SLC25A13 gene. Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) is the major pediatric CD phenotype, and its definite diagnosis relies on SLC25A13 genetic analysis. China is a vast country with a huge population, but the SLC25A13 genotypic features of CD patients in our country remains far from being well clarified. Via sophisticated molecular analysis, this study diagnosed 154 new CD patients in mainland China and identified 9 novel deleterious SLC25A13 mutations, i.e. c.103A > G, , c.493C > T, c.755 − 1G > C, c.845_c.848 + 1delG, c.933_c.933 + 1insGCAG, c.1381G > T, c.1452 + 1G > A and c.1706_1707delTA. Among the 274 CD patients diagnosed by our group thus far, 41 SLC25A13 mutations/variations were detected. The 7 mutations c.775C > T, c.851_854del4, c.1078C > T, IVS11 + 1G > A, c.1364G > T, c.1399C > T and IVS16ins3kb demonstrated significantly different geographic distribution. Among the total 53 identified genotypes, only c.851_854del4/c.851_854del4 and c.851_854del4/c.1399C > T presented different geographic distribution. The northern population had a higher level of SLC25A13 allelic heterogeneity than those in the south. These findings enriched the SLC25A13 mutation spectrum and brought new insights into the geographic distribution of the variations and genotypes, providing reliable evidences for NICCD definite diagnosis and for the determination of relevant molecular targets in different Chinese areas.Citrin deficiency (CD) is a Mendelian disease due to biallelic mutations of  SLC25A13 gene. With 18 exons and 17 introns, this gene is localized to chromosome 7q21.3, and encodes citrin, the liver-type mitochondrial aspartate/glutamate carrier isoform 2 (AGC2)12Citrin deficiency (CD) is a Mendelian disease entity due to biallelic mutations of Three age-dependent CD phenotypes have been described, which manifest in neonates or infants as neonatal intrahepatic cholestasis caused by citrin deficiency 567894111213141895171819SLC25A13 genetic testing has been recognized as a reliable method for the definitive diagnosis of NICCD. However, about 10–15% of the SLC25A13 mutations could not be detected by conventional DNA analysis2123SLC25A13 analysis to date2225261927SLC25A13 mutations worldwide demonstrate remarkable heterogeneity1222232427293031323334353637SLC25A13 genotypic features of CD patients, including the mutation spectrum and their geographic distribution, remains far from being well clarified in our country.The SLC25A13 mutations and genotypes, and evaluated the allelic heterogeneity in an attempt to provide reliable evidences for the determination of relevant molecular diagnostic targets in different geographic areas of China.In this study, besides the conventional DNA analyses, cDNA cloning and in silico prediction as well as functional analysis were carried out to definitely diagnose pediatric CD patients. In addition, we investigated the geographic distribution of the As shown in 23242733SLC25A13 mutations were identified in this study, and among them, c.103A > G (p.M35V), c.493C > T (p.Q165X), c.755−1G > C, c.845_c.848 + 1delG, c.933_c.933 + 1insGCAG, c.1381G > T (p.E461X), c.1452 + 1G > A and c.1706_1707delTA (p.S331fsX363) , r.845_848delG (p.D283fsX285) and r.933_934insGCAG (p.A312fsX317), respectively (As shown in 1fsX363)  were detectively .In patient C0360, four high-frequency mutation screening just detected the maternal mutation IVS16ins3kb. Further cDNA cloning analysis revealed that all the transcripts from the paternal allele featured exon 5 skipping . The fol39SLC25A13 allele harbored two mutations , while the paternal mutation was not detected by direct DNA sequencing. The subsequent cDNA cloning analysis also shown that all the transcripts were from the maternal allele with r.851_854del4; moreover, half of them (12/21) demonstrated exon 14 skipping   , indicatAlignment analysis in 11 different species indicated that the amino acid M35 in citrin protein is highly conserved . The proP = 0.341) from that of the empty vector pYX212 (vector). However, both of them had significantly lower (P = 0.000) growth ability in comparison to pYX212-citrin (citrin). These findings indicated that the mutation p.M35V, as a lack-of-function variation, caused the elimination of the AGC2 function of citrin protein.As depicted in SLC25A13 mutations/variations were detected in 522 out of the 528 independent alleles in SLC25A13 spectrum was composed of 41 mutations/variations, including 12 missense mutations, 8 deletion, 10 nonsense, 4 splice-site, 3 insertion, 1 duplication, 1 pathogenic SNP2941P all <0.05 (P = 0.001) and northern (P = 0.000) populations. By contrast, the relative frequency of IVS16ins3kb was lower in the south than in the border (P = 0.013) and the north (P = 0.005). Besides, when compared with the south region, the relative frequencies of c.1399C > T (p.R467X) (P = 0.003) in the border and those of c.1364G > T (p.R455L) (P = 0.012) and IVS11 + 1G > A (P = 0.015) in the north were higher. No significant difference were observed on the geographic distribution of the mutations IVS6 + 5G > A and c.1638_1660dup among different areas.Among all the 41 mutations/variations, as shown in ll <0.05 . In pairP < 0.05. Further pairwise comparisons revealed that the relative frequency of c.851_854del4/ c.851_854del4 in the south was much higher than that in the north (P = 0.000).Except the 6 patients with only one mutation detected and the 13 ones with parents of different geographic origins, in this paper, the remaining 245 unrelated individuals were enrolled for the comparison of the genotype distribution. As shown in P values of 0.001 and 0.012, respectively. In other words, the northern population had a higher level of allelic heterogeneity at the SLC25A13 locus than the southern population.The observed homozygosity calculated based on the genotype frequencies was 17.39% (4/23) in the north, 34.28% (12/35) in the border and 52.94% (99/187) in the south, respectively. In addition, the theoretical homozygosity value, which was calculated based on allele frequencies, was 15.24% in the north, 24.31% in the border and 44.81% in the south, respectively. In pairwise comparisons, both of the observed and theoretical homozygosity values were higher in the south than in the north, with the SLC25A13 genetic analysis in our department23242733SLC25A13 exons and their adjacent intronic regions could identified the remaining micro mutations, which accounted for 12.5%. Besides, the functional and bioinformatic tools also made substantial contribution to the pathogenicity confirmation of the novel missense mutation c.103A > G (p.M35V). As a result, the 154 new CD patients diagnosed in this paper, together with those reported in our department previously, constituted a 274-case cohort. So far as we know, this is the hitherto largest CD cohort in official references worldwide, laying a foundation for our subsequent clinical investigation. In particular, the 9 novel mutations identified in this study enriched the SLC25A13 mutation spectrum, and provided reliable laboratory evidences not only for the definite diagnosis of the corresponding individuals, but also for the genetic counseling of their families in the future.The first case of NICCD in mainland China was reported by our group in 2006SLC25A13 cDNA cloning analysis using PBLs had proved to be a feasible tool for the detection of the large insertion/deletion mutations2324r.329_468, p.E110fs127X) following the mutation IVS4ins6kb (GenBank accession number: KF425758)SLC25A13 alleles remained obscure in this study; however, the rate of unidentified mutations  in our study is much lower than those in previous publications using the conventional molecular approaches only. SLC25A13 cDNA cloning analysis using PBLs should be taken as an important tool for the molecular diagnosis of CD patients.It was noteworthy that, as an unique technique developed by our groupSLC25A13 mutations demonstrated different geographic distribution in this study. The relative frequencies of c.851_854del4 tended to decrease gradually from south to north, while that of IVS16ins3kb had an opposite tendency, as shown in the 44SLC25A13 locus in the present study are compatible with this presumed migration model. Actually, previous haplotype studyAlthough the relative frequencies of c.1638_1660dup and IVS6 + 5G > A were uniform in different regions of China, some vs 4.35%), as shown in SLC25A13 locus. This finding suggested that some CD patients might be missed while the CD prevalence be underestimated in the north, when the same high-frequency mutations as in the south were choosed as the molecular targets for the detection of CD patients and SLC25A13 carriers in the north area. Therefore, the exploration of additional SLC25A13 mutations should be regarded as an important issue in the north area in terms of CD molecular diagnosis and epidemic survey.Moreover, although a diversity of genotypes with a total number of 53 was discovered in the large CD cohort, c.851_854del4/c.851_854del4 was the unique genotype with higher relative frequency in the south than in the north  and was approved by the Medical Ethical Committee of the First Affiliated Hospital, Jinan University. SLC25A13 analyses were conducted with the written informed consents from the guardians of the patients.This research enrolled a total of 274 CD patients diagnosed by our group in the past over 10 years, including 154 new CD cases which were diagnosed by sophisticated molecular, functional and bioinformatic analysis of SLC25A13 exons and their adjacent intronic regions were amplified by PCR and analyzed by Sanger sequencing in patients with just one mutation was detected. Following that, if there was a SLC25A13 mutation remained obscure, IVS4ins6kb, another large insertion with relative high frequency in Chinese, would be screened by LA-PCR as in our previous publicationsGenomic DNA of the patients suspected to have CD and their parents was extracted from EDTA-anticoagulant peripheral venous blood. The 4 high-frequency mutations c.851_854del4, c.1638_1660dup, IVS6 + 5G > A and IVS16ins3kb were initially screened by using PCR/LA-PCR and PCR-RFLP, respectively. All the 18 RT-PCR and cDNA cloning analysis were subsequently carried out in patients still with undetected mutation by all the approaches above, and in those with novel mutations that might affect the splicing of pre-mRNA molecules. In brief, total RNA was extracted from peripheral blood lymphocytes (PBLs), which were collected from 2 ml fresh EDTA-anticoagulant peripheral venous blood. Then RT-PCR was performed to synthesized cDNA following the kit manufacture’s protocol . With the cDNA as template, nest-PCR was then performed for the target products, and the purified nest-PCR products were cloned into PMD-18T vector  and transformed into DH5α Escherichia coli competent cells. The positive clones were tested by LA-PCR with the universal primer set RVM (GAGCGGATAACAATTTCACACAGG) and M13-47 (CAGCACTGACCCTTTTGGGACCGC) and then sequenced, as previously describedhttp://www.ebi.ac.uk/Tools/msa/clustalo/)www.ncbi.nlm.nih.gov). Then the pathogenicity of the missense mutation was predicted by the softwares PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/)47http://mutationtaster.org/ MutationTaster/index.html)http://provean.jcvi.org/index.php)50The conservative property of the amino acid affected by the novel missense mutation was analyzed by the software Clustal Omega  to constitute the plasmid pYX212-citrin and pYX212-mutant, respectively. The BYagc1Δ strains were then transfected with the recombinant plasmids and the empty vector pYX212, respectively. The positive clones were screened using the uracil minus medium SD-URA and cultured in SA medium with acetate as the unique carbon source. After 96 hour of culture, the growth abilities of these three strains were assessed by the cell density measured at OD600.A diploid AGC1-disrupted yeast model, BYagc1Δ, which was constructed in our previous publicationSLC25A13 mutations was 1/48 in the south but 1/940 in the north of this riverThe Yangtze River has been considered as a historically significant boundary of the Chinese population5253Χi2, where ∑ stands for summation over all alleles, and Χi is the frequency of the ith allele55ith allele is m, Χi is calculated to be m/N, where N is the total number of the mutant alleles being investigated. The alleles harboring obscure mutations were counted as SLC25A13 alleles different from those with detected variations, and thus, each of them was defined to have a frequency of 1/N.The theoretical homozygosity (J) at a locus in a given population is measured by J = ∑SLC25A13 mutations and genotypes, as well as the homozygosity values among the three geographic areas, were compared by means of Chi-square test or Fisher’s exact tests, respectively. When the Chi-square test of 3 × 2 table was significant with P < 0.05, pairwise comparisons were then performed with Bonferroni corrections of the P values. There were 3 pairwise comparisons: (1) north vs border, (2) north vs south, and (3) border vs south; accordingly, the adjusted P values of 2 × 2 Chi-square test for significance was 0.017 (0.05/3)P < 0.05 as the significant criteria. All statistical calculations were performed on the software SPSS17.0.The frequencies of the How to cite this article: Lin, W.-X. et al. Molecular diagnosis of pediatric patients with citrin deficiency in China: SLC25A13 mutation spectrum and the geographic distribution. Sci. Rep.6, 29732; doi: 10.1038/srep29732 (2016).


Porcine circovirus 2 (PCV2) strain 102 belongs to the PCV2b-1C subtype, and its sole structural protein (Cap) exhibits high homology with that of other PCV2b isolates reported in South Korea, China, and the United States. Strain 102 contains a new mutation (R37H) in the domain of the nuclear localization signal (NLS) of the Cap. Circoviridae family (–-3 nucleotide substitutions per site every year (–Porcine circovirus 2 (PCV2) is a nonenveloped, icosahedral, single-stranded, circular DNA virus and belongs to the e family , 2. PCV2e family –7. The g family –. Previouery year , and PCVery year –11.In this study, various tissue samples  from postweaning multisystemic wasting syndrome (PMWS) pigs were collected in a field in Yiyang, Hunan Province, China. The complete genome of PCV2 strain 102 was amplified by PCR . Subsequently, PCR products were cloned into the pSP-72 cloning vector  and sequenced for double-stranded DNA. The resultant sequences were assembled and analyzed using DNAStar.The complete genome sequence of strain 102 consists of 1,767 nucleotides (nt), with a G+C content of 48.33%. It shares the highest sequence homology (99.9%) with another PCV2 strain, Han8 (GenBank accession no. JQ181600). Phylogenetic analysis suggests strain 102 can be clustered with PCV2b-1C. ORF1 of the strain consists of 945 nt, encoding 314 amino acid (aa) residues of Rep; ORF2 consists of 705 nt, encoding 235 aa residues of Cap, which shares high amino acid homology with that of PCV2b strains discovered in South Korea (GenBank accession no. KJ133547 and KJ437506), the United States (GenBank accession no. JX535296 and JX535297), and China (GenBank accession no. HM038030 and HM038017), with the exception that the Cap of strain 102 has a unique point mutation (R37H) in the nuclear localization signal (NLS). ORF3 of strain 102 consists of 315 nt, encoding an apoptosis-related protein containing 105 aa residues.Recently, a series of mutated strains of PCV2b were discovered worldwide –16. Some–KP112484.The complete genome sequence of strain 102 has been submitted to GenBank under the accession no.


Panax ginseng. Many research studies indicate that these deglycosylated, less-polar ginsenosides have better bioactivity than the major ginsenosides. In the present study, we sought to verify the enhanced anticancer effect of P. ginseng extract after undergoing the Maillard reaction as well as elucidate the underlying mechanism of action. The effects of 9 amino acids were tested; among them, the content of 20(S)-Rg3 in the ginseng extract increased to more than 30, 20, and 20% when processed with valine, arginine, and alanine, respectively, compared with that after normal heat processing. The ginseng extract that was heat-processed with arginine exhibited the most potent inhibitory effect on A2780 ovarian cancer cell proliferation. Therefore, the generation of 20(S)-Rg3 was suggested to be involved in this effect. Moreover, the inhibitory effect of 20(S)-Rg3 on A2780 cell proliferation was significantly stronger than that of 20(R)-Rg3. Protein expression levels of cleaved caspase-3, caspase-8, caspase-9, and PARP in the A2780 ovarian cancer cells markedly increased, whereas the expression of BID decreased after 20(S)-Rg3 treatment. Therefore, we confirmed that the anticancer effects of the products of ginseng that was heat-processed with arginine are mediated mainly via the generation of the less-polar ginsenoside 20(S)-Rg3.Ginsenosides are the active components of The use of naturally occurring compounds for the design of novel anticancer drugs has increased in recent years, especially owing to increasing demand for natural remedies among cancer patients –4. ExperThe roots of ginseng contain dammarane-based saponins, which are composed of 1 to 4 glycosyl moieties combined with a dammarane backbone. The saponins that are present in high concentrations in ginseng are the major ginsenosides Rb1, Rb2, Rc, Rd, and Re  9, 10]., 10.9, 1 Panax ginseng extract via the Maillard reaction as well as elucidate its mechanism of action.Heat processing and enzyme-based methods are two major techniques that can achieve deglycosylation of ginsenosides . The Maiβ-actin and polyclonal antibodies against cleaved caspase-3, cleaved caspase-9, BH3-interacting domain death (BID) agonist, and poly (ADP-ribose) polymerase (PARP) were purchased from Cell Signaling Technology, Inc. .Ginsenoside standards Rb1, Rb2, Rc, Rd, Re, 20(S)-Rg3, 20(R)-Rg3, Rk1, and Rg5 were purchased from Ambo Institute . L-Amino acid standards alanine, arginine, glutamine, glycine, leucine, lysine, serine, trans-4-hydroxy-L-proline, and valine were purchased from Sigma Aldrich . Monoclonal antibodies against cleaved caspase-8 and P. ginseng) was purchased from a local ginseng market in Seoul (Korea). Dried ginseng was ground until it could pass through an 80-mesh sieve. It was then extracted under reflux with 50% EtOH three times at 70°C for 2 h and filtered through filter paper . The solvent was evaporated in vacuo to give a 50% EtOH extract with a yield of about 20%, by weight, of the original ginseng powder. The heat processing conditions were designed such that the total ginsenosides contained in the ginseng extract reacted with the same molar concentrations of amino acids. The mixtures were steamed together at 120°C for 3 h as reported previously [Four-year-old ginseng  was used to determine cell proliferation according to the manufacturer's recommendations. Briefly, cells were seeded in 96-well plates at a density of 1 × 104 cells/well and incubated for 24 h at 37°C. The cells were treated with different concentrations of compounds. After incubation for 24 h, 10 μL of the kit reagent was added to each well, and the cells were incubated for an additional hour. Cell proliferation was measured by scanning with a microplate reader at 450 nm. Control cells were exposed to culture media containing 0.5% v/v dimethyl sulfoxide (DMSO).The human ovarian cancer A2780 cell line was purchased from the American Type Culture Collection . The cells were grown in RPMI1640 medium  supplemented with 10% fetal bovine serum , 100 units/mL penicillin, and 100 2 was used as the nebulizing gas at a pressure of 2.0 bar. A Phenomenex Luna C18 column  was used, and the mobile phase comprised binary gradient of solvent A (acetonitrile : water : 5% acetic acid in water = 15 : 80 : 5) and solvent B (acetonitrile : water = 80 : 20) at a flow rate of 1.0 mL/min. The gradient flow program was as follows: initial 0% B; 6 min, 30% B; 18 min, 50% B; 30 min, 100% B; 37 min, 100% B; 42 min, 0% B.The analytical reversed-phase high-performance liquid chromatography (HPLC) system comprised a solvent degasser , binary pump , an autosampler , and model ZAM 3000 Evaporative Light Scattering Detector (ELSD) . ELSD conditions were optimized in order to achieve maximum sensitivity; the temperature of the nebulizer was set for 50°C, and NμL of a propidium iodide (PI) staining solution containing 40 μg/mL PI and 20 μg/mL RNase A in PBS. Then, the cells were incubated at room temperature for 30 min in the dark and analyzed with a FACSCalibur flow cytometer  and the ModFit LT version 2.0 computer program.To detect changes in cell cycle distribution, the treated cells were collected, washed with cold PBS, and fixed in 70% ethanol at 4°C for 30 min. The cells were then washed twice with PBS and resuspended in 500 5 cells) grown in 60 mm dishes were treated with the indicated concentration of samples for 24 h. Whole-cell extracts were then prepared according to the manufacturer's instructions using RIPA buffer  supplemented with 1x protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride (PMSF) [μg/lane) were separated by electrophoresis in a precast 4–15% Mini-PROTEAN TGX gel , blotted onto PVDF transfer membranes, and analyzed with epitope-specific primary and secondary antibodies. Bound antibodies were visualized using ECL Advance Western Blotting Detection Reagents  and a LAS 4000 imaging system .Cells (8 × 10e (PMSF) . ProteinP values of less than 0.05 were considered statistically significant.Statistical significance was determined through analysis of variance (ANOVA) followed by a multiple comparison test with a Bonferroni adjustment. In the present study, we developed a novel method of converting ginsenosides, the major active components of ginseng, and enhancing the anticancer effect of ginseng by high-temperature amino acid processing.P. ginseng contains a high concentration of ginsenosides.  Panax ginseng extract after heat processing with arginine and leucine. These heat processing conditions were selected because they generated the greatest total amount of less-polar ginsenosides and were stereospecific in their generation of Rg3 epimers ( epimers .The percent viability of A2780 ovarian cancer cells after treatment with ginseng extract (with leucine or arginine) and compounds (20(R)-Rg3 and 20(S)-Rg3) is shown in Dysregulation of apoptosis leads to various human pathologies including cancer , 25. Int P. ginseng extract via Maillard reaction was verified and its mechanism of action was further outlined. The heat-processed ginseng had abundance of ginsenosides Rg3, Rg5, and Rk1. After treatment with amino acids, the ginseng had a higher concentration of 20(S)-Rg3 and 20(R)-Rg3 and thus had an increased medicinal effect. We demonstrated that 20(S)-Rg3 suppressed the growth of A2780 ovarian cancer cells in vitro. Therefore, heat processing by Maillard reaction is a useful method to enhance the anticancer effect of ginseng by increasing the content of 20(S)-Rg3.In conclusion, the modulation of the anticancer effect of a


Bothrops and Bothriechis, have independently evolved arboreal tendencies. Little is known regarding the composition and activity of their venoms. In order to close this knowledge gap, venom proteomics and toxin activity of species of Bothriechis, and Bothrops (including Bothriopsis) were investigated through established analytical methods. A combination of proteomics and bioactivity techniques was used to demonstrate a similar diversification of venom composition between large and small species within Bothriechis and Bothriopsis. Increasing our understanding of the evolution of complex venom cocktails may facilitate future biodiscoveries.Central and South American pitvipers, belonging to the genera  Snake venoms are complex secretions of various enzymes, peptides and small organic molecules that have diverse modes of action on both prey and accidental human bite victims ,3,4,5,6.Bothriechis and Bothrops that have independently evolved to occupy arboreal niches. That Bothriechis is a distinctive genus from the “bothropoid” genera [Bothriopsis, have proven more difficult due to limited sampling of taxa. Numerous studies have presented evidence of the paraphyly of Bothrops with respect to Bothriopsis [Bothriopsis and Bothrocophias with Bothrops [Bothriechis [Bothriopsis and Bothrocophias should remain distinct [New World pitvipers are believed to be descendants of an ancestral Asian pitviper species, by way of a single New World colonization event, via the Bering land bridge ,62,63,64” genera  has been” genera ,66,67; hhriopsis ,70,71,72hriechis , while odistinct ,74,75 , sampling 90% of the known “bothropoid” taxa and utilizing both morphological characters and mtDNA sequence data found support for several monophyletic clades within Bothrops (sensu lato) and proposed taxonomic revisions, including recognition of Bothriopsis, based on the distinct species groups recovered [et al. (2012) [et al. (2009) [Bothrops with respect to Bothriopsis (with the inclusion of Bothrops sanctaecrucis), they proposed following the recommendation of Salomão et al. (1997) [et al. (2002) [Bothriopsis with Bothrops [et al. (1997) [et al. (2002) [Bothrops, as the weight of current evidence (cited above) favors this classification.Recent studies utilizing a combined morphological/molecular approach and increased sampling of taxa have disagreed on generic assignment as well. Fenwick ecovered . Carrasc. (2012)  incorpor. (2009)  in addit. (1997)  and Wüst. (2002)  and synoBothrops . We have. (1997)  and Wüst. (2002)  in recogCrotalus [Bothrops [Bothriechis, in particular B. schlegelii, venom [Bothrops and Bothriechis are both medically relevant species across their range and together are responsible for a large number of severe envenomations [2 (PLA2) [Despite the medical significance of pitviper envenomations in the New World ,81,82,83Crotalus ,88,89,90Bothrops ,103,104.i, venom ,106,107 i, venom ,110,111 i, venom ,115,116 i, venom ,123,124 omations ,126. Ame2 (PLA2) ,127. Due2 (PLA2) ,129. The2 (PLA2) . Determi2 (PLA2) ,131 and Bothriechis and Bothrops genera that have independently invaded the tree canopies of South America [Bothriechis aurifer, Bothriechis lateralis and Bothriechis marchi typically exceeding Bothriechis schlegelli in adult size, while Bothrops bilineata is a much smaller species than Bothrops taeniata. For comparison, we have included two terrestrial Bothrops species that also differ markedly in maximum adult size, with Bothrops asper reaching adult sizes in excess of twice that of Bothrops neuwiedi bolivianus. While B. lateralis and B. schlegelli have been proteomically previously profiled, as has Bothriechis nigroviridis [In this study we investigate the venom proteomes of arboreal members of the  America ,77,107. oviridis ,132, theShotgun mass spectrometry recovered proteins of known pitviper toxin types  in agreeBothriechis species  contained more P-III, P-II and P-I SVMP than the venom of the smaller sized species B. schlegelii . A similar correlation between body size and SVMP/PLA2 content was found in arboreal Bothrops. However this trend was reversed in the terrestrial Bothrops. These results were complemented by the 2D gel analyses  may present differential results. Indeed, further investigation of metalloprotease activity yielded results that contrasted those gathered with the aforementioned analyses , with B. aurifer and B. marchi having only weak activity while B. lateralis and B. schlegelli were very active  may be more potent in these two species. Similarly, despite both being rich in PLA2, the two terrestrial Bothrops species displayed significantly different levels of PLA2 enzymatic activity. Bothrops asper displayed high levels of PLA2 enzymatic activity, consistent with myotoxic envenomation effects, while Bothrops neuwiedi bolivianus was much less potent in this regard.PLAy active . This isBothrops species tested, SVMP levels are correlated with consumed prey class percentages, reported by Martins et al. (2002) [B. bilineata and B. taeniata) possessing lower levels of SVMP activity than species that contained non-volant mammals as a higher percentage of their diet (B. asper and B. neuwiendi). Pitviper venoms have been dichotomously classified into type 1 (proteolytic or “tenderizer”) and type II (more toxic) venoms [B. asper  in regards to B. neuwiedi  may be due to size class of mammal taken, as pitvipers are gape-limited predators that consume their prey whole [Crotalus atrox, was found as the best predictor of overall maximum gape [B. asper is recorded as consuming less percentage of mammals than B. neuwiedi, B. asper obtains an average SVL length roughly twice that of B. neuwiedi [B. asper does indeed consume larger prey/ prey of lower surface area to volume ratio than B. neuwiedi.For the . (2002) , with sp) venoms , as anurey whole ,135,136.mum gape . Althougneuwiedi , suggestneuwiedi ,138,139 Bothriechis are lacking. As a whole the genus appears to be a generalist with an ontogenetic shift toward mammalian prey, with B. lateralis and B. schlegelii even documented as occasionally predating on bats and small birds (Campbell and Lamar 2004 and references therein) [B. aurifer in relation to other members of Bothriechis. This increased level of SVMP activity in combination with Campbell and Lamar (2004) [B. aurifer may contain a higher percentage of non-volant mammals in its diet than the other members of Bothriechis tested. In contrast to high activity levels of SVMP recorded in B. aurifer, B. schleglii and B. lateralis both present higher levels of sPLA2 activity in relation to other members of Bothriechis. The increased levels of sPLA2 activity present in the venom profiles of B. lateralis and B. schleglii is suggestive of their arboreal encounters with volant animals. As sPLA2 is a neurotoxin affecting the nervous system, recruiting such a toxin would aid in quickly immobilizing flying prey whilst ambushing from a tree branch high up in the forest canopy, therefore reducing the risk of escape and injury.Detailed dietary studies for members of therein) . We deter (2004)  noting tBothrops and Bothriechis venoms has the ability to improve treatment for those receiving an envenomation [Detailed knowledge of venom components present in nomation ,140,141.nomation ,81,82,83nomation ,126.Further research into the identification and activity of toxins present in the venoms of snakes within this clade will be conducted in order to elucidate the selection pressures that have shaped these fine-tuned weapon systems. This research will hopefully contribute towards filling existing knowledge gaps concerning the evolution of venom composition and activity in the Viperidae.Bothriechis aurifer , Bothriechis lateralis , Bothriechis marchi , Bothriechis schlegelii (Costa Rica), Bothrops bilineata (French Guyana), Bothrops taeniata , Bothrops asper (Costa Rica), and Bothrops neuwiedi bolivianus (Bolivia).A series of 8 snake species were chosen due to their phylogenetic positioning, genetic relatedness, body size/length variation, and lack of existing knowledge of their venom composition for proteomic analyses. Pooled venom samples were collected from both from adult wild and captive specimens residing in private collections. Venom samples were collected by inducing the snake to bite a sterile container and collected venom was immediately stored on dry ice or in liquid nitrogen during transport. Once in the laboratory, samples were stored at −80 °C after lyophilization. Species investigated were Reduced 1D Tris-glycine SDS-PAGE methodology followed that of the study conducted by Ali et al., 2013 (cf. ). SpecifA sample of 125 µL solubilization buffer was added to a pre-washed sample of 300 µg of protein from required venom stock, prepared in a 15 mL Falcon tube. A solution of 0.75 µL of Bio-Lyte Ampholyte was added to the buffer and gently mixed. A sterile IPG strip  was then added to the Corning Falcon Conical tube  and allowed to absorb the solution overnight.IPG strips were removed from Falcon tubes and placed gel side up an Isoelectric Focusing (IEF) machine . Strips were covered with mineral oil and run overnight. Running conditions followed that of Ali et al., 2013 (cf. ).Required mini gels (7 cm × 6 cm) were cast using the same protocol for 1D Tris-glycine SDS-PAGE resolving layers (without the stacking layer). A layer of gel-overlay buffer (1 mL) was placed on top of the resolving layer once dry. A solution of 38 mg dithiothreitol (DTT) and 2.5 mL Equilibration buffer was added to each strip and gently mixed for 10 min. DTT solution was removed and a solution of 50 mg iodoacetamide (IAA) and 2.5 mL Equilibration buffer, gently mixed for 20 min.Gel-overlay buffer was removed and 1 mL liquid agarose was laid over the gel. IPG strips were then embedded into the agarose layer, positive end on the left. A molecular marker gel bit was then inserted on the negative end. Gels were then placed in the electrophoresis container with a combination of 10× electrode buffer and Milli Q (80:800 mL). Power pack was set at 20 mA for 60 min and run at room temperature. Once gels were finished they were removed from their glass cast and placed in 10 mL Coomassie brilliant blue stain, and set on a rocker overnight.In order to identify the types of toxins present in each gel, bands/spots from 1D SDS-PAGE glycine gels were picked, digested and analysed by LC-MS/MS. Visible protein bands were cut and stored at −80 °C. De-stain solution (500 µL 50 mM Ammonium bicarbonate (ABC)/50% Acetonitrile (ACN)) was added to each picked band/spot and left overnight. The destain was removed and repeated.Once blue stain had disappeared from picked bands, destain was removed. For 1D bands, a solution of 40 µL of 10 mM DTT was added to each band. Bands were then incubated at 60 °C for 30 min reducing disulphide bonds, and DTT removed. A solution of 40 µL of 55 mM IAA was added to the bands and incubated for 30 min at room temperature in the dark, alkylating free cysteine residues. Once IAA was removed, 100 µL of 50 mM ABC wash was added. Tubes were then spun down using a vortex for 2 min, ABC wash was removed and process repeated, washing away any excess IAA . Remaining IAA was removed, and 100 µL of 100% ACN was added and left for minimum 5 min. ACN dehydrates the gel pieces, turning them white. Once gel pieces had turned white, ACN was removed.Gel pieces were rehydrated with 8 µL trypsin, brought up in hydrochloric acid . This was left for 10–20 min at 4 °C. Depending on gel piece size, an additional 6 µL to 16 µL 50 mM ABC buffer was added. Gel pieces were kept moist overnight during digestion, incubated at 37 °C.Digested peptides were resuspended in 10 µL of 2.5% ACN/0.1% FA. Samples were filtered via ziptips using the following protocol; tips were wetted with 100% ACN (3 × 10 µL), and equilibrated with 10 µL 5% ACN/0.1% TFA, also repeated 3 times. Samples were loaded and pipetted up and down at least twice the volume of sample. Tip was washed with 5% ACN/0.1% TFA (3 × 10 µL). Samples were eluted with 80% ACN/0.1% TFA (10 µL) in a new tube. Combined samples were transferred into glass vials and diluted with 0.1% FA .For LC-MS/MS analysis, parameters are as follows; samples were separated using RP-chromatography on a Dionex Ultimate 3000 RSLC nano-system . Samples were desalted on a Thermo PepMap 100 C18 trap   for 5 min with a flow rate of 30 µL/min. This was followed by separation on an Acclaim PepMap RSLC C18  (150 mm × 75 µm) column at a flow rate of 300 nL/min. A gradient of 10%–70% buffer B over 7 min where buffer A (1% ACN/0.1% FA) and buffer B (80% ACN/0.1% FA) was used to separate peptides. Eluted peptides were directly analysed on an Orbitap Elite mass spectrometer  using an NSI electrospray interface. Source parameters included a capillary temperature of 275 °C; S-Lens RF level at 60%; source voltage of 2 kV and maximum injections times of 200 ms for MS and 150 ms for MS2. Instrument parameters included an FTMS scan across m/z range 350–1800 at 60,000 resolution followed by information dependent acquisition of the top 10 peptides across m/z 40-1800. Dynamic ion exclusion was employed using a 15 s interval. Charge state screening was enabed with rejection of +1 charged ions and monoisotopic precursor selection enabled. Data was converted to mascot generic format (mgf) using the msConvert software (ProteoWizard v2.0) and searched using Protein Pilot™ v5.0 (Sciex). Pre washed 10 µg protein venom samples, solubilised with 40 µL of Milli Q, was added to 5 µL of 1 M Ammonium Carbonate. A total of 55 µL per sample  was added, with a total combined volume of 100 µL. Samples were then incubated at 37 °C for 2 h, and then freeze-dried. Samples were re-suspended in 25 µL of 40 mM ammonium bicarbonate. Trypsin (1 ng) was added to each re-suspended sample and incubated at 37 °C overnight. Samples were dried and re-suspended in 40 µL 5% acetonitrile and 1% formic acid in preparation for LC-MS-MS analysis according to methods described in detail elsewhere .Freeze dried venom was reconstituted in a buffer containing 150 mM NaCl and 50 mM Tri-HCl (pH 6.3). Metalloprotease activity in venom (10 ng/µL) was measured by adding quenched fluorescent substrate . Fluorescence was monitored (excitation at 320 nm and emission at 405 nm) over 100 min, and rate of substrate cleavage calculated from a standard curve of known fluorophore (7-methoxycoumarin) concentrations. The specificity of metalloprotease activity was confirmed by incubating venom for 24 h at 37 °C with EDTA (50 mM).2, 0.02% Brij-35 pH 7.5, supplied) and equilibrated for 30 min at room temperature. Development buffer was replaced with renaturing buffer and incubated overnight at 37 °C for maximum sensitivity.Dried samples of 30 µg were prepared for each species and reconstituted in 15 µL of Bio-Rad Sample Buffer (#161-0764 supplied). Bio-Rad Zymogram PAGE ready-made 10% gelatin and 12% casein gels  were run using standard PAGE protocol at 100V for 90 min in 1× Tris-Glycine SDS Running Buffer . Gel was removed from case and incubated at room temperature for 30 min in 100 mL 1× Zymogram Renaturing Buffer . Renaturing Buffer was replaced with 100 mL 1x zymogram Development Buffer  2 and MMP 9 can be detected via 10% Zymography, while MMP 1, MMP 7, MMP 12, and MMP 13 are detected via 12% Zymography. By performing both Zymography tests with the identical venom concentrations under identical conditions, direct comparison between venoms can be made.2, 100 mM KCl, 0.3 mM Triton X-100). Positive control wells were prepared using 10 uL DTNB, 10 uL provided bee venom (100 µg/mL) and 5 µL Assay buffer. Sample wells consisted of adding 10 µL DTNB, 10 µL sample and 5 µL Assay Buffer. Reactions were initiated by adding 200 µL Substrate Solution (supplied) to all wells. Samples were prepared in a 96 well plate and run at A-405 nm every minute for 10 min using Fluostar Optima absorbance . Data was analysed using MARS data analysis software  and results were published using PRISM software graphs.Triplicate venom concentrations of 1 µg were prepared, along with the of positive control bee venom supplied with the Cayman 765001 assay kit. Triplicates were used instead of duplicates to strengthen the statistical power. Supplied methods consisted of non-enzymatic controls, positive controls and sample wells. Non-enzymatic controls were prepared using 10 µL DTNB  (supplied) and 15 µL Assay Buffer (25 mM Tris-HCl, pH 7.5, 10 mM CaCl


After 12 months, the control group presented a V'O2max and a gas exchange threshold significantly lower than the intervention group. Plasma POVC and PGPC were significantly different from healthy subjects before the intervention, but not after. In conclusion, SET was effective in improving cardiorespiratory fitness, cardiometabolic risk and oxidative stress status in T2DM.To evaluate the effects of supervised exercise training (SET) on cardiometabolic risk, cardiorespiratory fitness and oxidative stress status in 2 diabetes mellitus (T2DM), twenty male subjects with T2DM were randomly assigned to an intervention group, which performed SET in a hospital-based setting, and to a control group. SET consisted of a 12-month supervised aerobic, resistance and flexibility training. A reference group of ten healthy male subjects was also recruited for baseline evaluation only. Participants underwent medical examination, biochemical analyses and cardiopulmonary exercise testing. Oxidative stress markers  were measured in plasma and in peripheral blood mononuclear cells. All investigations were carried out at baseline and after 12 months. SET yielded a significant modification (p < 0.05) in the following parameters: V'O Physiological levels of reactive oxygen species (ROS) are important to maintain various cell functions, although an overload of ROS that exceeds the capacity of the antioxidant system can induce oxidative stressper se can improve blood glucose control and cardiovascular risk in T2DM81112Cardiorespiratory fitness is the ability to transfer oxygen from ambient air to skeletal muscle mitochondria during sustained exercise with large muscle groups, whose criterion measure is the maximal oxygen consumption (Cardiopulmonary exercise testing (CPX) is the preferred tool to assess cardiorespiratory fitness and it is increasingly being used in a wide spectrum of clinical conditions affecting exercise capacity917Previous studies on the effects of exercise in T2DM were based mostly on short-term interventions, with mean duration of 15–24 weeks917182021We conducted a clinical trial involving the Sport and Exercise Medicine Centre and the Diabetology Service, “Spedali Civili di Brescia” Hospital Trust, Hospital of Montichiari, Italy.To avoid confounding factors that could affect oxidative stress status and cardiometabolic risk, we selected only male subjects, aged between 40 and 70 years, nonsmokers and not taking antioxidant supplements. Twenty subjects with T2DM were recruited and randomized into an intervention group that performed supervised exercise training for one year  and a control group  that received standard medical care only. Specific eligibility criteria included BMI between 25 and 34.9 (overweight and grade I obesity), diagnosis of T2DMResearch protocol was approved by the Ethics Committee of Spedali Civili di Brescia (12 May 2010) and participants gave written informed consent. All experiments were performed in accordance with the Declaration of Helsinki.Investigations consisted of medical examination, biochemical analyses, systemic oxidative stress evaluation and CPX. C and SET groups were examined at baseline (T0) and after twelve months (T12), while H group only at T0.Medical examination was intended to rule out any contraindication to a maximal exercise test and to assess blood pressure, waist circumference, BMI and dietary habits. Dietary intake was studied in SET and C group with the European Prospective Investigation into Cancer and Nutrition food frequency questionnaireBiochemical analyses were performed at the laboratories of “Spedali Civili di Brescia” Hospital Trust at T0 and T12. Venous blood samples were obtained from each subject at 8:00 a.m after at least 12 hours fasting and 48 hours from the last exercise bout; morning doses of antidiabetic medications were discontinued. The following parameters were evaluated: fasting plasma glucose, triglycerides, total and HDL cholesterol , HbA1c  and serum insulin . Insulin resistance was calculated using the Homeostasis Model of Assessment - Insulin Resistance (HOMA-IR) formula [(serum insulin in mUI/l) × (fasting plasma glucose in mMol/l)/22.5]. LDL cholesterol was estimated by the Friedewald formula. In addition, metabolic syndrome severity score (Z-score) was calculated starting from individual components of metabolic syndromeThe research laboratories of the University Hospital of Verona, Italy, evaluated systemic oxidative stress by measuring oxidized forms of the phospholipid 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (ox-PAPC). The following different oxPAPC were taken into consideration: 1-palmitoyl-2--sn-glycero-3-phosphorylcholine (POVPC); 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC). Plasma and peripheral blood mononuclear cells (PBMC) concentrations of oxPAPC at T0 and at T12 were tested. After appropriate storing and carriage, samples were drawn into pyrogen-free blood collection tubes. Multiple aliquots of serum were placed into sterile 1-ml screw-capped polypropylene vials with phenolic antioxidant 2,6- Di-tert-butyl-4-methylphenol 10 mmol/L (Sigma) added to inhibit lipid peroxidation and stored at 280 uC. Samples were frozen and thawed only once. PBMC were isolated as previously describedGET) and at VCP (VCP).CPX was performed on an electro-magnetically braked cycle ergometer , with an incremental ramp protocol (20 W/min) until volitional exhaustion or an evident plateau in the All subjects with T2DM received standard medical care aimed at achieving optimal glycemic, lipid, blood pressure and body weight targets, as established by existing guidelinesGET. From the third month, training heart rates have been allowed to temporary increase above HRGET, gradually reaching but not overcoming HRVCP, in an interval-training fashion. Time per session has been increased progressively in the first 3 months, starting from 15 minutes and reaching the target of 35 minutes.SET group's training program consisted of 12 months of aerobic, resistance and flexibility training, according to the most recent guidelinesResistance training consisted of 40 to 50 minutes of different exercises involving the major muscle groups . Exercises consisted both in calisthenics and repetitions with ankle weights, dumbbells and elastic bands. Subjects began with 3 sets of 8 repetitions, then progressively improved to 3 sets of 12–15 repetitions. For exercises requiring dumbbells, weights started from 1–3 kg and were increased up to 2–6 kg depending on the subject and the type of exercise. Flexibility training was composed of static stretching exercises that involved upper and lower body, before and after the resistance training sessions.p < 0.05 was considered statistically significant.Baseline to end-of-study changes (expressed as mean ± standard deviation) were analyzed using Student's t-test for paired samples. To assess the statistical significance of differences between groups at each time point we used Student's t-test for independent samples. A value of Mean age ± standard deviation of SET, C, and H group was respectively 60.56 ± 5.94, 57.50 ± 9.46 and 54.6 ± 8.7 years, with no significant difference. Among subjects with T2DM, 13 (7 in SET and 6 in C) took metformin 1.5–2.5 g/day, 4 (1 in SET and 3 in C) took metformin 1.7 g/day plus pioglitazone 30 mg/day, 3 (2 in SET and 1 in C) took metformin 2 g/day plus glimepiride 3 mg/day. As a consequence of inclusion criteria, all patients in SET and C group took a statin and either an ACE-inhibitor or an angiotensin receptor blocker.−1·min−1 for both groups. H group had a higher maximal aerobic power in comparison SET and C group  and a higher p = 0.006 and p = 0.003 respectively). In all participants HRmax and maximal work rate corresponded to more than 85% of predicted value based on an age- and sex-matched population, concomitant exercise ECG was negative for ischemia and there was no arterial oxygen desaturation during effort. In SET group, mean HRGET was 116 bpm, while mean HRVCP was 129 bpm. Mean endurance exercise intensity prescription was 79% of the measured HRmax  for the first two months and between 79% and 92% of HRmax since the third month, when interval training was started. These values correspond to 69%–87% of the HRmax estimated with the formula 220 – age.Cardiometabolic risk, cardiorespiratory parameters and total energy intake did not differ significantly between SET and C groups, while in H group felt within the range of normality . Mean , p = 0.02/p = 0.01 and p = 0.01/p = 0.02 respectively). No statistically significant variation are detected in PBMC (p = 0.21/p = 0.34 and p = 0.51/p = 0.14).oxPAPC levels are shown in  in PBMC , but thep = 0.01), LDL cholesterol from 102 ± 30 to 81 ± 24 mg/dl , HOMA-IR from 8.22 ± 5.59 to 4.22 ± 3.19  and similarly fasting insulinemia from 8.22 ± 5.59 to 4.22 ± 3.19 uUI/ml . There was a trend towards lower body weight, BMI, HbA1c and metabolic syndrome severity score , reaching a weight-adjusted value of 23.8 ml·kg−1·min−1. Higher values of absolute and weight-adjusted p = 0.02 and p = 0.01 respectively) and p = 0.005 and p = 0.007, respectively) were also found, without a significant change in the corresponding HRGET and HRRC. Work rate at GET, work rate at VCP and maximal work rate significantly improved, while HRmax did not vary significantly (−1·min−1 to 19.1 ± 3.6 ml·kg−1·min−1 (−9.0%). SET group achieved an absolute and weight-adjusted p = 0.04) and 24.6% (p = 0.01) greater, respectively, compared to C group. SET group achieved also a higher weight-adjusted p = 0.03 for both) and achieved a significantly higher maximal work rate (p = 0.0042).For what concerns cardiorespiratory parameters, SET group showed significant improvements . Absolutficantly . C groupork rate . At T12,p = 0.03 and−31.6%, p = 0.04 respectively) respect to C who obtained a decrease of 10% or lower . A statistically significant decrease was not achieved in PBMC, but the trend was similar to that of plasma.For what concerns oxPAPC , SET groOur data confirms that subjects with T2DM present a more oxidizing environment than healthy subjects1233Since evidence for oxidative stress as a key mechanism altering insulin resistance has been widely discussed2p = 0.08), despite a significant improvement of HOMA-IR. This is probably due to the low number of subjects enrolled in SET group (n = 10), the mean baseline HbA1c value <7.0% , the decrease of ant-diabetic medications in two subjects and the absence of a specific nutritional intervention.Previous studies and meta-analyses showed that structured exercise training improves physical fitness in subjects with T2DM, along with a reduction of HbA1c9Despite a slight decrease of body weight was observed in all groups, this difference is not statistically significant, while a significant reduction in waist circumference has been observed (−1.5 cm). This is consistent with other studies, where an increase in fat free mass and a decrease in fat mass is reported without changes in total body weight−1·min−1). We want to emphasize that Our findings strengthen the theory that subjects with T2DM and overweight have decreased exercise capacity compared to healthy subjectsGET and HRVCP with CPX permits to bypass this effect, in analogy to what happens with beta blockers. A theoretical risk of sub-optimal endurance intensity prescription is in any case present in the later phases of the intervention, since our study lacks of an intermediate CPX to better adapt the progression of training to individual physiological adaptations.It is to be remarked that all subjects with T2DM were on metformin, a drug that has been found to blunt several effects of exercise, like improved cardiorespiratory fitnessAlong with cardiorespiratory fitness, increased strength plays an important role in intramuscular glucose and fat uptake and oxidation, contributing along with cardiorespiratory fitness to an increased insulin sensitivity and a decreased visceral fat in subjects with T2DMThe long-term compliance of SET group subjects deserves a special mention, as no one dropped out from the study. This might be possible thanks to a personalized approach, a protected training environment, the supervision of qualified exercise professionals and the gratuitousness of the service offered. In contrast, physical activity advice alone or theory-based intervention gathered only poor results22In conclusion, this study demonstrates that personalized and supervised exercise training of 1-year duration can affect positively insulin sensitivity and blood levels of LDL cholesterol. Increased cardiorespiratory fitness and a healthier body composition are probably the underlining causes. All these factors, with complex interactions, might have brought to a reduced systemic oxidative stress, as revealed by plasmatic oxPAPC. Moreover, the reduction of systemic oxidative stress may have had a positive feedback on insulin sensitivity. This provides new insights into the molecular mechanisms of non-pharmacologic treatment of chronic diseases.G.V. and C.M. wrote the manuscript. G.V., D.A. and E.B. designed and performed the clinical test on subjects. P.D. and D.A. selected and examined the subjects. L.B. and I.L. designed and supervised the exercise training program. C.M., A.P., C.R. and L.C. designed and performed the oxidative stress markers dosage. All authors reviewed the manuscript.


In subsets of both groups, drinking water was replaced with TJ (diluted 50:50 in water) from late gestation to necropsy. At P7d and P56d, we analyzed total antioxidant capacity (TAC), markers of oxidative stress (nitrotyrosine and heme oxygenase-1 expression), inflammation (interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) expression), collagen (COL) and smooth muscle in the lungs; we also assessed lung structure. We quantified macrophages in lung tissue (at P7d) and leukocytes in BALF (at P56d). At P7d, TJ increased pulmonary TAC and COL1α1 expression and attenuated the hyperoxia-induced increase in nitrotyrosine and macrophage influx; however, changes in lung structure were not affected. At P56d, TJ increased TAC, decreased oxidative stress and reversed the hyperoxia-induced increase in bronchiolar smooth muscle. Additionally, TJ alone decreased IL-1β expression, but following hyperoxia TJ increased TNF-α expression and did not alter the hyperoxia-induced increase in leukocyte number. We conclude that TJ supplementation during and after neonatal exposure to hyperoxia protects the lung from some but not all aspects of hyperoxia-induced injury, but may also have adverse side-effects. The effects of TJ are likely due to elevation of circulating antioxidant concentrations.Many preterm infants require hyperoxic gas for survival, although it can contribute to lung injury. Experimentally, neonatal hyperoxia leads to persistent alterations in lung structure and increases leukocytes in bronchoalveolar lavage fluid (BALF). These effects of hyperoxia on the lungs are considered to be caused, at least in part, by increased oxidative stress. Our objective was to determine if dietary supplementation with a known source of antioxidants  could protect the developing lung from injury caused by breathing hyperoxic gas. Neonatal mice (C57BL6/J) breathed either 65% O Due to improvements in neonatal clinical care, most preterm infants born after 25 weeks of gestation are now capable of survival. However, many preterm infants, especially those born before 32 weeks of gestation, are at high risk of developing bronchopulmonary dysplasia (BPD) , 2. BPD In spite of advances in the respiratory management of very preterm infants, the incidence of BPD has not significantly decreased . ApproxiAn important factor contributing to lung injury and altered lung development induced by neonatal hyperoxia is thought to be an increase in oxidative stress. PretermWe hypothesized that dietary supplementation with a range of antioxidants contained in commercially available TJ would protect the developing lung from the injurious effects of hyperoxia by increasing antioxidant capacity and decreasing oxidative stress; as oxidative stress and inflammation are intimately linked, and neonatal inhalation of hyperoxic gas has been shown to adversely affect pulmonary immune status , we propOur study was approved by the Monash University Animal Ethics Committee (project number: MARP/2011/061) in accordance with the National Health and Medical Research Council of Australia’s Code of Practice for the Care and Use of Animals for Scientific Purposes.Mus musculus) were randomly assigned to four treatment groups: 1. a group that breathed room air from birth (Air); 2. a group that breathed 65% O2 for 7 days after birth ; 3. a group that breathed room air from birth and whose diet was supplemented with TJ (Air+TJ); 4. a group that breathed 65% O2 for 7 days after birth and whose diet was supplemented with TJ (Hyp+TJ). In the hyperoxia groups, concentrations of O2 and CO2 were continuously monitored  within the housing chambers . Each litter was housed individually. The mice were provided with food and water (or TJ) ad libitum. Mice were health checked daily throughout the experimental period. If mice lost weight (~20% reduction in body weight), stopped eating and/or drinking, or displayed reduced physical activity, they were removed from experimental conditions and euthanized via cervical dislocation. No unexpected deaths were encountered. No invasive experimental procedures were performed in this experiment that required the administration of analgesics or anesthetics to ameliorate animal suffering. Within each treatment group, dams and litters were allocated into short-term (7 days) and long-term (56 days) survival studies. Mice are considered to have reached adulthood by 56 days.Time-mated pregnant C57BL6/J mice , 19 females (F); Hyp+TJ, n = 24: 11M, 13F). The Air and Air+TJ groups breathed room air from birth to P7d . Necropsy was performed at P7d following cervical dislocation and the lungs were collected for analysis.Mice in the Hyp and Hyp+TJ groups were exposed to 65% O2 from birth until P7d, after which they were raised in room air until P56d . The Air and Air+TJ groups breathed room air from birth to P56d . Mice were weaned at P21d. At P56d, necropsy was performed following cervical dislocation for collection of blood, bronchoalveolar lavage fluid (BALF) and the lungs.Mice in the Hyp and Hyp+TJ long-term survival groups were exposed to 65% Oad libitum access to the diluted TJ in place of drinking water. The diluted TJ was prepared daily and the volumes consumed were recorded daily; the daily volume of water consumed by the Air and Hyp groups was also recorded. For the short-term survival cohort, drinking water was replaced with the diluted TJ from E15d to necropsy at P7d. For the long-term survival cohort, the diluted TJ was provided from day 15 of pregnancy (E15d) to P56d. Before birth, offspring were presumed to have received antioxidants from the TJ via the placenta and after birth they received antioxidants via maternal milk or from drinking TJ.Starting at 15 days after mating , pregnant mice from the TJ groups were provided with commercially available TJ . According to the manufacturer, the TJ contained the antioxidants lycopene (59mg/L) and vitamin C (300mg/L). The TJ was diluted in water at a ratio of 50:50; the dams and offspring in the TJ groups had At necropsy at P56d, blood was collected by cardiac puncture and plasma stored at -80°C. Plasma and TJ samples were analyzed to determine concentrations of the carotenoids lycopene, α-carotene, β-carotene, β-cryptoxanthin and lutein/zeaxanthin by high performance liquid chromatography (HPLC), which was conducted in a darkened laboratory under red light at The University of Newcastle, Australia. Plasma samples from offspring in each treatment group were pooled to obtain a sufficient volume for analysis; therefore, only one value per group was obtained. Ethanol:ethyl acetate (1:1) containing the internal standard canthaxanthin was added to each sample, and each sample was then vortexed and centrifuged at 3000xg for 10 minutes at 4°C. The supernatant was removed and collected, and the above process repeated three times, the first two with ethanol:ethyl acetate (1:1) and the third with hexane. The supernatants collected from each centrifugation were pooled and ultra-pure water was added to the mixture, which was then vortexed and centrifuged. The supernatant containing the organic solvents was decanted and any remaining solvents evaporated with nitrogen. The sample pellets were reconstituted in dichloromethane:methanol (1:2 v/v) and chromatography was performed on a Hypersil ODS column (100mm x 2.1m x 5μm) with a flow rate of 0.3mL/min. Carotenoids were analyzed using a mobile phase of acetonitrile:dichloromethane:methanol (containing 0.05% ammonium acetate) (85:10:5 v/v) and a diode array detector (450nm) .in situ via the trachea (at 25cmH2O) with 4% paraformaldehyde (PFA) in 0.1M phosphate buffered saline . The lungs were then immersion-fixed with 4% PFA in 0.1M PBS (pH 7.4) for 24h, post-fixed in Zamboni’s fixative  for another 24h, processed in ethanol and xylene and then embedded in paraffin wax. At P7d, the numbers of animals in each group were: Air, n = 24 ; Hyp, n = 29 ; Air+TJ, n = 19 ; Hyp+TJ, n = 16 . At P56d, animal numbers in each group were: Air, n = 27 ; Hyp, n = 26 ; Air+TJ, n = 19 ; Hyp+TJ, n = 16 .At necropsy, lungs of offspring used for histological, immunohistochemical and immunofluorescent analyses were fixed Separate animals from each treatment group (n = 6–8 per group) provided samples of lung tissue that were snap-frozen in liquid nitrogen at necropsy and stored at -80°C for subsequent quantitative real-time polymerase chain reaction (qPCR) analyses and determination of total antioxidant capacity (TAC).Single sagittal sections (5μm) of both the entire left and right paraffin-embedded lungs were used for histological and immunohistochemical analyses. Sagittal sections (5μm) through the entire right paraffin-embedded lung were used for immunofluorescent analysis. For histological and immunohistochemical analyses, stained sections were examined by light microscopy . Color images were captured at a final magnification of x400 using a digital camera . Images were analyzed using image analysis software . For immunofluorescent analysis, stained sections were examined by fluorescent microscopy  and images captured with a digital camera  linked to image analysis software . Images were collected at a final magnification of x200. All analyses were conducted in a blinded manner by the same researcher (S.B).Nitrotyrosine staining within lung tissue was used as a marker for oxidative stress . NitrotyHO-1 (GenBank accession number NM_010442.2) [HO-1 mRNA levels were normalized against the housekeeping gene β-actin (GenBank accession number NM_007393.3) using the comparative delta CT (cycle threshold) method; at P56d, HO-1 mRNA levels were normalized against the housekeeping gene 18S rRNA (GenBank accession number NC_000072.6) [Heme oxygenase-1 (HO-1) is an inducible catabolic enzyme that is upregulated in response to a state of oxidative stress . Using f10442.2) . At P7d,00072.6) . Values 2+ ions to Cu+. The Cu+ ion is chelated with a colorimetric probe producing an absorbance peak at 560nm. Samples were assayed in conjunction with a series of standards, and TAC determined by interpolation of the standard curve.At P7d and P56d, the TAC of small antioxidant molecules including dietary antioxidants and uric acid was measured in samples of frozen lung tissue using a TAC assay kit, according to the manufacturer’s instructions . The assay measures the ability of a sample to reduce CuWe assessed macrophages in the lung as they play a key role in inflammation . MacrophWe measured the number and type of leukocytes in BALF as they are an indicator of pulmonary innate immunity. BALF was collected at necropsy via cannulation of the trachea and lavaging the airway lumen with 2 x 1mL of saline. Leukocytes within the BALF were isolated, prepared and stained with either 0.4% trypan blue solution or May-Grünwald-Geimsa for the determination of the total number of leukocytes per mL of BALF and differential counts of macrophages, neutrophils, lymphocytes and eosinophils, respectively .IL-1β and TNF-α gene expression as an indicator of pulmonary inflammation. Using frozen lung tissue collected at P7d and P56d, we performed total RNA extraction followed by cDNA synthesis and qPCR [IL-1β and TNF-α . At P7d, mRNA levels for the genes of interest were normalized against the housekeeping gene β-actin (GenBank accession number NM_007393.3) using the comparative delta CT (cycle threshold) method; at P56d, the mRNA levels were normalized against the housekeeping gene 18S rRNA (GenBank accession number NC_000072.6) [We measured and qPCR  to deter00072.6) . Values Lung tissue fraction and mean linear intercept  were measured using sections of lung parenchyma stained with Masson’s Trichrome. We used 30 fields of view per offspring (15 each from the left and right lungs) that were free of large conducting airways and major blood vessels. The tissue fraction and MLI were determined by superimposing test grids over each field of view .BM) ranging from 200–1000μm (approximate diameter: 64–318μm). Bronchiolar wall variables were expressed in relation to the PBM to adjust for differences in bronchiolar size [BA); both were expressed in relation to the PBM [BM [BM.We assessed bronchiolar wall structure as bronchioles are an important determinant of lung function  by inflular size . Wall stlar size  and the  the PBM . Bronchi the PBM . Gordon  PBM [BM . Immunoh PBM [BM . The areCOL1α1, COL1α2 and COL3α1, and the smooth muscle marker α-SMA . At P7d and P56d, the mRNA levels for the genes of interest were normalized against the housekeeping genes β-actin (GenBank accession number NM_007393.3) and 18S rRNA (GenBank accession number NC_000072.6), respectively, using the comparative delta CT (cycle threshold) method [Using frozen lung tissue collected at P7d and P56d, we performed total RNA extraction followed by cDNA synthesis and qPCR  to deter) method . Values treatment) and sex (psex) as factors. If the ANOVA showed statistical significance, a least significant difference post-hoc test was performed to identify differences between individual treatment groups. Comparison between treatment groups for the volume of TJ or water consumed over time was made using two-way repeated measures ANOVA. TAC and gene expression of IL-1β, TNF-α, COL1α1, COL1α2, COL3α1 and α-SMA were analyzed using a one-way ANOVA using treatment as a factor. Statistical significance was accepted at p<0.05.Numerical data are expressed as mean ± SEM. Statistical outliers were identified using Grubbs’ outlier test and removed if detected  . The ShaThe volumes of either TJ or water consumed by the dams between birth (P0d) and weaning (P21d) were not significantly different between treatment groups. The mean volume of liquids consumed by all mice (dams and offspring) over this period was 436.7 ± 59.5mL/day/kg body weight. Between weaning and P56d, the volume of either TJ or water consumed by the offspring was not significantly different between groups. The mean volume of liquids consumed in all groups over this period was 261.9 ± 24.0mL/day/kg body weight.The lycopene concentration in the plasma of the TJ groups was higher than in the non-TJ groups . The concentration of lycopene in undiluted TJ was 40mg/L; therefore the concentration that was consumed was 20mg/L. Based on the mean volume of liquids consumed by all mice, the dose of TJ was 8.7 ± 1.2mg/day/kg body weight prior to weaning and 5.2 ± 0.5mg/day/kg body weight after weaning. Plasma concentrations of other carotenoids assayed  were 0.0μg/L (i.e. undetectable) in all groups.2+ reduced) were: Air, 0.29 ± 0.01; Hyp, 0.31 ± 0.01; Air+TJ, 0.31 ± 0.01; Hyp+TJ, 0.34 ± 0.01 (2+ reduced) were: Air, 0.21 ± 0.01; Hyp, 0.21 ± 0.01; Air+TJ, 0.24 ± 0.01; Hyp+TJ, 0.25 ± 0.01, At P7d, the TAC of lung tissue was significantly greater in the Hyp+TJ group than in the Air group (p = 0.001), and there was a trend for it to be greater in the Air+TJ group than in the Air group (p = 0.096). Values (nmol/μl Cu4 ± 0.01 . At P56dHO-1 was significantly greater in the hyperoxia groups than in the normoxia groups (p<0.001); however, the relative gene expression of HO-1 was significantly lower in the Hyp+TJ group compared to the Hyp group ; it was significantly lower in the Air+TJ group than in the Air group (p = 0.032), and lower in the Hyp+TJ group compared to the Hyp group (p = 0.007). Values were: Air, 1.7 ± 0.1%; Hyp, 2.2 ± 0.2%; Air+TJ, 1.2 ± 0.2%; Hyp+TJ, 1.6 ± 0.2% . No sex The total number of leukocytes per mL of BALF was significantly greater in the hyperoxia groups than in the normoxia groups (p<0.01). There were 150% more leukocytes in the Hyp group compared to the Air group (p = 0.004) and 109% more in the Hyp+TJ group compared to the Air+TJ group (p = 0.006). Values were: Air, 9,238 ± 1,304 cells/mL; Hyp, 23,119 ± 3,453 cells/mL; Air+TJ, 12,371 ± 2,130 cells/mL; Hyp+TJ, 25,878 ± 4,054 cells/mL . No sex Enumeration of the different populations of leukocytes in BALF showed that there were no significant differences between treatment groups for the proportion of macrophages , lymphocytes  or neutrophils   and sex-treatment interaction (psex*treatment = 0.040) effects were observed, with females in the Air  and Hyp+TJ  groups having greater epithelial cell proliferation than males. No significant differences were observed between the sexes in the Hyp and Air+TJ groups. At P56d, the proportion of proliferating epithelial cells did not differ between treatment groups  and COL3α1 (p = 0.052) to be greater in the TJ groups compared to the Hyp group, but this did not reach statistical significance . The gene expression of COL1α1, COL1α2 and COL3α1 in lung tissue was not significantly different between groups at P56d  do not persist to adulthood, apart from a reduction in the number of bronchiolar-alveolar attachments in the Hyp+TJ group. Overall, our findings indicate recovery from the hyperoxia-induced inhibition of alveolarization by adulthood and suggest that TJ may adversely affect the ability of alveolarization to recover from early impairment. As previous studies have observed changes in lung function in adulthood following exposure to neonatal hyperoxia [Our findings on the effects of hyperoxia on lung tissue fraction, MLI and bronchiolar-alveolar attachments are consistent with previous studies , 38, 63.yperoxia , furtherOur findings suggest that TJ does not prevent hyperoxia-induced proliferation of bronchiolar epithelial cells at P7d. As the observed increase in bronchiolar epithelial cell proliferation was not accompanied by an increase in epithelial area, it is possible that the rate of epithelial cell apoptosis was increased, leading to increased cell turnover. At P56d, the proportion of ciliated epithelial cells was decreased in both of the TJ groups, suggesting that TJ alone, but not hyperoxia, could be causing this effect. Thus, the TJ supplemented mice may have had a reduced ability to move mucus along their airways.COL1α1 in lung tissue and bronchiolar collagen deposition were elevated in the TJ groups relative to the Air group, at P7d and P56d respectively. This TJ-induced increase in collagen was unexpected and could be detrimental to lung function as increased collagen is indicative of pulmonary fibrosis [The gene expression of fibrosis  and is ofibrosis .α-SMA at either P7d or P56d; this finding may have been because α-SMA deposition was only measured in the outer airway wall whereas α-SMA gene expression was measured in samples of whole lung tissue. The observed increase in ASM in adult mice following neonatal hyperoxia is consistent with previous studies [An increase in bronchiolar smooth muscle was observed in the Hyp group at P56d, relative to the Air and Hyp+TJ groups. This increased bronchiolar smooth muscle was not accompanied by an increase in the gene expression of  studies , 66, 67. studies  and is a studies . As our  studies , 70.Our study revealed differences between the Air and Air+TJ groups at P7d and P56d, suggesting that TJ alone may alter lung development. The potentially adverse effects of TJ observed at P56d may be due to the long duration of TJ ingestion. Our findings suggest that TJ alone can alter cellular and structural development of the lungs, including increasing lung tissue fraction and collagen deposition, and markers of oxidative stress and inflammation under physiological conditions. Therefore, it is plausible that a reduction in ROS resulting from TJ ingestion during normal physiological conditions could adversely affect lung development.We conclude that, although TJ does not ameliorate hyperoxia-induced alterations in lung architecture immediately following exposure, it has the beneficial effects of increasing TAC and attenuating the hyperoxia-induced influx of macrophages and increase in oxidative stress in the lungs. Importantly, TJ also protected hyperoxia-exposed lungs from a persistent increase in bronchiolar smooth muscle. However, TJ may alter the persistent pulmonary inflammatory response to neonatal hyperoxia, and it appears that TJ alone adversely affects some aspects of lung cellular and architectural development, redox balance and innate immunity, and may induce fibrosis. In view of the potentially adverse effects of TJ observed in our study, future studies could trial lower dosages, different dosing regimens or other routes of administration.S1 FigIL-1β (A) and TNF-α (B) in lung tissue. Data points represent values from individual animals. Values with the same letter are not significantly different from each other (p<0.05).mRNA expression of (PDF)Click here for additional data file.S2 FigA), mean linear intercept (B), the number of bronchiolar-alveolar attachments (C), the bronchiolar epithelial area (D), proportion of proliferating (E) and ciliated (F) cells in the bronchiolar epithelium, bronchiolar collagen content (G), and bronchiolar α-SMA content (H). Data points represent values from individual animals. Values with different letters are significantly different from each other (p<0.05).Morphometric analyses of tissue fraction ((PDF)Click here for additional data file.S3 FigCOL1α2 (A), COL3α1 (B) and α-SMA (C) in lung tissue. Data points represent values from individual animals. Values with the same letter are not significantly different from each other (p<0.05).mRNA expression of (PDF)Click here for additional data file.S4 FigA), mean linear intercept (B), the number of bronchiolar-alveolar attachments (C), the bronchiolar epithelial area (D) and proportion of proliferating (E) and ciliated (F) cells in the bronchiolar epithelium. Data points represent values from individual animals. Values with different letters are significantly different from each other (p<0.05).Morphometric analyses of tissue fraction ((PDF)Click here for additional data file.S5 FigCOL1α1 (A), COL1α2 (B), COL3α1 (C) and α-SMA (D) in lung tissue. Data points represent values from individual animals. Values with the same letter are not significantly different from each other (p<0.05).mRNA expression of (PDF)Click here for additional data file.


AbstractAnthuriumandraeanum Linden ex André, 1877, Monsteradeliciosa Liebmann, 1849, Philodendronscandens Koch & Sello, 1853, Spathiphyllumwallisii Regel, 1877, Syngoniumauritum  Schott, 1829 and Zantedeschiaelliottiana  Engler, 1915 within the monocotyledonous family Araceae (aroids) were performed. Chromosome numbers varied between 2n=2x=24 and 2n=2x=60 and the chromosome length varied between 15.77 µm and 1.87 µm. No correlation between chromosome numbers and genome sizes was observed for the studied genera. The chromosome formulas contained only metacentric and submetacentric chromosomes, except for Philodendronscandens in which also telocentric and subtelocentric chromosomes were observed. The highest degree of compaction was calculated for Spathiphyllumwallisii (66.49Mbp/µm). B-chromosome-like structures were observed in Anthuriumandraeanum. Their measured size was 1.87 times smaller than the length of the shortest chromosome. After FISH experiments, two 45S rDNA sites were observed in 5 genera. Only in Zantedeschiaelliottiana, 4 sites were seen. Our results showed clear cytogenetic differences among genera within Araceae, and are the first molecular cytogenetics report for these genera. These chromosome data and molecular cytogenetic information are useful in aroid breeding programmes, systematics and evolutionary studies.Karyotype analysis and FISH mapping using 45S rDNA sequences on 6 economically important plant species PageBreak Araceae (commonly known as aroids) are a very widely distributed monocotyledonous family. Most aroids are tropical and subtropical species while some members are growing in temperate regions. There are about 117 genera and 3300 species (The  species . The lea species . HoweverPageBreakin situ hybridization (FISH) and based on DNA sequence information, chromatin regions of individual chromosomes can be addressed  Schott, 1829 and Zantedeschiaelliottiana  Engler, 1915. These six species were chosen for their economic importance as ornamental species.In our study, flow cytometric analysis for genome size measurements, karyotype construction, and FISH mapping using 45S rDNA sequences were performed for the first time on the Anthuriumandraeanum ‘061’ and Spathiphyllumwallisii ‘Domino’ were present in the ILVO collection; Monsteradeliciosa ‘Variegata’, Philodendronscandens and Syngoniumauritum were obtained from the greenhouse of Tsitsin RAS Botanical Garden, Moscow, Russia; Zantedeschiaelliottiana ‘068’ was provided by Sandegroup, the Netherlands. The plants used in this study are known ornamental cultivars (no hybrids). The plants were grown in greenhouse conditions  in terracotta pots, filled with potting soil  and watered two days before collecting the root tips.Genome size analysis was performed according to Pisumsativum Linnaeus, 1759 ‘Ctirad’  Merrill, 1917, ‘Polank’  water overnight. Anthuriumandraeanum ‘061’, Monsteradeliciosa ‘Variegata’, Philodendronscandens, Syngoniumauritum, and Zantedeschiaelliottiana ‘068’ root tips were pretreated in a α-bromonaphthalin solution overnight at 4 °C. α-Bromonaphthalin solution was prepared dissolving 10 µL of α-bromonaphthalin in 10 mL water. After the pretreatment, the root tips were fixed in Carnoy solution (3:1 absolute ethanol-acetic acid) at least 1 h at room temperature. They were either used immediately or stored at -20 °C until use. The Carnoy solution was removed by washing the root tips three times in tap water for 20 minutes. The root tips were digested in a pectolytic enzyme mixture  in 10 mM citrate buffer  at 37 °C for 1 h. Chromosome preparations were made according to the spreading method of Actively growing root tips were collected. The root tips of EcoRI fragment of the 45S rDNA from Triticumaestivum Linnaeus, 1753  paraformaldehyde for 10 min at room temperature and air dried after sequential washes in 70% (-20 °C), 90% and 100% ethanol for 3 min each . DNA denAnthuriumandraeanum, Monsteradeliciosa, Philodendronscandens, Spathiphyllumwallisii, Syngoniumauritum and 10 metaphases for Zantedeschiaelliottiana using MicroMeasure  and mounted with Vectashield® . Slides were examined under a Zeiss Axio Imager microscope . Images were captured by AxioCam and Axiovision 4.6 software, Zeiss. Karyotype analysis was done on five well-spread, DAPI stained metaphases for oMeasure  for WindPageBreakPageBreakPageBreakZantedeschiaelliottiana, Philodendronscandens and Syngoniumauritum while for Spathiphyllumwallisii the largest genome size (7.39 ± 0.04 pg/1C) was observed. Monsteradeliciosa (2n=60) had the highest chromosome number. The lowest chromosome number was found in Syngoniumauritum (2n=24). The total chromosome length at haploid chromosome level varied between 147.14 ± 0.39 µm and 44.64 ± 0.52 µm. Compared to the other species Anthuriumandraeanum and Spathiphyllumwallisii had the biggest chromosomes. Philodendronscandens possessed the smallest chromosomes and was the only species that contains subtelocentric and telocentric chromosomes besides metacentric and submetacentric chromosomes  genetically related. Moreover, differences between parent plants concerning chromosome number, genome size and morphology of pairing chromosomes decide the fate of hybrid chromosome pairing during meiosis. According to PageBreakAnthuriumandraeanum is 2n=30  agree with the counts of Monsteradeliciosa compared to other Araceae plants, might be due to either an ancient polyploidization origin of the genus or a high basic chromosome number.The first things we noticed were different chromosome numbers and genome sizes among the six genera. The commonly known chromosome number for is 2n=30  which isdraeanum . An equaiphyllum  and confof 2n=32 ; 1971b.  numbers . Differe numbers . Our couAraceae genome sizes are described to vary between 0.33 , and 7.11 pg/1C for Spathiphyllum . For Zantedeschiaelliottiana, the total genomic content calculated in our study (1.35 pg/1C) was clearly higher than the 0.59 pg/1C mentioned by Pisumsativum L. ‘Citrad’ as the reference plant. This additional analysis confirmed Zantedeschiaelliottiana genome size as 1.30 pg/1C.Spathiphyllumwallisii ‘Domino’ had the largest genome size, the total chromosome complement was lower than in Anthuriumandraeanum and Monsteradeliciosa. Zantedeschiaelliottiana had a higher chromosome length than Philodendronscandens and Syngoniumauritum although its DNA content was lower. A direct correlation between total chromosome complement and genomic content is reported  Engler, 1930 Philodendronradiatum Schott, 1853, Piptospathaburbidgei  Hotta, 1965, Schismatoglottis Zollinger & Moritzi, 1846 and Typhonium Schott, 1829  is composed of GC rich tandem repeats . Generalelements .Araceae which is a valuable addition to the phylogenetic differences demonstrated by In Situ Hybridization (GISH) in Araceae. Finally, the physical localization of the rRNA genes can provide a first link between (future) physical and genetic maps, which can lead to a better understanding of the genetic structure of the Aroids.In conclusion, our results give a clear first view on the cytogenetic differences among six genera within


Moorella thermoacetica DSM 2955T, an acetogenic bacterium, which uses the Wood–Ljungdahl pathway for reduction of H2 + CO2 or CO. The genome consists of a single circular chromosome (2.62 Mb).Here, we report the complete genome sequence of  Moorella thermoacetica DSM 2955T, originally named Clostridium thermoaceticum, which was isolated from horse feces in 1942 , and therefore M. thermoacetica DSM 2955T was deposited in “Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH” (DSMZ) as the second type strain of the species. However, since isolation of M. thermoacetica, the strains DSM 521T and ATCC 39073 were used as model acetogens for elucidating the biochemistry of the Wood-Ljungdahl pathway (M. thermoacetica ATCC 39073.We determined the genome sequence of one of the type strains of de novo assembly was performed by employing the SPAdes genome assembler software 3.5.0 (M. thermoacetica ATCC 39073 (CP000232) as reference. Remaining gaps were closed by PCR-based techniques, primer walking, and Sanger sequencing of the products using BigDye 3.0 chemistry and an ABI3730XL capillary sequencer . For this purpose, the Gap4 (v4.11) software of the Staden package  and used to create Illumina shotgun paired-end sequencing libraries. A MiSeq system and MiSeq reagent kit v3 600 cycles) were used for sequencing as recommended by the manufacturer . Sequencing resulted in 2,908,542 paired-end reads (2 × 300 bp). Trimmomatic 0.32  is smaller than that of strain ATCC 39073 , but larger than that of the recently published strain DSM 521T   and 936 SNPs, respectively.The genome of Prodigal . Genes cProdigal  and tRNAProdigal , respectProdigal  was usedProdigal . The gen,564 bp) . It harbCP012370.This complete genome project has been deposited at DDBJ/EMBL/GenBank under the accession number


An intraparticle diffusion model showed that the adsorption process occurred in three phases, suggesting that a boundary layer adsorption phenomena slightly affected the adsorption process, and that intraparticle diffusion was dominant. The adsorption process was thermodynamically unfavorable and non-spontaneous; temperature increases improved phosphorus removal. Total organic carbon (TOC) assays indicated that chemical modification reduced the release of soluble organic compounds from 135.6 mg/L to 7.76 mg/L. This new information about adsorption performances provides valuable information, and can inform future technological applications designed to remove phosphorus from aqueous solutions.The renewable pine needles was used as an adsorbent to remove phosphorus from aqueous solutions. Using batch experiments, pine needles pretreated with alkali-isopropanol (AI) failed to effectively remove phosphorus, while pine needles modified with lanthanum hydroxide (LH) showed relatively high removal efficiency. LH pine needles were effective at a wide pH ranges, with the highest removal efficiency reaching approximately 85% at a pH of 3. The removal efficiency was kept above 65% using 10 mg/L phosphorus solutions at desired pH values. There was no apparent significant competitive behavior between co-existing anions of sulfate, nitrate, and chloride (SO Phosphorus plays a critical role in the development of ecosystems, agriculture, and industry, but also becomes a pollutant in water bodies , 2. To mBiosorbents are important tools for chemical recover and recycling. They are environmentally friendly, cost-effective, and easily available from industrial, agricultural, and other types of biomasses , 8. BiosModified media including ammonium-type chemicals and metal hydroxides (oxides). Modifying ammonium chemicals (e.g. animated intermediate  and ureaLanthanum hydroxides (oxides) are environmentally friendly materials  that hav3)3·6H2O) was used to prepare the adsorbent LH pine needles. Phosphorus stock solutions were prepared using potassium dihydrogen phosphorus (KH2PO4).All chemicals used for this study were analytical grade, purchased from Tianjin Fuchen Chemical; all solutions were prepared with distilled water. Lanthanum nitrate hexhydrate (La(NONaOH/V isopropanol = 1:1) for 24 hours. The soaked pine needles were then washed again with distilled water and dried overnight, again at a temperature of 353 K. The cooled AI pine needles were then set aside for further use.Pine needles were gathered from the woods (shown in 3)3, stirred for 1 h, and deposited for 5 h. An ammonia solution  was then added drop by drop to adjust the pH to 8–9. The samples were again deposited for 10 h, washed 5 times with distilled water, and dried at 353 K for 720 min.LH pine needles were synthesized using the following procedures: 20 g of AI pine needles were added to 250 mL 0.1 M La for 720 min at temperatures of 288, 298, and 308 K. This was done to evaluate the effect of initial phosphorus concentrations and to support isotherm studies.42-, CO32-, NO3-, or Cl- ions, individually. The release of soluble organic compounds (determined by TOC) was conducted by mixing 0.1 g pine needles, AI or LH pine needles with 5 mg/L phosphorus solution for 720 min.For kinetics studies, 1 g of LH pine needles was added to 500 mL of 5 mg/L phosphorus solution for 1,440 min. Samples of 2 mL were then collected at different time intervals . The effect of initial pH was studied by mixing 0.1 g LH pine needles with 10 mg/L phosphorus solution at pH values ranging from 3 to 10 for 720 min. Solution pH was adjusted with 0.1 mol/L HCl and NaOH. To start the effect of coexisting anions, 0.1 g LH pine needles were added into 5 mg/L phosphrous solutions, which contained 1, 5, 10, 50 or 100 mg/L of SOAll the experiments were carried out in triplicate.2CO3 and 1.7 mM of NaHCO3 was used as a mobile phase, at a flow rate of 1.2 mL/min.Surface morphologies and TOC analyses were obtained using a PHILIPS XL30 ESEM with an SUTW-Sapphire system and TOC-L CPH , respectively. Phosphorus concentrations were determined using a Metrohm 881 ion chromatograph coupled with a Metrosep A Supp4 column. A solution with 1.8 mM of NaThe concentration of residual lanthanum ions in the solutions after the adsorption process was determined. The residual solution was collected into the syringe and filtered with 0.45 μm filter disk into a proper sample tube. Concentration of lanthanum in the filtered solutions was analyzed by an inductively coupled plasma atomic emission spectrometer (ICP) .qe, mg P/g) and removal efficiencies (%) were determined using the following equations:Phosphorus adsorption capacities (qe (mg P/g) was the phosphorus adsorption capacity, V (mL) was the volume of the solution, Co and Ce (mg/L) were the concentrations before and after adsoption, and m (g) was the adsorbent mass.In these equations, The kinetics of phosphorus removal by LH pine needles were assessed using the following models , 35; Figqt (mg P/g) is the phosphorus adsorption capacities at time t (min); k1 (min-1) is the adsorption rate constant of the pseudo first-order kinetic model; k2 (g·mg-1·min-1) is the adsorption rate constant of the pseudo second-order kinetic model; Kint (mg·g-1·min-0.5) is the rate constant of intra-particle diffusion model; C is the diffusion constant.In these equations, qe(exp) (2.33 mg P/g) were very close to the calculated value qe (2.43 mg P/g). This suggests that phosphorus adsorption onto LH pine needles was done through chemisorption, or there was chemical bonding between adsorbent active sites [As ve sites .C = 0.175), indicating that the boundary layer adsorption had some effect on the adsorption process [The adsorbate species may be transported from the bulk solution onto the sorbents through intra-particle diffusion/transport processes ; as such process , 37.According to Al-Degs’ study , thermodΔG°, ΔH°, and ΔS° are the free energy of sorption (kJ/mol), the standard enthalpy change (kJ/mol), and the standard entropy change (J/(mol·K)), respectively; T (K) is the absolute temperature in Kelvin; R (8.314 J/(mol·K)) was the universal gas constant; Kd is the thermodynamic equilibrium constant; α is the adsorbent dose (g·L-1); ΔH° and ΔS° identify the slope and intercept of the linear plot of lnKdvs. 1/T, respectively.In these equations, qm as temperature increases. The ΔG° values decreased with the increasing reaction temperature, a result of the positive entropic contribution (ΔG° = ΔH°—TΔS°). Unexpectedly, positive values of ΔG° resulted. These results are different from previous research examining phosphorus sorption to lanthanum (hydr)oxides modified materials [aterials , 32, it aterials . This suThe adsorption isotherms were also calculated using Langmuir models; qm (mg P/g) was the theoretical maximum phosphorus adsorption capacity; b (L/mg) was the affinity constant.In this equation, qm values may not provide an accurate estimate of the long term sorption capacity, but could still be useful for comparing alternative materials. Compared with the other biomass sorbents listed in The Langmuir model fit the data very well (r > 0.97) at the three different temperatures. In the case of the LH pine needles, hydroxyl groups (originating from Lanthanum hydroxide and phenolic-OH or carboxylic groups of the LH pine needles) may react with phosphorus oxygen (P-O) to form monolayer adsorption , 41. At The pH value is important in adsorption, because it affects the species of phosphorus ions and the ionization of active functional groups on sorbent surfaces . As Fig 2PO4-, HPO42- and PO43- ions, depending on the pH of the bulk solution [2PO4- with a small amount of H3PO4. Monovalent H2PO4- may be strongly adsorbed onto LH pine needles by the intensive electrostatic interaction, or exchanged with hydroxyl groups arising from ionized Lanthanum hydroxide (La(OH)2+) deposited on the LH pine needles working as active sites. This could release OH- to the solution, leading to the increased pH value [2PO4- could be protonated to H3PO4, which may replace the neutral water molecule from the hydrated lanthanum ions loaded onto the LH pine needles [Phosphorus can exist in different ionic species, including Hsolution . At pH 3 needles .- brought about competitive adsorption, blocking the adsorption process and decreasing the removal efficiency. Meanwhile, lanthanum hydroxide protonation became weaker, and the lanthanum was in the insoluble form of La(OH)3 when the pH value was higher than 7.6. This state was unfavorable for adsorption and precipitation [- ion could complex with functional groups, such as cellulose, to form cellulose-OH- links [- concentrations and ligand exchange to promote adsorption and keep the final pH increasing slowly from 6.8 to 7.9, while the initial solution pH increased from 4.0 to 9.5. In turn, this process could keep removal efficiency over 65% in the alkaline region.This could explain why the removal efficiency was higher in the lower pH range. When the pH value increased to 9.5, the removal efficiency did not dramatically decrease; this is not consistent with other adsorption research using La-modified adsorbents , 26, 32.pitation . HoweverH- links . This wo42-, NO3-, Cl-, CO32- were chosen as interfering anions to test LH pine needle selectivity; As shown in 42-, NO3-, Cl- and achieve complete removal when the interfering anions are at concentrations of 100 mg/L. For CO32-, the removal efficiency clearly decreases as the CO32- concentration increases. This may be because the Ksp of La2(CO3)3 (3.98×10−34) is lower than that of LaPO4 (3.7×10−23) forming La2(CO3)3 sediment more easily, and decreasing phosphorus removal efficiency [Testing the adsorption selectivity of a new adsorbent is vital before applying in practical use. Here, SOficiency .The ICP analysis was used to determine the concentration of the loosely bound La in the treated solution. As expected, the concentration of residual lanthanum ions was very low. The concentration of residual lanthanum ions was 1.61, 0.32 and 0.05 mg/L when the initial concentration of phosphate solution was 5, 20 and 50 mg P/L, respectively, and the corresponding concentration of lanthanum was thought nontoxic and environment-friendly by previous studies , 48. ThiIn addition, a TOC analysis determined the organic material release in the phosphorus removal process. As In this study, AI pine needles showed a poor ability to adsorb low concentrations of phosphorus; LH pine needles showed higher removal efficiency. SEM photographs and EDAX analysis clearly showed that chemical treatment and LH precipitation resulted in many pores and adsorption site production. Sorption kinetics followed a pseudo first-order model and intra-particle diffusion model; the endothermic adsorption process could be divided into three phases. The phosphorus adsorption on LH pine needles showed a successful fit agaist Langmuir models. The maximum phosphorus capacity for LH pine needles was approximately 4.8 mg P/g at 298 K. LH pine needles demonstrated effective removal abilities across a wide pH range, with particularly strong abilities in the low pH range. There was only a slight inhibition of nitrate, sulfate and chloride, with the exception of carbonate in the co-existing anions competition tests. After chemical treatment, concentrations of released soluble organic compounds were significantly reduced.S1 Fig(DOC)Click here for additional data file.