The RepeatMasker analysis unveiled that eleven. 17% of contigs harbored a repeat as well as most represented ele ments belong to SINE families. The latter end result is in line together with the studies performed during the Indonesian coelacanth genome, through which the activity of SINE aspects in Latimeria was inferred. The identification of LF SINEs and DeuSINEs in L. menadoensis transcriptome might verify that these components are basically energetic. In addition, since their conservation in increased vertebrates, this move ment may predate the common ancestor of Crossopter ygians, for in excess of 400 Myr. On the other hand the occurrence of total SINEs in contigs bearing protein coding sequence might reveal the gain of new practical roles, as previously described in tetrapod genomes.
Regarding the activity of LINEs, the second most rep resented interspersed factors, the InterProScan analysis recognized amino acidic domains linked to these autono mous retrotransposons. Obatoclax manufacturer Chicken Repeat 1 factors are the most abundant amongst LINEs. In contrast to the G. gallus genome wherever these aspects are predominant but, with extremely few exceptions, nonfunctional, in Latimeria they appear to be active. Fragmented LTRs and ERVs had been recognized while in the tran scriptome. This consequence is in agreement with the analyses on Foamy like retroviral elements just lately found in L. chalumnae genome by Han and Worobey exhibiting numerous frame shifts and halt codons. The abundance in the Harbinger DNA transposons in L. menadoensis genome suggests that Class II factors represent a exceptional fraction with the coelacanth TEs, having said that our examination indi cates that couple of DNA components are expressed.
This discord ance might be associated towards the lack of coelacanth specific sequences belonging to this class during the RM database or to their propagation mode. The identification of mobile ele ments in transcriptomes sheds light on an unexpected genome dynamicity in an organism regarded to be a liv ing fossil even from a molecular selleck chemical viewpoint. RNA seq mapping around the African coelacanth genome Greater than half from the sequence data produced by the RNA seq of L. menadoensis liver and testis mapped to the genes annotated by Ensembl within the L. chalumnae genome, revealing an general great annotation on the African coelacanth transcripts, although in some cases the RNA seq data developed within this research could provide some proof of further exons and al ternative splicing, provided that the 6.
97% of your reads corresponded to regions annotated as introns. Nevertheless, a rather high proportion of reads, near to 40%, could not be mapped around the genes annotated by Ensembl, consistently with all the method adopted by Ensembl for that annotation pipeline, that is automated and largely centered on protein coding gene versions. In fact, almost the 35% in the sequencing reads could map over the assembled genomic scaffolds outdoors from the an notated gene boundaries, revealing that a relevant por tion of the transcripts expressed from the Indonesian coelacanth liver and testis might correspond to genes which were not annotated through the Ensembl RNA seq annotation pipeline.

Sweetpotatoes are commercially propagated by vegetative cuttings. These cuttings produce adventitious roots that give rise on the SRs. Adventitious roots originate from primordia situated on the nodes, likewise as from your cut ends, i. e, wound roots. At first, white fibrous roots build and some of these subsequently produce into SRs. Based on the variety of FRs in duced to type SRs, sweetpotato plants will yield both a higher or minimal amount of SRs that may even be diminished to a single quite large SR per plant. Togari described the sequence of anatomical occasions resulting in SR initiation in types Okinawan and Beinakzi and reported that the regular vascular cambium layer very first seems 20 days right after transplanting, followed from the first advancement of secondary anomalous cambium options at 25 DAT.
Togari also documented the incidence of stele lignification and proposed that lignifica tion prevents SR initiation. Wilson and Lowe also recommended that only the look of AC can avert stele lignification. selleck chemicals Not long ago, we demonstrated in the two Georgia Jet and Beauregard sweetpotato varieties, the time period spanning 5 to 35 DAT is essential in figuring out regardless of whether adventitious roots come to be lignified or initiate as SRs, and that the appearance of AC marks the initial phase of SR formation. The molecular mechanisms underlying the induction of adventitious roots to turn out to be SRs are, having said that, poorly understood. Expression scientific studies are actually used in an energy to elucidate components involved in SR formation. You et al. constructed a cDNA library of early stage SRs, and identified 22 genes differentially expressed between FRs and SRs.
Among them had been a no apical meristem like and also a MADS box protein gene, each of which were down regulated in SRs. McGregor uncovered several NAC loved ones transcrip tional regulator proteins that have been down regulated in storage roots, similar to the NAM like protein described by You et al. McGregor also identified up regulated expression of two NAM like investigate this site genes, too as sporamin genes and genes concerned in starch biosynthesis, in storage roots that developed 6 weeks immediately after planting compared to fibrous roots. Many added MADS box genes expressed in root tissues have been isolated from sweetpotato, and their doable roles in root development are actually deduced. Tanaka et al. recognized 10 genes with differential expression between FRs, thick roots, and SRs.
One of the genes, SRF6, encoded a receptor like kinase with high expression close to the primary cambium and xylem meristem. Also, Tanaka et al. suggested 3 sweetpotato class one knotted1 like homeobox genes as you possibly can regulators of cytokinin ranges in SRs. De novo assembly of transcript sequences generated by subsequent generation sequencing technologies provides a fast strategy to obtaining expressed gene sequences for non model organisms.

Popgene program package by Yeh et al. was applied to determine heterozygosity. The polymorphism data content material of every marker was calculated according to Anderson et al, CP3M and vacuum dried for two hrs just before subjecting it to autoradiography for two 3 days at 70 C determined by the signal intensity. The size on the fragments was estimated using twenty bp DNA dimension common. Sequencing of PCR products PCR solutions had been separated on polyacralamide gel. Picked fragments were excised and dipped in 10 l nuclease totally free water for thirty min. One more round of PCR was manufactured following exactly the same protocol with extracted DNA as template. The PCR goods had been separated on 2% Seakem LE agarose gel and extracted working with kit. DNA concentration in each and every case was measured making use of Nano Drop one thousand. The PCR products had been ligated to pGEM T straightforward vector.
Sequencing was performed applying ABI 3730 xl DNA Analyzer in 20 l of sequencing reactions consisted of 250 ng of template DNA, 4. 0 pmol universal sequencing primer, 8 l of ready response combine BigDye ter minator. The base calling and submit processing in the sequence information had been finished using sequence analysis program. The nucleotide sequences had been aligned working with DNAS TAR application utilizing Clustal W algorithm inhibitor CX-4945 process. Information analysis The fragment dimension is reported for that most intensely ampli fied band for each UGMS locus or common stutter should the intensity was same applying twenty bp DNA size common. Null alleles had been assigned to genotypes with confirmed no amplification products beneath the standard ailments. The polymorphism established according to your presence or absence and data was entered in the binary data matrix as discrete variables.
Jaccards coefficient was calcu lated to develop a phylogenetic tree to the unweighted pair group method with arithmetic suggest. The personal computer package deal NTSYS computer Ver. two. 02e, Rohlf, was Where Pij would be the frequency in the jth pattern for marker i and summation extends selleck chemicals above n patterns. The match of every locus distribution to expected distribution below two different mutation designs, the IAM and SMM was examined working with the plan BOTTLENECK. Contemplating the locus limitations in data evaluation utilizing BOTTLENECK, especially forty UGMS loci having detected PIC 3. 0 have been picked. Observed allele frequency and sample sizes have been input parameters. These analyses offer a test statistic, the Wilcoxon signal rank test, to the probability that an observed allele distribution with a provided heterozygosity was produced under every on the two mutation models. Background Abiotic stresses are the principal lead to of decreasing the average yield of main crops by in excess of 50% resulting in losses really worth numerous million bucks every 12 months.

Therefore, up regulation of cdc18 and cdt1 in sgf73, meu29, sec65 and pab1 may possibly bring about DNA re replication, and that contributes to your observed diploidization. Meanwhile, disturbed replication initiation possibly compromises DDR while in early S phase. Correspon dingly, each of the members in W4C and S4C groups showed solid sensitivities to HU. Discussion In this review, six reagents had been applied from the screen plus they may cause distinctive varieties of DNA injury. The screen exposed 6 genes whose deletions displayed sturdy sensitivities to 5 reagents, as well as rad1, rhp55, ulp2, pst2, mlo3 and trk1. Broad sensitivities to diverse DNA harm reagents propose that these genes perform while in the universal pathway of DDR, one example is, in the conserved ATM/ATR pathway. As expected, rad1 plays a role while in the ATR pathway, and rhp55 while in the ATM pathway.
ulp2, encoding a SUMO protease, is needed for cell division soon after termination with the DNA damage the original source checkpoint. The higher sensitivity of ulp2 to a broad assortment of DNA injury reagents emphasizes the importance of silencing in the DNA injury checkpoint and restart on the cell cycle. pst2 encodes a subunit with the Clr6 histone deacetylase. Deletion of pst2 could result in hyperacetylation of histones and down regulation of histone H3 S10 phosphorylation, consequence ing in abnormal chromosome condensation as well as a defect in DNA damage repair. Identification of pst2 throughout the screen signifies the significance of chromatin condensation and decondensation in DDR. The protein encoded by mlo3 was necessary for export and high-quality management of mRNA, suggesting DDR is relevant on the level and top quality of mRNA. The display has revealed the novel link involving DDR and trk1, gene encoding a potassium ion transporter. Two calcium transporter genes, cch1, and pmr1, have also been identified on this research.
cch1, in conjunction with other ion transporter genes, together with zrg17, fep1, ctr4 and zhf1, had been recognized for the duration of past worldwide screens for DDR selelck kinase inhibitor genes. These outcomes imply a shut connection between ion transport and DDR. Ion transport controls a number of crucial physiological para meters, which includes membrane likely and ion stability. It will be intriguing to uncover the mechanism how ion transport influences the DDR in future studies. The display also identified genes whose deletion exhib ited sensitivity to just one type of DNA injury reagent. Characterization of these genes will help to elucidate the distinct DDR to get a sure DNA lesion. Such as, dele tion of psl1 displayed exact sensitivity to MMS. Previ ous screens have recognized very similar genes, which include cac2, mag1, rev3 and slx4. These genes, as well as psl1, could function collectively to get rid of the injury brought about by alkylated DNA.

0. However, it should be noted that whereas the triplicate data of the SRSF display means that errors existing inside a single offending replicate might be removed, the re moval of edge effects inside the HFA data leaves only one remaining information stage. Even though not optimum, the quantity of wells excluded from the HFA dataset for that reason are included in Table one to facilitate inter display comparisons. Evaluation to this point has only considered the FL/RL ratios of potential interactions. This can be a consequence in the display design that makes use of a reporter constitutively expressing Renilla luciferase like a proxy for cell amount. The prediction remaining that in the scenario the place JAK/STAT pathway exercise is unaffected but cells underneath proliferate, both FL and RL channels are proportionately diminished with all the FL/RL ratio remaining frequent.
How ever, this biological normalisation selleck chemicals relies on luciferase expression, activity and detection of each channels chan ging linearly with respect to each other. In scenarios the place linearity isn’t excellent or values in either channel are ex treme, the resulting FL/RL Z score ratio can indicate a possibly misleading important change. To visualise this relationship in between the FL and RL signals, we plotted FL and RL channels individually for each screen, an method that obviously visua lises the impact in the controls made use of on both FL and RL channels. In general acting as antici pated, the pathway regulators impact the FL channel, but not around the RL channel, although the technical handle targeting the Renilla luciferase mRNA has the inverse impact minimizing RL amounts but getting no impact on FL. By contrast, the DIAP1 control results each channels, whereas the non interacting management ZK686. 3 lies within the primary cluster of samples.
We also visualise wells that were excluded because of edge results or liquid handling errors during the first information visualisation. While a lot of of those excluded wells cluster inside of the middle of your graphs, some are plainly outliers that may are already picked as putative hits without the need of this amount of evaluation. inhibitor STA-9090 Even though the biological and technical controls act as anticipated, we observed that knockdown of the subset of genes initially characterised as hits in reality demonstrate big RL Z scores, but largely unaffected FL values. On top of that, visual monitoring of those wells in subsequent re screens didn’t propose that this raise in RL exercise was a consequence of elevated cell quantity. Closer examin ation on the predicted biological ontologies of these genes identified several heat shock proteins and com ponents with the proteolysis pathway. As this kind of, it is actually very likely that this interaction is actually a re sult of modifications to Renilla Luciferase protein stability and/or activity. As this kind of, added techniques to identify false constructive signals arising from substantial improvements during the RL signal can also be vital.

Metabolic pathways involved with formation of seedless fruit As big proportion of altered expressed genes have been involved with varieties of metabolic processes. Depending on the KEGG analysis, 36 distinctive metabolic pathways have been altered all through the 4 developmental phases. Amid these pathways, nine had been related to amino acid meta bolic pathway, and genes associated with carbohy drate and power metabolic process showed down regulated expression all through subsequent developmental phases of floral organs. Aside from, genes related to precise secondary metabolism this kind of as terpenoids and polyketides metabo lism have been also observed to be altered. Interestingly, a gene encoding fatty acyl CoA reductase, which could possibly be involved with lipid metabolic practice, was identi fied.
This gene was discovered really homologous with putative male sterile protein in castor bean, fatty acyl CoA reductase three in poplar and male sterile two like protein in Arabidopsis. Herein, selleck chemical this gene was named as male sterile like protein. And qRT PCR analysis showed its expression degree elevated from SF to BF phases then declined at OV stage. The expression pattern was equivalent in the two QS and EG, even so, it showed naturally higher expression degree in QS than in EG all through the developmental course of action. Differential expression of transcription factor genes Its noteworthy that amongst the 133 unigenes, twelve were assigned for the class of transcription element determined by plant TF database. Figure six showed the certain expression pattern of six AP2 ERF loved ones TFs, two zinc finger TFs, a single MYB TF and a single NAC TF utilizing qRT PCR assay.
These TFs had equivalent expression profile all through the four developmental phases among EG and QS. For in stance, amongst 6 AP2 ERF TFs, four showed co expression pattern like V type. It showed that the gene expression level in QS was increased than that in EG from SF stage to Wnt-C59 clinical trial MF stage, having said that, these genes have been subsequently repressed far more definitely in QS from MF stage to BF stage, plus the gene expression degree was down regulated mostly at BF stage. Two zinc finger TFs and one R2R3 MYB TF likewise showed very similar V sort variation tendency. Another two AP2 ERF TFs showed V like variety expression pattern in QS. Nevertheless, the expression pattern of AP2/ERF domain containing TF1 was by some means numerous from other individuals, since it showed relatively stabilized expression level for the duration of the 4 stages in EG. As for NAC TF, its expression degree was down regulated definitely at BF and OV stages in QS examine with EG. It had been notable that no expression was observed at OV stage in QS. The results advised that these TFs could play vital roles during the seedless phenotype for mation, as well as the relative expression degree in QS versus EG seemed to get crucial aspect on this process.

smegmatis necessitates even more investigation. 1 additional intriguing observation is the fact that GlnR in creases the expression of genes encoding enzymes pre dicted to become involved in processes that break down cellular elements into ammonium. One example is, an amine oxidase which break down amines into ammonia and an aldehyde, a urea amidolyase that converts urea to CO2 and ammonia, a deaminase which breaks down nucleotides selleckchem into nucleosides and ammonia, and numerous hydrolases which act on carbon nitrogen bonds are all GlnR regulated. These cellular elements may either originate from other organisms while in the natural environment, or from within the cell. By way of example, E. coli can use its very own peptidoglycan D Ala D Ala like a nitrogen source.
As a result it can be conceivable that in occasions of intense nitrogen austerity, mycobacteria could use cellular components to supply the ammonium essential for development and survival while in the quick phrase until eventually nitrogen again gets to be offered. Determination selleck inhibitor within the GlnR binding webpage motif and Key residues MEME recognized a 17 bp consensus GlnR binding se quence of Gn2AC n6GnAACA present after in all the GlnR binding web-sites. Streptomyces includes a 16 bp GlnR DNA binding motif, that is similar to the a single recognized right here. Tiffert et al. proposed the exist ence of two GlnR motifs inside the binding motif in S. coelicolor, an a web-site as well as a extremely conserved b web site located six bp apart in which the b internet site features a larger affinity for GlnR compared to the a webpage. Even so, the motif identified in S. venezuelae, GTnAC n6 GTnAC only incorporates two copies of an a web page. The M.
smegmatis GlnR binding motif is made up of two numerous web pages, a variable a variety web page separated by 6 bp from a b web-site that is definitely hugely similar to the S. coelicolor b web-site sequence. Pullan et al. suggested that conservation in the b web-site may be indicative of sturdy GlnR regulation when it comes to gene expression. Even so, we didn’t obtain any correlation amongst the presence or absence on the b webpage. Such as MSMEG4501 and MSMEG5358 have the b web-site, whilst MSMEG6816, MSMEG2982 and MSMEG2526 usually do not, however all exhibit equivalent differential gene expression. Consequently the conserved b internet site is not really the sole determinant from the strength of GlnR regulation, and further transcription variables may possibly contribute. However, there’s a really conserved AC n9 AC motif present in each M. smegmatis and Streptomyces, and we’ve shown that spacing involving the dinucleotides, likewise since the presence of adenosine are the two crucial for GlnR binding. The 9 bp distance involving these adeno sine residues represents a single flip in the big groove on the DNA helix, making sure the two can be found to interact with GlnR. Conclusions In summary, we have now demonstrated that GlnR may be the glo bal nitrogen response regulator in M.

About 25 freshly emerged whiteflies and 2nd instars aphid nymphs were released an common per leaf of plants. Total ex perimental plants were covered with perforated poly ethylene bags to stop the insects from escaping. The insect infestation experiments were carried out in three biological replicates. Remedies of ten mM MgCl2 had been spread as mock solution. In sects had been removed by a fine brush following two and 24 h of infestation, and instantly, two middle leaves had been fro zen in liquid N2 for total RNA isolation. Every one of the experi ments with Aphids and whiteflies had been performed with approval of IBSC. RNA isolation and sample preparation Complete RNA have been isolated through the use of Spectrum plant complete RNA Kit according for the manufac turers protocol and underwent DNaseI treatment method.
From 3 biological replicates, only one plants RNA was utilized for transcriptome sequencing. To amplify the mRNA, double stranded cDNA have been ready making use of oligo dT primers containing T7 promoter and SuperScriptW Double Stranded cDNA Synthesis Kit. These double stranded cDNA have been ampli fied applying an selelck kinase inhibitor in vitro amplification system of your Gene chip IVT labeling kit. Amplified cRNA underwent double stranded cDNA planning through the use of random hexamer primer and SuperScriptW Double Stranded cDNA Synthesis Kit.These double stranded cDNA have been purified through the QIAquick PCR purification column. The double stranded cDNA have been utilized for transcriptome sequencing. The transcriptome sequencing was performed as per the companies protocol of Roches GS Titanium pyrose quencing.
Further to confirm linearity in expression pattern amongst the 3 biological replicate, microarray experiments have been carried out with isolated RNA sample by way of Affymetrix kit as per the manufacturers Lenvatinib price protocol. Assembly and annotation of transcriptomes The reads from every library were assembled individually by Roche Newbler edition two. 3. The as sembly criteria had been set as forty bp overlap dimension and 90% identity concerning the reads. Contigs of every library have been pooled together and assembled to form a prevalent information set. The reads were counted from their respective librar ies to the newly assembled contigs, and their TPM was calculated. For further examination, TPM values were log2 transformed. Genes for example Actin, UBQ7, Gbpolyubiquitin 1, Gbpolyu biquitin two, Histone three, and 18S rRNA had been picked to the normalization of the expressed contigs in every single ailment. Digital gene Expression was carried out by DEGseq package in R bioconductor 2. 15 for each library with reference to regulate. Contigs with p worth 0. 05 were selected for differential gene expression. Two fold up and two fold down regulated contigs were picked. For that reason, 8 cat egories were manufactured. The differential genes for every transcripts were subjected to 2 by 2 Chi square check.

Chondrogenic differentiation The chondrogenic differentiation capacity of MSC is evi denced from the formation of shiny cell spheres expressing type II collagen in pellet cultures. Chondrogenic vary entiation of AT MSC and UC MSC continues to be described by lots of groups applying medium supplements this kind of as transforming growth factor b1, ascorbate 2 phosphate, and dexamethasone. Feng et al. professional moted chondrogenic differentiation of AT MSC by the addition of growth and differentiation aspect five and stimulation by FGF two or BMP six has also been reported. Productive chondrogenic differentiation is indicated through the detection with the further cellular matrix part glycosaminoglycan, by immunohistological staining e. g. of collagen II and aggrecan or by verification from the expression of common genes within the chondrogenic lineage through PCR.
Osteogenic differentiation Enhanced alkaline phosphatase expression and minerali zation assayed by von Kossa or alizarin red staining signifies the occurrence of osteogenic differentiation. Various groups reported differentiation protocols for AT MSC by using dexamethasone, b glyceropho sphate and ascorbic acid as medium dietary supplements. The identical medium composition this content was made use of for your thriving osteogenic differentiation of UC MSC. Medium supplementation by one,25 dihydroxyvita min D3 or BMPs has also been reported to enhance osteogenic differentiation. Results of oxidative anxiety and hypoxia in MSC Variations in cell functions amongst MSC populations derived from grownup or neonatal tissues may also be influ enced by the microenvironment.
Within the proper tissues in vivo, stem cells like MSC are selleck chemical often present in stem cell niches under hypoxic problems. Thus, in vitro principal culture inside a normoxic ambiance may be considered as an publicity to enhanced oxi dative anxiety and promotes the generation of metabolic radicals or reactive oxygen species. The intracel lular accumulation of ROS may cause protein and DNA injury if these compounds are insufficiently metabo lized by an proper anti oxidative defense technique. Consequently, ROS accumulation at substantial oxygen amounts induces elevated apoptosis and premature aging by STASIS. Certainly, MSC cultured beneath normoxic con ditions exhibit premature senescence along with a reduction in population doublings in comparison to cells cultured under hypoxia and can also demonstrate restricted cell division resulting from telomere shortening and replicative senescence.
The migratory capability of MSC cultured under hypoxic problems is also enhanced in contrast to that observed in normoxia. Hypoxic condi tions for this reason influence proliferation and cell fate com mitment, which means that gradients of oxygen tensions influence the prolonged upkeep of the stem cell phe notype and pluripotency. Also, serum starva tion and deprivation of development things can advertise premature aging in MSC and scientific studies of MSC in a hypoxic natural environment show that serum starvation might be linked with significant cell death.

As a member of the tumor necrosis aspect receptor super relatives, LMP1 expression can activate the nuclear factor kappa B, activator protein 1 and utilize ing Janus kinases or and signal transducers and activators of transcription pathways and regulate their substrates. LMP1 also targets the phosphatidylinositol 3 kinase AKT pathway to induce fibroblast transformation and enhance cell sur vival. Furthermore, LMP1 can promote epithelial cell motility and enhance invasiveness by activating the extra celluar signal regulated kinase mitogen activated protein kinase pathway. Mammalian target of rapamacin is definitely an evolu tionarily conserved serine threonine protein kinase with an important position in cell development and proliferation via regulation of ribosome biogenesis and protein translation. PI3K AKT is considered a vital upstream mediator from the mTOR signaling pathway.
The characterized downstream effectors of mTOR are ribo somal protein S6 kinases, and eukaryotic initia tion factor 4E binding protein, with eIF4E dissociating selleck Imatinib from 4E BP1 to initiate translation right after 4E BP1 phosphaorylation, even though P70S6K translates mRNA transcripts having a 5 Major motif following hyper phosphorylation by mTOR. To even further clarify the signaling pathways regulated by LMP1 in NPC, we investigated the association among the mTOR signaling pathway and LMP1, the expression of p mTOR, p P70S6K and p 4EBP1, and their relation ship to clinicopathologic parameters of NPC individuals. Products and strategies Sufferers and tissue samples For this retrospective research, archival formalin fixed, par affin embedded specimens from 230 key NPC patients admitted from 1992 2002 for the Sun Yat Sen University Cancer Center were recruited. All NPC samples were obtained before treat ment with normal curative radiotherapy, with or with out chemotherapy.
Sixty sufferers had been diagnosed as differentiated non keratinized, and 170 sufferers had undifferentiated carcinoma. In accordance on the Chinese 1992 staging procedure, patients were classified as six in stage I, 49 in stage II, 110 in stage III, 65 in stage IV. The vast majority of sufferers had been male, ranging from 86 to 14, which has a median age of 46. This examine was accredited from the selleckchem Investigation Ethics Committee of your Sun Yat Sen University Cancer Center. Tissue microarray construction Paraffin embedded specimens have been from a previously constructed tissue microarray. Protocols and instruments to the tissue array building had been described previ ously. Immunohistochemistry The immunohistochemistry protocol was as described previously. Briefly, tissue sections had been de waxed for antigen retrieval, and incubated with major antibodies LMP1 monoclonal antibody at a dilution of 1 50, or p mTOR, p P70S6K, or p 4EBP1 at dilutions of 1 100 overnight at four C.