Paenibacillus polymyxa A-8, which secretes β-mannosidase, was isolated from the soil sample under a pine tree located in the “Laoban” mountain region of Sichuan, China. The β-mannosidase gene (MANB) was isolated from P. polymyxa A-8, using primers according to the complete genome. The MANB  encoding 849 amino acid residues was expressed in Escherichia coli. The specific activities of β-mannosidase produced by P. polymyxa A-8 and E. coli pET30a-MANB were 12 nkat/mg and 635 nkat/mg respectively. SDS-PAGE analysis indicated that the molecular mass of the recombinant MANB was approximately 96 kDa. The recombinant MANB was active between pH 7.0–8.5 with the maximum activity at pH 7.0. It had good pH stability and adaptability. The MANB had the optimal temperature of 35°C and was relatively stable at 35–40°C. In addition, the MANB activity was enhanced by K+, Ca2+, Mn2+, and Mg2+ and inhibited by Zn2+, Cu2+, and Hg2+. Mannan is the second largest component of the hemicellulose and is distributed in plant cell walls β-Mannosidase  catalyzes the hydrolysis of β-1,4-mannosidic bonds by releasing a single β-D-mannose unit from the nonreducing end of mannooligo saccharides Escherichia coli under the control of the bacteriophage T7 promoter, which can greatly increase their production level The various microorganisms, including bacteria, archaea, fungi and actinomycetes, have been reported to secret β-mannosidases Paenibacillus polymyxa A-8 was isolated from the soil sample under a pine tree located in the “Laoban” mountain region of Sichuan, China. The β-mannosidase gene (MANB) from P. polymyxa A-8 was cloned and expressed in E. coli with the vector pET30α. In addition, the biochemical properties of the recombinant MANB were fully determined.In the present work, the bacterium P. polymyxa was isolated from the soil under a pine tree located in the “Laoban” mountain region near Sichuan Agricultural University . The P. polymyxa was cultivated at 30°C for 24 h in Konjac flour medium to induce β-mannosidase. E. coli BL21 was used to express the gene MANB. The pTG19-T  was the T-cloning plasmid. The expression plasmid, pET30a, was stored in our laboratory.This experiment was approved by Sichuan Agricultural University. 5′-AGAGTTTGATCCTGGCTCAG-3′ (forward) and 5′-TACCTTGTTACCACTT-3′ (reverse). The PCR reaction mixtures (25 µL) included approximately 10 ng of isolated strain DNA, 0.1 µM each primer, and 12.5 µl PCR premix . The thermal program was as follows: 6 min at 94°C: followed by 32 cycles of 94°C for 38 s, 59°C for 50 s, and 72°C for 1.5 min. The purified PCR products were ligated into the vector pTG19-T and sequenced by Generay.The 16S rRNA of the isolated microorganism was obtained by polymerase chain reaction (PCR) with the universal primers P. polymyxa A-8 was used as the template DNA for PCR. To amplify the MANB gene, both primers were designed according to complete genome of P. polymyxa M1 GGTACCATTAATGTGATAGGGTTGG -ATG-3′5′-GCT , and the reverse primer was GCGGCCGCCTAACCTTGTATCCAATTGATCA-3′5′-ATT . The PCR mixtures and program were similar to the above description. The resulting product was inserted into the pTG19-T vector.Genomic DNA extracted from MANB from P. polymyxa A-8 was performed by the ClustalW program, DNAstar 7.1 software and the BLAST server in GeneBank.The sequence analysis of KpnI and NotI, and the isolated MANB fragment was ligated into the pET30a vector. Next, the resulting vector pET30a-MANB was transformed into E. coli BL21.The vector pTG19-MANB was double-digested with E. coli pET30a-MANB harboring MANB was cultivated in 50 mL fresh LB medium kanamycin (30 µg/ml) and grown at 37°C for 4 h. Then, isopropyl β-D-thiogalactopyranoside (IPTG) was added into the culture broth at a final concentration of 1 mM. After cultivation for an additional 4 h, the cells were collected and fully resuspended in 10 mL of 0.05 M Tris/HCl buffer (pH 7.0), followed by ultrasonication. After centrifugation, the supernate was stored at −70°C.The single SDS–PAGE was performed on a 12% running gel and stained by Coomassie brilliant blue The supernatants containing the crude recombinant MANB were purified by a 6× His-Tagged Protein Purification kit  according to the manuals. The purified recombinant protein was stored at −70°C for analysis.The MANB activity was determined according to the methods described by Fliedrováet et al. The optimal pH of MANB was examined at 35°C for 30 min by using various buffers of pH 6–9 . The pH stability of MANB was tested by pre-incubating the enzyme in different buffers (pH 6–9) for 30 min without substrate before detecting its residual activity.The optimum temperature of the recombinant enzyme was determined at 20 to 50°C and pH 7.0. To examine its temperature stability, the MANB was incubated at 35°C, 40°C, and 45°C, respectively, for 30 min. In addition, its thermostability was determined by incubating the recombinant MANB for various times  at different temperatures (35°C and 45°C), and its residual mannosidase activity was then detected under the standard conditions as described above.2, KCl, FeCl3, CaCl2, MnCl2, MgCl2, CuCl2 or HgCl2. After the reaction, the mixture was centrifuged. Then the supernate was used to determine its activity. The mannosidase activity of the assay system in the absence of any additives was considered to be 100%.The effect of different metal ions on the activity of recombinant MANB was examined in 50 mM Tris/HCl buffer (pH 7.0) which contained 10 mM of ZnClThermotoga neapolitana 5068 reported by Duffaud et al. (1997) According to the forming of clear zones around the colonies, the plate technique was used to isolate mannosidase microorganisms P. polymyxa L1-9 than to the other microorganisms  residues with an evaluated MW of 96.3 kDa. Use of the BLAST program by NCBI showed that the nucleotide sequence of MANB gene had 94%, 80% and 77% identity to the Paenibacillus polymyxa M1 genome (HE577054), Arthroderma otae CBS MANB (XM_002846187) and Trichophyton verrucosum HKI MANB (XM_003022716). The sequence for MANB was deposited in the GeneBank database under the accession number of KF576220.The targeted DNA fragment was amplified from the genomic DNA of P. polymyxa A-8 belongs to the GHs family 2  family 2 . This faE. coli. The recombinant E. coli pET30a-MANB was cultivated in LB broth and induced by IPTG. SDS-PAGE analysis revealed that the MW of the expressed protein was apprx. 96 kDa corresponding with that evaluated from the amino acid sequence  and temperature (20–50°C) values. The recombinant MANB showed maximal activity at pH 7.0, and more than 70% of its optimal activity was maintained at pH 7.0–8.5  and Aspergillus niger ATCC-46890 (optimum pH: 2.4–5.0) E. coli pET30a-MANB had considerable pH stability and over 59% of the maximal activity remained after incubation at 35°C and pH 5.0–9.0 for 30 min . However, its activity was markedly inhibited by Zn2+, Cu2+ and Hg2+. Similar results have been reported for Streptomyces sp. S27 β-mannosidases activity which was increased by Mg and inhibited by Zn2+, Cu2+ and Hg2+The effect of metal ions on the recombinant MANB activity was also detected . The effMANB gene was successfully cloned from P. polymyxa A-8 and expressed in E. coli BL21. The recombinant MANB produced in E. coli pET30a-MANB had considerable pH and temperature stability. Moreover, the development of more powerful expression systems such as Picha pastoris will be considered in our laboratory for further research.The


The human Shwachman-Diamond syndrome (SDS) is an autosomal recessive disease caused by mutations in a highly conserved ribosome assembly factor SBDS. The functional role of SBDS is to cooperate with another assembly factor, elongation factor 1-like (Efl1), to promote the release of eukaryotic initiation factor 6 (eIF6) from the late-stage cytoplasmic 60S precursors. In the present work, we characterized, both biochemically and structurally, the interaction between the 60S subunit and SBDS protein (Sdo1p) from yeast. Our data show that Sdo1p interacts tightly with the mature 60S subunit in vitro through its domain I and II, and is capable of bridging two 60S subunits to form a stable 2:2 dimer. Structural analysis indicates that Sdo1p bind to the ribosomal P-site, in the proximity of uL16 and uL5, and with direct contact to H69 and H38. The dynamic nature of Sdo1p on the 60S subunit, together with its strategic binding position, suggests a surveillance role of Sdo1p in monitoring the conformational maturation of the ribosomal P-site. Altogether, our data support a conformational signal-relay cascade during late-stage 60S maturation, involving uL16, Sdo1p, and Efl1p, which interrogates the functional P-site to control the departure of the anti-association factor eIF6.The online version of this article (doi:10.1007/s13238-015-0242-5) contains supplementary material, which is available to authorized users. In eukaryotes, additional complexity is added, as the assembly starts in the nucleolus and involves the import of RPs and associated factors, as well as the export of premature ribosomal particles (66S and 43S pre-ribosomes) across nuclear membrane. Final maturation of the pre-ribosomes takes place in cytoplasm, and is coupled to the regulation of translation initiation  and 200 assembly factors (AFs)  and several complementary approaches, we performed structural and biochemical characterization of the interaction between the yeast SBDS homologue, Sdo1p and the 60S subunit. Our data reveal that Sdo1p binds to the ribosomal P-site and directly contacts H69 and H38 of the 25S rRNA. Moreover, owing to its structural flexibility, Sdo1p displays a dynamic behavior on the 60S subunit, with wobbling terminal domains (II and III). Very interestingly, Sdo1p is able to induce dimerization of the 60S subunits in a very specific manner. Together with published data, our results suggest that Sdo1p is an essential surveillance factor for the 60S maturation, which monitors the conformational status of the ribosomal P-site, including surrounding uL16, H69 and H38, and couples, through Efl1p, the maturation of the ribosomal P-site to the release of Tif6p.in vitro  or 80S ribosomes (12 mmol/L Mg2+) using SDGC. As shown in Fig. 2+), and as expected, Sdo1p only specifically dimerizes those 60S subunits without apparent effect on the 80S ribosomes . 80S ribosomes largely dissociate into 60S and 40S subunits in this dissociating condition  and 258 + 2T to C that produces premature truncation (84Cfs3) , comparable to the full-length protein (KD = 18 nmol/L) . The truncated domain I, II and II-III display modest level of affinities (KD ranging from 25–40 nmol/L) . In sharp contrast, domain III had no detectable binding .Firstly, we tested the ability of these mutants in the 60S subunit dimerization by SDGC in the presence of chemical crosslinker. As shown in Fig. From these results, a clear conclusion could be drawn: domain I of Sdo1p is the major RNA binding domain, while domain III has no RNA binding activity.Knowing that different oligomeric states of the 60S-Sdo1p complexes could form, we processed cryo-EM images according to the particle sizes. Particles representing dimers were selected and subjected to reference-free alignment and classification by RELION (See MATERIALS AND METHODS). As shown in Fig. From 2D image analysis, the 60S subunit and Sdo1p could form stable 60S-60S dimers, which should enable the 3D structural determination of dimers by single particle analysis. Towards this end, different 3D classification strategies were tested on particles of dimers, and both the symmetry-free and 2-fold symmetric reconstructions were explored . The 3D classification was done using RELION in a multi-round way. After the first round (C2-imposed), particles with 3D class structures showing relatively rigid dimers were kept for further analysis. Following steps were carried out without imposing two-fold symmetry. As a result, only 11% of particles displayed structural details for both 60S subunits, and structural refinement from these particles rendered a final density map at 14-Å resolution . This is expected, as 2D image analysis indicates that many dimers are not rigid dimmers Fig. , additioThe homology model of yeast Sdo1p was predicted by I-TASSER , exhaustive 3D classification did not reveal high-resolution densities for Sdo1p. When more particles were included for final refinement, as expected, we could only obtain a high-resolution structure for the 60S subunit. In contrast, when a small homogenous fraction of particles were used, an additional piece of density could be seen in the ribosomal P-site. . Based on our domain assignment in the 60S dimer, it is likely domain I of Sdo1p.3), and subjected the linked sample to mass spectrometry. The reactive sites of the cross-linkers interact with the primary amino group of lysine and N-terminal tail of the protein. Considering the 11.4-Å spacer arm between reactive sites, lysine side-chains within 30 Å could be potentially cross-linked.To further confirm our structural and biochemical data that Sdo1p binds to the ribosomal P-site, we employed cross-linking based mass spectrometry (CX-MS) -Lys184 (uL16) Figs. B and S5,Altogether, these results demonstrate that the interaction of Sdo1p with the 60S subunit is highly dynamic, which might be essential for its function in probing the conformational status of the ribosomal P-site during the late stage of cytoplasmic maturation of the 60S subunit.. More importantly, uL16 (R98S) mutant in yeast causes a defect in late-stage 60S subunit maturation and targets mutant ribosomes for degradation -p53-HDM2 pathway, and uL5 (RPL11) is one of RPs involved in stabilizing p53 following nucleolar stress , domain I (1–95), domain II (96–169), domain III (170–250), domain I-II (1–169) and domain II-III (96–250) constructs. The fragments were cloned into pET21b vector to yield pET21b-Sdo1p constructs with C-terminal 6× -His-tag for purification. The plasmids were transformed into E. coli BL21 (DE3) cells for overexpression.The genes encoding full-length and truncated Sdo1p were amplified using standard Polymerase Chain Reaction (PCR) from 600 of 1.0 and induced by 0.5 mmol/L IPTG for 4 h. Cells were pelleted and suspended in buffer A  containing 30 mmol/L imidazole and disrupted by an ultrasonic processor. Cell lysates were clarified by centrifugation at 13,000 rpm  for 1 h. The clarified lysates were load onto a Ni-NTA column  and eluted with buffer B (same as buffer A but containing 500 mmol/L imidazole). The target proteins were pooled, concentrated with Millipore Amicon Ultra Centrifugal filters and subjected to gel filtration chromatography  with buffer C . Purified proteins were split into aliquots and stored at −80°C.For protein purification, cells were cultured in LB media containing 50 μg/mL of ampicillin at 30°C to an ODS. cerevisiae S288C) were grown in 3 L of YEPD  medium to an OD600 of 1.0. Ribosome purification was carried out as previously described . Resuspended cells were disrupted with a high-pressure homogenizer (5000 p.s.i.) for three times. Cell lysates were centrifuged at 13,000 rpm for 1 h at 4°C in a JA 25.50 motor (Beckman Coulter). Supernatants were layered over a 10%–50% linear sucrose gradient , and centrifuged at 30,000 rpm for 5 h in an SW32 rotor (Beckman Coulter) at 4°C. The gradient profile was monitored at 254-nm wavelength and fractionated in a gradient collector (Teledyne Isco). Fractions of 80S ribosomes were collected, concentrated with Amicon Ultra Centrifugal filters (Millipore) and washed with separation buffer . After the buffer was changed to separation buffer, the mixture was incubated at 37°C for 10 min. Reaction mixtures were layered over a 10%–40% linear sucrose gradient  and centrifuged at 30,000 rpm in an SW32 rotor (Beckman Coulter) at 4°C for 7 h. The gradient was similar analyzed and fractions of 40S and 60S subunits were separately collected. After concentrating with Millipore Amicon Ultra Centrifugal filters, samples were split into aliquots and stored at −80°C for further use.Yeast cells (2), and incubated at 30°C for 15 min. The mixture was loaded onto a gel filtration column . The peak fractions were subjected to SDS-PAGE.The 60S subunit (150 pmol) and full-length Sdo1p (20-fold excess) were mixed in 500-µL buffer C  and centrifuged at 95,000 rpm for 2 h in a TLA-100 rotor (Beckman Coulter) at 4°C. The supernatants (1/18) and the pellets (1/3) were collected separately and resolved by Tricine-SDS-PAGE.60S ribosomal subunits 50 pmol) were incubated with full-length or truncated Sdo1p proteins in a ratio of 1:20 for 15 min at 30°C in 90 µL buffer. The mixtures were then loaded onto a sucrose cushion . 37.5 pmol ribosomes  were incubated with full-length or truncated Sdo1p proteins in a ratio of 1:20 for 15 min at 30°C in 200 µL buffer. The mixtures were incubated with or without 0.1% glutaraldehyde for another 15 min, layered onto a 10%–50% linear sucrose gradient , and centrifuged at 39,000 rpm for 4 h in an SW41 rotor (Beckman Coulter) at 4°C. The gradient was analyzed and fractionated in an ISCO gradient collector.Samples of ribosomes and Sdo1p variants were changed to a Hepes-KOH buffer system  were immobilized to the optic biosensors of Anti-Penta-HIS . Before each experiment, biosensors were pre-equilibrated with binding buffer, followed by equilibrium binding with Sdo1p variants (2 μg/mL). Pre-experiments were carried to determine the optimal concentration range of the 60S subunit suitable for the measurements. For the single concentration experiments, a final concentration of 48 nmol/L 60S subunit was used. For the concentration gradient experiments, 2-fold dilution series of the 60S subunit were used . Both the association and dissociation of each measurement were carried out for 240 s. The data was analyzed and dissociation constants were calculated by the software of the Data analysis 7.0 provided by ForteBio.The BLI-based experiments were performed with Octet RED96 System  according to the manufacturer’s instructions. Sdo1p variants (wild type and mutants) in Hepes-KOH buffer system  with AutoEMation package  was incubated with 20-fold or 10-fold excess of full-length Sdo1p at 30°C for 15 min. The mixture was diluted to a final concentration of 60 nmol/L for the 60S complex in binding buffer. 4-µL aliquots of samples were applied to 300-mesh 2/2 glow-discharged Quantifoil grids (Quantifoil Micro Tools) which were pre-coated with a thin layer of carbon. The grids were blotted and plunged into liquid ethane with an FEI Mark IV Vitrobot operated at 4°C. For the sample of dimeric 60S-Sdo1p complexes (20-fold excess of Sdo1p), data collection was performed with an FEI F20 at 80,000× magnification with a Gatan UltraScan 4000 CCD camera. For the sample of monomeric 60S-Sdo1p complexes, data collection was done with an FEI Titan Krios equipped with an FEI Eagle 4 K × 4 K CCD camera at 75,000× magnification. All the images were recorded under low-dose conditions   were classified into 10 classes using RELION. One class  displayed substantial additional density at the ribosomal P-site. After structural refinement, the final reported resolution is 9 Å based on the 0.143 cutoff criteria of the gold-standard FSC.The yeast homology model of Sdo1p was modelled using I-TASSER . The mixture was cross-linked with DSS or BS3 (Thermo Fisher Scientific) at 1:1 mass ratio at room temperature for 1 h. The reaction was then quenched with 20 mmol/L ammonium bicarbonate. Cross-linking products were analyzed by SDS-PAGE to evaluate the cross-linking efficiency. For MS analysis, proteins were precipitated with acetone, resuspended in 8 mol/L urea, 100 mmol/L Tris, pH 8.5, and digested with trypsin. LC-MS/MS analyses were carried on an EASY-nLC 1000 system (Thermo Fisher Scientific) interfaced with a Q-Exactive mass spectrometer (Thermo Fisher Scientific). Peptides were separated on an analytical capillary column  using a 60 min linear gradient at a flow rate of 200 nL/min. The mass spectrometer was operated in data-dependent mode with one full MS scan followed by ten HCD MS/MS scans with a dynamic exclusion time of 30 s. Precursors of the +1, +2, or unassigned charge states were rejected. pLink


Talaromyces pinophilus strain 1-95. The purified amylolytic enzyme, with an apparent molecular weight of 58 kDa on SDS-PAGE, hydrolyzed maltopentaose, maltohexaose, and maltoheptaose into mainly maltose and maltotriose and minor amount of glucose, confirming the endo-acting mode of the enzyme, and hence, was named Talaromyces pinophilus α-amylase (TpAA). TpAA was most active at pH 4.0–5.0 (with the temperature held at 37°C) and 55°C (at pH 5.0), and stable within the pH range of 5.0–9.5 (at 4°C) and below 45°C (at pH 5.0). Interestingly, the Ca2+ did not improve its enzymatic activity, optimal temperature, or thermostability of the enzyme, indicating that the TpAA was Ca2+-independent. TpAA displayed higher enzyme activity toward malto-oligosaccharides and dextrin than other previously reported α-amylases. This highly active Ca2+-independent α-amylase may have potential applications in starch-to-ethanol conversion process.Alpha-amylase is a very important enzyme in the starch conversion process. Most of the α-amylases are calcium-dependent and exhibit poor performance in the simultaneous saccharification and fermentation process of industrial bioethanol production that uses starch as feedstock. In this study, an extracellular amylolytic enzyme was purified from the culture broth of newly isolated  Starch is a kind of macromolecule carbohydrate that is composed of glucose units connected by α-1,4-glycosidic bonds in linear chains and α-1,6-glycosidic bonds in branching points . The α-aFossil oil is a kind of nonrenewable resource. One of the alternatives to fossil oil is ethanol, which is a clean and renewable fuel ,6. The p2+-dependent dextrinizing α-amylase from Bacillus licheniformis [2+ to enhance the enzyme activity and thermostability of the α-amylase [2+-independent α-amylases, which are active and stable at high temperature and near neutral pH, have been reported to be candidates for possible substitution of the commercial Ca2+-dependent dextrinizing α-amylases [2+, since Ca2+ accelerates the deterioration of industrial equipments by forming the precipitate calcium oxalate, which blocks pipes and heat exchangers, inhibits the isomerization of glucose, and accumulates in the end product in the fructose production industry [Aspergillus niger or related species at about 60°C and pH 4.0–5.0 [Saccharomyces cerevisiae at 30–35°C and pH 4.0–5.0 [The traditional ethanol-producing industry that uses starch as a feedstock employs a separate hydrolysis and fermentation (SHF) process. This process includes three sequential steps: liquefaction, saccharification and fermentation. In the first step of liquefaction, the gelatinized starch is liquefied to malto-oligosaccharides by the Caniformis –12 or re-amylase ,13–16. Ramylases –21. The industry ,15. In t 4.0–5.0 ,23. In t 4.0–5.0 ,24.During the last decade, the saccharification step and fermentation step of the SHF process were combined by directly adding glucoamylase and yeast after the malto-oligosaccharides syrup had been cooled down to 30–35°C, so that the hydrolysis of malto-oligosaccharides to glucose by glucoamylase and the fermentation of glucose to ethanol were simultaneously conducted at 30–35°C and pH 4.0–5.0 . This ne2+-dependent α-amylases can be added to accelerate the hydrolysis of malto-oligosaccharides as they act synergistically with glucoamylase [Since the present used glucoamylases exhibit poor performance at 30–35°C due to their high temperature optima ,29, a muoamylase ,30.2+-independent possible candidates [2+-independent α-amylases have been reported to display high relative activity at 30–35°C [The α-amylases used currently in industry –12,30 anndidates –21 exhib 30–35°C –34, thei 30–35°C ,24,27.Talaromyces pinophilus strain 1–95. This α-amylase showed collectively good properties as it is Ca2+-independent, has high specific and relative activity at 30–35°C and an optimal pH of 4.0–5.0, and exhibits higher enzyme activity than other reported α-amylases in hydrolyzing malto-oligosaccharides and dextrin.In this study, we purified and characterized one α-amylase from the newly isolated No animal or protected plants were involved in this study, and the ecosystem was not compromised by the collection of experimental samples. This study did not harm the natural environment or the health of humans.Microorganism-containing samples were collected from the suburbs of Wuzhou city . These samples included soil, rotten starchy materials, rotten fruits and plants, and fermented Chinese pickled vegetables.Soluble cassava starch was bought from the Mingyang Biochemistry Co., Ltd. , and other carbohydrates were all purchased from Sigma Co., Ltd . Unless otherwise stated, all chemicals used in this study were of analytical grade.2 column (4.6 mm×250 mm) was purchased from Dalian Elite Analytical Instrument Co., Ltd. .ÄKTA purifier 100 instruments, HiPrep 16/10 phenyl fast flow hydrophobic interaction chromatography (HIC) column, HiPrep Q XL 16/10 sepharose anion exchange chromatography (AEC) column, and Sephadex G25 HiPrep 26/10 desalting column were all purchased from GE Co., Ltd. . High performance liquid chromatography instrument was purchased from Shimadzu Co., Ltd. . Hypersil NH2HPO4 3, (NH4)2SO4 2.5, MgSO4·7H2O 0.2, CaCl2 0.13, FeSO4 0.0255, Trypan Blue 0.06, and agar 30, at pH 5.0. The liquid cultivation medium did not contain Trypan Blue or agar; other components were the same as in the isolating medium. To release the microorganisms in sample, one gram of sample was thoroughly ground and submerged in 50 mL of sterile distilled water, and then was shaken at 28°C and 180 rpm for 30 min. The suspension was serially diluted, and then 50 μL of each dilution were evenly spread onto one isolating medium agar plate. After cultivating at 28°C for 5 days, microorganisms with a clear halo around the colony were inoculated into the liquid cultivation medium (100 mL of medium in a 500-mL Erlenmeyer flask). After 5 days of cultivation at 28°C and 180 rpm, the amylolytic activity in the supernatant was measured at 30°C and pH 4.0 by using the dinitrosalicylic acid (DNS) method [The isolating medium used for isolation of amylolytic microorganisms contained (g/L): soluble starch 10, tryptone 5, K) method . Finally3 3, K2HPO4•3H2O 1.3, MgSO4•7H2O 0.5, KCl 0.5, FeSO4•7H2O 0.01, ZnSO4•7H2O 0.01, pH 7.0, agar 1.5% [The fungal strain 1–95 was identified by analyzing its morphological and molecular data. For morphological observation, strain 1–95 was three-point inoculated on Czapek-Dox agar plate in 9-cm petri dish and incubated in dark at 25°C and 37°C, and morphological and microscopic examinations  were performed. The Czapek-Dox agar media contained (g/L): sucrose 30, NaNOgar 1.5% .5’-CGCCAATCGAGGTCATGACATGG-3’), respectively. The PCR procedure was according to that described by Lin et al. [http://blast.ncbi.nlm.nih.gov/Blast.cgi).For molecular data analysis, the partial internal transcribed spacer (ITS) sequence, and the partial coding sequences of the β-tubulin gene and calmodulin gene of the fungal strain 1–95 were amplified using the corresponding primer sets of ITS1 and ITS4 , Bt2a ann et al. . The amp2SO3 0.5). Then the mixture was incubated in boiling water for 5 min followed by cooling down to room temperature. The absorbance value of the mixture under 540 nanometer was read and the amylolytic activity was calculated using a glucose standard curve constructed by using DNS method [The amylolytic activity was determined by measuring the reducing sugars released by substrate hydrolysis. Before enzyme and protein assays, the culture supernatant of fungal strain 1–95 and the purified amylolytic enzyme solutions were desalted using a HiPrep 26/10 desalting column with deionized water as eluent. In the enzyme assay process, 0.35 mL of 0.1 M citrate-phosphate buffer with a preset pH value containing 1% (w/v) substrate was preheated at the preset temperature for 5 min, and then 0.05 mL of appropriately diluted enzyme solution was added and mixed well. After 10 min of incubation, the hydrolytic reaction was stopped by adding 0.8 mL of DNS reagent (containing (g/L): rochelle salt 200, NaOH 20, dinitrosalicylic acid 10, crystalline phenol 2, NaProtein concentration was determined via the BCA method  using a 2 (W/V) and 2% KI (W/V), a transparent zone appeared with a deep blue background indicating the location of amylolytic enzyme.For zymogram analysis using native PAGE, denaturant and reductant were excluded in the electrophoresis system, and 1 g/L soluble starch was contained in the separating gel . When thThe culture broth of fungal strain 1–95 displaying amylolytic activity was obtained by liquid cultivation using the method described above. The cell-free culture supernatant was gently mixed with a 33% volume of 4 M ammonium sulfate solution (pH 6.5) and incubated at 4°C for 24 h. After centrifugation at 8000×g for 10 min (4°C) and followed by filtration with a 0.22-μm filter membrane, the supernatant was applied to HIC using a HiPrep 16/10 phenyl fast flow column, and the bound proteins were eluted with a linear gradient of ammonium sulfate from 1 M to 0 M . The eluted fractions displaying amylolytic activity were combined and desalted using a HiPrep 26/10 desalting column with 0.02 M Tris-HCl (pH 8.0) as eluent, and then subjected to AEC using a HiPrep Q XL 16/10 sepharose column. The bound proteins were eluted with a linear gradient of NaCl from 0 to 1 M , and the eluted fractions displaying amylolytic activity were subjected to native PAGE and zymogram analysis to determine the protein band corresponding to the amylolytic enzyme. To obtain large amount of the amylolytic enzyme, the remaining eluent fractions containing the amylolytic enzyme were combined and subjected to native PAGE at 4°C for 4 h. After electrophoresis, the zone of unstained gel containing the protein band of the amylolytic enzyme, which was localized by native PAGE and zymogram analysis, was cut and ground, and then dispersed in 0.02 M Tris-HCl (pH 8.0) at 4°C for 24 h to allow the amylolytic enzyme to permeate into the buffer.The enzyme solution was concentrated by using a 10 kDa cutoff ultra-filtration membrane, and then the purity of enzyme was determined by using SDS-PAGE. The purified enzyme was desalted and used for all the experiments described below.2 column. A water solution containing 74% acetonitrile (V/V) was used as the mobile phase with a flow rate of 1 mL/min. The hydrolysis products were detected using a RID-10A SHIMADZU refractive detector, with a solution containing glucose and DP2 to DP7 as standards.The purified enzyme was used at a dose of 0.2 U/mg substrate  to hydrolyze the linear malto-oligosaccharides with the degree of glucose polymerization from 3 to 7 (DP3 to DP7) at 30°C and pH 5.0 for 12 h. The hydrolysates were filtered through a 0.2-μm filter membrane and analyzed by high performance liquid chromatography (HPLC) using a Hypersil NH2) [The effect of pH on the enzyme activity was determined by measuring the enzyme activity at pH 3.0–7.0 (in 0.1 M citrate-phosphate buffer) and 37°C. The effect of temperature on the amylolytic activity was determined by measuring the enzyme activity at pH 5.0 and temperatures from 25°C to 75°C (with or without 5 mM CaCl2) . The hig2) [The effect of pH on enzyme stability was determined by mixing 0.1 mL of enzyme solution with 0.9 mL of 0.1 M buffers with different pH values. The buffers used were citrate-phosphate buffer (pH 3.0–7.0), Tris-HCl buffer (pH 7.0–9.0), and glycine-NaOH buffer (pH 8.5–11.0). After incubating at 4°C for 24 h, the residual enzyme activities were measured at pH 5.0 and 55°C. The effect of temperature on enzyme stability was tested by incubating the enzyme at 40–65°C for 1 h  ,19,34, aThe kinetic parameters of the α-amylase with respect to different soluble starches were measured at pH 5.0 and 55°C, with the concentration of the substrate ranging from 1.25 to 10 g/L, All experiments were performed in triplicate, and mean values were used to calculate the Km and Vmax values using the Lineweaver-Burk plotting method.2, CoCl2, CrCl3, CsCl2, CuCl2, FeCl2, FeCl3, KCl, LiCl, MgCl2, MnCl2, NaCl, NiCl2, Pb(NO3)2, ZnCl2, AgNO3, ethylenediaminetetraacetic acid (EDTA), disodium ethylenediaminetetraacetic acid (Na2EDTA), and ethylene glycol tetraacetic acid (EGTA). These additives were widely used in many previous reports of α-amylase, and thus were selected for this study. These additives were individually added into the enzyme assay system to reach final concentration of 1, 2.5, 5, and 10 mM, and then the enzyme assay was conducted. The enzyme activity without additives was defined as 100%, and the enzyme activities measured with different additives were calculated as relative activities.The enzyme activity was measured at pH 5.0 and 55°C in the presence of different individual additives of CaClThe organic solvents methanol, ethanol, ethylene glycol, acetonitrile, ethyl acetate, n-propanol, isopropyl alcohol, acetone, glycerol, and n-butanol were used to determine the effects of low-molecular-weight organic solvents on the enzyme activity at pH 5.0 and 55°C. The concentration of ethanol was 5, 10, or 15% (V/V), respectively. The concentration of other additives was 5% (V/V). The enzyme activity without additives was defined as 100%, and the enzyme activities measured with different additives were calculated as relative activities.Enzyme activities were measured toward different substrates at a concentration of 1% (w/v) at pH 5.0 and 55°C. The enzyme activity on soluble starch was defined as 100%, and the enzyme activities on other substrates were calculated as relative activities.The partial ITS sequence, partial coding sequences of the β-tubulin gene and calmodulin gene of the fungal strain 1–95 were deposited into the GenBank database under accession numbers FJ795356, KP342453, and KP342454, respectively.To obtain mesophilic and acidic amylolytic enzymes, amylolytic microorganisms were isolated from the 115 collected samples at pH 5 and 28°C. Among the 115 samples, 99 microbial strains showing a clear hydrolyzing halo around the colony were isolated and screened by inoculating into liquid cultivation medium at pH 5 and 28°C for 5 days. Among these microbial strains, a filamentous fungal strain 1–95 was found to produce the highest amylolytic activity of 1.49 U/mL (measured at 30°C and pH 4.0) in the culture broth and was selected for further study.Talaromyces. The conidia are globose to elliptical with smooth surface  [The fungal strain 1–95 grew well at both 25°C and 37°C, but for ease of comparison with other strains, the morphological data for its growth at 25°C was described here. The newborn hyphae of fungal strain 1–95 were white, loose and floccose, and then turned yellow after 3 days of cultivation. Yellow color dominated the colony after 4 days cultivation, then the strain entered conidia generation phase, and the center of the colony began to turn green. After 9 days of cultivation, the diameter of the colony reached 4.5 cm . During  surface . The morophilus) –47.Talaromyces pinophilus . The partial coding sequence of the β-tubulin gene of fungal strain 1–95 shared higher than 99% identity with those of the β-tubulin genes of eight strains of Talaromyces pinophilus . The partial coding sequence of the calmodulin gene of fungal strain 1–95 shared higher than 99% identity with those of the calmodulin genes of three strains of Talaromyces pinophilus .The ITS sequence of fungal strain 1–95 shared 100% identity with the ITS sequence of nophilus )  [Bacillus sp. KR-8104 (59 kDa) [Bacillus sp. TM1 (59 kDa) [Aspergillus niger CBS 513.88 (63 kDa) [T. pinophilus 1–95 had a specific activity of 353.41 U/mg protein toward soluble starch at 30°C and pH 4.0.As shown in analysis ; these r(58 kDa) , Bacillu(59 kDa) , Bacillu(59 kDa) , and Asp(63 kDa) . The purTalaromyces pinophilus α-amylase (TpAA).By comparing their retention times to those of the standard sugars in HPLC , it was Penicillium expansum [In the ethanol-producing industry using starch as feedstock, a dextrinizing α-amylase is required in order to reduce the DP of starch at high temperature ,23,24, aexpansum  showed sexpansum ,33, weakexpansum , or wereexpansum , TpAA shexpansum ,49–51. Aexpansum ,30.2, indicating that the temperature optimum [2+-independent.TpAA displayed pH optima within the range of 4.0–5.0  and was  optimum  and the  optimum ,19,34 ofAlpha-amylases having different pH and temperature characteristics have different applications. Thermostable α-amylases are used to degrade gelatinized starch at high temperature ,24,52, w2+-dependent α-amylase from Aspergillus oryzae S2 [TpAA showed Km values from 2.51 to 6.70 mg/L toward six kinds of soluble starches , which wryzae S2 ; and theryzae S2 . These r2+ did not enhance the enzyme activity, indicating that the enzyme activity of TpAA was Ca2+-independent [+, Mg2+, and Na+ did not affect the enzyme activity, which was similar to BsAA [2+ did not affect the enzyme activity, which was similar to TLYAA [3+ on the enzyme activity of TpAA was similar to that on BTMAA [2+ on the enzyme activity of TpAA was similar to that on McAA [2+, Cu2+, Ag+, and Pb2+ on the enzyme activity of TpAA were similar to those on AtAA [2EDTA and EGTA had no effects on the enzyme activity of TpAA.As shown in ependent ,20,32,56to TLYAA . The stion BTMAA , and the on McAA  and AnAA on McAA . The inh on AtAA . EDTA, N2+ and chelating reagents on the activity of α-amylase varied. In general, Ca2+ stimulates the enzyme activity of or stabilizes α-amylases [2+ insensitive [2+ inhibits the activity of α-amylase [2EDTA usually act as inhibitors of the activity of α-amylase [2EDTA [2+ and EDTA, similar to some α-amylases reported previously [In previous reports, the effect of Caamylases ,51,55,57ensitive ,33,50,56-amylase ,31,58,59-amylase ,48,56,58e [2EDTA ,50,54,55eviously ,33,50.2+, Co2+, Cr3+, Fe2+, K+, Li+, Mg2+, Na+, or Zn2+; stimulated by 5 mM Co2+, Cs2+, Fe2+, or Fe3+; insensitive to 1 mM and only partially inhibited by 10 mM Cu2+, Mn2+, Pb2+, or Ag+. Overall, most of the tested metal ions at various concentrations had no effect on the enzyme activity or even stimulated the enzyme activity of TpAA, indicating that the enzyme activity of TpAA was highly compatible with these metal ions. Some α-amylases were reported to be poorly compatible with metal ions because their enzyme activities were seriously inhibited by most of metal ions mentioned above [The enzyme activity of TpAA was insensitive to 10 mM Caed above ,31,51,56ed above ,31,51,56TpAA exhibited relative activity of 91.84%, 84.08%, and 69.72% in the presence of 5%, 10%, and 15% ethanol, respectively, and showed high enzyme performance in the presence of 5% of other low-molecular-weight organic solvents . This chTpAA showed higher activity toward amylose than amylopectin , which mTpAA could not hydrolyze α-CD (Cyclodextrin) and β-CD but could hydrolyzed γ-CD. This might be due to the cyclical shape of CD contributing to the configuration of substrate and making the substrate low accessibility to the active site of the enzyme, thus making enzyme catalysis difficult. The cyclical shape of CD with longer chain showed weaker effect on the configuration of the molecule than other CD with shorter chain. McAA displayed similar behavior with a high activity toward γ-CD and lower activity toward β-CD than γ-CD, and almost no activity toward α-CD . Many α-TpAA exhibited very low activity toward pulullan, perhaps because TpAA prefers α-1,4 glycosidic bond to α-1,6 glycosidic bond, and the abundance of α-1,6-D-glycosidic bonds contributes to the configuration of the substrate and makes enzyme catalysis difficult ,63. Most2+-independent α-amylases, TpAA displays excellent comprehensive characteristics.TpAA showed high specific activity toward soluble starches from corn, wheat, cassava, rice, and potato, and the highest enzyme activity was observed when cassava starch was the substrate. It is well known that corn, wheat, and cassava starches are widely used for fuel ethanol production –9. The dTalaromyces pinophilus 1–95, and biochemically characterized. The enzyme activity and thermostability of TpAA were both found to be Ca2+-independent. When compared with other Ca2+-independent α-amylases, TpAA displays excellent comprehensive characteristics including high activity at pH 4.0–5.0 and high specific and relative activities at 30–35°C; high enzyme activity toward malto-oligosaccharides, dextrin, and soluble starches from corn, wheat, cassava, rice, and potato; and high compatibility with metal ions and insensitive to low-molecular-weight organic solvents. Thus, TpAA has potential applications in the SSF of starch to ethanol.In this study, TpAA was purified from a newly isolated filamentous fungal strain,


Patients with type 2 diabetes mellitus (T2DM) have increased severity of periodontitis. Toll-like receptor (TLR)4, its co-receptors CD14 and MD-2, and adaptor MyD88 play pivotal roles in lipopolysaccharide (LPS)-triggered tissue inflammation and periodontitis. This study investigated the effects of T2DM and periodontitis on TLR4, CD14, MD-2 and MyD88 mRNA expression in surgically removed periodontal tissues.Periodontal tissue specimens were collected from 14 patients without periodontitis and T2DM (Group 1), 15 patients with periodontitis alone (Group 2), and 7 patients with both periodontitis and T2DM (Group 3). The mRNA of TLR4, CD14, MD-2 and MyD88 was quantified using real-time PCR and compared between the groups.p = 0.02) whereas the mRNA expression of TLR4, MD-2 and MyD88 was not significantly different among the groups. Furthermore, when patients in Groups 1 and 2 were combined (n = 22), the CD14 mRNA expression was significantly lower than that in patients of Group 1 (p = 0.04).Statistical analysis showed that periodontal expression of CD14 mRNA was significantly reduced across Groups 1, 2 and 3 (CD14 mRNA expression was downregulated across patients with neither periodontitis nor T2DM, patients with periodontitis alone and patients with both diseases, suggesting that CD14 mRNA expression is associated with a favorable host response or subjected to a negative feedback regulation. Gram-negative bacteria are the major pathogens involved in periodontitis, which is characterized by inflammatory tissue destruction of tooth-supporting structures . Studiesin vitro studies that high glucose enhances LPS-triggered expression of proinflammatory genes in macrophages and gingival fibroblasts [It has been well established that periodontitis is more prevalent and severe in patients with poorly controlled type 2 diabetes mellitus (T2DM) than nondiabetic individuals –11. Findroblasts –19, indiIn the studies to understand how T2DM increases host responsiveness to LPS, it has been shown that T2DM increases the expression of TLR4 in host cells such as monocytes , 21. GivIn this study, we hypothesized that the expression of TLR4 or TLR4-associated molecules such as CD14, MD-2 and MyD88 are regulated by periodontitis and T2DM. To test our hypothesis, we collected periodontal tissues from patients with or without periodontitis and T2DM at the time of necessary surgeries and determined the mRNA expression of TLR4, CD14, MD-2 and MyD88 using quantitative real-time polymerase chain reaction (PCR).Thirty-six patients were selected from a population referred to the Post-doctoral Periodontics Program in the College of Dental Medicine at the Medical University of South Carolina, Charleston, South Carolina. Patients recruited included 14 individuals who did not have periodontitis and T2DM (group 1), 15 patients with periodontitis only (group 2), and 7 patients with both diseases (group 3). The patients in group 1 who required non-periodontal disease-related surgeries, such as crown lengthening and extractions served as controls. All the tissues collected were periodontal tissues taken as collars from the gingival margin to include both epithelium and connective tissue.The patients from group 2 and group 3 met the following diagnostic criteria for periodontitis –14: A peTotal RNA was isolated from periodontal tissue specimens using the RNeasy minikit . The first-strand complementary DNA (cDNA) was synthesized with the iScript™ cDNA synthesis kit  by following the instruction provided by the manufacturer. The reaction was cycled for 5 min at 25 °C, 30 min at 42 °C and 5 min at 85 °C using a PTC-200 DNA Engine .Ct) of fluorescent units was used for analysis. Quantification was calculated using the Ct of the target signal relative to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signal in the same RNA sample.The RT reaction mixture from the above experiments was then subjected to real-time PCR amplification. The Beacon Designer Software  was used for designing primers Table . Primersp < 0.05 was considered significant. The correlation analysis on the relationship between CD14 mRNA expression and PD, AL or HbA1c was evaluated by Pearson’s method. Our previous studies have indicated that the sample size of 7 or greater provided sufficient power to detect the difference of periodontal mRNA expression of IL-6 and MMP-8 across control individuals who did not have periodontitis and T2DM, patients with periodontitis alone and patients with both periodontitis and diabetes [The age, gender and race of the study participants are presented as counts and the continuous variables are presented as medians. The GraphPad Instat 3 software  was used for statistical analysis. Nonparametric analyses testing for any differences in continuous variables among groups greater than two were performed using the Kruskal-Wallis procedure. Nonparametric analysis testing for any difference in variables between two groups was performed using the Mann-Whitney procedure. A value of diabetes , 14.The clinical data for patient age, gender, race, smoking status, HbA1c, periodontal PD, and AL were presented in Table - The ages of subjects in groups 1, 2, and 3 ranged from 42 to 79, 40 to 79, and 52 to 71 with a mean ± standard deviation of 58 ± 11, 56 ± 11, and 63 ± 6, respectively. No significant difference between groups was found.Age - The ratios of male/female gender in groups 1, 2, and 3 were 3.7 (11/3), 2 (10/5), and 7 (7/0), respectively. No significant difference was found between the groups.Gender - The ratios of Caucasians vs. African Americans in groups 1, 2, and 3 were 6.0 (12/2), 2.8 (11/4), and and 0.17 (1/6), respectively. Statistical analysis indicated that African Americans in group 3 were significantly more than those in group 1 or group 2, which is consistent with the previous reports that African Americans have a high risk of developing T2DM and periodontitis [Race dontitis , 23.- The smoker/nonsmoker ratios in groups 1, 2, and 3 were 0.4 (4/10), 1.5 (9/6), and 2.5 (5/2), respectively. Statistical analysis indicated that smokers in group 2 and group 3 were significantly more than those in group 1, which is consistent with the previous reports that smoking is associated with a high risk for periodontitis [Smoking status dontitis .Periodontitis - The PD in groups 1, 2 and 3 was 2.45 ± 0.86, 4.20 ± 2.00 and 4.80 ± 2.32 mm, respectively. The AL in groups 1, 2 and 3 was 2.48 ± 0.89, 5.03 ± 2.48 and 5.24 ± 2.39 mm, respectively. A significant difference of PD and AL was found between group 1 and group 2 or group 3, but no significant difference of PD and AL was found between group 2 and group 3.Diabetes - All patients in group 3 reported having T2DM. The mean HbA1c in Groups 3 ranged from 7.0 % to 10.7 % with a mean ± standard deviation of 8.19 ± 1.24 % that is significantly higher than 5.81 ± 0.55 % and 5.60 ± 0.56 %, respectively, in Group 1 and Group 2. Four of the seven patients were prescribed metformin alone or in combination with Glucotrol  or NPH insulin . One patient was prescribed insulin Glargine , one patient was prescribed insulin Aspart , and one patient did not report taking any medication for diabetes.p = 0.020). We also compared the CD14 expression between two groups. Results showed that although CD14 expression in Group 2  is lower than that in Group 1 (patients without both periodontitis and T2DM), the difference is not statistically significant. However, when the patients in Group 2 were combined with Group 3 (patients with both periodontitis and T2DM), the CD14 expression was significantly lower than that in Group 1 (p = 0.04) (Table p = 0.003), indicating that periodontal CD14 expression in patients with both periodontitis and T2DM is significantly lower than that in patients without periodontitis and T2DM.The expression of CD14, TLR4, MD-2, and MyD88 in surgically removed periodontal tissues was successfully quantified using real-time PCR. As shown in Table The relationship between CD14 mRNA expression and PD, AL or HbA1c was statistically analyzed and no significant correlations were found between CD14 mRNA expression and these clinical parameters Table .Table 5TOur current study demonstrated that CD14 mRNA level was significantly reduced across patients with neither periodontitis nor T2DM, patients with periodontitis alone, and patients with both periodontitis and T2DM, suggesting that periodontitis and T2DM negatively regulates CD14 gene expression in periodontal tissue. Our findings are consistent with those by Jin et al., who reported that the CD14 protein level in gingival biopsy tissues from patients with periodontitis was significantly lower than that in subjects without periodontitis . They alIn the current study, we employed real-time PCR technique to quantify the mRNA expression level of TLR4, CD14, MD-2 and MyD88 in periodontal tissue. It is well known that regulation of cytokine expression at the mRNA level via nuclear factor-kappa B (NFκB)-mediated transcriptional activation plays a crucial role in periodontitis . TherefoCD14 was characterized as LPS receptor in 1990 , almost Previous studies have shown that CD14 has dual roles in response to Gram-negative bacteria . For exaPrevious studies have also shown the dual roles of CD14 in T2DM. On one hand, it was reported that CD14 expression was significantly associated with T2DM and correlated with serum concentrations of C-reactive protein ; On the The finding that CD14 was downregulated in patients with periodontitis may also reveal a negative feedback mechanism by which periodontal tissue of patients with periodontitis prevent further damage from repeated attacks by bacteria-derived LPS. Interestingly, Nemoto et al. reported that CD14 expression on human gingival fibroblasts (HGFs) was markedly reduced by phorbol 12-myristate 13-acetate (PMA)-activated polymorphonuclear leukocytes (PMNs) in a coculture system of HGF and PMNs . Their fIn addition to CD14, our current study also examined the periodontal expression of TLR4, MD-2 and MyD88. Results showed that the periodontal mRNA expressions of TLR4, MD-2 and MyD88 had no significant difference across the 3 groups. While our current study is the first one focusing on TLR4 mRNA expression, Promsudthi et al. and Rojo-Botello et al. have reported the effect of T2DM and chronic periodontitis on TLR4 protein expression , 30. Proin vitro [It has been well documented that CD14 expression is upregulated by LPS and inflammatory cytokines in vitro –45. Howein vitro . Interesin vitro . TherefoCD14 expression was downregulated across patients with neither periodontitis nor T2DM, patients with periodontitis alone, and patients with both periodontitis and T2DM. This finding indicates that CD14 expression is associated with a favorable host response or subjected to a negative feedback regulation.


To evaluate efficacy and toxicity of hypofractionated intensity-modulated simultaneous integrated boost (IMRT-SIB) and image-guided (IGRT) radiotherapy in the treatment of high-risk prostate cancer patients.Eighty-two patients with high-risk prostate cancer were analysed. An IMRT treatment was planned delivering 68.75 Gy to the prostate, 55 Gy to the seminal vesicles and positive nodes and 45 Gy to the pelvis in 25 fractions. The first 59 patients received 4 weekly fractions whereas the last 23 patients received 5 weekly fractions. All patients were submitted to hormonal therapy.The median follow-up was 31 months. Acute grade 1–2 gastrointestinal (GI) toxicity rates were 13.4%. Grade 1–2 and grade 3 genitourinary (GU) toxicity rates were 22% and 1.2%, respectively.Grade 1 and 2 GI late toxicity rates were 1.2%. No grade ≥3 toxicity was recorded. Grade 1 GU late toxicity rate was 2.4%. No grade ≥2 toxicity was recorded.No significant difference was calculated in terms of acute and late toxicity between the group treated 4 or 5 times weekly.The actuarial 3-years Overall survival and Freedom from biochemical failure were 98.6% and 91.3%, respectively.The present study demonstrated that hypofractionated IGRT-IMRT-SIB in patients with high-risk prostate cancer is efficient with acceptable toxicity profile. Outcome in terms of survival are promising, but longer follow-up is needed. High-risk prostate cancer requires a multimodality approach therapy. One of the standard curative treatments is the definitive radiation therapy (RT) associated with androgen deprivation therapy (ADT) , 2. SeveRecent literature suggests that adenocarcinoma of the prostate gland is different from many other malignancies, with an average a/b ratio about 1.5 Gy . The a/b ratio for late rectal radiation effects is generally calculated between 3 and 4 Gy . The ratHypofractionated radiotherapy can be carried out safety after the development of image-guided radiotherapy (IGRT) and intensity-modulated radiotherapy (IMRT). IMRT has been widely accepted as an efficient technique for dose escalation in localized prostate cancer, and better conformed radiotherapy to pelvic lymph nodes for higher-risk prostate cancer. Although, the pelvic lymph node irradiation is still controversial, randomized data have supported its use . In addiIn 2009, according to these postulations, we started a prospective study at our institution using a hypofractionated schedule with SIB-IMRT-IGRT technique including patients with high-risk prostate cancer. The primary end-point was to evaluate late toxicity and preliminary results.Between November 2009 and September 2013, 105 patients with biopsy proven, high-risk prostate cancer were treated with hypofractionated radiation therapy with SIB-IMRT-IGRT. Under a S. Andrea Hospital IRB-approved protocol the data were prospectively collected and retrospectively analyzed to evaluate the efficacy and the tolerance. Written inform consent was obtained by all patients.A minimum follow up time of 12 months was required as inclusion criteria. For this reason, 82 patients were selected for the statistical analysis in the present study. Median age at diagnosis was 74 years (range 58–88 years). All patients presented cT3/4 N0/N1 M0 clinical stage and/or a Gleason score of ≥ 8 and/or a pre-treatment prostate-specific antigen (PSA) serum level ≥ 20 ng/ml.Pre-treatment evaluation included: a complete physical examination, PSA level, complete blood counts and standard biochemistry tests, bone scan, total body computed tomography (CT) with contrast medium and prostate magnetic resonance image (MRI) with diffusion and perfusion sequences. Three patients  presenting with clinical positive node  were submitted to PET scan with choline to confirm nodal involvement.Median of PSA value at diagnosis was 9.87 ng/ml (range 3–89.7 ng/ml). Patient characteristics are summarized in Table All patients underwent CT planning (2.5 mm slice thickness) in the supine position with feet rests for the implementation of treatment planning. The preparation for CT scan comprises the administration to a mini enema for rectal emptying and then patients were invited to next urination. Also, they were requested to drink 500 ml of water half an hour before the start of the procedure to fulfil the bladder. Planning CT was fused with MRI images  using point-to-point matching to help clinical target volume (CTV) delineation.Wide description of the treatment patterns was previously reported .Eclipse inverse planning system (Varian) was used to calculate IMRT plans with simultaneous integrated boost. The PTV1 (prostate) was planned to receive 68.75 Gy (2.75 Gy per fraction). The PTV2  and the PTV4 (positive nodes) were planned to receive 55 Gy (2.2 Gy per fraction) and the PTV3 (pelvis) was planned to receive 45 Gy (1.8 Gy per fraction) in 25 fractions. For patients with cT3b, the PTV2 received the same dose as the PTV1. The first 59 patients (72%) received 4 weekly fractions, with overall treatment time of 43 days. The last 23 patients (28%) received 5 weekly fractions, with overall treatment time of 33 days.Prior to each treatment, patients underwent a Kilo-voltage cone-beam CT that was compared with the planning CT to verify the correct position. The patients’ position was adjusted with an initial automatic bone alignment, followed by a soft tissue alignment using the prostate-rectum interface.The organ at risk (OAR) dose volume constraints were as follows: for the rectum V52Gy < 35% and V61Gy < 25%; for the bladder V45Gy < 50%; for the femoral head V50Gy < 10%; for peritoneal cavity V54Gy < 5%.The Normalized Total Dose (NTD) was 82,5 Gy assuming an α/β of 1,5 for the tumour with an iso-effective late complication total dose of 78 Gy delivered in 39 fractions considering an α/β of 3 for rectal late adverse effects.All patients were submitted to neoadjuvant (starting 3 months before radiation therapy), concomitant and subsequent adjuvant ADT for other 2–3 years using a luteinizing hormone-releasing hormone (LHRH) analogous plus anti-androgen .From the start of radiation therapy, all patients were advised to follow a low-fibre and low-fat diet and to assume a cranberry based integrator and lactic ferment once daily.Acute toxicities were recorded using the RTOG/EORTC scale  weekly d–Meier method. The chi-square test was performed to compare 4 times weekly and 5 times weekly treatment-related toxicities between these two groups. Statistical analyses were performed using SPSS statistical software package version 13.0 . A p value lower than 0.05 was considered as statistically significant.The biochemical failure was defined as the PSA nadir +2 ng/mL according to Phoenix criteria . OverallThe overall median follow-up was 31 months (range 12–58 months). The median follow-up for patients treated 4 times weekly was 40 months (range 13–58 months), whereas for patients treated 5 times weekly was 23,5 months (range 12–31 months).The median PSA value before radiotherapy was 0,64 ng/ml , at the first follow-up was 0.08 ng/ml  and at the last follow-up was 0.06 ng/ml .The actuarial 3-years overall survival (OS) was 98,6%. There was one death at the time of the statistical analysis. The patient died after 13 months from RT completion for cardio-pulmonary disorder and he also presented lumbar-aortic nodal disease progression. The actuarial 3-years freedom from biochemical failure (FFBF) was 91,3%  toxicity in 5/82 (6.1%) and 6/82 patients (7.3%), respectively; grade 1, 2 and 3 genitourinary (GU) toxicity in15/83 (18.3%), 3/82 (3.7%) and 1/82 patients (1.2%), respectively.At one month after treatment completion we observed grade 1 and 2 GI toxicities in 4/82 (4.9%) and in 1/82 patients (1.2%), respectively. Grade 1 and 2 GU toxicities were experienced by12/82 (14.6%) and 2/82 (2.4%) patients, respectively.At 3 months after radiation treatment grade 1 GI toxicity was observed in 4/82 patients (4.9%). Grade 1 GU toxicity was observed in 8/82 patients . No grade ≥ 2 GU and GI toxicities were recorded.No statistically significant difference was calculated between the two groups treated 4 and 5 times weekly, respectively during the treatment, at 1 and 3 from the end of therapy. Data are summarized in Table At 6 months from the end of therapy, 2 patients (2.4%) presented grade 1 GI toxicity and 6 patients (7.3%) presented grade 1 GU toxicity. No grade ≥ 2 toxicities were recorded.At the last follow-up, grade 1 GI and GU toxicities were observed in 1 patient (1.2%) and 2 patients (2.4%), respectively. Only 1 patient (1.2%) developed grade 2 GI toxicity No grade ≥ 3 toxicities were recorded.No statistically significant difference was recorded between two groups treated 4 and 5 times weekly, respectively. Data are summarized in Table The lower a/b ratio of prostate cancer compared to the adjacent OAR predicts for the sparing of normal tissues when hypofractionated RT is carried out . In addiMoreover, we reported low rates of acute toxicity during the treatment with grade 1 and grade 2 GI toxicity of 6.1% and 7.3%, respectively, and grade 1, 2 and 3 GU toxicity of 18.3%, 3.7% and 1.2%, respectively. At 3 months after radiation treatment only 4.9% presented grade 1 GI toxicity and 9.8% presented grade 1 GU toxicity. In the current study, we found that the overall treatment time (4 or 5 times weekly) had no impact on acute toxicity rates.The present study reports outcome regarding biochemical control and late toxicity after a median follow-up of 31 months. The actuarial 3-years OS was 98.6%. The actuarial 3-years DFS was 91.3% . None of those patients presented loco-regional relapse.In the literature, there are several randomized controlled trials with mature follow-up addressing moderate hypofractionated radiotherapy in clinically localized prostate cancer. Arcangeli et al.  reportedLimited data from prospective studies reported late toxicity dose-escalation with hypofractionated RT on prostate cancer patients after longer follow-up. These studies treated the prostate, the seminal vesicles, with or without the pelvic lymph nodes.Kupelian et al.  reportedIntensity-modulated radiation therapy is emerging as an important technique for more conformal targets of pelvic lymph nodes for higher-risk prostate cancer treatment . LimitedIn the present study the overall median follow up was 31 months. At 6 months after the end of RT 2.4% of the patients presented grade 1 GI toxicity and 7.3% grade 1 GU toxicity. No grade ≥ 2 toxicity was recorded. At last follow-up 1.2% of the patients presented grade 1 GI toxicity, 1.2% of the patients had grade 2 GI toxicity, and 2.4% of the patients had grade with 1 GU toxicity. No grade ≥ 3 toxicity was recorded. No significant difference was recorded between the two groups treated 4 times weekly and 5 times weekly. Compared to our study, Quon et al.  reportedIn our study, we treated 59 patients with four times weekly radiotherapy considering the hypothesis of Fowler et al.  to maintT, with the first day designated Day 0. The parameters were selected with a comprehensive review of clinical results from several dozen published schedules used for head and neck cancer treatment [-1; an onset time for repopulation in human oral mucosa of Tk = 7 days [Tp = 2.5 days thereafter. Although this formula is likely simple containing only two repopulation rates, zero and Tp, it has served well in comparing tumor BEDs in the head and neck schedules studied [Based on simple linear quadratic (LQ) model including cell proliferation . Specifireatment . They in= 7 days ; and an The purpose of this schedule is to moderately reduce the overall treatment time, in order to maintain acceptable acute toxicity rates. Based on the low rate of toxicity reported in the first 59 patients we decided to reduce the total treatment time up to 33 days and, then, the last 23 patients received five fractions per week, with no calculated difference in terms of observed toxicity rates between the two groups on the statistical analysis. Regard to the postulation of Fowler the reported low rate of toxicity also in patients treated in 33 days may be explained by the use of IMRT combined to IGRT and reduced margin (5 mm) from CTV to PTV. Our study also presented limits as the relatively short follow-up time.Our study demonstrated that hypofractionated radiotherapy using modern technique is efficient and safe. Good outcome in terms of survival and low toxicity rates were observed. The reduction of overall treatment time does no increase toxicity rates. Longer follow-up is necessary to confirm these data. Further studies with longer follow-up are needed to better define outcomes and late toxicity rates after hypofractionated radiotherapy in high-risk prostate cancer patients.


The base excision is not coupled with the strand breakage and yet causes restriction because the restriction enzyme action can impair transformation ability of unmethylated DNA even in the absence of strand breaks in vitro. The base excision of R.PabI is inhibited by methylation of the target adenine base. These findings expand our understanding of genetic and epigenetic processes linking those in prokaryotes and eukaryotes.The restriction-modification systems use epigenetic modification to distinguish between self and nonself DNA. A modification enzyme transfers a methyl group to a base in a specific DNA sequence while its cognate restriction enzyme introduces breaks in DNA lacking this methyl group. So far, all the restriction enzymes hydrolyze phosphodiester bonds linking the monomer units of DNA. We recently reported that a restriction enzyme (R.PabI) of the PabI superfamily with half-pipe fold has DNA glycosylase activity that excises an adenine base in the recognition sequence (5′-GTAC). We now found a second activity in this enzyme: at the resulting apurinic/apyrimidinic (AP) (abasic) site , its AP lyase activity generates an atypical strand break. Although the lyase activity is weak and lacks sequence specificity, its covalent DNA–R.PabI reaction intermediates can be trapped by NaBH Among tneb.com) ,3. Theseneb.com)  and inteneb.com) . RM systneb.com) . Bacterineb.com) .The biological significance of RM systems is not yet fully understood. RM systems were discovered through host controlled variation of bacterial viruses: a virus propagated in one host might not grow well in another host because of differences in DNA modification . RM systAll restriction enzymes examined so far hydrolyze phosphodiester bonds linking monomer nucleotide units leaving 3′-OH (hydroxyl) and 5′-P (phosphate) ends Figure . Type IIin vitro screening to identify a superfamily of restriction enzymes with a novel, half-pipe fold  limits transformation of DNA with an unmethylated form of this sequence . This roverhang . This waoverhang ,22.Escherichia coli lysate  .R.PabI with DNA cleavage activity was expressed in a strain co-expressing CviQI methyltransferase that generates 5′-GTm6AC-3′ as M.PabI . The M.CL21(DE3)  by electin vitro  in the presence of 0.5% arabinose (BYF72). The plasmid pET28a::pabIR was designated pYF46 .The R.PabI coding region was obtained from NdeI and BamHI-cut pMW40 constructed for expression in vitro . The frain vitro  by LigatBYF72 cells were spread on LB agar plates with 50 μg/ml ampicillin, 50 μg/ml kanamycin and 0.5% arabinose. After overnight incubation at 37°C, single colonies were picked up and transferred to 5 ml LB with 50 μg/ml ampicillin, 50 μg/ml kanamycin and 0.5% arabinose. After overnight with shaking at 37°C, cultures were added to 1 l LB with 50 μg/ml ampicillin, 50 μg/ml kanamycin and 0.5% arabinose. After 7 h with shaking at 37°C, IPTG was added to a 1 mM final concentration with incubation at 37°C for 4 h followed by centrifugation at 3.5 krpm for 10 min at 4°C. Cells were resuspended in 60 ml of 20 mM Tris–HCl (pH7.5) with 150 mM NaCl and divided into 30 ml aliquots that were sonicated  and centrifuged at 7 krpm for 20 min at 4°C. Supernatants were heated at 75°C for 10 min followed by centrifugation at 7 krpm for 20 min at 4°C.2. Histidine-tagged R.PabI was digested with thrombin (Novagen) at 20°C for 16 h. Tag-free R.PabI were obtained by the following purification with benzamidine Sepharose 6B (Amersham Biosciences) and dialysis against 10 mM 2-(N-morpholino)ethanesulfonic acid (MES) (pH 6.0), 100 mM NaCl, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM DTT and 50% glycerol. Resulting R.PabI preparations showed a single band by SDS-PAGE with the expected molecular weight as calculated from the amino acid sequence. From 1 l of E. coli culture, 400 μg of purified R.PabI was obtained. Storage of R.PabI in the above solution (10 mM MES (pH 6.0), 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT and 50% glycerol) at −80°C for 4 years has not resulted in noticeable changes to DNA cleavage activity.R.PabI was purified from supernatants through three columns. Supernatants were treated with HisTrap Kits (Amersham Biosciences) with a buffer change by dialysis with 10 mM Tris–HCl (pH7.5), 100 mM NaCl and 1 mM dithiothreitol (DTT). Enzymes were purified with heparin Sepharose CL-6B (Amersham Biosciences) followed by dialysis with 20 mM Tris–HCl (pH7.5), 150 mM NaCl and 2.5 mM CaClE. coli BMF235 ( = HST08 (pBAD30 cviQIM), Supplementary Table S1) was cultivated in LB broth with 0.0002, 0.002, 0.02 or 0.2% arabinose and 100 μg/ml ampicillin with aeration at 37°C. Culture containing 20 mM glucose instead of arabinose was prepared as a negative control to repress expression of the cviQIM gene. After 5 h or one overnight, plasmid DNA was purified from 5 ml culture using GenElute Plasmid Miniprep Kits (Sigma–Aldrich).A single colony of 32P]ATP (PerkinElmer), 50 mM Tris–HCl, 10 mM MgCl2, 5 mM DTT in 10 μl at 37°C for 30 min followed by kinase inactivation at 90°C for 2 min and purification with MicroSpin G-25 columns  , 740 kBq γ-2PATP (PeReactions for plasmids were 10 μl with 0.77 pmol (77 nM) of R.PabI and 0.11 pmol (11 nM) of plasmid DNA in 0.1 M sodium phosphate buffer (pH 6.5) at 85°C for 6 h. DNA was separated by agarose gel electrophoresis and visualized with GelRed Nucleic Acid Gel Stain (Biotium) and ultraviolet light  at 37°C for 1 h to cleave DNA at AP sites. Products were separated through 10% Long Ranger polyacrylamide gels (TaKaRa) by 50 V for 2 h  of R.PabI, 0.11 pmol (11 nM) of plasmid DNA in 0.1 M sodium phosphate buffer (pH 6.5), incubated at 37°C for 30 min. As indicated, reaction mixtures were treated with 5 μg/ml proteinase K in10 mM EDTA and 0.1% sodium dodecyl sulfate (SDS) overnight at 37°C and incubated at 85°C for 6 h  of R.PabI in 0.1 M phosphate buffer  at 70°C for 1 h. Half the reaction mixture was treated with 0.1 M NaOH at 70°C for 10 min to cleave DNA at AP sites and neutralized with HCl. Samples were separated by 18% denaturing PAGE  of a double-stranded oligonucleotide substrate containing the 5′-GTAC sequence and a 5′-Adenine nucleotides in the recognition sequence (5′-GTAC/3′-CATG) were replaced by AP sites by incubating uracil-containing (5′-GTUC/3′-CUTG) double-stranded oligonucleotides (Supplementary Table S2) with uracil N-glycosylase (UNG) (New England Biolabs). Complete conversion to AP sites was confirmed by NaOH cleavage.Pyrococcus horikoshii OT3 cellulase and Aeropyrum pernix K1 inositol 1-phosphate synthase; both were generous gifts from Thermostable Enzyme Laboratory, Co.), and the resulting products were analyzed as described for the AP lyase activity of R.PabI. The sequence specificity of R.PabI AP lyase activity was analyzed using 0–1 pmol (0–50 nM) of R.PabI and 0.2 pmol (10 nM) of the substrate duplexes containing two AP sites in cognate and non-cognate sequences (Supplementary Table S2).AP lyase reactions were performed in the presence of 0–3 pmol (0–150 nM) of R.PabI as described above for DNA glycosylase reactions at 70°C for 1 h except that the substrate duplexes  contained two AP sites and no postreaction NaOH treatment was included. To confirm that R.PabI has an intrinsic  AP lyase activity, 0.2 pmol (10 nM) of the substrate duplex containing two AP sites was incubated with 0–30 pmol (0–1500 nM) of bovine serum albumin (BSA) or a thermophylic enzyme  at 70°C for 20 min, and then with 100 mM NaBH4 (or 100 mM NaCl) at 25°C for 30 min. DNA–R.PabI complexes were denatured in gel loading buffer containing 3% SDS at 90°C for 10 min and separated by 10% SDS-PAGE. For controls, 20 units of Endo III (New England Biolabs) and 0.2 pmol (10 nM) a double-stranded substrate containing an AP site and a 5′-32P label on the top strand was incubated in 10 mM Tris–HCl (pH 7.5), 100 mM NaCl and 1 mM EDTA  at 37°C for 20 min followed by 100 mM NaBH4 (or 100 mM NaCl) as above. DNA–Endo III complexes were analyzed as described for R.PabI.R.PabI  and a double-stranded substrate DNA  containing 5′- GT#C (# = AP site) and a 5′-4-trapping reactions were also performed in a large scale (1 pmol of substrate DNA and 30 pmol of R.PabI or 100 units of Endo III). DNA–enzyme complexes were separated by 10% SDS-PAGE, electro-eluted from the gel bands, concentrated by evaporation, and desalted by Centri-Sep Spin columns. DNA–enzyme complexes (ca. 0.05 pmol) were digested with 0.1–1 μg of proteinase K (Wako) and separated by 18% denaturing PAGE.In addition, NaBH8 bacteria  thawed on ice. The mixture was kept on ice for 15 min, at 42°C for 60 s, and on ice for 5 min, and then 600 μl of SOC medium  was added to the mixture. Cells were incubated for 60 min at 37°C. Serial dilutions of the cell culture were spread on LB plates  containing 100 μg/ml ampicillin.Reactions of 10 μl contained 0.77 pmol (77 nM) of R.PabI and 0.11 pmol (11 nM) of plasmid DNA in 0.1 M sodium phosphate buffer (pH 6.5). After 0.5 h at 37°C, plasmid DNA was purified with a NucleoSpin Extract II (MACHEREY-NAGEL) and 0.11 pmol (200 ng) of plasmid DNA was mixed with a competent cell suspension with ∼10E. coli was difficult, probably because of its toxic effect on the genome. We earlier employed an in vitro expression system to determine the R.PabI crystal structure  was inhibited by expression of the methyltransferase before plasmid preparation Figure  as expecOH-3′ and 5′-PAC…) were efficiently ligated. This result suggested that R.PabI cleavage produced DNA-end structures that were not suitable substrates for the DNA ligase. We came to doubt whether they were 3′-OH and 5′-P ends, shared by all the restriction endonucleases examined .The ligation efficiency for DNA fragments produced by R.PabI was very low , while blunt ends generated by R.RsaI  that excises an adenine base in its recognition sequence (5′-GTAC) even at a low temperature (40°C)  Supplem, this fi-3′ ends .oC and products were analyzed by denaturing PAGE  ,30. To dPyrococcus.The glycosylase and AP lyase activities of R.PabI were not coupled because most AP sites generated by the glycosylase remained uncleaved in the absence of NaOH treatment Figure . R.PabI The sequence specificity of R.PabI AP lyase was analyzed using substrates containing two AP sites in sequence contexts other than GTAC Figure . R.PabI 4 ) (4 (or NaCl for control) and the products were analyzed by SDS-PAGE after heat denaturation in gel loading buffer containing 3% SDS  is initial release of adenine by substitution with water (glycosylase reaction)  and subsequent opportunistic Schiff-base formation between the resulting AP site and R.PabI ), leading to partial cleavage of the AP sites ). Schiff-base intermediates characteristic of bifunctional DNA glycosylases ) can be stabilized by reduction with NaBHii, xi)) –31. TherS Figure B. Both os Figure , and theE Figure .4 treatment resulted in shifted bands indicative of DNA (26-mer)-peptide crosslinks, together with free 26-mer products. This result indicated that NaBH4-treated DNA–R.PabI complexes indeed contained covalent DNA–R.PabI complexes (Schiff base) together with non-covalent complexes. The DNA-tethered R.PabI-derived peptides generated after digestion with 0.1 μg of proteinase K . The purified complexes were digested with proteinase K and the resulting products were analyzed by denaturing PAGE Figure . The NaCThe aforementioned results clearly demonstrated that R.PabI has two enzymatic activities: DNA glycosylase and AP lyase. R.PabI restriction mechanism could be different from DNA phosphodiester bond hydrolysis. Which of these activities, base excision and strand breakage, are important in the restriction process? We examined whether R.PabI impairs DNA activity in the absence of DNA strand breaks Figure . We usedin vitro.Plasmid DNA treated with R.PabI at a low temperature (37°C) mainly remained supercoiled   or an AP endonuclease (of the recipient E. coli). Although the presence of bi-stranded clustered AP sites on DNA in vivo is known to affect cellular processes involving DNA replication and repair  , althougs Figure  requiresd repair ,33, we drestriction phosphodiesterase class and the restriction glycosylase/lyase class. The former class would include all restriction enzymes that act through the hydrolysis of phosphodiester bonds; the latter class would be the PabI superfamily with the half-pipe fold ) ,30. SubsThe AP lyase activity of R.PabI was not coupled to its glycosylase activity Figure . Unlike 2 mechanism  and a stepwise SN1, or dissociative mechanism .Uncoupling of glycosylase and AP lyase activities has been reported for the bacterial MutY glycosylase that removes adenine from an adenine:8-oxoguanine pair , the humThe DNA cleavage and glycosylase reactions by R.PabI require a large molar ratio of the enzyme to the substrate DNA. Further analysis is necessary to find out whether the enzyme turns over or not.OH-3′ overhang  or its oxidized derivative ,41. AlthOur findings of a methyl-sensitive DNA glycosylase and AP lyase activities associated with a restriction-modification system expand our understanding of genetic and epigenetic processes.Supplementary Data are available at NAR Online.


Endometriosis is defined as the presence of endometrial glands and stroma at ectopic locations. Although the prevalence of endometriosis is as high as 35 %–50 %, its pathogenesis remains controversial. An increasing number of studies suggest that changes in immune reactivity may be primarily involved in the development of endometriosis development. In this sense, it has been strongly suggested that a fundamental part of immunologic system, the natural killer cells (NK cells), are an important part of this process. NK cells, a component of the innate immune system, have been extensively studied for their ability to defend the organism against infections and malignancy. Recent studies have shown that IL-2-activated NK (A-NK) cells are able to attack and destroy tumors in lungs and livers of mice, demonstrating the therapeutic potential of these cells. Similarly to metastatic tumor cells, endometrial cells are able to adhere, infiltrate and proliferate at ectopic locations. Therefore, in this study, we evaluated the ability of adoptively transferred and endogenous NK cells to infiltrate endometriosis lesions.As NK cells donors were used C57BL/6 B6. PL- Thy 1.1 female mice. As uterine horns donors were used C57/BL6+GFP female mice and as endometriosis recipients C57BL/6 Thy1.2 female mice. Endometriosis induction was made by injection of endometrial tissue fragments. After 4 weeks, necessary for endometriosis lesions establishment the animals were divided in 3 experimental groups with 10 animals each. Group 1 received i.v doses of 5x106 A-NK in 200μl RPMI; Group 2 received i.p dose of 5x106 A-NK in 200μl RPMI and Group 3 received i.p dose of IL2 (0.5 mL RPMI containing 5.000U of IL2).Our data show that exogenous A-NK cells injected via ip combined with endogenous A-NK cells seems to be the most efficient way for activated NK cells track and infiltrate endometriosis.For the first time, it was shown that both endogenous as exogenous A-NK cells are able to track, migrate and infiltrate endometriosis lesion. This seems to be a promising result, and if confirmed the efficiency of A-NK cells in killing endometriosis lesions, maybe in the future we could use this approach as an alternative treatment for women with endometriosis. Endometriosis is cracterized by presence of endometrial glands and stroma in ectopic locations, as pelvic peritoneum, ovaries, and rectovaginal septum, affecting 6 % to 10 % of women in reproductive age . The patNK cells are large granular lymphocytes, representing about 5 % to 15 % of peripheral blood lymphocytes . NK cellOur analyses show that exogenous A-NK cells injected via the i.v or i.p routes very efficiently infiltrate endometriosis lesions Figs. , 4. In nEndometriosis affects almost 10 % of women in the reproductive age. The pathogenesis of endometriosis remains controversial. However, many recent studies have implicated the immune system and especially NK cells in endometriosis development. On this basis, NK cells, a fundamental part of immunologic system seems to play an important role in endometriosis development. Several of these studies have reported a decrease in cytotoxicity of NK cells in the peritoneal fluid from women with endometriosis , 13, 18.Our results show at least in part that exogenous activated NK cells enhance trafficking of endogenous NK cells to endometriotic lesions. It seems to be a very promising result, and if confirmed that A-NK cells are efficient in killing endometriosis lesions, maybe in the future we could use this approach as an alternative treatment for women with endometriosis. Therefore, we believe it is necessary understand better decreasing of cytotoxicity of NK cells in peritoneal fluid of women with endometriosis and why endogenous NK cells seems not to be able to attack endometriosis and also if exogenous A-NK cells despite infiltrate the lesions, are really able to kill endometriosis.o 12091050.The study was performed at University of Pittsburgh Cancer Institute and approved by University ethics committee under the Protocol NSplenocytes from C57BL/6 B6. PL- Thy 1.1 female mice were used for production of A-NK cells. As uterine horn donors we used C57/BL6 + GFP female mice (transgenic for green fluorescent protein) Fig.  and endoEndometriosis was induced by i.p. injection of endometrial tissue fragments, according Wieser et al . To stim6 A-NK in 200 μl RPMI; Group 2 received i.p dose of 5 × 106 A-NK in 200 μl RPMI; Group 3 received i.p dose of IL2 (0.5 mL RPMI containing 30.000 IU/mL of pegylated (PEG-) rhIL2). To support the transferred A-NK cells, animals from group 1 and 2 received i.p. injections of 0.5 mL RPMI containing 30.000 IU/mL of pegylated (PEG-) rhIL2 at 12 hour intervals for 3 days. Group 3 received the same amount of Peg-IL-2 (IL-2 control), whereas control group received injections of 0.5 ml RPMI without IL-2 [Animals were divided in 3 experimental groups with 10 animals each. Group 1 received i.v doses of 5 × 10out IL-2 .2 in 50 ml of RPMI1640 supplemented with 5 % heat inactivated fetal calf serum and 5 % normal human serum, 10 ml/l non-essential amino acids (Life Technologies), 50 mM 2-mercaptoethanol, 2 mM glutamine, 20 mM Hepes buffer, 0.8 g/l streptomycin and 1.6x105u/l penicillin, hereafter referred to as complete medium (CM). Cells were stimulated with 6.000U/mL of human recombinant IL-2. After 3 days of incubation, CD8-positive cells were magnetically removed following incubation of the cell culture with rat anti-CD8 antibody  and subsequently with anti-rat coated magnetic beads . The CD8-depleted cells were resuspended in fresh CM containing 6000 IU/mL IL-2 to a final concentration of 1x105cells/ml and returned to culture flasks. After an additional 3 days of culture, non-adherent cells were decanted and the plastic-adherent cells were harvested after a brief treatment with 0.02 % EDTA and washed twice in RPMI1640 before use [Spleens were removed aseptically from C57BL/6 B6. PL- Thy 1.1 female mice and a single-cell suspension was prepared in RPMI1640. Erythrocytes were lysed by incubation with ammonium chloride-potassium buffer at room temperature for 3 min and the spleen cells were subsequently washed twice in RPMI1640. Cells were transferred to T150 plastic flasks  and cultured at 37 °C in an atmosphere of 5 % COfore use . Routinefore use , 14.A-NK cells were adjusted to appropriate concentrations and injected into C57BL/6 Thy1.2 mice via lateral tail vein (i.v.) or into the peritoneal cavity (i.p.). To support the transferred A-NK cells, animals from group 1 and 2 received i.p. injections of 0.5 mL RPMI containing 30.000 IU/mL of pegylated (PEG-) rhIL2 at 12 hour intervals for 3 days. Group 3 received the same amount of Peg-IL-2 (IL-2 control), whereas group 4 received injections of 0.5 ml RPMI without IL-2.At three days after injection of A-NK cells, animals were sacrificed and the lesions removed. The collected tissues were sectioned and fixed in ice-cold acetone for 5 minutes. Sections were stained with PE anti-rat CD90.1 to reveal the Thy1.1+, adoptively transferred A-NK cells in the Thy1.2+ recipients. Sections we also stained with anti-NKp46 antibody followed by Alexa 488 donkey anti-goat antibody to reveal endogenous NK cells (NKp46+/Thy1.1-). Before coverslipping, sections we stained with Hoechst 33342 to reveal all nuclei.The density of immune-stained cells were determined by image analysis (MetaMorph ®) .To compare all study groups, analysis of variance (ANOVA) was performed.