This phenotype is encountered in other conditions, defining Prader-Willi-like syndrome (PWLS).Prader-Willi syndrome dn. As far as we know, this is the first diagnosed case of PWLS associated with a complex sSMC, involving a 30.9 Mb gain in the 6q16.3q23.3 region and a 3.5 Mb gain in the 10p11.21p11.1 region. Several genes have been mapped to the 6q region including the TCBA1 gene, which is associated with developmental delay and recurrent infections, the ENPP1 gene, associated with insulin resistance and susceptibility to obesity and the BMIQ3 gene, associated with body mass index (BMI). No OMIM gene was found in the smallest 10p11.21p11.1 region.We report a 14-year-old boy with a complex small supernumerary marker chromosome (sSMC) associated with PWLS. The propositus presents clinical features commonly found in patients with PWLS, including growth hormone deficit. Banding karyotype analysis and fluorescence in situ hybridization (FISH) revealed a marker derived from chromosome 6 and a neocentromere as suspected, but array-CGH enabled us to characterize this marker as a der(10)t(6qter → 6q23.3::10p11.1 → 10p11.21)We suggest that the duplicated chromosome segment 6q16.3q23.3 may be responsible for the phenotype of our case and may also be a candidate locus of PWLS. SIM1), and 10q26 /46,XY[17].ish neo(6)dn. Array-CGH analysis confirmed the duplicated region of chromosome 6 ×2 ~ 3,10p11.21p11.1×2 ~ 3. Array-CGH mosaicism of chromosome 10 and chromosome 6 was estimated at about 25.0 % (log2 ratio: 0.14). The mosaicism rate found with array-CGH analysis was similar to that found with karyotyping. Maternal uniparental disomy of chromosome 15 was ruled out (data not shown).Conventional cytogenetic analysis on cultured T-lymphocytes revealed a male karyotype with a small supernumerary marker chromosome in 32 % of the cells analysed: mos 47, XY,+mar[8]/46,XY[17]  are a subgroup of sSMC derived from more than one chromosome  has collhttp://decipher.sanger.ac.uk and via email from decipher@sanger.ac.uk.) with the phenotype: case 4145 (7 Mb duplication) with brachycephaly, epicanthus, intellectual disability, intrauterine growth retardation, muscular hypotonia and ptosis; case 2068365 (2.3 Mb duplication) with hydrocephalus, macrocephaly and parietal foramina. Three cases with 6q21q23 duplications were described only at the cytogenetic level and associated with developmental delay, congenital hearts defects, depressed nasal bridge and epicanthal folds [To investigate whether the duplicated 6q16.3q23.3 and 10p11.21p11.1 regions could play a role in PWL syndrome, all cases with comparable chromosome 6q and 10p duplications documented in PubMed, the sSMC Liehr database  and DECIal folds , 16 and al folds . Regardial folds , 19, butal folds , 20, 21.al folds . In our n = 14), the second potentially relevant (n = 17) and the third probably benign (n = 29). The second group with potentially clinically relevant CNVs  contained genes of particular interest involved in insulin or adiponectin receptor and peroxisomal acid metabolism: SOC6, PLIN2, CDH13, CNTNAP2, SOX3 and ACOXL.A total of 60 CNVs have been found in 100 patients with syndromic obesity in a recent study  using arTCBA1, BMIQ3 and ENPP1 genes. Two balanced translocations showed that the TCBA1  gene was truncated in 1 patient with a severe neurological phenotype , and a second patient with developmental delay and recurrent infections [TCBA1 gene causes developmental delay and a distinct phenotype. BMIQ3 was linked with body mass index (BMI) using a genome-wide linkage analysis [ENPP1 was associated with insulin resistance and susceptibility to obesity [SIM1) at 6q16.2 [POU3F2 (BRN2) at 6q16.1 [SIM1 and POU3F2 genes were not included in the duplicated 6q16.3q23.2 region of our patient.The OMIM gene map search  of the dfections , 25. Theanalysis  using po obesity . Moreove obesity , 29 and t 6q16.2  and/or PTCBA1 as the candidate gene for mental retardation and BMIQ3 and ENPP1 implicated in the susceptibility to obesity. This case illustrates the importance of array-CGH to characterize more precisely sSMC not elucidated by FISH, and suggests that all neocentromere markers detected by karyotype must by analysed by array-CGH.According to these data and a review of the literature, our patient’s phenotype seems to be related to the euchromatin of chromosome 6 rather than chromosome 10. The duplicated 6q16.3q23.3 may be a candidate locus of PWLS with Written informed consent was obtained from the patient’s parents for publication and accompanying images of this case report. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


Protein kinases are attractive drug targets for numerous human diseases including cancers, diabetes and neurodegeneration. A number of kinase inhibitors that covalently target a cysteine residue in their target kinases have recently entered use in the cancer clinic. Despite the advantages of covalent kinases inhibitors, their inherent reactivity can lead to non-specific binding to other cellular proteins and cause off- target effects in cells. It is thus essential to determine the identity of these off targets in order to fully account for the phenotype and to improve the selectivity and efficacy of covalent inhibitors. Herein we present a detailed protocol for a chemoproteomic method to enrich and identify cellular targets of covalent kinase inhibitors. Protein kinases are a large family of enzymes that transfer the γ-phosphate group of ATP to the tyrosine, serine or threonine residues of substrate proteins thus modulating numerous biological processes in eukaryotes. There are approximately 530 protein kinases in human, which constitute about 1.7% of all human genes amine Copper (II) sulfate pentahydrate 2X Laemmli sample buffer MeOH - methanol (EMD cat. no. MXD475-1)Urea Thiourea DTT - dithiothreitol Iodoacetamide Streptavidin Agrose PBS - phosphate buffered saline, pH 7.4 SDS - sodium dodecyl sulfate Sodium dithionite Tris HCl - tris(hydroxymethyl) aminomethane Bromophenol blue (EMD cat. no. BX1410)Glycerol (EMD cat. no. GX0185-5)β-mercaptoethanol Colloidal blue staining kit 1% NP40 buffer 2O, prepare fresh and vortex prior to use)PMSF solution (125 mM stock in HBiotin-azo-azide stock solution (5 mM stock solution in DMSO)2O, prepare fresh prior to use)TCEP solution (50 mM stock solution in HTBTA stock solution (10 mM stock solution in DMSO)4 solution CuSOResuspension buffer 2O, prepare fresh prior to use)DTT solution Iodoacetamide solution (550 mM in H1% SDS in PBS (1% (wt/vol) SDS in PBS buffer)2S2O4and 1% SDS in PBS, prepare fresh prior to use)Sodium dithionite buffer 2X SDS-free loading buffer Seed cells in 15 cm petri dishes and grow to ∼90% confluency in appropriate medium. Three 15 cm petri dishes are typically required for each condition, e.g. probe treatment or control.Replenish with 20 mL of fresh medium and add 20 uL of probe solution . Gently swirl the plate to mix. Same volume of DMSO is added to the control cells.After incubation at 37°C for 1 h, aspirate medium, wash cells twice with DPBS, and harvest cells in 5 mL DPBS using a cell scraper. Combine cell suspension from three plates and transfer to a 15 mL centrifuge tube.Centrifuge cells at 2,000 × g and 4°C for 2 min. Carefully aspirate supernatant without disturbing the pellet. Proceed to cell lysis and biotin-azide conjugation.Dissolve protease inhibitor cocktail  in 500 μL of 1% NP40 buffer by bath sonication. Add 30 μL of 125 mM PMSF solution to prepare the lysis buffer. Resuspend the cell pellet in 530 uL of the above ice-cold lysis buffer. The resulting cell lysate is diluted with 800 μL of 1% NP40 buffer.Sonicate the cell lysate in bath sonicator for 5 min and incubate on ice for another 30 min before centrifugation at 20,000 × g at 4°C for 10 min . Take the supernatant and determine protein concentration using the BCA assay.Each sample is diluted to a final volume of 9.4 mL that contains 10 mg of total protein. Add the following reagents sequentially to reach a final volume of 10 mL and gently vortex the sample after each addition.100 μL of 5 mM biotin-azo-azide 200 μL of 50 mM TCEP 4 200 μL 50 mM CuSOIncubate at room temperature (RT) for 1 h in the dark. Add 40 mL of methanol to the reaction mixture. Keep at −20°C overnight. Proceed to target protein enrichment.Pellet proteins by centrifugation at 5,200 × g and 0°C for 30 min before decanting the supernatant. Wash the residue by resuspending the pellet with 40 mL ice-cold methanol, centrifuging at 5,200 × g and 0°C for 30 min, and removing supernatant. Repeat methanol wash twice more.Air-dry pellets by placing the tube upside down with an angle of 45 degree for 30 min. Resuspend protein pellet with bath sonication in 4 mL of resuspension buffer to achieve a final concentration of 1.25 mg/mL (assuming 50% protein loss).Add 40 uL of DTT solution and incubate for 40 min at RT. Subsequently add 40 uL of iodoacetamide solution and incubate for 30 min in the dark at RT.Pre-Wash streptavidin beads (250 μL) with PBS (2 × 1 mL) and resuspension buffer (1 × 1 mL). Centrifuge at 2,000 × g for 3 min at RT to collect the beads. Remove supernatant carefully. Add resuspended proteins to streptavidin beads in 15 mL centrifuge tube and incubate on a rotator for 2 h at RT.Collect beads by centrifugation at 2,000 × g for 3 min at RT. Wash beads sequentially with resuspension buffer (2 × 10 mL), PBS (2 × 10 mL), and 1% SDS in PBS (2 × 10 mL). Transfer beads to a 2 mL dolphin-nose tube using wide- bore pipette tips.Add 200 μL sodium dithionite buffer to beads and incubate at RT for 30 min with gentle rocking. Collect supernatant by centrifugation at 2,000 × g for 3 min at RT. Repeat the elution once more and combine supernatant.Add 1.6 mL ice-cold MeOH to the combined supernatant and incubate at −20°C overnight. Centrifuge at 10,000 × g for 10 min at 4°C to pellet protein before decanting supernatant. Place tube up side down with an angle of 45 degree to air-dry the pellet. Proceed to SDS- PAGE gel analysis.Resuspend protein pellet in 15 μL of 4% SDS buffer, bath sonicate briefly, and then add 15 μL of 2X SDS-free loading buffer.Boil the sample at 98°C for 5 min and centrifuge at 10,000 × g for 3 min at RT before loading all 30 μL of the sample into SDS-PAGE gel. 20 μg of whole cell lysate can be used as input for comparison.The gel is stained with Colloidal Blue for 3 h and then destained with water overnight according to manufacture's manual.Cut out the desired gel slices and send out for mass spectrometry analysis to identify the proteins.


In addition, the inhibitory potential of TCAS on major CYP450 drug metabolizing enzymes  was assessed. The results showed that CYP1A2 and CYP2C9 mediated TCAS hydroxylation. IC50 values for TCAS in rat and human liver microsomes were greater than 50 µM, and it demonstrated a weak inhibition of rat and human CYP450 enzymes. Finally, sandwiched hepatocytes were used to evaluate the induction of CYP1A and CYP3A to define the function of TCAS in vivo. The results showed that incubation of TCAS at different concentrations for 72 h failed to induce CYP1A and CYP3A. However, incubation of the cells with 50 and 100 µM TCAS caused a profound decrease in the activities of CYP1A and CYP3A, which was probably due to cytotoxic effects, suggesting that exposure to TCAS might be a health concern.In this study, we investigated the enzymes catalyzing the phaseⅠmetabolism of thiacalixarene (TCAS) based on  It is considered the representative of the third-generation supramolecular receptor compounds arene tetrasulfonate (TCAS) with purity greater than 98% was purchased from TCI America . α-Naphthoflavone and β-NADPH were purchased from Roche Molecular Biochemicals . Recombinant human cytochromes P450 , pooled human liver microsomes (HLMs) and CYP450 monoclonal antibodies  were provided by BD Gentest . All other reagents and solvents were of analytical grade and commercially available.−1. For quantification, the instrument was operated in the ESI positive ion multiple reaction monitoring (SIM) mode with the following optimized MS/MS conditions: transfer capillary temperature of 350 °C; spray voltage of 3500 V. The selected transitions were 271.6 for TCAS and 260 for PRO (IS). The linear quantitation ranges for TCAS ranged from 50 to 500 μg/mL. The lower limit of detection of TCAS was 50 μg/mL.The quantitative determination of TCAS was conducted using a new method. A Thermo TSQ Vantage triple quadrupole mass spectrometer  equipped with a Waters ACQUITY Ultra Performance HPLC system and a Thermo Syncronis C18 column  was used in this study. The mobile phase consisted of 0.1% formic acid water solution (A) and acetonitrile containing 0.1% formic acid (B). The gradient program used was as follows: 0 min 10% B, 0–1 min 20% B, 1–1.5 min 85% B, 1.5–2 min 85% B, and 2–3 min 10% B, for a total duration of 4.5 min. The column temperature was set at 25 °C and the flow rate was 0.25 mL·min2 and 100 mM of sodium phosphate buffer (pH 7.4). This experiment was performed in triplicate. After 5 min of preincubation at 37 °C, the reaction was initiated by adding 10 μL of 20 mM NADPH  and continued for 60 min with gentle shaking. Incubations in the absence of NADPH served as controls. Reactions were terminated using 400 μL of ice-cold stop solution (a 1:1 (v:v) mixture of acetonitrile and methanol) containing 50 ng/mL tolbutamide (IS), vortexing for 30 sec, and centrifugation at 16,000 g for 10 min. The supernatant was collected and analyzed by an LC-MS/MS method described above.A standard incubation mixture (200 µL) consisted of 2 pmol/mL of complementary DNA (cDNA)-expressed human cytochrome P450 enzymes, 100 µM of TCAS, 3.3 mM of MgClAll human liver microsomal (0.4 mg/mL) incubations were conducted in 100 mM phosphate buffer, pH 7.4, initiated by the addition of NADPH  for 5 min before incubation with TCAS (100 µM) and inhibitor of specific isoform of CYP450. The following CYP450 isoform-selective inhibitors were used: α-naphthoflavone ; sulfaphenazole ; tranylcypromine ; quinidine  and ketoconazole  ,9,10. InThe antibodies were preincubated with microsomes in an ice-bath for 20min, which contained enzymes of CYP1A2, CYP2C9, CYP2D6 and CYP3A4. Then, they were incubated with TCAS (100 µM) and the NADPH generating system at 37 °C for 30 min before stopping with 400 μL ice-cold stop solution (1:1 (v:v) mixture of acetonitrile and methanol) containing 50 ng/mL tolbutamide (IS). After vortexing for 30 seconds, and centrifugation at 16,000 g for 10 min, the supernatant was collected and analyzed by LC-MS/MS. Control incubations without monoclonal antibodies were conducted. All incubations were conducted in triplicate.The inhibitory effects of TCAS on the activities of CYP1A2, CYP2C9, CYP2D6, CYP2B6 and CYP3A were tested in RLMs and HLMs using probes selective for each isoform. The reaction probes used were as follows: phenacetin for CYP1A2; tolbutamide for CYP2C9; dextromethorphan for CYP2D6; midazolam for CYP3A and bupropion for CYP2B6. Incubation conditions specific to each isoform were linear with time, substrate, and protein concentrations as detailed in our previous studies. Isoform-specific substrate probes were incubated in duplicate at 37 °C with RLMs and HLMs and an NADPH-generating system in the absence (control) or presence of various concentrations  of TCAS. A 5-min pre-incubation was carried out before the reaction was initiated by adding 0.4 mg/mL of RLMs and HLMs. After incubation for 30 min, the reaction was terminated with 400 μL of ice-cold stop solution. After vortexing for 30 seconds, and centrifugation at 16,000 g for 10 minutes, the supernatant was collected and analyzed by LC-MS/MS .6 cells/well in 24-well plates. Cells were incubated at 37 °C in a humidified incubator with 95% O2/5% CO2 and allowed to attach for 4 h, during which the medium was aspirated to remove unattached cells and fresh medium was added. Twenty-four hours later, cells were overlaid with BD Matrigel basement membrane matrix at a concentration of 0.25 mg/mL in 0.5 mL of ice-cold DMEM for an additional 24 h at 37 °C in a humidified incubator with 95% O2/5% CO2. Subsequently, cells were treated with TCAS , phenobarbital  and 3- methylcholanthrene  from 24 to 96 h in culture. Medium was changed daily. On day 4, the cultured hepatocytes were washed twice with Hank’s balanced salt solution containing 25 mM HEPES and incubated with 0.5 mL of the same buffer containing 50 μM phenacetin and 5 μM midazolam at 37 °C for 1h. At the end of incubation, 0.5 mL aliquots of the media were transferred to 1.5-mL test tubes and stored at −20 °C until analysis. All incubations were conducted in triplicate.Rat primary hepatocytes were prepared according to a modified, two-step collagenase perfusion method . Viabilip < 0.05 was considered statistically significant. The inverse correlation between the formation of acetaminophen and 1-hydroxymidazolam in untreated hepatocytes and the fold increase in induced hepatocytes, was determined with regression analysis using SPSS. Microsoft Excel  was used to calculate IC50 estimates by linear transformation of the raw data. The data were corrected for both background and control activities.Results were analyzed by a two-factor ANOVA. A A panel of recombinant human metabolic enzymes (CYPs) was screened for activity against 100 μM TCAS. To account for drift in signal and spontaneous parent loss, each time point was compared with a TCAS sample (without enzyme) incubated under identical conditions. After 1-hour incubation, only CYP1A2 and 2C9 showed significant activity as measured by the conversion of TCAS (18.4% and 14.7%) . In contp < 0.01) and 51.7% (p < 0.01), respectively, at a substrate concentration of 100 µM (α-Naphthoflavone (6 µM) and sulfaphenazole (60 µM) inhibited TCAS formation by a mean 54.9%  or cells incubated in the presence of a typical inducer of CYP1A (3-MC) or CYP3A (PB). Cells incubated in the presence of 2.5 µM 3-MC and 1 mM PB demonstrated a mean 23.8-fold and 5.1-fold induction of CYP1A and CYP3A activity, respectively, compared with control cells. In contrast, TCAS failed to induce CYP1A and CYP3A at different concentrations for 72 h . Notablyin vitro, as recommended by the relevant guidelines of the United States [50 values (Incubation of liver microsomes is generally used to study drug metabolism d States . The livd States ,15,16. O0 values . The resCYP450 induction has been investigated in rat hepatocytes ,18. Thisin vitro studies. In addition, TCAS inhibited activities of CYP1A, CYP2C and CYP2D, but not CYP2B and CYP3A in rat liver microsomes. It also exhibited weak inhibition against the activities of CYP1A2, CYP2C9, CYP2B6, CYP2D6 and CYP3A4 in human liver microsomes in vitro. Taken together, the results suggested that TCAS manifested weak interactions with drugs that are metabolized by different CYP450 enzymes, as discussed above. Our results revealed that TCAS had no effect on activities of CYP1A and CYP3A. Conversely, it exerted inhibitory effect on activities of CYP1A and CYP3A when the concentration of TCAS was greater than 50 µM, suggesting that human exposure to TCAS is not entirely safe.In conclusion, TCAS was metabolized through CYP1A2 and CYP2C9, which was confirmed by a series of


Mapping the conditions of spider silk proteins along the silk gland, and combining with molecular studies, reveals a pH controlled switch between lock and trigger forms, providing insights into spider silk formation. 2) in distal parts of the gland, and a CO2 analogue interacts with buried regions in CT as determined by nuclear magnetic resonance (NMR) spectroscopy. Activity staining of histological sections and inhibition experiments reveal that the pH gradient is created by carbonic anhydrase. Carbonic anhydrase activity emerges in the same region of the gland as the opposite effects on NT and CT stability occur. These synchronous events suggest a novel CO2 and proton-dependent lock and trigger mechanism of spider silk formation.Spider silk fibers are produced from soluble proteins (spidroins) under ambient conditions in a complex but poorly understood process. Spidroins are highly repetitive in sequence but capped by nonrepetitive N- and C-terminal domains (NT and CT) that are suggested to regulate fiber conversion in similar manners. By using ion selective microelectrodes we found that the pH gradient in the silk gland is much broader than previously known. Surprisingly, the terminal domains respond in opposite ways when pH is decreased from 7 to 5: Urea denaturation and temperature stability assays show that NT dimers get significantly stabilized and then lock the spidroins into multimers, whereas CT on the other hand is destabilized and unfolds into ThT-positive β-sheet amyloid fibrils, which can trigger fiber formation. There is a high carbon dioxide pressure . Spiders manage both to store the highly aggregation-prone spidroins in solution at extreme concentrations in the silk glands and then to rapidly convert these spidroins into a solid fiber within fractions of a second as they spin fibres. This process has been extensively studied and is thought to involve a pH gradient, but how this pH gradient is generated and maintained was not resolved. Here, we measured the pH at locations along the ampullate gland and determined that the pH decreases to 5.7 in the middle of the spinning duct. We also observed that the carbon dioxide pressure is simultaneously increased and that its accumulation may affect the stability of CT. We find that active carbonic anhydrase (CA) is crucial to maintain the pH gradient along the gland. Detailed molecular studies of NT and CT under the disparate conditions present along the gland revealed a lock and trigger mechanism whereby in more neutral pH conditions, precocious spidroin aggregation is prevented, and when in more acidic pH conditions, NT dimers firmly interconnect the spidroins and the CT unfolds into β-sheet nuclei that can trigger rapid polymerization of the spidroins. We conclude that this mechanism enables temporal and spatial control of silk formation and may be harnessed in attempts to produce artificial silk replicas. Spider silk fibers contain regions of crystalline and noncrystalline β-sheets, which mediate mechanical stability Nephila clavipes, from the proximal part of the tail to the middle part of the second limb of the duct. Concentrations of CO32− were also determined at locations where pH was high enough to allow reliable measurements, and used to calculate HCO3− concentrations. We found that the pH decreases from 7.6±0.1 (n = 11) in the proximal tail to 5.7±0.0 (n = 6) in the second limb of the duct and that HCO3− concentration increases from 5 mM in the proximal tail to 21 mM in the distal part of the sac (2) could be calculated and was found to increase along the gland   the sac . With thhe gland . We obseric ISMs .3− and CO2 concentrations from the proximal to the distal parts of the gland  to study the backbone conformational dynamics of CT at pH 7.5 to 5.5. No major differences in HDX were seen between pH 7.5 and 6.5, but helices 2, 3, and 5 showed increased HDX at pH 5.5 compared to at pH 6.5 , indicat studied  is very  MiSp CT . Computa3− concentration and low CO2 permeability of the gland—imply that pCO2 is elevated along the sac and duct. For MaSp CT, it has been shown that shear forces induce conformational changes that result in increased exposure of nonpolar surfaces 2 interacts mainly with nonpolar regions in proteins 2 analogue CS2A. ventricosus MiSp CT. CS2 interacts specifically with a few, mainly hydrophobic, CT residues distributed in helices 2–4, of which many are partly buried  unfolded proteins in a nucleation-dependent reaction and are found in tissue deposits associated with disease but also in some functional protein aggregates in vitro is recapitulated in vivo. NT and CT are unique to spidroins and there are no known homologues. The lock (accomplished by NT) and trigger (accomplished by CT) mechanism proposed herein is therefore likely unique for spider silk formation, in contrast to the previously identified shear-induced polymerization mechanism that also apply to, for example, silk worm silk formation The spidroins' terminal domains are highly conserved, both between species and between different types of silks 2 and proton gradients that ensure temporal and spatial confinement of the divergent structural changes of CT and NT. This novel lock and trigger mechanism elegantly explains how silk formation can occur at a very high speed, more than 1 m/s In summary, the spidroin N- and C-terminal domains show synchronous and opposite structural changes in response to the physiological conditions of the spinning duct. CT unfolds into β-sheet nuclei that can trigger rapid polymerization of the spidroins, whereas gradually locked NT dimers alleviate the need for rapid diffusion N,N-dimethyltrimethylsilylamine (Fluka 41716), after which the barrel was mounted on a micromanipulator and heated using a hot air gun giving temperatures of 200–300°C for 60 s. Ion-selective cocktails for H+ (Fluka 95291), CO32−  +, K+, and Cl− were sucked into the tip to form a 100 to 200 µm long column, and a backfilling solution  was added in the middle of the outer barrel. The inner barrel  was pulled to a tip diameter of 1 µm and filled with 3 M KCl pH 7.4. The inner barrel was then inserted into and secured in the outer barrel, the inner glass tip being positioned 4–10 µm away from the outer barrel tip. A silver wire was inserted into the inner barrel and connected to an amplifier.Concentric ISMs + or Cl−, or using 1, 2, 4, and 8 mM K+, respectively. Carbonate electrodes were calibrated as described The ISMs were calibrated using pH 6.87 and pH 7.42 buffers, 50, 100, 200, and 400 mM NaN. clavipes collected in Florida from September to November were kept in individual containers and fed water. Spiders were anaesthetized with CO2 gas before severing at the pedicle. Dissection of the major ampullate glands was carried out in a modified spider Ringer 2, 2 mM CaCl2, 3 mM KCl, and 10 mM glucose) buffered with 26 mM bicarbonate and 5% CO2, yielding a pH of 7.4.Adult female 3− and CO2-buffered modified spider Ringer at room temperature. ISM measurements were performed in triplicates in different parts of the gland. The difference in potential between the bath and the inside of the gland was recorded on a chart recorder (Zipp and Konnen) and later translated into change in concentration of the ion of interest using the Nernst equation  or a modified Henderson–Hasselbalch equation 32−) to get the concentration of HCO3−. Determined pH values and HCO3− concentrations were used to calculate pCO2 according to the Henderson–Hasselbalch equation, assuming equilibrium.Major ampullate glands were mounted in a submersion-style incubation chamber and superfused with HCOTo study the influence of CA activity on the pH gradient, some glands were incubated for 1 h in 0.1 mM methazolamide , a membrane-permeable CA inhibitor, prior to pH measurements, after which the methazolamide was washed away for 30 min and pH measurements repeated.2 permeability studies. Glands were dissected, mounted, and superfused with HCO3− and CO2-buffered spider Ringer at room temperature as described above. A pH electrode was inserted into the gland, after which the surrounding Ringer solution was buffered by 26 mM bicarbonate and 100% CO2. pH measurements were continued up to 1 h to see if intraluminal pH changed in response to the elevated pCO2 surrounding the gland. The Ringer solution was then changed again, being buffered by 26 mM bicarbonate and 5% CO2, yielding a pH of 7.4, after which the pH electrode was removed from the gland and put in the Ringer and pH was recorded. This was made to ensure that the electrode had not been drifting.Some glands were subjected to COA. diadematus, N. clavipes, E. australis, and Tegenaria sp.) were anesthetized and sacrificed as described above. Dissection was carried out in 67 mM sodium phosphate buffer at pH 7.2 or in a modified Spider Ringer (see above). Some opisthosomas were fixed and embedded directly after removal of the exoskeleton, whereas others were dissected so that the major and minor ampullate glands could be isolated before fixation.Spiders  glutaraldehyde in 67 mM phosphate buffer, pH 7.2, for 24 h at 4°C and subsequently rinsed with phosphate buffer, pH 7.2. After fixation, tissues were dehydrated using increasing concentrations of ethanol, infiltrated and embedded in a water-soluble glycol methacrylate (Leica Historesin embedding kit).3, CoSO4, H2SO4, and KH2PO4, whereby carbon dioxide leaves, pH increases, and a cobalt–phosphate–carbonate complex is formed at sites with CA activity. This complex is then converted into a black cobalt–sulphide precipitate. The sections were counterstained with Azure blue. For control of unspecific staining, the CA inhibitor acetazolamide was included in the incubation medium.Historesin embedded major and minor ampullate glands and opisthosomas were sectioned at 2 µm in a microtome (Leica RM 2165) and stained for CA activity using a histochemical method A. ventricosus MiSp NT and CT coding gene fragments corresponding to (NT: GSGNSQPIWT NPNAAMTMTN NLVQCASRSG VLTADQMDDM GMMADSVNSQ MQKMGPNPPQ HRLRAMNTAM AAEVAEVVAT SPPQSYSAVL NTIGACLRES MMQATGSVDN AFTNEVMQLV KMLSADSANE VST) and (CT: GSGNSTVAAY GGAGGVATSS SSATASGSRI VTSGGYGYGT SAAAGAGVAA GSYAGAVNRL SSAEAASRVS SNIAAIASGG ASALPSVISN IYSGVVASGV SSNEALIQAL LELLSALVHV LSSASIGNVS SVGVDSTLNV VQDSVGQYVG) were amplified by PCR with the full-length MiSp gene as template Escherichia coli. The E. coli were grown at 37°C in LB medium containing 70 mg/l kanamycin until OD600 was about 0.9. The temperature was lowered to 30°C, IPTG was added to a final concentration of 0.3 mM, and the cells were incubated for about 4 h. The E. coli were then harvested by centrifugation at 6,400×g for 20 min at 4°C , after which the pellet was resuspended in 20 mM Tris pH 8.0, 1 mg/ml lysozyme was added, and the solution was incubated on ice for 30 min. Next, DNase and MgCl2 were added and the mixture was kept on ice for 30 min. The cell lysate was centrifuged  at 4°C for 20 min . For purification of CT, the supernatant was loaded on a Ni-NTA column and the fusion protein was eluted with 300 mM imidazole. For purification of NT, which is mainly found in the pellet after lysis, pellets were resuspended in 20 mM Tris pH 8.0 containing 2 M urea, sonicated for 2 min, and the supernatant was treated as for CT. The fusion proteins were then dialyzed against 20 mM Tris pH 8.0 overnight at 4°C, cleaved by 1/1,000 (w/w) thrombin, and run over a Ni-NTA column to remove the fusion tag. This resulted in essentially pure NT or CT (>90% purity as determined by SDS PAGE gel electrophoresis and Coomassie staining).A. ventricosus MiSp (full-length sequence above). The expressed protein was labeled with 15N, and the NMR spectrum showed that the first 25 residues adopt random coil fold. Therefore, A. ventricosus MiSp CT was truncated and residues 31–150 (marked in bold in the sequence above) were expressed in minimal medium and labeled by 15N and 13C/15N. The NMR sample was prepared by adding 8% (v/v) D2O and 0.02% (w/v) NaN3 to a 1 mM solution of uniformly 13C/15N-labelled protein in 20 mM sodium phosphate buffer (pH 6.8) with 20 mM NaCl.For NMR structure determination, we initially expressed a 150-amino-acid-residue-long C-terminal part of 15N-1H]-HSQC (1024×128), HNCA (1024×24×40), CBCA(CO)NH (2048×48×40), HNCO (1024×24×40), HN(CA)CO (1024×24×40), and for side-chain assignment and NOE restraint collection (mixing time given in parentheses): 15N-resolved NOESY-HSQC , 13C-resolved NOESY-HSQC , and 13C(aromatic)-resolved NOESY-HSQC . Additionally, in order to identify intermolecular NOEs, a 13C/15N-filtered 13C-resolved NOESY-HSQC spectrum  was recorded on a sample containing 50% 13C/15N-labelled and 50% unlabelled proteins 2. Aliquots of 20% CS2 in DMSO were added in a stepwise manner to the NMR sample of CT, yielding CS2 concentrations of 50 mM, 100 mM, and 200 mM, and a 2D [15N-1H]-HSQC spectrum was recorded each time. To account for perturbations due to DMSO, a reference experiment was performed by adding DMSO only in the same amounts. CS2-induced chemical shift perturbations were calculated by comparing the spectrum at 200 mM CS2 with the spectrum at the end of the reference titration with DMSO, and using the formula  as described for CT.All NMR experiments were carried out at 298 K on a 600-MHz Varian Unity Inova spectrometer equipped with an HCN triple-resonance pulsed-field-gradient cold probe. The following 2D and 3D spectra were acquired for backbone resonance assignment (number of complex points given in parentheses): [15N-1H]-HSQC NMR spectra of MiSp CT, samples at pH 5.0, 5.3, and 5.5 were prepared by diluting 50 µl of a concentrated stock solution of MiSp CT in 20 mM sodium phosphate buffer, 20 mM NaCl, 0.03% NaN3, pH 6.8 with 200 µl of 100 mM CD3COOD/CD3COONa, 20 mM NaCl, 0.03% NaN3 buffer, and adding 20 µl of D2O.For 2D 50% by KaleidaGraph.For NT, urea-induced denaturation was performed by diluting the protein to 5 µM in 20 mM HEPES/20 mM MES with 0–7 M urea in 0.25 M steps. Tryptophan fluorescence emission spectra were measured on a spectrofluorometer (Tecan Safire 2) using Costar black polystyrene assay plates with 96 flat bottom wells. The samples were excited at 280 nm using a 5 nm bandwidth, and emission spectra were recorded in 1 nm steps between 300 and 400 nm using a 10 nm bandwidth. Spectra were recorded at constant pH values ranging from 5.0 to 7.5 with 0.2–0.5 unit steps. For CT, CD spectroscopy at 222 nm was used to determine [den]A. ventricosus MiSp NT (110 µg/ml) or CT (90 µg/ml) in 20 mM sodium phosphate buffer at pH 7.5, pH 6.5, or pH 5.5 were recorded at 25, 45, 65, 85, and 95°C and at 25°C again after cooling. For temperature melting curves, the CD at 222 nm was monitored between 25 and 95°C with 1°C/min increase.CD spectra were recorded from 260 to 190 nm at 25°C in 0.1 mm path length quartz cuvettes using an Aviv 410 Spectrometer. The wavelength step was 0.5 nm, averaging time 0.3 s, scan speed 20 nm/min, time constant 100 ms, and bandwidth 1 nm. The spectra shown are subtracted for background and averaged over three consecutive scans. The HT voltages were always below 600 V during the entire scans. Spectra of 7.5 µM 2O (Cambridge Isotopes). A. ventricosus MiSp CT was diluted from 555 µM stock solution, pH 6.5, to 55.5 µM in deuterated phosphate buffer, pH 6.5 or 5.5. We removed 19.5 µl aliquots after 400 s, 50 min, 100 min, 200 min, or 300 min (pH 5.5) or after 40 s, 5 min, 10 min, 20 min, or 30 min (pH 6.5). Aliquots were placed in prechilled 500 µl Eppendorf tubes containing 0.5 µl 5% TFA (Merck), vortexed, and immediately frozen in liquid nitrogen. For a fully deuterated control, CT was incubated in deuterated phosphate buffer, pH 6.5, for 24 h at 25°C. Samples were stored at −80°C until ESI MS analysis.Deuteration buffers were prepared by freeze-drying 200 µl of 20 mM sodium phosphate buffer, pH 5.5 or 6.5, followed by reconstitution in 200 µl D18 trap column (Waters). Two 140D solvent delivery systems (Applied Biosystems) were employed, operating at 20 µl/min (for washing with 0.05% TFA) or at 15 µl/min . Digestion and desalting were carried out in a single step for 10 min, and peptides were then eluted in a single step and delivered to the mass spectrometer via a TaperTip emitter (Proxeon). The entire flow system was submerged in an ice bath.Samples were thawed and immediately injected into an HPLC system using a chilled 25 µl Hamilton syringe. CT protein was digested in a Porozyme pepsin cartridge (Applied Biosystems), and peptides were trapped and desalted in a Waters Symmetry CESI spectra were acquired in positive-ion mode with a Waters Ultima API mass spectrometer (Waters) equipped with a Z-spray source. The source temperature was 80°C, the capillary voltage was 2.5 kV, and the cone and radiofrequency lens 1 potentials were 100 and 38 V, respectively. The mass spectrometer was operated in single-reflector mode to achieve a resolution of 10,000 . The mass scale was calibrated using [Glu1]fibrinopeptide B. Peptic peptides were identified based on a map of pepsin-digested undeuterated CT using automated liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis with a Waters NanoAcquity system (Waters). Peptide sequences were identified by individual analysis of collision-induced dissociation (CID) spectra using the Waters MassLynx and ProteinLynx software packages (Waters).A. ventricosus MiSp CT equilibrated 10 min in 20 mM HEPES/MES pH 7.5 or 5.5 using Sephacryl S-100  in the same buffers and at a flow rate of 0.5 ml/min. Molecular mass standards aprotenin (6.5 kDa), ribonuclease (13.7 kDa), CA (29 kDa), ovalbumin (43 kDa), and conalbumin (75 kDa) were used for calibration.We analyzed 100 µl of 1 mg/ml Table S1http://dichroweb.cryst.bbk.ac.uk/html/home.shtml).Percentage of different secondary structures estimated from the CD spectra of NT and CT at 25 and 95°C , using t(PDF)Click here for additional data file.Table S2NMR and refinement statistics for protein structures.(PDF)Click here for additional data file.


Intracellular pH plays an important role in the response to cancer invasion. We have designed and synthesized a series of new fluorescent probes (Superior LysoProbes) with the capacity to label acidic organelles and monitor lysosomal pH. Unlike commercially available fluorescent dyes, Superior LysoProbes are lysosome-specific and are highly stable. The use of Superior LysoProbes facilitates the direct visualization of the lysosomal response to lobaplatin elicited in human chloangiocarcinoma (CCA) RBE cells, using confocal laser scanning microscopy. Additionally, we have characterized the role of lysosomes in autophagy, the correlation between lysosome function and microtubule strength, and the alteration of lysosomal morphology during apoptosis. Our findings indicate that Superior LysoProbes offer numerous advantages over previous reagents to examine the intracellular activities of lysosomes. Lysosomes are membrane bound organelles that contain digestive enzymes functioning to recycle damaged organelles as well as digest nucleic acids, polysaccharides, fats and proteins. In the maintenance of cellular homeostasis, lysosomes are active in intracellular signaling, energy metabolism, secretions, and plasma membrane repair. Dysfunction of lysosomal structure or function is associated with multiple pathologies, including inflammation, cancer, neurodegenerative disease, and specific lysosomal storage diseases such as Tay-Sachs123456789101112131415171819202122232425262728293031171819202122232425262728293031In earlier work, we developed several fluorescent probes that aggregate in lysosomes, and they were employed to monitor intracellular pH and localize lysosomes in cultured cells+]. As shown in 2HPO4–0.1 M citric acid buffer). As showed in + was rapid and its fluorescence intensity saturation was reached within a few minutes at the range of pH 4.80 ~ 6.00, an important advantage for real-time monitoring of lysosomal pH changes. Additionally, Superior LysoProbes maintain extensive fluorescence and high concentration at low pH, offering distinct advantages over other fluorescent probes.Superior LysoProbes I–IV were non-fluorescent under basic conditions, whereas a shift to acidic conditions yielded a pink chromophore with strong fluorescence. This indicates that these probes may serve as ‘naked-eye' indicators for . The concentration to achieve 50% inhibition (IC50) was calculated by nonlinear regression using the mean data values obtained in triplicate independent experiments. Cell morphologic changes were assessed using a phase-contrast microscope  following a 24 hours incubation with lobaplatin.The cytotoxicity of lobaplatin to RBE cells was examined using a cell proliferation assay. Cells were seeded in a 96-well microtiter plate at 5 × 10The effect of lobaplatin on RBE cell cycle distribution was determined by FACS analysis. Cells were exposed to different concentrations of lobaplatin for 24 hours, and then harvested, washed twice with PBS and fixed with 70% ethanol solution at 4°C overnight. Following centrifugation, cell pellets were stained with 5 μg/mL PI and 1 μg/mL RNase A at 4°C for 30 minutes. The samples were analyzed by FACScan flow cytometer with CellQuest software. The distribution of cells in various stages of the cell cycle was determined using ModFit LT software.Cell apoptosis was evaluated using the Annexin V-FITC Apoptosis Detection Kit . RBE cells were exposed to various concentrations of lobaplatin (0–25 μg/mL) for 24 hours and then cells were harvested and re-suspended in 1 × Binding Buffer. 5 μL of Annexin V-FITC and 5 μL of propidium iodide (PI) were added to RBE cells and the cells subsequently analyzed by flow cytometry.t test was used for statistical analyses and P < 0.05 was chosen as the threshold for significance.Statistical calculations were carried out with the SPSS 15 for Windows software package. Results are expressed as the mean ± SD. Student's L.B. designed the study. X.C. did the synthesis of these fluorescent probes, Y.B., T.W. and P.L. performed the cell assays. S.H. and X.Y. performed the probes characterization. L.B., C.E.B., K.M.G., J.J. and W.J.P. performed data analysis, wrote and edited the manuscript.Lysosomal Targeting with Stable and Sensitive Fluorescent Probes (Superior LysoProbes): Applications for Lysosome Labeling and Tracking during Apoptosis


KCND3 gene as the cause of spinocerebellar ataxia type 19/22 (SCA19/22), revealing a previously unknown role for the voltage-gated potassium channel, Kv4.3, in Purkinje cell survival. However, how mutant Kv4.3 affects wild-type Kv4.3 channel functioning remains unknown. We provide evidence that SCA19/22-mutant Kv4.3 exerts a dominant negative effect on the trafficking and surface expression of wild-type Kv4.3 in the absence of its regulatory subunit, KChIP2. Notably, this dominant negative effect can be rescued by the presence of KChIP2. We also found that all SCA19/22-mutant subunits either suppress wild-type Kv4.3 current amplitude or alter channel gating in a dominant manner. Our findings suggest that altered Kv4.3 channel localization and/or functioning resulting from SCA19/22 mutations may lead to Purkinje cell loss, neurodegeneration and ataxia.The dominantly inherited cerebellar ataxias are a heterogeneous group of neurodegenerative disorders caused by Purkinje cell loss in the cerebellum. Recently, we identified loss-of-function mutations in the The online version of this article (doi:10.1007/s00018-015-1894-2) contains supplementary material, which is available to authorized users. KCND3, which encodes the voltage-gated potassium channel Kv4.3 [IA) [IA currents control the spike frequency and the back-propagation of action potentials, processes which are particularly important for Purkinje cell firing [Spinocerebellar ataxia type 19/22 (SCA19/22) is a dominantly inherited neurodegenerative, clinically heterogeneous disorder caused by mutations in el Kv4.3 . Kv4.3 c.3 [IA) –12, hear.3 [IA) , 13–15. l firing . Alteratl firing –20. Thesl firing .The question remains whether the SCA19/22 mutations result in reduced Kv4.3 channel activity due to haploinsufficiency through pure loss of function and/or due to the mutant proteins acting as dominant negative suppressors of the wild-type Kv4.3 subunits. The latter has been proven for mutations in Kv1.1, which cause episodic ataxia type 1 (EA1) and which display dominant negative effects on potassium currents associated with different clinical phenotypes in EA1 patients . SimilarXenopus oocytes. In contrast, the ΔF227, M373I and S390N mutations altered the gating kinetics of the WT/ΔF227, WT/M373I and WT/S390N heterocomplexes in a dominant and different manner. Our work demonstrates how difficult it is to establish a common underlying pathological mechanism for SCA19/22, a problem that is reflected in the clinical heterogeneity of this disease. Further work is necessary to reveal the detailed contribution of the mutant Kv4.3 channel subunit in the native IA current composition and to elucidate its effects on Purkinje cell functioning.For SCA19/22, as a dominant disease, we aimed to elucidate whether its pathology is caused by haploinsufficiency through pure loss of Kv4.3 function and/or whether the mutant proteins act as dominant negative suppressors of the wild-type Kv4.3 subunits. We therefore studied wild-type (WT) Kv4.3 trafficking and channel complex formation and function in the presence of SCA19/22-mutant Kv4.3 subunits, to advance our understanding of the underlying disease mechanism(s). All SCA19/22-mutant Kv4.3 subunits exerted dominant negative effects on WT Kv4.3 trafficking leading to intracellular retention and enhanced protein instability of the WT/mutant Kv4.3 channel heterocomplexes that could be rescued by KChIP2. Notably, the trafficking deficit was caused by temperature-sensitive misfolding of the mutant subunits. Only the T352P mutation dominantly reduced the channel activity of WT/T352P heterocomplexes in a dose-dependent manner in XhoI and EcoRI restriction sites. An extracellular hemagglutinin tag (HA: YPYDVPDYA) was introduced into the cDNA of pcDNA3.1-Kv4.3 wild type (WT) and mutants  in the extracellular loop between transmembrane domains S1 and S2 (L216-P217) using site-directed mutagenesis via forward primer 5′-CCGGGCAGCAAGGAGCTGTACCCATACGACGTCCCAGACTACGCTCCGTGCGGGGAGCGTAC-3′ and reverse primer 5′-GTACGCTCCCCGCACGGAGCGTAGTCTGGGACGTCGTATGGGTACAGCTCCTTGCTGCCCGG-3′, as described previously for Kv4.2. All constructs were verified by Sanger sequencing. The addition of HA and EGFP tags to Kv4.3 did not affect the functional properties of the channel . The Emerald-KChIP2b was provided by Dr. K. Takimoto  and the cDNA of KChIP2b (referred to as KChIP2) was subcloned in pcDNA3.1 via NcoI.The pcDNA3.1-Kv4.3 WT and mutant plasmids was generated as previously described [2. Transfections were done using polyethylenimine (Polysciences), according to the manufacturer’s instructions. To generate WT/mutant heterocomplex Kv4.3 channels or to generate WT or mutant homocomplex Kv4.3–KChIP2 channels, all plasmids were expressed in 1:1 ratio. Additionally, to generate WT/mutant heterocomplex Kv4.3–KChIP2 channels, plasmids containing WT, mutant Kv4.3 and KChIP2 were transfected in 0.5:0.5:1 ratio. For example, the amount of DNA used for the protein stability experiments was 0.5 µg WT Kv4.3, 0.5 µg mutant Kv4.3 and 1 µg KChIP2 (6 well plate). For the immunocytochemistry and FACS experiments, the amount of plasmid DNA transfected was 0.25 µg WT Kv4.3, 0.25 µg mutant Kv4.3 and 0.5 µg KChIP2 (24 well plate). The protein stability was assessed treating the HeLa cells with a protein synthesis inhibitor cycloheximide  for 0, 3 and 6 h, and protein expression was analyzed by Western blot and quantified as described previously [HeLa cells were grown in Dulbecco’s Modified Eagle’s Medium (Invitrogen) supplemented with 10 % fetal bovine serum (Invitrogen) and 1 % penicillin–streptomycin (Gibco) in a 37 °C incubator with 5 % COImmunocytochemistry was performed as previously described with some modifications . BrieflyFor the cell surface quantification using flow cytometry of the WT/mutant heterocomplex Kv4.3 channels, HeLa cells were transfected with a combination of extracellular HA-tagged WT with EGFP-tagged Kv4.3 WT or mutants, and HA-tagged mutant with EGFP-tagged Kv4.3 WT, either with or without KChIP2. For quantification of cell surface expression of WT or mutant Kv4.3 subunits at 30 vs. 37 °C, HeLa cells were transfected with HA–WT or HA–mutant Kv4.3 together with GFP  and cultured at 30 or 37 °C for 24 h. Cells expressing WT Kv4.3 together with WT-Kv4.3-HA were used as positive control and non-transfected cells were used as a negative control. Non-permeabilized cells were incubated with anti-HA (3F10) antibody  for 2 h at 37 °C or at 30 °C and secondary Alexa647-conjugated anti-rat antibody . After washing, cells were re-suspended in 500 µl ice-cold 10 % fetal bovine serum in PBS. Cytometric analysis was performed using an FACS Calibur flow cytometer (BD Biosciences) with 488 and 635 nm lasers, and analyzed using the Cell Quest Pro software (BD Biosciences). Representative images for the FACS analysis are shown in Fig. S4.For protein analysis, cells were homogenized in 2 % SDS-PBS buffer containing protease inhibitor cocktail (Roche) and protein concentration was determined using the BCA method (BioRad). Then, 50 µg of protein were separated in an SDS-PAGE, blotted in nitrocellulose membrane, blocked and incubated with mouse anti-Kv4.3 , mouse anti-actin  and mouse anti-KChIP2 . Final quantification was performed using the program Quantity One (Bio-Rad).Xenopus laevis oocyte expression system was used to characterize the functional properties of WT and mutant Kv4.3 channels in the presence of KChIP2. All animal procedures were approved by the Chancellor’s Animal Research Committee at the University of California, Los Angeles. RNA was transcribed in vitro using the mMessage mMachine T7 Ultra kit (Ambion) and injected into stage V–VI oocytes. RNAs (160–200 ng) encoding WT or mutant Kv4.3 or a mixture of WT and mutant Kv4.3 were co-injected with RNA encoding KChIP2, which was included at equimolar amounts to WT Kv4.3. The oocytes were kept in an 18 °C incubator and all oocyte recordings were performed at room temperature (~18 °C). Potassium currents were recorded 1–3 days later using a Warner OC-725 two-electrode voltage clamp, as described previously with some modifications [2 and 5 mM HEPES, pH 7.5. Alternatively, in some experiments the bath solution contained 2 mM KCl, 96 mM NaCl, 1.8 mM CaCl2, 1 mM MgCl2 and 5 mM HEPES, pH 7.5 . Linear leak and capacitive currents were subtracted using a P/-4 protocol. The conductance/voltage relationship was determined by pulsing the membrane from a holding potential of −100 mV to voltages ranging from −80 to +70 mV in 10 mV increments. Conductance values were calculated from peak current amplitudes assuming a linear open channel current–voltage relationship and a reversal potential of −95 mV, and normalized to the maximum value obtained in the experiment. Normalized conductance values were plotted versus voltage and fitted with a single Boltzmann function to obtain values for V½,act and the slope factor. Mean current density plots were generated by measuring the peak current amplitudes as a function of voltage and normalized to that for wild-type Kv4.3 expressed in parallel in the same batch of oocytes. Data from different batches of oocytes were then averaged. RNA encoding KChIP2 was co-injected at an equimolar ratio with the total amount of Kv4.3 RNA.The ications , 33, 34.I/Imax). Data were plotted as a function of prepulse voltage and fitted with a Boltzmann function to determine the midpoint voltage, V½,inact and the slope factor. The kinetics of recovery from inactivation at −80 mV were assessed using a two-pulse protocol, in which two pulses to +60 mV were applied separated by an interpulse interval of varying duration at −80 mV. The fractional recovery was calculated as the ratio of the peak current amplitude during the second pulse to that of the first pulse (I/Imax). This ratio was plotted versus the interpulse duration. The time to reach peak amplitude was measured by evoking currents at +60 mV, which were then scaled to the same amplitude and overlaid. Additionally, the fraction of current remaining at the end of the pulse was measured. Data were fitted with one exponential component to estimate the recovery time constant, τrec.To characterize the steady-state properties of inactivation, the voltage was stepped from the holding potential (−100 mV) to prepulse potentials ranging from −130 to +10 mV for 1 s to allow inactivation to occur. The membrane was then stepped to the test potential of +60 mV for 1 s. The extent of inactivation during the prepulse was calculated as the ratio of peak current amplitude during the test pulse relative to the peak current amplitude at +60 mV in the absence of a prepulse  followed by Student’s KCND3 allele, we wondered whether the mutant  and WT Kv4.3 subunits would assemble into functional tetrameric WT/mutant heterocomplexes at the plasma membrane. We first studied the effect of the mutant subunits on the protein trafficking of WT Kv4.3. To study the subcellular localization of the WT/mutant heterocomplexes, HA-tagged WT Kv4.3 was co-expressed with either enhanced green fluorescent protein (EFGP)-fused T352P, M373I, S390N, ΔF227 or WT Kv4.3 in HeLa cells. Immunocytochemistry revealed that, in the presence of any mutant subunit, WT Kv4.3 was mainly detected intracellularly co-localizing with the mutant subunit, and almost no plasma membrane localization was seen  –37 or N4We have previously shown that KChIP2 was able to rescue the trafficking defects and protein instability of the single T352P and M373I subunits. However, KChIP2 was unable to rescue these deficits for S390N [These data show that KChIP2 can eliminate the dominant negative effect of the mutant subunits on the trafficking and stability of the WT/mutant heterocomplexes. Thus, in the presence of sustainable levels of KChIP2, stable and correctly assembled WT/mutant heterocomplex Kv4.3 channels are located at the plasma membrane in a heterologous system.Xenopus oocyte expression system was used to analyze the strength of any dominant effects of the mutant Kv4.3 subunits and to determine the effects of the mutations on the functional properties of WT/mutant heterocomplexes. Advantageously, oocytes are kept at 18 °C, which could enhance the rescue of temperature-dependent folding and trafficking defects in a manner similar to that observed for HeLa cells at 30 °C.Since correctly assembled macromolecular heterocomplexes consisting of KChIP2, WT and mutant Kv4.3 subunits are present at the plasma membrane, we wondered whether these channel complexes were functionally active. The + currents . Electrophysiological experiments were performed at ~18 °C. Representative current traces, 150 ms in duration, are shown in Fig. :1. Elect+ currents showed rapid activation and subsequent inactivation characteristic of Kv4.3 channels. In our experiments, inactivation was slower than in previous reports, because currents were recorded at ~18 °C rather than the more typical 22 °C . At the lower temperature, a slow component of inactivation dominates the kinetics of current decay, whereas at 22 °C fast and slow components make similar contributions to decay kinetics (Table S1). We compared the steady-state properties of activation in WT and mutant homo- and heterotetrameric channels by determining peak conductance/voltage relationships  and the slope factor  heterocomplexes was shifted by +9 mV  heterocomplexes had significantly reduced slope factors compared to WT   heterocomplexes compared to WT alone, consistent with the idea that these mutations increase the rate of inactivation  heterocomplexes  heterocomplexes did not show changes in the voltage dependence of inactivation and behaved like WT homocomplexes, whereas WT/M373I and WT/S390N (1:1) heterocomplexes exhibited the same shift in inactivating voltage as the respective mutant homocomplex channels. Alterations in the slope factor were only detected for ΔF227 and S390N homocomplexes , and the I/Imax was plotted versus the interpulse duration  effects may contribute to the disease etiology. We consider three potential scenarios leading to partial haploinsufficiency and/or dominant (negative) effects due to SCA19/22 mutations. In interpreting our data, we assume that the formation of WT/mutant heterocomplexes is a stochastic process in which WT and mutant subunits assemble randomly in tetrameric complexes when they are expressed in a 1:1 ratio. Additionally, we assume that the stoichiometry of Kv4.3/KChIP2 complex is variable and depends on the expression levels of KChIP2. This was supported by a recent publication that showed that the stoichiometry of Kv4.2/KChIP4 complex depends on the expression level of KChIP4 . In all In our first scenario, the assembly of S390N and ΔF227 mutant homocomplexes, even in the presence of KChIP2, causes partial haploinsufficiency due to almost complete endoplasmic reticulum retention and reduced channel complex stability. Dominant negative effects on the plasma membrane trafficking have also been seen in other potassium channelopathies, such as long QT syndrome type 1 and 2, nonsyndromic sensorineural deafness type 2 and SCA13, which are disorders caused by truncating and missense mutations in other voltage-gated potassium channels Kv7.1, Kv11.1, Kv7.4 and Kv3.3, respectively –44. The In our second scenario, loss of function of all WT/mutant heterocomplexes might occur with low KChIP2 levels, such as decline of expression during aging or due to SCA19/22 mutations. Although some studies have reported on KChIP expression and function in the adult rodent cerebellum –50, no aI150ms/Ipeak ratio compared to WT alone, indicating that a limited number of mutant subunits were able to be incorporated into active, cell surface channels. These results indicate that incorporation of one T352P subunit in an otherwise WT channel was not sufficient to abolish channel activity in oocytes, although the suppression of the current amplitude is dose dependent. Furthermore, dominant alterations in gating of the heterocomplex Kv4.3 channel were observed, since the activation and inactivation shifts of all WT/mutant heterocomplexes, except for WT/T352P heterocomplexes showed values between the WT and the mutant homocomplexes. Dominant suppression of channel activity and altered gating were also described for many of the mutant Kv3.3 heterocomplex channels underlying SCA13 [For the third scenario, we have shown that, in addition to the haploinsufficiency, all mutant Kv4.3 subunits also exhibit dominant effects on WT Kv4.3 channel function, even in the presence of KChIP2, which may contribute to disease onset and progression. This is especially true for T352P, which strongly suppressed current amplitude when this mutant subunit co-assembled with WT Kv4.3. The threonine to proline substitution at position 352 in the pore region is seemingly extremely damaging, and we suspect that this mutation leads to a full collapse of the channel pore and thereby complete inactive channels complexes despite its cellular localization. Consequently, WT/T352P heterocomplex channels exhibited significant changes in their time to reach peak amplitude and their ng SCA13 –29.V½,inact in the hyperpolarized direction of the WT/S390N heterocomplexes and the reduced activity of the WT/T352P heterocomplexes , may boOur data clearly show the complexity of establishing a common pathological mechanism underlying SCA19/22, a complexity consistent with the clinical heterogeneity of this disease. Based on our results, we can only establish a relationship between the loss of function of the T352P mutant channel and a more severe phenotype and an earlier age of disease onset in patients, and between channel gating deficits caused by M373I, S390N and ΔF227 and milder ataxic symptoms. However, future work should prove whether SCA19/22-induced alterations in synaptic activity are associated with reduced neuronal survival and will reveal the role that these alterations play in neuroprotection .Supplementary material 1 (DOC 11756 kb)Below is the link to the electronic supplementary material.


Gossypium hirsutum homoeologous chromosome 12 encodes important genes that contribute to fiber fuzz, lethality, gland development and male sterility. In this study a physical map of the cotton TM-1 chromosome 12 was constructed. A number of large-insert cotton genome libraries are available, and genome-wide physical mapping using large insert segments combined with bacterial cloning is a thriving area of genome research. However, sequencing of the cotton genome is difficult due to sequence repeats and homoeologous regions. In order to effectively distinguish the homologous segments, a new method for adjusting the parameters of the FPC software was applied for contig map construction.The Hind III fingerprinting and used for construction of the contig map. Seven pairs of SSR markers located on A12 and D12 were chosen for contig analysis. Following optimization of the tolerance (10) and cutoff (1e-12) parameters, combining all clones from A12 and D12 produced two separate contigs.All available markers on chromosomes A12 and D12 were used to screen the TM-1 BAC library by PCR. A total of 775 clones  were obtained using The BAC contig map of chromosomes A12 and D12 was constructed and FPC software parameters were optimized for analysis. The resulting approach is a powerful platform for genome-wide and evolutionary research on cotton. Maxxa [Suyuan7235 [Zhongmiansuo 12 [0-613-2R [Pima 90–53 [BACs  are important resources for map-based cloning and large-scale sequencing of complex genomes due to their ability to stably maintain large DNA fragments that facilitate easy manipulation , 2. BAC Maxxa , Suyuan7yuan7235 , Zhongmi0-613-2R , and PimGossypium consists of over 50 species, including 40–45 diploids (2n = 2 × = 26) and five allotetraploids (2n = 4 × = 52) [N1,n2) at naked seeds [Le1, Le2 at hybrid lethality [gl2, gl3 at gossypol glanding, ne1,ne2 at mecariless, Bw1, Bw2 at withering bract, ms8,ms9 at male sterile [G. hirsutum acc. TM-1 has been constructed [1 (TM-1 × Hai7124) cross [Cotton is the source of the world most important plant-derived fiber, and is also an important oilseed crop, as well as a model species for the study of plant polyploidy, cellulose biosynthesis and cell wall biogenesis . The gen × = 52)  that wer × = 52) –14. The  × = 52) –17 which × = 52) . A12 anded seeds , Le1, Leethality , 21, gl2 sterile –26. For structed , 28. Fur4) cross –34.G. hirsutum L. Acc. TM-1 homoeologous chromosomes A12 and D12, the molecular markers identified in the aforementioned genetic map were used to screen the BAC library. BACs containing genetic markers that produced characteristic fingerprints when digested with the restriction enzyme Hind III were used to build contigs using the FPC software. A BAC fingerprint-based contig map of both A12 and D12 was successfully constructed by adjusting the tolerance and cutoff parameters in FPC. This approach will facilitate future map-based cloning of important cotton genes and expand our understanding of the relationships between the genetic and physical maps of A12 and D12.In this study, to facilitate construction of BAC contig maps for the BAC library screening was based on the published linkage map , and a tG. barbadense cv. Hai7124 and two in G. hirsutum acc. TM-1 . A total of 77 contigs from A12 were generated, ranging in size from 53 kb to 155 kb, whereas 82 individual contigs were produced for D12 ranging from 61 kb to 139 kb  was used in this study [Hind III and incorporated into the BAC vector pIndigBAC-5. The library consists of 147,456 clones with an average insert size of 122.8 kb that ranges from 97 to 240 kb. Approximately 96 % of the clones contain inserts over 100 kb, therefore this library represents 7.4 haploid genome equivalents in theory, based on an AD genome size of 2425 Mb. Clones were stored in 384-well plates and arrayed into multiplex pools for rapid and reliable library screening. BAC screening was carried using four-round PCR using SSR markers selected from the A12 and D12 high-density genetic maps derived from populations of the tetraploid Gossypium species.A BAC library constructed from is study . The libHindIII, and subjected to 1 % agarose gel electrophoresis at 40 V for 16 h. Restriction fragment identification was performed using IMAGE 3.10b software [BAC clones were inoculated into 96-well 2.2 mL plates, and each well contained 1.5 mL of 2xYT medium (12.5 μg/mL CM). Plates were covered with sealing film and incubated at 37 °C for 20–24 h on a shaker. BAC DNA was isolated using standard alkaline lysis , digestehttp://www.agcol.arizona.edu/software/fpc/) was used to assemble the physical map contigs from the BAC fingerprints. A series of tests were conducted in which fingerprints of a set of overlapping clones were compared using different tolerance values (from 5 to 10) and cutoff values (from 1e-01 to 1e-12). Based on these results, a tolerance of 10 and a primary cutoff of 1e-12 were selected for contig assembly.The computer program FPC V9.3 (


Lomentospora (Scedosporium) prolificans is a life-threatening opportunistic pathogen of immunocompromised humans, resistant to anti-fungal drugs. Melanin has been shown to protect human pathogenic fungi against antifungal drugs, oxidative killing and environmental stresses. To determine the protective role of melanin in L. prolificans to oxidative killing (H2O2), UV radiation and the polyene anti-fungal drug amphotericin B, targeted gene disruption was used to generate mutants of the pathogen lacking the dihydroxynaphthalene (DHN)-melanin biosynthetic enzymes polyketide synthase (PKS1), tetrahydroxynapthalene reductase (4HNR) and scytalone dehydratase (SCD1). Infectious propagules (spores) of the wild-type strain 3.1 were black/brown, whereas spores of the PKS-deficient mutant ΔLppks1::hph were white. Complementation of the albino mutant ΔLppks1::hph restored the black-brown spore pigmentation, while the 4HNR-deficient mutant ΔLp4hnr::hph and SCD-deficient mutant ΔLpscd1::hph both produced orange-yellow spores. The mutants ΔLppks1::hph and ΔLp4hnr::hph showed significant reductions in spore survival following H2O2 treatment, while spores of ΔLpscd1::hph and the ΔLppks1::hph complemented strain ΔLppks1::hph:PKS showed spore survivals similar to strain 3.1. Spores of the mutants ΔLp4hnr::hph and ΔLpscd1::hph and complemented strain ΔLppks1::hph:PKS showed spore survivals similar to 3.1 following exposure to UV radiation, but survival of ΔLppks1::hph spores was significantly reduced compared to the wild-type strain. Strain 3.1 and mutants ΔLp4hnr::hph and ΔLppks1::hph:PKS were resistant to amphotericin B while, paradoxically, the PKS1- and SCD1-deficient mutants showed significant increases in growth in the presence of the antifungal drug. Taken together, these results show that while melanin plays a protective role in the survival of the pathogen to oxidative killing and UV radiation, melanin does not contribute to its resistance to amphotericin B.The dematiaceous (melanised) fungus  Lomentospora prolificans  is a dematiaceous (melanised) fungus that has emerged over recent years as a serious and often life-threatening pathogen of immunocompromised individuals including those with AIDS, haematological malignancies and bone marrow and solid organ transplants   3] of DHses step  of melanses step  and is ises step . For taromycin B . The lefPKS1 gene was performed using a similar experimental strategy. Primer pairs Lppks1-LFF/Lppks1-LFR and Lppks1-RFF/Lppks1-RFR were used for amplification of 1.9-kb fragments from 5′ and 3′ flanks of the ORF respectively. The left and right fragments of the HPH gene were amplified as described. In a second round of PCR, the left flank was fused with one half of hygromycin cassette (HY) (1.2-kb) and the right flank with the other half of the hygromycin cassette (YG) (0.8-kb). The second round PCR products LF+HY (3.1-kb) and RF+YG (2.7-kb) were used for protoplast transformation. Homologous recombination resulted in the replacement of the PKS1 ORF with the functional HPH gene. Putative ΔLppks1::hph mutants were similarly selected based on resistance to 600 µg/mL of the antibiotic hygromycin B.Targeted replacement of the Lpscd1::hph mutant and six ΔLppks1::hph putative mutants were analysed  compared to the grey wild-type strain 3.1.Hygromycin B resistant mutants were investigated further by Southern blot analysis. A single putative Δanalysed . GenomicSCD1 ORF C or a 1.PKS1 ORF H. The sih mutant C, lane 3h mutant C, lane 1 is grey D,I in coh mutant E, while h mutant F, generaLppks1::hph mutant  (p < 0.05) reductions in survival of the mutants ΔLppks1::hph and ΔLp4hnr::hph, with survival percentages relative to matched controls of 21% and 24% respectively. There was no significant difference in the effect of H2O2 on survival of ΔLppscd1::hph spores when compared to strain 3.1 with a percentage survival, relative to its matched control, of 45%. A similar result was found with the mutant ΔLppks1::hph:PKS, with a percentage survival relative to its matched control of 46%, indicating that complementation had restored survival to wild-type levels.Exposure of spores of strain 3.1 to 160 mM H spores) A. When c2 resulted in 51% survival of strain 3.1 spores relative to non-irradiated control spores (p < 0.05) decrease in percentage survival of the mutant ΔLppks1::hph compared to the wild-type, with a percentage survival of 28%. There was no significant difference in effect of UV irradiation on the percentage survival of the mutants ΔLpscd1::hph and ΔLp4hnr::hph when compared to strain 3.1, with percentages of 40% and 42% respectively. A similar result was found with the mutant ΔLppks1::hph:PKS, with a percentage survival relative to its matched control of 63%, indicating that complementation had restored survival to wild-type levels.A UV dose of 200 mJ/cml spores B. Under L. prolificans tested to date are resistant to the broad-spectrum antifungal drug amphotericin B [Aspergillus fumigatus (AF293). We chose a concentration of 32 µg/mL, having first established that strain 3.1 of the fungus is resistant to concentrations >16 µg/mL, consistent with other studies [Lp4hnr1::hph was unaffected by the drug. Similarly, there was no significant difference in the growth of the complemented strain ΔLppks1::hph:PKS in the presence and absence of the drug, showing that restoration of melanin biosynthesis re-established drug insensitivity of the fungus to wild-type levels. Unexpectedly, significant increases in growth were found in the albino mutant ΔLppks1::hph and mutant ΔLpscd1::hph in the presence of the drug, while the drug completely inhibited growth of the control pathogen Aspergillus fumigatus as expected.All strains of ericin B ,15. Here studies ,18. GrowLomentospora prolificans is a dematiaceous (melanised) fungus that causes life-threatening disseminated infections in immunocompromised humans and patients with underlying respiratory problems [L. prolificans to UV radiation, to oxidative killing by H2O2, and inhibition by amphotericin B. To do this, we used targeted disruption of melanin biosynthesis genes to generate mutants blocked at different stages in the DHN-melanin pathway.problems ,10,11,12problems ,16,17,18problems ,30,32,33problems ,25,41. IL. prolificans mutant (ΔLp4hnr::hph) lacking the DHN-melanin enzyme tetrahydroxynaphthalene reductase (4HNR) which catalyzes the reduction of 1,3,6,8-THN to scytalone  and scytalone dehydratase (SCD1) enzyme activities, respectively. Furthermore, we complemented the PKS-deficient mutant to restore melanin biosynthesis in the ΔLppks1::hph mutant. The split marker technique of homologous recombination used here enables exchange of the open reading frame (ORF) of the enzyme-encoding gene with the hygromycin B phosphotransferase (HPH)-encoding gene, a selectable marker conferring resistance to the antibiotic hygromycin B. The procedure was carried out by amplification and fusion of linear DNA fragments to create the hygromycin-resistance cassette. A previous study had shown that the size of the sequences upstream and downstream of the ORF affects transformation efficiency [PKS ORF (6.7-kb). In the case of the SCD-encoding gene, the flank sizes were shorter (approximately 1.0-kb and 0.9-kb for left and right flanks respectively), owing to the smaller size of the SCD ORF of approximately 0.7-kb.In a previous study , we usedficiency  and so t−) mutants of L. prolificans that had unstable white phenotypes that reverted to the olive-grey to black characteristic of the wild-type strain, or mutants that had stable white phenotypes that varied in colony morphologies. Unlike UV mutagenesis, which is a random mutation process, targeted gene disruption allowed us to mutate individual enzyme-encoding genes involved in melanin biosynthesis in a highly specific way and to generate stable enzyme-deficient mutants. In the case of ΔLpscd1::hph, colonies of the mutant were beige while its spore suspensions were orange-yellow. A similar spore pigmentation was shown in the ΔLp4hnr::hph mutant developed previously that accumulated flaviolin as a shunt product [SCD1 gene in the ascomycete Grosmannia clavigera and showed this color was due to accumulation of the intermediate scytalone. Targeted disruption of the L. prolificans PKS-encoding gene resulted in the production of albino (white) ΔLppks1::hph mutants, similar to the white PKS-deficient mutants of Bipolaris oryzae and A. fumigatus developed elsewhere [Lppks1::hph mutant was successfully achieved, resulting in a mutant ΔLppks1::hph:PKS with grey pigmentation similar to that of the wild-type strain.Using this strategy, we were able to generate enzyme-deficient mutants that had abnormal pigmentation due to disruption of normal DHN-melanin biosynthesis. Previously, Ruiz-Diez and co-workers  used UV  product . Wang an product  producedlsewhere ,46. CompMetarhizium anisopliae to express DHN-melanin biosynthesis genes increased spore germination, hyphal branching and appressorium production. Mutants of the fungus Grosmannia clavigera with PKS and SCD gene disruptions had albino and reddish-brown phenotypes respectively, but had no marked differences in morphology, sporulation, or in vegetative growth compared to the wild-type strain. In the current study, vegetative growth and was significantly reduced in the 4HNR-deficient mutant ΔLp4hnr::hph consistent with previous results [Lppks1::hph:PKS. Spore production was reduced in ΔLp4hnr::hph and increased in ΔLpscd1::hph.Changes to normal melanin production in fungi can alter their morphology. Tseng and co-workers  showed t results , while gAlternaria alternata compared to melanised strains as a consequence of UV exposure, while another study demonstrated resistance to UV light of melanised strains of the human pathogen Sporothrixschenckii compared to a melanin-deficient mutant [2O2 in Cryptococcus neoformans, Aspergillus spp., S. schenckii and Fonsecaea pedrosoi [Lppks1::hph mutant showed increased sensitivity to UV and H2O2 in spore survival assays. Restoration of melanisation in the ΔLppks1::hph:PKS complemented strain and increased survival of ΔLppks1::hph:PKS spores to wild-type levels following UV and H2O2 exposure, shows that melanin provides protection against UV damage and tolerance to oxidative killing in L. prolificans. In contrast, another study knocked down the polyketide synthase gene in Penicillium marneffei and found no difference between the wild-type and the mutant in rates of survival following exposure to UV light [Melanin is known to contribute to the stress tolerances of fungi, including both plant and human pathogens, conferring resistance to UV radiation  and to ot mutant . In humat mutant  and is apedrosoi ,28,29,30UV light .L. prolificans to antifungal agents is well documented [C. neoformans and Histoplasmacapsulatum against amphotericin B, with non-melanised C. neoformans strains showing greater sensitivity to the drug than melanised strains [L. prolificans generated here (ΔLppks1::hph) showed significantly increased growth in the presence of amphotericin B, while complementation of the PKS gene in the mutant ΔLppks1::hph:PKS bestowed insensitivity to the drug, similar to that of the wild-type strain 3.1. The unexpected increase in growth of the albino mutant in the presence of the drug, a phenomenon also found with the ΔLpscd1::hph mutant, is difficult to explain. Paradoxical increases in growth of the human pathogens Candida albicans and Aspergillus fumigatus due to elevated concentrations of the anti-fungal echinocandin drug caspofungin have been reported [−) strains L. prolificans generated by UV mutagenesis [Lppks1::hph and ΔLpscd1::hph might be compensation for DHN-melanin loss through alternative pathways of melanin biosynthesis. In addition to DHN-melanins, the ascomycete fungi Aspergillus fumigatus and A. nidulans are able to synthesize pyomelanin and DOPA-melanin [L. prolificans. Notwithstanding this, we have shown that melanin does not contribute to the resistance of L. prolificans to amphotericin B, consistent with the findings of Ruiz-Diez and co-workers [The resistance of cumented ,16,17,18cumented  demonstr strains . Contrarreported ,53, but agenesis  were shoagenesis . An alte-melanin . Similar-workers .L. prolificans with protection from oxidative killing by H2O2 and protection from UV radiation, but is not involved in its resistance to the anti-fungal drug amphotericin B.In conclusion, we have shown using targeted deletion of melanin biosynthesis genes that melanin confers Lomentospora prolificans wild-type strain 3.1 [Aspergillus fumigatus strain AF293 were grown routinely on oatmeal agar  or Sabouraud dextrose agar  containing 2% agar) at 30 °C under a 16 h fluorescent light regime to induce sporulation. Agar was sterilized by autoclaving at 121 °C for 15 min.The rain 3.1 , gene di2O) and suspension with a sterile L-shaped spreader . The suspension was filtered through sterile Miracloth  to remove hyphal tissue, centrifuged at 13,000× g to pellet spores and the spores re-suspended in sterile MQ-H2O. Sterile 75 cm2 tissue culture flasks containing 100 mL of tissue culture medium  containing 10% fetal bovine serum , 2 mM l-glutamine , penicillin and streptomycin) were inoculated with spore suspension to give a final concentration of 104 spores/mL and incubated with shaking (35 rpm) in an Innova 4000 rotary incubator  for 48 h at 30 °C. Hyphal biomass was harvested by filtration through sterile Miracloth, washed with sterile MQ-H2O, blotted dry with paper towel  and stored at −80 °C until required. Frozen mycelium was ground to a fine powder in liquid N2 using a mortar and pestle, the powder transferred to 1.5 mL micro-centrifuge tubes and 800 µL of extraction buffer  added followed by 800 µL of phenol:chloroform:isoamyl alcohol mixture (25:24:1). The mixture was shaken gently for 30 s and then centrifuged at 14,000× g for 15 min at 4 °C. The upper layer was transferred to a fresh tube and mixed with an equal volume of phenol:chloroform:isoamyl alcohol mixture (25:24:1), vortexed for 30 s and centrifuged as described. The upper layer was combined with an equal volume of chloroform:isoamyl alcohol (24:1) and vortexed for 30 s. Following centrifugation for 5 min at 14,000× g, the upper layer was transferred to a fresh tube, the DNA precipitated by adding chilled isopropanol and pelleted by centrifugation at 4 °C for 15 min at 14,000× g. The pellet was washed with 70% ethanol and centrifuged for a further 10 min at 14,000× g. The pellet was dried for 15 min at 23 °C, re-suspended in 30 µL MQ-H2O containing RNAse and then incubated for 1 h at 37 °C. DNA quality and quantity was determined by both agarose gel electrophoresis and by using a Nanodrop spectrophotometer .DNA extraction was carried out according to the procedures described in Alastruey-Izquierdo and co-workers . Spores Restriction enzymes were obtained from Promega UK Ltd.  or New England Biolabs . DNA (0.2–1.0 μg) was digested with 5 to 10 units of enzyme in a total volume of 30 µL. For Southern blot analysis, digestion was performed by incubating 50 μg of genomic DNA with 60 units of restriction enzyme in a total volume 50 µL. In both cases, the mixtures were incubated at 37 °C for 16 h.L. prolificans strain 3.1 archived at Biosciences, University of Exeter [http://depts.washington.edu/bakerpg/primertemp/primermelttemp.html) and for reverse complements (http://arep.med.harvard.edu/labgc/adnan/projects/Utilities/revcomp.html). The primers used in this study are shown in The full genome sequence of f Exeter  was used® PCR System 2400 cycler . GoTaq® Green Master Mix , Phusion High Fidelity DNA polymerase  or Long PCR enzyme mix  were used according to the manufacturer’s instructions. For GoTaq, the reaction mixture contained 12.5 µL GoTaq Green Master Mix, 10 μM of forward and reverse primer, 50 ng of genomic DNA and nuclease free water to a total volume of 50 µL and PCR cycling conditions were an initial denaturation step at 98 °C for 3 min followed by 35 cycles at 98 °C for 1 min, 53.5–20 °C  for 1 min and at 72 °C, followed by a final extension step at 72 °C for 10 min. Phusion High Fidelity DNA polymerase reaction mixture contained 5 µL of 5× Phusion HF buffer, 0.5 µL of 10 mM dNTPs, 0.25 µL Phusion, 10 μM of forward and reverse primer, 50 ng of genomic DNA and nuclease-free water to a final volume of 50 µL. PCR cycling conditions were an initial denaturation step at 98 °C for 30 s followed by 35 cycles of 98 °C for 10 s, 62 °C for 30 s and 72 °C for 45 s, with a final extension for 10 min at 72 °C. Long PCR enzyme mix was used for PKS1 gene amplification. The PCR reaction mixture contained 5 µL of 10× Long PCR buffer with 15 mM MgCl2, 1 µL of 10 mM dNTPs, 1 µL of 10 μM of forward and reverse primer, 50 ng of genomic DNA, Long PCR enzyme mix to a total volume of 50 µL with nuclease-free water. PCR cycling conditions were an initial denaturation step at 94 °C for 3 min, 10 cycles at 94 °C for 20 s, 50 °C for 30 s and 68 °C for 7 min, followed by 25 cycles of 94 °C for 20 s, 50 °C for 30 s, 68 °C for 7 min. The final extension step was at 68 °C for 1 min.Polymerase Chain Reaction (PCR) was used to amplify DNA fragments in an Applied Biosystems GeneAmp2O). Electrophoresis was carried out in 1× Tris-borate EDTA buffer  for 1 h at 100 V. The DNA fragments were visualized using a UV transilluminator and fragment sizes was determined by comparison with a 1-kb ladder (Promega). Gel images were captured by using an Image Master VDS-CL gel documentation system  fitted with a Fujifilm FTI-500 Thermal Imaging system . DNA fragments were purified from agarose gels using Wizard® SV gel and PCR Clean-Up Systems (Promega) according to the manufacturer’s instructions. Purified DNA was stored at −20 °C until required.Agarose gel electrophoresis was used to fractionate digested DNA. The DNA was separated in 0.8% agarose gels and stained with ethidium bromide after mixing with loading dye  and 45 mL MQ-H® HD Cloning kit  according to the manufacturer’s instructions. The ligation reaction components were combined in a PCR tube in the following order: 2 μL 5× In-Fusion HD enzyme premix, 50 ng linearized vector, 200 ng purified PCR fragment and MQ-H2O to a total volume of 10 μL. The cloning reaction was incubated at 50 °C for 15 min and then kept on ice until required.Ligation of DNA fragments was carried out using the In-FusionEscherichia coli cells (Clontech) were thawed, placed on ice and mixed gently for even distribution. Fifty-μL of component cells were combined gently with 5 ng (2.5 μL) of In-Fusion cloning reaction. The tubes were incubated on ice for 30 min, heat-shocked at 42 °C for 45 s and then transferred to ice for 1–2 min. SOC medium  preheated to 37 °C was added to a total volume of 500 μL. The tube was incubated for 1 h at 37 °C with shaking (200 rpm) in an Innova 4000 rotary incubator. One hundred-μL were streaked on LB agar plates  containing 18 g/L agar) overlaid with X-Gal (40 µg/mL) and supplemented with 1 mL of 100 μg/mL ampicillin. Plates were inverted and incubated at 37 °C for 16 h. Colonies were selected based on white-blue screening.Stellar competent g for 1 min, the supernatant was discarded and the pellet re-suspended in 100 µL lysis solution (250 μL 1 M Tris-HCl (pH8.0), 200 μL 0.5 M EDTA, 170 μL, 60% sucrose, 2.4 mL MQ-H2O) and vortexed. Two hundred-μL of alkaline solution  was added and the samples were placed on ice for 10 min with occasional shaking. One hundred and fifty-μL of 3 M sodium acetate (pH 5.2) were added and the samples were incubated on ice for a further 10 min and then centrifuged  for 10 min at 4 °C. The supernatant was transferred to a fresh tube containing 1 mL of 100% ethanol and stored on ice for 10 min before centrifugation at 13,000× g for 10 min. This process was repeated with 70% ethanol, the pellet dried at 23 °C and then re-suspended in 30 µL of MQ-H2O containing 7 µL RNase (10 mg/mL). The DNA was stored at −20 °C until used.A single colony of transformed bacteria was used to inoculate 10 mL of LB broth containing 10 µL of 100 μg/mL ampicillin. The culture was incubated overnight at 37 °C with shaking (180 rpm) in an Innova 4000 rotary incubator (Eppendorf). A 1 mL aliquot of culture was transferred to a micro-centrifuge tube and, after centrifugation at 13,000× g for 10 min and the supernatant discarded. The pellet was re-suspended in 3 mL of cell re-suspension solution (50 mM Tris (pH 7.5), 10 mM EDTA, and 100 μg/mL of RNase). Three-mL of cell lysis solution  SDS) were added and the contents of the tube were mixed by inverting 3–2 times. After 3 min incubation at 23 °C, 5 mL of neutralization solution  was added, the tube was inverted 5–20 times and the lysate centrifuged at 14,000× g for 15 min. The PureYield™ purification system was used to purify DNA according to the manufacturer’s instructions and DNA stored at −20 °C until required.High quality plasmid DNA was isolated using the PureYield™ Plasmid Midiprep System (Promega) according to the manufacturer’s instructions. A single colony of bacterial cells was grown in 50 mL LB containing 1 mL of a 100 μg/mL ampicillin solution for 16 h at 37 °C with shaking (180 rpm) in an Innova 4000 rotary incubator (Eppendorf). The cells were centrifuged at 5,000× 4·7H2O, 1.5 mL 10 mM NaPO4, 1.8 g of 5% Glucanex , pH 5.6) and incubated at 30 °C with shaking (75 rpm) for 3 h. The contents of the tube were transferred to sterile polycarbonate Nalgene Oakridge tubes (Thermo Fisher) and the protoplasts were overlaid with chilled ST buffer (0.6 M Sorbitol and 0.1 M Tris-HCl (pH 7.0)). The protoplasts were centrifuged at 5000× g for 15 min at 4 °C using a swinging bucket rotor  in a Beckman J2.MC centrifuge (Beckman Coulter). The protoplasts were recovered at the OM/ST interface and transferred to a sterile Oakridge tube, which was filled with cold STC buffer , 10 mM CaCl2). Protoplasts were pelleted at 3000× g for 10 min at 4 °C and washed twice with 10 mL STC, with complete re-suspension each time. Protoplasts were re-suspended in 1 mL STC and numbers quantified using a haemocytometer. Protoplasts were combined with 3 µg DNA in a sterile micro-centrifuge tube in a final volume of 150 µL and incubated for 15 min at 23 °C. One-mL of PTC , 10 mM CaCl2) was added and the contents were mixed by gentle inversion. The protoplasts were incubated at 23 °C for 15 min, added to 150 mL molten (45 °C) OCM agar  chloride tetrahydrate, 5 mg/L iron(II) sulphate heptahydrate, 1.7 mg/L cobalt(II) chloride hexahydrate, 1.6 mg/L copper(II) sulphate pentahydrate, 1.5 mg/L sodium molybdate dehydrate, 50 mg/L EDTA), 10 g glucose, 2 g peptone, 1 g yeast extract, 1 g casamino acids, 1 mL vitamin solution , 273.84 g sucrose and 15 g agar in a final volume of 1 L, adjusted to pH 6.5 with 1 M NaOH) and the protoplast suspension poured into sterile 9 cm plastic culture dishes for incubation in the dark at 30 °C for 16 h. The plates were then overlaid with complete medium , 1 g/L casamino acids, 0.1% trace elements, 0.1%, 0.1% vitamin solution, 6 g/L NaNO3, 0.5 g/L, KCl, 0.5 g/L, MgSO4, 1.5 g/L, KH2PO4, (pH to 6.5 with NaOH), 15 g/L agar) containing 600 µg/mL hygromycin B  and were then incubated for 1 week in the dark at 30 °C. Colonies resistant to hygromycin B were sub-cultured on CM containing 200 µg/mL hygromycin B and finally onto OA. For selection of sulfonylurea-resistant transformants, CM was replaced with BDCM medium  with chlorimuron ethyl at a concentration of 300 μg/mL in the overlay, chlorimuron ethyl at a concentration of 100 μg/mL in BDCM sub-cultures and finally growth on OA.Spores were harvested from 2-week-old OA cultures, inoculated into 100 mL TCM and incubated for 3 days at 30 °C with shaking (35 rpm) in an Innova 4000 rotary incubator. Hyphal biomass was harvested by filtration through Miracloth and washed and dried as described. The mycelium was transferred to a 50 mL Falcon tube containing 40 mL OM buffer/Glucanex (44 g MgSOSouthern blotting was carried out according to the protocol of Southern . Thirty-4 (pH 7.0), 350 mL 20% SDS and Seradest added to a final volume of 1 L) at 62 °C for 30 min. Probes were denatured by boiling for 10 min and then added to hybridization bottles containing the membranes. Following hybridization at 62 °C for 16 h, the membranes were washed twice (15 min each) at 62 °C with Southern wash buffer (100 mL 1 M NaPO4 (pH 7.0), 50 mL 20% SDS and MQ-H2O to 1 L) and once at 23 °C using 20 mL DIG wash buffer 1  and with MQ-H2O added to 2 L) for 5 min. Membranes were submerged in 50 mL of blocking buffer (1 g semi-skimmed milk powder and 100 mL DIG buffer 1) for 30 min at 23 °C and then incubated with 50 mL of antibody solution (2.5 µL anti-digoxigenin-AP Fab fragments (Sigma-Aldrich) and 50 mL blocking buffer) under the same conditions. After two 15 min washes with DIG wash buffer and equilibriation with 20 mL DIG buffer 3  with MQ-H2O added to 2 L) for 5 min, membranes were incubated for 5 min with 2 mL CDP-star® chemiluminescent substrate solution (Sigma-Aldrich) and dried completely. Finally, the membrane was exposed to X-ray film (Fuji Photo Film (UK) Ltd., Manchester, Cheshire, UK) placed in a film cassette and incubated at 37 °C for 15 min. Films were developed using an OPTIMA X-Ray Film Processor .DNA probes were amplified by using Phusion HF buffer and the appropriate primers shown in 2O using plastic L-shaped spreaders, filtered through Miracloth, and spore concentrations determined using a haemocytometer. There were 3 replicates for each experiment and experiments were repeated 3 times.Strains were inoculated centrally onto replicate OA plates and colony diameters were measured over a 2-week growth period at 30 °C, with spore production quantified after 14 day. Spores were suspended in 20 mL dH2O2 that resulted in a 50% reduction in survival of the wild-type strain 3.1. Replicate spore suspensions with a concentration of 103 spores/mL were suspended in 100 mM phosphate buffer (pH 7.0) comprising a range of mM concentrations of H2O2. Following exposure, 300 µL of spore suspensions were spread on the surface of SDA plates and incubated for 3 day at 30 °C in the dark. The control consisted of untreated spores (suspended in buffer only) and percentage survival was determined from the numbers of colonies that developed from treated spores compared to the untreated control. Through this process it was determined that exposure of spores to 160 mM H2O2 for 60 min resulted in 50% survival of treated spores of the wild-type compared to the matched untreated control. All subsequent experiments using mutant strains were conducted using this same treatment regime and percentage survival relative to matched controls determined as described. There were 3 replicates for each strain and experiments were conducted three times.Preliminary investigations set out to establish the molarity and exposure time to H3 spores/mL were exposed to UV light generated by a HL-2000 HybriLinker Hybridization oven (UVP) with doses of 100, 200, 300, 400 and 500 mJ/cm2. Following exposure, 300 µL of spore suspensions were spread on the surface of SDA plates and incubated for 3 days at 30 °C in the dark. The control consisted of non-irradiated spores and percentage survival was determined from the numbers of colonies that developed from irradiated spores compared to the non-irradiated control. Through this process it was determined that a UV dose of 200 mJ/cm2 resulted in 50% survival of irradiated spores of the wild-type compared to the matched non-irradiated control. All subsequent experiments using mutant strains were conducted at this dose and percentage survivals relative to matched controls determined as described. There were 3 replicates for each strain and experiments were conducted three times.Preliminary investigations aimed to establish the UV dose at 254 nm that resulted in a 50% reduction in survival of the wild-type strain 3.1. Replicate spore suspensions with a concentration of 102O only. Tissue culture flasks (75 cm2) containing AmB or control media were inoculated with spores of the L. prolificans strains, or the drug-sensitive pathogen Aspergillus fumigatus (strain AF293), to give a final concentration of 106 spores/mL. Flasks were incubated for 3 days at 30 °C with shaking (48 rpm) in an Innova 4000 rotary incubator and the mycelium was harvested, dried at 70 °C for 48 h and dry weights obtained. The experiment was repeated three times with three replicates for each strain and treatment.The sensitivities of the mutants and strain 3.1 to the polyene amphotericin B  were tested using a liquid culture method. Tissue culture medium was amended with a liquid formulation of AmB  to give final concentration of 32 µg/mL and the control consisted of TCM amended with an equivalent volume of sterile MQ-H®, Coventry, UK). Analysis of variance (ANOVA) was used to compare means of more than two data sets and Post-hoc Tukey-Kramer analysis was then performed to distinguish which sets were significantly different from one another. For comparisons of percentages, data was transformed using the arcsin−1 function prior to statistical analysis.Unless otherwise stated, numerical data were analyzed using the statistical program Minitab (Minitab 16, Minitab