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Warfarin, a commonly prescribed oral anticoagulant medication, is highly effective in treating deep vein thrombosis and pulmonary embolism. However, the clinical dosing of warfarin is complicated by high inter-individual variability in drug exposure and response and its narrow therapeutic index.

Bone metastases are common in advanced breast cancer (BC) patients and increase the risk for skeletal-related events (SREs), which present a significant health and economic burden. Bone targeting agents (BTAs) can improve health-related quality of life by delaying or preventing SREs; nevertheless, a significant portion of eligible BC patients are not receiving this therapy. A bone health education needs assessment survey was conducted to examine cancer-related bone health awareness and to identify opportunities to improve bone health education. Direct-to-patient outreach was used to recruit adult BC patients in the USA self-reporting a diagnosis of bone metastasis within the past 3 years. Of the 200 patients, 59% experienced at least one SRE prior to survey participation (44% radiation to bone, 29% bone fracture, 17% spinal cord compression, 15% surgery to bone), and 83% were currently receiving a BTA. Awareness of general cancer bone health, protection strategies against SREs, and screening tests were low to moderate. Patients currently not receiving a BTA were least knowledgeable about cancer bone health, with only 40% aware of BTAs as a protective strategy, and only 26% were very or extremely satisfied with the information received from healthcare providers. Sixty-two percent of patients wanted to receive information by more than one mode of communication. Notable gaps in bone health education were observed in bone metastatic BC patients at risk for SREs, suggesting the need for earlier and more effective communication and education strategies to promote appropriate BTA use and better health outcomes.

PURPOSE We surveyed oncologists who treat classic Hodgkin lymphoma (cHL) as part of the CONNECT study to understand the treatment decision‐making process, including the impact of positron emission tomography/computed tomography (PET/CT) imaging. METHODS US physicians self-identifying as oncologists, hematologists, or hematologists/oncologists with ≥2 years of practice experience who treated ≥1 adult with stage III/IV cHL in the frontline setting in the last year were surveyed (October 19-November 16, 2020). Physician demographics, guideline adherence, and PET/CT utilization, interpretation, and access barriers were assessed. RESULTS In total, 301 physicians participated in the survey. Eighty-eight percent of physicians gave somewhat-to-significant consideration to NCCN guidelines. Most physicians (94%; n = 284) reported obtaining a PET/CT scan at diagnosis; of these physicians, 97% reported obtaining an interim PET/CT scan for stage III/IV cHL, with 65% typically obtaining an interim PET/CT scan after cycle 2. The Deauville 5-point scale (5PS) was the primary scoring system used to review PET/CT results by 62% of physicians, with a positive score defined as ≥3 by 44%, ≥4 by 37%, and ≥2 by 12% of physicians. Fifty-five percent of physicians reported difficulty in obtaining PET/CT scans. CONCLUSION Although most physicians considered NCCN guidelines when treating patients with stage III/IV cHL, interim PET/CT scans after cycle 2 were not universally obtained. When PET/CT scans were obtained, Deauville 5PS scores were not always provided, and variability existed on what defined a positive score. These findings suggest that opportunities exist for education and improved PET-adapted treatment approaches.

Darcy R Flora,1 Katherine B Carlson,2 David C Fuehrer,1 Benoit Cadieux,3 Guy Boike,4 Jennifer Schenfeld,2 Kimberly A Lowe2 1GRYT Health Inc., Rochester, NY, USA; 2Center for Observational Research, Amgen Inc., Thousand Oaks, CA, USA; 3Global Medical Organization, Amgen Inc., Thousand Oaks, CA, USA; 4Karmanos Cancer Institute, Detroit, MI, USACorrespondence: Darcy R FloraGRYT Health Inc., 919 Winton Road South, Suite 307, Rochester, NY, 14618, USATel +1 844 487 4798Fax +1 585 486 3102Email darcy@grythealth.comPurpose: Cancer patients with bone metastasis (BM) from solid tumors or multiple myeloma (MM) have an increased risk of painful skeletal-related events (SREs), which can decrease quality of life and increase mortality. Bone targeting agents (BTAs) can help delay or prevent SREs; however, a significant portion of eligible patients are not receiving BTA therapy. This study was conducted to understand patient awareness of cancer-related bone health and to identify opportunities to improve bone health education in cancer patients at risk of SREs.Methods: The online BonE heAlth eduCatiOn Needs assessment (BEACON) survey included questions about patient demographics, cancer diagnosis and treatments (including BTA usage), and extent and satisfaction with bone health education received. Direct-to-patient outreach was used to recruit patients. Eligible patients were US adults with a diagnosis of self-reported MM or BM from a solid tumor (breast, lung, or prostate cancer) within the past three years.Results: Of 125 patients, 71% were diagnosed with solid tumors with BM and 29% with MM. At least one prior SRE was experienced by 57% of patients (38% radiation to bone, 32% bone fracture, 22% spinal cord compression, and 19% surgery to bone), and 74% were currently receiving BTA therapy. Awareness of cancer bone health, protection strategies, and screening tests was low to moderate; patients were least informed of the impact of lifestyle changes (38%) and specific cancer treatments (≤ 35%) on bone health. Sixty-two percent of patients were not completely satisfied with the bone health education received. Patients generally wanted more information (58%) and to receive information by more than one mode of communication.Conclusion: Notable gaps in bone health education were observed in cancer patients at risk for SREs indicating an important need for improved communication and education strategies to promote better health outcomes.Keywords: bone metastasis, bone targeting agents, fracture, radiation to bone, surgery to bone, spinal cord compression

PURPOSE Nearly 40% of patients with bone metastases from breast cancer experience a skeletal-related event (SRE) such as fracture, spinal cord compression, bone surgery, or radiation to bone, within 1 year of diagnosis (1). Bone-targeting agents (BTAs) significantly reduce the risk of SREs, but real-world data indicates that only 58% (Commercial) and 67% (Medicare) of eligible metastatic breast cancer patients receive treatment with a BTA (2, 3). We surveyed U.S. patients with bone metastases from breast cancer to ascertain their awareness regarding bone health, to describe the source and amount of bone health education received, and to identify potential gaps in bone health education. METHODS The BonE heAlth eduCatiOn Needs assessment (BEACON) survey was developed through a collaboration between GRYT Health Inc. and Amgen Inc. The online survey consisted of treatment-related questions, cancer-related bone health educational questions, and open-ended questions. U.S. adult patients who consented to participate, with self-reported bone metastasis from breast cancer, were recruited using a unique IRB-approved direct-to-patient approach pioneered by GRYT Health, which incorporates digital outreach, advocacy partnerships, and virtual app and conference-based communication. RESULTS Seventy-four breast cancer patients with bone metastasis completed the online survey. Thirty-eight (51%) had experienced at least one SRE, defined as bone fracture (n=19; 26%), spinal cord compression (n=11; 15%), or radiation (n=28; 38%) or surgery (n=9; 12%) on a bone. While the majority of patients were aware that bones are more fragile in individuals with cancer (n=48; 65%) and that treatments are available to help prevent broken bones caused by cancer (n=50; 68%), fewer understood that bones are more fragile after receiving chemotherapy (n=20; 27%) and radiation (n=28; 38%), or that lifestyle changes can help to prevent broken bones (n=23; 31%). Similarly, when asked about bone protection, the majority of patients had knowledge about calcium and/or vitamin D supplements (n=55; 74%) and BTAs (n=53; 72%), but only 24% had knowledge of lifestyles changes as a bone health protection strategy (n=18). Oncologist and nurses were the most commonly reported HCPs to provide cancer-related bone health information. Patient-reported satisfaction with the amount of bone cancer education was “low” or “moderate” (n=46; 62%); “low” satisfaction was more common in patients not receiving a BTA (n=6/13; 46%) compared to those currently receiving a BTA (n=17/61; 28%). More than half of patients reported receiving either no bone health information (n=8; 11%) or less information than desired (n=35; 47%). CONCLUSION Despite potential limitations in patient recall and population selection biases, there appears to exist important gaps in knowledge and education related to bone health and SRE prevention among metastatic breast cancer patients. Patient- and provider-oriented interventions that increase opportunities for communication and education on bone health may help ensure optimal SRE prevention strategies including appropriate BTA use. REFERENCES 1. Clemons M, Gelmon KA, Pritchard KI, Paterson AH. Curr Oncol 2012 Oct; 19(5):259-268. 2. Hernandez RK, Adhia A, Wade SW, et al. Clin Epidemiol 2015 Jul; 7:335-345. 3. McGrath LJ, Overman RA, Reams D, et al. Clin Epidemiol 2018 Sep; 10:1349-1358. Citation Format: Darcy R Flora, Katherine B Carlson, Kimberly Lowe, Benoit Cadieux, Guy Boike, Basia Bachmann, David C Fuehrer. Findings from the BonE heAlth eduCatiOn needs assessment (BEACON) study: A survey of bone metastatic breast cancer patients at risk for skeletal-related events [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS9-69.

There is a general lack of understanding on the detailed processes and mechanism of low-angle translational block slides. The September 20, 2018 Naga landslide, with a volume of 27 M m3 and a runout distance of 1.34 km, provided new insights on the initiation, transport, and deposition mechanisms of this type of landslide. Drone photogrammetry, video footage, satellite images, slope stability analyses, and field evidence revealed that the landslide occurred as multiple block detachments along a series of tension cracks that formed and grew progressively prior to the main failure. Predominance of intact upright blocks traceable to the distal end of the deposit indicates dominant translational motion. Facies within the Naga landslide deposit revealed that at least three processes (slide, dry flow, and fall) occurred during the main movement. Post-slide processes immediately after emplacement included small avalanches and rockfalls related to the instability of the landslide deposit and the main scarp. Preliminary slope stability analyses showed that the slopes were marginally unstable (FoS 0.67–3.96) even in dry conditions. The low-angle profile of the slip surface (6°) favors stability, but low shear strength of the limestone bedrock and unsupported cut slopes may have contributed to the failure.

e24004 Background: The health and work productivity burden informal cHL patient (pt) caregivers face is unknown. As part of the US-based CONNECT study, we evaluated caregivers’ burden and role in treatment (tx) decisions by relation to the pt: spouse/partner (SP) vs other (parent, child, friend, other relative). Methods: The CONNECT caregiver survey was an IRB-approved online survey administered from Dec 2020-Mar 2021 to self-identified current or former adult cHL pt caregivers. Health-related quality of life (HRQoL, PROMIS-Global), work impact (WPAI), decision-making, tx selection, and physician communication were assessed. Statistical significance was at the 95% confidence level. Results: 209 caregivers (58% women; median age 47 yrs; 54% employed; 53% SP) completed the survey. At completion, 69% cared for pts diagnosed in the past 1-2 yrs; 48% of pts cared for had stage III/IV cHL and 58% were in remission/not receiving active tx. While caregiver HRQoL was similar to that of the general population on the PROMIS-Global, employed caregivers had work impairment (29%) from caregiving activities (Table) which was higher when the pt was on vs off tx. Caregiving began at pt symptom onset for more SP vs other caregivers (61 vs 27%), and after the pt’s first tx for more other vs SP caregivers (34 vs 5%). 88% of caregivers discussed tx options with the pt. Cure, caregivers’ top tx goal (49%), was rated higher by SP vs other caregivers (56 vs 42%). Tx decisions with the pt (54 vs 23%) and tx option discussions with the doctor (52 vs 28%) were more common for SP vs other caregivers. More SP vs other caregivers had extensive tx option discussions with the pt (88 vs 68%), said it was important the doctor discussed managing side effects (94 vs 84%), felt the doctor provided adequate information about side effects (91 vs 71%), and felt aligned with the pt’s tx goals (93 vs 79%). Caregivers noted COVID-19 impacts like limiting daily activities to reduce COVID-19 risks (72%). Conclusions: Although cHL pt caregivers reported good HRQoL, caregiving impacted their work productivity regardless of relation to the pt. Cure was caregivers’ top tx goal. SP vs other caregivers were more involved and earlier, reporting alignment with pt tx goals and decision-making.[Table: see text]

Introduction Hodgkin lymphoma (HL) represents ~10% of all lymphomas in the United States (US) with classical HL (cHL) accounting for ~95% of all HL cases. cHL has a bimodal age distribution with peaks at ages 15-39 and ≥75 years. As part of CONNECT, the first real-world survey in cHL to include physicians, patients, and caregivers, patient treatment preferences for those with stage III or IV cHL were explored and differences evaluated between those aged Methods The CONNECT patient survey was a non-interventional patient-centered survey. Participants included were aged ≥18 years at the time of participation (aged ≥12 years at diagnosis), diagnosed with cHL within the past 10 years, and previously or currently being treated for cHL in the US. The CONNECT survey was reviewed and approved by the New England Institutional Review Board and administered from December 30, 2020, to March 1, 2021. Results In CONNECT, 182 participants had stage III or IV cHL (64% female; 77% Caucasian) with 64% aged Cure was ranked as the first or second goal of initial cHL treatment for 86% of participants aged At diagnosis, a significantly higher percentage of participants aged P < 0.001; Figure B). However, 44% of those aged A significantly higher percentage of participants aged When asked about long-term side effects of greatest concern, a significantly higher percentage of participants aged Conclusion Treatment goals differ significantly between participants with stage III or IV cHL based primarily on age, with those aged Figure 1 Figure 1. Disclosures Flora: Seagen, Inc: Research Funding. Parsons: SeaGen: Consultancy. Liu: Seagen, Inc: Current Employment, Current equity holder in publicly-traded company. Yu: Seagen, Inc: Current Employment, Current equity holder in publicly-traded company. Fanale: Seagen, Inc: Current Employment, Current equity holder in publicly-traded company. Kumar: Seagen, Inc: Consultancy. Byrd: Seagen, Inc: Research Funding.

Background Current NCCN guidelines recommend 1 of 3 first-line (1L) regimens for stage III or IV classical Hodgkin lymphoma (cHL): ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine), A+AVD (brentuximab vedotin, doxorubicin, vinblastine, dacarbazine), or escalated BEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, prednisone); preferred regimens vary by region (e.g., North America vs Europe). The NCCN recommends positron emission tomography/computerized tomography (PET/CT) imaging after cycle 2 (interim PET2) to guide ABVD escalation or de-escalation. We surveyed physicians on their cHL treatment decision-making process and how PET/CT scan access, reimbursement, and comprehension influence their choices as part of CONNECT, the first real-world survey of physicians, patients, and caregivers in cHL. Methods Medical oncologists, hematologist/oncologists, or hematologists who treat cHL were invited to participate in an Institutional Review Board-approved, 30-minute online anonymous survey. Eligible participants had ≥2 years of practice experience in the United States (US) and treated ≥1 adult (aged ≥18 years) with stage III or IV cHL and ≥1 adult with cHL in the 1L setting within the prior 12 months. Surveys were completed from October 19, 2020-November 16, 2020. Results Of 301 participating physicians, 80% were hematologist/oncologists with a median practice duration of 15 years; 62% practiced in community and 38% in academic settings. Participants were located in the US (South, 34%; Northeast, 26%; West, 21%; Midwest, 20%) and spent 90% of their professional time in direct patient care. In the preceding 12 months, participants treated a median (interquartile range) of 16 (7-40) patients with active cHL (stage III [median], 4; stage IV, 5) and 15 (8-40) cHL survivors. When treating cHL, 88% of participants reported giving NCCN guidelines somewhat/significant consideration. Overall, 94% of participants (n=284) reported using a PET/CT combined scan to diagnose/stage cHL, in line with current guideline recommendations. Of these participants, 97% reported typically getting an interim PET/CT scan for stage III or IV cHL with 65% typically getting the scan after cycle 2 (Figure A). Participants reported both escalating and de-escalating treatment based on interim PET/CT results (Figure B) with 61% making decisions after cycle 2. Of participants using a PET/CT scan, 42% reported receiving both a Deauville score and a standardized uptake value (SUV; Figure C) with 62% of participants noting that the Deauville score was the primary system used for reviewing PET/CT results (Figure D). However, 19% of participants reported challenges interpreting PET/CT results. Among participants using a Deauville score (n=209), consensus was limited on what defined a positive scan (≥3, 44%; ≥4, 37%). Challenges obtaining PET/CT scans were reported by 16% of participants using PET/CT scans. However, despite not reporting challenges 55% of participants on average were unable to obtain a PET/CT scan 20% of the time. Of participants using PET/CT scans, 86% reported typically receiving results within 2 business days and 14% within 3-5 business days. Twenty-one percent of participants reported that delays in PET/CT results affected their ability to use a PET-adaptive approach. Forty-nine percent of those using PET/CT scans reported increased difficulty in PET/CT access for stage III or IV cHL due to lack of insurance coverage. In absence of a PET/CT scan, 36% of participants reported using an interim biopsy and 63% an interim CT scan to inform treatment choices. Among all participants, 36% reported increased difficulty in getting patients with cHL access to PET/CT scans due to COVID-19. Conclusions Although participants consider NCCN guidelines when treating cHL, interim PET scans are not universally obtained after cycle 2 for stage III or IV cHL, with 65% of participants who use PET/CT scans obtaining an interim PET scan after cycle 2 for stage III or IV cHL. When PET/CT scans are obtained, Deauville scores are commonly provided; however, there is variability in what is termed a positive or negative Deauville score. Challenges in obtaining PET/CT scans, with increased difficulty during COVID-19, were reported. Also, there are other barriers, such as lack of insurance, that may prohibit the optimal adherence to guidelines on interim PET/CT utilization. Figure 1 Figure 1. Disclosures Parsons: SeaGen: Consultancy. Yu: Seagen, Inc: Current Employment, Current equity holder in publicly-traded company. Liu: Seagen, Inc: Current Employment, Current equity holder in publicly-traded company. Kumar: Seagen, Inc: Consultancy. Fanale: Seagen, Inc: Current Employment, Current equity holder in publicly-traded company. Flora: Seagen, Inc: Research Funding.

Background Mainstay therapies for patients with stage III or IV classical Hodgkin lymphoma (cHL) include several multiagent chemotherapy regimens. A combination of doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) is commonly administered in the first-line (1L) setting; however, ~30% of patients with stage III or IV cHL will be refractory to or relapse following ABVD treatment. Brentuximab vedotin, doxorubicin, vinblastine, and dacarbazine (A+AVD), a novel targeted therapy in combination with a standard chemotherapy regimen, is another option for 1L treatment of stage III or IV cHL. In the 5-year update of the ECHELON-1 trial (Straus, 2021), patients with stage III or IV cHL randomized to 1L A+AVD compared with ABVD continued to demonstrate progression-free survival (PFS) improvement with a 32% reduction in the risk of progression or death (HR=0.681, nominal P=0.002). To understand the decision-making process when selecting a 1L cHL treatment regimen for stage III or IV cHL, we surveyed physicians to gain insights into their preferred regimens and factors that influence their treatment choices as part of CONNECT, the first real-world observational survey in cHL that includes physicians, patients, and caregivers. Methods Physicians in the United States who treat patients with cHL were recruited to participate in an Institutional Review Board-approved, online anonymous survey from October 19, 2020-November 16, 2020. Eligible participants were medical oncologists, hematologist/oncologists, or hematologists with ≥2 years medical practice experience who, within the past 12 months, had treated ≥1 adult (aged ≥18 years) with stage III or IV cHL and ≥1 adult in the 1L setting. Results Of participating physicians (N=301), 62% were community-based and 80% identified themselves as a hematologist/oncologist, reporting a median of 15 years of experience. Participants saw a median (interquartile range) of 16 (7-40) patients with active, newly diagnosed cHL and 15 (8-40) cHL survivors in the 12 months preceding survey participation. The most important considerations (ranked 1 or 2) for cHL treatments were clinical trial efficacy and safety data (60%) and official guideline recommendations (58%). Specifically, efficacy attributes including overall survival (OS; 91%), long-term PFS (86%), curative potential (85%), and complete response (81%) were rated highest or as having the most essential impact. In contrast, patient personal goals (6%), treatment costs (4%), and patient financial support programs (4%) were endorsed by Conclusions Efficacy attributes, including OS and PFS; quality of life; and patient age were top reasons cited by surveyed participants for selecting a specific 1L treatment regimen in stage III or IV cHL. Treatment preferences for newly diagnosed stage III or IV cHL varied based on patient characteristics including presence of bulky mediastinal disease, disease stage, perceived risk of relapse, age, and comorbidities. Figure 1 Figure 1. Disclosures Yu: Seagen, Inc: Current Employment, Current equity holder in publicly-traded company. Liu: Seagen, Inc: Current Employment, Current equity holder in publicly-traded company. Kumar: Seagen, Inc: Consultancy. Fanale: Seagen, Inc: Current Employment, Current equity holder in publicly-traded company. Flora: Seagen, Inc: Research Funding. Parsons: SeaGen: Consultancy.

Multiple factors can impact warfarin therapy, including genetic variations in the drug-metabolizing enzyme cytochrome P450 2C9 (CYP2C9). Compared with individuals with the wild-type allele, CYP2C9*1, carriers of the common *3 variant have significantly impaired CYP2C9 metabolism. Genetic variations in CYP2C9, the primary enzyme governing the metabolic clearance of the more potent S-enantiomer of the racemic anticoagulant warfarin, may impact warfarin-drug interactions. To establish a baseline for such studies, plasma and urine concentrations of R- and S-warfarin and 10 warfarin metabolites were monitored for up to 360 hours following a 10-mg warfarin dose in healthy subjects with 4 different CYP2C9 genotypes: CYP2C9*1/*1 (n = 8), CYP2C9*1/*3 (n = 9), CYP2C9*2/*3 (n = 3), and CYP2C9*3/*3 (n = 4). Plasma clearance of S-warfarin, but not R-warfarin, decreased multiexponentially and in a CYP2C9 gene-dependent manner: 56%, 70%, and 75% for CYP2C9*1/*3, CYP2C9*2/*3, and CYP2C9*3/*3 genotypes, respectively, compared with CYP2C9*1/*1, resulting in pronounced differences in the S:R ratio that identified warfarin-sensitive genotypes. CYP2C9 was the primary P450 enzyme contributing to S-warfarin metabolism and a minor contributor to R-warfarin metabolism. In the presence of a defective CYP2C9 allele, switching of warfarin metabolism to other oxidative pathways and P450 enzymes for the metabolic elimination of S-warfarin was not observed. The 10-hydroxywarfarin metabolites, whose detailed pharmacokinetics are reported for the first time, exhibited a prolonged half-life with no evidence of renal excretion and displayed elimination rate-limited kinetics. Understanding the impact of CYP2C9 genetics on warfarin pharmacokinetics lays the foundation for future genotype-dependent warfarin-drug interaction studies.

Cytochrome P450 (P450) protein-protein interactions have been shown to alter their catalytic activity. Furthermore, these interactions are isoform specific and can elicit activation, inhibition, or no effect on enzymatic activity. Studies show that these effects are also dependent on the protein partner cytochrome P450 reductase (CPR) and the order of protein addition to purified reconstituted enzyme systems. In this study, we use controlled immobilization of P450s to a gold surface to gain a better understanding of P450-P450 interactions between three key drug-metabolizing isoforms (CYP2C9, CYP3A4, and CYP2D6). Molecular modeling was used to assess the favorability of homomeric/heteromeric P450 complex formation. P450 complex formation in vitro was analyzed in real time utilizing surface plasmon resonance. Finally, the effects of P450 complex formation were investigated utilizing our immobilized platform and reconstituted enzyme systems. Molecular modeling shows favorable binding of CYP2C9-CPR, CYP2C9-CYP2D6, CYP2C9-CYP2C9, and CYP2C9-CYP3A4, in rank order. KD values obtained via surface plasmon resonance show strong binding, in the nanomolar range, for the above pairs, with CYP2C9-CYP2D6 yielding the lowest KD, followed by CYP2C9-CYP2C9, CYP2C9-CPR, and CYP2C9-CYP3A4. Metabolic incubations show that immobilized CYP2C9 metabolism was activated by homomeric complex formation. CYP2C9 metabolism was not affected by the presence of CYP3A4 with saturating CPR concentrations. CYP2C9 metabolism was activated by CYP2D6 at saturating CPR concentrations in solution but was inhibited when CYP2C9 was immobilized. The order of addition of proteins (CYP2C9, CYP2D6, CYP3A4, and CPR) influenced the magnitude of inhibition for CYP3A4 and CYP2D6. These results indicate isoform-specific P450 interactions and effects on P450-mediated metabolism.

Neuroendocrine neoplasias (NEN) of the gastroenteropancreatic (GEP) system frequently present with metastatic deposits. The proliferation marker Ki-67 is used for diagnosis and to assess the prognosis of disease. The aim of our study was to evaluate the usefulness of Ki-67 % in the assessment of NEN patients with regard to their disease stage in clinical practice. Additionally, a comparative analysis of Ki-67 levels among different sites of disease was performed.This retrospective study included patients with GEP NEN referred to our center from 2010 to 2012. The NEN diagnosis was confirmed by standard histopathology. Ki-67 immunohistochemistry was done on paraffin-embedded sections using an automated Leica immunohistochemistry machine. NEN grading was carried out according to European Neuroendocrine Tumor Society recommendations (low grade [G1] to intermediate grade [G2], well to moderately differentiated neuroendocrine neoplasms; high-grade [G3], moderately to poorly differentiated neuroendocrine neoplasms). Results of tumor staging and grading were correlated. In a subgroup of cases, comparative analysis of Ki-67 levels in different sites of disease was carried out.One hundred sixty-one GEP NEN patients were included in the study. Metastatic disease was seen in 46.1 % (53/115) of G1 tumors, 77.8 % (28/36) of G2 tumors, and 100 % of (10/10) G3 tumors (p = 0.0002). When stratified according to primary tumor site, metastatic disease was documented in 42.9 % (36/84) of patients with pancreatic NEN and in 91.9 % (34/37) of those with small intestinal primary. Stage IV metastatic disease was present in 27.8 % (32/115) and 72.2 % (26/36) of the G1 and G2 tumors, respectively, and in 90 % (9/10) of the G3 tumors. Assessment of the Ki-67 index for a subset of cases at metastatic sites as well as the primary tumor site showed discrepancies in 35.3 % cases. In 7/9 (77.8 %) patients with liver metastases, Ki-67 % was higher in the liver lesions than in the primary tumor.Patients with GEP NEN exhibiting a high Ki-67 proliferation index present with metastatic disease in the vast majority of cases. Depending upon the primary tumor site, metastases are to be expected also in tumors with low Ki-67 %, although they are considered less aggressive. Different disease sites may express heterogeneous Ki-67 levels.

Electron transfer in cytochrome P450 enzymes is a fundamental process for activity. It is difficult to measure electron transfer in these enzymes because under the conditions typically used they exist in a variety of states. Using nanotechnology-based techniques, gold conducting nanopillars were constructed in an indexed array. The P450 enzyme CYP2C9 was attached to each of these nanopillars, and conductivity measurements made using conducting probe atomic force microscopy under constant force conditions. The conductivity measurements were made on CYP2C9 alone and with bound substrates, a bound substrate-effector pair, and a bound inhibitor. Fitting of the data with the Poole-Frenkel model indicates a correlation between the barrier height for electron transfer and the ease of CYP2C9-mediated metabolism of the bound substrates, though the spin state of iron is not well correlated. The approach described here should have broad application to the measurement of electron transfer in P450 enzymes and other metalloenzymes.

Polymorphisms in cytochrome P450 enzymes can significantly alter the rate of drug metabolism, as well as the extent of drug-drug interactions. Individuals who homozygotically express the CYP2C9*3 allele (I359L) of CYP2C9 exhibit ∼70 to 80% reductions in the oral clearance of drugs metabolized through this pathway; the reduction in clearance is ∼40 to 50% for heterozygotic individuals. Although these polymorphisms result in a decrease in the activity of individual enzyme molecules, we hypothesized that decreasing the total number of active enzyme molecules in an in vitro system (CYP2C9*1/*1 human liver microsomes) by an equivalent percentage could produce the same net change in overall metabolic capacity. To this end, the selective CYP2C9 mechanism-based inactivator tienilic acid was used to reduce irreversibly the total CYP2C9 activity in human liver microsomes. Tienilic acid concentrations were effectively titrated to produce microsomal preparations with 43 and 73% less activity, mimicking the CYP2C9*1/*3 and CYP2C9*3/*3 genotypes, respectively. With probe substrates specific for other major cytochrome P450 enzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C19, CYP2D6, CYP2E1, and CYP3A4), no apparent changes in the rate of metabolism were noted for these enzymes after the addition of tienilic acid, which suggests that this model is selective for CYP2C9. In lieu of using rare human liver microsomes from CYP2C9*1/*3 and CYP2C9*3/*3 individuals, a tienilic acid-created knockdown in human liver microsomes may be an appropriate in vitro model to determine CYP2C9-mediated metabolism of a given substrate, to determine whether other drug-metabolizing enzymes may compensate for reduced CYP2C9 activity, and to predict the extent of genotype-dependent drug-drug interactions.

Wastewater discharged from tannery industries is highly complex, concentrated, and toxic. In view of the varying nature of discharged wastewater and the numerous small industries in Egypt, there is a need for highly efficient treatment processes that are simple to operate and have low/reasonable construction and operation costs. This study investigated the possibility of applying innovative low cost biological treatment using upflow anaerobic sludge blanket (UASB) in providing adequate treatment for tannery wastewater. The anaerobic treatment application was thus evaluated through using two stage UASB reactors connecting in series, each with volume of 94 l. Five hydraulic retention times (HRT) were used along the experimental works, which lasted for a year, starting by HRT of 24 h then 18, 12, 8 and finally 5 h for each UASB reactor. The proposed process at 12 h HRTs could pre-treat the tannery wastewater to be disposed to the municipality sewers. The study created best fit equations to predict the efficiency of the system.

Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) bind natriuretic peptide receptor (NPR)-A and decrease blood pressure and cardiac hypertrophy by elevating cGMP concentrations. Physiological responses to ANP and BNP are diminished in congestive heart failure (CHF) by an unknown mechanism. C-type natriuretic peptide (CNP) binding to NPR-B decreases cardiac hypertrophy, but the effect of CHF on NPR-B is unknown. Here, we measured ANP/NPR-A-dependent and CNP/NPR-B-dependent guanylyl cyclase activities in membranes from failing and nonfailing hearts. Transaortic banding of mice resulted in marked CHF as indicated by increased heart/body weight ratios, increased left ventricular diameters, and decreased ejection fractions. In nonfailed hearts, saturating ANP concentrations increased particulate guanylyl cyclase activity almost 10-fold, whereas saturating CNP concentrations increased activity 6.9-fold, or to about 70% of the ANP response. In contrast, in failed heart preparations, CNP elicited twice as much activity as ANP due to dramatic reductions in NPR-A activity without changes in NPR-B activity. For the first time, these data indicate that NPR-B activity represents a significant and previously unappreciated portion of the natriuretic peptide-dependent guanylyl cyclase activity in the normal heart and that NPR-B accounts for the majority of the natriuretic peptide-dependent activity in the failed heart. Based on these findings, we suggest that drugs that target both NPRs may be more beneficial than drugs like nesiritide (Natrecor) that target NPR-A alone.

Gold nanopillars, functionalized with an organic self-assembled monolayer, can be used to measure the electrical conductance properties of immobilized proteins without aggregation. Measurements of the conductance of nanopillars with cytochrome P450 2C9 (CYP2C9) proteins using conducting probe atomic force microscopy demonstrate that a correlation exists between the energy barrier height between hopping sites and CYP2C9 metabolic activity. Measurements performed as a function of tip force indicate that, when subjected to a large force, the protein is more stable in the presence of a substrate. This agrees with the hypothesis that substrate entry into the active site helps to stabilize the enzyme. The relative distance between hopping sites also increases with increasing force, possibly because protein functional groups responsible for electron transport depend on the structure of the protein. The inhibitor sulfaphenazole, in addition to the previously studied aniline, increased the barrier height for electron transfer and thereby makes CYP2C9 reduction more difficult and inhibits metabolism. This suggests that P450 Type II binders may decrease the ease of electron transport processes in the enzyme, in addition to occupying the active site.

This paper discusses the virtual finite-state machine (VFSM) design and implementation paradigm and our experience in introducing VFSM on software development projects for several Lucent Technologies products. VFSM, which allows software developers to specify the control behavior of a module as a finite-state machine, is supported by a toolset that automates many tasks associated with producing an implementation, including aspects of code generation, documentation, and testing. VFSM has been used in the design of more than 75 software modules, and its application has resulted in shorter development intervals and the elimination of defects prior to testing. In this paper, we present an overview of the VFSM design and implementation paradigms and the capabilities provided by the VFSM toolset. We also discuss the technical and nontechnical issues that have had an impact on the successful introduction of VFSM.

At some point, anyone with knowledge of drug metabolism and enzyme kinetics started out knowing little about these topics. This chapter was specifically written with the novice in mind. Regardless of the enzyme one is working with or the goal of the experiment itself, there are fundamental components and concepts of every experiment using drug metabolism enzymes. The following case studies provide practical tips, techniques, and answers to questions that may arise in the course of conducting such experiments. Issues ranging from assay design and development to data interpretation are addressed. The goal of this section is to act as a starting point to provide the reader with key questions and guidance while attempting his/her own work.

At some point, anyone with knowledge of drug metabolism and enzyme kinetics started out knowing little about these topics. This chapter was specifically written with the novice in mind. Regardless of the enzyme one is working with or the goal of the experiment itself, there are fundamental components and concepts of every experiment using drug metabolism enzymes. The following case studies provide practical tips, techniques, and answers to questions that may arise in the course of conducting such experiments. Issues ranging from assay design and development to data interpretation are addressed. The goal of this section is to act as a starting point to provide the reader with key questions and guidance while attempting his/her own work.

Cytochrome P450 enzymes play a key role in the metabolism of pharmaceutical agents. To determine metabolite toxicity, it is necessary to obtain P450 metabolites from various pharmaceutical agents. Here, we describe a bioreactor that is made by immobilizing cytochrome P450 2C9 (CYP2C9) to a poly(methyl methacrylate) surface and, as an alternative to traditional chemical synthesis, can be used to biosynthesize P450 metabolites in a plug flow bioreactor. As part of the development of the CYP2C9 bioreactor, we have studied two different methods of attachment: (1) coupling via the N-terminus using N-hydroxysulfosuccinimide 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and (2) using the Ni(II) chelator 1-acetato-4-benzyl-triazacyclononane to coordinate the enzyme to the surface using a C-terminal histidine tag. Additionally, the propensity for metabolite production of the CYP2C9 proof-of-concept bioreactors as a function of enzyme attachment conditions (e.g., time and enzyme concentration) was examined. Our results show that the immobilization of CYP2C9 enzymes to a PMMA surface represents a viable and alternative approach to the preparation of CYP2C9 metabolites for toxicity testing. Furthermore, the basic approach can be adapted to any cytochrome P450 enzyme and in a high-throughput, automated process.

An attempt was made to establish a human self-control paradigm where impulsive response binges produced a greater reinforcement density at the molecular, or short term, level but where self-control responses produced a greater reinforcer density at the molar, or long term, level. Humans earned points by pressing two buttons. Pressing one of the buttons, the impulsive choice, produced 2 points over 1 s. Pressing the other button, the self-controlled choice, produced 10 points over 1 s after a delay of 19 s. In Experiment 1, subjects made 30 choices in a delay condition and 30 choices in the no delay condition. In the delay condition reinforcement of an impulsive response was followed by a 19-s postreinforcer delay that made trial durations equal for both impulsive and self-control choices. In the no delay condition there were no postreinforcer delays following impulsive choices. However, there was a wait period after every 10 trials equal to 19 s times the number of impulsive choices on the previous 10 trials. Thus, in the no delay condition at molecular level, impulsive responding produced a reinforcement density that was greater than the reinforcement density produced by self-controlled responding. However, because of the wait periods, at the molar level self-control responding produced the greatest density of reinforcement. In the delay condition self-controlled responding produced the greatest density of reinforcement at both the molar and molecular levels. Subjects exposed to the no delay condition first responded impulsively during the no delay condition but in a self-controlled fashion during the delay condition. Subjects who received the delay condition first were self-controlled during the delay condition and maintained self-controlled responding during the no delay condition. In Experiment 2 the no delay paradigm was used for 60 trials with punishment and no punishment conditions. In the punishment condition, which occurred either during the first or final 30 trials, following an impulsive response, “NO!” would appear on the screen concurrent with a 0.5-s, 92-db tone. Subjects who experienced the no punishment condition first tended to be impulsive during the no punishment phase but became self-controlled during the punishment condition. Subjects exposed to punishment in the first 30 trials were self-controlled during punishment and maintained self-controlled responding during the no punishment condition. These results suggest that once preference for self-control is developed, self-control responding may be maintained even when impulsive responding produces a greater density of reinforcement at the molecular level.

11598Background: Novel molecular analytes are needed in small bowel neuroendocrine tumors (SBNETs) to better determine disease aggressiveness and predict treatment response. In the present study, w...

This work describes an original and simple technique for protein immobilization into nanowells, fabricated using nanopatterned-array fabrication methods, while ensuring the protein retains the normal biological activity. Nanosphere-lithography was used to fabricate a nanowell array with nanowells that were 100 nm in diameter and a periodicity of 500 nm. The base of the nanowells was gold and the surrounding material was silicon dioxide. The different surface chemistries of these materials were used to attach two different self-assembled monolayers (SAM) with different affinities for the protein used here, cytochrome P450 (P450). The nanowell SAM, a methyl terminated thiol, had high affinity for the P450. The surrounding SAM, a polyethylene glycol silane, displayed very little affinity toward the P450 isozyme CYP2C9, as demonstrated by x-ray photoelectron spectroscopy and surface plasmon resonance. The regularity of the nanopatterned array was examined by scanning electron microscopy and atomic force microscopy. P450-mediated metabolism experiments of known substrates demonstrated that the nanowell bound P450 enzyme exceeded its normal activity, as compared to P450 solutions, when bound to the methyl terminated self-assembled monolayer. The nanopatterned array chips bearing P450 display long term stability and give reproducible results making them potentially useful for high throughput screening assays or as nanoelectrode arrays.

Atrial natriuretic peptide (ANP) binds guanylyl cyclase-A (GC-A) and natriuretic peptide receptor-C (NPR-C). Internalization of GC-A and NPR-C is poorly understood, in part, because previous studies used (125)I-ANP binding to track these receptors, which are expressed in the same cell. Here, we evaluated GC-A and NPR-C internalization using traditional and novel approaches. Although HeLa cells endogenously express GC-A, (125)I-ANP binding and cross-linking studies only detected NPR-C, raising the possibility that past studies ascribed NPR-C-mediated processes to GC-A. To specifically measure internalization of a single receptor, we developed an (125)I-IgG-binding assay that tracks extracellular FLAG-tagged versions of GC-A and NPR-C independently of each other and ligand for the first time. FLAG-GC-A bound ANP identically with wild-type GC-A and was internalized slowly (0.5%/min), whereas FLAG-NPR-C was internalized rapidly (2.5%/min) in HeLa cells. In 293 cells, (125)I-ANP and (125)I-IgG uptake curves were superimposable because these cells only express a single ANP receptor. Basal internalization of both receptors was 8-fold higher in 293 compared with HeLa cells and ANP did not increase internalization of FLAG-GC-A. For FLAG-NPR-C, neither ANP, BNP, nor CNP increased its internalization in either cell line. Prolonged ANP exposure concomitantly reduced surface and total GC-A levels, consistent with rapid exchange of extracellular and intracellular receptor pools. We conclude that ligand binding does not stimulate natriuretic peptide receptor internalization and that cellular environment determines the rate of this process. We further deduce that NPR-C is internalized faster than GC-A and that increased internalization is not required for GC-A down-regulation.

The measurement of the longitudinal behavior of the accelerated particle beams at Fermilab is crucial to the optimization and control of the beam and the maximizing of the integrated luminosity for the particle physics experiments. Longitudinal measurements in the Tevatron and Main Injector synchrotrons are based on the analysis of signals from resistive wall current monitors. This article describes the signal processing performed by a 2 GHz-bandwidth oscilloscope together with a computer running a LabVIEW program which calculates the longitudinal beam parameters.

Preparations and tests are underway at the N-15 site in Waxahachie, Texas, to obtain operational parameters and characteristics for the 100-m-long 50-mm-aperture half cell of the Superconducting Super Collider Laboratory (SSCL) collider. The first power phase of this test refers to the accelerator system string test. The experimental procedure is described. These measurements are being added to the previous information obtained during the thousands of hours of operating the 40-mm-aperture half cell minus the quadrupole string. These data are being compared to each other and to various computer models to obtain optimum operating scenarios for the final collider components and their use. The last run on the previous string emphasized the mid-cell spool piece recooler components; i.e., bypass leads, recooler, cool down valve, quench valve, etc., and their operation. Problems previously reported and their proposed solutions or proven fixes are discussed. Future tests with the present half cell and possible plans for its expansion to a full cell plus are presented. Heat loads from previous runs are compared with their design values. >

Human subjects performed a button-pressing task for three sessions on one of six different multiple variable interval, variable interval (mult VI VI) schedules, followed by one session of extinction on both schedule components. Separate groups were trained with schedule components of VI-5s vs VI-10s, VI-5s vs VI-20s, VI-5s vs VI-40s, VI-10 vs VI-20s, VI-10s vs VI-40s, and VI-20s vs VI-40s. During acquisition, the degree of component discrimination as well as how closely the data fitted Herrnstein′s Matching Law were controlled by both the absolute and relative differences between schedule components and the overall density of reinforcements. During extinction, most subjects responded more to the high-density component than to the low-density component, but responding also declined more rapidly in the higher density component. Differences during extinction were influenced by the absolute and relative differences in the schedule components experienced during acquisition as well as by which component was experienced first during extinction.

Natriuretic peptide receptor-A (NPR-A), also known as guanylyl cyclase-A, is a transmembrane receptor guanylyl cyclase that is activated by the cardiac hormones atrial natriuretic peptide and B-type natriuretic peptide. Although ligand-dependent NPR-A degradation (also known as down-regulation) is widely acknowledged in human and animal models of volume overload, down-regulation in cultured cells is controversial. Here, we examined the effect of ANP exposure on cellular NPR-A levels as a function of time. Relative receptor concentrations were estimated using guanylyl cyclase and immunoblot assays in a wide variety of cell lines that endogenously or exogenously expressed low or high numbers of receptors. ANP exposures of 1 h markedly reduced hormone-dependent but not detergent-dependent guanylyl cyclase activities in membranes from exposed cells. However, 1-h ANP exposures did not significantly reduce NPR-A concentrations in any cell line. In contrast, exposures of greater than 1 h reduced receptor concentrations in a time-dependent manner. The time required for half of the receptors to be degraded (t1/2) in primary bovine aortic endothelial and immortalized HeLa cells was approximately 8 h. In contrast, a 24-h exposure of ANP to 293T cells stably overexpressing NPR-A caused less than half of the receptors to be degraded. To our knowledge, this is the first report to directly measure NPR-A down-regulation in endogenously expressing cells. We conclude that down-regulation is a universal property of NPR-A but is relatively slow and varies with receptor expression levels and cell type.

Humans pressed two buttons for points that were exchangeable for money. Pressing one of the buttons, the impulsive choice, produced 2 points over 4 sec. Pressing the other button, the self-controlled choice, produced 10 points over 4 sec after a delay of 16 sec. In Experiment 1,12 subjects made 30 choices while the computer produced aversive noise and 12 subjects made 30 choices in the absence of noise. A wait period occurred after Trials 15 and 30 which prevented impulsive choices from reducing the length of the experiment. Subjects who were exposed to the noise made fewer self-control choices than those who were not exposed. Experiment 2 was a within-subject replication of Experiment 1, in which subjects made 30 choices in the presence of noise and 30 choices in the absence of noise counterbalanced across subjects. Subjects who first made choices in the absence of noise developed strong self-control preference and maintained that preference in the presence of noise. Self-control was attenuated for subjects who first made choices during noise and showed little, if any, increase in self-control preference when choices were made in the absence of noise. The results parallel those found in “learned helplessness” studies. The results suggest that contextual considerations, beyond traditional stimulus control issues need to be taken into consideration for a more complete understanding of human behaviors such as “self-control.” Practical implications are discussed.

Choice responding in adult humans on a discrete-trial button-pressing task was examined as a function of amount, delay, and overall density (points per unit time) of reinforcement. Reinforcement consisted of points that were exchangeable for money. In T 0 conditions, an impulsive response produced 4 points immediately and a self-control response produced 10 points after a delay of 15 s. In T 15 conditions, a constant delay of 15 s was added to both prereinforcer delays. Postreinforcer delays, which consisted of 15 s added to the end of each impulsive trial, equated trial durations regardless of choice, and was manipulated in both T 0 and T 15 conditions. In all conditions, choice was predicted directly from the relative reinforcement densities of the alternatives. Self-control was observed in all conditions except T 0 without postreinforcer delays, where the impulsive choices produced the higher reinforcement density. These results support previous studies showing that choice is a direct function of the relative reinforcement densities when conditioned (point) reinforcers are used. In contrast, where responding produces intrinsic (immediately consumable) reinforcers, immediacy of reinforcement appears to account for preference when density does not.

An attempt was made to verify that SSC (Superconducting Super Collider) magnets (dipoles) can be installed, leak checked, cooled to LHe temperature, energized, and quenched as a string in a tunnel type environment. The data given here were obtained during a controls and system check-out run. This run basically had all of the proper accelerator controls as well as, in the background, an independent magnet test facility monitor and protection. Early data are presented on the heat loads of some circuits and quench performance of the two magnet strings used. The heat loads found were high and the quench performance appeared to be better than expected. After disassembly occurred, obvious causes were present for some of the heat load. >

Background Natriuretic peptide receptor-A (NPR-A) is a transmembrane receptor guanylyl cyclase that binds and mediates the effects of atrial and B-type natriuretic peptides (ANP/ BNP). Internalization and ligand-dependent degradation of NPR-A is controversial, in part due to the use of ligand binding studies to predict the cellular location of the receptor. Here, we used a more direct sequential immunoprecipitation-western blot assay to demonstrate that longterm ANP exposure increases NPR-A degradation in primary, immortalized, and transfected cells.

Forensic dentistry generally addresses the problem of identifying individuals based on the properties of teeth or identifying individuals based on bite mark impressions. It is legally relevant to accurately and reliably match a bite mark impression to place a criminal at the scene of a crime. Therefore, a system which minimizes human interaction to conduct the comparison would be beneficial to ensure accuracy and reduce human bias. This paper describes experiments with developing a semi-automated method to compare 3D dental models taken from candidate humans and bite mark impression images left in the scene of the crime. Once the contours from the bite mark image and the 3-dimensional dental model are captured, the ideal alignment is calculated by finding the transformation which minimizes a distance measure. The best match is then identified by performing this comparison to a set of candidate dental models. The results are compared to identification results by human forensic odontology experts.

Natriuretic peptides are a family of three structurally related hormone/ paracrine factors. Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) are secreted from the cardiac atria and ventricles, respectively. ANP signals in an endocrine and paracrine manner to decrease blood pressure and cardiac hypertrophy. BNP acts locally to reduce ventricular fibrosis. C-type natriuretic peptide (CNP) primarily stimulates long bone growth but likely serves unappreciated functions as well. ANP and BNP activate the transmembrane guanylyl cyclase, natriuretic peptide receptor-A (NPR-A). CNP activates a related cyclase, natriuretic peptide receptor-B (NPR-B). Both receptors catalyze the synthesis of cGMP, which mediates most known effects of natriuretic peptides. A third natriuretic peptide receptor, natriuretic peptide receptor-C (NPR-C), clears natriuretic peptides from the circulation through receptor-mediated internalization and degradation. However, a signaling function for the receptor has been suggested as well. Targeted disruptions of the genes encoding all natriuretic peptides and their receptors have been generated in mice, which display unique physiologies. A few mutations in these proteins have been reported in humans. Synthetic analogs of ANP (anaritide and carperitide) and BNP (nesiritide) have been investigated as potential therapies for the treatment of decompensated heart failure and other diseases. Anaritide and nesiritide are approved for use in acute decompensated heart failure, but recent studies have cast doubt on their safety and effectiveness. New clinical trials are examining the effect of nesiritide and novel peptides, like CD-NP, on these critical parameters. In this review, the history, structure, function, and clinical applications of natriuretic peptides and their receptors are discussed.

The proposition that the use of contingent, “extrinsic” reinforcement undermines the rate at which “intrinsically interesting” behaviors occur is examined from a behavior-analytic viewpoint. Review of the literature suggests that environmental stimuli control the rates of behaviors rather than interest intrinsic to the organism. Reduced rates of behavior typically attributed to the undermining of intrinsic interest are more objectively accounted for by environmental stimuli functions, including instructional control and by Herrnstein’s (1970) matching law. When the hypothetical version of intrinsic motivation is contrasted with a physiological version of intrinsic motivation, the hypothetical version makes the opposite prediction of every effect that occurs. Recommendations based on the concept of undermining intrinsic interest are flawed and possibly dangerous.