Composition of soil Frankia assemblages across ecological drivers parallels that of nodule assemblages in Alnus incana ssp. tenuifolia in interior Alaska.

In root nodule symbioses (RNS) between nitrogen (N)-fixing bacteria and plants, bacterial symbionts cycle between nodule-inhabiting and soil-inhabiting niches that exert differential selection pressures on bacterial traits. Little is known about how the resulting evolutionary tension between host plants and symbiotic bacteria structures naturally occurring bacterial assemblages in soils. We used DNA cloning to examine soil-dwelling assemblages of the actinorhizal symbiont Frankia in sites with long-term stable assemblages in Alnus incana ssp. tenuifolia nodules. We compared: (1) phylogenetic diversity of Frankia in soil versus nodules, (2) change in Frankia assemblages in soil versus nodules in response to environmental variation: both across succession, and in response to long-term fertilization with N and phosphorus, and (3) soil assemblages in the presence and absence of host plants. Phylogenetic diversity was much greater in soil-dwelling than nodule-dwelling assemblages and fell into two large clades not previously observed. The presence of host plants was associated with enhanced representation of genotypes specific to A. tenuifolia, and decreased representation of genotypes specific to a second Alnus species. The relative proportion of symbiotic sequence groups across a primary chronosequence was similar in both soil and nodule assemblages. Contrary to expectations, both N and P enhanced symbiotic genotypes relative to non-symbiotic ones. Our results provide a rare set of field observations against which predictions from theoretical and experimental work in the evolutionary ecology of RNS can be compared.

Polaritonic Cross Feshbach Resonance.

We demonstrate the existence of a cross Feshbach resonance by strongly driving a lower polariton mode and by monitoring in time the transmission of a short optical pulse at the energy of the upper polariton mode in a semiconductor microcavity. From the signatures of the optical resonance, strength, and sign of the energy shift, we attribute the origin of the scattering process between polariton modes with opposite circular polarization to a biexciton bound state. From this study, we infer the conditions required for a strong enhancement of the generation of entangled photon pairs.

Effect of Treatment With Tabalumab, a B Cell-Activating Factor Inhibitor, on Highly Sensitized Patients With End-Stage Renal Disease Awaiting Transplantation.

B cell-activation factor (BAFF) is critical for B cell maturation. Inhibition of BAFF represents an appealing target for desensitization of sensitized end-stage renal disease (ESRD) patients. We conducted a Phase 2a, single-arm, open-label exploratory study investigating the effect of tabalumab (BAFF inhibitor) in patients with ESRD and calculated panel reactive antibodies (cPRAs) >50%. The treatment period duration was 24 weeks. Eighteen patients received tabalumab, at doses of 240-mg subcutaneous (SC) at Week 0 followed by 120-mg SC monthly for 5 additional months. Patients were followed for an additional 52 weeks. Immunopharmacologic effects were characterized through analysis of blood for HLA antibodies, BAFF concentrations, immunoglobulins, T and B cell subsets, as well as pre- and posttreatment tonsil and bone marrow biopsies. Significant reductions in cPRAs were observed at Weeks 16 (p = 0.043) and 36 (p = 0.004); however, absolute reductions were small (<5%). Expected pharmacologic changes in B cell subsets and immunoglobulin reductions were observed. Two tabalumab-related serious adverse events occurred (pneumonia, worsening of peripheral neuropathy), while the most common other adverse events were injection-site pain and hypotension. Three patients received matched deceased donor transplants during follow-up. Treatment with a BAFF inhibitor resulted in statistically significant, but not clinically meaningful reduction in the cPRA from baseline (NCT01200290, Clinicaltrials.gov).

Cross Sections for the Exclusive Photon Electroproduction on the Proton and Generalized Parton Distributions.

Unpolarized and beam-polarized fourfold cross sections (d^{4}σ/dQ^{2}dx_{B}dtdϕ) for the ep→e^{'}p^{'}γ reaction were measured using the CLAS detector and the 5.75-GeV polarized electron beam of the Jefferson Lab accelerator, for 110 (Q^{2},x_{B},t) bins over the widest phase space ever explored in the valence-quark region. Several models of generalized parton distributions (GPDs) describe the data well at most of our kinematics. This increases our confidence that we understand the GPD H, expected to be the dominant contributor to these observables. Through a leading-twist extraction of Compton form factors, these results support the model predictions of a larger nucleon size at lower quark-momentum fraction x_{B}.

Towards a resolution of the proton form factor problem: new electron and positron scattering data.

There is a significant discrepancy between the values of the proton electric form factor, G(E)(p), extracted using unpolarized and polarized electron scattering. Calculations predict that small two-photon exchange (TPE) contributions can significantly affect the extraction of G(E)(p) from the unpolarized electron-proton cross sections. We determined the TPE contribution by measuring the ratio of positron-proton to electron-proton elastic scattering cross sections using a simultaneous, tertiary electron-positron beam incident on a liquid hydrogen target and detecting the scattered particles in the Jefferson Lab CLAS detector. This novel technique allowed us to cover a wide range in virtual photon polarization (ϵ) and momentum transfer (Q(2)) simultaneously, as well as to cancel luminosity-related systematic errors. The cross section ratio increases with decreasing ϵ at Q(2)=1.45 GeV(2). This measurement is consistent with the size of the form factor discrepancy at Q(2)≈1.75 GeV(2) and with hadronic calculations including nucleon and Δ intermediate states, which have been shown to resolve the discrepancy up to 2-3 GeV(2).

Longitudinal target-spin asymmetries for deeply virtual compton scattering.

A measurement of the electroproduction of photons off protons in the deeply inelastic regime was performed at Jefferson Lab using a nearly 6 GeV electron beam, a longitudinally polarized proton target, and the CEBAF Large Acceptance Spectrometer. Target-spin asymmetries for ep→e^{'}p^{'}γ events, which arise from the interference of the deeply virtual Compton scattering and the Bethe-Heitler processes, were extracted over the widest kinematics in Q^{2}, x_{B}, t, and ϕ, for 166 four-dimensional bins. In the framework of generalized parton distributions, at leading twist the t dependence of these asymmetries provides insight into the spatial distribution of the axial charge of the proton, which appears to be concentrated in its center. These results also bring important and necessary constraints for the existing parametrizations of chiral-even generalized parton distributions.

Cerebrovascular collaterals correlate with disease severity in adult North American patients with Moyamoya disease.

BACKGROUND AND PURPOSE: Cerebrovascular collaterals have been increasingly recognized as predictive of clinical outcomes in Moyamoya disease in Asia. The aim of this study was to characterize collaterals in North American adult patients with Moyamoya disease and to assess whether similar correlations are valid. MATERIALS AND METHODS: Patients with Moyamoya disease (n = 39; mean age, 43.5 ±10.6 years) and age- and sex-matched control subjects (n = 33; mean age, 44.3 ± 12.0 years) were graded via angiography. Clinical symptoms of stroke or hemorrhage were graded separately by imaging. Correlations between collateralization and disease severity, measured by the modified Suzuki score, were evaluated in patients with Moyamoya disease by fitting a regression model with clustered ordinal multinomial responses. RESULTS: The presence of leptomeningeal collaterals (P = .008), dilation of the anterior choroidal artery (P = .01), and the posterior communicating artery/ICA ratio (P = .004) all correlated significantly with disease severity. The presence of infarct or hemorrhage and posterior steno-occlusive disease did not correlate significantly with the modified Suzuki score (P = .1). Anterior choroidal artery changes were not specific for hemorrhage. Patients with Moyamoya disease were statistically more likely than controls to have higher posterior communicating artery/ICA ratios and a greater incidence of leptomeningeal collaterals. CONCLUSIONS: As with Moyamoya disease in Asian patients, the presence of cerebrovascular collaterals correlated with the modified Suzuki score for disease severity in North American patients with Moyamoya disease. However, anterior choroidal artery changes, which correlated with increased rates of hemorrhage in Asian studies, were not specific to hemorrhage in North Americans.

Phylogeny and assemblage composition of Frankia in Alnus tenuifolia nodules across a primary successional sere in interior Alaska.

In nitrogen (N) fixing symbioses, host-symbiont specificity, genetic variation in bacterial symbionts and environmental variation represent fundamental constraints on the ecology, evolution and practical uses of these interactions, but detailed information is lacking for many naturally occurring N-fixers. This study examined phylogenetic host specificity of Frankia in field-collected nodules of two Alnus species (A. tenuifolia and A. viridis) in interior Alaska and, for A. tenuifolia, distribution, diversity, spatial autocorrelation and correlation with specific soil factors of Frankia genotypes in nodules collected from replicated habitats representing endpoints of a primary sere. Frankia genotypes most commonly associated with each host belonged to different clades within the Alnus-infective Frankia clade, and for A. tenuifolia, were divergent from previously described Frankia. A. tenuifolia nodules from early and late succession habitats harboured distinct Frankia assemblages. In early succession, a single genotype inhabited 71% of nodules with no discernable autocorrelation at any scale, while late succession Frankia were more diverse, differed widely among plants within a site and were significantly autocorrelated within and among plants. Early succession Frankia genotype occurrence was strongly correlated with carbon/nitrogen ratio in the mineral soil fraction, while in late succession, the most common genotypes were correlated with different soil variables. Our results suggest that phylogenetic specificity is a significant factor in the A. tenuifolia-Frankia interaction and that significant habitat-based differentiation may exist among A. tenuifolia-infective genotypes. This is consistent with our hypothesis that A. tenuifolia selects specific Frankia genotypes from early succession soils and that this choice is attenuated in late succession.

Effect of disease state on ionization during bioanalysis of MK-7009, a selective HCV NS3/NS4 protease inhibitor, in human plasma and human Tween-treated urine by high-performance liquid chromatography with tandem mass spectrometric detection.

HPLC-MS/MS methods for the determination of a Hepatitis C NS3/NS4 protease inhibitor (MK-7009) in human plasma and Tween-treated urine were developed and validated over the concentration range 1-1000 ng/mL and 0.2-100 microg/mL respectively. A stable isotope labeled internal standard (ISTD), D(4)-MK-7009, was employed. Analytes were chromatographed by reversed phase HPLC and quantified by an MS/MS system. Electrospray ionization in the positive mode was employed. Multiple reaction monitoring of the precursor to product ion pairs m/z 758.6-->637.4 MK-7009 and m/z 762.5-->637.4 ISTD was used for quantitation. Analyte and internal standard were extracted from 250 microL of plasma using an automated 96-well liquid-liquid extraction. Plasma pH adjustment prior to extraction minimized ionization suppression in plasma samples from patients with Hepatitis C. The urine method involved direct dilution in the 96-well format of 0.020 mL Tween-treated urine. These methods have supported several clinical studies. Incurred plasma sample reanalysis demonstrated adequate assay reproducibility and ruggedness.

Analysis of methionine oxides and nitrogen-transporting amino acids in chilled and acclimated maize seedlings.

In maize seedlings, chilling causes a reduction of glutamine synthetase (GS) activity, while acclimation protects GS (manuscript submitted). Since ROS can oxidize both protein-bound and free Met to methionine sulfoxide (MSO) and further to methionine sulfone (MSO2, a GS inhibitor), it was hypothesized that the chilling-induced oxidative stress may cause accumulation of MSO and MSO2, thus contributing to the inactivation of GS. MSO2 preferentially inhibited the chloroplastic isoform, GS2. HPLC analysis of polar amino acids from coleoptiles + leaves, mesocotyls and roots of control, chilled, acclimated, acclimated and chilled and chilled and rewarmed plants revealed that free MSO and MSO2 do not accumulate after low temperature treatments. Nevertheless, acclimation significantly increased the expression of putative protein methionine sulfoxide reductase (PMSR), especially in mesocotyls. Different low temperature treatments caused complex changes in the profiles of N-transporting amino acids, Asp, Glu, Asn and Gln.

Comparison of two assay methods for activities of uroporphyrinogen decarboxylase and coproporphyrinogen oxidase.

Uroporphyrinogen decarboxylase (UROD) and coproporphyrinogen oxidase (copro'gen oxidase) are two of the least well understood enzymes in the heme biosynthetic pathway. In the fifth step of the pathway, UROD converts uroporphyrinogen III to coproporphyrinogen III by the decarboxylation of the four acetic acid side chains. Copro'gen oxidase then converts coproporphyrinogen III to protoporphyrinogen IX via two sequential oxidative decarboxylations. Studies of these two enzymes are important to increase our understanding of their mechanisms. Assay comparisons of UROD and copro'gen oxidase from chicken blood hemolysates (CBH), using a newly developed micro-assay, showed that the specific activity of both enzymes is increased in the micro-assay relative to the large-scale assay. The micro-assay has distinct advantages in terms of cost, labor intensity, amount of enzyme required, and sensitivity.

Using QuickTime virtual reality objects in computer-assisted instruction of gross anatomy: Yorick--the VR Skull.

QuickTime virtual reality (QTVR) is a software technology that creates, on a normal computer screen, the illusion of holding and turning a three-dimensional object. QTVR is a practical photo-realistic virtual reality technology that is easily implemented on any current personal computer or via the Internet with no special hardware requirements. Because of its ability to present dynamic photo-quality images, we reasoned that QTVR can provide a more realistic presentation of anatomic structure than two-dimensional atlas pictures and facilitate study of specimens outside the dissection lab. We created QTVR objects, using portions of the skull, and incorporated them into an instructional program for first-year medical students. To obtain images, the bones of the skull were mounted on a rotating table, and a digital camera was positioned on a swinging arm so that the focal point remained coincident with the rotational center of the object as the camera was panned through a vertical arc. Digital images were captured at intervals of 10 degrees rotation of the object (horizontal pan). The camera was then swung through an arc with additional horizontal pan sequences taken at 10 degrees intervals of vertical pan. The images were edited to place the object on a solid black background, then assembled into a linear QuickTime movie. The linear movie was processed to yield a QTVR object movie that can be manipulated on vertical and horizontal axes using the mouse. QTVR movies were incorporated into an interactive environment that provided labeling, links to text-based information and self-testing capabilities. This program, Yorick-the VR Skull, has been used in our first-year medical and graduate gross anatomy courses for the past two years. Results of student evaluation of the program indicate that this QTVR-based program is an effective learning tool that is well received by students.

Effect of androgen administration during puberty on hepatic CYP2C11, CYP3A, and CYP2A1 expression in adult female rats.

This biochemical and pharmacokinetic investigation was undertaken to evaluate the effects of androgen administration during puberty on sex-dependent cytochrome P450 (CYP or P450) enzyme expression in adult female rats. Hepatic testosterone 2alpha-hydroxylase activity and CYP2C11 and CYP3A protein levels were elevated in prepubertally ovariectomized rats injected subcutaneously with testosterone enanthate at 35-49 days of age and killed 41 days after discontinuation of treatment. In contrast, testosterone 6beta- and 7alpha-hydroxylase activities and CYP2A1 protein content were not affected. The increase in CYP2C11 and CYP3A was likely not due to circulating testosterone because plasma testosterone was undetectable. The calculated elimination half-life was 51 +/- 6 hr (mean +/- SE) after testosterone enanthate administration. By 80 days after treatment, CYP2C11 and CYP3A levels were no longer increased. To determine if CYP2C11 expression was responsive to a more periodic pattern of androgen release, ovariectomized rats were injected subcutaneously once or twice daily with unesterified testosterone (elimination half-life was 2.0 +/- 0.3 hr, mean +/- SE). Once- or twice-daily dosing (5 or 2.5 micromol/kg/injection, respectively) during days 35-49 of age did not increase the mean CYP2C11 expression in 90-day-old female rats, although testosterone 2alpha-hydroxylase activity and CYP2C11 protein content were elevated in three of the eight rats injected twice daily. Neither dosing regimen increased CYP3A or decreased CYP2A1 expression. In summary, the results indicate that treatment with testosterone enanthate during puberty resulted in a prolonged but reversible increase in hepatic expression of CYP2C11 and CYP3A.

Effect of ovariectomy and androgen on phenobarbital induction of hepatic CYP2B1 and CYP2B2 in Sprague-Dawley rats.

The purpose of this study was to investigate the impact of prepubertal ovariectomy and postpubertal administration of testosterone on inducibility of rat hepatic CYP2B1 and CYP2B2 by phenobarbital. Intact adult male and female Sprague-Dawley rats were injected ip with sodium phenobarbital (10 mg/kg) or saline (control) once daily on days 129-135 of age and sacrificed one day after the last dose. Hepatic microsomal androstenedione 16beta-hydroxylase activity, benzyloxyresorufin O-dealkylase activity, pentoxyresorufin O-dealkylase activity, and CYP2B1 protein levels were lower in phenobarbital-treated female rats than in phenobarbital-treated male rats. In contrast, there was no sex difference in inducibility of CYP2B2. The lesser inducibility of CYP2B1 in adult female rats was attributed to the presence of an intact ovary because prepubertal ovariectomy (day 25 of age) resulted in increased induction of CYP2B1 and its associated activities (androstenedione 16beta-hydroxylase, benzyloxyresorufin O-dealkylase and pentoxyresorufin O-dealkylase) by phenobarbital. By comparison, postpubertal administration of testosterone enanthate (5 micromol/kg sc once daily on days 80-94 of age) did not enhance the inducibility of CYP2B1 or its associated activities in prepubertally ovariectomized adult (136-day-old) rats administered phenobarbital (10 mg/kg/day on days 129-135 of age). However, the androgen treatment did increase CYP2C11-dependent testosterone 2alpha-hydroxylase activity in the same microsomal samples. Overall, the results show a sex difference in phenobarbital induction of hepatic CYP2B1 but not CYP2B2 in adult Sprague-Dawley rats. They also indicate that prepubertal ovariectomy enhances the effect of phenobarbital on CYP2B1, whereas administration of testosterone enanthate postpubertally does not influence the inducibility of either CYP2B1 or CYP2B2 in prepubertally ovariectomized adult rats.

Laboratory metabolism and evaporative water loss of the aardwolf, Proteles cristatus.

We examined oxygen consumption and total evaporative water loss of aardwolves (Proteles cristatus) at temperatures within and below their thermal neutral zone during both summer and winter. During summer (December), body masses of aardwolves averaged 8.1 +/- 0.7 kg (+/-1 standard deviation). Within their thermal neutral zone, oxygen consumption was 2,194 +/- 443 mL O2 h-1, or 1,058 kJ d-1. The relationship between oxygen consumption (VO2, mL O2 h-1) and ambient temperature (Ta, degree C) below the lower critical temperature was VO2 = 6,310-178 (Ta). During winter (August), aardwolves had an average mass of 7.8 +/- 0.7 kg and a basal metabolic rate of 1,844 +/- 224 mL O2 h-1, or 889 kJ d-1. Below the thermal neutral zone, VO2 = 4,308-116 (Ta). Basal metabolic rate and the slope of the line relating oxygen consumption to ambient temperature were both significantly higher in summer than in winter. Evaporative water loss increased with air temperature for both seasons but was higher in summer than winter. Wet thermal conductance was relatively constant below the thermal neutral zone, but was significantly higher in summer (0.022 +/- 0.001 mL O2 g-1 h-1 degree C-1) than in winter (0.015 +/- 0.001 mL O2 g-1 h-1 degree C-1).

CPOM: alleviating the demand for ICU beds.

Technology is adapting to health care's changing environment as more acutely ill patients, including those with respiratory risks, are being placed on the general care units. Centralized noninvasive pulse oximetry, a method to measure oxygen saturation, provides a relatively inexpensive, efficient means to serve a broader spectrum of patients.

Changes in Isozyme Profiles of Catalase, Peroxidase, and Glutathione Reductase during Acclimation to Chilling in Mesocotyls of Maize Seedlings.

The response of antioxidants to acclimation and chilling in various tissues of dark-grown maize (Zea mays L.) seedlings was examined in relation to chilling tolerance and protection from chilling-induced oxidative stress. Chilling caused an accumulation of H2O2 in both the coleoptile + leaf and the mesocotyl (but not roots), and acclimation prevented this accumulation. None of the antioxidant enzymes were significantly affected by acclimation or chilling in the coleoptile + leaf or root. However, elevated levels of glutathione in acclimated seedlings may contribute to an enhanced ability to scavenge H2O2 in the coleoptile + leaf. In the mesocotyl (visibly most susceptible to chilling), catalase3 was elevated in acclimated seedlings and may represent the first line of defense from mitochondria-generated H2O2. Nine of the most prominent peroxidase isozymes were induced by acclimation, two of which were located in the cell wall, suggesting a role in lignification. Lignin content was elevated in mesocotyls of acclimated seedlings, likely improving the mechanical strength of the mesocotyl. One cytosolic glutathione reductase isozyme was greatly decreased in acclimated seedlings, whereas two others were elevated, possibly resulting in improved effectiveness of the enzyme at low temperature. When taken together, these responses to acclimation illustrate the potential ways in which chilling tolerance may be improved in preemergent maize seedlings.

Localization and Characterization of Peroxidases in the Mitochondria of Chilling-Acclimated Maize Seedlings.

We present evidence of two peroxidases in maize (Zea mays L.) mitochondria. One of these uses guaiacol and the other uses cytochrome c as the electron donor. Treatments of fresh mitochondria with protease(s) indicate that ascorbate and glutathione peroxidases are likely bound to the mitochondria as cytosolic contaminants, whereas guaiacol and cytochrome peroxidases are localized within the mitochondria. These two mitochondrial peroxidases are distinct from contaminant peroxidases and mitochondrial electron transport enzymes. Cytochrome peroxidase is present within the mitochondrial membranes, whereas guaiacol peroxidase is loosely bound to the mitochondrial envelope. Unlike other cellular guaiacol peroxidases, mitochondrial guaiacol peroxidase is not glycosylated. Digestion of lysed mitochondria with trypsin activated mitochondrial guaiacol peroxidase but inhibited cytochrome peroxidase. Isoelectric focusing gel analysis indicated guaiacol peroxidase as a major isozyme (isoelectric point 6.8) that is also activated by trypsin. No change in the mobility of guaiacol peroxidase due to trypsin treatment on native polyacrylamide gel electrophoresis was observed. Although both peroxidases are induced by chilling acclimation treatments (14[deg]C), only cytochrome peroxidase is also induced by chilling (4[deg]C). Because chilling induces oxidative stress in the maize seedlings and the mitochondria are a target for oxidative stress injury, we suggest that mitochondrial peroxidases play a role similar to catalase in protecting mitochondria from oxidative damage.