The construction of nanostructures from biodegradable precursors and shell/core crosslinking have been pursued as strategies to solve the problems of toxicity and limited stability, respectively. Polyphosphoester (PPE)-based micelles and crosslinked nanoparticles with non-ionic, anionic, cationic, and zwitterionic surface characteristics for potential packaging and delivery of therapeutic and diagnostic agents, were constructed using a quick and efficient synthetic strategy, and importantly, demonstrated remarkable differences in terms of cytotoxicity, immunotoxicity, and biofouling properties, as a function of their surface characteristics and also with dependence on crosslinking throughout the shell layers. For instance, crosslinking of zwitterionic micelles significantly reduced the immunotoxicity, as evidenced from the absence of secretions of any of the 23 measured cytokines from RAW 264.7 mouse macrophages treated with the nanoparticles. The micelles and their crosslinked analogs demonstrated lower cytotoxicity than several commercially-available vehicles, and their degradation products were not cytotoxic to cells at the range of the tested concentrations. PPE-nanoparticles are expected to have broad implications in clinical nanomedicine as alternative vehicles to those involved in several of the currently available medications.
The formation of covalent protein adducts by lipid electrophiles contributes to diseases and toxicities linked to oxidative stress, but analysis of the adducts presents a challenging analytical problem. We describe selective adduct capture using biotin affinity probes to enrich protein and peptide adducts for analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). One approach employs biotinamidohexanoic acid hydrazide to covalently label residual carbonyl groups on adducts. The other employs alkynyl analogs of lipid electrophiles, which form adducts that can be post labeled with azidobiotin tags by Cu+-catalyzed cycloaddition (Click chemistry). To enhance the selectivity of adduct capture we use an azidobiotin reagent with a photocleavable linker, which allows recovery of adducted proteins and peptides under mild conditions. This approach allows both the identification of protein targets of lipid electrophiles and sequence mapping of the adducts.
electrophile; Click chemistry; protein adducts; lipid oxidation; photocleavable biotin; shotgun proteomics
Glaucoma is a common cause of visual disability and affects ∼1.6% of individuals over 40 years of age (
1). Non-synonymous coding sequence variations in the ankyrin repeat and SOCS box containing gene 10 (ASB10) were recently associated with 6.0% of cases of primary open angle glaucoma (POAG) in patients from Oregon and Germany. We tested a cohort of POAG patients (n= 158) and normal control subjects (n= 82), both from Iowa, for ASB10 mutations. Our study had 80% power to detect a 4.9% mutation frequency in POAG patients. A total of 11 non-synonymous coding sequence mutations were detected in the cohort, but no association with POAG was detected when analyzed individually or as a group (P > 0.05). Furthermore, a survey of the National Heart, Lung, and Blood Institute's (NHLBI's) Exome Sequencing Project revealed that non-synonymous ASB10 mutations are present in the general population at a far higher frequency than the prevalence of POAG. These data suggest that non-synonymous mutations in ASB10 do not cause Mendelian forms of POAG.
4-1BB (CD137), an inducible costimulatory molecule, strongly enhances the proliferation and effector function of CD8+ T cells. Since the serine/threonine kinase, glycogen synthase kinase-3 (GSK-3), is involved in a variety of signaling pathways of cellular proliferation, migration, immune responses, and apoptosis, we examined whether 4-1BB signaling activates GSK-3/β-catenin signaling and downstream transcription factors to enhance the proliferation of CD8+ T cells. 4-1BB signaling induces rapid activation of ERK and IκB degradation, and shows delayed activation of AKT at 24 h post 4-1BB stimulation on anti-CD3 activated T cells. ERK and AKT signals were required for sustained β-catenin levels by inactivating GSK-3, which was also observed with delayed kinetics after 4-1BB stimulation. As a transcriptional partner of β-catenin, 4-1BB signaling decreased levels of FOXO1 and increased levels of stimulatory TCF1 in CD8+ T cells at 2–3 days but not at early time points after 4-1BB engagement. The enhanced proliferation of CD8+ T cells due to 4-1BB signaling was completely abolished by treatment with the TCF1/β-catenin inhibitor quercetin. These results show that 4-1BB signaling enhances the proliferation of activated CD8+ T cells by activating the TCF1/β-catenin axis via the PI3K/AKT/ERK pathway. As effects of 4-1BB on AKT, FOXO1, β-catenin and GSK-3β showed delayed kinetics it is likely that an intervening molecule induced by 4-1BB and ERK signaling in activated T cells is responsible for these effects. These effects were observed on CD8+ but not on CD4+ T cells. Moreover, 4-1BB appeared to be unique among several TNFRs tested in inducing increase in stimulatory over inhibitory TCF-1.
To test the hypothesis that the amount and distribution of glaucomatous damage along the entire retinal ganglion cell–axonal complex (RGC-AC) can be quantified and to map the RGC-AC connectivity in early glaucoma using automated image analysis of standard spectral-domain optical coherence tomography.
Spectral-domain optical coherence tomography volumes were obtained from 116 eyes in 58 consecutive patients with glaucoma or suspected glaucoma. Layer and optic nerve head (ONH) analysis was performed; the mean regional retinal ganglion cell layer thickness (68 regions), nerve fiber layer (NFL) thickness (120 regions), and ONH rim area (12 wedge-shaped regions) were determined. Maps of RGC-AC connectivity were created using maximum correlation between regions’ ganglion cell layer thickness, NFL thickness, and ONH rim area; for retinal nerve fiber bundle regions, the maximum “thickness correlation paths” were determined.
The mean (SD) NFL thickness and ganglion cell layer thickness across all macular regions were 22.5 (7.5) μm and 33.9 (8.4) μm, respectively. The mean (SD) rim area across all ONH wedge regions was 0.038 (0.004) mm2. Connectivity maps were obtained successfully and showed typical nerve fiber bundle connectivity of the RGC-AC cell body segment to the initial NFL axonal segment, of the initial to the final RGC-AC NFL axonal segments, of the final RGC-AC NFL axonal to the ONH axonal segment, and of the RGC-AC cell body segment to the ONH axonal segment.
In early glaucoma, the amount and distribution of glaucomatous damage along the entire RGC-AC can be quantified and mapped using automated image analysis of standard spectral-domain optical coherence tomography. Our findings should contribute to better detection and improved management of glaucoma.
To characterize the molecular and functional status of the rat retina and optic nerve after acute elevation of intraocular pressure (IOP).
Retinal ischemia was induced in rats by increasing the IOP (110 mmHg/60 minutes). Microarray analysis, quantitative RT-PCR (qRT-PCR) and immunohistochemistry were used to characterize retinal tissue. PLGA microspheres containing neurotrophic factors (BDNF, GDNF, or CNTF) or empty microspheres were injected into the vitreous of operated animals 1 day after elevation of IOP. Pupil light reflex (PLR) parameters and electroretinograms (ERG) were monitored at multiple time points during the 60-day postoperative recovery period.
Molecular analysis showed a significant intrinsic up-regulation of CNTF at 10 and 25 days after induction of the acute ocular hypertension (p=0.0067). Molecular tissue analysis of GDNF and its receptors (GDNFR1, GDNFR2), and BDNF and its receptor (trkB) showed no change in expression. Animals that received CNTF microspheres had no significant functional recovery compared to animals which received blank microspheres (p>0.05). Animals that received GDNF or BDNF microspheres showed significant PLR recovery (p<0.05 and p<0.001 respectively) compared to non-treated animals.
Continuous release of neurotrophic growth factors (NGFs) significantly protects optic nerve function in the experimental model of retinal ischemia observed by PLR analysis.
Neuroprotection; Retina; BDNF; GDNF; CNTF
Free radical co-oxidation of polyunsaturated lipids with tyrosine or phenolic analogs of tyrosine gave rise to lipid peroxide-tyrosine (phenol) adducts in both aqueous micellar and organic solutions. The novel adducts were isolated and characterized by 1D and 2D NMR as well as by mass spectrometry. The spectral data suggest that the polyunsaturated lipid peroxyl radicals give stable peroxide coupling products exclusively at the para position of the tyrosyl (phenoxy) radicals. These adducts have characteristic 13C chemical shifts at 185 ppm due to the cross-conjugated carbonyl of the phenol-derived cyclohexadienone. The primary peroxide adducts subsequently undergo intramolecular Diels-Alder (IMDA) cyclization, affording a number of diastereomeric tricyclic adducts that have characteristic carbonyl 13C chemical shifts at ~198 ppm. All NMR HMBC and HSQC correlations support the structure assignment of the primary and Diels-Alder adducts, as does MS collision induced dissociation. Kinetic rate constants and activation parameters for the IMDA reaction were determined and the primary adducts were reduced with cuprous ion giving a phenol-derived 4-hydroxycyclohexa-2,5-dienone. No products from adduction of peroxyls at the phenolic ortho position were found either in the primary or the cuprous reduction product mixtures. These studies provide a framework for understanding the nature of lipid-protein adducts formed by peroxyl-tyrosyl radical-radical termination processes. Coupling of lipid peroxyl radicals with tyrosyl radicals leads to cyclohexenone and cyclohexadienone adducts which are of interest in and of themselves since, as electrophiles, they are likely targets for protein nucleophiles. One consequence of lipid peroxyl reactions with tyrosyls may therefore be protein-protein crosslinks via interprotein Michael adducts.
Tyrosine oxidation; lipid peroxidation; para-hydroxy tyrosine; lipid protein adduct; Intramolecular Diels-Alder cyclization
TGF-β is well known to play a critical role in diabetic kidney disease, and ongoing clinical studies are testing the potential therapeutic promise of inhibiting TGF-β production and action. An aspect of TGF-β action that has not received much attention is its potential role in explaining sex-related proclivity for kidney disease. In this review, we discuss recent studies linking TGF-β signaling to sex-related effects in diabetic kidney disease and suggest targets for future studies.
Sex difference; kidney; diabetes
The present study examined the effect of dietary genistein, a soy isoflavone, on breast cancer patients who take tamoxifen, an antiestrogen treatment, using a preclinical model. The interaction of various doses of genistein with tamoxifen on the growth of estrogen receptor-positive breast cancer MCF-7 cells was investigated by subcutaneously injecting MCF-7 cells into the flank of ovariectomized athymic mice. Animals were randomized into eight experimental groups with 10–13 mice per group: control (C), estrogen (E) (0.08 mg E implant), tamoxifen (T) (3 mg T implant), estrogen + tamoxifen (E + T), tamoxifen + 500 p.p.m. genistein (T + G500), estrogen + tamoxifen + 250 p.p.m. genistein (E + T + G250), estrogen + tamoxifen + 500 p.p.m. genistein (E + T + G500) and estrogen + tamoxifen + 1000 p.p.m. genistein (E + T + G1000). Treatment of tamoxifen significantly reduced the estrogen-induced MCF-7 tumor prevalence and tumor size. This inhibitory effect of tamoxifen was significantly negated by the low doses of dietary genistein (250 and 500 p.p.m.), whereas the 1000 p.p.m. genistein did not have the same effect. Cells harvested from tamoxifen-treated tumors retained estrogen responsiveness of their progenitor MCF-7 cells, indicating that the abrogating effect of genistein on tamoxifen-treated tumor growth was not caused by a diminished tamoxifen response but directly by genistein. The low doses of dietary genistein abrogated the inhibitory effect of tamoxifen potentially by acting on the tumor cell proliferation/apoptosis ratio and the messenger RNA (mRNA) expression of cyclin D1 in addition to regulating the mRNA expression of progesterone receptor. Therefore, data from the current study suggest that caution is warranted regarding the consumption of dietary genistein by breast cancer patients while on tamoxifen therapy.
Age-related macular degeneration (AMD) is a complex genetic disease, with many loci demonstrating appreciable attributable disease risk. Despite significant progress toward understanding the genetic and environmental etiology of AMD, identification of additional risk factors is necessary to fully appreciate and treat AMD pathology. In this study, we investigated copy number variants (CNVs) as potential AMD risk variants in a cohort of 400 AMD patients and 500 AMD-free controls ascertained at the University of Iowa. We used three publicly available copy number programs to analyze signal intensity data from Affymetrix® GeneChip SNP Microarrays. CNVs were ranked based on prevalence in the disease cohort and absence from the control group; high interest CNVs were subsequently confirmed by qPCR. While we did not observe a single-locus “risk CNV” that could account for a major fraction of AMD, we identified several rare and overlapping CNVs containing or flanking compelling candidate genes such as NPHP1 and EFEMP1. These and other candidate genes highlighted by this study deserve further scrutiny as sources of genetic risk for AMD.
A robust multiscale stereo matching algorithm is proposed to find reliable correspondences between low contrast and weakly textured retinal image pairs with radiometric differences. Existing algorithms designed to deal with piecewise planar surfaces with distinct features and Lambertian reflectance do not apply in applications such as 3D reconstruction of medical images including stereo retinal images. In this paper, robust pixel feature vectors are formulated to extract discriminative features in the presence of noise in scale space, through which the response of low-frequency mechanisms alter and interact with the response of high-frequency mechanisms. The deep structures of the scene are represented with the evolution of disparity estimates in scale space, which distributes the matching ambiguity along the scale dimension to obtain globally coherent reconstructions. The performance is verified both qualitatively by face validity and quantitatively on our collection of stereo fundus image sets with ground truth, which have been made publicly available as an extension of standard test images for performance evaluation.
Depth from stereo; radiometric differences; pixel feature vector; fundus image; scale space
Modulation of neurogenesis that acts as an endogenous repair mechanism would have a significant impact on future therapeutic strategies for Parkinson’s disease (PD). Several studies demonstrated dopaminergic modulation of neurogenesis in the subventricular zone (SVZ) of the adult brain. Levodopa, the gold standard therapy for PD, causes an increase in homocysteine levels that induces neuronal death via N-methyl-D-aspartate (NMDA) receptor. The present study investigated whether elevated homocysteine by levodopa treatment in a parkinsonian model would modulate neurogenesis via NMDA receptor signal cascade and compared the effect of levodopa and pramipexol (PPX) on neurogenic activity.
Neurogenesis was assessed in vitro using neural progenitor cells (NPCs) isolated from the SVZ and in vivo with the BrdU-injected animal model of PD using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Modulation of homocysteine levels was evaluated using co-cultures of NPCs and astrocytes and PD animals. Immunochemical and Western blot analyses were used to measure neurogenesis and determine the cell death signaling. Levodopa treatment increased release of homocysteine on astrocytes culture media as well as in plasma and brain of PD animals. Increased homocysteine by levodopa led to increased apoptosis of NPCs through the NMDA receptor-dependent the extracellular signal-regulated kinase (ERK) signaling pathways. The administration of a NMDA antagonist significantly attenuated apoptotic cell death in levodopa-treated NPCs and markedly increased the number of BrdU-positive cells in the SVZ of levodopa-treated PD animals. Comparative analysis revealed that PPX treatment significantly increased the number of NPCs and BrdU-positive cells in the SVZ of PD animals compared to levodopa treatment. Our present study demonstrated that increased homocysteine by levodopa has a detrimental effect on neurogenesis through NMDA receptor-mediated ERK signaling pathway.
Modulation of levodopa-induced elevated homocysteine by NMDA antagonist or dopamine agonist has a clinical relevance for PD treatment in terms of adult neurogenesis.
The current standard for treating operable early stage non-small cell lung cancer is surgical resection and for inoperable cases it is external beam radiotherapy. Lung functions are adversely affected with both the above treatments. CyberKnife treatment limits radiation damage by tracking targets moving with each breath. The effect of CyberKnife treatment on pulmonary function tests has not been well documented.
Lung cancer patients who underwent CyberKnife treatment and had pre- and post-treatment pulmonary function tests were included. Paired t-tests were conducted. We also conducted subgroup analysis.
Thirty-seven patients were included. Median age was 73 years. No statistical difference between mean pre- and post-CyberKnife pulmonary function tests was found.
We observed that CyberKnife better preserves lung function status compared to current standards of care. It has shown to have very minimal side effects.
non-small cell lung cancer; radiation pneumonitis; radiotherapy; pulmonary function tests
4-1BB (CD137, TNFRSF9), a member of the activation-induced tumor necrosis factor receptor family, is a powerful T-cell costimulatory molecule. It generally enhances CD8+ T responses and even breaks the tolerance of CD8+ T cells in an antigen-specific manner. In the present study we found that it was expressed in the placentas of pregnant mice and that its expression coincided with that of the immunesuppressive enzyme indoleamine 2,3-dioxygenase (IDO). Therefore, we investigated whether 4-1BB signaling is involved in fetal rejection using agonistic anti-4-1BB mAb and 4-1BB-deficient mice. Treatment with agonistic anti-4-1BB mAb markedly increased the rate of rejection of allogeneic but not syngeneic fetuses, and this was primarily dependent on CD8+ T cells. Complement component 3 (C3) seemed to be the effector molecule because 4-1BB triggering resulting in accumulation of C3 in the placenta, and this accumulation was also reversed by anti-CD8 mAb treatment. These findings demonstrate that 4-1BB triggering breaks the tolerance of CD8+ T cells to alloantigens in the placenta. Moreover, triggering 4-1BB protected the pregnant mice from Listeria monocytogenes (LM) infection, but led to rejection of semi-allogeneic fetuses. Therefore, given the cross-recognition of alloantigen by pathogen-reactive CD8+ T cells, the true function of 4-1BB may be to reverse the hypo-responsiveness of pathogen-reactive CD8+ T cells in the placenta in cases of infection, even if that risks losing the fetus.
Through a structure-structure correlation analysis of focal macular and peripapillary SD-OCT regions in 57 subjects with glaucoma (or glaucoma suspicion), a color-coded map closely resembling the nerve fiber bundle pattern of retinal ganglion cells emerged.
To correlate the thicknesses of focal regions of the macular ganglion cell layer with those of the peripapillary nerve fiber layer using spectral-domain optical coherence tomography (SD-OCT) in glaucoma subjects.
Macula and optic nerve head SD-OCT volumes were obtained in 57 eyes of 57 subjects with open-angle glaucoma or glaucoma suspicion. Using a custom automated computer algorithm, the thickness of 66 macular ganglion cell layer regions and the thickness of 12 peripapillary nerve fiber layer regions were measured from registered SD-OCT volumes. The mean thickness of each ganglion cell layer region was correlated to the mean thickness of each peripapillary nerve fiber layer region across subjects. Each ganglion cell layer region was labeled with the peripapillary nerve fiber layer region with the highest correlation using a color-coded map.
The resulting color-coded correlation map closely resembled the nerve fiber bundle (NFB) pattern of retinal ganglion cells. The mean r2 value across all local macular-peripapillary correlations was 0.49 (± 0.11). When separately analyzing the 30 glaucoma subjects from the 27 glaucoma-suspect subjects, the mean r2 value across all local macular-peripapillary correlations was significantly larger in the glaucoma group (0.56 ± 0.13 vs. 0.37 ± 0.11; P < 0.001).
A two-dimensional (2-D) spatial NFB map of the retina can be developed using structure-structure relationships from SD-OCT. Such SD-OCT-based NFB maps may enhance glaucoma detection and contribute to monitoring change in the future.
We report identification of a novel genetic locus (GLC1P) for normal tension glaucoma (NTG) on chromosome 12q14 using linkage studies of an African-American pedigree (maximum non-parametric linkage score = 19.7, max LOD score = 2.7). Subsequent comparative genomic hybridization and quantitative polymerase chain reaction (PCR) experiments identified a 780 kbp duplication within the GLC1P locus that is co-inherited with NTG in the pedigree. Real-time PCR studies showed that the genes within this duplication [TBK1 (TANK-binding kinase 1), XPOT, RASSF3 and GNS] are all expressed in the human retina. Cohorts of 478 glaucoma patients (including 152 NTG patients), 100 normal control subjects and 400 age-related macular degeneration patients were subsequently tested for copy number variation in GLC1P. Overlapping duplications were detected in 2 (1.3%) of the 152 NTG subjects, one of which had a strong family history of glaucoma. These duplications defined a 300 kbp critical region of GLC1P that spans two genes (TBK1 and XPOT). Microarray expression experiments and northern blot analysis using RNA obtained from human skin fibroblast cells showed that duplication of chromosome 12q14 results in increased TBK1 and GNS transcription. Finally, immunohistochemistry studies showed that TBK1 is expressed in the ganglion cells, nerve fiber layer and microvasculature of the human retina. Together, these data link the duplication of genes on chromosome 12q14 with familial NTG and suggest that an extra copy of the encompassed TBK1 gene is likely responsible for these cases of glaucoma. However, animal studies will be necessary to rule out a role for the other duplicated or neighboring genes.
To promote cell survival, the antiapoptotic factor Bcl-xL both
inhibits Bax-induced mitochondrial outer membrane permeabilization and
stabilizes mitochondrial inner membrane ion flux and thus overall mitochondrial
Mammalian Bcl-xL protein localizes to the outer mitochondrial
membrane, where it inhibits apoptosis by binding Bax and inhibiting Bax-induced
outer membrane permeabilization. Contrary to expectation, we found by electron
microscopy and biochemical approaches that endogenous Bcl-xL also
localized to inner mitochondrial cristae. Two-photon microscopy of cultured
neurons revealed large fluctuations in inner mitochondrial membrane potential
when Bcl-xL was genetically deleted or pharmacologically inhibited,
indicating increased total ion flux into and out of mitochondria. Computational,
biochemical, and genetic evidence indicated that Bcl-xL reduces
futile ion flux across the inner mitochondrial membrane to prevent a wasteful
drain on cellular resources, thereby preventing an energetic crisis during
stress. Given that F1FO–ATP synthase directly
affects mitochondrial membrane potential and having identified the mitochondrial
ATP synthase β subunit in a screen for Bcl-xL–binding
partners, we tested and found that Bcl-xL failed to protect β
subunit–deficient yeast. Thus, by bolstering mitochondrial energetic
capacity, Bcl-xL may contribute importantly to cell survival
independently of other Bcl-2 family proteins.
DNA amplifications, leading to the overexpression of oncogenes, are a cardinal feature of lung cancer and directly contribute to its pathogenesis. To uncover novel such alterations, we performed an array-based comparative genomic hybridization survey of 128 non-small cell lung cancer cell lines and tumors. Prominent among our findings, we identified recurrent high-level amplification at cytoband 22q11.21 in 3% of lung cancer specimens, with another 11% of specimens exhibiting low-level gain spanning that locus. The 22q11.21 amplicon core contained eight named genes, only four of which were overexpressed (by transcript profiling) when amplified. Among these, CRKL encodes an adaptor protein functioning in signal transduction, best known as a substrate of the BCR-ABL kinase in chronic myelogenous leukemia. RNA interference-mediated knockdown of CRKL in lung cancer cell lines with (but not without) amplification led to significantly decreased cell proliferation, cell-cycle progression, cell survival, and cell motility and invasion. In addition, overexpression of CRKL in immortalized human bronchial epithelial cells led to EGF-independent cell growth. Our findings indicate that amplification and resultant overexpression of CRKL contributes to diverse oncogenic phenotypes in lung cancer, with implications for targeted therapy, and highlighting a role of adapter proteins as primary genetic drivers of tumorigenesis.
CRKL; lung cancer; DNA amplification; genomic profiling; adapter protein
This study has identified rare and recurrent deletions and duplications in POAG patients in the first large-scale, whole-genome study of structural variation performed in a sample of POAG patients and POAG-free subjects.
This study sought to investigate the role of rare copy number variation (CNV) in age-related disorders of blindness, with a focus on primary open-angle glaucoma (POAG). Data are reported from a whole-genome copy number screen in a large cohort of 400 individuals with POAG and 500 age-matched glaucoma-free subjects.
DNA samples from patients and controls were tested for CNVs using a combination of two microarray platforms. The signal intensity data generated from these arrays were then analyzed with multiple CNV detection programs including CNAG version 2.0, PennCNV, and dChip.
A total of 11 validated CNVs were identified as recurrent in the POAG set and absent in the age-matched control set. This set included CNVs on 5q23.1 (DMXL1, DTWD2), 20p12 (PAK7), 12q14 (C12orf56, XPOT, TBK1, and RASSF3), 12p13.33 (TULP3), and 10q34.21 (PAX2), among others. The CNVs presented here are exceedingly rare and are not found in the Database of Genomic Variants. Moreover, expression data from ocular tissue support the role of these CNV-implicated genes in vision-related processes. In addition, CNV locations of DMXL1 and PAK7 overlap previously identified linkage signals for glaucoma on 5p23.1 and 20p12, respectively.
The data are consistent with the hypothesis that rare CNV plays a role in the development of POAG.
Analysis of mutant mouse strains and linkage analysis with human families have both demonstrated that mutations influencing the podosomal adaptor protein SH3 and PX domains 2B (SH3PXD2B) can result in a congenital form of glaucoma. Here, we use immunohistochemistry to describe localization of the SH3PXD2B protein throughout the adult human eye and test whether sequence variants in SH3PXD2B occur in multiple other forms of glaucoma.
In immunohistochemical experiments, cryosections of human donor eyes were evaluated for SH3PXD2B immunoreactivity with a polyclonal antibody. In genetic experiments, exon sequences of SH3PXD2B from patients with primary congenital glaucoma (n=21), Axenfeld-Rieger syndrome (n=30), and primary open angle glaucoma (n=127) were compared to control subjects (n=89). The frequency of non-synonymous SH3PXD2B coding sequence variants were compared between patient cohorts and controls using Fisher’s exact test.
Varying intensities of SH3PXD2B immunoreactivity were detected in almost all ocular tissues. Among tissues important to glaucoma, immunoreactivity was detected in the drainage structures of the iridocorneal angle, ciliary body, and retinal ganglion cells. Intense immunoreactivity was present in photoreceptor inner segments. From DNA analysis, a total of 11 non-synonymous variants were detected. By Fisher’s Exact test, there was not a significant skew in the overall frequency of these changes in any patient cohort versus controls (p-value >0.05). Each cohort contained unique variants not detected in other cohorts or patients.
SH3PXD2B is widely distributed in the adult human eye, including several tissues important to glaucoma pathogenesis. Analysis of DNA variants in three forms of glaucoma detected multiple variants unique to each patient cohort. While statistical analysis failed to support a pathogenic role for these variants, some of them may be rare disease-causing variants whose biologic significance warrants investigation in follow up replication studies and functional assays.
Grapes (Vitis vinifera L.) are economically the most important fruit crop worldwide. However, the complexity of molecular and biochemical events that lead to the onset of ripening of nonclimacteric fruits is not fully understood which is further complicated in grapes due to seasonal and cultivar specific variation. The Portuguese wine variety Trincadeira gives rise to high quality wines but presents extremely irregular berry ripening among seasons probably due to high susceptibility to abiotic and biotic stresses.
Ripening of Trincadeira grapes was studied taking into account the transcriptional and metabolic profilings complemented with biochemical data. The mRNA expression profiles of four time points spanning developmental stages from pea size green berries, through véraison and mature berries (EL 32, EL 34, EL 35 and EL 36) and in two seasons (2007 and 2008) were compared using the Affymetrix GrapeGen® genome array containing 23096 probesets corresponding to 18726 unique sequences. Over 50% of these probesets were significantly differentially expressed (1.5 fold) between at least two developmental stages. A common set of modulated transcripts corresponding to 5877 unigenes indicates the activation of common pathways between years despite the irregular development of Trincadeira grapes. These unigenes were assigned to the functional categories of "metabolism", "development", "cellular process", "diverse/miscellanenous functions", "regulation overview", "response to stimulus, stress", "signaling", "transport overview", "xenoprotein, transposable element" and "unknown". Quantitative RT-PCR validated microarrays results being carried out for eight selected genes and five developmental stages (EL 32, EL 34, EL 35, EL 36 and EL 38). Metabolic profiling using 1H NMR spectroscopy associated to two-dimensional techniques showed the importance of metabolites related to oxidative stress response, amino acid and sugar metabolism as well as secondary metabolism. These results were integrated with transcriptional profiling obtained using genome array to provide new information regarding the network of events leading to grape ripening.
Altogether the data obtained provides the most extensive survey obtained so far for gene expression and metabolites accumulated during grape ripening. Moreover, it highlighted information obtained in a poorly known variety exhibiting particular characteristics that may be cultivar specific or dependent upon climatic conditions. Several genes were identified that had not been previously reported in the context of grape ripening namely genes involved in carbohydrate and amino acid metabolisms as well as in growth regulators; metabolism, epigenetic factors and signaling pathways. Some of these genes were annotated as receptors, transcription factors, and kinases and constitute good candidates for functional analysis in order to establish a model for ripening control of a non-climacteric fruit.