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PubMed2006

Shotgun proteomics of cyanobacteria--applications of experimental and data-mining techniques.

Cyanobacteria are photosynthetic bacteria notable for their ability to produce hydrogen and a variety of interesting secondary metabolites. As a result of the growing number of completed cyanobacterial genome projects, the development of post-genomics analysis for this important group has been accelerating. DNA microarrays and classical two-dimensional gel electrophoresis (2DE) were the first technologies applied in such analyses. In many other systems, 'shotgun' proteomics employing multi-dimensional liquid chromatography and tandem mass spectrometry has proven to be a powerful tool. However, this approach has been relatively under-utilized in cyanobacteria. This study assesses progress in cyanobacterial shotgun proteomics to date, and adds a new perspective by developing a protocol for the shotgun proteomic analysis of the filamentous cyanobacterium Anabaena variabilis ATCC 29413, a model for N(2) fixation. Using approaches for enhanced protein extraction, 646 proteins were identified, which is more than double the previous results obtained using 2DE. Notably, the improved extraction method and shotgun approach resulted in a significantly higher representation of basic and hydrophobic proteins. The use of protein bioinformatics tools to further mine these shotgun data is illustrated through the application of PSORTb for localization, the grand average hydropathy (GRAVY) index for hydrophobicity, LipoP for lipoproteins and the exponentially modified protein abundance index (emPAI) for abundance. The results are compared with the most well-studied cyanobacterium, Synechocystis sp. PCC 6803. Some general issues in shotgun proteome identification and quantification are then addressed.

Computational Biology↗
PubMedDate not supplied

Low peripheral blood viral HIV-2 RNA in individuals with high CD4 percentage differentiates HIV-2 from HIV-1 infection.

OBJECTIVES: To elucidate why the virulence of HIV-1 and HIV-2 infections differ in West African populations. STUDY DESIGN/METHOD: Peripheral blood plasma virion RNA and cellular proviral DNA levels were measured in a cross-section of 59 HIV-1 and 49 HIV-2 singly infected individuals representing all stages of infection in The Gambia, West Africa. Novel reverse transcriptase polymerase chain reaction (RT-PCR) assays specific and sensitive for virus quantification of non-clade B HIV-1 and HIV-2 infections were used. RESULTS: HIV-1 and HIV-2 proviral and plasma RNA levels were inversely correlated with CD4+ count for both infections with cellular proviral load similar at each stage of infection. Critically, up to three-fourths of HIV-2-infected individuals with high CD4 percentages (> 28%) had undetectable (< 500 copies/mL) levels of peripheral blood HIV-2 RNA in contrast to HIV-1-infected individuals who had readily detectable plasma virus at all stages of infection (P < .0001). Plasma RNA levels were similar in the intermediate and end stages of infection, indicating similar replication potential for both viruses. In the cross-section of HIV-1- and HIV-2-infected patients studied, the data indicate a wider dynamic range of HIV-2 RNA in vivo compared with HIV-1. DISCUSSION: Low levels of HIV-2 replication and virion expression characterize individuals with high CD4+ lymphocyte counts, suggesting that a very different dynamic equilibrium exists between virus and host for HIV-2 compared with HIV-1. By analogy with HIV-1, our data implicate a considerably lower turnover of HIV-2 virion RNA in vivo with a markedly reduced production of infectious genomes in individuals during the subclinical phase of infection. CONCLUSION: The lower levels of virion expression of HIV-2 infections in vivo are compatible with observed differences in the natural history of HIV-1 and HIV-2 infections, relating to overall differences in the pathogenesis and disease progression of the two infections.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Natural interferon-alpha versus its combination with 6-methyl-prednisolone in the therapy of type II mixed cryoglobulinemia: a long-term, randomized, controlled study.

Type II mixed cryoglobulinemia (MC) is an often progressive vasculitis characterized by circulating cold-precipitable proteins that usually consists of polyclonal IgG and monoclonal IgM kappa with rheumatoid factor (RF) activity. Its etiology is unknown, although recent evidence strongly suggests that hepatitis C virus (HCV) plays a major role. Plasmapheresis, corticosteroids, and cytotoxic drugs have been used in the therapy of MC patients. Recently, favorable results with recombinant interferon-alpha (rIFN alpha) have been reported. To further assess its effectiveness, we studied the effects of natural human interferon-alpha (nIFN alpha), alone and in combination with 6-methyl-prednisolone (PDN), in a prospective, randomized, controlled trial in patients with symptomatic MC. Sixty-five patients were enrolled onto the trial, 52 (80%) of whom presented serum anti-HCV antibodies and specific genomic RNA sequences. Fifteen patients received nIFN alpha (3 MU) intramuscularly (IM) three times weekly, whereas 17 patients also received 16 mg/d of PDN orally on non-IFN days. Moreover, 18 patients received 16 mg/d of PDN only, and 15 were untreated. Treatment was discontinued after 1 year and patients were monitored for 8 to 17 months (mean, 13). A complete response was achieved in eight of 15 patients (53.3%) treated with nIFN alpha and nine of 17 (52.9%) treated with nIFN alpha plus PDN, as compared with three of 18 patients (16.7%) who received PDN only (P < .05) and one of 15 (6.7%) untreated controls (P < .01). Partial response occurred in two of 15 (13.3%) patients treated with nIFN alpha, three of 17 (17.6%) who received nIFN alpha plus PDN, one of 18 (5.5%) who received PDN only, and one of 15 (6.7%) controls. A complete response in six patients (66.7%) was achieved within 3 months in the group that received nIFN alpha plus PDN, as compared with two patients (25%) of those who received nIFN alpha alone (P < .02). In anti-HCV-positive patients, the clinical response occurred in step with reduced or undetectable levels of HCV RNA and transaminase normalization. Quantification of circulating HCV RNA represented a good predictive response marker. The probability of relapse within 3 months after treatment was 100% (three of three patients) and 75% (six of eight patients), respectively, in patients who received PDN alone or nIFN alpha alone as compared with none of those who received nIFN alpha plus PDN (P < .001).(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed2003

Quantification of expression and methylation of the Igf2r imprinted gene in segmental trisomic mouse model.

Insulin-like growth factor 2 receptor (Igf2r), a maternally expressed imprinted gene, is triplicated in Ts43H trisomic mice. To verify its expression in a trisomic mode, we examined the allele-specific transcripts and relative levels of Igf2r mRNA in trisomic and control mice. Igf2r was expressed from the maternal allele(s) and silenced on the paternal allele(s) in most tissues of maternally or paternally derived Ts43H mice. The triallelic expression was observed in adult brain and testis and in embryonic head, but the antisense transcript, Air, was strictly paternal, and methylation of region 2 was strictly maternal in all tissues. The Igf2r expression in maternally derived trisomics, with two active copies of the gene, did not exceed the average mRNA levels of euploid controls with monoallelic expression. Thus, an indication is presented for a dosage compensation mechanism of Igf2r in the trisomic context.

Animals↗
PubMed2005

The negative regulator of Borna disease virus polymerase is a non-structural protein.

The X protein of Borna disease virus (BDV) negatively regulates viral polymerase activity. With a BDV mini-replicon system, 30 % inhibition of polymerase activity was observed at an X to phosphoprotein (P) plasmid ratio of 1:6 and 100 % inhibition at a ratio of 1:1. It was therefore hypothesized that (i) the X:P ratio in infected cells is not significantly higher than 1:6 to prevent complete inhibition of polymerase activity and (ii) X is not efficiently incorporated into viral particles, allowing efficient replication early in infection. To test these assumptions, a monoclonal antibody directed against BDV X was generated. Immunofluorescence analysis revealed co-localization of X with the nucleoprotein (N) and P in the nucleus, as well as in the cytoplasm of BDV-infected cells. Quantification of viral protein levels by Western blot analysis, using purified Escherichia coli-derived X, P and N as protein standards, revealed an X:P:N ratio in BDV-infected cells of approximately 1:6:40. However, only traces of X could be detected in purified BDV stock, suggesting that X is excluded from virus particles. These results indicate that X is a non-structural protein. The lack of X in virus particles may facilitate polymerase activity early in infection; however, the presence of X in persistently infected cells may result in partial inhibition of the polymerase and thus contribute to viral persistence.

Animals↗
PubMed2005

Detection of hepatitis B virus genotypes and mutants: current status.

Characterization results of the hepatitis B virus (HBV) genome before, during and after antiviral treatment have changed in the last year for a number of reasons. First of all, with the introduction of more nucleoside and nucleotide antiviral therapies, it has become clear that variants or mutants do emerge in time. Viral genomic changes in the HBV polymerase gene can result in a direct antiviral effect, but compensatory mutations can also be identified during prolonged treatment periods. Furthermore, there is an increasing number of reports suggesting that HBV genotypes can be related to, for example, disease progression or the effect of antiviral treatment itself (alpha-interferon or lamivudine). Combined with HBV DNA viral load monitoring, an increase in viral load or a limited reduction during treatment is indicative of genomic changes related to resistance. However, these genomic changes can also be present in the absence of an increase in HBV DNA. Methodologies for the detection of these variants, as well as the determination of genotypes, are rather straightforward. Sequence analysis is time-consuming and expensive, but provides the most information, particularly if not all information on mutations related to antiviral resistance is known. However, the sensitivity of direct sequencing for the presence of minor variant populations is poor, and no mixtures of variant populations are, in general, detected. The ability to detect minor populations and, if possible, even quantify them, gives more insightful information on the dynamic evolution of the virus itself. Antiviral treatment can result in the appearance of more than one population of variants, which can be present for a prolonged period of time, and even remain undetected with current technologies. However, screening specifically for these variant populations before starting treatment for so-called untreated patients (who have received no antiviral treatment for, e.g., 6 months) has already shown that the effects of treatment can be biased. Furthermore, the detection of more dynamic viral populations - including both wild-type and resistant variants - during, but also after therapy, does provide helpful information in the analysis of virological data. Technologies enabling the detection and quantification of these variant populations are presented and discussed.

Antiviral Agents↗
PubMed2025

Patterns of HIV-1 viral load suppression and drug resistance during the dolutegravir transition: a population-based longitudinal study.

BACKGROUND: Data on the population-scale impact of dolutegravir (DTG)-based HIV regimens in sub-Saharan Africa are extremely limited. We used data from a surveillance cohort in southern Uganda to assess viral suppression and antiretroviral (ART) resistance over 10-years alongside DTG scale-up. METHODS: Consenting participants in the population-based Rakai Community Cohort Study between August 2011 and March 2023 aged 15-59 completed questionnaires and provided samples for HIV testing, viral load quantification, and viral deep-sequencing. We collected data on DTG-utilization at HIV care clinics. We estimated the prevalence of HIV suppression (<1,000 copies/mL) and ART resistance using robust Poisson regression. Bayesian logistic regression quantified associations between resistance and individual-level suppression across surveys. FINDINGS: Among 20,383 people living with HIV (PLHIV), suppression increased from 57.1% (95% confidence interval [CI]: 55.4%-58.8%) to 90.3% (95%CI: 89.2%-91.4%) between 2014 and 2022. By 2020 84.4% (95%CI: 83.7%-85.2%) and 64.6% (95%CI: 63.9%-65.3%) of men and women were on DTG regimens. Among treatment-experienced viremic PLHIV, overall resistance decreased from 51.1% (95%CI: 40.7%-64.1%, 2014) to 27.9% (95%CI: 21.3%-36.5%, 2022). Only two participants harbored intermediate/high-level DTG resistance, attributable to inQ148R, inE138K, and inG140A. Low-level INSTI resistance (inS153Y) was observed in 23/207 (7.5%) of viremic individuals, with putative evidence of transmission. By 2022, suppression was unrelated to prior history of NNRTI/NRTI resistance (risk ratios: 1.14, 95%HPD: 0.96-1.32 and 1.12, 95%HPD: 0.88 - 1.35). INTERPRETATION: Viral suppression increased during the DTG-transition with minimal emerging intermediate/high-level resistance. Falling resistance among treatment-experienced PLHIV underscores the role of ART adherence in reducing viremia. The emergence of inS153Y justifies continued genomic surveillance of ART resistance. FUNDING: National Institutes of Health and the Gates Foundation.

Journal Article↗
PubMed1995

[The quantification of human immunodeficiency virus viremia in patients with rapid and slow progression of the disease].

BACKGROUND: The natural course of the human immunodeficiency virus type 1 (HIV-1) infection is very variable. The factors which appear to determine the speed of immunodeficiency progression are multiple, although the virulence of the predominant viral strain seems to be one the main factors. The plasmatic viremia in individuals with rapid and slow HIV-1 progression was analyzed in an attempt to establish the degree of correlation between HIV-1 replication and the natural course of the disease. METHODS: Forty-two samples from 34 seropositive patients, 11 with rapid progression criteria (< 5 years from acute infection and CD4+ lymphocytes < 0.2 x 10(9)/l) and 23 with slow progression (> 7 years from demonstrated infection and > 0.5 x 10(9) CD4+ lymphocytes/l) were studied. The plasmatic viremia was quantified by a new method of plasma DNA genetic amplification, denominated the branched DNA (bDNA) technique. As a reference circulating p24 was determined and the presence of several proviral regions were studied in peripheral blood lymphocytes by polymerase chain reaction (PCR). RESULTS: The presence of RNA molecules was detected in plasma of 7 (58.3%) out of 12 samples of rapid progression (RP) patients by bDNA. To the contrary, this was negative in 30 samples from slow progression (SP) patients. Four of the 5 negative RP samples corresponded to patients who had taken antiretroviral drugs at the time of the study. The p24 antigenemia was positive in 5 (41.6%) from the RP patients and in none of the SP patients. The presence of gag, pol and env sequences was positive by PCR in all RP patients and in most of the SP patients. However, repeatedly negative results by PCR were observed in 5 SP samples for all or some of the genomic regions studied. CONCLUSIONS: Patients with rapid progression of HIV-1 have higher plasmatic viremia than subjects with slow disease progression.

Adult↗
PubMed2003

Quantitative analysis and prognostic implication of SARS coronavirus RNA in the plasma and serum of patients with severe acute respiratory syndrome.

BACKGROUND: The availability of an early diagnostic tool for severe acute respiratory syndrome (SARS) would have major public health implications. We investigated whether the SARS coronavirus (SARS-CoV) can be detected in serum and plasma samples during the early stages of SARS and studied the potential prognostic implications of such an approach. METHODS: We developed two real-time quantitative reverse transcription-PCR (RT-PCR) assays, one for the polymerase and the other for the nucleocapsid region of the virus genome, for measuring the concentration of SARS-CoV RNA in serum/plasma samples from SARS patients. Plasma samples were obtained from 12 confirmed SARS patients on the day of hospital admission, as well as on days 7 and 14 after fever onset. Serum samples were also obtained from 23 confirmed SARS patients on the day of hospital admission, 11 of whom subsequently required intensive care. Viral RNA was extracted from the plasma/serum samples. The extracted RNA was subjected to analysis by the RT-PCR assays. RESULTS: The RT-PCR system for the polymerase region detected SARS-CoV RNA in 50% of plasma and 78% of serum samples from SARS patients during the first week of illness. The detection rates for plasma dropped to 25% at day 14 after fever onset. The median serum SARS-CoV concentrations in patients who required and did not require intensive care unit admission during the course of hospitalization were 5800 and 140 copies/mL, respectively (Mann-Whitney test, P <0.005). These data were confirmed by the RT-PCR system for the nucleocapsid region, which showed an even higher detection rate of 87%. The correlation between the results obtained by the two RT-PCR systems was high (Pearson correlation analysis, r = 0.998; P <0.001). CONCLUSION: Plasma/serum SARS-CoV quantification represents a potentially useful early diagnostic and prognostic tool for SARS.

Humans↗
PubMed2003

Investigating protein domain combinations in complete proteomes.

Protein-related information is more accumulated rather than reduced to a synthetic view. Itemising properties of protein sequences is informative, so is the list of ingredients to do some cooking, but without a recipe, that is, quantification and chronology, understanding is incomplete. If the goal of accumulating information is to discover or reveal the function and related biochemical mechanisms, information has to be weighed and ordered. As a guideline, the weight of a piece of information should reflect how often it consistently occurs in various contexts. We propose a common sense approach to quantify and put data and information into perspective. Complete bacterial proteomes are individually mapped with the Pfam-A database of domains and protein family signatures in an attempt to assess the modularity of proteins at the level of a single proteome and the implications of a modular description of proteins for a functional interpretation. Poorly annotated proteins in the most documented bacteria (E. coli and B. subtilis) were considered in an attempt to formulate hypothesis on the basis of domain/module content.

Bacillus subtilis↗
PubMed2002

Metabolomics: quantification of intracellular metabolite dynamics.

The rational improvement of microbial strains for the production of primary and secondary metabolites ('metabolic engineering') requires a quantitative understanding of microbial metabolism. A process by which this information can be derived from dynamic fermentation experiments is presented. By applying a substrate pulse to a substrate-limited, steady state culture, cellular metabolism is shifted away from its metabolic steady state. With the aid of a rapid sampling and quenching routine it is possible to take 4-5 samples per second during this process, thus capturing the metabolic response to this stimulus. Over 30 metabolites, nucleotides and cofactors from Escherichia coli metabolism can be extracted and analysed using a range of different techniques, for example enzymatic assays, HPLC and LC-MS methods. Using different substrates as limiting and pulse-substrates (glucose, glycerol), different metabolic pathways and substrate uptake systems are investigated. The resulting plots of intracellular metabolite concentrations against time serve as a data basis for modelling microbial metabolic networks.

Bioreactors↗
PubMed1993

Quantitative determination of human cytomegalovirus target sequences in peripheral blood leukocytes by nested polymerase chain reaction and temperature gradient gel electrophoresis.

A competitive nested PCR-temperature gradient gel electrophoresis protocol (nPCR/TGGE) has been established for the quantification of human cytomegalovirus (HCMV) target sequences. The measurement was achieved by co-amplification of a defined copy number of an internal standard (st) and separation of st and wild-type (wt) amplimers by temperature gradient gel electrophoresis (TGGE). The number of HCMV target sequences could be precisely determined within wt/st ratios of 0.1 to 10. With 50 copies of the st sequence the detection limit of nPCR/TGGE was found to be five to 10 copies of the target sequence. Effects of sample preparation on quantitative HCMV PCR were minimized by the additional quantification of beta-globin target sequences and calculation of the ratio of HCMV copies/beta-globin copies. Serial peripheral blood leukocyte specimens of 17 renal allograft recipients positive in a qualitative nested HCMV PCR were tested using nPCR/TGGE. Thirty healthy blood donors served as negative controls. Positive results were obtained by nPCR/TGGE in nine renal allograft recipients but in none of the healthy blood donors. Five of five patients with an HCMV pp65 antigenaemia and positive for HCMV IgM were positive in nPCR/TGGE. The highest HCMV/beta-globin ratios (10,000 to 8000 copies HCMV/10(6) copies beta-globin) were found in transplant recipients experiencing acute clinically symptomatic HCMV infection. HCMV DNA levels in asymptomatic patients ranged from 900 to 200 copies HCMV/10(6) beta-globin.

Base Sequence↗
PubMed2001

Molecular cytogenetic characterization of head and neck squamous cell carcinoma and refinement of 3q amplification.

We applied a combination of molecular cytogenetic methods, including comparative genomic hybridization (CGH), spectral karyotyping (SKY), and fluorescence in situ hybridization, to characterize the genetic aberrations in a panel of 11 cell lines derived from head and neck squamous cell carcinoma and 1 cell line derived from premalignant oral epithelium. CGH identified recurrent chromosomal losses at 1p, 3p, 4, 8p, 10p, and 18q; gains at 3q, 5p, 8q, 9q, and 14q; and high-level amplification at 3q13, 3q25-q26, 5q22-q23, 7q21, 8q24, 11q13-q14, 12p13, 14q24, and 20q13.1. Several recurrent translocations including t(1;13)(q10;q10), t(13;13)(q10;q10), t(14;14)(q10;q10), i(8)(q10), and i(9)(q10) and breakpoint clusters at 1p11, 1q21, 3p11, 5q11, 5q13, 6q23, 8p11, 8q11, 9p13, 9q13, 10q11, 11q13, 13q10, 14q10, and 15q10 were identified by SKY. There was a good correlation between the number of aberrations identified by CGH and SKY (r = 0.69), and the analyses were both confirmatory and complementary in their assessment of genetic aberrations. Amplification at 3q26-q27 was identified in 42% of cases. Although SKY defined the derivation of 3q gain, the precise breakpoint remained unassigned. Positional cloning efforts directed at the amplified region at 3q26-q27 identified three highly overlapping nonchimeric yeast artificial chromosome clones containing the apex of amplification. The use of these yeast artificial chromosome clones as a probe for fluorescence in situ hybridization analysis allowed a detailed characterization and quantification of the 3q amplification and refinement of unassigned SKY breakpoints.

Carcinoma, Squamous Cell↗
PubMed2006

Experimental infection of big brown bats (Eptesicus fuscus) with Eurasian bat lyssaviruses Aravan, Khujand, and Irkut virus.

Here we describe the results of experimental infections of captive big brown bats (Eptesicus fuscus) with three newly isolated bat lyssaviruses from Eurasia (Aravan, Khujand, and Irkut viruses). Infection of E. fuscus was moderate (total, 55-75%). There was no evidence of transmission to in-contact cage mates. Incubation periods for Irkut virus infection were significantly shorter (p < 0.05) than for either Aravan or Khujand virus infections. In turn, quantification of viral RNA by TaqMan PCR suggests that the dynamics of Irkut virus infection may differ from those of Aravan/Khujand virus infection. Although infectious virus and viral RNA were detected in the brain of every rabid animal, dissemination to non-neuronal tissues was limited. Levels of viral RNA in brain of Aravan/Khujand virus-infected bats was significantly correlated with the number of other tissues positive by TaqMan PCR (p < 0.05), whereas no such relationship was observed for Irkut virus infection (where viral RNA was consistently detected in all tissues other than kidney). Infectious virus was isolated sporadically from salivary glands, and both infectious virus and viral RNA were obtained from oral swabs. The detection of viral RNA in oral swabs suggests that viral shedding in saliva occurred <5 days before the onset of clinical disease.

Animals↗
PubMed1999

Pgk1 and Hprt gene activity in the peri-implantation mouse embryo is influenced by the parental origin of the X-chromosome.

The activity of two X-linked genes, Pgk1 and Hprt, that are localized on X-chromosomes of different parental origins in the XX mouse embryo was analyzed by the quantification of allele-specific transcripts. For the Pgk1 gene, the maternal allele-specific transcripts were consistently more abundant than the paternal transcripts in the blastocyst and the late gastrula. For the Hprt gene, the Hprt(b) allele was preferentially expressed in the blastocysts when it is present on the maternal X-chromosome. However, this skewed expression of the maternal allele was not observed in the reciprocal situation when the Hprt(a) allele was on the maternal X-chromosome. Like the Pgk1 locus, significantly more maternal Hprt transcripts were found in the gastrula-stage embryos irrespective of their genotypes. One possible interpretation of these results is that, in the XX mouse embryos, the genetic loci on maternal X-chromosome may be transcriptionally more active than their paternal counterparts during peri-implantation development.

Alleles↗
PubMed2006

Detection and quantification of Renibacterium salmoninarum DNA in salmonid tissues by real-time quantitative polymerase chain reaction analysis.

Renibacterium salmoninarum is an important salmonid pathogen that is difficult to culture. We developed and assessed a real-time, quantitative, polymerase chain reaction (qPCR) assay for the detection and enumeration of R. salmoninarum. The qPCR is based on TaqMan technology and amplifies a 69-base pair (bp) region of the gene encoding the major soluble antigen (MSA) of R. salmoninarum. The qPCR assay consistently detected as few as 5 R. salmoninarum cells per reaction in kidney tissue. The specificity of the qPCR was confirmed by testing the DNA extracts from a panel of microorganisms that were either common fish pathogens or reported to cause false-positive reactions in the enzyme-linked immunosorbent assay (ELISA). Kidney samples from 38 juvenile Chinook salmon (Oncorhynchus tshawytscha) in a naturally infected population were examined by real-time qPCR, a nested PCR, and ELISA, and prevalences of R. salmoninarum detected were 71, 66, and 71%, respectively. The qPCR should be a valuable tool for evaluating the R. salmoninarum infection status of salmonids.

Actinobacteria↗
PubMed2002

The gsalpha gene: predominant maternal origin of transcription in human thyroid gland and gonads.

Mutations in the guanine nucleotide binding alpha-subunit 1 gene (GNAS1) cause Albright's hereditary osteodistrophy, and the parent of transmission determines variable phenotypic expression of the disease. This has suggested that GNAS1 may be under tissue-specific imprinting control, although studies so far available have failed to clearly define the pattern of GNAS1 expression in humans. To establish if GNAS1 is imprinted in human endocrine tissues, we selected 14 thyroid, 10 granulosa cell, 13 pituitary (3 normal glands, 7 GH-secreting adenomas, and 3 nonfunctioning adenomas), 3 adrenal, and 11 lymphocyte samples shown to be heterozygous for a known polymorphism in exon 5. RNA from these tissues was analyzed by RT-PCR, and expression from both parental alleles was evaluated by enzymatic digestion and subsequent quantification of the resulting fragments. The parental origin of Gs alpha was assessed by evaluating neuroendocrine secretory protein 55 and extra large alphas-like protein transcripts, which have been shown to be monoallelically and parent-specifically expressed from the maternal and paternal allele, respectively. By this approach, the great majority of thyroid (n = 12), ovarian (n = 7), and pituitary (n = 11) samples showed an almost exclusive or significantly predominant expression of the maternal allele over the paternal one, whereas in lymphocyte and adrenal samples both alleles were equally expressed. Our results provide evidence for a predominant maternal origin of GNAS1 transcripts in different human adult endocrine tissues, particularly thyroid, ovary, and pituitary, and strongly suggest that this mechanism may play a crucial role in the determination of the phenotypic expression of Albright's hereditary osteodistrophy.

Adenoma↗
PubMed2005

Integrative neuroscience: the role of a standardized database.

Most brain related databases bring together specialized information, with a growing number that include neuroimaging measures. This article outlines the potential use and insights from the first entirely standardized and centralized database, which integrates information from neuroimaging measures (EEG, event related potential (ERP), structural/functional MRI), arousal (skin conductance responses (SCR)s, heart rate, respiration), neuropsychological and personality tests, genomics and demographics: The Brain Resource International Database. It comprises data from over 2000 "normative" subjects and a growing number of patients with neurological and psychiatric illnesses, acquired from over 50 laboratories (in the U.S.A, United Kingdom, Holland, South Africa, Israel and Australia), all with identical equipment and experimental procedures. Three primary goals of this database are to quantify individual differences in normative brain function, to compare an individual's performance to their database peers, and to provide a robust normative framework for clinical assessment and treatment prediction. We present three example demonstrations in relation to these goals. First, we show how consistent age differences may be quantified when large subject numbers are available, using EEG and ERP data from nearly 2000 stringently screened. normative subjects. Second, the use of a normalization technique provides a means to compare clinical subjects (50 ADHD subjects in this study) to the normative database with the effects of age and gender taken into account. Third, we show how a profile of EEG/ERP and autonomic measures potentially provides a means to predict treatment response in ADHD subjects. The example data consists of EEG under eyes open and eyes closed and ERP data for auditory oddball, working memory and Go-NoGo paradigms. Autonomic measures of skin conductance (tonic skin conductance level, SCL, and phasic skin conductance responses, SCRs) were acquired simultaneously with central EEG/ERP measures. The findings show that the power of large samples, tested using standardized protocols, allows for the quantification of individual differences that can subsequently be used to control such variation and to enhance the sensitivity and specificity of comparisons between normative and clinical groups. In terms of broader significance, the combination of size and multidimensional measures tapping the brain's core cognitive competencies, may provide a normative and evidence-based framework for individually-based assessments in "Personalized Medicine."

Animals↗