Atypical Presentation of Gout: A Case Report.

Gout is the most common inflammatory arthritis which is caused by the buildup of uric acid crystals in the joints, that leads to severe pain, swelling, and stiffness. The condition typically affects the first metatarsophalangeal joint but it can impact other joints in the body. We present a case in which a 43-year-old male with a past medical history of obesity, hypertension, osteoarthritis, and gout presented with bilateral leg pain and the inability to walk for the last two years. Labs showed persistent leukocytosis, elevated ESR (erythrocyte sedimentation rate), normal uric acid levels, with physical exam findings of bilateral tender nodular leg lesions. Chest X-ray, head CT without contrast, left hip X-ray and ultrasound of left lower extremity were performed which were all negative. Biopsy of the tender skin nodules confirmed the diagnosis of tophaceous gout. Acute and prophylactic treatment of tophaceous gout resulted in resolved inflammation and leukocytosis without any complications.

HTT (huntingtin) and RAB7 co-migrate retrogradely on a signaling LAMP1-containing late endosome during axonal injury.

ABBREVIATIONS: Atg5: Autophagy-related 5; Atg8a: Autophagy-related 8a; AL: autolysosome; AP: autophagosome; BAF1: bafilomycin A1; BDNF: brain derived neurotrophic factor; BMP: bone morphogenetic protein; Cyt-c-p: Cytochrome c proximal; CQ: chloroquine; DCTN1: dynactin 1; Dhc: dynein heavy chain; EE: early endosome; DYNC1I1: dynein cytoplasmic 1 intermediate chain 1; HD: Huntington disease; HIP1/Hip1: huntingtin interacting protein 1; HTT/htt: huntingtin; iNeuron: iPSC-derived human neurons; IP: immunoprecipitation; Khc: kinesin heavy chain; KIF5C: kinesin family member 5C; LAMP1/Lamp1: lysosomal associated membrane protein 1; LE: late endosome; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAP3K12/DLK: mitogen-activated protein kinase kinase kinase 12; MAPK8/JNK/bsk: mitogen-activated protein kinase 8/basket; MAPK8IP3/JIP3: mitogen-activated protein kinase 8 interacting protein 3; NGF: nerve growth factor; NMJ: neuromuscular junction; NTRK1/TRKA: neurotrophic receptor tyrosine kinase 1; NRTK2/TRKB: neurotrophic receptor tyrosine kinase 2; nuf: nuclear fallout; PG: phagophore; PtdIns3P: phosphatidylinositol-3-phosphate; puc: puckered; ref(2)P: refractory to sigma P; Rilpl: Rab interacting lysosomal protein like; Rip11: Rab11 interacting protein; RTN1: reticulon 1; syd: sunday driver; SYP: synaptophysin; SYT1/Syt1: synaptotagmin 1; STX17/Syx17: syntaxin 17; tkv: thickveins; VF: vesicle fraction; wit: wishful thinking; wnd: wallenda.

Excess Rab4 rescues synaptic and behavioral dysfunction caused by defective HTT-Rab4 axonal transport in Huntington's disease.

Huntington's disease (HD) is characterized by protein inclusions and loss of striatal neurons which result from expanded CAG repeats in the poly-glutamine (polyQ) region of the huntingtin (HTT) gene. Both polyQ expansion and loss of HTT have been shown to cause axonal transport defects. While studies show that HTT is important for vesicular transport within axons, the cargo that HTT transports to/from synapses remain elusive. Here, we show that HTT is present with a class of Rab4-containing vesicles within axons in vivo. Reduction of HTT perturbs the bi-directional motility of Rab4, causing axonal and synaptic accumulations. In-vivo dual-color imaging reveal that HTT and Rab4 move together on a unique putative vesicle that may also contain synaptotagmin, synaptobrevin, and Rab11. The moving HTT-Rab4 vesicle uses kinesin-1 and dynein motors for its bi-directional movement within axons, as well as the accessory protein HIP1 (HTT-interacting protein 1). Pathogenic HTT disrupts the motility of HTT-Rab4 and results in larval locomotion defects, aberrant synaptic morphology, and decreased lifespan, which are rescued by excess Rab4. Consistent with these observations, Rab4 motility is perturbed in iNeurons derived from human Huntington's Disease (HD) patients, likely due to disrupted associations between the polyQ-HTT-Rab4 vesicle complex, accessory proteins, and molecular motors. Together, our observations suggest the existence of a putative moving HTT-Rab4 vesicle, and that the axonal motility of this vesicle is disrupted in HD causing synaptic and behavioral dysfunction. These data highlight Rab4 as a potential novel therapeutic target that could be explored for early intervention prior to neuronal loss and behavioral defects observed in HD.

In Vivo Visualization of Moving Synaptic Cargo Complexes within Drosophila Larval Segmental Axons.

Identifying moving synaptic vesicle complexes and isolating specific proteins present within such complexes in vivo is challenging. Here we detail a protocol that we have developed that is designed to simultaneously visualize the axonal transport of two fluorescently tagged synaptic vesicle proteins in living Drosophila larval segmental nerves in real time. Using a beam-splitter and split view software, larvae expressing GFP-tagged Synaptobrevin (Syb) and mRFP-tagged Rab4-GTPase or YFP-tagged Amyloid Precursor protein (APP) and mRFP-tagged Rab4-GTPase are imaged simultaneously using separate wavelengths. Merged kymographs from the two wavelengths are evaluated for colocalization analysis. Vesicle velocity analysis can also be done. Such analysis enables us to visualize the motility behaviors of two synaptic proteins present on a single vesicle complex and identify candidate proteins moving on synaptic vesicles in vivo, under physiological conditions.

Excess active P13K rescues huntingtin-mediated neuronal cell death but has no effect on axonal transport defects.

High levels of oxidative stress is detected in neurons affected by many neurodegenerative diseases, including huntington's disease. Many of these diseases also show neuronal cell death and axonal transport defects. While nuclear inclusions/accumulations likely cause cell death, we previously showed that cytoplasmic axonal accumulations can also contribute to neuronal death. However, the cellular mechanisms responsible for activating cell death is unclear. One possibility is that perturbations in normal axonal transport alter the function of the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT)-pathway, a signal transduction pathway that promotes survival/growth in response to extracellular signals. To test this proposal in vivo, we expressed active PI3K in the context of pathogenic huntingtin (HTT-138Q) in Drosophila larval nerves, which show axonal transport defects and neuronal cell death. We found that excess expression of active P13K significantly suppressed HTT-138Q-mediated neuronal cell death, but had no effect on HTT-138Q-mediated axonal transport defects. Expression of active PI3K also rescued Paraquat-mediated cell death. Further, increased levels of pSer9 (inactive) glycogen synthase kinase 3ß was seen in HTT-138Q-mediated larval brains, and in dynein loss of function mutants, indicating the modulation of the pro-survival pathway. Intriguingly, proteins in the PI3K/AKT-pathway showed functional interactions with motor proteins. Taken together our observations suggest that proper axonal transport is likely essential for the normal function of the pro-survival PI3K/AKT-signaling pathway and for neuronal survival in vivo. These results have important implications for targeting therapeutics to early insults during neurodegeneration and death.

Melanocortin 4 Receptor Pathway Dysfunction in Obesity: Patient Stratification Aimed at MC4R Agonist Treatment.

Context: The hypothalamic melanocortin 4 receptor (MC4R) pathway serves a critical role in regulating body weight. Loss of function (LoF) mutations in the MC4R pathway, including mutations in the pro-opiomelanocortin (POMC), prohormone convertase 1 (PCSK1), leptin receptor (LEPR), or MC4R genes, have been shown to cause early-onset severe obesity. Methods: Through a comprehensive epidemiological analysis of known and predicted LoF variants in the POMC, PCSK1, and LEPR genes, we sought to estimate the number of US individuals with biallelic MC4R pathway LoF variants. Results: We predict ~650 α-melanocyte-stimulating hormone (MSH)/POMC, 8500 PCSK1, and 3600 LEPR homozygous and compound heterozygous individuals in the United States, cumulatively enumerating >12,800 MC4R pathway-deficient obese patients. Few of these variants have been genetically diagnosed to date. These estimates increase when we include a small subset of less rare variants: ß-MSH/POMC,PCSK1 N221D, and a PCSK1 LoF variant (T640A). To further define the MC4R pathway and its potential impact on obesity, we tested associations between body mass index (BMI) and LoF mutation burden in the POMC, PCSK1, and LEPR genes in various populations. We show that the cumulative allele burden in individuals with two or more LoF alleles in one or more genes in the MC4R pathway are predisposed to a higher BMI than noncarriers or heterozygous LoF carriers with a defect in only one gene. Conclusions: Our analysis represents a genetically rationalized study of the hypothalamic MC4R pathway aimed at genetic patient stratification to determine which obese subpopulations should be studied to elucidate MC4R agonist (e.g., setmelanotide) treatment responsiveness.

Axonal Transport and Neurodegeneration: How Marine Drugs Can Be Used for the Development of Therapeutics.

Unlike virtually any other cells in the human body, neurons are tasked with the unique problem of transporting important factors from sites of synthesis at the cell bodies, across enormous distances, along narrow-caliber projections, to distally located nerve terminals in order to maintain cell viability. As a result, axonal transport is a highly regulated process whereby necessary cargoes of all types are packaged and shipped from one end of the neuron to the other. Interruptions in this finely tuned transport have been linked to many neurodegenerative disorders including Alzheimer's (AD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS) suggesting that this pathway is likely perturbed early in disease progression. Therefore, developing therapeutics targeted at modifying transport defects could potentially avert disease progression. In this review, we examine a variety of potential compounds identified from marine aquatic species that affect the axonal transport pathway. These compounds have been shown to function in microtubule (MT) assembly and maintenance, motor protein control, and in the regulation of protein degradation pathways, such as the autophagy-lysosome processes, which are defective in many degenerative diseases. Therefore, marine compounds have great potential in developing effective treatment strategies aimed at early defects which, over time, will restore transport and prevent cell death.

Amyloid precursor protein-mediated endocytic pathway disruption induces axonal dysfunction and neurodegeneration.

The endosome/lysosome pathway is disrupted early in the course of both Alzheimer's disease (AD) and Down syndrome (DS); however, it is not clear how dysfunction in this pathway influences the development of these diseases. Herein, we explored the cellular and molecular mechanisms by which endosomal dysfunction contributes to the pathogenesis of AD and DS. We determined that full-length amyloid precursor protein (APP) and its ß-C-terminal fragment (ß-CTF) act though increased activation of Rab5 to cause enlargement of early endosomes and to disrupt retrograde axonal trafficking of nerve growth factor (NGF) signals. The functional impacts of APP and its various products were investigated in PC12 cells, cultured rat basal forebrain cholinergic neurons (BFCNs), and BFCNs from a mouse model of DS. We found that the full-length wild-type APP (APPWT) and ß-CTF both induced endosomal enlargement and disrupted NGF signaling and axonal trafficking. ß-CTF alone induced atrophy of BFCNs that was rescued by the dominant-negative Rab5 mutant, Rab5S34N. Moreover, expression of a dominant-negative Rab5 construct markedly reduced APP-induced axonal blockage in Drosophila. Therefore, increased APP and/or ß-CTF impact the endocytic pathway to disrupt NGF trafficking and signaling, resulting in trophic deficits in BFCNs. Our data strongly support the emerging concept that dysregulation of Rab5 activity contributes importantly to early pathogenesis of AD and DS.

Huntingtin differentially regulates the axonal transport of a sub-set of Rab-containing vesicles in vivo.

Loss of huntingtin (HTT), the Huntington's disease (HD) protein, was previously shown to cause axonal transport defects. Within axons, HTT can associate with kinesin-1 and dynein motors either directly or via accessory proteins for bi-directional movement. However, the composition of the vesicle-motor complex that contains HTT during axonal transport is unknown. Here we analyze the in vivo movement of 16 Rab GTPases within Drosophila larval axons and show that HTT differentially influences the movement of a particular sub-set of these Rab-containing vesicles. While reduction of HTT perturbed the bi-directional motility of Rab3 and Rab19-containing vesicles, only the retrograde motility of Rab7-containing vesicles was disrupted with reduction of HTT. Interestingly, reduction of HTT stimulated the anterograde motility of Rab2-containing vesicles. Simultaneous dual-view imaging revealed that HTT and Rab2, 7 or 19 move together during axonal transport. Collectively, our findings indicate that HTT likely influences the motility of different Rab-containing vesicles and Rab-mediated functions. These findings have important implications for our understanding of the complex role HTT plays within neurons normally, which when disrupted may lead to neuronal death and disease.

Presenilin influences glycogen synthase kinase-3 ß (GSK-3ß) for kinesin-1 and dynein function during axonal transport.

Within axons, molecular motors transport essential components required for neuronal growth and viability. Although many levels of control and regulation must exist for proper anterograde and retrograde transport of vital proteins, little is known about these mechanisms. We previously showed that presenilin (PS), a gene involved in Alzheimer's disease (AD), influences kinesin-1 and dynein function in vivo. Here, we show that these PS-mediated effects on motor protein function are via a pathway that involves glycogen synthase kinase-3ß (GSK-3ß). PS genetically interacts with GSK-3ß in an activity-dependent manner. Excess of active GSK-3ß perturbed axonal transport by causing axonal blockages, which were enhanced by reduction of kinesin-1 or dynein. These GSK-3ß-mediated axonal defects do not appear to be caused by disruptions or alterations in microtubules (MTs). Excess of non-functional GSK-3ß did not affect axonal transport. Strikingly, GSK-3ß-activity-dependent axonal transport defects were enhanced by reduction of PS. Collectively, our findings suggest that PS and GSK-3ß are required for normal motor protein function. Our observations propose a model, in which PS likely plays a role in regulating GSK-3ß activity during transport. These findings have important implications for our understanding of the complex regulatory machinery that must exist in vivo and how this system is coordinated during the motility of vesicles within axons.

Axonal transport and neurodegenerative disease: vesicle-motor complex formation and their regulation.

The process of axonal transport serves to move components over very long distances on microtubule tracks in order to maintain neuronal viability. Molecular motors - kinesin and dynein - are essential for the movement of neuronal cargoes along these tracks; defects in this pathway have been implicated in the initiation or progression of some neurodegenerative diseases, suggesting that this process may be a key contributor in neuronal dysfunction. Recent work has led to the identification of some of the motor-cargo complexes, adaptor proteins, and their regulatory elements in the context of disease proteins. In this review, we focus on the assembly of the amyloid precursor protein, huntingtin, mitochondria, and the RNA-motor complexes and discuss how these may be regulated during long-distance transport in the context of neurodegenerative disease. As knowledge of these motor-cargo complexes and their involvement in axonal transport expands, insight into how defects in this pathway contribute to the development of neurodegenerative diseases becomes evident. Therefore, a better understanding of how this pathway normally functions has important implications for early diagnosis and treatment of diseases before the onset of disease pathology or behavior.

Transcriptome analyses of the human retina identify unprecedented transcript diversity and 3.5 Mb of novel transcribed sequence via significant alternative splicing and novel genes.

BACKGROUND: The retina is a complex tissue comprised of multiple cell types that is affected by a diverse set of diseases that are important causes of vision loss. Characterizing the transcripts, both annotated and novel, that are expressed in a given tissue has become vital for understanding the mechanisms underlying the pathology of disease. RESULTS: We sequenced RNA prepared from three normal human retinas and characterized the retinal transcriptome at an unprecedented level due to the increased depth of sampling provided by the RNA-seq approach. We used a non-redundant reference transcriptome from all of the empirically-determined human reference tracks to identify annotated and novel sequences expressed in the retina. We detected 79,915 novel alternative splicing events, including 29,887 novel exons, 21,757 3' and 5' alternate splice sites, and 28,271 exon skipping events. We also identified 116 potential novel genes. These data represent a significant addition to the annotated human transcriptome. For example, the novel exons detected increase the number of identified exons by 3%. Using a high-throughput RNA capture approach to validate 14,696 of these novel transcriptome features we found that 99% of the putative novel events can be reproducibly detected. Further, 15-36% of the novel splicing events maintain an open reading frame, suggesting they produce novel protein products. CONCLUSIONS: To our knowledge, this is the first application of RNA capture to perform large-scale validation of novel transcriptome features. In total, these analyses provide extensive detail about a previously uncharacterized level of transcript diversity in the human retina.

GCOD - GeneChip Oncology Database.

BACKGROUND: DNA microarrays have become a nearly ubiquitous tool for the study of human disease, and nowhere is this more true than in cancer. With hundreds of studies and thousands of expression profiles representing the majority of human cancers completed and in public databases, the challenge has been effectively accessing and using this wealth of data. DESCRIPTION: To address this issue we have collected published human cancer gene expression datasets generated on the Affymetrix GeneChip platform, and carefully annotated those studies with a focus on providing accurate sample annotation. To facilitate comparison between datasets, we implemented a consistent data normalization and transformation protocol and then applied stringent quality control procedures to flag low-quality assays. CONCLUSION: The resulting resource, the GeneChip Oncology Database, is available through a publicly accessible website that provides several query options and analytical tools through an intuitive interface.

The Functional Genomics Experiment model (FuGE): an extensible framework for standards in functional genomics.

The Functional Genomics Experiment data model (FuGE) has been developed to facilitate convergence of data standards for high-throughput, comprehensive analyses in biology. FuGE models the components of an experimental activity that are common across different technologies, including protocols, samples and data. FuGE provides a foundation for describing entire laboratory workflows and for the development of new data formats. The Microarray Gene Expression Data society and the Proteomics Standards Initiative have committed to using FuGE as the basis for defining their respective standards, and other standards groups, including the Metabolomics Standards Initiative, are evaluating FuGE in their development efforts. Adoption of FuGE by multiple standards bodies will enable uniform reporting of common parts of functional genomics workflows, simplify data-integration efforts and ease the burden on researchers seeking to fulfill multiple minimum reporting requirements. Such advances are important for transparent data management and mining in functional genomics and systems biology.

TM4 microarray software suite.

Powerful specialized software is essential for managing, quantifying, and ultimately deriving scientific insight from results of a microarray experiment. We have developed a suite of software applications, known as TM4, to support such gene expression studies. The suite consists of open-source tools for data management and reporting, image analysis, normalization and pipeline control, and data mining and visualization. An integrated MIAME-compliant MySQL database is included. This chapter describes each component of the suite and includes a sample analysis walk-through.

Comprehensive EST analysis of tomato and comparative genomics of fruit ripening.

A large tomato expressed sequence tag (EST) dataset (152 635 total) was analyzed to gain insights into differential gene expression among diverse plant tissues representing a range of developmental programs and biological responses. These ESTs were clustered and assembled to a total of 31 012 unique gene sequences. To better understand tomato gene expression at a plant system level and to identify differentially expressed and tissue-specific genes, we developed and implemented a digital expression analysis protocol. By clustering genes according to their relative abundance in the various EST libraries, expression patterns of genes across various tissues were generated and genes with similar patterns were grouped. In addition, tissues themselves were clustered for relatedness based on relative gene expression as a means of validating the integrity of the EST data as representative of relative gene expression. Arabidopsis and grape EST collections were also characterized to facilitate cross-species comparisons where possible. Tomato fruit digital expression data was specifically compared with publicly available grape EST data to gain insight into molecular manifestation of ripening processes across diverse taxa and resulted in identification of common transcription factors not previously associated with ripening.

Comparative analyses of potato expressed sequence tag libraries.

The cultivated potato (Solanum tuberosum) shares similar biology with other members of the Solanaceae, yet has features unique within the family, such as modified stems (stolons) that develop into edible tubers. To better understand potato biology, we have undertaken a survey of the potato transcriptome using expressed sequence tags (ESTs) from diverse tissues. A total of 61,940 ESTs were generated from aerial tissues, below-ground tissues, and tissues challenged with the late-blight pathogen (Phytophthora infestans). Clustering and assembly of these ESTs resulted in a total of 19,892 unique sequences with 8,741 tentative consensus sequences and 11,151 singleton ESTs. We were able to identify a putative function for 43.7% of these sequences. A number of sequences (48) were expressed throughout the libraries sampled, representing constitutively expressed sequences. Other sequences (13,068, 21%) were uniquely expressed and were detected only in a single library. Using hierarchal and k means clustering of the EST sequences, we were able to correlate changes in gene expression with major physiological events in potato biology. Using pair-wise comparisons of tuber-related tissues, we were able to associate genes with tuber initiation, dormancy, and sprouting. We also were able to identify a number of characterized as well as novel sequences that were unique to the incompatible interaction of late-blight pathogen, thereby providing a foundation for further understanding the mechanism of resistance.