Human induced pluripotent stem cells generate light responsive retinal organoids with variable and nutrient dependent efficiency

The availability of in vitro models of the human retina in which to perform pharmacological and toxicological studies is an urgent and unmet need. An essential step for developing in vitro models of human retina is the ability to generate laminated, physiologically functional and light-responsive retinal organoids from renewable and patient specific sources. We investigated five different human induced pluripotent stem cell (iPSC) lines and showed a significant variability in their efficiency to generate retinal organoids. Despite this variability, by month 5 of differentiation, all iPSC-derived retinal organoids were able to generate light responses, albeit immature, comparable to the earliest light responses recorded from the neonatal mouse retina, close to the period of eye opening. All iPSC-derived retinal organoids exhibited at this time a well-formed outer nuclear like layer containing photoreceptors with inner segments, connecting cilium and outer like segments. The differentiation process was highly dependent on seeding cell density and nutrient availability determined by factorial experimental design. We adopted the differentiation protocol to a multiwell plate format which enhanced generation of retinal organoids with retinal pigmented epithelium (RPE) and improved ganglion cell development and the response to physiological stimuli. We tested the response of iPSC-derived retinal organoids to Moxifloxacin and showed that similarly to in vivo adult mouse retina, the primary affected cell types were photoreceptors. Together our data indicate that light responsive retinal organoids derived from carefully selected and differentiation efficient iPSC lines can be generated at the scale needed for pharmacology and drug screening purposes. © AlphaMed Press 2018

Additional file 6: of Comparative genomic analysis reveals the evolution and environmental adaptation strategies of vibrios

Figure S4. Neighbor-joining phylogenetic tree of the 20 vibrios genomes based on 16S rRNA gene. The number at each node denotes the bootstrap value based on 1000 replicates. S. denitrificans OS217 was used as the outgroup. Bar, 0.01 substitutions per site. (PDF 186 kb

Additional file 1: of Comparative genomic analysis reveals the evolution and environmental adaptation strategies of vibrios

Table S1. General features of the 20 complete genomes of Vibrio species analyzed. (DOCX 48 kb

Additional file 5: of Comparative genomic analysis reveals the evolution and environmental adaptation strategies of vibrios

Figure S3. Pan genome tree. The tree was created based on the presence or absence of gene clusters in the 20 complete Vibrio genomes. The number at each node denotes the bootstrap value based on 1000 replicates. The color red and yellow are corresponding to the core genome tree, suggesting the discrepancy between core and pan genome trees. (PDF 194 kb

Additional file 7: of Comparative genomic analysis reveals the evolution and environmental adaptation strategies of vibrios

Figure S5. Heatmap presentation of pairwise average nucleotide identity of the 19 Vibrio species with complete genomes. The genomes are hierarchical clustered according to the values of rows. The clusters present in blue and orange are corresponding to the core genome tree. (PDF 212 kb

Additional file 8: of Comparative genomic analysis reveals the evolution and environmental adaptation strategies of vibrios

Figure S6. Genes related to the chitin-degrading process in 20 vibrios with complete genomes. Each column indicates a chitin metabolism-related gene family, with the family name indicating the predicted function. The number in the box indicates the copy number of that gene family in the corresponding genome. (PDF 282 kb

Theory of Covalent Adsorbate Frontier Orbital Energies on Functionalized Light-Absorbing Semiconductor Surfaces

Functional hybrid interfaces between organic molecules and semiconductors are central to many emerging information and solar energy conversion technologies. Here we demonstrate a general, empirical parameter-free approach for computing and understanding frontier orbital energies – or redox levels – of a broad class of covalently bonded organic–semiconductor surfaces. We develop this framework in the context of specific density functional theory (DFT) and many-body perturbation theory calculations, within the GW approximation, of an exemplar interface, thiophene-functionalized silicon (111). Through detailed calculations taking into account structural and binding energetics of mixed-monolayers consisting of both covalently attached thiophene and hydrogen, chlorine, methyl, and other passivating groups, we quantify the impact of coverage, nonlocal polarization, and interface dipole effects on the alignment of the thiophene frontier orbital energies with the silicon band edges. For thiophene adsorbate frontier orbital energies, we observe significant corrections to standard DFT (∼1 eV), including large nonlocal electrostatic polarization effects (∼1.6 eV). Importantly, both results can be rationalized from knowledge of the electronic structure of the isolated thiophene molecule and silicon substrate systems. Silicon band edge energies are predicted to vary by more than 2.5 eV, while molecular orbital energies stay similar, with the different functional groups studied, suggesting the prospect of tuning energy alignment over a wide range for photoelectrochemistry and other applications

Lack of Bcr and Abr Promotes Hypoxia-Induced Pulmonary Hypertension in Mice

<div><h3>Background</h3><p>Bcr and Abr are GTPase activating proteins that specifically downregulate activity of the small GTPase Rac in restricted cell types <em>in vivo</em>. Rac1 is expressed in smooth muscle cells, a critical cell type involved in the pathogenesis of pulmonary hypertension. The molecular mechanisms that underlie hypoxia-associated pulmonary hypertension are not well-defined.</p> <h3>Methodology/Principal Findings</h3><p><em>Bcr</em> and <em>abr</em> null mutant mice were compared to wild type controls for the development of pulmonary hypertension after exposure to hypoxia. Also, pulmonary arterial smooth muscle cells from those mice were cultured in hypoxia and examined for proliferation, p38 activation and IL-6 production. Mice lacking Bcr or Abr exposed to hypoxia developed increased right ventricular pressure, hypertrophy and pulmonary vascular remodeling. Perivascular leukocyte infiltration in the lungs was increased, and under hypoxia <em>bcr−/−</em> and <em>abr−/−</em> macrophages generated more reactive oxygen species. Consistent with a contribution of inflammation and oxidative stress in pulmonary hypertension-associated vascular damage, Bcr and Abr-deficient animals showed elevated endothelial leakage after hypoxia exposure. Hypoxia-treated pulmonary arterial smooth muscle cells from Bcr- or Abr-deficient mice also proliferated faster than those of wild type mice. Moreover, activated Rac1, phosphorylated p38 and interleukin 6 were increased in these cells in the absence of Bcr or Abr. Inhibition of Rac1 activation with Z62954982, a novel Rac inhibitor, decreased proliferation, p38 phosphorylation and IL-6 levels in pulmonary arterial smooth muscle cells exposed to hypoxia.</p> <h3>Conclusions</h3><p>Bcr and Abr play a critical role in down-regulating hypoxia-induced pulmonary hypertension by deactivating Rac1 and, through this, reducing both oxidative stress generated by leukocytes as well as p38 phosphorylation, IL-6 production and proliferation of pulmonary arterial smooth muscle cells.</p> </div

Hypoxia-induced pulmonary vascular remodeling in <i>bcr−/−</i> and <i>abr−/−</i> mice.

<p><b>A,</b> Hematoxylin and eosin staining on representative lung specimens from <i>bcr−/−, abr−/−</i> and <i>wt</i> mice under normoxia or hypoxia. Note that the walls of the pulmonary arteries of the <i>bcr−/−</i> and <i>abr−/−</i> mice are remarkably thicker than those of the <i>wt</i> mice after hypoxia. Magnification, 200×. <b>B,</b> Quantification of changes in the pulmonary artery walls. Percent wall thickness was determined on H&E stained sections as described in Methods on nine vessels of comparable size per mouse, with 6 mice per genotype per condition. *p<0.05 compared with the same genotype at normoxia. # p<0.05 compared with WT mice in the same exposure condition. Bars, mean ± SD. <b>C,</b> Immunostaining with α-SMA antibodies on pulmonary vessels from representative normoxia or hypoxia-treated mice. <b>D,</b> Quantification of areas for α-SMA-positive cells. Areas of α-SMA-positive cells were calculated using ImageJ software as described in the Materials and Methods.</p

Struttura complessa di servizio urbano sull’asse flaminio a Roma: modalità di integrazione nell’ambito di un sistema cardo-decumano attrezzato, quale ipotesi alternativa al sistema direzionale tangenziale previsto dal PRG del 1962, 1972.

(1972, Contributo in rivista / Ricerca progettuale). “Struttura complessa di servizio urbano sull’asse flaminio a Roma: modalità di integrazione nell’ambito di un sistema cardo-decumano attrezzato, quale ipotesi alternativa al sistema direzionale tangenziale previsto dal PRG del 1962”, 1972. Un contributo (test dimostrativo, prova di studio, arrangiamento virtuale, esperimento di trasposizione, trasferimento di senso), formulato attraverso il riutilizzo e la trascrizione di due serie di elaborati estratte rispettivamente dalla Tesi di laurea di Luigi Calcagnile (figg. 5-10, pp. 80-82), e dalla Tesi di laurea di Rosalba Pillai (figg. 11-14, p. 83), traslocate e montate in libera successione, con una breve nota di presentazione (p. 79), inserito nel saggio critico di Antonino Terranova, “Note su alcune esperienze di composizione architettonica” (pp. 49-75/103), in Università degli Studi di Roma-Facoltà di Ingegneria, Rassegna dell’Istituto di Architettura e Urbanistica, a. VIII, n. 22-23 - agosto 1972