Ventilation-perfusion relationships following experimental pulmonary contusion.

Ventilation-perfusion changes after right-sided pulmonary contusion (PC) in swine were investigated by means of the multiple inert gas elimination technique (MIGET). Anesthetized swine (injury, n = 8; control, n = 6) sustained a right-chest PC by a captive-bolt apparatus. This was followed by a 12-ml/kg hemorrhage, resuscitation, and reinfusion of shed blood. MIGET and thoracic computed tomography (CT) were performed before and 6 h after injury. Three-dimensional CT scan reconstruction enabled determination of the combined fractional volume of poorly aerated and non-aerated lung tissue (VOL), and the mean gray-scale density (MGSD). Six hours after PC in injured animals, Pa(O(2)) decreased from 234.9 +/- 5.1 to 113.9 +/- 13.0 mmHg. Shunt (Q(S)) increased (2.7 +/- 0.4 to 12.3 +/- 2.2%) at the expense of blood flow to normal ventilation/perfusion compartments (97.1 +/- 0.4 to 87.4 +/- 2.2%). Dead space ventilation (V(D)/V(T)) increased (58.7 +/- 1.7% to 67.2 +/- 1.2%). MGSD increased (-696.7 +/- 6.1 to -565.0 +/- 24.3 Hounsfield units), as did VOL (4.3 +/- 0.5 to 33.5 +/- 3.2%). Multivariate linear regression of MGSD, VOL, V(D)/V(T), and Q(S) vs. Pa(O(2)) retained VOL and Q(S) (r(2) = .835) as independent covariates of Pa(O(2)). An increase in Q(S) characterizes lung failure 6 h after pulmonary contusion; Q(S) and VOL correlate independently with Pa(O(2)).     

High dimensional mediation analysis with latent variables

We propose a model for high dimensional mediation analysis that includes latent variables. We describe our model in the context of an epidemiologic study for incident breast cancer with one exposure and a large number of biomarkers (i.e., potential mediators). We assume that the exposure directly influences a group of latent, or unmeasured, factors which are associated with both the outcome and a subset of the biomarkers. The biomarkers associated with the latent factors linking the exposure to the outcome are considered “mediators.” We derive the likelihood for this model and develop an expectation‐maximization algorithm to maximize an L1‐penalized version of this likelihood to limit the number of factors and associated biomarkers. We show that the resulting estimates are consistent and that the estimates of the nonzero parameters have an asymptotically normal distribution. In simulations, procedures based on this new model can have significantly higher power for detecting the mediating biomarkers compared with the simpler approaches. We apply our method to a study that evaluates the relationship between body mass index, 481 metabolic measurements, and estrogen‐receptor positive breast cancer.     

Assessment of chemical-crosslink-assisted protein structure modeling in CASP13

With the advance of experimental procedures obtaining chemical crosslinking information is becoming a fast and routine practice. Information on crosslinks can greatly enhance the accuracy of protein structure modeling. Here, we review the current state of the art in modeling protein structures with the assistance of experimentally determined chemical crosslinks within the framework of the 13th meeting of Critical Assessment of Structure Prediction approaches. This largest-to-date blind assessment reveals benefits of using data assistance in difficult to model protein structure prediction cases. However, in a broader context, it also suggests that with the unprecedented advance in accuracy to predict contacts in recent years, experimental crosslinks will be useful only if their specificity and accuracy further improved and they are better integrated into computational workflows.     

Protein structure prediction assisted with sparse NMR data in CASP13

CASP13 has investigated the impact of sparse NMR data on the accuracy of protein structure prediction. NOESY and ¹⁵N-¹H residual dipolar coupling data, typical of that obtained for ¹⁵N,¹³C-enriched, perdeuterated proteins up to about 40 kDa, were simulated for 11 CASP13 targets ranging in size from 80 to 326 residues. For several targets, two prediction groups generated models that are more accurate than those produced using baseline methods. Real NMR data collected for a de novo designed protein were also provided to predictors, including one data set in which only backbone resonance assignments were available. Some NMR-assisted prediction groups also did very well with these data. CASP13 also assessed whether incorporation of sparse NMR data improves the accuracy of protein structure prediction relative to nonassisted regular methods. In most cases, incorporation of sparse, noisy NMR data results in models with higher accuracy. The best NMR-assisted models were also compared with the best regular predictions of any CASP13 group for the same target. For six of 13 targets, the most accurate model provided by any NMR-assisted prediction group was more accurate than the most accurate model provided by any regular prediction group; however, for the remaining seven targets, one or more regular prediction method provided a more accurate model than even the best NMR-assisted model. These results suggest a novel approach for protein structure determination, in which advanced prediction methods are first used to generate structural models, and sparse NMR data is then used to validate and/or refine these models.     

Downstream elements of mammalian pre-mRNA polyadenylation signals: primary,secondary and higher-order structures

Primary, secondary and higher-order structures of downstream elements of mammalian pre-mRNA polyadenylation signals [poly(A) signals] are re viewed. We have carried out a detailed analysis on our database of 244 human pre-mRNA poly(A) signals in order to characterize elements in their downstream regions. We suggest that the downstream region of the mammalian pre-mRNA poly(A) signal consists of various simple elements located at different distances from each other. Thus, the downstream region is not described by any precise consensus. Searching our database, we found that ~80% of pre-mRNAs with the AAUAAA or AUUAAA core upstream elements contain simple downstream elements, consisting of U-rich and/or 2GU/U tracts, the former occurring ~2-fold more often than the latter. Approximately one-third of the pre-mRNAs analyzed here contain sequences that may form G-quadruplexes. A substantial number of these sequences are located immediately downstream of the poly(A) signal. A possible role of G-rich sequences in the polyadenylation process is discussed. A model of the secondary structure of the SV40 late pre-mRNA poly(A) signal downstream region is presented.     

Influence of ischemic preconditioning on intracellular sodium,pH, and cellular energy status in isolated perfused heart

The possible relationships between intracellular Na(+) (Na(i)(+)), bioenergetic status and intracellular pH (pH(i)) in the mechanism for ischemic preconditioning were studied using (23)Na and (31)P magnetic resonance spectroscopy in isolated Langendorff perfused rat heart. The ischemic preconditioning (three 5-min ischemic episodes followed by two 5-min and one 10-min period of reperfusion) prior to prolonged ischemia (20 min stop-flow) resulted in a decrease in ischemic acidosis and faster and complete recovery of cardiac function (ventricular developed pressure and heart rate) after 30 min of reperfusion. The response of Na(i) during ischemia in the preconditioned hearts was characterized by an increase in Na(i)(+) at the end of preconditioning and an accelerated decrease during the first few minutes of reperfusion. During post-ischemic reperfusion, bioenergetic parameters (PCr/P(i) and betaATP/P(i) ratios) were partly recovered without any significant difference between control and preconditioned hearts. The reduced acidosis during prolonged ischemia and the accelerated decrease in Na(i)(+) during reperfusion in the preconditioned hearts suggest activation of Na(+)/H(+) exchanger and other ion transport systems during preconditioning, which may protect the heart from intracellular acidosis during prolonged ischemia, and result in better recovery of mechanical function (LVDP and heart rate) during post-ischemic reperfusion.     

Development of a transformation system for the flavinogenic yeast Candida famata

Riboflavin-overproducing mutants of the flavinogenic yeast Candida famata are used for industrial riboflavin production. This paper describes the development of an efficient transformation system for this species. Leucine-deficient mutants have been isolated from C. famata VKM Y-9 wild-type strain. Among them leu2 mutants were identified by transformation to leucine prototrophy with plasmids YEp13 and PRpL2 carrying the Saccharomyces cerevisiae LEU2 gene. DNA fragments (called CfARSs) conferring increased transformation frequencies and extrachromosomal replication were isolated from a C. famata gene library constructed on the integrative vector containing the S. cerevisiae LEU2 gene as a selective marker. The smallest cloned fragment (CfARS16) has been sequenced. This one had high adenine plus thymine (A+T) base pair content and a sequence homologous to the S. cerevisiae ARS Consensus Sequence. Methods for spheroplast transformation and electrotransformation of the yeast C. famata were optimized. They conferred high transformation frequencies (up to 10(5) transformants per microg DNA) with a C. famata leu2 mutant using replicative plasmids containing the S. cerevisiae LEU2 gene as a selective marker. Riboflavin-deficient mutants were isolated from the C. famata leu2 strain and their biochemical identification was carried out. Using the developed transformation system, several C. famata genomic fragments complementing mutations of structural genes for riboflavin biosynthesis (coding for GTP cyclohydrolase, reductase, dihydroxybutanone phosphate synthase and riboflavin synthase, respectively) have been cloned.     

Symmetry‐restrained molecular dynamics simulations improve homology models of potassium channels

Most crystallized homo‐oligomeric ion channels are highly symmetric, which dramatically decreases conformational space and facilitates building homology models (HMs). However, in molecular dynamics (MD) simulations channels deviate from ideal symmetry and accumulate thermal defects, which complicate the refinement of HMs using MD. In this work we evaluate the ability of symmetry constrained MD simulations to improve HMs accuracy, using an approach conceptually similar to Critical Assessment of techniques for protein Structure Prediction (CASP) competition: build HMs of channels with known structure and evaluate the efficiency of proposed methods in improving HMs accuracy (measured as deviation from experimental structure). Results indicate that unrestrained MD does not improve the accuracy of HMs, instantaneous symmetrization improves accuracy but not stability of HMs during subsequent unrestrained MD, while gradually imposing symmetry constraints improves both accuracy (by 5–50%) and stability of HMs. Moreover, accuracy and stability are strongly correlated, making stability a reliable criterion in predicting the accuracy of new HMs. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Association Thermodynamics and Conformational Stability of β-Sheet Amyloid β(17-42) Oligomers: Effects of E22Q (Dutch) Mutation and Charge Neutralization

Amyloid fibrils are associated with many neurodegenerative diseases. It was found that amyloidogenic oligomers, not mature fibrils, are neurotoxic agents related to these diseases. Molecular mechanisms of infectivity, pathways of aggregation, and molecular structure of these oligomers remain elusive. Here, we use all-atom molecular dynamics, molecular mechanics combined with solvation analysis by statistical-mechanical, three-dimensional molecular theory of solvation (also known as 3D-RISM-KH) in a new MM-3D-RISM-KH method to study conformational stability, and association thermodynamics of small wild-type Aβ_17-42 oligomers with different protonation states of Glu²², as well the E22Q (Dutch) mutants. The association free energy of small β-sheet oligomers shows near-linear trend with the dimers being thermodynamically more stable relative to the larger constructs. The linear (within statistical uncertainty) dependence of the association free energy on complex size is a consequence of the unilateral stacking of monomers in the β-sheet oligomers. The charge reduction of the wild-type Aβ_17-42 oligomers upon protonation of the solvent-exposed Glu²² at acidic conditions results in lowering the association free energy compared to the wild-type oligomers at neutral pH and the E22Q mutants. The neutralization of the peptides because of the E22Q mutation only marginally affects the association free energy, with the reduction of the direct electrostatic interactions mostly compensated by the unfavorable electrostatic solvation effects. For the wild-type oligomers at acidic conditions such compensation is not complete, and the electrostatic interactions, along with the gas-phase nonpolar energetic and the overall entropic effects, contribute to the lowering of the association free energy. The differences in the association thermodynamics between the wild-type Aβ_17-42 oligomers at neutral pH and the Dutch mutants, on the one hand, and the Aβ_17-42 oligomers with protonated Glu²², on the other, may be explained by destabilization of the inter- and intrapeptide salt bridges between Asp²³ and Lys²⁸. Peculiarities in the conformational stability and the association thermodynamics for the different models of the Aβ_17-42 oligomers are rationalized based on the analysis of the local physical interactions and the microscopic solvation structure.     

Ionizing radiation and hematopoietic malignancies: Altering the adaptive landscape

Somatic evolution, which underlies tumor progression, is driven by two essential components: (1) diversification of phenotypes through heritable mutations and epigenetic changes and (2) selection for mutant clones which possess higher fitness. Exposure to ionizing radiation (IR) is highly associated with increased risk of carcinogenesis. This link is traditionally attributed to causation of oncogenic mutations through the mutagenic effects of irradiation. On the other hand, potential effects of irradiation on altering fitness and increasing selection for mutant clones are frequently ignored. Recent studies bring the effects of irradiation on fitness and selection into focus, demonstrating that IR exposure results in stable reductions in the fitness of hematopoietic stem and progenitor cell populations. These reductions of fitness are associated with alteration of the adaptive landscape, increasing the selective advantages conferred by certain oncogenic mutations. Therefore, the link between irradiation and carcinogenesis might be more complex than traditionally appreciated: while mutagenic effects of irradiation should increase the probability of occurrence of oncogenic mutations, IR can also work as a tumor promoter, increasing the selective expansion of clones bearing mutations which become advantageous in the irradiation-altered environment, such as activated mutations in Notch1 or disrupting mutations in p53.Key words: Notch, p53, fitness, irradiation, hematopoietic, evolution