Analytical validation of quantitative SARS-CoV-2 subgenomic and viral load laboratory developed tests conducted on the Panther Fusion® (Hologic) with preliminary application to clinical samples.

OBJECTIVE: Validate the performance characteristics of two analyte specific, laboratory developed tests (LDTs) for the quantification of SARS-CoV-2 subgenomic RNA (sgRNA) and viral load on the Hologic Panther Fusion® using the Open Access functionality. METHODS: Custom-designed primers/probe sets targeting the SARS-CoV-2 Envelope gene (E) and subgenomic E were optimized. A 20-day performance validation following laboratory developed test requirements was conducted to assess assay precision, accuracy, analytical sensitivity/specificity, lower limit of detection and reportable range. RESULTS: Quantitative SARS-CoV-2 sgRNA (LDT-Quant sgRNA) assay, which measures intermediates of replication, and viral load (LDT-Quant VLCoV) assay demonstrated acceptable performance. Both assays were linear with an R2 and slope equal to 0.99 and 1.00, respectively. Assay precision was evaluated between 4-6 Log10 with a maximum CV of 2.6% and 2.5% for LDT-Quant sgRNA and LDT-Quant VLCoV respectively. Using negative or positive SARS-CoV-2 human nasopharyngeal swab samples, both assays were accurate (kappa coefficient of 1.00 and 0.92). Common respiratory flora and other viral pathogens were not detected and did not interfere with the detection or quantification by either assay. Based on 95% detection, the assay LLODs were 729 and 1206 Copies/mL for the sgRNA and VL load LDTs, respectively. CONCLUSION: The LDT-Quant sgRNA and LDT-Quant VLCoV demonstrated good analytical performance. These assays could be further investigated as alternative monitoring assays for viral replication; and thus, medical management in clinical settings which could inform isolation/quarantine requirements.

Viscoelastic Biomechanical Properties of the Conventional Aqueous Outflow Pathway Tissues in Healthy and Glaucoma Human Eyes.

BACKGROUND: Although the tissues comprising the ocular conventional outflow pathway have shown strong viscoelastic mechanical response to aqueous humor pressure dynamics, the viscoelastic mechanical properties of the trabecular meshwork (TM), juxtacanalicular connective tissue (JCT), and Schlemm's canal (SC) inner wall are largely unknown. METHODS: A quadrant of the anterior segment from two human donor eyes at low- and high-flow (LF and HF) outflow regions was pressurized and imaged using optical coherence tomography (OCT). A finite element (FE) model of the TM, the adjacent JCT, and the SC inner wall was constructed and viscoelastic beam elements were distributed in the extracellular matrix (ECM) of the TM and JCT to represent anisotropic collagen. An inverse FE-optimization algorithm was used to calculate the viscoelastic properties of the ECM/beam elements such that the TM/JCT/SC model and OCT imaging data best matched over time. RESULTS: The ECM of the glaucoma tissues showed significantly larger time-dependent shear moduli compared to the heathy tissues. Significantly larger shear moduli were also observed in the LF regions of both the healthy and glaucoma eyes compared to the HF regions. CONCLUSIONS: The outflow tissues in both glaucoma eyes and HF regions are stiffer and less able to respond to dynamic IOP.

Estimating Human Trabecular Meshwork Stiffness by Numerical Modeling and Advanced OCT Imaging.

Purpose: The purpose of this study was to estimate human trabecular meshwork (hTM) stiffness, thought to be elevated in glaucoma, using a novel indirect approach, and to compare results with direct en face atomic force microscopy (AFM) measurements. Methods: Postmortem human eyes were perfused to measure outflow facility and identify high- and low-flow regions (HF, LF) by tracer. Optical coherence tomography (OCT) images were obtained as Schlemm's canal luminal pressure was directly manipulated. TM stiffness was deduced by an inverse finite element modeling (FEM) approach. A series of AFM forcemaps was acquired along a line traversing the anterior angle on a radially cut flat-mount corneoscleral wedge with TM facing upward. Results: The elastic modulus of normal hTM estimated by inverse FEM was 70 ± 20 kPa (mean ± SD), whereas glaucomatous hTM was slightly stiffer (98 ± 19 kPa). This trend was consistent with TM stiffnesses measured by AFM: normal hTM stiffness = 1.37 ± 0.56 kPa, which was lower than glaucomatous hTM stiffness (2.75 ± 1.19 kPa). None of these differences were statistically significant. TM in HF wedges was softer than that in LF wedges for both normal and glaucomatous eyes based on the inverse FEM approach but not by AFM. Outflow facility was significantly correlated with TM stiffness estimated by FEM in six human eyes (P = 0.018). Conclusions: TM stiffness is higher, but only modestly so, in glaucomatous patients. Outflow facility in both normal and glaucomatous human eyes appears to associate with TM stiffness. This evidence motivates further studies to investigate factors underlying TM biomechanical property regulation.

Aqueous outflow regulation: Optical coherence tomography implicates pressure-dependent tissue motion.

Glaucoma is a leading cause of blindness worldwide and results from damage to the optic nerve. Currently, intraocular pressure is the only treatable risk factor. Changes in aqueous outflow regulate pressure; regulation becomes abnormal in glaucoma. From inside the eye aqueous flows out through the trabecular meshwork into a venous sinus called Schlemm's canal, next into collector channels and finally returns to the episcleral vessels of the venous system. The location of aqueous outflow regulation is unknown. Ex vivo and in vivo studies implicate both pressure-dependent trabecular tissue motion and tissues distal to Schlemm's canal in regulation of aqueous outflow. Technologies have not previously been available to study these issues. New ex vivo imaging in human eyes identifies hinged flaps or leaflets at collector channel entrances using a high-resolution spectral domain optical coherence tomography (SD-OCT) platform. The hinged flaps open and close in synchrony with pressure-dependent trabecular meshwork motion. The SD-OCT platform images from the trabecular meshwork surface while experimentally changing transtrabecular pressure gradients. New in vivo imaging in human eyes uses a motion sensitive technology, phase-sensitive OCT to quantitate real-time pulse-dependent trabecular tissue motion as well as absence of such motion when aqueous access to the outflow system is blocked. The recent studies suggest that aqueous outflow regulation results from synchronous pressure-dependent motion involving a network of interconnected tissues including those distal to Schlemm's canal. The new imaging technologies may shed light on glaucoma mechanisms and provide guidance in the management of medical, laser and surgical decisions in glaucoma.

Platform to investigate aqueous outflow system structure and pressure-dependent motion using high-resolution spectral domain optical coherence tomography.

The aqueous outflow system (AOS) is responsible for maintaining normal intraocular pressure (IOP) in the eye. Structures of the AOS have an active role in regulating IOP in healthy eyes and these structures become abnormal in the eyes with glaucoma. We describe a newly developed system platform to obtain high-resolution images of the AOS structures. By incorporating spectral domain optical coherence tomography (SD-OCT), the platform allows us to systematically control, image, and quantitate the responses of AOS tissue to pressure with a millisecond resolution of pulsed flow. We use SD-OCT to image radial limbal segments from the surface of the trabecular meshwork (TM) with a spatial resolution of ∼5 µm in ex vivo nonhuman primate eyes. We carefully insert a cannula into Schlemm's canal (SC) to control both pressures and flow rates. The experimental results demonstrate the capability of the platform to visualize the unprecedented details of AOS tissue components comparable to that delivered by scanning electron microscopy, as well as to delineate the complex pressure-dependent relationships among the TM, structures within the SC, and collector channel ostia. The described technique provides a new means to characterize the anatomic and pressure-dependent relationships of SC structures, particularly the active motion of collagenous elements at collector channel ostia; such relationships have not previously been amenable to study. Experimental findings suggest that continuing improvements in the OCT imaging of the AOS may provide both insights into the glaucoma enigma and improvements in its management.

Deletion of Dicer in late erythroid cells results in impaired stress erythropoiesis in mice.

MicroRNAs (miRNAs) have been shown to influence erythroid lineage commitment and differentiation; however, our knowledge of miRNA function in terminal erythropoiesis remains limited. To address this issue, we generated a novel animal model, where the miRNA-processing enzyme, Dicer, is selectively inactivated in erythropoietin receptor positive erythroid cells beginning with CFU-e/proerythroblast cells. This results in significant depletion of all miRNAs from the proerythroblast stage onwards, with one exception, miR-451, which is processed by Ago2 in a Dicer-independent manner. We observed that mature Dicer-dependent miRNAs, like miR-451, are dispensable under steady-state conditions, but these mutants have an impaired response to stress erythropoiesis, as demonstrated by a delay in recovery from anemia. This defect was specific to later maturing erythroid cells, as progenitor numbers were unaffected. In addition to generating a novel mouse model to study miRNA function in late erythroid cells, we conclude that miRNAs (both Dicer-dependent and independent) act primarily to regulate the optimal response to stress among late erythroid cells.

Stage-specific functional roles of integrins in murine erythropoiesis.

When the erythroid integrins α5ß1 and α4ß1 were each deleted previously at the stem cell level, they yielded distinct physiologic responses to stress by affecting erythoid expansion and terminal differentiation or only the latter, respectively. To test at what stage of differentiation the integrin effects were exerted, we created mice with α4- or α5-integrin deletions only in erythroid cells and characterized them at homeostasis and after phenylhydrazine-induced hemolytic stress. Unlike our prior data, the phenotype of mice with α5-erythroid deletions was similar to controls, especially after stress. These outcomes seem to reconcile divergent prior views on the role of α5-integrin in erythropoiesis. By contrast, α4 integrins whether deleted early or late have a dominant effect on bone marrow retention of erythroblasts and on terminal erythroid maturation at homeostasis and after stress.

Erythroid cells generated in the absence of specific ß1-integrin heterodimers accumulate reactive oxygen species at homeostasis and are unable to mount effective antioxidant defenses.

We have previously reported that ß1(Δ/Δ) mice have a markedly impaired response to hemolytic stress, but the mechanisms of this were unclear. In the present study we explored in detail quantitative, phenotypic and functional aspects of erythropoiesis at homeostasis in a large number of animals for each of 3 murine models with specific ß1 heterodimer integrin deficiencies. We found that, at homeostasis, ß1-deficient mice have a modest uncompensated anemia with ineffective erythropoiesis and decreased red blood cell survival. Mice lacking only α4 integrins (α4ß1/α4ß7) do not share this phenotype. There is an increased tendency for reactive oxygen species accumulation in ß1(Δ/Δ) erythroid cells with decreased anti-oxidant defenses at homeostasis which are exaggerated after stress. Furthermore, expansion of erythroid cells in spleen post-stress is dependent on α5ß1, likely through mechanisms activating focal adhesion kinase complexes that are distinct from α4ß1-mediated responses. In vivo inhibition of focal adhesion kinase activation partially recapitulates the ß1(Δ/Δ) stress response. Mice lacking all α4 and ß1 integrins (double knockouts) had, at homeostasis, the most severe phenotype with selective impairment of erythroid responses. The fact that integrins participate in mitigating stress in erythroid cells through redox activation of distinct signaling pathways by specific integrin heterodimers is a link that has not been appreciated until now.

Combinatorial and distinct roles of α5 and α4 integrins in stress erythropoiesis in mice.

To delineate the role of specific members of ß1 integrins in stress erythropoiesis in the adult, we compared the response to phenylhydrazine stress in 3 genetically deficient models. The survival of ß1-conditionally deficient mice after phenylhydrazine is severely compromised because of their inability to mount a successful life saving splenic erythroid response, a phenotype reproduced in ß1(Δ/Δ) reconstituted animals. The response of bone marrow to phenylhydrazine-induced stress was, unlike that of spleen, appropriate in terms of progenitor cell expansion and mobilization to peripheral blood although late differentiation defects qualitatively similar to those in spleen were present in bone marrow. In contrast to ß1-deficient mice, α4(Δ/Δ) mice showed only a kinetic delay in recovery and similar to ß1(Δ/Δ), terminal maturation defects in both bone marrow and spleen, which were not present in VCAM-1(Δ/Δ) mice. Convergence of information from these comparative studies lends new insight to the distinct in vivo roles of α4 and α5 integrins in erythroid stress, suggesting that the presence of mainly α5ß1 integrin in all hematopoietic progenitor cells interacting with splenic microenvironmental ligands/cells is instrumental for their survival and accumulation during hemolytic stress, whereas presence of α4 or of both α5 and α4, is important for completion of terminal maturation steps.

The phosphoinositide phosphatase Sjl2 is recruited to cortical actin patches in the control of vesicle formation and fission during endocytosis.

The Saccharomyces cerevisiae synaptojanin-like proteins (Sjl1, Sjl2, and Sjl3) are phosphoinositide (PI) phosphatases that regulate PI metabolism in the control of actin organization and membrane trafficking. However, the primary sites of action for each of the yeast synaptojanin-like proteins remain unclear. In this study, we show that Sjl2 is localized to cortical actin patches, sites of endocytosis. Cortical recruitment of Sjl2 requires the actin patch component Abp1. Consistent with this, the SH3 domain-containing protein Abp1 physically associates with Sjl2 through its proline-rich domain. Furthermore, abp1Delta mutations confer defects resembling loss of SJL2; sjl1Delta abp1Delta double-mutant cells exhibit invaginated plasma membranes and impaired endocytosis, findings similar to those for sjl1Delta sjl2Delta mutant cells. Thus, Abp1 acts as an adaptor protein in the localization or concentration of Sjl2 during late stages of endocytic vesicle formation. Overexpression of the Hip1-related protein Sla2 delayed the formation of extended plasma membrane invaginations in sjl2ts cells, indicating that Sla2 may become limiting or misregulated in cells with impaired PI phosphatase activity. Consistent with this, the cortical actin patch protein Sla2 is mislocalized in sjl1Delta sjl2Delta mutant cells. Together, our studies suggest that PI metabolism by the synaptojanin-like proteins coordinately directs actin dynamics and membrane invagination, in part by regulation of Sla2.

Retromer function in endosome-to-Golgi retrograde transport is regulated by the yeast Vps34 PtdIns 3-kinase.

A direct role for phosphoinositides in vesicular trafficking has been demonstrated by the identification of the yeast VPS34 gene encoding the phosphatidylinositol 3-kinase responsible for the synthesis of phosphatidylinositol 3-phosphate (PtdIns3P). Vps34p binds the protein kinase Vps15p, and it has recently been shown that Vps15p and Vps34p associate with Vps30p and Vps38p to form a multimeric complex, termed complex II. We observed that mutations in the VPS30 and VPS38 genes led to a selective sorting and maturation phenotype of the soluble vacuolar protease CPY. Localization studies revealed that the CPY receptor Vps10p and the Golgi-endoprotease Kex2p were mislocalized to vacuolar membranes in strains deficient for either Vps30p or Vps38p, respectively. Interestingly, we measured decreased PtdIns3P levels in Deltavps30 and Deltavps38 cells and observed redistribution of Vps5p and Vps17p to the cytoplasm in these mutants. Vps5p and Vps17p are subunits of the retromer complex that is required for endosome-to-Golgi retrograde transport. Both proteins contain the Phox homology (PX) domain, a recently identified phosphoinositide-binding motif. We demonstrate that the PX domains of Vps5p and Vps17p specifically bind to PtdIns3P in vitro and in vivo. On the basis of these and other observations, we propose that the PtdIns 3-kinase complex II directs the synthesis of a specific endosomal pool of PtdIns3P, which is required for recruitment/activation of the retromer complex, thereby ensuring efficient endosome-to-Golgi retrograde transport.