Antiviral activity and synthesis of quaternized promazine derivatives against HSV-1

N-(4-chlorobenzyl)triflupromazinium chloride, a known antitubercular agent, has been found to also be active against HSV-1. A preliminary structure-activity relation has been explored to determine which groups are crucial to viral inhibition. Antiviral assessments such as GFP reduction, plaque reduction, treatment timing and wash-out studies have also been probed to determine a mode of action for QPD-1. Based on this preliminary data, it appears that QPD-1 is a reversible inhibitor, suspected to inhibit early stages of viral replication of HSV-1 at 50μM, equipotent to acyclovir.     

Multi-locus species delimitation in closely related animals and fungi: one marker is not enough

Despite taxonomy’s 250‐year history, the past 20 years have borne witness to remarkable advances in technology and techniques, as well as debate. DNA barcoding has generated a substantial proportion of this debate, with its proposition that a single mitochondrial sequence will consistently identify and delimit species, replacing more evidence‐rich and time‐intensive methods. Although mitochondrial DNA (mtDNA) has since been the focus of voluminous discussion and case studies, little effort has been made to comprehensively evaluate its success in delimiting closely related species. We have conducted the first broadly comparative literature review addressing the efficacy of molecular markers for delimiting such species over a broad taxonomic range. By considering only closely related species, we sought to avoid confusion of success rates with those due to deeply divergent taxa. We also address whether increased population‐level or geographic sampling affects delimitation success. Based on the results from 101 studies, we found that all marker groups had approximately equal success rates (～70%) in delimiting closely related species and that the use of additional loci increased average delimitation success. We also found no relationship between increased sampling of intraspecific variability and delimitation success. Ultimately, our results support a multi‐locus integrative approach to species delimitation and taxonomy.     

Effects of energy content and energy density of pre-portioned entrées on energy intake

Pre-portioned entrées are commonly consumed to help control portion size and limit energy intake. The influence of entrée characteristics on energy intake, however, has not been well studied. We determined how the effects of energy content and energy density (ED, kcal/g) of pre-portioned entrées combine to influence daily energy intake. In a crossover design, 68 non-dieting adults (28 men and 40 women) were provided with breakfast, lunch, and dinner on 1 day a week for 4 weeks. Each meal included a compulsory, manipulated pre-portioned entrée followed by a variety of unmanipulated discretionary foods that were consumed ad libitum. Across conditions, the entrées were varied in both energy content and ED between a standard level (100%) and a reduced level (64%). Results showed that in men, decreases in the energy content and ED of pre-portioned entrées acted independently and added together to reduce daily energy intake (both P < 0.01). Simultaneously decreasing the energy content and ED reduced total energy intake in men by 16% (445 ± 47 kcal/day; P < 0.0001). In women, the entrée factors also had independent effects on energy intake at breakfast and lunch, but at dinner and for the entire day the effects depended on the interaction of the two factors (P < 0.01). Simultaneously decreasing the energy content and ED reduced daily energy intake in women by 14% (289 ± 35 kcal/day; P < 0.0001). Both the energy content and ED of pre-portioned entrées affect daily energy intake and could influence the effectiveness of such foods for weight management.     

Insights from Characterizing Extinct Human Gut Microbiomes

In an effort to better understand the ancestral state of the human distal gut microbiome, we examine feces retrieved from archaeological contexts (coprolites). To accomplish this, we pyrosequenced the 16S rDNA V3 region from duplicate coprolite samples recovered from three archaeological sites, each representing a different depositional environment: Hinds Cave (∼8000 years B.P.) in the southern United States, Caserones (1600 years B.P.) in northern Chile, and Rio Zape in northern Mexico (1400 years B.P.). Clustering algorithms grouped samples from the same site. Phyletic representation was more similar within sites than between them. A Bayesian approach to source-tracking was used to compare the coprolite data to published data from known sources that include, soil, compost, human gut from rural African children, human gut, oral and skin from US cosmopolitan adults and non-human primate gut. The data from the Hinds Cave samples largely represented unknown sources. The Caserones samples, retrieved directly from natural mummies, matched compost in high proportion. A substantial and robust proportion of Rio Zape data was predicted to match the gut microbiome found in traditional rural communities, with more minor matches to other sources. One of the Rio Zape samples had taxonomic representation consistent with a child. To provide an idealized scenario for sample preservation, we also applied source tracking to previously published data for Ötzi the Iceman and a soldier frozen for 93 years on a glacier. Overall these studies reveal that human microbiome data has been preserved in some coprolites, and these preserved human microbiomes match more closely to those from the rural communities than to those from cosmopolitan communities. These results suggest that the modern cosmopolitan lifestyle resulted in a dramatic change to the human gut microbiome.     

LOV to BLUF: flavoprotein contributions to the optogenetic toolkit

Optogenetics is an emerging field that combines optical and genetic approaches to non-invasively interfere with cellular events with exquisite spatiotemporal control. Although it arose originally from neuroscience, optogenetics is widely applicable to the study of many different biological systems and the range of applications arising from this technology continues to increase. Moreover, the repertoire of light-sensitive proteins used for devising new optogenetic tools is rapidly expanding. Light, Oxygen, or Voltage sensing (LOV) and Blue-Light-Utilizing flavin adenine dinucleotide (FAD) (BLUF) domains represent new contributors to the optogenetic toolkit. These small (100-140-amino acids) flavoprotein modules are derived from plant and bacterial photoreceptors that respond to UV-A/blue light. In recent years, considerable progress has been made in uncovering the photoactivation mechanisms of both LOV and BLUF domains. This knowledge has been applied in the design of synthetic photoswitches and fluorescent reporters with applications in cell biology and biotechnology. In this review, we summarize the photochemical properties of LOV and BLUF photosensors and highlight some of the recent advances in how these flavoproteins are being employed to artificially regulate and image a variety of biological processes.     

Hierarchal type III secretion of translocators and effectors from Escherichia coli O157:H7 requires the carboxy terminus of SepL that binds to Tir

Type III secretion (T3S) from enteric bacteria is a co-ordinated process with a hierarchy of secreted proteins. In enteropathogenic and enterohaemorrhagic Escherichia coli , SepL and SepD are essential for translocator but not effector protein export, but how they function to control this differential secretion is not known. This study has focused on the different activities of SepL including membrane localization, SepD binding, EspD export and Tir secretion regulation. Analyses of SepL truncates demonstrated that the different functions associated with SepL can be separated. In particular, SepL with a deletion of 11 amino acids from the C-terminus was able to localize to the bacterial membrane, export translocon proteins but not regulate Tir or other effector protein secretion. From the repertoire of effector proteins only Tir was shown to bind directly to full-length SepL and the C-terminal 48 amino acids of SepL was sufficient to interact with Tir. By synchronizing induction of T3S, it was evident that the Tir-binding capacity of SepL is important to delay the release of effector proteins while the EspADB translocon is secreted. The interaction between Tir and SepL is therefore a critical step that controls the timing of T3S in attaching and effacing pathogens.     

Crystal structure of glycogen synthase kinase 3 beta: structural basis for phosphate-primed substrate specificity and autoinhibition.

Glycogen synthase kinase 3 beta (GSK3 beta) plays a key role in insulin and Wnt signaling, phosphorylating downstream targets by default, and becoming inhibited following the extracellular signaling event. The crystal structure of human GSK3 beta shows a catalytically active conformation in the absence of activation-segment phosphorylation, with the sulphonate of a buffer molecule bridging the activation-segment and N-terminal domain in the same way as the phosphate group of the activation-segment phospho-Ser/Thr in other kinases. The location of this oxyanion binding site in the substrate binding cleft indicates direct coupling of P+4 phosphate-primed substrate binding and catalytic activation, explains the ability of GSK3 beta to processively hyperphosphorylate substrates with Ser/Thr pentad-repeats, and suggests a mechanism for autoinhibition in which the phosphorylated N terminus binds as a competitive pseudosubstrate with phospho-Ser 9 occupying the P+4 site.     

In vivo peroxidative activity of FALS-mutant human CuZnSODs expressed in yeast.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder leading to loss of motor neurons. We previously characterized the enhanced peroxidative activity of the human familial ALS (FALS) mutants of copper-zinc superoxide dismutase (CuZnSOD) A4V and G93A in vitro. Here, a similar activity is demonstrated for human FALS CuZnSOD mutants in an in vivo model system, the yeast Saccharomyces cerevisiae. Spin trap adducts of alpha-(pyridyl-4-N-oxide)-N-tert-butylnitrone (POBN) have been measured by electron paramagnetic resonance (EPR) in yeast expressing mutant (A4V, L38V, G93A, and G93C) and wild type CuZnSOD upon addition of hydrogen peroxide to the culture. The trapped radical is a hydroxyethyl adduct of POBN, identified by spectral parameters. Mutant CuZnSODs produced greater concentrations of the trapped adduct compared to the wild type enzyme. This observation provides evidence for an oxidative radical mechanism, whereby the mutants of CuZnSOD catalyze the formation of reactive oxygen species that may be related to the development or progression of FALS. This study also presents an in vivo model system to study free radical production in FALS-associated CuZnSOD mutations.