There was no overall distinction in protocol defined or TIMI key

There was no general difference in protocol defined or TIMI serious bleeding between clopidogrel and ticagrelor. Ticagrelor was connected with a increased rate of non CABG associated main bleeding and a higher number of fatal intracranial hemorrhages. There have been fewer episodes of CABG connected main bleeds and fatal bleeding with ticagrelor. Of all the subgroups, there was no important distinction in bleeding risk except for heterogeneity with BMI, with an interaction P value of Individuals having a BMI kg m had less major bleeding with clopidogrel. While ticagrelor is actually a extra potent antiplatelet agent than clopidogrel, no net expand in main bleeding was present in animal studies. It’s been proposed that there is a wider separation on the anithrombotic effects in the bleeding risk with ticagrelor in contrast with thienopyridines.
In rat and canine versions, ticagrelor was in a position to accomplish antithrombotic results that surpassed thienopyridines not having an equivalent elevation of the bleeding time. Typically, cardiovascular surgeons favor to wait days immediately after withdrawal of the thienopyridine prior to CABG. selleckchem additional reading The reversible inhibition of PY platelet receptors of ticagrelor has resulted in speculation that it may be a safer alternative for selleckchem kinase inhibitor individuals requiring earlier operations in contrast with thienopyridines. This advantage hasn’t been realized, as there was no variation to ticagrelor in excess of clopidogrel when it comes to threat of big CABG connected bleeding in PLATO trial. In DISPERSE examine, there were numerically fewer significant bleeds in individuals undergoing CABG concerning and days following discontinuing these drugs, though the main difference didn’t appear to get statistically substantial.
The primary finish stage for offset inside the ONSET OFFSET trial was the slope of your antiplatelet impact curve. This was assessed employing ?mol L of ADP by light transmittance aggregometry and unveiled a alot more fast offset for ticagrelor in contrast with clopidogrel. Nonetheless, hop over to this website it is also obvious that platelet inhibition after study drug was discontinued didn’t turn out to be statistically important till hours, and platelet inhibition assessed by VerifyNow and VASP P certainly not showed a statistical distinction amongst ticagrelor and clopidogrel inside the hrs and days immediately after study drug discontinuation. As a result, the hope that ticagrelor offers a substantial benefit over thienopyridines inside the to start with days after discontinuation hasn’t been supported by the readily available data.
Ventricular pauses have been seen extra commonly with ticagrelor in clinical trials. A post hoc analysis of DISPERSE demonstrated a greater variety of ventricular pauses during the ticagrelor group, which in most cases remained asymptomatic. Most pauses have been reported to get on account of sinus block or sinus exit block, despite the fact that there have been patients who knowledgeable full heart block.

In review , all subjects were white and of these, % had been of H

In examine , all topics were white and of these, % were of Hispanic or Latino ethnicity, whereas in research , percent of subjects have been white with only % Hispanic or Latino. Subjects in review also tended to have additional extreme disorder compared with topics in examine . Somewhere around % of subjects in review had moderate to extreme scores on ISGA in contrast with percent in review . Likewise, indicate baseline lesion counts were increased during the than the population. The mean variety of days subjects had been exposed to remedy was . days in study and . days in study . Neighborhood tolerability. During the split face review, each C BPO and also a BPO were very well tolerated, with low investigatorrated scores for erythema, dryness, and peeling . Then again, imply scores for these parameters have been considerably higher following application of a BPO than C BPO at Weeks and .
Imply topic ratings for indications and symptoms of nearby tolerability were also substantially decrease with C BPO than which has a BPO at Weeks and . The incidence and ratings as assessed by investigators for erythema, dryness, and peeling continued to decline from Week when C BPO therapy selleck chemicals PI3K pathway inhibitor only began, such that at Week mean scores for every of those indicators were negligible and, in each case, nearly two thirds or more of sufferers had no indications present . Subject ratings for tolerability parameters also continued to lessen while in full face treatment with C BPO, this kind of that at Week , the imply score for every parameter was , incredibly minimum . Acne severity. Mean ISGA enhanced for each sides from the encounter and there was no substantial distinction between the scores for C BPO and a BPO through the split encounter portion in the examine. Exclusively, imply ISGA scores have been .
and . for C BPO along with a BPO, respectively, at Week , and and , respectively, at Week . In excess of the course in the entire study, there was a substantial improvement in full face ISGA ratings . With regards to lesion counts, pooled data showed hydralazine a substantial reduction from the amount of inflammatory, noninflammatory, and complete lesions at Weeks and compared with baseline . No comparative analysis was undertaken for lesion counts throughout the split encounter phase of your research considering that baseline lesion counts had been undertaken over the complete encounter in review . Patient preference and QOL. Patient QOL improved in excess of the course in the research, with reductions in scores for all domains in the Skindex good quality of life questionnaire, as well as the total score .
Through the split face portion within the research, virtually all subjects rated C BPO along with a BPO as quick or really simple to use, even with make up, and there have been no among group differences. Similarly, each treatment options were rated equally productive at minimizing acne breakouts.

Picornavirus RNAs are naturally uncapped and translate by a cap a

Picornavirus RNAs are naturally uncapped and translate by a cap and eIFE independent mechanism, by which the ribosomes bind to an IRES . Enteroviruses and rhinoviruses disrupt eIFF by cleavage of the eIFG subunit by Apro. This cleavage has been reported for being direct or indirect . eIFG cleavage does not preclude but, rather, stimulates cap independent initiation of viral protein synthesis, since the cap binding subunit, eIFE, remains associated with the N terminal cleavage item . The C terminal cleavage fragment of eIFG interacts with eIFA and eIF to support IRES dependent, but not cap dependent, translation initiation . In contrast to enteroviruses and rhinoviruses, no cleavage of eIFG happens following infection of cells with cardioviruses, such as EMCV .
Also, the A protein of EMCV will not be much like the enterovirus and rhinovirus Apro and doesn’t possess protease purchase PP2 consensus motifs or detectable proteolytic activity . It has lengthy been assumed that the shutoff of host cell protein synthesis right after EMCV infection benefits in the capability of viral RNA to efficiently compete with capped cellular mRNAs for some limiting component on the translational machinery . Not long ago, it had been recommended that EMCV brings about the shutoff of host translation by dephosphorylation and activation of a suppressor of cap dependent translation, E BP . E BP in its underphosphorylated kind binds to eIFE and inhibits its association with eIFG . E BP doesn’t inhibit capindependent translation, for instance that of picornaviruses, given that this translation selleckchem kinase inhibitor is independent of eIFE .
An additional possible mechanism, which can be not mutually exclusive, certainly is the dephosphorylation of eIFE . Phosphorylation of E BP is decreased by rapamycin and wortmannin, which inhibit the phosphatidylinositol kinase FKBP rapamycin connected protein NPI-2358 signal transduction pathway . PI kinase is activated by development things and hormones to supply cell proliferation and survival signals. On activation, PI kinase phosphorylates the D position of PIs, which then act as second messengers to result the various functions of PI kinase . Wortmannin inhibits PI kinase by binding irreversibly to its catalytic subunit . The immunosuppressive drug rapamycin may be a potent inhibitor of FRAP , a member in the phosphatidylinositol kinase related household, and that is imagined for being a downstream target of PI kinase .
Rapamycin augments the shutoff of host cell protein synthe sis and the rate of synthesis of viral proteins just after infection with poliovirus and EMCV , presumably since it inhibits capdependent translation, and as a result confers an benefit for the viral mRNA. On the other hand, the observed effect of rapamycin is modest, most likely since the two EMCV and poliovirus replicate quickly.

However, injection of wortmannin strongly diminished AKT phosphor

Then again, injection of wortmannin strongly reduced AKT phosphorylation immediately after NMDA application but didn’t greatly reduce survival of ipRGCs suggesting that AKT signaling just isn’t the principle element of ipRGC resistance towards NMDA toxicity. Other published information point to an involvement of pituitary adenylate cyclase activating polypeptide , a peptide noticed particularly in ipRGCs within the retinohypothalamic tract . Exogenous administration of PACAP continues to be shown to be neuroprotective for common ganglion cells following optic nerve transection , intraocular hypertension , kainic acid treatment , and NMDA application . Inter-estingly, exogenous administration of PACAP stimulates IL six production by M?ller cells inside the retina in vitro and in vivo . IL 6 is a acknowledged activator on the JAK STAT pathway, which may confer protection for photoreceptors and ganglion cells . Quite a few members of this endogenous rescue pathway were activated in response to NMDA injection.
As reported by other folks , we observed strongly greater phosphorylation of STAT3 immediately after NMDA application. Also, Lif was expressed really early, followed by Edn2 and Fgf2 , which is similar to versions of photoreceptor injury . Therefore, a signaling mechanism involving M?ller glial cells may well be selleck chemical PHA 767491 activated not only by photoreceptor degeneration but additionally soon after NMDA injection. On the other hand, blocking JAK STAT signaling through the application of AG 490 didn’t decrease survival of ipRGCs soon after NMDA remedy. Considering the fact that we also observed elevated amounts of proapoptotic proteins just like pSTAT1 and CASP1, NMDA administration activated pro and antiapoptotic signaling . The nature with the cells that activated the individual signaling pathways nonetheless really need to be established in long term experiments.
Although RGCs and ipRGCs obtain signaling input from rods and cones by way of synaptic contacts with bipolar Everolimus and amacrine cells , survival of ganglion cells is mainly not affected in models of photoreceptor degeneration. Then again, some species differences appear to exist pertaining to Opn4 expression during the absence of photoreceptors. Studies in RCS rats suggest decreased Opn4 levels regardless of frequent numbers of ipRGCs from the degenerated retina . Moreover, N methyl N nitrosourea treatment reduced expression of Opn4 by 83 , whereas only about a single third of melanopsin expressing cells were lost right after MNU injection . While MNU primarily induces degeneration of photoreceptors, regardless of whether this loss of ipRGCs was a direct consequence of MNU or was indirectly brought about by photoreceptor degeneration remains to get proven.
In contrast, retinas of rod and cone less likewise as of rd10 mice display expression of Opn4 similar to wild type mice. As a result, ipRGCs in mice may not be straight influenced by phototransduction associated signaling from photoreceptors and or regulated glutamate release from 2nd purchase neurons.

Binding of p85 to tyrosine kinase receptors removes the inhibitor

Binding of p85 to tyrosine kinase receptors removes the inhibitory result of p85 on p110, leading to the total activation of PI3K. The activated kinase catalyzes the phosphorylation of phosphatidylinositol four,five biphosphate to phosphatidylinositol 3,four,5 triphosphate . PIP3 acts being a docking website for 3 phosphoinositide dependent kinase 1 and Akt that, in turn, phosphorylates their substrates, as well as mammalian target of rapamycin and glycogen synthase kinase . PDK1 is actually a cytoplasmic kinase that phosphorylates serine threonine residues within the activation section of AGC family members protein, at first identified because the kinase that phosphorylates Akt on threonine 308 on binding to PIP3 . Actually, PDK1 is able to identify the phosphoinositides phosphorylated in place three by PI3K, via its C terminal pleckstrin homology domain. This occasion localizes PDK1 for the plasma membrane the place it phosphorylates Akt .
PDK1 substrates lacking the PH domain, such as p70S6K , SGK , RSK , and PKC isoforms , demand a several mechanism for their activation: PDK1, as a result of its PIF binding pocket, binds the hydrophobic motif on these substrates, selleck chemical Dapivirine 244767-67-7 and this leads to their phosphorylation and complete activation . Moreover, it has been described that PDK1 binds and regulates other substrates by means of kinase independent mechanisms. PDK1 has been demonstrated to activate the Ral guanine nucleotide exchange factors through its noncatalytic N terminal 50 amino acids and uncovered to activate Rho related coiled coil containing protein kinase one by competing against its inhibitor RhoE . The PI3K pathway is often aberrantly activated in breast cancer with mutations selleckchem kinase inhibitor happening in up to a single quarter of breast cancers.
PIK3CA activating mutations and PTEN loss will be the most regular events in human breast tumors, whereas a significant position for Akt1 mutations can also be emerging . Furthermore, a lot of the aspects of this pathway are located hyperactive or amplified in breast tumors: PIK3CA , PIK3CB , Akt1 , Akt2 UNC0638 , PDK1 , p70S6 kinase , and IKBKE . Such alterations strongly correlate having a alot more aggressive phenotype as well as a bad prognosis. Recently, PDK1 was identified overexpressed both in the protein and mRNA levels in most human breast cancer with regular genomic amplifications. In addition, its Ser 241 phosphorylated type was discovered enriched in human breast carcinoma versus benign tumors . Regardless of this, forced PDK1 expression is described to be oncogenic only inside the Comma 1D murine mammary cell model , whereas in breast derived cell lines, it will be able to potentiate the oncogenic results of upstream lesions but not to transform per se .
In mice, its oncogenic effect looks to perform by altering the PI3K pathway considering that PTEN driven tumors were severely attenuated in PDK1 knockout and hypomorphic mice.

GLI2 induced myofibroblast differentiation needs TGF signaling Si

GLI2 induced myofibroblast differentiation involves TGF signaling Signaling by means of TGF has become implicated in selling fibrosis and differentiation of fibroblasts into myofibroblasts in tumor linked desmoplasia . To find out no matter if TGF signaling underlies the phenotypes observed while in the GLI2 expressing HaCaT organotypic cultures, we treated these cultures using the TGF RI II kinase inhibitor LY2109761 or DMSO. Each the LY2109761 and management, DMSO taken care of cultures displayed the basal like epithelial cells characteristic of GLI2 expressing HaCaT GLI2 cultures, likewise as related proportions of Ki67 and PHH3 good nuclei .
While in the dermal layer in the LY2109761 treated cultures, having said that, selleck read the article we observed noeither differentiation of fibroblasts into myofibroblasts nor keratinocyte invasion . We obtained related results when TGF signaling was abrogated in foreskin fibroblasts by introduction of the dominant adverse TGF type II receptor construct lacking the kinase domain or when the keratinocyte and fibroblast layers had been separated by a twenty forty m thick layer of acellular collagen . Hence, TGF signaling in fibroblasts is needed for their transdifferentiation and generation of a stromal natural environment permissive for invasion. Invading keratinocytes down regulate GLI2 responsive genes and therefore are only locally invasive in the myofibroblast modified collagen matrix Transdifferentiation of stromal myofibroblasts through TGF one is also linked to upregulated secretion of HGF, which acts in the paracrine method to activate its receptor, c Met on keratinocytes by inducing its autophosphorylation.
Activation of MET results in enhanced proliferation, motility and invasiveness of keratinocytes. We confirmed that HGF transcription was up regulated in GLI2 expressing HaCaT GLI2 reconstructs . In addition, the GLI2 expressing HaCaT GLI2 cells while in the tissue reconstructs stained positively for the two c Met and phosphorylated c Met, whereas manage HaCaT Tet cells CC-5013 were only good for c Met . Hence, c Met signaling is activated from the GLI2 expressing cells with the prospective to induce keratinocyte migration and invasion.
All keratinocytes invading into the collagen fibroblast layer of GLI2 expressing HaCaT GLI2 tissue reconstructs appeared to become beneficial for pancytokeratin and have been uniformly unfavorable for E cadherin , although cells during the epithelial layer showed membranous staining for each E cadherin and catenin , suggesting that invasion is dependent about the reduction of E cadherin as is frequently observed in other systems. Enhanced expression of catenin was also noted within the myofibroblasts in GLI2 expressing HaCaT GLI2 reconstructs.

Nonetheless, taken like a group, these proteins provide a rich Ra

However, taken being a group, these proteins present a rich Raf neighborhood of proteins that might plausibly be targeted to sensitize cells towards the result of Raf therapies. Although there’s a considerable gap involving developing therapeutic strategies, and obtaining the tools at once on the market to translate the strategies towards the clinic, several of the proteins thus linked to Raf have independently been of curiosity for growth of modest molecule therapeutics: agents to these proteins could be logical candidates to assess in blend with Raf inhibitors for synergistic impact.
selleck chemical dig this Past studies of physical interactions, the EGFR Ras Raf MEK ERK signaling pathway has been heavily exploited for molecular modeling; as of 2005, above thirty mathematical versions had analyzed dynamics of signal transmission , and efforts to produce productive predictive models carry on. In parallel, interaction selleckchem kinase inhibitor network research are developing a conceptual infrastructure to know the connection of disorder centered genes to your complete network of cellular interactions , whilst other researchers look for to define the total set of mutations associated with unique cancers , or to recognize transcriptional profiles marking genes with significantly altered expression in unique cancers . Collectively, these efforts will continue to boost the context for pondering about Raf and desirable close to neighbor targets to ablate.
Lastly, this concise assessment has centered specifically on a few of the most Secretase inhibitors necessary aspects of Raf kinase biology appropriate to state from the artwork therapies. For lengthier, in depth review of Raf construction and Raf pathway signaling, the interested reader is urged to talk to many excellent latest reviews addressing these subjects . Nuclear receptors have been a topic of interest in pharmacology and toxicology as a result of their vital purpose in cellular signalling and homeostasis. They are the largest recognized household of transcription components that function as modulators of tissue gene expression . Several xenobiotics might possibly serve as activators of these transcription aspects and as a result result in dramatic improvements in physiological processes . Two nuclear receptors, constitutive androstane receptor and peroxisome proliferator activated receptor , are actually implicated as major mediators accountable for non genotoxic hepatocarcinogenesis in rodents.
On top of that, activation of those nuclear receptors by diverse xenobiotics and resultant induction of metabolizing and various immediate response genes yields related toxicity phenotypes in rodent liver together with secondary oxidative stress, cell proliferation, and greatest growth of liver tumors.

The in vitro EC50 value of cyclosporine for inhibition of human P

The in vitro EC50 worth of cyclosporine for inhibition of human P gp in MDCK MDR1 cells employing loperamide being a substrate has become reported for being 0.78 0.04 M . Working with this value and the variety of vascular volume corrected values of fold change in brain distribution of loperamide reported in knock out mice , we quantitatively predicted the boost in loperamide brain distribution at five.6 M cyclosporine blood concentration . The raise in loperamide CNS distribution in humans predicted at this cyclosporine blood concentration ranged from 56 412 . The actual observed worth falls inside this assortment. Obviously, the sizeable variability from the in vivo brain distribution of loperamide suggests that extra research are required to much better define this value.
Nonetheless, these PD153035 very good to fantastic quantitative predictions give additional credence to the use of this strategy to quantitatively predict DDIs in the human BBB. However, to generalize beyond interactions with cyclosporine, it is actually critical that this strategy be tested with P gp inhibitors other than cyclosporine. Cyanide is usually a potent neurotoxicant that generates a quick onset of toxicity and death inside of minutes. In sub lethal toxicity, lesions within the central nervous process may well build which might manifest as being a Parkinson like syndrome . In these folks, decide on injury to your basal ganglia is obvious, with dopaminergic pathways displaying the highest sensitivity . We lately reported that mice exposed to cyanide above 9 days had selective loss of dopaminergic neurons in the substantia nigra mid brain spot .
Seeing that cyanide inhibits cytochrome oxidase to disrupt mitochondrial function, degeneration of dopaminergic neurons probable will involve mitochondrial mediated death pathways . Up regulation of uncoupling protein 2 , an anion carrier expressed within the inner mitochondrial membrane, is linked with quite a few designs of brain damage and neurodegeneration mercaptopurine by which the level of expression seems to manage the degree of cell damage . A lower level UCP two expression stimulates leakage of protons throughout the mitochondrial inner membrane, therefore reducing the mitochondrial membrane potential and reducing generation of reactive oxygen species . This action protects cells from oxidative worry . Alternatively, excess mitochondrial uncoupling, which occurs with UCP 2 above expression, sensitizes cells to cytotoxic agents, possibly by reducing cellular ATP levels .
We’ve proven that up regulation of UCP two can improve cyanide toxicity .

The stimulating present remained at 0 six mA and the catheter was

The stimulating current remained at 0.six mA plus the catheter was sophisticated five cm past the needle tip about the initially insertion, without a decrease in motor response, or want for supplemental needle manipulation. The needle was withdrawn over the catheter and the catheter stylet was removed. Working with the 17 gauge Tuohy needle, the catheter was tunneled subcutaneously beneath the hairline toward the contralateral side in order to avoid the surgical field, and affixed to the contralateral shoulder applying liquid adhesive and clear occlusive dressings . An preliminary 40 mL bolus of ropivacaine with epinephrine , injected underneath ultrasound visualization , created topical anesthesia to cold and light touch inside the distribution standard of an anterolateral interscalene single injection block inside of 15 minutes .
The patient underwent an uncomplicated surgical procedure beneath common anesthesia , receiving 150 g of fentanyl for induction devoid of subsequent opioid administration. A perineural infusion of ropivacaine was initiated intraoperatively using a moveable infusion pump . Benefits The patient emerged from ATP-competitive EGFR inhibitor general anesthesia ache totally free and was discharged in the recovery area following one hour with no requiring more analgesics. The patient was discharged household the morning of postoperative day 1, by using a full infusion pump , a prescription for oral oxycodone tablets for breakthrough soreness, thorough oral and written catheter related directions, and Acute Ache Service get hold of information. Throughout the perineural infusion, the patient reported a discomfort score of 0 to one by using a 0 to ten numeric selleckchem kinase inhibitor rating scale , while not supplemental analgesics required within the hospital or at home.
The patient was contacted each day by phone until eventually household catheter elimination from the patient?s caretaker inside the afternoon of postoperative day four . DISCUSSION selleck chemical ACY-1215 Whilst steady interscalene nerve blocks give dramatic postoperative patient gains, perineural catheter placement at this degree within the brachial plexus may perhaps be technically challenging and carry prospective dangers. The ultrasound guided technique presented on this report is conveniently mastered and, in our experience, has resulted within a substantial success fee with each residents and fellows doing procedures below attending supervision. The needle trajectory of this approach is very similar to the lately described single injection transscalene brachial plexus block that employs a posterior needle insertion along the lateral border from the middle scalene muscle .
19 The system described within the current report differs in 2 vital respects: ultrasound advice permits speedy identification of the brachial plexus, making it possible for a single needle pass during the overwhelming majority of cases, in addition to confirmation of satisfactory perineural area anesthetic distribution of the initial surgical block; as well as the perineural catheter placement lets extended duration of postoperative analgesia that has a nearby anesthetic infusion.

Alternatively, cells have been separated microbead conjugated CD1

Alternatively, cells were separated microbead conjugated CD133 antibodies and magnetic columns . CD133 positive cells are enriched for glioma stem cells defined through practical assays of self renewal and tumor propagation whereas CD133 detrimental cells are depleted for non stem glioma cells. GSCs had been cultured in Neurobasal media supplemented with B27 without having Vitamin A, L glutamine, sodium pyruvate , 10 ng ml simple fibroblast development factor , and 10 ng ml epidermal growth aspect . Non stem glioma cells have been cultured for no less than 12 hours in ten serum containing DMEM to allow cell survival. Right after recovery, DMEM media was eliminated as well as the cells cultured in supplemented Neurobasal medium so experiments were performed in identical media. Non stem glioma cells were cultured in Neurobasal media for a minimum of 12 hrs in advance of experiments have been carried out.
The cancer stem cell nature of your CD133 beneficial cells was confirmed by fluorescent selleck chemicals order Rocilinostat ACY-1215 in situ hybridization , serial neurosphere assays, and tumor formation assays, but cultures depleted of cancer stem cells did not self renew and or initiate tumors Immunofluorescence Staining Freshly frozen human glioma surgical biopsy samples were processed as previously described in accordance having a Duke University Medical Center Institutional Critique Board accredited protocol . Slides have been stained with polyclonal rabbit anti IL6R with monoclonal mouse anti CD133 or monoclonal mouse anti gp130 with rabbit polyclonal anti CD133 antibodies . For sections of xenografts handled with IL6 antibody, slides were stained with polyclonal anti Nestin or monoclonal PCAM . Primary antibodies had been incubated for 16 hours at 4 C followed by detection with Alexa 488 goat anti mouse and Alexa 568 goat anti rabbit secondary antibodies.
Nuclei were stained with Hoechst 33342 and slides were mounted making use of Fluoromount . Confocal z stacks have been taken by a 63 water immersion aim lens on a Leica TG-101348 SP5 confocal microscopre employing sequential scans . Complete RNA was prepared working with the RNeasy kit , and reverse transcribed into cDNA utilizing an iScript cDNA synthesis kit . mRNA ranges were measured working with probes from SABiosciences with SYBR Green and a ABI 7900 method . Lentiviral shRNA clones targeting IL6R , IL6 and scramble manage were bought from Sigma . These vectors were cotransfected using the packaging vectors psPAX2 and pCI VSVG into 293FT cells by lipofectamine 2000 to provide virus.
To assess the likely contribution of IL6 signals to glioma biology in the context in the not too long ago recognized tumor subpopulations, we measured IL6 receptor expression in freshly isolated GSCs and non stem glioma cells derived applying our previously described methodology .