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PDF file, 285K, Supplementary Figure 1. Synthetic scheme and purity of KCN1 3,4-dimethoxy-N-(2,2-dimethyl-2H-chromen-6-yl)methyl-N-phenylbenzenesulfonamide. Supplementary Figure 2. KCN1 is chemically stable in cell culture medium under normoxic or hypoxic conditions. Supplementary Figure 3. Effect of KCN1 in the NCI-60 Tumor Cell Line Screen.

Supplementary Data from Identification of a Novel Small Molecule HIF-1α Translation Inhibitor

Purpose: Hypoxia inducible factor-1 (HIF-1), the central mediator of the cellular response to low oxygen, functions as a transcription factor for a broad range of genes that provide adaptive responses to oxygen deprivation. HIF-1 is overexpressed in cancer and has become an important therapeutic target in solid tumors. In this study, a novel HIF-1α inhibitor was identified and its molecular mechanism was investigated.Experimental Design: Using a HIF-responsive reporter cell–based assay, a 10,000-member natural product–like chemical compound library was screened to identify novel HIF-1 inhibitors. This led us to discover KC7F2, a lead compound with a central structure of cystamine. The effects of KC7F2 on HIF-1 transcription, translation, and protein degradation processes were analyzed.Results: KC7F2 markedly inhibited HIF-mediated transcription in cells derived from different tumor types, including glioma, breast, and prostate cancers, and exhibited enhanced cytotoxicity under hypoxia. KC7F2 prevented the activation of HIF-target genes such as carbonic anhydrase IX, matrix metalloproteinase 2 (MMP2), endothelin 1, and enolase 1. An investigation into the mechanism of action of KC7F2 showed that it worked through the down-regulation of HIF-1α protein synthesis, an effect accompanied by the suppression of the phosphorylation of eukaryotic translation initiation factor 4E binding protein 1 and p70 S6 kinase, key regulators of HIF-1α protein synthesis.Conclusion: These results show that KC7F2 is a potent HIF-1 pathway inhibitor and its potential as a cancer therapy agent warrants further study. (Clin Cancer Res 2009;15(19):6128–36)

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PDF file, 591K, Supplementary Figure 4. KCN1 inhibits the growth of pre-established human glioblastoma s.c. xenografts. Supplementary Figure 5. Effect of KCN1 treatment on mice weight. Supplementary Figure 6. Chronic systemic KCN1 administration shows little effect on major organs, except liver. Supplementary Figure 7. Effect of KCN1 on the survival of mice with orthotopic (intracranial) brain tumor models. Supplementary Figure 8. Permeability coefficients (PC) of KCN1 calculated from the transfer across monolayers of brain endothelial cells.

CCR Translation for the Article from Identification of a Novel Small Molecule HIF-1α Translation Inhibitor

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Hepatitis A is a vaccine-preventable disease typically acquired through fecal-oral transmission. Hepatitis A virus (HAV) infection rates in the United States declined approximately 97% during 1995-2015 after the introduction and widespread pediatric use of hepatitis A vaccines (1). Since 2016, hepatitis A outbreaks have been reported in 37 states, involving approximately 44,650 cases, 27,250 hospitalizations, and 415 deaths as of September 23, 2022 (2). A report describing early outbreaks in four states during 2017 noted that most infections occurred among persons reporting injection or noninjection drug use or experiencing homelessness; this finding signaled a shift in HAV infection epidemiology from point-source outbreaks associated with contaminated food to large community outbreaks associated with person-to-person transmission (3). CDC analyzed interim data from 33 outbreak-affected states to characterize demographic, risk factor, and clinical outcome data from 37,553 outbreak-associated hepatitis A cases reported during August 1, 2016-December 31, 2020. Among persons with available risk factor or clinical outcome information, 56% reported drug use, 14% reported experiencing homelessness, and 61% had been hospitalized; 380 outbreak-associated deaths were reported. The most effective means to prevent and control hepatitis A outbreaks is through hepatitis A vaccination, particularly for persons at increased risk for HAV infection (4). The epidemiologic shifts identified during these outbreaks led to a 2019 recommendation by the Advisory Committee on Immunization Practices (ACIP) for vaccination of persons experiencing homelessness and reinforcement of existing vaccination recommendations for persons who use drugs (4). Substantial progress in the prevention and control of hepatitis A has been made; the number of outbreak-affected states has been reduced from 37 to 13 (2). Increased hepatitis A vaccination coverage, particularly through implementation of successful, nontraditional vaccination strategies among disproportionately affected populations (5), is needed to continue progress in halting current outbreaks and preventing similar outbreaks in the future.

US hepatitis B mortality has been described nationally, but examination subnationally may identify differences in mortality rates and decedent characteristics, including birthplace.To examine characteristics of decedents with hepatitis B-listed deaths during 2010 to 2019 and compare age-adjusted hepatitis B-listed death rates during 2010 to 2019 vs 2000 to 2009.This cross-sectional study used Multiple Cause of Death data from 50 US states and the District of Columbia (DC) from 2000 to 2019 to assess characteristics of US residents with hepatitis B listed as an underlying cause of death (UCOD) or contributing cause of death on death certificates. Data were analyzed from September 2019 to May 2022.Hepatitis B listed as underlying or contributing cause of death.Outcomes of interest were hepatitis B-listed death counts, age-adjusted rates, and characteristics of decedents during 2000 to 2019. The distribution of hepatitis B-listed deaths according to sociodemographic characteristics and UCOD among US- and non-US-born decedents were also examined.A total of 35 280 decedents with hepatitis B listed as the cause of death were identified, including 17 483 deaths during 2010 to 2019. Decedents were 63.3% US-born, and 25.8% of decedents were Asian or Pacific Islander and 46.5% of decedents were White; 28.4% of decedents were listed as having hepatitis C virus (HCV) or HIV coinfection. State-level rates significantly surpassed the overall US rate (0.47 deaths per 100 000 population) in DC (high, 1.78 deaths per 100 000 population), Hawaii, Oklahoma, California, Tennessee, West Virginia, Mississippi, Oregon, Washington, Louisiana, Kentucky, and New York (low, 0.61 deaths per 100 000 population). Median (IQR) age at hepatitis B death was significantly younger in Kentucky (54.0 [46.0-64.0] years), West Virginia (56.0 [47.0-65.0] years), Tennessee (57.0 [50.0-65.0] years), Mississippi (58.0 [50.0-65.0] years), and Ohio (59.0 [50.0-66.0] years) than the national median (60.0 [53.0-69.0] years), which itself was significantly younger than nonhepatitis B-listed deaths (77 [63.0-87.0] years; P .001). Hepatitis B was the UCOD among approximately 30% of US- and non-US-born decedents with hepatitis B COD. Irrespective of birthplace, most decedents had liver-related UCOD. Compared with non-US-born decedents, US-born decedents more frequently had nonliver conditions listed as UCOD. Liver cancer was the predominant UCOD among non-US-born decedents (37.9% of decedents). From 2000 to 2009 compared with 2010 to 2019, the hepatitis B-listed mortality rate significantly decreased nationally (change, -18.97%) and in 14 states; significant increases were observed in West Virginia (change, 83.78%) and Kentucky (change, 69.44%).These findings suggest that US-born decedents constituted two-thirds of all hepatitis B-listed deaths and median age at death was youngest in Appalachian states. Irrespective of birthplace, most decedents had liver-related UCOD; however, US-born decedents more frequently had nonliver UCOD than non-US-born decedents. In addition to addressing liver-related complications, US-born persons with chronic infection may also require diagnosis and management of multiple comorbidities.

Objective:Emergency departments (EDs) are critical settings for hepatitis C care in the United States. We assessed trends and characteristics of hepatitis C–associated ED visits during 2006-2014.Methods:We used data from the 2006-2014 Nationwide Emergency Department Sample to estimate numbers, rates, and costs of hepatitis C–associated ED visits, defined by either first-listed diagnosis of hepatitis C or all-listed diagnosis of hepatitis C. We assessed trends by demographic characteristics, liver disease severity, and patients’ disposition by using joinpoint analysis, and we calculated the average annual percentage change (AAPC) from 2006 to 2014.Results:During 2006-2014, the rate per 100 000 visits of first-listed and all-listed hepatitis C–associated ED visits increased significantly from 10.1 to 25.4 (AAPC = 13.0%; P < .001) and from 484.4 to 631.6 (AAPC = 3.4%; P < .001), respectively. Approximately 70% of these visits were made by persons born during 1945-1965 (baby boomers); 30% of visits were made by Medicare beneficiaries and 40% by Medicaid beneficiaries. Significant rate increases were among visits by baby boomers (first-listed: AAPC = 13.8%; all-listed: AAPC = 2.6%), persons born after 1965 (first-listed: AAPC = 14.3%; all-listed: AAPC = 9.2%), Medicare beneficiaries (first-listed: AAPC = 18.0%; all-listed: AAPC = 3.9%), and persons hospitalized after ED visits (first-listed: AAPC = 20.0%; all-listed: AAPC = 2.3%; all P < .001). Increasing proportions of compensated cirrhosis were among visits by baby boomers (first-listed: AAPC = 11.5%; all-listed: AAPC = 6.3%). Annual hepatitis C–associated total ED costs increased by 400.0% (first-listed) and 192.0% (all-listed) during 2006-2014.Conclusion:Public health efforts are needed to address the growing burden of hepatitis C care in the ED.

Background and aims In the USA, HBV is one of the leading causes of chronic liver disease and cirrhosis and is a major cause of liver cancer. We aimed to estimate the prevalence of past and present HBV infection, susceptibility to HBV infection, and vaccine-induced immunity to hepatitis B among the US population during 2013-2018. Approach and results Prevalence estimates and 95% CIs were analyzed using 2013-2018 data from the National Health and Nutrition Examination Survey. Serologic testing among noninstitutionalized persons aged ≥ 6 years was used for classifying persons as total hepatitis B core antibody (anti-HBc), indicative of current or previous (ever having had) HBV infection; HBsAg, indicative of current HBV infection; and antibody to ABsAg (anti-HBs), indicative of immunity attributable to hepatitis B vaccination. Persons who tested negative for anti-HBc, HBsAg, and anti-HBs were considered susceptible to HBV infection. Non-US-born residents accounted for 69.1% of the population with chronic HBV infection and were 9.1 times more likely to be living with chronic hepatitis B, compared with US-born persons. Among adults aged ≥ 25 years who resided in US households, an estimated 155.8 million persons (or 73.4%) were susceptible to HBV infection, and an estimated 45.4 million had vaccine-induced immunity to hepatitis B. Men who have sex with men (MSM) were 3.6 times more likely to have ever been infected with HBV; however, MSM were just as likely to have vaccine-induced immunity to hepatitis B as non-MSM. Conclusion Despite increasing immune protection among young persons vaccinated after birth, the estimated prevalence of persons living with chronic hepatitis B in the USA has remained unchanged at 0.3% since 1999.

Objectives To obtain timely and detailed data on COVID-19 cases in the United States, the Centers for Disease Control and Prevention (CDC) uses 2 data sources: (1) aggregate counts for daily situational awareness and (2) person-level data for each case (case surveillance). The objective of this study was to describe the sensitivity of case ascertainment and the completeness of person-level data received by CDC through national COVID-19 case surveillance. Methods We compared case and death counts from case surveillance data with aggregate counts received by CDC during April 5–September 30, 2020. We analyzed case surveillance data to describe geographic and temporal trends in data completeness for selected variables, including demographic characteristics, underlying medical conditions, and outcomes. Results As of November 18, 2020, national COVID-19 case surveillance data received by CDC during April 5–September 30, 2020, included 4 990 629 cases and 141 935 deaths, representing 72.7% of the volume of cases (n = 6 863 251) and 71.8% of the volume of deaths (n = 197 756) in aggregate counts. Nationally, completeness in case surveillance records was highest for age (99.9%) and sex (98.8%). Data on race/ethnicity were complete for 56.9% of cases; completeness varied by region. Data completeness for each underlying medical condition assessed was Conclusions Incompleteness in national COVID-19 case surveillance data might limit their usefulness. Streamlining and automating surveillance processes would decrease reporting burdens on jurisdictions and likely improve completeness of national COVID-19 case surveillance data.

INTRODUCTION Bidis are the most commonly smoked tobacco product in India. Understanding bidi smoking is important to reducing overall tobacco smoking and health-related consequences in India. We analyzed 2009–2010 and 2016–2017 Global Adult Tobacco Survey (GATS) India data to examine bidi smoking and its associated sociodemographic correlates and perceptions of dangers of smoking. METHODS GATS is a nationally representative household survey of adults aged ≥15 years, designed to measure tobacco use and tobacco control indicators. Current bidi smoking was defined as current smoking of one or more bidis during a usual week. We computed bidi smoking prevalence estimates and relative change during 2009–2010 and 2016–2017. Used pooled multilevel logistic regression to identify individual-level determinants of bidi smoking and neighborhood-level and state-level variations. RESULTS Overall, 9.2% and 7.7% of adults smoked bidis in India during 2009–2010 and 2016–2017, respectively, reflecting 16.4% significant relative decline. In pooled analysis, male, older age, rural residence, lower education level, lower wealth index, less knowledge about harms of smoking, and survey year were associated with increased odds of bidi smoking. Results also showed variance in odds of smoking bidis is associated with neighborhood (15.9%) and state (31.8%) level. CONCLUSIONS Higher odds of bidi smoking were found among males, older age groups, and among those with lower socioeconomic status. Accordingly, health education interventions designed for these groups across India and other population-level interventions, such as WHO recommendation on increasing price on tobacco products, could help reduce bidi smoking. In addition, state/neighborhood-specific interventions could also help address differential bidi smoking across India.

Hepatitis A virus (HAV) is primarily transmitted fecal-orally after close contact with an infected person (1); it is the most common cause of viral hepatitis worldwide, typically causing acute and self-limited symptoms, although rarely liver failure and death can occur (1). Rates of hepatitis A had declined by approximately 95% during 1996-2011; however, during 2016-2018, CDC received approximately 15,000 reports of HAV infections from U.S. states and territories, indicating a recent increase in transmission (2,3). Since 2017, the vast majority of these reports were related to multiple outbreaks of infections among persons reporting drug use or homelessness (4). In addition, increases of HAV infections have also occurred among men who have sex with men (MSM) and, to a much lesser degree, in association with consumption of imported HAV-contaminated food (5,6). Overall, reports of hepatitis A cases increased 294% during 2016-2018 compared with 2013-2015. During 2016-2018, CDC tested 4,282 specimens, of which 3,877 (91%) had detectable HAV RNA; 565 (15%), 3,255 (84%), and 57 (

Recent global (1) and national (2,3) health equity initiatives conclude that the elimination of health disparities requires improved understanding of social context (4,5) and ability to measure social determinants of health, including food and housing security (3). Food and housing security reflect the availability of and access to essential resources needed to lead a healthy life. The 2013 Behavioral Risk Factor Surveillance System (BRFSS) included two questions to assess perceived food and housing security in 15 states.* Among 95,665 respondents, the proportion who answered "never or rarely" to the question "how often in the past 12 months would you say you were worried or stressed about having enough money to buy nutritious meals?" ranged from 68.5% to 82.4% by state. Among 90,291 respondents living in housing they either owned or rented, the proportion who answered "never or rarely" to the question, "how often in the past 12 months would you say you were worried or stressed about having enough money to pay your rent/mortgage?" ranged from 59.9% to 72.8% by state. Food security was reported less often among non-Hispanic blacks (blacks) (68.5%) and Hispanics (64.6%) than non-Hispanic whites (whites) (81.8%). These racial/ethnic disparities were present across all levels of education; housing security followed a similar pattern. These results highlight racial/ethnic disparities in two important social determinants of health, food and housing security, as well as a substantial prevalence of worry or stress about food or housing among all subgroups in the United States. The concise nature of the BRFSS Social Context Module's single-question format for food and housing security makes it possible to incorporate these questions into large health surveys so that social determinants can be monitored at the state and national levels and populations at risk can be identified.

Chronic hepatitis C (CHC) is a leading cause of morbidity and mortality and has imposed a high health care burden in the United States. Direct-acting antiviral (DAA) regimens are well tolerated and highly effective for CHC therapy but were initially marketed at a high price. Studies of their real-world use with a nationwide population are limited.To examine patient characteristics, treatment adherence, effectiveness, and health care costs in a large U.S. population with commercial and Medicare supplemental insurance plans who received simeprevir (SIM), sofosbuvir (SOF), or ledipasvir/sofosbuvir (LED/SOF) during the years 2013-2015.Patients with at least 1 diagnosis code for CHC and at least 1 claim for SIM, SOF, or LED/SOF prescriptions were selected. The date of the first claim for SIM, SOF, or LED/SOF was defined as the index date. Analyses were stratified by 4 regimens: SOF + SIM ± ribavirin (RBV), SOF + peginterferon alpha-2a or 2b (PEG) + RBV, SOF + RBV, and LED/SOF ± RBV. Adherence was defined by the proportion of days covered (PDC) ≥ 80%. Sustained virologic response (SVR12) was defined as a hepatitis C virus (HCV) RNA load of ≤ 25 IU/mL measured at ≥ 12 weeks following the end of the days supply of the last DAA refill. Health care costs such as DAA drug costs and medical costs (inpatient costs plus outpatient costs) were described.Of 10,808 CHC patients, approximately two thirds were male, and mean age was 55 years. The proportion of patients with compensated cirrhosis among each regimen ranged from 7.4% in LED/SOF ± RBV to 13.8% in SOF + SIM ± RBV, and the proportion of patients with decompensated cirrhosis ranged from 3.9% in LED/SOF ± RBV to 10.7% in SOF + SIM ± RBV. The majority of patients (89.0%) used the newer regimen LED/SOF ± RBV in 2015. Adherence rates were estimated at 80.5%, 81.5%, 85.7%, and 91.4% for SOF + SIM ± RBV (n = 1,761); SOF + PEG + RBV (n = 1,314); SOF + RBV (n = 1,994); and LED/SOF ± RBV (n = 5,739), respectively. Regimen-specific adherence predictors included sex, age group, payer type, health plan, and treatment option with RBV. Being born during 1945-1965, liver disease severity, and Charlson Comorbidity Index levels did not predict adherence in any regimen. Overall SVR12 was 92.6% in 203 patients with available HCV RNA results: 100% (41/41) in SOF + SIM ± RBV; 83.3% (25/30) in SOF + PEG + RBV; 90.6% (29/32) in SOF + RBV; and 93% (93/100) in LED/SOF ± RBV. While the drug costs for these DAA regimens were initially high, they had decreased 18.9% (P0.001) during 2013-2015. Medical costs decreased 9.2% (P0.001) 1 year after the index dates.These results indicate that DAA drug costs decreased steadily during 2013-2015 and that 89% of patients on SOF-based DAA regimens took newer, lower-cost regimens with adherence rates above 80%. Available data show that SVR12 rates were close to those obtained in clinical studies. Medical costs also significantly decreased 1 year after the index dates.No outside funding supported this study. All authors are U.S. federal employees of the Centers for Disease Control and Prevention. The authors declare that they have no competing interests. The findings and conclusions in this research are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.

Supplemental Table Distributions of all-cause emergency department (ED) visits by characteristics in the United States, 2006 and 2014

Objectives. To assess the impact of a large-scale place-based intervention on obesity prevalence in Black communities. Methods. The Racial and Ethnic Approaches to Community Health across the United States (REACH US) project was conducted in 14 predominantly Black communities in California, Illinois, Massachusetts, New York, Ohio, Pennsylvania, South Carolina, Virginia, Washington, and West Virginia. We measured trends from 2009 to 2012 in the prevalence of obesity. We used Behavioral Risk Factor Surveillance System data to compare these trends with trends among non-Hispanic Whites and non-Hispanic Blacks in the United States and in the 10 states where REACH communities were located, and with a propensity score–matched national sample of non-Hispanic Blacks. Results. The age-standardized prevalence of obesity decreased in REACH US communities (P = .045), but not in the comparison populations (P = .435 to P = .996). The relative change was −5.3% in REACH US communities versus +2.4% in propensity score–matched controls (P value for the difference = .031). The net effect on the reduction of obesity prevalence was about 1 percentage point per year for REACH. Conclusions. Obesity prevalence was reduced in 14 disadvantaged Black communities that participated in the REACH project.

Background There is little information on cigarette-purchasing behaviour among smokers globally. Understanding cigarette purchase and point-of-sale patterns can help guide the development and implementation of tobacco-control strategies in retail environments. Objective The purpose of this study was to identify where adults in 19 countries last purchased cigarettes. Methods Data were from 19 low-income and middle-income countries that conducted the Global Adult Tobacco Survey (GATS) during 2008–2012. GATS is a nationally representative household survey of adults aged 15 years or older using a standardised protocol to measure tobacco-related behaviours. Data were weighted to yield nationally representative estimates within each country and summarised by using descriptive statistics. Results Overall prevalence of current cigarette smoking ranged from 3.7% in Nigeria to 38.5% in the Russian Federation. Among current cigarette smokers, locations of last purchase were as follows: stores, from 14.6% in Argentina to 98.7% in Bangladesh (median=66.8%); street vendors, from 0% in Thailand to 35.7% in Vietnam (median=3.0%); kiosks, from 0.1% in Thailand to 77.3% in Argentina (median=16.1%); other locations, from 0.3% in China and Egypt to 57.5% in Brazil (median=2.6%). Conclusion Cigarettes are purchased at various retail locations globally. However, stores and kiosks were the main cigarette purchase locations in 18 of the 19 countries assessed. Knowledge of where cigarette purchases occur could help guide interventions to reduce cigarette accessibility and use.

Background Hepatitis A virus (HAV) is primarily spread fecal-orally and causes acute illness including fever, jaundice, and diarrhea. After introduction and widespread use of the hepatitis A vaccine in the United States, infection with HAV decreased and outbreaks typically associated with a common-source were uncommon. Method CDC receives reports of hepatitis A infections from states through the National Notifiable Disease Surveillance System (NNDSS) and/or directly to the viral hepatitis outbreak response team. We analyzed NNDSS hepatitis A data for 2007–2016, and a combination of NNDSS data and cases directly reported to the CDC hepatitis A outbreak response team during 2017; excluding 2017 NNDSS data from the four states that directly reported outbreaks to the outbreak response team to eliminate the potential for double-counting cases. Result During 2007–2011, a total of 10,619 hepatitis A cases were reported; 521 (5%) were associated with outbreaks. Of the 274 outbreak-associated cases for whom clinical data were reported, 102 (37%) were hospitalized and one (0.3%) died. Of the 407 outbreak-associated cases for whom risk exposure data were reported, 210 (52%) were associated with a common source. Comparatively, during 2012–2017, a total of 11,483 hepatitis A cases were reported; 2,323 (20%) were associated with outbreaks. Of the outbreak-associated cases for whom clinical data were reported, 1,306/2,162 (60%) were hospitalized and 43/2,178 (2%) died. Of the outbreak-associated cases for whom risk exposure data were reported, 379/2,188 (17%) were associated with a common source. Conclusion In the United States, outbreaks of hepatitis A infections in the decade prior to 2017 were infrequent and typically associated with a common source. Reported cases associated with hepatitis A outbreaks are increasing, along with concurrent increases in hospitalizations and deaths among persons with outbreak-associated infections. Recent outbreaks indicate a decrease in cases associated with a common-source exposure. Decreasing the susceptible population through adherence to childhood vaccination recommendations and targeted vaccination of recommended at-risk groups can prevent future hepatitis A outbreaks of any transmission pattern. Disclosures All authors: No reported disclosures.

Tobacco use is a leading preventable cause of morbidity and mortality, with nearly 6 million deaths caused by tobacco use worldwide every year (1). Cigarette smoking is the most common form of tobacco use in most countries, and the majority of adult smokers initiate smoking before age 18 years (2,3). Limiting access to cigarettes among youths is an effective strategy to curb the tobacco epidemic by preventing smoking initiation and reducing the number of new smokers (3,4). CDC used the Global Youth Tobacco Survey (GYTS) data from 45 countries to examine the prevalence of current cigarette smoking, purchase of cigarettes from retail outlets, and type of cigarette purchases made among school students aged 13-15 years. The results are presented by the six World Health Organization (WHO) regions: African Region (AFR); Eastern Mediterranean Region (EMR); European Region (EUR); Region of the Americas (AMR); South-East Asian Region (SEAR); and Western Pacific Region (WPR). Across all 45 countries, the median overall current cigarette smoking prevalence among students aged 13-15 years was 6.8% (range = 1.7% [Kazakhstan]-28.9% [Timor-Leste]); the median prevalence among boys was 9.7% (2.0% [Kazakhstan]-53.5% [Timor-Leste]), and among girls was 3.5% (0.0% [Bangladesh]-26.3% [Italy]). The proportion of current cigarette smokers aged 13-15 years who reported purchasing cigarettes from a retail outlet such as a store, street vendor, or kiosk during the past 30 days ranged from 14.9% [Latvia] to 95.1% [Montenegro], and in approximately half the countries, exceeded 50%. In the majority of countries assessed in AFR and SEAR, approximately 40% of cigarette smokers aged 13-15 years reported purchasing individual cigarettes. Approximately half of smokers in all but one country assessed in EUR reported purchasing cigarettes in packs. These findings could be used by countries to inform tobacco control strategies in the retail environment to reduce and prevent marketing and sales of tobacco products to youths (5).

Weighted Prevalences of Health-related Quality of Life Measures by Year, BRFSS. (DOCX 20 kb)

The hypoxia inducible factor (HIF) pathway is an attractive target for cancer, as it controls tumor adaptation to growth under hypoxia and mediates chemotherapy and radiation resistance. We previously discovered 3,4-dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N-phenylbenzenesulfonamide as a novel, small-molecule HIF-1 pathway inhibitor in a high-throughput cell-based assay, but its in vivo delivery is hampered by poor aqueous solubility (0.009 μM in water; log P7.4 = 3.7). Here we describe the synthesis of 12 N-alkyl-N-[(8-R-2,2-dimethyl-2H-chromen-6-yl)methyl]heteroarylsulfonamides, which were designed to possess optimal lipophilicities and aqueous solubilities by in silico calculations. Experimental log P7.4 values of 8 of the 12 new analogs ranged from 1.2–3.1. Aqueous solubilities of three analogs were measured, among which the most soluble N-[(8-methoxy-2,2-dimethyl-2H-chromen-6-yl)methyl]-N-(propan-2-yl)pyridine-2-sulfonamide had an aqueous solubility of 80 μM, e.g., a solubility improvement ...

Aberrant protein aggregation causes numerous neurological diseases including Creutzfeldt-Jakob disease (CJD), but the aggregation mechanisms remain poorly understood. Here, we report AFM results on the formation pathways of β-oligomers and nonfibrillar aggregates from wild-type full-length recombinant human prion protein (WT) and an insertion mutant (10OR) with five additional octapeptide repeats linked to familial CJD. Upon partial denaturing, seeds consisting of 3-4 monomers quickly appeared. Oligomers of ~11-22 monomers then formed through direct interaction of seeds, rather than by subsequent monomer attachment. All larger aggregates formed through association of these β-oligomers. Although both WT and 10OR exhibited identical aggregation mechanisms, the latter oligomerized faster due to lower solubility and, hence, thermodynamic stability. This novel aggregation pathway has implications for prion diseases as well as others caused by protein aggregation.

Filamin A (FLNA) is an integrator of cell mechanics and signaling. The spreading and migration observed in FLNA sufficient A7 melanoma cells but not in the parental FLNA deficient M2 cells have been attributed to FLNA. In A7 and M2 cells, the normal prion (PrP) exists as pro-PrP, retaining its glycosylphosphatidyl-inositol (GPI) anchor peptide signal sequence (GPI-PSS). The GPI-PSS of PrP has a FLNA binding motif and binds FLNA. Reducing PrP expression in A7 cells alters the spatial distribution of FLNA and organization of actin and diminishes cell spreading and migration. Integrin β1 also binds FLNA. In A7 cells, FLNA, PrP, and integrin β1 exist as two independent, yet functionally linked, complexes; they are FLNA with PrP or FLNA with integrin β1. Reducing PrP expression in A7 cells decreases the amount of integrin β1 bound to FLNA. A PrP GPI-PSS synthetic peptide that crosses the cell membrane inhibits A7 cell spreading and migration. Thus, in A7 cells FLNA does not act alone; the binding of pro-PrP enhances association between FLNA and integrin β1, which then promotes cell spreading and migration. Pro-PrP is detected in melanoma in situ but not in melanocyte. Invasive melanoma has more pro-PrP. The binding of pro-PrP to FLNA, therefore, contributes to melanomagenesis.

Purpose: Hypoxia inducible factor-1 (HIF-1), the central mediator of the cellular response to low oxygen, functions as a transcription factor for a broad range of genes that provide adaptive responses to oxygen deprivation. HIF-1 is overexpressed in cancer and has become an important therapeutic target in solid tumors. In this study, a novel HIF-1α inhibitor was identified and its molecular mechanism was investigated.Experimental Design: Using a HIF-responsive reporter cell–based assay, a 10,000-member natural product–like chemical compound library was screened to identify novel HIF-1 inhibitors. This led us to discover KC7F2, a lead compound with a central structure of cystamine. The effects of KC7F2 on HIF-1 transcription, translation, and protein degradation processes were analyzed.Results: KC7F2 markedly inhibited HIF-mediated transcription in cells derived from different tumor types, including glioma, breast, and prostate cancers, and exhibited enhanced cytotoxicity under hypoxia. KC7F2 prevented the activation of HIF-target genes such as carbonic anhydrase IX, matrix metalloproteinase 2 (MMP2), endothelin 1, and enolase 1. An investigation into the mechanism of action of KC7F2 showed that it worked through the down-regulation of HIF-1α protein synthesis, an effect accompanied by the suppression of the phosphorylation of eukaryotic translation initiation factor 4E binding protein 1 and p70 S6 kinase, key regulators of HIF-1α protein synthesis.Conclusion: These results show that KC7F2 is a potent HIF-1 pathway inhibitor and its potential as a cancer therapy agent warrants further study. (Clin Cancer Res 2009;15(19):6128–36)

The cellular prion protein (PrP) is a highly conserved, widely expressed, glycosylphosphatidylinositol-anchored (GPI-anchored) cell surface glycoprotein. Since its discovery, most studies on PrP have focused on its role in neurodegenerative prion diseases, whereas its function outside the nervous system remains unclear. Here, we report that human pancreatic ductal adenocarcinoma (PDAC) cell lines expressed PrP. However, the PrP was neither glycosylated nor GPI-anchored, existing as pro-PrP and retaining its GPI anchor peptide signal sequence (GPI-PSS). We also showed that the PrP GPI-PSS has a filamin A-binding (FLNa-binding) motif and interacted with FLNa, an actin-associated protein that integrates cell mechanics and signaling. Binding of pro-PrP to FLNa disrupted cytoskeletal organization. Inhibition of PrP expression by shRNA in the PDAC cell lines altered the cytoskeleton and expression of multiple signaling proteins; it also reduced cellular proliferation and invasiveness in vitro as well as tumor growth in vivo. A subgroup of human patients with pancreatic cancer was found to have tumors that expressed pro-PrP. Most importantly, PrP expression in tumors correlated with a marked decrease in patient survival. We propose that binding of pro-PrP to FLNa perturbs FLNa function, thus contributing to the aggressiveness of PDAC. Prevention of this interaction could provide an attractive target for therapeutic intervention in human PDAC.

The human prion gene, PRNP , has two allelic forms that encode either a methionine or valine at codon 129. This polymorphism strongly influences the pathogenesis of prion disease. However, the underlying mechanism remains unclear. We compared the conformation between wild-type human prion protein (rPrP C ) with either a valine or methionine at position 129, using a panel of monoclonal antibodies that are specific for epitopes along the entire protein. We found that rPrP C(129M) has a more exposed helix 1 region compared to rPrP C(129V) . Helix 1 is important in the aggregation process. Accordingly, rPrP C(129M) aggregates at a faster rate and forms more aggregate than rPrP C(129V) . In addition, by using a rPrP with a pathogenic mutation of five additional octapeptide repeat insertions, rPrP (129M)/10OR , as “seeds”, we showed that rPrP (129M)/10OR promotes the aggregation of rPrP C(129M) more efficiently than rPrP C(129V) . These findings provide a possible mechanism underlying the influence of residue 129 on human prion disease.

The normal PrP(C) (cellular prion protein) contains sLe(X) [sialyl-Le(X) (Lewis X)] and Le(X). sLe(X) is a ligand of selectins. To examine whether PrP(C) is a ligand of selectins, we generated three human PrP(C)-Ig fusion proteins: one with Le(X), one with sLe(X), and the other with neither Le(X) nor sLe(X). Only Le(X)-PrP(C)-Ig binds E-, L- and P-selectins. Binding is Ca(2+)-dependent and occurs with nanomolar affinity. Removal of sialic acid on sLe(X)-PrP(C)-Ig enables the fusion protein to bind all selectins. These findings were confirmed with brain-derived PrP(C). The selectins precipitated PrP(C) in human brain in a Ca(2+)-dependent manner. Treatment of brain homogenates with neuraminidase increased the amounts of PrP(C) precipitated. Therefore the presence of sialic acid prevents the binding of PrP(C) in human brain to selectins. Hence, human brain PrP(C) interacts with selectins in a manner that is distinct from interactions in peripheral tissues. Alternations in these interactions may have pathological consequences.

Mutation in the prion gene, PRNP, accounts for approx. 10-15% of human prion diseases. However, little is known about the mechanisms by which a mutant prion protein (PrP) causes disease. We compared the biochemical properties of a wild-type human prion protein, rPrP(C) (recombinant wild-type PrP), which has five octapeptide-repeats, with two recombinant human prion proteins with insertion mutations, one with three more octapeptide repeats, rPrP(8OR), and the other with five more octapeptide repeats, rPrP(10OR). We found that the insertion mutant proteins are more prone to aggregate, and the degree and kinetics of aggregation are proportional to the number of inserts. The octapeptide-repeat and alpha-helix 1 regions are important in aggregate formation, because aggregation is inhibited with monoclonal antibodies that are specific for epitopes in these regions. We also showed that a small amount of mutant protein could enhance the formation of mixed aggregates that are composed of mutant protein and wild-type rPrP(C). Accordingly, rPrP(10OR) is also more efficient in promoting the aggregation of rPrP(C) than rPrP(8OR). These findings provide a biochemical explanation for the clinical observations that the severity of the disease in patients with insertion mutations is proportional to the number of inserts, and thus have implications for the pathogenesis of inherited human prion disease.

The role of neighborhood walkability and safety in mediating the association between education and physical activity has not been quantified. We used data from the 2010 and 2012 Communities Putting Prevention to Work Behavioral Risk Factor Surveillance System and structural equation modeling to estimate how much of the effect of education level on physical activity was mediated by perceived neighborhood walkability and safety. Neighborhood walkability accounts for 11.3% and neighborhood safety accounts for 6.8% of the effect. A modest proportion of the important association between education and physical activity is mediated by perceived neighborhood walkability and safety, suggesting that interventions focused on enhancing walkability and safety could reduce the disparity in physical activity associated with education level.

The conversion of the normal cellular prion protein, PrP C , into the protease-resistant, scrapie PrP Sc aggregate is the cause of prion diseases. We developed a novel enzyme-linked immunosorbent assay (ELISA) that is specific for PrP aggregate by screening 30 anti-PrP monoclonal antibodies (MAbs) for their ability to react with recombinant mouse, ovine, bovine, or human PrP dimers. One MAb that reacts with all four recombinant PrP dimers also reacts with PrP Sc aggregates in ME7-, 139A-, or 22L-infected mouse brains. The PrP Sc aggregate is proteinase K resistant, has a mass of 2,000 kDa or more, and is present at a time when no protease-resistant PrP is detectable. This simple and sensitive assay provides the basis for the development of a diagnostic test for prion diseases in other species. Finally, the principle of the aggregate-specific ELISA we have developed may be applicable to other diseases caused by abnormal protein aggregation, such as Alzheimer's disease or Parkinson's disease.

Purpose: The hypoxia-inducible factor-1 (HIF-1) plays a critical role in tumor adaptation to hypoxia, and its elevated expression correlates with poor prognosis and treatment failure in patients with cancer. In this study, we determined whether 3,4-dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N-phenylbenzenesulfonamide, KCN1, the lead inhibitor in a novel class of arylsulfonamide inhibitors of the HIF-1 pathway, had antitumorigenic properties in vivo and further defined its mechanism of action. Experimental Design: We studied the inhibitory effect of systemic KCN1 delivery on the growth of human brain tumors in mice. To define mechanisms of KCN1 anti-HIF activities, we examined its influence on the assembly of a functional HIF-1α/HIF-1β/p300 transcription complex. Results: KCN1 specifically inhibited HIF reporter gene activity in several glioma cell lines at the nanomolar level. KCN1 also downregulated transcription of endogenous HIF-1 target genes, such as VEGF, Glut-1, and carbonic anhydrase 9, in a hypoxia-responsive element (HRE)-dependent manner. KCN1 potently inhibited the growth of subcutaneous malignant glioma tumor xenografts with minimal adverse effects on the host. It also induced a temporary survival benefit in an intracranial model of glioma but had no effect in a model of melanoma metastasis to the brain. Mechanistically, KCN1 did not downregulate the levels of HIF-1α or other components of the HIF transcriptional complex; rather, it antagonized hypoxia-inducible transcription by disrupting the interaction of HIF-1α with transcriptional coactivators p300/CBP. Conclusions: Our results suggest that the new HIF pathway inhibitor KCN1 has antitumor activity in mouse models, supporting its further translation for the treatment of human tumors displaying hypoxia or HIF overexpression. Clin Cancer Res; 18(24); 6623–33. ©2012 AACR.

Converting normal prion protein (PrP(C)) to the pathogenic PrP(Sc) isoform is central to prion disease. We previously showed that, in the presence of lipids, recombinant mouse PrP (rPrP) can be converted into the highly infectious conformation, suggesting a crucial role of lipid-rPrP interaction in PrP conversion. To understand the mechanism of lipid-rPrP interaction, we analyzed the ability of various rPrP mutants to bind anionic lipids and to gain lipid-induced proteinase K (PK) resistance. We found that the N-terminal positively charged region contributes to electrostatic rPrP-lipid binding but does not affect lipid-induced PK resistance. In contrast, the highly conserved middle region of PrP, consisting of a positively charged region and a hydrophobic domain, is essential for lipid-induced rPrP conversion. The hydrophobic domain deletion mutant significantly weakened the hydrophobic rPrP-lipid interaction and abolished the lipid-induced C-terminal PK resistance. The rPrP mutant without positive charges in the middle region reduced the amount of the lipid-induced PK-resistant rPrP form. Consistent with a critical role of the middle region in lipid-induced rPrP conversion, both disease-associated P105L and P102L mutations, localized between lysine residues in the positively charged region, significantly affected lipid-induced rPrP conversion. The hydrophobic domain-localized 129 polymorphism altered the strength of hydrophobic rPrP-lipid interaction. Collectively, our results suggest that the interaction between the middle region of PrP and lipids is essential for the formation of the PK-resistant conformation. Moreover, the influence of disease-associated PrP mutations and the 129 polymorphism on PrP-lipid interaction supports the relevance of PrP-lipid interaction to the pathogenesis of prion disease.

Mutations in the TP53 gene or the p53 pathway are required for the formation of many human cancers, including brain tumors. The p53 protein is a multi-modal tumor suppressor that controls many processes that are disrupted during tumor formation, in large part by functioning as a transcription factor. p53 regulates the expression of many target genes, including those encoding anti-proliferative, pro-apoptotic, and anti-angiogenic proteins that mediate many of its intracellular tumor-suppressive activities. The p53 protein can also directly induce apoptosis by associating with mitochondrial proteins in a transcription-independent fashion. Furthermore, p53 exerts oncosuppressive effects through cell-extrinsic mechanisms that rely heavily on its ability to control exosome-mediated protein secretion. Here we provide an overview of p53 functions in tumor suppression and review its involvement in the formation of brain tumors.

Recombinant prion protein, rPrP, binds DNA. Both the KKRPK motif and the octapeptide repeat region of rPrP are essential for maximal binding. rPrP with pathogenic insertional mutations binds more DNA than wild-type rPrP. DNA promotes the aggregation of rPrP and protects its N terminus from proteinase K digestion. When rPrP is mixed with an expression plasmid and Ca2+, the rPrP·DNA complex is taken up by mammalian cells leading to gene expression. In the presence of Ca2+, rPrP by itself is also taken up by cells in a temperature- and pinocytosis-dependent manner. Cells do not take up rPrPΔKKRPK, which lacks the KKRPK motif. Thus, rPrP is the carrier for DNA and the KKRPK motif is essential for its uptake. When mixed with DNA, a pentapeptide KKRPK, but not KKKKK, is sufficient for DNA internalization and expression. In contrast, whereas the normal cellular prion protein, PrPC, on the cell surface can also internalize DNA, the imported DNA is not expressed. These findings may have relevance to the normal functions of PrPC and the pathogenic mechanisms of human prion disease.

Mutation in the prion gene PRNP accounts for 10–15% of human prion diseases. However, little is known about the mechanisms by which mutant prion proteins (PrPs) cause disease. Here we investigated the effects of 10 different pathogenic mutations on the conformation and ligand-binding activity of recombinant human PrP (rPrP). We found that mutant rPrPs react more strongly with N terminus-specific antibodies, indicative of a more exposed N terminus. The N terminus of PrP contains a glycosaminoglycan (GAG)-binding motif. Binding of GAG is important in prion disease. Accordingly, all mutant rPrPs bind more GAG, and GAG promotes the aggregation of mutant rPrPs more efficiently than wild-type recombinant normal cellular PrP (rPrP C ). Furthermore, point mutations in PRNP also cause conformational changes in the region between residues 109 and 136, resulting in the exposure of a second, normally buried, GAG-binding motif. Importantly, brain-derived PrP from transgenic mice, which express a pathogenic mutant with nine extra octapeptide repeats, also binds more strongly to GAG than wild-type PrP C . Thus, several rPrPs with distinct pathogenic mutations have common conformational changes, which enhance binding to GAG. These changes may contribute to the pathogenesis of inherited prion diseases.

We have developed a sensitive in vitro assay for detecting disease-associated prion aggregates by combining an aggregation-specific enzyme-linked immunosorbent assay (AS-ELISA) with the fluorescent amplification catalyzed by T7 RNA polymerase technique (FACTT). The new assay, named aggregation-specific FACTT (AS-FACTT), is much more sensitive than AS-ELISA and could detect prion aggregates in the brain of mice as early as 7 days after an intraperitoneal inoculation of PrP Sc . However, AS-FACTT was still unable to detect prion aggregates in blood of infected mice. To further improve the detection limit of AS-FACTT, we added an additional prion amplification step (Am) and developed a third-generation assay, termed Am-A-FACTT. Am-A-FACTT has 100% sensitivity and specificity in detecting disease-associated prion aggregates in blood of infected mice at late but still asymptomatic stages of disease. At a very early stage, Am-A-FACTT had a sensitivity of 50% and a specificity of 100%. Most importantly, Am-A-FACTT also detects prion aggregates in blood of mule deer infected with the agent causing a naturally occurring prion disease, chronic wasting disease. Application of this assay to cattle, sheep, and humans could safeguard food supplies and prevent human contagion.