TLR-3 is Present in Human Adipocytes,but Its Signalling is Not Required for Obesity-Induced Inflammation in Adipose Tissue In Vivo

Innate immunity plays a pivotal role in obesity-induced low-grade inflammation originating from adipose tissue. Key receptors of the innate immune system including Toll-like receptors-2 and -4 (TLRs) are triggered by nutrient excess to promote inflammation. The role of other TLRs in this process is largely unknown. In addition to double-stranded viral mRNA, TLR-3 can also recognize mRNA from dying endogenous cells, a process that is frequently observed within obese adipose tissue. Here, we identified profound expression of TLR-3 in adipocytes and investigated its role during diet-induced obesity. Human adipose tissue biopsies (n=80) and an adipocyte cell-line were used to study TLR-3 expression and function. TLR-3-/- and WT animals were exposed to a high-fat diet (HFD) for 16 weeks to induce obesity. Expression of TLR-3 was significantly higher in human adipocytes compared to the non-adipocyte cells part of the adipose tissue. In vitro, TLR-3 expression was induced during differentiation of adipocytes and stimulation of the receptor led to elevated expression of pro-inflammatory cytokines. In vivo, TLR-3 deficiency did not significantly influence HFD-induced obesity, insulin sensitivity or inflammation. In humans, TLR-3 expression in adipose tissue did not correlate with BMI or insulin sensitivity (HOMA-IR). Together, our results demonstrate that TLR-3 is highly expressed in adipocytes and functionally active. However, TLR-3 appears to play a redundant role in obesity-induced inflammation and insulin resistance.     

Aspergillus enzymes involved in degradation of plant cell wall polysaccharides.

Degradation of plant cell wall polysaccharides is of major importance in the food and feed, beverage, textile, and paper and pulp industries, as well as in several other industrial production processes. Enzymatic degradation of these polymers has received attention for many years and is becoming a more and more attractive alternative to chemical and mechanical processes. Over the past 15 years, much progress has been made in elucidating the structural characteristics of these polysaccharides and in characterizing the enzymes involved in their degradation and the genes of biotechnologically relevant microorganisms encoding these enzymes. The members of the fungal genus Aspergillus are commonly used for the production of polysaccharide-degrading enzymes. This genus produces a wide spectrum of cell wall-degrading enzymes, allowing not only complete degradation of the polysaccharides but also tailored modifications by using specific enzymes purified from these fungi. This review summarizes our current knowledge of the cell wall polysaccharide-degrading enzymes from aspergilli and the genes by which they are encoded.     

A Novel Method for Assessment of Natural Killer Cell Cytotoxicity Using Image Cytometry

Natural killer (NK) cells belong to the innate arm of the immune system and though activated NK cells can modulate immune responses through the secretion of cytokines, their primary effector function is through target cell lysis. Accordingly, cytotoxicity assays are central to studying NK cell function. The ⁵¹Chromium release assay, is the “gold standard” for cytotoxicity assay, however, due to concerns over toxicity associated with the use and disposal of radioactive compounds there is a significant interest in non-radioactive methods. We have previously used the calcein release assay as a non-radioactive alternative for studying NK cell cytotoxicity. In this study, we show that the calcein release assay varies in its dynamic range for different tumor targets, and that the entrapped calcein could remain unreleased within apoptotic bodies of lysed tumor targets or incompletely released resulting in underestimation of percent specific lysis. To overcome these limitations, we developed a novel cytotoxicity assay using the Cellometer Vision Image Cytometer and compared this method to standard calcein release assay for measuring NK cell cytotoxicity. Using tumor lines K562, 721.221, and Jurkat, we demonstrate here that image cytometry shows significantly higher percent specific lysis of the target cells compared to the standard calcein release assay within the same experimental setup. Image cytometry is able to accurately analyze live target cells by excluding dimmer cells and smaller apoptotic bodies from viable target cell counts. The image cytometry-based cytotoxicity assay is a simple, direct and sensitive method and is an appealing option for routine cytotoxicity assay.     

Predictive Spatiotemporal Manipulation of Signaling Perturbations Using Optogenetics

Recently developed optogenetic methods promise to revolutionize cell biology by allowing signaling perturbations to be controlled in space and time with light. However, a quantitative analysis of the relationship between a custom-defined illumination pattern and the resulting signaling perturbation is lacking. Here, we characterize the biophysical processes governing the localized recruitment of the Cryptochrome CRY2 to its membrane-anchored CIBN partner. We develop a quantitative framework and present simple procedures that enable predictive manipulation of protein distributions on the plasma membrane with a spatial resolution of 5 μm. We show that protein gradients of desired levels can be established in a few tens of seconds and then steadily maintained. These protein gradients can be entirely relocalized in a few minutes. We apply our approach to the control of the Cdc42 Rho GTPase activity. By inducing strong localized signaling perturbation, we are able to monitor the initiation of cell polarity and migration with a remarkable reproducibility despite cell-to-cell variability.     

Variability of mouth width in European eel,Anguilla anguilla,in relation to varying feeding conditions in three Dutch lakes

Synopsis Mouth widths of eels in three Dutch lakes were compared relative to feeding conditions over two years. Average mouth width varied by season and year and per lake. Of two groups of feeding specialists distinguished in each population chironomid feeders showed a smaller mouth width compared to fish feeders of the same body length. Proportions of these specialists differed between years, lakes, and seasons and changed in response to changing feeding conditions. The shift to fish feeding occurred when chironomid biomass decreased and young fish recruited. The shift was greatest in the year when chironomids collapsed completely and in the lakes with high eel densities. Mature males were narrow-headed and left the lakes at sizes between 30 and 40 cm. Larger eels were always broad-headed females. Implications for foraging efficiency and population structure are discussed.     

Effects of the fertility transition on birth seasonality in the Netherlands

Synchronous with the decline in fertility that took place in the post-war period in the Netherlands, patterns of birth seasonality changed as well. In this paper seasonal fluctuations in fertility in the Netherlands are examined using population register data for the period 1952 to 2005. The peak in births has changed from spring to summer and subsequently to August/September, thereby shifting from the European to the American pattern. The seasonal shift can be attributed to parity-specific changes. Before the transition, birth seasonality did not differ much between the different parities. In the transition period from higher to low fertility, differences between parities increased which persist up to today. At present, the overall seasonality pattern is determined by first births. Moreover, birth seasonality varies by maternal age. The findings stimulate the discussion on the role of planning as a cause of birth seasonality.     

Structure and kinetic characterization of human sperm-specific glyceraldehyde-3-phosphate dehydrogenase, GAPDS

hGAPDS (human sperm-specific glyceraldehyde-3-phosphate dehydrogenase) is a glycolytic enzyme essential for the survival of spermatozoa, and constitutes a potential target for non-hormonal contraception. However, enzyme characterization of GAPDS has been hampered by the difficulty in producing soluble recombinant protein. In the present study, we have overexpressed in Escherichia coli a highly soluble form of hGAPDS truncated at the N-terminus (hGAPDSΔN), and crystallized the homotetrameric enzyme in two ligand complexes. The hGAPDSΔN-NAD+-phosphate structure maps the two anion-recognition sites within the catalytic pocket that correspond to the conserved Ps site and the newly recognized Pi site identified in other organisms. The hGAPDSΔN-NAD+-glycerol structure shows serendipitous binding of glycerol at the Ps and new Pi sites, demonstrating the propensity of these anion-recognition sites to bind non-physiologically relevant ligands. A comparison of kinetic profiles between hGAPDSΔN and its somatic equivalent reveals a 3-fold increase in catalytic efficiency for hGAPDSΔN. This may be attributable to subtle amino acid substitutions peripheral to the active centre that influence the charge properties and protonation states of catalytic residues. Our data therefore elucidate structural and kinetic features of hGAPDS that might provide insightful information towards inhibitor development.     

The diagnosis of the sex chromosomes in human tissues

Summary The nuclei in the skin, and in the neutrophils, have been studied in men and women, in relation to a diagnosis of the sex chromosomes in non-dividing nuclei.It has been shown in the skin, that the appearance of chromocenters, which are presumably formed by the X and Y, can vary according to metabolic conditions, but that a determination of the percent of nuclei with different numbers of chromocenters and nucleoli in the young and old spinous cells, gives a characteristic distribution of nuclear types in each of the two sexes.Since such a determination includes cells with individual X and Y chromocenters, it should be possible to detect by this method not only cases that are XX or XY, but also cases with abnormal sex chromosome constitutions.     

Protein-responsive ribozyme switches in eukaryotic cells

Genetic devices that directly detect and respond to intracellular concentrations of proteins are important synthetic biology tools, supporting the design of biological systems that target, respond to or alter specific cellular states. Here, we develop ribozyme-based devices that respond to protein ligands in two eukaryotic hosts, yeast and mammalian cells, to regulate the expression of a gene of interest. Our devices allow for both gene-ON and gene-OFF response upon sensing the protein ligand. As part of our design process, we describe an in vitro characterization pipeline for prescreening device designs to identify promising candidates for in vivo testing. The in vivo gene-regulatory activities in the two types of eukaryotic cells correlate with in vitro cleavage activities determined at different physiologically relevant magnesium concentrations. Finally, localization studies with the ligand demonstrate that ribozyme switches respond to ligands present in the nucleus and/or cytoplasm, providing new insight into their mechanism of action. By extending the sensing capabilities of this important class of gene-regulatory device, our work supports the implementation of ribozyme-based devices in applications requiring the detection of protein biomarkers.