Phaidra - University of Veterinary Medicine Vienna
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Dietary carbohydrate sources differently prime the microbial ecosystem but not the epithelial gene expression profile along the complete gut of young calves
No full textRecent data indicated similar growth performance of young calves fed solely high-quality hay instead of a starter diet based on starchy ingredients. Yet, providing exclusively such distinct carbohydrate sources during early life might specifically prime the microbiota and gene expression along the gut of young calves, which remains to be explored. We investigated the effects of starter diets differing in carbohydrate composition, that is medium- or high-quality hay and without or with 70% concentrate supplementation (on fresh matter basis), across the gastrointestinal tract (GIT) of weaned Holstein calves (100 ± 4 days of age) using 16 S rRNA gene sequencing and analyses of short-chain fatty acids and host epithelial gene expressions.The concentrate supplementation drastically decreased microbial diversity throughout the gut, which was also true to a much lesser extent for high-quality hay when compared to medium-quality hay in the foregut. Similarly, the factor concentrate strongly shaped the diet-associated common core microbiota, which was substantially more uniform along the gut with concentrate supplementation. The fermentation profile shifted towards less acetate but more propionate with concentrate supplementation in almost all gut sections, corresponding with higher abundances of starch-utilizing bacteria, while major fibrolytic clusters declined. Noteworthy, the n-butyrate proportion decreased in the rumen and increased in the colon with concentrate, showing an opposite, gut site-dependent effect. Both dietary factors modestly influenced the host epithelial gene expression.Concentrate supplementation clearly primed the microbial ecosystem on a starch-targeted fermentation with characteristic genera occupying this niche along the entire GIT of calves, whereas the microbial differentiation due to hay quality was less distinct. Overall, changes in the microbial ecosystem were only marginally reflected in the targeted transcriptional profile of the host epithelium
The impact of opioid administration on the incidence of postanaesthetic colic in horses
No full textEffective management of postoperative pain is essential to ensure patient welfare, reduce morbidity and optimize recovery. Opioids are effective in managing moderate to severe pain in horses but concerns over their adverse effects on gastrointestinal (GI) motility and associated increased colic risk limit their widespread use. Studies investigating the impact of systemic opioids on both GI motility and colic incidence in horses have yielded inconclusive outcomes. Therefore, this retrospective study aims to assess the influence of systemic administration of butorphanol, morphine, and methadone on post-anaesthetic colic (PAC) incidence. Horses undergoing general anaesthesia for non-gastrointestinal procedures that were hospitalized for at least 72 h post-anaesthesia were included in this study. Anaesthetised horses were stratified by procedure type into horses undergoing diagnostic imaging without surgical intervention, emergency or elective surgery. In addition, patients were grouped by opioid treatment regime into horses receiving no opioids, intraanaesthetic, short- (24 h) postoperative opioids. Administered opioids encompassed butorphanol, morphine and methadone. The number of horses showing signs of colic in the 72 h after anaesthesia was assessed for each group. A total of 782 horses were included, comprising 659 undergoing surgical procedures and 123 undergoing diagnostic imaging. The overall PAC incidence was 15.1%. Notably, horses undergoing diagnostic imaging without surgery had a significantly lower PAC rate of 6.5% compared to those undergoing surgery (16.7%, p = 0.0146). Emergency surgeries had a significantly lower PAC rate of 5.8% compared to elective procedures (18%, p = 0.0113). Of the 782 horses, 740 received intraoperative opioids and 204 postoperative opioids, 102 of which long-term (≥24 h). Neither intraoperative (p = 0.4243) nor short-term postoperative opioids (p = 0.5744) increased PAC rates. Notably, only the long-term (≥24 h) administration of morphine significantly increased PAC incidence to 34% (p = 0.0038). In contrast, long-term butorphanol (5.3% PAC, p = 0.8482) and methadone (18.4% PAC, p = 0.6161) did not affect PAC rates. In summary, extended morphine administration was the only opioid treatment associated with a significantly increased risk of PAC
Lymphocyte-to-Monocyte Ratio Might Serve as a Prognostic Marker in Young Patients with Tongue Squamous Cell Carcinoma
No full textYoung patients with tongue squamous cell carcinoma (TSCC) mostly lack typical prognostic markers and face a dire prognosis. The aim of this study was to analyze the prognostic relevance of lymphocyte-to-monocyte ratio (LMR) in TSCC patients, with a special emphasis on patients under 45 years.This retrospective study included all patients primarily treated for TSCC. The prognostic relevance of LMR was investigated in terms of predicting the overallsurvival (OS) and disease-free survival (DFS).A total of 74 patients were included and the young cohort (<45 years) comprised 27 individuals. The mortality and recurrence rates were 39.2% (n = 29) and 37.8% (n = 28), respectively. OS and DFS were significantly shorter in the low LMR group within the whole cohort. Furthermore, low LMR was associated with worse prognosis, particularly inferior OS (median OS 1.7 vs. 14.6 years, p = 0.0156) and worse DFS (median DFS 0.8 years vs. not reached, p = 0.0405) in the young patient cohort.Our results reveal that pretreatment LMR might become a prognostic tool for young TSCC patients, especially due to its availability. However, further studies on larger cohorts are necessary to validate our results
Novel phylogenetic clade of avian Haemoproteus parasites (Haemosporida, Haemoproteidae) from Accipitridae raptors, with description of a new Haemoproteus species
No full textClonal evolution and cellular plasticity are the genetic and non-genetic driving forces of tumor heterogeneity, which in turn determine tumor cell responses towards therapeutic drugs. Several lines of evidence suggest that therapeutic interventions foster the selection of drug-resistant neural crest stem-like cells (NCSCs) that establish minimal residual disease (MRD) in melanoma. Here, we establish a dual-reporter system, enabling the tracking of NGFR expression and mRNA stability and providing insights into the maintenance of NCSC states. We observed that a transcriptional reporter that contained a 1-kilobase fragment of the human NGFR promoter was activated only in a minor subset (0.72 ± 0.49%, range 0.3-1.5), and ~2-4% of A375 melanoma cells revealed stable NGFR mRNA. The combination of both reporters provides insights into phenotype switching and reveals that both cellular subsets gave rise to cellular heterogeneity. Moreover, whole transcriptome profiling and gene-set enrichment analysis (GSEA) of the minor cellular subset revealed hypoxia-associated programs that might serve as potential drivers of an in vitro switching of NGFR-associated phenotypes and relapse of post-BRAF inhibitor-treated tumors. Concordantly, we observed that the minor cellular subset increased in response to dabrafenib over time. In summary, our reporter-based approach provides insights into plasticity and identified a cellular subset that might be responsible for the establishment of MRD in melanoma.Les parasites hémosporidies aviaires (ordre Haemosporida, phylum Apicomplexa) sont des parasites sanguins et tissulaires transmis par des insectes diptères hématophages. Trois genres (Plasmodium, Haemoproteus et Leucocytozoon) ont été le plus souvent trouvés chez les oiseaux, avec plus de 270 espèces décrites et nommées chez les hôtes aviaires en fonction principalement des caractères morphologiques des stades sanguins. Une grande diversité des Haemoproteus reste à identifier et à caractériser morphologiquement et génétiquement, en particulier ceux qui infectent les oiseaux de proie, un groupe d’oiseaux sous-représenté dans les études sur les hémosporidies. Le but de cette étude était d’étudier et d’identifier les Haemoproteus à partir d’un large échantillon comprenant des rapaces accipitriformes de 16 espèces, en combinant des protocoles morphologiques et de nouveaux protocoles moléculaires ciblant les gènes cytb de ce groupe de parasites. Cette étude fournit des descriptions morphologiques et des caractérisations moléculaires de deux espèces d’Haemoproteus, H. multivacuolatus n. sp. et H. nisi Peirce and Marquiss, 1983. Les Haemoproteus de ce groupe n’ont jusqu’à présent été trouvés que chez les rapaces accipitriformes et pourraient être classés dans un sous-genre ou même un genre distinct. Les séquences cytb de ces parasites divergent de plus de 15 % de celles de tous les autres genres d’hémosporidies aviaires connus et forment un clade phylogénétique unique. Cette étude souligne l’importance de développer de nouveaux outils de diagnostic pour détecter des parasites moléculairement très divergents qui pourraient être indétectables par les outils conventionnels couramment utilisés.Les parasites hémosporidies aviaires (ordre Haemosporida, phylum Apicomplexa) sont des parasites sanguins et tissulaires transmis par des insectes diptères hématophages. Trois genres (Plasmodium, Haemoproteus et Leucocytozoon) ont été le plus souvent trouvés chez les oiseaux, avec plus de 270 espèces décrites et nommées chez les hôtes aviaires en fonction principalement des caractères morphologiques des stades sanguins. Une grande diversité des Haemoproteus reste à identifier et à caractériser morphologiquement et génétiquement, en particulier ceux qui infectent les oiseaux de proie, un groupe d’oiseaux sous-représenté dans les études sur les hémosporidies. Le but de cette étude était d’étudier et d’identifier les Haemoproteus à partir d’un large échantillon comprenant des rapaces accipitriformes de 16 espèces, en combinant des protocoles morphologiques et de nouveaux protocoles moléculaires ciblant les gènes cytb de ce groupe de parasites. Cette étude fournit des descriptions morphologiques et des caractérisations moléculaires de deux espèces d’Haemoproteus, H. multivacuolatus n. sp. et H. nisi Peirce and Marquiss, 1983. Les Haemoproteus de ce groupe n’ont jusqu’à présent été trouvés que chez les rapaces accipitriformes et pourraient être classés dans un sous-genre ou même un genre distinct. Les séquences cytb de ces parasites divergent de plus de 15 % de celles de tous les autres genres d’hémosporidies aviaires connus et forment un clade phylogénétique unique. Cette étude souligne l’importance de développer de nouveaux outils de diagnostic pour détecter des parasites moléculairement très divergents qui pourraient être indétectables par les outils conventionnels couramment utilisés
Cryptosporidium parvum infection alters the intestinal mucosa transcriptome in neonatal calves: implications for immune function
No full textOne of the leading causes of infectious diarrhea in newborn calves is the apicomplexan protozoan Cryptosporidium parvum (C. parvum). However, little is known about its immunopathogenesis. Using next generation sequencing, this study investigated the immune transcriptional response to C. parvum infection in neonatal calves. Neonatal male Holstein-Friesian calves were either orally infected (N = 5) or not (CTRL group, N = 5) with C. parvum oocysts (gp60 subtype IIaA15G2R1) at day 1 of life and slaughtered on day 7 after infection. Total RNA was extracted from the jejunal mucosa for short read. Differentially expressed genes (DEGs) between infected and CTRL groups were assessed using DESeq2 at a false discovery rate < 0.05. Infection did not affect plasma immunohematological parameters, including neutrophil, lymphocyte, monocyte, leucocyte, thrombocyte, and erythrocyte counts as well as hematocrit and hemoglobin concentration on day 7 post infection. The immune-related DEGs were selected according to the UniProt immune system process database and were used for gene ontology (GO) and pathway enrichment analysis using Cytoscape (v3.9.1). Based on GO analysis, DEGs annotated to mucosal immunity, recognizing and presenting antigens, chemotaxis of neutrophils, eosinophils, natural killer cells, B and T cells mediated by signaling pathways including toll like receptors, interleukins, tumor necrosis factor, T cell receptor, and NF-KB were upregulated, while markers of macrophages chemotaxis and cytosolic pattern recognition were downregulated. This study provides a holistic snapshot of immune-related pathways induced by C. parvum in calves, including novel and detailed feedback and feedforward regulatory mechanisms establishing the crosstalk between innate and adaptive immune response in neonate calves, which could be utilized further to develop new therapeutic strategies
Epitope mapping of monoclonal antibodies against the structural proteins VP1, VP2 and VP3 of acute bee paralysis virus
No full textDiplomarbeit - Veterinärmedizinische Universität Wien - 2023Das Akute Bienenparalyse Virus hat vielfältige negative Einflüsse auf die Gesundheit von Bienenvölkern. Forschung an dieser Krankheit und weiteren Faktoren, die im Zusammenhang mit dem sogenannten „colony collaps disorder“ stehen, ist in Zeiten, in denen die Bienensterblichkeit ein geradezu dramatisches Ausmaß angenommen hat, von immenser Wichtigkeit. Das Wissen, welches durch Epitopkartierung erhoben wird, dient nicht nur der Grundlagenforschung, sondern kann auch in der Entwicklung von Schnelltests zur leichteren Feststellung von einem Befall mit dem besagten Virus oder neuen Therapieansätzen eingesetzt werden. In dieser Arbeit konnte über die Westernblot Methode gezeigt werden, dass das Strukturprotein VP3 von allen drei in dieser Arbeit untersuchten monoklonalen Antikörpern gebunden wird. Jedoch muss das vollständige Epitop noch erhoben werden. Durch Verkürzungs-PCRs konnte gezeigt werden, dass die letzte Aminosäure des Strukturproteins VP3 mit Sicherheit essenziell für die Antikörperbindung ist. Mögliche weiterführende Schritte wären beispielsweise die Verkürzungs-PCRs vom N-terminalen Ende der Sequenz durchzuführen oder den hinteren Teil des Strukturproteins VP3 in eine andere Sequenz hinein zu klonieren, um zu sehen ob die Antikörper die Sequenz immer noch erkennen können.The Acute bee paralysis virus has various negative impacts on bee colony health. Investigating this disease and other factors contributing to the “colony collapse disorder” in times of immense bee losses is of utter interest. The knowledge gathered by epitope mapping, is not only serving as basic research but can enable other scientists to discover new ways to diagnose this disease via rapid tests or help develop new treatment plans. Within the framework of this thesis, it was possible to show via the Western blot method, that all three monoclonal antibodies investigated in this study bind to the structural protein VP3. By shortening the template VP3 it was possible to show with certainty, that the very last part, especially the last amino acid of VP3 is essential for the antibody-binding capability. Further investigations need to be conducted to confirm the complete epitope or if the epitope consists only of the last amino acid of structure protein VP3. Possible further steps are n-terminal shortening of template VP3 or cloning the c-terminal end into another sequence and testing if the antibodies are still able to detect a binding site.Diploma thesis - University of Veterinary Medicine Vienna - 202
Dual therapeutic targeting of MYC and JUNB transcriptional programs for enhanced anti-myeloma activity
No full textDeregulation of transcription factors (TFs) leading to uncontrolled proliferation of tumor cells within the microenvironment represents a hallmark of cancer. However, the biological and clinical impact of transcriptional interference, particularly in multiple myeloma (MM) cells, remains poorly understood. The present study shows for the first time that MYC and JUNB, two crucial TFs implicated in MM pathogenesis, orchestrate distinct transcriptional programs. Specifically, our data revealed that expression levels of MYC, JUNB, and their respective downstream targets do not correlate and that their global chromatin-binding patterns are not significantly overlapping. Mechanistically, MYC expression was not affected by JUNB knockdown, and conversely, JUNB expression and transcriptional activity were not affected by MYC knockdown. Moreover, suppression of MYC levels in MM cells via targeting the master regulator BRD4 by either siRNA-mediated knockdown or treatment with the novel proteolysis targeting chimera (PROTAC) MZ-1 overcame bone marrow (BM) stroma cell/IL-6-induced MYC- but not MEK-dependent JUNB-upregulation and transcriptional activity. Consequently, targeting of the two non-overlapping MYC- and JUNB-transcriptoms by MZ-1 in combination with genetic or pharmacological JUNB-targeting approaches synergistically enhanced MM cell death, both in 2D and our novel dynamic 3D models of the BM milieu as well as in murine xenografts. In summary, our data emphasize the opportunity to employ MYC and JUNB dual-targeting treatment strategies in MM as another exciting approach to further improve patient outcomes
Diametral influence of deoxynivalenol (DON) and deepoxy-deoxynivalenol (DOM-1) on the growth of Campylobacter jejuni with consequences on the bacterial transcriptome
No full textDeoxynivalenol (DON) is a type B trichothecene mycotoxin that is commonly found in cereals and grains worldwide. The presence of this fungal secondary-metabolite raises public-health concerns at both the agriculture and food industry level. Recently, we have shown that DON has a negative impact on gut integrity, a feature also noticed for Campylobacter (C.) jejuni. We further demonstrated that DON increased the load of C. jejuni in the gut and inner organs. In contrast, feeding the less toxic DON metabolite deepoxy-deoxynivalenol (DOM-1) to broilers reduced the Campylobacter load in vivo. Consequently, it can be hypothesized that DON and DOM-1 have a direct effect on the growth profile of C. jejuni. The aim of the present study was to further resolve the nature of this interaction in vitro by co-incubation and RNA-sequencing.The co-incubation of C. jejuni with DON resulted in significantly higher bacterial growth rates from 30 h of incubation onwards. On the contrary, the co-incubation of C. jejuni with DOM-1 reduced the CFU counts, indicating that this DON metabolite might contribute to reduce the burden of C. jejuni
in birds, altogether confirming in vivo data. Furthermore, the transcriptomic profile of C. jejuni following incubation with either DON or DOM-1 differed. Co-incubation of C. jejuni with DON significantly increased the expression of multiple genes which are critical for Campylobacter growth, particularly members of the Flagella gene family, frr (ribosome-recycling factor), PBP2 futA-like (Fe3+ periplasmic binding family) and PotA (ATP-binding subunit). Flagella are responsible for motility, biofilm formation and host colonization, which may explain the high Campylobacter load in the gut of DON-fed broiler chickens. On the contrary, DOM-1 downregulated the Flagella gene family and upregulated ribosomal proteins.The results highlight the adaptive mechanisms involved in the transcriptional response of C. jejuni to DON and its metabolite DOM-1, based on the following effects: (a) ribosomal proteins; (b) flagellar proteins; (c) engagement of different metabolic pathways. The results provide insight into the response of an important intestinal microbial pathogen against DON and lead to a better understanding of the luminal or
environmental acclimation mechanisms in chickens
Dual-Probe Activity-Based Protein Profiling Reveals Site-Specific Differences in Protein Binding of EGFR-Directed Drugs
No full textComparative dose-dependent analysis of interactions between small molecule drugs and their targets, as well as off-target interactions, in complex proteomes is crucial for selecting optimal drug candidates. The affinity of small molecules for targeted proteins is largely dictated by interactions between amino acid side chains and these drugs. Thus, studying drug-protein interactions at an amino acid resolution provides a comprehensive understanding of the drug selectivity and efficacy. In this study, we further refined the site-specific activity-based protein profiling strategy (ABPP), PhosID-ABPP, on a timsTOF HT mass spectrometer. This refinement enables dual dose-dependent competition of inhibitors within a single cellular proteome. Here, a comparative analysis of two activity-based probes (ABPs), developed to selectively target the epidermal growth factor receptor (EGFR), namely, PF-06672131 (PF131) and PF-6422899 (PF899), facilitated the simultaneous identification of ABP-specific binding sites at a proteome-wide scale within a cellular proteome. Dose-dependent probe-binding preferences for proteinaceous cysteines, even at low nanomolar ABP concentrations, could be revealed. Notably, in addition to the intrinsic affinity of the electrophilic probes for specific sites in targeted proteins, the observed labeling intensity is influenced by several other factors. These include the efficiency of cellular uptake, the stability of the probes, and their intracellular distribution. While both ABPs showed comparable labeling efficiency for EGFR, PF131 had a broader off-target reactivity profile. In contrast, PF899 exhibited a higher labeling efficiency for the ERBB2 receptor and bound to catalytic cysteines in several other enzymes, which is likely to disrupt their catalytic activity. Notably, PF131 effectively labeled ADP/ATP translocase proteins at a concentration of just 1 nm, and we found this affected ATP transport. Analysis of the effect of PF131 and its parent inhibitor Afatinib on murine translocase SLC25A4 (ANT1)-mediated ATP transport strongly indicated that PF131 (10 μM) partially blocked ATP transport. Afatinib was less efficient at inhibiting ATP transport by SLC25A4 than PF131, and the reduction of ATP transport by Afatinib was not significant. Follow-up analysis is required to evaluate the affinity of these inhibitors for ADP/ATP translocase SLC25A4 in more detail. Additionally, the analysis of different binding sites within the EGF receptor and the voltage-dependent anion channel 2 revealed secondary binding sites of both probes and provided insights into the binding poses of inhibitors on these proteins. Insights from the PhosID-ABPP analysis of these two ABPs serve as a valuable resource for understanding drug on- and off-target engagement in a dose- and site-specific manner
In vitro assessment of the immunomodulatory effects of probiotic Bacillus strains on chicken PBMCs
No full textThe beneficial effects of feeding probiotic Bacillus subtilis DSM 32315 (BS) and Bacillus velezensis CECT 5940 (BV) to chickens in vivo are well-documented, with potential immune modulation as a key mechanism. In this study, we investigated the direct interactions of chicken peripheral blood mononuclear cells (PBMCs) with BS or BV in vitro through whole transcriptome profiling and cytokine array analysis. Transcriptome profiling revealed 20 significantly differentially expressed genes (DEGs) in response to both Bacillus treatments, with twelve DEGs identified in BS-treated PBMCs and eight in BV-treated PBMCs. Pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) indicated significant regulation of immune-related pathways by both BS and BV. Notably, BS treatment upregulated genes associated with immune cell surface markers (CD4, CD25, CD28), anti-inflammatory cytokine interleukin-10 (IL-10), and C-C motif chemokine ligand 5 (CCL5), while downregulating the gene encoding pro-inflammatory IL-16. BV treatment similarly affected genes associated with immune cell surface markers, IL-16, and CCL5, with no impact on the gene encoding IL-10. Both treatments induced higher expression of the gene encoding the avian β-defensin 1 (AvBD1). The results of this in vitro study indicate an immunomodulatory effect of BS and BV in chicken PBMCs by regulating genes involved in anti-inflammatory, bacteriostatic, protective, and pro-inflammatory responses. Consequently, BS and BV may serve to augment the immune system\u27s capacity to defend against infection by modulating immune responses and cytokine expression. Thus, the administration of these probiotics holds promise for reducing reliance on antimicrobials in farming practices