Integrated omics of Saccharomyces cerevisiae CENPK2-1C reveals pleiotropic drug resistance and lipidomic adaptations to cannabidiol.

Yeast metabolism can be engineered to produce xenobiotic compounds, such as cannabinoids, the principal isoprenoids of the plant Cannabis sativa, through heterologous metabolic pathways. However, yeast cell factories continue to have low cannabinoid production. This study employed an integrated omics approach to investigate the physiological effects of cannabidiol on S. cerevisiae CENPK2-1C yeast cultures. We treated the experimental group with 0.5 mM CBD and monitored CENPK2-1C cultures. We observed a latent-stationary phase post-diauxic shift in the experimental group and harvested samples in the inflection point of this growth phase for transcriptomic and metabolomic analysis. We compared the transcriptomes of the CBD-treated yeast and the positive control, identifying eight significantly overexpressed genes with a log fold change of at least 1.5 and a significant adjusted p-value. Three notable genes were PDR5 (an ABC-steroid and cation transporter), CIS1, and YGR035C. These genes are all regulated by pleiotropic drug resistance linked promoters. Knockout and rescue of PDR5 showed that it is a causal factor in the post-diauxic shift phenotype. Metabolomic analysis revealed 48 significant spectra associated with CBD-fed cell pellets, 20 of which were identifiable as non-CBD compounds, including fatty acids, glycerophospholipids, and phosphate-salvage indicators. Our results suggest that mitochondrial regulation and lipidomic remodeling play a role in yeast's response to CBD, which are employed in tandem with pleiotropic drug resistance (PDR). We conclude that bioengineers should account for off-target product C-flux, energy use from ABC-transport, and post-stationary phase cell growth when developing cannabinoid-biosynthetic yeast strains.

Foldseek reveals a CBGA prenylating enzyme GlyMa_02G168000 from Glycine max.

The present research provides an application for an aromatic prenyltransferase from Glycine max for use in heterologous microorganism expression to generate cannabinoids. The known cannabinoid prenyltransferase CsPT04 was queried in FoldSeek. An enzyme derived from Glycine max known as GLYMA_02G168000, which is a predicted homogentisate solanyltransferase, was identified and found to have affinity for the prenylation of geranyldiphosphate (GPP) and olivetolic acid (OA) to produce cannabigerolic acid (CBGA) and cannabigerol (CBG). The in vitro production of CBGA was accomplished through the heterologous expression of this prenyltransferase in Saccharomyces cerevisiae. After growing the yeast cells, a purified microsomal fraction was harvested, which was rich in the membrane-bound prenyltransferase GlyMa_02G168000. Addition of purified microsomal fraction to a reaction matrix facilitated the successful prenylation of externally supplied OA with GPP, culminating in the production of CBGA. Structural comparisons revealed a notably closer similarity between GLYMA_02G168000 and CsPT04, compared to the similarity of other cannabinoid prenyltransferases with CsPT04. Herein, a novel application for a homogentisate solanyltransferase has been established towards the production of cannabinoids.

Synthetic Strategies for Rare Cannabinoids Derived from Cannabis sativa.

Efficient syntheses of eight key cannabinoids were established and optimized. Predominant cannabinoids such as cannabigerol (CBG-C5) and cannabidiol (CBD-C5) were prepared from olivetol via regioselective condensation. Further treatments of CBD led to Δ9-tetrahydrocannabinol (THC-C5), Δ8-iso-tetrahydrocannabinol (iso-THC-C5), and cannabinol (CBN-C5). Alternatively, a [3 + 3] annulation between olivetol and citral yielded the minor cannabinoid cannabichromene (CBC-C5), which was converted into two very rare polycycles, cannabicyclol (CBL-C5) and cannabicitran (CBT-C5), in a one-pot reaction. Finally, all eight syntheses were extended by utilizing resorcinol and two phenolic analogues, achieving a cannabinoid group with more than 30 compounds through a facile synthesis strategy.

Cannabinoids as New Drug Candidates for the Treatment of Glaucoma.

Glaucoma is a blinding eye disease that affects about 70 million patients globally today. The cannabinoid receptors and the endocannabinoid system have found attention for new drug concepts. This review will analyze the potential of cannabinoids, primarily tetrahydrocannabinol, THCVS, and cannabinol, as drug candidates and the role of CB1/CB2 receptors with regard to the pathophysiology of glaucoma. The mode of action of cannabinoids as innovative drug candidates and recent formulations for topical delivery will be discussed. Cannabinoid receptors with associated TRPV channels will be evaluated for their potential as drug targets. Especially the role of the endocannabinoid system (fatty acid amide hydrolase, monoacylglycerol lipase) impacting the prostaglandin network (cyclooxygenase, PGE, PGF) and neuroprotection by inhibition of nitric oxide radical formation is in the focus of this review. Delivery systems, including recent clinical trials, will be analyzed to evaluate the potential for innovative future ophthalmological drugs.

Evaluating long-read de novo assembly tools for eukaryotic genomes: insights and considerations.

BACKGROUND: Assembly algorithm choice should be a deliberate, well-justified decision when researchers create genome assemblies for eukaryotic organisms from third-generation sequencing technologies. While third-generation sequencing by Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio) has overcome the disadvantages of short read lengths specific to next-generation sequencing (NGS), third-generation sequencers are known to produce more error-prone reads, thereby generating a new set of challenges for assembly algorithms and pipelines. However, the introduction of HiFi reads, which offer substantially reduced error rates, has provided a promising solution for more accurate assembly outcomes. Since the introduction of third-generation sequencing technologies, many tools have been developed that aim to take advantage of the longer reads, and researchers need to choose the correct assembler for their projects. RESULTS: We benchmarked state-of-the-art long-read de novo assemblers to help readers make a balanced choice for the assembly of eukaryotes. To this end, we used 12 real and 64 simulated datasets from different eukaryotic genomes, with different read length distributions, imitating PacBio continuous long-read (CLR), PacBio high-fidelity (HiFi), and ONT sequencing to evaluate the assemblers. We include 5 commonly used long-read assemblers in our benchmark: Canu, Flye, Miniasm, Raven, and wtdbg2 for ONT and PacBio CLR reads. For PacBio HiFi reads , we include 5 state-of-the-art HiFi assemblers: HiCanu, Flye, Hifiasm, LJA, and MBG. Evaluation categories address the following metrics: reference-based metrics, assembly statistics, misassembly count, BUSCO completeness, runtime, and RAM usage. Additionally, we investigated the effect of increased read length on the quality of the assemblies and report that read length can, but does not always, positively impact assembly quality. CONCLUSIONS: Our benchmark concludes that there is no assembler that performs the best in all the evaluation categories. However, our results show that overall Flye is the best-performing assembler for PacBio CLR and ONT reads, both on real and simulated data. Meanwhile, best-performing PacBio HiFi assemblers are Hifiasm and LJA. Next, the benchmarking using longer reads shows that the increased read length improves assembly quality, but the extent to which that can be achieved depends on the size and complexity of the reference genome.

Implications of the Conformationally Flexible, Macrocyclic Structure of the First-Generation, Direct-Acting Anti-Viral Paritaprevir on Its Solid Form Complexity and Chameleonic Behavior.

Direct-acting antiviral regimens have transformed therapeutic management of hepatitis C across all prevalent genotypes. Most of the chemical matter in these regimens comprises molecules well outside the traditional drug development chemical space and presents significant challenges. Herein, the implications of high conformational flexibility and the presence of a 15-membered macrocyclic ring in paritaprevir are studied through a combination of advanced computational and experimental methods with focus on molecular chameleonicity and crystal form complexity. The ability of the molecule to toggle between high and low 3D polar surface area (PSA) conformations is underpinned by intramolecular hydrogen bonding (IMHB) interactions and intramolecular steric effects. Computational studies consequently show a very significant difference of over 75 Å2 in 3D PSA between polar and apolar environments and provide the structural basis for the perplexingly favorable passive permeability of the molecule. Crystal packing and protein binding resulting in strong intermolecular interactions disrupt these intramolecular interactions. Crystalline Form I benefits from strong intermolecular interactions, whereas the weaker intermolecular interactions in Form II are partially compensated by the energetic advantage of an IMHB. Like Form I, no IMHB is observed within the receptor-bound conformation; instead, an intermolecular H-bond contributes to the potency of the molecule. The choice of metastable Form II is derisked through strategies accounting for crystal surface and packing features to manage higher form specific solid-state chemical reactivity and specific processing requirements. Overall, the results show an unambiguous link between structural features and derived properties from crystallization to dissolution, permeation, and docking into the protein pocket.

Chondroitin Sulfate Glycosaminoglycan Scaffolds for Cell and Recombinant Protein-Based Bone Regeneration.

Bone morphogenetic protein 2 (BMP-2)-loaded collagen sponges remain the clinical standard for treatment of large bone defects when there is insufficient autograft, despite associated complications. Recent efforts to negate comorbidities have included biomaterials and gene therapy approaches to extend the duration of BMP-2 release and activity. In this study, we compared the collagen sponge clinical standard to chondroitin sulfate glycosaminoglycan (CS-GAG) scaffolds as a delivery vehicle for recombinant human BMP-2 (rhBMP-2) and rhBMP-2 expression via human BMP-2 gene inserted into mesenchymal stem cells (BMP-2 MSC). We demonstrated extended release of rhBMP-2 from CS-GAG scaffolds compared to their collagen sponge counterparts, and further extended release from CS-GAG gels seeded with BMP-2 MSC. When used to treat a challenging critically sized femoral defect model in rats, both rhBMP-2 and BMP-2 MSC in CS-GAG induced comparable bone formation to the rhBMP-2 in collagen sponge, as measured by bone volume, strength, and stiffness. We conclude that CS-GAG scaffolds are a promising delivery vehicle for controlling the release of rhBMP-2 and to mediate the repair of critically sized segmental bone defects. Stem Cells Translational Medicine 2019;8:575-585.

Generation of Avian Induced Pluripotent Stem Cells.

Avian species are among the most diverse vertebrates on our planet and significantly contribute to the balance of the ecology. They are also important food source and serve as a central animal model to decipher developmental biology and disease principles. Derivation of induced pluripotent stem cells (iPSCs) from avian species would enable conservation of genetic diversity as well as offer a valuable cell source that facilitates the use of avian models in many areas of basic and applied research. In this chapter, we describe methods used to successfully reprogram quail fibroblasts into iPSCs by using human transcription factors and the techniques critical to the characterization of their pluripotency.

Generation of Chimeras from Porcine Induced Pluripotent Stem Cells.

Pig induced pluripotent stem cells (piPSCs) offer a great opportunity and a number of advantages in the generation of transgenic animals. These immortalized cells can undergo multiple rounds of genetic modifications (e.g., gene knock-in, knockout) and selection leading to animals that have optimized traits of biomedical or agricultural interests. In this chapter we describe the production and characterization of piPSCs, microinjection of piPSCs into embryos, embryo transfer and production of chimeric animals based on successful protocols.

Pig Induced Pluripotent Stem Cell-Derived Neural Rosettes Developmentally Mimic Human Pluripotent Stem Cell Neural Differentiation.

For diseases of the brain, the pig (Sus scrofa) is increasingly being used as a model organism that shares many anatomical and biological similarities with humans. We report that pig induced pluripotent stem cells (iPSC) can recapitulate events in early mammalian neural development. Pig iPSC line (POU5F1(high)/SSEA4(low)) had a higher potential to form neural rosettes (NR) containing neuroepithelial cells than either POU5F1(low)/SSEA4(low) or POU5F1(low)/SSEA4(high) lines. Thus, POU5F1 and SSEA4 pluripotency marker profiles in starting porcine iPSC populations can predict their propensity to form more robust NR populations in culture. The NR were isolated and expanded in vitro, retaining their NR morphology and neuroepithelial molecular properties. These cells expressed anterior central nervous system fate markers OTX2 and GBX2 through at least seven passages, and responded to retinoic acid, promoting a more posterior fate (HOXB4+, OTX2-, and GBX2-). These findings offer insight into pig iPSC development, which parallels the human iPSC in both anterior and posterior neural cell fates. These in vitro similarities in early neural differentiation processes support the use of pig iPSC and differentiated neural cells as a cell therapy in allogeneic porcine neural injury and degeneration models, providing relevant translational data for eventual human neural cell therapies.

National Collegiate Athletic Association Division I athletic trainers' concussion-management practice patterns.

CONTEXT: A cornerstone of the recent consensus statements on concussion is a multifaceted concussion-assessment program at baseline and postinjury and when tracking recovery. Earlier studies of athletic trainers' (ATs') practice patterns found limited use of multifaceted protocols; however, these authors typically grouped diverse athletic training settings together. OBJECTIVE: To (1) describe the concussion-management practice patterns of National Collegiate Athletic Association (NCAA) Division I ATs, (2) compare these practice patterns to earlier studies, and (3) objectively characterize the clinical examination. DESIGN: Cross-sectional study. SETTING: Online survey. PATIENTS OR OTHER PARTICIPANTS: A total of 610 ATs from NCAA Division I institutions, for a response rate of 34.4%. MAIN OUTCOME MEASURE(S): The survey had 3 subsections: demographic questions related to the participant's experiences, concussion-assessment practice patterns, and concussion-recovery and return-to-participation practice patterns. Specific practice-pattern questions addressed balance, cognitive and mental status, neuropsychological testing, and self-reported symptoms. Finally, specific components of the clinical examination were examined. RESULTS: We identified high rates of multifaceted assessments (i.e., assessments using at least 3 techniques) during testing at baseline (71.2%), acute concussion assessment (79.2%), and return to participation (66.9%). The specific techniques used are provided along with their adherence with evidence-based practice findings. Respondents endorsed a diverse array of clinical examination techniques that often overlapped objective concussion-assessment protocols or were likely used to rule out associated potential conditions. Respondents were cognizant of the Third International Consensus Statement, the National Athletic Trainers' Association position statement, and the revised NCAA Sports Medicine Handbook recommendations. CONCLUSIONS: Athletic trainers in NCAA Division I demonstrated widespread use of multifaceted concussion-assessment techniques and appeared compliant with recent consensus statements and the NCAA Sports Medicine Handbook.

Induced pluripotency in chicken embryonic fibroblast results in a germ cell fate.

Germ cells (GCs) are critically important as the vehicle that passes genetic information from one generation to the next. Correct development of these cells is essential and perturbation in their development often leads to reproductive failure and disease. Despite the importance of GCs, little is known about the mechanisms underlying the acquisition and maintenance of the GC character. Using a reprogramming strategy, we demonstrate that overexpression of ectopic transcription factors in embryonic fibroblasts can lead to the generation of chicken induced primordial germ cells (ciPGCs). These ciPGCs express pluripotent markers POU5F1, SSEA1, and the GC defining proteins, CVH and DAZL, closely resembling in vivo sourced PGCs instead of embryonic stem cells. Moreover, CXCR4 expressing ciPGCs were capable of migrating to the embryonic gonad after injection into the vasculature of stage 15 embryos, indicating the acquisition of a GC fate in these cells. Direct availability of ciPGCs in vitro would facilitate the study of GC development as well as provide a potential strategy for the conservation of important genetics of agricultural and endangered birds using somatic cells.

Rapid Heterotrophic Ossification with Cryopreserved Poly(ethylene glycol-) Microencapsulated BMP2-Expressing MSCs.

Autologous bone grafting is the most effective treatment for long-bone nonunions, but it poses considerable risks to donors, necessitating the development of alternative therapeutics. Poly(ethylene glycol) (PEG) microencapsulation and BMP2 transgene delivery are being developed together to induce rapid bone formation. However, methods to make these treatments available for clinical applications are presently lacking. In this study we used mesenchymal stem cells (MSCs) due to their ease of harvest, replication potential, and immunomodulatory capabilities. MSCs were from sheep and pig due to their appeal as large animal models for bone nonunion. We demonstrated that cryopreservation of these microencapsulated MSCs did not affect their cell viability, adenoviral BMP2 production, or ability to initiate bone formation. Additionally, microspheres showed no appreciable damage from cryopreservation when examined with light and electron microscopy. These results validate the use of cryopreservation in preserving the viability and functionality of PEG-encapsulated BMP2-transduced MSCs.

Avian-induced pluripotent stem cells derived using human reprogramming factors.

Avian species are important model animals for developmental biology and disease research. However, unlike in mice, where clonal lines of pluripotent stem cells have enabled researchers to study mammalian gene function, clonal and highly proliferative pluripotent avian cell lines have been an elusive goal. Here we demonstrate the generation of avian induced pluripotent stem cells (iPSCs), the first nonmammalian iPSCs, which were clonally isolated and propagated, important attributes not attained in embryo-sourced avian cells. This was accomplished using human pluripotency genes rather than avian genes, indicating that the process in which mammalian and nonmammalian cells are reprogrammed is a conserved process. Quail iPSCs (qiPSCs) were capable of forming all 3 germ layers in vitro and were directly differentiated in culture into astrocytes, oligodendrocytes, and neurons. Ultimately, qiPSCs were capable of generating live chimeric birds and incorporated into tissues from all 3 germ layers, extraembryonic tissues, and potentially the germline. These chimera competent qiPSCs and in vitro differentiated cells offer insight into the conserved nature of reprogramming and genetic tools that were only previously available in mammals.

Effect of macronutrients, age, and obesity on 6- and 24-h postprandial glucose metabolism in cats.

Obesity and age are risk factors for feline diabetes. This study aimed to test the hypothesis that age, long-term obesity, and dietary composition would lead to peripheral and hepatorenal insulin resistance, indicated by higher endogenous glucose production (EGP) in the fasted and postprandial state, higher blood glucose and insulin, and higher leptin, free thyroxine, and lower adiponectin concentrations. Using triple tracer-(2)H(2)O, [U-(13)C(3)] propionate, and [3,4-(13)C(2)] glucose infusion, and indirect calorimetry-we investigated carbohydrate and fat metabolic pathways in overnight-fasted neutered cats (13 young lean, 12 old lean, and 12 old obese), each fed three different diets (high protein with and without polyunsaturated fatty acids, and high carbohydrate) in a crossover design. EGP was lowest in fasted and postprandial obese cats despite peripheral insulin resistance, indicated by hyperinsulinemia. Gluconeogenesis was the most important pathway for EGP in all groups, but glycogen contributed significantly. Insulin and leptin concentrations were higher in old than in young lean cats; adiponectin was lowest in obese cats but surprisingly highest in lean old cats. Diet had little effect on metabolic parameters. We conclude that hepatorenal insulin resistance does not develop in the fasted or postprandial state, even in long-term obese cats, allowing the maintenance of euglycemia through lowering EGP. Glycogen plays a major role in EGP, especially in lean fasted cats, and in the postprandial state. Aging may predispose to insulin resistance, which is a risk factor for diabetes in cats. Mechanisms underlying the high adiponectin of healthy old lean cats need to be further explored.

The impact of obesity, sex, and diet on hepatic glucose production in cats.

Obesity is a risk factor for type 2 diabetes in cats. The risk of developing diabetes is severalfold greater for male cats than for females, even after having been neutered early in life. The purpose of this study was to investigate the role of different metabolic pathways in the regulation of endogenous glucose production (EGP) during the fasted state considering these risk factors. A triple tracer protocol using (2)H(2)O, [U-(13)C(3)]propionate, and [3,4-(13)C(2)]glucose was applied in overnight-fasted cats (12 lean and 12 obese; equal sex distribution) fed three different diets. Compared with lean cats, obese cats had higher insulin (P < 0.001) but similar blood glucose concentrations. EGP was lower in obese cats (P < 0.001) due to lower glycogenolysis and gluconeogenesis (GNG; P < 0.03). Insulin, body mass index, and girth correlated negatively with EGP (P < 0.003). Female obese cats had approximately 1.5 times higher fluxes through phosphoenolpyruvate carboxykinase (P < 0.02) and citrate synthase (P < 0.05) than male obese cats. However, GNG was not higher because pyruvate cycling was increased 1.5-fold (P < 0.03). These results support the notion that fasted obese cats have lower hepatic EGP compared with lean cats and are still capable of maintaining fasting euglycemia, despite the well-documented existence of peripheral insulin resistance in obese cats. Our data further suggest that sex-related differences exist in the regulation of hepatic glucose metabolism in obese cats, suggesting that pyruvate cycling acts as a controlling mechanism to modulate EGP. Increased pyruvate cycling could therefore be an important factor in modulating the diabetes risk in female cats.