C. elegans has become one of the preferred metazoans for studying cell biological processes, such as ERAD. However, a marker of ERAD activity comparable to CPY* has not been developed for this model system. We describe a mutant of pro-cathepsin L fused to YFP that no longer targets to the lysosome, but is efficiently eliminated by the ERAD pathway. Using this mutant pro-cathepsin L, we found that components of the mammalian ERAD system that participate in the degradation of ER luminal substrates were conserved in C. elegans. This transgenic line will facilitate high-throughput genetic or pharmacological screens for ERAD modifiers using widefield epifluorescence microscopy.Endoplasmic-reticulum associated degradation (ERAD) is a major cellular misfolded protein disposal pathway that is well conserved from yeast to mammals. In yeast, a mutant of carboxypeptidase Y (CPY*) was found to be a luminal ER substrate and has served as a useful marker to help identify modifiers of the ERAD pathway. Due to its ease of genetic manipulation and the ability to conduct a genome wide screen for modifiers of molecular pathways,  C. elegansC. elegans system has not been fully exploited due to the absence of well-defined luminal substrates that permit the visual, biochemical or genetic assessment of putative ERAD modifier genes.Biological pathways governing protein transcription, synthesis, folding, modification, trafficking and degradation maintain cellular protein homeostasis (proteostasis) C. elegans specific protein. In mammalian systems, mutations in the prepro-region of lysosomal papain-like cysteine peptidases induce protein misfolding and convert them to luminal ERAD substrates that are efficiently degraded by the ubiquitin-proteasome system (UPS) C. elegans, the best-described lysosomal cysteine peptidase is the cathepsin L-like protease, CPL-1 Thus, the purpose of this study was to generate a fluorescent luminal ERAD substrate using a C. elegans orthologue of yeast CPY. We identified 6 genes with approximately 30% and 48% similarity to yeast CPY and its human homologue, cathepsin A, respectively , Pnhx-2cpl-1::YFP (∼75-kDa), and a control line expressing human α1-antitrypsin, Pnhx-2sGFP::ATM (∼75-kDa)  changes in protein expression, whole animal lysates were analyzed under denaturing and non-denaturing conditions using PAGE and immunoblotting with anti-GFP/YFP antisera . Under d∼75-kDa)  [21]. Hoic lines . There aic lines , suggestthe case  and willW32A;Y35A::YFP band was only visible after ERAD inhibition (vide infra)  . These sW32A;Y35A::YFP if it was a luminal substrate. To test this hypothesis, we subjected the transgenic lines and their controls to ERAD(RNAi) by feeding a subset of bacterial clones from the Ahringer library that express double-stranded RNAs to different ERAD components  that appeared to have no effect on CPL-1W32A;Y35A::YFP accumulation, we tested their ability to activate the unfolded protein response (UPR) using the ire-1 activation sensor, Phsp-4GFP (hrdl-1(RNAi), significantly increased GFP expression in transgenic animals carrying the Phsp-4GFP transgene (hrdl-1(RNAi) was active, we performed semi-quantitative reverse transcriptase PCR and showed that steady-state HRDL-1 mRNA levels were decreased in Pnhx-2cpl-1W32A;Y35A::YFP animals treated with hrdl-1(RNAi) but not control(RNAi)  .W32A;Y35A::YFP fluorescence increased with ERAD inhibition, we determined whether ERAD activity accounted for the inability to detect CPL-1W32A;Y35A::YFP by immunoblotting. Protein lysates from CPL-1W32A;Y35A::YFP transgenic animals treated with hrd-1(RNAi) had detectable proteins levels, under both denaturing and non-denaturing conditions, comparable to those of the CPL-1::YFP expressing line , as a negative control, showed little accumulation of CPL-1W32A;Y35A::YFP (GFP(RNAi), reduced the YFP signal such that it is undetectable under these imaging conditions (cdc-48(RNAi) (hrd-1(RNAi) (sel-1(RNAi)  , hrd-1(R-1(RNAi)  or sel-1-1(RNAi) . Taken tW32A;Y35A::YFP accumulation. To test this hypothesis, we examined animals after proteasomal inhibition by both RNAi's directed at different proteasomal subunits (nhx2UB-V::mCherry. The UB-V::mCherry fusion protein contains a G76V mutation which blocks de-ubiquitination by de-ubquitinating enzymes (DUBs) and results in constitutive degradation by the proteasome myo-2GFP.Since proteasomal degradation is the end-point of the ERAD pathway, its inhibition should result in CPL-1subunits  and chemW32A;Y35A::YFP transgenic lines were exposed to RNAi's specific for different components of the 19S regulatory particle and the 20S catalytic core for 24 hours  had approximately 10-fold higher levels of total intestinal fluorescence within the intestine compared to animals expressing the UB-V::mCherry under the same conditions  controls  reduced the signal to nearly undetectable levels  and unc-51(RNAi) treated worms. These results suggested that these RNAi treatments blocked autophagosome formation. Additionally, treatment with lgg-1(RNAi) suppressed the expression of mCherry::LGG-1, showing that LGG-1 expression itself could be down-regulated. Although there was no significant increase in CPL-1W32A;Y35A::YFP accumulation after autophagy knockdown, there was a slight increase in YFP fluorescence compared to control animals. To confirm that the autophagy pathway was not a major means of CPL-1W32A;Y35A::YFP disposal, we crossed Pnhx-2cpl-1W32A;Y35A::YFP animals with an unc-51(e369) knockout strain to yield unc-51(e369);Pnhx-2CPL-1W32AY35A::YFP;Pmyo-2mCherry animals. Two independent lines were analyzed, and showed no significant differences in the level of CPL-1W32A;Y35A::YFP, as compared to the controls  and is known to function in embryogenesis, yolk protein processing, molting and lysosomal-dependent necrotic cell death pathway nhx-2 promoter does not drive embryonic expression) W32A;Y35A) abolished the ability of CPL-1 to traffic to these locations. Furthermore, the YFP signal was visible in the ER, either as a diffuse reticular pattern or within distended cisternae , with the most homologous, F13D12.6, showing the greatest similarity (33.4%) and identity (19.7%). However, F13D12.6::YFP did not traffic to the endo-lysosomal compartment. Since C. elegans intestinal cells contain both lysosomes and lysosomal-related organelles The canonical ERAD substrate is the mutated yeast vacuolar protease, CPY* W32A;Y35A::YFP accumulation was easily discernible by widefield epifluorescence microscopy. Thus, the animals expressing CPL-1W32A;Y35A::YFP should facilitate the use of high-throughput chemical or genome-wide genetic screens to further our understanding of the ERAD and UPS pathways.In summary, upon ERAD/UPS inhibition, CPL-1All amplifications were performed using the Expand high fidelity PCR system . Restriction enzymes used for cloning procedures were purchased from New England Biolabs  unless otherwise stated.myo-2 mCherry construct (pAV1944) (myo-2 promoter was amplified (primer set 2), and the fragment was ligated into the SphI and XbaI sites to yield the final construct, pAV1944.To generate the PpAV1944) , a 717 bpAV1944) , and thenhx-2mCherry (pAV1951) followed by gel purification to remove mCherry. Next, an 868 bp YFP cDNA containing synthetic introns was amplified (primer set 3) from pPD133.86 (a kind gift from Dr. Andrew Fire), and the fragment ligated into the NheI and SacI restriction sites to form Pnhx-2YFP (pKS2236). Pnhx-2cpl-1::YFP (pKS2301) was generated by amplifying a 2.6 kb cpl-1 genomic DNA fragment (primer set 4), and ligating the fragment into the NheI site of Pnhx-2YFP (pKS2236). Pnhx-2cpl-1W32A;Y35A::YFP (pKS2311) was created by site-directed mutagenesis of those codons corresponding to amino acids 32 and 35 of Pnhx-2 cpl-1::YFP using the Quikchange™ mutagenesis kit (primer set 5) according to manufacturers instructions .For YFP fusion proteins, an NheI/SacI double digest was performed on Pnhx-2F13D12.6::YFP (pNG2462) was generated by amplifying a 3.2 kb F13D12.6 genomic DNA fragment (primer set 6) and ligating the fragment into the NheI site of Pnhx-2YFP (pKS2236). Pnhx-2F13D12.6G166R::YFP (pNG2470) was generated by site-directed mutagenesis (primer set 7).Pnhx-2DsRed::KDEL (pAV1825), the nhx-2 promoter was amplified (primer set 8) and sub-cloned into the HindIII and XbaI sites of pPD95.85. The stop codon of GFP was mutated by Quikchange™ site-directed mutagenesis to a KasI site (primer set 9). Next, the 675 bp DsRed insert was amplified (primer set 10) from the pDsRed-Express-C1 vector  with KpnI and KasI sequences at the 5′ and 3′ ends, respectively. The reverse primer also contained the codons corresponding to the ER retention motif, KDEL. The GFP insert was replaced by ligating the DsRed::KDEL insert into the KpnI and KasI sites.To generate Pnhx-2UB-V::mCherry (pSG2142) construct, the 248 bp ubiquitin sequence was amplified (primer set 11) from the Punc-47UbA47V::DsRed expression construct nhx-2 promoter and amplified ubiquitin sequence were sub-cloned into the HindIII/XbaI and NheI sites, respectively, of mCherry_pPD49.26 (pAV1997). Quikchange™ site directed mutagenesis (primer set 12) of the Pnhx-2Ub-V::mCherry (pSG2142) yielded an intermediate construct, Pnhx-2UB-R::mCherry (pSG2143), which was not used for this investigation. A second round of Quikchange™ site directed mutagenesis (primer set 13) on Pnhx-2UB-R::mCherry (pSG2143) yielded Pnhx-2UB-M::mCherry (pSG2144). The Phsp-4GFP (pAV2021) construct was generated by amplifying the 1.2 kb hsp-4 promoter (primer set 14) from genomic DNA and ligating the fragment into the HindIII and XbaI sites of pPD95.77 To generate the Pnhx-2YFP and Pmyo-2mCherry with 20 ng/µl and 5 ng/µl, respectively, into the gonad of young adult N2 hermaphrodites. Strains VK1256, VK1258, VK1770 and VK1870 were generated by co-injecting 20 ng/µl of the expression plasmids Pnhx-2cpl-1::YFP, Pnhx-2 cpl-1W32A;Y35A::YFP, Pnhx-2F13D12.6::YFP and Pnhx-2F13D12.6G166R::YFP, respectively, with 20 ng/µl Pnhx-2DsRed::KDEL. Strain VK1879 was generated by co-injecting the Pnhx-2 cpl-1W32A;Y35A::YFP plasmid with Pmyo-2mCherry at 20 ng/µl and 5 ng/µl, respectively. Strains VK1244, VK1243 and VK1241 were generated by co-injecting 80 ng/µl of the plasmids Pnhx-2Ub-M::mCherry, Pnhx-2Ub-V::mCherry and Pnhx-2mCherry::lgg-1, respectively, with 5 ng/µl of myo-2GFPP. The worm strain VK737 was generated by co-injecting the Phsp-4GFP and Pmyo-2mCherry plasmids at 80 ng/µl and 5 ng/µl, respectively. Strain VK689 was generated as described by Gosai, et al. nhx-2CPL-1W32AY35A::YFP;Pmyo-2mCherry]unc-51(e369);vkEx1258, human cathepsin A [CAI20250.1], and six C. elegans homologues (identified by BLAST) using the ClustalW algorithm. Accession numbers for aligned potential C. elegans CPY homologues are as follows: F13D12.6 [CAA88947.1], C08H9.1 [CAA91143.1], K10B2.2a [CCD66392.1], F41C3.5 [CCD65861.1], F32A5.3 [CCD66273.1], and Y40D12A.2 [CCD64385.1]. Blue shading/highlighting is pre-pro domain, the green box is the excision peptide and red boxes are catalytic triad residues. (B–I) Transgenic animals expressing F13D12::YFP (B–E) or F13D12.6G166R::YFP (F–I) were examined by confocal microscopy. Both lines were co-injected with a DsRed::KDEL transgene to mark the ER , and were incubated with BSA::AlexaFluor647 to label the endo-lysosomal compartment . Both F13D12.6::YFP and F13D12.6G166R::YFP demonstrated a fine reticular pattern within intestinal cells that co-localized with DsRed::KDEL . The wild-type protein did not co-localize with BSA::AlexaFluor647, suggesting that this protein did not traffic to the endo-lysosomal compartment. Scale bar represents 10 µm. (J) Immunoblot of protein lysates from two different transgenic strains expressing F13D12.6G166R::YFP. Blot probed with anti-GFP/YFP antisera. Mr fusion protein = 77-kDa. (K) The mutant protein, CPL-1W32A;Y35A::YFP, but not F13D12.6G166R::YFP, accumulated in the ER after ERAD knockdown using cdc-48(RNAi), hrd-1(RNAi) or sel-2(RNAi); suggesting that the latter mutant protein was not an ERAD substrate under these experimental conditions.(TIF)Click here for additional data file.Figure S2W32A;Y35A::YFP does not affect Expression of CPL-1C. elegans lifespan. Kaplan-Meier survival curves were generated for N2 (blue), Pnhx-2cpl-1::YFP (green), or Pnhx-2cpl-1W32A;Y35A::YFP (red) animals to determine if animal longevity was affected by expression of either transgene. Individual strains had mean survival times of 300 h (N2), 360 h (CPL-1::YFP), and 312 h . Statistical significance compared to control was assessed by log-rank test, which indicated no statistical difference between strains . Assays were performed at 20°C and the total number of observations counted were equal to the number of animals that died plus the number of censored animals.(TIF)Click here for additional data file.Figure S3Controls for ERAD RNAi effectiveness. (A) Pnhx-2YFP or (B) Phsp-4GFP animals were exposed to the ERAD RNAi panel for 48 h and processed as described in vector(RNAi) control. Total intensity per animal was used in place of total area. Statistical analysis of the RNAi treated animals relative to vector was performed using an unpaired, 2-tailed t-test  (*p<0.05). No statistical difference in total YFP fluorescence was observed for all tested RNAi's, suggesting RNAi treatment did not alter levels of transgene expression by activating the nhx-2 promoter (A). All RNAi's tested significantly raised Phsp-4GFP expression levels except for hrdl-1(RNAi) as previously described hrdl-1(RNAi) was demonstrated by showing knockdown of steady-state HRDL-1 mRNA levels by semi-quantitative RT-PCR. Total RNA was isolated from CPL-1W32A;Y35A animals treated with either vector, hrd-1, or hrdl-1 RNAi. RT-PCR (+/−RT) reactions were performed on a 10-fold serial dilution of cDNA's from each RNAi condition to amplify HRD-1, HRDL-1, or AMA-1. HRDL-1 cDNA was not detected after the hrdl-1(RNAi). AMA-1 and genomic (g) DNA served as RT and amplification controls, respectively.(TIF)Click here for additional data file.Figure S4Autophagy RNAi controls. mCherry::LGG-1 expressing animals were exposed to an autophagy RNAi panel for 48 hours and starved for 4 hours to induce autophagy. Images of 5–10 animals were collected using a widefield epifluorescence microscopy. In vector(RNAi) animals the mCherry::LGG-1 expression profile shifted from a diffuse to a more punctate distribution, indicating autophagosome formation, while treatment with lgg-1(RNAi) successfully reduced the mCherry::LGG-1 fluorescence to below detectable levels. unc-51(RNAi) or bec-1(RNAi) also prevented puncta formation, indicating suppression of autophagosome formation.(TIF)Click here for additional data file.Table S1Transgene List.(DOCX)Click here for additional data file.Table S2PCR primer pairs for transgene construction.(DOCX)Click here for additional data file.Table S3C. elegans strain list.(DOCX)Click here for additional data file.


Age-associated decline in cardiovascular function is believed to occur from the deleterious effects of reactive oxygen species (ROS). However, failure of recent clinical trials using antioxidants in patients with cardiovascular disease, and the recent findings showing paradoxical role for NADPH oxidase-derived ROS in endothelial function challenge this long-held notion against ROS. Here, we examine the effects of endothelium-specific conditional increase in ROS on coronary endothelial function. We have generated a novel binary (Tet-ON/OFF) conditional transgenic mouse (Tet-Nox2:VE-Cad-tTA) that induces endothelial cell (EC)-specific overexpression of Nox2/gp91 (NADPH oxidase) and 1.8±0.42-fold increase in EC-ROS upon tetracycline withdrawal (Tet-OFF). We examined ROS effects on EC signaling and function. First, we demonstrate that endothelium-dependent coronary vasodilation was significantly improved in Tet-OFF Nox2 compared to Tet-ON (control) littermates. Using EC isolated from mouse heart, we show that endogenous ROS increased eNOS activation and nitric oxide (NO) synthesis through activation of the survival kinase AMPK. Coronary vasodilation in Tet-OFF Nox2 animals was CaMKKβ-AMPK-dependent. Finally, we demonstrate that AMPK activation induced autophagy and thus, protected ECs from oxidant-induced cell death. Together, these findings suggest that increased ROS levels, often associated with cardiovascular conditions in advanced age, play a protective role in endothelial homeostasis by inducing AMPK-eNOS axis. Increased levels of reactive oxygen species (ROS) are often associated with microvascular pathology in age-related cardiovascular diseases (CVD) including coronary artery disease (CAD) and ischemic heart disease (IHD) - the major causes of death and morbidity in the USA-3. ThesePrecise mechanisms by which ROS modulate signal transduction in ECs are not fully understood. Several studies have shown that ROS play critical roles in ECs by activating signaling intermediates including PI3K-Akt-eNOS, PLCγ1, PKC and ERK1/2 , 17, 18.in vitro, global knockdown animal models of NADPH oxidase (p47phox−/− or gp91phox−/−), or constitutive over-expression of NADPH oxidases (e.g. Nox4) in ECs were performed using escribed . VesselsMouse heart ECs (MHECs) were isolated from the heart specimens of Tet-ON and Tet-OFF animals, as previously described . For eacTet-ON and Tet-OFF MHEC grown on Lab-Tek Chamber slides were transduced with Ad- mRFP-EGFP-LC3 at 5 MOI (multiplicity of infection) , 40. Aftp < 0.05. Representative blots are shown in the Results section.Cell lysates were prepared from MHEC grown on 0.1% gelatin-coated plates to 80%-90% confluence. Western blot (WB) analyses using MHEC protein lysates were performed as previously described . Anti-ph2PO4 (pH 7.0), 1× protease mixture inhibitor (Sigma), 1 mM EGTA, 10 μg/mL aprotinin , 0.5 μg/mL leupeptin (Sigma), 0.7 μg/mL pepstatin (Sigma), and 0.5 mmol/L phenyl-methylsulfonyl fluoride . NADPH oxidase activity was measured using a modified assay arginine to L-[3H]citrulline as described previously [Tet-ON and Tet-OFF MHEC were lysed in ice-cold homogenization buffer . eNOS activity was determined using the NOS assay kit  that measures conversion of L-[eviously .All values are presented as mean ± SEM where appropriate. A value of P<0.05 between experimental groups was considered to represent a significant difference (*). Nonlinear regression modeling utilizing the extra sum-of-squares F test to compare slopes (Prism 5. Graph Pad Software) was used for vessel relaxation assays. ANOVA and Tukey's post-hoc test will be used for comparison between groups.


Pulmonary arterial hypertension (PAH) is a rare progressive, usually fatal lung disease of different ethiologies. Endothelial cell dysfunction contributes to the pathogenesis, although the specific mechanisms are not clear. Impaired vascular cGMP signaling seems to be involved in the development of pulmonary vascular remodeling and constriction, because drugs preventing cGMP degradation or stimulating cGMP production showed beneficial effects. Atrial (ANP) and B-type natriuretic peptides (BNP) both act through the guanylyl cyclase-A (GC-A) receptor and stimulate cGMP production in all vascular cell types , smooth muscle cells (SMC) and fibroblasts). The purpose of the study was to analyze whether mice with global or conditional, EC- or SMC-restricted GC-A ablation develop PH under normoxic conditions and which genes are differentially expressed.Mice with global or conditional, EC-restricted GC-A deletion develop PH even under normoxic conditions. Right ventricular (RV) systolic pressure is increased.This is accompanied by RV hypertrophy and dysfunction, enhanced muscularization of small pulmonary arteries and arteriolar rarefaction. At difference, deletion of GC-A in SMC did not result in PH. Because mice with EC-restricted ablation of GC-A (EC GC-A KO) develop PH, a microarray analysis was performed to identify differentially expressed genes. Precapillary pulmonary arteries (21-87 µm diameter) were dissected by Laser microdissection, RNA was isolated and transcribed into cDNA and applied to Mouse 430_2 Affimetrix chips. The mRNA expression of 34 genes was up- and of 13 genes was downregulated in precapillary pulmonary arteries of EC-GC-A-KO mice. The top ten overexpressed mRNAs were Epac2 (3.6 times), adenosine deaminase, tRNA-specific 2 (3.1 times), UBX domain protein 7 (3.0 times), ring finger protein 128 (2.8 times), DAZ interacting protein 3 zinc finger (2.8 times), UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 5 (2.7 times), LEM domain containing 3 (2.6 times), retinol dehydrogenase 10 (2.2 times), glypican 4 (2.2 times) and splicing factor proline/glutamine rich (2.2 times). The top ten downregulated mRNAs were charged multivesicular body protein 4C (-3.2 times), family with sequence similarity 199, X-linked (-3.1 times), transient receptor potential cation channel, subfamily M, member 7 (-2.8 times), pecanex homolog (Drosophila) (-2.7 times), serine/arginine-rich splicing factor 18 (-2.6 times), zinc finger and BTB domain containing 1 (-2.5 times), importin 5 (-2.4 times), adenomatosis polyposis coli (-2.3 times), myogenic factor 5 (-2.3 times) and solute carrier family 35 member B3 (-2.2 times).These observations illustrate that ANP/GC-A/cGMP signaling critically affects pulmonary EC proliferation/viability and the paracrine crosstalk of pulmonary EC with SMC. Furthermore, the pulmonary GC-A/cGMP responsiveness to ANP was mitigated in PAH patients. Here, we provide new mechanistic insights into PAH and genes involved in its development, i.e. the role of altered endothelial natriuretic peptide signaling.


We explored the apoptosis resistance properties of nickel chloride-transformed human lung epithelial cells and the underlying mechanisms. The results showed that transformed BEAS-2B human lung epithelial cells are resistant to NiCl2-induced apoptosis. They have increased Bcl-2, Bcl-xL and catalase protein levels over the passage matched non-transformed counterparts. The mechanisms of apoptosis resistance are mitochondria-mediated and caspase-dependent. Forced overexpression of Bcl-2, Bcl-xL and catalase proteins reduced NiCl2-induced cell death; siRNA-mediated knockdown of their expression sensitized the cells to nickel-induced apoptosis, suggesting that Bcl-2, Bcl-xl and catalase protein expression plays a critical role in apoptosis resistance. Akt also participates in this process, as its overexpression increases Bcl-xL protein expression levels and attenuates NiCl2-induced apoptosis. Furthermore, transformed cells are tumorigenic in a xenograft model. Together, these results demonstrate that nickel-transformed cells are apoptosis-resistant and tumorigenic. Increased expression of Bcl-2, Bcl-xL and catalase proteins are important mechanisms contributing to transformed cell oncogenic properties.Chronic exposure to nickel compounds is associated with increased incidence of certain types of human cancer, including lung and nasal cancers. Despite intensive investigation, the oncogenic processes remain poorly understood. Apoptosis resistance is a key feature for tumor cells to escape physiological surveillance and acquire growth advantage over normal cells. Although NiCl Nickel is a ubiquitous environmental metal, widely used in industrial processes. Nickel compounds are important human carcinogens. Epidemiologically, chronic and professional exposure to nickel compounds is associated with increased incidence of certain human cancer, including lung and nasal cancers ,2. Both Several mechanisms have been proposed for nickel-induced carcinogenesis. These include production of reactive oxygen species (ROS), induction of DNA damage ,4, alter2)-induced epithelial cell oncogenic transformation accompanied by enhanced Bcl-2, Bcl-xL protein expression (Apoptosis (programmed cell death) is a key mechanism to maintain normal cell homeostasis, it removes cells that carry abnormal genetic information and keeps the functional integrity of organ or cell populations . Aberranpression .Bcl-2, and Bcl-xL protein expression has been implicated in cancer and cell transformation ,12, ther2-transformed BEAS-2B cell apoptosis resistance. The mechanisms are mitochondria-mediated and caspase-dependent. Transformed cells are also tumorigenic with accompanied oncogenic protein expression. The results characterize the oncogenic properties of nickel-transformed human lung epithelial cells and provide insight into nickel-induced carcinogenesis.We demonstrate in this study that increased Bcl-2, Bcl-xL and ROS scavenging enzyme expression are important in conferring NiCl2-transformed BEAS-2B cells were isolated from soft agar by low dose NiCl2 exposure with BEAS-2B cells for 6 months . NiCl6 months . These c6 months . BEAS-2B6 months ,14. Tiss2) was purchased from Sigma Chemical Company . Polyclonal and monoclonal antibodies against phospho-Akt at Ser473 (#9275), phospho-Stat3 (#9145), Bcl-xL (#2764), catalase (#8841), HIF-1α (#3434), cleaved caspase-3 (#9661), cleaved caspase-7 (#9491), cleaved PARP (#9542), peroxiredoxin 1 (#8499) were purchased from Cell Signaling Technology . Bcl-2 was from Dako . SOD1 (sc-11407), SOD2 (sc-130345), β-actin (sc-47778), lamin A/C (sc-6215), Akt1 (sc-5298) and NF-κB p50 (sc-7178) were purchased from Santa Cruz Biotechnology .Nickel chloride (NiCl6) treated with or without NiCl2 were collected and washed once with cold PBS, they were stained with 5 μl of Annexin V-FITC or Hoechst 33342 followed by 10 μl of PI (5 μg/ml) in binding buffer  for 15 min at room temperature in dark. The apoptotic cells were determined using a Becton-Dickinson FACScan cytofluorometer. Both early apoptotic (Annexin V or Hoechst 33342-positive and PI-negative) and late apoptotic (Annexin V or Hoechst 33342-positive and PI-positive) cells were included in cell death determinations.Apoptosis was determined by Annexin V/propidium iodide (PI) staining kit . In some experiments, due to the expression of green fluorescence protein (GFP) interfering with Annexin V-FITC reading, a double stain apoptosis detection kit using Hoechst 33342/PI staining  was chosen to replace Annexin V-FITC. Briefly, cells . Proteins (20 μg) were separated on 10% SDS polyacrylamide gels. Proteins were subsequently transferred from gel onto nitrocellulose membrane . Membranes were blocked for 1 h at room temperature in Tris-buffered saline plus 0.01% Tween-20 (TBS-T) in 5% non-fat dry milk (pH 7.4). Various antibodies were diluted at 1:1,000 in TBS-T with 5% non-fat dry milk solution, incubated with membrane at 4°C overnight and washed three times with TBS-T. The secondary antibody, horseradish peroxidase (HRP)-conjugated antibodies, diluted at 1:2,500 in TBS-T with 5% non-fat dry milk were incubated with membrane at room temperature for 2–3 h. Signal was detected with an enhanced chemiluminescence detection kit . In each experiment, either anti-β-actin or anti-lamin A/C antibody was reprobed to monitor protein loading. Image quantification was processed by ImageJ software . In some experiments, mean densitometry values were adjusted with β-actin and expressed as fold changes over the appropriate controls.(Rt-Et); where Rt is the mean threshold cycle for the reference gene HPRT and Et is the mean threshold cycle for experimental gene. Data are expressed as arbitrary units and adjusted to fold changes over the non-stimulated control cells. Primer sequences are provided in Total RNA from BEAS-2B cells was extracted using TRIzol reagents as described previously . Reverse5) were seeded into 60-mm culture dishes and treated with NiCl2 for 16 h. Cells were trypsinized, washed in ice-cold PBS and incubated with 2 μM JC-1 at 37°C for 20 min. Cells were washed once with PBS and analyzed by FACScan cytofluorometer.Mitochondrial membrane potential (ΔΨm) was monitored using MitoProbe JC-1 assay kit (Invitrogen). 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1), a lipophilic cationic fluorescence dye, is capable of selectively entering mitochondria, where it forms monomers, and emits green fluorescence (FL-1) when ΔΨm is relatively low. At high ΔΨm, JC-1 aggregates and gives red fluorescence (FL-2) . Thus th® 3/7 assay system  following manufacturer’s instructions. Briefly, BEAS-2B or transformed BEAS-2B cells were treated with or without NiCl2 at 1.5 mM for 16 h. Caspase-Glo 3/7 reagent (100 μl) was added into 96-well plates and incubated for 1 h at room temperature. The luminescence was measured using a Glomax™ 96 microplate luminometer (Promega). Z-VAD-FMK , a cell-permeant pan-caspase inhibitor that irreversibly binds to the catalytic site of caspase proteases that inhibit induction of apoptosis was added 30 min before nickel stimulation at 20 μM solution in DMSO (Promega). Data were collected and expressed as fold change over the control without NiCl2 treatment.Caspase activity was assessed using the luminescent Caspase-Glo2 for another 24 h. Cell lysates or cells were collected either for western blot or apoptosis analysis as mentioned above.Transfection procedures followed manufacturer’s recommended protocol. Small interference RNA (siRNA) for Bcl-2 (sc-29214), Bcl-xL (sc-43630) were purchased from Santa Cruz Biotechnology . Scrambled control and siRNA for catalase (s-2443) were obtained from Ambion . Briefly, siRNA for Bcl-2, Bcl-xL, catalase and controls were incubated with Lipofectamine™ RNAiMAX in Opti-MEM I medium (Invitrogen) for 20 min at room temperature. They were then added to cell culture media without antibiotics. Media were replaced 24 h post-transfection. Cells were treated with or without NiClGFP labeled p-MIG, p-MIG-Bcl-2, pMIG-Bcl-xL retroviral vector  were kin6) were mixed with Matrigel and injected in both side of flank region in 50 μl volume. Five animals in each group gave 10 injection sites. Mice were fed normally, the body weight, and tumor growth was monitored twice weekly until the tumor size reached 1 × 1 × 1 cm3 in size or 8 weeks post inoculation.Athymic nude (nu/nu) female mice, 5 weeks old and weighing about 18 to 20 g were purchased from Jackson Laboratory . Mice were handled following the University of Kentucky guidelines for care and use of laboratory animals and approved by the ethical committee. BEAS-2B or transformed BEAS-2B cells . Student’s t-test was used to compare the difference between various controls and NiCl2 for 24 h for apoptosis induction. At 0.5, and 1 mM doses, only BEAS-2B cells showed apoptosis induction in 24 h, T-BEAS-2B cell showed little or no apoptosis (data not shown), therefore, 1.5 mM of NiCl2 was selected for apoptosis induction in both types of cells.To evaluate Bcl-2, Bcl-xL protein expression and their role in nickel-induced apoptosis, BEAS-2B and transformed BEAS-2B (T-BEAS-2B) cells were first treated with or without 0.5, 1 and 1.5 mM of NiCl2 for 24 h to analysis apoptosis. The results indicated that T-BEAS-2B cells had significantly lower level of apoptosis . These results suggested a mechanism of NiCl2-induced gene transcription repression  enhanced apoptosis . These results indicated that enhanced Bcl-2, Bcl-xL and catalase protein expression are important mechanisms for reduced mitochondria damage in transformed cells.To investigate whether transformed cells are resistant to mitochondria damage. We evaluated NiCl2B cells . Forced 2B cells . Signifi2-induced apoptosis resistance and mitochondria damage; thus we further studied the underlying molecular mechanisms. We compared caspase activation in both types of cells. The result in 2 stimulation, BEAS-2B cells had much stronger caspase activation, presented as increased c-caspase 3, 7 and c-PARP protein expression when compared with T-BEAS-2B cells  and dominant negative Akt (DN-Akt) stable cell lines to determine the effects of Akt on NiCl2-induced apoptosis. The results indicated that Akt stable cells had persistently enhanced p-Akt level (2 challenge induced marginally higher levels of apoptosis in control and DN-Akt cells over the Akt stable cells (2-induce BEAS-2B cell apopotsis.p-Akt expression was upregulated in T-BEAS-2B cells in a previous study . To undekt level , and inckt level . In addile cells , pointin2-transformed cells (To evaluate if the transformed cells are tumorigenic, we injected both control BEAS-2B and T-BEAS-2B cells into nude mice and observed the tumor growth. The results indicated that among five injected mice in each group, only T-BEAS-2B cells induced tumor growth, and tumors occurred at multiple injection sites, grew into various sizes within 8 weeks . Furthered cells .2-transformed human lung epithelial cells, and explored the potential molecular mechanisms. The results demonstrate that NiCl2 transformed BEAS-2B cells are resistant to apoptosis and tumorigenic. Higher levels of Bcl-2, Bcl-xL and catalase protein expression are important mechanisms contributing to the transformed cell oncogenic properties.Nickel is a known human carcinogen and induces genotoxic stress. A number of mechanisms have been proposed for its carcinogenic effects; such as generation of ROS, induction of DNA damage, and activation of oncogenic pathways . Howeverin vitro studies have shown that water-insoluble nickel compounds such as nickel oxide, nickel subsulfide are more readily phagocytized than the water-soluble nickel compounds such as nickel chloride. They can reach the nucleus of the cell in greater amounts than that of water-soluble nickel compounds and induce more cell damage (2 was selected due to the BEAS-2B cells being transformed by NiCl2 exposure (2-induced apoptosis resistance in transformed cells by attenuating the detrimental effects from ROS generated by NiCl2 exposure. However, the biological implications of these changes during transformation have yet to be explored, as well as the molecular mechanisms that result in their increased expression in transformed cells. The data provide evidence that transformation involves multiple cellular signal alternations, which deserve further investigation.Previously l damage ,20. In texposure . One impexposure ,4. Increexposure . Howeverexposure ,18, the exposure , suggestexposure ,18. High2-induced apoptosis can be affected by both Bcl-2 and Bcl-xL expressing vector and siRNA transfection, indicating that their relative levels regulate cell apoptosis resistance in T-BEAS-2B cells. The question of how transformed cell evolve into higher level of Bcl-2, and Bcl-xL protein expression remain to be investigated, we hypothesis that either genetic or epigenetic modifications may be responsible during the transformation and selection processes.Bcl-2 and Bcl-xL proteins are overexpressed in a variety of human cancers , and are2 treatment reduced the cellular level of Bcl-2 and Bcl-xL proteins in both transformed and non-transformed cells (2 exposure, therefore, confer these cells the apoptosis resistant property.In our study, NiCled cells , but the2-induced apoptosis. The results uncovered anti-apoptotic protein-dependent mechanism in nickel-induced apoptosis resistance in transformed cells.Several signaling pathways such as PI3K, NF-κB, Akt, and Sonic hedgehog have been shown to regulate Bcl-2 and Bcl-xL protein expression and apoptosis in different cell types ,24,25. I2-mediated cell death resistance program in transformed BEAS-2B cells. Future investigations will be required to clarify if other mechanisms might also be involved in transformed cell resistance to apoptosis.It is worth noting that overexpression of Akt could not completely block the nickel-induced apoptosis , and eveOne important feature of transformed cells is their tumorigenic property. These cells also have enhanced p-Stat3, NF-κB-p50 subunit, and HIF-1α protein expression. The altered oncogene expression probably reflects extensive genetic or epigenetic rearrangements during the transformation processes which allow transformed cells to adopt and grow in a new environment. It is also possible that they acquire cancer stem cell trait to allow cell resistant apoptosis. Future studies focusing on nickel-induced apoptosis resistance and cell transformation are of great interest to understand nickel-induced carcinogenic mechanisms and to provide options for prevention.


Levels of the selective autophagy substrate p62 have been established in recent years as a specific readout for basal autophagic activity. Here we compared different experimental approaches for using this assay in Drosophila larvae. Similar to the more commonly used western blots, quantifying p62 dots in immunostained fat body cells of L3 stage larvae detected a strong accumulation of endogenous p62 aggregates in null mutants for Atg genes and S6K. Importantly, genes whose mutation or silencing results in early stage lethality can only be analyzed by microscopy using clonal analysis. The loss of numerous general housekeeping genes show a phenotype in large-scale screens including autophagy, and the p62 assay was potentially suitable for distinguishing bona fide autophagy regulators from silencing of a DNA polymerase subunit or a ribosomal gene that likely has a non-specific effect on autophagy. p62 accumulation upon RNAi silencing of known autophagy regulators was dependent on the duration of the knockdown effect, unlike in the case of starvation-induced autophagy. The endogenous p62 assay was more sensitive than a constitutively overexpressed p62-GFP reporter, which showed self-aggregation and large-scale accumulation even in control cells. We recommend western blots for following the conversion of overexpressed p62-GFP reporters to estimate autophagic activity if sample collection from mutant larvae or adults is possible. In addition, we also showed that overexpressed p62 or Atg8 reporters can strongly influence the phenotypes of each other, potentially giving rise to false or contradicting results. Overexpressed p62 aggregates also incorporated Atg8 reporter molecules that might lead to a wrong conclusion of strongly enhanced autophagy, whereas expression of an Atg8 reporter transgene rescued the inhibitory effect of a dominant-negative Atg4 mutant on basal and starvation-induced autophagy. Human p62/SQSTM1 (sequestosome-1) is a multidomain scaffold protein involved in various signaling pathways regulating a number of processes including apoptosis, stress responses, and cell growth. Different p62-based assays have been implemented in recent years in various experimental systems, including immunostaining of cultured cells or tissue samples, western blots, and GFP-tagged reporters. Given the importance of the p62 assay in characterizing autophagy phenotypes of selected mutants and RNAi treatments, we have generated polyclonal antibodies against endogenous Drosophila p62. The antibody recognized a single prominent band of about 100 kDa on western blots prepared form L3 stage larvae or adult heads , Vps34 (encoding the phosphatidyl-inositol 3-kinase), Atg2 and Atg18a (encoding binding partners of the transmembrane protein Atg9), Atg7 and Atg8a (encoding the E1-like enzyme and one of the ubiquitin-like proteins in the autophagy-specific protein conjugation systems)  enhanced p62 aggregation, similar to overexpression of dominant-negative Vps34 . SilenciNumerous genes are required for fundamental cellular processes such as transcription or translation. It is not so surprising that these genes are often identified as putative hits in various genome-wide RNAi screens, although their effect is likely indirect in most cases. While knockdown of many of these genes reduces cell size to an extent that evaluation of autophagy phenotypes becomes practically impossible, silencing of the genes coding for the RNA polymerase II. complex member Atms or the ribosomal subunit RpS8 strongly interfered with starvation-induced mCherry-Atg8 and Lysotracker puncta formation . On the A GFP-tagged p62 reporter was previously used successfully in Drosophila to detect basal autophagy defects in Atg13 mutant cells. JF showed better suppression than Atg18aKK in both L3 and L2 stage animals, likely explaining the quantitatively different p62 results , one striking observation was that the p62 assay occasionally showed large differences in case of two different RNAi lines targeting the same gene. For example, two different transgenic RNAi lines for Atg18a had quantitatively different effects on p62 aggregation, potentially reflecting differences in knockdown efficiencies. Interestingly, one of two different RNAi lines against Atg16 (encoding a protein required for Atg8a lipidation) failed to enhance p62 aggregation, similar to the case of PTEN, inactivation of which strongly enhances insulin signaling to promote cell growth and suppress autophagy . As both animals . AltogetFinally, we have evaluated the impact of overexpressed p62 and Atg8a reporters  on autophagy phenotypes, as the possibility of genetic interactions could not be excluded. In fact, when p62-GFP was co-expressed with mCherry-Atg8a, large colocalizing GFP- and mCherry-positive aggregates were formed even in well-fed animals , further supporting our hypothesis that partial loss of Atg4a function can be rescued by overexpression of Atg8a.Atg4 encodes a cysteine protease required for the cleavage of the C-terminal amino acids of Atg8a, which is necessary for its subsequent activation and conjugation to the membrane lipid phosphatidyl-ethanolamine. Atg8a needs to be delipidated after successful completion of an autophagosome to allow lysosomal fusion. A mutant form of human Atg4B with a substitution of cysteine 74 to alanine was shown to act in a dominant-negative fashion, inhibiting proper formation and clearence of autophagosomes. f p62 , . As exprf p62 , , b, we tf p62 , , d. UltrDetermining the levels of the specific autophagy substrate p62 is becoming a standard assay to estimate basal autophagy rates. In case of autophagy induced by a certain stimulus such as starvation, levels of proteins required for autophagy at the time of the stimulus and during the response are critical determinants of the magnitude of autophagy that follows. In contrast, p62 levels are also strongly influenced by basal autophagic activity, so formation of excess p62 aggregates is a progressive, time-dependent process. Therefore, duration of a certain treatment or effective gene silencing by RNAi has a major impact on this type of assay. In line with that, we have shown here that p62 levels may remain normal despite a strong block of starvation-induced autophagy if the onset of efficient gene knockdown is delayed during larval Drosophila development. Based on these observations, the perdurance of maternally supplied gene products during development may also account for some of the differences seen in p62 levels in different Atg mutants.Additionally, certain problems may arise when using overexpressed reporter transgenes for autophagy. First, overexpressed Atg8a reporters were found to be captured in overexpressed p62 aggregates, giving a false impression of increased autophagy. Second, overexpression of an Atg8a reporter may genetically rescue the partial loss of a gene as seen in case of Atg4a, again complicating the interpretation of potentially contradicting results. While the examples shown here may be rare, we have seen several instances when inactivation of certain genes led to such a high accumulation of endogenous p62 aggregates that sequestered Atg8a reporter molecules, and overexpressed Atg8a reporters also seemed to genetically rescue the effect of a number of RNAi lines on autophagy.Finally, although our experiments involved fat body cells of Drosophila larvae, we are convinced that the results and limitations presented here will be applicable to most cells and organisms used in autophagy research.UAS-p62-GFP, Atg13[Δ81],p62[KK108193], Atg8a[KK109654], Atg18a[KK105366], Atg14[KK108559], Atg16[KK105993], TSC2[KK103417], Pten[KK101475], RpS8[KK106835], Atms[GD20876],Atg7[d77]/Atg7[d14],Vps34[Δm22], UAS-Vps34[KD],p62[HMS00551], p62[HMS00938], Atg1[JF02273], Atg18a[JF02898], Atg9[JF02891], Atg7[JF02787], Atg16[HMS01347], Atg2[JF02786], Pten[JF01987],Atg2[EP3697]/Df(3L)BSC119], Atg18a[KG03090]/Df(3L)Exel6112], S6K[l-1], UAS-Rheb[EP50.084cre(w-)]/TM6B, w [1118],Atg1 [25].Atg8a[d4] mutant used in this study was generated by imprecise excision of the P element KG07569 and harbors a deletion removing the first 25 codons of Atg8a and the first 53 codons of CG1826, a gene with an unknown function (kindly provided by Tom Neufeld). The mutants are homozygous viable and fertile with no gross morphological or developmental abnormalities. UAS-Atg4[C98A] transgenics were established as described below.Drosophila stocks were maintained on a standard cornmeal/sugar/agar medium at 25°C and 50% humidity on a 12 h light/12 h dark cycle. Thestocks used in this study were: The full-length coding sequence of Atg4a/CG4428 with a Cysteine 98 to Alanine mutation changing the coding triplet from TGC to GCC was chemically synthesized (Genscript), and cloned into pUAST using EcoRI and XhoI restriction sites. Drosophila embryo transformation was carried out according to standard methods (Bestgene).The polyclonal affinity-purified p62 antibody was raised in rabbits using the peptide antigene PRTEDPVTTPRSTQ corresponding to amino acids 297–311 (Genscript). Polyclonal anti-GFP antibodies were raised using standard procedures by immunizing rats with bacterially expressed His-tagged eGFP purified on Ni affinity columns (Sigma). Samples were separated by SDS-PAGE on an 8% acrylamide gel and transferred to Immobilon-P PVDF membrane (Millipore). Membranes were blocked in 3% milk/TBS for 1 h at room temperature and washed three times for 10 min each in TBST (TBS +0.1% Tween-20). Blots were incubated with primary antibodies: rabbit polyclonal anti-p62 , mouse monoclonal anti-tubulin AA4.3 (DSHB) [1∶200], rat polyclonal anti-GFP  in 1.5% milk/TBST for 1 h at room temperature, followed by three 10-min washes in TBST. Blots were incubated in AP-conjugated goat anti-rat (Sigma), anti-rabbit or anti-mouse secondary antibody (Millipore) diluted 1∶10,000 in 1.5% milk/TBST for 1 h at room temperature. Blots were washed for 3×10 min in TBST and then incubated with Immobilon Western Chemiluminescent AP Substrate (Millipore), followed by exposure to Super RX film (Fuji).Clonal analysis using the spontaneously activated Gal4/UAS system in the larval fat body was carried out as described previously. 2) were noted from the summary of masked images. At least 10 images were evaluated from 4–6 animals per genotype. The number/size data were summarized in Excel, and normalized to control . For clonal analysis, image type was again changed from RGB Color to RGB Stack. Then the threshold was adjusted for the adequte channel, and clone cells and neighbouring control cells were evaluated. First, randomly selected GFP-positive or control cells were manually encircled with the Freehand selection tool, and then the selected area was analyzed in the channel of interest , and the count and average size (in pixel2) were noted from the summary of masked images. At least 10 images from 4–6 animals were evaluated for each genotype. These number/size data were processed as in the case of mutants, except that GFP-positive cells were compared to controls cells in the same image, and data was also normalized to cell size. Two-tailed two-sample unequal Student’s t test was used to estimate p values in all cases.For western blots, the image of the scanned film was inverted in Adobe Photoshop, saved and loaded in ImageJ. The strongest band was selected first, and individual bands were measured  using a selection area with the same width and height as for the strongest band. Mean p62 values were normalized to tubulin mean scores first, and then expression levels were calculated relative to the control sample . For immunostainings, images were loaded in ImageJ, and the image type was changed from RGB Color to RGB Stack. In the case of mutants, a 300×300 dpi area was chosen randomly from the red channel, and the threshold was adjusted to select dots. Each selected area was analyzed  and the count and average size , Atg7 (B), Atg8a (C), Atg13 (D), Atg18a (E) and Vps34 (F) all increase p62 aggregation. Scalebar in panel A equals 30 µm for all images. Genotypes are: (A) Atg2[EP3697]/Df(3L)BSC119]; (B) Atg7[d77]/Atg7[d14]; (C) Atg8a[d4]; (D) Atg13[Δ81]; (E) Atg18a[KG03090]/Df(3L)Exel6112]; (F) Vps34[Δm22].(TIF)Click here for additional data file.Figure S2The effect of additional RNAi and overexpression lines on p62 aggregation. The effect of overexpressed dominant-negative Atg4 (B), dominant-negative Vps34 (G), wild-type Rheb (H), and knockdown of Atg2 (A), Atg7 (C), Atg8a (D), Atg9 (E), Atg14 (F), Atg16 , Atg18a , Pten  and Atms (O) on p62 aggregation. Scalebar in panel A equals 40 µm for all images. Genotypes are: (A) hsFlp; UAS-Dcr2/+; Act>CD2>Gal4, UAS-GFPnls/Atg2[JF02786]; (B) hsFlp; UAS-Dcr2/+; Act>CD2>Gal4, UAS-GFPnls/UAS-Atg4[C98A]; (C) hsFlp; UAS-Dcr2/+; Act>CD2>Gal4, UAS-GFPnls/Atg7[JF02787]; (D) hsFlp; UAS-Dcr2/Atg8a[KK109654]; Act>CD2>Gal4, UAS-GFPnls/+; (E) hsFlp; UAS-Dcr2/+; Act>CD2>Gal4, UAS-GFPnls/Atg9[JF02891]; (F) hsFlp; UAS-Dcr2/Atg14[KK108559]; Act>CD2>Gal4, UAS-GFPnls/+; (G) hsFlp; UAS-Dcr2/UAS-Vps34[KD]; Act>CD2>Gal4, UAS-GFPnls/+; (H) hsFlp; UAS-Dcr2/+; Act>CD2>Gal4, UAS-GFPnls/UAS-Rheb[EP50.084cre(w-)]; (I) hsFlp; UAS-Dcr2/Atg16[KK105993]; Act>CD2>Gal4, UAS-GFPnls/+; (J) hsFlp; UAS-Dcr2/+; Act>CD2>Gal4, UAS-GFPnls/Atg16[HMS01347]; (K) hsFlp; UAS-Dcr2/Atg18a[KK105366]; Act>CD2>Gal4, UAS-GFPnls/+; (L) hsFlp; UAS-Dcr2/+; Act>CD2>Gal4, UAS-GFPnls/Atg18a[JF02898]; (M) hsFlp; UAS-Dcr2/Pten[KK101475]; Act>CD2>Gal4, UAS-GFPnls/+; (N) hsFlp; UAS-Dcr2/+; Act>CD2>Gal4, UAS-GFPnls/Pten[JF01987]; (O) hsFlp; UAS-Dcr2/Atms[GD20876]; Act>CD2>Gal4, UAS-GFPnls/+.(TIF)Click here for additional data file.Figure S3Knockdown of RpS8 or Atms inhibits starvation-induced mCherry-Atg8a and Lysotracker puncta formation. Silencing of RpS8 or Atms strongly interferes with starvation-induced mCherry-Atg8a dot formation  and blocks punctate Lysotracker staining . Scalebar in panel A equals 30 µm for all images. Genotypes are: (A) hs-Flp; UAS-Dcr2/RpS8[KK106835]; Act>CD2>Gal4, UAS-GFPnls, r4-mCherry-Atg8a/+; (B) hs-Flp; UAS-Dcr2/Atms[GD20876]; Act>CD2>Gal4, UAS-GFPnls, r4-mCherry-Atg8a/+; (C) hs-Flp; UAS-LampGFP/RpS8[KK106835]; Act>CD2>Gal4, UAS-Dcr2; (D) hs-Flp; UAS-LampGFP/Atms[GD20876]; Act>CD2>Gal4, UAS-Dcr2.(TIF)Click here for additional data file.Figure S4p62-GFP aggregation in additional RNAi and overexpression lines. Knockdown of Atg7 (A), Atg8a (B), Atg9 (C), Atg14 (D), Atg18a (E), Pten (G), Tsc2 (H), RpS8(I), Atms (J) and expression of dominant-negative Vps34 (F) in p62-GFP expressing cell clones. Exposure times are indicated in the top right corner for each image. Scalebar in panel A equals 30 µm for all images. Genotypes are: (A) hsFlp; UAS-p62-GFP/+; Act>CD2>Gal4, UAS-Dcr2/Atg7[JF02787]; (B) hsFlp; UAS-p62-GFP/Atg8a[KK109654]; Act>CD2>Gal4, UAS-Dcr2/+; (C) hsFlp; UAS-p62-GFP/+; Act>CD2>Gal4, UAS-Dcr2/Atg9[JF02891]; (D) hsFlp; UAS-p62-GFP/Atg14[KK108559]; Act>CD2>Gal4, UAS-Dcr2/+; (E) hsFlp; UAS-p62-GFP/Atg18a[KK105366]; Act>CD2>Gal4, UAS-Dcr2/+; (F) hsFlp; UAS-p62-GFP/UAS-Vps34[KD]; Act>CD2>Gal4, UAS-Dcr2/+; (G) hsFlp; UAS-p62-GFP/+; Act>CD2>Gal4, UAS-Dcr2/Pten[JF01987]; (H) hsFlp; UAS-p62-GFP/TSC2[KK103417]; Act>CD2>Gal4, UAS-Dcr2/+; (I) hsFlp; UAS-p62-GFP/RpS8[KK106835]; Act>CD2>Gal4, UAS-Dcr2/+; (J) hsFlp; UAS-p62-GFP/Atms[GD20876]; Act>CD2>Gal4, UAS-Dcr2/+.(TIF)Click here for additional data file.Figure S5The effect of Atg16 RNAi lines on starvation-induced punctate Lysotracker staining in L3 and L2 larval stages. Both RNAi lines for Atg16 show a similar block of Lysotracker puncta formation in L3 (panels A and C), while the size of these dots is only reduced significantly by Atg16HMS in L2 . * indicates a significant difference (p<0.05), ** indicates a very significant difference (p<0.01), based on two-tailed two-sample unequal Student’s t tests. Scalebar in panel A equals 30 µm for panels A, C, and scalebar in panel B equals 30 µm for panels B, D. Genotypes are:  hs-Flp; UAS-LampGFP/Atg16[KK105993]; Act>CD2>Gal4, UAS-Dcr2;  hs-Flp; UAS-LampGFP/+; Act>CD2>Gal4, UAS-Dcr3/Atg16[HMS01347].(TIF)Click here for additional data file.Figure S6Overexpression of mCherry-Atg8a rescues the effect of Atg8a mutation or expression of dominant-negative Atg4 on p62 accumulation. Expression of mCherry-Atg8a reduces the size and number of p62 aggregates in Atg8a null mutants . No accumulation of p62 dots is observed in fat body cells coexpressing mCherry-Atg8a and dominant-negative Atg4 relative to control cells . Scalebar in panel A equals 30 µm. Genotypes are: (A)Atg8a[d4]; +/+; r4-mCherry-Atg8a/+; (B) hs-Flp; UAS-Dcr2/+; Act>CD2>Gal4, UAS-GFPnls, r4-mCherry-Atg8a/UAS-Atg4[C98A].(TIF)Click here for additional data file.


High cut-off membranes are proposed for blood purification therapy in septic shock. However, albumin loss related to these membranes is a major drawback limiting their clinical acceptance. Super High-Flux membranes with an optimized cut-off may combine enhanced middle molecule clearances (inflammatory mediators) with limited albumin loss. The aim of our study was to compare small, middle molecule clearances and albumin loss between continuous hemodialysis using a Super High-Flux membrane (SHF-HD) and conventional continuous hemofiltration (CVVH).® filter; Fresenius Medical Care)  or conventional CVVH . Each patient received a maximum of three sessions of 48 hours each. Creatinine (113 Da), β2-microglobulin (β2-M) (11.8 kDa), kappa free light chain of immunoglobulins (κ-FLC) (23 kDa) and albumin (68 kDa) clearances were measured at 15 minutes, 1 hour, 4 hours, 12 hours, 24 hours and 48 hours. β2-M and κ-FLC were chosen as a middle molecular weight marker. A linear mixed-effects model compared clearances between groups.After approval by the ethics committee, patients were enrolled in a single-blind RCT. Patients with septic shock and acute kidney injury received either SHF-HD (EMiC2P = 0.18) and β2-M (P = 0.98) clearances. Plasma albumin levels and the amount of albumin infused did not differ between groups. See Figure Twenty-four patients were included, 12 in the SHF-HD group (32 sessions) and 12 in CVVH (30 sessions). κ-FLC and albumin clearances were higher in the SHF-HD group over time. No difference was observed for creatinine (The removal of middle molecular weight molecules is higher with SHF-HD. Albumin loss was limited in both groups, even with SHF-HD. Therefore, SHF membranes seem to represent an alternative to high cut-off membranes for blood purification therapies.


The presence of late gadolinium enhancement (LGE) is related to cardiac remodeling and poorer prognosis in ischemic and non-ischemic cardiomyopathies. Non-invasive LGE- cardiovascular magnetic resonance (LGE-CMR) is able to detect infarct-typical and -atypical patterns of LGE after heart transplantation (HTX), mainly due to post HTX coronary artery vasculopathy (CAV) and myocarditis. In this study, we sought to describe the influence of the presence and patterns of both LGE forms on left ventricular (LV) remodeling after HTX.153 HTX patients were divided into 3 groups depending on the presence and pattern of LGE . 146 patients received coronary angiography within 4 weeks after CMR for assessment of CAV. Vector-ECG gated 32-channel parallel imaging cine SSFP sequences for standard imaging plane and LV volumetry acquisition were acquired on a 1.5T Whole Body MRI scanner . LGE-CMR (Gadolinium:0.2 mmol/kg) imaging was performed and analyzed blindly by two experienced observers. Patients were divided according to the presence and patterns of LGE. CAV was divided into three groups:1=mild;2=moderate;3=severe post transplant coronary artery disease. Data were expressed as mean and standard deviation or percentage. Groups were compared using Chi Square for categorical data and ANOVA or Kruskal-Wallis tests for continuous variables. P-values ≤ 0.05 were considered statistically significant.Overall, 135 patients (88%) presented patterns of LGE, whereas infarct-atypical patterns were more prevalent (66%). Group II showed significantly lower ejection fractions (56±12%) when compared to group I (63 ± 8%) and III  but significantly increased end-diastolic (83±26ml/m2) volumes/BSA  and end-systolic volumes/BSA (38±21ml/m2 vs. group I=26±8ml/m2 and III=28±5ml/m2; p<0,001, Figure The presence of infarct-typical LGE-CMR is related to negative post HTX remodeling revealing lower ejection fractions and increased volumes in patients after HTX. Future studies are warranted to evaluate the long-term effect of infarct-typical and atypical patterns on future post HTX outcomes.none


C. elegans smg-8 is not required to degrade nonsense-containing mRNAs. We examine the genetic requirement for smg-8 to destabilize the endogenous, natural NMD targets produced by alternative splicing of rpl-7a and rpl-12. We test smg-8 for degradation of the endogenous, NMD target generated by unc-54(r293), which lacks a normal polyadenylation site. We probe the effect of smg-8 on the exogenous NMD target produced by myo-3::GFP, which carries a long 3′ untranslated region that destabilizes mRNAs. None of these known NMD targets is influenced by smg-8 mutations. In addition, smg-8 animals lack classical Smg mutant phenotypes such as a reduced brood size or abnormal vulva. We conclude that smg-8 is unlikely to encode a component critical for NMD.The nonsense mediated decay (NMD) pathway degrades mRNAs bearing premature translation termination codons. In mammals, SMG-8 has been implicated in the NMD pathway, in part by its association with SMG-1 kinase. Here we use four independent assays to show that  C. elegans, mRNAs that have acquired a nonsense mutation or an extended 3′ untranslated region (UTR) are targeted for NMD S. cerevisiaeC. elegansDrosophila and mammals, the NMD core components are modulated by additional SMG factors. Recent screens have also identified and confirmed new candidate NMD proteins in C. elegans, H. sapiens and D. rerioThe nonsense mediated decay (NMD) pathway is an evolutionarily conserved mRNA surveillance mechanism that recognizes and degrades transcripts bearing premature translation termination codons C. elegans, EJC components are dispensable for NMD, and PTCs are instead distinguished from normal stop codons by the size of the 3′UTR Drosophila and probably worms) Elegant studies in several organisms have suggested a model in which NMD reflects a competition between SMG-2/UPF1 and Poly(A) Binding Protein (PABP) for ribosome-associated translation release factors. In mammals, the locations of mRNA splice junctions are marked by exon-junction complexes (EJC) during mRNA processing in vitro. Inactivation of SMG-8 and SMG-9 lead to a partial stabilization of ß-globin mRNAs in mammalian cell culture, suggesting they might play a role in NMD. The authors also used RNA interference (RNAi) to inactivate C. elegans smg-8 and smg-9, and concluded that smg-8, but not smg-9, contributed to NMD in worms Recently, Yamashita and colleagues used immunoprecipation of HeLa cell lysates to identify proteins that interact with SMG-1, a phosphatidylinositol kinase-related protein kinase C. elegans smg-8 using a newly generated mutant allele. smg-8(tm2937) contains a 272 bp deletion and a 1 bp insertion within smg-8 (smg-8 in the NMD pathway. Our findings suggest that smg-8 is unlikely to be a key component for NMD in C. elegans.Here we examine in smg-8 . This desmg genes. Hodgkin and colleagues reported a reduced brood size of 174 (range 147–211) for smg-1, which was lower than the mean brood size of wild-type animals  smg-8(2937), similar to the mean brood size of our wild-type strain   worms (smg-8(tm2937) animals lack two overt phenotypes associated with canonical smg mutants.For our first assay, we examined two of the classical phenotypes associated with  n = 10) . In addi7) worms . These drpl-7a and rpl-12, which are natural NMD targets smg-8 shows no NMD phenotype by this assay. When a known component of the NMD pathway, such as smg-1, smg-2 or smg-3, is mutated, the mRNA isoform containing the PTC is stabilized, generating a robust upper band (smg-8(tm2937), a very faint upper band was observed, comparable to that of the wild-type strain , which enhances RNAi tm2937 allele was hypomorphic, we treated smg-8(tm2937) mutants with smg-8 RNAi or smg-9 RNAi, but again we observed no NMD phenotype . A virtually identical result was obtained with smg-8 RNAi treatment of smg-8 or eri-6/7 mutant worms (0.7 and 0.4 fold enrichment respectively)  . Togetheunc-54(r293)unc-54 gene generates a muscle myosin heavy chain (MHC) in C. elegans, and the unc-54(r293) allele contains a 256 bp deletion that removes the normal 3′ cleavage/polyadenylation site and most of the 3′UTR unc-54 mRNA transcript that terminates at a cryptic poly(A) site and renders unc-54 an NMD target. Without the MHC, unc-54 mutant worms are paralyzed  mRNA is stabilized, wild-type protein is produced and the Unc phenotype is suppressed unc-54(r293); smg-8(tm2937) double mutants and observed no suppression of the paralysis phenotype  accumulated very little GFP compared to the positive controls myo-3::GFP; smg-1(r861) or myo-3::GFP; smg-3(r867)  , indicatsmg-8 for a role in NMD using four different assays: i) anatomical phenotype and brood size, ii) accumulation of natural NMD targets rpl-7a and rpl-12, iii) rescue of the paralysis phenotype caused by the endogenous NMD target unc-54(r293) and iv) GFP accumulation of the exogenous NMD target reporter myo-3::GFP. The discrepancy between our study and the results presented by Yamashita and colleagues is due in part to the use of an allele (our study) vs. RNAi smg-8 inactivation on NMD in C. elegans was not robust in the Yamashita study smg-8 mutants resembled wild-type worms and differed from classical smg mutants. We detected no accumulation of mRNA or protein in smg-8 mutants, even when the smg-8 mutation was combined with RNAi. We suggest that smg-8 in C. elegans is a novel, conserved gene whose function remains to be elucidated.In summary, we tested unc-54(r293)I, SM456 smg-1(r861)I, SM436 smg-2(e2008)I, SM196 smg-3(r867)IV, smg-1(r861);unc-54(r293)I, smg-8(tm2937)II;unc-54(r293)I, CL724 dvIs38 [pCL60 (Pmyo-3::GFP::degron/long 3′ UTR)+pRF4], SM1944 smg-1(r861)I;myo-3::GFP, SM1929 smg-3(r867)IV;myo-3::GFP, SM1937 smg-8(tm2937)II;myo-3::GFP, SM1881 smg-8(tm2937)II eight times outcrossed.Worm growth and maintenance were performed as described before smg-1, smg-8 or smg-9 grown for ∼8 hours at 37°C were plated using 1 mM IPTG (Sigma) and 50 mg/ml of Carbenicillin (Sigma). RNAi clones were derived from the Ahringer library smg-8(tm2937) or eri-6/7(tm1917) worms were transferred at the L4 stage to RNAi plates and allowed to lay embryos for one day. The progeny was collected ∼48 hours later, when most worms had grown at least to the L4 stage, by rinsing with water and frozen at -80°C for subsequent RNA extraction. Nine 35 mm plates were used per strain per experiment.HT115 bacteria expressing double stranded RNA targeting For total RNA extraction, glass beads (Sigma) and 1 ml of Trizol Reagent (GibcoBRL) were added to frozen worm pellets. Pellets were lysed by vortex followed by chloroform extraction. RNA was precipitated with isopropanol and washed with 70% ethanol. Resuspended RNA was extracted with phenol:chloroform and precipitated with ethanol. A first-strand reaction kit (NEB) was used to perform the reverse transcriptase reaction, following the manufacturers protocol.rpl-7a from the cDNA was performed as described in GACATCCAGCCAAAGAAGGA and rpl-7a-rv AACGGTGTTTGGTCTCTTGG.Amplification of ACCCAAGACTGGAAGGGTCT and Rpl-12 R1 GCCATCGATCTTGGTCTCAT.Primers for rpl-12 are Rpl-12-F1 rpl-7a, mRNA levels were calculated using the delta-delta-CT method, relative to the control gene pmp-3smg-8 were smg8-fw-4348 GCTGCCAATATTTCCATCGT and smg8-rv-5165 TGACCACGGGAACATTCATA.For smg-8 and 10 worms at the L4 stage were picked into individual plates at 20°C. Their progeny was counted everyday until no more progeny was generated. The number of progeny per plate was averaged (n = 10).


The correct compound should have been tris(1-chloro-2-propyl) phosphate (TCPP).The February 2011 article EHP regrets the error.


C. elegans lacking EGL-4 function are hypersensitive in their behavioral response to low concentrations of the bitter tastant quinine and exhibit an elevated calcium flux in the ASH sensory neurons in response to quinine. We provide the first direct evidence for cGMP/PKG function in ASH and propose that ODR-1, GCY-27, GCY-33 and GCY-34 act in a non-cell-autonomous manner to provide cGMP for EGL-4 function in ASH. Our data suggest that activated EGL-4 dampens quinine sensitivity via phosphorylation and activation of the regulator of G protein signaling (RGS) proteins RGS-2 and RGS-3, which in turn downregulate Gα signaling and behavioral sensitivity.Signaling levels within sensory neurons must be tightly regulated to allow cells to integrate information from multiple signaling inputs and to respond to new stimuli. Herein we report a new role for the cGMP-dependent protein kinase EGL-4 in the negative regulation of G protein-coupled nociceptive chemosensory signaling.  C. elegans  depend heavily upon their ability to taste and smell chemical information in their soil environment to find food and avoid danger. While similar signal transduction pathways are implicated in both C. elegans and vertebrate chemosensation, there are still large gaps in our understanding of the mechanisms used to regulate signaling in these systems. We have identified a new role for the C. elegans cGMP-dependent protein kinase (PKG) EGL-4 in the negative regulation of nociceptive chemosensory signaling. Our data suggest that EGL-4 negatively regulates signaling and behavior by activating known inhibitors of G protein-coupled signal transduction, RGS proteins. Using C. elegans behavioral response to aversive stimuli as the readout for neuronal activity, we provide the first evidence for PKG regulation of RGS function in sensory neurons in any system.All animals rely on their ability to sense and respond to their constantly changing environments to survive.  Caenorhabditis elegans, the pair of polymodal nociceptive head sensory neurons termed ASH respond to a broad range of aversive stimuli, including soluble chemicals , odorants (e.g. octanol), ions (e.g. Na+), osmotic stress and mechanosensory stimulation (nose touch) The ability to detect and avoid noxious stimuli in the environment is critical to an organism's survival. Nociceptive sensory systems mediate detection of harmful stimuli, allowing rapid initiation of protective behavioral responses. In the nematode C. elegans genome encodes >500 predicted functional chemosensory GPCRs C. elegans stimulatory Gα subunits ODR-3 and GPA-3 (both most similar to Gαi/o) are used by ASH to mediate avoidance of a variety of stimuli In general, olfaction and some forms of taste (including bitter) are mediated by G protein-coupled signal transduction pathways i protein that is coupled to T2R bitter receptors Regulator of G protein signaling (RGS) proteins are important negative regulators of G protein-coupled signal transduction. They bind to Gα-GTP and accelerate the intrinsic GTPase activity of the Gα subunits. Once GTP is hydrolyzed (back to GDP), signaling via Gα is attenuated C. elegans lacking RGS-3 function are defective in their response to a subset of strong sensory stimuli detected by the ASH sensory neurons rgs-3 mutant animals, we note that feeding status and, consequently, biogenic amine (e.g. dopamine and serotonin) levels modulate rgs-3 behavioral responses rgs-3 animals responded to 100% octanol (odorant) and 10 mM quinine (tastant) when they were assayed in the presence of food , and were only defective when assayed after a short (10 minute) period of starvation C. elegans to an environmental stimulus is ultimately coordinated by proteins (e.g. RGS-3) that directly regulate the sensory G protein-coupled signaling cascade, in conjunction with signals modulated by nutritional status. All of the tastant avoidance experiments presented herein were performed 30 minutes after animals were removed from food, in contrast to the previous study focused on rgs-3, where “off food” assays were performed 10 minutes after animals were removed from the bacterial food source q and consequently enhancing GTPase activity and the dampening of Gα signaling cGMP-dependent protein kinases (PKGs) are serine/threonine kinases that are activated upon the binding of cGMP C. elegans, EGL-4 is a cGMP-dependent protein kinase with physiological roles including egg laying, life span, cell growth, quiescence and dauer formation C. elegans sensory responses. For example, egl-4(lof) animals are strongly defective for chemotaxis to the AWA-detected odorant diacetyl In C. elegans EGL-4 has two allosteric cGMP-binding domains in its amino-terminal half C. elegans genome C. elegans sensory neurons, no GCYs are known to be expressed in or function in ASH.In mammalian systems, cGMP binding activates PKG C. elegans cGMP-dependent protein kinase EGL-4 as a negative regulator of nociceptive chemosensory signaling. Animals lacking EGL-4 function respond better than wild-type animals to dilute concentrations of several ASH-detected chemical stimuli, including the bitter tastant quinine. We provide evidence that a subset of GCYs function non-cell-autonomously to generate cGMP to stimulate EGL-4 phosphorylation and activation of RGS proteins in ASH to dampen G protein-coupled chemosensory signal transduction and behavioral sensitivity.Herein we describe a new role for the C. elegans and egl-4(n479) loss-of-function (lof) animals effectively avoid the bitter tastant 10 mM quinine (egl-4(lof) animals appeared to respond to 10 mM quinine slightly better than wild-type animals (p<0.01). To determine whether EGL-4 might contribute to chemosensory signaling in a way that might not be fully revealed by this high concentration of quinine, which elicits a robust behavioral response even in wild-type animals, we challenged both genotypes with dilute (1 mM) quinine. At this lower concentration, egl-4(lof) animals were clearly hypersensitive, with significantly more egl-4(lof) animals responding to the dilute quinine than wild-type animals (ad450 is a gain-of-function (gof) allele of egl-4. Consistent with our prediction, egl-4(gof) animals were no longer hypersensitive to quinine and responded worse than wild-type animals to 10 mM quinine  srb-6 (ASH) srbc-66 (ASK) egl-4(lof) response to wild-type levels so that animals were no longer hypersensitive to the dilute stimulus  animals  animals to the bitter tastants amodiaquine and primaquine (egl-4(lof) animals were hypersensitive to amodiaquine, with more animals responding than wild-type across a range of concentrations (egl-4(lof) animals responded to primaquine similarly to wild-type animals and did not appear hypersensitive  animals responded better than wild-type animals  animals was similar to that of wild-type animals  that are thought to signal in a G protein-independent manner  animals . We concosm-10 promoter Upon prolonged exposure to attractive odorants, EGL-4 translocates from the cytoplasm into the nucleus of the AWC olfactory neurons to regulate long-term adaptation in a manner downstream of primary sensory transduction osm-10 promoter egl-4(lof) animals. Deletion of the endogenous nuclear localization sequence, GFP−EGL-4(ΔNLS), lead to cytoplasmic accumulation and had no effect on the ability of EGL-4 to rescue the quinine hypersensitivity of egl-4(lof) animals (egl-4(lof) animals expressing the constitutively nuclear NLS−GFP−EGL-4 remained hypersensitive in their response to 1 mM quinine  and egl-4(gof) animals expressing G-CaMP3 in ASH (sra-6p::G-CaMP3) egl-4(lof) animals displayed quinine-evoked ASH calcium fluxes of greater amplitude in response to 1 mM quinine, while expression of wild-type egl-4 in ASH, using the osm-10 promoter egl-4(gof) animals showed a diminished calcium flux in response to 10 mM quinine  and rgs-3(lof) animals displayed a statistically significant hypersensitive response, with more animals than wild-type responding to the dilute stimulus. Although an RGS-2 reporter construct did not show expression in ASH osm-10 promoter rgs-2 and rgs-3 in the ASH sensory neurons of wild-type animals and in both cases, knock-down also resulted in hypersensitivity to dilute quinine (PKGs have been shown to down-regulate Gα quinine . Further quinine .egl-4(lof);rgs-2(lof) and egl-4(lof);rgs-3(lof) double mutant animals should display a behavioral phenotype similar to egl-4(lof) animals and the hypersensitivity should not be additive. We found that the double mutant animals' response to dilute (1 mM) quinine was indistinguishable from animals lacking only EGL-4 function (egl-4(gof) animals, which display reduced sensitivity to quinine (egl-4(gof) allele should relieve the dampened sensitivity of egl-4(gof) mutants in response to 10 mM quinine. Indeed, egl-4(gof);rgs-2(lof) and egl-4(gof);rgs-3(lof) double mutants both responded to 10 mM quinine similarly to the rgs single mutants  animals expressing RGS-2(S126A) and rgs-3(lof) animals expressing RGS-3(S154A) remained hypersensitive to quinine, suggesting that these putative PKG phosphorylation sites are required for RGS-2 and RGS-3 function. We conclude that EGL-4 functions upstream of RGS-2 and RGS-3, and propose that phosphorylation by EGL-4 may be required for the function of these regulatory proteins in ASH.The characterized consensus sequence of mammalian PKG, seen in 75% of targets surveyed, consists of (R/K)egl-4(lof) quinine hypersensitivity, egl-4(lof) animals expressing EGL-4(T276A) remained hypersensitive to dilute quinine (A single point mutation (T276A) within the cGMP-binding domain of EGL-4 abolished its function in AWC-mediated adaptation  quinine . This suC. elegans genome odr-1, gcy-27, gcy-33 and gcy-34, responded better than wild-type animals (egl-4(gof) animals, which display reduced sensitivity to 10 mM quinine (egl-4(gof) mutation and double mutant animals were assayed for quinine avoidance. Each double mutant remained less sensitive to both 10 mM and 1 mM quinine, similar to the egl-4(gof) single mutant animals . However, gcy-27 is expressed in the ASK head sensory neurons osm-10srb-6odr-1(lof) animals was rescued by srb-6p::odr-1, but not osm-10p::odr-1 expression, and expression using neither promoter was sufficient to rescue gcy-33(lof) or gcy-34(lof) hypersensitivity (gcy-27(lof) animals was rescued by both osm-10p::gcy-27 and srb-6p::gcy-27, as well as by ASK-selective expression using the srbc-66 promoter gcy-27 genomic sequence, we cannot rule out the possibility that regulatory information within the introns could direct expression in addition cells beyond those predicted by the cell-selective promoters used. Taken together, and consistent with the reported expression patterns, these results suggest that the primary site of GCY function is in cells other than the ASHs, and that the cyclases may function in a non-cell-autonomous manner to provide cGMP to regulate EGL-4 function in ASH.GCY expression has not previously been reported in ASH, consistent with our own analysis of sitivity . InteresC. elegans genome encodes only two PKGs, EGL-4  and PKG-2, it provides an excellent model environment in which to further elucidate the unique physiological roles of PKGs in different cellular contexts. In the AWC olfactory neurons, EGL-4 has been shown to regulate both short-term and long-term adaption. At the cellular level, adaptation is thought to serve as a protective measure against prolonged stimulation, as in the case of photoreceptor signaling for long-term light adaptation C. elegans, short-term olfactory adaptation (<30 minutes) diminishes odorant sensitivity for up to 60 minutes and utilizes cytoplasmic EGL-4, whereas long-term adaptation to prolonged odor exposure (>80 minutes) requires the translocation of EGL-4 to the nucleus, where it can alter gene expression and decrease sensitivity to attractive scents for the lifetime of the animal  PKGs regulate the physiological responses of a variety of cell types, and have a wide range of known and predicted protein targets C. elegans. For example, EGL-4 likely functions upstream of the TGF-β pathway SMAD transcription factors DAF-3 and DAF-5, as mutations in DAF-3 and DAF-5 suppress several egl-4 mutant phenotypes, including chemosensory, dauer formation, egg laying and body size defects C. elegans Notch signaling in sensory neurons, acting directly or indirectly downstream of Notch receptor activation. Animals lacking the DOS-motif Notch co-ligand OSM-11 do not effectively avoid 100% octanol, and this defect is partially suppressed by the gain-of-function egl-4(ad450) allele Similar to PKGI and PKGII in mammalian systems, whose targets include components of G protein-coupled signaling egl-4(lof) animals respond better than wild-type animals to 10 mM quinine and also avoid dilute levels of quinine (1 mM) that wild-type animals do not respond to (egl-4(lof) animals  animals exhibited an elevated ASH calcium flux, when compared to wild-type animals (3R(Ser1755) potentiates IP3R-mediated calcium release from internal stores C. elegans. This difference highlights the diversity of mechanisms by which PKGs can regulate signaling in different cellular contexts.Calcium influx is essential for neuronal function and increased intracellular calcium levels trigger exocytosis of neurotransmitter-containing synaptic vesicles  animals . CombineC. elegans response to quinine utilizes the Gα subunits ODR-3 and GPA-3 C. elegans rgs-2(lof) and rgs-3(lof) animals were assayed 30 minutes after being removed from their bacterial food source, they also displayed a significant hypersensitivity phenotype  animals are defective in their avoidance of 10 mM quinine when assayed just 10 minutes after removal from food (rgs-3(lof) animals responding better than wild-type animals to quinine (10 mM and 1 mM) when they are assayed 30 minutes after removal from food (rgs-2(lof) animals are defective in response to 10 mM quinine when assayed after only the short (10 minute) period of starvation (rgs-2(lof);rgs-3(lof) double mutant animals reach the level of hypersensitivity seen in the single mutants at 30 minutes off food (rgs-3(lof) single mutant animals to 10 mM quinine at 10 minutes off food egl-4(lof) animals and rgs-2(lof);rgs-3(lof) animals, EGL-4 may have additional targets within the ASH sensory neurons. Alternatively, RGS-2 and RGS-3 may retain residual function in egl-4(lof) animals, such that loss of EGL-4 function does not affect cellular signaling as adversely as loss of the RGS proteins themselves.The sensitivity of rom food  [28]. Throm food . Similararvation , but arearvation . Interesoff food . It is pC. elegans genome, the physiological roles of most are unknown. Our analysis revealed that EGL-4 requires cGMP binding in order to negatively regulate quinine sensitivity , DA521 egl-4(ad450), CX10979 kyEx2865 , FG414 , FG417 egl-4(n479);kyEx2865 , FG413 egl-4(ad450);kyEx2865 , FG454 egl-4(n479);udEx208 ;kyEx2865 , LX147 rgs-1(nr2017), LX160 rgs-2(vs17), LX242 rgs-3(vs19), LX533 rgs-6(vs62), FG105 rgs-10(ok1039), FG108 rgs-10/11(vs109), MT8504 egl-10(md176), LX1226 eat-16(tm761), FG329 egl-4(n479);rgs-2(vs17), FG330 rgs-3(vs19);egl-4(n479), FG275 egl-4(ad450);rgs-2(vs17), FG276 rgs-3(vs19);egl-4(ad450), FG269 rgs-3(vs19);rgs-2(vs17), FG376 rgs-3(vs19);egl-4(n479);rgs-2(vs17), VC2796 gcy-3(gk1154), RB1010 gcy-5(ok930), IK800 gcy-8(oy44), VC2675 gcy-15(gk1102), VC2450 gcy-17(gk1155), VC2321 gcy-18(ok3047);nT1[q1351], RB1909 gcy-19(ok2472), RB1935 gcy-20(ok2538), RB924 gcy-23(ok797), VC2375 gcy-25(gk1187), CZ3714 gcy-31(ok296), RB1048 gcy-32(ok995), AX1295 gcy-35(ok769), RB626 gcy-37(ok384), IK597 gcy-23(nj37);gcy-8(oy44);gcy-18(nj38), FG290 odr-1(n1936), FG280 gcy-27(ok3653), FG278 gcy-33(ok232), FG279 gcy-34(ok2953), FG294 egl-4(ad450);odr-1(n1936), FG288 gcy-27(ok3653);egl-4(ad450), FG289 egl-4(ad450);gcy-33(ok232) and FG285 egl-4(ad450);gcy-34(ok2953).Strains used in this study include: N2 Bristol wild-type, MT1074 egl-4, rgs-2, rgs-3, odr-1, gcy-27, gcy-33, and gcy-34 rescue experiments, 50 ng/µl of pJM67 elt-2::gfp plasmid rgs-2 and rgs-3 overexpression, 250 ng/µl of the rgs plasmid was co-injected with 50 ng/µl of pJM67 elt-2::gfp plasmid egl-4 rescue with G-CaMP3 expression, 5 ng/µl of myo-3p::mCherry plasmid (Yamamoto Lab) was used as the co-injection marker, along with 50 ng/µl of the osm-10p::egl-4 rescuing plasmid. Animals expressing the egl-4 rescuing array (udEx208) were then crossed with kyEx2865-expressing  animals, and egl-4(n479) animals co-expressing both arrays were isolated. Cell-specific RNAi knock-down experiments were performed as previously described elt-2::gfp plasmid Germline transformations were performed as previously described osm-10 promoter was isolated from CR142 C. elegans Vector Kit, Addgene).The ∼900 bp srbc-66 promoter was isolated from srbc-66::gfpC. elegans Vector Kit, Addgene).The ∼2.1 kb egl-4 cDNA (isoform 2a.1) was PCR amplified from odr-3::egl-4The srb-6 promoter was isolated from pFG10 osm-10 promoter).The ∼1.3 kb egl-4 cDNA (isoform 2a.1) was PCR amplified from odr-3::egl-4The rgs-2 cDNA was PCR amplified from pMD6 The C. elegans Vector Kit, Addgene) to create pHA443. The ∼900 bp osm-10 promoter was isolated from CR142 cDNA encoding the “A” isoform of RGS-3, including the initiator methionine, was amplified from yk252h2 (kind gift of Y. Kohara), incorporating a 5′ NheI and a 3′ NcoI site. This fragment was inserted into the Fire vector pPD49.26 (Fire Lab rgs-2(S126A) cDNA was then PCR amplified, incorporating a 5′ SalI site and a 3′ NcoI site, and subcloned into these sites of pFG1.Site-directed mutagenesis  was used to incorporate the S126A change into pMD6 rgs-3(S154A) cDNA was then PCR amplified, incorporating a 5′ NheI site and a 3′ NcoI site, and subcloned into these sites of pFG1.Site-directed mutagenesis  was used to incorporate the S154A change into pHA443 . The odr-3 promoter from odr-3::gfp::egl-4osm-10 promoter was isolated from pFG1 using SphI/XmaI and inserted into these sites upstream of gfp::egl-4.SphI/NgoMIV digest was used to remove 2496 bp of the 2678 bp odr-3 promoter from odr-3::gfp::egl-4(ΔNLS)osm-10 promoter was isolated from pFG1 using SphI/XmaI and inserted into these sites upstream of gfp::egl-4(ΔNLS).SphI/NgoMIV digest was used to remove 2496 bp of the 2678 bp odr-3 promoter from odr-3::NLS::gfp::egl-4osm-10 promoter was isolated from pFG1 using SphI/XmaI and inserted into these sites upstream of NLS::gfp::egl-4.SphI/NgoMIV digest was used to remove 2496 bp of the 2678 bp odr-3 promoter from odr-3::gfp::egl-4(T276A)osm-10 promoter was isolated from pFG1 using SphI/XmaI and inserted into these sites upstream of gfp::egl-4(T276A).SphI/NgoMIV digest was used to remove 2496 bp of the 2678 bp gcy-31p::gcy-33cDNA encoding the “A” isoform of GCY-33, including the initiator methionine, was isolated from gcy-31p::gcy-33cDNA encoding the “A” isoform of GCY-33, including the initiator methionine, was isolated from gcy-34 sequence was PCR amplified from N2 lysate, incorporating a 5′ NheI site and a 3′ Asp718I site, and subcloned into these sites of pFG1.Genomic gcy-34 genomic sequence was isolated from pFG138 using NheI/Asp718I and subcloned into these sites of pFG10 The odr-1 genomic region from 2.6 kb upstream of the start site for odr-1b to 79 bp of the last exon (22nd) of odr-1b. This construct is missing the last 46 nucleotides of the 22nd exon of odr-1b and the 3′ UTR, as predicted by PACdb ID: R01E6.3_216. We added back the last 46 nt and all of the 3′ UTR (449 nt) by first amplifying the 3′ end of odr-1b and all of the 3′ UTR from N2 genomic DNA, adding a KpnI site on the 3′ end of the amplicon. This was Topo cloned and cut from the Topo cloning vector with KpnI. This KpnI fragment was inserted into the Kpn1 site of R01E6.1SC odr-1b (before stop) so that gfp could be inserted in frame. This construct was called KC2. Finally, odr-1p::odr-1b::gfp (KC3) was generated by cutting KC2 with AgeI/NruI and inserting gfp .R01E6.1SC Genomic sequence encoding ODR-1 was PCR amplified from KC3 (above), incorporating a 5′ NheI site and a 3′ NcoI site, and subcloned into these sites of pFG1.odr-1 sequence was isolated from pFG140 using NheI/NcoI and subcloned into these sites of pFG10 Genomic gcy-27 sequence was PCR amplified from N2 lysate, incorporating a 5′ NheI site and a 3′ BsrGI site, and subcloned into the NheI/Asp718 sites of pFG1.Genomic gcy-27 genomic sequence was isolated from pFG142 using NheI/ApaI and subcloned into these sites of pFG10 The gcy-27 genomic sequence was isolated from pFG142 using NheI/ApaI and subcloned into these sites of pFG50.The All constructs were verified by sequencing when appropriate.kyEx2865 -expressing worm for imaging while streams of buffer or quinine (or buffer for controls) under laminar flow were alternatively presented to the nose of the worm. To reduce the influence of neuronal activation by blue light, we pre-exposed the worm to blue light for 5–8 seconds. The worm was imaged one minute later using a 40X air objective on an inverted Axiovert 200 microscope . Images were captured with an exposure time of 20 milliseconds every 500 milliseconds with an ORCA-Flash 2.8 camera  and recorded over 15 seconds with µManager software 0) was calculated. The ROI was centered on the cell body of the ASH neuron. After 5 seconds of imaging with M13 buffer (pH 7.4), the quinine stimulus was given for the last 10 seconds of imaging. Quinine was dissolved as a 1 or 10 mM solution in M13 and the pH was adjusted to 7.4 after dissolving. Ten worms for each condition were tested and averaged. A one-tailed unpaired Student's t-Test for each point in all conditions was performed. The maximum change in fluorescence before and after exposure to quinine was determined and used for statistics.Neuronal calcium changes were recorded using the GFP-based fluorescent calcium reporter G-CaMP3 Well-fed young adults were used for analysis, and all behavioral assays were performed on at least three separate days, in parallel with controls. The number of transgenic animals assayed in each experiment is indicated within the figure legends, and in all cases n≥58 for non-transgenic animals. Response to the soluble tastant quinine was scored as the percentage of animals that initiated backward locomotion within 4 seconds of encountering a quinine drop placed on the agar plate in front of a forward moving animal Figure S1C. elegans sensitivity to quinine. Animals lacking EGL-4, RGS-2, RGS-3 or RGS-2 and RGS-3 function were tested for response to both (A) 10 mM quinine and (B) 1 mM quinine when starved for increasing lengths of time. (A) rgs-2(lof) and rgs-3(lof) animals responded to 10 mM similarly to wild-type animals when assayed 30 minutes after removal from their bacterial food source . rgs-2(lof);rgs-3(lof) double mutant animals did not respond similarly to wild-type animals until 60 minutes after removal from food (p>0.5). (B) The behavioral sensitivity of rgs-2(lof), rgs-3(lof) and rgs-2(lof);rgs-3(lof) animals to dilute quinine increased with longer periods off food. The percentage of animals responding is shown. The combined data of n≥40 animals is shown. Error bars represent the standard error of the mean (SEM). Alleles used: egl-4(n479), rgs-2(vs17), and rgs-3(vs19) loss-of-function. WT = the N2 wild-type strain. lof = loss-of-function, min = minutes.Length of starvation alters (PDF)Click here for additional data file.