Lignin-modifying processes in the rhizosphere of arid land grasses.

Genes associated with elevated oxidative enzyme activities in arid systems have not been well characterized. To link measured oxidative activities with specific enzymes, we assembled protein-coding reads from the rhizospheres (RHZ) of two arid land grasses. Targeted gene scans for open reading frames, encoding genes potentially involved in lignin modification, resulted in 127 distinct assembly products. The putative genes included those significantly similar to Class II secretory fungal peroxidases. These genes are expressed at sufficiently high levels for assembly, annotation and differentiation across experimental conditions, and they demonstrate the interplay of root systems, environment and plant microbiomes. The genes assembled also included copper-dependent lytic polysaccharide monooxygenases. We detail the enzymes in the host grass RHZs and present a preliminary taxonomic microhabitat characterization. Our findings provide support for biologically mediated Fenton chemistry in the root zones of desert grasses, and provide insight into arid land carbon flow. These results also demonstrate a hyperdiverse microbial community. Both ribosomal RNA and messenger RNA sequences were dominated by bacteria, followed by fungal sequence abundance. Among the notable fungal sequences were those from the members of the arbuscular mycorrhizal fungi (Glomeromycota), which though abundant in this study, we rarely observed in previous PCR-based surveys.

Physicians' responses to clinical decision support on an intensive care unit--comparison of four different alerting methods.

BACKGROUND: In intensive care environments, technology is omnipresent whereby ensuring constant monitoring and the administration of critical drugs to unstable patients. A clinical decision support system (CDSS), with its widespread possibilities, can be a valuable tool in supporting adequate patient care. However, it is still unclear how decision support alerts should be presented to physicians and other medical staff to ensure that they are used most effectively. OBJECTIVE: To determine the effect of four different alert presentation methods on alert compliance after the implementation of an advanced CDSS on the intensive care unit (ICU) in our hospital. METHODS: A randomized clinical trial was executed from August 2010 till December 2011, which included all patients admitted to the ICU of our hospital. The CDSS applied contained a set of thirteen locally developed clinical rules. The percentage of alert compliance was compared for four alert presentation methods: pharmacy intervention, physician alert list, electronic health record (EHR) section and pop-up alerts. Additionally, surveys were held to determine the method most preferred by users of the CDSS. RESULTS: In the study period, the CDSS generated 902 unique alerts, primarily due to drug dosing during decreased renal function and potassium disturbances. Alert compliance was highest for recommendations offered in pop-up alerts (41%, n=68/166), followed by pharmacy intervention (33%, n=80/244), the physician alert list (20%, n=40/199) and the EHR section (19%, n=55/293). The method most preferred by clinicians was pharmacy intervention, and pop-up alerts were found suitable as well if applied correctly. The physician alert list and EHR section were not considered suitable for CDSSs in the process of this study. CONCLUSION: The alert presentation method used for CDSSs is crucial for the compliance with alerts for the clinical rules and, consequently, for the efficacy of these systems. Active alerts such as pop-ups and pharmacy intervention were more effective than passive alerts, which do not automatically appear within the clinical workflow. In this pilot study, ICU clinicians also preferred pharmacy intervention and pop-up alerts. More research is required to expand these results to other departments and other hospitals, as well as to other types of CDSSs and different alert presentation methods.

Functional capacity of XRCC1 protein variants identified in DNA repair-deficient Chinese hamster ovary cell lines and the human population.

XRCC1 operates as a scaffold protein in base excision repair, a pathway that copes with base and sugar damage in DNA. Studies using recombinant XRCC1 proteins revealed that: a C389Y substitution, responsible for the repair defects of the EM-C11 CHO cell line, caused protein instability; a V86R mutation abolished the interaction with POLbeta, but did not disrupt the interactions with PARP-1, LIG3alpha and PCNA; and an E98K substitution, identified in EM-C12, reduced protein integrity, marginally destabilized the POLbeta interaction, and slightly enhanced DNA binding. Two rare (P161L and Y576S) and two frequent (R194W and R399Q) amino acid population variants had little or no effect on XRCC1 protein stability or the interactions with POLbeta, PARP-1, LIG3alpha, PCNA or DNA. One common population variant (R280H) had no pronounced effect on the interactions with POLbeta, PARP-1, LIG3alpha and PCNA, but did reduce DNA-binding ability. When expressed in HeLa cells, the XRCC1 variants-excluding E98K, which was largely nucleolar, and C389Y, which exhibited reduced expression-exhibited normal nuclear distribution. Most of the protein variants, including the V86R POLbeta-interaction mutant, displayed normal relocalization kinetics to/from sites of laser-induced DNA damage: except for E98K and C389Y, and the polymorphic variant R280H, which exhibited a slightly shorter retention time at DNA breaks.

Clay nanoparticle-supported single-molecule fluorescence spectroelectrochemistry.

Here we report that clay nanoparticles allow formation of a modified transparent electrode, spontaneous adsorption of fluorescent redox molecules on the clay layer, and thus the subsequent observation of single-molecule fluorescence spectroelectrochemistry. We can trace single-molecule fluorescence spectroelectrochemistry by probing the fluorescence intensity change of individually immobilized single redox molecules modulated via cyclic voltammetric potential scanning. This work opens a new approach to explore interfacial electron transfer mechanisms of redox reactions.

Single-molecule fluorescence spectroelectrochemistry of cresyl violet.

Here we report a new path to study single molecule electron transfer dynamics by coupling scanning fluorescence microscopy with a potentiostat via a conventional electrochemical cell to enable single-molecule fluorescence spectroelectrochemistry of cresyl violet in aqueous solution, demonstrating that the single-molecule fluorescence intensity of cresyl violet is modulated synchronously with the cyclic voltammetric potential scanning.

Enzyme specific activity in functionalized nanoporous supports.

Here we reveal that enzyme specific activity can be increased substantially by changing the protein loading density (P(LD)) in functionalized nanoporous supports so that the enzyme immobilization efficiency (I(e), defined as the ratio of the specific activity of the immobilized enzyme to the specific activity of the free enzyme in solution) can be much higher than 100%. A net negatively charged glucose oxidase (GOX) and a net positively charged organophosphorus hydrolase (OPH) were entrapped spontaneously in NH(2)- and HOOC-functionalized mesoporous silica (300 Å, FMS) respectively. The specific activity of GOX entrapped in FMS increased with decreasing P(LD). With decreasing P(LD), I(e) of GOX in FMS increased from<35% to>150%. Unlike GOX, OPH in HOOC-FMS showed increased specific activity with increasing P(LD). With increasing P(LD), the corresponding I(e) of OPH in FMS increased from 100% to>200%. A protein structure-based analysis of the protein surface charges directing the electrostatic interaction-based orientation of the protein molecules in FMS demonstrates that substrate access to GOX molecules in FMS is limited at high P(LD), consequently lowering the GOX specific activity. In contrast, substrate access to OPH molecules in FMS remains open at high P(LD) and may promote a more favorable confinement environment that enhances the OPH activity.

Synergetic effects of nanoporous support and urea on enzyme activity.

We report synergetic effects of functionalized mesoporous silica (FMS) and urea to promote favorable protein conformational changes. The specific activity of glucose isomerase (GI) entrapped in FMS in the presence of urea was approximately double that of GI in solution in the absence of urea. Rather than losing all activity in a denaturing solution of 8.0 M urea, the specific activity of GI entrapped in FMS remained higher than the highest specific activity of GI free in solution.

Biosensing paraoxon in simulated environmental samples by immobilized organophosphorus hydrolase in functionalized mesoporous silica.

There is a critical need for highly sensitive, cost-effective sensors to conduct ecological analyses for environmental and homeland security-related applications. Enzyme biosensors, which are currently gaining acceptance for environmental monitoring applications, need improvements to deliver faster measurements with stabilized sensing elements, e.g., enzymes. We report here on a method which significantly overcomes this difficulty, and demonstrate its application in a biosensor for aquatic environmental applications. A fast-responding and stable biosensor was developed via immobilization of organophosphorus hydrolase (OPH) in functionalized mesoporous silica (FMS) with pore sizes in tens of nanometers. The OPH-FMS composite was held on glassy carbon electrode by a dried Nafion gel and FMS protected OPH from Nafion-resulted activity loss. The resulting enzyme biosensor, when integrated with an electrochemical instrument, responded rapidly to low paraoxon concentration and achieved steady-state current in less than 10 s, with a detection limit of 4.0x10(-7) M paraoxon. The biosensor was tested for detection of paraoxon in simulated environmental samples, under wide-ranging physicochemical conditions. Results clearly indicate high recovery efficiencies in aqueous solutions (96 to 101%) at different pH, total organic carbon, total dissolved solids, and total suspended solids, and demonstrate the ability of the biosensor unit to continuously monitor paraoxon in aqueous conditions similar to those found in river and lake systems.

Characterization of functionalized nanoporous supports for protein confinement.

Here we characterize a highly efficient approach for protein confinement and enzyme immobilization in NH(2)- or HOOC- functionalized mesoporous silica (FMS) with pore sizes as large as tens of nanometres. We observed a dramatic increase of enzyme loading in both enzyme activity and protein amount when using appropriate FMS in comparison with unfunctionalized mesoporous silica and normal porous silica. With different protein loading density in NH(2)-FMS, the negatively charged glucose oxidase (GOX) displayed an immobilization efficiency (I(e), the ratio of the specific activity of the immobilized enzyme to the specific activity of the free enzyme in stock solution) in a range from 30% to 160%, while the same charged glucose isomerase (GI) showed an I(e) of 100% to 120%, and the positively charged organophosphorus hydrolase (OPH) exhibited I(e) of more than 200% in HOOC-FMS. The enzyme-FMS composite was stained with the charged gold nanoparticles and imaged by transmission electron microscopy (TEM). Fourier transform infrared (FTIR) spectroscopy showed no major secondary structural change for the enzymes entrapped in FMS. Thanks to the large, rigid, open pore structure of FMS, the reaction rate and K(m) of the entrapped enzymes in FMS were comparable to those of the free enzymes in solution. In principle, the general approach described here should be applicable to many enzymes, proteins, and protein complexes since both pore sizes and functional groups of FMS are controllable.

Entrapping enzyme in a functionalized nanoporous support.

The enzyme organophosphorus hydrolase (OPH) was spontaneously entrapped in carboxylethyl- or aminopropyl-functionalized mesoporous silica with rigid, uniform open-pore geometry (30 nm). This approach yielded larger amounts of protein loading and much higher specific activity of the enzyme when compared to the unfunctionalized mesoporous silica and normal porous silica with the same pore size. When OPH was incubated with the functionalized mesoporous silica, protein molecules were sequestered in or excluded from the porous material, depending on electrostatic interaction with the charged functional groups. OPH entrapped in the organically functionalized nanopores showed an exceptional high immobilization efficiency of more than 200% and enhanced stability far exceeding that of the free enzyme in solution. The combination of high protein loading, high immobilization efficiency and stability is attributed to the large and uniform pore structure, and to the optimum environment introduced by the functional groups.

Equilibrium and stop-flow kinetic studies of fluorescently labeled DNA substrates with DNA repair proteins XPA and replication protein A.

Nucleotide excision repair (NER) is a crucial pathway in the maintenance of genome stability requiring at least two dozen proteins. XPA and RPA have essential roles in the damage recognition step of NER. To better understand the mechanism of their interactions with DNA, we utilized equilibrium and stop-flow kinetic approaches with fluorescently labeled oligonucleotides. Fluorescein is a bona fide NER lesion because a circular plasmid with a single defined fluorescein was repaired by efficient extracts from Xenopus oocyte nuclei. Single-stranded and double-stranded oligonucleotides 5'-labeled with fluorescein were used in the subsequent studies. Oligonucleotide fluorescence was quenched upon specific binding to full-length recombinant Xenopus XPA (xXPA) and/or human RPA. The binding was highly sensitive to the buffer conditions. Analysis of equilibrium binding data with ds DNA and xXPA revealed a single dissociation constant (K(d)) of 24.4 nM. Stopped-flow kinetic experiments were described by a first-order on-rate constant k(on) of 9.03 x 10(8) M(-1) s(-1) and k(off) of 26.1 s(-1). From the ratio of off-rate to on-rate, a calculated K(d) of 28.9 nM was obtained, revealing that the kinetic and equilibrium studies were consistent. The affinity of xXPA for ds undamaged DNA determined in our spectrofluorometry experiments was up to 3 orders of magnitude higher than previously reported values using different substrates, conditions, and assays [gel-shifts (EMSA), filter-binding, anisotropy, and surface plasmon resonance]. The same substrate DNA containing a 4-bp mismatch in the middle yielded a K(d) five times higher (158 nM), indicating weaker binding by xXPA. The differences in K(d) values for these two substrates were mainly attributable to the k(on), rather than k(off) rates. Fluorescence intensity changes upon interaction of xXPA with ss 50-mer were too low to calculate an accurate K(d). Although recombinant human RPA binding to the ds 50-mer was very weak (K(d) > 1 mM), stop-flow and equilibrium measurements to ss oligonucleotide yielded K(d) values of 96 and 20.3 nM, respectively, which correlated with previously reported values using gel mobility shift assays and a similarly sized poly-dT. Equilibrium and stop-flow measurements to the cognate and mismatched ds oligonucleotides using both xXPA and hRPA yielded a 2- to 3-fold increase in the K(d).

Surgical treatment of parotid tumors in the general community hospital.

We reviewed 51 consecutive parotidectomies done at a general community hospital and gathered data on age and sex of the patients, length of stay in the hospital, perioperative outcome, complications, and surgical pathology. Statistical method included logistic regression and ANOVA to determine relationship and dependence of the variables. There were 24 male and 27 female patients. Thirty-six (70.6%) of the parotid tumors were benign. There were no major complications. Treatment of parotid tumors in the community hospital is feasible and safe if basic guidelines are implemented. The epidemiology of parotid tumors in the relatively smaller hospital should resemble that reported in the literature. Our data suggest that parotid malignancy may occur more frequently in elderly men and that length of stay in the hospital may be longer for patients having surgery for malignant than for benign parotid tumors.