A theoretical study of myoglobin working as a nitric oxide scavenger

The mechanism for the reaction between nitric oxide (NO) and O₂ bound to the heme iron of myoglobin (Mb), including the following isomerization to nitrate, has been investigated using hybrid density functional theory (B3LYP). Myoglobin working as a NO scavenger could be of importance, since NO reversibly inhibits the terminal enzyme in the respiration chain, cytochrome c oxidase. The concentration of NO in the cell will thus affect the respiration and thereby the synthesis of ATP. The calculations show that the reaction between NO and the heme-bound O₂ gives a peroxynitrite intermediate whose O–O bond undergoes a homolytic cleavage, forming a NO₂ radical and myoglobin in the oxo-ferryl state. The NO₂ radical then recombines with the oxo-ferryl, forming heme-bound nitrate. Nine different models have been used in the present study to examine the effect on the reaction both by the presence and the protonation state of the distal His64, and by the surroundings of the proximal His93. The barriers going from the oxy-Mb and nitric oxide reactant to the peroxynitrite intermediate and further to the oxo-ferryl and NO₂ radical are around 10 and 7 kcal/mol, respectively. Forming the product, nitrate bound to the heme iron has a barrier of less than ~7 kcal/mol. The overall reaction going from a free nitric oxide and oxy-Mb to the heme bound nitrate is exergonic by more than 30 kcal/mol.     

TfoX-Based Genetic Mapping Identifies Vibrio fischeri Strain-Level Differences and Reveals a Common Lineage of Laboratory Strains

Bacterial strain variation exists in natural populations of bacteria and can be generated experimentally through directed or random mutation. The advent of rapid and cost-efficient whole-genome sequencing has facilitated strain-level genotyping. Even with modern tools, however, it often remains a challenge to map specific traits to individual genetic loci, especially for traits that cannot be selected under culture conditions (e.g., colonization level or pathogenicity). Using a combination of classical and modern approaches, we analyzed strain-level variation in Vibrio fischeri and identified the basis by which some strains lack the ability to utilize glycerol as a carbon source. We proceeded to reconstruct the lineage of the commonly used V. fischeri laboratory strains. Compared to the wild-type ES114 strain, we identify in ES114-L a 9.9-kb deletion with endpoints in tadB2 and glpF; restoration of the missing portion of glpF restores the wild-type phenotype. The widely used strains ESR1, JRM100, and JRM200 contain the same deletion, and ES114-L is likely a previously unrecognized intermediate strain in the construction of many ES114 derivatives. ES114-L does not exhibit a defect in competitive squid colonization but ESR1 does, demonstrating that glycerol utilization is not required for early squid colonization. Our genetic mapping approach capitalizes on the recently discovered chitin-based transformation pathway, which is conserved in the Vibrionaceae; therefore, the specific approach used is likely to be useful for mapping genetic traits in other Vibrio species.     

Levels of Alpha-Toxin Correlate with Distinct Phenotypic Response Profiles of Blood Mononuclear Cells and with agr Background of Community-Associated Staphylococcus aureus Isolates

Epidemiological studies of Staphylococcus aureus have shown a relation between certain clones and the presence of specific virulence genes, but how this translates into virulence-associated functional responses is not fully elucidated. Here we addressed this issue by analyses of community-acquired S. aureus strains characterized with respect to antibiotic resistance, ST types, agr types, and virulence gene profiles. Supernatants containing exotoxins were prepared from overnight bacterial cultures, and tested in proliferation assays using human peripheral blood mononuclear cells (PBMC). The strains displayed stable phenotypic response profiles, defined by either a proliferative or cytotoxic response. Although, virtually all strains elicited superantigen-mediated proliferative responses, the strains with a cytotoxic profile induced proliferation only in cultures with the most diluted supernatants. This indicated that the superantigen-response was masked by a cytotoxic effect which was also confirmed by flow cytometry analysis. The cytotoxic supernatants contained significantly higher levels of α-toxin than did the proliferative supernatants. Addition of α-toxin to supernatants characterized as proliferative switched the response into cytotoxic profiles. In contrast, no effect of Panton Valentine Leukocidin, δ-toxin or phenol soluble modulin α-3 was noted in the proliferative assay. Furthermore, a significant association between agr type and phenotypic profile was found, where agrII and agrIII strains had predominantly a proliferative profile whereas agrI and IV strains had a predominantly cytotoxic profile. The differential response profiles associated with specific S. aureus strains with varying toxin production could possibly have an impact on disease manifestations, and as such may reflect specific pathotypes.     

A unique histone 3 lysine 14 chromatin signature underlies tissue-specific gene regulation

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Resilience of ecosystem processes: a new approach shows that functional redundancy of biological control services is reduced by landscape simplification

Functional redundancy can increase the resilience of ecosystem processes by providing insurance against species loss and the effects of abundance fluctuations. However, due to the difficulty of assessing individual species’ contributions and the lack of a metric allowing for a quantification of redundancy within communities, few attempts have been made to estimate redundancy for individual ecosystem processes. We present a new method linking interaction metrics with metabolic theory that allows for a quantification of redundancy at the level of ecosystem processes. Using this approach, redundancy in the predation on aphids and other prey by natural enemies across a landscape heterogeneity gradient was estimated. Functional redundancy of predators was high in heterogeneous landscapes, low in homogeneous landscapes and scaled with predator specialisation. Our approach allows quantifying functional redundancy within communities and can be used to assess the role of functional redundancy across a wide variety of ecosystem processes and environmental factors.     

SubCellBarCode: Proteome-wide Mapping of Protein Localization and Relocalization

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Sk?ne Emergency Department Assessment of Patient Load (SEAL)—A Model to Estimate Crowding Based on Workload in Swedish Emergency Departments

Objectives

Emergency department (ED) crowding is an increasing problem in many countries. The purpose of this study was to develop a quantitative model that estimates the degree of crowding based on workload in Swedish EDs.

Methods

At five different EDs, the head nurse and physician assessed the workload on a scale from 1 to 6 at randomized time points during a three week period in 2013. Based on these assessments, a regression model was created using data from the computerized patient log system to estimate the level of crowding based on workload. The final model was prospectively validated at the two EDs with the largest census.

Results

Workload assessments and data on 14 variables in the patient log system were collected at 233 time points. The variables Patient hours, Occupancy, Time waiting for the physician and Fraction of high priority (acuity) patients all correlated significantly with the workload assessments. A regression model based on these four variables correlated well with the assessed workload in the initial dataset (r2 = 0.509, p < 0.001) and with the assessments in both EDs during validation (r2 = 0.641; p < 0.001 and r2 = 0.624; p < 0.001).

Conclusions

It is possible to estimate the level of crowding based on workload in Swedish EDs using data from the patient log system. Our model may be applicable to EDs with different sizes and characteristics, and may be used for continuous monitoring of ED workload. Before widespread use, additional validation of the model is needed.     

Local but Not Systemic Administration of Uridine Prevents Development of Antigen-Induced Arthritis

Objective

Uridine has earlier been show to down modulate inflammation in models of lung inflammation. The aim of this study was to evaluate the anti-inflammatory effect of uridine in arthritis.

Methods

Arthritis was induced by intra-articular injection of mBSA in the knee of NMRI mice pre-immunized with mBSA. Uridine was either administered locally by direct injection into the knee joint or systemically. Systemic treatment included repeated injections or implantation of a pellet continuously releasing uridine during the entire experimental procedure. Anti-mBSA specific immune responses were determined by ELISA and cell proliferation and serum cytokine levels were determined by Luminex. Immunohistochemistry was used to identify cells, study expression of cytokines and adhesion molecules in the joint.

Results

Local administration of 25–100 mg/kg uridine at the time of arthritis onset clearly prevented development of joint inflammation. In contrast, systemic administration of uridine (max 1.5 mg uridine per day) did not prevent development of arthritis. Protection against arthritis by local administration of uridine did not affect the anti-mBSA specific immune response and did not prevent the rise in serum levels of pro-inflammatory cytokines associated with the triggering of arthritis. In contrast, local uridine treatment efficiently inhibited synovial expression of ICAM-1 and CD18, local cytokine production and recruitment of leukocytes to the synovium.

Conclusion

Local, but not systemic administration of uridine efficiently prevented development of antigen-induced arthritis. The protective effect did not involve alteration of systemic immunity to mBSA but clearly involved inhibition of synovial expression of adhesion molecules, decreased TNF and IL-6 production and prevention of leukocyte extravasation. Further, uridine is a small, inexpensive molecule and may thus be a new therapeutic option to treat joint inflammation in RA.     

Association of CMV-Specific T Cell-Mediated Immunity with CMV DNAemia and Development of CMV Disease in HIV-1–Infected Individuals

Background

Among HIV-1–infected individuals, cytomegalovirus (CMV) reactivation and disease occur in the setting of advanced immunosuppression. The value of a standardized assessment of CMV-specific T-cell mediated immunity by the CMV QuantiFERON assay (CMV-QFT) has not yet been thoroughly investigated in HIV-1–infected subjects.

Methods

Prospective, longitudinal study in 153 HIV-1–infected subjects with a CD4⁺ T cell count < 350/μL who simultaneously underwent CMV-QFT, CMV serology testing and CMV-DNA quantification. Factors associated with CMV-QFT were evaluated. Clinical screening for CMV manifestations was then performed every 3 months.

Results

Among the 141 CMV IgG-seropositive individuals the CMV-QFT assay yielded reactive results in 84% (118/141), negative results in 15% (21/141) and indeterminate (negative mitogen IFN-gamma response) results in 1% (2/141) of subjects. The mean actual CD4⁺ T cell count was significantly higher in CMV-QFT reactive subjects, when compared to CMV-QFT non-reactive individuals (183 ± 102 vs. 126 ± 104 cells/μL, P = 0.015). A significantly lower proportion of CMV-QFT reactive vs. non-reactive patients displayed CMV DNAemia > 100 copies/mL (23% (27/118) vs. 48% (11/23), P = 0.02). Furthermore, a statistically significant inverse association between mitogen IFN-gamma response and CMV-DNAemia > 1000 copies/mL was observed (P < 0.001). During the observational period, 5 CMV end-organ manifestations were observed. In three of the CMV cases the CMV-QFT yielded indeterminate results.

Conclusions

While CMV-QFT reactivity indicates CMV-specific immunity, indeterminate results due to negative mitogen IFN-gamma response might reflect HIV-1-induced immunodeficiency. Thus, dependency upon CD4⁺ T cell count should be considered when interpreting CMV-QFT results.