Sphingomonas sp. PAMC 26621. SpG6PD1 was expressed in Escherichia coli as a soluble protein, having optimum activity at pH 7.5–8.5 and 30 °C for NADP+ and 20 °C for NAD+. SpG6PD1 utilized both NADP+ and NAD+, with the preferential utilization of NADP+. A high Km value for glucose 6-phosphate and low activation enthalpy (ΔH‡) compared with the values of mesophilic counterparts indicate the psychrophilic nature of SpG6PD1. Despite the secondary structure of SpG6PD1 being maintained between 4–40 °C, its activity and tertiary structure were better preserved between 4–20 °C. The results of this study indicate that the SpG6PD1 that has a flexible structure is most suited to a psychrophilic bacterium that is adapted to a permanently cold habitat.Glucose 6-phosphate dehydrogenase (G6PD) (EC 1.1.1.363) is a crucial regulatory enzyme in the oxidative pentose phosphate pathway that provides reductive potential in the form of NADPH, as well as carbon skeletons for the synthesis of macromolecules. In this study, we report the cloning, expression, and characterization of G6PD (SpG6PD1) from a lichen-associated psychrophilic bacterium  NADPH provides reducing power for the synthesis of lipid and aromatic amino acids [Glucose 6-phosphate dehydrogenase (G6PD) is a ubiquitous enzyme from prokaryotes to eukaryotes that catalyzes the first reaction in the pentose phosphate pathway (PPP) wherein glucose 6-phosphate (G6P) is converted to 6-phosphoglucono-δ-lactone while reducing NAD(P)no acids , and alsno acids ,3.Chlorella ellipsoidea markedly increased under freezing conditions, and also stimulated the freezing tolerance of Chlorella vulgaris [Saccharomyces cerevisiae harboring the G6PD gene from Chlorella vulgaris C-27 (CvG6PD) was observed during the freeze-thaw process as compared with control yeast cells lacking the CvG6PD gene [While numerous G6PDs from thermophiles and mesophiles are well characterized ,5,6,7,8,vulgaris ,13. Inte6PD gene . Convers6PD gene . These rSphingomonas sp. PAMC 26621, which was isolated from an Arctic lichen Cetraria sp. on Svalbard islands [To gain insight into the cold adaptation mechanism of psychrophilic G6PDs, we cloned, expressed, and characterized a psychrophilic G6PD (SpG6PD1) from the  islands . zwf gene with a 1467-bp open reading frame was cloned from the genome of Sphingomonas sp. PAMC 26621. The SpG6PD1 has a calculated molecular weight of 55.6 kDa and an isoelectric point of 5.71. The amino acid sequence alignment of SpG6PD1 with those of 10 other G6PDs showed the highest identity with Sphingomonas elodea (88% identity) followed by Pseudomonas fluorescens (49% identity) and Escherichia coli (46% identity) (The dentity) .L. mesenteroides [L. mesenteroides G6PD as a template using the Swiss model, which predicted that SpG6PD1 was a homodimer enzyme.Moreover, the catalytic triad , the substrate-binding site (RIDHYLGK), and the cofactor-binding pockets were conserved in SpG6PD1 as reported in teroides ,17,18,19E. coli BL21(DE3) as a soluble protein  had no effect on SpG6PD1 activity. These results illustrate that metal ions are not required for the activity of SpG6PD1. Effects of metal ions on G6PD activity have been reported previously in numerous studies [SpG6PD1 activity was slightly enhanced by adding divalent cations Mgand 5 mM ; to our  studies ,29,30,31+ as a cofactor, and incubation at various temperatures (4–60 °C). We observed that the activity of SpG6PD1 maintained between 4–20 °C for 2 h. However, at 30 °C and 40 °C, the activity decreased to 68% and 58% of the initial activity, respectively. Interestingly, when exposed for 20 min, the SpG6PD1 activity rapidly reduced at 50 °C and completely lost at 60 °C  with NADPat 60 °C .The determined kinetic and thermodynamic parameters of SpG6PD1 are presented in +-linked and NAD+-linked reactions, the Km and kcat values at 30 °C are higher than those at 20 °C. In NADP+-linked reactions, SpG6PD1 shows a Km value of 145 µM for G6P (KmG6P) and a kcat value of 8081 min−1 , whereas the kcat value (1196 min−1) is 6.8-fold lower than that obtained with the NADP+ reaction (kcat/Km) of the NADP+-linked reaction is 31-fold higher than that of the NAD+-linked reaction  for the NADP+ reaction (30.3 kJ·mol−1) is less than twice the Ea for the NAD+-linked reaction (56.5 kJ·mol−1). Conversely, the ΔH‡ and ΔS‡ values obtained for SpG6PD1 are lower than its cold-adapted, mesophilic, and thermophilic counterparts  spectra in the far-UV region (200–250 nm) and the intrinsic protein fluorescence upon excitation at 280 nm, respectively. The spectroscopic results show that SpG6PD1 maintains the tertiary structure at 4–20 °C, whereas the secondary structure is maintained between 4–40 °C; both structures are completely denatured at 60 °C after 1 h of incubation .This data suggests that SpG6PD1 is a thermolabile enzyme. Furthermore, the CD spectra show maximum absorption peaks at 222 and 208 nm, indicating that SpG6PD1 is a protein containing an abundant α-helix .Sphingomonas sp. PAMC 26621 maintains activity and structure between 4–20 °C as a psychrophilic enzyme. The kinetic and thermodynamic data reveal high Km values and low activation enthalpy compared to its mesophilic and thermophilic counterparts. These are typical characteristics of a psychrophilic enzyme that enables the adaptation of the SpG6PD1 to the icy environment [Numerous G6PDs isolated from mesophiles and thermophiles have been characterized ,27,30,33ironment ,35.Leuconostoc mesenteroides, Cosgrove et al. have proposed the catalytic mechanism of LmG6PD. They report that His240 acts as a general base that abstracts the proton from the C1-OH of G6P, and the Asp177 was hydrogen bonded to the His240 to stabilize the positive charge forming in the transition state, while His178 interacts with the phosphate moiety of G6P to hold G6P during the reaction [In reaction . The psyreaction . Km values for G6P in NADP+-linked and NAD+-linked reactions; the (KmG6PNAD+)/(KmG6PNADP+) ratio of SpG6PD1 was 4.7, which is higher than that obtained for Streptomyces aureofaciens [Leuconostoc mesenteroides [Methylomonas M15 [Thermoanaerobacter tengcongensis [+-preferring G6PD enzymes [+ and NAD+ as cofactors. Moreover, the kcat/KmG6P for NADP+-linked reactions was found to be considerably higher than that for NAD+-linked reactions (31-fold)   , indicatt 30 °C) . This re to NAD+ .Sphingomonas sp. PAMC 26621, the NADPH demand is also expected from the presence of antioxidant enzymes in the genome; glutathione reductase and four isoforms of NADPH-dependent thioredoxin reductase indicate that the anti-oxidative potential is truly essential for the survival of this bacterium in cold-induced oxidative stress conditions. Thus, G6PD is expected to play a key role in Sphingomonas sp. PAMC 26621 survival under extremely cold habitats such as the Arctic, by generating NADPH.The role of G6PD in protecting cells from oxidants has also been reported. Notably, the activity of G6PDs is remarkably enhanced under conditions containing oxidants such as paraquat, lipid hydroperoxide, and tellurite, which result in an increase of NADPH concentration to combat the oxidative stress ,40,41. IKmG6P values are markedly higher, whereas the thermal stability and activation enthalpy (ΔH‡) are lower than those of mesophilic and thermophilic counterparts  of the Korea Polar Research Institute  [, Korea) . The pETL. mesenteroides  [A homology search was carried out using the BLAST program . Sequenc1003420) .zwf gene (Accession number: NZ_AIDW01000018) encoding SpG6PD1 was amplified from the genome of Sphingomonas sp. PAMC 26621 using a polymerase chain reaction (PCR) with a forward primer (5′-GTCGCATGCCAATCCGCAC-3′) and a reverse primer (5′-GAAATCAATCGTCCTGCCAGG-3′). The amplified product was subcloned in a TA vector and transformed into E. coli DH5α. Next, the zwf gene in the TA vector was amplified by PCR using a forward primer: 5′-GTCGATGGCCGGCCAATC-3′  and a reverse primer: 5′- GAAATCAAGCTTGTCCTGCCAG-3′ . The amplified product was digested with Hind III and Nco I, and subsequently subcloned in a pET28b (+) vector followed by transformation into Escherichia coli BL21 (DE3). The construct was confirmed by DNA sequencing.The E. coli BL21 (DE3) cells harboring pET28b-zwf were cultured at 37 °C in Luria-Bertani (LB) broth containing kanamycin (50 µg/mL). Following isopropyl β-d-1-thiogalactopyranoside induction at cell density OD = 0.6–0.8, the cells were cultured for an additional 10 h at 30 °C and subsequently harvested by centrifugation, washed, and resuspended in buffer A , and 5% (w/v) glycerol). Then, the harvested cells were disrupted by sonication in an ice-water bath, followed by centrifugation of the cell extract at 13000 × g for 30 min at 4 °C. The resultant supernatant was loaded on a 1-mL HisTrap column , washed with 10 mL of buffer A, and eluted by using a linear gradient of 20–500 mM imidazole in buffer B  glycerol). The fractions containing G6PD activity were pooled and desalted in buffer A using a 5-mL HiTrap desalting column . Next, the recombinant SpG6PD1 protein was purified by anion-exchange column chromatography with a 1-mL Q Sepharose column  using a linear gradient of 25–1000 mM KCl in buffer A. All of the purification steps were carried out at 4 °C. The purified enzyme was frozen in liquid N2 and stored at –80 °C until further experiments.The +, 2 mM of glucose 6-phosphate, and an appropriate amount of SpG6PD1 enzyme in buffer A. The mixture was incubated at 30 °C for 2 min, and then measured 340A in a 1-cm path length cuvette on a Shimadzu UV-1800 spectrophotometer. One enzyme unit is defined as the amount of enzyme catalyzing the formation of 1.0 µmol of NAD(P)H per min at 30 °C. The G6PD activity was determined by measuring the production of NAD(P)H at 340 nm. The assay mixture  consisted of 100 µM of NAD(P)Protein concentration was determined by the Bradford method , with bo2HPO4 (pH 5.0–5.5), sodium phosphate buffer (pH 6.0–7.0), Tris⋅HCl (pH 7.5–8.5), and Glycine–NaOH (pH 9.0–10.0) for both NADP+-linked and NAD+-linked reactions. The apparent optimum temperature was determined by measuring the enzyme activity at 4−60 °C in the reaction mixture for 2 min. After measuring the optimum pH and temperature of SpG6PD1 with either NADP+ or NAD+ as a cofactor, NADP+ was employed for characterizing SpG6PD1 throughout the experiments due to the low activity with NAD+. To elucidate the effects of different metal ions on the activity of SpG6PD1, 1 mM or 5 mM  of EDTA, Mn2+, Zn2+, Ni2+, Mg2+, Cu2+, Ca2+, and Fe3+ was added into the reaction mixture separately, and SpG6PD1 activity was measured at 30 °C with NADP+ as a cofactor. Also, the thermal stability of SpG6PD1 was determined by measuring the residual activity in the reaction mixture containing NADP+ at 30 °C for 2 min at various temperatures . Residual enzyme activity was measured every 20 min up to 2 h. The catalytic activity at 30 °C before incubation was considered to be 100%.The optimum pH of SpG6PD1 was determined by performing the enzyme assay in the buffer systems of citric acid–NaKm) and catalytic rate constant (kcat) were determined by measuring the activity of SpG6PD1 at varying concentrations of G6P (500–2000 µM) at 20 °C and 30 °C for 2 min. The Vmax and Km parameters were calculated by applying the Lineweaver–Burk plot. The activation energy (Ea) of the reaction was determined from the slope of the Arrhenius plot (lnkcat versus 1/T). Thermodynamic parameters (Gibbs free energy of activation (ΔG‡), enthalpy (ΔH‡), and entropy (ΔS‡)) were calculated using the following equations, as described in a previous study [R is the gas constant (8.314 J·mol−1K−1), Bk is the Boltzmann constant (1.3805 × 10−23 J·K−1), h is the Plank constant (6.6256 × 10−34 Js), and k is the catalytic rate constant.The Michaelis−Menten constant (us study :(1)ΔG‡=RA 200-µL aliquot of sample (0.3 mg/mL) in buffer A was incubated at the indicated temperature  for 1 h. The temperature-induced unfolding of SpG6PD1 was investigated at the Korea Basic Science Institute  by circular dichroism (CD) spectra using a JASCO J-1500 spectropolarimeter. The spectra were the averages of two scans and were plotted as residual ellipticity (mdeg) against wavelength (nm) using the GraphPad Prism 5 software. The fluorescence emission spectra of SpG6PD1 (1 mM) were measured at 25 °C upon incubation at various temperatures for 1 h, with excitation at 280 nm and monitoring the emission spectra between 300–400 nm by using a SCINCO FS-2 fluorescence spectrometer.


Both butorphanol + diprenorphine + oxygen and butorphanol + oxygen, at the doses used, significantly improved the etorphine-induced hypoxaemia in both boma- and field-immobilised white rhinoceroses. Clinically acceptable oxygenation in field-immobilised white rhinoceroses can be achieved by using either treatment regimen, provided that it is combined with oxygen insufflation.Opioid-induced immobilisation results in severe respiratory impairment in the white rhinoceros. It has therefore been attempted in the field to reverse this impairment with the use of opioid agonist-antagonists, such as nalorphine, nalbuphine, butorphanol and diprenorphine; however, the efficacy of some of these treatments has yet to be determined. The efficacy of butorphanol, either alone or in combination with diprenorphine both with and without oxygen insufflation, in alleviating opioid-induced respiratory impairment was evaluated. The study was performed in two parts: a boma trial and a field trial. Rhinoceroses were immobilised specifically for the study, according to a strict protocol to minimise confounding variables. A two-way analysis of variance was used to compare the physiological responses of the rhinoceroses to the different treatments and their effects over time. The intravenous administration of butorphanol (at 3.3 mg per mg etorphine) plus diprenorphine (at 0.4 mg per mg etorphine) did not offer any advantage over butorphanol (at 15 mg per mg etorphine) alone with regard to improving P The ability to safely immobilise white rhinoceroses for management procedures such as translocation, veterinary treatments and fitting of tracking devices is essential for the long-term survival of these mega-herbivores, especially with increased poaching pressure. The advent of potent mu-opioid agonists, such as etorphine hydrochloride, significantly decreased the mortality rate of rhinoceroses during capture events  and housed individually in bomas. Husbandry procedures as per the South African National Parks (SANParks) standard operating procedures (SOPs) for boma-kept rhinoceroses were carried out throughout the experimental period. The trial started after a 1-month adaptation period and data collection occurred from August to December 2012. All immobilisations took place in the morning (between 05:20 and 10:50) and barometric pressure ranged from 737.0 mmHg to 745.9 mmHg.µg/kg – 2.6 µg/kg) , 30 mg – 45 mg azaperone (dose range 30 µg/kg – 40 µg/kg)  and 2500 international units (i.u). hyaluronidase . The dosage of the drug was calculated from the previously measured body mass according to a standardised dose table  as described previously  (But), butorphanol IV  + diprenorphine IV  (But+M5050), butorphanol IV (15 mg per mg etorphine) + oxygen intra-tracheal insufflation  (But+O2), butorphanol IV (3.3 mg per mg etorphine) + diprenorphine IV (0.4 mg per mg etorphine) + oxygen intra-tracheal insufflation (30 L/min) (But+M5050+O2) and sterile water (control) were evaluated and compared as measures to support the respiratory physiology of the immobilised white rhinoceros.The experiment consisted of five trials with the following interventions : butorphEach rhinoceros received each intervention for the five trials in a randomised order at 2-week intervals. The interventions were administered 6 min after the rhinoceros became laterally recumbent. Clinical data and samples were collected at 5 min after the animal became laterally recumbent (before the intervention) and every 5 min thereafter for a 20-min immobilisation period. In the control trial, butorphanol (15 mg per mg etorphine) was administered at 21 min to facilitate arousal and loading into a crate.Respiratory rate (breaths per minute) was monitored by counting thoracic and abdominal excursions and feeling for expired air at the nares. An immobilisation score, ranging from 1 (no immobilising or sedative effect) to 6 (excessive immobilisation depth with respiration < 3 breaths/min), was used to assess the level of immobilisation at 5, 10, 15 and 20 min. Level 3 indicated standing sedation that allowed handling, while levels 4 and 5 indicated recumbent immobilisation with or without ear movement, respectively.aCO2) and oxygen (PaO2) were measured immediately using a portable pre-calibrated blood gas analyser with pre-calibrated blood gas cassettes . The blood gas variables are reported at a body temperature of 37 °C. Body temperature was measured with a thermocouple thermometer  probe inserted 10 cm into the rectum.Arterial blood samples for blood gas analyses were collected from the medial auricular artery catheterised with a 22G × 1″ IV catheter . A 0.5 mL sample was collected anaerobically into 1 mL heparinised syringes at 5, 10, 15 and 20 min after the rhinoceros became laterally recumbent. Partial pressures of carbon dioxide  as described by Bush et al. .Twenty-one minutes into the recumbent period, the rhinoceros was stimulated to stand and was guided into a crate for weighing using a scale suspended from a vehicle-mounted crane. Approximately 35 min after the animal became laterally recumbent, the effects of etorphine were reversed using naltrexone  administered intravenously into an auricular vein at 20 times the etorphine dose. The rhinoceros was then released from the crate into its boma.All rhinoceroses used in these trials were monitored using a standardised boma scoring system on a daily basis for changes in appetite, defaecation and behaviour .Clinical monitoring was carried out as in the boma study, but the immobilisation period was extended to 25 min; thus, an extra arterial blood sample and a set of recordings were taken at that time. The arterial blood samples were analysed using a portable pre-calibrated blood gas analyser with recalibrated test cards  followed by Tukey’s multiple comparisons for two separate analyses. In the first analysis, we looked for differences between responses to But, But+M5050 and sterile water (control) at 5, 10, 15 and 20 min , while in the second analysis we looked for differences between responses to But+O2, But+M5050+O2 and sterile water (control) at 5, 10, 15 and 20 min . For the field study, we used a two-way ANOVA followed by Sidak’s multiple comparisons test to test for differences between responses to But+O2 and But+M5050+O2 at 5, 10, 15, 20 and 25 min. A Tukey’s post-hoc test was used to test for differences in values between time points within each trial.We used GraphPad Prism version 6.00 for Mac OS X  for statistical analyses. All results are reported as mean ± SD, and This project was approved by the Animal Use and Care Committee  of South African National Parks (SANParks) as well as the Animal Ethics Committees of the University of the Witwatersrand  and the University of Pretoria .2 group (n = 14) was 1272 kg ± 227 kg, while that for the But+M5050+O2 group (n = 8) was 1336 kg ± 339 kg. Most field rhinoceroses were estimated to be in the correct body mass bracket according to the dose table . No acute mortality occurred in the rhinoceroses that were studied.All boma-housed rhinoceroses were in good health, indicated by normal demeanour and normal eating and defaecation habits throughout the study period. Field rhinoceroses were also in good health at the time of darting, determined by observing normal behaviour from the helicopter before darting and evaluating body condition score. Each animal’s health status was confirmed by subsequent haematology and biochemistry analysis of blood samples collected at the time of each immobilisation. Rectal temperatures for boma-immobilised rhinoceroses ranged from 36.7 °C to 39.0 °C and 37.6 °C to 38.5 °C for field-immobilised rhinoceroses. The average body mass of the boma rhinoceroses was 1220 kg ± 148 kg. In the field study, the average body mass for the But+OaO2 values (p > 0.05) of 27 mmHg ± 7 mmHg (control), 28 mmHg ± 4 mmHg (But), 30 mmHg ± 6 mmHg (But+M5050), 31 mmHg ± 6 mmHg (But+O2) and 30 mmHg ± 7 mmHg (But+M5050+O2). PaO2 in the rhinoceroses then changed over time in both sets of trials, namely, the trials without oxygen  = 216.3, p < 0.0001) and with oxygen  = 81.64, p < 0.0001). At the time points following the treatment interventions, PaO2 differed amongst the trials without oxygen  = 125.2, p < 0.0001) as well as those with oxygen supplementation  = 49.52, p < 0.0001). Both But and But+M5050 led to improved oxygenation at 10, 15 and 20 min compared to the control trial, but the rhinoceroses were still hypoxaemic at 20 min . Similarly, both But+O2 and But+M5050+O2 treatments led to improved oxygenation at 10, 15 and 20 min compared to the control trial, but unlike the treatments without oxygen, these treatments completely corrected the etorphine-induced hypoxaemia. In these two trials with oxygen, the rhinoceroses had PaO2 values of 154 mmHg ± 53 mmHg (But+O2) and 152 mmHg ± 21 mmHg (But+M5050+O2) at 20 min. Throughout the immobilisation period, both the But+O2 and But+M5050+O2 treatment interventions led to similar PaO2 values in the rhinoceroses (p > 0.05). On the other hand, PaO2 values in the But and But+M5050 trials were statistically different at 10 min  and 20 min , but not at 15 min (p > 0.05).Chemical immobilisation resulted in severe hypoxaemia in all trials . At 5 miaCO2 values at 5 min of 82 mmHg ± 6 mmHg (control), 83 mmHg ± 9 mmHg (But), 81 mmHg ± 7 mmHg (But+M5050), 79 mmHg ± 7 mmHg (But+O2) and 72 mmHg ± 5 mmHg (But+M5050+O2)  = 20.8, p < 0.0001) and those with oxygen  = 4.61, p = 0.01). PaCO2 in the rhinoceroses also changed amongst treatment groups in the two sets of trials  = 34.16, p < 0.0001 [trials without oxygen]) and  = 8.78, p = 0.003 [trials with oxygen]). The treatment interventions led to lower PaCO2 values in the rhinoceroses compared to when no treatment was administered (control) at all time points following the interventions, and by the end of the immobilisation period, PaCO2 values in the rhinoceroses that received treatments were lower compared to those at 5 min (p < 0.05). At 20 min, the rhinoceroses had PaCO2 values of 82 mmHg ± 4 mmHg (control), 68 mmHg ± 3 mmHg (But), 64 mmHg ± 7 mmHg (But+M5050), 72 mmHg ± 13 mmHg (But+O2) and 72 mmHg ± 5 mmHg (But+M5050+O2). Throughout the immobilisation period, there were no differences in the rhinoceroses’ PaCO2 values between the But and But+M5050 trials , nor the But+O2 and But+M5050+O2 trials (p > 0.05).All rhinoceroses had similarly elevated P5050+O2) . PaCO2 tp > 0.05) with values of 7 ± 2 breaths/min (control), 6 ± 1 breaths/min (But), 7 ± 1 breaths/min (But+M5050), 7 ± 1 breaths/min (But+O2) and 7 ± 2 breaths/min (But+M5050+O2)  = 14.76, p < 0.0001 [trials without oxygen] and F = 9.85, p = 0.0003 [trials with oxygen]). However, the different treatment interventions did not lead to differences in respiratory rates in the rhinoceroses  = 3.4, p = 0.06 [trials without oxygen] and F = 0.1449, p = 0.87 [trials with oxygen]), except at 10 min, where the post-hoc test indicates that But+O2 led to improved respiratory rates compared to But+M5050+O2 (p < 0.05). At 20 min, the rhinoceroses had respiratory rates that were similar to those at 5 min across all trials, namely 6 ± 2 breaths/min (control), 7 ± 1 breaths/min (But), 7 ± 2 breaths/min (But+M5050), 6 ± 1 breaths/min (But+O2) and 6 ± 1 breaths/min (But+M5050+O2).At 5 min, the respiratory rates in the rhinoceroses were similar across trials (5050+O2) ., which aO2 = 35 mmHg ± 7 mmHg in the But+O2 trial and 34 mmHg ± 6 mmHg in the But+M5050+O2 trial)  and 62 mmHg ± 10 mmHg  in the But+O2 and But+M5050+O2 trials, respectively. Indeed, PaO2 was greater at all time points following the interventions, compared to that at 5 min, in both trials  = 25.49, p < 0.0001) and there were no differences in the responses to the different treatments  = 1.37, p = 0.244). Oxygenation in the rhinoceroses remained elevated throughout the immobilisation period with PaO2 values at 25 min of 81 mmHg ± 27 mmHg (But+O2) and 80 mmHg ± 20 mmHg (But+M5050+O2).Similar to the boma study, chemical immobilisation with a combination of etorphine, azaperone and hyaluronidase led to severe hypoxaemia in all the rhinoceroses at 5 min post-recumbency  . Both coaCO2 values of 65 mmHg ± 8 mmHg (But+O2) and 69 mmHg ± 7 mmHg (But+M5050+O2)  = 26.04, p < 0.0001) to the extent that PaCO2 was lower (p < 0.05) than at 5 min at 10, 15 and 20 min, but not at 25 min. In the But+M5050+O2 trial, PaCO2 values were statistically similar at all time points (p > 0.05). At 10 min (p < 0.01) and 15 min (p < 0.05), the PaCO2 in the rhinoceroses that received But+O2 was lower than in those that received But+M5050+O2. At 10 min, PaCO2 values in the rhinoceroses were 58 mmHg ± 9 mmHg (But+O2) and 64 mmHg ± 7 mmHg (But+M5050+O2). By the end of the immobilisation period, PaCO2 values in the rhinoceroses were 60 mmHg ± 7 mmHg and 63 mmHg ± 6 mmHg in the But+O2 and But+M5050+O2 trials, respectively.Together with the hypoxaemia, the rhinoceroses were also hypercapnic 5 min after they became recumbent, with P5050+O2) . The PaCF = 23.72, p < 0.0001) and across time  = 7.98, p < 0.0001) (2 administration led to increased respiratory rates in the rhinoceroses compared to those that received But+M5050+O2 at 10 min (p < 0.01) and 15 min (p < 0.05). But+M5050+O2 administered to the rhinoceroses led to a decrease in respiratory rates at 20 min  and 25 min  compared to that at 5 min. On the other hand, But+O2 led to an initial clinically relevant but statistically insignificant increase in the respiratory rates of the rhinoceroses at 10 min , which was followed by a gradual decrease to the extent that the respiratory rates at 20 min  and 25 min  were lower than the respiratory rates at 10 min.Corresponding with the observed hypercapnia, the respiratory rates were also different between treatments ( 0.0001) . Immobil2 and But+M5050+O2), with no differences between the use of a single or combination of partial agonist-antagonist treatments. Although the interventions without oxygen (But and But+M5050) improved but did not correct the etorphine-induced hypoxaemia, the responses to the treatments were clinically similar in the boma trials. Similarly, in field-immobilised rhinoceroses, there were no differences in the rhinoceroses’ PaO2 values between the two treatments (But+O2 and But+M5050+O2), but the blood oxygenation was not completely corrected despite the animals receiving oxygen insufflation, although it was significantly improved, leading to a clear clinical benefit from the treatments.Previous work has demonstrated that butorphanol with oxygen insufflation corrects the etorphine-induced hypoxaemia in boma-immobilised white rhinoceroses  as a result of increasing PaO2 levels  is supplied with etorphine (M99®) by its current manufacturer, Voluplex, and is registered as the standard antidote for etorphine-induced immobilisation. To reverse the effects of etorphine, diprenorphine is given at 2–3 mg per mg etorphine  with oxygen. Some veterinarians are using diprenorphine on its own to reverse the etorphine-induced respiratory impairment in the white rhinoceros. Based on the results of these trials, it may be worthwhile to determine if a higher dose of diprenorphine used alone could offer any physiological benefit over butorphanol alone. However, this study confirms that arterial oxygen levels can be restored to clinically acceptable values in immobilised white rhinoceroses administered either But+O


ESR experiments, excluding effects of the oils’ matrices and other minor constituents, confirmed that a food representative tocopherol ratio of (γ + δ)/α = 4.77, as represented in soybean oil, led to a more pronounced delay of lipid oxidation than a lower ratio in canola (1.39) and sunflower oil (0.06). An optimum (γ + δ)/α -tocopherol ratio contributing to the oxidative quality of vegetable oils extending their shelf life has to be investigated.The antioxidant activity of tocopherols in vegetable oils was shown to chiefly depend on the amount and the tocopherol homolog present. However, the most effective ratio of tocopherol homologs with regard to the antioxidant capacity has not been elucidated so far. The present study analyzed the effect of different tocopherol concentrations, homologs and ratios of homologs on markers of lipid oxidation in the most commonly consumed vegetable oils  stored in a 12 h light/dark cycle at 22 ± 2 °C for 56 days under retail/household conditions. After 56 days of storage, the α-tocopherol-rich canola and sunflower oil showed the strongest rise in lipid peroxides, yielding 25.1 ± 0.03 meq O Tocopherols are characterized by a chromane ring with a hydroxyl group and a hydrophobic side chain. Depending on the number and position of methyl groups on the chromanol ring, tocopherols occur as α-, β-, γ- and δ-homologs, showing structure-specific chemical and biological activities. The hydroxyl group of the chromanol ring chiefly determines the antioxidant activity, as it can donate a hydrogen atom to reduce free radicals. In vivo, the relative antioxidant activity of the tocopherol homologs is generally agreed to follow the order of α > β > γ > δ ,2,3,4. ITherefore, this study aimed to (i) quantitate tocopherol homologs in canola, corn, olive, soybean and sunflower oil, as commonly consumed dietary oils , purchasTo evaluate the oxidative status of a subset of most commonly consumed commercial vegetable oils  at the tThe fatty acid composition of the sunflower, canola, and soybean oil was analyzed after purchase . Stored Since household storage conditions of soybean oil were demonstrated to decrease contents in tocopherols , the stap < 0.05), whereas the overall mean loss of γ-tocopherol, ranging from −23.8 ± 2.52% to −29.0 ± 2.20%, was less pronounced (p < 0.05). Delta-tocopherol, which could be quantitated in soybean oil solely, showed a mean decrease (p < 0.05) of 10.9 ± 3.15% and 17.6 ± 2.00% in non- and tocopherol-fortified oil, respectively.Concentrations of tocopherol homologs also decreased in all oils after 56 days of storage , with α-2/kg (+2395%), in canola oil to 25.1 ± 0.03 meq O2/kg (+2425%), in soybean oil to 19.1 ± 0.05 meq O2/kg (+1659%) and fortified soybean oil to 20.6 ± 0.51 meq O2/kg (+1631%) compared to day 1 (p < 0.05) (p < 0.05).After 56 days of storage, the POV increased in sunflower oil to 24.7 ± 0.05 meq O < 0.05) . SunflowPearson correlation analyses of total tocopherols, tocopherol homologs and POVs on the last day of the storage period revealed a significant association between an increase in the POV and the decrease in total tocopherols as well as α-tocopherol after 56 days of storage . The higp < 0.05), but there was no statistically significant difference to that of 10 µM γ-tocopherol. Furthermore, it was shown that tocopherols in a tocopherol homolog ratio (γ + δ)/α = 1 did not show a significant difference in scavenging the DPPH radicals compared to δ-tocopherol when applied in concentrations of 50 µM. The highest scavenging activities were demonstrated for 50 µM of γ-tocopherol (−86.5%) (p < 0.05). Examining whether the tocopherol homolog ratio of (γ + δ)/α = 5 showed a stronger effect in scavenging the DPPH radicals than only γ-tocopherol, the scavenging activities of γ-tocopherol were also compared in the ratio of 5:1. However, the tocopherol homolog ratio of (γ + δ)/α = 5 showed a decrease of the spin counts of the DPPH radical of 24.2% compared to tocopherol homolog ratio (γ + δ)/α = 1, whereas 50 µM γ-tocopherol showed only a 19.2% decrease compared to 10 µM γ-tocopherol.To confirm the antioxidant properties of the tocopherol homolog ratio (γ + δ)/α and to exclude possible effects of the oils’ matrices including potential effects of minor constituents, the free radical scavenging activity of individual tocopherol homologs and combinations thereof was investigated by means of electron spin resonance (ESR) spectroscopy . At the Hexanal, mainly formed by decomposition of linoleic acid hydroperoxides, was analyzed as a secondary lipid peroxidation marker . After np < 0.05). The formation of hexanal [ng/mg LA] was shown to be statistically significant higher in sunflower (+140% and +142%) and canola oil (+126% and +129%) compared to soybean and fortified soybean oil showing the highest hexanal concentrations in the following order sunflower = canola > soybean = fortified soybean oil at the end of the experimental period (p < 0.05).Over the storage period accelerated contents of hexanal [ng/mg LA] could be shown in all four oil types compared to their initial concentrations . Since it has been shown that the fatty acid composition in sunflower  and canola  oil did not change under accelerated storage conditions , canola , corn [AT: n = 2], sunflower [AT: n = 5], soybean ) was purchased. Tocopherol contents and peroxide values (POVs) of the oil samples were analyzed. After quantitation of the tocopherol homologs in the market samples, we chose a refined sunflower oil , a refined canola oil , and a refined soybean oil  and δ-tocopherol (64.8 ± 0.87 mg/kg oil) for a storage experiment under typical household conditions [From retail stores in Austria (AT), Bangladesh (BD), Brazil (BR) and the United States (US), a total of 55 vegetable oils of five oil types , 0.5 mL of a saturated potassium iodide solution was added and mixed well for 1 min. Afterwards, 30 mL of double-distilled water was added and an end point titration with 0.1 N sodium thiosulfate, with starch as an indicator, was performed.Determination of the peroxide value (POV) was performed according to the AOCS official method Cd 8-53 . Brieflyv/v), and transferred to a capillary tube (100 µL), which was placed into a high sensitivity resonator (Bruker ER 4122SHQE). The stability of DPPH was examined after 3 min by a cw-ESR spectrometer  operating at 9 GHz with 100 kHz modulation frequency at 25 °C. DPPH spectra were recorded by setting the following parameters: center field, 3518.35 G; sweep field, 144 G; sweep time, 300 s; conversion time, 292.97 ms; modulation amplitude, 4 G; microwave power, 4 mW; receiver gain, 99 dB; resolution, 1024 points; scan time, 5 min. The two neighboring nitrogen atoms resulted in five hyperfine splitting lines with isotropic hyperfine coupling constants of approximately 8.2 and 9.3 G. For quantitative measurements of the spin counts of DPPH (Ns), the DPPH spectrum was double-integrated (DI). A reference-free quantitation of the number of spins was performed according to the following equation considering the sample volume (V), the microwave power (P), the modulation amplitude (Bm), the Q-factor (Q) of the resonator, the calibration factor (c) of the resonator, the electron spin (S), the Boltzmann factor (nB) and the resonator field profile ) [The free radical-scavenging activity of the tocopherol homologs was evaluated by ESR (electron spin resonance) spectroscopy. The stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) at a concentration of 100 µM was monitored in the presence or absence of 50 µM α-tocopheryl acetate, 50 µM α-tocopherol, 50 µM γ-tocopherol, 50 µM δ-tocopherol, 10 µM γ-tocopherol, 50 µM tocopherol ratio (γ + δ/α) = 1 and 50 µM tocopherol ratio (γ + δ/α) = 5 in the dark. DPPH, tocopherol and α-tocopheryl acetate were dissolved in ethanol/water ) :Ns = DI v/v) and 0.4 µg/mL hexanal-d12 was added as an internal standard. After sealing airtight the vial, the sample was incubated on a CTC Combi Pal  and analysis was performed by means of a GC-MS  using a capillary column  for separation. Detailed measurement parameters are given by Pignitter et al. [m/z 72 for hexanal and m/z 80 for hexanal-d12 in SIM mode. Hexanal and hexanal-d12 in the sample were identified by comparing their retention times and fragmentation patterns with their corresponding reference standards. In addition, hexanal was identified using the NIST11 database by comparing its fragmentation pattern with a reference spectrum. Limit of detection was determined by a signal-to-noise ratio of 3 with 0.01 µg/mL.Hexanal was quantitated as reported previously . Brieflyr et al. . Quantitn = 3–4) and two technical replicates . Outliers were eliminated using the Nalimov outlier test. p values below 0.05 indicate statistical significance. Data are displayed as mean ± standard deviation (SD).Statistical analyses were carried out using SigmaPlot 11 . One-way ANOVA followed by Holm-Sidak post hoc test was used for determining statistical difference. Correlation analyses were done using the Pearson calculation method. Statistical analyses were carried out with three to four independent replicates per oil type  -tocopherol to optimize the oxidative stability of the oil during household storage conditions in order to guarantee not only sensory properties but also the nutritional quality of the product. Measures to improve the oxidative stability of vegetable oils by optimizing the tocopherol homolog composition towards an ideal (γ + δ/α) -tocopherol ratio should be implemented in plant production and processing, although the contribution of other intrinsic factors, e.g., fatty acid composition, other antioxidants, mineral contents or pro-oxidants, on the oxidative stability of vegetable oils must still be elucidated in future studies.The here-presented results as well as recent findings of our group in soybean oil  clearly


This effect is independent of NEDD8 conjugation onto substrates. The study indicates that the NEDD8 cycle is a regulatory module of HSP70 function. These findings may be important in tumorigenesis, as we find decreased NEDP1 levels in hepatocellular carcinoma with concomitant accumulation of NEDD8 conjugates.Ubiquitin and ubiquitin-like chains are finely balanced by conjugating and de-conjugating enzymes. Alterations in this balance trigger the response to stress conditions and are often observed in pathologies. How such changes are detected is not well understood. We identify the HSP70 chaperone as a sensor of changes in the balance between mono- and poly-NEDDylation. Upon DNA damage, the induction of the de-NEDDylating enzyme NEDP1 restricts the formation of NEDD8 chains, mainly through lysines K11/K48. This promotes APAF1 oligomerization and apoptosis induction, a step that requires the HSP70 ATPase activity. HSP70 binds to NEDD8, and,   •Restriction of NEDD8 chains by NEDP1 is required for DNA damage-induced apoptosis•The HSP70 chaperone is a sensor of the balance between mono- and NEDD8 chains•Mono-NEDD8 stimulates HSP70 activity, which allows the formation of the apoptosome•NEDP1 levels are downregulated in mouse hepatocellular carcinoma The formation of ubiquitin and/or ubiquitin-like polymers is dynamically balanced by conjugating and de-conjugating enzymes. Bailly et al. report that the HSP70 chaperone is a sensor of the NEDD8 cycle. Conversion of NEDD8 chains into mono-NEDD8 by the de-NEDDylating enzyme NEDP1 upon DNA damage activates HSP70, which allows the formation of the apoptosome and apoptosis induction. A key characteristic of ubiquitin and ubiquitin-like (Ubl) molecules is their ability to modify substrates as single moieties or in the form of polymeric chains. The extent and topology of polymeric chains is finely balanced by the coordinated action of conjugating and de-conjugating enzymes . The actC. elegans and in human cells, we found a conserved role of the NEDD8 cycle in the DNA damage-induced apoptosis. The induction of NEDP1 upon DNA damage restricts the formation of NEDD8 chains mainly through lysines K11/K48 in the cytoplasm. This promotes the oligomerization of the apoptotic protease activating factor 1 (APAF1) and apoptosis induction. We found that de-NEDDylation is required for the release of the heat shock protein 70 (HSP70) chaperone from APAF1, a required step toward APAF1 oligomerization. HSP70 binds to NEDD8 and we mapped the ATPase domain as the binding site for NEDD8 on HSP70. Biochemical analysis shows that the balance between mono-NEDD8 and NEDD8 chains is a regulatory module for HSP70 function; mono-NEDD8 activates the ATPase activity of HSP70, which is counteracted upon NEDD8 polymerization. Restriction of poly-NEDDylation by NEDP1 restores the stimulatory effect of NEDD8 on HSP70 ATPase activity. The studies reveal that HSP70 is a sensor of changes in the NEDD8 cycle controlled by NEDP1. They also provide mechanistic insights on the role of poly-NEDDylation restriction as an activation signal for HSP70 function and apoptosis induction upon DNA damage. These findings may be relevant in pathology, as we found that NEDP1 protein levels are downregulated in a mouse model system for hepatocellular carcinoma with concomitant accumulation of NEDD8 conjugates. Collectively, the data provide a molecular basis for a potential suppressive role of NEDP1 in tumorigenesis through restriction of NEDD8 chains.The Ubl molecule NEDD8 is highly conserved and essential in almost all tested organisms. Its functions have been characterized mainly through mono-NEDDylation of the cullin family of proteins and stimulation of the activity of cullin-RING ligases (CRLs), but also through modification of non-cullin substrates . Defectsulp-3  as the C. elegans homologous gene of human NEDP1 by reciprocal BLAST analysis ), within the reading frame, which abolishes ulp-3 expression, shown by RNA and protein-level analysis (ulp-3 knockout (KO) animals are viable, and further systematic phenotypic characterization shows no defects in cell cycle progression, growth, and fertility compared to wild-type animals (data not shown). However, worms deleted or silenced for ulp-3, in contrast to wild-type animals, are almost completely resistant to the induction of apoptosis upon ionizing radiation (IR) in germ cells , in which apoptotic corpse engulfment is severely impaired, allowing quantitation of developmental apoptosis. No difference in the number of apoptotic cells that persist in the head of L1 larvae between the ced-1 single mutant and the ulp-3; ced-1 double mutant was observed, suggesting a specific role for ULP-3 in germ cells apoptosis upon IR  , the main cellular circuitry involved in apoptosis induction is the DNA-damage checkpoint signaling pathway that ultimately triggers the highly conserved transcription activator p53, called CEP-1 in -like 1) . We anal upon IR C and S2Dulp-3 deletion on CED-4 localization using a C. elegans strain stably expressing CED-4::GFP under its endogenous promoter and 3′UTR. CED-4::GFP displays perinuclear localization consistent with the pattern of endogenous CED-4 (ulp-3 significantly reduces the CED-4::GFP mobile fraction by nearly 40% (12.18% ± 1.03%)  into the apoptosome homomeric complex is the critical step for caspase 3-like protein (CED-3) activation and apoptosis induction . We monius CED-4 D. By usius CED-4 . Deletio± 1.03%) D and 1E.3 mutant F. FRAP areatment E and 1G.ulp-3-deleted worms were labeled with light and heavy isotopes, respectively  remnants left on lysine residues upon trypsin digestion of proteins by mass spectrometry demonstrates their modification with ubiquitin, NEDD8, or ISG15 . We hypoectively A. To speectively A. A totaafter IR . Interesafter IR B, consisafter IR B, indicaafter IR . This ma K11/K48 B.Figure C. elegans, we investigated in human cells the role of the ULP-3 homolog NEDP1 in the DNA damage-induced apoptosis. Knockdown of NEDP1 by small interfering RNA (siRNA) increased overall protein NEDDylation . This mutant is deficient in modifying substrates but itself can be modified by endogenous wild-type NEDD8, allowing the isolation of NEDD8 chains that are not attached to substrates (unanchored). Similarly to the data obtained with the wild-type NEDD8 chains, unanchored NEDD8 chains inhibited HSP70 ATPase activity, which was induced upon de-conjugation by NEDP1 . Unanchored ubiquitin chains have been reported to control several processes, including innate immune response, viral uncoating, and aggresome formation (While we cannot exclude that the NEDDylation of a yet unidentified target(s) is part of the presented signaling cascade, the ormation . The preormation  suggest The observed downregulation of NEDP1 levels in HCC indicates that NEDP1 is a target during tumorigenesis. This finding defines a molecular basis for the reported defects in the NEDD8 cycle in tumors as evidenced by an increase in protein NEDDylation . As inhidimitris.xirodimas@crbm.cnrs.fr). Plasmids newly generated in this study will be made readily available to the scientific community. We will honor requests in a timely fashion.Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Dimitris P. Xirodimas (ced-1(e1935), ced-3(n717), ced-9(n1653ts), cep-1(lg12501), gla-3(op216), glp-4(bn2ts), hpr-17(tm1579), lig-4(ok716), opls219 (CED-4::GFP), orn-1(tm5454), ulp-3(tm1287). The ulp-3(tm1287) and orn-1(tm5454) deletion alleles were obtained from the Japanese Consortium (NBRP) and backcrossed five times against our N2 wild-type genetic background. Double and triple mutants generated in this study will be available at the CGC and/or available on request. Apoptosis, cell cycle arrest assays and genotoxic sensitivity testing were performed as previously described , MCF7 , H1299  were originally obtained from the ATCC bioresource. Hepatoma cell line derived from GNMT-KO mice liver tumors (OKER cells) , were emad libitum access to food and water.Animal experiments were performed according to the Spanish Guide for Care and Use of laboratory animals and the European Research Council animal care and use guidelines. Protocols were approved by the CIC bioGUNE Animal Care and Use Committee and the local authority (Diputación de Bizkaia). In the present study, animals were between 5 to 12-month-old males. Animals were bred and housed in the animal unit of CIC bioGUNE (AAALAC-accredited facility) with controlled conditions of temperature (22°C) and humidity, in a 12 h light/dark cycle with For Western Blots, worm proteins were extracted from synchronized adult worms in 50mM HEPES-NaOH pH8.0, Urea 9M, SDS 2% supplemented with zirconium 0.7mm beads (BioSpec Product) and disrupted in a bead beater . Debris were removed by quick centrifugation cycles  and soluble proteins were then separated by a 12000 g, 30min centrifugation. Supernatant protein concentration was measured by BCA following manufacturer’s instructions (Life technologies). 2xSDS Laemmli buffer was added before boiling samples for 5min.orn-1 gene (tm5454). We backcrossed the orn-1(tm5454) deletion five times against our N2 wild-type background then against ulp-3(tm1287). For isotopic labeling, SILAC bacteria (SLE1) were labeled as in To minimize Arginine-to-Proline conversion we obtained a deletion allele from the Japanese NBRP consortium that disrupts the C. elegans database with mass accuracy of ± 50ppm for precursor ions and 1Da for product ions. Results were filtered with mass accuracy of ± 5 ppm on precursor ions and presence of the intended motif (K-ε-GG). LTQ-Orbitrap Velos was used (Thermoscientific). A 5% default false positive rate was used to filter the SORCERER results.Di-Glycine motif peptide identification was performed by Cell Signaling Technology, following UbiScan protocols and instructions  using Ubiquitin Branch Motif Antibody (K-ε-GG) #3925. Peptides were loaded onto 10cm x 75 μm PicoFrit Capillary column packed with Magic C18 AQ reversed-phase resin. The column was developed with a 90min linear gradient of acetonitrile in 0.125% formic acid delivered at 280nL/min. MS parameters settings: MS run time 96min, MS1 Scan Range (300.0-1500.00), Top 20 MS/MS . MS/MS spectra were evaluated using SEQUEST 3G and the SORCERER 2 platform from Sage-N Research . Searches were performed against the most recent update of the NCBI 2∗2H2O, 2mM MgCl2∗6H2O, 25mM HEPES pH 7.3). Worm gonads were dissected with a 23G syringe and immediately covered with a coverslip sealed with nail polish. Acquisitions were performed immediately after (less than 5min).For FRAP-based acquisitions, worms were individually picked on an unseeded NGM plate to wash off bacteria and transferred on a glass slide in a 10 μL drop of egg buffer  and every 10sec nine times during the recovery (post-bleached 2). Intensities data of bleach, background and non-bleached nucleus regions  were extracted from the Leica software and analyzed with excel to calculate fluorescent protein mobility. The fraction of the fluorescent protein that was mobile was calculated by comparing the fluorescence intensities of non-bleached and bleached nucleus.Cell cycle arrest was scored as described in E. coli HT115 RNAi bacteria were grown in M9 minimal supplemented with glucose 0.4% (wt/vol), 2mM MgSO4, 0.1mM CaCl2, thiamine 0.01% (wt/vol) and 100 μg/ml ampicillin. A single bacteria colony streaked on LB plate was used to start the culture at 37°C under agitation until OD600nm = 1. Double-stranded RNA expression was induced by adding IPTG to a final concentration of 0.5mM for 3hrs. Bacteria culture was seeded on NGM plates complemented with ampicillin. Worms were placed on NGM plates seeded with RNAi bacteria at late L1 stage for ulp-3 RNAi and L3 stage for ned-8 RNAi. Late L4 to young adult stage worms were analyzed. We found that growing HT115 bacteria in minimal medium greatly increases RNAi efficiency even for loci reported to be problematic to achieve appropriate depletion level.We adapted the RNAi bacterial feeding method from p-3 and ned-8 RNAi, the following oligos were used to clone cDNA into the L4440 vector:ulp-3:For 5′ to 3′: TATATATAGCGGCCGCATGTCAGTTCCCCCGG5′ to 3′: TATATATAGCGGCCGCTTATTTCGCTTCAAAATTAACAATCned-8:For 5′ to 3′: AGGCGGCCGCTTAAAATCCTCCGCGG5′ to 3′: AAGGCGGCCGCATGCTCATCAAAGTTAFor ulcDNA were prepared as described in the qRT-PCR section.egl-1) were normalized with internal level of control transcript (tubulin tbg-1) relatively stably expressed between all experiments. For data analysis, maximum quantitative point method was applied and induction was calculated with the equation:Gravid adult worms were collected from the plates and washed three times to remove bacteria with PBS before introduction in Trizol and frozen at −80°C. Total RNA was extracted by Trizol/chloroform followed by RNeasy® Kit (QIAGEN) and cDNA synthesis performed on 300ng of RNA with QuantiTech® Reverse Transcription Kit (QIAGEN). qRT-PCR reactions were performed in Roche LC480 qPCR in triplicates, and cDNA was diluted in Mesa Green qPCR Master Mix (Eurogentec) with specific primers. The mean values for the levels of transcript of interest (5′-TATATATAGGCGCGCCATGTCAGTTCCCCCGGATTTCCGC (forward)5′-GAAGCGAAATAAGGCCGGCCTATATATA (reverse)Full length ulp-3 was amplified from cDNA derived from adult worms using the same protocol as used in qRT-PCR experiments section using the following oligo:6) and pMAL-c2 (MBP). Protein expression of MBP-ULP-3 and His6-ULP-3 was performed in BL21(DE3) CodonPlus bacteria grown and induced at 37°C for 2hrs with 0.5mM IPTG. MBP-ULP-3 was purified according to manufacturer standard protocols for amylose resin (New England Biolabs). His6-ULP-3 was purified on Cobalt Talon beads (Clontech) following manufacturer instructions for soluble protein and finally dialysed three times against 50mM Tris-HCl pH7.5, 150mM NaCl, 10% glycerol, 0.5mM DTT. MBP-ULP-3 was used to immunize rabbits (BioGenes GmbH). For affinity purification of the antibodies, His6-ULP-3 was covalently coupled to AffiGel-15 resin (BioRad) and used to purify final bleeds serum.AscI was introduced at the 5′ of the start codon and FseI at the 3′ of the stop codon and cloned into appropriately modified pQE-80L , the hydrophobic patch (I44A) NEDD8 mutant and all KR NEDD8 mutants were generated by site directed mutagenesis and sequences were verified by automated sequencing.RNA from cells in 6cm dishes was isolated with the Promega SV Total RNA Isolation kit. cDNA synthesis was performed using the Invitrogen SuperScript III First-Strand Synthesis SuperMix using 300ng of RNA. Real-time PCR was performed as described in Approximately, 50μg of liver tissue were homogenized by using Precellys 24 (Bertin Technologies) with 1ml of lysis buffer  supplemented with protease and phosphatase inhibitor cocktail, 20mM iodoacetamide and 20mM N-Ethylmaleimide. Lysates were centrifuged  and total protein content in the supernatant was measured by Bradford. 20μg of protein was used for SDS-PAGE and western blot analysis.All cell lines were originally obtained from the ATCC bioresourse. Cell lines were maintained in DMEM in 10% FCS and standard antibiotics, in 5% CO2 and 37°C and regularly tested for mycoplasma contamination. For siRNA transfections 5nM of oligos were transfected with RNAiMAX lipofectamine. All siRNAs are from Dharmacon as ON-TARGET-Plus pools of 4 individual oligos. For the experiment in 6-NEDD8 or His6-NEDD8 K11/48R mutant was performed as described in U2OS cells stably expressing wild-type His6-NEDD8 or His6-ubiquitin, 2 μg of Flag-L11, 3 μg of Flag-ULP-3 or NEDP1 with Fugene. 48hrs post transfection Ni2+-pull downs and total cell extracts (input 2xSDS Laemmli buffer) were prepared as described in H1299 cells in 10cm dishes were transfected with 1 μg of His6 cells/ml  and FITC-Annexin V was added at 1:100 dilution. Samples were kept at RT in the dark for 20min before 7-ADD viability dye  was added and samples were analyzed by flow cytometry. For both assays data represent the average of 3 different experiments ± SEM.U2OS cells were transfected in 6 well plates. Next day cells were trypsinised and seeded in 24well plates (7000 cells/well) in duplicates. 24hrs later cells were treated as indicated and one set of cells was used to measure cell survival and the other caspase 3/7 activity. The ratio between caspase 3/7 activity over survival was measured and represented as fold change to the control untreated cells. For Annexin V staining 1x10cm 80% confluent U2OS cells was used for each condition. Cells were collected by careful trypsinisation , washed in PBS, before resuspended in binding buffer, 1x10The U6gRNA-Cas9-2A-GFP plasmid for NEDP1 was purchased from SIGMA. 2 plasmids with different guide sequences were obtained . U2OS cells were transfected in 10cm dishes with the plasmids and 24hrs later cells were collected and GFP positive cells were selected and separated in 96-well plates as single cells by flow cytometry. Several clones were screened for NEDP1 and NEDD8 expression. Total DNA from the used clone in this study (H6) was used to amplify the NEDP1 locus by PCR and sequencing of the PCR product confirmed the deletion in NEDP1 gene (305-314bp).2) with protease inhibitor  and 10mM iodoacetamide. Cells were lysed by adding Triton X-100 at a final concentration of 0.1% for 1min at 4°C, then centrifuged for 5min at 1300 g, 4°C. The supernatant (cytoplasmic fraction) was mixed with equal volume of 2xSDS Laemmli buffer. The pellet (nuclear fraction) was washed 3x with buffer A, then resuspended with 300 μL of buffer B  and incubated 30min on ice. Lysates were centrifuged at 10800rpm for 20 s. The supernatant (nucleoplasmic fraction) was mixed with equal volume of 2xSDS Laemmli buffer. The pellet was washed 3 times with buffer B then resuspended in 600 μL of 2xSDS Laemmli buffer (pellet fraction).2x10cm dishes of 80% confluent parental or H6 U2OS cells were used for fractionation. Cells were washed and collected in 1ml PBS and 100 μL of the sample were pelleted at 13000rpm for 1min and lysed with 100 μL of 2xSDS Laemmli buffer (input). The remaining 900 μL were pelleted at 10800rpm for 20 s and resuspended in 300 μL buffer A  for 15min on ice. Lysates were cleared by centrifugation at 13000rpm for 15min at 4°C and extracts of equal amount of total protein were used for immunoprecipitations overnight at 4°C with 2 μg of APAF1 antibody and 20 μL of protein G beads (prewashed with lysis buffer). Next day beads were washed 2x with 500 μL of lysis buffer (centrifuged at 5000rpm at 4°C in each wash), resuspended in 100 μL of 2xSDS Laemmli buffer and boiled for 5min. Immunoprecipitates were analyzed by western blotting. For Flag pull downs 20 μL of pre-washed Flag beads were used.6-GST fusion before cleavage with thrombin as described in 2, 1mM DTT. Reactions were terminated with the addition of 10 μL of 2xSDS Laemmli buffer, samples were boiled for 5min, separated on a 4%–12% precast SDS-PAGE and Coomassie blue stained.Protein induction in bacteria was performed with 0.5mM IPTG at 16°C overnight and induced proteins were purified as MBP fusions using standard methods. NEDP1 was purified as His4x10cm dishes of 80% confluent cells were washed 2x with PBS before collected and lysed in 50mM Tris, pH 7.4, 100mM KCl, 1% NP-40, 2mM EDTA, protease inhibitors (Roche tablets) for 15min on ice. After centrifugation at 14000rpm for 15min at 4°C, equal amount of extracts were loaded on 10%–50% sucrose gradient. Samples were centrifuged for 24hrs at 36000rpm at 4°C in SW41 Ti swing out rotor. 1ml fractions were collected using the Brandel density gradient fractionation system with upward displacement. Proteins were TCA precipitated, pellets washed 3x with cold acetone resuspended in 100 μL of 2xSDS Laemmli buffer, boiled for 5min and samples were analyzed by western blotting. On a separate experiment samples were spiked with aldolase (158kDa) and thyroglobulin (690kDa) as migration markers for the fractionation.in vitro experiments were performed with freshly produced/purified His6-NEDD8 proteins each time, with no prior freezing. Constructs were expressed in BL21 codon plus bacteria grown and induced at 37°C for 2hrs with 0.5mM IPTG and lysed immediately in 50mM Tris-HCl pH7.5, 0.5M NaCl, 10% Glycerol, 10mM Imidazole, 0.2% Tween-20 and 5mM β-mercaptoethanol at 4°C for 30min then sonicated  and centrifuged at 20000 g for 30min at 4°C. Supernatant was incubated with 300 μL of pre-washed nickel beads for at least 4hrs at 4°C. Beads were extensively washed in lysis buffer and finally washed in 50mM Tris-HCl pH7.5, 150mM NaCl, 5mM β-mercaptoethanol. Material was then eluted in 20mM Tris-HCl pH7.5, 150mM NaCl, 0.5M Imidazole, 5mM β-mercaptoethanol. After analysis by PAGE followed by Coomassie Blue staining, fractions were dialysed in 20mM Tris-HCl pH7.5, 150mM NaCl, 0.5mM DTT and stored at 4°C for subsequent in vitro analysis.All Constructs were expressed in BL21 codon plus bacteria grown and induced at 37°C for 2hrs with 0.5mM IPTG. Cells were harvested and lysed in 50mM Tris-HCl pH7.5, 50mM NaCl, 0.5mM DTT and sonicated . After 20000 g centrifugation supernatant was incubated with 100 μL of pre-washed GST beads for at least 4hrs at 4°C. Beads were then extensively washed with 50mM Tris-HCl pH7.5, 150mM NaCl, 0.1% Triton X-100 with a final wash in 50mM Tris-HCl pH7.5, 150mM NaCl, 0.5mM DTT. Sequential elutions were performed with 50mM Glutathione pH7.5, 50mM Tris-HCl pH7.5, 150mM NaCl, 0.5mM DTT. Eluted fractions were analyzed by SDS-PAGE stained with Quick Coomassie and best fractions were pooled and dialysed against 50mM Tris-HCl pH7.5, 150mM NaCl, 0.5mM DTT. Extracts were finally cleared by 30000 g centrifugation.6-HSP70 ATPase domain  was prepared  in assay buffer  in 50 μL final volume. This construct contains the linker domain which stimulates the ATPase activity of HSP70  at 4°C for 30min then sonicated  and centrifuged at 20000 g for 30min at 4°C. Supernatant was incubated with 50 μL of pre-washed nickel beads for at least 4hrs at 4°C. Beads were extensively washed in high salt washing buffer  and finally washed three times with washing buffer without Tween-20. Material was then eluted in elution buffer  twice in 100 μL then dialysed against 100mM Tris-HCl pH7.5, 20mM KCl, 6mM MgCl2 at least 10000 fold. For subsequent ATPase activity measurements, 20 μL were incubated with or without 500ng of bacterially expressed His6-NEDP1 at 37°C for 2hrs in 100 μL final of 100mM Tris-HCl pH7.5, 20mM KCl, 6mM MgCl2. Reactions were then transferred at 4°C and 2 μM of HSP70 ATPase domain (1-402) were added and incubated for 2hrs to allow binding. Reactions were started by adding ATP at 200 μM final concentration and incubated at 37°C for 2hrs. ATPase activity was measured as described above.U2OS cell lines stably expressing His6-NEDD8, His6-HSP40 in 300 μL binding buffer  for 1hr at 4°C then 20 μL of GST beads were added to each reaction for 1hr at 4°C under gentle rotation. Beads were washed 3x with binding buffer and 50 μL of SDS loading buffer was added before boiling. Supernatants were analyzed by western blotting. In the experiment in 100nM of bacterially expressed GST or various GST-HSP70 constructs were incubated with 100nM of bacterially expressed His6-NEDD8 in 50mM Tris-HCl, pH 7.5, 2mM ATP, 5mM MgCl2, 2mM DTT. Reactions were incubated at 37°C for 5hrs. His6-NEDD8 conjugates were isolated with 10 μL of pre-washed Ni+2-agarose beads in 500 μL of 50mM Tris-HCl, pH 7.5, 0.5M NaCl for 2hrs at room temperature. Beads were extensively washed with Tris-HCl, pH 7.5, 0.5M NaCl before elution (2x100 μl) in Tris-HCl, pH 7.5, 0.5M NaCl, 250mM imidazole. Dialysis and ATPase activity measurements were performed as with the isolation of poly-NEDD8 conjugates from cell extracts (see above).50 μL reactions were set up including recombinant proteins: 300ng GST-NAE, 5 μg Ubc12 and 10 μg His2) at 4°C for 30min and sonicated  then centrifuged at 20000 g for 30min at 4°C. Supernatant was incubated with 10 μg of GST or GST-HSP70 constructs at 4°C at least 2hrs. Then 50 μL of pre-cleared (binding buffer supplemented with 20 μg of molecular grade BSA) GST beads were added and incubated for 1hr under gentle rotation. Beads were washed 3x with binding buffer supplemented with 0.5% NP-40 and 50 μL of SDS loading buffer was added before boiling. Eluates were analyzed by western blotting.Harvested cells were incubated in binding buffer , Leica LAS , SEQUEST 3G and the SORCERER 2  were used. Statistical details including the statistical tests used can be found in figure legends. Values represent the average ± SEM. P values were calculated using two-tailed unpaired Student’s t test, n values represent the number of independent experiments or number of animals used, as indicated in figure legends.ftp://massive.ucsd.edu/MSV000084214/.The SILAC proteomics data generated during this study are available at MassIVE in the following link:


Here, using a combined bioinformatics and top-down discovery proteomics approach, coupled with lipidomic analyses, PLA2 were found to be associated with release-ready cortical secretory vesicles (CV) that possess the minimal molecular machinery for docking, Ca2+ sensing and membrane fusion. Tightly coupling the molecular analyses with well-established quantitative fusion assays, we show for the first time that inhibition of a CV surface calcium independent intracellular PLA2 and a luminal secretory PLA2 significantly reduce docking/priming in the late steps of regulated exocytosis, indicating key regulatory roles in the critical step(s) preceding membrane merger.The fundamental molecular mechanism underlying the membrane merger steps of regulated exocytosis is highly conserved across cell types. Although involvement of Phospholipase A Quantification of the generated NBD-FFA and NBD-PA was done using 1-oleoyl-2-{12-[ amino Arachidonic acid 20:4 (NBD-ARA) and 1-oleoyl-2-{12-[ amino] dodecanoyl}-sn-glycero-3-phosphate (NBD-PA) as reference standards. ARA (20:4) was purchased from Matreya LLC  while all other lipids were purchased from Avanti Polar Lipids . Quantitative lipid analyses were carried out using High Performance Thin Layer Chromatography (HPTLC) ,50,51,562 antibody (PA5-27945) and anti-human mouse monoclonal sPLA2 antibody (ab-24498) were from Invitrogen and Abcam , respectively. Blocking agents―non-fat dry milk powder and BSA―were from Devondale  and Sigma Aldrich, respectively. HRP linked anti-mouse IgG (NA93IV) and anti-rabbit IgG (NA934VS) antibodies came from GE Healthcare . Lumunata Cresendo Western HRP substrate was purchased from Merck Millipore. The blots and gels were imaged using the Image Quant™ LAS 4000 Biomolecular Imager .Amicon Ultra-4 centrifugal filters (3kDa cut-off) came from Merck Millipore. Constituents of the Protease inhibitor cocktail ,57 were 2 activities in intact unlabelled CSC suspensions  were assessed by measuring changes in FFA following treatments with the indicated concentrations of BEL, LY311727 and FKGK-11; exploratory experiments (n = 1–2) with 40 µM BEL, 10 µM FKGK-11, 100 nM LY311727 and higher concentrations were first carried out. Only those concentrations that significantly reduced endogenous FFA relative to control were selected for further study. CSC were isolated according to established protocols free in quadruplicate, consistent with established protocol and this was repeated in separate experiments as indicated (n) [2+]free were measured with a Ca2+ sensitive electrode  as previously described free undergo exocytosis in vitro yielding a classic sigmoidal Ca2+ activity curve with an EC50 of 38.9 ± 6.4 µM [Ca2+]free (n = 14) [n = 3–4). On treating CSC with 100 µM and 500 µM BEL [2+ sensitivity to an EC50 of 230.6 ± 101.5 µM [Ca2+]free (n = 3). CSC treated with 50 µM and 200 µM FKGK-11, another iPLA2 inhibitor free ; n = 14 (n = 14) ,52,69,70(n = 14) ,68 signi0 µM BEL ,64,71,72a2+]free ; n = 3. nhibitor , also si2 isozyme on CSC and their potential role in regulating late steps of regulated exocytosis, we sought to identify these isozymes, beginning with sequence alignment to identify regions of homology in putative urchin PLA2 and characterized human and murine forms. The sequence alignment of sPLA2 and iPLA2 in the selected species clearly indicated conserved amino acid residues throughout the primary amino acid sequences, consistent with a high overall conservation from urchin to human ; this region also carries highly conserved GXSXG and GGGVKG catalytic and nucleotide binding consensus sequences . The NBD-PE in controls after 10 min incubation was 12.3 ± 0.9 picomoles which was significantly reduced to 9.0 ± 1.2, 7.4 ± 1.2, 6.8 ± 1.0 and 3.8 ± 2.3 picomoles following 30 s, 2 min, 5 min and 10 min incubations, respectively . Notably, a very large increase in NBD-FFA in CV aliquots that were pre-incubated with 104.0 µM [Ca2+]free confirmed Ca2+ promoted CV luminal PLA2 activity (n = 3). In contrast, 18.4 ± 2.0 picomoles NBD-PC was detected in buffer controls and no significant change was detected even after 10 min incubation with the CV luminal protein . Thus, a decrease in NBD-PE and parallel increase in NBD-FFA over time and a large increase in the generation of NBD-FFA in the presence of higher [Ca2+]free confirmed the presence of a Ca2+ dependent catalytically active sPLA2 in the isolated CV luminal fraction, which is selective for PE over PC.To further and more specifically localize the PLAinterval A; n = 3.ectively A. The geactivity B, n = 3. protein A. This c2 was also investigated by incubating isolated CV membrane fragments with NBD-PE or -PC (n = 2–3). Buffer without CV membrane was used as the control to which the indicated amount of NBD-PC or -PE was added and incubated for 10 min. In all incubations, no significant change in the NBD-labelled lipids nor generation of NBD-FFA was observed. Therefore, to confirm whether the apparent membrane associated iPLA2 was lost or became inactive during CV membrane isolation, intact CV were assayed for PLA2 activity following trypsin treatment and loss of iPLA2 was confirmed by western blotting. Loss of the ~63 kDa iPLA2 protein band following trypsin treatment confirmed the presence of iPLA2 on the CV surface . Absorbance measurements of parallel samples confirmed that there was no major loss of CV over the course of these incubations (not shown).The substrate preference of the ~63kDa CV membrane iPLAad group ,80. The rn blots C; n = 3.2 from the external membrane of intact CV following trypsin treatment and the corresponding loss of activity assessed using the selective PED6 substrate (n = 2–3). In the standard assay, there was no significant change in the fusion extent although Ca2+ sensitivity appeared right-shifted following trypsin treatment; however, the shift was not significant relative to the untreated controls. Notably, in the settle assay used to assess docking and priming free (n = 19), which was translationally invariant with CV-PM fusion (n = 14) free and 79.8 ± 7.5 µM [Ca2+]free was also observed following treatment with 50 µM and 200 µM FKGK-11.As in previous work, to enable a tight focus on the late steps of Caa2+]free A; n = 19M fusion ; n = 14 (n = 14) ,45,49,512 isozymes in promoting and maintaining CV priming and/or docking . FFA levels decreased to 12.9 ± 1.3 and 8.9 ± 3.5 fmoles/μg MP in CV treated with 20 µM and 200 µM LY311727, respectively. Additionally, a significant increase in the endogenous PE levels in the CV treated with 200 µM LY311727  and increased TAG levels to 376.7 ± 22.5 picomoles/μg MP, relative to the basal level of 217.2 ± 40.9 picomoles in the control .When tested in the settle assay, CV treated with all doses of the inhibitors showed further decreases in fusion extent. These concentration-dependent decreases in fusion were to 89.9 ± 4.6% and 53.5 ± 13.4% in 20 µM and 200 µM LY311727 treated CV, respectively; 91.3 ± 4.6% and 87.6 ± 6.6% in 100 µM and 500 µM BEL treated CV, respectively; and 77.0 ± 16.0% and 58.9 ± 10.9% in 50 µM and 200 µM FKGK-11 treated CV, respectively, confirming the role(s) of PLA docking B. To cones/μg MP ; n = 3–4LY311727 , confirm2—a sPLA2 in the CV lumen and an iPLA2 on the CV surface—and also demonstrated that their activities play a role in promoting and maintaining the docked and/or primed state of secretory vesicles. The results are consistent with previous findings in platelets that PLA2 activity is not essential for fusion per se but has clear modulatory roles late in the exocytotic pathway /[aqueous] . Hence,kylation  and the kylation . Therefo2 activity produces both negative and positive curvature lipids—LPC and FFA, respectively—which are known to inhibit or promote fusion depending on their site of incorporation free is ~1–10 µM, occurring as transients, linked in part to the opening of p-type Ca2+ channels [2+]free at the CV membrane would be much higher. This is also consistent with the estimates of luminal [Ca2+]free in chromaffin granules under basal conditions [2+ transients in vivo, the activity of localized luminal sPLA2 would increase, preferentially hydrolysing PE on the inner CV monolayer  membrane, near docking/fusion sites, would block spontaneous merger of the vesicle and PM due to high local levels of LPC at membrane contact sites, in particular considering the substantially higher positive intrinsic curvature of LPC relative to LPE [2+-triggered fusion mechanism [2 activity  of the CV luminal sPLA2 and outer membrane bound iPLA2 in maintaining the docked, fusion-ready state of vesicles on the PM is evident as both interventions used here―pharmacological inhibition of CV luminal sPLA2 and membrane associated iPLA2 and proteolytic removal of iPLA2―significantly impaired priming and/or docking.According to the Stalk-pore hypothesis, the fusion of two distinct membrane bilayers proceeds through sequential steps resulting in the localized mixing of proximal monolayers followed by fusion of the distal monolayers ,95,96,97poration ,94,95,98vesicles ,100,101;channels . As thesonolayer A,B , at echanism , in whicactivity . In thisturation ,106,107.vesicles ,108,109.V fusion A,B; whet2 isozymes―a CV luminal sPLA2 and an iPLA2 associated with the outer CV membrane; and (ii) that proteolytic removal of CV surface proteins including iPLA2 or the pharmacological inhibition of both the isozymes significantly reduces priming and/or docking. Based on this as well as previously measured vesicular Ca2+ transients, basal Ca2+ dependent CV luminal sPLA2 activity and CV membrane attached iPLA2 would appear to act together to support efficient vesicle priming and/or docking. While intriguing, this warrants further study, particularly relating to the apparent effects of FFA on the formation of SNARE complexes and/or in modulating membrane biophysical properties, as FFA is also known to induce the transition from the bilayer to the hexagonal phase upon its incorporation in the membrane [The data confirm (i) the presence of endogenous, catalytically active CV associated PLAmembrane ,107,110.


Glycosaminoglycans (GAGs) are known to play pivotal roles in physiological processes and pathological conditions. To study interactions of GAGs with proteins, immobilization of GAGs is often required. Current methodologies for immobilization involve modification of GAGs and/or surfaces, which can be time-consuming and may involve specialized equipment. Here, we use an efficient and low-cost method to immobilize GAGs without any  modification using highly concentrated salt solutions. A number of salts from the Hofmeister series were probed for their capacity to immobilize heparin and chondroitin-6-sulfate on microtiter plates applying single chain antibodies against GAGs for detection (ELISA). From all salts tested, the cosmotropic salt ammonium sulfate was most efficient, especially at high concentrations (80–100% (v/v) saturation). Immobilized GAGs were bioavailable as judged by their binding of FGF2 and VEGF, and by their susceptibility towards GAG lyases . Using 80% (v/v) saturated ammonium sulfate, block and continuous gradients of heparin were established and a gradient of FGF2 was created using a heparin block gradient as a template. In conclusion, high concentrations of ammonium sulfate are effective for immobilization of GAGs and for the establishment of gradients of both GAGs and GAG-binding molecules, which enables the study to the biological roles of GAGs.The online version of this article (10.1007/s10719-019-09872-4) contains supplementary material, which is available to authorized users. Glycosaminoglycans (GAGs) are an important class of biomolecules, consisting of repeating disaccharide units. The highly negatively charged GAGs are ubiquitously present on cell surfaces and within the extracellular matrix (ECM), where they interact with a variety of structural and signaling proteins, including collagens, growth factors and chemokines . ThroughThe mechanisms underlying the biological roles of GAGs, including their interactions with biomolecules and cells, largely remain to be elucidated. To be able to study these interactions, GAGs need to be immobilized on surfaces, for example on a microtiter plate to perform an enzyme-linked immunosorbent assay (ELISA). Due to their negative charges GAGs poorly bind to apolar surfaces like polystyrene microtiter plates. A variety of GAG immobilization methods have been developed by modifying GAGs themselves –14, and/We previously reported on the use of high salt solutions for linking negatively charged macrobiomolecules, including glycosaminoglycans, to various surfaces  , 21. Thi4)2SO4), ammonium chloride (NH4Cl), sodium sulfate (Na2SO4), magnesium sulfate (MgSO4), sodium chloride (NaCl), lithium chloride (LiCl), magnesium chloride (MgCl2), magnesium sulfate (MgSO4), magnesium acetate (Mg(Ac)2), potassium chloride (KCl), guanidinium chloride (GuHCl), calcium acetate (CaAc) and bovine serum fraction V (BSA) were purchased from Merck . Calcium sulfate (CaSO4), calcium chloride (CaCl2), Tween-20® and diethanolamine were provided by Sigma-Aldrich . p-Nitrophenyl phosphate disodium salt was obtained from MP Biomedicals . UltraPure® Tris was received from Invitrogen™ . Heparin sodium was from Organon , heparan sulfate (HS) sodium salt from bovine kidney and chondroitin-6-sulfate (CSC) sodium salt from shark cartilage from Sigma-Aldrich and dermatan sulfate (DS) sodium salt from Celsus laboratories, Inc. . Heparinase I, II and II were provided by IBEX Technologies, Inc.  and chondroitinase ABC by Sigma-Aldrich. Recombinant human VEGF 165 protein was from R&D systems . Recombinant rat FGF2 was produced in Escherichia coli using a FGF2 expression construct cloned in the prokaryotic vector pQE16, which was a gift from the Department of Pathology, Radboud university medical center, Nijmegen, The Netherlands [Ammonium sulfate 2SO4, NH4Cl, Na2SO4, MgSO4, NaCl, LiCl, MgCl2, MgSO4, KCl, GuHCl, CaSO4, CaCl2 and MilliQ water. Incubation was overnight on a wet cloth to minimize fringe effects. The plates were rinsed six times with PBS containing 0.1% (v/v) Tween-20® (PBST). After blocking with 1% (w/v) BSA in PBST for 60 min, the scFv antibodies HS4C3V and IO3H10 in 1% (w/v) BSA in PBST were allowed to bind to the coated heparin and CSC respectively for 60 min. Subsequently, the plates were incubated with mouse anti-VSV antibody P5D4 in 1% (w/v) BSA in PBST and anti-mouse IgG-AP in 1% (w/v) BSA in PBST for 60 min. The bound AP conjugated antibody was detected by addition of 100 μl to each well of 1 mg p-nitrophenyl phosphate/ml in 1 M diethanolamine with 0.5 mM MgCl2, pH 9.8. The conversion of p-nitrophenyl phosphate into of p-nitrophenol by AP produces a yellow color, which was measured at 405 nm.The optimal saturated salt solution to coat GAG to 96-wells microtiter plates was determined using an indirect ELISA. First, microtiter plates were incubated with various amounts of heparin or CSC (500–0 ng/well) dissolved in 80% (v/v) saturated solutions of various salts i.e. (NH4)2SO4 using the same method as described above and compared to NaCl as a less effective salt. The assessed degrees of saturation used were 100% (v/v), 90% (v/v), 80% (v/v), 70% (v/v), 60% (v/v), 50% (v/v) and 25% (v/v).The optimal degree of salt saturation (v/v) to immobilize heparin, CSC and HS was determined for (NH4)2SO4 solution, which was added to each plate as an internal reference.Each condition was performed in duplicate and the experiment was repeated three times (n = 3) for heparin and CSC and four times (n = 4) for HS. Data were normalized against the absorbance value at 405 nm observed for 500 ng GAGs in 80% (v/v) saturated (NH4)2SO4 solution overnight. The plates were rinsed six times with PBST after each incubation. After blocking the plate with 1% (w/v) BSA in PBST for 90 min, the GAG-coated wells were either incubated with a combination of heparinase I, II and III (0.02 IU of each enzyme in 1 ml 100 mM NaAc with 0.2 mM CaCl2 pH 7.2), with chondroitinase ABC (0.02 IU in 1 ml 25 mM Tris and 2 mM Mg(Ac)2 pH 8), or with digestion buffer only, overnight at 37 °C. Subsequently, the remaining GAGs were detected with the scFv antibodies HS4C3V, IO3H10 and GD3A12 using the indirect ELISA method described above. Each condition was performed in duplicate and the experiment was repeated three times (n = 3).A 96-wells microtiter plate was incubated with 1 μg/well of heparin, HS, CSC or DS in 80% (v/v) saturated (NH4)2SO4 overnight. The plates were rinsed six times with PBST after each incubation. After blocking with 1% (w/v) BSA in PBST for 60 min, the GAG-coated microtiter plates coated were incubated with 25 ng/well of either rat FGF2 or human VEGF in 1% (w/v) BSA in PBST for 60 min. The bound growth factors were detected with rabbit anti-fibroblast growth factor-basic (1–24) and goat anti-human VEGF 165 antibody and subsequently rabbit anti-goat and goat anti-rabbit IgG-AP using the indirect ELISA method described above. Each condition was performed in duplicate and the experiment was repeated three times (n = 3).Microtiter plates were incubated with 500 ng/well of heparin, HS, CSC or DS in 80% (v/v) saturated (NH4)2SO4 each hour with an additional 100 μl of 80% (v/v) saturated (NH4)2SO4. To create a FGF2 gradient, a heparin block gradient was incubated with 400 μl of 200 ng/ml FGF2 in 0.4% (w/v) BSA in PBS for 60 min. The plate was rinsed six times with PBST, blocked with 1% (w/v) BSA in PBST for 60 min and incubated with anti-fibroblast growth factor-basic (1–24) antibody and goat anti rabbit IgG conjugated IRDye 800CW, 60 min each, and rinsing with PBST after each incubation. To create a continuous gradient of heparin, 2 μg of heparin in 100 μl of 80% (v/v) saturated (NH4)2SO4 was placed at one side of a well of a 24-wells cell suspension plate. The heparin was immediately diluted with 80% (v/v) saturated (NH4)2SO4 solution at a rate of 0.1 ml/h using a pump system . After the whole surface of the well was covered with (NH4)2SO4, the plates were washed six times with PBST and blocked with 1% (w/v) BSA in PBST 60 min. The gradient of heparin was visualized by successive incubations with HS4C3V, P5D4 and goat anti mouse IgG conjugated with IRDye 800CW in 1% (w/v) BSA in PBST for 60 min each and rinsing with PBST after each incubation. The IRDye staining was visualized using the Odyssey CLx imaging system, which was further processed and analyzed with Fiji 1.51n software.A block gradient of heparin was created in a 24-wells Greiner CELLSTAR® cell suspension plate by diluting 2 μg of heparin in 100 μl of 80% (v/v) saturated (NH4)2SO4 was placed inside the mold and incubated overnight. After removing the mold and rinsing six times with PBST, the plate was blocked with 1% BSA in PBST. The pattern of immobilized heparin was visualized using the antibodies HS4C3V, P5D4 and goat anti mouse IgG conjugated with IRDye 800CW in 1% (w/v) BSA in PBST. The reverse pattern was created by performing the same experiment with CSC placed on the outside of the mold in a new well of the 24-wells plate. The reverse clover pattern of CSC was detected with IO3H10, polyclonal rabbit anti-VSV-G antibody and goat anti rabbit IgG conjugated with IRDye 680CW in 1% (w/v) BSA in PBST. The IRDye staining was visualized using the Odyssey CLx imaging system, which was further processed and analyzed with Fiji 1.51n.A clover-shaped mold was designed with Autodesk® Tinkercad® and 3D printed with polylactic acid using an adapted Anet® A8 3D printer  Fig. . The mol4)2SO4) was superior in immobilizing heparin and CSC 2SO4 was dose-dependent. Increasing the concentration of heparin in the coating solution resulted in a near linear increase of immobilized heparin, whereas for CSC an initial steep increase was observed followed by a more gradual increase towards a maximum value. Use of other (cosmotropic) salts resulted in only minor amounts of GAGs immobilized at the given GAG concentration range. Only at the highest amount of CSC (500 ng/well), 80% (v/v) saturated magnesium sulfate (MgSO4) gave a positive signal.Heparin and chondroitin-6-sulfate (CSC) were immobilized on polystyrene plates using 80% (v/v) saturated solutions of various salts applying 0–500 ng GAG/well, and immobilization was assessed by an indirect ELISA assay using single chain antibodies against heparin and CSC . In comparison with other salts of the Hofmeister series ammonium sulfate 2SO4 as the most efficient salt for the immobilization of GAGs, the optimum salt saturation percentage was assessed. Heparin, CSC and heparan sulfate (HS) were immobilized using various saturation percentages of (NH4)2SO4 and of sodium chloride (NaCl) as a less effective salt. The 100, 90, 80 and 70% (v/v) saturated solutions of (NH4)2SO4 were more efficient in immobilizing heparin compared to solutions of with lesser degrees of saturation and all solutions of NaCl 2SO4 of 50, 60, 70, 80, 90 and 100% (v/v) saturation were most efficient, where none of the NaCl solutions showed more than 20% of the maximum binding using (NH4)2SO4 2SO4, while for CSC and HS 2SO4 solution for immobilization of all GAGs.Having identified  were incubated with chondroitinase ABC for this reason. After digestion, a major reduction in immobilized GAGs was observed Fig. , indicatTo evaluate the capacity of the immobilized GAGs to bind growth factors, FGF2 and VEGF were allowed to bind to immobilized heparin, HS, CSC and DS. All immobilized GAGs were able to bind FGF2 and VEGF, with heparin resulting in the highest amounts of detected growth factors Fig. . Further4)2SO4 at one side of the well in a 24-wells plate followed by a stepwise dilution the heparin stock at the same position with the immobilization solution over time 2SO4 followed by 1-h incubation. The hydrophobicity of the well and surface tension causes the heparin solution to maintain its droplet shape during al dilution steps. This expanding droplet pattern is clearly visible when visualizing the obtained block gradient of heparin with immunohistochemical IRDye staining, with each consecutive “block” with decreasing intensity of the IRDye representing one dilution step. Using the heparin block gradient as a template, a gradient of FGF2 was obtained by incubation with FGF2 in the presence of a low amount of BSA to prevent aspecific binding to the plate 2SO4 was pipetted to one side of a well and continuously diluted with the immobilization solution from the same starting position using a pump system  saturated 2SO4 was pipetted inside the mold, while CSC in 80% (v/v) saturated (NH4)2SO4 was pipetted in another well on the outside of the mold. In this manner, a clover pattern of heparin and a reverse clover pattern of CSC were obtained as visualized by immunohistochemistry 2SO4, with both ions at the cosmotropic end of the Hofmeister series, was the most efficient salt. Vlachy et al. proposed a Hofmeister-like ordering of anionic headgroups and their interaction with cations, suggesting that ammonium will form closer ion pairs with sulfate compared with other cations e.g. sodium [4)2SO4 has a higher GAG immobilization efficiency even compared to salts with a higher molarity at saturation, such as LiCl, which would mean a larger number of effective anions and cations to immobilize the GAGs


A subset of Rab GTPases have been implicated in cilium formation in cultured mammalian cells , 4, 5, 6  •Inturned and Fuzzy are tri-longin domain proteins•Inturned and Fuzzy form a Rab23 GEF complex•Rab23 GEF localizes to cilia and promotes cilium formation•DrosophilaRab23 acts downstream of Inturned and Fuzzy to regulate planar polarity in  Gerondopoulos et al. show that the CPLANE components Inturned and Fuzzy form a conserved Rab23 GEF involved in the establishment of planar polarity and in cilium formation. Inturned and Fuzzy each contain 3 longin domains, with the first pair required for Rab23 GEF activity. Phylogenetic analysis shows that Rab23 is a member of the Rab7/32/38 family of Rabs acting in lysosome and lysosome-related organelle trafficking . Rab7 faThis pattern of homology indicates that Intu-Fuz are likely to form a functional pair, equivalent to Mon1-Ccz1 and Hps1-Hps4. Interaction mapping revealed that Intu and Fuz form a binary complex C and S1Ccat/KM ∼6 × 103 M−1s−1  are thought to act in a complex with the WD40-repeat protein Fritz (Frtz) (Wdpcp in vertebrates). In, Fy, and Frtz localize to proximal cell ends, where they regulate the phosphorylation and localization of the atypical formin multiple wing hairs (Mwh). Mwh then inhibits actin polymerization and restricts production of actin-rich trichomes to distal cell edges  GEFs to distinguish them from complexes such as the Rab1 GEF TRAPP, which have a dimeric longin domain catalytic site or single longin domain DENN-family Rab GEFs  bovine calf serum (Thermo Fisher Scientific) supplemented with 2.5 mM Glutamax™, and 1.2g/l sodium bicarbonate. For passaging, cells were washed in PBS, and then removed from the dish by incubation with TripLE Express (Thermo Fisher Scientific).HEK293T cells were cultured at 37°C and 5% COBL21(DE3) were grown in LB and induced at 37°C overnight for expression of recombinant Rab proteins.Drosophila melanogaster flies were grown on standard cornmeal/agar/molasses media at 18°C or 25°C, in plastic vials in a controlled humidity environment, on a 12 hr/12 hr light-dark cycle. For pupal wing dissections, pupae were aged for 28 hr after puparium formation (APF) at 25°C, or for 32 hr APF for trichome staining.fy2 and frtz33 are null mutations and Rab23T69A carries a point mutation in the GTPase domain of Rab23. RNAi lines Rab23GD13145 and Rab23GD13147 were obtained from the Vienna Drosophila Resource Centre (VDRC). EYFP-Rab23 is a knock-in of EYFP into the endogenous locus of Rab23 -GDP  in a total volume of 200μl for 20min at 4°C. For standard GDP-releasing GEF assays 100μl of the loading reaction was mixed with 10μl 10mM Mg-GTP, 10-100nM GEF or a buffer control, and adjusted with assay buffer to 120μl final volume. The GEF reaction occurred for 20min at 30°C. After this, 2.5μl were taken for a specific activity measurement, the remainder was split into two tubes, then incubated with 500μl ice-cold assay buffer containing 1mM MgCl2, and 20μl packed glutathione-Sepharose for 60min at 4°C. After 3 washes with 500μl ice-cold assay buffer the Sepharose was transferred to a vial containing 4ml scintillation fluid and counted. The amount of nucleotide exchange was calculated in pmoles GDP-released. For GTP-binding assays the following modifications were made: only unlabelled GDP was used in the loading reaction; in the GEF reaction 0.5μl 10mM GTP and 1μCi [35S]-GTPγS  were used. The amount of nucleotide exchange was calculated in pmoles GTP-bound.Nucleotide binding and endpoint assays for GEF activity were carried out as follows , 23. FirO-(N-methylanthraniloyl)-GDP (Mant-GDP) (Jena Bioscience) in 20mM HEPES, pH 6.8, 1mg/ml BSA (protease and Ig-G free), 20mM EDTA, pH8.0, 40mM Mant-GDP at 30°C for 30min. After loading, 25mmol MgCl2 was added and the sample was exchanged into reaction buffer , 150mM NaCl, 1mM MgCl2) using Zeba spin columns (Thermo Scientific). Nucleotide exchange was measured using 1nmol of the loaded Rab and the amount of GEF specified in the figure legends in a final volume of 100μl reaction buffer by monitoring the quenching of fluorescence after release of Mant-GDP using a Tristar LB 941 plate reader (Berthold Technologies) under control of MikroWin Software. Samples were excited at 350nm and emission monitored at 440nm. GTP was added to a final concentration of 0.1mM to start the exchange reaction at 30°C. Curve fitting and extraction of pseudo first order rate constants (kobs) was carried out using Microsoft Excel [obs = (kcat/Km)x[GEF]+kbasal where kbasal is the rate constant measured in the absence of GEF, catalytic efficiency (kcat/Km) can be obtained.For analysis of Rab GEF kinetics, 10nmol of hexahistidine-GST-Rab23 was loaded with 2′-(3′)-bis-2O, 10μl 10 mM EDTA, pH 8.0, 5μl of 1 mM GTP, 2μl γ-[32P]GTP , and 100pmol Rab protein were mixed on ice. A 2.5 μL aliquot of the assay mix was scintillation counted to measure the specific activity in cpm/pmol GTP. Reactions were then incubated at 30°C for 60min. The 5 μL aliquots were immediately added to 795μl of ice-cold 5% [wt/vol] activated charcoal slurry in 50mM NaH2PO4, left for 1h on ice, and centrifuged at 16,100 g in a benchtop microfuge  to pellet the charcoal. A 400μl aliquot of the supernatant was scintillation counted, and the amount of GTP hydrolysed was calculated from the specific activity of the reaction mixture.For Rab-loading reactions, 10μl of assay buffer, 73μl HCells were grown on No. 1.5 glass coverslips in 6-well plates. IMCD3 cells were plated at 30,000 cells per well and transfected with siRNA duplexes for 3 days. hTERT-RPE1 were plated at 20,000 cells per well and transfected with siRNA duplexes for 2 days or DNA for 1 day. To promote cilium formation, medium was replaced with growth medium lacking serum for 14h for IMCD3 or 48h for hTERT-RPE1 cells. For plasmid and siRNA transfection, Mirus TransIT-X2 (Mirus Bio LLC) and Oligofectamine (Invitrogen), respectively, were used according to the manufacturers’ instructions.After the treatments described in the figure legends, cells were washed twice with 2ml of PBS at room temperature prior to fixation with either trichloracetic acid (TCA) or paraformaldehyde (PFA). For TCA-glycine fixation, cells were incubated in 2ml 10% [wt/vol] TCA at 4°C for 15min, and then washed five times in 2ml 30mM glycine, PBS pH7.4. For PFA fixation, cells were incubated in 2ml 3% [wt/vol] PFA in PBS for 15min, and then washed with 2ml 50mM ammonium chloride in PBS for 10min. Following fixation, all cells were permeabilised for 7min with 0.2% [vol/vol] Triton X-100 in PBS, followed by three washes in PBS. In all cases primary and secondary antibody staining was carried out in PBS for 60min at room temperature. Affinity purified antibodies were used at 1μg/ml while commercial antibodies were used as directed by the manufacturers. DAPI was added to the secondary antibody staining solution at 0.3μg/ml to stain DNA. Coverslips were mounted in Moviol 4-88 mounting medium . Fixed samples on glass slides were imaged using a 60x NA1.35 oil immersion objective on an Olympus BX61 upright microscope with filtersets for DAPI, GFP/Alexa 488, Alexa-555, Alexa-568, and Alexa-647 (Chroma Technology Corp.), a CoolSNAP HQ2 camera (Roper Scientific), and Metamorph 7.5 imaging software (Molecular Dynamics Inc.). A Lumen 200 Watt metal halide light source (Prior Scientific Instruments Ltd) was used to illuminate the samples. Image stacks of up to 4 planes with a spacing of 0.3μm through the cell volume were taken. Image stacks were maximum intensity projected and then merged to create 24-bit RGB TIFF files in Metamorph. Images in 24-bit RGB format were then cropped in Photoshop CS3 and placed into Illustrator CS3 (Adobe Systems Inc.) to produce the figures.For electron microscopy, hTERT-RPE1 cells were grown in 2cm dishes and treated as described in the legend to fy mutants are cold sensitive, pupae were raised at 18°C for 64.5 hr for Actin labeling. Pupae expressing RNAi against Rab23 under GAL4-UAS control were raised at 29°C to enhance the expression of dsRNA. Briefly, pupae were removed from their pupal case and fixed for 25-35 min in 4% [wt/vol] paraformaldehyde in PBS, depending on antibody combinations. Wings were then dissected and the outer cuticle removed, and were blocked for 1 hr in PBS containing 0.2% Triton X-100 (PTX) and 10% normal goat serum. Primary and secondary antibodies were incubated overnight at 4°C in PTX with 10% normal goat serum, and all washes were in PTX. Antibodies against Mwh (rat or rabbit) and Frtz (rabbit) have been described previously  paraformaldehyde in PBS for 30 min. Wings were mounted in 25 μl of PBS containing 10% [vol/vol] glycerol and 2.5% DABCO, pH7.5.Pupal wings were imaged on a Leica SP1 confocal microscope using a 40x NA1.4 apochromatic lens or a Nikon A1R GaAsP confocal microscope using a 60x NA1.4 apochromatic lens. 9 Z-slices separated by 150 nm were imaged at a pixel size of 70-100 nm, and the 3 brightest slices around apicolateral junctions were selected and averaged for each channel in ImageJ.Adult wings were dehydrated in isopropanol and mounted in GMM , and incubated overnight on a 60°C hot plate to clear. Wings were photographed at 20x magnification.For pupal wing westerns, female larvae were raised at 19°C, and then shifted to 29°C for 27 hr APF before dissection of pupal wings directly into sample buffer. One pupal wing equivalent was used per lane.Details of the number of experimental repeats, numbers of cells analyzed and the relevant statistics are detailed in the figure legends. Data were plotted and statistical analysis performed using GraphPad Prism software.Nucleotide exchange and Rab GAP activity was measured in duplicate in 3 independent experiments. Mean values were plotted in bar graphs and error bars indicate the SEM. No statistical tests were performed.obs was obtained by fitting an exponential decay curve to the individual curves in Microsoft Excel. The mean kobs value obtained was then plotted as a function of Rab GEF or GEF subunit concentration in GraphPad Prism. No statistical tests were performed.Nucleotide exchange over time was measured as a function of Rab GEF or GEF subunit concentration in 3 independent experiments. kFor cilium length, the freehand measuring tool of FIJI (ImageJ) was used to measure the length of individual cilia for 100 cells in each of 3 independent experiments. Acetylated tubulin and Arl13b were used as markers for this purpose. GraphPad Prism was used to calculate the mean length and SEM in μm for each experimental condition. These values were plotted in bar graphs.To quantify the frequency of cilium formation, the number of elongated cilia and punctate ciliary vesicles were identified and counted using Arl13b staining as a marker for 100 cells in each of 3 independent experiments. GraphPad Prism was used to calculate the mean frequency and SEM for each experimental condition. These values were plotted in bar graphs. No statistical tests were performed.This study did not generate or analyze datasets or code.