Ecto-CD38-NADase inhibition modulates cardiac metabolism and protects mice against doxorubicin-induced cardiotoxicity.

AIMS: Doxorubicin (DXR) is a chemotherapeutic agent that causes dose-dependent cardiotoxicity. Recently, it has been proposed that the NADase CD38 may play a role in doxorubicin-induced cardiotoxicity (DIC). CD38 is the main NAD+-catabolizing enzyme in mammalian tissues. Interestingly, in the heart, CD38 is mostly expressed as an ecto-enzyme that can be targeted by specific inhibitory antibodies. The goal of the present study is to characterize the role of CD38 ecto-enzymatic activity in cardiac metabolism and the development of DIC. METHODS AND RESULTS: Using both a transgenic animal model and a non-cytotoxic enzymatic anti-CD38 antibody, we investigated the role of CD38 and its ecto-NADase activity in DIC in pre-clinical models. First, we observed that DIC was prevented in the CD38 catalytically inactive (CD38-CI) transgenic mice. Both left ventricular systolic function and exercise capacity were decreased in wild-type but not in CD38-CI mice treated with DXR. Second, blocking CD38-NADase activity with the specific antibody 68 (Ab68) likewise protected mice against DIC and decreased DXR-related mortality by 50%. A reduction of DXR-induced mitochondrial dysfunction, energy deficiency, and inflammation gene expression were identified as the main mechanisms mediating the protective effects. CONCLUSION: NAD+-preserving strategies by inactivation of CD38 via a genetic or a pharmacological-based approach improve cardiac energetics and reduce cardiac inflammation and dysfunction otherwise seen in an acute DXR cardiotoxicity model.

Long-range repression by ecdysone receptor on complex enhancers of the insulin receptor gene.

The insulin signalling pathway is evolutionarily conserved throughout metazoans, playing key roles in development, growth, and metabolism. Misregulation of this pathway is associated with a multitude of disease states including diabetes, cancer, and neurodegeneration. The human insulin receptor gene (INSR) is widely expressed throughout development and was previously described as a 'housekeeping' gene. Yet, there is abundant evidence that this gene is expressed in a cell-type specific manner, with dynamic regulation in response to environmental signals. The Drosophila insulin-like receptor gene (InR) is homologous to the human INSR gene and was previously shown to be regulated by multiple transcriptional elements located primarily within the introns of the gene. These elements were roughly defined in ~1.5 kbp segments, but we lack an understanding of the potential detailed mechanisms of their regulation. We characterized the substructure of these cis-regulatory elements in Drosophila S2 cells, focusing on regulation through the ecdysone receptor (EcR) and the dFOXO transcription factor. By identifying specific locations of activators and repressors within 300 bp subelements, we show that some previously identified enhancers consist of relatively compact clusters of activators, while others have a distributed architecture not amenable to further reduction. In addition, these assays uncovered a long-range repressive action of unliganded EcR. The complex transcriptional circuitry likely endows InR with a highly flexible and tissue-specific response to tune insulin signalling. Further studies will provide insights to demonstrate the impact of natural variation in this gene's regulation, applicable to human genetic studies.

Increasing capacity by moving away from one-to-one clinical supervision: using peer-assisted learning and a group model of student placements in community paediatric speech and language therapy to enable student-led service delivery.

BACKGROUND: Health Education England (HEE) and the Royal College of Speech and Language Therapists (RCSLT) have identified the need to increase placement capacity. Speech and language therapy is a shortage profession in the UK, so services need to consider innovative placement models to increase their placement offers without increasing the time burden on speech and language therapists (SLTs). AIMS: To increase capacity for pre-registration practice-based learning by using peer-assisted learning (PAL) in a group model of student placement to enable student-led service delivery which provides high standards of clinical care and student experience and is an efficient use of SLT time. METHODS & PROCEDURES: A paediatric speech and language therapy service hosted eight student speech and language therapists (SSLTs) for their final pre-registration placement. SSLTs completed pre- and post-placement confidence ratings for a range of clinical skills; SSLTs and SLTs rated how useful different types of support were, and education settings provided feedback about working with the SSLTs. The number of clinical sessions completed by the SSLTs and the percentage of outcomes achieved for children with speech, language and communication needs were calculated. SLTs completed time-trackers for placement-related activities. OUTCOMES & RESULTS: The impact of the placements was assessed using a tri-vector methodology consisting of: self-reporting by the student (using an evaluation form), feedback from the placement sites (schools) and analysis of targets set for individual children. SSLTs reported increased confidence in all clinical areas in their post-placement evaluation form. SLTs reported increased confidence in SSLTs working independently and an increase in the perceived benefit to the service for having SSLTs in comparison with the time invested in supporting SSLTs. SSLTs and SLTs found all types of support provided during the placement useful. Schools reported high levels of satisfaction for working with SSLTs. SSLTs completed more clinical sessions than an SLT would have been able to in the time SLTs invested in placement-related activities. Children achieved 60% of the targets set by SSLTs. CONCLUSIONS & IMPLICATIONS: This placement model increased the capacity for SSLT placements by using PAL in a group model of student placement to enable student-led service delivery. The model provided high standards of clinical care and student experience and was an efficient use of SLT time. Wider use of this placement model would increase placement capacity and could also address vacancies in services. WHAT THIS PAPER ADDS: What is already known on the subject SSLTs and SLTs are positive about the benefits of paired placements in comparison with individual placements. Other allied health professions have demonstrated that larger placements can be an effective way to support students and have used students to deliver student-led services. What this study adds to existing knowledge This paper is the first to look at whether PAL in a group model of student placements can be used in speech and language therapy to enable student-led service delivery which provides high standards of clinical care, maintains high standards of student experience and is an efficient use of SLT time. What are the potential or actual clinical implications of this work? This paper demonstrates that PAL can be used effectively in a group model of student placements in a paediatric SLT service to increase student capacity and enable student-led service delivery. The proposed placement model provides a high-quality placement for SSLTs and the children they work with, and is also an efficient use of SLT time.

A nitroalkene derivative of salicylate alleviates diet-induced obesity by activating creatine metabolism and non-shivering thermogenesis.

Obesity-related type II diabetes (diabesity) has increased global morbidity and mortality dramatically. Previously, the ancient drug salicylate demonstrated promise for the treatment of type II diabetes, but its clinical use was precluded due to high dose requirements. In this study, we present a nitroalkene derivative of salicylate, 5-(2-nitroethenyl)salicylic acid (SANA), a molecule with unprecedented beneficial effects in diet-induced obesity (DIO). SANA reduces DIO, liver steatosis and insulin resistance at doses up to 40 times lower than salicylate. Mechanistically, SANA stimulated mitochondrial respiration and increased creatine-dependent energy expenditure in adipose tissue. Indeed, depletion of creatine resulted in the loss of SANA action. Moreover, we found that SANA binds to creatine kinases CKMT1/2, and downregulation CKMT1 interferes with the effect of SANA in vivo. Together, these data demonstrate that SANA is a first-in-class activator of creatine-dependent energy expenditure and thermogenesis in adipose tissue and emerges as a candidate for the treatment of diabesity.

Long-range repression by ecdysone receptor on complex enhancers of the insulin receptor gene.

The insulin signaling pathway is evolutionarily conserved throughout metazoans, playing key roles in development, growth, and metabolism. Misregulation of this pathway is associated with a multitude of disease states including diabetes, cancer, and neurodegeneration. Genome-wide association studies indicate that natural variants in putative intronic regulatory elements of the human insulin receptor gene ( INSR) are associated with metabolic conditions, however, this gene's transcriptional regulation remains incompletely studied. INSR is widely expressed throughout development and was previously described as a 'housekeeping' gene. Yet, there is abundant evidence that this gene is expressed in a cell-type specific manner, with dynamic regulation in response to environmental signals. The Drosophila insulin-like receptor gene ( InR ) is homologous to the human INSR gene and was previously shown to be regulated by multiple transcriptional elements located primarily within the introns of the gene. These elements were roughly defined in ∼1.5 kbp segments, but we lack an understanding of the potential detailed mechanisms of their regulation, as well as the integrative output of the battery of enhancers in the entire locus. Using luciferase assays, we characterized the substructure of these cis-regulatory elements in Drosophila S2 cells, focusing on regulation through the ecdysone receptor (EcR) and the dFOXO transcription factor. The direct action of EcR on Enhancer 2 reveals a bimodal form of regulation, with active repression in the absence of the ligand, and positive activation in the presence of 20E. By identifying the location of activators of this enhancer, we characterized a long-range of repression acting over at least 475 bp, similar to the action of long-range repressors found in the embryo. dFOXO and 20E have contrasting effects on some of the individual regulatory elements, and for the adjacent enhancers 2 and 3, their influence was/was not found to be additive, indicating that enhancer action on this locus can/cannot be characterized in part by additive models. Other characterized enhancers from within this locus exhibited "distributed" or "localized" modes of action, suggesting that predicting the joint functional output of multiple regulatory regions will require a deeper experimental characterization. The noncoding intronic regions of InR have demonstrated dynamic regulation of expression and cell type specificity. This complex transcriptional circuitry goes beyond the simple conception of a 'housekeeping' gene. Further studies are aimed at identifying how these elements work together in vivo to generate finely tuned expression in tissue- and temporal-specific manners, to provide a guide to understanding the impact of natural variation in this gene's regulation, applicable to human genetic studies.

Endogenous metabolism in endothelial and immune cells generates most of the tissue vitamin B3 (nicotinamide).

In mammals, nicotinamide (NAM) is the primary NAD precursor available in circulation, a signaling molecule, and a precursor for methyl-nicotinamide (M-NAM) synthesis. However, our knowledge about how the body regulates tissue NAM levels is still limited. Here we demonstrate that dietary vitamin B3 partially regulates plasma NAM and NAM-derived metabolites, but not their tissue levels. We found that NAD de novo synthesis from tryptophan contributes to plasma and tissue NAM, likely by providing substrates for NAD-degrading enzymes. We also demonstrate that tissue NAM is mainly generated by endogenous metabolism and that the NADase CD38 is the main enzyme that produces tissue NAM. Tissue-specific CD38-floxed mice revealed that CD38 activity on endothelial and immune cells is the major contributor to tissue steady-state levels of NAM in tissues like spleen and heart. Our findings uncover the presence of different pools of NAM in the body and a central role for CD38 in regulating tissue NAM levels.

Low NAD+ Levels Are Associated With a Decline of Spermatogenesis in Transgenic ANDY and Aging Mice.

Advanced paternal age has increasingly been recognized as a risk factor for male fertility and progeny health. While underlying causes are not well understood, aging is associated with a continuous decline of blood and tissue NAD+ levels, as well as a decline of testicular functions. The important basic question to what extent ageing-related NAD+ decline is functionally linked to decreased male fertility has been difficult to address due to the pleiotropic effects of aging, and the lack of a suitable animal model in which NAD+ levels can be lowered experimentally in chronologically young adult males. We therefore developed a transgenic mouse model of acquired niacin dependency (ANDY), in which NAD+ levels can be experimentally lowered using a niacin-deficient, chemically defined diet. Using ANDY mice, this report demonstrates for the first time that decreasing body-wide NAD+ levels in young adult mice, including in the testes, to levels that match or exceed the natural NAD+ decline observed in old mice, results in the disruption of spermatogenesis with small testis sizes and reduced sperm counts. ANDY mice are dependent on dietary vitamin B3 (niacin) for NAD+ synthesis, similar to humans. NAD+-deficiency the animals develop on a niacin-free diet is reversed by niacin supplementation. Providing niacin to NAD+-depleted ANDY mice fully rescued spermatogenesis and restored normal testis weight in the animals. The results suggest that NAD+ is important for proper spermatogenesis and that its declining levels during aging are functionally linked to declining spermatogenesis and male fertility. Functions of NAD+ in retinoic acid synthesis, which is an essential testicular signaling pathway regulating spermatogonial proliferation and differentiation, may offer a plausible mechanism for the hypospermatogenesis observed in NAD+-deficient mice.

CD38 inhibitor 78c increases mice lifespan and healthspan in a model of chronological aging.

Nicotinamide adenine dinucleotide (NAD) levels decline during aging, contributing to physical and metabolic dysfunction. The NADase CD38 plays a key role in age-related NAD decline. Whether the inhibition of CD38 increases lifespan is not known. Here, we show that the CD38 inhibitor 78c increases lifespan and healthspan of naturally aged mice. In addition to a 10% increase in median survival, 78c improved exercise performance, endurance, and metabolic function in mice. The effects of 78c were different between sexes. Our study is the first to investigate the effect of CD38 inhibition in naturally aged animals.

The CD38 glycohydrolase and the NAD sink: implications for pathological conditions.

Nicotinamide adenine dinucleotide (NAD) acts as a cofactor in several oxidation-reduction (redox) reactions and is a substrate for a number of nonredox enzymes. NAD is fundamental to a variety of cellular processes including energy metabolism, cell signaling, and epigenetics. NAD homeostasis appears to be of paramount importance to health span and longevity, and its dysregulation is associated with multiple diseases. NAD metabolism is dynamic and maintained by synthesis and degradation. The enzyme CD38, one of the main NAD-consuming enzymes, is a key component of NAD homeostasis. The majority of CD38 is localized in the plasma membrane with its catalytic domain facing the extracellular environment, likely for the purpose of controlling systemic levels of NAD. Several cell types express CD38, but its expression predominates on endothelial cells and immune cells capable of infiltrating organs and tissues. Here we review potential roles of CD38 in health and disease and postulate ways in which CD38 dysregulation causes changes in NAD homeostasis and contributes to the pathophysiology of multiple conditions. Indeed, in animal models the development of infectious diseases, autoimmune disorders, fibrosis, metabolic diseases, and age-associated diseases including cancer, heart disease, and neurodegeneration are associated with altered CD38 enzymatic activity. Many of these conditions are modified in CD38-deficient mice or by blocking CD38 NADase activity. In diseases in which CD38 appears to play a role, CD38-dependent NAD decline is often a common denominator of pathophysiology. Thus, understanding dysregulation of NAD homeostasis by CD38 may open new avenues for the treatment of human diseases.

Alcohol Craving and Psychiatric Disorders Among Current Drinkers.

BACKGROUND AND OBJECTIVES: Previous research has shown that alcohol craving is associated with psychiatric comorbidities. However, no population studies have examined the odds of psychiatric disorders in cravers and noncravers. The purpose of this study was to investigate current prevalence rates and odds ratios of psychiatric disorders among alcohol drinkers with and without alcohol craving in a population-based sample. We also compared four craving groups (cravers with and without alcohol use disorder [AUD], noncravers with and without AUD) for psychiatric comorbidities. METHODS: The study data were drawn from the National Epidemiologic Survey on Alcohol and Related Conditions (NESARC). A subset of the NESARC sample (N = 22 000) who reported alcohol use during the past 12 months was included. Prevalence rates of psychiatric disorders were compared among current drinkers with alcohol craving (N = 900) and without alcohol craving (N = 21 500). RESULTS: Cravers had higher prevalence rates of current psychiatric disorders than noncravers. Even after adjustment for other psychiatric disorders including AUD, cravers had significantly higher odds of any substance use disorder (adjusted odds ratio [AOR], 9.01), any mood disorder (AOR, 1.78), any anxiety disorder (AOR, 1.86), and any personality disorder (AOR, 1.92) than noncravers. Interestingly, cravers without AUD had even higher rates of any anxiety disorder and any personality disorder than noncravers with AUD. CONCLUSIONS AND SCIENTIFIC SIGNIFICANCE: Alcohol craving is associated with a higher prevalence of various psychiatric disorders. These findings suggest that alcohol craving may be related to transdiagnostic features that are present across various psychiatric disorders. (Am J Addict 2021;30:34-42).

A Theory of Planned Behavior Scale for Adherence to Trauma-Focused Posttraumatic Stress Disorder Treatments.

Evidence-based psychotherapies for posttraumatic stress disorder (PTSD), such as cognitive processing therapy and prolonged exposure (CPT/PE), greatly reduce suffering for veterans, but many veterans fail to complete treatment. Developing a theory-based understanding of adherence is necessary to inform interventions to improve treatment retention. We developed and tested a series of scales applying the theory of planned behavior (TPB) to CPT/PE adherence. The scales were administered in mailed surveys as part of a larger mixed-methods study of veteran adherence to PE/CPT. Surveys were sent to 379 veterans who were initiating CPT/PE across four U.S. Veterans Affairs (VA) hospitals and 207 of their loved ones. Subsequent session attendance and homework compliance were coded via a review of electronic medical records. We examined item-level characteristics, factor structure, and the convergent and discriminant validity of the resultant scales. The findings support four subscales: two related to attitudes (i.e., Treatment Makes Sense and Treatment Fits Needs), one related to perceived behavioral control over participation (i.e., Participation Control), and one related to perceived family attitudes about CPT/PE participation (i.e., Subjective Norms). Scale validity was supported through significant associations with theoretically relevant constructs, including intentions to persist in CPT/PE, rs = .19-.38; treatment completion, rs = .21-.25; practical treatment barriers, rs = -.19 to -.24; and therapeutic alliance, rs = .39-.57.

CD38 ecto-enzyme in immune cells is induced during aging and regulates NAD+ and NMN levels.

Decreased NAD+ levels have been shown to contribute to metabolic dysfunction during aging. NAD+ decline can be partially prevented by knockout of the enzyme CD38. However, it is not known how CD38 is regulated during aging, and how its ecto-enzymatic activity impacts NAD+ homeostasis. Here we show that an increase in CD38 in white adipose tissue (WAT) and the liver during aging is mediated by accumulation of CD38+ immune cells. Inflammation increases CD38 and decreases NAD+. In addition, senescent cells and their secreted signals promote accumulation of CD38+ cells in WAT, and ablation of senescent cells or their secretory phenotype decreases CD38, partially reversing NAD+ decline. Finally, blocking the ecto-enzymatic activity of CD38 can increase NAD+ through a nicotinamide mononucleotide (NMN)-dependent process. Our findings demonstrate that senescence-induced inflammation promotes accumulation of CD38 in immune cells that, through its ecto-enzymatic activity, decreases levels of NMN and NAD+.

Differential Contributions of DNA-Binding Proteins to Polycomb Response Element Activity at the Drosophila giant Gene.

Polycomb-group (PcG) proteins are evolutionarily conserved epigenetic regulators whose primary function is to maintain the transcriptional repression of target genes. Recruitment of Drosophila melanogaster PcG proteins to target genes requires the presence of one or more Polycomb Response Elements (PREs). The functions or necessity for more than one PRE at a gene are not clear and individual PREs at some loci may have distinct regulatory roles. Various combinations of sequence-specific DNA-binding proteins are present at a given PRE, but only Pleiohomeotic (Pho) is present at all strong PREs. The giant (gt) locus has two PREs, a proximal PRE1 and a distal PRE2. During early embryonic development, Pho binds to PRE1 ∼30-min prior to stable binding to PRE2. This observation indicated a possible dependence of PRE2 on PRE1 for PcG recruitment; however, we find here that PRE2 recruits PcG proteins and maintains transcriptional repression independently of Pho binding to PRE1. Pho-like (Phol) is partially redundant with Pho during larval development and binds to the same DNA sequences in vitro Although binding of Pho to PRE1 is dependent on the presence of consensus Pho-Phol-binding sites, Phol binding is less so and appears to play a minimal role in recruiting other PcG proteins to gt Another PRE-binding protein, Sp1/Kruppel-like factor, is dependent on the presence of Pho for PRE1 binding. Further, we show that, in addition to silencing gene expression, PcG proteins dampen transcription of an active gene.

Association of poorer dietary quality and higher dietary inflammation with greater symptom severity in depressed individuals with appetite loss.

BACKGROUND: Major depressive disorder (MDD) is the leading cause of years lived with disability; however, little is known about its etiology to inform treatment. For a subset of MDD patients, appetite change and/or bodily inflammation may play a role in exacerbating symptoms. The goal of this study is to examine whether, relative to healthy comparisons (HC), MDD individuals with increased versus decreased appetite symptoms show a differential relationship between diet quality and inflammation. METHODS: Unmedicated current MDD (n = 61) varying in appetite change (decrease (MDD-DE): n = 39; increase (MDD-IN): n = 22) and HC (n = 42) completed 24-hour dietary recall and state depression/anxiety measures. Healthy eating and dietary inflammatory indices were calculated from dietary reports. Blood samples measured five inflammation-related biomarkers. Analyses investigated between- and within-group differences in the Healthy Eating Index (HEI), the Dietary Inflammatory Index (DII), inflammation-related blood biomarkers, and symptom severity. RESULTS: While both MDD-DE and MDD-IN exhibited lower HEI scores than HC, only MDD-IN showed higher plasma interleukin-1 receptor antagonist (IL-1RA) and interleukin-6 (IL-6) levels than HC. In contrast, MDD-DE exhibited higher DII scores than MDD-IN and HC. Within MDD-DE, greater symptom severity was associated with lower HEI and higher DII. LIMITATIONS: Modest sample sizes and the cross-sectional study design limited power to detect within-MDD effects. CONCLUSIONS: Although MDD, regardless of appetite change, is linked to poorer dietary quality, depression severity was related to dietary characteristics only in subjects who reported appetite loss. Thus, increasing the quality of dietary intake could be a treatment target for some individuals with depression.

A Simple Intervention to Increase Human Papillomavirus Vaccination in a Family Medicine Practice.

Human papillomavirus (HPV) is a sexually transmitted virus that is the leading cause of cervical cancer world- wide. It is vaccine-preventable. According to the Centers for Disease Control, only 60 percent of girls have started the HPV vaccination series countrywide and only 50 percent of boys have started. South Dakota is below this national average. In an effort to assess - and improve - HPV vaccination rates in our practice a quality improvement effort was undertaken. Two interventions were implemented a month apart: the first was a mailing to the parent(s) of all patients 11-12 years of age during the time period of the intervention; the second was an in-office reminder system for both patients and physicians at the time of an office encounter. After each of the interventions, the immunization rate for one injection was significantly greater than baseline; while slightly higher than baseline, that for those receiving both injections was not statistically different for either intervention.

Methods of Symptomatic Relief of Teething in Infants and Young Children Recommended by South Dakota Physicians.

Varieties of different methods for managing the symptoms of teething are available, and are of variable efficacy and safety. This study surveyed family physicians and pediatricians in the state of South Dakota to find out what they are recommending in their current clinical practices for the relief of symptoms associated with tooth eruption. The most common recommendation given by primary care physicians was acetaminophen or ibuprofen (80 percent). The next most common recommendation was the use of teething rings, both rubber (59 percent) and cold or frozen rings (67 percent). Nearly 20 percent recommended modalities that professional organizations and governmental agencies advise against using due to the potential for significant harm.

Including People with Dementia in Research: An Analysis of Australian Ethical and Legal Rules and Recommendations for Reform.

Research is crucial to advancing knowledge about dementia, yet the burden of the disease currently outpaces research activity. Research often excludes people with dementia and other cognitive impairments because researchers and ethics committees are concerned about issues related to capacity, consent, and substitute decision-making. In Australia, participation in research by people with cognitive impairment is governed by a national ethics statement and a patchwork of state and territorial laws that have widely varying rules. We contend that this legislative variation precludes a consistent approach to research governance and participation and hinders research that seeks to include people with impaired capacity. In this paper, we present key ethical principles, provide a comprehensive review of applicable legal rules in Australian states and territories, and highlight significant differences and ambiguities. Our analysis includes recommendations for reform to improve clarity and consistency in the law and reduce barriers that may exclude persons with dementia from participating in ethically approved research. Our recommendations seek to advance the national decision-making principles recommended by the Australian Law Reform Commission, which emphasize the rights of all adults to make their own decisions and for those with impaired capacity to have access to appropriate supports to help them make decisions that affect their lives.

Health Beliefs and Perceptions of Trachoma in Communities on the Bijagos Archipelago of Guinea Bissau.

PURPOSE: The World Health Organization aims to eliminate blinding trachoma by 2020 using the SAFE strategy: Surgery for trichiasis, Antibiotics, Facial cleanliness and Environmental improvement. Trachoma is hyperendemic on the remote Bijagos Archipelago of Guinea-Bissau, West Africa. Sociocultural factors remain unexplored here, despite their potential impact on disease control, particularly through the "F" and "E" aspects. By examining these, we aim to illuminate this population's unreported health beliefs, hygiene behaviors and disease perceptions. This understanding will help to optimize future public health interventions, and guide the distribution of limited healthcare resources. METHODS: Two unmatched interview series were conducted 1 year apart on Bubaque Island in the Bijagos Archipelago; one in rural villages using purposive snowball sampling, the other in a semi-urban settlement, using random-cluster sampling. Interviews were conducted and recorded in Kriolu, the local dialect, by a supervised local field assistant before translation into English for conventional content analysis. RESULTS: Trachoma was unheard of in either series, despite ongoing local trachoma research. A heterogeneous range of disease etiology and preventative measures were suggested, but the importance of hygiene was more widely reported by semi-urban interviewees. Although western medicine was well regarded, traditional practices continued, particularly in the rural populations. CONCLUSIONS: Differences in knowledge, beliefs and behaviors were apparent between the two series. Despite widespread rudimentary knowledge of disease prevention, targeted education might benefit both communities, particularly basic hygiene education for rural communities. Healthcare access should also be improved for rural populations. The impact of these measures could be assessed by future fieldwork.

Phosphorylation sites of Epstein-Barr virus EBNA1 regulate its function.

Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis and a risk factor for developing a variety of lymphomas and carcinomas. EBV nuclear antigen 1 (EBNA1) is the only viral protein found in all EBV-related malignancies. It plays a key role in establishing and maintaining the altered state of cells transformed with EBV. EBNA1 is required for a variety of functions, including gene regulation, replication and maintenance of the viral genome, but the regulation of EBNA1's functions is poorly understood. We demonstrate that phosphorylation affects the functions of EBNA1. By using electron-transfer dissociation tandem mass spectrometry, ten specific phosphorylated EBNA1 residues were identified. A mutant derivative preventing the phosphorylation of all ten phosphosites retained the unusually long half-life and the ability to translocate into the nucleus of wild-type EBNA1. This phosphorylation-deficient mutant, however, had a significantly reduced ability to activate transcription and to maintain EBV's plasmids in cells.