Search

Apical-out intestinal organoids are a relatively simple method of gaining access to the apical cell surface and have faced increasing scientific interest over the last few years. Apical-out organoids can thus be used for disease modelling to compare differing effects on the basolateral versus the apical cell surface. However, these 'inside-out' organoids die relatively quickly and cannot be propagated as long as their basal-out counterparts. Here, we show that apical-out organoids have drastically reduced proliferative potential, as evidenced by immunohistochemical staining and the incorporation of the thymidine analogue EdU. At the same time, cell death levels are increased. Nevertheless, these phenomena cannot be explained by an induction of differentiation, as the gene expression of key marker genes for various cell types does not change over time., (© 2023 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.)

Introduction: Diseases of the digits often occur in cattle on larger cattle mountain pastures. In the late spring 2020, at the time of the ascent of 1554 cattle to 11 high altitude alpine pastures in the Lower Engadine region, lesions in the area of the digits were clinically assessed and documented. 254 cattle were of non-cantonal and 1300 of local origin (Lower Engadine; postal code CH-75XX). Skin lesions in the area of the digits, identified as digital dermatitis (DD; Mortellaro's disease), were further classified according to the DD scoring system. Nonspecific skin lesions with clinical evidence of granulation tissue formation were termed chronic penetrating skin lesions (CPSL). At the end of the alpine pasturing season, in the early fall (descent of cattle from the alpine pastures), the procedure was repeated, and biopsies were taken from randomly selected cattle with CPSL. Digital dermatitis lesions were found in 34 of 1551 cattle at ascent, but no case of CPSL was found at that time. At descent, 19 of 1529 cattle had DD lesions and 88 cattle had CPSL. The clinical appearance of the CPSL was consistent with chronic skin lesions caused by penetrating skin lacerations. Histologically, the majority of the CPSL were classified as chronic hyperplastic dermatitis with granulation tissue formation. In all CPSL biopsies examined by PCR, Fusobacterium necrophorum and Porphyromonas levii, but neither Dichelobacter nodosus nor the tested Treponema species were detected. Fluorescence in situ hybridization revealed a negative result for Treponema species in all biopsies. In the regression analysis, cattle in the age group of 365 to 730 days had an increased risk for the presence of CPSL compared to the age group of 160 to 365 days (odds ratio (OR) = 4,95; confidence interval (CI) = 1,97-12,43). Holstein cattle had an increased risk of developing CPSL compared to Brown cattle (OR = 2,92; CI = 1,46-5,86) and cattle of non-cantonal origin showed a massively higher risk compared to local cattle (OR = 10,59; CI = 5,79 - 19,37). The statistically significant associations found in the present study can be taken into account in the selection of animals for summer pasturing on high altitudes in the future in order to reduce the prevalence of CPSL and consequently reduce the antimicrobial use. Spread of DD during the alpine pasturing season within the cattle groups examined was not found.

Heterothermic thermoregulation requires intricate regulation of metabolic rate and activation of pro-survival factors. Eliciting these responses and coordinating the necessary energy shifts likely involves retrograde signalling by mitochondrial-derived peptides (MDPs). Members of the group were suggested before to play a role in heterothermic physiology, a key component of hibernation and daily torpor. Here we studied the mitochondrial single-nucleotide polymorphism (SNP) m.3017C>T that resides in the evolutionarily conserved gene MT-SHLP6. The substitution occurring in several mammalian orders causes truncation of SHLP6 peptide size from twenty to nine amino acids. Public mass spectrometric (MS) data of human SHLP6 indicated a canonical size of 20 amino acids, but not the use of alternative translation initiation codons that would expand the peptide. The shorter isoform of SHLP6 was found in heterothermic rodents at higher frequency compared to homeothermic rodents ( p < 0.001). In heterothermic mammals it was associated with lower minimal body temperature ( T b , p < 0.001). In the thirteen-lined ground squirrel, brown adipose tissue-a key organ required for hibernation, showed dynamic changes of the steady-state transcript level of mt-Shlp6 . The level was significantly higher before hibernation and during interbout arousal and lower during torpor and after hibernation. Our finding argues to further explore the mode of action of SHLP6 size isoforms with respect to mammalian thermoregulation and possibly mitochondrial retrograde signalling., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Emser, Spielvogel, Millesi and Steinborn.)

Haemonchus contortus is the most pathogenic nematode in small ruminants and anthelmintic resistance (AR) hampers its efficient control. Early detection of AR status is required to reduce selection for AR and cannot be achieved using phenotypic tests. For benzimidazoles (BZs), the detection of AR-associated alleles characterised by single nucleotide polymorphisms (SNPs) in the isotype 1 β-tubulin gene allows early AR detection in strongyles. The F200Y, F167Y, E198A and E198L polymorphisms have been described in BZ-resistant populations with a clear variation in frequencies between regions. A novel digital PCR (dPCR) enables the detection of all of the above-described polymorphisms in H. contortus. Assays were validated using synthetic DNA fragments containing these SNPs. Then, larvae obtained and pooled at farm level from 26 Austrian and 10 Italian sheep farms were analysed. For all assays a detection limit of 15 copies/μl of resistance alleles and a high level of accuracy were demonstrated, allowing to detect allele frequencies of 1% in most samples. In Austrian samples, elevated frequencies of F200Y resistance alleles were detected on all farms. Polymorphisms in codon 167 and codon 198 were identified in H. contortus from Austria for the first time. In Italian samples, the frequency of resistance alleles was still comparatively low, but F200Y resistance alleles were traceable. In conclusion we developed for the first time dPCR assays that target all SNPs of relevance associated with BZ-resistance in H. contortus. Future research on AR development could benefit from an early onset of SNP-based surveillance that would include the developed assays for all SNPs of relevance. Improved surveillance in the long term will include other important, though less pathogenic, nematode genera in the analyses., 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., (Copyright © 2023. Published by Elsevier Ltd.)

The diversity of plant monoterpenes is largely based on the catalytic activity of monoterpene synthases. Additionally, copy number variation of monoterpene synthase genes may contribute to the quantity of transcripts and hence to the essential oil profile. This study used whole-genome sequencing and digital PCR for the measurement of copy number variation and quantification of gene expression in three closely related Salvia species, namely Salvia officinalis, Salvia pomifera and Salvia fruticosa. Twelve, 13 and 15 monoterpene synthase-encoding open-reading frames were predicted for Salvia officinalis, Salvia pomifera and Salvia fruticosa, respectively. In Salvia officinalis, one of the open reading frames was disrupted indicating a pseudogene. Monoterpene synthase genes were generally single copy per haploid genome, only a few were double or triple copy genes. Expression levels of monoterpene synthases in leaves corresponded generally well with essential oil composition. In some cases, a higher expression level of a certain monoterpene synthase could be explained by its duplication or triplication. The very high content of thujones in Salvia pomifera, for example, was accompanied by gene duplication and increased gene expression of (+)-sabinene synthase responsible for the thujone precursor sabinene. In Salvia officinalis, three individuals different in their essential oil profile showed significant differences in their monoterpene synthase expression levels corresponding roughly to the profile of the essential oils. Transcript expression of monoterpene synthase genes were measured in leaf, calyx and corolla. The corolla differed significantly from leaves, while calyces usually showed a profile intermediary between leaf and corolla., 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., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

We aimed at extending the repertoire of high-quality miRNA normalizers for reverse transcription-quantitative PCR (RT-qPCR) of human plasma with special emphasis on the extremely guanine-cytosine-rich portion of the miRNome. For high-throughput selection of stable candidates, microarray technology was preferred over small-RNA sequencing (sRNA-seq) since the latter underrepresented miRNAs with a guanine-cytosine (GC) content of at least 75% ( p = 0.0002, n = 2). miRNA abundances measured on the microarray were ranked for consistency and uniformity using nine normalization approaches. The eleven most stable sequences included miRNAs of moderate, but also extreme GC content (45%-65%: miR-320d, miR-425-5p, miR-185-5p, miR-486-5p; 80%-95%: miR-1915-3p, miR-3656-5p, miR-3665-5p, miR-3960-5p, miR-4488-5p, miR-4497 and miR-4787-5p). In contrast, the seven extremely GC-rich miRNAs were not found in the two plasma miRNomes screened by sRNA-seq. Stem-loop RT-qPCR was employed for stability verification in 32 plasma samples of healthy male Caucasians (age range: 18-55 years). In general, inter-individual variance of miRNA abundance was low or very low as indicated by coefficient of variation ( CV ) values of 0.6%-8.2%. miR-3665 and miR-1915-3p outperformed in this analysis ( CV s: 0.6 and 2.4%, respectively). The eight most stable sequences included four extremely GC-rich miRNAs (miR-1915-3p, miR-3665, miR-4787-5p and miR-4497). The best-performing duo normalization factor ( NF ) for the condition of human plasma, miR-320d and miR-4787-5p, also included a GC-extreme miRNA. In summary, the identification of extremely guanine-cytosine-rich plasma normalizers will help to increase accuracy of PCR-based miRNA quantification, thus raise the potential that miRNAs become markers for psychological stress reactions or early and precise diagnosis of clinical phenotypes. The novel miRNAs might also be useful for orthologous contexts considering their conservation in related animal genomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Baumann, Athanasiou, Faridani, Schwerdtfeger, Wallner and Steinborn.)

Enriching mitochondrial DNA (mtDNA) for sequencing entire mitochondrial genomes (mitogenomes) can be achieved by single long-range PCR. This avoids interference from the omnipresent nuclear mtDNA sequences (NUMTs). The approach is currently restricted to the use of samples collected from humans and ray-finned fishes. Here, we extended the use of single long-range PCR by introducing back-to-back oligonucleotides that target a sequence of extraordinary homology across vertebrates. The assay was applied to five hibernating rodents, namely alpine marmot, Arctic and European ground squirrels, and common and garden dormice, four of which have not been fully sequenced before. Analysis of the novel mitogenomes focussed on the prediction of mitochondrial-derived peptides (MDPs) providing another level of information encoded by mtDNA. The comparison of MOTS-c, SHLP4 and SHLP6 sequences across vertebrate species identified segments of high homology that argue for future experimentation. In addition, we evaluated four candidate polymorphisms replacing an amino acid in mitochondrially encoded subunits of the oxidative phosphorylation (OXPHOS) system that were reported in relation to cold-adaptation. No obvious pattern was found for the diverse sets of mammalian species that either apply daily or multiday torpor or otherwise cope with cold. In summary, our single long-range PCR assay applying a pair of back-to-back primers that target a consensus sequence motif of Vertebrata has potential to amplify (intact) mitochondrial rings present in templates from a taxonomically diverse range of vertebrates. It could be promising for studying novel mitogenomes, mitotypes of a population and mitochondrial heteroplasmy in a sensitive, straightforward and flexible manner., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Emser, Schaschl, Millesi and Steinborn.)

Alimentary lymphomas arising from T cells are rare and aggressive malignancies in humans. In comparison, they represent the most common anatomical form of lymphoma in cats. Due to the low prevalence in humans, the underlying pathomechanism for these diseases is poorly characterised, limiting experimental analysis and therapeutic exploration. To date, activating mutations of the JAK/STAT core cancer pathway and particularly the STAT5B oncoprotein have been identified in human enteropathy-associated T cell lymphoma. Here, we describe a high homology of human and feline STAT3 and STAT5B proteins and strong conservation at the genomic level. Analysis of 42 samples of feline T cell alimentary lymphoma reveals broad activation of STAT3 and STAT5B. Screening for known activating mutations in STAT3 or STAT5B identifies the presence of the STAT5B N642H driver mutation in feline enteropathy-associated T cell lymphoma in 7 out of 42 (16.67%) samples in total. Regarding lymphoma subtypes, the majority of mutations with 5 out of 17 (29.41%) cases were found in feline enteropathy-associated lymphoma type II (EATL II). This identification of an oncogenic STAT5B driver mutation in felines recapitulates the genetic situation in the corresponding human disease, thereby establishing the cat as a potential new model for a rare and incurable human T cell disease.

There is strong evidence that equine parvovirus-hepatitis (EqPV-H) is associated with the onset of Theiler's disease, an acute hepatic necrosis, in horses. However, the impact of this virus on other hepatopathies remains unknown. The objective of this retrospective study was to evaluate the prevalence and quantify the viral loads of EqPV-H in formalin-fixed, paraffin-embedded equine and donkey livers with various histopathologic abnormalities. The pathologies included cirrhosis, circulatory disorders of the liver, toxic and metabolic hepatic diseases as well as neoplastic and inflammatory diseases ( n = 84). Eight normal liver samples were included for comparison as controls. EqPV-H DNA was qualitatively and quantitatively measured by real-time PCR and digital PCR, respectively. The virus was detected in two livers originating from horses diagnosed with abdominal neoplasia and liver metastasis (loads of 5 × 10 3 and 9.5 × 10 3 genome equivalents per million cells). The amount of viral nucleic acids measured indicates chronic infection or persistence of EqPV-H, which might have been facilitated by the neoplastic disease. In summary, this study did not provide evidence for EqPV-H being involved in hepatopathies other than Theiler's disease.

Heteroplasmic mice represent a valuable tool to study the segregation of different mtDNA haplotypes (mtDNAs with differing alleles) in vivo against a defined nuclear background. We describe two methods for the creation of such models, differing in the resulting initial heteroplasmy levels: (a) transfer of ooplasm and (b) fusion of two blastomeres. These methods result in typical heteroplasmy of 5% and 50% donor mtDNA , respectively. The choice of method depends on the aim of the study. By means of breeding even 100% donor mtDNA can be reached within a few generations.

Intrinsic biological fluctuation and/or measurement error can obscure the association of gene expression patterns between RNA and protein levels. Appropriate normalization of reverse-transcription quantitative PCR (RT-qPCR) data can reduce technical noise in transcript measurement, thus uncovering such relationships. The accuracy of gene expression measurement is often challenged in the context of cancer due to the genetic instability and "splicing weakness" involved. Here, we sequenced the poly(A) cancer transcriptome of canine osteosarcoma using mRNA-Seq. Expressed sequences were resolved at the level of two consecutive exons to enable the design of exon-border spanning RT-qPCR assays and ranked for stability based on the coefficient of variation (CV). Using the same template type for RT-qPCR validation, i.e. poly(A) RNA, avoided skewing of stability assessment by circular RNAs (circRNAs) and/or rRNA deregulation. The strength of the relationship between mRNA expression of the tumour marker S100A4 and its proportion score of quantitative immunohistochemistry (qIHC) was introduced as an experimental readout to fine-tune the normalization choice. Together with the essential logit transformation of qIHC scores, this approach reduced the noise of measurement as demonstrated by uncovering a highly significant, strong association between mRNA and protein expressions of S100A4 (Spearman's coefficient ρ = 0.72 (p = 0.006)). KEY MESSAGES: • RNA-seq identifies stable pairs of consecutive exons in a heterogeneous tumour. • Poly(A) RNA templates for RT-qPCR avoid bias from circRNA and rRNA deregulation. • HNRNPL is stably expressed across various cancer tissues and osteosarcoma. • Logit transformed qIHC score better associates with mRNA amount. • Quantification of minor S100A4 mRNA species requires poly(A) RNA templates and dPCR.

In this study, levels of inflammatory protein biomarkers in venous plasma, plasma derived from capillary blood from the earlobe, and capillary plasma stored as dried plasma spots (DPS) were compared. Samples from 12 male individuals were assessed with a panel of 92 inflammation-related proteins using multiplex proximity extension assay. Correlations between sample types varied greatly between analytes. A high correlation of ρ > 0.8 was observed between capillary plasma and DPS for 32 analytes. At this level of correlation, 13 analytes correlated between venous and capillary plasma and 5 analytes in the comparison of venous blood with DPS., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The human amniotic membrane (hAM) has been used for tissue regeneration for over a century. In vivo ( in utero ), cells of the hAM are exposed to low oxygen tension (1-4% oxygen), while the hAM is usually cultured in atmospheric, meaning high, oxygen tension (20% oxygen). We tested the influence of oxygen tensions on mitochondrial and inflammatory parameters of human amniotic mesenchymal stromal cells (hAMSCs). Freshly isolated hAMSCs were incubated for 4 days at 5% and 20% oxygen. We found 20% oxygen to strongly increase mitochondrial oxidative phosphorylation, especially in placental amniotic cells. Oxygen tension did not impact levels of reactive oxygen species (ROS); however, placental amniotic cells showed lower levels of ROS, independent of oxygen tension. In contrast, the release of nitric oxide was independent of the amniotic region but dependent on oxygen tension. Furthermore, IL-6 was significantly increased at 20% oxygen. To conclude, short-time cultivation at 20% oxygen of freshly isolated hAMSCs induced significant changes in mitochondrial function and release of IL-6. Depending on the therapeutic purpose, cultivation conditions of the cells should be chosen carefully for providing the best possible quality of cell therapy.

G protein receptor kinases (GRKs) and β-arrestins are key regulators of μ-opioid receptor (MOR) signaling and trafficking. We have previously shown that high-efficacy opioids such as DAMGO stimulate a GRK2/3-mediated multisite phosphorylation of conserved C-terminal tail serine and threonine residues, which facilitates internalization of the receptor. In contrast, morphine-induced phosphorylation of MOR is limited to Ser 375 and is not sufficient to drive substantial receptor internalization. We report how specific multisite phosphorylation controlled the dynamics of GRK and β-arrestin interactions with MOR and show how such phosphorylation mediated receptor desensitization. We showed that GRK2/3 was recruited more quickly than was β-arrestin to a DAMGO-activated MOR. β-Arrestin recruitment required GRK2 activity and MOR phosphorylation, but GRK recruitment also depended on the phosphorylation sites in the C-terminal tail, specifically four serine and threonine residues within the 370 TREHPSTANT 379 motif. Our results also suggested that other residues outside this motif participated in the initial and transient recruitment of GRK and β-arrestins. We identified two components of high-efficacy agonist desensitization of MOR: a sustained component, which required GRK2-mediated phosphorylation and a potential soluble factor, and a rapid component, which was likely mediated by GRK2 but independent of receptor phosphorylation. Elucidating these complex receptor-effector interactions represents an important step toward a mechanistic understanding of MOR desensitization that leads to the development of tolerance and dependence., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Classical opioid analgesics, including morphine, mediate all of their desired and undesired effects by specific activation of the μ-opioid receptor (μ receptor). The use of morphine for treating chronic pain, however, is limited by the development of constipation, respiratory depression, tolerance and dependence. Analgesic effects can also be mediated through other members of the opioid receptor family such as the κ-opioid receptor (κ receptor), δ-opioid receptor (δ receptor) and the nociceptin/orphanin FQ peptide receptor (NOP receptor). Currently, a new generation of opioid analgesics is being developed that can simultaneously bind with high affinity to multiple opioid receptors. With this new action profile, it is hoped that additional analgesic effects and fewer side effects can be achieved. Recent research is mainly focused on the development of bifunctional μ/NOP receptor agonists, which has already led to novel lead structures such as the spiroindole-based cebranopadol and a compound class with a piperidin-4-yl-1,3-dihydroindol-2-one backbone (SR16835/AT-202 and SR14150/AT-200). In addition, the ornivol BU08028 is an analogue of the clinically well-established buprenorphine. Moreover, the morphinan-based nalfurafine exerts its effect with a dominant κ receptor-component and is therefore utilized in the treatment of pruritus. The very potent dihydroetorphine is a true multi-receptor opioid ligand in that it binds to μ, κ and δ receptors. The main focus of this review is to assess the paradigm of opioid ligands targeting multiple receptors with a single chemical entity. We reflect on this rationale by discussing the biological actions of particular multi-opioid receptor ligands, but not on their medicinal chemistry and design., Linked Articles: This article is part of a themed section on Emerging Areas of Opioid Pharmacology. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.14/issuetoc., (© 2017 The British Pharmacological Society.)

Vital mitochondrial DNA (mtDNA) populations exist in cells and may consist of heteroplasmic mixtures of mtDNA types. The evolution of these heteroplasmic populations through development, ageing, and generations is central to genetic diseases, but is poorly understood in mammals. Here we dissect these population dynamics using a dataset of unprecedented size and temporal span, comprising 1947 single-cell oocyte and 899 somatic measurements of heteroplasmy change throughout lifetimes and generations in two genetically distinct mouse models. We provide a novel and detailed quantitative characterisation of the linear increase in heteroplasmy variance throughout mammalian life courses in oocytes and pups. We find that differences in mean heteroplasmy are induced between generations, and the heteroplasmy of germline and somatic precursors diverge early in development, with a haplotype-specific direction of segregation. We develop stochastic theory predicting the implications of these dynamics for ageing and disease manifestation and discuss its application to human mtDNA dynamics.

The major cow's milk allergen Bos d 5 belongs to the lipocalin protein family, with an intramolecular pocket for hydrophobic ligands. We investigated whether Bos d 5 when loaded with the active vitamin A metabolite retinoic acid (RA), would elicit differential immune responses compared to the unloaded state. By in silico docking an affinity energy of -7.8 kcal/mol was calculated for RA into Bos d 5. Loading of RA to Bos d 5 could be achieved in vitro, as demonstrated by ANS displacement assay, but had no effect on serum IgE binding in tolerant or challenge-positive milk allergic children. Bioinformatic analysis revealed that RA binds to the immunodominant T-cell epitope region of Bos d 5. In accordance, Bos d 5 significantly suppressed the CD3+ CD4+ cell numbers, proliferative response and IL-10, IL-13 and IFN-γ secretion from stimulated human PBMCs only when complexed with RA. This phenomenon was neither associated with apoptosis of T-cells nor with the activation of Foxp3+ T-cells, but correlated likely with enhanced stability to lysosomal digestion due to a predicted overlap of Cathepsin S cleavage sites with the RA binding site. Taken together, proper loading of Bos d 5 with RA may suppress its immunogenicity and prevent its allergenicity.

Over a century ago, clinicians started to use the human amniotic membrane for coverage of wounds and burn injuries. To date, literally thousands of different clinical applications exist for this biomaterial almost exclusively in a decellularized or denuded form. Recent reconsiderations for the use of vital human amniotic membrane for clinical applications would take advantage of the versatile cells of embryonic origin including the entirety of their cell organelles. Recently, more and more evidence was found, showing mitochondria to be involved in most fundamental cellular processes, such as differentiation and cell death. In this study, we focused on specific properties of mitochondria of vital human amniotic membrane and characterized bioenergetical parameters of 2 subregions of the human amniotic membrane, the placental and reflected amnion. We found significantly different levels of adenosine triphosphate (ATP) and extracellular reactive oxygen species, concentrations of succinate dehydrogenase, and lactate upon inhibition of ATP synthase in placental and reflected amnion. We also found significantly different rates of mitochondrial respiration in isolated human amniotic epithelial cells and human amniotic mesenchymal stromal cells, according to the subregions. Differences in metabolic activities were inversely related to mitochondrial DNA copy numbers in isolated cells of placental and reflected amnion. Based on significant differences of several key parameters of energy metabolism in 2 subregions of vital amnion, we propose that these metabolic differences of vital placental and reflected amnion could have critical impact on therapeutic applications. Inclusion of region-specific metabolic properties could optimize and fine-tune the clinical application of the human amniotic membrane and improve the outcome significantly.

In horses, squamous cell carcinomas (SCC) commonly affect the external genitals. There is growing evidence that equine papillomavirus type 2 (EcPV2) infection promotes disease development. To assess the possible association of EcPV2 with equine SCCs of the head (HSCC), 15 HSCC DNA samples were screened by E6/E7, E2, and LCR PCR and amplicons were analysed for sequence variations. The physical form of EcPV2 in HSCC, genital lesions, and smegma from horses with SCC was then addressed using EcPV2 immunocapture PCR (IC/PCR) for detection of virion, and E6 vs. E2 qPCR to investigate possible integration events. Four of 15 HSCC tested positive for EcPV2 DNA and harboured known or novel genetic variants of E6, E7, E2 and the LCR. Eighteen of 35 sample extracts including 3/4 smegma samples scored positive by IC/PCR, suggesting that about 51% of tested extracts harboured virions. E6/E2 qPCR from tumour DNA revealed E2/E6 copies/cell ranging between <1 (E2; E6) and 797 (E2) or 1434 (E6). IC/PCR-positive smegma samples contained higher E2 and E6 copy numbers, ranging between 1490 and 4.95×10 5 (E2) or 2227 and 8.54×10 5 (E6) copies/cell. Together with IC/PCR results, this finding suggests that smegma can serve as a rich EcPV2 reservoir. HSCCs harboured significantly lower viral DNA amounts (<1-25 copies/cell) than most genital tumour and smegma DNA isolates. The majority of samples contained more E6 than E2 DNA, with E6:E2 ratios ranging between 0.88 and 4.12. Although not statistically significant (P>0.05), this finding suggests that EcPV2 can integrate into the equine host cell genome., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Purpose: To determine the prognostic factors of long-term visual outcome in birdshot retinochoroidopathy (BRC)., Methods: Design: Retrospective case series. Study Population: Successive HLA-A29+ BRC patients whose latest visit was between May and August 2013 at a single tertiary center (Pitié-Salpétrière Hospital, Paris)., Observation Procedure: Endpoint visual status (remission or deterioration) was determined for each patient based on clinical and ancillary data from the latest visit including optical coherence tomography (OCT), automated visual field (AVF), and angiograms. Main Outcome Measure: Epidemiologic, clinical, OCT, AVF, angiographic, and electrophysiological data at baseline were correlated to final visual status., Results: Fifty-five patients were included. Mean observation period was 8 years (range: 0.6-23 years). Mean disease duration was 9.8 years (range: 1.2-32.7 years). Female-to-male sex ratio was 1.6:1. Factors of good visual prognosis (remission vs deterioration) included at baseline: late age of disease onset (49.5 vs 45 years, P = .05), presence of vitreous inflammatory reactions >2+ (35.9% vs 6.2%, P = .04), vascular leakage on fluorescein angiograms (FA) (44.4% vs 12.5%, P = .03), absence of macular pigment epithelium atrophy on FA (88.9% vs 62.5%, P = .05), and presence of macular edema on OCT (33.3% vs 6.2%, P = .04). Preserved electrooculography light peak and Arden ratio (P = .06) and presence of choroidal spots on infracyanine green angiograms (80.0% vs 53.3%, P = .08) seemed associated with the best prognoses., Conclusion: This study suggests a series of prognostic factors of long-term visual outcome in BRC. Keeping in mind the insidious evolution of the disease, knowledge of such prognostic factors should help tailor the treatment and monitoring of birdshot patients., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Osteosarcoma is considered the most common bone cancer in cats and dogs, with cats having a much better prognosis than dogs, since the great majority of dogs with osteosarcoma develop distant metastases. In search of a factor possibly contributing to this disparity, the stem cell growth factor receptor KIT was targeted, and the messenger (m)RNA and protein expression levels of KIT were compared in canine vs. feline osteosarcomas, as well as in normal bone. The mRNA expression of KIT was quantified by reverse transcription-quantitative polymerase chain reaction, and was observed to be significantly higher in canine (n=14) than in feline (n=5) osteosarcoma samples (P<0.001). KIT protein expression was evaluated by immunohistochemistry, which revealed that 21% of canine osteosarcoma samples did not exhibit KIT staining in their neoplastic cells, while in 14% of samples, a score of 1 (<10% positive tumour cells) was observed, and in 50% and 14% of samples, a score of 2 (10-50% positivity) and 3 (>50% positivity), respectively, was observed. By contrast, the cancer cells of all the feline bone tumour samples analysed were entirely negative for KIT. Notably, canine and feline osteocytes of healthy bone tissue lacked any KIT expression. These results could be the first evidence that KIT may be involved in the higher aggressiveness of canine osteosarcoma compared with feline osteosarcoma.

Cell fate decisions in many physiological processes, including embryogenesis, stem cell niche homeostasis and wound healing, are regulated by secretion of small signaling proteins, called cytokines, from source cells to their neighbors or into the environment. Concentration level and steepness of the resulting paracrine gradients elicit different cell responses, including proliferation, differentiation or chemotaxis. For an in-depth analysis of underlying mechanisms, in vitro models are required to mimic in vivo cytokine gradients. We set up a microparticle-based system to establish short-range cytokine gradients in a three-dimensional extracellular matrix context. To provide native binding sites for cytokines, agarose microparticles were functionalized with different glycosaminoglycans (GAG). After protein was loaded onto microparticles, its slow release was quantified by confocal microscopy and fluorescence correlation spectroscopy. Besides the model protein lysozyme, SDF-1 was used as a relevant chemokine for hematopoietic stem and progenitor cell (HSPC) chemotaxis. For both proteins we found gradients ranging up to 50μm from the microparticle surface and concentrations in the order of nM to pM in dependence on loading concentration and affinity modulation by the GAG functionalization. Directed chemotactic migration of cells from a hematopoietic cell line (FDCPmix) and primary murine HSPC (Sca-1(+) CD150(+) CD48(-)) toward the SDF-1-laden microparticles proved functional short-range gradients in a two-dimensional and three-dimensional setting over time periods of many hours. The approach has the potential to be applied to other cytokines mimicking paracrine cell-cell interactions in vitro., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Sequence variations in genes of the monoamine neurotransmitter system and their common function in human and non-human primate species are an ongoing issue of investigation. However, the COMT gene, coding for the catechol-O-methyltransferase, has not yet attracted much scientific attention regarding its functional role in non-human primates. Considering that a polymorphism of the human COMT gene affects the enzyme activity and cortisol level in response to a social stressor, this study investigated the impact of COMT on endocrine stress and behavioural parameters in Japanese macaques (Macaca fuscata). The species exemplifies a despotic hierarchy in which males' social rank positions require an adaptation of behaviour strategies. During the mating period steroid secretion and the frequency of aggressive encounters between males increase. We addressed i) whether this species exhibits potential functional COMT variants, ii) whether these variants are associated with faecal cortisol excretion of males, iii) how they are distributed among different social rank positions and iv) whether they are associated with behavioural strategies during times of mate competition. By genotyping 26 males we identified three COMT haplotypes (HT), including a putative splice mutant (HT3). This variant was associated with increased cortisol excretion. Given the observed inverse correlation between cortisol and physical aggression, we assume that different COMT haplotypes may predispose individuals to pursue more or less aggressive strategies. How these gene-stress effects might favour a specific social role is discussed. Our study of non-invasive genotyping in combination with behavioural and endocrine parameters represents an important step towards the understanding of gene-stress effects in a hierarchically organised primate society., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Vertebrate evolution is accompanied by a substantial conservation of transcriptional programs with more than a third of unique orthologous genes showing constrained levels of expression. Moreover, there are genes and exons exhibiting excellent expression stability according to RNA-seq data across a panel of eighteen tissues including the ovary (Human Body Map 2.0)., Results: We hypothesized that orthologs of these exons would also be highly uniformly expressed across neonatal ovaries of the horse, which would render them appropriate reference genes (RGs) for normalization of reverse transcription quantitative PCR (RT-qPCR) data in this context. The expression stability of eleven novel RGs (C1orf43, CHMP2A, EMC7, GPI, PSMB2, PSMB4, RAB7A, REEP5, SNRPD3, VCP and VPS29) was assessed by RT-qPCR in ovaries of seven neonatal fillies and compared to that of the expressed repetitive element ERE-B, two universal (OAZ1 and RPS29) and four traditional RGs (ACTB, GAPDH, UBB and B2M). Expression stability analyzed with the software tool RefFinder top ranked the normalization factor constituted of the genes SNRPD3 and VCP, a gene pair that is not co-expressed according to COEXPRESdb and GeneMANIA. The traditional RGs GAPDH, B2M, ACTB and UBB were only ranked 3rd and 12th to 14th, respectively., Conclusions: The functional diversity of the novel RGs likely facilitates expression studies over a wide range of physiological and pathological contexts related to the neonatal equine ovary. In addition, this study augments the potential for RT-qPCR-based profiling of human samples by introducing seven new human RG assays (C1orf43, CHMP2A, EMC7, GPI, RAB7A, VPS29 and UBB).

Cells of the human amniotic membrane (hAM) have stem cell characteristics with low immunogenicity and anti-inflammatory properties. While hAM is an excellent source for tissue engineering, so far, its sub-regions have not been taken into account. We show that placental and reflected hAM differ distinctly in morphology and functional activity, as the placental region has significantly higher mitochondrial activity, however significantly less reactive oxygen species. Since mitochondria may participate in processes such as cell rescue, we speculate that amniotic sub-regions may have different potential for tissue regeneration, which may be crucial for clinical applications., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The discovery of expression of the erythropoietin receptor (EPO-R) on neoplastic cells has led to concerns about the safety of treating anaemic cancer patients with EPO. In addition to its endocrine function, the receptor may play a role in tumour progression through an autocrine mechanism. In this study, the expression of EPO, EPO-R and platelet-derived growth factor BB (PDGF-BB) was analysed in five feline and 13 canine osteosarcomas using immunohistochemistry (IHC) and reverse transcription polymerase chain reaction (RT-PCR). EPO expression was positive in all tumours by IHC, but EPO mRNA was only detected in 38% of the canine and 40% of the feline samples. EPO-R was expressed in all samples by quantitative RT-PCR (RT-qPCR) and IHC. EPO-R mRNA was expressed at higher levels in all feline tumours, tumour cell lines, and kidney when compared to canine tissues. PDGF-BB expression was variable by IHC, but mRNA was detected in all samples. To assess the functionality of the EPO-R on tumour cells, the proliferation of canine and feline osteosarcoma cell lines was evaluated after EPO administration using an alamarBlue assay and Ki67 immunostaining. All primary cell lines responded to EPO treatment in at least one of the performed assays, but the effect on proliferation was very low indicating only a weak responsiveness of EPO-R. In conclusion, since EPO and its receptor are expressed by canine and feline osteosarcomas, an autocrine or paracrine tumour progression mechanism cannot be excluded, although in vitro data suggest a minimal role of EPO-R in osteosarcoma cell proliferation., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In Austria, vaccination coverage against Bordetella pertussis infections during infancy is estimated at around 90%. Within the last years, however, the number of pertussis cases has increased steadily, not only in children but also in adolescents and adults, indicating both insufficient herd immunity and vaccine coverage. Waning immunity in the host and/or adaptation of the bacterium to the immunised hosts could contribute to the observed re-emergence of pertussis. In this study we therefore addressed the genetic variability in B. pertussis strains from several Austrian cities. Between the years 2002 and 2008, 110 samples were collected from Vienna (n = 32), Linz (n = 63) and Graz (n = 15) by nasopharyngeal swabs. DNA was extracted from the swabs, and bacterial sequence polymorphisms were examined by MLVA (multiple-locus variable number of tandem repeat analysis) (n = 77), by PCR amplification and conventional Sanger sequencing of the polymorphic regions of the prn (pertactin) gene (n = 110), and by amplification refractory mutation system quantitative PCR (ARMS-qPCR) (n = 110) to directly address polymorphisms in the genes encoding two pertussis toxin subunits (ptxA and ptxB), a fimbrial adhesin (fimD), tracheal colonisation factor (tcfA), and the virulence sensor protein (bvgS). Finally, the ptxP promoter region was screened by ARMS-qPCR for the presence of the ptxP3 allele, which has been associated with elevated production of pertussis toxin. The MLVA analysis revealed the highest level of polymorphisms with an absence of MLVA Type 29, which is found outside Austria. Only Prn subtypes Prn1/7, Prn2 and Prn3 were found with a predominance of the non-vaccine type Prn2. The analysis of the ptxA, ptxB, fimD, tcfA and bvgS polymorphisms showed a genotype mixed between the vaccine strain Tohama I and a clinical isolate from 2006 (L517). The major part of the samples (93%) displayed the ptxP3 allele. The consequences for the vaccination strategy are discussed.

Background: A single nucleotide polymorphism (SNP) in the first intron of the myostatin gene (MSTN) is associated with aptness of elite Thoroughbreds to race over sprint, middle or long distances. This intronic marker (g.66493737 T ≻ C), a short interspersed nuclear element (SINE) of 227 bp (Ins227bp) insertion polymorphism in the MSTN promoter, and the adjacent SNP BIEC2-417495 have not been studied for their association with racing aptness of the average Thoroughbreds raced in countries with lower status of the racing industry. This study investigated these markers regarding their prevalence and association with performance in common race horses. Markers were genotyped by amplification refractory mutation system-quantitative PCR (ARMS-qPCR) or amplicon melting. Furthermore, we asked whether the Ins227bp marker might theoretically regulate the expression of myostatin by generating a novel target for DNA methylation or by changing binding sites for transcription factors. Putative sites for DNA methylation or binding of transcription factors were predicted by MethPrimer and by the softwares JASPAR, MatInspector and UniPROBE, respectively., Results: Pairwise linkage disequilibrium between g.66493737 T ≻ C and Ins227bp was high (r (2) = 0.93). A lower linkage was determined for g.66493737 T ≻ C and BIEC2-417495 (r (2) = 0.69) as well as for BIEC2-417495 and Ins227bp (r (2) = 0.76). The estimated frequencies for the presence of Ins227bp (I) indel and the C alleles at g.66493737 T ≻ C and BIEC2-417495 were 0.46, 0.47 and 0.43, respectively. Heterozygotes represented the most abundant genotype at each locus. The best racing distance (BRD) was significantly different between the homozygotes of each SNP (p = 0.01 to 0.03). C allele homozygotes at BIEC2-417495 or g.66493737 T ≻ C, as well as Ins227bp homozygotes earned most money on a mean distance ranging from 1211 to 1230 m. Heterozygotes earned most money on races over 1690 to 1709 m. The BRD for the T/T carriers at both SNP loci and for the SINE-free genotype was 1812 to 1854 m. Other performance parameters were not significantly different between the genotypes, except of the relative success score (RSS). The RSS was significantly slightly better on a distance of ≤ 1300 m for all carriers of the C allele and the Ins227bp compared to homozygous T genotypes and SINE-negative horses (p = 0.037 to 0.046). For distances of more than 1300 m the RSS was not significantly different between genotypes. In silico assessment indicated that the Ins227bp promoter insertion might have generated a CpG island and a few novel putative binding sites for transcription factors., Conclusions: All three target polymorphisms (Ins227bp, g.66493737 T ≻ C, BIEC2-417495) are suitable markers to assess the ability of non-elite Thoroughbreds to race at short or longer distances. The CpG island generated by Ins227bp may cause training-induced silencing of MSTN expression.

Heteroplasmic mice represent a valuable tool to study the segregation of different mtDNA haplotypes (mtDNAs with differing alleles) in vivo against a defined nuclear background. We describe two methods for the creation of such models, differing in the resulting initial heteroplasmy levels: (1) transfer of ooplasm and (2) fusion of two blastomeres. These methods result in typical heteroplasmy of 5 % and 50 % donor mtDNA, respectively. The choice of method depends on the aim of the study. By means of breeding, even 100 % donor mtDNA can be reached within few generations.

Gizzard erosion in broilers due to an infection with virulent fowl adenovirus serotype 1 (FAdV-1) is an emerging disease. Although experimental studies were performed, a possible prevention strategy was not reported so far. The present study was set up to determine (i) a possible influence of birds' age at time of inoculation on the pathogenicity of a European FAdV-1 field strain (PA7127), (ii) the virulence of a apathogenic FAdV-1 strain (CELO), and (iii) its capability to protect SPF broilers from adenoviral gizzard erosion caused by the field virus. Oral infection of birds with PA7127 at 1-, 10- and 21-days of life, resulted in reduced weight gain compared to non-infected birds, with significance for birds infected at day-old. Independent of the birds' age at time of inoculation, clinical signs appearing approximately one week after challenge coincided with gizzard lesions. Birds infected exclusively with CELO at the first day of life did not show any clinical signs or pathological changes in the gizzard, confirming the apathogenicity of this European FAdV-1. A similar result was obtained for birds orally infected at the first day of life with CELO and challenged three weeks later with the pathogenic PA7127 strain. Therefore, complete protection of adenoviral gizzard erosion in broilers by vaccination of day-old birds could be demonstrated for the first time, although virus excretion was detected post challenge. Establishment of an amplification refractory mutation system quantitative PCR (ARMS-qPCR) facilitated the identification of the FAdV-1 strain and presence of challenges virus was confirmed in one sample., (Copyright © 2014 Elsevier B.V. All rights reserved.)