A Frameshift Variant of GluN2A Identified in an Epilepsy Patient Results in NMDA Receptor Mistargeting.

N-methyl-D-aspartate receptors (NMDARs) are crucial for neuronal development and synaptic plasticity. Dysfunction of NMDARs is associated with multiple neurodevelopmental disorders, including epilepsy, autism spectrum disorder, and intellectual disability. Understanding the impact of genetic variants of NMDAR subunits can shed light on the mechanisms of disease. Here, we characterized the functional implications of a de novo mutation of the GluN2A subunit (P1199Rfs*32) resulting in the truncation of the C-terminal domain. The variant was identified in a male patient with epileptic encephalopathy, multiple seizure types, severe aphasia, and neurobehavioral changes. Given the known role of the CTD in NMDAR trafficking, we examined changes in receptor localization and abundance at the postsynaptic membrane using a combination of molecular assays in heterologous cells and rat primary neuronal cultures. We observed that the GluN2A P1199Rfs*32-containing receptors traffic efficiently to the postsynaptic membrane but have increased extra-synaptic expression relative to WT GluN2A-containing NMDARs. Using in silico predictions, we hypothesized that the mutant would lose all PDZ interactions, except for the recycling protein Scribble1. Indeed, we observed impaired binding to the scaffolding protein postsynaptic protein-95 (PSD-95); however, we found the mutant interacts with Scribble1, which facilitates the recycling of both the mutant and the WT GluN2A. Finally, we found that neurons expressing GluN2A P1199Rfs*32 have fewer synapses and decreased spine density, indicating compromised synaptic transmission in these neurons. Overall, our data show that GluN2A P1199Rfs*32 is a loss-of-function variant with altered membrane localization in neurons and provide mechanistic insight into disease etiology.

Cross-regulation of Connexin43 and ß-catenin influences differentiation of human neural progenitor cells.

Connexin43 (Cx43) is the most widely and abundantly expressed gap junction (GJ) protein and it is strongly associated with the regulation of cell cycle progression. Emerging roles for Cx43 in cell adhesion and migration during neural differentiation have also been recently recognized, and this has emphasized the involvement of Cx43 in different physiological process beyond its role as a GJ protein. In this study, we explore the function of Cx43 in the differentiation of human neural progenitor cells (hNPCs) using viral vectors that mediate the overexpression or knockdown of the protein. Results showed that in the absence of this protein fetal cortex-derived hNPCs differentiated toward a neuronal phenotype at expenses of a glial phenotype. Furthermore, the silencing of Cx43 did not affect hNPC proliferation rate or numbers of apoptotic cells. The increase in the number of neurons was not recapitulated when GJ intercellular communications were pharmacologically blocked, and this suggested that Cx43 was influencing hNPCs differentiation with a GJ-independent effect. In addition, Cx43 knockdown significantly increased ß-catenin signaling, which has been shown to regulate the transcription of pro-neuronal genes during embryonic neural development. Our results add further support to the hypothesis that Cx43 protein itself regulates key signaling pathways during development and neurogenesis beyond its role as GJ protein.

Parkinson's disease in a dish - Using stem cells as a molecular tool.

Parkinson's disease (PD) is the second most common neurodegenerative disease, with a strong genetic component to both the familial and sporadic forms. The cardinal motor symptoms of the disease result from the loss of dopamine (DA) neurons in the midbrain. There is currently no cure for PD and improved methods for modelling the disease are required in order to develop more effective therapeutic interventions. Patient-derived induced pluripotent stem cells (iPSCs) carry the genetic background of the donor, enabling accurate modelling of genetic diseases in vitro. Various human iPSCs from patients suffering different genetic forms of PD have been differentiated into DA neurons and demonstrated signs of the pathophysiology of PD in vitro. The examination of key cellular pathways such as calcium regulation and autophagy indicate that disease-associated genetic variants may have important implications for cellular function. This review examines and critiques how DA neurons from patient iPSCs have been used to model PD in vitro, and what iPSCs might hold for the future of PD research. This article is part of the Special Issue entitled 'The Synaptic Basis of Neurodegenerative Disorders'.

Enhancing a cancer prevention and control curriculum through interactive group discussions.

The Principles and Practice of Cancer Prevention and Control course (Principles course) is offered annually by the National Cancer Institute Cancer Prevention Fellowship Program. This 4-week postgraduate course covers the spectrum of cancer prevention and control research (e.g., epidemiology, laboratory, clinical, social, and behavioral sciences) and is open to attendees from medical, academic, government, and related institutions across the world. In this report, we describe a new addition to the Principles course syllabus, which was exclusively a lecture-based format for over 20 years. In 2011, cancer prevention fellows and staff designed and implemented small group discussion sessions as part of the curriculum. The goals of these sessions were to foster an interactive environment, discuss concepts presented during the Principles course, exchange ideas, and enhance networking among the course participants and provide a teaching and leadership opportunity to current cancer prevention fellows. Overall, both the participants and facilitators who returned the evaluation forms (n=61/87 and 8/10, respectively) reported a high satisfaction with the experience for providing both an opportunity to explore course concepts in a greater detail and to network with colleagues. Participants (93%) and facilitators (100%) stated that they would like to see this component remain a part of the Principles course curriculum, and both groups provided recommendations for the 2012 program. The design, implementation, and evaluation of this initial discussion group component of the Principles course are described herein. The findings in this report will not only inform future discussion group sessions in the Principles course but may also be useful to others planning to incorporate group learning into large primarily lecture-based courses.

16 kDa prolactin reduces angiogenesis, but not growth of human breast cancer tumors in vivo.

Prolactin (PRL) is a peptide hormone necessary for normal growth and development of the human breast. In addition, high levels of PRL in plasma correlate with increased risk of breast cancer, especially among postmenopausal women. Several isoforms of PRL exist in human circulation, including a 16 kDa isoform that is an N-terminal fragment of the full-length 23 kDa PRL. 16 kDa PRL has been shown to be anti-angiogenic in vitro and in vivo, and to reduce formation of tumors from prostate, colon and melanoma cancer cell lines. Here we explore the effect of 16 kDa PRL expression in vitro and in vivo using two breast cancer cell line models (MCF-7 and MDA-MB-231) and also the HCT-116 colon cancer cell line. In all three cell lines, 16 kDa PRL expression inhibited cell proliferation in vitro compared to empty vector controls. In vivo results were markedly different between the two types of cell lines. HCT-116 cells expressing 16 kDa PRL exhibited reduced vascularization and tumor formation, consistent with published results. The breast cancer cell lines expressing 16 kDa PRL also exhibited inhibition of angiogenesis in vivo but no reduction in tumor size or formation. These results suggest that the effects of 16 kDa PRL on tumor formation may vary across tissue types. The unique sensitivity of breast cancer to PRL as a mitogen and/or additional factors in the mammary gland environment (e.g. local hormone/mitogen concentration) may play a dominant role in tumor formation in vivo, thus outweighing the anti-angiogenesis effects and in vitro reduction in cell proliferation induced by 16 kDa PRL.

Prolactin serum levels and breast cancer: relationships with risk factors and tumour characteristics among pre- and postmenopausal women in a population-based case-control study from Poland.

BACKGROUND: Previous prospective studies have found an association between prolactin (PRL) levels and increased risk of breast cancer. Using data from a population-based breast cancer case-control study conducted in two cities in Poland (2000-2003), we examined the association of PRL levels with breast cancer risk factors among controls and with tumour characteristics among the cases. METHODS: We analysed PRL serum levels among 773 controls without breast cancer matched on age and residence to 776 invasive breast cancer cases with available pretreatment serum. Tumours were centrally reviewed and prepared as tissue microarrays for immunohistochemical analysis. Breast cancer risk factors, assessed by interview, were related to serum PRL levels among controls using analysis of variance. Mean serum PRL levels by tumour characteristics are reported. These associations also were evaluated using polytomous logistic regression. RESULTS: Prolactin levels were associated with nulliparity in premenopausal (P=0.05) but not in postmenopausal women. Associations in postmenopausal women included an inverse association with increasing body mass index (P=0.0008) and direct association with use of recent/current hormone therapy (P=0.0006). In case-only analyses, higher PRL levels were more strongly associated with lobular compared with ductal carcinoma among postmenopausal women (P=0.02). Levels were not different by tumour size, grade, node involvement or oestrogen receptor, progesterone receptor, or human epidermal growth factor receptor 2 status. CONCLUSIONS: Our analysis demonstrates that PRL levels are higher among premenopausal nulliparous as compared with parous women. Among postmenopausal women, levels were higher among hormone users and lower among obese women. These results may have value in understanding the mechanisms underlying several breast cancer risk factor associations.

A new approach to measuring estrogen exposure and metabolism in epidemiologic studies.

Endogenous estrogen plays an integral role in the etiology of breast and endometrial cancer, and conceivably ovarian cancer. However, the underlying mechanisms and the importance of patterns of estrogen metabolism and specific estrogen metabolites have not been adequately explored. Long-standing hypotheses, derived from laboratory experiments, have not been tested in epidemiologic research because of the lack of robust, rapid, accurate measurement techniques appropriate for large-scale studies. We have developed a stable isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS(2)) method that can measure concurrently all 15 estrogens and estrogen metabolites (EM) in urine and serum with high sensitivity (level of detection=2.5-3.0fmol EM/mL serum), specificity, accuracy, and precision [laboratory coefficients of variation (CV's) < or =5% for nearly all EM]. The assay requires only extraction, a single chemical derivatization, and less than 0.5mL of serum or urine. By incorporating enzymatic hydrolysis, the assay measures total (glucuronidated+sulfated+unconjugated) EM. If the hydrolysis step is omitted, the assay measures unconjugated EM. Interindividual differences in urinary EM concentrations (pg/mL creatinine), which reflect total EM production, were consistently large, with a range of 10-100-fold for nearly all EM in premenopausal and postmenopausal women and men. Correlational analyses indicated that urinary estrone and estradiol, the most commonly measured EM, do not accurately represent levels of total urinary EM or of the other EM. In serum, all 15 EM were detected as conjugates, but only 5 were detected in unconjugated form. When we compared our assay methods with indirect radioimmunoassays for estrone, estradiol, and estriol and enzyme-linked immunosorbent assays for 2-hydroxyestrone and 16alpha-hydroxyestrone, ranking of individuals agreed well for premenopausal women [Spearman r (r(s))=0.8-0.9], but only moderately for postmenopausal women (r(s)=0.4-0.8). Our absolute readings were consistently lower, especially at the low concentrations characteristic of postmenopausal women, possibly because of improved specificity. We are currently applying our EM measurement techniques in several epidemiologic studies of premenopausal and postmenopausal breast cancer.

Candidate gene analysis of 21q22: support for S100B as a susceptibility gene for bipolar affective disorder with psychosis.

A genome-wide scan in 60 bipolar affective disorder (BPAD) affected sib-pairs (ASPs) identified linkage on chromosome 21 at 21q22 (D21S1446, NPL = 1.42, P = 0.08), a BPAD susceptibility locus supported by multiple studies. Although this linkage only approaches significance, the peak marker is located 12 Kb upstream of S100B, a neurotrophic factor implicated in the pathology of psychiatric disorders, including BPAD and schizophrenia. We hypothesized that the linkage signal at 21q22 may result from pathogenic disease variants within S100B and performed an association analysis of this gene in a collection of 125 BPAD type I trios. S100B single nucleotide polymorphisms (SNPs) rs2839350 (P = 0.022) and rs3788266 (P = 0.031) were significantly associated with BPAD. Since variants within S100B have also been associated with schizophrenia susceptibility, we reanalyzed the data in trios with a history of psychosis, a phenotype in common between the two disorders. SNPs rs2339350 (P = 0.016) and rs3788266 (P = 0.009) were more significantly associated in the psychotic subset. Increased significance was also obtained at the haplotype level. Interestingly, SNP rs3788266 is located within a consensus-binding site for Six-family transcription factors suggesting that this variant may directly affect S100B gene expression. Fine-mapping analyses of 21q22 have previously identified transient receptor potential gene melastatin 2 (TRPM2), which is 2 Mb upstream of S100B, as a possible BPAD susceptibility gene at 21q22. We also performed a family-based association analysis of TRPM2 which did not reveal any evidence for association of this gene with BPAD. Overall, our findings suggest that variants within the S100B gene predispose to a psychotic subtype of BPAD, possibly via alteration of gene expression.

Genome-wide scan of bipolar disorder and investigation of population stratification effects on linkage: support for susceptibility loci at 4q21, 7q36, 9p21, 12q24, 14q24, and 16p13.

Bipolar disorder (BPD) is a complex genetic disorder with cycling symptoms of depression and mania. Despite the extreme complexity of this psychiatric disorder, attempts to localize genes which confer vulnerability to the disorder have had some success. Chromosomal regions including 4p16, 12q24, 18p11, 18q22, and 21q21 have been repeatedly linked to BPD in different populations. Here we present the results of a whole genome scan for linkage to BPD in an Irish population. Our most significant result was at 14q24 which yielded a non-parametric LOD (NPL) score of 3.27 at the D14S588 marker with a nominal P-value of 0.0006 under a narrow (bipolar type I only) model of affection. We previously reported linkage to 14q22-24 in a subset of the families tested in this analysis. We also obtained suggestive evidence for linkage at 4q21, 9p21, 12q24, and 16p13, chromosomal regions that have all been previously linked to BPD. Additionally, we report on a novel approach to linkage analysis, STRUCTURE-Guided Linkage Analysis (SGLA), which is designed to reduce genetic heterogeneity and increase the power to detect linkage. Application of this technique resulted in more highly significant evidence for linkage of BPD to three regions including 16p13, a locus that has been repeatedly linked to numerous psychiatric disorders.

Structural analysis of a set of proteins resulting from a bacterial genomics project.

The targets of the Structural GenomiX (SGX) bacterial genomics project were proteins conserved in multiple prokaryotic organisms with no obvious sequence homolog in the Protein Data Bank of known structures. The outcome of this work was 80 structures, covering 60 unique sequences and 49 different genes. Experimental phase determination from proteins incorporating Se-Met was carried out for 45 structures with most of the remainder solved by molecular replacement using members of the experimentally phased set as search models. An automated tool was developed to deposit these structures in the Protein Data Bank, along with the associated X-ray diffraction data (including refined experimental phases) and experimentally confirmed sequences. BLAST comparisons of the SGX structures with structures that had appeared in the Protein Data Bank over the intervening 3.5 years since the SGX target list had been compiled identified homologs for 49 of the 60 unique sequences represented by the SGX structures. This result indicates that, for bacterial structures that are relatively easy to express, purify, and crystallize, the structural coverage of gene space is proceeding rapidly. More distant sequence-structure relationships between the SGX and PDB structures were investigated using PDB-BLAST and Combinatorial Extension (CE). Only one structure, SufD, has a truly unique topology compared to all folds in the PDB.

Deposit3D: a tool for automating structure depositions to the Protein Data Bank.

Almost all successful protein structure-determination projects in the public sector culminate in a structure deposition to the Protein Data Bank (PDB). In order to expedite the deposition process, Deposit3D has been developed. This command-line script calculates or gathers all the required structure-deposition information and outputs this data into a mmCIF file for subsequent upload through the RCSB PDB ADIT interface. Deposit3D might be particularly useful for structural genomics pipeline projects because it allows workers involved with various stages of a structure-determination project to pool their different categories of annotation information before starting a deposition session.

EGF/ErbB receptor family in ovarian cancer.

In summary, the EGF/ErbB family of receptor tyrosine kinases has been shown to play a key role in normal ovarian follicle development, and cell growth regulation of the ovarian surface epithelium. Disregulation of these normal growth regulatory pathways, including overexpression and/or mutation of EGFR/ErbB receptor family members, as well as elements of their downstream signalling pathways, have been shown to contribute to the etiology and progression of epithelial ovarian cancer. It is, therefore, not surprising that these gene products, and their related soluble receptor isoforms may have clinical utility as tumor and/or serum biomarkers of disease activity. Moreover, since several of these soluble receptor isoforms have potent growth inhibitory activity, and are naturally occurring in the circulation, they are ideal candidates for the development of novel therapeutics for the treatment of ovarian cancer patients.

Escherichia coli K1 purA and sorC are preferentially expressed upon association with human brain microvascular endothelial cells.

In order to better understand the events that allow Escherichia coli K1 to cross the blood-brain barrier we used differential fluorescence induction to identify bacterial genes that are preferentially expressed when associated with human brain microvascular endothelial cells (HBMEC), which comprise the blood-brain barrier. Random gene fusions of E. coli K1 DNA were created in a promoterless gfp vector and gene fusion libraries were incubated with and without HBMEC. The cells were subjected to a series of fluorescence-activated cell sorting screens to identify promoter fusions which lead to fluorescence when bacteria were associated with HBMEC, yet not fluorescent when grown in media alone. Two genes were identified, purA (encodes adenylosuccinate synthetase) and a sorC homologue (encodes a member of the sorC family of transcriptional regulators), whose expression were preferentially induced when bacteria were associated with eukaryotic cells. Individual gene disruption mutants of E. coli K1 purA and sorC demonstrated significantly decreased HBMEC invasion phenotype in vitro, when compared to the wild-type strain, and could be complemented when the respective wild-type sequences were supplied in trans. The purA and sorC mutants were deficient in their ability to grow in defined minimal media, without adenine, and with sorbose as sole carbon source, respectively, yet capable of normal growth in complex media. We have identified novel phenotypes associated with E. coli K1 purA and sorC, which provide evidence that these genes contribute to the invasion of HBMEC.

Analysis of codon usage patterns of bacterial genomes using the self-organizing map.

Codon usage varies both between organisms and between different genes in the same organism. This observation has been used as a basis for earlier work in identifying highly expressed and horizontally transferred genes in Escherichia coli. In this work, we applied Kohonen's self-organizing map to analysis of the codon usage pattern of the Escherichia coli, Aquifex aeolicus, Archaeoglobus fulgidus, Haemophilus influenzae RD:, Methanococcus jannaschii, Methanobacterium thermoautotrophicum, and Pyrococcus horikoshii genomes for evidence of highly expressed genes and horizontally transferred genes. All of the analyzed genomes had a clear category of horizontally transferred genes, and their apparent percentages ranged from 7.7% to 21.4%. The apparent percentage of highly expressed genes ranges from 0% to 11.8%. A clustering of average codon usage of main gene categories of the seven genomes showed an interesting mixing of gene classes in four thermophilic/hyperthermophilic organisms, A. aeolicus, A. fulgidus, M. thermoautotrophicum, and P. horikoshii, which suggests possible origins of their horizontally transferred genes as well as the need for adaptation to a specific environment. Further classification of the three gene categories in E. coli and H. influenzae according to gene function revealed that genes involved in communication (such as regulation and cell process) and structure (cell structure and structural proteins) are more likely to be horizontally transferred than are genes involved in information (transcription, translation, and related processes) and in some groups of energy (such as energy metabolism and carbon compound catabolism).

An information-based sequence distance and its application to whole mitochondrial genome phylogeny.

MOTIVATION: Traditional sequence distances require an alignment and therefore are not directly applicable to the problem of whole genome phylogeny where events such as rearrangements make full length alignments impossible. We present a sequence distance that works on unaligned sequences using the information theoretical concept of Kolmogorov complexity and a program to estimate this distance. RESULTS: We establish the mathematical foundations of our distance and illustrate its use by constructing a phylogeny of the Eutherian orders using complete unaligned mitochondrial genomes. This phylogeny is consistent with the commonly accepted one for the Eutherians. A second, larger mammalian dataset is also analyzed, yielding a phylogeny generally consistent with the commonly accepted one for the mammals. AVAILABILITY: The program to estimate our sequence distance, is available at http://www.cs.cityu.edu.hk/~cssamk/gencomp/GenCompress1.htm. The distance matrices used to generate our phylogenies are available at http://www.math.uwaterloo.ca/~mli/distance.html.

Bacterial invasion and transcytosis in transfected human brain microvascular endothelial cells.

Most cases of neonatal bacterial meningitis develop as a result of a hematogenous spread, but it is not clear how circulating bacteria cross the blood-brain barrier. Attempts to answer these questions have been hampered by the lack of a reliable model of the human blood-brain barrier. Human brain microvascular endothelial cells (HBMEC) were isolated and transfected with a pBR322 based plasmid containing simian virus 40 large T antigen (SV40-LT). The transfected HBMEC exhibited similar brain endothelial cell characteristics as the primary HBMEC, i.e. gamma glutamyl transpeptidase and a high transendothelial electrical resistance. Escherischia coli and Citrobacter spp, two important Gram-negative bacilli causing neonatal meningitis, were found to transcytose across primary and transfected HBMEC, without affecting the integrity of the monolayer. In addition, E. coli and C. freundii invaded transfected HBMEC as shown previously with primary HBMEC. We conclude that E. coli and C. freundii are able to invade and transcytose HBMEC and these bacterial-HBMEC interactions are similar between primary and transfected HBMEC. Therefore, our transfected HBMEC should be useful for studying pathogenesis of CNS infections.

Yersinia enterocolitica ClpB affects levels of invasin and motility.

Expression of the Yersinia enterocolitica inv gene is dependent on growth phase and temperature. inv is maximally expressed at 23 degrees C in late-exponential- to early-stationary-phase cultures. We previously reported the isolation of a Y. enterocolitica mutant (JB1A8v) that shows a decrease in invasin levels yet is hypermotile when grown at 23 degrees C. JB1A8v has a transposon insertion within uvrC. Described here is the isolation and characterization of a clone that suppresses these mutant phenotypes of the uvrC mutant JB1A8v. This suppressing clone encodes ClpB (a Clp ATPase homologue). The Y. enterocolitica ClpB homologue is 30 to 40% identical to the ClpB proteins from various bacteria but is 80% identical to one of the two ClpB homologues of Yersinia pestis. A clpB::TnMax2 insertion mutant (JB69Qv) was constructed and determined to be deficient in invasin production and nonmotile when grown at 23 degrees C. Analysis of inv and fleB (flagellin gene) transcript levels in JB69Qv suggested that ClpB has both transcriptional and posttranscriptional effects. In contrast, a clpB null mutant, BY1v, had no effect on invasin levels or motility. A model accounting for these observations is presented.

Application of signature-tagged mutagenesis for identification of escherichia coli K1 genes that contribute to invasion of human brain microvascular endothelial cells.

Escherichia coli K1 is the leading cause of gram-negative bacterial meningitis in neonates. It is principally due to our limited understanding of the pathogenesis of this disease that the morbidity and mortality rates remain unacceptably high. To identify genes required for E. coli K1 penetration of the blood-brain barrier (BBB), we used the negative selection strategy of signature-tagged transposon mutagenesis (STM) to screen mutants for loss or decreased invasion of human brain microvascular endothelial cells (HBMEC) which comprise the BBB. A total of 3,360 insertion mutants of E. coli K1 were screened, and potential HBMEC invasion mutants were subjected to a secondary invasion screen. Those mutants that failed to pass the serial invasion screens were then tested individually. Seven prototrophic mutants were found to exhibit significantly decreased invasive ability in HBMEC. We identified traJ and five previously uncharacterized loci whose gene products are necessary for HBMEC invasion by E. coli K1. In addition, cnf1, a gene previously shown to play a role in bacterial invasion, was identified. More importantly, a traJ mutant was attenuated in penetration of the BBB in the neonatal rat model of experimental hematogenous meningitis. This is the first in vivo demonstration that traJ is involved in the pathogenesis of E. coli K1 meningitis.