Antifungal chemical compounds identified using a C. elegans pathogenicity assay

There is an urgent need for the development of new antifungal agents. A facile in vivo model that evaluates libraries of chemical compounds could solve some of the main obstacles in current antifungal discovery. We show that Candida albicans, as well as other Candida species, are ingested by Caenorhabditis elegans and establish a persistent lethal infection in the C. elegans intestinal track. Importantly, key components of Candida pathogenesis in mammals, such as filament formation, are also involved in nematode killing. We devised a Candida-mediated C. elegans assay that allows high-throughput in vivo screening of chemical libraries for antifungal activities, while synchronously screening against toxic compounds. The assay is performed in liquid media using standard 96-well plate technology and allows the study of C. albicans in non-planktonic form. A screen of 1,266 compounds with known pharmaceutical activities identified 15 (approximately 1.2%) that prolonged survival of C. albicans-infected nematodes and inhibited in vivo filamentation of C. albicans. Two compounds identified in the screen, caffeic acid phenethyl ester, a major active component of honeybee propolis, and the fluoroquinolone agent enoxacin exhibited antifungal activity in a murine model of candidiasis. The whole-animal C. elegans assay may help to study the molecular basis of C. albicans pathogenesis and identify antifungal compounds that most likely would not be identified by in vitro screens that target fungal growth. Compounds identified in the screen that affect the virulence of Candida in vivo can potentially be used as "probe compounds" and may have antifungal activity against other fungi.     

Weight loss on the web: A pilot study comparing a structured behavioral intervention to a commercial program

Objective: Internet weight loss programs have become widely available as alternatives to standard treatment, but few data are available on their efficacy. This study aimed to investigate the effectiveness of a structured behavioral weight loss website (VTrim) vs. a commercial weight loss website ( eDiets.com ). Research Methods and Procedures: A randomized, controlled trial was conducted from February 2003 to March 2005, in 124 overweight and obese subjects ages 18 years and older with a BMI of 25 to 39.9 kg/m² (mean age, 47 ± 9 years; BMI, 32 ± 3 kg/m²; 20% men). Analyses were performed for the 88 subjects who had complete follow‐up data. Participants were randomly assigned to 12‐month VTrim (n = 62) or eDiets.com (n = 62) intervention. VTrim participants had access to a therapist‐led structured behavioral weight loss program delivered on‐line. eDiets.com subjects had access to a self‐help commercial on‐line weight loss program. Body weight, social support, and use of website components were measured at 0, 6, and 12 months. Results: Repeated‐measures analyses showed that the VTrim group lost significantly more weight than the eDiets.com group at 6 months (8.3 ± 7.9 kg vs. 4.1 ± 6.2 kg; p = 0.004) and maintained a greater loss at 12 months (7.8 ± 7.5 kg vs. 3.4 ± 5.8 kg; p = 0.002). More participants in the VTrim group maintained a 5% weight loss goal (65% vs. 37.5%; p = 0.01) at 12 months. Discussion: An on‐line, therapist‐led structured behavioral weight loss website produced greater weight loss than a self‐help commercial website. Because commercial sites have great potential public health impact, future research should investigate the feasibility of incorporating a more structured behavioral program into a commercial application.     

Amyotrophic lateral sclerosis: an emerging era of collaborative gene discovery

Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease (MND). It is currently incurable and treatment is largely limited to supportive care. Family history is associated with an increased risk of ALS, and many Mendelian causes have been discovered. However, most forms of the disease are not obviously familial. Recent advances in human genetics have enabled genome-wide analyses of single nucleotide polymorphisms (SNPs) that make it possible to study complex genetic contributions to human disease. Genome-wide SNP analyses require a large sample size and thus depend upon collaborative efforts to collect and manage the biological samples and corresponding data. Public availability of biological samples (such as DNA), phenotypic and genotypic data further enhances research endeavors. Here we discuss a large collaboration among academic investigators, government, and non-government organizations which has created a public repository of human DNA, immortalized cell lines, and clinical data to further gene discovery in ALS. This resource currently maintains samples and associated phenotypic data from 2332 MND subjects and 4692 controls. This resource should facilitate genetic discoveries which we anticipate will ultimately provide a better understanding of the biological mechanisms of neurodegeneration in ALS.     

Effects of experimental harvesting of an invasive hybrid cattail on wetland structure and function

Invasive plants, such as the hybrid cattail Typha × glauca, can reduce biodiversity and alter the ability of wetlands to provide critical ecosystem services, including nutrient cycling and carbon storage. Several approaches have been used to reduce Typha dominance and restore invaded wetlands, but long‐term studies assessing benefits of these restoration efforts are limited. A previous study demonstrated that aboveground harvesting of Typha × glauca stems and litter reduced Typha dominance 2 years post‐treatment in a Great Lakes coastal wetland. In the current study, we extended monitoring of experimental aboveground Typha harvest to 4 years post‐treatment and added assessments of treatment effects on soil nutrients, carbon emissions, and microbial community composition. Aboveground harvest treatment resulted in a dramatic reduction in Typha litter cover that persisted for 4 years, increased soil temperature, and increased abundance of the native plant genus Carex. However, aboveground harvest treatment did not significantly reduce Typha abundance, nor did it have significant effects on soil nutrient concentrations, carbon fluxes, or the taxonomic composition of soil microbial communities. We did observe differences in bacterial community composition between plots based on time since Typha invasion, which may indicate some legacy effects of Typha invasion. At the scale of this experiment (4 × 4 m plots), our results indicate that a single aboveground removal of Typha × glauca is not sufficient to restore a heavily invaded freshwater wetland ecosystem, and that periodic harvesting of Typha stems and litter may be required to maintain native plant abundance.     

Enzyme-mediated hydrogel encapsulation of single cells for high-throughput screening and directed evolution of oxidoreductases

Directed evolution of oxidoreductases to improve their catalytic properties is being ardently pursued in the industrial, biotechnological, and biopharma sectors. Hampering this pursuit are current enzyme screening methods that are limited in terms of throughput, cost, time, and complexity. We present a directed evolution strategy that allows for large-scale one-pot screening of glucose oxidase (GOx) enzyme libraries in well-mixed homogeneous solution. We used GOx variants displayed on the outer cell wall of yeasts to initiate a cascade reaction with horseradish peroxidase (HRP), resulting in peroxidase-mediated phenol cross-coupling and encapsulation of individual cells in well-defined fluorescent alginate hydrogel shells within ~10 min in mixed cell suspensions. Following application of denaturing stress to whole-cell GOx libraries, only cells displaying GOx variants with enhanced stability or catalytic activity were able to carry out the hydrogel encapsulation reaction. Fluorescence-activated cell sorting was then used to isolate the enhanced variants. We characterized three of the newly evolved Aspergillus niger GOx enzyme sequences and found up to ~5-fold higher specific activity, enhanced thermal stability, and differentiable glycosylation patterns. By coupling intracellular gene expression with the rapid formation of an extracellular hydrogel capsule, our system improves high-throughput screening for directed evolution of H ₂O ₂-producing enzymes many folds.     

A test of an antipredatory function of conspicuous plastron coloration in hatchling turtles

Bright colorations in animals are sometimes an antipredatory signal meant to startle, warn, or deter a predator from consuming a prey organism. Freshwater turtle hatchlings of many species have bright ventral coloration with high internal contrast that may have an antipredator function. We used visual modeling and field experiments to test whether the plastron coloration of Chrysemys picta hatchlings deters predators. We found that bird predators can easily distinguish hatchling turtles from their backgrounds and can easily see color contrast within the plastron. Raccoons cannot easily discriminate within-plastron color contrast but can see hatchlings against common backgrounds. Despite this, we found that brightly-colored, high contrast, replica turtles were not attacked less than low contrast replica turtles, suggesting that the bright coloration is not likely to serve an antipredatory function in this context. We discuss the apparent lack of innate avoidance of orange coloration in freshwater turtles by predators and suggest that preference and avoidance of colors are context-dependent. Since the bright colors are likely not a signal, we hypothesize that the colors may be caused by pigments deposited in tissue from maternal reserves during development. In most species, these pigments fade ontogenetically but they may have important physiological functions in species that maintain the bright coloration throughout adulthood.     

Rodent and germplasm trafficking: risks of microbial contamination in a high-tech biomedical world

High-tech biomedical advances have led to increases both in the number of mice used for research and in exchanges of mice and/or their tissues between institutions. The latter are associated with the risk of dissemination of infectious agents. Because of the lack of international standardization of health surveillance programs, health certificates for imported rodents may be informative but may not address the needs of the importing facility. Preservation of mouse germplasm is achieved by cryopreservation of spermatozoa, embryos, or ovaries, and embryonic stem cells are used for the production of genetically engineered mice. After embryo transfer, recipients and rederived pups that test negative in microbiological screening for relevant microorganisms are released into full barrier holding areas. However, current research shows that embryos may also transmit microorganisms, especially viruses, to the recipient mice. In this article, we discuss regulations and practical issues in the shipping of live mice and mouse tissues, including spermatozoa, embryos, ovaries, and embryonic stem cells, and review work on microbial contamination of these biological materials. In addition, we present ways to reduce the risk of transmission of pathogens to mice under routine conditions.     

DJ‐1 deficiency impairs autophagy and reduces alpha‐synuclein phagocytosis by microglia

Parkinson's disease (PD) is a progressive neurodegenerative disorder, of which 1% of the hereditary cases are linked to mutations in DJ‐1, an oxidative stress sensor. The pathological hallmark of PD is intercellular inclusions termed Lewy Bodies, composed mainly of α‐Synuclein (α‐Syn) protein. Recent findings have shown that α‐Syn can be transmitted from cell to cell, suggesting an important role of microglia, as the main scavenger cells of the brain, in clearing α‐Syn. We previously reported that the knock down (KD) of DJ‐1 in microglia increased cells’ neurotoxicity to dopaminergic neurons. Here, we discovered that α‐Syn significantly induced elevated secretion of the proinflammatory cytokines IL‐6 and IL‐1β and a significant dose‐dependent elevation in the production of nitric oxide in DJ‐1 KD microglia, compared to control microglia. We further investigated the ability of DJ‐1 KD microglia to uptake and degrade soluble α‐Syn, and discovered that DJ‐1 KD reduces cell‐surface lipid raft expression in microglia and impairs their ability to uptake soluble α‐Syn. Autophagy is an important mechanism for degradation of intracellular proteins and organelles. We discovered that DJ‐1 KD microglia exhibit an impaired autophagy‐dependent degradation of p62 and LC3 proteins, and that manipulation of autophagy had less effect on α‐Syn uptake and clearance in DJ‐1 KD microglia, compared to control microglia. Further studies of the link between DJ‐1, α‐Syn uptake and autophagy may provide useful insights into the role of microglia in the etiology of the PD.

     

Comparative genomics: tracking chromosome evolution in the family ursidae using reciprocal chromosome painting

The Ursidae family includes eight species, the karyotype of which diverges somewhat, in both chromosome number and morphology, from that of other families in the order Carnivora. The combination of consensus molecular phylogeny and high-resolution trypsin G-banded karyotype analysis has suggested that ancestral chromosomal fissions and at least two fusion events are associated with the development of the different ursid species. Here, we revisit this hypothesis by hybridizing reciprocal chromosome painting probes derived from the giant panda (Ailuropoda melanoleuca), domestic cat (Felis catus), and man (Homo sapiens) to representative bear species karyotypes. Comparative analysis of the different chromosome segment homologies allowed reconstruction of the genomic composition of a putative ancestral bear karyotype based upon the recognition of 39 chromosome segments defined by painting as the smallest conserved evolutionary unit segments (pSCEUS) among these species. The different pSCEUS combinations occurring among modern bear species support and extend the postulated sequence of chromosomal rearrangements and provide a framework to propose patterns of genome reorganization among carnivores and other mammal radiations.