Much more operate has to be carried out to below stand how youthful adult bees react to poor eating plan and to fully grasp the basic biology of nutrition in this procedure. We aimed to additional understand the transcriptional changes connected with starvation in the course of early adult de velopment. To this end, we assayed three day previous and eight day previous bees stored in ordinarily working hives and caged over only honey or honey and bee bread and allowed trophallaxis using the rest of their col ony. Full transcriptomes from stomach carcasses were analyzed to focus on changes from the A. mellifera abdomen unique in the digestive tract. We hypothesized that starvation and aging would considerably impact gene expression and physi ology, that starvation would impact youthful bees differently than older bees, and that early grownup devel opment will be impacted by diet regime.
We also aimed to provide testable predictions for study on improvement and nutrition in the. mellifera and develop targets for im proving the productivity of bee colonies under dietary pressure. braf inhibitor Right here, we report that starvation tremendously has an effect on the ex pression of genes associated to nurse physiology and develop ment and the processes that take place as adults produce into nurse bees are altered by sub optimal diet regime. Effects Bees fed pollen had more substantial hypopharyngeal glands compared to those who were not fed pollen We began by assessing no matter whether bees that were fed only honey had reduced hypopharyngeal glands, a traditional signature of starvation. Hypopharyngeal gland size was measured in twelve courses of bees, For each of these twelve deal with ment combinations, HGs had been dissected from approxi mately five bees and ten randomly picked acini have been measured for each gland.
Hypopharyngeal gland dimension was significantly impacted by all things tested inside the model eating plan, host colony, age, diet by colony, age by food plan, age by colony, and the 3 kinase inhibitor EMD 121974 way age by diet program by colony interaction, Post hoc analyses showed significant vary ences between 3d old bees fed the rich versus poor eating plan, 8d outdated bees fed the wealthy versus bad food plan, and 3d versus 8d bees fed the rich diet regime, but no big difference in between 3d and 8d previous bees fed only honey, Because of the major interaction concerning food plan and col ony from the full model mentioned above, the effect of eating plan and age on HG dimension was also investigated for each colony individually.
For colony A, the result of diet and the interaction involving age and food plan had been considerable however the ef fect of age was not, For colony B, the effect of diet program as well as interaction in between age and food plan had been major but the result of age was not, For colony C, the result of age and eating plan were signifi cant, however the effect of your age ?? diet plan interaction was not, The HG sizes of bees that were utilized to evaluate the predictive electrical power of the mRNA Seq outcomes with qPCR, were also measured.

Finally, the precipitate was dis solved in carbonate buffer and submitted to polymyxin agarose affinity column chromatography as described by Kannenberg and Carlson, The LPS preparations eluted from polymyxin column by carbonate buffer con taining 1% deoxycholate were implemented for GC MS evaluation, and have been separated by twelve. 5% Tricine SDS Page and visualized by silver staining, EPS and LPS examination The sugar composition within the degraded polysaccharides liberated from LPSs in the wild style and Rt2440 by mild acid hydrolysis was established by GC MS analysis of their alditol acetates. For this, water soluble degraded polysaccharide obtained following lipid A centrifugation was subjected to reduction, For that determination of acid sugars, the samples were subjected to methanolysis at 85 C for sixteen h in one M methanolic HCl, carboxyl reduc tion with NaBD4, hydrolysis with 2M acid for 4 h at 100 C, reduction with NaBD4, and acetylation.
To the neutral and amino sugar analysis, the samples had been hydrolyzed with 2 M TFA, N acety lated before reduction with NaBD4, and acetylated. The glycosyl composition Lenalidomide solubility examination of EPS samples was performed just after methanolysis, followed by trimethylsily lation as described in Vanderlinde et al, Part of the methanolysates was subjected to carboxyl reduction, hydrolysis in two N TFA, reduction and acetyla tion, as within the method described above for the acidic sugar determination in LPS. Monosaccharides within the type of alditol acetates and methyl glycosides of tri methylsilyl ethers had been analysed by GC MS to the Hew lett Packard fuel chromatograph interfaced to the 5971 mass selective detector making use of the 30 m HP 5MS capillary column, NMR spectroscopy 1H experiments had been recorded with the Varian Unity plus 500 instrument in D2O solu tions at 70 C with acetone as an inner common employing standard Varian software program.
Motility assay R. leguminosarum motility assay was performed in 0. 3% M1 agar medium. five ul culture grown in liquid TY med ium at 28 C for 24 h to an OD600 of 0.four was stabbed into plates with M1 medium. To get rid of the floccula tion of the rosR mutants, cell clumps were wiped and broken up on the inner surface of a glass tube implementing a sterile wooden stick. Then, the tube was left standing for 15 min so that the remaining clumps selleckchem signaling inhibitor sunk to the bottom. The suspended cells in the major have been taken cautiously and, if needed, diluted down into TY to get the sought after cell density, The plates had been incubated at 28 C for 3 days, and bacterial development from your point of inoculation was measured. Motility assay was executed twice in triplicate. Biofilm formation assay microtiter plate approach The biofilm formation assay was carried out according to approach described by Rinaudi and Gonzalez, Briefly, R.

coli as recombinant polypeptides with six histidine residues at their N termini by traditional strategies. The purified polypeptides are shown in the lower panel of Figure 3B. The concentration with the His polypeptides was to start with determined from Coomassie stained SDS Web page gels by examination of complete band intensity within the corre sponding polypeptide making use of picture evaluation with an inter nal protein normal of known concentration. The polypeptides had been then assessed for binding to immobi lized target molecules by ELISA and surface plasmon resonance evaluation, His NarG and His FnBPA polypeptides have been applied as inner detrimental and favourable controls, respectively. Since the His SCOR and His IspD polypeptides remained insoluble inside the E. coli cytoplasm, these proteins could not be purified in non denaturing ailments and could regretably not be included inside the verification.
In the ELISA assay, the His Coa and His Ebh poly peptides interacted with all the similar immobilized target molecules as individuals of your corre sponding Ftp library clones, The His PurK polypeptide bound to Fn but interacted poorly with Fg, whereas His Usp showed only a low level interaction with Fn. Similarly as the adverse handle poly peptide His NarG, the His find more info FnBPA and His PBP poly peptides showed no binding to Fn or Fg while in the ELISA. While in the SPR evaluation, the His PurK, His Coa, and His Usp polypeptides bound to immobilized Fg whereas the His FnBPA, His PurK, and His Ebh polypeptides showed affinity to Fn similarly as did the cell free of charge growth media of corresponding Ftp library clones tested by ELISA, In contrast to your ELISA final results, the His Ebh polypeptide reacted also with Fg within the SPR evaluation. The His PBP polypeptide along with the adverse manage peptide His NarG showed no binding adequate ties while in the SPR analysis.
Having said that, the SPR effects primarily confirmed the outcomes obtained with culture supernatants of Ftp clones. The affinity constants obtained from the SPR evaluation VEGFR Inhibitors are proven in Table two. Discussion S. aureus NCTC 8325, the parental strain with the proph age cured S. aureus NCTC 8325 four employed for construction with the extracelluar secretion library, carries 22 of the genes encoding the 24 surface proteins implicated in adhesion and all of the 13 genes to the secretable proteins implicated in immune response evasion as lately described by McCarthy and Lindsay, In accordance towards the literature, only eight of these proteins are actually reported to bind Fn and or Fg and 5 interact with all the ECM. Cna, the only collagen binding protein during the record of adhesins, isn’t current in S. aureus NCTC 8325 4, Taking into consideration the above data as well as the proven fact that we deliberately screened for binding to only a handful of model targets of S.

The sPLA2 is often a 13 to 15 kDa protein while the cPLA2 can be a 85 kDa selelck kinase inhibitor protein in human macrophages. The cPLA2 possesses characteristics that sug gest that it’s associated to receptor activated signal trans duction cascades, This PLA2 is identified to translocate for the membrane in response to a rise in intracellu lar calcium concentration, Cytosolic PLA2 hydrolyses the sn 2 place of phospholipids, leading to the release of lysophospholipids and cost-free fatty acids. Quite possibly the most com monly released fatty acid is arachidonic acid, which in turn is converted to eicosanoids that regulate various processes like calcium channels, mitogenic signals and most pathway inhibitors significant, the inflammatory response of mac rophages, The present study was undertaken to determine the presence of and characterize added G subunits in S.
schenckii, to identify any significant interacting partners of your new G subunit, and eventually to find out the involvement if any in the interacting protein, in this instance cPLA2, while in the manage of dimorphism within this fungus. Here we give details of the identification and sequencing in the ssg two gene, including gene organization, the presence and position of introns, derived amino acid sequence and conserved polypeptide encoded domains. Making use of SSG 2 as bait, we recognized a cPLA2 homologue interacting with this G professional tein subunit. We give the genomic sequence of this gene and the complete derived amino acid sequence. We also report the results within the yeast to mycelium transition as well as the yeast cell cycle of phopholipase effectors in S. schenckii. This do the job constitutes the very first report on the presence of many G protein subunits in S. schenckii, the presence of the cPLA2 homologue interacting with this G protein subunit, along with the involvement of cPLA2 in the control of dimorphism in S.

The per centage of assigned transcripts and their overall mean coverage expression level for every on the top rated 37 KEGG pathways are proven in Figure 4A. The major KEGG meta bolic pathways included carbohydrate metabolism, vitality and lipid metabolism, amino acid metabolic process as well as biosynthesis of secondary metabolites. Inside the secondary metabolic process, 270 transcripts have been classified into 27 subcategories, and most of them had been mapped to terpenoid backbone and diterpenoid biosynthesis, car or truck otenoid biosynthesis, flavone and flavonol biosynthesis, zeatin and glucosinolate biosynthesis, These final results indicated the active metabolic processes in E. fischeriana root, but in addition imply that a variety of metabo lites are synthesized in the root.
you can check here Interestingly as antici pated the terpenoid and diterpenoid biosynthesis pathways encompasses the biggest quantity of transcripts associated with secondary metabolites, which could relate using the capability of E. fischeriana root to produce prostratin and relevant diterpenoid compounds. We up coming established the general expression levels for every KEGG pathway as described above for GO codes. The pathways with the highest expression amounts were Translation and Signaling molecules and interaction with imply coverage values of 327 and 148, respectively, The vast majority of all other pathways dis played similar expression amounts with an overall suggest coverage close to 50. Comparison of E. fischeriana transcriptome with related species To evaluate the conservation with the E. fischeriana genes in related species we compared transcripts for each locus against NCBI Expressed Sequence Tags of Hevea brasiliensis, E.
esula and R. communis. ESTs have been made use of, as they’re quite possibly the most comprehensive obtainable resource for comparative analyses. Non redundant sequence information sets for E. fischeriana, E. esula, H. brasiliensis, and R. communis have been generated and clustered to determine orthologous gene clusters as described in approaches. A complete of ten,131 gene clusters have been recognized amongst E. fischeriana, E. esula, H. brasi selleck chemicals LY2835219 liensis and R. communis. Figure five exhibits the overlapping orthologous genes between all evaluated species. As anticipated we uncovered that E. fischeriana shares the largest amount of orthologous genes with E. esula as in contrast to R. communis and H. brasi liensis, but in terms of proportion of shared orthologous sequences as compared on the total quantity of obtainable genes for each species, H. brasiliensis shared the largest fraction of their genes as com pared for the equality very similar fractions of 20. 6% and twenty. 7% for E. esula and R. communis, respectively. We also identified one,145 genes which are shared by all species repre senting a likely core orthologous gene set for Euphor biaceae species.

The information had the exact same temporal pattern as our earlier scientific studies, but there in younger grownups, CPLCG4 was similar to CPLCG3 and CPF4 was reduce than CPF3, Adult eclosion in An. gambiae is gated to take place soon after the dark time period begins. Some pharate adults at P24 will likely be only some hrs selleck inhibitor away from eclosion, other individuals will wait substantially longer. The animals we were evaluating among our published function and this examination were kept under different photoperiods and collected at unique instances with the day, so quantitative variations in relative transcript ranges will not be surprising, and without a doubt we ob served this difference. Transcript localization Results from in situ hybridization are in accord together with the mRNA temporal patterns, but reveal that the condition is far more complex.
The CPF3 probe hybridized selleck chemical Stattic most effective in pha fee adults, though CPLCG3 was most abundant in youthful adults, Nonetheless, for some specimens, there was solid hybridization at the other stage, Transcripts have been also detected in other tissues. Transcripts from all 4 genes were current while in the thorax where muscle and cuticle came in speak to, In pharate adults of the two sexes, all 4 genes had tran scripts inside the genitalia, None of your probes were detected during the eyes, using the excep tion of artifactual labeling from the acellular lens, a com mon challenge with RNA probes which also frequently react with all the previous, acellular, pupal cuticle, Although CPF3 and CPF4 had identical patterns of hybridization to appendages, only the probe for CPF4 reacted with all the standard epidermis from the pharate grownup abdomen, and here, just the sclerites and never intersegmental membranes, We carried out RT qPCR for CPF3 and CPF4 on anterior and posterior areas.
Transcript amounts had been larger in the abdomen for CPF4 than they had been for CPF3, a pleasant verify ation on the in situ outcomes. The presence of CPLCG3 and CPLCG4 in limbs is in accord with their function in insecticide resistance since these would be the parts of the body that can be found in get hold of with insecticides that had been utilized to surfaces. Likewise, the presence of CPF3 within the genitalia may well reflect a function in mating. But, the fact that the 2 CPLCGs were existing in genitalia and each CPFs were present in limbs, complicates a simplistic story. Rather these benefits seem to suggest that CPF3 four and CPLCG3 4 play complementary roles in formation of appendage cuticles.

most abun dant allele minimum go through coverage. 10. 2nd most abundant allele minimum read coverage. 10. minimum regular coverage. ten. minimum flanking length. 100 nt. minimum high-quality score. 0. 99. minimal absolute isola tion. 30 nt. Primer style and amplification Primer pairs flanking the SSR motifs have been constructed making use of MISA with all the following parameters. end stability. 250. minimal dimension. a hundred nt. greatest dimension. 300 nt. In complete we synthesized 114 primer pairs, Primer pairs flanking the SNPs have been constructed utilizing Primer3 with all the following parameters. finish stability. 250. optimum Tm. 60 C. minimal dimension. 120 nt. maxi mum dimension. 201 nt. In complete we synthesized 354 primer pairs, Primer validation was carried out on genomic DNA from the B493 ? QAL wild carrot derivatives and 3 culti vated genotypes, B493, B6272 and B7262.
Microsatellite flanking primers were tested in the PCR of twenty ul volume containing 13 ul water, 2 ul 10X DNA polymerase buffer, 0. eight ul dNTPs, one ul 5 uM of each primer, 0. two ul Taq polymerase and two ul of genomic DNA, PCR problems had been. initial denaturation at 94 C for 2 min, followed by 33 cycles of 94 C for 45 sec, Tm selleckchem custom peptide synthesis for one. 0 min, 72 C for 1. 0 min. and 20 sec, and a last phase at 72 C for 10. 0 min, Elec trophoresis was carried out for two three hrs at 100 V on 2% agarose TAE gels supplemented with 0. 2 ug ml of ethi dium bromide. To verify the predicted polymorphism of SSRs, 31 pri mer pairs were tested employing a fluorescent approach, PCR was carried out in the twenty ul last volume which includes twelve. 5 ul water, two ul 10X DNA polymerase buffer, 0. 8 ul dNTPs, one ul 5 uM of reverse primer, 0.
5 ul five uM of M13 tailed forward primer, one ul five uM of M13 labelled either with 6 FAM, HEX or NED fluorochromes, 0. 2 ul Taq polymerase and 2 ul of genomic DNA, The amplification circumstances have been 94 C for two min. ten cycles of 94 C for forty sec, 60 C for 1 min that has a reduction of 0. 5 C each cycle, 72 C for 1 min. 40 cycle of 94 GDC0199 C for 45 sec, 60 C for 1. 0 min, 72 C for 1. 0 min. and twenty sec, as well as a final step at 72 C for 10. 0 min. Amplicon lengths were estimated using an ABI 3730xl capillary sequencer obtainable on the University of Wisconsin Biotechnology Center and analyzed with Gene Marker software program version one. five, Single Nucleotide Polymorphism validation was carried out on a PCR of twenty ul volume containing twelve. 2 ul water, two ul 10X DNA polymerase buffer, 1. six ul dNTPs, one ul 5 uM of each primer, 0.
2 ul Taq polymerase and 2 ul of genomic DNA, PCR situations had been. original denaturation at 94 C for 2 min, followed by 25 cycles of 94 C for thirty sec, appro priate annealing temperature for thirty sec, and 72 C for 45 sec, and also a ultimate phase at 72 C for 10 min. Pre sence and length with the amplicon was detected on 2% agarose TAE gels supplemented with 0. two ug ml of ethi dium bromide, and separated for two 3 hrs at one hundred V.

PCR and statistical evaluation of EST SSRs For primer pairs resulting from your read2Marker pipe line, 96 with the 111 primer pairs had been selected in an arbi trary and random style. For anyone resulting in the CMiB pipeline, 96 from the two,371 primer pairs that showed no similarity with previously reported EST SSR markers had been chosen at random right after the exclusion of primer pairs that had by now been chosen for the read2Marker pipeline. In complete, 192 primer pairs have been synthesized, PCR was 1st carried out for two persons in ten uL response mixtures consist of ing ca. 5 ng genomic DNA, one ? PCR buffer, 200 uM of every dNTP, 1. 5 mM MgCl2, 0. two uM of each synthesized primer, and 0. 25 U of Taq polymerase, making use of the next program.
94 C for five min, then 40 cycles of 94 C for 30 s, fifty five 62 C for thirty s and 72 C for thirty s, followed by a last extension at 72 C for five min. The PCR solutions were electrophoretically sepa selleckchem tsa inhibitor rated on 2% agarose gels and stained with ethidium bromide to check out for effective amplification. The utility of EST SSR primers that created visible bands within the agarose gel was demonstrated by analyzing polymorph isms amongst sixteen individuals of C. japonica from numerous places across Japan, PCR was carried out in ten uL reaction mixtures under the circumstances described over using the annealing tempera tures listed in Further file 5. Table S3. PCR products have been labelled with ChromaTide Rhodamine Green five dUTP in accordance to a procedure described elsewhere, and analyzed utilizing a 3100 Genetic Analyzer with GeneScan software package, For every locus, the num ber of alleles was counted plus the observed and expected heterozygosity was calculated.
Poly morphism data articles was calculated applying the Excel Microsatellite Toolkit, selleckchem Deviation from Hardy Weinberg equilibrium was examined implementing GenepopV4, To analyze components affecting effective PCR amplification, we fitted a generalized linear model by using a binomial error distribution, the logit hyperlink function plus the PCR amplifica tion since the binary dependent variable, The explanatory variables were the pipeline made use of to style and design the primers, estimated primer location, the sum in the melting tem peratures of your forward and reverse primers as well as anticipated PCR solution length in base pairs. The primer lo cation was estimated during the identical way as for SSRs, as described inside the Mining of microsatellites part over.
We also analyzed the relationships concerning the degree of polymorphism for every locus and elements that may have an impact on it by fitting a generalized linear model which has a Poisson error distribution, log link function along with the amount of alleles because the dependent variable. The explanatory variables were the pipeline utilized to design and style the primers, estimated SSR spot, maximum quantity of SSR repeats within the amplified region, as well as length of your SSR repeat unit corre sponding towards the optimum repeat within the expected PCR items.

having said that, once the anxiety was continued further for thirty days, we observed that the two accessions possess a related response to severe drought, except for RWC and predawn water potential. Underneath extreme drought situations, each the accessions showed a significant enhance in water possible. Vagad showed a 5 fold improve, whereas RAHS 14 showed a ten fold boost in water potential, but RWC was decreased by only 10%, Leaf dehydration curves for Vagad and RAHS 14 are shown in Figure 3a, b. The preliminary sharp drop of curves represents the stoma tal transpiration that was closing for the duration of leaf dehydra tion. The steady state decline underneath the irrigated problem in both the accessions exhibited a related slope.
on the other hand, as drought progressed, the slope in Vagad remained exactly the same underneath reasonable drought and after that decreased beneath selleck chemicals PCI-32765 significant drought, whereas in RAHS 14, the slope remained continual beneath irrigated and drought circumstances. Transcriptional profiling during drought and irrigated problems in Vagad and RAHS 14 We implemented Affymetrix microarray and cotton chip for comparative expression profiling of leaves of irrigated and water stressed plants of Vagad and RAHS 14 at a p value 0. 05 plus a fold change 2. 0. We identified 656 and 535 genes as becoming differentially up regulated in Vagad and RAHS 14, respectively, all through the irrigated issue, Similarly, 430 and 411 genes had been differentially up regulated in Vagad and RAHS 14, respectively, below the drought affliction, These differentially up regu lated genes have been further annotated with the SEA method and identified enriched gene ontology terms.
We considered only these GO terms that had been enriched at the very least twice above background cotton learn this here now data and observed various substantial variations inside the metabolic pathways in the two sensitive and tolerant accession. Singular Enrichment Evaluation for identification of enriched GO terms in Vagad and RAHS 14 while in irrigated problem Gene ontology utilizing the SEA method uncovered distinct distinctions from the total metabolic process of Vagad and RAHS 14 even beneath the irrigated problem. Vagad has quite a few enriched biological processes compared with RAHS 14, The phenyl propanoid pathways leading to coumarin and similarly, a lignin biosynthesis flavonoid pathway leading to polyketide biosynthesis this kind of as stilbene biosynthesis have been enriched all through the irrigated affliction in Vagad, Genes were more mapped towards the KEGG metabolic pathway of these processes, Other biologi cal processes this kind of as ketone biosynthesis, pigment metabolic process, and reductive pentose phosphate were also enriched in Vagad through the irrigated affliction.
In molecular functions and cellular components, numerous membrane transporters, ligases, chalcone synthase, gly ceraldheyde three phosphate dehydrogenase, oxidoreductase, and detrimental regulation of transcription relevant actions have been enriched in Vagad, In RAHS 14, in con trast to Vagad, different biological processes such as membrane lipid metabolic process involving fatty acid bio synthesis, glycolipid, sphingolipid, and glycosaminogly can metabolism linked processes had been enriched, RAHS 14 also invests its power in carbohydrate metabolism, several homeostasis linked processes, defense response, auxin metabolic process, and root development related biological processes, In RAHS 14, molecular function and cellular parts had been enriched with numerous activities such as hydrolases, beta galactosidase, lipases, and esterases.

g, individuals associated with sexual reproduction. From the samples of M. brunnea, obtained in the east ern area of China, we did observe the asexual state but not the sexual state. Likewise, no sexual reproduc tion was located for Marssonina species in New Zealand Farm Forestry, To identity irrespective of whether this fungus undergoes a sexual cycle, we implemented BLAST hunting for orthologues of all genes linked to sexual reproduction and meiosis, Most of these intercourse connected genes were not found in M. brunnea. Some genes demanded for meiosis were present in M. brunnea, nevertheless they were involved in regulation as transcription factors or as supplementaries in syngenesis. For example, DMC1 linked to meiosis had been observed in M. brunnea, whereas those genes necessary to the formation of DMC1 containing nucleoprotein filaments have been absent, Each one of these supported that M.
brunnea might possibly have no capability to execute sex ual reproduction. Transcriptome analysis To review gene gene interactions concerning the patho gen and host, we utilized parallel massive sequencing of cDNA to estimate the relative expression ranges of genes from M. brunnea and the host, poplar clone NL895, Three cDNA libraries had been constructed, find out this here like sample M6 of M. brunnea spores collected from potato dextrose agor, sample 895 M6 of clone NL895 leaves immediately after 96 hrs of infection by M. brunnea, and sample 895 of clone NL895 leaves with no infection. RNA seq reads from 3 diverse samples have been aligned towards the genome sequences of M. brunnea and Populus applying TopHat, Further file twelve gives the mapping final results of RNA seq reads.
Of your ten,040 predicted genes in M. brunnea as well as the 45,554 predicted genes in Populus, 9,340 and 31,794 were identified via great post to read RNA seq, respectively, suggesting a fairly higher coverage of the transcriptome. The relative amount of expression was calculated by using the quantity of uniquely mapped reads for that annotated genes, One can find 2,559 M. brunnea predicted genes that display distinct levels of expres sion from Sample M6 to Sample 895 M6, of which one,898 are up regulated. Most up regulated genes come through the LysM household that functions effectors to suppress plant basal immunity throughout the colonization of plants, with 30 predicted genes from a total of 33 currently being significantly up regulated, Other families that have up regulated genes are SNF2 household proteins, involved in such biological processes as transcription, DNA repair, chromatin remodeling and hyphae de velopment, AMP binding enzymes, enjoying a crucial position in degradation and synthesis of amino acids and lipids, and GDSL like lipase acylhydrolases household proteins, The numbers of up regulated genes for these three households are 11, eleven, and 10 from a complete of 25, 26, and 15, respectively.