Search

Fusarium head blight (FHB) is a devastating disease of wheat, causing yield losses, reduced grain quality, and mycotoxin contamination. Breeding can mitigate the severity of FHB epidemics, especially with genomics-assisted methods. The mechanisms underlying resistance to FHB in wheat have been extensively studied, including phenological traits and genome-wide markers associated with FHB severity. Here, we aimed to improve genomic prediction for FHB resistance across breeding programs by incorporating FHB-correlated traits and FHB-associated loci as model covariates. We combined phenotypic data on FHB severity, anthesis date, and plant height with genome-wide marker data from five Central European winter wheat breeding programs for genome-wide association studies (GWAS) and genomic prediction. Within all populations, FHB was correlated with anthesis date and/or plant height, and a marker linked to the semi-dwarfing locus Rht-D1 was detected with GWAS for FHB. Including the Rht-D1 marker, anthesis date, and/or plant height as covariates in genomic prediction modeling improved prediction accuracy not only within populations but also in cross-population scenarios., Competing Interests: Authors JH and HK were employed by the company Secobra Saatzucht GmbH. Authors FL, AN, and MS were employed by the company Saatzucht Donau GmbH & CoKG. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Morales, Akdemir, Girard, Neumayer, Reddy Nannuru, Shahinnia, Stadlmeier, Hartl, Holzapfel, Isidro-Sánchez, Kempf, Lillemo, Löschenberger, Michel and Buerstmayr.)

Key Message: Stem rust resistance was mainly based on a few, already known resistance genes; for yellow rust resistance there was a combination of designated genes and minor QTLs. Yellow rust (YR) caused by Puccinia striiformis f. sp. tritici (Pst) and stem rust (SR) caused by Puccinia graminis f. sp. tritici (Pgt) are among the most damaging wheat diseases. Although, yellow rust has occurred regularly in Europe since the advent of the Warrior race in 2011, damaging stem rust epidemics are still unusual. We analyzed the resistance of seven segregating populations at the adult growth stage with the parents being selected for YR and SR resistances across three to six environments (location-year combinations) following inoculation with defined Pst and Pgt races. In total, 600 progenies were phenotyped and 563 were genotyped with a 25k SNP array. For SR resistance, three major resistance genes (Sr24, Sr31, Sr38/Yr17) were detected in different combinations. Additional QTLs provided much smaller effects except for a gene on chromosome 4B that explained much of the genetic variance. For YR resistance, ten loci with highly varying percentages of explained genetic variance (pG, 6-99%) were mapped. Our results imply that introgression of new SR resistances will be necessary for breeding future rust resistant cultivars, whereas YR resistance can be achieved by genomic selection of many of the detected QTLs., (© 2024. The Author(s).)

Introduction: The development of paediatric medical devices continues to lag adult medical devices and contributes to issues of inequity, safety, quality and patient outcomes. New legislation and funding mechanisms have been introduced over the past two decades, but the gap remains. Clinical trials have been identified as a pain point, but components of effective clinical research infrastructure are poorly understood. As part of a multimodal research strategy, the Pediatric Device Consortia (PDC) will conduct a scoping review to better understand infrastructural barriers to and facilitators of paediatric medical device clinical research identified in the health sciences literature., Methods and Analysis: The following databases will be included for this review: Medline, Embase, Cochrane CENTRAL, Web of Science and IEEE Xplore. Additional grey literature will be sought out through Google Scholar and reviewing the citations of included studies. Included studies will discuss medical devices according to the U.S. Food and Drug Administration classification, focus on the paediatric population (ages 0-21 years) and involve human premarket or postmarket research. All study types that were published in 2007-present in English, Spanish, French or Italian will be included. Using Covidence web-based software, two independent reviewers will screen the resulting titles, abstracts and the full text of potential studies. Conflicts will be resolved by the primary investigator during both phases. REDCap will be used for quantitative and qualitative data charting, generating data tables and narrative synthesis., Ethics and Dissemination: This research did not require research ethics board consideration as it does not involve human participants and all data will be collected from published literature. We will share our findings through peer-reviewed manuscripts, clinical and research conference presentations and professional networks available to the PDC., Study Registration: Open Science Framework (https://osf.io/k72bn)., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

In order to recapitulate complex eukaryotic compartmentalization, synthetic biology aims to recreate cellular membrane-lined compartments from the bottom-up. Many important cellular organelles and cell-produced extracellular vesicles are in the size range of several hundreds of nanometers. Although attaining a fundamental characterization and mimicry of their cellular functions is a compelling goal, the lack of methods for controlled vesicle formation in this size range has hindered full understanding. Here, we show the optimization of a simple and efficient protocol for the production of large unilamellar vesicles (LUVs) with a median diameter in the range of 450-550 nm with high purity. Importantly, we rely on commercial reagents and common laboratory equipment. We thoroughly characterize the influence of different experimental parameters on the concentration and size of the resulting vesicles and assess changes in their lipid composition and surface charge. We provide guidance for researchers to optimize LUV production further to suit specific applications.

Osteoarthritis (OA) is the most prevalent form of arthritis and a major cause of pain and disability. The pathology of OA involves the whole joint in an inflammatory and degenerative process, especially in articular cartilage. OA may be divided into distinguishable phenotypes including one associated with the metabolic syndrome (MetS) of which dyslipidemia and hyperglycemia have been individually linked to OA. Since their combined role in OA pathogenesis remains to be elucidated, we investigated the chondrocyte response to these metabolic stresses, and determined whether a n-3 polyunsaturated fatty acid (PUFA), i.e., eicosapentaenoic acid (EPA), may preserve chondrocyte functions. Rat chondrocytes were cultured with palmitic acid (PA) and/or EPA in normal or high glucose conditions. The expression of genes encoding proteins found in cartilage matrix (type 2 collagen and aggrecan) or involved in degenerative (metalloproteinases, MMPs) or in inflammatory (cyclooxygenase-2, COX-2 and microsomal prostaglandin E synthase, mPGES) processes was analyzed by qPCR. Prostaglandin E 2 (PGE 2 ) release was also evaluated by an enzyme-linked immunosorbent assay. Our data indicated that PA dose-dependently up-regulated the mRNA expression of MMP-3 and -13 . PA also induced the expression of COX-2 and mPGES and promoted the synthesis of PGE 2 . Glucose at high concentrations further increased the chondrocyte response to PA. Interestingly, EPA suppressed the inflammatory effects of PA and glucose, and strongly reduced MMP-13 expression. Among the free fatty acid receptors (FFARs), FFAR4 partly mediated the EPA effects and the activation of FFAR1 markedly reduced the inflammatory effects of PA in high glucose conditions. Our findings demonstrate that dyslipidemia associated with hyperglycemia may contribute to OA pathogenesis and explains why an excess of saturated fatty acids and a low level in n-3 PUFAs may disrupt cartilage homeostasis.

The lack of physiological parity between 2D cell culture and in vivo culture has led to the development of more organotypic models, such as organoids. Organoid models have been developed for a number of tissues, including the liver. Current organoid protocols are characterized by a reliance on extracellular matrices (ECMs), patterning in 2D culture, costly growth factors and a lack of cellular diversity, structure, and organization. Current hepatic organoid models are generally simplistic and composed of hepatocytes or cholangiocytes, rendering them less physiologically relevant compared to native tissue. We have developed an approach that does not require 2D patterning, is ECM independent, and employs small molecules to mimic embryonic liver development that produces large quantities of liver-like organoids. Using single-cell RNA sequencing and immunofluorescence, we demonstrate a liver-like cellular repertoire, a higher order cellular complexity, presenting with vascular luminal structures, and a population of resident macrophages: Kupffer cells. The organoids exhibit key liver functions, including drug metabolism, serum protein production, urea synthesis and coagulation factor production, with preserved post-translational modifications such as N-glycosylation and functionality. The organoids can be transplanted and maintained long term in mice producing human albumin. The organoids exhibit a complex cellular repertoire reflective of the organ and have de novo vascularization and liver-like function. These characteristics are a prerequisite for many applications from cellular therapy, tissue engineering, drug toxicity assessment, and disease modeling to basic developmental biology., (© 2023. The Author(s).)

Key Message: We found two loci on chromosomes 2BS and 6AL that significantly contribute to stripe rust resistance in current European winter wheat germplasm. Stripe or yellow rust, caused by the fungus Puccinia striiformis Westend f. sp. tritici, is one of the most destructive wheat diseases. Sustainable management of wheat stripe rust can be achieved through the deployment of rust resistant cultivars. To detect effective resistance loci for use in breeding programs, an association mapping panel of 230 winter wheat cultivars and breeding lines from Northern and Central Europe was employed. Genotyping with the Illumina® iSelect® 25 K Infinium® single nucleotide polymorphism (SNP) genotyping array yielded 8812 polymorphic markers. Structure analysis revealed two subpopulations with 92 Austrian breeding lines and cultivars, which were separated from the other 138 genotypes from Germany, Norway, Sweden, Denmark, Poland, and Switzerland. Genome-wide association study for adult plant stripe rust resistance identified 12 SNP markers on six wheat chromosomes which showed consistent effects over several testing environments. Among these, two marker loci on chromosomes 2BS (RAC875_c1226_652) and 6AL (Tdurum_contig29607_413) were highly predictive in three independent validation populations of 1065, 1001, and 175 breeding lines. Lines with the resistant haplotype at both loci were nearly free of stipe rust symptoms. By using mixed linear models with those markers as fixed effects, we could increase predictive ability in the three populations by 0.13-0.46 compared to a standard genomic best linear unbiased prediction approach. The obtained results facilitate an efficient selection for stripe rust resistance against the current pathogen population in the Northern and Central European winter wheat gene pool., (© 2022. The Author(s).)

Idiopathic pulmonary fibrosis (IPF) is a fatal disease with limited treatment options. In this study, we focus on the properties of airway basal cells (ABC) obtained from patients with IPF (IPF-ABC). Single cell RNA sequencing (scRNAseq) of bronchial brushes revealed extensive reprogramming of IPF-ABC towards a KRT17 high PTEN low dedifferentiated cell type. In the 3D organoid model, compared to ABC obtained from healthy volunteers, IPF-ABC give rise to more bronchospheres, de novo bronchial structures resembling lung developmental processes, induce fibroblast proliferation and extracellular matrix deposition in co-culture. Intratracheal application of IPF-ABC into minimally injured lungs of Rag2 -/- or NRG mice causes severe fibrosis, remodeling of the alveolar compartment, and formation of honeycomb cyst-like structures. Connectivity MAP analysis of scRNAseq of bronchial brushings suggested that gene expression changes in IPF-ABC can be reversed by SRC inhibition. After demonstrating enhanced SRC expression and activity in these cells, and in IPF lungs, we tested the effects of saracatinib, a potent SRC inhibitor previously studied in humans. We demonstrate that saracatinib modified in-vitro and in-vivo the profibrotic changes observed in our 3D culture system and novel mouse xenograft model., (© 2022. The Author(s).)

Background: Stunting is a major health problem in low-income countries. We aimed to describe the implementation of a lipid-based nutrient supplement (LNS) program in a rural neighborhood in Cambodia and to assess its impact on statural growth., Method: This was a before-after comparative study. The program was promoted by the Pédiatres du Monde (PDM) organization between 2011 and 2019 in six villages in a rural area in Cambodia. The supplementation program consisted of daily administration of LNS during the third semester of pregnancy for the mothers and then between 6 and 24 months of age for the toddlers. Anthropometric data of the children were recorded during PDM visits before and after the program implementation, which allowed us to compare child growth in the two groups: control and intervention groups. Primary outcome was height-for-age between 24 and 35 months of age., Results: Overall, 198 data were collected for children between 24 and 35 months of age in the control group. A total of 347 pregnant women were enrolled in the intervention phase. A total of 188 data were collected for children between 24 and 35 months of age in the intervention group. The mean height-for-age z-score in the population receiving LNS was higher than in the control group (-1.14 vs. -1.60, p < 0.001). There was no significant difference between the two groups regarding the weight-for-height z-score (WHZ; -1.11 vs. -1.26, p = 0.18) and children in the intervention group had a higher middle upper-arm circumference z-score (MUACZ; -0.75 vs.. -1.1, p < 0.001)., Conclusion: LNS supplementation significantly and increased the HAZ between 24 and 35 months of age. However, the fight against malnutrition is complex and needs intervention on multiple levels., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 French Society of Pediatrics. Published by Elsevier Masson SAS. All rights reserved.)

Matrix Gla protein (MGP) is mostly known to be a calcification inhibitor, as its absence leads to ectopic calcification of different tissues such as cartilage or arteries. MGP deficiency also leads to low bone mass and delayed bone growth. In the present contribution, we investigate the effect of MGP deficiency on the structural and material mechanical bone properties by focusing on the elastic response of femurs undergoing three-points bending. To this aim, biomechanical tests are performed on femurs issued from Mgp-deficient mice at 14, 21, 28, and 35 days of postnatal life and compared to healthy control femurs. µCT acquisitions enable to reconstruct bone geometries and are used to construct subject-specific finite element models avoiding some of the reported limitations concerning the use of beam-like assumptions for small bone samples. Our results indicate that MGP deficiency may be associated to differences in both structural and material properties of femurs during early stages of development. MGP deficiency appears to be related to a decrease in bone dimensions, compensated by higher material properties resulting in similar structural bone properties at P35. The search for a unique density-elasticity relationship based on calibrated bone mineral density (BMD) indicates that MGP deficiency may affect bone tissue in several ways, that may not be represented uniquely from the quantification of BMD. Despite of its limitation to elastic response, the present preliminary study reports for the very first time the mechanical skeletal properties of Mgp-deficient mice at early stages of development.

The low efficiency in transfecting rat- and human-derived chondrocytes have been hampering developments in the field of cartilage biology. Transforming growth factor (TGF)-β1 has shown positive effects on chondrocytes, but its applications remain limited due to its short half-life, low stability and poor penetration into cartilage. Naturally derived liposomes have been shown to be promising delivery nanosystems due to their similarities with biological membranes. Here, we used agro-based rapeseed liposomes, which contains a high level of mono- and poly-unsaturated fatty acids, to efficiently deliver encapsulated TGF-β1 to rat chondrocytes. Results showed that TGF-β1 encapsulated in nano-sized rapeseed liposomes were safe for chondrocytes and did not induce any alterations of their phenotype. Furthermore, the controlled release of TGF-β1 from liposomes produced an improved response in chondrocytes, even at low doses. Altogether, these outcomes demonstrate that agro-based nanoliposomes are promising drug carriers.

Macrophages derived from human induced pluripotent stem cells (iPSCs) have the potential to enable the development of cell-based therapies for numerous disease conditions. We here provide a detailed protocol for the mass production of iPSC-derived macrophages (iPSC-Mac) in scalable suspension culture on an orbital shaker or in stirred-tank bioreactors (STBRs). This strategy is straightforward, robust and characterized by the differentiation of primed iPSC aggregates into 'myeloid-cell-forming-complex' intermediates by means of a minimal cytokine cocktail. In contrast to the 'batch-like differentiation approaches' established for other iPSC-derived lineages, myeloid-cell-forming-complex-intermediates are stably maintained in suspension culture and continuously generate functional and highly pure iPSC-Mac. Employing a culture volume of 120 ml in the STBR platform, ~1-4 × 10 7 iPSC-Mac can be harvested at weekly intervals for several months. The STBR technology allows for real-time monitoring of crucial process parameters such as biomass, pH, dissolved oxygen, and nutrition levels; the system also promotes systematic process development, optimization and linear upscaling. The process duration, from the expansion of iPSC until the first iPSC-Mac harvest, is 28 d. Successful application of the protocol requires expertise in pluripotent stem cell culture, differentiation and analytical methods, such as flow cytometry. Fundamental know-how in biotechnology is also advantageous to run the process in the STBR platform. The continuous, scalable production of well-defined iPSC-Mac populations is highly relevant to various fields, ranging from developmental biology, immunology and cell therapies to industrial applications for drug safety and discovery., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Moth-eye structures are patterned onto gallium selenide surfaces with sub-micrometer precision. In this way, Fresnel reflection losses are suppressed to below one percent within an ultrabroad optical bandwidth from 15 to 65 THz. We tune the geometry by rigorous coupled-wave analysis. Subsequently, ablation with a Ga + ion beam serves to write optimized structures in areas covering 30 by 30 μm. The benefits are demonstrated via optical rectification of femtosecond laser pulses under tight focusing, resulting in emission of phase-stable transients in the mid-infrared. We analyze the performance of antireflection coating directly in the time domain by ultrabroadband electro-optic sampling.

The effects of an injectable anesthesia with 0.05 mg/kg medetomidine, 5 mg/kg ketamine, and 0.5 mg/kg butorphanol administered together intramuscularly were evaluated in 22 captive Humboldt penguins ( Spheniscus humboldti , 10 male and 12 female), with a mean age of 8.5 ± 8.23 years. The birds fasted for18-24 hours prior to the procedure. Induction was followed by 4 distinct progressive responses of the birds to the anesthetic effect, including onset of initial effects at 2.0 ± 1.7 minutes (x̄ ± SD), sternal recumbency with the head still elevated at 2.2 ± 1.6 minutes, lowering and placing the beak tip to the ground at 3.6 ± 3.4 minutes, and lateral positioning of the neck and head at 4.2 ± 3.4 minutes. A general state of sedation, muscle relaxation, and analgesia were noted 10.0 ± 2.8 minutes postinjection. However, according to an established scoring system for the assessment of anesthetic depth in avian patients, a surgical plane of anesthesia was not achieved. Muscle relaxation determined by the same scoring system lasted for 31.4 ± 17.1 minutes. The penguins' mean respiratory rate did not demonstrate significant change and spontaneous ventilation was present throughout the procedure. Relative peripheral arterial oxygen saturation decreased significantly from 92.83 ± 5.77% at 10 minutes to 90.91 ± 5.77% at 40 minutes following induction. The birds' heart rate also decreased significantly from 112.55 ± 23.97 beats/min at 10 minutes to 101.65 ± 25.42 beats/min at 40 minutes. The measured cloacal temperatures were maintained within normal range despite ambient temperatures of up to 28.3°C (82.9°F). Reversal of medetomidine with 0.25 mg/kg atipamezole was conducted after 45.1 ± 7.3 minutes. Recovery was smooth but of variable duration with patients being able or willing to stand steadily in an upright position after 50.1 ± 34.6 minutes. One penguin died during recovery from a ruptured left ventricle and consecutive pericardial tamponade, but no predisposing factors were identified. The anesthetic protocol proved to be effective for noninvasive and minor painful procedures (eg, diagnostic imaging, blood collection). Disadvantages to the administration of the combined anesthetic agents in the penguins included a short period of muscle relaxation and smooth but potentially prolonged recovery. The safety of the anesthetic protocol described for Humboldt penguins in this report has to be evaluated critically against the the death of 1 penguin during recovery.

Organoid models of early tissue development have been produced for the intestine, brain, kidney and other organs, but similar approaches for the heart have been lacking. Here we generate complex, highly structured, three-dimensional heart-forming organoids (HFOs) by embedding human pluripotent stem cell aggregates in Matrigel followed by directed cardiac differentiation via biphasic WNT pathway modulation with small molecules. HFOs are composed of a myocardial layer lined by endocardial-like cells and surrounded by septum-transversum-like anlagen; they further contain spatially and molecularly distinct anterior versus posterior foregut endoderm tissues and a vascular network. The architecture of HFOs closely resembles aspects of early native heart anlagen before heart tube formation, which is known to require an interplay with foregut endoderm development. We apply HFOs to study genetic defects in vitro by demonstrating that NKX2.5-knockout HFOs show a phenotype reminiscent of cardiac malformations previously observed in transgenic mice.

Hematopoietic development is spatiotemporally tightly regulated by defined cell-intrinsic and extrinsic modifiers. The role of cytokines has been intensively studied in adult hematopoiesis; however, their role in embryonic hematopoietic specification remains largely unexplored. Here, we used induced pluripotent stem cell (iPSC) technology and established a 3-dimensional, organoid-like differentiation system (hemanoid) maintaining the structural cellular integrity to evaluate the effect of cytokines on embryonic hematopoietic development. We show, that defined stages of early human hematopoietic development were recapitulated within the generated hemanoids. We identified KDR+/CD34high/CD144+/CD43-/CD45- hemato-endothelial progenitor cells (HEPs) forming organized, vasculature-like structures and giving rise to CD34low/CD144-/CD43+/CD45+ hematopoietic progenitor cells. We demonstrate that the endothelial to hematopoietic transition of HEPs is dependent on the presence of interleukin 3 (IL-3). Inhibition of IL-3 signalling blocked hematopoietic differentiation and arrested the cells in the HEP stage. Thus, our data suggest an important role for IL-3 in early human hematopoiesis by supporting the endothelial to hematopoietic transition of hemato-endothelial progenitor cells and highlight the potential of a hemanoid-based model to study human hematopoietic development.

Keutel syndrome (KS) is a rare autosomal recessive genetic disorder that was first identified in the beginning of the 1970s and nearly 30 years later attributed to loss-of-function mutations in the gene coding for the matrix Gla protein (MGP). Patients with KS are usually diagnosed during childhood (early onset of the disease), and the major traits include abnormal calcification of cartilaginous tissues resulting in or associated with malformations of skeletal tissues (e.g., midface hypoplasia and brachytelephalangism) and cardiovascular defects (e.g., congenital heart defect, peripheral pulmonary artery stenosis, and, in some cases, arterial calcification), and also hearing loss and mild developmental delay. While studies on Mgp -/- mouse, a faithful model of KS, show that pathologic mineral deposition (ectopic calcification) in cartilaginous and vascular tissues is the primary cause underlying many of these abnormalities, the mechanisms explaining how MGP prevents abnormal calcification remain poorly understood. This has negative implication for the development of a cure for KS. Indeed, at present, only symptomatic treatments are available to treat hypertension and respiratory complications occurring in the KS patients. In this review, we summarize the results published in the last 50 years on Keutel syndrome and present the current status of the knowledge on this rare pathology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Cancela, Laizé, Conceição, Kempf and Murshed.)

Fibrillar collagens and proteoglycans (PGs) are quantitatively the major constituents of extracellular matrices (ECM). They carry numerous crucial post-translational modifications (PTMs) that tune the resulting biomechanical properties of the corresponding tissues. The mechanisms determining these PTMs remain largely unknown, notably because available established cell lines do not recapitulate much of the complexity of the machineries involved. ATDC5 cells are a model of chondrogenesis widely used for decades, but it remains described mostly at histological and transcriptional levels. Here, we asked to what extent this model recapitulates the events of ECM synthesis and processing occurring in cartilage. Insulin-stimulated ATDC5 cells exhibit up- or down-regulation of more than one-hundred proteins, including a number of known participants in chondrogenesis and major markers thereof. However, they also lack several ECM components considered of significant, yet more subtle, function in cartilage. Still, they assemble the large PG aggrecan and type II collagen, both carrying most of their in vivo PTMs, into an ECM. Remarkably, collagen crosslinking is fully lysyl oxidase (LOX)-dependent. The ATDC5 model recapitulates critical aspects of the cartilage ECM-processing machinery and should be useful to decipher the mechanisms involved. Proteomics data are available via ProteomeXchange with identifier PXD014121. SIGNIFICANCE: The present work provides the first proteome characterization of the ATDC5 chondrogenesis model, which has been used for decades in the field of cartilage biology. The results demonstrate the up- and down-regulation of more than one hundred proteins. Overall, specific drawbacks of the model are pointed out, that will be important to take into consideration for future studies. However, major cartilage components are massively assembled into an extracellular matrix and carry most of their post-translational modifications occurring in cartilage tissue. Unlike other available established cell lines, the ATDC5 model recapitulates major aspects of cartilage biosynthesis and should be useful in investigating the mechanisms that regulate collagen maturation events., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Investigations in cartilage biology have been hampered by the limited capacity of chondrocytes, especially in rats and humans, to be efficiently transfected. Liposomes are a promising delivery system due to their lipid bilayer structure similar to a biological membrane. Here we used natural rapeseed lecithin, which contains a high level of mono- and poly-unsaturated fatty acids, to evaluate the cytocompatibility of these phospholipids as future potential carriers of biomolecules in joint regenerative medicine. Results show that appropriate concentrations of nanoliposome rapeseed lecithin under 500 µg/mL were safe for chondrocytes and did not induce any alterations of their phenotype. Altogether, these results sustain that they could represent a novel natural carrier to deliver active substances into cartilage cells.

Aiming at clinical translation, robust directed differentiation of human pluripotent stem cells (hPSCs), preferentially in chemically defined conditions, is a key requirement. Here, feasibility of suspension culture based hPSC-cardiomyocyte (hPSC-CM) production in low-cost, xeno-free media compatible with good manufacturing practice standards is shown. Applying stirred tank bioreactor systems at increasing dimensions, our advanced protocol enables routine production of about 1 million hPSC-CMs/mL, yielding ∼1.3 × 10 8  CM in 150 mL and ∼4.0 × 10 8 CMs in 350-500 mL process scale at >90% lineage purity. Process robustness and efficiency is ensured by uninterrupted chemical WNT pathway control at early stages of differentiation and results in the formation of almost exclusively ventricular-like CMs. Modulated WNT pathway regulation also revealed the previously unappreciated role of ROR1/CD13 as superior surrogate markers for predicting cardiac differentiation efficiency as soon as 72 h of differentiation. This monitoring strategy facilitates process upscaling and controlled mass production of hPSC derivatives., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Loss-of-function mutations of the SCN5A gene encoding for the sodium channel α-subunit Na V 1.5 result in the autosomal dominant hereditary disease Brugada Syndrome (BrS) with a high risk of sudden cardiac death in the adult. We here engineered human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the CRISPR/Cas9 introduced BrS-mutation p.A735V-Na V 1.5 (g.2204C > T in exon 14 of SCN5A) as a novel model independent of patient´s genetic background. Recent studies raised concern regarding the use of hiPSC-CMs for studying adult-onset hereditary diseases due to cells' immature phenotype. To tackle this concern, long-term cultivation of hiPSC-CMs on a stiff matrix (27-42 days) was applied to promote maturation. Patch clamp recordings of A735V mutated hiPSC-CMs revealed a substantially reduced upstroke velocity and sodium current density, a prominent rightward shift of the steady state activation curve and decelerated recovery from inactivation as compared to isogenic hiPSC-CMs controls. These observations were substantiated by a comparative study on mutant A735V-Na V 1.5 channels heterologously expressed in HEK293T cells. In contrast to mutated hiPSC-CMs, a leftward shift of sodium channel inactivation was not observed in HEK293T, emphasizing the importance of investigating mechanisms of BrS in independent systems. Overall, our approach supports hiPSC-CMs' relevance for investigating channelopathies in a dish.

The physicochemical deposition of calcium-phosphate in the arterial wall is prevented by calcification inhibitors. Studies in cohorts of patients with rare genetic diseases have shed light on the consequences of loss-of-function mutations for different calcification inhibitors, and genetic targeting of these pathways in mice have generated a clearer picture on the mechanisms involved. For example, generalized arterial calcification of infancy (GACI) is caused by mutations in the enzyme ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (eNPP1), preventing the hydrolysis of ATP into pyrophosphate (PP i ). The importance of PP i for inhibiting arterial calcification has been reinforced by the protective effects of PP i in various mouse models displaying ectopic calcifications. Besides PP i , Matrix Gla Protein (MGP) has been shown to be another potent calcification inhibitor as Keutel patients carrying a mutation in the encoding gene or Mgp -deficient mice develop spontaneous calcification of the arterial media. Whereas PP i and MGP represent locally produced calcification inhibitors, also systemic factors contribute to protection against arterial calcification. One such example is Fetuin-A, which is mainly produced in the liver and which forms calciprotein particles (CPPs), inhibiting growth of calcium-phosphate crystals in the blood and thereby preventing their soft tissue deposition. Other calcification inhibitors with potential importance for arterial calcification include osteoprotegerin, osteopontin, and klotho. The aim of the present review is to outline the latest insights into how different calcification inhibitors prevent arterial calcification both under physiological conditions and in the case of disturbed calcium-phosphate balance, and to provide a consensus statement on their potential therapeutic role for arterial calcification.

The ideal cell culture model should mimic the cell physiology and the mechanical and the chemical cues that are present in specific tissues and organs, within a convenient high-throughput format. A possible key feature for such models is to recapture the cell polarity, the interactions between cells, and the interactions between the cells and the elastic extracellular matrix (ECM) by orienting the cells in a three-dimensional (3D) matrix. A common method to create 3D cell environments is to let the cells aggregate into spheroids with a diameter of around 200 μm. A major challenge for 3D cell cultures is to perform quick and easy imaging of the dense cell population, especially noninvasively. This protocol explains how to take advantage of the number of cells growing out from cell spheroids over time as a readout of the effect of a drug. The assay is compatible with standard imaging techniques and can be performed noninvasively using light microscopy or as a complement to other fluorescent imaging assays.

The increasing number of severe infections with multi-drug-resistant pathogens worldwide highlights the need for alternative treatment options. Given the pivotal role of phagocytes and especially alveolar macrophages in pulmonary immunity, we introduce a new, cell-based treatment strategy to target bacterial airway infections. Here we show that the mass production of therapeutic phagocytes from induced pluripotent stem cells (iPSC) in industry-compatible, stirred-tank bioreactors is feasible. Bioreactor-derived iPSC-macrophages (iPSC-Mac) represent a highly pure population of CD45 + CD11b + CD14 + CD163 + cells, and share important phenotypic, functional and transcriptional hallmarks with professional phagocytes, however with a distinct transcriptome signature similar to primitive macrophages. Most importantly, bioreactor-derived iPSC-Mac rescue mice from Pseudomonas aeruginosa-mediated acute infections of the lower respiratory tract within 4-8 h post intra-pulmonary transplantation and reduce bacterial load. Generation of specific immune-cells from iPSC-sources in scalable stirred-tank bioreactors can extend the field of immunotherapy towards bacterial infections, and may allow for further innovative cell-based treatment strategies.

With their capability to self-renew and differentiate into derivatives of all three germ layers, human pluripotent stem cells (hPSCs) offer a unique model to study aspects of human development in vitro. Directed differentiation towards mesendodermal lineages is a complex process, involving transition through a primitive streak (PS)-like stage. We have recently shown PS-like patterning from hPSCs into definitive endoderm, cardiac as well as presomitic mesoderm by only modulating the bulk cell density and the concentration of the GSK3 inhibitor CHIR99021, a potent activator of the WNT pathway. The patterning process is modulated by a complex paracrine network, whose identity and mechanistic consequences are poorly understood. To study the underlying dynamics, we here applied mathematical modeling based on ordinary differential equations. We compared time-course data of early hPSC differentiation to increasingly complex model structures with incremental numbers of paracrine factors. Model simulations suggest at least three paracrine factors being required to recapitulate the experimentally observed differentiation kinetics. Feedback mechanisms from both undifferentiated and differentiated cells turned out to be crucial. Evidence from double knock-down experiments and secreted protein enrichment allowed us to hypothesize on the identity of two of the three predicted factors. From a practical perspective, the mathematical model predicts optimal settings for directing lineage-specific differentiation. This opens new avenues for rational stem cell bioprocessing in more advanced culture systems, e.g. in perfusion-fed bioreactors enabling cell therapies., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Induced pluripotent stem cell (iPSC)-derived hematopoietic cells represent a highly attractive source for cell and gene therapy. Given the longevity, plasticity, and self-renewal potential of distinct macrophage subpopulations, iPSC-derived macrophages (iPSC-Mφ) appear of particular interest in this context. We here evaluated the airway residence, plasticity, and therapeutic efficacy of iPSC-Mφ in a murine model of hereditary pulmonary alveolar proteinosis (herPAP). We demonstrate that single pulmonary macrophage transplantation (PMT) of 2.5-4 × 10 6 iPSC-Mφ yields efficient airway residence with conversion of iPSC-Mφ to an alveolar macrophage (AMφ) phenotype characterized by a distinct surface marker and gene expression profile within 2 months. Moreover, PMT significantly improves alveolar protein deposition and other critical herPAP disease parameters. Thus, our data indicate iPSC-Mφ as a source of functional macrophages displaying substantial plasticity and therapeutic potential that upon pulmonary transplantation will integrate into the lung microenvironment, adopt an AMφ phenotype and gene expression pattern, and profoundly ameliorate pulmonary disease phenotypes., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Multi-parent advanced generation intercross (MAGIC) populations are a newly established tool to dissect quantitative traits. We developed the high resolution MAGIC wheat population WM-800, consisting of 910 F 4:6 lines derived from intercrossing eight recently released European winter wheat cultivars., Results: Genotyping WM-800 with 7849 SNPs revealed a low mean genetic similarity of 59.7% between MAGIC lines. WM-800 harbours distinct genomic regions exposed to segregation distortion. These are mainly located on chromosomes 2 to 6 of the wheat B genome where founder specific DNA segments were positively or negatively selected. This suggests adaptive selection of individual founder alleles during population development. The application of a genome-wide association study identified 14 quantitative trait loci (QTL) controlling plant height in WM-800, including the known semi-dwarf genes Rht-B1 and Rht-D1 and a potentially novel QTL on chromosome 5A. Additionally, epistatic effects controlled plant height. For example, two loci on chromosomes 2B and 7B gave rise to an additive epistatic effect of 13.7 cm., Conclusion: The present study demonstrates that plant height in the MAGIC-WHEAT population WM-800 is mainly determined by large-effect QTL and di-genic epistatic interactions. As a proof of concept, our study confirms that WM-800 is a valuable tool to dissect the genetic architecture of important agronomic traits.

Endothelial cells (ECs) are involved in a variety of cellular responses. As multifunctional components of vascular structures, endothelial (progenitor) cells have been utilized in cellular therapies and are required as an important cellular component of engineered tissue constructs and in vitro disease models. Although primary ECs from different sources are readily isolated and expanded, cell quantity and quality in terms of functionality and karyotype stability is limited. ECs derived from human induced pluripotent stem cells (hiPSCs) represent an alternative and potentially superior cell source, but traditional culture approaches and 2D differentiation protocols hardly allow for production of large cell numbers. Aiming at the production of ECs, we have developed a robust approach for efficient endothelial differentiation of hiPSCs in scalable suspension culture. The established protocol results in relevant numbers of ECs for regenerative approaches and industrial applications that show in vitro proliferation capacity and a high degree of chromosomal stability., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Three-dimensional (3D) models with cells arranged in clusters or spheroids have emerged as valuable tools to improve physiological relevance in drug screening. One of the challenges with cells cultured in 3D, especially for high-throughput applications, is to quickly and non-invasively assess the cellular state in vitro. In this article, we show that the number of cells growing out from human induced pluripotent stem cell (hiPSC)-derived cardiac spheroids can be quantified to serve as an indicator of a drug&rsquo;s effect on spheroids captured in a microfluidic device. Combining this spheroid-on-a-chip with confocal high content imaging reveals easily accessible, quantitative outgrowth data. We found that effects on outgrowing cell numbers correlate to the concentrations of relevant pharmacological compounds and could thus serve as a practical readout to monitor drug effects. Here, we demonstrate the potential of this semi-high-throughput &ldquo;cardiac cell outgrowth assay&rdquo; with six compounds at three concentrations applied to spheroids for 48 h. The image-based readout complements end-point assays or may be used as a non-invasive assay for quality control during long-term culture.

Competing Interests: Competing interests: None declared.

Biologically active human bone morphogenetic protein-4 (hBMP-4) was successfully produced in a prokaryotic host. For this aim, hBMP-4 cDNA was cloned in Escherichia coli ( E. coli ) and the protein was produced in a non-active aggregated form. After washing and solubilization, in vitro refolding of the rhBMP-4 monomer was performed using rapid dilution. In this study, different refolding conditions were tested for the dimerization of rhBMP-4 by one-factor-at-a-time variation. The dimerization process was found to be sensitive to pH, protein concentration and the presence of aggregation suppressors. In contrast, redox conditions and ionic strength did not impact refolding as expected. The dimer was separated from the remaining monomer, aggregates and host cell contaminants in a single step using cation-exchange membrane chromatography. The rhBMP-4 dimer produced in E. coli was biologically active as demonstrated by its capability to induce trophoblast differentiation and primitive streak induction of human pluripotent stem cells (hPSCs).

Fundamental research and drug development for personalized medicine necessitates cell cultures from defined genetic backgrounds. However, providing sufficient numbers of authentic cells from individuals poses a challenge. Here, we present a new strategy for rapid cell expansion that overcomes current limitations. Using a small gene library, we expanded primary cells from different tissues, donors, and species. Cell-type-specific regimens that allow the reproducible creation of cell lines were identified. In depth characterization of a series of endothelial and hepatocytic cell lines confirmed phenotypic stability and functionality. Applying this technology enables rapid, efficient, and reliable production of unlimited numbers of personalized cells. As such, these cell systems support mechanistic studies, epidemiological research, and tailored drug development.

The envisioned routine application of human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) for therapies and industry-compliant screening approaches will require efficient and highly reproducible processes for the mass production of well-characterized CM batches.On their way toward beating CMs, hPSCs initially undergo an epithelial-to-mesenchymal transition into a primitive-streak (PS)-like population that later gives rise to all endodermal and mesodermal lineages, including cardiovascular progenies (CVPs). CVPs are multipotent and possess the capability to give rise to all major cell types of the heart, including CMs, endothelial cells, cardiac fibroblasts, and smooth muscle cells. This article provides an historical overview and describes the stepwise development of protocols that typically result in the appearance of beating CMs within 7-12 days of hPSC differentiation.We describe the development of directed and closely controlled cardiomyogenic differentiation, which now enables the induction of >90% CM purity without further lineage enrichment. Although secreted lineage specifiers (revealed from developmental biology) were initially used, we outline the advantages of chemical pathway modulators, as defined by more recent screening approaches. Subsequently, we discuss the use of defined culture media for upscaling the production of hPSC-CMs in controlled bioreactors and how this, in principle, unlimited source of human CMs can be used to progress heart regeneration and stimulate the drug discovery pipeline. Graphical Abstract.

A 24-year-old female secretary bird ( Sagittarius serpentarius) was presented with acute, mild dyspnea occurring only during feeding times. Despite initial conservative therapy consisting of antibiotics and antifungal, antiparasitic, and anti-inflammatory drugs, the dyspnea worsened progressively, resulting in severe respiratory distress. Radiographs of the trachea suggested stenosis in the caudal one-third of the trachea. Tracheal endoscopy revealed an obstruction of approximately 90% of the tracheal lumen, in addition to mild suspected aspergillosis of the air sacs. Tracheal resection and anastomosis were performed, during which 1.5 cm of abnormal trachea was removed. Histopathologic examination showed severe granulomatous tracheitis, most likely induced by foreign body material. Respiratory signs resolved immediately postoperatively. Antibiotic and anti-inflammatory therapy continued for another 7 days and the bird was treated with antifungals for a total of 45 days. The bird recovered uneventfully. We encourage tracheal resection and anastomosis for severe tracheal stenosis even in aged, large birds of prey that are managed in large aviaries.

Controlled large-scale production of human pluripotent stem cells (hPSCs) is indispensable for their envisioned clinical translation. Aiming at advanced process development in suspension culture, the sensitivity of hPSC media to continuous peristaltic pump-based circulation, a well-established technology extensively used in hydraulically-driven bioreactors, was investigated. Unexpectedly, conditioning of low protein media (i.e. E8 and TeSR-E8) in a peristaltic pump circuit induced severe viability loss of hPSCs cultured as aggregates in suspension. Optical, biochemical, and cytological analyses of the media revealed that the applied circulation mode resulted in the reduction of the growth hormone insulin by precipitation of micro-sized particles. Notably, in contrast to insulin depletion, individual withdrawal of other medium protein components (i.e. bFGF, TGFβ1 or transferrin) provoked minor reduction of hPSC viability, if any. Supplementation of the surfactant glycerol or the use of the insulin analogue Aspart did not overcome the issue of insulin precipitation. In contrast, the presence of bovine or human serum albumin (BSA or HSA, respectively) stabilized insulin rescuing its content, possibly by acting as molecular chaperone-like protein, ultimately supporting hPSC maintenance. This study highlights the potential and the requirement of media optimization for automated hPSC processing and has broad implications on media development and bioreactor-based technologies.

With an increased number of pet reptiles, many diseases occur due to nutritional disorders. Between 2013 and 2015, irregular gallbladder contents (sludge/choleliths) in adult bearded dragons (Pogona vitticeps) were recorded in many of the routinely conducted necropsies at the reptile rescue station in Munich (Auffangstation für Reptilien, München e.V., Munich, Germany). Nine animals, six from the rescue station and three from an associated veterinary practice (Tierärztliche Praxis für Exoten, Augsburg, Germany), were studied. Gallbladder contents from all animals were analysed at the Institute for Clinical Chemistry in Zurich, Switzerland. In three of nine animals, one cholelith composed of 100% calcium carbonate (CaCO 3 ) was detected and it precipitated either as pure calcite or as a calcite:vaterite combination. In the remaining six animals, analyses suggested a protein-based material. The detection of choleliths/sludge was not anticipated at necropsy or surgery in eight of nine animals. The diet of the six animals from the rescue station was retrospectively described as mainly insects, whereas the diet of the three animals from the veterinary practice also contained little plant matter. Fed insect species were mealworm larva (Tenebrio molitor), house cricket (Acheta domestica), migratory locust (Locusta migratoria) and zophobas larva (Zophobas morio), all high in protein and fat. In other species, a nidus must be present for CaCO 3 to precipitate. As a protein-based sludge was detected in six gallbladders, it is possible that a high-protein diet could lead to such a nidus and subsequently to cholelith formation. Cholelithiasis seems to be a rising problem in adult bearded dragons and is likely underdiagnosed, as many choleliths were found at necropsy. This rise in cholelithiasis may correlate with an unnatural high-protein, high-fat insect-based diet instead of a balanced plant-based diet., (Journal of Animal Physiology and Animal Nutrition © 2017 Blackwell Verlag GmbH.)

Objectives: Fibroblast Growth Factor 23 (FGF23) is well documented as a crucial player in the systemic regulation of phosphate homeostasis. Moreover, loss-of-function experiments have revealed that FGF23 also has a phosphate-independent and local impact on skeletogenesis. Here, we used ATDC5 cell line to investigate the expression of FGF23 and the role it may play locally during the differentiation of these cells., Methods: ATDC5 cells were differentiated in the presence of insulin, and treated with recombinant FGF23 (rFGF23), inorganic phosphate (Pi) and/or PD173074, an inhibitor of FGF receptors (FGFRs). The mRNA expressions of FGF23, FGFRs and markers of hypertophy, Col X and MMP13, were determined by qPCR analysis and FGF23 production was assessed by ELISA. FGFR activation was determined by immunoprecipitation and immunoblotting., Results: FGF23 mRNA expression and production were increased during ATDC5 differentiation. At D28 in particular, rFGF23 stimulation increased hypertrophic markers expression, as Col X and MMP13, and mineralization. A synergic effect of Pi and rFGF23 stimulation was observed on these markers and on the mineralization process. The use of PD173074, a pan-FGFR inhibitor, decreased terminal differentiation of ATDC5 by preventing rFGF23 pro-hypertrophic effects., Conclusions: Altogether, our results provide evidence that FGF23 plays an important role during differentiation of ATDC5 cell line, by promoting both hypertrophy and mineralization.

Human pluripotent stem cells (hPSCs), both embryonic (hESCs) and induced (hiPSCs), can be differentiated into derivatives of the three germ layers and are promising tools in regenerative medicine. Cardiovascular diseases are the top-ranking cause of premature death worldwide, and cell replacement therapies based on in vitro differentiated cardiomyocytes might provide a promising perspective to cure patients in the future. The molecular processes during hPSC cardiomyogenesis are far from being fully understood, and we thus have focused here on characterizing the proteome along hESC in vitro differentiation into cardiomyocytes (CMs). Stable isotope labeling of amino acids in cell culture was applied to quantitatively assess the proteome throughout defined stages of hESC cardiomyogenesis. Genetically enriched, >90% pure CM populations were used for shotgun proteomics, leading to the identification and quantitative determination of several thousand proteins. Pathway analysis revealed alterations in energy metabolism during cardiomyogenesis. Enzymes of glycolysis were identified as up-regulated upon differentiation, whereas enzymes involved in oxidative phosphorylation were down-regulated in aggregates on day 20 of differentiation (<10% CMs) and reconstituted on day 35 in >90% pure CMs. A structural protein that attracted our attention was the PDZ and LIM domain containing protein 5 (PDLIM5), which was strongly up-regulated during cardiomyogenesis and for which we detected novel stage-specific isoforms. Notably, expression of the 53 kDa isoforms b and g (corresponding to transcript variants 2 and 7) of PDLIM5 occurred simultaneously to the onset of expression of the early cardiac transcription factor NKX2.5, known to play a key role in cardiac development.

Subtype-specific human cardiomyocytes (CMs) are valuable for basic and applied research. Induction of cardiomyogenesis and enrichment of nodal-like CMs was described for mouse pluripotent stem cells (mPSCs) in response to 1-ethyl-2-benzimidazolinone (EBIO), a chemical modulator of small-/intermediate-conductance Ca 2+ -activated potassium channels (SKs 1-4). Investigating EBIO in human pluripotent stem cells (PSCs), we have applied three independent differentiation protocols of low to high cardiomyogenic efficiency. Equivalent to mPSCs, timed EBIO supplementation during hPSC differentiation resulted in dose-dependent enrichment of up to 80% CMs, including an increase in nodal- and atrial-like phenotypes. However, our study revealed extensive EBIO-triggered cell loss favoring cardiac progenitor preservation and, subsequently, CMs with shortened action potentials. Proliferative cells were generally more sensitive to EBIO, presumably via an SK-independent mechanism. Together, EBIO did not promote cardiogenic differentiation of PSCs, opposing previous findings, but triggered lineage-selective survival at a cardiac progenitor stage, which we propose as a pharmacological strategy to modulate CM subtype composition., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

In vitro differentiation of human pluripotent stem cells (hPSCs) recapitulates early aspects of human embryogenesis, but the underlying processes are poorly understood and controlled. Here we show that modulating the bulk cell density (BCD: cell number per culture volume) deterministically alters anteroposterior patterning of primitive streak (PS)-like priming. The BCD in conjunction with the chemical WNT pathway activator CHIR99021 results in distinct paracrine microenvironments codifying hPSCs towards definitive endoderm, precardiac or presomitic mesoderm within the first 24 h of differentiation, respectively. Global gene expression and secretome analysis reveals that TGFß superfamily members, antagonist of Nodal signalling LEFTY1 and CER1, are paracrine determinants restricting PS progression. These data result in a tangible model disclosing how hPSC-released factors deflect CHIR99021-induced lineage commitment over time. By demonstrating a decisive, functional role of the BCD, we show its utility as a method to control lineage-specific differentiation. Furthermore, these findings have profound consequences for inter-experimental comparability, reproducibility, bioprocess optimization and scale-up.

Objective: Fibroblast Growth Factor 23 (FGF23) may represent an attractive candidate that could participate to the osteoarthritic (OA)-induced phenotype switch of chondrocytes. To address this hypothesis, we investigated the expression of FGF23, its receptors (FGFRs) and co-receptor (Klotho) in human cartilage and studied the effects of rhFGF23 on OA chondrocytes., Method: Gene expression or protein levels were analysed by RT-PCR and immunohistochemistry. Collagenase 3 (MMP13) activity was measured by a fluorescent assay. MAPK signalling pathways were investigated by phosphoprotein array, immunoblotting and the use of selective inhibitors. RNA silencing was performed to confirm the respective contribution of FGFR1 and Klotho., Results: We showed that the expression of FGF23, FGFR1 and Klotho was up-regulated at both mRNA and protein levels in OA chondrocytes when compared to healthy ones. These overexpressions were markedly elevated in the damaged regions of OA cartilage. When stimulated with rhFGF23, OA chondrocytes displayed an extended expression of FGF23 and of markers of hypertrophy such as MMP13, COL10A1, and VEGF. We demonstrated that FGF23 auto-stimulation was both FGFR1-and Klotho-dependent, whereas the expression of markers of hypertrophy was mainly dependent on FGFR1 alone. Finally, we showed that FGF23-induced MMP13 expression was strongly regulated by the MEK/ERK cascade and to a lesser extent, by the PI-3K/AKT pathway., Conclusion: These results demonstrate that FGF23 sustains differentiation of OA chondrocytes in a Klotho-independent manner., (Copyright © 2016 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Human pluripotent stem cell (hPSC)-derived cardiomyocytes hold great potential for in vitro modeling of diseases like cardiomyopathies. Yet, knowledge about expression and functional impact of sarcomeric protein isoforms like the myosin heavy chain (MyHC) in hPSC-cardiomyocytes is scarce. We hypothesized that ventricular β-MyHC expression alters contraction and calcium kinetics and drives morphological and electrophysiological differentiation towards ventricular-like cardiomyocytes. To address this, we (1) generated human embryonic stem cell-derived cardiomyocytes (hESC-CMs) that switched towards exclusive β-MyHC, and (2) functionally and morphologically characterized these hESC-CMs at the single-cell level. MyHC-isoforms and functional properties were investigated during prolonged in vitro culture of cardiomyocytes in floating cardiac bodies (soft conditions) vs. culture on a stiff matrix. Using a specific anti-β-MyHC and a newly generated anti-α-MyHC-antibody, we found individual cardiomyocytes grown in cardiac bodies to mostly express both α- and β-MyHC-protein isoforms. Yet, 35 and 75 days of cultivation on laminin-coated glass switched 66 and 87 % of all cardiomyocytes to exclusively express β-MyHC, respectively. Twitch contraction and calcium transients were faster for CMs on laminin-glass. Surprisingly, both parameters were only little affected by the MyHC-isoform, although hESC-CMs with only β-MyHC had much lower ATP-turnover and tension cost, just as in human ventricular cardiomyocytes. Spontaneous contractions and no strict coupling of β-MyHC to ventricular-like action potentials suggest that MyHC-isoform expression does not fully determine the hESC-CM differentiation status. Stiff substrate-induced pure β-MyHC-protein expression in hESC-CMs, with several contractile parameters close to ventricular cardiomyocytes, provides a well-defined in vitro system for modeling of cardiomyopathies and drug screening approaches.

: The routine application of human pluripotent stem cells (hPSCs) and their derivatives in biomedicine and drug discovery will require the constant supply of high-quality cells by defined processes. Culturing hPSCs as cell-only aggregates in (three-dimensional [3D]) suspension has the potential to overcome numerous limitations of conventional surface-adherent (two-dimensional [2D]) cultivation. Utilizing single-use instrumented stirred-tank bioreactors, we showed that perfusion resulted in a more homogeneous culture environment and enabled superior cell densities of 2.85 × 10 6 cells per milliliter and 47% higher cell yields compared with conventional repeated batch cultures. Flow cytometry, quantitative reverse-transcriptase polymerase chain reaction, and global gene expression analysis revealed a high similarity across 3D suspension and 2D precultures, underscoring that matrix-free hPSC culture efficiently supports maintenance of pluripotency. Interestingly, physiological data and gene expression assessment indicated distinct changes of the cells' energy metabolism, suggesting a culture-induced switch from glycolysis to oxidative phosphorylation in the absence of hPSC differentiation. Our data highlight the plasticity of hPSCs' energy metabolism and provide clear physiological and molecular targets for process monitoring and further development. This study paves the way toward more efficient GMP-compliant cell production and underscores the enormous process development potential of hPSCs in suspension culture., Significance: Human pluripotent stem cells (hPSCs) are a unique source for the, in principle, unlimited production of functional human cell types in vitro, which are of high value for therapeutic and industrial applications. This study applied single-use, clinically compliant bioreactor technology to develop advanced, matrix-free, and more efficient culture conditions for the mass production of hPSCs in scalable suspension culture. Using extensive analytical tools to compare established conditions with this novel culture strategy, unexpected physiological features of hPSCs were discovered. These data allow a more rational process development, providing significant progress in the field of translational stem cell research and medicine., (©AlphaMed Press.)

Connective tissue calcifying diseases (CTCs) are characterized by abnormal calcium deposition in connective tissues. CTCs are caused by multiple factors including chronic diseases (Type II diabetes mellitus, chronic kidney disease), the use of pharmaceuticals (e.g. warfarin, glucocorticoids) and inherited rare genetic diseases such as pseudoxanthoma elasticum (PXE), generalized arterial calcification in infancy (GACI) and Keutel syndrome (KTLS). This review explores our current knowledge of these rare inherited CTCs, and highlights the most promising avenues for pharmaceutical intervention. Advancing our understanding of rare inherited forms of CTC is not only essential for the development of therapeutic strategies for patients suffering from these diseases, but also fundamental to delineating the mechanisms underpinning acquired chronic forms of CTC., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Nine Humboldt penguins ( Spheniscus humboldti ), between 1 and 1.5 years old and kept at Zoo Dresden, developed local and systemic infections with various opportunistic pathogens within a period of 4 months. Affected birds died peracutely without preceding symptoms or showed various clinical signs, including separation from conspecifics, reduced food intake, lethargy, dyspnea, swelling of the salt glands, and ocular discharge. One bird showed central nervous signs, including seizures. Pathologic examination of deceased birds revealed severe necrotizing inflammation of the mucous membranes and deep structures of the glottis, trachea, nasal sinus, and conchae and granulomatous inflammation of the salt glands. Further findings were airsacculitis, pneumonia, hepatitis, conjunctivitis, and myositis. Pseudomonas aeruginosa was the predominant pathogen in 7 cases. Six penguins died or were euthanatized, whereas 3 penguins that received systemic antibiotic treatment with tobramycin (10 mg/kg IM q24h for 10 days) showed rapid clinical improvement. Insufficient turnover rate of the filtration system, biofilm formation on pipe surfaces, and other factors are assumed to have promoted pathogen buildup in the pool water and subsequent infection.