Molecular abnormalities in autopsied brain tissue from the inferior horn of the lateral ventricles of nonagenarians and Alzheimer disease patients.

BACKGROUND: The ventricular system plays a vital role in blood-cerebrospinal fluid (CSF) exchange and interstitial fluid-CSF drainage pathways. CSF is formed in the specialized secretory tissue called the choroid plexus, which consists of epithelial cells, fenestrated capillaries and the highly vascularized stroma. Very little is currently known about the role played by the ventricles and the choroid plexus tissue in aging and Alzheimer's disease (AD). METHODS: In this study, we used our state-of-the-art proteomic platform, a liquid chromatography/mass spectrometry (LC-MS/MS) approach coupled with Tandem Mass Tag isobaric labeling to conduct a detailed unbiased proteomic analyses of autopsied tissue isolated from the walls of the inferior horn of the lateral ventricles in AD (77.2 ± 0.6 yrs), age-matched controls (77.0 ± 0.5 yrs), and nonagenarian cases (93.2 ± 1.1 yrs). RESULTS: Ingenuity pathway analyses identified phagosome maturation, impaired tight-junction signaling, and glucose/mannose metabolism as top significantly regulated pathways in controls vs nonagenarians. In matched-control vs AD cases we identified alterations in mitochondrial bioenergetics, oxidative stress, remodeling of epithelia adherens junction, macrophage recruitment and phagocytosis, and cytoskeletal dynamics. Nonagenarian vs AD cases demonstrated augmentation of oxidative stress, changes in gluconeogenesis-glycolysis pathways, and cellular effects of choroidal smooth muscle cell vasodilation. Amyloid plaque score uniquely correlated with remodeling of epithelial adherens junctions, Fc γ-receptor mediated phagocytosis, and alterations in RhoA signaling. Braak staging was uniquely correlated with altered iron homeostasis, superoxide radical degradation and phagosome maturation. CONCLUSIONS: These changes provide novel insights to explain the compromise to the physiological properties and function of the ventricles/choroid plexus system in nonagenarian aging and AD pathogenesis. The pathways identified could provide new targets for therapeutic strategies to mitigate the divergent path towards AD.

An independent validation of a gene expression signature to differentiate malignant melanoma from benign melanocytic nevi.

BACKGROUND: Recently, a 23-gene signature was developed to produce a melanoma diagnostic score capable of differentiating malignant and benign melanocytic lesions. The primary objective of this study was to independently assess the ability of the gene signature to differentiate melanoma from benign nevi in clinically relevant lesions. METHODS: A set of 1400 melanocytic lesions was selected from samples prospectively submitted for gene expression testing at a clinical laboratory. Each sample was tested and subjected to an independent histopathologic evaluation by 3 experienced dermatopathologists. A primary diagnosis (benign or malignant) was assigned to each sample, and diagnostic concordance among the 3 dermatopathologists was required for inclusion in analyses. The sensitivity and specificity of the score in differentiating benign and malignant melanocytic lesions were calculated to assess the association between the score and the pathologic diagnosis. RESULTS: The gene expression signature differentiated benign nevi from malignant melanoma with a sensitivity of 91.5% and a specificity of 92.5%. CONCLUSIONS: These results reflect the performance of the gene signature in a diverse array of samples encountered in routine clinical practice. Cancer 2017;123:617-628. © 2016 American Cancer Society.

Lipidomic analyses identify injury-specific phospholipid changes 3 mo after traumatic brain injury.

Phospholipid (PL) abnormalities are observed in the cerebrospinal fluid of patients with traumatic brain injury (TBI), suggesting their role in TBI pathology. Therefore, PL levels were examined in a TBI mouse model that received 1.8 mm deep controlled cortical impact injury or craniectomy only (control). The rotarod and Barnes maze acquisition and probe tests were performed within 2 wk after injury, with another probe test performed 3 mo postinjury. Liquid chromatography/mass spectrometry analyses were performed on lipid extracts from several brain regions and plasma from injured and control mice collected at 3 mo postinjury. Compared to controls, injured mice with sensorimotor and learning deficits had decreased levels of cortical and cerebellar phosphatidylcholine (PC) and phosphatidylethanolamine (PE) levels, while hippocampal PC, sphingomyelin and PE levels were elevated. Ether PE levels were lower in the cortices and plasma of injured animals. Polyunsaturated fatty acid-containing PC and PE species, particularly ratios of docosahexaenoic acid (DHA) to arachidonic acid, were lower in the hippocampi and cortices and plasma of injured mice. Given the importance of DHA in maintaining neuronal function and resolving inflammation and of peroxisomes in synthesis of ether PLs, normalizing these PLs may be a useful strategy for treating the chronic pathology of TBI.

Exposure to an organophosphate pesticide, individually or in combination with other Gulf War agents, impairs synaptic integrity and neuronal differentiation, and is accompanied by subtle microvascular injury in a mouse model of Gulf War agent exposure.

Gulf War illness (GWI) is a currently untreatable multi-symptom disorder experienced by 1990-1991 Persian Gulf War (GW) veterans. The characteristic hallmarks of GWI include cognitive dysfunction, tremors, migraine, and psychological disturbances such as depression and anxiety. Meta-analyses of epidemiological studies have consistently linked these symptomatic profiles to the combined exposure of GW agents such as organophosphate-based and pyrethroid-based pesticides (e.g. chlorpyrifos (CPF) and permethrin (PER) respectively) and the prophylactic use of pyridostigmine bromide (PB) as a treatment against neurotoxins. Due to the multi-symptomatic presentation of this illness and the lack of available autopsy tissue from GWI patients, very little is currently known about the distinct early pathological profile implicated in GWI (including its influence on synaptic function and aspects of neurogenesis). In this study, we used preclinical models of GW agent exposure to investigate whether 6-month-old mice exposed to CPF alone, or a combined dose of CPF, PB and PER daily for 10 days, demonstrate any notable pathological changes in hippocampal, cortical (motor, piriform) or amygdalar morphometry. We report that at an acute post-exposure time point (after 3 days), both exposures resulted in the impairment of synaptic integrity (reducing synaptophysin levels) in the CA3 hippocampal region and altered neuronal differentiation in the dentate gyrus (DG), demonstrated by a significant reduction in doublecortin positive cells. Both exposures also significantly increased astrocytic GFAP immunoreactivity in the piriform cortex, motor cortex and the basolateral amygdala and this was accompanied by an increase in (basal) brain acetylcholine (ACh) levels. There was no evidence of microglial activation or structural deterioration of principal neurons in these regions following exposure to CPF alone or in combination with PB and PER. Evidence of subtle microvascular injury was demonstrated by the reduction of platelet endothelial cell adhesion molecule (PECAM)-1 levels in CPF+PB+PER exposed group compared to control. These data support early (subtle) neurotoxic effects on the brain following exposure to GW agents.

Depletion of CXCR2 inhibits γ-secretase activity and amyloid-ß production in a murine model of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder that leads to progressive cognitive decline. Recent studies from our group and others have suggested that certain G-protein coupled receptors (GPCRs) can influence the processing of the amyloid precursor protein (APP). Earlier, we demonstrated that stimulation of a chemokine receptor, CXCR2, results in enhanced γ-secretase activity and in increased amyloid-beta (Aß) production. Taken together, results obtained from in vitro studies indicate that therapeutic targeting of CXCR2 might aid in lowering Aß levels in the AD brain. To better understand the precise function and to predict the consequences of CXCR2 depletion in the AD brain, we have crossed CXCR2 knockout mice with mice expressing presenilin (PS1 M146L) and APPsw mutations (PSAPP). Our present study confirms that CXCR2 depletion results in reduction of Aß with concurrent increases of γ-secretase substrates. At the mechanistic level, the effect of CXCR2 on γ-secretase was not found to occur via their direct interaction. Furthermore, we provide evidence that Aß promotes endocytosis of CXCR2 via increasing levels of CXCR2 ligands. In conclusion, our current study confirms the regulatory role of CXCR2 in APP processing, and poses it as a potential target for developing novel therapeutics for intervention in AD.

APOE genotype dependent molecular abnormalities in the cerebrovasculature of Alzheimer's disease and age-matched non-demented brains.

Cerebrovascular dysfunction is a hallmark feature of Alzheimer's disease (AD). One of the greatest risk factors for AD is the apolipoprotein E4 (E4) allele. The APOE4 genotype has been shown to negatively impact vascular amyloid clearance, however, its direct influence on the molecular integrity of the cerebrovasculature compared to other APOE variants (APOE2 and APOE3) has been largely unexplored. To address this, we employed a 10-plex tandem isobaric mass tag approach in combination with an ultra-high pressure liquid chromatography MS/MS (Q-Exactive) method, to interrogate unbiased proteomic changes in cerebrovessels from AD and healthy control brains with different APOE genotypes. We first interrogated changes between healthy control cases to identify underlying genotype specific effects in cerebrovessels. EIF2 signaling, regulation of eIF4 and 70S6K signaling and mTOR signaling were the top significantly altered pathways in E4/E4 compared to E3/E3 cases. Oxidative phosphorylation, EIF2 signaling and mitochondrial dysfunction were the top significant pathways in E2E2 vs E3/E3cases. We also identified AD-dependent changes and their interactions with APOE genotype and found the highest number of significant proteins from this interaction was observed in the E3/E4 (192) and E4/E4 (189) cases. As above, EIF2, mTOR signaling and eIF4 and 70S6K signaling were the top three significantly altered pathways in E4 allele carriers (i.e. E3/E4 and E4/E4 genotypes). Of all the cerebrovascular cell-type specific markers identified in our proteomic analyses, endothelial cell, astrocyte, and smooth muscle cell specific protein markers were significantly altered in E3/E4 cases, while endothelial cells and astrocyte specific protein markers were altered in E4/E4 cases. These proteomic changes provide novel insights into the longstanding link between APOE4 and cerebrovascular dysfunction, implicating a role for impaired autophagy, ER stress, and mitochondrial bioenergetics. These APOE4 dependent changes we identified could provide novel cerebrovascular targets for developing disease modifying strategies to mitigate the effects of APOE4 genotype on AD pathogenesis.

Molecular Pathobiology of the Cerebrovasculature in Aging and in Alzheimers Disease Cases With Cerebral Amyloid Angiopathy.

Cerebrovascular dysfunction and cerebral amyloid angiopathy (CAA) are hallmark features of Alzheimer's disease (AD). Molecular damage to cerebrovessels in AD may result in alterations in vascular clearance mechanisms leading to amyloid deposition around blood vessels and diminished neurovascular-coupling. The sequelae of molecular events leading to these early pathogenic changes remains elusive. To address this, we conducted a comprehensive in-depth molecular characterization of the proteomic changes in enriched cerebrovessel fractions isolated from the inferior frontal gyrus of autopsy AD cases with low (85.5 ± 2.9 yrs) vs. high (81 ± 4.4 yrs) CAA score, aged-matched control (87.4 ± 1.5 yrs) and young healthy control (47 ± 3.3 yrs) cases. We employed a 10-plex tandem isobaric mass tag approach in combination with our ultra-high pressure liquid chromatography MS/MS (Q-Exactive) method. Enriched cerebrovascular fractions showed very high expression levels of proteins specific to endothelial cells, mural cells (pericytes and smooth muscle cells), and astrocytes. We observed 150 significantly regulated proteins in young vs. aged control cerebrovessels. The top pathways significantly modulated with aging included chemokine, reelin, HIF1α and synaptogenesis signaling pathways. There were 213 proteins significantly regulated in aged-matched control vs. high CAA cerebrovessels. The top three pathways significantly altered from this comparison were oxidative phosphorylation, Sirtuin signaling pathway and TCA cycle II. Comparison between low vs. high CAA cerebrovessels identified 84 significantly regulated proteins. Top three pathways significantly altered between low vs. high CAA cerebrovessels included TCA Cycle II, Oxidative phosphorylation and mitochondrial dysfunction. Notably, high CAA cases included more advanced AD pathology thus cerebrovascular effects may be driven by the severity of amyloid and Tangle pathology. These descriptive proteomic changes provide novel insights to explain the age-related and AD-related cerebrovascular changes contributing to AD pathogenesis. Particularly, disturbances in energy bioenergetics and mitochondrial biology rank among the top AD pathways altered in cerebrovessels. Targeting these failed mechanisms in endothelia and mural cells may provide novel disease modifying targets for developing therapeutic strategies against cerebrovascular deterioration and promoting cerebral perfusion in AD. Our future work will focus on interrogating and validating these novel targets and pathways and their functional significance.

Optimization of a transient antibody expression platform towards high titer and efficiency.

Transient gene expression (TGE) using mammalian cells is an extensively used technology for the production of antibodies and recombinant proteins and has been widely adopted by both academic and industrial labs. Chinese Hamster Ovary (CHO) cells have become one of the major workhorses for TGE of recombinant antibodies due to their attractive features: post-translational modifications, adaptation to high cell densities, and use of serum-free media. In this study, we describe the optimization of parameters for TGE for antibodies from CHO cells. Through a matrix evaluation of multiple factors including inoculum, transfection conditions, amount and type of DNA used, and post-transfection culture conditions, we arrived at an uniquely optimized process with higher titer and reduced costs and time, thus increasing the overall efficiency of early antibody material supply. We further investigated the amount of coding DNA used in TGE and the influence of kinetics and size of the transfection complex on the in vitro efficiency of the transfection. We present here the first report of an optimized TGE platform using Filler DNA in an early drug discovery setting for the screening and production of therapeutic mAbs.

Lymphotropism of Merkel cell polyomavirus infection, Nova Scotia, Canada.

To test the hypothesis that Merkel cell polyomavirus (MCPyV) can infect cells of the lymphoid system, we analyzed 353 specimens, including 152 non-Hodgkin lymphomas, 44 Hodgkin lymphomas, 110 benign lymph nodes, 27 lymph nodes with metastasis, and 20 extranodal tissue samples. MCPyV DNA was detected by quantitative PCR in 13 (6.6%) of 196 lymphomas, including 5 (20.8%) of 24 chronic lymphocytic leukemia specimens, and in 11 (10%) of 110 benign lymph nodes, including 8 (13.1%) of 61 samples of reactive hyperplasia and 3 (10.3%) of 29 normal lymph nodes. Other samples were MCPyV negative. Sequence analysis of 9 virus-positive samples confirmed the identity of MCPyV; 3 viral strains were represented. Immunohistochemical testing showed that 1 T-cell lymphoma expressed MCPyV T-antigen. These findings suggest that the lymphoid system plays a role in MCPyV infection and may be a site for MCPyV persistence.

The SUMO conjugation pathway in Populus: genomic analysis, tissue-specific and inducible SUMOylation and in vitro de-SUMOylation.

Covalent attachment of the small ubiquitin-like modifier (SUMO) to proteins in eukaryotic cells can regulate an assortment of cellular processes including transcription, and DNA-protein and protein-protein interactions. We identified gene models and found evidence for expression of genes involved in SUMOylation and SUMO deconjugation in Populus. We detected SUMOylated proteins in diverse organ and tissue types. SUMOylation was altered during responses to heat shock, desiccation, peroxide and irrigation of roots with high salt solution. SUMO deconjugation from substrates was sensitive to cysteine protease inhibitors. Product sizes and sensitivity to inhibitors are consistent with poly-SUMO chain formation as an intermediate step in SUMO redistribution to substrates in plant cells responding to treatments. The SUMOylation pathway is active in Populus and substrate conjugation to SUMO is a rapid response to multiple inducers.

Adipophilin expression in sebaceous tumors and other cutaneous lesions with clear cell histology: an immunohistochemical study of 117 cases.

Adipophilin is a monoclonal antibody against a protein on the surface of intracellular lipid droplets, and it was recently shown to be expressed in sebocytes and sebaceous lesions. This study examines adipophilin expression in various sebaceous lesions and other cutaneous tumors with a clear cell histology that may mimic sebaceous differentiation. A total of 117 cutaneous clear cell lesions including 16 sebaceous adenomas, 25 sebaceous carcinomas, 8 basal cell carcinomas, 12 squamous cell carcinomas, 6 xanthomas, 10 xanthelasmas, 10 xanthogranulomas, 4 balloon cell nevi, 5 trichilemmomas, 8 clear cell hidradenomas, and 13 metastatic renal cell carcinomas were examined using immunohistochemistry for the expression of adipophilin. Of these 117 lesions, 42 (36%) were from the periocular region. Adipophilin was expressed in 16 of 16 (100%) sebaceous adenomas, 23 of 25 (92%) sebaceous carcinomas, 10 of 10 (100%) xanthelasmas, 9 of 10 (90%) xanthogranulomas, 6 of 6 (100%) xanthomas, and 9 of 13 (62.5%) metastatic renal cell carcinomas. The characteristic staining pattern differed between sebaceous and non-sebaceous tumors with the former showing a membranous vesicular pattern and the latter being more granular. Adipophilin expression was not seen in any of the other lesions with clear cell histology, basal cell carcinomas, or squamous cell carcinomas, including cases that had focal clear cell differentiation. Adipophilin can be valuable in an immunohistochemical panel when evaluating cutaneous lesions with clear cell histology as it identifies intracytoplasmic lipid vesicles in sebaceous and xanthomatous lesions. In periocular lesions, it is effective in helping to exclude basal cell carcinoma and squamous cell carcinoma when sebaceous carcinoma is under consideration. Adipophilin expression is not as useful for the differential diagnosis that includes metastatic renal cell carcinoma, a rare but important, diagnostic differential. The pattern of adipophilin reactivity is important to observe as membranous vesicular staining is suggestive of intracellular lipids whereas granular cytoplasmic reactivity is not.

COL1A1:PDGFB chimeric transcripts are not present in indeterminate fibrohistiocytic lesions of the skin.

Indeterminate fibrohistiocytic lesions of the skin share histological and immunohistochemical features of both benign fibrous histiocytoma/dermatofibroma and dermatofibrosarcoma protuberans (DFSP). Unlike dermatofibroma, DFSP harbors recurrent genetic aberrations resulting in the fusion of COL1A1 on chromosome 17 and PDGFB on chromosome 22. Because indeterminate fibrohistiocytic lesions share some features with DFSP, they were evaluated for the possible presence of COL1A1-PDGFB chimeric transcripts. Twelve formalin-fixed paraffin-embedded cases were examined for COL1A1-PDGFB chimeric transcripts using a previously validated sensitive multiplex reverse transcriptase-polymerase chain reaction assay. The median patient age was 52.5 years (33-70 years) with 9 females and 3 males. The most common site was the extremities (n = 8) followed by the trunk (n = 2) and the head and neck region (n = 2). All demonstrated the expected reactivity for both CD34 and factor XIIIa, and the majority focally infiltrated into subcutaneous fat. Of the 6 patients with follow-up, 2 had residual tumor excised, but no patient developed a recurrence. None of the tumors harbored COL1A1-PDGFB fusion transcripts identified by reverse transcriptase-polymerase chain reaction. Although indeterminate fibrohistiocytic lesions share some features with DFSP, the lack of COL1A1-PDGFB chimeric transcripts suggests that they are distinct entities.

Correction to: Mild TBI Results in a Long-Term Decrease in Circulating Phospholipids in a Mouse Model of Injury.

The original version of this article unfortunately contained a mistake. Gary S. Laco should not be listed as an author in the author group.

Structural optimization of a CXCR2-directed antagonist that indirectly inhibits gamma-secretase and reduces Abeta.

Amyloid beta (Abeta), a key molecule in the pathogenesis of Alzheimer's disease (AD), is derived from the amyloid precursor protein (APP) by sequential proteolysis via beta- and gamma-secretases. Because of their role in generation of Abeta, these enzymes have emerged as important therapeutic targets for AD. In the case of gamma-secretase, progress has been made towards designing potent inhibitors with suitable pharmacological profiles. Direct gamma-secretase inhibitors are being evaluated in clinical trials and new strategies are being explored to block gamma-secretase activity indirectly as well. In this regard, we have previously reported an indirect regulation of gamma-secretase through antagonism of CXCR2, a G-protein coupled receptor (GPCR). We demonstrated that N-(2-hydroxy-4-nitrophenyl)-N'-(2-bromophenyl)urea (SB225002), a selective inhibitor of CXCR2 also plays a role in an indirect inhibition of gamma-secretase. Furthermore, we reported a approximately 5-fold difference in the selective inhibition of APP versus Notch processing via gamma-secretase following treatment with SB225002. Herein we describe the synthesis and optimization of SB225002. By determination of the structure-activity relationship (SAR), we derived small molecules that inhibit Abeta40 production with IC(50) values in the sub-micromolar range in a cell-based assay and also validated the potential of CXCR2 as a new target for therapeutic intervention in AD.

Different expression patterns of p27 and p57 proteins in benign and malignant melanocytic neoplasms and in cultured human melanocytes.

BACKGROUND: The p27(KIP1) and p57(KIP2) proteins belong to the CIP/KIP family of cyclin-dependent kinase inhibitors involved in the growth arrest and cellular senescence. High levels of p27(KIP1) unexpectedly have been detected in invasive malignant melanomas (MM), whereas the role of p57(KIP2) in melanocytic lesions is unknown. We therefore chose to study the expression of p27(KIP1) and p57(KIP2) in melanocytic neoplasms. DESIGN: The expression of p27(KIP1) and p57(KIP2) were examined by immunohistochemistry in 40 melanocytic neoplasms and by Western blot analysis in cultured human melanocytes. RESULTS: Expression of both nuclear p27(KIP1) and p57(KIP2) (> 10% of cells with nuclear labeling) was observed in most cases with non-proliferating melanocytes (8/10, benign nevi and 9/10 DN, dysplastic nevi), but in only a few cases containing proliferating melanocytes (3/11 RN, recurrent nevi and 2/9 MM, melanoma) (p < 0.002). In proliferating melanocytes, there was an inverse correlation of nuclear expression of p27(KIP1) and p57(KIP2) in both RN (p27(KIP1) = 3/11 RN and p57(KIP2) = 8/11 RN) and MM (p27(KIP1) = 7/9 MM and p57(KIP2) = 2/9 MM) (p < 0.05). Western blot analysis detected p57(KIP2) only in proliferating melanocytes. p27(KIP1) was detected in both proliferating and senescent melanocytes. CONCLUSION: The difference in expression patterns of p27(KIP1) and p57(KIP2) in proliferating and senescent melanocytes suggests the interplay between these proteins may play a functional role in melanocytic tumorigenesis.

Proteomic Identification of Pathways Responsible for the Estradiol Therapeutic Window in AD Animal Models.

Benefits and risks were reported for hormone therapy (HT) to prevent chronic disease, including Alzheimer's disease (AD). While the Women's Health Initiative (WHI) found no protective effect of HT on the cognitive function of women whose treatment was initiated far past the onset of menopause, other studies showed reduced risk of AD with midlife treatment, versus increased risk of AD with late treatment. These suggest a critical window during which estradiol must be administered to prevent cognitive decline and AD in women. Our published work supports this, by demonstrating that early and long-term estradiol treatment improves cognitive function and reduce Aß accumulation in AD mouse models with estradiol deficiency, while there is no effect of late and short-term estradiol treatment on AD neuropathogenesis. However, little is known about the molecular mechanisms underlying the critical window and whether different protein networks are responsible for the brain estradiol deficiency-associated risk of AD in females. In this study, we used proteomics to identify target protein pathways that are activated during the estradiol therapeutic window in AD mouse model. Our results showed that different signaling pathways were involved in the regulatory effects of estradiol on MAP1A and hemoglobin α. Estradiol treatment increased the level of MAP1A through the phosphorylation of ERK1/2 and increased the level of hemoglobin α through the phosphorylation of AKT. This study has provided molecular insights into the "critical window" theory and identifies specific target proteins of therapeutic responsiveness that may lead to improved treatment strategies and optimal estradiol therapy.

Deciphering the melanoma interactome.

Late-stage malignant melanoma continues to pose a significant therapeutic challenge, despite numerous recent advances in our understanding of the molecular and genetic pathways leading to tumor development and progression. Dr Scott McNutt was among the first researchers to employ the cutting edge technology, electron microscopy, to the study of cutaneous neoplasms. This work provided the foundation for more recent studies using molecular pathology to examine disease in the context of aberrant interactions between cellular signaling pathways in the so-called 'interactome'. Understanding the functional interrelationships of aberrant signaling networks in melanoma may lead to the development of novel therapies for advanced disease. This mini review will focus on few of the proteins that likely significantly contribute to the melanoma disease interactome.

Clear cell sarcoma of soft tissue: diagnostic utility of fluorescence in situ hybridization and reverse transcriptase polymerase chain reaction.

A 7-year-old girl presented with pain and progressive swelling on the left plantar surface. Biopsy of a 2.5 cm mass showed nests of large round to oval neoplastic cells with abundant amphophilic to clear cytoplasm, prominent nucleoli and high mitotic activity. Occasional cells showed spindled morphology. Infrequent melanin pigment was present. Melanocytic markers (HMB45, S-100) were diffusely positive. A diagnosis of clear cell sarcoma of soft tissue (CCSS) was made, and the mass was re-excised with negative margins. 28 months later, a 1.0 cm pulmonary nodule was identified and wedge excision showed metastatic CCSS. Cytogenetics showed a complex karyotype (unbalanced translocation der(12;14)(q10;q10), additional chromosome 22 material of unknown origin). Although the CCSS translocation t(12;22)(q13;q12) was not identified, EWSR1 gene rearrangement was detected by fluorescence in situ hybridization (FISH). Reverse transcription polymerase chain reaction (RT-PCR) showed an EWS-ATF1 fusion transcript, confirmed by direct sequencing. CCSS requires differentiation from malignant melanoma, because of overlapping clinical presentations, sites of involvement, histomorphology, immunocytochemical profiles and ultrastructure. In many circumstances, definitive diagnosis is only possible with confirmation of the CCSS-defining translocation.

An unusual presentation of atypical fibroxanthoma.

Atypical fibroxanthoma (AFX) is a rare cutaneous spindle-cell neoplasm. The tumor occurs most commonly in sun-damaged skin of the head and neck in elderly patients. A small subset of patients (approximately one in five cases) is middle-aged and presents with AFX of the trunk or extremities in areas without evidence of actinic damage. We report an unusual case of AFX of the lower leg in an 81-year-old woman with extensive actinic damage.

Hippocampal Proteomic Analysis Reveals Distinct Pathway Deregulation Profiles at Early and Late Stages in a Rat Model of Alzheimer's-Like Amyloid Pathology.

The cerebral accumulation and cytotoxicity of amyloid beta (Aß) is central to Alzheimer's pathogenesis. However, little is known about how the amyloid pathology affects the global expression of brain proteins at different disease stages. In order to identify genotype and time-dependent significant changes in protein expression, we employed quantitative proteomics analysis of hippocampal tissue from the McGill-R-Thy1-APP rat model of Alzheimer-like amyloid pathology. McGill transgenic rats were compared to wild-type rats at early and late pathology stages, i.e., when intraneuronal Aß amyloid burden is conspicuous and when extracellular amyloid plaques are abundant with more pronounced cognitive deficits. After correction for multiple testing, the expression levels of 64 proteins were found to be considerably different in transgenic versus wild-type rats at the pre-plaque stage (3 months), and 86 proteins in the post-plaque group (12 months), with only 9 differentially regulated proteins common to the 2 time-points. This minimal overlap supports the hypothesis that different molecular pathways are affected in the hippocampus at early and late stages of the amyloid pathology throughout its continuum. At early stages, disturbances in pathways related to cellular responses to stress, protein homeostasis, and neuronal structure are predominant, while disturbances in metabolic energy generation dominate at later stages. These results shed new light on the molecular pathways affected by the early accumulation of Aß and how the evolving amyloid pathology impacts other complex metabolic pathways.