Phaidra - University of Veterinary Medicine Vienna
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Toxic effects of the emerging Alexandrium pseudogonyaulax (Dinophyceae) on multiple trophic levels of the pelagic food web
No full textThe dinoflagellate Alexandrium pseudogonyaulax, a harmful algal bloom species, is currently appearing in increasing frequency and abundance across Northern European waters, displacing other Alexandrium species. This mixotrophic alga produces goniodomins (GDs) and bioactive extracellular substances (BECs) that may pose a threat to coastal ecosystems and other marine resources. This study demonstrated the adverse effects of A. pseudogonyaulax on four marine trophic levels, including microalgae (Rhodomonas salina), microzooplankton (Polykrikos kofoidii) and mesozooplankton (Acartia tonsa), as well as fish gill cells (RTgill-W1, Oncorhynchus mykiss), ultimately leading to enhanced mortality and cell lysis. Furthermore, cell-free supernatants collected from A. pseudogonyaulax cultures caused complete loss of metabolic activity in the RTgill-W1 cell line, indicating ichthyotoxic properties, while all tested GDs were much less toxic. In addition, cell-free supernatants of A. pseudogonyaulax led to cell lysis of R. salina, while all tested GDs were non-lytic. Finally, reduced egg hatching rates of A. tonsa eggs exposed to cell-free supernatants of A. pseudogonyaulax and impaired mobility of P. kofoidii and A. tonsa exposed to A. pseudogonyaulax were also observed. Altogether, bioassay results suggest that the toxicity of A. pseudogonyaulax is mainly driven by BECs and not by GDs, although further research into factors modulating the lytic activity of Alexandrium spp. are needed
Electrophilic MiniFrags Revealed Unprecedented Binding Sites for Covalent HDAC8 Inhibitors
No full textScreening of ultra-low-molecular weight ligands (MiniFrags) successfully identified viable chemical starting points for a variety of drug targets. Here we report the electrophilic analogues of MiniFrags that allow the mapping of potential binding sites for covalent inhibitors by biochemical screening and mass spectrometry. Small electrophilic heterocycles and their N-quaternized analogues were first characterized in the glutathione assay to analyze their electrophilic reactivity. Next, the library was used for systematic mapping of potential covalent binding sites available in human histone deacetylase 8 (HDAC8). The covalent labeling of HDAC8 cysteines has been proven by tandem mass spectrometry measurements, and the observations were explained by mutating HDAC8 cysteines. As a result, screening of electrophilic MiniFrags identified three potential binding sites suitable for the development of allosteric covalent HDAC8 inhibitors. One of the hit fragments was merged with a known HDAC8 inhibitor fragment using different linkers, and the linker length was optimized to result in a lead-like covalent inhibitor
Targeting TGF beta receptor 1 in head and neck squamous cell carcinoma
No full textThe transforming growth factor-Beta (TGF-ß) pathway may be involved in the radioresistance of head and neck squamous cell carcinoma (HNSCC). This study analyzed TGF-ß receptor 1 (TGFBR1) expression in HNSCC patients and evaluated the antineoplastic and radiosensitizing effects of vactosertib, a novel TGFBR1 inhibitor, in vitro.TGFBR1 expression was examined in HNSCC patients at the mRNA level in silico and the protein level by immunohistochemistry, including surgical specimens of primary tumors, matched lymph node metastasis, and recurrent disease. Furthermore, a novel small molecule TGFBR1 inhibitor was evaluated in HNSCC cell lines. Finally, an indirect coculture model using patient-derived cancer-associated fibroblasts was applied to mimic the tumor microenvironment.Patients with high TGFBR1 mRNA levels showed significantly worse overall survival in silico (OS, p = 0.024). At the protein level, an association between TGFBR1+ tumor and OS was observed for the subgroup with TGFBR1-stroma (p = 0.001). Those results prevailed in multivariable analysis. Inhibition of TGFBR1 showed antineoplastic effects in vitro. In combination with radiation, vactosertib showed synergistic effects.Our results indicate a high risk of death in tumorTGFBR1+|stromaTGFBR1- expressing patients. In vitro data suggest a potential radiosensitizing effect of TGFBR1 inhibition by vactosertib
Reduced bacterial load in stallion semen by modified single layer centrifugation or sperm washing
No full textThe presence of bacteria poses a significant challenge to the quality of stallion semen used in artificial insemination. The bacterial content of insemination doses arises from various sources, such as the healthy stallion, environment, and collection equipment, and is implicated in fertility problems as well as reduced sperm quality during storage. The conventional approach of adding antibiotics to semen extenders raises concerns about antimicrobial resistance and potential negative effects on sperm characteristics, and may not be effective in inhibiting all bacteria. The objective of this study was to determine whether an innovative alternative to antibiotic usage - centrifugation through a single layer of a low density colloid (SLC) - could reduce the bacterial load in stallion semen, and to compare sperm characteristics in samples arising from this procedure, or simple extension of the ejaculate in semen extender, or from sperm washing, i.e. adding extender and then centrifuging the sample to allow the removal of most of the seminal plasma and extender. Eighteen semen samples were collected from six stallions. The semen samples were split and extended prior to washing or SLC, or received no further treatment other than extension. After preparation aliquots from each type of sample were sent for bacteriological examination; the remaining samples were stored for up to 72 h, with daily checks on sperm quality. The low density colloid SLC outperformed sperm washing or extension for bacterial reduction, effectively removing several bacterial species. The bacterial load in the samples was as follows: extended semen, 16 ± 6.7 × 105; washed, 5.8 ± 2.0 × 105; SLC, 2.3 ± 0.88 × 105, p < 0.0001. In addition, SLC completely removed some bacterial species, such as Staphylococcus xylosus. Although there is no selection for robust spermatozoa with the low density colloid, sperm motility, membrane integrity, and DNA fragmentation were not different to washed sperm samples. These findings suggest that SLC with a low density colloid offers a promising method for reducing bacterial contamination in stallion semen without resorting to antibiotics
The Alteration of Proteomic Profiles in Hippocampus of Type 2 Diabetic Mice Associated With Cognitive Impairment
No full textClinical and experimental studies have demonstrated that type 2 diabetes mellitus (T2DM) affects the brain structure and function, in particular the hippocampus, leading to cognitive impairments. However, the molecular mechanisms underlying cognitive deficits induced by T2DM are not fully understood. In this study, we aimed to investigate the effects of T2DM on behavior, the proteome profile in the hippocampus, and the potential molecular pathways involved in the development of cognitive dysfunction in T2DM mice. We found that the diabetic mice exhibited cognitive impairment in the novel object location recognition test and the novel object recognition test. The proteomic analysis revealed that various molecular pathways were involved in this context. These included the upregulation of proteins in the protein synthesis and folding pathway (EIF5A, RSP24, and PPIB), endocytosis and cellular trafficking (VPS24, SNX12, and ARP2/3), cannabinoid receptor interacting (CRIP1), ubiquitination (SKP1), and oxidative stress response (NUDT3). Downregulated proteins were related to mitochondria function (ANT1), neuronal development (ELP1), protein glycosylation (RPN2), and endocytosis (VPS4). Our study shows that T2DM mice exhibit neurocognitive impairment, which is linked to the dysregulation of hippocampal proteins involved in various molecular pathways. These findings contribute to a better understanding of the pathophysiology of T2DM-related cognitive impairment and may identify molecular targets for drug development to treat T2DM-associated cognitive impairment conditions
Evaluation of factors associated with immunoglobulin, protein, fat and lactose concentrations in colostrum of dairy cows from Austria
No full textCalves rely on the passive transfer with immunoglobulins derived from colostrum. Currently, there is a lack of knowledge on colostrum management practices and colostrum quality on small scale family-owned dairy farms in Austria. The objectives of this study were to describe factors that are associated with immunoglobulin, protein, fat and lactose concentrations in dairy cow colostrum from the federal state of Salzburg. Therefore, an online questionnaire was designed to gather information on general farm characteristics. Further, the farmers collected individual colostrum samples and completed a detailed accompanying questionnaire for each sample. Immunoglobulin levels were determined by using a Brix refractometer and protein, fat and lactose by standardized laboratory methods. Linear mixed effect models were built to test factors associated with colostrum immunoglobulin, fat, protein and lactose concentrations.In total, 1,050 colostrum samples from 72 dairy farms were collected. The number of calvings per year was distributed as follows: ?10 calvings: 8.3% of the farms, 11 to 20: 31.9%, 21 to 30: 29.2%, 31 to 40: 15.3% and ??41 calvings: 15.3%. Overall, the median Brix value was 22.0% (7.3-36.1%). The number of samples with good and poor-quality colostrum was 517 and 528, respectively. Cow-level factors significantly affecting colostrum Brix% were parity, calving season, ante partum colostrum leakage, time lag between parturition and colostrum collection. In total, a subset of 307 colostrum samples from 39 farms from pure-breed dual-purpose Simmental cows were further analysed for protein, fat and lactose concentration. The median concentration for fat was 5.1% (0.5-18.5%), protein 14.6% (4.2-27.5%) and lactose 2.3% (0.2-5.0%). The cow-level factors affecting protein concentration were similar to the factors influencing Brix%. Fat concentration was influenced by the time lag between calving and colostrum collection and by parity.The present study confirmed the factors, which are currently known to have an impact on colostrum quality. This was the first large scale approach in the federal state of Salzburg to survey colostrum management including colostrum sample collection. The range of colostrum quality was wide (7.3% Brix to 36.1% Brix) therefore many calves will be at risk of receiving poor quality colostrum as defined by a Brix of ??22%
Nano-encapsulated Yucca extract as feed additives: Ruminal greenhouse gas emissions of three forages
No full textReducing greenhouse gas (GHG) emissions from livestock is a crucial step towards mitigating the impact of climate change and improving environmental sustainability in agriculture. This study aimed to evaluate the effects of Yucca schidigera extract, chitosan, and chitosan nanoparticles as feed additives on in vitro GHG emissions and fermentation profiles in ruminal fluid from bulls. Total gas, CH4, CO, and H2S emissions (up to 48 h), rumen fermentation profiles, and CH4 conversion efficiency were measured using standard protocols. The experiments involved supplementing 0.25, 0.5, and 1 mL/g dry matter (DM) of additives in different forages (alfalfa hay, corn silage, and oats hay). The chemical composition of forage showed suitable levels of DM, ash, crude protein, acid detergent fiber, neutral detergent fiber, lignin, and metabolizable energy. The addition of these supplements increased asymptotic gas production across all forages while simultaneously reducing CH4, CO, and H2S emissions, though the extent of reduction varied depending on forage type. Moreover, the treatments improved fermentation profiles, including pH and dry matter digestibility, and significantly influenced CH4 conversion efficiency (CH4:ME, CH4:OM, and CH4:SCFA; P?<?0.05). These results underscore the potential of Y. schidigera extract, chitosan, and chitosan nanoparticles as effective strategies for mitigating GHG emissions from ruminants given these promising in vitro findings. Further in vivo studies are recommended to validate their efficacy under real-world conditions, which could pave the way for practical applications in the field
Molecular basis for thiocarboxylation and release of Urm1 by its E1-activating enzyme Uba4
No full textUbiquitin-related modifier 1 (Urm1) is a highly conserved member of the ubiquitin-like (UBL) family of proteins. Urm1 is a key component of the eukaryotic transfer RNA (tRNA) thiolation cascade, responsible for introducing sulfur at wobble uridine (U34) in several eukaryotic tRNAs. Urm1 must be thiocarboxylated (Urm1-SH) by its E1 activating enzyme UBL protein activator 4 (Uba4). Uba4 first adenylates and then thiocarboxylates the C-terminus of Urm1 using its adenyl-transferase (AD) and rhodanese (RHD) domains. However, the detailed mechanisms of Uba4, the interplay between the two domains, and the release of Urm1 remain elusive. Here, we report a cryo-EM-based structural model of the Uba4/Urm1 complex that reveals the position of its RHD domains after Urm1 binding, and by analyzing the in vitro and in vivo consequence of mutations at the interface, we show its importance for the thiocarboxylation of Urm1. Our results confirm that the formation of the Uba4-Urm1 thioester and thiocarboxylation of Urm1\u27s C-terminus depend on conserved cysteine residues of Uba4 and that the complex avoids unwanted side-reactions of the adenylate by forming a thioester intermediate. We show how the Urm1-SH product can be released and how Urm1 interacts with upstream (Tum1) and downstream (Ncs6) components of the pathway. Our work provides a detailed mechanistic description of the reaction steps that are needed to produce Urm1-SH, which is required to thiolate tRNAs and persulfidate proteins
Dark brooder pullets are less active than Non-brooder pullets, but show similar level of synchronisation at the resource areas
No full textMaternal care in commercial chickens can partially be replaced with dark brooders that offer heat and a dark area to rest and possibly avoid negative social interactions. Previous research has established the positive effects of dark brooders on reducing disturbance during resting in young pullets and injurious pecking in pullets and laying hens, which could reduce overall activity levels. The objective of this study was to employ precision livestock farming techniques to compare the overall activity levels and behavioural synchrony at resources in pullets reared with dark brooders until 41 days of age and those reared with whole house heating. Four brooder treatments, varying in size (Small/Large) and deployment method (Raised/Fixed), which could have implications for commercial use, were employed. Activity of the whole pen floor area and behavioural synchrony in drinker and feeder areas were automatically assessed over a 12 -hour photoperiod at 10 and 60 days of age. Pixel change detection gauged overall activity across the pen, while an object detection model evaluated pullet behavioural synchrony. The analysis revealed increased activity levels in non -Brooder pullets compared to those in most Brooder treatments at both 10 and 60 days of age. However, no significant differences in behavioural synchrony were observed between Brooder and non -Brooder pullets. The underlying mechanism of dark brooder\u27s effects on the activity levels and behaviour synchrony remains unclear, but the observed reduction in activity levels in brooded pullets likely corresponds to increased resting behaviour and reduced injurious pecking. Furthermore, it is noteworthy that only a small percentage of pullets (up to 57.14%) were observed to use the resources simultaneously. This underscores the importance of conducting additional research to delve into the effects of resource allocation on both behavioural synchrony and activity levels in pullets. We observed minimal differences in the behaviour of pullets raised under different brooder types, suggesting that the simplest (Small -Fixed) brooders may be suitable for commercial use
Unexpected absence of exo-erythrocytic merogony during high gametocytaemia in two species of Haemoproteus (Haemosporida: Haemoproteidae), including description of Haemoproteus angustus n. sp. (lineage hCWT7) and a report of previously unknown residual bodies during in vitro gametogenesis
No full textNeglected avian blood parasites of the genus Haemoproteus (Haemoproteidae) have recently attracted attention due to the application of molecular diagnostic tools, which unravelled remarkable diversity of their exo-erythrocytic (or tissue) stages both regarding morphology and organ tropism levels. The development of haemoproteids might result in pathologies of internal organs, however the exo-erythrocytic development (EED) of most Haemoproteus species remains unknown. Seven individual birds - Curruca communis (1) and Phylloscopus trochilus (6) - with high gametocytaemia (between 1% and 24%) of Haemoproteus angustus n. sp. (hCWT7) and Haemoproteus palloris (lineage hWW1) were sampled in Lithuania, and their internal organs were examined extensively by parallel application of histology and chromogenic in situ hybridization methods. Tissue stages were apparently absent, suggesting that the parasitaemia was not accompanied by detectable tissue merogony. Haemoproteus angustus n. sp. was described and characterized morphologically and molecularly. Sexual process and ookinete development of the new species readily occurred in vitro, and a unique character for Haemoproteus parasites was discovered - the obligatory development of several tiny residual bodies, which were associated with intracellular transformation of both macrogametocytes and microgametocytes before their escape from the host cells and formation of gametes. A DNA haplotype network was constructed with lineages that cluster in one clade with the lineage hCWT7. This clade consists of lineages mostly found in Curruca birds, indicating specificity for birds of this genus. The lineage hCWT7 is mainly a parasite of C. communis. Most reports of this lineage came from Turkey, with only a few records in Europe, mostly in birds wintering in Africa where transmission probably occurs. This study highlights unexpected difficulties in the research of EED even when using sensitive molecular diagnostic tools and extends information about transformation in early stages of gametogenesis in haemosporidian parasites