COVID-19 in Joint Ageing and Osteoarthritis: Current Status and Perspectives.

COVID-19 is a trending topic worldwide due to its immense impact on society. Recent trends have shifted from acute effects towards the long-term morbidity of COVID-19. In this review, we hypothesize that SARS-CoV-2 contributes to age-related perturbations in endothelial and adipose tissue, which are known to characterize the early aging process. This would explain the long-lasting symptoms of SARS-CoV-2 as the result of an accelerated aging process. Connective tissues such as adipose tissue and musculoskeletal tissue are the primary sites of aging. Therefore, current literature was analyzed focusing on the musculoskeletal symptoms in COVID-19 patients. Hypovitaminosis D, increased fragility, and calcium deficiency point towards bone aging, while joint and muscle pain are typical for joint and muscle aging, respectively. These characteristics could be classified as early osteoarthritis-like phenotype. Exploration of the impact of SARS-CoV-2 and osteoarthritis on endothelial and adipose tissue, as well as neuronal function, showed similar perturbations. At a molecular level, this could be attributed to the angiotensin-converting enzyme 2 expression, renin-angiotensin system dysfunction, and inflammation. Finally, the influence of the nicotinic cholinergic system is being evaluated as a new treatment strategy. This is combined with the current knowledge of musculoskeletal aging to pave the road towards the treatment of long-term COVID-19.

Non-neuronal Role of Acetylcholinesterase in Bone Development and Degeneration.

Acetylcholinesterase (AChE), an enzyme catalyzing the degradation of acetylcholine, plays an important suppressive role in the cholinergic regulation by terminating the action of acetylcholine. The expression of acetylcholinesterase and other cholinergic components is not restricted to only brain and nerve tissues but can also be found in non-neuronal tissues like the immune system and bone tissue. Primary identification of these components has been achieved. However, the information about their specific functions and underlying molecular mechanisms in bone remains scattered. Here, the physiological process of bone development, homeostasis, and degeneration are introduced. Next, the cholinergic system and its expression in bone tissue is documented. Among them, special attention goes to AChE, as the structure of this enzyme suggests diverse binding affinities, enabled by a peripheral site and a catalytic site. The peripheral site supports the non-enzymatic function of AChE in non-neuronal systems. Based on recent studies, the non-neuronal roles of acetylcholinesterase, both enzymatically and non-enzymatically, in bone development, homeostasis and degeneration are summarized briefly together with potential mechanisms to support these functions. We conclude that AChE may be a potential therapeutic target for bone diseases like osteoporosis.

Assessment of Dried Blood Spots for Multi-Mycotoxin Biomarker Analysis in Pigs and Broiler Chickens.

Dried blood spots (DBSs), a micro-sampling technique whereby a drop of blood is collected on filter paper has multiple advantages over conventional blood sampling regarding the sampling itself, as well as transportation and storage. This is the first paper describing the development and validation of a method for the determination of 23 mycotoxins and phase I metabolites in DBSs from pigs and broiler chickens using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The targeted mycotoxins belong to groups for which the occurrence in feed is regulated by the European Union, namely, aflatoxins, ochratoxin A and several Fusarium mycotoxins, and to two groups of unregulated mycotoxins, namely Alternaria mycotoxins and Fusarium mycotoxins (enniatins and beauvericin). The impact of blood haematocrit, DBS sampling volume and size of the analysed DBS disk on the validation results was assessed. No effects of variation in size of the analysed disk, haematocrit and spotted blood volume were observed for most mycotoxins, except for the aflatoxins and ß-zearalanol (BZAL) at the lowest haematocrit (26%) level and for the enniatins (ENNs) at the lowest volume (40 µL). The developed method was transferred to an LC-high resolution mass spectrometry instrument to determine phase II metabolites. Then, the DBS technique was applied in a proof-of-concept toxicokinetic study including a comparison with LC-MS/MS data from plasma obtained with conventional venous blood sampling. A strong correlation (r > 0.947) was observed between plasma and DBS concentrations. Finally, DBSs were also applied in a pilot exposure assessment study to test their applicability under field conditions.

Multi LC-MS/MS and LC-HRMS Methods for Determination of 24 Mycotoxins including Major Phase I and II Biomarker Metabolites in Biological Matrices from Pigs and Broiler Chickens.

A reliable and practical multi-method was developed for the quantification of mycotoxins in plasma, urine, and feces of pigs, and plasma and excreta of broiler chickens using liquid chromatography⁻tandem mass spectrometry. The targeted mycotoxins belong to the regulated groups, i.e., aflatoxins, ochratoxin A and Fusarium mycotoxins, and to two groups of emerging mycotoxins, i.e., Alternaria mycotoxins and enniatins. In addition, the developed method was transferred to a LC-high resolution mass spectrometry instrument to qualitatively determine phase I and II metabolites, for which analytical standards are not always commercially available. Sample preparation of plasma was simple and generic and was accomplished by precipitation of proteins alone (pig) or in combination with removal of phospholipids (chicken). A more intensive sample clean-up of the other matrices was needed and consisted of a pH-dependent liquid⁻liquid extraction (LLE) using ethyl acetate (pig urine), methanol/ethyl acetate/formic acid (75/24/1, v/v/v) (pig feces) or acetonitrile (chicken excreta). For the extraction of pig feces, additionally a combination of LLE using acetone and filtration of the supernatant on a HybridSPE-phospholipid cartridge was applied. The LC-MS/MS method was in-house validated according to guidelines defined by the European and international community. Finally, the multi-methods were successfully applied in a specific toxicokinetic study and a screening study to monitor the exposure of individual animals.

Biomarkers for Exposure as A Tool for Efficacy Testing of A Mycotoxin Detoxifier in Broiler Chickens and Pigs.

Applying post-harvest control measures such as adding mycotoxin detoxifying agents is a frequently-used mitigation strategy for mycotoxins. EFSA states that the efficacy of these detoxifiers needs to be tested using specific biomarkers for exposure. However, the proposed biomarkers for exposure are not further optimized for specific target species. Hence, the goal of this study was a) to evaluate the most suitable biomarkers for deoxynivalenol (DON) and zearalenone (ZEN) in porcine plasma, urine and feces; and DON, aflatoxin B1 (AFB1) and ochratoxin A (OTA) in plasma and excreta of broiler chickens and b) to determine the efficacy of a candidate detoxifier, as a proof-of-concept study. Therefore, a mixture of mycotoxins was administered as a single oral bolus with or without detoxifying agent. In accordance with literature AFB1, OTA, and DON-sulphate (DON-S) proved optimal biomarkers in broilers plasma and excreta whereas, in pigs DON-glucuronide (DON-GlcA) and ZEN-glucuronide (ZEN-GlcA) proved the optimal biomarkers in plasma, DON and ZEN-GlcA in urine and, ZEN in feces. A statistically significant reduction was seen between control and treatment group for both AFB1 and DON in broiler plasma, under administration of the mycotoxin blend and detoxifier dose studied suggesting thus, beneficial bioactivity.

Insights into In Vivo Absolute Oral Bioavailability, Biotransformation, and Toxicokinetics of Zearalenone, α-Zearalenol, ß-Zearalenol, Zearalenone-14-glucoside, and Zearalenone-14-sulfate in Pigs.

The aim of this study was to determine the toxicokinetic characteristics of ZEN and its modified forms, α-zearalenol (α-ZEL), ß-zearalenol (ß-ZEL), zearalenone-14-glucoside (ZEN14G), and zearalenone-14-sulfate (ZEN14S), including presystemic and systemic hydrolysis in pigs. Crossover pig trials were performed by means of intravenous and oral administration of ZEN and its modified forms. Systemic plasma concentrations of the administered toxins and their metabolites were quantified and further processed via tailor-made compartmental toxicokinetic models. Furthermore, portal plasma was analyzed to unravel the site of hydrolysis, and urine samples were analyzed to determine urinary excretion. Results demonstrate complete presystemic hydrolysis of ZEN14G and ZEN14S to ZEN and high oral bioavailability for all administered compounds, with further extensive first-pass glucuronidation. Conclusively, the modified-ZEN forms α-ZEL, ß-ZEL, ZEN14G, and ZEN14S contribute to overall ZEN systemic toxicity in pigs and should be taken into account for risk assessment.

Hemocompatibility assessment of two siRNA nanocarrier formulations.

PURPOSE: Since the discovery of RNAi and its therapeutic potential, carrier systems have been developed to deliver small RNAs (particularly siRNA) for modulation of gene expression at the post-transcriptional level. An important factor determining the fate and usability of these systems in vivo is interaction with blood components, blood cells, and the immune system. In this study, a lipid-based and a polymer-based carrier system containing siRNA have been investigated in vitro in terms of their hemocompatibility. METHODS: The nanocomplexes studied were Angiplex, a targeted lipid-based system, and pHPMA-MPPM polyplex, a formulation based on a cationic polymer. siVEGFR-2 was encapsulated in both carriers and activation of platelets, coagulation, and complement cascade as well as induction of platelet aggregation were evaluated in vitro. RESULTS: Both systems had been shown before to cause significant silencing in vitro. Our findings indicated that pHPMA-MPPM polyplex triggered high platelet activation and aggregation although it did not stimulate coagulation substantially. Angiplex, on the other hand, provoked insignificant activation and aggregation of platelets and activated coagulation minimally. Complement system activation by Angiplex was in general low but stronger than pHPMA-MPPM polyplex. CONCLUSIONS: Taken together, these in vitro assays may help the selection of suitable carriers for systemic delivery of siRNA in early preclinical investigations and reduce the use of laboratory animals significantly.