Dissection of the signal transduction machinery responsible for the lysyl oxidase-like 4-mediated increase in invasive motility in triple-negative breast cancer cells: mechanistic insight into the integrin-ß1-NF-κB-MMP9 axis.

Background: Triple-negative breast cancer (TNBC) cells are a highly formidable cancer to treat. Nonetheless, by continued investigation into the molecular biology underlying the complex regulation of TNBC cell activity, vulnerabilities can be exposed as potential therapeutic targets at the molecular level. We previously revealed that lysyl oxidase-like 4 (LOXL4) promotes the invasiveness of TNBC cells via cell surface annexin A2 as a novel binding substrate of LOXL4, which promotes the abundant localization of integrin-ß1 at the cancer plasma membrane. However, it has yet to be uncovered how the LOXL4-mediated abundance of integrin-ß1 hastens the invasive outgrowth of TNBC cells at the molecular level. Methods: LOXL4-overexpressing stable clones were established from MDA-MB-231 cells and subjected to molecular analyses, real-time qPCR and zymography to clarify their invasiveness, signal transduction, and matrix metalloprotease (MMP) activity, respectively. Results: Our results show that LOXL4 potently promotes the induction of matrix metalloprotease 9 (MMP9) via activation of nuclear factor-κB (NF-κB). Our molecular analysis revealed that TNF receptor-associated factor 4 (TRAF4) and TGF-ß activated kinase 1 (TAK1) were required for the activation of NF-κB through Iκß kinase kinase (IKKα/ß) phosphorylation. Conclusion: Our results demonstrate that the newly identified LOXL4-mediated axis, integrin-ß1-TRAF4-TAK1-IKKα/ß-Iκßα-NF-κB-MMP9, is crucial for TNBC cell invasiveness.

Lysyl oxidase-like 4 promotes the invasiveness of triple-negative breast cancer cells by orchestrating the invasive machinery formed by annexin A2 and S100A11 on the cell surface.

Background: Our earlier research revealed that the secreted lysyl oxidase-like 4 (LOXL4) that is highly elevated in triple-negative breast cancer (TNBC) acts as a catalyst to lock annexin A2 on the cell membrane surface, which accelerates invasive outgrowth of the cancer through the binding of integrin-ß1 on the cell surface. However, whether this machinery is subject to the LOXL4-mediated intrusive regulation remains uncertain. Methods: Cell invasion was assessed using a transwell-based assay, protein-protein interactions by an immunoprecipitation-Western blotting technique and immunocytochemistry, and plasmin activity in the cell membrane by gelatin zymography. Results: We revealed that cell surface annexin A2 acts as a receptor of plasminogen via interaction with S100A10, a key cell surface annexin A2-binding factor, and S100A11. We found that the cell surface annexin A2/S100A11 complex leads to mature active plasmin from bound plasminogen, which actively stimulates gelatin digestion, followed by increased invasion. Conclusion: We have refined our understanding of the role of LOXL4 in TNBC cell invasion: namely, LOXL4 mediates the upregulation of annexin A2 at the cell surface, the upregulated annexin 2 binds S100A11 and S100A10, and the resulting annexin A2/S100A11 complex acts as a receptor of plasminogen, readily converting it into active-form plasmin and thereby enhancing invasion.

Successful treatment of streptococcal toxic shock syndrome complicated by primary peritonitis and bilateral empyema in a healthy young woman: Identification of uncommon clone emm103 and novel sequence type 1363.

Streptococcal toxic shock syndrome (STSS) has a dramatic clinical course and high mortality rate. Here, we report a case of STSS complicated by primary peritonitis and bilateral empyema. A previously healthy young woman was diagnosed with STSS complicated by primary peritonitis and bilateral empyema. Blood culture results on admission were negative. Sever shock, respiratory failure, systemic inflammation, thrombocytopenia, renal failure, ascites, and pleural effusion occurred, mimicking thrombocytopenia, anasarca, fever, reticulin fibrosis/renal failure and organomegaly (TAFRO) syndrome. Retesting blood cultures identified Streptococcus pyogenes. Gram staining of ascites and pleural fluid indicated gram-positive cocci in chains. Antibiotics, immunoglobulins, and surgical intervention led to recovery without complications. Ex-post genotypic analyses showed uncommon emm103.0 (cluster E3) of emm long sequence (784 base) and novel sequence type 1363. STSS diagnosis can be difficult as it mimics other systemic inflammatory diseases. Therefore, it is crucial for clinicians to perform microbiological examinations from infection foci, even if the initial culture is negative.

Identification of three distinct cell populations for urate excretion in human kidneys.

In humans, uric acid is an end-product of purine metabolism. Urate excretion from the human kidney is tightly regulated by reabsorption and secretion. At least eleven genes have been identified as human renal urate transporters. However, it remains unclear whether all renal tubular cells express the same set of urate transporters. Here, we show renal tubular cells are divided into three distinct cell populations for urate handling. Analysis of healthy human kidneys at single-cell resolution revealed that not all tubular cells expressed the same set of urate transporters. Only 32% of tubular cells were related to both reabsorption and secretion, while the remaining tubular cells were related to either reabsorption or secretion at 5% and 63%, respectively. These results provide physiological insight into the molecular function of the transporters and renal urate handling on single-cell units. Our findings suggest that three different cell populations cooperate to regulate urate excretion from the human kidney, and our proposed framework is a step forward in broadening the view from the molecular to the cellular level of transport capacity.

Metabolic Dysfunction-Associated Fatty Liver Disease on Distinct Microbial Communities at the Bacterial Phylum Level.

INTRODUCTION: Limited data are available on the correlation between microbial communities and metabolic dysfunction-associated fatty liver disease (MAFLD). This study aimed to evaluate the influence of MAFLD on diverse microbial communities. METHODS: We recruited 43 patients with a nonviral liver disease. Enrolled patients were divided into two groups according to MAFLD criteria. The fecal microbial composition was evaluated using the variable V3-V4 region of the 16S ribosomal RNA region, which was amplified using polymerase chain reaction. First, we assessed the influence of MAFLD on distinct microbial communities at the bacterial phylum level. Next, the correlation between the microbial communities and diversity in patients with MAFLD was evaluated. RESULTS: Among the enrolled participants, the non-MAFLD and MAFLD groups consisted of 21 and 22 patients, respectively. Sequences were distributed among ten bacterial phyla. The relative abundance of Firmicutes was significantly higher in the MAFLD group than in the non-MAFLD group (p = 0.014). The microbial diversity was not significantly influenced by the presence of MAFLD (Chao-1 index: p = 0.215 and Shannon index: p = 0.174, respectively); nonetheless, the correlation coefficient between the abundances of Firmicutes and microbial diversity was higher in the non-MAFLD group than in the MAFLD group. CONCLUSION: The presence of MAFLD increased the relative abundances of Firmicutes at the bacterial phylum level, which may cause the discrepancy between the abundances of Firmicutes and diversity in patients with MAFLD.

Cellular Uptake and Cytotoxicity of Clostridium perfringens Iota-Toxin.

Clostridium perfringens iota-toxin is composed of two separate proteins: a binding protein (Ib) that recognizes a host cell receptor and promotes the cellular uptake of a catalytic protein and (Ia) possessing ADP-ribosyltransferase activity that induces actin cytoskeleton disorganization. Ib exhibits the overall structure of bacterial pore-forming toxins (PFTs). Lipolysis-stimulated lipoprotein receptor (LSR) is defined as a host cell receptor for Ib. The binding of Ib to LSR causes an oligomer formation of Ib in lipid rafts of plasma membranes, mediating the entry of Ia into the cytoplasm. Ia induces actin cytoskeleton disruption via the ADP-ribosylation of G-actin and causes cell rounding and death. The binding protein alone disrupts the cell membrane and induces cytotoxicity in sensitive cells. Host cells permeabilized by the pore formation of Ib are repaired by a Ca2+-dependent plasma repair pathway. This review shows that the cellular uptake of iota-toxin utilizes a pathway of plasma membrane repair and that Ib alone induces cytotoxicity.

Impact of body composition for patients with hepatocellular carcinoma who received atezolizumab plus bevacizumab therapy.

OBJECTIVE: To investigate the association between body composition and prognosis in patients with advanced hepatocellular carcinoma treated with atezolizumab plus bevacizumab. METHODS: This cohort study analysed 119 patients who received atezolizumab plus bevacizumab for unresectable hepatocellular carcinoma. We investigated the association between body composition and progression-free survival and overall survival. Body composition was quantified by the visceral fat index, subcutaneous fat index, and skeletal muscle index. A high or low index score was defined as that above or below the median of these indices. RESULTS: Poor prognosis was observed in the low visceral fat index and low subcutaneous fat index groups. The mean progression-free survival in the low visceral fat index and low subcutaneous fat index groups vs. the other groups were 194 and 270 days, respectively [95% confidence interval (CI), 153-236 and 230-311 days, respectively; P = 0.015], while the mean overall survival was 349 vs. 422 days, respectively (95% CI, 302-396 and 387-458 days, respectively; P = 0.027). In the multivariate analysis, both a low subcutaneous fat index and low visceral fat index were statistically associated with lower progression-free and overall survival rates [hazard ratio (HR) 1.721; 95% CI, 1.101-2.688; P = 0.017; and HR 2.214; 95% CI, 1.207-4.184; P = 0.011, respectively]. CONCLUSION: Low visceral fat index and subcutaneous fat index scores were independent predictors of poor prognosis in patients with unresectable hepatocellular carcinoma treated with atezolizumab plus bevacizumab.

Impact of liver fibrosis on the relative abundance of a urease-positive Streptococcus salivarius group from saliva in patients with chronic liver disease.

AIM: We performed genomic analysis to study the relative abundance of a urease-positive Streptococcus salivarius group isolated from the saliva of patients with chronic liver disease. METHODS: Male and female patients with chronic liver disease aged over 20 years were included. First, we assessed the frequency and type of the S. salivarius group isolated from oral saliva using molecular biology techniques based on 16S rRNA and dephospho-coenzyme A kinase gene sequencing. Next, we assessed the correlation between the urease positivity rate in the S. salivarius group isolated from oral saliva and liver fibrosis based on chronic liver disease. Urease-positive strains were identified by the urease test using urea broth (Difco, Franklin Lakes, NJ, USA). Liver fibrosis was evaluated by the liver stiffness measurement value based on magnetic resonance elastography. RESULTS: A total of 45 patients identified using the multiplex polymerase chain reaction for the 16S rRNA gene were tested using the multiplex polymerase chain reaction for the dephospho-coenzyme A kinase gene. Confirming the strains detected in each of the 45 patients, urease-positive S. salivarius was detected in 28 patients (62%), urease-negative S. salivarius in 25 patients (56%), and urease-positive Streptococcus vestibularis in 12 patients (27%). There was no patient with urease-negative S. vestibularis. The urease-positive rate of the S. salivarius group in the cirrhosis and non-cirrhosis groups were 82.2% and 39.2%, respectively. The liver cirrhosis group had a higher urease positivity rate than the non-cirrhotic group (p < 0.001). CONCLUSIONS: Liver fibrosis influences the frequency of a urease-positive S. salivarius group isolated from oral saliva.

Clostridium perfringens epsilon-toxin requires acid sphingomyelinase for cellular entry.

OBJECTIVES: Clostridium perfringens epsilon-toxin is considered to be a crucial agent in enterotoxemia in domestic animals. Epsilon-toxin enters host cells via endocytosis and results in the formation of late endosome/lysosome-derived vacuoles. In the present study, we found that acid sphingomyelinase promotes the internalization of epsilon-toxin in MDCK cells. METHODS: We measured the extracellular release of acid sphingomyelinase (ASMase) by epsilon-toxin. We examined the role of ASMase in epsilon-toxin-induced cytotoxicity using selective inhibitors and knockdown of ASMase. Production of ceramide after toxin treatment was determined by immunofluorescence technique. RESULTS: Blocking agents of ASMase and exocytosis of lysosomes inhibited this epsilon-toxin-induced vacuole formation. Lysosomal ASMase was liberated to extracellular space during treatment of the cells with epsilon-toxin in the presence of Ca2+. RNAi-mediated attenuation of ASMase blocked epsilon-toxin-induced vacuolation. Moreover, incubation of MDCK cells with epsilon-toxin led to production of ceramide. The ceramide colocalized with lipid raft-binding cholera toxin subunit B (CTB) in the cell membrane, indicating that conversion of lipid raft associated sphingomyelin to ceramide by ASMase facilitates lesion of MDCK cells and internalization of epsilon-toxin. CONCLUSIONS: Based on the present results, ASMase is required for efficient internalization of epsilon-toxin.

Lysyl oxidase-like 4 exerts an atypical role in breast cancer progression that is dependent on the enzymatic activity that targets the cell-surface annexin A2.

Background: LOX family members are reported to play pivotal roles in cancer. Unlike their enzymatic activities in collagen cross-linking, their precise cancer functions are unclear. We revealed that LOXL4 is highly upregulated in breast cancer cells, and we thus sought to define an unidentified role of LOXL4 in breast cancer. Methods: We established the MDA-MB-231 sublines MDA-MB-231-LOXL4 mutCA and -LOXL4 KO, which stably overexpress mutant LOXL4 that loses its catalytic activity and genetically ablates the intrinsic LOXL4 gene, respectively. In vitro and in vivo evaluations of these cells' activities of cancer outgrowth were conducted by cell-based assays in cultures and an orthotopic xenograft model, respectively. The new target (s) of LOXL4 were explored by the MS/MS analytic approach. Results: Our in vitro results revealed that both the overexpression of mutCA and the KO of LOXL4 in cells resulted in a marked reduction of cell growth and invasion. Interestingly, the lowered cellular activities observed in the engineered cells were also reflected in the mouse model. We identified a novel binding partner of LOXL4, i.e., annexin A2. LOXL4 catalyzes cell surface annexin A2 to achieve a cross-linked multimerization of annexin A2, which in turn prevents the internalization of integrin ß-1, resulting in the locking of integrin ß-1 on the cell surface. These events enhance the promotion of cancer cell outgrowth. Conclusions: LOXL4 has a new role in breast cancer progression that occurs via an interaction with annexin A2 and integrin ß-1 on the cell surface.

Antibiotic-resistant status and pathogenic clonal complex of canine Streptococcus canis-associated deep pyoderma.

BACKGROUND: Streptococcus canis causes deep pyoderma in canines, which raises concerns about the risk of isolates from lesions acquiring an antibiotic-resistant phenotype. It is necessary to identify effective antibiotics and the characteristics of the pathogenic cluster for S. canis-associated deep pyoderma. RESULTS: The signalment, molecular typing, and antibiotic-resistant status of S. canis isolated from deep pyoderma lesions (27 strains) and oral cavities (26 strains) were analyzed. Older dogs tended to have S. canis-associated deep pyoderma (15 of 27 dogs over 10 years old). Veterinarians chose quinolones for 10/16 cases (63%), even though the rate of quinolone-resistant strains of S. canis is 38-59%. Although 70% of the strains showed resistance to three or more antibiotic classes (37/53), 94% (50/53) strains showed sensitivity for penicillins. We also identified ß-lactamase activity among penicillin-resistant strains of S. canis. Clonal complex 13 (CC13) was detected only in lesions and formed independent clusters in the phylogenetic tree. One strain of CC13 was resistant to the anti-methicillin-resistant Staphylococcus aureus drugs, vancomycin and linezolid. CONCLUSION: Although antibiotic-resistant strains of S. canis are isolated at a high rate, they can currently be treated with ß-lactamase-inhibiting penicillins. CC13 may be a pathogenic cluster with high levels of antibiotics resistance.

Evaluation of Skeletal Muscle Mass in Patients with Chronic Liver Disease Shows Different Results Based on Bioelectric Impedance Analysis and Computed Tomography.

OBJECTIVE: We aimed to evaluate the difference between computed tomography (CT)-based and bioelectrical impedance analysis (BIA)-based assessment of sarcopenia in patients with chronic liver disease (CLD). METHODS: We enrolled a total of 257 patients who were evaluated with or without sarcopenia. Sarcopenia was defined as a low skeletal muscle mass index (SMI) with low muscular strength by the Japan Society of Hepatology. To evaluate whether or not the different methods influence the diagnosis of sarcopenia for patients with CLD, we assessed the number and characteristics of mismatches between the low SMI using BIA and CT. We also compared the overall survival (OS) in patients with and without sarcopenia based on CT and BIA to evaluate the appropriate methods. RESULTS: The numbers of patients with low SMI using BIA or CT were 53 (20.6%) and 114 (44.3%) patients, respectively. Multivariate analysis revealed that hepatic ascites and body weight were independent factors of mismatch between SMI using BIA versus CT (hazard ratio [HR] 3.232, p < 0.001; HR 2.347, p = 0.005, respectively). The median OS in patients with sarcopenia based on CT was significantly lower than that in patients without sarcopenia (p = 0.006). In contrast, there was no difference between patients with sarcopenia based on BIA (p = 0.217). CONCLUSION: Muscle mass in patients with CLD may be overestimated by the BIA method compared to CT when assessing sarcopenia, especially in cases of fluid retention.

The change rate in serum nitric oxide may affect lenvatinib therapy in hepatocellular carcinoma.

BACKGROUND: Lenvatinib is appropriate for reducing the production of nitric oxide (NO) and facilitating as block angiogenesis. However, to our knowledge, there are no data that support the correlation between NO and clinical response in patients who received lenvatinib therapy for HCC. Therefore, we investigated the correlation between the change rate of NO levels and clinical responses including adverse events (AEs) after lenvatinib therapy for unresectable hepatocellular carcinoma (HCC). METHODS: This study was conducted using previously collected data from another study. We enrolled 70 patients who received lenvatinib for advanced or unresectable HCC. NO was measured by converting nitrate (NO3-) to nitrite (NO2-) with nitrate reductase, followed by quantitation of NO2- based on Griess reagent. To determine whether lenvatinib influences NO in unresectable HCC, we evaluated the influence of the change rate of NO from baseline after administration of lenvatinib on maximal therapeutic response and SAE. RESULTS: After lenvatinib administration, a change rate in the NO from 0.27 to 4.16 was observed. There was no difference between the clinical response to lenvatinib and the change rate of NO (p = 0.632). However, the change rate of NO was significantly lower in patients with AEs than in those without AEs (p = 0.030). When a reduction in NO rate of < 0.8 was defined as a clinically significant reduction of NO (CSRN), the CSRN group had significantly worse progression-free survival (PFS) and overall survival (OS) than the non-CSRN group (p = 0.029 and p = 0.005, respectively). CONCLUSION: Decreased NO levels were associated with the occurrence of AEs and worse prognosis after lenvatinib administration. Change rate in serum NO can be used as predictive markers in patients receiving lenvatinib therapy for HCC.

Phylogenic analysis of new viral cluster of large phages with unusual DNA genomes containing uracil in place of thymine in gene-sharing network, using phages S6 and PBS1 and relevant uncultured phages derived from sewage metagenomics.

Bacteriophages (phages) are the most diverse and abundant life-form on Earth. Jumbophages are phages with double-stranded DNA genomes longer than 200 kbp. Among these, some jumbophages with uracil in place of thymine as a nucleic acid base, which we have tentatively termed "dU jumbophages" in this study, have been reported. Because the dU jumbophages are considered to be a living fossil from the RNA world, the evolutionary traits of dU jumbophages are of interest. In this study, we examined the phylogeny of dU jumbophages. First, tBLASTx analysis of newly sequenced dU jumbophages such as Bacillus phage PBS1 and previously isolated Staphylococcus phage S6 showed similarity to the other dU jumbophages. Second, we detected the two partial genome sequences of uncultured phages possibly relevant to dU jumbophages, scaffold_002 and scaffold_007, from wastewater metagenomics. Third, according to the gene-sharing network analysis, the dU jumbophages, including phages PBS1 and S6, and uncultured phage scaffold_002 formed a cluster, which suggested a new viral subfamily/family. Finally, analyses of the phylogenetic relationship with other phages showed that the dU jumbophage cluster, which had two clades of phages infecting Gram-negative and Gram-positive bacteria, diverged from the single ancestral phage. These findings together with previous reports may imply that dU jumbophages evolved from the same origin before divergence of Gram-negative and Gram-positive bacteria.

Fecal microbiota transplantation as therapy for recurrent Clostridioides difficile infection is associated with amelioration of delirium and accompanied by changes in fecal microbiota and the metabolome.

Recurrent Clostridioides difficile infection (rCDI) is a frustrating condition that may affect a person's quality of life for months. Microbiome-based therapy such as fecal microbiota transplantation (FMT) has been effective for the treatment of rCDI by correcting the imbalance of the gut microbiota. Appropriate antibiotic treatment is recommended for at least two recurrences before offering FMT. Here, we report the case of a 92-year-old woman who experienced five recurrences of Clostridioides difficile infection (CDI) (six episodes in total) complicated by dementia and delirium, both of which were dramatically improved by FMT, which was associated with alterations in fecal microbiota and the metabolome. Analyses of whole microbial communities and metabolomic analyses were performed on stool specimens collected from the patient on the first episode, the third episode, the day of FMT (before FMT), and 2, 8, and 23 weeks after the FMT and from the donor. The patient had various fecal dysbioses on the first and third episodes and on the day of FMT. Two weeks after FMT, diversity of the gut bacteriome as well as the virome increased dramatically and was reflected in a positive clinical outcome for this patient. Metabolomic analysis revealed that short-chain fatty acids, which have been reported to be associated with improved memory function, were increased after FMT.

Gene Expression Profiles of Human Cerebral Organoids Identify PPAR Pathway and PKM2 as Key Markers for Oxygen-Glucose Deprivation and Reoxygenation.

Ischemic stroke is one of the most common neurological diseases. However, the impact of ischemic stroke on human cerebral tissue remains largely unknown due to a lack of ischemic human brain samples. In this study, we applied cerebral organoids derived from human induced pluripotent stem cells to evaluate the effect of oxygen-glucose deprivation/reoxygenation (OGD/R). Pathway analysis showed the relationships between vitamin digestion and absorption, fat digestion and absorption, peroxisome proliferator-activated receptor (PPAR) signaling pathway, and complement and coagulation cascades. Combinational verification with transcriptome and gene expression analysis of different cell types revealed fatty acids-related PPAR signaling pathway and pyruvate kinase isoform M2 (PKM2) as key markers of neuronal cells in response to OGD/R. These findings suggest that, although there remain some limitations to be improved, our ischemic stroke model using human cerebral organoids would be a potentially useful tool when combined with other conventional two-dimensional (2D) mono-culture systems.

27-Hydroxycholesterol regulates human SLC22A12 gene expression through estrogen receptor action.

The excretion and reabsorption of uric acid both to and from urine are tightly regulated by uric acid transporters. Metabolic syndrome conditions, such as obesity, hypercholesterolemia, and insulin resistance, are believed to regulate the expression of uric acid transporters and decrease the excretion of uric acid. However, the mechanisms driving cholesterol impacts on uric acid transporters have been unknown. Here, we show that cholesterol metabolite 27-hydroxycholesterol (27HC) upregulates the uric acid reabsorption transporter URAT1 encoded by SLC22A12 via estrogen receptors (ER). Transcriptional motif analysis showed that the SLC22A12 gene promoter has more estrogen response elements (EREs) than other uric acid reabsorption transporters such as SLC22A11 and SLC22A13, and 27HC-activated SLC22A12 gene promoter via ER through EREs. Furthermore, 27HC increased SLC22A12 gene expression in human kidney organoids. Our results suggest that in hypercholesterolemic conditions, elevated levels of 27HC derived from cholesterol induce URAT1/SLC22A12 expression to increase uric acid reabsorption, and thereby, could increase serum uric acid levels.

Analysis of a plasmid encoding botulinum neurotoxin type G gene in Clostridium argentinense.

Clostridium argentinense produces botulinum neurotoxin type G (BoNT/G). We sequenced and analyzed the plasmid harboring the bont/G gene, designated pCAG, in C. argentinense strain 2740. The pCAG consisted of 140,070 bp containing the bont/G gene cluster. Although this gene cluster showed high similarities in its DNA sequence and ORF arrangement to those of other bont gene clusters, the other regions of the plasmid did not. A phylogenetic study suggested that pCAG had a unique evolutionary history compared with other clostridial bont-harboring plasmids. This suggests that pCAG is possibly a novel type of plasmid expressing the bont/G gene in C. argentinense.

Inhibition of the ATR kinase enhances 5-FU sensitivity independently of nonhomologous end-joining and homologous recombination repair pathways.

The anticancer agent 5-fluorouracil (5-FU) is cytotoxic and often used to treat various cancers. 5-FU is thought to inhibit the enzyme thymidylate synthase, which plays a role in nucleotide synthesis and has been found to induce single- and double-strand DNA breaks. ATR Ser/Thr kinase (ATR) is a principal kinase in the DNA damage response and is activated in response to UV- and chemotherapeutic drug-induced DNA replication stress, but its role in cellular responses to 5-FU is unclear. In this study, we examined the effect of ATR inhibition on 5-FU sensitivity of mammalian cells. Using immunoblotting, we found that 5-FU treatment dose-dependently induced the phosphorylation of ATR at the autophosphorylation site Thr-1989 and thereby activated its kinase. Administration of 5-FU with a specific ATR inhibitor remarkably decreased cell survival, compared with 5-FU treatment combined with other major DNA repair kinase inhibitors. Of note, the ATR inhibition enhanced induction of DNA double-strand breaks and apoptosis in 5-FU-treated cells. Using gene expression analysis, we found that 5-FU induced the activation of the intra-S cell-cycle checkpoint. Cells lacking BRCA2 were sensitive to 5-FU in the presence of ATR inhibitor. Moreover, ATR inhibition enhanced the efficacy of the 5-FU treatment, independently of the nonhomologous end-joining and homologous recombination repair pathways. These findings suggest that ATR could be a potential therapeutic target in 5-FU-based chemotherapy.

Brainstem Organoids From Human Pluripotent Stem Cells.

The brainstem is a posterior region of the brain, composed of three parts, midbrain, pons, and medulla oblongata. It is critical in controlling heartbeat, blood pressure, and respiration, all of which are life-sustaining functions, and therefore, damages to or disorders of the brainstem can be lethal. Brain organoids derived from human pluripotent stem cells (hPSCs) recapitulate the course of human brain development and are expected to be useful for medical research on central nervous system disorders. However, existing organoid models are limited in the extent hPSCs recapitulate human brain development and hence are not able to fully elucidate the diseases affecting various components of the brain such as brainstem. Here, we developed a method to generate human brainstem organoids (hBSOs), containing midbrain/hindbrain progenitors, noradrenergic and cholinergic neurons, dopaminergic neurons, and neural crest lineage cells. Single-cell RNA sequence (scRNA-seq) analysis, together with evidence from proteomics and electrophysiology, revealed that the cellular population in these organoids was similar to that of the human brainstem, which raises the possibility of making use of hBSOs in investigating central nervous system disorders affecting brainstem and in efficient drug screenings.