The Role of the Size and Location of the Tumors and of the Vertebral Anatomy in Determining the Structural Stability of the Metastatically Involved Spine: a Finite Element Study

Vertebral fractures associated with the loss of structural integrity of neoplastic vertebrae are common, and determined to the deterioration of the bone quality in the lesion area. The prediction of the fracture risk in metastatically involved spines can guide in deciding if preventive solutions, such as medical prophylaxis, bracing, or surgery are indicated for the patient. In this study, finite element models of 22 thoracolumbar vertebrae were built based on CT scans of three spines, covering a wide spectrum of possible clinical scenarios in terms of age, bone quality and degenerative features, taking into account the local material properties of bone tissue. Simulations were performed in order to investigate the effect of the size and location of the tumoral lesion, the bone quality and the vertebral level in determining the structural stability of the neoplastic vertebrae. Tumors with random size and positions were added to the models, for a total of 660 simulations in which a compressive load was simulated. Results highlighted the fundamental role of the tumor size, whereas the other parameters had a lower, but non-negligible impact on the axial collapse of the vertebra, the vertebral bulge in the transverse plane and the canal narrowing under the application of the load. All the considered parameters are radiologically measurable, and can therefore be translated in a straightforward way to the clinical practice to support decisions about preventive treatment of metastatic fractures.     

SlyA, a regulatory protein from Salmonella typhimurium, induces a haemolytic and pore-forming protein in Escherichia coli

A chromosomal fragment from Salmonella typhimurium, when cloned in Escherichia coli, generates a haemolytic phenotype. This fragment carries two genes, termed slyA and slyB. The expression of slyA is sufficient for the haemolytic phenotype. The haemolytic activity of E. coli carrying multiple copies of slyA is found mainly in the cytoplasm, with some in the periplasm of cells grown to stationary phase, but overexpression of SlyB, a 15 kDa lipoprotein probably located in the outer membrane, may lead to enhanced, albeit unspecific, release of the haemolytic activity into the medium. Polyclonal antibodies raised against a purified SlyA-HlyA fusion protein identified the over-expressed monomeric 17 kDa SlyA protein mainly in the cytoplasm of E. coli grown to stationary phase, although smaller amounts were also found in the periplasm and even in the culture supernatant. However, the anti-SlyA antibodies reacted with the SlyA protein in a periplasmic fraction that did not contain the haemolytic activity. Conversely, the periplasmic fraction exhibiting haemolytic activity did not contain the 17 kDa SlyA protein. Furthermore, S. typhimurium transformed with multiple copies of the slyA gene did not show a haemolytic phenotype when grown in rich culture media, although the SlyA protein was expressed in amounts similar to those in the recombinant E. coli strain. These results indicate that SlyA is not itself a cytolysin but rather induces in E. coli (but not in S. typhimurium) the synthesis of an uncharacterised, haemolytically active protein which forms pores with a diameter of about 2.6 nm in an artificial lipid bilayer. The SlyA protein thus seems to represent a regulation factor in Salmonella, as is also suggested by the similarity of the SlyA protein to some other bacterial regulatory proteins. slyA- and slyB-related genes were also obtained by PCR from E. coli, Shigella sp. and Citrobacter diversus but not from several other gram-negative bacteria tested.     

SlyA,a regulatory protein from Salmonella typhimurium,induces a haemolytic and pore-forming protein in Escherichia coli

A chromosomal fragment from Salmonella typhimurium, when cloned in Escherichia coli, generates a haemolytic phenotype. This fragment carries two genes, termed slyA and slyB. The expression of slyA is sufficient for the haemolytic phenotype. The haemolytic activity of E. coli carrying multiple copies of slyA is found mainly in the cytoplasm, with some in the periplasm of cells grown to stationary phase, but overexpression of SlyB, a 15 kDa lipoprotein probably located in the outer membrane, may lead to enhanced, albeit unspecific, release of the haemolytic activity into the medium. Polyclonal antibodies raised against a purified SlyA-HlyA fusion protein identified the over-expressed monomeric 17 kDa SlyA protein mainly in the cytoplasm of E. coli grown to stationary phase, although smaller amounts were also found in the periplasm and even in the culture supernatant. However, the anti-SlyA antibodies reacted with the SlyA protein in a periplasmic fraction that did not contain the haemolytic activity. Conversely, the periplasmic fraction exhibiting haemolytic activity did not contain the 17 kDa SlyA protein. Furthermore, S. typhimurium transformed with multiple copies of the slyA gene did not show a haemolytic phenotype when grown in rich culture media, although the SlyA protein was expressed in amounts similar to those in the recombinant E. coli strain. These results indicate that SlyA is not itself a cytolysin but rather induces in E. coli (but not in S. typhimurium) the synthesis of an uncharacterised, haemolytically active protein which forms pores with a diameter of about 2.6 nm in an artificial lipid bilayer. The SlyA protein thus seems to represent a regulation factor in Salmonella, as is also suggested by the similarity of the SlyA protein to some other bacterial regulatory proteins. slyA- and slyB-related genes were also obtained by PCR from E. coli, Shigella sp. and Citrobacter diversus but not from several other gram-negative bacteria tested.     

Degradation of 4-aminobenzenesulfonate by a two-species bacterial coculture

The mutualistic interactions in a 4-aminobenzenesulfonate (sulfanilate) degrading mixed bacterial culture were studied. This coculture consisted of Hydrogenophaga palleronii strain S1 and Agrobacterium radiobacter strain S2. In this coculture only strain S1 desaminated sulfanilate to catechol-4-sulfonate, which did not accumulate in the medium but served as growth substrate for strain S2. During growth in batch culture with sulfanilate as sole source of carbon, energy, nitrogen and sulfur, the relative cell numbers (colony forming units) of both strains were almost constant. None of the strains reached a cell number which was more than threefold higher than the cell number of the second strain. A mineral medium with sulfanilate was inoculated with different relative cell numbers of both strains (relative number of colony forming units S1:S2 2200:1 to 1:500). In all cases, growth was found and the proportion of both strains moved towards an about equal value of about 3:1 (strain S1:strain S2). In contrast to the coculture, strain S1 did not grow in a mineral medium in axenic culture with 4-aminobenzenesulfonate or any other simple organic compound tested. A sterile culture supernatant from strain S2 enabled strain S1 to grow with 4-aminobenzenesulfonate. The same growth promoting effect was found after the addition of a combination of 4-aminobenzoate, biotin and vitamin B₁₂. Strain S1 grew with 4-aminobenzenesulfonate plus the three vitamins with about the same growth rate as the mixed culture in a mineral medium. When (resting) cells of strain S1 were incubated in a pure mineral medium with sulfanilate, up to 30% of the oxidized sulfanilate accumulated as catechol-4-sulfonate in the culture medium. In contrast, only minor amounts of catechol-4-sulfonate accumulated when strain S1 was grown with 4ABS in the presence of the vitamins.     

MEGAN Community Edition - Interactive Exploration and Analysis of Large-Scale Microbiome Sequencing Data

There is increasing interest in employing shotgun sequencing, rather than amplicon sequencing, to analyze microbiome samples. Typical projects may involve hundreds of samples and billions of sequencing reads. The comparison of such samples against a protein reference database generates billions of alignments and the analysis of such data is computationally challenging. To address this, we have substantially rewritten and extended our widely-used microbiome analysis tool MEGAN so as to facilitate the interactive analysis of the taxonomic and functional content of very large microbiome datasets. Other new features include a functional classifier called InterPro2GO, gene-centric read assembly, principal coordinate analysis of taxonomy and function, and support for metadata. The new program is called MEGAN Community Edition (CE) and is open source. By integrating MEGAN CE with our high-throughput DNA-to-protein alignment tool DIAMOND and by providing a new program MeganServer that allows access to metagenome analysis files hosted on a server, we provide a straightforward, yet powerful and complete pipeline for the analysis of metagenome shotgun sequences. We illustrate how to perform a full-scale computational analysis of a metagenomic sequencing project, involving 12 samples and 800 million reads, in less than three days on a single server. All source code is available here: https://github.com/danielhuson/megan-ce     

The conserved GTPase HflX is a ribosome splitting factor that binds to the E-site of the bacterial ribosome

Using a combination of biochemical, structural probing and rapid kinetics techniques we reveal for the first time that the universally conserved translational GTPase (trGTPase) HflX binds to the E-site of the 70S ribosome and that its GTPase activity is modulated by peptidyl transferase centre (PTC) and peptide exit tunnel (PET) binding antibiotics, suggesting a previously undescribed mode of action for these antibiotics. Our rapid kinetics studies reveal that HflX functions as a ribosome splitting factor that disassembles the 70S ribosomes into its subunits in a nucleotide dependent manner. Furthermore, our probing and hydrolysis studies show that the ribosome is able to activate trGTPases bound to its E-site. This is, to our knowledge, the first case in which the hydrolytic activity of a translational GTPase is not activated by the GTPase activating centre (GAC) in the ribosomal A-site. Furthermore, we provide evidence that the bound state of the PTC is able to regulate the GTPase activity of E-site bound HflX.     

Perturbation of the molecular clockwork in the SCN of non-obese diabetic mice prior to diabetes onset

Circadian disruption is associated with the development of diabetes. Non-obese diabetic (NOD) mice show abnormal diurnal profiles in energy balance and locomotor activity suggesting circadian misalignment. Therefore, we analyzed cFos and mPER1 as markers for rhythmic neuronal activity within the suprachiasmatic nucleus (SCN) of wildtype (WT) and non-diabetic (nNOD) as well as acutely diabetic NOD (dNOD) mice. cFos levels show a day/night difference in both WT and nNOD but not in dNOD. mPER1 levels did not show a day/night difference in both nNOD and dNOD. This suggests that disruption of SCN rhythmicity in NOD mice precedes the actual onset of diabetes.     

Characterization of γ-glutamyl transpeptidase activity of cultured endothelial cells from porcine brain capillaries

Summary Endothelial cells were isolated with high viability (>93%) from porcine brain capillaries by Percoll gradient centrifugation after purely enzymatic digestion. Primary cultures were grown to confluent cell monolayers and quantitated for the activity of -glutamyl transpeptidase. The -glutamyl transpeptidase activity starts from a high enzymatic level, decreases with time in culture to about 15% of the initial value, and remains constant at this level after day 10 in culture. The activity progression depends on surface conditions. In the presence of collagen, an exponential decrease starts immediately after seeding, with a time constant of 70±10h. In the absence of collagen, -glutamyl transpeptidase activity first decreases on day 1 after plating, recovers to the initial value on day 2 and 3 and afterwards declines exponentially to a low and constant activity level. Ethanol added to the cell culture at a time when low constant activity is reached, reactivates the -glutamyl transpeptidase to 30% of the initial value.     

Microbial metabolism of chlorosalicylates: accelerated evolution by natural genetic exchange

Methylsalicylate-grown cells of Pseudomonas sp. WR 401 cometabolized 3-, 4- and 5-substituted halosalicylates to the corresponding halocatechols. Further degradation was unproductive due to the presence of high levels of catechol 2,3-dioxygenase. This strain acquired the ability to utilize 3-chlorobenzoate following acquisition of genes from Pseudomonas sp. B 13 which are necessary for the assimilation of chlorocatechols. This derivative (WR 4011) was unable to use 4- or 5-chlorosalicylates. Derivatives able to use these compounds were obtained by plating WR 4011 on 5-chlorosalicylate minimal medium; one such derivative was designated WR 4016. The acquisition of this property was accompanied by concomitant loss of the methylsalicylate phenotype. During growth on 4- or 5-chlorosalicylate the typical enzymes of chlorocatechol assimilation were detected in cell free extracts, whereas catechol 2,3-dioxygenase activity was not induced. Repeated subcultivation of WR 4016 in the presence of 3-chlorosalicylate produced variants (WR 4016-1) which grew on all three isomers.Abbreviations CS chlorosalicylate - MS methylsalicylate - 3CB 3-chlorobenzoate - nal^r nalidixin-resistant - str^r streptomycin-resistant - C230 catechol-2,3-dioxygenase - C120 catechol-1,2-dioxygenase - HMSH 2-hydroxymuconic semialdehyde hydrolase or 2-hydroxy-6-oxo-hexa-2,4-dienoic acid-hydrolase - HMSD 2-hydroxymuconic semialdehyde dehydrogenase - Dienlacton hydrolase 4-carboxymethylenebut-2-en-4-olide hydrolase