Molecular dynamics simulation of β₂-microglobulin in denaturing and stabilizing conditions

β₂‐Microglobulin has been a model system for the study of fibril formation for 20 years. The experimental study of β₂‐microglobulin structure, dynamics, and thermodynamics in solution, at atomic detail, along the pathway leading to fibril formation is difficult because the onset of disorder and aggregation prevents signal resolution in Nuclear Magnetic Resonance experiments. Moreover, it is difficult to characterize conformers in exchange equilibrium. To gain insight (at atomic level) on processes for which experimental information is available at molecular or supramolecular level, molecular dynamics simulations have been widely used in the last decade. Here, we use molecular dynamics to address three key aspects of β₂‐microglobulin, which are known to be relevant to amyloid formation: (1) 60 ns molecular dynamics simulations of β₂‐microglobulin in trifluoroethanol and in conditions mimicking low pH are used to study the behavior of the protein in environmental conditions that are able to trigger amyloid formation; (2) adaptive biasing force molecular dynamics simulation is used to force cis‐trans isomerization at Proline 32 and to calculate the relative free energy in the folded and unfolded state. The native‐like trans‐conformer (known as intermediate 2 and determining the slow phase of refolding), is simulated for 10 ns, detailing the possible link between cis‐trans isomerization and conformational disorder; (3) molecular dynamics simulation of highly concentrated doxycycline (a molecule able to suppress fibril formation) in the presence of β₂‐microglobulin provides details of the binding modes of the drug and a rationale for its effect. Proteins 2011. © 2010 Wiley‐Liss, Inc.     

Phylogenomic evidence for the presence of a flagellum and cbb(3) oxidase in the free-living mitochondrial ancestor

The initiation of the intracellular symbiosis that would give rise to mitochondria and eukaryotes was a major event in the history of life on earth. Hypotheses to explain eukaryogenesis fall into two broad and competing categories: those proposing that the host was a phagocytotic proto-eukaryote that preyed upon the free-living mitochondrial ancestor (hereafter FMA), and those proposing that the host was an archaebacterium that engaged in syntrophy with the FMA. Of key importance to these hypotheses are whether the FMA was motile or nonmotile, and the atmospheric conditions under which the FMA thrived. Reconstructions of the FMA based on genome content of Rickettsiales representatives-generally considered to be the closest living relatives of mitochondria-indicate that it was nonmotile and aerobic. We have sequenced the genome of Candidatus Midichloria mitochondrii, a novel and phylogenetically divergent member of the Rickettsiales. We found that it possesses unique gene sets found in no other Rickettsiales, including 26 genes associated with flagellar assembly, and a cbb(3)-type cytochrome oxidase. Phylogenomic analyses show that these genes were inherited in a vertical fashion from an ancestral α-proteobacterium, and indicate that the FMA possessed a flagellum, and could undergo oxidative phosphorylation under both aerobic and microoxic conditions. These results indicate that the FMA played a more active and potentially parasitic role in eukaryogenesis than currently appreciated and provide an explanation for how the symbiosis could have evolved under low levels of oxygen.     

Synthesis and biological evaluation of tetracyclic thienopyridones as antibacterial and antitumor agents

A facile synthesis of model 4-oxopyrido[3',2':4,5]thieno[3,2-b]indole-3-carboxylic acids 9a-e was achieved via Stille arylation of 2-chloro-3-nitro-4-oxothieno[2,3-b]pyridine-5-carboxylate and a subsequent microwave-assisted phosphite-mediated Cadogan reaction. The new compounds were tested for their in vitro antimicrobial and antiproliferative activity. Compounds 9a-c and 9e exhibited very high potency against Gram positive Bacillus subtilis and Bacillus megaterium at concentrations 0.000015-0.007 μg/mL. They also displayed excellent activity towards other Gram positive bacilli and staphylococci and Gram negative Haemophilus influenzae, being in most cases superior or equal to commercial fluoroquinolones. Both 9a and 9c were inhibitors of the DNA gyrase activity. As concerns antitumor properties, compounds 9b-e showed growth inhibition of MCF-7 breast tumor and A549 non-small cell lung cancer cells with IC(50) 1.6-2.8 μM and 2.6-6.9 μM, respectively, coupled with absence of cytotoxicity towards normal cells. These compounds are promising as dual acting chemotherapeutics.     

An evaluation of the concept of innateness

The concept of innateness is often used in explanations and classifications of biological and cognitive traits. But does this concept have a legitimate role to play in contemporary scientific discourse? Empirical studies and theoretical developments have revealed that simple and intuitively appealing ways of classifying traits (e.g. genetically specified versus owing to the environment) are inadequate. They have also revealed a variety of scientifically interesting ways of classifying traits each of which captures some aspect of the innate/non-innate distinction. These include things such as whether a trait is canalized, whether it has a history of natural selection, whether it developed without learning or without a specific set of environmental triggers, whether it is causally correlated with the action of certain specific genes, etc. We offer an analogy: the term ‘jade’ was once thought to refer to a single natural kind; it was then discovered that it refers to two different chemical compounds, jadeite and nephrite. In the same way, we argue, researchers should recognize that ‘innateness’ refers not to a single natural kind but to a set of (possibly related) natural kinds. When this happens, it will be easier to progress in the field of biological and cognitive sciences.     

IRF6 is a mediator of Notch pro-differentiation and tumour suppressive function in keratinocytes

While the pro-differentiation and tumour suppressive functions of Notch signalling in keratinocytes are well established, the underlying mechanisms remain poorly understood. We report here that interferon regulatory factor 6 (IRF6), an IRF family member with an essential role in epidermal development, is induced in differentiation through a Notch-dependent mechanism and is a primary Notch target in keratinocytes and keratinocyte-derived SCC cells. Increased IRF6 expression contributes to the impact of Notch activation on growth/differentiation-related genes, while it is not required for induction of 'canonical' Notch targets like p21(WAF1/Cip1), Hes1 and Hey1. Down-modulation of IRF6 counteracts differentiation of primary human keratinocytes in vitro and in vivo, promoting ras-induced tumour formation. The clinical relevance of these findings is illustrated by the strikingly opposite pattern of expression of Notch1 and IRF6 versus epidermal growth factor receptor in a cohort of clinical SCCs, as a function of their grade of differentiation. Thus, IRF6 is a primary Notch target in keratinocytes, which contributes to the role of this pathway in differentiation and tumour suppression.     

Nesfatin-1 stimulates glucagon and insulin secretion and beta cell NUCB2 is reduced in human type 2 diabetic subjects

Nesfatin-1 is a novel anorexigenic regulatory peptide. The peptide is the N-terminal part of nucleobindin 2 (NUCB2) and is expressed in brain areas regulating feeding. Outside the brain, nesfatin-1 expression has been reported in adipocytes, gastric endocrine cells and islet cells. We studied NUCB2 expression in human and rodent islets using immunocytochemistry, in situ hybridization and western blot. Furthermore, we investigated the potential influence of nesfatin-1 on secretion of insulin and glucagon in vitro and in vivo in mice and in INS-1 (832/13) cells. The impact of type 2 diabetes (T2D) and glucolipotoxicity on NUCB2 gene expression in human islets and its relationship to insulin secretory capacity and islet gene expression was studied using microarray. Nesfatin-1 immunoreactivity (IR) was abundant in human and rodent beta cells but absent in alpha, delta, PP and ghrelin cells. Importantly, in situ hybridization showed that NUCB2 mRNA is expressed in human and rat islets. Western blot analysis showed that nesfatin-1 IR represented full length NUCB2 in rodent islets. Human islet NUCB2 mRNA was reduced in T2D subjects but upregulated after culture in glucolipotoxic conditions. Furthermore, a positive correlation between NUCB2 and glucagon and insulin gene expression, as well as insulin secretory capacity, was evident. Nesfatin-1 enhanced glucagon secretion but had no effect on insulin secretion from mouse islets or INS-1 (832/13) cells. On the other hand, nesfatin-1 caused a small increase in insulin secretion and reduced glucose during IVGTT in mice. We conclude that nesfatin-1 is a novel glucagon-stimulatory peptide expressed in the beta cell and that its expression is decreased in T2D islets.     

Biocompatibility analysis of high molecular weight chitosan obtained from Pleoticus muelleri shrimps. Evaluation in prokaryotic and eukaryotic cells

The search for the exploitation and recycling of biomaterials is increasing for reducing the use of non-renewable resources and minimizing environmental pollution caused by synthetic materials. In this context, Chitosan (CS) being a naturally occurring biopolymer becomes relevant. The aim of the present work was to explore the effects of High Molecular Weight CS (H-CS) from Argentinean shrimp's wastes in prokaryotic and eukaryotic in vitro cell cultures. Ultrastructure of H-CS was analysed by SEM and TEM. In vitro studies were performed in prokaryotic (Lactobacillus casei BL23) and eukaryotic (Caco-2, ARPE-19, EA.hy926 and 3T3-L1) culture cells. High performance microscopic techniques were applied to examine culture cells. No changes in morphology were found in any of the cell types. In addition, fluorescent-dyed H-CS revealed that eukaryotic cells could internalize it optimally. Viability was maintained and proliferation rate even increased for Caco-2, ARPE-19 and 3T3-L1 cells under H-CS treatment. Besides, viability was neither altered in L. casei nor in EA.hy926 cells after H-CS exposure. In conclusion, H-CS could be a suitable biopolymer to be exploited for biomedical or food industry applications.     

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.     

Genetic population structure and distribution of a fungal polypore, Datronia caperata (Polyporaceae), in mangrove forests of Central America

Aim We examine the genetic structure of a fungal polypore, Datronia caperata (Berk.) Ryvarden (Polyporaceae), colonizing white mangrove, Laguncularia racemosa (L.) Gaertn. f. (Combretaceae), of Central America. Location Mangrove forests of Costa Rica and Panama. Methods Sequences of elongation factor alpha (EFA), beta tubulin (BTUB) and nuclear ribosomal internal transcribed spacer (ITS) regions were obtained from 54 collections of D. caperata collected from Caribbean and Pacific L. racemosa forests in Central America. Measures of haplotype and nucleotide diversity, nested clade analyses and coalescent analyses were used to estimate the direction and extent of migration of the fungus, and the factors promoting population divergence. We also conducted phylogenetic analyses using Bayesian estimation to test whether putative D. caperata collected from L. racemosa was conspecific with D. caperata colonizing other hosts from diverse Neotropical forests. Results Our results demonstrate that there is genetic isolation between D. caperata populations from Caribbean mangroves and those from Pacific mangroves. Our data suggest that the best explanation for the observed haplotype distribution is a recent range expansion from the Caribbean to the Pacific coasts, with subsequent isolation. This is supported by the infrequent overlap of haplotypes, unidirectional migration estimates from the Caribbean to the Pacific and the older estimated age of mutations in the Caribbean low‐copy BTUB and EFA loci. In addition, our data suggest that D. caperata from mangroves are not conspecific with collections from other hosts found in diverse Neotropical forests. Main conclusions The low frequency of shared haplotypes between coasts, coupled with the incomplete lineage sorting after cessation of gene flow, is consistent with isolation during the last Pleistocene glaciation. We hypothesize that the greater haplotype and nucleotide diversity in the Pacific occurs either because larger effective population sizes of D. caperata are maintained in Pacific mangroves or because D. caperata populations underwent a significant bottleneck as a result of local extinction followed by recolonization. In addition, we found that D. caperata found on L. racemosa was not conspecific with D. caperata from non‐mangrove hosts and suggest that D. caperata found on L. racemosa may be a host specialist.