Characterization of Tg(Etv4-GFP) and Etv5RFP Reporter Lines in the Context of Fibroblast Growth Factor 10 Signaling During Mouse Embryonic Lung Development

Members of the PEA3 transcription factors are emerging as bone fide targets for fibroblast growth factor (FGF) signaling. Among them, ETV4 and ETV5 appear to mediate FGF10 signaling during early embryonic lung development. In this paper, recently obtained Tg(Etv4-GFP) and Etv5CreERT2−RFP fluorescent reporter lines were generally characterized during early embryonic development and in the context of FGF10 signaling, in particular. We found that both Tg(Etv4-GFP) and Etv5CreERT2−RFP were primarily expressed in the epithelium of the lung during embryonic development. However, the expression of Etv5CreERT2−RFP was much higher than that of Tg(Etv4-GFP), and continued to increase during development, whereas Tg(Etv4-GFP) decreased. The expression patterns of the surrogate fluorescent protein GFP and RFP for ETV4 and ETV5, respectively, agreed with known regions of FGF10 signaling in various developing organs, including the lung, where ETV4-GFP was seen primarily in the distal epithelium and to a lesser extent in the surrounding mesenchyme. As expected, ETV5-RFP was restricted to the lung epithelium, showing a decreasing expression pattern from distal buds to proximal conducting airways. FGF10 inhibition experiments confirmed that both Etv4 and Etv5 are downstream of FGF10 signaling. Finally, we also validated that both fluorescent reporters responded to FGF10 inhibition in vitro. In conclusion, these two reporter lines appear to be promising tools to monitor FGF10/FGFR2b signaling in early lung development. These tools will have to be further validated at later stages and in other organs of interest

1,4-Bis(3-pyridylmethyl­eneamino­meth­yl)benzene

The title compound, C20H18N4, is a flexible 3,3′-bipyridyl-type ligand with a long spacer group between the two pyridyl functions. The mol­ecule crystallizes around an inversion center, with one half-mol­ecule in the asymmetric unit and a dihedral angle of 71.85 (8)° between the pyridine ring and the central benzene ring

Effective gene expression in the rat dorsal root ganglia with a non-viral vector delivered via spinal nerve injection

Delivering gene constructs into the dorsal root ganglia (DRG) is a powerful but challenging therapeutic strategy for sensory disorders affecting the DRG and their peripheral processes. The current delivery methods of direct intra-DRG injection and intrathecal injection have several disadvantages, including potential injury to DRG neurons and low transfection efficiency, respectively. This study aimed to develop a spinal nerve injection strategy to deliver polyethylenimine mixed with plasmid (PEI/DNA polyplexes) containing green fluorescent protein (GFP). Using this spinal nerve injection approach, PEI/DNA polyplexes were delivered to DRG neurons without nerve injury. Within one week of the delivery, GFP expression was detected in 82.8% ± 1.70% of DRG neurons, comparable to the levels obtained by intra-DRG injection (81.3% ± 5.1%, p = 0.82) but much higher than those obtained by intrathecal injection. The degree of GFP expression by neurofilament(+) and peripherin(+) DRG neurons was similar. The safety of this approach was documented by the absence of injury marker expression, including activation transcription factor 3 and ionized calcium binding adaptor molecule 1 for neurons and glia, respectively, as well as the absence of behavioral changes. These results demonstrated the efficacy and safety of delivering PEI/DNA polyplexes to DRG neurons via spinal nerve injection.National Science Council of Taiwan (100-2321-B-002-007)National Science Council of Taiwan (100-2320-B-002-083-MY3)Taiwan. Ministry of Science and Technology (104-2300-B-002-019-MY3)National Taiwan University. College of Medicine (Translational Medicine Project)National Taiwan University Hospital (101C101-201

Endoscopic Submucosal Dissection for Gastric Epithelial Tumors: A Multicenter Study in Taiwan

Background/PurposeEndoscopic submucosal dissection (ESD) is an advanced endoscopic procedure to resect early gastric cancer (EGC). The purpose of this study was to determine the effectiveness and complications of ESD for gastric epithelial tumors in Taiwan.MethodsWe retrospectively analyzed the efficacy and outcome of ESD in patients who received ESD for gastric epithelial tumors between June 2004 and August 2007.ResultsA total of 70 patients with gastric epithelial tumors were treated by ESD. The mean age was 66.5 ±12.9 years (range, 35–84 years). The mean size of the gastric epithelial tumors was 1.85 ± 0.81 cm. The mean size of resected specimens was 3.26 ± 1.39 cm. The one-piece resection rate was 91.4% (64/70). The median operation time was 92.4 minutes. The complicating bleeding and perforation rates were 5.7% (4/70) and 4.3% (3/70), respectively. Emergency surgery was performed for three patients with perforations. The local recurrence rate of gastric cancer was 2.8%. Except for one patient who died of congestive heart failure and another who died of stroke, the remaining 68 patients (97.1%) survived.ConclusionESD is a promising local curative treatment option for EGC in Taiwan but it still carries risks of perforation and bleeding. The education and learning curve of endoscopists will improve the outcome of this procedure

The Potentials and Caveats of Mesenchymal Stromal Cell-Based Therapies in the Preterm Infant

Preponderance of proinflammatory signals is a characteristic feature of all acute and resulting long-term morbidities of the preterm infant. The proinflammatory actions are best characterized for bronchopulmonary dysplasia (BPD) which is the chronic lung disease of the preterm infant with lifelong restrictions of pulmonary function and severe consequences for psychomotor development and quality of life. Besides BPD, the immature brain, eye, and gut are also exposed to inflammatory injuries provoked by infection, mechanical ventilation, and oxygen toxicity. Despite the tremendous progress in the understanding of disease pathologies, therapeutic interventions with proven efficiency remain restricted to a few drug therapies with restricted therapeutic benefit, partially considerable side effects, and missing option of applicability to the inflamed brain. The therapeutic potential of mesenchymal stromal cells (MSCs)-also known as mesenchymal stem cells-has attracted much attention during the recent years due to their anti-inflammatory activities and their secretion of growth and development-promoting factors. Based on a molecular understanding, this review summarizes the positive actions of exogenous umbilical cord-derived MSCs on the immature lung and brain and the therapeutic potential of reprogramming resident MSCs. The pathomechanistic understanding of MSC actions from the animal model is complemented by the promising results from the first phase I clinical trials testing allogenic MSC transplantation from umbilical cord blood. Despite all the enthusiasm towards this new therapeutic option, the caveats and outstanding issues have to be critically evaluated before a broad introduction of MSC-based therapies

Comparative Safety of the BNT162b2 Messenger RNA COVID-19 Vaccine vs Other Approved Vaccines in Children Younger Than 5 Years.

Importance SARS-CoV-2 vaccines are authorized for use in most age groups. The safety of SARS-CoV-2 vaccines is unknown in children younger than 5 years. Objective To retrospectively evaluate the safety of the BNT162b2 vaccine used off-label in children younger than 5 years compared with the safety of non-SARS-CoV-2 vaccines in the same sample. Design, Setting, and Participants This investigator-initiated retrospective cohort study included parents or caregivers who registered children for SARS-CoV-2 vaccination in outpatient care facilities in Germany. The study was performed as an authenticated online survey. A total of 19 000 email addresses were contacted from vaccination registration databases between April 14 and May 9, 2022. Inclusion criteria were child age younger than 5 years at the first BNT162b2 vaccination and use of a correct authentication code to prove invitation. Exposures Off-label BNT162b2 vaccination and on-label non-SARS-CoV-2 vaccinations. Main Outcomes and Measures Reported short-term safety data of 1 to 3 doses of 3 to 10 μg BNT162b2 in children from birth to younger than 60 months are presented. Coprimary outcomes were the frequencies of 11 categories of symptoms after vaccination with bivariate analyses and regression models adjusting for age, sex, weight, and height. Results The study included 7806 children (median age, 3 years [IQR, 2-4 years]; 3824 [49.0%] female) who were followed up of for a mean (SD) of 91.4 (38.8) days since first BNT162b2 vaccination (survey response rate, 41.1%). A 10-μg dosage was more frequently associated with local injection-site symptoms compared with lower dosages. In the active-comparator analysis, the probability of any symptoms (odds ratio [OR], 1.62; 95% CI, 1.43-1.84), local symptoms (OR, 1.68; 95% CI, 1.38-2.05), musculoskeletal symptoms (OR, 2.55; 95% CI, 1.32-4.94), dermatologic symptoms (OR, 2.18; 95% CI, 10.7-4.45), or otolaryngologic symptoms (OR, 6.37; 95% CI, 1.50-27.09) were modestly elevated after BNT162b2 compared with non-SARS-CoV-2 vaccines, whereas the probabilities of general symptoms (OR, 0.77; 95% CI, 0.63-0.95) and fever (OR, 0.42; 95% CI, 0.32-0.55) were lower after BNT162b2. Symptoms requiring hospitalization (n = 10) were reported only at BNT162b2 dosages above 3 μg. Conclusions and Relevance In this cohort study, the symptoms reported after BNT162b2 administration were comparable overall to those for on-label non-SARS-CoV-2 vaccines in this cohort of children younger than 5 years. The present data may be used together with prospective licensure studies of BNT162b2 efficacy and safety and could help guide expert recommendations about BNT162b2 vaccinations in this age group

Calcium oscillations coordinate feather mesenchymal cell movement by SHH dependent modulation of gap junction networks

Collective cell migration mediates multiple tissue morphogenesis processes. Yet how multi-dimensional mesenchymal cell movements are coordinated remains mostly unknown. Here we report that coordinated mesenchymal cell migration during chicken feather elongation is accompanied by dynamic changes of bioelectric currents. Transcriptome profiling and functional assays implicate contributions from functional voltage-gated Ca^(2+) channels (VGCCs), Connexin-43 based gap junctions, and Ca^(2+) release activated Ca^(2+) (CRAC) channels. 4-Dimensional Ca^(2+) imaging reveals that the Sonic hedgehog-responsive mesenchymal cells display synchronized Ca^(2+) oscillations, which expand progressively in area during feather elongation. Inhibiting VGCCs, gap junctions, or Sonic hedgehog signaling alters the mesenchymal Ca^(2+) landscape, cell movement patterns and feather bud elongation. Ca^(2+) oscillations induced by cyclic activation of opto-cCRAC channels enhance feather bud elongation. Functional disruption experiments and promoter analysis implicate synergistic Hedgehog and WNT/β-Catenin signaling in activating Connexin-43 expression, establishing gap junction networks synchronizing the Ca^(2+) profile among cells, thereby coordinating cell movement patterns

A Comprehensive Analysis of Fibroblast Growth Factor Receptor 2b Signaling on Epithelial Tip Progenitor Cells During Early Mouse Lung Branching Morphogenesis

This study demonstrates that FGF10/FGFR2b signaling on distal epithelial progenitor cells, via ß-catenin/EP300, controls, through a comprehensive set of developmental genes, morphogenesis, and differentiation. Fibroblast growth factor (FGF) 10 signaling through FGF receptor 2b (FGFR2b) is mandatory during early lung development as the deletion of either the ligand or the receptor leads to lung agenesis. However, this drastic phenotype previously hampered characterization of the primary biological activities, immediate downstream targets and mechanisms of action. Through the use of a dominant negative transgenic mouse model (Rosa26rtTA; tet(o)sFgfr2b), we conditionally inhibited FGF10 signaling in vivo in E12.5 embryonic lungs via doxycycline IP injection to pregnant females, and in vitro by culturing control and experimental lungs with doxycycline. The impact on branching morphogenesis 9 h after doxycycline administration was analyzed by morphometry, fluorescence and electron microscopy. Gene arrays at 6 and 9 h following doxycycline administration were carried out. The relationship between FGF10 and ß-catenin signaling was also analyzed through in vitro experiments using IQ1, a pharmacological inhibitor of ß-catenin/EP300 transcriptional activity. Loss of FGF10 signaling did not impact proliferation or survival, but affected both adherens junctions (up-regulation of E-cadherin), and basement membrane organization (increased laminin). Gene arrays identified multiple direct targets of FGF10, including main transcription factors. Immunofluorescence showed a down-regulation of the distal epithelial marker SOX9 and mis-expression distally of the proximal marker SOX2. Staining for the transcriptionally-active form of ß-catenin showed a reduction in experimental vs. control lungs. In vitro experiments using IQ1 phenocopied the impacts of blocking FGF10. This study demonstrates that FGF10/FGFR2b signaling on distal epithelial progenitor cells via ß-catenin/EP300 controls, through a comprehensive set of developmental genes, cell adhesion, and differentiation

Thrombomodulin Regulates Keratinocyte Differentiation and Promotes Wound Healing

The membrane glycoprotein thrombomodulin (TM) has been implicated in keratinocyte differentiation and wound healing, but its specific function remains undetermined. The epidermis-specific TM knockout mice were generated to investigate the function of TM in these biological processes. Primary cultured keratinocytes obtained from TMlox/lox; K5-Cre mice, in which TM expression was abrogated, underwent abnormal differentiation in response to calcium induction. Poor epidermal differentiation, as evidenced by downregulation of the terminal differentiation markers loricrin and filaggrin, was observed in TMlox/lox; K5-Cre mice. Silencing TM expression in human epithelial cells impaired calcium-induced extracellular signal–regulated kinase pathway activation and subsequent keratinocyte differentiation. Compared with wild-type mice, the cell spreading area and wound closure rate were lower in keratinocytes from TMlox/lox; K5-Cre mice. In addition, the lower density of neovascularization and smaller area of hyperproliferative epithelium contributed to slower wound healing in TMlox/lox; K5-Cre mice than in wild-type mice. Local administration of recombinant TM (rTM) accelerated healing rates in the TM-null skin. These data suggest that TM has a critical role in skin differentiation and wound healing. Furthermore, rTM may hold therapeutic potential for the treatment of nonhealing chronic wounds