5-Methyltetrahydrofolate is photosensitive in the presence of riboflavin.

5-Methyltetrahydrofolate (5MTHF) is the main form of folate in human plasma, and an important vitamin for human health. Photodegradation of folates may have played a role in the development of different human skin colours. 5MTHF can be degraded directly by exposure to ultraviolet radiation or by exposure to visible light in the presence of endogenous sensitizers like riboflavin (RF). These photochemical reactions were studied by absorption spectroscopy. While 5MTHF is stable under UV and visible light exposure in pure aqueous media, it is quickly degraded in the presence of RF during UVA and blue light exposure. The degradation of 5MTHF is dependent on the concentration of RF, but not on the concentration of 5MTHF itself. UVA and blue light gave similar reactions. Further investigations are necessary to evaluate the consequences of large light exposures in vivo in humans. Our findings should be taken into the ongoing discussion about the development of human skin colours. Due to the presence of RF in human blood, folate can be significantly degraded during prolonged or intense blue light exposure. Thus, a dark skin colour may be favourable for prevention of folate degradation under high solar fluence rates, such as in equatorial areas.     

Detection and Characterization of Clade 1 Reassortant H5N1 Viruses Isolated from Human Cases in Vietnam during 2013

Highly pathogenic avian influenza (HPAI) H5N1 is endemic in Vietnamese poultry and has caused sporadic human infection in Vietnam since 2003. Human infections with HPAI H5N1 are of concern due to a high mortality rate and the potential for the emergence of pandemic viruses with sustained human-to-human transmission. Viruses isolated from humans in southern Vietnam have been classified as clade 1 with a single genome constellation (VN3) since their earliest detection in 2003. This is consistent with detection of this clade/genotype in poultry viruses endemic to the Mekong River Delta and surrounding regions. Comparison of H5N1 viruses detected in humans from southern Vietnamese provinces during 2012 and 2013 revealed the emergence of a 2013 reassortant virus with clade 1.1.2 hemagglutinin (HA) and neuraminidase (NA) surface protein genes but internal genes derived from clade 2.3.2.1a viruses (A/Hubei/1/2010-like; VN12). Closer analysis revealed mutations in multiple genes of this novel genotype (referred to as VN49) previously associated with increased virulence in animal models and other markers of adaptation to mammalian hosts. Despite the changes identified between the 2012 and 2013 genotypes analyzed, their virulence in a ferret model was similar. Antigenically, the 2013 viruses were less cross-reactive with ferret antiserum produced to the clade 1 progenitor virus, A/Vietnam/1203/2004, but reacted with antiserum produced against a new clade 1.1.2 WHO candidate vaccine virus (A/Cambodia/W0526301/2012) with comparable hemagglutination inhibition titers as the homologous antigen. Together, these results indicate changes to both surface and internal protein genes of H5N1 viruses circulating in southern Vietnam compared to 2012 and earlier viruses.     

Glucocorticoids Suppress Renal Cell Carcinoma Progression by Enhancing Na,K-ATPase Beta-1 Subunit Expression

Glucocorticoids are commonly used as palliative or chemotherapeutic clinical agents for treatment of a variety of cancers. Although steroid treatment is beneficial, the mechanisms by which steroids improve outcome in cancer patients are not well understood. Na,K-ATPase beta-subunit isoform 1 (NaK-β₁) is a cell-cell adhesion molecule, and its expression is down-regulated in cancer cells undergoing epithelial-to mesenchymal-transition (EMT), a key event associated with cancer progression to metastatic disease. In this study, we performed high-throughput screening to identify small molecules that could up-regulate NaK-β₁ expression in cancer cells. Compounds related to the glucocorticoids were identified as drug candidates enhancing NaK-β₁ expression. Of these compounds, triamcinolone, dexamethasone, and fluorometholone were validated to increase NaK-β₁ expression at the cell surface, enhance cell-cell adhesion, attenuate motility and invasiveness and induce mesenchymal to epithelial like transition of renal cell carcinoma (RCC) cells in vitro. Treatment of NaK-β₁ knockdown cells with these drug candidates confirmed that these compounds mediate their effects through up-regulating NaK-β₁. Furthermore, we demonstrated that these compounds attenuate tumor growth in subcutaneous RCC xenografts and reduce local invasiveness in orthotopically-implanted tumors. Our results strongly indicate that the addition of glucocorticoids in the treatment of RCC may improve outcome for RCC patients by augmenting NaK-β₁ cell-cell adhesion function.     

Low Incidence of Renal Dysfunction among HIV-Infected Patients on a Tenofovir-Based First Line Antiretroviral Treatment Regimen in Myanmar

Background

Since 2004, Médecins Sans Frontières-Switzerland has provided treatment and care for people living with HIV in Dawei, Myanmar. Renal function is routinely monitored in patients on tenofovir (TDF)-based antiretroviral treatment (ART), and this provides an opportunity to measure incidence and risk factors for renal dysfunction.

Methods

We used routinely collected program data on all patients aged ≥15 years starting first-line TDF-based ART between January 2012 and December 2013. Creatinine clearance (CrCl) was assessed at base line and six-monthly, with renal dysfunction defined as CrCl < 50ml/min/1.73m². We calculated incidence of renal dysfunction and used Cox regression analysis to identify associated risk factors.

Results

There were 1391 patients, of whom 1372 had normal renal function at baseline. Of these, 86 (6.3%) developed renal dysfunction during a median time of follow-up 1.14 years with an incidence rate of 5.4 per 100 person-years: 78 had CrCl between 30–50ml/min/1.73m² and were maintained on TDF–based ART, but 5 were changed to another regimen: 4 because of CrCl <30ml/min/1.73m². Risk factors for renal dysfunction included age ≥45 years, diagnosed diabetes, underlying renal disease, underweight and CD4 count <200cells/mm³. There were 19 patients with baseline renal dysfunction and all continued on TDF-based ART: CrCl stayed between 30–49 ml/min/1.73m² in five patients while the remainder regained normal renal function.

Conclusions

In a resource-poor country like Myanmar, the low incidence of renal toxicity in our patient cohort suggests that routine assessment of CrCl may not be needed and could be targeted to high risk groups if resources permit.     

Counting the Cost of Diabetes in the Solomon Islands and Nauru

Aim

To determine the costs associated with diabetes to governments, people with diabetes and their carers, and its impact on quality of life in two Pacific Island countries—the Solomon Islands and Nauru.

Materials and Methods

This cross-sectional cost of illness study was conducted on 330 people with type 2 diabetes (197 from the Solomon Islands and 133 from Nauru) using a structured cost of illness survey questionnaire adapted from the Australian DiabCo$t study. Quality of life was measured by the EQ-5D Visual Analogue Scale.

Results

There were 330 respondents (50% female; mean duration of diabetes 10.9 years; mean age 52.6 years). The estimated annual national cost of diabetes incurred by the Solomon Islands government was AUD12.8 million (AUD281 per person/year) and by Nauru government was AUD1.2 million (AUD747 per person/year). The major contribution to the government costs was inpatient services cost (71% in the Solomon Islands and 83% in Nauru). Annual expenditure for diabetes was approximately 20% of the governments’ annual health care expenditure. Considerable absenteeism and retirement from work due to diabetes was found.

Conclusions

This study found substantial public and personal costs associated with diabetes. The findings provide objective data on which health policy, funding and planning decisions about the prevention and control of diabetes in the Solomon Islands and Nauru can be reliably based and subsequently evaluated.     

FORGE Canada Consortium: Outcomes of a 2-Year National Rare-Disease Gene-Discovery Project

Inherited monogenic disease has an enormous impact on the well-being of children and their families. Over half of the children living with one of these conditions are without a molecular diagnosis because of the rarity of the disease, the marked clinical heterogeneity, and the reality that there are thousands of rare diseases for which causative mutations have yet to be identified. It is in this context that in 2010 a Canadian consortium was formed to rapidly identify mutations causing a wide spectrum of pediatric-onset rare diseases by using whole-exome sequencing. The FORGE (Finding of Rare Disease Genes) Canada Consortium brought together clinicians and scientists from 21 genetics centers and three science and technology innovation centers from across Canada. From nation-wide requests for proposals, 264 disorders were selected for study from the 371 submitted; disease-causing variants (including in 67 genes not previously associated with human disease; 41 of these have been genetically or functionally validated, and 26 are currently under study) were identified for 146 disorders over a 2-year period. Here, we present our experience with four strategies employed for gene discovery and discuss FORGE’s impact in a number of realms, from clinical diagnostics to the broadening of the phenotypic spectrum of many diseases to the biological insight gained into both disease states and normal human development. Lastly, on the basis of this experience, we discuss the way forward for rare-disease genetic discovery both in Canada and internationally.     

AMP-activated protein kinase (AMPK) activators from Myristica fragrans (nutmeg) and their anti-obesity effect

AMP-activated protein kinase (AMPK) is a potential therapeutic target for the treatment of metabolic syndrome including obesity and type-2 diabetes. As part of an ongoing search for new AMPK activators from plants, this study found that the total extract of Myristica fragrans (nutmeg) activated the AMPK enzyme in differentiated C2C12 cells. As active constituents, seven 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignans, tetrahydrofuroguaiacin B (1), saucernetindiol (2), verrucosin (3), nectandrin B (4), nectandrin A (5), fragransin C₁ (6), and galbacin (7) were isolated from this extract. Among the isolates, compounds 1, 4, and 5 at 5 μM produced strong AMPK stimulation in differentiated C2C12 cells. In addition, the preventive effect of a tetrahydrofuran mixture (THF) on weight gain in a diet-induced animal model was further examined. These results suggest that nutmeg and its active constituents can be used not only for the development of agents to treat obesity and possibly type-2 diabetes but may also be beneficial for other metabolic disorders.     

Localized glucoprivation of hindbrain sites elicits corticosterone and glucagon secretion

Glucose is required for brain energy metabolism. Decerebration, aqueduct occlusion, and cannula mapping studies have established that glucose-sensing cells capable of eliciting feeding and adrenal medullary responses to glucoprivation are localized in the hindbrain. Glucoprivation also evokes corticosterone and glucagon secretion, but the location of receptors mediating these responses is unknown. To determine whether glucoreceptive sites controlling these responses are present in the hindbrain, we administered the antiglycolytic agent, 5-d-thioglucose (5TG, 24 microg in 200 nl) into brain stem sites through implanted cannulas and examined plasma concentrations of corticosterone and glucagon. Both hindbrain and hypothalamic sites were tested. Blood was collected remotely from intra-atrial catheters at 0, 30, 60, 90, 120, 180, and 240 min after 5TG or control injection. Caudal hindbrain 5TG injections potently increased circulating corticosterone and glucagon concentrations. For corticosterone, the mean peak response (maximum concentration minus time 0 concentration) elicited at positive sites (23 of 40 sites) was 391 ng/ml (SE = 16). For glucagon, the mean peak response at positive sites (27 of 40 sites) was 46 pg/ml (SE = 6). Glucoprivically evoked glucagon secretion was abolished by the ganglionic blocker, hexamethonium, but not by adrenal denervation. Six of twenty-five hypothalamic sites were positive for corticosterone secretion, yielding plasma levels of 279 +/- 23 ng/ml, but none of the hypothalamic injection sites elevated glucagon concentrations. Results demonstrate that receptor cells responsive to glucose deficit and capable of increasing corticosterone and glucagon concentrations exist within the hindbrain, thus further delineating central glucoregulatory neural circuitry.     

The inhibition of endocytosis affects HDL-lipid uptake mediated by the human scavenger receptor class B type I

The scavenger receptor SR-BI plays an important role in the hepatic clearance of HDL cholesterol and other lipids, driving reverse cholesterol transport and contributing to protection against atherosclerosis in mouse models. We characterized the role of endocytosis in lipid uptake from HDL, mediated by the human SR-BI, using a variety of approaches to inhibit endocytosis, including hypertonic shock, potassium or energy depletion and disassembly of the actin cytoskeleton. Our studies revealed that unlike mouse SR-BI, human SR-BI-mediated HDL-lipid uptake was reduced by inhibition of endocytosis. This was not dependent on the cytoplasmic C-terminus of SR-BI. Monitoring the uptake of both the protein and lipid components of HDL revealed that although overall lipid uptake was decreased, the degree of selective lipid uptake was increased. These data suggest that that endocytosis is a dynamic regulator of SR-BI's selective lipid uptake activity.