Identification of novel small-molecule histone deacetylase inhibitors by medium-throughput screening using a fluorigenic assay

Cationic peptides, known to disrupt bacterial membranes, are being developed as promising agents for therapeutic intervention against infectious disease. In the present study, we investigate structure-activity relationships in the bacterial membrane disruptor betapep-25, a peptide 33-mer. For insight into which amino acid residues are functionally important, we synthesized alanine-scanning variants of betapep-25 and assessed their ability to kill bacteria (Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus) and to neutralize LPS (lipopolysaccharide). Activity profiles were found to vary with the bacterial strain examined. Specific cationic and smaller hydrophobic alkyl residues were crucial to optimal bactericidal activity against the Gram-negative bacteria, whereas larger hydrophobic and cationic residues mediated optimal activity against Gram-positive Staph. aureus. Lysine-substituted norleucine (n-butyl group) variants demonstrated that both charge and alkyl chain length mediate optimal activity. In terms of LPS neutralization, activity profiles were essentially the same against four species of LPS (E. coli 055 and 0111, Salmonella enterica serotype Typhimurium and Klebsiella pneumoniae), and different for two others (Ps. aeruginosa and Serratia marcescens), with specific hydrophobic, cationic and, surprisingly, anionic residues being functionally important. Furthermore, disulfide-bridged analogues demonstrated that an anti parallel beta-sheet structure is the bioactive conformation of betapep-25 in terms of its bactericidal, but not LPS endotoxin neutralizing, activity. Moreover, betapep-25 variants, like the parent peptide, do not lyse eukaryotic cells. This research contributes to the development and design of novel antibiotics.     

Acceptance and perceived barriers of implementing a guideline for managing low back in general practice

Background

There is considerable interest today in shared decision-making (SDM), defined as a decision-making process jointly shared by patients and their health care provider. However, the data show that SDM has not been broadly adopted yet. Consequently, the main goal of this proposal is to bring together the resources and the expertise needed to develop an interdisciplinary and international research team on the implementation of SDM in clinical practice using a theory-based dyadic perspective.

Methods

Participants include researchers from Canada, US, UK, and Netherlands, representing medicine, nursing, psychology, community health and epidemiology. In order to develop a collaborative research network that takes advantage of the expertise of the team members, the following research activities are planned: 1) establish networking and on-going communication through internet-based forum, conference calls, and a bi-weekly e-bulletin; 2) hold a two-day workshop with two key experts (one in theoretical underpinnings of behavioral change, and a second in dyadic data analysis), and invite all investigators to present their views on the challenges related to the implementation of SDM in clinical practices; 3) conduct a secondary analyses of existing dyadic datasets to ensure that discussion among team members is grounded in empirical data; 4) build capacity with involvement of graduate students in the workshop and online forum; and 5) elaborate a position paper and an international multi-site study protocol.

Discussion

This study protocol aims to inform researchers, educators, and clinicians interested in improving their understanding of effective strategies to implement shared decision-making in clinical practice using a theory-based dyadic perspective.     

Initiation of mammalian O-mannosylation in vivo is independent of a consensus sequence and controlled by peptide regions within and upstream of the alpha-dystroglycan mucin domain

To reveal insight into the initiation of mammalian O-mannosylation in vivo, recombinant glycosylation probes containing sections of human alpha-dystroglycan (hDG) were expressed in epithelial cell lines. We demonstrate that O-mannosylation within the mucin domain of hDG occurs preferentially at Thr/Ser residues that are flanked by basic amino acids. Protein O-mannosylation is independent of a consensus sequence, but strictly dependent on a peptide region located upstream of the mucin domain. This peptide region cannot be replaced by other N-terminal peptides, however, it is not sufficient to induce O-mannosylation on a structurally distinct mucin domain in hybrid constructs. The presented in vivo evidence for a more complex regulation of mammalian O-mannosylation contrasts with a recent in vitro study of O-mannosylation in human alpha-dystroglycan peptides indicating the existence of an 18-meric consensus sequence. We demonstrate in vivo that the entire region p377-417 is necessary and sufficient for O-mannosylation initiation of hDG, but not of MUC1 tandem repeats. The feature of a doubly controlled initiation process distinguishes mammalian O-mannosylation from other types of O-glycosylation, which are largely controlled by structural properties of the substrate positions and their local peptide environment.     

Thinking about RNA? MicroRNAs in the brain

MicroRNAs (miRNAs) are a recently discovered class of small RNA molecules implicated in a wide range of diverse gene regulatory mechanisms. Interestingly, numerous miRNAs are expressed in a spatially and temporally controlled manner in the nervous system. This suggests that gene regulation networks based on miRNA activities may be particularly relevant in neurons. Recent studies show the involvement of RNA-mediated gene silencing in neurogenesis, neural differentiation, synaptic plasticity, and neurologic and psychiatric diseases. This review focuses on the roles of miRNAs in the gene regulation of the nervous system.     

KnotInFrame: prediction of -1 ribosomal frameshift events

Programmed -1 ribosomal frameshift (-1 PRF) allows for alternative reading frames within one mRNA. First found in several viruses, it is now believed to exist in all kingdoms of life. Strong stimulators for -1 PRF are a heptameric slippery site and an RNA pseudoknot. Here, we present a new algorithm KnotInFrame, for the automatic detection of -1 PRF signals from genomic sequences. It finds the frameshifting stimulators by means of a specialized RNA-pseudoknot folding program, fast enough for genome-wide analyses. Evaluations on known -1 PRF signals demonstrate a high sensitivity.     

Non‐destructive dry matter estimation of Alhagi sparsifolia vegetation in a desert oasis of Northwest China

Question: Can above‐ground biomass of naturally growing Alhagi sparsifolia shrubs be estimated non‐destructively? Location: Qira oasis (37° 01′N, 80° 48′E, 1365 ma.s.l.) at the southern fringe of the Taklamakan desert, Xinjiang, NW China. Methods: Two methods were compared to estimate above‐ground biomass (AGB) of Alhagi. At first shrub AGB was estimated by manual ground measurements (called ‘allometric approach’) of length, width and height of 50 individuals. Subsequently regression equations were established between calculated shrub canopy volume and shrub AGB (r²= 0.96). These equations were used to calculate AGB from manual ground measurements in 20 sample plots within the Alhagi field. Secondly, kite‐based colour aerial photography coupled with the use of a Geographic Information System (called ‘GIS approach’) was tested. First and second order polynomial regressions between AGB data of the 50 individual shrubs and their respective canopy area allowed to automatically calculate the AGB of all remaining shrubs covered by the photograph (r²= 0.92 to 0.96). The use of non‐linear AGB regression equations required an automatised separation of shrubs growing solitary or in clumps. Separation criteria were the size and shape of shrub canopies. Results: The allometric approach was more reliable but also more time‐consuming than the GIS‐based approach. The latter led to an overestimation of Alhagi dry matter in densely vegetated areas. However, this systematic error decreased with increasing size of the surveyed area. Future research in this field should focus on improvements of AGB estimates in areas of high shrub density.     

A fluorimetric assay for real-time monitoring of adenylyl cyclase activity based on terbium norfloxacin

Adenylyl cyclases catalyze the production of the second messenger cyclic AMP from ATP. Until now, there has been no fluorescent adenylyl cyclase assay known that is applicable to high-throughput screening and kinetic determinations that can directly monitor the turnover of the unmodified substrate ATP. In this study, a fluorescence-based assay is described using the Ca(II)- and calmodulin-dependent adenylyl cyclase edema factor (EF) from Bacillus anthracis and Tb(III)-norfloxacin as probe for the enzyme activity. This assay can be used to study enzyme regulators, allows real-time monitoring of adenylyl cyclase activity, and does not substitute ATP by fluorescent derivatives. These derivatives must be judged critically due to their interference on the activity of enzymes. Furthermore, the new assay makes redundant the application of radioactively labeled substrates such as [α-³²P]ATP or fluorescently labeled antibodies such as anti-cyclic AMP. We determined the Michaelis-Menten constant (K_M), the v₀^max value of ATP turnover, and the IC₅₀ values for three inhibitors of EF by this newly developed fluorescent method.     

Nighttime Wakefulness Associated with Infant Rearing in Callithrix kuhlii

Parent-infant cosleeping occurs in human and nonhuman primates, yet studies on the impact of cosleeping on parental sleep patterns have been limited to human mothers. We examined the effects of cosleeping on the nighttime wakefulness of a biparental New World primate, Wied's black tufted-ear marmoset (Callithrix kuhlii). We compared the sleep patterns of marmoset parents caring for young infants to those without infants, using an 8 mm videocamera and timelapse VCR under infrared illumination. The presence of young infants significantly impacted the sleep of mothers but not fathers. In fact, mothers rearing young infants were awake >3 times as often as mothers without infants. We also examined the nighttime wakefulness of marmoset parents across the first 9 weeks of infant life (birth through weaning). Although callitrichid mothers tend to reduce their daytime investment in offspring very early in infant life by relinquishing the care of infants to fathers and alloparents, increased nighttime wakefulness was not limited to the early postpartum period for the mothers. Instead, mothers exhibited more nighttime wakefulness than fathers did across the first 9 weeks of infant life. Our results indicate that the presence of infants has a greater impact on the sleep patterns of Callithrix kuhlii mothers than fathers, suggesting that mothers are more involved in infant care than previously realized and that fathers are not nearly as involved in nighttime care as their behavior during the day would suggest.