A novel intracoronary hypothermia device reduces myocardial reperfusion injury in pigs.

BACKGROUND: Hypothermia therapy has been suggested to attenuate myocardial necrosis; however, the clinical implementation as a valid therapeutic strategy has failed, and new approaches are needed to translate into clinical applications. This study aimed to assess the feasibility, safety, and efficacy of a novel selective intracoronary hypothermia (SICH) device in mitigating myocardial reperfusion injury. METHODS: This study comprised two phases. The first phase of the SICH was performed in a normal porcine model for 30 minutes ( n = 5) to evaluate its feasibility. The second phase was conducted in a porcine myocardial infarction (MI) model of myocardial ischemia/reperfusion was performed by balloon occlusion of the left anterior descending coronary artery for 60 minutes and maintained for 42 days. Pigs in the hypothermia group ( n = 8) received hypothermia intervention onset reperfusion for 30 minutes and controls ( n = 8) received no intervention. All animals were followed for 42 days. Cardiac magnetic resonance analysis (5 and 42 days post-MI) and a series of biomarkers/histological studies were performed. RESULTS: The average time to lower temperatures to a steady state was 4.8 ± 0.8 s. SICH had no impact on blood pressure or heart rate and was safely performed without complications by using a 3.9 F catheter. Interleukin-6 (IL-6), tumor necrosis factor-α, C-reactive protein (CRP), and brain natriuretic peptide (BNP) were lower at 60 min post perfusion in pigs that underwent SICH as compared with the control group. On day 5 post MI/R, edema, intramyocardial hemorrhage, and microvascular obstruction were reduced in the hypothermia group. On day 42 post MI/R, the infarct size, IL-6, CRP, BNP, and matrix metalloproteinase-9 were reduced, and the ejection fraction was improved in pigs that underwent SICH. CONCLUSIONS: The SICH device safely and effectively reduced the infarct size and improved heart function in a pig model of MI/R. These beneficial effects indicate the clinical potential of SICH for treatment of myocardial reperfusion injury.

Are baseline conditions of coronary arteries sufficient for calculating angio-based index of microcirculatory resistance and fractional flow reserve?

Background: Angio-based index of microcirculatory resistance (IMR) and fractional flow reserve (FFR) have been developed, however, the differences between baseline and hyperemic data and their effects on their computation have not yet been discussed. This study aimed to compare the diagnostic performance of a novel method for calculating IMR and FFR from coronary angiography under baseline and hyperemic conditions. Methods: We performed a retrospective study to investigate the diagnostic performance of angiography-derived IMR (AccuIMR) and FFR (AccuFFRangio) computed from the hyperemic condition (AccuIMRhyp, AccuFFRangiohyp) and baseline condition (AccuIMRbase, AccuFFRangiobase) in 101 consecutive patients with chronic coronary syndrome (CCS) who underwent measurements of IMR and FFR at a single center, using wire-based IMR and FFR as the reference standard. Results: AccuIMRhyp showed much better correlation with IMR than AccuIMRbase (r=0.77 vs. 0.47, P<0.001). The diagnostic accuracy and area under the curve (AUC) for identifying significant microvascular dysfunction was higher for AccuIMRhyp than AccuIMRbase [92.1% (95% CI: 85.0-96.5%) vs. 83.2% (95% CI: 74.4-89.9%), P=0.012; 0.942 (95% CI: 0.877-0.979) vs. 0.815 (95% CI: 0.726-0.886), P=0.003]. The computed AccuFFRangio showed good correlations with FFR and good diagnostic performance under both hyperemic and baseline conditions [r=0.68 vs. 0.68, P>0.99; diagnostic accuracy =95.9% (95% CI: 89.8-98.9%) vs. 94.9% (95% CI: 88.4-98.3%), P=0.728; AUC =0.989 (95% CI: 0.942-1.000) vs. 0.973 (95% CI: 0.919-0.995), P=0.381]. The net reclassification index (NRI) demonstrated that hyperemic group had improved reclassification ability compared to the baseline group in identification of IMR >25 (NRI =0.20, P<0.001) and FFR ≤0.8 (NRI =0.11, P<0.001). Conclusions: By comparing the calculated angio-derived IMR and FFR under the baseline and hyperemic conditions, this study demonstrates that AccuIMR calculation is more accurate using the hyperemic condition, while AccuFFRangio calculation is accurate under both conditions.

Current development of pyrazole-azole hybrids with anticancer potential.

Chemotherapy is a critical treatment modality for cancer patients, but multidrug resistance remains one of the major challenges in cancer therapy, creating an urgent need for the development of novel potent chemical entities. Azoles, particularly pyrazole, could interact with different biological targets and exhibit diverse biological properties including anticancer activity. Many clinically used anticancer agents own an azole moiety, demonstrating that azoles are privileged and pivotal templates in the discovery of novel anticancer chemotherapeutics. The present article is an attempt to highlight the recent advances in pyrazole-azole hybrids with anticancer potential and discuss the structure-activity relationships, covering articles published from 2018 to present, to facilitate the rational design of more effective anticancer candidates.

Structural Feature Analyzation Strategies toward Discovery of Orally Bioavailable PROTACs of Bruton's Tyrosine Kinase for the Treatment of Lymphoma.

Bruton's tyrosine kinase proteolysis-targeting chimeras (BTK-PROTACs) have emerged as a promising approach to address the limitations of BTK inhibitors. However, conducting the rational discovery of orally bioavailable BTK-PROTACs presents significant challenges. In this study, dimensionality reduction analysis and model molecule validation were utilized to identify some key structural features for improving the oral absorption of BTK-PROTACs. The results were applied to optimize the newly discovered BTK-PROTACs B1 and B2. Compound C13 was discovered with improved oral bioavailability, high BTK degradation activity, and selectivity. It exhibited inhibitory effects against different hematologic cancer cells and attenuated the BTK-related signaling pathway. The oral administration of C13 effectively reduced BTK protein levels and suppressed tumor growth. This study led to the discovery of a new orally bioavailable BTK-PROTAC for the treatment of lymphoma, and we hope that the strategy will find wide utility.

Structure-based rational design enables efficient discovery of a new selective and potent AKT PROTAC degrader.

AKT and associated signaling pathways have been recognized as promising therapeutic targets for decades, and growing evidence indicates that inhibition or degradation of cellular AKT are viable strategies to treat cancer. Guided by an in silico modeling approach for rational linker design and based on our previous work in this field, we herein efficiently synthesized a small group of cereblon-recruiting AKT PROTAC molecules and identified a highly potent AKT degrader B4. Compared to the existing AKT degraders, B4 has a structurally unique AKT targeting warhead derived from the pyrazole-furan conjugated piperidine derivatives. It induces selective degradation of all three isoforms of AKT and exhibits efficacious anti-proliferation against several human hematological cancers. Notably, B4 demonstrates potent inhibition of AKT downstream signaling superior to its parental inhibitor. Together with its active analogs, B4 expands the arsenal of AKT chemical degraders as a valuable probe to uncover AKTs new functions and as a potential drug candidate to treat cancer.

The current scenario on anticancer activity of artemisinin metal complexes, hybrids, and dimers.

Cancer, the most significant cause of morbidity and mortality, has already posed a heavy burden on health care systems globally. In recent years, cancer treatment has made a significant breakthrough, but cancer cells inevitably acquire resistance, and the efficacy of the treatment is greatly reduced as the tumor progresses. To overcome the above issues, novel chemotherapeutics are needed urgently. Artemisinin and its derivatives-sesquiterpene lactone compounds possessing a unique peroxy bridge moiety-exhibit excellent safety and tolerability profiles. Mechanistically, artemisinin derivatives can promote cancer cell apoptosis, induce cell cycle arrest and autophagy, and inhibit cancer cell invasion and migration. Accordingly, artemisinin derivatives demonstrate promising anticancer efficacy both in vitro and in vivo, and even in clinical Phase I/II trials. The purpose of the present review article is to provide an emphasis on the current scenario (January 2017-January 2022) of artemisinin derivatives with potential anticancer activity, inclusive of artemisinin metal complexes, hybrids, and dimers. The structure-activity relationships and mechanisms of action are also discussed to facilitate the further rational design of more effective candidates.

Novel Fe(III)-Polybasic acid coordination polymer nanoparticles with targeted retention for photothermal and chemodynamic therapy of tumor.

The development of Fe-coordination polymer-based nanoparticles, with safe and high anti-tumor effects, for the treatment of tumor is facing challenges such as limited resources and poor targeting. In this study, we prepared Fe-polyhydroxy coordination polymer nanoparticles (TA-Fe@MNPs), based on tartaric acid (TA)-Fe(III) coordination polymer as the new photothermal agent, mannose (M) as the target, and bovine serum albumin (BSA) and polyethyleneimine (PEI) as the carrier materials, and investigated them for targeting the multifunctional therapy of tumors. The TA-Fe@MNPs synthesized via a simple coordination of Fe3+ with TA, bovine serum albumin, and polyethyleneimine under ambient conditions exhibited an appropriate size (~125 nm), electrically neutral surfaces, good biocompatibility, and low normal cell toxicity. The TA-Fe@MNPs are the first to exhibit a remarkable photothermal performance. They also showed a pH-sensitive Fenton-like response that was further enhanced via glutathione response. Interestingly, after a single injection, the TA-Fe@MNPs could be retained at the tumor site for 36 h with an effective photothermal dose, which was attributed to the reduced protein adsorption and slow elimination in tumor cells with the aid of M modification and carrier materials, while that for the TA-Fe@NPs did so for only 2 h. Tumor ablation was demonstrated by in vivo photothermal and chemokinetic therapy using TA-Fe@MNPs, and their safety was evident from the weight changes and blood parameters. These results indicated that the TA-Fe@MNPs, as new photothermal and CDT agents, have the potential to be used in clinical tumor therapy nanoplatforms.

Recent advance of peptide-based molecules and nonpeptidic small-molecules modulating PD-1/PD-L1 protein-protein interaction or targeting PD-L1 protein degradation.

Tumor immunotherapy has made great progress in recent years. In the tumor microenvironment, the binding of PD-1 and its ligand PD-L1 can promote tumor immune escape and tumor survival. Clinical studies have indicated that antibodies blocking PD-1 and PD-L1 have reliable effects on many advanced malignant tumors. However, no small-molecule inhibitors have been approved so far, indicating that the development of marketable small-molecules PD-1/PD-L1 targeted therapy drugs is a challenging process. Small-molecule inhibitors can overcome the limitations of monoclonal antibodies, including poor oral bioavailability, high cost, poor tissue and tumor penetration and long half-life, which prompt researchers to turn their attention to the development of peptide molecules and small-molecule inhibitors modulating PD-1/PD-L1 to overcome some disadvantages of monoclonal antibodies or targeting PD-L1 protein degradation as potential alternatives or supplements. In this review, we will focus on the peptide-based and nonpeptidic molecules against PD-1/PD-L1 base on the structural classification. More importantly, we also focus on the latest research progress of small-molecules mediated PD-L1 degradation mechanism.

Bruton's tyrosine kinase inhibitors in primary central nervous system lymphoma-evaluation of anti-tumor efficacy and brain distribution.

BACKGROUND: Primary central nervous system lymphoma (PCNSL) is an aggressive lymphoma confined to central nervous system. Current treatments including surgery, chemotherapy and whole-brain radiotherapy often fail to achieve satisfactory effect, especially in elderly. As a regimen in targeted therapy, Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has been tested in several clinical trials against PCNSL, offering hope for patients unfit for chemotherapy. We aim to evaluate and compare the anti-PCNSL ability of three different BTK inhibitors, ibrutinib, zanubrutinib and tirabrutinib, providing direct evidence for the targeted therapy of PCNSL. METHODS: Retrospective study was done on patients who received ibrutinib-based therapy in our hospital. Cerebrospinal fluid (CSF) from one patient was collected to measure the concentration of ibrutinib. Inhibition assay and apoptosis assay were done on lymphoma cells to determine the anti-tumoral effects of three inhibitors. Pharmacokinetic study was conducted to evaluate their ability in penetrating blood brain barrier and distributing in brain. RESULTS: In retrospective study, we found three patients with PCNSL who had good clinical response to ibrutinib-based therapy (2 complete remission, 1 partial remission), which further support the use of BTK inhibitors in PCNSL. In vitro studies show that ibrutinib has the best anti-tumoral ability among three inhibitors. In vivo study on pharmacokinetic profiles indicate that both ibrutinib and tirabrutinib are good in distributing in brain parenchyma. CONCLUSIONS: In conclusion, our study results suggest that BTK inhibitors can be promising candidates for PCNSL treatment, preferring the use of ibrutinib and tirabrutinib as anti-PCNSL agents among the three inhibitors.

Isocitrate Dehydrogenase 2 Inhibitors for the Treatment of Hematologic Malignancies: Advances and Future Opportunities.

Tumor cells frequently reprogram cellular metabolism from oxidative phosphorylation to glycolysis. Isocitrate dehydrogenase 2 (IDH2) has been intensively studied due to its involvement in the metabolic activity of cancer cells. Mutations in IDH2 promote neomorphic activity through the generation of oncometabolite 2-hydroxyglutarate (2-HG). The overproduced 2-HG can competitively inhibit α-KG-dependent dioxygenases to trigger cell differentiation disorders, a major cause of blood tumors. This review outlines recent progress in the identification of IDH2 inhibitors in blood cancer to provide a reference for ongoing and future clinical studies.

Development of differentiation modulators and targeted agents for treating neuroblastoma.

Neuroblastoma (NB) is one of the most common pediatric malignancies. Easy metastasis, poor prognosis, and a high degree of heterogeneity of NB hinder its successful treatment. Several different therapeutic strategies have been developed to overcome these problems, including differentiation and targeted therapy. In this review, we summarize the recent development of differentiation modulators and targeted agents for treating NB. Several promising targets of NB were also discussed.

High nitrogen stainless steel drug-eluting stent - Assessment of pharmacokinetics and preclinical safety in vivo.

Pharmacokinetic analyses were performed using 20 pigs for 120-days implantation, while one sirolimus-eluting stent was implanted into one of their coronary artery. At different time points, the residual sirolimus on the stent, delivered locally (to artery wall), regionally (to adjacent and downstream muscle) and systemically (to plasma and visceral organs), was detected throughout 120 days. Preclinical safety evaluation was performed using 32 pigs for 180-days implantation to study the safety of metal platform material and the effectiveness of sirolimus eluting coating on the HNS stent. The neointima area, restenosis rate and inflammatory grade for HNS and control group stents were detected and analyzed. Approximately 80% sirolimus was eluted from the sirolimus-eluting stents after 30-days implantation in vivo. Additionally, there was sustained sirolimus in the artery wall, cardiac muscle and heart throughout 120-days implantation, and sirolimus accumulated to the peak at 90-days implantation. It was inferred that the sirolimus eluting stent in this study was covered by neointima before 90-days implantation, indicating that the sirolimus eluting coating on the HNS stent was safe and effective. Very little sirolimus was distributed in visceral organs after 14-days implantation. HNS sirolimus-eluting stent exhibited lower restenosis rate and lower inflammatory grade than control group, which verified that the sirolimus-eluting coating design in this study was reasonable and practical. In addition, there were no significant difference in restenosis rate and inflammatory score between HNS bare-metal stent and drug-eluting stents, illustrating that HNS has good bio-compatibility and is suitable to use as coronary artery stent material.

Discovery of 3,4,6-Trisubstituted Piperidine Derivatives as Orally Active, Low hERG Blocking Akt Inhibitors via Conformational Restriction and Structure-Based Design.

A series of 3,4-disubstituted piperidine derivatives were obtained based on a conformational restriction strategy and a lead compound, A12, that exhibited potent in vitro and in vivo antitumor efficacies; however, obvious safety issues limited its further development. Thus, systematic exploration of the structure-activity relationship of compound A12, involving the phenyl group, hinge-linkage, and piperidine moiety, led to the discovery of the superior 3,4,6-trisubstituted piperidine derivative E22. E22 showed increased potency in Akt1 and cancer cell inhibition, remarkably reduced human ether-a-go-go-related gene blockage, and significantly improved safety profiles. Compound E22 also exhibited good kinase selectivity, had a good pharmacokinetic profile, and displayed very potent in vivo antitumor efficacy, with over 90% tumor growth inhibition in the SKOV3 xenograft model. Further mechanistic studies were conducted to demonstrate that compound E22 could significantly inhibit the phosphorylation of proteins downstream of Akt kinase in cells and tumor tissue from the xenograft model.

Discovery of pyrazole-thiophene derivatives as highly Potent, orally active Akt inhibitors.

A series of pyrazole-thiophene derivatives exhibiting good Akt inhibitory activities were obtained on the basis of conformational restriction strategy, leading to the discovery of compound 1d and 1o which showed excellent in vitro antitumor effect against a variety of hematologic cancer cells and their potential of inducing apoptosis, blocking the cell cycles at S phase and significantly inhibiting the phosphorylation of downstream biomarkers of Akt kinase of cancer cells. Amongst, compound 1o also exhibited good PK profiles and inhibited about 40% tumor growth in MM1S xenograft model. Compound 1o might be a potential candidate for further development.

Chemical Proteomic Profiling of Bromodomains Enables the Wide-Spectrum Evaluation of Bromodomain Inhibitors in Living Cells.

Bromodomains, epigenetic "readers" of lysine acetylation marks, exist in different nuclear proteins with diverse biological functions in chromatin biology. Malfunctions of bromodomains are associated with the pathogenesis of human diseases, such as cancer. Bromodomains have therefore emerged as therapeutic targets for drug discovery. Given the high structural similarity of bromodomains, a critical step in the development of bromodomain inhibitors is the evaluation of their selectivity to avoid off-target effects. While numerous bromodomain inhibitors have been identified, new methods to evaluate the inhibitor selectivity toward endogenous bromodomains in living cells remain needed. Here we report the development of a photoaffinity probe, photo-bromosporine (photo-BS), that enables the wide-spectrum profiling of bromodomain inhibitors in living cells. Photo-BS allowed light-induced cross-linking of recombinant bromodomains and endogenous bromodomain-containing proteins (BCPs) both in vitro and in living cells. The photo-BS-induced labeling of the bromodomains was selectively competed by the corresponding bromodomain inhibitors. Proteomics analysis revealed that photo-BS captured 28 out of the 42 known BCPs from the living cells. Assessment of the two bromodomain inhibitors, bromosporine and GSK6853, resulted in the identification of known as well as previously uncharacterized bromodomain targets. Collectively, we established a chemical proteomics platform to comprehensively evaluate bromodomain inhibitors in terms of their selectivity against endogenous BCPs in living cells.

Design, synthesis, and molecular docking study of 3H-imidazole[4,5-c]pyridine derivatives as CDK2 inhibitors.

A novel series of imidazo[4,5-c]pyridine-based CDK2 inhibitors were designed from the structure of CYC202 via scaffold hopping strategy. These compounds were synthesized and biologically evaluated for their CDK2 inhibitory and in vitro anti-proliferation potential against cancer cell lines. Several compounds exhibited potent CDK2 inhibition with IC50 values of less than 1 µM. The most potent compound 5b showed excellent CDK2 inhibitory (IC50 = 21 nM) and in vitro anti-proliferation activity against three different cell lines (HL60, A549, and HCT116). The molecular docking and dynamic studies portrayed the potential binding mechanism between 5b and CDK2, and several key interactions between them were observed, which would be the reason for its potent CDK2 inhibitory and anti-proliferation activities. Therefore, the pyridin-3-ylmethyl moiety would serve as an excellent pharmacophore for the development of novel CDK2 inhibitors for targeted anti-cancer therapy.

Inverse Relationship between Serum VEGF Levels and Late In-Stent Restenosis of Drug-Eluting Stents.

Late in-stent restenosis (ISR) has raised concerns regarding the long-term efficacy of drug-eluting stents (DES). The role of vascular endothelial growth factor (VEGF) in the pathological process of ISR is controversial. This retrospective study aimed to investigate the relationship between serum VEGF levels and late ISR in patients with DES implantation. A total of 158 patients who underwent angiography follow-up beyond 1 year after intervention were included. The study population was classified into ISR and non-ISR groups. The ISR group was further divided according to follow-up duration and Mehran classification. VEGF levels were significantly lower in the ISR group than in the non-ISR group [96.34 (48.18, 174.14) versus 179.14 (93.59, 307.74) pg/mL, p < 0.0001]. Multivariate regression revealed that VEGF level, procedure age, and low-density lipoprotein cholesterol were independent risk factors for late ISR formation. Subgroup analysis demonstrated that VEGF levels were even lower in the very late (≥5 years) and diffuse ISR group (Mehran patterns II, III, and IV) than in the late ISR group (1-4 years) and the focal ISR group (Mehran pattern I), respectively. Furthermore, significant difference was found between diffuse and focal ISR groups. Serum VEGF levels were inversely associated with late ISR after DES implantation.

Role of KCa3.1 Channels in Macrophage Polarization and Its Relevance in Atherosclerotic Plaque Instability.

OBJECTIVE: Emerging evidence indicates that proinflammatory macrophage polarization imbalance plays a key role in atherosclerotic plaque progression and instability. The calcium-activated potassium channel KCa3.1 is critically involved in macrophage activation and function. However, the role of KCa3.1 in macrophage polarization is unknown. This study investigates the potential role of KCa3.1 in transcriptional regulation in macrophage polarization and its relationship to plaque instability. APPROACH AND RESULTS: Human monocytes were differentiated into macrophages using macrophage colony-stimulating factor. Macrophages were then polarized into proinflammatory M1 cells by interferon-γ and lipopolysaccharide and into alternative M2 macrophages by interleukin-4. A model for plaque instability was induced by combined partial ligation of the left renal artery and left common carotid artery in apolipoprotein E knockout mice. Significant upregulation of KCa3.1 expression was observed during the differentiation of human monocytes into macrophages. Blocking KCa3.1 significantly reduced the expression of proinflammatory genes during macrophages polarization. Further mechanistic studies indicated that blocking KCa3.1 inhibited macrophage differentiation toward the M1 phenotype by downregulating signal transducer and activator of transcription-1 phosphorylation. In animal models, KCa3.1 blockade therapy strikingly reduced the incidence of plaque rupture and luminal thrombus in carotid arteries, decreased the expression of markers associated with M1 macrophage polarization, and enhanced the expression of M2 markers within atherosclerotic lesions. CONCLUSIONS: These results suggest that blocking KCa3.1 suppresses plaque instability in advanced stages of atherosclerosis by inhibiting macrophage polarization toward an M1 phenotype.

Short-term safety and efficiency of cryoablation for renal sympathetic denervation in a swine model.

BACKGROUND: Renal sympathetic nerves are involved in the reflective activation of the sympathetic nervous system in circulatory control. Catheter-based renal denervation (RDN) ameliorated treatment-resistant hypertension safely, but 10%-20% of treated patients are nonresponders to radiofrequency denervation. The purpose of this study was to investigate the safety and efficiency of cryoablation for sympathetic denervation in a swine model and to explore a new way of RDN. METHODS: Seven swines randomly assigned to two groups: Renal cryoablation (CR) group and control group. The control group underwent renal angiogram only. The CR group underwent renal angiogram plus bilateral renal cryoablation. Renal angiograms via femoral were performed before denervation, after denervation and prior to the sacrifice to access the diameter of renal arterial and the pressure of aorta abdominalis. Euthanasia of the swine was performed on 28-day to access norepinephrine (NE) changes of the renal cortex and the changes of renal nerves. RESULTS: Cryoablation did not induce severe complications at any time point. There was no significant change in diameter of renal artery. CR reduced systolic blood pressure (BP) from 145.50 ± 9.95 mmHg at baseline to 119.00 ± 14.09 mmHg. There was a slight but insignificant decrease in diastolic BP. The main nerve changes at 28-day consisted of necrosis with perineurial fibrosis at the site of CR exposure in conjunction with the nerve vacuolation. Compared with the control group, renal tissue NE of CR group decreased by 89.85%. CONCLUSIONS: Percutaneous catheter-based cryoablation of the renal artery is safe. CR could effectively reduce NE storing in the renal cortex, and the efficiency could be maintained 28-day at least.

Design, synthesis, biological evaluation, and docking studies of (s)-phenylalanine derivatives with a 2-cyanopyrrolidine moiety as potent dipeptidyl peptidase 4 inhibitors.

A novel series of (S)-phenylalanine derivatives with a 2-cyanopyrrolidine moiety were designed and synthesized through a rational drug design strategy. Biological evaluation revealed that most tested compounds were potent dipeptidyl peptidase 4 (DPP-4) inhibitors; among them, the cyclopropyl-substituted phenylalanine derivative 11h displayed the most potent DPP-4 inhibitory activity with an IC50 value of 0.247 µm. In addition, molecular docking analysis of the representative compounds 11h, 11k, and 15a were performed, which not only revealed the impact of binding modes on DPP-4 inhibitory activity but also provided additional methodological values for design and optimization.