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Ferroptosis is an iron-dependent form of programmed cell death that is influenced by biological processes such as iron metabolism and senescence. As brain iron levels increase with aging, ferroptosis is also implicated in the development of age-related pathologic conditions such as Alzheimer's disease (AD) and related dementias (ADRD). Indeed, inhibitors of ferroptosis have been shown to be protective in models of degenerative brain disorders like AD/ADRD. Given the inaccessibility of the living human brain for metabolic studies, the goal of this work was to characterize an in vitro model for understanding how aging and iron availability influence neuronal iron metabolism and ferroptosis. First, the human (SH-SY5Y) and mouse (Neuro-2a) neuroblastoma lines were terminally differentiated into mature neurons by culturing in all-trans-retinoic acid for at least 72 h. Despite demonstrating all signs of neuronal differentiation and maturation, including increased expression of the iron storage protein ferritin, we discovered that differentiation conferred ferroptosis resistance in both cell lines. Gene expression data indicates differentiated neurons increase their capacity to protect against iron-mediated oxidative damage by augmenting cystine import, and subsequently increasing intracellular cysteine levels, to promote glutathione production and glutathione peroxidase activity (GPX). In support of this hypothesis, we found that culturing differentiated neurons in cysteine-depleted media sensitized them to GPX4 inhibition, and that these effects are mitigated by cystine supplementation. Such findings are important as they provide guidance for the use of in vitro experimental models to investigate the role of ferroptosis in neurodegeneration in pathologies such as ADRD.

The focus is on the demographic drivers and demographic implications of urban health and wellbeing in towns and cities across the globe. The aim is to identify key linkages between demographic change and urban health - subjects of two largely disparate fields of research and practice - with a view to informing arguments and advocacy for urban health while identifying research gaps and priorities. The core arguments are threefold. First, urban health advocates should express a globalized perspective on demographic processes, encompassing age-structural shifts in addition to population growth and decrease, and acknowledging their uneven spatial distributions within and between urban settings in different contexts. Second, advocates should recognize the dynamic and transformational effects that demographic forces will exert on economic and political systems in all urban settings. While demographic forces underpin the production of (intra)urban inequities in health, they also present opportunities to address those inequities. Third, a demographic perspective may help to extend urban health thinking and intervention beyond a biomedical model of disease, highlighting the need for a multi-generational view of the changing societal bases for urban health, and enjoining significant advances in how interested parties collect, manage, analyse, and use demographic data. Accordingly, opportunities are identified to increase the availability of granular and accurate data to enable evidence-informed action on the demographic/health nexus., Competing Interests: No competing interests were disclosed., (Copyright: © 2023 Duminy J et al.)

Cells require iron for essential functions like energy production and signaling. However, iron can also engage in free radical formation and promote cell proliferation thereby contributing to both tumor initiation and growth. Thus, the amount of iron within the body and in individual cells is tightly regulated. At the cellular level, iron homeostasis is maintained post-transcriptionally by iron regulatory proteins (IRPs). Ferroptosis is an iron-dependent form of programmed cell death with vast chemotherapeutic potential, yet while IRP-dependent targets have established roles in ferroptosis, our understanding of the contributions of IRPs themselves is still in its infancy. In this review, we present the growing circumstantial evidence suggesting that IRPs play critical roles in the adaptive response to ferroptosis and ferroptotic cell death and describe how this knowledge can be leveraged to target neoplastic iron dysregulation more effectively., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Cardona and Montgomery.)

While most cases of cutaneous squamous cell carcinoma (cSCC) are benign, invasive cSCC is associated with higher mortality and is often more difficult to treat. As such, understanding the factors that influence the progression of cSCC are important. Aggressive cancers metastasize through a series of evolutionary changes, collectively called the epithelial-to-mesenchymal transition (EMT). During EMT, epithelial cells transition to a highly mobile mesenchymal cell type with metastatic capacities. While changes in expression of TGF-β, ZEB1, SNAI1, MMPs, vimentin, and E-cadherin are hallmarks of an EMT process occurring within cancer cells, including cSCC cells, EMT within tissues is not an "all or none" process. Using patient-derived cSCC and adjacent normal tissues, we show that cells within individual cSCC tumors are undergoing a hybrid EMT process, where there is variation in expression of EMT markers by cells within a tumor mass that may be facilitating invasion. Interestingly, cells along the outer edges of a tumor mass exhibit a more mesenchymal phenotype, with reduced E-cadherin, β-catenin, and cytokeratin expression and increased vimentin expression. Conversely, cells in the center of a tumor mass retain a higher expression of the epithelial markers E-cadherin and cytokeratin and little to no expression of vimentin, a mesenchymal marker. We also detected inverse expression changes in the miR-200 family and the EMT-associated transcription factors ZEB1 and SNAI1, suggesting that cSCC EMT dynamics are regulated in a miRNA-dependent manner. These novel findings in cSCC tumors provide evidence of phenotypic plasticity of the EMT process occurring within patient tissues, and extend the characterization of a hybrid EMT program occurring within a tumor mass. This hybrid EMT program may be promoting both survival and invasiveness of the tumors. A better understanding of this hybrid EMT process may influence therapeutic strategies in more invasive disease.

The tumor suppressor TP53 is the most commonly mutated gene in human cancers, and iron is necessary for cancer cell growth and proliferation, but there is a significant gap in knowledge for how the two cooperate to affect cellular physiology. Elucidating this role is complicated, however, because each TP53 mutation subtype exhibits unique phenotypic responses to changes in iron availability. The goal of this work was to determine how cells expressing distinct TP53 mutation subtypes respond to iron restriction. Utilizing a reverse genetics approach, we generated eight isogenic cell lines that either lacked TP53 expression, expressed wild-type TP53, or expressed one of the six most common TP53 "hotspot" mutations. We then employed isobaric peptide labeling and mass spectrometry to quantitively measure changes in global protein expression, both in response to induction of mutant TP53 expression, and in response to iron chelation. Our findings indicate that mutant TP53-dependent sensitivities to iron restriction are not driven by differences in responsiveness to iron chelation, but more so by mutant TP53-dependent differences in cellular antioxidant and lipid handling protein expression. These findings reinforce the importance of distinguishing between TP53 mutation subtypes when investigating approaches to target mutant TP53. We also identify unique TP53-dependent perturbances in protein expression patterns that could be exploited to improve iron-targeted chemotherapeutic strategies.

By 2050, two-thirds of the world's population is expected to be living in cities and towns, a marked increase from today's level of 55 percent. If the general trend is unmistakable, efforts to measure it precisely have been beset with difficulties: the criteria defining urban areas, cities and towns differ from one country to the next and can also change over time for any given country. The past decade has seen great progress toward the long-awaited goal of scientifically comparable urbanization measures, thanks to the combined efforts of multiple disciplines. These efforts have been organized around what is termed the "statistical urbanization" concept, whereby urban areas are defined by population density, contiguity and total population size. Data derived from remote-sensing methods can now supply a variety of spatial proxies for urban areas defined in this way. However, it remains to be understood how such proxies complement, or depart from, meaningful country-specific alternatives. In this paper, we investigate finely resolved population census and satellite-derived data for the United States, Mexico and India, three countries with widely varying conceptions of urban places and long histories of debate and refinement of their national criteria. At the extremes of the urban-rural continuum, we find evidence of generally good agreement between the national and remote sensing-derived measures (albeit with variation by country), but identify significant disagreements in the middle ranges where today's urban policies are often focused., Competing Interests: Conflicts of Interest: The authors declare no conflict of interest.

Health agendas for low- and middle-income countries (LMICs) should embrace and afford greater priority to urban family planning to help achieve a number of the global Sustainable Development Goals. The urgency of doing so is heightened by emerging evidence of urban fertility stalls and reversals in some sub-Saharan African contexts as well as the significance of natural increase over migration in driving rapid urban growth. Moreover, there is new evidence from evaluations of large programmatic interventions focused on urban family planning that suggest ways to inform future programmes and policies that are adapted to local contexts. We present the key dimensions and challenges of urban growth in LMICs, offer a critical scoping review of recent research findings on urban family planning and fertility dynamics, and highlight priorities for future research., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Duminy, Cleland, Harpham, Montgomery, Parnell and Speizer.)

The SLC39A8 gene encodes a divalent metal transporter, ZIP8. SLC39A8 is associated with pleiotropic effects across multiple tissues, including the brain. We determine the different brain magnetic resonance imaging (MRI) phenotypes associated with SLC39A8. We used a phenome-wide association study approach followed by joint and conditional association analysis. Using the summary statistics datasets from a brain MRI genome-wide association study on adult United Kingdom (UK) Biobank participants, we systematically selected all brain MRI phenotypes associated with single-nucleotide polymorphisms (SNPs) within 500 kb of the SLC39A8 genetic locus. For all significant brain MRI phenotypes, we used GCTA-COJO to determine the number of independent association signals and identify index SNPs for each brain MRI phenotype. Linkage equilibrium for brain phenotypes with multiple independent signals was confirmed by LDpair. We identified 24 brain MRI phenotypes that vary due to MRI type and brain region and contain a SNP associated with the SLC39A8 locus. Missense ZIP8 polymorphism rs13107325 was associated with 22 brain MRI phenotypes. Rare ZIP8 variants present in a published UK Biobank dataset are associated with 6 brain MRI phenotypes also linked to rs13107325. Among the 24 datasets, an additional 4 association signals were identified by GCTA-COJO and confirmed to be in linkage equilibrium with rs13107325 using LDpair. These additional association signals represent new probable causative SNPs in addition to rs13107325. This study provides leads into how genetic variation in SLC39A8 , a trace mineral transport gene, is linked to brain structure differences and may affect brain development and nervous system function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hermann, Chambers, Davis, Montgomery, Lin and Chowanadisai.)

The tumor suppressor gene TP53 is the most commonly mutated gene in human cancer. In addition to loss of tumor suppressor functions, mutations in TP53 promote cancer progression by altering cellular iron acquisition and metabolism. A newly identified role for TP53 in the coordination of iron homeostasis and cancer cell survival lies in the ability for TP53 to protect against ferroptosis, a form of iron-mediated cell death. The purpose of this study was to determine the extent to which TP53 mutation status affects the cellular response to ferroptosis induction. Using H1299 cells, which are null for TP53, we generated cell lines expressing either a tetracycline inducible wild-type (WT) TP53 gene, or a representative mutated TP53 gene from six exemplary "hotspot" mutations in the DNA binding domain (R273H, R248Q, R282W, R175H, G245S, and R249S). TP53 mutants (R273H, R248Q, R175H, G245S, and R249S) exhibited increased sensitivity ferroptosis compared to cells expressing WT TP53. As iron-mediated lipid peroxidation is critical for ferroptosis induction, we hypothesized that iron acquisition pathways would be upregulated in mutant TP53-expressing cells. However, only cells expressing the R248Q, R175H, and G245S TP53 mutation types exhibited statistically significant increases in spontaneous iron regulatory protein (IRP) RNA binding activity following ferroptosis activation. Moreover, changes in the expression of downstream IRP targets were inconsistent with the observed differences in sensitivity to ferroptosis. These findings reveal that canonical iron regulatory pathways are bypassed during ferroptotic cell death. These results also indicate that induction of ferroptosis may be an effective therapeutic approach for tumor cells expressing distinct TP53 mutation types.

While the population of the United States has been predominantly urban for nearly 100 years, periodic transformations of the concepts and measures that define urban places and population have taken place, complicating over-time comparisons. We compare and combine data series of officially-designated urban areas, 1990-2010, at the census block-level within Metropolitan Statistical Areas (MSAs) with a satellite-derived consistent series on built-up area from the Global Human Settlement Layer to create urban classes that characterize urban structure and provide estimates of land and population. We find considerable heterogeneity in urban form across MSAs, even among those of similar population size, indicating the inherent difficulties in urban definitions. Over time, we observe slightly declining population densities and increasing land and population in areas captured only by census definitions or low built-up densities, constrained by the geography of place. Nevertheless, deriving urban proxies from satellite-derived built-up areas is promising for future efforts to create spatio-temporally consistent measures for urban land to guide urban demographic change analysis.

The most commonly mutated gene in all human cancers is the tumor suppressor gene TP53 ; however, in addition to the loss of tumor suppressor functions, mutations in TP53 can also promote cancer progression by altering cellular iron acquisition and metabolism. The primary objective of this work was to determine how TP53 mutation status influences the molecular control of iron homeostasis. The effect of TP53 mutation type on cellular iron homeostasis was examined using cell lines with inducible versions of either wild-type TP53 or a representative mutated TP53 gene from exemplary "hotspot" mutations in the DNA binding domain (R248, R273, and R175) as well as H193Y. The introduction of distinct TP53 mutation types alone was sufficient to disrupt cellular iron metabolism. These effects were mediated, at least in part, due to differences in the responsiveness of iron regulatory proteins (IRPs) to cellular iron availability. IRPs are considered the master regulators of intracellular iron homeostasis because they coordinate the expression of iron storage (ferritin) and iron uptake (transferrin receptor) genes. In response to changes in iron availability, cells harboring either a wild-type TP53 or R273H TP53 mutation displayed canonical IRP-mediated responses, but neither IRP1 RNA binding activity nor IRP2 protein levels were affected by changes in iron status in cells harboring the R175H mutation type. However, all mutation types exhibited robust changes in ferritin and transferrin receptor protein expression in response to iron loading and iron chelation, respectively. These findings suggest a novel, IRP-independent mode of iron regulation in cells expressing distinct TP53 mutations. As TP53 is mutated in nearly half of all human cancers, and iron is necessary for cancer cell growth and proliferation, the studies have implications for a wide range of clinically important cancers.

India is the world's most populous country, yet also one of the least urban. It has long been known that India's official estimates of urban percentages conflict with estimates derived from alternative conceptions of urbanization. To date, however, the detailed spatial and settlement boundary data needed to analyze and reconcile these differences have not been available. This paper presents gridded estimates of population at a resolution of 1 km along with two spatial renderings of urban areas-one based on the official tabulations of population and settlement types (i.e., statutory towns, outgrowths, and census towns) and the other on remotely-sensed measures of built-up land derived from the Global Human Settlement Layer. We also cross-classified the census data and the remotely-sensed data to construct a hybrid representation of the continuum of urban settlement. In their spatial detail, these materials go well beyond what has previously been available in the public domain, and thereby provide an empirical basis for comparison among competing conceptual models of urbanization., Competing Interests: Conflicts of Interest: The authors declare no conflict of interest.

Background: Defects in the type and degree of cellular glycosylation impact oncogenesis on multiple levels. Although the type of glycosylation is determined by protein sequence encoded by the genome, the extent and modifications of glycosylation depends on the activity of biosynthetic enzymes and recent data suggests that the glycome is also subject to epigenetic regulation. This study focuses on the ability of HDAC inhibition to alter glycosylation and to lead to pro-oncogenic alterations in the glycome as assessed by metastatic potential and chemoresistance., Methods: Epigenetically plastic SW13 adrenocortical carcinoma cells were treated with FK228, an HDAC inhibitor with high affinity for HDAC1 and, to a lesser extent, HDAC2. In comparing HDAC inhibitor treated and control cells, differential expression of glycome-related genes were assessed by microarray. Differential glycosylation was then assessed by lectin binding arrays and the ability of cellular proteins to bind to glycans was assessed by glycan binding arrays. Differential sensitivity to paclitaxel, proliferation, and MMP activity were also assessed., Results: Treatment with FK228 alters expression of enzymes in the biosynthetic pathways for a large number of glycome related genes including enzymes in all major glycosylation pathways and several glycan binding proteins. 84% of these differentially expressed glycome-related genes are linked to cancer, some as prognostic markers and others contributing basic oncogenic functions such as metastasis or chemoresistance. Glycan binding proteins also appear to be differentially expressed as protein extracts from treated and untreated cells show differential binding to glycan arrays. The impact of differential mRNA expression of glycosylation enzymes was documented by differential lectin binding. However, the assessment of changes in the glycome is complicated by the fact that detection of differential glycosylation through lectin binding is dependent on the methods used to prepare samples as protein-rich lysates show different binding than fixed cells in several cases. Paralleling the alterations in the glycome, treatment of SW13 cells with FK228 increases metastatic potential and reduces sensitivity to paclitaxel., Conclusions: The glycome is substantially altered by HDAC inhibition and these changes may have far-reaching impacts on oncogenesis.

Most of future population growth will take place in the world's cities and towns. Yet, there is no well-established, consistent way to measure either urban land or people. Even census-based urban concepts and measures undergo frequent revision, impeding rigorous comparisons over time and place. This study presents a new spatial approach to derive consistent urban proxies for the US. It compares census-designated urban blocks with proxies for land-based classifications of built-up areas derived from time-series of the Global Human Settlement Layer (GHSL) for 1990-2010. This comparison provides a new way to understand urban structure and its changes: Most land that is more than 50% built-up, and people living on such land, are officially classified as urban. However, 30% of the census-designated urban population and land is located in less built-up areas that can be characterized as mainly suburban and peri-urban in nature. Such insights are important starting points for a new urban research program: creating globally and temporally consistent proxies to guide modelling of urban change., Competing Interests: The authors have declared that no competing interests exist.

Emerging evidence suggests that the enzymes in the biosynthetic pathway for the synthesis of heparan sulfate moieties of heparan sulfate proteoglycans (HSPGs) are epigenetically regulated at many levels. As the exact composition of the heparan sulfate portion of the resulting HSPG molecules is critical to the broad spectrum of biological processes involved in oncogenesis, the epigenetic regulation of heparan sulfate biosynthesis has far-reaching effects on many cellular activities related to cancer progression. Given the current focus on developing new anti-cancer therapeutics focused on epigenetic targets, it is important to understand the effects that these emerging therapeutics may have on the synthesis of HSPGs as alterations in HSPG composition may have profound and unanticipated effects. As an introduction, this review will briefly summarize the variety of important roles which HSPGs play in a wide-spectrum of cancer-related cellular and physiological functions and then describe the biosynthesis of the heparan sulfate chains of HSPGs, including how alterations observed in cancer cells serve as potential biomarkers. This review will then focus on detailing the multiple levels of epigenetic regulation of the enzymes in the heparan sulfate synthesis pathway with a particular focus on regulation by miRNA and effects of epigenetic therapies on HSPGs. We will also explore the use of lectins to detect differences in heparan sulfate composition and preview their potential diagnostic and prognostic use in the clinic., Competing Interests: The authors declare no conflict of interest.

The incidence of skin cancer (e.g., squamous cell carcinoma, basal cell carcinoma, and melanoma) has been increasing over the past several years. It is expected that there will be a parallel demand for cutaneous tumor samples for biomedical research studies. Tissue availability, however, is limited due the cost of establishing a biorepository and the lack of protocols available for obtaining clinical samples that do not interfere with clinical operations. A protocol was established to collect and process cutaneous tumor and associated blood and saliva samples that has minimal impact on routine clinical procedures on the date of a Mohs surgery. Tumor samples are collected and processed from patients undergoing their first layer of Mohs surgery for biopsy-proven cutaneous malignancies by the Mohs histotechnologist. Adjacent normal tissue is collected at the time of surgical closure. Additional samples that may be collected are whole-blood and buccal swabs. By utilizing tissue samples that are normally discarded, a biorepository was generated that offers several key advantages by being based in the clinic versus the laboratory setting. These include a wide range of collected samples; access to de-identified patient records, including pathology reports; and, for the typical donor, access to additional samples during follow-up visits.

Histone deacetylase (HDAC) inhibitors are powerful epigenetic regulators that have enormous therapeutic potential and have pleiotropic effects at the cellular and systemic levels. To date, HDAC inhibitors are used clinically for a wide variety of disorders ranging from hematopoietic malignancies to psychiatric disorders, are known to have anti-inflammatory properties, and are in clinical trials for several other diseases. In addition to influencing gene expression, HDAC enzymes also function as part of large, multisubunit complexes which have many nonhistone targets, alter signaling at the cellular and systemic levels, and result in divergent and cell-type specific effects. Thus, the effects of HDAC inhibitor treatment are too intricate to completely understand with current knowledge but the ability of HDAC inhibitors to modulate the immune system presents intriguing therapeutic possibilities. This review will explore the complexity of HDAC inhibitor treatment at the cellular and systemic levels and suggest strategies for effective use of HDAC inhibitors in biomedical research, focusing on the ability of HDAC inhibitors to modulate the immune system. The possibility of combining the documented anticancer effects and newly emerging immunomodulatory effects of HDAC inhibitors represents a promising new combinatorial therapeutic approach for HDAC inhibitor treatments.

Objective: To determine if vitamin D intake is associated with acute lower respiratory infections (ALRI) in children., Methods: The vitamin D intakes of children younger than 5 years of age admitted to hospital with either bronchiolitis or pneumonia were compared to an unmatched control group of the same age without respiratory infection. Caregivers of 197 children completed a questionnaire collecting information on demographic variables, ALRI risk factors and diet. Associations of ALRI with vitamin D intake and other ALRI risk factors were determined., Results: The mean vitamin D intake of children with ALRI was 48 IU/kg/d compared to 60 IU/kg/d in the control group. When controlling for age, ethnicity, socio-economic status, northern residence, breastfeeding, immunizations and smoking contact, children with a vitamin D intake of less than 80 IU/kg/d were greater than 4 times more likely to have ALRI compared to children with a vitamin D intake exceeding 80 IU/kg/day (OR 4.9, 95% CI: 1.5, 16.4)., Conclusions: A higher vitamin D intake than currently recommended might be needed to offer protection against diseases such as ALRI. Increased vitamin D supplementation could have important public health consequences, as bronchiolitis and pneumonia are the most common reasons for hospitalization in young children.

The use of Global Positioning Systems (GPS) and Geographical Information Systems (GIS) in disease surveys and reporting is becoming increasingly routine, enabling a better understanding of spatial epidemiology and the improvement of surveillance and control strategies. In turn, the greater availability of spatially referenced epidemiological data is driving the rapid expansion of disease mapping and spatial modeling methods, which are becoming increasingly detailed and sophisticated, with rigorous handling of uncertainties. This expansion has, however, not been matched by advancements in the development of spatial datasets of human population distribution that accompany disease maps or spatial models.Where risks are heterogeneous across population groups or space or dependent on transmission between individuals, spatial data on human population distributions and demographic structures are required to estimate infectious disease risks, burdens, and dynamics. The disease impact in terms of morbidity, mortality, and speed of spread varies substantially with demographic profiles, so that identifying the most exposed or affected populations becomes a key aspect of planning and targeting interventions. Subnational breakdowns of population counts by age and sex are routinely collected during national censuses and maintained in finer detail within microcensus data. Moreover, demographic and health surveys continue to collect representative and contemporary samples from clusters of communities in low-income countries where census data may be less detailed and not collected regularly. Together, these freely available datasets form a rich resource for quantifying and understanding the spatial variations in the sizes and distributions of those most at risk of disease in low income regions, yet at present, they remain unconnected data scattered across national statistical offices and websites.In this paper we discuss the deficiencies of existing spatial population datasets and their limitations on epidemiological analyses. We review sources of detailed, contemporary, freely available and relevant spatial demographic data focusing on low income regions where such data are often sparse and highlight the value of incorporating these through a set of examples of their application in disease studies. Moreover, the importance of acknowledging, measuring, and accounting for uncertainty in spatial demographic datasets is outlined. Finally, a strategy for building an open-access database of spatial demographic data that is tailored to epidemiological applications is put forward.

For 18 months in 2009-2010, the Rockefeller Foundation provided support to establish the Roundtable on Urban Living Environment Research (RULER). Composed of leading experts in population health measurement from a variety of disciplines, sectors, and continents, RULER met for the purpose of reviewing existing methods of measurement for urban health in the context of recent reports from UN agencies on health inequities in urban settings. The audience for this report was identified as international, national, and local governing bodies; civil society; and donor agencies. The goal of the report was to identify gaps in measurement that must be filled in order to assess and evaluate population health in urban settings, especially in informal settlements (or slums) in low- and middle-income countries. Care must be taken to integrate recommendations with existing platforms (e.g., Health Metrics Network, the Institute for Health Metrics and Evaluation) that could incorporate, mature, and sustain efforts to address these gaps and promote effective data for healthy urban management. RULER noted that these existing platforms focus primarily on health outcomes and systems, mainly at the national level. Although substantial reviews of health outcomes and health service measures had been conducted elsewhere, such reviews covered these in an aggregate and perhaps misleading way. For example, some spatial aspects of health inequities, such as those pointed to in the 2008 report from the WHO's Commission on the Social Determinants of Health, received limited attention. If RULER were to focus on health inequities in the urban environment, access to disaggregated data was a priority. RULER observed that some urban health metrics were already available, if not always appreciated and utilized in ongoing efforts (e.g., census data with granular data on households, water, and sanitation but with little attention paid to the spatial dimensions of these data). Other less obvious elements had not exploited the gains realized in spatial measurement technology and techniques (e.g., defining geographic and social urban informal settlement boundaries, classification of population-based amenities and hazards, and innovative spatial measurement of local governance for health). In summary, the RULER team identified three major areas for enhancing measurement to motivate action for urban health-namely, disaggregation of geographic areas for intra-urban risk assessment and action, measures for both social environment and governance, and measures for a better understanding of the implications of the physical (e.g., climate) and built environment for health. The challenge of addressing these elements in resource-poor settings was acknowledged, as was the intensely political nature of urban health metrics. The RULER team went further to identify existing global health metrics structures that could serve as platforms for more granular metrics specific for urban settings.

This paper examines the reporting of sexual and other risk behaviors within a randomized experiment using a computerized versus face-to-face interview mode. Biomarkers for sexually transmitted infection (STI) were used to validate self-reported behavior by interview mode. As part of a parent study evaluating home versus clinic screening and diagnosis for STIs, 818 women aged 18-40 years were recruited in 2004 at or near a primary care clinic in São Paulo, Brazil, and were randomized to a face-to-face interview or audio computer-assisted self-interviewing. Ninety-six percent of participants were tested for chlamydia, gonorrhea, and trichomoniasis. Reporting of STI risk behavior was consistently higher with the computerized mode of interview. Stronger associations between risk behaviors and STI were found with the computerized interview after controlling for sociodemographic factors. These results were obtained by using logistic regression approaches, as well as statistical methods that address potential residual confounding and covariate endogeneity. Furthermore, STI-positive participants were more likely than STI-negative participants to underreport risk behavior in the face-to-face interview. Results strongly suggest that computerized interviewing provides more accurate and reliable behavioral data. The analyses also confirm the benefits of using data on prevalent STIs for externally validating behavioral reporting.

Sometime in the next 20 to 30 years, developing countries in Asia and Africa are likely to cross a historic threshold, joining Latin America in having a majority of urban residents. The urban demographic transformation is described here, with an emphasis on estimates and forecasts of urban population aggregates. To provide policy-makers with useful scientific guidance in the upcoming urban era, demographic researchers will need to refine their data sets to include spatial factors as well as urban vital rates and to make improvements to forecasting methods currently in use.

In the United States and other high-income countries, there is intense scholarly and programmatic interest in the effects of household and neighborhood living standards on health. Yet few studies of developing-country cities have explored these issues. We investigated whether the health of urban women and children in poor countries is influenced by both household and neighborhood standards of living. Using data from the urban samples of 85 Demographic and Health Surveys and modeling living standards using factor-analytic MIMIC methods, we found that the neighborhoods of relatively poor households are more heterogeneous than is often asserted. Our results indicated that poor urban households do not tend to live in uniformly poor neighborhoods: about 1 in 10 of a poor household's neighbors is relatively affluent, belonging to the upper quartile of the urban distribution of living standards. Do household and neighborhood living standards influence health? Using multivariate models, we found that household living standards are closely associated with three health measures: unmet need for modern contraception, attendance of a trained health care provider at childbirth, and young children's height for age. Neighborhood living standards exert a significant additional influence in many of the surveys we examined, especially for birth attendance.

Very few demographic surveys in developing countries have gathered information on household incomes or consumption expenditures. Researchers interested in living standards therefore have had little alternative but to rely on simple proxy indicators. The properties of these proxies have not been analyzed systematically. We ask what hypotheses can be tested using proxies, and compare these indicators with consumption expenditures per adult, our preferred measure of living standards. We find that the proxies employed in much demographic research are very weak predictors of consumption per adult. Nevertheless, hypothesis tests based on proxies are likely to be powerful enough to warrant consideration.

As part of a study on mechanisms modulating ozone-induced surfactant perturbations, we used the electron paramagnetic resonance (EPR) spin trapping technique to determine the type and origin of radicals generated following interaction of ozone with aqueous solutions and cell-free bronchoalveolar lavage fluid (BAL) fractions. All aqueous media were exposed to ozone at 25 degrees C with or without added chelator, 1 mM diethylenetriaminepentaacetic acid, and spintrap, 100 mM 5,5'-dimethyl-1-pyrroline-1-oxide (DMPO). Exposure of distilled water to 0.5, 1.0, 2.0, and 3.0 ppm ozone for 1 h yielded four-line spectra, 1:2:2:1, consistent with hydroxyl radical adduct formation (DMPO-OH), the amplitudes of which increased with the ozone concentration. No signals were obtained from air-exposed samples. Similar four-line spectra were also produced following interaction of 3 ppm ozone with Hank's balanced salt solution (HBSS) alone or containing BAL fractions. Addition of the hydroxyl radical scavenger dimethyl sulfoxide (DMSO) to the incubation medium strongly inhibited formation of DMPO-OH adduct during ozone exposure. As an alternate method of demonstrating the generation of hydroxyl radicals, aqueous solutions of 1 mM L-phenylalanine were exposed to high concentrations of ozone and shown, using ion-exchange chromatography, to contain small amounts of L-tyrosine. Production of hydroxyl radicals upon interaction of ozone and water was further substantiated using the spintrap PBN (phenyl-N-tert-butylnitrone) in the presence of DMSO which reacts with the hydroxyl radical resulting in the formation of methyl radical. The methyl radical subsequently reacts with spintrap PBN, yielding PBN-methyl adduct. In the absence of DMSO there was no detectable formation of methyl radical adduct. EPR double distilled water containing DMPO showed a small amount of DMPO-OH adduct upon exposure to ozone. Addition of 10 microM ferrous sulfate to this mixture produced a 10-fold increase of the signal, which was attenuated in the presence of 1500 U catalase, strongly attenuated with 50-500 microM deferoxamine or 8000 U catalase and abolished by higher concentration of deferoxamine (1 mM). The signal was not influenced by 1000 U superoxide dismutase. These results indicate that hydroxyl radicals are produced via iron-dependent reactions during the initial interaction of ozone with aqueous media, including bronchoalveolar fluid.

The nonspecificity of symptoms and exposures commonly reported as "sick building syndrome" is very similar to that reported for the condition termed "multiple chemical sensitivity (MCS)." In many instances health care practitioners are divided on the reality of the latter. As in all scientific cases of possible cause-and-effect relationships, an organized, dispassionate approach should be used to evaluate each claim of MCS. Most obviously, known medical conditions must be ruled out before chemical "sensitivity" is proposed. Also, several questions on the toxicology of the chemical exposure must be addressed. Although temporality may be apparent (toxicity followed exposure), the time frame for the appearance of symptoms must be appropriate to the exposure conditions and chemical(s) involved. The chemical(s) must have known or reasonably inferred properties to cause the claimed effect. The dose(s) received must be adequate to effect a response (dose-response characteristics). Alternate causation must be evaluated thoroughly, that is, are there other more logical explanations for the symptoms? In contrast to single-chemical exposures, exposure to mixtures of chemicals, as commonly found in sick buildings, is very difficult to evaluate. However, a rational, scientific approach to cause-and-effect questions will help avoid erroneous and subjective decision making.

Ozone can cause pulmonary edema and simultaneously decrease blood pressure. Atrial natriuretic factor (ANF) may mediate both of these effects in that it increases pulmonary capillary permeability resulting in edema formation and vasodilates the vascular system. The present investigation was designed to test directly the effect of ozone on the content of ANF in the lungs, heart, and the circulatory system. Ozone (0.5 ppm) exposure for 8 h increased ANF content in the lungs fourfold (13.33 +/- 2.7 ng/g tissue) in six Fischer 344 rats compared with ANF content (3.03 +/- 0.2 ng/g tissue) in six Fischer 344 rats exposed only to room air. Ozone increased ANF content in the heart fivefold in the same animals from 288 +/- 39.6 ng/g tissue in the rats exposed to room air to 1,473 +/- 234.8 ng/g tissue in those exposed to ozone. Ozone also doubled the concentration of ANF in the circulatory system. This study demonstrates that ozone does increase ANF in the lungs, heart, and circulatory system, suggesting that ANF may mediate the decreased blood pressure and pulmonary edema observed with ozone exposure.

Ozone can cause pulmonary edema and simultaneously decrease blood pressure. Atrial natriuretic peptides may mediate both of these effects in that they increase pulmonary capillary permeability resulting in edema formation and are potent vasodilating peptides. To examine this possibility, the lungs of Fischer 344 rats were exposed to ozone (0.5 ppm) for 8 hours which resulted in a three- to fourfold increase in atrial natriuretic peptides. Ozone also increased atrial natriuretic peptides in the heart two- to fivefold from 266 +/- 25, 226 +/- 22, and 288 +/- 40 ng/g (room air) to 716 +/- 26, 471 +/- 14, and 1473 +/- 235 ng/g recognized by the proANFs 1-30 and 31-67 and atrial natriuretic factor radioimmunoassays, respectively. Ozone also doubled the concentrations of pro-ANFs 1-30, 31-67, and 1-98 and ANF in the circulation. This study demonstrates that ozone increases atrial natriuretic peptides within the heart, lung, and circulation, suggesting that atrial natriuretic peptides may mediate the decreased blood pressure and pulmonary edema observed with ozone exposure. Since the proANF 31-67 radioimmunoassay exclusively recognizes the ANF prohormone within the heart, this study further indicates that ozone can increase the synthesis of the ANF prohormone.

Ozone can cause pulmonary edema and simultaneously decrease blood pressure. Atrial natriuretic peptides may mediate both of these affects since they increase pulmonary capillary permeability and are potent vasodilating peptides. To examine this possibility and determine if aged animals respond differently to ozone, adult (4-6 months old) and aged (24-26 months old) Fischer 344 rats were exposed to ozone (0.5 parts per million) for 8 h. Ozone increased atrial natriuretic peptides recognized by the proANF 1-30, proANF 31-67, and atrial natriuretic factor (ANF) radioimmunoassays in lung from 3.7 +/- 0.2, 3.5 +/- 0.1, and 3.0 +/- 0.2 ng/g of lung, respectively, to 9.7 +/- 1.0, 9.2 +/- 0.4, and 13.3 +/- 2.7 ng/g in adult rats (n = 6) and from 3.6 +/- 0.1, 3.5 +/- 0.1, and 3.2 +/- 0.1 ng/g of lung of aged rats (n = 6) to 10.2 +/- 0.3, 10.0 +/- 0.2, and 12.6 +/- 0.2 ng/g. Ozone increased the content of these peptides in the heart 2- to 5-fold from 266 +/- 25, 226 +/- 22, and 288 +/- 40 ng/g of heart to 716 +/- 26, 471 +/- 14, and 1,473 +/- 235 ng/g in the same adult animals and from 495 +/- 17, 483 +/- 22, and 501 +/- 18 ng/g to 903 +/- 16,879 +/- 21, and 1,489 +/- 31 ng/g of heart in the aged animals. Ozone also doubled the concentration of these atrial natriuretic peptides in the circulatory system. This study demonstrates that ozone increases atrial natriuretic peptides in the lung, heart, and circulation of equal magnitude in aged versus adult animals, indicating an equal response to ozone with aging. This study further suggests that atrial natriuretic peptides may mediate the decreased blood pressure and pulmonary edema observed with ozone exposure.

Atrial natriuretic factor (ANF) has been found to be increased in the circulation of aged rats. The present investigation was designed to determine if the N-terminal ANF prohormone peptides with blood pressure lowering properties are increased in the circulation of "aged" (24-26 months) versus "adult" (4-6 month old) Fisher 344 rats. We utilized two sensitive and specific radioimmunoassays that immunologically recognize the 98 amino acid (a.a.) N-terminus (proANF 1-98) and a.a. 31-67 (proANF 31-67, Vessel dilator) from the midportion of the N-terminus of the 126 a.a. ANF prohormone. ProANF 1-98, and proANF 31-67, as well as ANF, were found to be elevated in the circulation of aged versus adult animals with proANF 31-67 and ANF elevations being significant at p < 0.05. To determine the potential source of the increased atrial natriuretic peptides in aged animals four tissue sources (liver, lung, heart, and intestine) from both adult and aged animals were examined. Hearts of aged animals contained significantly (P < 0.05; ANOVA) more proANF 1-98, proANF 31-67, and ANF than hearts of adult animals. There was no significant difference in the concentration of proANF 1-98, proANF 31-67, or ANF in the lung, liver, or intestine of the aged versus the adult animals. These results indicate that aged animals contained increased N-terminal and C-terminal ANF prohormone peptides in the circulation. The source of these increased peptides appears to be the heart.

As part of a study on the effects of acute ozone stress on the lung surfactant system, we correlated morphometric, biochemical, and functional indices of lung injury using male rats exposed to 3 ppm ozone for 1, 2, 4, and 8 hr. Evaluation of lung mechanics, using the Pulmonary Evaluation and Diagnostic Laboratory System, revealed a significant decrease in dynamic lung compliance (ml/cmH2O/kg) from a control value of 0.84 +/- 0.02 (SEM) to 0.72 +/- 0.04 and 0.57 +/- 0.06 at 4 and 8 hr, respectively. At 2 hr there was a transient increase in PaO2 to 116 torr (control = 92 torr) followed by a decrease at 4 hr (65 torr) and 8 hr (55 torr). Morphometry of lung tissue, fixed by perfusion of fixative via the pulmonary artery at 12 cm H2O airway distending pressure, demonstrated an increase in the area of the intravascular compartment at 8 hr, in association with a 65 and 39% replacement of the alveolar area by fluid in ventral and dorsal lung regions, respectively. There was a positive correlation (r = 0.966) between alveolar edema and transudated proteins in lavage fluid. A stepwise multiple regression model, with edema as the dependent variable, suggested that pulmonary vasodilatation, hypoxemia, and depletion of surfactant tubular myelin in lavage fluid were indices for predicting alveolar edema. In a second model, with lavage protein concentration as the dependent variable, decreasing dynamic compliance and hypoxemia were predictors of progressive, intraalveolar transudation of plasma proteins. The above structural-functional relationships support the concept that ozone-induced high-protein alveolar edema is pathogenetically linked to pulmonary hyperemia, deficiency of surfactant tubular myelin, and associated lung dysfunctions.

Using immunogold labeling of fixed, cryosubstituted tissue sections, we compared the distribution of lysozyme, an oxidant-sensitive lamellar body protein, with that of surfactant protein A (SP-A) in rat Type II cells, extracellular surfactant forms, and alveolar macrophages. Morphometric analysis of gold particle distribution revealed that lysozyme and SP-A were present throughout the secretory and endosomal pathways of Type II cells, with prominent localization of lysozyme in the peripheral compartment of lamellar bodies. All extracellular surfactant forms were labeled for both proteins with preferential labeling of tubular myelin and unilamellar vesicles. Labeling of tubular myelin for SP-A was striking when compared with that of lamellar bodies and other extracellular surfactant forms. Lamellar body-like forms and multilamellar structures were uniformly labeled for lysozyme, suggesting that this protein is rapidly redistributed within these forms after secretion of lysozyme-laden lamellar bodies. By contrast, increased labeling for SP-A was observed over peripheral membranes of lamellar body-like forms and multilamellar structures, apparently reflecting progressive SP-A enrichment of these membranes during tubular myelin formation. The results indicate that lysozyme is an integral component of the lamellar body peripheral compartment and secreted surfactant membranes, and support the concept that lysozyme may participate in the structural organization of lung surfactant.

Retrograde extrapolation is a mathematical process, based on sound scientific principles, that is used routinely in pharmacology, toxicology, and clinical medicine. This process may be applied to the situation of ethyl alcohol consumption with reliability when reasonable assumptions are made concerning absorption rates, elimination rates, and patterns of alcohol consumption, including drinking duration and volume consumed. By utilizing an established range of values for the elimination rate of alcohol of 0.015-0.020 g/dl/h, a relatively narrow range of extrapolated blood alcohol concentrations (BACs) can be determined in situations where the time frame in question is after peak alcohol absorption into the blood. A wider range of elimination rates of 0.01-0.03 g/dl/h may be applied and will satisfy the possibility of nonlinear kinetics within an individual; however, this wider range will have little practical effect on the predicted BACs. When the time point in question is prior to peak absorption, a wider range of predicted BAC values will result. The extent of this range will be influenced by the amount of information available concerning the temporal pattern of alcohol consumption. Reported drinking volumes are notoriously inaccurate and, in fact, are of little practical use. Given the parameters of body weight and time duration between initiation of drinking and determination of the BAC, the number of "drinks" consumed may be reliability calculated. Retrograde extrapolation is applicable in the forensic setting with scientific reliability when reasonable and justifiable assumptions are utilized.

Male, pathogen-free Fischer 344 rats aged 6 and 24 mo were exposed to 1.5 or 3.0 ppm for 8 h and recovery rates of diphosphonucleotides (NAD+ and NADH) and triphosphonucleotides (NADP+ and NADPH) were measured and compared to controls. Recovery after 0.5 ppm was not examined because no significant changes occurred in either age group after this lower exposure. At zero time (immediately after exposures) both concentrations are depressed in adults and aged animals except for NADH in aged animals at 3.0 ppm; NADP+ in adults at 1.5 and 3.0 ppm was decreased, but not significantly. For NAD+ and NADH, recovery of whole lung concentrations is complete by 24 h following an 8-h exposure to 1.5 or 3.0 ppm of ozone. Only after 3.0 ppm of ozone was the ratio of the reduced to oxidized form (NADH/NAD+) still elevated after 24 h; however, it also returned to control levels by 96 h. For the triphosphonucleotides, an 8-h exposure to 1.5 ppm of ozone resulted in a sustained depression of whole lung concentrations of NADPH throughout the 96-h recovery period. Also, only after the 1.5 ppm exposure was the reduced to oxidized ratio (NADPH/NADP+) significantly depressed throughout the 96-h recovery period. Unexpectedly, recovery of whole lung levels returned to normal within 24 h after the 8-h exposure to both the 1.5 and the 3.0 ppm concentrations. With the exception of the sustained effect on NADPH levels, these data indicate that di- and triphosphonucleotide concentrations rapidly return to normal in the lung after severe, acute oxidant injury. There were no differences in recovery rates between the adult and the aged groups.

To identify the early changes of surfactant secretion in response to acute oxidant stress, the authors evaluated morphometrically centriacinar type II cells and lavage fluid surfactant forms obtained immediately after exposure of adult rats to 3 ppm ozone for 1, 2, 4, or 8 hours. In this model, the rat lung develops progressive alveolar edema with significant elevation of lavage fluid proteins at 2 to 8 hours of exposure. Ultrastructural changes in type II cells at 1 and 2 hours included enhanced lamellar body (LB) fusion with significant increase in the compound and vacuolated LB compartments. Parallel changes of lavage fluid surfactant membranes included a sustained, twofold increase in the proportion of loosely coiled multilamellar structures at 1 to 8 hours, with reciprocal decrease in the proportion of tubular myelin from control value of 56% to 34%. The proportion of densely coiled LB-like forms in lavage fluid increased significantly at 4 and 8 hours, whereas the proportions of unilamellar structures remained unchanged. The results indicate that ozone-induced alveolar injury initiates time-dependent defects in the organization of stored and secreted surfactant membranes. The acute ozone stress inhibits unfolding of secreted lamellar body membranes as well as their organization into tubular myelin, thereby perturbing the proportions of extracellular surfactant membranes that are available for adsorption onto the surface film.

1. During investigation of microsomal xenobiotic N-demethylation in the presence of ascorbic acid, large increases in apparent enzymic activity were observed. 2. Examination of incubation components indicated that a non-enzymic interaction between ascorbic acid and Tris buffer, in the presence of acetylacetone and ammonium acetate (Nash reagent), was occurring. 3. The resulting chromophore had an absorption maximum at 412 nm that coincided with the absorption for the chromophore resulting from the interaction of the Nash reagent with the product (formaldehyde) of the enzymic reaction. 4. Strict controls of ascorbic acid potential chemical interaction with incubation components are required in enzymic studies.

Displacement of spin-labeled drugs from drug-specific antibodies in human serum can be measured by use of electron spin resonance. This "spin immunoassay" has advantages of speed, simplicity, and small volume requirements over other standard techniques such as gas-liquid, liquid-liquid, or thin-layer chromatography. Unlike radioimmunoassay, there is no need to separate free and bound label and there are no radiation hazards. We present clinical and experimental data showing how this method can be applied to the routine determination of morphine and diphenylhydantoin concentrations in serum.

Guinea pigs were subjected to dietary control of ascorbic acid intake to produce deficient, normal, and supplemented tissue ascorbate concentrations. Paraquat, a potent pulmonary toxin, was then administered. Tissue distribution and covalent binding of paraquat were not affected significantly by ascorbate nutritional status except for decreased 24-hr kidney burdens in deficient animals. Paraquat increased pulmonary thiobarbituric acid reactivity and mixed disulfide formation, but no evidence of either a protective or a potentiative interaction of endogenous ascorbate and paraquat was observed. This study indicates that alteration of endogenous ascorbic acid levels does not alter the biochemical response to paraquat.

Monoamine oxidase (MAO) activity was determined in rat tissues following in vivo treatments with chlorphentermine (CP). Oxidation of seven amine substrates by liver, lung or brain mitochondrial MAO was investigated at 2 h after a single i.p. injection (60 mg/kg) or after repeated injection (once daily for 3 days, 20 mg/kg, i.p.). Deamination of the type A substrates, norepinephrine, serotonin and octopamine, was decreased significantly in liver, lung and brain after both single and repeated injections. Oxidative deamination of tyramine and dopamine (type A + B substrates) was also lowered in all organs after single and repeated exposure to CP, but to a lesser degree than the type A substrates. However, oxidation of the type B substrates, benzylamine and tryptamine, was unaffected by CP administration in comparison to control. These data indicate that CP is a specific inhibitor of mitochondrial MAO, form A.