Copper Supplementation at 8 mg Neither Affects Circulating Lipids nor Liver Function in Apparently Healthy Chilean Men

There is a close relationship between selenium deficiency and Kaschin–Beck disease (KBD). Although the etiology of KBD is not known and selenium deficiency is not its actual cause, it is an important environmental risk factor. In particular, in the Qing-Tibet Plateau, a selenium-deficient region, the prevalence of KBD is serious and still increasing and continues to damage public health. By providing selenium to the population in appropriate amounts, and especially to children, KBD can be effectively controlled and prevented.     

Analysis of the local organization and dynamics of cellular actin networks

A ctin filaments, with the aid of multiple accessory proteins, self-assemble into a variety of network patterns. We studied the organization and dynamics of the actin network in nonadhesive regions of cells bridging fibronectin-coated adhesive strips. The network was formed by actin nodes associated with and linked by myosin II and containing the formin disheveled-associated activator of morphogenesis 1 (DAAM1) and the cross-linker filamin A (FlnA). After Latrunculin A (LatA) addition, actin nodes appeared to be more prominent and demonstrated drift-diffusion motion. Superresolution microscopy revealed that, in untreated cells, DAAM1 formed patches with a similar spatial arrangement to the actin nodes. Node movement (diffusion coefficient and velocity) in LatA-treated cells was dependent on the level and activity of myosin IIA, DAAM1, and FlnA. Based on our results, we developed a computational model of the dynamic formin-filamin-actin asters that can self-organize into a contractile actomyosin network. We suggest that such networks are critical for connecting distant parts of the cell to maintain the mechanical coherence of the cytoplasm.     

Regulation of Phospholipase D Activity and Phosphatidic Acid Production after Purinergic (P2Y6) Receptor Stimulation

Phosphatidic acid (PA) is a lipid second messenger located at the intersection of several lipid metabolism and cell signaling events including membrane trafficking, survival, and proliferation. Generation of signaling PA has long been primarily attributed to the activation of phospholipase D (PLD). PLD catalyzes the hydrolysis of phosphatidylcholine into PA. A variety of both receptor-tyrosine kinase and G-protein-coupled receptor stimulations have been shown to lead to PLD activation and PA generation. This study focuses on profiling the PA pool upon P2Y₆ receptor signaling manipulation to determine the major PA producing enzymes. Here we show that PLD, although highly active, is not responsible for the majority of stable PA being produced upon UDP stimulation of the P2Y₆ receptor and that PA levels are tightly regulated. By following PA flux in the cell we show that PLD is involved in an initial increase in PA upon receptor stimulation; however, when PLD is blocked, the cell compensates by increasing PA production from other sources. We further delineate the P2Y₆ signaling pathway showing that phospholipase Cβ3 (PLCβ3), PLCδ1, DGKζ and PLD are all downstream of receptor activation. We also show that DGKζ is a novel negative regulator of PLD activity in this system that occurs through an inhibitory mechanism with PKCα. These results further define the downstream events resulting in PA production in the P2Y₆ receptor signaling pathway.     

Dynamics of Influenza Virus and Human Host Interactions During Infection and Replication Cycle

The replication and life cycle of the influenza virus is governed by an intricate network of intracellular regulatory events during infection, including interactions with an even more complex system of biochemical interactions of the host cell. Computational modeling and systems biology have been successfully employed to further the understanding of various biological systems, however, computational studies of the complexity of intracellular interactions during influenza infection is lacking. In this work, we present the first large-scale dynamical model of the infection and replication cycle of influenza, as well as some of its interactions with the host’s signaling machinery. Specifically, we focus on and visualize the dynamics of the internalization and endocytosis of the virus, replication and translation of its genomic components, as well as the assembly of progeny virions. Simulations and analyses of the models dynamics qualitatively reproduced numerous biological phenomena discovered in the laboratory. Finally, comparisons of the dynamics of existing and proposed drugs, our results suggest that a drug targeting PB1:PA would be more efficient than existing Amantadin/Rimantaine or Zanamivir/Oseltamivir.     

An Automated Live Imaging Platform for Studying Merozoite Egress-Invasion in Malaria Cultures

Most cases of severe and fatal malaria are caused by the intraerythrocytic asexual reproduction cycle of Plasmodium falciparum. One of the most intriguing and least understood stages in this cycle is the brief preinvasion period during which dynamic merozoite–red-cell interactions align the merozoite apex in preparation for penetration. Studies of the molecular mechanisms involved in this process face formidable technical challenges, requiring multiple observations of merozoite egress-invasion sequences in live cultures under controlled experimental conditions, using high-resolution microscopy and a variety of fluorescent imaging tools. Here we describe a first successful step in the development of a fully automated, robotic imaging platform to enable such studies. Schizont-enriched live cultures of P. falciparum were set up on an inverted stage microscope with software-controlled motorized functions. By applying a variety of imaging filters and selection criteria, we identified infected red cells that were likely to rupture imminently, and recorded their coordinates. We developed a video-image analysis to detect and automatically record merozoite egress events in 100% of the 40 egress-invasion sequences recorded in this study. We observed a substantial polymorphism of the dynamic condition of pre-egress infected cells, probably reflecting asynchronies in the diversity of confluent processes leading to merozoite release.     

EVOLUTION OF SEX DETERMINATION SYSTEMS WITH HETEROGAMETIC MALES AND FEMALES IN SILENE

The plant genus Silene has become a model for evolutionary studies of sex chromosomes and sex‐determining mechanisms. A recent study performed in Silene colpophylla showed that dioecy and the sex chromosomes in this species evolved independently from those in Silene latifolia, the most widely studied dioecious Silene species. The results of this study show that the sex‐determining system in Silene otites, a species related to S. colpophylla, is based on female heterogamety, a sex determination system that is unique among the Silene species studied to date. Our phylogenetic data support the placing of S. otites and S. colpophylla in the subsection Otites and the analysis of ancestral states suggests that the most recent common ancestor of S. otites and S. colpophylla was most probably dioecious. These observations imply that a switch from XX/XY sex determination to a ZZ/ZW system (or vice versa) occurred in the subsection Otites. This is the first report of two different types of heterogamety within one plant genus of this mostly nondioecious plant family.     

Evaluation of a D-amino-acid-containing fluorescence resonance energy transfer peptide library for profiling prokaryotic proteases

Bacterial proteases play an important role in a broad spectrum of processes, including colonization, proliferation, and virulence. In this respect, bacterial proteases are potential biomarkers for bacterial diagnosis and targets for novel therapeutic protease inhibitors. To investigate these potential functions, the authors designed and used a protease substrate fluorescence resonance energy transfer (FRET) library comprising 115 short d- and l-amino-acid-containing fluorogenic substrates as a tool to generate proteolytic profiles for a wide range of bacteria. Bacterial specificity of the d-amino acid substrates was confirmed using enzymes isolated from both eukaryotic and prokaryotic organisms. Interestingly, bacterial proteases that are known to be involved in housekeeping and nutrition, but not in virulence, were able to degrade substrates in which a d-amino acid was present. Using our FRET peptide library and culture supernatants from a total of 60 different bacterial species revealed novel, bacteria-specific, proteolytic profiles, although in-species variation was observed for Pseudomonas aeruginosa, Porphyromonas gingivalis, and Staphylococcus aureus. Overall, the specific characteristic of our substrate peptide library makes it a rapid tool to high-throughput screen for novel substrates to detect bacterial proteolytic activity.     

Expression profile and function of Wnt signaling mechanisms in malignant mesothelioma cells

Malignant mesothelioma (MM) is an uncommon and particularly aggressive cancer associated with asbestos exposure, which currently presents an intractable clinical challenge. Wnt signaling has been reported to play a role in the neoplastic properties of mesothelioma cells but has not been investigated in detail in this cancer. We surveyed expression of Wnts, their receptors, and other key molecules in this pathway in well established in vitro mesothelioma models in comparison with primary mesothelial cultures. We also tested the biological response of MM cell lines to exogenous Wnt and secreted regulators, as well as targeting β-catenin. We detected frequent expression of Wnt3 and Wnt5a, as well as Fzd 2, 4 and 6. The mRNA of Wnt4, Fzd3, sFRP4, APC and axin2 were downregulated in MM relative to mesothelial cells while LEF1 was overexpressed in MM. Functionally, we observed that Wnt3a stimulated MM proliferation while sFRP4 was inhibitory. Furthermore, directly targeting β-catenin expression could sensitise MM cells to cytotoxic drugs. These results provide evidence for altered expression of a number of Wnt/Fzd signaling molecules in MM. Modulation of Wnt signaling in MM may prove a means of targeting proliferation and drug resistance in this cancer.     

Laboratory and microcosm experiments testing the toxicity of chlorinated hydrocarbons on a cyanobacterium strain (Synechococcus PCC 6301) and on natural phytoplankton assemblages

In the last few years, halogenated hydrocarbons have been detected in the soil, in the aquatic environment, in organisms, and even in drinking water. The toxic effects of three chlorinated aliphatic hydrocarbons (trichloroethylene, tetrachloroethylene and tetrachloroethane) were studied in laboratory experiments (using the cyanobacterium Synecococcus elongatus PCC 6301 as test organism) and in field-like circumstances (natural phytoplankton assemblages enclosed in microcosms). The results of the laboratory experiments showed that all of the tested compounds significantly inhibited the growth of the cultures within the first 4 h. Enzymatic changes of the treated cultures suggested that oxidative stress occured—all of the three compounds caused an increase in the activity of peroxidases and superoxide dismutase, and also increased the levels of lipid peroxidation. Observed changes in microcosms were comparable with the results of the laboratory experiments: the number of individuals and chlorophyll contents decreased in the treated assemblages. The elevated levels of peroxidation on the second day in the assemblages treated with tetrachloroethane and tetrachloroetylene suggest that oxidative stress could occur in field conditions. One of the most important findings is the decrease in species number. Our results showed that cryptomonads, some green algae species and the cyanobacterium Limnothrix gradually disappeared from the treated beakers during the experiment.