One of the biggest challenges in probing the atmospheric boundary layer with small unmanned aerial vehicles is the turbulent 3D wind vector measurement. Several approaches have been developed to ...estimate the wind vector without using multi-hole flow probes. This study compares commonly used wind speed and direction estimation algorithms with the direct 3D wind vector measurement using multi-hole probes. This was done using the data of a fully equipped system and by applying several algorithms to the same data set. To cover as many aspects as possible, a wide range of meteorological conditions and common flight patterns were considered in this comparison. The results from the five-hole probe measurements were compared to the pitot tube algorithm, which only requires a pitot-static tube and a standard inertial navigation system measuring aircraft attitude (Euler angles), while the position is measured with global navigation satellite systems. Even less complex is the so-called no-flow-sensor algorithm, which only requires a global navigation satellite system to estimate wind speed and wind direction. These algorithms require temporal averaging. Two averaging periods were applied in order to see the influence and show the limitations of each algorithm. For a window of 4 min, both simplifications work well, especially with the pitot-static tube measurement. When reducing the averaging period to 1 min and thereby increasing the temporal resolution, it becomes evident that only circular flight patterns with full racetracks inside the averaging window are applicable for the no-flow-sensor algorithm and that the additional flow information from the pitot-static tube improves precision significantly.
The use of human pluripotent stem cells (hPSCs) in cell therapy is hindered by the tumorigenic risk from residual undifferentiated cells. Here we performed a high-throughput screen of over 52,000 ...small molecules and identified 15 pluripotent cell-specific inhibitors (PluriSIns), nine of which share a common structural moiety. The PluriSIns selectively eliminated hPSCs while sparing a large array of progenitor and differentiated cells. Cellular and molecular analyses demonstrated that the most selective compound, PluriSIn #1, induces ER stress, protein synthesis attenuation, and apoptosis in hPSCs. Close examination identified this molecule as an inhibitor of stearoyl-coA desaturase (SCD1), the key enzyme in oleic acid biosynthesis, revealing a unique role for lipid metabolism in hPSCs. PluriSIn #1 was also cytotoxic to mouse blastocysts, indicating that the dependence on oleate is inherent to the pluripotent state. Finally, application of PluriSIn #1 prevented teratoma formation from tumorigenic undifferentiated cells. These findings should increase the safety of hPSC-based treatments.
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► High-throughput screen identifies selective cytotoxic inhibitors of hPSCs ► The most potent and selective compound inhibits stearoyl-coA desaturase (SCD1) ► Pluripotent cells uniquely depend on oleate metabolism for their viability ► SCD1 inhibition rapidly and robustly eliminates undifferentiated cells from culture
A high-throughput screen identifies a specific role for oleate metabolism in human pluripotent cells, and a selective small molecule-based route for their elimination.
Little is known about the role of islet delta cells in regulating blood glucose homeostasis in vivo. Delta cells are important paracrine regulators of beta cell and alpha cell secretory activity, ...however the structural basis underlying this regulation has yet to be determined. Most delta cells are elongated and have a well-defined cell soma and a filopodia-like structure. Using in vivo optogenetics and high-speed Ca
imaging, we show that these filopodia are dynamic structures that contain a secretory machinery, enabling the delta cell to reach a large number of beta cells within the islet. This provides for efficient regulation of beta cell activity and is modulated by endogenous IGF-1/VEGF-A signaling. In pre-diabetes, delta cells undergo morphological changes that may be a compensation to maintain paracrine regulation of the beta cell. Our data provides an integrated picture of how delta cells can modulate beta cell activity under physiological conditions.
Familial adenomatous polyposis (FAP) is a precancerous, colorectal disease characterized by hundreds to thousands of adenomatous polyps caused by mutations in the tumor suppressor gene adenomatous ...polyposis coli (APC). Approximately 30% of these mutations are premature termination codons (PTC), resulting in the production of a truncated, dysfunctional APC protein. Consequently, the β-catenin degradation complex fails to form in the cytoplasm, leading to elevated nuclear levels of β-catenin and unregulated β-catenin/wnt-pathway signaling. We present in vitro and in vivo data demonstrating that the novel macrolide, ZKN-0013, promotes read through of premature stop codons, leading to functional restoration of full-length APC protein. Human colorectal carcinoma SW403 and SW1417 cells harboring PTC mutations in the APC gene showed reduced levels of nuclear β-catenin and c-myc upon treatment with ZKN-0013, indicating that the macrolide-mediated read through of premature stop codons produced bioactive APC protein and inhibited the β-catenin/wnt-pathway. In a mouse model of adenomatous polyposis coli, treatment of APC
min
mice with ZKN-0013 caused a significant decrease in intestinal polyps, adenomas, and associated anemia, resulting in increased survival. Immunohistochemistry revealed decreased nuclear β-catenin staining in the epithelial cells of the polyps in ZKN-0013-treated APC
min
mice, confirming the impact on the β-catenin/wnt-pathway. These results indicate that ZKN-0013 may have therapeutic potential for the treatment of FAP caused by nonsense mutations in the APC gene.
Key messages
• ZKN-0013 inhibited the growth of human colon carcinoma cells with APC nonsense mutations.
• ZKN-0013 promoted read through of premature stop codons in the APC gene.
• In APC
min
mice, ZKN-0013 treatment reduced intestinal polyps and their progression to adenomas.
• ZKN-0013 treatment in APC
min
mice resulted in reduced anemia and increased survival.
The blood brain barrier (BBB) has evolved unique characteristics such as dense coverage of the endothelial cells by pericytes and interactions with astrocytes through perivascular endfeet. We study ...BBB formation in a 3-dimensional multicellular spheroid system of human primary brain endothelial cells (hpBECs), primary pericytes (hpPs) and primary astrocytes (hpAs). We show for the first time that hpBECs, hpPs and hpAs spontaneously self-organize into a defined multicellular structure which recapitulates the complex arrangement of the individual cell types in the BBB structure. Pericytes play a crucial role mediating the interaction between hpBECs and hpAs. This process is not dependent on a scaffold support demonstrating that formation and cellular architecture of the BBB is intrinsically programmed within each specific cell type. In a matrigel setup the hpBECs, hpPs and hpAs also undergo self-arrangement to form endothelial tube-like structures tightly covered by hpPs and loosely attached hpAs mainly at the junctions.
Abstract In rats bearing an intracranial T9 glioma, immunization with tumor antigens induces myeloid suppressor cells, which express neutrophil (His48) and monocyte (CD11bc) markers, to infiltrate ...the tumors. The His48+ /CD11bc+ cells were not derived from CNS microglia but were hematogenous; suppressed multiple T cell effector functions; and are myeloid-derived suppressor cells (MDSC). The glioma-infiltrating MDSC expressed arginase I, iNOS, indoleamine 2,3-dioxygenase and TGF-β; however, inhibitor/blocking studies demonstrated that NO production was the primary mechanism of suppression which induced T cell apoptosis. These findings suggest that neuro-immunomodulation by MDSC in rat gliomas maybe mediated by a pathway requiring NO production.
The blood–retina barrier and blood–brain barrier (BRB/BBB) are selective and semipermeable and are critical for supporting and protecting central nervous system (CNS)-resident cells. Endothelial ...cells (ECs) within the BRB/BBB are tightly coupled, express high levels of Claudin-5 (CLDN5), a junctional protein that stabilizes ECs, and are important for proper neuronal function. To identify novel CLDN5 regulators (and ultimately EC stabilizers), we generated a CLDN5-P2A-GFP stable cell line from human pluripotent stem cells (hPSCs), directed their differentiation to ECs (CLDN5-GFP hPSC-ECs), and performed flow cytometry-based chemogenomic library screening to measure GFP expression as a surrogate reporter of barrier integrity. Using this approach, we identified 62 unique compounds that activated CLDN5-GFP. Among them were TGF-β pathway inhibitors, including RepSox. When applied to hPSC-ECs, primary brain ECs, and retinal ECs, RepSox strongly elevated barrier resistance (transendothelial electrical resistance), reduced paracellular permeability (fluorescein isothiocyanate-dextran), and prevented vascular endothelial growth factor A (VEGFA)-induced barrier breakdown in vitro. RepSox also altered vascular patterning in themouse retina during developmentwhen delivered exogenously. To determine the mechanism of action of RepSox, we performed kinome-, transcriptome-, and proteome-profiling and discovered that RepSox inhibited TGF-β, VEGFA, and inflammatory gene networks. In addition, RepSox not only activated vascular-stabilizing and barrier-establishing Notch and Wnt pathways, but also induced expression of important tight junctions and transporters. Taken together, our data suggest that inhibitingmultiple pathways by selected individual small molecules, such as RepSox, may be an effective strategy for the development of better BRB/BBB models and novel EC barrier-inducing therapeutics.
Hyperfunction of the mTORC1 pathway has been associated with idiopathic and syndromic forms of autism spectrum disorder (ASD), including tuberous sclerosis, caused by loss of either TSC1 or TSC2. It ...remains largely unknown how developmental processes and biochemical signaling affected by mTORC1 dysregulation contribute to human neuronal dysfunction. Here, we have characterized multiple stages of neurogenesis and synapse formation in human neurons derived from TSC2-deleted pluripotent stem cells. Homozygous TSC2 deletion causes severe developmental abnormalities that recapitulate pathological hallmarks of cortical malformations in patients. Both TSC2+/− and TSC2−/− neurons display altered synaptic transmission paralleled by molecular changes in pathways associated with autism, suggesting the convergence of pathological mechanisms in ASD. Pharmacological inhibition of mTORC1 corrects developmental abnormalities and synaptic dysfunction during independent developmental stages. Our results uncouple stage-specific roles of mTORC1 in human neuronal development and contribute to a better understanding of the onset of neuronal pathophysiology in tuberous sclerosis.
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•TSC2 deletion causes gene-dosage-dependent mTORC1 hyperactivity in neurodevelopment•TSC2 deletion causes alterations in pathways and genes associated with autism•TSC2+/− and TSC2−/− human neurons show stage-specific cellular and synaptic defects•mTORC1 inhibition corrects synaptic defects independently of early neurodevelopment
The neurodevelopmental disorder tuberous sclerosis is caused by loss of TSC1/2, negative regulators of mTORC1. Combining genome editing technology and human stem cell differentiation, Costa et al. characterize stage-specific molecular, cellular, and synaptic alterations in neurons with TSC2 deletion and show that mTORC1 inhibition corrects defects in synaptic function independently of early neurodevelopmental abnormalities.
Spinal muscular atrophy (SMA) is a progressive neurodegenerative disease affecting lower motor neurons. SMA is caused by mutations in the Survival Motor Neuron 1 (SMN1) gene, which result in reduced ...levels of functional SMN protein. Biochemical studies have linked the ubiquitously expressed SMN protein to the assembly of pre-mRNA processing U snRNPs, raising the possibility that aberrant splicing is a major defect in SMA. Accordingly, several transcripts affected upon SMN deficiency have been reported. A second function for SMN in axonal mRNA transport has also been proposed that may likewise contribute to the SMA phenotype. The underlying etiology of SMA, however, is still not fully understood. Here, we have used a combination of genomics and live Ca(2+) imaging to investigate the consequences of SMN deficiency in a zebrafish model of SMA. In a transcriptome analyses of SMN-deficient zebrafish, we identified neurexin2a (nrxn2a) as strongly down-regulated and displaying changes in alternative splicing patterns. Importantly, the knock-down of two distinct nrxn2a isoforms phenocopies SMN-deficient fish and results in a significant reduction of motor axon excitability. Interestingly, we observed altered expression and splicing of Nrxn2 also in motor neurons from the Smn(-/-);SMN2(+/+) mouse model of SMA, suggesting conservation of nrxn2 regulation by SMN in mammals. We propose that SMN deficiency affects splicing and abundance of nrxn2a. This may explain the pre-synaptic defects at neuromuscular endplates in SMA pathophysiology.
Human induced pluripotent stem cells (hiPSCs) are invaluable to study developmental processes and disease mechanisms particularly in the brain. hiPSCs can be differentiated into mature and functional ...dopaminergic (DA) neurons. Having robust protocols for the generation of differentiated DA neurons from pluripotent cells is a prerequisite for the use of hiPSCs to study disease mechanisms, for drug discovery, and eventually for cell replacement therapy. Here, we describe a protocol for generating and expanding large numbers of homogeneous midbrain floor plate progenitors (mFPPs) that retain efficient DA neurogenic potential over multiple passages and can be cryobanked. We demonstrate that expanded mFPPs have increased DA neuron potential and differentiate more efficiently and rapidly than progenitors generated by standard protocols. In addition, this novel method results in increased numbers of DA neurons that in vitro show characteristic electrophysiological properties of nigrostriatal DA neurons, produce high levels of dopamine, and integrate into host mice when grafted in vivo. Thus, we describe a robust method for producing human mesencephalic DA neurons from hiPSCs.
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