NF-κB drives epithelial-mesenchymal mechanisms of lung fibrosis in a translational lung cell model.

In the progression phase of idiopathic pulmonary fibrosis (IPF), the normal alveolar structure of the lung is lost and replaced by remodeled fibrotic tissue and by bronchiolized cystic airspaces. Although these are characteristic features of IPF, knowledge of specific interactions between these pathological processes is limited. Here, the interaction of lung epithelial and lung mesenchymal cells was investigated in a coculture model of human primary airway epithelial cells (EC) and lung fibroblasts (FB). Single-cell RNA sequencing revealed that the starting EC population was heterogenous and enriched for cells with a basal cell signature. Furthermore, fractions of the initial EC and FB populations adopted distinct pro-fibrotic cell differentiation states upon cocultivation, resembling specific cell populations that were previously identified in lungs of patients with IPF. Transcriptomic analysis revealed active NF-κB signaling early in the cocultured EC and FB, and the identified NF-κB expression signatures were found in "HAS1 High FB" and "PLIN2+ FB" populations from IPF patient lungs. Pharmacological blockade of NF-κB signaling attenuated specific phenotypic changes of EC and prevented FB-mediated interleukin-6, interleukin-8, and CXC chemokine ligand 6 cytokine secretion, as well as collagen α-1(I) chain and α-smooth muscle actin accumulation. Thus, we identified NF-κB as a potential mediator, linking epithelial pathobiology with fibrogenesis.

More than the oocyte source, egg donors as patients: a national picture of United States egg donors.

PURPOSE: To characterize national oocyte donation practice patterns from the perspective of individual donors rather than of recipients. METHODS: Retrospective cohort including all donor oocyte retrievals and transfers reported to SARTCORS in 2016 and 2017 in the USA. Primary outcomes include characteristics of oocyte donors and of donor oocyte cycles. Secondary outcomes include overall pregnancy rates, elective single embryo transfer (eSET) rates, and perinatal outcomes among donor oocyte recipient transfers. RESULTS: During the study period, 49,193 donor oocyte retrievals were performed, of which the largest proportion were in the Western US. For all reported retrievals, there were 17,099 unique donors, each of whom underwent an average of 2.4 retrievals (range 1-22). Average donor age was 26.3 years (range 18-48). On average, 24.6 oocytes (SD 12.4) were retrieved each cycle, ranging from 0 to 102. Among 37,657 donor oocyte recipient transfers, 20,159 (53.5%) involved eSET, and 17,725 (47.1%) resulted in live birth. Miscarriage rates were 17.5%, and good perinatal outcome (GPO), defined as full-term normal birthweight delivery, was more likely among singleton (75.7%) than multiple (23.8%) pregnancies. CONCLUSION: The average number of retrievals that donors underwent and oocyte yield mirrored national guidelines; however outliers, exist that may unnecessarily increase donor risk. Additionally, among resultant donor transfers, 46.5% transferred more than one embryo despite national recommendations for eSET. The significantly higher likelihood of GPO among singleton pregnancies points to the need to further increase donor recipient eSET rates.

Novel high-throughput myofibroblast assays identify agonists with therapeutic potential in pulmonary fibrosis that act via EP2 and EP4 receptors.

Pathological features of pulmonary fibrosis include accumulation of myofibroblasts and increased extracellular matrix (ECM) deposition in lung tissue. Contractile α-smooth muscle actin (α-SMA)-expressing myofibroblasts that produce and secrete ECM are key effector cells of the disease and therefore represent a viable target for potential novel anti-fibrotic treatments. We used primary normal human lung fibroblasts (NHLF) in two novel high-throughput screening assays to discover molecules that inhibit or revert fibroblast-to-myofibroblast differentiation. A phenotypic high-content assay (HCA) quantified the degree of myofibroblast differentiation, whereas an impedance-based assay, multiplexed with MS / MS quantification of α-SMA and collagen 1 alpha 1 (COL1) protein, provided a measure of contractility and ECM formation. The synthetic prostaglandin E1 (PGE1) alprostadil, which very effectively and potently attenuated and even reversed TGF-ß1-induced myofibroblast differentiation, was identified by screening a library of approved drugs. In TGF-ß1-induced myofibroblasts the effect of alprostadil was attributed to activation of prostanoid receptor 2 and 4 (EP2 and EP4, respectively). However, selective activation of the EP2 or the EP4 receptor was already sufficient to prevent or reverse TGF-ß1-induced NHLF myofibroblast transition. Our high-throughput assays identified chemical structures with potent anti-fibrotic properties acting through potentially novel mechanisms.

Serotonin biosynthesis as a predictive marker of serotonin pharmacodynamics and disease-induced dysregulation.

The biogenic amine serotonin (5-HT) is a multi-faceted hormone that is synthesized from dietary tryptophan with the rate limiting step being catalyzed by the enzyme tryptophan hydroxylase (TPH). The therapeutic potential of peripheral 5-HT synthesis inhibitors has been demonstrated in a number of clinical and pre-clinical studies in diseases including carcinoid syndrome, lung fibrosis, ulcerative colitis and obesity. Due to the long half-life of 5-HT in blood and lung, changes in steady-state levels are slow to manifest themselves. Here, the administration of stable isotope labeled tryptophan (heavy "h-Trp") and resultant in vivo conversion to h-5-HT is used to monitor 5-HT synthesis in rats. Dose responses for the blockade of h-5-HT appearance in blood with the TPH inhibitors L-para-chlorophenylalanine (30 and 100 mg/kg) and telotristat etiprate (6, 20 and 60 mg/kg), demonstrated that the method enables robust quantification of pharmacodynamic effects on a short time-scale, opening the possibility for rapid screening of TPH1 inhibitors in vivo. In the bleomycin-induced lung fibrosis rat model, the mechanism of lung 5-HT increase was investigated using a combination of synthesis and steady state 5-HT measurement. Elevated 5-HT synthesis measured in the injured lungs was an early predictor of disease induced increases in total 5-HT.

Novel S1P1 receptor agonists - Part 4: Alkylaminomethyl substituted aryl head groups.

In a previous communication we reported on the discovery of alkylamino pyridine derivatives (e.g. 1) as a new class of potent, selective and efficacious S1P1 receptor (S1PR1) agonists. However, more detailed profiling revealed that this compound class is phototoxic in vitro. Here we describe a new class of potent S1PR1 agonists wherein the exocyclic nitrogen was moved away from the pyridine ring (e.g. 11c). Further structural modifications led to the identification of novel alkylaminomethyl substituted phenyl and thienyl derivatives as potent S1PR1 agonists. These new alkylaminomethyl aryl compounds showed no phototoxic potential. Based on their in vivo efficacy and ability to penetrate the brain, the 5-alkyl-aminomethyl thiophenes appeared to be the most interesting class. Potent and selective S1PR1 agonist 20e, for instance, maximally reduced the blood lymphocyte count (LC) for 24 h after oral administration of 10 mg/kg to rat and its brain concentrations reached >500 ng/g over 24 h.

Novel S1P1 receptor agonists - Part 5: From amino-to alkoxy-pyridines.

In a previous communication we reported on the discovery of aminopyridine 1 as a potent, selective and orally active S1P1 receptor agonist. More detailed studies revealed that this compound is phototoxic in vitro. As a result of efforts aiming at eliminating this undesired property, a series of alkoxy substituted pyridine derivatives was discovered. The photo irritancy factor (PIF) of these alkoxy pyridines was significantly lower than the one of aminopyridine 1 and most compounds were not phototoxic. Focused SAR studies showed, that 2-, 3-, and 4-pyridine derivatives delivered highly potent S1P1 receptor agonists. While the 2-pyridines were clearly more selective against S1PR3, the corresponding 3- or 4-pyridine analogues showed significantly longer oral half-lives and as a consequence longer pharmacological duration of action after oral administration. One of the best compounds, cyclopentoxy-pyridine 45b lacked phototoxicity, showed EC50 values of 0.7 and 140 nM on S1PR1 and S1PR3, respectively, and maximally reduced the blood lymphocyte count for at least 24 h after oral administration of 10 mg/kg to Wistar rats.

A Genetic Screen for Mutations Affecting Cell Division in the Arabidopsis thaliana Embryo Identifies Seven Loci Required for Cytokinesis.

Cytokinesis in plants involves the formation of unique cellular structures such as the phragmoplast and the cell plate, both of which are required to divide the cell after nuclear division. In order to isolate genes that are involved in de novo cell wall formation, we performed a large-scale, microscope-based screen for Arabidopsis mutants that severely impair cytokinesis in the embryo. We recovered 35 mutations that form abnormally enlarged cells with multiple, often polyploid nuclei and incomplete cell walls. These mutants represent seven genes, four of which have previously been implicated in phragmoplast or cell plate function. Mutations in two loci show strongly reduced transmission through the haploid gametophytic generation. Molecular cloning of both corresponding genes reveals that one is represented by hypomorphic alleles of the kinesin-5 gene RADIALLY SWOLLEN 7 (homologous to tobacco kinesin-related protein TKRP125), and that the other gene corresponds to the Arabidopsis FUSED ortholog TWO-IN-ONE (originally identified based on its function in pollen development). No mutations that completely abolish the formation of cross walls in diploid cells were found. Our results support the idea that cytokinesis in the diploid and haploid generations involve similar mechanisms.

Redundancy and specialization among plant microRNAs: role of the MIR164 family in developmental robustness.

In plants, members of microRNA (miRNA) families are often predicted to target the same or overlapping sets of genes. It has thus been hypothesized that these miRNAs may act in a functionally redundant manner. This hypothesis is tested here by studying the effects of elimination of all three members of the MIR164 family from Arabidopsis. It was found that a loss of miR164 activity leads to a severe disruption of shoot development, in contrast to the effect of mutation in any single MIR164 gene. This indicates that these miRNAs are indeed functionally redundant. Differences in the expression patterns of the individual MIR164 genes imply, however, that redundancy among them is not complete, and that these miRNAs show functional specialization. Furthermore, the results of molecular and genetic analyses of miR164-mediated target regulation indicate that miR164 miRNAs function to control the transcript levels, as well as the expression patterns, of their targets, suggesting that they might contribute to developmental robustness. For two of the miR164 targets, namely CUP-SHAPED COTYLEDON1 (CUC1) and CUC2, we provide evidence for their involvement in the regulation of growth and show that their derepression in miR164 loss-of-function mutants is likely to account for most of the mutant phenotype.

Patterns of auxin transport and gene expression during primordium development revealed by live imaging of the Arabidopsis inflorescence meristem.

BACKGROUND: Plants produce leaf and flower primordia from a specialized tissue called the shoot apical meristem (SAM). Genetic studies have identified a large number of genes that affect various aspects of primordium development including positioning, growth, and differentiation. So far, however, a detailed understanding of the spatio-temporal sequence of events leading to primordium development has not been established. RESULTS: We use confocal imaging of green fluorescent protein (GFP) reporter genes in living plants to monitor the expression patterns of multiple proteins and genes involved in flower primordial developmental processes. By monitoring the expression and polarity of PINFORMED1 (PIN1), the auxin efflux facilitator, and the expression of the auxin-responsive reporter DR5, we reveal stereotypical PIN1 polarity changes which, together with auxin induction experiments, suggest that cycles of auxin build-up and depletion accompany, and may direct, different stages of primordium development. Imaging of multiple GFP-protein fusions shows that these dynamics also correlate with the specification of primordial boundary domains, organ polarity axes, and the sites of floral meristem initiation. CONCLUSIONS: These results provide new insight into auxin transport dynamics during primordial positioning and suggest a role for auxin transport in influencing primordial cell type.

The early extra petals1 mutant uncovers a role for microRNA miR164c in regulating petal number in Arabidopsis.

BACKGROUND: MicroRNAs (miRNAs) are small 20-25 nucleotide non-protein-coding RNAs that negatively regulate expression of genes in many organisms, ranging from plants to humans. The MIR164 family of miRNAs in Arabidopsis consists of three members that share sequence complementarity to transcripts of NAC family transcription factors, including CUP-SHAPED COTYLEDON1 (CUC1) and CUC2. CUC1 and CUC2 are redundantly required for the formation of boundaries between organ primordia. The analysis of transgenic plants that either overexpress miR164a or miR164b or express a miRNA-resistant version of CUC1 or CUC2 has shown that miRNA regulation of CUC1 and CUC2 is necessary for normal flower development. A loss-of-function allele of MIR164b did not result in a mutant phenotype, possibly because of functional redundancy among the three members of the MIR164 family. RESULTS: In this study, we describe the characterization of the early extra petals1 (eep1) Arabidopsis mutant, whose predominant phenotype is the formation of extra petals in early-arising flowers. We demonstrate that eep1 is a loss-of-function allele of MIR164c, one of three known members of the MIR164 family. Our analyses of miR164c function and eep1 mir164b double mutants reveal that miR164c controls petal number in a nonredundant manner by regulating the transcript accumulation of the transcription factors CUC1 and CUC2. CONCLUSIONS: The data presented in this study indicate that closely related miRNA family members that are predicted to target the same set of genes can have different functions during development, possibly because of nonoverlapping expression patterns.

The gravitropism defective 2 mutants of Arabidopsis are deficient in a protein implicated in endocytosis in Caenorhabditis elegans.

The gravitropism defective 2 (grv2) mutants of Arabidopsis show reduced shoot phototropism and gravitropism. Amyloplasts in the shoot endodermal cells of grv2 do not sediment to the same degree as in wild type. The GRV2 gene encodes a 277-kD polypeptide that is 42% similar to the Caenorhabditis elegans RME-8 protein, which is required for endocytosis. We hypothesize that a defect in endocytosis may affect both the initial gravity sensing via amyloplasts sedimentation and the subsequent more general tropic growth response.

Organ polarity in Arabidopsis. NOZZLE physically interacts with members of the YABBY family.

Plant lateral organs exhibit proximal-distal and adaxial-abaxial polarity. In Arabidopsis, abaxial cell fate is regulated in part by putative transcription factors of the YABBY family, such as FILAMENTOUS FLOWER (FIL) and INNER NO OUTER (INO), by a mechanism that currently is not fully understood. NOZZLE (NZZ) encodes a plant-specific nuclear protein. Genetic evidence has shown that NZZ is involved in the positive feedback regulation of INO, thereby acting both as a temporal and spatial repressor of INO transcription. This mechanism allows the ovule primordium to complete its proximal-distal organization, prior to the onset of adaxial-abaxial development in the chalaza. During our study, we isolated FIL in a yeast two-hybrid screen using NZZ as bait. In vitro pull-down experiments confirmed the NZZ-FIL interaction. NZZ also bound INO and YABBY3, suggesting that NZZ generally interacts with YABBY proteins in vitro. The polar-charged region of NZZ was necessary and sufficient to bind to the zinc finger of INO and to interact with its C terminus carrying the high mobility group-like domain. We suggest that NZZ coordinates proximal-distal patterning and adaxial-abaxial polarity establishment in the developing ovule by directly binding to INO.

Pattern formation during early ovule development in Arabidopsis thaliana.

Ovules of higher plants are the precursors of seeds. Ovules emerge from placental tissue inside the gynoecium of flowers. Three elements, funiculus, chalaza, and nucellus, can be distinguished along the proximal-distal axis of the outgrowing radially symmetrical ovule primordium. The asymmetric initiation of the outer integument marks the switch to adaxial-abaxial development, which leads to the formation of a bilaterally symmetrical ovule. The putative transcriptional regulator NOZZLE (NZZ) plays a role in mediating this transition by controlling the timing of expression of the putative transcriptional regulator INNER NO OUTER (INO) in an abaxial domain of the chalaza, from where the outer integument initiates. Integument formation depends on the homeobox gene WUSCHEL (WUS), which is expressed in the nucellus and is sufficient to induce integuments non-cell autonomously from a region adjacent to its expression domain. In this study, we describe the expression pattern of the homeobox-leucine zipper gene PHABULOSA (PHB) during ovule development, demonstrating that adaxial-abaxial polarity is established from the very beginning of ovule development. Furthermore, we examined the expression pattern of PHB, INO, and WUS in ovules of plants, which are affected in integument initiation and thus defective in the transition from proximal-distal to adaxial-abaxial development. We found that NZZ is required to restrict PHB expression to the distal chalaza, from where the inner integument initiates. PHB expression is not established in the distal chalaza of two mutants, aintegumenta (ant) and wus, which fail to form integuments. Furthermore, we suggest that one mechanism by which WUS controls integument formation is by establishing the chalaza and that outer and inner integument identity determination depends on additional region-specific factors. In addition, we present evidence that NZZ is essential for the normal nucellar expression pattern of WUS. Thus, both WUS and PHB affect processes downstream of NZZ action during the transition from proximal--distal to adaxial--abaxial ovule development.