Peptoids advance multidisciplinary research and undergraduate education in parallel: Sequence effects on conformation and lipid interactions

Peptoids are versatile peptidomimetic molecules with wide-ranging applications from drug discovery to materials science. An understanding of peptoid sequence features that contribute to both their three-dimensional structures and their interactions with lipids will expand functions of peptoids in varied fields. Furthermore, these topics capture the enthusiasm of undergraduate students who prepare and study diverse peptoids in laboratory coursework and/or in faculty led research. Here, we present the synthesis and study of 21 peptoids with varied functionality, including 19 tripeptoids and 2 longer oligomers. We observed differences in fluorescence spectral features for 10 of the tripeptoids that correlated with peptoid flexibility and relative positioning of chromophores. Interactions of representative peptoids with sonicated glycerophospholipid vesicles were also evaluated using fluorescence spectroscopy. We observed evidence of conformational changes effected by lipids for select peptoids. We also summarize our experiences engaging students in peptoid-based projects to advance both research and undergraduate educational objectives in parallel.     

Nitrogen isotope values of Pennisetum glaucum (pearl millet) grains: towards a reconstruction of past cultivation conditions in the Sahel,West Africa

Vegetation History and Archaeobotany - The nitrogen isotope compositions of charred wheat and barley grains reflect manuring intensity and have been used to reconstruct past manuring practices at...     

Amiloride-sensitive Na+ channels contribute to regulatory volume increases in human glioma cells

Despite intensive research, brain tumors remain among the most difficult type of malignancies to treat, due largely to their diffusely invasive nature and the associated difficulty of adequate surgical resection. To migrate through the brain parenchyma and to proliferate, glioma cells must be capable of significant changes in shape and volume. We have previously reported that glioma cells express an amiloride- and psalmotoxin-sensitive cation conductance that is not found in normal human astrocytes. In the present study, we investigated the potential role of this ion channel to mediate regulatory volume increase in glioma cells. We found that the ability of the cells to volume regulate subsequent to cell shrinkage by hyperosmolar solutions was abolished by both amiloride and psalmotoxin 1. This toxin is thought to be a specific peptide inhibitor of acid-sensing ion channel (ASIC1), a member of the Deg/ENaC superfamily of cation channels. We have previously shown this toxin to be an effective blocker of the glioma cation conductance. Our data suggest that one potential role for this conductance may be to restore cell volume during the cell's progression thorough the cell cycle and while the tumor cell migrates within the interstices of the brain.     

Asking about Sex in General Health Surveys: Comparing the Methods and Findings of the 2010 Health Survey for England with Those of the Third National Survey of Sexual Attitudes and Lifestyles

Objectives

Including questions about sexual health in the annual Health Survey for England (HSE) provides opportunities for regular measurement of key public health indicators, augmenting Britain''s decennial National Survey of Sexual Attitudes and Lifestyles (Natsal). However, contextual and methodological differences may limit comparability of the findings. We examine the extent of these differences between HSE 2010 and Natsal-3 and investigate their impact on parameter estimates.

Methods

Complex survey analyses of data from men and women in the 2010 HSE (n = 2,782 men and 3,588 women) and Natsal-3 undertaken 2010–2012 (n = 4,882 men and 6,869 women) aged 16-69y and resident in England, both using probability sampling, compared their characteristics, the amount of non-response to, and estimates from, sexual health questions. Both surveys used self-completion for the sexual behaviour questions but this was via computer-assisted self-interview (CASI) in Natsal-3 and a pen-and-paper questionnaire in HSE 2010.

Results

The surveys achieved similar response rates, both around 60%, and demographic profiles largely consistent with the census, although HSE participants tended to be less educated, and reported worse general health, than Natsal-3 participants. Item non-response to the sexual health questions was typically higher in HSE 2010 (range: 9–18%) relative to Natsal-3 (all <5%). Prevalence estimates for sexual risk behaviours and STI-related indicators were generally slightly lower in HSE 2010 than Natsal-3.

Conclusions

While a relatively high response to sexual health questions in HSE 2010 demonstrates the feasibility of asking such questions in a general health survey, differences with Natsal-3 do exist. These are likely due to the HSE’s context as a general health survey and methodological limitations such as its current use of pen-and-paper questionnaires. Methodological developments to the HSE should be considered so that its data can be interpreted in combination with those from dedicated sexual health surveys, thus improving our ability to monitor trends in sexual health.     

Development of the CRISPR/Cas9 System for Targeted Gene Disruption in Aspergillus fumigatus

Low rates of homologous recombination have broadly encumbered genetic studies in the fungal pathogen Aspergillus fumigatus. The CRISPR/Cas9 system of bacteria has recently been developed for targeted mutagenesis of eukaryotic genomes with high efficiency and, importantly, through a mechanism independent of homologous repair machinery. As this new technology has not been developed for use in A. fumigatus, we sought to test its feasibility for targeted gene disruption in this organism. As a proof of principle, we first demonstrated that CRISPR/Cas9 can indeed be used for high-efficiency (25 to 53%) targeting of the A. fumigatus polyketide synthase gene (pksP), as evidenced by the generation of colorless (albino) mutants harboring the expected genomic alteration. We further demonstrated that the constitutive expression of the Cas9 nuclease by itself is not deleterious to A. fumigatus growth or virulence, thus making the CRISPR system compatible with studies involved in pathogenesis. Taken together, these data demonstrate that CRISPR can be utilized for loss-of-function studies in A. fumigatus and has the potential to bolster the genetic toolbox for this important pathogen.     

Paroxetine suppresses recombinant human P2X7 responses

P2X7 receptor (P2X7) activity may link inflammation to depressive disorders. Genetic variants of human P2X7 have been linked with major depression and bipolar disorders, and the P2X7 knockout mouse has been shown to exhibit anti-depressive-like behaviour. P2X7 is an ATP-gated ion channel and is a major regulator of the pro-inflammatory cytokine interleukin 1β (IL-1β) secretion from monocytes and microglia. We hypothesised that antidepressants may elicit their mood enhancing effects in part via modulating P2X7 activity and reducing inflammatory responses. In this study, we determined whether common psychoactive drugs could affect recombinant and native human P2X7 responses in vitro. Common antidepressants demonstrated opposing effects on human P2X7-mediated responses; paroxetine inhibited while fluoxetine and clomipramine mildly potentiated ATP-induced dye uptake in HEK-293 cells stably expressing recombinant human P2X7. Paroxetine inhibited dye uptake mediated by human P2X7 in a concentration-dependent manner with an IC₅₀ of 24 μM and significantly reduces ATP-induced inward currents. We confirmed that trifluoperazine hydrochloride suppressed human P2X7 responses (IC₅₀ of 6.4 μM). Both paroxetine and trifluoperazine did not inhibit rodent P2X7 responses, and mutation of a known residue (F 95L) did not alter the effect of either drug, suggesting neither drug binds at this site. Finally, we demonstrate that P2X7-induced IL-1β secretion from lipopolysaccharide (LPS)-primed human CD14⁺ monocytes was suppressed with trifluoperazine and paroxetine.     

Development of a small peptide tag for covalent labeling of proteins

A 21-mer peptide that can be used to covalently introduce synthetic molecules into proteins has been developed. Phage-displayed peptide libraries were subjected to reaction-based selection with 1,3-diketones. The peptide was further evolved by addition of a randomized region and reselection for improved binding. The resulting 21-mer peptide had a reactive amino group that formed an enaminone with 1,3-diketone and was used as a tag for labeling of maltose binding protein. Using this peptide tag and 1,3-diketone derivatives, a variety of molecules such as reporter probes and functionalities may be covalently introduced into proteins of interest.     

Structure of a hexameric RNA packaging motor in a viral polymerase complex

Packaging of the Cystovirus varphi8 genome into the polymerase complex is catalysed by the hexameric P4 packaging motor. The motor is located at the fivefold vertices of the icosahedrally symmetric polymerase complex, and the symmetry mismatch between them may be critical for function. We have developed a novel image-processing approach for the analysis of symmetry-mismatched structures and applied it to cryo-electron microscopy images of P4 bound to the polymerase complex. This approach allowed us to solve the three-dimensional structure of the P4 in situ to 15-A resolution. The C-terminal face of P4 was observed to interact with the polymerase complex, supporting the current view on RNA translocation. We suggest that the symmetry mismatch between the two components may facilitate the ring opening required for RNA loading prior to its translocation.     

The two-component system sivS/R regulates virulence in Streptococcus iniae

Streptococcus iniae causes invasive disease and death in fish, and to a lesser extent, sporadic cases of soft-tissue infections in humans. A two-component system termed sivS/R, which regulates capsule expression, was previously identified and characterized. In this study, it is shown that a sivS/R deletion-insertion mutant, termed 9117Deltasiv, causes transient bacteremia and reduced virulence compared with the parent strain when tested in a murine model of bacteremic infection. Furthermore, real-time PCR studies indicated that SivS/R regulates the expression levels of the streptolysin S structural gene, sagA, as well as the CAMP factor gene, cfi. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of S. iniae spheroplasts revealed downregulation of three surface proteins in the mutant strain compared with the parent strain. These proteins were identified by MS to be a putative lipoprotein, a hyaluronate-associated protein and a pyruvate kinase. This study demonstrates that SivS/R regulates virulence in vivo, and controls the expression of a number of genes in S. iniae.