Cloning of the koi herpesvirus (KHV) gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis

BACKGROUND: Outbreaks with mass mortality among common carp Cyprinus carpio carpio and koi Cyprinus carpio koi have occurred worldwide since 1998. The herpes-like virus isolated from diseased fish is different from Herpesvirus cyprini and channel catfish virus and was accordingly designated koi herpesvirus (KHV). Diagnosis of KHV infection based on viral isolation and current PCR assays has a limited sensitivity and therefore new tools for the diagnosis of KHV infections are necessary. RESULTS: A robust and sensitive PCR assay based on a defined gene sequence of KHV was developed to improve the diagnosis of KHV infection. From a KHV genomic library, a hypothetical thymidine kinase gene (TK) was identified, subcloned and expressed as a recombinant protein. Preliminary characterization of the recombinant TK showed that it has a kinase activity using dTTP but not dCTP as a substrate. A PCR assay based on primers selected from the defined DNA sequence of the TK gene was developed and resulted in a 409 bp amplified fragment. The TK based PCR assay did not amplify the DNAs of other fish herpesviruses such as Herpesvirus cyprini (CHV) and the channel catfish virus (CCV). The TK based PCR assay was specific for the detection of KHV and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions. The TK based PCR was compared to previously described PCR assays and to viral culture in diseased fish and was shown to be the most sensitive method of diagnosis of KHV infection. CONCLUSION: The TK based PCR assay developed in this work was shown to be specific for the detection of KHV. The TK based PCR assay was more sensitive for the detection of KHV than previously described PCR assays; it was as sensitive as virus isolation which is the golden standard method for KHV diagnosis and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions

Vitamin D Supplementation in Chronic Schizophrenia Patients Treated with Clozapine:A Randomized, Double-Blind, Placebo-controlled Clinical Trial

Background: While accumulating evidence suggests that vitamin D deficiency may be involved in the risk to develop schizophrenia and its outcome, there are no studies on vitamin D supplementation in this context. We sought to assess the effect of vitamin D supplementation on psychiatric, cognitive and metabolic parameters in chronic clozapine-treated schizophrenia patients. Methods: This eight-week, randomized, double-blind, placebo-controlled clinical trial, recruited schizophrenia patients who had been maintained on clozapine treatment for at least 18 weeks and had low levels of vitamin D (<75 nmol/l) and total PANSS scores >70 (to ascertain the presence of residual symptoms). Patients were randomly allocated to either weekly oral drops of vitamin D (14,000 IU) or placebo and subsequently assessed at two-week intervals for psychosis severity, mood, cognition and metabolic profile. Results: Twenty four patients were randomly assigned to vitamin D (aged 39.4 ± 9.6 years, 75% males) and the other 23 patients to the placebo arm (aged 42.5 ± 11.2 years, 60.9% males). After eight weeks, the vitamin D group exhibited a significant increase in vitamin D levels (31.4 vs −0.4 nmol/l, p < 0.0001). There was no significant effect of vitamin D on psychotic, depressive or metabolic parameters. However, in the vitamin D group, there was a trend towards improved cognition (effect size = 0.17, significance lost following Bonferroni correction). Conclusions: Vitamin D supplementation was associated with a trend towards improved cognition, but did not affect psychosis, mood or metabolic status. It is possible that the robust decrease in the PANSS scores in both groups may have obscured an effect of vitamin D supplementation

HiRISE views enigmatic deposits in the Sirenum Fossae region of Mars

HiRISE images together with other recent orbital data from Mars define new characteristics of enigmatic Hesperian-aged deposits in Sirenum Fossae that are mostly 100–200 m thick, drape kilometers of relief, and often display generally low relief surfaces. New characteristics of the deposits, previously mapped as the “Electris deposits,” include local detection of meter-scale beds that show truncating relationships, a generally light-toned nature, and a variably blocky, weakly indurated appearance. Boulders shed by erosion of the deposits are readily broken down and contribute little to talus. Thermal inertia values for the deposits are ~200 J m^(−2) K^(−1) s^(−1/2) and they may incorporate hydrated minerals derived from weathering of basalt. The deposits do not contain anomalous amounts of water or water ice. Deflation may dominate degradation of the deposits over time and points to an inventory of fine-grained sediment. Together with constraints imposed by the regional setting on formation processes, these newly resolved characteristics are most consistent with an eolian origin as a loess-like deposit comprised of redistributed and somewhat altered volcanic ash. Constituent sediments may be derived from airfall ash deposits in the Tharsis region. An origin directly related to airfall ash or similar volcanic materials is less probable and emplacement by alluvial/fluvial, impact, lacustrine, or relict polar processes is even less likely

«Stien blir til mens man går den». En kvalitativ studie av erfaring med organisering og deltakelse i tverrfaglige team

Temaet for denne masteroppgaven er erfaring med organisering og deltakelse i tverrfaglig samarbeid. Som case har det blitt brukt demensteam i små kommuner for å studere dette. «Tverrfaglighet» er et begrep som ofte dukker opp i forbindelse med velferdstjenester, spesielt innenfor helse- og omsorgssektoren. Å utvikle tverrfaglighet blir sett på som et samfunnsoppdrag i forsøket på å løse utfordringene som følger av demografisk endring og endret sykdomsbilde. I studiet kommer det frem at teamene må organisere etter de ressurser de har tilgjengelig. De viser at organiserer innovativt og medlemmene av teamet kommer sammen fram til løsninger for å utnytte de ressursene de har til rådighet, på mest mulig effektiv måte. Teamet utvikler seg som følge av de ulike profesjonenes kompetanse og erfaringer. «Stien blir til mens man går den» som gir et godt bilde av hvordan teamet utvikler seg gjennom medlemmenes erfaringer, over tid. Informantene opplever det som interessant og lærerikt å delta i et tverrfaglig team, de lærer av og med hverandre. Men at det må ligge noe forutsetninger av uformell og formell karakter

Cloning of the koi herpesvirus (KHV) gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis-0

<p><b>Copyright information:</b></p><p>Taken from "Cloning of the koi herpesvirus (KHV) gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis"</p><p>BMC Microbiology 2005;5():13-13.</p><p>Published online 17 Mar 2005</p><p>PMCID:PMC1079851.</p><p>Copyright © 2005 Bercovier et al; licensee BioMed Central Ltd.</p>orithm

Establishment of Homozygote Mutant Human Embryonic Stem Cells by Parthenogenesis

<div><p>We report on the derivation of a diploid 46(XX) human embryonic stem cell (HESC) line that is homozygous for the common deletion associated with Spinal muscular atrophy type 1 (SMA) from a pathenogenetic embryo. By characterizing the methylation status of three different imprinted loci (MEST, SNRPN and H19), monitoring the expression of two parentally imprinted genes (SNRPN and H19) and carrying out genome-wide SNP analysis, we provide evidence that this cell line was established from the activation of a mutant oocyte by diploidization of the entire genome. Therefore, our SMA parthenogenetic HESC (pHESC) line provides a proof-of-principle for the establishment of diseased HESC lines without the need for gene manipulation. As mutant oocytes are easily obtained and readily available during preimplantation genetic diagnosis (PGD) cycles, this approach should provide a powerful tool for disease modelling and is especially advantageous since it can be used to induce large or complex mutations in HESCs, including gross DNA alterations and chromosomal rearrangements, which are otherwise hard to achieve.</p></div

Methylation levels at <i>H19</i>, <i>SNRPN</i> and <i>MEST</i> imprinted loci.

<p>Bisulfite single colony sequencing was performed on 3 different imprinted regions (<i>H19</i>, SNRPN and <i>MEST</i>) to determine methylation levels in normal (WT) and SZ-SMA5 HESC lines. Each line represents a single DNA molecule. Full circles correspond to methylated CpGs while empty circles represent unmethylated CpGs.</p

Whole genome view of Cytoscan SNP Array data.

<p>Genome wide SNP array results obtained from (A) reference DNA from a male with a normal karyotype (46; XY); and (B) SZ-SMA5 HESC line DNA. The X axis represents chromosomes 1–22, X and Y. (I) The Y axis represents the copy number, determined by the log2 ratio (grey dots) on the left side of the graph, and it's smoothed ratio (red line) on the right. The expected copy number is 2 for autosomal chromosomes (log2 of 0 and smooth signal of 2). The log2 ratio and the smooth signal are determined from both the nonpolymorphic copy number probes and the polymorphic SNP probes. (II) The Y-axis corresponds to homozygote calls (AA or BB) and heterozygote calls (AB). Allele peaks of 1, 0, and -1 indicate a copy number of two, while allele peaks of 0.5 and -0.5 indicate a copy number of one. Allele peaks are calculated from SNP probes. The distinction between XY (reference DNA) and XX (SZ-SMA5) cells is clearly illustrated by the difference in X chromosome copy number. In addition, the overall 0.49% inherent heterozygote call error rate in SZ-SMA5 is below even the expected array genotyping error of ~1% (as determined by dividing the number of heterozygous calls by the total number of SNP probes on the array). Therefore, these data indicate that SZ-SMA5 features a completely homozygous diploid genome. (C) Fraction of SNP heterozygote calls in WT male reference and SZ-SMA5 DNA. Chromosomes are indicated in the X axis and the Y axis indicates the fraction of heterozygous SNP calls per total SNP calls on each chromosome.</p

Characterization of SZ-SMA5.

<p>(A) Expression of undifferentiated cell specific markers by immunostaining for <i>OCT4</i> (red nuclear staining, merged onto Hoechst (blue)), for the cell surface marker <i>Tra 1–60</i> (red, merged onto Hoechst (blue)); and (B) for alkaline phosphatase activity (AP). (C) Expression of undifferentiated cell specific markers: <i>OCT4</i>, <i>REX1</i>, <i>NANOG</i> and <i>SOX2</i> in SZ-SMA5 and a wild-type (WT) HESC control by RT-PCR. <i>GAPDH</i> expression served as a loading control. (D) Teratoma sections stained by H&E from SZ-SMA5 demonstrating multi-cellular structures derived from the three different embryonic germ layers. (E) A representative normal 46(XX) karyotype in SZ-SMA5 as identified by Giemsa staining of 20 different spreads of metaphase chromosomes.</p