George P. Cobb

Evidence that miRNAs are different from other RNAs

Abstract.An examination of 513 known pre-miRNAs and 237 other RNAs (tRNA, rRNA, and mRNA) revealed that miRNAs were significantly different from other RNAs (p < 0.001). miRNA genes were less conserved than other RNA genes, although their mature miRNA sequences were highly conserved. The A+U content of pre-miRNAs was higher than non-coding RNA (p < 0.001), but lower than mRNAs. The nucleotides in pre-miRNAs formed more hydrogen bonds and base pairs than in other RNAs. miRNAs had higher negative adjusted minimal folding free energies than other RNAs except tRNAs (p < 0.001). The MFE index (MFEI) was a sufficient criterion to distinguish miRNAs from all coding and non-coding RNAs (p < 0.001). The MFEI for miRNAs was 0.97, significantly higher than tRNAs (0.64), rRNAs (0.59), or mRNAs (0.65). Our findings should facilitate the prediction and identification of new miRNAs using computational and experimental strategies.

Identification and characterization of new plant microRNAs using EST analysis.

ABSTRACTSeventy-five previously known plant microRNAs (miRNAs) were classified into 14 families according to their gene sequence identity. A total of 18,694 plant expressed sequence tags (EST) were found in the GenBank EST databases by comparing all previously known Arabidopsis miRNAs to GenBanks plant EST databases with BLAST algorithms. After removing the EST sequences with high numbers (more than 2) of mismatched nucleotides, a total of 812 EST contigs were identified. After predicting and scoring the RNA secondary structure of the 812 EST sequences using mFold software, 338 new potential miRNAs were identified in 60 plant species. miRNAs are widespread. Some microRNAs may highly conserve in the plant kingdom, and they may have the same ancestor in very early evolution. There is no nucleotide substitution in most miRNAs among many plant species. Some of the new identified potential miRNAs may be induced and regulated by environmental biotic and abiotic stresses. Some may be preferentially expressed in specific tissues, and are regulated by developmental switching. These findings suggest that EST analysis is a good alternative strategy for identifying new miRNA candidates, their targets, and other genes. A large number of miRNAs exist in different plant species and play important roles in plant developmental switching and plant responses to environmental abiotic and biotic stresses as well as signal transduction. Environmental stresses and developmental switching may be the signals for synthesis and regulation of miRNAs in plants. A model for miRNA induction and expression, and gene regulation by miRNA is hypothesized.

Review: Computational identification of microRNAs and their targets

MicroRNAs (miRNAs) are one class of newly identified riboregulators of gene expression in many eukaryotic organisms. They play important roles in multiple biological and metabolic processes, including developmental timing, signal transduction, cell maintenance and differentiation, diseases and cancers. miRNAs regulate gene expression at the posttranscriptional level by directly cleaving targeted mRNAs or repressing translation. Although the founding members of miRNAs were discovered by genetic screening approaches, experimental approaches were limited by their low efficiency, time consuming, and high cost. As an alternative, computational approaches were developed. Computational approaches for identifying miRNAs are based on the following major characteristics of miRNAs: hairpin-shaped secondary structures, high conservation for some miRNAs, and high minimal folding free energy index (MFEI). Computational approaches also play an important role in identifying miRNA targets. A majority of known miRNAs and their targets were identified by computational approaches. Several web-based or non-web-based computer software programs are publicly available for predicting miRNAs and their targets.

Acute effects of Fe2O3, TiO2, ZnO and CuO nanomaterials on Xenopus laevis

Metal oxide nanomaterials have exhibited toxicity to a variety of aquatic organisms, especially microbes and invertebrates. To date, few studies have evaluated the toxicity of metal oxide nanomaterials on aquatic vertebrates. Therefore, this study examined effects of ZnO, TiO(2), Fe(2)O(3), and CuO nanomaterials (20-100 nm) on amphibians utilizing the Frog Embryo Teratogenesis Assay Xenopus (FETAX) protocol, a 96 h exposure with daily solution exchanges. Nanomaterials were dispersed in reconstituted moderately hard test medium. These exposures did not increase mortality in static renewal exposures containing up to 1,000 mg L(-1) for TiO(2), Fe(2)O(3), CuO, and ZnO, but did induce developmental abnormalities. Gastrointestinal, spinal, and other abnormalities were observed in CuO and ZnO nanomaterial exposures at concentrations as low as 3.16 mg L(-1) (ZnO). An EC(50) of 10.3 mg L(-1) ZnO was observed for total malformations. The minimum concentration to inhibit growth of tadpoles exposed to CuO or ZnO nanomaterials was 10 mg L(-1). The results indicate that select nanomaterials can negatively affect amphibians during development. Evaluation of nanomaterial exposure on vertebrate organisms are imperative to responsible production and introduction of nanomaterials in everyday products to ensure human and environmental safety.

Uptake of perchlorate in terrestrial plants.

Cucumber (Cucumis sativus L.), lettuce (Lactuca sativa L.), and soybean (Glycine max) were used to determine uptake of the perchlorate anion (100 ppb) from sand. Plants were watered with different ratios of Hydrosol (a diluted solution of Peters All-Purpose Plant Food) to Milli-Q water (18 MOmega) to determine if the presence of other nutrients (such as nitrate) influenced perchlorate uptake. Perchlorate concentrations in sand and plant tissues were determined weekly. Perchlorate uptake was observed in all three plant species. In most experiments, perchlorate was completely depleted from sand in which plants were growing. Perchlorate concentrations in lettuce were also significantly higher than those in cucumber and soybean (P < 0.0001). Perchlorate concentrations in sand decreased at a higher rate at lower ratios of Hydrosol to Milli-Q, indicating that plant (cucumber) uptake of perchlorate is influenced by the presence of external nutrients. The results of an 8-week uptake study in cucumber and a 6-week uptake study in lettuce suggest that a threshold perchlorate concentration is reached: for cucumber, 150 ppm and for lettuce, 750 ppm. Although the presence of external nutrients decreases the rate of perchlorate uptake by plants, significant concentrations of perchlorate occur in aboveground plant tissues even after relatively short periods of growth. The potential for trophic transfer of perchlorate from soil to higher organisms through plants exists.

Monocrotophos-Induced Mass Mortality of Swainson's Hawks in Argentina, 1995–96

Swainsons hawks (Buteo swainsoni) were observed in the grasslands (pampas) of Argentina during the austral summer of 1995–96. Widespread agrochemical use to control insects had a profound effect on flocks of hawks foraging on grasshoppers and caterpillars throughout the pampas. We describe 19 mortality incidents and an estimated 5,095 dead hawks. Forensic analysis indicated that the organophosphate insecticide monocrotophos was responsible for the deaths at six separate sites, accounting for over 4,000 of the mortalities. Brain cholinesterase was lethally inhibited (>95%) and monocrotophos residues (n=31/45, mean=0.20 ppm, range 0.05–1.08 ppm) were found in the contents of the gastrointestinal tract. No other insecticide residues were found. Sample analyses, combined with interviews with farmers, indicated that monocrotophos was responsible for deaths at 17 of 19 mortality incidents.

Mercury speciation and biomagnification in the food web of Caddo Lake, Texas and Louisiana, USA, a subtropical freshwater ecosystem

We studied the biomagnification of total mercury and methylmercury in a subtropical freshwater lake, Caddo Lake, Texas and Louisiana, USA. The present study is unique in that it not only included invertebrates (seven species) and fish (six species) but also an amphibian (one species), reptiles (three species), and mammals (three species). Nonfish vertebrates such as those included in the present study are often not included in assessments of trophic transfer of Hg. Mean trophic position (determined using stable isotopes of nitrogen) ranged from 2.0 (indicative of a primary consumer) to 3.8 (indicative of a tertiary consumer). Mean total Hg concentrations ranged from 36 to 3,292 ng/g dry weight in muscle and whole body and from 150 to 30,171 ng/g dry weight in liver. Most of the Hg in muscle and whole-body tissue was found as methylmercury, and at least 50% of the Hg found in liver was in the inorganic form (with the exception of largemouth bass, Micropterus salmoides). Mercury concentrations were positively correlated with trophic position, indicating that biomagnification occurs in the food web of Caddo Lake. The food web magnification factors (FWMFs; slope of the relationship between mean Hg concentration and trophic position) for both total Hg and methylmercury were similar to those observed in other studies. Because most of the total Hg in consumers was methylmercury, the FWMF for methylmercury was not significantly different from the FWMF for total Hg. Some vertebrates examined in the present study had low Hg concentrations in their tissues similar to those observed in invertebrates, whereas others had concentrations of Hg in their tissues that in previous studies have been associated with negative health consequences in fish.

Occurrence of synthetic musk fragrances in effluent and non-effluent impacted environments.

Synthetic musk fragrances (SMFs) are considered micropollutants and can be found in various environmental matrices near wastewater discharge areas. These emerging contaminants are often detected in wastewater at low concentrations; they are continuously present and constitute a constant exposure source. Objectives of this study were to investigate the environmental fate, transport, and transformation of SMFs. Occurrence of six polycyclic musk compounds (galaxolide, tonalide, celestolide, phantolide, traseolide, cashmeran) and two nitro musk compounds (musk xylene and musk ketone) was monitored in wastewater, various surface waters and their sediments, as well as groundwater, soil cores, and plants from a treated wastewater land application site. Specifically, samples were collected quarterly from (1) a wastewater treatment plant to determine initial concentrations in wastewater effluent, (2) a storage reservoir at a land application site to determine possible photolysis before land application, (3) soil cores to determine the amount of sorption after land application and groundwater recharge to assess lack thereof, (4) a lake system and its sediment to assess degradation, and (5) non-effluent impacted local playa lakes and sediments to assess potential sources of these compounds. All samples were analyzed using gas chromatography coupled with mass spectrometry (GC-MS). Data indicated that occurrence of SMFs in effluent-impacted environments was detectable at ng/L and ng/g concentrations, which decreased during transport throughout wastewater treatment and land application. However, unexpected concentrations, ng/L and ng/g, were also detected in playa lakes not receiving treated effluent. Additionally, soil cores from land application sites had ng/g concentrations, and SMFs were detected in plant samples at trace levels. Galaxolide and tonalide were consistently found in all environments. Information on occurrence is critical to assessing exposure to these potential endocrine disrupting compounds. Such information could provide a scientific framework for establishing the need for environmental regulations.

The decline of mink in Georgia, North Carolina, and South Carolina: The role of contaminants

Since the 1960s, mink (Mustela vison) populations in Georgia, North Carolina, and South Carolina have declined, especially in the coastal plain. A prior study suggested that the decline may stem from environmental contaminants. Based on water quality data from each state, we identified 17 substances potentially related to the decline: aldrin, dieldrin, endrin, DDD, DDE, DDT, PCBs, chlordane, alpha-BHC, toxaphene, dibenzofuran, copper, chromium, cadmium, lead, arsenic, and mercury. Mink livers were analyzed for PCB and organochlorine pesticides, and kidneys and femurs were analyzed for metals. Reference sample concentrations from piedmont, mountain, and foothill locations were compared to state coastal plain totals and counties. PCBs for Georgia, dieldrin for South Carolina, and endrin and aldrin for North Carolina were significantly higher than the piedmont reference group. Liver PCB concentrations were higher than those known to cause mink reproductive dysfunction. Mercury concentrations were significantly higher in coastal plain mink from all three states and were in the range of those known to cause impacts to reproduction, growth, and behavior to wild mink. It is unknown what concentrations of cyclodienes cause reduced reproduction or other physiological effects in mink, but the levels reported here probably indicate background concentrations that do not contribute to the decline.

Development of a method for the determination of 9 currently used cotton pesticides by gas chromatography with electron capture detection.

A reliable, sensitive, and reproducible method was developed for quantitative determination of nine new generation pesticides currently used in cotton agriculture. Injector temperature significantly affected analyte response as indicated by electron capture detector (ECD) chromatograms. A majority of the analytes had an enhanced response at injector temperatures between 240 and 260 degrees C, especially analytes such as acephate that overall had a poor response on the ECD. The method detection limits (MDLs) were 0.13, 0.05, 0.29, 0.35, 0.08, 0.10, 0.32, 0.05, and 0.59 ng/mL for acephate, trifuralin, malathion, thiamethozam, pendimethalin, DEF6, acetamiprid, brifenthrin, and lambda-cyhalothrin. This study provides a precision (0.17-13.1%), accuracy (recoveries=88-107%) and good reproducible method for the analytes of interest. At relatively high concentrations, only lambda-cyhalothrin was unstable at room temperature (20-25 degrees C) and 4 degrees C over 10 days. At relatively low concentrations, acephate and acetamiprid were also unstable regardless of temperature. After 10 days storage at room temperature, 30-40% degradation of lambda-cyhalothrin was observed. It is recommended that acephate, acetamiprid, and lambda-cyhalothrin be stored at -20 degrees C or analyzed immediately after extraction.