MicroRNA-34a is a tumor suppressor in choriocarcinoma via regulation of Delta-like1

Background: Choriocarcinoma is a gestational trophoblastic tumor which causes high mortality if left untreated. MicroRNAs (miRNAs) are small non protein-coding RNAs which inhibit target gene expression. The role of miRNAs in choriocarcinoma, however, is not well understood. In this study, we examined the effect of miR-34a in choriocarcinoma.Methods: MiR-34a was either inhibited or ectopically expressed transiently in two choriocarcinoma cell lines (BeWo and JEG-3) respectively. Its actions on cell invasion, proliferation and colony formation at low cell density were examined. The miR-34a putative target Notch ligand Delta-like 1 (DLL1) was identified by adoption of different approaches including: in-silico analysis, functional luciferase assay and western blotting. Real-time quantitative polymerase chain reaction was used to quantify changes in the expression of matrix proteinase in the treated cells. To nullify the effect of miR-34a ectopic expression, we activated Notch signaling through force-expression of the Notch intracellular domain in the miR-34a force-expressed cells. In addition, we studied the importance of DLL1 in BeWo cell invasion through ligand stimulation and antibody inhibition. Furthermore, the induction in tumor formation of miR-34a-inhibited BeWo cells in SCID mice was investigated.Results: Transient miR-34a force-expression significantly suppressed cell proliferation and invasion in BeWo and JEG-3 cells. In silicon miRNA target prediction, luciferase functional assays and Western blotting analysis demonstrated that miR-34a regulated DLL1 expression in both cell lines. Although force-expression of miR-34a suppressed the expression of DLL1 and NOTCH1, the extent of suppression was higher in DLL1 than NOTCH1 in both cell lines. MiR-34a-mediated DLL1 suppression led to reduced matrix metallopeptidase 9 and urokinase-type plasminogen activator expression. The effect of miR-34a on cell invasion was partially nullified by Notch signaling activation. DLL1 ligand stimulated while anti-DLL1 antibody treatment suppressed cell invasion. Mice inoculated with BeWo cells transfected with miR-34a inhibitor had significantly larger xenografts and stronger DLL1 expression than those with cells transfected with the control inhibitor.Conclusions: MiR-34a reduced cell proliferation and invasiveness, at least, partially through its inhibitory effect on DLL1. © 2013 Pang et al.; licensee BioMed Central Ltd.published_or_final_versio

Assessment of the imprinting efficiency of an imide with a “stoichiometric” pyridine-based functional monomer in precipitation polymerisation

The efficiency of the stoichiometric non-covalent imprinting of the imide 2,3,5-tri-O-acetyluridine (TAU) with 2,6-bis(acrylamido)pyridine (BAAPy) as functional monomer due to their strong donor-acceptor-donor/acceptor-donor-acceptor (DAD/ADA) hydrogen bond array interaction has been evaluated by bulk imprinting. This study is the first to investigate the imprinting and template rebinding efficiencies of the TAU/BAAPy molecularly imprinted polymeric (MIP) system prepared by precipitation polymerisation. We found that the stoichiometric 1:1 T:FM ratio has not been maintained in precipitation polymerisation and an optimal TAU:BAAPy ratio of 1:2.5 was obtained in acetonitrile without agitation affording an affinity constant (1.7 × 104 M?1) and a binding capacity (3.69 ?mol/g) higher than its bulk counterpart. Molecular modelling, NMR studies, and selectivity assays against analogues uridine and 2,3,5-tri-O-acetyl cytidine (TAC) indicate that, aside from the DAD/ADA hydrogen bond interaction, BAAPy also interacts with the acetyl groups of TAU. Template incorporation and rebinding in precipitation MIPs are favoured by a moderate initiator concentration, ie, initiator:total monomer (I:TM) ratio of 1:131, while low I:TM ratio (ie, 1:200) drastically reduced template incorporation and binding capacity. Vigorous agitation by stirring showed higher template incorporation but significantly lower template rebinding compared to that prepared without agitation. While the imprinting efficiencies for the best performing bulk and precipitation TAU MIPs generated in this study were moderate, 41% and 60%, respectively, their rebinding capacities were only between 3 and 4% of the incorporated template. We also present quantitative nuclear magnetic resonance spectroscopy as an efficient method for MIP characterisation

Notch signaling during human T cell development

Notch signaling is critical during multiple stages of T cell development in both mouse and human. Evidence has emerged in recent years that this pathway might regulate T-lineage differentiation differently between both species. Here, we review our current understanding of how Notch signaling is activated and used during human T cell development. First, we set the stage by describing the developmental steps that make up human T cell development before describing the expression profiles of Notch receptors, ligands, and target genes during this process. To delineate stage-specific roles for Notch signaling during human T cell development, we subsequently try to interpret the functional Notch studies that have been performed in light of these expression profiles and compare this to its suggested role in the mouse

Syndromic Recognition of Influenza A Infection in a Low Prevalence Community Setting

BACKGROUND: With epidemics of influenza A virus infection, people and medical professionals are all concerned about symptoms or syndromes that may indicate the infection with influenza A virus. METHODOLOGY/PRINCIPAL FINDINGS: A prospective study was performed at a community clinic of a metropolitan area. Throat swab was sampled for 3-6 consecutive adult patients with new episode (<3 days) of respiratory tract infection every weekday from Dec. 8, 2005 to Mar. 31, 2006. Demographic data, relevant history, symptoms and signs were recorded. Samples were processed with multiplex real time PCR for 9 common respiratory tract pathogens and by virus culture. Throat swab samples were positive for Influenza A virus with multiplex real time PCR system in 12 of 240 patients. The 12 influenza A positive cases were with more clusters and chills than the other 228. Certain symptoms and syndromes increased the likelihood of influenza A virus infection. The syndrome of high fever plus chills plus cough, better with clustering of cases in household or workplace, is with the highest likelihood (positive likelihood ratio 95; 95% CI 12-750). Absence of both cluster and chills provides moderate evidence against the infection (negative likelihood ratio 0.51; 95% CI 0.29-0.90). CONCLUSIONS/SIGNIFICANCE: Syndromic recognition is not diagnostic but is useful for discriminating between influenza A infection and common cold. In addition to relevant travel history, confirmatory molecular test can be applied to subjects with high likelihood when the disease prevalence is low

'Fat mass and obesity associated' gene (FTO): No significant association of variant rs9939609 with weight loss in a lifestyle intervention and lipid metabolism markers in German obese children and adolescents

<p>Abstract</p> <p>Background</p> <p>We have previously identified strong association of six single nucleotide polymorphisms (SNPs) in <it>FTO </it>(fat mass and obesity associated gene) to early onset extreme obesity within the first genome wide association study (GWA) for this phenotype. The aim of this study was to investigate whether the obesity risk allele of one of these SNPs (rs9939609) is associated with weight loss in a lifestyle intervention program. Additionally, we tested for association of rs9939609 alleles with fasting blood parameters indicative of glucose and lipid metabolism.</p> <p>Methods</p> <p>We initially analysed rs9939609 in a case-control study comprising 519 German overweight and obese children and adolescents and 178 normal weight adults. In 207 of the obese individuals who took part in the outpatient obesity intervention program 'Obeldicks' we further analysed whether carrier status of the obesity risk A-allele of rs9939609 has a differential influence on weight loss after the intervention program. Additionally, we investigated in 480 of the overweight and obese patients whether rs9939609 is associated with fasting blood levels of glucose, triglycerides and HDL and LDL-cholesterol. Genotyping was performed using allele specific polymerase chain reaction (ARMS-PCR). For the association study (case-control approach), the Cochran-Armitage trend test was applied. Blood parameters were analysed using commercially available test kits and the log10-transformed blood parameters and changes in BMI-standard deviation scores (BMI-SDS) were analysed by linear regression with sex and age as covariates under an additive mode of inheritance with the rs9939609 A-allele as risk allele.</p> <p>Results</p> <p>We confirmed the association of the risk A-allele of rs9939609 with overweight and early onset obesity (one sided p = 0.036). However, we observed no association of rs9939609 alleles with weight loss or fasting levels of blood glucose, triglycerides and cholesterol.</p> <p>Conclusion</p> <p>We confirmed the rs9939609 A-allele as a risk factor for early onset obesity whereas its impact on weight loss or on serum levels of glucose, triglycerides and cholesterol could not be detected in our samples.</p> <p>Trial Registration</p> <p><b>This study is registered </b>at clinicaltrials.gov (NCT00435734).</p

The Proteolipid Protein Promoter Drives Expression outside of the Oligodendrocyte Lineage during Embryonic and Early Postnatal Development

The proteolipid protein (Plp) gene promoter is responsible for driving expression of one of the major components of myelin – PLP and its splice variant DM-20. Both products are classically thought to express predominantly in oligodendrocytes. However, accumulating evidence suggests Plp expression is more widespread than previously thought. In an attempt to create a mouse model for inducing oligodendrocyte-specific gene deletions, we have generated transgenic mice expressing a Cre recombinase cDNA under control of the mouse Plp promoter. We demonstrate Plp promoter driven Cre expression is restricted predominantly to mature oligodendrocytes of the central nervous system (CNS) at postnatal day 28. However, crosses into the Rosa26LacZ and mT/mG reporter mouse lines reveal robust and widespread Cre activity in neuronal tissues at E15.5 and E10.5 that is not strictly oligodendrocyte lineage specific. By P28, all CNS tissues examined displayed high levels of reporter gene expression well outside of defined white matter zones. Importantly, our study reinforces the emerging idea that Plp promoter activity is not restricted to the myelinating cell lineage, but rather, has widespread activity both during embryonic and early postnatal development in the CNS. Specificity of the promoter to the oligodendrocyte cell lineage, as shown through the use of a tamoxifen inducible Plp-CreERt line, occurs only at later postnatal stages. Understanding the temporal shift in Plp driven expression is of consequence when designing experimental models to study oligodendrocyte biology

Differential Functional Constraints on the Evolution of Postsynaptic Density Proteins in Neocortical Laminae

The postsynaptic density (PSD) is a protein dense complex on the postsynaptic membrane of excitatory synapses that is implicated in normal nervous system functions such as synaptic plasticity, and also contains an enrichment of proteins involved in neuropsychiatric disorders. It has recently been reported that the genes encoding PSD proteins evolved more slowly than other genes in the human brain, but the underlying evolutionary advantage for this is not clear. Here, we show that cortical gene expression levels could explain most of this effect, indicating that expression level is a primary contributor to the evolution of these genes in the brain. Furthermore, we identify a positive correlation between the expression of PSD genes and cortical layers, with PSD genes being more highly expressed in deep layers, likely as a result of layer-enriched transcription factors. As the cortical layers of the mammalian brain have distinct functions and anatomical projections, our results indicate that the emergence of the unique six-layered mammalian cortex may have provided differential functional constraints on the evolution of PSD genes. More superficial cortical layers contain PSD genes with less constraint and these layers are primarily involved in intracortical projections, connections that may be particularly important for evolved cognitive functions. Therefore, the differential expression and evolutionary constraint of PSD genes in neocortical laminae may be critical not only for neocortical architecture but the cognitive functions that are dependent on this structure

The bubble snails (Gastropoda, Heterobranchia) of Mozambique: an overlooked biodiversity hotspot

This first account, dedicated to the shallow water marine heterobranch gastropods of Mozambique is presented with a focus on the clades Acteonoidea and Cephalaspidea. Specimens were obtained as a result of sporadic sampling and two dedicated field campaigns between the years of 2012 and 2015, conducted along the northern and southern coasts of Mozambique. Specimens were collected by hand in the intertidal and subtidal reefs by snorkelling or SCUBA diving down to a depth of 33 m. Thirty-two species were found, of which 22 are new records to Mozambique and five are new for the Western Indian Ocean. This account raises the total number of shallow water Acteonoidea and Cephalaspidea known in Mozambique to 39 species, which represents approximately 50 % of the Indian Ocean diversity and 83 % of the diversity of these molluscs found in the Red Sea. A gap in sampling was identified in the central swamp/mangrove bio-region of Mozambique, and therefore, we suggest that future research efforts concentrate on or at least consider this region.publishedVersio

Construction of non-polar mutants in Haemophilus influenzae using FLP recombinase technology

Background Nontypeable Haemophilus influenzae (NTHi) is a gram-negative bacterium that causes otitis media in children as well as other infections of the upper and lower respiratory tract in children and adults. We are employing genetic strategies to identify and characterize virulence determinants in NTHi. NTHi is naturally competent for transformation and thus construction of most mutants by common methodologies is relatively straightforward. However, new methodology was required in order to construct unmarked non-polar mutations in poorly expressed genes whose products are required for transformation. We have adapted the lambda red/FLP-recombinase-mediated strategy used in E. coli for use in NTHi. Results A cassette containing a spectinomycin resistance gene and an rpsL gene flanked by FRT sites was constructed. A PCR amplicon containing 50 base pairs of DNA homologous to the 5' and 3' ends of the gene to be disrupted and the cassette was generated, then recombineered into the target NTHi gene, cloned on a plasmid, using the lambda recombination proteins expressed in E. coli DY380. Thus, the gene of interest was replaced by the cassette. The construct was then transformed into a streptomycin resistant NTHi strain and mutants were selected on spectinomycin-containing growth media. A plasmid derived from pLS88 with a temperature sensitive replicon expressing the FLP recombinase gene under the control of the tet operator/repressor was constructed. This plasmid was electroporated into the NTHi mutant at the permissive temperature and FLP expression was induced using anhydrotetracycline. The recombinase recognizes the FRT sites and eliminates the antibiotic cassette by site-specific recombination, creating the unmarked non-polar mutation. The plasmid is cured by growth of cells at the restrictive temperature. Conclusion The products of the genes in the NTHi pilABCD operon are required for type IV pilus biogenesis and have a role in transformation. We demonstrated the utility of our methodology by the construction of a non-polar pilA mutation in NTHi strain 2019 and complementation of the mutation with a plasmid containing the pilA gene. Utilization of this approach allowed us to readily generate unmarked non-polar mutations in NTHi genes.This work was supported by NIH grants R01DC007464 to RSM, R01DC003915 to Lauren Bakaletz and a subcontract from N01AI30040 to Michael Apicella. We thank Michael Apicella for the gifts of NTHi strains 2019 and 2019 rpsL