Isolation, structural determination, and antiviral activities of a novel alanine-conjugated polyketide from Talaromyces sp.

Antiviral agents are highly sought after. In this study, a novel alkylated decalin-type polyketide, alaspelunin, was isolated from the culture broth of the fungus Talaromyces speluncarum FMR 16671, and its structure was determined using spectroscopic analyses (1D/2D NMR and MS). The compound was condensed with alanine, and its absolute configuration was determined using Marfey's method. Furthermore, the antiviral activity of alaspelunin against various viruses was evaluated, and it was found to be effective against both severe acute respiratory syndrome coronavirus 2 and pseudorabies (Aujeszky's disease) virus, a pathogen affecting pigs. Our results suggest that this compound is a potential broad-spectrum antiviral agent.

Reduced Relapse-Free Survival in Colorectal Cancer Patients with Elevated Soluble CD40 Ligand Levels Improved by Vitamin D Supplementation.

Although elevated serum levels of soluble CD40 ligand (sCD40L) were reported in patients with cancer, the importance of high sCD40L levels in clinical oncology remains unknown. We conducted a post hoc analysis of the AMATERASU randomized clinical trial of vitamin D3 supplementation (2000 IU/day) in patients with digestive tract cancer to assess its significance. Serum sCD40L levels were measured by ELISA in 294 residual samples, and were divided into tertiles. In patients with colorectal cancer (CRC), 5-year relapse-free survival (RFS) rates in the middle and highest tertiles were 61.6% and 61.2%, respectively, which was significantly lower than 83.8% in the lowest tertile. A Cox proportional hazard analysis showed that the lowest tertile had a significantly lower risk of relapse or death than the highest tertile even with multivariate adjustment (hazard ratio (HR), 0.30; 95% confidence interval (CI), 0.11-0.80; p = 0.016). In the subgroup of CRC patients with the highest tertile of sCD40L, the 5-year RFS rate in the vitamin D group was 77.9%, which was significantly higher than 33.2% in the placebo group (HR, 0.30; 95% CI, 0.11-0.81; p = 0.018 [Pinteraction = 0.04]). In conclusion, elevated sCD40L might be a biomarker of poor prognosis in patients with CRC, but vitamin D supplementation might improve RFS in patients with high sCD40L.

Effect of Vitamin D Supplements on Relapse or Death in a p53-Immunoreactive Subgroup With Digestive Tract Cancer: Post Hoc Analysis of the AMATERASU Randomized Clinical Trial.

Importance: Recent meta-analyses of randomized clinical trials found that daily vitamin D3 supplementation had beneficial effects on cancer mortality, although the results are still controversial. Objective: To examine whether vitamin D supplementation reduces the risk of relapse or death in a supgroup of patients with digestive tract cancer who were p53 immunoreactive. Design, Setting, and Participants: This was a post hoc subgroup analysis of the AMATERASU randomized, double-blind, placebo-controlled clinical trial. This trial included patients at a single university hospital in Japan with digestive tract cancers between January 2010 and February 2018 followed up for a median (IQR) of 3.5 (2.5-5.3) years to compare the effects of vitamin D supplementation with placebo and was reported in 2019. Patients from among 417 participants in the AMATERASU trial whose residual serum samples were available were included. Data were analyzed from October 20 to November 24, 2022. Interventions: Vitamin D3 (2000 IU/d) supplementation or placebo. Main Outcomes and Measures: The primary outcome was 5-year relapse or death. The subgroup of patients who were p53 immunoreactive was defined by positivity for anti-p53 antibodies in serum and nuclear accumulation of p53 oncosuppressor protein in more than 99% of cancer cells, which is considered a biomarker for p53 missense mutations. Anti-p53 antibody levels were measured using chemiluminescent enzyme immune assay. Immunohistochemical staining data of p53 protein in cancer tissue in pathologic specimens were obtained from a previous study and divided into 4 grades. Results: Among 392 patients with digestive tract cancer (mean [SD] age, 66 [10.7] years; 260 males [66.3%]), there were 37 patients with esophageal cancer (9.4%), 170 patients with gastric cancer (43.4%), 2 patients with small bowel cancer (0.5%), and 183 patients with colorectal cancer (46.7%). Serum anti-p53 antibody was detectable in 142 patients (36.2%), and p53-immunohistochemistry grade showed a positive association with serum anti-p53 antibody levels (coefficient = 0.19; P < .001). In the p53-immunoreactive subgroup (80 patients), relapse or death occurred in 9 of 54 patients (16.7%) in the vitamin D group and 14 of 26 patients (53.8%) in the placebo group; 5-year relapse-free survival (RFS) was significantly higher in the vitamin D group (13 patients [80.9%]) than the placebo group (1 patient [30.6%]; hazard ratio [HR], 0.27; 95% CI, 0.11-0.61; P = .002). This was significantly different from 272 patients in the non-p53 immunoreactive subgroup, in which vitamin D had no effect on 5-year RFS (vitamin D: 35 of 158 patients [22.2%] vs placebo: 24 of 114 patients [21.1%]; HR, 1.09; 95% CI, 0.65-1.84) (P for interaction = .005). Conclusions and Relevance: This study found that vitamin D supplementation reduced the risk of relapse or death in the subgroup of patients with digestive tract cancer who were p53 immunoreactive. Trial Registration: Identifier: UMIN000001977.

Time Analysis of Recidivism in Acquired Cholesteatoma Using the Kaplan-Meier Method.

OBJECTIVE: To analyze the time trends of recidivism of acquired cholesteatoma using the Kaplan-Meier method. STUDY DESIGN: We conducted a retrospective, observational study of 256 patients having their first cholesteatoma surgery. The cumulative recidivism-free rate was calculated using Kaplan-Meier survival analysis related to the follow-up period, pathophysiology, the extent of the disease, and recidivism pathologies. RESULTS: Pars flacida cholesteatoma with tympanic cavity progression had a high likelihood of recurrence disease. Pars tensa cholesteatoma led to more recurrence of the disease than the residual disease. In both pars flacida and pars tensa cholesteatoma, the incidence of disease recurrence increased even 3 years after surgery. On the contrary, the incidence of residual disease peaked within 3 years after surgery, and thereafter, the incidence of residual disease tended to be small. In particular, pars flacida cholesteatoma extending into the mastoid cavity or tympanic cavity tended to recur up to 5 years postoperatively. CONCLUSIONS: We calculated the cumulative recidivism-free rates of 256 patients with cholesteatoma using Kaplan-Meier survival analysis. These results can lead to better estimates of the length of the follow-up period. LEVEL OF EVIDENCE: Level IV evidence from case-control studies.

Identification of Methylsulochrin as a Partial Agonist for Aryl Hydrocarbon Receptors and Its Antiviral and Anti-inflammatory Activities.

Although aryl hydrocarbon receptors (AhRs) are related to the metabolic pathway of xenobiotics, recent studies have revealed that this receptor is also associated with the life cycle of viruses and inflammatory reactions. For example, flutamide, used to treat prostate cancer, inhibits hepatitis C virus proliferation by acting as an AhR antagonist, and methylated-pelargonidin, an AhR agonist, suppresses pro-inflammatory cytokine production. To discover a novel class of AhR ligands, we screened 1000 compounds derived from fungal metabolites using a reporter assay and identified methylsulochrin as a partial agonist of the aryl hydrocarbon receptor. Methylsulochrin was found to inhibit the production of hepatitis C virus (HCV) in Huh-7.5.1 cells. Methylsulochrin also suppressed the production of interleukin-6 in RAW264.7 cells. Furthermore, a preliminary structure-activity relationship study using sulochrin derivatives was performed. Our findings suggest the use of methylsulochrin derivatives as anti-HCV compounds with anti-inflammatory activity.

Isolation, structural determination, and antiviral activities of metabolites from vanitaracin A-producing Talaromyces sp.

Vanitaracin A, an anti-hepatitis B virus polyketide, has been previously isolated from Talaromyces sp. In the present study, we searched for novel compounds in the culture broth obtained from a vanitaracin A-producing fungus under various conditions. Three novel compounds (vanitaracin C, vanitaraphilone A, and 2-hydroxy-4-(hydroxymethyl)-6-methylbenzaldehyde) were isolated, and their structures were determined using spectroscopic methods (1D/2D NMR and MS). In addition, the antiviral spectrum of vanitaracin A was examined by measuring its antiviral activities against rabies virus, Borna disease virus 1, and bovine leukemia virus. This compound exhibited antiviral activity against bovine leukemia virus, which is the causative agent of enzootic bovine leukosis. The anti-bovine leukemia virus effects of other compounds isolated from the vanitaracin A-producing fungus, namely, vanitaracins B and C, vanitaraphilone A, and 2-hydroxy-4-(hydroxymethyl)-6-methylbenzaldehyde, were also evaluated. Vanitaracin B, vanitaraphilone A and 2-hydroxy-4-(hydroxymethyl)-6-methylbenzaldehyde were also found to exhibit activity against bovine leukemia virus. These findings reveal the broad-spectrum antiviral activity of the vanitaracin scaffold and suggest several candidates for the development of anti-bovine leukemia virus drugs.

Applying Machine Learning to Determine 25(OH)D Threshold Levels Using Data from the AMATERASU Vitamin D Supplementation Trial in Patients with Digestive Tract Cancer.

Some controversy remains on thresholds for deficiency or sufficiency of serum 25-hydroxyvitamin D (25(OH)D) levels. Moreover, 25(OH)D levels sufficient for bone health might differ from those required for cancer survival. This study aimed to explore these 25(OH)D threshold levels by applying the machine learning method of multivariable adaptive regression splines (MARS) in post hoc analyses using data from the AMATERASU trial, which randomly assigned Japanese patients with digestive tract cancer to receive vitamin D or placebo supplementation. Using MARS, threshold 25(OH)D levels were estimated as 17 ng/mL for calcium and 29 ng/mL for parathyroid hormone (PTH). Vitamin D supplementation increased calcium levels in patients with baseline 25(OH)D levels ≤17 ng/mL, suggesting deficiency for bone health, but not in those >17 ng/mL. Vitamin D supplementation improved 5-year relapse-free survival (RFS) compared with placebo in patients with intermediate 25(OH)D levels (18−28 ng/mL): vitamin D, 84% vs. placebo, 71%; hazard ratio, 0.49; 95% confidence interval, 0.25−0.96; p = 0.04. In contrast, vitamin D supplementation did not improve 5-year RFS among patients with low (≤17 ng/mL) or with high (≥29 ng/mL) 25(OH)D levels. MARS might be a reliable method with the potential to eliminate guesswork in the estimation of threshold values of biomarkers.

Determining the absolute configuration of vanitaracin A, an anti-hepatitis B virus agent.

Vanitaracin A is an anti-hepatitis B virus (anti-HBV) compound isolated from the culture broth of the fungus Talaromyces sp. Vanitaracin A inhibits the entry of HBV into target cells with sub-micromolar IC50 values. While a structure-activity relationship study is highly desirable, a synthetic approach still needs to be developed, which is difficult because the absolute configurations of the six stereogenic centers in the structure of vanitaracin A have not yet been determined. In the present study, we used the crystalline sponge method to clarify the configuration of the compound after determining the absolute configuration of the secondary alcohol using a modified Mosher ester method. Combining these analyses with the NOESY spectrum of vanitaracin A and NMR analyses of the crude side-chain carboxylic acid obtained by the alkaline hydrolysis of vanitaracin A revealed the absolute configurations of all stereogenic centers in this important compound.

Effect of Vitamin D Supplements on Relapse of Digestive Tract Cancer with Tumor Stromal Immune Response: A Secondary Analysis of the AMATERASU Randomized Clinical Trial.

The aim was to examine whether vitamin D supplementation (2000 IU/day) reduces the risk of relapse in a subgroup of patients with digestive tract cancer, showing a sufficient immune response in tumor stroma by conducting secondary subgroup analyses of the AMATERASU randomized, double-blind, placebo-controlled trial (UMIN000001977). A total of 372 patients were divided into two subgroups stratified by the median density of immune cells infiltrating in tumor stroma into higher and lower halves. In the higher-half subgroup of CD56+ cells, the relapse ratio was significantly lower in the vitamin D group (7.4%) than in the placebo group (20.5%) (subdistribution hazard ratio (SHR), 0.35; 95% confidence interval (CI), 0.15-0.82), but it was equivalent (25.2% vs. 22.7%) in the lower-half subgroup of CD56+ cells (SHR, 1.21; 95% CI, 0.68-2.19) with a significant interaction (Pinteraction = 0.02). Although there were no significant differences, the risk of relapse was lower in the vitamin D group than in the placebo group in the higher half of CD45RO+ memory T cells (8.9% vs. 19.2%), and of CD8+ cytotoxic T cells (11.3% vs. 22.5%). In patients with digestive tract cancer, vitamin D supplementation was hypothesized to reduce the risk of relapse in the subgroup of patients who already have an adequate infiltration of immune cells in their tumor stroma.

Novel neuroprotective hydroquinones with a vinyl alkyne from the fungus, Pestalotiopsis microspora.

New hydroquinone derivatives bearing a vinyl alkyne, pestalotioquinols A and B, were isolated from a fungal culture broth of Pestalotiopsis microspora. The structures of these novel compounds were determined by interpretation of spectroscopic data (1D/2D NMR, MS, and IR), and the absolute configuration of the stereogenic center of pestalotioquinol A was assigned using the modified Mosher's method. Nerve growth factor-differentiated neuronal PC12 cells were pretreated with pestalotioquinols A and B and removed from the medium, and then treated with a generator of peroxynitrite (ONOO-), a reactive nitrogen species, to induce cell death. The cytotoxicity of the treated cells was assessed by measuring lactate dehydrogenase leakage. As a result, 1-3 µM pretreatment of pestalotioquinols A and B rescued neuronal PC12 cells from peroxynitrite-induced cytotoxicity and the protective activity was sustained after removing each compound from the medium. These results demonstrate that pestalotioquinol derivatives are a new class of hydroquinones possessing a vinyl alkyne and exhibiting relatively high neuroprotective effects.

Photo images, 3D/CT data and mtDNA of the freshwater mussels (Bivalvia: Unionidae) in the Kyushu and Ryukyu Islands, Japan, with SEM/EDS analysis of the shell.

BACKGROUND: Freshwater mussels (Bivalvia: Unionidae), which are keystone species of freshwater ecosystems, are in global decline. In addition to ecological/genetic studies, morphological examinations are needed to help provide information for the development of additional freshwater mussel studies and eventually conservation efforts for freshwater ecosystems.The microscopic structure, which can be obtained by scanning electron microscopy (SEM) and elemental composition, which can be obtained with energy dispersive X-ray spectrometry (EDS), of mollusc shells are of interest to malacologists. However, information about freshwater mussels is still limited.Kyushu Island is the southernmost island of the four major islands of Japan. Kyushu Island is a hotspot of bitterling fishes in Japan, which simultaneously means that the island is a hotspot of freshwater mussels. The Ryukyu Islands stretch southwest from Kyushu Island to Taiwan; a freshwater mussel of unknown origin was reported from the Ryukyu Islands.Digital archiving for biology and ecology is a continuing challenge for open science. This data paper describes online published photo images, 3D/CT and mtDNA data and SEM/EDS analyses of the shell of freshwater mussels that inhabit the Kyushu and Ryukyu Islands, Japan. Our data will provide basic information regarding freshwater biology and be of public interest as open science. NEW INFORMATION: Photo images, 3D/CT data, mtDNA data, SEM images and EDS elemental analysis of freshwater mussels that inhabit the Kyushu and Ryukyu Islands (61 individuals, nine species/subspecies) were published online in a local database (http://ffish.asia/Unionidae3D), GBIF (http://ipt.pensoft.net/resource?r=unionidae3d) and DDBJ/EMBL/Genbank (LC431810-LC431840).