Improving Access to Diagnostics for Schistosomiasis Case Management in Oyo State, Nigeria: Barriers and Opportunities.

Schistosomiasis is one of the Neglected Tropical Diseases that affects over 200 million people worldwide, of which 29 million people in Nigeria. The principal strategy for schistosomiasis in Nigeria is a control and elimination program which comprises a school-based Mass Drug Administration (MDA) with limitations of high re-infection rates and the exclusion of high-risk populations. The World Health Organization (WHO) recommends guided case management of schistosomiasis (diagnostic tests or symptom-based detection plus treatment) at the Primary Health Care (PHC) level to ensure more comprehensive morbidity control. However, these require experienced personnel with sufficient knowledge of symptoms and functioning laboratory equipment. Little is known about where, by whom and how diagnosis is performed at health facilities within the case management of schistosomiasis in Nigeria. Furthermore, there is a paucity of information on patients' health-seeking behaviour from the onset of disease symptoms until a cure is obtained. In this study, we describe both perspectives in Oyo state, Nigeria and address the barriers using adapted health-seeking stages and access framework. The opportunities for improving case management were identified, such as a prevalence study of high-risk groups, community education and screening, enhancing diagnostic capacity at the PHC through point-of-care diagnostics and strengthening the capability of health workers.

Five-year mortality in patients with diabetic foot ulcer during 2009-2010 was lower than expected.

AIM: Mortality rates are decreasing in patients with diabetes. However, as this observation also concerns patients with diabetic foot ulcer (DFU), additional data are needed. For this reason, our study evaluated the 5-year mortality rate in patients with DFU during 2009-2010 and identified risk factors associated with mortality. METHODS: Consecutive patients who attended a clinic for new DFU during 2009-2010 were followed until healing and at 1 year. Data on mortality were collected at year 5. Multivariate Cox proportional-hazards model was used to identify mortality risk factors. RESULTS: A total of 347 patients were included: mean age was 65±12 years, diabetes duration was 16 [10; 27] years; 13% were on dialysis; and 7% had an organ transplant. At 5 years, 49 patients (14%) were considered lost to follow-up. Total mortality rate at 5 years was 35%, and 16% in patients with neuropathy. On multivariate analyses, mortality was positively associated with: age [hazard ratio (HR): 1.05 (1.03-1.07), P<0.0001]; duration of diabetes [HR: 1.02 (1.001-1.03], P=0.03]; PEDIS perfusion grade 2 vs. 1 [HR: 2.35 (1.28-4.29), P=0.006)]; PEDIS perfusion grade 3 vs. 1 [HR: 3.14 (1.58-6.24), P=0.001); and ulcer duration at year 1 [HR 2.09 (1.35-3.22), P=0.0009]. CONCLUSION: Mortality rates were not as high as expected despite the large number of comorbidities, suggesting that progress has been made in the health management of these patients. In particular, patients with neuropathic foot ulcer had a survival rate of 84% at 5 years.

Concentric photothermal coagulation with basket-integrated optical device for treatment of tracheal stenosis.

A basket-integrated optical device is developed to consistently treat tubular tissue by centering an optical diffuser in the lumen. Four nitinol arms in conjunction with the optical diffusing applicator are deployed to induce homogeneous circumferential light emission and concentric photothermal coagulation on tracheal tissue. A 1470-nm laser light is employed for the tissue testing at various irradiation conditions and evaluated in terms of thermal gradient and temperature evolution. Preliminary experiments on liver tissue demonstrate the concentric development of the radial thermal coagulation in the tissue (eccentric ratio = ~5.5%). The interstitial tissue temperature increases with the total amount of energy delivery (around 65°C). Ex vivo trachea testing yields up to 16.5% tissue shrinkage due to dehydration as well as uniform ablation of the cilia and goblet cells in a mucosa layer under 7-W irradiation for 10 s. The proposed optical device may be a feasible therapeutic method to entail the circumferential coagulation in the tubular tissues in a reliable manner.

Introduction to Sumoylation.

Reversible post-translational modification is a rapid and efficient system to control the activity of pre-existing proteins. Modifiers range from small chemical moieties, such as phosphate groups, to proteins themselves as the modifier. The patriarch of the protein modifiers is ubiquitin which plays a central role in protein degradation and protein targeting. Over the last 20 years, the ubiquitin family has expanded to include a variety of ubiquitin-related small modifier proteins that are all covalently attached to a lysine residue on target proteins via series of enzymatic reactions. Of these more recently discovered ubiquitin-like proteins, the SUMO family has gained prominence as a major regulatory component that impacts numerous aspects of cell growth, differentiation, and response to stress. Unlike ubiquitinylation which often leads to proteins turn over, sumoylation performs a variety of function such as altering protein stability, modulating protein trafficking, directing protein-protein interactions, and regulating protein activity. This chapter will introduce the basic properties of SUMO proteins and the general tenets of sumoylation.

Sumoylation in Development and Differentiation.

Tissue morphogenesis is a fascinating aspect of both developmental biology and regeneration of certain adult organs, and timely control of cellular differentiation is a key to these processes. During development, events interrupting cellular differentiation and leading to organ failure are embryonic lethal; likewise, perturbation of differentiation in regenerating tissues leads to dysfunction and disease. At the molecular level, cellular differentiation is orchestrated by a well-coordinated cascade of transcription factors (TFs) and chromatin remodeling complexes that drive gene expression. Altering the localization, stability, or activity of these regulatory elements can affect the sequential organization of the gene expression program and result in failed or abnormal tissue development. An accumulating body of evidence shows that the sumoylation system is a critical modulator of these regulatory cascades. For example, inhibition of the sumoylation system during embryogenesis causes lethality and/or severe abnormalities from invertebrates to mammals. Mechanistically, it is now known that many of the TFs and components of chromatin remodeling complexes that are critical for development and differentiation are targets for SUMO modification, though the specific functional consequences of the modifications remain uncharacterized in many cases. This chapter will address several of the models systems that have been examined for the role of sumoylation in differentiation and development. Understanding the profound regulatory role of SUMO in different tissues should lead not only to a better understanding of developmental biology, stem cell linage control, and the mechanisms of cellular differentiation, but may also lead to the identification of new targets for drug therapy and/or therapeutic manipulation of damaged organs and tissues.

Viral Interplay with the Host Sumoylation System.

Viruses have evolved elaborate means to regulate diverse cellular pathways in order to create a cellular environment that facilitates viral survival and reproduction. This includes enhancing viral macromolecular synthesis and assembly, as well as preventing antiviral responses, including intrinsic, innate, and adaptive immunity. There are numerous mechanisms by which viruses mediate their effects on the host cell, and this includes targeting various cellular post-translational modification systems, including sumoylation. The wide-ranging impact of sumoylation on cellular processes such as transcriptional regulation, apoptosis, stress response, and cell cycle control makes it an attractive target for viral dysregulation. To date, proteins from both RNA and DNA virus families have been shown to be modified by SUMO conjugation, and this modification appears critical for viral protein function. More interestingly, members of the several viral families have been shown to modulate sumoylation, including papillomaviruses, adenoviruses , herpesviruses, orthomyxoviruses, filoviruses , and picornaviruses . This chapter will focus on mechanisms by which sumoylation both impacts human viruses and is used by viruses to promote viral infection and disease.

The role of ubiquitin and ubiquitin-like modification systems in papillomavirus biology.

Human papillomaviruses (HPVs) are small DNA viruses that are important etiological agents of a spectrum of human skin lesions from benign to malignant. Because of their limited genome coding capacity they express only a small number of proteins, only one of which has enzymatic activity. Additionally, the HPV productive life cycle is intimately tied to the epithelial differentiation program and they must replicate in what are normally non-replicative cells, thus, these viruses must reprogram the cellular environment to achieve viral reproduction. Because of these limitations and needs, the viral proteins have evolved to co-opt cellular processes primarily through protein-protein interactions with critical host proteins. The ubiquitin post-translational modification system and the related ubiquitin-like modifiers constitute a widespread cellular regulatory network that controls the levels and functions of thousands of proteins, making these systems an attractive target for viral manipulation. This review describes the interactions between HPVs and the ubiquitin family of modifiers, both to regulate the viral proteins themselves and to remodel the host cell to facilitate viral survival and reproduction.

Growth and differentiation of HaCaT keratinocytes.

HaCaT cells are a spontaneously immortalized, human keratinocyte line that has been widely used for studies of skin biology and differentiation. Under typical culture conditions HaCaT cells have a partially to fully differentiated phenotype due to the high calcium content of both standard media and fetal bovine serum. This chapter describes low-calcium culture conditions for reverting HaCaT cells to the fully basal state followed by subsequent controlled differentiation using calcium induction.

Analysis of global sumoylation changes occurring during keratinocyte differentiation.

Sumoylation is a highly dynamic process that plays a role in a multitude of processes ranging from cell cycle progression to mRNA processing and cancer. A previous study from our lab demonstrated that SUMO plays an important role in keratinocyte differentiation. Here we present a new method of tracking the sumoylation state of proteins by creating a stably transfected HaCaT keratinocyte cell line expressing an inducible SNAP-SUMO3 protein. The SNAP-tag allows covalent fluorescent labeling that is denaturation resistant. When combined with two-dimensional gel electrophoresis, the SNAP-tag technology provides direct visualization of sumoylated targets and can be used to follow temporal changes in the global cohort of sumoylated proteins during dynamic processes such as differentiation. HaCaT keratinocyte cells expressing SNAP-SUMO3 displayed normal morphological and biochemical features that are consistent with typical keratinocyte differentiation. SNAP-SUMO3 also localized normally in these cells with a predominantly nuclear signal and some minor cytoplasmic staining, consistent with previous reports for untagged SUMO2/3. During keratinocyte differentiation the total number of proteins modified by SNAP-SUMO3 was highest in basal cells, decreased abruptly after induction of differentiation, and slowly rebounded beginning between 48 and 72 hours as differentiation progressed. However, within this overall trend the pattern of change for individual sumoylated proteins was highly variable with both increases and decreases in amount over time. From these results we conclude that sumoylation of proteins during keratinocyte differentiation is a complex process which likely reflects and contributes to the biochemical changes that drive differentiation.

Cell culture assay for transient replication of human and animal papillomaviruses.

This unit contains protocols for evaluation of replication functionality of papillomavirus genomes or subgenomic fragments. Replication is measured after transient cotransfection of the genome (or subgenomic fragment) with expression vectors encoding the viral E1 and E2 proteins. Input DNA is methylated at the adenine of GATC sequences by propagation in E. coli. DNA that replicates in mammalian cells will lose the adenine methylation and become DpnI-resistant, while residual methylated input DNA will remain DpnI-sensitive. After transfection, DNA extraction, and DpnI digestion, replicated DNA can be detected by Southern blotting as a full-length plasmid, since it is resistant to digestion. This assay can be used to map the genomic location of a functional origin or to evaluate replication activity of mutations in either the origin DNA sequences or the E1 or E2 proteins.

Sumoylation at the host-pathogen interface.

Many viral proteins have been shown to be sumoylated with corresponding regulatory effects on their protein function, indicating that this host cell modification process is widely exploited by viral pathogens to control viral activity. In addition to using sumoylation to regulate their own proteins, several viral pathogens have been shown to modulate overall host sumoylation levels. Given the large number of cellular targets for SUMO addition and the breadth of critical cellular processes that are regulated via sumoylation, viral modulation of overall sumoylation presumably alters the cellular environment to ensure that it is favorable for viral reproduction and/or persistence. Like some viruses, certain bacterial plant pathogens also target the sumoylation system, usually decreasing sumoylation to disrupt host anti-pathogen responses. The recent demonstration that Listeria monocytogenes also disrupts host sumoylation, and that this is required for efficient infection, extends the plant pathogen observations to a human pathogen and suggests that pathogen modulation of host sumoylation may be more widespread than previously appreciated. This review will focus on recent aspects of how pathogens modulate the host sumoylation system and how this benefits the pathogen.

A fast method using a new hydrophilic-lipophilic balanced sorbent in combination with ultra-high performance liquid chromatography for quantification of significant bioactive metabolites in wines.

This manuscript describes the development and validation of an ultra-fast, efficient, and high throughput analytical method based on ultra-high performance liquid chromatography (UHPLC) equipped with a photodiode array (PDA) detection system, for the simultaneous analysis of fifteen bioactive metabolites: gallic acid, protocatechuic acid, (-)-catechin, gentisic acid, (-)-epicatechin, syringic acid, p-coumaric acid, ferulic acid, m-coumaric acid, rutin, trans-resveratrol, myricetin, quercetin, cinnamic acid and kaempferol, in wines. A 50-mm column packed with 1.7-µm particles operating at elevated pressure (UHPLC strategy) was selected to attain ultra-fast analysis and highly efficient separations. In order to reduce the complexity of wine extract and improve the recovery efficiency, a reverse-phase solid-phase extraction (SPE) procedure using as sorbent a new macroporous copolymer made from a balanced ratio of two monomers, the lipophilic divinylbenzene and the hydrophilic N-vinylpyrrolidone (Oasis™ HLB), was performed prior to UHPLC-PDA analysis. The calibration curves of bioactive metabolites showed good linearity within the established range. Limits of detection (LOD) and quantification (LOQ) ranged from 0.006 µg mL(-1) to 0.58 µg mL(-1), and from 0.019 µg mL(-1) to 1.94 µg mL(-1), for gallic and gentisic acids, respectively. The average recoveries ± SD for the three levels of concentration tested (n=9) in red and white wines were, respectively, 89 ± 3% and 90 ± 2%. The repeatability expressed as relative standard deviation (RSD) was below 10% for all the metabolites assayed. The validated method was then applied to red and white wines from different geographical origins (Azores, Canary and Madeira Islands). The most abundant component in the analysed red wines was (-)-epicatechin followed by (-)-catechin and rutin, whereas in white wines syringic and p-coumaric acids were found the major phenolic metabolites. The method was completely validated, providing a sensitive analysis for bioactive phenolic metabolites detection and showing satisfactory data for all the parameters tested. Moreover, was revealed as an ultra-fast approach allowing the separation of the fifteen bioactive metabolites investigated with high resolution power within 5 min.

HPV E6 proteins target Ubc9, the SUMO conjugating enzyme.

The human papillomavirus oncogenic protein, E6, interacts with a number of cellular proteins, and for some targets, E6 directs their degradation through the ubiquitin-proteasome pathway. Post-translational modification with ubiquitin-like modifiers, such as SUMO, also influences protein activities, protein-protein interactions, and protein stability. We report that the high risk HPVE6 proteins reduce the intracellular quantity of the sole SUMO conjugation enzyme, Ubc9, concomitant with decreased host sumoylation. E6 did not significantly influence transcription of Ubc9, indicating that the effects were likely at the protein level. Consistent with typical E6-mediated proteasomal degradation, E6 bound to Ubc9 in vitro, and required E6AP for reduction of Ubc9 levels. Under stable E6 expression conditions in differentiating keratinocytes there was a decrease in Ubc9 and a loss of numerous sumoylated targets indicating a significant perturbation of the normal sumoylation profile. While E6 is known to inhibit PIASy, a SUMO ligase, our results suggest that HPV E6 also targets the Ubc9 protein to modulate host cell sumoylation, suggesting that the sumoylation system may be an important target during viral reproduction and possibly the subsequent development of cervical cancer.

Common importin alpha specificity for papillomavirus E2 proteins.

Papillomaviruses infect keratinocytes and their reproduction is tied to differentiation of the skin. The E2 protein of papillomaviruses is a multifunctional early protein that binds specifically to the viral DNA to regulate genome transcription, replication, and segregation. All of these are nuclear events that require specific transport of E2 into the host nucleus. Nuclear localization signal (NLS) sequences have been mapped for several E2 proteins, and these sequences resemble motifs that interact with cellular transport adaptor molecules termed alpha importins. To determine which importins could carry E2 proteins, in vitro binding studies were performed with three different E2 proteins and the five ubiquitous alpha importins. The E2 proteins preferentially interacted with alpha importins 3 and 5, and showed very weak or no interaction with the other three widely expressed alpha importins (alpha1, alpha 4, and alpha 7). While all five alpha importins appear to be constitutively expressed in keratinocytes, during differentiation of a human keratinocyte line (HaCaT) we observed a specific increase in expression of alphas 3 and 5. This differentiation-specific increase in alpha 3 and alpha 5 expression suggests that preferential usage of these two importins by E2 may facilitate E2 nuclear uptake during terminal differentiation.

Cloning the human SUMO1 promoter.

Regulation of the sumoylation system at the level of gene expression has not yet been explored. To begin to define transcriptional regulatory features, the promoter region for the SUMO1 gene was cloned from human genomic DNA and characterized. Initially, a 532 base pair fragment upstream of and including the predicted SUMO1 transcription start site (TSS) was cloned and shown to possess promoter activity. Subsequent deletion analysis showed that a smaller fragment containing 158 bp upstream of the TSS region exhibited basal promoter activity in both human and rodent cell lines. Within this basal promoter fragment, there were predicted binding sites for numerous transcription factors, including the nude mouse gene product, Whn (FoxN1). Electrophoretic mobility shift assays showed that Whn could bind to an ACGC motif adjacent to the TSR, and in transfection studies Whn stimulated a 3-fold increase in transcription from this cloned promoter in keratinocytes (HaCaT cells). Mutation of the ACGC motif abrogated both Whn binding and transcriptional activation, indicating that the Whn effect is likely due to direct interaction with this promoter element. Consistent with these observations on the cloned promoter region, Whn also modestly stimulated transcription from the endogenous, genomic SUMO1 promoter in HaCaT cells, consistent with Whn potentially playing a regulatory role for SUMO1 transcription in keratinocytes.

Incidence and characteristics of lower limb amputations in people with diabetes.

AIMS: To estimate the incidence, characteristics and potential causes of lower limb amputations in France. METHODS: Admissions with lower limb amputations were extracted from the 2003 French national hospital discharge database, which includes major diagnoses and procedures performed during hospital admissions. For each patient, diabetes was defined by its record in at least one admission with or without lower limb amputation in the 2002-2003 databases. RESULTS: In 2003, 17 551 admissions with lower limb amputation were recorded, involving 15 353 persons, which included 7955 people with diabetes. The crude incidence of lower limb amputation in people with diabetes was 378/100 000 (349/100 000 when excluding traumatic lower limb amputation). The sex and age standardized incidence was 12 times higher in people with than without diabetes (158 vs. 13/100 000). Renal complications and peripheral arterial disease and/or neuropathy were reported in, respectively, 30% and 95% of people with diabetes with lower limb amputation. Traumatic causes (excluding foot contusion) and bone diseases (excluding foot osteomyelitis) were reported in, respectively, 3% and 6% of people with diabetes and lower limb amputation, and were 5 and 13 times more frequent than in people without diabetes. CONCLUSIONS: We provide a first national estimate of lower limb amputation in France. We highlight its major impact on people with diabetes and its close relationship with peripheral arterial disease/neuropathy and renal complications in the national hospital discharge database. We do not suggest the exclusion of traumatic causes when studying the epidemiology of lower limb amputation related to diabetes, as diabetes may contribute to amputation even when the first cause appears to be traumatic.

Host cell sumoylation level influences papillomavirus E2 protein stability.

The stability of papillomavirus E2 proteins is regulated by proteasomal degradation, and regulation of degradation could contribute to the higher expression levels of E2 proteins observed in suprabasal layers of differentiated skin. We have recently shown that the E2 proteins are modified by sumoylation [Wu Y-C, Roark AA, Bian X-L, Wilson, VG (2008) Virol 378:329-338], and that sumoylation levels are up-regulated during keratinocyte differentiation [Deyrieux AF, Rosas-Acosta G, Ozbun MA, Wilson VG (2007) J Cell Sci 120:125-136]. These observations, coupled with the known ability of sumoylation to prevent proteasomal degradation of certain proteins, suggested that this modification might contribute to stabilizing E2 proteins in suprabasal keratinocytes. Conditions that increased overall sumoylation were found to increase the intracellular amounts of the HPV11, 16, and 18 E2 proteins. No effect of sumoylation was seen on E2 transcripts, and the increased levels of E2 proteins resulted from a greatly increased half-life for the E2 proteins. In vitro studies confirmed that sumoylation could block the proteasomal degradation of the 16E2 protein. Interestingly, this stabilization effect was indirect as it did not require sumoylation of 16E2 itself and must be acting through sumoylation of a cellular target(s). This sumoylation-dependent, indirect stabilization of E2 proteins is a novel process that may couple E2 levels to changes in the cellular environment. Specifically, our results suggest that the levels of papillomavirus E2 protein could be up-regulated in differentiating keratinocytes in response to the increased overall sumoylation that accompanies differentiation.

Signalling of the BCR is regulated by a lipid rafts-localised transcription factor, Bright.

Regulation of BCR signalling strength is crucial for B-cell development and function. Bright is a B-cell-restricted factor that complexes with Bruton's tyrosine kinase (Btk) and its substrate, transcription initiation factor-I (TFII-I), to activate immunoglobulin heavy chain gene transcription in the nucleus. Here we show that a palmitoylated pool of Bright is diverted to lipid rafts of resting B cells where it associates with signalosome components. After BCR ligation, Bright transiently interacts with sumoylation enzymes, blocks calcium flux and phosphorylation of Btk and TFII-I and is then discharged from lipid rafts as a Sumo-I-modified form. The resulting lipid raft concentration of Bright contributes to the signalling threshold of B cells, as their sensitivity to BCR stimulation decreases as the levels of Bright increase. Bright regulates signalling independent of its role in IgH transcription, as shown by specific dominant-negative titration of rafts-specific forms. This study identifies a BCR tuning mechanism in lipid rafts that is regulated by differential post-translational modification of a transcription factor with implications for B-cell tolerance and autoimmunity.

Mice overexpressing murine oncostatin M (OSM) exhibit changes in hematopoietic and other organs that are distinct from those of mice overexpressing human OSM or bovine OSM.

Oncostatin M (OSM) and leukemia inhibitory factor (LIF) belong to the interleukin-6 family of cytokines. The authors' previous in vitro work demonstrated that in mouse cells mouse OSM (mOSM) signals through a heterodimeric receptor complex incorporating the mOSM-specific receptor mOSMRbeta while human OSM (hOSM) and bovine OSM (bOSM) use the mouse LIF receptor mLIFRbeta rather than mOSMRbeta. These in vitro data suggest that prior studies in mouse systems with hOSM or bOSM (the usual molecules used in early studies) reflect LIF rather than OSM biology. The current work assessed whether or not this divergence in actions among these three OSMs also occurs in vivo in mouse models. Adult female (C57BL/6J x DBA/2J) F(1) mice were engineered to stably overexpress mOSM, hOSM, or bOSM by retrovirus-mediated gene transfer (n = 10 or more per group). After 4 weeks, molecular and hematologic profiles and anatomic phenotypes in multiple organs were assessed by standard techniques. Animals overexpressing either hOSM or bOSM had an identical phenotype resembling that associated with LIF activation, including significant hematologic abnormalities (anemia, neutrophilia, lymphopenia, eosinopenia, and thrombocytosis); weight loss; profound enlargement (lymph node, spleen) and/or structural reorganization (lymph node, spleen, thymus) of lymphoid organs; and severe osteosclerosis. In contrast, mice overexpressing mOSM did not develop hematologic changes, weight loss, or osteosclerosis and exhibited more modest and anatomically distinct restructuring of lymphoid organs. These data indicate that activities imputed to OSM and the mOSMRbeta signaling pathway using in vitro and in vivo mouse experimental systems are unique to mOSM.

[Management of a diabetic foot ulcer]. / Conduite à tenir face à une plaie du pied chez un diabétique.

A chronic diabetic foot ulcer requires a search for the etiology. The three main causes to search for are poor off-loading compliance, osteomyelitis, and peripheral vascular disease. The level of severity is measured with the U.T. classification and the level of infection with the classification of the International Consensus on the Diabetic Foot. Peripheral vascular disease must be precisely evaluated by Doppler ultrasound, which describes all the arteries of the lower limb. Angiography is required only in case of revascularization. Treatment of the ulcer includes strict off-loading, topical treatment, optimal treatment of hyperglycemia, and antibiotic therapy on a case-by-case basis for osteomyelitis and/or, angioplasty or by-pass procedures. Osteomyelitis can be treated by associating conservative surgery, antibiotic therapy, and off-loading. No amputation, even of one toe, must be done without a previous vascular check-up. Off-loading of the ulcer must be regularly checked. Poor off-loading compliance must be systematically investigated if the ulcer worsens or healing is delayed.