Carriers of Loss-of-Function Mutations in EXT Display Impaired Pancreatic Beta-Cell Reserve Due to Smaller Pancreas Volume

Exotosin (EXT) proteins are involved in the chain elongation step of heparan sulfate (HS) biosynthesis, which is intricately involved in organ development. Loss of function mutations (LOF) in EXT1 and EXT2 result in hereditary exostoses (HME). Interestingly, HS plays a role in pancreas development and beta-cell function, and genetic variations in EXT2 are associated with an increased risk for type 2 diabetes mellitus. We hypothesized that loss of function of EXT1 or EXT2 in subjects with hereditary multiple exostoses (HME) affects pancreatic insulin secretion capacity and development. We performed an oral glucose tolerance test (OGTT) followed by hyperglycemic clamps to investigate first-phase glucose-stimulated insulin secretion (GSIS) in HME patients and age and gender matched non-affected relatives. Pancreas volume was assessed with magnetic resonance imaging (MRI). OGTT did not reveal significant differences in glucose disposal, but there was a markedly lower GSIS in HME subjects during hyperglycemic clamp (iAUC HME: 0.72 [0.46–1.16] vs. controls 1.53 [0.69–3.36] nmol·l⁻¹·min⁻¹, p<0.05). Maximal insulin response following arginine challenge was also significantly attenuated (iAUC HME: 7.14 [4.22–10.5] vs. controls 10.2 [7.91–12.70] nmol·l⁻¹·min⁻¹ p<0.05), indicative of an impaired beta-cell reserve. MRI revealed a significantly smaller pancreatic volume in HME subjects (HME: 72.0±15.8 vs. controls 96.5±26.0 cm³ p = 0.04). In conclusion, loss of function of EXT proteins may affect beta-cell mass and insulin secretion capacity in humans, and render subjects at a higher risk of developing type 2 diabetes when exposed to environmental risk factors.     

Quantification of Left Ventricular Torsion and Diastolic Recoil Using Cardiovascular Magnetic Resonance Myocardial Feature Tracking

Objectives

Cardiovascular magnetic resonance feature tracking (CMR-FT) offers quantification of myocardial deformation from routine cine images. However, data using CMR-FT to quantify left ventricular (LV) torsion and diastolic recoil are not yet available. We therefore sought to evaluate the feasibility and reproducibility of CMR-FT to quantify LV torsion and peak recoil rate using an optimal anatomical approach.

Methods

Short-axis cine stacks were acquired at rest and during dobutamine stimulation (10 and 20 µg·kg⁻¹·min⁻¹) in 10 healthy volunteers. Rotational displacement was analysed for all slices. A complete 3D-LV rotational model was developed using linear interpolation between adjacent slices. Torsion was defined as the difference between apical and basal rotation, divided by slice distance. Depending on the distance between the most apical (defined as 0% LV distance) and basal (defined as 100% LV distance) slices, four different models for the calculation of torsion were examined: Model-1 (25–75%), Model-2 (0–100%), Model-3 (25–100%) and Model-4 (0–75%). Analysis included subendocardial, subepicardial and global torsion and recoil rate (mean of subendocardial and subepicardial values).

Results

Quantification of torsion and recoil rate was feasible in all subjects. There was no significant difference between the different models at rest. However, only Model-1 (25–75%) discriminated between rest and stress (Global Torsion: 2.7±1.5°cm⁻¹, 3.6±2.0°cm⁻¹, 5.1±2.2°cm⁻¹, p<0.01; Global Recoil Rate: −30.1±11.1°cm⁻¹s⁻¹,−46.9±15.0°cm⁻¹s⁻¹,−68.9±32.3°cm⁻¹s⁻¹, p<0.01; for rest, 10 and 20 µg·kg⁻¹·min⁻¹ of dobutamine, respectively). Reproducibility was sufficient for all parameters as determined by Bland-Altman analysis, intraclass correlation coefficients and coefficient of variation.

Conclusions

CMR-FT based derivation of myocardial torsion and recoil rate is feasible and reproducible at rest and with dobutamine stress. Using an optimal anatomical approach measuring rotation at 25% and 75% apical and basal LV locations allows effective quantification of torsion and recoil dynamics. Application of these new measures of deformation by CMR-FT should next be explored in disease states.     

Effects of T-Type Calcium Channel Blockers on Renal Function and Aldosterone in Patients with Hypertension: A Systematic Review and Meta-Analysis

Background

High blood pressure can cause kidney damage, which can increase blood pressure, leading to a vicious cycle. It is not clear whether the protective effects of T-type calcium channel blockers (T-type CCBs) on renal function are better than those of L-type CCBs or renin-angiotensin system (RAS) antagonists in patients with hypertension.

Methods and Findings

PUBMED, MEDLINE, EMBASE, OVID, Web of Science, Cochrane, CNKI, MEDCH, VIP, and WANFANG databases were searched for clinical trials published in English or Chinese from January 1, 1990, to December 31, 2013. The weighted mean difference (WMD) and 95% confidence interval (CI) were calculated and reported. A total of 1494 reports were collected, of which 24 studies with 1,696 participants (including 809 reports comparing T-type CCBs versus L-type CCBs and 887 reports comparing T-type CCB versus RAS antagonists) met the inclusion criteria. Compared with L-type CCBs, T-type CCBs resulted in a significant decline in aldosterone (mean difference = −15.19, 95% CI −19.65–−10.72, p<1×10⁻⁵), proteinuria (mean difference = −0.73, 95% CI −0.88–−0.57, p<1×10⁻⁵), protein to creatinine ratio (mean difference = −0.22, 95% CI −0.41–−0.03, p = 0.02), and urinary albumin to creatinine ratio (mean difference = −55.38, 95% CI −86.67–−24.09, p = 0.0005); no significant difference was noted for systolic blood pressure (SBP) (p = 0.76) and diastolic blood pressure (DBP) (p = 0.16). The effects of T-type CCBs did not significantly differ from those of RAS antagonists for SBP (p = 0.98), DBP (p = 0.86), glomerular filtration rate (p = 0.93), albuminuria (p = 0.97), creatinine clearance rate (p = 0.24), and serum creatinine (p = 0.27) in patients with hypertension.

Conclusion

In a pooled analysis of data from 24 studies measuring the effects of T-type CCBs on renal function and aldosterone, the protective effects of T-type CCBs on renal function were enhanced compared with L-type CCBs but did not differ from RAS antagonists. Their protective effects on renal function were independent of blood pressure.     

Fetal DNA Methylation Associates with Early Spontaneous Preterm Birth and Gestational Age

Spontaneous preterm birth (PTB, <37 weeks gestation) is a major public health concern, and children born preterm have a higher risk of morbidity and mortality throughout their lives. Recent studies suggest that fetal DNA methylation of several genes varies across a range of gestational ages (GA), but it is not yet clear if fetal epigenetic changes associate with PTB. The objective of this study is to interrogate methylation patterns across the genome in fetal leukocyte DNA from African Americans with early PTB (24^1/7–34^0/7 weeks; N = 22) or term births (39^0/7–40^6/7weeks; N = 28) and to evaluate the association of each CpG site with PTB and GA. DNA methylation was assessed across the genome with the HumanMethylation450 BeadChip. For each individual sample and CpG site, the proportion of DNA methylation was estimated. The associations between methylation and PTB or GA were evaluated by fitting a separate linear model for each CpG site, adjusting for relevant covariates. Overall, 29 CpG sites associated with PTB (FDR<.05; 5.7×10⁻¹⁰<p<2.9×10⁻⁶) independent of GA. Also, 9637 sites associated with GA (FDR<.05; 9.5×10⁻¹⁶<p<1.0×10⁻³), with 61.8% decreasing in methylation with shorter GA. GA-associated CpG sites were depleted in the CpG islands of their respective genes (p<2.2×10⁻¹⁶). Gene set enrichment analysis (GSEA) supported enrichment of GA-associated CpG sites in genes that play a role in embryonic development as well as the extracellular matrix. Additionally, this study replicated the association of several CpG sites associated with gestational age in other studies (CRHBP, PIK3CD and AVP). Dramatic differences in fetal DNA methylation are evident in fetuses born preterm versus at term, and the patterns established at birth may provide insight into the long-term consequences associated with PTB.     

Effect of Salicylic Acid Formulations on Induced Plant Defense against Cassava Anthracnose Disease

This study was to investigate defense mechanisms on cassava induced by salicylic acid formulation (SA) against anthracnose disease. Our results indicated that the SA could reduce anthracnose severity in cassava plants up to 33.3% under the greenhouse condition. The β-1,3-glucanase and chitinase enzyme activities were significantly increased at 24 hours after inoculation (HAI) and decrease at 48 HAI after Colletotrichum gloeosporioides challenge inoculation, respectively, for cassava treated with SA formulation. Synchrotron radiation–based Fourier-transform infrared microspectroscopy spectra revealed changes of the C=H stretching vibration (3,000–2,800 cm⁻¹), pectin (1,740–1,700 cm⁻¹), amide I protein (1,700–1,600 cm⁻¹), amide II protein (1,600–1,500 cm⁻¹), lignin (1,515 cm⁻¹) as well as mainly C–O–C of polysaccharides (1,300–1,100 cm⁻¹) in the leaf epidermal and mesophyll tissues treated with SA formulations, compared to those treated with fungicide carbendazim and distilled water after the challenged inoculation with C. gloeosporioides. The results indicate that biochemical changes in cassava leaf treated with SA played an important role in the enhancement of structural and chemical defense mechanisms leading to reduced anthracnose severity.     

Cardiac Compression of Lung Lower Lobes after Coronary Artery Bypass Graft with Cardiopulmonary Bypass

Background

Atelectasis is a major cause of hypoxemia after coronary artery bypass grafting (CABG) and is commonly ascribed to general anesthesia, high inspiratory oxygen concentration and cardiopulmonary bypass (CPB). The objective of this study was to evaluate the role of heart-induced pulmonary compression after CABG with CPB.

Methods

Seventeen patients without pre-operative cardiac failure who were scheduled for coronary artery bypass graft underwent pre- and postoperative thoracic computed tomography. The cardiac mass, the pressure exerted on the lungs by the right and left heart and the fraction of collapsed lower lobe segments below and outside of the heart limits were evaluated on a computed tomography section 1 cm above the diaphragmatic cupola.

Results

In the postoperative period, cardiac mass increased by 32% (117±31 g versus 155±35 g, p<0.001), leading to an increase in the pressure that was exerted on the lungs by the right (2.2±0.6 g.cm⁻² versus 3.2±1.2 g.cm⁻², p<0.05) and left heart (2.4±0.7 g.cm⁻² versus 4.2±1.8 g.cm⁻², p<0.001). The proportion of collapsed lung segments beneath the heart markedly increased [from 6.7% to 32.9% on the right side (p<0.001) and from 6.2% to 29% on the left side (p<0.001)], whereas the proportion of collapsed lung segments outside of the heart limits slightly increased [from 0.7% to 10.8% on the right side (p<0.001) and from 1.5% to 12.6% on the left side (p<0.001)].

Conclusion

The pressure that is exerted by the heart on the lungs increased postoperatively and contributed to the collapse of subjacent pulmonary segments.     

Functional Enhancement of Electrofusion-derived BRIN-BD11 Insulin-secreting Cells After Implantation into Diabetic Mice

Electrofusion-derived BRIN-BD11 cells are glucosesensitive insulin-secreting cells which provide an archetypal bioengineered surrogate β-cell for insulin replacement therapy in diabetes mellitus, 5x10⁶ BRIN-BD11 cells were implanted intraperitoneally into severely hyperglycaemic (>24mmol/l) streptozotocin-induced insulin-treated diabetic athymic nude (nu/nu) mice. The implants reduced hyperglycaemia such that insulin injections were discontinued by 5–16 days (<17mmol/l) and normoglycaemia (<9mmol/l) was achieved by 7–20 days. Implanted cells were removed after 28 days and re-established in culture. After re-culture for 20 days, glucose-stimulated (16.7mmol/l) insulin release was enhanced by 121% (p<0.001) compared to non-implanted cells. Insulin responses to glucagon-like peptide-1 (10⁻⁹mol/l), cholecystokinin-8 (10⁻⁸ mol/l) and L-alanine (10 mmol/l) were increased by 32%, 31% and 68% respectively (p<0.05–0.01). Insulin content of the cells was 148% greater at 20 days after re-culture than before implantation (p<0.001), but basal insulin release (at 5.6 mmol/l glucose) was not changed. After re-culture for 40 days, insulin content declined to 68% of the content before implantation (p<0.01), although basal insulin release was unchanged. However, the insulin secretory responses to glucose, glucagonlike peptide-1, cholecystokinin-8 and L-alanine were decreased after 40 days of re-culture to 65%, 72%, 73% and 42% respectively of the values before implantation (p<0.05–0.01). The functional enhancement of electrofusion-derived surrogate β-cells that were re-cultured for 20 days after implantation and restoration of normoglycaemia indicates that the in vivo environment could greatly assist β-cell engineering approaches to therapy for diabetes.     

t-10, c-12 CLA Dietary Supplementation Inhibits Atherosclerotic Lesion Development Despite Adverse Cardiovascular and Hepatic Metabolic Marker Profiles

Animal and human studies have indicated that fatty acids such as the conjugated linoleic acids (CLA) found in milk could potentially alter the risk of developing metabolic disorders including diabetes and cardiovascular disease (CVD). Using susceptible rodent models (apoE^−/− and LDLr^−/− mice) we investigated the interrelationship between mouse strain, dietary conjugated linoleic acids and metabolic markers of CVD. Despite an adverse metabolic risk profile, atherosclerosis (measured directly by lesion area), was significantly reduced with t-10, c-12 CLA and mixed isomer CLA (Mix) supplementation in both apoE^−/− (p<0.05, n = 11) and LDLr^−/− mice (p<0.01, n = 10). Principal component analysis was utilized to delineate the influence of multiple plasma and tissue metabolites on the development of atherosclerosis. Group clustering by dietary supplementation was evident, with the t-10, c-12 CLA supplemented animals having distinct patterns, suggestive of hepatic insulin resistance, regardless of mouse strain. The effect of CLA supplementation on hepatic lipid and fatty acid composition was explored in the LDLr^−/− strain. Dietary supplementation with t-10, c-12 CLA significantly increased liver weight (p<0.05, n = 10), triglyceride (p<0.01, n = 10) and cholesterol ester content (p<0.01, n = 10). Furthermore, t-10, c-12 CLA also increased the ratio of 18∶1 to 18∶0 fatty acid in the liver suggesting an increase in the activity of stearoyl-CoA desaturase. Changes in plasma adiponectin and liver weight with t-10, c-12 CLA supplementation were evident within 3 weeks of initiation of the diet. These observations provide evidence that the individual CLA isomers have divergent mechanisms of action and that t-10, c-12 CLA rapidly changes plasma and liver markers of metabolic syndrome, despite evidence of reduction in atherosclerosis.     

Sinupret Activates CFTR and TMEM16A-Dependent Transepithelial Chloride Transport and Improves Indicators of Mucociliary Clearance

Introduction

We have previously demonstrated that Sinupret, an established treatment prescribed widely in Europe for respiratory ailments including rhinosinusitis, promotes transepithelial chloride (Cl⁻) secretion in vitro and in vivo. The present study was designed to evaluate other indicators of mucociliary clearance (MCC) including ciliary beat frequency (CBF) and airway surface liquid (ASL) depth, but also investigate the mechanisms that underlie activity of this bioflavonoid.

Methods

Primary murine nasal septal epithelial (MNSE) [wild type (WT) and transgenic CFTR^−/−], human sinonasal epithelial (HSNE), WT CFTR-expressing CFBE and TMEM16A-expressing HEK cultures were utilized for the present experiments. CBF and ASL depth measurements were performed. Mechanisms underlying transepithelial Cl⁻ transport were determined using pharmacologic manipulation in Ussing chambers, Fura-2 intracellular calcium [Ca²⁺]_i imaging, cAMP signaling, regulatory domain (R-D) phosphorylation of CFTR, and excised inside out and whole cell patch clamp analysis.

Results

Sinupret-mediated Cl⁻ secretion [ΔI_SC(µA/cm²)] was pronounced in WT MNSE (20.7+/−0.9 vs. 5.6+/−0.9(control), p<0.05), CFTR^−/− MNSE (10.1+/−1.0 vs. 0.9+/−0.3(control), p<0.05) and HSNE (20.7+/−0.3 vs. 6.4+/−0.9(control), p<0.05). The formulation activated Ca²⁺ signaling and TMEM16A channels, but also increased CFTR channel open probability (Po) without stimulating PKA-dependent pathways responsible for phosphorylation of the CFTR R-domain and resultant Cl⁻ secretion. Sinupret also enhanced CBF and ASL depth.

Conclusion

Sinupret stimulates CBF, promotes transepithelial Cl⁻ secretion, and increases ASL depth in a manner likely to enhance MCC. Our findings suggest that direct stimulation of CFTR, together with activation of Ca²⁺-dependent TMEM16A secretion account for the majority of anion transport attributable to Sinupret. These studies provide further rationale for using robust Cl⁻ secretagogue based therapies as an emerging treatment modality for common respiratory diseases of MCC including acute and chronic bronchitis and CRS.     

Neutrophil-Derived Myeloperoxidase Aggravates Non-Alcoholic Steatohepatitis in Low-Density Lipoprotein Receptor-Deficient Mice

Background

Chronic inflammation and oxidative stress play fundamental roles in the pathogenesis of non-alcoholic steatohepatitis (NASH). Previously, we reported that myeloperoxidase (MPO), an aggressive oxidant-generating neutrophil enzyme, is associated with NASH severity in man. We now investigated the hypothesis that MPO contributes to the development and progression of NASH.

Methodology

Low-density lipoprotein receptor-deficient mice with an MPO-deficient hematopoietic system (LDLR^−/−/MPO^−/−tp mice) were generated and compared with LDLR^−/−/MPO^+/+tp mice after induction of NASH by high-fat feeding.

Results

High-fat feeding caused a ∼4-fold induction of liver MPO in LDLR^−/−/MPO^+/+ mice which was associated with hepatic sequestration of MPO-positive neutrophils and high levels of nitrotyrosine, a marker of MPO activity. Importantly, LDLR^−/−/MPO^−/−tp mice displayed markedly reduced hepatic neutrophil and T-lymphocyte infiltration (p<0.05), and strong down regulation of pro-inflammatory genes such as TNF-α and IL-6 (p<0.05, p<0.01) in comparison with LDLR^−/−/MPO^+/+tp mice. Next to the generalized reduction of inflammation, liver cholesterol accumulation was significantly diminished in LDLR^−/−/MPO^−/−tp mice (p = 0.01). Moreover, MPO deficiency appeared to attenuate the development of hepatic fibrosis as evident from reduced hydroxyproline levels (p<0.01). Interestingly, visceral adipose tissue inflammation was markedly reduced in LDLR^−/−/MPO^−/−tp mice, with a complete lack of macrophage crown-like structures. In conclusion, MPO deficiency attenuates the development of NASH and diminishes adipose tissue inflammation in response to a high fat diet, supporting an important role for neutrophils in the pathogenesis of metabolic disease.     

Central Nervous System PET-CT Imaging Reveals Regional Impairments in Pediatric Patients with Wolfram Syndrome

Wolfram syndrome (WFS) is inherited as an autosomal recessive disease with main clinical features of diabetes mellitus, optic atrophy, diabetes insipidus and deafness. However, various neurological defects may also be detected. The aim of this study was to evaluate aspects of brain structure and function using PET-CT (positron emission tomography and computed tomography) and MRI (magnetic resonance imaging) in pediatric patients with WFS. Regional changes in brain glucose metabolism were measured using standardized uptake values (SUVs) based on images of (¹⁸F) fluorodeoxyglucose (FDG) uptake in 7 WFS patients aged 10.1–16.0 years (mean 12.9±2.4) and in 20 healthy children aged 3–17.9 years (mean 12.8±4.1). In all patients the diagnosis of WFS was confirmed by DNA sequencing of the WFS1 gene. Hierarchical clustering showed remarkable similarities of glucose uptake patterns among WFS patients and their differences from the control group. SUV data were subsequently standardized for age groups <13 years old and>13 years old to account for developmental differences. Reduced SUVs in WFS patients as compared to the control group for the bilateral brain regions such as occipital lobe (−1.24±1.20 vs. −0.13±1.05; p = 0.028) and cerebellum (−1.11±0.69 vs. −0.204±1.00; p = 0.036) were observed and the same tendency for cingulate (−1.13±1.05 vs. −0.15±1.12; p = 0.056), temporal lobe (−1.10±0.98 vs. −0.15±1.10; p = 0.057), parietal lobe (−1.06±1.20 vs. −0.08±1.08; p = 0.058), central region (−1.01±1.04 vs. −0.09±1.06; p = 0.060), basal ganglia (−1.05±0.74 vs. −0.20±1.07; p = 0.066) and mesial temporal lobe (−1.06±0.82 vs. −0.26±1.08; p = 0.087) was also noticed. After adjusting for multiple hypothesis testing, the differences in glucose uptake were non-significant. For the first time, regional differences in brain glucose metabolism among patients with WFS were shown using PET-CT imaging.     

Low Concentrations of o,p’-DDT Inhibit Gene Expression and Prostaglandin Synthesis by Estrogen Receptor-Independent Mechanism in Rat Ovarian Cells

o,p’-DDT is an infamous xenoestrogen as well as a ubiquitous and persistent pollutant. Biomonitoring studies show that women have been internally exposed to o,p’-DDT at range of 0.3–500 ng/g (8.46×10⁻¹⁰ M−1.41×10⁻⁶ M) in blood and other tissues. However, very limited studies have investigated the biological effects and mechanism(s) of o,p’-DDT at levels equal to or lower than current exposure levels in human. In this study, using primary cultures of rat ovarian granulosa cells, we determined that very low doses of o,p’-DDT (10⁻¹²−10⁻⁸ M) suppressed the expression of ovarian genes and production of prostaglandin E2 (PGE2). In vivo experiments consistently demonstrated that o,p’-DDT at 0.5–1 mg/kg inhibited the gene expression and PGE2 levels in rat ovary. The surprising results from the receptor inhibitors studies showed that these inhibitory effects were exerted independently of either classical estrogen receptors (ERs) or G protein-coupled receptor 30 (GPR30). Instead, o,p’-DDT altered gene expression or hormone action via inhibiting the activation of protein kinase A (PKA), rather than protein kinase C (PKC). We further revealed that o,p’-DDT directly interfered with the PKA catalytic subunit. Our novel findings support the hypothesis that exposure to low concentrations of o,p’-DDT alters gene expression and hormone synthesis through signaling mediators beyond receptor binding, and imply that the current exposure levels of o,p’-DDT observed in the population likely poses a health risk to female reproduction.     

Human Spermatogenic Failure Purges Deleterious Mutation Load from the Autosomes and Both Sex Chromosomes,including the Gene DMRT1

Gonadal failure, along with early pregnancy loss and perinatal death, may be an important filter that limits the propagation of harmful mutations in the human population. We hypothesized that men with spermatogenic impairment, a disease with unknown genetic architecture and a common cause of male infertility, are enriched for rare deleterious mutations compared to men with normal spermatogenesis. After assaying genomewide SNPs and CNVs in 323 Caucasian men with idiopathic spermatogenic impairment and more than 1,100 controls, we estimate that each rare autosomal deletion detected in our study multiplicatively changes a man''s risk of disease by 10% (OR 1.10 [1.04–1.16], p<2×10⁻³), rare X-linked CNVs by 29%, (OR 1.29 [1.11–1.50], p<1×10⁻³), and rare Y-linked duplications by 88% (OR 1.88 [1.13–3.13], p<0.03). By contrasting the properties of our case-specific CNVs with those of CNV callsets from cases of autism, schizophrenia, bipolar disorder, and intellectual disability, we propose that the CNV burden in spermatogenic impairment is distinct from the burden of large, dominant mutations described for neurodevelopmental disorders. We identified two patients with deletions of DMRT1, a gene on chromosome 9p24.3 orthologous to the putative sex determination locus of the avian ZW chromosome system. In an independent sample of Han Chinese men, we identified 3 more DMRT1 deletions in 979 cases of idiopathic azoospermia and none in 1,734 controls, and found none in an additional 4,519 controls from public databases. The combined results indicate that DMRT1 loss-of-function mutations are a risk factor and potential genetic cause of human spermatogenic failure (frequency of 0.38% in 1306 cases and 0% in 7,754 controls, p = 6.2×10⁻⁵). Our study identifies other recurrent CNVs as potential causes of idiopathic azoospermia and generates hypotheses for directing future studies on the genetic basis of male infertility and IVF outcomes.     

GIS-Based Multi-Criteria Analysis for Arabica Coffee Expansion in Rwanda

The Government of Rwanda is implementing policies to increase the area of Arabica coffee production. Information on the suitable areas for sustainably growing Arabica coffee is still scarce. This study aimed to analyze suitable areas for Arabica coffee production. We analyzed the spatial distribution of actual and potential production zones for Arabica coffee, their productivity levels and predicted potential yields. We used a geographic information system (GIS) for a weighted overlay analysis to assess the major production zones of Arabica coffee and their qualitative productivity indices. Actual coffee yields were measured in the field and were used to assess potential productivity zones and yields using ordinary kriging with ArcGIS software. The production of coffee covers about 32 000 ha, or 2.3% of all cultivated land in the country. The major zones of production are the Kivu Lake Borders, Central Plateau, Eastern Plateau, and Mayaga agro-ecological zones, where coffee is mainly cultivated on moderate slopes. In the highlands, coffee is grown on steep slopes that can exceed 55%. About 21% percent of the country has a moderate yield potential, ranging between 1.0 and 1.6 t coffee ha⁻¹, and 70% has a low yield potential (<1.0 t coffee ha⁻¹). Only 9% of the country has a high yield potential of 1.6–2.4 t coffee ha⁻¹. Those areas are found near Lake Kivu where the dominant soil Orders are Inceptisols and Ultisols. Moderate yield potential is found in the Birunga (volcano), Congo-Nile watershed Divide, Impala and Imbo zones. Low-yield regions (<1 t ha⁻¹) occur in the eastern semi-dry lowlands, Central Plateau, Eastern Plateau, Buberuka Highlands, and Mayaga zones. The weighted overlay analysis and ordinary kriging indicated a large spatial variability of potential productivity indices. Increasing the area and productivity of coffee in Rwanda thus has considerable potential.     

Detection of Melamine in Feed Using Liquid-Liquid Extraction Treatment Combined with Surface-Enhanced Raman Scattering Spectroscopy

A rapid, selective, and sensitive method to determine the melamine content in animal feeds was developed using surface-enhanced Raman scattering spectroscopy on aggregated 55 nm Au nanoparticles with liquid–liquid extraction sample preparation. Butyl alcohol was used as the initial extraction solvent, and liquid–liquid extraction was performed twice using HCl (pH 3–4) and 6∶1 (v/v) n-butyl alcohol/ethyl acetate. The intensity of the matrix-based peak at 731 cm⁻¹ was set at 100 as a basis for the feeds, and the peak at 707 cm⁻¹ was the characteristic peak of melamine used in the calculations. Sufficient linearity was obtained in the range 2–10 µg·g⁻¹ (R ² = 0.991). Limits of detection and quantification in the feeds were 0.5 and 2 µg·g⁻¹, respectively. The recovery rates were 82.5–90.2% with coefficients of variation below 4.02%. This new protocol could be easily developed for the routine monitoring of on-site feed quality and market surveillance.     

Quantitative Assessment of the Association between rs2046210 at 6q25.1 and Breast Cancer Risk

Genome-wide association studies (GWAS) have identified several genetic susceptibility loci for breast cancer (BC). One of them, conducted among Chinese women, found an association of rs2046210 at 6q25.1 with the risk of BC recently. Since then, numerous association studies have been carried out to investigate the relationship between this polymorphism and BC risk in various populations. However, these have yielded contradictory results. We therefore performed a meta-analysis to clarify this inconsistency. Overall, a total of 235003 subjects based on 13 studies were included in our study. Significantly increased BC risk was detected in the pooled analysis [allele contrast: OR = 1.13, 95%CI = 1.10–1.17, P(Z) <10⁻⁵, P(Q) <10⁻⁴; dominant model: OR = 1.21, 95%CI = 1.14–1.27, P(Z) <10⁻⁵, P(Q) <10⁻⁴; recessive model: OR = 1.18, 95%CI = 1.12–1.24, P(Z) <10⁻⁵, P(Q) = 0.04]. In addition, our data revealed that rs2046210 conferred greater risk in estrogen receptor (ER)-negative tumors [OR = 1.27, 95%CI = 1.15–1.40, P(Z) <10⁻⁵, P(Q) <10⁻⁴] than in ER-positive ones [OR = 1.18, 95%CI = 1.09–1.28, P(Z) <10⁻⁴, P(Q) = 0.0003]. When stratified by ethnicity, significant associations were found in Caucasian and Asian populations, but not detected among Africans. There was evidence of heterogeneity (P<0.05), however, the heterogeneity largely disappeared after stratification by ethnicity. The present meta-analysis demonstrated that the rs2046210 polymorphism may be associated with increased BC susceptibility, but this association varies in different ethnicities.     

Influence of Acidic pH on Hydrogen and Acetate Production by an Electrosynthetic Microbiome

Production of hydrogen and organic compounds by an electrosynthetic microbiome using electrodes and carbon dioxide as sole electron donor and carbon source, respectively, was examined after exposure to acidic pH (∼5). Hydrogen production by biocathodes poised at −600 mV vs. SHE increased>100-fold and acetate production ceased at acidic pH, but ∼5–15 mM (catholyte volume)/day acetate and>1,000 mM/day hydrogen were attained at pH ∼6.5 following repeated exposure to acidic pH. Cyclic voltammetry revealed a 250 mV decrease in hydrogen overpotential and a maximum current density of 12.2 mA/cm² at −765 mV (0.065 mA/cm² sterile control at −800 mV) by the Acetobacterium-dominated community. Supplying −800 mV to the microbiome after repeated exposure to acidic pH resulted in up to 2.6 kg/m³/day hydrogen (≈2.6 gallons gasoline equivalent), 0.7 kg/m³/day formate, and 3.1 kg/m³/day acetate ( = 4.7 kg CO₂ captured).     

Reduction of Selenite to Red Elemental Selenium by Rhodopseudomonas palustris Strain N

The trace metal selenium is in demand for health supplements to human and animal nutrition. We studied the reduction of selenite (SeO₃ ⁻²) to red elemental selenium by Rhodopseudomonas palustris strain N. This strain was cultured in a medium containing SeO₃ ⁻² and the particles obtained from cultures were analyzed using transmission electron microscopy (TEM), energy dispersive microanalysis (EDX) and X ray diffraction analysis (XRD). Our results showed the strain N could reduce SeO₃ ⁻² to red elemental selenium. The diameters of particles were 80–200 nm. The bacteria exhibited significant tolerance to SeO₃ ⁻² up to 8.0 m mol/L concentration with an EC₅₀ value of 2.4 m mol/L. After 9 d of cultivation, the presence of SeO₃ ²⁻ up to 1.0 m mol/L resulted in 99.9% reduction of selenite, whereas 82.0% (p<0.05), 31.7% (p<0.05) and 2.4% (p<0.05) reduction of SeO₃ ⁻² was observed at 2.0, 4.0 and 8.0 m mol/L SeO₃ ²⁻ concentrations, respectively. This study indicated that red elemental selenium was synthesized by green technology using Rhodopseudomonas palustris strain N. This strain also indicated a high tolerance to SeO₃ ⁻². The finding of this work will contribute to the application of selenium to human health.     

The Metabolic and Performance Effects of Caffeine Compared to Coffee during Endurance Exercise

There is consistent evidence supporting the ergogenic effects of caffeine for endurance based exercise. However, whether caffeine ingested through coffee has the same effects is still subject to debate. The primary aim of the study was to investigate the performance enhancing effects of caffeine and coffee using a time trial performance test, while also investigating the metabolic effects of caffeine and coffee. In a single-blind, crossover, randomised counter-balanced study design, eight trained male cyclists/triathletes (Mean±SD: Age 41±7y, Height 1.80±0.04 m, Weight 78.9±4.1 kg, VO₂ max 58±3 ml•kg⁻¹•min⁻¹) completed 30 min of steady-state (SS) cycling at approximately 55% VO₂max followed by a 45 min energy based target time trial (TT). One hour prior to exercise each athlete consumed drinks consisting of caffeine (5 mg CAF/kg BW), instant coffee (5 mg CAF/kg BW), instant decaffeinated coffee or placebo. The set workloads produced similar relative exercise intensities during the SS for all drinks, with no observed difference in carbohydrate or fat oxidation. Performance times during the TT were significantly faster (∼5.0%) for both caffeine and coffee when compared to placebo and decaf (38.35±1.53, 38.27±1.80, 40.23±1.98, 40.31±1.22 min respectively, p<0.05). The significantly faster performance times were similar for both caffeine and coffee. Average power for caffeine and coffee during the TT was significantly greater when compared to placebo and decaf (294±21 W, 291±22 W, 277±14 W, 276±23 W respectively, p<0.05). No significant differences were observed between placebo and decaf during the TT. The present study illustrates that both caffeine (5 mg/kg/BW) and coffee (5 mg/kg/BW) consumed 1 h prior to exercise can improve endurance exercise performance.