Pseudomonas putida Bet001 isolated from palm oil mill effluent was studied. The biosynthesis of mcl-PHA in this newly isolated microorganism follows a growth-associated trend. Mcl-PHA accumulation ranging from 49.7 to 68.9% on cell dry weight (CDW) basis were observed when fatty acids ranging from octanoic acid (C8∶0) to oleic acid (C18∶1) were used as sole carbon and energy source. Molecular weight of the polymer was found to be ranging from 55.7 to 77.7 kDa. Depending on the type of fatty acid used, the 1H NMR and GCMSMS analyses of the chiral polymer showed a composition of even and odd carbon atom chain with monomer length of C4 to C14 with C8 and C10 as the principal monomers. No unsaturated monomer was detected. Thermo-chemical analyses showed the accumulated PHA to be semi-crystalline polymer with good thermal stability, having a thermal degradation temperature (Td) of 264.6 to 318.8 (±0.2) oC, melting temperature (Tm) of 43. (±0.2) oC, glass transition temperature (Tg) of −1.0 (±0.2) oC and apparent melting enthalpy of fusion (ΔHf) of 100.9 (±0.1) J g−1.The biosynthesis and characterization of medium chain length poly-3-hydroxyalkanoates (mcl-PHA) produced by  The current increase in the utilization of polyhydroxyalkanoates (PHAs) in various industrial and biomedical applications is due to their biodegradability, compatibility, resorbability and piezoelectricity PseudomonasThe low level of medium-chain-length PHA (mcl-PHA) and limited substrate utilization in different bacterial species, poses limitation to industrial production of these polymers 18∶1) to produce mcl-PHA with unsaturated side-chain monomers Pseudomonas putida isolated from palm oil mill effluent are reported, along with characterization of the mcl-PHA copolymer produced when the bacterium is fed with oleic acid (C18∶1). For comparison, selected fatty acids viz. octanoic acid (C8∶0), dodecanoic acid (C12∶0) and hexadecanoic acid (C16∶0) were also tested as substrates.A number of published works use unsaturated fatty acids such as oleic acid : peptone 20; K2SO4 10; MgCl2.6H2O 1.4; irgasan 0.025; glycerol 25.2 and agar 13.6. Preliminary biochemical characterization was done using api® 20 NE (bioMerieux® USA) biochemical typing kits as stated in manufacturer’s guideline. Intracellular PHA accumulation was screened using both Sudan Black B and Nile red staining techniques according to literatures −1): yeast extract 10.0 (Bacto™ USA), nutrient broth 15.0  and ammonium sulfate 5.0  was used as inoculum cultivation medium for the bacterium. 3% (v/v) of P. putida Bet001 stock inoculum was introduced into this medium aseptically and incubated in shaking incubator  at 30°C, 250 rpm for 24 hours. The biomass was harvested from 3 ml culture broth via centrifugation at 4°C, 9000 ×g for 10 min. The recovered biomass was seeded aseptically into PHA production medium. The medium contains sterile minerals of (g L−1) sodium ammonium hydrogen phosphate tetrahydrate [NaNH4HPO4.4H2O] 3.5, K2HPO4 5.7, KH2PO4 3.7, and 10 mM oleic acid (unless otherwise stated) at a pH of 7.0 (±0.1). Separate sterile solutions of 0.1 M MgSO4.7H2O and trace elements (MT) containing (g L−1): CaCl2.2H2O 1.47, CoCl2.6H2O 2.38, CuCl2.2H2O 0.17, FeSO4.7H2O 2.78, MnCl2.4H2O 1.98 and ZnSO4.7H2O 0.29 dissolved in 1 M HCl were aseptically added to the PHA production media prior to inoculation at 1.0% (v/v) and 0.1% (v/v), respectively. 5 g L−1 of harvested biomass from inoculum growth media was seeded into this medium  aseptically and incubated as described previously for a total period of 48 hours unless otherwise stated. PHA extraction was done according to literature Sterile growth medium  containing  at the absorbance of 600 nm. Aliquote samples were withdrawn aseptically at regular intervals and centrifuged at 9000 ×n-heptane (3 ml) and centrifuge at 9000 × g to separate the residual fatty acid. 2 ml of the top layer of n-heptane was withdrawn into a test tube, to this solution, 200 µl of 5% (w/v) copper (II) acetate monohydrate solution (5 g copper (II) acetate monohydrate dissolved in 90 ml distilled water; pyridine and distilled water were respectively used to adjust the solution pH to 6 and bring the final volume to100 ml) was added and vortexed for 60 sec. This mixture was allowed to stand for 20 sec before it was read at 705 nm against distilled water blank using Jasco V-630 UV/VIS spectrophotometer . A standard calibration was performed using a stock of different fatty acid concentrations (0.5 to 1 mM) that were made by dissolving the specified mass of fatty acid in n-heptane and treated as mentioned earlier.The residual fatty acid estimation was done according to Marseno et al. Residual ammonium was quantified using phenol hypochlorite method as described by Solorzano spectrum-400 spectrometer  at room temperature (27°C) and a scan range of 400 to 4000 cm−1 was applied for 10 scans. A paste of 0.01 g of the sample was prepared in dichloromethane. A liquid film of the mixture was then applied on to NaCl crystal window and the spectrum was read after the solvent has been dried.Attenuated total reflectance (ATR) FTIR was used to record the PHA spectrum on Perkin-Elmer FTIR 1H NMR spectrum at 250 MHz against tetramethylsilane (TMS) internal reference standard. Approximately 5 mg PHA sample was dissolved in 2 ml deuterated chloroform (CDCl3) and filtered into NMR tube using borosilicate glass syringe equipped with 0.22 µm PTFE disposable filter .JEOL JNM-GSX 270 FT-NMR  spectrometer was used to record the −1, hold for 2 min, and increase to 250°C at 10°C min−1 hold for 15 min. Helium was used as carrier gas at 48.3 ml min−1 and 0.41 bar pressure. Mass spectra were acquired at 1250 scan speed using electron impact energy of 70 eV at 200°C ion-source and 280oC interface temperatures respectively. Standard monomers of methyl hydroxyalkanoates  were used as reference for peak retention time and ionization mass determination.GC-MSMS was recorded on Agilent triple quadrupole 7000B , equipped with GCMSMS triple axis detector carrying Agilent HP-5 ms column . PHA sample in the form of hydroxyalkanoic acids methyl esters were used for the GC-MSMS analysis according to method reported in literatures 2, 2.63×103, 9.10×103, 3.79×104, 3.55×105, 7.06×105, 3.84×106 and 6.77×106 Da). The PHA samples were dissolved in tetrahydrofuran (THF) at a concentration of 2.0 mg mL−1, filtered through a 0.22 µm PTFE filter. 100 µL aliquot of the sample was injected at 40°C using THF as a mobile phase at a flow rate of 1.0 mL min−1.Waters 600  equipped with a Waters refractive index detector (model 2414) having the following gel columns  in series: HR1, HR2, HR5E and HR5E Waters Styragel HR-THF was used to record the chromatogram relative to calibration curve of standard monodisperse polystyrenes (3.72 x10−1 under a nitrogen flow rate of 0.12 L min−1at a head pressure of 1.5 bar. The melting temperature (Tm) was taken at the endothermic peak of the DSC thermogram. The endothermic melting enthalpy (ΔHm) was used to calculate the polymer crystallinity (Xp) on the basis of melting enthalpy (ΔHmo) of 100% crystalline PHB according to −1 as the melting enthalpy of 100% crystalline PHB as cited in literature The extracted PHA was subjected to differential thermal analysis using Mettler-Toledo differential scanning calorimeter  running STARe DSC ver 8.10 software; equipped with HAAKE EK90/MT digital immersion cooler . Scans were made at a temperature range of −60°C to 180°C with a heating rate of 10°C min−1 under a nitrogen flow rate of 20 ml min−1.TGA analysis was performed on a Perkin-Elmer TGA 4000 instrument. The sample was heated from 50°C to 900°C at a rate of 10°C minPseudomonas putida. This result was corroborated by RDP blast neighbor-joining phylogenetic analysis  ester bond stretching vibration according to Randriamahefa et al. −1 have been assigned to bacterial intracellular amide (–CO–N–) I and II respectively  and triplet peaks 2 (δ 5.2 ppm) are assigned to methylene and methine protons of the α and β-carbon respectively Tm) of this polymer to be 43.0 (±0.2) oC, glass transition temperature (Tg) of −1.0 (±0.2) oC and apparent melting enthalpy of fusion (ΔHf) of 100.9 (±0.1) J g−1. The relatively low melting and glass transition temperatures were attributed to the random composition of 3HA monomers in the polymer (see later discussion). This observation was found to be in good agreement with what has been reported by Matsusaki et al. Pseudomonas sp. 6 1–3 strain fed with dodecanoic acid (C12∶0). The bacterium produced random copolymer of PHA with endothermic melting temperature of 42°C. The observed high apparent heat of fusion could be due to the presence of long chain monomer fraction which in turn was reported to favor side-chain crystallization resulting in polymer with high apparent enthalpy of fusion. This observation was found to agree with reported literatures The calorimetric scan of the extracted PHA showed the endothermic melting temperature .This is attributed to the increase in the fractions of longer monomer such as 3HDD and 3HTD that became incorporated into the PHA from the metabolism of the longer fatty acid substrates  to 3HTD (fourteen carbon atom length) except when oleic acid was used as substrate, a small amount of odd chain monomer i.e. 3HHp (seven carbon atom length) was also detected. At first, this was considered as possible contamination of the extracted polymer sample by cellular components. Thus, the purification step was repeated at least three times and the polymer re-extracted for GCMSMS analysis. The persistence of the strong signal detected by mass spectrometry for the presence of 3HHp entails re-consideration of the contamination hypothesis with non-PHA components. From the basic knowledge about β-oxidation of even carbon atom length of fatty acids, it is not clear at this point the exact reason for the incorporation of an odd carbon atom length monomer like 3HHp into the polymer when oleic acid (C18∶1) was used as a sole carbon and energy source for the bacterial growth and PHA accumulation, and not with other fatty acids tested.The effect of different carbon source on PHA composition has been studied. The composition of PHA accumulated by P. entomophila L48 to accumulate PHA having four different monomer units when fed with dodecanoic acid, with monomer fractions of 3HD (38.6%), 3HO (44.5%) as the dominant fractions. Feeding hexadecanoic acid as a carbon and energy source to P. putida Bet001 resulted in PHA with five different monomers . When oleic acid (C18∶1) was fed under the same conditions, copolymer with up to seven different monomers of 3HB (1.52 mole%), 3HHx (5.01 mole%), 3HO (31.81 mole%), 3HD (24.07 mole%), 3HDD (22.86 mole%), 3HTD (14.05 mole%) and a small amount of odd carbon chain length monomer of 3-hydroxyheptanoate (3HHp) (0.68 mole mole%) were obtained. In contrast to other fatty acids tested, when oleic acid was fed, a short-chain length monomer of 3HB (four carbon atom length) accumulated. The accumulation of 3HB monomer was not detected in the bacterium when fatty acids C8∶0, C12∶0 and C16∶0 were fed. Again, similar to the accumulation of 3HHp, the exact reason for this observation is not clear at this stage. It was also observed that when oleic acid (C18∶1) was fed, no unsaturated PHA monomer was detected in the polymer produced. The most abundant monomers were C8 and C10 in all the samples tested, and this is in agreement with the hypothesis that PHA formed by Pseudomonads belonging to rRNA homology group I have C8 and C10 monomers as dominant components when grown on C8 to C18 fatty acids substrate Based on mass spectrometric analyses, feeding dodecanoic acid as sole carbon source resulted in PHA composed of four different monomers . Similarly, Chung et al. From the results presented in P. putida Bet001 is depicted in −1, the specific growth (µ) also increases from 0.005 to 0.036 h−1with corresponding PHA yield of 13.4 to 68.9% (w/w) respectively. Increasing nitrogen source beyond this point resulted in a gradual plateau in the specific growth rate, probably due to the carbon source concentration began to be limited as in this study where the ammonium ion concentration was varied and the carbon source concentration was kept fixed. In a similar study, Annuar et al. P. putida PGA1 from 0 to 0.18 h−1 by increasing the ammonium ion concentration from 0 to 0.1 g L−1; further increase in ammonium ion concentration (0.6 to 0.8 g L−1) resulted in a decrease in the specific growth rate to 0.14 h−1. Interestingly, the PHA fraction from the total biomass also increases as the specific growth rate increases, indicating a growth associated PHA biosynthesis in this bacterium. The PHA fraction reached a constant value when specific growth rate became constant at high ammonium ion concentrations. This is in contrast with the recognized PHA accumulation in many Pseudomonas species, where the highest accumulation is observed when the specific growth rate of the organism is low under nutrient limitation/starvation −1) could probably be due to high ammonium uptake by the cells The effect of nitrogen source concentration on specific growth rate and PHA yield during PHA biosynthesis in −1 while varying the carbon source concentration; it is observed that increase in C:N molar ratio from 10 to 30 was followed by increase in percentage PHA accumulation and biomass from 16 to 69% and from 9.6 to 15.3 g L−1, respectively. The proportional increase in biomass with increase of C:N ratio up to 20, could indicate that below 20 carbon is limiting growth. Additional increase in C:N molar ratio after 20 however, did not results in further increase in biomass amount, as it is expected that the ammonium concentration no longer sufficient to sustain extended growth. The results from this experiment offer additional support for the growth-linked PHA accumulation in this bacterium. Maximum PHA accumulation at approximately 70% was found for C:N molar ratios of 25 and 30. The molecular weight of the PHA from the different C:N molar ratios tested fell within a narrow range of 37 to 75 kDa. The crystallinity (Xp) and poly dispersity index (PDI) remained constant at all the C:N molar ratio tested.It has been observed that PHA content of the cell can be increased by changing the molar ratio between carbon-to-nitrogen (C:N) source . By fixiP. putida Bet001 when fed with different fatty acids. The biosynthesis of the PHA by this new isolate is growth-associated. When fed with oleic acid (C18∶1), the PHA extracted from the isolate showed the presence of a monomer with odd carbon atom length i.e. 3-hydroxyheptanoate (3HHp). However, no unsaturated monomer was detected when oleic acid (C18∶1) was fed. P. putida Bet001 accumulated about 69% PHA on cell dry weight basis with good thermal stability, relatively high molecular weight and crystallinity.A chiral PHA copolymer consisting of monomers ranging from 3-hydroxybutyrate (3HB) to 3-hydroxytetradecanoate (3HTD) were synthesized by a new isolate


To better define the role that these post-translational modifications play, we created homozygous LKB1S431A/S431A and LKB1C433S/C433S knockin mice. These animals were viable, fertile and displayed no overt phenotypes. Employing a farnesylation-specific monoclonal antibody that we generated, we established by immunoprecipitation that the vast majority, if not all, of the endogenous LKB1 is prenylated. Levels of LKB1 localized at the membrane of the liver of LKB1C433S/C433S mice and their fibroblasts were reduced substantially compared with the wild-type mice, confirming that farnesylation plays a role in mediating membrane association. Although AMPK was activated normally in the LKB1S431A/S431A animals, we unexpectedly observed in all of the examined tissues and cells taken from LKB1C433S/C433S mice that the basal, as well as that induced by the AMP-mimetic AICAR (5-amino-4-imidazolecarboxamide riboside), AMPK activation, phenformin and muscle contraction were significantly blunted. This resulted in a reduced ability of AICAR to inhibit lipid synthesis in primary hepatocytes isolated from LKB1C433S/C433S mice. The activity of several of the AMPK-related kinases analysed  was not affected in tissues derived from LKB1S431A/S431A or LKB1C433S/C433S mice. Our observations reveal for the first time that farnesylation of LKB1 is required for the activation of AMPK. Previous reports have indicated that a pool of AMPK is localized at the plasma membrane as a result of myristoylation of its regulatory AMPKβ subunit. This raises the possibility that LKB1 farnesylation and myristoylation of AMPKβ might promote the interaction and co-localization of these enzymes on a two-dimensional membrane surface and thereby promote efficient activation of AMPK.The LKB1 tumour suppressor protein kinase functions to activate two isoforms of AMPK (AMP-activated protein kinase) and 12 members of the AMPK-related family of protein kinases. The highly conserved C-terminal residues of LKB1 are phosphorylated (Ser 431 was essential for orchestrating neuronal cell polarity and embryo development.Our observations reveal a significant role for the farnesylation of LKB1 being required for the efficient activation of AMPK. Our findings also cast doubt on previous claims that phosphorylation of Ser LKB1 kinase tumour suppressor gene cause the inherited Peutz–Jeghers cancer syndrome, in which patients are predisposed to developing benign and malignant tumours  possesses a residue at its C-terminus that is equivalent Ser431, but lacks the CAAX prenylation motif glucose and D-[14C]mannitol were from PerkinElmer, and [γ-32P]ATP and Protein G–Sepharose were purchased from GE Healthcare. Phosphocellulose P81 paper was from Whatman. [1-14C]Acetic acid was obtained from American Radiolabeled Chemicals. Phenformin, forskolin and PMA were purchased from Sigma. All plasmids, antibodies and recombinant proteins that we have generated for the present study are available on request from our reagents website (http:s://mrcppureagents.dundee.ac.uk/).Complete protease inhibitor cocktail tablets were obtained from Roche, AICAR (5-amino-4-imidazolecarboxamide riboside) was from Apollo Scientific, 2-deoxy-237, anti-TBC1D1 , anti-BRSK1  and anti-BRSK2 . The anti-HA (haemagglutinin) antibody was from Roche. The anti-LKB1 , anti-phospho-LKB1 Ser431 , anti-GAPDH , anti-HSP90 , anti-Na+/K+-ATPase , anti-phospho-AMPK Thr172 , anti-AMPKα1/α2  anti-phospho-raptor [regulatory associated protein of mTOR ] Ser792 , anti-raptor , anti-phospho-ACC (acetyl-CoA carboxylase) Ser79 , anti-ACC , anti-NUAK1/ARK5 , anti-lamin A/C , anti-EEA1 , anti-BiP , anti-LAMP1 , anti-phospho-ERK1/2 Thr202/Tyr204  and anti-ERK1/2  were purchased from Cell Signaling Technology. Secondary antibodies coupled to HRP (horseradish peroxidase) were from Thermo Scientific. We also attempted to analyse the expression of endogenous mouse LKB1 by immunofluorescence. However, we were unable to identify an antibody that was capable of specifically localizing LKB1 expression in wild-type MEFs (mouse embryonic fibroblasts) in which no signal was observed in parallel experiments with LKB1-knockout MEFs. The antibodies tested were anti-LKB1  from Cell Signaling Technology and anti-LKB1 [Ley 37D/G6]  from Abcam. The protocol used was as follows. Cells for immunofluorescence studies were grown on glass coverslips. They were fixed in 4%paraformaldehyde for 10 min followed by permeabilization with 1% Nonidet P40 for 15 min. Blocking was performed with donkey serum for 1 h and the cells were then incubated with primary antibodies diluted in PBS containing 0.2% BSA and 0.02% sodium azide for 1 h at room temperature (20°C). Cells were washed with PBS containing 0.2% BSA and 0.02% sodium azide and incubated with Alexa Fluor®-conjugated secondary antibodies (raised in donkeys) for 1 h at room temperature. Cells were then washed with PBS containing 0.2% BSA and 0.02% sodium azide and the slides mounted with a mounting medium followed by visualization with a confocal microscope.The following antibodies were raised by the DSTT  at the University of Dundee in sheep and affinity-purified against the indicated antigens: anti-AMPKα1  and anti-AMPKα2  , and antEscherichia coli BL21 cells and cultured at 37°C with shaking until a D value of 0.8 was achieved. Protein expression was induced with 100 μM IPTG for 4 h at 37°C with shaking. Bacteria were pelleted by centrifugation at 6500 g for 30 min at 4°C. Cells were then resuspended in 17 ml of lysis buffer  per litre of culture. Cells were lysed by sonication at 70% amplitude for 1 min on ice and the resulting supernatant was clarified by centrifugation at 31500 g. The supernatant was passed over a 10 ml chelating Sepharose IDA column (GE Life Sciences) loaded with NiSO4. Unbound protein was washed from the column using 10 bed volumes of lysis buffer or until the UV 280 value returned to base levels. His-tagged proteins were eluted over a 0–500 mM imidazole gradient and the fractions containing the protein pooled. Following buffer exchange on a Hiprep 26/10 desalting column (GE Life Sciences) into 25 mM Hepes (pH 7.2), the pooled fraction was loaded on to a Hitrap Q column (GE Life Sciences). After washing with 5 bed volumes of the loading buffer, the protein was eluted over a 0–500 mM NaCl gradient. A final purification across a G200 Sephadex column into 25 mM Hepes (pH 7.2), 40 mM NaCl and 1 mM DTT resulted in a highly pure and active enzyme.The expression plasmid encoding the His-tagged recombinant farnesyl transferase α and β subunits was from Dr Aymelt Itzen . It was transformed into Farnesyl pyrophosphate was synthesized as described previously .°C for 3 h in a prenylation buffer containing 25 mM Hepes (pH 8.5), 40 mM NaCl, 2 mM MgCl2, 20 μM ZnCl2, 1 mM TCEP [tris(2-carboxyethyl)phosphine], 160 μM farnesyl pyrophosphate and 5 μM farnesyl transferase.The LKB1 peptide (CKIRRLSACKQQ) (synthesized by GL Biochem) was farnesylated at 3718 sorbent (for strong hydrophobic retention by the active silanol groups) cartridges (Phenomenex) at 25% (non-farnesylated), 60% (farnesylated) and 100% acetonitrile/0.1% trifluoroacetic acid. The presence and purity of the peptide in the different fractions were tested by MALDI–TOF-MS  using a mixture of α-cyano-4-hydroxycinnamic acid and 2,5-dyhydroxybenzoic acid (1:1) as a matrix, in reflectron positive mode. For external calibration, a mix of nine mono-isotopic masses were used, i.e. bradykinin, [M+H]+=757.3992 Da; angiotensin II, [M+H]+=1046.5418 Da; angiotensin I, [M+H]+=1296.6848 Da; substance P, [M+H]+=1347.7354 Da; bombesin, [M+H]+=1619.8223 Da; renin substrate, [M+H]+=1758.9326 Da; ACTH (adrenocorticotropic hormone) clip 1–17, [M+H]+=2093.0862 Da; ACTH clip 18–39, [M+H]+=2465.1983 Da; and somatostatin 28, [M+H]+=3147.4710 Da .Isolation of the non-farnesylated from the farnesylated peptide was done by SPE (solid-phase extraction) using STRATA™-C18-E polymeric reversed phase end-capped C18 column  and peptide separation was achieved with a flow rate of 0.20 ml/min . A gradient (slope 5) spanning 0–100% mobile phase B over 130 min was used.To assess the efficiency of the reaction and thus the purity of the peptide after the farnesylation reaction, the product was run on an off-line HPLC Ultimate 3000 system (Thermo Scientific) and the chromatograms were acquired using a VWD-3400RS UV–Vis photometer at a wavelength of 214 nM. Samples were run on a highly resolving CThe purified LKB1 farnesylated peptide (10 mg) was used by Dundee Cell Products to produce a mouse monoclonal antibody that strongly recognized this antigen. The hybridoma expressing the most sensitive immunoblotting antibody that recognized farnesylated LKB1 was selected and antibody purified accordingly to the company's protocol.C433S/C433S and LKB1S431A/S431A mice. The mice were generated and maintained on a C57BL/6J background. Genotyping was performed by PCR using genomic DNA isolated from ear biopsies. Primer 1 (5′-CTAGTGTGGC-CAAGTCAGAGG-3′) and primer 2 (5′-AGACCAGCTTGCTC-TGTTGG-3′) were used to detect the wild-type and knockin alleles. The PCR program consisted of 5 min at 95°C, then 35 cycles of 30 s at 95°C, 30 s at 60°C, 1 min at 72°C and 10 min at 72°C.TaconicArtemis generated the LKB1All animal studies and breeding was approved by the University of Dundee ethical committee and performed under a U.K. Home Office project licence.Mice were fasted for 6 h and injected intraperitoneally with glucose (2 g/kg of body mass) or AICAR (250 mg/kg of body mass) diluted in PBS. Blood was collected from the tail tip before and at various times after injection. Blood glucose levels were measured by an AlphaTrak glucometer (Abbott Laboratories).C433S/C433S and LKB1S431A/S431A MEFs isolated from mouse embryos at E13.5 (embryonic day 13.5) were generated as described previously  containing 5% non-fat dried skimmed milk powder for 1 h. The membranes were then probed with the primary antibody  for 16 h at 4°C for 1 h on a shaking platform with 5 μl of protein G–Sepharose coupled to anti-LKB1, anti-AMPKα1, anti-AMPKα2, anti-SIK3, anti-NUAK1, anti-MARK4, anti-BRSK1 and anti-BRSK2 (sheep) antibodies. The immunoprecipitates were washed twice with 1 ml of lysis buffer containing 0.5 M NaCl and twice with buffer A . Phosphotransferase activity towards the LKBtide peptide (SNLYHQGKFLQTFCGSPLYRRR) for LKB1, AMARA peptide (AMARAASAAALARRR) for AMPKα1 and AMPKα2, or Sakamototide substrate (ALNRTSSDSALHRRR) [32P]ATP and 200 μM LKBtide peptide, AMARA peptide or Sakamototide substrate. The assays were carried out at 30°C with continuous shaking to keep the immunoprecipitates in suspension and were terminated after 20 min by applying 40 μl of the reaction mixture on to p81 paper. These were washed in phosphoric acid and the incorporated radioactivity was measured by scintillation counting. One milliunit of activity was defined as that which catalysed the incorporation of 1 pmol of 32P into the substrate per min. To assess LKB1 activity by measuring the activation of an AMPK complex, endogenous LKB1 was immunoprecipitated from mouse tissue lysates (0.5 mg) as described above and incubated with 0.3 μg of dephosphorylated inactive E. coli-expressed recombinant AMPK complex (α1β2γ1 subunits) as described previously [Tissues or cell lysates (0.05–2 mg) were incubated at 4SALHRRR)  for SIK314C]acetic acid in the presence or absence of increasing concentrations of AICAR. Cells were washed three times with ice-cold PBS and then scraped into chilled PBS. Primary hepatocytes were pelleted by centrifugation at 2000 g for 2 min. A methanol/chloroform lipid extraction was performed on the cell pellets. After the extraction, the insoluble material (pellet) was dissolved in 0.5 M NaOH/0.5% SDS in order to quantify the amount of protein per well. The solvent found in the supernatant was evaporated and the incorporation of [14C] into the lipid was measured by scintillation counting. The incorporation rate was defined in units of μmol of acetate/g of protein per h.Primary hepatocytes were seeded in six-well plates. After an overnight incubation in serum-free M199 medium containing 100 nM dexamethasone, hepatocytes were labelled for 1 h with 1 mM acetate and 0.5 μCi/ml  that, to our knowledge, has not been previously reported to be farnesylated (Supplementary Tables S1 and S2 at http://www.biochemj.org/bj/458/bj4580041add.htm). The anti-LKB1 farnesylation-specific antibody may also recognize geranylgeranylated prenylated proteins, as we found at least eight Rab GTPase isoforms co-immunoprecipitating with the anti-LKB1 farnesylation-specific antibody that are modified by introduction of two geranylgeranyl groups on to two cysteine residues at the C-terminal consensus sequence, XCXC or XXCC, which is introduced by the Rab geranylgeranyl transferase  mutant was expressed at similar levels to the wild-type LKB1 in the liver, muscle, spleen, kidney and MEFs . Moreover, the fraction labelled membrane was also contaminated with ER as identified using immunoblot analysis with a GRP78/BiP marker . We also attempted to analyse the expression of endogenous mouse LKB1 by immunofluorescence. However, we were unable to identify an antibody that was capable of specifically localizing LKB1 expression in wild-type MEFs in which no signal was also observed in the LKB1-knockout MEFs examined in parallel experiments .To assess whether loss of LKB1 farnesylation affected membrane localization, we fractionated liver and MEF , an analogue of AMP capable of stimulating AMPK via an LKB1-dependent mechanism  phosphorylated the artificial substrate LKBtide peptide and activated the recombinant heterotrimeric AMPK complex to the same extent as wild-type LKB1 E and 2F.n AMPKβ1  and AMPKn AMPKβ1  could fun AMPKβ1 . If LKB1n AMPKβ1 . Moreove of AMPK .2 of AMPKβ1 and AMPKβ2 is mutated to ablate myristoylation. If a pool of AMPK was activated by LKB1 at the plasma membrane, there is some analogy with other signalling systems, such as the PDK1 (phosphoinositide-dependent kinase 1) network. The PDK1 pro-tein kinase possesses a PH (pleckstrin homology) domain that promotes membrane association through its ability to interact with the 3-phosphoinositide second messenger product of the class 1 PI3K (phosphoinositide 3-kinase) pathway PtdInsP3 [P3-binding PH domain at the plasma membrane. Binding of Akt to PtdInsP3 induces a marked conformational change that exposes Thr308, the residue that PDK1 phosphorylates [In vitro-reconstitution studies showed that co-localization of PDK1 and Akt on lipid vesicles containing PtdInsP3 markedly increased in the efficiency at which PDK1 can phosphorylate and activate Akt [in vitro studies, a knockin mutation that prevents PDK1 from interacting with PtdInsP3 was expected to have a major effect on Akt activation, but instead only reduced Akt activation ~2-fold in the tissues and cell lines investigated [P3 is probably due to a second mechanism that brings Akt and PDK1 together and that is mediated by PDK1 possessing a docking site that recognizes Akt after it is phosphorylated at Ser473 by mTORC2 (mTOR complex 2) [Further work is warranted to establish the role that the myristoylation of AMPKβ subunits plays in regulating membrane localization and LKB1 phosphorylation. This might be best addressed by creating knockin mice in which Gly3,4,5)P3 . This heorylates . In vitrvate Akt ,64. On tstigated ,66. Thismplex 2) . It is pmplex 2) , emphasiP5 and InsP6 (inositol hexakisphosphate), which are present at micromolar levels serve to anchor a portion of cellular PDK1 in the cytosol as they interact with the PH domain of PDK1 at nanomolar affinities, thereby preventing PDK1 from interacting with 3-phosphoinositides at the membrane [C433S/C433S animals.As for LKB1, a significant proportion of PDK1 is localized in the cytoplasm even following the activation of PI3K pathways, where it plays a critical role in phosphorylating cytosolic substrates, such as p70 S6K (S6 kinase) and RSK isoforms, that are not known to reside within membranes. Evidence suggests that the soluble inositol phosphates, such as inositolmembrane . It woul431 controlled axon specification in the developing nervous system in response to BDNF by promoting the activation of the AMPK-related kinases BRSK1/BRSK2 (SAD-B/SAD-A) [431 was a focal point of a signalling network that linked extracellular determinants of neuronal morphogenesis through a cascade of at least five kinases, TrkB (tropomyosin-related kinase B), PKA, LKB1, SAD-A and SAD-B, to effectors that polarize neurons [S431A/S431A mice are viable and display no obvious overt phenotype or co-ordination/balance abnormalities does cast doubt on whether phosphorylation of Ser431 is critical in controlling the regulation of neuronal polarity. For example, BRSK1/BRSK2 (SAD-B/SAD-A) double-knockout mice that exhibit a major neuronal polarity phenotype die within 2 h of birth, display little spontaneous movement and have a weak response to tactile stimulation [S431A/S431A mice. Our finding that BRSK1/BRSK2 (SAD-B/SAD-A), as well as other AMPK-related kinases assayed , as well as AMPK are normally active in the brain and other tissues of LKB1S431A/S431A mice also suggests that phosphorylation of Ser431 is not critical for the activation of these enzymes.Previous work has suggested that LKB1 and its phosphorylation of SerB/SAD-A) ,38. This neurons ,38. The mulation . Further431 has no effect on the catalytic activity or ability of LKB1 to associate with STRAD and MO25 [Drosophila loss-of-function mutations in the LKB1 gene caused defects in polarity of the oocyte, and this was rescued by low level expression in the germ line of wild-type LKB1, but not by mutation of the residue homologous with Ser431 (Ser535) [S431A/S431A embryos; however, we observe that these mice were born at the expected Mendelian frequency, suggesting that mammalian embryonic development is not significantly affected by ablation of this phosphorylation site  . This obion site B. IntereS431A/S431A or LKB1C433S/C433S mice of up to 1 year of age revealed no detectable spontaneous tumour formation, suggesting that neither of these mutations alone is sufficient to inhibit the LKB1 tumour-suppressor function and lead to spontaneous tumour development. This conclusion is consistent with the data from Fogarty and Hardie [S431A/S431A or LKB1C433S/C433S mice with tumour-prone mice to investigate whether these mutations have any effect on the ability of LKB1 to operate as a tumour suppressor.Much previous work has also focused on the loss of LKB1 in tumours. Pathological examination of organs from LKB1d Hardie  that sho


Mycobacterium bovis strains at wild and domestic animals interphase area in Zambia, where bovine tuberculosis was diagnosed in wildlife seemed to be important.In Zambia, the presence of bovine tuberculosis in both wild and domestic animals has long been acknowledged and mutual transmission between them has been predicted without any direct evidence. Elucidation of the circulating M. bovis isolates from lechwe and cattle, respectively. Spoligotype analysis revealed that M. bovis strains from lechwe and cattle in Kafue basin clustered into a major node SB0120, where isolates outside the Kafue basin clustered into different nodes of SB0131 and SB0948. The comparatively higher variety of strains in cattle compared to lechwe elucidated by Mycobacterial Interspersed Repetitive Units–Variable Number Tandem Repeats analyses are consistent with cattle being the probable source of M. bovis in wild and domestic animals interphase area in Zambia.A PCR identified 15 and 37 M. bovis strains transfer between cattle and lechwe, with the latter having developed into a sylvatic reservoir host.These results provide strong evidence of  Mycobacterium tuberculosis complex (MTC) , Fab fragments (Roche), with TBSTE-E at room temperature for 30 min. Then the membranes were sequentially washed in TBST-E at room temperature for 1 min, 10 min and 1 min. Then, POD on the membranes was detected by TMB solution  according to the manufacturer’s protocol.Spoligotyping was performed according to the procedure by Kamerbeek and co-workers with slight modifications . Forty-t2O, 0.4 μL of dNTP (10 mM each), 3.0 μL of 5x colorless Go Taq buffer, 3.0 μL of betaine (5 M), 0.6 μL of Primer 1 and 0.6 μL of Primer 2, 0.1 μL of Go Taq (5 units/ μL) DNA Polymerase, and 1 μL of DNA sample. An initial denaturation step of 95°C for 5 min was followed by 32 cycles at 95°C for 15 sec, 58°C for 20 sec and 72°C for 1 min with a final elongation step at 72°C for 1 min in a thermalcycler. The PCR reaction mixture under the GCII buffer consisted of 4.8 μL H2O, 0.4 μL of dNTP (10 mM each), 7.5 μL of 2 x GCII buffer, 0.6 μL of primers, 0.1 μL of Go Taq (5 units/ μL) DNA Polymerase (Promega), and 1 μL of DNA sample. An initial denaturation step at 95°C for 5 min was followed by 32 cycles of 95°C for 15 sec, 50°C for 20 sec and 72°C for 45 sec with a final elongation step at 72°C for 1 min in a thermalcycler. All the samples were electrophoresed on a 2% agarose gel to identify the repeat numbers.The isolates were further genotyped by PCR amplification using primers targeting 26 VNTR loci . Two difMycobacterium tuberculosis complex-discriminating multiplex PCR; MIRU-VNTR: Mycobacterial interspersed repetitive units–variable-number tandem repeats; RD: Regions of difference.PCR: Polymerase chain reaction; BTB: Bovine tuberculosis; MTCD-MPCR: All authors have no competing interests.HBM and MM undertook sample collection, laboratory experiments, analyzing the results and drafting the manuscript. CN contributed the data analysis. YF, HS and AI were responsible for laboratory experiments. WM contributed to the design of field data collection. ASM and YS contributed to the design, writing of the manuscript and coordinated the study. All authors read and approved the manuscript.HBM, MM, CN, WM and ASM are veterinarians.


Myod1, Mef2c, Esx1, Foxa1, Hnf4a, Gata2, Gata3, Myc, Elf5, Irf2, Elf1, Sfpi1, Ets1, Smad7, Nr2f1, Sox11, Dmrt1, Sox9, Foxg1, Sox2, or Ascl1—can direct efficient, specific, and rapid differentiation into myocytes, hepatocytes, blood cells, and neurons. Furthermore, transfection of synthetic mRNAs of TFs generates their appropriate target cells. These results demonstrate both the utility of this approach to identify potent TFs for cell differentiation, and the unanticipated capacity of single TFs directly guides differentiation to specific lineage fates.A network of transcription factors (TFs) determines cell identity, but identity can be altered by overexpressing a combination of TFs. However, choosing and verifying combinations of TFs for specific cell differentiation have been daunting due to the large number of possible combinations of ∼2,000 TFs. Here, we report the identification of individual TFs for lineage-specific cell differentiation based on the correlation matrix of global gene expression profiles. The overexpression of identified TFs— •Lineage-determining single TFs are identified based on the correlation matrix•A proof of concept is demonstrated for ESC differentiation by 21 TFs•TFs orchestrate global gene expression changes via direct binding to target genes•Transfections of synthetic TF mRNAs generate desired differentiated cells Here, Ko and colleagues report that the correlation matrix of global gene expression profiles can identify individual TFs whose overexpression directs efficient, specific, and rapid differentiation into myocytes, hepatocytes, blood cells, and neurons. These results demonstrate that the unanticipated capacity of single TFs directly guides differentiation to specific lineage fates. One of the goals of regenerative medicine is to generate the desired types of differentiated cells from pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) . Common To facilitate the TF network analysis, we and others have applied a systems biology approach  to the lHere, we propose and test the use of the correlation matrix to predict, validate, and identify potent TFs for cell differentiation. We show a proof of concept by demonstrating direct cell differentiation into target organ cells such as myocytes, hepatocytes, blood cells, and neurons by overexpression of identified single TFs.Previously, we reported gene expression profiling data that have been generated 48 hr after overexpressing 137 TFs individually in mouse ESCs  and S1 . AlthougMyod1 was a top-ranked TF, followed by Mef2c, Tcf3, and Esx1, whose overexpression shifted a transcriptome of ESCs toward skeletal muscles  in the absence of LIF. The overexpression of Myod1 dramatically increased the number of cells stained with antibodies against myosin heavy chain (MHC) and MYOGENIN by day 5, confirming the rapid differentiation of ESCs into myocytes  overexpression gained the ability to form cell fusion with C2C12 mouse myoblast cells prestained with a red PKH26 dye 40 hr after coculture, indicating some functional maturity of the differentiated cells + cells measured by the immunofluorescence analyses  staining, the uptake of low-density lipoproteins (LDLs), and the secretion of ALBUMIN in culture medium on day 7  were two top-ranked TFs, followed by Myc, Elf5, Irf2, Tgif1, and Ets1, whose overexpression shifted a transcriptome of ESCs toward multiple-lineage blood cells ; a synaptic marker (SYNAPSIN); dopaminergic neuron markers (tyrosine hydroxylase [TH] and dopamine transporter [DAT]); a motor neuron marker (ISL1/ISL2); and an inhibitory neurotransmitter (GABA)   C and 5H.Ascl1-induced neurons on day 11: 5 out of the 11 examined cells fired action potentials that reached 0 mV upon injection of depolarizing currents  that the correlation matrix indicated as potential neural differentiation factors also turned out to be interesting examples. Nr2f1  has been implicated for specifying neural stem cells (NSCs)    and the s (NSCs) . Howevernd Smad7 F and 5G. < 0.05) H and 5I.Ascl1, toward an endoderm fate after induction of Hnf4a and Foxa1, and toward a blood cell fate after induction of Sfpi1 , the FLAG-tagged exogenous HNF4A protein bound to the HNF4A-binding motif in the promoter regions of hepatocyte-related genes , and the FLAG-tagged exogenous SFPI1 protein bound to the SFPI1-binding motif in the promoter regions of hematopoietic-related genes   . Taken tMyod1, Hnf4a, Sfpi1, or Ascl1-encoding RNA, followed by 3 days of cell culture in the DMs  TF activation cascades for cell differentiation and impel the further downstream events to complete the differentiation process. This could explain why the cell differentiation by the overexpression of a TF is faster than the cell differentiation by the manipulation of environmental conditions. Of course, direct reprogramming of fibroblast cells to specific cell types can be considered, in a sense, examples of bypassing the cell differentiation cascades; however, most cases require the combinations of multiple TFs  except fMyod1  and Ascl1   and then subjected to analysis using Accuri C6 or FACSCanto II (Becton Dickinson) . For intAldrich) . Fixed cImmunostaining for cultured cells was carried out as described previously . PrimaryMyod1-IRES-Venus. On day 7, medium was replaced with DMEM supplemented with 2% horse serum, and 1 × 105 C2C12 cells were seeded onto myocytes. After 16 and 40 hr, C2C12 cell-stained PKH26 and myocyte-expressed Myod1-IRES-Venus were photographed with inverted fluorescent microscopy  with the use of NIS-Elements Software (Nikon).C2C12 cells were stained with a red PKH26 dye (Sigma-Aldrich) before coculturing with myocytes derived from ESCs. ESCs were differentiated into myocytes for 7 days by induction of 3 V microplate reader (PerkinElmer).Cells on day 7 of differentiation were fixed by 4% paraformaldehyde and stained by PAS (Sigma-Aldrich) according to the manufacturer’s instructions. LDL uptake by cells was assessed by fluorescent microscopy after incubation of the differentiated cells with 10 μg/ml acetylated LDL labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindo-carbocyanine perchlorate (DiI-Ac-LDL)  for 4 hr at 37°C and DAPI. The amounts of mouse ALBUMIN secreted in the culture media (DMEM without glucose or phenol red [Invitrogen] supplemented with 2 mM sodium pyruvate [Sigma-Aldrich] and 20 mM sodium lactate [Sigma-Aldrich]) were measured after culture of differentiated cells from ESCs or adult mouse primary hepatocytes for 24 hr using a Mouse Albumin ELISA Kit (Bethyl Laboratory) according to the manufacturer’s instructions. The absorbance was measured with a VICTOR5 differentiated cells in the presence (Dox+) or absence (Dox−) of doxycycline (1 μg/ml). ESCs were plated on day 3 of differentiation in 1.4 ml of hematopoietic colony-forming assay medium and cultivated for an additional 11 days (Hematopoietic colony-forming cell assays were performed in MethoCult H3434 semisolid medium (STEMCELL Technologies) supplemented with 50 ng/ml human TPO and 10 ng/ml G-CSF. A total of 2 × 10 11 days . The col2PO4, 24 mM NaHCO3, 2 mM CaCl2, and 1 mM MgSO4 and was oxygenated continuously, and osmolality was set at 315 mOsm. The same internal solution was used in all the recordings, and it contained 125 mM KCl, 10 mM NaCl, 4 mM Mg-ATP, 0.3 mM Na-GTP, and 1.6 mM KHCO3 with a pH of 7.3, and osmolality was set at 270–290 mOsm. The recording solution was oxygenated continuously, and osmolality was set at 315 mOsm and kept at room temperature. Input resistance and membrane time constant were measured in current clamp conditions. Spontaneous events were collected semiautomatically during 30 s long periods at different holding potentials, and the ten largest ones observed at least 2× SD above the baselines were chosen to calculate the reversal potential values after fitting a linear line across points. Liquid junction potential was measured −5.5 mV  for 11 days. For visual guidance, we used an upright microscope equipped with DIC infrared camera as described previously by  −5.5 mV  and subtMicroarray analyses were carried out as described previously by Differentiated cells after 7 days were subjected to crosslinking with 1% formaldehyde. Chromatin was digested in the buffer containing 0.1% sodium deoxycholate and then sheared to DNA fragments with an average length of 100–500 bp. Sonicated DNA was subjected to immunoprecipitation using anti-FLAG antibody (Sigma-Aldrich). Immunoprecipitated DNA was reverse crosslinked and then performed to qPCR using Power SYBR Green PCR Master Mix (Applied Biosystems). Sets of primers were used to amplify DNA sequences . PCR ampMyod1, Sfpi1, Hnf4a, Ascl1-encoding RNA, or GFP-encoding RNA (control RNA) (Stemgent) with RNAiMAX from the third day of differentiation. After 5 consecutive days of transfection, cells were cultured in the organ-specific medium for 3 days. Differentiated cells were examined by immunostaining and flow cytometric analysis.mRNA synthesis was performed as reported previously by At least three independent experiments were performed. Statistical analysis of the data was performed with ANOVA. p < 0.05 was considered significant. Values are reported as mean ± SEM.K.Y. performed all experiments and wrote the manuscript. Y.P. performed the microarray procedure. A.A.S. analyzed microarray data. V.Z. and K.N. carried out electrophysiology. Y.P. and H.Y. helped with mRNA synthesis. D.S. supervised the project. M.S.H.K. supervised all experiments and wrote the manuscript.


The purpose of the current study was to identify potential ligands and develop a novel diagnostic test to highly pathogenic avian influenza A virus (HPAI), subtype H5N1 viruses using phage display technology. The H5N1 viruses were used as an immobilized target in a biopanning process using a 12-mer phage display random peptide library. After five rounds of panning, three phages expressing peptides HAWDPIPARDPF, AAWHLIVALAPN or ATSHLHVRLPSK had a specific binding activity to H5N1 viruses were isolated. Putative binding motifs to H5N1 viruses were identified by DNA sequencing. In terms of the minimum quantity of viruses, the phage-based ELISA was better than antiserum-based ELISA and a manual, semi-quantitative endpoint RT-PCR for detecting H5N1 viruses. More importantly, the selected phages bearing the specific peptides to H5N1 viruses were capable of differentiating this virus from other avian viruses in enzyme-linked immunosorbent assays. Since the first evidence regarding direct transmission of highly pathogenic avian influenza A virus (HPAI), subtype H5N1 from poultry to human in 1997 and resulted in the death of 6 of the 18 infected individuals In addition to vaccination and administration of antiviral drugs against H5N1 viruses, development of effective detection approaches is required to manage and control the deadly disease. Phage display is a recently developed technology and phage random peptide library consists of a pool of billions of heterologous peptides that can be produced by the fusion of random nucleic acid sequences to the N terminus of one of the capsid protein genes of a filamentous bacteriophage l-glutamine and 10% fetal bovine serum at 37°C and 5% CO2 in air. HPAI H5N1 strain A/goose/Jilin/hb/2003 were propagated in the MDCK cells in the absence of serum and purified by differential centrifugation conventionally. The concentration of the purified viruses diluted in PBS was measured by Thermo Scientific NANODROP 2000 Spectrophotometer  and calculated by the molar absorbance coefficient A260/A280 according to the manufacturer's instructions.Madin-Darby canine kidney (MDCK) cells  were grown in Dulbecco's MEM with 1 mM 3 (pH 8.6) buffer overnight at 4°C. The next day, the plates were blocked for 1 h at 4°C with 5% skimmed milk diluted in 0.05% (vol/vol) Tween 20 in phosphate-buffered saline (PBST). Following six washes with TBST , the viruses were incubated with the phage library at a final concentration of 2×1011 (100 µl/well) at room temperature for 30 min with gently rocking. Subsequently, unbound phages were removed by 10 times wash with TBST and the bound phages were eluted by adding 100 µL elution buffer (0.2 M glycine-HCl [pH 2.2]) at room temperature for 30 min. The eluate neutralized with 15 µL 1 M Tris-HCl (pH 9.1) was collected and tittered. The phages were amplified in Escherichia coli ER2738 and purified by polyethylene glycol precipitation.Phage display was done according to the manufacturer's instructions (New England Biolabs) with minor modifications. For the first round of panning, 96-well plates were coated with the H5N1 viruses at a concentration of 14 µg/well in 0.1 M NaHCOE.coli were determined to evaluate the enrichment efficiency.The second and third rounds of panning were repeated under similar panning conditions in addition to the increased concentration of Tween 20 (0.5% [vol/vol]) in TBST. In the fourth round of panning, the coated viruses were replaced by the supernatant form MDCK culture. After incubation of the phages to the supernatant at room temperature for 30 min, the resulting phages were subjected to the fifth round of panning. The titer of the phages in input, elute buffer (output) and that after amplification in 3 (pH8.6) at a concentration of 10 µg/well. Seven controls were set: phage library coating group; phage-free group; secondary antibody-free group; porcine transmissible gastroenteritis virus coating group; avian infectious bronchitis virus coating group; blocking buffer group and virus dilution solution coating group. The coating process was done overnight at 4°C. The next day, the plates were blocked with 1% bovine serum albumin (BSA) in TBS buffer (TBSB) for 2 h at room temperature. The plates were washed six times with TBST and then incubated with individual phage from the last round of biopanning at a concentration of 2×1011 in 0.1 M NaHCO3 (pH 8.6) for 1 h at 37°C. After six washes with TBST, the M13 polyclonal antibody  was added to these wells for 1 h at 37°C. After six washes with TBST, the wells were incubated with the horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody . The color was developed using o-phenylenediamine (OPD), and the optical density (OD) value was read using an ELISA plate reader at a wavelength of 405 nm. The experiments were performed in triplicate.Positive phage clones were identified by indirect ELISA. Briefly, ELISA plates were coated with H5N1 viruses diluted in 0.1 M NaHCO5′-TCACCTCGAAAGCAAGCTGA and antisense primer: 5′-CCCTCATAGTTAGCGTAACG were used to PCR amplify the gene encoding the exogenous peptides of M13. The PCR profile included 95°C for 5 min, 30 cycles of 95°C for 30 s, 57°C for 30 s, 72°C for 30 s. There was a final extension of 72°C for 7 min. The corresponding amino acid sequences were deduced, based on subsequent DNA sequencing.Ten positive phage clones were amplified and precipitated with polyethylene glycol-NaCl. Each phage clone DNA was purified using a plasmid extraction kit . The purified DNA template, sense primers: 12 was used as primary antibody. After triple washes with TBST, the wells were incubated with anti-M13 antibody (1∶1600 dilution in PBS) for 1 h followed by another incubation with HRP-conjugated goat anti-rabbit antibody (1∶5000 dilution in PBS) for 1 h. The OD405 value of detected phage wells (P)/negative control (N)>2 was judged as positive results. For conventional ELISA, the H5N1 viruses were serially diluted in DMEM medium and coated into ELISA plates as above. Rabbit antiserum against H5N1 viruses was serially diluted in PBS buffer and incubated with the coated H5N1 viruses for 1 h. After triple washes with TBST, the wells were incubated with HRP-conjugated secondary antibody (1∶5000 dilution in PBS) for another 1 h. The normal rabbit serum was used as negative control for ELISA and the OD450 of detection well (P)/that of control well (N)>2 was judged as positive results.The sensitivity of phage–based detection was compared with antibody-based ELISA and reverse transcription (RT)-PCR to determine the minimum quantity of the virus detected. For phage-based ELISA, the H5N1 viruses serially diluted in DMEM were coated into ELISA plates overnight at 4°C. The next day, the wells were blocked with 5% skimmed milk for 3 h at room temperature. Then the selected phages and phage complex from the phage display library (control phage) diluted in PBS at a final concentration of 1.5×105′-GATACGCTGCAGACAAAGAA) and antisense primer (P2: 5′-TTCTGCATTGTAACGATCCA), and sterile water (16.7 µl). PCR parameters were composed of 94°C for 5 min, 30 cycles of 94°C for 30 s, 52.3°C for 30 s and 72°C for 40 s followed by 72°C for 10 min. The authenticity of the PCR product was confirmed by sequencing.A conventional RT-PCR amplifying partial hemagglutinin (HA) gene (561 bp in length) of H5N1 virus was performed. The H5N1 viruses were diluted in PBS buffer and the final concentration of the viruses was adjusted to 0.647 mg/ml. Firstly, 3 ml of the viruses were subjected to viral RNA extraction using a RNA extraction kit  according to the manufacturer's instructions. The extracted viral RNA was dissolved in diethyl procarbonate treated sterile water in a volume of 40 µl. The reverse transcription system included 5 µl of viral RNA (1 µg), 7.5 µl of sterile water, 1 µl of M-MLV reverse transcriptase , 0.5 µl of RNase Inhibitor (40 U/ml), 1 µl of Oligo dT, 1 µl of dNTP Mixture (10 mM) and 5×MLV Buffer (4 µl). The reaction was performed at 42°C for 30 min, 99°C for 5 min and 5°C for 5 min. The resulting cDNA was serially diluted and subjected to PCR. The PCR mixture included cDNA (2.5 µl), 10×Easy Taq polymerase , 2 µl of dNTP Mixture (2.5 mM), 10×PCR Buffer (2.5 µl), 0.5 µl of sense primer (P1: 3 (pH8.6) to a final concentration of 15 µg/well and coated onto ELISA plates overnight at 4°C. Subsequent ELISA steps were performed as above-mentioned protocols. The OD405 values were recorded. At least three independent experiments were carried out. Each data point was presented as mean ± SD. Statistical significance was evaluated using the t-test. “*” means a value of P<0.01 was considered statistically highly significant. Furthermore, the identified phages were used to detect an H9N2 avian influenza virus coated in ELISA plates to further analyze the specificity of the phage-based ELISA using the above-mentioned procedure.The specificities of the selected phages were evaluated by using them as diagnostic reagents to detect a panel of avian viruses. These viruses included duck plague virus (DPV) a vaccine strain, avian bronchitis virus (IBV) strain Beaudette, fowlpox virus (FPV) isolate HH2008, avian infectious bursal disease virus (IBDV) strain UK661, avian infectious laryngotracheitis virus (AILV) strain K317 and newcastle disease virus (NDV) strain La Sota. The viruses were diluted in 0.1 M NaHCOAt present, antiviral drugs are available in fighting influenza, such as the M2 inhibitors and the neuraminidase inhibitors. Nonetheless, the emergence of drug resistant influenza strains raises concern over their effectiveness. It has been reported that M2 inhibitors resistant H5N1 viruses are widespread One purpose of the current study was to use phage display to identify specific ligands of H5N1 viruses. There are reports regarding the phage-displayed peptides selected from combinatorial libraries that interacting with hepatitis B virus, adenovirus type 2, Andes virus, Sin Nombre virus and Hantaan virus and coronavirus Following extraction of DNA of the selected phages, the genes encoding the peptides expressed on the surfaces of the recombinant phages were amplified by PCR. The PCR products were approx. 250 bp in size as expected . DNA seq−3 = 0.243 µg. These results indicate that the phage-based ELISA is as good as if not better than antiserum-based ELISA and RT-PCR for detecting H5N1 viruses.The sensitivity of phage-based detection was firstly analyzed by ELISA. As shown in Since another purpose of this study was to develop a novel diagnostic assay to H5N1 viruses, it is clearly important to be able to distinguish influenza A virus from other viruses that might cause mixed infections. Therefore, the selected phages were analyzed for their specificities in recognizing H5N1 viruses and other avian viruses. The avian viruses selected in this study are very common in China and some of which may cause co-infection with H5N1 viruses


Non-invasive ocular ultrasonography has recently been proposed to detect elevated intracranial pressure (ICP). On the other hand, the effect of positive end-expiratory pressure (PEEP) on ICP is well documented. The aim of the present ongoing study is to record the effect of changes of airway pressures on optical sheath nerve diameter (OSND).2O increments, to a maximum of 12. Two measurements were performed with a 7.5 MHz probe  in each eye before and after PEEP changes. Demographic data  Ppeak and Pm were also recorded. Exclusion criteria were intracranial pathology, ocular neuritis and trauma. Normality with P-P plots and the Kolmogorov-Smirnov test were calculated followed by bivariate and single linear regression analysis .In this prospective observational study, measurements of OSND were carried out in 21 patients of a polyvalent 10-bed adult ICU before and 2 minutes after changes of PEEP from a baseline of 5 with 1 cmHSeventy-two measurements were included for further analysis. Mean ± SD values were: PEEP (7.9 ± 2.3), Ppeak (24.35 ± 5), Pm (12.03 ± 3.16), OSNDright (4.03 ± 0.79), OSNDleft (4.1 ± 0.79), ΔOSND (0.24 ± 0.2), while values for age and APACHE II score were 62 ± 4 years and 14.4 ± 1.4 respectively. PEEP and OSND seem to have a moderate relation (tendency equation: OSND = 2.454 + 0.581×PEEP) compared with the weaker effect of Ppeak and Pm (Table Our study identified a moderate relation between PEEP and OSND and a weaker one between Ppeak, Pm and OSND. Thus, in selected cases OSND could serve as a bedside marker of effect of airway pressure to ICP. Yet, larger studies are needed to come to a safer conclusion.


Human papillomaviruses (HPVs) are agents of the most common sexually transmitted diseases in females and males. Precise data about the presence, mechanism of infection and clinical significance of HPV in the male reproductive tract and especially in sperm are not available. Here we show that HPV can infect human sperm, it localizes at the equatorial region of sperm head through interaction between the HPV capsid protein L1 and syndecan-1. Sperm transfected with HPV E6/E7 genes and sperm exposed to HPV L1 capsid protein are capable to penetrate the oocyte and transfer the virus into oocytes, in which viral genes are then activated and transcribed. These data show that sperm might function as vectors for HPV transfer into the oocytes, and open new perspectives on the role of HPV infection in males and are particularly intriguing in relation to assisted reproduction techniques. Papillomaviridae family Human papillomaviruses (HPVs) comprise a highly diverse group of small, non-enveloped double-stranded DNA viruses that belong to the In males, HPV DNA and RNA have been well documented not only in the anal region, perineal area, scrotum, glans, penile shaft, and urethra However, precise data about the presence and significance of HPV in sperm are not available. In particular, the exact localization and mechanism of infection by HPV in sperm, as well as the role of infected sperm cells as a transmission vector for the virus are still unknown. It is not known whether HPV-infected sperm are able to infect the partner and whether they are able to fertilize. This is also of crucial importance in relation to in vitro fertilization techniques because of the possibility that sperm infected with HPV injected in the oocyte cytoplasm (like during the intra-cytoplasmic sperm injection procedure) could interfere with fertilization, implantation, embryo development, premature abortion, and definitively with outcome and safety of assisted reproduction techniques (ART). Infertile patients seeking ART are at higher risk to carry HPV in sperm (10% with respect to 0% of fertile controls) In this study we aimed at better understand the mechanism of infection by HPV in human sperm and the ability of HPV-infected sperm to fertilize and transfer HPV DNA and capsid proteins to the oocyte. We used different in vitro methods to localize HPV DNA in sperm and to show the interaction between the capsid protein L1 and the primary attachment receptor syndecan-1 on sperm. Then, we used the hamster egg-human sperm penetration test to show the ability of sperm to transfer the capsid protein L1 to the oocyte and the transfer of E6/E7 viral genes to the oocyte by transfected sperm and the expression of these genes by the oocyte.Human sperm from a male previously found to carry HPV16 in semen Cell entry of HPV is initiated by binding of the virus particle to cell surface receptors, a prerequisite for subsequent interaction with the elusive uptake receptor, internalization and deliver of the genetic material into the nucleus of the target cell We then studied the fertilizing ability of HPV infected sperm and the possible transfer of the virus into oocytes. To this aim we used the hamster egg-human sperm penetration test (HEPT) using sperm transfected with a plasmid containing the HPV genes E6/E7 linked to GFP and using sperm exposed to the HPV capsid protein L1. The E6/E7 genes were amplified from plasmid p1321 HPV-16 E6/E7 and subcloned into plasmid pIRES-AcGFP1 to construct recombinant plasmid pIRES-AcGFP1-E6E7 , which wTaken together, the findings of the present study show that sperm might be infected by HPV and that the mechanism of entry of the virus in the sperm is mediated by interaction of the HPV L1 capsid protein with syndecan-1 that is mainly localized in the equatorial region of the sperm head. Other viruses found in association with sperm, such as HIV and HSV, have been shown to interact with HSPGs These findings confirm previous studies that showed that HPV16 capsids bind at the equatorial region of the sperm head The study was approved by the Institutional Ethics Committee of the Hospital of Padova and written informed consent was provided by participants.2 incubator), the collected spermatozoa were used for subsequent analyses. Subjects have been described before Human semen samples from volunteers were obtained by masturbation after 2–5 days of sexual abstinence in sterile containers. Samples were allowed to liquefy for 30 minutes at room temperature and semen volume, pH, sperm concentration, viability, motility, and morphology were determined following World Health Organization guidelines for semen analysis 6 sperm were fixed in a methanol-acetic acid solution for at least 1 hour at −20°C. To permeabilize sperm membrane, samples were treated with pepsin  in prewarmed HCl 1 mol. for 10 minutes at 37°C. Permeabilization of the specimens was stopped with 4 min. washes in PBS 1×. Samples were then dehydrated in 70%, 80%, and absolute ethanol for 2 minutes and finally air-dried. Samples were then overlaid with 20 μL of hybridization solution containing biotin (BIO)-labeled HPV DNA probe  . Each slide was covered with a glass coverslip and the edges were sealed with nail polish to prevent loss of the mixture during denaturation and hybridization. After denaturation of cellular target DNA and HPV DNA probe on a heating block for 5 min. at 95°C, hybridization was performed by incubating the samples at 37°C overnight in a humidified chamber. Thereafter, the coverslips were carefully removed and the slides were washed in PBS 1× for 10 minutes. After 15 minutes of incubation at 37°C with a wash solution to eliminate non-specific bound probe for use in situ hybridization (ISH) , the slides were washed three times in PBS 1× for 3 minutes. The negative control was processed in the same way, but omitting the viral probe. The biotin-labeled HPV probe was detected by incubation with 1∶200 streptavidin Texas red  for 40 minutes at room temperature. After detection, the slides were washed twice in PBS 1×/0.01% Triton and then twice in PBS 1× and mounted with a solution containing 4,6-diamino-2-phenylindole (DAPI 5 mg/mL) and antifade . Samples were analyzed using a fluorescence microscope  equipped with a triple band-pass filter set . For each slide 200 spermatozoa were analyzed. When nuclei were completely and homogeneously stained and when multiple small spots or single large signals were present, the sperm cells were classified as positive. The method was tested on control slides containing CaSki cells , human cervical carcinoma cell line with stably integrated and transcriptionally active HPV genomes that served as control for the specific probe.Sperm samples containing at least 2×106 sperm/mL in sperm washing medium and incubated with 20 µg/ml HPV16-L1  for 1 h at 37°C. The reaction volume was 300 µL. After incubation, motile spermatozoa were harvested by the swim-up technique as described above. The swim-up sperm fraction was divided in two samples used for later HPV16-L1 capsid detection after or not induction of acrosome reaction by calcium Ionophore III (Sigma-Aldrich) at 10 µM. Sperm without incubation with HPV16-L1 were used as negative control.Sperm were resuspended at a concentration of 20×10For immunofluorescence, 10 µl of sperm sample was smeared on clean, grease-free slides, air-dried, and fixed in PBS/paraformaldehyde 4% for 15 minutes. The glasses were washed three times in PBS for 5 minutes at room temperature and then used for the HPV capsid (HPV16-L1) and acrosome (pisum sativum) detection.For HPV16-L1 detection the sperm were incubated with mouse monoclonal antibody HPV 16-L1 (CAMVIR-1)  for 120 minutes at room temperature, and then washed in 0.2% tween-PBS for 5 minutes. Immunoreaction was detected by sequential incubation with biotinylated goat anti-mouse immunoglobulin secondary antibody  and streptavidin Texas red  both for 60 minutes at room temperature. After that the slides were incubated with the acrosome specific Pisum Sativum (1 µg/ml) for 30 minutes at room temperature and then washed twice in PBS. Nuclei were counterstained with DAPI 5 mg/mL, slides were mounted with anti-fade buffer and 24×24 mm coverslip. Immunostaining was evaluated with Nikon ViCo Video Confocal Microscope.DNA extraction from swim-upped sperm was performed by QIAamp DNA mini kit . The presence of HPV DNA sequence (L1 gene) was investigated by nested polymerase chain reaction (PCR) using MY09/MY11 as outer primers and GP5þ/GP6þ as inner primers. The amplification products were analyzed by staining with SYBR safe DNA gel stain, after electrophoresis on 2% agarose gel. PCR amplification was carried also with a positive control  and a negative control (no template).6 sperm cells with and without incubation with 3.5 U/ml Heparinase-III  for 60 minutes at 37°C. Sperm were incubated with HPV16-L1 protein (20×106 sperm/mL were resuspended in sperm washing medium and incubated with 20 µg/ml HPV16-L1 protein for 1 h at 37°C).To evaluate the interaction between syndecan-1 and HPV16 capsid, fresh spermatozoa were washed twice in sperm washing medium and centrifuged (300 g for 10 minutes). The pellet was resuspended in PBS and divided into two aliquots with 40×10To detect CD138 (Syndecan-1) and HPV16-L1 in cytofluorimetry sperm were incubated with mouse anti-human CD138 monoclonal antibody, FITC conjugated, clone 1D4  or with mouse monoclonal antibody HPV16-L1 (CAMVIR-1) (1 µg/mL)  both on ice for 45 minutes in the dark. Sperm were washed twice in phosphate-buffered saline (PBS) and centrifuged at 300 g for 5 minutes. For HPV16-L1, the second layer phycoerythrin anti-mouse antibody  was added to the cells and incubated for 45 minutes on ice. A multiple myeloma cell line U266  were used as positive controls for syndecan-1, and the samples incubated with secondary antibody alone were used as negative controls for HPV16-L1. Samples were washed twice in PBS and centrifuged at 300 g for 5 minutes. The pellet was resuspended in 300 µl of PBS and then analyzed on a FACScalibur  using Cellquest to acquire and analyze the data.To detect CD138 (Syndecan-1) and HPV16-L1 colocalization in immunofluorescence, 10 µl of sperm samples were smeared on clean, grease-free slides, air-dried, and fixed in PBS/paraformaldehyde 4% for 15 minutes. The glasses were washed three times in PBS for 5 minutes, incubated with mouse anti-human CD138 monoclonal antibody, FITC conjugated, clone 1D4  overnight at 4°C, washed twice in PBS and incubated with mouse monoclonal antibody HPV 16-L1 (CAMVIR-1)  for 120 minutes at room temperature. The slides were washed twice in 0.2% tween-PBS for 5 minutes. Immunoreaction for the HPV16-L1 was detected by sequential incubation with biotinylated goat anti-mouse immunoglobulin secondary antibody  and streptavidin Texas red  both for 60 minutes at room temperature. The slides were washed twice in PBS, and nuclei were counterstained with (DAPI 5 mg/mL). Slides were mounted with anti-fade buffer and 24×24 mm coverslip. Immunostaining was evaluated with Nikon ViCo Video Confocal Microscope. Sperm without incubation with CD138 (Syndecan-1) and HPV16-L1 were used as negative control.2, 1 µL of PCR grade nucleotide mix (10×), 20 pmol of each primer with SalI and BamHI restriction sites  including forward: 5′-GTGCGCGTCGACGTGATGCACCAAAAGAGAACTG-3′ and reverse: 5′-GTGCGCGGATCCGTGTGGTTTCTGAGAACAGATG-3′, 0,75 µL Expand high fidelity enzyme mix  and sterile H2O in a final volume of 50 μL. The amplification program was as follows: denaturing at 94°C for 2 minutes, 10 cycles each at 94°C for 15 seconds, annealing at 55°C for 30 seconds and extension at 72°C for 1 minute, 20 cycles each at 94°C for 15 seconds, annealing at 55°C for 30 seconds and extension at 72°C for 1 minute plus 5 seconds cycle elongation for each successive cycle, followed by a final extension at 72°C for 7 minutes. The amplification products were analyzed by staining with SYBR safe DNA gel stain, after electrophoresis on 1% agarose gel and then purified using Microcon YM-100 . Then the purified amplification products and plasmid pIRES2-AcGFP1 were digested by restriction enzyme SalI and BamHI (10 U at 37°C for 120 minutes). These digested PCR products were purified from a 0.8% agarose gel using the S.N.A.P. Gel Purification Kit , according to the manufacturer's protocol. The purified PCR products and plasmid pIRES2-AcGFP1 were analyzed on a second 0.8% agarose gel to confirm recovery. Finally, these purified PCR products were recombined into plasmid pIRES2-AcGFP1 using T4 DNA ligase kit  and subjected transformation into E. coli DH5α  according to manufacturer instruction. The successful construction was confirmed by PCR, BamHI, and Sal I digestion and DNA sequencing.The expression vector for HPV E6/E7 genes and GFP was constructed as Motile sperm were harvested by the swim-up technique as described above. The swim-up sperm fraction was divided in four samples: one used for the transfection with plasmid pIRES2-AcGFP1-E6E7, the second one used for the transfection with 2 µL Lipofectamine 2000 alone , the third one used for the transfection with plasmid pIRES2-AcGFP1 and the last one as negative control.2 for 4 hours. Three hours after the beginning of capacitation, sperm were exposed to plasmid pIRES2-AcGFP1-E6E7. Briefly, a total of 100 µL mixture containing 800 ng plasmid pIRES2-AcGFP1-E6E7 and 2 μL Lipofectamine 2000  in BWW was incubated at room temperature for 20 minutes, and then was added to sperm sample and kept in the incubator for another 1 hour. After that, the sperm sample was washed at least five times in 5 mL of fresh BWW.Sperm were incubated in BWW supplemented with HEPES  and 3.5 mg/ml HSA (Human Serum Albumin) for capacitation at 37°C and 5% CO2O were used as the positive and negative controls, respectively. The PCR mixture consisted of 5 µL 10× TaqGold buffer, 3 µL of MgCl2 25 mM, 5 µL of each deoxynucleosidetriphosphate 200 µmol/L, 20 pmol of each primer (forward CMV: 5′-CGCAAATGGGCGGTAGGCGTG-3′ and reverse IRES: 5′-CCTCACATTGCCAAAAGACG-3′), 2.5 U TaqGold polymerase and sterile H2O in a final volume of 50 µL. The amplification program was as follows: denaturing at 94°C for 10 minutes, 37 cycles each at 94°C for 1 minute, annealing at 55°C for 1 minute and extension at 72°C for 2 minutes, followed by a final extension at 72°C for 10 minutes. The amplification products were analyzed by staining with SYBR safe DNA gel stain, after electrophoresis on 1% agarose gel.The successful of human sperm transfection with plasmid pIRES2-AcGFP1-E6E7 was confirmed by PCR. The DNA was extracted from sperm by QIAamp DNA Mini Kit , according to the manufacturer's protocol. Plasmid pIRES2-AcGFP1-E6E7 and sterile ddH6 motile spermatozoa under mineral oil. After 3 hours incubation at 37°C in a 5% CO2 atmosphere oocytes were recovered from the droplets and washed to loose adherent spermatozoa. To evaluate the successful sperm penetration, 40 oocytes (10 for each condition) were analyzed with the nuclear acid staining SYBRGreen  under phase contrast microscopy and under the fluorescence microscope at 400× magnifications . Sperm egg penetration test was expressed as mean number of sperm penetrated for oocyte (HSA). Twenty-four hours after insemination, the remaining oocytes were investigated under the fluorescence microscope  at 400× magnifications. The oocytes were collected and classified into two groups: oocytes with green fluorescence and oocytes without green fluorescence. The oocytes from two groups were washed three times in cold 1× PBS to remove serum from the medium and then each oocyte was transferred singly into a PCR tube for PCR and RT-PCR.The HEPT was performed according to the World Health Organization 2O as the positive and negative controls, respectively. The first round multiplex PCR was performed with mixture consisted of 5 µL 10× TaqGold buffer, 3 µL of MgCl2 25 mM, 5 µL of each deoxynucleosidetriphosphate 200 µmol/L, 20 pmol of each primer (forward CMV: 5′-CGCAAATGGGCGGTAGGCGTG-3′ and reverse IRES: 5′-CCTCACATTGCCAAAAGACG-3′), 2.5 U TaqGold polymerase and sterile H2O in a final volume of 50 μL. The amplification program was as follows: denaturing at 94°C for 10 minutes, 37 cycles each at 94°C for 1 minute, annealing at 55°C for 1 minute and extension at 72°C for 2 minutes, followed by a final extension at 72°C for 10 minutes. This first round multiplex PCR is followed by a nested PCR reaction with one of the same outer primer (forward CMV) combined with a unique inner primer (reverse: 5′-GCATAAATCCCGAAAAGCAA-3′). The amplification products were routinely analyzed by staining with SYBR safe DNA gel stain, after electrophoresis on 1,5% agarose gel.5 μL of cell lysis buffer were added to each oocyte, mixed then incubated at 65°C for 10 minutes. The cell lysate of oocyte was used as a DNA template. Plasmid pIRES2-AcGFP1-E6E7 and sterile ddH5′-GTCGACGTGATGCACCAAAAGAGAACTG-3′ and reverse: 5′- GCATAAATCCCGAAAAGCAA-3′) and two negative controls (no template and no reverse transcription). The amplification step was carried out using these conditions: denaturing at 94°C for 10 minutes, 37 cycles each at 94°C for 1 minute, annealing at 55°C for 1 minute and extension at 72°C for 1 minute, followed by a final extension at 72°C for 10 minutes. About 20 μL of each PCR product were made visible by staining with SYBR safe after electrophoresis on 1.5% agarose gel. This experiment was repeated three times under the same condition.RNA extraction from each oocyte followed by RT-PCR was performed using the cells-to-cDNA II kit . 10 µL of cold cell lysis buffer II were added to each oocyte, and incubated at 75°C for 10 minutes. 0.12 U DNase I was added to the mixture at 37°C for 30 minutes. Reverse transcription was preformed according to the kit protocol. PCR amplification was carried out with 10 µL cDNA of each sample from reverse transcription reaction as a template, E6/E7 specific primer pair  at room temperature. All oocyte were then incubated with mouse monoclonal antibody HPV 16-L1 (CAMVIR-1) (0.8 µg/mL)  for 120 minutes at room temperature. Immunoreaction was then detected by sequential incubation with FITC-anti-IgG mouse secondary antibody  for 60 minutes at room temperature. The slides were washed twice in PBS, and nuclei were counterstained with DAPI (5 mg/mL). Slides were mounted with anti-fade buffer and 24×24 mm coverslip. Finally, the oocytes were placed on to the slides and the immunostaining was evaluated with Nikon ViCo Video Confocal Microscope.The sperm were incubated or not with HPV16-L1 20 µg/ml and then were selected by swim-up for 45 minutes at 37°C, capacitated for 3 hours, and washed in BWW. Both samples were resuspended in fresh medium and the concentration of motile sperm was adjusted to 5 mil/mL. Meanwhile, the oocytes were prepared as described before. Fifteen eggs were placed in 100 µL containing 10


The effect of SMe1EC2 (1×10–6 and 1×10–5 mol/l) on high potassium-, phenylephrine- or serotonin-induced contraction or acetylcholine-induced relaxation was also determined in aortic rings. We found that SMe1EC2 (1×10–8–1×10–4 mol/l) elicited significant dilatations in both cerebral and coronary arterioles (max dilatation: 25±8% and 18±5% respectively). Yet, SMe1EC2 (1×10–6 and 1×10–5 mol/l) did not influence the tone of aortic rings nor did it affect high potassium-, phenylephrine- or serotonin -induced contractions and acetylcholine-induced relaxation. Thus SMe1EC2 was able to dilate resistance arteries but did not affect aortic contractility. It is likely that SMe1EC2 does not possess α1-adrenolytic and anti-serotoninergic activity in the vascular wall.The antioxidant and reactive-oxygen-species-scavenging activity of stobadine has been demonstrated in previous studies. Recently, chemical modification of this leading structure led to the synthesis of other pyridoindole derivatives with significantly increased intrinsic antioxidant efficacy. Further structural modifications of stobadine provided the opportunity to increase bioavailability and attenuate unwanted side effects, such as α-adrenolytic activity. The aim of the work was to evaluate the direct effect of a novel pyridoindole, SMe1EC2, on the vascular wall ex vivo. The vasomotor effect of SMe1EC2 (1×10 H-pyrido indole has been recognized to have significant antioxidant properties and the ability to scavenge free radicals, such as hydroxyl, peroxyl, and alkoxyl radicals  (–)-cis-2,8-dimethyl-2,3,4,4a,5,9b-hexahydro-1H-pyrido indole has been recognized to have significant antioxidant properties and the ability to scavenge free radicals, such as hydroxyl, peroxyl, and alkoxyl radicals  (–)-cis-2,8-dimethyl-2,3,4,4a,5,9b-hexahydro-1et al., H-pyridoindole, has already shown a potent protective effect in various animal models, such as acute head trauma  were used in the experiments. The rats were housed separately, fed standard rat chow, allowed free access to drinking water, and treated according to institutional guidelines. All protocols were approved by the Institutional Animal Care and Use Committees.2, followed by subsequent cervical dislocation. Heart and brain were removed and stored in ice-cold MOPS solution. With the use of microsurgery instruments and an operating microscope, a branch of the septal coronary artery or superficial branch of the middle cerebral artery (approximately 1 mm in length) was isolated and transferred into an organ chamber containing two glass micropipettes and filled with cold MOPS solution (0–4 °C). The arteries were then cannulated on both ends on micropipettes, secured with sutures, and both micropipettes were connected with silicone tubing to an adjustable hydrostatic reservoir. The vessel chamber (15 ml) was continuously supplied with heated Krebs solution composed of (in mmol/l) NaCl 120, KCl 5.9, NaH2PO4 1.2, MgCl2 1.2, NaHCO3 15.4, CaCl2 2.5 (coronary)/1.25 , and glucose 5.5, bubbled with 21% O2 and 5% CO2 to reach the physiological pH. The temperature was set by a temperature controller at 37 °C and the vessel was allowed to develop spontaneous tone in response to an intraluminal pressure of 60 mmHg under no flow conditions (equilibration period of 1 h). The internal arteriolar diameter (at the midpoint of the arteriolar segment) was measured by videomicroscopy .The rats were anesthetized with CO–8 to maximum 1×10–5 mol/l) to test the viability of the vessel. Afterwards, SMe1EC2.2HCl was applied in increasing concentrations (1×10–8 to 1×10–4 mol/l). Arteriolar responses to agonists were expressed as percentage of the maximal dilation of the vessel.After 1-h incubation period, acetylcholine (Ach) was applied abluminally in increasing concentrations (from 1×102 and 5% CO2 at pH 7.4. The composition of PSS was (in mmol/l): NaCl 118.0, KCl 4.7, KH2PO4 1.2, MgSO4 1.2, CaCl2 2.5, NaHCO3 25.0 and glucose 11.0. The preparations were connected to an isometric force transducer (Experimetria Hungary), stretched passively to 20 mN and equilibrated for 60 minutes.The rat thoracic aorta was excised and transferred into oxygenated physiological salt solution (PSS). The aorta was cleaned of adherent tissue and cut into 8 rings, each approx. 2–3 mm long. Special care was taken not to damage the endothelium. The rings were mounted between two hooks in water-jacketed (37 °C) chambers containing PSS bubbled with a mixture of 95% O–8–1×10–4 mol/l. Then the drug was washed out and the preparations were precontracted with high potassium PSS  in which NaCl in normal PSS was exchanged for an equimolar concentration of KCl. When the contraction reached a steady state, SMe1EC2 was added in cumulative manner. In a different set of experiments, contraction was induced by supramaximal concentration of phenylephrine . At the plateau of the contraction, the effect of acetylcholine in the cumulative concentrations of 1×10–8–1×10–5 mol/l was tested in absence or presence of SMe1EC2 in the concentration of 1×10–6 or 1×10–5mol/l. After washing with PSS and reaching the initial tension value, concentration-response curves of phenylephrine (1×10–9–1×10–5 mol/l) were performed in absence or presence of SMe1EC2 (1×10–6 or 1×10–5mol/l). The same protocol was used with concentration-response curves of serotonin (1×10–8–1×10–4 mol/l). Values of pD2 (logarithm of IC50 which represents the concentration of a drug required for 50% inhibition in vitro) were calculated on using GraphPad Prism.After the equilibration period, SMe1EC2 was applied to the bath in cumulative concentrations of 1×10p<0.05 was considered statistically significant.The results are presented as the mean ± SEM. Statistical analyses were performed by using the unpaired Student's t-test or the two-way ANOVA with Bonferroni posttest. A value of –4 mol/l) (–5 and 1×10–4 mol/l (–4 mol/l) of SMe1EC2 was reached after 4.5 minutes.Abluminimal application of SMe1EC2 elicited dose-dependent dilatations of isolated cerebral arterioles reaching statistically significant dilatation (25±8%) at the highest concentration (1×104 mol/l) . In the –4 mol/l . The max–6 or 1×10–5mol/l did not influence the concentration-response curves either of phenylephrine or serotonin. The maximal responses to these agents and the pD2 values  leads to increased production of reactive oxygen species (ROS) , which are responsible for maintaining resting tone in arteries. Increase in one of these mediators leads to the changes in arterial tone. Endothelium-dependent relaxation induced by acetylcholine was found to be slightly increased by superoxide dismutase (SOD) , the diameter of isolated cerebral arterioles was increased only by the highest concentration used (1×10–4 mol/l).Further we evaluated the effect of SMe1EC2 on resistance arteries. Our results showed a slight but significant vasodilator effect of SMe1EC2 on rat microvessels, yet the effect was observed only at the highest concentrations studied. While the internal diameter of isolated coronary arterioles increased in response to exogenous administration of SMe1EC2  and β2-adrenergic–mediated relaxation, which appears to be partially endothelium-mediated . We speculate that the vasodilation response of arterioles to high concentrations of the pyridoindole tested may be explained by its action on β2-adrenergic receptors, however the exact mechanism of this effect needs to be elucidated. The effect of the highest concentration (1×10–4 mol/l) on the diameter of isolated coronary and cerebral arterioles may be operative by its non-specific membrane-stabilizing action.In the contradiction to the aorta, which contains an excess of α


Nucella lapillus by reciprocal transplantation of snails between two shores differing in wave action and rearing snails of the same provenance in a common garden. Results were compared with those reported for similar experiments conducted elsewhere. Microsatellite variation indicated limited gene flow between the populations. Intrinsic growth rate was greater in exposed-site than sheltered-site snails, but the reverse was true of absolute growth rate, suggesting heritable compensation for reduced foraging opportunity at the exposed site. Shell morphology of reciprocal transplants partially converged through plasticity toward that of native snails. Shell morphology of F2s in the common garden partially retained characteristics of the P-generation, suggesting genetic control. A maternal effect was revealed by greater resemblance of F1s than F2s to the P-generation. The observed synergistic effects of plastic, maternal and genetic control of shell-shape may be expected to maximise fitness when environmental characteristics become unpredictable through dispersal.Assessment of plastic and heritable components of phenotypic variation is crucial for understanding the evolution of adaptive character traits in heterogeneous environments. We assessed the above in relation to adaptive shell morphology of the rocky intertidal snail  The relative contributions of plasticity and inheritance to phenotypic expression are crucial quantities for understanding the evolution of adaptive character traits in spatially and temporally variable habitats Nucella lapillus (L.) have been studied in relation to contrasting selection regimes associated with high and low wave exposure N. lapillus is a predatory snail with limited dispersal ability owing to direct development and a restricted crawling range N. lapillus. The shell of N. lapillus is more globular at sites exposed to wave action and more elongated at sheltered sites Littorina saxatilisN. lapillus.Aquatic snails have become popular models for investigating environmental and heritable components of phenotypic expression because they are easily reared in the laboratory, readily transplanted between habitats in the field, and have measurable phenotypic characters whose variation is closely correlated with physical and biological environmental factors critical to survival Nucella lapillus from sites differing in exposure to wave action. Snails from the more exposed site had a relatively larger foot, affording greater adhesion, than those from the more sheltered. Progeny of exposed- and sheltered-site snails developed a similar relative foot size when reared under calm-water conditions in the laboratory, indicating plastic rather than heritable phenotypic variation. Reciprocal transplantation of progeny between sites, however, revealed strong asymmetry in the response to environment. Progeny from the sheltered site transplanted to the exposed came to resemble the native snails by developing a relatively much larger foot  than controls replanted on the sheltered site. Progeny transplanted from the exposed to the sheltered site, on the other hand, developed a pedal area only slightly smaller than that of controls. Trussell Littorina obtusata, again finding asymmetric plasticity with sheltered-site snails developing a relatively larger foot in response to exposure to wave action, but not vice versa.Etter Etter N. lapillus, other studies have linked phenotypic variation (shell shape) to genotype In contrast to Etter's N. lapillus, we performed reciprocal-transplant and common-garden experiments, complemented by population genetic analysis, on an exposed- and a sheltered-site population free of karyotype polymorphism. While substantially corroborating previous studies of N. lapillus, our data also reveal new insights on the plastic and heritable components of phenotypic variation of this species.To further assess the symmetry of plasticity and heritable variation in adaptive morphology of N. lapillus , they were labelled with a waterproof pen. Labels were covered with superglue (Loctite™) to protect against abrasion and the marked juveniles were released as summarized in Each of the above treatments was given a unique colour-code. On release, individuals were gently irrigated with seawater to encourage them to emerge from their shells and grip the substratum. In early November 2008, marked animals were recaptured (two visits to each site), photographed as described below, re-marked and returned to the field. In September 2009 the experiment was completed by returning marked individuals to the laboratory where they were photographed.Nucella lapillus juveniles (≤12 mm shell length) were collected from each shore . Each experiment incorporated a duplicated common garden lacking the effects of wave exposure typical of the exposed field site and of crab predation typical of the sheltered field site. Quantitative comparison of traits shown by successive generations would potentially distinguish plastic, maternal and genetic components of variation.Experiments were run for F1 generation) were apportioned between two tanks: 25 of sheltered site ancestry and 90 of exposed site ancestry per tank . Seawater was supplied via two constant-head cisterns at a rate of 3 ml s−1 with permanent aeration supplied from air-diffusion stones. Ambient temperature fluctuated seasonally between 8–16°C. To avoid position effects, tanks were transposed each month. Subjects were photographed, as below, after 6 months and again after 12 months from the beginning of the experiment. Since eggs were laid in both tanks, opportunity was taken to continue the experiment through the F2 generation, retrospectively assigning parentage by genetic analysis (below).Adults from the exposed and the sheltered site were collected in February 2008 and allowed to spawn separately in aquaria as for the reciprocal transplant experiment (above). Having grown large enough for marking (≤12 mm shell length), hatched juveniles  and 16SNucRV (5′-CTCAGTCGGCCCAACTAAAA-3′) . PCR amplifications were carried out in 25 µl reactions containing 1 µl of cDNA, 0.3 pmol of each primer, 1× PCR Buffer, 1.5 mM MgCl2, 0.2 mM of each dNTP (Promega) and 0.5 U Taq DNA polymerase (Promega) on an Biorad DNA engineTetrad2 Thermal cycler. An initial denaturation step of 5 min at 95°C was followed by 35 cycles at 95°C for 1 min, 53°C for 1 min, and 72°C for 1 min followed by a final extension at 72°C for 10 min. PCR results for both genes were sequenced using the Macrogen™ (www.macrogen.com) sequencing facility and subsequently aligned and compared using the software Bioedit Chromosome counting was complemented by comparison between populations of mitochondrial (16S) and nuclear (mMDH) genes, which vary in association with karyotypic and phenotypic polymorphism, in turn correlated with environmental variables such as wave exposure N. lapillus from Cable Bay (N = 96) and Llanfairfechan (N = 96) (0) and expected heterozygosity (HE) under Hardy-Weinberg assumptions, estimates of STF, ISF, and their significance per population over all loci were calculated according to Weir and Cockerham Microsatellites were used to assess gene flow between the two populations, separated by approximately 45 km or 90 km of coastline depending on dispersal route. Adult (N = 96)  were col1, n = 112) and offspring  were genotyped as described above, using seven microsatellite markers . Parental analysis was performed using CERVUS Potential parents of known gender . Shell growth at 3 months after initial release was ranked ES>EE>SS>SE , scores were standardized as residuals from linear regression. All paired comparisons  were sta1 parents comprised 36 males and 54 females of exposed-site ancestry and 10 males and 14 females of sheltered-site ancestry. Of the F2 progeny, 112 snails survived for 12 mo and genotyping unequivocally assigned 85 of these to known parentage, 5 having sheltered-site ancestry, 52 exposed-site ancestry and 28 mixed ancestry. The P generation was represented by controls from the reciprocal transplant experiment.F1 generations but not in the F2 generation except by planned comparison  and exposed-site snails (n = 51) differed on RW1 but not on RW2 or RW3, although difference on the latter was marginally non-significant . For the F2 generation, RWs 1–3 accounted for 30%, 20% and 11% of variance respectively. RW1 was correlated with centroid size and therefore corrected scores were used for subsequent analysis. Uncorrected scores were used for RW2 and RW3. Difference in mean score between sheltered-site (n = 5) and exposed-site snails (n = 27) was marginally non-significant on RW1 and RW2, and non-significant on RW3 .To gain resolution within generations, GM analyses were run separately for the full FE) per locus ranged from 0.332 to 0.892 and the observed heterozygosity (HO) from 0.313 to 0.917 , revealing significant structuring between the two sampling sites . There was therefore no evidence of karyotype polymorphism. All microsatellite loci were polymorphic for both populations. The number of alleles per population per locus ranged from 2 to 19, with a total number of 108 alleles in the global sample. The expected heterozygosity .Nucella lapillus is reported to have higher size-specific somatic growth rate but similar size-specific shell-growth rate at exposed sites than at sheltered Littorina obtusata, in which snails from exposed sites grew faster than those from sheltered under laboratory conditions of low flow velocity L. saxatilis where snails transplanted from high to low shore grew faster than low-shore residents Nucella spp. observed at exposed sites  persisted into the Fite loci  coincideite loci , S5 suggite loci  is probaN. lapillus aggregate in protective microhabitats in order to mate and spawn and although lacking specific homing behaviour they tend to use a restricted number of spawning sites distributed within a radius of up to about10 m that also encompasses their foraging range  and recaptured in November 2008; Jul-09: percentage of snails released at the beginning of the experiment and recaptured at the end of the experiment in July 2009; Jul-09b = percentage snails re-released in November 2008 and recaptured at the end of the experiment. E: exposed; S:sheltered; f: juveniles collected from the field; l: laboratory-hatched juveniles; fSS ; fES ; fEE ; fSE ; lSS ; lES ; lEE ; lSE .(TIF)Click here for additional data file.Figure S2Reciprocal transplant experiment: extreme deformation grids – Rw1/Rw2 (top), Rw2/Rw3 (bottom).(TIF)Click here for additional data file.Figure S3Location of study sites along the North Wales coastline. CB = Cable Bay , Thomas Exposure Index (TEI) = 14; CAE = Caethle , TEI = 15; FB = Friars Bay , TEI = 3; LL = Llanfairfechan , TEI = 2; MB = Menai Bridge , TEI = 0; RP = Ravens Point , TEI = 14; RWB = Redwharf Bay , TEI = 8.(TIF)Click here for additional data file.Figure S4Experiment 1: ontogenetic changes in shell morphology. Mean aperture external width adjusted to shell length (ANCOVA). For each size class, the first group represents snails reared in the laboratory and the second group snails collected from the ancestral field-population. Sample size (N) and covariate value (CV) for adjusting mean aperture width were as follows. Group 1: N exposed 1 = 10, N exposed 2 = 15, N sheltered 1 = 11, N sheltered 2 = 12; CV = 2.264 mm. Group 2: N exposed 1 = 11, N exposed 2 = 12, N sheltered 1 = 15, N sheltered 2 = 15; CV = 2.726 mm. Group 3: N exposed 1 = 13, N exposed 2 = 14, N sheltered 1 = 12, N sheltered 2 = 6; CV = 7.915 mm. Group 4: N exposed 1 = 15, N exposed 2 = 15, N sheltered 1 = 15, N sheltered 2 = 15; CV = 7.670 mm. Group 5: N exposed 1 = 17, N exposed 2 = 17, N sheltered 1 = 15, N sheltered 2 = 18; CV = 12.331 mm. Group 6: N exposed 1 = 15, N exposed 2 = 15, N sheltered 1 = 15, N sheltered 2 = 15; CV = 12.753 mm. Group 7: N exposed 1 = 35, N exposed 2 = 30, N sheltered 1 = 28, N sheltered 2 = 22; CV = 18.066 mm. Group 8: N exposed 1 = 39, N exposed 2 = 30, N sheltered 1 = 30, N sheltered 2 = 30; CV = 16.872 mm.(TIF)Click here for additional data file.Figure S5Comparison of relative aperture width of adult laboratory-reared and field-collected N. lapillus from shores differing in exposure to wave action. Adjusted means (see text) are shown with standard errors. Shores are ranked in increasing order of wave exposure (see sure see : Menai B(TIF)Click here for additional data file.Data S1Supplementary data. Experiment 1: ontogenetic changes in shell morphology; Experiment 2: wave-exposure gradient.(DOC)Click here for additional data file.