unc-45 gene of Caenorhabditis elegans encodes a muscle-specific tetratricopeptide repeat-containing protein

The unc-45 gene of the nematode, Caenorhabditis elegans, is essential for muscle organization and embryonic development. Genetic evidence suggests the unc-45 gene product controls muscle thick filament assembly. We report here on the determination of the gene's chromosomal location and the isolation and sequencing of its cDNA. The amino terminus of the predicted unc-45 protein contains three tandem repeats that belong in the tetratricopeptide repeat family. Tetratricopeptide motifs have been shown to be involved in protein interactions, and some of the closest homologues have chaperone-like activity. The carboxy terminus of the protein has homology with the related fungal proteins, CRO1 and She4p, which have been postulated to play a role in assembly of or interactions with a cytoplasmic myosin. We have also determined the sequence of the homologous gene from C. briggsae, which demonstrates a high level of conservation. We show that the unc-45 gene promoter can drive reporter gene expression, which is limited to muscle tissues (pharyngeal, body wall, vulval, and anal muscles), consistent with a role for the unc-45 gene in muscle development or function.     

The cover of living and dead corals from airborne remote sensing

Trends in coral cover are widely used to indicate the health of coral reefs but are costly to obtain from field survey over large areas. In situ studies of reflected spectra at the coral surface show that living and recently dead colonies can be distinguished. Here, we investigate whether such spectral differences can be detected using an airborne remote sensing instrument. The Compact Airborne Spectrographic Imager (Itres Research Ltd, Canada) was flown in two configurations: 10 spectral bands with 1-m² pixels and 6 spectral bands with 0.25-m² pixels. First, we show that an instrument with 10 spectral bands possesses adequate spectral resolution to distinguish living Porites, living Pocillopora spp., partially dead Porites, recently dead Porites (total colony mortality within 6 months), old dead (>6 months) Porites, Halimeda spp., and coralline red algae when there is no water column to confuse spectra. All substrata were distinguished using fourth-order spectral derivatives around 538 nm and 562 nm. Then, at a shallow site (Tivaru) at Rangiroa Atoll, Tuamotu Archipelago (French Polynesia), we show that live and dead coral can be distinguished from the air to a depth of at least 4 m using first- and fourth-order spectral derivatives between 562–580 nm. However, partially dead and recently dead Porites colonies could not be distinguished from an airborne platform. Spectral differences among substrata are then exploited to predict the cover of reef substrata in ten 25-m² plots at nearby Motu Nuhi (max depth 8 m). The actual cover in these plots was determined in situ using quadrats with a 0.01-m² grid. Considerable disparity occurred between field and image-based measures of substrate cover within individual 25-m² quadrats. At this small scale, disparity, measured as the absolute difference in cover between field and remote-sensing methods, reached 25% in some substrata but was always less than 10% for living coral (99% of which consisted of Porites spp.). At the scale of the reef (all ten 25-m² quadrats), however, disparities in percent cover between imagery and field data were less than 10% for all substrata and extremely low for some classes (e.g. <3% for living Porites, recently dead Porites and Halimeda). The least accurately estimated substrata were sand and coralline red algae, which were overestimated by absolute values 7.9% and 6.6%, respectively. The precision of sampling was similar for field and remote-sensing methods: field methods required 19 plots to detect a 10% difference in coral cover among three reefs with a statistical power of 95%. Remote-sensing methods required 21 plots. However, it took 1 h to acquire imagery over 92,500 m² of reef, which represents 3,700 plots of 25 m² each, compared with 3 days to survey 10 such plots underwater. There were no significant differences in accuracy between 1-m² and 0.25-m² image resolutions, suggesting that the advantage of using smaller pixels is offset by reduced spectral information and an increase in noise (noise was observed to be 1.6–1.8 times greater in 0.25-m² pixels). We show that airborne remote sensing can be used to monitor coral and algal cover over large areas, providing that water is shallow and clear, and that brown fleshy macroalgae are scarce, that depth is known independently (e.g. from sonar survey).     

Reaction kinetics and modeling of the enzyme-catalyzed production of lactosucrose using beta-fructofuranosidase from Arthrobacter sp. K-1

Lactosucrose synthesis from sucrose and lactose was carried out by using beta-fructofuranosidase from Arthrobacter sp. K-1. The transfructosylation mechanism was found to be of an ordered bi-bi type in which sucrose was bound first to the enzyme and lactosucrose was released last. Hydrolysis side-reaction experiments indicated that the reactions were uncompetitively inhibited by glucose and lactose, while no inhibition by fructose was apparent. The overall reaction rates were formulated. The reaction rate constants, equilibrium constant, and dissociation and Michaelis constants were determined at 35 degrees C and 50 degrees C by fitting the experimental concentration changes with the calculated values by a nonlinear least-square method. The average relative derivation for the concentrations was 9.67%. The kinetic parameters were also calculated for 43 degrees C and 60 degrees C by assuming the Arrhenius law, and the course of reaction was predicted. The obtained reaction rate equations well represented the concentration changes during the experiment at all temperatures.     

Caenorhabditis elegans UNC-45 is a component of muscle thick filaments and colocalizes with myosin heavy chain B, but not myosin heavy chain A

In the nematode Caenorhabditis elegans, animals mutant in the gene encoding the protein product of the unc-45 gene (UNC-45) have disorganized muscle thick filaments in body wall muscles. Although UNC-45 contains tetratricopeptide repeats (TPR) as well as limited similarity to fungal proteins, no biochemical role has yet been found. UNC-45 reporters are expressed exclusively in muscle cells, and a functional reporter fusion is localized in the body wall muscles in a pattern identical to thick filament A-bands. UNC-45 colocalizes with myosin heavy chain (MHC) B in wild-type worms as well as in temperature-sensitive (ts) unc-45 mutants, but not in a mutant in which MHC B is absent. Surprisingly, UNC-45 localization is also not seen in MHC B mutants, in which the level of MHC A is increased, resulting in near-normal muscle thick filament structure. Thus, filament assembly can be independent of UNC-45. UNC-45 shows a localization pattern identical to and dependent on MHC B and a function that appears to be MHC B-dependent. We propose that UNC-45 is a peripheral component of muscle thick filaments due to its localization with MHC B. The role of UNC-45 in thick filament assembly seems restricted to a cofactor for assembly or stabilization of MHC B.     

Effects of time and rainfall on PCR success using DNA extracted from deer fecal pellets

Non-invasive wildlife research using DNA from feces has become increasingly popular. Recent studies have attempted to solve problems associated with recovering DNA from feces by investigating the influence of factors such as season, diet, collection method, preservation method, extraction protocol, and time. To our knowledge, studies of this nature have not addressed DNA degradation over time in wet environments, and have not been performed on fecal pellets of ungulates. Therefore, our objective was to determine the length of time a fecal pellet from a Sitka black-tailed deer (Odocoileus hemionus sitkensis) could remain in the field in a temperate rainforest environment before the DNA became too degraded for individual identification. Pellets were extracted from the rectum of recently killed deer and placed in an environment protected from rainfall and in an environment exposed to rainfall. Pellets from each treatment group were sampled at intervals of 2, 7, 14, 21, and 28 days after deer harvest. DNA was extracted from sampled pellets and individual samples were genotyped using microsatellite markers. Amplification failure and errors (dropout and false alleles) were recorded to determine extent of DNA degradation. Eighty percent of samples in the protected environment and 22% of samples in the exposed environment were successfully genotyped during the 28-day experiment. With no samples being successfully genotyped in the exposed environment after 7 days, our study showed that rainfall significantly increases degradation rates of DNA from ungulate pellets.     

Gene transfer to pre-hematopoietic and committed hematopoietic precursors in the early mouse Yolk Sac: a comparative study between <Emphasis Type="Italic">in situ</Emphasis> electroporation and retroviral transduction

Background  

Hematopoietic development in vertebrate embryos results from the sequential contribution of two pools of precursors independently generated. While intra-embryonic precursors harbour the features of hematopoietic stem cells (HSC), precursors formed earlier in the yolk sac (YS) display limited differentiation and self-renewal potentials. The mechanisms leading to the generation of the precursors in both sites are still largely unknown, as are the molecular basis underlying their different potential. A possible approach to assess the role of candidate genes is to transfer or modulate their expression/activity in both sites. We thus designed and compared transduction protocols to target either native extra-embryonic precursors, or hematopoietic precursors.     

European society of intensive care medicine study of therapeutic hypothermia (32-35°C) for intracranial pressure reduction after traumatic brain injury (the Eurotherm3235Trial)

Background

Severe malaria remains a major cause of global morbidity and mortality. Despite the use of potent anti-parasitic agents, the mortality rate in severe malaria remains high. Adjunctive therapies that target the underlying pathophysiology of severe malaria may further reduce morbidity and mortality. Endothelial activation plays a central role in the pathogenesis of severe malaria, of which angiopoietin-2 (Ang-2) has recently been shown to function as a key regulator. Nitric oxide (NO) is a major inhibitor of Ang-2 release from endothelium and has been shown to decrease endothelial inflammation and reduce the adhesion of parasitized erythrocytes. Low-flow inhaled nitric oxide (iNO) gas is a US FDA-approved treatment for hypoxic respiratory failure in neonates.

Methods/Design

This prospective, parallel arm, randomized, placebo-controlled, blinded clinical trial compares adjunctive continuous inhaled nitric oxide at 80 ppm to placebo (both arms receiving standard anti-malarial therapy), among Ugandan children aged 1-10 years of age with severe malaria. The primary endpoint is the longitudinal change in Ang-2, an objective and quantitative biomarker of malaria severity, which will be analysed using a mixed-effects linear model. Secondary endpoints include mortality, recovery time, parasite clearance and neurocognitive sequelae.

Discussion

Noteworthy aspects of this trial design include its efficient sample size supported by a computer simulation study to evaluate statistical power, meticulous attention to complex ethical issues in a cross-cultural setting, and innovative strategies for safety monitoring and blinding to treatment allocation in a resource-constrained setting in sub-Saharan Africa.

Trial Registration

ClinicalTrials.gov Identifier: NCT01255215     

Chimeras of the agouti-related protein: insights into agonist and antagonist selectivity of melanocortin receptors

The specific melanocortin receptors, MC3R and MC4R, are directly linked to metabolism and body weight control. These receptors are activated by the peptide hormone alpha-MSH and antagonized by the agouti-related protein (AGRP). Whereas alpha-MSH acts broadly on most members of the MCR family (with the exception of MC2R), AGRP is highly specific for only MC3R and MC4R. AGRP is a complex ligand of approximately 100 amino acids. Within AGRP, MCR recognition and antagonism is localized to a 34 residue, cysteine-rich domain that adopts an inhibitor cystine knot (ICK) fold. An oxidatively folded peptide corresponding to this domain, referred to as mini-AGRP, exhibits full antagonist function and selectivity for MC3R and MC4R. Here we investigate a series of chimera proteins based on the mini-AGRP scaffold. Amino acid sequences derived from peptide agonists are grafted into the mini-AGRP active loop, implicated in receptor recognition, with the goal of producing ICK based agonists specific for MC3R and MC4R. Several constructs indeed exhibited potent agonist activity; however, with all chimeras, receptor selectivity is significantly altered. Pharmacologic data indicate that the chimeras do not interact with MC receptors through native AGRP like contacts. A model to explain the data suggest that there is only partial overlap of the agonist versus antagonist binding surfaces within MC receptors. Moreover, accessibility to the binding pocket is highly receptor specific with MC3R being the least tolerant of ligand alterations.     

Bacterial delivery of functional messenger RNA to mammalian cells

The limited access to the nuclear compartment may constitute one of the major barriers after bacteria-mediated expression plasmid DNA delivery to eukaryotic cells. Alternatively, a self-destructing Listeria monocytogenes strain was used to release translation-competent mRNA directly into the cytosol of epithelial cells, macrophages and human dendritic cells. Enhanced green fluorescent protein (EGFP)-encoding mRNA, adapted for translation in mammalian cells by linking an IRES element to the 5'-end of the egfp coding sequence, was produced by T7 RNA polymerase in the carrier bacteria upon entry into the cytosol where the mRNA is efficiently released from the lysed bacteria and immediately translated in eukaryotic host cells. Besides the much earlier expression of EGFP being detectable already 4 h after infection, the number of EGFP expressing mammalian cells obtained with this novel RNA delivery technique is comparable to or - especially in phagocytic cells - even higher than that obtained with the expression plasmid DNA delivery strategy. Accordingly, bacteria-mediated delivery of ovalbumin-encoding mRNA to macrophages resulted in efficient antigen processing and presentation in vitro indicating that this approach may also be adapted for the in vivo delivery of antigen-encoding mRNA leading to a more efficient immune response when applied to vaccine development.