Mustn1 is essential for craniofacial chondrogenesis during Xenopus development

Mustn1 is a vertebrate-specific protein that, in vitro, was showed to be essential for prechondrocyte function and thus it has the potential to regulate chondrogenesis during embryonic development. We use Xenopus laevis as a model to examine Mustn1 involvement in chondrogenesis. Previous work suggests that Mustn1 is necessary but not sufficient for chondrogenic proliferation and differentiation, as well as myogenic differentiation in vitro. Mustn1 was quantified and localized in developing Xenopus embryos using RT-PCR and whole mount in situ hybridization. Xenopus embryos were injected with either control morpholinos (Co-MO) or one designed against Mustn1 (Mustn1-MO) at the four cell stage. Embryos were scored for morphological defects and Sox9 was visualized via in situ hybridization. Finally, Mustn1-MO-injected embryos were co-injected with Mustn1-MO resistant mRNA to confirm the specificity of the observed phenotype. Mustn1 is expressed from the mid-neurula stage to the swimming tadpole stages, predominantly in anterior structures including the pharyngeal arches and associated craniofacial tissues, and the developing somites. Targeted knockdown of Mustn1 in craniofacial and dorsal axial tissues resulted in phenotypes characterized by small or absent eye(s), a shortened body axis, and tail kinks. Further, Mustn1 knockdown reduced cranial Sox9 mRNA expression and resulted in the loss of differentiated cartilaginous head structures (e.g. ceratohyal and pharyngeal arches). Reintroduction of MO-resistant Mustn1 mRNA rescued these effects. We conclude that Mustn1 is necessary for normal craniofacial cartilage development in vivo, although the exact molecular mechanism remains unknown.     

Engineered bacterial outer membrane vesicles with enhanced functionality

We have engineered bacterial outer membrane vesicles (OMVs) with dramatically enhanced functionality by fusing several heterologous proteins to the vesicle-associated toxin ClyA of Escherichia coli. Similar to native unfused ClyA, chimeric ClyA fusion proteins were found localized in bacterial OMVs and retained activity of the fusion partners, demonstrating for the first time that ClyA can be used to co-localize fully functional heterologous proteins directly in bacterial OMVs. For instance, fusions of ClyA to the enzymes β-lactamase and organophosphorus hydrolase resulted in synthetic OMVs that were capable of hydrolyzing β-lactam antibiotics and paraoxon, respectively. Similarly, expression of an anti-digoxin single-chain Fv antibody fragment fused to the C terminus of ClyA resulted in designer “immuno-MVs” that could bind tightly and specifically to the antibody's cognate antigen. Finally, OMVs displaying green fluorescent protein fused to the C terminus of ClyA were highly fluorescent and, as a result of this new functionality, could be easily tracked during vesicle interaction with human epithelial cells. We expect that the relative plasticity exhibited by ClyA as a fusion partner should prove useful for: (i) further mechanistic studies to identify the vesiculation machinery that regulates OMV secretion and to map the intracellular routing of ClyA-containing OMVs during invasion of host cells; and (ii) biotechnology applications such as surface display of proteins and delivery of biologics.     

Structural and functional consequences of tyrosine phosphorylation in the LRP1 cytoplasmic domain

The cytoplasmic domain of LRP1 contains two NPXY motifs that have been shown to interact with signaling proteins. In previous work, we showed that Tyr(4507) in the distal NPXY motif is phosphorylated by v-Src, whereas denaturation of the protein was required for phosphorylation of Tyr(4473) in the membraneproximal NPXY motif. Amide H/D exchange studies reveal that the distal NPXY motif is fully solvent-exposed, whereas the proximal one is not. Phosphopeptide mapping combined with in vitro and in vivo kinase experiments show that Tyr(4473) can be phosphorylated, but only if Tyr(4507) is phosphorylated or substituted with glutamic acid. Amide H/D exchange experiments indicate that solvent accessibility increases across the entire LRP1 cytoplasmic region upon phosphorylation at Tyr(4507); in particular the NPXY(4473) motif becomes much more exposed. This differential phosphorylation is functionally relevant: binding of Snx17, which is known to bind at the proximal NPXY motif, is inhibited by phosphorylation at Tyr(4473). Conversely, Shp2 binds most strongly when both of the NPXY motifs in LRP1 are phosphorylated.     

Risk factor analysis and diagnoses of coronary heart disease in patients with hypercholesterolemia from Croatian Zagorje County

Our aim is to determine if there exists a difference in risk factors and diagnosis between patients being treated on internal medicine ward for coronary heart disease who have higher levels of cholesterol in their blood and other patients, without proved higher levels of cholesterol, hospitalized for coronary heart disease. We followed patients hospitalized in General Hospital Zabok for coronary heart disease for the period between 2004-2006y. On admission patients were diagnosed with coronary heart disease based on laboratory markers specific for the disease (CK, troponin, LDH,CRP), ECG and history taking. We analyzed two groups of patients for diagnosis and risk factors on discharge from the hospital: one group with proven hypercholesterolemia, the other with coronary heart disease without hypercholesterolemia. For the duration of the study there were no significant alternations concerning risk factors for coronary heart disease, and hypertension was the most prevalent of these factors in both groups. Values of HDL, as an indirect indicator of coronary heart disease, were lower in both groups for the duration of the study. In group of patients with hypercholesterolemia myocardial infarction with a ST segment elevation, as a discharge diagnosis, was a more prevalent complication of the disease, while for the group of patients without hypercholesterolemia stable angina pectoris was more prevalent and this is explained as atheroma plaque stabilization when there are normal values of blood cholesterol.     

Successional Changes in an Evolving Anaerobic Chlorophenol-Degrading Community Used To Infer Relationships between Population Structure and System-Level Processes

The response of a complex methanogenic sediment community to 2-chlorophenol (2-CP) was evaluated by monitoring the concentrations of this model contaminant and important metabolic intermediates and products and by using rRNA-targeted probes to track several microbial populations. Key relationships between the evolving population structure, formation of metabolic intermediates, and contaminant mineralization were identified. The nature of these relationships was intrinsically linked to the metabolism of benzoate, an intermediate that transiently accumulated during the mineralization of 2-CP. Before the onset of benzoate fermentation, reductive dehalogenation of 2-CP competed with methanogenesis for endogenous reducing equivalents. This suppressed H₂ levels, methane production, and archaeal small-subunit (SSU)-rRNA concentrations in the sediment community. The concentrations of bacterial SSU rRNA, including SSU rRNA derived from “Desulfovibrionaceae” populations, tracked with 2-CP levels, presumably reflecting changes in the activity of dehalogenating organisms. After the onset of benzoate fermentation, the abundance of Syntrophus-like SSU rRNA increased, presumably because these syntrophic organisms fermented benzoate to methanogenic substrates. Consequently, although the parent substrate 2-CP served as an electron acceptor, cleavage of its aromatic nucleus also influenced the sediment community by releasing the electron donors H₂ and acetate. Increased methane production and archaeal SSU-rRNA levels, which tracked with the Syntrophus-like SSU-rRNA concentrations, revealed that methanogenic populations in particular benefited from the input of reducing equivalents derived from 2-CP.     

Hormonal Control of Egg Dumping and Guarding in the Lace Bug, Gargaphia solani (Hemiptera: Tingidae)

This study examines the effect of juvenile hormone (JH) or a similarly acting juvenoid on the expression of maternal egg guarding and its life history alternative, egg dumping, in the lace bug, Gargaphia solani Heidemann. JH manipulations were indirect: methoprene, a synthetic JH analog, and precocene II, an allatocidal phytochemical commonly used to reduce JH synthesis, were applied exogenously to test the hypothesis that high JH titers promote egg production and egg dumping behavior, while low titers terminate egg production and initiate maternal care. As predicted, egg dumpers treated with precocene II ignored dumping opportunities and became egg guarders. Similarly, egg guarders that were treated with methoprene became gravid within 2 days and abandoned their eggs to become egg dumpers. These manipulations suggest that hormones can trigger the expression of both egg dumping and egg guarding in G. solani even when environmental conditions are inappropriate.     

Frequency of Educational Computer Use as a Longitudinal Predictor of Educational Outcome in Young People with Specific Language Impairment

Computer use draws on linguistic abilities. Using this medium thus presents challenges for young people with Specific Language Impairment (SLI) and raises questions of whether computer-based tasks are appropriate for them. We consider theoretical arguments predicting impaired performance and negative outcomes relative to peers without SLI versus the possibility of positive gains. We examine the relationship between frequency of computer use (for leisure and educational purposes) and educational achievement; in particular examination performance at the end of compulsory education and level of educational progress two years later. Participants were 49 young people with SLI and 56 typically developing (TD) young people. At around age 17, the two groups did not differ in frequency of educational computer use or leisure computer use. There were no associations between computer use and educational outcomes in the TD group. In the SLI group, after PIQ was controlled for, educational computer use at around 17 years of age contributed substantially to the prediction of educational progress at 19 years. The findings suggest that educational uses of computers are conducive to educational progress in young people with SLI.