Stabilizing the Proteomes of Acute Myeloid Leukemia Cells: Implications for Cancer Proteomics.

Previous work has shown that inhibition of abundant myeloid azurophil granule-associated serine proteases (ELANE [neutrophil elastase], PRTN3 [protease 3], and CTSG [Cathepsin G]) is required to stabilize some proteins in myeloid cells. We therefore hypothesized that effective inhibition of these proteases may be necessary for quantitative proteomic analysis of samples containing myeloid cells. To test this hypothesis, we thawed viably preserved acute myeloid leukemia cells from cryovials in the presence or the absence of diisopropyl fluorophosphate (DFP), a cell-permeable and irreversible serine protease inhibitor. Global proteomic analysis was performed, using label-free and isobaric peptide-labeling quantitation. The presence of DFP resulted in an increase of tryptic peptides (14-57%) and proteins (9-31%). In the absence of DFP, 11 to 31% of peptide intensity came from nontryptic peptides; 52 to 75% had cleavage specificity consistent with activities of ELANE-PRTN3. Treatment with DFP reduced the intensity of nontryptic peptides to 4-8% of the total. ELANE inhibition was 95%, based on diisopropyl phosphate modification of active site serine residue. Overall, the relative abundance of 20% of proteins was significantly altered by DFP treatment. These results suggest that active myeloid serine proteases, released during sample processing, can skew quantitative proteomic measurements. Finally, significant ELANE activity was also detected in Clinical Proteomics Tumor Analysis Consortium datasets of solid tumors (many of which have known myeloid infiltration). In the pancreatic cancer dataset, the median percentage of nontryptic intensity detected across patient samples was 34%, with many patient samples having more than half of their detected peptide intensity from nontryptic cleavage events consistent with ELANE-PRTN3 cleavage specificity. Our study suggests that in vitro cleavage of proteins by myeloid serine proteases may be relevant for proteomic studies of any tumor that contains infiltrating myeloid cells.

Alterations in protein regulators of neurodevelopment in the cerebrospinal fluid of infants with posthemorrhagic hydrocephalus of prematurity.

Neurological outcomes of preterm infants with posthemorrhagic hydrocephalus are among the worst in newborn medicine. There remains no consensus regarding the diagnosis or treatment of posthemorrhagic hydrocephalus, and the pathological pathways leading to the adverse neurological sequelae are poorly understood. In the current study, we developed an innovative approach to simultaneously identify potential diagnostic markers of posthemorrhagic hydrocephalus and investigate novel pathways of posthemorrhagic hydrocephalus-related neurological disability. Tandem multi-affinity fractionation for specific removal of plasma proteins from the hemorrhagic cerebrospinal fluid samples was combined with high resolution label-free quantitative proteomics. Analysis of cerebrospinal fluid obtained from infants with posthemorrhagic hydrocephalus demonstrated marked differences in the levels of 438 proteins when compared with cerebrospinal fluid from age-matched control infants. Amyloid precursor protein, neural cell adhesion molecule-L1, neural cell adhesion molecule-1, brevican and other proteins with important roles in neurodevelopment showed profound elevations in posthemorrhagic hydrocephalus cerebrospinal fluid compared with control. Initiation of neurosurgical treatment of posthemorrhagic hydrocephalus resulted in resolution of these elevations. The results from this foundational study demonstrate the significant promise of tandem multi-affinity fractionation-proteomics in the identification and quantitation of protein mediators of neurodevelopment and neurological injury. More specifically, our results suggest that cerebrospinal fluid levels of proteins such as amyloid precursor protein or neural cell adhesion molecule-L1 should be investigated as potential diagnostic markers of posthemorrhagic hydrocephalus. Notably, dysregulation of the levels these and other proteins may directly affect ongoing neurodevelopmental processes in these preterm infants, providing an entirely new hypothesis for the developmental disability associated with posthemorrhagic hydrocephalus.

Comparative proteomic analysis identifies age-dependent increases in the abundance of specific proteins after deletion of the small heat shock proteins αA- and αB-crystallin.

Mice with deletion of genes for small heat shock proteins αA- and αB-crystallin (αA/αB(-/-)) develop cataracts. We used proteomic analysis to identify lens proteins that change in abundance after deletion of these α-crystallin genes. Wild-type (WT) and αA/αB(-/-) knockout (DKO) mice were compared using two-dimensional difference gel electrophoresis and mass spectrometric analysis, and protein identifications were validated by Mascot proteomic software. The abundance of histones H2A, H4, and H2B fragment, and a low molecular weight ß1-catenin increased 2-3-fold in postnatal day 2 lenses of DKO lenses compared with WT lenses. Additional major increases were observed in abundance of ßB2-crystallin and vimentin in 30-day-old lenses of DKO animals compared with WT animals. Lenses of DKO mice were comprised of nine protein spots containing ßB2-crystallin at 10-40-fold higher abundance and three protein spots containing vimentin at ≥2-fold higher abundance than in WT lenses. Gel permeation chromatography identified a unique 328 kDa protein in DKO lenses, containing ß-crystallin, demonstrating aggregation of ß-crystallin in the absence of α-crystallins. Together, these changes provide biochemical evidence for possible functions of specific cell adhesion proteins, cytoskeletal proteins, and crystallins in lens opacities caused by the absence of the major chaperones, αA- and αB-crystallins.

Respiratory uncoupling in skeletal muscle delays death and diminishes age-related disease.

Age-related disease, not aging per se, causes most morbidity in older humans. Here we report that skeletal muscle respiratory uncoupling due to UCP1 expression diminishes age-related disease in three mouse models. In a longevity study, median survival was increased in UCP mice (animals with skeletal muscle-specific UCP1 expression), and lymphoma was detected less frequently in UCP female mice. In apoE null mice, a vascular disease model, diet-induced atherosclerosis was decreased in UCP animals. In agouti yellow mice, a genetic obesity model, diabetes and hypertension were reversed by induction of UCP1 in skeletal muscle. Uncoupled mice had decreased adiposity, increased temperature and metabolic rate, elevated muscle SIRT and AMP kinase, and serum characterized by increased adiponectin and decreased IGF-1 and fibrinogen. Accelerating metabolism in skeletal muscle does not appear to impact aging but may delay age-related disease.

Identification of potential mediators of retinotopic mapping: a comparative proteomic analysis of optic nerve from WT and Phr1 retinal knockout mice.

Retinal ganglion cells (RGCs) transmit visual information topographically from the eye to the brain, creating a map of visual space in retino-recipient nuclei (retinotopy). This process is affected by retinal activity and by activity-independent molecular cues. Phr1, which encodes a presumed E3 ubiquitin ligase (PHR1), is required presynaptically for proper placement of RGC axons in the lateral geniculate nucleus and the superior colliculus, suggesting that increased levels of PHR1 target proteins may be instructive for retinotopic mapping of retinofugal projections. To identify potential target proteins, we conducted a proteomic analysis of optic nerve to identify differentially abundant proteins in the presence or absence of Phr1 in RGCs. 1D gel electrophoresis identified a specific band in controls that was absent in mutants. Targeted proteomic analysis of this band demonstrated the presence of PHR1. Additionally, we conducted an unbiased proteomic analysis that identified 30 proteins as being significantly different between the two genotypes. One of these, heterogeneous nuclear ribonucleoprotein M (hnRNP-M), regulates antero-posterior patterning in invertebrates and can function as a cell surface adhesion receptor in vertebrates. Thus, we have demonstrated that network analysis of quantitative proteomic data is a useful approach for hypothesis generation and for identifying biologically relevant targets in genetically altered biological models.

Identification of a unique TLR2-interacting peptide motif in a microbial leucine-rich repeat protein.

Pathogenesis of many bacterially-induced inflammatory diseases is driven by Toll-like receptor (TLR) mediated immune responses following recognition of bacterial factors by different TLRs. Periodontitis is a chronic inflammation of the tooth supporting apparatus often leading to tooth loss, and is caused by a Gram-negative bacterial consortium that includes Tannerella forsythia. This bacterium expresses a virulence factor, the BspA, which drives periodontal inflammation by activating TLR2. The N-terminal portion of the BspA protein comprises a leucine-rich repeat (LRR) domain previously shown to be involved in the binding and activation of TLR2. The objective of the current study was to identify specific epitopes in the LRR domain of BspA that interact with TLR2. Our results demonstrate that a sequence motif GC(S/T)GLXSIT is involved in mediating the interaction of BspA with TLR2. Thus, our study has identified a peptide motif that mediates the binding of a bacterial protein to TLR2 and highlights the promiscuous nature of TLR2 with respect to ligand binding. This work could provide a structural basis for designing peptidomimetics to modulate the activity of TLR2 in order to block bacterially-induced inflammation.

Serum markers may distinguish biliary atresia from other forms of neonatal cholestasis.

BACKGROUND: Biliary atresia (BA) is the most serious liver disease in infants. Diagnosis currently depends on surgical exploration of the biliary tree. Noninvasive tests that distinguish BA from other types of neonatal liver disease are not available. PATIENTS AND METHODS: To identify potential serum biomarkers that classify children with neonatal cholestasis, we performed 2-dimensional difference gel electrophoresis, statistical analysis, and tandem mass spectrometry using serum samples from 19 infants with BA and 19 infants with non-BA neonatal cholestasis. RESULTS: Eleven potential serum biomarkers were found that could in combination classify children with neonatal cholestasis. CONCLUSIONS: Although no single biomarker or imaging test adequately distinguishes BA from other types of neonatal cholestasis, combinations of biomarkers, imaging tests, and noninvasive clinical criteria should be further explored as potential tests for rapid and accurate diagnosis of BA.

A novel surgeon credentialing and quality assurance process using transoral surgery for oropharyngeal cancer in ECOG-ACRIN Cancer Research Group Trial E3311.

PURPOSE: Understanding the role of transoral surgery in oropharyngeal cancer (OPC) requires prospective, randomized multi-institutional data. Meticulous evaluation of surgeon expertise and surgical quality assurance (QA) will be critical to the validity of such trials. We describe a novel surgeon credentialing and QA process developed to support the ECOG-ACRIN Cancer Research Group E3311 (E3311) and report outcomes related to QA. PATIENTS AND METHODS: E3311 was a phase II randomized clinical trial of transoral surgery followed by low- or standard-dose, risk-adjusted post-operative therapy with stage III-IVa (AJCC 7th edition) HPV-associated OPC. In order to be credentialed to accrue to this trial, surgeons were required to demonstrate active hospital credentials and technique-specific surgical expertise with ≥20 cases of transoral resection for OPC. In addition, 10 paired operative and surgical pathology reports from the preceding 24 months were reviewed by an expert panel. Ongoing QA required <10% rate of positive margins, low oropharyngeal bleeding rates, and accrual of at least one patient per 12 months. Otherwise surgeons were placed on hold and not permitted to accrue until re-credentialed using a new series of transoral resections. RESULTS: 120 surgeons trained in transoral minimally invasive surgery applied for credentialing for E3311 and after peer-review, 87 (73%) were approved from 59 centers. During QA on E3311, positive final pathologic margins were reported in 19 (3.8%) patients. Grade III/IV and grade V oropharyngeal bleeding was reported in 29 (5.9%) and 1 (0.2%) of patients. CONCLUSIONS: We provide proof of concept that a comprehensive credentialing process can support multicenter transoral head and neck surgical oncology trials, with low incidence of positive margins and *grade III/V oropharyngeal bleeding.

Type I collagen N-telopeptides adopt an ordered structure when docked to their helix receptor during fibrillogenesis.

The in vitro rate and specificity of fibrillogenesis in type I collagen depends on the integrity of the amino (N)-telopeptide domain. In vivo an intact N-telopeptide domain is also required for normal fibril assembly. Although Chou-Fasman predictions and NMR studies suggested that a type I beta-turn could be induced in alpha1(I) N-telopeptide chains, computer modeling did not identify ordered structures. Nevertheless, X-ray analysis and electron tomography studies have shown that the N-telopeptide is in one of the most highly ordered fibril domains. This study was undertaken to determine if the docking of the N-telopeptide to its helix receptor domain could induce the telopeptides to take up a specific conformation. With use of molecular modeling suite of programs, a (Gly-Pro-Pro)(n) triple-helical structure was built on the basis of high-resolution X-ray crystallographic coordinates and then replaced with the actual bovine collagen residues 924-938, the triple-helical alpha1(I)-N-telopeptide-receptor sequences. Energy minimization produced a modified triple-helical conformation. The bovine alpha1(I) N-telopeptide sequence was similarly minimized and docked to this receptor. The docking induced an ordered conformation with a stabilizing hydrogen bond in the N-telopeptide and, importantly, a reciprocal reordering of the triple-helical conformation in the binding domain. This docked structure placed Lys residues in both telopeptide and helix in the correct locations for cross-link formation. The modeling has been extended to the three-chain N-telopeptide domain and finally to the construction of the Hulmes-Miller quasi-hexagonal packing structure. Each N-telopeptide domain can form linkages with two adjacent, aligned helix receptor domains. The telopeptides and the order of staggering of the three chains in the helix play crucial roles in the packing and intrafibrillar cross-linking patterns and the relative azimuthal orientations of adjacent molecules in the fibril. The models confirm the high order in the N-telopeptide 4D overlap zone.

Hyperparathyroidism and multiple endocrine neoplasia.

Multiple endocrine neoplasia (MEN) syndromes comprise the group of heritable endocrinopathies, MEN 1, MEN 2A, and MEN 2B. Primary hyperparathyroidism caused by multiglandular involvement is usually the initial manifestation in MEN 1, occurring in more than 90% of patients. In patients with MEN 2A, hyperparathyroidism develops less commonly and is usually milder than in MEN 1. Advances in genetics and molecular biology aid in confirming the diagnosis and screening relatives who are carriers or at risk for the disease. Surgery plays an important role in the management of hyperparathyroidism in both MEN 1 and MEN 2A,although the timing and extent of surgery are areas of controversy.Long-term follow-up reveals a high rate of recurrent hyperparathyroidism in MEN 1 despite surgical intervention.

Second malignant tumors after treatment of nasopharyngeal carcinoma: four case reports and literature review.

The purpose of this study was to identify the histopathology, location, and latency interval for the development of second malignant tumors (SMT) after successful treatment for nasopharyngeal carcinoma (NPC). Of 55 patients, four developed SMT after successful treatment of NPC in a single institutional series for an incidence of 7%. An additional 31 patients with SMT after treatment for NPC were identified from the literature. At minimum, all patients were treated with radiotherapy to the primary site. The histopathology of SMT included sarcoma (69%), squamous cell carcinoma (17%), adenocarcinoma (6%), meningioma (6%), and lymphoma (3%). SMT occurred at various sites in the head and neck, but most (51%) arose in the sinonasal cavity. For the entire group, the mean latency interval between treatment for NPC and the development of SMT was 11.8 years. These findings indicate that the development of SMT in patients achieving long-term survival after treatment for NPC may be radiation induced. Long-term follow-up for these patients is important to assess for this potentially late complication.

In vivo substrates of the lens molecular chaperones αA-crystallin and αB-crystallin.

αA-crystallin and αB-crystallin are members of the small heat shock protein family and function as molecular chaperones and major lens structural proteins. Although numerous studies have examined their chaperone-like activities in vitro, little is known about the proteins they protect in vivo. To elucidate the relationships between chaperone function, substrate binding, and human cataract formation, we used proteomic and mass spectrometric methods to analyze the effect of mutations associated with hereditary human cataract formation on protein abundance in αA-R49C and αB-R120G knock-in mutant lenses. Compared with age-matched wild type lenses, 2-day-old αA-R49C heterozygous lenses demonstrated the following: increased crosslinking (15-fold) and degradation (2.6-fold) of αA-crystallin; increased association between αA-crystallin and filensin, actin, or creatine kinase B; increased acidification of ßB1-crystallin; increased levels of grifin; and an association between ßA3/A1-crystallin and αA-crystallin. Homozygous αA-R49C mutant lenses exhibited increased associations between αA-crystallin and ßB3-, ßA4-, ßA2-crystallins, and grifin, whereas levels of ßB1-crystallin, gelsolin, and calpain 3 decreased. The amount of degraded glutamate dehydrogenase, α-enolase, and cytochrome c increased more than 50-fold in homozygous αA-R49C mutant lenses. In αB-R120G mouse lenses, our analyses identified decreased abundance of phosphoglycerate mutase, several ß- and γ-crystallins, and degradation of αA- and αB-crystallin early in cataract development. Changes in the abundance of hemoglobin and histones with the loss of normal α-crystallin chaperone function suggest that these proteins also play important roles in the biochemical mechanisms of hereditary cataracts. Together, these studies offer a novel insight into the putative in vivo substrates of αA- and αB-crystallin.

Quantitative label-free proteomics for discovery of biomarkers in cerebrospinal fluid: assessment of technical and inter-individual variation.

BACKGROUND: Biomarkers are required for pre-symptomatic diagnosis, treatment, and monitoring of neurodegenerative diseases such as Alzheimer's disease. Cerebrospinal fluid (CSF) is a favored source because its proteome reflects the composition of the brain. Ideal biomarkers have low technical and inter-individual variability (subject variance) among control subjects to minimize overlaps between clinical groups. This study evaluates a process of multi-affinity fractionation (MAF) and quantitative label-free liquid chromatography tandem mass spectrometry (LC-MS/MS) for CSF biomarker discovery by (1) identifying reparable sources of technical variability, (2) assessing subject variance and residual technical variability for numerous CSF proteins, and (3) testing its ability to segregate samples on the basis of desired biomarker characteristics. METHODS/RESULTS: Fourteen aliquots of pooled CSF and two aliquots from six cognitively normal individuals were randomized, enriched for low-abundance proteins by MAF, digested endoproteolytically, randomized again, and analyzed by nano-LC-MS. Nano-LC-MS data were time and m/z aligned across samples for relative peptide quantification. Among 11,433 aligned charge groups, 1360 relatively abundant ones were annotated by MS2, yielding 823 unique peptides. Analyses, including Pearson correlations of annotated LC-MS ion chromatograms, performed for all pairwise sample comparisons, identified several sources of technical variability: i) incomplete MAF and keratins; ii) globally- or segmentally-decreased ion current in isolated LC-MS analyses; and iii) oxidized methionine-containing peptides. Exclusion of these sources yielded 609 peptides representing 81 proteins. Most of these proteins showed very low coefficients of variation (CV<5%) whether they were quantified from the mean of all or only the 2 most-abundant peptides. Unsupervised clustering, using only 24 proteins selected for high subject variance, yielded perfect segregation of pooled and individual samples. CONCLUSIONS: Quantitative label-free LC-MS/MS can measure scores of CSF proteins with low technical variability and can segregate samples according to desired criteria. Thus, this technique shows potential for biomarker discovery for neurological diseases.

Inhibition of lens photodamage by UV-absorbing contact lenses.

PURPOSE: To determine whether class 1 UV-blocking contact lenses protect against UVB radiation-induced damage in a human lens epithelial cell line (HLE B-3) and postmortem human lenses using a proteomics approach. METHODS: HLE B-3 cells were exposed to 6.4 mW/cm(2) UVB radiation at 302 nm for 2 minutes (768 mJ/cm(2)) with or without covering by senofilcon A class 1 UV-blocking contact lenses or lotrafilcon A non-UV-blocking (lotrafilcon A has some UV-blocking ability, albeit minimal) contact lenses. Control cells were not exposed to UVB radiation. Four hours after treatment, cells were analyzed by two-dimensional difference gel electrophoresis and tandem mass spectrometry, and changes in protein abundance were quantified. F-actin and microtubule cytoskeletons were examined by fluorescence staining. In addition, human donor lenses were exposed to UVB radiation at 302 nm for 4 minutes (1536 mJ/cm(2)). Cortical and epithelial cell proteins were scraped from lens surfaces and subjected to the same protein analyses. RESULTS: Senofilcon A lenses were beneficial for protecting HLE B-3 cells against UVB radiation-induced changes in caldesmon 1 isoform, lamin A/C transcript variant 1, DEAD (Asp-Glu-Ala-Asp) box polypeptide, ß-actin, glyceraldehyde 3-phosphate dehydrogenase (G3PDH), annexin A2, triose phosphate isomerase, and ubiquitin B precursor. These contact lenses also prevented actin and microtubule cytoskeleton changes typically induced by UVB radiation. Conversely, non-UV-blocking contact lenses were not protective. UVB-irradiated human lenses showed marked reductions in αA-crystallin, αB-crystallin, aldehyde dehydrogenase 1, ßS-crystallin, ßB2-crystallin, and G3PDH, and UV-absorbing contact lenses significantly prevented these alterations. CONCLUSIONS: Senofilcon A class 1 UV-blocking contact lenses largely prevented UVB-induced changes in protein abundance in lens epithelial cells and in human lenses.

Identification and validation of novel cerebrospinal fluid biomarkers for staging early Alzheimer's disease.

BACKGROUND: Ideally, disease modifying therapies for Alzheimer disease (AD) will be applied during the 'preclinical' stage (pathology present with cognition intact) before severe neuronal damage occurs, or upon recognizing very mild cognitive impairment. Developing and judiciously administering such therapies will require biomarker panels to identify early AD pathology, classify disease stage, monitor pathological progression, and predict cognitive decline. To discover such biomarkers, we measured AD-associated changes in the cerebrospinal fluid (CSF) proteome. METHODS AND FINDINGS: CSF samples from individuals with mild AD (Clinical Dementia Rating [CDR] 1) (n = 24) and cognitively normal controls (CDR 0) (n = 24) were subjected to two-dimensional difference-in-gel electrophoresis. Within 119 differentially-abundant gel features, mass spectrometry (LC-MS/MS) identified 47 proteins. For validation, eleven proteins were re-evaluated by enzyme-linked immunosorbent assays (ELISA). Six of these assays (NrCAM, YKL-40, chromogranin A, carnosinase I, transthyretin, cystatin C) distinguished CDR 1 and CDR 0 groups and were subsequently applied (with tau, p-tau181 and Aß42 ELISAs) to a larger independent cohort (n = 292) that included individuals with very mild dementia (CDR 0.5). Receiver-operating characteristic curve analyses using stepwise logistic regression yielded optimal biomarker combinations to distinguish CDR 0 from CDR>0 (tau, YKL-40, NrCAM) and CDR 1 from CDR<1 (tau, chromogranin A, carnosinase I) with areas under the curve of 0.90 (0.85-0.94 95% confidence interval [CI]) and 0.88 (0.81-0.94 CI), respectively. CONCLUSIONS: Four novel CSF biomarkers for AD (NrCAM, YKL-40, chromogranin A, carnosinase I) can improve the diagnostic accuracy of Aß42 and tau. Together, these six markers describe six clinicopathological stages from cognitive normalcy to mild dementia, including stages defined by increased risk of cognitive decline. Such a panel might improve clinical trial efficiency by guiding subject enrollment and monitoring disease progression. Further studies will be required to validate this panel and evaluate its potential for distinguishing AD from other dementing conditions.

Cross-species comparison of orthologous gene expression in human bladder cancer and carcinogen-induced rodent models.

Genes differentially expressed by tumor cells represent promising drug targets for anti-cancer therapy. Such candidate genes need to be validated in appropriate animal models. This study examined the suitability of rodent models of bladder cancer in B6D2F1 mice and Fischer-344 rats to model clinical bladder cancer specimens in humans. Using a global gene expression approach cross-species analysis showed that 13-34% of total genes in the genome were differentially expressed between tumor and normal tissues in each of five datasets from humans, rats, and mice. About 20% of these differentially expressed genes overlapped among species, corresponding to 2.6 to 4.8% of total genes in the genome. Several genes were consistently dysregulated in bladder tumors in both humans and rodents. Notably, CNN1, MYL9, PDLIM3, ITIH5, MYH11, PCP4 and FM05 were found to commonly down-regulated; while T0P2A, CCNB2, KIF20A and RRM2 were up-regulated. These genes are likely to have conserved functions contributing to bladder carcinogenesis. Gene set enrichment analysis detected a number of molecular pathways commonly activated in both humans and rodent bladder cancer. These pathways affect the cell cycle, HIF-1 and MYC expression, and regulation of apoptosis. We also compared expression changes at mRNA and protein levels in the rat model and identified several genes/proteins exhibiting concordant changes in bladder tumors, including ANXA1, ANXA2, CA2, KRT14, LDHA, LGALS4, SERPINA1, KRT18 and LDHB. In general, rodent models of bladder cancer represent the clinical disease to an extent that will allow successful mining of target genes and permit studies on the molecular mechanisms of bladder carcinogenesis.

YKL-40: a novel prognostic fluid biomarker for preclinical Alzheimer's disease.

BACKGROUND: Disease-modifying therapies for Alzheimer's disease (AD) would be most effective during the preclinical stage (pathology present, cognition intact) before significant neuronal loss occurs. Therefore, biomarkers that detect AD pathology in its early stages and predict dementia onset and progression will be invaluable for patient care and efficient clinical trial design. METHODS: AD-associated changes in cerebrospinal fluid (CSF) were measured using two-dimensional difference gel electrophoresis and liquid chromatography tandem mass spectrometry. Subsequently, CSF YKL-40 was measured by enzyme-linked immunosorbent assay in the discovery cohort (n = 47), validation cohort (n = 292) with paired plasma samples (n = 237), frontotemporal lobar degeneration (n=9) [corrected], and progressive supranuclear palsy (PSP; n = 6). Immunohistochemistry was performed to identify source(s) of YKL-40 in human AD brain. RESULTS: Discovery and validation cohorts, showed higher mean CSF YKL-40 in very mild and mild AD-type dementia (Clinical Dementia Rating [CDR] 0.5 and 1) versus control subjects (CDR 0) and PSP subjects. Importantly, CSF YKL-40/Aß42 ratio predicted risk of developing cognitive impairment (CDR 0 to CDR > 0 conversion), as well as the best CSF biomarkers identified to date, tau/Aß42 and p-tau 181/Aß42. Mean plasma YKL-40 was higher in CDR 0.5 and 1 versus CDR 0, and correlated with CSF levels. YKL-40 immunoreactivity labeled astrocytes near a subset of amyloid plaques, implicating YKL-40 in the neuroinflammatory response to Aß deposition. CONCLUSIONS: These data demonstrate that YKL-40, a putative indicator of neuroinflammation, is elevated in AD and, together with Aß42, has potential prognostic utility as a biomarker for preclinical AD.

Proteomic analysis of anoxia tolerance in the developing zebrafish embryo.

While some species and tissue types are injured by oxygen deprivation, anoxia tolerant organisms display a protective response that has not been fully elucidated and is well-suited to genomic and proteomic analysis. However, such methodologies have focused on transcriptional responses, prolonged anoxia, or have used cultured cells or isolated tissues. In this study of intact zebrafish embryos, a species capable of >24 h survival in anoxia, we have utilized 2D difference in gel electrophoresis to identify changes in the proteomic profile caused by near-lethal anoxic durations as well as acute anoxia (1 h), a timeframe relevant to ischemic events in human disease when response mechanisms are largely limited to post-transcriptional and post-translational processes. We observed a general stabilization of the proteome in anoxia. Proteins involved in oxidative phosphorylation, antioxidant defense, transcription, and translation changed over this time period. Among the largest proteomic alterations was that of muscle cofilin 2, implicating the regulation of the cytoskeleton and actin assembly in the adaptation to acute anoxia. These studies in an intact embryo highlight proteomic components of an adaptive response to anoxia in a model organism amenable to genetic analysis to permit further mechanistic insight into the phenomenon of anoxia tolerance.

Expression of a novel marker, Ubc9, in squamous cell carcinoma of the head and neck.

BACKGROUND: Ubiquitin-conjugating enzyme (Ubc9) is a novel enzyme involved in posttranslational modification of cellular proteins. The objective of this study was to determine the expression of Ubc9 in squamous cell carcinoma of the head and neck (SCCHN). METHODS: SCCHN specimens were stained with anti-Ubc9 antibodies, scored using a semiquantitative method, and statistically analyzed. RESULTS: Forty-six tumors were stained, 26 of which included adjacent mucosa. Ubc9 was significantly upregulated in the malignant and peritumoral tissues compared with mucosa from normal individuals. In peritumoral tissues, Ubc9 expression was detected in the basal and suprabasal epithelial layers. No Ubc9 was detected in epithelial cells in normal mucosa. These differences in Ubc9 expression were statistically significant (p < .0001). Tumor Ubc9 expression significantly correlated with clinical and pathologic stage. CONCLUSIONS: Ubc9 is significantly overexpressed in the primary SCCHN tumors and peritumoral mucosa compared with normal epithelial cells. These findings suggest that Ubc9 may play an important role in tumorigenesis and tumor progression of SCCHN.

Early prediction of response to chemoradiotherapy for head and neck cancer: reliability of restaging with combined positron emission tomography and computed tomography.

OBJECTIVE: To assess the role of combined positron emission tomography and computed tomography (PET-CT) in predicting early treatment response at the primary site and in the neck after chemoradiotherapy (CRT) for advanced squamous cell carcinoma of the head and neck (SCCHN). DESIGN: Retrospective analysis with a median follow-up of 24 months. SETTING: Academic, tertiary referral center. PATIENTS AND INTERVENTIONS: Thirty-one patients who were treated with concomitant intra-arterial CRT underwent PET-CT 6 to 8 weeks after the completion of treatment. Patients with findings on the physical examination, CT, or PET-CT indicative of persistent disease underwent appropriate surgical intervention for pathological assessment. Patients with a complete clinical response were observed with routine follow-up physical examination for disease recurrence. No evidence of disease at least 6 months after the completion of PET-CT was considered confirmation of complete clinical response. MAIN OUTCOME MEASURES: Presence or absence of residual or recurrent disease during the follow-up period was used to calculate the sensitivity, specificity, and positive and negative predictive values of PET-CT for the primary site and the neck. RESULTS: Assessment of tumor response at the primary site with PET-CT had a sensitivity, specificity, and positive and negative predictive values of 83%, 54%, 31%, and 92%, respectively. In patients with pretreatment N1 to N3 disease, the sensitivity, specificity, and positive and negative predictive values of posttreatment PET-CT were 75%, more than 94%, more than 75%, and 94%, respectively, and the specificity and negative predictive value for patients with pretreatment N0 disease in the neck were 92% and more than 92%, respectively. CONCLUSIONS: Negative PET-CT findings accurately determine early disease response at the primary site and in the neck. False-positive findings are common at the primary site. Patients with a negative PET-CT finding after the completion of intra-arterial CRT do not require surgical intervention.