Polyphosphate-kinase-1 dependent polyphosphate hyperaccumulation for acclimation to nutrient loss in the cyanobacterium, Synechocystis sp. PCC 6803.

Polyphosphate is prevalent in living organisms. To obtain insights into polyphosphate synthesis and its physiological significance in cyanobacteria, we characterize sll0290, a homolog of the polyphosphate-kinase-1 gene, in the freshwater cyanobacterium Synechocystis sp. PCC 6803. The Sll0290 protein structure reveals characteristics of Ppk1. A Synechocystis sll0290 disruptant and sll0290-overexpressing Escherichia coli transformant demonstrated loss and gain of polyphosphate synthesis ability, respectively. Accordingly, sll0290 is identified as ppk1. The disruptant (Δppk1) grows normally with aeration of ordinary air (0.04% CO2), consistent with its photosynthesis comparable to the wild type level, which contrasts with a previously reported high-CO2 (5%) requirement for Δppk1 in an alkaline hot spring cyanobacterium, Synechococcus OS-B'. Synechocystis Δppk1 is defective in polyphosphate hyperaccumulation and survival competence at the stationary phase, and also under sulfur-starvation conditions, implying that sulfur limitation is one of the triggers to induce polyphosphate hyperaccumulation in stationary cells. Furthermore, Δppk1 is defective in the enhancement of total phosphorus contents under sulfur-starvation conditions, a phenomenon that is only partially explained by polyphosphate hyperaccumulation. This study therefore demonstrates that in Synechocystis, ppk1 is not essential for low-CO2 acclimation but plays a crucial role in dynamic P-metabolic regulation, including polyP hyperaccumulation, to maintain physiological fitness under sulfur-starvation conditions.

A Mature Tertiary Lymphoid Structure with a Ki-67-Positive Proliferating Germinal Center Is Associated with a Good Prognosis and High Intratumoral Immune Cell Infiltration in Advanced Colorectal Cancer.

Tertiary lymphoid structures (TLSs) are complex lymphocyte clusters that arise in non-lymphoid tissues due to inflammation or cancer. A mature TLS with proliferating germinal centers is associated with a favorable prognosis in various cancers. However, the effect of TLS maturity on advanced colorectal cancer (CRC) remains unexplored. We analyzed the significance of TLS maturity and tumor Ki-67 expression in surgically resected tumors from 78 patients with pathological T4 CRC. Mature TLS was defined as the organized infiltration of T and B cells with Ki-67-positive proliferating germinal centers. We analyzed the relationship between TLS maturity and intratumoral immune cell infiltration. Mature TLS with germinal center Ki-67 expression was associated with microsatellite instability and improved survival; however, high tumor Ki-67 expression was associated with poor survival in the same cohort. Multivariate analysis identified the absence of mature TLS as an independent predictor of poor post-recurrence overall survival. Intratumoral infiltration of T lymphocytes and macrophages was significantly elevated in tumors with mature TLS compared to those lacking it. High Ki-67 levels and absent mature TLS were identified as poor prognostic factors in advanced CRC. Mature TLS could serve as a promising marker for patients at high-risk of CRC.

The Gustave Roussy Immune score is a powerful biomarker for predicting therapeutic resistance to chemotherapy in gastric cancer patients.

PURPOSE: It is highly important to be able to predict the therapeutic efficacy of chemotherapy on patients with unresectable advanced or recurrent gastric cancer (GC). The Gustave Roussy Immune Score (GRIm-s) is a predictor of therapeutic sensitivity to chemotherapy and immune checkpoint inhibitors (ICIs) in other cancers. The present study aimed to analyze the association of the GRIm-s with the therapeutic sensitivity of first-line chemotherapy in GC patients. METHODS: We included 156 patients receiving primary chemotherapy treatment for unresectable or advanced recurrent GC between January 2012 and December 2021 at our institution. We evaluated the correlation between the GRIm-s and therapeutic sensitivities to chemotherapy. The GRIm-s was assessed before the start of first-line chemotherapy. RESULTS: Among the 156 patients, 138 (88.5%) and 18 (11.5%) were classified in the low- and high-risk groups, respectively. The GRIm-s high-risk group was significantly older (p = 0.013), had more advanced unresectable cancer (p = 0.0098), and was significantly less likely to progress to second-line chemotherapy (p = 0.014). The overall survival rate (OS) (p = 0.039) and the progression free survival rate (PFS) (p = 0.017) were significantly worse in the GRIm-s high-risk group. The high GRIm-s was an independent prognostic factor for poor survival in multivariate analysis (p = 0.0094). CONCLUSIONS: Focusing on the GRIm-s before first-line chemotherapy initiation for unresectable advanced or postoperative recurrent GC was useful in predicting the therapeutic resistance to chemotherapy, transition to second-line chemotherapy, and poor prognosis.

Risks and Outcomes of Early Dose Reduction in Adjuvant CAPOX Therapy for Colorectal Cancer.

BACKGROUND/AIM: Adjuvant capecitabine and oxaliplatin (CAPOX) therapy is standard strategy for colorectal cancer with risk of recurrence. Early dose reduction (EDR) of CAPOX therapy is commonly used in real-world practice. However, there is limited evidence regarding the effectiveness of CAPOX for patients who had EDR. Therefore, this study aimed to clarify the risks of EDR and its effect on long-term outcomes and body composition factors. PATIENTS AND METHODS: Patients who received CAPOX therapy after radical surgery for colorectal cancer between June 2013 and December 2021 were included. EDR was defined as dose reduction within four courses of CAPOX therapy. Body composition factors were measured for 1 year following surgery to determine the EDR effects. RESULTS: Eighty-four patients were included; 35 (42%) of them had EDR. The multivariate analysis revealed that underweight [odds ratio (OR)=4.95, 95% confidence interval (CI)=1.13-21.7, p=0.03] was a risk factor for EDR. Relapse-free survival (RFS) was significantly better in the non-EDR group (p=0.01). The 5-year RFS rates for the non-EDR and EDR groups were 88.7% and 65.4%, respectively. The multivariate analysis revealed that age >65 years [hazard ratio (HR)=3.97; 95% CI=1.16-13.62, p=0.03] and EDR (HR=7.62; 95% CI=1.71-33.91, p=0.005) were associated with poorer RFS. The 1-year body composition analysis revealed decreases in all factors in the EDR group. CONCLUSION: Preoperative underweight status was associated with EDR, which resulted in decreased RFS and body composition factors when compared with the non-EDR group. Therefore, avoiding EDR and early nutritional intervention after EDR may improve outcomes.

A case of hepatosplenic cat scratch disease with hemophagocytic lymphohistiocytosis.

Cat scratch disease (CSD) is associated with Bartonella henselae (B. henselae) infection caused by cat scratches or bites. It typically presents with lymphadenitis and fever. However, there are atypical cases such as hepatosplenic CSD, which presents with specific lesions in the liver and spleen. Hemophagocytic lymphohistiocytosis (HLH) is a rare and severe multisystem disorder triggered by infections, cancers, or autoimmune diseases. We experienced a rare case of hepatosplenic CSD with HLH in a non-immunocompromised adult. A 78-year-old woman complained of fever and fatigue. Laboratory tests revealed anemia and liver dysfunction; abdominal contrast-enhanced computed tomography (CT) revealed splenomegaly and nodular hypodense areas in the spleen. In addition, the levels of ferritin and serum soluble IL-2R were markedly elevated, so clinical diagnosis of HLH was made. Positron emission tomography/CT revealed diffuse fluorodeoxyglucose uptake in the liver and spleen suggesting malignant lymphoma, while the pathological findings from liver biopsy suggested infectious diseases. Although she had no cat bites and scratches, she had many cats; therefore, serum B. henselae antibody titers were measured. The B. henselae IgG and IgM titer were 1:128 and 1:20; thus, she was diagnosed with hepatosplenic CSD. Patients with hepatosplenic nodular lesions and contact with cats should be considered for this disease.

Tocilizumab-induced mucosal injury in the terminal ileum mimicking intestinal Behçet's disease: A case report.

RATIONALE: Tocilizumab, a humanized anti-interleukin-6 (IL-6) receptor monoclonal antibody, is used for the treatment of adult-onset Still disease (AOSD). Despite its efficacy in many clinical situations, concerns have been raised regarding intestinal mucosal injury in patients receiving tocilizumab. PATIENT CONCERNS: A 64-year-old woman with a history of AOSD was admitted to our hospital with hematochezia. She had AOSD for 15 years and underwent treatment with biweekly tocilizumab 9 months prior to admission. Colonoscopy revealed a large punched-out ulcer in the terminal ileum. On pathological evaluation, nonspecific enteritis with lymphocytes and eosinophils were seen. Based on the location and shape of the lesion, we suspected intestinal Behçet's disease. However, the ulcer reduced in size over time by discontinuation of tocilizumab without additional drug treatment, indicating that it was a drug-induced ulcer. DIAGNOSIS: The patient was diagnosed with tocilizumab-induced small intestinal ulcer. INTERVENTIONS: The patient treated with the discontinuation of tocilizumab. OUTCOMES: The discontinuation of tocilizumab resulted in ulcer scarring. There was no recurrence of hematochezia. LESSONS: Tocilizumab can cause deep ulcerative lesions in the terminal ileum, which may resemble intestinal Behçet's disease. It is important to continuously monitor abdominal symptoms during tocilizumab therapy and aggressively perform colonoscopy when hematochezia or abdominal pain is observed.

Optogenetic decoding of Akt2-regulated metabolic signaling pathways in skeletal muscle cells using transomics analysis.

Insulin regulates various cellular metabolic processes by activating specific isoforms of the Akt family of kinases. Here, we elucidated metabolic pathways that are regulated in an Akt2-dependent manner. We constructed a transomics network by quantifying phosphorylated Akt substrates, metabolites, and transcripts in C2C12 skeletal muscle cells with acute, optogenetically induced activation of Akt2. We found that Akt2-specific activation predominantly affected Akt substrate phosphorylation and metabolite regulation rather than transcript regulation. The transomics network revealed that Akt2 regulated the lower glycolysis pathway and nucleotide metabolism and cooperated with Akt2-independent signaling to promote the rate-limiting steps in these processes, such as the first step of glycolysis, glucose uptake, and the activation of the pyrimidine metabolic enzyme CAD. Together, our findings reveal the mechanism of Akt2-dependent metabolic pathway regulation, paving the way for Akt2-targeting therapeutics in diabetes and metabolic disorders.

Acute Cholecystitis Associated with Eosinophilic Granulomatosis with Polyangiitis That Responded to Glucocorticoid Therapy.

Eosinophilic granulomatosis with polyangiitis (EGPA) is a systemic disorder characterized by tissue eosinophilic infiltration and vasculitis. Although EGPA causes multiple organ damage, it causes cholecystitis less frequently. We herein report a case of acute cholecystitis associated with EGPA in which successful treatment with glucocorticoid therapy allowed surgery to be avoided. EGPA can present as acute cholecystitis. It is important not to overlook acute cholecystitis associated with EGPA in patients with abdominal pain with peripheral eosinophilia. Furthermore, in cases of mild cholecystitis associated with EGPA that are diagnosed preoperatively, cholecystectomy might be avoided with conservative glucocorticoid treatment.

Systematic Interrogation of the Temperature Perturbation in the Insulin Signaling Pathway for Optogenetic Stimulation.

The application of NIR to optogenetic systems is in great demand due to its superior properties enabling in vivo deep tissue penetration. Irradiation of NIR to tissue samples or cells rapidly generates heat locally. The resultant elevation in temperature affects cells at the molecular level because of the activation of the heat shock pathway and ROS generation. Nevertheless, few reports have presented detailed comparisons of the effects of the temperature change rate on signaling pathway biomolecules, especially those of rapid heat changes. Aiming at broadening the understanding of temperature sensitivity, we investigated seven insulin signaling pathway biomolecules (INSR, IRS1, Akt, GSK3ß, p70S6K, FoxO1, and ERK1/2) in three cell lines (C2C12, HepG2, and Fao) at temperatures between 25 and 45 °C. The results show that, except for INSR, pAkt(T308), and FoxO1, biomolecules are sensitive to rapid temperature changes at temperatures higher than 42 °C, at which they are significantly phosphorylated. At 25 °C, around a 50% reduction in phosphorylation occurred. Moreover, p70S6K is sensitive over time. It dephosphorylates quickly (5 min) and then phosphorylates over time. Our findings extend the temperature range to 45 °C, while providing additional time course information about the signaling pathway biomolecule response necessary to advance NIR optogenetic research.

LI-RADS Classification and Outcomes of Hepatocellular Carcinoma Treated With Transcatheter Arterial Chemoembolization Plus Radiofrequency Ablation.

AIM: The aim of this study was to clarify the usefulness of the Liver Imaging Reporting and Data System (LI-RADS) for predicting a patient's prognosis after transcatheter arterial chemoembolization (TACE) combined with radiofrequency ablation (TACE-RFA) for hepatocellular carcinoma (HCC) of Barcelona-Clinic Liver Cancer (BCLC) stage 0 or A. PATIENTS AND METHODS: We retrospectively analyzed cases of patients with HCC who underwent TACE-RFA (Jan 2005 to Dec 2015). Nodules were categorized based on their LI-RADS v2018 core. The LI-RADS category was assigned to each nodule using dynamic contrast-enhanced computed tomography. LR-3, LR-4 and LR-5 nodules were extracted. The overall (OS) and recurrence-free (RFS) survival was assessed among patients with BCLC 0 and BCLC A disease. RESULTS: Of the 64 nodules extracted, 22 were LR-3 or -4 (mean±standard deviation=14.8±6.7 mm) and 42 were LR-5 (17.1±6.9 mm). Regarding OS, there was no significant difference between those with LR-3 or -4 and LR-5 (p=0.278). In terms of RFS, there was a significant difference between those with LR-3 or -4 and those with LR-5 (p=0.03). In particular, patients with BCLC A with LR-5 nodules had significantly poorer RFS than those with LR-3 or -4 (p=0.016) nodules. CONCLUSION: For patients with BCLC A, LR-3 or -4 nodules are associated with a better prognosis than LR-5 nodules.

Maximum Plasma Concentration of Lenvatinib Is Useful for Predicting Thrombocytopenia in Patients Treated for Hepatocellular Carcinoma.

BACKGROUND: Although lenvatinib treatment has a favorable efficacy for unresectable hepatocellular carcinoma (HCC), it is associated with adverse events (AEs) that must be closely monitored and managed. Thrombocytopenia is one of the major AEs. The aim of this study was to clarify whether thrombocytopenia can be predicted by the plasma concentration of lenvatinib. METHODS: This was a single-center retrospective observational study. Twenty-three patients with unresectable HCC and pharmacokinetics data at the initial lenvatinib administration between May 2018 and September 2020 at Oita University Hospital were enrolled. The AEs during the 4 weeks after the initiation of treatment were evaluated, and the correlations between the thrombocytopenia and the plasma concentration of lenvatinib were examined. Spearman's correlation was used to evaluate the correlation between two continuous variables. RESULTS: The rate of platelet count decrease correlated with the maximum plasma concentration (Cmax) (r = 0.65, P = 0.001), whereas it did not with the minimum plasma concentration (Cmin) (r = 0.29, P = 0.206). After stepwise multiple linear regression analysis, the starting dose of lenvatinib and the serum albumin concentration were identified as independent explanatory variables. Next, a formula for predicting the Cmax using these two variables was created. The predicted Cmax was strongly correlated with the Cmax (r = 0.87, P < 0.0001) and the rate of platelet count decrease (r = 0.67, P = 0.001). CONCLUSIONS: This study identified the usefulness of the drug Cmax to predict the rate of platelet count decrease within 4 weeks after the initiation of treatment. Although it is difficult to measure the plasma concentration of lenvatinib in community hospitals, the predicted Cmax is useful for predicting the rate of platelet count decrease with this treatment.

A Broad Range High-Throughput Assay for Lenvatinib Using Ultra-High Performance Liquid Chromatography Coupled to Tandem Mass Spectrometry With Clinical Application in Patients With Hepatocellular Carcinoma.

BACKGROUND: Lenvatinib is increasingly being selected as the first-line treatment for unresectable hepatocellular carcinoma (HCC) based on the results of the REFLECT trial. However, early discontinuation of lenvatinib because of adverse effects is a frequent occurrence. Hence, lenvatinib is a difficult drug for use in the clinical setting. One of the causes is that the dose of lenvatinib is mainly determined by body weight alone, despite high interindividual variability. To overcome this problem, a dosing regimen of lenvatinib based on a population pharmacokinetic (PPK) model for HCC patients is proposed. The aim of this study was to develop a high-throughput quantification method for lenvatinib using ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) that can be applied to a PPK analysis of HCC patients in the future. METHODS: After a simple solid-phase extraction step using a 96-well plate, lenvatinib was analyzed by UHPLC-MS/MS in a positive electrospray ionization mode. RESULTS: The novel method fulfilled the requirements of the US Food and Drug Administration guidance on bioanalytical method validation. The calibration curve was linear over the 0.2-1000 ng/mL concentration range. The average recovery rate was 98.63 ± 4.55% (mean ± SD). The precision was below 6.05%, and the accuracy was within 12.96% for all quality control levels. The matrix effect varied between 103.33% and 134.61%. This assay was successfully applied to the measurement of plasma concentrations in 6 HCC patients receiving lenvatinib. CONCLUSIONS: A novel high-throughput UHPLC-MS/MS assay for quantification of lenvatinib in human plasma was successfully developed. This method can be applied to PPK analysis for patients receiving lenvatinib in the clinical setting.

Photocleavable Cadherin Inhibits Cell-to-Cell Mechanotransduction by Light.

Precise integration of individual cell behaviors is indispensable for collective tissue morphogenesis and maintenance of tissue integrity. Organized multicellular behavior is achieved via mechanical coupling of individual cellular contractility, mediated by cell adhesion molecules at the cell-cell interface. Conventionally, gene depletion or laser microsurgery has been used for functional analysis of intercellular mechanotransduction. Nevertheless, these methods are insufficient to investigate either the spatiotemporal dynamics or the biomolecular contribution in cell-cell mechanical coupling within collective multicellular behaviors. Herein, we present our effort in adaption of PhoCl for attenuation of cell-to-cell tension transmission mediated by E-cadherin. To release intercellular contractile tension applied on E-cadherin molecules with external light, a genetically encoded photocleavable module called PhoCl was inserted into the intracellular domain of E-cadherin, thereby creating photocleavable cadherin (PC-cadherin). In response to light illumination, the PC-cadherin cleaved into two fragments inside cells, resulting in attenuating mechanotransduction at intercellular junctions in living epithelial cells. Light-induced perturbation of the intercellular tension balance with surrounding cells changed the cell shape in an epithelial cell sheet. The method is expected to enable optical manipulation of force-mediated cell-to-cell communications in various multicellular behaviors, which contributes to a deeper understanding of embryogenesis and oncogenesis.

Risk factors for deterioration of long-term liver function after radiofrequency ablation therapy.

AIM: To identify factors that influence long-term liver function following radiofrequency ablation (RFA) in patients with viral hepatitis-related hepatocellular carcinoma. METHODS: A total of 123 patients with hepatitis B virus- or hepatitis C virus-related hepatocellular car-cinoma (HCC) (n = 12 and n = 111, respectively) were enrolled. Cumulative rates of worsening Child-Pugh (CP) scores (defined as a 2-point increase) were examined. RESULTS: CP score worsening was confirmed in 22 patients over a mean follow-up period of 43.8 ± 26.3 mo. Multivariate analysis identified CP class, platelet count, and aspartate aminotransferase levels as signi-ficant predictors of a worsening CP score (P = 0.000, P = 0.011 and P = 0.024, respectively). In contrast, repeated RFA was not identified as a risk factor for liver function deterioration. CONCLUSION: Long-term liver function following RFA was dependent on liver functional reserve, the degree of fibrosis present, and the activity of the hepatitis condition for this cohort. Therefore, in order to maintain liver function for an extended period following RFA, suppression of viral hepatitis activity is important even after the treatment of HCC.

TNF-alpha induces hepatic steatosis in mice by enhancing gene expression of sterol regulatory element binding protein-1c (SREBP-1c).

We investigated the effect of tumor necrosis factor-alpha (TNF-alpha), a member of the proinflammatory cytokine family, on steatosis of the mouse liver by analyzing morphological changes and hepatic triglyceride content in response to TNF-alpha. We also examined expression of the sterol regulatory element binding protein-1c gene. Intraperitoneal injection of TNF-alpha acutely and dramatically accelerated the accumulation of fat in the liver, as evidenced by histological analysis and hepatic triglyceride content. This treatment increased liver weight, increased serum levels of free fatty acids, and increased fatty acid synthase and sterol regulatory element binding protein-1c mRNA expression. Furthermore, intraperitoneal injection of lipopolysaccaride (LPS) to induce TNF-alpha expression also accelerated hepatic fat accumulation. Pretreatment with anti-TNF-alpha antibody attenuated the development of LPS-induced fatty change in the liver. Antibody pretreatment not only decreased sterol regulatory element binding protein-1c expression in LPS-treated mice but also attenuated the expression of suppressors of cytokine signaling-3 mRNA. This study suggests that TNF-alpha, acting downstream of LPS, increases intrahepatic fat deposition by affecting hepatic lipogenetic metabolism involving sterol regulatory element binding protein-1c.

Involvement of stomach ghrelin and hypothalamic neuropeptides in tumor necrosis factor-alpha-induced hypophagia in mice.

This study aimed to clarify the interaction of tumor necrosis factor-alpha (TNF-alpha), an anorexigenic cytokine, with ghrelin, an orexigenic peptide secreted by the stomach lining, and hypothalamic neuropeptides in the regulation of food intake in mice. The peripheral administration of TNF-alpha dose-dependently decreased the 24-h cumulative food intake compared with the administration of saline. Reduced food intake was observed at 6 h and 24 h. The same TNF-alpha treatment significantly decreased the plasma level of ghrelin at 6 h and 24 h after treatment compared with the control levels. These changes were accompanied by a significant reduction in the expression of ghrelin mRNA in the stomach at 24 h after treatment. TNF-alpha treatment also resulted in a significant increase in expression of pro-opiomelanocortin (POMC) mRNA and a significant decrease in expression of agouti-related protein (AGRP) mRNA in the hypothalamus at 6 h after treatment. Finally, the pre-administration of ghrelin, reversed the TNF-alpha-induced hypophagia in mice at 6 and 24 h. Taken together, these findings suggest that hypothalamic POMC and AGRP and stomach ghrelin may be involved in TNF-alpha-induced hypophagia in mice.

The role of histamine H1 receptor and H2 receptor in LPS-induced liver injury.

To examine the role of histamine H1 and H2 receptors in the regulation of lipopolysaccharide (LPS)-induced liver injury, a combination of D-galactosamine and LPS (GalN/LPS) was administered to histamine H1 receptor knockout (H1-R KO) and H2 receptor knockout (H2-R KO) mice. The numbers of necrotic and apoptotic hepatocytes in the liver, as well as the levels of serum aspartate transaminase (AST) and alanine transaminase (ALT), were increased significantly by GalN/LPS treatment compared to the appropriate controls. Pretreatment with histamine ameliorated the GalN/LPS-induced necrotic and apoptotic changes in the hepatocytes and inhibited the elevation of serum AST and ALT levels. Histamine attenuated the GalN/LPS-induced increases in the levels of TNF-alpha, but augmented those of IL-10 both in the liver and serum. Histamine inhibited the GalN/LPS-induced caspase-3 activity in the liver. Furthermore, these effects of histamine were completely or partially attenuated in H2-R KO mice, but not in H1-R KO mice. Peritoneal macrophages from H2-R KO mice exhibited blunted changes in the effects of histamine on LPS-induced TNF-alpha and IL-10 production in vitro compared to the wild-type (WT) controls. In summary, the present findings suggest that the histamine H2-R-TNF-alpha and -IL-10 pathways play protective roles in endotoxin-induced hepatic injury.