These samples are processed and stored in 384-tube formats and entered into the bio-storage repository by robotic processing. Resulting examination information from genomic and protein sequence assays are linked to your 2D barcode from the sample, and that is coupled towards the patient in the study. The MM database captures all samples as well as the corresponding data, and may be reached by MM consortia members anyplace in the world. Mass spectrometry imaging Drug characterization is an ever-increasing analysis place that at this time holds a central position in each drug developments as well as within hospitals in which individuals are getting diagnosed. Classical X-ray is accompanied with MRI, PET and CT for many within the most typical disorders including MM. Mass spectrometry imaging is usually a swiftly developing approach that gives you information around the spatial distribution of molecular profiles on the tissue sample.
In terms of MM, the MSI may be used to provide proof of drug presence in tissue microenvironments. The distributions special info of drug and metabolite are measured straight in patient tissue samples using a resolving power of thirty ?m . This technologies platform enables direct drug measurement wherever pharmaceuticals and metabolites may be analyzed, as being a cold compound, without any labeling. It has been slowly accepted that MSI gives you rich molecular data offered by this mass spectrometry imaging platform. It interfaces nicely with basic histology, as the staining approaches of tissues are compatible with MSI. This will supply complementary information, and so supplies new possibilities within the clinical area.
This ranges from new insights into the molecular adjustments connected with pathogenesis. Oral squamous cell carcinoma could be the most typical neoplasm of the head and neck. Carcinoma cells accumulate a series of genetic and/or epigenetic changes and altered phenotypes for the duration of tumor progression. Reduction of epithelial morphology and acquisition of mesenchymal qualities, termed the epithelial-to-mesenchymal transition , are typical for carcinoma cells all through tumor progression and correlate using the community invasiveness and metastatic possible on the tumor . Between the mechanisms largely linked together with the metastatic conversion of epithelial cells as well as the EMT, the loss of Ecadherin- mediated cell adhesion is prominent . The Akt/PKB family members of kinases is actually a downstream effector of phosphatidylinositol 3-kinase and is frequently activated in human cancers, as well as OSCC .
Recently, activation with the PI3K/Akt axis is emerging as a central attribute of EMT. Akt-induced EMT entails downregulation of E-cadherin, which appears to outcome from upregulation from the transcription repressor Snail. Akt action is induced by ligand stimulation of growth issue receptors for example the insulin-like growth factor-I receptor and also the EGF loved ones of receptors .

The promoter area of the CGRP gene contains a consensus sequence responsive towards the transcription issue CREB . In L6 DRG in the course of cystitis, a significant population of CGRP neurons consists of phospho-CREB. This suggests that CREB could possibly also be involved with NGF signaling in the course of cystitis. It has been reported that retrograde NGF regulates CREB activation in cultured rat sympathetic neurons, and plays a crucial purpose in neuronal plasticity . Steady with this particular notion, our effects demonstrate that in cystitis endogenous NGF facilitates CREB activation in major sensory neurons due to the fact NGF antibody remedy blocks cystitis-induced CREB activation in L6 DRG. You will find also parallel decreases while in the CGRP expression in addition to CREB activation in DRG neurons co-expressing the two molecules following NGF antibody remedy of your cystitis animals.
Taken with each other, these results propose that NGF regulates sensory activity and CGRP expression calls for CREB activation while in cystitis. CREB is usually activated by a number of kinases MDV3100 structure as well as the Ca2+/CaMdependent kinase II, PKA, and MAPK and Akt , and occupies about four,000 promoter websites in human tissues . Consequently, together with CGRP, other neuropeptides and ion channels may well also be regulated by CREB in sensory neurons . This really is proven regularly in our research that from the L6 DRG in the course of cystitis countless phospho-CREB neurons tend not to express CGRP. Examination of retrograde pathways which might be initiated by NGF leading to CGRP expression in DRG demonstrates that application of exact inhibitors towards the MEK/ERK pathway blocks retrograde NGF-induced CGRP upregulation in the sensory neuronal cell entire body, even though inhibition of your PI3K/Akt pathway has no effect.
Upregulation of CGRP from the ERK MAPK pathway has also been demonstrated in trigeminal ganglia neurons . It really is noteworthy the recent review doesn’t preclude the chance of other variables in regulating CGRP expression from the DRG. These variables consist of but usually are not constrained to growth variables, cytokines, purinergic technique, and glutamate and receptors which are WP1066 solubility also elevated during the inflamed bladder and/or sensory pathways during cystitis . Cytokine activin is able to increase CGRP expression in sensory neurons in culture and in vivo after peripheral irritation . It is actually proven that activin acts synergistically with NGF in inducing CGRP expression in sensory neurons .
In conclusion, the current examine demonstrates that activation of the completely unique signaling involving activation of ERK5 but not Akt in cystitis- and NGF-induced CGRP expression in the DRG suggests that target of ERK pathway may be a probable therapeutic techniques in remedy of bladder ache with cystitis. Lately, new medicines are actually created for that treatment of HIV-1 patients that act at diverse procedures in the viral replication cycle .

The alter in Mcl-1 protein ranges was even further quantified by densitometry analysis and showed a statistically considerable decline in all sufferers tested following sorafenib publicity . Given that Mcl-1 reduction was a popular phenomenon in all culture conditions, these benefits demonstrate that sorafenib-induced apoptosis is connected with a reduction of Mcl-1, that’s recognized to perform a critical function in CLL cell survival . Sorafenib Reduced the Ranges of Lively B-RAF, C-RAF and ERK in CLL Cells Sorafenib was at first discovered for its ability to inhibit the kinase action of RAF by binding to its inactive kind and sequestering it into inactive complexes , and its cytotoxic exercise is believed to become mediated not less than in part by its impact on RAF action. Hence, we investigated the result of sorafenib for the amounts within the activated phosphorylated varieties of B- and C-RAF, that are quite possibly the most lively isoforms involved in the activation/phosphorylation of ERK from the context of your cellular microenvironment.
A sorafenib-mediated reduction in phospho-B-RAF, phospho-C-RAF and phospho-ERK levels was observed in CLL cells cocultured selleck chemical Inhibitor Libraries with NLCs and MSCs . Given that sorafenib won’t right inhibit ERK phosphorylation , these outcomes indicate that sorafenib can actively inhibit RAF likewise as its downstream effectors in CLL cells inside the presence of a supportive cellular microenvironment. Collectively, these results propose that sorafenib induces apoptosis of CLL cells as a result of Mcl-1 downregulation. To even further investigate if your inhibition of the RAF/MEK/ERK pathway can lead to Mcl-1 downregulation in CLL cells, we examined regardless if inhibition of MEK prospects to downregulation of Mcl-1 applying the MEK inhibitor PD98059.
Exposure of CXCL12-stimulated CLL cells to PD98059 induced apoptosis , which was accompanied by a downregulation of Mcl-1 . Moreover, publicity of CLL cells to PD98059 and to the RAF inhibitor GW5074 while in the presence of MSCs also caused apoptosis and resulted within a downregulation of Mcl-1 protein amounts . These outcomes show that in CLL cells, the RAF/MEK/ERK pathway can regulate Mcl-1 expression amounts and that this axis is important for CLL viability. It further suggests that sorafenib might lead to apoptosis of CLL cells via the inhibition in the RAF/MEK/ERK pathway and Mcl-1 downregulation. CLL Cells Will not be Sensitive to Inhibition of KIT, PDGFR and FLT3 but Are Delicate to Inhibition of RAF and VEGFR Sorafenib is a multikinase inhibitor, targeting not just RAF, but additionally plateletderived development factor receptor , KIT, FMS-like tyrosine kinase three and vascular endothelial growth issue receptor .
To identify which other targets of sorafenib are vital for CLL cell viability, and hence may perhaps contribute to sorafenib-mediated cytotoxicity, we in contrast a set of inhibitors sharing overlapping targets with sorafenib for their ability to abrogate CLL cell viability .

Numerous studies propose that indolinones and anilinophthalazines. In particular, the anticancer drug Sutent displayed equally potent inhibition of VEGFR2 and FGFR1 tyrosine kinase activity in vitro and the two VEGF-A- and bFGF-mediated responses in endothelial cells. For the initially time we also showed that these medicines exert their results by modulating receptor tyrosine kinase trafficking. The results of those drugs on receptor tyrosine kinase action is usually translated into all the more potent effects on endothelial perform, cell migration and tubulogenesis. Hence, we produce a large physique of proof that indolinones and anilinophthalazines are real multi-kinase inhibitors. Tipifarnib is often a potent, selective and competitive inhibitor in the enzyme farnesyltransferase .
This enzyme is essential inside the processing and activation of signalling molecules linked to cell proliferation and malignant transformation, such as Ras, Rho-B, Rac, and lamin proteins special info . Inhibition of FTase by tipifarnib induces antileukaemic and anti tumoral activity, which has been demonstrated in both in vitro and in vivo animal versions . The nature within the cellular and tumour tissue responses elicited by tipifarnib treatment method in vivo is consistent using the hypothesis that the antitumour effects are staying derived from disruption of multiple effectors downstream of FTase inhibition. A variety of phase one dose-escalation studies in patients with innovative solid tumours happen to be carried out with oral tipifarnib being a single agent, utilizing twice-daily schedules ranging from 5 days every two weeks, to steady dosing .
These scientific studies have been made to determine the maximum tolerated dose and to characterize the safety plus the pharmacokinetics of tipifarnib. Those research NPS-2143 calcium channel blocker demonstrated that the pharmacokinetics of tipifarnib are linear in doses of as much as 600 mg twice each day and allowed the measurement on the oral bioavailability within the alternative, capsule and tablet formulation in cancer sufferers. Very little or no evidence of time-dependent pharmacokinetics was observed soon after repeated administration . Tipifarnib is swiftly absorbed immediately after oral consumption, using a peak plasma concentration becoming reached inside of two?4 h . Plasma tipifarnib concentrations decline in the biexponential method, and the half-life linked with all the to start with disposition phase is two?five h. Its terminal half-life is sixteen?20 h and exposure for the drug over this period constituted only a tiny portion with the all round spot beneath the plasma concentration vs.
time curve. Minimal plasma accumulation is noticed upon twice-daily administration, indicating the initially disposition phase dominates the plasma concentration-time profile of tipifarnib . The drug is nearly thoroughly bound to plasma protein and binding is independent on the plasma drug concentrations .

Nevertheless, to date, the blockade of GFs-receptor binding has not been sufficiently exploited with all the exception on the recombinant humanized IgG monoclonal antibody bevacizumab that binds VEGF avoiding its interaction to its receptors . Amid RTKs, EGFR, which belo ngs to Erb-B receptor loved ones, plays a important role in cell development and differentiation; it can be overexpressed in many strong tumours together with hMPM . The extra-cytoplasmatic portion of EGFR is the molecular target of MoAbs, including cetuximab, panitumumab and trastuzumab. Cetuximab, a chimeric MoAb , recognizes EGFR with affinity 5- to 10-fold increased than endogenous ligands inhibiting the receptor function . Panitumumab is usually a completely human MoAb precise to EGFR: it will work by binding the extracellular domain with the EGFR stopping its activation.
Trastuzumab can be a humanized MoAb that binds to your domain IV with the extracellular segment of your HER2/neu receptor . Cells treated with trastuzumab undergo arrest throughout the G1 phase from the cell cycle lowering the proliferation. It’s been advised that trastuzumab induces some of its effeexplanation cts by down-regulating HER2/neu top to disruption of receptor dimerization and signalling by means of the downstream PI-3 K cascade. Trastuzumab also suppresses angiogenesis by induction of anti-angiogenic things and repression in the pro-angiogenic ones. Upstream steps of signal transduction pathways GF binding and activation of RTK may be the to start with fundamental stage that triggers the sequences of intracellular signals regulating cell proliferation, survival, progression, metabolic process, angiogenesis, metastasis and drug resistance.
Typically, RTKs are constituted of an extracellular ligand-binding portion connected to your cytoplasmic domain by just one transmembrane peptide. The cytoplasmic domain contaRidaforolimus ins a conserved TK core and quite a few regulatory sequences that undergo autophosphorylation and phosphorylation by heterologous kinases, after precise ligand binding. GF-RTKs docking 1st prompts receptor steric alterations resulting in homo/ eterodimerization, then induces the phosphorylation of precise residues of TKs while in the cytoplasmatic tails; the activated receptors recruit and phosphorylate intracellular effectors that initiate a downstream signalling cascade main to many biological responses . Quite a few agents are synthesized to target RTKs and block their kinase activity by competing with ATP binding: gefitinib , lapatinib and erlotinib , compact MW 4-anilino-quinazoline derivatives, are EGFR TK inhibitors; sorafenib , sunitinib and vandetanib are bis-aryl urea, L-malate salt and 4-anilino-quinazoline, respectively, and have been developed as anti-VEGFRs ; VEGFRs , PDGFRa-b, c-kit; and anti-VEGFRs, respectively.

Considering the fact that contacts with the extrahelical base stabilize AGT binding, we propose that variations within the stabilities of helical base conformations at inner sites and DNA ends account to the preferential end binding discovered with AGT. If correct, this outcome predicts that AGTs binding and repair functions may well be enhanced, not just at DNA ends, but wherever base stacking or hydrogen bonding is impaired. DISCUSSION The at present readily available crystal structures of AGTDNA complexes present single protein molecules bound at isolated DNA web pages . In contrast, solution binding and crosslinking research indicate that AGT types cooperative complexes on single- and double-stranded substrates Cooperative binding is additionally witnessed with structurally similar alkyltransferase-like proteins, which have been proposed to bridge amongst direct base restore and nucleotide excision repair pathways .
Mutations situated from the proteinprotein interface and far through the DNA-contact surface have sturdy effects on binding cooperativity in vitro and resistance to alkylating agents in vivo , suggesting that DNA-binding cooperativity plays a function within the physiological activities in the enzyme. To better comprehend the cooperative binding selleckchem supplier SB 203580 mechanism, we employed ultracentrifugation and AFM to characterize the binding of AGT to one thousand and 2686 bp duplex DNAs. Binding affinities found for these DNAs were much like ones previously measured, within the exact same buffer, for short duplexes . That is consistent together with the plan that proteinDNA and proteinprotein interactions are comparable on short and extended substrates. However, the limiting binding website sizes had been drastically greater on lengthy DNAs than on short duplexes ).
It seems very likely that these distinctions reflect packing inhomogeneities read review that are anticipated once the binding substrate is substantial and that are largely absent when short substrates are applied. The McGheevon Hippel binding model is often employed to characterize cooperative proteinDNA interactions . This model envisions a single binding mode characterized by a exceptional binding internet site dimension , just one class of proteinDNA contacts that has a single equilibrium frequent K and also a single class of cooperative interactions . Although this model may possibly not capture all characteristics of intricate biological programs, it provides a beneficial benchmark for the comparison of their properties.
In our AFM scientific studies, AGT gave normal cluster sizes that agree properly with predictions of your McGheevon Hippel model at low binding occupancies, but at higher occupancies cluster sizes fell below the predicted selection . The presence of AGT molecules defective in proteinprotein interaction could have restricted the dimension of cooperative clusters. On the other hand, our preparations had been >95% energetic in cooperative DNA binding and in alkyltransferase reactions that require cooperative binding .

Preoperative PET might in fact understage the patient, nevertheless it is helpful for identifying distant metastases.eleven Several paraneoplastic syndromes have been described with mesothelioma. These include hypercalcemia, hypoglycemia, autoimmune hemolytic anemia, hypercoagulable states, and disseminated intravascular coagulation. These syndromes are nonspecific and can be viewed that has a variety of malignancies. PATHOLOGY Diagnosis of mesothelioma could be tricky. The disorder is relatively uncommon, and many pathologists might not have substantial practical experience with it. The quantity of tissue obtained is often minimum and may not be sufficient to complete the necessary battery of tests that could distinguish mesothelioma from other pleural-based malignancies. Histologic variability may perhaps make diagnosis challenging. Quite possibly the most widespread histologic kind is epithelioid and it is connected with the finest prognosis.
Sarcomatoid variants with characteristic spindle morphology are associated which has a worse prognosis. Normally, mixed epithelioid and sarcomatoid i was reading this histologies could be viewed. Tissue obtained by cytologic analysis of pleural fluid or blind pleural biopsy is limited and underclassifies the right histology up to 25% with the time. If pleural fluid is obtained, big volume collections need to be performed as well as a cytospin evaluation carried out to increase diagnostic accuracy. Thoracoscopic biopsies with direct visualization of pleural nodules give the most effective yield. Immunohistochemical staining is vital to distinguish mesothelioma from adenocarcinomas of lung origin or metastatic from other sites. Calretinin is usually constructive in mesothelioma, by using a reported sensitivity of 95% and specificity of 87%.12 Thrombomodulin has the top specificity at 92% but is significantly less delicate at 68%.
Other valuable antibodies directed against mesothelial-associated antigens contain Seliciclib mesothelin, cytokeratin 5, Wilms? tumor-1 gene solution, and HBME-1 as well as the nonmesothelial antigens Lewis-Y blood group , MOC-31, BerEp4, CD15, as well as carcinoembryonic antigen family. A consensus statement from an expert panel of sixteen pathologists from the International Mesothelioma Interest Group established suggestions for diagnosing mesothelioma and distinguishing it from other tumors such as adenocarcinoma by using a panel of histochemical markers with no less than 80% or more sensitivity.13 In accordance on the panel of specialists, by utilizing this strategy a pathologist can make the diagnosis of mesothelioma 95% in the time.
While in the remaining 5%, tissue might not be sufficient, not representative in the tumor, or, in some instances, so poorly differentiated that diagnosis is complicated. DIAGNOSIS Correct diagnosis of mesothelioma is dependent upon ample tissue.

Studies have shown that insertion within the lipophilic prenyl moiety to the plasma membrane isn’t a simple random occasion, but that certain prenyl receptors facilitate prenylated protein binding . These and also other research lend assistance on the hypothesis that prenylation also provides specificity for interaction partners. Therefore, ongoing get the job done is targeted on inhibiting the binding of farnesylated Ras to web pages around the inner surface of your plasma membrane. Two farnesyl isoprenoid containing tiny molecules are described, salirasib and TLN 4601 , which are proposed to antagonize Ras function by competitors for membrane bound farnesyl binding docking proteins. Whereas clinical trial analyses of TLN 4601 have a short while ago been terminated, one can find continued preclinical and clinical analyses of salirasib. Salirasib, a farnesylcysteine mimetic, selectively disrupts the association of chronically energetic Ras proteins using the plasma membrane .
The proposed mechanism of Salirasib is compete with Ras for binding to membrane related Ras escort proteins , which possess putative farnesyl binding domains, thereby dislodging hif 1 alpha inhibitor Ras in the plasma membrane and disrupting effector signaling. Studies demonstrate that galectin 1 interacts with mutant H Ras and K Ras, and that this interaction is needed for membrane localization from the GTPases and subsequent transforming exercise in human and rat epithelial cells . Salirasib blocks the membrane association of H , K , and N Ras proteins in both transformed cells and cancer cells with oncogenic mutant Ras or hyperactivated wild kind Ras, which includes pancreatic, melanoma, glioblastoma, neuroblastoma and neurofibromatosis cancer cells .
Additionally, Kloog and colleagues demonstrated that signaling from 3 from the most studied effector pathways downstream of Ras, Raf MEK ERK , RalGEF Ral , and PI3K AKT , may very well be suppressed by therapy with salirasib. Inhibition of aberrant Ras activation in cells by salirasib may possibly alter a number of cellular properties, which include cell selleck chemical OSI-027 mTOR inhibitor survival , proliferation , and migration . In one more study, tumor growth was inhibited by salirasib and was linked to a reduction in the abundance of Ras inside the tumor tissue in pancreatic and neurofibromatosis xenograft tumor models . Yet, salirasib has also been shown to block mTOR exercise directly , an unexpected exercise in light from the fact that mTOR is not farnesylated. Thus, salirasib could possess several mechanisms of action that may contribute to inhibition of tumor development.
Ultimately, Phase I clinical trials have proven that salirasib was nicely tolerated and a few Phase I II trials are ongoing . Activated Ras binds preferentially to a spectrum of functionally varied downstream effectors through which most are characterized by Ras binding or Ras association domains that directly interact with Ras .

This collagen bundle may possibly serve being a highway for later immigration of other cells in to the pillar. The deposition of an extra extracellular matrix by these cells can lead to the enlargement on the pillar. The driving force behind the formation within the protrusion while in the vessel lumen while in intussusceptive microvascular development stays elusive. Although it truly is believed for being exerted by perivascular cells, this kind of as pericytes or myofibroblasts, four?6 a cell?s pushing force is only while in the 10 to 100 pN range.9 In contrast, contractile forces created by cells lie from the a number of hundred nanonormal variety.10 In accordance to our model, no extraluminal force is critical to the formation with the pillar. Slender EC processes floating from the lumen can make contact with other components with the endothelial tube . Additional development of cytoplasmic processes of ECs can lead to the repositioning of this original speak to to achieve farther components of the lumen.
This may well be followed by the formation of endothelial bridges consisting of several ECs. The contractile force exerted through the microfilaments current at a substantial density in the ECs forming the bridge may well be solid enough to pull a collagen bundle into and with the lumen. The remarkably edematous and loosely organized peritumoral connective tissue may possibly enable this operation. Nevertheless, the presence chemical screening of adhesions at a higher density for the myofibroblasts in the pericapillary connective tissue suggests the collagen matrix is below stress, as a result of both indirect or direct attachment of those cells for the collagen bundles. These attachments might possibly counteract the motion in the collagen bundles.
Whilst we do not have direct evidence for the movement Stigmasterol of your collagen bundles, the observed similarity involving the diameter from the collagen bundles within the pillar and inside the connective tissue, and, also, the discovery of collagen bundles extending only halfway in to the lumen have led us to conclude that pre-existing collagen bundles are transferred by these ECs through the lumen. The observation that collagen bundles are transferred inside a handover- hand cycle during the case of fibroblasts in vitro supports this hypothesis. This approach was dependent on integrin _-2/_-1?mediated adhesion and to the contractile action of the actomyosin cytoskeleton.twenty Then again, in our situation, the adhesion receptor responsible to the binding of collagen I while in the pillar stays unknown.
We could not detect integrin _-1 or _-11 expression amounts, and integrin _-2 was current only sometimes at a very low density, which did not correlate together with the quantity of adhesion spots containing vinculin in the pillar. This calls into question the role of these integrin subunits during the transport from the collagen bundle.

As shown in kinase 1, the inhibitory result of HKa on tumor migration is extra potent than that of D5 but both considerably The O6 alkylguanine DNA alkyltransferase1 repairs O6 alkylguanine and O4 alkylthymine adducts that come about in DNA that has been exposed to alkylating agents1; 2; three. Each adducts are mutagenic and carcinogenic1; 4; five and O6 alkylguanine adducts may also be cytotoxic6. AGT also protects tumor cells towards chemotherapeutic drugs that methylate or chloroethylate DNA6; seven, and clinical trials are underway to find out regardless if AGT inhibitors can boost the efficacy of DNAalkylating drugs8; 9. This relevance to cancer etiology and chemotherapy has stimulated exploration over the structure of AGT10; eleven; twelve, its synthesis and degradation13; 14 and its mechanisms of DNA repair15; sixteen, but crucial gaps continue to be in our comprehending of how AGT interacts with DNA and with proteins bound to DNA.
Human AGT can be a monomeric protein that is expressed constitutively in usual cells3; 7; 17. It binds single stranded and duplex DNAs with little sequence or lesion specificity, modest affinity price LY2886721 and vital cooperativity18,19; 20. Within the fix response, just one alkyl group is transferred from your O6 position of guanine or O4 position of thymine to an lively website cysteine . This returns the DNA base to its unmodified state, however the alkylated form of the enzyme is usually a dead end state that is rapidly degraded21; 22. Because fix by AGT is stoichiometric, the quantity of O6 alkylguanine and O4 alkylthymine adducts that can be repaired at one time is dependent upon the cellular concentration from the un alkylated type of AGT two; three and on its distribution involving alkylated and competing undamaged sites throughout the genome.
This reality motivates our examine of AGT DNA interactions. AGT repairs the two single stranded and duplex DNAs23; 24; 25 and recent information indicate that the equilibrium constants, cooperativity parameters and limiting binding densities are remarkably related for complexes formed with single and double stranded templates18; 26. The simplest PF-4708671 designs that account for these effects are ones through which the protein protein contacts are related in complexes formed with single and double stranded DNAs and by which the helical twist of double stranded DNA in complex with AGT is similar to that of totally free DNA. Here we describe and test structural versions based on these properties.
These designs predict the identities of residues in the protein protein interface, the conformation of DNAs during the complexes and the numbers of ionic contacts formed in between AGT and substrate DNAs. Outcomes Models of cooperative AGT DNA complexes An AGT monomer occupies a DNA surface that spans eight bp11 but the occluded binding webpage sizes in cooperative complexes are substantially smaller .