Identification of amino acid residues critical for the B cell growth-promoting activity of HIV-1 matrix protein p17 variants

Background

HIV-1 matrix protein p17 variants (vp17s) detected in HIV-1-infected patients with non-Hodgkin's lymphoma (HIV-NHL) display, differently from the wild-type protein (refp17), B cell growth-promoting activity. Biophysical analysis revealed that vp17s are destabilized as compared to refp17, motivating us to explore structure-function relationships.

Post-translational modifications in MeHg-induced neurotoxicity

Mercury (Hg) exposure remains a major public health concern due to its widespread distribution in the environment. Organic mercurials, such as MeHg, have been extensively investigated especially because of their congenital effects. In this context, studies on the molecular mechanism of MeHg-induced neurotoxicity are pivotal to the understanding of its toxic effects and the development of preventive measures. Post-translational modifications (PTMs) of proteins, such as phosphorylation, ubiquitination, and acetylation are essential for the proper function of proteins and play important roles in the regulation of cellular homeostasis. The rapid and transient nature of many PTMs allows efficient signal transduction in response to stress. This review summarizes the current knowledge of PTMs in MeHg-induced neurotoxicity, including the most commonly PTMs, as well as PTMs induced by oxidative stress and PTMs of antioxidant proteins. Though PTMs represent an important molecular mechanism for maintaining cellular homeostasis and are involved in the neurotoxic effects of MeHg, we are far from understanding the complete picture on their role, and further research is warranted to increase our knowledge of PTMs in MeHg-induced neurotoxicity.     

The "ins" and "outs" of the high-affinity choline transporter CHT1

Maintenance of acetylcholine (ACh) synthesis depends on the activity of the high-affinity choline transporter (CHT1), which is responsible for the reuptake of choline from the synaptic cleft into presynaptic neurons. In this review, we discuss the current understanding of mechanisms involved in the cellular trafficking of CHT1. CHT1 protein is mainly found in intracellular organelles, such as endosomal compartments and synaptic vesicles. The presence of CHT1 at the plasma membrane is limited by rapid endocytosis of the transporter in clathrin-coated pits in a mechanism dependent on a dileucine-like motif present in the carboxyl-terminal region of the transporter. The intracellular pool of CHT1 appears to constitute a reserve pool of transporters, important for maintenance of cholinergic neurotransmission. However, the physiological basis of the presence of CHT1 in intracellular organelles is not fully understood. Current knowledge about CHT1 indicates that stimulated and constitutive exocytosis, in addition to endocytosis, will have major consequences for regulating choline uptake. Future investigations of CHT1 trafficking should elucidate such regulatory mechanisms, which may aid in understanding the pathophysiology of diseases that affect cholinergic neurons, such as Alzheimer's disease.     

Irreversible Electroporation of Malignant Hepatic Tumors - Alterations in Venous Structures at Subacute Follow-Up and Evolution at Mid-Term Follow-Up

Purpose

To evaluate risk factors associated with alterations in venous structures adjacent to an ablation zone after percutaneous irreversible electroporation (IRE) of hepatic malignancies at subacute follow-up (1 to 3 days after IRE) and to describe evolution of these alterations at mid-term follow-up.

Materials and Methods

43 patients (men/women, 32/11; mean age, 60.3 years) were identified in whom venous structures were located within a perimeter of 1.0 cm of the ablation zone at subacute follow-up after IRE of 84 hepatic lesions (primary/secondary hepatic tumors, 31/53). These vessels were retrospectively evaluated by means of pre-interventional and post-interventional contrast-enhanced magnetic resonance imaging or computed tomography or both. Any vascular changes in flow, patency, and diameter were documented. Correlations between vascular change (yes/no) and characteristics of patients, lesions, and ablation procedures were assessed by generalized linear models.

Results

191 venous structures were located within a perimeter of 1.0 cm of the ablation zone: 55 (29%) were encased by the ablation zone, 78 (41%) abutted the ablation zone, and 58 (30%) were located between 0.1 and 1.0 cm from the border of the ablation zone. At subacute follow-up, vascular changes were found in 19 of the 191 vessels (9.9%), with partial portal vein thrombosis in 2, complete portal vein thrombosis in 3, and lumen narrowing in 14 of 19. At follow-up of patients with subacute vessel alterations (mean, 5.7 months; range, 0 to 14 months) thrombosis had resolved in 2 of 5 cases; vessel narrowing had completely resolved in 8 of 14 cases, and partly resolved in 1 of 14 cases. The encasement of a vessel by ablation zone (OR = 6.36, p<0.001), ablation zone being adjacent to a portal vein (OR = 8.94, p<0.001), and the usage of more than 3 IRE probes (OR = 3.60, p = 0.035) were independently associated with post-IRE vessel alterations.

Conclusion

Venous structures located in close proximity to an IRE ablation zone remain largely unaffected by this procedure, and thrombosis is rare.     

Olive flowering trends in a large Mediterranean area (Italy and Spain)

The aim of this study was to investigate the main climatic and biological trends related to olive flowering in central-southern Italy compared to those in Andalusia, Spain. Results since 1982 were compared for the two long-series monitoring areas of Cordoba and Perugia, and since 1992–1999 for the short-series areas. The relationship between climatic trends and the biological response of the olive, a widespread culture in the Mediterranean basin, were investigated. An aerobiological method involving capturing pollen released into the atmosphere was utilised as a bioindicator of flowering phenology. The study results confirm the strong relationship between flowering periods and spring temperature trends for the olive. Temperature during March, April and May was the parameter most related to flowering date in the study areas, particularly in Italy. In some cases we found a significant correlation between flowering and past autumn temperatures, probably due to their effect on floral bud dormancy induction, but this phenomenon appeared to be of minor importance in the studied areas. The phenological trend results show the continuous advance of flowering dates to the late 1990s, followed by a relatively stationary time series related to a short-term temperature fluctuation in the Mediterranean area. This latter period probably represents a mesoscale event forced by a macroscale event—the North Atlantic Oscillation. The results reveal that the trend towards increased temperatures, and the consequent flowering advance of some species, indicated some years ago is nowadays not as clear as was expected and should be confirmed over the next few years in the Mediterranean areas under investigation.     

Lineage-specific evolution and gene flow in Listeria monocytogenes are independent of bacteriophages

Listeria monocytogenes is a foodborne pathogen causing systemic infection with high mortality. To allow efficient tracking of outbreaks a clear definition of the genomic signature of a cluster of related isolates is required, but lineage-specific characteristics call for a more detailed understanding of evolution. In our work, we used core genome MLST (cgMLST) to identify new outbreaks combined to core genome SNP analysis to characterize the population structure and gene flow between lineages. Whilst analysing differences between the four lineages of L. monocytogenes we have detected differences in the recombination rate, and interestingly also divergence in the SNP differences between sub-lineages. In addition, the exchange of core genome variation between the lineages exhibited a distinct pattern, with lineage III being the best donor for horizontal gene transfer. Whilst attempting to link bacteriophage-mediated transduction to observed gene transfer, we found an inverse correlation between phage presence in a lineage and the extent of recombination. Irrespective of the profound differences in recombination rates observed between sub-lineages and lineages, we found that the previously proposed cut-off of 10 allelic differences in cgMLST can be still considered valid for the definition of a foodborne outbreak cluster of L. monocytogenes.     

Arterially perfused neurosphere-derived cells distribute outside the ischemic core in a model of transient focal ischemia and reperfusion in vitro

BACKGROUND: Treatment with neural stem cells represents a potential strategy to improve functional recovery of post-ischemic cerebral injury. The potential benefit of such treatment in acute phases of human ischemic stroke depends on the therapeutic viability of a systemic vascular delivery route. In spite of the large number of reports on the beneficial effects of intracerebral stem cells injection in experimental stroke, very few studies demonstrated the effectiveness of the systemic intravenous delivery approach. METODOLOGY/PRINCIPAL FINDINGS: We utilized a novel in vitro model of transient focal ischemia to analyze the brain distribution of neurosphere-derived cells (NCs) in the early 3 hours that follow transient occlusion of the medial cerebral artery (MCA). NCs obtained from newborn C57/BL6 mice are immature cells with self-renewal properties that could differentiate into neurons, astrocytes and oligodendrocytes. MCA occlusion for 30 minutes in the in vitro isolated guinea pig brain preparation was followed by arterial perfusion with 1x10(6) NCs charged with a green fluorescent dye, either immediately or 60 minutes after reperfusion onset. Changes in extracellular pH and K(+) concentration during and after MCAO were measured through ion-sensitive electrodes. CONCLUSION/SIGNIFICANCE: It is demonstrated that NCs injected through the vascular system do not accumulate in the ischemic core and preferentially distribute in non-ischemic areas, identified by combined electrophysiological and morphological techniques. Direct measurements of extracellular brain ions during and after MCA occlusion suggest that anoxia-induced tissue changes, such as extracellular acidosis, may prevent NCs from entering the ischemic area in our in vitro model of transitory focal ischemia and reperfusion suggesting a role played by the surrounding microenviroment in driving NCs outside the ischemic core. These findings strongly suggest that the potential beneficial effect of NCs in experimental focal brain ischemia is not strictly dependent on their homing into the ischemic region, but rather through a bystander mechanism possibly mediated by the release of neuroprotective factors in the peri-infarct region.     

A Bacillus megaterium Plasmid System for the Production, Export, and One-Step Purification of Affinity-Tagged Heterologous Levansucrase from Growth Medium

A multiple vector system for the production and export of recombinant affinity-tagged proteins in Bacillus megaterium was developed. Up to 1 mg/liter of a His₆-tagged or Strep-tagged Lactobacillus reuteri levansucrase was directed into the growth medium, using the B. megaterium esterase LipA signal peptide, and recovered by one-step affinity chromatography.     

The New York Consortium on Membrane Protein Structure (NYCOMPS): a high-throughput platform for structural genomics of integral membrane proteins

The New York Consortium on Membrane Protein Structure (NYCOMPS) was formed to accelerate the acquisition of structural information on membrane proteins by applying a structural genomics approach. NYCOMPS comprises a bioinformatics group, a centralized facility operating a high-throughput cloning and screening pipeline, a set of associated wet labs that perform high-level protein production and structure determination by x-ray crystallography and NMR, and a set of investigators focused on methods development. In the first three years of operation, the NYCOMPS pipeline has so far produced and screened 7,250 expression constructs for 8,045 target proteins. Approximately 600 of these verified targets were scaled up to levels required for structural studies, so far yielding 24 membrane protein crystals. Here we describe the overall structure of NYCOMPS and provide details on the high-throughput pipeline.