16,37 To examine this, we treated fibroblasts isolated from old lungs by using a demethylating agent, AZA. We did not locate modifications inThy one mRNA expression with this particular treatment method. These data propose that age linked alterations in lung fibroblast Thy one expression may not be a result of hypermethylation of Thy 1 gene promoter areas as previously described. sixteen On the other hand, it has been shown that irritation could alter endothelial cell Thy 1 expression and vice versa. These cells secrete distinct amounts of inflammatory cytokines such as interleukin 1, Prostaglandin E2 and IL one. 38 40 IL 1B stimulated PGE2 expression in orbital Thy one constructive fibroblasts, whereas stimulating IL eight expression in Thy one damaging fibroblasts. 40 Far more recently, following stimulation with tumor necrosis aspect alpha, Thy one constructive fibroblasts showed reduction in MMP 9 and intercellular adhesion molecule one, and that is believed to be brought on by interference with Src kinase activation.
41 This is exciting offered that we located increases in Thy one damaging fibroblasts while in the lungs of selleck outdated mice in conjunction with an increase in MMP 9 expression. Also, it isn’t clear whether or not the adjust in lung fibroblast Thy 1 expression learn this here now leads to every one of the profibrotic modifications described in aged lung or no matter if the relative modifications in lung extracellular matrix composition result in alterations in lung fibroblast phenotype. The latter is supported by a review displaying that alterations in culture surface composition could influence epithelial cell phenotype and TGF B1 expression in vitro. 42 No matter whether the improvements we uncovered here are immediately linked to each other is unknown, and the actual mechanisms of loss of Thy one expression with age will call for additional investigation.
In summary, we noticed that old lungs are additional vulnerable to development of injury and fibrosis from the bleomycin induced lung injury model. We believe that this can be caused by a profibrotic phenotype existing in outdated lungs, and that is characterized by greater expression

of TGF B1, TGF BR1 and Smad3 and improved expression with the Fn EDA splice variant and MMPs. Other likely mechanisms involved in the advancement of fibrosis while in the bleomycin model relate to your tissue expression of bleomycin hydrolase. 43 Now, there are no published scientific studies examining the activity or degree of BH in aged tissues. 1 study showed a transform of BH amounts throughout growth with a rise in BH levels in a wide variety of rat tissues up to the age of six weeks, afterward, the ranges decreased. Unfortunately, that specific research didn’t evaluate BH amounts in older animals. 44 Other individuals have proven alterations in BH related with Alzheimers disorder, and that is a sickness of older people. 45,46 For this reason, presumably, there could possibly be some changes of BH in lungs of old mice compared with young ones.

It’s been shown that a tyrosine phosphatase exercise is concerned during the down regula tion of IFN induced gene expression. Treatment with sodium vanadate, a PTPase inhibitor, can retain the tyrosine phosphorylation of Stat1 and protect against the transcrip tional down regulation of IFN induced genes. Considering that the actions of STATs are unquestionably dependent on tyrosine phos phorylation, the precise recognition and dephosphorylation of STATs by their PTPases are expected to get essential for gene regulation. We report right here that the Stat1 N terminal domain, which can be highly conserved between all STAT household members isolated to date, is needed for that tyrosine dephosphorylation of Stat1. An N terminal deletion mutant Stat1 protein was constitutively phosphorylated on tyrosine and tremendously en hanced the antiproliferative exercise of IFN. Supplies AND Strategies Construction of plasmids and mutagenesis.
pGST Stat1, encoding GST Stat1, was constructed by inserting Stat1 cDNA in to the BamHI web-site within the eukaryotic expression vector pEBG, which consists of a glutathione S transferase tag upstream in the cloning internet sites. pGST mStat1, encoding GST mStat1, was designed by subclon ing a PCR fragment encoding amino acids 62 to 750 of Stat1 in to the BamHI website of pEBG. pGST myStat1 was derived from pGST mStat1 by which the their explanation TAT codon for Tyr 701 was transformed to TTT. pGST pStat1 was derived from pGST Stat1 in which the AGA codon for Arg 31 was mutated to GCA. For expressing the mutant Stat1 protein devoid of a GST tag, the corresponding cDNA was cloned in to the BamHI website within the pEBB vector. Mutagenesis was achieved by a standard PCR approach. Transfection and establishment of cell lines. Transient transfection in 293 cells was performed as previously described.
For secure transfection, expression constructs collectively together with the vector pMLTR containing the neo resistance gene had been launched into NIH 3T3 cells. At 48 h soon after transfection, secure clones were selected during the presence of 0. 5 mg of G418 per ml as previously described. Secure cell lines had been primary tained in Dulbeccos modied Eagles Doripenem medium containing 10% donor bovine serum. Very similar approaches have been employed for that transfection and assortment of stable clones in U3A cells. Gel mobility shift evaluation. Gel mobility shift examination was performed as pre viously described. The Stat1 binding webpage from your promoter of the human Fc RI receptor was utilised as

the probe. Preparation of cell extracts, Western blotting, and immu noprecipitation. Cells have been washed with cold phosphate buffered saline and lysed in a buffer containing 50 mM Tris, 300 mM NaCl, 0. 5% Nonidet P forty, 10% glycerol, one mM EDTA, 1 mM dithiothreitol, 0. one mM sodium vanadate, 1 mM phenylmethylsulfonyl uoride, 0. five g of leupeptin per ml, and 3 g of aprotinin per ml. Immunoprecipitation was performed as previously described.

HPLC evaluation of plasma tocopherols indicates that substantial conversion of dietary plant derived / tocopherols into tocopherol happens in H. virescens. Larval nutrition and juvenile hormone regulate molecular parts from the amino acid target of rapamycin signaling pathway within the anautogenous mosquito, Aedes aegypti S. H. Shiao, I. A. Hansen, plus a. S. Raikhel Center for Illness Vector Analysis and Department of Entomology, University of California, Riverside, The amino acid target of rapamycin signaling pathway plays a critical function in blood meal activation of vitellogenesis and egg maturation, which even further define the anautogenic nature of your mosquito Aedes aegypti. Here we show the expression of vital molecular components inside the AA/TOR pathway depend upon attaining sufficient nutritional reserves throughout larval development and this was even more established to be beneath the management with the juvenile hormone III.
By manipulating the amount of larval meals, we selleck produced two size phenotypes. normal, nicely nourished mosquitoes, which generate eggs following the to begin with blood meal, and smaller, malnourished mosquitoes, which call for a 2nd blood meal for you to produce eggs. Inside the modest mosquito, mRNA and protein expression profiles within the yolk protein vitellogenin inside of the fat physique were drastically delayed compare to that observed in typical mosquitoes. By topical application of JHIII shortly immediately after eclosion, little mosquitoes were capable to produce eggs that has a single blood meal in conjunction with a constructive shift in Vg mRNA and protein profiles that resemble that displayed in typical mosquitoes. We even more display that the quantity of nutrients attained during larval advancement straight influences expression profiles IOX2 manufacturer with the AA/TOR pathway parts.
The mRNA and protein expression PS-341 of the insect cationic amino acid transporter two, which is with the top rated on the AA/TOR pathway, is delayed in minor mosquitoes. This phenotype is rescued by JHIII application. Additionally, phosphorylation of S6 kinase, a significant downstream target of your AA TOR pathway, is stimulated after just one blood meal in typical mosquitoes. This impact was only observed in little mosquitoes with JHIII application. Our effects revealed that the AA TOR signaling pathway regulates vitellogenesis immediately via mosquito larval nutrition and it is mediated by means of JHIII. Hence, our findings produce molecular proof on how dietary problems while in larval growth mediate the anautogenic nature of adult female mosquitoes. Mosquito homologues of Drosophila dorso ventral patterning protease Easter and its inhibitor Serpin 27A are involved with the signaling from the Toll immune pathway within the mosquito, Aedes aegypti Sang Woon Shin, Guowu Bian and Alexander S.

These terms are of interest for CD4 T cell differentiation, as well as the likelihood that tick feeding suppresses transcription through key infestation. This is a possible mechanism behind the late induction of host responses for the duration of principal infestation. Secondary infestation A 2nd exposure to feeding by I. scapularis nymphs resulted inside a a lot quicker and stronger host response as proven in Figures 3A and 3B. In contrast for the main infesta tion, rather significant gene modulation was evident by 12 hrs p. i. The genes modulated during primary infesta tions have been also modulated all through secondary infestations and have been, normally, the genes with all the highest fold alterations. Thus we postulate that genes upregulated dur ing the primary infestation form a core host response that drives anti tick immunity even on repeated publicity. Migration The migration of cells into an inflammatory concentrate is a crucial facet of host immunity.
Resident cells need to realize skin injury from the feeding tick and secrete things that enhance the recruitment of immune effectors towards the bite internet site. Gene ontology analyses of upregulated genes through the secondary infestation strongly support the necessary position of chemotaxis in the anti tick immune response. Precise GO terms recommended the migration selleckchem of neutrophils, monocytes, other leuko cytes, and lymphocytes into the bite web page. The upregulation of CCL1 was the only observed transform in chemokine expression concerning main and secondary infestation. Interestingly, this chemokine has become shown to appeal to Th2 and T regulatory cells. Other upregulated genes recognized to support cell migration incorporated selectins, integrins, and the integrin ligand ICAM1. When quite a few alpha chain integrins had been upregulated, the sole beta chain upregulated was b two.
In support GW3965 of preceding reports that I. scapularis saliva inhibited endothelial cell expression of P selectin, our examine showed only minimal upregulation of SELP that was not supported by later on validation. Cytokines A lot of added cytokines were modulated throughout the secondary infestation when when compared with the main publicity. These transcripts group together to type the cytokine cluster on gene ontology evaluation, lending formal assistance to their relevance in the anti tick response. In particular, IL four and IL 13 were upregu lated, these cytokines can be created by Th2 cells, but additionally by basophils, eosinophils, and mast cells. Basophils have already been shown to get indispensible for anti tick immu nity in models of infestation wherever acquired resistance happens, and their migration into the bite web site was supported through the upregulation of CCL chemokines and IL 3, which are chemotactic variables for basophils. It truly is interesting to note the important grow in expression of IL four and IL 13 throughout the secondary expo confident in spite of the down regulation of IL 25, a crucial inducer of form 2 immunity.

Mailing tackle. Utilized Tumor Virology Program, Div. F010, Inserm U701, Deutsches Krebsforschungszen trum, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany. Telephone. 6221 424969. Published ahead of print on 28 October 2009. modulated by oncogenic transformation, thereby favoring virus multiplication in proliferating neoplastic tissues. How ever, the nature and perform of a few of these characterized factors are to date not sufcient to entirely clarify the parvovirus oncotropism, indicating that still unknown further cellular aspects have to contribute to some extent to this virus property. The rst line of defense produced by cells towards a viral invasion includes the activation of an innate antiviral im mune response by way of the manufacturing and release of sort I inter ferons.
These antiviral cytokines are produced by invaded cells on detection of pathogen connected molec ular patterns consisting of nucleic acids derived from viruses, like double stranded RNA, single stranded PD173074 solubility RNA, or DNA, by cellular pathogen recog nition receptors which have been both membrane bound or current from the cytoplasm. On activation, PRRs stimulate various downstream latent transcription variables, which includes NF B, ATF2 c jun, and interferon regulatory factor three, which then cooperate selleck inhibitor to induce the expression of IFN molecules. This step denes the early phase in the antiviral response. Subse quently, the cytokine is launched from infected hosts and inter acts in an autocrine and paracrine trend with specic mem brane bound receptors, thereby stimulating the downstream JAK/STAT pathway. The latter activation is characterized, particularly, through the phosphorylation with the transcription variables STAT1 and STAT2, their heterodimerization, and more as sociation with IRF 9.
This heterotrimer translocates to your nucleus, binds on the IFN stimulated response element current in the promoters of IFN stimulated genes,

and enhances their transcription. Among the ISGs targeted, numerous code for polypeptides which exert antiviral actions, like PKR or two 5 oligoadenylate synthetase. Other ISGs encode proteins that further improve the antiviral response, this kind of as STAT1 and STAT2, IRF 9, or the tran scription component IRF 7. The latter issue is of major significance for your development with the defense mechanism, because it sets in movement a favourable suggestions regulation with the JAK/STAT path way by inducing the transcription of the 2nd wave of kind I antiviral cytokines belonging each for the likewise as to your subtype. Seeing that IFN s and IFN bind on the exact same receptors, they more activate the JAK/STAT pathway and thereby the antiviral response. Thus, release of form I IFNs by invaded hosts is critical to block viral replication, limit infection, and facilitate virus clearance.

Depending over the distinct Gab family members member, tyrosine phosphorylation may well deliver recruitment internet sites for your SH2 domains from the tyrosine phosphatase SHP2, adaptors from the Crk household, PLC as well as regulatory subunit of PI3K, p85. Nevertheless, the kinases and phosphatases controlling the phosphorylation standing of these tyrosine residues are in lots of cases nonetheless unwell defined. Even though, at the very least in vitro, RTKs such since the EGFR can phosphorylate Gab1 directly, it is actually getting increasingly evident that a variety of systems this kind of as RTKs, antigen receptors, cytokine recep tors and also the Bcr Abl oncoprotein sub contract PTKs within the Src, Syk/ZAP 70 and JAK families to drive the tyrosine phosphorylation of Gab1/2. In some instances, a cascade of PTKs regulates Gab phosphoryla tion, this kind of as the Bcr Abl/JAK2/Lyn pathway in human CML cells. Also, it is actually feasible that personal PTKs might possibly target distinct tyrosine residues in Gab proteins.
By recruiting numerous effectors with SH2 directory domains, Gab pro teins mediate not only signal amplification, AMG208 but, being a func tion of your recruitment of distinct enzymatic routines, also channel the receptor derived signals into pathways with distinct biological properties. Consequently, Gab professional teins act like a nucleation core of an entire signalling sub technique, which we will dissect while in the following sections. The SHP2/Ras/ERK pathway Almost certainly the ideal characterized effector arm of Gab professional teins is mediated by means of the protein tyrosine phosphatase SHP2. SHP2 is made up of tandem SH2 domains, the most N terminal of which confers automobile inhibition from the C termi nal phosphatase domain. Countless SHP2 interaction partners as well as the Gab proteins incorporate two SHP2 binding sites, which, if phosphorylated, will act being a bi phosphoryl tyrosine activation motif and confer simultaneous binding of the two SH2 domains, therefore relieving auto inhibition.
Consequently, SHP2 interaction partners like Gab proteins may not only act as recruit ment platforms, but also as allosteric activators. But what exactly are the functional consequences of Gab mediated SHP2 recruitment and activation The top understood result mediated from the Gab/SHP2 interaction is definitely the sustained and/or improved activation on the ERK/MAPK pathway. This effect takes place in response to several different stim uli, which include treatment of cells with EGF, VEGF, HGF and LPA. Yet, in specific cellular contexts, the Gab/SHP2 complicated also positively regulates other downstream pathways. These consist of c Kit induced Rac activation also as 1 integrin and development issue induced PI3K activation. The thorough mechanisms involved in Gab/SHP2 mediated regulation of Rac and PI3K have however for being resolved. In cultured mam malian cells, recruitment of SHP2 to unique Gab professional teins regulates diverse biological endpoints, like PDGF induced cytoskeletal organization and VEGF induced migration in endothelial cells, cell adhe sion and migration of Ba/F3 haematopoietic cells, epithelial morphogenesis in MDCK cells and acinar development of MCF 10A mammary epithelial cells.

Current review demonstrated a significant correlation among HPV16 positivity and overexpression of STAT3 and pSTAT3 in cervical precancer and cancer lesions. Interestingly, many of the HPV16 precancers and might cer instances specifically the lesions with WDSCC histo pathology showed a lower level of STAT3/pSTAT3 expression and nuclear positivity. Despite the fact that HPV16 posi tivity amid diverse histopathological grades differed only marginally it didn’t similarly correlate with STAT3 expression or nuclear localization. Histopatholo gically even more superior HPV16 PDSCC scenarios expressed greater amounts of nuclear localized STAT3 in comparison to HPV16 WDSCC instances. This displays that mere pre sence of viral DNA in host cells will not induce STAT3 exercise but calls for expression of viral genes/oncogenes to interact with host cell signaling that governs activa tion of STAT3 signaling cascade as well as absence of those components could possibly lead to diminished STAT3 activity.
Furthermore, bodily state of viral DNA plus the copy amount of the virus that influ ence the magnitude of viral oncogene expression can be the critical aspects accountable for variable Selumetinib clinical trial cellular response with respect to amounts of STAT3 activ NPS-2143 structure ity. On the other hand, these upstream mechanisms are still to be investigated. Constitutively active STAT3 has been proven for being connected with larger histological grades and invasive cancer in various epithelial as well as other malignancies. Though the causes for aberrant STAT3 activ ity in cervical precancer and cancer lesions stays to become investigated, its association with HPV16 infection in cer vical carcinogenesis is evident from data presented from the current examine. Research indicate substantial viral oncogenes E6/ E7 mRNA expression or improved viral genomes/cell strongly relate to advanced histopathological grades that favor poor prognosis.
Similarly, STAT3 has also been proven to become a bad prognostic issue in cervical cancer by other investigators. The relation among enhanced STAT3 expression with HPV16 copy variety or its oncogene expression is at the moment not acknowledged, although numerous lines of proof suggest a achievable inter action concerning these two regulatory arms of cervical vehicle cinogenesis. Just like HPV E7 induced transformation

in cervical cells by focusing on retinoblastoma exercise, Simian Virus forty induced transformation triggered by inactivation of pRB by means of its significant T antigen has become shown to lead to upregulation of STAT3. On the other hand, p53 and STAT3 happen to be shown to antago nize expression of each other as p53 prevents the result of STAT3 on cell transformation and STAT3 down regulates the expression and perform of p53 by binding to the p53 promoter and resulting in decreased de novo expression of p53.

Latest preclinical data demonstrated that anti VEGF agents can transiently nor malize the elevated permeability and interstitial stress of brain tumor vessels, enhancing on this way the pene tration of concurrently administered medicines. Moreover direct VEGF or VEGFR2 inhi bition for glioblastoma, clinical scientific studies are remaining con ducted or planned with agents focusing on more downstream or different pathways frequently altered in brain tumors, which include the mTOR/Akt and EGFR pathways. Nonetheless, the accomplishment together with the present compounds from the management of brain tumors is very constrained. It really is likely that blend of therapeutic agents focusing on different pathways, mainly angiogenic pathways, will make more substantial clinical results. On this context, we targeted on leptin, a multifunctional hormone which is ready to exert angiogenic activity in numerous in vitro and in vivo model programs.
Leptin continues to be implicated in neoplastic processes, primarily in weight problems relevant cancers, where the hormone has become proven to selleckchem compound libraries stimulate cancer cells development, survi val, resistance to different chemothera peutic agents as well as migration, invasion and angiogenesis. Inside the central nervous strategy leptin regulates numerous physiological brain functions, like hippo campal and cortex dependent understanding, memory and cognitive perform, neuronal stem cells servicing, and neuronal and glial growth. In addi tion, current analysis suggests the prospective position of this hormone from the progression of brain tumors. We previously demonstrated that the expression of leptin and ObR in human brain tumor tissues corre lates with all the degree of malignancy, as well as highest levels of each markers are detected in GBM. Specifi cally, and in relevance for the current review, leptin and ObR were expressed in over 80% and 70% of 15 GBM tissues analyzed.
Other studies demonstrated lep tin mRNA expression in rat glioma tissues and cell lines. Simply because leptin and ObR in human brain tumors are often coexpressed, leptin results are possible for being mediated by autocrine pathways. Applying in vitro versions, we found that LN18 and LN229 ObR positive GBM cells respond to leptin with cell growth and induction of U-95666E the oncogenic pathways of Akt and STAT3, and also inactivation of your

cell cycle sup pressor Rb. Nonetheless, the probable role of intra tumoral leptin in glioma progression, specifically during the regulation of angiogenesis, has never been addressed. Here we investigated in case the hormone will be expressed by human GBM cell cultures, if it could have an impact on angio genic and mitogenic likely of endothelial cells, and if its action may be inhibited with precise ObR antagonists. The outcomes were in contrast with that induced by the perfect characterized angiogenic regula tor, VEGF.

Therefore, our rationale was if we try to recognize gene signatures inside of well defined pathways, not merely does this strategy alleviate the dimensionality trouble, but the mechanism based mostly gene signatures need to also be even more biologically appropriate compared to the signatures derived through the whole human tran scriptome. Unsupervised hierarchical clustering examination was 1st utilized to divide cancer patients into separate groups dependant on expression patterns of genes in a known pathway. Patient survival during the various groups was then compared. If a specific pathway plays a critical position in tumor progression and metastasis, individuals with distinct gene expression patterns in the pathway may possibly have incredibly numerous clinical outcomes. The outcomes presented right here indicate the pattern of gene expression from the cell cycle pathway can indeed serve as a robust biomarker for breast cancer prognosis.
We even further built a predictive model for prognosis based on the cell cycle gene signature and observed selleckchem our model to be far more precise compared to the Amsterdam 70 gene signature when examined with various gene expression datasets generated from many patient populations. Approaches Information supply 5 distinct gene expression profiling datasets on breast cancers have been analyzed in this research. Various datasets have been utilised to demonstrate repeatability from the analysis. Particular particulars on each dataset are summarized in Table one. For each gene expression dataset, twenty molecular pathways have been analyzed. The twenty pathways were assembled in the Inge nuity Pathway databases and also the SuperArray cancer pathway array annotations. The list of twenty pathways and genes inside of every single pathway are supplied in additional files. Data preprocessing For every array study according to Affymetrix oligonucleotide platforms, we downloaded the.
CEL files and produced gene expression values working with the Affymetrix MAS5 algo rithm with trimmed mean values normalized to 500. A trimmed imply is the normal value immediately after getting rid of the lowest 2% and also the highest 2% of all expression values for the array. Prior to examination, each information set was preprocessed selleck chemicals with a log2 transformation and subsequently expression of each gene was standardized

employing median centering. Data transformation and standardization have been performed applying scripts written while in the R statistical programming lan guage. Whenever a gene is represented by various probe sets on Affymetrix oligonucleotide arrays, the average expres sion value was made use of for more examination. Hierarchical Clustering Every pathway specific information set was analyzed by hierarchi cal regular linkage clustering. The clustering was per formed making use of Gene Cluster three. 0 or applying R packages. The resulting numerical output was utilized by Java Treeview v1. 1 to gener ate the connected heatmaps and clustering dendrograms.

An fascinating obtaining within this review was that curcumin appeared for being sparing the regular epithelial cells by arresting them on the G0 phase of the cell cycle by means of down regulation of cyclin D1 and its linked protein kinases or up regulation of the inhibitory protein. The experiments with cyclin D1 deregulated cells showed that curcumin did not alter cyclin D1 expression degree in cancer cells, but in normal cells, wherever cyclin D1 expression is tightly reg ulated by mitogenic signaling, its expression is inhibited by curcumin. This inability of curcumin to inhibit cyclin D1 expression in cyclin D1 deregulated cells may serve because the basis for differential regulation of cancerous and nor mal cells. Furthermore, curcumin was noticed to inhibit the association of cyclin D1 with CDK4/CDK6 or phosphor ylation of pRb in some cancer cells wherever the expression of cyclin D1 will not be deregulated and thus arrest them at G0/ G1 phase.
This yellow pigment has been shown to inhibit neoplastic cell proliferation by decreasing Cdk1 kinase exercise and arresting cells at G2/ M test level. Ectopically in excess of expression of cyclin D1 renders susceptibility of those cells in the direction of PP242 clinical trial curcumin toxicity. These final results may well nicely clarify why in cancer cells, regardless of up regulation of p53 and maximize in Cip1 degree, there was no cell cycle arrest. The fact is, the degree of cyc lin D1 is very large in these cells and remained unchanged upon curcumin treatment. Consequently, the quantity of Cip1, as up regulated by curcumin, was still not enough to in excess of power cyclin D1 and also to end cell cycle progression. For the other hand, in non malignant cells, the level of Cip1 elevated radically with parallel down regulation of cyclin D1, thereby creating the ratio of Cip1 to cyclin D1 one and this may be one particular in the triggers of cell cycle arrest with out apoptosis.
The over discussion not simply relates curcumin exercise with cell cycle regulation but also explains the mechanism underlying the differential result of this phytochemical in regular and malignant cells. Curcumin regulating guardian of genome The tumor suppressor gene p53, acknowledged since the guardian of genome, is situated at the crossroads KRN-633 of a net perform of signaling pathways that happen to be very important for cell growth regulation and apoptosis. In regular unstressed cells, these upstream pathways predominantly contain the binding by proteins this kind of as Mdm2 that professional mote p53 degradation by way of the ubiquitin 26S proteasome

pathway. COP9 signalosome exact phos phorylation targets p53 to ubiquitin 26S proteasome dependent degradation.