Pathogenic role of NAMPT in the perivascular regions after ischemic stroke in mice with type 2 diabetes mellitus.

Ischemic stroke in patients with abnormal glucose tolerance results in poor outcomes. Nicotinamide phosphoribosyltransferase (NAMPT), an adipocytokine, exerts neuroprotective effects. However, the pathophysiological role of NAMPT after ischemic stroke with diabetes and the relationship of NAMPT with cerebrovascular lesions are unclear. The purpose of this study was to clarify the pathophysiological role of NAMPT in cerebral ischemia with diabetes, using db/db mice as a type 2 diabetes animal model. The number of degenerating neurons increased after middle cerebral artery occlusion and reperfusion (MCAO/R) in db/db mice compared with the degenerating neurons in db/+ mice. Extracellular NAMPT (eNAMPT) levels, especially monomeric eNAMPT, increased significantly in db/db MCAO/R mice but not db/+ mice in isolated brain microvessels. The increased eNAMPT levels were associated with increased expression of inflammatory cytokine mRNA. Immunohistochemical analysis demonstrated that NAMPT colocalized with GFAP-positive cells after MCAO/R. In addition, both dimeric and monomeric eNAMPT levels increased in the conditioned medium of primary cortical astrocytes under high glucose conditions subsequent oxygen/glucose deprivation. Our findings are the first to demonstrate the ability of increased monomeric eNAMPT to induce inflammatory responses in brain microvessels, which may be located near astrocyte foot processes.

Risk Factors of Proteinuria in Patients with Hepatocellular Carcinoma Receiving Lenvatinib.

Proteinuria is one of the most frequently reported adverse events leading to the discontinuation of lenvatinib treatment in patients with advanced hepatocellular carcinoma (HCC). However, there are no reports regarding the risk factors of proteinuria in patients with HCC or patients receiving lenvatinib. We retrospectively reviewed the medical records of patients with HCC receiving lenvatinib at the Kobe City Medical Center General Hospital between April 2018 and December 2020. The severity of proteinuria was graded based on the Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. A multivariate Cox proportional hazards model was employed to identify the risk factors of developing grade ≥2 proteinuria. Among the 37 patients included, 3 patients had grade-1 proteinuria at baseline and 10 patients had estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m2 at baseline. Grades 1, 2, and 3 proteinuria were observed in 15 (40.5%), 10 (27.0%), and 2 (5.4%) patients, respectively, during lenvatinib treatment. The median value of eGFR was significantly lower in patients who developed grade ≥2 proteinuria than those with grade ≤1 proteinuria (59.6 vs. 78.1 mL/min/1.73 m2, p = 0.045). Multivariate analysis revealed that pre-existing proteinuria at baseline (hazard ratio (HR), 9.72; 95% confidence interval (CI), 1.29-52.21; p = 0.030), and eGFR <60 mL/min/1.73 m2 at baseline (HR, 4.49; 95% CI, 1.32-16.07; p = 0.017) were significantly associated with developing grade ≥2 proteinuria. These patients should be monitored carefully, and our preliminary data should be confirmed by further studies.

Effects of antioxidant capacity on micronucleus induction by cigarette smoke in mammalian cells.

We have compared micronucleus (MN) induction by cigarette smoke in the L5178Y, TK6, and CHL/IU cell lines. The test sample was total particulate matter of 3R4F reference cigarette smoke, suspended in DMSO. After 3-h treatment, with or without a rat liver S9 metabolic activation system, followed by 24-h recovery, dose-dependent MN increases were seen in all cell lines. However, CHL/IU and TK6 cells were more resistant than L5178Y cells (comparison by Benchmark Doses with PROAST software). 3R4F smoke generates reactive oxygen species (ROS). Therefore, we explored the relationship between the sensitivities to 3R4F smoke and the antioxidant capacities of the cell lines. While the total antioxidant capacities were not significantly different among the cell lines, cellular glutathione (GSH) was higher in CHL/IU cells than in L5178Y cells. Pretreatment of CHL/IU cells with a GSH precursor, N-acetylcysteine (NAC), reduced the genotoxicity/cytotoxicity of 3R4F, whereas an inhibitor of GSH biosynthesis, buthionine sulfoximine (BSO), enhanced it. The effects of NAC and BSO were also seen after treatment with allyl isothiocyanate, a ROS-generating chemical, but not with mitomycin C, a ROS-independent genotoxicant. Pretreatment with NAC increased cellular thiol levels. From the present results, the genotoxicity and cytotoxicity of cigarette smoke differs among these cell lines in a manner that may be related to their antioxidant thiol levels.

Relationship between tongue pressure and dysphagia diet in patients with acute stroke.

A dysphagia diet is important for patients with stroke to help manage their nutritional state and prevent aspiration pneumonia. Tongue pressure measurement is a simple, non-invasive, and objective method for diagnosing dysphagia. We hypothesized that tongue pressure may be useful in making a choice of diet for patients with acute stroke. Using balloon-type equipment, tongue pressure was measured in 80 patients with acute stroke. On admission, a multidisciplinary swallowing team including doctors, nurses, speech therapists, and management dietitians evaluated and decided on the possibility of oral intake and diet form; the tongue pressure was unknown to the team. Diet form was defined and classified as dysphagia diet Codes 0 to 4 and normal form (Code 5 in this study) according to the 2013 Japanese Dysphagia Diet Criteria. In multivariate analysis, only tongue pressure was significantly associated with the dysphagia diet form (p<0.001). Receiver operating characteristic analyses revealed that the optimal cutoff tongue pressure for predicting diet Codes 1, 2, 3, 4, and 5 was 3.6 (p<0.001, area under the curve [AUC] = 0.997), 9.6 (p<0.001, AUC = 0.973), 12.8 (p<0.001, AUC = 0.963), 16.5 (p<0.001, AUC = 0.979), and 17.3 kPa (p<0.001, AUC = 0.982), respectively. Tongue pressure is one of the sensitive indicators for choosing dysphagia diet forms in patients with acute stroke. A combination of simple modalities will increase the accuracy of the swallowing assessment and choice of the diet form.

Weight of evidence approach using a TK gene mutation assay with human TK6 cells for follow-up of positive results in Ames tests: a collaborative study by MMS/JEMS.

BACKGROUND: Conflicting results between bacterial mutagenicity tests (the Ames test) and mammalian carcinogenicity tests might be due to species differences in metabolism, genome structure, and DNA repair systems. Mutagenicity assays using human cells are thought to be an advantage as follow-up studies for positive results in Ames tests. In this collaborative study, a thymidine kinase gene mutation study (TK6 assay) using human lymphoblastoid TK6 cells, established in OECD TG490, was used to examine 10 chemicals that have conflicting results in mutagenicity studies (a positive Ames test and a negative result in rodent carcinogenicity studies). RESULTS: Two of 10 test substances were negative in the overall judgment (20% effective as a follow-up test). Three of these eight positive substances were negative after the short-term treatment and positive after the 24 h treatment, despite identical treatment conditions without S9. A toxicoproteomic analysis of TK6 cells treated with 4-nitroanthranilic acid was thus used to aid the interpretation of the test results. This analysis using differentially expressed proteins after the 24 h treatment indicated that in vitro specific oxidative stress is involved in false positive response in the TK6 assay. CONCLUSIONS: The usefulness of the TK6 assay, by current methods that have not been combined with new technologies such as proteomics, was found to be limited as a follow-up test, although it still may help to reduce some false positive results (20%) in Ames tests. Thus, the combination analysis with toxicoproteomics may be useful for interpreting false positive results raised by 24 h specific reactions in the assay, resulting in the more reduction (> 20%) of false positives in Ames test.

Alternative Oxidase Capacity of Mitochondria in Microsporophylls May Function in Cycad Thermogenesis.

Cone thermogenesis is a widespread phenomenon in cycads and may function to promote volatile emissions that affect pollinator behavior. Given their large population size and intense and durable heat-producing effects, cycads are important organisms for comprehensive studies of plant thermogenesis. However, knowledge of mitochondrial morphology and function in cone thermogenesis is limited. Therefore, we investigated these mitochondrial properties in the thermogenic cycad species Cycas revoluta Male cones generated heat even in cool weather conditions. Female cones produced heat, but to a lesser extent than male cones. Ultrastructural analyses of the two major tissues of male cones, microsporophylls and microsporangia, revealed the existence of a population of mitochondria with a distinct morphology in the microsporophylls. In these cells, we observed large mitochondria (cross-sectional area of 2 µm2 or more) with a uniform matrix density that occupied >10% of the total mitochondrial volume. Despite the size difference, many nonlarge mitochondria (cross-sectional area <2 µm2) also exhibited a shape and a matrix density similar to those of large mitochondria. Alternative oxidase (AOX) capacity and expression levels in microsporophylls were much higher than those in microsporangia. The AOX genes expressed in male cones revealed two different AOX complementary DNA sequences: CrAOX1 and CrAOX2 The expression level of CrAOX1 mRNA in the microsporophylls was 100 times greater than that of CrAOX2 mRNA. Collectively, these results suggest that distinctive mitochondrial morphology and CrAOX1-mediated respiration in microsporophylls might play a role in cycad cone thermogenesis.

Evaluation of pharmaceutical intervention in direct-acting antiviral agents for hepatitis C virus infected patients in an ambulatory setting: a retrospective analysis.

BACKGROUND: Direct-acting antivirals (DAAs) are known to improve tolerability and have higher efficacy and shorter treatment durations compared with conventional interferon (IFN)-based treatments for hepatitis C virus (HCV) infection. Management of drug interactions and maintenance of patient adherence are important to achieve adequate therapeutic effects, sustained virological response (SVR). In order to maximize the benefits of oral DAA therapy, we established an ambulatory care pharmacy practice, a model of integrated collaboration between physicians and pharmacists, for patients receiving IFN-free DAA therapy. In this study, we evaluated pharmaceutical intervention for patients visiting the ambulatory care pharmacy practice. METHODS: HCV-infected outpatients who visited our ambulatory care pharmacy practice between September 2014 and May 2017 were eligible for inclusion in the study. When IFN-free DAAs were first prescribed, the physicians recommended all patients to visit the ambulatory care pharmacy practice after their clinical examination. Subsequently, at the second visit or later, the patients visited the pharmacy service before the physician's examination. The primary endpoint was SVR, defined as HCV RNA below the lower limit of quantification after the completion of treatment. We also evaluated the adherence rate to DAAs, suggestions to the physicians by the pharmacists, and questions from the patients. All data were obtained retrospectively using an electronic medical record system. RESULTS: Among the 401 study subjects, 386 patients completed the IFN-free DAA therapy. A total of 365 patients have reached 12 or 24 weeks after completing the treatment. The overall SVR rate was 98.1% (358/365). The proportion of patients with adherence ≥90% was 99.3% (398/401). Two-hundred and sixty-seven (84%) among 318 suggestions of prescription made by the pharmacists mainly to manage the adverse events were accepted by the physicians. The pharmacists received and answered 1072 questions on DAA therapy from the patients. CONCLUSIONS: This study indicates that the pharmaceutical intervention may contribute to enhanced adherence to DAAs and higher SVR rates in comparison with previous reports. This study also demonstrates that collaboration between physicians and pharmacists in an ambulatory setting provides favorable outcomes for patients receiving IFN-free DAAs.

Successful use of regional anesthesia in non-intubated video-assisted thoracic surgery in patients with cardiopulmonary failure: two case reports.

BACKGROUND: One-lung ventilation under general anesthesia is necessary for thoracic surgery, but this procedure is often difficult in surgery for patients with cardiopulmonary failure. Non-intubated video-assisted thoracic surgery (VATS) is performed under local anesthesia for patients with respiratory failure, but has not been performed for patients with circulatory failure. Here, we report management of two patients with cardiopulmonary failure who underwent non-intubated VATS with paravertebral block and infiltration anesthesia. CASE PRESENTATION: Case 1 was a 79-year-old male with dyspnea at rest due to left large pleural effusion and cardiac dysfunction who underwent thoracoscopic pleural biopsy with paravertebral block under spontaneous breathing. The patient was also receiving dialysis. Case 2 was a 53-year-old male who developed empyema due to large pleural effusion, resulting in a poor general condition and cardiac dysfunction, and underwent video-assisted empyema curettage only with infiltration anesthesia under spontaneous breathing. In both patients, intraoperative respiration and circulation remained stable with values similar to those present preoperatively, and there were no problems after surgery. CONCLUSIONS: We safely anesthetized two patients with difficulty to general anesthesia by ensuring sufficient regional anesthesia during VATS under spontaneous breathing. These cases suggest that regional anesthesia for non-intubated VATS can contribute to maintain intra- and postoperative respiration and circulation in patients with cardiopulmonary failure.

Imbalanced expression of polycistronic miRNA in acute myeloid leukemia.

miR-1 and miR-133 are clustered on the same chromosomal loci and are transcribed together as a single transcript that is positively regulated by ecotropic virus integration site-1 (EVI1). Previously, we described how miR-133 has anti-tumorigenic potential through repression of EVI1 expression. It has also been reported that miR-1 is oncogenic in the case of acute myeloid leukemia (AML). Here, we show that expression of miR-1 and miR-133, which have distinct functions, is differentially regulated between AML cell lines. Interestingly, the expression of miR-1 and EVI1, which binds to the promoter of the miR-1/miR-133 cluster, is correlative. The expression levels of TDP-43, an RNA-binding protein that has been reported to increase the expression, but inhibits the activity, of miR-1, were not correlated with expression levels of miR-1 in AML cells. Taken together, our observations raise the possibility that the balance of polycistronic miRNAs is regulated post-transcriptionally in a hierarchical manner possibly involving EVI1, suggesting that the deregulation of this balance may play some role in AML cells with high EVI1 expression.

miR-133 regulates Evi1 expression in AML cells as a potential therapeutic target.

The Ecotropic viral integration site 1 (Evi1) is a zinc finger transcription factor, which is located on chromosome 3q26, over-expression in some acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Elevated Evi1 expression in AML is associated with unfavorable prognosis. Therefore, Evi1 is one of the strong candidate in molecular target therapy for the leukemia. MicroRNAs (miRNAs) are small non-coding RNAs, vital to many cell functions that negatively regulate gene expression by translation or inducing sequence-specific degradation of target mRNAs. As a novel biologics, miRNAs is a promising therapeutic target due to its low toxicity and low cost. We screened miRNAs which down-regulate Evi1. miR-133 was identified to directly bind to Evi1 to regulate it. miR-133 increases drug sensitivity specifically in Evi1 expressing leukemic cells, but not in Evi1-non-expressing cells The results suggest that miR-133 can be promising therapeutic target for the Evi1 dysregulated poor prognostic leukemia.

Novel functional small RNAs are selectively loaded onto mammalian Ago1.

Argonaute (Ago) proteins function in RNA silencing as components of the RNA-induced silencing complex (RISC). In lower organisms, the small interfering RNA and miRNA pathways diverge due in part to sorting mechanisms that direct distinct small RNA (sRNA) duplexes onto specific Ago-RISCs. However, such sorting mechanisms appear to be lost in mammals. miRNAs appear not to distinguish among Ago1-4. To determine the effect of viral infection on the sorting system, we compared the content of deep-sequenced RNA extracted from immunoprecipitation experiments with the Ago1 and Ago2 proteins using Epstein-Barr virus (EBV)-infected cells. Consistent with previous observations, sequence tags derived from miRNA loci in EBV and humans globally associate in approximately equivalent amounts with Ago1 and Ago2. Interestingly, additional sRNAs, which have not been registered as miRNAs, were associated with Ago1. Among them, some unique sequence tags derived from tandem loci in the human genome associate exclusively with Ago1 but not, or rarely, with Ago2. This is supported by the observation that the expression of the unique sRNAs in the cells is highly dependent on Ago1 proteins. When we knocked down Ago1, the expression of the Ago1-specific sRNAs decreased dramatically. Most importantly, the Ago1-specific sRNAs bound to mRNAs and regulated target genes and were dramatically upregulated, depending on the EBV life cycle. Therefore, even in mammals, the sorting mechanism in the Ago1-4 family is functional. Moreover, the existence of Ago1-specific sRNAs implies vital roles in some aspects of mammalian biology.

[Validation study on a rapid multi-residue method for determination of pesticide residues in vegetables and fruits by LC-MS/MS].

A validation study was conducted on a rapid multiresidue method for determination of pesticide residues in vegetables and fruits by LC-MS/MS. Pesticide residues in the vegetables or fruits were extracted with acetonitrile in a disposable tube using a homogenizer, followed by salting out with anhydrous magnesium sulfate and sodium chloride in the presence of citrate salts for buffering. The extract was purified with a double-layered cartridge column (graphite carbon black/primary secondary amine silica gel; GCB/PSA). For citrus fruits a purification step with a C18 column was added (this column was connected to the GCB/PSA column). After removal of the solvent, the extract was resolved in methanol/water and analyzed by means of LC-MS/MS. The method was validated according to the method validation guideline of the Ministry of Health, Labour and Welfare of Japan; recovery tests were performed on 8 kinds of vegetables and fruits [cabbage, cucumber, Japanese radish, onion, potato, spinach, Amanatsumikan (a citrus fruit) and apple] by fortification of 161 pesticide residues at the concentrations 0.01 and 0.05 µg/g (each concentration of pesticide residue was extracted from 2 samples on 5 separate days). The trueness of the method for 127 pesticides in all 8 commodities was 70-120% with satisfactory repeatability and within-run reproducibility. This method is concluded to be applicable for determination of pesticide residues in vegetables and fruits.

Antiangiogenic activity of flavonoids from Melia azedarach.

Three flavonoid glycosides, 1 (rutin: quercetin 3-O-rutinoside), 2 (kaempferol 3-O-robinobioside) and 3 (kaempferol 3-O-rutinoside) were isolated from the subcritical water extracts of Melia azedarach leaves. Strong antiangiogenic activity of these compounds was observed in the in vivo assay using the chorioallantoic membrane (CAM) from growing chick embryos.

Multiple angiogenetic factors are upregulated in POEMS syndrome.

Polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes (POEMS) syndrome is a multisystem disorder associated with plasma cell dyscrasia. Elevated serum levels of vascular endothelial growth factor (VEGF), which strongly promotes neovascularization and vasopermeability, are considered to be responsible for the characteristic symptoms such as angiomata, pleural effusion/ascites, edema, and organomegaly in the disorder. To study whether other angiogenetic factors are upregulated in POEMS syndrome, we measured serum levels of basic fibroblast growth factor and hepatocyte growth factor (HGF), as well as VEGF, in 17 patients with POEMS syndrome. All these factors were significantly upregulated in the POEMS syndrome patients. After the treatment with anti-VEGF antibody, the levels of HGF did not change, suggesting that elevation of HGF levels is not secondary to VEGF overproduction. These results suggest that different angiogenetic factors might contribute to the pathogenesis of POEMS syndrome, and this fact might contribute to the insufficient clinical effects obtained by suppression of VEGF alone.

Decreases in the serum VLDL-TG/non-VLDL-TG ratio from early stages of chronic hepatitis C: alterations in TG-rich lipoprotein levels.

BACKGROUND: The liver secretes very-low-density lipoproteins (VLDLs) and plays a key role in lipid metabolism. Plasma total triglyceride (TG) level variations have been studied in patients with hepatitis C virus (HCV)-related chronic hepatitis (CH-C). However, the results of these studies are variable. A homogenous assay protocol was recently proposed to directly measure the TG content in VLDL (VLDL-TG) and VLDL remnants. METHODOLOGY/PRINCIPAL FINDINGS: Using the assay protocol, we determined serum VLDL-TG levels in 69 fasting patients with biopsy-proven HCV-related chronic liver disease and 50 healthy subjects. Patients were classified into stages F0-F4 using the 5-point Desmet scale. Serum total TG levels in patients with non-cirrhotic (F1-F3) CH-C did not demonstrate significant differences compared with healthy subjects, but serum VLDL-TG levels did demonstrate significant differences. Mean serum VLDL-TG levels tended to decrease with disease progression from F1 to F4 (cirrhosis). Compared with healthy subjects, serum non-VLDL-TG levels significantly increased in patients with stages F2 and F3 CH-C; however, we observed no significant difference in patients with liver cirrhosis. Furthermore, the serum VLDL-TG/non-VLDL-TG ratio, when taken, demonstrated a significant decrease in patients with CH-C from the mildest stage F1 onward. CONCLUSIONS/SIGNIFICANCE: The decrease in serum VLDL-TG levels was attenuated by increase in non-VLDL-TG levels in patients with non-cirrhotic CH-C, resulting in comparable total TG levels. Results of previous studies though variable, were confirmed to have a logical basis. The decrease in the serum VLDL-TG/non-VLDL-TG ratio as early as stage F1 demonstrated TG metabolic alterations in early stages of CH-C for the first time. The involvement of TG metabolism in CH-C pathogenesis has been established in experimental animals, while conventional TG measurements are generally considered as poor indicators of CH-C progression in clinical practice. The serum VLDL-TG/non-VLDL-TG ratio, which focuses on TG metabolic alterations, may be an early indicator of CH-C.

Rad18 is required for long-term maintenance of spermatogenesis in mouse testes.

Maintaining the integrity of spermatogenic stem cells is essential to transfer genetic information to a descendant. However, knowledge of maintenance of genetic stability in stem cells is still limited. RAD18 is critical for postreplication repair through mono- and multi-ubiquitination of proliferating cell nuclear antigen (PCNA) to maintain genomic stability. Mammalian RAD18 is highly expressed in the spermatocytes and the nuclei of a few spermatogonia in adult mice. To elucidate the physiological function of RAD18, we analyzed a phenotype of Rad18-/- mice. The mice were born and appeared to grow normally. Although the mice were fertile, fertility and testis weight decreased with age. Histological examination revealed normal spermatogenesis in almost all seminiferous tubules in Rad18-/- testes at 2 months old, and abnormal sperm could not be detected in the epididymis. However, 25% of the tubules lost almost all germ cells at 12 months. The seminiferous tubules frequently retained only late differentiated phase germ cells, suggesting that the exhaustion of spermatogonial stem cells leads to the loss of all germ cells in the seminiferous tubules. Wild-type germ cells were successfully transplanted into and colonized in the seminiferous tubules of aged Rad18-/- mice, indicating that Sertoli cells have a normal supportive function even in aged testes. We conclude that RAD18 is intrinsically required for the long-term maintenance of spermatogenesis.