Systematic Mapping of WNT-FZD Protein Interactions Reveals Functional Selectivity by Distinct WNT-FZD Pairs

The seven-transmembrane-spanning receptors of the FZD_1–10 class are bound and activated by the WNT family of lipoglycoproteins, thereby inducing a complex network of signaling pathways. However, the specificity of the interaction between mammalian WNT and FZD proteins and the subsequent signaling cascade downstream of the different WNT-FZD pairs have not been systematically addressed to date. In this study, we determined the binding affinities of various WNTs for different members of the FZD family by using bio-layer interferometry and characterized their functional selectivity in a cell system. Using purified WNTs, we show that different FZD cysteine-rich domains prefer to bind to distinct WNTs with fast on-rates and slow off-rates. In a 32D cell-based system engineered to overexpress FZD₂, FZD₄, or FZD₅, we found that WNT-3A (but not WNT-4, -5A, or -9B) activated the WNT-β-catenin pathway through FZD_2/4/5 as measured by phosphorylation of LRP6 and β-catenin stabilization. Surprisingly, different WNT-FZD pairs showed differential effects on phosphorylation of DVL2 and DVL3, revealing a previously unappreciated DVL isoform selectivity by different WNT-FZD pairs in 32D cells. In summary, we present extensive mapping of WNT-FZD cysteine-rich domain interactions complemented by analysis of WNT-FZD pair functionality in a unique cell system expressing individual FZD isoforms. Differential WNT-FZD binding and selective functional readouts suggest that endogenous WNT ligands evolved with an intrinsic natural bias toward different downstream signaling pathways, a phenomenon that could be of great importance in the design of FZD-targeting drugs.     

Exploration of the Arrest Peptide Sequence Space Reveals Arrest-enhanced Variants

Translational arrest peptides (APs) are short stretches of polypeptides that induce translational stalling when synthesized on a ribosome. Mechanical pulling forces acting on the nascent chain can weaken or even abolish stalling. APs can therefore be used as in vivo force sensors, making it possible to measure the forces that act on a nascent chain during translation with single-residue resolution. It is also possible to score the relative strengths of APs by subjecting them to a given pulling force and ranking them according to stalling efficiency. Using the latter approach, we now report an extensive mutagenesis scan of a strong mutant variant of the Mannheimia succiniciproducens SecM AP and identify mutations that further increase the stalling efficiency. Combining three such mutations, we designed an AP that withstands the strongest pulling force we are able to generate at present. We further show that diproline stretches in a nascent protein act as very strong APs when translation is carried out in the absence of elongation factor P. Our findings highlight critical residues in APs, show that certain amino acid sequences induce very strong translational arrest and provide a toolbox of APs of varying strengths that can be used for in vivo force measurements.     

A review of the subspecies status of the Icelandic Purple Sandpiper Calidris maritima littoralis

The Icelandic Purple Sandpiper Calidris maritima littoralis (C.L. Brehm, 1831) represents one member of a poorly understood subspecies complex. Currently, differences in size define two other subspecies: Calidris maritima belcheri Engelmoer & Roselaar, 1998, which breeds in north‐eastern Canada along the Hudson Bay and James Bay, and Calidris maritima maritima (Brunnich, 1764), which breeds along the Arctic coasts elsewhere in northern Canada, Greenland, Svalbard, Scotland, and Fennoscandia, to northern central Siberia. There are large size differences amongst populations of C. m. maritima, however. As an Arctic/Alpine breeding bird, C. m. littoralis could provide an interesting perspective on the evolutionary changes following a northwards expansion of a species after glacial retreat. Considering the extent of the ice sheet in the northern hemisphere during the last glaciation, and the short period of time since it ended, the correct attribution of subspecies status for C. m. maritima may reflect either rapid diversification from a single population or ancestral splits of distinct evolutionary lineages that survived in isolation at southern latitudes. We applied morphometric subspecies criteria, diagnosability by Amadon's rule, and genetic analysis of five nuclear introns, and the mitochondrial DNA markers cytochrome oxidase c subunit I (COI) and NADH dehydrogenase subunit 2 (ND2), to geographically separate breeding populations in order to examine the subspecies status of the Icelandic population. The results do not provide support for the subspecies status of the Icelandic population because the nominate and Icelandic subspecies fail to uphold Amadon's rule, and genetic analyses indicate that the study populations derive from a single shared refugium. © 2015 The Linnean Society of London     

Three new species of Hagnagora Druce, 1885 (Lepidoptera,Geometridae, Larentiinae) from Ecuador and Costa Rica and a concise revision of the genus

Three new Hagnagora Druce species (Geometridae, Larentiinae) are described: Hagnagora richardi Brehm, sp. n. from Ecuador, Hagnagora hedwigae Brehm, sp. n. from Ecuador, and Hagnagora mirandahenrichae Brehm, sp. n. from Costa Rica. A checklist of taxa assigned to Hagnagora is provided. Hagnagora is provisionally divided into six clades: the anicata clade (6 species), the buckleyi clade (3 species), the croceitincta clade (3 species), the ephestris clade (3 species), the mortipax clade (4 species) and Hagnagora subrosea (1 species). Two taxa are revived from synonymy: Hagnagora catagrammina Druce, stat. rev. and Hagnagora luteoradiata Thierry-Mieg, stat. rev. Two taxa are reinstated from subspecies to species level: Hagnagora acothysta Schaus, stat. rev. and Hagnagora jamaicensis Schaus, stat. rev. Four taxa are provisionally removed from Hagnagora: “Hagnagora” ignipennis, “Hagnagora” mesenata, “Hagnagora” vittata, and “Hagnagora” ceraria. After these changes, the genus Hagnagora now comprises 20 valid species.     

Substitution at carbon 2 of 19-nor-1α,25-dihydroxyvitamin D3 with 3-hydroxypropyl group generates an analogue with enhanced chemotherapeutic potency in PC-3 prostate cancer cells

The active form of vitamin D(3), 1α,25-dihydroxyvitamin D(3)(1α,25(OH)(2)D(3)), has anti-proliferative and anti-invasive activities in prostate cancer cells. Because of 1α,25(OH)(2)D(3) therapeutic potential in treating cancers, numerous analogues have been synthesized with an attempt to increase anti-proliferative and/or decrease calcemic properties. Among these analogues, 19-nor-1α,25(OH)(2)D(2) while being less calcemic has equivalent potency as 1α,25(OH)(2)D(3) in several in vitro and in vivo systems. We recently showed that 19-nor-2α-(3-hydroxypropyl)-1α,25(OH)(2)D(3) (MART-10) was at least 500-fold and 10-fold more active than 1α,25(OH)(2)D(3) in inhibiting the proliferation of an immortalized normal prostate PZ-HPV-7 cells and the invasion of androgen insensitive PC-3 prostate cancer cells, respectively. In this study, we further investigated the effects of MART-10 and 1α,25(OH)(2)D(3) on the dose- and time-dependent induction of CYP24A1 gene expression in PC-3 prostate cancer cells. We found that MART-10 induced CYP24A1 gene expression at a lower concentration with a longer duration compared to 1α,25(OH)(2)D(3), suggesting that MART-10 is less susceptible to CYP24A1 degradation. Molecular docking model of human CYP24A1 and MART-10 indicates that its side chain is far away from the heme ion and is less likely to be hydroxylated by the enzyme. Furthermore, MART-10 was a more potent inhibitor of PC-3 cell proliferation and invasion compared to 1α,25(OH)(2)D(3). In addition, MART-10 down-regulated matrix metalloproteinase-9 (MMP-9) expression which could be one mechanism whereby MART-10 influences cancer cell invasion. Finally, we observed that subcutaneous administration of MART-10 up-regulated the CYP24A1 mRNA expression in rat kidneys without affecting their plasma calcium levels. Thus, our findings demonstrate that MART-10 is biologically active in vivo and may be an effective vitamin D analogue for clinical trials to treat prostate cancer.     

P2-P1' macrocyclization of P2 phenylglycine based HCV NS3 protease inhibitors using ring-closing metathesis

Macrocyclization is a commonly used strategy to preorganize HCV NS3 protease inhibitors in their bioactive conformation. Moreover, macrocyclization generally leads to greater stability and improved pharmacokinetic properties. In HCV NS3 protease inhibitors, it has been shown to be beneficial to include a vinylated phenylglycine in the P2 position in combination with alkenylic P1' substituents. A series of 14-, 15- and 16-membered macrocyclic HCV NS3 protease inhibitors with the linker connecting the P2 phenylglycine and the alkenylic P1' were synthesized by ring-closing metathesis, using both microwave and conventional heating. Besides formation of the expected macrocycles in cis and trans configuration as major products, both ring-contracted and double-bond migrated isomers were obtained, in particular during formation of the smaller rings (14- and 15-membered rings). All inhibitors had K(i)-values in the nanomolar range, but only one inhibitor type was improved by rigidification. The loss in inhibitory effect can be attributed to a disruption of the beneficial π-π interaction between the P2 fragment and H57, which proved to be especially deleterious for the d-phenylglycine epimers.     

Salmonella bongori provides insights into the evolution of the Salmonellae

The genus Salmonella contains two species, S. bongori and S. enterica. Compared to the well-studied S. enterica there is a marked lack of information regarding the genetic makeup and diversity of S. bongori. S. bongori has been found predominantly associated with cold-blooded animals, but it can infect humans. To define the phylogeny of this species, and compare it to S. enterica, we have sequenced 28 isolates representing most of the known diversity of S. bongori. This cross-species analysis allowed us to confidently differentiate ancestral functions from those acquired following speciation, which include both metabolic and virulence-associated capacities. We show that, although S. bongori inherited a basic set of Salmonella common virulence functions, it has subsequently elaborated on this in a different direction to S. enterica. It is an established feature of S. enterica evolution that the acquisition of the type III secretion systems (T3SS-1 and T3SS-2) has been followed by the sequential acquisition of genes encoding secreted targets, termed effectors proteins. We show that this is also true of S. bongori, which has acquired an array of novel effector proteins (sboA-L). All but two of these effectors have no significant S. enterica homologues and instead are highly similar to those found in enteropathogenic Escherichia coli (EPEC). Remarkably, SboH is found to be a chimeric effector protein, encoded by a fusion of the T3SS-1 effector gene sopA and a gene highly similar to the EPEC effector nleH from enteropathogenic E. coli. We demonstrate that representatives of these new effectors are translocated and that SboH, similarly to NleH, blocks intrinsic apoptotic pathways while being targeted to the mitochondria by the SopA part of the fusion. This work suggests that S. bongori has inherited the ancestral Salmonella virulence gene set, but has adapted by incorporating virulence determinants that resemble those employed by EPEC.     

A validated method for the detection and quantitation of 50 drugs of abuse and medicinal drugs in oral fluid by gas chromatography-mass spectrometry

Oral fluid is of increasing interest as an alternative sample matrix in drugs of abuse analysis. Compared to the conventional sample matrix blood, sample volumes of oral fluid are smaller and the concentrations of some drugs can be much lower. This imposes some restrictions on the analysis method, which has to be able to detect and quantify multiple analytes from a small sample volume at low concentrations. A sensitive multi-component method for quantitative determination of 50 drug compounds from oral fluid samples collected with the StatSure SalivaSampler? device was developed. The compounds analysed include cannabis, cocaine, amphetamines, opioids, benzodiazepines and other psychoactive medicines. Both liquid–liquid-extraction (LLE) and solid-phase-extraction (SPE) are employed in the sample pre-treatment and the samples are analysed using gas chromatography–mass spectrometry (GC–MS) with the mass selective detector (MSD) operating in either electron ionization (EI) or negative-ion chemical ionization (NICI) mode. The method was fully validated, and the validated parameters included linearity, selectivity, accuracy, precision, and extraction efficiency. Stability of the collected samples during storage at ?18 °C was also studied, and even after over a year's storage all analyte concentrations were more than 60% of the original concentrations. The described method is suitable for routine analysis of oral fluid samples and it has been applied to analysis of more than 4000 oral fluid samples collected anonymously from volunteer road users in Finland during 2007–2009 as a part of the EU project DRUID (Driving under the Influence of Drugs, Alcohol and Medicines). At the moment the developed method is the most comprehensive validated analysis method for oral fluid samples.     

Born to cope with climate change? Experimentally manipulated hatching time does not affect duckling survival in the mallard <Emphasis Type="Italic">Anas platyrhynchos</Emphasis>

Two main hypotheses proposed to explain the seasonal decline in reproductive performance in birds are (1) deterioration of environmental conditions and (2) lower parental quality of late breeders. Previous experimental work addressing these hypotheses generally have problematic biases pertaining to delay of hatching, costs of re-laying and incubation, as well as variation in the quality of eggs, territories, offspring and parental traits. We address these biases in an experimental test of the timing hypothesis (i.e. (1) above) in a precocial bird. Using a 2-year cross-over design and game-farm mallard (Anas platyrhynchos) eggs originating from a number of hens and a standardised delay procedure, we introduced early and late broods with a foster female onto boreal oligotrophic lakes and monitored subsequent duckling survival. Standardised invertebrate sampling was done concurrently to get a measure of lake-level abundance of aquatic prey, a likely causative agent of putative seasonal difference in duckling survival. Survival data and covariates (duckling age; days) were analysed by an information theoretic approach. There was no effect of treatment (i.e. manipulation of hatching date) on duckling survival, which was higher in 2005 than in 2004. In contrast to observational studies from more seasonal wetlands, our experiment demonstrates that duckling survival on boreal lakes was not affected by a 12-day delay in hatching date. Since we did not find any consistent trends in abundance of aquatic prey, i.e. neither clear peaks nor differences between treatment periods, we hypothesise that moderate climate change has minor effects on resource abundance and hence also on mallard duckling survival in boreal environments.