High-Speed Fluorescence Imaging Corroborates Biological Data on the Influence of Different Nozzle Types on Cell Spray Viability and Formation.

Treating severe dermal disruptions often presents significant challenges. Recent advancements have explored biological cell sprays as a promising treatment, but their success hinges on efficient cell delivery and complete wound coverage. This requires a good spray distribution with a small droplet size, high particle number, and ample surface coverage. The type of nozzle used with the spray device can impact these parameters. To evaluate the influence of different nozzles on spray characteristics, we compared air-assisted and unassisted nozzles. The unassisted nozzle displayed small particle size, high particle number, good overall coverage, high cell viability, preserved cell metabolic activity, and low cytotoxicity. Air-assisted nozzles did not perform well regarding cell viability and metabolic activity. Flow visualization analysis comparing two different unassisted nozzles using high-speed imaging (100 kHz frame rate) revealed a tulip-shaped spray pattern, indicating optimal spray distribution. High-speed imaging showed differences between the unassisted nozzles. One unassisted nozzle displayed a bi-modal distribution of the droplet diameter while the other unassisted nozzle displayed a mono-modal distribution. These findings demonstrate the critical role of nozzle selection in successful cell delivery. A high-quality, certified nozzle manufactured for human application omits the need for an air-assisted nozzle and provides a simple system to use with similar or better performance characteristics than those of an air-assisted system.

Sculpting nuclear envelope identity from the endoplasmic reticulum during the cell cycle.

The nuclear envelope (NE) regulates nuclear functions, including transcription, nucleocytoplasmic transport, and protein quality control. While the outer membrane of the NE is directly continuous with the endoplasmic reticulum (ER), the NE has an overall distinct protein composition from the ER, which is crucial for its functions. During open mitosis in higher eukaryotes, the NE disassembles during mitotic entry and then reforms as a functional territory at the end of mitosis to reestablish nucleocytoplasmic compartmentalization. In this review, we examine the known mechanisms by which the functional NE reconstitutes from the mitotic ER in the continuous ER-NE endomembrane system during open mitosis. Furthermore, based on recent findings indicating that the NE possesses unique lipid metabolism and quality control mechanisms distinct from those of the ER, we explore the maintenance of NE identity and homeostasis during interphase. We also highlight the potential significance of membrane junctions between the ER and NE.

Fatty acid synthesis suppresses dietary polyunsaturated fatty acid use.

Dietary polyunsaturated fatty acids (PUFA) are increasingly recognized for their health benefits, whereas a high production of endogenous fatty acids - a process called de novo lipogenesis (DNL) - is closely linked to metabolic diseases. Determinants of PUFA incorporation into complex lipids are insufficiently understood and may influence the onset and progression of metabolic diseases. Here we show that fatty acid synthase (FASN), the key enzyme of DNL, critically determines the use of dietary PUFA in mice and humans. Moreover, the combination of FASN inhibition and PUFA-supplementation decreases liver triacylglycerols (TAG) in mice fed with high-fat diet. Mechanistically, FASN inhibition causes higher PUFA uptake via the lysophosphatidylcholine transporter MFSD2A, and a diacylglycerol O-acyltransferase 2 (DGAT2)-dependent incorporation of PUFA into TAG. Overall, the outcome of PUFA supplementation may depend on the degree of endogenous DNL and combining PUFA supplementation and FASN inhibition might be a promising approach to target metabolic disease.

Loss of Polycystin-1 causes cAMP-dependent switch from tubule to cyst formation.

Autosomal dominant polycystic kidney disease is the most common monogenic disease that causes end-stage renal failure. It primarily results from mutations in the PKD1 gene that encodes for Polycystin-1. How loss of Polycystin-1 translates into bilateral renal cyst development is mostly unknown. cAMP is significantly involved in cyst enlargement but its role in cyst initiation has remained elusive. Deletion of Polycystin-1 in collecting duct cells resulted in a switch from tubule to cyst formation and was accompanied by an increase in cAMP. Pharmacological elevation of cAMP in Polycystin-1-competent cells caused cyst formation, impaired plasticity, nondirectional migration, and mis-orientation, and thus strongly resembled the phenotype of Polycystin-1-deficient cells. Mis-orientation of developing tubule cells in metanephric kidneys upon loss of Polycystin-1 was phenocopied by pharmacological increase of cAMP in wildtype kidneys. In vitro, cAMP impaired tubule formation after capillary-induced injury which was further impaired by loss Polycystin-1.

Cyst growth in ADPKD is prevented by pharmacological and genetic inhibition of TMEM16A in vivo.

In autosomal dominant polycystic kidney disease (ADPKD) multiple bilateral renal cysts gradually enlarge, leading to a decline in renal function. Transepithelial chloride secretion through cystic fibrosis transmembrane conductance regulator (CFTR) and TMEM16A (anoctamin 1) are known to drive cyst enlargement. Here we demonstrate that loss of Pkd1 increased expression of TMEM16A and CFTR and Cl- secretion in murine kidneys, with TMEM16A essentially contributing to cyst growth. Upregulated TMEM16A enhanced intracellular Ca2+ signaling and proliferation of Pkd1-deficient renal epithelial cells. In contrast, increase in Ca2+ signaling, cell proliferation and CFTR expression was not observed in Pkd1/Tmem16a double knockout mice. Knockout of Tmem16a or inhibition of TMEM16A in vivo by the FDA-approved drugs niclosamide and benzbromarone, as well as the TMEM16A-specific inhibitor Ani9 largely reduced cyst enlargement and abnormal cyst cell proliferation. The present data establish a therapeutic concept for the treatment of ADPKD.

Lipid Peroxidation Drives Renal Cyst Growth In Vitro through Activation of TMEM16A.

BACKGROUND: Transepithelial chloride- secretion, through the chloride channels cystic fibrosis transmembrane conductance regulator (CFTR) and TMEM16A (anoctamin 1), drives cyst enlargement in polycystic kidney disease (PKD). Polycystic kidneys are hypoxic, and oxidative stress activates TMEM16A. However, mechanisms for channel activation in PKD remain obscure. METHODS: Using tissue samples from patients with autosomal dominant PKD, embryonic kidney cultures, and an MDCK in vitro cyst model, we assessed peroxidation of plasma membrane phospholipids in human and mouse polycystic kidneys. We also used electrophysiologic Ussing chamber and patch clamp experiments to analyze activation of TMEM16A and growth of renal cysts. RESULTS: Peroxidation of phospholipids in human and mouse kidneys as well as MDCK cysts in vitro is probably due to enhanced levels of reactive oxygen species. Lipid peroxidation correlated with increased cyst volume as shown in renal cultures and MDCK cysts in three-dimensional cultures. Reactive oxygen species and lipid peroxidation strongly activated TMEM16A, leading to depletion of calcium ion stores and store-operated calcium influx. Activation of TMEM16A- and CFTR-dependent chloride secretion strongly augmented cyst growth. Exposure to scavengers of reactive oxygen species, such as glutathione, coenzyme Q10, or idebenone (a synthetic coenzyme Q10 homolog), as well as inhibition of oxidative lipid damage by ferrostatin-1 largely reduced activation of TMEM16A. Inhibition of TMEM16A reduced proliferation and fluid secretion in vitro. CONCLUSIONS: These findings indicate that activation of TMEM16A by lipid peroxidation drives growth of renal cysts. We propose direct inhibition of TMEM16A or inhibition of lipid peroxidation as potentially powerful therapeutic approaches to delay cyst development in PKD.

Inflammatory Biomarkers and Clinical Judgment in the Emergency Diagnosis of Urgent Abdominal Pain.

BACKGROUND: The early diagnosis of urgent abdominal pain (UAP) is challenging. Most causes of UAP are associated with extensive inflammation. Therefore, we hypothesized that quantifying inflammation using interleukin-6 and/or procalcitonin would provide incremental value in the emergency diagnosis of UAP. METHODS: This was an investigator-initiated prospective, multicenter diagnostic study enrolling patients presenting to the emergency department (ED) with acute abdominal pain. Clinical judgment of the treating physician regarding the presence of UAP was quantified using a visual analog scale after initial clinical and physician-directed laboratory assessment, and again after imaging. Two independent specialists adjudicated the final diagnosis and the classification as UAP (life-threatening, needing urgent surgery and/or hospitalization for acute medical reasons) using all information including histology and follow-up. Interleukin-6 and procalcitonin were measured blinded in a central laboratory. RESULTS: UAP was adjudicated in 376 of 1038 (36%) patients. Diagnostic accuracy for UAP was higher for interleukin-6 [area under the ROC curve (AUC), 0.80; 95% CI, 0.77-0.82] vs procalcitonin (AUC, 0.65; 95% CI, 0.62-0.68) and clinical judgment (AUC, 0.69; 95% CI, 0.65-0.72; both P < 0.001). Combined assessment of interleukin-6 and clinical judgment increased the AUC at presentation to 0.83 (95% CI, 0.80-0.85) and after imaging to 0.87 (95% CI, 0.84-0.89) and improved the correct identification of patients with and without UAP (net improvement in mean predicted probability: presentation, +19%; after imaging, +15%; P < 0.001). Decision curve analysis documented incremental value across the full range of pretest probabilities. A clinical judgment/interleukin-6 algorithm ruled out UAP with a sensitivity of 97% and ruled in UAP with a specificity of 93%. CONCLUSIONS: Interleukin-6 significantly improves the early diagnosis of UAP in the ED.

ESBL-plasmid carriage in E. coli enhances in vitro bacterial competition fitness and serum resistance in some strains of pandemic sequence types without overall fitness cost.

BACKGROUND: Extended spectrum beta lactamase (ESBL)-producing extraintestinal pathogenic Escherichia coli infections are of global interest because of their clinical and economic impact. The ESBL resistance genes disseminate through plasmids, and are found in successful global lineages such as ST131 and ST648. The carriage of plasmids has been suggested to result in a fitness burden, but recently it was shown that ESBL-plasmids enhanced virulence in pandemic ST131 and ST648 lineages without affecting their fitness. Herein, we investigated the influence of ESBL-plasmids on bacterial competition and serum resistance, both of which are essential characteristics of ExPEC during infections. METHODS: Triplets of ESBL-plasmid-carrying wildtype (WT), plasmid-cured variant (PCV) and transformant (T) of five ExPEC strains of ST131 and ST648 were used for bacterial competition experiments with colicin-producing commensal E. coli, competitive adhesion experiments and serum survival. In addition, resilience after SDS, acid, osmotic challenges and RNA sequence data were analyzed. RESULTS: In all five strains tested, ESBL-plasmid carriage did not negatively influence E. coli fitness in direct bacterial competition with commensal E. coli in vitro. That is, WTs did not show any disadvantages when compared to their isogenic plasmid-free PCV. For one strain we even found the opposite as PCV17433 was out-competed by a commensal strain, which suggests an even protective role of the ESBL-plasmid carried by the WT17433. Similarly, in the serum-resistance experiments, the PCVs of two strains (PCV17433 and PCV17887) were more sensitive to serum, unlike WTs and Ts. The observed inter-strain differences could be explained by the different genetic content of plasmids carried in those strains. CONCLUSIONS: Overall, we found no compelling evidence for an increased burden resulting from the carriage of ESBL-plasmids in the absence of antimicrobial selection pressure in the strains of pandemic ST131 and ST648; rather, the possession of certain ESBL-plasmids was beneficial for some strains in regarding competition fitness and serum survival.

A large-scale peer teaching programme - acceptance and benefit.

INTRODUCTION: The involvement of students in the embodiment of university teaching through peer-assisted learning formats is commonly applied. Publications on this topic exclusively focus on strictly defined situations within the curriculum and selected target groups. This study, in contrast, presents and evaluates a large-scale structured and quality-assured peer teaching programme, which offers diverse and targeted courses throughout the preclinical part of the medical curriculum. METHODS: The large-scale peer teaching programme consists of subject specific and interdisciplinary tutorials that address all scientific, physiological and anatomic subjects of the preclinical curriculum as well as tutorials with contents exceeding the formal curriculum. In the study year 2013/14 a total of 1,420 lessons were offered as part of the programme. Paper-based evaluations were conducted over the full range of courses. Acceptance and benefit of this peer teaching programme were evaluated in a retrospective study covering the period 2012 to 2014. Usage of tutorials by students who commenced their studies in 2012/13 (n=959) was analysed from 2012 till 2014. Based on the results of 13 first assessments in the preclinical subjects anatomy, biochemistry and physiology, the students were assigned to one of five groups. These groups were compared according to participation in the tutorials. To investigate the benefit of tutorials of the peer teaching programme, the results of biochemistry re-assessments of participants and non-participants of tutorials in the years 2012 till 2014 (n=188, 172 and 204, respectively) were compared using Kolmogorov-Smirnov- and Chi-square tests as well as the effect size Cohen's d. RESULTS: Almost 70 % of the students attended the voluntary additional programme during their preclinical studies. The students participating in the tutorials had achieved different levels of proficiency in first assessments. The acceptance of different kinds of tutorials appears to correlate with their performance in first assessments. 94% of the students participating in tutorials offered in the study year 2013/14 rated the tutorials as "excellent" or "good". An objective benefit has been shown by a significant increase in re-assessment scores with an effect size between the medium and large magnitudes for participants of tutorials compared to non-participants in the years 2012, 2013 and 2014. In addition, significantly higher pass rates of re-assessments could be observed. CONCLUSION: Acceptance, utilisation and benefit of the assessed peer teaching programme are high. Beyond the support of students, a contribution to the individualisation of studies and teaching is made. Further studies are necessary to investigate possible influences of large-scale peer teaching programmes, for example on the reduction of study length and drop-off rates, as well as additional effects on academic achievements.

WITHDRAWN: Structural and mechanistic studies of hydroperoxide conversions catalyzed by a CYP74 clan epoxy alcohol synthase from amphioxus (Branchiostoma floridae).

This manuscript has been withdrawn by the Author.

Biosynthesis of allene oxides in Physcomitrella patens.

BACKGROUND: The moss Physcomitrella patens contains C18- as well as C20-polyunsaturated fatty acids that can be metabolized by different enzymes to form oxylipins such as the cyclopentenone cis(+)-12-oxo phytodienoic acid. Mutants defective in the biosynthesis of cyclopentenones showed reduced fertility, aberrant sporophyte morphology and interrupted sporogenesis. The initial step in this biosynthetic route is the conversion of a fatty acid hydroperoxide to an allene oxide. This reaction is catalyzed by allene oxide synthase (AOS) belonging as hydroperoxide lyase (HPL) to the cytochrome P450 family Cyp74. In this study we characterized two AOS from P. patens, PpAOS1 and PpAOS2. RESULTS: Our results show that PpAOS1 is highly active with both C18 and C20-hydroperoxy-fatty acid substrates, whereas PpAOS2 is fully active only with C20-substrates, exhibiting trace activity (~1000-fold lower kcat/KM) with C18 substrates. Analysis of products of PpAOS1 and PpHPL further demonstrated that both enzymes have an inherent side activity mirroring the close inter-connection of AOS and HPL catalysis. By employing site directed mutagenesis we provide evidence that single amino acid residues in the active site are also determining the catalytic activity of a 9-/13-AOS - a finding that previously has only been reported for substrate specific 13-AOS. However, PpHPL cannot be converted into an AOS by exchanging the same determinant. Localization studies using YFP-labeled AOS showed that PpAOS2 is localized in the plastid while PpAOS1 may be found in the cytosol. Analysis of the wound-induced cis(+)-12-oxo phytodienoic acid accumulation in PpAOS1 and PpAOS2 single knock-out mutants showed that disruption of PpAOS1, in contrast to PpAOS2, results in a significantly decreased cis(+)-12-oxo phytodienoic acid formation. However, the knock-out mutants of neither PpAOS1 nor PpAOS2 showed reduced fertility, aberrant sporophyte morphology or interrupted sporogenesis. CONCLUSIONS: Our study highlights five findings regarding the oxylipin metabolism in P. patens: (i) Both AOS isoforms are capable of metabolizing C18- and C20-derived substrates with different specificities suggesting that both enzymes might have different functions. (ii) Site directed mutagenesis demonstrated that the catalytic trajectories of 9-/13-PpAOS1 and PpHPL are closely inter-connected and PpAOS1 can be inter-converted by a single amino acid exchange into a HPL. (iii) In contrast to PpAOS1, PpAOS2 is localized in the plastid where oxylipin metabolism takes place. (iv) PpAOS1 is essential for wound-induced accumulation of cis(+)-12-oxo phytodienoic acid while PpAOS2 appears not to be involved in the process. (v) Knock-out mutants of neither AOS showed a deviating morphological phenotype suggesting that there are overlapping functions with other Cyp74 enzymes.

Crystal structures of Physcomitrella patens AOC1 and AOC2: insights into the enzyme mechanism and differences in substrate specificity.

In plants, oxylipins regulate developmental processes and defense responses. The first specific step in the biosynthesis of the cyclopentanone class of oxylipins is catalyzed by allene oxide cyclase (AOC) that forms cis(+)-12-oxo-phytodienoic acid. The moss Physcomitrella patens has two AOCs (PpAOC1 and PpAOC2) with different substrate specificities for C18- and C20-derived substrates, respectively. To better understand AOC's catalytic mechanism and to elucidate the structural properties that explain the differences in substrate specificity, we solved and analyzed the crystal structures of 36 monomers of both apo and ligand complexes of PpAOC1 and PpAOC2. From these data, we propose the following intermediates in AOC catalysis: (1) a resting state of the apo enzyme with a closed conformation, (2) a first shallow binding mode, followed by (3) a tight binding of the substrate accompanied by conformational changes in the binding pocket, and (4) initiation of the catalytic cycle by opening of the epoxide ring. As expected, the substrate dihydro analog cis-12,13S-epoxy-9Z,15Z-octadecadienoic acid did not cyclize in the presence of PpAOC1; however, when bound to the enzyme, it underwent isomerization into the corresponding trans-epoxide. By comparing complex structures of the C18 substrate analog with in silico modeling of the C20 substrate analog bound to the enzyme allowed us to identify three major molecular determinants responsible for the different substrate specificities (i.e. larger active site diameter, an elongated cavity of PpAOC2, and two nonidentical residues at the entrance of the active site).