Human monocyte‐derived macrophages spontaneously differentiated in vitro show distinct phenotypes

Tissue macrophages are resident phagocytes that acquire specific phenotypes according to the microenvironment. Morphological and functional heterogeneity has been evidenced in different homeostatic and pathological conditions. Indeed, the nature of macrophage subsets may have either harmful or beneficial functions in disease progression/resolution. Therefore the possibility to pharmacologically manipulate heterogeneity represents a relevant challenge. Since human tissue macrophages are not easily obtained, various in vitro models are currently used that do not adequately reflect the heterogeneity and plasticity of tissue macrophages. We had previously reported that two dominant and distinct macrophage morphotypes co‐exist in the same culture of human monocytes spontaneously differentiated for 7 days in autologous serum. The present study was aimed to the phenotypic characterization of these morphotypes, that is, round‐ and spindle‐shaped. We observed that, besides substantial differences in cytoskeleton architecture, round monocyte‐derived macrophages (MDMs) showed higher lipid content, increased macropinocytosis/efferocytosis capacity, and overexpression of CD163, interleukin (IL)‐10, and transforming growth factor (TGF) β2. Conversely, spindle MDMs exhibited enhanced respiratory burst and higher expression of the chemokine (C‐C motif) ligands 18 and 24 (CCL18 and CCL24). Overall, round MDMs show functional traits reminiscent of the non‐inflammatory and reparative M2 phenotype, whereas spindle MDMs exhibit a pro‐inflammatory profile and express genes driving lymphocyte activation and eosinophil recruitment. MDMs obtained in the culture condition herein described represent a valuable model to disentangle and manipulate the functional heterogeneity of tissue macrophages that has been disclosed in scenarios spanning from inflammatory and wounding responses to atherosclerotic lesions. J. Cell. Physiol. 228: 1464–1472, 2013. © 2012 Wiley Periodicals, Inc.     

The Ebola Virus Matrix Protein Penetrates into the Plasma Membrane: A KEY STEP IN VIRAL PROTEIN 40 (VP40) OLIGOMERIZATION AND VIRAL EGRESS*

Ebola, a fatal virus in humans and non-human primates, has no Food and Drug Administration-approved vaccines or therapeutics. The virus from the Filoviridae family causes hemorrhagic fever, which rapidly progresses and in some cases has a fatality rate near 90%. The Ebola genome encodes seven genes, the most abundantly expressed of which is viral protein 40 (VP40), the major Ebola matrix protein that regulates assembly and egress of the virus. It is well established that VP40 assembles on the inner leaflet of the plasma membrane; however, the mechanistic details of plasma membrane association by VP40 are not well understood. In this study, we used an array of biophysical experiments and cellular assays along with mutagenesis of VP40 to investigate the role of membrane penetration in VP40 assembly and egress. Here we demonstrate that VP40 is able to penetrate specifically into the plasma membrane through an interface enriched in hydrophobic residues in its C-terminal domain. Mutagenesis of this hydrophobic region consisting of Leu²¹³, Ile²⁹³, Leu²⁹⁵, and Val²⁹⁸ demonstrated that membrane penetration is critical to plasma membrane localization, VP40 oligomerization, and viral particle egress. Taken together, VP40 membrane penetration is an important step in the plasma membrane localization of the matrix protein where oligomerization and budding are defective in the absence of key hydrophobic interactions with the membrane.     

The Outer Membrane TolC-like Channel HgdD Is Part of Tripartite Resistance-Nodulation-Cell Division (RND) Efflux Systems Conferring Multiple-drug Resistance in the Cyanobacterium Anabaena sp. PCC7120

The TolC-like protein HgdD of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 is part of multiple three-component “AB-D” systems spanning the inner and outer membranes and is involved in secretion of various compounds, including lipids, metabolites, antibiotics, and proteins. Several components of HgdD-dependent tripartite transport systems have been identified, but the diversity of inner membrane energizing systems is still unknown. Here we identified six putative resistance-nodulation-cell division (RND) type factors. Four of them are expressed during late exponential and stationary growth phase under normal growth conditions, whereas the other two are induced upon incubation with erythromycin or ethidium bromide. The constitutively expressed RND component Alr4267 has an atypical predicted topology, and a mutant strain (I-alr4267) shows a reduction in the content of monogalactosyldiacylglycerol as well as an altered filament shape. An insertion mutant of the ethidium bromide-induced all7631 did not show any significant phenotypic alteration under the conditions tested. Mutants of the constitutively expressed all3143 and alr1656 exhibited a Fox⁻ phenotype. The phenotype of the insertion mutant I-all3143 parallels that of the I-hgdD mutant with respect to antibiotic sensitivity, lipid profile, and ethidium efflux. In addition, expression of the RND genes all3143 and all3144 partially complements the capability of Escherichia coli ΔacrAB to transport ethidium. We postulate that the RND transporter All3143 and the predicted membrane fusion protein All3144, as homologs of E. coli AcrB and AcrA, respectively, are major players for antibiotic resistance in Anabaena sp. PCC 7120.     

Historical trends in the epidemiology of candidaemia: analysis of an 11-year period in a tertiary care hospital in Brazil

Candida species are an important cause of bloodstream infections (BSI). To evaluate the epidemiological, clinical and microbiological aspects of two cohorts {1994-1999 [period 1 (P1) ]; 2000-2004 [period 2 (P2) ]} of candidaemic patients, we performed a retrospective analysis from a laboratory-based survey. A total of 388 candidaemias were identified, with an incidence of 0.20/1,000 patient-days and a significant increase in P2 vs. P1 (0.25 vs. 0.15, p = 0.04). Cancer and prior antibiotic use were frequent and Candida albicans was the most prevalent species found (42.4%). Resistance to fluconazole was found in 2.47% of the strains. No differences were observed in the species distribution of Candida during the study periods. In the P2 cohort, there were higher prevalence of elderly individuals, cardiac, pulmonary and liver diseases, renal failure, central venous catheters and antibiotic therapy. In P1, there were higher prevalence of neurological diseases and chemotherapy. The crude mortality was 55.4%. In conclusion, our incidence rates remained high. Furthermore, the distribution pattern of Candida species and the fluconazole resistance profile remained unchanged. Moreover, we found a clear trend of higher prevalence of candidaemia among the elderly and among patients with comorbidities. Finally, it is necessary to discuss strategies for the prevention and control of Candida BSI in Brazil.     

Mdm10 is an ancient eukaryotic porin co-occurring with the ERMES complex

Mitochondrial β-barrel proteins fulfill central functions in the outer membrane like metabolite exchange catalyzed by the voltage-dependent anion channel (VDAC) and protein biogenesis by the central components of the preprotein translocase of the outer membrane (Tom40) or of the sorting and assembly machinery (Sam50). The mitochondrial division and morphology protein Mdm10 is another essential outer membrane protein with proposed β-barrel fold, which has so far only been found in Fungi. Mdm10 is part of the endoplasmic reticulum mitochondria encounter structure (ERMES), which tethers the ER to mitochondria and associates with the SAM complex. In here, we provide evidence that Mdm10 phylogenetically belongs to the VDAC/Tom40 superfamily. Contrary to Tom40 and VDAC, Mdm10 exposes long loops towards both sides of the membrane. Analyses of single loop deletion mutants of Mdm10 in the yeast Saccharomyces cerevisiae reveal that the loops are dispensable for Mdm10 function. Sequences similar to fungal Mdm10 can be found in species from Excavates to Fungi, but neither in Metazoa nor in plants. Strikingly, the presence of Mdm10 coincides with the appearance of the other ERMES components. Mdm10's presence in both unikonts and bikonts indicates an introduction at an early time point in eukaryotic evolution.     

Measuring Actin Flow in 3D Cell Protrusions

Actin dynamics is important in determining cell shape, tension, and migration. Methods such as fluorescent speckle microscopy and spatial temporal image correlation spectroscopy have been used to capture high-resolution actin turnover dynamics within cells in two dimensions. However, these methods are not directly applicable in 3D due to lower resolution and poor contrast. Here, we propose to capture actin flow in 3D with high spatial-temporal resolution by combining nanoscale precise imaging by rapid beam oscillation and fluctuation spectroscopy techniques. To measure the actin flow along cell protrusions in cell expressing actin-eGFP cultured in a type I collagen matrix, the laser was orbited around the protrusion and its trajectory was modulated in a clover-shaped pattern perpendicularly to the protrusion. Orbits were also alternated at two positions closely spaced along the protrusion axis. The pair cross-correlation function was applied to the fluorescence fluctuation from these two positions to capture the flow of actin. Measurements done on nonmoving cellular protrusion tips showed no pair-correlation at two orbital positions indicating a lack of flow of F-actin bundles. However, in some protrusions, the pair-correlation approach revealed directional flow of F-actin bundles near the protrusion surface with flow rates in the range of ∼1 μm/min, comparable to results in two dimensions using fluorescent speckle microscopy. Furthermore, we found that the actin flow rate is related to the distance to the protrusion tip. We also observed collagen deformation by concomitantly detecting collagen fibers with reflectance detection during these actin motions. The implementation of the nanoscale precise imaging by rapid beam oscillation method with a cloverleaf-shaped trajectory in conjunction with the pair cross-correlation function method provides a quantitative way of capturing dynamic flows and organization of proteins during cell migration in 3D in conditions of poor contrast.     

The defensin–lipid interaction: Insights on the binding states of the human antimicrobial peptide HNP-1 to model bacterial membranes

Antimicrobial peptides are an important component of innate immunity and have generated considerable interest as a new potential class of natural antibiotics. The biological activity of antimicrobial peptides is strongly influenced by peptide–membrane interactions. Human Neutrophil Peptide 1 (HNP-1) is a 30 aminoacid peptide, belonging to the class of α-defensins. Many biophysical studies have been performed on this peptide to define its mechanism of action. Combining spectroscopic and thermodynamic analysis, insights on the interaction of the α-defensin with POPE:POPG:CL negative charged bilayers are given. The binding states of the peptide below and above the threshold concentration have been analyzed showing that the interaction with lipid bilayers is dependent by peptide concentration. These novel results that indicate how affinity and biological activities of natural antibiotics are depending by their concentration, might open new way of investigation of the antimicrobial mode of action.     

Diversity and expression of P450 genes from Dendroctonus valens LeConte (Curculionidae: Scolytinae) in response to different kairomones

Bark beetles (Curculionidae: Scolytinae) are major cause of woody plants death in the world. They colonize the stem and other parts of trees recognizing host-produced specific compounds (kairomones) and insect pheromones. Bark beetle's antennae and alimentary canal participate in the host selection identifying chemical compounds produced by trees and insects, and also in the metabolism and detoxification of these compounds. The red turpentine beetle (RTB), Dendroctonus valens LeConte, is an unaggressive species that colonize > 40 pine species (Pinaceae) in North and Central America. Several studies suggest that bark beetle cytochrome P450 enzymes are involved in monoterpene oxidation. In this study we identified by means of PCR, cloning, sequencing, and bioinformatic analysis, eleven full-length genes: five CYP4, four CYP6, and two CYP9 in the antennae and gut region of RTB, after stimulation with vapors of monoterpenes: (±)-α-pinene, (R)-(+)-α-pinene, (S)-(?)-β-pinene, (S)-(?)-α-pinene and (+)-3-carene; pine trees volatiles used by RTB as kairomones. The recovered cDNA of these genes vary from 1.5 kb to 1.8 kb and the open frame encodes from 496 to 562 amino acid proteins. The bioinformatic analysis suggests that the majority of P450 proteins encoded by these genes are membrane anchored in the endoplasmic reticulum. RT-qPCR assays showed differential expression of all CYP genes between male and female. The gene expression was dependent of monoterpenes and exposure time, with some of them sex, antennae and gut region specific. Significant differences among monoterpenes, gut region, antennae and exposure time were found. Our results suggest that some of these genes may be involved in the detoxification process of these compounds during tree colonization.