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Current approaches to cancer immunotherapy include immune checkpoint inhibitors, cancer vaccines, and adoptive cellular therapy. These therapies have produced significant clinical success for specific cancers, but their efficacy has been limited. Oncolytic virotherapy (OVT) has emerged as a promising immunotherapy for a variety of cancers. Furthermore, the unique characteristics of OVs make them a good choice for delivering tumor peptides/antigens to induce enhanced tumor-specific immune responses. The first oncolytic virus (OV) approved for human use is the attenuated herpes simplex virus type 1 (HSV-1), Talimogene laherparepvec (T-VEC) which has been FDA approved for the treatment of melanoma in humans. In this study, we engineered the recombinant oncolytic HSV-1 (oHSV) VC2-OVA expressing a fragment of ovalbumin (OVA) as a fusion protein with VP26 virion capsid protein. We tested the ability of VC2-OVA to act as a vector capable of stimulating strong, specific antitumor immunity in a syngeneic murine melanoma model. Therapeutic vaccination with VC2-OVA led to a significant reduction in colonization of tumor cells in the lungs of mice intravenously challenged B16cOVA cells. In addition, VC2-OVA induced a potent prophylactic antitumor response and extended survival of mice that were intradermally engrafted with B16cOVA tumors compared with mice immunized with control virus.

Current oncolytic virotherapies possess limited response rates. However, when certain patient selection criteria are used, oncolytic virotherapy response rates have been shown to increase., Oncolytic virotherapy (OVT) is now understood to be an immunotherapy that uses viral infection to liberate tumor antigens in an immunogenic context to promote the development of antitumor immune responses. The only currently FDA-approved oncolytic virotherapy, T-Vec, is a modified type 1 herpes simplex virus (HSV-1). While T-Vec is associated with limited response rates, its modest efficacy supports the continued development of novel OVT viruses. Herein, we test the efficacy of a recombinant HSV-1, VC2, as an OVT in a syngeneic B16F10-derived mouse model of melanoma. VC2 possesses mutations that block its ability to enter neurons via axonal termini. This greatly enhances its safety profile by precluding the ability of the virus to establish latent infection. VC2 has been shown to be a safe, effective vaccine against both HSV-1 and HSV-2 infection in mice, guinea pigs, and nonhuman primates. We found that VC2 slows tumor growth rates and that VC2 treatment significantly enhances survival of tumor-engrafted, VC2-treated mice over control treatments. VC2-treated mice that survived initial tumor engraftment were resistant to a second engraftment as well as colonization of lungs by intravenous introduction of tumor cells. We found that VC2 treatment induced substantial increases in intratumoral T cells and a decrease in immunosuppressive regulatory T cells. This immunity was critically dependent on CD8+ T cells and less dependent on CD4+ T cells. Our data provide significant support for the continued development of VC2 as an OVT for the treatment of human and animal cancers. IMPORTANCE Current oncolytic virotherapies possess limited response rates. However, when certain patient selection criteria are used, oncolytic virotherapy response rates have been shown to increase. This, in addition to the increased response rates of oncolytic virotherapy in combination with other immunotherapies, suggests that oncolytic viruses possess significant therapeutic potential for the treatment of cancer. As such, it is important to continue to develop novel oncolytic viruses as well as support basic research into their mechanisms of efficacy. Our data demonstrate significant clinical potential for VC2, a novel type 1 oncolytic herpes simplex virus. Additionally, due to the high rates of survival and the dependence on CD8+ T cells for efficacy, our model will enable study of the immunological correlates of protection for VC2 oncolytic virotherapy and oncolytic virotherapy in general. Understanding the mechanisms of efficacious oncolytic virotherapy will inform the rational design of improved oncolytic virotherapies.

Bovine herpesvirus type 1 (BoHV-1) is the viral causative agent of infectious bovine rhinotracheitis and a component of the bovine respiratory complex commonly referred to as shipping fever in calves. BoHV-1 is also responsible for losses of aborted calves and reductions in dairy productivity. BoHV-1 belongs to the neurotropic alphaherpesviruses which have a predilection to infect and establish latency in sensory neurons. Neuronal cell cultures provide a useful platform for experiments investigating neuronal entry, retrograde and anterograde transport, and the establishment of latency. Rodent neuronal cell lines and primary rabbit neuronal cells have been utilized for BoHV-1, though a reliable host-specific neuronal cell culture system has not been developed. In this study, BoHV-1 readily infected bovine-derived immortalized neuronal progenitor cells (FBBC-1) differentiated in cell culture producing neurite-like projections and exhibiting neuronal cell markers NeuN and L1CAM. FBBC-1 cells expressed both nectin-1 and nectin-2 alphaherpesvirus receptors on their cell surfaces, however, nectin-2 was detected in much greater abundance than nectin-1. To facilitate investigations of BoHV-1 infection, a recombinant BoHV-1 virus expressing the green fluorescent protein (GFP) cloned into a bacterial artificial chromosome (BAC) was used to generate an mCherry-VP26 fusion protein. The BoHV-1 GFP expressing VP26mCherry labeled virus infected differentiated FBBC-1 cells as evidenced by the production of infectious virions and the expression of both GFP and mCherry fluorophores. Time-lapse live cell microscopy revealed the presence of mCherry fluorescent capsids in neuronal projections immediately after virus entry moving retrograde in a saltatory manner. Proximity ligation assays (PLA) using MDBK cells demonstrated that BoHV-1 glycoprotein D (gD) interacted more efficiently with nectin-1 than nectin-2. However, the gD interaction with nectin-2 predominated in differentiated FBBC-1 cells in comparison to the gD nectin-1 interaction. The efficiently differentiated FBBC-1 neuronal cell line and fluorescently labeled BoHV-1 virions will assist experimentation aiming to elucidate specific mechanisms of virus entry and transport in a homologous bovine neuronal cell culture system.

Latanoprost is a common glaucoma medication. Here, we study longitudinal effects of sustained latanoprost treatment on intraocular pressure (IOP) in C57BL/6J mice, as well as two potential side-effects, changes in iris pigmentation and central corneal thickness (CCT). Male C57BL/6J mice were treated daily for 16 weeks with latanoprost. Control mice were treated on the same schedule with the preservative used with latanoprost, benzalkonium chloride (BAK), or handled, without ocular treatments. IOP and CCT were studied at pre-treatment, 2 “early” time points, and 2 “late” time points; slit-lamp analysis performed at a late time point; and expression of corneal and iridial candidate genes analyzed at the end of the experiment. Latanoprost lowered IOP short, but not long-term. Sustained application of BAK consistently resulted in significant corneal thinning, whereas sustained treatment with latanoprost resulted in smaller and less consistent changes. Neither treatment affected iris pigmentation, corneal matrix metalloprotease expression or iridial pigment-related genes expression. In summary, latanoprost initially lowered IOP in C57BL/6J mice, but became less effective with sustained treatment, likely due to physiological adaptation. These results identify a new resource for studying changes in responsiveness associated with long-term treatment with latanoprost and highlight detrimental effects of commonly used preservative BAK.

IMPACT: Our data demonstrate that VC2 oncolytic virotherapy has significant clinical potential. OBJECTIVES/GOALS: Use our novel oncolytic herpes simplex virus type I (HSV-1), VC2, to understand how oncolytic virotherapy affects the immunosuppressive tumor microenvironment as a mechanism of efficacy. METHODS/STUDY POPULATION: We tested the efficacy of VC2 as an oncolytic virotherapy (OVT) in a syngeneic B16F10-derived mouse model of melanoma. We modified the B16F10 to express nectin-1 (B16F10n-1), the major receptor for HSV-1. Engrafted B16F10n-1 tumors were intratumorally treated with either phosphate-buffered saline (PBS) or 1x10^6 pfu VC2. At indicated time points, treated tumors were excised and processed for immunohistochemistry or flow cytometry analysis. For our experimental metastasis studies, mice were intravenously challenged with B16F10n-1 cells. For our depletion studies, CD4+ and CD8+ T cells were depleted in mice by treatment with mouse anti-CD4 and anti-CD8 monoclonal antibodies respectively, while the control mice were given Rat IgG2b isotype. RESULTS/ANTICIPATED RESULTS: We found that VC2 slowed tumor growth rates and significantly enhanced survival times over control treated mice. VC2-treated mice that survived initial tumor engraftment were able to reject a second tumor challenge and were also resistant to lung colonization (experimental metastasis) of tumor cells. Furthermore, VC2 treatment promoted increased intratumoral T cell infiltration and induced a strong antitumor effect that decreased growth rates of distant, untreated tumors. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our data demonstrate that VC2 OVT has significant clinical potential. Furthermore, due to the increased survival rates and CD8+ T cells dependence, our model will enable study of the immunological correlates of protection for VC2 OVT and OVT in general, as well as to inform the rational design of future OVs with improved therapeutic potentials.

Herpes simplex virus type-1 (HSV-1) and type-2 (HSV-2) are prototypical alphaherpesviruses that are characterized by their unique properties to infect trigeminal and dorsal root ganglionic neurons, respectively, and establish life-long latent infections. These viruses initially infect mucosal epithelial tissues and subsequently spread to neurons. They are associated with a significant disease spectrum, including orofacial and ocular infections for HSV-1 and genital and neonatal infections for HSV-2. Viral glycoproteins within the virion envelope bind to specific cellular receptors to mediate virus entry into cells. This is achieved by the fusion of the viral envelope with the plasma membrane. Similarly, viral glycoproteins expressed on cell surfaces mediate cell-to-cell fusion and facilitate virus spread. An interactive complex of viral glycoproteins gB, gD/gH/gL, and gK and other proteins mediate these membrane fusion phenomena with glycoprotein B (gB), the principal membrane fusogen. The requirement for the virion to enter neuronal axons suggests that the heterodimeric protein complex of gK and membrane protein UL20, found only in alphaherpesviruses, constitute a critical determinant for neuronal entry. This hypothesis was substantiated by the observation that a small deletion in the amino terminus of gK prevents entry into neuronal axons while allowing entry into other cells via endocytosis. Cellular receptors and receptor-mediated signaling synergize with the viral membrane fusion machinery to facilitate virus entry and intercellular spread. Unraveling the underlying interactions among viral glycoproteins, envelope proteins, and cellular receptors will provide new innovative approaches for antiviral therapy against herpesviruses and other neurotropic viruses.

In addition to needing acute emergency management, blast-mediated traumatic brain injury (TBI) is also a chronic disorder with delayed-onset symptoms that manifest and progress over time. While the immediate consequences of acute blast injuries are readily apparent, chronic sequelae are harder to recognize. Indeed, the identification of individuals with mild-TBI or TBI-induced symptoms is greatly impaired in large part due to the lack of objective and robust biomarkers. The purpose of this study was to address these need by identifying candidates for serum-based biomarkers of blast TBI, and also to identify unique or differentially regulated protein expression in the thalamus in C57BL/6J mice exposed to blast using high throughput qualitative screens of protein expression. To identify thalamic proteins differentially or uniquely associated with blast exposure, we utilized an antibody-based affinity-capture strategy (referred to as “proteomics-based analysis of depletomes”; PAD) to deplete thalamic lysates from blast-treated mice of endogenous thalamic proteins also found in control mice. Analysis of this “depletome” detected 75 unique proteins, many with associations to the myelin sheath. To identify blast-associated proteins eliciting production of circulating autoantibodies, serum antibodies of blast-treated mice were immobilized, and their immunogens subsequently identified by proteomic analysis of proteins specifically captured following incubation with thalamic lysates (a variant of a strategy referred to as “proteomics-based expression library screening”; PELS). This analysis identified 46 blast-associated immunogenic proteins, including 6 shared in common with the PAD analysis (ALDOA, PHKB, HBA-A1, DPYSL2, SYN1, and CKB). These proteins and their autoantibodies are appropriate for further consideration as biomarkers of blast-mediated TBI., Biochemistry; Neuroscience; Proteins; Retinal ganglion cell; Biomarker; Blast injury; Traumatic brain injury.

Key Clinical Message Childhood-onset schizophrenia is rare, comprising 1% of known schizophrenia cases. Here, we report a patient with childhood-onset schizophrenia who has three large chromosomal abnormalities: an inherited 2.2 Mb deletion of chromosome 3p12.2–p12.1, a de novo 16.7 Mb duplication of 16q22.3–24.3, and a de novo 43 Mb deletion of Xq23–q28.

Autism is a neurodevelopmental disorder characterized by three core symptom domains: ritualistic-repetitive behaviors, impaired social interaction, and impaired communication and language development. Recent studies have highlighted etiologically relevant recurrent copy number changes in autism, such as 16p11.2 deletions and duplications, as well as a significant role for unique, novel variants. We used Affymetrix 250K GeneChip Microarray technology (either NspI or StyI) to detect microdeletions and duplications in a subset of children from the Autism Genetic Resource Exchange (AGRE). In order to enrich our sample for potentially pathogenic CNVs we selected children with autism who had additional features suggestive of chromosomal loss associated with developmental disturbance (positive criteria filter) but who had normal cytogenetic testing (negative criteria filter). We identified families with the following features: at least one child with autism who also had facial dysmorphology, limb or digit abnormalities, or ocular abnormalities. To detect changes in copy number we used a publicly available program, Copy Number Analyser for GeneChip® (CNAG) Ver. 2.0. We identified novel deletions and duplications on chromosomes 1q24.2, 3p26.2, 4q34.2, and 6q24.3. Several of these deletions and duplications include new and interesting candidate genes for autism such as syntaxin binding protein 5 (STXBP5 also known as tomosyn) and leucine rich repeat neuronal 1 (LRRN1 also known as NLRR1). Lastly, our data suggest that rare and potentially pathogenic microdeletions and duplications may have a substantially higher prevalence in children with autism and additional developmental anomalies than in children with autism alone.

Autism spectrum disorders (ASDs) are childhood neurodevelopmental disorders with complex genetic origins. Previous studies focusing on candidate genes or genomic regions have identified several copy number variations (CNVs) that are associated with an increased risk of ASDs. Here we present the results from a whole-genome CNV study on a cohort of 859 ASD cases and 1,409 healthy children of European ancestry who were genotyped with approximately 550,000 single nucleotide polymorphism markers, in an attempt to comprehensively identify CNVs conferring susceptibility to ASDs. Positive findings were evaluated in an independent cohort of 1,336 ASD cases and 1,110 controls of European ancestry. Besides previously reported ASD candidate genes, such as NRXN1 (ref. 10) and CNTN4 (refs 11, 12), several new susceptibility genes encoding neuronal cell-adhesion molecules, including NLGN1 and ASTN2, were enriched with CNVs in ASD cases compared to controls (P = 9.5 x 10(-3)). Furthermore, CNVs within or surrounding genes involved in the ubiquitin pathways, including UBE3A, PARK2, RFWD2 and FBXO40, were affected by CNVs not observed in controls (P = 3.3 x 10(-3)). We also identified duplications 55 kilobases upstream of complementary DNA AK123120 (P = 3.6 x 10(-6)). Although these variants may be individually rare, they target genes involved in neuronal cell-adhesion or ubiquitin degradation, indicating that these two important gene networks expressed within the central nervous system may contribute to the genetic susceptibility of ASD.

The present article introduces RetFM-J, a semi-automated ImageJ-based module that detects, counts, and collects quantitative data on nuclei of the inner retina from H&E-stained whole-mounted retinas. To illustrate performance, computer-derived outputs were analyzed in inbred C57BL/6J mice. Automated characterization yielded computer-derived outputs that closely matched manual counts. As a method using open-source software that is freely available, inexpensive staining reagents that are robust, and imaging equipment that is routine to most laboratories, RetFM-J could be utilized in a wide variety of experiments benefiting from high-throughput, quantitative, uniform analyses of total cellularity in the inner retina.

Promoters often contain multiple binding sites for a single factor. The yeast HO gene contains nine highly conserved binding sites for the SCB (Swi4/6-dependent cell cycle box) binding factor (SBF) complex (composed of Swi4 and Swi6) in the 700-bp upstream regulatory sequence 2 (URS2) promoter region. Here, we show that the distal and proximal SBF sites in URS2 function differently. Chromatin immunoprecipitation (ChIP) experiments show that SBF binds preferentially to the left side of URS2 (URS2-L), despite equivalent binding to the left-half and right-half SBF sites in vitro. SBF binding at URS2-L sites depends on prior chromatin remodeling events at the upstream URS1 region. These signals from URS1 influence chromatin changes at URS2 but only at sites within a defined distance. SBF bound at URS2-L, however, is unable to activate transcription but instead facilitates SBF binding to sites in the right half (URS2-R), which are required for transcriptional activation. Factor binding at HO, therefore, follows a temporal cascade, with SBF bound at URS2-L serving to relay a signal from URS1 to the SBF sites in URS2-R that ultimately activate gene expression. Taken together, we describe a novel property of a transcription factor that can have two distinct roles in gene activation, depending on its location within a promoter.

Summary Gene-to-gene variation in the frequency of single nucleotide polymorphisms (SNPs) has been observed in humans, mice, rats, primates and pigs, but a relationship across species in this variation has not been described. Here, the frequency of porcine coding SNPs (cSNPs) identified by in silico methods, and the frequency of murine cSNPs, were compared with the frequency of human cSNPs across homologous genes. From 150 000 porcine expressed sequence tag (EST) sequences, a total of 452 SNP-containing sequence clusters were found, totalling 1394 putative SNPs. All the clustered porcine EST annotations and SNP data have been made publicly available at http://sputnik.btk.fi/project?name¼swine. Human and murine cSNPs were identified from dbSNP and were characterized as either validated or total number of cSNPs (validated plus nonvalidated) for comparison purposes. The correlation between in silico pig cSNP and validated human cSNP densities was found to be 0.77 (p < 0.00001) for a set of 25 homologous genes, while a correlation of 0.48 (p < 0.0005) was found for a primarily random sample of 50 homologous human and mouse genes. This is the first evidence of conserved gene-to-gene variability in cSNP frequency across species and indicates that site-directed screening of porcine genes that are homologous to cSNP-rich human genes may rapidly advance cSNP discovery in pigs. J. Anim. Breed. Genet. ISSN 0931-2668

Purpose Traumatic brain injury (TBI) frequently leads to chronic visual dysfunction. The purpose of this study was to investigate the effect of TBI on retinal ganglion cells (RGCs), and to test whether treatment with the novel neuroprotective compound P7C3-S243 could prevent in vivo functional deficits in the visual system. Methods Blast-mediated TBI was modeled using an enclosed over-pressure blast chamber. The RGC physiology was evaluated using a multielectrode array and pattern electroretinogram (PERG). Histological analysis of RGC dendritic field and cell number were evaluated at the end of the study. Visual outcome measures also were evaluated based on treatment of mice with P7C3-S243 or vehicle control. Results We show that deficits in neutral position PERG after blast-mediated TBI occur in a temporally bimodal fashion, with temporary recovery 4 weeks after injury followed by chronically persistent dysfunction 12 weeks later. This later time point is associated with development of dendritic abnormalities and irreversible death of RGCs. We also demonstrate that ongoing pathologic processes during the temporary recovery latent period (including abnormalities of RGC physiology) lead to future dysfunction of the visual system. We report that modification of PERG to provocative postural tilt testing elicits changes in PERG measurements that correlate with a key in vitro measures of damage: the spontaneous and light-evoked activity of RGCs. Treatment with P7C3-S243 immediately after injury and throughout the temporary recovery latent period protects mice from developing chronic visual system dysfunction. Conclusions Provocative PERG testing serves as a noninvasive test in the living organism to identify early damage to the visual system, which may reflect corresponding damage in the brain that is not otherwise detectable by noninvasive means. This provides the basis for developing an earlier diagnostic test to identify patients at risk for developing chronic CNS and visual system damage after TBI at an earlier stage when treatments may be more effective in preventing these sequelae. In addition, treatment with the neuroprotective agent P7C3-S243 after TBI protects from visual system dysfunction after TBI.

Schizophrenia is a chronic and severe psychiatric disorder that is highly heritable. While both common and rare genetic variants contribute to disease risk, many questions still remain about disease etiology. We performed a genome-wide analysis of copy number variants (CNVs) in 166 schizophrenia subjects and 52 psychiatrically healthy controls. First, overall CNV characteristics were compared between cases and controls. The only statistically significant finding was that deletions comprised a greater proportion of CNVs in cases. High interest CNVs were then identified as conservative using the following filtering criteria: (i) known deleterious CNVs; (ii) CNVs1 Mb that were novel (not found in a database of control individuals); and (iii) CNVs1 Mb that were novel and that overlapped the coding region of a gene of interest. Cases did not harbor a higher proportion of conservative CNVs in comparison to controls. However, similar to previous reports, cases had a slightly higher proportion of individuals with clinically significant CNVs (known deleterious or conservative CNVs1 Mb) or with multiple conservative CNVs. Two case individuals with the highest burden of conservative CNVs also share a recurrent 15q11.2 BP1-2 deletion, indicating a role for a potential multiple-hit CNV model for schizophrenia. In total, we report three 15q11.2 BP1-2 deletion individuals with schizophrenia, adding to a growing body of evidence that this CNV is involved in disease etiology.

Glaucoma is a group of diseases characterized by progressive excavation of the optic disc caused by loss of the retinal ganglion cell axons. It causes peripheral visual field loss and if untreated can lead to blindness; it is the second leading cause of legal blindness in the United States. Primary open-angle glaucoma (POAG), the most common form in Western populations, is insidious in onset and affects 1% to 2% of the population over age 40. When POAG is observed in individuals under the age of 40 it is called juvenile open-angle glaucoma (JOAG). Increased intraocular pressure is a well-documented risk factor, but not a diagnostic criterion, for POAG.1 More recently, reduced central corneal thickness (CCT) has been recognized as an important risk factor for glaucoma.2 Medical and surgical treatments aimed at reducing intraocular pressure may be effective in preventing progressive visual loss in POAG patients, but treatment is often not implemented until significant, unrecoverable vision loss has occurred due to a lack of symptoms in early disease and delayed diagnosis (see Ref. 3 for a review). Heritability estimates range from 0.36 to 0.57 for features of glaucoma, such as intraocular pressure and optic disc diameter, supporting the assertion that POAG has a strong genetic component.4 Linkage analysis studies in large families segregating POAG in Mendelian fashion have identified 14 loci for the disease5–23 (Table 1). Two causative genes have been identified through fine mapping of such linkage regions including myocilin (MYOC) at 1q23-q245,6 and optineurin (OPTN) at 10p13.11 Together, variants in these genes are estimated to account for approximately 5% of POAG in the population at large.11,24 Table 1. Known Glaucoma Loci Identified through Linkage Analysis In this study, we investigated copy number variants (CNVs) as potential glaucoma-causing variation in individuals with POAG ascertained at the University of Iowa. CNVs are operationally defined as genomic insertions or deletions that are larger than 1 kb and not a result of transposable elements. CNVs often escape detection in traditional studies of genetic variation, such as direct sequencing, single-strand conformation polymorphism analysis, and single-nucleotide polymorphism (SNP) association studies. CNVs are now recognized as a major contributor to human genetic variation and have been associated with other genetically complex disorders including autism, schizophrenia, HIV/AIDS susceptibility, and Crohn's disease.25–34 The eye is a highly specialized organ that has shown significant sensitivity to dosage changes of key developmental and regulatory genes, making glaucoma an excellent phenotype for CNV screening. Select copy number mutations have been known to play a role in glaucoma and related disorders of vision for some time, including deletions of LMXB1, FOXC1, and 4q34, among others.35–37 Gross karyotypic abnormalities of 9p23 and trisomies of chromosome 13 have been associated with developmental glaucoma.38–40 Recent fine mapping of 6p25, the FOXC1 locus, has provided evidence of a spectrum of mechanisms by which deletions and duplications of the FOXC1 gene occur.37,41,42 The initial clue that FOXC1 is involved in glaucoma was based on a chromosomal abnormality found in a single patient, demonstrating the value of identifying rare variants that may identify candidate genes and loci for disease causation. One recent study of 27 glaucoma patients and 12 controls analyzed by array comparative genome hybridization (CGH) methods found no CNVs in either patients or controls.43 However, the ability to detect disease-associated CNVs in this sample was limited due to (1) array CGH probe density (2) small sample size, and (3) single algorithm copy number calls. Before the present study, there have been no large-scale studies of copy number variation (CNV) in glaucoma. The development of software to analyze signal intensity data from high-density SNP-based array platforms, coupled with confirmation by quantitative PCR, enabled us to undertake a detailed cataloguing of CNVs in 400 POAG patients and 500 control subjects.

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.

DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues is difficult and requires special protocols in order to extract small amounts of DNA suitable for amplification. Most described methods report an amplification success rate between 60 and 80%; therefore, there is a need to improve molecular detection and identification of fungi in FFPE tissue. Eighty-one archived FFPE tissues with a positive Gomori methenamine silver (GMS) stain were evaluated using five different commercial DNA extraction kits with some modifications. Three different panfungal PCR assays were used to detect fungal DNA, and two housekeeping genes were used to assess the presence of amplifiable DNA and to detect PCR inhibitors. The sensitivities of the five extraction protocols were compared, and the quality of DNA detection (calculated for each kit as the number of housekeeping gene PCR-positive samples divided by the total number of samples) was 60 to 91% among the five protocols. The efficiencies of the three different panfungals used (calculated as the number of panfungal-PCR-positive samples divided by the number of housekeeping gene PCR-positive samples) were 58 to 93%. The panfungal PCR using internal transcribed spacer 3 (ITS3) and ITS4 primers yielded a product in most FFPE tissues. Two of the five DNA extraction kits (from TaKaRa and Qiagen) showed similar and promising results. However, one method (TaKaRa) could extract fungal DNA from 69 of the 74 FFPE tissues from which a housekeeping gene could be amplified and was also cost-effective, with a nonlaborious protocol. Factors such as sensitivity, cost, and labor will help guide the selection of the most appropriate method for the needs of each laboratory.

Schizophrenia is a neurodevelopmental psychiatric disorder characterized by a variety of structural brain abnormalities that appear to progress across the course of illness. Schizophrenia also is highly heritable, and one gene that has emerged as a possible susceptibility factor is G72. G72 influences brain development and activity by an as-yet unclear mechanism, and multiple studies have reported associations between G72 and schizophrenia. We were interested in linking these domains of investigation by determining whether G72 also influences the rate of longitudinal structural brain changes in individuals with schizophrenia. As part of the Iowa Longitudinal Study of Recent Onset Psychoses, we genotyped four G72 polymorphisms previously associated with schizophrenia in 110 subjects with schizophrenia or schizoaffective disorder from whom we had obtained two brain MRI scans an average of three years apart. The four polymorphisms captured three haplotypes, one of which was strongly associated with an increased rate of frontal lobe volume decrement. This same haplotype was also associated with more severe psychotic symptoms at the time of the second scan. These data thus suggest that variation in G72 modulates the progressive brain changes that characterize schizophrenia.

The rice genome has proven a valuable resource for comparative approaches to address individual genomic regions in Triticeae species at the molecular level. To exploit this resource for rye genetics and breeding, an inventory was made of EST-derived markers with known genomic positions in rye, which were related with those in rice. As a first inventory set, 92 EST-SSR markers were mapped which had been drawn from a non-redundant rye EST collection representing 5,423 unigenes and 2.2 Mb of DNA. Using a BC1 mapping population which involved an exotic rye accession as donor parent, these EST-SSR markers were arranged in a linkage map together with 25 genomic SSR markers as well as 131 AFLP and four STS markers. This map comprises seven linkage groups corresponding to the seven rye chromosomes and covers 724 cM of the rye genome. For comparative studies, additional inventory sets of EST-based markers were included which originated from the rye-mapping data published by other authors. Altogether, 502 EST-based markers with known chromosomal localizations in rye were used for BlastN search and 334 of them could be in silico mapped in the rice genome. Additionally, 14 markers were included which lacked sequence information but had been genetically mapped in rice. Based on the 348 markers, each of the seven rye chromosomes could be aligned with distinct portions of the rice genome, providing improved insight into the status of the rye–rice genome relationships. Furthermore, the aligned markers provide genomic anchor points between rye and rice, enabling the identification of conserved ortholog set markers for rye. Perspectives of rice as a model for genome analysis in rye are discussed.

The PAX6 gene is a transcription factor expressed early in development, predominantly in the eye, brain and gut. It is well known that mutations in PAX6 may result in aniridia, Peter’s anomaly and kertatisis. Here, we present mutation analysis of a patient with aniridia, autism and mental retardation. We identified and characterized a 1.3 Mb deletion that disrupts PAX6 transcriptional activity and deletes additional genes expressed in the brain. Our findings provide continued evidence for the role of PAX6 in neural phenotypes associated with aniridia.

The frequent comorbidity of Autism Spectrum Disorders (ASDs) with epilepsy suggests a shared underlying genetic susceptibility; several genes, when mutated, can contribute to both disorders. Recently, PRICKLE1 missense mutations were found to segregate with ASD. However, the mechanism by which mutations in this gene might contribute to ASD is unknown. To elucidate the role of PRICKLE1 in ASDs, we carried out studies in Prickle1(+/-) mice and Drosophila, yeast, and neuronal cell lines. We show that mice with Prickle1 mutations exhibit ASD-like behaviors. To find proteins that interact with PRICKLE1 in the central nervous system, we performed a yeast two-hybrid screen with a human brain cDNA library and isolated a peptide with homology to SYNAPSIN I (SYN1), a protein involved in synaptogenesis, synaptic vesicle formation, and regulation of neurotransmitter release. Endogenous Prickle1 and Syn1 co-localize in neurons and physically interact via the SYN1 region mutated in ASD and epilepsy. Finally, a mutation in PRICKLE1 disrupts its ability to increase the size of dense-core vesicles in PC12 cells. Taken together, these findings suggest PRICKLE1 mutations contribute to ASD by disrupting the interaction with SYN1 and regulation of synaptic vesicles.

The genome of the model plant Arabidopsis thaliana has been sequenced by an international collaboration, The Arabidopsis Genome Initiative. Here we report the complete sequence of chromosome 5. This chromosome is 26 megabases long; it is the second largest Arabidopsis chromosome and represents 21% of the sequenced regions of the genome. The sequence of chromosomes 2 and 4 have been reported previously and that of chromosomes 1 and 3, together with an analysis of the complete genome sequence, are reported in this issue. Analysis of the sequence of chromosome 5 yields further insights into centromere structure and the sequence determinants of heterochromatin condensation. The 5,874 genes encoded on chromosome 5 reveal several new functions in plants, and the patterns of gene organization provide insights into the mechanisms and extent of genome evolution in plants.

Each REXX implementation has a number of functions pertaining to the host environment. The MVS (OS/390) host environment commands described in this chapter are, with the exception of the GETDSN and STORAGE commands, equivalent CLIST statements. Other host environment commands may be invoked in the normal (REXX) way, see Sections 13.2 through 13.4.