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The C. elegans MELK ortholog PIG-1 regulates cell size asymmetry and daughter cell fate in asymmetric neuroblast divisions
Shaun Cordes, C. Andrew Frank, Gian Garriga
Development 2006 133: 2747-2756; doi: 10.1242/dev.02447
Shaun Cordes
C. Andrew Frank
Gian Garriga
In the nematode Caenorhabditis elegans, neurons are generated from asymmetric divisions in which a mother cell divides to produce daughters that differ in fate. Here, we demonstrate that the gene pig-1 regulates the asymmetric divisions of neuroblasts that divide to produce an apoptotic cell and either a neural precursor or a neuron. In pig-1 mutants, these neuroblasts divide to produce daughters that are more equal in size, and their apoptotic daughters are transformed into their sisters, leading to the production of extra neurons. PIG-1 is orthologous to MELK, a conserved member of the polarity-regulating PAR-1/Kin1/SAD-1 family of serine/threonine kinases. Although MELK has been implicated in regulating the cell cycle, our data suggest that PIG-1, like other PAR-1 family members, regulates cell polarity.
Asymmetric cell division
PIG-1
HAM-1
Cell polarity
Cell fate
Programmed cell death
Much of metazoan cellular diversity is generated by asymmetric cell divisions in which a polarized mother cell divides to produce daughters that adopt different fates (Horvitz and Herskowitz, 1992). The C. elegans zygote requires the serine/threonine kinase PAR-1 to establish polarity along the presumptive anteroposterior (AP) axis. Loss of PAR-1 activity disrupts asymmetric positioning of the mitotic spindle and the asymmetric distribution of cell fate determinants to daughter cells (Guo and Kemphues, 1995; Kemphues et al., 1988). PAR-1 orthologs in other organisms also regulate cell polarity. In Drosophila, PAR-1 polarizes the microtubule cytoskeleton in the germ line and establishes the embryonic AP axis by regulating the asymmetric localization of cell fate determinants in the oocyte (Cox et al., 2001; Shulman et al., 2000; Tomancak et al., 2000). PAR-1 also regulates epithelial polarity, both in the Drosophila follicular epithelium (Doerflinger et al., 2003) and in cultured MDCK cells (Cohen et al., 2004).
PAR-1 is a member of a superfamily of serine/threonine kinases that regulate cell polarity. In addition to PAR-1, this group includes Kin1p from Schizosaccharomyces pombe and the conserved SAD-1 family. Kin1p regulates bipolar growth and the correct positioning of the nucleus and cell cleavage plane (Drewes and Nurse, 2003; La Carbona et al., 2004). Nematode SAD-1 may regulate neural polarity (Crump et al., 2001), and the mouse homologs of SAD-1 are required for neurons to develop processes with normal dendritic and axonal properties (Kishi et al., 2005). Expression of vertebrate PAR-1 homologs in S. pombe can partially rescue kin1 phenotypes, suggesting an ancestral role for this kinase family in regulating cell polarity (Drewes and Nurse, 2003).
Another member of the PAR-1/Kin1/SAD-1 superfamily of serine/threonine kinases is Maternal Embryonic Leucine zipper Kinase (MELK), also known as pEg3 kinase and MPK38 (Blot et al., 2002; Gil et al., 1997; Heyer et al., 1997). MELK proteins share sequence homology and a similar domain architecture with PAR-1 family members. Both contain an N-terminal kinase domain and a C-terminal kinase-associated region. MELK was originally identified in a screen for maternal genes expressed during preimplantation of the mouse embryo (Heyer et al., 1997). MELK is also expressed in other vertebrate tissues, especially in proliferating cell populations, including the blast cells of the early embryo (Heyer et al., 1999), embryonic stem cells (Nakano et al., 2005), adult germ cells (ovaries and spermatogonia) (Heyer et al., 1999), hematopoietic stem cells (Easterday et al., 2003; Saito et al., 2005) and neural stem cells (Easterday et al., 2003; Nakano et al., 2005). MELK has been implicated in the regulation of spliceosome assembly (Vulsteke et al., 2004), gene expression (Saito et al., 2005) and cell proliferation (Davezac et al., 2002; Gray et al., 2005; Nakano et al., 2005). Its role in regulating cell division is controversial, with conflicting evidence suggesting it has both stimulatory and inhibitory functions.
In a screen for C. elegans mutants with defective asymmetric neuroblast divisions, we identified alleles of the gene pig-1 (par-1-like gene), which encodes the worm ortholog of MELK. We show that PIG-1 controls cell size asymmetry and neuroblast daughter cell fate in certain neuroblast lineages. PIG-1 acts cell autonomously in affected lineages, and we propose that PIG-1 regulates asymmetric neuroblast divisions by controlling neuroblast polarity. These data are the first to implicate MELK family proteins in regulating cell polarity.
C. elegans genetics
Nematodes were cultured as previously described (Brenner, 1974). N2 Bristol was the wild-type strain used in this study, and experiments were performed at 20°C. The following mutations and transgenic arrays were used.
LG I: ynIs45 [Pflp-15::gfp] (Kim and Li, 2004), zdIs5 [mec-4::gfp] (Clark and Chiu, 2003), kyIs39 [Psra-6::gfp] (Troemel et al., 1995); LG II: ynIs25 [Pflp-12::gfp] (Kim and Li, 2004; Li et al., 1999a), rrf-3(pk1426) (Simmer et al., 2002), juIs76 [Punc-25:gfp] (Jin et al., 1999), gmIs20 [hlh-14::gfp] (Frank et al., 2003);
LG III: gmIs12 [Psrb-6::gfp] (Hawkins et al., 2005; Troemel et al., 1995);
LG IV: pig-1 (gm280, gm300, gm301, gm344) (this study), ced-3(n717) (Ellis and Horvitz, 1986), ham-1(n1811, n1438, gm214, gm279) (Desai et al., 1988; Frank et al., 2005; Guenther and Garriga, 1996);
LG V: egl-1(n1084 n3082) (Conradt and Horvitz, 1998), ayIs9 [Pegl-17::gfp] (Branda and Stern, 2000), muIs102 [Pgcy-32::gfp] (Yu et al., 1997) (Y. Lie and C. Kenyon, personal communication). gmIs22 [nlp-1::gfp] (Frank et al., 2003; Li et al., 1999b);
Unmapped: mgIs21 [Plin-11::gfp] (Hobert et al., 1998); and Extrachromosomal arrays: gmEx320 and gmEx321 [Ppig-1::gfp; rol-6(d)], gmEx326 [Ppig-1::pig-1::gfp; rol-6(d)], gmEx394, -95, and -96 [Pmab-5::pig-1::gfp; Pdpy-30::NLS::dsRed2; rol-6(d)] (this study).
Isolation of pig-1 mutations
The pig-1 alleles gm280, gm300 and gm301 were isolated in forward genetic screens for mutations that altered the number of PHB neurons. Mutagenesis was carried out as described by Brenner (Brenner, 1974). Mutagenized gmIs12 P0 worms were allowed to lay eggs. After several days, we transferred 50 F1 animals onto fresh plates. From each plate we transferred 20 F2 hermaphrodites to individual plates and screened their progeny. We screened the progeny of 2400 F2 hermaphrodites.
pig-1(gm344) was isolated from a library of worms mutagenized with UV-trimethylpsoralen. The construction of this mutant library has been described (Withee et al., 2004). The gm344 breakpoints are defined by the following two sequences that flank the 524 bp deletion: 5′-AGCTCGTGTCAGGACGCGAA-3′ and 5′-TGGCCACCTTTTGATTTGTC-3′.
Detection and analysis of specific neurons
The HSN and NSM neurons were detected by staining adult hermaphrodites with rabbit anti-serotonin antibodies as previously described (Garriga et al., 1993). All other neurons were detected with the following integrated transcriptional reporters that express GFP under control of the indicated C. elegans promoter. PHB neurons: gmIs12 [Psrb-6::gfp] (Hawkins et al., 2005; Troemel et al., 1995), which is expressed both in PHB and the adjacent PHA neurons, and gmIs22 [nlp-1::gfp], which is PHB-specific (Frank et al., 2003; Li et al., 1999b). PHA, I2 and CAN neurons: ynIs45 [flp-15::gfp] (Kim and Li, 2004). AQR, PQR and URX neurons: muIs102 [Pgcy-32::gfp] (Yu et al., 1997) (Y. Lie and C. Kenyon, personal communication). ALM, AVM, PVM and PLM neurons: zdIs5 [mec-4::gfp] (Clark and Chiu, 2003). SDQ, SMBD and SMBV neurons: ynIs25 [flp-12::gfp] (Kim and Li, 2004; Li et al., 1999a). M4 neuron: ayIs9 [Pegl-17::gfp] (Branda and Stern, 2000). PVQ neuron: kyIs39 [Psra-6::gfp] (Troemel et al., 1995). VC neurons: mgIs21 [Plin-11::gfp] (Hobert et al., 1998). VD and DD neurons: juIs76 [unc-25::gfp] (Jin et al., 1999).
Immunostaining, dye filling and microscopy
Live animals were observed as previously described (Sulston and Horvitz, 1977). Embryos were fixed and stained as described by Guenther and Garriga (Guenther and Garriga, 1996). Dye filling of the phasmid neurons with DiI was performed as previously described (Hedgecock et al., 1987). Worms were examined using a Zeiss Axioskop 2 microscope. Images were collected using an ORCA-ER CCD camera (Hammamatsu) and Openlab imaging software (Improvision). Images were prepared for publication using Adobe PhotoShop (Adobe Systems).
Lineage analysis
L1 animals expressing zdIs5[mec-4::gfp] were mounted and followed by Nomarski optics as previously described (Sulston and Horvitz, 1977). We began following the left or right Q lineage in animals where the Q cell had divided once. After observing an animal, it was transferred to a plate with food. When the animal reached the L4 stage, we used epifluorescence to score the number of A/PVM neurons.
Cloning PIG-1
Single nucleotide polymorphism mapping was used to position pig-1 on the left arm of LG IV between snp_Y38C1A.2 and snp_K11H12.1 (Wicks et al., 2001). We then used RNAi to systematically inactivate each of the ORFs in this region by feeding bacterial strains expressing dsRNA complementary to these ORFs to gmIs12; ced-3(n717) hermaphrodites (Kamath et al., 2001; Timmons and Fire, 1998). RNAi feeding strains used to inactivate genes in this region were obtained from the library designed by the Ahringer Laboratory (Fraser et al., 2000).
Analysis of pig-1 transcripts
We characterized the transcript produced by the pig-1 open reading frame by sequencing eight cDNA clones (yk554F7, yk338H11, yk400B12, yk150E3, yk13C10, yk88F11, yk516B10, yk167G5) kindly provided by Y. Kohara (National Institute of Genetics, Mishima, Japan). To characterize the 5′ terminus of the pig-1 message, we amplified a pig-1 cDNA from an oligo(dT)-primed embryonic cDNA library using the SL1 splice-leader-specific forward primer pSL1 (5′-GGTTTAATTACCCAAGTTTG-3′) and a pig-1-specific reverse primer W03G16N (5′-gagatccacaacacgtatcc-3′). Sequencing this PCR product revealed that SL1 is trans-spliced to the pig-1 message one nucleotide upstream of the pig-1 start codon.
Sequence alignments were performed using ClustalX (Thompson et al., 1997) and dendograms were generated with NJplot (Perriere and Gouy, 1996). The GenBank Accession numbers for sequences used in phylogenetic analyses are: Homo sapiens MELK, NP_055606; MARK2 (PAR-1), NP_059672; SAD1A, AAS86442; SAD1B, AAS86443; Mus musculus MELK, NP_034920; Xenopus laevis pEG3 (MELK), CAA78913; Danio rerio MELK, BAC75706; C. elegans PAR-1, NP_001024019; PIG-1, NP_001023420; SAD-1, NP_510253; Drosophila melanogaster PAR-1, AAF69801; CG6114 (SAD-1), NP_648814; S. pombe kin1, P22987.
Plasmid construction and transgenic strains
To generate the pig-1 transcriptional fusion (Ppig-1::gfp), we amplified the intergenic region between pig-1 and the nearest upstream gene, and cloned the product into the GFP vector pPD95.77 (A. Fire, S. Xu, J. Ahnn and G. Seydoux, personal communication). gmEx320 and gmEx321 were generated by injecting pPD95.77::Ppig-1 (50 ng/μl) and the co-injection marker pRF4 [rol-6(d); 50 ng/μl] (Mello et al., 1991) into N2 hermaphrodites.
To generate the pig-1 translational fusion (Ppig-1::pig-1::gfp), we amplified a full-length pig-1 cDNA from plasmid yk400B12 and cloned the product between the pig-1 promoter region and GFP coding sequences. gmEx326 was generated by injecting this plasmid into N2 hermaphrodites at 50 ng/μl with 50 ng/μl pRF4.
The Pmab-5::pig-1::gfp construct was generated by ligating a full-length pig-1 cDNA into plasmid pPD95.77::Pmab-5. pPD95.77::Pmab-5 contains the entire mab-5 promoter driving expression of GFP (Fleming et al., 2005). gmEx394, gmEx395 and gmEx396 were generated by injecting this plasmid into zdIs5; pig-1(gm344) hermaphrodites at 10 ng/μl with 50 ng/μl of pRF4 and 50 ng/μl of plasmid Pdpy-30::NLS::DsRed2.
The Pdpy-30::NLS::DsRed2 plasmid was constructed by first amplifying the dpy-30 promoter from plasmid pTY1003 (a gift from Barbara Meyer, University of California, Berkeley, CA, USA) using a reverse primer that contained a start codon followed by the SV40 T antigen nuclear localization signal (NLS) (Kalderon et al., 1984). This product was cloned into vector pPD95.75::DsRed2 (Bulow et al., 2004).
Analysis of neuroblast daughter size
The daughters of the HSN/PHB neuroblast were identified using gmIs20, a nuclear-localized translational fusion between hlh-14 and GFP (Frank et al., 2003). The size of nuclei in these cells is proportional to cell size: these cells have very little cytoplasm and are roughly spherical (Frank et al., 2005). Nuclear diameter was calculated by averaging the length of the long axis of a nucleus with the length of the perpendicular axis of the same nucleus.
The daughters of the Q.p neuroblast were identified using ayIs9 (Pegl-17::gfp) (Branda and Stern, 2000). We measured cell area in a single plane of focus. These cells are extremely flat, and thus measurements of cell area are good approximations of cell size. We calculated cell area by circumscribing the cell and measuring its interior area with Openlab software (Improvision). We averaged two measurements per cell.
Isolation and initial characterization ofpig-1 mutants
To identify molecules that regulate asymmetric neuroblast divisions in C. elegans, we screened for mutants with defective asymmetric division of the HSN/PHB neuroblast. In wild-type embryos, the bilaterally symmetric HSN/PHB neuroblast divides to produce a smaller anterior daughter that undergoes programmed cell death and a larger posterior daughter, the HSN/PHB precursor (Fig. 1D). Each precursor subsequently divides to produce an HSN, a serotonergic motoneuron that stimulates egg laying by the hermaphrodite, and a PHB, a chemosensory neuron. The primary goal of our screen was to identify mutations that transform one neuroblast daughter into the other, and thereby alter the number of HSN and PHB neurons produced.
pig-1 mutants have extra neurons derived from multiple lineages. (A) Schematic diagram of an L1 hermaphrodite: anterior to the left, dorsal up. Neurons produced from lineages affected in pig-1 mutants are indicated. (B) Lineages affected in pig-1 mutants. NB, neuroblast. PQR, PVM and SDQL are produced by the left Q neuroblast, and AQR, AVM and SDQR are produced by the right Q neuroblast. Note that the PVQ lineage is not affected in pig-1 mutants but is shown to indicate its relationship to the HSN/PHB lineage. (C) Lineages unaffected in pig-1 mutants. Neurons that were examined are shaded black. DD1,3,5 are produced on the left side of the body, and DD2,4,6 on the right. (B,C) Neurons were identified as described in the Materials and methods. Cells that were not scored are unshaded. (D-H) pig-1 mutants have extra neurons derived from the HSN/PHB neuroblast lineage. (D) Schematic diagram of the wild-type HSN/PHB neuroblast lineage. (E,F) Images of wild-type (E) and pig-1(gm301) (F) hermaphrodites stained with an anti-serotonin antiserum to visualize the HSNs (arrows). (G,H) Composite fluorescence and DIC images of wild-type (G) and pig-1(gm301) (H) hermaphrodites expressing the PHB-specific reporter gmIs22 [nlp-1::gfp] (arrows). Scale bar: 10 μm.
We screened for mutants with abnormal numbers of PHBs using the reporter gmIs12, which expresses GFP in two neurons in the tail: PHA and PHB (Hawkins et al., 2005; Troemel et al., 1995). Three maternal-effect recessive alleles of pig-1 were isolated in these screens: gm280, gm300 and gm301. A deletion allele, gm344, was subsequently isolated using a reverse-genetic approach. These mutants are relatively healthy but have a low penetrance of extra gmIs12- positive cells in the tail that we determined were extra PHB neurons by using PHA- or PHB-specific GFP reporters (Fig. 1G,H; Fig. 2A,B). If the extra PHB phenotype of pig-1 mutants resulted from the production of an extra HSN/PHB precursor, pig-1 mutants should also produce a low frequency of extra HSNs. Staining pig-1 mutants with an anti-serotonin antibody verified this prediction (Fig. 1E,F; Fig. 2A).
Could pig-1 act prior to the birth of the HSN/PHB neuroblast to regulate the production of HSNs and PHBs? The transcription factor hlh-14, for example, specifies the fate of the progenitor that generates the HSN/PHB neuroblast and its sister the PVQ neuroblast (Fig. 1B). In hlh-14 mutants, the HSN, PHB and PVQ neurons are lost (Frank et al., 2003). We reasoned that pig-1 mutant lineages would produce extra PVQs if the progenitor that produces the HSN/PHB and PVQ neuroblasts were duplicated, or fewer PVQs if the PVQ neuroblast were transformed into its sister, the HSN/PHB neuroblast. Using the PVQ-specific GFP reporter kyIs39 [Psra-6::gfp] (Troemel et al., 1995), we scored the number of PVQs in pig-1 mutants and found no deviation from wild type [pig-1(gm280), n=138; pig-1(gm301), n=118]. These observations suggest that the extra HSNs and PHBs in pig-1 mutants arise because of defects in the HSN/PHB neuroblast or its descendants and not because of earlier lineage defects. The data are consistent with a model where pig-1 mutations transform the anterior daughter of the HSN/PHB neuroblast, which is normally destined to die, into a second HSN/PHB precursor.
pig-1 mutants have extra neurons derived from other lineages
To address whether pig-1 mutations perturb other lineages, we scored the number of cells produced using cell-specific GFP reporters and identified six lineages that produce extra neurons in pig-1 mutants: the HSN/PHB, I2, M4 and PLM/ALN precursors divide during embryogenesis, and the Q.a and Q.p precursors divide during the first larval stage (Fig. 1, Fig. 2A, Fig. 3). Many other lineages appear to be unaffected (Fig. 1C). The only feature shared by the affected lineages is the presence of an apoptotic cell. Our observations are consistent with a model where mutations in pig-1 transform these cells into their sisters.
pig-1 ced-3 and ham-1 HSN and PHB phenotypes. (A) pig-1 mutants have extra phasmid (PHA or PHB) neurons and extra HSNs. Mutations in pig-1 interact synergistically with mutations in ced-3 to control the penetrance of extra neurons. The number of PHA/Bs was scored using the integrated reporter gmIs12 [srb-6::gfp], and the number of HSNs was scored by immunostaining with an anti-serotonin antiserum. (B) The extra phasmid neurons in pig-1 mutants are PHBs. The numbers of PHA and PHB neurons were determined using the reporters gmIs22[nlp-1::gfp] and ynIs45[flp-15::gfp], respectively. (C) Mutations in pig-1 are epistatic to ham-1. (D) pig-1 is epistatic to ham-1 in a ced-3 background. (C,D) The numbers of HSNs and PHBs were scored as described in A. For all genotypes, the number of lineages scored is shown below the histogram in parentheses.
pig-1 regulates cell fate independent of the canonical programmed cell death pathway
The core programmed cell death machinery in C. elegans consists of four conserved proteins: EGL-1, CED-9, CED-4 and CED-3 (for reviews, see Conradt and Xue, 2005; Kaufmann and Hengartner, 2001). In wild-type animals, the BCL2-like protein CED-9 sequesters the Apaf1-like protein CED-4 to mitochondria, preventing it from interacting with the caspase CED-3. Somatic cells that undergo programmed cell death express the BH3-domain protein EGL-1. EGL-1 binds to CED-9, resulting in the release of CED-4 from mitochondria and CED-3 activation.
Two observations suggested that pig-1 could be a component of the programmed cell death pathway. First, the low frequency of extra HSNs and PHBs observed in pig-1 mutants closely approximates that observed in ced-3 and ced-4 mutants (Guenther and Garriga, 1996). Second, all of the lineages affected by pig-1 mutations produce apoptotic cells. We hypothesized that if pig-1 acted in a linear pathway with ced-3, animals with mutations in both pig-1 and ced-3 would have the same phenotype as the single mutants. Alternatively, if pig-1 and ced-3 acted in parallel, then the double mutant would have a stronger phenotype than the individual single mutants. We found that pig-1 ced-3 double mutants had a dramatically stronger phenotype than either single mutant, resulting in nearly 100% of lineages producing extra HSNs and PHBs (Fig. 2A,B). Synergistic interactions between mutations in pig-1 and ced-3 were also observed in three other lineages (Q.p, I2 and PLM/ALN) that we analyzed (Fig. 3A,C,D). pig-1 mutations also interacted synergistically with a loss-of-function mutation in egl-1 in the Q.p and PLM lineages (Fig. 3A,D). Together, these data indicate that pig-1 acts at least partly in parallel to the cell death pathway. Our favored interpretation of these results is that mutations in pig-1 almost always transform the apoptotic daughter of the neuroblast into its sister, but that this transformation is usually masked by apoptosis. When this mask is removed by a mutation in a cell death gene like ced-3, the underlying transformation is revealed.
Lineage analysis of pig-1
We postulated that pig-1 mutants have extra neurons because the cells normally fated to die in affected lineages occasionally survive and are transformed into their sister cells. To test this hypothesis, we followed the divisions of the left Q.p neuroblast, which divides to produce a posterior cell that dies (Q.pp) and the precursor to the PVM and SDQ neurons (Fig. 1B). In four out of seven Q.p divisions that we followed in pig-1(gm344) larvae, both neuroblast daughters survived and divided to produce two extra neurons. Because we followed the divisions in zdIs5 [mec-4::gfp]; pig-1 mutants, we determined whether the lineages that we followed produced extra PVM neurons. There was a perfect correlation between the ability of Q.pp to survive and divide and the presence of extra PVMs. In the four divisions where Q.pp survived and divided, the lineage produced an extra PVM. In the three divisions where Q.pp died, the lineage produced a single PVM. We also noted that the sizes of the pig-1 Q.p daughter nuclei were similar, which is different from wild type where the Q.pp nucleus is much smaller than the Q.pa nucleus.
To verify that the ectopic divisions of the surviving cells in pig-1 mutants could not be explained merely by defective apoptosis, we followed the divisions of six Q.p neuroblasts in zdIs5; ced-3 mutants. In all of these lineages, Q.pp survived but did not divide, and none produced an extra AVM or PVM. As in wild type, the ced-3 Q.p division was highly asymmetric, producing a large Q.pa nucleus and a small Q.pp nucleus. These observations are consistent with previous analyses of the Ced-3 phenotype in other lineages (Ellis and Horvitz, 1986; Guenther and Garriga, 1996). Together, they indicate that the apoptotic daughters of neuroblasts have different fates than their sisters, independent of their ability to survive. These data support the model that the extra neurons observed in pig-1 mutants were generated by the inappropriate survival and transformation of cells that normally die into their sisters.
pig-1 regulates additional neuroblast lineages. (A-D) Neurons were identified as described in the Materials and methods. The number of lineages scored per genotype is shown in parentheses; ND, not determined. (A) pig-1 mutants have extra AVM, PVM and SDQ neurons, all descendants of the Q.p neuroblast. (B) pig-1 mutants have extra AQR and PQR neurons, and extra M4 neurons. (C) pig-1 mutants have extra I2 neurons. The average number of I2 neurons per lineage is shown above the bars of the histogram. (D) pig-1 mutants have extra PLMs and mutations in pig-1 are epistatic to mutations in ham-1. As in the HSN/PHB lineage, mutations in pig-1 interact synergistically with mutations in the pro-apoptotic genes ced-3 and egl-1 to control the penetrance of extra neurons in the Q.p (A), I2 (C) and PLM/ALN (D) lineages. (We did not examine pig-1 interactions with ced-3 or egl-1 in the lineages that produce the A/PQR and M4 neurons.)
pig-1 regulates neuroblast daughter cell size asymmetry
The similar sizes of the Q.p daughter nuclei in pig-1 mutants suggested that pig-1 regulates the asymmetric division of neuroblasts into daughters of unequal size. To confirm a role for pig-1 in regulating cell size, we quantified daughter cell size for both the HSN/PHB and Q.p neuroblast divisions. In the HSN/PHB neuroblast division, we used the hlh-14::gfp transgene gmIs20 to measure the size of the neuroblast daughter nuclei (Frank et al., 2003). The HSN/PHB neuroblast normally divides to produce a smaller anterior daughter, which is destined to die, and a larger posterior HSN/PHB precursor. In wild-type embryos, the diameter of the nucleus of the anterior daughter was approximately three-quarters that of the nucleus of its sister, whereas, in pig-1 mutants, the daughter nuclei were more equal in size (Fig. 4). The size differences between pig-1 and wild-type HSN/PHB neuroblast daughter nuclei cannot be explained merely by inappropriate survival of the anterior daughter, as a mutation in ced-3 has no phenotype in this assay (Fig. 4E). We also quantified the size of the Q.p neuroblast daughters using the cytoplasmic reporter ayIs9 (Pegl-17::gfp) (Branda and Stern, 2000). Although the anterior daughter of Q.p (a neural precursor) is normally larger than its sister (which undergoes apoptosis), the daughters are more symmetric in size in pig-1 mutants (Fig. 4F). Similar effects on daughter cell size were also observed in the Q.a division (data not shown). Together, these data indicate that pig-1 controls daughter cell size in asymmetric neuroblast divisions.
PIG-1 encodes a conserved serine/threonine kinase
We mapped pig-1 to a 500-kbp interval on LGIV containing approximately 100 genes. Three observations indicate that the open reading frame (ORF) W03G1.6 from this region is pig-1. First, we inactivated each ORF in this region using RNA-mediated interference (RNAi) (Kamath et al., 2001; Timmons and Fire, 1998). Only RNAi-mediated inactivation of W03G1.6 in gmIs12; ced-3(n717) animals induced a PHB duplication phenotype (Fig. 2A). Second, we sequenced the gene in three pig-1 mutant strains and identified a single missense mutation for each allele (see below). Third, we generated a deletion allele of W03G1.6 and found that the mutant displayed Pig-1 phenotypes.
Sequence analysis of pig-1 cDNAs indicates the gene transcribes a single SL1 trans-spliced mRNA that encodes a conserved serine/threonine kinase that is homologous to vertebrate MELK (Fig. 5). Like MELK family members, PIG-1 has an N-terminal kinase domain, a middle region of reduced homology, and a kinase-associated domain at its C terminus (Fig. 5B). Protein sequence comparisons indicate that PIG-1 and its vertebrate homologs form a separate subfamily of serine/threonine kinases distinct from the Kin1p, PAR-1 and SAD-1 families, indicating that PIG-1 is the C. elegans ortholog of MELK (Fig. 5C). We found that gm300 and gm301 alleles are missense mutations in the kinase domain that change conserved glycines to charged amino acids (Fig. 5B). The weaker allele gm280 is a missense mutation in the kinase-associated domain that changes a conserved glycine to arginine (Fig. 5B). This analysis indicates that both the kinase and kinase-associated domains are important for pig-1 activity.
pig-1 regulates the positioning of the cleavage plane in the HSB/PHB and Q.p neuroblasts. (A-E) pig-1 disrupts the size asymmetry of the daughters of the HSN/PHB neuroblast. The nuclear-localized reporter gmIs20 (hlh-14::gfp) was used to identify the daughters of the HSN/PHB neuroblast and measure the size of their nuclei (Frank et al., 2005). All images show a ventral view of a single embryo with anterior to the left. In each image, the anterior (arrow) and posterior (arrowhead) daughters of the HSN/PHB neuroblast are magnified in the inset. (A) Wild type, (B) pig-1(gm301), (C) ham-1(gm279) and (D) pig-1(gm301) ham-1(gm279). Scale bars: 5 μm. (E) Quantification of the ratio in nuclear diameter between the anterior and posterior daughters of the HSN/PHB neuroblast in multiple genotypes; n, number of pairs of neuroblast daughters scored. (F) Quantification of the ratio in cell size between the posterior and anterior daughters of the Q.p neuroblast; n, number of pairs of neuroblast daughters scored. The transcriptional reporter ayIs9 (Pegl-17::gfp) was used to identify and measure the size of the daughters of the Q.p neuroblast. Like the HSN/PHB neuroblast division, the Q.p neuroblast division is more symmetric in pig-1 mutants.
Molecular characterization of pig-1. (A) Schematic diagram of the pig-1 transcript. White boxes represent exons, and the location of the SL1-splice leader is indicated. The terminal intron of pig-1 contains two genes, depicted as grey arrows, that are transcribed in the opposite orientation to pig-1. (B) Schematic diagram of the domain architecture of the PIG-1 protein, depicting the N-terminal kinase domain (unshaded) and the C-terminal kinase-associated domain (black). The locations and identities of the gm300, gm301 and gm280 missense mutations are shown above the diagram. Below the diagram is a table comparing each domain in PIG-1 to orthologs in frog, fish, mouse and human. (C) Phylogenetic relationship of PIG-1 to selected members of the PAR-1/SAD-1/Kin1 superfamily of serine/threonine kinases. PIG-1 clusters with other MELK family members.
The pig-1 deletion allele gm344 is a 524 bp deletion that removes the pig-1 promoter, all of the first exon, and part of the second exon of the pig-1 coding sequence. gm344 appears to be a molecular null based on RT-PCR and western blot assays (data not shown). Like the other pig-1 mutants, gm344 mutants are viable and the strength of Pig-1 mutant phenotypes is identical to what we observed in pig-1(gm301) mutants (Fig. 3A,D). These comparisons suggest that the alleles gm300, gm301 and gm344 eliminate PIG-1 function, and that the allele gm280 reduces it.
PIG-1 expression and localization
Pig-1 phenotypes are maternally rescued, consistent with the presence of pig-1 transcripts in the hermaphrodite germ line (NEXT database, Y. Kohara, personal communication). To characterize zygotic pig-1 expression, we examined transgenic worms expressing a transcriptional reporter containing 850 bp upstream of the pig-1 start codon fused to GFP coding sequences. This construct contained all of the noncoding sequence between pig-1 and the next predicted upstream gene, and expressed GFP ubiquitously in early embryos. The expression became progressively more restricted in older embryos and young larvae, and was not observed in adults (data not shown). In larvae, we observed expression in dividing cells: ventral nerve cord neuroblasts, vulval precursors, dividing hypodermal seam cells, and the Q neuroblasts and their descendants (Fig. 6A-F; data not shown). To investigate the subcellular distribution of the PIG-1 protein, we examined transgenic worms expressing a full-length pig-1::gfp translational fusion. This construct contains the same promoter used in the transcriptional reporter fused to a full-length pig-1 cDNA tagged with GFP. This transgene rescued Pig-1 phenotypes in the PLM/ALN and Q.p lineages (data not shown). In all cells, the fusion protein was localized to the cytoplasm and excluded from nuclei (Fig. 6G,H).
Expression and subcellular localization of PIG-1. (A-F) Images of larvae expressing Ppig-1::gfp: anterior, left; ventral, up. (A,C,E) DIC microscopy; (B,D,F) GFP fluorescence. (A,B) L1 larva expressing Ppig-1::gfp in the Q neuroblast (arrow) and V5 seam cell (arrowhead). (C,D) L1 larva expressing Ppig-1::gfp in the Q neuroblast daughters Q.a (arrow) and Q.p (arrowhead). (E,F) L1 larva expressing Ppig-1::gfp in the Q lineage descendants AQR (asterisk), AVM (arrow), and SQDR (arrowhead). (G,H) DIC (G) and fluorescence (H) images of an embryo expressing Ppig-1::pig-1::gfp. pig-1::gfp is present throughout the cytoplasm and excluded from the nucleus. Scale bars: 10μ m.
PIG-1 acts cell-autonomously to regulate the asymmetric division of the Q.p neuroblast. The numbers of AVM and PVM neurons were scored using zdIs5 [mec-4::gfp]; the number of lineages scored is shown in parentheses. Expression of pig-1 from the mab-5 promoter does not rescue the extra AVM phenotype of pig-1(gm344) but does rescue the extra PVM phenotype. Statistical analysis was performed using a one-tailed two-sample Z-test (using the normal approximation to the binomial distribution) by the StatCrunch program (www.statcrunch.com).
pig-1 acts cell autonomously in the Q.p lineage
To determine whether pig-1 acts cell autonomously in neuroblasts to regulate their divisions, we focused on the Q.p neuroblast, which produces the A/PVM and SDQ neurons (Fig. 1B). The pig-1 promoter drives expression in the Q neuroblast and its descendents (Fig. 6A-F), consistent with a cell autonomous role for pig-1. To test this hypothesis, we expressed a pig-1 cDNA from the mab-5 promoter and asked whether its expression would rescue the Q.p defects of pig-1 mutants. mab-5 encodes a homeobox transcription factor expressed in a bilaterally symmetric group of posterior cells with one exception: it is expressed in the left Q lineage (which produces PVM), but not the right Q lineage (which produces AVM) (Cowing and Kenyon, 1992; Salser and Kenyon, 1992). We reasoned that if pig-1 acts in the Q lineage, pig-1 expression from the mab-5 promoter should rescue the extra PVM but not the extra AVM phenotype of pig-1 mutants. Consistent with the hypothesis that PIG-1 acts in Q.p to promote its asymmetric division, the Pmab-5::pig-1 transgene rescued the Pig-1 PVM but not the AVM phenotype (Fig. 7).
pig-1 and ham-1 genetic interactions
The C. elegans HAM-1 protein regulates the asymmetric divisions of many embryonic neuroblasts, including the HSN/PHB neuroblast (Frank et al., 2005; Guenther and Garriga, 1996). Like mutations in pig-1, mutations in ham-1 alter both HSN/PHB neuroblast daughter cell size asymmetry and cell fate. In ham-1(gm279) null mutants, the relative sizes of the neuroblast daughters are reversed, and the anterior daughter is larger than its sister. The anterior daughter is transformed into its sister, resulting in the production of extra HSNs and PHBs. ham-1 HSNs and PHBs also often fail to differentiate properly, and occasionally mutant lineages fail to produce HSNs or PHBs that express cell-type specific markers; this latter phenotype may be a consequence of the failure of the posterior daughter of the HSN/PHB neuroblast to divide or differentiate correctly because of its small size. ham-1 encodes a novel protein that is asymmetrically distributed to the posterior cortex of the dividing neuroblast and is inherited specifically by the precursor. HAM-1 is hypothesized to control the asymmetric segregation of developmental potential into the neuroblast daughters, in part by regulating the volume of neuroblast cytoplasm distributed to each daughter cell (Frank et al., 2005).
The similarity of pig-1 and ham-1 phenotypes led us to determine whether the two genes act together to regulate neuroblast divisions. To address this possibility, we examined the output of the HSN/PHB neuroblast lineage in pig-1 ham-1 double mutants. While pig-1 mutants have a low penetrance of extra PHBs, ham-1 mutants have a higher penetrance of extra PHBs, as well as missing PHBs (Fig. 2C). We were surprised to discover that although ham-1 mutants have a stronger phenotype, pig-1 mutations were epistatic to ham-1 mutations: pig-1 ham-1 double mutants had a low penetrance of extra PHBs and were not missing PHBs. This relationship was observed with multiple pig-1 and ham-1 alleles (Fig. 2C; data not shown). We obtained similar results when we scored the number of HSNs (Fig. 2C) and the number of PLMs (Fig. 3D), indicating that the genetic interaction between the two genes is not PHB or lineage specific. Mutations in pig-1 are also epistatic to ham-1 in a ced-3 background: whereas ham-1(n1438) ced-3 double mutants have extra PHBs about 60% of the time, pig-1 ced-3 and pig-1 ham-1(n1438) ced-3 mutants almost always have extra cells (Fig. 2D).
Modeling the HSN/PHB lineage in pig-1 and ham-1 mutants. In wild-type animals, the HSN/PHB neuroblast is polarized along the AP axis, with an anteriorly displaced cleavage plane (vertical dotted line) and posteriorly localized determinants of neural precursor fate (gray circles). The neuroblast divides to produce a small anterior daughter that inherits no determinants and undergoes apoptosis, and a larger posterior daughter that inherits the determinants and becomes a neural precursor. In pig-1 mutants, the neuroblast does not polarize and this results in a symmetrically localized cleavage plane and uniformly distributed determinants. Both neuroblast daughters inherit these determinants resulting in the production of two neural precursors. In ham-1 mutants, neuroblast polarity is partially inverted along the AP axis resulting in a posteriorly displaced cleavage plane and an anteriorly enriched distribution of cell fate determinants. Extra neurons are produced when a sufficient concentration of determinants enters both neuroblast daughters, resulting in the production of two precursors. No neurons are produced when the anterior daughter undergoes apoptosis and the posterior daughter receives an insufficient concentration of determinants to develop as a precursor. The concentration of determinants in the posterior daughter could fall below a threshold required for precursor fate, either because of stochastic differences in cleavage position or determinant distribution. Neuroblasts in pig-1 ham-1 double mutants are defective for polarity, resulting in pig-1 phenotypes and pig-1 epistasis to ham-1.
pig-1 mutations also suppressed Ham-1 differentiation phenotypes. The PHB phasmid neurons in ham-1 mutants frequently fail to fill with the dye DiI because of defective sensilla development (Guenther and Garriga, 1996). pig-1 mutants have no dye-filling defects, and mutations in pig-1 suppress the dye-filling defects of ham-1 mutants (data not shown). pig-1 mutations also suppress the HSN migration defects observed in ham-1 mutants (data not shown).
Mutations in both pig-1 and ham-1 perturb the asymmetric division of the HSN/PHB neuroblast into daughters of unequal size. While strong ham-1 mutations reverse the relative sizes of the daughters (Frank et al., 2005), mutations in pig-1 cause the neuroblast to divide more symmetrically. When we compared the sizes of the HSN/PHB neuroblast daughters in pig-1 ham-1 double mutants, we found that they were similar to pig-1 mutants (Fig. 4). In summary, pig-1 ham-1 double mutants seem to phenocopy pig-1 single mutants in assays that examine the cell lineage, cell size, and the differentiation of neuroblast descendants in the HSN/PHB neuroblast lineage.
pig-1 may regulate neuroblast polarity
Many genes that regulate asymmetric cell divisions control cell polarity. For example, the PAR proteins are required in the C. elegans zygote for the asymmetric positioning of the mitotic spindle and the asymmetric segregation of cell fate determinants to daughter cells (Kemphues et al., 1988; Rose and Kemphues, 1998). pig-1 mutants share phenotypic features with these cell polarity mutants, and PIG-1 is related molecularly to PAR-1. However, although pig-1 and par-1 phenotypes have similar features, PAR-1 localizes asymmetrically in the mother cell, whereas PIG-1 and its vertebrate orthologs do not (Chartrain et al., 2005; Heyer et al., 1999). The symmetric distribution of PIG-1 suggests that it may play a permissive role in the regulation of neuroblast polarity. In this sense, PIG-1 may resemble the symmetrically distributed 14-3-3 protein PAR-5, which regulates both spindle positioning and cell fate in the C. elegans zygote (Morton et al., 2002).
We propose that PIG-1 controls the asymmetric positioning of the mitotic spindle and asymmetric segregation of neural fate determinants to daughter cells. In this model, loss of pig-1 activity disrupts neuroblast polarity, resulting in symmetric positioning of the cleavage plane and mislocalization of cell fate determinants. The neuroblast therefore divides symmetrically, and cell fate determinants are symmetrically distributed to daughter cells. The segregation of these determinants into the normally apoptotic daughter of the neuroblast transforms it into its sister.
HAM-1 is also thought to regulate asymmetric neuroblast divisions by regulating neuroblast polarity, and mutations in ham-1 also disrupt cell size asymmetry and cell fate in the HSN/PHB neuroblast division. HAM-1 has previously been proposed to regulate neuroblast daughter cell fate in part by controlling cell size (Frank et al., 2005). One important question that arises from our analysis is whether HAM-1 and PIG-1 could function solely to regulate the position of the neuroblast cleavage plane, and the ensuing cell fate transformation could result from altered daughter cell size. This model predicts that determinants are symmetrically distributed in the neuroblast and that cell fate is determined by the concentration of determinants in a neuroblast daughter. Alternatively, PIG-1 and HAM-1 could regulate the position of the neuroblast cleavage plane and the asymmetric segregation of cell fate determinants to daughter cells independently. In this model, determinants are asymmetrically distributed in the neuroblast, and their segregation into daughter cells is mostly independent of cleavage plane position.
Two observations lead us to favor a modified version of the second model, in which the size of daughter cells does not directly specify their fate, but might influence their survival (Fig. 8). First, cell size alone cannot control the apoptotic fate of the daughter cells. Although the posterior daughter of the HSN/PHB neuroblast is smaller in ham-1 mutants, it is still the larger anterior daughter that dies (Frank et al., 2005). Second, if cleavage plane position were to regulate quantitative differences in the segregation of cell fate determinants to the anterior daughter of the HSN/PHB neuroblast, the frequency at which this cell is transformed into its sister in pig-1 and ham-1 mutants should be proportional to the deviation of the cleavage plane from its normal position. This does not seem to be the case, as analyses of pig-1 ced-3 and ham-1 ced-3 double mutants suggest that pig-1 and ham-1 mutations always transform the anterior daughter of the HSN/PHB neuroblast into its sister, independent of daughter cell size. However, the penetrance of extra cells in pig-1, ham-1 and pig-1 ham-1 mutants does appear to be proportional to the deviation of the cleavage plane from its normal position, so cell size could affect the frequency at which the anterior daughter of the neuroblast escapes programmed cell death. Thus, increasing the size of the anterior daughter of the HSN/PHB neuroblast may increase the frequency at which this cell escapes programmed cell death.
The epistatic interactions between the pig-1 and ham-1 mutations are intriguing. If daughter cell size influences the penetrance of extra and missing neurons, then pig-1's epistasis to ham-1 at the level of daughter size is sufficient to explain why pig-1 mutations suppress the extra neuron and missing neuron phenotypes of ham-1 mutants (Fig. 8). pig-1 might be epistatic to ham-1 because HAM-1 inhibits PIG-1-dependent positioning of the mitotic spindle. HAM-1 may restrict PIG-1 activity to the anterior of the HSN/PHB neuroblast, and the mispositioning of the cleavage plane in ham-1 mutants could be a consequence of ectopic PIG-1 activity in the posterior of the neuroblast. Alternatively, PIG-1 might be a general factor required for spindle positioning. PIG-1, for example, could regulate, or be regulated by, both HAM-1 and an activity that has been postulated to antagonize HAM-1 (Frank et al., 2005).
PIG-1 and MELK
The vertebrate orthologs of PIG-1 have been implicated in regulating cell proliferation, although the nature of this regulation has been the subject of debate. MELK kinase activity exhibits cell cycle dependence, with maximal activity during mitosis (Blot et al., 2002; Davezac et al., 2002). MELK has been shown to bind and phosphorylate CDC25B, a phosphatase that promotes G2/M progression by removing inhibitory phosphate groups from the cyclin-dependent kinase CDC2 (Davezac et al., 2002). MELK phosphorylation was proposed to inhibit CDC25B, as several groups have shown that MELK overexpression results in a block at the G2/M boundary (Davezac et al., 2002; Gray et al., 2005; Vulsteke et al., 2004). The C. elegans genome encodes three CDC25 homologs. To determine whether Pig-1 phenotypes require CDC-25 activity, we used RNAi (Fraser et al., 2000) to inactivate these genes in pig-1 and pig-1 ced-3 animals. We examined the output of the HSN/PHB and PLM/ALN lineages, and found that RNAi of cdc-25.2 suppressed the extra PHB and PLM phenotypes of these mutants (A. Singhvi and G.G., unpublished). While these observations are consistent with a role for PIG-1 as a negative regulator of CDC-25.2, it is also possible that the cdc-25.2 RNAi effects reflect a nonspecific effect on cell division.
MELK has also been proposed to positively regulate the cell cycle. Overexpression of MELK in mouse neurosphere cultures results in increased proliferation, whereas MELK knockdown by siRNA has the opposite effect (Nakano et al., 2005). MELK has also been shown to positively regulate the growth rate of cultured human tumor cell lines, both in vitro and after injection into a murine host (Gray et al., 2005). Nakano et al. (Nakano et al., 2005) proposed that MELK positively regulates the cell cycle through its association with the zinc-finger protein ZPR9 and the proto-oncogene B-myb. MELK binds and phosphorylates ZPR9 (Seong et al., 2002), which in turn binds B-myb and increases its transcriptional activity (Seong et al., 2003). Although an ortholog of ZPR9 exists in C. elegans (ORF C16A3.4), RNAi of this gene does not generate a Pig-1-like phenotype (S.C. and G.G., unpublished).
Altered cell cycle progression in the neuroblast could lead to the phenotypes observed in pig-1 mutants, as cell cycle regulators are known to control asymmetric divisions. In Drosophila, Cdc2/B type cyclins regulate asymmetric divisions of neuroblasts in the central nervous system. Loss of zygotic cdc2 activity results in both defective spindle positioning and sister-sister cell fate transformations (Tio et al., 2001). In C. elegans, the anaphase-promoting complex (APC), the APC activator cdc20 and the APC effector separin are required during the first cell division for proper positioning of the cleavage plane and the asymmetric distribution of cell fate determinants (Rappleye et al., 2002). PIG-1 could regulate the timing of cell cycle events in the neuroblast, which in turn, could indirectly regulate neuroblast polarity. In conclusion, our analysis of PIG-1 suggests that MELK could regulate polarity and daughter cell fate in asymmetrically dividing cell lineages such as stem cells.
We thank A. Singhvi and N. Hawkins for comments on the manuscript, A. Singhvi for sharing unpublished observations, and members of the Garriga, Meyer and Dernburg labs for helpful discussions. We also thank Y. Kohara for cDNAs and C. Li, Y. Lie, C. Kenyon, O. Hobert and B. Meyer for bacterial and C. elegans strains. Some of the C. elegans strains used in this study were obtained from the C. elegans Genetics Center, which is funded by the NIH National Center for Research Resources. This work was supported by an Ernest Brown Babcock Scholarship to S.C., a National Science Foundation predoctoral fellowship to C.A.F., and by National Institutes of Health grant NS42213 to G.G.
↵* Present address: Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143-2822, USA
Accepted May 16, 2006.
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Wicks, S. R., Yeh, R. T., Gish, W. R., Waterston, R. H. and Plasterk, R. H. (2001). Rapid gene mapping in Caenorhabditis elegans using a high density polymorphism map. Nat. Genet. 28,160 -164.
Withee, J., Galligan, B., Hawkins, N. and Garriga, G. (2004). Caenorhabditis elegans WASP and Ena/VASP proteins play compensatory roles in morphogenesis and neuronal cell migration. Genetics 167,1165 -1176.
Yu, S., Avery, L., Baude, E. and Garbers, D. L. (1997). Guanylyl cyclase expression in specific sensory neurons: a new family of chemosensory receptors. Proc. Natl. Acad. Sci. USA 94,3384 -3387.
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Still more edits and additions all over the place. There are a couple of things that deserve a mention here. First, I have updated the page of spells from the films and games to correct the entry on the spell Arresto Momentum. Penny begged me to get to that one because of all the email she was getting.
Secondly, and more importantly, I have added a new editor to the Lexicon staff. As many of you have probably realized, the job of editing a site this large was just too massive for Michele and I to keep up with. We have received plenty of emails pointing out corrections that needed to be made but we've had no time to get to them. Our new editor, Josh Santilli, will be helping to handle some of the backlog of editing jobs and also working on some of our newer projects. His staff email address is [email protected] Feel free to drop him an email to welcome him. If he doesn't respond right away, it's probably because we're going to be keeping him busy! Welcome aboard, Josh.


Water - Waterborne Environmental, Inc.
Water quality is increasingly becoming a focus of interest from diverse stakeholders. Whether for protection of aquatic species or for human consumption, a reliable understanding of the potential for exposure in both surface and groundwater is essential. Waterborne's experience in being able to understand and model the fate and transport of chemicals in surface water provides reliable estimates of potential exposure. Regulatory models for surface water such as VVWM, EXAMS, AGRO, TOXSWA are utilized to best represent these environmental conditions.
Geographic Information Systems (GIS) have long been used in combination with specialized models to simulate the fate and transport of chemicals and other constituents in watersheds. Incorporating the entire upstream land area in an exposure assessment is an important component in ensuring water quality for years to come.
Groundwater represents a different environmental compartment for protection. Factors such as soil, weather and chemical properties influence the potential for movement to groundwater. Often groundwater modeling is paired with prospective or retrospective monitoring for comparison or validation. Exposure models using PRZM, PEARL, or MACRO are often applied in regulatory settings, however other models can be applied.


Cutting polystyrene foam is easy with a hot wire cutter. But the wire sags when it gets hot and it's hard to get accurate, straight and nicely jointed edges. To overcome this, I'll show you how to make an adjustable, self-tensioning, accurate and reliable hot-wire foam cutter.
Now hot wire foam cutters are ten-a-penny on YouTube and there's dozens of sites with their own versions. However I've got a couple of projects coming up where an accurate, reliable foam cutter will come in very handy, and these other vids don't seem to cover adjustability, I've decided to share my ideas. But in deference to the other videos, I don't want to cover the build in too much detail, but what follows will enable a complete novice to build one from scratch. It will also help someone like me, who has an existing cutter, get greater adjustability and accuracy from it.
So let's get started… here's my cutter – and yes, I know – it doesn't look very impressive. It's a bit shabby and stained, but it's a number of years old and has been assembled and disassembled dozens of times for easy storage.
This isn't an exact science as you can build the project very much to your own requirements, but the materials shown here and dimensions are a good guide.
As with most of my builds, I work with whatever materials I have to hand and you certainly can do the same.
What you probably won't have to hand is the wire… you'll need Ni-Chrome wire but fortunately this is very cheap and easily available on eBay. I got a 4 metre length for just a few pounds and that will probably last me forever.
You're also going to need an electrical transformer – something that reduces mains power in your neck of the woods to safe and dependable voltage. I'm going to cover the transformer in more details later.
This inverted L forms the arm of the cutter that holds the hot wire. Glue and screw it for strength and ideally cut a small slot or rebate towards the bottom that's the same height as the thickness of the board.
I used a square piece of MDF. This is nice, flat, stable material. To the bottom of this I attached a couple of wooden blocks to act as legs. It's worth noting here, leave yourself a good clear section around the perimeter of the board to accept clamps.
It's a simple matter to glue and screw the arm onto the board. If the rebate you cut is a good fit, this should be a doddle. You'll see I've used brackets on my build, but that's because I haven't used glue. I like to take my cutter apart for easy storage, but ideally gluing and screwing is the way to maximise strength. However, before you glue anything, you'll need to drill a clearance hole Ni-Chrome wire.
To establish where the wire will pass through the board, you can use a carpenters' square, but if you don't have one large enough, a simple plumb line will do just as well as long as you're on a nice level surface. Notice that line does NOT begin at the end of the arm but actually a couple of centimetres or an inch away from the arm. This is to allow for adjustment later.
With the spot marked where the wire needs to go, using the finest drill bit you've probably got, drill perpendicular hole.
Looking at the underside of the board now, you can see that I took an ordinary steel washer and drilled a hole through it with the same drill bit. Any piece of metal will do. I just happened to have a washer. The wire passes through the face of the board, through the hole that's been drilled and through the hole in the washer. The washer is fixed in place and this prevents the thin wire slicing through the soft MDF or even scorching it.
A terminal block is screwed nearby with a length of electrical wire within it. The Ni-Chrome wire is fed through the drilled hole and screwed into this. The Ni-chrome wire will be under tension and could pull free from terminal block, so I clamped the wire in place using a small metal bracket. Again anything will do for this purpose, even another washer, as long as it holds the wire firmly.
A terminal block is attached to the lower section of the arm and the electrical wire from the underside of the base connected to it. The power adapter is connected into this same terminal block. There should be two wires, a positive and a negative, and this should be low DC (direct current) voltage and NOT mains voltage. That's critical to understand. If you get this part wrong, you could be creating something that could easily kill you.
For the cutter to work, you don't really need to understand which of the transformer wires is positive or negative (though the positive is often red or white and the negative generally black). But this knowledge is helpful as you'll see later.
Now's the time to add a switch and I to had an old domestic light switch to hand, so I used that. You will need a switch and you shouldn't leave the cutter turned on if not in use as it will burn out the Ni-Chrome wire.
An electrical wire is attached to second terminal of the terminal block, that's the brown wire on my diagram, which is NOT the wire from the base of the cutter. This brown wire simply goes into the switch and out again. You can see the reverse side of the light switch here. Typically there's two terminals or connection points on such switches and the brown wire goes into one of these. A second brown wire is screwed into the other terminal.
This second brown cable is attached to the adjusting bracket that we'll see in a moment. A simple ring connector can be used for this.
Let's just briefly look at the electrical circuit we've now made.
The first part of the circuit is the Ni-Chrome wire and this passed through the board, into a terminal block, through an electrical wire and to a second terminal block. This is connected to one of the wires from the power transformer. From the transformer, power can pass through the second transformer cable, into the terminal block, into the switch, through the switch if it's turned on, out the other side, along to out adjustable bracket and the circuit is complete.
So let's look a little more closely now at the adjustment bracket.
Now this really is simplicity itself. It's simply a length of metal ideally, maybe four or five inches long, but crucially it has a slot running down its length. It's through this slot that the bracket is attached to arm of the cutter. I made use of an old radiator bracket which I crudely cut down. Sorry if the aesthetics aren't very appealing folks, but at the time of making I was more interested in functionality, and this bracket was a gift from the DIY gods.
If you can't find something suitable and have to fabricate a bracket, make sure that the slot is about 50% wider than the width of the bolts you use to fix it in place. A loose fitting is crucial to this design. As I say, metal is ideal due to its strength and conductivity, but you could use plywood or similar – though remember you'd need to connect the switched brown wire directly to the Ni-Chrome wire.
Towards the end of this bracket, you need to drill a fine hole for the Ni-Chrome wire to pass through.
With the bracket resting on the arm, drill a couple of pilot holes with a drill bit that just a little smaller than the diameter of your bolts. You can then turn the bolts with a spanner into the timber and you should find they grip very well.
You only need to sink roughly half the length of the bolt into the arm. The other 50% holds two nuts per bolt. The lowest bolt is tightened by hand so the bracket can move, but only just. Later when you're happy with the placement of the Ni-Chrome wire, you can fully tighten the lower nut. One of the upper nuts can clamp down on the ring connector, securing the electrical connection. This nut on nut practice is a good method of preventing unwanted loosening.
This clamp is of course the secret to the vertical adjustment. The bracket can be moved to a desired location and this in turn adjusts the vertical angle of the Ni-Chrome wire. Ideally you want this to be perfectly perpendicular to the base board, helping you achieve a nice ninety degree cut when in use.
With careful adjustment, a hell of a lot of patience and the use of a good square, a perfectly true wire is achievable.
But of course, that's not really doable until the Ni-Chrome wire is under tension.
There's two ways of tensioning the wire that I've used, Gravity and Spring. Let's start with Gravity.
Gravity is constant and free, so to use it we need to find a way of having gravity pull on our wire, and we can do that by attaching a weight.
A simple groove filed into the bracket allows a weight to be suspended, but care needs to be taken so that the weight doesn't collide with the wire. Heavier is best as it will tighten the wire better, but if the weight you apply is too heavy it will snap the wire.
A tiny spot of oil on the groove point will help the weight pull nicely on the wire, but keep the main drop clean of oil otherwise it will likely smoke and smell.
This process does work and I did use it briefly. It holds the wire taught and can enable fine adjustment, but as I say, heavier is best. Personally I prefer to use a compression spring.
The spring needs to be quite strong. If you can squash it easily with your fingers, it's probably too weak. But if you can't budge it, it might be too strong.
The Ni-Chrome wire is simply fed through the spring and clamped at the top. I used a small bolt and a couple of nuts to achieve this. Importantly you need to compress the spring a little before tightening the nuts and clamping the wire in place. It's fiddly and you feel like you need four extra hands, but it can be done. Once held in place, the Ni-Chrome wire should be permanently under tension from the spring. Again, not too much tension as the wire will snap, but enough that you can play a tune on it.
So there you have it, an adjustable, and self-tensioning hot-wire foam cutter. Surprisingly simple isn't it.
And you can leave it there. But the light in my shed isn't great and I fancied adding a light to mine.
If you've seen my video on homemade, custom Daytime Running Lights, you'll remember I made use of these Eagle-Eye LED's. These are bright and have a low power consumption. They also happily work between 6 and 12 volts and as my transformer is 12 volts, I couldn't resist.
Wiring the LED into the circuit is easy, but you will need to know which wire is positive and which is negative. On my diagram, the brown wire is the positive connection and this is the one that passes through the switch. So that's the perfect place to connect the positive lead of the LED, on the switched terminal of the switch. That way the light will only be on when in use. This then acts as a reminder that the power is on… helping you avoid burning out the Ni-Chrome wire if the cutter's not in use.
The negative lead of the LED joins the blue cables in the terminal block, completing the circuit. So now, when the switch is turned on, the wire gets hot, ready for use and the light shines down on the work.
I left my LED suspended. This means I can alter its position if I need to. But if you prefer to fix yours in place, that's down to you.
Now all we need to do is discuss the power transformers in a little more detail. This is probably the most difficult part for me to comfortably narrate. I've been making hot wire cutters since I was a kid so I have no fear of them and probably I'm much too flippant in my choice of power supply for my own good, so please DO CHECK OUT what other people say on the subject.
So what is a transformer?
As I said earlier, it's a device to convert the mains voltage in your area to a safer, lower voltage. Here in the UK, mains voltage tends to be between 220 and 240 volts. In the US, I believe it's around 110 volts. To be honest it doesn't matter the national voltage is as long as you use a transformer that's meant to be used in your region of the world. So when you source one, make sure it's locally obtained.
I didn't buy my transformer… well I did, but not for this project. If you're like me you've probably got a draw full of these things left over from answerphones, kid's games, etc. The device breaks, you throw it away, but somehow the transformer stays behind, and for THIS project that's just what you want. I sorted through my box of old transformers and I found a 12 volt direct current transformer. Reading the label, it said it could handle 2 amps of current and that made it perfect for this project. And that's the sort of thing you're looking for… low voltage, decent current handling. If the current rating is too low, you're transformer will burn out.
There are lots of clever people out there talking in terms of Ohms Law, resistance, ampage and voltage calculations and quite honestly, they're right. The problem is I'm too damn casual about the whole thing to be bothered. I know that's wrong, but at least I'm being honest with you. For me the voltage and current were okay, so I wired the whole thing together and gave it a blast. The wire didn't glow – which it SHOULDN'T as that's a sign of overpowering – but it cut nicely. So for me that was enough.
You can be similarly flippant if you wish, BUT just make sure your voltage is nice and low first. It doesn't have to be direct current for a wire cutter, but these sort of transformers generally are DC as they're used to power devices that normally run on batteries.
If your transformer fails to heat the wire enough to cut foam, you may have to shorten you wire (reducing its resistance) or you'll to increase the voltage of your transformer.
If you're lucky enough to have a model railway controller, this might be ideal. These usually allow you to vary voltage between zero and 12 voltage and tend to be good for around 2.5 amps. I've got an old Clipper Controller that's got to be 40 years old and still going strong. But DO check the ampage of yours. If it's not 2 amps or above, don't risk it. You'll probably burn it out.
Also in the description underneath this video I've included a few links to excellent websites that cover this topic in the sort of detail that's ideal. If you find a good one, let me know and I'll tack it on to the description, but for now folks that's me done on the subject of transformers and hot wire cutters.
So that's it folks. We're all done.
If you have any questions, feel free to get it touch. I'm no expert, but I'm happy to offer whatever help I can.


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3112 W Brigantine Ave
Brigantine, NJ 08203 • View Phone NumberTel. (609) 266-3481
Obituary for Genevieve Shulski
Mays Landing
Beloved Wife, Mother, Grandmother and Sister
Genevieve Shulski, 93, of Mays Landing formerly of Philadelphia passed away on Friday October 1, 2021. Genevieve was born in Fontoy, France to the late Johann and Apollonia (Chrzanowska) Czarmuch. Genevieve met her late husband Theodore during World War II. After the war Theodore returned to France to bring her back to the United States where they were married and raised their family in Philadelphia.
Genevieve was employed as secretary at Crathern and Smith Engineers in Philadelphia. She was also employed by Sears and Roebuck Co. on the Boulevard and FMC.
Genevieve was the beloved wife of the late Theodore F. Shulski. She is survived by her son Kenneth Shulski and her grandsons Michael Shulski, William Shulski (Marissa) and Chad Cappuccilli. She is also survived by her sister Wanda Czarmuch and her brothers Edmond and John Czarmuch. In addition to her parents and her husband she is predeceased by her son Gary.
Relatives and friends are invited to the Life Celebration and viewing for Genevieve on Sunday October 10, 2021 from 6 to 8PM at the Keates-Plum Funeral Home, 3112 Brigantine Ave Brigantine and on Monday from 10 to 10:50AM at St Thomas Church, 331 8th Street South, Brigantine. Mass of Christian Burial will follow at 11am. Interment will be private at Holy Sepulchre Cemetery, Wyndmoor, PA.
To share your fondest memory of Genevieve please visit www.keatesplum.com.
Add your thoughts, prayers or a condolence to Genevieve's guestbook.
Keates-Plum Funeral Home
Brigantine, NJ 08203

redpajama_set_name: RedPajamaCommonCrawl


You feel like you are going crazy. They turn everything around. They will make you feel like you are the one that is going crazy instead of them. You might become paranoid. You might worry about what you wear and what you say and freak out if someone changes your plans or something unexpected happens that you will have to explain later. If you are a peaceful person, you might find yourself constantly fighting. You might explode when you get too frustrated. You feel like there is something seriously wrong with you. You feel like you are walking on eggshells.
In Tasogare no Udewa Densetsu, one of the fetished-up outfits that Shugo discovers in a treasure chest is a sailor fuku. The character designer for the. In Amakusa , Natsuki Hayami and her female friends wore seifuku at school. The all-girl high school attended by Akira, Fujigaya, has relatively realistically depicted sailor fukus, with skirts way below the knee.
Mar 25, · I was dating a senior when I was a junior and he dumped me towards the end of the school year faster than you can say break my heart. He had no desire to be attached to a HS girl while he was in college.
Jan 27, 4: I get that you want to come off cool, casual, and down for whatever. But there are too many ladies out there lying to themselves about what they want from a man just to keep a man. Does he periodically lose his phone? He might invite you to hang out with his friends or proudly show them your Instagram.
You might even start being greeted by his friends. If a guy considers you a booty-call, he will, more often than not, refuse to go out with you. Booty-calls get, well, booty-calls. But if this has been going on for months, your presence should be evident in some way. People are often proud of their significant other and want to show them off, whether that be a tweet or two every once in a while, or a picture on Instagram together.
Think Bridesmaids, when Annie called Ted to pick her up after her car broke down. He did it with the disgustingly obvious expectation that he was getting something from her. Or any other important facts about me? I later found out this was a girl he really liked.
Feb 19, · Dating a College Boy Sign in to add this video to a playlist Dating A College Guy While In High School. Sign in Share Sign in to report inappropriate content.
Posted on February 28, by Staff Writer Anyone who has been in a relationship knows that it's not easy — relationships take time and hard work. On that same note, we all know college is difficult — it, too, takes time and hard work. If we plug this simple math into an equation it looks something like this: So, what could be more difficult than having a relationship in college? How about having a long distance relationship in college? Whether you and your high school sweetheart attend different schools in different states or you and your significant other go to the same college together but spend summers apart, you know that maintaining a long distance relationship can be one of the most difficult tasks assigned to you during your college years.
Does he make jokes? Knowing the difference between friendly conversation and playful banter can sometimes be tricky. Luckily, we talked to college guys and girls to get the scoop on what it all means. The same goes for his responses. The effort he puts into responding is quite telling, even though at first glance it might seem platonic.
He definitely knows you're someone worth pursuing and since that's based on your intelligence and your interesting conversation, he's most likely a keeper!
A Freshman Girl's Guide to College Dating By Lauren Kaplan • Dating August 10, at pm Say so long to your high school guys, and hello to college boys.
If he still puts up with all your crazy after all these years and STILL loves you fiercely, you two will end up riding everything out until the very end. While this might seem somewhat appealing in the beginning of the relationship because the drama makes for interesting bedroom play , once you enter adulthood and want to start a family, this can only mean appending doom is in the horizon.
You Both Fight Fair Fighting fair with your significant other shows signs of maturity on many different levels. This is a sign of adulthood and stability in a relationship. So you thought it was cool the first time you and your college sweetheart appeared on the show Cops. Appearing on it the next three times you two manage to get into a fight. Sure, he may not lay a hand on you, but you two fight so loud and long that the neighbors become involved… which then involve the cops.
Okay, you both have problems. I want to be with someone who can make me laugh at the drop of a hat and keep me laughing and smiling for hours after the fact. Same goes both ways for men at least men with substance.
Go Au Naturel Are you finding strategies to plan your first date? Are you worried about how to create a unique first date that will impress your girlfriend? Maybe you know that not any first date idea can work for any type of girl. Therefore, before planning some kinds of outdoorsy activities, you will want to make sure that she is the outdoorsy type.
Before planning your first date, you should think about her interests as well as her personality to decide which first date idea you can try. Keep reading this writing to learn more. Why does every first date seem to be an endless boring, actually cycle of grabbing dinner or drinks at restaurants and bars even when you have been experienced this several times before? Do not get me wrong — I really love drinking just as much as any other college freshman. However, there is actually something a little bit boring in my mind when I do the same things, hear the same song even when dancing with a brand new dating partner.
I know many people who think and feel exactly like me! Not to mention, do you really want to wake up hangover after hanging out with someone you did not hit it off with particularly if there is not even an interesting thing to talk about after all? Thanks, but I do not want this! And I know you too!
Share Shares 22K Since we are currently in the middle of the school year, it seemed a good time for a list of top movies about High School. For criteria, I tried to focus on movies set in or on the school grounds and classrooms, about High School life, including cliques and bullies, and involving the teachers when possible.
Cady is the new redhead that falls in with The Plastics, an A-list clique of supermodel-esque blondes. The Plastics plan their revenge, but Cady uses her jungle-environment home-schooling to get the last laugh. Also stars Tina Fey as the hot but socially awkward teacher, Ms.
Okay, so a girl who is a junior in highschool started talking with me today. I'm one of those people who feels really bad if I ignore someone, so I talked with her. I'm a junior in college, as well.


Last weekend was so packed with stuff, I feel obligated to write a post about it!
On friday we planned to go to Game City, a free gaming event in the city hall of Vienna where big publishers present their new games. While games are nice, the real motivation to go there was of course to meet Ulrich Schröder, a great artist from Germany who draws a lot of the "Donald Duck Sonderheft" covers. On the way there we ran into the Neubaugassenflohmarkt, a big flea market where a lot of stores sell things such as art supplies, cameras, postcards and useless junk. We looked around a bit and then stopped by Haus des Meeres, the big Aquarium in Vienna which is located in an old anti-aircraft tower. They just finished their new rooftop cafe, which we had to see!
A really nice view over Vienna.
The cafe on top of Haus des Meeres.
Later at Game City we met Ulrich after waiting in line for a while. It was quite busy there already and we had a short chat and agreed on meeting after Game City closes, which gave us about 1.5 hours to kill. At the same time on Stephansplatz, there was the campaign finale of the right-winged party FPÖ along with a protest against the party and their policies, so we went there and met up with Chris. Sadly, a lot of the protesters were too extreme for our tastes and while I would have liked to protest against the racism and intolerance that FPÖ spreads, we didn't quite feel like protesting against the police. So we stayed between the 2 sides and tried to take a few good photos capturing the atmosphere.
The protest behind a wall of policemen.
Back at Game City, we met up with Ulrich and Henrieke showed some of her drawings before going home.
On saturday we got up early to sell at a flea market. My whole family was there and we got rid of quite some junk! At around 9:30 we went to Game City again to get drawings by Ulrich. We stood in line for about 2 hours, but it was worth it for getting a great drawing!
The beutiful city hall where Game City was held.
This is a first person virtual reality thingie that was developed at my University!
The drawing I got from Ulrich.
We hurried to get back to the flea market, had some quick lunch before and tried to sell as much stuff as possible. Despite the market being open until 5pm, most people packed at 3 already and so did we as no more customers were coming in. Henrieke and me qent on a quest to get a cake and we had a nice afternoon with my family then.
Vienna Comix gets a separate post!


Coldsore tracking, in which a detailed deep-sequencing analysis of the virus causing the condition reveals both the timeframe and geographical region in which it was contracted.
Coming soon to divorce proceedings near you.
The genomes of two distinct strains of the virus that causes the common lip cold sore, herpes simplex virus type 1 (HSV-1), have been identified within an individual person — an achievement that could be useful to forensic scientists for tracing a person's history. The research also opens the door to understanding how a patient's viruses influence the course of disease. The research by an international team led by Moriah L. Szpara, assistant professor of biochemistry and molecular biology at Penn State University, will be published in the May 2016 issue of the journal Virology.
Most people harbor HSV-1, frequently as a strain acquired from their mothers shortly after birth and carried for the rest of their lives. The new discovery was made with the help of a volunteer from the United States. The research revealed that one strain of the HSV-1 virus harbored by this individual is of a European/North American variety and the other is an Asian variety — likely acquired during the volunteer's military service in the Korean War in the 1950s.
"It's possible that more people have their life history documented at the molecular level in the HSV-1 strains they carry," said Derek Gatherer, a lecturer in the Division of Biomedical and Life Sciences at Lancaster University in the United Kingdom and a member of the research team, which also includes scientists at Georgia State University, the University of Pittsburgh, and Princeton University.
Earlier research by the same team has demonstrated that the geographical origin of HSV-1 can be predicted, as well. Since Asian, African, and European/North American varieties of the virus exist, and the virus is often acquired early in life, the research implies that a personal strain of HSV-1 can reflect a person's origin. Another implication is that two individuals who have identical strains of HSV-1 are more likely to be related than those who have different strains.
"Using similar genetic fingerprinting of HSV-1 could help flesh out a person's life story, adding an extra layer of genetic information not provided by our genomes alone. Forensic virology could be on the way in the same way in which we use genetic fingerprinting of our human DNA to locate perpetrators at the scene of a crime and to help trace the relatives of unidentified bodies," Gatherer said.


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Wildfires burning across Greece; 2 arrested for arson
Posted: Aug 25, 2019 / 08:35 AM EDT / Updated: Aug 25, 2019 / 09:20 AM EDT
In this Saturday, Aug. 24, 2019 photo shows an helicopter fills its bucket with water during a wildfire near Pythagorio town on the eastern Greek island of Samos. Authorities evacuated two hotels and house the customers in an arena in the town of Pythagorio. Hot, dry weather and high winds helped fuel a total of 68 wildfires across Greece Saturday and the situation will remain dangerous Sunday. (AP Photo/Michael Svarnias)
ATHENS, Greece (AP) — Greece's fire service says it has arrested two people in southern Greece suspected of starting fires, one of them who did so three times.
The suspects, 47 and 43 years old, were arrested by firefighters in separate cases Saturday, in the Peloponnese. One is charged with starting a fire in a forest early Saturday and the other of starting three, including one last Sunday and two in less than an hour Saturday.
Hot, dry, windy weather has helped fuel dozens of wildfires across the country and the fire service has deployed almost 700 firefighters in the past 24 hours. The most serious fire, on the eastern island of Samos, is now under control, but authorities had to evacuate four hotels Saturday and house the guests in an indoor stadium overnight.
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Cyprus blasts 'pirate state' Turkey's new gas drilling bid
by MENELAOS HADJICOSTIS, Associated Press / Jan 19, 2020
NICOSIA, Cyprus (AP) — Cyprus on Sunday denounced Turkey as a "pirate state" that flouts international law as Turkey's bid to drill for natural gas in waters where Cyprus has economic rights rekindled tensions over energy reserves in the eastern Mediterranean.
Cyprus said Turkey was now attempting to drill inside an exploration area, or block, south of the ethnically split Mediterranean island nation that's already licensed to energy companies Eni of Italy and Total of France.
by JAKE COYLE, Associated Press / Jan 19, 2020
Scarlett Johansson and Nicole Kidman will have two chances to win Sunday night at the 26th Screen Actors Guild Awards, but the award show's ability to forecast the Oscars looks cloudy.
Among the films vying for the screen actors' top honor, best ensemble, are Oscar heavyweights "Once Upon a Time ... in Hollywood" and "The Irishman." They are competing with "Parasite" (only the second foreign language nominee after "Life Is Beautiful," which received a nod in 1999), "Jojo Rabbit" and "Bombshell."
StormTracker 13 Forecast Details


Acoustics play a crucial role in air-conditioning applications, because people are stressed by loud or persistent noise. But for process applications in production buildings too, workplaces have to comply with sound limit values as defined in occupational safety guidelines.
Knowing the emitted acoustic power level of flow technology components, and their interaction with the installation situation, is therefore of central importance. Measurement of the acoustic power level LwA of the volumetric flow or pressure is therefore in many cases the basis for optimisation of an air-conditioning or process air system. In other cases it is also used for testing of a product development.
As part of LTG's engineering services, we conduct in our echo chamber measurements conforming to accuracy class I as per DIN 45635 /2 and supplying meaningful results. The excellent infrastructure comprises various orifice and nozzle measuring sections, permitting measurement of a wide range of volumetric air flows. For acoustic measurements, we have six independent and variously positioned microphones. Their averaged measurements achieve standard divergences above the limit frequency of the room and at 110 Hz considerably below the DIN limit values. Exact measured data recording also enables us to measure octave and third-octave spectra with a two-channel real-time frequency analyser, to make assessments of the acoustic power levels (e.g. A-weighting), to calculate total acoustic power levels and to make idle level corrections.
We are therefore excellently equipped for measuring real volumetric flows and checking the acoustic power levels of individual products or planned product combinations – for example of an shut-off damper plus constant volumetric flow controller plus corridor wall outlet – to verify planning or to identify weak points. Furthermore, valid measurement results also secure the (further) development of your product.
Put us to the test – we look forward to receiving your inquiry!


text:
Charles Darling was a college football and baseball player for the Boston College Eagles. A triple threat, he played quarterback and fullback on the football team. Darling was captain of the 1923 team. Darling made various All-American selections.

References

American football quarterbacks
American football fullbacks
Boston College Eagles football players
Boston College Eagles baseball players
Baseball outfielders
All-American college football players

redpajama_set_name: RedPajamaWikipedia