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The absence of a standardised method to quantify canine DNA recovered from livestock attacks leaves forensic providers without an important quality control step to help support their decision making. Typically used to normalise the amount of DNA for STR amplification, modern forensic DNA quantification approaches use qPCR of target genes and can also include an Internal Positive Controls (IPC) to determine the presence of PCR inhibitors. The co-amplification of livestock DNA alongside canine DNA has meant that previously developed qPCR methods are not suitable for use so a standardised approach is needed. This research describes the development of a Taq-man multiplex qPCR assay that simultaneously quantifies the autosomal MC1R and Y-specific SRY gene to determine the concentration of canine DNA recovered from attacked livestock. Data suggests that the method is robust and reproducible with no significant difference in the standard curves produced from multiple runs or from different DNA standards derived from different canines. Assay sensitivity of between 15 and 31 pg is consistent with other forensic quantification assays and also in line with the sensitivity of the two tested canine STR kits, Canine Genotype 2.1 Kit and CaDNAP Panels 1 and 2. The assay is highly specific to canines when tested against 163 different dogs representing 33 different breeds and no cross-amplification of non-target species' DNA was observed even from livestock DNA tested at 31.25 ng/µl. This strongly suggests that any DNA detected on evidence collected from attacked livestock is canine. The assay also shows robust tolerance to common livestock inhibitors continuing to amplify when inhibitor-spiked DNA samples were tested. Both mixed and inhibited DNA samples underwent STR typing using two canine forensic STR kits with data showing the Canine Genotype 2.1 Kit displaying pronounced cross-amplification of livestock DNA and and/or extensive PCR inhibition leading to the complete loss of amplification when using this kit. Conversely the CaDNAP Panels 1 and 2 showed little cross-amplification of livestock DNA and improved inhibitor tolerance suggesting that this approach was better suited for the analysis of livestock attack samples. Findings are discussed and the impact of the observations on future work in this area are explored., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024. Published by Elsevier B.V. All rights reserved.)

Background: Anaphylaxis is a severe allergic reaction that can be lethal if not treated adequately. The underlying molecular mechanisms responsible for the severity are mostly unknown., Objective: This study is based on a clinical case of a patient with extremely severe anaphylaxis to paper wasp venom. This patient has a mutation in the KARS gene, which encodes lysyl-tRNA synthetase (LysRS), a moonlight protein with a canonical function in protein synthesis and a noncanonical function in antigen dependent-FcεRI activation in mast cells. In this study, the objective was to characterize the mutation at the molecular level., Methods: Analysis of the KARS mutation was carried out using biochemical and functional approaches, cell transfection, Western blot, confocal microscopy, cell degranulation, prostaglandin D 2 secretion, and proteases gene transcription. Structural analysis using molecular dynamics simulations and well-tempered metadynamics was also performed., Results: The mutation found, P542R (proline was replaced by arginine at aminoacid 542), affects the location of the protein as we show in biochemical and structural analyses. The mutation resembles active LysRS and causes a constitutive activation of the microphthalmia transcription factor, which is involved in critical mast cell functions such as synthesis of mediators and granule biogenesis. Moreover, the structural analysis provides insights into how LysRS works in mast cell activation., Conclusions: A link between the aberrant LysRS-P542R function and mast cell-exacerbated activation with increase in proinflammatory mediator release after antigen-IgE-dependent response could be established., (Copyright © 2020 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Stimulator of interferon genes (STING) plays a central role in innate immune responses to viral and intracellular bacterial infections, and cellular damage. STING is a cytosolic sensor of cyclic dinucleotides (CDNs) including those produced by pathogenic bacteria and those arising endogenously as products of the DNA sensor cGAS (e.g., 2'3' cGAMP). The two most common alternative allelic variants of STING in humans are STING-R71H-G230A-R293Q (STING-HAQ) and STING-R232H that are found in 20.4% and 13.7-17.6% of the population, respectively. To determine the biologic consequences of these genotypic variations, we generated knock-in mice containing the murine equivalents of each variant and studied their responsiveness to CDNs. Homozygous STING-HAQ (R71H-I229A-R292Q) and STING-R231H mice were found to be unresponsive to all exogenous CDNs tested (ci-di-GMP, ci-di-AMP, 3'3' cGAMP and Rp,Rp-CDA). Responses of homozygous STING-HAQ mice to endogenous 2'3' cGAMP was also greatly impaired. However, homozygous STING-R231H mice are fully responsive to 2'3' cGAMP. Analysis of heterozygous mice revealed reduced responsiveness to exogenous and endogenous CDNs in mice carrying a single copy of STING-HAQ, while STING-R231H heterozygous mice exhibit reduced responsiveness to exogenous but not endogenous CDNs. These findings confirm and extend previous reports by demonstrating differing impact of allelic variation of STING on the ability to sense and respond to exogenous vs. endogenous CDNs. Finally, the STING-R231H variant mouse represents a useful tool with which to examine the relative contributions of STING sensing of exogenous and endogenous CDNs in the context of bacterial infections and CDN-based cancer immunotherapeutics., (Copyright © 2020 Walker, Kim, Crisler, Nguyen, Lenz, Cambier and Getahun.)

Objective: Attacks on humans by dogs in a pack, though uncommon, do happen, and result in severe, sometimes fatal, injuries. We describe the role that canine genetic markers played during the investigation of a fatal dog-pack attack involving a 50-year-old male truck driver in a parking lot in Tuscany (Italy). Using canine specific STR genetic markers, the local authorities, in the course of their investigations, reconstructed the genetic relationships between the dogs that caused the deadly aggression and other dogs belonging to the owner of the parking who, at the moment of the aggression, was located in another region of Italy., Results: From a Bayesian clustering algorithm, the most likely number of clusters was two. The average relatedness among the dogs responsible for the aggression was higher than the average relatedness among the other dogs or between the two groups. Taken together, all these results indicate that the two groups of dogs are clearly distinct. Genetic relationships showed that the two groups of dogs were not related. It was therefore unlikely that the murderous dogs belonged to the owner of the parking lot who, on grounds of this and additional evidence, was eventually acquitted.

Little is known about the immunomodulation by tick saliva during a natural tick bite in human skin, the site of the tick-host interaction. We examined the expression of chemokines, cytokines and leucocyte markers on the mRNA levels and histopathologic changes in human skin biopsies of tick bites (n=37) compared to unaffected skin (n=9). Early tick-bite skin lesions (<24 hours of tick attachment) were characterized by a predominance of macrophages and dendritic cells, elevated mRNA levels of macrophage chemoattractants (CCL2, CCL3, CCL4) and neutrophil chemoattractants (CXCL1, CXCL8), of the pro-inflammatory cytokine, IL-1β, and the anti-inflammatory cytokine, IL-5. In contrast, the numbers of lymphocytes and mRNA levels of lymphocyte cell markers (CD4, CD8, CD19), lymphocyte chemoattractants (CXCL9, CXCL10, CXCL11, CXCL13, CCL1, CCL22), dendritic cell chemoattractants (CCL20), and other pro- (IL-6, IL-12p40, IFN-γ, TNF-α) and anti-inflammatory cytokines (IL-4, IL-10, TGF-β) did not differ from normal skin. With longer tick attachment (>24 hours), the numbers of innate immune cells and mediators (not significantly) declined, whereas the numbers of lymphocytes (not significantly) increased. Natural tick bites by Ixodes ricinus ticks initially elicit a strong local innate immune response in human skin. Beyond 24 hours of tick attachment, this response usually becomes less, perhaps because of immunomodulation by tick saliva., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

STING (stimulator of interferon genes) has recently been found to play an important role in host defenses against virus and intracellular bacteria via the regulation of type-I IFN signaling and innate immunity. Chronic infection with Helicobacter pylori is identified as the strongest risk factor for gastric cancer. Thus, we aim to explore the function of STING signaling in the development of gastric cancer. Immunohistochemistry was used to detect STING expression in 217 gastric cancer patients who underwent surgical resection. STING protein expression was remarkably decreased in tumor tissues compared to non-tumor tissues, and low STING staining intensity was positively correlated with tumor size, tumor invasion depth, lymph mode metastasis, TNM stage, and reduced patients' survival. Multivariate analysis identified STING as an independent prognostic factor, which could improve the predictive accuracy for overall survival when incorporated into TNM staging system. In vitro studies revealed that knock-down of STING promoted colony formation, viability, migration and invasion of gastric cancer cells, and also led to a defect in cytosolic DNA sensing. Besides, chronic H. pylori infection up-regulated STING expression and activated STING signaling in mice. In conclusion, STING was proposed as a novel independent prognostic factor and potential immunotherapeutic target for gastric cancer., Competing Interests: The authors declare no competing financial interests.

The capsaicin receptor TRPV1 ion channel is a polymodal nociceptor that responds to heat with exquisite sensitivity through an unknown mechanism. Here we report the identification of a novel toxin, RhTx, from the venom of the Chinese red-headed centipede that potently activates TRPV1 to produce excruciating pain. RhTx is a 27-amino-acid small peptide that forms a compact polarized molecule with very rapid binding kinetics and high affinity for TRPV1. We show that RhTx targets the channel's heat activation machinery to cause powerful heat activation at body temperature. The RhTx-TRPV1 interaction is mediated by the toxin's highly charged C terminus, which associates tightly to the charge-rich outer pore region of the channel where it can directly interact with the pore helix and turret. These findings demonstrate that RhTx binding to the outer pore can induce TRPV1 heat activation, therefore providing crucial new structural information on the heat activation machinery.

In Pavlovian conditioning, animals learn to associate initially neutral stimuli with positive or negative outcomes, leading to appetitive and aversive learning respectively. The honeybee (Apis mellifera) is a prominent invertebrate model for studying both versions of olfactory learning and for unraveling the influence of genotype. As a queen bee mates with about 15 males, her worker offspring belong to as many, genetically-different patrilines. While the genetic dependency of appetitive learning is well established in bees, it is not the case for aversive learning, as a robust protocol was only developed recently. In the original conditioning of the sting extension response (SER), bees learn to associate an odor (conditioned stimulus - CS) with an electric shock (unconditioned stimulus - US). This US is however not a natural stimulus for bees, which may represent a potential caveat for dissecting the genetics underlying aversive learning. We thus first tested heat as a potential new US for SER conditioning. We show that thermal stimulation of several sensory structures on the bee's body triggers the SER, in a temperature-dependent manner. Moreover, heat applied to the antennae, mouthparts or legs is an efficient US for SER conditioning. Then, using microsatellite analysis, we analyzed heat sensitivity and aversive learning performances in ten worker patrilines issued from a naturally inseminated queen. We demonstrate a strong influence of genotype on aversive learning, possibly indicating the existence of a genetic determinism of this capacity. Such determinism could be instrumental for efficient task partitioning within the hive.

Many haematophagous insects produce factors that help their blood meal and coincidently favor pathogen transmission. However nothing is known about the ability of Culicoides midges to interfere with the infectivity of the viruses they transmit. Among these, Bluetongue Virus (BTV) induces a hemorrhagic fever- type disease and its recent emergence in Europe had a major economical impact. We observed that needle inoculation of BTV8 in the site of uninfected C. nubeculosus feeding reduced viraemia and clinical disease intensity compared to plain needle inoculation. The sheep that developed the highest local inflammatory reaction had the lowest viral load, suggesting that the inflammatory response to midge bites may participate in the individual sensitivity to BTV viraemia development. Conversely compared to needle inoculation, inoculation of BTV8 by infected C. nubeculosus bites promoted viraemia and clinical symptom expression, in association with delayed IFN- induced gene expression and retarded neutralizing antibody responses. The effects of uninfected and infected midge bites on BTV viraemia and on the host response indicate that BTV transmission by infected midges is the most reliable experimental method to study the physio-pathological events relevant to a natural infection and to pertinent vaccine evaluation in the target species. It also leads the way to identify the promoting viral infectivity factors of infected Culicoides in order to possibly develop new control strategies against BTV and other Culicoides transmitted viruses.

Indirect Genetic Effects (IGEs), also known as associative effects, are the heritable effects that an individual has on the phenotype of its social partners. Selection for IGEs has been proposed as a method to reduce harmful behaviours, in particular aggression, in livestock and aquaculture. The mechanisms behind IGEs, however, have rarely been studied. The objective was therefore to assess aggression in pigs which were divergently selected for IGEs on growth (IGEg). In a one generation selection experiment, we studied 480 offspring of pigs (Sus scrofa) that were selected for relatively high or low IGEg and housed in homogeneous IGEg groups in either barren or enriched environments. Skin lesion scores, a proxy measure of aggression, and aggressive behaviours were recorded. The two distinct IGEg groups did not differ in number of skin lesions, or in amount of reciprocal fighting, both under stable social conditions and in confrontation with unfamiliar pigs in a 24 h regrouping test. Pigs selected for a positive effect on the growth of their group members, however, performed less non-reciprocal biting and showed considerably less aggression at reunion with familiar group members after they had been separated during a 24 h regrouping test. The enriched environment was associated with more skin lesions but less non-reciprocal biting under stable social conditions. Changes in aggression between pigs selected for IGEg were not influenced by G×E interactions with regard to the level of environmental enrichment. It is likely that selection on IGEg targets a behavioural strategy, rather than a single behavioural trait such as aggressiveness.

Knowledge about gene expression in animals involved in abnormal behaviors can contribute to the understanding of underlying biological mechanisms. This study aimed to explore the motivational background to tail biting, an abnormal injurious behavior and severe welfare problem in pig production. Affymetrix microarrays were used to investigate gene expression differences in the hypothalamus and prefrontal cortex of pigs performing tail biting, pigs receiving bites to the tail and neutral pigs who were not involved in the behavior. In the hypothalamus, 32 transcripts were differentially expressed (P < 0.05) when tail biters were compared with neutral pigs, 130 when comparing receiver pigs with neutrals, and two when tail biters were compared with receivers. In the prefrontal cortex, seven transcripts were differently expressed in tail biters when compared with neutrals, seven in receivers vs. neutrals and none in the tail biters vs. receivers. In total, 19 genes showed a different expression pattern in neutral pigs when compared with both performers and receivers. This implies that the functions of these may provide knowledge about why the neutral pigs are not involved in tail biting behavior as performers or receivers. Among these 19 transcripts were genes associated with production traits in pigs (PDK4), sociality in humans and mice (GTF2I) and novelty seeking in humans (EGF). These are in line with hypotheses linking tail biting with reduced back fat thickness and explorative behavior., (© 2012 The Authors. Genes, Brain and Behavior © 2012 Blackwell Publishing Ltd and International Behavioural and Neural Genetics Society.)

The objective of this study was to identify loci associated with tail biting or being a victim of tail biting in Norwegian crossbred pigs using a genome-wide association study with PLINK case-control analysis. DNA was extracted from hair or blood samples collected from 98 trios of crossbred pigs located across Norway. Each trio came from the same pen and consisted of one pig observed to initiate tail biting, one pig which was the victim of tail biting and a control pig which was not involved in either behaviour. DNA was genotyped using the Illumina PorcineSNP60 BeadChip whole-genome single-nucleotide polymorphism (SNP) assay. After quality assurance filtering, 53,952 SNPs remained comprising 74 animals (37 pairs) for the tail biter versus control comparison and 53,419 SNPs remained comprising 80 animals (40 pairs) for the victim of tail biting versus control comparison. An association with being a tail biter was observed on Sus scrofa chromosome 16 (SSC16; p = 1.6 × 10(-5)) and an unassigned chromosome (p = 3.9 × 10(-5)). An association with being the victim of tail biting was observed on Sus scrofa chromosomes 1 (SSC1; p = 4.7 × 10(-5)), 9 (SSC9; p = 3.9 × 10(-5)), 18 (SSC18; p = 7 × 10(-5) for 9,602,511 bp, p = 3.4 × 10(-5) for 9,653,881 bp and p = 5.3 × 10(-5) for 29,577,783 bp) and an unassigned chromosome (p = 6.1 × 10(-5)). An r(2) = 0.96 and a D' = 1 between the two SNPs at 9 Mb on SSC18 indicated extremely high linkage disequilibrium, suggesting that these two markers represent a single locus. These results provide evidence of a moderate genetic association between the propensity to participate in tail-biting behaviour and the likelihood of becoming a victim of this behaviour.

Microsatellite markers are a useful tool for ecological monitoring of natural and managed populations. A technical limitation is the necessity for investment in the development of primers. Heterologous primers can provide an alternative to searching for new loci. In bees, these markers have been used in populational and intracolonial genetic analyses. The genus Melipona has the largest number of species among bee genera, about 70, occurring throughout the Neotropical region. However, only five species of the genus Melipona have specific microsatellite markers. Given the great diversity of this genus, this number is not representative. We analyzed the transferability of 49 microsatellite loci to four other species of the genus Melipona (M. scutellaris, M. mondury, M. mandacaia, and M. quadrifasciata). Four individuals of each species, from different localities, were used in amplification tests. Primer pairs described for five Melipona species and for Trigona carbonaria were tested. Among the 49 loci, 22 gave amplification products for all four species, while three gave nonspecific bands and five showed no amplification products. The remaining loci varied in the pattern of amplification, according to the species examined. The number of alleles ranged from 1 to 6. The results demonstrate the possibility of using these heterologous markers in other Melipona species, increasing the number of loci that can be analyzed and contributing to further genetic analyses of intra- and intercolonial structure, which is required for conservation measure planning, genetic improvement and resolution of taxonomic problems.

The association of filaggrin mutations with atopic eczema (atopic dermatitis, AD) is well established and it is thought that filaggrin dysfunction impairs the skin's barrier function allowing allergen penetration and subsequent cutaneous sensitisation and inflammation. However, as most forms of barrier dysfunction are not associated with allergic sensitisation to common allergens, the possibility that filaggrin itself is involved in Th1/Th2 polarisation remains. We tested the hypothesis that allergen delivered to the skin independently of the stratum corneum is not associated with filaggrin mutations. Wasp stings bypass the stratum corneum and deliver antigen to the dermis. We found that European individuals with AD (n = 32) have an increased frequency of the 2 commonest filaggrin null mutations (R501X and 2282del4) compared to those with vespid allergy (n = 56) and healthy controls (n = 30). Thus, filaggrin does not appear to have a downstream effect on the development of allergic disease, and it is indeed filaggrin's role in the epithelial function that is likely to determine the link between filaggrin mutations and allergic sensitisation., (Copyright © 2010 S. Karger AG, Basel.)

We have sequenced an 81-kb genomic region from the honey bee, Apis mellifera, associated with a quantitative trait locus (QTL) sting-2 for aggressive behavior. This sequence represents the first extensive study of the honey-bee genome structure encompassing putative genes in a QTL for a behavioral trait. Expression of 13 putative genes, as well as two transcripts that were present in a honey-bee EST database, was confirmed through reverse transcription analysis of mRNA from the honey-bee head. Whereas most transcripts exhibited little or no variation between European and Africanized honey-bee alleles, one transcript demonstrated significant nonsynonymous substitutions, deletions, and insertions. All 13 putative genes lacked similarity to known invertebrate or vertebrate proteins or transcripts. This observation may be reflective of the processes that determine the genomic evolution of an insect with social behavior and/or haplo-diploidy and are an indication of the unique nature of the honey-bee genome. These results make this sequence an invaluable research tool for the ongoing honey-bee whole-genome sequencing effort.

To study the behavioral role of neurons containing the D1 dopamine receptor (D1+), we have used a genetic neurostimulatory approach. We generated transgenic mice that express an intracellular form of cholera toxin (CT), a neuropotentiating enzyme that chronically activates stimulatory G-protein (Gs) signal transduction and cAMP synthesis, under the control of the D1 promoter. Because the D1 promoter, like other CNS-expressed promoters, confers transgene expression that is regionally restricted to different D1+ CNS subsets in different transgenic lines, we observed distinct but related psychomotor disorders in different D1CT-expressing founders. In a D1CT line in which transgene expression was restricted to the following D1+ CNS regions-the piriform cortex layer II, layers II-III of somatosensory cortical areas, and the intercalated nucleus of the amygdala-D1CT mice showed normal CNS and D1+ neural architecture but increased cAMP content in whole extracts of the piriform and somatosensory cortex. These mice also exhibited a constellation of compulsive behavioral abnormalities that strongly resembled human cortical-limbic-induced compulsive disorders such as obsessive-compulsive disorder (OCD). These compulsive behaviors included episodes of perseverance or repetition of any and all normal behaviors, repetitive nonaggressive biting of siblings during grooming, and repetitive leaping. These results suggest that chronic potentiation of cortical and limbic D1+ neurons thought to induce glutamatergic output to the striatum causes behaviors reminiscent of those in human cortical-limbic-induced compulsive disorders.

Human bite mark evidence is often found in violent crimes. Due to the difficulties of physically comparing an injury site on elastic and curved skin surfaces to the teeth of a suspect, the authors have considered using salivary DNA evidence to identify the bite perpetrator. Several techniques were evaluated to determine the best method of recovering saliva from human skin before extracting genomic DNA from the collection substrate. A classical stain recovery technique using a wet cotton swab was tested against one utilizing a wet filter paper. Additionally, a new method, referred to as the double swab technique, using a wet cotton swab followed by a dry cotton swab was also evaluated. After recovering a dried saliva stain, DNA was extracted using the modified Chelex method, quantified using the slot-blot procedure, and amplified at three polymorphic loci. The double swab technique showed the highest percentage recovery of saliva from human skin among the three methods studied. This technique is suggested as an improvement over the classical single wet cotton swab technique.