TgaA,a VirB1-Like Component Belonging to a Putative Type IV Secretion System of Bifidobacterium bifidum MIMBb75

Bifidobacterium bifidum MIMBb75 is a human intestinal isolate demonstrated to be interactive with the host and efficacious as a probiotic. However, the molecular biology of this microorganism is yet largely unknown. For this reason, we undertook whole-genome sequencing of B. bifidum MIMBb75 to identify potential genetic factors that would explain the metabolic and probiotic attributes of this bacterium. Comparative genomic analysis revealed a 45-kb chromosomal region that comprises 19 putative genes coding for a potential type IV secretion system (T4SS). Thus, we undertook the initial characterization of this genetic region by studying the putative virB1-like gene, named tgaA. Gene tgaA encodes a peptidoglycan lytic enzyme containing two active domains: lytic murein transglycosylase (LT, cd00254.3) and cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP, pfam05257.4). By means of several in vitro assays, we experimentally confirmed that protein TgaA, consistent with its computationally assigned role, has peptidoglycan lytic activity, which is principally associated to the LT domain. Furthermore, immunofluorescence and immunogold labeling showed that the protein TgaA is abundantly expressed on the cell surface of B. bifidum MIMBb75. According to the literature, the T4SSs, which have not been characterized before in bifidobacteria, can have important implications for bacterial cell-to-cell communication as well as cross talk with host cells, justifying the interest for further studies aimed at the investigation of this genetic region.     

Elucidating the Mechanism of Substrate Recognition by the Bacterial Hsp90 Molecular Chaperone

Hsp90 is a conformationally dynamic molecular chaperone known to promote the folding and activation of a broad array of protein substrates (“clients”). Hsp90 is believed to preferentially interact with partially folded substrates, and it has been hypothesized that the chaperone can significantly alter substrate structure as a mechanism to alter the substrate functional state. However, critically testing the mechanism of substrate recognition and remodeling by Hsp90 has been challenging. Using a partially folded protein as a model system, we find that the bacterial Hsp90 adapts its conformation to the substrate, forming a binding site that spans the middle and C-terminal domains of the chaperone. Cross-linking and NMR measurements indicate that Hsp90 binds to a large partially folded region of the substrate and significantly alters both its local and long-range structure. These findings implicate Hsp90's conformational dynamics in its ability to bind and remodel partially folded proteins. Moreover, native-state hydrogen exchange indicates that Hsp90 can also interact with partially folded states only transiently populated from within a thermodynamically stable, native-state ensemble. These results suggest a general mechanism by which Hsp90 can recognize and remodel native proteins by binding and remodeling partially folded states that are transiently sampled from within the native ensemble.     

The rolD oncogene promotes axillary bud and adventitious root meristems in Arabidopsis

The expression of the Agrobacterium rhizogenes rolD oncogene induces precocious floral transition and strong flowering potential in tobacco and tomato. Here, we describe specific developmental effects induced by expression of rolD in Arabidopsis. We show that floral transition, as histologically monitored, occurred in rolD- plants earlier than in wild type, and this was coupled with a premature and enhanced formation of vegetative and reproductive axillary bud meristems. Furthermore, CYP79F1/SUPERSHOOT/BUSHY (SPS), a gene that negatively controls shoot branching in Arabidopsis and involved in glucosinolate metabolism and in cytokinin and auxin homeostasis, was down-regulated in rolD plants. The multiplication of post-embryonic meristems was also observed in the root system, with enhanced adventitious root formation. This result was confirmed by thin cell layer response in vitro, both under hormone-free and standard rooting conditions. However, the formation of lateral root meristems was not affected by rolD expression. Our results show that rolD accelerates and enhances specific post-embryonic meristems in Arabidopsis.     

Experimental support of the scaling rule for demographic stochasticity

A scaling rule of ecological theory, accepted but lacking experimental confirmation, is that the magnitude of fluctuations in population densities due to demographic stochasticity scales inversely with the square root of population numbers. This supposition is based on analyses of models exhibiting exponential growth or stable equilibria. Using two quantitative measures, we extend the scaling rule to situations in which population densities fluctuate due to nonlinear deterministic dynamics. These measures are applied to populations of the flour beetle Tribolium castaneum that display chaotic dynamics in both 20-g and 60-g habitats. Populations cultured in the larger habitat exhibit a clarification of the deterministic dynamics, which follows the inverse square root rule. Lattice effects, a deterministic phenomenon caused by the discrete nature of individuals, can cause deviations from the scaling rule when population numbers are small. The scaling rule is robust to the probability distribution used to model demographic variation among individuals.     

Structural Features of Transiently Modified Beta-Lactoglobulin Relevant to the Stable Binding of Large Hydrophobic Molecules

Binding sites for hydrophobic molecules on bovine β-lactoglobulin, and their susceptibility to temperature, were studied by using various spectroscopic probes. Binding of probes carrying a single fluorophore moiety, a single nitroxide moiety, or both moieties on the same molecule, was followed by EPR and fluorescence. The presence of a fatty acid side chain in the dual probes was found to be required for binding to β-lactoglobulin. Binding occurred only after the protein was heated at temperatures below the threshold for its irreversible denaturation. Binding became extremely tight and stable upon cooling of the protein–probe mixture. Comparison among the various probes suggests that multiple binding sites for hydrophobes are present in the native protein, and in the partially—and reversibly—modified form of β-lactoglobulin present in solution at neutral pH and subdenaturing temperatures. Thus, the specificity of hydrophobes binding to β-lactoglobulin may be modulated by simple physical treatment of the protein.     

Diagnostic performance of multi-organ ultrasound with pocket-sized device in the management of acute dyspnea

Background

The availability of ultra-miniaturized pocket ultrasound devices (PUD) adds diagnostic power to the clinical examination. Information on accuracy of ultrasound with handheld units in immediate differential diagnosis in emergency department (ED) is poor. The aim of this study is to test the usefulness and accuracy of lung ultrasound (LUS) alone or combined with ultrasound of the heart and inferior vena cava (IVC) using a PUD for the differential diagnosis of acute dyspnea (AD).

Methods

We included 68 patients presenting to the ED of “Maurizio Bufalini” Hospital in Cesena (Italy) for AD. All patients underwent integrated ultrasound examination (IUE) of lung-heart-IVC, using PUD. The series was divided into patients with dyspnea of cardiac or non-cardiac origin. We used 2 × 2 contingency tables to analyze sensitivity, specificity, positive predictive value and negative predictive value of the three ultrasonic methods and their various combinations for the diagnosis of cardiogenic dyspnea (CD), comparing with the final diagnosis made by an independent emergency physician.

Results

LUS alone exhibited a good sensitivity (92.6%) and specificity (80.5%). The highest accuracy (90%) for the diagnosis of CD was obtained with the combination of LUS and one of the other two methods (heart or IVC).

Conclusions

The IUE with PUD is a useful extension of the clinical examination, can be readily available at the bedside or in ambulance, requires few minutes and has a reliable diagnostic discriminant ability in the setting of AD.

     

Blood trace metals in a sporadic amyotrophic lateral sclerosis geographical cluster

Amyotrophic lateral sclerosis (ALS) is a fatal disorder with unknown etiology, in which genetic and environmental factors interplay to determine the onset and the course of the disease. Exposure to toxic metals has been proposed to be involved in the etiology of the disease either through a direct damage or by promoting oxidative stress. In this study we evaluated the concentration of a panel of metals in serum and whole blood of a small group of sporadic patients, all living in a defined geographical area, for which acid mine drainage has been reported. ALS prevalence in this area is higher than in the rest of Italy. Results were analyzed with software based on artificial neural networks. High concentrations of metals (in particular Se, Mn and Al) were associated with the disease group. Arsenic serum concentration resulted lower in ALS patients, but it positively correlated with disease duration. Comet assay was performed to evaluate endogenous DNA damage that resulted not different between patients and controls. Up to now only few studies considered geographically well-defined clusters of ALS patients. Common geographical origin among patients and controls gave us the chance to perform metallomic investigations under comparable conditions of environmental exposure. Elaboration of these data with software based on machine learning processes has the potential to be extremely useful to gain a comprehensive view of the complex interactions eventually leading to disease, even in a small number of subjects.     

Regulation of prostaglandin production by inhibition of poly (ADP-ribose) synthase in carrageenan-induced pleurisy.

The aim of the present study was to investigate the effect of two inhibitors (3-aminobenzamide and nicotinamide) of poly (ADP-ribose) synthetase on the production of the inflammatory mediator prostaglandins in a model of acute inflammation, carrageenan-induced pleurisy, where prostaglandins are known to play a crucial role. The results show that the poly (ADP-ribose) synthetase inhibitors, 3-aminobenzamide (2.5, 5 and 10 mg/kg) and nicotinamide (12.5, 25 and 50 mg/kg), inhibit the inflammatory response (pleural exudate formation, polymorphonuclear cell infiltration and prostaglandin production). The present results demonstrate that inhibition of poly (ADP-ribose) synthetase exerts potent anti-inflammatory effects. Part of these anti-inflammatory effects may be related to a reduction of prostaglandin production during the inflammatory process.