Development of a diatom index of biotic integrity for acid mine drainage impacted streams

Acid mine drainage (AMD) resulting from extensive coal mining throughout Appalachia since the 1800s has caused a legacy of severe acid and dissolved metal loads to thousands of stream miles, which has critically impacted aquatic life and ecological attributes. Relationships of diatoms and macroinvertebrates with AMD have been established, but no index specifically designed to quantify AMD impacts using diatoms has been created, nor have the response of multiple organism groups been compared for their utility as indices assessing AMD severity.For the purpose of developing an effective assessment and management strategy for AMD impacted streams, this study created and tested a multi-metric AMD-diatom index of biotic integrity (AMD-DIBI) and compared its response to AMD severity with an already established multi-metric macroinvertebrate community index (ICI). In 2006, 41 sites in southeast Ohio were sampled that represented an AMD impact gradient and non-AMD impacted reference sites. Metrics comprising the AMD-DIBI were selected based on their responsiveness to AMD and nutrient impacts. In the following year, the AMD-DIBI and its metrics were tested on a validation dataset consisting of 18 sites in an AMD impacted watershed. Results indicated a significant correlation between AMD-DIBI and ICI scores, and both indices and all metrics were strongly correlated with water chemistry variables indicative of AMD pollution (P < 0.05). Stepwise multiple regression selected alkalinity and conductivity as most influential to AMD-DIBI (adjusted r² = 0.70) and ICI scores (adjusted r² = 0.66). Narrative classes (e.g., Poor, Fair, Good, and Excellent) defined by index scores provided effective classifications of AMD severity. When tested on the watershed scale, AMD-DIBI and its metrics very successfully quantified AMD gradients and coal mining impacts as indicated by canonical correspondence analysis. This newly developed AMD-DIBI will be very useful for assessing impairment, sensitivity, and recovery of diatom communities in streams damaged or threatened by coal mining activities. In addition, because the AMD-DIBI was very responsive to a gradient of AMD pollution, it could be used in future studies measuring the long-term status of streams and effectiveness of various remediation methods. This study highlights the responsive power of diatom-based metrics.     

In situ proteo-metabolomics reveals metabolite secretion by the acid mine drainage bio-indicator,Euglena mutabilis

Euglena mutabilis is a photosynthetic protist found in acidic aquatic environments such as peat bogs, volcanic lakes and acid mine drainages (AMDs). Through its photosynthetic metabolism, this protist is supposed to have an important role in primary production in such oligotrophic ecosystems. Nevertheless, the exact contribution of E. mutabilis in organic matter synthesis remains unclear and no evidence of metabolite secretion by this protist has been established so far. Here we combined in situ proteo-metabolomic approaches to determine the nature of the metabolites accumulated by this protist or potentially secreted into an AMD. Our results revealed that the secreted metabolites are represented by a large number of amino acids, polyamine compounds, urea and some sugars but no fatty acids, suggesting a selective organic matter contribution in this ecosystem. Such a production may have a crucial impact on the bacterial community present on the study site, as it has been suggested previously that prokaryotes transport and recycle in situ most of the metabolites secreted by E. mutabilis. Consequently, this protist may have an indirect but important role in AMD ecosystems but also in other ecological niches often described as nitrogen-limited.     

Diversity of the Sediment Microbial Community in the Aha Watershed (Southwest China) in Response to Acid Mine Drainage Pollution Gradients

Located in southwest China, the Aha watershed is continually contaminated by acid mine drainage (AMD) produced from upstream abandoned coal mines. The watershed is fed by creeks with elevated concentrations of aqueous Fe (total Fe > 1 g/liter) and SO₄²⁻ (>6 g/liter). AMD contamination gradually decreases throughout downstream rivers and reservoirs, creating an AMD pollution gradient which has led to a suite of biogeochemical processes along the watershed. In this study, sediment samples were collected along the AMD pollution sites for geochemical and microbial community analyses. High-throughput sequencing found various bacteria associated with microbial Fe and S cycling within the watershed and AMD-impacted creek. A large proportion of Fe- and S-metabolizing bacteria were detected in this watershed. The dominant Fe- and S-metabolizing bacteria were identified as microorganisms belonging to the genera Metallibacterium, Aciditerrimonas, Halomonas, Shewanella, Ferrovum, Alicyclobacillus, and Syntrophobacter. Among them, Halomonas, Aciditerrimonas, Metallibacterium, and Shewanella have previously only rarely been detected in AMD-contaminated environments. In addition, the microbial community structures changed along the watershed with different magnitudes of AMD pollution. Moreover, the canonical correspondence analysis suggested that temperature, pH, total Fe, sulfate, and redox potentials (E_h) were significant factors that structured the microbial community compositions along the Aha watershed.     

Use of 16S rRNA Gene Terminal Restriction Fragment Analysis To Assess the Impact of Solids Retention Time on the Bacterial Diversity of Activated Sludge

Terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes was used to investigate the reproducibility and stability in the bacterial community structure of laboratory-scale sequencing batch bioreactors (SBR) and to assess the impact of solids retention time (SRT) on bacterial diversity. Two experiments were performed. In each experiment two sets of replicate SBRs were operated for a periods of three times the SRT. One set was operated at an SRT of 2 days and another set was operated at an SRT of 8 days. Samples for T-RFLP analysis were collected from the two sets of replicate reactors. HhaI, MspI, and RsaI T-RFLP profiles were analyzed using cluster analysis and diversity statistics. Cluster analysis with Ward's method using Jaccard distance and Hellinger distance showed that the bacterial community structure in both sets of reactors from both experimental runs was dynamic and that replicate reactors were clustered together and evolved similarly from startup. Richness (S), evenness (E), the Shannon-Weaver index (H), and the reciprocal of Simpson's index (1/D) were calculated, and the values were compared between the two sets of reactors. Evenness values were higher for reactors operated at an SRT of 2 days. Statistically significant differences in diversity (H and D) between the two sets of reactors were tested using a randomization procedure, and the results showed that reactors from both experimental runs that were operated at an SRT of 2 days had higher diversity (H and D) at the 5% level. T-RFLP analysis with diversity indices proved to be a powerful tool to analyze changes in the bacterial community diversity in response to changes in the operational parameters of activated-sludge systems.     

No evidence of association between variant rs2075650 in lipid metabolism-related locus APOE/TOMM40 and advanced age-related macular degeneration in Han Chinese population

Age-related macular degeneration (AMD) is a late-onset, neurodegenerative disease. Genes related to lipid metabolism are important in AMD pathogenesis. Recently, a variant rs2075650 located in lipid metabolism-related locus APOE/TOMM40 was identified to be associated with advanced AMD and early AMD, respectively, in two genome-wide association studies with European ancestry, while no association study between rs2075650 and overall advanced AMD in Chinese population has been conducted before. We evaluated the potential effect of this variant on advanced AMD in a Han Chinese cohort with 204 advanced AMD patients and 1536 healthy controls. The results suggested that rs2075650 was neither associated with advanced AMD in allele level (P = 0.348) nor in genotype level (P = 0.890 under additive model with age and sex adjusted). In conclusion, our study did not confirm the impact of rs2075650 on advanced AMD risk, indicating that rs2075650 is unlikely a superior marker for APOE/TOMM40 susceptible region with advanced AMD in Han Chinese population.     

To include or not to include (the invader in community analyses)? That is the question

An increasing number of studies report impacts from invasive species on community metrics or ecosystem functions. We draw attention to an issue arising whenever impact is measured on a community where the invader is an integrated part: should or shouldn’t the attributes of the invader itself be included in the data-analysis? We identify many examples from the published literature showing inconsistency in whether or not data for the invader is included or excluded, and discuss potential implications for ecological interpretations. We also provide a case study to show that the invasive seaweed Undaria pinnatifida can be interpreted to have strong or no impact on seaweed communities, depending on its inclusion or exclusion in the data analysis. We conclude that it is critical for studies to (1) clearly state in the methods section, if the invaders are included or excluded from the data-analysis, (2) acknowledge potential differences in outcomes when comparing results based on different methods, and (3) analyze, if possible, impacts both with and without the invader. Finally, we note that this ‘inclusion versus exclusion’ conundrum is not only relevant to invasion biology, but to any field where the test-object of interest can be an integrated part of the response, such as when impact of seaweed blooms are analysed on community productivity or community effects are quantified over time from ecological pulse-perturbation experiments.     

Proof of Concept,Randomized, Placebo-Controlled Study of the Effect of Simvastatin on the Course of Age-Related Macular Degeneration

Background

HMG Co-A reductase inhibitors are ubiquitous in our community yet their potential role in age-related macular degeneration (AMD) remains to be determined.

Methodology/Principal Findings

Objectives: To evaluate the effect of simvastatin on AMD progression and the effect modification by polymorphism in apolipoprotein E (ApoE) and complement factor H (CFH) genes. Design: A proof of concept double-masked randomized controlled study. Participants: 114 participants aged 53 to 91 years, with either bilateral intermediate AMD or unilateral non-advanced AMD (with advanced AMD in fellow eye), BCVA≥20/60 in at least one eye, and a normal lipid profile. Intervention: Simvastatin 40 mg/day or placebo, allocated 1∶1. Main outcome measures: Progression of AMD either to advanced AMD or in severity of non-advanced AMD. Results. The cumulative AMD progression rates were 70% in the placebo and 54% in the simvastatin group. Intent to treat multivariable logistic regression analysis, adjusted for age, sex, smoking and baseline AMD severity, showed a significant 2-fold decrease in the risk of progression in the simvastatin group: OR 0.43 (0.18–0.99), p = 0.047. Post-hoc analysis stratified by baseline AMD severity showed no benefit from treatment in those who had advanced AMD in the fellow eye before enrolment: OR 0.97 (0.27–3.52), p = 0.96, after adjusting for age, sex and smoking. However, there was a significant reduction in the risk of progression in the bilateral intermediate AMD group compared to placebo [adjusted OR 0.23 (0.07–0.75), p = 0.015]. The most prominent effect was observed amongst those who had the CC (Y402H) at risk genotype of the CFH gene [OR 0.08 (0.02–0.45), p = 0.004]. No evidence of harm from simvastatin intervention was detected.

Conclusion/Significance

Simvastatin may slow progression of non-advanced AMD, especially for those with the at risk CFH genotype CC (Y402H). Further exploration of the potential use of statins for AMD, with emphasis on genetic subgroups, is warranted.

Trial Registration

Australian New Zealand Clinical Trial Registry (ANZCTR) ACTRN1260500032065     

Cumulative association between age-related macular degeneration and less studied genetic variants in PLEKHA1/ARMS2/HTRA1: a meta and gene-cluster analysis

The objective of this study is to examine the cumulative effect of the less studied genetic variants in PLEKHA1/ARMS2/HTRA1 on age-related macular degeneration (AMD). We performed an extensive literature search for studies on the association between AMD and the less studied genetic variants in PLEKHA1/ARMS2/HTRA1. Multiple meta-analyses were performed to evaluate the association between individual genetic variants and AMD. A gene-cluster analysis was used to investigate the cumulative effect of these less studied genetic variants on AMD. A total of 23 studies from 20 published papers met the eligibility criteria and were included in our analyses. Several genetic variants in the gene cluster are significantly associated with AMD in our meta-analyses or in individual studies. Gene-cluster analysis reveals a strong cumulative association between these genetic variants in this gene cluster and AMD (p < 10^?5). However, two previously suspected SNPs in ARMS2, including rs2736911, the SNP having the largest number of studies in our meta-analyses; and rs3793917, the SNP with the largest sample size, were not significantly associated with AMD (both p’s > 0.12). Sensitivity analyses reveal significant association of AMD with rs2736911 in Chinese but not in Caucasian, with c.372_815del443ins54 in Caucasian but not in Chinese, and with rs1049331 in both ethnic groups. These less studied genetic variants have a significant cumulative effect on wet AMD. Our study provides evidence of the joint contribution of genetic variants in PLEKHA1/ARMS2/HTRA1 to AMD risk, in addition to the two widely studied genetic variants whose association with AMD was well established.     

Loss of CD34 Expression in Aging Human Choriocapillaris Endothelial Cells

Structural and gene expression changes in the microvasculature of the human choroid occur during normal aging and age-related macular degeneration (AMD). In this study, we sought to determine the impact of aging and AMD on expression of the endothelial cell glycoprotein CD34. Sections from 58 human donor eyes were categorized as either young (under age 40), age-matched controls (> age 60 without AMD), or AMD affected (>age 60 with early AMD, geographic atrophy, or choroidal neovascularization). Dual labeling of sections with Ulex europaeus agglutinin-I lectin (UEA-I) and CD34 antibodies was performed, and the percentage of capillaries labeled with UEA-I but negative for anti-CD34 was determined. In addition, published databases of mouse and human retinal pigment epithelium-choroid were evaluated and CD34 expression compared between young and old eyes. Immunohistochemical studies revealed that while CD34 and UEA-I were colocalized in young eyes, there was variable loss of CD34 immunoreactivity in older donor eyes. While differences between normal aging and AMD were not significant, the percentage of CD34 negative capillaries in old eyes, compared to young eyes, was highly significant (p = 3.8×10⁻⁶). Endothelial cells in neovascular membranes were invariably CD34 positive. Published databases show either a significant decrease in Cd34 (mouse) or a trend toward decreased CD34 (human) in aging. These findings suggest that UEA-I and endogenous alkaline phosphatase activity are more consistent markers of aging endothelial cells in the choroid, and suggest a possible mechanism for the increased inflammatory milieu in the aging choroid.     

Nutrient limitation of algal periphyton in streams along an acid mine drainage gradient

Metal oxyhydroxide precipitates that form from acid mine drainage (AMD) may indirectly limit periphyton by sorbing nutrients, particularly P. We examined effects of nutrient addition on periphytic algal biomass (chl a), community structure, and carbon and nitrogen content along an AMD gradient. Nutrient diffusing substrata with treatments of +P, +NP and control were placed at seven stream sites. Conductivity and SO₄ concentration ranged over an order of magnitude among sites and were used to define the AMD gradient, as they best indicate mine discharge sources of metals that create oxyhydroxide precipitates. Aqueous total phosphorous (TP) ranged from 2 to 23 μg · L^?1 and significantly decreased with increasing SO₄. Mean chl a concentrations at sites ranged from 0.2 to 8.1 μg · cm^?2. Across all sites, algal biomass was significantly higher on +NP than control treatments (Co), and significantly increased with +NP. The degree of nutrient limitation was determined by the increase in chl a concentration on +NP relative to Co (response ratio), which ranged from 0.6 to 9.7. Response to nutrient addition significantly declined with increasing aqueous TP, and significantly increased with increasing SO₄. Thus, nutrient limitation of algal biomass increased with AMD impact, indicating metal oxyhydroxides associated with AMD likely decreased P availability. Algal species composition was significantly affected by site but not nutrient treatment. Percent carbon content of periphyton on the Co significantly increased with AMD impact and corresponded to an increase in the relative abundance of Chlorophytes. Changes in periphyton biomass and cellular nutrient content associated with nutrient limitation in AMD streams may affect higher trophic levels.     

Genetic analysis of typical wet-type age-related macular degeneration and polypoidal choroidal vasculopathy in Japanese population

Age-related macular degeneration (AMD) is a common cause of blindness in the elderly. Caucasian patients are predominantly affected by the dry form of AMD, whereas Japanese patients have predominantly the wet form of AMD and/or polypoidal choroidal vasculopathy (PCV). Although genetic association in the 10q26 (ARMS2/HTRA1) region has been established in many ethnic groups for dry-type AMD, typical wet-type AMD, and PCV, the contribution of the 1q32 (CFH) region seem to differ among these groups. Here we show a single nucleotide polymorphism (SNP) in the ARMS2/HTRA1 locus is associated in the whole genome for Japanese typical wet-type AMD (rs10490924: , OR = 4.16) and PCV (rs10490924: , OR = 2.72) followed by CFH (rs800292: , OR = 2.08; , OR = 2.00), which differs from previous studies in Caucasian populations. Moreover, a SNP (rs2241394) in complement component C3 gene showed significant association with PCV (, OR = 3.47). We conclude that dry-type AMD, typical wet-type AMD, and PCV have both common and distinct genetic risks that become apparent when comparing Japanese versus Caucasian populations.

Electronic supplementary material

The online version of this article (doi:10.1007/s12177-009-9047-1) contains supplementary material, which is available to authorized users.     

Contribution of organic anion transporting polypeptide OATP2B1 to amiodarone accumulation in lung epithelial cells

The accumulation mechanisms of amiodarone (AMD) involving transporters in lung alveolar epithelial type II cells were studied. The uptake of AMD was examined using human alveolar epithelial-derived cell line A549 as a model. AMD was transported by the carrier-mediated system, and the apparent K_m and V_max values were 66.8 ± 30.3 μM and 49.7 ± 9.7 nmol/mg protein/5 min, respectively. The uptake of AMD by A549 cells was Na⁺-independent and was inhibited by substrates of human organic anion transporting polypeptide (OATP). The inhibition profiles were similar to the inhibitory effects of several compounds on OATP2B1-mediated E-3-S transport, and RT-PCR analysis showed mRNA expression of OATP2B1 and 1B3 in A549 cells. SiRNAs targeted to the OATP2B1 gene decreased the OATP2B1 mRNA expression level in A549 cells up to about 50% and reduced the uptake of AMD up to about 40%. These results indicate that AMD uptake mediated by carriers, including OATP2B1, might lead to accumulation of AMD in the lung and AMD-induced pulmonary toxicity (AIPT).     

Association between Variant Y402H in Age-Related Macular Degeneration (AMD) Susceptibility Gene CFH and Treatment Response of AMD: A Meta-Analysis

Purpose

To investigate the association between polymorphism rs1061170 (T1277C, Y402H) in age-related macular degeneration (AMD) susceptibility gene Complement Factor H (CFH) and treatment response of neovascular AMD.

Methods

We performed a literature-based meta-analysis including 10 published association studies involving 1,510 patients. Treatments included anti-VEGF (bevacizumab and ranibizumab) or photodynamic therapy. Summary odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using fixed- and random-effects models. Q-statistic test was used to assess heterogeneity.

Results

Polymorphism rs1061170 showed a significant summary OR of 1.68 (95% CI, 1.09 to 2.60; P = 0.020; CC versus TT; random-effects) for treatment response of neovascular AMD with heterogeneity of 0.09. In subgroup analysis, rs1061170 was more likely to be a predictor of response to anti-VEGF therapy (P = 0.011). However, heterozygous TC genotype was not associated with altered treatment response (OR = 1.18, 95% CI, 0.95 to 1.47; P = 0.145; fixed-effects). Influence analysis indicated the robustness of our findings.

Conclusions

rs1061170 might be associated with treatment response of neovascular AMD, especially for the anti-VEGF agents. It might be the first meta-analytically confirmed genetic marker predictive for AMD treatment response though a further validation in larger studies is needed.     

Weed communities of rain-fed lowland rice vary with infestation by Rhamphicarpa fistulosa

The facultative hemiparasitic plant Rhamphicarpa fistulosa (Orobanchaceae) thrives in seasonally wet soils in sub-Saharan Africa, mainly in marginal lowland rice growing environments where weeds are already a major constraint for rice production. Because lowland rice production is increasing in tropical Africa, it is important to ascertain the influence of R. fistulosa on weed plant communities in these rice-growing habitats. We investigated weed plant community richness and composition at four different levels of R. fistulosa infestation across two years of surveys from lowland rice fields in northern Togo (West Africa). Despite a lack of significant differences in community richness among sites with different R. fistulosa infestation levels, there were significant differences in community composition, both when estimated from presence-absence data and from relative abundance data, after controlling statistically for geographic proximity among sites. Rhamphicarpa fistulosa infestation, therefore, may influence the competitive balance between rice and its weeds and shape weed community structure. However, experimental studies are required to elucidate the weed host range of R. fistulosa and the direct and indirect effects of this hemiparasite in rice fields in order to predict its net impact on rice and its weed species.     

The effect of water pollution on biological control of water hyacinth

South Africa has one of the world’s biggest gold mining regions with an associated problem of acid mine drainage (AMD), which increases the bioavailability of heavy metal contaminants in water. The prevalence of water hyacinth (Eichhornia crassipes) in South African water systems, despite the release of seven biocontrol agents since 1974, is often attributed to high levels of eutrophication. Metal concentration in plant shoots is known to affect insect herbivory. Nevertheless, little is known about the effect of heavy metals or AMD on Neochetina eichhorniae and Neochetina bruchi, which are the most widely established biocontrol agents on E. crassipes in South Africa. Herein, the effect of eight different heavy metals common in AMD (arsenic (As), gold (Au), copper (Cu), iron (Fe), mercury (Hg), manganese (Mn), uranium (U) and zinc (Zn)), as well as three different simulated AMD concentration treatments (low, medium and high), on the performance of Neochetina weevils were investigated by releasing adults on plants growing in tubs and pools, three weeks after the addition of individual metal or AMD treatments. After six weeks, the number of weevil larvae per plant, the number of adult survivors per plant, the number of adult feeding scars on leaves, and the number of larval mines per plant were recorded. Two females of N. eichhorniae and N. bruchi from each tub were dissected and the number of ovariole follicles was counted. Adult feeding in Neochetina was significantly reduced on plants exposed to both Cu and As while larval feeding was significantly reduced on plants exposed to Hg, Zn, As and Cu, with Cu causing the greatest effect. Similarly, weevil feeding and reproduction were reduced in the medium and high concentration AMD treatments. Larval development was significantly impaired by both metal and AMD treatments. These negative effects disagree with published data which showed no effect of metals on Neochetina weevils. The disparity is explained by long exposure of the weevils on whole plants, rather than short exposure to excised leaves, as recorded in the literature. Finally these findings provide evidence that some heavy metals and AMD might be constraining biocontrol agents of water hyacinth in South Africa.