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Selenophene and thiophene-core estrogen receptor ligands that inhibit motility and development of parasitic stages of Haemonchus contortus
Sarah Preston1,
Junjie Luo2,
Yuezhou Zhang1, 3, 4,
Abdul Jabbar1,
Simon Crawford5,
Jonathan Baell3,
Andreas Hofmann1, 6,
Min Hu4,
Hai-Bing Zhou2Email author and
Robin B. Gasser1, 2Email author
Parasites & Vectors20169:346
Accepted: 27 May 2016
Parasitic worms represent a substantial disease burden in animals and humans worldwide. The control of parasitic roundworms (nematodes) relies heavily on the use of anthelmintic drugs. However, widespread drug resistance in nematodes seriously compromises the effectiveness of many anthelmintics around the world. Thus, there is a need to discover new drugs, with unique modes of action, against parasites.
Here, we synthesised and tested 74 selective estrogen receptor modulators (SERMs) for in vitro-activity on parasitic larvae of Haemonchus contortus (barber's pole worm), one of the most important nematode pathogens of small ruminants (including sheep and goats) and a key representative of one of the largest groups of parasitic nematodes (the Strongylida) of animals. We also studied the morphology of treated and untreated larvae using scanning electron microscopy (SEM), and assessed the agonistic/antagonistic activity of SERMs in a human embryonic kidney cell line using a luciferase reporter assay system.
We identified three SERMs (one selenophene and two thiophene-core compounds) with potent inhibitory activities (at 3–25 μM) on the motility and development of parasitic stages of H. contortus. An SEM examination of treated H. contortus revealed considerable damage to the cuticle of fourth- but not exsheathed, third-stage larvae; this damage appeared to be consistent with that observed upon treatment with monepantel but not moxidectin (control compounds).
The potency of the three SERMs compared favourably with commercially available anthelmintics, such that they warrant further assessment as nematocides. Future studies could focus on assessing the selectivity of these SERMs to parasites, characterising their target(s) and/or designing analogs that are parasite-specific.
Selective estrogen receptor modulators (SERMs)
Selenophene and thiophene-core SERMs
Inhibitory activity
Haemonchus contortus
Nematode
Despite their major socioeconomic impact globally, parasitic worms (helminths) of animals and humans are seriously neglected, in terms of funding for the research and development of chemotherapeutics, vaccines and diagnostics. The current economic losses caused by such worms to agriculture worldwide have a substantial adverse impact on farm profitability and exacerbate the global food shortage. For example, roundworms (nematodes) of livestock cause major losses to farmers due to disease, reduced weight gain, weight loss, poor productivity (e.g. in terms of meat or milk yields) and mortality in animals. Nematodes of the order Strongylida are of paramount importance as pathogens of livestock animals, such as sheep, goats, cattle and pigs, causing gastrointestinal or respiratory diseases and associated complications. In particular, Haemonchus contortus (the barber's pole worm) is a highly significant pathogen of livestock worldwide, affecting hundreds of millions of small ruminants (including sheep and goats) and causing economic losses [1, 2] estimated at tens of billions of dollars per annum. This parasite feeds on blood in the stomach (abomasum) and causes gastritis, anaemia and associated complications as well as mortality. It is transmitted orally from contaminated pasture to the host through a direct life-cycle [3]: eggs are excreted in host faeces; the first-stage larvae (L1s) develop inside eggs to then hatch (usually within one day) and develop through to the second (L2)- and third (L3)-stage larvae in about a week; infective L3s are then ingested by the host, exsheath (xL3) and, after a histotropic phase, develop through fourth (L4)-stage larvae to dioecious adults (within three weeks) in the abomasum.
The control of H. contortus and related nematodes has relied heavily on treatment with anthelmintic drugs, including benzimidazoles, imidazothiazole, macrocyclic lactones and amino-acetonitrile derivatives. However, the excessive use of such drugs has led or is leading to widespread resistance in these nematodes to many anthelmintics of these classes [4–11], seriously compromising their effectiveness in many regions around the world. Although a vaccine (Barbervax®, Wormvax, Albany, Australia) was released to support treatment programs against haemonchosis, there is a continual need to work toward identifying new drug targets, and developing new or repurposing existing compounds [12, 13].
In public-private and cross-disciplinary partnerships, we have developed an efficient whole-organism drug-screening assay for H. contortus for the discovery and subsequent repurposing of compounds to parasitic nematodes [14–16]. Interestingly, recent reports have shown that some selective estrogen receptor modulators (SERMs) have considerable activity against protistan parasites, such as Leishmania [17–19] and cestodes, including Echinococcus granulosus, Taenia crassiceps and T. solium [20, 21]. In these cestodes, estrogen receptor-like proteins have been identified, and the mammalian ligand (17-β-estradiol) has been shown to enhance the (asexual) reproductive rate of T. crassiceps [22, 23]. These studies suggest that estrogen receptors (ERs) and associated pathways exist in parasitic helminths and might represent possible anthelmintic targets.
In studies directed at human applications, in order to obtain structurally novel compounds that are relatively straight forward to synthesize or produce, we have sought to expand the chemical diversity of ligands for ERs by replacing their internal scaffolding with various heterocycles and other structurally related elements and, in the process, we have produced and evaluated a series of selenophene and thiophene-derived SERMs with selective activities on ERs (including ERα and ERβ) (Hai-Bing Zhou et al., unpublished). These findings indicated that changes to the core structure, although remote from close contact with residues in the ligand-binding pocket, can have a major impact on activity; in addition, some structural alterations can lead to super-agonism, resulting in the compound having a greater response than the endogenous ligand [24]. Although our focus has been on selective ligands for ERs of mammalian cells [25–27], we were keen to assess whether selenophene- or thiophene-core ligands, particularly if they do not have an effect on mammalian ERs, have an agonistic or super-agonistic activity on parasitic larval stages of H. contortus.
Unless otherwise stated, reagents and materials were obtained from commercial suppliers and were used without further purification. The tetrahydrofuran (THF) and dichloromethane (DCM) were dried over Na and CaCl2, respectively, and distilled prior to use. Reactions were monitored by thin layer chromatography (TLC), and column chromatography purification was performed using silica gel (230–400 mesh). NMR spectra were measured using Bruker DRX and DMX spectrometers at 400 MHz for 1H spectra and 100 MHz for 13C spectra and calibrated from residual solvent signal. In the following, the procedures for the de novo synthesis of selenophene- and thiophene-core compounds are described below.
General procedure for the synthesis of 2,5-dibromoselenophene
In the absence of light, selenophene (5.09 g, 38.9 mmol) was dissolved in dry N,N-dimethylformamide (DMF), and the solution degassed. N-bromosuccinimide (NBS, 2 equiv) was added in four portions within 30 min, and the orange solution then stirred at room temperature (22–24 °C) for 18 h. The reaction mixture was poured into ice water and extracted with DCM. The combined organic phases were washed with water and brine, and dried over Na2SO4. The removal of the solvent yielded 10.1 g of orange liquid, which was purified by column chromatography (silica; n-hexane); the pure product was a colourless liquid.
General procedure for the synthesis of 2,3,5-trisbromoselenophene
Bromine (3.1 equiv) in CHCl3 was added dropwise to a stirred solution of selenophene (1 equiv) in CHCl3 and AcOH at 0 °C over the course of 1 h. The reaction mixture was warmed to room temperature and stirred for 12 h, and then heated to 70 °C for 5 h. Upon completion of the reaction, the mixture was allowed to cool to room temperature and transferred to a large beaker. Excess bromine was evaporated at room temperature, and the resultant mixture was diluted with CHCl3. The organic phase was successively washed with water, diluted in aqueous NaOH solution and brine, and then concentrated. The crude crystalline product was further purified by column chromatography using hexane as an eluent to give an orange liquid.
General procedure for the Suzuki coupling reaction of bromoselenophene with arylboronic acid
Under Ar atmosphere, a mixture of bromoselenophene (1 equiv), arylboronic acid (3 equiv for dibromoselenophenes, 4 equiv for tribromoselenophenes), Pd catalyst (0.1 equiv), sodium carbonate (1 equiv per bromine) in an oxygen-free toluene/water (1:1) solution was stirred at 120 °C for 24 h, after which the reaction mixture was cooled to room temperature. The aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous MgSO4/Na2SO4 and then filtered and concentrated in vacuum. The product was purified by column chromatography.
General procedure for ether cleavage
Under Ar atmosphere, to a solution of methoxyphenyl derivative 2 (1 equiv) in dry DCM at -20 °C, boron tribromide (3 equiv per methoxy function) was added dropwise. The reaction mixture was stirred at room temperature. After 4 h, water was added to quench the reaction, and ethyl acetate was used to extract the aqueous layer. The combined organic layers were washed with brine, dried over anhydrous MgSO4/Na2SO4, and then filtered and concentrated in vacuum. The product was purified by column chromatography.
In total, we synthesized 52 thiophene-core analogues using the method reported by Min et al. [24] and 22 selenophene-core analogues using established procedures (Additional file 1).
Biological assay - H. contortus
The selenophene and thiophene-core ligands (n = 74) (Additional file 1) were screened at a concentration of 20 μM on exsheathed third-stage larvae (xL3s) of H. contortus in 96-well microculture plates (cat. no. 3635; Corning 3650, Life Sciences, Corning, USA) using relevant control compounds (i.e. moxidectin and monepantel), as described previously [15]. In brief, compounds were dissolved to a stock concentration of 10 mM in either dimethyl sulfoxide (DMSO) or methanol (Sigma-Adrich Scientific, St. Louis, USA). These ligands were then individually diluted to the final concentration of 20 μM using Luria Bertani medium (LB) supplemented with 100 IU/ml of penicillin, 100 μg/ml of streptomycin and 2.5 μg/ml of amphotericin (LB*). Compounds were dispensed (in triplicate) into the wells of a 96-well microculture plate using a multichannel pipette. In addition, the negative controls (LB*, LB* + 0.5 % solvent; six wells each), and positive controls (20 μM of monepantel; Zolvix, Novartis Animal Health, Basel, Switzerland and 20 μM of moxidectin; Cydectin, Virbac, Carros, France; triplicate wells) and xL3s (~300/well) were dispensed into wells of the plate using an automated multichannel pipette (Viaflo Assist/II, Integra Biosciences, Zizers, Switzerland). Following a 72 h incubation at 38 °C and 10 % CO2, a video recording (5 s) was taken of each well of the 96-well microculture plate (containing xL3s) using a greyscale camera (Rolera Bolt CMOS, Qimaging Scientific, Surrey, Canada) and a motorised X-Y axis stage (BioPoint 2, Ludl Electronics Products, Hawthorne, USA). Individual videos were processed to calculate a motility index (MI) using an algorithm described previously [14]. MIs were normalised to the negative and positive controls (to remove plate-to-plate variation) using the program Prism (v.6 GraphPad Software, USA). Z'-scores were calculated to validate the performance of the screening assay; reliable assays achieve Z'-scores of between 0.5 and 1 [28]. A compound was recorded as having an activity if it reduced xL3 motility by ≥ 70 % following incubation for 72 h. Subsequently, the anti-xL3 activity of individual compounds was confirmed, and half maximum inhibitory concentration (IC50) values estimated from dose–response curves (24 h, 48 h and 72 h). Compounds that reduced the motility of xL3s were also tested for their ability to inhibit the motility of L4s (same protocol as for xL3s) and/or to affect the development of xL3s to L4s [14]. Following the measurement of L4 motility, larvae were re-incubated for 4 more days and fixed with 1 % iodine (50 μl). Then, L4 development was assessed based on the presence of a well-developed mouth/pharynx using a light microscope [14] and expressed as a percentage of the total number of larvae examined (n = 30).
All assays (to assess xL3 motility, L4 development and L4 motility) were performed in triplicate, three times, on separate days. Data (MI) from each assay were converted to a percentage compared with the negative control (LB* + 0.5 % solvent), and IC50 values determined using a variable slope four-parameter equation, constraining the top value to 100 % and using a least squares (ordinary) fit model (v.6 GraphPad Software). Significant differences in IC50 values were established using the extra sum-of-squares F-test, employing a P-value of 0.05 (v.6 GraphPad software).
Scanning electron microscopy (SEM)
SEM was used to assess whether compounds that reduced motility by ≥ 70 % caused structural damage to the larval stages. The xL3s and L4s were cultivated as described previously [14, 15]. Compounds were diluted to a final concentration of 100 μM in LB*, and 50 μl were transferred to wells of a 96-well microculture plate; six wells were used for each treatment and for the negative control which contained LB* and 1 % methanol (AR1115-P2.5 L, RCI; Labscan Limited). The xL3s and L4s were resuspended in LB* at a density of ~6,000 per ml, and 50 μl were transferred into individual wells. Compounds were incubated with the larvae for 24 h at 38 °C and 10 % CO2. Following incubation, for each treatment, the larvae from each of the six wells were pooled into 1.5 ml Eppendorf tubes, washed 3 times in 0.9 % saline at 9,000 g and resuspended in 1 ml of phosphate-buffered saline (PBS). Subsequently, the larvae were fixed in 2.5 % glutaraldehyde in PBS for 2 h at room temperature, rinsed three times in PBS for 15 min, and then post-fixed in 1 % osmium tetroxide for 2 h at room temperature. Fixed larvae were rinsed again 3 times in PBS for 15 min. Then, aliquots of concentrated larvae (200 μl) were incubated on polyethyleneimine (PEI)-coated glass coverslips (pre-prepared by smearing 22 mm square glass coverslips with a 0.1 % solution of PEI and dried by heat under a cool flame). Following this incubation, the excess supernatant was removed, and the coverslips with adhered nematodes were dehydrated in increasing concentrations of ethanol (10, 30, 50, 70, 90 and 100 %) in water for 30 min. The coverslips were dried in a critical point dryer (EM CPD030, Leica, Wetzlar, Germany) and mounted on to aluminium stubs (25 mm) with double-sided carbon tabs. The coverslips were then coated with gold using a Xenosput sputter coater (Dynavac, Australia). The larvae on the coverslips were imaged using a field-emission scanning electron microscope (XL30 Philips, Netherlands) at 2.0 kV using a spot size of two; 12 representative images were taken of each sample.
Estrogen receptor binding affinity
Relative binding affinities were determined by a competitive fluorometric binding assay, as described previously [29]. Briefly, 40 nM fluorescence tracer (coumestrol, Sigma-Aldrich) and 0.8 μM purified human ERα or ERβ ligand binding domain were diluted in 100 mM potassium phosphate buffer (pH 7.4), containing 100 μg/ml bovine γ-globulin (Sigma-Aldrich), and an equal volume of test compound was added. Incubation was for 2 h at 25 °C. The fluorescence polarisation values were measured. Binding affinities are expressed as relative binding affinity (RBA) values, with the RBA of 17-β estradiol set to 100 %. The values given are the average ± range of two independent determinations. IC50 values were calculated using a published formula [29].
Gene transcriptional activity
The human embryonic kidney cell line, HEK 293 T, was maintained in Dulbecco's Minimum Essential Medium (DMEM) (Gibco, Thermo Fisher Scientific, Scoresby, Australia) with 10 % foetal bovine serum (FBS) (HyClone, GE Healthcare Life Sciences, Pittsburgh, USA). Cells in phenol red-free DMEM with 10 % FBS were plated. HEK 293 T cells were transfected with 25 μl (per well) containing 300 ng of 3 × estrogen response element (ERE)-luciferase reporter, 100 ng of either ERα or ERβ expression vector, 125 mM calcium chloride (GuoYao, China) and 12.5 μl HEPES-buffered saline (2× HBS). The next day, the cells were treated with increasing doses of ER ligands diluted in phenol red-free DMEM with 10 % FBS. After 24 h, luciferase activity was measured using the dual-luciferase reporter assay system (Promega, Fitchburg, USA) performed according to the manufacturer's protocol.
Primary screening results, and half maximum inhibitory concentrations (IC50) values of compounds with activity on xL3 and L4 stages of H. contortus
In the primary screen of the 74 compounds (Fig. 1; Additional file 1), one selenophene-core compound (WZY-2) and two thiophene-core compounds (MJ-17 and MJ-22) were found to inhibit xL3 motility by > 70 %. Dose–response experiments showed that the IC50 values of these compounds ranged from 6.6 μM to 25.7 μM, with WZY-2 and MJ-22 being significantly more potent at inhibiting xL3 motility than MJ-17 at all three time points examined (24, 48 and 72 h) (Fig. 2; Table 1). Similar to moxidectin, all three compounds did not become significantly more potent over time, with no significant difference in the IC50 values of compounds at 24, 48 and 72 h (Table 1); however, although the three test compounds and moxidectin (control) had a more rapid effect on the nematode than monepantel (control), as indicated by lower IC50 values at the earlier time points (24 h and 48 h), monepantel was most potent at inhibiting xL3 motility at 72 h (Table 1). Subsequently, we assessed the development of xL3 to L4 in the presence of each of the three test compounds (WZY-2, MJ-22, MJ-17). All three compounds as well as moxidectin and monepantel had an inhibitory effect on L4 development, which was similar to that of moxidectin, with IC50 values for WZY-2, MJ-22 and MJ-17 ranging from 10.5 to 13.7 μM, and with the monepantel control compound being the most potent at inhibiting L4 development (Table 1). Compounds WZY-2, MJ-22 and MJ-17 were also found to inhibit the motility of L4s, with IC50 values ranging from 3.2 to 13.9 μM. Although there was no observable difference in potency between WZY-2 and MJ-22 on xL3 and L4 motility at all three time points examined, MJ-17 was significantly more potent at inhibiting the motility of L4 (IC50: 6.1–6.2 μM) than xL3 (IC50: 23.8–25.6 μM) at both 48 h and 72 h (extra sum-of-a-squares F-test: F = 5.947, df = 108, P = 0.0164; Table 1; Fig. 2).
a Inhibitory properties of the 74 compounds at 20 μM on exsheathed third-stage larvae (xL3s) are displayed as the percent of motility compared with the positive (+) and negative (−) controls. Negative controls were xL3 treated with Luria Bertani (LB) medium supplemented with 100 IU/ml of penicillin, 100 μg/ml of streptomycin and 2.5 μg/ml of amphotericin (LB*) containing 0.5 % dimethyl sulfoxide (DMSO) or methanol (MeOH); LB* + 0.5 % DMSO/MeOH. b The structures and motility indices of compounds that have a similar structure to the active compounds, MJ-17, MJ-22 and WZY-2 (bold), are also shown for comparative purposes. The motility indices for individual compounds were calculated from the mean of triplicate motility indices, with the variance represented by the standard error of the mean (± SEM)
Dose–response curves for the effects of each of the selenophene-core (WZY-2) and thiophene-core (MJ-17 and MJ-22) compounds on parasitic stages of Haemonchus contortus in vitro. Graphs showing the inhibition of motility of third-stage larvae (xL3s) at 24 h, 48 h and 72 h (a-c) for individual compounds; inhibition of development (d-f) and motility (g-i) of fourth-stage larvae (L4s) after seven days. Each data point represents the mean of three experiments (± standard error of the mean, SEM)
Testing of selenophene and thiophene-core ligands (WZY-2 and MJ-22, MJ-17) on Haemonchus contortus in the biological assay. A comparison of the 'half of the maximum inhibitory concentration' (IC50) values of individual ligands with those of the two reference anthelmintics (monepantel and moxidectin)
WZY-2
MJ-22
Monepantel
Moxidectin
xL3 motility (IC50s in μM)
25.7a,b
25.6c,d,g
12.1e
9.6f
23.8e,f,h
L4 motility (IC50s in μM)
L4 development (IC50s in μM)
IC50 values with the same superscript letter are significantly different from each other (P < 0.05)
SEM of the xL3 and L4 stages of H. contortus
We examined by SEM the morphology of xL3 and L4 stages of H. contortus following incubation with 100 μM of WZY-2, MJ-22 and MJ-17 for 24 h. Although no structural damage was observed in xL3s compared with the untreated control worms, distinct damage was evident on the surface of L4s treated with each of these three compounds (Fig. 3). Compared with the surface of L4s incubated for 24 h in the solvent (DMSO) control, all three compounds disrupted the cross-sectional striations of the cuticle, induced the appearance of horizontal ridges and indentations and, in some cases, resulted in severe perturbation of the outer cuticle (Fig. 3). These features were similar to the cuticle damage observed following the treatment with 100 μM of monepantel. Moxidectin did not induce any cuticle damage in L4s at the concentrations tested, which seems consistent with a distinct mode of action (binding to ligand-gated chloride channels), resulting in influx of chloride ions and flaccid paralysis of the parasite [30].
Representative scanning electron micrographs (magnification: 8,000×) of fourth-stage larvae (L4s) incubated for 24 h in either a solvent (DMSO) control, or 100 μM of each WZY-2, MJ-22, MJ-17, monepantel or moxidectin. Scale-bars: 5 μm
Transcriptional activation assays/estrogen receptor (ER) binding affinity
The binding affinities of the selenophene-core and thiophene-core ligands for both ERα and ERβ were determined by a competitive fluorometric receptor-binding assay. These affinities are presented as relative binding affinity (RBA) values, with estradiol having an affinity of 100 %. The thiophene-core compound, MJ-17, had the highest affinity to both ERs, with RBA values of 13.07 and 17.5 for ERα and ERβ, respectively, and exhibited ERβ selectivity. Compound MJ-22, which is an asymmetrically substituted compound, had lower binding affinities to these ERs (RBA values of 0.97 and 0.91 for ERα and ERβ, respectively) than corresponding symmetrical compounds. Compound WZY-2, a selenophene-core compound, had RBA values of 6.11 and 12.7 for ERα and ERβ, respectively. In the ER-responsive luciferase reporter gene assay, the ligands WZY-2 MJ-22 and MJ-17 induced transcriptional activities of genes encoding ERα and ERβ, based on comparison with 17β estradiol (E2). The luciferase assay was conducted in HEK 293 T cells transfected with the 3 × ERE-luciferase reporter. The result showed that compound WZY-2 was shown to act as an antagonist on ERβ with an IC50 value of 3.02 μM and a high efficacy (Table 2). Additionally MJ-17 displayed a > 25-fold higher EC50 value on ERα and ERβ than the corresponding 2,3-diaryl thiophenes in the micromolar range, whereas MJ-22 profiled as a super-agonist on both ERα and ERβ, being at least twice as efficacious on ERβ than estradiol (Table 2).
Effects of selenophene- and thiophene-core ligand conjugates on the transcriptional activities of estrogen receptors α and β
Agonist modea
Antagonist modeb
ERα
ERβ
EC50 (μM)
Efficacy (%E2)
IC50 (μM)
Efficacy (%E2)c
Eff (%E2)
-21 ± 13
73 ± 8
13.9 ± 5.3
117 ± 8
277 ± 15
aLuciferase activity was measured in HEK 293 T cells transfected with 3 × estrogen response element (ERE)-driven luciferase reporter and expression vectors encoding estrogen receptor α (ERα) or ERβ and treated in triplicate with increasing doses (up to 10-5 M) of the compounds. Half maximal effective concentrations (EC50) and standard deviation (mean ± SD), shown as a percentage of 10-8 M 17β-estradiol (E2), were determined
bHalf maximal inhibitory concentrations (IC50) and standard deviation (mean ± SD) were determined in the percentage of 10-8 M 17β-estradiol (E2) on ERα or ERβ
cERs have considerable basal activity in HEK 293 T cells. Omitted EC50 or IC50 values were too high to be determined accurately
Infections and diseases caused by parasitic worms impose a substantial economic burden to the livestock production industry worldwide, and cause a loss of > 5 million disability-adjusted life years (DALYs) in humans [2, 31, 32]. The control of parasitic nematodes of animals relies heavily on the use of chemical treatments [33]. Consequently, the identification of new anthelmintics is pivotal to circumvent the rapid spread and widespread occurrence of drug resistance in parasitic nematodes populations, particularly of small ruminants [4–11]. In an effort to work toward identifying possible new candidates, we screened, using a recently developed and validated in vitro-screening platform [14, 15], a compound library containing 74 compounds, and identified three hetero-cyclic core SERMs, designated WZY-2, MJ-22 and MJ-17, which have a potent inhibitory effect on both the motility and development as well as the structural integrity of parasitic larvae of H. contortus in vitro.
SERMs are defined as molecules that act as antagonist or agonist upon binding to ERs [34]. There are two well-known ER subtypes in humans, ERα and ERβ, which are proposed to contribute to the high selectivity and target-site specificity of SERMs [34–37]. For instance, the SERM tamoxifen, used to treat breast cancer, acts as an antagonist of the ERα in breast parenchyma. However, in the endometrial and skeletal tissues, this SERM acts as an agonist on the ERβ, and can increase the risk of uterine cancer [34, 38]. The three SERMs identified in this screen are structurally quite distinct from tamoxifen. MJ-17 and MJ-22 are thiophene-core compounds that act as agonists on human receptors ERα and ERβ. By contrast, WZY-2, a selenophene-core compound that differs from MJ-17 at only one position on the pentane ring (selenium instead of sulphur), acts as an antagonist on the ERβ receptor (Table 1; Fig. 1).
Of the 74 compounds screened, nine SERMs had structural similarity to WZY-2, MJ-22 and MJ-17, but did not have any effect on the motility of xL3s of H. contortus. The structures of these SERMs and their ability to reduce xL3 motility at 20 μM are shown in Fig. 1. Since selenium is, in many respects, similar to sulfur in its properties, from a medicinal chemistry perspective, two structural activity relationship (SAR) classes can be categorized, depending on the position of diphenyl substitution on the heteroaryl ring, being either a 2,5-substitution to give more "linear" molecules, such as WZY-2 and MJ-22, or a 3,4-substitution to provide a more "compact" system, such as is the case for MJ-17.
Considering first the 2,5-diphenyl substituted series, as exemplified by selenophene WZY-2 and thiophene MJ-22, replacing the chloro substituents in WZY-2 with fluoro substituents results in WZY-3, with a greatly increased xL3 motility index of 100 %. Thus, the chloro groups are highly favourable. Since WZY-3 is already very hydrophobic, with a CLogP of 5.5, the improvement in activity is unlikely to relate to the hydrophobicity-induced improved transport to the parasite, but rather an occupation of local hydrophobic binding pockets. The ortho substitution will cause the biaryl system to be non-planar, but this conformation should be readily achievable in WZY-3, such that conformational preferences are unlikely to be the cause of the differential biological activities. When the selenium atom of WZY-2 is replaced with a sulfur atom, to give MJ-29, there is a decrease in inhibitory activity, with an xL3 motility index of 67 %. Further, the potent activity of MJ-22 (xL3 motility index: 6.7 %; Fig. 1) is quite remarkable, since it possesses phenolic substituents (methyl and fluoro) that would be expected, on the basis of the above observations for WZY-2 and WZY-3, to be significantly less active. These two observations are suggestive of an SAR sub-stream for 2,5-diphenylthiophenes that is different from 2,5-diphenylselenophenes, perhaps as a result of the smaller bond lengths involved with sulfur compared with selenium, causing a less linear overall topography. Little more can be added to the discussion at this stage, without an analysis of a more extensive set of 2,5-diphenylthiophenes. That at least one of the phenolic hydroxy groups of WZY-2 is important for activity is witnessed by the limited reduction in motility of H. contortus caused by WZY-1 (Fig. 1), the direct analogue of WZY-2, for which both phenolic hydroxyl groups have been methylated. WZY-4 and WZY-5 have both "lost" activity, but multiple substituent changes in the phenyl and selenophene rings make it challenging to explain the cause of this loss of activity and to establish whether it is due to the bromination of the selenophene or the altered substitutions in the phenyl rings.
An analysis of the 3,4-diphenyl-substituted thiophene series, as exemplified by MJ-17, immediately reveals that the position of the sulfur atom is crucial for good activity, since MJ-32, with the sulfur atom effectively rotated one place anti-clockwise in this resultant 3,4-diphenylthiophene, has lost all activity. As one would not expect a large conformational effect on these rigid scaffolds, this result suggests that the sulfur atom in MJ-17 plays a key role in the binding interaction with the host target, possibly through the ability of sulfur to polarize, to a great degree, in order to "favourably adapt" to a protein's "local environment" [39, 40]. That the chloro groups assume an important role is illustrated by the fact that their absence from MJ-35 results in a complete loss of activity. As the chloro groups are in ortho positions, an obvious influence on biological activity could be the direction of a favourable binding conformation, but this could be combined with a specific hydrophobic interaction with the target protein (ER). It seems unlikely that these groups contribute to transport to the site, as MJ-35 is already relatively hydrophobic, with a CLogP of 4.7. Poor activity for MJ-4 reveals the importance of at least one hydroxyl group for better activity, while similarly poor activity for MJ-33 would be consistent with a suboptimal engagement of an ortho-chloro binding pocket.
Potent activity in two topographically distinct series can often suggest non-specific modes of action and, indeed, polyphenols are known to non-specifically alter membranes at micromolar concentrations [41]. However, poor activity for analogs, such as WZY-3, MJ-29 and WZY-4 of the 2,5-substituted series, and MJ-32 and MJ-35 of the 3,4-disubstitued series is reassuring, and suggests that the activity of MJ-17 is specific. Furthermore, ERs in mammalian cell lines have been shown to bind these two types of topographically distinct series, and a similar SAR is observed (Table 2.). This information is supportive of a specific SERM mode of action, but also raises the challenge of obtaining divergent SAR between host and parasite ERs.
Previously, SERMs, such as tamoxifen and raloxifene, have been shown to reduce the burden of parasitic cestodes in experimental infections in hamsters or mice [18, 20, 21, 23]. In particular, tamoxifen is believed to interfere with the ER pathway in the parasite, as high levels of estrogen have been shown to increase the viability and reproductive rate of the asexual (cysticercus) stage of T. crassiceps, and treatment with this drug was shown to reduce the production of ER-like proteins [22, 23]. It is possible that the SERMs studied here (i.e. WZY-2, MJ-17 and MJ-22) might block a similar pathway, leading to reduced motility/viability as well as fertility in the ensuing H. contortus adults, an aspect warranting future study.
Thiophene compounds, similar to MJ-22 and MJ-17, but differing mainly by the loss of one of the phenyl groups have been associated with anti-parasitic activity in worms, such as H. contortus and the closely related species, Trichostrongylus colubriformis [42]. Gonzalez et al. [42] reported a series of thiophene analogues with potent in vitro activity, inhibiting the motility of ensheathed L3s of H. contortus and T. colubriformis. However, activity against these two nematode species was not detected in vivo in infected gerbils. This lack of in vivo activity of these thiophene compounds was hypothesized to relate to differences in how the drugs interact with free-living L3s in vitro compared with parasitic L4s and adults in vivo [42]. An advantage of the present screening platform [14, 15] is that compounds can be tested in vitro for activity on the parasitic (i.e. xL3s and L4s) rather than free-living stages, overcoming possible variation in efficacy linked to free-living stage usually tested in vitro (i.e. L1-L3) vis-à-vis the parasitic stages in vivo [42].
While WZY-2, MJ-17 and MJ-22 were initially identified in a primary screen against the xL3 stage, all compounds had inhibitory activity on L4s. WZY-2 and MJ-22 were found to be significantly more potent than MJ-17 at impeding xL3 motility, suggesting a clear delineation in efficacy between the "linear" SERM series and "compact" SERM series. However, all compounds were found to have a similar potency at inhibiting both motility and development of the L4 stage. When comparing the activity of the compounds on the two parasitic larval stages (xL3 and L4), MJ-17 was found to be more potent at inhibiting the motility of L4s than of xL3s. However, this was not the case for WZY-2 and MJ-22, which were shown to have a similar inhibitory activity on both of these larval stages. This differential efficacy against these stages was reflected in scanning electron micrographs, showing distinctiveness in the phenotypes of the two larval stages following incubation with each of the three SERMs (WZY-2, MJ-22 and MJ-17) for 24 h. While no adverse effect of individual compounds was observed on the morphology of xL3s, despite an inhibition of their motility, treated L4s were severely damaged, to a similar extent to that observed using the equivalent dose of monepantel (Fig. 3). The differences in the phenotype between xL3s and L4s, despite the same loss in motility at the same concentration, could relate to a number of reasons, such as a differing magnitude of drug uptake by the parasite, binding to the target(s) and accumulation therein. Indeed, a key morphological difference between xL3 and L4s is the presence of a prominent mouth/pharynx in the L4s compared with xL3s [43, 44]. Therefore, drug uptake (via ingestion) might be considerably higher in L4s through active feeding [44] than in xL3s, in which absorption/diffusion could be mainly trans-cuticular. Moreover, it is possible that the drug target(s), presently proposed to be ERs, might be expressed at a higher level in the L4 stage, allowing greater binding and accumulation of the compounds, thus effecting reduced motility and pronounced morphological alterations in this developmental stage.
This study has identified three SERMs with potent anti-nematode activity on parasitic larvae of H. contortus. These SERMs might have potential as drugs against this and related nematodes, provided that analogs can be designed that act specifically on the nematode and not the host animal. Further work needs to confirm that the target(s) of these SERMs in H. contortus are indeed ERs and explore their structures. The design of nematode-specific SERMs, as probes, would also allow for explorations of ERs and associated pathways in H. contortus and related parasites.
Support from the Victoria Life Sciences Computation Initiative, Australia (VLSCI; grant no. VR0007) on its Peak Computing Facility at the University of Melbourne, Australia, an initiative of the Victorian Government, Australia is gratefully acknowledged (RBG) as is other support from the Medicines for Malaria Venture (MMV), YourGene Bioscience (Taiwan) and Melbourne Water (RBG).
The present study was funded by the Australian Research Council (ARC), the National Health and Medical Research Council of Australia (NHMRC) and the Wellcome Trust (Pathfinder) (RBG). JB, who is a Larkins Fellow and in receipt of an NHMRC Senior Research Fellowship, also receives funding support from NHMRC and ARC. MH's research is supported by grants from the National Natural Science Foundation of China (NSFC) (no. 31172310), National Key Basic Research Program (973 program) of China (no. 2015CB150300) and the Special Fund for Agro-scientific Research in the Public Interest (no. 201303037). HBZ's research is supported by grants from the NSFC (nos. 81373255, 31371331 and 81573279), Key Scientific Research Project of Ministry of Education of China (313040) and Hubei Province's Outstanding Medical Academic Leader Program. The funding bodies played no role in the design of the study or collection, analysis or interpretation of data, or in the writing of the manuscript.
Conceived and designed the study and supervised the project: RBG and HBZ. Undertook the study and data analysis: SP, JL, YZ and AJ. Undertook electron microscopy: SC. Reagents or project consultation: MH, JB and AH. Contributed to analysis using various tools: SP, AJ and SC. Wrote the paper: RBG and SP. All authors read and approved the final version of the manuscript.
Animal ethics approval (AEC no. 0707258) was granted by The University of Melbourne.
Additional file 1: Details of the synthetic routes for the 22 selenophene-core and 52 thiophene-core analogues screened against Haemonchus contortus in the present study. The characteristics of these compounds are also given. (DOC 2062 kb)
Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, VIC, 3010, Australia
Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Wuhan University School of Pharmaceutical Sciences, Wuhan, 430072, China
Medicinal Chemistry, Monash University Institute of Pharmaceutical Sciences (MIPS), Monash University, Parkville, VIC, 3052, Australia
State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei, 430070, China
School of Biosciences, The University of Melbourne, Parkville, VIC, 3010, Australia
Structural Chemistry Program, Eskitis Institute for Drug Discovery, Griffith University, Brisbane, QLD, 4111, Australia
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text:
<?php

namespace hiapi\event;

use DateTimeImmutable;
use Ramsey\Uuid\UuidInterface;

/**
 * Interface EventInterface
 *
 * @author Dmytro Naumenko <d.naumenko.a@gmail.com>
 */
interface EventInterface
{
    /**
     * Provides the event id
     *
     * @return UuidInterface
     */
    public function uuid(): UuidInterface;

    /**
     * @return DateTimeImmutable
     */
    public function createdAt(): DateTimeImmutable;

    /**
     * @return string The event name
     */
    public function type(): string;

    /**
     * Stop event propagation.
     */
    public function stopPropagation();

    /**
     * Check whether propagation was stopped.
     *
     * @return bool
     */
    public function isPropagationStopped();
}

redpajama_set_name: RedPajamaGithub


Old Dominion Stout is brewed with a combination of 2-row pale, Carapils, Chocolate, & Caramel malts. Immediately, you should detect a deep rich roasted nose indicative of the full-flavored, creamy dark brew that is to follow. Jerry hops this full-bodied stout with Perle hops up front to add a slight bitterness and then later with Willamette hops for their aromatic contributions. Old Dominion's Stout is brewed as an Irish cream stout. Did someone say St. Patrick's Day? Yes, we planned it that way. Pretty slick, huh? The complex flavor is well balanced yet mellow, with coffee and chocolate notes throughout. You might consider pairing it with grilled mahi-mahi, seared ahi tuna, or some dark flavorful bread and a creamy, aged Stilton. For other unique cheese and beer pairings, you might want to check out our Gourmet Cheese of the Month Club!
Don't drink more ' Drink Better'. So goes the slogan of the Old Dominion Brewing Company. Old Dominion, established in 1989, has the proud honor of being the first microbrewery to serve our nation's Capital. Jerry Bailey's home brewing career ended when he started Old Dominion Brewing Company in 1989. Thank the powers that be for this sweet moment in time. His brewing philosophy is simple and pure: to make the best beer possible. A man has to have goals and we support this one!
Old Dominion has invested in state of the art brewing equipment, purchased the finest ingredients, and used the most appropriate brewing techniques to make world-class beers. The Old Dominion Brewpub is located in the brewery and serves up mouth-watering burgers, salads, soups, and other pub fare. Boasting with a well deserved air of self confidence, Jerry states,' I don't know of a single thing we could do that we are not doing to make better beer'.
Like Penn, Old Dominion uses only malted barley, hops, east and, the inevitable, water to craft their brews. No preservatives or adjuncts are used and the beers are never pasteurized. The brewery sold its first beer back in May of 1990 and by 1992, they were selling just under 6,000 barrels of beer annually. Dedicated to producing fresh, flavorful, full-bodied beers, Old Dominion offers not only a high quality product but a plethora of styles as well. A privately owned brewery located in Loudoun County about three miles north of Dulles Airport, the brewery creates over twenty beers, many of which are seasonal beers only brewed several months out of the year. The beers are sold exclusively only in Washington, D.C., Virginia, and in parts of Maryland. Of course if you're really nice and visit our site at www.beermonthclub.com, we'll gladly ship you out some more of these tasty treats for a modest monetary pittance.
For more information about the brewery and scheduled tours, call (703) 724-9100 or check out their web site at www.olddominion.com.


text:
Vital Signs Issues
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50 Plus Program
Essential oils have gone mainstream, offering proven therapeutic value
For many people, the fragrant scent of essential oils derived from flowers, trees or spices are a soothing balm to calm nerves and promote relaxation and wellbeing. A growing body of research now attests to the therapeutic value of these plant-derived elixirs. "Essential oils have made their way into mainstream practice," says Elizabeth Ko, MD, medical director of the UCLA Health Integrative Medicine Collaborative.
"And it begs the question of the larger practice of integrative medicine, which blends conventional medicine with these nonpharmacologic, evidence-based practices, like essential oils, massage, yoga and acupuncture."
Dr. Elizabeth Ko. Photo: UCLA Health
Dr. Ko: "Essential oils are volatile liquid substances extracted from plants, usually flowers, herbs or trees. The liquid is extracted through steam distillation or by machine, and during that process, the fragrance is released, hence the word 'aromatic.' These oils are highly concentrated. For example, it takes about 220 pounds of lavender flowers to make one pound of essential oil. Essential oils are quickly absorbed by smell receptors that are linked to the limbic system, which controls heart rate, blood pressure, breathing and stress. Every plant has a different chemical makeup that affects its smell absorption and its effect."
How are essential oils used?
Dr. Ko: "These potent plant oils can be inhaled directly — taking a whiff from the bottle or from a cotton ball or tissue drizzled with a couple of drops — or indirectly, through a diffuser that spreads atomized fragrance through the air. Diluted essential oils may also be used topically by adding a few drops to a carrier oil, such as a vegetable or nut oil, as is often done for aromatherapy massage. While essential oils are not regulated by the U.S. Food and Drug Administration, they are generally recognized as safe when used as recommended. Aromatherapy has a relatively low toxicity profile when administered by inhalation or dilute topical application. It is important to note that we do not ingest essential oils."
What is the role of essential oils in the hospital setting?
Dr. Ko: "Units throughout our hospitals have sets of three essential oils — lavender, lemon and peppermint — that medical personnel can offer to patients. Members of our integrative therapy team provide patients with relaxation services that include aromatherapy with essential oils, guided meditation and breath work, gentle in-bed movement and Reiki, a light touch stress-reduction technique. Aromatherapy, which has been offered at UCLA Health for many years, may be the most accessible of the available relaxation modalities. Lavender oil appears to be the most popular in the hospital. Patients associate lavender with relaxation, and that's why they tend to pick it more often. Lemon is more uplifting, while peppermint is invigorating.
How do essential oils affect people?
Dr. Ko: "The smell of lavender is familiar and calming to many, and it's also the moststudied essential oil. Lavender interacts with the neurotransmitter GABA to help quiet the mind and the nervous system. Studies show that lavender oil can improve sleep quality and increase time spent in deep, slow-wave sleep. Another study suggests lavender may work as effectively as anti-anxiety medicines, such as Xanax or Ativan. I encourage my patients who have anxiety disorder or panic disorder to carry a vial of essential oil close at hand. When the temptation hits to pop a pill, I ask them to pause and take a deep breath with a touch of lavender oil. If it's effective, then great. If they do need something more afterward, at least they tried a lower-risk, lower-cost intervention. Aromatherapy with bergamot essential oil, a type of citrus, also has been shown to ease feelings of anxiety and stress. Lemon oil, which smells like ripe fruit, has been found to boost mood. Peppermint oil, which has an eye-opening, bright scent, appears to increase memory and alertness.
What are some other popular oils?
Dr. Ko: "Cinnamon, chamomile, tea tree and eucalyptus, which one study found effective in reducing pain and inf lammation. When shopping for essential oils, I recommend that people look for formulations that are 100% pure. Beware of ultra-low prices, as pure oils generally cost at least $10 for a half-ounce bottle.
Dr. Ko: "Effects from essential-oil aromatherapy tend to be short-lived. It's not on the order of hours; probably more on the order of minutes. Still, a half-ounce bottle will likely last months, even with frequent use, since only a drop or two is needed to produce perceptible fragrance. With the variety, general safety and increasingly documented efficacy of essential oils, I suggest that those who are cur ious take an exper imental approach to see what smells they like and what feelings are elicited. It can be a nice way to explore."
For more information about the UCLA Health Integrative Medicine Collaborative >
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redpajama_set_name: RedPajamaCommonCrawl


The invention provides a method and device for transmitting and receiving services. The method includes: determining the transmission technology corresponding to the service to be sent, in which the transmission technology includes at least the first transmission technology and the second transmission technology, which are different from the corresponding UE version of the receiving user equipment, and determining the transmission resources corresponding to the transmission technology. And sends the service to be sent on the determined sending resources. The invention solves the problem of poor compatibility of service transmission caused by different versions of UE can not work together in related technology, and achieves the technical effect of improving the compatibility of service transmission.


Turun Lilja (The Lily of Turku) by Wäinö Aaltonen (1894-1966) stands in Runeberg Park next to the Aura Bridge. It was the first outdoor sculpture acquired by the City of Turku. This modernist sculpture was Aaltonen's third monument, and it was realised in granite in the stoneworks of J.E. Forsman in Helsinki. The sculpture was completed in 1924-1926 and unveiled in 1928.
The sculpture was originally named "The Lily of France", but Aaltonen requested the name be changed. The new name and the lily (pictured in the Turku coat of arms) at the feet of the woman have made this sculpture a true symbol of Turku.
Turun Lilja has been a part of the spring celebrations in Turku since 1929. Every May Day Eve she is capped and then cleaned with a giant tooth brush by the dental students. These activities have been approved by the Museum Centre of Turku so as not to damage the sculpture.
The outdoor sculptures can be found on the Citynomadi app. While the texts presenting the artworks are in Finnish, the app will guide you through the city and help you locate the art. Enjoy!


text:
Class Roster
Archived Rosters
MAE 4530
Schedule of Classes - January 29, 2023 7:36PM EST
Course Catalog - January 29, 2023 7:15PM EST
Computer-Aided Engineering: Applications to Biological Processes
Course information provided by the Courses of Study 2022-2023.
Introduction to simulation-based design as an alternative to prototype-based design; modeling and optimization of complex real-life processes for design and research, using industry-standard physics-based computational software. Emphasis is on problem formulation, starting from a real process and developing its computer model. Modeling application (project) can be biomedical (thermal therapy and drug delivery) or broader biological and bioenvironmental applications that involve heat transfer, mass transfer, and fluid flow. Computational topics introduce the finite-element method, model validation, pre- and post-processing, and pitfalls of using computational software. Students choose their own semester-long project, which is a major part of the course (no final exam).
When Offered Spring.
Prerequisites/Corequisites Prerequisite: BEE 3500, MAE 3240, CHEME 3240 or equivalent.
Satisfies Requirement Fulfills technical elective requirement for M.E. students. Satisfies capstone design requirement for BE students. Satisfies College of Engineering technical communication requirement.
Students will improve their ability to identify, formulate, and solve complex engineering problems by applying principles of engineering, science, and mathematics (ABET 1).
Students will demonstrate improved ability to apply engineering design to produce solutions that meet specified needs with consideration of public health, safety and welfare, as well as relevant global, cultural, social, environmental and economic factors (ABET 2).
Students will display an ability to communicate effectively with a range of audiences (ABET 3).
Students will demonstrate an ability to recognize ethical and professional responsibilities in engineering situations and make informed judgements which consider the impact of engineering solutions in global, economic, environmental, and societal contexts (ABET 4).
Students will demonstrate an ability to function effectively on a team whose members together provide leadership, create a collaborative and inclusive environment, establish goals, plan tasks and meet objectives (ABET 5).
Students will demonstrate the ability to acquire and apply new knowledge as needed, using appropriate learning strategies (ABET 7).
Students will improve their capacity to integrate modern biology with engineering principles (ABET-BE).
View Enrollment Information
Syllabi:
Regular Academic Session. Combined with: BEE 4530, BEE 5530, MAE 5530
Credits and Grading Basis
3 Credits Graded(Letter grades only)
Class Number & Section Details
9342 MAE 4530 LEC 001
Meeting Pattern
MWF 10:10am - 11:00am Phillips Hall 407
Jan 23 - May 9, 2023
Datta, A
To be determined. There are currently no textbooks/materials listed, or no textbooks/materials required, for this section. Additional information may be found on the syllabus provided by your professor.
For the most current information about textbooks, including the timing and options for purchase, see the Cornell Store.
Instruction Mode: In Person
Prerequisite/Corequisites: heat and mass transfer (BEE 3500 or equivalent).
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About the Class Roster
The schedule of classes is maintained by the Office of the University Registrar. Current and future academic terms are updated daily. Additional detail on Cornell University's diverse academic programs and resources can be found in the Courses of Study. Visit The Cornell Store for textbook information.
Please contact coursenroll@cornell.edu with questions or feedback.


This extraordinary double bill brings together two leading Nordic ensembles, one Swedish and one Danish, that will include a 400 year range of folk music traditions.
Swedish trio Väsen's primary influence is the folk music and traditional instruments of their homeland, namely the nyckelharpa, and their music stretches boundaries to incorporate fluid improvisation and compositional additives. The Danish String Quartet's love of traditional Scandinavian folk music is becoming as well known as the quartet's masterly approach to the classical repertoire. The four strings present their favorite melodies from Nordic folk music, including selections from their recent release, Wood Works, a critically acclaimed, sonically lustrous and cohesively conceived collection of music that is rooted in their ancient melodies from Nordic folk music.


Massive year-on-year value gains can be witnessed across Australia's real estate market, as new sales data begins to reveal the true extent of property price growth experienced in 2015.
Median house prices in at least one suburb in every state and territory but for the ACT grew by more than 40 per cent in the 12 months to October 2015, according to the latest CoreLogic RP Data.
Leading the charge is the NSW suburb of Brewarrina, located in the state's north west.
The median house price in the tiny town of 1,300 people grew by 50 per cent over 12 months, to sit at a current figure of $123,750.
The northern Wollongong suburb of Wombarra was the second highest-growing suburb in the country, with house prices recording growth of 49.88 per cent to sit on a current median price of $1,259,000.
While NSW made up the bulk of the 41 high-growth suburbs – eight of which achieved growth above 45 per cent – suburbs in other states and the Northern Territory returned equally impressive figures.
Ripponlea, in Melbourne's south-east, recorded growth of 48.69 per cent based on a median house price of $1.42 million.
House prices in Murchison, a small village 40 kilometres from Shepparton, grew by 45.61 per cent to $215,500.
Queensland was the state to record the second highest number of suburbs growing in excess of 40 per cent over 12 months – with six suburbs meeting the mark.
Harlaxton was the state's best performer, unit prices growing by 44.55 per cent to $305,000.
Other top performers included Coolangatta, with house price growth of 43.68 per cent, and Brookwater, with unit price growth of 43.15 per cent.
In South Australia, the Yorke Peninsula town of Stansbury returned the best performance. House prices there grew by 47.83 per cent to $340,000.
The median house price in Albany, Western Australia, grew to $730,000 – a 45.27 per cent increase that secured its spot as that state's highest growth suburb.
Bayview in the Northern Territory recorded median house price growth of 46.07 per cent to sit on a current figure of $1,152,500.
The town of Strahan – on Tasmania's west coast – recorded a median house price of $245,000, representing a 44.12 per cent spike.
The only jurisdiction to miss out on recording growth in excess of 40 per cent was the ACT.
It's highest growth suburb was Wright, on 37.77 per cent.
To see more results, visit our Best Suburbs section.
What does 2016 have in store for property investors?
Is apartment living the new normal?


Stories that tackle head-on social media in fiction seem few and far between despite it being something most of us interact with daily. This is a story about a relationship and what happens when a relationship ends and what happens when you still go on Instagram to look at their Instagram and maybe their new girlfriend's Instagram and maybe when you're making your Instagram posts you're really thinking about what they will think when they look at their Instagram. Sherwood captures so accurately the pressure of watching and being watched online, the careful, carefree performance of it, the rhythm it becomes in our daily lives.


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Classic Tales to Mirror When Telling Your Brand Story
Are you more of an Odysseus or a Scarlett O'Hara? Does your journey mimic that of Frodo's in "The Lord of the Rings" saga, or is it closer to Luke Skywalker's in "Star Wars"?
These questions aren't as trivial as they may seem. Storytelling has connected civilizations for millennia, bringing a sense of adventure into the most mundane voyages. Who wants to read about a guy on a boat with an obsession for a sea creature? All the readers of "Moby Dick," that's who.
Weaving tales makes you more memorable and could save your company from languishing in the annals of entrepreneurial history. And so the question becomes: How does your brand story find its happy ending?
A Progressive Policy Institute study shows the rate of startups rose to 2.5 percent in fall 2016, indicating a bounceback in entrepreneurial ventures. Yet those positive numbers are tempered by reports like the Statistic Brain study that noted more than half of companies fail before their five-year anniversaries; fewer than 30 percent even see the 10-year mark.
A sobering fact, but in a world of trendy startups with cutesy names and cool apps, someone should stand out from a sea of startup copycats. The trick? Mirror classic stories to develop your brand story, connect with your audience and provide a product or service they won't find anywhere else. The result? The "happily ever after" you need to succeed.
Was it a dark and stormy night?
A hook is needed to set apart your startup wheat from the chaff; that means weaving your journey into a classic, unforgettable tale.
Each entrepreneur's path takes a one-of-a-kind trajectory. By harnessing timeless storytelling strategies, inspire your industry and consumers. Consider those classic frameworks that make the foundation of everything from Aesop's Fables to "War and Peace."
Many well-known anecdotes have roots in the mythology of our ancestors, which means a few tried-and-true setups: "Overcoming the monster" is a common one — "War of the Worlds" comes to mind. Then, there's "rags to riches," perfectly exemplified by "Great Expectations." And of course "rebirth," a traditional way to move a character through life's epiphanies and come out on top.
Chances are strong your startup's message could be transformed with one of these storylines.
A true tale about the benefits of zigging and zagging.
My own journey falls into the "voyage and return" category, a series of zigs and zags all its own.
I skipped out of a corporate path to receive my master's degree in creative writing after discovering the rich history of storytelling while studying literature at the University of Oxford. This deep dive into the literary world — and obsession with tales we tell to connect and build community — took me around the globe, studying with mystic teachers from India to Israel. The storytelling tools I discovered were the missing link for brands struggling to define themselves and build passionate communities around a product or service. After all, it's the stories we tell that move us into action. And it's the mythological frameworks of ancient cultures that provide the soul-level stories that stand the test of time.
Seeing this need in the market, I wanted to share it with companies around the world so they could create mission-driven brands. When I launched my own creative agency, I was faced with challenges — hustling against competitors and launching a business solo — but I had the storytellers' tools to guide me. This transformed the way I created meaningful and powerful content to build strong, passionate communities. Harnessing narratives was an incredible way to take ownership of a story.
Want to capture your own startup saga in words that will transfix and inspire partners, investors and clients? It all starts when you mirror your story with a classic tale and follow a few breadcrumbs:
1. Determine which tale suits you best.
Did you rise above circumstances ("tragedy") to help others and use the experience as a springboard to change others' lives through science? Or were you the class clown in college who loved making people laugh and became compelled to create a social media app that generates fun memes ("comedy")? Consider how you got to where you are today.
Dell is the perfect "rags to riches" example, as are all "I started this business in my garage" tales. Don't worry about seeming trite; people appreciate storylines they can easily follow. No need to reinvent the wheel.
And the Dells of the world aren't alone — everyone has a unique story shaped by the context of life. By acknowledging your story, you can tap into the power that exists in your own experiences. No one else has seen the world the way you have; share your experiences to help and empower others. You're already a hero, but in the sea of mass media, it's easy to forget we're equipped with gifts to share. Believe you're the hero of your story.
2. Find other businesses successfully mirroring the same tale type.
Consider the companies you trust most: Are they already utilizing storytelling elements? Do any of theirs dovetail with yours? What can you learn?
First, you can discern where you are on your path and how to communicate that to others; you may find you're not alone. Countless others have taken the same path before you, and in connecting with their stories, you can build one that will resonate with an even wider audience.
In order to tell a successful story, make sure the hero is identified, as well as any challenges (company or otherwise). Lastly, a "magic power" — a value proposition — should be identified.
Sunrun, the largest U.S. residential solar company, is a perfect example of "overcoming the monster," aka beating "Big Electric." Sunrun offers its solution so consumers can be heroes for family and community. And Tesla is doing something similar with its storytelling about Solar Roof panels, adding a commercial component to save American Samoa through innovation. It's a palpable representation of Tesla as hero, and the heroism extends to individuals and businesses.
3. Flesh out your storytelling adventure.
You'll not only set up your company as the star of your corporate journey, but you'll also include visitors and customers into your tale.
What you need to determine is the following: your characters (target audience); their challenges (pain points); your and their motivations (best outcomes); a setting (connecting the dots); obstacles (what's getting in prospects' way of your great solution); the climax (how you're rescuing individuals, businesses, societies, etc.); and a conclusion (what's necessary to achieve that happy ending?).
After you flesh these out, construct a story concept. Still not sure which story relates best to your company? Consider tales that captivated you and provided your greater mission in life. Don't worry if your answer seems silly or overly complex. Get your creative juices flowing, then write.
The mythology of the corporate landscape may be ever-evolving and seem a little tough to break into for startups, but heroes will always be welcome. Your company's tale is ready to tell.
Source: Entrepreneur.com
Fintech: Africa's payments and remittances space becoming crowded
Why Startups Like Uber Stumble Over Problems They Could Have Avoided
The World's Most Valuable Brands, Africa continues to elude ranking
The one thing that your competitors cannot take from you
Thinking of a business logo? Check out the stories behind these top brand logos, as a guide
The psychology of colour and how it can grow and develop your business brand
How employee experience enhances employer branding
3 WAYS YOUR BRAND REPUTATION CAN EARN YOU MILLIONS
Fintech waiting game is on as players line up to capture Africa's unbanked market


First, if placed in handcuffs by a member of law enforcement, accept it. Period. Trying to get out of cuffs in that scenario never goes well. And, actually escaping makes things go even worse. Worser! Worser should be a word and it would be used here.
However, this blog is geared toward helping writers and we writers, oh, we love escaping stuff. We love "worser," in all its forms. So, this post is for your character who finds theirself (yes, I use theirself as a gender neutral singular possessive) in cuffs.
Ok, last week we discussed how dislocating the thumb to escape handcuffs is not a "thing." In fact, manipulating your body in any manner won't help you. It might allow you to bring your hands to the front of you which will make the actual escape method easier. But, that presumes that your arms can loop under your rump. However, that alone doesn't free you.
Option one: Break the chain between the cuffs like this guy!
What I think he is doing is twisting the chain with enough torque to snap the hinge.
If you look at the circle labeled "keyhole," you will see that a little arm comes off of it. Lift that little arm and you lift the pawl (the green thingy) which frees up the teeth holding the cuffs in place.
How do you lift the pawl? Well, it ain't so hard. All you need is a paper clip or bobby pin. Slip it into the keyhole and move it around until it turns and lifts the pawl.
Option three: Make a shim. You can use a common hair barrette.
See the V shape in the top. The two legs on it can be turned into shims. The shim can be slipped between the ratchet and teeth and allow the cuff to slide loose. A paperclip or any other slim piece of metal can be used the same way. In order to get the shim far enough in, you will have to tighten the cuff a bit to feed it down under the pawl. So, if they cuffs are as tight as they can be, this might not work.
Now that you know how to escape, your focus can be on finding the correct tools to get the job done. They can be in the room or your character can always carry something on their person for this exact purpose. There are shoe strings that house shims and/or handcuff keys under the aglets. What's an aglet? Well, clearly you don't watch Phineas and Ferb. It's that little plastic thing at the end of the shoestring that keeps it from fraying. And, here's a song about it!
Recently, at a writer's conference, I was on a panel called "Blood and Guts." Panels are a group of folks who happen to know a little extra about a subject. They field questions from the audience which, in this case, happened to be writers.
One writer asked if we thought it was possible to dislocate your thumb to escape handcuffs. I said I didn't believe so and asked a few police officers after the fact. They agreed, probably not. However, they did said women escape cuffs more often. If it was because they were dislocating their thumbs, they couldn't say for sure. But, they kinda doubted it. More likely they escape because officers are required to leave a bit of room between the handcuffs and the wrist, enough to be able to slip a finger between the two.
From the videos I've viewed, all featuring women, the cuff is pulled over the hand. The obligatory amount of space police officers must leave can facilitate that. The women all folded their hand together vertically. And, in every case, the thumb joint required a bit of pulling to pass.
That is the type of escape we are going to consider: pulling the cuff over the entire hand. And, if you can't fit it over your hand, will dislocating your thumb help? First, which joint are we even talking about?
The 1st CMC actually controls where the 1st metacarpal (the next joint up), and thus the 1st MCP, moves in space. As the 1st metacarpal rolls over the socket created by the carpal bones (at the CMC joint), the 1st metacarpal rolls forward and brings the 1st MCP with it. So, in theory, dislocating the 1st CMC would, in theory, shift the whole 1st metacarpal out of place. So yes, the CMC is what would need to be dislocated to get the metacarpal out of the way, which is required to slip out of a properly tightened set of cuffs (which get caught first on the head of the 1st metacarpal at the 1st CMC).
...the first CMC is a saddle joint, so the only real way to traumatically dislocate it is to break one of the bony components of the saddle (or pull the thumb so far straight out that you distract the joint past those ridges, thus ripping every ligament in the joint).
Even if it were the next joint up, the MCP joint, that held the cuffs at bay, dislocating it wouldn't be much help either.
However, it is common for folks with Ehler-Danlos Syndrome to also have heart issues. So, even if they remain calm enough in such an emergency situation to escape the handcuffs, a speedy getaway on foot might be an issue.
To really get out of handcuffs, check back next week! Until then, that's it for this round at FightWrite.net. Get blood on your pages.
More than once an author has asked me how their character could beat a robot. And, they do so with a tone that suggests it's impossible. Folks, what have I always said about fighting? There are no absolutes. That includes robots.
Ok, so let's break this down. First, we need a clear picture of the thing. What is the robot designed to do? That will tell you about its physical build, strength, capabilities and programming. And, don't make it a do-it-all robot that does construction work and performs surgery and can make a killer latte in its chest cavity. Give it a specific purpose and limitations like you would any human character.
Next, rather than focusing on beating it, aim to best it.
Robots are stronger and more resilient than humans. That's kind of their thing. Muscling it doesn't make sense. Find the thing's weakness and exploit it. And, yes, it has to have a weakness otherwise the story is over.
"Red lights flickered to a low glow then brightened, piercing the darkness. Activated, the robot scanned the room, identifying us all. Then it sat down and read a book because it knew we couldn't defeat it.
Folks, even Ultron could be defeated. Do not make your robot invincible. You have to be able to "vince" it. That should be the opposite of invincible: vincible. Or, if you like, Vince Vaugnable.
Sometimes, however, finding a weakness is difficult. Your character may not have that kind of time. In that case, I suggest the exact opposite. Use their strength to their undoing. Examine what makes them awesome and consider how it can be used to against them.
will point out how some might limit them. Others I will let you figure out. Then, you can decide how any can be used in your character's favor. I don't know the specifics of every story so listing all the possibilities is, in fact, impossible. What I can do is get you to look at the robot a little differently which might allow you to see options that you didn't before. Any time I point out an attribute, look at the problem it presents for the robot.
Android - I'm going over this one first because it seems common. If a robot moves like a human, it can be taken down like a human. Don't assume that it will be too heavy. It has to be similar in weight to a human or it wouldn't be able to use human furniture. If it is especially heavy, it will be easier to trip. On that same note, once it hits the ground, it will have to right itself like a human which might be cumbersome depending on how it was resting.
It will be also be vulnerable in the eyes and joints and damaging the throat will limit its communication. Turning its head all the way around won't be possible because of the skin so its field of vision is limited. Lastly, it likely has a fluid necessary to function which means it can bleed. Think Bishop in Aliens.
using only what it has learned and there is no "deep learning." It cannot recognize the hidden layers in a concept or the ethics of it. It will not change its course of action until a particular situation teaches it a new course. And again, that new course of action has to make sense. If its intelligence requires updates, then it can be hacked.
access low items without telescoping features. Also, it is likely to be bottom heavy and wide for stability which might limit areas it can access. And, although wheels are very fast, they can't maneuver a turn quickly – especially when they are moving quickly. Speed is a double-edged sword. The faster something is moving, the more hazardous it can be to be taken off course. Speaking of off course, robots on wheels have a tough time off road. Sometimes all it takes to best them is step off the sidewalk.
have difficulty flying in small or narrow places. And, as with wheels, its speed can be problematic.
Telescoping features - Any time you lengthen something away from its center of mass, the structure is more unstable. Also, it's easier to disable a small part of something rather than the whole.
Power Source - Circuitry is the nervous system of a robot. Disrupt it and you disable the whole. To disrupt electronics, electricity has to flow across it in a way that it is not designed flow.
Non-AI - A non AI only does what it does. They're a bit like zombies in that they are single minded. That's not to say they can't problem solve. It's just that they can only solve problems they're programmed to recognize. We've all seen enough movies to know that to get past a retinal scan, you just have to have the right eyeball in your hand. Why is that? Because the robot isn't looking for what's around the eye, only at the retina itself. The fact that the organ isn't in a head isn't a problem the software is designed to recognize or address. Which, by the way, is pretty stupid. As with AIs, non-AIs are susceptible to being hacked.
Armor/Weaponry - Both of these tend to add a good bit of weight. The heavier something is, the harder it is to get it moving. And, once it gets moving, the harder it is to stop it. That's all because of inertia. The heavier something is, the greater the tendency it has to stay still. Once you get it moving, inertia will want it to continue in a straight line which makes quick turns an issue.
one! When dealing with a disparity of numbers, look for ways to even the odds. In my post The Site is Part of the Fight, I wrote about bottlenecking large numbers. That way, you only deal with as many as can fit through the bottleneck.
Ok, I know I've only scratched the surface here, but my goal wasn't to write your scene. It was to help you look at your scene differently, to get your brain working.
And, because it does pertain to robots taking over and also because I can't stop laughing about it... This is the moment you realize the robots have taken your job.
Can You Carry a Katana on Your back?
This past weekend at the Realm Makers convention, I had the privilege of speaking to a great group of writers about creating fight scenes. I was asked some really good questions, a couple of which stood out. So, over the next few weeks, I will be discussing a few of those.
Can you really carry a katana behind your back? When I first posted this, I had no katana training. Thanks to iaido at Haru Dojo, I now have a bit and hope to have more. So, I have changed this a wee bit from the original post. Now, it is more correct.
Any who, a katana on the back: I've been asked this question a bit and I'm sure much of that is owed to The Walking Dead's Michonne, who is awesome. Period.
Traditionally, the samurai wore their katana at their side. If they were in armor, it was worn cutting edge down. Without armor, cutting edge up. Other cultures had different customs but I was specifically asked about a katana which is Japanese.
Only swords too long for the side were generally worn on the back. However, they were often carried in such a way that they weren't drawn from over the shoulder. Rather, they were carried horizontally at the belt.
But, back to the question. Can you carry a katana, or, let's say any sword, on your back? Yes. But, really, the better question is, can you DRAW a sword from your back? If it's longer than your reach, then your outstretched arm won't unsheathe it. You will have to pull the "saya" or sheath away which you have to do if the katana is on the hip as well. However, on the hip you don't offer your brachial artery as you would extending your arm up over the shoulder. Either way, the blade will be toward the hand. When on the hip, you are pulling that blade away from the body. There's nothing in the way. On the back, you might give the back of your head a shave. Also, putting a katana back in the saya on one's back would be a titch harder than at the side.
The first video shows how to draw and sheathe. The second video will show you why this method for drawing a sword doesn't always work.
If you would like to learn to use a katana, call your local aikido dojo. Iaido (ee-EYE-doe)is a branch of aikido that covers proper drawing and use of the blade. Also, aikido is a FANTASTIC martial arts. Give it a look!


text:
Q: Entity Framework data insertion with nested object having auto increment primary keys with one request I am just curious to see if anyone knows of a way to insert nested objects that have auto increment keys without having to insert object one at a time.
For example, let's say I have the following tables in the database.
CREATE TABLE Analysis
(
    Analysis     bigint IDENTITY(1,1),
    AnalysisGuid uniqueidentifier NULL,
    Date         date(50)         NOT NULL,

    CONSTRAINT PK_Analysis PRIMARY KEY(AnalysisId)
);

CREATE TABLE Scenarios
(
    ScenarioId   bigint IDENTITY(1,1),
    AnalysisId   bigint           NOT NULL,
    ScenarioGuid uniqueidentifier NULL,
    HVACNumber   int              NULL,

    CONSTRAINT FK_Scenarios_Analysis
        FOREIGN KEY(Analysis) REFERENCES Analysis(AnalysisId)
                ON DELETE CASCADE,
    CONSTRAINT PK_Scenarios PRIMARY KEY(ScenarioId)
);

Where Scenario to Analysis is a M:1 relationship. And there are some other nested objects with auto increment primary key underneath the Scenarios with M:1 relationship as well.
The object that comes through the API represents an nested Analysis Object at the top level. As speed is a concern for this application, is there a way to insert the data using Entity Framework Core without going through
Insert Analysis object --> SaveChanges() --> get the Analysis primary key --> go through the Scenarios list --> insert one by one --> SaveChanges() --> get the Scenario primary key --> ...
Any help would be appreciated. Thanks!

A: If your EF is properly/fully set up (nav properties between parent/child, keys declared to be db generated etc) you just add the child object to the parent's nav property, add the parent to the context set and save it..
var p = new Parent();
var c1 = new Child();
var c2 = new Child();
p.Children.Add(c1);
p.Children.Add(c2);

context.Parents.Add(p);
context.SaveChanges();

After the save is done, the p.Id/c1.ParentId/c2.ParentId will be the parent ID calc'd by the db, and the c1.Id/c2.Id will also be their calc'd values.
It'll work the other way round too (adding parent to children); the change tracker isn't fussy, so long as it can reach your changes somehow

redpajama_set_name: RedPajamaStackExchange


text:
A flying mouse comes to life, in the glass and on the page
by Karl H. Kazaks
TROUTVILLE, VA — The location is a steel-framed building set in a clearing in a thicket of woods in rural Botetourt County, just outside of Roanoke — the perfect habitat for a brewery with a name like Flying Mouse.
"I grew up a big fan of comic books," said Frank Moeller, Flying Mouse's owner and brewmaster. When Moeller decided to take the plunge and turn a decades-long home-brewing hobby into a commercial business, he was determined to create a brewery that had at the center of its brand its very own comic book character, with unique story lines and imagery.
So Moeller huddled with his brother Chris, a graphic designer and the brewery's Creative Director, and the two created the brewery's mascot.
Bartleby Hopsworth (the name is a play off of barley and hops) is an inventive and adventuresome mouse. Driven by the experience of seeing a bat fly, and wanting to fly himself, Bartleby, with the help of some time travel, designs a device which allows him to take wing.
The comic is drawn with a steampunk style — an artistic school that fuses Victorian-era fashion and engineering with inventiveness and modern-day technological advances.
For example, the comic's imagery includes details such as brass buckles, goggles and top hats. But also important — to steampunk and to Bartleby — is the emphasis on a forward-thinking mindset.
That ethic is found in one of the brewery's mottos, "Celebrate the spirit of invention."
After all, Bartleby was able to design and build his own wings.
The adventures of Bartleby are not yet fully written, but the Moeller brothers plan to write a graphic novel about the mouse and his adventures.
The history of Flying Mouse, too, is still being written, but this much is known so far: Moeller did take a home-brewing dream and turn it into a business complete with a 20-barrel brewhouse.
The brewery is located in a steel-frame building that formerly served as a precast concrete plant. The front part of the space is the taproom, with the brewery itself located further back.
"It has a nice open skeleton," Moeller said. "It's probably the only brewery with a 10-ton bridge crane." Moeller used the crane, which can move up and down the central space of the building, to set the brewery's tanks.
The space came with good infrastructure — connection to utilities and two wells. It also came with years of accumulated concrete dust.
"We went through several times blowing the whole thing clean with compressed air," Moeller said. "Then we did the same thing with a power washer."
Today the brewhouse has three 20-barrel fermenters and a 40-barrel fermenter (for double batches) from Specific Mechanical Systems.
"They've got a good reputation," Moeller said. "They make good quality equipment at a good price."
The day of Flying Mouse's grand opening, in September of 2013, Moeller felt, he recalled, "terror, stress. Then again my wife could tell you that's pretty much every day."
Kidding aside, Moeller added, "It's like anyone else trying to get ready for a big party."
Customers must like the parties Moeller and his wife Debbie put on because the place has become a popular gathering spot.
There is live music most Saturdays, plus food from a food truck or caterers. They host regular special events like paint night, where patrons taste beer while working on a painting, as well as four main events each year: St. Patrick's Day, a summer shrimp fest, Oktoberfest and a holiday sweets party (a dessert potluck).
Over time, Moeller has improved the location's amenities, adding, for example, a fire pit outside, and creating an indoor party space in the back of the brewery. He has plans to turn an unused bay of the building into a dedicated party space, thanks to the demand the brewery has for private events.
"We've hosted class reunions, a retirement party, wedding receptions — even a wedding," Moeller said.
For his regular customers, Moeller keeps things fresh coming up with new activities and by making new beers.
"We make pretty much session beers," Moeller said, in describing Flying Mouse's house style. "Beers that people drink by the gallon, not by the glass."
In addition to selling retail from the taproom, the brewery beers are distributed in Virginia and West Virginia.
Moeller sees the future of craft beer wholesaling becoming like wine wholesaling, with sales people adept at talking about the distinct qualities of each craft beer.
Even though the brewery has a quiet country setting, it's also located near a major thoroughfare.
"We get lots of out-of-town, out-of-state traffic," Moeller said. "From Tennessee and Arkansas to Florida, Connecticut, New Jersey."
He was describing traffic that comes off of nearby Interstate 81, but he could very well have described the foot traffic that comes to the brewery from the Appalachian Trail, which passes by just a half-mile from the brewery.
In choosing his packaging, Moeller says he has consciously "aligned with the outdoor lifestyle," which works for the hikers on the AT. The packaged beer comes in cans, not bottles, and even the growlers are stainless steel or polymer, not glass.
Soon, this part of southwest Virginia will see two big additions to the brewing scene. Ballast Point and Deschutes are both building East Coast breweries just a few miles from Flying Mouse.
"We'll be in the brewing epicenter," Moeller said.
Prior to opening the brewery, Moeller was a mechanical engineer, working in building design for two decades. Being a full-time brewer still allows him to satisfy the engineer inside of him.
"The gadgets and moving parts have a lot of appeal to me," he said.
But the new career has also allowed him to embrace the creativity inherent with brewing, catching inspiration for a new brew — or a new adventure for Bartleby Hopsworth.
It's that spirit of adventure which binds the brewery to its customers. Even though the brewery is near a lot of things, it's still a destination and something you have to decide to go to.
Once they get there, customers get to share the adventure of Frank Moeller's life — expressed both through his beers and the still-evolving saga of Bartleby Hopsworth.
Lee Newspapers2017-01-20T14:23:00-05:00Wine and Craft Beverage News Articles|0 Comments