Relationship between Heat-Labile Enterotoxin Secretion Capacity and Virulence in Wild Type Porcine-Origin Enterotoxigenic Escherichia coli Strains

Heat-labile enterotoxin (LT) is an important virulence factor secreted by some strains of enterotoxigenic Escherichia coli (ETEC). The prototypic human-origin strain H10407 secretes LT via a type II secretion system (T2SS). We sought to determine the relationship between the capacity to secrete LT and virulence in porcine-origin wild type (WT) ETEC strains. Sixteen WT ETEC strains isolated from cases of severe diarrheal disease were analyzed by GM1ganglioside enzyme-linked immunosorbent assay to measure LT concentrations in culture supernatants. All strains had detectable LT in supernatants by 2 h of culture and 1 strain, which was particularly virulent in gnotobiotic piglets (3030-2), had the highest LT secretion level all porcine-origin WT strains tested (P<0.05). The level of LT secretion (concentration in supernatants at 6-h culture) explained 92% of the variation in time-to-a-moribund-condition (R² = 0.92, P<0.0001) in gnotobiotic piglets inoculated with either strain 3030-2, or an ETEC strain of lesser virulence (2534-86), or a non-enterotoxigenic WT strain (G58-1). All 16 porcine ETEC strains were positive by PCR analysis for the T2SS genes, gspD and gspK, and bioinformatic analysis of 4 porcine-origin strains for which complete genomic sequences were available revealed a T2SS with a high degree of homology to that of H10407. Maximum Likelihood phylogenetic trees constructed using T2SS genes gspC, gspD, gspE and homologs showed that strains 2534-86 and 3030-2 clustered together in the same clade with other porcine-origin ETEC strains in the database, UMNK88 and UMN18. Protein modeling of the ATPase gene (gspE) further revealed a direct relationship between the predicted ATP-binding capacities and LT secretion levels as follows: H10407, -8.8 kcal/mol and 199 ng/ml; 3030-2, -8.6 kcal/mol and 133 ng/ml; and 2534-86, -8.5 kcal/mol and 80 ng/ml. This study demonstrated a direct relationship between predicted ATP-binding capacity of GspE and LT secretion, and between the latter and virulence.     

Synchronous primary mammary osteosarcoma and invasive breast cancer. A case report – Pathohistological and immunohistochemical analysis

Primary osteogenic sarcoma of the breast is a rare neoplasm, diagnosed mainly by pathohistological and immunohistochemical analysis.We hereby present a case of primary osteogenic sarcoma in the right breast of a 62-year-old woman with synchronous appearance of an invasive ductal carcinoma. Clinical findings are manifested with two separate painless formations 2.5 cm/2 cm and 1.5 cm/1 cm in size, located on the border of the upper and lower lateral quadrant of the right breast. No axillary lymphadenopathy was diagnosed. The pathohistological and immunohistochemistry findings of both tumors revealed a synchronous manifestation of two distinct neoplasms – epithelial and non-epithelial. Multimodality treatment consisted of Patey''s radical mastectomy; 3 cycles of adjuvant chemotherapy; postoperative 50 Gy radiotherapy to the chest wall followed by additional 3 cycles of chemotherapy and anti-estrogen hormonotherapy.Due to the rarity of osteogenic mammary sarcoma, even more so in a combination with epithelial breast tumors, its clinical features are unclear and optimal treatment remains controversial. Considering the poor prognosis of the combination of both malignomas, we discuss a number of diagnostic and therapeutic issues.     

Barn Owl Productivity Response to Variability of Vole Populations

We studied the response of the barn owl annual productivity to the common vole population numbers and variability to test the effects of environmental stochasticity on their life histories. Current theory predicts that temporal environmental variability can affect long-term nonlinear responses (e.g., production of young) both positively and negatively, depending on the shape of the relationship between the response and environmental variables. At the level of the Czech Republic, we examined the shape of the relationship between the annual sum of fledglings (annual productivity) and vole numbers in both non-detrended and detrended data. At the districts’ level, we explored whether the degree of synchrony (measured by the correlation coefficient) and the strength of the productivity response increase (measured by the regression coefficient) in areas with higher vole population variability measured by the s-index. We found that the owls’ annual productivity increased linearly with vole numbers in the Czech Republic. Furthermore, based on district data, we also found that synchrony between dynamics in owls’ reproductive output and vole numbers increased with vole population variability. However, the strength of the response was not affected by the vole population variability. Additionally, we have shown that detrending remarkably increases the Taylor’s exponent b relating variance to mean in vole time series, thereby reversing the relationship between the coefficient of variation and the mean. This shift was not responsible for the increased synchrony with vole population variability. Instead, we suggest that higher synchrony could result from high food specialization of owls on the common vole in areas with highly fluctuating vole populations.     

Neural Network Prediction of ICU Length of Stay Following Cardiac Surgery Based on Pre-Incision Variables

Background

Advanced predictive analytical techniques are being increasingly applied to clinical risk assessment. This study compared a neural network model to several other models in predicting the length of stay (LOS) in the cardiac surgical intensive care unit (ICU) based on pre-incision patient characteristics.

Methods

Thirty six variables collected from 185 cardiac surgical patients were analyzed for contribution to ICU LOS. The Automatic Linear Modeling (ALM) module of IBM-SPSS software identified 8 factors with statistically significant associations with ICU LOS; these factors were also analyzed with the Artificial Neural Network (ANN) module of the same software. The weighted contributions of each factor (“trained” data) were then applied to data for a “new” patient to predict ICU LOS for that individual.

Results

Factors identified in the ALM model were: use of an intra-aortic balloon pump; O₂ delivery index; age; use of positive cardiac inotropic agents; hematocrit; serum creatinine ≥ 1.3 mg/deciliter; gender; arterial pCO₂. The r² value for ALM prediction of ICU LOS in the initial (training) model was 0.356, p <0.0001. Cross validation in prediction of a “new” patient yielded r² = 0.200, p <0.0001. The same 8 factors analyzed with ANN yielded a training prediction r² of 0.535 (p <0.0001) and a cross validation prediction r² of 0.410, p <0.0001. Two additional predictive algorithms were studied, but they had lower prediction accuracies. Our validated neural network model identified the upper quartile of ICU LOS with an odds ratio of 9.8(p <0.0001).

Conclusions

ANN demonstrated a 2-fold greater accuracy than ALM in prediction of observed ICU LOS. This greater accuracy would be presumed to result from the capacity of ANN to capture nonlinear effects and higher order interactions. Predictive modeling may be of value in early anticipation of risks of post-operative morbidity and utilization of ICU facilities.     

Crystal structure of a truncated urease accessory protein UreF from Helicobacter pylori

Urease plays a central role in the pathogenesis of Helicobacter pylori in humans. Maturation of this nickel metalloenzyme in bacteria requires the participation of the accessory proteins UreD (termed UreH in H. pylori), UreF, and UreG, which form sequential complexes with the urease apoprotein as well as UreE, a metallochaperone. Here, we describe the crystal structure of C‐terminal truncated UreF from H. pylori (residues 1–233), the first UreF structure to be determined, at 1.55 Å resolution using SAD methods. UreF forms a dimer in vitro and adopts an all‐helical fold congruent with secondary structure prediction. On the basis of evolutionary conservation analysis, the structure reveals a probable binding surface for interaction with other urease components as well as key conserved residues of potential functional relevance. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Identification of novel chromosomal regions associated with airway hyperresponsiveness in recombinant congenic strains of mice

Airway responsiveness is the ability of the airways to respond to bronchoconstricting stimuli by reducing their diameter. Airway hyperresponsiveness has been associated with asthma susceptibility in both humans and murine models, and it has been shown to be a complex and heritable trait. In particular, the A/J mouse strain is known to have hyperresponsive airways, while the C57BL/6 strain is known to be relatively refractory to bronchoconstricting stimuli. We analyzed recombinant congenic strains (RCS) of mice generated from these hyper- and hyporesponsive parental strains to identify genetic loci underlying the trait of airway responsiveness in response to methacholine as assessed by whole-body plethysmography. Our screen identified 16 chromosomal regions significantly associated with airway hyperresponsiveness (genome-wide P ≤ 0.05): 8 are supported by independent and previously published reports while 8 are entirely novel. Regions that overlap with previous reports include two regions on chromosome 2, three on chromosome 6, one on chromosome 15, and two on chromosome 17. The 8 novel regions are located on chromosome 1 (92–100 cM), chromosome 5 (>73 cM), chromosome 7 (>63 cM), chromosome 8 (52–67 cM), chromosome 10 (3–7 cM and >68 cM), and chromosome 12 (25–38 cM and >52 cM). Our data identify several likely candidate genes from the 16 regions, including Ddr2, Hc, Fbn1, Flt3, Utrn, Enpp2, and Tsc.     

An Iris-Like Mechanism of Pore Dilation in the CorA Magnesium Transport System

Magnesium translocation across cell membranes is essential for numerous physiological processes. Three recently reported crystal structures of the CorA magnesium transport system revealed a surprising architecture, with a bundle of giant α-helices forming a 60-Å-long pore that extends beyond the membrane before widening into a funnel-shaped cytosolic domain. The presence of divalent cations in putative intracellular regulation sites suggests that these structures correspond to the closed conformation of CorA. To examine the nature of the conduction pathway, we performed 110-ns molecular-dynamics simulations of two of these structures in a lipid bilayer with and without regulatory ions. The results show that a 15-Å-long hydrophobic constriction straddling the membrane-cytosol interface constitutes a steric bottleneck whose location coincides with an electrostatic barrier opposing cation translocation. In one of the simulations, structural relaxation after the removal of regulatory ions led to concerted changes in the tilt of the pore helices, resulting in iris-like dilation and spontaneous hydration of the hydrophobic neck. This simple and robust mechanism is consistent with the regulation of pore opening by intracellular magnesium concentration, and explains the unusual architecture of CorA.     

BANMOKI: a searchable database of homology-based 3D models and their electrostatic properties of five bacterial nucleoside monophosphate kinase families

The nucleoside monophosphate kinases (NMPK) are important enzymes that control the ratio of mono- and di-phosphate nucleosides and participate in gene regulation and signal transduction in the cell. However, despite their importance only several 3D structures were experimentally determined in contrast to the wealth of sequences available for each of the NMPK families. To fill this gap we present a Web-based database containing structural models for all proteins of the five bacterial nucleoside monophosphate kinase (bNMPK) families. The models were computed by means of homology-based approach using a few experimentally determined bNMPK structures. The database also contains pK(a) values and their components calculated for the homology-based 3D models, which is a unique feature of the database. The BActerial Nucleoside MOnophosphate KInases (BANMOKI) database is freely accessible (http://www.ces.clemson.edu/compbio/banmoki) and offers an easy user-friendly interface for browsing, searching and downloading content of the database. The users can investigate, using the searching tools of the database, the properties of the bNMP kinases in respect to sequence composition, electrostatic interactions and structural differences.     

Growth hormone receptor deficiency in mice results in reduced systolic blood pressure and plasma renin, increased aortic eNOS expression, and altered cardiovascular structure and function

To study the role of the growth hormone receptor (GHR) in the development of cardiovascular structure and function, female GHR gene-disrupted or knockout (KO) and wild-type (WT) mice at age 18 wk were used. GHR KO mice had lower plasma renin levels (12 +/- 2 vs. 20 +/- 4 mGU/ml, P < 0.05) and increased aortic endothelial NO synthase (eNOS) expression (146%, P < 0.05) accompanied by a 25% reduction in systolic blood pressure (BP, 110 +/- 4 vs. 147 +/- 3 mmHg, P < 0.001) compared with WT mice. Aldosterone levels were unchanged, whereas the plasma potassium concentration was elevated by 14% (P < 0.05) in GHR KO. Relative left ventricular weight was 14% lower in GHR KO mice (P < 0.05), and cardiac dimensions as analyzed by echocardiography were similarly reduced. Myograph studies revealed a reduced maximum contractile response in the aorta to norepinephrine (NE) and K(+) (P < 0.05), and aorta media thickness was decreased in GHR KO (P < 0.05). However, contractile force was normal in mesenteric arteries, whereas sensitivity to NE was increased (P < 0.05). Maximal acetylcholine-mediated dilatation was similar in WT and GHR KO mice, whereas the aorta of GHR KO mice showed an increased sensitivity to acetylcholine (P < 0.05). In conclusion, loss of GHR leads to low BP and decreased levels of renin in plasma as well as increase in aortic eNOS expression. Furthermore, GHR deficiency causes functional and morphological changes in both heart and vasculature that are beyond the observed alterations in body size. These data suggest an important role for an intact GH/IGF-I axis in the maintenance of a normal cardiovascular system.