in clinical practice.
The identification of serum biomarkers would be much more desirable, but is technically challenging. However, our laboratory has identified a number of putative serum biomarkers, which are currently being followed up and validated (unpublished data).
Once sensitive and specific markers are established, diagnostic assays can be developed to validate the identified biomarkers on a larger sample set and, ultimately, in the clinical setting. A likely scenario could be a multi-analyte array configuration based on up to ten indicative biomarkers. Such systems will be configured initially into bench-top instruments and, subsequently, into disposable, easy-to-use diagnostic platforms for the physician's office or the patient's bedside based on hand-held readers. The sensors will be designed with an in-built ability to allow the collected data to be electronically transmitted directly to the physician for interpretation.
Supporting Information
Figure S1. The m/z = 6,800–7,000 Region of CSF Spectra Was under a Strong Influence of Doubly Charged Ions from the Transthyretin Proteins
Figure S2. Identification of the 3,959-Da Schizophrenia-Specific Peptide as a VGF Fragment
Figure S3. Patients with Depression who Presented with Acute Psychotic Symptoms (n = 3) at the Time of Sample Collection Did Not Co-Cluster with Schizophrenia Patients and Did Not Show a Significant Difference when Compared to Non-Psychotic Patients with Depression
Table S1. History of Antipsychotic Drug Use for Patients Included in Figure 2C
Table S2. History of Antipsychotic Drug Use for Patients Included in Figure 5C
Table S3. Correlation Analyses of CSF Expression Levels of VGF23–62 (m/z = 3,959), TTR Proteins (m/z = 13,765, 13,853, and 13,936), and Demographic Variables
Table S4. Summary of Potential Peptide/Protein Biomarkers for Four Neurological/Neuropsychiatric Disorders Investigated in This Study
Thanks to all other members of the Bahn Laboratory for discussions, help, and encouragement. Most of all, thanks to all patients and healthy volunteers for their selfless donation of samples used in this study. SB holds a NARSAD Essel Independent Investigator Fellowship.
Author Contributions
SB initiated and conceptualized the study. JTJH, FML, and SB designed the study. JTJH, FML, and LW analyzed the data. FML, DK, CWG, BMN, SG, and DS enrolled patients. JTJH, FML, and SB contributed to writing the paper. JTJH, FML, DO, LW, NH, DK, CWG, and SG collected data or carried out experiments for the study. DO performed the ESI-MS/MS and data analyses that identified the VGF peptide and the transthyretin. NH and BR helped design the initial pilot studies and collected some data from the experiments, as well as providing technical assistance and input regarding the identification of the proteins described in this paper. BR also coordinated all collaborative interactions with Ciphergen Biosystems.
- 1. Thaker GK, Carpenter WT Jr (2001) Advances in schizophrenia. Nat Med 7: 667–671.
- 2. Susser E, Wanderling J (1994) Epidemiology of nonaffective acute remitting psychosis vs. schizophrenia. Sex and sociocultural setting. Arch Gen Psychiatry 51: 294–301.
- 3. Sullivan PF (2005) The genetics of schizophrenia. PLoS Med 2: e212..
- 4. Tang N, Tornatore P, Weinberger SR (2004) Current developments in SELDI affinity technology. Mass Spectrom Rev 23: 34–44.
- 5. Goldstein LE, Muffat JA, Cherny RA, Moir RD, Ericsson MH, et al. (2003) Cytosolic beta-amyloid deposition and supranuclear cataracts in lenses from people with Alzheimer's disease. Lancet 361: 1258–1265.
- 6. Petricoin EF, Liotta LA (2004) SELDI-TOF-based serum proteomic pattern diagnostics for early detection of cancer. Curr Opin Biotechnol 15: 24–30.
- 7. Rosty C, Christa L, Kuzdzal S, Baldwin WM, Zahurak ML, et al. (2002) Identification of hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein I as a biomarker for pancreatic ductal adenocarcinoma by protein biochip technology. Cancer Res 62: 1868–1875.
- 8. Swatton JE, Sellers LA, Faull RL, Holland A, Iritani S, et al. (2004) Increased MAP kinase activity in Alzheimer's and Down syndrome but not in schizophrenia human brain. Eur J Neurosci 19: 2711–2719.
- 9. Hahm S, Mizuno TM, Wu TJ, Wisor JP, Priest CA, et al. (1999) Targeted deletion of the VGF gene indicates that the encoded secretory peptide precursor plays a novel role in the regulation of energy balance. Neuron 23: 537–548.
- 10. Alder J, Thakker-Varia S, Bangasser DA, Kuroiwa M, Plummer MR, et al. (2003) Brain-derived neurotrophic factor-induced gene expression reveals novel actions of VGF in hippocampal synaptic plasticity. J Neurosci 23: 10800–10808.
- 11. Ruetschi U, Zetterberg H, Podust VN, Gottfries J, Li S, et al. (2005) Identification of CSF biomarkers for frontotemporal dementia using SELDI-TOF. Exp Neurol 196: 273–281.
- 12. Schweigert FJ, Wirth K, Raila J (2004) Characterization of the microheterogeneity of transthyretin in plasma and urine using SELDI-TOF-MS immunoassay. Proteome Sci 2: 5.
- 13. Reiber H (2001) Dynamics of brain-derived proteins in cerebrospinal fluid. Clin Chim Acta 310: 173–186.
- 14. Ranganathan S, Williams E, Ganchev P, Gopalakrishnan V, Lacomis D, et al. (2005) Proteomic profiling of cerebrospinal fluid identifies biomarkers for amyotrophic lateral sclerosis. J Neurochem 95: 1461–1471.
- 15. Carrette O, Demalte I, Scherl A, Yalkinoglu O, Corthals G, et al. (2003) A panel of cerebrospinal fluid potential biomarkers for the diagnosis of Alzheimer's disease. Proteomics 3: 1486–1494.
- 16. Levi A, Ferri GL, Watson E, Possenti R, Salton SR (2004) Processing, distribution, and function of VGF, a neuronal and endocrine peptide precursor. Cell Mol Neurobiol 24: 517–533.
- 17. Salton SR, Ferri GL, Hahm S, Snyder SE, Wilson AJ, et al. (2000) VGF: A novel role for this neuronal and neuroendocrine polypeptide in the regulation of energy balance. Front Neuroendocrinol 21: 199–219.
- 18. Succu S, Cocco C, Mascia MS, Melis T, Melis MR, et al. (2004) Pro-VGF-derived peptides induce penile erection in male rats: Possible involvement of oxytocin. Eur J Neurosci 20: 3035–3040.
- 19. Wisor JP, Takahashi JS (1997) Regulation of the VGF gene in the golden hamster suprachiasmatic nucleus by light and by the circadian clock. J Comp Neurol 378: 229–238.
- 20. Volz H, Gaser C, Hager F, Rzanny R, Ponisch J, et al. (1999) Decreased frontal activation in schizophrenics during stimulation with the continuous performance test—A functional magnetic resonance imaging study. Eur Psychiatry 14: 17–24.
- 21. Meyer-Lindenberg A, Miletich RS, Kohn PD, Esposito G, Carson RE, et al. (2002) Reduced prefrontal activity predicts exaggerated striatal dopaminergic function in schizophrenia. Nat Neurosci 5: 267–271.
- 22. Holmes E, Tsang TM, Huang TJ, Leweke FM, Koethe D, et al. (2006) Metabolic profiling of CSF: Evidence that early intervention may impact on disease progression and outcome in schizophrenia. PLoS Med 3: e327..
- 23. Prabakaran S, Swatton JE, Ryan MM, Huffaker SJ, Huang JT, et al. (2004) Mitochondrial dysfunction in schizophrenia: Evidence for compromised brain metabolism and oxidative stress. Mol Psychiatry 9: 684–697.
- 24. Watson E, Hahm S, Mizuno TM, Windsor J, Montgomery C, et al. (2005) VGF ablation blocks the development of hyperinsulinemia and hyperglycemia in several mouse models of obesity. Endocrinology 146: 5151–5163.
- 25. Schreiber G (2002) The evolutionary and integrative roles of transthyretin in thyroid hormone homeostasis. J Endocrinol 175: 61–73.
- 26. Southwell BR, Duan W, Alcorn D, Brack C, Richardson SJ, et al. (1993) Thyroxine transport to the brain: Role of protein synthesis by the choroid plexus. Endocrinology 133: 2116–2126.
- 27. Morley JE, Shafer RB (1982) Thyroid function screening in new psychiatric admissions. Arch Intern Med 142: 591–593.
- 28. Ryan WG, Roddam RF, Grizzle WE (1994) Thyroid function screening in newly admitted psychiatric inpatients. Ann Clin Psychiatry 6: 7–12.
- 29. Kirkegaard C, Faber J (1998) The role of thyroid hormones in depression. Eur J Endocrinol 138: 1–9.
- 30. Hall RCW, Stickney S, Beresford TP (1986) Endocrine disease and behavior. Integr Psychiatry 4: 122–135.
- 31.
Chen ML, Chen CH (2005) Comparative proteome analysis revealed up-regulation of transthyretin in rat brain under chronic clozapine treatment. J Psychiatr Res. E-pub 31 May 2005.
- 32. Sullivan GM, Mann JJ, Oquendo MA, Lo ES, Cooper TB, et al. (2006) Low cerebrospinal fluid transthyretin levels in depression: Correlations with suicidal ideation and low serotonin function. Biol Psychiatry 60: 500–506.


text: Public Private Partnerships across the GCC – taking stock Share this 21 NOV 2016 By: Trevor Butcher Adam Haque Many long-term observers of the public private partnership (PPP) market in the GCC have developed a healthy degree of scepticism over the years. Too many false dawns have unsurprisingly led them to believe that the PPP model of procurement is one that simply won't ever take off in this market - at least outside of the power and water sectors. The increased interest in PPP over the past 24 months is, in the minds of the sceptics, a short term blip that will be forgotten as soon as the oil price begins to climb or structural changes on the expenditure side allow regional governments to balance their budgets by other means. Despite this, approaching the end of 2016, it appears that most if not all GCC governments are seeking to implement PPP procurements or have announced their intention to do so. The oil price remains stubbornly low and expectations of future rises have been dampened. There is also an increased awareness of the need for structural change in many of the local economies. Although many challenges remain before there is a healthy pipeline of PPP deals across the region – and we don't expect many of the sceptics to have been converted – there is a real feeling that the stars have never been better aligned to allow that pipeline to develop. Of course, the GCC is not a single market and the situation varies markedly between the different countries. It is increasingly clear that each country will develop its own approach, including as to the legislative framework, implementing bodies and types of projects undertaken. Sponsors, funders, contractors and consultants need to be aware of these differences to ensure they are best placed to capitalise on the opportunities presented. This paper takes a high-level look at each of the GCC counties, considers the progress they have made to date in developing a PPP market, and explores how they might progress in 2017. One of the challenges in addressing this topic is that there are many different understandings of what constitutes a PPP. Whilst a variety of different models is healthy, in this paper we are primarily referring to long-term contracts (typically 15 years or more) between a private partner and a government partner in which the private partner provides a public asset or service, including financing, design, construction, operation and maintenance, with the bulk of its remuneration being based on the availability or condition of the asset and/or the quality of the service over the operating period. This paper does not address the power (including renewables), water or natural resources markets in any detail, as these are generally progressing – very successfully – along a parallel track, often based on sector specific legislation.

subset: commoncrawl


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A look back at France's second World Cup championship win in photos. View gallery »
Several dozen people are confirmed dead after a truck driver plowed through the... View gallery »
The world grieves alongside France with vigils, rallies and displays in cities... View gallery »
Multiple attacks hit Paris on Friday, including a hostage situation at the... View gallery »
Getty France became the 14th country to legalize same-sex weddings after French President Francois Hollande signed a bill into law extending the right to marry to gay couples, according to NBC News. The legislation is one of the biggest social reforms the country has witnessed since abolishing the death penalty in 1981. The bill, which had been pledged by the Socialist president, had sparked much debating from conservatives in France. Opponents of the bill have engaged in heated protests, some of which have been violent. Frigide Barjot, a political activist and humorist, has said a protest is slated for May 29 will draw millions into the streets. However, polls show that more than half of the country supported gay marriage. Get More at NBC News


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Americus, Ga. – The Georgia Southwestern men's basketball team is set to return to the Peach Belt Conference Tournament. The Hurricanes make their second consecutive tournament appearance, third in the last four years, and have their highest seed since capturing the top seed in 2011
All the action is set to take place on March 7-9 at the Frank G. Lumpkin Center in Columbus, Ga. The Hurricanes, seeded second in the West Division, will take on the East Division's third seed Lander University at 5 p.m. in the third quarterfinal matchup on Friday afternoon (March 7).
Fans can watch Friday's game, and all the games of the tournament, free of charge on the PBC's Ustream channel at ustream.tv/peachbeltconference. Live stats will also be provided, as well as in-game updates on the PBC Twitter feed.
The PBC will also provide a stream of every post-game press conference. Links to all the live coverage along with ticket information can be found on the official tournament website here. A PDF bracket can be found here.
The Hurricanes finished the regular season with an overall record of 19-9, and 11-8 in conference. The Hurricanes are currently on a won game winning streak after the win on the road at North Georgia. GSW is fortunate to have seven seniors taking leading roles for the Hurricane roster.
Alan Arnett is leading GSW with 466 points so far this season. Arnett, a second-team All-Conference selection, is fourth in the conference in points scored and has 16.6 points per game. He is shooting 48.8 percent from the field, and 78.2 percent from the free throw stripe. The Kokomo, Ind. native has the third highest playing time in conference, being one of nine players to reach over 900 minutes.
The Hurricanes consistently see double-double figures from senior Johathan Brooks, who is nationally ranked 23rd with 11 double-doubles during the season. During the ninth week of the season, Brooks was named PBC Player of the Week after a week of achieving two double-doubles averaging 12.5 points and 16 rebounds. He also added 2.5 assists and two rebounds. Entering the tournament Brooks averages the most rebounds in conference with 282 rebounds, averaging 10.1 per game. Macon, Ga. native is also among the nine PBC players to reach over 900 minutes of play. Brooks is shooting 46 percent from the field, 44.3 percent from beyond the arch, and 65.1 from the free throw line. Brooks is fourth in the league with 44 blocks.
GSW is expecting senior Daryl Davis to produce high numbers as well during the tournament. Davis was rewarded with being named PBC Player of the Week during the fifth week of the season. This was awarded after Davis shot 72.2% (13-of-18) from the field in two games and added two blocks per game. Davis is averaging 13.6 points per game and 6.6 rebounds per game. He is second in conference with a field goal percentage of 58.8. The Mobile, Ala. native is also eighth in the PBC with 34 blocks. Davis is shooting 71.3 percent from the free throw line.
Senior Jamon Hawkins will be expected to start for GSW in the guard position. Hawkins is a solid player from the floor as he adds 11.2 points per game. Hawkins is shooting 42.6 percent from the field, 37.3 from behind the arch and 76.6 percent from the free throw line. Senior Adrian Knight is also expected to join the starting five during the tournament as he has for the past four games. Knight contributes about 2.5 points per game and 1.6 rebounds per game.
Derrick Dawkins, Bobby Glover, Josh Birchfield, and Kevin Burnell add depth to the Hurricane bench each producing quality game time minutes.
Lander (17-11, 11-8 PBC), is led by PBC All-Conference team member, Michael Panaggio. Panaggio is ranked second in minutes per game, fourth in total assists with 132, and seventh in three-pointers made and assist-to-turnover ratio.
The last game between Lander and GSW was January 30th. Lander came out on top with a 62-58 win. Lander had double-digit scoring by Darius Carrier, Panaggio, Mario Pendarvis, and Jr Washington. This game was GSW's fourth loss of the season.
The winner of the Georgia Soutwestern/Lander quarterfinal will take on the winner of the quarterfinal between Columbus State and USC Aiken in Saturday's (March 8) second semifinal at 8 p.m.
The tournament championship game will take place on Sunday, March 9, at 4:30 p.m. The champion will receive an automatic bid into the NCAA Division II Tournament.


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Martin to Fill In for Hamlin
Mark Martin will replace Denny Hamlin in the No. 11 Toyota during Hamlin's recovery from a back injury, according to a news release provided by the Joe Gibbs Racing team Thursday.
Hamlin suffered an L1 compression fracture in a crash on the final lap of the NASCAR Sprint Cup Series Auto Club 400 at Auto Club Speedway in Fontana, Calif., on Sunday.
Upon his return to North Carolina, Hamlin was evaluated by Dr. Jerry Petty of Carolina Neurosurgery & Spine Associates. Dr. Petty's diagnosis was that Hamlin, 32, would not require surgery, but would be sidelined to give his injury time to heal. Petty estimated the length of Hamlin's recovery at six weeks.
"We've been real fortunate to have never been in this situation with the need to find someone to fill in for an injured driver," said J.D. Gibbs, president of JGR. "A lot goes into a decision like this but we are really pleased to have someone of the character and caliber of Mark Martin to fill in while Denny is out.
Martin is currently competing for Michael Waltrip Racing, which also fields Toyotas, on a limited schedule. He was not scheduled to be in the team's No. 55 Camry for the April 7 event at Martinsville (Va.) Speedway; however, he was slated to drive for MWR at the next three events after next week's race -- Texas, Kansas and Richmond.
Brian Vickers is scheduled to compete for MWR at Martinsville, while team co-owner Michael Waltrip returns to the seat for the May 5 Cup race at Talladega, Ala.
Rodney Childers, Martin's crew chief at MWR, tweeted: "This will be like me loaning my girlfriend to my best friend for 4 weeks to see if I like this other girl moving fwd.. It will be very hard. But overall, this will show a lot of people that we work a lot closer with JGR than most people think we do."
If Hamlin is out for the entire six weeks, he would return the following week, May 10-11, when the series heads to Darlington (S.C.) Raceway.
According to JGR officials, Martin has one previous start with the team, a runner-up finish in a Nationwide Series event in 2012 at Las Vegas Motor Speedway.
A veteran with more than 850 career starts in Cup, Martin is a 40-time winner in the series. His last Cup win came in 2009 while competing for Hendrick Motorsports.
"I'm happy to have this opportunity to help out JGR, FedEx and Denny," Martin said. "Hopefully I can keep those guys up front and compete for wins while Denny heals up."
He has 48 career starts at Martinsville, an 0.526-mile track, with two wins (1992 and 2000). He has a career average finish of 13.3 there.
Hamlin, 10th in the points standings after the season's first five races, said he was supportive of Martin's willingness to step in for him.
"I just hate not being able to be in the car right now, but I really appreciate Mark being able to fill in for me," he said. "Like everyone in NASCAR, I have a tremendous amount of respect for Mark and I know he will keep that (car) up front until I get back behind the wheel."


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The Adams Gallery at Suffolk University presents The Mirror of Race: Seeing Ourselves through History, a photography exhibit that challenges viewers to think about what they see when they look into this mirror onto an earlier time. Interactive elements suggest reflection about how one sees race – in oneself and in others.
The exhibit includes daguerreotypes, tintypes, ambrotypes and other forms of early photography as well as prints from tintypes made by an Air National Guardsman deployed to Afghanistan. Ed Drew's modern-day tintypes are believed to be the first battle zone photographs using this method since the American Civil War.


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Volume 17, Number 33 | May 06 - 12, 2005
Downtown Express photo by Lincoln Anderson
Construction workers were building the Gansevoort garage addition last Friday, but the city has now put a hold on the project.
City halts garage work in Hudson Park
By Albert Amateau
In a deal brokered last week by State Supreme Court Justice Michael Stallman, the Department of Sanitation has agreed to voluntarily cease construction of the temporary garage that it began in January on the Gansevoort Peninsula.
The agreement, pending further hearings on the lawsuit by Friends of Hudson River Park seeking to get the Sanitation Department off Pier 97 as well as the Gansevoort Peninsula, could be the first step in the broader long-sought goal of freeing the full 5 miles of the Hudson River waterfront park for park use.
This is something that cries out for a reasonable solution, Stallman said to Daniel Alterman, attorney for Friends, and Susan E. Amron, representing the city, at the April 29 hearing on the Friends application for a temporary restraining order against further work on the two-story garage. Sanitation has said the temporary two-story structure could serve until 2012 or beyond.
It seems to me that eight years is somewhat long, said Stallman. But it also seems fair to everybody to have a finite term of [Sanitation] use, known to everybody and legally enforceable, he added.
We also want to make sure we dont create a short-term problem by precipitously forcing these uses onto other communities, Stallman said. He set Thursday May 7 for more discussion on the issue, and asked Amron for Sanitations voluntary agreement to cease work on the garage. The department agreed on Monday afternoon to cease work as of Wednesday.
Alterman said on Tuesday that he was gratified that Sanitation has agreed to cease construction on the garage. Id be pleased if this leads to a more global agreement to make the Hudson waterfront free of Sanitation uses much earlier that 2012, he added.
Albert Butzel, president of Friends of Hudson River Park, a community group advocating for the 5-mile riverfront park being built between the Battery and 59th Sts., said, Its early in the action but its a step forward to get Sanitation off the waterfront and get some rent for the park in the meantime.
The suit also wants the court to set a reasonable rent or use and occupancy payment that the city must pay to the Hudson Park Trust for the continued occupancy of Pier 97 and most of the peninsula from the Dec. 31, 2003, deadline set in legislation until Sanitation leaves.
The suit charges that the Sanitation Departments continued use of the peninsula to park garbage trucks violates the 1998 Hudson River Park Act by not using its best efforts to get off the waterfront before the deadline. Sanitations response to the Friends complaint says the department has been diligently working to relocate the garbage truck parking. The effort involves six garages for Manhattan districts in Lower Manhattan, the Village, Chelsea, Midtown and the East Side and the Upper West Side.
The District 2 garage now at the Gansevoort Peninsula, the District 5 garage now at E. 73rd St. and the overflow from District 1 [Lower Manhattan] will eventually be relocated at a new site, the reply says.
One possibility for the new site, according to Sanitation Department spokesperson Kathy Dawkins, is the 800,000-square-foot U.P.S. parking lot between Washington and West Sts. stretching north of Spring St.
Conjecture among real estate people is that U.P.S. has selected the Brodsky Organization to develop the site but the city is moving to take it for Sanitation under eminent domain. Bob Godlewski, U.P.S. spokesperson, said, Were continuing to try to maximize the value of our property there and we have to protect the facilities that we have there already. We have a staging area for tractor-trailers there.
However, the southern part of the Hudson Sq. neighborhood, formerly known as the Printing District, is increasingly becoming a high-end residential area, and was recently rezoned for residential use. Just north of the site and a block east on Greenwich St., more than 200 units of luxury housing have gone on the market with condos selling for more than $2 million. More luxury apartments are on the way, including one on Washington St. just south of Spring St., planned by Vendome Realty to be designed by the firm of the late Philip Johnson.
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the carolina watchman vol xv.—thibd series salisbury n g june 26 1304 no 37 tht c 0 l<li r :'-" 1 lll<l virgin mary ., lini'u r divine ucky apple ■ine : • ind sm ill ept i ii — ■: . ■- m.;i tlumn . do ■tier ■atlon ■i praj ing ■• ar i inks i ink s ■11 h .' him ■■• ' him : ' ! mj will i 1 1 1 1 ■. pale v now ii v i r ii . lo • lei'l : «. - i inert : i wlszi 15 vuqi/k kialto ./ ii uiainglon a ( '., i exit ans in 31 . . ;' ( fibraltar that white _ that nobls bay is ca diz the point of departure of three of yages it was there . rannical i ton ( arlos com i mil : ; unate shoemaker to cat - .-, . h he had made for did not lit internal application of shoe • icted upon the luckless disci f si < rispin we are no inform i i n-'told in confidence til of ai ry inclinations nally ap i it hurts al 1 ; dog or a shot ame and with it head ad blowing directly straits this compelled up so the rialto be : vfrico-european ferry in the sombi e night f the moroccon mountains tit ship and standing . raining light on r then hack to cape tin scarcely gaining a ca ■the whole manuvre ■. >. : - turaging but ( ■is the si raits be uarrow we w ei e obliged to tack ■■i iur and all hands being red for i hat opera ion neither ( go below the morn bright and beautiful i in rock of gibraltar just nld rise in this much sung 1 1 would be highly repreheu in it to rise in any other man - i much in bi en said sung • ii about the cosmic glorii s hie tii ivas fresh i'l w ind w a reg . lid though it continued y in the same quarter as the ore our situation peemed liter in the rosy light of v were as much ■x liilerating morn - _: : of land as if ; : - ight's sleep stn ng current - the straits from the mi ditenanean and n own i xertions we had ■progn is dm ing '. we were at the narrow betwi i n the i and the a:i m h city of tangiers this n c'icrops laid the ' " ; ■ol atln i and was hoary hen troy fell it was 1 don s bastian king of por - i a battle with the m i rs lisappeaied so mysteriously - i seeu nor heard from for twenty years then he turned up like a royal enoch arden an old white haired man in venice the air is so fresh the skies so blue and the landscape so charming that there is exhileration even in tacking away we speed toward the african ehofe little white specks appear along the water-sidej soon these are tiaii-formed to houses and little black specks moving around among them in turn change to people ere long the whole city of tan giers is plainly visible and its streets swarming with men women and don key are clearly discernable still we fly on the wrinkled brown faced old mussulman standing guard on the rampart of the fort gazes sto lidly at us with true oriental indiffer ence allah is great soliloquizes thir did heathen warrior if it be the will of kismet that this dog of a christian should run into the fort and knock me into the north-west corner of paradise then he must do so a boat laden with fruit and mann ed by dusky rascals filled with fraud and spec ulative intentions pulls off from tht shore a his fur their plans we fly p:ist them having no time to be cozened a pretty good-natured little moorish maiden among their number lands up in the bow of the i kit and cleverly throws a handful of dates on our deck as we pass now : not a stone's throw separates us from he walls of tiie fort we can look down the throats of the rusty old u r uns peeping through the embrasures — can eiuiiit the patches on the corn i inander's pantaloons surely we will dash into the fort ! suddenly the order ring out hard a lee ! main top sail haul ! let fly lore bowline and round all the heavy yards fly round the ship wheels gracefully about assumes an even keel for a j moment careens to the other side and speeds away like an arrow to the oth er shore leaving the enterprising ; arabs and the martial old fatalist far astern the straits are filled with shipping some are tediously beat ■ing up like ourselves ; others run ning westward with square yards among the latter is a stately man of war as it passes us there is a mutual display of colors followed by a mu tual disappointment we supposed them to be danes and they evident ly took us for italians like themselves i again we are on the european side the green vineyards and olive groves with the cottages dotted about on the mountain sides form a panorama very pleasant to eves satiated with sea and sky for forty-two days every crest and crag is crowned with a little building in the shape of a light house these arc watch towers built for the purpose of keeping anil eye on the uncertain movements of the erratic moors in order to dissuade them from a naughty habit they had of running over the way when the a igerinp stock of houris ran short and borrowing a few senorita visigoths while old padre visigoth was out grubbing in his olive orchard from the spanish side a splendid view of the atlas j range is obtained and of all grotesque a:ul fantastic shapes they take the lead one is almost ready to credit the legend that they are a petrified menagerie some old ethiopian john robinson come to grief perhaps in this gorgon ic col lection there is a i monkey which only needs rascality i and breath to be alive ; a cimel gre cian bend idiotic expression profuse minder lip and all towering majes tically above them as befits his regal - i itv lies the hoary old weather-beaten moorish lion with his shaggy grey head resting on his paws he seems grimly contemplaing t lie land of his leonine rival the brittish roarer from which lie is forever excluded in the ravine between this remarkable mountain and the african pillar of hercules lies the largo moorish city of ccnta here dwelt the bold trai tor count julian here was the home of the pr ud countess and her daughter the beautiful and un fortunate florinda from this port in tlu set out the expedition which subdued spain commanded by the doughty toric or tojic el tuerto as he was called by the spanish on ac count c f a deficiency in optics and guided by the traitor julian that great white building on the edge of the precipice is the castle from the highest turret of which the crafty and cruel hnrled the five year old son of count julian while the dis tracted mother held by two moorish j soldiers beheld the scene from below , one moe tack and the grim rock of gibraltar the key of the levant | towering 1400 feet above the sea lies on our weather bow the history of this stern stronghold is one of the most interesting extant and nov j we are almost beneath the shadow of j this ancient of days this furrowed and rrim old sentinel who for count less ages has etood guard at the door ; of the world's theatre who has beheld unmoved the crash of mighty empires looked down upon the smoking wake of hannibal's victorious army seen the bloody frays of turbaned moor j and christian knight and received : the yearning glances of the undaunt > ed columbus as he sallied forth with ' his frail ships to verify the theories ' ' of the learned and give a new world to ferdinand the noble bay y now opens to view above the for est of masts the cathedral spire tow ers like a giant of the yosemite amid saplings it is soon apparent that it will be an exceedingly close shave to weather europa point but as it is our only chance we crowd all sail and press on the interminable rows of barracks appeared nestling around the foot of the rock rising above them terrace ; upon terrace gallery upon gallery are countless batteries of the most formid able guns the world possesses on the extreme summit is seen what from our deck appears to be a pen holder with a diminutive red rag on the end but which in reality is a flag-staff bearing the standard of ! brittania one of her patriotic poets assures us she needs no bulwark no towers along her steep she nevertheless ! possesses herself by what have i naively calls policie of any stray ; bulwarks towers etc that fortune ! may throw in her way l>y this time we are within pistol shot of the mole affairs begin to assume an intensely ; interesting appearance opinions as ! to whether we will weather the point or not are about equally divided like toric we have gone too far to ! retreat and as a yankee skipper re marked off cape cod we've got to weather that air p'int or put our an gel plumage on now not a ship's length separates us from the light house on europa point the keeper [ surveys us with a calculating eye and i wonders how the wreckage will pan i out some of the men cast earnest glances at the life preservers a minute more and the kialto sweeps ' past the southern extremity not three i fathoms distant a little group of ' english officers standing on the jetty swing their caps a danish man o war in the bay dips his colors the j light-house keeper turns disappoint edly away and the rialto at last ! floats in the azure waters of the med iteranean at our nearest point to the mole we could have thrown a cat ashore not being possessed of the necessary feline however we could only content ourselves with observ ing that such a tiling was possible 2o be continued ugly spots on their record blame's use of the cipher — logan in a snap — his anii war record new bork sun's special washington june 18 — a very careful examination of the records of i blaine and logan hao already begun 1 in logan's c.'ise it is susceptible of proof by gentleman in this city that at the beginning of the war he was as 1 bitter in his opposition to what lie called the coercing of the southern states as any secessionist to one gentlemen now living here who met i logan on march 9 1s61 logan said that he regarded the attemp of lin colu to coerce sovereigu states as un constitutional and infamous ami if it j was persisted in he would go to his home in southern illinois and raise a regiment to help those states defend themselves against federal encroach ment logan's early secession pro clivities in southern illinois will be made public logan expects to spend the summer months in maine prob ably taking a cottage at mt desert this will give him the opportunity to be close to blaine when mr blaine was returning from garfield's funeral he said to a friend who suggested that he would be the next republican nominee : no i am discharged cured yet notwithstanding this assertion little facts arc leaking out which show that the white house fever is never cured mr blaine for instance was in con stant communion by cypher telegrams with his managers in chicago he followed their advice in all things and it is asserted that when he went to the railway station on monday he had not fully decided to go north un til a cypher dispatch was handed to him at the depot telling him to go that mr blaine not only knew who the agents were who went through the south but also suggested some of them as certain assertions about the use of money to secure his nomi nation are being made in greater and greater detail the editor of the sun day capital asserts that money in large quantities was used to secure blai nc's nomination nor was there anything haphazard in the way in which the bribery was done a man to do the buying was assigned to each of the several delegations from the south thirty votes were wanted not later than the third ballot and prices were arranged for the first second and third ballots it has been asser ted in detail that 10,000 was sent to the sherman house for six votes from south carolina but smalls watched his delegation so closely that only two were obtained the same sum was ready for six votes from north carolina and tennessee it was asserted by a texas delegate that from 750 to 4,000 had been des tributed among the delegates from his state it is probable that the story of blaine's attempt to have garfield's cabinet during his illness declare that the president was unable to per form the duties of the office and urge arthur to assume the functions of the oifice during the disability will be told it was believed at the time that nothing but the threat of mac vcagh prevented blaine from over ruling the rest of the cabinet who were opposed to this dangerous line of policy one of blaine's leading managers said yesterday that arrangements were being made for a meeting between blaine and conkling early in august through the intervention of friends a reconciliation has been effected ac cording to this man's assertfon conk ling is willingto makeseveral speeches for blaine after the october elections this assertion agrees with an opinion expressed by collector robertson at chicago it was said as a result of such action conkling would probably be returned to the senate as lap ham's successor it is apparent that the blaine man agers will have no difficulty in rais ing all the money they want the star-routers with a lively sense of favors to come will contribute liber ally and it is reported that william walter phe!p's individual subscrip tion of 8100,000 has been promised levity with letters out of print uie letter b greatly in demand the letter i always in debt and disgrace the letter e never out of office the letter i always first and last in the river the letter it frequently late u for it frequent ly co i:es alter t good for naught the letter o always in use — the letter u the most welcome letter of all — the one with an x in it always away from home — u and i forever in bed — e always cross — x always in drink but never intoxi cated — k — chicago sun baton rouge june 20 the bill appropriating 100,000 to the new orleans world's exposition has final ly passed the house for elector at large it li more than probable that the ! name of hon w . m liobbins of statesvilc will be presented to the state convention as a suitable candi date for presidential elector on the democratic ticket from the state at large as it is understood that his friends are anxious to bring him i back into active politics m:ij liobbins if appointed and we understand that he would accept the appointment would make an admira i ble effective and energetic canvass of the state for the democratic nominee of the chicago convention he is probably as well posted on national politics as any man in north carolina and he is one of the best public speakers in the state jf the coming canvass as many suppose is to be hot and heavy m:ij robbins can hit as many and as hard blows as any man we know of we believe uo better selection could be made maj robbins never fails to interest and rouse his audi tors some of the english medical jour ; mils have already begun to point out | the great importance of not over-feed i ing infants with starcy foods such as , bread farina gruel etc as the warm season approaches according to au ! thorities like sir james paget such over-feeding is a fruitful cause of the large infant mortality in warm weath \ er the one article most necessary to : the life of a child is water i _ london june 20 — a berlin dis patch to the morning post says that ; several important arrests were made yesterday in connection with an in cendiary conspiracy this conspira cy it is asserted has ramifications in america should inquiry prove that i the german conspirators have receiv ed funds from america immediate j diplomatic action will be taken by i the german representative at wash , ington .«». washington june 20 — win ii { dt'mpsev a well known bookseller i and stationer of this city has been arrested on a charge of being connect ed with navy department frauds ! detective win p wood has repre sented to the secretary of the navy that he has found evidence of even more extensive frauds in other bu reaus of the navy department than in that of medicine and surgery nashville june 20 — the dem ocratic state convention adjourned last night after re-nominating gov bate by acclamation the platform endorses the administration of gov bate regards the adjustment of the debt f tennessee as final and pledges faithful maintenance of its conditions and favors a tariff for sufficient reve nue to defray the expenses of the gov ernment i will not allow any one to say that any man served in the war with more zeal than i did is senator lo gan'.i blustering way of extolling his own ardent patriotism perhaps no body wants to say that but we take the liberty without the senator's per mission of saying that some men made up their minds on which bide to serve with considerate more promptness than john a logan did boston globe nordenskjoki the arctic explorer is asking aritish assistance for an ex pedition to the south ships have never approached the south pole nearer than 2,000 miles this lost re gion 4,000 miles in diameter re mains a scaled mystery to the out side world washington june 20 secretary chandler has ordered a court of in quiry to investigate the frauds recent ly practiced by means of false vouch ers in the bureau of medicine and surgery of the navy another war rani was sworn out to-day against a prominent washington stationer nam ed dempsey,implieat€d in these frauds new york june 20 cornelius k garrison has assigued to john 1 perry preferences 631,000 paris ky june 20 rev geo t gould president millersburg fe male college has assigned liabilities unknown assets 13,000 among other tribulations that fall upon this devoted country the cam paign poet is beginning to got in hid work jolin sheppard ix v swink j 31 monroe kluttzs warehouse for tiic sale of leaf tobacco salisbury north carolina farmer's remember klutt's warehouse has sold three fourths of all the tobacco sold on tin markel this seaso . t , show the highest averages for crops and a general average second w in the state for the same grades of tobac <>. kluttz's warehouse s the best lighted best arranged and the only house in the place that has storage room for planters tobacco if you want the highest prices for your tobacco sell ai kluttz's warehouse where you will always find a r ] l turn-out of anxious buyers john sheppard the champion tobacco aucxioitkeb of westrrk north carolina has orders for tobaccos and will pay highest prices i for all grades from the ground leaves to fancy lemon wrappers daily sales highest prices guaranteed your friends truly sheppard swink & monroe salisbury n <'.. june lr!i iss4 pig^f|||f>ppu||meg|j 1 a and will completely change ...■■blood ; n tha cn<;re s.ritem in three months any person who will tike 1 till each iii l from 1 it vi weeks n\ay bo restored to bound health if such a tbiupr lie po.-..-iii -. ';'■■_• female complaints these i'ills linve no equal physicians use them for the can if livkb and hjdney diseases sold everywhere or s?nt by mail for o in stamps c':r ilara free i ;. johns n t co , botton mas o e3 fp§k ej -'-'■i -" r j fj crocp a>ihm bronchitis ttcnral rllalls *'■■' r -^ -■'• k • r - - : " i n johnsons no id c ti - ■•<; "~ ■i [■' i — • ' ■' • > u in«:aiitjncoii»iy relieve bew icrribla '.'■i ■i ■■s ., : • ■\ ■' ■■■-. -:.: « i positive cure nine cases i ri ►■'.' •.- i ' , • n -.... ■•• inttrantlon tlml will rave many l s m sg ?■" i '* m ' j - - l ' '""""* by mail don t delay * momen johnson's akodykh li . r -!' * < '■■■: - ■lenza bleedlnir t the lnmn ii.«r r,r 1 i ■.■.:■• ush i ! i it cholera morbui kidney itvouei.aai ui : us s lever n-herc • i s j itlnson .': co bntdu m •-. der dose one tpasp ifiil i tch pint of fool itwill nls positively prevent an.j enre ni>jrrn.],ra soldotcrywherp.orsr'-t liv triaiir.irr.'r.ia mnn^t/r-iiis et k r 1 fy ed stamps furnished in large cans,price$l hvmuil.sijoli vnlwivkev wnvfacka orculanftee j s juilnao & to ko.uii mas , dec 20 l3s3 t 10:ly pacers warehouse ! union steet - - - danville va is now opened and ready for business we have one of the largest and most complete warehouse ever built mm the sal.e of leaf tobacco in the best leaf market in the united states v trial i all we a*k ' pace bros & co ) moms wip ml w a w w si furie dealer upboisterer ""• and undertaker v fine walnut suits - • ■50 w$je$jh cottage suits 20,25 and$30 i ■£! i i . k ? 4^p woven wire mattresses 7.5(1 parlor suits 35 to 100 cheap bed3 2.50 fine line of carpets sewing machines weed and hartford uu pl^it l t t trff ■■■■■■■retime ■> not t illy oocupi d will bod i t to thtir inwrert ,', v i : i v j , f " .,>- r ram ju.t coming on the ii-ir..f aot.od thm " ■••■*■"'" "•,•...:; "','.,.. ■.• ■- a i fmakinirid ey und of ielf cultnre write for mdll to ttis1 b f jobrasos & <«»., 1,<»13 main street richmond ffe m '. - ■■'..■■■:; ; -,:;- ; to.vxe psss . total assets 710,745 2 a home < ompany seeking i h mie i 1 ronagc steong ppwo libeeal term 1 preini ani c in i ' is j allen brown agt 23-gui ~ n ' '. . f 3 ■eceta3lepill3 5 r 7 ft ip eft l i ¥ e r i id ill 3 ileus complaints ...-,. no srip ■. ii . v.su save your fbtjit ! scarr's fruit preservative ? without the use of sealed cans tlic cheapest and only sure kind known perfectly harmless call ' j '■'■at enniss dkcc store l:tf notice j ■will jc i meeting of the stock the western n c railroad com j x ( '.. on tuesday the 25th j me mm bj order»of the i'resi dent geo p kuwi.s scc'j & trcas'jr salvbiirj x c . may 21 lb»*4 j
Object Description
|Carolina Watchman
|The Carolina Watchman
|Technical Metadata
|Image was scanned by OCLC at the Preservation Service Center in Bethlehem, PA. Archivial image is an 8-bit greyscale tiff that was scanned from microfilm at 400 dpi. The original file size was
|[T. K. Bruner and J. J. Bruner]
|Date Digital
|[T. K. Bruner and J. J. Bruner]
|United States, North Carolina, Rowan County, Salisbury
|Digital Format
|Project Subject
|State Archives of North Carolina Historic Newspaper Archive
|The June 26, 1884 issue of the Carolina Watchman a weekly newspaper from Salisbury, North Carolina
|The SA of NC considers this item in the public domain by U.S. law but responsibility for permissions rests with researchers.
|OCLC number
|Carolina Watchman
|The Carolina Watchman
|Technical Metadata
|Image was scanned by OCLC at the Preservation Service Center in Bethlehem, PA. Archivial image is an 8-bit greyscale tiff that was scanned from microfilm at 400 dpi. The original file size was 5291848 Bytes
|Date Digital
|Hamilton C. Jones
|United States, North Carolina, Rowan County, Salisbury
|Digital Format
|Project Subject
|State Archives of North Carolina Historic Newspaper Archive
|An archive of the Carolina Watchman a weekly and semi weekly newspaper from Salisbury, North Carolina
|The SA of NC considers this item in the public domain by U.S. law but responsibility for permissions rests with researchers.
the carolina watchman vol xv.—thibd series salisbury n g june 26 1304 no 37 tht c 0 l


text:
Mug Shot | Stokely Carmichael
Now that they've taken Dr. King off, it's time to end this nonviolence bullshit.
– Stokely Carmichael
Stokely Carmichael was the youngest of the Freedom Riders in the summer of 1961, arrested for entering an "all white" cafeteria and disturbing the peace. He was nineteen years old.