The Complex and Important Cellular and Metabolic Functions of Saturated Fatty Acids

This review summarizes recent findings on the metabolism and biological functions of saturated fatty acids (SFA). Some of these findings show that SFA may have important and specific roles in the cells. Elucidated biochemical mechanisms like protein acylation (N-myristoylation, S-palmitoylation) and regulation of gene transcription are presented. In terms of physiology, SFA are involved for instance in lipogenesis, fat deposition, polyunsaturated fatty acids bioavailability and apoptosis. The variety of their functions demonstrates that SFA should no longer be considered as a single group

Evaluation of multi-exponential curve fitting analysis of oxygen-quenched phosphorescence decay traces for recovering microvascular oxygen tension histograms

Although it is generally accepted that oxygen-quenched phosphorescence decay traces can be analyzed using the exponential series method (ESM), its application until now has been limited to a few (patho)physiological studies, probably because the reliability of the recovered oxygen tension (pO2) histograms has never been extensively evaluated and lacks documentation. The aim of this study was, therefore, to evaluate the use of the ESM to adequately determine pO2 histograms from phosphorescence decay traces. For this purpose we simulated decay traces corresponding to uni- and bimodal pO2 distributions and recovered the pO2 histograms at different signal-to-noise ratios (SNRs). Ultimately, we recovered microvascular pO2 histograms measured in the rat kidney in a model of endotoxemic shock and fluid resuscitation and showed that the mean microvascular oxygen tension, 〈pO2〉, decreased after induction of endotoxemia and that after 2 h of fluid resuscitation, 〈pO2〉 remained low, but the hypoxic peak that had arisen during endotoxemia was reduced. This finding illustrates the importance of recovering pO2 histograms under (patho)physiological conditions. In conclusion, this study has characterized how noise affects the recovery of pO2 histograms using the ESM and documented the reliability of the ESM for recovering both low- and high-pO2 distributions for SNRs typically found in experiments. This study might therefore serve as a frame of reference for investigations focused on oxygen (re)distribution during health and disease and encourage researchers to (re-)analyze data obtained in (earlier) studies possibly revealing new insights into complex disease states and treatment strategies

Comparing methods to estimate treatment effects on a continuous outcome in multicentre randomized controlled trials: A simulation study

<p>Abstract</p> <p>Background</p> <p>Multicentre randomized controlled trials (RCTs) routinely use randomization and analysis stratified by centre to control for differences between centres and to improve precision. No consensus has been reached on how to best analyze correlated continuous outcomes in such settings. Our objective was to investigate the properties of commonly used statistical models at various levels of clustering in the context of multicentre RCTs.</p> <p>Methods</p> <p>Assuming no treatment by centre interaction, we compared six methods (ignoring centre effects, including centres as fixed effects, including centres as random effects, generalized estimating equation (GEE), and fixed- and random-effects centre-level analysis) to analyze continuous outcomes in multicentre RCTs using simulations over a wide spectrum of intraclass correlation (ICC) values, and varying numbers of centres and centre size. The performance of models was evaluated in terms of bias, precision, mean squared error of the point estimator of treatment effect, empirical coverage of the 95% confidence interval, and statistical power of the procedure.</p> <p>Results</p> <p>While all methods yielded unbiased estimates of treatment effect, ignoring centres led to inflation of standard error and loss of statistical power when within centre correlation was present. Mixed-effects model was most efficient and attained nominal coverage of 95% and 90% power in almost all scenarios. Fixed-effects model was less precise when the number of centres was large and treatment allocation was subject to chance imbalance within centre. GEE approach underestimated standard error of the treatment effect when the number of centres was small. The two centre-level models led to more variable point estimates and relatively low interval coverage or statistical power depending on whether or not heterogeneity of treatment contrasts was considered in the analysis.</p> <p>Conclusions</p> <p>All six models produced unbiased estimates of treatment effect in the context of multicentre trials. Adjusting for centre as a random intercept led to the most efficient treatment effect estimation across all simulations under the normality assumption, when there was no treatment by centre interaction.</p

Gene Expression Changes Associated with the Airway Wall Response to Injury

Understanding the way in which the airway heals in response to injury is fundamental to dissecting the mechanisms underlying airway disease pathology. As only limited data is available in relation to the in vivo characterisation of the molecular features of repair in the airway we sought to characterise the dynamic changes in gene expression that are associated with the early response to physical injury in the airway wall.We profiled gene expression changes in the airway wall using a large animal model of physical injury comprising bronchial brush biopsy in anaesthetised sheep. The experimental design featured sequential studies in the same animals over the course of a week and yielded data relating to the response at 6 hours, and 1, 3 and 7 days after injury. Notable features of the transcriptional response included the early and sustained preponderance of down-regulated genes associated with angiogenesis and immune cell activation, selection and differentiation. Later features of the response included the up-regulation of cell cycle genes at d1 and d3, and the latter pronounced up-regulation of extracellular matrix-related genes at d3 and d7.It is possible to follow the airway wall response to physical injury in the same animal over the course of time. Transcriptional changes featured coordinate expression of functionally related genes in a reproducible manner both within and between animals. This characterisation will provide a foundation against which to assess the perturbations that accompany airway disease pathologies of comparative relevance

A Topical Microbicide Gel Formulation of CCR5 Antagonist Maraviroc Prevents HIV-1 Vaginal Transmission in Humanized RAG-hu Mice

For prevention of HIV infection many currently licensed anti-HIV drugs and new ones in the pipeline show potential as topically applied microbicides. While macaque models have been the gold standard for in vivo microbicide testing, they are expensive and sufficient numbers are not available. Therefore, a small animal model that facilitates rapid evaluation of potential candidates for their preliminary efficacy is urgently needed in the microbicide field. We previously demonstrated that RAG-hu humanized mouse model permits HIV-1 mucosal transmission via both vaginal and rectal routes and that oral pre-exposure chemo-prophylactic strategies could be tested in this system. Here in these proof-of-concept studies, we extended this system for topical microbicide testing using HIV-1 as the challenge virus. Maraviroc, a clinically approved CCR5 inhibitor drug for HIV treatment, was formulated as a microbicide gel at 5 mM concentration in 2.2% hydroxyl ethyl cellulose. Female RAG-hu mice were challenged vaginally with HIV-1 an hour after intravaginal application of the maraviroc gel. Our results showed that maraviroc gel treated mice were fully protected against vaginal HIV-1 challenge in contrast to placebo gel treated mice which all became infected. These findings highlight the utility of the humanized mouse models for microbicide testing and, together with the recent data from macaque studies, suggest that maraviroc is a promising candidate for future microbicide clinical trials in the field

Genes Selectively Up-Regulated by Pheromone in White Cells Are Involved in Biofilm Formation in Candida albicans

To mate, MTL-homozygous strains of the yeast pathogen Candida albicans must switch from the white to opaque phase. Mating-competent opaque cells then release pheromone that induces polarization, a G1 block and conjugation tube formation in opaque cells of opposite mating type. Pheromone also induces mating-incompetent white cells to become adhesive and cohesive, and form thicker biofilms that facilitate mating. The pheromone response pathway of white cells shares the upstream components of that of opaque cells, but targets a different transcription factor. Here we demonstrate that the genes up-regulated by the pheromone in white cells are activated through a common cis-acting sequence, WPRE, which is distinct from the cis-acting sequence, OPRE, responsible for up-regulation in opaque cells. Furthermore, we find that these white-specific genes play roles in white cell biofilm formation, and are essential for biofilm formation in the absence of an added source of pheromone, suggesting either an autocrine or pheromone-independent mechanism. These results suggest an intimate, complex and unique relationship between switching, mating and MTL-homozygous white cell biofilm formation, the latter a presumed virulence factor in C. albicans

Apoptosis-like cell death in Leishmania donovani treated with KalsomeTM10, a new liposomal amphotericin B

The present study aimed to elucidate the cell death mechanism in Leishmania donovani upon treatment with KalsomeTM10, a new liposomal amphotericin B. Methodology/Principal findings We studied morphological alterations in promastigotes through phase contrast and scanning electron microscopy. Phosphatidylserine (PS) exposure, loss of mitochondrial membrane potential and disruption of mitochondrial integrity was determined by flow cytometry using annexinV-FITC, JC-1 and mitotraker, respectively. For analysing oxidative stress, generation of H2O2 (bioluminescence kit) and mitochondrial superoxide O2 − (mitosox) were measured. DNA fragmentation was evaluated using terminal deoxyribonucleotidyl transferase mediated dUTP nick-end labelling (TUNEL) and DNA laddering assay. We found that KalsomeTM10 is more effective then Ambisome against the promastigote as well as intracellular amastigote forms. The mechanistic study showed that KalsomeTM10 induced several morphological alterations in promastigotes typical of apoptosis. KalsomeTM10 treatment showed a dose- and time-dependent exposure of PS in promastigotes. Further,study on mitochondrial pathway revealed loss of mitochondrial membrane potential as well as disruption in mitochondrial integrity with depletion of intracellular pool of ATP. KalsomeTM10 treated promastigotes showed increased ROS production, diminished GSH levels and increased caspase-like activity. DNA fragmentation and cell cycle arrest was observed in KalsomeTM10 treated promastigotes. Apoptotic DNA fragmentation was also observed in KalsomeTM10 treated intracellular amastigotes. KalsomeTM10 induced generation of ROS and nitric oxide leads to the killing of the intracellular parasites. Moreover, endocytosis is indispensable for KalsomeTM10 mediated anti-leishmanial effect in host macrophag

Glycation marker glucosepane increases with the progression of osteoarthritis and correlates with morphological and functional changes of cartilage in vivo

Background: Changes of serum concentrations of glycated, oxidized, and nitrated amino acids and hydroxyproline and anticyclic citrullinated peptide antibody status combined by machine learning techniques in algorithms have recently been found to provide improved diagnosis and typing of early-stage arthritis of the knee, including osteoarthritis (OA), in patients. The association of glycated, oxidized, and nitrated amino acids released from the joint with development and progression of knee OA is unknown. We studied this in an OA animal model as well as interleukin-1β-activated human chondrocytes in vitro and translated key findings to patients with OA. Methods: Sixty male 3-week-old Dunkin-Hartley guinea pigs were studied. Separate groups of 12 animals were killed at age 4, 12, 20, 28 and 36 weeks, and histological severity of knee OA was evaluated, and cartilage rheological properties were assessed. Human chondrocytes cultured in multilayers were treated for 10 days with interleukin-1β. Human patients with early and advanced OA and healthy controls were recruited, blood samples were collected, and serum or plasma was prepared. Serum, plasma, and culture medium were analyzed for glycated, oxidized, and nitrated amino acids. Results: Severity of OA increased progressively in guinea pigs with age. Glycated, oxidized, and nitrated amino acids were increased markedly at week 36, with glucosepane and dityrosine increasing progressively from weeks 20 and 28, respectively. Glucosepane correlated positively with OA histological severity (r = 0.58, p < 0.0001) and instantaneous modulus (r = 0.52–0.56; p < 0.0001), oxidation free adducts correlated positively with OA severity (p < 0.0009–0.0062), and hydroxyproline correlated positively with cartilage thickness (p < 0.0003–0.003). Interleukin-1β increased the release of glycated and nitrated amino acids from chondrocytes in vitro. In clinical translation, plasma glucosepane was increased 38% in early-stage OA (p < 0.05) and sixfold in patients with advanced OA (p < 0.001) compared with healthy controls. Conclusions: These studies further advance the prospective role of glycated, oxidized, and nitrated amino acids as serum biomarkers in diagnostic algorithms for early-stage detection of OA and other arthritic disease. Plasma glucosepane, reported here for the first time to our knowledge, may improve early-stage diagnosis and progression of clinical OA

Aerosols in the Pre-industrial Atmosphere

Purpose of Review: We assess the current understanding of the state and behaviour of aerosols under pre-industrial conditions and the importance for climate. Recent Findings: Studies show that the magnitude of anthropogenic aerosol radiative forcing over the industrial period calculated by climate models is strongly affected by the abundance and properties of aerosols in the pre-industrial atmosphere. The low concentration of aerosol particles under relatively pristine conditions means that global mean cloud albedo may have been twice as sensitive to changes in natural aerosol emissions under pre-industrial conditions compared to present-day conditions. Consequently, the discovery of new aerosol formation processes and revisions to aerosol emissions have large effects on simulated historical aerosol radiative forcing. Summary: We review what is known about the microphysical, chemical, and radiative properties of aerosols in the pre-industrial atmosphere and the processes that control them. Aerosol properties were controlled by a combination of natural emissions, modification of the natural emissions by human activities such as land-use change, and anthropogenic emissions from biofuel combustion and early industrial processes. Although aerosol concentrations were lower in the pre-industrial atmosphere than today, model simulations show that relatively high aerosol concentrations could have been maintained over continental regions due to biogenically controlled new particle formation and wildfires. Despite the importance of pre-industrial aerosols for historical climate change, the relevant processes and emissions are given relatively little consideration in climate models, and there have been very few attempts to evaluate them. Consequently, we have very low confidence in the ability of models to simulate the aerosol conditions that form the baseline for historical climate simulations. Nevertheless, it is clear that the 1850s should be regarded as an early industrial reference period, and the aerosol forcing calculated from this period is smaller than the forcing since 1750. Improvements in historical reconstructions of natural and early anthropogenic emissions, exploitation of new Earth system models, and a deeper understanding and evaluation of the controlling processes are key aspects to reducing uncertainties in future

Low Dosage of Histone H4 Leads to Growth Defects and Morphological Changes in Candida albicans

Chromatin function depends on adequate histone stoichiometry. Alterations in histone dosage affect transcription and chromosome segregation, leading to growth defects and aneuploidies. In the fungal pathogen Candida albicans, aneuploidy formation is associated with antifungal resistance and pathogenesis. Histone modifying enzymes and chromatin remodeling proteins are also required for pathogenesis. However, little is known about the mechanisms that generate aneuploidies or about the epigenetic mechanisms that shape the response of C. albicans to the host environment. Here, we determined the impact of histone H4 deficit in the growth and colony morphology of C. albicans. We found that C. albicans requires at least two of the four alleles that code for histone H4 (HHF1 and HHF22) to grow normally. Strains with only one histone H4 allele show a severe growth defect and unstable colony morphology, and produce faster-growing, morphologically stable suppressors. Segmental or whole chromosomal trisomies that increased wild-type histone H4 copy number were the preferred mechanism of suppression. This is the first study of a core nucleosomal histone in C. albicans, and constitutes the prelude to future, more detailed research on the function of histone H4 in this important fungal pathogen