Abstractcox1 and nad4) DNA markers in a study of several populations of Xiphinemaamericanum-group from Europe and Morocco, two cryptic species Xiphinemabrownisp. n. (formerly reported as Xiphinemapachtaicum) and Xiphinemapenevisp. n. were revealed. The species are described, illustrated and their phylogenetic relationships discussed. The first species is most similar to Xiphinemaparasimile and is a member of Xiphinemasimile species complex. The phylogenetic reconstructions inferred from three molecular markers  showed that Xiphinemapenevisp. n. is part of Xiphinemapachtaicum-subgroup and is closely related to Xiphinemaincertum, Xiphinemapachtaicum, Xiphinemaparapachydermum, Xiphinemaplesiopachtaicum, Xiphinemaastaregiense and Xiphinemapachydermum. Also, a separate “Xiphinemasimile-subgroup”, outside the Xiphinemapachtaicum-subgroup and so far consisting only of the parthenogenetic species Xiphinemasimile, Xiphinemaparasimile, Xiphinemabrownisp. n. and probably Xiphinemavallense was formed. New primers for amplification and sequencing of part of the nad4 mitochondrial gene were designed and used.Using ribosomal  and mitochondrial (partial  Xiphinemaamericanum-group is a well defined natural complex of species  and one population from Morocco provisionally also determined as Xiphinemapachtaicum. The objectives of the present study were: i) to characterise populations from the Czech Republic, Slovakia and Morocco both morphologically and genetically; ii) to sequence populations of Xiphinemapachtaicum and Xiphinemaparasimile Barsi & Lamberti, 2004 from Bulgaria for comparison; iii) to clarify phylogenetic relationships of identified species using ribosomal and mithochondrial DNA.The  species  with hig species , althoug species . Even fo species , 2004 do species . This wa species , 2010b oXiphinema specimens examined originated from various localities in the Czech Republic , Slovakia , Bulgaria  and Morocco . Details of the soil sampling, nematode isolation and processing for Czech and Slovakian populations are given in Xiphinema specimens recovered were heat killed at 55°C for two minutes, fixed in a 4% formalin, 1% glycerol solution, processed to anhydrous glycerol . Photographs were taken using an Axio Imager.M2-Carl Zeiss compound microscope with a digital camera (ProgRes C7) and specialised software (CapturePro Software 2.8). Measurements were made using an Olympus BX41 light microscope, a digitising tablet , and computer Digitrak 1.0f programme .The glycerol , and mouPageBreakIndividual nematodes from Bulgaria, Morocco (DESS-preserved), Czech Republic and Slovakia (1M NaCl-preserved) were mounted on temporary slides containing glass beads and after taking measurements and photomicrographs the slides were dismantled, individual nematodes removed, and added in 0.25 M NaOH to digest overnight and thereafter heated to 99°C for 3 min. Afterwards 10 μl of 0.25 M HCl, and 5 μl each of 0.5 M Tris-HCl (pH 8) and 2% Triton X-100 were added and the mixture was incubated for another 3 min at 99°C . Finallycox1 and nad4) of ribosomal and mitochondrial DNA were amplified and sequenced. Primer sequences and references to the primers are given in Table Six regions  was designed using online software PRIMER 3 (http://frodo.wi.mit.edu/) from the sequences which were amplified by (CDF+CDR). For final analysis all specimens and populations of Xiphinemabrowni sp. n. from the Czech Republic and Slovakia were amplified and sequenced by using nadpachF + nadpachR primers.Initially partial TM 4.8  was used to assemble and view each sequence and check for base-calling errors. Accession numbers of all sequences are given in Table The PCR reaction was performed in 25 μl total volume containing 1 PCR bead , 20.5 μl double distilled sterile water, 2.0 μl of each primer (10pmol/μl) , and 0.5 μl of DNA added as a template for PCR. A negative control (sterilized water) was included in all PCR experiments. The cycling profile for all ribosomal DNA and mtDNA markers was as described by PageBreakto confirm their nematode origin and to identify the most closely related nematode sequences. Sequences revealing high similarity to those obtained here were included in the phylogenetic analyses of both ribosomal and mitochondrial gene regions (http://guidance.tau.ac.il/ (PageBreakand posterior probabilities (PP) of branch support. Convergence diagnostic values were calculated every 1000 generations with a predefined stop value equal to 0.01. A single strict consensus tree was visualised using FigTree v1.4.2 graphical viewer. Posterior probabilities values of ≥0.80 were considered as credible support values for nodes.A BLAST  search at NCBI  was performed using the obtained sequences as queries  regions , etc. Seu.ac.il/ . All thru.ac.il/ . The phyu.ac.il/  using thTaxon classificationAnimaliaDorylaimidaLongidoridaehttp://zoobank.org/E385F7F7-2C78-4D54-BC57-0D24EDD43CB8Xiphinemapachtaicum  Kirjanova, 1951 apud See Tables Females. Body slender C to open spiral shaped. Cuticle with fine transverse striae. Thickness of the cuticle at postlabial region 1–1.5 μm, 1.5 rarely 2 μm at mid-body and 2 μm at post-anal region. Labial region set-off from the rest of the body by a constriction, expanded, rounded laterally, 5.0±1.1 (4–7) μm high. Amphideal fovea hardly visible, funnel-shaped, its opening c. 5 μm (50%) wide visible posterior the constriction level. Distance between first and second guide ring in specimens with retracted odonostyle 5–10 μm long. Odontophore with moderately developed basal flanges 6.1±0.6 (5.5–7) μm wide. A small vestigium observed occasionally in slender part of pharynx. Pharyngeal characters presented at Table c 1.3 times anal body diameter. Reproductive system amphidelphic, symbiont bacteria present in the ovaries. Separate uteri and ovejector present  of Xiphinemabrowni sp. n. are deposited in GenBank , Wageningen, the Netherlands. The ribosomal and mtDNA sequences  were sequenced by GU222424). The Slovakian population was sequenced in this study and it was identical to previously published sequence of Kurdějov the population identified as Xiphinemapachtaicum  and two Spanish populations of Xiphinemaopisthohysterum Siddiqi, 1961 (JQ990040 and KP268967), respectively. The esPageBreakPageBreaktimated divergences (p-distance) between the 18S rDNA sequences of the new species and the closest species, Xiphinemaparasimile from Bulgaria (this study) and Xiphinemasimile from Serbia (AM086681) were 0.3 (6 nt) and 1.2% (21 nt), respectively. Again, the new D2-D3 PageBreakPageBreakPageBreakPageBreakPageBreakPageBreakPageBreaksequence of Xiphinemaparasimile from Bulgaria was most similar (p-distance = 4.6%), followed by the Serbian populations of Xiphinemaparasimile  and various populations of Xiphinemasimile (14.1–14.7%). The partial cox1 sequences of Xiphinemabrowni sp. n revealed highest similarity to Xiphinemasimile from Slovakia (AM086708). Surprisingly, these two species showed very high similarity 99% (2 nts difference) in cox1 sequences and higher dissimilarity in 18S rDNA . Other authors  and Xiphinemaopistohysterum (JQ990054) and clear separation in D2-D3 28S sequences (or 96 % identity). Further, the cox1 sequences of Xiphinemabrowni sp. n. and the closest species Xiphinemaparasimile, Xiphinemasimile  and Xiphinemapachtaicum  were translated to amino acids and aligned  to the corresponding sequences of  authors  have als9 and 87%cox1) Xiphinemaparasimile from Bulgaria was a sister species of Xiphinemabrowni sp. n. and both species were part of a well supported clade with other European populations of Xiphinemasimile  could help to better clarify its relationships. The position of the new species in the phylogeny trees based on ITS1 and ITS2 sequences was unstable  and odontostyle (73–85 μm), lip region expanded, laterally rounded, separated from the rest of body by a constriction, post-equatorial vulva position (V=52–58 %), symbiotic bacteria present, female tail conical dorsally convex, with narrow rounded to pointed tip, 24–35 μm long, , and specific ribosomal and mtDNA sequences , somewhat longer odontostyle av. 79–83 (73–85) μm vs av. 70 (64–74) μm in the type population, avs. 69–70 (63–74) in Bulgarian populations and avs. 68–70 (67–72) μm in females from Romania  vs av. 66 (62–69) in type population, avs. 68.5–70 (66–72.5) in other Bulgarian populations, 67.5 (65–70) μm in a population from Bosna and Herzegovina, and avs. 67–68 (61–73) μm in females from the Czech Republic (Xiphinemabrowni sp. n. and Xiphinemasimile for this character (Xiphinemabrowni sp. n. differs from Xiphinemasimile in the length and structure of uteri (in the new species separate uteri and ovejector present vs separate uteri not present), different tail shape (conoid vs bluntly conoid), and in the shorter bulbus (53–69 vs 76–92 μm)   ; higher lip region (4–7 μm vs 2–3.5 μm); presence of symbiont bacteria in ovaries vs ovaries without symbionts; somewhat higher c’ values (c’=1.8 (1.53–2.07) vs c’=1.6 (1.4–1.7); the different tail shape ; shorter spicules in males (29 μm vs 38 μm).Xiphinemabrowni sp. n. can be differentiated from:Additionally, Xiphinemapachtaicum by the different vagina shape , more posterior location of the dorsal nucleus (DN=13–21% vs 9–10 %) (Table vs subdigitate). Illustrations of selected features of the closest species Xiphinemapachtaicum, Xiphinemaparasimile and Xiphinemapenevi sp. n. are presented in Figs ed, Figs , 18 and %) Table , differePageBreakXiphinemaparatenuicutis in having symbionts in its ovaries vs absent, males rare vs abundant, higher values for c’ (1.8 (1.5–2.1) vs 1.4 (1.2–1.6), different location of dorsal nucleus ; somewhat shorter bulbus (avs. 59–60 (53–69) vs av. 73 (60–86); shorter uteri (av. 81 vs av. 138 μm); higher c’ values (c’=1.8 (1.53–2.07) vs c’=1.4 (1.3–1.7); and differently shaped vagina (bell-shaped vs funnel shaped).Xiphinemabrowni sp. n. and Xiphinemapenevi sp. n. see below.For comparison between The species is named after Prof Derek JF Brown, an outstanding nematologist, for his significant contributions to the knowledge of plant parasitic nematodes and the development of nematology in Bulgaria.PageBreakTaxon classificationAnimaliaDorylaimidaLongidoridaehttp://zoobank.org/C98CE5B3-9BAE-423C-B887-9BFFFD489798See Tables Females. Body open spiral to C shaped. Thickness of the cuticle at postlabial region 1 μm, 1–1.5 μm at mid-body and 2–2.5 μm at post-anal region, outer cuticle layer not reaching the tail end. Labial region flat anteriorly, laterally rounded, set off from the rest of the body by constriction, 2.5–4 μm high. Amphideal fovea hardly visible, its opening 4 μm in a paratype specimen (40–47 % of the corresponding body width); Distance between first and second guide ring in specimens with retracted odonostyle, 2.5–5 μm long. Odontophore with well developed flanges, 6–9 μm wide, often a small vestigium located in odonthophore area. Pharyngeal characters presented at Table PageBreakible, 2–2.5 μm. Prerectum indistinct, rectum 21.6±1.8 (19–24) μm, n=8, c 1.3 of corresponding body width. Reproductive system amphidelphic, symbiotic bacteria present in the ovaries. Uteri short, ovejector not developed, only in one specimen a structure resembling ovejector was observed , Wageningen, the Netherlands. Three ribosomal sequences  of Xiphinemapenevi sp. n. are deposited in GenBank , two populations of Xiphinemaincertum Lamberti, Choleva & Agostinelli, 1983 from Spain  and Xiphinemapachtaicum (90% for ITS2). In both 18S and D2-D3 phylogeny reconstructions Xiphinemapenevi sp. n. was part of well supported clades with other species of Xiphinemapachtaicum-subgroup . In the phylogeny reconstruction based on ITS2 sequences, the species grouped with two other Xiphinemapachtaicum populations.Sequences for three gene regions were obtained . BLAST at NCBI using any of these sequences as queries revealed highest similarity to Xiphinemapenevi sp. n. is characterised by specific combination of traits: slender body of medium size (1.54–1.85 mm), lip region rounded laterally, flattened anteriorly, separated from the body by a constriction, odontostyle 72–79 μm long, post-equatorial vulva position (V=56–58%), symbiont bacteria present in ovaria, female tail 26–32 μm long (c=50.8–61.2 and c’=1.7–1.9), conoid dorsally convex ventrally slightly concave with pointed tip, and specific ribosomal sequences (18S and ITS2). The alpha-numeric codes based on average values (ranges given in parentheses) using the polytomous key by Xiphinemaparasimile, Xiphinemaparabrevicolle, Xiphinemaparapachydermum, Xiphinemaparatenuicutis  vs 83 μm in holotype, av. 84 (78–88.5) in the present study, 89 (85–97) in females from Ethiopia, and distance of oral aperture to guide ring (68 (66–71) vs 78 in holotype, 77 (73–80) in the present study; shorter pharyngeal bulb (65–72 vs 75–80 μm) in the present study; different tail shape , outer cuticular layer not reaching vs reaching tail tip. ; its somewhat shorter odontostyle (72–79 vs 77–89 μm) and uteri PageBreak(104 vs 138 μm); different position of the dorsal nucleus (DN in front of or at the level of DO (beginning of cuticular lining of the bulb) vs DN below the level of DO); different tail shape , smaller values for c and larger for c’ ratios ;Xiphinemavallense by the position of amphideal fovea (posterior constriction vs on the lips); its shorter body (L=1.69 (1.5–1.85) vs 2.01 (1.83–2.22), different position of dorsal PageBreakPageBreakPageBreakPageBreaknucleus (DN in front or at the level of DO vs DN below the level of DO); different tail shape  smaller values for c and larger values for c’ ratios , longer hyaline part (8–10 μm vs 6.5–8.5 μm);Xiphinemabrowni sp. n. by its somewhat shorter body (L=1.69 (1.5–1.85) vs 2.03 (1.8–2.40) mm and longer bulbus (65–72 vs 53–69) μm; lower (2.5–4 vs 4–7 μm) and differently shaped lip region (not expanded vs expanded); different location of the dorsal nucleus (DN=9.9–12.9 % vs DN=12.7–21.1%); different vagina shape  vs 1.99 (1.75–2.26) mm in type population and avs. 1.78 -1.82 (1.56–2.04) in females from Bulgaria), different lip region shape , the different location of dorsal nucleus (DN 9.9–12.9 % vs 13.6–18.6 %), longer bulbus (65–72 PageBreakPageBreakPageBreakvs 55.5–63 μm) (Table vs bell-like), structure of uteri (ovejector not present vs ovejector and separate uteri present) and length of uterus (36–68 vs 27–46 μm in type population and 27–39 μm in population from Bulgaria (Table vs 33 (30.3–37.1) in the type population and 30–32 (27–35) in females from Bulgaria, c’=1.8 (1.6–1.9) vs 2.02 (1.79–2.28) in the type population and 2.0 (1.7–2.3) in females from Bulgaria)  ;Xiphinemaincertum by its different tail shape  and larger c’ values (c’=1.8 (1.6–1.9) vs c’=1.5 (1.4–1.7) in type material and 1.2 (0.9–1.3) in specimens from Spain, larger a values (a=61 (57–2-65) vs a=57 (56–58) in type population and a=49.7 (44.6–52.5) in the population from Spain and different vagina shape compared with females from Spain, this character not described for the type population  mm vs 2.24 (2.08–2.44) mm), different location of dorsal nucleus (DN=10–13 % vs DN=15–20%), presence of symbiont bacteria in ovaria vs not present; males occurrence (not present vs abundant);PageBreakPageBreakXiphinemaparapachydermum by its different tail tip  and in having symbionts in its ovaries vs absent, males occurrence (not present vs abundant).The new species is named after Dr Lyubomir Penev, an internationally recognised publisher and authority in entomology and ecology as acknowledgement of his invaluable help and support provided to one of the authors (VP) in her research activities.Taxon classificationAnimaliaDorylaimidaLongidoridae Kirjanova, 1951Tables Xiphinemapachtaicum has been recorded from Bulgaria and data on its morphology are available in previous studies  with BLAST showing identity or very high similarity to other Xiphinemapachtaicum populations available at NCBI . Further, the DNA sequences of Xiphinemaparasimile from Vinogradets  showed highest similarity to Xiphinemasimile from Serbia (99% for 18S), various other populations of Xiphinemasimile and Xiphinemaopisthohysterum  and 78% two cox1 sequences – Xiphinemapachtaicum from the Czech Republic (GU222424) and Xiphinemasimile from Slovakia (AM086708). The first one is the previously published sequence of Xiphinemabrowni sp. n. identified as Xiphinemapachtaicum  seems to occur in central European countries. The second group of closely related species consists of Xiphinemapachtaicum, Xiphinemapenevi sp. n., Xiphinemaincertum, Xiphinemaparapachydermum, Xiphinemaplesiopachtaicum, Xiphinemaastaregiense and Xiphinemapachydermum. Again, one of these species (Xiphinemapachtaicum) has a much wider distribution in Europe, Asia and Africa  consisted of 8 species with five being described from Europe , one from North America , and one from Asia (Xiphinemaopisthohysterum). Our analyses using ribosomal and mitochondrial DNA sequences currently available in GenBank and the two new species described in this study supports the delimitation of the “Xiphinemapachtaicum-subgroup”, however it also includes Xiphinemaincertum, Xiphinemapachtaicum, Xiphinemapachydermum and the recently described species Xiphinemaparapachydermum, Xiphinemaastaregiense, Xiphinemaplesiopachtaicum and Xiphinemapenevi sp. n. Phylogenetic reconstructions showed that Xiphinemamadeirense, Xiphinemaopisthohysterum, Xiphinemasimile and Xiphinemauthahense are not part of this group, for Xiphinemafortutium no sequences are available. These results are in line with the findings of other recent studies on the Xiphinemaamericanum-group (Xiphinemasimile (presented by two types of sequences for populations from Serbia and the Czech Republic in 18S rDNA and cox1 trees), Xiphinemaparasimile and Xiphinemabrowni sp. n. formed a separate subgroup outside the Xiphinemapachtaicum-subgroup, so far consisting only of parthenogenetic species. Therefore we proposed this clade to be referred as the Xiphinemasimile-subgroup. The recently described species Xiphinemavallense seems also evolutionary very closely related to this subgroup because of its high morphometric and DNA similarity, however amplifying additional sequences for other molecular markers (e.g. 18S and cox1) could help to clarify its relationships.Based on a hierarchical cluster analysis of morphometrics um-group . XiphinePageBreak


Background. Cardiopulmonary bypass (CPB) causes release of matrix metalloproteinase- (MMP-) 9, contributing to pulmonary infiltration and dysfunction. The aims were to investigate MMP-9 production and associated perioperative variables and oxygenation following CPB. Methods. Thirty patients undergoing elective cardiac surgery were included. Arterial blood was sampled at 6 sequential points  for plasma MMP-9 concentrations by ELISA. The perioperative laboratory data and variables, including bypass time, PaO2/FiO2, and extubation time, were also recorded. Results. The plasma MMP-9 concentrations significantly elevated at 2–6 h after beginning CPB (P < 0.001) and returned to the preanesthesia level at 24 h (P = 0.23), with predominant neutrophil counts after surgery (P < 0.001). The plasma MMP-9 levels at 4 and 6 h were not correlated with prolonged CPB time and displayed no association with postoperative PaO2/FiO2, regardless of reduced ratio from preoperative 342.9 ± 81.2 to postoperative 207.3 ± 121.3 mmHg (P < 0.001). Conclusion. Elective cardiac surgery with CPB induced short-term elevation of plasma MMP-9 concentrations within 24 hours, however, without significant correlation with CPB time and postoperative pulmonary dysfunction, despite predominantly increased neutrophils and reduced oxygenation. Cardiopulmonary bypass (CPB) induces a systemic inflammatory response  and the 2/FiO2 among patients undergoing elective cardiac surgery.The MMPs are a family of more than 25 species of zinc-dependent proteases that are essential for normal tissue remodeling in processes including bone growth, wound healing, and reproduction  or coronThe study was approved by the ethics committee (TSGHIRB-1-101-05-115) and written informed consent was obtained from 30 patients undergoing elective cardiac surgery. Patients with cancers, patients with autoimmune diseases, patients receiving steroids, and patients with preoperative respiratory or hepatic failure were excluded.μg/kg, propofol 0.5–1.5 mg/kg, and cisatracurium 0.1–0.2 mg/kg and maintained with sevoflurane or isoflurane after tracheal intubation. A pulmonary artery catheter was placed through right internal jugular vein and transesophageal echocardiography was used to monitor real-time cardiac performance throughout the whole procedure. Routine median sternotomy and standard cardiopulmonary bypass  with an extracorporeal membrane oxygenator  were carried out in sequence to maintain the body temperature at 28–30°C during surgery. The perfusionist adjusted sevoflurane or isoflurane concentration on the vaporizer to keep mean arterial blood pressure between 50 and 80 mmHg during bypass period. Following standard rewarming and deairing, the pump was weaned at the end of the procedure. Routine inotropic support including dopamine or dobutamine infusion was added for acceptable cardiac output, if necessary. All patients were transferred to the cardiovascular surgical ICU with endotracheal intubation after surgery.In the operation room, patients were premedicated with fentanyl and midazolam for arterial catheterization. General anesthesia was induced with fentanyl 1.5–3 Each 20 mL of arterial blood was sampled from the arterial line at 6 sequential time points  into EDTA-containing tubes. After immediate centrifugation of blood samples at speed 1500 rpm for 10 min at room temperature, the plasma samples were divided into Eppendorf tubes and frozen at −70°C for later plasma MMP-9 concentration testing.2, expressed in mmHg) to inspired oxygen fraction (FiO2) was also calculated before and after operation, as well as before extubation. The severity of hypoxemia is defined as mild (200 < PaO2/FiO2 ≤ 300 mmHg), moderate (100 < PaO2/FiO2 ≤ 200 mmHg), and severe (PaO2/FiO2 ≤ 100 mmHg) by the Berlin definition of acute respiratory distress syndrome [Arterial blood gas analysis was routinely examined perioperatively and immediately upon ICU admission by GEM Premier 3000 . Perioperative variables, including general anesthesia, operation, and bypass times, as well as preoperative and postoperative laboratory data, were also recorded. The ratio of arterial oxygen tension  according to the manufacturer's protocol . The absorbance of the color at 450 nm was recorded using a TECAN Sunrise ELISA Reader .2/FiO2 ratios was analyzed by paired t-test or Pearson correlation analysis. P value <0.05 was considered statistically significant.The results were analyzed through SPSS software version 17 . The demographic data, perioperative variables, postoperative leukocyte counts, arterial blood gas data, and plasma MMP-9 concentrations were presented as mean ± SD. The correlation between MMP-9 concentrations, CPB time, and pulmonary PaOP < 0.001). The blood glucose levels were significantly elevated from 135.7 ± 37.1 (range 83–221) to 210.2 ± 44.1 (range 126–338) mg/dL postoperatively (P < 0.001). To examine alveolar oxygen exchange, PaO2/FiO2 ratio was calculated. There were only 12 patients having a preoperative ratio within 200–300 mmHg (mild hypoxemia), while turning to 3 patients with ratio ≤100 mmHg (severe hypoxemia), 16 within 100–200 mmHg (moderate hypoxemia), and 6 within 200–300 mmHg (mild hypoxemia) postoperatively. The mean PaO2/FiO2 ratio reduced from preoperative 342.9 ± 81.2 (range 232.0–563.0) to postoperative 207.3 ± 121.3 (range 67.0–538.3) mmHg (P < 0.001) (n = 30) but recovered before extubation 325.7 ± 129.8 (range 156.0–597.5) mmHg (P = 0.456), with extubation time 38.0 ± 28.6 (range 3.5–140) hours after arriving at ICU (n = 27), except for 3 patients with pneumonia or mediastinitis-related septic shock, and expired at postoperative 10, 25, and 31 days in the ICU. The mean ICU stay (n = 27) was 3.5 ± 1.4 (range 2–9) days, with mean hospital stay 11.7 ± 3.9 (range 7–23) days after surgery.Demographic data of 30 patients were summarized in n = 30) rose significantly at 2, 4, and 6 hours after the start of CPB, with 462.6 ± 247.2 (range 65.5–1102.4), 381.1 ± 174.3 (range 144.1–843.8), and 331.4 ± 288.4 (range 19.9–1266.4) ng/mL, respectively, as compared with 85.7 ± 88.8 ng/mL before anesthesia induction . The mean level returned closely to the preanesthesia value at 24 hours after the start of CPB .As shown in P = 0.60 and 0.83, resp.). Besides, the MMP-9 levels at 4 hours displayed no differences between patients with less or more than 121 min CPB time . To test the MMP-9 effect on pulmonary dysfunction, the MMP-9 levels at 4 and 6 hours were not associated with postoperative PaO2/FiO2 ratio  and neither was CPB time for PaO2/FiO2 (P = 0.25).Using Pearson correlation analysis, plasma MMP-9 concentrations at 4 and 6 hours after initiation of CPB were not correlated with CPB time , moderate (100 < PaO2/FiO2 ≤ 200 mmHg), and severe (PaO2/FiO2 ≤ 100 mmHg). In our patients, postoperative depression of oxygenation was observed, including reduced PaO2/FiO2 ratio and patients shifting from preoperative mild hypoxemia (n = 12) to postoperative severe (n = 3), moderate (n = 16), and mild (n = 6) hypoxemia. Based on the previous evidence of predominant neutrophil recruitment and activation following CPB [2/FiO2 ratio. Eventually, our results demonstrated transient enhancement of MMP-9 concentrations at 4–6 hours following CPB, which, however, was not correlated with reduced postoperative PaO2/FiO2, indicating clinically short-term and insignificant influence on pulmonary function in our patients with elective cardiac surgery.Cardiac surgery using CPB may cause postoperative pulmonary dysfunction, including acute lung injury and/or acute respiratory distress syndrome (ARDS) . Accordiate n = 1, and milwing CPB , neutropwing CPB , 20, andwing CPB , 12, we Two limitations should be addressed. First, only the plasma MMP-9 concentrations and clinical parameters were analyzed during the acute phase in this study, needing further ex vivo or experimental data to verify the biological changes of acute lung injury and the following myocardial remodeling. Second, limited case number in this study may diminish the clinical manifestation and its significance of correlation. More participants with various groups of severity are needed to verify MMP-9 as a clinical indicator for pulmonary dysfunction after cardiac surgery.In conclusion, we identified that elective cardiac surgery with CPB induced a short-term elevation of MMP-9 concentrations at 2–6 hours after beginning CPB, with predominant increase of neutrophils. The postoperative MMP-9 levels upon arriving at ICU were not correlated with bypass time and reduced oxygenation after cardiac surgery.


Cladosporium oxysporum GQ-3 producing extracellular xylanase was isolated from decaying agricultural waste and identified based on the morphology and comparison of internal transcribed spacer (ITS) rDNA gene sequence. C. oxysporum produced maximum xylanase activity of 55.92 U/mL with wheat bran as a substrate and NH4Cl as a nitrogen source. Mg2+ improved C. oxysporum xylanase production. Partially purified xylanase exhibited maximum activity at 50°C and pH 8.0, respectively, and showed the stable activity after 2-h treatment in pH 7.0–8.5 or below 55°C. Mg2+ enhanced the xylanase activity by 2% while Cu2+ had the highest inhibition ratio of 57.9%. Furthermore, C. oxysporum xylanase was resistant to most of tested neutral and alkaline proteases. Our findings indicated that Cladosporium oxysporum GQ-3 was a novel xylanase producer, which could be used in the textile processes or paper/feed industries. A new fungus Generalosidases , 5 and hosidases –9. Aspergillus, Penicillium, and Trichoderma have been proved to have a significant capability of secreting a wide range of xylanases. Our previous work also found that the screened fungi Penicillium sp. WX-Z1, Penicillium oxalicum ZH-19, and Aspergillus sp. Zh-26 could produce xylanases with the highest activity of 46.50 U/mL, 80.23 U/mL, and 47.30 U/mL under their optimized fermentation conditions, respectively [Xylanases are produced by bacteria , fungi , actinomectively , 15. For3 3.0, K2HPO4 0.5, MgSO4·7H2O 0.5, and KCl 0.5. After being cultured at 30°C for 2-d, the isolate was inoculated on Petri dishes containing (g/L) wheat bran 5.0, KH2PO4 0.5, and agar 10.0, at 30°C for 6-d. The developed colonies were seeded to the fermentation media consisting of (g/L) wheat bran 5.0, NH4Cl 10.0, KH2PO4 1.0, NaCl 1.0, MgSO4·7H2O 1.0, CaCl2·2H2O 0.5, and yeast extract 1.0, for determining xylanase production. The chemicals of reagent grade for preparing media or determining xylanase activity were obtained from Sinopharm Chemical Reagent Co., Ltd. . One fungal isolate with highest xylanolytic activity was designated as GQ-3 and ultimately chosen for further study.For screening xylanase secreting strains, about 1.0 g of decaying wheat straw, collected in the Maoshan Town, Zhenjiang City, Jiangsu Province, was added to the screening media (g/L): KNOGenomic DNA of fungal isolate was extracted from fresh cultures and used as template for PCR amplification using a method described previously . The ITS6 spores/mL with sterile distilled water.The culture seed was prepared by growing fungal cells on PDA slopes at 30°C for 6 d and adjusted the cells concentration to 102PO4 1.0, MgSO4·7H2O 1.0, CaCl2·2H2O 0.3, and yeast extract 1.0. Effect of carbon sources and nitrogen sources on xylanase production was evaluated by substituting glucose or yeast extract in the basal medium, respectively. Four fermentation temperatures from 25°C to 42°C, and 7 initial pHs from 3.0 to 9.0 were used for selecting the optimal temperature and initial pH with the media containing wheat bran . Fermentation was conducted at 30°C on a rotary shaker (150 rpm) for 5 d if not further specified. The sample was withdrawn at regular intervals and centrifuged at 14,000 rpm (4°C) for 10 min to collect supernatant as the crude enzyme for determining xylanase activity.The basal medium for one-factor-at-a-time experiments was composed of (g/L) wheat bran 5.0, KHμmol of xylose from xylan in 1 min.The xylanase activity was expressed by release of reducing sugars from oat spelt xylan  using the dinitrosalicylic acid method . ReactioProtein concentration was determined using a Bradford Protein Assay Kit  with bovine serum albumin as a standard protein. Specific activity was expressed as the number of xylanase units per milligram of enzyme protein.The culture supernatant was concentrated 10-fold by ultrafiltration membrane with molecular weight cutoff (MWCO) of 10 kDa  and precipitated with 40% saturation ammonium sulfate. The protein precipitate was dissolved in potassium phosphate buffer solution (PBS), loaded onto a DEAE-Sepharose Fast Flow column (1.2 cm × 60 cm) , and step-wisely washed with a discontinuous gradient of PBS, PBS containing 0.3 mol/L NaCl, and PBS containing 0.5 mol/L NaCl. The elution fractions having xylanolytic activity were collected and lyophilized for biochemical characterization.The enzymes were mixed with buffers with varying pH at 1 : 10 (v/v) ratio and incubated for 60 min to identify the optimal pH and pH stability. The buffers used were as follows: 0.2 M acetate buffer (pH 3.0–5.0), 0.2 M phosphate buffer (pH 6.0–7.5), and 0.2 M Tris–HCl buffer (pH 7.5–9.0).The optimum catalytic temperature of partially purified xylanase was selected by determining the enzyme activity in the range of 30–70°C. The temperature stability was determined by measuring the residual xylanolytic activity after being treated with temperatures from 40 to 70°C for 0.0, 0.5, 1.0, 2.0, and 3.0 h.+, Mn2+, Na+, Mg2+, Fe2+, Cu2+, Li+, Co2+, Hg2+, and Zn2+ at the concentrations of 1.0 mM. Inhibition/activation degree of xylanase activity was expressed as percentage of a control sample incubated in absence of any additive.Effect of metal ions on xylanase activity was evaluated by incubating reaction mixtures containing 1.0% oat spelt xylan with K Tritirachium album), pepsin, trypsin, and collagenase (from Clostridium histolyticum) with unit of 10.0 μg/mL. The residual xylanolytic activity was measured after 3-h preincubation at 37°C. The reaction mixture without proteases was used as control.Resistance of partially purified xylanase to proteases was tested by adding neutral and alkaline proteases including proteinase K , followed by Duncan's multiple-comparison test. Differences were considered significant when the  Cladosporium oxysporum , Coccus aethiopicus De Lotto, and Trioza erytreae (del Guercio) [ C. oxysporum GQ-3 was the new record to produce xylanase.The colony morphology of fungal isolate GQ-3 after 7-d cultivation on potato dextrose agar (PDA) at 30°C was shown in xysporum . The nucGuercio)  and prodGuercio) . To the  Cladosporium oxysporum. Generally, the lignocellulosic material, such as barley husk, corn cobs, hay, wheat bran, or straw, had the advantage of pure xylan or xylose due to the low cost to produce xylanase in a larger scale. Wheat bran is rich in carbohydrates , proteins, fats, and vitamins and has been proved as an effective xylanase inducer. For example, comparing with corncob, cassava bran, corn straw, or sugar cane bagasse, Neosartorya spinosa strain P2D19 produced highest xylanase activity (15.1 U/mL) from wheat bran [−1 by Pseudomonas sp. WLUN024 [From eat bran . Xu et a WLUN024 . Aureobasidium pullulans and Trichosporon cutaneum. In the present study, C. oxysporum GQ-3 fermented xylose  effectively to produce xylanase of 20.32 U/mL. Other pure sugars such as glucose, fructose, and sobiol were not preferable for C. oxysporum with xylanase production of below 3.00 U/mL, which might be due to their prevention of enzyme synthesis [Xylose has also been proved as a xylanase inducer in some organisms such asynthesis . C. oxysporum GQ3. Tryptone and yeast extract gave the high xylanase activities of 40.82 U/mL and 42.93 U/mL, respectively. Similarly, Trichoderma harzianum PPDDN10 NFCCI-2925 produced maximum xylanase activity of 2137.75 IU/gds with mycological peptone (MYP) [ T. harzianum, S. commune, and T. lanuginosus [4Cl, (NH4)2SO4, ammonium citrate, and NH4H2PO4 improved xylanase production. The xylanase activity reached the highest level of 55.92 U/mL with NH4Cl as a nitrogen source (P < 0.05).As shown in ne (MYP) . In someuginosus . In the  C. oxysporum GQ-3 was cultivated in basal medium at pH values ranging from 3.0 to 9.0 to find the optimal initial pH for xylanase production. As shown in  C. oxysporum xylanase production. C. oxysporum had a higher xylanase production with the activity of over 44.2 U/mL at slightly acidic pH (pH 6.0), neutral pH (pH 7.0), and slight alkaline pH (pH 7.0–8.0). Our previous study also found that Penicillium oxalicum ZH-30 produced the highest xylanase activity of 14.91 U/mL at pH 7.72 [Generally, initial pH and fermentation temperature were regarded as the significant factors affecting cell growth and xylanase production by influencing nutrients transport and corresponding enzymatic systems. Herein, pH 7.72 . C. oxysporum with wheat bran concentration of 1.0% (w/v). Temperature from 28°C to 30°C benefited C. oxysporum xylanase production with the highest activity of 52.84 U/mL at 30°C. With temperature increase to 42°C, xylanase production decreased significantly to 1.74 U/mL (P < 0.05). Nawel et al. also found that temperatures from 30°C to 37°C were suitable for cell growth and xylanase production of J. denitrificans BN-13 L [ C. oxysporum. BN-13 L . Therefo C. oxysporum xylanase production. Compared with control result, supplementation of Zn2+, Fe2+, Mg2+, Mn2+, or Ca2+ was positive for xylanase production with activity of over 36.00 U/mL. Mg2+ at the concentration of 5.0 mM gave the maximum xylanase activity of 53.04 U/mL. However, Cu2+ completely inhibited xylanase production with the activity of 0.24 U/mL (P < 0.05). A similar result also was found by Reis et al. with 17% of xylanase activity improvement (30.50 U/mL) after addition of MgSO4 and CaCl2 [Fungi require a unique combination of several unusual nutrient conditions, that is, hydrogen ions, dissolved oxygen certain trace metals, or phosphate for sufficient growth or xylanase production. nd CaCl2 . C. oxysporum xylanase yield and activity during 2-step purification processes. Ammonium sulfate precipitation led to xylanase specific activity of 10.12 U/mg protein and increased purification fold to 3.61. During the DEAE-Sepharose Fast column chromatography, fraction eluted with PBS containing 0.3 mol/L NaCl had the highest xylanase activity of 48.56 U/mL (elution profile not shown). The partially purified xylanase had the increased specific activity of 55.29 U/mg protein and purification fold of 20.98. C. oxysporum GQ3. Too high (over 9.0) or low (below 6.0) pH conditions significantly inhibited xylanase activity (P < 0.05). Hence, it could be concluded that C. oxysporum xylanase could tolerate alkaline conditions and is possibly classified as an alkaline xylanase.  C. oxysporum xylanase had the optimal activity with the relative value of 100% at pH 8.0. Recently, alkali xylanases attract increasing interest for their applications in paper and feed industries, textile processes, enzymatic saccharification and waste treatments. For example, alkaline xylanase could directly treat the pulp with high pH and avoid the cost/time consuming steps of pH readjustments. Similarly, Geobacillus thermodenitrificans also produced an alkali xylanase with the maximum activity at pH 7.5 and 70°C [Catalytic pH markedly affects enzymes activity by dissociating bind of substrate and catalysis and even destroying their molecular structures. and 70°C . C. oxysporum xylanase activity increased from 32.64 U/mL to 56.90 U/mL with increase of temperature from 20°C to 50°C and then decreased to 37.64 U/mL at 50°C and even 14.44 U/mL at 70°C. Hence, the optimum catalytic temperature of C. oxysporum xylanase was regarded as 50°C.From C. oxysporum xylanase had the stable activity at below 50°C ( C. oxysporum xylanase.low 50°C  and appr2+ had the highest inhibition ratio of 57.9%. Mg2+ slightly enhanced partially purified xylanase activity by 2.8%, indicating its possible role as a metal ion cofactor. Previously references have revealed that xylanases activity depended on their producers and metal ion types. For example, Na+ and Mn2+ strongly inhibited 33.2% and 80.5% of Alternaria mali xylanase activity while K+, Li+, Fe2+, Cu2+, and Zn2+ showed no significant effect [2+ significantly inhibited Streptomyces sp. xylanase [2+ strongly inhibited 66.0% and Ca2+ stimulated 51.3% of C. algeriensis xylanase [As shown in t effect . Mn2+ sixylanase , while Hxylanase . C. oxysporum GQ3 showed the resistance to neutral and alkaline proteases when incubated with pepsin, trypsin, collagenase, and proteinase K at 37°C for 3 h. The enzyme retained unchanged activity with collagenase treatment while residual activity was 95.8% and 91.3% of the maximum xylanase activity after the incubation with proteinase K and trypsin, respectively. Interestingly, xylanase produced by Streptomyces sp. TN119 GH 11 also exhibited significant re sistance to protease digestion and even increased its activity by 5.8%, 9.4%, 21.6%, and 129.0% after treatments of trypsin, collagenase, α-chymotrypsin, and proteinase K, respectively [The partially purified xylanase from C. oxysporum GQ-3 was proved as an efficient xylanase producer with the agroindustrial residue wheat bran as substrate. The C. oxysporum partially purified xylanase activity has the optimal catalytic pH and temperature of 8.0 and 50°C, respectively. It showed the pH and thermostable activities while influenced by metal ions. Our study will provide a reference for better understanding of xylanase from C. oxysporum and further improving the strain for large scale production and industrial application such as textile processes or paper and feed industries.In conclusion, the isolated fungus


Psychrobacter piscatorii strain LQ58, isolated from a deep-sea hydrothermal vent on the East Pacific Rise. The sequence will provide further insight into the environmental adaptation of psychrotolerant bacteria and the development of novel cold-active enzymes for industrial application.Here, we report the 3.1-Mb draft genome sequence of  Psychrobacter are psychrotolerant or psychrophilic bacteria, which are widely distributed in marine and terrestrial environments (––Psychrobacter piscatorii strain LQ58 (=MCCC 1A10701) is an aerobic, psychrotolerant, chemoorganotrophic bacterium, isolated from a deep-sea hydrothermal vent sediment sample collected on the East Pacific Rise at a depth of 2,906 m . Growth was observed at temperatures between 0°C and 35°C with an optimum at 22°C in the presence of 2% NaCl. Strain LQ58 showed highest 16S rDNA sequence similarity with P. piscatorii T-3-2 (99.3%) (Psychrobacter aquaticus CMS56 (98.6%)  (http://www.ncbi.nlm.nih.gov/genome/annotation_prok). Genes of interest were manually evaluated.Extraction of genomic DNA from strain LQ58 was carried out using a bacterial DNA kit (Omega) according to the manufacturer’s instructions. The genome of strain LQ58 was sequenced by whole-genome shotgun sequencing using the Illumina MiSeq system at Shanghai Majorbio Bio-pharm Technology Co., Ltd. . A total of 804,651,728 reads were generated, representing an approximately 260-fold coverage of the genome. These reads were assembled using SOAPdenovo version 2.0  and resuThe draft genome sequence of strain LQ58 was 3,089,314 bp in length, with a G+C content of 44.1%. The genome contains 2,676 predicted open reading frames and 2,562 predicted protein-coding sequences). There were 33 tRNA genes, 3 rRNA genes, and 1 noncoding RNA (ncRNA) gene predicted from this assembly.In silico DNA-DNA hybridization (DDH) and average nucleotide identity values of LQ58 against P. aquaticus CMS56 were 22.6 ± 2.36% and 79.3% , 1 capsular polysaccharide synthesis gene cluster, and 1 GroES-GroEL operon associated with cold stress . In addiase gene , which mLNDJ00000000. The version described in this paper is the first version, LNDJ01000000.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number


The Brief Pain Inventory (BPI) was used to measure pain intensity prior to treatment (T0), 2, 4, 6, 8 and 12 weeks  after treatment in a 24-hour period. Analgesic, quality of life (QOL) scores and complications were also recorded. Four patients were excluded as they were lost to follow-up or had incomplete data.89 patients with painful bone metastases secondary to lung cancer were consented and enrolled in this study from June 2013 to May 2015. All patients had failed or refused conventional treatments underwent percutaneous CT-guided 0. At T2, T4, T6, T8 and T12, the score in a 24-hour period decreased to 4.9±1.2 (P<0.01), 3.7±1.3 (P<0.01), 3.4±1.2 (P<0.01), 2.6±0.9 (P<0.01), and 1.4±0.8 (P<0.01) respectively. Comparison of QOL scores showed improvements including sleep, appetite, spiritual state, and fatigue at T2, T4, T6, T8 and T12 when compared to T0. No serious complications or massive bleeding were observed.85 patients with 126 bone metastases from lung cancer were treated. There were significantly lower scores after treatment in the visual analog scale (VAS) and analgesic. The VAS scores for worst pain was 6.3±1.8 at T125I brachytherapy is a safe and effective method for palliation of painful bone metastases from lung cancer after failure or rejection of conventional treatments. Bone was a common site of metastasis for patients with lung cancer, particularly non-small cell lung cancer, approximately 30-40% would appear skeletal metastases . At the 125I brachytherapy [Currently, conventional treatments were defined as bone metastases secondary to lung cancer had received local control therapies , chemotherapy, analgesics and so on) before ytherapy . Palliatytherapy . For focytherapy . For difAlthough a majority of traditional treatments have been used to treat painful bone metastases, the results can be disappointing . Recent 125I seed implantation was an acceptable and useful minimally invasive therapy for primary lung tumor or metastatic lymph node resulting in a high local control rate making it an alternate approach for achieving remission [125I brachytherapy for the palliation of painful bone metastases from lung cancer after failure or rejection of conventional treatments has not been reported. The purpose of this study is to report such data in patients with skeletal metastases secondary to lung cancer.Previous studies had shown that emission , 14. HowThe patient, tumor, and brachytherapy characteristics were delineated in Table 2, T4, T6, T8 and T12, the score in a 24-hour period decreased to 4.9±1.2, 3.7±1.3, 3.4±1.2, 2.6±0.9, and 1.4±0.8 respectively. Similarity, the score for average pain were 4.6±1.5, 3.5±1.2, 2.6±0.9, 2.1±0.7, 1.4±1.1 and 0.9±0.5 in a 24-hour period at T0, T2 (p<0.01), T4 (p<0.01), T6 (p<0.01), T8 (p<0.01) and T12 (p<0.01) respectively. The relief from pain was significantly improved from T0 to T12, whereas there was no significant difference in scores between T0 and T2 (P=0.27). The mean morphine equivalent 24-hour doses of 136.2±33.9mg at T0 and 109.4±29.8mg(p=0.57), 96.8±22.1mg(p=0.32), 108.4±27.2mg(p=0.64), 76.3±19.8mg(p=0.80), 39±6.8 mg(p=0.87) in the first 24 hours following T2, T4, T6, T8, T12 were not significantly different.The effectiveness of pain palliation is summarized in Table 4(p<0.01), T6(p<0.01), T8(p<0.01), T12(p<0.01) than T0, whereas there was no significant difference in scores between T0 and T2 (P=0.32) , spiritual state(p=0.33), and fatigue(p=0.28) categories  at 2 weeks on CT or PET-CT image and the range of tumor necrosis was gradually increasing with the follow-up prolonged. In addition, patients treated with 125I brachytherapy did not experience increased pain.All local bone tumors afterSeveral complications related to the procedure occurred during or after brachytherapy Table . Small vUp to 30 % of patients with lung cancer have bone metastases, and half of these develop intractable pain . The etiConventional therapies for painful bone metastases included systemic and local interventions. Chemotherapy and analgesic were systemically administered in select patients for palliation, but the effectiveness was variable , 19, 20.125I brachytherapy did not experience increased pain. A second advantage is providing complete irradiation to the local tumor, while it provided a lower dose to normal adjacent tissue.Previously, multiple minimally invasive image-guided ablation strategies have been studied as treatments for painful bone metastases , 24. Cry125I brachytherapy was an effective, accurate method for palliation of painful bone metastases. We found that the scores for worst pain were 6.3±1.8 before brachytherapy, and 4.9±1.2, 3.7±1.3, 3.4±1.2, 2.6±0.9, and 1.4±0.8 at 2, 4, 6, 8 and 12 weeks respectively, the symptom of pain was decreased obviously from T2 to T12. These corresponding results further illustrated that 125I brachytherapy for palliation of painful bone metastases painful bone metastases was significant and feasible.In this study, we demonstrated that 125I brachytherapy was effective at improving QOL. Comparison of QOL scores, the quality of sleep, appetite, spiritual state, fatigue and KPS at T2, T4, T6, T8 and T12 were all significantly better than at T0. We found that the treatment of pain was valuable, because the bone metastases were the only evidence of recurrence since lung cancer was controlled. Particularly encouraging were outcomes in five patients who were alive without pain or recurrence after125I brachytherapy. For these patients, 125I brachytherapy had been a curative treatment thus far.Another significant finding in this study was that 125I brachytherapy for pain palliation depended on the accurate placement of radioactive seeds within a known volume of tumor [125I seed dropped off with distance, our study showed that TPS can help peripheral tumor doses reach the MPD of 100-140 Gy and ensure that more than 95% of the tumor get 100% prescription dose [125I brachytherapy was safe and effective.The success ofof tumor . Americaof tumor . Becauseion dose . Thus, t125I brachytherapy. Thus, the next research is to explore the responses and sensibility about different pathology sub-types of lung cancer metastases for 125I brachytherapy. Last, responses to the pain score and QOL rating systems were provided by patients who were not previously familiar with these surveys introducing subjectivity into their assessments.The main limitations of this study were small sample size and a short duration of follow up. Also, a control group consisting of patients treated with EBRT or chemotherapy should be needed, further comparison and longer follow-up were desired for future analysis. Although pathology sub-types of lung cancer metastases with the 85 patients were clarified, it is not clear which pathology sub-type of metastasis is more sensitive to 125I seed implantation is a safe, effective, and accurate treatment for patients with painful bone metastases from lung cancer, who had failed or refused conventional therapies. Both QOL and pain palliation are achieved by this locoregional approach to skeletal metastases. Studies, in larger cohorts, can help substantiate this claim and may help broaden application of this therapy to primary bone tumors.In conclusion, CT-guided 125I brachytherapy After excluding patients lost to follow-up or for whom data was missing (n=4), data for the remaining 85 patients with 126 treated metastases was analyzed.The study was approved by the ethics committees at Sun Yat-sen University Cancer Center. Between June 2013 and May 2015, a total of 89 lung cancer patients with painful bone metastases  were recruited and consented for participation in this study. Most patients had previously pain management to bone metastases with external beam radiation therapy(EBRT), chemotherapy, analgesics, or patients had refused therapies, but the remission of pain was unsatisfied. These patients underwent CT-guided 125I brachytherapy with CT-imaging; (i) Absence of impending fracture; (j) No severe cardiopulmonary dysfunction.All enrolled patients met the following criteria: (a) Bone metastases from lung cancer were histologically or cytologically proven with imaging concordance; (b) Score of ≥4 worst pain over the past 24 hours according to BPI-Short Form; (c) Failure or refusal of conventional treatments ; (d) The number of bone lesions ≤3 with the size of each lesion ≤6cm; (e) Expected survival time ≥3 months; (f) Karnofsky performance status of ≥70; (g) Absence of coagulopathy ; (h) Ability to tolerate 125I seed  was 6711–1985, with a diameter of 0.8 mm, and a length of 4.5 mm. The central source of the particles was a 125I radionuclide silver rod, with a diameter of 0.5 mm, and a length of 3.0 mm. The matched peripheral dose was 110–140 Gy, and the average energy was 27–32 KeV. Each deposit had an initial activity of 0.8 mCi and a half-life of 59.6 days, release of continuous low-dose γ-ray and soft X-ray  after decaying into the organization. The 125I seeds had effective anti-tumor activity at a radius of 1.7 cm. Within 8–10 months, 93–97% of the brachytherapy dose was delivered.The type of each implanted 125I brachytherapy, 5mm axial enhanced CT images were obtained in all patients , planned target volume (PTV) and surrounding vital organs  was sought in every CT slice. PTV is defined as a 1.5 cm of expansion external to the GTV. The prescribed dose was averaged 120 Gy (range 100-140 Gy). Based on three orthogonal diameters within the target tumor and a prescribed matched peripheral dose (MPD) of averaging 120 Gy, TPS generated a dose-volume histogram (DVH), isodose curves of different percentages, and calculated the position of brachytherapy applicator, dose and number of implanted seeds Figure . The PTV125I seeds according to TPS , an 18 G spinal needle  reached the farthest tumor edge, but was kept at approximately or less than 5 mm of the border Figure . A turntS Figure . If a le0, T2, T4, T6, T8 and T12, respectively. The worst pain, least pain, and average pain scores from 0 to 10  were recorded. Relief of pain through the use of pain treatments or medications were scored on a 0–100 % scale in 24 hours . To further evaluate the results, pain scores of 0–3 were categorized as mild; 4–6, as moderate; and 7–10, as severe pain. The percentage distribution of patients whose worst pain score decreased in each category was compared during various periods.A dynamic enhanced CT and clinical hematological examination  were performed every two weeks for the first two months, then once every month after 2 months. Each patient was asked to answer relative questions with respect to the lesion that was to be treated by interview or telephone. we assessed pain intensity using the BPI-Short Form  and personal analgesic use in a 24-hour period at T0, T2, T4, T6, T8 and T12 by using a five-point categoric scale , similarly, KPS also was recorded. The changes in scores during these intervals, indicating recovery of QOL, were calculated and analyzed.Quality-of-life factors including sleep, appetite, spiritual state, and fatigue were rated by patients at T125I seed implantation underwent cardiac monitoring. All postoperative symptoms and complatations were recorded in detail, including a small amount of subcutaneous hemorrhage, local skin reaction, minor displacement of radioactive seeds, fever, granulocytopenia, pathologic fracture, hydropneumothorax, radiation pneumonia and massive bleeding. We calculated the percentage of various complications in order to further evaluated the safety of 125I seed implantation.Those patients who underwent percutaneous 0. Scores for QOL factors were compared using the same methods. The percentage distribution of patients in each pain category during each interal was analyzed with the Cochran-Mantel- Haenszel x2 test. A P value of less than 0.05 was considered an indicator of a statistically significant difference.The statistical software package SPSS version 10.0 was used for statistical analyses. Brachytherapy effectiveness was assessed by comparing the mean pain scores and the percentage distribution of patients reporting scores in each pain category  for each time period. The mean pain scores differences among various treatment periods were analyzed by paired t tests supplemented with repeated-measures analysis of variance (ANOVA), comparing them with scores at T


In the report “Ebola Virus Disease Cluster in the United States — Dallas County, Texas, 2014” two errors occurred.(37.8°C) but increased to 102.9°F (39.4°C), abdominal pain, and headache .”On page 1087, in the second paragraph, the first sentence should read, “On September 25, a man aged 45 years (patient 1), who had arrived in the United States from Liberia 5 days earlier, went to a Dallas County, Texas, emergency department with fever, initially 100.1°F On October 10, a nurse (patient 2) previously involved in direct care of patient 1 had a sore throat; on October 11, patient 2 went to the emergency department with fever (100.6°F [38.1°C]) and was confirmed to have Ebola by real-time PCR later that day.”In the fourth paragraph, the first sentence should read, “In the report, “Arthritis Among Veterans — United States, 2011–2013,” several errors occurred.three veterans reported that they had arthritis (34.7%) and that that age-standardized prevalence was higher among veterans than nonveterans across most sociodemographic categories, including sex .”On page 999, in the first paragraph, the fourth sentence should read as follows: “This report summarizes the results of these analyses, which found that one in crude prevalence of reported arthritis than nonveterans, 34.7% (CI = 34.3–35.1) versus 24.0 (CI = 23.8–24.1).”Also on page 999, in the fourth paragraph, the first sentence should read as follows: “Veterans had a higher overall 1,420,290),” Nonveterans ,” and “Veterans .”On page 1000, in Table 1, the characteristics of race/ethnicity, highest educational attainment, employment status, annual household income, and body mass index should have been footnoted using the existing * footnote . Also in Table 1, the n’s in the right sided column headings under “Overall” should read as “Overall  . Among male veterans, the median age-standardized state-specific prevalence was 24.7% (range = 18.4% in Hawaii to 32.7% in West Virginia); among women the median was 30.3% (range = 22.4% in Hawaii to 42.7% in Oregon) (Table 2).”Also on page 1000, in the first paragraph, the first two sentences should read as follows: “Among the 50 states and DC, the median state-specific 34.7% of veterans reported having arthritis (35.0% among men and 31.3% among women) and that prevalence was higher among veterans than nonveterans across most sociodemographic categories.”Finally, in the summary box on page 1003, in the “What is added by this report?” section, the second sentence should read as follows: “The analysis found that


Lactobacillus paracasei HD1-7 isolated from Chinese sauerkraut juice, was studied. Following partial purification with ammonium sulfate precipitation, CM Sepharose Fast Flow, and Sephadex G-10 chromatography, the molecular weight of Paracin 1.7 was about 10 kDa based on Tricine-SDS-PAGE results. A 2.87 fold purified bacteriocin was produced, reaching a final yield of 39.93% and the specific activity of 1.56 × 103 AU/mg. The N-terminal amino acid sequence of Paracin 1.7 was VSNTFFA, and the LC/LTQ results revealed that the N-terminal amino acid sequence was similar to that of ABC-type oligopeptide transport system protein and N-acetylmuramoyl-L-alanine amidase. Paracin 1.7 was sensitive to protease K, had antimicrobial activities at a broad pH range (3.0–8.0), and was heat resistant (121 °C for 20 min). Paracin 1.7 from Lactobacillus paracasei HD1-7 is a novel bacteriocin that has potential applications in food preservation.Bacteriocins have antimicrobial activities against food-spoiling bacteria and food-borne pathogens. Paracin 1.7, a bacteriocin synthesized by  Lactobacillus rhamnosus and bacteriocin Paracin 1.7 produced by L. paracasei HD1-7. The class-II bacteriocins are further divided into four subclasses, class- IIa, -IIb, -IIc, and -IId2. Class-IIa contains the antilisterial one-peptide pediocin-like bacteriocins that have similar amino acid sequences234Bacteriocins are ribosomally synthesized low-molecular weight peptides or proteins with potential use in food preservation due to their bactericidal effects on food spoilage and pathogenic organisms2345L. lactis, has been used in several countries to extend the shelf life of food productsBesides bacteriocins, LAB produce many other inhibitory compounds, such as organic acids, free fatty acids, ammonia, diacetyl, hydrogen peroxide, and enzymesL. paracasei HD1-7 from Chinese sauerkraut juiceL. paracasei HD1-7 growth and Paracin 1.7 synthesisStaphylococcus, Micrococcus, Bacillus, and Lactobacillus) and Gram negative bacteria . This study purified and characterized Paracin 1.7 from L. paracasei HD1-7. The amino acid analysis of this bacteriocin will provide assistance for gene cloning and expression.In previous studies, we isolated Following ammonium sulfate precipitation, the antibacterial activities of the supernatant and precipitate were assessed . There wThe bacteriocin was further purified by CM Sepharose Fast Flow . There was one peak, which was assessed for antibacterial activity. After cation-exchange chromatography, the specific activity of the bacteriocin increased to 457 AU/ml and the final yield of the bacteriocin was 88% of the original yield. The purification fold was 1.32. The active fraction collected from CM Sepharose Fast Flow was further purified by Sephadex G-10 and tested for antibacterial activity. The specific activity of the bacteriocin increased to 365 AU/ml and the final yield was 40% of the original. The purification fold was 2.87.The antibacterial activity, yield, fold purification, and purification method of Paracin 1.7 are summarized in Purified Paracin1.7 was extracted using a protein extraction kit  and analyzed by tricine-SDS-PAGE, which yielded a single band at about 10 kDa.The N-terminal amino acid sequence was VSNTFFA. This partial sequence of Paracin 1.7 was compared to those present in the database. However, there were no significant similarities, revealing that Paracin 1.7 is a novel peptide.The liquid chromatography/LTQ mass spectrometry results revealed that the partial sequences were homologous with those of the ABC-type oligopeptide transport system and N-acetylmuramoyl-L-alanine amidase. ABC-transporter is a specific protein, which belongs to class II bacteriocins; N-acetylmuramoyl-L-alanine amidase destroys microbial cell walls leading to cell rupture and death.The physicochemical characteristics of Paracin 1.7 were determined. The results revealed that the antibacterial activity of Paracin 1.7 was affected by temperature, pH, hydrolytic enzymes, and organic solvents .L. paracasei HD1-7 is resistant to heat.The antibacterial activity of Paracin 1.7 was not significantly affected following heat treatment at 40, 50, 60, 70, or 100 °C for 20 min. The inhibitory activity of bacteriocin was 82.48% of its original activity at 121 °C after 20 min. These results suggest that the bacteriocin produced by At pH 3–6, the antibacterial activity of the bacteriocin was higher than that of the control. Additionally, the antibacterial activity was the highest at pH 3, decreased at pH 7–8, and disappeared at pH 9. These results suggest that the bacteriocin is affected by pH.L. paracasei HD1-7 was sensitive to protease K and trypsin, moderately sensitive to papainase and insensitive to α-amylase. Therefore, the characteristic of the bacteriocin produced by L. paracasei HD1-7 are those of a peptide.The effects of a number of enzymes  on bactericin were evaluated. Following treatment with them at their optimal temperature and pH, the antibacterial activity decreased. The bacteriocin from The effects of organic solvents on bacteriocin activity were assessed. Treatment with n-butanol did not affect the inhibitory activity of bacteriocin. The bacteriocin activity retained >97% when treated with methanol, ethanol, acetone, and benzene.Bacteria can produce two different types of antimicrobial peptides: 1) ribosomally synthesized peptides or bacteriocins, which exhibit a relatively narrow range of antimicrobial activity, i.e., inhibit closely related bacteria and 2) non-ribosomally synthesized peptides, which exhibit broader antimicrobial activity. These antimicrobial peptides, however, have no bactericidal effects on bacteriocin-producing bacteria1L. paracasei HD1-7 were purified by a three-step method. Following partial purification with 70% ammonium sulfate, CM Sepharose Fast Flow, and Sephadex G-10, the final yield of Paracin 1.7 was 39.93% with an specific activity and fold purification of 1.56 × 103 AU/mg and 2.87, respectively. Based on the Tri-glycine-SDS-PAGE results, the molecular weight of Paracin 1.7 was about 10 kDa. The purification method used in this study was fast and easy to perform.In this study, bacteriocins synthesized by L. paracasei HD1-7 are those of a peptide.The N-terminal amino acid sequence of Paracin 1.7 was VSNTFFA . The enzymatic results revealed that Paracin 1.7 was sensitive to protease K and trypsin, and relatively sensitive to α-amylase and papainase, suggesting that the characteristic of the bacteriocin produced by The LC\LTQ results revealed that the bacteriocin contains an ABC-transporter protein that belongs to class II bacteriocins. The ABC-transporter protein is an ATP enzyme which is anchored to the bacterial membrane. It can catalyze the flip of lipid in the bilayer and change their conformation. N-acetylmuramoyl-L-alanine amidase binds to the ABC-transporter protein and is transferred into the cell, affecting synthesis and transport of macromolecules and contributing to cell lysis and apoptosisThe bacteriocin Paracin 1.7 does not contain YGNGVXC, which is specific to class IIa. This sequence can promote non-specific binding between bacteriocins and the target cell surface. This sequence may penetrate the helical structure of the cytomembrane, change the permeability of mannose phosphate molecules embedded in the membrane, cause conformational changes to the protein, and contribute to membrane-leakage and cell death12Based on comparisons of the N-terminal sequence, there were no bacteriocins similar to Paracin 1.7. While the N-terminal sequence does not contain cationic amino acids, such as lysine or arginine, it still has antibacterial activity. Therefore, Paracin 1.7 might be similar to the bacteriocin reported by SebeiParacin 1.7 was stable to heat treatment at 40–70 °C for 20 min. Following heat treatment at 121 °C for 20 min, Paracin 1.7 retained 82% of its original antibacterial activity. Following storage 4 °C for six months, there was no loss in antibacterial activity of Paracin 1.7 (data not shown); therefore, this bacteriocin has broad applications in the food processing industry. The antibacterial activity of Paracin 1.7 was stable at pH 3–8. Paracin 1.7 was more stable in acidic conditions and inactivated in alkaline conditions. In contrast, nisin exhibits a strong antibacterial activity at low pH and is inactivated at pH close to 7Leuconostoc paramesenteroides are not sensitive to protease but are sensitive to amylaseParacin 1.7 was degraded by trypsin and proteinase K, moderately sensitive to papainase and insensitive to amylase. Studies have reported that mesenterocin E131 is completely inactivated by proteinase K, trypsin, and chymotrypsinWith the addition of organic solvents, large-scale production of Paracin 1.7 dry powder can be produced. The effects of organic solvents on bacteriocin activity was investigated. The results revealed that organic solvents had a relatively weak effect on bacteriocin activity; Paracin 1.7 retained >97% of its original antibacterial activity following treatment with ethanol, acetone, benzene, n-butanol, and methanol. Experiments currently undergoing in our laboratory are focusing on the applications of Paracin 1.7 in food processing technologies.L. paracasei HD1-7 (CCTCCM205015), isolated from Chinese sauerkraut juice, was grown in MRS broth  at pH 5.5: soya peptone, 10 g/L; beef extract, 10 g/L; yeast extract, 5 g/L; glucose, 20 g/L; K2HPO4, 2 g/L; Na2SO3, 0.1 g/L; sodium acetate, 5 g/L; MgSO4.7H2O, 0.2 g/L; MnSO4, 0.05 g/L; ammonium citrate, 0.4 g/L; and Tween 80, 1 ml. B. subtilis (ATCC 11774) was used as the indicator strain in antimicrobial activity assays. The strain was grown in LB broth (Aoboxing) at pH 7.0: peptone, 10 g/L; yeast extract, 5 g/L; and NaCl, 10 g/L. Both media were autoclaved at 0.122 MPa for 15 min. For long time storage, the strains were stored at −80 °C in MRS or BP broth  supplemented with 20% (v/v) glycerol, and propagated three times in MRS or BP broth prior to use. For antimicrobial activity assays, we used BP broth supplemented with 0.75% (w/v) agar for the upper medium and water agar supplemented with 2% (w/v) agar for the lower medium.The bacteriocin-producing strain L. paracasei HD1-7 was grown in sterile MRS broth and incubated at 30 °C under continuous shaking (180 rpm) for 18 h until the cell density reached 107 CFU/ml. The bacterial suspension was used to inoculate fresh, sterile MRS broth at 5% (v/v) and incubated for 24 h.L. paracasei HD1-7 was obtained following centrifugation  at 4,500 rpm for 15 min at 4 °C. CFS was further filtered with an air pump (0.2-mm pore size water filter) and concentrated in a rotary evaporator  to 1/10th its original volume.Cell free supernatant (CFS) of B. subtilis was 107 CFU/ml. Antimicrobial activity was determined by measuring the diameter of the zone of inhibition around the wells and it was expressed as arbitrary units (AU) per ml. To eliminate the antimicrobial effect of lactic acid, the pH of the supernatants was adjusted to 5.5 with 1 M NaOH. Titers were defined as the reciprocal of the highest dilution that inhibited the growth of the indicator strain. Bacteriocin activity was expressed as AU/mlThe antimicrobial activity of Paracin 1.7 was assessed by an agar-well diffusion methodL. paracasei HD1-7 (30 ml) was loaded at 3 mL/min into a CM Sepharose Fast Flow cation-exchange column (16 mm × 200 mm), previously equilibrated with 0.05 mol/L ammonium acetate buffer (pH 6.0). Elution was performed using a stepwise ammonium acetate gradient (0.05–1.0 mol/L) at 1 ml/min and detected at 254 nm with a UV detector. Fractions were assessed for antimicrobial activity.Bacteriocin was purified by CM Sepharose Fast Flow, Sephadex, and HPLC. CFS of The active fractions (fraction I) obtained from cation-exchange chromatography were pooled, concentrated, and loaded into Sephadex columns (1000 mm × 10 mm) containing G50, G25, and G10 packing materials to further fractionate the active fraction. The columns were equilibrated with 0.05 mol/L ammonium acetate (pH 6.0) at 0.8 ml/min. Eluted fractions were detected at 254 nm; active fractions were concentrated by vacuum evaporation. Antimicrobial activity was assayed using the agar-well diffusion method. Concentrated active fractions obtained from Sephadex G10 were further purified in a Waters HPLC system  equipped with a Delta-Pak TM300 C18 column ; the flow rate was 0.8 ml/min. Elution was performed with a gradient from 100% buffer A  to 100% buffer B  in 30 min.L. paracasei HD 1–7 was mixed with ammonium sulfate, resulting in different concentrations  at 4 °C under constant stirring overnight. The cell suspensions were subsequently centrifuged at 10,000 RPM for 15 min (4 °C); both the supernatant and precipitate were harvested and bacteriocin activity was measured.First, supernatant of The solution obtained following ammonium sulfate precipitation was loaded into a CM Sepharose Fast Flow  cation-exchange column equilibrated with ammonium acetate buffer (pH 6.0). Active fractions  were eluted using an ammonium acetate buffer gradient (0.05–1 mol/L) at 1.3 ml/min. Finally, partially purified Paracin 1.7 from CM Sepharose was purified by Sephadex G-10  chromatography (12 mm × 200 mm) using an AKTA purifier 100 equilibrated with the same buffer. Elution was performed with ammonium acetate (pH 6.0) at 1 ml/min. Bacteriocin activity and protein concentration were measured at each step of the purification process. All experiments were conducted in triplicate.Purity and molecular weight of the active fractions obtained from Sephadex G-10 were determined by tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)24Following tricine-SDS-PAGE, the protein of interest was excised from the gel. An LTQ linear ion trap mass spectrometry  coupled to an Agilent 1100 HPLC system was used for amino acid sequencing. The results were compared with a database. Partial sequencing was determined according to the spectral library.http://aps.unmc.edu/AP/main.php.The bacteriocin was electrophoretically transferred to a 0.2-μm pore size PVDF membraneThe physicochemical characteristics of Paracin 1.7 that were evaluated included its temperature, pH, enzymatic, and organic solvent sensitivities. Additionally, the preservation life of Paracin 1.7 was determined. To determine its temperature sensitivity, Paracin 1.7 was heated to 40, 50, 60, 70, 100, and 121 °C for 20 min. Residual bacteriocin activity was measured at each temperature using unheated Paracin 1.7 as the control.Paracin 1.7 was adjusted to pH 3, 4, 5, 6, 7, 8, and 9 using sterile 5 mol/ml NaOH or HCl and maintained at the corresponding pH for 20 min. Residual bacteriocin activity at each pH was measured using unadjusted Paracin 1.7 as the control.The sensitivity of Paracin 1.7 to different enzymes, including protease K , trypsin , α-amylase , and papainase , was evaluated by incubating Paracin 1.7 in the presence of each enzyme (1 mg/ml) at the optimal temperature and pH for 1 h. The mixture was subsequently heated to 100 °C for 5 min to inactivate the enzymes. Residual bacteriocin activity was measured using Paracin 1.7 without enzymatic treatment as the control.The tolerance of bacteriocin to different organic solvents was assessed by incubating Paracin 1.7 for 1 h in the presence of 10% (w/v) organic solvents  at 25 °C. Residual bacteriocin activity was measured using Paracin 1.7 without organic solvents as control.The preservation life of bacteriocin was assessed by storing Paracin 1.7 for 6 months at 4 °C. Residual bacteriocin activity was determined every 15 days.Residual bacteriocin activity was assayed by the agar well diffusion method and calculated based on the ratio of inhibitory activity with treatment and inhibitory activity without treatment . Statistical analyses were performed by SPSS 13.0.How to cite this article: Ge, J. et al. Purification and Partial Characterization of a Novel Bacteriocin Synthesized by Lactobacillus paracasei HD1-7 Isolated from Chinese Sauerkraut Juice. Sci. Rep. 6, 19366; doi: 10.1038/srep19366 (2016).


Globally, the development of a cost-effective long-term renewable energy infrastructure is one of the most challenging problems faced by society today. Microalgae are rich in potential biofuel substrates such as lipids, including triacylglycerols (TAGs). Some of these algae also biosynthesize small molecule hydrocarbons. These hydrocarbons can often be used as liquid fuels, often with more versatility and by a more direct approach than some TAGs. However, the appropriate TAGs, accumulated from microalgae biomass, can be used as substrates for different kinds of renewable liquid fuels such as biodiesel and jet fuel.Stichococcus bacillaris strain siva2011, together with its bioprocessing, hydrocarbon and fatty acid methyl ester (FAME) profiles. The S. bacillaris strain siva2011 was scaled-up in an 8 L bioreactor with 0.2% CO2. The C16:0, C16:3, C18:1, C18:2 and C18:3 were 112.2, 9.4, 51.3, 74.1 and 69.2 mg/g dry weight (DW), respectively. This new strain produced a significant amount of biomass of 3.79 g/L DW on day 6 in the 8 L bioreactor and also produced three hydrocarbons.This article describes the isolation and identification of a lipid-rich, hydrocarbon-producing alga, S. bacillaris strain siva2011 was discovered and its biomass has been scaled-up in a newly designed balloon-type bioreactor. The TAGs and hydrocarbons produced by this organism could be used as substrates for jet fuel or biodiesel.A new oil-rich microalga  Over a large number of cycles, the net effect could be a significant reduction in the addition of CO2 into the atmosphere compared to continued reliance only on fossil fuels. A wide variety of existing biofuel technologies have been tested, but none have proven to provide a suitable source of replacement liquid fuels. Although current alternatives such as ethanol and biodiesel can provide carbon neutrality, fuels derived largely from normally edible plant sources affect the food supply negatively . A neighbor-joining tree was created using a Clustal X2.0.12 set to exclude positions with gaps and correct for multiple substitutions. Based on the 18S rDNA sequences, 1,000 bootstrap trials were used to show the relationship of the S. bacillaris and the strain siva2011  and requires room temperature. The newly designed reactor has a larger headspace, which efficiently captures light and also has good media circulation to enhance photosynthesis. In other words, this unique bioreactor configuration could minimize the factors limiting the overall rate of photosynthesis in a high density culture.To further explore the potential for enhanced fuel application, production of the S. bacillaris strain siva2011 grown for 6 days in 4 L and 8 L airlift balloon bioreactors with 0.05, 0.1, 0.2 or 0.5% CO2, respectively. Of the four concentrations of CO2 tested, 0.2% yielded the highest biomass. The exponential growth phase was noticed on day 4 and stationary phase on day 6. Although the culture medium nutrients did not deplete in 6 days, the pH dropped to highly acidic levels  (data not shown).After autoclaving the media, the pH dropped from 6.0 to 4.2 because of fructose degradation. The sugar degradation could be minimized by sterilizing filtration; however, this procedure increases the percentage of culture contamination. Figure 2, a maximum biomass of 3.79 g/L dry weight (DW) was achieved in 8 L and 3.45 g/L DW in 4 L, respectively. Since the new strain requires a very low 0.2% of CO2, the input cost on large-scale could be minimized. When compared to 0.5% CO2, algal cells grown in a 4 L bioreactor were higher in biomass, with 0.55, 0.986 and 1.45 g/L DW in the 0.05, 0.1 and 0.2% CO2, respectively. Similarly, in the 8 L bioreactor, biomass accumulation was 0.793, 1.107 and 1.79 g/L DW after 6 days of culture. Both growth and pH kinetic trends were similar in 4 L and 8 L bioreactors. The data supports the notion that this strain does not require light intensity over 15 to 30 μE m-2 s-1; therefore, achieving high density biomass may not be a problem. However, the optimization of air flow is needed for better media circulation and growth. The media circulation is critical for efficient photosynthesis. The oil-rich alga Ettlia oleoabundans accumulates 2.28 g/L dry biomass in BBM in approximately 22 to 27 days [S. bacillaris strain siva2011 accumulates approximately 1.5 g/L higher biomass within 6 days in modified Murashige and Skoog (MS) medium.On day 6 with 0.2% CO 27 days , while SS. bacillaris strain siva2011 has been regarded as an indicator for productivity in the bioprocess. Prediction of DW is needed and helpful for scale-up of S. bacillaris strain siva2011 biomass in large-scale reactors. Quinn et al. [2 in 4 L and 8 L bioreactors compared with experimental/measured data obtained from each test. An overall trend of predicted DW for 8 L was similar to the 4 L data. The R2 value has been estimated in the 8 L as 85.8%, while the 4 L exhibits 87.4%. Both tests give an error under 15%. This suggests that the selected variables are not adequate for accurate validation and mapping within an acceptable error. Application of polynomial equations with more variables in the modeling or another mathematical approach might be warranted for future optimization if better accuracy is needed.For scale-up studies, modeling is necessary because it provides detailed estimates regarding prediction and validation. For instance, DW of n et al.  modeled S. bacillaris strain siva2011 biomass. This chromatogram shows that this strain biosynthesizes three free hydrocarbons, namely n-nonadecane (C19H40), nonacosane (C29H60) and heptadecane (C17H36). These alkanes are also found in traditional and non-traditional liquid fuels. The S. bacillaris strain siva2011 contained 1.36% of total hydrocarbons: the C19H40, C29H60 and C17H36 were 6.3, 4.1 and 3.2 mg/g DW, respectively, with 0.2% CO2. In addition, it was unchanged in both the 4 L and 8 L bioreactor studies. Some of these hydrocarbons were previously reported in other algal species [Saccharomyces cerevisiae cells, while C8 to C11 alkanes were cytotoxic [S. bacillaris strain siva2011 biosynthesize longer chain hydrocarbons, the alkane level is lower than in the TAGs. Since the S. bacillaris strain siva2011 is a new species, a reference genome is necessary for transcriptome analysis, and subsequent metabolic engineering is unavailable.Figure  species ,28. In cacosane CH60 and h species . The ove species . In geneytotoxic . AlthougS. bacillaris strain siva2011 biomass contains two free fatty acids (FFAs) , linolenic acid (C18:3)) and phytol  cultivation of small molecules [et al. [S. bacillaris 158/11 contains C16:0 6.5%, C18:3 5.2%, C18:2 4.6% and C18:1 13.8%. This suggests that the Stichococcus species has unique fatty acid profiles which could be used for high-quality liquid fuel production. However, the actual large-scale feasibility test for algal biomass scale-up is needed for aviation fuel production.The c acid C1:0, linolf phytol . The resnd NJ-17 . When S.olecules . It was olecules ,34. Thus [et al.  showed tS. bacillaris strain siva2011 is rich in lipids, presumably TAGs, with a suitable carbon range for aviation or other liquid fuels. Indeed, scaling studies showed that at 0.2% CO2 supplementation S. bacillaris strain siva2011 had better growth and increased FAMEs in the 8 L bioreactor than 4 L. It is likely that the 20 L bioreactor could have substantially lower hydrodynamic stress. However, further studies on mass transfer at larger scales seem to be warranted. The culture conditions vary from alga species to species. S. bacillaris strain siva2011 can grow in conditions mentioned in this study. Irrespective of the need to further characterize the biochemical pathways for this organism, it is nevertheless important to point out that there is already sufficient empirical evidence that it will likely be a possible candidate for renewable production of light liquid fuels based on the copious production of lipids and hydrocarbons, and especially the relatively high degree of unsaturation found therein.Long-term energy demands will eventually greatly outweigh the world supply of fossil fuels, and their use increases greenhouse gases. Therefore, alternative sources and methods of producing fuels must be found. Although algae can capture greenhouse gas emissions while producing oxygen, the need for high biomass and oil accumulation are challenging for algal-based bioenergy production. S. bacillaris strain siva2011 cells were isolated from in vitro Lagerstroemia seedlings. The morphology of algal cells was identified by light microscopy. The genus and species were identified by the 18S rDNA region of the nuclear chromosome and the 23S region of the chloroplast rDNA. PCR was performed using primers to amplify the 23S and the 18S region of the rDNA. The products were then sequenced. The genus Stichococcus was identified based on the 18S rDNA sequence in the NCBI database. The identification was confirmed and authenticated by The Culture Collection of Algae at the University of Texas at Austin (UTEX), Austin, TX, USA. The phylogenetic tree was created based on the 18S rDNA sequence using a Clustal X2.0.12 set to exclude positions with gaps, correct for multiple substitutions and run 1,000 bootstrap trials.Axenic S. bacillaris strain siva2011 was cultured in 5 L and 20 L liquid-phase airlift balloon-type bioreactors [4NO3 0.6 g, KNO3 1.5 g [-2 s-1 for 10 hours followed by 14 hours of dark and 23 to 25°C culture conditions were used. After autoclaving the medium and the bioreactor, the axenic algal cells were cultured into the bioreactor. The inoculum was active cells that were 3 days old and 0.05 g fresh weight (FW)/L. The bioreactor cultures were supplemented with different concentrations of sterile filtered CO2 such as 0.05, 0.1, 0.2 or 0.5%. The input air mixture CO2 gas flow was set at 0.1 vvm (volume (of air) per volume (of liquid) per minute). To screen growth kinetics of S. bacillaris strain siva2011, algal biomass was harvested, and medium pH was measured after 1, 2, 3, 4, 5 and 6 days. Algal cells were harvested by centrifugation at 10,000 rpm for 5 minutes. After harvesting, algal biomass were frozen in liquid nitrogen and freeze-dried. DW was recorded after the samples were freeze-dried to a constant weight.The new alga reactors -34 with reactors  liquid mreactors . To evalreactors  for rootO3 1.5 g ,33 with 2 experimental data to predict DW of S. bacillaris strain siva2011 production ranging from 4 L to 8 L. Related variables including DW, yCO2, pH, time (t) and volume (V) are listed from constituents in S. bacillaris strain siva2011 production tests, as follows:A linear regression model has been developed based on 0.2% COTo test its feasibility, it has been simplified into a linear equation with two variables, t and V, among the whole variables as shown in Equation (2):Experimental data and variables have been normalized using maximum DW and t to establish a mathematical model as shown in Equations (3) and (4).f is maximum DW (g/L) produced from 8 L test under 0.2% CO2 fraction and tf is maximum t to reach the maximum DW. The DWf and tf were 3.79 g/L on day 6. V has been standardized by a baseline 8 L in this model. Constants a, b and c in Equation (2) have been determined using test data and a linear least squares method. The 4 L and 8 L data were used to determine the constants: a, b and c. The final non-dimensional equation is suggested by Equation (5):Where DW1 was base volume at 4 L and V2 was extended at 8 L volume.Vet al. [et al. [One gram of 6-day-old freeze-dried algal cells were used for analysis of hydrocarbons and FAMEs. The total lipids were evaluated according to Jones et al. . FAMEs wet al. ,40. Each [et al. . QuantitAll experiments were repeated at least three times, each with three replications except sequencing. The experimental variations were expressed as a mean standard error.ACP: Acyl-acyl carrier protein; BBM: Bold’s Basal Medium; CO2: Carbon dioxide; DW: Dry weight; FA: Fatty acid; FAME: Fatty acid methyl ester; FFA: Free fatty acid; FW: Fresh weight; GC-MS: Gas chromatography–mass spectrometry; GDH: Glutamate dehydrogenase; HNO3: Nitric acid; KNO3: Potassium nitrate; MS: Murashige and Skoog; NADPH: Nicotinamide adenine dinucleotide phosphate; NCBI: National Center for Biotechnology Information; NH4NO3: Ammonium nitrate; PCR: Polymerase chain reaction; TAG: Triacylglycerol; UTEX: The Culture Collection of Algae at the University of Texas at Austin.The authors declare that they have no competing interests.GS, JL and KJ made substantial contributions to experimental design or analysis or interpretation of data. Specifically, GS led and designed the experiments and performed the organism isolation, bioprocessing and GC-MS quantitative studies. JL performed the gas chromatography separation and mass spectrometry experiments for compound identification. KJ performed the mathematical model to validate the variables. GS wrote the manuscript, which was reviewed and approved by all authors. All authors read and approved the final manuscript.NCBI basic local alignment search tool comparison of the S. bacillaris strain siva2011 18S rDNA sequences with different Stichococcus species. A large difference was found in the Clustal X2.0.12 multiple sequence alignment between the S. bacillaris strain siva2011 and the existing strains at nucleotides 610 to 980. SIVA, S. bacillaris strain siva2011.Click here for file


In the present study we constructed multiple Apoe-/- mouse strains to find diabetes-related phenotyptic variations that might be linked to atherosclerosis development. Evaluation of both early and advanced lesion formation in aortic root revealed that C57BL/6, SWR/J, and SM/J Apoe-/- mice were susceptible to atherosclerosis and that C3H/HeJ and BALB/cJ Apoe-/- mice were relatively resistant. On a chow diet, fasting plasma glucose varied among strains with C3H/HeJ having the highest (171.1 ± 9.7 mg/dl) and BALB/cJ the lowest level (104.0 ± 6.6 mg/dl). On a Western diet, fasting plasma glucose rose significantly in all strains, with C57BL/6, C3H/HeJ and SWR/J exceeding 250 mg/dl. BALB/cJ and C3H/HeJ were more tolerant to glucose loading than the other 3 strains. C57BL/6 was sensitive to insulin while other strains were not. Non-fasting blood glucose was significantly lower in C3H/HeJ and BALB/cJ than C57BL/6, SM/J, and SWR/J. Glucose loading induced the 1st and the 2nd phase of insulin secretion in BALB/cJ, but the 2nd phase was not observed in other strains. Morphological analysis showed that BALB/cJ had the largest islet area  and C57BL/6 had the smallest one . This study has demonstrated strain-specific variations in the metabolic and atherosclerotic phenotypes, thus laying the basis for future genetic characterization.We recently have found that apolipoprotein E-deficient (Apoe We . We –/- ckground 14]. The. The–/- ckground 16]17].[17].–/- .[17].–/--/- mouse strains were evaluated for lesion formation in the aortic root when fed the chow or Western diet. Atherosclerotic lesion sizes differed greatly among the 5 Apoe-/- mouse strains with either diet. On the chow diet, B6, SWR, and SM Apoe-/- mice formed significantly larger atherosclerotic lesions than C3H and BALB Apoe-/- mice with P values in the range of 0.012 to 0.00013 , followed by SWR.Apoe-/- with a lesion area of 17,756 ± 2,707 μm2/section (n = 11) and SM.Apoe-/- with an area of 15,575 ± 3,412 μm2/section (n = 8). In contrast, C3H.Apoe-/- and BALB.Apoe-/- mice developed small aortic lesions, with an area of 879 ± 311 (n = 6) and 1,510 ± 823 μm2/section (n = 10), respectively.6 to 18 female mice from each of the 5 Apoe 0.00013 . B6.Apoe-/- mice developed large aortic lesions, with an average lesion area of 518,648 ± 27,979 μm2/section (n = 18) . BALB.Apoe-/- mice were also relatively resistant to atherosclerosis, with a lesion area of 148,667 ± 32,000 μm2/section (n = 9). SM.Apoe-/- and SWR.Apoe-/- mice were relatively susceptible to atherosclerosis, having lesion areas of 310,020 ± 15,083 (n = 10) and 206,202 ± 29,432 μm2/section (n = 15), respectively.After being fed the Western diet for 12 weeks, B6.Apoe(n = 18) . In cont-/- mice had the highest fasting plasma glucose level among the 5 strains, with an average of 171.1 ± 9.7 mg/dl (n = 6), and BALB.Apoe-/- mice had the lowest level, with an average of 104.0 ± 6.6 mg/dl (n = 9) , 146.4 ± 8.7 (n = 13), and 133.3 ± 7.2 mg/dl (n = 9), respectively. Compared to B6.Apoe-/- mice, BALB and SM Apoe-/- mice had significantly lower fasting glucose levels (P<0.0028). After 12 weeks on the Western diet, all 5 strains showed significant rises in fasting glucose levels (P<0.004). B6, C3H, and SWR Apoe-/- mice had fasting glucose levels exceeding 250 mg/dl, with C3H having a level of 346.5 ± 14.4 mg/dl (n = 13), followed by SWR with 304.0 ± 9.9 mg/dl (n = 11) and B6 with a level of 294.0 ± 8.7 mg/dl (n = 17). BALB and SM Apoe-/- mice had relatively low glucose levels, with values of 186.0 ± 19.7 mg/dl (n = 8) and 196.2 ± 7.9 mg/dl (n = 10), respectively. The difference between the high and the low glucose groups was statistically significant, with P values in the range of 0.0053 to 3.15 x 10-8.On the chow diet, C3H.Apoe (n = 9) . The othth min for C3H or at the 20th min for other 4 strains (P<0.0067). On the contrary, SM and SWR mice displayed significant increases or a trend of significant increases in blood glucose at the peak and over the entire curve on GTT . In response to insulin, B6.Apoe-/- mice showed a deeper and longer-lasting fall in blood glucose levels .GTT and ITT were performed on 5 to 16 mice per strain after 10 to 11 weeks of Western diet consumption. In response to intraperitoneally injected glucose, blood glucose levels quickly rose to the peak at the 10 strains . Once ree levels . The oth-/- mice exhibited both the first phase and the second phase of insulin secretion while the second phase of insulin secretion was not observed in other 4 strains  > B6  > SM  > C3H  > SWR .The test was performed on 4 to 12 mice fed the Western diet for each strain. In response to glucose, BALB.Apoe strains . For BAL-/- mice had the largest total cross-sectional area of islets , which was approximately 2-fold as large as that of B6.Apoe-/- mice  (P = 1.28x10-6). The other 3 strains of mice were also different in their islet cross-sectional areas, with the order of SM  > C3H  > SWR . BALB and B6 mice also had the largest and the smallest cross-sectional area , respectively. The cross-sectional islet areas of C3H and SM mice were 20,461 ± 1,594 and 19,174 ± 343 μm2, respectively, significantly larger than the area of B6 mice (P< 0.019). SWR mice also had a larger cross-sectional islet area  than B6 mice, but the difference did not reach statistical significance (P = 0.058).BALB.Apoe n = 11) . The dif-/- mice had a body weight of 24.5 ± 0.6 (n = 9) and, significantly larger than that of B6.Apoe-/- mice  (-/- mice had body weight of 22.8 ± 1.4 (n = 10), 22.2 ± 1.0 (n = 11), and 21.7 ± 0.7 (n = 15) g, larger than the weight of B6.Apoe-/- mice, but the difference was not statistically significant .BALB.Apoe< 0.012) . SM, C3H-/- mice had the highest non-HDL cholesterol level among the 5 strains, with an average of 1,304.8 ± 48.8 mg/dl (n = 10), and BALB.Apoe-/- mice had the lowest non-HDL level, with an average of 410.1 ± 43.0 mg/dl (n = 12), significantly different from that of B6.Apoe-/- mice . C3H.Apoe-/- mice had an average non-HDL level of 930.2 ± 39.8 mg/dl (n = 13) and SWR.Apoe-/- mice had an non-HDL level of 699.7 ± 43.1 mg/dl (n = 11), both of which were not significantly different from that of B6.Apoe-/- mice . HDL cholesterol levels also varied considerably among the 5 strains, with BALB having a level of 283.0 ± 22.2 mg/dl and with B6 having a level of 21.9 ± 4.1 mg/dl. SWR.Apoe-/- mice also had a HDL level of 23.0 ± 4.6 mg/dl, comparable to the level of B6 (P = 0.20) but lower than the levels of 50.0 ± 5.6 and 42.8 ± 2.9 mg/dl in SM and C3H mice (P<0.0012). The triglyceride levels also varied among the 5 strains with C3H and SM mice having significantly higher levels  than B6 (80.3 ± 8.1 mg/dl), SWR (117.1 ± 12.1 mg/dl) and BALB (81.9 ± 5.7 mg/dl) (P<0.05).Plasma non-HDL (LDL and VLDL) cholesterol, HDL cholesterol and triglyceride levels were measured for 6 to 18 mice per strain fed the Western diet . SM.Apoe-/- mouse strains to find phenotypic interaction between atherosclerosis and type 2 diabetes. B6, SM, and SWR Apoe-/- mice were found to be susceptible to atherosclerosis, intolerant to glucose loading and have impairments in non-fasting blood glucose on the Western diet. On the contrary, C3H and BALB Apoe-/- mice were found to be resistant to atherosclerosis, tolerant to glucose loading and have low non-fasting blood glucose. However, no distinctive patterns were found between the atherosclerosis-susceptible strains and the resistant strains in fasting glucose, insulin resistance, body weight, islet mass, and fasting lipids.In this study, we created multiple Apoe-/- mouse strains to atherosclerosis was determined by assessing plaque formation in the aortic root. At 12 weeks of age on a chow diet, B6, SM, and SWR Apoe-/- mice developed much larger lesions than C3H and BALB Apoe-/- mice. This finding is in agreement with the previous observations that mouse strains B6, SM and SWR are susceptible to fatty streak lesion formation and that C3H and BALB are resistant . In . In -/- how diet . After 1 lesions . The relOne major finding of the present study was the distinct patterns displayed between atherosclerosis-susceptible strains and resistant strains in non-fasting glucose levels and glucose tolerance on the Western diet. The atherosclerosis-resistant strains C3H and BALB had significantly lower non-fasting blood glucose and displayed greater glucose tolerance than the susceptible B6, SM and SWR strains. Epidemiological studies have revealed a close link between postprandial hyperglycemia and cardiovascular mortality in different ethnic groups 19]20].[20].19][.[20].19]In this study, we found no distinction between atherosclerosis-prone and resistant strains in fasting glucose. Rather, atherosclerosis-resistant C3H mice had as high a fasting glucose level as that of atherosclerosis-prone B6 and SWR mice, and atherosclerosis-prone SM mice had a fasting plasma glucose level that was almost as low as that of atherosclerosis-resistant BALB mice. In humans, fasting glucose levels are often found to be less strongly associated with cardiovascular risks than postprandial fasting glucose values .A paradoxical finding is that C3H mice had an extremely high fasting glucose level but a low non-fasting glucose level on the Western diet. In contrast, BALB mice had low fasting and non-fasting glucose levels on the same diet. Both strains also exhibited high insulin resistance, but C3H mice showed low basal and stimulated insulin secretion. These findings suggest that low insulin secretion was probably responsible for fasting hyperglycemia observed in C3H mice. Differential effects of defective insulin resistance versus secretion on fasting and postprandial glucose have been investigated on identical twins with type 2 diabetes , hemipan-/- mouse strains except for BALB had impairments in insulin secretion on the Western diet. Insulin secretion following glucose loading is usually biphasic, consisting of a temporary initial peak of insulin release, followed by a prolonged second phase. The first phase insulin secretion was quite comparable among the strains, but the second phase insulin response was only observed in BALB mice. The basal insulin level also varied greatly among the mouse strains. BALB/c mice had a relatively large islet mass, which may contribute to the high level of insulin secretion, the strong tolerance to glucose and the high resistance to diabetes compared to other strains.We have found that the ApoeDespite insulin resistance, BALB mice developed no diabetes due to normal β-cell function. In contrast, B6 mice had no impairment in insulin sensitivity but developed diabetes. These results suggest that defective β cell function is essential to the development of diabetes. The recent genome-wide association studies have identified much more new loci that are implicated in β-cell development and function than those implicated in insulin action, highlighting insulin secretion in the development of type 2 diabetes in humans as well .In mice fasting plasma glucose levels exceeding 250 mg/dl is considered diabetic . Based o-/- strains. C3H and SM mice also had higher HDL levels than B6 and SWR mice. Besides its anti-atherogenic properties, HDL has anti-diabetic effects, including promotion of the uptake of glucose by skeletal muscle . The. The35].-/- mouse model. The clinical observation that nearly 80% of Asian patients with coronary artery disease had abnormal glucose tolerance 42].[42].-/- .[42].-/-All procedures were carried out in accordance with current National Institutes of Health guidelines and approved by the University of Virginia Animal Care and Use Committee .-/- strains were backcrossed 9 generations or more to their respective background strains. B6.Apoe-/- mice were purchased from the Jackson Laboratory, Bar Harbor, ME. C3H/HeJ (C3H), BALB, SM/J (SM) and SWR/J (SWR) Apoe-/- mice were created in our laboratory using the classical congenic breeding protocol . The. The47].Evaluation of insulin secretion was performed as we described . BrieflyPancreatic islet mass was evaluated with a histological method as we described . Brieflyn indicating the number of mice. The distribution of data was examined for normality with SPSS software. ANOVA or Student's t test were used for determining statistical significance between groups when data were roughly normally distributed or at least symmetric. Differences were considered statistically significant at P < 0.05.Values were expressed as means ± SE, with S1 Text(XLSX)Click here for additional data file.