Serum bta-miRNA-375 as a potential biomarker for the early diagnosis of enzootic bovine leukosis.

To identify a biomarker for the early diagnosis of enzootic bovine leukosis (EBL) caused by bovine leukemia virus (BLV), we investigated the expression of a microRNA, bta-miR-375, in cattle serum. Using quantitative reverse-transcriptase PCR analysis, we measured bta-miR-375 levels in 27 samples from cattle with EBL (EBL cattle), 45 samples from animals infected with BLV but showing no clinical signs (NS cattle), and 30 samples from cattle uninfected with BLV (BLV negative cattle). In this study, we also compared the kinetics of bta-miR-375 with those of the conventional biomarkers of proviral load (PVL), lactate dehydrogenase (LDH), and thymidine kinase (TK) from the no-clinical-sign phase until EBL onset in three BLV-infected Japanese black (JB) cattle. Bta-miR-375 expression was higher in NS cattle than in BLV negative cattle (P < 0.05) and greater in EBL cattle than in BLV negative and NS cattle (P < 0.0001 for both comparisons). Receiver operating characteristic curves demonstrated that bta-miR-375 levels distinguished EBL cattle from NS cattle with high sensitivity and specificity. In NS cattle, bta-miR-375 expression was increased as early as at 2 months before EBL onset-earlier than the expression of PVL, TK, or LDH isoenzymes 2 and 3. These results suggest that serum miR-375 is a promising biomarker for the early diagnosis of EBL.

Multiple endocrine neoplasia type 2B diagnosed after small intestinal volvulus with progressive megacolon in an adolescent.

Multiple endocrine neoplasia type 2B is a rare autosomal dominant disease characterized by the presence of medullary thyroid carcinoma, pheochromocytoma, Marfan-like fatigue, a peculiar face with thickening of the lips, mucosal neuromas on the lips and tongue, and gastrointestinal phenomena. Most patients harbor pathological variants of the RET gene. Herein, we present the first case of a 14 year-old boy who experienced small intestinal volvulus along with a megacolon, and he was diagnosed with multiple endocrine neoplasia type 2B. The patient complained of constipation since he was 2 years old and slowly progressive abdominal distension at school age. At 14 years of age, he presented with remarkable megacolon mimicking Hirschsprung's disease and complicated with small intestinal volvulus. The volvulus was successfully repaired, and the particularly dilated transverse colon was resected following a rectal biopsy. Histopathological evaluation of the resected transverse colon revealed to be compatible with ganglioneuromatosis. After emergency surgery, the patient was diagnosed with multiple endocrine neoplasia type 2B with medullary thyroid carcinoma, and a de novo variant of RET was confirmed. Gastroenterologists should consider it when treating patients with constipation, especially those with megacolon. Therefore, timely diagnosis may lead to appropriate treatment of medullary thyroid carcinoma and improve mortality.

A first-in-human clinical study of laparoscopic autologous myoblast sheet transplantation to prevent delayed perforation after duodenal endoscopic mucosal dissection.

BACKGROUND: The detection rate of superficial non-ampullary duodenal epithelial tumors (SNADETs) has recently been increasing. Large tumors may contain malignant lesions and early therapeutic intervention is recommended. Endoscopic mucosal dissection (ESD) is considered a feasible treatment modality, however, the anatomical and physiological characteristics of the duodenum create a risk of postoperative perforation after ESD. METHODS: To explore whether myoblast sheet transplantation could prevent delayed perforation after ESD, a first-in-human (FIH) clinical trial of laparoscopic autologous myoblast sheet transplantation after duodenal ESD was launched. Autologous myoblast sheets fabricated from muscle tissue obtained seven weeks before ESD were transplanted laparoscopically onto the serous side of the ESD. The primary endpoints were the onset of peritonitis due to delayed perforation within three days after surgery and all adverse events during the follow-up period. RESULTS: Three patients with SNADETs ≥ 20 mm in size underwent transplantation of a myoblast sheet onto the serous side of the duodenum after ESD. In case 1, The patient's postoperative course was uneventful. Endoscopy and abdominal computed tomography revealed no signs of delayed perforation. Despite incomplete mucosal closure in case 2, and multiple micro perforations during ESD in case 3, cell sheet transplantation could prevent the postoperative massive perforation after ESD, and endoscopy on day 49 after transplantation revealed no stenosis. CONCLUSIONS: This clinical trial showed the safety, efficacy, and procedural operability of this novel regenerative medicine approach involving transplanting an autologous myoblast sheet laparoscopically onto the serosa after ESD in cases with a high risk of delayed perforation. This result indicates the potential application of cell sheet medicine in treating various abdominal organs and conditions with minimal invasiveness in the future. TRIAL REGISTRATION: jRCT, jRCT2073210094. Registered November 8 2021, https://jrct.niph.go.jp/latest-detail/jRCT2073210094 .

Establishment of a novel canine soft tissue sarcoma cell line and comparison of its characteristics with other soft tissue sarcoma cell lines.

Soft tissue sarcoma (STS) is a relatively common tumor in dogs. However, very few canine STS cell lines are available. This study aimed to establish a new cell line, STS-YU1, derived from a recurrence of myxosarcoma in an 11-year-old mixed-breed dog. We examined STS-YU1 for in vitro cell proliferation, migration, anticancer drug sensitivity, transcriptome analysis using next-generation sequencing (RNA-seq), and in vivo tumorigenicity in mice and compared it with previously established STS cell lines, MUMA-G and A72. The cell proliferation and migration of STS-YU1 were higher than MUMA-G although MUMA-G only exhibited tumorigenicity in mice. STS-YU1 showed dose-dependent cytotoxicity to anticancer drugs, but with weak effects. RNA-seq analysis revealed the molecular phenotype of STS-YU1 was different from that of a previously reported cell line, A72. Hence, the use of STS-YU1 would help in efficient drug screening against canine STS in vitro.

Mice, myeloid cells, and dengue: a new model for unraveling vascular leakage mysteries.

Introduction: Severe dengue is thought to be caused by an excessive host immune response. Methods: To study the pathogenesis of severe dengue, we developed a novel model using LysM Cre+Ifnarflox/flox mice carrying depleted Ifnar expression only in subsets of murine myeloid cells. Results: Although dengue virus (DENV) clinical isolates were not virulent in LysM Cre+Ifnarflox/flox mice, mouse-adapted DV1-5P7Sp and DV3P12/08P4Bm, which were obtained by passaging the spleen or bone marrow of mice, demonstrated 100% lethality with severe vascular leakage in the liver and small intestine. DV1-5P7Sp and DV3P12/08P4Bm harbored five and seven amino acid substitutions, respectively. Infection also induced neutrophil infiltration in the small intestine, and increased expression of IL-6 and MMP-8 and blockade of TNF-α signaling protected the mice, as demonstrated in a previous severe dengue mouse model using C57/BL6 mice lacking both IFN-α/ß and IFN-γ receptors. Notably, the new models with DV1-5P7Sp and DV3P12/08P4Bm showed an increased proliferative capacity of the adapted viruses in the thymus and bone marrow. Discussion: These observations suggest that myeloid cell infection is sufficient to trigger cytokine storm-induced vascular leakage. This model can refine the factors involved in the pathology of severe dengue leading to vascular leakage.

Vero cell-adapted SARS-CoV-2 strain shows increased viral growth through furin-mediated efficient spike cleavage.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes several host proteases to cleave the spike (S) protein to enter host cells. SARS-CoV-2 S protein is cleaved into S1 and S2 subunits by furin, which is closely involved in the pathogenicity of SARS-CoV-2. However, the effects of the modulated protease cleavage activity due to S protein mutations on viral replication and pathogenesis remain unclear. Herein, we serially passaged two SARS-CoV-2 strains in Vero cells and characterized the cell-adapted SARS-CoV-2 strains in vitro and in vivo. The adapted strains showed high viral growth, effective S1/S2 cleavage of the S protein, and low pathogenicity compared with the wild-type strain. Furthermore, the viral growth and S1/S2 cleavage were enhanced by the combination of the Δ68-76 and H655Y mutations using recombinant SARS-CoV-2 strains generated by the circular polymerase extension reaction. The recombinant SARS-CoV-2 strain, which contained the mutation of the adapted strain, showed increased susceptibility to the furin inhibitor, suggesting that the adapted SARS-CoV-2 strain utilized furin more effectively than the wild-type strain. Pathogenicity was attenuated by infection with effectively cleaved recombinant SARS-CoV-2 strains, suggesting that the excessive cleavage of the S proteins decreases virulence. Finally, the high-growth-adapted SARS-CoV-2 strain could be used as the seed for a low-cost inactivated vaccine; immunization with this vaccine can effectively protect the host from SARS-CoV-2 variants. Our findings provide novel insights into the growth and pathogenicity of SARS-CoV-2 in the evolution of cell-cell transmission. IMPORTANCE: The efficacy of the S protein cleavage generally differs among the SARS-CoV-2 variants, resulting in distinct viral characteristics. The relationship between a mutation and the entry of SARS-CoV-2 into host cells remains unclear. In this study, we analyzed the sequence of high-growth Vero cell-adapted SARS-CoV-2 and factors determining the enhancement of the growth of the adapted virus and confirmed the characteristics of the adapted strain by analyzing the recombinant SARS-CoV-2 strain. We successfully identified mutations Δ68-76 and H655Y, which enhance viral growth and the S protein cleavage by furin. Using recombinant viruses enabled us to conduct a virus challenge experiment in vivo. The pathogenicity of SARS-CoV-2 introduced with the mutations Δ68-76, H655Y, P812L, and Q853L was attenuated in hamsters, indicating the possibility of the attenuation of excessive cleaved SARS-CoV-2. These findings provide novel insights into the infectivity and pathogenesis of SARS-CoV-2 strains, thereby significantly contributing to the field of virology.

Mpox Neutralizing Antibody Response to LC16m8 Vaccine in Healthy Adults.

Mpox Neutralizing Antibody Response to LC16m8 VaccineIn this study of 50 healthy volunteers in Japan, a smallpox vaccine (LC16m8) exhibited a robust neutralizing antibody response against two strains of the mpox virus. With a 94% "take" rate by day 14, seroconversion rates on day 28 were 72 and 70% against the Zr599 and Liberia strains, respectively, decreasing to 30% for both on day 168; no serious adverse events occurred.

Proof-of-concept study of the caninized anti-canine programmed death 1 antibody in dogs with advanced non-oral malignant melanoma solid tumors.

BACKGROUND: The anti-programmed death 1 (PD-1) antibody has led to durable clinical responses in a wide variety of human tumors. We have previously developed the caninized anti-canine PD-1 antibody (ca-4F12-E6) and evaluated its therapeutic properties in dogs with advance-staged oral malignant melanoma (OMM), however, their therapeutic effects on other types of canine tumors remain unclear. OBJECTIVE: The present clinical study was carried out to evaluate the safety profile and clinical efficacy of ca-4F12-E6 in dogs with advanced solid tumors except for OMM. METHODS: Thirty-eight dogs with non-OMM solid tumors were enrolled prospectively and treated with ca-4F12-E6 at 3 mg/kg every 2 weeks of each 10-week treatment cycle. Adverse events (AEs) and treatment efficacy were graded based on the criteria established by the Veterinary Cooperative Oncology Group. RESULTS: One dog was withdrawn, and thirty-seven dogs were evaluated for the safety and efficacy of ca-4F12-E6. Treatment-related AEs of any grade occurred in 13 out of 37 cases (35.1%). Two dogs with sterile nodular panniculitis and one with myasthenia gravis and hypothyroidism were suspected of immune-related AEs. In 30 out of 37 dogs that had target tumor lesions, the overall response and clinical benefit rates were 6.9% and 27.6%, respectively. The median progression-free survival and overall survival time were 70 days and 215 days, respectively. CONCLUSIONS: The present study demonstrated that ca-4F12-E6 was well-tolerated in non-OMM dogs, with a small number of cases showing objective responses. This provides evidence supporting large-scale clinical trials of anti-PD-1 antibody therapy in dogs.

Prednisolone improves hippocampal regeneration after trimethyltin-induced neurodegeneration in association with prevention of T lymphocyte infiltration.

The endogenous regenerative capacity of the brain is quite weak; however, a regenerative reaction, the production of new neurons (neurogenesis), has been reported to occur in brain lesions. In addition, leukocytes are well known to infiltrate brain lesions. Therefore, leukocytes would also have a link with regenerative neurogenesis; however, their role has not been fully elucidated. In this study, we investigated leukocyte infiltration and its influence on brain tissue regeneration in a trimethyltin (TMT)-injected mouse model of hippocampal regeneration. Immunohistochemically, CD3-positive T lymphocytes were found in the hippocampal lesion of TMT-injected mice. Prednisolone (PSL) treatment inhibited T lymphocyte infiltration and increased neuronal nuclei (NeuN)-positive mature neurons and doublecortin (DCX)-positive immature neurons in the hippocampus. Investigation of bromodeoxyuridine (BrdU)-labeled newborn cells revealed the percentage of BrdU/NeuN- and BrdU/DCX-positive cells increased by PSL treatment. These results indicate that infiltrated T lymphocytes prevent brain tissue regeneration by inhibiting hippocampal neurogenesis.

Machine learning algorithm-based estimation model for the severity of depression assessed using Montgomery-Asberg depression rating scale.

AIM: Depressive disorder is often evaluated using established rating scales. However, consistent data collection with these scales requires trained professionals. In the present study, the "rater & estimation-system" reliability was assessed between consensus evaluation by trained psychiatrists and the estimation by 2 models of the AI-MADRS (Montgomery-Asberg Depression Rating Scale) estimation system, a machine learning algorithm-based model developed to assess the severity of depression. METHODS: During interviews with trained psychiatrists and the AI-MADRS estimation system, patients responded orally to machine-generated voice prompts from the AI-MADRS structured interview questions. The severity scores estimated from two models of the AI-MADRS estimation system, the max estimation model and the average estimation model, were compared with those by trained psychiatrists. RESULTS: A total of 51 evaluation interviews conducted on 30 patients were analyzed. Pearson's correlation coefficient with the scores evaluated by trained psychiatrists was 0.76 (95% confidence interval 0.62-0.86) for the max estimation model, and 0.86 (0.76-0.92) for the average estimation model. The ANOVA ICC rater & estimation-system reliability with the evaluation scores by trained psychiatrists was 0.51 (-0.09 to 0.79) for the max estimation model, and 0.75 (0.55-0.86) for the average estimation model. CONCLUSION: The average estimation model of AI-MADRS demonstrated substantially acceptable rater & estimation-system reliability with trained psychiatrists. Accumulating a broader training dataset and the refinement of AI-MADRS interviews are expected to improve the performance of AI-MADRS. Our findings suggest that AI technologies can significantly modernize and potentially revolutionize the realm of depression assessments.

A three-dimensional model with two-body interactions for endothelial cells in angiogenesis.

We introduce a three-dimensional mathematical model for the dynamics of vascular endothelial cells during sprouting angiogenesis. Angiogenesis is the biological process by which new blood vessels form from existing ones. It has been the subject of numerous theoretical models. These models have successfully replicated various aspects of angiogenesis. Recent studies using particle-based models have highlighted the significant influence of cell shape on network formation, with elongated cells contributing to the formation of branching structures. While most mathematical models are two-dimensional, we aim to investigate whether ellipsoids also form branch-like structures and how their shape affects the pattern. In our model, the shape of a vascular endothelial cell is represented as a spheroid, and a discrete dynamical system is constructed based on the simple assumption of two-body interactions. Numerical simulations demonstrate that our model reproduces the patterns of elongation and branching observed in the early stages of angiogenesis. We show that the pattern formation of the cell population is strongly dependent on the cell shape. Finally, we demonstrate that our current mathematical model reproduces the cell behaviours, specifically cell-mixing, observed in sprouts.

Dengue virus infection induces selective expansion of Vγ4 and Vγ6TCR γδ T cells in the small intestine and a cytokine storm driving vascular leakage in mice.

Dengue is a major health problem in tropical and subtropical regions. Some patients develop a severe form of dengue, called dengue hemorrhagic fever, which can be fatal. Severe dengue is associated with a transient increase in vascular permeability. A cytokine storm is thought to be the cause of the vascular leakage. Although there are various research reports on the pathogenic mechanism, the complete pathological process remains poorly understood. We previously reported that dengue virus (DENV) type 3 P12/08 strain caused a lethal systemic infection and severe vascular leakage in interferon (IFN)-α/ß and γ receptor knockout mice (IFN-α/ß/γRKO mice), and that blockade of TNF-α signaling protected mice. Here, we performed transcriptome analysis of liver and small intestine samples collected chronologically from P12/08-infected IFN-α/ß/γRKO mice in the presence/absence of blockade of TNF-α signaling and evaluated the cytokine and effector-level events. Blockade of TNF-α signaling mainly protected the small intestine but not the liver. Infection induced the selective expansion of IL-17A-producing Vγ4 and Vγ6 T cell receptor (TCR) γδ T cells in the small intestine, and IL-17A, together with TNF-α, played a critical role in the transition to severe disease via the induction of inflammatory cytokines such as TNF-α, IL-1ß, and particularly the excess production of IL-6. Infection also induced the infiltration of neutrophils, as well as neutrophil collagenase/matrix metalloprotease 8 production. Blockade of IL-17A signaling reduced mortality and suppressed the expression of most of these cytokines, including TNF-α, indicating that IL-17A and TNF-α synergistically enhance cytokine expression. Blockade of IL-17A prevented nuclear translocation of NF-κB p65 in stroma-like cells and epithelial cells in the small intestine but only partially prevented recruitment of immune cells to the small intestine. This study provides an overall picture of the pathogenesis of infection in individual mice at the cytokine and effector levels.

Activation of the Akt signalling pathway as a prognostic indicator in canine soft tissue sarcoma.

Canine soft tissue sarcoma (STS) is relatively common in dogs and is the generic term for tumours that originate from mesenchymal cells. While histopathological grade and immunolabelling with Ki-67 have been used for estimating prognosis, additional indicators are needed for predicting prognosis. Aberrant cell signalling pathways may contribute to disease activity and, therefore, prognostic markers. However, their role in canine STS remains poorly understood. The aim of this study was to investigate expression of phosphorylated Akt (phospho-Akt) and phosphorylated S6 (phospho-S6) as potential prognostic indicators. Immunohistochemical labelling was conducted on clinical samples of canine STS (n = 67). We found that phospho-Akt expression was positively correlated with histopathological grade (P = 0.001) and Ki-67 index (P <0.01). There was no apparent relationship between the type of STS and the expression of phospho-Akt. The number of cases that expressed phospho-S6, which is the downstream molecule of the Akt signalling pathway, was higher in immunopositive phospho-Akt cases than in immunonegative phospho-Akt cases (P <0.0001). Furthermore, phospho-Akt expression was significantly higher in recurrent and metastatic cases. We also confirmed that phosphorylation of Akt occurred in conjunction with S6 phosphorylation in three canine STS cell lines. These results suggest that immunolabelling for phospho-Akt, phospho-S6 and Ki-67 could potentially be used as a prognostic indicator and therapeutic target in canine STS.

Dose difference between anisotropic analytical algorithm (AAA) and Acuros XB (AXB) caused by target's air content for volumetric modulated arc therapy of head and neck cancer.

Background: We clarified the dose difference between the anisotropic analytical algorithm (AAA) and Acuros XB (AXB) with increasing target's air content using a virtual phantom and clinical cases. Materials and methods: Whole neck volumetric modulated arc therapy (VMAT) plan was transferred into a virtual phantom with a cylindrical air structure at the center. The diameter of the air structure was changed from 0 to 6 cm, and the target's air content defined as the air/planning target volume (PTV) in percent (air/PTV) was varied. VMAT plans were recalculated by AAA and AXB with the same monitor unit (MU) and multi-leaf collimator (MLC) motions. The dose at each air/PTV (5%-30%) was compared between each algorithm with D98%, D95%, D50% and D2% for the PTV. In addition, MUs were also compared with the same MLC motions between the D95% prescription with AAA (AAA_D95%), AXB_D95%, and the prescription to 100% minus air/PTV (AXB_D100%-air/PTV) in clinical cases of head and neck (HNC). Results: When air/PTV increased (5-30%), the dose differences between AAA and AXB for D98%, D95%, D50% and D2% were 3.08-15.72%, 2.35-13.92%, 0.63-4.59%, and 0.14-6.44%, respectively. At clinical cases with air/PTV of 5.61% and 28.19%, compared to AAA_D95%, the MUs differences were, respectively, 2.03% and 6.74% for AXB_D95% and 1.80% and 0.50% for AXB_D100%-air/PTV. Conclusion: The dose difference between AAA and AXB increased as the target's air content increased, and AXB_D95% resulted in a dose escalation over AAA_D95% when the target's air content was ≥ 5%. The D100%-air/PTV of PTV using AXB was comparable to the D95% of PTV using AAA.

Bimodal artificial intelligence using TabNet for differentiating spinal cord tumors-Integration of patient background information and images.

We proposed a bimodal artificial intelligence that integrates patient information with images to diagnose spinal cord tumors. Our model combines TabNet, a state-of-the-art deep learning model for tabular data for patient information, and a convolutional neural network for images. As training data, we collected 259 spinal tumor patients (158 for schwannoma and 101 for meningioma). We compared the performance of the image-only unimodal model, table-only unimodal model, bimodal model using a gradient-boosting decision tree, and bimodal model using TabNet. Our proposed bimodal model using TabNet performed best (area under the receiver-operating characteristic curve [AUROC]: 0.91) in the training data and significantly outperformed the physicians' performance. In the external validation using 62 cases from the other two facilities, our bimodal model showed an AUROC of 0.92, proving the robustness of the model. The bimodal analysis using TabNet was effective for differentiating spinal tumors.

Subcutaneous angiogenesis induced by transdermal delivery of gel-in-oil nanogel dispersion.

Subcutaneous transplantation aims to enhance the growth and functionality of transplanted cells for therapeutic outcomes in tissue engineering. However, the limited subcutaneous vascular network poses a challenge. Conventional methods involve co-transplantation with endothelial cells or angiogenic scaffold implantation, but they have drawbacks like tissue inflammation, compromised endothelial cell functionality, and the risk of repeated scaffold transplantation. Effective techniques are needed to overcome these challenges. This study explores the potential of G/O-NGD, a gel-in-oil nanogel dispersion, as a transdermal carrier of proliferative factors to promote angiogenesis in subcutaneous graft beds before cell transplantation. We observed robust subcutaneous angiogenesis by delivering varying amounts of bFGF using the G/O-NGD emulsion. Quantitative analysis of several parameters confirmed the efficacy of this method for building a subcutaneous vascular network. G/O-NGD is a biodegradable material that facilitates localized transdermal delivery of bFGF while maintaining its activity. The findings of this study have significant implications in both medical and industrial fields.

Evaluation of In-room Volumetric Imaging Doses for Image-guided Radiotherapy: A Multi-institutional Study.

Aims: We investigated imaging dose and noise under clinical scan conditions at multiple institutions using a simple and unified method, and demonstrated the need for diagnostic reference levels in image-guided radiotherapy (IGRT). Materials and Methods: Nine cone-beam and helical computed tomography (CT) scanners (Varian, Elekta, Accuray Inc., and BrainLAB) from seven institutions were investigated in this study. The weighted cone-beam dose index (CBDIw) was calculated for head and pelvic protocols using a 100 mm pencil chamber under the conditions used in actual clinical practice at each institution. Cone-beam CT image noise was evaluated using polymethylmethacrylate head and body phantoms with diameters of 16 and 32 cm, respectively. Results: For head and pelvic protocols, CBDIw values ranged from 0.94-6.59 and 1.47-20.9 mGy, respectively. Similarly, standard deviation (SD) values ranged from 9.3-34.0 and 26.9-97.4 HU, respectively. The SD values tended to increase with decreasing imaging dose (r = -0.33 and -0.61 for the head and pelvic protocols, respectively). Conclusions: Among the nine machines, the imaging dose for high imaging dose institutions was approximately 20 mGy to the pelvic phantom, and there was a 14-fold difference in dose compared with the other institutions. These results suggest the need to establish DRLs for IGRT to guide clinical decision-making.

An inhaled ACE2 decoy confers protection against SARS-CoV-2 infection in preclinical models.

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.

An efficient evaluation system for factors affecting the genome editing efficiency in mouse.

Genome editing technology is widely used in the field of laboratory animal science for the production of genetic disease models and the analysis of gene function. One of the major technical problems in genome editing is the low efficiency of precise knock-in by homologous recombination compared to simple knockout via non-homologous end joining. Many studies have focused on this issue, and various solutions have been proposed; however, they have yet to be fully resolved. In this study, we established a system that can easily determine the genotype at the mouse (Mus musculus) Tyr gene locus for genome editing both in vitro and in vivo. In this genome editing system, by designing the Cas9 cleavage site and donor template, wild-type, knockout, and knock-in genotypes can be distinguished by restriction fragment length polymorphisms of PCR products. Moreover, the introduction of the H420R mutation in tyrosinase allows the determination of knock-in mice with specific coat color patterns. Using this system, we evaluated the effects of small-molecule compounds on the efficiency of genome editing in mouse embryos. Consequently, we successfully identified a small-molecule compound that improves knock-in efficiency in genome editing in mouse embryos. Thus, this genome editing system is suitable for screening compounds that can improve knock-in efficiency.

Cellular senescence induction leads to progressive cell death via the INK4a-RB pathway in naked mole-rats.

Naked mole-rats (NMRs) have exceptional longevity and are resistant to age-related physiological decline and diseases. Given the role of cellular senescence in aging, we postulated that NMRs possess unidentified species-specific mechanisms to prevent senescent cell accumulation. Here, we show that upon induction of cellular senescence, NMR fibroblasts underwent delayed and progressive cell death that required activation of the INK4a-retinoblastoma protein (RB) pathway (termed "INK4a-RB cell death"), a phenomenon not observed in mouse fibroblasts. Naked mole-rat fibroblasts uniquely accumulated serotonin and were inherently vulnerable to hydrogen peroxide (H2 O2 ). After activation of the INK4a-RB pathway, NMR fibroblasts increased monoamine oxidase levels, leading to serotonin oxidization and H2 O2 production, which resulted in increased intracellular oxidative damage and cell death activation. In the NMR lung, induction of cellular senescence caused delayed, progressive cell death mediated by monoamine oxidase activation, thereby preventing senescent cell accumulation, consistent with in vitro results. The present findings indicate that INK4a-RB cell death likely functions as a natural senolytic mechanism in NMRs, providing an evolutionary rationale for senescent cell removal as a strategy to resist aging.