Evaluation of ddRADseq for reduced representation metagenome sequencing

Background. Profiling of microbial communities via metagenomic shotgun sequenc- ing has enabled researches to gain unprecedented insight into microbial community structure and the functional roles of community members. This study describes a method and basic analysis for a metagenomic adaptation of the double digest restriction site associated DNA sequencing (ddRADseq) protocol for reduced representation metagenome profiling. Methods. This technique takes advantage of the sequence specificity of restriction endonucleases to construct an Illumina-compatible sequencing library containingDNA fragments that are between a pair of restriction sites located within close proximity. This results in a reduced sequencing library with coverage breadth that can be tuned by size selection. We assessed the performance of the metagenomic ddRADseq approach by applying the full method to human stool samples and generating sequence data. Results. The ddRADseq data yields a similar estimate of community taxonomic profile as obtained from shotgun metagenome sequencing of the same human stool samples. No obvious bias with respect to genomic G + C content and the estimated relative species abundance was detected. Discussion. Although ddRADseq does introduce some bias in taxonomic representa- tion, the bias is likely to be small relative to DNA extraction bias. ddRADseq appears feasible and could have value as a tool for metagenome-wide association studies

Microbial ecological response of the intestinal flora of Peromyscus maniculatus and P. leucopus to heavy metal contamination

Heavy metal contamination negatively affects natural systems including plants, birds, fish and bacteria by reducing biodiversity at contaminated sites. At the Tri-State Mining District, efforts have been made to remediate sites to mitigate the detrimental effects that contamination has caused on human health. While the remediation effort has returned the site to within federal safety standards, it is unclear if this effort is sufficient to restore floral and faunal communities. Intrinsic to ecosystem and organism health is the biodiversity and composition of microbial communities. We have taken advantage of recent advances in sequencing technology and surveyed the bacterial community of remediated and reference soils as well as the intestinal microbial community of two ubiquitous rodent species to provide insight on the impacts of residual heavy metal contamination on the ecosystem. Rodents found on the remediated site had reduced body mass, smaller body size and lower body fat than animals on reference sites. Using bar-coded, massively parallel sequencing, we found that bacterial communities in both the soil and Peromyscus spp. gastrointestinal tracts had no difference in diversity between reference and remediated sites but assemblages differed in response to contamination. These results suggest that niche voids left by microbial taxa that were unable to deal with the remnant levels of heavy metals on remediated sites were replaced by taxa that could persist in this environment. Whether this replacement provided similar ecosystem services as ancestral bacterial communities is unknown.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/79374/1/MEC_4485_sm_Supporting.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/79374/2/j.1365-294X.2009.04485.x.pd

Nanoparticles within WWTP sludges have minimal impact on leachate quality and soil microbial community structure and function

One of the main pathways by which engineered nanoparticles (ENPs) enter the environment is through land application of waste water treatment plant (WWTP) sewage sludges. WWTP sludges, enriched with Ag and ZnO ENPs or their corresponding soluble metal salts during anaerobic digestion and subsequently mixed with soil (targeting a final concentration of 1400 and 140 mg/kg for Zn and Ag, respectively), were subjected to 6 months of ageing and leaching in lysimeter columns outdoors. Amounts of Zn and Ag leached were very low, accounting for <0.3% and <1.4% of the total Zn and Ag, respectively. No differences in total leaching rates were observed between treatments of Zn or Ag originally input to WWTP as ENP or salt forms. Phospholipid fatty acid profiling indicated a reduction in the fungal component of the soil microbial community upon metal exposure. However, overall, the leachate composition and response of the soil microbial community following addition of sewage sludge enriched either with ENPs or metal salts was very similar

Environmental detection of Penicillium marneffei and growth in soil microcosms in competition with Talaromyces stipitatus

Accepted versio

Rapid detection of Ganoderma-infected oil palms by microwave ergosterol extraction with HPLC and TLC

Detection of basal stem rot (BSR) by Ganoderma of oil palms was based on foliar symptoms and production of basidiomata. Enzyme-Linked Immunosorbent Assays-Polyclonal Antibody (ELISA-PAB) and PCR have been proposed as early detection methods for the disease. These techniques are complex, time consuming and have accuracy limitations. An ergosterol method was developed which correlated well with the degree of infection in oil palms, including samples growing in plantations. However, the method was capable of being optimised. This current study was designed to develop a simpler, more rapid and efficient ergosterol method with utility in the field that involved the use of microwave extraction. The optimised procedure involved extracting a small amount of Ganoderma, or Ganoderma-infected oil palm suspended in low volumes of solvent followed by irradiation in a conventional microwave oven at 70 °C and medium high power for 30 s, resulting in simultaneous extraction and saponification. Ergosterol was detected by thin layer chromatography (TLC) and quantified using high performance liquid chromatography with diode array detection. The TLC method was novel and provided a simple, inexpensive method with utility in the field. The new method was particularly effective at extracting high yields of ergosterol from infected oil palm and enables rapid analysis of field samples on site, allowing infected oil palms to be treated or culled very rapidly. Some limitations of the method are discussed herein. The procedures lend themselves to controlling the disease more effectively and allowing more effective use of land currently employed to grow oil palms, thereby reducing pressure to develop new plantations.This project was supported by the Fundamental Research Grant Scheme (FRGS), administered through the Ministry of Higher Education, Malaysia (Grant No: 5524175)

Avaliação da aterosclerose subclínica e de níveis plasmáticos de LDL minimamente modificada em pacientes com espondilite anquilosante e sua correlação com a atividade da doença

AbstractIntroductionAccelerated atherosclerosis has been shown in some autoimmune diseases, mainly in Systemic Lupus Erythematosus and Rheumatoid Arthritis. Although high preva- lence of corticosteroids use may be a confounding factor due to their detrimental effects on several risk factors, systemic inflammation per se is supposed to play an important role in atherogenesis in these patients.MethodsWe have evaluated sub-clinical atherosclerosis and plasma levels of circulating electronegative LDL, which represents the fraction of LDL that is minimally modified, in patients with ankylosing spondylitis (AS). Fourteen patients who fulfilled the modified New York criteria for AS were compared with 13 paired controls. Carotid intimal-media thick- ness (IMT) was assessed by ultrasonography bilaterally in common carotid artery, internal carotid artery and in the bifurcation. Groups were homogeneous regarding cardiovascular risk factors. Only a single patient in AS group was in use of corticosteroid.ResultsThe presence of active inflammation was demonstrated by elevated BASDAI and higher CRP levels and in patients versus controls (12.36 vs. 3.45mg/dl, P = 0.002). No dif- ference was found in carotid IMT between both groups, in any site of artery. Averaged IMT (6 measurements, at 3 pre-specified sites bilaterally) was 0.72 ± 0.28 in AS group and 0.70 ± 0.45mm in controls (P = 0.91). Minimally modified LDL did not differ significantly either between patients and controls (14.03 ± 17.40 vs. 13.21 ± 10.21; P = 0.88).ConclusionsPatients with AS did not show increased carotid IMT in comparison to con- trols. In the same way, circulating plasma levels of LDL (-), did not differ significantly in both groups