Mono-UFMylation promotes misfolding-associated secretion of α-synuclein.

Stressed cells secret misfolded proteins lacking signaling sequence via an unconventional protein secretion (UcPS) pathway, but how misfolded proteins are targeted selectively in UcPS is unclear. Here, we report that misfolded UcPS clients are subject to modification by a ubiquitin-like protein named ubiquitin-fold modifier 1 (UFM1). Using α-synuclein (α-Syn) as a UcPS model, we show that mutating the UFMylation sites in α-Syn or genetic inhibition of the UFMylation system mitigates α-Syn secretion, whereas overexpression of UFBP1, a component of the endoplasmic reticulum-associated UFMylation ligase complex, augments α-Syn secretion in mammalian cells and in model organisms. UFM1 itself is cosecreted with α-Syn, and the serum UFM1 level correlates with that of α-Syn. Because UFM1 can be directly recognized by ubiquitin specific peptidase 19 (USP19), a previously established UcPS stimulator known to associate with several chaperoning activities, UFMylation might facilitate substrate engagement by USP19, allowing stringent and regulated selection of misfolded proteins for secretion and proteotoxic stress alleviation.

Analysis of Changes in Sleep Quality and Patterns after Hip Fracture Using Real Evidence of Artificial Intelligence Linked (REAL) Hip Cohort Data.

Background and Objectives: Hip fractures are commonly found in elderly patients, and often result in chronic pain and decreased physical function, as well as worsening of overall health. It is known that early surgical intervention during the acute phase and rehabilitation are important for improving clinical outcomes for these patients. However, the importance of management for improving the quality of life of these patients is becoming more emphasized. Studies on changes in sleep patterns after hip fractures are rare overseas. Therefore, the aim of this study is to investigate the prevalence of sleep disturbance in patients with hip fractures and to analyze the changes in sleep disturbance after surgery by comparing the preoperative and postoperative results. Materials and Methods: During the period from August 2022 to January 2023, patients who underwent surgical treatment for hip fractures and were recruited into the REAL Hip Cohort were selected as research subjects. The sleep survey was conducted using the Pittsburgh Sleep Quality Index (PSQI). The PSQI is composed of 18 questions, each divided into areas of sleep quality, sleep latency, duration, efficiency, disturbance, use of medication, and daytime dysfunction. Each area is scored 0-3 points and the total is 0-21. A score greater than five indicates sleep disorder. The PSQI was surveyed during hospitalization and three months after surgery for post-fracture sleep status. To analyze changes before and after the fracture, paired T-tests and chi-square tests were performed. Results: From August 2022 to January 2023, a total of 40 patients who were recruited into the REAL Hip Cohort responded to the PSQI survey. The average age was 77.4 years and 36 were female. Sleep quality worsened from 0.75 ± 1.0 before surgery to 1.4 ± 1.0 three months after surgery (p = 0.019), and sleep efficiency also worsened from 0.4 ± 0.6 to 1.4 ± 1.0 (p < 0.001). The PSQI increased from an average of 5.2 ± 2.8 before surgery to 8.2 ± 4.2 three months after surgery (p = 0.007), and the number of patients who could be diagnosed with sleep disorders also increased from 12 (40%) to 24 (60%) (p = 0.030). Conclusions: A decline in overall sleep status was observed in patients in a survey on sleep patterns three months after hip fracture. Additional management is needed to improve their sleep patterns.

APOL1-G2 accelerates nephrocyte cell death by inhibiting the autophagy pathway.

People of African ancestry who carry the APOL1 risk alleles G1 or G2 are at high risk of developing kidney diseases through not fully understood mechanisms that impair the function of podocytes. It is also not clear whether the APOL1-G1 and APOL1-G2 risk alleles affect these cells through similar mechanisms. Previously, we have developed transgenic Drosophila melanogaster lines expressing either the human APOL1 reference allele (G0) or APOL1-G1 specifically in nephrocytes, the cells homologous to mammalian podocytes. We have found that nephrocytes that expressed the APOL1-G1 risk allele display accelerated cell death, in a manner similar to that of cultured human podocytes and APOL1 transgenic mouse models. Here, to compare how the APOL1-G1 and APOL1-G2 risk alleles affect the structure and function of nephrocytes in vivo, we generated nephrocyte-specific transgenic flies that either expressed the APOL1-G2 or both G1 and G2 (G1G2) risk alleles on the same allele. We found that APOL1-G2- and APOL1-G1G2-expressing nephrocytes developed more severe changes in autophagic pathways, acidification of organelles and the structure of the slit diaphragm, compared to G1-expressing nephrocytes, leading to their premature death. We conclude that both risk alleles affect similar key cell trafficking pathways, leading to reduced autophagy and suggesting new therapeutic targets to prevent APOL1 kidney diseases.

A SNARE protective pool antagonizes APOL1 renal toxicity in Drosophila nephrocytes.

BACKGROUND: People of Sub-Saharan African ancestry are at higher risk of developing chronic kidney disease (CKD), attributed to the Apolipoprotein L1 (APOL1) gene risk alleles (RA) G1 and G2. The underlying mechanisms by which the APOL1-RA precipitate CKD remain elusive, hindering the development of potential treatments. RESULTS: Using a Drosophila genetic modifier screen, we found that SNARE proteins (Syx7, Ykt6, and Syb) play an important role in preventing APOL1 cytotoxicity. Reducing the expression of these SNARE proteins significantly increased APOL1 cytotoxicity in fly nephrocytes, the equivalent of mammalian podocytes, whereas overexpression of Syx7, Ykt6, or Syb attenuated their toxicity in nephrocytes. These SNARE proteins bound to APOL1-G0 with higher affinity than APOL1-G1/G2, and attenuated APOL1-G0 cytotoxicity to a greater extent than either APOL1-RA. CONCLUSIONS: Using a Drosophila screen, we identified SNARE proteins (Syx7, Ykt6, and Syb) as antagonists of APOL1-induced cytotoxicity by directly binding APOL1. These data uncovered a new potential protective role for certain SNARE proteins in the pathogenesis of APOL1-CKD and provide novel therapeutic targets for APOL1-associated nephropathies.

A Critical Role for ERO1α in Arterial Thrombosis and Ischemic Stroke.

BACKGROUND: Platelet adhesion and aggregation play a crucial role in arterial thrombosis and ischemic stroke. Here, we identify platelet ERO1α (endoplasmic reticulum oxidoreductase 1α) as a novel regulator of Ca2+ signaling and a potential pharmacological target for treating thrombotic diseases. METHODS: Intravital microscopy, animal disease models, and a wide range of cell biological studies were utilized to demonstrate the pathophysiological role of ERO1α in arteriolar and arterial thrombosis and to prove the importance of platelet ERO1α in platelet activation and aggregation. Mass spectrometry, electron microscopy, and biochemical studies were used to investigate the molecular mechanism. We used novel blocking antibodies and small-molecule inhibitors to study whether ERO1α can be targeted to attenuate thrombotic conditions. RESULTS: Megakaryocyte-specific or global deletion of Ero1α in mice similarly reduced platelet thrombus formation in arteriolar and arterial thrombosis without affecting tail bleeding times and blood loss following vascular injury. We observed that platelet ERO1α localized exclusively in the dense tubular system and promoted Ca2+ mobilization, platelet activation, and aggregation. Platelet ERO1α directly interacted with STIM1 (stromal interaction molecule 1) and SERCA2 (sarco/endoplasmic reticulum Ca2+-ATPase 2) and regulated their functions. Such interactions were impaired in mutant STIM1-Cys49/56Ser and mutant SERCA2-Cys875/887Ser. We found that ERO1α modified an allosteric Cys49-Cys56 disulfide bond in STIM1 and a Cys875-Cys887 disulfide bond in SERCA2, contributing to Ca2+ store content and increasing cytosolic Ca2+ levels during platelet activation. Inhibition of Ero1α with small-molecule inhibitors but not blocking antibodies attenuated arteriolar and arterial thrombosis and reduced infarct volume following focal brain ischemia in mice. CONCLUSIONS: Our results suggest that ERO1α acts as a thiol oxidase for Ca2+ signaling molecules, STIM1 and SERCA2, and enhances cytosolic Ca2+ levels, promoting platelet activation and aggregation. Our study provides evidence that ERO1α may be a potential target to reduce thrombotic events.

Aspergillus Galactomannan Titer as a Diagnostic Marker of Invasive Pulmonary Aspergillosis in Lung Transplant Recipients: A Single-Center Retrospective Cohort Study.

Invasive pulmonary aspergillosis (IPA) can occur in immunocompromised patients, and an early detection and intensive treatment are crucial. We sought to determine the potential of Aspergillus galactomannan antigen titer (AGT) in serum and bronchoalveolar lavage fluid (BALF) and serum titers of beta-D-glucan (BDG) to predict IPA in lung transplantation recipients, as opposed to pneumonia unrelated to IPA. We retrospectively reviewed the medical records of 192 lung transplant recipients. Overall, 26 recipients had been diagnosed with proven IPA, 40 recipients with probable IPA, and 75 recipients with pneumonia unrelated to IPA. We analyzed AGT levels in IPA and non-IPA pneumonia patients and used ROC curves to determine the diagnostic cutoff value. The Serum AGT cutoff value was 0.560 (index level), with a sensitivity of 50%, specificity of 91%, and AUC of 0.724, and the BALF AGT cutoff value was 0.600, with a sensitivity of 85%, specificity of 85%, and AUC of 0.895. Revised EORTC suggests a diagnostic cutoff value of 1.0 in both serum and BALF AGT when IPA is highly suspicious. In our group, serum AGT of 1.0 showed a sensitivity of 27% and a specificity of 97%, and BALF AGT of 1.0 showed a sensitivity of 60% and a specificity of 95%. The result suggested that a lower cutoff could be beneficial in the lung transplant group. In multivariable analysis, serum and BALF AGT, with a minimal correlation between the two, showed a correlation with a history of diabetes mellitus.

In vivo longitudinal 920†nm two-photon intravital kidney imaging of a dynamic 2,8-DHA crystal formation and tubular deterioration in the adenine-induced chronic kidney disease mouse model.

Chronic kidney disease (CKD) is one of the most common renal diseases manifested by gradual loss of kidney function with no symptoms in the early stage. The underlying mechanism in the pathogenesis of CKD with various causes such as high blood pressure, diabetes, high cholesterol, and kidney infection is not well understood. In vivo longitudinal repetitive cellular-level observation of the kidney of the CKD animal model can provide novel insights to diagnose and treat the CKD by visualizing the dynamically changing pathophysiology of CKD with its progression over time. In this study, using two-photon intravital microscopy with a single 920 nm fixed-wavelength fs-pulsed laser, we longitudinally and repetitively observed the kidney of an adenine diet-induced CKD mouse model for 30 days. Interestingly, we could successfully visualize the 2,8-dihydroxyadenine (2,8-DHA) crystal formation with a second-harmonics generation (SHG) signal and the morphological deterioration of renal tubules with autofluorescence using a single 920 nm two-photon excitation. The longitudinal in vivo two-photon imaging results of increasing 2,8-DHA crystals and decreasing tubular area ratio visualized by SHG and autofluorescence signal, respectively, were highly correlated with the CKD progression monitored by a blood test showing increased cystatin C and blood urea nitrogen (BUN) levels over time. This result suggests the potential of label-free second-harmonics generation crystal imaging as a novel optical technique for in vivo CKD progression monitoring.

Effect of Propofol versus Sevoflurane Anesthesia on Acute Kidney Injury after Lung Transplantation Surgery: A Prospective Randomized Controlled Trial.

This prospective randomized controlled trial aimed to compare the effects of sevoflurane and propofol anesthesia on the occurrence of acute kidney injury (AKI) following lung transplantation (LTx) surgery. Sixty adult patients undergoing bilateral LTx were randomized to receive either inhalation of sevoflurane or continuous infusion of propofol for general anesthesia. The primary outcomes were AKI incidence according to the Acute Kidney Injury Network (AKIN) criteria and blood biomarker of kidney injury, including neutrophil gelatinase-associated lipocalin (NGAL) and cystatin C levels within 48 h of surgery. Serum interleukin (IL)-1ß, IL-6, tumor necrosis factor-α, and superoxide dismutase were measured before and after surgery. The post-operative 30-day morbidity and long-term mortality were also assessed. Significantly fewer patients in the propofol group developed AKI compared with the sevoflurane group (13% vs. 38%, p = 0.030). NGAL levels were significantly lower in the propofol group at immediately after, 24 h, and 48 h post-operation. IL-6 levels were significantly lower in the propofol group immediately after surgery. AKI occurrence was significantly associated with a lower 5-year survival rate. Total intravenous anesthesia with propofol reduced the AKI incidence in LTx compared with sevoflurane, which is understood to be mediated by the attenuation of inflammatory responses.

SARS-CoV-2 Nsp6 damages Drosophila heart and mouse cardiomyocytes through MGA/MAX complex-mediated increased glycolysis.

SARS-CoV-2 infection causes COVID-19, a severe acute respiratory disease associated with cardiovascular complications including long-term outcomes. The presence of virus in cardiac tissue of patients with COVID-19 suggests this is a direct, rather than secondary, effect of infection. Here, by expressing individual SARS-CoV-2 proteins in the Drosophila heart, we demonstrate interaction of virus Nsp6 with host proteins of the MGA/MAX complex (MGA, PCGF6 and TFDP1). Complementing transcriptomic data from the fly heart reveal that this interaction blocks the antagonistic MGA/MAX complex, which shifts the balance towards MYC/MAX and activates glycolysis-with similar findings in mouse cardiomyocytes. Further, the Nsp6-induced glycolysis disrupts cardiac mitochondrial function, known to increase reactive oxygen species (ROS) in heart failure; this could explain COVID-19-associated cardiac pathology. Inhibiting the glycolysis pathway by 2-deoxy-D-glucose (2DG) treatment attenuates the Nsp6-induced cardiac phenotype in flies and mice. These findings point to glycolysis as a potential pharmacological target for treating COVID-19-associated heart failure.

Intravital longitudinal cellular visualization of oral mucosa in a murine model based on rotatory side-view confocal endomicroscopy.

Oral mucosa is a soft tissue lining the inside of the mouth, protecting the oral cavity from microbiological insults. The mucosal immune system is composed of diverse types of cells that defend against a wide range of pathogens. The pathophysiology of various oral mucosal diseases has been studied mostly by ex vivo histological analysis of harvested specimens. However, to analyze dynamic cellular processes in the oral mucosa, longitudinal in vivo observation of the oral mucosa in a single mouse during pathogenesis is a highly desirable and efficient approach. Herein, by utilizing micro GRIN lens-based rotatory side-view confocal endomicroscopy, we demonstrated non-invasive longitudinal cellular-level in vivo imaging of the oral mucosa, visualizing fluorescently labeled cells including various immune cells, pericytes, nerve cells, and lymphatic and vascular endothelial cells. With rotational and sliding movement of the side-view endomicroscope on the oral mucosa, we successfully achieved a multi-color wide-area cellular-level visualization in a noninvasive manner. By using a transgenic mouse expressing photoconvertible protein, Kaede, we achieved longitudinal repetitive imaging of the same microscopic area in the buccal mucosa of a single mouse for up to 10 days. Finally, we performed longitudinal intravital visualization of the oral mucosa in a DNFB-derived oral contact allergy mouse model, which revealed highly dynamic spatiotemporal changes of CSF1R or LysM expressing immune cells such as monocytes, macrophages, and granulocytes in response to allergic challenge for one week. This technique can be a useful tool to investigate the complex pathophysiology of oral mucosal diseases.

In vivo observation of multi-phase spatiotemporal cellular dynamics of transplanted HSPCs during early engraftment.

Hematopoietic stem cell transplantation (HSCT) is commonly used to treat patients with various blood disorders, genetic and immunological diseases, and solid tumors. Several systemic complications following HSCT are critical limiting factors for achieving a successful outcome. These systemic complications are mainly due to the lack of initial engraftment after transplantation. However, the detailed underlying cellular dynamics of early engraftment have not been fully characterized yet. We performed in vivo longitudinal visualization of early engraftment characteristics of transplanted hematopoietic stem and progenitor cells (HSPCs) in the mouse calvarial bone marrow (BM). To achieve this, we utilized an in vivo laser-scanning confocal microscopy imaging system with a cranial BM imaging window and stereotaxic device. We observed two distinct cellular behaviors of HSPCs in vivo, cluster formation and cluster dissociation, early after transplantation. Furthermore, we successfully identified three cellular phases of engraftment with distinct cellular distances which are coordinated with cell proliferation and cell migration dynamics during initial engraftment.

Rhomboid protease RHBDL4 promotes retrotranslocation of aggregation-prone proteins for degradation.

Protein degradation is fundamentally important to ensure cell homeostasis. In the endoplasmic reticulum (ER), the ER-associated degradation (ERAD) pathway targets incorrectly folded and unassembled proteins for turnover by the cytoplasmic proteasome. Previously, we showed that the rhomboid protease RHBDL4, together with p97, mediates membrane protein degradation. However, whether RHBDL4 acts in concert with additional ERAD components is unclear, and its full substrate spectrum remains to be defined. Here, we show that, in addition to membrane proteins, RHBDL4 cleaves aggregation-prone luminal ERAD substrates. Since mutations of the RHBDL4 rhomboid domain led to stabilization of substrates at the cytoplasmic side, we hypothesize that, analogous to the homolog ERAD factor derlin, RHBDL4 is directly involved in substrate retrotranslocation. RHBDL4's interaction with the erlin ERAD complex and reciprocal interaction of rhomboid substrates with erlins suggest that RHBDL4 and erlins form a complex that clips substrates and thereby rescues aggregation-prone peptides in the ER from aggregation.

Seamless Capacitive Body Channel Wireless Power Transmission Toward Freely Moving Multiple Animals in an Animal Cage.

Unstable wireless power transmission toward multiple living animals in an animal cage is one of the significant barriers to performing long-term and real-time neural monitoring in preclinical research. Here, seamless capacitive body channel (SCB) wireless power transmission (WPT) along with power management integrated circuit (PMIC) is designed using a standard 65 nm CMOS process. The SCB WPT enables stable wireless power transmission toward multiple 35 mm×20 mm×2 mm sized receivers (RXs) attached to freely moving animals in a 600 mm×600 mm×120 mm sized animal cage. By utilizing fringe-field capacitance and a body channel for wireless power link between the cage and RXs, the maximum difference in all measured power efficiencies in diverse scenarios is only 6.66 % with a 20 mW load. Even with a 90 ° RX rotation against the cage, power efficiency marks 17.76 %. Furthermore, an in-vivo experiment conducted with three untethered rats demonstrates the capability of continuous long-term power delivery in practical situations.

Longitudinal intravital imaging of cerebral microinfarction reveals a dynamic astrocyte reaction leading to glial scar formation.

Cerebral microinfarct increases the risk of dementia. But how microscopic cerebrovascular disruption affects the brain tissue in cellular-level are mostly unknown. Herein, with a longitudinal intravital imaging, we serially visualized in vivo dynamic cellular-level changes in astrocyte, pericyte and neuron as well as microvascular integrity after the induction of cerebral microinfarction for 1 month in mice. At day 2-3, it revealed a localized edema with acute astrocyte loss, neuronal death, impaired pericyte-vessel coverage and extravascular leakage of 3 kDa dextran (but not 2 MDa dextran) indicating microinfarction-related blood-brain barrier (BBB) dysfunction for small molecules. At day 5, the local edema disappeared with the partial restoration of microcirculation and recovery of pericyte-vessel coverage and BBB integrity. But brain tissue continued to shrink with persisted loss of astrocyte and neuron in microinfarct until 30 days, resulting in a collagen-rich fibrous scar surrounding the microinfarct. Notably, reactive astrocytes expressing glial fibrillary acidic protein (GFAP) appeared at the peri-infarct area early at day 2 and thereafter accumulated in the peri-infarct until 30 days, inducing glial scar formation in cerebral cortex. Our longitudinal intravital imaging of serial microscopic neurovascular pathophysiology in cerebral microinfarction newly revealed that astrocytes are critically susceptible to the acute microinfarction and their reactive response leads to the fibrous glial scar formation.

3D Visualization of Dynamic Cellular Reaction of Pulpal CD11c+ Dendritic Cells against Pulpitis in Whole Murine Tooth.

In dental pulp, diverse types of cells mediate the dental pulp immunity in a highly complex and dynamic manner. Yet, 3D spatiotemporal changes of various pulpal immune cells dynamically reacting against foreign pathogens during immune response have not been well characterized. It is partly due to the technical difficulty in detailed 3D comprehensive cellular-level observation of dental pulp in whole intact tooth beyond the conventional histological analysis using thin tooth slices. In this work, we validated the optical clearing technique based on modified Murray's clear as a valuable tool for a comprehensive cellular-level analysis of dental pulp. Utilizing the optical clearing, we successfully achieved a 3D visualization of CD11c+ dendritic cells in the dentin-pulp complex of a whole intact murine tooth. Notably, a small population of unique CD11c+ dendritic cells extending long cytoplasmic processes into the dentinal tubule while located at the dentin-pulp interface like odontoblasts were clearly visualized. 3D visualization of whole murine tooth enabled a reliable observation of these rarely existing cells with a total number less than a couple of tens in one tooth. These CD11c+ dendritic cells with processes in the dentinal tubule were significantly increased in the dental pulpitis model induced by mechanical and chemical irritation. Additionally, the 3D visualization revealed a distinct spatial 3D arrangement of pulpal CD11c+ cells in the pulp into a front-line barrier-like formation in the pulp within 12 h after the irritation. Collectively, these observations demonstrated the unique capability of optical clearing-based comprehensive 3D cellular-level visualization of the whole tooth as an efficient method to analyze 3D spatiotemporal changes of various pulpal cells in normal and pathological conditions.

The Prediction and Prognosis of Fungal Infection in Lung Transplant Recipients-A Retrospective Cohort Study in South Korea.

(1) Background: Lung transplant recipients (LTRs) are at substantial risk of invasive fungal disease (IFD), although no consensus has been reached on the use of antifungal agents (AFAs) after lung transplantation (LTx). This study aimed to assess the risk factors and prognosis of fungal infection after LTx in a single tertiary center in South Korea. (2) Methods: The study population included all patients who underwent LTx between January 2012 and July 2019 at a tertiary hospital. It was a retrospective cohort study. Culture, bronchoscopy, and laboratory findings were reviewed during episodes of infection. (3) Results: Fungus-positive respiratory samples were predominant in the first 90 days and the overall cumulative incidence of Candida spp. was approximately three times higher than that of Aspergillus spp. In the setting of itraconazole administration for 6 months post-LTx, C. glabrata accounted for 36.5% of all Candida-positive respiratory samples. Underlying connective tissue disease-associated interstitial lung disease, use of AFAs before LTx, a longer length of hospital stay after LTx, and old age were associated with developing a fungal infection after LTx. IFD and fungal infection treatment failure significantly increased overall mortality. Host factors, antifungal drug resistance, and misdiagnosis of non-Aspergillus molds could attribute to the breakthrough fungal infections. (4) Conclusions: Careful bronchoscopy, prompt fungus culture, and appropriate use of antifungal therapies are recommended during the first year after LTx.

χ-separation: Magnetic susceptibility source separation toward iron and myelin mapping in the brain.

Obtaining a histological fingerprint from the in-vivo brain has been a long-standing target of magnetic resonance imaging (MRI). In particular, non-invasive imaging of iron and myelin, which are involved in normal brain functions and are histopathological hallmarks in neurodegenerative diseases, has practical utilities in neuroscience and medicine. Here, we propose a biophysical model that describes the individual contribution of paramagnetic (e.g., iron) and diamagnetic (e.g., myelin) susceptibility sources to the frequency shift and transverse relaxation of MRI signals. Using this model, we develop a method, χ-separation, that generates the voxel-wise distributions of the two sources. The method is validated using computer simulation and phantom experiments, and applied to ex-vivo and in-vivo brains. The results delineate the well-known histological features of iron and myelin in the specimen, healthy volunteers, and multiple sclerosis patients. This new technology may serve as a practical tool for exploring the microstructural information of the brain.

Intravital Imaging of Circulating Red Blood Cells in the Retinal Vasculature of Growing Mice.

Purpose: To establish a custom-built, high-speed 90 frame-per-second laser-scanning confocal microscope for real-time in vivo retinal imaging of individual flowing red blood cells (RBCs) in retinal vasculature of live mouse model. Methods: Fluorescently labeled RBCs were injected into mice of different ages (3 to 62 weeks old). Anti-CD31 antibody conjugated with Alexa Fluor 647 was injected to visualize retinal endothelial cells (ECs). Longitudinal and cross-sectional intravital retinal imaging of flowing RBCs and ECs was performed in two strains (C57BL/6 and Balb/c) by using the custom-built confocal microscope. Results: Simultaneous tracking of the routes of many fluorescently labeled individual RBCs flowing from a large artery and vein to a single capillary in the retina of live mice was achieved, which enabled in vivo measurement of retinal RBC flow velocities in each vessel type in growing mice from 3 to 62 weeks after birth. Average RBC flow velocities were gradually increased during growing from 3 to 14 weeks by more than two times. Then the average RBC flow velocity was maintained at about 20 mm/s in artery and 16 mm/s in vein until 62 weeks. Conclusions: Our study successfully established a custom-built high-speed 90-Hz retinal confocal microscope for measuring RBC flow velocity at the single cell level. It could be a useful tool to investigate the pathophysiology of various retinal diseases associated with blood flow impairment. Translational Relevance: This technological method could be a valuable assessment tool to help the development of novel therapeutics for retinal diseases.

Characterization of SARS-CoV-2 proteins reveals Orf6 pathogenicity, subcellular localization, host interactions and attenuation by Selinexor.

BACKGROUND: SARS-CoV-2 causes COVID-19 which has a widely diverse disease profile. The mechanisms underlying its pathogenicity remain unclear. We set out to identify the SARS-CoV-2 pathogenic proteins that through host interactions cause the cellular damages underlying COVID-19 symptomatology. METHODS: We examined each of the individual SARS-CoV-2 proteins for their cytotoxicity in HEK 293 T cells and their subcellular localization in COS-7 cells. We also used Mass-Spec Affinity purification to identify the host proteins interacting with SARS-CoV-2 Orf6 protein and tested a drug that could inhibit a specific Orf6 and host protein interaction. RESULTS: We found that Orf6, Nsp6 and Orf7a induced the highest toxicity when over-expressed in human 293 T cells. All three proteins showed membrane localization in COS-7 cells. We focused on Orf6, which was most cytotoxic and localized to the endoplasmic reticulum, autophagosome and lysosomal membranes. Proteomics revealed Orf6 interacts with nucleopore proteins (RAE1, XPO1, RANBP2 and nucleoporins). Treatment with Selinexor, an FDA-approved inhibitor for XPO1, attenuated Orf6-induced cellular toxicity in human 293 T cells. CONCLUSIONS: Our study revealed Orf6 as a highly pathogenic protein from the SARS-CoV-2 genome, identified its key host interacting proteins, and Selinexor as a drug candidate for directly targeting Orf6 host protein interaction that leads to cytotoxicity.

Functional analysis of SARS-CoV-2 proteins in Drosophila identifies Orf6-induced pathogenic effects with Selinexor as an effective treatment.

BACKGROUND: SARS-CoV-2 causes COVID-19 with a widely diverse disease profile that affects many different tissues. The mechanisms underlying its pathogenicity in host organisms remain unclear. Animal models for studying the pathogenicity of SARS-CoV-2 proteins are lacking. METHODS: Using bioinformatic analysis, we found that 90% of the virus-host interactions involve human proteins conserved in Drosophila. Therefore, we generated a series of transgenic fly lines for individual SARS-CoV-2 genes, and used the Gal4-UAS system to express these viral genes in Drosophila to study their pathogenicity. RESULTS: We found that the ubiquitous expression of Orf6, Nsp6 or Orf7a in Drosophila led to reduced viability and tissue defects, including reduced trachea branching as well as muscle deficits resulting in a "held-up" wing phenotype and poor climbing ability. Furthermore, muscles in these flies showed dramatically reduced mitochondria. Since Orf6 was found to interact with nucleopore proteins XPO1, we tested Selinexor, a drug that inhibits XPO1, and found that it could attenuate the Orf6-induced lethality and tissue-specific phenotypes observed in flies. CONCLUSIONS: Our study established Drosophila as a model for studying the function of SARS-CoV2 genes, identified Orf6 as a highly pathogenic protein in various tissues, and demonstrated the potential of Selinexor for inhibiting Orf6 toxicity using an in vivo animal model system.