Microscopic and molecular characterization of ovarian follicle atresia in Rhodnius prolixus Stahl under immune challenge

In this work we characterized the degenerative process of ovarian follicles of the bug Rhodnius prolixus challenged with the non-entomopathogenic fungus Aspergillus niger. An injection of A. niger conidia directly into the hemocoel of adult R. prolixus females at the onset of vitellogenesis caused no effect on host lifespan but elicited a net reduction in egg batch size. Direct inspection of ovaries from the mycosed insects revealed that fungal challenge led to atresia of the vitellogenic follicles. Light microscopy and DAPI staining showed follicle shrinkage, ooplasm alteration and disorganization of the monolayer of follicle cells in the atretic follicles. Transmission electron microscopy of thin sections of follicle epithelium also showed nuclei with condensed chromatin, electron dense mitochondria and large autophagic vacuoles. Occurrence of apoptosis of follicle cells in these follicles was visualized by TUNEL labeling. Resorption of the yolk involved an increase in protease activities (aspartyl and cysteinyl proteases) which were associated with precocious acidification of yolk granules and degradation of yolk protein content. The role of follicle atresia in nonspecific host-pathogen associations and the origin of protease activity that led to yolk resorption are discussed.     

Structural characterization of the lipid A region of Aeromonas salmonicida subsp. salmonicida lipopolysaccharide

The lipid A components of Aeromonas salmonicida subsp. salmonicida from strains A449, 80204-1 and an in vivo rough isolate were isolated by mild acid hydrolysis of the lipopolysaccharide. Structural studies carried out by a combination of fatty acid, electrospray ionization-mass spectrometry and nuclear magnetic resonance analyses confirmed that the structure of lipid A was conserved among different isolates of A. salmonicida subsp. salmonicida. All analyzed strains contained three major lipid A molecules differing in acylation patterns corresponding to tetra-, penta- and hexaacylated lipid A species and comprising 4'-monophosphorylated beta-2-amino-2-deoxy-d-glucopyranose-(1-->6)-2-amino-2-deoxy-d-glucopyranose disaccharide, where the reducing end 2-amino-2-deoxy-d-glucose was present primarily in the alpha-pyranose form. Electrospray ionization-tandem mass spectrometry fragment pattern analysis, including investigation of the inner-ring fragmentation, allowed the localization of fatty acyl residues on the disaccharide backbone of lipid A. The tetraacylated lipid A structure containing 3-(dodecanoyloxy)tetradecanoic acid at N-2',3-hydroxytetradecanoic acid at N-2 and 3-hydroxytetradecanoic acid at O-3, respectively, was found. The pentaacyl lipid A molecule had a similar fatty acid distribution pattern and, additionally, carried 3-hydroxytetradecanoic acid at O-3'. In the hexaacylated lipid A structure, 3-hydroxytetradecanoic acid at O-3' was esterified with a secondary 9-hexadecenoic acid. Interestingly, lipid A of the in vivo rough isolate contained predominantly tetra- and pentaacylated lipid A species suggesting that the presence of the hexaacyl lipid A was associated with the smooth-form lipopolysaccharide.     

Intermittent hypoxia treatments cause cellular priming in human microglia

Obstructive sleep apnoea syndrome (OSAS) is a sleep-disordered breathing characterized by nocturnal collapses of the upper airway resulting in cycles of blood oxygen partial pressure oscillations, which lead to tissue and cell damage due to intermittent hypoxia (IH) episodes. Since OSAS-derived IH may lead to cognitive impairment through not fully cleared mechanisms, herein we developed a new in vitro model mimicking IH conditions to shed light on its molecular effects on microglial cells, with particular attention to the inflammatory response. The in vitro model was set-up and validated by measuring the hypoxic state, HIF-1α levels, oxidative stress by ROS production and mitochondrial activity by MTS assay. Then, the mRNA and protein levels of certain inflammatory markers (NF-κB and interleukin 6 (IL-6)) after different IH treatment protocols were investigated. The IH treatments followed by a normoxic period were not able to produce a high inflammatory state in human microglial cells. Nevertheless, microglia appeared to be in a state characterized by increased expression of NF-κB and markers related to a primed phenotype. The microglia exposed to IH cycles and stimulated with exogenous IL-1β resulted in an exaggerated inflammatory response with increased NF-κB and IL-6 expression, suggesting a role for primed microglia in OSAS-driven neuroinflammation.     

Microwave-assisted sample preparation for rapid and sensitive analysis of H. pylori lipid A applicable to a single colony

The lipid A of Gram-negative bacteria plays a major role in the pathogenesis of bacterial infections. Lipid A diversity is observed both in the number and length of fatty-acid side chains and in the presence of terminal phosphate residues and associated modifications. In this report, we describe a new sample preparation method based on microwave-assisted enzymatic digestion and detergent-free mild hydrolysis, in conjunction with a MALDI-time-of-flight (TOF)/TOF analysis, to determine the structures of lipid A from Helicobacter pylori. The total time for sample preparation and mass spectrometric analysis is within 2 h and applicable to profiling the lipid A structures from dried bacterial cells on as little as 1 μg. The reliability of the technique was further demonstrated through the analysis of the lipid A from bacterial cells of different H. pylori strains. The phosphorylation and acylation patterns of lipid A could be elucidated using material from a single colony. Furthermore, we found unusual heptaacyl lipid A species present in H. pylori mutant that have not been previously reported, although the abundance was relatively low. The present study provides the first characterization of the lipid A component from a single bacterial colony sample by mass spectrometry.     

Recombinant expression and biochemical characterization of the unique elongating beta-ketoacyl-acyl carrier protein synthase involved in fatty acid biosynthesis of Plasmodium falciparum using natural and artificial substrates

The human malaria parasite Plasmodium falciparum synthesizes fatty acids by using a type II synthase that is structurally different from the type I system found in eukaryotes. Because of this difference and the vital role of fatty acids, the enzymes involved in fatty acid biosynthesis of P. falciparum represent interesting targets for the development of new antimalarial drugs. beta-Ketoacyl-acyl carrier protein (ACP) synthase (PfFabBF), being the only elongating beta-ketoacyl-ACP synthase in P. falciparum, is a potential candidate for inhibition. In this study we present the cloning, expression, purification, and characterization of PfFabBF. Soluble protein was obtained when PfFabBF was expressed as a NusA fusion protein in Escherichia coli BL21(DE3)-CodonPlus-RIL cells under conditions of osmotic stress. The fusion protein was purified by affinity and ion exchange chromatography. Various acyl-P. falciparum acyl carrier protein (PfACP) substrates were tested for their specific activities, and their kinetic parameters were determined. Activity of PfFabBF was highest with C(4:0)- to C(10:0)-acyl-PfACPs and decreased with use of longer chain acyl-PfACPs. Consistent with the fatty acid synthesis profile found in the parasite cell, no activity could be detected with C(16:0)-PfACP, indicating that the enzyme is lacking the capability of elongating acyl chains that are longer than 14 carbon atoms. PfFabBF was found to be specific for acyl-PfACPs, and it displayed much lower activities with the corresponding acyl-CoAs. Furthermore, PfFabBF was shown to be sensitive to cerulenin and thiolactomycin, known inhibitors of beta-ketoacyl-ACP synthases. These results represent an important step toward the evaluation of P. falciparum beta-ketoacyl-ACP synthase as a novel antimalaria target.     

The sexual phase of the diatom <Emphasis Type="Italic">Pseudo-nitzschia multistriata</Emphasis>: cytological and time-lapse cinematography characterization

Pseudo-nitzschia is a thoroughly studied pennate diatom genus for ecological and biological reasons. Many species in this genus, including Pseudo-nitzschia multistriata, can produce domoic acid, a toxin responsible for amnesic shellfish poisoning. Physiological, phylogenetic and biological features of P. multistriata were studied extensively in the past. Life cycle stages, including the sexual phase, fundamental in diatoms to restore the maximum cell size and avoid miniaturization to death, have been well described for this species. P. multistriata is heterothallic; sexual reproduction is induced when strains of opposite mating type are mixed, and proceeds with cells producing two functionally anisogamous gametes each; however, detailed cytological information for this process is missing. By means of confocal laser scanning microscopy and nuclear staining, we followed the nuclear fate during meiosis, and using time-lapse cinematography, we timed every step of the sexual reproduction process from mate pairing to initial cell hatching. The present paper depicts cytological aspects during gametogenesis in P. multistriata, shedding light on the chloroplast behaviour during sexual reproduction, finely describing the timing of the sexual phases and providing reference data for further studies on the molecular control of this fundamental process.     

Huntingtin-associated Protein 1 (HAP1) Is a cGMP-dependent Kinase Anchoring Protein (GKAP) Specific for the cGMP-dependent Protein Kinase Iβ Isoform

Protein-protein interactions are important in providing compartmentalization and specificity in cellular signal transduction. Many studies have hallmarked the well designed compartmentalization of the cAMP-dependent protein kinase (PKA) through its anchoring proteins. Much less data are available on the compartmentalization of its closest homolog, cGMP-dependent protein kinase (PKG), via its own PKG anchoring proteins (GKAPs). For the enrichment, screening, and discovery of (novel) PKA anchoring proteins, a plethora of methodologies is available, including our previously described chemical proteomics approach based on immobilized cAMP or cGMP. Although this method was demonstrated to be effective, each immobilized cyclic nucleotide did not discriminate in the enrichment for either PKA or PKG and their secondary interactors. Hence, with PKG signaling components being less abundant in most tissues, it turned out to be challenging to enrich and identify GKAPs. Here we extend this cAMP-based chemical proteomics approach using competitive concentrations of free cyclic nucleotides to isolate each kinase and its secondary interactors. Using this approach, we identified Huntingtin-associated protein 1 (HAP1) as a putative novel GKAP. Through sequence alignment with known GKAPs and secondary structure prediction analysis, we defined a small sequence domain mediating the interaction with PKG Iβ but not PKG Iα. In vitro binding studies and site-directed mutagenesis further confirmed the specificity and affinity of HAP1 binding to the PKG Iβ N terminus. These data fully support that HAP1 is a GKAP, anchoring specifically to the cGMP-dependent protein kinase isoform Iβ, and provide further evidence that also PKG spatiotemporal signaling is largely controlled by anchoring proteins.     

Individual Differences in Scotopic Visual Acuity and Contrast Sensitivity: Genetic and Non-Genetic Influences

Despite the large amount of variation found in the night (scotopic) vision capabilities of healthy volunteers, little effort has been made to characterize this variation and factors, genetic and non-genetic, that influence it. In the largest population of healthy observers measured for scotopic visual acuity (VA) and contrast sensitivity (CS) to date, we quantified the effect of a range of variables on visual performance. We found that young volunteers with excellent photopic vision exhibit great variation in their scotopic VA and CS, and this variation is reliable from one testing session to the next. We additionally identified that factors such as Circadian preference, iris color, astigmatism, depression, sex and education have no significant impact on scotopic visual function. We confirmed previous work showing that the amount of time spent on the vision test influences performance and that laser eye surgery results in worse scotopic vision. We also showed a significant effect of intelligence and photopic visual performance on scotopic VA and CS, but all of these variables collectively explain <30% of the variation in scotopic vision. The wide variation seen in young healthy volunteers with excellent photopic vision, the high test-retest agreement, and the vast majority of the variation in scotopic vision remaining unexplained by obvious non-genetic factors suggests a strong genetic component. Our preliminary genome-wide association study (GWAS) of 106 participants ruled out any common genetic variants of very large effect and paves the way for future, larger genetic studies of scotopic vision.     

Targeting farnesoid X receptor for liver and metabolic disorders

The farnesoid X receptor (FXR) is a metabolic nuclear receptor expressed in the liver, intestine, kidney and adipose tissue. By regulating the expression and function of genes involved in bile acid (BA) synthesis, uptake and excretion, FXR has emerged as a key gene involved in the maintenance of cholesterol and BA homeostasis. FXR ligands are currently under clinical investigation for the treatment of cholestasis, dyslipidemic disorders and conditions of insulin resistance in type 2 diabetes and non-alcoholic steatohepatitis (NASH). Because activation of FXR impacts a considerable number of genes, development of FXR modulators that selectively regulate specific pathways will limit potentially undesirable side effects. Interaction of FXR with other BAs and xenobiotics sensors such as the constitutive androstane receptor and the pregnane X receptor might allow the development of combination therapies for liver and metabolic disorders.