The ascidian natural product eusynstyelamide B is a novel topoisomerase II poison that induces DNA damage and growth arrest in prostate and breast cancer cells

As part of an anti-cancer natural product drug discovery program, we recently identified eusynstyelamide B (EB), which displayed cytotoxicity against MDA-MB-231 breast cancer cells (IC50 = 5 μM) and induced apoptosis. Here, we investigated the mechanism of action of EB in cancer cell lines of the prostate (LNCaP) and breast (MDA-MB-231). EB inhibited cell growth (IC50 = 5 μM) and induced a G2 cell cycle arrest, as shown by a significant increase in the G2/M cell population in the absence of elevated levels of the mitotic marker phospho-histone H3. In contrast to MDA-MB-231 cells, EB did not induce cell death in LNCaP cells when treated for up to 10 days. Transcript profiling and Ingenuity Pathway Analysis suggested that EB activated DNA damage pathways in LNCaP cells. Consistent with this, CHK2 phosphorylation was increased, p21CIP1/WAF1 was up-regulated and CDC2 expression strongly reduced by EB. Importantly, EB caused DNA double-strand breaks, yet did not directly interact with DNA. Analysis of topoisomerase II-mediated decatenation discovered that EB is a novel topoisomerase II poison.

Genetic control of bone density and turnover: Role of the collagen 1α1, estrogen receptor, and vitamin D receptor genes

Genetic factors are known to influence both the peak bone mass and probably the rate of change in bone density. A range of regulatory and structural genes has been proposed to be involved including collagen 1α1 (COL1A1), the estrogen receptor (ER), and the vitamin D receptor (VDR), but the actual genes involved are uncertain. We therefore studied the role of the COL1A1 and VDR loci in control of bone density by linkage in 45 dizygotic twin pairs and 29 nuclear families comprising 120 individuals. The influences on bone density of polymorphisms of COL1A1, VDR, and ER were studied by association both cross-sectionally and longitudinally in 193 elderly postmenopausal women (average age, 69 years) over a mean follow-up time of 6.3 years. Weak linkage of the COL1A1 locus with bone density was observed in both twins and families (p = 0.02 in both data sets), confirming previous observations of linkage of this locus with bone density. Association between the MscI polymorphism of COL1A1 and rate of lumbar spine bone loss was observed with significant gene-environment interaction related to dietary calcium intake (p = 0.0006). In the lowest tertile of dietary calcium intake, carriers of "s" alleles lost more bone than "SS" homozygotes (p = 0.01), whereas the opposite was observed in the highest dietary calcium intake (p = 0.003). Association also was observed between rate of bone loss at both the femoral neck and the lumbar spine and the TaqI VDR polymorphism (p = 0.03). This association was strongest in those in the lowest tertile of calcium intake, also suggesting the presence of gene-environment interaction involving dietary calcium and VDR, influencing bone turnover. No significant association was observed between the PvuII ER polymorphism alone or in combination with VDR or COL1A1 genotypes, with either bone density or its rate of change. These data support the involvement of COL1A1 in determination of bone density and the interaction of both COL1A1 and VDR with calcium intake in regulation of change of bone density over time.

Suggestive linkage of the parathyroid receptor type 1 to osteoporosis

We have investigated the role of 23 candidate genes in the control of bone mineral density (BMD) by linkage studies in families of probands with osteoporosis (lumbar spine [LS] or femoral neck [FN] BMD T score < -2.5) and low BMD relative to an age- and gender-matched cohort (Z score < -2.0). One hundred and fifteen probands (35 male, 80 female) and 499 of their first- or second-degree relatives (223 males and 276 females) were recruited for the study. BMD was measured at the LS and FN using dual-energy X-ray absorptiometry and expressed as age- and gender-matched Z scores corrected for body mass index. The candidate genes studied were the androgen receptor, type I collagen A1 (COLIA1), COLIA2, COLIIA1, vitamin D receptor (VDR), colony-stimulating factor 1, calcium-sensing receptor, epidermal growth factor (EGF), estrogen receptor 1 (ESR1), fibrillin type 1, insulin-like growth factor 1, interleukin-1 alpha (IL-1α), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-11 (IL-11), osteopontin, parathyroid hormone (PTH), PTH-related peptide, PTH receptor type 1 (PTHR1), transforming growth factor-beta 1, and tumor necrosis factors alpha and beta. Sixty-four microsatellites lying close to or within these genes were investigated for linkage with BMD. Using the program MapMaker/Sibs there was suggestive evidence of linkage between BMD and PTHR1 (maximum LOD score obtained [MLS] 2.7-3.5). Moderate evidence of linkage was also observed with EGF (MLS 1.8), COLIA1 (MLS 1.7), COLIIA1/VDR (MLS 1.7), ESR1 (MLS 1.4), IL-1α (MLS 1.4), IL-4 (MLS 1.2), and IL-6 (MLS 1.2). Variance components analysis using the program ACT, correcting for proband-wise ascertainment, also showed evidence of linkage (p ≤0.05) at markers close to or within the candidate genes IL- 1α, PTHR1, IL-6, and COLIIA1/VDR. Further studies will be required to confirm these findings, to refine the location of gene responsible for the observed linkage, and to screen the candidate genes targeted at these loci for mutations.

The nature of malnutrition in children with end-stage liver disease awaiting orthotopic liver transplantation

To evaluate malnutrition in chronic liver disease, and its relationship to nutrient deficiencies and hepatic dysfunction. 27 children with end-stage liver disease were studied. Mean protein-energy intakes were 70% of recommended daily intakes. The patients were underweight and stunted with reduced mean triceps and subscapular skinfold thicknesses and midupper arm circumference. Mean total body potassium was only 63 ± 18% of that expected for age and sex. Deficiency of essential fatty acids (32%), and low concentrations of fat-soluble vitamins (A, 92%; E, 32%), iron (32%), zinc (42%), and selenium (13%) were common. Serum ammonia concentrations were raised in all patients, and increased methionine, tyrosine, and glutamic acid, and reduced glutamine concentrations were noted. There was no correlation between the degree of malnutrition and the degree of liver synthetic function, the degree of cholestasis, or the degree of liver injury. We suggest that potentially correctable factors in addition to liver failure (eg, inadequate absorbed intake) were important determinants of malnutrition in these patients.

Inverse Correlation between Vitamin D and C-Reactive Protein in Newborns

Some studies suggested that adequate vitamin D might reduce inflammation in adults. However, little is known about this association in early life. We aimed to determine the relationship between cord blood 25-hydroxyvitamin D (25(OH)D) and C-reactive protein (CRP) in neonates. Cord blood levels of 25(OH)D and CRP were measured in 1491 neonates in Hefei, China. Potential confounders including maternal sociodemographic characteristics, perinatal health status, lifestyle, and birth outcomes were prospectively collected. The average values of cord blood 25(OH)D and CRP were 39.43 nmol/L (SD = 20.35) and 6.71 mg/L (SD = 3.07), respectively. Stratified by 25(OH)D levels, per 10 nmol/L increase in 25(OH)D, CRP decreased by 1.42 mg/L (95% CI: 0.90, 1.95) among neonates with 25(OH)D <25.0 nmol/L, and decreased by 0.49 mg/L (95% CI: 0.17, 0.80) among neonates with 25(OH)D between 25.0 nmol/L and 49.9 nmol/L, after adjusting for potential confounders. However, no significant association between 25(OH)D and CRP was observed among neonates with 25(OH)D ≥50 nmol/L. Cord blood 25(OH)D and CRP levels showed a significant seasonal trend with lower 25(OH)D and higher CRP during winter-spring than summer-autumn. Stratified by season, a significant linear association of 25(OH)D with CRP was observed in neonates born in winter-spring (adjusted β = −0.11, 95% CI: −0.13, −0.10), but not summer-autumn. Among neonates born in winter-spring, neonates with 25(OH)D <25 nmol/L had higher risk of CRP ≥10 mg/L (adjusted OR = 3.06, 95% CI: 2.00, 4.69), compared to neonates with 25(OH)D ≥25 nmol/L. Neonates with vitamin D deficiency had higher risk of exposure to elevated inflammation at birth.

Effects of methyl eugenol feeding on mating compatibility of Asian population of Bactrocera dorsalis (Diptera: Tephritidae) with African population and with B. carambolae

Males of some species included in the Bactrocera dorsalis complex are strongly attracted to methyl eugenol (ME) (1,2-dimethoxy-4-(2-propenyl) benzene), a natural compound occurring in a variety of plant species. ME feeding of males of the B. dorsalis complex is known to enhance their mating competitiveness. Within B. dorsalis, recent studies show that Asian and African populations of B. dorsalis are sexually compatible, while populations of B. dorsalis and Bactrocera carambolae are relatively incompatible. The objectives of this study were to examine whether ME feeding by males affects mating compatibility between Asian and African populations of B. dorsalis and ME feeding reduces male mating incompatibility between B. dorsalis (Asian population) and B. carambolae. The data confirmed that Asian and African populations of B. dorsalis are sexually compatible for mating and showed that ME feeding only increased the number of matings. Though ME feeding also increased the number of matings of B. dorsalis (Asian population) and B. carambolae males but the sexual incompatibility between both species was not reduced by treatment with ME. These results conform to the efforts resolving the biological species limits among B. dorsalis complex and have implications for fruit fly control programs in fields and horticultural trade.

Phylogenetic lineage and pilus protein Spb1/SAN1518 affect opsonin-independent phagocytosis and intracellular survival of Group B Streptococcus

Opsonin-independent phagocytosis of Group B Streptococcus (GBS) is important in defense against neonatal GBS infections. A recent study indicated a role for GBS pilus in macrophage phagocytosis (Maisey et al Faseb J 22 2008 1715-24). We studied 163 isolates from different phylogenetic backgrounds and those possessing or lacking the gene encoding the pilus backbone protein, Spb1 (SAN1518, PI-2b) and spb1-deficient mutants of wild-type (WT) serotype III-3 GBS 874391 in non-opsonic phagocytosis assays using J774A.1 macrophages. Numbers of GBS phagocytosed differed up to 23-fold depending on phylogenetic background; isolates possessing spb1 were phagocytosed more than isolates lacking spb1. Comparing WT GBS and isogenic spb1-deficient mutants showed WT was phagocytosed better compared to mutants; Spb1 also enhanced intracellular survival as mutants were killed more efficiently. Complementation of mutants restored phagocytosis and resistance to killing in J774A.1 macrophages. Spb1 antiserum revealed surface expression in WT GBS and spatial distribution relative to capsular polysaccharide. spb1 did not affect macrophage nitric oxide and TNF-alpha responses; differences in phagocytosis did not correlate with N-acetyl d-glucosamine (from GBS cell-wall) according to enzyme-linked lectin-sorbent assay. Together, these findings support a role for phylogenetic lineage and Spb1 in opsonin-independent phagocytosis and intracellular survival of GBS in J774A.1 macrophages.

Acute fluid shifts influence the assessment of serum vitamin D status in critically ill patients

Introduction Recent reports have highlighted the prevalence of vitamin D deficiency and suggested an association with excess mortality in critically ill patients. Serum vitamin D concentrations in these studies were measured following resuscitation. It is unclear whether aggressive fluid resuscitation independently influences serum vitamin D. Methods Nineteen patients undergoing cardiopulmonary bypass were studied. Serum 25(OH)D3, 1α,25(OH)2D3, parathyroid hormone, C-reactive protein (CRP), and ionised calcium were measured at five defined timepoints: T1 - baseline, T2 - 5 minutes after onset of cardiopulmonary bypass (CPB) (time of maximal fluid effect), T3 - on return to the intensive care unit, T4 - 24 hrs after surgery and T5 - 5 days after surgery. Linear mixed models were used to compare measures at T2-T5 with baseline measures. Results Acute fluid loading resulted in a 35% reduction in 25(OH)D3 (59 ± 16 to 38 ± 14 nmol/L, P < 0.0001) and a 45% reduction in 1α,25(OH)2D3 (99 ± 40 to 54 ± 22 pmol/L P < 0.0001) and i(Ca) (P < 0.01), with elevation in parathyroid hormone (P < 0.0001). Serum 25(OH)D3 returned to baseline only at T5 while 1α,25(OH)2D3 demonstrated an overshoot above baseline at T5 (P < 0.0001). There was a delayed rise in CRP at T4 and T5; this was not associated with a reduction in vitamin D levels at these time points. Conclusions Hemodilution significantly lowers serum 25(OH)D3 and 1α,25(OH)2D3, which may take up to 24 hours to resolve. Moreover, delayed overshoot of 1α,25(OH)2D3 needs consideration. We urge caution in interpreting serum vitamin D in critically ill patients in the context of major resuscitation, and would advocate repeating the measurement once the effects of the resuscitation have abated.

The role of 25-hydroxyvitamin D deficiency in promoting insulin resistance and inflammation in patients with chronic kidney disease: A randomised controlled trial

BACKGROUND Approximately 50% of patients with stage 3 Chronic Kidney Disease are 25-hydroxyvitamin D insufficient, and this prevalence increases with falling glomerular filtration rate. Vitamin D is now recognised as having pleiotropic roles beyond bone and mineral homeostasis, with the vitamin D receptor and metabolising machinery identified in multiple tissues. Worryingly, recent observational data has highlighted an association between hypovitaminosis D and increased cardiovascular mortality, possibly mediated via vitamin D effects on insulin resistance and inflammation. The main hypothesis of this study is that oral Vitamin D supplementation will ameliorate insulin resistance in patients with Chronic Kidney Disease stage 3 when compared to placebo. Secondary hypotheses will test whether this is associated with decreased inflammation and bone/adipocyte-endocrine dysregulation. METHODS/DESIGN This study is a single-centre, double-blinded, randomised, placebo-controlled trial. Inclusion criteria include; estimated glomerular filtration rate 30-59 ml/min/1.73 m(2); aged >or=18 on entry to study; and serum 25-hydroxyvitamin D levels <75 nmol/L. Patients will be randomised 1:1 to receive either oral cholecalciferol 2000IU/day or placebo for 6 months. The primary outcome will be an improvement in insulin sensitivity, measured by hyperinsulinaemic euglycaemic clamp. Secondary outcome measures will include serum parathyroid hormone, cytokines (Interleukin-1beta, Interleukin-6, Tumour Necrosis Factor alpha), adiponectin (total and High Molecular Weight), osteocalcin (carboxylated and under-carboxylated), peripheral blood mononuclear cell Nuclear Factor Kappa-B p65 binding activity, brachial artery reactivity, aortic pulse wave velocity and waveform analysis, and indirect calorimetry. All outcome measures will be performed at baseline and end of study. DISCUSSION To date, no randomised controlled trial has been performed in pre-dialysis CKD patients to study the correlation between vitamin D status with supplementation, insulin resistance and markers of adverse cardiovascular risk. We remain hopeful that cholecalciferol may be a safe intervention, with health benefits beyond those related to bone-mineral homeostasis. TRIAL REGISTRATION Australian and New Zealand Clinical Trials Registry ACTRN12609000246280.

Defects in the IFT-B component IFT172 cause Jeune and Mainzer-Saldino syndromes in humans

Intraflagellar transport (IFT) depends on two evolutionarily conserved modules, subcomplexes A (IFT-A) and B (IFT-B), to drive ciliary assembly and maintenance. All six IFT-A components and their motor protein, DYNC2H1, have been linked to human skeletal ciliopathies, including asphyxiating thoracic dystrophy (ATD; also known as Jeune syndrome), Sensenbrenner syndrome, and Mainzer-Saldino syndrome (MZSDS). Conversely, the 14 subunits in the IFT-B module, with the exception of IFT80, have unknown roles in human disease. To identify additional IFT-B components defective in ciliopathies, we independently performed different mutation analyses: candidate-based sequencing of all IFT-B-encoding genes in 1,467 individuals with a nephronophthisis-related ciliopathy or whole-exome resequencing in 63 individuals with ATD. We thereby detected biallelic mutations in the IFT-B-encoding gene IFT172 in 12 families. All affected individuals displayed abnormalities of the thorax and/or long bones, as well as renal, hepatic, or retinal involvement, consistent with the diagnosis of ATD or MZSDS. Additionally, cerebellar aplasia or hypoplasia characteristic of Joubert syndrome was present in 2 out of 12 families. Fibroblasts from affected individuals showed disturbed ciliary composition, suggesting alteration of ciliary transport and signaling. Knockdown of ift172 in zebrafish recapitulated the human phenotype and demonstrated a genetic interaction between ift172 and ift80. In summary, we have identified defects in IFT172 as a cause of complex ATD and MZSDS. Our findings link the group of skeletal ciliopathies to an additional IFT-B component, IFT172, similar to what has been shown for IFT-A.

Ratios of T-cell immune-effectors with tumour associated macrophages and PD-1/PD-L1 axis immune-checkpoint molecules, as prognosticators in diffuse large B cell lymphoma: A population-based study

Background Risk-stratification of diffuse large B-cell lymphoma (DLBCL) requires identification of patients with disease that is not cured despite initial R-CHOP. Although the prognostic importance of the tumour microenvironment (TME) is established, the optimal strategy to quantify it is unknown. Methods The relationship between immune-effector and inhibitory (checkpoint) genes was assessed by NanoString™ in 252 paraffin-embedded DLBCL tissues. A model to quantify net anti-tumoural immunity as an outcome predictor was tested in 158 R-CHOP treated patients, and validated in tissue/blood from two independent R-CHOP treated cohorts of 233 and 140 patients respectively. Findings T and NK-cell immune-effector molecule expression correlated with tumour associated macrophage and PD-1/PD-L1 axis markers consistent with malignant B-cells triggering a dynamic checkpoint response to adapt to and evade immune-surveillance. A tree-based survival model was performed to test if immune-effector to checkpoint ratios were prognostic. The CD4*CD8:(CD163/CD68)*PD-L1 ratio was better able to stratify overall survival than any single or combination of immune markers, distinguishing groups with disparate 4-year survivals (92% versus 47%). The immune ratio was independent of and added to the revised international prognostic index (R-IPI) and cell-of-origin (COO). Tissue findings were validated in 233 DLBCL R-CHOP treated patients. Furthermore, within the blood of 140 R-CHOP treated patients immune-effector:checkpoint ratios were associated with differential interim-PET/CT+ve/-ve expression.

Novel synthesis of superparamagnetic Ni-Co-B nanoparticles and their effect on superconductor properties of MgB2

A new procedure for the preparation of amorphous Ni-Co-B nanoparticles is reported, with a detailed investigation of their morphology by X-ray diffraction and transmission electron microscopy, as well as their magnetic properties. Many factors, such as chemical composition, anisotropy, size and shape of the particles, were controlled through chemical synthesis, resulting in the control of morphological and magnetic properties of the nanoparticles. Controlling pH values with ethylenediamine and using sodium dodecyl sulfate surfactant lowered the size of the nanoparticles to below 10 nm. Such a small structure and chemical disorder in nanocrystalline materials lead to magnetic properties that are different from those in their bulk-sized counterparts. The obtained nanoparticles can be used for different purposes, from pharmaceutical applications to implementations in different materials technology. The focus of this research is the synthesis of Ni-Co-B nanoparticles in a new way and studying the reaction of Ni-Co-B nanoparticles with Mg and B precursors and their effect on MgB2 properties. New nanostructures are formed in the reaction of Ni-Co-B nanoparticles with Mg: Mg2Ni, Co2Mg and possibly Mg2Co.

Nano-confined synthesis of highly ordered mesoporous carbon and its performance as electrode material for electrochemical behavior of riboflavin (vitamin B2) and dopamine

Highly ordered mesoporous carbon (MC) has been synthesized from sucrose, a non-toxic and costeffective source of carbon. X-ray diffraction, N2 adsorption–desorption isotherm and transmission electron micrograph (TEM) were used to characterize the MC. The XRD patterns show the formation of highly ordered mesoporous structures of SBA15 and mesoporous carbon. The N2 adsorptiondesorption isotherms suggest that the MC exhibits a narrow pore-size distribution with high surface area of 1559 m2/g. The potential application of MC as a novel electrode material was investigated using cyclic voltammetry for riboflavin (vitamin B2) and dopamine. MC-modified glassy carbon electrode (MC/GC) shows increase in peak current compared to GC electrode in potassium ferricyanide which clearly suggest that MC/GC possesses larger electrode area (1.8 fold) compared with bare GC electrode. The electrocatalytic behavior of MC/GC was investigated towards the oxidation of riboflavin (vitamin B2) and dopamine using cyclic voltammetry which show larger oxidation current compared to unmodified electrode and thus MC/GC may have the potential to be used as a chemically modified electrode.