Predominant Target Research Articles

Assessment of Joint Health Status of Haemophilia Patients in a Tertiary Care Hospital of Bangladesh

Background: Haemophilia is a sex-linked bleeding disorder. Affected patients suffer spontaneous or post-traumatic bleeding into various sites of the body, mainly into joints, depending on the level of coagulation factor deficiency. Recurrent joint bleeds lead to progressive disability. Susceptibility to joint haemorrhage in persons with haemophilia suggests that the routine assessment of joint health is an important aspect of clinical management and outcome studies assessing the efficacy of treatment. Aim: The aim of the study was to assess of joint health status in haemophilia patients attending in a tertiary care hospital, Bangladesh. Methodology: A descriptive observational study was carried out in the department of Haematology, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka. A total number of 45 patients who were attended in OPD and IPD, meeting inclusion and exclusion criteria of enrolled haemophilia patients, were included in the study. Joint assessment or any kind of joint impairment was assessed by the Haemophilia Joint Health Score (HJHS) score 2.1 version (an eight-item scoring tool for assessing joint impairments of the six index joints in boys with haemophilia aged 4–18 years. Clinical and laboratory information was recorded with a semi structured questionnaire. Result: Among 45 haemophilia patients, the mean age was 12.9±3.4 years while males were predominant (91.1%). Majority (80.0%) of the patients (36) were diagnosed as haemophilia A. This study observed that, 48.9% of the patients had moderate and 28.9% had severe haemophilia. Knee joint was the predominant target joint. Left knee joint involved in 80% cases and right knee joint were involved in 55.6% of patients. The mean global gait score was found 1.49±0.66 and mean HJHS score was 15.8±4.4. There was no significant correlation between HJHS score and severity of haemophilia (r= -0.081; p=0.596) while significant correlation between the ages of patient with HJHS score (r=0.536; p=0.001) was observed. Conclusion: Haemophilia is a debilitating and life-threatening disease that affects mostly knee, ankle and elbow joints. The joint evaluation system is of paramount importance in clinical practice which is capable to prevent major haemarthrosis and chronic haemophilic synovitis. Haemophilia Joint Health Score (HJHS) is an effective and reliable tool to detect early and subtle changes in joint health and function. This study finding will help in future to assess early the joint conditions and complications, which will assist to modify the treatment approach on demand and start prophylaxis.

Perioperative Practices in Moyamoya Syndrome Revascularization: An International Transdisciplinary Survey

Background There are no evidence‐based resources guiding the perioperative management of patients with moyamoya syndrome who are undergoing revascularization surgery. We investigated practice patterns among a transdisciplinary group aiming at identifying possible heterogeneity of practices on key components of care that warrant prospective studies. Methods We disseminated a web‐based Qualtrics survey internationally to physician members of the following: Neurocritical Care Society, Society of Critical Care Medicine, American Academy of Neurology, Society for Neuroscience in Anesthesiology and Critical Care, American Association of Neurological Surgeons, Asian Society of Neuroanesthesia and Critical Care, Indian Society of Neuroanesthesia and Critical Care, Japanese Society for Neuroscience in Anesthesiology and Critical Care, and World Federation of Neurosurgical Societies. The survey contained questions on demographics and aspects of preoperative, intraoperative, and postoperative care. Results Among the 175 physicians who managed at least 1 adult (aged ≥18 years) undergoing moyamoya revascularization in the preceding 24 months, 18 countries (United States, 84.6%) and 4 disciplines (anesthesiology [44.7%, 76/170], critical care medicine [30.6%, 52/170], neurology [32.4%, 55/170], and neurosurgery [15.3%, 26/170]) were represented. Anesthesiologists preferred total intravenous over volatile anesthesia (56.3%, 40/71 versus 42.3%, 30/71) and arterial line zeroing at the circle of Willis/tragus over the phlebostatic axis/right atrium intraoperatively (84.3%, 59/70 versus 11.4%, 8/70) and postoperatively (68.9%, 42/61 versus 24.6%, 15/61). Intraoperative blood pressure goals were primarily targeted to baseline blood pressure (34.8%, 48/138), whereas postoperative blood pressure goals were mainly determined by neurosurgeon preference (48.9%, 65/133). The predominant hemodynamic target was mean arterial pressure intraoperatively (50.4%, 68/135) and systolic blood pressure postoperatively (48.5%, 63/130). Crystalloid infusion was the preferred method to achieve perioperative hemodynamic goals (median rank, 1.0), followed by colloid infusion (median rank, 2.0) and phenylephrine (median rank, 2.0); however, colloid infusion and phenylephrine were considered contraindicated by 18.2% (10/55) and 20.0% (11/55), respectively. Conclusions We demonstrate perioperative practice heterogeneity for moyamoya syndrome revascularization among physicians for both methods and targets of hemodynamic management, constituting equipoise for prospective studies targeting optimal management strategies.

A neutralizing epitope on the SD1 domain of SARS-CoV-2 spike targeted following infection and vaccination.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is the target for neutralizing antibodies elicited following both infection and vaccination. While extensive research has shown that the receptor binding domain (RBD) and, to a lesser extent, the N-terminal domain (NTD) are the predominant targets for neutralizing antibodies, identification of neutralizing epitopes beyond these regions is important for informing vaccine development and understanding antibody-mediated immune escape. Here, we identify a class of broadly neutralizing antibodies that bind an epitope on the spike subdomain 1 (SD1) and that have arisen from infection or vaccination. Using cryo-electron microscopy (cryo-EM) and hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS), we show that SD1-specific antibody P008_60 binds an epitope that is not accessible within the canonical prefusion states of the SARS-CoV-2 spike, suggesting a transient conformation of the viral glycoprotein that is vulnerable to neutralization.

Nanoencapsulation of Ru(p-cymene) Complex Bearing Ginger-based Natural Product into Liposomal Nanoformulation to Improve Its Cellular Uptake and Antiproliferative Activity.

The organometallic compounds are prospective candidates in the row of developing metallochemotherapeutics with the aim of overcoming the limitations of platinum drugs. In order to explore the anticancer properties of organometallic compounds with the natural medicines, two Ru(II)-p-cymene complexes containing the natural products, viz., 6-gingerol (6G) and benzylated-6-gingerdione (B-6GD) have been synthesized and characterized well. The phenolic group of the Ru(6G) complex facilitates its higher cell-free antioxidant activity than its analogue complex. Also, the same complex shows higher cytotoxicity toward A549 lung and HeLa-S3 cervical cancer cells than the Ru(B-6GD) complex but lower cytotoxicity toward A2058 metastatic melanoma cancer cells. Both complexes are shown to easily accumulate in melanoma cancer cells, and their degree of cytotoxicity in the same cells is found to be positively correlated with cell uptake. The cytotoxicity of complexes arises from their intracellular activity, mainly due to the induction of singlet oxygen production in cancer cells. The subcellular fractionation study shows that mitochondria and ER-Golgi membranes might be their predominant targets. Also, the mechanistic investigation revealed that Ru(B-6GD) induces caspase-dependent non-apoptotic cell death whereas Ru(6G) can induce caspase-independent non-apoptotic cell death. Furthermore, both complexes are found to moderately alter the adhesion properties of cancer cells, which is beneficial for antimetastatic treatment. Despite the potential pharmacological activity, Ru(6G) is encapsulated into polymer-supported liposomes to reduce its toxicity and further improve its anticancer potency. The π-conjugated yne-ene chain of polydiacetylene aids in the development of a stable nanoformulation, which achieved a slow release of the complex. Most importantly, the cancer cell uptake of the liposome-encapsulated Ru(6G) complex is 20 times enhanced and the total ROS formation in cancer cells is significantly increased compared to the non-encapsulated complex. However, the nanoformulation does not alter the antimetastatic potency of the encapsulated complex.

Abstract 5848: Inhibition of the CRM1 nuclear export protein can treat mutant KRAS tumors through stabilizing the DLC1 tumor suppressor protein

Abstract Mutant KRAS is found in >30% of lung adenocarcinomas, and tumors with mutant KRAS usually have poor outcomes with current therapies. The rationale for the new treatment approach described here for tumors with mutant KRAS arises from the critical antitumor role that we have uncovered for reactivating the DLC1 tumor suppressor protein, which is frequently downregulated in a wide range of human cancers. Although several mechanisms account for DLC1 downregulation, here we have focused on a subset of mutant KRAS lung tumor lines that express wild type DLC1 mRNA but lack detectable DLC1 protein. We have recently reported that cytoplasmic EZH2 protein directly methylates Lysine-678 of DLC1 protein leading to its ubiquitination by CUL-4A and consequently, proteasomal degradation. In this study, we have found a specific inhibitor of CRM1 protein (Chromosomal Region Maintenance 1, also known as Exportin 1 [XPO1]) substantially reduces cytoplasmic EZH2 level in several mutant KRAS lung cancer cell lines demonstrating that cytoplasmic EZH2 depends on its nuclear export by CRM1. CRM1, which is the major mammalian nuclear export regulator, is frequently overexpressed and/or mutated in human cancer, including lung adenocarcinomas, and these phenotypes are associated with poor prognosis. Endogenous CRM1 and EZH2 protein colocalized and formed a complex in several lung cancer lines and patient-derived tumor xenografts implying the cancer relevance of this interaction. In addition to blocking the cytoplasmic export of EZH2, pharmacologic inhibition of CRM1 increased the steady-state level of DLC1 protein in several mutant KRAS lung cancer lines and patient-derived xenografts. The tumor suppressor activity of the post-translationally stabilized DLC1 protein was further increased by kinase inhibitors of AKT and SRC, which reverse the direct phosphorylation and attenuation of DLC1 tumor suppressor functions by these kinases. The three-drug combination of inhibitors of CRM1 (Selinexor), AKT (MK-2206), and SRC (Saracatinib), which is well-tolerated in mice, induced potent inhibition of tumor xenograft growth and was associated with induction of markers of apoptosis and senescence. Although each of these inhibitors has multiple targets, CRISPR/Cas9 knockout of DLC1 abolished most of the tumor growth inhibitory activity of the three-drug combination, strongly suggesting that DLC1 is the predominant target for the antitumor activity of the drug combination. Interestingly, KRAS siRNA knockdown phenocopied CRM1 inhibition, as it reduced cytoplasmic EZH2, DLC1 methylation, and DLC1 degradation. Our findings suggest CRM1 inhibitors, when given in conjunction with AKT and/or SRC kinase inhibitors, may have clinical efficacy against lung tumors with mutant KRAS. Citation Format: Brajendra K. Tripathi, Nicole Hirsh, Xiaolan Qian, Dunrui Wang, Marian E. Durkin, Fernando J. de Miguel, Katerina Politi, James H. Doroshow, Douglas R. Lowy. Inhibition of the CRM1 nuclear export protein can treat mutant KRAS tumors through stabilizing the DLC1 tumor suppressor protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5848.

Calibration of an Extended Eddy Viscosity Turbulence Model Using Bayesian Update

The present work aims at improving the performance of the eddy viscosity-based Menter - Shear Stress Transport turbulence model for complex aircraft flows by adding physical extensions to the length-scale equation along with calibration aided by uncertainty quantification methods. Three different extensions based on rotation and curvature correction, wake flow correction, and irrotational strain correction are added to the original model, resulting in a parametric model, which is then calibrated using Bayesian updates. A series of test cases is employed to assess the model performance, whereby delta wings with deflected flaps and vortical flows are the predominant target application.

In situ identification of cellular drug targets in mammalian tissue

The lack of tools to observe drug-target interactions at cellular resolution in intact tissue has been a major barrier to understanding invivo drug actions. Here, we develop clearing-assisted tissue click chemistry (CATCH) to optically image covalent drug targets in intact mammalian tissues. CATCH permits specific and robust in situ fluorescence imaging of target-bound drug molecules at subcellular resolution and enables the identification of target cell types. Using well-established inhibitors of endocannabinoid hydrolases and monoamine oxidases, direct or competitive CATCH not only reveals distinct anatomical distributions and predominant cell targets of different drug compounds in the mouse brain but also uncovers unexpected differences in drug engagement across and within brain regions, reflecting rare cell types, as well as dose-dependent target shifts across tissue, cellular, and subcellular compartments that are not accessible by conventional methods. CATCH represents a valuable platform for visualizingin vivointeractions of small molecules in tissue.

Identification of Phosphorylation and Other Post-Translational Modifications in the Central C4C5 Domains of Murine Cardiac Myosin Binding Protein C.

Cardiac myosin binding protein C (cMyBPC) is a critical multidomain protein that modulates myosin cross bridge behavior and cardiac contractility. cMyBPC is principally regulated by phosphorylation of the residues within the M-domain of its N-terminus. However, not much is known about the phosphorylation or other post-translational modification (PTM) landscape of the central C4C5 domains. In this study, the presence of phosphorylation outside the M-domain was confirmed in vivo using mouse models expressing cMyBPC with nonphosphorylatable serine (S) to alanine substitutions. Purified recombinant mouse C4C5 domain constructs were incubated with 13 different kinases, and samples from the 6 strongest kinases were chosen for mass spectrometry analysis. A total of 26 unique phosphorylated peptides were found, representing 13 different phosphorylation sites including 10 novel sites. Parallel reaction monitoring and subsequent mutagenesis experiments revealed that the S690 site (UniProtKB O70468) was the predominant target of PKA and PKG1. We also report 6 acetylation and 7 ubiquitination sites not previously described in the literature. These PTMs demonstrate the possibility of additional layers of regulation and potential importance of the central domains of cMyBPC in cardiac health and disease. Data are available via ProteomeXchange with identifier PXD031262.

Antibiogram of burn wound isolates at Masina hospital, Mumbai, India: A 12-year descriptive cross sectional study

: To study the antibiogram of Klebsiella, Pseudomonas and Staphylococcus and its change in sensitivities with time. : 4909 swabs were taken from790 of patients admitted to the Burns unit of Masina Hospital, Mumbai, over a period of 12 years (2008–2019). The swabs were cultured and percentage antibiotic sensitivity of 6835 predominate isolates to different class of antibiotic was determined and reviewed. : Klebsiella was the predominant organism in our set-up, followed by Pseudomonas and Staphylococcus aureus. The antibiotic sensitivities of the most predominant organisms are discussed in detail in this article. : Gram negative nosocomial infection predominate a burn injury. Knowing the predominant target pathogens and their sensitivity pattern towards different antibiotics will avoid misuse of antibiotic, contribute to prescribing the correct antibiotics and timely clinical treatment. A routine microbiological surveillance prior to administrating an antibiotic, a well established infection control department and regular reporting of changing antibiotic trends will help us overcome our battle against emerging multi drug resistant organism, thereby having more successful treatment outcome in burn patients.

Citrin mediated metabolic rewiring in response to altered basal subcellular Ca2+ homeostasis

In contrast to long-term metabolic reprogramming, metabolic rewiring represents an instant and reversible cellular adaptation to physiological or pathological stress. Ca2+ signals of distinct spatio-temporal patterns control a plethora of signaling processes and can determine basal cellular metabolic setting, however, Ca2+ signals that define metabolic rewiring have not been conclusively identified and characterized. Here, we reveal the existence of a basal Ca2+ flux originating from extracellular space and delivered to mitochondria by Ca2+ leakage from inositol triphosphate receptors in mitochondria-associated membranes. This Ca2+ flux primes mitochondrial metabolism by maintaining glycolysis and keeping mitochondria energized for ATP production. We identified citrin, a well-defined Ca2+-binding component of malate-aspartate shuttle in the mitochondrial intermembrane space, as predominant target of this basal Ca2+ regulation. Our data emphasize that any manipulation of this ubiquitous Ca2+ system has the potency to initiate metabolic rewiring as an instant and reversible cellular adaptation to physiological or pathological stress.

Application of the individualized objectives defined by the European 2019 lipid guidelines in patients with type 2 diabetes

Background and objectivecardiovascular disease is one of the main complications of people with type 2 diabetes (T2D). The ESC/ESA 2019 lipid guide has led to a change in dyslipidemia control. We analyse the evolution of the lipid profile, the fulfillment of the LDL-C targets, how patients are classified and the impact of this guide on lipid control in T2D patients. Materials and methodsA prospective cohort study from 2017 to 2020 from a cohort of 297 T2D out of a total of 1229 (95% confidence level). We classified patients according to their cardiovascular risk (CVR) and whether they met or their low-density cholesterol (LDL-C) goal. ResultsAge: 62.58 ± 10.68; 52.79% men. Mean LDL-C levels 116.2 at baseline and 100.2 mg/dl at four years (p < .001). They met their individualized LDL-C target after publication of the guide: 57 (21.67%). There were more controls who were under 65 (57.9% vs 36.9% p < .01; RR 0.83), men (66.7% vs 49.5% p < .05; RR 0, 86) and smokers (17.5% vs 7.8% p < .05). 74.23% had a high CVR and a target LDL-C <70 mg/dl. ConclusionsSince the publication of the ESC/ESA 2019 lipid guide, a decrease in LDL-c levels has been observed. Only one in five patients fulfill their individualized LDL-C target. Male patients, under 65 years of age and smokers presented an advantage in meeting their goal. Most T2D patients have a high CVR, and the predominant LDL-C target is less than 70 mg/dl.

Lesion characteristics and procedural complications of chronic total occlusion percutaneous coronary intervention in patients with prior bypass surgery: A meta‐analysis

Coronary artery bypass graft (CABG) accelerates the prevalence of native coronary chronic total occlusion (CTO), and this kind of CTO shows extensive challenging and complex atherosclerotic pathology. As a result, the procedural success rate of percutaneous coronary intervention (PCI) is inferior to another kind of lesions. The present meta‐analysis aims to compare the lesion characteristics and procedural complications of CTO‐PCI in patients with or without prior CABG. A total of 8 studies, comprising of 13439 patients, published from inception to August 2021 were included in this meta‐analysis. Results were pooled using random effects model and are presented as odds ratio (OR) with 95% confidence intervals (95% CIs). From the 13439 patients enrolled, 3349 (24.9%) patients had previous CABG and 10090 (75.1%) formed the control group in our analysis. For the clinical characteristic, compared to the non‐CABG patients, prior CABG patients were older (OR, 3.98; 95% CI, 3.19–4.78; p < .001; I 2 = 72%), had more male (OR, 1.30; 95% CI, 1.14–1.49; p < .001; I 2 = 6%), diabetes mellitus (OR, 1.54; 95% CI, 1.36–1.73; p < .001; I 2 = 37%), dyslipidemia (OR, 1.89; 95% CI, 1.33–2.69; p < .001; I 2 = 81%), hypertension (OR, 1.88; 95% CI, 1.46–2.41; p < .001; I 2 = 71%), previous myocardial infarction (OR, 1.94; 95% CI, 1.48–2.56; p < .001; I 2 = 85%), and previous PCI (OR, 1.74; 95% CI, 1.52–1.98; p < .001; I 2 = 22%). Non‐CABG patents had more current smoker (OR, .45; 95% CI, 0.27–0.74; p < .001; I 2 = 91%). BMI (OR, −0.01; 95% CI, −0.07–0.06; p = .85; I 2 = 36%) were similar in both groups. For lesions location, the right coronary artery (RCA) was predominant target vessel in both groups (50.5% vs 48.7%; p＝.49), although, the left circumflex (LCX) was more frequently CTO in the prior CABG group (27.3% vs 18.9%; p＜.01), while left anterior descending artery (LAD) in non‐CABG ones (16.0% vs 29.1%; p＜0.01). For lesions characteristics, prior CABG patients had more blunt stump (OR, 1.71; 95% CI, 1.46–2.00; p < .001; I 2 = 40%), proximal cap ambiguity (OR, 1.45; 95% CI, 1.28–1.64; p < .001; I 2 = 0.0%), severe calcifications (OR, 2.91; 95% CI, 2.19–3.86; p < .001; I 2 = 83%), more bending (OR, 3.07; 95% CI, 2.61–3.62; p < .001; I 2 = 0%), lesion length > 20 mm (OR, 1.59; 95% CI, 1.10–2.29; p = .01; I 2 = 83%), inadequate distal landing zone (OR, 1.95; 95% CI, 1.75–2.18; p＜.001; I 2 = 0.0%), distal cap at bifurcation (OR, 1.65; 95% CI, 1.46–1.88; p < .001; I 2 = 0.0%), and higher J‐CTO score (SMD, 0.52; 95% CI, 0.42–0.63; p < .001; I 2 = 65%). But side branch at proximal entry (OR, 0.88; 95% CI, 0.72–1.07; p = .21; I 2 = 45%), in‐stent CTO (OR, 0.99; 95% CI, 0.86–1.14; p = .88; I 2 = 0.0%), lack of interventional collaterals (OR, 0.80; 95% CI, 0.55–1.15; p = .23; I 2 = 78%), and previously failed attempt (OR, 0.73; 95% CI, 0.48–1.11; p = .14; I 2 = 89%) were similar in both groups. For complication, prior CABG patients had more perforation with need for intervention (OR, 1.91; 95% CI, 1.36–2.69; p < 0.001; I 2 = 34%), contrast‐induced nephropathy (OR, 3.40; 95% CI, 1.31–8.78; p = .01; I 2 = 0.0%). Non‐CABG patents had more tamponade (OR, 0.25; 95% CI, 0.09–0.72; p = .01; I 2 = 0.0%), and the major bleeding complication (OR, 1.18; 95% CI, 0.57–2.44; p = .65; I 2 = 0%) were no significant difference in both groups. In conclusion, Patients with prior CABG undergoing CTO‐PCI have more complex lesion characteristics, though procedural complication rates were comparable.

Aplicación de los objetivos individualizados definidos por la guía europea 2019 de lípidos en pacientes con diabetes tipo 2

Background and objectiveCardiovascular disease is one of the main complications of people with type 2 diabetes (T2D). The ESC/ESA 2019 lipid guide has led to a change in dyslipidemia control. We analyze the evolution of the lipid profile, the fulfillment of the low-density cholesterol (LDL-C) targets, how patients are classified and the impact of this guide on lipid control in T2D patients. Materials and methodsA prospective cohort study from 2017 to 2020 from a cohort of 297 T2D out of a total of 1229 (95% confidence level). We classified patients according to their cardiovascular risk and whether they met or not their LDL-C goal. ResultsAge 62.58±10.68 years; 52.79% men. Mean LDL-C levels 116.2 at baseline and 100.2mg/dL at 4 years (P<.001). They met their individualized LDL-C target after publication of the guide: 57 (21.67%). There were more controls who were under 65 years (57.9 vs. 36.9%; P<.01; RR 0.83), men (66.7 vs. 49.5%; P<.05; RR 0.86) and smokers (17.5 vs. 7.8%; P<.05). A percentage of 74.23 had a high cardiovascular risk and a target LDL-C<70mg/dL. ConclusionsSince the publication of the ESC/ESA 2019 lipid guide, a decrease in LDL-C levels has been observed. Only one in 5 patients fulfill their individualized LDL-C target. Male patients, under 65 years of age and smokers presented an advantage in meeting their goal. Most T2D patients have a high cardiovascular risk, and the predominant LDL-C target is less than 70mg/dL.

Pharmacological Poly (ADP-Ribose) Polymerase Inhibitors Decrease Mycobacterium tuberculosis Survival in Human Macrophages.

Diabetes mellites (DM) is correlated with increased susceptibility to and disease progression of tuberculosis (TB), and strongly impairs effective global TB control measures. To better control the TB-DM co-epidemic, unravelling the bidirectional interactivity between DM-associated molecular processes and immune responses to Mycobacterium tuberculosis (Mtb) is urgently required. Since poly (ADP-ribose) polymerase (PARP) activation has been associated with DM and with Mtb infection in mouse models, we have investigated whether PARP inhibition by pharmacological compounds can interfere with host protection against Mtb in human macrophage subsets, the predominant target cell of Mtb. Pharmacological inhibition of PARP decreased intracellular Mtb and MDR-Mtb levels in human macrophages, identifying PARP as a potential target for host-directed therapy against Mtb. PARP inhibition was associated with modified chemokine secretion and upregulation of cell surface activation markers by human macrophages. Targeting LDH, a secondary target of the PARP inhibitor rucaparib, resulted in decreased intracellular Mtb, suggesting a metabolic role in rucaparib-induced control of Mtb. We conclude that pharmacological inhibition of PARP is a potential novel strategy in developing innovative host-directed therapies against intracellular bacterial infections.

Clinical Implication of E2F Transcription Factor 1 in Hepatocellular Carcinoma Tissues.

Background: To date, the clinical management of advanced hepatocellular carcinoma (HCC) patients remains challenging and the mechanisms of E2F transcription factor 1 (E2F1) underlying HCC are obscure. Materials and Methods: Our study integrated datasets mined from several public databases to comprehensively understand the deregulated expression status of E2F1. Tissue microarrays and immunohistochemistry staining was used to validate E2F1 expression level. The prognostic value of E2F1 was assessed. In-depth subgroup analyses were implemented to compare the differentially expressed levels of E2F1 in HCC patients with various tumor stages. Functional enrichments were used to address the predominant targets of E2F1 and shedding light on their potential roles in HCC. Results: We confirmed the elevated expression of E2F1 in HCC. Subgroup analyses indicated that elevated E2F1 level was independent of various stages in HCC. E2F1 possessed moderate discriminatory capability in differentiating HCC patients from non-HCC controls. Elevated E2F1 correlated with Asian race, tumor classification, neoplasm histologic grade, eastern cancer oncology group, and plasma AFP levels. Furthermore, high E2F1 correlated with poor survival condition and pooled HR signified E2F1 as a risk factor for HCC. Enrichment analysis of differentially expressed genes, coexpressed genes, and putative targets of E2F1 emphasized the importance of cell cycle pathway, where CCNE1 and CCNA2 served as hub genes. Conclusions: We confirmed the upregulation of E2F1 and explored the prognostic value of E2F1 in HCC patients. Two putative targeted genes (CCNE1 and CCNA2) of E2F1 were identified for their potential roles in regulating cell cycle and promote antiapoptotic activity in HCC patients.

Conceptions of Choice, Equity, &amp; Rurality in Educational Research

Although school choice advocates often promote a vision of additional schooling options for all students, the predominant target of school choice researchers has been densely populated, urban cores in the United States. However, this belies the fact that many rural communities have similarly engaged in forms of school choice decision-making. While we do not argue for further encroachment of school choice policies in rural contexts, we believe there are myriad, novel opportunities for meaningful education research regarding school choice, equity, and conceptions of rurality. To advance toward a robust agenda for rural school choice, we review the existing literature on school choice and rural education, provide key recommendations, and assert the need for additional consideration of the following: critical socio-political histories and theories; methodological diversity; issues of race, racism, sexual orientation, and equity; social-emotional learning and development; impacts of the COVID-19 global pandemic; and broadened understandings of rurality.

Inhibitory Potential of Dietary Nutraceuticals on Cellular PI3K/Akt Signaling: Implications in Cancer Prevention and Therapy.

The phosphatidyl inositol-3 kinase (PI3K)/protein kinase B (Akt)/mechanistic target of rapamycin (mTOR) signaling has been associated with many cellular physiological events, such as proliferation, maturation, survival, and metabolism. Besides its role in normal cells, the pathway is often upregulated in various cancers. Due to its prominent role in the cancer progression events, it is now being considered as a target for cancer therapy and cancer chemoprevention. The present review provides a concise outline of the role of the PI3K/Akt/mTOR pathway in carcinogenesis and progression events, including metastasis, drug resistance and stemness. Further, emphasis needs to be placed on the PI3K/Akt/mTOR pathway inhibitory potentials of various food-derived bioactive components in cancer prevention. Data on the PI3K/Akt/mTOR inhibiting natural products and their bioactive compounds have been obtained from PubMed/Medline, Scopus, Eurekaselect, etc. Findings from the above citation databases from 2000-2021 are included in the manuscript. Numerous compounds from plants have been isolated and identified as anticancer agents; among these, a predominant class is nutraceuticals. The PI3K pathway is the predominant target of these natural products, and many of these drug candidates are under various stages of drug development. These compounds have shown a significant inhibitory effect on the kinase activities of PI3K and Akt, resulting in the abrogation of cancer initiation and progression events. In addition, these compounds have been shown to reverse the resistance to chemotherapeutic drugs and also reduce the population of cancer stem cells. The nutraceuticals are promising candidates as anticancer agents by blocking PI3K signaling cascades. As the PI3K is a central pathway to various receptors signaling, the dietary intervention may prove to be highly effective.

Polymer Nanodiscs and Their Bioanalytical Potential.

Membrane proteins (MPs) play a pivotal role in cellular function and are therefore predominant pharmaceutical targets. Although detailed understanding of MP structure and mechanistic activity is invaluable for rational drug design, challenges are associated with the purification and study of MPs. This review delves into the historical developments that became the prelude to currently available membrane mimetic technologies before shining a spotlight on polymer nanodiscs. These are soluble nanosized particles capable of encompassing MPs embedded in a phospholipid ring. The expanding range of reported amphipathic polymer nanodisc materials is presented and discussed in terms of their tolerance to different solution conditions and their nanodisc properties. Finally, the analytical scope of polymer nanodiscs is considered in both the demonstration of basic nanodisc parameters as well as in the elucidation of structures, lipid-protein interactions, and the functional mechanisms of reconstituted membrane proteins. The final emphasis is given to the unique benefits and applications demonstrated for native nanodiscs accessed through a detergent free process.

Abstract 1308: Development and validation of a flow cytometric assay for measuring glycoprotein A repetitions predominant protein GARP target engagement by ABBV-151

Abstract Introduction: GARP is a cell surface protein expressed post activation on regulatory T-cells (Treg), monocytes, B cells, and platelets. GARP binds to latent transforming growth factor beta (TGF-β1) and regulates the release of active TGF-β1. TGF-β1 modulates Treg-mediated immunosuppression and represses effector T cell responses. ABBV-151 is a first-in-class monoclonal antibody that binds to the GARP-TGF-β1 complex and blocks active TGF-β1 release. Here we describe the validation of a TE assay for ABBV-151 using human platelets to measure receptor occupancy (RO) and binding saturation, that will inform dosing decisions during a first-in-human Phase I dose escalation study. Procedure: A competing (LHG10.6 that recognizes the GARP-TGF-β1 complex) and non-competing (1E7 that recognizes total GARP) anti-GARP antibodies were used to measure TE on platelets. Human whole blood was incubated with increasing concentrations of ABBV-151, followed by platelet-rich plasma (PRP) preparation and storage at -80°C. Platelets were thawed, washed, and stained with a fluorescently labeled antibody cocktail (LHGH10.6, 1E7 and CD61) and analyzed on a flow cytometer. The analytical measurement range, inter-operator and inter-assay precision and sample stability at -80°C (up to 12 months) for this TE assay was determined. Results: ABBV-151 TE assay demonstrated a dose response relationship over a range of ABBV-151 concentrations (0.08 to 400 ng/mL) that corresponded to 0-80% occupancy, with an EC50 of 6.2 ± 2.1 ng/mL, and 0-100% saturation with an EC50 of 6.7 ± 1.9 ng/mL. Inter-assay precision demonstrated ≤3% CV at low ABBV-151 doses, and ≤14% in samples at high doses. Inter-operator precision for untreated and treated samples was between 3 to 9% CV, respectively. The stability data indicated frozen platelets could be stored up to 12 months with highly reproducible assay performance. Conclusions: This TE assay for ABBV-151 on platelets demonstrates desirable analytical performance characteristics. The percent saturation (PS) showed higher precision than the RO measurements for this assay and PS was determined to be a reliable metric for measuring TE. Frozen platelets samples were suitable for the TE assay for up to a year allowing for batch processing of samples at a central lab and for minimizing the variability in test results. This novel TE assay overcomes key limitations of implementing TE assays in clinical trials, and quantification of PS using this TE assay in clinical trials is currently ongoing to assess the pharmacodynamic/pharmacokinetic relationship of ABBV-151 therapy. Citation Format: Juan M. Cardenas, Jayaprakash Kotha, Sridevi Kotha, Lisa K. Jennings, Michelle Graham, Stacie Lambert, Kinjal Hew. Development and validation of a flow cytometric assay for measuring glycoprotein A repetitions predominant protein GARP target engagement by ABBV-151 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1308.

Abstract 2508: Targeting mutant KRAS tumors by inhibiting cytoplasmic EZH2, which directly methylates and destabilizes the DLC1 tumor suppressor protein

Abstract Tumors with mutant KRAS, which occurs in ~95% of pancreatic cancers, ~45% of colorectal cancers, and ~30% of lung adenocarcinomas, usually have a poor response to current therapies, resulting in poor outcomes. The rationale for the new treatment approach described here for tumors with mutant KRAS arises from the critical antitumor role that we have found for the DLC1 tumor suppressor protein. DLC1 protein is frequently downregulated in a wide range of cancers. Although several mechanisms account for this downregulation, we have focused on a subset of tumor lines that express DLC1 mRNA but lack detectable DLC1 protein. Using the model system of lung adenocarcinomas with mutant KRAS that have this DLC1 phenotype, we have found that in 5 out of 5 human lung adenocarcinoma cell lines (4 with codon-12 and 1 with a codon-61 KRAS mutation), KRAS siRNAs knockdown results in readily detectable steady-state level of DLC1 protein. This phenotype of detectable steady-state DLC1 protein is also seen in these mutant KRAS lines when treated with the EZH2 inhibitor Tazemetostat. Although EZH2 is an important regulator of mRNA expression, secondary to its methylation of Lysine 27 on histone H3, the effect of EZH2 on DLC1 is mainly attributable to the EZH2 methylation of K678 on DLC1 protein in the cytoplasm, which leads to CUL-4A ubiquitin-dependent proteasomal degradation of DLC1 protein. EZH2 inhibition prevents this degradation process, resulting in at least a 10-fold increase in DLC1 protein half-life. KRAS siRNAs knockdown phenocopies CRM1 inhibition, a major nuclear export protein, as it reduces both cytoplasmic EZH2 and DLC1 methylation. Although pharmacologic inhibition of EZH2 by Tazemetostat in the lung cancer lines stabilizes the DLC1 protein, the antitumor activity is greatly increased by the addition of the AKT inhibitor MK-2206 and/or the SRC inhibitor Saracatinib, which reverse the direct phosphorylation of DLC1 by these kinases and increase the tumor suppressor activity of DLC1. The above three-drug combination, which is well-tolerated in mice, induces potent inhibition of tumor xenograft growth that is highly dependent on DLC1 protein and is associated with induction of markers of apoptosis (Annexin V) and senescence (beta-galactosidase). Although each of these inhibitors has multiple targets, siRNAs knockdown of DLC1 abolishes ~80% of the growth inhibitory activity of the three-drug combination, strongly suggesting that DLC1 is the predominant antitumor target of the drug combination. In summary, we identify DLC1 protein is a new critical substrate for cytoplasmic EZH2; our findings suggest that EZH2 inhibitors should be considered for their potential impact on post-translational regulation in addition to their effects on gene transcription; and provide evidence that the drug combination identified here may have clinical efficacy against tumors with mutant KRAS. Citation Format: Brajendra K. Tripathi, Disha Bhargava, Xiaolan Qian, Dunrui Wang, Marian E. Durkin, Fernando J. de Miguel, Katerina Politi, Kylie J. Walters, James H. Doroshow, Douglas R. Lowy. Targeting mutant KRAS tumors by inhibiting cytoplasmic EZH2, which directly methylates and destabilizes the DLC1 tumor suppressor protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2508.