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Gene editing using CRISPR/Cas9 is a promising method to cure many human genetic diseases. We have developed an efficient system to deliver Cas9 into the adeno-associated virus integration site 1 (AAVS1) locus, known as a safe harbor, using lentivirus and AAV viral vectors, as a step toward future in vivo transduction. First, we introduced Cas9v1 (derived from Streptococcus pyogenes ) at random into the genome using a lentiviral vector. Cas9v1 activity was used when the N-terminal 1.9 kb, and C-terminal 2.3 kb fragments of another Cas9v2 (human codon-optimized) were employed sequentially with specific single-guide RNAs (sgRNAs) and homology donors carried by AAV vectors into the AAVS1 locus. Then, Cas9v1 was removed from the genome by another AAV vector containing sgRNA targeting the long terminal repeat of the lentivirus vector. The reconstituted Cas9v2 in the AAVS1 locus was functional and gene editing was efficient.

The COVID-19 pandemic has profoundly affected societies around the world. Although the emergency phase of coronavirus disease 2019 (COVID-19) has ended, the threat it poses remains persistent. This review aims to clarify the mechanisms of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection to support effective management of the disease. A central focus is the host cellular response to the viral infection, with particular emphasis on the role of cytokines. Cytokines play a dual role in antiviral defense: they contribute to the inhibition of viral replication and facilitate the clearance of pathogens, yet dysregulated cytokine responses can result in severe immunopathology. Interferons (type I, type II, and type III) and other cytokines are pivotal in activating intracellular antiviral mechanisms and in orchestrating the recruitment of immune cells through extracellular signaling. Effective immune responses to viral infections are governed not only by primary immune cells—such as dendritic cells, T lymphocytes, and B lymphocytes—but also by the local cytokine milieu shaped by infected and neighboring cells. Given the presence of endogenous inhibitors and autoantibodies in vivo, it is essential to evaluate the functional activity of cytokines in clinical samples. We propose a novel approach to quantify biologically active cytokine levels.

Chikungunya virus (CHIKV) is an enveloped virus that enters host cells and transits within the endosomes before starting its replication cycle, the precise mechanism of which is yet to be elucidated. Endocytosis and endosome acidification inhibitors inhibit infection by CHIKV, murine leukemia virus (MLV), or SARS-coronavirus, indicating that these viral entries into host cells occur through endosomes and require endosome acidification. Although endosomal cathepsin B protease is necessary for MLV, Ebola virus, and SARS-CoV infections, its role in CHIKV infection is unknown. Our results revealed that endocytosis inhibitors attenuated CHIKV-pseudotyped MLV vector infection in 293T cells but not in TE671 cells. In contrast, macropinocytosis inhibitors attenuated CHIKV-pseudotyped MLV vector infection in TE671 cells but not in 293T cells, suggesting that CHIKV host cell entry occurs via endocytosis or macropinocytosis, depending on the cell lines used. Cathepsin B inhibitor and knockdown by an shRNA suppressed CHIKV-pseudotyped MLV vector infection both in 293T and TE671 cells. These results show that cathepsin B facilitates CHIKV infection regardless of the entry pathway.

Background The neutrophil-to-lymphocyte ratio (NLR) and the platelet-to-lymphocyte ratio (PLR) are associated with poor prognoses in patients with gastric cancer; however, few studies have focused on the dynamic changes in these ratios during the treatment of patients with gastric cancer. Here, we assessed the clinical utility of changes in these ratios as prognostic indicators in patients with stage II or III gastric cancer who received adjuvant chemotherapy. Methods We retrospectively reviewed 100 patients who received S-1 adjuvant chemotherapy at ≥70% of the relative dose intensity, and their NLRs and PLRs were evaluated at different times: prior to gastrectomy and upon commencement and termination of adjuvant chemotherapy. To assure the clinical utility of the changes in NLR and PLR as prognostic indicators, other clinical factors were assessed as well. Results Disease recurred in 35 patients as follows: lymph node metastasis (17 patients, 17.0%), peritoneal metastasis (12 patients, 12.0%), and hematogenous metastasis (6 patients, 6.0%); 24 patients died. An increase in the NLR during adjuvant chemotherapy with S-1 was identified as an independent indicator associated with overall survival (hazard ratio [HR] 6.736, 95% confidence interval [CI] 2.420–18.748; P  < 0.001), and relapse-free survival (HR 5.309, 95% CI 2.585–10.901; P  < 0.001). Conclusion An increase in the NLR during S-1 adjuvant chemotherapy may be a useful prognostic indicator in patients with stage II or III gastric cancer.

Progranulin (PGRN) plays a crucial role in diverse biological processes, including cell proliferation and embryonic development. PGRN can be cleaved by neutrophil elastase to release granulin (GRN). PGRN has been found to inhibit inflammation. Whereas, GRN plays a role as a pro-inflammatory factor. However, the pathophysiological roles of PGRN and GRN, at early stages after cerebral ischemia, have not yet been fully understood. The aim of this study was to obtain further insight into the pathologic roles of PGRN and GRN. We demonstrated that the amount of PGRN was significantly increased in microglial cells after cerebral ischemia in rats and that neutrophil elastase activity was also increased at an early stage after cerebral ischemia, resulting in the production of GRN. The inhibition of neutrophil elastase activity suppressed PGRN cleavage and GRN production, as well as the increase in pro-inflammatory cytokines, after cerebral ischemia. The administration of an elastase inhibitor decreased the number of injured cells and improved the neurological deficits test scores. Our findings suggest that an increase in the activity of elastase to cleave PGRN, and to produce GRN, was involved in an inflammatory response at the early stages after cerebral ischemia, and that inhibition of elastase activity could suppress the progression of cerebral ischemic injury.

The combined use of a vital dye and radioactive colloid reportedly performs better in detecting sentinel lymph nodes (SLNs) for cancers than the use of either of them alone. However, especially for gastric cancer, two endoscopic procedures are required to administer these two tracers, which burdens the patients and practitioners. Here we propose the use of stannous colloid (SnC) mixed with indocyanine green (ICG) as a new mixed tracer (SnC–ICG); its characteristics were investigated in vivo and in vitro to estimate its usefulness for SLN navigation. The tracers were administered to rats and the accumulation of radioactivity and/or near-infrared fluorescence were evaluated in the regional lymph nodes (LNs) using single positron emission computed tomography and near-infrared fluorescence imaging, respectively. SnC–ICG showed significantly better clearance from the injection site and better migration to primary LNs than the single administration of SnC or ICG aqueous solution. SnC–ICG demonstrated a wide particle size variability, stabilized to 1200-nm upon the addition of albumin in vitro; These properties could contribute to its behavior in vivo. The use of SnC–ICG could contribute better performance to detect SLNs for gastric cancer with less burden on both patients and medical practitioners.

Cerebral ischemia induces neuronal cell death and causes various kinds of brain dysfunction. Therefore, prevention of neuronal cell death is most essential for protection of the brain. On the other hand, it has been reported that epigenetics including DNA methylation plays a pivotal role in pathogenesis of some diseases such as cancer. Accumulating evidences indicate that aberrant DNA methylation is related to cell death. However, DNA methylation after cerebral ischemia has not been fully understood yet. The aim of this present study was to investigate the relationships between DNA methylation and neuronal cell death after cerebral ischemia. We examined DNA methylation under the ischemic condition by using transient middle cerebral artery occlusion and reperfusion (MCAO/R) model rats and N-methyl-D-aspartate (NMDA)-treated cortical neurons in primary culture. In this study, we demonstrated that DNA methylation increased in these neurons 1 day after MCAO/R and that DNA methylation, possibly through activation of DNA methyltransferases (DNMT) 3a, increased in such neurons immediately after NMDA treatment. Furthermore, NMDA-treated neurons were protected by treatment with a DNMT inhibitor. Our results showed that DNA methylation would be an initiation factor of neuronal cell death and that inhibition of such methylation could become an effective therapeutic strategy for stroke.

Glioblastoma is the most common and lethal intracranial tumor type, characterized by high angiogenic and infiltrative capacities. To provide a novel insight into therapeutic strategies against glioblastoma, the cytotoxicity of arenobufagin and hellebrigenin was investigated in the human glioblastoma cell line, U-87. Similar dose-dependent cytotoxicity was observed in the cells, whereas no detectable toxicity was confirmed in mouse primary astrocytes. Treatment with each drug downregulated the expression levels of Cdc25C, Cyclin B1 and survivin, which occurred in parallel with G2/M phase arrest. Necrotic-like cell death was only observed in the cells treated with a relatively high concentration (>100 ng/ml). These results indicate that the two drugs exhibited distinct cytotoxicity against cancerous glial cells with high potency and selectivity, suggesting that growth inhibition associated with G2/M phase arrest and/or necrosis were attributed to their toxicities. Activation of the p38 mitogen activated protein kinase (MAPK) signaling pathway was also observed in treated cells. Notably, a specific inhibitor of p38 MAPK, SB203580, itself caused a significant decrease in cell viability, and further enhanced the cytotoxicity of the two drugs, suggesting an important pro-survival role for p38 MAPK. Given that p38 MAPK serves an essential role in promoting glioblastoma cell survival, developing a novel combination regimen of arenobufagin/hellebrigenin plus a p38 MAPK inhibitor may improve the efficacy of the two drugs, and may provide more therapeutic benefits to patients with glioblastoma. The qualitative assessment demonstrated the existence of arenobufagin in the cerebrospinal fluid of arenobufagin-treated rats, supporting its clinical application.

A high-risk infectious disease or a Category A pathogen, Lassa virus (LASV), requires strict containment, classified as biosafety level 4 (BSL-4) conditions, which restricts research on the virus due to the scarcity of BSL-4 facilities. Thus, replication-defective pseudotyped retroviral vectors have been widely used as safe materials for neutralizing activity assays of drugs and antibodies in BSL-2. Here, we established a novel retroviral vector system encoding LASV glycoprotein complex (GPC) that can exclusively replicate in cells expressing the Gag-Pol protein of murine leukemia virus (MLV) under BSL-2 conditions. Using this conditional replication system, we successfully isolated LASV GPC variants resistant to either an anti-LASV compound, lamellarin α 20-sulfate, or a neutralizing antibody derived from a Lassa fever survivor. In the lamellarin α 20-sulfate-resistant variants, K125E and H13R amino acid substitutions cooperatively conferred resistance. The K125E enhanced infectivity and simultaneously conferred a lethal effect on cells in the conditional replication system, while the H13R mitigated the latter effect, thereby enabling stable expression of LASV GPC in cells. In the neutralizing antibody-resistant variants, I403T substitution was responsible for the resistance by impairing antibody binding. This study provides a valuable BSL-2-based platform for isolating LASV GPC variants resistant to inhibitors and characterizing their mutations.

Intramedullary spinal cord metastasis (ISCM) is a rare manifestation of renal cell carcinoma (RCC). A 73-year-old man presented with left shoulder pain and left upper extremity weakness for two months. Magnetic resonance imaging (MRI) revealed intramedullary and intradural extramedullary lesions at the C5 level, compressing the spinal cord from the center of the cord and the left ventral side. Contrast-enhanced CT revealed a right renal mass and brain MRI showed no other lesions. Digital subtraction angiography showed a tumor stain from the anterior spinal artery and subsequent angioarchitecture of the intra- and extramedullary tumors. Following exoscopic resection of the extramedullary tumor, the intramedullary tumor was removed via a posterior midline myelotomy. The tumor surrounding the anterior spinal artery was intentionally left to prevent neurological deficits. The histopathological examination revealed metastatic clear cell RCC. Postoperative MRI revealed a small residual tumor ventral to the spinal cord. The nephrectomy for the right RCC was performed one month after the initial spinal surgery. Within the subsequent one month, the residual tumor rapidly increased in size. Reoperation with exoscopic-endoscopic techniques achieved complete tumor resection. The patient underwent radiotherapy to the C3-6 levels (30 Gy in 10 fractions) and pembrolizumab therapy. Postoperative MRI demonstrated no recurrence for four months, and the patient's symptoms remained in the same preoperative state. This case highlights the successful use of advanced minimally invasive techniques for treating ventrally exophytic ISCM from RCC.