Genetic transformation of novel isolates of chicken Lactobacillus bearing probiotic features for expression of heterologous proteins: a tool to develop live oral vaccines

Background  

The use of lactic acid bacteria as vehicles to delivery antigens to immunize animals is a promising issue. When genetically modified, these bacteria can induce a specific local and systemic immune response against selected pathogens. Gastric acid and bile salts tolerance, production of antagonistic substances against pathogenic microorganisms, and adhesive ability to gut epithelium are other important characteristics that make these bacteria useful for oral immunization.     

Effects of the known pathogenic mutations on the aggregation pathway of the amyloidogenic peptide of apolipoprotein A-I

The 93-residue N-terminal fragment of apolipoprotein A-I (ApoA-I) is the major constituent of fibrils isolated from patients affected by the amyloidosis caused by ApoA-I mutations. We have prepared eight polypeptides corresponding to all the currently known amyloidogenic variants of the N-terminal region of ApoA-I, other than a truncation mutation, and investigated their aggregation kinetics and the associated structural modifications. All the variants adopted a monomeric highly disordered structure in solution at neutral pH, whereas acidification of the solution induced an unstable α-helical conformation and the subsequent aggregation into the cross-β structure aggregate. Two mutations (Δ70-72 and L90P) almost abrogated the lag phase of the aggregation process, three mutations (Δ60-71, L75P, and W50R) significantly accelerated the aggregation rate by 2- to 3-fold, while the remaining three variants (L64P, L60R, and G26R) were not significantly different from the wild type. Therefore, an increase in aggregation propensity cannot explain per se the mechanism of the disease for all the variants. Prediction of the protection factors for hydrogen exchange in the native state of full-length protein reveals, in almost all the variants, an expansion of the conformational fluctuations that could favour the proteolytic cleavage and the release of the amyloidogenic peptide. Such an event seems to be a necessary prerequisite for ApoA-I fibrillogenesis in vivo, but the observed increased aggregation propensity of certain variants can have a strong influence on the severity of the disease, such as an earlier onset and a faster progression.     

Decoding the Structural Bases of D76N ?2-Microglobulin High Amyloidogenicity through Crystallography and Asn-Scan Mutagenesis

D76N is the first natural variant of human β-2 microglobulin (β2m) so far identified. Contrary to the wt protein, this mutant readily forms amyloid fibres in physiological conditions, leading to a systemic and severe amyloidosis. Although the Asp76Asn mutant has been extensively characterized, the molecular bases of its instability and aggregation propensity remain elusive. In this work all Asp residues of human β2m were individually substituted to Asn; D-to-N mutants (D34N, D38N, D53N, D59N, D96N and D98N) were characterised in terms of thermodynamic stability and aggregation propensity. Moreover, crystal structures of the D38N, D53N, D59N and D98N variants were solved at high-resolution (1.24–1.70 Å). Despite showing some significant variations in their thermal stabilities, none showed the dramatic drop in melting temperature (relative to the wt protein) as observed for the pathogenic mutant. Consistently, none of the variants here described displayed any increase in aggregation propensity under the experimental conditions tested. The crystal structures confirmed that D-to-N mutations are generally well tolerated, and lead only to minor reorganization of the side chains in close proximity of the mutated residue. D38N is the only exception, where backbone readjustments and a redistribution of the surface electrostatic charges are observed. Overall, our results suggest that neither removing negative charges at sites 34, 38, 53, 59, 96 and 98, nor the difference in β2m pI, are the cause of the aggressive phenotype observed in D76N. We propose that the dramatic effects of the D76N natural mutation must be linked to effects related to the crucial location of this residue within the β2m fold.     

An international survey in Latin America on the practice of interventional cardiology during the COVID-19 pandemic,with a particular focus on myocardial infarction

Netherlands Heart Journal - A reduction in the number of interventional cardiology procedures has emerged as a result of the COVID-19 pandemic. A survey was performed to...     

Modulation of RAGE Isoforms Expression in the Brain and Plasma of Rats Exposed to Transient Focal Cerebral Ischemia

Activation of RAGE (receptor for advanced glycation endproducts) and of its subtypes may play a role in neuronal damage and neuroinflammation associated with brain ischemia, though the underlying mechanisms remain unclear. In this study, we have examined by Western blotting the expression of RAGE isoforms in the cerebral cortex and striatum of Wistar rats subjected to transient (1 or 2 h) middle cerebral artery occlusion (tMCAo). The findings show that the full-length RAGE (~50 kDa) and its isoforms in the 26-43 kDa range are significantly decreased in the ischemic cortex, but not in the striatum, after 1 and 2 h tMCAo when compared to the sham group. By contrast, in the striatum, ischemia-reperfusion injury caused a significant increase of full-length RAGE and its isoforms in the 72-100 kDa range. We also investigated the soluble form of RAGE, which was significantly decreased in the plasma of rats subjected to transient or permanent MCAo. In conclusion, the present data demonstrate that regional brain expression of RAGE is differentially affected by tMCAo in rat. These modifications are accompanied by a decrease in the plasma levels of soluble RAGE, thereby suggesting a potential role for soluble RAGE as a peripheral biomarker of focal ischemia.     

On the molecular structure of human neuroserpin polymers

The polymerization of serpins is at the root of a large class of diseases; the molecular structure of serpin polymers has been recently debated. In this work, we study the polymerization kinetics of human neuroserpin by Fourier Transform Infra Red spectroscopy and by time-lapse Size Exclusion Chromatography. First, we show that two distinct neuroserpin polymers, formed at 45 and 85°C, display the same isosbestic points in the Amide I' band, and therefore share common secondary structure features. We also find a concentration independent polymerization rate at 45°C suggesting that the polymerization rate-limiting step is the formation of an activated monomeric species. The polymer structures are consistent with a model that predicts the bare insertion of portions of the reactive center loop into the A β-sheet of neighboring serpin molecule, although with different extents at 45 and 85°C.     

The Eberth–Katschenko layer in three species of ceratophryines anurans (Anura: Ceratophrydae)

Mangione, S., Garcia, G. and Cardozo, O.M. 2011. The Eberth–Katschenko layer in three species of ceratophryines anurans (Anura: Ceratophrydae). —Acta Zoologica (Stockholm) 92 : 21–26. In this study, we made a morphological analysis by optic microscopy of the dorsal and ventral skin of the body of Ceratophrys cranwellii, Lepidobatrachus llanensis and Chacophrys pierotti to verify if there exist shared integumental characters in relation to the Eberth–Katschenko layer (E–K layer). The presence of cells associated with this layer, historically considered acellular, is one of the leading characters. This study contributes to support that the E–K layer would be the remnant of an ancestral dermal skeleton and not a physiologically significant formation. Hence, the presence of cells associated with the E–K layer would represent a synapomorphy for Ceratophrydae.     

Class I Major Histocompatibility Complex,the Trojan Horse for Secretion of Amyloidogenic β2-Microglobulin

To form extracellular aggregates, amyloidogenic proteins bypass the intracellular quality control, which normally targets unfolded/aggregated polypeptides. Human D76N β₂-microglobulin (β₂m) variant is the prototype of unstable and amyloidogenic protein that forms abundant extracellular fibrillar deposits. Here we focus on the role of the class I major histocompatibility complex (MHCI) in the intracellular stabilization of D76N β₂m. Using biophysical and structural approaches, we show that the MHCI containing D76N β₂m (MHCI₇₆) displays stability, dissociation patterns, and crystal structure comparable with those of the MHCI with wild type β₂m. Conversely, limited proteolysis experiments show a reduced protease susceptibility for D76N β₂m within the MHCI₇₆ as compared with the free variant, suggesting that the MHCI has a chaperone-like activity in preventing D76N β₂m degradation within the cell. Accordingly, D76N β₂m is normally assembled in the MHCI and circulates as free plasma species in a transgenic mouse model.