The second coordination sphere of FIH controls hydroxylation

The factor inhibiting HIF (FIH) is a proximate oxygen sensor for human cells, hydroxylating Asn(803) within the α-subunit of the hypoxia inducible factor (HIF). FIH is an α-ketoglutatrate (αKG)-dependent, non-heme Fe(II) dioxygenase, in which Fe(II) is coordinated by a (His(2)Asp) facial triad, αKG, and H(2)O. Hydrogen bonding among the facial triad, the HIF-Asn(803) side chain, and various second-sphere residues suggests a functional role for the second coordination sphere in tuning the chemistry of the Fe(II) center. Point mutants of FIH were prepared to test the functional role of the αKG-centered (Asn(205) and Asn(294)) or HIF-Asn(803)-centered (Arg(238) and Gln(239)) second-sphere residues. The second sphere was tested for local effects on priming Fe(II) to react with O(2), oxidative decarboxylation, and substrate positioning. Steady-sate kinetics were used to test for overall catalytic effects; autohydroxylation rates were used to test for priming and positioning, and electronic spectroscopy was used to assess the primary coordination sphere and the electrophilicity of αKG. Asn(205) → Ala and Asn(294) → Ala mutants exhibited diminished rates of steady-state turnover, while minimally affecting autohydroxylation, consistent with impaired oxidative decarboxylation. Blue-shifted metal to ligand charge transfer transitions for (Fe+αKG)FIH indicated that these point mutations destabilized the π* orbitals of αKG, further supporting a slowed rate of oxidative decarboxylation. The Arg(238) → Met mutant exhibited steady-state rates too low to measure and diminished product yields, suggesting impaired substrate positioning or priming; the Arg(238) → Met mutant was capable of O(2) activation for the autohydroxylation reaction. The Gln(239) → Asn mutant exhibited significantly slowed steady-state kinetics and diminished product yields, suggesting impaired substrate positioning or priming. As HIF binding to the Gln(239) → Asn mutant stimulated autohydroxylation, it is more likely that this point mutant simply mispositions the HIF-Asn(803) side chain. This work combines kinetics and spectroscopy to show that these second-sphere hydrogen bonds play roles in promoting oxidative decarboxylation, priming Fe(II) to bind O(2), and positioning HIF-Asn(803).     

Verbascum eskisehirensis sp. nov. (Scrophulariaceae) from central Anatolia, Turkey

Verbascum eskisehirensis Karavel., Ocak & Ekici sp. nov. (sect. Bothrosperma Murb.) is described and illustrated from Turkey. This new species is confined to B3 Eskisehir in central Anatolia. A morphological comparison is made with the closely related species; V. oreophilum K. Koch and V. pyramidatum M. Bieb. In addition, a cluster analysis was conducted and a distribution map of the new species and the related species is also given.     

Accuracy of Doppler-echocardiographic mean pulmonary artery pressure for diagnosis of pulmonary hypertension

Background

The validity of Doppler echocardiographic (DE) measurement of systolic pulmonary artery pressure (sPAP) has been questioned. Recent studies suggest that mean pulmonary artery pressure (mPAP) might reflect more accurately the invasive pressures.

Methodology/Principal Findings

241 patients were prospectively studied to evaluate the diagnostic accuracy of mPAP for the diagnosis of PH. Right heart catheterization (RHC) and DE were performed in 164 patients mainly for preoperative evaluation of heart valve dysfunction. The correlation between DE and RHC was better when mPAP (r = 0.93) and not sPAP (r = 0.81) was assessed. Bland-Altman analysis revealed a smaller variation of mPAP than sPAP. The following ROC analysis identified that a mPAP≥25.5 mmHg is useful for the diagnosis of PH. This value was validated in an independent cohort of patients (n = 50) with the suspicion of chronic-thromboembolic pulmonary hypertension. The calculated diagnostic accuracy was 98%, based on excellent sensitivity of 98% and specificity of 100%. The corresponding positive and negative predictive values were 100%, respectively 88%.

Conclusion

mPAP has been found to be highly accurate for the initial diagnosis of PH. A cut-off value of 25.5 mmHg might be helpful to avoid unnecessary RHC and select patients in whom RHC might be beneficial.     

Atomic force microscope elastography reveals phenotypic differences in alveolar cell stiffness.

To understand the connection between alveolar mechanics and key biochemical events such as surfactant secretion, one first needs to characterize the underlying mechanical properties of the lung parenchyma and its cellular constituents. In this study, the mechanics of three major cell types from the neonatal rat lung were studied; primary alveolar type I (AT1) and type II (AT2) epithelial cells and lung fibroblasts were isolated using enzymatic digestion. Atomic force microscopy indentation was used to map the three-dimensional distribution of apparent depth-dependent pointwise elastic modulus. Histograms of apparent modulus data from all three cell types indicated non-Gaussian distributions that were highly skewed and appeared multimodal for AT2 cells and fibroblasts. Nuclear stiffness in all three cell types was similar (2.5+/-1.0 kPa in AT1 vs. 3.1+/-1.5 kPa in AT2 vs. 3.3+/-0.8 kPa in fibroblasts; n=10 each), whereas cytoplasmic moduli were significantly higher in fibroblasts and AT2 cells (6.0+/-2.3 and 4.7+/-2.9 kPa vs. 2.5+/-1.2 kPa). In both epithelial cell types, actin was arranged in sparse clusters, whereas prominent actin stress fibers were observed in lung fibroblasts. No systematic difference in actin or microtubule organization was noted between AT1 and AT2 cells. Atomic force microscope elastography, combined with live-cell fluorescence imaging, revealed that the stiffer measurements in AT2 cells often colocalized with lamellar bodies. These findings partially explain reported heterogeneity of alveolar cell deformation during in situ lung inflation and provide needed data for better understanding of how mechanical stretch influences surfactant release.     

Small Molecule Diffusion into Swelling Iota-Carrageenan Gels: A Fluorescence Study

Abstract

Small molecule diffusion into Iota-Carrageenan gel was studied by using steady-state fluorescence (SSF) technique. Pyranine, dissolved in water was used as fluorescence probe. Fluorescence emission intensity, I_p, and scattered light intensity, I_sc, were monitored to study diffusion and swelling processes at various temperatures respectively. Fickian and Li-Tanaka models were elaborated to produce diffusion, D, and collective diffusion, D ₀, coefficients. Diffusion and swelling activation energies were also obtained and found to be 20.5 kj mol^?1 and 28.2 kj mol^?1, respectively.     

Health Transformation Project and Defensive Medicine Practice among Neurosurgeons in Turkey

Background

The term “Defensive” medicine was coined in the early 1970′s and has been an important topic of scientific investigation and professional debate ever since.

Objective

The aim of this study was to investigate the characteristics of defensive medicine, its reasons, and the extent to which it is practiced in the Turkish health care system. This is the first national survey to study the practice of defensive medicine among neurosurgeons in Turkey.

Methods

The present cross-sectional study on defensive medicine assessed neurosurgeons registered at the Turkish Neurosurgical Society, who are actively working in various centers and hospitals within the Turkish health care system. A 40-question survey was adapted from existing measures described in the literature and was completed by a total of 404 neurosurgeons, representing 36.7% of the neurosurgeons registered at the Turkish Neurosurgical Society.

Results

Seventy-two percent of the participants in the current study reported practicing defensive medicine. This practice was mainly reported among inexperienced neurosurgeons (74.4%). Most were younger than 40 years of age (75.2%), working in state hospitals/universities (72.7%), and living in the Marmara region (38%). Respondents reported engaging in defensive medicine by avoiding high-risk surgery (62.6%), ordering additional imaging studies (60.9%) and laboratory tests (33.7%), and referring patients to consultants (31.2%). Most participants consider every patient as a potential threat in terms of a medical lawsuit (68.3%) and do not believe the courts can distinguish malpractice from complications (89.6%).

Conclusion

Concerns and perceptions about medical liability lead neurosurgeons to practice defensive medicine. By avoiding high-risk surgery, ordering unnecessary diagnostic tests, and referring the patients to consultants, neurosurgeons try to minimize the risk of malpractice and protect themselves from legal risks, resulting in higher healthcare expenditure and longer treatment periods.     

Effect of sulfide on nitrate reduction in mixed methanogenic cultures

The effect of sulfide on nitrate reduction and methanogenesis was investigated in two mixed, mesophilic (35 degrees C) methanogenic cultures: sulfide-free and sulfide-acclimated (67 mg S/L total sulfide). A mixture of dextrin/peptone served as the carbon/electron donor source for the two stock cultures, as well as in all assays reported here. The sulfide-free enriched culture was amended with both nitrate (75-350 mg N/L) and sulfide (10-100 mg S/L). Denitrification was the predominant pathway at all sulfide levels tested and methanogenesis did not recover in any of the sulfide- and nitrate-amended cultures, except in the 10 mg S/L culture. Accumulation of denitrification intermediates such as NO and N(2)O took place, which irreversibly inhibited the methanogens and resulted in the complete cessation of methane production. In contrast, conversion of nitrate to nitrite and then to ammonia via dissimilatory nitrate reduction to ammonia (DNRA) prevented the accumulation of denitrification intermediates and led to the recovery of methanogenesis in the nitrate-amended, sulfide-acclimated, mixed methanogenic culture. The effect of the COD/N value on nitrate reduction was assessed with the sulfide-acclimated, methanogenic culture at COD/N values of 10, 20, and 60. As the COD/N value increased, the fraction of nitrate reduced through DNRA also increased. The results of this study have significant implications relative to the combined anaerobic treatment of carbon-, nitrogen-, and/or sulfur-bearing wastes.