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In this study, the viral genome extraction performance of automatic nucleic acid extractors and manual nucleic acid extraction kits was compared. We showed that compared with manual kits, the automatic extractors showed superior genome extraction performance using bovine viral diarrhea virus (BVDV) genome-positive cattle sera and bovine coronavirus/infectious bovine rhinotracheitis virus-spiked cattle nasal swabs. In addition, the subgenotyping of BVDV strains detected in Tokachi Province in Japan during 2016-2017 was performed. Results showed that most of these BVDV strains belonged to subgenotype 1b, while few strains belonged to subgenotypes 1a and 2a. This study showed the high applicability of automatic nucleic acid extractors in extracting multiple viral genomes and the dominant subgenotype of BVDV in Tokachi.

application/pdf, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a threat to human health. Acidic electrolyzed water (AEW) has recently been suggested to demonstrate virucidal activity. Many types of AEW with different pH values, generated by the electrolysis of different chemicals, such as sodium chloride, potassium chloride, and hydrochloric acid, are commercially available. In this study, we compared the virucidal activities of these types of AEW against SARS-CoV-2, including the ancestral strain and variant Alpha, Beta, Gamma, Delta, and Omicron strains. Virus solution (viral titer, 6.9 log10 50% tissue culture infective dose [TCID50]/mL) was mixed with AEW (free available chlorine concentration, 34.5 ppm) at mixing ratios of 1:9, 1:19, and 1:49. At mixing ratios of 1:9 and 1:19, AEW with a pH of 2.8 showed stronger virucidal activities than AEW with a pH of 4.1 to 6.5 against the SARS-CoV-2 ancestral strain in 20 s. From the strongest to the weakest virucidal activity, the AEW pH levels were as follows: pH 2.8, pH 4.1 to 5.4, pH 6.4 to 6.5. At a ratio of 1:49, the viral titers of viruses treated with all AEW solutions at pH 2.8 to 6.5 were almost below the detection limit, which was 1.25 log10 TCID50/mL. The virus inactivation efficiency of AEW was reduced in the presence of fetal bovine serum and other substances contained in the virus solution used in this study. AEW with pH values of 2.8 to 6.5 showed virucidal activity against all of the tested SARS-CoV-2 strains, including the ancestral and variant strains. These results provide useful knowledge for the effective application of AEW as a SARS-CoV-2 disinfectant. Copyright © 2022 Takeda et al.

We evaluated the SARS-CoV-2-inactivation activity of ozonated glycerol (OG). When a viral solution with 1% fetal bovine serum (FBS) was mixed with test solutions at a ratio of 1:19 and incubated for 20 s, OG with ozone concentrations of over 1000 ppm inactivated ≥ 94.38% of the virus. Extension of the reaction time to 1 h led to the inactivation of ≥ 99.82% of the virus (the viral titer was below the detection limit). Extension to 24 h resulted in concentrations over 200 ppm OG inactivating ≥ 99.87% of the virus (the viral titers were below the detection limit). Next, viral solutions with 1, 20, and 40% FBS were mixed with test solutions at a ratio of 1:19 and incubated for 5 min. Whereas the virucidal activity of 500 ppm OG was very limited in the presence of 1% FBS (79.47% inactivation), it increased in the presence of 20 and 40% FBS (95.13 and 97.95% inactivation, respectively; the viral titers were not below the detection limit). Meanwhile, over 1000 ppm OG inactivated ≥ 99.44% of the virus regardless of the FBS concentration (the viral titers were below the detection limit). Extension of the reaction time to 1 h led to 500 ppm OG inactivating ≥ 99.91 and ≥ 99.95% of the virus with 20 and 40% FBS, respectively (the viral titers were below the detection limit). These results suggested that OG might be useful as a virucidal agent against SARS-CoV-2.

The use of effective disinfectants is a key method of controlling the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Hypochlorous acid water (HAW) has a broad spectrum of virucidal activities. We previously reported that acidic electrolyzed water, one of the HAW products, had potent SARS-CoV-2-inactivating activity and showed promise as a disinfectant. However, different manufacturing methods have produced several HAW products with various pH values. Here, we compared the SARS-CoV-2-inactivating activities of various HAW products. At sufficiently high volume and residual chlorine concentration (RCC), the HAW products inactivated SARS-CoV-2 efficiently regardless of pH or manufacturing method. However, although HAW products at pH 5.0–6.4 maintained high RCC and sustained virucidal activity for 21 days, the RCC rapidly decreased in HAW products at pH ≤ 3.0. Our results may guide in choosing appropriate HAW products for different usage situations. HIGHLIGHTS Hypochlorous acid water (HAW) showed virucidal activity against SARS-CoV-2.; Various HAW products showed virucidal activity regardless of manufacturing methods.; Virucidal activity of HAWs depends on the amount of residual chlorine (RC).; HAWs with pH 5.0–6.4 maintained high RC concentration for 21 days.; RC concentration was rapidly decreased in HAW with pH ≤ 3.0.

BACKGROUND/AIM The inflammatory cytokine IL-8 and its receptor CXCR2 are key signalling pathway molecules in cancer development. We hypothesized that IL-8/CXCR2 signalling promotes tumour progression in oesophageal squamous cell carcinoma (ESCC) patients. MATERIALS AND METHODS We examined the relationship between IL-8/CXCR2 expression and clinicopathological factors by immunohistochemistry in samples from 63 patients with resectable ESCC. The effects of IL-8/CXCR2 signalling on cell proliferation and gene expression were examined in vitro and in vivo using ESCC cell lines. RESULTS Increased IL-8/CXCR2 signalling was associated with shorter overall survival (p

Background & Aims Organ-level research using an animal model lacking Il2rg, the gene responsible for X-linked severe combined immunodeficiency (X-SCID), is clinically unavailable and would be a powerful tool to gain deeper insights into the symptoms of patients with X-SCID. Methods We used an X-SCID animal model, which was first established in our group by the deletion of Il2rg gene in pigs, to understand the clinical signs from multiple perspectives based on pathology, immunology, microbiology, and nutrition. We also treated the X-SCID pigs with bone marrow transplantation (BMT) for mimicking a current therapeutic treatment for patients with X-SCID and investigated the effect at the organ-level. Moreover, the results were confirmed using serum and fecal samples collected from patients with X-SCID. Results We demonstrated that X-SCID pigs completely lacked Peyer’s patches (PPs) and IgA production in the small intestine, but possessed some dysfunctional intestinal T and B cells. Another novel discovery was that X-SCID pigs developed a heterogeneous intestinal microflora and possessed abnormal plasma metabolites, indicating that X-SCID could be an immune disorder that affects various in vivo functions. Importantly, the organogenesis of PPs in X-SCID pigs was not promoted by BMT. Although a few isolated lymphoid follicles developed in the small intestine of BMT-treated X-SCID pigs, there was no evidence that they contributed to IgA production and microflora formation. Consistently, most patients with X-SCID who received BMT possessed abnormal intestinal immune and microbial environments regardless of the presence of sufficient serum IgG. Conclusions These results indicate that the current BMT therapies for patients with X-SCID may be insufficient to induce the organogenesis of intestinal lymphoid tissues that are associated with numerous functions in vivo., Graphical abstract

Simple Summary Screening, monitoring, and diagnostic methods in oncology are a critical part of treatment. The currently used clinical methods have limitations, most notably the time, cost, and special facilities required for radioisotope-based techniques. The use of magnetic nanoparticles is an alternative approach that offers faster analyses with safer materials over a wide range of oncological applications, such as the detection of cancer biomarkers and immunostaining. Furthermore, magnetic nanoparticles, such as superparamagnetic iron oxide nanoparticles, can detect sentinel lymph nodes for breast cancer in a clinical setting, as well as those for gallbladder cancer in animal models within a timeframe that would enable them to be used during surgery with a magnetic probe. Abstract Screening, monitoring, and diagnosis are critical in oncology treatment. However, there are limitations with the current clinical methods, notably the time, cost, and special facilities required for radioisotope-based methods. An alternative approach, which uses magnetic beads, offers faster analyses with safer materials over a wide range of oncological applications. Magnetic beads have been used to detect extracellular vesicles (EVs) in the serum of pancreatic cancer patients with statistically different EV levels in preoperative, postoperative, and negative control samples. By incorporating fluorescence, magnetic beads have been used to quantitatively measure prostate-specific antigen (PSA), a prostate cancer biomarker, which is sensitive enough even at levels found in healthy patients. Immunostaining has also been incorporated with magnetic beads and compared with conventional immunohistochemical methods to detect lesions; the results suggest that immunostained magnetic beads could be used for pathological diagnosis during surgery. Furthermore, magnetic nanoparticles, such as superparamagnetic iron oxide nanoparticles (SPIONs), can detect sentinel lymph nodes in breast cancer in a clinical setting, as well as those in gallbladder cancer in animal models, in a surgery-applicable timeframe. Ultimately, recent research into the applications of magnetic beads in oncology suggests that the screening, monitoring, and diagnosis of cancers could be improved and made more accessible through the adoption of this technology.

As a result of the novel coronavirus disease 2019 pandemic, strengthening control measures against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become an urgent global issue. In addition to antiviral therapy and vaccination strategies, applying available virucidal substances for SARS-CoV-2 inactivation is also a target of research to prevent the spread of infection. Here, we evaluated the SARS-CoV-2 inactivation activity of a copper iodide (CuI) nanoparticle dispersion, which provides Cu+ ions having high virucidal activity, and its mode of actions. In addition, the utility of CuI-doped film and fabric for SARS-CoV-2 inactivation was evaluated. The CuI dispersion exhibited time-dependent rapid virucidal activity. Analyses of the modes of action of CuI performed by Western blotting and real-time reverse transcription-PCR targeting viral proteins and the genome revealed that CuI treatment induced the destruction of these viral components. In this setting, the indirect action of CuI-derived reactive oxygen species contributed to the destruction of viral protein. Moreover, the CuI-doped film and fabric demonstrated rapid inactivation of the SARS-CoV-2 solution in which the viral titer was high. These findings indicated the utility of the CuI-doped film and fabric as anti-SARS-CoV-2 materials for the protection of high-touch environmental surfaces and surgical masks/protective clothes. Throughout this study, we demonstrated the effectiveness of CuI nanoparticles for inactivating SARS-CoV-2 and revealed a part of its virucidal mechanism of action. IMPORTANCE The COVID-19 pandemic has caused an unprecedented number of infections and deaths. As the spread of the disease is rapid and the risk of infection is severe, hand and environmental hygiene may contribute to suppressing contact transmission of SARS-CoV-2. Here, we evaluated the SARS-CoV-2 inactivation activity of CuI nanoparticles, which provide the Cu+ ion as an antiviral agent, and we provided advanced findings of the virucidal mechanisms of action of Cu+. Our results showed that the CuI dispersion, as well as CuI-doped film and fabric, rapidly inactivated SARS-CoV-2 with a high viral titer. We also demonstrated the CuI’s virucidal mechanisms of action, specifically the destruction of viral proteins and the genome by CuI treatment. Protein destruction largely depended on CuI-derived reactive oxygen species. This study provides novel information about the utility and mechanisms of action of promising virucidal material against SARS-CoV-2.

Background/aim Pancreatic cancer, which exhibits resistance to cytotoxic and molecular targeted drugs, has an extremely poor prognosis. Nuclear factor-κB (NF-κB) is constitutively activated in many pancreatic cancer cases. Although the NF-κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) has exhibited anti-cancer effects in pancreatic cancer models, its poor solubility limits its use to intraperitoneal administration. Materials and methods Poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB) forms stable polymer aggregates with DHMEQ. The stability of DHMEQ aggregated with PMB in the human blood was measured by high-performance liquid chromatography-mass spectrometry (HPLC-MS) ex vivo. Anti-pancreatic cancer effects in AsPC-1 and MIA PaCa-2 pancreatic cancer cells were evaluated by cell growth inhibition assay in vitro and tumor growth inhibition assay in vivo. Results DHMEQ aggregated with PMB (PMB-DHMEQ) remained detectable after 60 min of incubation in the human blood, whereas DHMEQ aggregated with carboxymethyl cellulose (CMC-DHMEQ) was barely detectable. PMB-DHMEQ significantly inhibited AsPC-1 and MIA PaCa-2 cell growth in vitro compared to CMC-DHMEQ. Intravenous administration of PMB-DHMEQ reduced the tumor volume and liver metastasis compared to untreated or CMC-DHMEQ-treated mice. Conclusion Aggregation with PMB improved the solubility of DHMEQ, and effectively inhibited pancreatic cancer cell growth both in vitro and in vivo.

This study aimed to compare the SARS-CoV-2-inactivation activity and virucidal mechanisms of ozonated water (OW) with those of slightly acidic electrolyzed water (SAEW) and 70% ethanol (EtOH). SARS-CoV-2-inactivation activity was evaluated in a virus solution containing 1%, 20% or 40% fetal bovine serum (FBS) with OW, SAEW or EtOH at a virus-to-test solution ratio of 1:9, 1:19 or 1:99 for a reaction time of 20 s. EtOH showed the strongest virucidal activity, followed by SAEW and OW. Even though EtOH potently inactivated the virus despite the 40% FBS concentration, virus inactivation by OW and SAEW decreased in proportion to the increase in FBS concentration. Nevertheless, OW and SAEW showed potent virucidal activity with 40% FBS at a virus-to-test solution ratio of 1:99. Real-time PCR targeting the viral genome revealed that cycle threshold values in the OW and SAEW groups were significantly higher than those in the control group, suggesting that OW and SAEW disrupted the viral genome. Western blotting analysis targeting the recombinant viral spike protein S1 subunit showed a change in the specific band into a ladder upon treatment with OW and SAEW. OW and SAEW may cause conformational changes in the S1 subunit of the SARS-CoV-2 spike protein.

Current immunohistochemistry methods for diagnosing abnormal cells, such as cancer cells, require multiple steps and can be relatively slow compared with intraoperative frozen hematoxylin and eosin staining, and are therefore rarely used for intraoperative examination. Thus, there is a need for novel rapid detection methods. We previously demonstrated that functionalized fluorescent ferrite beads (FF beads) magnetically promoted rapid immunoreactions. The aim of this study was to improve the magnetically promoted rapid immunoreaction method using antibody-coated FF beads and a magnet subjected to a magnetic field. Using frozen sections of xenograft samples of A431 human epidermoid cancer cells that express high levels of epidermal growth factor receptor (EGFR) and anti-EGFR antibody-coated FF beads, we reduced the magnetically promoted immunohistochemistry procedure to a 1-min reaction and 1-min wash. We also determined the optimum magnetic force for the antibody reaction (from 7.79 × 10−15 N to 3.35 × 10−15 N) and washing (4.78 × 10−16 N), which are important steps in this technique. Furthermore, we stained paraffin-embedded tissue arrays and frozen sections of metastatic breast cancer lymph nodes with anti-pan-cytokeratin antibody-coated FF beads to validate the utility of this system in clinical specimens. Under optimal conditions, this ultra-rapid immunostaining method may provide an ancillary method for pathological diagnosis during surgery. (J Histochem Cytochem 58:XXX–XXX, 2010)

The number of viable bacteria was investigated in pre-cut vegetables and vegetable salads with mayonnaise. First, no discernible difference was found in the viable bacteria in either bagged or packaged pre-cut ones. Second, Pre-cut vegetable and dressed salads were stored at 5 ℃ and 25℃ for 48 hours to see how temperature difference would affect the behavior of bacteria. In all samples, the bacterial growth was slightly observed at 5℃ . In the pre-cut vegetables, at 25℃ , the number of viable bacteria was found to increase rapidly. However, in the dressed salads, the difference was observed depending upon different pH level. The dressed salads at pH5.8 showed the bacteria rapid increase, but the slow bacterial growth was observed at pH5.2. Finally, Pre-cut vegetables and dressed salads using antibacterial sheet stored at 25℃ for 26 hours to examine how the antibacterial sheet would affect the behavior of bacteria. It was found also that, for the dressed salads using antibacterial sheet, the growth of bacteria was suppressed during storage at 25℃. Therefore, commercial vegetables need to be eaten soon after purchase or they need to store at low temperature. Also, using antibacterial sheet, vegetables with mayonnaise can be taken more safely.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally. Although measures to control SARS-CoV-2, namely, vaccination, medication, and chemical disinfectants are being investigated, there is an increase in the demand for auxiliary antiviral approaches using natural compounds. Here we have focused on hydroxytyrosol (HT)-rich aqueous olive pulp extract (HIDROX®) and evaluated its SARS-CoV-2-inactivating activity in vitro. We showed that the HIDROX solution exhibits time- and concentration-dependent SARS-CoV-2-inactivating activities, and that HIDROX has more potent virucidal activity than pure HT. The evaluation of the mechanism of action suggested that both HIDROX and HT induced structural changes in SARS-CoV-2, which changed the molecular weight of the spike proteins. Even though the spike protein is highly glycosylated, this change was induced regardless of the glycosylation status. In addition, HIDROX or HT treatment disrupted the viral genome. Moreover, the HIDROX-containing cream applied on film showed time- and concentration-dependent SARS-CoV-2-inactivating activities. Thus, the HIDROX-containing cream can be applied topically as an antiviral hand cream. Our findings suggest that HIDROX contributes to improving SARS-CoV-2 control measures.

Mapping of sentinel lymph nodes (SLNs) can enable less invasive surgery. However, mapping is challenging for cancers of difficult-to-access visceral organs, such as the gallbladder, because the standard method using radioisotopes (RIs) requires preoperative tracer injection. Indocyanine green (ICG) and superparamagnetic iron oxide (SPIO) have also been used as alternative tracers. In this study, we modified a previously reported magnetic probe for laparoscopic use and evaluated the feasibility of detecting SLNs of the gallbladder using a laparoscopic dual tracer method by injecting ICG and SPIO into five swine and one cancer-bearing swine. The laparoscopic probe identified SPIO nanoparticles in the nodes of 4/5 swine in situ, the magnetic field counts were 2.5–15.9 μT, and fluorescence was detected in SLNs in all five swine. ICG showed a visual lymph flow map, and SPIO more accurately identified each SLN with a measurable magnetic field quite similar to the RI. We then developed an advanced gallbladder cancer model with lymph node metastasis using recombination activating gene 2-knockout swine. We identified an SLN in the laparoscopic investigation, and the magnetic field count was 3.5 μT. The SLN was histologically determined to be one of the two metastatic lymph nodes. In conclusion, detecting the SLNs of gallbladder cancer in situ using a dual tracer laparoscopic technique with ICG and SPIO was feasible in a swine model.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally. Although measures to control SARS-CoV-2, namely, vaccination, medication, and chemical disinfectants are being investigated, there is an increase in the demand for auxiliary antiviral approaches using natural compounds. Here we have focused on hydroxytyrosol (HT)-rich aqueous olive pulp extract (HIDROX®) and evaluated its SARS-CoV-2-inactivating activity in vitro. We showed that the HIDROX solution exhibits time- and concentration-dependent SARS-CoV-2-inactivating activities, and that HIDROX has more potent virucidal activity than pure HT. The evaluation of the mechanism of action suggested that both HIDROX and HT induced structural changes in SARS-CoV-2, which changed the molecular weight of the spike proteins. Even though the spike protein is highly glycosylated, this change was induced regardless of the glycosylation status. In addition, HIDROX or HT treatment disrupted the viral genome. Moreover, the HIDROX-containing cream applied on film showed time- and concentration-dependent SARS-CoV-2-inactivating activities. Thus, the HIDROX-containing cream can be applied topically as an antiviral hand cream. Our findings suggest that HIDROX contributes to improving SARS-CoV-2 control measures.

Hepatocellular carcinoma (HCC) is the fifth most common primary tumor and the third leading cause of cancer-related deaths worldwide. Rodent models of HCC have contributed to the advancement of studies investigating liver carcinogenesis, tumor-host interactions, and drug screening. However, their small size renders them unsuitable for surgical or clinical imaging studies, necessitating the development of larger-size HCC models. Here, we developed a xenograft model of human HCC in X-linked interleukin-2 receptor gamma chain gene (Il2rg)-targeted severe combined immunodeficient (SCID) pigs. HepG2 cell suspension in serum-free medium containing 50% membrane matrix was directly injected into the liver parenchyma of eight X-linked Il2rg-targeted SCID pigs (6.6–15.6 kg) via ultrasonography-guided percutaneous puncture. Tumor engraftment was evaluated weekly using ultrasonography, and cone-beam computed tomography was performed during arterial portography (CTAP) and hepatic arteriography (CTHA) to evaluate the hemodynamics of engrafted tumors. The engrafted tumors were histologically analyzed following necropsy and assessed for pathological similarities to human HCCs. Macroscopic tumor formation was observed in seven of the eight pigs (simple nodular tumors in three and multinodular tumors in four). Engrafted tumors were identified as low-echoic upon ultrasonography and as perfusion-defect nodules on the CTAP images. Meanwhile, CTHA showed that the tumors were hyperattenuating. Further, histopathological findings of the engrafted tumors were consistent with those of human HCC. In conclusion, the porcine model of human HCC, successfully generated herein, might help develop more effective therapeutic strategies for HCC.

Objectives Cryoablation is a potentially less invasive locoregional ablation modality. Although cryoablation has been used to treat malignancy in various organs, a limited application of this modality in the pancreas has been reported. Methods Acute phase response assessments and postprocedural course evaluations of 2 experimental locoregional ablation methods were conducted. In one method, open and laparoscopic cryoablation of a porcine pancreas using an argon-helium gas-based cryoablation system and monitoring of tissue temperature during the procedure were performed. In the other method, open cryoablation of a porcine pancreas using liquid nitrogen was performed. The animals were evaluated postoperatively. Results The size of the cryolesion was larger in the second treatment than in the first. Laparoscopic cryoablation was associated with the formation of an iceball, which possibly affected the surrounding structures. The ablated region was adequately cooled with 10 minutes of freeze/repeat cycles. The area cooled to a temperature of less than -40°C was approximately half the size of the cryolesion in diameter. The swine used for the evaluation of the postprocedural course survived 3 weeks after the procedure with a temporal elevation of the serum lipase level. Conclusions Cryoablation of the pancreas was experimentally practicable without severe complications under direct or laparoscopic vision.

Simple Summary Pancreatic cancer (PC) is among the most lethal malignancies due to an often delayed and difficult initial diagnosis. Therefore, the development of a novel, early stage, diagnostic PC marker in liquid biopsies is of great significance. In this study, we analyzed the differential glycomic profiling of extracellular vesicles (EVs) derived from serum using lectin microarray. The glyco-candidates of PC-specific EVs were quantified using a high-sensitive exosome-counting system, ExoCounter. An absolute quantification system for altered glycan-containing EVs elevated in PC serum was established. EVs recognized by O-glycan-binding lectins ABA or ACA were identified as candidate markers by lectin microarray. Quantitative analyses using ExoCounter revealed that the ABA- or ACA-positive EVs were significantly increased in the serum of PC patients. These specific EVs with O-glycans can act as potential biomarkers in a liquid biopsy for PC patients screening. Abstract Pancreatic cancer (PC) is among the most lethal malignancies due to an often delayed and difficult initial diagnosis. Therefore, the development of a novel, early stage, diagnostic PC marker in liquid biopsies is of great significance. In this study, we analyzed the differential glycomic profiling of extracellular vesicles (EVs) derived from serum (two cohorts including 117 PC patients and 98 normal controls) using lectin microarray. The glyco-candidates of PC-specific EVs were quantified using a high-sensitive exosome-counting system, ExoCounter. An absolute quantification system for altered glycan-containing EVs elevated in PC serum was established. EVs recognized by O-glycan-binding lectins ABA or ACA were identified as candidate markers by lectin microarray. Quantitative analyses using ExoCounter revealed that the ABA- or ACA-positive EVs were significantly increased in the culture of PC cell lines or in the serum of PC patients including carbohydrate antigen 19-9 negative patients with high area under curve values. The elevated numbers of EVs in PC serum returned to normal levels after pancreatectomy. Histological examination confirmed that the tumors stained with ABA/ACA. These specific EVs with O-glycans recognized by ABA/ACA are elevated in PC sera and can act as potential biomarkers in a liquid biopsy for PC patients screening.

Alcohol-based disinfectant shortage is a serious concern in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Acidic electrolyzed water (EW) with a high concentration of free available chlorine (FAC) shows strong antimicrobial activity against bacteria, fungi, and viruses. Here, we assessed the SARS-CoV-2-inactivating efficacy of acidic EW for use as an alternative disinfectant. The quick virucidal effect of acidic EW depended on the concentrations of contained-FAC. The effect completely disappeared in acidic EW in which FAC was lost owing to long-time storage after generation. In addition, the virucidal activity increased proportionately with the volume of acidic EW mixed with the virus solution when the FAC concentration in EW was same. These findings suggest that the virucidal activity of acidic EW against SARS-CoV-2 depends on the amount of FAC contacting the virus., Graphical abstract Image 1, Highlights • Acidic electrolyzed water (EW) shows virucidal activity against SARS-CoV-2. • Virucidal activity of acidic EW depends on free available chlorine (FAC). • Acidic solution without FAC does not inactivate SARS-CoV-2 in a 1-min reaction. • Large amounts of FAC are required to inactivate virus containing many proteins.

New laparoscopic sentinel lymph node navigation using a dedicated magnetic probe and magnetic nanoparticle tracer for gastric cancer patients allows minimally invasive surgeries. By identifying the sentinel lymph nodes containing magnetic nanoparticles, patients can avoid excessive lymph node extraction without nuclear facilities and radiation exposure. This paper describes the development of the laparoscopic magnetic probe, ACDC-probe, for laparoscopic sentinel lymph node identification utilizing the nonlinear response of the magnetic nanoparticles magnetized by an alternating magnetic field with a static magnetic field. For highly sensitive detection, the ratio of static to alternating magnetic fields was optimized to approximately 5. The longitudinal detection length was approximately 10 mm for 140 μg of iron, and the detectable amount of iron was approximately 280 ng at a distance of 1 mm. To demonstrate the feasibility of laparoscopic detection using the ACDC-probe and magnetic tracers, an experiment was performed on a wild swine. The gastric sentinel lymph node was clearly identified during laparoscopic navigation. These results suggest that the newly developed ACDC-probe is useful for laparoscopic sentinel lymph node detection and this magnetic technique appears to be a promising method for future sentinel lymph node navigation of gastric cancer patients.

Background: Pancreatic ductal adenocarcinoma (PDAC) has very poor prognosis despite existing multimodal therapies. This study aimed to investigate whether KRAS mutations at codons 12/13 in cell-free DNA (cfDNA) from preoperative and postoperative sera from patients with PDAC can serve as a predictive biomarker for treatment response and outcomes after surgery. Methods: Preoperative and postoperative serum samples obtained from 45 patients with PDAC whom underwent curative pancreatectomy at our institution between January 2013 and July 2016 were retrospectively analysed. Peptide nucleic acid-directed PCR clamping was used to identify KRAS mutations in cfDNA. Results: Among the 45 patients enrolled, 11 (24.4%) and 20 (44.4%) had KRAS mutations in cfDNA from preoperative and postoperative sera, respectively. Multivariate analysis revealed that KRAS mutations in postoperative serum (hazard ratio (HR)=2.919; 95% confidence interval (CI)=1.109–5.621; P=0.027) are an independent prognostic factor for disease-free survival. Furthermore, the shift from wild-type KRAS in preoperative to mutant KRAS in postoperative cfDNA (HR=9.419; 95% Cl=2.015–44.036; P=0.004) was an independent prognostic factor for overall survival. Conclusions: Changes in KRAS mutation status between preoperative and postoperative cfDNA may be a useful predictive biomarker for survival and treatment response.

Aim. The usefulness of photodynamic therapy (PDT) for treating sentinel lymph node (SLN) metastasis was evaluated.Materials and Methods. Verteporfin, a hydrophobic photosensitizer, forms a soluble aggregate with poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB). The concentrations of verteporfin were determined by measuring the fluorescence emitted at 700 nm. Seven days after the inoculation of A431 cells at the forearm of BALB/c nude mice, PMB-verteporfin was injected at dorsum manus and 75 J of light energy was delivered for 1 minute. Fifty-three mice were randomly assigned to the combination of PMB-verteporfin injection and light exposure, light exposure alone, PMB-verteporfin injection alone, and no treatment groups. Ten days after PDT, brachial lymph nodes, which were considered as SLNs, were harvested and evaluated.Results. The concentration of verteporfin in SLN was significantly higher than other organs. The combination of PMB-verteporfin injection and light exposure group significantly reduced the SLN metastasis (13%) comparing with no treatment group (52%), light exposure alone group (57%), and PMB-verteporfin injection alone group (46%).Conclusions. These data suggested that PDT using PMB as a nanotransporter of verteporfin could be a minimally invasive treatment of SLN metastasis in breast cancer and represent a potential alternative procedure to SLNB.

There are increased opportunities in oncology clinics to identify multiple pancreatic ductal adenocarcinomas (PDAC) that co‐occur simultaneously or arise metachronously in the pancreatic parenchyma, yet their pathogenesis remains elusive. We hypothesized that two potential pathways, multicentric carcinogenesis and intrapancreatic metastasis, might contribute to forming multiple PDAC. Among 241 resected cases, we identified 20 cancer nodules from nine patients with multiple PDAC (six with synchronous PDAC, one with metachronous PDAC, and two with both synchronous and metachronous PDAC). Integrated clinical, pathological, and mutational analyses, using TP53 and SMAD4 immunostaining and targeted next‐generation sequencing of 50 cancer‐related genes, were conducted to examine the intertumor relationships. Four of the nine patients were assessed as having undergone multicentric carcinogenesis because of heterogeneity of immunohistochemical and/or mutation characteristics. In contrast, tumors in the other five patients showed intertumor molecular relatedness. Two of these five patients, available for matched sequencing data, showed two or more shared mutations. Moreover, all the smaller nodules in these five patients showed identical TP53 and SMAD4 expression patterns to the corresponding main tumors. Consequently, these five patients were considered to have undergone intrapancreatic metastasis. None of the five smaller nodules arising from intrapancreatic metastasis was accompanied by pancreatic intraepithelial neoplasia, and three of them were tiny (≤1mm). Patients whose tumors resulted from intrapancreatic metastasis appeared to have higher disease stages and worse outcome than those with tumors from multicentric carcinogenesis. Our results provide insight into pancreatic carcinogenesis, showing that the development of multiple PDAC involves distinct evolutionary paths that potentially affect patient prognosis., Integrated clinical, pathological, and mutational analyses on multiple pancreatic cancers identify distinct evolutionary paths: multicentric carcinogenesis and intrapancreatic metastasis.

Background Oesophageal squamous cell carcinoma is an aggressive disease owing to early and widespread lymph node metastases. Multimodal therapy and radical surgery may improve prognosis. Few studies have investigated the efficacy of radical lymph node and thoracic duct resection. Methods Patients with oesophageal squamous cell carcinoma who underwent transthoracic minimally invasive oesophagectomy (TMIE) for cancer at Keio University Hospital between January 2004 and December 2016 were selected. Between 2004 and 2008, TMIE was performed in the lateral decubitus position without thoracic duct resection (standard TMIE). From 2009 onwards, TMIE with extended lymph node and thoracic duct resection was introduced (extended TMIE). Demographics, co-morbidity, number of retrieved lymph nodes, pathology, postoperative complications and recurrence-free survival (RFS) were compared between groups. Results Forty-four patients underwent standard TMIE and 191 extended TMIE. There were no significant differences in clinical and pathological tumour stage or postoperative complications. The extended-TMIE group had more lymph nodes removed at nodal stations 106recL and 112. Among patients with cT1 N0 disease, RFS was better in the extended-TMIE group (P < 0·001), whereas there was no difference in RFS between groups in patients with advanced disease. Conclusion Extended TMIE including thoracic duct resection increased the number of lymph nodes retrieved and was associated with improved survival in patients with cT1 N0 oesophageal squamous cell carcinoma.

Lymph node metastasis occurs via the migration of cancer cells through the lymphatic system. Sentinel lymph node (SLN) biopsy is a common diagnostic strategy. SLNs have been studied using healthy rodents and large animals without metastasis. Here we used immunodeficient swine to establish a model of lymph node metastasis. We used RAG2-knockout immunodeficient swine. A431 human epithelial carcinoma cells expressing green fluorescent protein were injected subcutaneously into the posterior sides of the auricle, forelimb and hindlimb of knockout swine. Indigo carmine dye was injected subcutaneously 8 weeks after tumour cell transplantation. SLNs were extracted, observed using a stereoscopic fluorescence microscope and analysed histologically using haematoxylin and eosin staining, and immunohistochemistry. Lymphoid follicles were found in wild-type swine, and a few aggregated lymphocytes and immature lymphoid follicles were observed in knockout swine. Fluorescence in the lymph nodes indicated metastasis of tumour cells to the lymph nodes. Tumour cells replaced lymph node architectures, showed high-grade nuclear atypia and formed irregular tumour nests. Our model may be useful for the preclinical validation of diagnostic methods and minimally invasive treatment of metastatic cancer.

Surgical site infections continue to be a common complication affecting surgical prognosis. Reactive oxygen species (ROS) are generated by ultrasound-irradiated titanium dioxide (TiO2) (UIT). Although excessive ROS production can cause cell damage, ROS at physiological levels mediate beneficial cellular responses, including angiogenesis. This study investigated whether UIT can promote healing of Escherichia coli-infected wounds. We used TiO2 and ultrasound irradiation using an ultrasonography machine at a frequency of 1.0 MHz and intensity of 0.4 W cm−2. These levels are not bactericidal in vitro; therefore, we could study the effect of UIT on E. coli survival without interference of ultrasound effects. The number of cluster of differentiation 31-positive blood vessels, which are indicative of angiogenesis, was decreased by bacterial infection, and increased at the wound edges in the UIT-treated infected wounds, suggesting upregulation of neovascularization by UIT. Although UIT treatment did not decrease E. coli survival in vivo, it promoted healing of the infected wounds as evidenced by a significant decrease in the wound area in the UIT-treated mice. Our findings demonstrate that UIT promotes wound healing in surgical site infections and suggest beneficial use of the UIT-based approach as a novel therapeutic method to treat infected surgical wounds. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2344–2351, 2017.

Magnetic immunostaining is a technique used to accelerate the antigen-antibody immunoreaction by increasing the local density of antibody on the surface of a tissue specimen using a magnetic field. The high density of antibody is achieved by applying a magnetic force to antibody-labeled ferrite beads toward the specimen. A technical challenge of using conventional magnets for this technique has been the inhomogeneous accumulation of magnetic beads on the specimen in accordance with the distribution of the magnetic field. Thus, in this study, a dome-shaped magnet that generated a strong and uniform magnetic force distribution was proposed and demonstrated. Numerical analysis was used to optimize the shape of the magnet. Analysis of the motion of magnetic beads showed that the accumulation of beads on the sample was complete within one minute and that the resulting homogeneity was sufficient for rapid and accurate immunostaining. Finally, experiments showed that the homogeneity of the bead distribution was improved by the use of a prototype dome-shaped magnet compared to conventional cylindrical magnets.Magnetic immunostaining is a technique used to accelerate the antigen-antibody immunoreaction by increasing the local density of antibody on the surface of a tissue specimen using a magnetic field. The high density of antibody is achieved by applying a magnetic force to antibody-labeled ferrite beads toward the specimen. A technical challenge of using conventional magnets for this technique has been the inhomogeneous accumulation of magnetic beads on the specimen in accordance with the distribution of the magnetic field. Thus, in this study, a dome-shaped magnet that generated a strong and uniform magnetic force distribution was proposed and demonstrated. Numerical analysis was used to optimize the shape of the magnet. Analysis of the motion of magnetic beads showed that the accumulation of beads on the sample was complete within one minute and that the resulting homogeneity was sufficient for rapid and accurate immunostaining. Finally, experiments showed that the homogeneity of the bead distribution was ...

Magnetic immunostaining provides a novel technique for rapid intraoperative diagnosis of cancer metastasis and its characteristics in lymph nodes. Previous studies have shown the effectiveness of this technique using the manual staining process. However, to make this technique feasible for use in hospitals, establishing an automatic process is important. Thus, the objective of this study is to realize a device for immunostaining using magnetic fields that could not only automatically stain the samples, but also wash the specimen and change experimental solutions as well. The specifications for our prototype device were determined based on the manually performed experiments. We successfully fabricated a prototype device and demonstrated automatic immunostaining using it. The device completed, within 10 minutes, processes of blocking the specimen, injection of the magnetic beads, antigen-antibody reaction, and washing out the beads. The reaction took 1 minute, which was significantly shorter than that of conventional immunostaining. The device is expected to provide improved reproducibility of staining because of its automated processes.

Bats of the genus Eptesicus have several non-retroviral RNA virus-derived sequences in their genomes, among which an endogenous bornavirus-like L element, named eEBLL-1, was suggested to encode functional proteins in the hosts. However, the function of eEBLL-1 remains unclear due to a lack of appropriate investigation tools, such as cultured cells expressing eEBLL-1. Here, we established a continuous cell line, named HAMOI-EnK cells, from kidney of Eptesicus nilssonii. HAMOI-EnK cells are robust and could be passaged for at least 10 months. eEBLL-1 in the genomes of HAMOI-EnK cells retains an intact open reading frame. Additionally, eEBLL-1 is transcribed in the sense-orientation in cells. To our knowledge, this is the first report to demonstrate that eEBLL-1 is transcribed in cultured cells.

BACKGROUND Accurate detection and monitoring of disease-related biomarkers is important in understanding pathophysiology. We devised a rapid immunoreaction system that uses submicrometer polymer-coated fluorescent ferrite (FF) beads containing both ferrites (magnetic iron oxide) and fluorescent europium complexes. METHODS FF beads were prepared by encapsulation of hydrophobic europium complexes into the polymer layers of affinity magnetic beads using organic solvent. A sandwich immunoassay using magnetic collection of antibody-coated FF beads to a specific place was performed. Brain natriuretic peptide and prostate-specific antigen were selected as target detection antigens to demonstrate the feasibility of this approach. An immunohistochemical staining using magnetic collection of antibody-coated FF beads onto carcinoma cell samples was also performed. RESULTS The sandwich immunoassays, taking advantage of the magnetic collection of antibody-coated FF beads, detected target antigens within 5 min of sample addition. Without magnetic collection, the sandwich immunoassay using antibody-coated FF beads required long times, similar to conventional immunoassays. Using the magnetic collection of antibody-coated FF beads, immunohistochemical staining enabled discrimination of carcinoma cells within 20 min. CONCLUSIONS This proof of principle system demonstrates that immunoreactions involving the magnetic collection of antibody-coated FF beads allow acceleration of the antigen–antibody reaction. The simple magnetic collection of antibody-coated FF beads to a specific space enables rapid detection of disease-related biomarkers and identification of carcinoma cells.

Summary Among multidisciplinary therapies developed for advanced esophageal cancer, neoadjuvant chemotherapy and chemoradiotherapy have been established as standard treatments. To deliver cautious follow up and intense treatment for high-risk patients, a simple and instructive biomarker for the postoperative recurrence needs to be identified. Fibrinogen, a common component of hemostasis, has been suggested to not only play an important role in cancer metastasis, but also correlate with tumor recurrence. We aim to clarify the validity of plasma fibrinogen as a marker for predicting the postoperative recurrence of esophageal squamous cell carcinoma patients who received neoadjuvant treatment. We reviewed 72 consecutive patients with esophageal squamous cell carcinoma who received neoadjuvant chemotherapy or chemoradiotherapy, followed by esophagectomy at the Keio University Hospital from 2001 to 2010. Of them, we retrospectively examined 68 patients who underwent plasma fibrinogen examination before and after neoadjuvant treatment and underwent transthoracic radical esophagectomy. We investigated patient characteristics, clinicopathological factors, neoadjuvant treatment effects, postoperative course, and plasma fibrinogen levels. We investigated pretreatment and preoperative (postneoadjuvant treatment) plasma fibrinogen levels, as well as changes in fibrinogen levels before and after neoadjuvant treatment. Patients with preoperative hyperfibrinogenemia (>350 mg/dL) and patients with increased plasma fibrinogen levels during neoadjuvant treatment showed significantly shorter postoperative disease-free survival (DFS) (P = 0.002 and P = 0.037, respectively). Moreover, we classified these patients into three classes on the basis of their preoperative fibrinogen levels and changes in fibrinogen levels during neoadjuvant treatment. Patients who had both high preoperative plasma fibrinogen and increased fibrinogen levels showed significantly shorter DFS than others. In contrast, patients who had normal preoperative plasma fibrinogen and decreased fibrinogen levels showed significantly longer DFS. Based on this fibrinogen classification, we could differentiate between significantly favorable and poor prognosis patients group. Overall, this classification (hazard ratio = 1.812, P = 0.013) and the response to neoadjuvant treatment (hazard ratio = 0.350, P = 0.007) were found to be significant determining factors for postoperative DFS. With the validity of preoperative plasma fibrinogen levels and changes in fibrinogen levels during neoadjuvant treatment, the plasma fibrinogen level was found to be a possible biomarker for postoperative recurrence in advanced esophageal cancer patients who received neoadjuvant treatment. Moreover, plasma fibrinogen classification could be a simple and valuable predictive marker for postoperative follow up.