Lipid raft redistribution and morphological cell polarization are separable processes providing a basis for hematopoietic stem and progenitor cell migration

Freshly isolated human hematopoietic stem and progenitor cells (HSPCs) are small and round cells which upon cultivation adopt a polarized morphology and redistribute certain cell surface antigens. To functionally dissect this polarization process, we addressed impacts of protein synthesis, HSPC trafficking, cytoskeleton organization or lipid raft integrity on the establishment and maintenance of the cell polarity of human HSPCs. Effects on the morphology, sub-cellular distribution of lipid raft-associated molecular polarization markers (Flotillin-1, Flotillin-2, ICAM-3) and in vitro migration capabilities of treated cells were studied. We could distinguish two levels of cellular polarization, a molecular and a morphological level. Our data suggest that protein synthesis, lipid raft integrity and enzymatic activities of PI3K and aPKC are required to organize the molecular cell polarity. The morphological cell polarization process, however, also depends on actin polymerization and rho-GTPase activities. In summary, our data qualify HSPC polarization processes as new pharmaceutical target to interfere with migratory and with homing capabilities of HSPCs.     

Echinococcus granulosus tegumental enzymes as in vitro markers of pharmacological damage: A biochemical and molecular approach

Cystic echinococcosis is a chronic, complex, and neglected disease. Novel therapeutical tools are needed to optimize human treatment. A number of compounds have been investigated, either using in vitro cultured parasites and/or applying in vivo rodent models. Although some of these compounds showed promising activities in vitro, and to some extent also in the rodent models, they have not been translated into clinical applications. Membrane enzyme activities in culture supernatants of treated protoscoleces with calcium modulator drugs and anthelmintic drugs were measured and provided an indication of compound efficacy. This work describes for the first time the detection of alkaline phosphatase, gamma-glutamyl-transpeptidase and acetylcholinesterase activities in supernatants of in vitro treated Echinococcus granulosus protoscoleces. Marked differences on the enzymatic activities in supernatants from drug treated cultures were detected. We demonstrated that those genes that show the highest degree of conservation when compared to orthologs, are constitutively and highly expressed in protoscoleces and metacestodes. Due to high sensibility and the lack of activity in supernatants of intact protoscoleces, gamma-glutamyl-transpeptidase is proposed as the ideal viability marker during in vitro pharmacological studies against E. granulosus protoscoleces.     

Continuous and noninvasive recording of cardiovascular parameters with the Finapres finger cuff enhances undergraduate student understanding of physiology

The Finapres finger cuff recording system provides continuous calculations of beat-to-beat variations in cardiac output (CO), total peripheral resistance, heart rate (HR), and blood pressure (BP). This system is unique in that it allows experimental subjects to immediately, continuously, and noninvasively visualize changes in CO at rest and during exercise. This study provides evidence that using the Finapres system improves undergraduate student engagement, understanding, and learning of how the cardiovascular system responds to exercise. Second-year science students undertaking a physiology practical class in 2009 (n = 243) and 2010 (n = 263) used the Finapres system to record CO, BP, and HR during graded exercise on a cycle ergometer. Student experiences with the Finapres was evaluated with a survey (a 5-point scale from strongly disagree to strongly agree). This indicated that students appreciated the immediacy of the recordings (88% of students agreed or strongly agreed, average for 2009 and 2010), gained an understanding of how to record physiological data (84%), enjoyed the practical (81%), and would recommend the Finapres to other students (81%). To determine if the practical enhanced student learning of cardiovascular physiology, identical tests were given to the students at the beginning (pretest) and end (posttest) of the class. There was a significant improvement from the pretest to the posttest (4% in 2009 and 20% in 2010). In summary, the ability of the Finapres to continuously display CO, BP, and HR during experimental protocols provides students with immediate feedback and improves their understanding of cardiovascular physiology.     

Comparing the hyper-variable V4 and V9 regions of the small subunit rDNA for assessment of ciliate environmental diversity

The hyper-variable V4 and V9 regions of the small subunit (SSU) rDNA have been targeted for assessing environmental diversity of microbial eukaryotes using next generation sequencing technologies. Here, we explore how the genetic distances among these short fragments compare with the distances obtained from near full-length SSU-rDNA sequences by comparing all pairwise estimates, as well as within and among species of ciliates. Results show that pairwise distances from V4 more closely match the near full-length SSU-rDNA and are more comparable with previous studies based on much longer SSU-rDNA fragments, then pairwise distances from V9. Thus, studies that use the V4 will estimate similar values of phylotype richness and community structure as would have been estimated using the full-length SSU-rDNA.     

Configuration of a high-content imaging platform for hit identification and pharmacological assessment of JMJD3 demethylase enzyme inhibitors

The biological complexity associated with the regulation of histone demethylases makes it desirable to configure a cellular mechanistic assay format that simultaneously encompasses as many of the relevant cellular processes as possible. In this report, the authors describe the configuration of a JMJD3 high-content cellular mechanistic imaging assay that uses single-cell multiparameter measurements to accurately assess cellular viability and the enzyme-dependent demethylation of the H3K27(Me)3 mark by exogenously expressed JMJD3. This approach couples robust statistical analyses with the spatial resolving power of cellular imaging. This enables segregation of expressing and nonexpressing cells into discrete subpopulations and consequently pharmacological quantification of compounds of interest in the expressing population at varying JMJD3 expression levels. Moreover, the authors demonstrate the utility of this hit identification strategy through the successful prosecution of a medium-throughput focused campaign of an 87 500-compound file, which has enabled the identification of JMJD3 cellular-active chemotypes. This study represents the first report of a demethylase high-content imaging assay with the ability to capture a repertoire of pharmacological tools, which are likely both to inform our mechanistic understanding of how JMJD3 is modulated and, more important, to contribute to the identification of novel therapeutic modalities for this demethylase enzyme.     

The three isoforms of nitric oxide synthase distinctively affect mouse nocifensive behavior

Nitric oxide synthases (NOSs) have been shown to modulate thermal hyperalgesia and mechanical hypersensitivity in inflammatory and neuropathic pain. However, little is known about the effect of NOSs on baseline function of sensory nerve fibers. Using genetic deficiency and pharmacologic inhibition of NOSs, we examined the impact of the three isoforms NOS1, NOS2, and NOS3 on baseline nocifensive behavior by measuring current vocalization threshold in response to electrical stimulation at 5, 250, 2000 Hz that preferentially stimulate C, Aδ, and Aβ fibers. In response to 5, 250 and 2000 Hz, NOS1-deficient animals had significantly higher current vocalization thresholds compared with wild-type. Genetic deficiency of NOS2 was associated with higher current vocalization thresholds in response to 5 Hz (C-fiber) stimulation. In contrast, NOS3-deficient animals had an overall weak trend toward lower current vocalization thresholds at 5 Hz and significantly lower current vocalization threshold compared with wild-type animals at 250 and 2000 Hz. Therefore, NOSs distinctively affect baseline mouse current vocalization threshold and appear to play a role on nocifensive response to electrical stimulation of sensory nerve fibers.     

Role of the SEL1L:LC3-I complex as an ERAD tuning receptor in the mammalian ER

Several regulators of endoplasmic reticulum (ER)-associated degradation (ERAD) have a shorter half-life compared to conventional ER chaperones. At steady state, they are selectively removed from the ER by poorly defined events collectively referred to as ERAD tuning. Here we identify the complex comprising the type-I transmembrane protein SEL1L and the cytosolic protein LC3-I as an ERAD tuning receptor regulating the COPII-independent, vesicle-mediated removal of the lumenal ERAD regulators EDEM1 and OS-9 from the ER. Expression of?folding-defective polypeptides enhances the lumenal content of EDEM1 and OS-9 by inhibiting their SEL1L:LC3-I-mediated segregation. This raises ERAD activity in the absence of UPR-induction. The mouse hepatitis virus (MHV) subverts ERAD tuning for replication. Consistently, SEL1L or LC3 silencing impair the MHV life cycle. Collectively, our data provide new molecular information about the ERAD tuning mechanisms that regulate ERAD in mammalian cells at the post translational level and how these mechanisms are hijacked by a pathogen.     

Eco-label conveys reliable information on fish stock health to seafood consumers

Concerns over fishing impacts on marine populations and ecosystems have intensified the need to improve ocean management. One increasingly popular market-based instrument for ecological stewardship is the use of certification and eco-labeling programs to highlight sustainable fisheries with low environmental impacts. The Marine Stewardship Council (MSC) is the most prominent of these programs. Despite widespread discussions about the rigor of the MSC standards, no comprehensive analysis of the performance of MSC-certified fish stocks has yet been conducted. We compared status and abundance trends of 45 certified stocks with those of 179 uncertified stocks, finding that 74% of certified fisheries were above biomass levels that would produce maximum sustainable yield, compared with only 44% of uncertified fisheries. On average, the biomass of certified stocks increased by 46% over the past 10 years, whereas uncertified fisheries increased by just 9%. As part of the MSC process, fisheries initially go through a confidential pre-assessment process. When certified fisheries are compared with those that decline to pursue full certification after pre-assessment, certified stocks had much lower mean exploitation rates (67% of the rate producing maximum sustainable yield vs. 92% for those declining to pursue certification), allowing for more sustainable harvesting and in many cases biomass rebuilding. From a consumer's point of view this means that MSC-certified seafood is 3-5 times less likely to be subject to harmful fishing than uncertified seafood. Thus, MSC-certification accurately identifies healthy fish stocks and conveys reliable information on stock status to seafood consumers.