Nautilus, Inc.: Dealing With a Lone Wolf in the Sales Team

Headquartered in Vancouver, Washington, Nautilus Inc. is a global fitness products company providing innovative, quality solutions to help people achieve a healthy lifestyle. With a brand portfolio including Nautilus®, Bowflex®, Schwinn®, and Universal®, Nautilus markets innovative fitness products through direct and retail channels (Bean 2013).” Nautilus, Inc. has a strong sales organization with remarkably low turnover of salespeople. They take a consultative approach to sales. Among the group, however, is a “lone wolf” who generally makes transactional sales rather than being consultative and focuses on her individual results rather than the objectives of the company. At the same time, she has the top sales results of the group. She also has an outstanding work ethic and works very hard. She is good at multi-tasking and never takes breaks between calls to rest or socialize with co-workers. There are several potential issues in this case. The objective is to determine how to optimize the effectiveness of the sales organization. Part of that is determining how to deal with the lone wolf. How do her methods and results affect the rest of the sales team? Do her results justify her methods, or is it more important to have everyone following the company direction of consultative selling

Functional Requirements for Heparan Sulfate Biosynthesis in Morphogenesis and Nervous System Development in C. elegans

The regulation of cell migration is essential to animal development and physiology. Heparan sulfate proteoglycans shape the interactions of morphogens and guidance cues with their respective receptors to elicit appropriate cellular responses. Heparan sulfate proteoglycans consist of a protein core with attached heparan sulfate glycosaminoglycan chains, which are synthesized by glycosyltransferases of the exostosin (EXT) family. Abnormal HS chain synthesis results in pleiotropic consequences, including abnormal development and tumor formation. In humans, mutations in either of the exostosin genes EXT1 and EXT2 lead to osteosarcomas or multiple exostoses. Complete loss of any of the exostosin glycosyltransferases in mouse, fish, flies and worms leads to drastic morphogenetic defects and embryonic lethality. Here we identify and study previously unavailable viable hypomorphic mutations in the two C. elegans exostosin glycosyltransferases genes, rib-1 and rib-2. These partial loss-of-function mutations lead to a severe reduction of HS levels and result in profound but specific developmental defects, including abnormal cell and axonal migrations. We find that the expression pattern of the HS copolymerase is dynamic during embryonic and larval morphogenesis, and is sustained throughout life in specific cell types, consistent with HSPGs playing both developmental and post-developmental roles. Cell-type specific expression of the HS copolymerase shows that HS elongation is required in both the migrating neuron and neighboring cells to coordinate migration guidance. Our findings provide insights into general principles underlying HSPG function in development

WISER deliverable D3.1-4: guidance document on sampling, analysis and counting standards for phytoplankton in lakes

Sampling, analysis and counting of phytoplankton has been undertaken in European lakes for more than 100 years (Apstein 1892, Lauterborn 1896, Lemmermann 1903, Woloszynska 1912, Nygaard 1949). Since this early period of pioneers, there has been progress in the methods used to sample, fix, store and analyse phytoplankton. The aim of the deliverable D3.1-4 is to select, harmonize and recommend the most optimal method as a basis for lake assessment. We do not report and review the huge number of European national methods or other published manuals for phytoplankton sampling and analysis that are available. An agreement on a proper sampling procedure is not trivial for lake phytoplankton. In the early 20th century, sampling was carried out using plankton nets. An unconcentrated sample without any pre-screening is required for quantitative phytoplankton analysis, for which various water samplers were developed. Sampling of distinct water depths or an integral sample of the euphotic zone affects the choice of the sampler and sampling procedure. The widely accepted method to quantify algal numbers together with species determination was developed by Utermöhl (1958), who proposed the counting technique using sediment chambers and inverse microscopy. This is the basis for the recently agreed CEN standard “Water quality - Guidance standard on the enumeration of phytoplankton using inverted microscopy (Utermöhl technique)” (CEN 15204, 2006). This CEN standard does not cover the sampling procedure or the calculation of biovolumes for phytoplankton species, although Rott (1981), Hillebrand et al (1999) and Pohlmann & Friedrich (2001) have contributed advice on how to calculate taxa biovolumes effectively. Willén (1976) suggested a simplified counting method, when counting 60 individuals of each species. For the Scandinavian region an agreed phytoplankton sampling and counting manual was compiled, which has been in use for about 20 years (Olrik et al. 1998, Blomqvist & Herlitz 1998). It is very unfortunate that no European guidance on sampling of phytoplankton in lakes was agreed before the phytoplankton assessment methods for the EU-WFD were developed and intercalibrated by Member States. In 2008 an initiative by the European Commission (Mandate M424) for two draft CEN standards on sampling in freshwaters and on calculation of phytoplankton biovolume was unfortunately delayed by administrative difficulties. Recently a grant agreement was signed between the Commission and DIN (German Institute for Standardization) in January 2012 to develop these standards. We believe this WISER guidance document can usefully contribute to these up-coming standards

Causal networks of phytoplankton diversity and biomass are modulated by environmental context

Untangling causal links and feedbacks among biodiversity, ecosystem functioning, and environmental factors is challenging due to their complex and context-dependent interactions (e.g., a nutrient-dependent relationship between diversity and biomass). Consequently, studies that only consider separable, unidirectional effects can produce divergent conclusions and equivocal ecological implications. To address this complexity, we use empirical dynamic modeling to assemble causal networks for 19 natural aquatic ecosystems (N24◦~N58◦) and quantified strengths of feedbacks among phytoplankton diversity, phytoplankton biomass, and environmental factors. Through a cross-system comparison, we identify macroecological patterns; in more diverse, oligotrophic ecosystems, biodiversity effects are more important than environmental effects (nutrients and temperature) as drivers of biomass. Furthermore, feedback strengths vary with productivity. In warm, productive systems, strong nitrate-mediated feedbacks usually prevail, whereas there are strong, phosphate-mediated feedbacks in cold, less productive systems. Our findings, based on recovered feedbacks, highlight the importance of a network view in future ecosystem management

WISER deliverable D3.1-3, part 2: WISER temporal uncertainty analysis for phytoplankton

The broad objective of this analysis has been to quantify and compare the degree of temporal (inter-annual and monthly) and spatial (among countries and waterbodies) variation in lake phytoplankton metrics. The three focal metrics have been chlorophyll a concentration, PTI and total cyanobacterial biovolume. Though some previous studies (e.g. SNIFFER work) have aimed to quantify temporal variation in phytoplankton at the scale of a single lake system, we have attempted the complementary approach of conducting a large-scale (pan- European) analysis that will give a more integrated picture of the degree of temporal uncertainty in phytoplankton metrics

Glypican Is a Modulator of Netrin-Mediated Axon Guidance.

Netrin is a key axon guidance cue that orients axon growth during neural circuit formation. However, the mechanisms regulating netrin and its receptors in the extracellular milieu are largely unknown. Here we demonstrate that in Caenorhabditis elegans, LON-2/glypican, a heparan sulfate proteoglycan, modulates UNC-6/netrin signaling and may do this through interactions with the UNC-40/DCC receptor. We show that developing axons misorient in the absence of LON-2/glypican when the SLT-1/slit guidance pathway is compromised and that LON-2/glypican functions in both the attractive and repulsive UNC-6/netrin pathways. We find that the core LON-2/glypican protein, lacking its heparan sulfate chains, and secreted forms of LON-2/glypican are functional in axon guidance. We also find that LON-2/glypican functions from the epidermal substrate cells to guide axons, and we provide evidence that LON-2/glypican associates with UNC-40/DCC receptor-expressing cells. We propose that LON-2/glypican acts as a modulator of UNC-40/DCC-mediated guidance to fine-tune axonal responses to UNC-6/netrin signals during migration

<i>lon-2</i>/glypican functions in the attractive <i>unc-6</i>/netrin guidance pathway.

<p>(A) During the first larval stage of <i>C</i>. <i>elegans</i>, the pioneer neuron AVM extends ventrally along the body wall until it reaches the ventral nerve cord. Its migration results from the combined attractive response to UNC-6/netrin (secreted at the ventral midline) via the UNC-40/DCC receptor and the repulsive response to SLT-1/Slit (secreted by the dorsal muscles) via its SAX-3/Robo receptor. We visualized the morphology of the AVM axon using the transgene P<i>mec-4</i>::<i>gfp</i>. (B) The heparan sulfate proteoglycans <i>lon-2</i>/glypican and <i>sdn-1</i>/syndecan cooperate to guide the axon of AVM, as their simultaneous loss enhances guidance defects. The role of <i>lon-2</i>/glypican in axon guidance is specific, as the loss of <i>lon-2</i>/glypican, but not the loss of the other <i>C</i>. <i>elegans</i> glypican, <i>gpn-1</i>, enhances the defects of <i>sdn-1</i>/syndecan mutants. (C) Complete loss of <i>lon-2</i>/glypican enhances the axon guidance defects resulting from disrupted <i>slt-1</i>/Slit signaling in mutants for <i>slt-1</i>/Slit or its receptor <i>sax-3</i>/Robo, as well as in animals misexpressing <i>slt-1</i> in all body wall muscles (using a P<i>myo-3</i>::<i>slt-1</i> transgene). Data for wild type and <i>lon-2</i> are the same as in (B). (D) Complete loss of <i>lon-2</i>/glypican does not enhance the AVM guidance defects of <i>unc-6</i>/netrin mutants or of mutants for its receptor <i>unc-40</i>/DCC, suggesting that <i>lon-2</i>/glypican functions in the same genetic pathway as <i>unc-6</i>/netrin. Data for wild type and <i>lon-2</i> are the same as in (B). (E) Loss of <i>sdn-1</i>/syndecan function does not enhance the defects of <i>slt-1</i>/Slit or sax-3/Robo mutants but enhances the defects of <i>unc-</i>40/DCC mutants. Data for wild type, <i>sdn-1</i>, <i>slt-1</i>, <i>sax-3</i>, and <i>unc-40</i> are the same as in (B–D). Error bars are standard error of the proportion. Asterisks denote significant difference\: *** <i>p</i> ≤ 0.001,** <i>p</i> ≤ 0.01, and * <i>p</i> ≤ 0.05 (<i>z</i>-tests, <i>p</i>-values were corrected by multiplying by the number of comparisons). ns, not significant.</p

LON-2/glypican associates with UNC-40/DCC-expressing cells.

<p>(A) Experimental design. Each construct was individually and transiently transfected in S2 cells. After 2 d, cells from independent single transfections were mixed and incubated overnight and then immunostained for the corresponding tags. HA::LON-2-conditioned medium was mixed with UNC-40::FLAG-expressing cells. (B) HA::LON-2 is released from cells that produce it and associates with UNC-40-expressing cells. HA::LON-2 fills the cytoplasm of the cells that produce it (indicated by an asterisk, see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002183#pbio.1002183.s010" target="_blank">S8 Fig</a>). Notably, HA::LON-2 is observed decorating the outline of UNC-40::FLAG-expressing cells (experiments 1, 6, 7, and 8). HA::LON-2ΔGAG also associates with UNC-40::FLAG-expressing cells (experiment 2). Cells expressing UNC-40ΔNt::FLAG that lacks the extracellular domain do not have HA::LON-2 signal, indicating that the association of LON-2 with UNC-40-expressing cells requires the extracellular domain of UNC-40 (experiment 3). HA::LON-2-conditioned medium contains HA::LON-2 that associates with UNC-40::FLAG-expressing cells, indicating that HA::LON-2 is released from the cells that produce into a diffusible form that interacts with UNC-40::FLAG cells (experiment 8). HA::LON-2 does not associate with cells expressing SfGFP::UNC-6 (experiments 4, 6, and 7) or with untransfected cells. UNC-40-FLAG-expressing cells can simultaneously associate with HA::LON-2 and SfGFP::UNC-6 (experiment 6). HA::LON-2 associates with cells expressing a mutant form of UNC-40/DCC that is unable to bind SfGFP::UNC-6, as it lacks the Fn4/5 UNC-6 binding domains (UNC-40ΔFn4/5::FLAG; experiment 7). Scale bars, 10 μm. (C) Quantification of the association of HA::LON-2 (from expressing cells, from medium of expressing cells, or from cells expressing HA::LON-2ΔGAG) with cells expressing UNC-40::FLAG, UNC-40ΔNt::FLAG, SfGFP::UNC-6, or UNC-40ΔFn4/5::FLAG and untransfected cells. Ten different optical fields containing ~300 cells from three independent experiments were quantified and averaged. (D) Cells expressing UNC-40::FLAG can display irregular morphology, which is enhanced by the presence of HA::LON-2. Images of the different morphologies displayed by UNC-40::FLAG-expressing cells: with a smooth edge, with an irregular edge, or with membrane extensions. The morphology of S2 cells expressing mCherry alone or coexpressing UNC-40::FLAG and mCherry, which were mixed with control untransfected cells or with HA::LON-2-expressing cells, were quantified for irregular edges (grey bars) or membrane extensions (black bars). A higher percentage of UNC-40::FLAG-expressing cells show membrane extensions or irregular edges when mixed with HA::LON-2-expressing cells, as compared to when they are mixed with control mCherry cells. Error bars are standard error of the mean. Asterisks denote significant difference: *** <i>p</i> ≤ 0.001, * <i>p</i> ≤ 0.05. ns, not significant. In (D), significant differences in irregular cell shape are indicated by grey asterisks, and significant difference in membrane extensions is indicated by the black asterisk.</p

A secreted form of LON-2/glypican that lacks the heparan sulfate chain attachments is functional in axon guidance.

<p>(A) The HSPG LON-2/glypican is composed of a core protein and three HS chains. The core protein is predicted to fold into a globular domain on its N-terminal region and to be GPI-anchored. (B) Schematics of the engineered forms of LON-2 that we used: LON-2ΔGAG, in which the HS chain attachment sites are mutated; LON-2ΔGPI, in which the GPI anchor is deleted; N-LON-2, in which the C-terminus is deleted; and C-LON-2, in which the N-terminal globular domain is deleted. Western blot analysis of protein extracts of worms expressing LON-2::GFP or LON-2ΔGAG::GFP confirms that deleting the HS attachment sites on LON-2 affects HS addition on LON-2. Protein extracts from wild type (N2) and an unrelated GFP strain (<i>lqIs4</i>) are negative controls. (C) A form of LON-2/glypican lacking HS chain attachment sites (LON-2ΔGAG) functions in axon guidance. LON-2ΔGAG rescues the AVM guidance defects of double mutants <i>lon-2 slt-1</i> back to the level of <i>slt-1</i> single mutants. Secreted globular LON-2/glypican is functional in axon guidance. LON-2/glypican was engineered to be secreted by deleting its GPI anchor (LON-2ΔGPI) or by deleting the C-terminus, thus lacking the GPI anchor and the HS attachment sites (N-LON-2). Both LON-2ΔGPI and N-LON-2 function in axon guidance, as assayed by their ability to rescue axon guidance defects of <i>lon-2 slt-1</i> back down to the level of <i>slt-1</i> single mutants. In contrast, a form of LON-2/glypican containing its C-terminus including the three HS attachment sites, but lacking its N-terminal globular domain (C-LON-2), is not functional (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002183#pbio.1002183.s013" target="_blank">S2</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002183#pbio.1002183.s014" target="_blank">S3</a> Tables), indicating that the N-terminal globular domain of the core protein is key to the function of LON-2/glypican in axon guidance. For each rescued transgenic line, transgenic animals were compared to nontransgenic sibling controls (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002183#pbio.1002183.s013" target="_blank">S2</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002183#pbio.1002183.s014" target="_blank">S3</a> Tables). Data for wild type, <i>lon-2</i>, <i>slt-1</i>, and <i>lon-2 slt-1</i> are the same as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002183#pbio.1002183.g001" target="_blank">Fig 1B and 1C</a>. (D) A form of LON-2/glypican lacking HS chain attachment sites is functional in DTC guidance. The DTC migration of <i>lon-2</i> mutants carrying the transgene P<i>lon-2</i>::LON-2ΔGAG is rescued back to wild-type levels. Secreted N-terminus globular LON-2/glypican (N-LON-2) is functional in DTC guidance, as DTC guidance defects of <i>lon-2</i> mutants are rescued by N-LON-2. Transgenic animals were compared to nontransgenic sibling controls (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002183#pbio.1002183.s018" target="_blank">S7 Table</a>). Data for wild type and <i>lon-2</i> are the same as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002183#pbio.1002183.g002" target="_blank">Fig 2B</a>. Error bars are standard error of the proportion. Asterisks denote significant difference: *** <i>p</i> ≤ 0.001, ** <i>p</i> ≤ 0.01 (<i>z</i>-tests, <i>p</i>-values were corrected by multiplying by the number of comparisons).</p

A model for the role of LON-2/glypican in UNC-6/netrin-UNC-40/DCC-mediated axon guidance.

<p>HSPG LON-2/glypican (red) is expressed from the hyp7 epidermal cells (pink) underlying the migrating growth cone of the AVM neuron (tan). LON-2/glypican is released from the hypodermal cell surface and may associate with the developing axon expressing the receptor UNC-40/DCC (blue), directly or indirectly interacting with UNC-40/DCC, to modulate UNC-6/netrin (green) signaling. A second HSPG, SDN-1/syndecan (black), acts in the SLT-1/Slit-SAX-3/Robo (grey) axon guidance pathway.</p