Implementation of a multidisciplinary nutritional support team and clinical outcomes in critically ill patients with COVID-19.

BACKGROUND & AIMS: The South Korean government established the multidisciplinary nutritional support teams (NST) system for enhancing the evaluation and adequate supply of nutritional support to patients at high risk of malnutrition. However, the impact of the NST on clinical outcomes in critically ill patients with coronavirus disease 2019 (COVID-19) remains unclear. We aimed to investigate whether NST implementation affects survival outcomes in patients with COVID-19 requiring intensive care unit (ICU) admission. METHODS: Using data from the National Health Insurance Service and Korea Disease Control and Prevention Agency in South Korea, adult patients with COVID-19 admitted to the ICU between October 8, 2020, and December 31, 2021, were included. The NST comprised four professional personnel (physicians, full-time nurses, full-time pharmacists, and full-time clinical dietitians). Patients admitted to ICUs with and without the NST system were assigned to the NST and non-NST groups, respectively. RESULTS: A total of 13,103 critically ill adult patients were included in the final analysis; among them, 10,103 (77.1 %) and 3,000 (22.9 %) patients were included in the NST and non-NST groups, respectively. In the NST group, 2,803 (27.7 %) critically ill patients with COVID-19 were prescribed enteral or parenteral nutrition by the NST. In a covariate-adjusted multivariable model, the NST group showed a 40 % lower in-hospital mortality rate than the non-NST group (odds ratio: 0.60, 95 % confidence interval: 0.51, 0.71; P < 0.001). In subgroup analyses, compared with the non-NST group, the NST group showed significantly lower in-hospital mortality rates at 2, 3, 4, and 5 points on the World Health Organization clinical progression scale among patients with acute respiratory distress and mechanical ventilatory support. CONCLUSIONS: NST implementation was associated with improved survival outcomes in critically ill patients with COVID-19; accordingly, it may be recommended for improving adequate nutritional support and evaluation in critically ill patients.

Environmental factors affecting akinete germination and resting cell awakening of two cyanobacteria.

Globally, cyanobacteria frequently cause blooms that outcompete other species in the waterbody, affecting the diversity, decreasing water exchange rates, and promoting eutrophication that leads to excessive algal growth. Here, Dolichospermum circinale (akinetes) and Microcystic aeruginosa (resting cells), were isolated from the sediment in the Uiam Dam in the North Han River and near Ugok Bridge in the Nakdong River, respectively. The morphology, germination process and rates, and growth was evaluated in different environmental conditions. D. cercinalis germination began on day two of culturing, with maximum cell growth observed on day ten. In contrast, M. aeruginosa exhibited daily increase in cell density and colony size, with notable density increase on day six. Next, different environmental conditions were assessed. Akinetes exhibited high germination rates at low light intensity (5-30 µmol/m2/s), whereas resting cells exhibited high growth rates at high light intensity (50-100 µmol/m2/s). Furthermore, both cell types exhibited optimum germination and growth in media containing N and P at 20-30° at a pH of 7-9. Our study reveals the optimum conditions for the germination and growth of cyanobacterial akinetes and resting cells isolated from river sediment, respectively, and will assist in predicting cyanobacterial blooms for appropriate management.

The Relation Between eHealth Literacy and Health-Related Behaviors: Systematic Review and Meta-analysis.

BACKGROUND: With widespread use of the internet and mobile devices, many people have gained improved access to health-related information online for health promotion and disease management. As the health information acquired online can affect health-related behaviors, health care providers need to take into account how each individual's online health literacy (eHealth literacy) can affect health-related behaviors. OBJECTIVE: To determine whether an individual's level of eHealth literacy affects actual health-related behaviors, the correlation between eHealth literacy and health-related behaviors was identified in an integrated manner through a systematic literature review and meta-analysis. METHODS: The MEDLINE, Embase, Cochrane, KoreaMed, and Research Information Sharing Service databases were systematically searched for studies published up to March 19, 2021, which suggested the relationship between eHealth literacy and health-related behaviors. Studies were eligible if they were conducted with the general population, presented eHealth literacy according to validated tools, used no specific control condition, and measured health-related behaviors as the outcomes. A meta-analysis was performed on the studies that could be quantitatively synthesized using a random effect model. A pooled correlation coefficient was generated by integrating the correlation coefficients, and the risk of bias was assessed using the modified Newcastle-Ottawa Scale. RESULTS: Among 1922 eHealth literacy-related papers, 29 studies suggesting an association between eHealth literacy and health-related behaviors were included. All retrieved studies were cross-sectional studies, and most of them used the eHealth Literacy Scale (eHEALS) as a measurement tool for eHealth literacy. Of the 29 studies, 22 presented positive associations between eHealth literacy and health-related behaviors. The meta-analysis was performed on 14 studies that presented the correlation coefficient for the relationship between eHealth literacy and health-related behaviors. When the meta-analysis was conducted by age, morbidity status, and type of health-related behavior, the pooled correlation coefficients were 0.37 (95% CI 0.29-0.44) for older adults (aged ≥65 years), 0.28 (95% CI 0.17-0.39) for individuals with diseases, and 0.36 (95% CI 0.27-0.41) for health-promoting behavior. The overall estimate of the correlation between eHealth literacy and health-related behaviors was 0.31 (95% CI 0.25-0.34), which indicated a moderate correlation between eHealth literacy and health-related behaviors. CONCLUSIONS: Our results of a positive correlation between eHealth literacy and health-related behaviors indicate that eHealth literacy can be a mediator in the process by which health-related information leads to changes in health-related behaviors. Larger-scale studies with stronger validity are needed to evaluate the detailed relationship between the proficiency level of eHealth literacy and health-related behaviors for health promotion in the future.

Alterations of vital signs as prognostic factors after intraprocedural rupture of intracranial aneurysms during endovascular treatment.

OBJECTIVE: To report our experience with intraprocedural rupture (IPR) of intracranial aneurysms during endovascular treatment and evaluate alterations in vital signs as independent prognostic factors to predict the outcomes of IPR. METHODS: Between January 2008 and August 2021, 34 patients (8 ruptured and 26 unruptured) were confirmed to have IPR based on our dataset with 3178 endovascular coiling procedures. The patients who underwent additional surgeries related to IPR were classified as the OP group (n=9), while those who did not receive additional surgeries were classified as the non-OP group (n=25). Vital signs were recorded during the procedure by anesthesiologists and analyzed. RESULTS: Of the 34 patients included in this study, eight initially presented with subarachnoid hemorrhage due to a ruptured aneurysm. The clinical outcomes at discharge were significantly different between the two groups (p=0.046). In the OP group, five patients showed favorable outcomes at discharge, while four showed unfavorable outcomes. In the non-OP group, 23 patients showed favorable outcomes at discharge while two patients showed unfavorable outcomes. Maximal (MAX) systolic blood pressure (SBP) (odds ratio [OR] 1.520, 95% confidence interval [CI] 1.084-2.110; p=0.037) and higher differential value MAX-median blood pressure (MBP) (OR 1.322, 95% CI 1.029-1.607; p=0.044) remained independent risk factors for poor prognosis after IPR on multivariate logistic regression analysis. CONCLUSIONS: The MAX SBP and the difference between the maximal and baseline values of MBP are key factors in predicting the prognosis of patients after IPR, as well as providing useful information for predicting the outcome. Further research is required to confirm the relationship between naive pressure and prognosis.

Influence of Zooplankton and Environmental Factors on Clear-Water Phase in Lake Paldang, South Korea.

Lake Paldang is a complex water system with both fluvial and lacustrine characteristics and the largest artificial dam lake in South Korea. In this study, the different occurrence patterns and causes of the clear-water phase (CWP) were investigated using water quality and hydrological factors at four sites in Lake Paldang. Among the environmental and other factors associated with CWP occurrence, secchi depth and turbidity exhibited significant correlations with precipitation, hydraulic retention time (HRT), and individual and relative abundance (RA) of zooplankton (Cladocera) (p < 0.01). Hence, a change in the HRT because of precipitation could alter the CWP. The Cladocera individuals and RA showed significant correlations with the water temperature, indicating that the emergence of Cladocera in spring as the water temperature rises could cause the CWP. Sensitivity assessments conducted using Bayesian models demonstrated different CWP occurrence sensitivity relationships for the river-type, lake-type, and shallow and deep sites. Turbidity, secchi depth, and zooplankton factors also showed sensitive relationships with CWP occurrence for all sites. The sensitivity to precipitation and HRT was higher in the river-type sites. The lake-type sites, with common Cladocera emergence and long HRT, favored CWP occurrence. Thus, CWP occurrence was dependent on the site characteristics and climate conditions.

Production and Characterization of High Value Prebiotics From Biorefinery-Relevant Feedstocks.

Hemicellulose, a structural polysaccharide and often underutilized co-product stream of biorefineries, could be used to produce prebiotic ingredients with novel functionalities. Since hot water pre-extraction is a cost-effective strategy for integrated biorefineries to partially fractionate hemicellulose and improve feedstock quality and performance for downstream operations, the approach was applied to process switchgrass (SG), hybrid poplar (HP), and southern pine (SP) biomass at 160°C for 60 min. As a result, different hemicellulose-rich fractions were generated and the chemical characterization studies showed that they were composed of 76-91% of glucan, xylan, galactan, arabinan, and mannan oligosaccharides. The hot water extracts also contained minor concentrations of monomeric sugars (≤18%), phenolic components (≤1%), and other degradation products (≤3%), but were tested for probiotic activity without any purification. When subjected to batch fermentations by individual cultures of Lactobacillus casei, Bifidobacterium bifidum, and Bacteroides fragilis, the hemicellulosic hydrolysates elicited varied responses. SG hydrolysates induced the highest cell count in L. casei at 8.6 log10 cells/ml, whereas the highest cell counts for B. fragilis and B. bifidum were obtained with southern pine (5.8 log10 cells/ml) and HP hydrolysates (6.4 log10 cells/ml), respectively. The observed differences were attributed to the preferential consumption of mannooligosaccharides in SP hydrolysates by B. fragilis. Lactobacillus casei preferentially consumed xylooligosaccharides in the switchgrass and southern pine hydrolysates, whereas B. bifidum consumed galactose in the hybrid poplar hydrolysates. Thus, this study (1) reveals the potential to produce prebiotic ingredients from biorefinery-relevant lignocellulosic biomass, and (2) demonstrates how the chemical composition of hemicellulose-derived sources could regulate the viability and selective proliferation of probiotic microorganisms.

Establishing the validity of surface topography for assessment of scoliosis: a prospective study.

STUDY DESIGN: Descriptive survey. OBJECTIVES: Compare radiographic parameters measured using surface topography (ST) with those obtained radiographically to determine the validity of ST for scoliotic assessment. METHODS: While anterior-posterior radiography is the gold standard for diagnosing scoliosis, repeated radiographic use is associated with increased carcinogenicity. Studies have thus focused on radiation-free systems, including ST, to calculate the scoliotic angle. Seventeen patients ages 25-76 were included. Each patient received one AP radiograph and three repeated ST measurements over two months. Values were analyzed by two raters to determine comparability between ST and radiographic measurements. Interobserver reliability (ICC) was calculated and statistical significance was determined by the p-value of a paired two-tailed t-test. RESULTS: ICC showed excellent reliability (> 0.90). There was no significant difference (p > 0.40) in apical vertebral deviation/translation between conventional radiography (0.9 ± 0.8) and ST (1.2 ± 1.0). There was no significant difference (p > 0.30) in sagittal balance radiographic (4.0 ± 3.1) and ST (4.4 ± 3.3), and coronal balance radiographic (1.4 ± 1.3) and ST (1.1 ± 1.1) measurements. Significant difference (p < .001) was found between lumbar lordosis radiographic (52.6 ± 18.4) and ST (37.9 ± 16.6), kyphotic angle radiographic (35.1 ± 16.0) and ST (50.0 ± 11.9), and scoliotic angle radiographic (11.3 ± 12.4) and ST (17.7 ± 10.2) measurements. CONCLUSIONS: No significant difference was observed between various ST and radiographic measurements, including apical vertebral deviation, sagittal balance, and coronal balance. While a larger prospective study is needed to further assess the validity of ST, these initial measurements suggest the possibility of an effective and radiation-free adjunctive method of assessing balance in the coronal plane.

Combined effects of temperature and macronutrient balance on life-history traits in Drosophila melanogaster: implications for life-history trade-offs and fundamental niche.

Temperature and nutrition are amongst the most influential environmental determinants of Darwinian fitness in ectotherms. Since the ongoing climate warming is known to alter nutritional environments encountered by ectotherms, a precise understanding of the integrated effects of these two factors on ectotherm performance is essential for improving the accuracy of predictions regarding how ectotherms will respond to climate warming. Here we employed response surface methodology to examine how multiple life-history traits were expressed across a grid of environmental conditions representing full combinations of six ambient temperatures (13, 18, 23, 28, 31, 33 °C) and eight dietary protein:carbohydrate ratios (P:C = 1:16, 1:8, 1:4, 1:2, 1:1, 2:1, 4:1, 8:1) in Drosophila melanogaster. Different life-history traits were maximized in different regions in the two-dimensional temperature-nutrient space. The optimal temperature and P:C ratio identified for adult lifespan (13 °C and 1:16) were lower than those for early-life female fecundity (28 °C and 4:1). Similar divergence in thermal and nutritional optima was found between body mass at adult emergence (18 °C and P:C 1:1) and the rate of pre-adult development (28 °C and P:C 4:1). Pre-adult survival was maximized over a broad range of temperature (18-28 °C) and P:C ratio (1:8-8:1). These results indicate that the occurrence of life-history trade-offs is regulated by both temperature and dietary P:C ratio. The estimated measure of fitness was maximized at 23 °C and P:C 2:1. Based on the shape of the response surface constructed for this estimated fitness, we characterized the fundamental thermal and nutritional niche for D. melanogaster with unprecedented detail.

Molecular Probes to Evaluate the Synthesis and Production Potential of an Odorous Compound (2-methylisoborneol) in Cyanobacteria.

The volatile metabolite, 2-Methylisoborneol (2-MIB) produced by cyanobacterial species, causes odor and taste problems in freshwater systems. However, simple identification of cyanobacteria that produce such off-flavors may be insufficient to establish the causal agent of off-flavor-related problems as the production-related genes are often strain-specific. Here, we designed a set of primers for detecting and quantifying 2-MIB-synthesizing cyanobacteria based on mibC gene sequences (encoding 2-MIB synthesis-catalyzing monoterpene cyclase) from various Oscillatoriales and Synechococcales cyanobacterial strains deposited in GenBank. Cyanobacterial cells and environmental DNA and RNA were collected from both the water column and sediment of a eutrophic stream (the Gong-ji Stream, Chuncheon, South Korea), which has a high 2-MIB concentration. Primer sets mibC196 and mibC300 showed universality to mibC in the Synechococcales and Oscillatoriales strains; the mibC132 primer showed high specificity for Pseudanabaena and Planktothricoides mibC. Our mibC primers showed excellent amplification efficiency (100-102%) and high correlation among related variables (2-MIB concentration with water RNA r = 689, p < 0.01; sediment DNA r = 0.794, p < 0.01; and water DNA r = 0.644, p < 0.05; cyanobacteria cell density with water RNA and DNA r = 0.995, p < 0.01). These primers offer an efficient tool for identifying cyanobacterial strains possessing mibC genes (and thus 2-MIB-producing potential) and for evaluating mibC gene expression as an early warning of massive cyanobacterial occurrence.

Effects of Microbial Activity and Environmental Parameters on the Degradation of Extracellular Environmental DNA from a Eutrophic Lake.

Extracellular DNA (exDNA) pool in aquatic environments is a valuable source for biomonitoring and bioassessment. However, degradation under particular environmental conditions can hamper exDNA detectability over time. In this study, we analyzed how different biotic and abiotic factors affect the degradation rate of extracellular environmental DNA using 16S rDNA sequences extracted from the sediment of a eutrophic lake and Anabaena variabilis cultured in the laboratory. We exposed the extracted exDNA to different levels of temperature, light, pH, and bacterial activity, and quantitatively analyzed the concentration of exDNA during 4 days. The solution containing bacteria for microbial activity treatment was obtained from the lake sediment using four consecutive steps of filtration; two mesh filters (100 µm and 60 µm mesh) and two glass fiber filters (2.7 µm and 1.2 µm pore-sized). We found that temperature individually and in combination with bacterial abundance had significant positive effects on the degradation of exDNA. The highest degradation rate was observed in samples exposed to high microbial activity, where exDNA was completely degraded within 1 day at a rate of 3.27 day-1. Light intensity and pH had no significant effects on degradation rate of exDNA. Our results indicate that degradation of exDNA in freshwater ecosystems is driven by the combination of both biotic and abiotic factors and it may occur very fast under particular conditions.

Spinal Epidural Lipomatosis: A Review of Pathogenesis, Characteristics, Clinical Presentation, and Management.

STUDY DESIGN: Narrative review of available literature. OBJECTIVE: To summarize current trends in pathogenesis and management of spinal epidural lipomatosis (SEL) and suggest areas where more research would be of benefit. METHODS: The available literature relevant to SEL was reviewed. PubMed, Medline, OVID, EMBASE, Cochrane, and Google Scholar were used to review the literature. Institutional review board approval is not applicable for this study. RESULTS: This article clearly summarizes current trends in the pathogenesis and management of SEL. CONCLUSIONS: Possible etiologies of SEL include exogenous steroid use, endogenous steroid hormonal disease, obesity, surgery induced, and idiopathic disease. Comorbidities such as acquired immunodeficiency syndrome and Scheuermann's disease have also been implicated in the pathogenesis of SEL. Steroid-induced SEL seems to have a proclivity for the thoracic region of the spine and has a higher incidence of paraplegia when compared with other forms. Several treatment modalities exist for SEL and are dictated by the underlying cause of the disorder. These include weight reduction, cessation of steroid medications, treatment of underlying endocrine abnormalities, and surgical decompression. Conservative treatments generally aim to decrease the thickness of adipose tissue in the epidural space, but the majority of patients tend to undergo surgical decompression to relieve neurologic symptoms. Surgical decompression provides a statistically significant reduction in symptoms, but postoperative mortality is high, influenced primarily by the patient's preoperative comorbidities. Physicians should consider the underlying cause of SEL in a given patient before pursuing specific treatment modalities, but alarm symptoms, such as the development of acute cauda equina syndrome, should likely be treated with urgent surgical decompression.

Structural changes in lignocellulosic biomass during activation with ionic liquids comprising 3-methylimidazolium cations and carboxylate anions.

BACKGROUND: Lignocellulosic biomass requires either pretreatment and/or fractionation to recover its individual components for further use as intermediate building blocks for producing fuels, chemicals, and products. Numerous ionic liquids (ILs) have been investigated for biomass pretreatment or fractionation due to their ability to activate lignocellulosic biomass, thereby reducing biomass recalcitrance with minimal impact on its structural components. In this work, we studied and compared 1-allyl-3-methylimidazolium formate ([AMIM][HCOO]) to the commonly used 1-ethyl-3-methylimidazolium acetate ([EMIM][CH3COO]) for its potential to activate hybrid poplar biomass and enable high cellulose and hemicellulose enzymatic conversion. Although [EMIM][CH3COO] has been widely used for activation, [AMIM][HCOO] was recently identified to achieve higher biomass solubility, with an increase of 40% over [EMIM][CH3COO]. RESULTS: Since IL activation is essentially an early stage of IL dissolution, we assessed the recalcitrance of [EMIM][CH3COO] and [AMIM][HCOO]-activated biomass through a suite of analytical tools. More specifically, Fourier transform infrared spectroscopy and X-ray diffraction showed that activation using [AMIM][HCOO] does not deacetylate hybrid poplar as readily as [EMIM][CH3COO] and preserves the crystallinity of the cellulose fraction, respectively. This was supported by scanning electron microscopy and enzymatic saccharification experiments in which [EMIM][CH3COO]-activated biomass yielded almost twice the cellulose and hemicellulose conversion as compared to [AMIM][HCOO]-activated biomass. CONCLUSION: We conclude that the IL [AMIM][HCOO] is better suited for biomass dissolution and direct product formation, whereas [EMIM][CH3COO] remains the better IL for biomass activation and fractionation.

Functional Analysis of Cellulose Synthase CesA4 and CesA6 Genes in Switchgrass (Panicum virgatum) by Overexpression and RNAi-Mediated Gene Silencing.

Switchgrass (Panicum virgatum L.) is a leading lignocellulosic bioenergy feedstock. Cellulose is a major component of the plant cell walls and the primary substrate for saccharification. Accessibility of cellulose to enzymatic breakdown into fermentable sugars is limited by the presence of lignin in the plant cell wall. In this study, putatively novel switchgrass secondary cell wall cellulose synthase PvCesA4 and primary cell wall PvCesA6 genes were identified and their functional role in cellulose synthesis and cell wall composition was examined by overexpression and knockdown of the individual genes in switchgrass. The endogenous expression of PvCesA4 and PvCesA6 genes varied among including roots, leaves, stem, and reproductive tissues. Increasing or decreasing PvCesA4 and PvCesA6 expression to extreme levels in the transgenic lines resulted in decreased biomass production. PvCesA6-overexpressing lines had reduced lignin content and syringyl/guaiacyl lignin monomer ratio accompanied by increased sugar release efficiency, suggesting an impact of PvCesA6 expression levels on lignin biosynthesis. Cellulose content and cellulose crystallinity were decreased, while xylan content was increased in PvCesA4 and PvCesA6 overexpression or knockdown lines. The increase in xylan content suggests that the amount of non-cellulosic cell wall polysaccharide was modified in these plants. Taken together, the results show that the manipulation of the cellulose synthase genes alters the cell wall composition and availability of cellulose as a bioprocessing substrate.

Harmful Cyanobacterial Material Production in the North Han River (South Korea): Genetic Potential and Temperature-Dependent Properties.

Cyanobacteria synthesize various harmful materials, including off-flavor substances and toxins, that are regarded as potential socio-economic and environmental hazards in freshwater systems, however, their production is still not well understood. In this study, we investigated the potential and properties of harmful materials produced by cyanobacteria, depending on temperature, and undertook a phylogenetic analysis of cyanobacteria present in the North Han River (South Korea). Production potentials were evaluated using gene-specific probes, and the harmful material production properties of strains showing positive potentials were further characterized at different temperatures in the range 15 to 30 °C. We identified six cyanobacterial strains based on 16S rDNA analysis: two morphological types (coiled and straight type) of Dolichospermum circinale, Aphanizomenon flos-aquae, Oscillatoria limosa, Planktothricoides raciborskii, Pseudanabaena mucicola, and Microcystis aeruginosa. We confirmed that cyanobacterial strains showing harmful material production potential produced the corresponding harmful material, and their production properties varied with temperature. Total harmful material production was maximal at 20~25 °C, a temperature range optimal for cell growth. However, harmful material productivity was highest at 15 °C. These results indicate that the expression of genes related to synthesis of harmful materials can vary depending on environmental conditions, resulting in variable harmful material production, even within the same cyanobacterial strains.

Chemical and anatomical changes in Liquidambar styraciflua L. xylem after long term exposure to elevated CO2.

The anatomical and chemical characteristics of sweetgum were studied after 11 years of elevated CO2 (544 ppm, ambient at 391 ppm) exposure. Anatomically, branch xylem cells were larger for elevated CO2 trees, and the cell wall thickness was thinner. Chemically, elevated CO2 exposure did not impact the structural components of the stem wood, but non-structural components were significantly affected. Principal component analysis (PCA) was employed to detect differences between the CO2 treatments by considering numerous structural and chemical variables, as well as tree size, and data from previously published sources (i.e., root biomass, production and turnover). The PCA results indicated a clear separation between trees exposed to ambient and elevated CO2 conditions. Correlation loadings plots of the PCA revealed that stem structural components, ash, Ca, Mg, total phenolics, root biomass, production and turnover were the major responses that contribute to the separation between the elevated and ambient CO2 treated trees.

Chimeric cytochromes P450 engineered by domain swapping and random mutagenesis for producing human metabolites of drugs.

Human drug metabolites produced by cytochrome P450 enzymes are critical for safety testing and may themselves act as drugs or leads in the drug discovery and development process. Here, highly active chimeric fusion proteins (chimeras) were obtained by reductase domain swapping of mutants at key catalytic residues of the heme domain with that of a natural variant (CYP102A1.2) of P450 BM3 (CYP102A1.1) from Bacillus megaterium. Random mutagenesis at the heme domain of the chimera was also used to generate chimeric mutants that were more active and diverse than the chimeras themselves. To determine whether the chimeras and several mutants of the highly active chimera displayed enhanced catalytic activity and, more importantly, whether they acquired activities of biotechnological importance, we measured the oxidation activities of the chimeras and chimeric mutants toward human P450 substrates, mainly drugs. Some of the chimeric mutants showed high activity toward typical human P450 substrates including drugs. Statin leads, especially chiral products, with inhibitory effects toward HMG-CoA reductase could be obtained from metabolites of statin drugs generated using these chimeric mutants. This study reveals the critical role of the reductase domain for the activity of P450 BM3 and shows that chimeras generated by domain swapping can be used to develop industrial enzymes for the synthesis of human metabolites from drugs and drug leads.

Sequence-specific interaction between ABD-B homeodomain and castor gene in Drosophila.

We have examined the effect of bithorax complex genes on the expression of castor gene. During the embryonic stages 12-15, both Ultrabithorax and abdominal-A regulated the castor gene expression negatively, whereas Abdominal-B showed a positive correlation with the castor gene expression according to real-time PCR. To investigate whether ABD-B protein directly interacts with the castor gene, electrophoretic mobility shift assays were performed using the recombinant ABD-B homeodomain and oligonucleotides, which are located within the region 10 kb upstream of the castor gene. The results show that ABD-B protein directly binds to the castor gene specifically. ABD-B binds more strongly to oligonucleotides containing two 5'-TTAT-3' canonical core motifs than the probe containing the 5'-TTAC-3' motif. In addition, the sequences flanking the core motif are also involved in the protein-DNA interaction. The results demonstrate the importance of HD for direct binding to target sequences to regulate the expression level of the target genes.

Directed evolution reveals requisite sequence elements in the functional expression of P450 2F1 in Escherichia coli.

Cytochrome P450 2F1 (P450 2F1) is expressed exclusively in the human respiratory tract and is implicated in 3-methylindole (3MI)-induced pneumotoxicity via dehydrogenation of 3MI to a reactive electrophilic intermediate, 3-methyleneindolenine (3-MEI). Studies of P450 2F1 to date have been limited by the failure to express this enzyme in Escherichia coli. By contrast, P450 2F3, a caprine homologue that shares 84% sequence identity with P450 2F1 (86 amino acid differences), has been expressed in E. coli at yields greater than 250 nmol/L culture. We hypothesized that a limited number of sequence differences between P450s 2F1 and 2F3 could limit P450 2F1 expression in E. coli and that problematic P450 2F1 sequence elements could be identified by directed evolution. A library of P450 2F1/2F3 mutants was created by DNA family shuffling and screened for expression in E. coli. Three generations of DNA shuffling revealed a mutant (named JH_2F_F3_1_007) with 96.5% nucleotide sequence identity to P450 2F1 and which expressed 119 ± 40 pmol (n = 3, mean ± SD) hemoprotein in 1 mL microaerobic cultures. Across all three generations, two regions were observed where P450 2F3-derived sequence was consistently substituted for P450 2F1 sequence in expressing mutants, encoding nine amino acid differences between P450s 2F1 and 2F3: nucleotides 191-278 (amino acids 65-92) and 794-924 (amino acids 265-305). Chimeras constructed to specifically test the importance of these two regions confirmed that P450 2F3 sequence is essential in both regions for expression in E. coli but that other non-P450 2F1 sequence elements outside of these regions also improved the expression of mutant JH_2F_F3_1_007. Mutant JH_2F_F3_1_007 catalyzed the dehydrogenation of 3MI to 3-MEI as indicated by the observation of glutathione adducts after incubation in the presence of glutathione. The JH_2F_F3_1_007 protein differs from P450 2F1 at only 20 amino acids and should facilitate further studies of the structure-activity relationships of P450s of the 2F subfamily.

Identification of diverse modulators of central and peripheral circadian clocks by high-throughput chemical screening.

The circadian clock coordinates daily oscillations of essential physiological and behavioral processes. Conversely, aberrant clocks with damped amplitude and/or abnormal period have been associated with chronic diseases and aging. To search for small molecules that perturb or enhance circadian rhythms, we conducted a high-throughput screen of approximately 200,000 synthetic compounds using Per2lucSV reporter fibroblast cells and validated 11 independent classes of molecules with Bmal1:luciferase reporter cells as well as with suprachiasmatic nucleus and peripheral tissue explants. Four compounds were found to lengthen the period in both central and peripheral clocks, including three compounds that inhibited casein kinase Iε in vitro and a unique benzodiazepine derivative acting through a non-GABA(A) receptor target. In addition, two compounds acutely induced Per2lucSV reporter bioluminescence, delayed the rhythm, and increased intracellular cAMP levels, but caused rhythm damping. Importantly, five compounds shortened the period of peripheral clocks; among them, four compounds also enhanced the amplitude of central and/or peripheral reporter rhythms. Taken together, these studies highlight diverse activities of drug-like small molecules in manipulating the central and peripheral clocks. These small molecules constitute a toolbox for probing clock regulatory mechanisms and may provide putative lead compounds for treatment of clock-associated diseases.

Activation of lignocellulosic biomass by ionic liquid for biorefinery fractionation.

Fractionation of lignocellulosic biomass is an attractive solution to develop an economically viable biorefinery by providing a saccharide fraction to produce fuels and a lignin stream that can be converted into high value products such as carbon fibers. In this study, the analysis of ionic liquid-activated biomass demonstrates that in addition of decreasing crystallinity, the selected ILs (1-butyl-3-methylimidazolium acetate, 1-butyl-3-methylimidazolium chloride and 1-ethyl-3-methylimidazolium acetate) deacetylate Yellow poplar under mild conditions (dissolution at 60-80 °C), and lower the degradation temperature of each biomass polymeric component, thereby reducing the recalcitrance of biomass. Among the three tested ILs, 1-ethyl-3-methylimidazolium acetate performed the best, providing a strong linear relationship between the level of deacetylation and the rate of enzymatic saccharification for Yellow poplar.