We conclude that SRY isn’t going to appear to require DNA binding properties to inhibit catenin signaling. In addition, the choosing that de novo SRY mutants examined, all of which have recognized nuclear import defects, never inhibit catenin function as efficiently as wild form protein implies that the nuclear localization of SRY is vital for inhibition of catenin signaling. The loss of potential of SRY clinical mutants to inhibit catenin signaling implies that this repressive function of SRY is very likely to get a requisite for male intercourse determination in humans Discussion Vertebrate SOX proteins are potent effectors of catenin signaling demanded for crucial functions just like chondrogenesis, intestinal epithelium differentiation and neuronal differentiation. Here we extend these observations by linking the master sex determining gene SRY, the founding member with the SOX relatives, using the Wnt catenin pathway by showing that SRY can inhibit catenin signaling in HEK2T cells. Whilst the inhibitory result of various SOX proteins on Wnt catenin pathway is known in these cells, the molecular mechanism of inhibition is still unclear.
Probably Tivozanib selleck this confusion resides in our inabil ity to clearly discriminate in between the transcriptional and non transcriptional functions of SOX proteins. A non transcriptional function of SRY We’ve demonstrated that SRY is ready to interact directly with catenin in vitro and strongly reduce the catenin protein levels within HEK2T cells. These observations are constant with routines of SOX1 and SOX which could each interact with, and cut down catenin ranges . Because the reduction of catenin levels due to SOX1 or SOX was attributed to an greater proteasomal degradation of catenin, it truly is also probable that SRY acted within a comparable vogue and thiswarrants even further investigation. Interestingly,although the two the C terminus and N terminus of SRY interact with catenin in vitro, only the C terminus within the protein was important in mediating its inhibitory activity as unveiled by L1X sex reversed mutant.
That is analogous on the catenin interaction domains of SOX1 and SOX proteins which also lie within the C terminus of each protein, despite the fact that these domains share little similarity in amino acid sequence . These data suggest that SRY antagonizes the Wnt catenin pathway on the non genomic degree by way of a direct protein protein interaction concerning SRY and catenin rather than as a result of standard DNA binding and transactivation functions. This is supported from the observation that the Proteasome inhibitor DNA binding activity of SRY is just not necessary for inhibition with the Wnt catenin pathway in HEK2T cells and through the fact that an SRY fusion protein harboring a strong transcriptional activation domain did not modify SRY activity.

Using ECM fragments is usually a promising approach to decrease the risks of unpredictable effects, limiting the complications associated to the functional complexity of ECM proteins. Each viral and non viral vectors for gene delivery have been proposed. Effective antiangiogenic and antineoplastic therapeutic effects were obtained with recombinant adeno related virus mediated delivery of endostatin or the P12A endostatin variant with all the IgG kappa chain leader sequence utilized like a secretory signal . Similarly, the Lister vaccine strain of oncolytic vaccinia virus and also a prostate restricted adenovirus armed with a gene formed by endostatin fused to angiostatin reportedly produced sustained ranges of fusion protein and inhibited angiogenesis and experimental tumor growth. AAV mediated delivery on the 3 TSP 1 kind 1 repeats resulted in the expression with the transgene in usual tissues, diminished VEGF induced angiogenesis, and lowered tumor development and microvessel density each locally and at distant online sites . Fibroblasts retrovirally transduced to produce large levels of TSP 2 and seeded in polymer scaffolds were utilised as being a cell based mostly approach to supply constant in vivo manufacturing of TSP 2.
This program was profitable in inhibiting angiogenesis plus the development of various tumor designs . The production of endogenous inhibitors of angiogenesis mTOR inhibitor selleckchem is usually modified by acting for the genetic regulation of their synthesis. As an example, considering the fact that inactivation of oncosuppressor genes is associated with the acquisition of angiogenic capacity by tumor cells, therapeutic approaches aimed at restoring the functions of those genes could restore the angiogenic stability. Topical delivery of p DNA on the lung upregulated TSP 1, lowered lung tumor burden and tumor microvessel density and prolonged survival of tumor bearing mice . MicroRNAs are an additional instance . Silencing Dicer, the terminal endonuclease accountable for your generation of miRNAs, reduced TSP 1 expression in endothelial cells too as tumor induced angiogenesis and tumor development . Inhibition of miR 1 two improved TSP 1 expression . While nevertheless preliminary, these research set the bases for that advancement of antiangiogenic techniques acting on miRNA.
The synthesis of ECM derived Y-27632 price kinase inhibitor inhibitors of angiogenesis is stimulated by regular very low dose chemotherapy . The antiangiogenic and antineoplastic action of metronomic cyclophosphamide, FU, doxorubicin and paclitaxel in preclinical designs was related with increased ranges of TSP 1 . In patients, minimal dose metronomic irinotecan raised plasma levels of TSP one in metastatic colorectal cancer patients and elevated TSP one gene expression in PBMC Modulation of fragment release Angiogenesis may also be inhibited by stopping the proteolytic release of pro angiogenic ECM fragments and development components, which occurs through matrix degradation.

In addition, overexpression of Aurora A, which acts as an oncogene, has become demonstrated to end result in an abrogation from the spindle checkpoint leading to resistance in direction of taxol . Given that colon carcinomas exhibit a really large incidence of chromosomal instability, which could be associated with spindle checkpoint malfunction, a weakened checkpoint may possibly make clear the bad efficacy of paclitaxel or connected medication on this entity. In addition, survivin is commonly overexpressed in cancer cells and this may possibly contribute not simply to spindle checkpoint malfunction, but also to a hyperactive mitotic survival checkpoint rendering tumor cells resistant to paclitaxel remedy . An additional reason for resistance towards anti microtubule medicines may well be a modulation of your microtubule composition as well as a adjust in microtubule dynamics. Resistant tumor cells have been proven to express mutant varieties of and tubulin, by which the drug binding web-sites are mutated . Alternatively, resistant tumor cells have been shownto overexpress a specific isoform of tubulin , which success in considerable greater microtubule dynamics .
The exact same result is made by mutations in tubulin or by overexpression of Nilotinib distributor kinase inhibitor microtubule destabilizing proteins or by reduction of microtubule stabilizing proteins . The reality is, a deregulated expression of microtubule associated proteins is detected in cancer cells . Whilst adjustments inside the composition and dynamics of microtubules can obviously contribute to resistance in the direction of taxanes and also other anti microtubule drugs in vitro, it isn’t clear no matter if these mechanisms without a doubt account for resistance in individuals . Importantly, taxanes and Vinca alkaloids are very superior substrates for your P glycoprotein drug efflux pump, the solution in the multidrug resistance gene, which right contributes to a reduced cellular concentration within the drug . Then again, epothilones escape from MDR mediated efflux and therefore are as a result lively even in lots of taxol resistant tumor cell lines. Hence, quite a few other microtubule binding medicines which might be not substrates to the Pglycoprotein are now below investigation Mitotic kinesin: KSP Eg The function of KSP Eg Provided the truth that anti microtubule drugs drastically interfere with the perform of microtubules in resting and differentiated cells, which can bring about e.
g. peripheral neuropathies, there exists an urgent will need to determine novel drug targets that interfere using the usual progression of mitosis without the need of modulating the function of microtubules. Kinesin proteins signify promising candidates. Kinesins are Tivozanib a relatives of proteins that bind to and move along microtubules via their ATP dependent motor domain. In interphase, kinesin family members are responsible to the transport of cargo and, through mitosis, several kinesins are essential for the right chromosome alignment, segregation and centrosome separation .

We have found that PIs have markedly heterogeneous effects on eIF phosphorylation in the cells. In some , PIs fail to induce significantly, if any eIF phosphorylation or inhibition of worldwide protein synthesis, though in other people , PIs activate the UPR and downregulate translation really effectively. There is a suggestion that baseline levels of eIF are greater within the cells that fail to activate the UPR, but otherwise we’ve not yet identified the molecular mechanisms involved in these variations. Then again, preceding work has demonstrated that phosphorylation of eIF activates autophagy in cells infected by viruses or exposed to variety I interferons or through nutrient deprivation . Because autophagy is an option route of degradation for toxic protein aggregates, it might most likely play a cytoprotective part in some tumors. Certainly, preceding studies have demonstrated that combinations of PIs plus chemical inhibitors of autophagy induce larger levels of cell death than either sort of agent alone , an observation that we’ve reproduced in human prostate cancer cells .
Furthermore, we’ve got found that PIs trigger increases in the transcription of a variety of autophagy pathway genes , and these effects are also dependent on eIF phophorylation . In addition, the effects of international translational suppression probably have an even more important and quick effect on protein toxicity, given that it would TAK-875 swiftly shut off the input protein synthesis that compounds the tension. Function of aggresome formation in PI induced cell death As discussed above, an additional protein that mediates the coupling amongst the proteasome and autophagy is HDAC, which facilitates the transport of protein aggregates to perinuclear aggresomes . Proteasome inhibitor induced aggresome formation can be blocked by silencing HDAC or by exposing cells to pan particular chemicalHDACinhibitors like trichostatinAorSAHA . Thus, chemical pan HDAC inhibitors are amongst by far the most potent PI sensitizing agents we have identified to date, and they are capable of restoring PI sensitivity in cells which can be totally resistant towards the chemical substances at baseline .
Pan HDAC inhibitors do exhibit some toxicity, especially after they are combined with other agents, so it might be preferable to target HDAC more selectively. Schreiber?s group identified tubacin as a selective HDAC inhibitor, and perform from Anderson?s group showed that tubacin can also be a potent inhibitor of PI induced L-Shikimic acid aggresome formation . Led by James Bradner in the Broad Institute , investigators are synthesizing analogs of tubacin that display even higher selectivity for HDAC with a lot more desirable in vivo pharmacokinetic properties. There might be excellent interest in evaluating these compounds in relevant preclinical models prior to introducing them in to the clinic.

G checkpoint regulation is mediated by ATM and ATR kinases leading to inhibitory phosphorylation of CDK . Two distinct parts involving G arrest are defined, one particular of which involves an early ATM dependent, NBS independent transient reduction during the frequency of mitotic cells , which reflects arrest of cells in G on the time of irradiation . This response is independent of dose from to Gy and calls for the BRCA CtIPS complex talked about in Part The second G arrest will involve a prolonged accumulation of cells in G M that may be strongly dose dependent and more pronounced in cells lacking ATM, and in cells defective in NBS or BRCA . This G accumulation reflects broken cells defective in the S phase checkpoint undergoing prolonged arrest in G and calls for BRCA acting in concert with BACH as an alternative to CtIP . The mechanism of this BACH dependent arrest is not however clear. It truly is noteworthy that the NBS SA mutation as well as the BRCA SA mutation demonstrate no apparent impairment of IR survival in regular colonyformation assays on asynchronous populations . Earlier perform cause a similar conclusion regarding the status of Tp during the G checkpoint .
Synchronous cell populations are necessary to properly assess altered sensitivity. An in depth research making use of isogenic MEFs showed that ATR assists reduce mitotic entry in a time dependent manner by cooperating with ATM at early times right after IR and contributing alot more radically at later occasions . These differential contributions may reflect the immediate activation of ATM by DSBs along with the subsequent activation of ATR through the RPA coated ssDNA HRR intermediate Neratinib selleck chemicals . ATM?s contribution acts by way of Chk via Thr phosphorylation while ATR acts through Chk by phosphorylating Ser and Ser . Double mutant atm atr cells experience little or no G checkpoint in response to a substantial IR dose of Gy . In response to spontaneous or IR injury, the transition from G phase to mitosis is delayed through quite a few submit translational protein modifications likewise as Tp mediated transcriptional regulation . Upon completion of repair of most DSBs, the checkpoint must then be inactivated .
The Chk kinase, an very important protein for cell proliferation , is required for checkpoint activation in S and G phases in response to IR injury whereas Chk activation happens all through the cell cycle and is carried out by ATM and secondarily by DNA PK . In contrast to activated ATM , activated ATR might Cytisine not exist other than its interacting proteins within chromatin . As comprehensive below, Chk activation via IR induced DSBs necessitates the two ATM MRN and ATR with ATM acting upstream inside the identical “pathway” as ATR . ATR, not like ATM, is an necessary gene for cell viability in dividing cells due to its position in repairing broken replication forks .

Strand invasion of a sister chromatid by the RAD filament, resulting in displacement loop formation and heteroduplex DNA, needs the concerted action of the RAD ATPase, RADAP, and PALB. Crossover events, detectable by SCE analysis, happen independently of DNA replication in G irradiated cells. Despite the fact that Rad is really a pivotal HRR protein within the yeast S. cerevisiae, a necessity for human RAD is only evident during the context of BRCA deficiency. Initiation of end resection by ubiquitylation and deacetylation of CtIP on BRCA CtIP MRN complicated formation Effective fix of DNA DSBs by HRR needs BRCA acting as a result of mechanisms now becoming exposed. The Nterminus of BRCA protein and its partner BARD form a constitutive heterodimeric E ubiquitin ligase complex that will conjugate ubiquitin at Lys . IR induced BRCA foci co localize with conjugated ubiquitin foci , which display a dependence on ubiquitin Lys . These foci come up in parallel inside min postirradiation, and conjugated ubiquitin foci rely strongly over the presence of BRCA BARD complicated .
Exogenous expression of the ubiquitin KR substitution mutant particularly eliminates the conjugated ubiquitin foci detected making use of the Kinase Inhibitor Libraries kinase inhibitor FK antibody, but not when applying the FK antibody that detects K and K ubiquitin linkages, implying that BRCA connected conjugated ubiquitin relies on K linkage. These findings assistance the thought that BRCA facilitates HRR by way of ubiquitin conjugation of target protein this kind of as CtIP . As mentioned in Area SUMOylation of BRCA may be a prerequisite for BRCA?s efficient in vitro and in vivo ubiquitylation exercise , and autoubiquitylation may well market this action. Apart from the RAP BRCC ABRA BRCA BARD complicated currently described, BRCA resides in at least two other complexes , with specificity becoming established by the BRCT domain?s interaction with the pSPxF motif in the companion protein. In response to IR harm, BRCA is reported to promote ssDNA formation , likewise as RPA concentrate formation as a result of an interaction with CtIP and MRN , determined by evaluation of HCC brca mutant cells.
These final results propose that BRCA is needed for DNA end resection. Having said that, a review in avian DT cells finds standard Zoledronic Acid recruitment of RPA to web-sites of laser microirradiation in brca mutant cells . Therefore, BRCA might possibly have tiny or no purpose in promoting end resection in avian cells, in contrast to human cells. BRCA associates directly using the MRN complex in an ATM and Chk dependent manner that is definitely strongly enhanced by publicity to Gy IR . BRCA also interacts straight with CtIP by means of BRCA?s C terminal BRCT area especially in G phase where CtIP is phosphorylated at Ser positioned inside of the BRCA binding region .

Knockdown of RNF, and particularly expression in the dominant unfavorable HBKR mutant histone, success in impaired recruitment of NHEJ and HRR proteins to web pages of DSBs . Likewise, rs. The components desired for productive fix of an IR induced DSB vary together with the exact nature with the break, the standing of the chromatin, and maybe cell cycle place. Null mutations in proteins mediating DNA DSB signaling and DNA repair only partially inhibit DSB rejoining. In many instances pronounced sensitivity to killing by IR is accompanied by a quantitatively modest deficiency in DSB restore, as illustrated by atm mutant cells from ataxia telangiectasia sufferers. At physiologically relevant very low doses of cGy, AT fibroblasts continually fix of IRinduced DSBs as measured by persistent gHAX foci after h or longer . This fraction is independent of dose, as well as residual breaks persist for a lot of days in G arrested cultures, implying that they are qualitatively distinct in the repaired breaks.
Lig null human Nalm pre B cells have comparable radiosensitivity as AT cells , but inside h they fix only within the DSBs created by a dose of cGy . LIG null MEFs display a equivalent degree of radiosensitivity as dna pkcs MEFs . There is a impressive degree of apparently “excess” or redundant DSB signaling with respect to particular participants this kind of as the ATM kinase and its HAX substrate . For DSBs created by lower IR doses, ATM activation necessitates transautophosphorylation of inactive veliparib clinical trial kinase inhibitor ATM dimers to provide ATMS P monomers , which are recruited to the break sites with high efficiency even though most breaks are effectively repaired in its absence. HAX, that’s phosphorylated predominately by activated ATM , is now often accepted as an correct marker of IR induced breaks underneath selected conditions . Null mutations in HAX confer IR sensitivity and defects in DSB repair similar in magnitude to people of atm null cells.
As an example, hax null mouse ES cells are fold more delicate to IR killing than wild kind controls . In MEF cultures, hax null immortalized MEFs are fold sensitive despite the fact that in comparison ku MEFs are fold hypersensitive . It truly is noteworthy that hax MEFs proliferate poorly in culture but are able to rejoin most DSBs, even after a dose of Gy . HAX and MDC play a central part in recruiting many proteins to DSB websites, this kind of as BP PD 0332991 and BRCA ; while in the absence of either a single, BP and BRCA are recruited only transiently . The recruitment of other major downstream proteins that participate in HRR, this kind of as MRE RAD NBS and CtIP , are only modestly affected through the absence of HAX or MDC . These findings imply that there can be complex, overlapping hierarchies of signaling events and that a missing upstream component may only modestly diminish the overall efficiency of repair. Nuclear foci at injury websites An abundance of harm response proteins that come to be concentrated at web pages of DSBs is talked about during this evaluation.

On transition from anaphase I to telophase I and cytokinesis, AURKB was also connected to the mid spindle , consistent with its localization in mitotic cells as part of the CPC . Gentle spreading of oocytes to expose centromeres to antibody showed that AURKB is existing on entire chromosomes and gets preferentially enriched and localized to a centromere domain in the course of metaphase I of meiosis that is certainly also recognized by CREST antibody reacting with centromere proteins like CENP A and CENP C . The spatial separation of AURKB from MCAK residing at centromeres at anaphases could possibly contribute to help microtubule depolymerization all through chromosome segregation at anaphase I telophase I. However, not like mitotic cells progressing to interphase, in which AURKB is degraded, staining was yet again found on chromosomes at telophase I, as soon as homologues had separated to opposite spindle poles . At this stage, AURKB was preferentially observed at chromosomes retained in the oocyte without any or only faint staining by antibody of chromosomes from the primary polar body.
In metaphase II arrested mouse oocytes, AURKB occupied a centromere domain overlapping with CREST positive foci , comparable to metaphase I. MCAK was closely linked to the centromere and in addition occupied web sites recognized by CREST antibody , steady with some overlap in localization of AURKB and MCAK. This might regulate phosphorylation and inactivation Screening Library of MCAK at centromeres. The research suggests that there’s a failure in loss of cohesion concerning sister chromatid arms and resolution of chiasmata in oocytes with AURKB inhibition, which have been blocked from cytokinesis. The existing findings for this reason imply that alterations in AURKB exercise influence the reduction of cohesion concerning sister chromatid arms as implicated by sequential instead of instantaneous chiasma resolution, and in failure of sister chromatid disjunction in oocytes escaping the nuclear maturation block. The AURKB orthologue AIR has become implicated in phosphorylating and focusing on the meiotic cohesin protein Rec for cleavage by separase at the starting of anaphase I at meiosis I in C.
elegans . Depletion of AIR suggests that it promotes separation of homologues and reduction of cohesion Pazopanib distal on the single chiasma on meiotic chromosomes. Inactivation from the phosphatase that antagonizes AIR induced premature separation of chromatids in the course of meiosis I in C. elegans . In yeast, Aurora kinase is required for recruitment of centromeric phosphatase PPA to centromeres . Thus, the CPC containing lively AURKB might have dual functions in differentially regulating reduction of cohesion on sister chromatid arms and avoiding indirectly loss of cohesion in between centromeres of sister chromatids in meiosis I chromosomes by recruiting phosphatase to centromeres.

Inhibits AIR Kinase Action In Vivo To determine regardless of whether CDC . regulates AIR action in vivo, the phosphorylation and activation state of AIR was monitored in handle and cdc . treated air embryos using a commercial phospho specified pAurora antibody that recognizes Aurora A and B autophosphorylation and kinase activation . Immunostaining exposed solid AIR dependent mitotic centrosome staining and an AIR dependent chromosomal passenger complicated stainingpattern . In each control and cdc . handled air embryos , similar levels of pAIR CPC staining had been present on condensing chromosomes from early prophase to prometaphase . Even so, from metaphase via late telophase, there have been improved amounts of pAIR CPC staining in cdc . embryos as compared to controls . The exact same trend was uncovered for pAUR ranges through the entire entire embryo , and for pAIR CPC immunostaining in embryos reared at temperatures ranging from C . As pAIR ranges drop in management air embryos with raising temperature, cdc . embryos retain pAIR levels that exceed or are comparable to those in wt embryos reared at C or air embryos reared at C .
A related grow in pAIR ranges was uncovered in wt embryos taken care of with control and cdc indicating the kinase action of wt AIR is additionally topic to CDC . regulation . To verify these effects, the phosphorylation of ICP , a substrate and potent activator from the AIR kinase , was monitored by immunostaining wt and air embryos treated with manage and cdc . with a phospho certain antibody that recognizes peptide synthesis selleck the AIR phosphorylation web site . In all problems, pICP localized to chromosomes in early mitosis, and also to the spindle midzone and midbody in late mitosis . Centrosome and p granule pICP staining was not abolished by icp or air and consequently was not precise . In both manage and cdc . embryos, pICP faintly stained condensing chromosomes from early prophase to prometaphase . Nevertheless, as over, from metaphase by way of late telophase, there have been greater ranges of pICP staining on chromosomes and spindle midzone midbody microtubules in cdc . embryos as in contrast to controls .
A very similar trend was uncovered when pICP ranges have been measured throughout the entire embryo . In sum, these findings reveal that from the absence of CDC AIR kinase exercise is upregulated in C. elegans embryos from metaphase through late telophase G. Importantly, this maximize in AIR kinase activity isn’t going to correlate with the stabilization enzyme inhibitor selleck of AIR in late mitosis , suggesting that CDC . may possibly inhibit AIR kinase exercise and protein ranges through distinct mechanisms. CDC . Depleted Embryos Show Prolonged Cell Division and Mitotic Defects Dwell imaging of GFP AIR transgenic animals uncovered significant delays in chromosome alignment, anaphase onset, and cleavage furrow formation in cdc . embryos , steady together with the slow growth phenotype of cdc . embryos .

IGF R has acquired growing interest as being a promising target in cancer treatment, but its part as a therapeutic target in melanoma has not been systematically explored. IGF R can activate the two the MAPK and PIK pathways, both of which play vital roles in melanomagenesis. We examined the effect of IGF R inhibition on MAPK and PIK mediated signaling. Treatment method with PPP or AG had no result on ERK activation in resistant cells . Then again, phosphorylation of AKT was inhibited by treatment method with PPP . Consistent with our effects working with IGF R compact molecule inhibitors, expression of dominant unfavorable IGF R in resistant cells didn’t inhibit MEK and ERK phosphorylation , but had an inhibitory impact on AKT phosphorylation . Overexpression of your IGF R ligand, IGF , in Mel parental cells led to greater phosphorylation of AKT, but had no vital result on ERK phosphorylation . Together these information recommend that persistent IGF R signaling induces PIK AKT activation in VE mutant melanomas resistant to BRAF inhibitors. Nevertheless, our information never preclude the possibility that more things could also affect interregulation of IGF R and PIK in BRAF inhibitor resistant cells.
Thinking about that IGF R and PIK AKT perform very important roles mediating cell survival, we examined the result of MEK and IGF R inhibition about the expression of some Bcl family members identified to become very important for melanoma Nafamostat survival, which include Mcl , Awful, and BIM . Mel R cells expressed large amounts of phospho Poor and Mcl , neither of which have been thoroughly inhibited by treatment method with . Unphosphorylated Poor binds and inactivates the prosurvival factors Bcl and Bcl xl marketing apoptosis; phosphorylated Bad associates with permitting unbound Bcl Bcl xl to promote survival. Even though inactivation of MEK ERK by or AZD was adequate to inhibit Lousy phosphorylation and to induce BIM, inhibition of IGF R signaling did not have any considerable impact on these professional apoptotic components . Inhibition of either MEK or IGF R led to a partial downregulation in the pro survival element Mcl . In addition, concomitant inhibition of MEK and IGF R AKT mediated signaling had an additive result downregulating Mcl in Mel R cells .
MEK and IGF R appear to cooperate and promote survival of melanomas resistant to BRAF inhibitors; finasteride whereas MEK alone regulates BIM and Poor, both pathways jointly regulate Mcl expression. Overexpression of IGF decreased BIM expression, but it did not preclude the skill of to induce BIM . Whilst treatment of Mel cells with resulted in partial downregulation of Mcl , overexpression of IGF led to enhanced Mcl amounts, which couldn’t be downregulated by alone. These outcomes suggest that MEK and IGF R cooperate to promote cell survival in part by way of the coordinated regulation of Mcl .