A single-cell atlas of mouse lung development.

Lung organogenesis requires precise timing and coordination to effect spatial organization and function of the parenchymal cells. To provide a systematic broad-based view of the mechanisms governing the dynamic alterations in parenchymal cells over crucial periods of development, we performed a single-cell RNA-sequencing time-series yielding 102,571 epithelial, endothelial and mesenchymal cells across nine time points from embryonic day 12 to postnatal day 14 in mice. Combining computational fate-likelihood prediction with RNA in situ hybridization and immunofluorescence, we explore lineage relationships during the saccular to alveolar stage transition. The utility of this publicly searchable atlas resource (www.sucrelab.org/lungcells) is exemplified by discoveries of the complexity of type 1 pneumocyte function and characterization of mesenchymal Wnt expression patterns during the saccular and alveolar stages - wherein major expansion of the gas-exchange surface occurs. We provide an integrated view of cellular dynamics in epithelial, endothelial and mesenchymal cell populations during lung organogenesis.

Age-determined expression of priming protease TMPRSS2 and localization of SARS-CoV-2 in lung epithelium.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) novel coronavirus 2019 (COVID-19) global pandemic has led to millions of cases and hundreds of thousands of deaths. While older adults appear at high risk for severe disease, hospitalizations and deaths due to SARS-CoV-2 among children have been relatively rare. Integrating single-cell RNA sequencing (scRNA-seq) of developing mouse lung with temporally resolved immunofluorescence in mouse and human lung tissue, we found that expression of SARS-CoV-2 Spike protein primer TMPRSS2 was highest in ciliated cells and type I alveolar epithelial cells (AT1), and TMPRSS2 expression increased with aging in mice and humans. Analysis of autopsy tissue from fatal COVID-19 cases detected SARS-CoV-2 RNA most frequently in ciliated and secretory cells in airway epithelium and AT1 cells in peripheral lung. SARS-CoV-2 RNA was highly colocalized in cells expressing TMPRSS2. Together, these data demonstrate the cellular spectrum infected by SARS-CoV-2 in lung epithelium and suggest that developmental regulation of TMPRSS2 may underlie the relative protection of infants and children from severe respiratory illness.

Age-determined expression of priming protease TMPRSS2 and localization of SARS-CoV-2 infection in the lung epithelium.

The SARS-CoV-2 novel coronavirus global pandemic (COVID-19) has led to millions of cases and hundreds of thousands of deaths around the globe. While the elderly appear at high risk for severe disease, hospitalizations and deaths due to SARS-CoV-2 among children have been relatively rare. Integrating single-cell RNA sequencing (scRNA-seq) of the developing mouse lung with temporally-resolved RNA-in-situ hybridization (ISH) in mouse and human lung tissue, we found that expression of SARS-CoV-2 Spike protein primer TMPRSS2 was highest in ciliated cells and type I alveolar epithelial cells (AT1), and TMPRSS2 expression was increased with aging in mice and humans. Analysis of autopsy tissue from fatal COVID-19 cases revealed SARS-CoV-2 RNA was detected most frequently in ciliated and secretory cells in the airway epithelium and AT1 cells in the peripheral lung. SARS-CoV-2 RNA was highly colocalized in cells expressing TMPRSS2. Together, these data demonstrate the cellular spectrum infected by SARS-CoV-2 in the lung epithelium, and suggest that developmental regulation of TMPRSS2 may underlie the relative protection of infants and children from severe respiratory illness.

Efficacy and safety of 5-mm-diameter bipolar and ultrasonic shears for cutting carotid arteries of the hybrid pig.

BACKGROUND: Experimental data about the efficacy and safety of sealing devices are rare. Therefore, this study investigated these parameters for three commercially available energy-based vascular sealing and cutting systems. METHODS: In male hybrid pigs, 487 carotid artery segments were sealed and cut using the harmonic scalpel or several bipolar sealing devices. The sealing failure rate, burst pressure, process time, and extent of lateral thermal damage were analyzed. RESULTS: A regular sealing and cutting process in more than 90% of the carotid arteries was found using the following instruments: LS1520, ACE (level 1), ACE (level 3), CS14C (level 1), WAVE (level 1), and WAVE (level 5). The largest failure rate was found for the CS14C device (level 5: initial sealing failure, 21.5%). The maximal mean burst pressure (1727±453 mmHg) was reached using the ACE device (level 1). Significant differences were found in the size of the lateral thermal damage, which a ranged from 2.5 mm (LS1520) to 1.51 mm (CS14C, level 1). The process time ranged widely from 6.8 s (ACE, level 5) to 31.83 s (WAVE, level 1). CONCLUSION: The current study demonstrated that all the tested devices are efficacious and safe in sealing and cutting arteries up to 5 mm in diameter. All the devices showed supraphysiologic mean burst pressures. Differences in failure rate, thermal damage, and process time lead to an advised application of the different systems.

[Complications after lid loading. Is the platinum chain really effective?]. / Komplikationen nach Implantation von Lidgewichten. Ist die Platinkette wirklich effektiv?

INTRODUCTION: Numerous studies on the operative therapy for lagophthalmos exist. Reports about postoperative results, however, are rare and most studies include just a small number of patients. Additionally, since 1999a flexible platinum chains has been available as an alternative to the rigid gold implant. The authors are convinced that the platinum chain matches much more the anatomic prerequisites of the upper eye lid. Therefore, the aim of this study was to compare the complication rate of rigid gold implants with that of the flexible platinum chain. To increase the amount of patient data, a statistical meta-analysis after implantation of gold implants and the data form a survey of all surgeons who do implantations of the platinum chain have been compared. MATERIALS AND METHODS: To collect a greater number of patients a survey with focus on postoperative complications has been sent to all colleagues who perform implantations with the flexible platinum chain in patients with paralytic lagophthalmos. Complications included a corneal astigmatism, a migration of the implant, a bulging, an extrusion or an infection. In a further step the statistical comparison between the survey and the results of the meta-analysis about complications after application of rigid gold implants was performed. RESULTS: Through the survey, data for 212 implantations of the flexible platinum chains could be collected. The comparison showed a statistically significant lower rate of postoperative complications concerning astigmatism, bulging, and postoperative infections in the platinum chain group. Only the postoperative extrusion rates (6.8% after gold implantations and 3.3% after platinum chain implantations) showed no statistically significant difference (p=0.0612). In general, complications occurred in 45.1% of the cases after use of gold implants and in 12.8% after use of the platinum chain, respectively. CONCLUSION: Lid loading is a relatively simple and reversible procedure for the operative therapy of lagophthalmos. By using the platinum chain as an implant, postoperative complications can be decreased and thus the effectiveness of the treatment can be enhanced.

[Ultrasound of the upper eyelid to evaluate tarsal curvature in facial palsy]. / Sonographie des Oberlids zur Beurteilung der Tarsuskrümmung bei Fazialisparese.

BACKGROUND: Treatment of paralytic lagophthalmos involves surgical repair, requiring exact knowledge of eyelid anatomy. While there are extensive studies on anatomical eyelid measurements in healthy eyes, no data exist on the changes in the functional anatomy of the upper eyelid in paralytic lagophthalmos. The aim of this study, was to examine by ultrasound the upper lid tarsus during changes in the patient's line of vision, and to answer the question of whether there are changes in the curvature radius of the tarsus, caused by facial paralysis with resultant lagophthalmos. MATERIAL AND METHODS: Two groups were formed. The first group consisted of 50 subjects with healthy eyes, and the second contained 47 patients with paralytic lagophthalmos. The upper lid tarsal radius, when looking straight ahead and in the abducted position, was determined by ultrasound with a 7.5 MHz scanner in the non-contact mode, and then compared statistically. RESULTS: Both groups showed a highly significant difference in the tarsus curvature, when looking straight ahead and in the abduction position. While there was no significant difference between both groups in the abduction position, they differed significantly when looking straight ahead. Also, a significant difference was noted, between the eyes of healthy subjects and the healthy eye in patients with facial palsy. CONCLUSION: The changes in curvature of the tarsal plate, when looking straight ahead, can be explained by the loss of tone in the paralyzed Orbicularis Occuli muscle. This means, that in addition to the lagophthalmos resulting from facial paralysis, changes occur in the functional anatomy of the upper eyelid, which must be considered during surgical correction. Additionally, the physiological loss of tone of the upper eyelid tarsal plate, which comes with age, has a certain influence.

[Sensorineural hearing loss in LEOPARD syndrome]. / Innenohrschwerhörigkeit beim LEOPARD-Syndrom.

The rare multiple lentigines (LEOPARD) syndrome represents a complex of skin, cardiac, skeletal, inner ear and other malformations. There is marked variability in expression of the syndrome. We report on a 20 year old man, showing typical lentiginosis, a retardation of growth, tachycardiac conduction abnormality, ophthalmologic manifestations and a sensorineural hearing loss. Pathogenesis, clinical and differential diagnostic aspects are discussed in this case report. The early diagnosis of a senosorineural hearing loss is useful in order to provide appropriate rehabilitation. When lentiginosis is diagnosed, it is important to consider further abnormalities such as cardiomyopathy, which can be associated with a high mortality.

[Implantation of lid weights for therapy of lagophthalmos]. / Implantation von Lidgewichten zur Therapie des Lagophthalmus.

BACKGROUND: The aim of this study was to compare the clinical outcome and complication rate of rigid gold implants and flexible platinum chains in 96 patients treated for lagophthalmos. PATIENTS AND METHODS: We treated 50 patients with peripheral facial palsy and lagophthalmos using rigid gold implants and 46 using a flexible platinum chain. Besides subjective assessment by the patients, reduction of lagophthalmos, improvement of visual acuity, and pre- and postoperative grade of keratopathy were evaluated. RESULTS: Both groups showed a statistically significant reduction of lagophthalmos and keratopathy and increase of visual acuity. Postoperative complications were observed in both groups. Two extrusions occurred in the group receiving gold implants. The incidence of corneal astigmatism and bulging of the implant was statistically significantly lower in the platinum chain group. CONCLUSIONS: The use of flexible platinum chains instead of rigid gold implants for therapy of lagophthalmos leads to a reduction of the complication rate, thus improving the clinical outcome.

[Lidloading and intraocular pressure]. / Lidloading und Augeninnendruck.

BACKGROUND: Implantation of rigid gold or platinum lid weights (lidloading) represents the most common method for therapy of patients with lagophthalmus as a result of facial palsy. In a more recent approach also flexible platinum chains have been used. Glaucoma has frequently been described as a contraindication for lidloading. The aim of this study was to proof if lid weights lead to an increase of the intraocular pressure (IOP). PATIENTS AND METHODS: In a prospective study the IOP has been measured before and after implantation of 46 rigid gold implants and 39 flexible platinum chains in patients with paralytic lagophthalmos. RESULTS: Neither the implantation of rigid gold implants nor the use of the flexible platinum chain lead to a statistically significant change of the IOP. There was also no difference between the two groups. CONCLUSION: With this study we could demonstrate that glaucoma is not an absolute contraindication for the use of lid weights in patients with paralytic lagophthalmos. Moreover a lid weight is easily removable if complications should occur.

[Contact lens fitting as a possibility for visual rehabilitation in patients after open globe injuries]. / Kontaktlinsenversorgung als Möglichkeit der visuellen Rehabilitation bei Patienten nach offenen Augenverletzungen.

INTRODUCTION: Visual rehabilitation after open globe injury may be a challenging process because of ametropia following aphakia, corneal scarring with high or irregular corneal astigmatism or loss of contrast sensitivity due to traumatic aniridia. We report on contact lens fitting for visual rehabilitation in patients after open globe injury. PATIENTS: From 2000 to 2003, contact lenses were fitted unilaterally for the visual rehabilitation in 13 patients after open globe injury. In three patients we found unilateral aphakia, in 8 patients a high or irregular astigmatism after penetrating or autorotation keratoplasty and in two patients a traumatic aniridia, in one case combined with aphakia. RESULTS: 11 rigid contact lenses were fitted with different designs of the front and back surface as well as two iris-print lenses. In 11 patients (86 %) a good visual rehabilitation was achieved with an increase of visual acuity up to 9 lines while obtaining a good contact lens tolerance. One patient wearing an iris-print contact lens was unable to tolerate the contact lens due to its thickness and its weight. In another patient fitting of a contact lens was not possible because of the complicated corneal condition. We did not observed severe contact lens complications at any time. CONCLUSIONS: In addition to operative procedures for visual rehabilitation after open globe injuries, the use of contact lenses is another possible procedure for refractive correction. Different problems such as ametropia following aphakia, irregular or high astigmatism or aniridia can be solved with good visual results, good tolerance and less complications.

Cell vacuolization induced by Helicobacter pylori VacA cytotoxin does not depend on late endosomal SNAREs.

Cellular vacuoles induced by the Helicobacter pylori vacuolating cytotoxin VacA originate from late endosomal compartments. Their biogenesis requires the activity of both rab7 GTPase and the ATPase proton pump. The toxin has been suggested to cause an increased luminal osmotic pressure via its anion-specific channel activity localized on late endosomal compartments after endocytosis. Here, we show that the extensive membrane fusion that takes place in the transition from the small late endosomal compartments to the large vacuoles does not depend on soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) proteins. The process of vacuolization leads to disappearance of the large array of internal membranes of late endosomes. We suggest that most of the vacuole-limiting membrane derives from internal membranes.

Human cofilin forms oligomers exhibiting actin bundling activity.

Human cofilin possesses the tendency for self-association, as indicated by the rapid formation of dimers and oligomers when reacted with water-soluble carbodiimide, Ellman's reagent, or glutathione disulfide. Intermolecular disulfide bonds involve Cys(39) and probably Cys(147) of two adjacent cofilin units. The disulfide-linked dimers and oligomers exhibit a biological activity distinct from the monomer. While monomeric cofilin decreased viscosity and light-scattering of F-actin solutions, dimers and oligomers caused an increase in viscosity and light scattering. Electron microscopy revealed that cofilin oligomers induce the formation of highly ordered actin bundles with occasionally blunt ends similar to actin-cofilin rods observed in cells under oxidative stress. Bundling activity of the disulfide-linked oligomers could be completely reversed into severing activity by dithiothreitol. Formation of cofilin oligomers occurred also in the presence of actin at pH 8, but not at pH 6.6, and was significantly enhanced in the presence of phosphatidylinositol 4,5-bisphosphate. Our data are consistent with the idea that cofilin exists in two forms in vivo also: as monomers exhibiting the known severing activity and as oligomers exhibiting actin bundling activity. However, stabilization of cofilin oligomers in cytoplasm is probably achieved not by disulfide bonds but by a local increase in cofilin concentration and/or binding of regulatory proteins.

Characterization of the intracellular survival of Mycobacterium avium ssp. paratuberculosis: phagosomal pH and fusogenicity in J774 macrophages compared with other mycobacteria.

The phagosomes containing viable pathogenic mycobacteria, such as Mycobacterium (M.) tuberculosis and Mycobacterium avium ssp. avium (M. avium), are known to be limited in their ability to both acidify and fuse with late (but not early) endocytic organelles. Here, we analysed the pH and fusogenicity of phagosomes containing M. avium ssp. paratuberculosis (M. ptb), the causative agent of paratuberculosis in ruminants. Using the murine J774 macrophage cell line, we compared viable and heat-killed M. ptb and, in addition, viable or dead M. avium, as well as two non-pathogenic mycobacteria, Mycobacterium smegmatis and Mycobacterium gordonae. Electron microscopic analysis revealed that M. ptb persisted intracellularly in phagosomes for up to 15 days. The phagosomes containing live M. ptb and M. avium were significantly reduced in their ability to acquire some markers for the endocytic pathway, such as internalized calcein, BSA-gold or the membrane protein Lamp 2. However, they were almost completely accessible to 70 kDa fluorescein isothiocyanate (FITC)-dextran and Lamp 1. Overall, the phagosomes containing dead pathogenic mycobacteria behaved similarly to the ones containing live non-pathogenic mycobacteria in all experiments. Using FITC-dextran in a novel fluorescence-activated cell sorting (FACS)-based method, we could also show that the bulk of endocytic compartments, including phagosomes, were only very mildly acidified to approximately pH 6.3 over at least 72 h in J774 cells infected with live M. ptb and M. avium. In contrast, J774 cells treated with heat-killed M. ptb or BSA-coated latex beads showed substantial acidification of the phagosome/endocytic compartments to a pH value of approximately 5.2. After infection with M. smegmatis and M. gordonae, acidification was initially (1-5 h after infection) inhibited, but increased after longer infection to levels similar to those with dead mycobacteria.

Remodelling of the actin cytoskeleton is essential for replication of intravacuolar Salmonella.

Maturation and maintenance of the intracellular vacuole in which Salmonella replicates is controlled by virulence proteins including the type III secretion system encoded by Salmonella pathogenicity island 2 (SPI-2). Here, we show that, several hours after bacterial uptake into different host cell types, Salmonella induces the formation of an F-actin meshwork around bacterial vacuoles. This structure is assembled de novo from the cellular G-actin pool in close proximity to the Salmonella vacuolar membrane. We demonstrate that the phenomenon does not require the Inv/Spa type III secretion system or cognate effector proteins, which induce actin polymerization during bacterial invasion, but does require a functional SPI-2 type III secretion system, which plays an important role in intracellular replication and systemic infection in mice. Treatment with actin-depolymerizing agents significantly inhibited intramacrophage replication of wild-type Salmonella typhimurium. Furthermore, after this treatment, wild-type bacteria were released into the host cell cytoplasm, whereas SPI-2 mutant bacteria remained within vacuoles. We conclude that actin assembly plays an important role in the establishment of an intracellular niche that sustains bacterial growth.

ATP-dependent membrane assembly of F-actin facilitates membrane fusion.

We recently established an in vitro assay that monitors the fusion between latex-bead phagosomes and endocytic organelles in the presence of J774 macrophage cytosol (). Here, we show that different reagents affecting the actin cytoskeleton can either inhibit or stimulate this fusion process. Because the membranes of purified phagosomes can assemble F-actin de novo from pure actin with ATP (), we focused here on the ability of membranes to nucleate actin in the presence of J774 cytosolic extracts. For this, we used F-actin sedimentation, pyrene actin assays, and torsional rheometry, a biophysical approach that could provide kinetic information on actin polymerization and gel formation. We make two major conclusions. First, under our standard in vitro conditions (4 mg/ml cytosol and 1 mM ATP), the presence of membranes actively catalyzed the assembly of cytosolic F-actin, which assembled into highly viscoelastic gels. A model is discussed that links these results to how the actin may facilitate fusion. Second, cytosolic actin paradoxically polymerized more under ATP depletion than under high-ATP conditions, even in the absence of membranes; we discuss these data in the context of the well described, large increases in F-actin seen in many cells during ischemia.

Immunolocalization of cytoplasmic dynein and dynactin subunits in cultured macrophages: enrichment on early endocytic organelles.

Cytoplasmic dyneins and their cofactor, dynactin, work together to mediate the movement of numerous cargo organelles toward the minus-ends of microtubules. In many cases, there is compelling evidence that dynactin functions in part to attach dyneins to cargo organelles, but this may not always be the case. We have localized three dynactin subunits (Arp1, p62 and p150(Glued)) and two subunits of conventional cytoplasmic dynein (dynein intermediate chain and dynein heavy chain 1) in murine macrophages using immunogold labeling of thawed cryosections. Using stereological techniques, we have quantified the relative distributions of each of these subunits on specific membrane organelles to generate a comprehensive analysis of the distribution of these proteins in a single cell type. Our results show that each of the subunits tested exhibits the same distribution with respect to different membrane organelles, with highest levels present on early endosomes, and lower levels present on later endocytic organelles, the mitochondrial outer membrane, the plasma membrane and vesicles in the Golgi region. An additional pool of punctate dynactin labeling was detected in the cell periphery, in the absence of dynein labeling. Even when examined closely, membrane organelles could not be detected in association with these dynactin-positive sites; however, double labeling with anti-tubulin antibody revealed that at least some of these sites represent the ends of microtubules. The similarities among the labeling profiles with respect to membrane organelles suggest that dynein and dynactin bind to membrane organelles as an obligate unit. In contrast, our results show that dynactin can associate with microtubule ends in the absence of dynein, perhaps providing sites for subsequent organelle and dynein association to form a functional motility complex.

Preferential association of syntaxin 8 with the early endosome.

Members of the syntaxin family play a fundamental role in vesicle docking and fusion of diverse transport events. We have molecularly characterized syntaxin 8, a novel member of the syntaxin family. The nucleotide sequence of cloned rat cDNA predicts a polypeptide of 236 residues with a carboxyl-terminal 18-residue hydrophobic domain that may function as a membrane anchor. Characteristic of syntaxins, syntaxin 8 also contain regions that have the potential to form coiled-coil structures. Among the known syntaxins, syntaxin 8 is most homologous to syntaxin 6 which is predominantly associated with the trans-Golgi network (TGN). The syntaxin 8 transcript is detected in all rat tissues examined by northern blot. Antibodies against recombinant syntaxin 8 recognize a 27 kDa protein that is enriched in membrane fractions containing the Golgi apparatus and the endosomal/lysosomal compartments. Syntaxin 8 in membrane extract could be incorporated into a 20S protein complex in a way that is dependent on the soluble N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein ((alpha)-SNAP), suggesting that syntaxin 8 is indeed a SNAP receptor (SNARE). Indirect immunofluorescence microscopy reveals that the majority of syntaxin 8 is localized to the early endosome marked by Rab5. This is corroborated by immunogold labeling experiments showing enrichment of syntaxin 8 in the early endosome and its co-labeling with Rab5.

Involvement of ezrin/moesin in de novo actin assembly on phagosomal membranes.

The current study focuses on the molecular mechanisms responsible for actin assembly on a defined membrane surface: the phagosome. Mature phagosomes were surrounded by filamentous actin in vivo in two different cell types. Fluorescence microscopy was used to study in vitro actin nucleation/polymerization (assembly) on the surface of phagosomes isolated from J774 mouse macrophages. In order to prevent non-specific actin polymerization during the assay, fluorescent G-actin was mixed with thymosin beta4. The cytoplasmic side of phagosomes induced de novo assembly and barbed end growth of actin filaments. This activity varied cyclically with the maturation state of phagosomes, both in vivo and in vitro. Peripheral membrane proteins are crucial components of this actin assembly machinery, and we demonstrate a role for ezrin and/or moesin in this process. We propose that this actin assembly process facilitates phagosome/endosome aggregation prior to membrane fusion.

In vitro fusion of phagosomes with different endocytic organelles from J774 macrophages.

We describe novel biochemical and electron microscopy assays to investigate in vitro fusion of latex bead phagosomes with three different endocytic organelle fractions from J774 macrophages. After formation, early phagosomes fuse avidly with early and late endosomes and for a longer period of time with lysosomes, but they subsequently become fusion-incompetent. The fusion of early, but not late, phagosomes with all three endocytic fractions could be significantly stimulated by Rab5. In contrast to other cell types investigated, this Rab is uniquely enriched on both early and late endosomes in J774 macrophages. Moreover, exogenous Rab5 stimulates homotypic fusion between both sets of organelles. This was shown by a quantitative electron microscopy fusion assay that can directly assay fusion between any combination of morphologically defined organelles. By the same approach, we discovered an unexpected Rab5-stimulatable fusion between early and late endosomes in J774, but not in BHK cells. Thus, in J774 cells both Rab5 and the endocytic pathway seem to have evolved additional functions not yet seen in nonphagocytic cells.