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1.
J Vis Exp ; (204)2024 Feb 16.
Article in English | MEDLINE | ID: mdl-38436378

ABSTRACT

Microphysiological systems are miniaturized cell culture platforms used to mimic the structure and function of human tissues in a laboratory setting. However, these platforms have not gained widespread adoption in bioscience laboratories where open-well, membrane-based approaches serve as the gold standard for mimicking tissue barriers, despite lacking fluid flow capabilities. This issue can be primarily attributed to the incompatibility of existing microphysiological systems with standard protocols and tools developed for open-well systems. Here, we present a protocol for creating a reconfigurable membrane-based platform with an open-well structure, flow enhancement capability, and compatibility with conventional protocols. This system utilizes a magnetic assembly approach that enables reversible switching between open-well and microfluidic modes. With this approach, users have the flexibility to begin an experiment in the open-well format using standard protocols and add or remove flow capabilities as needed. To demonstrate the practical usage of this system and its compatibility with standard techniques, an endothelial cell monolayer was established in an open-well format. The system was reconfigured to introduce fluid flow and then switched to the open-well format to conduct immunostaining and RNA extraction. Due to its compatibility with conventional open-well protocols and flow enhancement capability, this reconfigurable design is expected to be adopted by both engineering and bioscience laboratories.


Subject(s)
Cell Culture Techniques , Microphysiological Systems , Humans , Endothelial Cells , Laboratories , Microfluidics
2.
J Biol Chem ; 299(12): 105369, 2023 Dec.
Article in English | MEDLINE | ID: mdl-37865311

ABSTRACT

Cardiac MyBP-C (cMyBP-C) interacts with actin and myosin to fine-tune cardiac muscle contractility. Phosphorylation of cMyBP-C, which reduces the binding of cMyBP-C to actin and myosin, is often decreased in patients with heart failure (HF) and is cardioprotective in model systems of HF. Therefore, cMyBP-C is a potential target for HF drugs that mimic its phosphorylation and/or perturb its interactions with actin or myosin. We labeled actin with fluorescein-5-maleimide (FMAL) and the C0-C2 fragment of cMyBP-C (cC0-C2) with tetramethylrhodamine (TMR). We performed two complementary high-throughput screens (HTS) on an FDA-approved drug library, to discover small molecules that specifically bind to cMyBP-C and affect its interactions with actin or myosin, using fluorescence lifetime (FLT) detection. We first excited FMAL and detected its FLT, to measure changes in fluorescence resonance energy transfer (FRET) from FMAL (donor) to TMR (acceptor), indicating binding. Using the same samples, we then excited TMR directly, using a longer wavelength laser, to detect the effects of compounds on the environmentally sensitive FLT of TMR, to identify compounds that bind directly to cC0-C2. Secondary assays, performed on selected modulators with the most promising effects in the primary HTS assays, characterized the specificity of these compounds for phosphorylated versus unphosphorylated cC0-C2 and for cC0-C2 versus C1-C2 of fast skeletal muscle (fC1-C2). A subset of identified compounds modulated ATPase activity in cardiac and/or skeletal myofibrils. These assays establish the feasibility of the discovery of small-molecule modulators of the cMyBP-C-actin/myosin interaction, with the ultimate goal of developing therapies for HF.


Subject(s)
Carrier Proteins , Drug Discovery , Heart Failure , Myofibrils , Small Molecule Libraries , Humans , Actins/metabolism , Drug Discovery/methods , Heart Failure/drug therapy , Heart Failure/metabolism , Myocardium/metabolism , Myosins/metabolism , Phosphorylation/drug effects , Protein Binding/drug effects , Small Molecule Libraries/pharmacology , Drug Evaluation, Preclinical , Myofibrils/drug effects , Carrier Proteins/metabolism , Biosensing Techniques , Adenosine Triphosphatases/metabolism , Muscle, Skeletal/metabolism , Recombinant Proteins/metabolism , Enzyme Activation/drug effects , Fluorescence Resonance Energy Transfer
3.
bioRxiv ; 2023 Apr 05.
Article in English | MEDLINE | ID: mdl-37066417

ABSTRACT

Cardiac MyBP-C (cMyBP-C) interacts with actin-myosin to fine-tune cardiac muscle contractility. Phosphorylation of cMyBP-C, which reduces binding of cMyBP-C to actin or myosin, is often decreased in heart failure (HF) patients, and is cardioprotective in model systems for HF. Therefore, cMyBP-C is a potential target for HF drugs that mimic phosphorylation and/or perturb its interactions with actin or myosin. We labeled actin with fluorescein-5-maleimide (FMAL), and the C0-C2 fragment of cMyBP-C (cC0-C2) with tetramethyl rhodamine (TMR). We performed two complementary high-throughput screens (HTS) on an FDA-approved drug library, to discover small molecules that specifically bind to cMyBP-C and affect its interactions with actin or myosin, using fluorescence lifetime (FLT) detection. We first excited FMAL and detected its FLT, to measure changes in fluorescence resonance energy transfer (FRET) from FMAL (donor) to TMR (acceptor), indicating binding and/or structural changes in the protein complex. Using the same samples, we then excited TMR directly, using a longer wavelength laser, to detect the effects of compounds on the environmentally sensitive FLT of TMR, to identify compounds that bind directly to cC0-C2. Secondary assays, performed on selected modulators with the most promising effects in the primary HTS assays, characterized specificity of these compounds for phosphorylated versus unphosphorylated cC0-C2 and for cC0-C2 versus C1-C2 of fast skeletal muscle (fskC1-C2). A subset of identified compounds modulated ATPase activity in cardiac and/or skeletal myofibrils. These assays establish feasibility for discovery of small-molecule modulators of the cMyBP-C-actin/myosin interaction, with the ultimate goal of developing therapies for HF.

4.
J Biol Chem ; 299(3): 102956, 2023 03.
Article in English | MEDLINE | ID: mdl-36731793

ABSTRACT

ß-III-Spectrin is a key cytoskeletal protein that localizes to the soma and dendrites of cerebellar Purkinje cells and is required for dendritic arborization and signaling. A spinocerebellar ataxia type 5 L253P mutation in the cytoskeletal protein ß-III-spectrin causes high-affinity actin binding. Previously we reported a cell-based fluorescence assay for identification of small-molecule actin-binding modulators of the L253P mutant ß-III-spectrin. Here we describe a complementary, in vitro, fluorescence resonance energy transfer (FRET) assay that uses purified L253P ß-III-spectrin actin-binding domain (ABD) and F-actin. To validate the assay for high-throughput compatibility, we first confirmed that our 50% FRET signal was responsive to swinholide A, an actin-severing compound, and that this yielded excellent assay quality with a Z' value > 0.77. Second, we screened a 2684-compound library of US Food and Drug Administration-approved drugs. Importantly, the screening identified numerous compounds that decreased FRET between fluorescently labeled L253P ABD and F-actin. The activity and target of multiple Hit compounds were confirmed in orthologous cosedimentation actin-binding assays. Through future medicinal chemistry, the Hit compounds can potentially be developed into a spinocerebellar ataxia type 5-specific therapeutic. Furthermore, our validated FRET-based in vitro high-throughput screening platform is poised for screening large compound libraries for ß-III-spectrin ABD modulators.


Subject(s)
Actins , Spectrin , Spinocerebellar Ataxias , Humans , Actins/genetics , Actins/metabolism , Drug Discovery , Neurons/metabolism , Spectrin/metabolism , Spinocerebellar Ataxias/drug therapy , Spinocerebellar Ataxias/genetics , Spinocerebellar Ataxias/metabolism
5.
J Biol Chem ; 298(12): 102675, 2022 12.
Article in English | MEDLINE | ID: mdl-36372234

ABSTRACT

Duchenne muscular dystrophy is a lethal muscle disease, caused by mutations in the gene encoding dystrophin, an actin-binding cytoskeletal protein. Absence of functional dystrophin results in muscle weakness and degeneration, eventually leading to cardiac and respiratory failure. Strategies to replace the missing dystrophin via gene therapy have been intensively pursued. However, the dystrophin gene is too large for current gene therapy approaches. Currently available micro-dystrophin constructs lack the actin-binding domain 2 and show decreased actin-binding affinity in vitro compared to full-length dystrophin. Thus, increasing the actin-binding affinity of micro-dystrophin, using small molecules, could be a beneficial therapeutic approach. Here, we have developed and validated a novel high-throughput screening (HTS) assay to discover small molecules that increase the binding affinity of dystrophin's actin-binding domain 1 (ABD1). We engineered a novel FRET biosensor, consisting of the mClover3, fluorescent protein (donor) attached to the C-terminus of dystrophin ABD1, and Alexa Fluor 568 (acceptor) attached to the C-terminal cysteine of actin. We used this biosensor in small-molecule screening, using a unique high-precision, HTS fluorescence lifetime assay, identifying several compounds from an FDA-approved library that significantly increase the binding between actin and ABD1. This HTS assay establishes feasibility for the discovery of small-molecule modulators of the actin-dystrophin interaction, with the ultimate goal of developing therapies for muscular dystrophy.


Subject(s)
Actins , Dystrophin , Muscular Dystrophy, Duchenne , Humans , Actins/metabolism , Dystrophin/genetics , Dystrophin/chemistry , Genetic Therapy , Muscular Dystrophy, Duchenne/genetics , Muscular Dystrophy, Duchenne/therapy , Protein Binding/drug effects , Small Molecule Libraries/chemistry , Small Molecule Libraries/metabolism , Small Molecule Libraries/pharmacology , Protein Domains
6.
Glob Health Action ; 15(1): 2029335, 2022 12 31.
Article in English | MEDLINE | ID: mdl-35323105

ABSTRACT

The HIV pandemic has long revealed the inequities and fault lines in societies, one of the most tenacious being the pandemic's disproportionate impact on adolescent girls and young women. In east and southern Africa, renewed global action is needed to invigorate an effective yet undervalued approach to expanding HIV prevention and improving women's health: integration of quality HIV and sexual and reproductive health (SRH) services. The urgency of advancing effective integration of these services has never been clearer or more pressing. In this piece, national health officials from Kenya, Malawi, and Zimbabwe and global health professionals have joined together in a call to catalyze actions by development partners in support of national strategies to integrate HIV and SRH information and services. This agenda is especially vital now because these adolescent girls and young women are falling through the cracks due to the cascading effects of COVID-19 and disruptions in both SRH and HIV services. In addition, the scale-up of pre-exposure prophylaxis (PrEP) has been anemic for this population. Examining the opportunities and challenges of HIV/SRH integration implemented recently in three countries - Kenya, Malawi, and Zimbabwe - provides lessons to spur integration and investments there and in other nations in the region, aimed at improving health outcomes for adolescent girls and young women and curbing the global HIV epidemic. While gaps remain between strong national integration policies and program implementation, the experiences of these countries show opportunities for expanded, quality integration. This commentary draws on a longer comparative analysis of findings from rapid landscaping analyses in Kenya, Malawi, and Zimbabwe, which highlighted cross-country trends and context-specific realities around HIV/SRH integration.


Subject(s)
COVID-19 , HIV Infections , Adolescent , Female , HIV Infections/epidemiology , HIV Infections/prevention & control , Humans , Kenya/epidemiology , Malawi/epidemiology , Zimbabwe/epidemiology
7.
Lancet HIV ; 8(5): e306-e310, 2021 05.
Article in English | MEDLINE | ID: mdl-33577781

ABSTRACT

Indicators for the measurement of programmes for the primary prevention of HIV are less aligned than indicators for HIV treatment, which results in a high burden of data collection, often without a clear vision for its use. As new evidence becomes available, the opportunity arises to critically evaluate the way countries and global bodies monitor HIV prevention programmes by incorporating emerging data on the strength of the evidence linking various factors with HIV acquisition, and by working to streamline indicators across stakeholders to reduce burdens on health-care systems. Programmes are also using new approaches, such as targeting specific sexual networks that might require non-traditional approaches to measurement. Technological advances can support these new directions and provide opportunities to use real-time analytics and new data sources to more effectively understand and adapt HIV prevention programmes to reflect population movement, risks, and an evolving epidemic.


Subject(s)
Delivery of Health Care/organization & administration , HIV Infections/prevention & control , National Health Programs/organization & administration , Preventive Health Services/organization & administration , Data Collection/methods , Global Health/trends , Humans , Quality Indicators, Health Care/statistics & numerical data
8.
Gates Open Res ; 5: 145, 2021.
Article in English | MEDLINE | ID: mdl-37794965

ABSTRACT

Background: Though substantial progress has helped curb the HIV epidemic, high rates of new HIV infections persist among adolescent girls and young women (AGYW) in sub-Saharan Africa, reflecting critical gaps in reaching them with integrated HIV prevention and sexual and reproductive health (SRH) services. The scale-up of oral pre-exposure prophylaxis (PrEP) and multiple novel HIV prevention products on the horizon offer countries a unique opportunity to expand innovative approaches to deliver comprehensive, integrated HIV prevention/SRH services. Methods: This article comparatively analyzes findings from rapid assessments in Kenya, Malawi and Zimbabwe across key themes to highlight cross-country trends and contextual realities around HIV prevention/SRH integration, with a focus on oral PrEP and contraception. In Kenya and Zimbabwe, assessments were completed by Ministries of Health (MOH) and the HIV Prevention Market Manager and include 20 health facility assessments, 73 key informant interviews (KIIs) and six community dialogues. In Malawi, the assessment was completed by the MOH and Georgetown University Center for Innovation in Global Health and includes 70 KIIs and a review of national policies and program implementation in Blantyre. Findings were contextualized through a review of literature and policies in each country. Results: Across countries, the policy environment is conducive to HIV prevention/SRH integration, though operationalization presents ongoing challenges, with most policies preceding and not accounting for oral PrEP rollout. National coordination mechanisms, youth-friendly health services and prevention of mother-to-child transmission programs are promising practices, while siloed and resource-constrained health systems, limited provider capacity, underfunded demand generation and structural factors exacerbate barriers to achieving integration. Conclusions: As new HIV prevention products are introduced, demand for integrated HIV prevention/SRH services is likely to grow. Investing in HIV prevention/SRH integration can help to ensure a sustainable response to the HIV epidemic, streamline service delivery and improve the health outcomes and lives of AGYW.

9.
J Int AIDS Soc ; 23(12): e25645, 2020 12.
Article in English | MEDLINE | ID: mdl-33345450

ABSTRACT

INTRODUCTION: While there is a global consensus on monitoring Human Immunodeficiency Virus (HIV) treatment progress, there has been less attention to the degree of consistency of the measurement of HIV prevention programmes-and the global prevention response is not on-track to achieve 2020 goals. In this paper, we assess the degree of variability in primary prevention indicators selected by national strategic plans (NSPs) and global stakeholder monitoring and evaluation (M&E) strategies. METHODS: We obtained the most recent NSPs from low and middle income Joint United Nations Programme on HIV/AIDS (UNAIDS) Fast-Track countries, and M&E documents from The Global Fund to Fight AIDS, Tuberculosis and Malaria (The Global Fund), President's Emergency Plan for AIDS Relief (PEPFAR), UNAIDS, the Global HIV Prevention Coalition and the World Health Organization (WHO). We extracted HIV primary prevention indicators from each document, standardized and aggregated them by age/ sex, categorized indicators by topic, and evaluated the frequency of matched indicators between countries and stakeholders. Data were collected between February and April of 2019. RESULTS: Twenty-one NSPs and five global stakeholder documents were assessed; 736 primary prevention indicators were identified; 284 remained following standardization and aggregation. NSPs contained from 3 to 48 primary prevention indicators, with an average of 23; categories included: HIV education and outreach (17.6%), testing (17.3%) and condom use (16.2%). Of unique national indicators, only 34% was shared between two or more countries. Sixty-nine per cent was applied in a single country only. 56% of NSP indicators did not appear in any global stakeholder document. Conversely, 42% of global indicators did not appear in any surveyed NSPs. Within global indicators, 63% was only measured by one global body, and no single indicator was measured by all five. CONCLUSIONS: These analyses reveal a lack of consensus both between and within countries' and global stakeholders' measurement of HIV prevention. Though some variability is expected, these findings point to a need to refocus attention on achieving greater consensus on a global measurement framework for HIV prevention.


Subject(s)
HIV Infections/prevention & control , Primary Prevention , Female , Global Health , Humans , Male
10.
J Hepatol ; 70(1): 142-150, 2019 01.
Article in English | MEDLINE | ID: mdl-30315827

ABSTRACT

BACKGROUND & AIMS: Lysosomal acid lipase deficiency (LAL-D) is an autosomal recessive condition that may present in a mild form (cholesteryl ester storage disease [CESD]), which mimics non-alcoholic fatty liver disease (NAFLD). It has been suggested that CESD may affect 1 in 40,000 and is under-diagnosed in NAFLD clinics. Therefore, we aimed to estimate the prevalence of LAL-D using analysis of genetic variation in LIPA. METHODS: MEDLINE and EMBASE were systematically searched for previously reported disease variants and prevalence estimates. Previous prevalence estimates were meta-analysed. Disease variants in LIPA were annotated with allele frequencies from gnomAD and combined with unreported major functional variants found in humans. Pooled ethnicity-specific prevalences for LAL-D and CESD were calculated using the Hardy-Weinberg equation. RESULTS: Meta-analysis of existing genetic studies estimated the prevalence of LAL-D as 1 per 160,000 (95% CI 1 per 65,025-761,652) using the allele frequency of c.894G>A in LIPA. A total of 98 previously reported disease variants in LIPA were identified, of which 32/98 were present in gnomAD, giving a prevalence of 1 per 307,482 (95% CI 257,672-366,865). Wolman disease was associated with more loss-of-function variants than CESD. When this was combined with 22 previously unreported major functional variants in LIPA identified in humans, the pooled prevalence of LAL-D was 1 per 177,452 (95% CI 149,467-210,683) with a carrier frequency of 1 per 421. The prevalence is lowest in those of East Asian, South Asian, and Finnish ancestry. CONCLUSION: Using 120 disease variants in LIPA, these data can reassure clinicians that LAL-D is an ultra-rare disorder. Given the therapeutic capability of sebelipase alpha, investigation for LAL-D might be included in second-line metabolic screening in NAFLD. LAY SUMMARY: Lysosomal Acid Lipase Deficiency (LAL-D) is a rare genetic condition that can cause severe liver disease, but it is difficult to diagnose and sometimes can look like simple fatty liver. It was not clear how common LAL-D was and whether many cases were being missed. To study this, we searched for all genetic mutations that could cause LAL-D, calculated how common those mutations were, and added them up. This let us estimate that LAL-D affects roughly 1 in 175,000 people. We conclude that LAL-D is a very rare condition, but it is treatable so may be included in a 'second-line' of tests for causes of fatty liver.


Subject(s)
Liver/pathology , Sterol Esterase/genetics , Wolman Disease/genetics , DNA Mutational Analysis , Diagnosis, Differential , Gene Frequency , Global Health , Humans , Liver/metabolism , Mutation , Non-alcoholic Fatty Liver Disease , Prevalence , Rare Diseases , Sterol Esterase/metabolism , Wolman Disease/diagnosis , Wolman Disease/epidemiology , Wolman Disease
11.
Dig Liver Dis ; 50(12): 1299-1304, 2018 12.
Article in English | MEDLINE | ID: mdl-30077465

ABSTRACT

BACKGROUND: Real-life data on vedolizumab effectiveness in inflammatory bowel disease (IBD) are still emerging. Data on the comparative safety of the gut selective profile are of particular interest. AIMS: To assess clinical outcome and safety in IBD patients treated with vedolizumab. METHODS: We retrospectively collected data of patients treated with vedolizumab at eight UK hospitals (August 2014-January 2018). Clinical response and remission at 14 and 52 weeks evaluated through Physician Global Assessment (PGA) and adverse events were recorded. Possible predictors of clinical response were examined. RESULTS: Two hundred and three IBD patients (mean treatment 16 ±â€¯8 months) were included. Of these, 135 patients (mean age 40.6 ±â€¯16.0 years; F:M 1.9:1) had CD and 68 (mean age 44.5 ±â€¯18.1 years; F:M 1:1.2) had UC. According to PGA, 106/135 (78.5%) CD and 62/68 (91.2%) UC patients (p = 0.02) had a clinical response/remission at 14 weeks, whereas 76/119 (63.9%) CD and 52/63 (82.5%) UC patients (p < 0.01) showed a sustained response or remission at 52 weeks, with a high adherence rate (97%). No predictors of clinical response were found. The cumulative incidence of infectious diseases was 11.9 per 100 person-years. CONCLUSION: Vedolizumab is an effective therapy for inducing and maintaining remission of IBD, with better results for UC, and with a good safety profile.


Subject(s)
Antibodies, Monoclonal, Humanized/therapeutic use , Gastrointestinal Agents/therapeutic use , Inflammatory Bowel Diseases/drug therapy , Medication Adherence/statistics & numerical data , Adult , Female , Humans , Male , Middle Aged , Remission Induction , Retrospective Studies , United Kingdom , Young Adult
12.
J Exp Zool A Ecol Integr Physiol ; 329(6-7): 373-381, 2018 07.
Article in English | MEDLINE | ID: mdl-29939491

ABSTRACT

The nest environment for eggs of reptiles has lifelong implications for offspring performance and success, and, ultimately, for population viability and species distributions. However, understanding the various abiotic and biotic drivers of nesting is complex, particularly regarding variation in nesting behavior of females and consequences for sex ratios in species with temperature-dependent sex determination (TSD). We investigated how nest construction and nesting phenology affect the incubation environment of a reptile with TSD, the tuatara (Sphenodon punctatus), a species that is at risk from climate-mediated male bias in population sex ratios. Using longitudinal behavioral data, we addressed the following questions. (1) Does nesting behavior vary with seasonal or location cues? (2) Does variation in nesting phenology or nest construction affect the incubation environment? We aimed to investigate whether female tuatara could modify nesting behavior to respond to novel environments, including a warming climate, allowing for successful incubation and balanced population sex ratios, maintaining population viability throughout their historic range. We predicted that earlier nesting after warm winters increased the likelihood that females will be produced, despite the sex determining system where males are produced from warmer temperatures. Further research is needed to understand the extent to which nesting behavior varies by individual through time, and across the range of tuatara, and the importance of habitat variability in maintaining production of females under future climate warming.


Subject(s)
Nesting Behavior/physiology , Reptiles/physiology , Sex Ratio , Animals , Climate Change , Ecosystem , Female , Male , Seasons , Temperature
14.
Tissue Eng Regen Med ; 13(6): 701-712, 2016 Dec.
Article in English | MEDLINE | ID: mdl-30603451

ABSTRACT

Diabetes is a major risk factor for the progression of vascular disease, contributing to elevated levels of glycoxidation, chronic inflammation and calcification. Tissue engineering emerges as a potential solution for the treatment of vascular diseases however there is a considerable gap in the understanding of how scaffolds and stem cells will perform in patients with diabetes. We hypothesized that adipose tissue-derived stem cells (ASCs) by virtue of their immunosuppressive potential would moderate the diabetes-intensified inflammatory reactions and induce positive construct remodeling. To test this hypothesis, we prepared arterial elastin scaffolds seeded with autologous ASCs and implanted them subdermally in diabetic rats and compared inflammatory markers, macrophage polarization, matrix remodeling, calcification and bone protein expression to control scaffolds implanted with and without cells in nondiabetic rats. ASC-seeded scaffolds exhibited lower levels of CD8+ T-cells and CD68+ pan-macrophages and higher numbers of M2 macrophages, smooth muscle cell-like and fibroblast-like cells. Calcification and osteogenic markers were reduced in ASCseeded scaffolds implanted in non-diabetic rats but remained unchanged in diabetes, unless the scaffolds were first pre-treated with penta-galloyl glucose (PGG), a known anti-oxidative elastin-binding polyphenol. In conclusion, autologous ASC seeding in elastin scaffolds is effective in combating diabetes-related complications. To prevent calcification, the oxidative milieu needs to be reduced by elastin-binding antioxidants such as PGG.

15.
Gigascience ; 4: 46, 2015.
Article in English | MEDLINE | ID: mdl-26445670

ABSTRACT

BACKGROUND: There is an increasing demand for rapid biodiversity assessment tools that have a broad taxonomic coverage. Here we evaluate a suite of environmental DNA (eDNA) markers coupled with next generation sequencing (NGS) that span the tree of life, comparing them with traditional biodiversity monitoring tools within ten 20×20 meter plots along a 700 meter elevational gradient. RESULTS: From six eDNA datasets (one from each of 16S, 18S, ITS, trnL and two from COI) we identified sequences from 109 NCBI taxonomy-defined phyla or equivalent, ranging from 31 to 60 for a given eDNA marker. Estimates of alpha and gamma diversity were sensitive to the number of sequence reads, whereas beta diversity estimates were less sensitive. The average within-plot beta diversity was lower than between plots for all markers. The soil beta diversity of COI and 18S markers showed the strongest response to the elevational variation of the eDNA markers (COI: r=0.49, p<0.001; 18S: r=0.48, p<0.001). Furthermore pairwise beta diversities for these two markers were strongly correlated with those calculated from traditional vegetation and invertebrate biodiversity measures. CONCLUSIONS: Using a soil-based eDNA approach, we demonstrate that standard phylogenetic markers are capable of recovering sequences from a broad diversity of eukaryotes, in addition to prokaryotes by 16S. The COI and 18S eDNA markers are the best proxies for aboveground biodiversity based on the high correlation between the pairwise beta diversities of these markers and those obtained using traditional methods.


Subject(s)
Biodiversity , DNA/genetics , Multigene Family , Animals
16.
Mar Pollut Bull ; 54(7): 955-62, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17448504

ABSTRACT

In September 1969, the Florida barge spilled 700,000L of No. 2 fuel oil into the salt marsh sediments of Wild Harbor (Buzzards Bay, MA). Today the aboveground environment appears unaffected, but a substantial amount of moderately degraded petroleum still remains 8-20cm below the surface. The salt marsh fiddler crabs, Uca pugnax, burrow into the sediments at depths of 5-25cm, and are chronically exposed to the spilled oil. Behavioral studies conducted with U. pugnax from Wild Harbor and a control site, Great Sippewissett marsh, found that crabs exposed to the oil avoided burrowing into oiled layers, suffered delayed escape responses, lowered feeding rates, and achieved lower densities. The oil residues are therefore biologically active and affect U. pugnax populations. Our results add new knowledge about long-term consequences of spilled oil, a dimension that should be included when assessing oil-impacted areas and developing management plans designed to restore, rehabilitate, or replace impacted areas.


Subject(s)
Brachyura/drug effects , Disasters , Environmental Monitoring , Marine Biology , Petroleum/toxicity , Water Pollutants, Chemical/toxicity , Wetlands , Animals , Brachyura/metabolism , Geography , Petroleum/metabolism , Risk Assessment , Seawater , Water Pollutants, Chemical/metabolism
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