Sequestration of microfibers and other microplastics by green algae, Cladophora, in the US Great Lakes.

Daunting amounts of microplastics are present in surface waters worldwide. A main category of microplastics is synthetic microfibers, which originate from textiles. These microplastics are generated and released in laundering and are discharged by wastewater treatment plants or enter surface waters from other sources. The polymers that constitute many common synthetic microfibers are mostly denser than water, and eventually settle out in aquatic environments. The interaction of these microfibers with submerged aquatic vegetation has not been thoroughly investigated but is potentially an important aquatic sink in surface waters. In the Laurentian Great Lakes, prolific growth of macrophytic Cladophora creates submerged biomass with a large amount of surface area and the potential to collect and concentrate microplastics. To determine the number of synthetic microfibers in Great Lakes Cladophora, samples were collected from Lakes Erie and Michigan at multiple depths in the spring and summer of 2018. After rinsing and processing the algae, associated synthetic microfibers were quantified. The average loads of synthetic microfibers determined from the Lake Erie and Lake Michigan samples were 32,000 per kg (dry weight (dw)) and 34,000 per kg (dw), respectively, 2-4 orders of magnitude greater than loads previously reported in water and sediment. To further explore this sequestration of microplastics, fresh and aged Cladophora were mixed with aqueous mixtures of microfibers or microplastic in the laboratory to simulate pollution events. Microscopic analyses indicated that fresh Cladophora algae readily interacted with microplastics via adsorptive forces and physical entanglement. These interactions mostly cease upon algal senescence, with an expected release of microplastics in benthic sediments. Collectively, these findings suggest that synthetic microfibers are widespread in Cladophora algae and the affinity between microplastics and Cladophora may offer insights for removing microplastic pollution. Macroalgae in the Laurentian Great Lakes contain high loads of synthetic microfibers, both entangled and adsorbed, which likely account for an important fraction of microplastics in these surface waters.

Tracking the distribution of microfiber pollution in a southern Lake Michigan watershed through the analysis of water, sediment and air.

Microplastic waste is a worldwide problem, heavily afflicting marine and freshwater environments; the loading of this pollution in water, sediment and living organisms continues to escalate. Synthetic microfibers, resulting from the release of microscopic fibers from synthetic textiles, constitute the most prevalent type of microplastics pollution in aquatic environments. This study investigated the origin and distribution of synthetic microfibers in a representative Lake Michigan watershed in Indiana (USA) by analyzing water, sediment and air samples above and below wastewater treatment plant discharges, downstream in the watershed and water from the Lake Michigan shoreline. Synthetic microfibers were also quantified in wastewater from a local wastewater treatment plant (WWTP) and in laundry effluent. Laboratory testing of numerous fabrics suggests that Fenton oxidation, used to break down natural fibers, effectively eliminates non-polluting, natural fibers from the samples. However, the hydroxyl radical-mediated oxidation bleaches the dye from certain synthetic microfibers, which likely leads to under-reported values for these microplastics in natural samples. The data collected from the watershed samples indicate that approximately 4 billion synthetic microfibers are transported daily through the Lake Michigan tributary. Wastewater effluent is not the only source of synthetic microfibers, since surface water samples above the WWTP contained a similar load to downstream samples. Repeated sampling exhibited variability in the number of microfibers detected, substantiating the heterogeneous distribution of these pollutants and the requirement for multiple samples for a given site. The average load of synthetic microfibers from water sampled at the Lake Michigan shoreline was higher than the tributary water, suggesting the shoreline functions as a repository for the microfibers. Given the extent and potential consequences of this pollution, quantification of the ubiquitous plastic fibers can be instituted as part of the traditional total suspended solids (TSS) water quality monitoring parameter.

A cell culture adapted HCV JFH1 variant that increases viral titers and permits the production of high titer infectious chimeric reporter viruses.

The unique properties of the hepatitis C virus (HCV) JFH1 isolate have made it possible to produce and study HCV in an infectious cell culture system. However, relatively low virus titers restrict some of the uses of this system and preparing infectious chimeric reporter viruses have been difficult. In this study, we report cell culture-adapted mutations in wild-type JFH1 yielding higher titers of infectious particles of both JFH1 and chimeric JFH1 viruses carrying reporter genes. Sequencing analyses determined that ten of the sixteen nonsynonymous mutations were in the NS5A region. Individual viruses harboring specific adaptive mutations were prepared and studied. The mutations in the NS5A region, which included all three domains, were most effective in increasing infectious virus production. Insertion of two reporter genes in JFH1 without the adaptive mutations ablated the production of infectious HCV particles. However, the introduction of specific adaptive mutations in the NS5A region permitted reporter genes, Renilla luciferase (Rluc) and EGFP, to be introduced into JHF1 to produce chimeric HCV-NS5A-EGFP and HCV-NS5A-Rluc reporter viruses at relatively high titers of infectious virus. The quantity of hyperphosphorylated NS5A (p58) was decreased in the adapted JFH1 compared wild type JFH1 and is likely be involved in increased production of infectious virus based on previous studies of p58. The JFH1-derived mutant viruses and chimeric reporter viruses described here provide new tools for studying HCV biology, identifying HCV antivirals, and enable new ways of engineering additional infectious chimeric viruses.

ENCODE tiling array analysis identifies differentially expressed annotated and novel 5' capped RNAs in hepatitis C infected liver.

Microarray studies of chronic hepatitis C infection have provided valuable information regarding the host response to viral infection. However, recent studies of the human transcriptome indicate pervasive transcription in previously unannotated regions of the genome and that many RNA transcripts have short or lack 3' poly(A) ends. We hypothesized that using ENCODE tiling arrays (1% of the genome) in combination with affinity purifying Pol II RNAs by their unique 5' m7GpppN cap would identify previously undescribed annotated and unannotated genes that are differentially expressed in liver during hepatitis C virus (HCV) infection. Both 5'-capped and poly(A)+ populations of RNA were analyzed using ENCODE tiling arrays. Sixty-four annotated genes were significantly increased in HCV cirrhotic as compared to control liver; twenty-seven (42%) of these genes were identified only by analyzing 5' capped RNA. Thirty-one annotated genes were significantly decreased; sixteen (50%) of these were identified only by analyzing 5' capped RNA. Bioinformatic analysis showed that capped RNA produced more consistent results, provided a more extensive expression profile of intronic regions and identified upregulated Pol II transcriptionally active regions in unannotated areas of the genome in HCV cirrhotic liver. Two of these regions were verified by PCR and RACE analysis. qPCR analysis of liver biopsy specimens demonstrated that these unannotated transcripts, as well as IRF1, TRIM22 and MET, were also upregulated in hepatitis C with mild inflammation and no fibrosis. The analysis of 5' capped RNA in combination with ENCODE tiling arrays provides additional gene expression information and identifies novel upregulated Pol II transcripts not previously described in HCV infected liver. This approach, particularly when combined with new RNA sequencing technologies, should also be useful in further defining Pol II transcripts differentially regulated in specific disease states and in studying RNAs regulated by changes in pre-mRNA splicing or 3' polyadenylation status.

Measuring antiviral activity of benzimidazole molecules that alter IRES RNA structure with an infectious hepatitis C virus chimera expressing Renilla luciferase.

Major progress has been made in developing infectious HCV cell culture systems and these systems have been useful in identifying novel HCV antivirals. However, more rapid and sensitive assays using infectious cell based HCV systems would facilitate the development of additional antivirals, including small molecules directed at unique targets such as the HCV RNA internal ribosomal entry site (IRES). We have found that the V3 region (28 aa) of NS5A of HCV JFH1 can be deleted from the genome with only modest effects on the titer of infectious virus produced in cell culture. Moreover, the V3 region can be replaced with the Renilla reniformis luciferase (Rluc) gene resulting in an infectious virus that stably expresses an NS5A-Rluc fusion protein. Infected cells cultured in 96-well plates provided a robust luciferase signal that accurately reflected the production of infectious virus. This infectious HCV reporter system was used to test the activity of three benzimidazole compounds that bind the HCV RNA IRES. Compounds in this chemical class of small molecules bind and alter the IRES RNA structure at low to sub-micromolar concentrations and interfere with viral replication. The current study shows that these compounds inhibit HCV replication in an infectious HCV cell culture system, defines their IC(50) in this system, and provides a platform for the rapid testing of next generation inhibitors.

Genome-wide characterization of methylguanosine-capped and polyadenylated small RNAs in the rice blast fungus Magnaporthe oryzae.

Small RNAs are well described in higher eukaryotes such as mammals and plants; however, knowledge in simple eukaryotes such as filamentous fungi is limited. In this study, we discovered and characterized methylguanosine-capped and polyadenylated small RNAs (CPA-sRNAs) by using differential RNA selection, full-length cDNA cloning and 454 transcriptome sequencing of the rice blast fungus Magnaporthe oryzae. This fungus causes blast, a devastating disease on rice, the principle food staple for over half the world's population. CPA-sRNAs mapped primarily to the transcription initiation and termination sites of protein-coding genes and were positively correlated with gene expression, particularly for highly expressed genes including those encoding ribosomal proteins. Numerous CPA-sRNAs also mapped to rRNAs, tRNAs, snRNAs, transposable elements and intergenic regions. Many other 454 sequence reads could not be mapped to the genome; however, inspection revealed evidence for non-template additions and chimeric sequences. CPA-sRNAs were independently confirmed using a high affinity variant of eIF-4E to capture 5'-methylguanosine-capped RNA followed by 3'-RACE sequencing. These results expand the repertoire of small RNAs in filamentous fungi.

Human RNA polymerase III transcriptomes and relationships to Pol II promoter chromatin and enhancer-binding factors.

RNA polymerase (Pol) III transcribes many noncoding RNAs (for example, transfer RNAs) important for translational capacity and other functions. We localized Pol III, alternative TFIIIB complexes (BRF1 or BRF2) and TFIIIC in HeLa cells to determine the Pol III transcriptome, define gene classes and reveal 'TFIIIC-only' sites. Pol III localization in other transformed and primary cell lines reveals previously uncharacterized and cell type-specific Pol III loci as well as one microRNA. Notably, only a fraction of the in silico-predicted Pol III loci are occupied. Many occupied Pol III genes reside within an annotated Pol II promoter. Outside of Pol II promoters, occupied Pol III genes overlap with enhancer-like chromatin and enhancer-binding proteins such as ETS1 and STAT1. Moreover, Pol III occupancy scales with the levels of nearby Pol II, active chromatin and CpG content. These results suggest that active chromatin gates Pol III accessibility to the genome.