Introduction and enhancement of vegetative cover at Lake Mead

Studies done by the Nevada Department of Wildlife (NDOW) and the Arizona Fish and Game between 1978 and 1981 indicate that inadequate cover may be limiting the production and survival of largemouth bass at the Lake Mead National Recreation Area (LMNRA). As a result of these studies, NDOW initiated a contract in 1986 with the Lake Mead Research Center (LMRC) to investigate means of improving habitat for game fish by introducing natural and/or artificial cover. During Phase I (1986-1987) of this contract, the shoreline of Lake Mead was surveyed for aquatic and terrestrial plant growth. Also during this time, submerged Christmas trees and Berkley Fish Habitat Modules were evaluated for their effectiveness in providing cover. Christmas trees appeared to provide cover for juvenile bluegill, a largemouth bass prey species. However, submerged trees lost their structure in about three years. Berkley Habitat Modules did not appear to be an effective form of cover. The National Park Service (NPS) asked that the introduction of artificial forms of cover not be continued until research was completed on the use of native plant material. Methods for aquatic and terrestrial plant introductions were determined during Phase II (1987-1988) of this contract. Other agencies and individuals involved in revegetation of reservoir inundation zones were contacted, and the literature was reviewed for plant material collecting procedures, planting guidelines, and site maintenance. A Plant Introduction Manual was compiled based on this information and was approved by the National Park Service for use in the Lake Mead National Recreation Area. During the winter, dormant cuttings were taken of two woody species, Goodding\u27s Willow and seepwillow baccharis (Salix gooddingii and Baccharis glutinosa) to be planted in the spring of 1988. In addition, collections were made of three emergent species (Typha angustifolia, Scirpus robustus, and Phragmites australis). Plant material was transported to the Nevada Division of Forestry (NDF) nursery where it was rooted and placed on pots for planting in May 1988. Sago pondweed (Potamogeton pectinatus) tubers were introduced into small study plots in the spring of 1987. More tubers became established and were healthier in fertilized plots than in unfertilized plots. During Phase III (1988-1989) of this study, plant material was introduced into one cove in the lower basin and two coves in the upper basin of Lake Mead in April and May of 1988. Unpredicted low lake levels resulted in the loss of many plants. Survival rates of rooted material, however, were better than those of direct cuttings of woody plants. Site selection, particularly the soils of the site, appears to play a large part in survival. Seepwillow baccharis had the highest survival rates. In addition, greenhouse studies indicate that emergent plant tubers have some tolerance to dessication. Twelve hundred sago pondweed tubers were planted in April 1988 in one cove in the upper basin, and 1,200 tubers were planted in a cove in the lower basin. Tubers had 100 percent germination success and provided 70 percent cover for fish by July 1988. Approximately 10,000 sago pondweed tubers were planted in April 1989 in Waterbarge Cove in the lower basin. Tubers were planted in water depths ranging from very shallow to 12-15 meters deep. Germination and establishment of tubers were very good in depths less than 7 meters; however, little or no germination of tubers was noted at depths greater than 7 meters. A one-acre area of shoreline was hydroseeded in October 1988. Germination and establishment of seedlings was highest where soil moisture was between 20 and 30 percent. However, many seedlings were lost when water levels began to rise in January 1989

Deep-sequencing of endothelial cells exposed to hypoxia reveals the complexity of known and novel microRNAs

In order to understand the role of microRNAs (miRNAs) in vascular physiopathology, we took advantage of deep-sequencing techniques to accurately and comprehensively profile the entire miRNA population expressed by endothelial cells exposed to hypoxia. SOLiD sequencing of small RNAs derived from human umbilical vein endothelial cells (HUVECs) exposed to 1% O or normoxia for 24 h yielded more than 22 million reads per library. A customized bioinformatic pipeline identified more than 400 annotated microRNA/ microRNA*species with a broad abundance range: miR-21 and miR-126 totaled almost 40% of all miRNAs. A complex repertoire of isomiRs was found, displaying also 5′ variations, potentially affecting target recognition. Highstringency bioinformatic analysis identified microRNA candidates, whose predicted pre-miRNAs folded into a stable hairpin. Validation of a subset by qPCR identified 18 high-confidence novel miRNAs as detectable in independent HUVEC cultures and associated to the RISC complex. The expression of two novel miRNAs was significantly down-modulated by hypoxia, while miR- 210 was significantly induced. Gene ontology analysis of their predicted targets revealed a significant association to hypoxiainducible factor signaling, cardiovascular diseases, and cancer. Overexpression of the novel miRNAs in hypoxic endothelial cells affected cell growth and confirmed the biological relevance of their down-modulation. In conclusion, deep-sequencing accurately profiled known, variant, and novel microRNAs expressed by endothelial cells in normoxia and hypoxia

Efficient extraction of small and large RNAs in bacteria for excellent total RNA sequencing and comprehensive transcriptome analysis.

BACKGROUND: Next-generation transcriptome sequencing (RNA-Seq) has become the standard practice for studying gene splicing, mutations and changes in gene expression to obtain valuable, accurate biological conclusions. However, obtaining good sequencing coverage and depth to study these is impeded by the difficulties of obtaining high quality total RNA with minimal genomic DNA contamination. With this in mind, we evaluated the performance of Phenol-free total RNA purification kit (Amresco) in comparison with TRI Reagent (MRC) and RNeasy Mini (Qiagen) for the extraction of total RNA of Pseudomonas aeruginosa which was grown in glucose-supplemented (control) and polyethylene-supplemented (growth-limiting condition) minimal medium. All three extraction methods were coupled with an in-house DNase I treatment before the yield, integrity and size distribution of the purified RNA were assessed. RNA samples extracted with the best extraction kit were then sequenced using the Illumina HiSeq 2000 platform. RESULTS: TRI Reagent gave the lowest yield enriched with small RNAs (sRNAs), while RNeasy gave moderate yield of good quality RNA with trace amounts of sRNAs. The Phenol-free kit, on the other hand, gave the highest yield and the best quality RNA (RIN value of 9.85 ± 0.3) with good amounts of sRNAs. Subsequent bioinformatic analysis of the sequencing data revealed that 5435 coding genes, 452 sRNAs and 7 potential novel intergenic sRNAs were detected, indicating excellent sequencing coverage across RNA size ranges. In addition, detection of low abundance transcripts and consistency of their expression profiles across replicates from the same conditions demonstrated the reproducibility of the RNA extraction technique. CONCLUSIONS: Amresco\u27s Phenol-free Total RNA purification kit coupled with DNase I treatment yielded the highest quality RNAs containing good ratios of high and low molecular weight transcripts with minimal genomic DNA. These RNA extracts gave excellent non-biased sequencing coverage useful for comprehensive total transcriptome sequencing and analysis. Furthermore, our findings would be useful for those interested in studying both coding and non-coding RNAs from precious bacterial samples cultivated in growth-limiting condition, in a single sequencing run

A transcriptional sketch of a primary human breast cancer by 454 deep sequencing

Background: The cancer transcriptome is difficult to explore due to the heterogeneity of quantitative and qualitative changes in gene expression linked to the disease status. An increasing number of "unconventional" transcripts, such as novel isoforms, non-coding RNAs, somatic gene fusions and deletions have been associated with the tumoral state. Massively parallel sequencing techniques provide a framework for exploring the transcriptional complexity inherent to cancer with a limited laboratory and financial effort. We developed a deep sequencing and bioinformatics analysis protocol to investigate the molecular composition of a breast cancer poly(A)+ transcriptome. This method utilizes a cDNA library normalization step to diminish the representation of highly expressed transcripts and biology-oriented bioinformatic analyses to facilitate detection of rare and novel transcripts. Results: We analyzed over 132,000 Roche 454 high-confidence deep sequencing reads from a primary human lobular breast cancer tissue specimen, and detected a range of unusual transcriptional events that were subsequently validated by RT-PCR in additional eight primary human breast cancer samples. We identified and validated one deletion, two novel ncRNAs (one intergenic and one intragenic), ten previously unknown or rare transcript isoforms and a novel gene fusion specific to a single primary tissue sample. We also explored the non-protein-coding portion of the breast cancer transcriptome, identifying thousands of novel non-coding transcripts and more than three hundred reads corresponding to the non-coding RNA MALAT1, which is highly expressed in many human carcinomas. Conclusion: Our results demonstrate that combining 454 deep sequencing with a normalization step and careful bioinformatic analysis facilitates the discovery and quantification of rare transcripts or ncRNAs, and can be used as a qualitative tool to characterize transcriptome complexity, revealing many hitherto unknown transcripts, splice isoforms, gene fusion events and ncRNAs, even at a relatively low sequence sampling