Enhanced SARS-CoV-2 entry via UPR-dependent AMPK-related kinase NUAK2.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remodels the endoplasmic reticulum (ER) to form replication organelles, leading to ER stress and unfolded protein response (UPR). However, the role of specific UPR pathways in infection remains unclear. Here, we found that SARS-CoV-2 infection causes marginal activation of signaling sensor IRE1α leading to its phosphorylation, clustering in the form of dense ER-membrane rearrangements with embedded membrane openings, and XBP1 splicing. By investigating the factors regulated by IRE1α-XBP1 during SARS-CoV-2 infection, we identified stress-activated kinase NUAK2 as a novel host-dependency factor for SARS-CoV-2, HCoV-229E, and MERS-CoV entry. Reducing NUAK2 abundance or kinase activity impaired SARS-CoV-2 particle binding and internalization by decreasing cell surface levels of viral receptors and viral trafficking likely by modulating the actin cytoskeleton. IRE1α-dependent NUAK2 levels were elevated in SARS-CoV-2-infected and bystander non-infected cells, promoting viral spread by maintaining ACE2 cell surface levels and facilitating virion binding to bystander cells.

Non-canonical autophagy functions of ATG16L1 in epithelial cells limit lethal infection by influenza A virus.

Influenza A virus (IAV) and SARS-CoV-2 (COVID-19) cause pandemic infections where cytokine storm syndrome and lung inflammation lead to high mortality. Given the high social and economic cost of respiratory viruses, there is an urgent need to understand how the airways defend against virus infection. Here we use mice lacking the WD and linker domains of ATG16L1 to demonstrate that ATG16L1-dependent targeting of LC3 to single-membrane, non-autophagosome compartments - referred to as non-canonical autophagy - protects mice from lethal IAV infection. Mice with systemic loss of non-canonical autophagy are exquisitely sensitive to low-pathogenicity IAV where extensive viral replication throughout the lungs, coupled with cytokine amplification mediated by plasmacytoid dendritic cells, leads to fulminant pneumonia, lung inflammation and high mortality. IAV was controlled within epithelial barriers where non-canonical autophagy reduced IAV fusion with endosomes and activation of interferon signalling. Conditional mouse models and ex vivo analysis showed that protection against IAV infection of lung was independent of phagocytes and other leucocytes. This establishes non-canonical autophagy in airway epithelial cells as a novel innate defence that restricts IAV infection and lethal inflammation at respiratory surfaces.

ESCPE-1 mediates retrograde endosomal sorting of the SARS-CoV-2 host factor Neuropilin-1.

Endosomal sorting maintains cellular homeostasis by recycling transmembrane proteins and associated proteins and lipids (termed "cargoes") from the endosomal network to multiple subcellular destinations, including retrograde traffic to the trans-Golgi network (TGN). Viral and bacterial pathogens subvert retrograde trafficking machinery to facilitate infectivity. Here, we develop a proteomic screen to identify retrograde cargo proteins of the endosomal SNX-BAR sorting complex promoting exit 1 (ESCPE-1). Using this methodology, we identify Neuropilin-1 (NRP1), a recently characterized host factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as a cargo directly bound and trafficked by ESCPE-1. ESCPE-1 mediates retrograde trafficking of engineered nanoparticles functionalized with the NRP1-interacting peptide of the SARS-CoV-2 spike (S) protein. CRISPR-Cas9 deletion of ESCPE-1 subunits reduces SARS-CoV-2 infection levels in cell culture. ESCPE-1 sorting of NRP1 may therefore play a role in the intracellular membrane trafficking of NRP1-interacting viruses such as SARS-CoV-2.

A widespread viral entry mechanism: The C-end Rule motif-neuropilin receptor interaction.

Many phylogenetically distant animal viruses, including the new coronavirus severe acute respiratory syndrome coronavirus 2, have surface proteins with polybasic sites that are cleaved by host furin and furin-like proteases. Other than priming certain viral surface proteins for fusion, cleavage generates a carboxy-terminal RXXR sequence. This C-end Rule (CendR) motif is known to bind to neuropilin (NRP) receptors on the cell surface. NRPs are ubiquitously expressed, pleiotropic cell surface receptors with important roles in growth factor signaling, vascular biology, and neurobiology, as well as immune homeostasis and activation. The CendR-NRP receptor interaction promotes endocytic internalization and tissue spreading of different cargo, including viral particles. We propose that the interaction between viral surface proteins and NRPs plays an underappreciated and prevalent role in the transmission and pathogenesis of diverse viruses and represents a promising broad-spectrum antiviral target.

Peptide-Directed Attachment of Hydroxylamines to Specific Lysines of IgG Antibodies for Bioconjugations with Acylboronates.

The role of monoclonal antibodies as vehicles to deliver payloads has evolved as a powerful tool in cancer therapy in recent years. The clinical development of therapeutic antibody conjugates with precise payloads holds great promise for targeted therapeutic interventions. The use of affinity-peptide mediated functionalization of native off-the-shelf antibodies offers an effective approach to selectively modify IgG antibodies with a drug-antibody ratio (DAR) of 2. Here, we report the traceless, peptide-directed attachment of two hydroxylamines to native IgGs followed by chemoselective potassium acyltrifluoroborate (KAT) ligation with quinolinium acyltrifluoroborates (QATs), which provide enhanced ligation rates with hydroxylamines under physiological conditions. By applying KAT ligation to the modified antibodies, conjugation of small molecules, proteins, and oligonucleotides to off-the-shelf IgGs proceeds efficiently, in good yields, and with simultaneous cleavage of the affinity peptide-directing moiety.

A method for the generation of pseudovirus particles bearing SARS coronavirus spike protein in high yields.

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has threatened human health and the global economy. Development of additional vaccines and therapeutics is urgently required, but such development with live virus must be conducted with biosafety level 3 confinement. Pseudotyped viruses have been widely adopted for studies of virus entry and pharmaceutical development to overcome this restriction. Here we describe a modified protocol to generate vesicular stomatitis virus (VSV) pseudotyped with SARS-CoV or SARS-CoV-2 spike protein in high yield. We found that a large proportion of pseudovirions produced with the conventional transient expression system lacked coronavirus spike protein at their surface as a result of inhibition of parental VSV infection by overexpression of this protein. Establishment of stable cell lines with an optimal expression level of coronavirus spike protein allowed the efficient production of progeny pseudoviruses decorated with spike protein. This improved VSV pseudovirus production method should facilitate studies of coronavirus entry and development of antiviral agents.Key words: severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, pseudovirus, vesicular stomatitis virus (VSV), spike protein.

Colonoscopic evaluation of diarrhea/colitis occurring as an immune-related adverse event.

OBJECTIVE: Diarrhea is often observed as an immune-related adverse event. In this study, we conducted a retrospective review of the severity of diarrhea, its treatment and the endoscopic findings in patients developing diarrhea as an immune-related adverse event. METHODS: From August 2015 to June 2019, a total of 369 patients received treatment with immune checkpoint inhibitors at our hospital. For this study, development of grade 2 or more diarrhea in these patients was defined as an immune-related adverse event. We analyzed the histopathological severity of the bowel lesions according to the Nancy histological index for ulcerative colitis. RESULTS: Of the 369 patients, 27 (7.3%) developed diarrhea as an immune-related adverse event. Of these 27 patients, 18 received steroid treatment. Colonoscopy was performed in 17 patients and culture of the feces in 18. The tests revealed evidence of bacterial colitis (Aeromonas hydrophila) in two patients. The Nancy histological index was 4, 3, 2, 1 and 0 in two, three, two, two and seven patients, respectively. No findings on colonoscopy were observed in 7 of the 17 patients (41%) who underwent colonoscopy, and most of these patients recovered without steroid treatment. Patients with lower values of the Nancy histological index tended to show better responses to steroid treatment. CONCLUSIONS: To avoid unnecessary steroid administration, colonoscopic evaluation is essential in patients receiving treatment with immune checkpoint inhibitors who present with diarrhea as an immune-related adverse event. In addition, the endoscopic findings could be useful to predict the response to steroid treatment.

Correction to: Increased prevalence of cerebral microbleeds in patients with low left ventricular systolic function.

In the original publication of the article, under the results section, the following sentence.

Increased prevalence of cerebral microbleeds in patients with low left ventricular systolic function.

Gradient-echo T2-star (T2*)-weighted magnetic resonance imaging (MRI) is a sensitive method to detect cerebral microbleeds (CMBs). The presence of CMBs was reported to be a marker of future cardiovascular mortality and is associated with various cardiovascular risk factors, use of antithrombotic drugs, and cognitive dysfunction. However, the relationship between cardiac function and CMBs remains unclear. We investigated the association between cardiac function and presence of CMBs in patients with cardiovascular diseases. This single-center retrospective study included a total of 424 participants (mean age 70 ± 12 years; men 286 (67%); mean left ventricular ejection fraction (LVEF) 61% ± 12%] who underwent echocardiography and brain T2*-weighted MRI within 1 month without neurologic abnormality. CMBs were found in 118 (28%) patients. There was no significant relationship between CMBs and anticoagulant or antiplatelet therapy. LVEF was significantly lower in patients with CMBs than in those without CMBs (59% ± 13% vs. 62% ± 11%, P < 0.05). On multivariate logistic analysis, lower LVEF [odds ratio (OR) 0.98, 95% confidence interval (CI) 0.96-1.00; P < 0.05] and age (OR 1.02, 95% CI 1.00-1.05; P < 0.05) were significantly associated with CMBs. The presence of CMBs was frequently observed in the patients with cardiovascular disease and was significantly associated with age and LVEF.

Improved diagnosis of the number of stenosed coronary artery vessels by segmentation with scatter and photo-peak window data for attenuation correction in myocardial perfusion SPECT.

BACKGROUND: Attenuation correction using segmentation of scatter and photo-peak window data (SSPAC) enables an evaluation of the attenuation map in a patient-specific manner without additional radiation exposure. We compared the accuracy of SSPAC and non-corrected myocardial perfusion scintigraphy methods for diagnosing the number of stenosed coronary artery vessels. METHODS AND RESULTS: We retrospectively reviewed the data from 183 consecutive patients who underwent 99mTc-tetrofosmin stress/rest SPECT examination and a coronary angiography within 3 months. The MPS images were reconstructed with and without SSPAC attenuation correction. We examined the accuracy of the quantitative interpretation using summed differential score in the detection of coronary artery disease (CAD). The attenuation maps were successfully determined in 179 of 183 patients (98%). In terms of the vessel-based diagnostic ability, sensitivity, specificity, positive predictive and negative predictive values of the SSPAC and non-correction methods for diagnosing CAD in individual coronary territories were 77%*, 89%, 74%*, and 90%* vs 51%, 87%, 62%, and 82%, respectively (*P < .05). In 35 patients with multi-vessel CAD, those values were 78%*, 81%, 93%, and 55%* vs 49%, 81%, 89%, and 34%, respectively (*P < .05; AUC: 0.82 vs 0.62, P < .05). CONCLUSION: SSPAC-corrected SPECT myocardial perfusion images exhibit improved accuracy in the detection of the number of stenosed coronary artery vessels, even in patients with multi-vessel CAD.

Principles of Virus Uncoating: Cues and the Snooker Ball.

Viruses are spherical or complex shaped carriers of proteins, nucleic acids and sometimes lipids and sugars. They are metastable and poised for structural changes. These features allow viruses to communicate with host cells during entry, and to release the viral genome, a process known as uncoating. Studies have shown that hundreds of host factors directly or indirectly support this process. The cell provides molecules that promote stepwise virus uncoating, and direct the virus to the site of replication. It acts akin to a snooker player who delivers accurate and timely shots (cues) to the ball (virus) to score. The viruses, on the other hand, trick (snooker) the host, hijack its homeostasis systems, and dampen innate immune responses directed against danger signals. In this review, we discuss how cellular cues, facilitators, and built-in viral mechanisms promote uncoating. Cues come from receptors, enzymes and chemicals that act directly on the virus particle to alter its structure, trafficking and infectivity. Facilitators are defined as host factors that are involved in processes which indirectly enhance entry or uncoating. Unraveling the mechanisms of virus uncoating will continue to enhance understanding of cell functions, and help counteracting infections with chemicals and vaccines.

IgG4-positive cell infiltration in various cardiovascular disorders - results from histopathological analysis of surgical samples.

BACKGROUND: The diagnosis of Immunoglobulin G4 (IgG4)-related disease (IgG4-RD), in general, depends on serum IgG4 concentrations and histopathological findings; therefore, diagnosis of IgG4-RD in cardiovascular organs/tissues is often difficult owing to the risk of tissue sampling. METHODS: Prevalence of IgG4-positive lymphoplasmacytic infiltration in 103 consecutive cardiovascular surgical samples from 98 patients with various cardiovascular diseases was analyzed immunohistochemically. RESULTS: The diagnoses of the enrolled patients included aortic aneurysm (abdominal, n = 8; thoracic, n = 9); aortic dissection (n = 20); aortic stenosis (n = 24), aortic regurgitation (n = 10), and mitral stenosis/regurgitation (n = 17). In total, 10 (9.7%) of the 103 specimens showed IgG4-positive cell infiltration with various intensities; five of these were aortic valve specimens from aortic stenosis, and IgG4-positive cell infiltration was present at >10 /HPF in three of them. In one aortic wall sample from an abdominal aortic aneurysm, various histopathological features of IgG4-RD, such as IgG4-positive cell infiltration, obliterating phlebitis, and storiform fibrosis, were observed. CONCLUSIONS: IgG4-positive cell infiltration was observed in 9.7% of the surgical cardiovascular specimens, mainly in the aortic valve from aortic stenosis and in the aortic wall from aortic aneurysm. Whether IgG4-positive cell infiltration has pathophysiological importance in the development or progression of cardiovascular diseases should be investigated in future studies.

ACAP3 regulates neurite outgrowth through its GAP activity specific to Arf6 in mouse hippocampal neurons.

ACAP3 (ArfGAP with coiled-coil, ankyrin repeat and pleckstrin homology domains 3) belongs to the ACAP family of GAPs (GTPase-activating proteins) for the small GTPase Arf (ADP-ribosylation factor). However, its specificity to Arf isoforms and physiological functions remain unclear. In the present study, we demonstrate that ACAP3 plays an important role in neurite outgrowth of mouse hippocampal neurons through its GAP activity specific to Arf6. In primary cultured mouse hippocampal neurons, knockdown of ACAP3 abrogated neurite outgrowth, which was rescued by ectopically expressed wild-type ACAP3, but not by its GAP activity-deficient mutant. Ectopically expressed ACAP3 in HEK (human embryonic kidney)-293T cells showed the GAP activity specific to Arf6. In support of this observation, the level of GTP-bound Arf6 was significantly increased by knockdown of ACAP3 in hippocampal neurons. In addition, knockdown and knockout of Arf6 in mouse hippocampal neurons suppressed neurite outgrowth. These results demonstrate that ACAP3 positively regulates neurite outgrowth through its GAP activity specific to Arf6. Furthermore, neurite outgrowth suppressed by ACAP3 knockdown was rescued by expression of a fast cycle mutant of Arf6 that spontaneously exchanges guanine nucleotides on Arf6, but not by that of wild-type, GTP- or GDP-locked mutant Arf6. Thus cycling between active and inactive forms of Arf6, which is precisely regulated by ACAP3 in concert with a guanine-nucleotide-exchange factor(s), seems to be required for neurite outgrowth of hippocampal neurons.

Is Serum Uric Acid Independently Associated With Left Ventricular Mass Index, Ejection Fraction, and B-Type Natriuretic Peptide Among Female and Male Cardiac Patients?

Mean serum uric acid (SUA) levels are higher in men than women. In addition, recent studies have suggested that the SUA threshold at which the cardiovascular risk might increase may vary between women and men. In the current retrospective study, by analyzing the data from 219 female and 519 male patients who were free from uric acid-lowering medication, we investigated whether SUA is associated with left ventricular mass index (LVMI), left ventricular ejection fraction (LVEF), and plasma levels of B-type natriuretic peptide (BNP) independent of confounding factors, such as serum calcium, inorganic phosphate, and fibroblast growth factor 23 (FGF23), in a gender-specific manner.In multivariate stepwise linear regression analysis in which age, blood pressure, eGFR, corrected calcium, inorganic phosphate, and FGF23 were entered as potential covariates, SUA was selected as a factor significantly associated with LVEF, LVMI, and plasma levels of BNP in both genders. On the other hand, however, after adding diuretic use as a potential covariate, the association between SUA and LVEF lost statistical significance in both genders, and that between SUA and BNP lost significance among female patients. These findings suggest that diuretic use is a non-negligible confounder in understanding the observed association between SUA and cardiac dysfunction and heart failure.In summary, SUA is associated with left ventricular hypertrophy independent of confounding factors including FGF23 and diuretic use in female and male patients. Whether lowering SUA can influence the progression of cardiac remodeling awaits further investigation.

An electrically driven, ultrahigh-speed, on-chip light emitter based on carbon nanotubes.

The integration of high-speed light emitters on silicon chips is an important issue that must be resolved in order to realize on-chip or interchip optical interconnects. Here, we demonstrate the first electrically driven ultrafast carbon nanotube (CNT) light emitter based on blackbody radiation with a response speed (1-10 Gbps) that is more than 10(6) times higher than that of conventional incandescent emitters and is either higher than or comparable to that of light-emitting diodes or laser diodes. This high-speed response is explained by the extremely fast temperature response of the CNT film, which is dominated by the small heat capacity of the CNT film and its high heat dissipation to the substrate. Moreover, we experimentally demonstrate 140 ps width pulsed light generation and real-time optical communication. This CNT-based emitter with the advantages of ultrafast response speeds, a small footprint, and integration on silicon can enable novel architectures for optical interconnects, photonic, and optoelectronic integrated circuits.

Novel attenuation correction of SPECT images using scatter photopeak window data for the detection of coronary artery disease.

BACKGROUND: Attenuation correction using segmentation with scatter and photopeak window data (SSPAC) may enable evaluation of the attenuation map in a patient-specific manner without the need for additional radiation exposure and more acquisition time. We examined the feasibility of SSPAC and compared the sensitivity, specificity, and accuracy of this new correction method with that of conventional non-corrected myocardial perfusion single-photon emission computed tomography (SPECT) among patients with suspected or diagnosed coronary artery disease. METHODS AND RESULTS: One hundred sixty-one patients who underwent both (99m)Tc-tetrofosmin stress/rest SPECT examination and invasive coronary angiography were enrolled in the study. Data from the SSPAC-corrected and non-corrected methods were analyzed quantitatively using summed stress scores. Attenuation maps were obtained successfully for 150 (93%) of the patients. The SSPAC-corrected and non-corrected methods accurately predicted coronary artery disease defined as >50% luminal stenosis verified by coronary artery angiography and/or prior myocardial infarction, for 91% and 77% patients, respectively (P < .05). For diagnosis of coronary artery disease, SSPAC improved sensitivity in the left anterior descending artery territory and specificity in the right coronary artery territory. CONCLUSIONS: Attenuation correction with SSPAC may be a feasible method of correction for myocardial perfusion SPECT and in some cases may provide better accuracy for diagnosing coronary artery disease.

The α-dystroglycan N-terminus is a broad-spectrum antiviral agent against SARS-CoV-2 and enveloped viruses.

The COVID-19 pandemic has shown the need to develop effective therapeutics in preparedness for further epidemics of virus infections that pose a significant threat to human health. As a natural compound antiviral candidate, we focused on α-dystroglycan, a highly glycosylated basement membrane protein that links the extracellular matrix to the intracellular cytoskeleton. Here we show that the N-terminal fragment of α-dystroglycan (α-DGN), as produced in E. coli in the absence of post-translational modifications, blocks infection of SARS-CoV-2 in cell culture, human primary gut organoids and the lungs of transgenic mice expressing the human receptor angiotensin I-converting enzyme 2 (hACE2). Prophylactic and therapeutic administration of α-DGN reduced SARS-CoV-2 lung titres and protected the mice from respiratory symptoms and death. Recombinant α-DGN also blocked infection of a wide range of enveloped viruses including the four Dengue virus serotypes, influenza A virus, respiratory syncytial virus, tick-borne encephalitis virus, but not human adenovirus, a non-enveloped virus in vitro. This study establishes soluble recombinant α-DGN as a broad-band, natural compound candidate therapeutic against enveloped viruses.

Herpes simplex virus requires poly(ADP-ribose) polymerase activity for efficient replication and induces extracellular signal-related kinase-dependent phosphorylation and ICP0-dependent nuclear localization of tankyrase 1.

Tankyrase 1 is a poly(ADP-ribose) polymerase (PARP) which localizes to multiple subcellular sites, including telomeres and mitotic centrosomes. Poly(ADP-ribosyl)ation of the nuclear mitotic apparatus (NuMA) protein by tankyrase 1 during mitosis is essential for sister telomere resolution and mitotic spindle pole formation. In interphase cells, tankyrase 1 resides in the cytoplasm, and its role therein is not well understood. In this study, we found that herpes simplex virus (HSV) infection induced extensive modification of tankyrase 1 but not tankyrase 2. This modification was dependent on extracellular signal-regulated kinase (ERK) activity triggered by HSV infection. Following HSV-1 infection, tankyrase 1 was recruited to the nucleus. In the early phase of infection, tankyrase 1 colocalized with ICP0 and thereafter localized within the HSV replication compartment, which was blocked in cells infected with the HSV-1 ICP0-null mutant R7910. In the absence of infection, ICP0 interacted with tankyrase 1 and efficiently promoted its nuclear localization. HSV did not replicate efficiently in cells depleted of both tankyrases 1 and 2. Moreover, XAV939, an inhibitor of tankyrase PARP activity, decreased viral titers to 2 to 5% of control values. We concluded that HSV targets tankyrase 1 in an ICP0- and ERK-dependent manner to facilitate its replication.

Histone deacetylase 8 is required for centrosome cohesion and influenza A virus entry.

Influenza A virus (IAV) enters host cells by endocytosis followed by acid-activated penetration from late endosomes (LEs). Using siRNA silencing, we found that histone deacetylase 8 (HDAC8), a cytoplasmic enzyme, efficiently promoted productive entry of IAV into tissue culture cells, whereas HDAC1 suppressed it. HDAC8 enhanced endocytosis, acidification, and penetration of the incoming virus. In contrast, HDAC1 inhibited acidification and penetration. The effects were connected with dramatic alterations in the organization of the microtubule system, and, as a consequence, a change in the behavior of LEs and lysosomes (LYs). Depletion of HDAC8 caused loss of centrosome-associated microtubules and loss of directed centripetal movement of LEs, dispersing LE/LYs to the cell periphery. For HDAC1, the picture was the opposite. To explain these changes, centrosome cohesion emerged as the critical factor. Depletion of HDAC8 caused centrosome splitting, which could also be induced by depleting a centriole-linker protein, rootletin. In both cases, IAV infection was inhibited. HDAC1 depletion reduced the splitting of centrosomes, and enhanced infection. The longer the distance between centrosomes, the lower the level of infection. HDAC8 depletion was also found to inhibit infection of Uukuniemi virus (a bunyavirus) suggesting common requirements among late penetrating enveloped viruses. The results established class I HDACs as powerful regulators of microtubule organization, centrosome function, endosome maturation, and infection by IAV and other late penetrating viruses.