Far-Ultraviolet Light at 222 nm Affects Membrane Integrity in Monolayered DLD1 Colon Cancer Cells.

222 nm far-ultraviolet (F-UV) light has a bactericidal effect similar to deep-ultraviolet (D-UV) light of about a 260 nm wavelength. The cytotoxic effect of 222 nm F-UV has not been fully investigated. DLD-1 cells were cultured in a monolayer and irradiated with 222 nm F-UV or 254 nm D-UV. The cytotoxicity of the two different wavelengths of UV light was compared. Changes in cell morphology after F-UV irradiation were observed by time-lapse imaging. Differences in the staining images of DNA-binding agents Syto9 and propidium iodide (PI) and the amount of cyclobutane pyrimidine dimer (CPD) were examined after UV irradiation. F-UV was cytotoxic to the monolayer culture of DLD-1 cells in a radiant energy-dependent manner. When radiant energy was set to 30 mJ/cm2, F-UV and D-UV showed comparable cytotoxicity. DLD-1 cells began to expand immediately after 222 nm F-UV light irradiation, and many cells incorporated PI; in contrast, PI uptake was at a low level after D-UV irradiation. The amount of CPD, an indicator of DNA damage, was higher in cells irradiated with D-UV than in cells irradiated with F-UV. This study proved that D-UV induced apoptosis from DNA damage, whereas F-UV affected membrane integrity in monolayer cells.

Exploratory study of factors associated with probable respiratory sarcopenia in elderly subjects.

BACKGROUND: The diagnostic criteria for respiratory sarcopenia have been recently reported. However, no studies have clarified the characteristics of skeletal muscle impairment of the limbs in subjects with respiratory sarcopenia. This study aimed to explore the factors, including skeletal muscle, associated with probable respiratory sarcopenia in elderly subjects. METHODS: Subjects were classified into the probable respiratory sarcopenia group and nonrespiratory sarcopenia group. Probable respiratory sarcopenia was defined as the concurrent presence of respiratory muscle weakness (as less than the predicted value calculated from age, sex, and height) and low skeletal muscle mass (<7.0 kg/m2 in males and 5.7 kg/m2 in females). The following factors were measured: respiratory muscle strength, skeletal muscle mass index, muscle thickness and echo intensity of the rectus femoris, extracellular-to-intracellular water ratio, hand grip strength, 5 sit-to-stand, knee extension strength, bone mineral density, age, sex, body mass index, degree of frailty, presence or absence of medical history, presence or absence of habitual exercise, period of time since the start of exercise, and number of hours of exercise at a time. The association subjects with probable respiratory sarcopenia were analyzed using hierarchical logistic regression analysis. RESULTS: Twenty-six with probable respiratory sarcopenia and 54 with nonrespiratory sarcopenia were included. Hierarchical logistic regression analysis revealed that echo intensity was a significant predictor of probable respiratory sarcopenia. The odds ratio for echo intensity was 2.54 (95% confidence interval: 1.04-6.23). CONCLUSIONS: Our results suggest that a decrease in muscle quality in the lower extremity is associated with probable respiratory sarcopenia.

A case of asymptomatic infection of Ascaris suum identified by PCR-restriction fragment length polymorphism and DNA sequence analysis.

Human ascariasis is a soil-transmitted helminthiasis and remains a neglected tropical disease. Ascaris suum has the potential to cause cross-infections between humans and pigs. In this study, we present a rare case of a patient with asymptomatic infection by Ascaris suum. A 66-year-old male underwent colonoscopy, and a white linear worm body was found in the hepatic curvature. The worm was collected by aspiration and submitted to the laboratory for parasite identification. The patient had no symptoms related to parasitic infection. The worm was highly suspected to be of the genus Ascaris. Because of the difficulty of morphological classification, genetic analysis was performed. From PCR-restriction fragment length polymorphism results and sequence analysis of the internal transcribed spacer-1 region, it was determined to be A. suum. The experience with rapid differentiation of A. suum by performing genetic analysis will be useful for future examinations of parasitic infections.

Far-ultraviolet irradiation at 222 nm destroys and sterilizes the biofilms formed by periodontitis pathogens.

BACKGROUND: Periodontal disease is the leading cause of tooth loss, and an association between periodontal disease and non-oral systemic diseases has been shown. Formation of biofilm by periodontal pathogens such as Fusobacterium nucleatum, Porphyromonas gingivalis, and Streptococcus mutans and their resistance to antimicrobial agents are at the root of persistent and chronic bacterial infections. METHODS: The bactericidal effect of far-ultraviolet (F-UV) light irradiation at 222 nm on periodontal bacteria was assessed qualitatively and quantitatively. The effect of biofilm disruption by F-UV light on periodontal bacteria was examined by crystal violet staining, and the morphologic changes of the biofilm after F-UV irradiation were explored by confocal laser microscopy and scanning electron microscopy. We developed a thin fiber-type 222 nm F-UV irradiator and studied its safety and effect of reducing bacteria in rodent models. RESULTS: F-UV light at 222 nm had a bactericidal effect on F. nucleatum, P. gingivalis, and S. mutans. Irradiation with F-UV light reduced the biofilm formed by the bacteria and sterilized them from within. Confocal laser microscopy showed a clear reduction in biofilm thickness, and scanning electron microscopy confirmed disintegration of the biofilm architecture. F-UV irradiation was less damaging to DNA and less cytotoxic than deep-ultraviolet light, and it reduced bacterial counts on the tooth surface. CONCLUSION: F-UV irradiation has the potential to destroy biofilm and act as a bactericide against pathogenic bacteria in the biofilm.

SARS-CoV-2 infection status in corneal preservation solution and COVID-19 prevalence after corneal transplantation.

The potential risks associated with organs from COVID-19-infected donors were unclear. To determine the SARS-CoV-2 infection status of corneas transplanted during the COVID-19 pandemic, we performed a polymerase chain reaction (PCR) using the corneal preservation solution that was used for corneal transplantation. We also examined the postoperative health status of the recipients. This study included 144 transplants in 143 eyes. Ninety-nine eyes of imported corneas and 10 of the 14 corneas donated in the prefecture were PCR tested at our hospital, and all were SARS-CoV-2 negative. All corneal transplants were performed after confirming their SARS-CoV-2 negativity by a PCR using a corneal preservation solution at our hospital or a nasopharyngeal swab at a previous facility. Despite postoperative steroid administration, no patient developed COVID-19 infection until discharge. Hence, if the donor's nasopharyngeal swab test is SARS-CoV-2 negative, COVID-19 infection in the recipient due to corneal transplantation may be prevented. Since corneal transplant recipients are susceptible to infection due to prolonged steroid administration and are at high risk for severe diseases if infection occurs, SARS-CoV-2 detection testing using nasopharyngeal swabs in donors should be performed.

Varying impact of lenvatinib or sorafenib therapy on skeletal muscle loss in patients with hepatocellular carcinoma.

BACKGROUND: Lenvatinib and sorafenib are key therapeutic agents for hepatocellular carcinoma. However, there are no useful biomarkers for selecting molecular-targeted agents (MTAs). Skeletal muscle volume is associated with the clinical outcomes in these patients. We investigated the effects of lenvatinib and sorafenib on the skeletal muscles of patients with HCC. METHODS: We evaluated the impact of skeletal muscle changes over a 3-month period for each MTA (n = 117; lenvatinib/sorafenib, 45/72). The skeletal muscle mass index (SMI) was measured at the third lumbar vertebra. Furthermore, we evaluated the direct effect of each MTA on primary human skeletal muscle cells by estimating muscle protein synthesis using western blot analysis. RESULTS: The median change in SMI was -0.7% (p = 0.959) and -5.9% (p <0.001) for the lenvatinib and sorafenib groups, respectively. Sorafenib had a greater effect on skeletal muscle loss than lenvatinib (p < 0.001). Additionally, SMI significantly decreased in the sorafenib group regardless of initial skeletal muscle volume (p < 0.001), whereas no significant differences were observed in the lenvatinib group. Sorafenib therapy (odds ratio [OR], 2.98; p = 0.023) and non-muscle depletion (OR, 3.31; p = 0.009) were associated with a decreased SMI. In vitro analysis showed that sorafenib negatively affected muscle synthesis compared to lenvatinib. CONCLUSIONS: Sorafenib may have a more negative effect on skeletal muscle than lenvatinib.

Hydrolytic activity of yeast oligosaccharyltransferase is enhanced when misfolded proteins accumulate in the endoplasmic reticulum.

It is known that oligosaccharyltransferase (OST) has hydrolytic activity toward dolichol-linked oligosaccharides (DLO), which results in the formation of free N-glycans (FNGs), i.e. unconjugated oligosaccharides with structural features similar to N-glycans. The functional importance of this hydrolytic reaction, however, remains unknown. In this study, the hydrolytic activity of OST was characterized in yeast. It was shown that the hydrolytic activity of OST is enhanced in ubiquitin ligase mutants that are involved in endoplasmic reticulum-associated degradation. Interestingly, this enhanced hydrolysis activity is completely suppressed in asparagine-linked glycosylation (alg) mutants, bearing mutations related to the biosynthesis of DLO, indicating that the effect of ubiquitin ligase on OST-mediated hydrolysis is context-dependent. The enhanced hydrolysis activity in ubiquitin ligase mutants was also found to be canceled upon treatment of the cells with dithiothreitol, a reagent that potently induces protein unfolding in the endoplasmic reticulum (ER). Our results clearly suggest that the hydrolytic activity of OST is enhanced under conditions in which the formation of unfolded proteins is promoted in the ER in yeast. The possible role of FNGs on protein folding is discussed.

Influence of filtering on the effective concentration and sterility of a 2% cyclosporine ophthalmic solution: a quality improvement perspective.

BACKGROUND: Pharmaceutical companies do not sell formulations for all diseases; thus, healthcare workers have to treat some diseases by concocting in-hospital preparations. An example is the high-concentration 2% cyclosporine A (CyA) ophthalmic solution. Utilizing a filter in sterility operations is a general practice for concocting in-hospital preparations, as is the case for preparing a 2% CyA ophthalmic solution. However, whether filtering is appropriate concerning the active ingredient content and bacterial contamination according to the post-preparing quality control of a 2% CyA ophthalmic solution is yet to be verified. METHODS: We conducted particle size, preparation concentration, and bacterial contamination studies to clarify aforementioned questions. First, we measured the particle size of CyA through a laser diffraction particle size distribution. Next, we measured the concentration after preparation with or without a 0.45-µm filter operation using an electrochemiluminescence immunoassay. Finally, bacterial contamination tests were conducted using an automated blood culture system to prepare a 2% CyA ophthalmic solution without a 0.45 µm filtering. Regarding the pore size of the filter in this study, it was set to 0.45 µm with reference to the book (the 6th edition) with recipes for the preparation of in-hospital preparations edited by the Japanese Society of Hospital Pharmacists. RESULTS: CyA had various particle sizes; approximately 30% of the total particles exceeded 0.45 µm. The mean ± standard deviation of filtered and non-filtered CyA concentrations in ophthalmic solutions were 346.51 ± 170.76 and 499.74 ± 76.95ng/mL, respectively (p = 0.011). Regarding bacterial contamination tests, aerobes and anaerobes microorganisms were not detected in 14 days of culture. CONCLUSIONS: Due to the results of this study, the concentration of CyA may be reduced by using a 0.45-µm filter during the preparation of CyA ophthalmic solutions, and furthermore that the use of a 0.45-µm filter may not contribute to sterility when preparing CyA ophthalmic solutions.

Metabolic Analysis of DFO-Resistant Huh7 Cells and Identification of Targets for Combination Therapy.

Hepatocellular carcinoma (HCC) is one of the most refractory cancers with a high rate of recurrence. Iron is an essential trace element, and iron chelation has garnered attention as a novel therapeutic strategy for cancer. Since intracellular metabolism is significantly altered by inhibiting various proteins by iron chelation, we investigated combination anticancer therapy targeting metabolic changes that are forcibly modified by iron chelator administration. The deferoxamine (DFO)-resistant cell lines were established by gradually increasing the DFO concentration. Metabolomic analysis was conducted to evaluate the metabolic alterations induced by DFO administration, aiming to elucidate the resistance mechanism in DFO-resistant strains and identify potential novel therapeutic targets. Metabolom analysis of the DFO-resistant Huh7 cells revealed enhanced glycolysis and salvage cycle, alternations in glutamine metabolism, and accumulation of dipeptides. Huh7 cultured in the absence of glutamine showed enhanced sensitivity to DFO, and glutaminase inhibitor (CB839) showed a synergistic effect with DFO. Furthermore, the effect of DFO was enhanced by an autophagy inhibitor (chloroquine) in vitro. DFO-induced metabolic changes are specific targets for the development of efficient anticancer combinatorial therapies using DFO. These findings will be useful for the development of new cancer therapeutics in refractory liver cancer.

Determination of the Optimal Wavelength of the Hemolysis Index Measurement.

Many biochemical auto-analyzers have methods that measure the hemolysis index (HI) to quantitatively assess the degree of hemolysis. Past reports on HI are mostly in vitro studies. Therefore, we evaluated the optimal wavelength of HI measurement ex vivo using clinical samples. Four different wavelengths (410/451 nm: HI-1, 451/478 nm: HI-2, 545/596 nm: HI-3 and 571/596 nm: HI-4) were selected for HI measurement, and correlations were examined from the measurement results of 3890 clinical samples. Another set of 9446 clinical samples was used to examine the correlation of HI with lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and potassium (K). Strong correlations were found between HI-4 and HI-1 and between HI-4 and HI-3. HI-1 and HI-2 cannot correctly assess hemolysis for high bilirubin samples, and HI-3 cannot correctly assess hemolysis for high triglyceride samples. LDH, AST and K correlated positively with HI-4 in clinical samples. For every 1-unit increase in HI-4, LDH increased by 19.51 U/L, AST by 1.03 U/L and K by 0.061 mmol/L, comparable to reports of other studies. In clinical samples, HI-4 was less susceptible to bilirubin and chyle and reflected well the changes in LDH, AST and K caused by hemolysis. This suggested that the optimal wavelength for HI measurement is 571 nm.

Early Prediction of Response Focused on Tumor Markers in Atezolizumab plus Bevacizumab Therapy for Hepatocellular Carcinoma.

Despite the promising efficacy of atezolizumab plus bevacizumab (atezo/bev), some patients with unresectable hepatocellular carcinoma (HCC) experience disease progression. This retrospective study, which included 154 patients, aimed to evaluate predictors of treatment efficacy of atezo/bev for unresectable HCC. Factors associated with treatment response were examined, focusing on tumor markers. In the high-alpha-fetoprotein (AFP) group (baseline AFP ≥ 20 ng/mL), a decrease in AFP level > 30% was an independent predictor of objective response (odds ratio, 5.517; p = 0.0032). In the low-AFP group (baseline AFP < 20 ng/mL), baseline des-gamma-carboxy prothrombin (DCP) level < 40 mAU/mL was an independent predictor of objective response (odds ratio, 3.978; p = 0.0206). The independent predictors of early progressive disease were an increase in AFP level ≥ 30% at 3 weeks (odds ratio, 4.077; p = 0.0264) and the presence of extrahepatic spread (odds ratio, 3.682; p = 0.0337) in the high-AFP group and up-to-seven criteria, OUT (odds ratio, 15.756; p = 0.0257) in the low-AFP group. In atezo/bev therapy, focusing on early AFP changes, baseline DCP, and tumor burden of up-to-seven criteria are useful in predicting response to treatment.

Methylated SEPT9 assay-based liquid biopsy as a biomarker in molecular targeted agent-treated hepatocellular carcinoma.

BACKGROUND: The development of molecular targeted agents (MTAs) has changed the treatment strategy for hepatocellular carcinoma (HCC). However, currently, there are no established predictive biomarkers for the treatment efficacy of MTAs. Previously, we developed a novel liquid biopsy test for HCC screening using sensitive methylated DNA testing of septin 9 gene (SEPT9). Here, we hypothesized that SEPT9 could be used as a biomarker for MTA treatment efficacy. METHODS: We enrolled 157 patients receiving sorafenib or lenvatinib as a first-line therapy and allocated 85 and 72 patients to the training and validation cohorts, respectively. For the methylation assay, DNA was treated with methylation-sensitive restriction enzymes, followed by multiplex droplet digital PCR. Various clinical parameters were compared with clinical outcomes. RESULTS: The multivariate analysis revealed Eastern Cooperative Oncology Group performance status (≥ 1; p = 0.048), alpha-fetoprotein (AFP) (≥ 400 ng/mL; p < 0.001), and methylated-septin-9 (m-SEPT9) (≥ 205 copies/mL; p = 0.018) as significant predictors of poor overall survival (OS) in the training cohort. m-SEPT9 was identified as a predictor of poor OS in the validation cohort. We developed a predictive score, called the MTA score, consisting of these three significant OS parameters (two points were added for AFP and one point for each of the other predictors). Patients with MTA scores ≥ 2 showed a significantly poor prognosis compared to those with MTA scores ≤ 1 in both the training and validation cohorts. CONCLUSIONS: m-SEPT9 could be a potential predictive biomarker for survival in patients with HCC treated with MTAs.

Impact of skeletal muscle volume on patients with BCLC stage-B hepatocellular carcinoma undergoing sorafenib therapy.

AIM: Skeletal muscle volume has been reported to be an important factor that determines overall survival (OS) and post-progression survival (PPS) in patients with hepatocellular carcinoma (HCC). However, the impact of skeletal muscle volume on HCC with Barcelona Clinic Liver Cancer (BCLC) stage B (BCLC-B) remains unclear. We conducted sub-analyses of a previous study on BCLC-B and compared our findings with data on HCC with BCLC stage C (BCLC-C). METHODS: We retrospectively enrolled 356 patients with HCC (BCLC-B, n = 78; and BCLC-C, n = 278) undergoing sorafenib therapy. Prognostic factors were analyzed using various parameters, including skeletal muscle volume. Muscle volume (MV) depletion was designated as less than the median value of the skeletal muscle index for each gender (cutoff value: 45.0 cm2 /m2 for male and 38.0 cm2 /m2 for female participants). RESULTS: Both OS and PPS showed no significant differences in patients with non-MV depletion and those with MV depletion in the BCLC-B group (Median OS [MST] 19.3 vs. 13.5 months [p = 0.348]; median PPS 9.7 vs. 10.8 months [p = 0.578]). In the BCLC-C group, patients with non-MV depletion had a significantly longer OS and PPS compared to patients with MV depletion (MST 12.4 vs. 9.0 months [p = 0.001] and median PPS 7.9 vs. 5.4 months [p = 0.002]). Multivariate analysis revealed that MV depletion was an independent prognostic factor of OS and PPS in the BCLC-C group but not in the BCLC-B group. CONCLUSIONS: Skeletal muscle volume showed little impact on the clinical outcomes of patients with BCLC-B undergoing sorafenib therapy.

Double cancer of primary hepatic angiosarcoma and hepatocellular carcinoma treated with atezolizumab plus bevacizumab.

AIM: Primary hepatic angiosarcoma (PHA) is extremely rare, and its imaging findings are similar to those of other liver tumors including hepatocellular carcinoma (HCC). Here, we report a case of hepatitis C virus (HCV)-related HCC followed by PHA that showed remarkable clinical response to atezolizumab plus bevacizumab (Atezo/Bev) therapy. CASE PRESENTATION: A 78-year-old man with recurrent HCC had a liver tumor with lymphadenopathy. Although considered as HCC recurrence, microscopic examination of the resected liver and lymph node showed PHA. Three months later, a solitary lung nodule was newly detected and subsequently resected. The pathological diagnosis was poorly differentiated HCC. Therefore, the patient was finally diagnosed with double cancer of PHA and HCC. Thereafter, he developed a new liver tumor with lymphadenopathy and received Atezo/Bev therapy. Liver tumor biopsy was carried out before the treatment. The pathological diagnosis was angiosarcoma. The patient showed a partial response after two courses of Atezo/Bev therapy. CONCLUSION: To our best knowledge, this report is the first case to present HCV-related HCC followed by PHA and to show that Atezo/Bev therapy is beneficial for PHA.

Reduced upper and lower limb muscle strengths without reduced skeletal muscle in elderly patients with heart failure.

Objective: This study aimed to characterize the muscle strength and skeletal muscle mass of patients with heart failure by investigating hand-grip strength, five times sit-to-stand (5STS) results, and skeletal muscle mass index (SMI). Materials and Methods: Muscle strength was assessed based on hand-grip strength and 5STS, while skeletal muscle mass was assessed using a bioelectrical impedance analyzer. Hierarchical logistic regression analysis was performed to explore the association between patients with heart failure and healthy elderly individuals. Results: Hierarchical logistic regression analysis was performed to examine the muscle strength and skeletal muscle mass characteristics in patients with heart failure. Hand-grip strength and 5STS responses but not SMI outcomes differed significantly between the two groups. The results of the hierarchical logistic regression analysis revealed that the hand-grip strength and 5STS were significant predictors of heart failure. The odds ratios for hand-grip strength and 5STS were 1.44 and 0.53, respectively. Conclusion: Our results suggested that upper and lower limb muscle strengths (handgrip strength and 5STS) in elderly patients with heart failure worsened significantly without a decrease in skeletal muscle mass.

CA19-9 in Combination with Methylated HOXA1 and SST Is Useful to Diagnose Stage I Pancreatic Cancer.

INTRODUCTION: We previously developed a novel methylation assay, the combined restriction digital PCR (CORD) assay, consisting of treatment of DNA with methylation-sensitive restriction enzymes and droplet digital PCR. METHODS: In this study, we assessed the diagnostic performance of serum methylated Homeobox A1 (mHOXA1) and methylated somatostatin (mSST) using the CORD assay in combination with CA19-9 for pancreatic cancer using serum samples from 82 healthy individuals, 13 patients with benign pancreatic disease, 3 patients with branched-duct intraductal papillary mucinous neoplasm, and 91 patients with pancreatic cancer. RESULTS: For the single marker tests, sensitivity for all stages of pancreatic cancer, stage I cancer, and specificity were, respectively, 71.4%, 50.0%, and 94.9% for CA19-9; 51.6%, 68.8%, and 90.8% for mHOXA1; and 50.1%, 68.8%, and 94.9% for mSST. Those for the combined marker tests were, respectively, 86.8%, 81.3%, and 85.7% for combined mHOXA1 and CA19-9; 86.8%, 87.5%, and 89.8% for combined mSST and CA19-9; and 89.0%, 87.5%, and 85.7% for all three markers combined. CONCLUSION: The combination of mHOXA1 and mSST with CA19-9 appears to be useful to detect pancreatic cancer even at an early stage.

MC180295 Inhibited Epstein-Barr Virus-Associated Gastric Carcinoma Cell Growth by Suppressing DNA Repair and the Cell Cycle.

DNA methylation of both viral and host DNA is one of the major mechanisms involved in the development of Epstein-Barr virus-associated gastric carcinoma (EBVaGC); thus, epigenetic treatment using demethylating agents would seem to be promising. We have verified the effect of MC180295, which was discovered by screening for demethylating agents. MC180295 inhibited cell growth of the EBVaGC cell lines YCCEL1 and SNU719 in a dose-dependent manner. In a cell cycle analysis, growth arrest and apoptosis were observed in both YCCEL1 and SNU719 cells treated with MC180295. MKN28 cells infected with EBV were sensitive to MC180295 and showed more significant inhibition of cell growth compared to controls without EBV infection. Serial analysis of gene expression analysis showed the expression of genes belonging to the role of BRCA1 in DNA damage response and cell cycle control chromosomal replication to be significantly reduced after MC180295 treatment. We confirmed with quantitative PCR that the expression levels of BRCA2, FANCM, RAD51, TOP2A, and CDC45 were significantly decreased by MC180295. LMP1 and BZLF1 are EBV genes with expression that is epigenetically regulated, and MC180295 could up-regulate their expression. In conclusion, MC180295 inhibited the growth of EBVaGC cells by suppressing DNA repair and the cell cycle.