This review will target around the Aurora kinase relatives, members of which management processes ranging from centrosome perform to chromosome segregation and cytokinesis. On a cellular level, dysfunction of those kinases can lead to aneuploidy and/or tetraploidy, and misregulation of these kinases is related with tumourigenesis. Aurora kinases Mammalian genomes incorporate 3 genes encoding Aurora kinases, denoted Aurora-A, Aurora-B, Aurora-C. The three Aurora kinases are serine?threonine protein kinases that with each other type a smaller kinase relatives phylogenetically linked to the branch in the AGC protein kinases . The founding member with the Aurora kinases stands out as the Drosophila Aurora kinase that was found within a display to determine genes involved in mitotic spindle function .
Fungi have only one Aurora kinase that’s functionally a lot more related to the Aurora-B kinases in increased organisms. Mammals have a third Aurora gene named Aurora-C. Aurora kinase perform is managed ATP-competitive MEK inhibitor by a number of mechanisms. Firstly, gene transcription from the Aurora kinases is cell cycle regulated. The promoters of Aurora-B and Aurora-A contain precise sequences demanded for transcription in G2 . Quite a few transcription aspects, such as E2F-1, E2F- 4, DP-2 and FoxM1 are already implicated in cell cycle regulated transcription of Aurora-B G2 . The Aurora-C gene seems to be mainly expressed in meiotically dividing cells, and its transcription is at the very least in portion controlled by a testis-specific transcription aspect called Testis Zinc Finger Protein . Regulation by activation and localisation Structurally, the Aurora kinases are closely linked to the family members of AGC kinases.
The AGC kinases are selleck chemicals high throughput chemical screening activated by an intramolecular interaction involving two distinct, extremely conserved sequences. Folding of the COOH-terminal sequence in a hydrophobic motif pocket is needed for full kinase activation and a few of the AGC kinases require phosphorylation within this pocket for activation . Even so, the Aurora kinases lack the common COOH-terminal motif, nevertheless they are nonetheless activated in the related molecular method, albeit together with the help of interacting cofactors that include these important motifs. Various cofactors are described for the Aurora kinases that are essential for right activation and function. .
Aurora-A For Aurora-A, the most beneficial characterised cofactor is TPX TPX2 was at first recognized like a microtubule-associated protein necessary for bipolar spindle assembly and it had been later on proven to bind and target Aurora-A on the mitotic spindle, but not to the centrosomes . TPX2?Aurora-A binding is promoted by an active Ran-GTP signalling pathway .
The original hit record consisted of a different series of compounds that had a substituted , diamino mercapto H pyrimidin 1 core. Because of the novelty from the core and their viability for a number of hydrogen bonds using the protein, two compounds and have been picked that has a representative substitution pattern. The efficiency of those compounds in inhibiting the VEGFR kinase was measured by their ability to stabilize or slow down the progression of angiogenesis. There are numerous assays in existence which have been acknowledged for being representative of in vivo angiogenesis. A study of formation with the 3 dimensional world wide web like structures of interconnected cells by the endothelial cells is amongst the widely used and extensively employed qualitative assays. The formation of your cellular networks progresses inside a stepwise manner together with the preliminary migration and alignment of cells, growth of capillary tube like structures, sprouting of new branches, and ultimately formation of cellular networks. The compounds had been subjected to the over assay to comprehend their capability to inhibit the activity of endothelial cells.
The outcomes are depicted in Figure and Table . As might be viewed, compounds and inhibit progression of angiogenesis to a major extent at lM concentration. The cells Sirt inhibitors don’t migrate and align, and they usually do not sprout branches for network formation. Compounds , as well as demonstrate inhibition of angiogenesis, but to a lesser extent. Compound showed lesser inhibition of angiogenetic method, perhaps, showed only the final stage of your system. Compound has negligible inhibitory effect. The effectiveness of angiogenesis inhibition shown by compounds and existing these as the most promising lead compounds. They make the necessary two hydrogen bonds with all the hinge region residues Glu and Cys of VEGFR kinase. The dose response curve for that compound is shown in Figure . This compound has inhibition at . lM concentration. Compound makes additional hydrogen bond with Cys by the side chain amide NH along with the terminal amino group . A single supplemental hydrogen bond is formed among the side chain amide carbonyl of compound and Gly of your protein .
These more interactions could be the contributing factors for the more effective inhibition MK-4827 of angiogenesis. Novel cores have already been recognized containing pthalimide and , diamino mercapto H pyrimidin a single. Additional modifications of those cores can be carried out for acquiring alot more potent angiogenesis inhibitors. Different substitutions and heterocyclic ring extensions for your side chain will probably be studied to enhance the potency of these lead compounds. Additionally, it is actually necessary to verify that these compounds inhibit immediately the function of VEGFR kinase in an effort to establish a connection amongst VEGFR kinase targets and angiogenesis.
The cellular Aurora A and Aurora B IC values for had been determined as . and . lM, respectively, indicating an approximately fold selectivity in inhibiting Aurora A. Encouraged by the sizeable increase in Aurora A inhibitory potency with analogues and , we studied their cell growth inhibitory activity in two cancer cell lines . All three compounds displayed cellular potency with GI values among and lM . The binding mode of this class of imidazo pyrazine based kinase inhibitors was elucidated by co crystallisation of Aurora A catalytic domain DN mutant with to a resolution of . As we hypothesised, occupies the ATP binding webpage with the imidazo pyrazine N and C NH forming hydrogen bonding interactions together with the Ala while in the hinge region in the kinase.
Imidazo pyrazine N is hydrogen bonded on the principal chain NH of Ala as well as the C NH on the carbonyl of Ala as proven in Figure A. The chlorine atom from the inhibitor sits inside a hydrophobic pocket formed from the gatekeeper residue , and in addition Val, Leu and Leu. Importantly, the C pyridyl yl substituent order StemRegenin 1 resides in near proximity to Thr of Aurora A , whereas the equivalent residue in Aurora B C may be a glutamic acid . This is often 1 of the three active website sequence distinctions in between Aurora A and Aurora B C. The Leu side chain in Aurora A points away from the lively web-site. There is tiny selectivity to get acquired from focusing on the side chain of Arg and that is extremely mobile and disordered in our co crystal construction. We exploited this observation during the layout of compounds with considerably enhanced selectivity in inhibiting Aurora A more than isoforms B and C.
It was envisaged that isoform selectivity for Aurora A may very well be attained from the introduction of the C phenyl ring bearing an electron wealthy substituent capable of forming a hydrogen bond with Thr in Aurora A, which would sterically clash with the equivalent residue in Aurora B C. On this basis, the sulfonamide derivative was ready telomerase inhibitors from , by initially reacting with phenylboronic acid beneath the ailments described in Scheme , after which treating the cross coupling product or service with CHSOCl in pyridine CHCl. Compound inhibited recombinant human Aurora A and Aurora C with IC values of . and lM, respectively. In cells, displayed a fold selectivity in inhibiting Aurora A over Aurora B, the cellular IC values determined as . and . lM, respectively.
While in the same cellular assay, we identified that the Aurora A inhibitor MLN was fold selective for Aurora A above Aurora B. The crystal framework of bound to Aurora A was established to a resolution of ?, and displays that occupies the ATPbinding site inside a mode similar to that observed for .
The compounds that had been capable of delivering exactly the same scaffold molecules by using a possible hydrogen bond donor at these positions also showed higher exercise, and these two positions have been also mapped onto pharmacophoric options. On the other hand, red areas have been observed in the carboxyl group with the C ring in compound , leading to a reduce in activity . Inhibitor C illustrates one of the most important favorable hydrophobic interactions that arose once the QSAR model was applied to compound , imatinib. Within the instruction set , additional pronounced blue regions were visible throughout the D ring, which was present in all the most energetic ligands. In particular, the more attachment of an aromatic ring to your D ring showed enhanced pursuits, thus generating these capabilities extra crucial for action regardless of these benefits remaining missing through the pharmacophore. Yet, within the context of a single or far more inactive ligands, the QSAR model can offer knowledge about structural benefits that may be detrimental to exercise.
Such as, Inhibitor D shows clear purchase Palomid 529 unfavorable interactions for your inactive education set compound ; red regions found on the ether chain of the C ring, indicating hydrophilic groups, such as CF CHF, usually are not favorable for exercise. Inhibitors E and F assess the favorable and unfavorable hydrogen bond acceptor and electron withdrawing qualities that arose once the QSAR model was utilized towards the most lively compound and inactive compound , which showed intermediate exercise. For compound , blue areas associated with electron withdrawing groups were noticeable around 1 nitrogen in the E ring, around the N group, and across the oxygen atom of your carbonyl group between the C ring and D ring, indicating their value for activity.
In compound , red regions have been linked with NH groups Fluorouracil situated in the meta position in the D ring, and red areas have been situated at the oxygen atom from the carbonyl group in between the C ring and D ring, thereby leading to a lessen in exercise . In this examine, a pharmacophore model was formulated for PAP derivatives by using training set molecules and additional cross validated by a test set of molecules. The seven characteristic CPH was noticed to get far more predictive compared with other typical pharmacophore hypothesis. The model effectively classified active and inactive molecules according to the action scale as well as gave satisfactory results for your cross validation tactics.
Producing a pharmacophore can supply essential knowledge for insight into the inhibitory likely of different chemotypes, this kind of as PAP derivative activities, and may be used within the design of novel BCR ABL inhibitors. In addition, the contours generated from QSAR research supply specifics to the connection involving structure and action and therefore are valuable for that additional design of extra potent inhibitors just before synthesis.
In flip, AKT regulates cell division by improving cyclin D stability and or by inactivating pkip either by regulating pkip mRNA expression, or by controlling the subcellular localization of pkip. Right here we report the PIK AKT pathway is activated in of thyroid carcinomas, and that this signaling cascade contributes to inactivation of pkip as a result of AKTdependent cytoplasmic sequestration of pkip. Materials and Systems Cell Lines and Reagents 5 human thyroid carcinoma cell lines have been utilized within this review: TPC and NPA , WRO , and FRO and FB All cell lines have been grown in Dulbecco?s modified Eagle?s medium containing fetal calf serum. LY and cycloheximide had been from Sigma Aldrich . Wortmannin was from Calbiochem . Tissue Specimens Thyroid carcinomas were collected in the Surgical procedure B segment on the National Cancer Institute Fondazione G. Pascale or retrieved from the files from the Division of Functional and Biomorphological Sciences at the University of Naples Federico II. Diagnosis was depending on common histological criteria. We chosen paraffin blocks that were zero cost of oxyphilic changes, that are sources of aspecific cytoplasmic staining, and that integrated both the tumor as well as the rim of usual thyroid tissue about it; the latter serving as manage of immunohistochemical staining.
Immunostaining Serial sections had been stained implementing monoclonal antipkip antibody and also the polyclonal antibody antiphospho AKT from Cell Signaling Engineering at a dilution of Following incubation with primary antibodies, the sections selleck pop over here had been incubated with biotinylated anti mouse rabbit immunoglobulins, and with peroxidase labeled streptavidin . Diaminobenzidine was put to use to visualize the signal. Sections incubated while not the distinct antibody and sections incubated with unrelated antibodies served as controls of the strategy. Antibody specificity was assessed by competition with antigens employed for antibody manufacturing . Tumors were scored as pkip optimistic or pkip unfavorable dependent on the staining cutoff of , as described elsewhere. pkip expression was evaluated from both nuclear and cytoplasmic staining.
pkip constructive tumors with cytoplasmic plus nuclear staining or with only cytoplasmic selleck great post to read staining have been designated cytoplasmic and tumors with only nuclear staining have been designated nuclear. If of cells have been stained, the tumor was thought about AKT good. BrdU Incorporation and Indirect Immunofluorescence To the bromo deoxyuridine monophosphate incorporation assay cells were grown to subconfluence on coverslips, incubated with mol L BrdU for hrs, fixed in paraformaldehyde, and permeabilized with . Triton X . We put to use Texas Red conjugated secondary antibodies to reveal BrdU optimistic cells, and fluorescein isothiocyanate conjugated secondary antibodies to reveal pkip good cells. Cell nuclei have been recognized by Hoechst staining. Fluorescence was visualized by using a Zeiss epifluorescent microscope outfitted with filters that discriminated between Texas Red and fluorescein.
Each set of data factors was curve fitted by a non linear regression, greatest fit, sigmoidal dose response curve without constraints, using the use of GraphPad Prism . Every single curve represents at the least individual preparations from a minimal of rats. Analysis was performed to the regular within the variety of preparations, since the variance amid preparations through the exact same artery was just like variance amongst preparations from unique animals. In addition, each time it was conceivable, arterial segments from your very same animal have been represented from the different experimental situations. By way of example, often, 4 segments from one femoral artery had been incubated with car , another 3 with 3 distinctive concentrations of troglitazone.
Whole curves were compared by two way evaluation of variance , with significance set at p A a single way ANOVA followed by Bonferroni t check for significance was utilized to densitometry evaluation Final results Impact of thiazolidinediones on vasomotor responses To investigate the impact of thiazolidinediones on vasomotor responses, we primary challenged isolated segments selleck chemicals small molecule inhibitors of rat femoral artery using a depolarizing mmol L KCl resolution; this to start with contraction, obtained while in the absence of thiazolidinedione, was taken as inner manage . The preparations have been then incubated for min inside the presence of different concentration of troglitazone, before staying challenged once more with KCl or with phenylephrine , an aadrenergic vasoconstrictor agonist. As proven in Inhibitor vasomotor responses to PE and KCl have been inhibited by troglitazone, inside a concentration dependent method ; mM troglitazone blocked by vasoconstriction induced by both PE or by KCl. The results of troglitazone, rosiglitazone and pioglitazone had been also tested on contractile responses induced by a single challenge with mM PE; on this paradigm, we analyzed the effect of thiazolidinediones within the phasic peak of contractile tone, taking place within min, and over the tonic contraction, as assessed at min.
As VX-950 shown in Inhibitor. C, every one of the 3 thiazolidinediones examined inhibited the contraction to PE, rosiglitazone currently being much less potent than troglitazone, and pioglitazone very much significantly less potent than both rosiglitazone and troglitazone; on top of that, the inhibition of tonic contraction to PE by all the three thiazolidinediones was significantly a lot more pronounced compared to the inhibition of phasic peak. So as to elucidate whether or not PPARg receptor stimulation was related to the observed inhibitory effect of thiazolidinediones on vascular contraction, we implemented GW, a PPARg antagonist .
WT and SD Bcl xL proteins had been overexpressed to a related extent and SA Bcl xL protein to a reduce degree . Only Bcl xL.SD was acknowledged through the phospho specified anti Bcl xL antibody . As expected, overexpression on the WT Bcl xL absolutely prevented oxaliplatin TRAIL induced apoptosis , confirming that it largely relies over the mitochondrial pathway. Interestingly, ectopic expression within the phospho mimic Bcl xL SD enhanced apoptosis induced by TRAIL alone and in combina tion with oxaliplatin, whereas HT Bcl xL.SA cells had been clearly even more resistant to oxaliplatin TRAIL blend. Due to the fact Bcl xL is proven to inhibit apoptosis by interacting with Bax and or Bak, we next examined no matter if combined treatment options, by means of Bcl xL phosphorylation, may well alter their interactions with Bcl xL. An interaction in between Bcl xL and Bax was detected in manage and TRAIL handled cells . This interaction was diminished considerably following oxaliplatin remedy, independently of TRAIL stimulation . About the other hand, no important interaction was detected among Bcl xL and Bak in the two cell lines .
Furthermore, phosphomimic Bcl xL showed a weaker interaction with Bax in management cells, as in contrast with NPI-2358 solubility the WT Bcl xL . In addition, though Bcl xL Bax heterodimers have been maintained on therapy in HT BclxL. WT , this interaction was impaired while in the presence of oxaliplatin in HT Bcl xL.SD . These observations have been confirmed in reciprocal anti Bax immunoprecipitation . The lack of interaction amongst Bcl xL and Bak was confirmed in HT.EV, HT.WT, and HT BclxL. SD cells , suggesting the function of Bak concerning oxaliplatin TRAIL induced apoptosis is not really linked to Bcl xL Bak interaction. We subsequent sought to find out the activation status of Bax launched from Bcl xL Bax heterodimers by immunoprecipitation experiments with the anti Bax A antibody, which especially recognizes lively Bax. Bax was activated solely using the combined remedy in HT.EV cells , but not in HT Bcl xL.WT . In agreement with all the hypothesis that Bcl xL phosphorylation prevents Bax sequestration, Bax activation was detected after the two TRAIL and oxaliplatin TRAIL solutions in HT BclxL.
SD cells . Moreover, as shown by co immunoprecipitation, Bak only read full report interacted with active Bax in HT EV cells handled with all the oxaliplatin TRAIL combination or HT Bcl xL.SD cells treated with TRAIL alone irrespective of oxaliplatin pretreatment . Bax Bak interaction was dependent entirely on Bax activation for the reason that Bak was immunoprecipitated only in cell extracts corresponding to active Bax . These success indicate that oxaliplatin primes mitochondrial activation by inhibiting Bcl xL induced Bax sequestration, therefore making it possible for Bax Bak interaction on apoptosis engagement.
In RT PCR assays performed on tissue samples this quantity corresponds to cells carrying the fusion gene if expression was equivalent to your H cell line. Very low fusion transcript expression in some usual and tumor samples, even though FISH detected the fusion gene in to of cells, suggests that fusion favourable cells in tissues express reduced amounts of fusion transcript than the H cell line. Our success indicate that EML ALK transcripts are certainly not tumor precise for NSCLC, given that they are really detected in about of distant non tumor lung tissues and are not retained while in the paired NSCLCs. EML ALK Protein Expression in NSCLC Harboring the EML ALK Transcript Scientific studies on EML ALK protein expression in NSCLC harboring EML ALK mRNA are scarce. To deal with this matter, we initially assessed the capability of anti ALK mAbs to acknowledge the EML ALK protein by Western blot and immunoprecipitation in lysates from the H cell line and EML ALK transfected Phoenix cells. All anti ALK mAbs acknowledged the EML ALK merchandise on the expected molecular weights .
A representative illustration employing the ALKc mAb is proven in Inhibitorsure A . Exactly the same antibody also immunoprecipitated the fusion protein from EML ALK transfected Phoenix cells . In manage lysates from Karpas and Rh cell lines, anti ALK antibodies recognized proteins with all the anticipated molecular weights of NPM ALK and total length ALK , respectively . We then sought the EML ALK protein in NSCLCs carrying the EML ALK transcript variant , for which sufficient pop over to this site material was readily available for examination. Neither Western blotting nor immunoprecipitation of NSCLC lysates with ALKc mAb and subsequent Western blot ting with ALKc or ALK p mAb uncovered the EML ALK protein in cancer specimens . Identical outcomes had been obtained in one particular non tumor lung sample with EML ALK transcript variant . Similarly, no particular EML ALK band was detected while in the single NSCLC specimen or in two non tumor tissues harboring the EML ALK variant transcript by both Western blot or immunoprecipitation .
In contrast, hybrid EML ALK proteins in the anticipated molecular weight were strongly expressed in, and immunoprecipitated from, the H cell line and EML ALK transfected Phoenix cells . These results demonstrate that Western blot and immunoprecipitation didn’t detect the EML ALK protein in NSCLC and non tumor lung samples expressing EML ALK transcripts. FISH Examination of EML ALK Gene in NSCLC Raltegravir Harboring EML ALK Transcript Inability to detect the EML ALK protein can be on account of: i tumor cells generating a lower amount of, or no, fusion protein; ii a minority of tumor cells carrying the EML ALK gene; or iii a combination of the two occasions.
Subsequently, sections had been washed for hrs in ethanol DEPC treated water followed by staining with Harris hemalaun. Color was developed by incubation with DEPC taken care of tap water for hours. Lastly, slides have been dipped in pure ethanol and xylol, air dried, and subjected to get in touch with absolutely free laser microdissection at a magnification of . The dissected normal epithelia and carcinoma epithelia had been instantly transferred into lids of PCR vials and collected in RLT buffer substituted with mercaptoethanol. LCM derived tissue samples from stromal cells, from matched usual epithelial cells and from BPH were isolated and taken care of as described above. Complete cellular RNA from LCM samples was extracted with the RNeasy Mini Kit . RNA integrity and quantity was assessed using the Agilent Bioanalyzer using the RNA Pico LabChip Kit . Added controls have been performed by analyzing unstained tissue sections and the remaining tissue following microdissection. Reverse transcription with random hexamer primers was carried out with all the Sensiscript RT Kit .
Total RNA was isolated from tissue samples, Pc cells, LNCaP cells, and DU cells together with the RNeasy Mini Kit according to the manufacturer?s directions, separated on a denaturing agarose gel and transferred to a Hybond N nylon membrane . The cDNA probes for human actin and human BI have been obtained from Deutsches Ressourcenzentrum fu? r Genomforschung GmbH . The probes have been labeled with dCTP implementing the rediprime II labeling kit and hybridized to your membrane hop over to this website in Speedy hyb buffer collectively with g ml denatured salmon sperm DNA at C for hrs. The filters were washed at area temperature for minutes in X SSC followed by to minutes in .X SSC and . SDS at C. The hybridization signals have been quantified with a Molecular Imager FX through the use of the Amount A single software program . Western Blot Examination The goat polyclonal antibody against human BI was purchased from Santa Cruz Biotechnology Inc as well as mouse monoclonal antibody towards tubulin was obtained from Aldrich.
Parental and transfected Pc , LNCaP, and DU cells have been incubated from the appropriate medium as described above and full cell lysates have been ready from lysis buffer containing mmol L NaCl, mmol L ethylenediaminetetraacetic acid, mmol L Tris HCl pH Triton X , g ml leupeptin, g ml aprotinin, and g ml phenylmethylsulfonyl fluoride. Fifty g of complete cell lysates U0126 were boiled and denatured in sample buffer containing SDS and dithiothreitol followed by gel electrophoresis making use of the NuPage Bis Tris pre cast gel in MES buffer . The proteins had been electrotransferred to nitrocellulose membrane Hybond C . The resulting protein bound membrane was blotted with selected antibodies as described over and visualized using bromo chloro indolyl phosphate nitroblue tetrazolium reagents . Trypan Blue Staining Cell death was established by trypan blue exclusion. Just after treatment with siRNA duplex or management nucleotides l of the . alternative of trypan blue were added to . ml of a Pc cell suspension .
Moreover, HPLC chromatography of this protein on a C column yielded just one peak . SBTI hemagglutinated rabbit erythrocytes taken care of with trypsin; this exercise was also inhibited by mucin, thyroglobulin, fetuin, N acetylneuraminic acid, and heparin . Nb lymphoma cell and splenocyte viability Nb lymphoma cell viability assays with raising concentrations of PDTI are shown in Inhibitors A. Final results demonstrated that this protein brought on a lessen of viability of those cells and that there was an optimum concentration in which this impact was observed elg mlT. Once the similar assay was carried out with SBTI a equivalent result was obtained but the optimal concentration was increased elg mlT. To find out whether or not the result is a consequence of your trypsin inhibitory or the lectin like action of PDTI, either heparin or N glycolylneuraminic acid was additional on the cell culture along with lg ml of PDTI. The addition of mg ml heparin didn’t lead to any significant variation with respect to your results for PDTI alone. However, heparin at mg ml was toxic for your cells.
N glycolylneuraminic acid at mM enhanced the PDTI impact of reducing Nb lymphoma cells viability. At mM this substance was toxic for the cells . Next, a feasible result of PDTI and SBTI on mouse splenocytes was evaluated. To selleck chemicals additional reading examine the action of these proteins on splenocytes viability, the same assays have been carried out with escalating concentrations of PDTI or SBTI and, as shown in Inhibitors C or D, respectively, no substantial variation was observed in any situation. Taking benefit of your preferential activation of T lymphocytes with concanavalin A, very similar assays had been carried out with these cells. The outcomes obtained with PDTI showed a pattern very similar to those obtained on Nb cells. To the other hand, SBTI was capable of reducing viability even at substantial concentrations elg mlT . However, neither PDTI nor SBTI brought about this kind of a high degree of cell death as that observed on lymphoma cells.
Electrophoretic examination of DNA fragmentation Using the aim of characterizing an apoptotic event in the two lymphoma cells and Con A activated splenocytes, an agarose gel electrophoresis was carried out on DNA obtained from cells taken care of with PDTI or SBTI . The classic attribute of apoptosis, cleavage of genomic DNA into nucleosomal fragments represented by multiples of bp, Dorzolamide was observed in lymphoma cells as a consequence of the presence of SBTI or PDTI . The exact same ??ladder?? pattern was observed with DNA obtained from Con A activated splenocytes treated with dexamethasone, a glucocorticoid regarded to induce lymphocytes apoptosis , SBTI , or PDTI . PDTI at a concentration of :lg ml didn’t lead to noticeable DNA fragmentation. The presence of serine protease inhibitors has been detected in microorganisms and in animal and plant tissues .