Istaroxime treatment ameliorates calcium dysregulation in a zebrafish model of phospholamban R14del cardiomyopathy.

The heterozygous Phospholamban p.Arg14del mutation is found in patients with dilated or arrhythmogenic cardiomyopathy. This mutation triggers cardiac contractile dysfunction and arrhythmogenesis by affecting intracellular Ca2+ dynamics. Little is known about the physiological processes preceding induced cardiomyopathy, which is characterized by sub-epicardial accumulation of fibrofatty tissue, and a specific drug treatment is currently lacking. Here, we address these issues using a knock-in Phospholamban p.Arg14del zebrafish model. Hearts from adult zebrafish with this mutation display age-related remodeling with sub-epicardial inflammation and fibrosis. Echocardiography reveals contractile variations before overt structural changes occur, which correlates at the cellular level with action potential duration alternans. These functional alterations are preceded by diminished Ca2+ transient amplitudes in embryonic hearts as well as an increase in diastolic Ca2+ level, slower Ca2+ transient decay and longer Ca2+ transients in cells of adult hearts. We find that istaroxime treatment ameliorates the in vivo Ca2+ dysregulation, rescues the cellular action potential duration alternans, while it improves cardiac relaxation. Thus, we present insight into the pathophysiology of Phospholamban p.Arg14del cardiomyopathy.

Tomo-seq: A method to obtain genome-wide expression data with spatial resolution.

To improve our understanding of pattern formation during development and disease we heavily rely on the identification of novel regulators and pathways. While RNA sequencing yields genome-wide expression data that suit this purpose, it lacks spatial resolution. Such spatial resolution can be obtained by microscopy-based methods like in situ hybridization, but these fail to provide information on more than a few genes at a time. Here, we describe tomo-seq, a technique that combines the advantages of the above-mentioned approaches and provides genome-wide expression data with spatial information. The tomo-seq technique is based on cryosectioning of an embryo or tissue of interest and performing RNA-seq on individual sections. Using this method, we have generated genome-wide transcriptomics with high spatial resolution of the whole zebrafish embryo at various stages of development (Junker et al., 2014) and of adult zebrafish hearts after injury (Wu et al., 2016).

Sox4 mediates Tbx3 transcriptional regulation of the gap junction protein Cx43.

Tbx3, a T-box transcription factor, regulates key steps in development of the heart and other organ systems. Here, we identify Sox4 as an interacting partner of Tbx3. Pull-down and nuclear retention assays verify this interaction and in situ hybridization reveals Tbx3 and Sox4 to co-localize extensively in the embryo including the atrioventricular and outflow tract cushion mesenchyme and a small area of interventricular myocardium. Tbx3, SOX4, and SOX2 ChIP data, identify a region in intron 1 of Gja1 bound by all tree proteins and subsequent ChIP experiments verify that this sequence is bound, in vivo, in the developing heart. In a luciferase reporter assay, this element displays a synergistic antagonistic response to co-transfection of Tbx3 and Sox4 and in vivo, in zebrafish, drives expression of a reporter in the heart, confirming its function as a cardiac enhancer. Mechanistically, we postulate that Sox4 is a mediator of Tbx3 transcriptional activity.

Identification of Pseudallescheria and Scedosporium species by three molecular methods.

The major clinically relevant species in Scedosporium (teleomorph Pseudallescheria) are Pseudallescheria boydii, Scedosporium aurantiacum, Scedosporium apiospermum, and Scedosporium prolificans, while Pseudallescheria minutispora, Petriellopsis desertorum, and Scedosporium dehoogii are exceptional agents of disease. Three molecular methods targeting the partial ß-tubulin gene were developed and evaluated to identify six closely related species of the S. apiospermum complex using quantitative real-time PCR (qPCR), PCR-based reverse line blot (PCR-RLB), and loop-mediated isothermal amplification (LAMP). qPCR was not specific enough for the identification of all species but had the highest sensitivity. The PCR-RLB assay was efficient for the identification of five species. LAMP distinguished all six species unambiguously. The analytical sensitivities of qPCR, PCR-RLB, and LAMP combined with MagNAPure, CTAB (cetyltrimethylammonium bromide), and FTA filter (Whatman) extraction were 50, 5 × 10(3), and 5 × 10(2) cells/µl, respectively. When LAMP was combined with a simplified DNA extraction method using an FTA filter, identification to the species level was achieved within 2 h, including DNA extraction. The FTA-LAMP assay is therefore recommended as a cost-effective, simple, and rapid method for the identification of Scedosporium species.

Human papillomavirus infection in Honduran women with normal cytology.

OBJECTIVE: This study was aimed at estimating type-specific HPV prevalence and its cofactors among Honduran women with normal cytology in order to provide valuable information to health policymakers about the epidemiology of this important sexually transmitted infection. METHODS: A total of 591 women with normal cytology from Tegucigalpa, Honduras were interviewed and tested for HPV using the SPF10 LiPA25. A structured epidemiological questionnaire was administered to each woman. RESULTS: The overall HPV prevalence was 51%. Twenty-three types of HPV were detected; HPV 16, 51, 31, 18, and 11 were the most common. The highest prevalence of cancer associated HPV types (15.0%) was found in the women less than 35 years. Besides the association with age, the main independent predictors of HPV infection were the lifetime number of sexual partners and having a low socioeconomic status and less than 5 previous Pap smears. CONCLUSIONS: In the population studied, there was a broad diversity of HPV infections, with high-risk types being the most common types detected. The establishment of a well-characterized population with regard to the community prevalence of type-specific HPV infection will provide a valuable baseline for monitoring population effectiveness of an HPV vaccine.

Influenza-associated encephalopathy: no evidence for neuroinvasion by influenza virus nor for reactivation of human herpesvirus 6 or 7.

During 2 consecutive influenza seasons we investigated the presence of influenza virus, human herpesvirus (HHV) type 6, and HHV-7 in cerebrospinal fluid samples from 9 white children suffering from influenza-associated encephalopathy. We conclude that it is unlikely that neuroinvasion by influenza virus or reactivation of either HHV-6 or HHV-7 is involved.

Cross-talk between orientation-dependent recognition determinants of a complex control RNA element, the enterovirus oriR.

The coxsackie B3 virus oriR is an element of viral RNA thought to promote the assembly of a ribonucleoprotein complex involved in the initiation of genome replication. The mutual orientation of its two helical domains X and Y is determined by a kissing interaction between the loops of these domains. Here, a genetic approach was worked out to identify spatial orientation-dependent recognition signals in these helices. Spatial orientation changes (due to linear and rotational shifts) were introduced by appropriate insertions/deletions of a single base pair into one or both of the domains, and phenotypic consequences caused by these mutations were studied. The insertion of a base pair into domain Y caused a defect in viral reproduction that could be suppressed by a base-pair insertion into domain X. Similarly, a defect in viral replication caused by a base-pair deletion from domain X could be suppressed by a base-pair deletion from domain Y. Thus, certain areas of the two domains should cross-talk to one another in the sense that a change of space position of one of them required an adequate reply (change of space position) from the other. Phenotypic effects of the local rotation of one or more base pairs (and of some other mutations) in either domain X or domain Y suggested that the two most distal base pairs of these domains served as orientation-dependent recognizable signals. The results were also consistent with the notion that the recognition of the distal base pair of domain Y involved a mechanism similar to the intercalation of an amino acid residue.

Short fragment polymerase chain reaction reverse hybridization line probe assay to detect and genotype a broad spectrum of human papillomavirus types. Clinical evaluation and follow-up.

The purpose of this study was to detect and genotype 16 different human papilloma virus (HPV) types simultaneously using a short fragment polymerase chain reaction (SPF) hybridization line probe assay (LiPA). 152 women who were referred to the gynecologist because of abnormal cervical smear underwent colposcopic examination and repeat cervical smear. In addition, the cervical scrapes were analyzed for the presence of HPV by a novel general HPV polymerase chain reaction assay followed by a single reaction genotyping assay allowing for a simultaneous detection and identification of 16 different HPV types. HPV DNA was detected in 38% of normal follow-up cervical scrapes, 51% of scrapes with atypical squamous cells of undetermined significance, 78% of scrapes with mild dysplasia (low grade squamous intraepithelial lesions), 86% of scrapes with moderate dysplasia (high grade squamous intraepithelial lesions), and in 88% of scrapes with severe dysplasia and carcinoma in situ. One case of invasive squamous cell carcinoma was positive for HPV 16. Overall, a single HPV type was detected in 56% of HPV positive scrapes, with HPV 16 being the most common and accounting for 45% of all single infections. Forty-four percent of the positive scrapes contained multiple HPV types, of which double infections prevailed. Follow-up results proved the reproducibility and reliability of SPF HPV LiPA. In conclusion, we have used and evaluated the SPF-HPV-LiPA system for the detection and genotyping of HPV infections. The combined detection-typing method proved to be sensitive, specific, simple, and fast, making mass screening of cervical scrapes accessible for routine practice and facilitating individual patient management.

Chitin oligosaccharide synthesis by rhizobia and zebrafish embryos starts by glycosyl transfer to O4 of the reducing-terminal residue.

Lipochitin oligosaccharides are organogenesis-inducing signal molecules produced by rhizobia to establish the formation of nitrogen-fixing root nodules in leguminous plants. Chitin oligosaccharide biosynthesis by the Mesorhizobium loti nodulation protein NodC was studied in vitro using membrane fractions of an Escherichia coli strain expressing the cloned M. loti nodC gene. The results indicate that prenylpyrophosphate-linked intermediates are not involved in the chitin oligosaccharide synthesis pathway. We observed that, in addition to N-acetylglucosamine (GlcNAc) from UDP-GlcNAc, NodC also directly incorporates free GlcNAc into chitin oligosaccharides. Further analysis showed that free GlcNAc is used as a primer that is elongated at the nonreducing terminus. The synthetic glycoside p-nitrophenyl-beta-N-acetylglucosaminide (pNPGlcNAc) has a free hydroxyl group at C4 but not at C1 and could also be used as an acceptor by NodC, confirming that chain elongation by NodC takes place at the nonreducing-terminal residue. The use of artificial glycosyl acceptors such as pNPGlcNAc has not previously been described for a processive glycosyltransferase. Using this method, we show that also the DG42-directed chitin oligosaccharide synthase activity, present in extracts of zebrafish embryos, is able to initiate chitin oligosaccharide synthesis on pNPGlcNAc. Consequently, chain elongation in chitin oligosaccharide synthesis by M. loti NodC and zebrafish DG42 occurs by the transfer of GlcNAc residues from UDP-GlcNAc to O4 of the nonreducing-terminal residue, in contrast to earlier models on the mechanism of processive beta-glycosyltransferase reactions.

[Genotyping of human papillomavirus genotyes in cervical scrapes by line probe assay].

OBJECTIVE: To pursue the further development of a practical HPV genotyping system applicable for mass screening. METHODS: Cervical scrapes from 155 women were tested for the presence of HPV using a general-primer based PCR. A reverse hybridization assayor, the line probe assay (LiPA) was used for genotyping 16 different HPV types(HPV 6, 11, 16, 18, 31, 33, 35, 40, 42, 43, 44, 45, 51, 52, 56, 58) simultaneously. RESULTS: 93(60%) out of 155 specimens tested were positive for HPV DNA by PCR. The HPV positivity amongst the group of women with no cervical abnormality was 43%, while that in women with cervical dysplasia and cervical cancer was 81.2% PCR products from 89 HPV positive cases were genotyped by LiPA, of which 49 cases were single HPV infected concerning 11 different HPV types and the commonest type was HPV-16, occurring in 25(51%) of all single HPV infected cases. The other 40 specimens contained multiple HPV types comprising 16 different HPV types. Although double infections(n = 25) prevailed, triple(n = 9), quadruple(n = 3) and quintuple (n = 3) infections were also found. PCR products of four HPV positive cases failed to be classified by LiPA. Sequencing analysis revealed that these 4 cases were HPV-66 which was not included in LiPA probes. Follow-up data of 12 cases confirmed the HPV genotyping results. CONCLUSION: The LiPA for HPV genotyping is easy to perform. It allows accurate and rapid identification of 16 different HPV types in cervical scrapes and will facilitate HPV detection as well as genotyping in cervical cancer screening programs.

Function of chitin oligosaccharides in plant and animal development.

In plant development chitin oligosaccharides have been studied intensively as part of the communication between leguminous plants and Rhizobium bacteria. The Rhizobium bacteria synthesize and secrete lipochitin oligosaccharides (LCOs) to induce the development of a root nodule, in which the bacteria will infiltrate to start a symbiotic relation with the plant. Here we will give an overview of the biosynthetic route used by the bacteria to synthesize these LCOs. Perception by the plant will also be discussed as well as early responses to the LCOs. By working with the genes from the biosynthetic route, other genes were identified that share homology with the chitin synthase genes from Rhizobium. These genes are now isolated from human, mouse, chick, Xenopus and zebrafish and can be divided into three classes. They are mainly expressed during early development at the same stage as chitin oligosaccharide synthase activity can be detected. A controversy has been risen about their biochemical activity and will be further discussed here.

Structural requirements of the higher order RNA kissing element in the enteroviral 3'UTR.

The origin of replication ( oriR ) involved in the initiation of (-) strand enterovirus RNA synthesis is a quasi-globular multi-domain RNA structure which is maintained by a tertiary kissing interaction. The kissing interaction is formed by base pairing of complementary sequences within the predominant hairpin-loop structures of the enteroviral 3' untranslated region. In this report, we have fully characterised the kissing interaction. Site-directed mutations which affected the different base pairs involved in the kissing interaction were generated in an infectious coxsackie B3 virus cDNA clone. The kissing interaction appeared to consist of 6 bp. Distortion of the interaction by mispairing of each of the base pairs involved in this higher order RNA structure resulted in either temperature sensitive or lethal phenotypes. The nucleotide constitution of the base which gaps the major groove of the kissing domain was not relevant for virus growth. The reciprocal exchange of the complete sequence involved in the kissing resulted in a mutant virus with wild type virus growth characteristics arguing that the base pair constitution is of less importance for the initiation of (-) strand RNA synthesis than the existence of the tertiary structure itself.

Expression of Rhizobium chitin oligosaccharide fucosyltransferase in zebrafish embryos disrupts normal development.

In this report we present data about the effect of the Rhizobium NodZ enzyme on zebrafish development. We injected zebrafish embryos with a plasmid expressing NodZ protein, and we confirmed that the enzyme is active and has chitin oligosaccharide fucosyltransferase (NodZ) activity in vitro. In addition, the embryos injected with the NodZ-expressing plasmid, but not with a control plasmid, showed malformations or bends in the tail, and in some cases shunted tail structures and fused somites. These results clearly indicate that the likely substrates for this enzyme, chitin oligosaccharides and free N-glycans, have essential functions during early vertebrate embryogenesis.

An important developmental role for oligosaccharides during early embryogenesis of cyprinid fish.

Derivatives of chitin oligosaccharides have been shown to play a role in plant organogenesis at nanomolar concentrations. Here we present data which indicate that chitin oligosaccharides are important for embryogenesis in vertebrates. We characterize chitin oligosaccharides synthesized in vitro by zebrafish and carp embryos in the late gastrulation stage by incorporation of radiolabeled N-acetyl-D-[U14C]glucosamine and by HPLC in combination with enzymatic conversion using the Bradyrhizobium NodZ alpha-1, 6-fucosyltransferase and chitinases. A rapid and sensitive bioassay for chitin oligosaccharides was also used employing suspension-cultured plant cells of Catharanthus roseus. We show that chitin oligosaccharide synthase activity is apparent only during late gastrulation and can be inhibited by antiserum raised against the Xenopus DG42 protein. The DG42 protein, a glycosyltransferase, is transiently expressed between midblastula and neurulation in Xenopus and zebrafish embryogenesis. Microinjection of the DG42 antiserum or the Bradyrhizobium NodZ enzyme in fertilized eggs of zebrafish led to severe defects in trunk and tail development.

Abnormal nephrogram pattern as an adverse reaction to contrast material. Case report.

Up to now two abnormal nephrographic patterns have been described as a result of arterial hypotension as an adverse response to urographic contrast material. We would like to describe a third pattern.