Nailfold Capillaroscopy: A Promising, Noninvasive Approach to Predict Retinopathy of Prematurity.

OBJECTIVE: To test the hypothesis that nailfold capillaroscopy can noninvasively detect dysregulated retinal angiogenesis and predict retinopathy of prematurity (ROP) in infants born premature before its development. METHODS: In a cohort of 32 infants born <33 weeks of gestation, 1386 nailfold capillary network images of the 3 middle fingers of each hand were taken during the first month of life. From these, 25 infants had paired data taken 2 weeks apart during the first month of life. Images were analyzed for metrics of peripheral microvascular density using a machine learning-based segmentation approach and a previously validated microvascular quantification platform (REAVER vascular analysis). Results were correlated with subsequent development of ROP based on a published consensus ROP severity scale. RESULTS: In total, 18 of 32 (56%) (entire cohort) and 13 of 25 (52%) (2-time point subgroup) developed ROP. Peripheral vascular density decreased significantly during the first month of life. In the paired time point analysis, vessel length density, a key metric of peripheral vascular density, was significantly greater at both time points among infants who later developed ROP (15 563 and 11 996 µm/mm2, respectively) compared with infants who did not (12 252 and 8845 µm/mm2, respectively) (P < .001, both time points). A vessel length density cutoff of >15 100 at T1 or at T2 correctly detected 3 of 3 infants requiring ROP therapy. In a mixed-effects linear regression model, peripheral vascular density metrics were significantly correlated with ROP severity. CONCLUSIONS: Nailfold microvascular density assessed during the first month of life is a promising, noninvasive biomarker to identify premature infants at highest risk for ROP before detection on eye exam.

SeqStain is an efficient method for multiplexed, spatialomic profiling of human and murine tissues.

Spatial organization of molecules and cells in complex tissue microenvironments provides essential organizational cues in health and disease. A significant need exists for improved visualization of these spatial relationships. Here, we describe a multiplex immunofluorescence imaging method, termed SeqStain, that uses fluorescent-DNA-labeled antibodies for immunofluorescent staining and nuclease treatment for de-staining that allows selective enzymatic removal of the fluorescent signal. SeqStain can be used with primary antibodies, secondary antibodies, and antibody fragments to efficiently analyze complex cells and tissues. Additionally, incorporation of specific endonuclease restriction sites in antibody labels allows for selective removal of fluorescent signals while retaining other signals that can serve as marks for subsequent analyses. The application of SeqStain on human kidney tissue provided a spatialomic profile of the organization of >25 markers in the kidney, highlighting it as a versatile, easy-to-use, and gentle new technique for spatialomic analyses of complex microenvironments.

Protocol for live enhanced resolution confocal imaging of dendritic spinule dynamics in primary mouse cortical neuron culture.

Dendritic spinules are fine membranous protrusions of neuronal spines that play a role in synaptic plasticity, but their nanoscale requires resolution beyond conventional confocal microscopy, hindering live studies. Here, we describe how to track individual spinules in live dissociated cortical pyramidal neurons utilizing fluorescence labeling, optimized confocal imaging parameters, and post-acquisition iterative 3D deconvolution, employing NIS Elements software. This approach enables investigations of spinule structural dynamics and function without using super-resolution microscopy, which involves special fluorophores and/or high laser power. For complete details on the use and execution of this protocol, please refer to Zaccard et al. (2020).

A spatially restricted fibrotic niche in pulmonary fibrosis is sustained by M-CSF/M-CSFR signalling in monocyte-derived alveolar macrophages.

Ontologically distinct populations of macrophages differentially contribute to organ fibrosis through unknown mechanisms.We applied lineage tracing, single-cell RNA sequencing and single-molecule fluorescence in situ hybridisation to a spatially restricted model of asbestos-induced pulmonary fibrosis.We demonstrate that tissue-resident alveolar macrophages, tissue-resident peribronchial and perivascular interstitial macrophages, and monocyte-derived alveolar macrophages are present in the fibrotic niche. Deletion of monocyte-derived alveolar macrophages but not tissue-resident alveolar macrophages ameliorated asbestos-induced lung fibrosis. Monocyte-derived alveolar macrophages were specifically localised to fibrotic regions in the proximity of fibroblasts where they expressed molecules known to drive fibroblast proliferation, including platelet-derived growth factor subunit A. Using single-cell RNA sequencing and spatial transcriptomics in both humans and mice, we identified macrophage colony-stimulating factor receptor (M-CSFR) signalling as one of the novel druggable targets controlling self-maintenance and persistence of these pathogenic monocyte-derived alveolar macrophages. Pharmacological blockade of M-CSFR signalling led to the disappearance of monocyte-derived alveolar macrophages and ameliorated fibrosis.Our findings suggest that inhibition of M-CSFR signalling during fibrosis disrupts an essential fibrotic niche that includes monocyte-derived alveolar macrophages and fibroblasts during asbestos-induced fibrosis.

Ooplasmic flow cooperates with transport and anchorage in Drosophila oocyte posterior determination.

The posterior determination of the Drosophila melanogaster embryo is defined by the posterior localization of oskar (osk) mRNA in the oocyte. Defects of its localization result in a lack of germ cells and failure of abdomen specification. A microtubule motor kinesin-1 is essential for osk mRNA posterior localization. Because kinesin-1 is required for two essential functions in the oocyte-transport along microtubules and cytoplasmic streaming-it is unclear how individual kinesin-1 activities contribute to the posterior determination. We examined Staufen, an RNA-binding protein that is colocalized with osk mRNA, as a proxy of posterior determination, and we used mutants that either inhibit kinesin-driven transport along microtubules or cytoplasmic streaming. We demonstrated that late-stage streaming is partially redundant with early-stage transport along microtubules for Staufen posterior localization. Additionally, an actin motor, myosin V, is required for the Staufen anchoring to the actin cortex. We propose a model whereby initial kinesin-driven transport, subsequent kinesin-driven streaming, and myosin V-based cortical retention cooperate in posterior determination.

Resolving new ultrastructural features of cytokinetic abscission with soft-X-ray cryo-tomography.

Mammalian cytokinetic abscission is mediated by the ESCRT membrane fission machinery. While much has been clarified on the topology and kinetics of abscission through high-resolution microscopy, key questions regarding the mechanism of abscission remain open. Here we apply cryogenic soft-X-ray tomography to elucidate new ultrastructural details in the intercellular membrane bridge connecting cells undergoing abscission. In particular, we resolve defined ring-like structures inside the midbody dark zone that have been inaccessible to EM, and identify membrane extrusions at the abscission sites. In cells at late stages of abscission we resolve a complex array of helical spirals, extending the structural information obtained by EM. Our results highlight the advantages of soft-X-ray tomography and emphasize the importance of using complementary approaches for characterizing cellular structures. Notably, by providing new structural data from intact cells we present a realistic view on the topology of abscission and suggest new mechanistic models for ESCRT mediated abscission.

Biomineralization pathways in a foraminifer revealed using a novel correlative cryo-fluorescence-SEM-EDS technique.

Foraminifera are marine protozoans that are widespread in oceans throughout the world. Understanding biomineralization pathways in foraminifera is particularly important because their calcitic shells are major components of global calcium carbonate production. We introduce here a novel correlative approach combining cryo-SEM, cryo-fluorescence imaging and cryo-EDS. This approach is applied to the study of ion transport processes in the benthic foraminifer genus Amphistegina. We confirm the presence of large sea water vacuoles previously identified in intact and partially decalcified Amphistegina lobifera specimens. We observed relatively small vesicles that were labelled strongly with calcein, and also identified magnesium (Mg)-rich mineral particles in the cytoplasm, as well as in the large sea water vacuoles. The combination of cryo-microscopy with elemental microanalysis and fluorescence imaging reveals new aspects of the biomineralization pathway in foraminifera which are, to date, unique in the world of biomineralization. This approach is equally applicable to the study of biomineralization pathways in other organisms.

Correlation between cationic lipid-based transfection and cell division.

We evaluate the temporal relation between protein expression by cationic lipid-mediated transfection and cell division using time lapse fluorescence microscopy. Detailed image analysis provides new insights on the single cell level while simultaneously achieving appropriate statistics. Earlier evidence by less direct methods such as flow cytometry indicates a primary route for transfection involving nuclear envelope breakdown, but also suggests the existence of a pathway independent of mitosis. We confirm and quantify both mechanisms. We found the timing for successful transfection to be unexpectedly flexible, contrary to assertions of a narrow time window. Specifically, cells dividing more than 24h after exposure to the transfection medium express the probed protein at a comparable level to cells in a mitotic state during or shortly after transfection. This finding can have a profound impact on the guidance and development of non-viral gene delivery materials.