The use of thermostable fluorescent proteins for live imaging in .

Introduction: Among hyperthermophilic organisms, protein localization is challenging due to the high growth temperatures that can disrupt proper folding and function of mostly mesophilic-derived fluorescent proteins. While protein localization in the thermophilic model archaeon has been achieved using antibodies with fluorescent probes in fixed cells, the use of thermostable fluorescent proteins for live imaging in thermophilic archaea has so far been unsuccessful. Given the significance of live protein localization in the field of archaeal cell biology, we aimed to identify fluorescent proteins for use in .

Metabolomic profiling of pheochromocytomas in dogs: Catecholamine phenotype and tricarboxylic acid cycle metabolites.

Background: In humans with pheochromocytomas (PCCs), targeted metabolomics is used to determine the catecholamine phenotype or to uncover underlying pathogenic variants in tricarboxylic acid (TCA) cycle genes such as succinate dehydrogenase subunits (SDHx).

Hypothesis/objectives: To analyze catecholamine contents and TCA cycle metabolites of PCCs and normal adrenals (NAs).

Animals: Ten healthy dogs, 21 dogs with PCC.

Adhesion pilus retraction powers twitching motility in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius.

Type IV pili are filamentous appendages found in most bacteria and archaea, where they can support functions such as surface adhesion, DNA uptake, aggregation, and motility. In most bacteria, PilT-family ATPases disassemble adhesion pili, causing them to rapidly retract and produce twitching motility, important for surface colonization. As archaea do not possess PilT homologs, it was thought that archaeal pili cannot retract and that archaea do not exhibit twitching motility.

Distinct life cycle stages of an ectosymbiotic DPANN archaeon.

DPANN archaea are a diverse group of microorganisms that are thought to rely on an ectosymbiotic lifestyle; however, the cell biology of these cell-cell interactions remains largely unknown. We applied live-cell imaging and cryo-electron tomography to the DPANN archaeon Nanobdella aerobiophila and its host, revealing two distinct life cycle stages. Free cells possess archaella and are motile.

Whole transcriptome analysis of canine pheochromocytoma and paraganglioma.

Pheochromocytomas and paragangliomas (PPGLs) are neuroendocrine tumors arising from the chromaffin cells in the adrenal medulla and extra-adrenal paraganglia, respectively. Local invasion, concurrent disorders, and metastases prevent surgical removal, which is the most effective treatment to date. Given the current lack of effective medical treatment, there is a need for novel therapeutic strategies.

adhesion pili power twitching motility in the absence of a dedicated retraction ATPase.

Type IV pili are ancient and widespread filamentous organelles found in most bacterial and archaeal phyla where they support a wide range of functions, including substrate adhesion, DNA uptake, self aggregation, and cell motility. In most bacteria, PilT-family ATPases disassemble adhesion pili, causing them to rapidly retract and produce twitching motility, important for surface colonization. As archaea do not possess homologs of PilT, it was thought that archaeal pili cannot retract.

Prediction of outcome after ligation or thin film banding of extrahepatic shunts, based on plasma albumin concentration and hematologic expression of 8 target genes in 85 dogs.

Background: In dogs with a congenital extrahepatic portosystemic shunt (EHPSS), outcome after surgical attenuation is difficult to predict.

Objectives: Develop a minimally invasive test to predict outcome after surgical EHPSS attenuation and establish risk factors for postattenuation seizures (PAS).

Animals: Eighty-five client-owned dogs referred for surgical attenuation of a single EHPSS.

A human kidney and liver organoid-based multi-organ-on-a-chip model to study the therapeutic effects and biodistribution of mesenchymal stromal cell-derived extracellular vesicles.

Mesenchymal stromal cell (MSC)-derived small extracellular vesicles (sEVs) show therapeutic potential in multiple disease models, including kidney injury. Clinical translation of sEVs requires further preclinical and regulatory developments, including elucidation of the biodistribution and mode of action (MoA). Biodistribution can be determined using labelled sEVs in animal models which come with ethical concerns, are time-consuming and expensive, and may not well represent human physiology.

The cell biology of archaea.

The past decade has revealed the diversity and ubiquity of archaea in nature, with a growing number of studies highlighting their importance in ecology, biotechnology and even human health. Myriad lineages have been discovered, which expanded the phylogenetic breadth of archaea and revealed their central role in the evolutionary origins of eukaryotes. These discoveries, coupled with advances that enable the culturing and live imaging of archaeal cells under extreme environments, have underpinned a better understanding of their biology.

Methods to Analyze Motility in Eury- and Crenarchaea.

Many archaea display swimming motility in liquid medium, which is empowered by the archaellum. Directional movement requires a functional archaellum and a sensing system, such as the chemotaxis system that is used by Euryarchaea. Two well-studied models are the euryarchaeon Haloferax volcanii and the crenarchaeon Sulfolobus acidocaldarius.

Progress and Challenges in Archaeal Cell Biology.

Over the past decades there has been a growing interest in the domain of archaea. In this chapter we highlight the recent advances that have been made in studying the cell biology of archaea. We particularly focus on methods for genetic manipulation and imaging of different archaeal species and discuss the technical limitations at the often-extreme growth conditions.

Methods for Markerless Gene Deletion and Plasmid-Based Expression in Sulfolobus acidocaldarius.

A well-functioning genetic system, which is important for studying gene functions in vivo, requires a transformation method, a vector system and a selection system. Sulfolobus acidocaldarius is a crenarchaeal model organism that grows optimally at 75 °C and a pH of 3. These extreme growth conditions cause some difficulties in developing a genetic system.

High level of polarized engraftment of porcine intrahepatic cholangiocyte organoids in decellularized liver scaffolds.

In Europe alone, each year 5500 people require a life-saving liver transplantation, but 18% die before receiving one due to the shortage of donor organs. Whole organ engineering, utilizing decellularized liver scaffolds repopulated with autologous cells, is an attractive alternative to increase the pool of available organs for transplantation. The development of this technology is hampered by a lack of a suitable large-animal model representative of the human physiology and a reliable and continuous cell source.

Volumetric Bioprinting of Organoids and Optically Tuned Hydrogels to Build Liver-Like Metabolic Biofactories.

Organ- and tissue-level biological functions are intimately linked to microscale cell-cell interactions and to the overarching tissue architecture. Together, biofabrication and organoid technologies offer the unique potential to engineer multi-scale living constructs, with cellular microenvironments formed by stem cell self-assembled structures embedded in customizable bioprinted geometries. This study introduces the volumetric bioprinting of complex organoid-laden constructs, which capture key functions of the human liver.

Bioprinting of Human Liver-Derived Epithelial Organoids for Toxicity Studies.

There is a need for long-lived hepatic in vitro models to better predict drug induced liver injury (DILI). Human liver-derived epithelial organoids are a promising cell source for advanced in vitro models. Here, organoid technology is combined with biofabrication techniques, which holds great potential for the design of in vitro models with complex and customizable architectures.

The Role of Polyphosphate in Motility, Adhesion, and Biofilm Formation in .

Polyphosphates (polyP) are polymers of orthophosphate residues linked by high-energy phosphoanhydride bonds that are important in all domains of life and function in many different processes, including biofilm development. To study the effect of polyP in archaeal biofilm formation, our previously described polyP (-) strain and a new polyP (-) strain generated in this report were used. These two strains lack the polymer due to the overexpression of their respective exopolyphosphatase gene ().

A Chemically Defined Hydrogel for Human Liver Organoid Culture.

End-stage liver diseases are an increasing health burden, and liver transplantations are currently the only curative treatment option. Due to a lack of donor livers, alternative treatments are urgently needed. Human liver organoids are very promising for regenerative medicine; however, organoids are currently cultured in Matrigel, which is extracted from the extracellular matrix of the Engelbreth-Holm-Swarm mouse sarcoma.

Live Imaging of a Hyperthermophilic Archaeon Reveals Distinct Roles for Two ESCRT-III Homologs in Ensuring a Robust and Symmetric Division.

Live-cell imaging has revolutionized our understanding of dynamic cellular processes in bacteria and eukaryotes. Although similar techniques have been applied to the study of halophilic archaea [1-5], our ability to explore the cell biology of thermophilic archaea has been limited by the technical challenges of imaging at high temperatures. Sulfolobus are the most intensively studied members of TACK archaea and have well-established molecular genetics [6-9].

Species-Specific Recognition of Mediated by UV-Inducible Pili and S-Layer Glycosylation Patterns.

The UV-inducible pili system of (Ups) mediates the formation of species-specific cellular aggregates. Within these aggregates, cells exchange DNA to repair DNA double-strand breaks via homologous recombination. Substitution of the pilin subunits UpsA and UpsB with their homologs from showed that these subunits facilitate species-specific aggregation.

Cellulose Nanofibril Hydrogel Promotes Hepatic Differentiation of Human Liver Organoids.

To replicate functional liver tissue in vitro for drug testing or transplantation, 3D tissue engineering requires representative cell models as well as scaffolds that not only promote tissue production but also are applicable in a clinical setting. Recently, adult liver-derived liver organoids are found to be of much interest due to their genetic stability, expansion potential, and ability to differentiate toward a hepatocyte-like fate. The current standard for culturing these organoids is a basement membrane hydrogel like Matrigel (MG), which is derived from murine tumor material and apart from its variability and high costs, possesses an undefined composition and is therefore not clinically applicable.

Long-Term Survival of Transplanted Autologous Canine Liver Organoids in a -Deficient Dog Model of Metabolic Liver Disease.

The shortage of liver organ donors is increasing and the need for viable alternatives is urgent. Liver cell (hepatocyte) transplantation may be a less invasive treatment compared with liver transplantation. Unfortunately, hepatocytes cannot be expanded in vitro, and allogenic cell transplantation requires long-term immunosuppression.

Identification of potential drugs for treatment of hepatic lipidosis in cats using an in vitro feline liver organoid system.

Background: Hepatic lipidosis is increasing in incidence in the Western world, with cats being particularly sensitive. When cats stop eating and start utilizing their fat reserves, free fatty acids (FFAs) increase in blood, causing an accumulation of triacylglycerol (TAG) in the liver.

Objective: Identifying potential new drugs that can be used to treat hepatic lipidosis in cats using a feline hepatic organoid system.

Large-Scale Production of LGR5-Positive Bipotential Human Liver Stem Cells.

Background And Aims: The gap between patients on transplant waiting lists and available donor organs is steadily increasing. Human organoids derived from leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5)-positive adult stem cells represent an exciting new cell source for liver regeneration; however, culturing large numbers of organoids with current protocols is tedious and the level of hepatic differentiation is limited.

Approach And Results: Here, we established a method for the expansion of large quantities of human liver organoids in spinner flasks.

Reduced FXR Target Gene Expression in Copper-Laden Livers of COMMD1-Deficient Dogs.

Wilson's disease (WD), an autosomal recessive disorder, results in copper accumulation in the liver as a consequence of mutations in the gene ATPase copper transporting beta (). The disease is characterized by chronic hepatitis, eventually resulting in liver cirrhosis. Recent studies have shown that dysregulation of nuclear receptors (NR) by high hepatic copper levels is an important event in the pathogenesis of liver disease in WD.