Pinus strobus, Pinus resinosa, Pinus banksiana, Picea mariana, Picea glauca, Larix laricina, and Abies balsamea. The total phenolic content, as well as ORACFL potency and cellular antioxidant activity (IC50), were evaluated for all bark extracts, and compared with the standardized water extract of Pinus maritima bark (Pycnogenol), which showed clinical efficiency to prevent ROS deleterious effects. The best overall phenolic extraction yield and antioxidant potential was obtained with Picea glauca and Picea mariana. Interestingly, total phenolic content of these bark extracts was similar to Pycnogenol but their antioxidant activity were higher. Moreover, most of the extracts did not inhibit the growth of human skin fibroblasts, WS1. A significant correlation was found between the total phenolic content and the antioxidant activity for water extracts suggesting that these compounds are involved in the activity.The bark of boreal forest conifers has been traditionally used by Native Americans to treat various ailments and diseases. Some of these diseases involve reactive oxygen species (ROS) that can be prevented by the consumption of antioxidants such as phenolic compounds that can be found in medicinal plants. In this study, ultrasonic assisted extraction has been performed under various solvent conditions (water:ethanol mixtures) on the bark of seven boreal forest conifers used by Native Americans including:  Pinus strobus (white pine), Picea glauca (white spruce), Larix laricina (Larch tamarack), and Abies balsamea  was used. The bark was prepared as decoctions, infusions or poultices to treat gonorrhea, tuberculosis, diarrhea, pain, cold, kidney troubles, burns, inflammation, and rheumatoid arthritis , [25:75], [50:50], [75:25], [100:0]. Extraction mixtures were then filtered and dried under vacuum at room temperature for 3 days up to constant weight.NUNC). All manipulations were performed in a light shielded environment. After 5 min of reaction, 125 μL of sodium carbonate decahydrate solution (20 g/100 mL) was added to each well. Absorbance was then measured at 758 nm using an automated Varioskan Ascent plate reader (Thermo Electron). Analysis was performed in triplicate, and the results were expressed in gallic acid equivalents.The total phenolic content was determined using the Folin-Ciocalteu reagent according to the procedure reported by Singleton and Rossi , with soet al. , Picea glauca-[100:0] and Picea mariana-[75:25] were found the richest in phenolic compounds with respectively 61 ± 4 g, 59 ± 5 g and 55 ± 6 g per 100 g of extract. These values are similar to Pycnogenol. For the other tested species, the highest total phenol content measured in the extracts were in the following order: Pinus banksiana-[25:75] (45 ± 1 g/100 g), Larix laricina-[100:0] (34 ± 2 g/100 g), Abies balsamea-[50:50] (32 ± 2 g/100 g) and Pinus strobus-[75:25] (26 ± 1 g/100 g). The cytotoxicity of all bark extracts was evaluated on human skin fibroblast WS1. The cells were incubated in the presence or absence of growing concentrations of extracts for 24 h. In Larix laricina-[75:25] extract, do not inhibit cell growth in doses of 50 μg/mL and less, indicating that the extracts are not cytotoxic for WS1 in these dose ranges. For the Picea mariana and Pinus banksiana extracts, no cytotoxicity was found in doses as high as 100 to 200 μg/mL. Royer et al., reported no cytotoxicity of hot water extract of Picea mariana on normal keratinocytes at concentrations lower than 55 μg/mL [The bark of each conifer was extracted using sonication with five solvent conditions including water:ethanol [100:0]; [75:25]; [50:50]; [25:75]; [0:100]. The total extraction yields, presented in  g/100 g . Of all 55 μg/mL .Abies balsamea, Pinus banksiana, Picea mariana) has been reported previously by another research group which used different extraction methods such as hot water reflux and ethanol maceration  possesses the highest ORACFL value with 29 μmol TE/mg. According to the cell-based assay, the results show that the extracts of Pinus banksiana-[50:50] and [25:75], Picea mariana-[75:25] and Picea glauca-[100:0], have a higher antioxidant activity with an IC50 smaller than 0.4 μg/mL. The correlation between ORACFL and cell-based assay for all species is weak but significant (P < 0.05) with a coefficient of determination R2 of 0.24. This weak correlation between both methods can possibly be explained by the cellular context of cell-based assay which is not present in ORAC assay.The antioxidant activity of bark extracts was evaluated using ORAC and a cell-based assay. ORACl., 2009 . ResultsPinus banksiana, the water:ethanol extracts [50:50] and [25:75] possess the highest antioxidant activity in comparison with other conditions of extraction. The ORACFL values for Pinus banksiana-[50:50] and [25:75] are of 16 ± 3 μmol TE/mg and 14 ± 2 μmol TE/mg, respectively, and their IC50 are of 0.30 ± 0.07 and 0.3 ± 0.2 μg/mL. Both extracts contain higher gallic acid equivalent phenolic concentrations with 37 g/100 g for [50:50] extract and 45 g/100 g for [25:75] extract. The most active extract of Pinus resinosa is the water:ethanol [50:50] with an ORAC value of 29 ± 5 μmol TE/mg and an IC50 of 0.7 ± 0.1. This extract is very rich in phenolic compounds, with 61 ± 4 g GAE/100 g, suggesting that phenolic compounds are in part responsible for the antioxidant activity. However, the [75:25] extract is also rich in phenolic compounds (61 ± 5 g/100 g) but significantly less active than the [50:50] extract. For Pinus strobus, no significant difference was found between the three most active conditions which include the water:ethanol [75:25], [50:50], and [25:75], with ORAC values ranging from 5.0 to 8.5 μmol TE/mg and IC50 ranging from 0.91 to 2.7 μg/mL. The total phenolic content is significantly correlated with cell-based assay but not with ORACFL values. The extracts of Picea glauca and Picea mariana possess similar antioxidant activities, using both assays. Indeed, the ORACFL values ranged from 11 to 17 μmol TE/mg and the IC50 ranged from 0.3 to 0.73 μg/mL. The total phenolic contents of these extracts were also relatively high with concentrations ranging from 36 g to 59 g GAE/100 g. The Larix laricina-[75:25] possesses a significantly higher antioxidant activity in comparison with other conditions of extraction. For this extract, the ORACFL value is 16 μmol TE/mg and the IC50 is of 0.878 μg/mL. However, the total phenolic content for all conditions tested are not significantly different with values ranging from 27 ± 2 g GAE to 34 ± 2 g GAE/100 g. For Abies balsamea, the highest antioxidant activity was obtained with water:ethanol [50:50] according to cell-based assay with IC50 of 0.3 μg/mL. This extract is also significantly richer in phenolic compounds (32 g GAE/100 g) in comparison with other conditions of extraction. In contrast, the highest ORACFL value was obtained with a [0:100] extract (11 μmol TE/mg) which contained 20 g GAE of phenolic compounds by 100 g of extract.The antioxidant activity of bark extracts was analysed for each species in order to evaluate the best conditions of extraction. For FL and cell-based assay were analyzed.Altogether, these results indicate that the total phenolic content do not always correlate with antioxidant activity. Therefore, the relationship between total phenolic content and ORACFL and cell-based assay) was determined by a Pearson correlation analysis for all conifer barks and conditions of extraction. Significant but moderate correlations were found between phenolic contents and ORAC as well as phenolic contents and cell based IC50 results, with correlation coefficient of r = 0.745 (P < 0.001) and r = −0.670 (P < 0.001) respectively. Once the correlation was established between phenolic contents and antioxidant activities, their relation was described using linear regression analyses  and r = −0.9030 (P = 0.005) respectively. Both relations are shown by linear regression in Native Americans used water infusion of conifer bark to relieve various ailments and diseases . TherefoPicea glauca and Picea mariana extracts possess the most significant (P < 0.05) total phenolic content and antioxidant activities in comparison with other species. Garcia-Pérez et al., also reported that hot water extract of Picea mariana bark are richer in phenolic compounds and also more antioxidant than bark extracts of Abies balsamea and Pinus banksiana [et al., have isolated and characterized most bioactive polyphenols in hot water extract of Picea mariana bark including trans-resveratrol which has a strong antioxidant activity [Picea glauca and Picea mariana possess, in comparison with Pycnogenol, similar concentration in phenolic compounds but are about 2 to 3 times more active considering ORAC values and about 3 times more active considering cell-based assay. Pinus banksianna, Pinus resinosa and Larix laricina possess similar antioxidant activity to Pycnogenol. Finally, Abies balsamea reveals lower antioxidant activity and total phenolic content.Considering only water extracts, anksiana . In addiactivity . On the Picea mariana and Pinus banksiana were higher than those reported in literature using reflux and maceration extraction methods. Among the seven species studied, only bark extracts of Pinus resinosa, Picea glauca, and Picea mariana shown phenolic contents similar or higher than Pycnogenol, a standardized commercial extract from Pinus maritima bark. The best overall antioxidant activities with both cellular and ORAC assays were obtained with Picea glauca and Picea mariana, and appear to vary less in regard to extraction solvent conditions used. A good correlation between total phenolic content and antioxidant activity is found for all water extracts of barks, suggesting that phenolic compounds are responsible for the activity.In conclusion, ultrasonic extraction of barks of boreal forest conifers yielded extracts that are not toxic, rich in phenolic compounds and having a strong antioxidant activity. Ultrasonic assisted extraction yields and phenolic contents of bark extracts from


Streptococcus agalactiae were isolated from Nile tilapia (Oreochromis niloticus) farms in Thailand. Draft genome sequences of two clinical isolates comprise 2,048,343 and 2,105,006 bp, while environmental isolates comprise 2,097,115 bp, having 1,573 to 1,578 coding sequences, respectively.During 2009–2011, two clinical and one environmental strains of  Streptococcus agalactiae is the Gram-positive bacterial pathogen that produces serious disease in several mammalian and aquatic animal species (–Oreochromis niloticus), have been reported to be susceptible to S. agalactiae infection, causing tremendous economic loss  were collected from diseased Nile tilapia in 2009 and 2010, while another strain (ENC06) was recovered from water collected from an earthen tilapia pond in 2011. The genome sequencing process was carried out using the Illumina MiSeq platform with 251 paired-end run cycles . Once the sequencing process was accomplished, paired-end reads of poor quality were filtered out using the CLC Genomic Workbench version 6.9  and de novo assembly was conducted later. A total of 80 to 106 contigs with 25× average coverage were obtained after the assembly process. The order and orientation of contigs were determined by aligning to S. agalactiae strain GD201008-001 (accession number CP003810) using the Contiguator version 2.0 web-based service , 16S (n = 1), and 23S rRNA (n = 1) contained in the S. agalactiae genomes. Annotation of the genome assemblies was performed using RAST version 4.0 . The MLST version 1.7 web tool (Two strains of rain ENC0 was recoS. agalactiae strains FNA07, FPrA02, and ENC06 have been deposited at GenBank under the accession numbers JMBI00000000, JMBJ00000000, and JMBK00000000, respectively. The BioProject identification number is PRJNA244605.The draft genome sequences of


Skeletal muscle apoptosis and autophagy are catabolic processes that contribute to muscle atrophy during aging, disease, and following muscle injury. In this article, we present data on skeletal muscle apoptosis, autophagy, and morphology in C57BL/6 mice following doxorubicin administration. More specifically, time-course data on caspase-3, caspase-8, caspase-9, calpain, and cathepsin activity are presented, along with data on ATG7, p62, LC3-I, and LC3-II protein expression. Data on skeletal muscle reactive oxygen species (ROS) production, muscle morphology, as well as body and muscle weights are also presented. Specification TableValue of the data●Provides a simultaneous assessment of skeletal muscle apoptosis, autophagy, and morphology in response to systemic doxorubicin administration.●Provides time-course data on skeletal muscle morphology and degradative processes following doxorubicin administration.●Valuable for researchers interested in the relationship between apoptosis and autophagy in skeletal muscle.●Help other researchers determine the utility of doxorubicin as a myotoxic agent.1Here we present data regarding the ability of systemic doxorubicin administration to induce skeletal muscle atrophy and morphological changes. Given that apoptosis and autophagy can impact muscle morphology and wasting 1.1p=0.07) towards a significant decrease in body weight after doxorubicin administration (p<0.05) difference in gastrocnemius weight when normalized to body weight at day 1 compared to day 9  increase in LC3-I protein in soleus on day 3 post-doxorubicin injection compared to day 1 (p<0.05) higher on day 3 compared to day 0, 1, 5, and 9. There were no significant differences in LC3-I protein  showed no obvious signs of muscle damage or changes in muscle morphology at any time point after doxorubicin administration . Additio22.1n=3 for each time point) following a single intraperitoneal injection of doxorubicin at a concentration of 20 mg/kg body weight. This dose has previously been shown to induce atrophy, damage, oxidative stress, and apoptotic and autophagic signaling in skeletal muscle at 24 h or 48 h following administration Male C57BL/6 mice between the ages of 13–17 weeks were sacrificed by cervical dislocation prior to (i.e. day 0) and 1, 3, 5, 7, or 9 days  were extracted from both hindlimbs. A sample of the entire circumference from the middle portion of one soleus and one plantaris was mounted in Optimal Cutting Temperature (OCT) compound and frozen in liquid nitrogen-cooled isopentane. The remaining soleus and plantaris, as well as the mixed gastrocnemius, were individually frozen in liquid nitrogen and stored at −80 °C.2, 1 mM DTT, 20% glycerol and 0.1% Triton X-100; pH 7.4) containing protease inhibitors . Mixed gastrocnemius muscle samples used for caspase, calpain, and cathepsin activity assays were homogenized in lysis buffer but without protease inhibitors. Homogenates were then centrifuged for 10 min at 1000g at 4 °C and supernatants collected and stored at −80 °C. Mixed gastrocnemius samples for ROS assays were homogenized in ice-cold buffer  containing protease inhibitors . Total protein concentrations were determined by the BCA protein assay.Soleus and plantaris muscle samples used for immunoblotting were homogenized in ice-cold lysis buffer , Ac-IETD-AMC (Sigma-Aldrich), and Ac-LEHD-AMC (Enzo Life Sciences), respectively, as previously performed 2.4Reactive oxygen species (ROS) generation was assessed in mixed gastrocnemius using dichlorofluorescein-diacetate (DCFH-DA) (Life Technologies) 2.5Immunoblotting was performed using soleus and plantaris muscle samples as previously described 2.6Soleus and plantaris muscle cross-sections mounted on microscope slides were first stained with hematoxylin for 30 s, after which excess hematoxylin was quickly removed, and slides washed in distilled water. Cross-sections were then stained with eosin for 90 s, after which excess eosin was quickly removed. Slides were then treated with graded ethanol incubations, a xylene clearing step, and finally coverslips mounted with permount.2.7p<0.05 being considered statistically significant.All quantitative data was analyzed using 1-way ANOVAs and Tukey׳s post hoc tests, with


Aspergillus nidulans, we found that it forms a large complex with several other proteins that share weak but significant homology to known augmin subunits. In A. nidulans, augmin was enriched at the SPB and also associated with spindle MTs during mitosis. However, the augmin gene disruptants did not exhibit growth defects under normal, checkpoint-deficient, or MT-destabilised conditions. Moreover, we obtained no evidence that A. nidulans augmin plays a role in γ-tubulin recruitment or in mitotic cell division. Our study uncovered the conservation of the augmin complex in the fungal species, and further suggests that augmin has several functions, besides mitotic spindle MT nucleation, that are yet to be identified.Augmin is a protein complex that binds to spindle microtubules (MTs), recruits the potent MT nucleator, γ-tubulin, and thereby promotes the centrosome-independent MT generation within mitotic and meiotic spindles. Augmin is essential for acentrosomal spindle assembly, which is commonly observed during mitosis in plants and meiosis in female animals. In many animal somatic cells that possess centrosomes, the centrosome- and augmin-dependent mechanisms work cooperatively for efficient spindle assembly and cytokinesis. Yeasts have lost the augmin genes during evolution. It is hypothesized that their robust MT nucleation from the spindle pole body (SPB), the centrosome-equivalent structure in fungi, compensates for the lack of augmin. Intriguingly, however, a gene homologous to an augmin subunit (Aug6/AUGF) has been found in the genome of filamentous fungi, which has the SPB as a robust MT nucleation centre. Here, we aimed to clarify if the augmin complex is present in filamentous fungi and to identify its role in mitosis. By analysing the Aug6-like gene in the filamentous fungus  Drosophila or fungi, γ-TuC at the centrosome can be present in 2 forms: a smaller complex γ-TuSC consisting of γ-tubulin and GCP2/3 subunits, and a larger ring complex of γ-TuRC that additionally contains GCP4–6 and NEDD1 subunits Formation of microtubules (MTs) is essential for various cellular events such as mitosis, organelle transport, and cell growth. MTs are nucleated at multiple locations. In animal somatic cells, the dominant MT nucleation site is the centrosome, at which the potent MT nucleator, the γ-tubulin complex (γ-TuC), accumulates and serves as the seed for new MT formation Drosophila S2 cells, depletion of augmin was found to have an effect on mitotic spindle formation or chromosome segregation. However, the defect became more pronounced when centrosomal nucleation was co-inhibited by using RNAi against centrosomin, a γ-tubulin recruiter/activator at the centrosome; cells could no longer form a bipolar spindle or properly segregate chromosomes Caenorhabditis elegans and yeast have failed to identify genes homologous to any augmin subunits (Aug1–Aug8). Conceivably, these species have robust MT nucleation activity associated with the centrosome or spindle pole body (SPB), and the augmin-mediated mechanism is dispensable for preparing MTs for spindle formation, chromosome segregation, and cytokinesis. In fact, electron microscopy of the spindle of yeast or the filamentous fungus Ashbya gossypii indicated that all the spindle MTs originate from the SPB region Several studies have shown that augmin is particularly important for cell division in cells that lack centrosomes. In Interestingly, a BLAST homology search has identified an Aug6-like gene in the genome of various non-yeast fungal species, including many species of the filamentous fungi, in which spindle assembly is generally believed to be exclusively mediated by SPB-originated MTs, similar to that in yeast A. nidulans, a popularly used fungal species for cell division research A. nidulans. However, we have obtained no evidence that augmin recruits γ-tubulin or mediates MT-dependent MT nucleation in the spindle. Fungal augmin might have uniquely evolved and possibly plays a yet unknown role.In this study, we began with the characterisation of the Aug6-like gene in A. nidulans. To this end, we tagged GFP in frame to the C-terminus of the endogenous AN6187 gene, followed by time-lapse microscopy (We sought to determine the intracellular location of the Aug6-like protein (AUGF/AN6187) in croscopy . GFP sigcroscopy . As expeA. nidulans, we performed gel filtration chromatography using the Aug6-GFP strain. Interestingly, anti-GFP immunoblotting indicated that Aug6 is a member of a large complex of ≥8.5 nm Stokes radius, reminiscent of the animal or plant augmin ; these genes are conserved in many other fungal species, but as of yet have not been identified in yeast . To inveTo confirm that Aug3 and Aug6 are co-precipitated, a reciprocal immunoprecipitation was performed using asynchronous culture; Aug3-HA was immunoprecipitated with the anti-HA antibody, followed by mass spectrometry or immunoblotting. In this experiment, Aug6 was co-precipitated with Aug3-HA, whereas γ-tubulin was not , right.In the mass spectrometry and localisation analyses, we identified another protein (AN0286) that co-localises with Aug6-GFP at the mitotic SPB but does not show significant homology to known augmin subunits in other species . This geA. nidulans has the augmin complex consisting of at least 5 conserved subunits.From these results, we conclude that A. nidulans, we deleted the aug6 gene by using homologous recombination . We conclude that augmin is dispensable for γ-tubulin localisation to the SPB or spindle MT formation in the hyphae of A. nidulans.Augmin was identified as a γ-tubulin-localisation factor, and this feature has been conserved in every model system previously investigated 6Δ cells . Time-laA. nidulans, 3 outer subunits of the γ-TuRC  are also dispensable for γ-tubulin localisation to the SPB aug/mad2 disruptant. Surprisingly, we found no growth defect even in this SAC-deficient condition , a centrosome component required for γ-tubulin-mediated nucleation A. nidulans. We deleted the apsB gene, which is homologous to Drosophila Cnn and plays an important role in cytoplasmic MT nucleation aug6/apsB disruptant did not further exaggerate the phenotype . The genes homologous to these 4 are found in other fungal species (but not in yeast). We have identified another uncharacterised protein (AN0286) as being a likely augmin subunit, as it was co-precipitated with Aug3 or Aug6 and co-localised with Aug6 at the SPB. It should be noted that the amino acid sequences of the augmin subunits are highly divergent. Even between 2 metazoan species, flies and humans, no clear sequence homologies have been identified for a few subunits A. nidulans genome, which we failed to identify via the BLAST search. Despite the lack of significant amino acid identity, structural similarities have been proposed between fly and human augmin subunits A. nidulans. However, the possibility cannot be excluded that fungal augmin consists of fewer number of subunits.The presence of Aug6-like proteins in the fungal genome has been reported in a previous study A. nidulans augmin was also found to be enriched at the SPB and spindle during mitosis. However, in the current study, we could not identify these reported phenotypes. A conserved function of augmin in animal and plant cells is MT amplification via γ-TuRC recruitment aug6 and/or aug3 genes did not cause these defects in A. nidulans hyphae, even under sensitised conditions such as in the presence of a MT-destabilising drug or absence of Mad2 (md2A) or Cnn (apsB). This is in contrast to the case of outer subunit genes of the γ-TuRC; depletion of these γ-TuRC-specific subunits (GCP4–6) showed a synthetic growth phenotype with Mad2 A. nidulans. Interestingly, a structure-function analysis of augmin and γ-TuC in human cells suggested that the NEDD1 component of γ-TuRC is responsible for augmin association A. nidulans does not seem to possess this particular subunit of γ-TuRC The reported phenotypes of augmin in every animal and plant species are related to the mitotic cell division process, such as spindle formation or cytokinesis. A. nidulans cells? We think this possibility is less likely. For example, in Drosophila, prominent phenotypes were identified in the meiotic spindle in augmin mutants A. nidulans augmin plays a role in meiotic spindle formation and chromosome segregation. As another example, a chemical genomics analysis in budding yeast S. cerevisiae showed that nearly all genes (≥97%) are important for optimal growth in certain growth conditions A. nidulans. Alternatively, A. nidulans augmin might play a yet unidentified role, which was not detected in our assays using normal or benomyl-containing culture media. In this regard, it is noteworthy that although augmin is enriched at the centrosome during the interphase of human cells A. nidulans might have the advantage of identifying the unknown function of augmin, as null strains are available. For example, by identifying the condition under which the augmin disruptant specifically shows growth retardation , a clue to the unidentified augmin function in A. nidulans might be obtained. This approach would be difficult using RNAi-based studies in animal or plant cells.Do these data imply that augmin plays no role in A. nidulans, has uncovered the evolutional conservation of the augmin complex. It also implies functional versatility of the augmin complex and/or presence of the unidentified machinery redundant with augmin. Furthermore, our study presents a new and simple genetically/biochemically tractable model for studying functions or molecular structure of the augmin complex.In summary, this study performed using the filamentous fungus, The plasmids and PCR primers used in this study are listed in A. nidulans strains used in this study are listed in The A. nidulans germlings and hyphae were grown in complete medium  or synthetic minimal medium , supplemented with 1 g/L uridine, 0.525 g/L arginine, 10 mg/L biotin, and/or 2.5 mg/L pyridoxine ViB6. Conidia  were collected and stored in a solution containing 0.09% NaCl and 0.01% Tween-80.Using a modified version of a procedure described previously Genomic DNA for PCR or Southern hybridisation was prepared using NucleoSpin Plant Midi kits (Macherey Nagel). Southern blot analysis was performed using the AlkPhos Direct kits . Both procedures were performed according to the manufacturer's instructions. In some cases, PCR genotyping was performed using the ‘colony PCR’ method, in which conidia were picked out with a toothpick, suspended in TE buffer, and added to the reaction mixture.6 cells/mL) and cultured at 37°C for 12 h (with shaking at 150 rpm). Cells were washed with 0.8 M NaCl solution and flash frozen in liquid nitrogen, followed by extract preparation by grinding in the presence of the lysis buffer . The extracts were centrifuged , and the supernatant was incubated for 90 min with 50 µL agarose beads conjugated with anti-GFP (or HA) antibody . The beads were washed 3 times with the extraction buffer, followed by elution of the precipitated proteins with urea solution . The eluate was precipitated with TCA, followed by boiling in the SDS sample buffer. In one case, we omitted the elution process and added the SDS sample buffer directly to the beads. For smaller-scale immunoprecipitation, conidia were inoculated into 50 mL complete medium (3×106 cells/mL) and cultured at 37°C for 12 h. Mitotic cells (prometaphase or metaphase) were accumulated by treating cells with 1.2 µg/mL benomyl for 60 min, followed by drug washout and culturing for 2.5 min. Cells were then flash frozen with liquid nitrogen.For large-scale immunoprecipitation for mass spectrometry, conidia were inoculated into 500 mL complete medium (3×10A. nidulans protein database extracted from the Aspergillus Genome Database (AspGD: http://www.aspergillusgenome.org/).Immunoprecipitants were separated on a 10–20% gradient gel and size-fractionated by horizontally slicing the gel into 16 pieces. Each piece of the gel was digested with trypsin for the LC/MS/MS analysis, as described previously 5 conidia were inoculated into 300 µL minimal medium in 8-well, glass-bottom chambers (IWAKI), and incubated for 7 h at 37°C or 18 h at room temperature. For the second, more recently developed method, we inoculated 3×105 conidia onto 1 mL of agar medium in 3.5-cm cell culture dishes and incubated them for 7 h at 37°C. We then transferred the agar pad upside down onto 6-well, glass-bottom plates (IWAKI) on which 40 µL minimal media had been spotted. Imaging was performed at 23–25°C using a spinning-disc confocal microscope equipped with an EMCCD camera . Immunostaining was performed for hyphae grown on the 22×22 mm coverslips placed in the 3.5-cm cell culture dishes. Prior to fixation, 3×106 conidia were inoculated into 2 mL complete medium and cultured for 7 h at 37°C or 18 h at room temperature. Cells were fixed for 30 min at 37°C using 1.5 mL of 3.2% paraformaldehyde, 50 mM PIPES-KOH (pH = 6.8), 5 mM MgSO4, 5% DMSO, and 25 mM EGTA. The fixed cells were washed twice with PE Buffer , followed by cell wall digestion . Anti-α-tubulin  and anti-γ-tubulin  were used for staining, and the samples were imaged using a spinning-disc confocal microscope.Time-lapse imaging of the hyphae was performed using 2 methods. For the first method, 3×10g, followed by glass-fibre filtration (Millex-AP 0.20 µm). The cleared extract (1 mL) was fractionated using a Superdex 200 10/300 column attached to a Biorad BioLogicDuoFlow system, followed by immunoblotting using anti-GFP . The size marker used in the chromatography analysis was described in For gel filtration chromatography, whole-cell extracts of the Aug6-GFP strain (cultured in 50 mL for 12 h) was centrifuged for 20 min at 106,000×2, 1 mM EGTA) . Unpolymerised tubulins were removed by centrifugation at 106,000×g for 10 min at 30°C, and the MT pellet was resuspended with 80 µL MRB80 containing 20 µM taxol. A. nidulans extracts in 900 µL lysis buffer  were prepared from the 30-mL cultures. Protein aggregates were removed by centrifugation at 208,000×g for 20 min at 4°C. Taxol-stabilised MTs (20 µL) and the cell extracts (90 µL) were mixed for 15 min at room temperature, followed by centrifugation at 106,000×g for 10 min at 25°C. The supernatant and pellet were immunoblotted with the anti-GFP antibody .MT was formed by incubating 260 µg pig tubulins with 2 mM GTP, 1 mM DTT, 20 µM taxol in 200 µL of MRB80 buffer . Bar, 2 µm.(MOV)Click here for additional data file.Movie S2Time-lapse imaging of Aug6-GFP and mCherry-tagged Aug1, Aug2, Aug3, Aug4, or AN0286. Cells were imaged every 15–30 s using a spinning-disc confocal microscope . Location of the mitotic spindle is indicated by arrows. Granule signals are autofluorescence, which were detected regardless of the expression of GFP/mCherry. Bar, 2 µm.(MOV)Click here for additional data file.Movie S3Time-lapse imaging of mCherry-tubulin in WT and aug6Δ. Control and aug6Δ cells expressing mCherry-tubulin were imaged every 20 s using a spinning-disc confocal microscope . Displayed are the maximum projections of 5 z-section images (every 0.25 µm). Bar, 2 µm.(MOV)Click here for additional data file.


Acute lung injury (ALI) and its most severe form acute respiratory distress syndrome (ARDS) have been the leading cause of morbidity and mortality in intensive care units (ICU). Currently, there is no effective pharmacological treatment for acute lung injury. Curcumin, extracted from turmeric, exhibits broad anti-inflammatory properties through down-regulating inflammatory cytokines. However, the instability of curcumin limits its clinical application.In vivo, SD rats were pretreatment with c26 for seven days and then intratracheally injected with LPS to induce ALI. Pulmonary edema, protein concentration in BALF, injury of lung tissue, inflammatory cytokines in serum and BALF, inflammatory cell infiltration, inflammatory cytokines mRNA expression, and MAPKs phosphorylation were analyzed. We also measured the inflammatory gene expression in human pulmonary epithelial cells.A series of new curcumin analogs were synthesized and screened for their inhibitory effects on the production of TNF-α and IL-6 in mouse peritoneal macrophages by ELISA. The evaluation of stability and mechanism of active compounds was determined using UV-assay and Western Blot, respectively. a17, a18, c9 and c26, exhibited their anti-inflammatory activity in a dose-dependent manner and exhibited greater stability than curcumin in vitro. Furthermore, the active compound c26 dose-dependently inhibited ERK phosphorylation. In vivo, LPS significantly increased protein concentration and number of inflammatory cells in BALF, pulmonary edema, pathological changes of lung tissue, inflammatory cytokines in serum and BALF, macrophage infiltration, inflammatory gene expression, and MAPKs phosphorylation . However, pretreatment with c26 attenuated the LPS induced increase through ERK pathway in vivo. Meanwhile, compound c26 reduced the LPS-induced inflammatory gene expression in human pulmonary epithelial cells.In the study, we synthesized 30 curcumin analogs. The bioscreeening assay showed that most compounds inhibited LPS-induced production of TNF-α and IL-6. The active compounds, c26 has remarkable protective effects on LPS-induced ALI in rat. These effects may be related to its ability to suppress production of inflammatory cytokines through ERK pathway. Compound c26, with improved chemical stability and bioactivity, may have the potential to be further developed into an anti-inflammatory candidate for the prevention and treatment of ALI.These results suggest that the novel curcumin analog The online version of this article (doi:10.1186/s12931-015-0199-1) contains supplementary material, which is available to authorized users. Acute lung injury (ALI) is defined as acute inflammatory lung injuries associated with histopathological changes including neutrophilic alveolar infiltrates, impaired alveolar fluid clearance, fibrin deposition and lung edema. Despite advances in therapies, the outcomes of ALI in critically ill patients remain dismal with a morbidity and mortality rate around 40% -3. With Numerous pharmacological agents were investigated in an effort to attenuate the release of these pro-inflammatory cytokines involved in ALI. In pre-clinical experiments, these anti-inflammatory agents have demonstrated potent inhibitory effects on the release of inflammatory mediators and protective effects on ALI -10. Howein vitro and in vivo. However, the poor solubility and chemical instability of curcumin, under physiological conditions, limit its bioavailability and clinical efficacy [in vivo and in vitro [Curcumin, a natural product isolated from turmeric, has been found to have broad anti-inflammatory activities both efficacy -18. Curcefficacy -21. We pin vitro ,23. Recein vitro -26. Howein vivo experiments were performed in accordance with procedures approved by Wenzhou Medical University Animal Policy and Welfare Committee .Male ICR mice  and Sprague–Dawley (SD) rats  were obtained from Shanghai SLAC Laboratory Animal Center, CAS (SLACCAS). Mice were housed under specific pathogen-free conditions with a 12-hour/12-hour light–dark cycle and maintained on a normal diet at Wenzhou Medical University Animal Center. All mice and St. Louis, MO). Enzyme-linked immunosorbent assay (ELISA) kits of TNF-α and IL-6 were obtained from eBioscience, Inc. . Extracellular signal-regulated kinase (ERK), p-ERK and CD68 antibodies were purchased from Santa Cruz Biotechnology, Inc., . Antibodies of p-P38, P38, p-Jun N-terminal kinase (JNK), and JNK were obtained from Cell Signaling Technology, Inc., .LPS was purchased from Sigma  for 3 days before sacrificed for MPMs harvest. MPMs were then centrifuged and suspended in RPMI-1640 medium  supplemented with 10% FBS and 1% penicillin/streptomycin. The cells were incubated overnight at 37°C in a 5% CO2-humidified air.ICR mice were stimulated by intraperitoneal injection of 6% thioglycolate broth , compounds (10 μM), or DMSO (control) for 30 minutes, which was followed by the treatment of 0.5 μg/mL LPS. After treatment, the cells were incubated for 24 hours. The media were collected to measure the amount of TNF-α and IL-6 through the use of ELISA kit  according to manufacturer’s protocol. The total protein concentrations in viable cell pellets were measured. The amounts of TNF-α and IL-6 were normalized to the total proteins in cells.Curcumin or the active compounds were dissolved in DMSO (1 mM) and diluted with PBS (pH 7.4) to 20 μM. The absorbance spectra were taken from 250 to 600 nm at 25°C on SpectraMax® M5 . Absorbance spectral readings were recorded for over a time span of 25 minutes at 5 minute intervals.The macrophages harvested from mice were pretreated with the active compounds or DMSO (control) for 30 minutes and then incubated with 0.5 μg/mL LPS for 20 minutes. After treatment, protein samples were collected, separated by SDS-PAGE, and transferred to PVDF membranes. Membranes were incubated with a blocking solution of 5% non-fat milk for 1.5 h at room temperature. Proteins on membranes were then separately probed with the primary antibodies overnight. Protein samples were further incubated with horseradish peroxidase-conjugated (HRP) secondary antibodies for 1 h, and visualized using enhanced chemiluminescence reagents (Bio-Rad Laboratories).CUR + LPS group) and Group 4 (c26 + LPS group) received an intra-gastric administration of compound c26 (20 mg/kg/day) daily for seven days prior to the administration of LPS . Six hours after LPS administration, all animals were anesthetized by chloral hydrate and sacrificed. Broncho-alveolar lavage fluids (BALF) were collected for determination of total protein concentration and inflammatory cell infiltration. The lobes of the right lung were harvested for the study of curcumin and c26 on LPS induced lung injury analysis.SD rats were randomly placed into three groups . The rats were anesthetized and the tracheas were surgically exposed. Group 1 (CON group) received an intratracheal injection of saline. Group 2 (LPS group) received a drop-wise intratracheal injection of LPS , Group 3  in order to obtain BALF as previously described . The colThe right upper lobe of lung was excised. After removal of the excessive water on the tissue surface, the wet weight was recorded. The sample was then dried at 60°C for 48 h until no weight change to record the dry weight. The wet weight/dry weight ratio (W/D) was calculated and used as an index of lung edema.The right lower lobe of lung was excised and fixed with 4% formalin. The lung tissues were embedded with paraffin, sliced to 5 μm sections, and stained with hematoxylin and eosin (HE). Rat lung histopathology images were acquired using a microscope . The immunohistochemistry analysis was performed following the anti-CD68 antibody staining protocol.The lung injury was evaluated by Lung injury scoresas described previously . In brieTotal RNA was isolated from Beas-2B cells or lung tissue of rats with ALI using Trizol, then reversely transcribed to cDNA using M-MLV according to guidelines of the manufacturer. Gene specific primers used for TNF-α, IL-6, IL-1β, COX-2 and β-actin are listed in Additional file In vitro experiments were performed with n ≥ 3 independent repeats. In vivo experiments were performed with n ≥ 5 rats in each group.Data collected from experiments were analyzed using Graphpad prism 5.0 software. Values were expressed as mean ± SEM. One way ANOVA test was employed to analyze the differences between sets of data. A p-value of < 0.05 was considered as statistically significant and denoted as*. 1H-NMR. The detailed synthetic routes, synthetic yields, melting points, 1H-NMR, and ESI-MS analysis of novel and unpublished compounds are being described in Additional file In the present study, curcumin analogs were obtained from the Claisen–Schmidt condensation of substituted 3-phenyl-ketone and various aromatic aldehydes. The synthetic roots were listed in Scheme a9, a10, a17, a18, c9 or c26 (10 μM) for 30 minutes significantly suppressed the release of IL-6 with an inhibition rate of above 80%  markedly attenuated the expression of TNF-α , or DMSO (control) for 30 minutes, followed by the treatment of 0.5 μg/mL LPS. As shown in Figure a17, a18, c9, and c26 significantly inhibited LPS-induced TNF-α and IL-6 release in a dose dependent manner. All four active compounds showed better inhibitory activity than curcumin even at 5 μM. The most potent compound, c26 (5 μM), demonstrated potent inhibition (>90% inhibition rate) of IL-6 and TNF-α expressions induced by LPS. The results further validate that these compounds possess significant anti-inflammatory activity.The most active compounds, a17, a18, c9, and c26 in PBS (5% DMSO and pH 7.4) were measured at different time points (0–25 min). Figure Previous report showed that 90% of curcumin rapidly decomposes in the physiological buffer solution pH 7.4) which limits its bioavailability in clinical application . To exam which lic26 exerts its anti-inflammatory activity in vitro, we measured the effects of c26 on phosphorylation of ERK, JNK, and p38. Figure c26 (10 μM) for 30 minutes significantly inhibited the phosphorylation of ERK, however, it had little effect on the phosphorylation of JNK, and p38  down-regulated the phosphorylation of ERK in a dose dependent manner.Previous studies have shown that multiple signaling pathways are involved in stimulating the releases of inflammatory cytokines. One of the critical signaling pathways is the Mitogen-activated protein kinase (MAPKs) pathway, which is comprised by ERK, c-Jun amino-terminal kinases (JNK), and p38. To investigate the possible mechanism by which the representative compound 8 Figure A. Figurein vivo. Figure c26 (c26 + LPS). Lung wet/dry weight ratio is an index of lung edema. Figure c26 (c26 + LPS) reduced lung wet/dry weight ratio, while pretreatment with curcumin (CUR + LPS) showed no obvious effect. Figure c26 on the histopathological features of rat lungs with ALI. The control group shows normal structure of rat lung tissues. In the LPS group, remarkable alveolar wall thickness, hemorrhage, alveolar collapse, and inflammatory infiltration were observed in the lungs six hours after LPS administration  plays a pivotal role in the death of patients in intensive care unit. There is considerable experimental and clinical evidence indicates that inflammatory cytokines play a major role in the pathogenesis of LPS-induced lung injury ,30. A vaStreptococcus pneumonia [a17, a18, c9 and c26 showed a dose-dependent inhibitory activity (Figure TNF-α and IL-6 are important pro-inflammatory cytokines that can stimulate production of a host of other cytokines . They haneumonia . Previouneumonia -38. In oc26 exhibited no inhibitory ability on the phosphorylation of JNK and p38, however, it dose-dependently inhibited ERK phosphorylation induced by LPS (Figure c26 may be mediated by the down-regulation of the ERK signaling. The underlying mechanism of the anti-inflammatory action of c26 needs to be further investigated.After LPS incubation, TLR4 signaling was initiated, thus leading to the activation of NF-kB and MAPK . NF-κB iS Figure . These rin vivo study, the model of acute lung injury was induced by intratracheal instillation LPS. LPS significantly increased protein concentration and the number of inflammatory cells in BALF, pulmonary edema, pulmonary histopathological changes, inflammatory cytokines in serum and BALF, macrophages infiltration, inflammatory cytokine mRNA levels and inflammatory pathway (Figures c26 attenuated the increase of these markers induced by LPS through inhibited the phosphorylation of ERK. In summary, the most active compound, c26, exhibited its anti-inflammatory activity in vitro and attenuated LPS-induced acute lung injury by reducing inflammatory responses in vivo through ERK pathway. This article provides a potential compound for the treatment of acute lung injury.It has been reported that sepsis, especially gram negative bacteria infection, is the main cause of ALI/ARDS ,47. LPS, Figures , 7 and 8In vitro, most curcumin analogs showed better activity on LPS-induced production of TNF-α and IL-6 than C66. Active compounds, a17, a18, c9, and c26, exhibited their anti-inflammatory activity in a dose-dependent manner and showed high chemical stability in vitro. From the perspective mechanisms, compound c26 dose-dependently inhibited LPS-induced ERK phosphorylation in macrophages. In rat models with ALI, pretreatment with c26 significantly attenuated LPS-induced pulmonary edema, pathological changes, inflammatory cytokines in serum and BALF, inflammatory cell infiltration, inflammatory cytokine mRNA expression and ERK phosphorylation. This presents the possibility that curcumin analogs might serve as potential agents for the treatment of ALI. Although the anti-inflammatory mechanism and underlying targets are still unknown, the beneficial effects of these compounds on LPS-induced inflammation make c26 one of important leads in the continuing drug development and research.In conclusion, we designed and synthesized 30 curcumin analogs based on the structure of curcumin and C66.


Eliglustat is an investigational oral substrate reduction therapy for Gaucher disease type 1 (GD1). Its skeletal effects were evaluated by prospective monitoring of bone mineral density (BMD), fractures, marrow infiltration by Gaucher cells, focal bone lesions, and infarcts during an open-label, multi-site, single-arm phase 2 trial (NCT00358150).Institutional review board approval and patient informed consent were obtained. Eliglustat (50 or 100 mg) was self-administered by mouth twice daily; 19 patients completed 4 years of treatment. All were skeletally mature . DXA and MRI assessments were conducted at baseline and annually thereafter. X-rays were obtained annually until month 24, and then every other year.p = 0.02; n = 15) by a mean (SD) of 9.9 % (14.2 %) from baseline to year 4; corresponding T-scores increased significantly (p = 0.01) from a mean (SD) of −1.6 (1.1) to −0.9 (1.3). Mean femur T-score remained normal through 4 years. Femur MRI showed that 10/18 (56 %) patients had decreased Gaucher cell infiltration compared to baseline; one patient with early improvement had transient worsening at year 4. There were no lumbar spine or femoral fractures and no reported bone crises during the study. At baseline, 8/19 (42 %) patients had focal bone lesions, which remained stable, and 7/19 (37 %) patients had bone infarctions, which improved in one patient by year 2. At year 4, one new asymptomatic, indeterminate bone lesion was discovered that subsequently resolved.Lumbar spine BMD increased significantly (Eliglustat may be a therapeutic option for treating the skeletal manifestations of GD1. Skeletal complications are a major cause of morbidity in patients with Gaucher disease type 1 (GD1) . In GD1,Imaging of the skeleton provides a means of evaluating GD1 severity and monitoring responsiveness to treatment. On magnetic resonance (MR) imaging, areas of reduced bone marrow signal intensity (“dark marrow”) occur in T1-weighted sequences. Progressive accumulation of Gaucher cells displaces normal adipocytes from the marrow compartment , 8, leadTreatment of GD1 with enzyme replacement therapy (ERT) has been available since 1991. Enzyme replacement enhances the body’s ability to metabolize the glycolipid. It is effective, but costly, difficult to produce, and requires intravenous infusion. In 2003, miglustat , a substrate reduction therapy (SRT), became available for GD1 –12, but www.clinicaltrials.gov. The Ethics Committee or Institutional Review Board at each site approved the protocol. Patients provided written informed consent in accordance with the Declaration of Helsinki. Patient privacy was maintained in compliance with the Health Insurance Portability and Accountability Act. The main inclusion criteria were a diagnosis of GD1 confirmed by deficient acid β-glucosidase activity and Gaucher genotype, age 18 to 65 years, the presence of splenomegaly with anemia, and/or thrombocytopenia [The phase 2 trial was a multi-site, open-label, single-arm study sponsored by Genzyme, a Sanofi company, and registered as NCT00358150 at ytopenia –18. Patin = 2), pregnancy (n = 3), administrative withdrawal (n = 1), and bone lesion (n = 1). The patient with the bone lesion was withdrawn after the first year because of worsening osteonecrosis of the femoral head, which was asymptomatic and was detected retrospectively on screening images.Of 26 patients enrolled, six discontinued before or at year 1, and one patient discontinued at year 2 , 17. TheThe remaining 19 patients completed 4 years of treatment. Eliglustat capsules (50 or 100 mg) were self-administered twice daily; dose selection was based on day-10 plasma drug concentrations. Spleen volume, liver volume, platelet counts, and hemoglobin values were determined as previously described , 15. Pat2) and as T- and Z-scores [MR and dual-energy X-ray absorptiometry (DXA) images were obtained at study sites during screening and every 12 months during treatment. Plain radiography (X-ray) was done annually until year 2, and then every other year. All images were evaluated by two central reviewers who compared each result to the previous result to assess changes over time  and were not blinded to patients or timepoints . Images Z-scores . Anteriot test or Wilcoxon signed-rank test, as appropriate. To evaluate associations between the change in BMD T-score and changes in other clinical parameters, Pearson’s correlation coefficients were calculated. A p value of 0.05 was considered statistically significant.To evaluate differences between baseline and year 4, changes or percent changes from baseline were analyzed using a paired For the 19 study patients, the median age at initiation of treatment was 31.4 years. All patients were skeletally mature . The extent of splenomegaly, cytopenia, and skeletal defects at baseline was consistent with a moderate-to-severe stage of GD1 . There was dark marrow in all six zones in five patients and in at least three zones in the other 13 patients. The intertrochanteric region, shaft, and distal metaphysis were the most commonly affected zones. During 4 years of eliglustat treatment, 10/18 (56 %) patients showed improvement in one to six zones, while the other eight (44 %) patients remained stable. At year 4, one patient exhibited worsening within one of six zones that had previously improved; however, this patient resumed improvement in dark marrow 1 year later. The patients who responded with dark marrow improvements tended to be male and to have more zones with marrow involvement, larger spleen volumes, and lower hemoglobin levels at baseline. The dark marrow responders also showed greater improvements in spleen volume, hemoglobin levels, and platelet counts at year 4 than non-responders Table .Table 2CFigure 2) increased significantly (p = 0.02) from baseline to year 4 by a mean (SD) of 9.9 % (14.2 %) in the 15 patients with data. This increase in mean (SD) BMD from 0.99 (0.15) to 1.09 (0.19) g/cm2 corresponded to an increase in mean (SD) T-score from −1.6 (1.1) (osteopenia) to −0.9 (1.3) , and to an increase in mean (SD) Z-score from −1.17 (0.9) to −0.48 (1.1). Figure Lumbar spine BMD n = 7), proximal metaphysis (n = 3), distal metaphysis (n = 2), and condyles (n = 1), remained stable during treatment, and no new lesions developed in any patient. Bone infarctions were present in 7/19 (37 %) patients at baseline, with a total of 12 infarctions detected in the diaphysis (n = 6), distal metaphysis (n = 2), femoral head (n = 2), intertrochanteric region (n = 1), and condyles (n = 1). After 2 years of treatment, two (of four) infarctions improved in a 24-year-old patient. All other pre-existing infarctions remained stable.No existing  fractures or new (incident) fractures were detected in the spine or femurs of any patient at baseline or during 4 years of eliglustat treatment. Thirteen focal bone lesions were observed in the femurs of 8/19 (42 %) patients at baseline. These pre-existing lesions, located in the diaphysis  . For theMarrow infiltration, or “dark marrow,” was evaluated by MRI of both femurs. With treatment, the highest percentage of dark marrow reductions occurred in zones expected to be most recently infiltrated, the condyles and greater trochanter, whereas relatively few reductions were observed in the early infiltrated intertrochanteric region. Patients with marrow improvement tended to have more extensive marrow involvement, more severe splenomegaly, and lower hemoglobin levels at baseline than patients who did not show improvement Table . InfiltrImprovement in bone density occurred earlier than detectable marrow improvement. Eliglustat treatment produced substantial BMD gains in the first year, which were furthered or maintained through the fourth year. An increase in BMD was reported at 6 months for miglustat . The autr = −0.29; p = 0.29), but did correlate with changes in platelet counts  and spleen volume . The lack of association between marrow involvement and bone density has been previously reported [Of 15 patients with data for lumbar spine BMD, 12 were osteopenic or osteoporotic at baseline, and some T-score improvement was seen in all but three patients by year 4. T-score changes did not correlate with marrow improvement  Fig. , top. ThThe main limitations of this trial are the small number of patients, especially given the clinical heterogeneity of GD1; the uncontrolled study design of this proof-of-concept phase 2 trial; and the unblinded reviews of the bone images. However, this trial provides useful and objective information about the long-term effect of eliglustat on bone manifestations of Gaucher disease. Additional data from an ongoing phase 3 randomized, controlled trial in treatment-naive adults with GD1 (ENGAGE) will provide further insight into the effect of eliglustat on hematologic, visceral, and bone parameters.The results of radiologic assessments of bone suggest that eliglustat, an investigational oral SRT, may be a therapeutic option for treating the skeletal manifestations of GD1. An initial comprehensive radiologic evaluation of GD1 patients for marrow and osseous complications should form the basis for monitoring the effects of eliglustat treatment at regular intervals.


We found evidence for 13C-incorporation into DNA from 13C-xylose and 13C-cellulose in 49 and 63 operational taxonomic units (OTUs), respectively. The types of microorganisms that assimilated 13C in the 13C-xylose treatment changed over time being predominantly Firmicutes at day 1 followed by Bacteroidetes at day 3 and then Actinobacteria at day 7. These 13C-labeling dynamics suggest labile C traveled through different trophic levels. In contrast, microorganisms generally metabolized cellulose-C after 14 days and did not change to the same extent in phylogenetic composition over time. Microorganisms that metabolized cellulose-C belonged to poorly characterized but cosmopolitan soil lineages including Verrucomicrobia, Chloroflexi, and Planctomycetes.We explored microbial contributions to decomposition using a sophisticated approach to DNA Stable Isotope Probing (SIP). Our experiment evaluated the dynamics and ecological characteristics of functionally defined microbial groups that metabolize labile and structural C in soils. We added to soil a complex amendment representing plant derived organic matter substituted with either  Soil mi Chapin, . Despite Chapin,  and we s Chapin, .Acidobacteria, Chloroflexi, Planctomycetes, and Verrucomicrobia. These phyla combined can comprise 32% of soil microbial communities  links genetic identity and activity without the need of diagnostic genetic markers or cultivation and has expanded our knowledge of microbial processes  and structural C (cellulose) components of plant derived organic matter, to characterize aspects of their ecological niche, and to characterize the temporal dynamics of xylose and cellulose metabolism in soil.We added to soil a complex amendment representative of organic matter derived from fresh plant biomass. All treatments received the same amendment but the identity of isotopically labeled substrates was varied between treatments. Specifically, we set up a control treatment where all components were unlabeled, a treatment with −1 dry soil and 1.16 (± s.d. 0.13) mg N g−1 dry soil  and equilibrated at room temperature for 2 weeks until the respiration rate normalized (as determined by GCMS measurement of headspace CO2). Sieving causes a transient increase in soil respiration , 20% xylose, 3% arabinose, 1% galactose, 1% glucose, 0.5% mannose, 10.6% amino acids (Teknova C9795), and 2.9% Murashige Skoog basal salt mixture . The amendment had a C:N ratio of 10. This mixture approximates the molecular composition of switchgrass biomass with hemicellulose replaced by its constituent monomers  and lignin (1.2 mg lignin g−1 d.w. soil) were uniformly distributed over the soil surface as a powder and the remaining constituents were added in solution in a volume of 0.12 ml g−1 d.w. soil. The volume of liquid was determined in relation to soil moisture to achieve 50% water holding capacity. Water holding capacity of 50% was chosen, in relation to the texture for this soil, to achieve ~70% water filled pore space, which is the optimal water content for respiration  which is representative of natural concentrations in soil during early phases of decomposition  and xylose-C (0.42 mg C g−1 soil d.w.) in the amendment comprised 6 and 3% of the total C in the soil, respectively.We amended soils with a complex amendment representing plant derived organic matter substituted with either 12C Control Plant Simulant (12CCPS) which included only unlabeled components, (2) 13C-Cellulose Plant Simulant (13CCPS) which included 13C-cellulose  instead of unlabeled cellulose, and (3) 13C-Xylose Plant Simulant (13CXPS) which included 13C5-D-xylose  instead of unlabeled xylose. A total of 12 microcosms were established for the 13C-xylose and control treatments and 10 for the 13C-cellulose treatment. Microcosms were sampled destructively at days 1 (control and xylose only), 3, 7, 14, and 30 and soils were stored at −80°C until nucleic acid extraction. Isotopic analysis was performed at the Cornell University Stable Isotope Laboratory. While microcosm experiments are useful for evaluating microbial activity under defined experimental conditions, results from such experiments need to be verified in the field before drawing conclusions about microbial activity in the natural environment.We set up three parallel treatments varying the isotopically labeled component in each treatment  was used. Cellulose was produced in 1L Erlenmeyer flasks containing 100 mL HS medium inoculated with three colonies of G. xylinus grown on HS agar plates. Flasks were incubated statically in the dark at 30°C for 2–3 weeks. Cellulose pellicules were decanted, rinsed with deionized water, suspended in two volumes of 1% Alconox, and then autoclaved. Cellulose pellicules were purified by dialysis for 12 h in 1 L deionized water and dialysis was repeated 10 times. Harvested pellicules were dried overnight (60°C), cut into pieces, and ground using a 5100 Mixer/Mill , and dry sieved to 53–250 μm. The particulate size range was selected to be representative of particulate organic matter in soils.Bacterial cellulose was produced by  and Son  minimal E. coli is not able to use cellulose as a C source but is capable of growth on a variety of nutrients available in the HS medium. The biological assay consisted of E. coli inoculated into minimal M9 media which lacked a C source and was supplemented with either: (1) 0.01% glucose, (2) 2.5 mg purified, ground cellulose, (3) 25 mg purified, ground cellulose, (4) 25 mg purified, ground cellulose and 0.01% glucose. Growth in media was checked by spectrometer (OD450). No measurable growth was observed with either 2 mg or 25 mg cellulose, indicating absence of contaminating nutrients that can support growth of E. coli. In addition, the presence of 25 mg cellulose did not inhibit the growth of E. coli cultures provided with glucose (relative to control), indicating the absence of compounds in the purified cellulose that could inhibit microbial growth (data not shown). Purified cellulose was also assayed for residual proteins and sugars using Bradford and Benedict's assays, respectively. Bradford assay was performed as in Bradford . Reducing sugars were not detected in cellulose using Benedict's reducing sugar assay . Finally, 13C-cellulose had an average 96% ± 5 (s.d.) degree of 13C labeling as determined by isotopic analysis (UCDavis Stable Isotope Facility).The purity of ground cellulose was checked by biological assay, Benedict's reducing sugars assay, Bradford assay, and isotopic analysis. Bradford . Ground,−1 in 2 mL lysis tubes containing 0.5 g of 0.1 mm diameter silica/zirconia beads (treated at 300°C for 4 h to remove RNAses), 0.5 mL extraction buffer (240 mM Phosphate buffer 0.5% N-lauryl sarcosine), and 0.5 mL phenol-chloroform-isoamyl alcohol (25:24:1) for 1 min at 5.5 m s−1. After lysis, 85 μL 5 M NaCl and 60 μL 10% hexadecyltriammonium bromide (CTAB)/0.7 M NaCl were added to lysis tube, vortexed, chilled for 1 min on ice, and centrifuged at 16,000 x g for 5 min at 4°C. The aqueous layer was transferred to a new tube and reserved on ice. To increase DNA recovery, the pellet was back extracted with 85 μL 5 M NaCl and 0.5 mL extraction buffer. The aqueous extract was washed with 0.5 mL chloroform:isoamyl alcohol (24:1). Nucleic acids were precipitated by addition of 2 volumes polyethylene glycol solution  on ice for 2 h, followed by centrifugation at 16,000 x g, 4°C for 30 min. The supernatant was discarded and pellets were washed with 1 mL ice cold 70% EtOH. Pellets were air dried, resuspended in 50 μL TE and stored at −20°C. To prepare nucleic acid extracts for isopycnic centrifugation as previously described Buckley et al. , 13C-cellulose treatments , and control treatments . A total of 5 μg DNA was added to each 4.7 mL CsCl density gradient. Density gradient were composed of 1.69 g ml−1 CsCl ml−1 in gradient buffer solution . Centrifugation was performed at 55,000 rpm 20°C for 66 h using a TLA-110 rotor in a Beckman Coulter Optima MAX-E ultracentrifuge. Fractions of ~100 μL were collected from below by displacing the DNA-CsCl-gradient buffer solution in the centrifugation tube with water using a syringe pump at a flow rate of 3.3 μL s−1 , η is the measured refractive index, and a and b are coefficient values of 10.9276 and 13.593, respectively, for CsCl at 20°C . A total of 35 fractions were collected from each gradient and the average density between fractions was 0.0040 g mL−1. The DNA was desalted by washing with TE (5X 200 μL) in the Acroprep filter wells. DNA was resuspeneded in 50 μL TE.We fractionated DNA on density gradients for  Birnie, . The refn = 20 per gradient) and from non-fractionated DNA from soil. Barcoded primers consisted of: 454-specific adapter B, a 10 bp barcode, a 2 bp linker (5′-CA-3′), and 806R primer for reverse primer (BA806R); and 454-specific adapter A, a 2 bp linker (5′-TC-3′), and 515F primer for forward primer (BA515F). Each PCR contained 1.25 U μl−1 AmpliTaq Gold , 1X Buffer II , 2.5 mM MgCl2, 200 μM of each dNTP, 0.5 mg ml−1 BSA, 0.2 μM BA515F, 0.2 μM BA806R, and 10 μL of 1:30 DNA template in 25 μl total volume. The PCR conditions were 95°C for 5min followed by 22 cycles of 95°C for 10 s, 53°C for 30 s, and 72°C for 30 s, followed by a final elongation at 72°C for 5 min. Amplification products were checked by 1% agarose gel. Reactions were performed in triplicate and pooled. Amplified DNA was gel purified (1% low melt agarose) using the Wizard SV gel and PCR clean-up system  per manufacturer's protocol. Samples were normalized by SequalPrep normalization plates  or based on PicoGreen DNA quantification and pooled in equimolar concentration. Amplicons were sequenced on Roche 454 FLX system using titanium chemistry at Selah Genomics .SSU rRNA genes were amplified from gradient fractions , an RNA-Seq differential expression statistical framework  and Nearest Taxon Index (NTI) were calculated using the “picante” R package  but do not provide precise quantification of isotope incorporation. Note, that these 13C into DNA .We calculated the change in center of mass for buoyant density, s Figure . DNA buohttps://github.com/chuckpr/CSIP_succession_data_analysis. DNA sequences are available at the NCBI Short Read Archive (Accession SRP072785).All code to take raw SSU rRNA gene sequencing reads to final publication figures and through all presented analyses is located at the following URL: −1. The sequencing effort recovered a total of 5940 OTUs. 2943 of the total 5940 OTUs were observed in non-fractionated samples. We observed 33 unique phylum and 340 unique genus annotations. Analysis of SSU rRNA gene amplicons from non-fractionated soil DNA  and 265 samples had over 1000 sequences. We sequenced SSU rRNA gene amplicons from an average of 20 fractions per CsCl gradient (s.d. 0.57). The average density between fractions was 0.0040 g mLA Figure  indicate13C from xylose remained in the soil at day 30 . In contrast, the microbial community could not be shown to change with treatment  to conclude they changed in relative abundance in the non-fractionated DNA over time  were the Bacilli (decreased), Flavobacteria (decreased), Gammaproteobacteria (decreased), and Herpetosiphonales (increased) . Of the 29 OTUs that changed in relative abundance over time, 14 putatively incorporated 13C into DNA . The SSU rRNA gene sequences produced in this study were binned into 5940 OTUs and we assessed evidence of 13C-labeling from both 13C-cellulose and 13C-xylose for each OTU. Forty-one OTUs responded to 13C-xylose, 55 OTUs responded to 13C-cellulose, and 8 OTUs responded to both xylose and cellulose  of OTUs that responded to both cellulose and xylose .The phylogenetic composition of xylose responders changed with time Figures , 5 and m1 Figure . On day Proteobacteria (46%), Verrucomicrobia (16%), Planctomycetes (16%), Chloroflexi (8%), Bacteroidetes (8%), Actinobacteria (3%), and Melainabacteria , Beta (4 OTUs), Gamma (5 OTUs), and Delta-proteobacteria (6 OTUs). Most proteobacterial cellulose responders were closely related (>97% identity) to isolated strains, including representatives of the genera: Cellvibrio, Devosia, Rhizobium, and Sorangium, which are all known for their ability to degrade cellulose ] had higher relative abundance than cellulose responders [1.2 × 10−3 (s.d. 3.8 × 10−3)] . Consistent with this observation, seven of the ten most abundant responders were xylose responders, and six of the ten most abundant OTUs overall  were xylose responders.In the non-fractionated DNA, xylose responders [3.5 × 103 s.d. 3. × 10−3] 13C. Hence, the extent of 13C incorporation into DNA can be evaluated by the difference in BD between 13C-labeled and unlabeled DNA. We calculated for each OTU its mean BD weighted by relative abundance to determine its “center of mass” within a given density gradient. We then quantified for each OTU the difference in center of mass between control gradients and gradients from 13C-xylose or 13C-cellulose treatments ] was greater than that of xylose responders (0.0097 g mL−1 (s.d. 0.0094); Figure P = 1.8610 × 10−6, Wilcoxon Rank Sum test).DNA buoyant density (BD) increases in proportion to atom % rrn gene copy number for responders as described ] than xylose responders [6.2 (3.4 s.d.)]  while xylose responders clustered terminally . The consenTRAIT clade depth for xylose and cellulose responders was 0.012 and 0.028 SSU rRNA gene sequence dissimilarity, respectively. As reference, the average clade depth as inferred from genomic analyses or growth in culture is ~0.017, 0.013, and 0.034 SSU rRNA gene sequence dissimilarity for arabinase (arabinose like xylose is a five C sugar found in hemicellulose), glucosidase and cellulase, respectively  to Bacteroidetes (day 3) and finally Micrococcales (day 7). Notably, Paenibacillus have been previously shown by DNA-SIP to metabolize glucose . The phylogenetic position of Melainabacteria is debated but Melainabacteria have been proposed to be a non-phototrophic sister phylum to Cyanobacteria. An analysis of a Melainabacteria genome  phylogenetic clustering, and (4) density shift in response to 13C-labeling. Xylose was consumed before cellulose and these substrates were consumed by different microorganisms  time of activity, (2) estimated  time of  number,  phylogens Figure . rrn coping, and  density 13C directly from 13C-xylose or 13C-cellulose, and in many ways, knowledge of secondary C-transformations and/or microbial biomass turnover may be more interesting with respect to the soil C-cycle than knowledge of primary degradation. In the case of cellulose degradation, non-cellulolytic bacteria may gain access to 13C from 13C-cellulose via ecological interactions including mutualism, parasitism, predation, and ‘cheating’. Cheaters in particular, who benefit from the activities of cellulolytic extracellular enzymes without bearing the costs of producing these enzymes, have been hypothesized to impact rates of cellulose degradation  representing 84% of day 1 xylose responders , viral lysis, and/or predation.The response to Actinobacteria and Bacteroidetes xylose responders provides further evidence for C transfer by saprotrophy and/or predation. Certain Bacteroidetes have been shown to become 13C-labeled after the addition of live 13C-labeled Escherichia coli to soil , Chloroflexi, Bacteroidetes, and Planctomycetes. Spartobacteria in particular are globally cosmopolitan soil microorganisms and are often the most abundant Verrucomicrobia in soil (Bergmann et al., Microorganisms govern C-transformations in soil and thereby influence global climate but we still do not know the specific identities of microorganisms that carry out critical C transformations. In this experiment, cellulose responders included members of the CP conceived, developed and implemented data analysis and interpreted data, AC designed and performed the experiments, and both CP and AC performed data analysis, generated figures, and wrote and edited the manuscript. CP and AC contributed equally to the manuscript and should be considered co-first authors. CK and SB assisted with sampling and experimental analyses including development and implementation of the DNA sequencing pipeline. DB conceived the experimental design and supervised all aspects of experimentation, data analysis, and manuscript preparation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Tolypothrix campylonemoides VB511288, isolated from building facades in Santiniketan, India. The members of this genus produce several compounds of commercial importance. The draft assembly is 10,627,177 bases in 135 scaffolds, and it contains 7,886 protein-coding genes, 994 pseudogenes, 18 rRNA genes, and 76 tRNA genes.We report here the draft genome sequence of  Tolypothrix is a genus of aerophytic heterocystous cyanobacteria and belongs to the order Nostocales . Organisms belonging to the genus Tolypothrix produce several metabolites having commercial importance. Some Tolypothrix species can be directly used as biofertilizers  under 16-h light/8-h dark conditions with periodic (once a day) shaking. DNA isolation and purification were done using the UniFlex bacterial DNA isolation kit (Genei). A total of 4 µg of genomic DNA was provided for sequencing, of which 1 µg was used for shotgun sequencing and 3 µg for mate-pair sequencing.N50 of 1,097,626 bp. The largest and smallest scaffolds are 1,728,859 bp and 10,190 bp long, respectively, and the total G+C content is 48%.Genome sequencing was carried out using an Illumina HiSeq platform. Two different libraries were constructed, a paired-end library with an insert size of 300 bp and a mate-pair library with an insert size 3 kb in opposite orientations. Sequencing was done, generating 151-bp read fragments for the paired-end library, at 103× coverage , and 101 bp fragments for the mate-pair library, at 72.4× coverage . The raw reads from both libraries were filtered and preprocessed using the SGA (http://www.ncbi.nlm.nih.gov/genome/annotation_prok/). The total number of predicted genes was 8,794, out of which 994 are pseudogenes. There were 14 clustered regularly interspaced short palindromic repeat (CRISPR) arrays, 1 noncoding RNA (ncRNA) gene, 76 tRNA genes, and 132 frameshifted genes predicted from this assembly. Several antibiotic resistance genes , genes associated with the degradation of toxic compounds , and some nonribosomal proteins with antifungal activities  were annotated in this assembly. Several proteins, such as arsenic transporters, were found in the genome, which need to be further investigated for their role in bioremediation.The genome was annotated using the NCBI PGAAP (T. campylonemoides VB511288 genome sequence and annotation data have been deposited in GenBank under the accession no. JXCB00000000.The


Deletions on the distal portion of the long arm of chromosome 6 are relatively uncommon, and only a small number occurs in the paternal copy, causing growth abnormalities. As a result, extensive clinical descriptions are lacking.. Genetic evaluation by Single Nucleotide Polymorphism arrays showed a de novo 6q24.2-q25.2 deletion on paternal chromosome 6.We describe a male of Italian descent born at 35 weeks by elective caesarean delivery presenting hypoplastic left colon, bilateral inguinal hernia, dysplastic tricuspid and pulmonary valves, premature ventricular contractions, recurrent otitis media, poor feeding, gastro-oesophageal reflux, bilateral pseudopapilledema, and astigmatism. He also showed particular facial dysmorphisms and postnatal growth failure. Early psychomotor development was mildly delayed. At 3.75 years, he was evaluated for severe short stature (−2.98 SD) and delayed bone age. He showed an insulin-like growth factor 1 concentration (IGF-1) in the low-normal range. Growth hormone stimulation tests showed a low response to clonidine and insulin. Magnetic resonance imaging showed hypophyseal hypoplasiaWe confirm that this is a new congenital malformation syndrome associated with a deletion of 6q24.2-q25.2 on paternal chromosome 6. We suggest evaluating the growth hormone axis in children with 6q24.2-q25.2 deletions and growth failure. Interstitial and terminal deletions of the long arm of chromosome 6 have been known since 1975 –10.The variability of size and location of specific deletions and the lack of molecular mapping of breakpoints have made it difficult to establish genotype–phenotype correlations , even thWith one notable exception , all patde novo, paternal, interstitial deletions involving the 6q24.3 region; one of these children was a subject of a previous publication [Kumar et al. , and Nowlication . Some palication , 14. Thelication , which slication . While tde novo 6q24.2-q25.2 deletion on paternal chromosome 6 with growth hormone (GH) deficiency. We evaluated the association between the deletion and the patient’s symptoms and followed up with recombinant human growth hormone (rhGH) treatment. Finally, we compared previously published cases with deletions overlapping our patient’s . Two sisters of the propositus were growing in the target range.I-9V.The patient was born by elective cesarean delivery to a 29-year-old gravida after normal conception and 35 weeks of gestation. The foetus was suspected of IUGR from the 27th week. His birth weight was 1740 g (−1.6 SDS), length was 43.5 cm (−1.1 SDS), and head circumference was 32.2 cm (0.0 SDS). The child’s Apgar score was 7Prenatal ultrasounds also showed apparent cardiomegaly, which was not confirmed postnatally. Following amniocentesis, the karyotype was found to be 46,XY. Microsatellite analysis of chromosomes 2, 11, and 16 showed biparental inheritance, excluding uniparental paternal disomy.As a newborn, the propositus was hospitalized for hypoplastic left colon, which caused constipation in the first few months of life.Early psychomotor development was mildly delayed. He rolled over at 5 months, sat alone and pulled to stand at 10 months, said his first words at 18 months, walked alone at 19 months, and said 25 single words at 2 years of age, but used no sentences.He was referred for genetic evaluation at age 13.5 months because of his poor growth. His medical problems included dysplastic tricuspid and pulmonary valves with mild regurgitation, mitral valve prolapse, chronic otitis media, poor feeding, and gastro-oesophageal reflux. Ophthalmoscopic examination showed bilateral pseudopapilledema and hyperopic astigmatism. He showed a relative macrocrania with particular facial features such as prominent forehead, epicanthic folds, upslanting palpebral fissures, large ears and broad nasal bridge , his weight was 11.500 Kg (−3.34 SDS), his body mass index (BMI) was 14.78 (−0.81 SDS), and his occipitofrontal circumference was 50 cm (0.33 SDS)  and 1.3 μg/ml  for IGF-1 and IGFBP-3, respectively.At 5 years 8 months of age, his height was 96 cm (−3.66 SDS), his weight was 13.500 kg (−3.67 SDS), and his BMI was 14.65 (−0.91) Fig. . A blood. Neuropsychological evaluation showed that deficits persisted, particularly in auditory-language association, auditory pattern recognition, and interhemispheric integration.At 6 yrs of age, an echocardiogram showed a bicuspid aortic valve. A magnetic resonance imaging (MRI) scan of the brain showed peritrigonal T2 white matter hyperintensity, in the absence of midline anomalies or anatomic defects of the central nervous system. The hypothalamic-hypophyseal region also showed pituitary hypoplasia with a cyst in the pars intermediaBecause of the persistent and severe growth failure, the child was evaluated with GH stimulation tests, which showed a low response after clonidine (GH peaked at 4.46 μg/l) and insulin (GH peaked at 3.14 μg/l) tests. Consequently, GH therapy (0.23 mg/kg/wk) was started.The child experienced a good response to GH therapy Fig. . At 7 yeHis pubertal development started normally: at 11 years 4 months, his height was 137.6 cm −1.27 SDS), his weight was 31.000 kg (−1.38 SDS), his BMI was 16.4 (−1.04 SDS), and his pubertal staging was: testicular volume of 5–6 ml bilaterally, PH2, and AH2  of the UCSC Genome Browser, this analysis showed a deletion of approximately 10,741 Mbp that involved the 6q24.2q25.2 region, with the breakpoint falling between 143,297,976 bp (first deleted oligomer) and 154,039,064 bp (last deleted oligomer) Fig. . HoweverWe described a patient with interstitial deletion of the chromosomal region 6q24.2-q25.2 on paternal chromosome 6, consistent with the observations of Kumar et al.  and NowaInterstitial and terminal deletions of chromosome 6q are rare disorders, commonly associated with intellectual disability, dysmorphic features, growth failure, and medical complications , 5, 11.Paternal interstitial deletions at 6q24.2-q25.2 are rarely described, but some patients can share significant IUGR and postnatal growth retardation, redundant skin, joint laxity, and strikingly similar facial features, associated with early developmental delay , 14. We Our case report strongly suggests that an impairment of the GH-IGF-1 axis is a possible cause of short stature in this syndrome. Therefore, these patients should be tested for GH secretion.Interestingly, Nowaczyk et al. reported that one in three patients with paternal deletion of 6q24.3 showed reduced IGF-1 and IGFBP-3 secretion but subnormal GH secretion after GH provocative testing  . In thisPLAG1  or ZAC1 (the zinc finger gene involved in apoptosis), a gene member of the network of coregulated genes comprising other imprinted genes involved in the control of embryonic growth [PLAGL1 is an imprinted gene, paternally expressed. Generally, in mouse inactivation of the maternally repressed Plagl1 transcription factor results in intrauterine growth restriction, altered bone formation, and neonatal lethality [PLAGL1 may be associated with foetal and post-natal weight and BMI but not with length [A 6q24.3 deletion of paternal origin is frequently found with c growth . PLAGL1 ethality . This ash length .de novo in one female case, but parent of origin is not determined; the inheritance is unknown in other three cases, while in two males is inherited from a non specified parent. Interestingly in three male cases the deletion is also present and constitutional in their fathers, two of which are referred with similar phenotype to child, i.e. short stature or growth delay, delayed speech and language development. In our case the deletion has occurred on paternal chromosome 6. In some patients, PLAGL1 gene is not included in the deletions, while in other patients with more centromeric 6q deletions, comprising PLAGL1 gene, growth impairment is not described as a clinical feature. Anyway the role of this gene in human growth process, remains to be elucidated.Our 6q24.2-25.2 deletion partially overlaps those reported in Decipher , which sGrowth deviations are a common complication in imprinting disorders. Imprinted genes are not bi-allelically expressed, and diseases arise when an individual inherits two copies of a chromosome that contains imprinted genes from one parent  .Nevertheless, our patient also showed pituitary hypoplasia as a possible cause of GH deficiency. In fact, some patients with 6q deletions may show congenital midline abnormalities, such as undescended testicles or very small genitalia, heart defects , imperforate anus, diaphragmatic hernia, cleft palate, and umbilical hernia [In conclusion, we report a child with a remarkable facial features, a history of IUGR and postnatal growth failure due to GH deficiency, disproportionate short stature, and minimal developmental delay associated with a 6q24.3 deletion of paternal origin. For the first time, we report the presence of GH deficiency and a long-term follow-up, hypothesizing that dysregulation of the GH-IGF-1 axis may be a frequent characteristic of this syndrome. Therefore, we suggest evaluating the GH axis in children with 6q24.3 deletions and growth failure. Identification of imprinted genes or further defining the expression of genes in the region of deletion may lead to the identification of specific genes important in the regulation of growth.The parents of the patient provided written informed consent for publication of this Case Report and any accompanying images. A copy of the written consent is available for review by the Editor of this Journal. This study was approved by the Anna Meyer Children’s Hospital Ethics Committee.


Bradyrhizobium japonicum Is-34, which is incompatible with Rj4 genotype soybeans. A candidate gene involved in this incompatibility was found to be present in this genome.We report here the draft genome sequence of  Bradyrhizobium bacteria establish a symbiosis with soybeans (Glycine max) by forming root nodules. In the root nodules, Bradyrhizobium bacteria provide fixed nitrogen to the soybean, and in return acquire carbohydrates from the soybean. However, some soybean cultivars restrict nodulation of specific strains such as B. japonicum Is-34 and B. elkanii USDA61 , and fragmented to 400 bp using the Covaris S2-A system . A library for sequencing was prepared by a NEBNext DNA library prep master mix set for Illumina , and paired-end sequenced (2 × 300 bp) on a MiSeq sequencer using a MiSeq version 3 reagent kit . Raw reads were trimmed and de novo assembled using the CLC Genomics Workbench version 7.5 . Parameters for the trimming were as follows: ambiguous limit, 2; quality limit, 0.001; number of 5′ terminal nucleotides, 10; and number of 3′ terminal nucleotides, 40. Parameters for the de novo assembly were as follows: update contigs, yes; bubble size, 600; minimum contig length, 1,000; word size, 51; perform scaffolding, yes; auto-detect paired distances, yes; mismatch cost, 2; insertion cost, 3; deletion cost, 3; length fraction, 0.5; and similarity fraction, 0.8.Genomic DNA of B. japonicum Is-34 was assembled into 248 contigs, with an accumulated length of 10,326,597 bp  and an average GC content of 63.1%. The genome was annotated by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP version 2.8), and a total of 9,145 coding sequences (CDSs), 6 rRNAs, and 69 tRNAs were predicted.The draft genome of Rj4 genotype soybeans, an effector protein of type III secretion system (T3SS) was postulated because disruptants of the T3SS structural genes in the incompatible Bradyrhizobium strain overcame the nodulation restriction of Rj4 genotype soybeans (B. japonicum Is-34. A tts box motif (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) revealed that this gene belongs to the C48 peptidase  family. In addition, the candidate gene (MA20_12780)-disrupted mutant of B. japonicum Is-34 overcame the nodulation restriction of Rj4 genotype soybeans. Detailed characterization of this disruptant will be reported in the near future.As a candidate gene for the incompatibility with  system TSS was po system TSS was poJRPN00000000. The version described in this paper is version JRPN01000000.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no.