Gut microbiome dysbiosis is associated with host genetics in the Norwegian Lundehund.

A group of diseases have been shown to correlate with a phenomenon called microbiome dysbiosis, where the bacterial species composition of the gut becomes abnormal. The gut microbiome of an animal is influenced by many factors including diet, exposures to bacteria during post-gestational growth, lifestyle, and disease status. Studies also show that host genetics can affect microbiome composition. We sought to test whether host genetic background is associated with gut microbiome composition in the Norwegian Lundehund dog, a highly inbred breed with an effective population size of 13 individuals. The Lundehund has a high rate of a protein-losing enteropathy in the small intestine that is often reported as Lundehund syndrome, which negatively affects longevity and life-quality. An outcrossing project with the Buhund, Norrbottenspets, and Icelandic sheepdog was recently established to reintroduce genetic diversity to the Lundehund and improve its health. To assess whether there was an association between host genetic diversity and the microbiome composition, we sampled the fecal microbiomes of 75 dogs of the parental (Lundehund), F1 (Lundehund x Buhund), and F2 (F1 x Lundehund) generations. We found significant variation in microbiome composition from the parental Lundehund generation compared to the outcross progeny. The variation observed in purebred Lundehunds corresponded to dysbiosis as seen by a highly variable microbiome composition with an elevated Firmicutes to Bacteroidetes ratio and an increase in the prevalence of Streptococcus bovis/Streptococcus equinus complex, a known pathobiont that can cause several diseases. We tracked several other environmental factors including diet, the presence of a cat in the household, living in a farm and the use of probiotics, but we did not find evidence of an effect of these on microbiome composition and alpha diversity. In conclusion, we found an association between host genetics and gut microbiome composition, which in turn may be associated with the high incidence of Lundehund syndrome in the purebred parental dogs.

Rapid In Situ Detection of THC and CBD in Cannabis sativa L. by 1064 nm Raman Spectroscopy.

The need to find a rapid and worthwhile technique for the in situ detection of the content of delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) in Cannabis sativa L. is an ever-increasing problem in the forensic field. Among all the techniques for the detection of cannabinoids, Raman spectroscopy can be identified as the most cost-effective, fast, noninvasive, and nondestructive. In this study, 42 different samples were analyzed using Raman spectroscopy with 1064 nm excitation wavelength. The use of an IR wavelength laser showed the possibility to clearly identify THC and CBD in fresh samples, without any further processing, knocking out the contribution of the fluorescence generated by visible and near-IR sources. The results allow assigning all the Raman features in THC- and CBD-rich natural samples. The multivariate analysis underlines the high reproducibility of the spectra and the possibility to distinguish immediately the Raman spectra of the two cannabinoid species. Furthermore, the ratio between the Raman bands at 1295/1440 and 1623/1663 cm-1 is identified as an immediate test parameter to evaluate the THC content in the samples.

Genetic Rescue of the Highly Inbred Norwegian Lundehund.

Augmenting the genetic diversity of small, inbred populations by the introduction of new individuals is often termed "genetic rescue". An example is the Norwegian Lundehund, a small spitz dog with inbreeding-related health problems that is being crossed with three Nordic breeds, including the Norwegian Buhund. Conservation breeding decisions for the (typically) small number of outcrossed individuals are vital for managing the rescue process, and we genotyped the Lundehund (n = 12), the Buhund (n = 12), their crosses (F1, n = 7) and first-generation backcrosses to the Lundehund (F2, n = 12) with >170,000 single nucleotide polymorphism loci to compare their levels of genetic diversity. We predicted that genome-wide diversity in F2 dogs would be higher than in the Lundehund but lower than in the F1 and the Buhund, and the heterozygosity values showed the expected patterns. We also found that runs of homozygosity, extended chromosomal regions of homozygous genotypes inherited from a common ancestor, were reduced in F2 individuals compared with Lundehund individuals. Our analyses demonstrate the benefits of outcrossing but indicate that some of the acquired genetic diversity is lost following immediate backcrossing. Additional breeding among F2 crosses could therefore merit from further consideration in genetic rescue management.

New Dihydrothiazole Benzensulfonamides: Looking for Selectivity toward Carbonic Anhydrase Isoforms I, II, IX, and XII.

In the present study we investigated the structure-activity relationships of a new series of 4-[(3-ethyl-4-aryl-2,3-dihydro-1,3-thiazol-2-ylidene)amino]benzene-1-sulfonamides (EMAC10101a-m). All synthesized compounds, with the exception of compound EMAC10101k, preferentially inhibit off-target hCA II isoform. Within the series, compound EMAC10101d, bearing a 2,4-dichorophenyl substituent in position 4 of the dihydrothiazole ring, was the most potent and selective toward hCA II with an inhibitory activity in the low nanomolar range.

Exploring new structural features of the 4-[(3-methyl-4-aryl-2,3-dihydro-1,3-thiazol-2-ylidene)amino]benzenesulphonamide scaffold for the inhibition of human carbonic anhydrases.

A library of 4-[(3-methyl-4-aryl-2,3-dihydro-1,3-thiazol-2-ylidene)amino]benzene-1-sulphonamides (EMAC8002a-m) was designed and synthesised to evaluate the effect of substituents in the positions 3 and 4 of the dihydrothiazole ring on the inhibitory potency and selectivity toward human carbonic anhydrase isoforms I, II, IX, and XII. Most of the new compounds preferentially inhibit the isoforms II and XII. Both electronic and steric features on the aryl substituent in the position 4 of the dihydrothiazole ring concur to determine the overall biological activity of these new derivatives.

Tuning the Dual Inhibition of Carbonic Anhydrase and Cyclooxygenase by Dihydrothiazole Benzensulfonamides.

A novel series of of 4-[(3-phenyl-4-aryl-2,3-dihydro-1,3-thiazol-2-ylidene)amino]benzene-1-sulfonamides (EMAC10111a-g) was synthesized and assayed toward both human carbonic anhydrase isozymes I, II, IX, and XII and cyclooxygenase isoforms. The majority of these derivatives preferentially inhibit hCA isoforms II and XII and hCOX-2 isozyme, indicating that 2,3,4-trisubstituted 2,3-dihydrothiazoles are a promising scaffold for the inhibition of hCA isozymes and of hCOX-2 enzyme. The nature of the substituent at the dihydrothiazole ring position 4 influenced the activity and selectivity toward both enzyme families. EMAC10111g resulted as the best performing compound toward both enzyme families and exhibited preferential activity toward hCA XII and hCOX-2 isozymes.

Targeting Tumor Associated Carbonic Anhydrases IX and XII: Highly Isozyme Selective Coumarin and Psoralen Inhibitors.

A small library of psoralen carboxylic acids and their corresponding benzenesulfonamide derivatives were designed and synthesized to evaluate their activity and selectivity toward tumor associated human carbonic anhydrase (hCA) isoforms IX and XII. Both psoralen acids and sulfonamides exhibited potent inhibition of IX and XII isozymes in the nanomolar concentration range. However, psoralen acids resulted as the most selective in comparison with the corresponding benzenesulfonamide derivatives. Our data indicate that the psoralen scaffold is a promising starting point for the design of highly selective tumor associated hCA inhibitors.

Phenylpropenoids from Bupleurum fruticosum as Anti-Human Rhinovirus Species A Selective Capsid Binders.

The dichloromethane extract of the leaves of Bupleurum fruticosum was found to inhibit the replication of human rhinovirus (HRV) serotypes 14 and 39. Bioassay-guided fractionation led to the isolation of seven phenylpropenol derivatives (3-9), two polyacetylenes (1 and 2), and one monoterpene (10). Compounds 1 and 10 were identified as previously undescribed secondary metabolites after extensive 1D and 2D NMR experiments as well as high-resolution mass spectrometry. Compounds 2, 4, and 5 showed a selective inhibition of viral replication against HRV39 serotype, with 2 and 4 being the most active, with EC50 values of 1.8 ± 0.02 and 2.4 ± 0.04 µM. Mechanism of action studies indicated that 4 behaves not only as a capsid binder, interfering with the early phases of virus replication, but also as a late-phase replication inhibitor. Docking experiments were performed to confirm the ability of the antiviral phenylpropenoids to selectively fit into the hydrophobic pocket of VP1-HRV39.

N-Acylbenzenesulfonamide Dihydro-1,3,4-oxadiazole Hybrids: Seeking Selectivity toward Carbonic Anhydrase Isoforms.

A series of N-acylbenzenesulfonamide dihydro-1,3,4-oxadiazole hybrids (EMAC8000a-m) was designed and synthesized with the aim to target tumor associated carbonic anhydrase (hCA) isoforms IX and XII. Most of the compounds were selective inhibitors of the tumor associated hCA XII. Moreover, resolution of EMAC8000d racemic mixture led to the isolation of the levorotatory eutomer exhibiting an increase of hCA XII inhibition potency and selectivity with respect to hCA II. Computational studies corroborated these data. Overall our data indicate that both substitution pattern and stereochemistry of dihydro-1,3,4-oxadiazole could be considered as key factors to determine activity and selectivity toward hCA isozymes. These results can provide further indication for the design and optimization of selective hCA inhibitors.

Genetic rescue of an endangered domestic animal through outcrossing with closely related breeds: A case study of the Norwegian Lundehund.

Genetic rescue, outcrossing with individuals from a related population, is used to augment genetic diversity in populations threatened by severe inbreeding and extinction. The endangered Norwegian Lundehund dog underwent at least two severe bottlenecks in the 1940s and 1960s that each left only five inbred dogs, and the approximately 1500 dogs remaining world-wide today appear to descend from only two individuals. The Lundehund has a high prevalence of a gastrointestinal disease, to which all remaining dogs may be predisposed. Outcrossing is currently performed with three Nordic Spitz breeds: Norwegian Buhund, Icelandic Sheepdog, and Norrbottenspets. Examination of single nucleotide polymorphism (SNP) genotypes based on 165K loci in 48 dogs from the four breeds revealed substantially lower genetic diversity for the Lundehund (HE 0.035) than for other breeds (HE 0.209-0.284). Analyses of genetic structure with > 15K linkage disequilibrium-pruned SNPs showed four distinct genetic clusters. Pairwise FST values between Lundehund and the candidate breeds were highest for Icelandic Sheepdog, followed by Buhund and Norrbottenspets. We assessed the presence of outlier loci among candidate breeds and examined flanking genome regions (1 megabase) for genes under possible selection to identify potential adaptive differences among breeds; outliers were observed in flanking regions of genes associated with key functions including the immune system, metabolism, cognition and physical development. We suggest crossbreeding with multiple breeds as the best strategy to increase genetic diversity for the Lundehund and to reduce the incidence of health problems. For this project, the three candidate breeds were first selected based on phenotypes and then subjected to genetic investigation. Because phenotypes are often paramount for domestic breed owners, such a strategy could provide a helpful approach for genetic rescue and restoration of other domestic populations at risk, by ensuring the involvement of owners, breeders and managers at the start of the project.

Through scaffold modification to 3,5-diaryl-4,5-dihydroisoxazoles: new potent and selective inhibitors of monoamine oxidase B.

3,5-Diaryl-4,5-dihydroisoxazoles were synthesized and evaluated as monoamine oxidase (MAO) enzyme inhibitors and iron chelators. All compounds exhibited selective inhibitory activity towards the B isoform of MAO in the nanomolar concentration range. The best performing compound was preliminarily evaluated for its ability to bind iron II and III cations, indicating that neither iron II nor iron III is coordinated. The best compounds racemic mixtures were separated and single enantiomers inhibitory activity evaluated. Furthermore, none of the synthesised compounds exhibited activity towards MAO A. Overall, these data support our hypothesis that 3,5-diaryl-4,5-dihydroisoxazoles are promising scaffolds for the design of neuroprotective agents.

Isatin thiazoline hybrids as dual inhibitors of HIV-1 reverse transcriptase.

A series of 3-3-{2-[2-3-methyl-4-phenyl-2,3-dihydro-1,3-thiazol-2-ylidene]hydrazin-1-ylidene-2,3-dihydro-1H-indol-2-one derivatives has been designed and synthesized to study their activity on both HIV-1 (Human Immunodeficiency Virus type 1) RT (Reverse Transcriptase) associated functions. These derivatives are analogs of previously reported series whose biological activity and mode of action have been investigated. In this work we investigated the influence of the introduction of a methyl group in the position 3 of the dihydrothiazole ring and of a chlorine atom in the position 5 of the isatin nucleus. The new synthesized compounds are active towards both DNA polymerase and ribonuclease H in the µM range. The nature of the aromatic group in the position 4 of the thiazole was relevant in determining the biological activity.

Isatin: a privileged scaffold for the design of carbonic anhydrase inhibitors.

The isatin scaffold is the constitutive fragment of several natural and synthetic bioactive molecules. Albeit several benzene sulphonamide-based carbonic anhydrase inhibitors (CAIs) have been reported, only recently isatin benzene sulphonamides have been studied and proposed as CAIs. In this study we have designed, synthesised, and evaluated the biological activity of a series of differently substituted isatin-based benzene sulphonamides which have been designed for the inhibition of carbonic anhydrase isoforms. The activity of all the synthesised compounds was evaluated towards human carbonic anhydrase I, II, IX, and XII isozymes. Our results indicate that the nature and position of substituents on the isatin ring can modulate both activity and isozyme selectivity.

Lack of sex-specific movement patterns in an alien species at its invasion front - consequences for invasion speed.

Efficient targeting of actions to reduce the spread of invasive alien species relies on understanding the spatial, temporal, and individual variation of movement, in particular related to dispersal. Such patterns may differ between individuals at the invasion front compared to individuals in established and dense populations due to differences in environmental and ecological conditions such as abundance of conspecifics or sex-specific dispersal affecting the encounter rate of potential mates. We assessed seasonal and diurnal variation in movement pattern (step length and turning angle) of adult male and female raccoon dog at their invasion front in northern Sweden using data from Global Positioning System (GPS)-marked adult individuals and assessed whether male and female raccoon dog differed in their movement behavior. There were few consistent sex differences in movement. The rate of dispersal was rather similar over the months, suggesting that both male and female raccoon dog disperse during most of the year, but with higher speed during spring and summer. There were diurnal movement patterns in both sexes with more directional and faster movement during the dark hours. However, the short summer nights may limit such movement patterns, and long-distance displacement was best explained by fine-scale movement patterns from 18:00 to 05:00, rather than by movement patterns only from twilight and night. Simulation of dispersing raccoon dogs suggested a higher frequency of male-female encounters that were further away from the source population for the empirical data compared to a scenario with sex differences in movement pattern. The lack of sex differences in movement pattern at the invasion front results in an increased likelihood for reproductive events far from the source population. Animals outside the source population should be considered potential reproducing individuals, and a high effort to capture such individuals is needed throughout the year to prevent further spread.

Exploring the thiazole scaffold for the identification of new agents for the treatment of fluconazole resistant Candida.

Cyclohexyliden- and 2-methylcyclohexyliden-hydrazo-4-arylthiazoles were synthesized and tested as antifungal agents. All compounds exhibited minimal inhibitory concentration (MIC) values comparable with those of fluconazole (FLC). Moreover, some compounds showed fungicidal activity at low concentration. Worth noting five out of nine compounds were active towards Candida albicans 25 FLC resistant isolated from clinical specimens. The cellular toxicity was evaluated and none of the compounds is toxic at the MIC. On the basis of our data we can conclude that these derivatives are promising agents for the treatment of resistant C. albicans.

New 4-[(3-cyclohexyl-4-aryl-2,3-dihydro-1,3-thiazol-2-ylidene)amino]benzene-1-sulfonamides, synthesis and inhibitory activity toward carbonic anhydrase I, II, IX, XII.

A series of 4-[(3-cyclohexyl-4-aryl-2,3-dihydro-1,3-thiazol-2-ylidene)amino]benzene-1-sulfonamides was synthesised and the activity of the new compounds as inhibitors of hCA I, II, IX, and XII was evaluated. These new derivatives exhibited some peculiarities with respect to previously reported sulfonamide based inhibitors of CA. We observed that the nature of the substituents in the position 3 and 4 of the dihydro-thiazole ring was relevant in determining both activity and selectivity profiles.

Molecular Genetics of Sex Identification, Breed Ancestry and Polydactyly in the Norwegian Lundehund Breed.

The Norwegian Lundehund breed of dog has undergone a severe loss of genetic diversity as a result of inbreeding and epizootics of canine distemper. As a consequence, the breed is extremely homogeneous and accurate sex identification is not always possible by standard screening of X-chromosomal loci. To improve our genetic understanding of the breed we genotyped 17 individuals using a genome-wide array of 170 000 single nucleotide polymorphisms (SNPs). Standard analyses based on expected homozygosity of X-chromosomal loci failed in assigning individuals to the correct sex, as determined initially by physical examination and confirmed with the Y-chromosomal marker, amelogenin. This demonstrates that identification of sex using standard SNP assays can be erroneous in highly inbred individuals.

Individual and temporal variation in habitat association of an alien carnivore at its invasion front.

Gathering information on how invasive species utilize the habitat is important, in order to better aim actions to reduce their negative impact. We studied habitat use and selection of 55 GPS-marked raccoon dogs (30 males, 25 females) at their invasion front in Northern Sweden, with particular focus on differences between males and females, between movement states, and between seasons and times of the day. Daily movement pattern was used to classify GPS-locations into dispersing and settled. We focused on both anthropogenic and natural landscape characteristics. Since we did not have any a priori knowledge about the spatial scale of raccoon dog habitat selection, we first assessed how landscape characteristics of random points changed with distance from the GPS-location they were paired to. Because changes in habitat use became less pronounced at approximately 5 km for all variables, we focused on habitat use at two spatial scales: fine (500 m) and coarse (5 km). Habitat selection was strongest at the coarse scale, and reflected the results found for habitat use. Raccoon dogs selected agricultural areas and wetlands, lower altitudes, and shallow slopes, and avoided forests, open natural areas, and areas close to water and roads. There were no differences in habitat selection between males and females, or between movement states. This lack of sexual segregation increases the probability of encountering potential mates during dispersal, and therefore the likelihood for reproduction in new areas. The seasonal and diurnal pattern of habitat use may provide guidance for where and when to aim management efforts.

Genetic variability and structure of the water vole Arvicola amphibius across four metapopulations in northern Norway.

Water vole Arvicola amphibius populations have recently experienced severe decline in several European countries as a consequence of both reduction in suitable habitat and the establishment of the alien predator American mink Neovison vison. We used DNA microsatellite markers to describe the genetic structure of 14 island populations of water vole off the coast of northern Norway. We looked at intra- and inter-population levels of genetic variation and examined the effect of distance among pairs of populations on genetic differentiation (isolation by distance). We found a high level of genetic differentiation (measured by F ST) among populations overall as well as between all pairs of populations. The genetic differentiation between populations was positively correlated with geographic distance between them. A clustering analysis grouped individuals into 7 distinct clusters and showed the presence of 3 immigrants among them. Our results suggest a small geographic scale for evolutionary and population dynamic processes in our water vole populations.

Low neutral genetic variability in a specialist puffin hunter: the Norwegian Lundehund.

The genetic variability of 125 Norwegian Lundehund and 27 Nova Scotia Duck Tolling Retriever was analysed using a set of 26 microsatellite markers. In Lundehund, the average number of alleles per locus was 1.73, and average observed (H(O)) and expected (H(E)) heterozygosity were 0.07. In Toller, all measures of genetic diversity were much higher than in Lundehund and similar to studies on other dog breeds. The cluster analysis correctly assigned individuals to their respective breed. The low genetic variability in Lundehund was not surprising, given the two strong bottlenecks in the 1940s and the 1960s. The relatedness of Lundehund to other Nordic small spitzes should be investigated in the view of possible outcrossing.