fras1 shapes endodermal pouch 1 and stabilizes zebrafish pharyngeal skeletal development

Lesions in the epithelially expressed human gene FRAS1 cause Fraser syndrome, a complex disease with variable symptoms, including facial deformities and conductive hearing loss. The developmental basis of facial defects in Fraser syndrome has not been elucidated. Here we show that zebrafish fras1 mutants exhibit defects in facial epithelia and facial skeleton. Specifically, fras1 mutants fail to generate a late-forming portion of pharyngeal pouch 1 (termed late-p1) and skeletal elements adjacent to late-p1 are disrupted. Transplantation studies indicate that fras1 acts in endoderm to ensure normal morphology of both skeleton and endoderm, consistent with well-established epithelial expression of fras1. Late-p1 formation is concurrent with facial skeletal morphogenesis, and some skeletal defects in fras1 mutants arise during late-p1 morphogenesis, indicating a temporal connection between late-p1 and skeletal morphogenesis. Furthermore, fras1 mutants often show prominent second arch skeletal fusions through space occupied by late-p1 in wild type. Whereas every fras1 mutant shows defects in late-p1 formation, skeletal defects are less penetrant and often vary in severity, even between the left and right sides of the same individual. We interpret the fluctuating asymmetry in fras1 mutant skeleton and the changes in fras1 mutant skeletal defects through time as indicators that skeletal formation is destabilized. We propose a model wherein fras1 prompts late-p1 formation and thereby stabilizes skeletal formation during zebrafish facial development. Similar mechanisms of stochastic developmental instability might also account for the high phenotypic variation observed in human FRAS1 patients.     

Drosophila melanogaster p24 trafficking proteins have vital roles in development and reproduction

p24 proteins comprise a family of type-I transmembrane proteins of ~24kD that are present in yeast and plants as well as metazoans ranging from Drosophila to humans. These proteins are most commonly localized to the endoplasmic reticulum (ER)-Golgi interface and are incorporated in anterograde and retrograde transport vesicles. Little is known about how disruption of p24 signaling affects individual tissue function or whole animals. Drosophila melanogaster express nine p24 genes, grouped into four subfamilies. Based upon our mRNA and protein expression data, Drosophila p24 family members are expressed in a variety of tissues. To identify functions for particular Drosophila p24 proteins, we used RNA interference (RNAi) to reduce p24 expression. Ubiquitous reduction of most p24 genes resulted in complete or partial lethality during development. We found that reducing p24 levels in adults caused defects in female fecundity (egg laying) and also reduced male fertility. We attributed reduced female fecundity to decreased neural p24 expression. These results provide the first genetic analysis of all p24 family members in a multicellular animal and indicate vital roles for Drosophila p24s in development and reproduction, implicating neural expression of p24s in the regulation of female behavior.     

Ecdysone signaling in adult Drosophila melanogaster

The steroid hormone 20-hydroxyecdysone and its EcR/USP receptor are vital during arthropod development for coordinating molting and metamorphosis. Traditionally, little attention has been given to potential post-developmental functions for this hormone signaling system. However, recent studies in Drosophila melanogaster indicate that the hormone and receptor are present and active in adults and that mutations decreasing hormone or receptor levels affect diverse processes such as reproduction, behavior, stress resistance, and lifespan. We review the current state of knowledge regarding adult hormone production and titers and discuss receptor expression and activity in order to identify potential mechanisms which explain the observed mutant phenotypes. Finally, we describe future research directions focused on identifying isoform-specific functions of EcR, distinguishing effects from EcR/USP gene activation and repression, and determining how ecdysone signaling impacts different tissue types.     

Influence of needle insertion speed on backflow for convection-enhanced delivery

Fluid flow back along the outer surface of a needle (backflow) can be a significant problem during the direct infusion of drugs into brain tissues for procedures such as convection-enhanced delivery (CED). This study evaluates the effects of needle insertion speed (0.2 and 1.8 mm/s) as well as needle diameter and flow rate on the extent of backflow and local damage to surrounding tissues. Infusion experiments were conducted on a transparent tissue phantom, 0.6% (w/v) agarose hydrogel, to visualize backflow. Needle insertion experiments were also performed to evaluate local damage at the needle tip and to back out the prestress in the surrounding media for speed conditions where localized damage was not excessive. Prestress values were then used in an analytical model of backflow. At the higher insertion speed (1.8 mm/s), local insertion damage was found to be reduced and backflow was decreased. The compressive prestress at the needle-tissue interface was estimated to be approximately constant (0.812 kPa), and backflow distances were similar regardless of needle gauge (22, 26, and 32 gauge). The analytical model underestimated backflow distances at low infusion flow rates and overestimated backflow at higher flow rates. At the lower insertion speed (0.2 mm/s), significant backflow was measured. This corresponded to an observed accumulation of material at the needle tip which produced a gap between the needle and the surrounding media. Local tissue damage was also evaluated in excised rat brain tissues, and insertion tests show similar rate-dependent accumulation of tissue at the needle tip at the lower insertion speed. These results indicate that local tissue damage and backflow may be avoided by using an appropriate insertion speed.     

Expanding the zebrafish embryo proteome using multiple fractionation approaches and tandem mass spectrometry

The proteome of zebrafish, Danio rerio, embryos has not been studied in great detail mainly due to the presence of high abundance yolk proteins in embryos. Here we report the highest number of the zebrafish embryo proteins identified so far to our knowledge, through a combination of a protein-level fractionation approach (1D SDS-PAGE) and two different peptide-level fractionation approaches (IEF and strong anion exchange (SAX)) of deyolked zebrafish embryos followed by LC-MS/MS. We detected 5267 proteins in total of which 3464 proteins were identified with at least two peptides (less than 1% peptide false discovery rate). The analysis of proteome coverage from each method showed that 56% of detected proteins were common to all approaches and 95% of the detected proteome was obtained from 1D SDS-PAGE approach alone. Bioinformatics analysis of the detected proteome demonstrated that nucleocytoplasmic transport (biological process) and ribosomal proteins (cellular component) were the most over-represented proteins, whereas cell-cell signaling (biological process) and extracellular space proteins (cellular component) were the most under-represented proteins in the identified proteome.     

Toxicokinetic Profile of N‐(2‐Aminoethyl)ethanolamine in the Female Wistar Rat and Distribution into the Late Gestation Fetus and Milk

N‐(2‐aminoethyl)ethanolamine (AEEA) caused aneurysms of the great vessels in rats exposed in utero and during the first days post partum, exacerbated by postnatal treatment of the lactating dams (Moore et al., 2012. Birth Defects Res B Dev Reprod Toxicol [95:116‐122]). The purpose of this work was to examine the systemic availability of AEEA during gestation and early lactation. The absorption of AEEA was determined following oral administration to nonpregnant and pregnant female Wistar rats. A single dose administered by gavage (0.5 or 50 mg/kg) on gestation day 18 was rapidly and extensively (>90%) absorbed from the gastrointestinal tract (absorption t_1/2 = 0.1–0.2 hr). Elimination from the plasma followed a biphasic pattern, with a rapid elimination phase (t_1/2 α = 1.6–1.8 hr) followed by a slower phase (t_1/2 β = 16.7–17.3 hr). Following repeated gavage administration during gestation day 17 to 19, ¹⁴C‐AEEA–derived radioactivity readily partitioned into the fetus and was evenly distributed therein, but cleared approximately twofold slower from the fetal blood and tissues than the maternal blood and chorioallantoic placenta. When administered to lactating dams during lactation days 1 to 12, ¹⁴C‐AEEA–derived radioactivity preferentially partitioned into the milk reaching levels that were between 1.6‐ and 2.5‐fold higher than the maternal blood. Although the concentration of AEEA equivalents in the maternal blood remained quite consistent, the concentration in the milk fell by almost 40% between lactation days 4 and 12, probably reflecting an increase in milk production over this same period. We confirm exposure of the offspring to AEEA both in utero and during lactation, but that AEEA does not appear to specifically concentrate in the great vessels.     

Increased temperature and altered summer precipitation have differential effects on biological soil crusts in a dryland ecosystem

Biological soil crusts (biocrusts) are common and ecologically important members of dryland ecosystems worldwide, where they stabilize soil surfaces and contribute newly fixed C and N to soils. To test the impacts of predicted climate change scenarios on biocrusts in a dryland ecosystem, the effects of a 2–3 °C increase in soil temperature and an increased frequency of smaller summer precipitation events were examined in a large, replicated field study conducted in the cold desert of the Colorado Plateau, USA. Surface soil biomass (DNA concentration), photosynthetically active cyanobacterial biomass (chlorophyll a concentration), cyanobacterial abundance (quantitative PCR assay), and bacterial community composition (16S rRNA gene sequencing) were monitored seasonally over 2 years. Soil microbial biomass and bacterial community composition were highly stratified between the 0–2 cm depth biocrusts and 5–10 cm depth soil beneath the biocrusts. The increase in temperature did not have a detectable effect on any of the measured parameters over 2 years. However, after the second summer of altered summer precipitation pattern, significant declines occurred in the surface soil biomass (avg. DNA concentration declined 38%), photosynthetic cyanobacterial biomass (avg. chlorophyll a concentration declined 78%), cyanobacterial abundance (avg. gene copies g^?1 soil declined 95%), and proportion of Cyanobacteria in the biocrust bacterial community (avg. representation in sequence libraries declined 85%). Biocrusts are important contributors to soil stability, soil C and N stores, and plant performance, and the loss or reduction of biocrusts under an altered precipitation pattern associated with climate change could contribute significantly to lower soil fertility and increased erosion and dust production in dryland ecosystems at a regional scale.     

Quality control in cytopathology. A system for simultaneous monitoring of accuracy and education and for proficiency testing

Because of its very nature, cytopathology has not lent itself well to quality control. At the University of North Carolina, we practice an internal quality control system based on peer review, interprofessional dialogue and utilization of follow-up information. Recently reported cytology specimens are rescreened and correlated with related surgical specimens. This review is summarized in a weekly conference, at which retrospective accuracy judgments are made for each case under the supervision of the medical director and head cytotechnologist. Diagnostic accuracy is thus monitored. Accuracy is also improved since education is simultaneously monitored. A practical proficiency-testing program built into this system is described.     

Transient association of MCM complex proteins with the nuclear matrix during initiation of mammalian DNA replication

The minichromosome maintenance complex (MCM2-7) is the putative DNA helicase in eukaryotes, and essential for DNA replication. By applying serial extractions to mammalian cells synchronized by release from quiescence, we reveal dynamic changes to the sub-nuclear compartmentalization of MCM2 as cells pass through late G1 and early S phase, identifying a brief window when MCM2 becomes transiently attached to the nuclear-matrix. The data distinguish 3 states that correspond to loose association with chromatin prior to DNA replication, transient highly stable binding to the nuclear-matrix coincident with initiation, and a post-initiation phase when MCM2 remains tightly associated with chromatin but not the nuclear-matrix. The data suggests that functional MCM complex loading takes place at the nuclear-matrix.     

3,7-Dimethyl-1-propargylxanthine: a potent and selective in vivo antagonist of adenosine analogs

3,7-Dimethyl-1-propargylxanthine (DMPX), a caffeine analog that exhibits in vitro selectivity for A2-adenosine receptors, compared to A1-adenosine receptors, has now been investigated with respect to in vivo potency and selectivity. DMPX potently and selectively blocked the actions of the potent A2 adenosine agonist, 5'-N-ethylcarboxamidoadenosine (NECA), in DBA/2 mice, compared to blockade of the same responses elicited by the selective A1-adenosine agonist, N6-cyclohexyladenosine (CHA). DMPX was 57-fold more potent versus NECA-induced hypothermia than versus CHA-induced hypothermia and 11-fold more potent versus NECA-induced behavioral depression than versus CHA-induced behavioral depression. The hypothermia is mediated by peripheral receptors, based on blockade by 8-(p-sulfophenyl)theophylline (PSPT), while the behavioral depression is centrally mediated, based on lack of blockade by PSPT. DMPX was 28- and 15-fold more potent than caffeine in blocking peripheral and central NECA-responses, respectively. DMPX was equipotent with caffeine versus CHA-induced hypothermia and 2.5-fold more potent than caffeine versus CHA-induced behavioral depression. The motor stimulating potency of DMPX (ED50 10 mumol/kg) was slightly greater than caffeine.