Supplementary Material for: Hochuekkito, a Japanese Herbal Medicine, Restores Metabolic Homeostasis between Mitochondrial and Glycolytic Pathways Impaired by Influenza A Virus Infection

<p><b><i>Background:</i></b> Hochuekkito (HKT), a traditional Japanese herbal medicine (Kampo), has been used to treat symptoms of several diseases. In a recent clinical study, HKT was shown to be protective against the influenza virus infection. However, the underlying mechanism of the prophylactic effect is not clear. Mitochondrial and glycolytic pathways play important roles in cellular energy metabolism to maintain biological functions. These metabolic pathways are affected by the influenza virus infection. In this study, we examined the relationship between the preventive effects of HKT against the influenza virus infection and cellular energy metabolism in mitochondria and glycolysis using Madin-Darby canine kidney cells and influenza A/PR/8/34 (H1N1) virus (IAV). <b><i>Methods:</i></b> Mitochondrial and glycolytic metabolic pathways were evaluated on the basis of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), respectively, using the XF24 Extracellular Analyzer. <b><i>Results:</i></b> The OCR/ECAR ratio in IAV-infected cells was lower than that in control cells. Cells that were treated with HKT before IAV infection showed a metabolic pattern similar to that in the control cells (increase in both OCR and ECAR). <b><i>Conclusions:</i></b> Our results suggest that HKT not only activates both mitochondrial and glycolytic energy metabolism in IAV-infected cells but also helps maintain metabolic homeostasis similar to that in noninfected cells.</p

Supplementary Material for: The Preventive Effect of the Traditional Japanese Herbal Medicine, Hochuekkito, against Influenza A Virus via Autophagy in vitro

<p><b><i>Background:</i></b> Hochuekkito (HKT), a traditional Japanese herbal medicine, enhances the immunity of elderly or weak individuals. It is also known to have preventive effects against influenza clinically. However, the detailed mechanisms of the preventive effects have not been clarified. We examined the relationship between the preventive effects of HKT and autophagy, a known stress response and quality control mechanism, using Madin-Darby canine kidney cells and influenza A/PR/8/34 (H1N1) virus. <b><i>Methods:</i></b> The effect of HKT on autophagy in influenza A virus (IAV)-infected cells was assessed by Western blotting and fluorescence microscopy using an RFP-GFP-LC3B sensor kit. <b><i>Results:</i></b> In Western blotting, treatment with HKT before IAV infection (pre-HKT) tended to induce autophagy in IAV-infected cells at an early stage of infection, eventually suppressing IAV-induced autophagy. Moreover, several autolysosomes, indicative of normal autophagosome-lysosome fusion, were observed in Pre-HKT cells transduced with RFP-GFP-LC3B but not in untreated IAV-infected cells. <b><i>Conclusions:</i></b> These findings indicated that IAV-mediated inhibition of the fusion of autophagosomes with lysosomes was prevented by HKT treatment before infection. According to these results, we propose that this phenomenon is one of the preventive effects of HKT against IAV.</p

Supplementary Material for: Usefulness of the Preoperative Administration of Tegafur Suppositories as Alternative Adjuvant Chemotherapy for Patients with Resectable Stage II or III Colorectal Cancer: A KODK4 Multicenter Randomized Control Trial

<b><i>Objective:</i></b> The aim of this study was to evaluate the feasibility and conferred protection against recurrence of preoperatively administered tegafur suppositories following the intravenous and oral administration of fluoropyrimidine in a multicenter randomized control trial. <b><i>Methods:</i></b> Patients with clinical T3/4 colorectal cancer were randomly assigned to receive the preoperative administration of tegafur suppositories (group A) or no preoperative treatment (group B). The primary end points were disease-free survival (DFS) and overall survival (OS). <b><i>Results:</i></b> The mean follow-up periods were 80.9 ± 31.0 months in group A and 64.5 ± 28.8 months in group B. The 5-year DFS rates were 89.3% in group A and 70.3% in group B (p = 0.045), whereas the 5-year OS rates were 91.4% in group A and 73.2% in group B (p = 0.051). Furthermore, a significant difference in the cumulative distant metastatic rate was observed (group A, 7.4% vs. group B, 23.4%; p = 0.03). However, no significant difference in the cumulative local recurrence rate was seen (group A, 4.6% vs. group B, 8.2%; p = 0.68). <b><i>Conclusion: </i></b>Despite a relatively small sample size, preoperative tegafur suppositories might protect recurrences and improve survival rates, mainly by preventing distant metastasis. These findings suggest the utility of tegafur suppositories as an alternative neoadjuvant treatment in modern chemotherapy for colorectal cancer

Supplementary Material for: Low Birthweight and Premature Birth Are Risk Factors for Podocytopenia and Focal Segmental Glomerulosclerosis

<b><i>Background:</i></b> Recent reports suggest that low birthweight (LBW) is a risk factor for kidney diseases, including focal segmental glomerulosclerosis (FSGS), although the underlying pathological mechanism remains unknown. Podocyte loss triggers glomerulosclerosis; however, whether FSGS in LBW children is associated with podocytopenia is unclear. <b><i>Methods:</i></b> We reviewed the birthweights and gestational age of all patients who underwent renal biopsies from 1995 to 2011 at our Institute. Sixteen patients had FSGS, of which 6 (37.5%) had LBW; this LBW rate was significantly higher than the overall LBW rate in Japan (9.7%). The incidence of LBW was also high in patients with minimal change nephrotic syndrome (MCNS; 12.5%). The glomerular cell numbers in biopsy sections were calculated using computer image analysis and compared with FSGS of normal birthweight (NBW-FSGS). Biopsy specimens from age-matched patients with MCNS were also compared. Wilms' tumor-1 (WT1) immunohistochemistry was performed to enumerate the podocytes. <b><i>Results:</i></b> All patients in the LBW-FSGS group were also preterm, with an average gestational age of 25.8 weeks. The number of podocytes per glomerulus in the LBW-FSGS patients was 34 and 24% lower as compared to that in the MCNS patients (p < 0.01) and the NBW-FSGS patients (p < 0.05), respectively. Similar results were observed for the WT1-positive glomerular cell number. <b><i>Conclusion:</i></b> LBW and premature birth were associated with FSGS development. The possibility that LBW and premature birth may be predisposing factors for severe podocytopenia in children with FSGS warrants further investigation