pixOL: pixel-wise dipole-spread function engineering for simultaneously measuring the 3D orientation and 3D localization of dipole-like emitters

Interactions between biomolecules are characterized by both where they occur and how they are organized, e.g., the alignment of lipid molecules to form a membrane. However, spatial and angular information are mixed within the image of a fluorescent molecule-the microscopy's dipole spread function (DSF). We demonstrate the pixOL algorithm for simultaneously optimizing all pixels within a phase mask to produce an engineered Green's tensor-the dipole extension of point-spread function engineering. The pixOL DSF achieves optimal precision for measuring simultaneously the 3D orientation and 3D location of a single molecule, i.e., 1.14 degree orientation, 0.24 sr wobble angle, 8.17 nm lateral localization, and 12.21 nm axial localization precisions over an 800-nm depth range using 2500 detected photons. The pixOL microscope accurately and precisely resolves the 3D positions and 3D orientations of Nile red within a spherical supported lipid bilayer, resolving both membrane defects and differences in cholesterol concentration, in 6 dimensions.

CV-containing vesicle budding from chloroplast inner envelope membrane is mediated by clathrin but inhibited by GAPC

Clathrin-mediated vesicular formation and trafficking are highly conserved in eukaryotic cells and are responsible for molecular cargo transport and signal transduction among organelles. It remains largely unknown whether clathrin-coated vesicles can be generated from chloroplasts. CHLOROPLAST VESICULATION (CV)-containing vesicles (CVVs) generate from chloroplasts and mediate chloroplast degradation under abiotic stress. In this study, we showed that CV interacted with the clathrin heavy chain (CHC) and induced vesicle budding from the chloroplast inner envelope membrane. Defects on CHC2 and the dynamin-encoding DRP1A gene affected CVV budding and releasing from chloroplast. CHC2 is also required for CV-induced chloroplast degradation and hypersensitivity to water stress. Moreover, GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPC) interacts with CV and impairs the CV-CHC2 interaction. GAPC1 overexpression inhibited CV-mediated chloroplast degradation and hypersensitivity to water stress. CV silencing alleviated the hypersensitivity of gapc1gapc2 plant to water stress. Together, our work revealed a pathway of clathrin-assisted CVV budding from the chloroplast inner envelope membrane, which mediated the stress-induced chloroplast degradation and stress response.

PIEZO1 Mutation May Determine Early Onset of Clinical Manifestation of Anemia of Myelodysplastic Syndromes

BACKGROUND: Anemia is the most frequent cytopenia in myelodysplastic syndromes (MDS), neoplastic diseases characterized by ineffective hematopoiesis and bone marrow dysplasia. MDS usually affect elderly individuals (median age in Italy 74 years), and quite rarely concern younger patients. It is of increasing interest to verify whether "young", not therapy-related MDS patients carry germline (GM) predisposition, in order to optimize therapeutic and transplant choices. The best approach is to perform Whole Exome Sequencing (WES) instead of targeted next-generation sequencing (t-NGS) panel in cases in which MDS patients have no syndromic signs or any indicative sign of predisposing alterations. AIM: We considered a cohort of 21 consecutive MDS cases with age at diagnosis < 60 years and anemia, referred to our MDS Unit for a second opinion. METHODS: After bone marrow (BM) re-evaluation and morphological confirmation of dysplasia compatible with MDS, we screened the 21 MDS patients by WES on BM-DNA or peripheral blood DNA (PB-DNA). For DNA sequencing, DNA libraries were sequenced using the NextSeq 550 Illumina System. Variant calling was carried out using GATK caller. T-NGS myeloid panel was in some cases applied to confirm somatic nature of mutations. Samples of 5/21 MDS patients and their relatives were further analyzed after WES by applying high-coverage t-NGS 86-gene custom panel for hereditary anemias on PB-DNA. WES of DNA extracted from saliva (S-DNA) samples was performed to provide a GM confirmation. In order to confirm pathogenic role in red blood cell (RBC) membrane defects of identified variants, we also performed the ektacytometry analysis that evaluates the erythrocyte deformability and the hydration status by subjecting them to an increasing osmotic gradient with constant shear stress. RESULTS: Five of 21 MDS cases (23.8%) with symptomatic anemia (3 MDS-MLD, 1 MDS-SLD, 1 MDS with isolated del5q) were identified as carriers of variants suspected responsible of RBC membrane defects in both BM/PB-DNA and S-DNA samples, where no other germline myeloid predisposing alterations were demonstrated. MDS-associated somatic variants were present in 4/5 patients (KRAS, TET2, CBL, SRSF2, U2AF1, IDH2, with VAF >25 %). High coverage t-NGS analysis confirmed the presence of a heterozygous rare missense variant in PIEZO1 in probands 1 and 5; a heterozygous rare missense variant in PIEZO1 and ANK1 in proband 2; a heterozygous rare missense variant in PIEZO1 and SPTA1 in proband 3; a complex mode of inheritance in proband 4 with causative variants in G6PD, KCCN4, SPTB, and ABCB6 genes. PIEZO1 or KCNN4 gene variants result in erythrocyte dehydration and determine dehydrated hereditary stomatocytosis (DHS). Ektacytometry analysis showed a significant difference in the osmolarity curve of these patients, confirming the pathogenic role of the variants identified. Among these MDS suspected cases, only proband 4 was diagnostically re-categorized because of lack of acquired recurrent somatic mutations and presence of multiple alterations in erythroid genes causing dysplasia of erythroid precursors, masquerading as MDS. None of the patients presented a history of unexplained anemia before the onset of MDS, consistent with what shown for DHS1 patients with well-compensated hemolysis. Inheritance pattern of the identified variants according the study of the families confirmed the segregation of the pathogenic variants. CONCLUSION: Onset of de novo MDS in unusually young age should always prompt investigation of predisposing conditions. We suggest here that co-existence of inherited RBC membrane defects with MDS may determine earlier clinical manifestation of the clonal disease. Hereditary anemias, in particular xerocytosis, may mimic MDS morphology. In our study, among the 5 "young" MDS with concomitant inherited RBC membrane defect, one case did not finally confirm MDS diagnosis by karyotype or NGS myeloid mutation analysis. This case fulfilled the diagnostic criteria for combined hereditary hemolytic anemia: DHS2, G6PD deficiency, spherocytosis, familial pseudohyperkalemia 2, with multi-locus mode of inheritance that justifies the clinical condition (splenomegaly, hemolytic crisis, ascites). Addition of genetic testing for hereditary anemias beside assessment of germline predisposing variants may allow a more precise diagnosis in controversial cases of "young" MDS. Disclosures Sanna: Janssen: Consultancy; Abbvie: Consultancy; Astra Zeneca: Consultancy. Iolascon: Celgene: Other: Advisory Board; Bluebird Bio: Other: Advisory Board. Santini: Menarini: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Geron: Membership on an entity's Board of Directors or advisory committees; BMS/Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astex: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees.

Dome-Shaped Macula versus Ridge-Shaped Macula Eyes in High Myopia Based on the 12-line Radial Optical Coherence Tomography Scan Pattern. Differences in Clinical Features

Purpose: To study clinical features in patients with ridge-shaped macula (RSM) compared with those with dome-shaped macula (DSM) having been previously classified by the number of swept-source optical coherence tomography (SS-OCT) radial scans affected. Methods: Retrospective observational study including 49 highly myopic eyes from 31 patients who underwent SS-OCT. DSM eyes were defined as those that showed a complete round inward convexity in all their axes, presenting an inward convexity ≥50 µm in the 12-line radial OCT scans. Eyes that did not meet this criterion and had at least one flat radial scan were grouped into the RSM group, defined as a macular inward convexity in some meridians across the fovea, whereas the opposite perpendicularly oriented meridians were flat. Age, spherical equivalent, axial length (AL), and best-corrected visual acuity (BCVA) were collected. Height of the bulge, scleral and choroidal thicknesses, Bruch´s membrane defects, and presence of perforating scleral vessels were recorded. Results: Thirty-seven (75.5%) eyes were classified into the RSM group and 12 (24.5%) into the DSM group. Twenty-six (53.0%) eyes showed macular elevation only in the horizontal direction. Mean AL showed statistically significant differences (28.8 ± 2.7 vs. 30.5 ± 1.5 mm in the RMS vs. DSM group, respectively) and the presence of Bruch´s membrane defects was more frequently seen in DSM (p < 0.001). Mean age, spherical equivalent, BCVA, height of the inward convexity, retinal foveal thickness, foveal scleral thickness, subfoveal choroidal thickness, and the presence of perforating scleral vessels did not show significant differences between groups. Conclusion: This study shows the reliability of using the 12 equal radial OCT scans as an objective method to define and differentiate DSM versus RSM. Patients with RSM showed differences in AL compared with those with DSM, being longer in DSM, and regarding the presence of Bruch´s membrane defects, being more common in DSM. This may contribute to identifying those patients that, in daily clinical practice, have a higher risk of developing complications due to their myopia.

Cx43 mediates changes in myofibroblast contraction and collagen release in human amniotic membrane defects after trauma

The wound healing capacity of the fetal membranes after spontaneous or iatrogenic membrane rupture is unclear. We examined the healing mechanisms in amniotic membrane (AM) defects after trauma. Traumatised human AM defects were cultured for 4 days. Markers for nuclear (DAPI), cell type (vimentin, αSMA) and healing (Cx43, TGFβ1, collagen) were examined by immunofluorescence (IMF) confocal microscopy, Second Harmonic Generation (SHG) imaging and RT-qPCR. After trauma, AMCs and myofibroblasts migrated to the AM wound edge. Within four days, αSMA expressing myofibroblasts showed abundant Cx43 localized in the cytoplasmic processes. The highly contractile spindle-shaped myofibroblasts were present in the defect site and released collagen. In contrast, AMCs expressed vimentin and formed Cx43 plaques between cells found in the outer edges of the wound. Whilst AMCs were absent in the defect site, αSMA expressing myofibroblasts continued to elongate and polarize the collagen fibres. Both TGFβ1 and Cx43 gene expression were significantly increased after trauma. Cx43 has differential effects on AM cell populations that increase cellularity, contraction and potentially migration to the wound edge resulting in collagen polarisation in the AM defect site. Establishing how Cx43 regulates AM cell function could be an approach to repair defects in the membranes after trauma.

The role of basement membranes in cardiac biology and disease

Basement membranes are highly specialised extracellular matrix structures that within the heart underlie endothelial cells and surround cardiomyocytes and vascular smooth muscle cells. They generate a dynamic and structurally supportive environment throughout cardiac development and maturation by providing physical anchorage to the underlying interstitium, structural support to the tissue, and by influencing cell behaviour and signalling. While this provides a strong link between basement membrane dysfunction and cardiac disease, the role of the basement membrane in cardiac biology remains under-researched and our understanding regarding the mechanistic interplay between basement membrane defects and their morphological and functional consequences remain important knowledge-gaps. In this review we bring together emerging understanding of basement membrane defects within the heart including in common cardiovascular pathologies such as contractile dysfunction and highlight some key questions that are now ready to be addressed.

Semiquantitative Criteria in the Eye Bank That Correlate with Cornea Guttata in Donor Corneas

Background Cornea guttata may not be recognized in the eye bank and recent studies have displayed that guttae are transplanted in about 15% of cases in varying severities. The purpose of this study was to establish semiquantitative criteria for the detection of cornea guttata in donor corneas in the eye bank. Methods In this retrospective cohort study, preoperative endothelial pictures of donor corneas were collected and classified according to the post-penetrating keratoplasty cornea guttata grade into three distinct groups: group 1 consists of healthy corneas with no guttae (guttata grade 0); group 2 constitutes corneas with mild asymptomatic cornea guttata (guttata grade +); and group 3 comprises corneas with advanced widespread cornea guttata (guttata grade ++/+++/++++). The preoperative pictures of each group were then individually analyzed using the following five semiquantitative criteria: The number and the area of the cell-depleted surfaces, the presence of less than 50% of the cells having a hexagonal or a circular shape, the presence of cell membrane defects and interruptions, the presence of blebs in the cell membrane, and the presence of groups of cells with a distinct whitish color. Results In total, 262 patients were included in this study, with a total number of 1582 preoperative donor corneal endothelial pictures. Out of those pictures, groups 1, 2, and 3 encompassed 995 (62.9%), 411 (26.0%), and 176 (11.1%) pictures, respectively. Three out of the five eye bank criteria were found to correlate with postoperative cornea guttata with a highly significant p value of < 0.001. These three criteria are the presence of less than 50% of the cells having a hexagonal or a circular shape, the presence of cell membrane defects and interruptions and, the presence of blebs. The presence of groups of cells with a distinct whitish color was only a weak predictive factor for cornea guttata (p = 0.069). There was no statistically significant correlation between the number and the area of cell-depleted surfaces and postoperative cornea guttata with a p = 0.181. Conclusion Three semiquantitative criteria that can be detected in the eye bank using inverted light microscopy seem to correlate with postoperative cornea guttata: The presence of blebs, the presence of cell membrane defects and interruptions, as well as endothelial pictures with less than 50% of the cells having a hexagonal of circular shape. The presence of groups of cells with a distinct whitish color appears to be a weak predictor of cornea guttata.