Trauma Program Value Assessment at an Academic Health Network System Over 12 Years.

BACKGROUND: Trauma is a leading cause of global death, with 200 000 deaths and over 3 million non-fatal injuries/year in the United States. We aim to assess trauma care value for patients who underwent urgent laparotomies (LAP) and thoracotomies (THO) in our Health Network System. METHODS: Clinical variables (v = 84) from trauma patients (>18 yo) were retrieved retrospectively (Jan-2010 to July-2016) and prospectively (Aug-2016 to Sept-2021) from a Health System warehouse under IRB-approved protocols. Patients were divided according to their Injury Severity Score (ISS) into mild/moderate cases (ISS <15) and severe cases (ISS >15). Value was assessed using quality and cost domains. Quality surrogates included graded postoperative complications (PCs), length of stay (LOS), 30-day readmission (RA), patient satisfaction (PS), and textbook (TB) cases. Total charges (TCs) and reimbursement index (RI) were included as surrogates for cost. Value domains were displayed in scorecards comparing Observed (O) with Expected (E) (using the ACS risk calculator) outcomes. Uni-/multivariate analyses were performed using SPSS. RESULTS: 41,927 trauma evaluations were performed, leading to 16 044 admissions, with 528 (3.2%) patients requiring urgent surgical procedures (LAP = 413 and THO = 115). Although the M:F ratio (7:3) was similar in LAP vs THO groups, age and BMI were significantly different (41.8 ± 19.1 vs 51.8 ± 19.9 years, 28.6 ± 9.9 vs 27.4 ± 7 Kg/m2, respectively, P < .05). Blunt trauma was involved in 68.8/77.3% of the LAP/THO procedures, respectively (P < .05). Multivariate analyses showed ISS, age, ASA class, and medical center as factors significantly predicting PC (P < .05). Postoperative complication grades from the LAP/THO groups showed above-average outcomes; nonetheless, LOS was higher than the national averages. CONCLUSIONS: The Trauma Program holds high value in our Health Network System. Protocols for decreasing LOS are being implemented.

Preclinical specificity & activity of a fully human 41BB-expressing anti-CD19 CART- therapy for treatment-resistant autoimmune disease.

Over 4% of the global population is estimated to live with autoimmune disease, necessitating immunosuppressive treatment that is often chronic, not curative, and carries associated risks. B cells have emerged as key players in disease pathogenesis, as evidenced by partial responsiveness to B cell depletion by antibody-based therapies. However, these treatments often have transient effects due to incomplete depletion of tissue-resident B cells. Chimeric antigen receptor (CAR) T cells targeting B cells have demonstrated efficacy in refractory systemic lupus erythematosus. To this end, we developed an anti-CD19 CAR T cell product candidate, CABA-201, containing a clinically evaluated fully human CD19 binder (IC78) with a 4-1BB costimulatory domain and CD3 zeta stimulation domain for treatment refractory autoimmune disease. Here, we demonstrate specific cytotoxic activity of CABA-201 against CD19+ Nalm6 cells with no off-target effects on primary human cells. Novel examination of CABA-201 generated from primary T cells from multiple patients with autoimmune disease displayed robust CAR surface expression and effective elimination of the intended target autologous CD19+ B cells in vitro. Together, these findings support the tolerability and activity of CABA-201 for clinical development in patients with autoimmune disease.

Water quality and antibiotic resistance in the recreational waters.

The overuse and improper disposal of antibiotics results in antibiotic resistance. This raises concern over the presence of antibiotic resistant bacteria (ARB) in waterways and pose health risks of antibiotic resistant infections to water recreationists. The purpose of this study was to monitor water quality, microbial ecology, and antibiotic resistance in water and biofilm on submerged plastics at two public boat launches in southeastern Louisiana. Water and biofilm samples were collected once a month, in triplicate, from two public boat launches in Louisiana, USA for a year. Water quality metrics included nitrate, ammonia, sulfate, phosphate, and organic carbon. Water samples were tested for total and fecal coliform abundance and the presence of ARB. Out of 131 bacterial isolates studied from these two sites, 86% of them tested positive for antibiotic resistance with multi-drug resistance. Antibiotic resistance genes (ARGs) for sulfonamide (sul2), bacitracin (bacA) and ampicillin (ampA) were identified in bacterial isolates from water and biofilm samples at both sites. Molecular genetic diversity analysis identified distinct taxonomic diversity differences in biofilm bacteria compared to the planktonic bacteria in the surrounding water. Biofilm samples showed increased diversity at the phylum, genus, and species levels.

Growth responses of Avicennia germinans and Batis maritima seedlings to weathered light sweet crude oil applied to soil and aboveground tissues.

Oil spills are a significant stressor to coastal and maritime environments worldwide. The growth responses of Batis maritima and Avicennia germinans seedlings to weathered Deepwater Horizon oiling were assessed through a mesocosm study using a factorial arrangement of 4 soil oiling levels (0 L m-2, 1 L m-2, 2 L m-2, 4 L -m-2) × 3 tissue oiling levels (0% of stem height, 50% of stem height, 100% of stem height). Overall, growth metrics of B. maritima displayed much greater sensitivity to both tissue and soil oiling than A. germinans, which exhibited a relatively high tolerance to both routes of oiling exposure. Batis maritima in the 4 L m-2 soil oiling treatment demonstrated significant reductions in cumulative stem height and leaf number, whereas no significant effects of soil oiling on A. germinans were detected. This was reflected in the end of the study biomass partitioning, where total aboveground and live aboveground biomass were significantly reduced for B. maritima with 4 L m-2 soil oiling, but no impacts to A. germinans were found. Tissue oiling of 100% did appear to reduce B. maritima stem diameter, but no effect of tissue oiling was discerned on biomass partitioning, suggesting that there were no impacts to integrated growth. These findings suggest that B. maritima would be more severely affected by moderate soil oiling than A. germinans.

Meta-analysis of salt marsh vegetation impacts and recovery: a synthesis following the Deepwater Horizon oil spill.

Marine oil spills continue to be a global issue, heightened by spill events such as the 2010 Deepwater Horizon spill in the Gulf of Mexico, the largest marine oil spill in US waters and among the largest worldwide, affecting over 1,000 km of sensitive wetland shorelines, primarily salt marshes supporting numerous ecosystem functions. To synthesize the effects of the oil spill on foundational vegetation species in the salt marsh ecosystem, Spartina alterniflora and Juncus roemerianus, we performed a meta-analysis using data from 10 studies and 255 sampling sites over seven years post-spill. We examined the hypotheses that the oil spill reduced plant cover, stem density, vegetation height, aboveground biomass, and belowground biomass, and tracked the degree of effects temporally to estimate recovery time frames. All plant metrics indicated impacts from oiling, with 20-100% maximum reductions depending on oiling level and marsh zone. Peak reductions of ~70-90% in total plant cover, total aboveground biomass, and belowground biomass were observed for heavily oiled sites at the marsh edge. Both Spartina and Juncus were impacted, with Juncus affected to a greater degree. Most plant metrics had recovery time frames of three years or longer, including multiple metrics with incomplete recovery over the duration of our data, at least seven years post-spill. Belowground biomass was particularly concerning, because it declined over time in contrast with recovery trends in most aboveground metrics, serving as a strong indicator of ongoing impact, limited recovery, and impaired resilience. We conclude that the Deepwater Horizon spill had multiyear impacts on salt marsh vegetation, with full recovery likely to exceed 10 years, particularly in heavily oiled marshes, where erosion may preclude full recovery. Vegetation impacts and delayed recovery is likely to have exerted substantial influences on ecosystem processes and associated species, especially along heavily oiled shorelines. Our synthesis affords a greater understanding of ecosystem impacts and recovery following the Deepwater Horizon oil spill, and informs environmental impact analysis, contingency planning, emergency response, damage assessment, and restoration efforts related to oil spills.

Utilization of West Virginia Pediatric Medicaid Claims Data to Guide Outpatient Antimicrobial Stewardship Interventions.

West Virginia consistently ranks in the top two nationally for per capita antibiotic prescription rates in both pediatric and adult outpatient populations. We present the first descriptive analysis evaluating outpatient prescription rates within the WV pediatric Medicaid population, which demonstrated significant antibiotic prescribing variability by provider type, specialty, and geography.

Is titanium alloy Ti-6Al-4 V cytotoxic to gingival fibroblasts-A systematic review.

OBJECTIVES: Grade V titanium alloy (Ti-6Al-4 V) is a well-recognized metallic biomaterial for medical implants. There has been some controversy regarding the use of this alloy in medical devices in relation to the toxicity of vanadium. In Dentistry, Ti-6Al-4 V remains prevalent. This systematic review aims to evaluate the effects of Ti-6Al-4 V on cells relevant to oral environments such as gingival fibroblasts. MATERIALS AND METHODS: A literature search was undertaken for relevant English language publications in the following databases: Dental and Oral Science, Medline and Web of Science. The electronic search was supplemented with a search of references. RESULTS: After application of inclusion and exclusion criteria. A total of eight papers are included in this review. These papers were all in vitro studies and were categorized into whole implant, discs, or implant particles based on the type of test materials used in the studies. CONCLUSION: Based on the analyses of the eight included studies in this review, if Ti-6Al-4 V as a material is unchallenged, i.e., as a whole implant in pH neutral environments, there appears to be little effect on fibroblasts. If Ti-6Al-4 V is challenged through corrosion or wear (particle release), the subsequent release of vanadium and aluminium particles has an increased cytotoxic effect in vitro in comparison to commercially pure titanium, hence concerns should be raised in the clinical setting.

Embryogenic cell suspensions for high-capacity genetic transformation and regeneration of switchgrass (Panicum virgatum L.).

BACKGROUND: Switchgrass (Panicum virgatum L.), a North American prairie grassland species, is a potential lignocellulosic biofuel feedstock owing to its wide adaptability and biomass production. Production and genetic manipulation of switchgrass should be useful to improve its biomass composition and production for bioenergy applications. The goal of this project was to develop a high-throughput stable switchgrass transformation method using Agrobacterium tumefaciens with subsequent plant regeneration. RESULTS: Regenerable embryogenic cell suspension cultures were established from friable type II callus-derived inflorescences using two genotypes selected from the synthetic switchgrass variety 'Performer' tissue culture lines 32 and 605. The cell suspension cultures were composed of a heterogeneous fine mixture culture of single cells and aggregates. Agrobacterium tumefaciens strain GV3101 was optimum to transfer into cells the pANIC-10A vector with a hygromycin-selectable marker gene and a pporRFP orange fluorescent protein marker gene at an 85% transformation efficiency. Liquid cultures gave rise to embryogenic callus and then shoots, of which up to 94% formed roots. The resulting transgenic plants were phenotypically indistinguishable from the non-transgenic parent lines. CONCLUSION: The new cell suspension-based protocol enables high-throughput Agrobacterium-mediated transformation and regeneration of switchgrass in which plants are recovered within 6-7 months from culture establishment.

The TcEG1 beetle (Tribolium castaneum) cellulase produced in transgenic switchgrass is active at alkaline pH and auto-hydrolyzes biomass for increased cellobiose release.

BACKGROUND: Genetically engineered biofuel crops, such as switchgrass (Panicum virgatum L.), that produce their own cell wall-digesting cellulase enzymes would reduce costs of cellulosic biofuel production. To date, non-bioenergy plant models have been used in nearly all studies assessing the synthesis and activity of plant-produced fungal and bacterial cellulases. One potential source for cellulolytic enzyme genes is herbivorous insects adapted to digest plant cell walls. Here we examine the potential of transgenic switchgrass-produced TcEG1 cellulase from Tribolium castaneum (red flour beetle). This enzyme, when overproduced in Escherichia coli and Saccharomyces cerevisiae, efficiently digests cellulose at optima of 50 °C and pH 12.0. RESULTS: TcEG1 that was produced in green transgenic switchgrass tissue had a range of endoglucanase activity of 0.16-0.05 units (µM glucose release/min/mg) at 50 °C and pH 12.0. TcEG1 activity from air-dried leaves was unchanged from that from green tissue, but when tissue was dried in a desiccant oven (46 °C), specific enzyme activity decreased by 60%. When transgenic biomass was "dropped-in" into an alkaline buffer (pH 12.0) and allowed to incubate at 50 °C, cellobiose release was increased up to 77% over non-transgenic biomass. Saccharification was increased in one transgenic event by 28%, which had a concurrent decrease in lignin content of 9%. Histological analysis revealed an increase in cell wall thickness with no change to cell area or perimeter. Transgenic plants produced more, albeit narrower, tillers with equivalent dry biomass as the control. CONCLUSIONS: This work describes the first study in which an insect cellulase has been produced in transgenic plants; in this case, the dedicated bioenergy crop switchgrass. Switchgrass overexpressing the TcEG1 gene appeared to be morphologically similar to its non-transgenic control and produced equivalent dry biomass. Therefore, we propose TcEG1 transgenics could be bred with other transgenic germplasm (e.g., low-lignin lines) to yield new switchgrass with synergistically reduced recalcitrance to biofuel production. In addition, transgenes for other cell wall degrading enzymes may be stacked with TcEG1 in switchgrass to yield complementary cell wall digestion features and complete auto-hydrolysis.

Assessing coastal wetland vulnerability to sea-level rise along the northern Gulf of Mexico coast: Gaps and opportunities for developing a coordinated regional sampling network.

Coastal wetland responses to sea-level rise are greatly influenced by biogeomorphic processes that affect wetland surface elevation. Small changes in elevation relative to sea level can lead to comparatively large changes in ecosystem structure, function, and stability. The surface elevation table-marker horizon (SET-MH) approach is being used globally to quantify the relative contributions of processes affecting wetland elevation change. Historically, SET-MH measurements have been obtained at local scales to address site-specific research questions. However, in the face of accelerated sea-level rise, there is an increasing need for elevation change network data that can be incorporated into regional ecological models and vulnerability assessments. In particular, there is a need for long-term, high-temporal resolution data that are strategically distributed across ecologically-relevant abiotic gradients. Here, we quantify the distribution of SET-MH stations along the northern Gulf of Mexico coast (USA) across political boundaries (states), wetland habitats, and ecologically-relevant abiotic gradients (i.e., gradients in temperature, precipitation, elevation, and relative sea-level rise). Our analyses identify areas with high SET-MH station densities as well as areas with notable gaps. Salt marshes, intermediate elevations, and colder areas with high rainfall have a high number of stations, while salt flat ecosystems, certain elevation zones, the mangrove-marsh ecotone, and hypersaline coastal areas with low rainfall have fewer stations. Due to rapid rates of wetland loss and relative sea-level rise, the state of Louisiana has the most extensive SET-MH station network in the region, and we provide several recent examples where data from Louisiana's network have been used to assess and compare wetland vulnerability to sea-level rise. Our findings represent the first attempt to examine spatial gaps in SET-MH coverage across abiotic gradients. Our analyses can be used to transform a broadly disseminated and unplanned collection of SET-MH stations into a coordinated and strategic regional network. This regional network would provide data for predicting and preparing for the responses of coastal wetlands to accelerated sea-level rise and other aspects of global change.

Downregulation of a UDP-Arabinomutase Gene in Switchgrass (Panicum virgatum L.) Results in Increased Cell Wall Lignin While Reducing Arabinose-Glycans.

Background: Switchgrass (Panicum virgatum L.) is a C4 perennial prairie grass and a dedicated feedstock for lignocellulosic biofuels. Saccharification and biofuel yields are inhibited by the plant cell wall's natural recalcitrance against enzymatic degradation. Plant hemicellulose polysaccharides such as arabinoxylans structurally support and cross-link other cell wall polymers. Grasses predominately have Type II cell walls that are abundant in arabinoxylan, which comprise nearly 25% of aboveground biomass. A primary component of arabinoxylan synthesis is uridine diphosphate (UDP) linked to arabinofuranose (Araf). A family of UDP-arabinopyranose mutase (UAM)/reversible glycosylated polypeptides catalyze the interconversion between UDP-arabinopyranose (UDP-Arap) and UDP-Araf. Results: The expression of a switchgrass arabinoxylan biosynthesis pathway gene, PvUAM1, was decreased via RNAi to investigate its role in cell wall recalcitrance in the feedstock. PvUAM1 encodes a switchgrass homolog of UDP-arabinose mutase, which converts UDP-Arap to UDP-Araf. Southern blot analysis revealed each transgenic line contained between one to at least seven T-DNA insertions, resulting in some cases, a 95% reduction of native PvUAM1 transcript in stem internodes. Transgenic plants had increased pigmentation in vascular tissues at nodes, but were otherwise similar in morphology to the non-transgenic control. Cell wall-associated arabinose was decreased in leaves and stems by over 50%, but there was an increase in cellulose. In addition, there was a commensurate change in arabinose side chain extension. Cell wall lignin composition was altered with a concurrent increase in lignin content and transcript abundance of lignin biosynthetic genes in mature tillers. Enzymatic saccharification efficiency was unchanged in the transgenic plants relative to the control. Conclusion: Plants with attenuated PvUAM1 transcript had increased cellulose and lignin in cell walls. A decrease in cell wall-associated arabinose was expected, which was likely caused by fewer Araf residues in the arabinoxylan. The decrease in arabinoxylan may cause a compensation response to maintain cell wall integrity by increasing cellulose and lignin biosynthesis. In cases in which increased lignin is desired, e.g., feedstocks for carbon fiber production, downregulated UAM1 coupled with altered expression of other arabinoxylan biosynthesis genes might result in even higher production of lignin in biomass.

Thresholds in marsh resilience to the Deepwater Horizon oil spill.

Ecosystem boundary retreat due to human-induced pressure is a generally observed phenomenon. However, studies that document thresholds beyond which internal resistance mechanisms are overwhelmed are uncommon. Following the Deepwater Horizon (DWH) oil spill, field studies from a few sites suggested that oiling of salt marshes could lead to a biogeomorphic feedback where plant death resulted in increased marsh erosion. We tested for spatial generality of and thresholds in this effect across 103 salt marsh sites spanning ~430 kilometers of shoreline in coastal Louisiana, Alabama, and Mississippi, using data collected as part of the natural resource damage assessment (NRDA). Our analyses revealed a threshold for oil impacts on marsh edge erosion, with higher erosion rates occurring for ~1-2 years after the spill at sites with the highest amounts of plant stem oiling (90-100%). These results provide compelling evidence showing large-scale ecosystem loss following the Deepwater Horizon oil spill. More broadly, these findings provide rare empirical evidence identifying a geomorphologic threshold in the resistance of an ecosystem to increasing intensity of human-induced disturbance.

Transgenic Plant-Produced Hydrolytic Enzymes and the Potential of Insect Gut-Derived Hydrolases for Biofuels.

Various perennial C4 grass species have tremendous potential for use as lignocellulosic biofuel feedstocks. Currently available grasses require costly pre-treatment and exogenous hydrolytic enzyme application to break down complex cell wall polymers into sugars that can then be fermented into ethanol. It has long been hypothesized that engineered feedstock production of cell wall degrading (CWD) enzymes would be an efficient production platform for of exogenous hydrolytic enzymes. Most research has focused on plant overexpression of CWD enzyme-coding genes from free-living bacteria and fungi that naturally break down plant cell walls. Recently, it has been found that insect digestive tracts harbor novel sources of lignocellulolytic biocatalysts that might be exploited for biofuel production. These CWD enzyme genes can be located in the insect genomes or in symbiotic microbes. When CWD genes are transformed into plants, negative pleiotropic effects are possible such as unintended cell wall digestion. The use of codon optimization along with organelle and tissue specific targeting improves CWD enzyme yields. The literature teaches several important lessons on strategic deployment of CWD genes in transgenic plants, which is the focus of this review.

Impacts of the Deepwater Horizon oil spill on the salt marsh vegetation of Louisiana.

The coastal wetland vegetation component of the Deepwater Horizon oil spill Natural Resource Damage Assessment documented significant injury to the plant production and health of Louisiana salt marshes exposed to oiling. Specifically, marsh sites experiencing trace or greater vertical oiling of plant tissues displayed reductions in cover and peak standing crop relative to reference (no oiling), particularly in the marsh edge zone, for the majority of this four year study. Similarly, elevated chlorosis of plant tissue, as estimated by a vegetation health index, was detected for marsh sites with trace or greater vertical oiling in the first two years of the study. Key environmental factors, such as hydrologic regime, elevation, and soil characteristics, were generally similar across plant oiling classes (including reference), indicating that the observed injury to plant production and health was the result of plant oiling and not potential differences in environmental setting. Although fewer significant impacts to plant production and health were detected in the latter years of the study, this is due in part to decreased sample size occurring as a result of erosion (shoreline retreat) and resultant loss of plots, and should not be misconstrued as indicating full recovery of the ecosystem.

Field assessment of the impacts of Deepwater Horizon oiling on coastal marsh vegetation of Mississippi and Alabama.

The Deepwater Horizon incident, which occurred in April 2010, resulted in significant oiling of coastal habitats throughout the northern Gulf of Mexico. Although the most substantial oiling of coastal salt marshes occurred in Louisiana, oiling of salt marshes in Mississippi and Alabama was documented as well. A field study conducted in Mississippi and Alabama salt marshes as a component of the Deepwater Horizon Natural Resource Damage Assessment determined that >10% vertical oiling of plant tissues reduced live vegetation cover and aboveground biomass (live standing crop) relative to reference sites in this region through fall 2012. This reduction of live vegetation cover and aboveground biomass appears to have largely resulted from diminished health and vigor of Juncus roemerianus, a key salt marsh species in Mississippi and Alabama. Fewer significant reductions in live vegetation cover and aboveground biomass were detected by the fall 2013 sampling, suggesting that vegetation in oiled salt marshes in this region may have begun to recover. This is corroborated by low levels of Deepwater Horizon oil contamination in these salt marsh soils. However, these findings should be interpreted in the context of the restricted sampling intensity of the present study. Environ Toxicol Chem 2016;35:2791-2797. © 2016 The Authors. Environmental Toxicology and Chemistry Published by Wiley Periodicals, Inc. on behalf of SETAC.

Ccr4-Not and TFIIS Function Cooperatively To Rescue Arrested RNA Polymerase II.

Expression of the genome requires RNA polymerase II (RNAPII) to transcribe across many natural and unnatural barriers, and this transcription across barriers is facilitated by protein complexes called elongation factors (EFs). Genetic studies in Saccharomyces cerevisiae yeast suggest that multiple EFs collaborate to assist RNAPII in completing the transcription of genes, but the molecular mechanisms of how they cooperate to promote elongation are not well understood. The Ccr4-Not complex participates in multiple steps of mRNA metabolism and has recently been shown to be an EF. Here we describe how Ccr4-Not and TFIIS cooperate to stimulate elongation. We find that Ccr4-Not and TFIIS mutations show synthetically enhanced phenotypes, and biochemical analyses indicate that Ccr4-Not and TFIIS work synergistically to reactivate arrested RNAPII. Ccr4-Not increases the recruitment of TFIIS into elongation complexes and enhances the cleavage of the displaced transcript in backtracked RNAPII. This is mediated by an interaction between Ccr4-Not and the N terminus of TFIIS. In addition to revealing insights into how these two elongation factors cooperate to promote RNAPII elongation, our study extends the growing body of evidence suggesting that the N terminus of TFIIS acts as a docking/interacting site that allows it to synergize with other EFs to promote RNAPII transcription.

Mercury concentrations in oligohaline wetland vegetation and associated soil biogeochemistry.

Concentrations of mercury were determined in above- and below-ground tissues of dominant plant species, as well as soils, in the wetlands of Lake Maurepas, Louisiana. Indicators of wetland soil biogeochemical status, such as soil redox potential, pore-water nutrient concentrations, and pore-water total sulfides, were also determined. Total mercury concentrations in plant tissues were within the typical range for vegetation not exposed to mercury contamination. Similarly, total mercury concentrations in soils were typical of uncontaminated wetlands within this geographic region. Soil methyl mercury levels in this study are slightly lower than those reported in other studies of nearby wetlands. This may reflect the less extensive geographic sampling in this study, or the low water levels in the Lake Maurepas system immediately prior to and during this study, which would have altered soil biogeochemical status. This is corroborated by measurements of soil redox potential and soil pore-water nitrogen and sulfur constituents conducted during this study that suggest minimal sulfate reduction was occurring in surficial soils. This study indicates that the wetlands surrounding Lake Maurepas are typical of many uncontaminated oligohaline wetlands in the southeastern U.S. in regard to mercury concentrations.

Identification, cloning, and expression of a GHF9 cellulase from Tribolium castaneum (Coleoptera: Tenebrionidae).

The availability of sequenced insect genomes has allowed for discovery and functional characterization of novel genes and proteins. We report use of the Tribolium castaneum (Herbst) (red flour beetle) genome to identify, clone, express, and characterize a novel endo-ß-1,4-glucanase we named TcEG1 (T. castaneum endoglucanase 1). Sequence analysis of a full-length TcEG1 cDNA clone (1356bp) revealed sequence homology to enzymes in glycosyl hydrolase family 9 (GHF9), and verified presence of a change (Gly for Ser) in the conserved catalytic domain for GHF9 cellulases. This TcEG1 cDNA clone was predicted to encode a 49.5kDa protein with a calculated pI of 5.39. Heterologous expression of TcEG1 in Drosophila S2 cell cultures resulted in secretion of a 51-kDa protein, as determined by Western blotting. The expressed protein was used to characterize TcEG1 enzymatic activity against two cellulose substrates to determine its specificity and stability. Our data support that TcEG1 as a novel endo-ß-1,4-glucanase, the first functional characterization of a cellulase enzyme derived from an insect genome with potential applications in the biofuel industry due to its high relative activity at alkaline pH.

Growth response and tissue accumulation trends of herbaceous wetland plant species exposed to elevated aqueous mercury levels.

The impacts of elevated aqueous mercury levels (0, 2, and 4 ppm) on the growth status and mercury tissue concentrations of Eleocharis parvula, Saururus cernuus, Juncus effuses, Typha latifolia, and Panicum hemitomon were determined. Both short-term (net CO2 assimilation) and long-term (biomass) indicators of plant growth status suggest that Eleocharis parvula, Saururus cernuus, and Juncus effuses were relatively unimpacted by elevated mercury levels, whereas Typha latifolia and Panicum hemitomon were somewhat impacted at elevated mercury levels. Eleocharis parvula, Panicum hemitomon, and Typha latifolia generally had the greatest overall belowground tissue concentrations of mercury (2 ppm treatment: 7.21, 7.32, and 9.64 ppm respectively; 4 ppm treatment: 16.23, 18.23, and 13.98 ppm, respectively) and aboveground tissue concentrations of mercury (2 ppm treatment: 0.01, 0.04, 0.02; 4 ppm treatment: 0.26; 0.11; 0.17 ppm, respectively). However, the species investigated in this study demonstrated lower levels of mercury accumulation into tissues when compared with similar investigations of other aquatic plants, suggesting that the above species are not optimal for phytoremediation efforts.

Characterization of cellulolytic activity from digestive fluids of Dissosteira carolina (Orthoptera: Acrididae).

Previous screening of head-derived and gut fluid extracts of Carolina grasshoppers, Dissosteira carolina (L.) revealed relatively high activity against cellulase substrates when compared to other insect groups. In this work we report on the characterization and identification of enzymes involved in cellulolytic activity in digestive fluids of D. carolina. In zymograms using carboxymethylcellulose (CMC) as substrate, we detected four distinct cellulolytic protein bands in D. carolina gut fluids, common to all developmental stages. These cellulolytic enzymes were localized to foregut and midgut regions of the D. carolina digestive tract. Cellulases were purified from D. carolina head and gut fluid extracts by liquid chromatography to obtain N-terminal amino acid sequence tags. Database searches with sequence tags from head fluids indicated high similarity with invertebrate, bacterial and plant beta1,4-endoglucanases, while no homologues were identified for the gut-derived protein. Our data demonstrate the presence of cellulolytic activity in the digestive system of D. carolina and suggest that cellulases of endogenous origin are present in this organism. Considering that this grasshopper species is a pest of grasses, including switchgrass that has been suggested bioethanol feedstock, characterization of insect cellulolytic systems may aid in developing applications for plant biomass biodegradation for biofuel production.