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Mateusz Michał Tomczyk,1 Sławomir Boncel,1 Artur Herman,2 Tomasz Krawczyk,1 Agata Jakóbik-Kolon,1 Mirosława Pawlyta,3 Maciej Krzywiecki,4 Artur Chrobak,5 Masafumi Minoshima,6 Fuminori Sugihara,7 Kazuya Kikuchi,6 Nikodem Kuźnik1 1Faculty of Chemistry, Silesian University of Technology, Gliwice, Poland; 2Department of Semiconductor Materials Engineering, Faculty of Fundamental Problems of Technology, Wroclaw University of Science and Technology, Wrocław, Poland; 3Faculty of Mechanical Engineering, Silesian University of Technology, Gliwice, Poland; 4Institute of Physics – Center for Science and Education, Silesian University of Technology, Gliwice, Poland; 5August Chełkowski Institute of Physics, University of Silesia, Katowice, Poland; 6Graduate School of Engineering, Osaka University, Osaka, Japan; 7Immunology Frontier Research Center, Osaka University, Osaka, JapanCorrespondence: Nikodem Kuźnik; Sławomir Boncel Email nikodem.kuznik@polsl.pl; slawomir.boncel@polsl.plPurpose: Salicyl (Sal) – among other oxygen functionalities – multi-walled carbon nanotubes (MWCNTs) and their nanohybrids are investigated as promising contrast agents (CA) in magnetic resonance imaging (MRI) or drug delivery platforms, due to their unique properties. The preliminary results and the literature reports were the motivation to endow high r2 relaxivities, excellent dispersibility in water, and biocompatibility to superparamagnetic MWCNTs nanohybrids. It was hypothesized that these goals could be achieved by, not described in the literature yet, two-stage oxygen functionalization of MWCNTs.Results: Two structurally different MWCNT materials differing in diameters (44 and 12 nm) and the iron content (4.7% and 0.5%) are studied toward the functionalization effect on the T2 relaxometric properties. MWCNT oxidation is typically the first step of functionalization resulting in “first generation” oxygen functional groups (OFGs) on the surface. Until now, the impact of OFGs on the relaxivity of MWCNT was not truly recognized, but this study sheds light on this issue. By follow-up functionalization of oxidized MWCNT with 4-azidosalicylic acid through [2+1] cycloaddition of the corresponding nitrene, “second generation” of oxygen functional groups is grafted onto the nanohybrid, ie, Sal functionality.Conclusion: The introduced OFGs are responsible for an almost 30% increase in the relaxivity, which leads to remarkable r2 relaxivity of 951 mM− 1s− 1 (419 (mg/mL)− 1s− 1), the unprecedented value reported to date for this class of CAs. Also, the resulting nanohybrids express low cytotoxicity and superb diffusion after subcutaneous injection to a mouse.Keywords: multi-walled carbon nanotubes, magnetic resonance imaging, MRI contrast agents, carbon nanotube functionalization, T2 relaxivity

This paper presents the design and development of robotic tweezers with a force- and displacement-sensing capability driven by piezoelectric stack actuators. In order to satisfy sufficient stroke and tip force for future medical operations, a rhombus strain amplification mechanism is adopted. One of the serially connected piezoelectric stack actuators nested in the end effector is used as a force sensor. The force-displacement characteristics at the outermost layer with respect to the forces of the innermost PZT actuators (i.e., forward model) is obtained from a lumped parameter model of the rhombus strain amplification mechanism and a Bernoulli-Euler beam model of the tweezer-style end effector. The end-effector tip force and displacement are measured using an inverse model of the nested multilayer structure relating these quantities to an induced voltage across the innermost PZT actuator. The prototype end effector has the size of 69 mm (length) × 14 mm (height) × 13 mm (width). The performance test shows that the prototype has 1.0-N force and 8.8-mm displacement at the tip. The sensing accuracy was also evaluated through experiments. The experimental results show that the prototype has mean error of 0.086 N for force and 0.39 mm for displacement, which are equivalent to 11% of their maximum measurable values. [ABSTRACT FROM PUBLISHER]

Circular RNAs (circRNA) lack 5' or 3' ends; their unique covalently closed structures prevent RNA degradation by exonucleases. These characteristics provide circRNAs with high pharmaceutical stability and biostability relative to current standard-of-care linear mRNAs. CircRNA levels are reportedly associated with certain human diseases, making them novel disease biomarkers and a noncanonical class of therapeutic targets. In this study, the endogenous circRNAs underlying the response to BNT162b2 mRNA vaccination were evaluated. To this end, peripheral blood samples were subjected to full-length sequencing of circRNAs via nanopore sequencing and transcriptome sequencing. Fifteen samples, comprising pre-, first, and second vaccination cohorts, were obtained from five healthcare workers with no history of SARS-CoV-2 infection or previous vaccination. A total of 4706 circRNAs were detected; following full-length sequencing, 4217 novel circRNAs were identified as being specifically expressed during vaccination. These circRNAs were enriched in the binding motifs of stress granule assemblies and SARS-CoV-2 RNA binding proteins, namely poly(A) binding protein cytoplasmic 1 (PABPC1), pumilio RNA binding family member 1 (PUM1), and Y box binding protein 1 (YBX1). Moreover, 489 circRNAs were identified as previously reported miRNA sponges. The differentially expressed circRNAs putatively originated from plasma B cells compared to circRNAs reported in human blood single-cell RNA sequencing datasets. The pre- and post-vaccination differences observed in the circRNA expression landscape in response to the SARS-CoV-2 BNT162b2 mRNA vaccine., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Whereas severe COVID-19 is often associated with elevated autoantibody titers, the underlying mechanism behind their generation has remained unclear. Here we report clonal composition and diversity of autoantibodies in humoral response to SARS-CoV-2. Immunoglobulin repertoire analysis and characterization of plasmablast-derived monoclonal antibodies uncovered clonal expansion of plasmablasts producing cardiolipin (CL)-reactive autoantibodies. Half of the expanded CL-reactive clones exhibited strong binding to SARS-CoV-2 antigens. One such clone, CoV1804, was reactive to both CL and viral nucleocapsid (N), and further showed anti-nucleolar activity in human cells. Notably, antibodies sharing genetic features with CoV1804 were identified in COVID-19 patient-derived immunoglobulins, thereby constituting a novel public antibody. These public autoantibodies had numerous mutations that unambiguously enhanced anti-N reactivity, when causing fluctuations in anti-CL reactivity along with the acquisition of additional self-reactivities, such as anti-nucleolar activity, in the progeny. Thus, potentially CL-reactive precursors may have developed multiple self-reactivities through clonal selection, expansion, and somatic hypermutation driven by viral antigens. Our results revealed the nature of autoantibody production during COVID-19 and provided novel insights into the origin of virus-induced autoantibodies., (© 2024 Sakakibara et al.)

Regulated neural-metabolic-inflammatory responses are essential for maintaining physiological homeostasis. However, the molecular machinery that coordinates neural, metabolic, and inflammatory responses is largely unknown. Here, we show that semaphorin 6D (SEMA6D) coordinates anxiogenic, metabolic, and inflammatory outputs from the amygdala by maintaining synaptic homeostasis. Using genome-wide approaches, we identify SEMA6D as a pleiotropic gene for both psychiatric and metabolic traits in human. Sema6d deficiency increases anxiety in mice. When fed a high-fat diet, Sema6d -/- mice display attenuated obesity and enhanced myelopoiesis compared with control mice due to higher sympathetic activity via the β3-adrenergic receptor. Genetic manipulation and spatial and single-nucleus transcriptomics reveal that SEMA6D in amygdalar interneurons is responsible for regulating anxiogenic and autonomic responses. Mechanistically, SEMA6D is required for synaptic maturation and γ-aminobutyric acid transmission. These results demonstrate that SEMA6D is important for the normal functioning of the neural circuits in the amygdala, coupling emotional, metabolic, and inflammatory responses., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Resting memory B cells can be divided into classical or atypical groups, but the heterogenous marker expression on activated memory B cells makes similar classification difficult. Here, by longitudinal analysis of mass cytometry and CITE-seq data from cohorts with COVID-19, bacterial sepsis, or BNT162b2 mRNA vaccine, we observe that resting B cell memory consist of classical CD45RB + memory and CD45RB lo memory, of which the latter contains of two distinct groups of CD11c + atypical and CD23 + non-classical memory cells. CD45RB levels remain stable in these cells after activation, thereby enabling the tracking of activated B cells and plasmablasts derived from either CD45RB + or CD45RB lo memory B cells. Moreover, in both COVID-19 patients and mRNA vaccination, CD45RB lo B cells formed the majority of SARS-CoV2 specific memory B cells and correlated with serum antibodies, while CD45RB + memory are activated by bacterial sepsis. Our results thus identify that stably expressed CD45RB levels can be exploited to trace resting memory B cells and their activated progeny, and suggest that atypical and non-classical CD45RB lo memory B cells contribute to SARS-CoV-2 infection and vaccination., (© 2024. The Author(s).)

Purpose: To compare the safety and effectiveness of n-butyl cyanoacrylate (nBCA)-ethiodized oil (NE) mixture and nBCA-ethiodized oil-ethanol (NEE) mixture embolization for Type II endoleak (T2EL) after endovascular aortic repair., Materials and Methods: This study included 32 patients with 49 procedures who underwent T2EL embolization between January 2008 and June 2022. Cases with no follow-up after embolization, technical failure, treatment with coil only, Type I endoleak at the embolization, and re-embolization were excluded. The resultant final cohort included 24 patients (14 men and 10 women; mean age, 83.3 years [interquartile range, 77-89 years]) who underwent initial T2EL embolization, with 15 patients in the NE group and 9 patients in the NEE group. The 2 groups were compared in terms of adverse events (AEs), freedom from sac enlargement, and freedom from reintervention., Results: The follow-up period after embolization for T2EL was 960 days (SD ± 1,007) in the NE group and 484 days (SD ± 192) in the NEE group, without significant differences. No AEs above moderate were observed in either group. The rate of freedom from sac enlargement at 1 year was 65.0% in the NE group and 87.5% in the NEE group (P = .03). The rate of freedom from reintervention at 1 year was 69.2% in the NE group and 100.0% in the NEE group (P = .02)., Conclusions: The NEE group had significantly higher rates of freedom from sac enlargement and reintervention at 1 year compared with the NE group. These results suggest that T2EL embolization with NEE may be more effective than that with NE., (Copyright © 2024 SIR. Published by Elsevier Inc. All rights reserved.)

Background: The association between the occlusion rate of the side branch arteries branching from the abdominal aortic aneurysm sac and aneurysm sac shrinkage is unclear. We aimed to evaluate the efficacy of preemptive embolization of multiple side branch arteries branching from the abdominal aortic aneurysm sac in early aneurysm sac shrinkage after endovascular aneurysm repair., Methods: Patients undergoing endovascular aneurysm repair of abdominal aortic aneurysms, with or without preemptive embolization of multiple side branch arteries, including the inferior mesenteric artery and lumbar arteries, between January 2016 and August 2021, were retrospectively evaluated. Preemptive embolization was introduced at our institution in January 2018 and has been performed in all patients who undergo endovascular aneurysm repair since then. We compared occlusion rates of the side branch arteries, frequency of type 2 endoleaks, changes in aneurysm sac size, percentage of aneurysm sac size decrease, and frequency of reduction in the aneurysm sac diameter by > 5 mm., Results: The study included 43 patients in the embolization group and 20 in the nonembolization group. Preemptive embolization was successfully performed without any ischemic complications. The total occlusion rate of side branch arteries was significantly higher in the embolization group than in the nonembolization group (70.2% vs. 29.3%, P < 0.05). At 24 months of follow-up, the type 2 endoleak frequency was significantly lower in the embolization group than in the nonembolization group (6.9% vs. 31.6%, P < 0.05). The frequency of reduction in the aneurysm sac diameter by > 5 mm was significantly higher in the embolization group than in the nonembolization group at 24 months (62.1% vs. 31.6% P < 0.05). The optimal cutoff value for the total occlusion rate of the side branch arteries to achieve reduction in the aneurysm sac diameter by > 5 mm at 24 months, after endovascular aneurysm repair, was 66.7% in all patients (area under the curve = 0.634; sensitivity = 62.5%; specificity = 70.8%). These findings suggest that occluding 66.7% or more of the side branch arteries may result in early aneurysmal shrinkage., Conclusions: Preemptive embolization of multiple side branch arteries, branching from the abdominal aortic aneurysm sac, may contribute to early aneurysm sac shrinkage; this may serve as a marker for fewer late complications after endovascular aneurysm repair., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Actinomycosis is a rare chronic suppurative granulomatous disease. Surgical biopsy is often performed in patients with chest actinomycosis because malignancy is suspected in most cases. A 62-year-old man presented to our hospital with fever and exertional dyspnea that had persisted for several months. Contrast-enhanced computed tomography showed an irregularly shaped mass with contrast enhancement in the anterior mediastinum and consolidation in the left upper lung lobe contiguous with this mass, as well as multiple nodules in both lungs. The pulmonary artery trunk was stenotic and surrounded by the mass, and the right heart system was enlarged. Thoracoscopic biopsy was performed but failed to yield a diagnosis. Contrast-enhanced computed tomography after one month revealed an increased mass and worsening right heart strain. 18 F-FDG (fluorodeoxyglucose) positron emission tomography/computed tomography and contrast-enhanced magnetic resonance imaging also suggested a malignant tumor, and an open chest biopsy was performed. No malignant cells were identified and actinomycetes were detected by histopathology and bacterial culture. The patient was treated with antibiotics, following which his contrast-enhanced computed tomography findings and general condition improved., (© 2024 The Authors. Published by Elsevier Inc. on behalf of University of Washington.)

Irgb6 is a priming immune-related GTPase (IRG) that counteracts Toxoplasma gondii. It is known to be recruited to the low virulent type II T. gondii parasitophorous vacuole (PV), initiating cell-autonomous immunity. However, the molecular mechanism by which immunity-related GTPases become inactivated after the parasite infection remains obscure. Here, we found that Thr95 of Irgb6 is prominently phosphorylated in response to low virulent type II T. gondii infection. We observed that a phosphomimetic T95D mutation in Irgb6 impaired its localization to the PV and exhibited reduced GTPase activity in vitro. Structural analysis unveiled an atypical conformation of nucleotide-free Irgb6-T95D, resulting from a conformational change in the G-domain that allosterically modified the PV membrane-binding interface. In silico docking corroborated the disruption of the physiological membrane binding site. These findings provide novel insights into a T. gondii-induced allosteric inactivation mechanism of Irgb6., (© 2023 The Authors. Genes to Cells published by Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Pulmonary fibrosis (PF), a condition characterized by inflammation and collagen deposition in the alveolar interstitium, causes dyspnea and fatal outcomes. Although the bleomycin-induced PF mouse model has improved our understanding of exogenous factor-induced fibrosis, the mechanism governing endogenous factor-induced fibrosis remains unknown. Here, we find that Ifngr1 -/- Rag2 -/- mice, which lack the critical suppression factor for group 2 innate lymphoid cells (ILC2), develop PF spontaneously. The onset phase of fibrosis includes ILC2 subpopulations with a high Il1rl1 (IL-33 receptor) expression, and fibrosis does not develop in ILC-deficient or IL-33-deficient mice. Although ILC2s are normally localized near bronchioles and blood vessels, ILC2s are increased in fibrotic areas along with IL-33 positive fibroblasts during fibrosis. Co-culture analysis shows that activated-ILC2s directly induce collagen production from fibroblasts. Furthermore, increased IL1RL1 and decreased IFNGR1 expressions are confirmed in ILC2s from individuals with idiopathic PF, highlighting the applicability of Ifngr1 -/- Rag2 -/- mice as a mouse model for fibrosis research., (© 2023. The Author(s).)

The entry of SARS-CoV-2 into host cells is mediated by the interaction between the spike receptor-binding domain (RBD) and host angiotensin-converting enzyme 2 (ACE2). Certain human antibodies, which target the spike N-terminal domain (NTD) at a distant epitope from the host cell binding surface, have been found to augment ACE2 binding and enhance SARS-CoV-2 infection. Notably, these antibodies exert their effect independently of the antibody fragment crystallizable (Fc) region, distinguishing their mode of action from previously described antibody-dependent infection-enhancing (ADE) mechanisms. Building upon previous hypotheses and experimental evidence, we propose that these NTD-targeting infection-enhancing antibodies (NIEAs) achieve their effect through the crosslinking of neighboring spike proteins. In this study, we present refined structural models of NIEA fragment antigen-binding region (Fab)-NTD complexes, supported by molecular dynamics simulations and hydrogen-deuterium exchange mass spectrometry (HDX-MS). Furthermore, we provide direct evidence confirming the crosslinking of spike NTDs by NIEAs. Collectively, our findings advance our understanding of the molecular mechanisms underlying NIEAs and their impact on SARS-CoV-2 infection.

Importance: In this study, whole-blood RNAs (prolactin and toll-like receptor 3) involved in the prognosis of patients with COVID-19 were identified. The RNA endotypes classified by these important RNAs highlight the possibility of stratifying the COVID-19 patient population and the need for targeted therapy based on these phenotypes., Competing Interests: The authors declare no conflict of interest.

COVID-19 is linked to endotheliopathy and coagulopathy, which can result in multi-organ failure. The mechanisms causing endothelial damage due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain elusive. Here, we developed an infection-competent human vascular organoid from pluripotent stem cells for modeling endotheliopathy. Longitudinal serum proteome analysis identified aberrant complement signature in critically ill patients driven by the amplification cycle regulated by complement factor B and D (CFD). This deviant complement pattern initiates endothelial damage, neutrophil activation, and thrombosis specific to organoid-derived human blood vessels, as verified through intravital imaging. We examined a new long-acting, pH-sensitive (acid-switched) antibody targeting CFD. In both human and macaque COVID-19 models, this long-acting anti-CFD monoclonal antibody mitigated abnormal complement activation, protected endothelial cells, and curtailed the innate immune response post-viral exposure. Collectively, our findings suggest that the complement alternative pathway exacerbates endothelial injury and inflammation. This underscores the potential of CFD-targeted therapeutics against severe viral-induced inflammathrombotic outcomes., Competing Interests: Declaration of interests N.S. and T.T. are patent holders associated with the technology described in this project., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The Omicron variant continuously evolves under the humoral immune pressure exerted by vaccination and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and the resulting Omicron subvariants display further immune evasion and antibody escape. An engineered angiotensin-converting enzyme 2 (ACE2) decoy composed of high-affinity ACE2 and an IgG1 Fc domain could offer an alternative modality to neutralize SARS-CoV-2. We previously reported its broad spectrum and therapeutic potential in rodent models. Here, we demonstrate that the engineered ACE2 decoy retains neutralization activity against Omicron subvariants, including the currently emerging XBB and BQ.1 strains, which completely evade antibodies currently in clinical use. SARS-CoV-2, under the suboptimal concentration of neutralizing drugs, generated SARS-CoV-2 mutants escaping wild-type ACE2 decoy and monoclonal antibodies, whereas no escape mutant emerged against the engineered ACE2 decoy. Furthermore, inhalation of aerosolized decoys improved the outcomes of rodents infected with SARS-CoV-2 at a 20-fold lower dose than that of intravenous administration. Last, the engineered ACE2 decoy exhibited therapeutic efficacy for cynomolgus macaques infected with SARS-CoV-2. These results indicate that this engineered ACE2 decoy represents a promising therapeutic strategy to overcome immune-evading SARS-CoV-2 variants and that liquid aerosol inhalation could be considered as a noninvasive approach to enhance the efficacy of COVID-19 treatments.

Recent advancements in proteomics allow for the concurrent identification and quantification of multiple proteins. This study aimed to identify proteins associated with severe burn pathology and establish a clinically useful molecular pathology classification. In a retrospective observational study, blood samples were collected from severe burn patients. Proteins were measured using mass spectrometry, and prognosis-related proteins were extracted by comparing survivors and non-survivors. Enrichment and ROC analyses evaluated the extracted proteins, followed by latent class analysis. Measurements were performed on 83 burn patients. In the non-survivor group, ten proteins significantly changing on the day of injury were associated with metabolic processes and toxin responses. ROC analysis identified HBA1, TTR, and SERPINF2 with AUCs > 0.8 as predictors of 28-day mortality. Latent class analysis classified three molecular pathotypes, and plasma mass spectrometry revealed ten proteins associated with severe burn prognosis. Molecular pathotypes based on HBA1, TTR, and SERPINF2 significantly correlated with outcomes., Competing Interests: The authors declare that they have no competing interests., (© 2023 The Author(s).)

Purpose: Completely occlusive acute-subacute portal and mesenteric vein thrombosis (PVMVT) with severe complications is fatal. Endovascular treatments (EVTs) of acute-subacute PVMVT are not standardized. Thrombectomy combined with continuous catheter-directed thrombolysis is considered an effective treatment. Here, we aimed to evaluate the outcome of EVTs of completely occlusive acute-subacute PVMVT with severe complications in patients without cirrhosis., Materials and Methods: Nineteen patients (nine men and 10 women; age, 60.1 ± 16.8 years) with completely occlusive acute-subacute PVMVT were retrospectively assessed. Acute-subacute PVMVT was defined as symptom onset within 40 days, with no cavernous transformation observed on contrast-enhanced computed tomography. The patients were treated with EVTs, a combination of thrombectomy (including aspiration thrombectomy, plain old balloon angioplasty, single injection of thrombolytic agents, and stent placement) and continuous catheter-directed thrombolysis. Kaplan-Meier analyses were performed to assess all-cause mortality, acute-subacute PVMVT-related mortality, and portal vein (PV) patency. The degree of recanalization and patency of PV, complications, factors related to acute-subacute PVMVT-related mortality, and factors related to patency of PV were also evaluated., Results: The all-cause and acute-subacute PVMVT-related mortality rates were 36.8% (7/19) and 31.6% (6/19), respectively. Seven (36.8%) and 11 (57.9%) patients achieved complete and partial recanalization, respectively. Among the 18 patients who achieved recanalization, follow-up images after 608.7 ± 889.5 days confirmed recanalization in 83.3% (15/18) patients, and 53.3% (8/15) of these patients achieved patency of PV. Seven patients (36.8%) developed complications, and two (10.5%) required interventional treatment for complications. Deterioration of liver function significantly worsened the prognosis (P = 0.046), while anticoagulation therapy significantly maintained portal patency (P = 0.03)., Conclusion: This endovascular method for acute-subacute PVMVT, which combines thrombectomy and continuous catheter-directed thrombolysis EVT approach was effective for thrombus resolution. However, further studies must define conditions that improve patient prognosis., (© 2023. The Author(s).)

Mechanisms underpinning the dysfunctional immune response in severe acute respiratory syndrome coronavirus 2 infection are elusive. We analyzed single-cell transcriptomes and T and B cell receptors (BCR) of >895,000 peripheral blood mononuclear cells from 73 coronavirus disease 2019 (COVID-19) patients and 75 healthy controls of Japanese ancestry with host genetic data. COVID-19 patients showed a low fraction of nonclassical monocytes (ncMono). We report downregulated cell transitions from classical monocytes to ncMono in COVID-19 with reduced CXCL10 expression in ncMono in severe disease. Cell-cell communication analysis inferred decreased cellular interactions involving ncMono in severe COVID-19. Clonal expansions of BCR were evident in the plasmablasts of patients. Putative disease genes identified by COVID-19 genome-wide association study showed cell type-specific expressions in monocytes and dendritic cells. A COVID-19-associated risk variant at the IFNAR2 locus (rs13050728) had context-specific and monocyte-specific expression quantitative trait loci effects. Our study highlights biological and host genetic involvement of innate immune cells in COVID-19 severity., (© 2023. The Author(s).)

Background: COVID-19 is now a common disease, but its pathogenesis remains unknown. Blood circulating proteins reflect host defenses against COVID-19. We investigated whether evaluation of longitudinal blood proteomics for COVID-19 and merging with clinical information would allow elucidation of its pathogenesis and develop a useful clinical phenotype., Methods: To achieve the first goal (determining key proteins), we derived plasma proteins related to disease severity by using a first discovery cohort. We then assessed the association of the derived proteins with clinical outcome in a second discovery cohort. Finally, the candidates were validated by enzyme-linked immunosorbent assay in a validation cohort to determine key proteins. For the second goal (understanding the associations of the clinical phenotypes with 28-day mortality and clinical outcome), we assessed the associations between clinical phenotypes derived by latent cluster analysis with the key proteins and 28-day mortality and clinical outcome., Results: We identified four key proteins (WFDC2, GDF15, CHI3L1, and KRT19) involved in critical pathogenesis from the three different cohorts. These key proteins were related to the function of cell adhesion and not immune response. Considering the multicollinearity, three clinical phenotypes based on WFDC2, CHI3L1, and KRT19 were identified that were associated with mortality and clinical outcome., Conclusion: The use of these easily measured key proteins offered new insight into the pathogenesis of COVID-19 and could be useful in a potential clinical application., (© 2022. The Author(s).)

The cellular activation of the NLRP3 inflammasome is spatiotemporally orchestrated by various organelles, but whether lysosomes contribute to this process remains unclear. Here, we show the vital role of the lysosomal membrane-tethered Ragulator complex in NLRP3 inflammasome activation. Deficiency of Lamtor1, an essential component of the Ragulator complex, abrogated NLRP3 inflammasome activation in murine macrophages and human monocytic cells. Myeloid-specific Lamtor1-deficient mice showed marked attenuation of NLRP3-associated inflammatory disease severity, including LPS-induced sepsis, alum-induced peritonitis, and monosodium urate (MSU)-induced arthritis. Mechanistically, Lamtor1 interacted with both NLRP3 and histone deacetylase 6 (HDAC6). HDAC6 enhances the interaction between Lamtor1 and NLRP3, resulting in NLRP3 inflammasome activation. DL-all-rac-α-tocopherol, a synthetic form of vitamin E, inhibited the Lamtor1-HDAC6 interaction, resulting in diminished NLRP3 inflammasome activation. Further, DL-all-rac-α-tocopherol alleviated acute gouty arthritis and MSU-induced peritonitis. These results provide novel insights into the role of lysosomes in the activation of NLRP3 inflammasomes by the Ragulator complex., (© 2022 The Authors.)

Characterization of protein-protein interactions (PPI) is a key to understanding the functions of proteins of interest. Recently developed proximity-dependent biotin identification (BioID) has been actively investigated as an alternative PPI mapping method because of its usefulness in uncovering transient PPI. Here, as an example of proximity labeling proteomics application in the testis, we generated two transgenic mouse lines expressing two biotin ligases (BioID2 or TurboID) fused with TESMIN, which translocates from the cytosol to the nucleus during meiotic progression and is required for reproduction. The BioID2 transgene, albeit not the TurboID transgene, rescued fertility defects of the Tesmin KO male mice, indicating that the TESMIN-BioID2 fusion can physiologically replace TESMIN. Furthermore, biotinylated protein pull-down and affinity-purification followed by mass spectrometry using the TESMIN-BioID2 transgenic mice captured components of the MYBL1-MuvB complex that regulate cell-cycle gene expression. Thus, our study shows that proximity labeling proteomics can be applied in male germ cells, although the choice of biotin ligase needs to be carefully tested., (© 2022. The Author(s).)

Background: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2, has led to major public health crises worldwide. Several studies have reported the comprehensive mRNA expression analysis of immune-related genes in patients with COVID-19, using blood samples, to understand its pathogenesis; however, the characteristics of RNA expression in COVID-19 and bacterial sepsis have not been compared. The current study aimed to address this gap., Methods: RNA-sequencing and bioinformatics analyses were used to compare the transcriptome expression of whole blood samples from patients with COVID-19 and patients with sepsis who were admitted to the intensive care unit of Osaka University Graduate School of Medicine., Results: The COVID-19 and sepsis cohorts showed upregulation of mitochondrial- and neutrophil-related transcripts, respectively. Compared with that in the control cohort, neutrophil-related transcripts were upregulated in both the COVID-19 and sepsis cohorts. In contrast, mitochondrial-related transcripts were upregulated in the COVID-19 cohort and downregulated in the sepsis cohort, compared to those in the control cohort. Moreover, transcript levels of the pro-apoptotic genes BAK1, CYCS, BBC3, CASP7, and CASP8 were upregulated in the COVID-19 cohort, whereas those of anti-apoptotic genes, such as BCL2L11 and BCL2L1, were upregulated in the sepsis cohort., Conclusions: This study clarified the differential expression of transcripts related to neutrophils and mitochondria in sepsis and COVID-19 conditions. Mitochondrial-related transcripts were downregulated in sepsis than in COVID-19 conditions, and our results indicated suboptimal intrinsic apoptotic features in sepsis samples compared with that in COVID-19 samples. This study is expected to contribute to the development of specific treatments for COVID-19., (© 2022. The Author(s).)

Antibodies make up an important and growing class of compounds used for the diagnosis or treatment of disease. While traditional antibody discovery utilized immunization of animals to generate lead compounds, technological innovations have made it possible to search for antibodies targeting a given antigen within the repertoires of B cells in humans. Here we group these innovations into four broad categories: cell sorting allows the collection of cells enriched in specificity to one or more antigens; BCR sequencing can be performed on bulk mRNA, genomic DNA or on paired (heavy-light) mRNA; BCR repertoire analysis generally involves clustering BCRs into specificity groups or more in-depth modeling of antibody-antigen interactions, such as antibody-specific epitope predictions; validation of antibody-antigen interactions requires expression of antibodies, followed by antigen binding assays or epitope mapping. Together with innovations in Deep learning these technologies will contribute to the future discovery of diagnostic and therapeutic antibodies directly from humans., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Xu, Ismanto, Zhou, Saputri, Sugihara and Standley.)

We evaluated mRNA and miRNA in COVID-19 patients and elucidated the pathogenesis of COVID-19, including protein profiles, following mRNA and miRNA integration analysis. mRNA and miRNA sequencing was done on admission with whole blood of 5 and 16 healthy controls (HCs) and 10 and 31 critically ill COVID-19 patients (derivation and validation cohorts, respectively). Interferon (IFN)-α2, IFN-β, IFN-γ, interleukin-27, and IFN-λ1 were measured in COVID-19 patients on admission (day 1, 181 critical/22 non-critical patients) and days 6-8 (168 critical patients) and in 19 HCs. In the derivation cohort, 3,488 mRNA and 31 miRNA expressions were identified among differentially expressed RNA expressions in the patients versus those in HCs, and 2,945 mRNA and 32 miRNA expressions in the validation cohort. Canonical pathway analysis showed the IFN signaling pathway to be most activated. The IFN-β plasma level was elevated in line with increased severity compared with HCs, as were IFN-β downstream proteins, such as interleukin-27. IFN-λ1 was higher in non-critically ill patients versus HCs but lower in critical than non-critical patients. Integration of mRNA and miRNA analysis showed activated IFN signaling. Plasma IFN protein profile revealed that IFN-β (type I) and IFN-λ1 (type III) played important roles in COVID-19 disease progression., Competing Interests: All authors of this manuscript have no conflicts of interest to disclose., (© 2022 The Authors.)

Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge 1-5 . Here we conducted a genome-wide association study (GWAS) involving 2,393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3,289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target., (© 2022. The Author(s).)

Background: Trauma is a heterogeneous condition, and specific clinical phenotypes may identify target populations that could benefit from certain treatment strategies. In this retrospective study, we determined clinical phenotypes and identified new target populations of trauma patients and their treatment strategies., Methods: We retrospectively analyzed datasets from the Japan Trauma Data Bank and determined trauma death clinical phenotypes using statistical machine learning techniques and evaluation of biological profiles., Results: The analysis included 71,038 blunt trauma patients [median age, 63 (interquartile range [IQR], 40-78) years; 45,479 (64.0%) males; median Injury Severity Score, 13 (IQR, 9-20)], and the derivation and validation cohorts included 42,780 (60.2%) and 28,258 (39.8%) patients, respectively. Of eight derived phenotypes (D-1-D-8), D-8 (n = 2178) had the highest mortality (48.6%) with characteristic severely disturbed consciousness and was further divided into four phenotypes: D-8α, multiple trauma in the young (n = 464); D-8β, head trauma with lower body temperature (n = 178); D-8γ, severe head injury in the elderly (n = 957); and D-8δ, multiple trauma, with higher predicted mortality than actual mortality (n = 579). Phenotype distributions were comparable in the validation cohort. Biological profile analysis of 90 trauma patients revealed that D-8 exhibited excessive inflammation, including enhanced acute inflammatory response, dysregulated complement activation pathways, and impaired coagulation, including downregulated coagulation and platelet degranulation pathways, compared with other phenotypes., Conclusions: We identified clinical phenotypes with high mortality, and the evaluation of the molecular pathogenesis underlying these clinical phenotypes suggests that lethal trauma may involve excessive inflammation and coagulation disorders., (© 2022. The Author(s).)

Introduction: Resistin is reported to form a cytokine network and cause endothelial damage. The pathogenesis of coronavirus disease 2019 (COVID-19) remains unknown, but the association between cytokine storm and endothelial damage is crucial. This study aimed to evaluate resistin in COVID-19 pathogenesis compared with sepsis., Materials and Methods: First, we evaluated the association of plasma resistin levels and disease severity and clinical outcome in two large cohorts: a publicly available cohort including 306 COVID-19 patients in the United States (MGH cohort) and our original cohort including only intubated 113 patients in Japan (Osaka cohort 1). Second, to understand pathogenesis, we evaluate resistin, cytokines and endothelial cell adhesion molecules in COVID-19 compared with sepsis. Blood samples were collected from 62 ICU-treated COVID-19 patients and 38 sepsis patients on day 1 (day of ICU admission), days 2-3, days 6-8, and from 18 healthy controls (Osaka cohort 2). The plasma resistin, inflammatory cytokines (IL-6, IL-8, MCP-1 and IL-10) and endothelial cell adhesion molecules (ICAM-1 and VCAM-1) were compared between patients and control. Correlations among resistin, inflammatory cytokines and endothelial cell adhesion molecules were evaluated in COVID-19 and sepsis., Results: In the MGH cohort, the day 1 resistin levels were associated with disease severity score. The non-survivors showed significantly greater resistin levels than survivors on days 1, 4 and 8. In the Osaka cohort 1, 28-day non-survivors showed significantly higher resistin levels than 28-day survivors on days 6-8. Patients with late recovery (defined as the day of weaning off mechanical ventilation >12 or death) had significantly higher resistin levels than those with early recovery on day 1 and days 6-8. In the Osaka cohort 2, plasma resistin levels were elevated in COVID-19 and sepsis patients compared to controls at all measurement points and were associated with inflammatory cytokines and endothelial cell adhesion molecules., Conclusion: Resistin was elevated in COVID-19 patients and was associated with cytokines and endothelial cell adhesion molecules. Higher resistin levels were related to worse outcome., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ebihara, Matsumoto, Matsubara, Togami, Nakao, Matsuura, Onishi, Kojima, Sugihara, Okuzaki, Hirata, Yamamura and Ogura.)

The lack of established biomarkers which reflect dynamic neuropathological alterations in multiple sclerosis (MS) makes it difficult to determine the therapeutic response to the tested drugs and to identify the key biological process that mediates the beneficial effect of them. In the present study, we applied high-field MR imaging in locally-induced experimental autoimmune encephalomyelitis (EAE) mice to evaluate dynamic changes following treatment with a humanized anti-repulsive guidance molecule-a (RGMa) antibody, a potential drug for MS. Based on the longitudinal evaluation of various MRI parameters including white matter, axon, and myelin integrity as well as blood-spinal cord barrier (BSCB) disruption, anti-RGMa antibody treatment exhibited a strong and prompt therapeutic effect on the disrupted BSCB, which was paralleled by functional improvement. The antibody's effect on BSCB repair was also suggested via GeneChip analysis. Moreover, immunohistochemical analysis revealed that EAE-induced vascular pathology which is characterized by aberrant thickening of endothelial cells and perivascular type I/IV collagen deposits were attenuated by anti-RGMa antibody treatment, further supporting the idea that the BSCB is one of the key therapeutic targets of anti-RGMa antibody. Importantly, the extent of BSCB disruption detected by MRI could predict late-phase demyelination, and the predictability of myelin integrity based on the extent of acute-phase BSCB disruption was compromised following anti-RGMa antibody treatment. These results strongly support the concept that longitudinal MRI with simultaneous DCE-MRI and DTI analysis can be used as an imaging biomarker and is useful for unbiased prioritization of the key biological process that mediates the therapeutic effect of tested drugs., Competing Interests: TH and AS are employees of Mitsubishi Tanabe Pharma Corporation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hirata, Itokazu, Sasaki, Sugihara and Yamashita.)

Bone metabolism is regulated by the cooperative activity between bone-forming osteoblasts and bone-resorbing osteoclasts. However, the mechanisms mediating the switch between the osteoblastic and osteoclastic phases have not been fully elucidated. Here, we identify a specific subset of mature osteoblast-derived extracellular vesicles that inhibit bone formation and enhance osteoclastogenesis. Intravital imaging reveals that mature osteoblasts secrete and capture extracellular vesicles, referred to as small osteoblast vesicles (SOVs). Co-culture experiments demonstrate that SOVs suppress osteoblast differentiation and enhance the expression of receptor activator of NF-κB ligand, thereby inducing osteoclast differentiation. We also elucidate that the SOV-enriched microRNA miR-143 inhibits Runt-related transcription factor 2, a master regulator of osteoblastogenesis, by targeting the mRNA expression of its dimerization partner, core-binding factor β. In summary, we identify SOVs as a mode of cell-to-cell communication, controlling the dynamic transition from bone-forming to bone-resorbing phases in vivo., (© 2022. The Author(s).)

Purpose: The purpose of the study is to evaluate the initial and midterm efficacy and safety of endovascular treatment (EVT) using Viabahn stent-graft (SG) for arterial injury and bleeding (AIB) at the visceral arteries., Materials and Methods: Consecutive patients with visceral AIB who underwent EVT using Viabahn between January 2017 and February 2021 were retrospectively reviewed. Technical success, clinical success, peripheral organ ischemia, peri-procedural complications, bleeding-related mortality, 30-day mortality, neck length, re-bleeding, endoleaks, and patency of the SGs at 1, 3, 6, and 12 months were evaluated., Results: EVT using Viabahn was performed in 14 patients (mean age: 68.6 years; 12 males) and 15 arteries. The technical and clinical success rates were 100%. The rates of peripheral organ ischemia, peri-procedural complications, bleeding-related mortality, and 30-day mortality were all 0%. The mean neck length was 9.9 mm. No endoleaks or re-bleeding occurred during the follow-up (mean: 732 days). The SG patency was confirmed after 1, 3, 6, and 12 months in 78.6%, 78.6%, 78.6%, and 56.1% of the patients, respectively., Conclusion: EVT using Viabahn for AIB at the visceral arteries was safe and effective. SG occlusions without ischemia often occurred after 12 months., (© 2021. The Author(s).)