DNA methyltransferase inhibitor RG108 and histone deacetylase inhibitors cooperate to enhance NB4 cell differentiation and E‐cadherin re‐expression by chromatin remodelling

Epigenetic silencing of cancer‐related genes by abnormal methylation and the reversal of this process by DNA methylation inhibitors represents a promising strategy in cancer therapy. As DNA methylation affects gene expression and chromatin structure, we investigated the effects of novel DNMT (DNA methyltransferase) inhibitor, RG108, alone and in its combinations with structurally several HDAC (histone deacetylase) inhibitors [sodium PB (phenyl butyrate) or BML‐210 (N‐(2‐aminophenyl)‐N′phenyloctanol diamine), and all‐trans RA (retinoic acid)] in the human PML (promyelocytic leukaemia) NB4 cells. RG108 at different doses from 20 to 100 μM caused time‐, but not a dose‐dependent inhibition of NB4 cell proliferation without cytotoxicity. Temporal pretreatment with RG108 before RA resulted in a dose‐dependent cell growth inhibition and remarkable acceleration of granulocytic differentiation. Prolonged treatments with RG108 and RA in the presence of HDAC inhibitors significantly increased differentiation. RG108 caused time‐dependent re‐expression of methylation‐silenced E‐cadherin, with increase after temporal or continuous treatments with RG108 and RA, or RA together with PB in parallel, in cell maturation, suggesting the role of E‐cadherin as a possible therapeutic marker. These processes required both PB‐induced hyperacetylation of histone H4 and trimethylation of histone H3 at lysine 4, indicating the cooperative action of histone modifications and DNA methylation/demethylation in derepression of E‐cadherin. This work provides novel experimental evidence of the beneficial role of the DNMT inhibitor RG108 in combinations with RA and HDACIs in the effective differentiation of human PML based on epigenetics.     

Financing HIV Programming: How Much Should Low- And Middle-Income Countries and their Donors Pay?

Global HIV control funding falls short of need. To maximize health outcomes, it is critical that national governments sustain reasonable commitments, and that international donor assistance be distributed according to country needs and funding gaps. We develop a country classification framework in terms of actual versus expected national domestic funding, considering resource needs and donor financing. With UNAIDS and World Bank data, we examine domestic and donor HIV program funding in relation to need in 84 low- and middle-income countries. We estimate expected domestic contributions per person living with HIV (PLWH) as a function of per capita income, relative size of the health sector, and per capita foreign debt service. Countries are categorized according to levels of actual versus expected domestic contributions, and resource gap. Compared to national resource needs (UNAIDS Investment Framework), we identify imbalances among countries in actual versus expected domestic and donor contributions: 17 countries, with relatively high HIV prevalence and GNI per capita, have domestic funding below expected (median per PLWH $143 and $376, respectively), yet total available funding including from donors would exceed the need ($368 and $305, respectively) if domestic contribution equaled expected. Conversely, 27 countries have actual domestic funding above the expected (medians $294 and $149) but total (domestic+donor) funding does not meet estimated need ($685 and $1,173). Across the 84 countries, in 2009, estimated resource need totaled $10.3 billion, actual domestic contributions $5.1 billion and actual donor contributions $3.7 billion. If domestic contributions would increase to the expected level in countries where the actual was below expected, total domestic contributions would increase to $7.4 billion, turning a funding gap of $1.5 billion into a surplus of $0.8 billion. Even with imperfect funding and resource-need data, the proposed country classification could help improve coherence and efficiency in domestic and international allocations.     

Dual Effects of Hydrogen Sulfide Donor on Meiosis and Cumulus Expansion of Porcine Cumulus-Oocyte Complexes

Hydrogen sulfide (H₂S) has been revealed to be a signal molecule with second messenger action in the somatic cells of many tissues, including the reproductive tract. The aim of this study was to address how exogenous H₂S acts on the meiotic maturation of porcine oocytes, including key maturation factors such as MPF and MAPK, and cumulus expansion intensity of cumulus-oocyte complexes. We observed that the H₂S donor, Na₂S, accelerated oocyte in vitro maturation in a dose-dependent manner, following an increase of MPF activity around germinal vesicle breakdown. Concurrently, the H₂S donor affected cumulus expansion, monitored by hyaluronic acid production. Our results suggest that the H₂S donor influences oocyte maturation and thus also participates in the regulation of cumulus expansion. The exogenous H₂S donor apparently affects key signal pathways of oocyte maturation and cumulus expansion, resulting in faster oocyte maturation with little need of cumulus expansion.     

Relationship of Circulating Soluble Urokinase Plasminogen Activator Receptor (suPAR) Levels to Disease Control in Asthma and Asthmatic Pregnancy

Asthma has a high burden of morbidity if not controlled and may frequently complicate pregnancy, posing a risk for pregnancy outcomes. Elevated plasma level of the inflammatory biomarker soluble urokinase plasminogen activator receptor (suPAR) is related to a worse prognosis in many conditions such as infectious, autoimmune, or pregnancy-related diseases; however the value of suPAR in asthma and asthmatic pregnancy is unknown. The present study aimed to investigate the suPAR, CRP and IL-6 levels in asthma (asthmatic non-pregnant, ANP; N = 38; female N = 27) and asthmatic pregnancy (AP; N = 15), compared to healthy non-pregnant controls (HNP; N = 29; female N = 19) and to healthy pregnant women (HP; N = 58). The relationship between suPAR levels and asthma control was also evaluated. The diagnostic efficacy of suPAR in asthma control was analyzed using ROC analysis. IL-6 and CRP levels were comparable in all study groups. Circulating suPAR levels were lower in HP and AP than in HNP and ANP subjects, respectively (2.01 [1.81–2.38] and 2.39 [2.07–2.69] vs. 2.60 [1.82–3.49] and 2.84 [2.33–3.72] ng/mL, respectively, p = 0.0001). suPAR and airway resistance correlated in ANP (r = 0.47, p = 0.004). ROC analysis of suPAR values in ANP patients with PEF above and below 80% yielded an AUC of 0.75 (95% CI: 0.57–0.92, p = 0.023) and with ACT total score above and below 20 an AUC of 0.80 (95% CI: 0.64–0.95, p = 0.006). The cut-off value of suPAR to discriminate between controlled and not controlled AP and ANP was 4.04 ng/mL. In conclusion, suPAR may help the objective assessment of asthma control, since it correlates with airway resistance and has good sensitivity in the detection of impaired asthma control. Decrease in circulating suPAR levels detected both in healthy and asthmatic pregnant women presumably represents pregnancy induced immune tolerance.     

Studies on retinal microangiopathy and coronary macroangiopathy in rats with streptozotocin-induced diabetes

Diabetes mellitus was induced in male rats by streptozotocin injection. After 4, 8 and 12 months, retinal and coronary vessels were prepared and their structure was studied. An age-dependent increase in thickness of the capillary basement membrane of retinal vessels was found both in controls and diabetics. Furthermore, diabetics had an added 20%, disease-related thickening, present after 4 months and thereafter. No significant change in coronary arteries was found. After 8 and 12 months, plasma lipids, triiodothyronin and thyroxin were measured. In diabetics, triglycerides were higher at both time intervals, high density lipoprotein was slightly increased and thyroxin was reduced after 8 months, and triiodothyronin was reduced after both 8 and 12 months. Diabetes of 12 months duration appears insufficient to induce significant arteriosclerotic changes in rats.     

Biochemical characterisation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from the liver fluke, Fasciola hepatica

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) catalyses one of the two steps in glycolysis which generate the reduced coenzyme NADH. This reaction precedes the two ATP generating steps. Thus, inhibition of GAPDH will lead to substantially reduced energy generation. Consequently, there has been considerable interest in developing GAPDH inhibitors as anti-cancer and anti-parasitic agents. Here, we describe the biochemical characterisation of GAPDH from the common liver fluke Fasciola hepatica (FhGAPDH). The primary sequence of FhGAPDH is similar to that from other trematodes and the predicted structure shows high similarity to those from other animals including the mammalian hosts. FhGAPDH lacks a binding pocket which has been exploited in the design of novel antitrypanosomal compounds. The protein can be expressed in, and purified from Escherichia coli; the recombinant protein was active and showed no cooperativity towards glyceraldehyde 3-phosphate as a substrate. In the absence of ligands, FhGAPDH was a mixture of homodimers and tetramers, as judged by protein–protein crosslinking and analytical gel filtration. The addition of either NAD⁺ or glyceraldehyde 3-phosphate shifted this equilibrium towards a compact dimer. Thermal scanning fluorimetry demonstrated that this form was considerably more stable than the unliganded one. These responses to ligand binding differ from those seen in mammalian enzymes. These differences could be exploited in the discovery of reagents which selectively disrupt the function of FhGAPDH.