Extracellular N6 -isopentenyladenosine (i6A) addition induces cotranscriptional i6A incorporation into ribosomal RNAs.

N6 -isopentenyladenosine (i6A), a modified adenosine monomer, is known to induce cell death upon its addition to the culture medium. However, the molecular fate of extracellularly added i6A has yet to be identified. Here we show that i6A addition to cell culture medium results in i6A incorporation into cellular RNA in several cell lines, including the 5-fluorouracil (5-FU)-resistant human oral squamous cell carcinoma cell line FR2-SAS and its parental 5-FU-sensitive cell line SAS. i6A was predominantly incorporated into 18S and 28S rRNAs, and i6A incorporation into total RNA was mostly suppressed by treating these cell lines with an RNA polymerase I (Pol I) inhibitor. i6A was incorporated into RNA even upon inactivation of TRIT1, the only cellular i6A-modifying enzyme. These results indicate that upon cellular uptake of i6A, it is anabolized to be used for Pol I transcription. Interestingly, at lower i6A concentrations, the cytotoxic effect of i6A was substantially more pronounced in FR2-SAS cells than in SAS cells. Moreover, in FR2-SAS cells, i6A treatment decreased the rate of cellular protein synthesis and increased intracellular protein aggregation, and these effects were more pronounced than in SAS cells. Our work provides insights into the molecular fate of extracellularly applied i6A in the context of intracellular nucleic acid anabolism and suggests investigation of i6A as a candidate for a chemotherapy agent against 5-FU-resistant cancer cells.

Enhanced accumulation of γ-aminobutyric acid by deletion of aminotransferase genes involved in γ-aminobutyric acid catabolism in engineered Halomonas elongata.

Halomonas elongata OUT30018 is a moderately halophilic bacterium that synthesizes and accumulates ectoine as an osmolyte by activities of the enzymes encoded by the high salinity-inducible ectABC operon. Previously, we engineered a γ-aminobutyric acid (GABA)-producing H. elongata GOP-Gad (ΔectABC::mCherry-HopGadmut) from an ectoine-deficient mutant of this strain due to its ability to use high-salinity biomass waste as substrate. Here, to further increase GABA accumulation, we deleted gabT, which encodes GABA aminotransferase (GABA-AT) that catalyzes the first step of the GABA catabolic pathway, from the H. elongata GOP-Gad genome. The resulting strain H. elongata ZN3 (ΔectABC::mCherry-HopGadmut ΔgabT) accumulated 291 µmol/g cell dry weight (CDW) of GABA in the cells, which is a 1.5-fold increase from H. elongata GOP-Gad's 190 µmol/g CDW. This result has confirmed the role of GABA-AT in the GABA catabolic pathway. However, redundancy in endogenous GABA-AT activity was detected in a growth test, where a gabT-deletion mutant of H. elongata OUT30018 was cultured in a medium containing GABA as the sole carbon and nitrogen sources. Because L-2,4-diaminobutyric acid aminotransferase (DABA-AT), encoded by an ectB gene of the ectABC operon, shares sequence similarity with GABA-AT, a complementation analysis of the gabT and the ectB genes was performed in the H. elongata ZN3 genetic background to test the involvement of DABA-AT in the redundancy of GABA-AT activity. Our results indicate that the expression of DABA-AT can restore GABA-AT activity in H. elongata ZN3 and establish DABA-AT's aminotransferase activity toward GABA in vivo. IMPORTANCE: In this study, we were able to increase the yield of GABA by 1.5 times in the GABA-producing H. elongata ZN3 strain by deleting the gabT gene, which encodes GABA-AT, the initial enzyme of the GABA catabolic pathway. We also report the first in vivo evidence for GABA aminotransferase activity of an ectB-encoded DABA-AT, confirming a longstanding speculation based on the reported in vitro GABA-AT activity of DABA-AT. According to our findings, the DABA-AT enzyme can catalyze the initial step of GABA catabolism, in addition to its known function in ectoine biosynthesis. This creates a cycle that promotes adequate substrate flow between the two pathways, particularly during the early stages of high-salinity stress response when the expression of the ectB gene is upregulated.

Metabolic engineering of high-salinity-induced biosynthesis of γ-aminobutyric acid improves salt-stress tolerance in a glutamic acid-overproducing mutant of an ectoine-deficient Halomonas elongata.

A moderately halophilic eubacterium, Halomonas elongata, has been used as cell factory to produce fine chemical 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine), which functions as a major osmolyte protecting the cells from high-salinity stress. To explore the possibility of using H. elongata to biosynthesize other valuable osmolytes, an ectoine-deficient salt-sensitive H. elongata deletion mutant strain KA1 (ΔectABC), which only grows well in minimal medium containing up to 3% NaCl, was subjected to an adaptive mutagenesis screening in search of mutants with restored salt tolerance. Consequently, we obtained a mutant, which tolerates 6% NaCl in minimal medium by overproducing L-glutamic acid (Glu). However, this Glu-overproducing (GOP) strain has a lower tolerance level than the wild-type H. elongata, possibly because the acidity of Glu interferes with the pH homeostasis of the cell and hinders its own cellular accumulation. Enzymatic decarboxylation of Glu to γ-aminobutyric acid (GABA) by a Glu decarboxylase (GAD) could restore cellular pH homeostasis; therefore, we introduced an engineered salt-inducible HopgadBmut gene, which encodes a wide pH-range GAD mutant, into the genome of the H. elongata GOP strain. We found that the resulting H. elongata GOP-Gad strain exhibits higher salt tolerance than the GOP strain by accumulating high concentration of GABA as an osmolyte in the cell (176.94 µmol/g cell dry weight in minimal medium containing 7% NaCl). With H. elongata OUT30018 genetic background, H. elongata GOP-Gad strain can utilize biomass-derived carbon and nitrogen compounds as its sole carbon and nitrogen sources, making it a good candidate for the development of GABA-producing cell factories.IMPORTANCEWhile the wild-type moderately halophilic H. elongata can synthesize ectoine as a high-value osmolyte via the aspartic acid metabolic pathway, a mutant H. elongata GOP strain identified in this work opens doors for the biosynthesis of alternative valuable osmolytes via glutamic acid metabolic pathway. Further metabolic engineering to install a GAD system into the H. elongata GOP strain successfully created a H. elongata GOP-Gad strain, which acquired higher tolerance to salt stress by accumulating GABA as a major osmolyte. With the ability to assimilate biomass-derived carbon and nitrogen sources and thrive in high-salinity environment, the H. elongata GOP-Gad strain can be used in the development of sustainable GABA-producing cell factories.

Metabolic pathway engineering of high-salinity-induced overproduction of L-proline improves high-salinity stress tolerance of an ectoine-deficient Halomonas elongata.

Halophilic bacteria have adapted to survive in high-salinity environments by accumulating amino acids and their derivatives as organic osmolytes. L-Proline (Pro) is one such osmolyte that is also being used as a feed stimulant in the aquaculture industry. Halomonas elongata OUT30018 is a moderately halophilic bacterium that accumulates ectoine (Ect), but not Pro, as an osmolyte. Due to its ability to utilize diverse biomass-derived carbon and nitrogen sources for growth, H. elongata OUT30018 is used in this work to create a strain that overproduces Pro, which could be used as a sustainable Pro-rich feed additive. To achieve this, we replaced the coding region of H. elongata OUT30018's Ect biosynthetic operon with the artificial self-cloned proBm1AC gene cluster that encodes the Pro biosynthetic enzymes: feedback-inhibition insensitive mutant γ-glutamate kinase (γ-GKD118N/D119N), γ-glutamyl phosphate reductase, and pyrroline-5-carboxylate reductase. Additionally, the putA gene, which encodes the key enzyme of Pro catabolism, was deleted from the genome to generate H. elongata HN6. While the Ect-deficient H. elongata KA1 could not grow in minimal media containing more than 4% NaCl, H. elongata HN6 thrived in the medium containing 8% NaCl by accumulating Pro in the cell instead of Ect, reaching a concentration of 353.1 ± 40.5 µmol/g cell fresh weight, comparable to the Ect accumulated in H. elongata OUT30018 in response to salt stress. With its genetic background, H. elongata HN6 has the potential to be developed into a Pro-rich cell factory for upcycling biomass waste into single-cell feed additives, contributing to a more sustainable aquaculture industry.IMPORTANCEWe report here the evidence for de novo biosynthesis of Pro to be used as a major osmolyte in an ectoine-deficient Halomonas elongata. Remarkably, the concentration of Pro accumulated in H. elongata HN6 (∆ectABC::mCherry-proBm1AC ∆putA) is comparable to that of ectoine accumulated in H. elongata OUT30018 in response to high-salinity stress. We also found that among the two γ-glutamate kinase mutants (γ-GKD118N/D119N and γ-GKD154A/E155A) designed to resemble the two known Escherichia coli feedback-inhibition insensitive γ-GKD107N and γ-GKE143A, the γ-GKD118N/D119N mutant is the only one that became insensitive to feedback inhibition by Pro in H. elongata. As Pro is one of the essential feed additives for the poultry and aquaculture industries, the genetic makeup of the engineered H. elongata HN6 would allow for the sustainable upcycling of high-salinity waste biomass into a Pro-rich single-cell eco-feed.

E3 Ubiquitin Ligase Riplet Is Expressed in T Cells and Suppresses T Cell-Mediated Antitumor Immune Responses.

The E3 ubiquitin ligase Riplet mediates retinoic acid-inducible gene-I polyubiquitination and is essential for viral-induced expression of type I IFNs in dendritic cells and macrophages. The function of Riplet in innate immunity has been well demonstrated; however, its role in adaptive immunity during the antitumor immune response is unclear. In this study, we examined the role of Riplet in the T cell-mediated antitumor immune response. Riplet was expressed in T cells and upregulated in CD8+ T cells in response to TCR-mediated stimulation. Furthermore, PR domain containing 1, eomesodermin, and killer cell lectin-like receptor G1 expression was increased in effector CD8+ T cells by Riplet knockout in vitro, which suggests that Riplet is involved in the effector function of CD8+ T cells. Our results indicated that Riplet deficiency augmented the antitumor response of MO4 (OVA-expressing melanoma)-bearing mice treated with OVA peptide-pulsed dendritic cells. Moreover, both CD4+ and CD8+ T cells played important roles in Riplet-mediated augmentation of the antitumor immune response. In tumor-draining lymph nodes, the Th1 response was promoted, and the induction of OVA-specific CD8+ T cells and IFN-γ production were enhanced by Riplet deficiency. Furthermore, the IFN-γ response and OVA-specific cytotoxicity of CD8+ T cells in tumor tissue were augmented by Riplet deficiency. The expression of Cxcl9fluorescence-minus-one and Cxcl10 mRNA was also enhanced in the tumor microenvironment by Riplet knockout, consistent with the augmented recruitment of CTLs. Overall, we clarified a function of Riplet in T cells, which is to suppress the antitumor immune response through modulating Th1 and CTLs.

Expression of an engineered salt-inducible proline biosynthetic operon in a glutamic acid over-producing mutant, Halomonas elongata GOP, confers increased proline yield due to enhanced growth under high-salinity conditions.

L-Proline (Pro) is an essential amino acid additive in livestock and aquaculture feeds. Previously, we created a Pro overproducing Halomonas elongata HN6 by introducing an engineered salt-inducible Pro biosynthetic mCherry-proBm1AC operon and deleting a putA gene that encoded a Pro catabolic enzyme in the genome of H. elongata OUT30018. Here, we report a generation of a novel Pro overproducing H. elongata HN10 strain with improved salt tolerance and higher Pro yield by expressing the mCherry-proBm1AC operon and deleting the putA gene in the genome of a spontaneous mutant H. elongata Glutamic acid Over-Producing, which overproduces glutamic acid (Glu) that is a precursor for Pro biosynthesis. The optimal salt concentration for growth of H. elongata HN10 was found to be 7% to 8% w/v NaCl, and the average Pro yield of 166 mg/L was achieved when H. elongata HN10 was cultivated in M63 minimal medium containing 4% w/v glucose and 8% w/v NaCl.

FOXM1-Mediated Regulation of Reactive Oxygen Species and Radioresistance in Oral Squamous Cell Carcinoma Cells.

Radioresistance is a major obstacle to the successful treatment of oral squamous cell carcinoma (OSCC). To help overcome this issue, we have developed clinically relevant radioresistant (CRR) cell lines generated by irradiating parental cells over time, which are useful for OSCC research. In the present study, we conducted gene expression analysis using CRR cells and their parental lines to investigate the regulation of radioresistance in OSCC cells. Based on gene expression changes over time in CRR cells and parental lines subjected to irradiation, forkhead box M1 (FOXM1) was selected for further analysis in terms of its expression in OSCC cell lines, including CRR cell lines and clinical specimens. We suppressed or upregulated the expression of FOXM1 in OSCC cell lines, including CRR cell lines, and examined radiosensitivity, DNA damage, and cell viability under various conditions. The molecular network regulating radiotolerance was also investigated, especially the redox pathway, and the radiosensitizing effect of FOXM1 inhibitors was examined as a potential therapeutic application. We found that FOXM1 was not expressed in normal human keratinocytes but was expressed in several OSCC cell lines. The expression of FOXM1 was upregulated in CRR cells compared with that detected in the parental cell lines. In a xenograft model and clinical specimens, FOXM1 expression was upregulated in cells that survived irradiation. FOXM1-specific small interfering RNA (siRNA) treatment increased radiosensitivity, whereas FOXM1 overexpression decreased radiosensitivity, and DNA damage was altered significantly under both conditions, as well as the levels of redox-related molecules and reactive oxygen species production. Treatment with the FOXM1 inhibitor thiostrepton had a radiosensitizing effect and overcame radiotolerance in CRR cells. According to these results, the FOXM1-mediated regulation of reactive oxygen species could be a novel therapeutic target for the treatment of radioresistant OSCC; thus, treatment strategies targeting this axis might overcome radioresistance in this disease.

In planta evidence that the HAK transporter OsHAK2 is involved in Na+ transport in rice.

HAK family transporters primarily function as K+ transporters and play major roles in K+ uptake and translocation in plants, whereas several HAK transporters exhibit Na+ transport activity. OsHAK2, a rice HAK transporter, was shown to mediate Na+ transport in Escherichia coli in a previous study. In this study, we investigated whether OsHAK2 is involved in Na+ transport in the rice plant. Overexpression of OsHAK2 increased Na+ translocation from the roots to the shoots of transgenic rice. It also increased both root and whole-plant Na+ content, and enhanced shoot length under low Na+ and K+ conditions. Meanwhile, OsHAK2 overexpression increased salt sensitivity under a long-term salt stress condition, indicating that OsHAK2 is not involved in salt tolerance, unlike in the case of ZmHAK4 in maize. These results suggest that OsHAK2 is permeable to Na+ and contributes to shoot growth in rice plants under low Na+ and K+ conditions.

The antioxidative stress regulator Nrf2 potentiates radioresistance of oral squamous cell carcinoma accompanied with metabolic modulation.

Nuclear factor erythroid 2-related factor 2 (Nrf2), which regulates the expression of critical antioxidant proteins, was recently demonstrated to play a key role in cancer progression. Resistance to radiotherapy is a major obstacle in treating oral squamous cell carcinoma (OSCC). However, little is known about the association between Nrf2 and radioresistance in OSCC. Two OSCC cell lines (SAS and HSC-2) and their clinically relevant radioresistant (CRR) clones (SAS-R, HSC-2-R) were used. The effects of Nrf2 downregulation on radiosensitivity and the involvement of glycolysis in Nrf2-mediated radioresistance were evaluated. Immunohistochemistry of phosphorylated Nrf2 (p-Nrf2) was performed in 110 patients with OSCC who underwent preoperative chemoradiotherapy and surgery. Nrf2 was stably upregulated in CRR cells in vitro and in a mouse xenograft model. Moreover, elevated Nrf2 expression was associated with radioresistance. The enhancement of Nrf2-dependent glycolysis and glutathione synthesis was involved in the development of radioresistance. Additionally, p-Nrf2 expression was closely related to the pathological response to chemoradiotherapy, and its expression was predictive of prognosis in patients with advanced OSCC. Our results suggest that Nrf2 plays an important role in the radioresistance of OSCC accompanied by metabolic reprogramming. Targeting Nrf2 antioxidant pathway may represent a promising treatment strategy for highly malignant OSCC.

Naringenin potentiates anti-tumor immunity against oral cancer by inducing lymph node CD169-positive macrophage activation and cytotoxic T cell infiltration.

The CD169+ macrophages in lymph nodes are implicated in cytotoxic T lymphocyte (CTL) activation and are associated with improved prognosis in several malignancies. Here, we investigated the significance of CD169+ macrophages in oral squamous cell carcinoma (OSCC). Further, we tested the anti-tumor effects of naringenin, which has been previously shown to activate CD169+ macrophages, in a murine OSCC model. Immunohistochemical analysis for CD169 and CD8 was performed on lymph node and primary tumor specimens from 89 patients with OSCC. We also evaluated the effects of naringenin on two murine OSCC models. Increased CD169+ macrophage counts in the regional lymph nodes correlated with favorable prognosis and CD8+ cell counts within tumor sites. Additionally, naringenin suppressed tumor growth in two murine OSCC models. The mRNA levels of CD169, interleukin (IL)-12, and C-X-C motif chemokine ligand 10 (CXCL10) in lymph nodes and CTL infiltration in tumors significantly increased following naringenin administration in tumor-bearing mice. These results suggest that CD169+ macrophages in lymph nodes are involved in T cell-mediated anti-tumor immunity and could be a prognostic marker for patients with OSCC. Moreover, naringenin is a new potential agent for CD169+ macrophage activation in OSCC treatment.

Potential use of EGFR-targeted molecular therapies for tumor suppressor CYLD-negative and poor prognosis oral squamous cell carcinoma with chemoresistance.

BACKGROUND: Tumor suppressor CYLD dysfunction by loss of its expression, triggers malignant transformation, especially drug resistance and tumor invasion/metastasis. Although loss of CYLD expression is significantly associated with poor prognosis in a large variety of tumors, no clinically-effective treatment for CYLD-negative cancer patients is available. METHODS: We focused on oral squamous cell carcinoma (OSCC), and sought to develop novel therapeutic agents for CYLD-negative cancer patients with poor prognosis. CYLD-knockdown OSCC cells by using CYLD-specific siRNA, were used to elucidate and determine the efficacy of novel drug candidates by evaluating cell viability and epithelial-mesenchymal transition (EMT)-like change. Therapeutic effects of candidate drug on cell line-derived xenograft (CDX) model and usefulness of CYLD as a novel biomarker using patient-derived xenograft (PDX) model were further investigated. RESULTS: CYLD-knockdown OSCC cells were resistant for all currently-available cytotoxic chemotherapeutic agents for OSCC, such as, cisplatin, 5-FU, carboplatin, docetaxel, and paclitaxel. By using comprehensive proteome analysis approach, we identified epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, played key roles in CYLD-knockdown OSCC cells. Indeed, cell survival rate in the cisplatin-resistant CYLD-knockdown OSCC cells was markedly inhibited by treatment with clinically available EGFR tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib. In addition, gefitinib was significantly effective for not only cell survival, but also EMT-like changes through inhibiting transforming growth factor-ß (TGF-ß) signaling in CYLD-knockdown OSCC cells. Thereby, overall survival of CYLD-knockdown CDX models was significantly prolonged by gefitinib treatment. Moreover, we found that CYLD expression was significantly associated with gefitinib response by using PDX models. CONCLUSIONS: Our results first revealed that EGFR-targeted molecular therapies, such as EGFR-TKIs, could have potential to be novel therapeutic agents for the CYLD-negative OSCC patients with poor prognosis.

Multicenter retrospective study of nivolumab for recurrent/metastatic oral squamous cell carcinoma.

OBJECTIVES: Immunotherapy with nivolumab for patients with recurrent/metastatic oral squamous cell carcinoma has not been evaluated. Here, we aimed to examine the efficacy, safety, and prognostic factors of nivolumab in these patients. MATERIALS AND METHODS: This multicenter retrospective observational study involved patients who received nivolumab between April 2017 and June 2019. The patient characteristics were evaluated for association with progression-free and overall survival. Progression-free and overall survival rates were calculated; parameters that were significant in the univariate analysis were used as explanatory variables. Independent factors for progression-free and overall survival were identified using multivariate analysis. RESULTS: Totally, 143 patients were included. The overall response and disease control rates were 27.3% and 46.2%, respectively. The median, 1- and 2-year progression-free survival rates were 2.7 months, 25.4%, and 19.2%, respectively; those for overall survival were 11.2 months, 47.3%, and 33.6%, respectively. The independent factors affecting progression-free survival were performance status and immune-related adverse event occurrence, whereas those affecting overall survival were performance status, target disease, and number of previous lines of systemic cancer therapy. Eight patients reported grade ≥3 immune-related adverse events. CONCLUSION: Nivolumab was effective for recurrent/metastatic oral squamous cell carcinoma treatment and was well tolerated by patients.

Factors Affecting Human Damage in Heavy Rains and Typhoon Disasters.

Floods due to heavy rains or typhoons are frequent annual hazards in Japan. This study aims to reduce disaster fatalities and contribute to disaster risk reduction. This retrospective observational study analyzed fatalities caused by heavy rains or typhoons. In Japan, 578 fatalities, related to seven occurrences of heavy rains and 16 typhoons, occurred between 2016 and 2020. Moreover, 13,195 houses collapsed due to hazards. Furthermore, 334 (73.2%) of the 456 fatalities were > 60 years old. Heavy rains caused more local area destruction due to floods and landslides than typhoons although wind- and disaster-related mortalities were found to be caused by typhoons. Human damage was eminent in older people because of their vulnerabilities and possibly dangerous behavior. Many fatalities were due to floods (46.9%) and landslides (44.1%). Indoor and outdoor mortalities due to heavy rains or typhoons were 157 (55.9%) and 124 (44.1%), respectively, and 24 (21.8%) of 124 outdoor mortalities occurred in vehicles. The number of recent flood mortalities in Japan correlates with the number of destroyed houses. Analyzing the victim's locations in the 2020 Kumamoto Heavy Rain using hazard and inundation maps suggested the difficulty of ensuring the safety of people living in dangerous areas. This study showed the characteristics of flood damage by heavy rains and typhoons in Japan and reports that flood damage is increasing because of the hazard size and community aging. Disaster risk reduction, disaster education, and evacuation safety plans for the elderly using hazard maps were important for strengthening disaster resilience.

Air Plasma-Activated Medium Evokes a Death-Associated Perinuclear Mitochondrial Clustering.

Intractable cancers such as osteosarcoma (OS) and oral cancer (OC) are highly refractory, recurrent, and metastatic once developed, and their prognosis is still disappointing. Tumor-targeted therapy, which eliminates cancers effectively and safely, is the current clinical choice. Since aggressive tumors are substantially resistant to multidisciplinary therapies that target apoptosis, tumor-specific activation of another cell death modality is a promising avenue for meeting this goal. Here, we report that a cold atmospheric air plasma-activated medium (APAM) can kill OS and OC by causing a unique mitochondrial clustering. This event was named monopolar perinuclear mitochondrial clustering (MPMC) based on its characteristic unipolar mitochondrial perinuclear accumulation. The APAM caused apoptotic and nonapoptotic cell death. The APAM increased mitochondrial ROS (mROS) and cell death, and the antioxidants such as N-acetylcysteine (NAC) prevented them. MPMC occurred following mitochondrial fragmentation, which coincided with nuclear damages. MPMC was accompanied by mitochondrial lipid peroxide (mLPO) accumulation and prevented by NAC, Ferrostatin-1, and Nocodazole. In contrast, the APAM induced minimal cell death, mROS generation, mLPO accumulation, and MPMC in fibroblasts. These results suggest that MPMC occurs in a tumor-specific manner via mitochondrial oxidative stress and microtubule-driven mitochondrial motility. MPMC induction might serve as a promising target for exerting tumor-specific cytotoxicity.

MADGAN: unsupervised medical anomaly detection GAN using multiple adjacent brain MRI slice reconstruction.

BACKGROUND: Unsupervised learning can discover various unseen abnormalities, relying on large-scale unannotated medical images of healthy subjects. Towards this, unsupervised methods reconstruct a 2D/3D single medical image to detect outliers either in the learned feature space or from high reconstruction loss. However, without considering continuity between multiple adjacent slices, they cannot directly discriminate diseases composed of the accumulation of subtle anatomical anomalies, such as Alzheimer's disease (AD). Moreover, no study has shown how unsupervised anomaly detection is associated with either disease stages, various (i.e., more than two types of) diseases, or multi-sequence magnetic resonance imaging (MRI) scans. RESULTS: We propose unsupervised medical anomaly detection generative adversarial network (MADGAN), a novel two-step method using GAN-based multiple adjacent brain MRI slice reconstruction to detect brain anomalies at different stages on multi-sequence structural MRI: (Reconstruction) Wasserstein loss with Gradient Penalty + 100 [Formula: see text] loss-trained on 3 healthy brain axial MRI slices to reconstruct the next 3 ones-reconstructs unseen healthy/abnormal scans; (Diagnosis) Average [Formula: see text] loss per scan discriminates them, comparing the ground truth/reconstructed slices. For training, we use two different datasets composed of 1133 healthy T1-weighted (T1) and 135 healthy contrast-enhanced T1 (T1c) brain MRI scans for detecting AD and brain metastases/various diseases, respectively. Our self-attention MADGAN can detect AD on T1 scans at a very early stage, mild cognitive impairment (MCI), with area under the curve (AUC) 0.727, and AD at a late stage with AUC 0.894, while detecting brain metastases on T1c scans with AUC 0.921. CONCLUSIONS: Similar to physicians' way of performing a diagnosis, using massive healthy training data, our first multiple MRI slice reconstruction approach, MADGAN, can reliably predict the next 3 slices from the previous 3 ones only for unseen healthy images. As the first unsupervised various disease diagnosis, MADGAN can reliably detect the accumulation of subtle anatomical anomalies and hyper-intense enhancing lesions, such as (especially late-stage) AD and brain metastases on multi-sequence MRI scans.

Predisposition to prolonged neutropenia after chemotherapy for paediatric acute myeloid leukaemia is associated with better prognosis in the Japanese Paediatric Leukaemia/Lymphoma Study Group AML-05 study.

The variability in myelosuppression after chemotherapy for acute myeloid leukaemia (AML) can affect its prognosis; however, the underlying mechanism remains controversial. In the Japanese Paediatric Leukaemia/Lymphoma Study Group AML-05 study, we showed that prolonged neutropenia was associated with high overall survival (P = 0·011) and low frequency of relapse (P = 0·042) in patients without granulocyte-colony stimulating factor (G-CSF) who completed the indicated treatment protocol. Our data indicate that predisposition to prolonged neutropenia after chemotherapy is correlated with a better outcome of AML treatment. Our results promote the usage of individualised drug dosing strategies to improve the therapeutic outcome in AML patients.

Alcohol dependence severity determines the course of treatment-seeking patients.

BACKGROUND: While accumulating evidence suggests a relation between the severity of alcohol dependence and the risk of its recurrence, the impact of dependence severity on the course of the disorder has not been carefully evaluated. The present study examined the impact of several severity indices of alcohol dependence on the drinking course after inpatient treatment. METHODS: This prospective study was conducted over a 12-month period following alcohol treatment at a specialized hospital. A total of 712 consecutively admitted alcohol-dependent patients were targeted for enrollment at the time of their hospitalization, with 637 patients registered and followed. The characteristics and severity of the subjects were assessed using multiple methods at admission, with their course after discharge followed continuously using mailed questionnaires that queried them regarding their drinking behavior. RESULTS: Greater severity of dependence, assessed using the number of ICD-10 diagnostic criteria met, was associated with a lower rate of abstinence during the study period (p = 0.035). The rate of abstinence also decreased significantly as the baseline blood gamma-glutamyl transferase value and Alcohol Dependence Scale (ADS) score increased (p = 0.031 and p = 0.0002, respectively). In multivariate Cox proportional hazards analyses, the group with the most severe ADS scores had a significantly greater risk of relapse to drinking than the group with the least severe scores (HR = 2.67, p = 0.001). Dependence severity also associated with the drinking pattern; participants in both the controlled drinking group and the abstinence group had lower ADS scores at admission and a later age at first drinking (p = 0.001 and p < 0.001, respectively) than those with poorer drinking outcomes. CONCLUSIONS: The present study showed that more severe alcohol dependence predicts a poorer course after alcohol treatment, as reflected by findings on multiple measures. These results suggest that assessing the dependence severity at the outset of treatment could be useful both in predicting treatment outcome and targeting interventions to alcohol-dependent individuals who need additional support in their recovery.

The outcomes of relapsed acute myeloid leukemia in children: Results from the Japanese Pediatric Leukemia/Lymphoma Study Group AML-05R study.

BACKGROUND: The prognosis of children with acute myeloid leukemia (AML) has improved with the efficacy of hematopoietic cell transplantation (HCT) as a second-line therapy and improvements in supportive care following anthracycline- and cytarabine-based chemotherapy; however, the outcomes of children with relapsed AML still remain unsatisfactory. PROCEDURE: In order to identify prognostic factors and improve their prognosis, we analyzed 111 patients who relapsed after treatment with the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 protocol and who were registered in the retrospective JPLSG AML-05R study. RESULTS: The 5-year overall survival rate was 36.1%. The major determinant of survival was duration from the diagnosis to relapse. The mean duration in the nonsurviving group (10.1 ± 4.1 months) was shorter than that in the surviving group (16.3 ± 8.3 months) (P < .01). Moreover, achieving a second complete remission (CR2) prior to HCT was associated with a good prognosis (P < .01). Etoposide, cytarabine, and mitoxantrone (ECM)- or fludarabine, cytarabine, and granulocyte colony-stimulating factor (FLAG)-based regimens were therefore recommended for reinduction therapy (P < .01). A genetic analysis also revealed the prognostic significance of FMS-like tyrosine kinase 3 (FLT3)-internal tandem duplication as a poor prognostic marker (P = .04) and core binding factor-AML, t(8;21), and inv(16) as good prognostic markers (P < .01). CONCLUSIONS: Achieving a CR2 prior to HCT is important in order to improve the prognosis of relapsed pediatric AML. Recent molecular targeted therapies, such as FLT3 inhibitors, may contribute to overcome their prognoses. Larger prospective investigations are necessary to establish individualized treatment strategies for patients with relapsed childhood AML.

Sarcopenia and obesity in long-term survivors of childhood leukemia/lymphoma: a report from a single institution.

BACKGROUND: The incidence and background factors of sarcopenia and obesity in long-term survivors of childhood leukemia/lymphoma were not clear in Japan. METHODS: Between August 2018 and September 2019, we recruited adults aged ≥18 years who had childhood leukemia/lymphoma. Blood sampling, body composition measurement by bioelectrical impedance analysis and grip strength test were performed. RESULTS: Among 81 adult survivors (34 men and 47 women) with a median age of 25.0 years, 9 (11%) had sarcopenia and 10 (12%) had obesity, of whom, 3 had metabolic syndrome. Sarcopenia was observed in 7 (21%) of 33 survivors with hematopoietic stem cell transplantation (HSCT) and 2 (4%) of 48 survivors without hematopoietic stem cell transplantation (P = 0.012). The incidence of obesity was significantly higher in the cranial radiotherapy (P = 0.021) and non-transplanted cases (P = 0.042). Univariate logistic regression analysis revealed that hematopoietic stem cell transplantation for sarcopenia (odds ratio, 6.19; 95% confidence interval, 1.2-32.0; P = 0.03) and cranial radiotherapy for obesity (odds ratio, 5.6; 95% confidence interval, 1.4-22.4; P = 0.015) were significantly associated. Hypertension was more prevalent among the obese survivors, and higher transaminase levels were found more in both the sarcopenia and obese survivors than in others. CONCLUSIONS: Young adult survivors of childhood leukemia/lymphoma could be at risk of developing sarcopenia after hematopoietic stem cell transplantation and obesity after cranial radiotherapy. Further studies are required to assess the body composition of long-term survivors to find detailed risk factors of sarcopenia and metabolic syndrome.

Role of serum amylase and salivary cytokines in oral complications during chemoradiotherapy.

OBJECTIVES: To investigate whether serum amylase can predict the recovery of salivary volume and determine the correlation of the level of cytokines, including epidermal growth factor, hepatocyte growth factor and keratinocyte growth factor, with oral mucositis during chemoradiotherapy for oral cancer. SUBJECTS AND METHODS: This study included 84 patients treated with preoperative chemoradiotherapy followed by curative surgery, following a phase II study protocol. We measured and analysed the correlation of the stimulated saliva volume, serum amylase and cytokines in resting saliva at baseline and 1 month after chemoradiotherapy with oral mucositis levels. RESULTS: We observed a negative correlation between the serum amylase level at the beginning of chemoradiotherapy and the stimulated saliva volume at 1 month after chemoradiotherapy (p = .03). Epidermal growth factor in resting saliva was significantly reduced after chemoradiotherapy (p < .01). The incidence of severe oral mucositis during chemoradiotherapy was significantly higher and negatively associated with the epidermal growth factor and keratinocyte growth factor levels (p = .04, p = .05). CONCLUSIONS: The serum amylase level at the beginning of chemoradiotherapy may be a predictor of the recovery of the saliva volume. Furthermore, cytokines such as epidermal growth factor and keratinocyte growth factor in resting saliva affect the development of oral mucositis during chemoradiotherapy.